Studies on the Surface Properties of Biodegradable Polymer ... · Rinku Tuli B.Pharm, M.Pharm This thesis is submitted in fulfilment of the requirements for the degree of Doctor of

Studies on the Surface Properties of Biodegradable Polymer

Carriers in Respiratory Delivery of Drug from

Dry Powder Inhaler Formulations

Rinku Tuli

B.Pharm, M.Pharm

This thesis is submitted in fulfilment of the requirements for the degree of

Doctor of Philosophy

Institute of Health and Biomedical Innovation

School of Clinical Sciences

Faculty of Health

Queensland University of Technology

Brisbane, Australia

2012

Abstract

III

Abstract

Dry Powder Inhaler (DPI) technology has a significant impact in the treatment of

various respiratory disorders. DPI formulations consist of a micronized drug (<5µm)

blended with an inert coarse carrier, for which lactose is widely used to date. DPIs

are one of the inhalation devices which are used to target the delivery of drugs to the

lungs. Drug delivery via DPI formulations is influenced by the physico-chemical

characteristics of lactose particles such as size, shape, surface roughness and

adhesional forces. Commercially available DPI formulations, which utilise lactose as

the carrier, are not efficient in delivering drug to the lungs. The reasons for this are

the surface morphology, adhesional properties and surface roughness of lactose.

Despite several attempts to modify lactose, the maximum efficient drug delivery to

the lungs remains limited; hence, exploring suitable alternative carriers for DPIs is of

paramount importance. Therefore, the objective of the project was to study the

performance of spherical polymer microparticles as drug carriers and the factors

controlling their performance.

This study aimed to use biodegradable polymer microspheres as alternative carriers

to lactose in DPIs for achieving efficient drug delivery into the lungs. This project

focused on fabricating biodegradable polymer microparticles with reproducible

surface morphology and particle shape. The surface characteristics of polymeric

carriers and the adhesional forces between the drug and carrier particles were

investigated in order to gain a better understanding of their influence on drug

dispersion. For this purpose, two biodegradable polymers- polycaprolactone (PCL)

and poly (DL-lactide-co-glycolide) (PLGA) were used as the carriers to deliver the

anti-asthmatic drug - Salbutamol Sulphate (SS).

The first study conducted for this dissertation was the aerosolization of SS from

mixtures of SS and PCL or PLGA microparticles. The microparticles were fabricated

using an emulsion technique and were characterized by laser diffraction for particle

size analysis, Scanning Electron Microscopy (SEM) for surface morphology and X-

ray Photoelectron Spectroscopy (XPS) to obtain surface elemental composition. The

dispersion of the drug from the DPI formulations was determined by using a Twin

Stage Impinger (TSI). The Fine particle Fraction (FPF) of SS from powder mixtures

was analyzed by High Performance Liquid Chromatography (HPLC).

Abstract

IV

It was found that the drug did not detach from the surface of PCL microspheres. To

overcome this, the microspheres were coated with anti-adherent agents such as

magnesium stearate and leucine to improve the dispersion of the drug from the

carrier surfaces. It was found that coating the PCL microspheres helped in

significantly improving the FPF of SS from the PCL surface. These results were in

contrast to the PLGA microspheres which readily allowed detachment of the SS from

their surface. However, coating PLGA microspheres with antiadherent agents did not

further improve the detachment of the drug from the surface. Thus, the first part of

the study demonstrated that the surface-coated PCL microspheres and PLGA

microspheres can be potential alternatives to lactose as carriers in DPI formulations;

however, there was no significant improvement in the FPF of the drug.

The second part of the research studied the influence of the size of the microspheres

on the FPF of the drug. For this purpose, four different sizes (25 µm, 48 µm, 100 µm

and 150 µm) of the PCL and PLGA microspheres were fabricated and characterized.

The dispersion of the drug from microspheres of different sizes was determined. It

was found that as the size of the carrier increased there was a significant increase in

the FPF of SS. This study suggested that the size of the carrier plays an important

role in the dispersion of the drug from the carrier surface.

Subsequent experiments in the third part of the dissertation studied the surface

properties of the polymeric carrier. The adhesion forces existing between the drug

particle and the polymer surfaces, and the surface roughness of the carriers were

quantified using Atomic Force Microscopy (AFM). A direct correlation between

adhesion forces and dispersion of the drug from the carrier surface was observed

suggesting that adhesion forces play an important role in determining the detachment

potential of the drug from the carrier surface. However, no direct relationship

between the surface roughness of the PCL or PLGA carrier and the FPF of the drug

was observed.

In conclusion, the body of work presented in this dissertation demonstrated the

potential of coated PCL microspheres and PLGA microspheres to be used in DPI

formulations as an alternative carrier to sugar based carriers. The study also

emphasized the role of the size of the carrier particles and the forces of interaction

Abstract

V

prevailing between the drug and the carrier particle surface on the aerosolization

performances of the drug.

Keywords

VI

Keywords

Dry Powder Inhalers

Biodegradable polymers

Polycaprolactone

Poly (DL-lactide-co-glycolide)

Polymeric drug carrier

Adhesion forces

Microparticles

Table of Contents

VII

Table of Contents

Abstract III

Keywords VI

Table of Contents VII

List of Figures XV

List of Tables XX

List of Abbreviations XXII

Statement of Original Authorship XXV

Acknowledgements XXVI

Dedications XXVIII

Journal Publications XXIX

Chapter 1 Introduction 1

1.1. Background 3

1.2. Aims of the project 6

1.2.1. Key aims 6

1.2.2. Specific aims 7

Chapter 2 Literature Review 9

2.1. Introduction 11

2.2. Respiratory delivery 11

2.2.1. Introduction 11

2.2.2. Organization of the Respiratory System 11

2.2.3. Bronchial Asthma 13

2.2.3.1. Background on asthma 13

2.2.3.2. Drugs commonly used to treat asthma 13

2.2.4. Drug deposition 14

Table of Contents

VIII

2.2.4.1. Inertial impaction 14

2.2.4.2. Gravitational sedimentation 15

2.2.4.3. Brownian diffusion 15

2.2.4.4. Electrostatic precipitation 15

2.2.4.5. Interception 15

2.3. Drug delivery from dry powder inhalers 15

2.3.1. Devices 16

2.3.2. Formulation 17

2.3.3. Drug detachment 18

2.3.3.1. Impact based detachment (Mechanical forces) 18

2.3.3.2. Fluid based detachment 19

2.3.4. Particle characteristics 19

2.3.4.1. Size of the drug 19

2.3.4.1.1. Aerodynamic diameter and Dynamic shape factor 19

2.3.4.2. Carrier size 20

2.3.4.2.1. Polydispersity 21

2.3.4.3. Carrier shape 22

2.3.4.4. Crystallinity and Polymorphism 22

2.3.4.5. Moisture Content and Hygroscopicity 22

2.4. Surface properties 22

2.4.1. Surface area 22

2.4.2. Surface Morphology and Roughness 23

2.4.3. Adhesion force / Forces of Interaction 24

2.4.3.1. Electrical forces 24

2.4.3.1.1. Contact potential 24

2.4.3.1.2. Coulombic forces 25

2.4.3.2. Non-electrical forces 25

Table of Contents

IX

2.4.3.2.1. Intermolecular forces 25

2.4.3.2.2. Capillary forces 26

2.4.3.2.3. Solid bridging between particles 26

2.4.4. Measurement of adhesion forces 27

2.4.4.1. Atomic Force Microscope (AFM) 27

2.4.4.2. Factors affecting adhesion force and drug dispersion 32

2.4.4.2.1. Particle size and shape 32

2.4.4.2.2. Surface free energy 33

2.4.4.2.3. Relative Humidity (RH) 34

2.4.4.2.4. Surface roughness 35

2.5. Ternary components 38

2.5.1. Magnesium stearate 38

2.5.2. Leucine 39

2.5.3. Other ternary components 40

2.6. Use of modified lactose as carriers in DPIs 40

2.7. Reasons for inefficient drug delivery with lactose 42

2.8. Alternative carriers 44

2.8.1. Sugars other than lactose 44

2.8.2. Lipidic vehicles 45

2.9. Proposing polymers as alternative to sugars 45

2.10. Polymers in pulmonary drug delivery 46

2.11. Hypothesis that polymers can fill in the gap 49

Chapter 3 General Methods 51

3.1. Materials 53

3.1.1. Model drug 53

3.1.2. Carrier 54

3.1.2.1. Polycaprolactone (PCL) 54

Table of Contents

X

3.1.2.2. Poly (DL-lactide-co- glycolide) (PLGA) 54

3.1.2.3. Lactose 55

3.1.3. Ternary Components 56

3.1.4. Solvents and Chemicals 56

3.2. General methods 56

3.2.1. Microparticle preparation 56

3.2.1.1. Oil in water solvent evaporation technique 56

3.2.1.1.1. Using an overhead stirrer 56

3.2.1.1.2. Using a homogenizer 57

3.2.1.2. Electrospraying 57

3.2.2. Coating of microspheres 58

3.2.2.1. Dry powder coating 58

3.2.2.2. Solution coating 58

3.2.3. Particle size measurement 58

3.2.3.1. For microspheres prepared by emulsion technique 58

3.2.3.2. For microspheres prepared by electrospraying 59

3.2.4. Scanning electron microscopy (SEM) 59

3.2.5. Energy Dispersive X-ray Analysis (EDX) 59

3.2.6. X-Ray Photoelectron Spectroscopy (XPS) 60

3.2.7. Adhesion force 60

3.2.7.1. Measurement of spring constant 60

3.2.7.2. Sample preparation 60

3.2.7.2.1. Plasma cleaning of the cover slips 60

3.2.7.2.2. Cleaning of the glass slides 61

3.2.7.2.3. Polymer microspheres 61

3.2.7.2.4. Polymer films (Spin coating) 61

3.2.7.2.5. Functionalization of silica probe with SS 62

Table of Contents

XI

3.2.7.3. Force Measurement 62

3.2.8. Surface Roughness 63

3.2.8.1. Imaging of polymer particles and films 63

3.2.8.2. Roughness measurement 63

3.2.9. Surface energy determination 64

3.3. Analytical methods 65

3.3.1. UV spectrophotometric assay 65

3.3.2. HPLC assay 65

3.4. Powder formulation 65

3.4.1. Powder mixing 65

3.4.2. Homogeneity tests 65

3.4.3. In vitro aerosol deposition 66

3.4.4. Statistical Analysis 67

Chapter 4 Method Validation 69

4.1. Summary 71

4.2. Analytical validation 71

4.2.1. Spectrophotometric assay 71

4.2.2. HPLC 72

4.3. Powder Formulations 74

4.3.1. Homogeneity test by UV 74

4.3.2. In vitro aerosol deposition 75

4.3.2.1. Inter-batch and intra-batch variability 75

4.4. Adhesion Force measurements 76

4.4.1. Functionalization of silica probe with SS 76

4.4.2. Individual adhesion forces by silica probe 79

4.4.3. Individual adhesion forces by SS probe 80

Table of Contents

XII

Chapter 5 Drug Dispersion from PCL and PLGA Microspheres 83


5.2. Results and Discussion 85

5.2.1. Particle sizing and drug dispersion from lactose 85

5.2.2. Characterization of microspheres and drug dispersion 92

5.2.2.1. PCL microspheres 92

5.2.2.2. PLGA microspheres 93

5.2.3. XPS analysis of microspheres 93



5.2.4. Characterization of microspheres after surface-coating and drug

dispersion 101



5.3. Conclusion 111

Chapter 6 Effect of the Size of the Carrier on the Dispersion of SS

113



6.2.1. Particle size and Morphology 115

6.2.1.1. Salbutamol Sulfate (SS) 115

6.2.1.2. Carriers 116

6.2.1.2.1. PCL microspheres 116

6.2.1.2.2. PLGA microspheres 119

6.2.1.3. Interactive mixtures of SS and Carriers 121

6.2.1.3.1. Interactive mixtures of SS and PCL carrier 121

6.2.1.3.2. Interactive mixtures of SS and PLGA carriers 123

Table of Contents

XIII

6.2.2. Effect of carrier size on drug dispersion 127

6.2.2.1. In vitro TSI deposition of SS from the carrier 127

6.2.2.1.1. Drug dispersion from PCL carrier 127

6.2.2.1.2. Drug dispersion from PLGA carrier 130

6.2.2.2. Influence of inherent carrier size on dispersion 131

6.3. Conclusion 133

Chapter 7 Characterization of PCL and PLGA Surfaces and their

Relationship with SS dispersion 135



7.2.1. Determination of adhesion forces 138

7.2.1.1. Adhesion forces with the PCL carrier 139

7.2.1.2. Adhesion forces with the PLGA carrier 143

7.2.2. XPS analysis of the films 147

7.2.2.1. XPS analysis of coated PCL films 147

7.2.2.2. XPS analysis of coated PLGA films 148

7.2.3. Relationship between adhesion force and FPF 149

7.2.3.1. PCL carriers 149

7.2.3.2. PLGA carriers 151

7.2.4. Surface Roughness 151

7.2.4.1. Surface roughness of PCL microspheres 152

7.2.4.2. Surface roughness of PCL films 153

7.2.4.3. Surface roughness of PLGA microspheres 154

7.2.4.4. Surface roughness of PLGA films 156

7.2.5. Relationship between FPF and RMS 157

7.2.5.1. PCL carriers 157

7.2.5.2. PLGA carriers 158

Table of Contents

XIV

7.2.6. Surface free energy determination 158

7.2.7. Difference between the PCL and PLGA polymers 160

7.2.7.1. Surface free energy 161

7.2.7.2. Glass transition temperature (Tg) 161

7.2.7.3. Elasticity of the polymer and the inhaler wall 162

7.3. Conclusion 162

Chapter 8 Overall Conclusions and Further Directions 163

8.1. Summary and Conclusions 165

8.2. Future Directions 167

Bibliography 169

List of Figures

XV

List of Figures

Figure 2.1. The Components of the Respiratory System [58] ..................................12

Figure 2.2 Principles of DPI design ........................................................................17

Figure 2.3 Schematic diagram of AFM where (1) Laser diode, (2) Cantilever, (3)

Mirror, (4) Position sensitive photodetector, (5) Electronics and (6)

Scanner with sample .............................................................................29

Figure 2.4 Anatomy of a force-distance curve [131] ...............................................30

Figure 2.5 The effect of surface roughness on the contact area between particles and

surfaces. (a)The ideal sphere on flat surface (b) Asperities much smaller

than the particle lead to reduction in the contact area (c) Asperities

comparable in size to the particles can lead to increase or decrease in the

actual contact area [166] .......................................................................36

Figure 3.1 Chemical structure of Salbutamol Sulfate (SS) ......................................53

Figure 3.2 Structure of Polycaprolactone (PCL) .....................................................54

Figure 3.3 Structure of Poly (DL-lactide-co- glycolide) (PLGA) ............................55

Figure 3.4 Structure of Lactose ..............................................................................55

Figure 3.5 Twin Stage Impinger (TSI) apparatus ....................................................67

Figure 4.1 Ultraviolet scan of SS in water over the range of 190-400 nm for the

determination of the wavelength of maximum absorbance (λmax= 276

nm) .......................................................................................................71

Figure 4.2 Beer‟s Law calibration curve of SS (n=3) ..............................................72

Figure 4.3 A representative HPLC chromatogram of Salbutamol Sulfate (0.6 µg/ml)

showing retention time at 4.224 minutes ...............................................73

Figure 4.4 A representative HPLC chromatogram of Salbutamol Sulfate (100 µg/ml)

showing retention time at 4.314 minutes ...............................................73

Figure 4.5 HPLC calibration curve of SS (n=5) ......................................................74

Figure 4.6 Inter-batch comparison of TSI deposition of SS for 2.5% mixture of SS

and PCL microspheres coated with 1% magnesium stearate solution

(n=5).....................................................................................................76

Figure 4.7 Intra-batch comparison of TSI deposition of SS for 2.5% mixture of SS

and PCL microspheres coated with 1% magnesium stearate solution

(n=5).....................................................................................................76

List of Figures

XVI

Figure 4.8 XPS survey scan of (A) Silica spheres (B) Silica spheres coated with SS

for 5 minutes (C) Silica spheres coated with SS for 10 minutes and (D)

Silica spheres coated with SS for 30 minutes ........................................ 77

Figure 4.9 SEM images of (A)Silica sphere at 6250×, (B)Silica sphere coated with

SS for 5 minutes at 10,000×, (C) Silica sphere coated with SS for 10

minutes at 10,000× and (D) Silica sphere coated with SS for 30 minutes

at 9375× ............................................................................................... 78

Figure 4.10 SEM image of the uncoated cantilever ................................................ 79

Figure 4.11 SEM image of the cantilever coated with SS ....................................... 79

Figure 4.12 Adhesion forces measured at a particular site on three different PCL

microspheres over a period of 30 minutes determined by AFM using

3.5 µm silica probe ............................................................................. 80


microspheres over a period of 30 minutes determined by AFM using

3.5 µm silica probe functionalized with SS ......................................... 81

Figure 5.1 Particle size distribution of Lactose particles, PCL microspheres and

PLGA microspheres ........................................................................... 86

Figure 5.2 SEM images of (A) Lactose (Aeroflo-95) at 500X , (B) SS at 10,000X

and (C) SS-Lactose mixture at 4000X .................................................. 87

Figure 5.3 EDX spectrum of SS-Lactose mixture ................................................... 88

Figure 5.4 SEM images of (A) PCL microspheres at 2188X and (B) SS-PCL mixture

at 2187X. Note the high surface coverage of SS on the PCL particle

compared to the lactose particle in Figure 5.2C..................................... 92

Figure 5.5 SEM images of (A) PLGA microspheres at 2344X and (B) SS- PLGA

mixture at 2813X. Note that the surface coverage of SS on the PLGA

particle which is to a lesser extent than the PCL particle in Figure

5.4C..........................................................................................................93

Figure 5.6 XPS scan of (A) PCL powder and (B) PCL microspheres ..................... 94

Figure 5.7 Particle size distribution of PCL microspheres fabricated by

electrospraying ..................................................................................... 96

Figure 5.8 SEM images of (A) PCL microspheres prepared by electrospraying at

15,994X and (B) SS- PCL mixture at 8000X. ....................................... 97

Figure 5.9 XPS scan of (A) PLGA powder and (B) PLGA microspheres ............. 100

List of Figures

XVII

Figure 5.10 SEM images of (A) 1% MgSt solution coated PCL microspheres at

1852X, (B) 2% MgSt solution coated PCL microspheres at 1250X, (C)

1% Leucine solution coated PCL microspheres at 1250X, (D) 2%

Leucine solution coated PCL microspheres at 938X, (E) 1% MgSt

powder coated PCL microspheres at 1000X (F) 2% MgSt powder

coated PCL microspheres at 700X, (G) 1% Leucine powder coated PCL

microspheres at 1099X and (H) 2% Leucine powder coated PCL

microspheres at 1583X ..................................................................... 103

Figure 5.11 SEM images of mixtures of 2.5% SS and (A) 1% MgSt solution coated

PCL microspheres at 1500X, (B) 2% MgSt solution coated PCL

microspheres at 1875X, (C) 1% Leucine solution coated PCL

microspheres at 1327X, (D) 2% Leucine solution coated PCL

microspheres at 2400X, (E) 1% MgSt powder coated PCL microspheres

at 1500X, (F) 2% MgSt powder coated PCL microspheres at 1828X,

(G) 1% Leucine powder coated PCL microspheres at 938X and (H) 2%

Leucine powder coated PCL microspheres at 847X .......................... 104

Figure 5.12 EDX spectrum of interactive mixture of 2.5% SS and PCL microspheres

coated with 1% MgSt solution .......................................................... 105

Figure 5.13 FPF of coated PCL microspheres ....................................................... 107

Figure 5.14 SEM images of (A) 1% MgSt solution coated PLGA microspheres at

3906X, (B) 1% Leucine solution coated PLGA microspheres at 8000X,

(C) 1% MgSt powder coated PLGA microspheres at 4405X and (D) 1%

Leucine powder coated PLGA microspheres at 2400X ..................... 108


PLGA microspheres at 4501X, (B) 1% Leucine solution coated PLGA

microspheres at 3750X, (C) ) 1% MgSt powder coated PLGA

microspheres at 5244X and (D) 1% Leucine powder coated PLGA

microspheres at 5859X........................................................................109

Figure 5.16 FPF of uncoated and coated PLGA microspheres .............................. 110

Figure 6.1 Particle size distribution of SS, n=5 ..................................................... 116

Figure 6.2 SEM images of SS powder at 2000× and 10,000× respectively ............ 116

Figure 6.3 Particle size distribution of four different batches of PCL microspheres,

n=5 ..................................................................................................... 117

List of Figures

XVIII

Figure 6.4 SEM images of PCL microspheres coated with MgSt of various sizes: (A)

25 µm (4687×), (B) 48 µm (4442×), (C) 104 µm (1852×) and (D) 150

µm (946×) .......................................................................................... 118

Figure 6.5 SEM images of PCL microspheres coated with leucine of various sizes:

(A) 25 µm (6392×), (B) 48 µm (10,535×), (C) 104 µm (1250×) and (D)

150 µm (1200×) ................................................................................. 119

Figure 6.6 Particle size distribution of four different batches of PLGA microspheres,

n=5 ..................................................................................................... 120

Figure 6.7 SEM images of PLGA microspheres of various sizes: (A) 20 µm

(10,000×), (B) 45 µm (3662×), (C) 90 µm (2344×) and (D) 150 µm

(1500×) ............................................................................................ 120

Figure 6.8 SEM images of interactive mixtures of 2.5% SS and PCL microspheres

coated with MgSt of various sizes: (A) 25 µm (3418×), (B) 48 µm

(4194×), (C) 104 µm (1500×) and (D) 150 µm (800×) ........................ 122


coated with leucine of various sizes: (A) 25 µm (7786×), (B) 48 µm

(6662×), (C) 104 µm (1327×) and (D) 150 µm (1131×) ...................... 123

Figure 6.10 SEM images of interactive mixtures of 2.5% SS and PLGA

microspheres of various sizes: (A) 20 µm (7500×),(B) 45 µm

(4000×), (C) 90 µm (2813×) and (D) 150 µm (1200×) ................... 124

Figure 6.11 The relationship between the FPF of SS and the size of the PCL

microspheres coated with 1% and 2% MgSt solution respectively, n=5

...................................................................................................... 130


microspheres coated with 1% and 2% leucine solution respectively,

n=5 ................................................................................................ 130

Figure 6.13 The relationship between the FPF of SS and the size of the PLGA

microspheres, n=5 .......................................................................... 131

Figure 7.1 Force distribution map of the adhesion forces from AFM with (A) 8X8

force points for films and (B) 32X32 force points for microspheres .... 138

Figure 7.2 An example of force-distance curve obtained from AFM. .................. 138

Figure 7.3 Adhesion force measurements of (A) Uncoated silica sphere and PCL

microspheres, (B) Silica sphere coated with SS and PCL microspheres,

List of Figures

XIX

(C) Uncoated silica sphere and PCL films and (D) Silica sphere coated

with SS and PCL films, n=5 ............................................................. 139

Figure 7.4 Adhesion force measurements of (A) Uncoated silica sphere and PLGA

microspheres, (B) Silica sphere coated with SS and PLGA microspheres,

(C) Uncoated silica sphere and PLGA films and (D) Silica sphere coated

with SS and PLGA films, n=5 ............................................................. 144

Figure 7.5 Survey spectra of (A) three layered film of PCL, PVA and MgSt and (B)

three layered film of PCL, PVA and Leucine ...................................... 148

Figure 7.6 Survey spectra of (A) three layered film of PLGA, PVA and MgSt and

(B) three layered film of PLGA, PVA and Leucine ............................ 149

Figure 7.7 Surface topography of PCL microspheres in (A) 2D view, (B) 3D view

and (C) Section view........................................................................... 153

Figure 7.9 Surface topography of PLGA microspheres in (A) 2D view, (B) 3D view,

(C) Section view and (D) Close view of the specs on the surface of the

PLGA microspheres............................................................................ 156

Figure 7.10 Surface roughness measurements of coated PLGA films and

microspheres, n=5.....................................................................................................157

Figure 7.11 Dispersive surface free energies of PCL and PLGA microspheres ..... 159

List of Tables

XX

List of Tables

Table 3.1 Speed of the spin coater and time intervals of spinning of each sample....62

Table 4.1 Accuracy and precision of the UV assay for SS (n=3)..............................72

Table 4.2 Accuracy and precision of the HPLC assay for SS (n=5)..........................74

Table 4.3 Homogeneity tests on five batches of 2.5% Drug-Carrier mixture (n=20)

.............................................................................................................................. .75

Table 5.1 Homogeneity tests on different batches of 2.5% Drug-Carrier mixture

(n=20)..........................................................................................................................88

Table 5.2 Fine Particle Fraction (FPF) of SS .......................................................... 90

Table 5.3 TSI data of SS from different carriers ..................................................... 91

Table 5.4 XPS results of PCL and PLGA microspheres coated with MgSt and

Leucine solution ................................................................................... 99


(n=20) .................................................................................................................. 125

Table 6.2 The % FPF of SS from 1% and 2% MgSt coated PCL microspheres of four

different sizes having different surface areas ......................................... 126

Table 6.3 The % FPF of SS from 1% and 2% Leucine coated PCL microspheres of

four different sizes having different surface areas ................................. 126

Table 6.4 The % FPF of SS from PLGA microspheres of four different sizes having

different surface areas ........................................................................... 127

Table 6.5 TSI data of SS from different carriers ................................................... 129

Table 7.1 Adhesion force measurements of PCL and coated PCL microspheres with

uncoated and coated silica sphere, n=5 ............................................... 143

Table 7.2 Adhesion force measurements of PCL and coated PCL films with


Table 7.3 Adhesion force measurements of PLGA and coated PLGA microspheres

with uncoated and coated silica sphere, n=5 ....................................... 146

Table 7.4 Adhesion force measurements of PLGA and coated PLGA films with


Table 7.5 Surface roughness measurements of the PCL and coated PCL

microspheres, n=5..................................................................................153

List of Tables

XXI

Table 7.6 Surface roughness measurements of the PCL and coated PCL films, n=5

............................................................................................................................. 154

Table 7.7 Surface roughness measurements of the PLGA and coated PLGA

microspheres, n=5 ............................................................................... 155

Table 7.8 Surface roughness measurements of the PLGA and coated PLGA films,

n=5 ..................................................................................................... 157

List of Abbreviations

XXII


% Percent

µ Micron

µg Microgram

µL Microliter

µm Micrometer

AFM Atomic Force Microscopy

BDP Beclomethasone dipropionate

BP British Pharmacopoeia

cm centimetre

CM Chitosan Microparticles

CV Coefficient of Variation

DCM Dichloromethane

DPI Dry Powder Inhaler

DSCG Disodium Cromoglycate

ED Emitted Dose

EDX Energy Dispersive X-ray Analysis

E-SPART Electrical Single Particle Aerodynamic Relaxation Time

eV Electron Volts

FCA Force Control Agents

FDA Food and Drug Administration

FPF Fine Particle Fraction

gm Gram

HPLC High Performance Liquid Chromatography

IGC Inverse Gas Chromatography

IPA Isopropyl Alcohol

IR Infrared

k Spring Constant

kV kilovolts


XXIII

LMWH Low Molecular Weight Heparin

LOD Limit of Detection

LOQ Limit of Quantification

m Meter

MDI Metered Dose Inhaler

MgSt Magnesium stearate

min minute

mL Millilitre

MMAD Mass Median Aerodynamic Diameter

MSLI Multi-Stage Liquid Impinger

MW Molecular Weight

N Particle Number

nm Nanometre

nN Nano Newton

o/w Oil in Water

PCL Polycaprolactone

PGA Poly (glycolic acid)

PLA Poly (lactic acid)

PLGA Poly (DL-lactide-co-glycolide)

PVA Polyvinyl Alcohol

r2 Regression Coefficient

RD Recovered Dose

RF Respirable Fraction

RH Relative Humidity

RMS Root Mean Square

rpm Revolution Per Minute

S1 Stage one

S2 Stage two

sccm Standard Cubic Centimetre per Minute


XXIV

SCFH Standard Cubic Feet per Hour

SEM Scanning Electron Microscopy

SLM Solid Lipid Microparticles

SS Salbutamol Sulfate

SX Salmeterol Xinafoate

Tg Glass transition temperature

TSI Twin Stage Impinger

UV Ultraviolet

VMD Volume Mean Diameter

WHO World Health Organization

XPS X-Ray Photoelectron Spectroscopy

XRD X-Ray Diffraction

λmax Wavelength of maximum absorbance

Statement of Original Authorship

XXV

Statement of Original Authorship

The work contained in this thesis has not been previously submitted to meet

requirements for an award at this or any other higher education institution. To the

best of my knowledge and belief, the thesis contains no material previously

published or written by another person except where due reference is made.

Rinku Tuli

Date:

Acknowledgements

XXVI

Acknowledgements

Knowledge is in the end based on acknowledgement

- Ludwig Wittgenstein

This piece of work would not have seen the light of the day without the support of

many individuals who have been instrumental in the completion of this project and

whom I would like to acknowledge.

First and foremost I thank Lord Almighty with folded hands for showering all His

blessings on me without which I would have not been what I am today.

I owe my deepest gratitude to my principal supervisor, Dr. Nazrul Islam for

providing me with an opportunity to pursue a PhD under him. I am thankful to him

for providing me with his constant support, encouragement and constructive criticism

to overcome the challenges encountered during this period. His help for securing the

scholarship from the University and his valuable inputs throughout the research is

highly appreciated. I am deeply indebted to Prof. Graeme George for his assiduous

guidance, inspiration and motivation throughout the tenure of my research work. He

is an embodiment of knowledge and his presence during our regular meetings and

discussions were of extreme help to me. His ideas, undying spirit and infusing the

positivity in the environment always charged me up during my low moments. The

organizational and analytical abilities and enthusiasm of Dr. Tim Dargaville inspired

me tremendously. I am highly grateful to him to help me getting initiated in the lab

during the beginning of my PhD when I was new to all the things around. He always

advised me to strike a balance between work and life. Mere words of thanks would

never be equated to the help and guidance which I have received from my

supervisory team time and again.

My earnest thanks to Queensland University of Technology (QUT) for providing me

with the opportunity and facilities to carry out research work. My heartfelt gratitude

to Faculty of Science and Technology for providing me the financial support for

undertaking this research work.

Many thanks to Mr. Matthew Mackay and Mr. Nathaniel Raup for training me on the

Malvern Mastersizer instrument for particle size analysis. I would also like to thank

Acknowledgements

XXVII

Dr. Chris Carvalho for inducting me on the HPLC instrument. My special thanks and

gratitude to Dr. Llew Rintoul for his expertise rendered in the IR analysis of the

samples. I offer my thanks to Miss Aurelie Muller for her help offered in the

electrospraying technique. My sincere thanks are due to Dr. Shayamal Das from

Monash University for conducting the IGC experiments of the samples.

I am grateful to Dr. Thor Bostrom, Dr. Loc Duong and Dr. Christina Theodoropoulos

for their help with SEM and Dr. Peter Hines for his help with EDX analysis of the

samples.

I am indebted to Dr. Barry Wood from University of Queensland (UQ) for his help

with the XPS analysis and data interpretation of the samples. My sincere thanks to

Mr. George Ganakas from UQ for all his support and help provided during the AFM

experiments.

My thanks are due to the technical staff in QUT especially Mr. David Smith for the

co-operation rendered to me.

My acknowledgment will be incomplete if I do not thank all my peers and my dear

friends for their constant support and their valuable help throughout my research

project. Their presence during the ups and downs of PhD journey was extremely

important to me.

Last but not the least I thank my family members who have been with me always as

pillars of support through the thick and thin of life. I owe my deepest gratitude for

their unflagging and unconditional support throughout my life. Without their support

I would have not reached this stage of my life.

It‟s my pleasure to thank all those who made this thesis possible. My gratitude,

respect and thanks to one and all who have helped me directly or indirectly and have

co-operated with me in my research work.

Dedications

XXVIII

Dedications

To,

Lord Almighty

and

My very loving family

Journal Publications

XXIX

Journal Publications

1. Tuli R, Dargaville T, George G, Islam N, Polycaprolactone Microspheres as

Carriers for Dry Powder Inhalers: Effect of Surface Coating on

Aerosolization of Salbutamol Sulfate, Journal of Pharmaceutical Sciences,

2012, 101 (2), 733-745.

2. Tuli R, George G, Dargaville T, Islam N, Studies on the Effect of the Size of

Polycaprolactone Microspheres for the Dispersion of Salbutamol Sulfate

from Dry Powder Inhaler Formulations, Pharmaceutical Research, DOI

10.1007/s1 1095-012-0772-y

Conference Abstracts

1. Poster presentation entitled “Effect of the Size of the Polycaprolactone

Carrier on the Dispersion of Salbutamol Sulfate in Dry Powder Inhalers” in

IHBI Inspires Postgraduate Student Conference 2011 held from 24th-25

th

November 2011 at Brisbane, Australia.

2. Poster presentation entitled “Surface coated Polycaprolactone Microspheres

as Carriers for Dry Powder Inhalers” in 2011 American Association of

Pharmaceutical Scientists (AAPS) Annual Meeting and Exposition held

from 23rd

-27th

October 2011 at Washington DC, USA

3. Oral presentation entitled “Polycaprolactone Microspheres as Carrier for Dry

Powder Inhalers: Effect of Surface Coating on Aerosolization of Salbutamol

Sulfate” was presented in Australian Pharmaceutical Science Association

(APSA) Annual Conference 2010 held from 6th

-9th December, 2010 at

Brisbane, Australia.

4. Oral presentation entitled “Studies on Efficient Respiratory Delivery of Drugs

Using Biodegradable Polycaprolactone Microspheres as Carriers for Dry

Powder Inhalers” was presented in IHBI Inspires Postgraduate Student

Conference 2010 held from 25th

-26th November 2010 at Gold Coast,

Australia.

Chapter 1 Introduction

1

CChhaapptteerr 11

IInnttrroodduuccttiioonn



3

1.1. Background

Pulmonary drug delivery is an important research area in the field of drug delivery

technology. It is the preferred mode of drug delivery in the treatment of various

disorders such as asthma and chronic obstructive pulmonary disease. The alveoli

region in the lungs has a large surface area and a highly permeable membrane for the

absorption of drugs into the blood. An advantage of pulmonary drug delivery

includes direct access of the drug to the lungs, hence it is one of the routes of choice

of drug administration of large molecules which degrade in the gastrointestinal fluid

and are subjected to first-pass metabolism in the liver. Pulmonary delivery also

utilizes minimal drug dose to produce the desired effect and provides a rapid

pharmacological response with minimal side effects [1]. It is a needle-free delivery

system capable of administering a variety of therapeutic substances [2].

Lung delivery is applicable not only for pulmonary disorders but also finds

application in the treatment of various diseases such as cancer, cystic fibrosis,

diabetes, osteoporosis and thrombosis [3-10]. Recently Islam et al have reviewed the

strategies and future prospects of pulmonary drug delivery for the management of

various neurological disorders including Parkinson‟s and Alzheimer‟s disease [11].

There are large numbers of devices available to target the delivery of drugs to the

lungs. Currently three major types of inhalers that are widely used for pulmonary

drug delivery are Nebulizers, Metered Dose Inhalers (MDIs) and Dry Powder

Inhalers (DPIs) and these devices use different mechanisms of delivering the drug

into the lungs [12].

The majority of dry powder inhalers are breath-actuated devices [13]. They are easy

to use and do not require co-ordination of actuation and inhalation. Unlike other

inhalers, DPIs do not use liquid propellants. They are portable, patient friendly and

do not require spacers [14-15]. DPIs today are an expanding area of interest of

pharmaceutical companies and are seen as the most promising mechanism for

pulmonary drug delivery.

A DPI formulation may consist of either only the drug as agglomerates or

agglomerates of drug and fine excipients with controlled flow property or the drug

blended with a suitable large carrier i.e. lactose. One of the major advantages of

formulating the system with the carrier includes increasing the bulk of the


4

formulation. This helps in delivering accurate doses of potent drugs. It contributes to

overcoming the cohesiveness of the micronized drug and improving the flow

properties of the drug-carrier mixture [16]. In carrier-based interactive mixtures the

drug particles are adhered onto the surface of lactose [17]. Efficient delivery of the

powder into the lungs from these interactive mixtures depends on decreased adhesion

between the carrier and drug, increased dispersion of the drug particles and

deposition of these drug particles into the lungs [18]. Hence, pulmonary delivery of

pharmacological agents from DPIs is dependent on the design of the device, the

formulation and the inhalation manoeuvres of the patient [19-20].

Most commercially available carrier-based DPI formulations only deliver about 20–

30% of the total dose to the lungs [21-22]. This low efficiency of DPIs is attributed

to the complex physiology of the respiratory tract, the characteristics of the powder

formulations for inhalation and the inhalation devices. In interactive mixtures,

micronized particles adhere to the surface of the carriers and produce agglomerates.

There is poor detachment of drug particles from the surface of the carrier particles

resulting in poor delivery efficiency. Effective respiratory delivery requires the

dispersion of drug from these agglomerates [23].

In the formulation aspects, drug delivery is influenced by the physico-chemical

characteristics of carrier particles such as particle size, shape and surface

morphology. Therefore, any disparities in the physical properties of the carrier lead

to variability in the Fine Particle Fraction (FPF) of the drug from the DPI

formulation which may, in turn, lead to variability in clinical performances. Thus the

physicochemical properties of carriers are important parameters in efficient delivery

of drugs from DPIs [24-26].

Commercially available inhalation grade lactose is irregular in shape with rough

surfaces which affects drug detachment during inspiration. Controlling the size,

shape and surface roughness of the lactose particles is difficult, and in turn, affects

the detachment of drug from the particles [24-26]. Thus, lactose as a carrier has its

own limitations. Hence, a number of studies have been conducted to improve the

delivery of drugs into the lung. These studies have focused on improving dispersion

of the drugs by optimizing the physico-chemical properties of the lactose carrier.

Such studies have included optimizing the carrier size [27], smoothing the carrier


5

surface [24], mixing different grades of carriers [28-29] and using lactose carriers

with different surface morphologies [24, 30]. Alternatively, modification of the

particle surfaces have been reported to improve the dispersibility of the drug from the

carrier surface using different technologies. One of the approaches is to coat the

surface of the coarse lactose by blending it with fine lactose, magnesium stearate

(MgSt) or leucine [31-35]. Common techniques for surface modification of carrier

particles include spray drying [36], encapsulation using supercritical carbon dioxide

[37], physical vapour deposition of particles in an aerosol flow reactor [38-39] and

dry process like mechanofusion [40]. However, drug dispersion from these powders

is still not satisfactory.

One of the formulation strategies is to use alternative sugars such as mannitol and

maltitol in DPIs [41]. Other sugars such as glucose, sorbitol and xylitol have also

been explored but they are hygroscopic and are not able to efficiently generate the

desirable FPF of the drug. Solid lipid microparticles (SLM) have also been

investigated in pulmonary administration as vehicles wherein the drug is

incorporated within the SLM. The traditional approach in DPI formulations is that

the drug is on the surface of the carrier but in SLMs the drug is incorporated within

the carrier thereby it acts as vehicle. However, these SLMs are preferable for high

entrapment of hydrophobic drugs and are suitable for long term treatments with an

aim to sustain the release of the drug into the lungs [42-43].

Currently, all DPI products existing in the market or which are to be launched in the

market utilize lactose as a carrier material [44]. Owing to the limitations of lactose

there is a pressing need to explore alternative carriers for DPI formulations which

may markedly improve the respiratory delivery of drugs into the lungs.

It is well-known that a curved surface with small asperities and low surface energy

generally reduces the contact area between adjacent surfaces [45-48]. Irregularly

shaped particle have more points of contact, which, in turn, leads to an increase in

cohesive and frictional forces in comparison to spherical and smooth particles.

Irregularly shaped particles also have a greater tendency for mechanical interlocking

of particles, which leads to a decrease in powder fluidity. Spherical particles have

minimal interparticulate contact, which will improve flow properties. Therefore,

spherical particles may be useful as carrier particles in DPI formulations. Indeed,


6

spherical spray-dried lactose particles have been shown to have higher deposition of

the drug prankulast hydrate (FPF: 17.8%) as compared with non-spherical lactose

with irregular surface morphologies (FPF: 3.4% - 14.7%) [30]. Hence, engineering of

carrier particles to provide a well-defined shape will be one of the important

strategies to improvise the efficacy of drug delivery with DPIs [24].

Polymers have long been used in various drug delivery technologies [49]. However,

the use of polymers as carriers in DPIs is still an unexplored area. As alternative

materials for use as carriers in DPI formulations, polymers are an attractive option.

Controlling the particle size, shape and surface roughness of polymers is much easier

[50] as compared to sugars. There are no DPIs in the market currently using

biodegradable polymers as carriers. Hence, the use of polymeric systems is an

approach that holds promise for improving the effectiveness of inhaled drugs for

both local and systemic action. They have been investigated widely in pulmonary

drug delivery to sustain the release of drugs [51-55] but have not been exploited as

carriers in DPIs. Thus, it would be worthwhile to research the use of biodegradable

polymers with controlled surface functionality as carriers for the pulmonary delivery

of drugs from powder formulation.

1.2. Aims of the project

1.2.1. Key aims

The principal aim of this study is to explore the potential of biodegradable polymers

as an alternative carrier to lactose with a view to gain a better understanding on the

polymer carrier surfaces for the development of dry powder inhaler formulations for

achieving efficient and maximum drug delivery deep into the lungs. The specific

objective of this research is to formulate microparticles of the polymers and to

investigate the effect of size and surface characteristics (morphology, surface

roughness and adhesional properties of the carriers) on the dispersion of the drug

from powder formulations where the drug is adhered on the surface of the large

polymer carriers.


7

1.2.2. Specific aims

This project focuses on the following objectives:

To produce microparticles of biodegradable polymer carrier with controlled

surface properties (size, shape, surface roughness, adhesional properties)

To investigate the surface properties of polymer carriers and relate the

outcome to drug dispersion

To investigate intrinsic adhesional forces of the polymer and drug using

Atomic Force Microscopy

In order to achieve these objectives, an anti-asthmatic drug, Salbutamol Sulfate (SS)

(inhalation grade) and biodegradable polymers, polycaprolactone (PCL) and poly

(DL-lactide-co-glycolide) (PLGA), were used. These two polymers were selected

because of the differences in their physicochemical properties.

Chapter 2 Literature Review

9

CChhaapptteerr 22

LLiitteerraattuurree RReevviieeww



11

2.1. Introduction

This chapter provides a review of the literature pertinent to the pulmonary delivery of

drugs. It outlines the organization of respiratory system and discusses the mechanism

of drug deposition. It also talks about the asthma disease and the drugs commonly

used to treat it. This chapter also details the drug delivery aspects from dry powder

inhalers which include the device and formulation aspects. It outlines the important

particle surface characteristics which affect drug delivery. It discusses the ternary

components used in DPI formulations and details the use of modified lactose as

carriers in DPIs. This chapter explores the reasons for not achieving maximum drug

delivery with lactose and describes the alternative carriers which have been used in

DPIs. It proposes the use of polymers as alternative to sugars, outlines various

polymers which have been used till date in pulmonary drug delivery and finally state

the hypothesis of the study.

2.2. Respiratory delivery

2.2.1. Introduction

Research in the field of pulmonary drug delivery has generated interest in the last

decade by using the lung as a means of delivering drugs systemically. The lung is

believed to be an ideal target for drug delivery because of its large surface area, good

vascularization, thinness of the alveolar epithelium and good capacity for solute

exchange. However the absorption of the inhaled substances depends on the

molecular weight of the substance, pH value, electrical charge, solubility and

stability of the inhaled substance [56-57].

2.2.2. Organization of the Respiratory System

The respiratory system can be divided into two components: Upper respiratory

system and Lower respiratory system (Figure 2.1). The upper respiratory system

consists of nose, nasal cavity, paranasal sinuses and pharynx. These passageways

filter, warm and humidify incoming air thereby protecting the more delicate surfaces

of the lower respiratory system and it cools and dehumidifies outgoing air. The lower

respiratory system includes the larynx, trachea, bronchi, bronchioles and alveoli of

the lungs [58].


12

Figure 2.1. The Components of the Respiratory System [58]

The respiratory tract consists of a conducting portion and respiratory portion. The

conducting portion begins at the entrance to the nasal cavity and extends through

passageways of pharynx, larynx, trachea, bronchi and bronchioles to the terminal

bronchioles. The respiratory portion of the tract includes the delicate respiratory

bronchioles and the alveoli, air-filled pockets within the lungs where all gas

exchange between air and blood occurs.

Gas exchange can occur quickly and efficiently because the distance between the

blood in an alveolar capillary and the air inside an alveolus is generally less than 1

µm and in some cases as small as 0.1 µm. The surface area of the lungs involved in

gas exchange ranges from 70 m2 to 140 m

2 which is roughly 35 times the surface

area of the body.

Filtering, warming and humidification of the inhaled air begin at the entrance to the

upper respiratory system and continue throughout the rest of the conducting system.

By the time the air reaches the alveoli, most foreign particles and pathogens have

been removed and the humidity and temperature are within acceptable limits [58].


13

The main function of the respiratory system is to provide an extensive surface area

for gas exchange between air and circulating blood and moving air to and fro from

the exchange surfaces of the lungs across the respiratory passageways (Martini

2006). Thus the disorders and diseases associated with the lungs essentially restrict

or impair the ability to breathe. Respiratory diseases range from mild and self-

limiting such as the common cold to life-threatening disorders such as bacterial

pneumonia or pulmonary embolism. However the scope of this project is confined to

anti-asthmatic drug Salbutamol Sulfate (SS) and hence asthma disease will be

discussed in the next section.

2.2.3. Bronchial Asthma

Asthma is a chronic disease characterized by recurrent attacks of breathlessness and

wheezing. It is a reversible obstructive airway disease that reverses either

spontaneously or with treatment. Attacks are brought on by spasms of smooth muscle

that lie in the walls of the smaller bronchi and bronchioles causing the passageways

to close partially. The patient has trouble exhaling and the alveoli may remain

inflated during expiration. Usually the mucous membranes which line the respiratory

passageways become irritated and secrete excessive amounts of mucus that may clog

the bronchi and bronchioles and worsen the attack [59].

Asthma varies in severity and frequency from person to person. Symptoms may

occur several times in a day or week in affected individuals, and for some people

become worse during physical activity or at night.

2.2.3.1. Background on asthma

According to World Health Organization (WHO), around 235 million people suffer

from asthma and about 255,000 cases of deaths have been reported in 2005 because

of asthma attacks. It is the most common chronic disease among children. It is not

just a public health problem for high income countries; in fact it is prevalent in all the

countries regardless of their level of development. Besides, it is a disorder which is

under-diagnosed and under-treated and hence it is a substantial burden to individuals

and families.

2.2.3.2. Drugs commonly used to treat asthma

Drugs used to treat asthma are bronchodilators and are classified as β2 adrenoreceptor

agonists, Xanthines, Muscarinic receptor antagonists and Corticosteroids.


14

β2 adrenoreceptor agonists dilate the bronchi by a direct action on β2 adrenoreceptors

on the smooth muscle. Two categories of β2 adrenoreceptor agonists are used in

asthma, Short acting agents which have onset of action within 30 minutes and

duration of action lasts for 4-6 hours. e.g. Salbutamol and Terbutaline. Long acting

agents have the duration of action for 12 hours. e.g. Salmeterol. The three naturally

occurring pharmacologically active Xanthine drugs used in the treatment of asthma

are Theophylline, Theobromine and Caffeine. The muscarinic receptor antagonists

widely used as an anti-asthmatic drug is Ipratropium. Other classes of drugs which

can be used for the treatment of asthma are anti-inflammatory drugs and

corticosteroids such as Fluticasone, Budesonide and Beclomethasone. These inhaled

corticosteroids reduce swelling and tightening in the airways. However β2

adrenoreceptor agonists form the first line drugs of choice for the treatment of

asthma [60-61].

2.2.4. Drug deposition

There are several mechanisms by which inhaled particles get deposited in the

respiratory tract; the primary mechanisms being inertial impaction, gravitational

sedimentation and Brownian diffusion. The other mechanisms by which deposition

may occur are electrostatic precipitation and interception.

Larger particles are deposited by impaction and sedimentation whereas smaller

particles are deposited primarily by diffusive transport. Particle size is the major

factor affecting drug deposition in the lung but it is also influenced by other factors

like particle characteristics (particle shape, density, electrostatic charge and

hygroscopicity), physiological factors (breathing patterns, breathing frequency), lung

anatomy (airway length, diameter, branching angles) and environmental factors

(temperature and humidity).

2.2.4.1. Inertial impaction

A particle carried in the air stream has its own momentum and when the aerosol

stream meets an obstacle or bends in the respiratory tract, the direction of the gas

flow changes. Hence the particles with high momentum may impact with the object

in front of them and leads to the deposition of the particles in the respiratory tract.

This is the main mechanism of the deposition of particles larger than 5 µm in


15

diameter. It usually occurs in the upper respiratory tract (entrance of trachea) and at

the conducting airway bifurcations [62-64].

2.2.4.2. Gravitational sedimentation

This mechanism means the settling of the particles under the action of gravity. It

occurs primarily for particles with a diameter ranging between 0.5 and 5 µm. It is a

predominant mechanism of deposition in the smaller airways, bronchi, bronchioles

and alveoli but it can also occur in the upper respiratory tract [62-64].

2.2.4.3. Brownian diffusion

Random motion of particles caused by collision with the gas molecules results in

deposition of the inhaled particle by Brownian diffusion. As the particle size

decreases, deposition by Brownian diffusion increases and is the dominant

mechanism of deposition for particles less than 0.5 µm. It mainly occurs in the acinar

region of lung but can also be found in the nose, mouth and pharyngeal airways for

very small particles (< 0.01 µm) [62-64].

2.2.4.4. Electrostatic precipitation

This occurs when the charged particles induces charges of opposite signs onto the

surfaces of the airways that are electrically conducting when normally uncharged.

Hence the charged particles become electrostatically attracted to the walls of

airways. Therefore the deposition of charged particles will be greater than the neutral

particles. It is a less common mechanism of deposition in the lungs [62-64].

2.2.4.5. Interception

This occurs when the dimensions of the particles are similar to the diameter of the

airway through which it is passing. It mostly occurs in small airways and alveoli and

is significant for particles like fibres for which the ratio between the length and

diameter is large. Such elongated particles get captured in the airways and gets

deposited there [62-64].

2.3. Drug delivery from dry powder inhalers

For the treatment of the respiratory disorders it is essential that the drugs reach the

deep lungs to elucidate the therapeutic response. For optimal efficacy the drug

delivery should be reproducible and this can be achieved by the combination of

judicious selection of the inhaler device and formulation metering [65].


16

2.3.1. Devices

The main function of the DPI device is to provide adequate delivery of inhaled drug

to the lungs. The DPI should be able to deliver high FPF of the drug and the carrier

should ideally remain in the upper airways [66].

Many different devices are available to aid in inhalation delivery. Three major types

of inhalers widely used for pulmonary drug delivery are Nebulizers, Metered Dose

Inhalers (MDIs) and Dry Powder Inhalers (DPIs) and they use different mechanisms

of delivering the drugs into the lungs [1, 12].

The first MDI (Medihaler®, Riker) was introduced in 1956 which used

chlorofluorocarbon propellants [67], later ultrasonic nebulizers dominated the market

in 1960s and about 10 years later the first DPI (Fisons Spinhaler®) was launched in

the market [68]. However, today DPIs are preferred devices for inhalation delivery

because they do not use liquid propellants, do not require spacers, do not require co-

ordination of actuation and inhalation, are portable, easy to use and patient friendly

[14-15, 69].

Based on their design, DPI devices are classified into three generation of DPIs. The

first generation DPIs are breath activated single unit dose systems. The second

generation of DPI is multi-dose DPIs (measurement of dose from a powder reservoir)

or multi-unit dose (they disperse individual doses which are pre-metered into blisters,

disks, dimples, tubes and strip by the manufacturers). The third generation DPIs

known as active devices use compressed gas or motor driven impellers or use

electronic vibration to disperse drug from the formulation [70]. Efficient delivery of

the drug from the DPIs is dependent on the design of the device, inspiratory flow rate

and deagglomeration of the drug particles [71-73].

There are number of DPI devices available on the market and few of them are in the

pipeline. There is not a single DPI device which has been significantly effective in

delivering the drugs from the formulation into the lungs. Hence researchers are trying

to improve this by changing the design of the DPI device and also by improving the

formulation [70].


17

2.3.2. Formulation

Dry powder formulations for inhalation consist of fine drug particles and coarse

carrier particles like lactose. The fine drug particles adhere to the carrier surface to

form ordered mixtures [17]. The carrier particles are used to improve the flow of the

drug particles which are usually present in a low concentration, with a usual drug-

carrier ratio of 1:67.5 (w/w) [24, 74]. The improvement in the flowability of the

powders helps in the reproducible dose metering. The carrier particles also help to

reduce the high cohesive forces among micron sized drug particles which prevents

the aggregation of the particles. Interactions between the drug and carrier particles

are mainly dependent on the physicochemical characteristics such as particle size,

shape, surface morphology, contact area and hygroscopicity [16, 75-76].

Figure 2.2 Principles of DPI design

The adhesion between carrier and drug must be sufficient for the drug-carrier blend

to be stable. Simultaneously, the adhesion between the drug-carrier has to be weak

enough to enable the detachment of drug from carrier during patient inspiration [77].

If the above two criteria are fulfilled then the drug will be able to reach the lungs

efficiently.


18

The carrier widely used in DPI formulations is lactose monohydrate [17, 27]. Lactose

in solid form can be crystalline or amorphous. Crystalline lactose can exist in one of

two distinct forms: β-lactose and α-lactose monohydrate. Crystals of α-lactose

monohydrate have a characteristic tomahawk-like shape. Crystals of pure β-lactose

have a characteristic kite-like form. They do not contain crystal water and often

referred to as anhydrous lactose. The advantages of lactose as a carrier are its ease of

availability, low price, its well-investigated toxicity profile and well-established

stability profile [65, 78].

2.3.3. Drug detachment

There are two major mechanisms by which the particles of drugs are detached from

the surface of the carrier particles. They are classified as impact based detachment

and fluid based detachment [79-82].

2.3.3.1. Impact based detachment (Mechanical forces)

This type of detachment of the drug from the carrier surface occurs when collisions

occurs between the carrier particles and the inhaler wall. As the particle impinges on

the wall surface and rebounds, it experiences a normal force due to the elasticity of

the wall and carrier material and it also experiences a tangential force due to friction.

This force is usually transmitted to the adhered drug particle through the carrier-drug

bond to impose acceleration.

The location of the drug particle on the carrier surface is an important factor which

determines the amount of force that will be required to detach the drug particle from

the carrier surface. For example if a carrier particle is travelling vertically downward

to collide with the horizontal wall of the inhaler then the acceleration is upwards.

Hence the drug particle which is adhered on the top of the carrier particle will be

pressed further onto the surface. If the drug particle is located near the bottom of the

carrier particle but not colliding with the wall then the drug particle can be

effectively detached from the carrier surface on account of the tensile force. If the

drug particle is located on the equator of the carrier particle, it will experience an

upward acceleration due to shear of carrier-drug bond and then the drug particles will

detach by shearing or twisting of the carrier [79-80].


19

2.3.3.2. Fluid based detachment

The fluid based detachment occurs when the flow stream (air) has unobstructed path

to access and remove the drug from the carrier surface. The mechanisms which can

help in the detachment of the drug from the carrier particles are described below.

Drag: If the drug-carrier mixture has a different velocity from the surrounding fluid,

then the airflow will cause a velocity gradient at the surface of the carrier particle

which in turn will generate the forces on the drug particles called as drag force. The

tangential drag force tends to shear or twist the drug particle away from the carrier.

Acceleration: If the carrier particle accelerates due to the fluid forces then a force is

created on the drug particle which enables it to remain attached on the carrier

surface. The direction of this force is opposite to the drag force.

Shear: If the drug-carrier particle is in the region of fluid shear, then the carrier

particle experiences it on its surface as the velocity gradient which causes the force

on the drug particles called as normal lift force. The normal lift forces tend to pull the

drug particle away from the carrier. This force is similar to drag forces [79, 82].

2.3.4. Particle characteristics

The physicochemical properties of the powder have a profound effect on the drug

dispersion and these properties are discussed below.

2.3.4.1. Size of the drug

Particle size of the drug plays a very important role in the formulation of dry powder

inhalers. Separate studies have been carried out to determine the optimal particle size

of the aerosol in patients with varying degree of asthma and with different drugs. It

was found that the optimum particle size for the dry powder inhalers should be

between 1-5 µm. If the particles are smaller than 0.5 µm then they may not deposit

because of the Brownian motion and if the particles are larger than 5 µm, they may

impact in the pharynx from where they are easily cleared [83-87].

2.3.4.1.1. Aerodynamic diameter and Dynamic shape factor

In general pharmaceutical powders are irregular in shape and are not spherical; hence

measurement of the actual geometric diameters of the irregular particles is difficult.

Any deviation from the sphericity is called as the dynamic shape factor.

Aerodynamic diameter (Dae) is the diameter of an equivalent volume sphere of unit


20

density (Deq) with the same terminal settling velocity as the actual particle. For

particles larger than 1 µm, the following equation describes the relationship between

these dimensions [88].

aeD eqD0

p 2.1

Where: Dae and Deq are aerodynamic and equivalent diameters respectively, ρp and ρ0

are particle and unit densities and χ is the dynamic shape factor.

2.3.4.2. Carrier size

The particle size of the carrier is an important parameter in the design of DPI

formulations and various studies demonstrating the effect of carrier size on drug

dispersion has been reported [28, 89-91]. The smaller the size of the carrier particles,

the more easy it is for the drug to get redispersed. The detachment of the drug

particle occurs laterally to the carrier surface i.e., the drug particle slides along the

surface until it reaches the edge and falls off [90]. The longer the travelling distance,

the greater is the drag force which is needed to overcome adhesion and friction

between drug particle and carrier particle surface. Hence particle size of the carrier to

a larger extent influences the drug dispersion [92-93]. It is also observed that

variation in the particle size of the carrier could significantly increase the FPF of the

particles in the lungs [18, 21, 25, 94].

Fine Particle Fraction (FPF) represents the fraction of the drug as a function of the

recovered dose. Researchers have carried out various studies with the lactose carrier

to study the effect of the size of the lactose carrier on the FPF of the drug. Podczeck

reported the effect of the size, shape and surface roughness of ten different grades of

lactose monohydrate on the dispersion of Salmeterol Xinafoate (SX) [90]. The author

found that the smaller size of the lactose carrier was efficient in obtaining higher FPF

of the drug. Steckel et al in their study found that the smallest carrier size of < 32µm

resulted in the highest FPF (37.46%) of Budesonide [28]. Louey et al determined the

dispersion of SS from ten different grades of lactose and a similar trend was

observed; the FPF of the carrier increased with the decreasing particle diameter [89].

However, the increased FPF of the drug was associated with the presence of <10% of


21

fine particles of lactose (< 5 µm). Similarly an increased dispersion of SS was

observed with decreasing size of coarse lactose [91]. Recently Ooi et al have

demonstrated the use of different sizes of polystyrene spheres as the carriers for the

aerosolization of SS. They also found that as the size of the carrier was increased the

aerosol performance decreased [48]. Hence in most of the studies, increased

dispersion of the drugs from the interactive mixtures was observed with the

decreased carrier size [94-97]. Islam et al also found that the dispersion of SX

increased with decrease in the size of the lactose carrier [18]. However the drug

dispersion from these mixtures is complicated by the fact that the FPF is not solely

dependent on the size of the lactose carrier but is also dependent on the presence of

associated fine particles of lactose on the surface of coarse carrier [23]. Other

parameters such as morphology, surface roughness, surface area and surface energy

of the carrier particles come into play in controlling the dispersion of the drug [30,

98-99].

On the other hand, in one of the study, a higher respirable fraction of terbutaline

sulfate was obtained from coarser lactose as compared to fine particles of lactose

[100]. Hamishehkar et al found that increasing the size of the sieved mannitol

(carrier) increased the aerosolization properties of insulin-loaded PLGA

microcapsules. Thus an increase in the size of the mannitol carrier increased the FPF

of the insulin-loaded PLGA. This occurred due to the presence of larger surface

discontinuities on the surface of the mannitol carrier. These surface discontinuities

provided the active site on the surface of the carrier for deagglomeration of PLGA

microcapsules and their deposition on the carrier surface [101].

Therefore some researchers found that a decrease in the size of the carrier resulted in

an increase in the FPF of the drug but some contradictory results have also been

reported in the literature.

2.3.4.2.1. Polydispersity

Along with the particle size, the degree of polydispersity is also important which is

defined as the range of the particle sizes around the mode. The difference in the

carrier sizes will lead to differences in the regional drug deposition which results in

variation in the therapeutic response of the drug [102].


22

2.3.4.3. Carrier shape

The shape of the particles is another important variable in dry powder inhalers

because it influences aerodynamic behaviour of the particle [103]. The shape of the

carrier particles might influence the mixing force due to increased friction and thus

indirectly affect the dispersion of the drug due to increased or decreased adhesion

forces [104].

2.3.4.4. Crystallinity and Polymorphism

A crystal is a solid in which the molecules or ions are arranged in an ordered,

repeating pattern. The ability of the solid to exist in more than one crystal form is

called polymorphism. Different polymorphs have different solubility, stability,

density, melting point, bioavailability and different energy states. A non crystalline

material is amorphous material and it has higher free energy than crystals. Crystal

habit describes the morphology of the particles and is important parameter as the

particle shape affects the aerodynamic behaviour and finally the lung deposition of

the particles [65].

2.3.4.5. Moisture Content and Hygroscopicity

Hygroscopicity is the intrinsic capability of the material to absorb moisture from its

surroundings. This in turn is affected by the crystallinity of the material and the

morphology of the particles. Moisture uptake leads to the aggregation of the particles

through solid bridge formation [105] which adversely affects the dispersion of the

particles and finally the lung deposition [106]. Hygroscopicity can increase the

particle size by aggregation of the particle and it can also alter the adhesive and

cohesive properties of the powder [107]. Thus the moisture content of the powder

influences flow and dispersion of the drug from the carrier [71].

2.4. Surface properties

The surfaces of particles are important factors in the determination of particle

interactions and the ease of dispersion.

2.4.1. Surface area

If the particles are small, the total surface area of the powder is very large. Also

surface morphology contributes to the surface area of the particles. Rough or

corrugated particles will have more surface area as compared to smooth particles. A

larger surface area renders the particles to greater potential for charging and moisture


23

uptake which in turn affects drug dispersion [65]. Low surface energy is needed to

avoid particle agglomeration in the DPI formulation [108].

2.4.2. Surface Morphology and Roughness

Particle morphology refers to the external shape and surface texture of a particle

[109]. The carrier morphology and the surface roughness are the major contributing

factors to affect the FPF of the drug in various studies.

It was observed by Ganderton that decrease in the lactose surface roughness resulted

in an increase in the respirable fraction of SS [95]. Another study by Zeng et al

showed that increasing the surface smoothness of lactose crystals increases the

respirable fraction of SS from DPIs [24]. Heng et al determined that the rougher

grades of lactose led to lower delivery of the SS because the rougher particles

hindered with the detachment of the SS from the lactose carrier surfaces [110].

Larhrib et al crystallized lactose under various conditions to obtain variations in the

morphologies of lactose. It was observed that the smooth surfaces of lactose provided

with an efficient drug delivery as compared to rougher surfaces [111]. Another

investigation by Young et al proved that the “particle smoothing” of lactose surfaces

led to an improvement in the FPF of the drug Beclomethasone dipropionate [112]. A

study conducted by Kawashima et al proved that spherical spray dried lactose

particles had higher deposition of the drug Prankulast hydrate (FPF: 17.8%)

compared with non-spherical lactose with irregular surface morphologies (FPF: 3.4%

- 14.7%) [30]. Thus, according to these above-listed investigations, decrease in the

surface roughness improved the aerosolization efficiency of a drug-carrier blend.

However, contrary results have been reported in the literature regarding the surface

roughness of the carrier particle. Chan et al observed higher deposition of SS with

increased surface roughness of the lactose carrier. They deposited fine particles of

lactose on the surface of the large lactose carrier which made microscopic

undulations on the surface of particles. These undulations were not able to

accommodate drug particles on their surface thereby facilitating easier detachment of

SS and improvised drug dispersion [113]. Extensive studies carried out by Adi et al

proved that changing the morphology of the drug particle to a rough surface,

decreases the contact area between drug and carrier which in turn decreases the

adhesion and increases the liberation of the drug particle from the carrier [114]. It


24

was found that when disodium cromoglycate powders were treated with lauric acid it

changed the morphology of the drug particles which in turn improved the FPF of the

drug [103, 115]. In a separate study, enhanced drug delivery of BSA was achieved

from corrugated particles as compared to smooth particles. The surface asperities

helped in reducing the contact area between the adjacent particles and this in turn

helped in the reduction of cohesion [116]. The particle surface corrugation was

further quantified in a separate study and it was found that a certain small degree of

roughness led to considerably enhanced performance of the DPI formulations by

reducing the contact area between the drug and the carrier [117]. Kawashima and his

co-workers modified the surface of the drug Prankulast hydrate by coating it with

ultrafine hydrophilic hydroxypropylymethyl cellulose phthalate. These surface

modified particles led to three-fold increase in the delivery of the drugs to the lungs

as compared to unmodified powder due to increased surface roughness and

hydrophilicity of the particles [118].

Thus, some studies support that the smooth carrier particles will help in improving

the drug delivery while some researchers consider that a certain amount of surface

roughness is a desirable feature for achieving better drug dispersion. However no

consensus has been achieved with respect to this aspect.

2.4.3. Adhesion force / Forces of Interaction

The most important factor influencing the behaviour of the formulation is the

adhesion force acting between the active ingredient and the carrier particles [75,

119]. Adhesion force is defined as the forces of interaction between two unlike

particles whereas cohesion is the forces of interaction between two similar particles.

Particle adhesion occurs when the forces of interaction between the particle and the

surface exceeds the detachment forces exerted on the particle. The types of solid

interactions occurring between the particles are broadly classified as electrical and

non-electrical forces and include contact potential, coulombic forces, intermolecular

forces, capillary forces and forces due to solid bridge formation.

2.4.3.1. Electrical forces

2.4.3.1.1. Contact potential

This type of particle interaction occurs between contiguous bodies which are

dissimilar and uncharged. Electrons have different energy levels. The difference in


25

the energy states between the outermost conduction band of electrons and the

vacuum energy level is called as work function (φ). The transfer of electrons between

two dissimilar materials occurs upon contact with each other until the equilibrium is

established. This produces the contact potential between the two which is the

difference between the work functions of the two materials. The force of interaction

occurring due to contact potential can be given by the following equation [120]:

S

qFe

22 2.2

Where:

Fe: Interactive force due to contact, q is the particle charge on detachment and S is

the contact area between the particle and the carrier surface.

2.4.3.1.2. Coulombic forces

This type of particle interaction occurs between charged particle and an uncharged

surface. Charged particles induces equal and opposite charges on the surface. The

Coulombic force of interaction is expressed by the following equation [120]:

2

2

l

QFim 2.3

Where Fim is the Coulombic image force, Q is the charge on the particle and l is the

distance between the centres of these charges.

2.4.3.2. Non-electrical forces

2.4.3.2.1. Intermolecular forces

These types of forces include van der Waals forces (dipole-dipole, dipole-induced

dipole and induced dipole-induced dipole forces), ion-dipole and ion-induced dipole

forces and hydrogen bonding. This interactive force between the two bodies

(spherical particle-plane interaction) can be represented by the following equation

[120]:


26

33

4

d

rBFm

2.4

Where Fm: Molecular forces of interaction (van der Waals forces), r is the radius of

the approaching spherical particle, d is the distance of separation, B is the constant of

molecular interaction with allowance of electromagnetic lag.

Thus the van der Waals adhesion force is inversely proportional to the distance of the

separation and will be most significant over the very short separation distances.

2.4.3.2.2. Capillary forces

Condensation of water vapour between adjacent particles leads to formation of the

liquid bridge and the surface tension force arising due to this produces an adhesive

force called capillary forces.

These forces can be represented by the following equation [120]:

rCosFc 4 2.5

Where:

Fc is the capillary interactive force between particles; r is the radius of the particle, γ

is the surface tension and θ is the contact angle between particular solids and liquids.

These interactions do not occur immediately on contact but it increases with time to

its maximum value. Also hydrophilic compounds will experience higher capillary

interaction than the hydrophobic compounds.

2.4.3.2.3. Solid bridging between particles

This type of interaction is not the primary interaction mechanism but occurs over a

period of time. Solid bridge interactions occurs between particles in different ways

like direct chemical interaction between the particles or reaction of particulate solids,

partial melting of low melting point solids caused by friction of heat and subsequent

cooling, crystallization of solid in the liquid film separating particles due to

temperature fluctuations or formation of mineral bridges between particles of the

same materials.


27

Thus the total adhesional force (F) is given by the following equation [120]:

smimec FFFFFF 2.6

Where:

Fc, Fe, Fim, Fm and Fs are capillary, contact potential, coulombic, intermolecular and

solid bridge interaction force component respectively.

Of all the above listed forces, van der Waals forces are considered to be the principal

force existing between drugs and carrier or the container surfaces [75]. In humid

environments, the adhesion forces increase due to the capillary force. When the

relative humidity (RH) is above 50% then the capillary forces dominate [121].

2.4.4. Measurement of adhesion forces

There are various techniques to determine the adhesion force between the particles.

The methods which have been used are vibration [122], centrifugation [123-124] and

impact separation methods [125]. These methods determine the forces of adhesion

between the two bodies by measuring the amount or number of particles detached

from the surface. However one disadvantage of these methods is that the adhesion

force between a single particle and the substrate cannot be determined. Hence in

order to measure this other methods like microbalance [126] and AFM colloid probe

techniques [127] are available. Currently, AFM is the most sophisticated and widely

used technique for determining adhesion forces between an individual particle and

the substrate and this is discussed in detail in the following section.

2.4.4.1. Atomic Force Microscope (AFM)

AFM technique was first invented by Binnig, Quate and Gerber in 1986 and is used

for exploring the sample surfaces by means of mechanical scanning [128]. Tri-

dimensional mapping of the surfaces is possible with the resolution available in the

subnanometer range. This technique is widely used to determine the surface

roughness of various samples and the forces of adhesion between two samples [129-

130]. AFM can be used to investigate various materials like ceramics, composites,

glass, synthetic and biological membranes, metals, polymers and semiconductors. It

can be used to study various phenomena like abrasion, adhesion, cleaning, corrosion,

etching, friction, lubrication, plating and polishing [129]. The advantages of this


28

technique are high magnification and superior resolution as compared to electron

microscopes. Also no expensive sample preparation is required as compared to SEM.

e.g. Gold coating of the sample is required to make the sample electrically

conductive in SEM.

Basic principles of AFM

A schematic diagram of AFM is shown in Figure 2.3. The microscope consists of

sharp tips, flexible cantilevers, a sensitive deflection sensor and high resolution tip

sample positioning.

Tip and cantilever: The tip is mounted at the end of a small cantilever. The tip is in

close contact with the sample and gives rise to image through its force interaction

with the surface of the sample.

Deflection sensor: The vertical deflection of the cantilever can be measured by AFM

with picometer resolution. A laser beam is reflected from the backside of the

cantilever onto a position sensitive photodetector consisting of two side by side

photodiodes. Even a small deflection of the cantilever will tilt the reflected beam and

change the position of the beam on the photodetector. The difference between the

two photodiode signals indicates the position of the laser spot on the detector and

thus the angular deflection of the cantilever.

Image formation: Images are formed by recording the effects of the interaction

forces between the tip and surface as the cantilever is scanned over the sample. The

scanner and the electronic feedback circuit together with sample, cantilever and

optical lever form a feedback loop set up for the purpose. This feedback system

ensures that the AFM measures not only the force of the sample but also controls it,

allowing acquisition of images at very low tip-to-sample forces.


29

Figure 2.3 Schematic diagram of AFM where (1) Laser diode, (2) Cantilever, (3)

Mirror, (4) Position sensitive photodetector, (5) Electronics and (6) Scanner with

sample

There are three different modes of imaging in AFM: Contact mode, Intermittent

mode and Non-contact mode.

Contact mode AFM: In contact mode, the force between the probe and the sample is

maintained constant by maintaining a constant cantilever deflection and an image of

the surface is thus obtained. An advantage of this mode is that it provides fast

scanning and can be used for friction analysis. But the disadvantages of this mode

includes that sometimes the force can damage or deform the soft samples.

Intermittent mode (Tapping mode): In tapping mode, the cantilever is oscillated at

its resonant frequency. Constant oscillation amplitude is maintained which maintains

a constant tip-sample interaction and hence the image of the surface is obtained. It is


30

suitable for samples which are easily damaged or loosely held to surface.

Disadvantages of this mode includes that it is quite challenging to image in liquid

systems.

Non-contact mode: The probe is not in contact with the sample surface but oscillates

above the adsorbed fluid layer on the surface during scanning. The feedback loop is

measured to monitor changes in the amplitude and the surface topography can be

measured. Very low forces are exerted on the sample and hence extended probe

lifetime is obtained. However the disadvantages of this technique includes that it has

lower resolution and need ultra high vacuum to have best imaging.

Force Curves

Force curves measure the amount of force experienced by the cantilever as the probe

tip is brought close to the surface and then pulled away. Force curves (force-versus-

distance curve) show the deflection of the free end of the AFM cantilever as the fixed

end of the cantilever is brought vertically towards and then away from the sample

surface. An illustration of a typical force-distance curve is given in Figure 2.4.

Figure 2.4 Anatomy of a force-distance curve [131]


31

Anatomy of a Force Curve

The idealized force distance curve describes a single approach-retract cycle of the

AFM tip (Figure 2.4).

A: As the cantilever is approaching the surface it will feel a long-range attractive (or

repulsive) force and it will deflect downwards (or upwards).

B: When the probe tip comes very close to the surface, it will feel sufficient

attractive force from the sample it will make contact with the surface.

C: As the fixed end of the cantilever is brought further closer to the sample, the

cantilever deflection will increase. The probe tip may indent into the surface at this

stage if the cantilever is sufficiently stiff.

D, E: Subsequently the tip breaks loose from the surface and retract back (D).

Various adhesive forces between the sample and the tip hamper the tip retraction.

These adhesive forces can be measured from the force-distance curve (E).

F: The tip finally withdraws and loses contact to the surface upon overcoming the

adhesive forces. It is a key measurement of the AFM force curve because it is the

point at which the adhesion is broken and the cantilever comes free from the surface.

As mentioned earlier the colloid probe technique is widely used in AFM. In the

colloid probe technique, silica spheres are glued onto cantilevers [127]. The smooth

spherical particles make the force measurements very sensitive and more

quantitative. A variety of probes of different chemical compositions can be used for

force measurements. The AFM colloid probe technique can be used to measure the

adhesion forces either in air, liquids, different gases and vacuum [129]. Adhesional

properties of different lactose carriers have been studied extensively via AFM colloid

probe technique [119]. Adhesion forces between Zanamivir particles and lactose

carriers [75], between SS particles and lactose carriers [132], between

beclomethasone dipropionate and a series of untreated and treated lactose [112] have

been reported. All previous studies have determined the influence of surface

roughness on the adhesion forces; however they have not focused on the distinction

between the contribution of extrinsic adhesion and true intrinsic adhesion to the


32

surface roughness. Islam et al reported the adhesion forces between SX and lactose

carriers and they took into account the forces due to intrinsic and extrinsic adhesion.

The estimate of the intrinsic force distribution was done by eliminating the effects of

surface roughness by spin coating the lactose samples to produce atomically smooth

surfaces. They found that the adhesion force distributions on the spin-coated films

were much narrower as compared to the lactose particles indicating that the surface

roughness of the lactose particles played a major role in affecting the adhesion forces

[133]. The adhesion forces between various sugar carriers (β cyclodextrin, lactose,

raffinose, trehalose and xylitol) and SS have been determined using AFM and the

adhesion force data was used to correlate with the in vitro delivery performance of

the drug from these sugar carriers [134]. The adhesion forces between lactose and

erythritol carriers and SS have been investigated using AFM. Less aerosolization of

SS was observed from erythritol carrier when compared to lactose carrier which was

attributed to the high adhesiveness of SS to erythritol [135]. Forces of interaction

between insulin microspheres and five different polymer surfaces which are used as

device materials for DPI have been determined using colloidal probe AFM [136].

All these studies addressed the application of AFM techniques as a tool for the

characterization of particle interaction in pharmaceutical powders.

2.4.4.2. Factors affecting adhesion force and drug dispersion

Adhesion force between the drug and carrier is affected by various parameters like

particle size and shape [137], surface roughness, surface free energy, geometry of

contact area [138], relative humidity [92], electrostatic charge [139], temperature

[140], contact time [141] and applied load [142] which in turn affects drug

dispersion. The major factors affecting the drug dispersion are explained below.

2.4.4.2.1. Particle size and shape

It was observed by Corn using the centrifuge technique that as the particle size

increased the adhesion force also increased [126]. In an another investigation using a

microbalance method Corn found that the forces of detachment of a particle from the

surface was directly proportional to particle diameter (d) and using air jet methods

and centrifugation methods it was found proportional to d2 and d

3 respectively [143].

Lam et al studied the effect of the particle size on the adhesion behaviour of

powders. They found that the adhesive forces between the steel substrate and starch


33

and spray dried particles in the increasing size range of 32-75 µm determined by

centrifugation method is directly proportional to the radius of the particles [137].

Kulvanich et al used the centrifuge technique and found that as the particle size of

the adherent increased, the adhesive tendency of the powders decreased [144].

Another study by Katainen et al confirmed that the particle size had a significant

effect on the adhesion forces between the two substrates [45]. Podczeck determined

that as the particle size of the carrier decreased the adhesion forces in an interactive

mixture increases [145].

2.4.4.2.2. Surface free energy

The effect of surface free energy on the adhesion forces has been investigated by

many researchers [112, 146-151]. Modification of the lactose surface by smoothing it

with magnesium stearate (MgSt) resulted in the decrease in the surface free energy of

the lactose carriers which lowers the energy barrier between the drug

Beclomethasone dipropionate (BDP) and the carrier and hence resulted in increased

FPF of BDP [112]. It was found that supercritically produced SX particles possessed

lower surface free energy which was measured by Inverse Gas Chromatography

(IGC) and this resulted in better FPF of the drug when compared with micronized SX

[146, 149-150]. In a study by Vasu et al, increased FPF from Rifampicin was

observed than Rifampicin loaded PLGA microparticles due to decreased surface

energy of Rifampicin [148]. James et al characterized two excipients, sub-micron α

lactose and sub-micron sucrose and found that the surface free energy of sub-micron

sucrose was higher when compared with sub-micron α lactose and hence the lactose

had lower adhesive interactions with the active drugs [147]. Traini et al studied the

surface characteristics of different pseudopolymorphs of lactose which had different

surface chemistries and surface energies and correlated it with aerosolization studies.

They established that an inverse relationship existed between the surface energy and

aerosolization studies due to increased carrier particle adhesion which reduced the

detachment of drug during aerosolization process [151]. Similarly an inverse

relationship was observed between the surface free energies of supercritical fluid

engineered albuterol or budesonide particles and their aerosolization from the lactose

blends. The reduced surface free energies from these powders minimized the drug-

carrier particle interactions which resulted in efficient drug detachment from the

carrier surface during aerosolization [152]. A similar kind of relationship was also


34

observed between three SX samples (SX, SX-I and SX-II) [153] and lactose and

between lactose and a new drug (IFNa-2b) [154]. The negative correlation between

surface free energy of spray dried and milled lactose and FPF of budesonide and

fluoroscein was observed [155]. Recently, Das et al investigated the relationship

between surface free energy of lactose and SX and its dispersibility from mixtures at

75% relative humidity (RH). It was observed that increase in the RH, caused an

increase in the surface free energies of both the powders due to surface moisture and

this in turn decreased the FPF of SX due to increased capillary interaction and/or

solid bridging [156].

In all the above investigations a decrease in the surface energy of the carrier resulted

in an increase in the FPF of the drug which was attributed to the fewer interactions

between the drug and the carrier. On the contrary, in a separate study by Cline et al

an increased FPF of Albuterol and Ipratropium bromide was observed when it was

blended with carriers (trehalose, lactose and mannitol carriers) in increasing order of

the magnitude of surface energies. This anomaly was explained by the authors that a

certain amount of minimum surface energy interaction was necessary between carrier

and drug particles to pull the highly cohesive drug particles apart during the blending

and aerosolization process [98].

2.4.4.2.3. Relative Humidity (RH)

Relative humidity plays an important role in affecting the adhesion forces which

subsequently affects the dispersion of the drug from the surface. Berard et al

investigated the effect of increasing humidity on the adhesion forces between lactose

and Zanamivir crystals. It was found that as the RH increased from 0% to 85%, the

surface topology of both the materials was modified progressively due to capillary

forces which led to the increased adhesion forces between the two [75, 157]. Tsukada

et al studied the effect of humidity on the adhesion forces between a spherical

polycrystalline drug particle and lactose monohydrate. It was found that as the

humidity increased from 30% to 90%, the adhesion forces also increased between the

two materials [158]. Price et al determined the adhesion forces between lactose

carrier and SS and Budesonide drugs using the AFM colloid probe technique at 15%

and 75% RH. They observed that the adhesion forces increased with the increase in

RH due to the capillary forces acting between then which can reduce the

deaggregation and dispersion of the drug [159]. The thickness of the adsorbed


35

moisture layers on the surface of lactose were also measured by AFM which in turn

affected the adhesion force [160]. Podczeck et al investigated the influence of

changing humidity on the autoadhesion of lactose and SX particles. It was observed

that the increase in humidity caused an increase in the autoadhesion forces [161].

Hooton et al studied the influence of different humidity (<10% to 65%) on the

adhesion forces of SS prepared by two different methods which yielded different

surface chemistries and geometry. They observed that as the humidity increased

there can be an increase or decrease in the adhesion forces which is attributed to the

varying geometry of the surfaces and the distribution of water on those asperities

[162]. Young et al determined the cohesion between drugs as a function of humidity

using the AFM. They observed increased cohesion as the humidity was raised from

15% to 75%. It depended on the type of drug, the presence of predominant capillary

forces at high RH or the presence of the attractive electrostatic interactions which

were dissipated at high RH [163-164]. Young et al later investigated the cohesion

forces between the three drugs (SS, Triamcinolone acetonide, Di-sodium

cromoglycate) as a function of RH and correlated the separation energy

measurements with the aerosolization performance. It was observed that the adhesion

forces were inversely proportional to aerosolization performance which was

measured as FPF of the drug [165].

Thus all these studies indicated that the relative humidity is capable of influencing

the forces of adhesion between two materials predominantly due to capillary forces

acting between the two which can in turn affect the FPF of the drug.

2.4.4.2.4. Surface roughness

Surface roughness is another important factor to be considered in particle to surface

adhesion. The increased surface roughness can increase the force of adhesion

between particles and surfaces if the particles are small, as they are able to slip into

the valleys between individual asperities. This was very well demonstrated by Beach

et al who had carried out adhesion force measurements between pharmaceutical

particles (Beclomethasone dipropionate, lactose) and rough polymeric surfaces of

polypropylene coatings, polycarbonate and acrylonitrile-butadiene-styrene. The

measurements revealed that roughness of the interacting surfaces had a significant

impact on the pull-off forces [166] (Figure 2.5). Meine et al also measured the

adhesion forces between polymer balls of polyethylene and silicon wafer under ultra


36

high vacuum conditions with the aid of AFM. The surface was made rough by

etching nanometre sized structures onto the surface. The results revealed the

influence of surface roughness on the adhesion force. However one interesting

outcome from the study was that the adhesion force was not only dependent on the

contact area between the particles but also on the circumference of the asperities

[167]. It was reported by Mizes that the particles which remain on the surface of the

bumps experience less forces of adhesion as compared to the particles situated deep

in the valleys and pits on the surface [168]. Tsukada et al carried out the adhesion

force measurements between spherical polycrystalline drug particle (3 µm in

diameter) and a plate of lactose or stainless steel using colloid probe technique under

varying humidity conditions (30% - 90% RH) and smooth or rough surface. It was

found that the adhesion force was decreased when the surface roughness was low,

the curvature of the particles is small or the humidity was low [158].

(A) (B)

(C)

Figure 2.5 The effect of surface roughness on the contact area between particles

and surfaces. (a)The ideal sphere on flat surface (b) Asperities much smaller than

the particle lead to reduction in the contact area (c) Asperities comparable in size

to the particles can lead to increase or decrease in the actual contact area [166]


37

Adi et al conducted a study to examine the effect of surface morphology and particle

adhesion on the FPF of the spherical model drug particles of bovine serum albumin

with different degrees of surface corrugation. It was found that as the degree of

corrugation (surface roughness) increased the particle adhesion was reduced which

resulted in a concomitant increase in the FPF of the drug [169]. Eve et al determined

the adhesion forces between Salbutamol and four different substrates and it was

found that surface roughness was a dominant factor in controlling the adhesion of

salbutamol to either lactose or salbutamol [170]. In a separate study by Young et al,

the lactose particles were made smooth and they resulted in lower energy of

separation and increased FPF of the drug as compared to rougher particles suggesting

that the smoother particles yielded better dispersion of the drug [112]. Rabinovich et

al determined the adhesion forces between glass spheres and silicon wafer substrate

and found that as the surface roughness of the silicon wafer substrate increased the

adhesion forces between the two decreased [171-172]. However increased surface

roughness increased the adhesion forces between gold and mica in presence of high

relative humidity due to capillary forces acting between them [173].

Podczeck et al determined the area of contact between SX and lactose and flat

surfaces of polyethylene, polyoxyethylene and aluminium and found out that rough

irregular particles had smaller areas of contact and hence preferred for DPI

formulations [174]. Iida et al found out that increasing the smoothness of the carrier

particle (from 0.70 µm to 0.42 µm) increased the FPF of the drug but only up to a

certain level, any further increase in the smoothness of the carrier (0.37 µm) later

resulted in the decrease in the FPF of the drug. This could be because initially as the

macroscopic roughness decreased the number of drug particles remaining in

macroscopic depressions decreased which facilitated drug separation from the carrier

particles. As the carrier particles became smoother there was a larger in-contact area

between drug particles and lactose carrier which resulted in lower FPF of the drug

[175]. Flament et al studied the influence of surface roughness of lactose carrier on

the adhesion of terbutaline sulfate and found that as the surface roughness increased

there was a decrease in the FPF of the drug. This occurred because the smoother

surfaces were capable of detaching higher percentage of drug from their surfaces

[176]. Podczeck et al determined the adhesion forces between lactose and SX using

the centrifugation technique and found that the surface morphology played an


38

important role in the contribution to the adhesion forces however they could not

assess quantitatively the effect of surface roughness on the adhesion forces [145,

177-179].

Hence the drug delivery from DPIs is influenced by the physicochemical

characteristics of carrier particles such as surface texture, particle size and particle

shape and the adhesion forces existing between the drug and the carrier [180].

2.5. Ternary components

Improvement in the dispersibility of the drug from the carrier surface has been

reported by modification of the particle surfaces using different technologies. One of

the approaches is to coat the surface of the coarse lactose by blending it with fine

lactose, MgSt or leucine [31-35].

2.5.1. Magnesium stearate

The inclusion of MgSt in the DPI formulation as a ternary additive helped in

improving the performance of the formulations [95]. Stewart found that the addition

of MgSt in DPI formulations helped in improving the performance by modifying the

interaction between carrier surface and drug particles [181]. Bolhuis et al found that

MgSt can form film layers which can adhere to drug-excipient particles and can

interfere with inter-particle bonding as a result of hydrophobic coating [182]. Gold et

al found that MgSt can reduce adhesion between the particles in powder bed [183].

Guchardi et al reported that the addition of 0.5% MgSt to the lactose blend helped in

increasing the FPF of Formoterol fumarate by 10% [184]. This occurred probably

because MgSt has a higher affinity to lactose (no electrostatic repulsion) [185].

Hence the active sites on lactose were readily occupied with MgSt and hence caused

easy liberation of drug from the lactose surface. The lubricant activity of MgSt had

been quantified using AFM [186]. Begat et al had proposed the co-processing of

Force Control Agents (FCA) (MgSt, leucine or lecithin) with SS or Budesonide by

mechanofusion process. They found that the FPF of the drugs increased considerably

because the FCAs helped in deagglomeration and aerosolization of the cohesive

powders in the system [187]. AFM studies conducted later suggested that the

processing of lactose with these FCA helped in significantly reducing the adhesive

interactions between SS and modified lactose samples [40]. Recently Islam et al

reported that there was an increase in the FPF of SS (18-33%) with the increase in


39

concentrations of ternary component MgSt from 0.1 to 1.5%, however, the FPF of SS

decreased with further increase in the concentrations of MgSt over 2% [188]. Young

et al reported a significant increase in the FPF of Beclomethasone dipropionate when

the surface of the lactose was modified by smoothing it with MgSt [112]. Similarly

Iida et al reported an increase in the FPF of SS by layering the surface of the lactose

with MgSt [189]. The improved dispersion in the above studies was obtained as a

result of smoothing because of the high affinity of MgSt on the active sites of lactose

which forms a layer to cover the depressions and hence aid in drug separation.

Recently Tay et al in his comprehensive review has proposed three various

mechanisms by which MgSt can increase the dispersion of SS. They proposed that

MgSt can form micro-interactive units containing adhered SS and the detachment of

SS from these micro-interactive units is more favourable than SS deagglomeration.

Secondly it can be that the coating of SS particles with nano-laminates of MgSt

decreased SS particle interaction which enhances deagglomeration of SS. Thirdly the

presence of fine particles of MgSt within SS agglomerate acts as an agglomerate

modifier and improves the dispersion of SS [190].

2.5.2. Leucine

The use of leucine in the DPI formulation as a ternary additive has helped in

improving the performance of the DPI formulations [31, 191]. This is possibly due to

antiadherent action of the material [192]. Lucas et al found that the addition of spray

dried leucine to lactose carrier helped in the improvement of FPF of SS [32].

Najafabadi et al co-spray dried disodium cromoglycate (DSCG) with L-leucine

which resulted in improved deposition profiles of the drug due to anti-adherent

properties of leucine [193]. Chew et al found that co-spray drying of disodium

cromoglycate (DSCG) powders with hydrophobic amino acids (leucine,

phenylalanine, tryptophan, methionine, asparagine and arginine) helped in improving

the dispersion of DSCG. The FPF increased significantly with leucine which is

attributed to reduced intermolecular interactions between leucine and DSCG

molecules [36]. Leucine has been co-spray dried with various drugs like insulin

[194], Ambroxol hydrochloride [195] and Naringin [196] which helped in the

improvement of the aerosolization properties of the drugs. Raula et al have coated

the SS particles with leucine by using the gas-phase coating technique in an aerosol


40

flow reactor which helped in improving the aerosolization properties of the drug

[197-200].

2.5.3. Other ternary components

In addition to the above listed ternary components, some researchers have also used

other ternary components which had resulted in improvement in the dispersion of

drug and its delivery.

It was found by Tee et al that the use of mannitol and sorbitol as ternary components

for DPIs increased the FPF of SS. In fact mannitol and sorbitol were found to

improve the FPF better as compared to the fine particles of lactose [41]. Similarly

micronized glucose were found to increase the FPF of SS [201]. The addition of fines

of erythritol [202] and polyethylene glycol [73] have also helped in the improvement

of the dispersion of the drugs.

Contrary finding was reported by Hamishehkar et al which established that the

addition of spray dried fine mannitol and spray dried leucine as ternary agents

decreased the dispersibility and deposition of PLGA microcapsules. This occurred

because spray dried mannitol or leucine particles formed agglomerates with the

PLGA microcapsules, entrapped them forming a massive net and consequently

inhibited the microcapsules to deposit in the air stream as the single particles [101].

Common techniques for surface modification of carrier particles include spray drying

[36], encapsulation using supercritical carbon dioxide [37], physical vapour

deposition of particles in an aerosol flow reactor [38-39] and dry coating of active

drug particles such as mechanofusion [40]. However, drug dispersion from these

powders is still not satisfactory.

2.6. Use of modified lactose as carriers in DPIs

As discussed above, the surface properties of lactose in inhalation products are very

critical. Lactose by itself has a highly irregular surface which affects the dispersion

of drugs from DPIs. To date various modifications have been carried out on the

lactose particles to provide efficient drug dispersion from the DPIs which are as

discussed below:

Iida et al carried out extensive studies to smooth the surfaces of the lactose carrier

particles. In one of the technique the protuberances on the surfaces of the lactose


41

particles were smoothed by stirring the mixture of lactose in 70% v/v ethanolic

solution. In another technique, the surface of the lactose particles was covered with

sucrose tristearate or with MgSt. Another modification of the method involved

application of intermittent shear force to the core particle surface for uniform surface

processing using a high speed elliptical rotor type powder. The in vitro deposition

was tested using a Twin Stage Impinger (TSI) apparatus and Spinhaler® DPI device

or Jethaler® DPI device. An improved in vitro deposition of the drug was obtained

with surface treated lactose particles as compared to untreated lactose particles

indicating improved inhalation properties of DPI [175, 189, 203-204]. Young et al

smoothed the surface of lactose by etching it with a wetting solvent (water:ethanol =

5:3) with and without the presence of MgSt in a high speed mixer. The drug and

treated lactose blends were tested for aerosolization efficiency using the TSI and

multidose DPI. It was found that the engineered lactose provided an improved

aerosolization efficiency as compared to untreated lactose which was confirmed by

AFM studies [112]. In another technique, lactose was smoothed by the process of

surface dissolution by using the saturated solution of lactose in water and by ramping

the temperature in the dissolution vessel at controlled rate. SS-carrier blends were

tested using TSI and Cyclohaler® DPI device. The surface smoothing of lactose

resulted in increased aerosolization performance at lower drug concentrations [205].

In another attempt to modify lactose, composite carriers of lactose were prepared by

spray drying aqueous lactose solution which provided homogenous surface

roughness to the particles. The aerosol performance of these composite carriers was

tested with micronized SS using the Next Generation Impactor (NGI) (eight stage

inertial impactor) and Cyclohaler® DPI device. The FPF varied between 21.3 ± 5.4%

and 31.3 ± 1.3% between regular lactose and the composite lactose [206].

Some researchers have worked on the technique of developing a new method of

crystallizing lactose to obtain crystals with desirable size (63-90 µm), better particle

size distribution, regular shape, smooth surface, higher elongation ration and better

flow. Dhumal et al developed a new method for engineering lactose crystals with the

aid of ultrasound under different crystallization conditions whereas Zeng et al

developed a new technique for crystallization of lactose from carbopol gel. The

aerosol performance of ultrasound engineered carriers was tested with micronized SS


42

using the Anderson cascade impactor (eight stage impactor) and Rotahaler® DPI

device and the results indicated improved aerosol performance [180, 207-208].

One important study by Zeng et al brought into light the fact that particle size and

surface smoothness of the carrier are not the sole factors for improving the dispersion

of drugs from dry powder inhalers. In fact the addition of a fine fraction of lactose

and its characteristics (micronized lactose or recrystallized fine lactose) had a

dominant effect in controlling the drug dispersion in DPI formulations. Addition of

micronized lactose increased the FPF of albuterol sulfate from 14.6 and 17.1% to

20.8 and 21.6% for the solvent-treated and untreated lactose respectively which was

determined using a TSI and a Rotahaler® DPI device [209]. This was further proved

by Islam et al in a study where the addition of fine lactose improved the dispersion of

SX and played a dominating role in controlling FPF of the drug [23].

All the above listed studies indicate the attempts made by various researchers to

smooth the surface of the lactose particles or the addition of ternary components in

an attempt to achieve efficient drug delivery. However there are a number of

variables and the data could not be considered conclusive because different

researchers have used different apparatus (TSI, 8-stage Next Generation Impactor, 8-

stage Anderson cascade impactor and 5-stage Marple Miller Impactor) and various

devices (Spinhaler®, Jethaler®, Cyclohaler®, Rotahaler® and Aerolizer®) for

determining drug dispersion.

Nevertheless as stated earlier, the aim of a DPI formulation is to have a pulmonary

drug with uniform distribution, limited dose variation, good flowability, adequate

physical stability in the device and good performance in terms of emitted dose and

FPF. A great deal of research has focused on fabricating particles with reduced

interparticulate adhesion and increased stability to improve the performance of dry

powder aerosol systems [114]. Thus it is evident that many modifications are

required to be carried out on lactose carrier particles to obtain an efficient drug

delivery. However there is a further scope for improvement in terms of drug delivery

efficiency.

2.7. Reasons for inefficient drug delivery with lactose

Most commercially available carrier-based dry powder inhalers deliver only about

20–30% of the total dose to the lungs and the rest of the drug remain adhered on the


43

carrier surface and is subsequently swallowed, resulting in poor performance of DPI

devices with respect to drug delivery [21]. The reasons for this inefficient delivery

have not been fully elucidated. It could be attributed to irregular shape and rough

surfaces of commercially available inhalation grade lactose which affects drug

detachment during inspiration. However the difficulty associated with the use of

sugars as carriers is controlling the size, shape and surface roughness of the particle

which in turn affects the detachment of drug from the particle [25].

Another common issue with DPIs is the sensitivity of the carrier formulation to

elevated temperature and humidity. Although α-lactose monohydrate is not a

hygroscopic material, moisture ingress into DPI formulations will influence

formulation performance by various mechanisms. As the coarse carrier particles in a

DPI formulation are typically <100 µm, whereas the drug particles are 1–10 µm, the

adsorbed moisture layers will promote cohesion between particles in the bulk

formulation. Another factor to consider is the presence of amorphous regions on the

surface or within the bulk of a particle. Produced by comminution, or as a natural

feature of the production process for the material, amorphous material represents a

metastable thermodynamic state with high free energy which means a high amount

of energy would be needed to separate drug particles from carrier surfaces [210]. In

addition, lactose cannot be used for compounds that interact with the reducing sugar

function of the lactose, such as formoterol, budesonide or peptides and proteins [44].

Additionally, a number of different grades or brands of lactose are currently available

in the market. A study has been conducted by Larhrib et al and they found that

different grades of lactose produced varying delivery profiles of the inhaled drug

(SS) thereby indicating that the source and grade of lactose has a substantial effect

on the drug delivery [27]. A study by Podczeck investigated the relationship between

the physical properties of the lactose monohydrate carrier particles and the

aerodynamic behaviour of the adhered drug particles. It was found that the

relationship was very complex and any interchange of the carrier material with

respect to brand or grade was not feasible [90].

The other contributing factor to the above problem is the complex physiology of the

respiratory tract. The characteristics of the powder formulations for inhalation and a

variety of inhalation devices further aggravate the problem. Further research in this


44

field is being conducted to improve the performance characteristics of the DPI device

in an attempt to achieve better drug dispersion [70]. To add to this, micronized drug

particles adhere around the surface of carriers and produce agglomerates and there is

poor detachment of the drug particles from the carrier surface. All these factors result

in poor delivery of the drug from the DPI.

Thus lactose as a carrier has its own limitations. The majority of previous studies

have focused on lactose-based formulations. These formulation challenges have

resulted in a significant research focus on DPI carrier technology. Hence it is

pertinent to study the potential application of other entities as carriers for dry powder

for inhalation.

2.8. Alternative carriers

2.8.1. Sugars other than lactose

Due to several drawbacks of lactose and modified lactose as a carrier for dry powder

inhalers, there is an urgent need to find suitable alternative carriers for better drug

dispersibility in DPIs.

Steckel et al explored alternative carriers like mannitol, glucose, sorbitol, maltitol

and xylitol as potential carriers in DPI formulations. Of all the sugars evaluated,

mannitol seemed to be a promising carrier for DPIs whereas sorbitol, maltitol and

xylitol sugars were not able to generate desirable FPF due to their hygroscopic

nature. However the problem of supplier variability was observed in all the carriers

evaluated which led to significant variations in the fine particle fraction of the drug

[44].

Lorant et al studied four different carriers‟ viz. crystallized mannitol (Pearlitol 110

C), spray-dried mannitol (Pearlitol 100 SD), crystallized maltitol (Maltisorb P90) and

spray-dried lactose (Lactopress SD 250) for two drugs: micronized terbutaline sulfate

and micronized formoterol fumarate. It was found that crystallized forms of the

carrier offered lower adhesion and better release of the active ingredient than spray-

dried forms. The crystallized mannitol produced maximal fine particle dose [78].

Tee et al have investigated the use of lactose, mannitol and sorbitol in the coarse and

fine form as carriers for DPI formulations for the delivery of SS [41]. Momin et al

explored the use of mannitol, dextrose, sorbitol, xylitol and maltitol for the


45

pulmonary delivery of Budesonide and found that mannitol was the favourable

carrier for the dispersion of the drug [211].

2.8.2. Lipidic vehicles

Some researchers have investigated the use of solid lipid microparticles (SLM) as

vehicles for pulmonary administration wherein the drug was incorporated within the

SLM and was not on the surface of SLM [42-43, 212]. Sanna et al proposed that

SLM may be used as a potential carrier for antiasthmatic or antimicrobial drugs.

They formulated SLM using Compitrol® (glyceryl behenate) as the lipophilic

component and Poloxamer as the emulsifying agent. However these type of

formulations are suitable for long term treatments [43]. Jaspart et al also formulated

SLM using Compitrol® as the lipid with an aim to sustain the release of the drug into

the lungs and they propose that these formulations can be used to prepare inhalation

powders containing suitable excipients. However these SLMs are suitable for high

entrapment of the hydrophobic drugs. Hydrophilic drugs like widely used anti-

asthmatic drug SS cannot be incorporated into the lipid matrix and hence it needed to

be converted into a hydrophobic derivative like Salbutamol acetonide [42]. SLM of

Budesonide drug and cholesterol and phospholipon as the lipidic carriers have been

prepared by means of spray drying. This formulation has been tested using Multi

stage liquid impinger and Cyclohaler ® as the inhalation device. However they

propose that additional studies needs to be conducted to determine the stability of

such products [212].

2.9. Proposing polymers as alternative to sugars

All DPI formulations available in the market today utilize lactose as carrier material

[44]. The culmination of the formulation challenges with the use of lactose and its

modified counterparts have led to the exploration of new efficient alternative carriers

for inhaled therapy.

Polymers have long been used in various drug delivery technologies due to their

versatility. Polymers have been used for decades as excipient in tablet and capsule

formulations and now they are being essentially used for advanced functions like

drug targeting. They have found widespread use in drug delivery systems such as

matrices, microparticles, hydrogels and dendrimers [213]. Advanced pharmaceutical

dosage forms utilize polymers for taste masking, drug protection, increased drug


46

bioavailability, as rheology modifiers in liquid dosage forms and as implants in

biomedical arena.

Several polymers that have been investigated for drug-delivery applications can be

broadly classified into natural and synthetic, biodegradable and non-biodegradable

polymers. Varieties of biodegradable polymers have been synthesized to deliver

drugs, macromolecules, cells and enzymes. The biodegradability of these polymers

can be manipulated by incorporating a variety of labile groups such as esters,

orthoesters, anhydrides, carbonate, amides, ureas, and urethanes in their backbone

structure [214-215]. Polyester-based polymers are one of the most widely

investigated for drug delivery. Poly(lactic acid) (PLA), poly(glycolic acid) (PGA)

and their copolymers poly(lactic acid-co-glycolic acid) are widely used biomaterials

in terms of design and performance for drug-delivery applications [214, 216]. Other

synthetic polymers used for pulmonary applications include polyanhydrides [217-

219]. Zhang et al have used polybutylcyanoacrylate to prepare nanoparticles for

intratracheal delivery [220].

Recently a large number of biodegradable polymer nanoparticles were reported to be

used in improving pulmonary delivery of drugs from powder formulations [221].

Over ten years ago the use of controlled release polymeric systems was proposed

improving the duration and effectiveness of inhaled drugs for both local and systemic

action [222] but as yet there are no DPIs on the market using biodegradable polymers

as carriers.

2.10. Polymers in pulmonary drug delivery

The below listed studies discuss the use of polymers for drug delivery to the lungs.

Polycaprolactone (PCL) polymer has been widely used in drug delivery because it is

biodegradable and undergoes hydrolysis by degradation of its ester linkages. PCL

nanoparticles have been used to encapsulate variety of drugs by nanoprecipitation,

solvent displacement and solvent evaporation techniques. Drugs such as anticancer

(Tamoxifen [223], Taxol [224], Vinblastine [225] and Docetaxel [226]), peptide

(insulin) [227], antiretroviral (Saquinavir) [228] and antifungal (Amphotericin B)

[229] have been incorporated within PCL. Recently Kho et al have formulated

nanoparticle aggregates of antibiotic-loaded PCL by a spray drying technique for

pulmonary delivery. Mannitol, lactose and leucine were used as drying adjuvants in


47

this process to maintain the structural integrity of PCL and aqueous re-dispersibility

of the aggregates formed. Further they proposed that these Levofloxacin-loaded PCL

nanoparticle aggregates could be potentially employed for inhaled therapy. The

spherical aggregates were produced in the size range of approximately 270 nm; the

drug was encapsulated inside the nano-aggregates formed and was intended to

exhibit a longer retention time in the lungs [230]. Thus the drug was dissolved in

PCL and was not used as a surface carrier for the drug as in our present study.

Poly (lactic acid-co-glycolic acid) is a widely investigated polymer in drug delivery.

PLGA 50/50 (MW 10.6kDa) has been used as a biodegradable polymeric carrier for

pulmonary delivery of low molecular weight heparin (LMWH). The in vitro drug-

release properties of the microspheres were tested using an eight stage Anderson

Cascade impactor and the results revealed that these porous microspheres of LMWH

could be delivered via DPI as an alternative to multiple parenteral administrations of

LMWH [231]. Microspheres of SS with PLGA 85/15, PLGA 50/50 (MW: 83,000

Da) and PLGA 75/25 (MW: 151,000 Da) (particle size: 92-207 µm) have been

formulated to achieve controlled drug delivery (extended release for 96 hours with

one formulation) but the polymer has not been evaluated for the purpose of carrier in

a DPI device [52, 54]. In another study, Ciprofloxacin nanoparticles (nanoCipro)

were encapsulated in large porous PLGA particles [PLGA 50/50 (MW 31,000)]

which resulted in a steady release of ciprofloxacin in the lungs. [51]. In another

attempt, Poly lactic acid (PLA) (MW 75,000-125,000 Da) polymer was used to

incorporate two glucocorticoid drugs Triamcinolone acetonide and Budesonide for a

slow-release formulation for pulmonary targeting. The powders were coated with

nanometre-thin layers of biodegradable polymers prepared using pulse laser

deposition technique. The respirable fraction of the micronized particles of the drugs

coated using optimum laser energy density were determined using an eight stage

cascade impactor and Aerolizer® inhaler device [53, 55]. Insulin had been

successfully encapsulated in the PLGA 75/25 nanospheres and intended for

administration via the lungs [232]. PLGA microspheres have been formulated in the

respirable size range of 3-7 µm and treated with polyamino acids and isopropanol to

obtain particles of surface charge suitable for use in DPIs [233-234]. The

electrostatic charge on the microspheres was determined by Electrical single particle

aerodynamic relaxation time (E-SPART) analysis and it was found that isopropanol-


48

treated PLGA microspheres exhibited the highest surface charge and also the higher

respirable fraction than untreated and the polyamino acids-treated microspheres.

Recombinant tuberculosis antigen has been encapsulated in PLGA 75/25

microparticles (3.3 µm) to extend the release of the antigen up to 10 days [235].

Chitosan is another polymer which has been extensively studied in pulmonary drug

delivery. Learoyd et al prepared a DPI formulation by spray drying 30% v/v aqueous

ethanol formulation containing terbutaline sulfate, lactose, leucine and chitosan. The

aerosolization of the powder was investigated using a Multi-Stage Liquid Impinger

(MSLI) aerosolized from a Spinhaler® DPI device. The FPF of the drug was found

to be dependent on the molecular weight of chitosan. The low MW formulation

exhibited an FPF of 64% whereas the high MW formulation demonstrated an FPF of

54%. The higher MW chitosan increased the viscosity of spray dried solution which

influenced the ability of the leucine to migrate to the surface during the spray drying

process. This in turn affected the interparticulate cohesion and decreased the

deaggregation of the particles during aerosolization [236]. In another study by

Corrigan et al, salbutamol sulfate/chitosan systems with and without the crosslinking

agent formaldehyde were prepared using spray drying. Chitosan–salbutamol sulfate

composites were compressed into discs and the drug release was quantified which

demonstrated delayed release of the drug. TSI analysis indicated good in vitro

deposition of the microparticulates as the respirable fraction obtained was 36.4%

[237]. Huang et al carried out in vivo studies on Betamethasone-loaded

microparticles which were prepared by a spray drying method using chitosan as raw

material, type-A gelatin and ethylene oxide-propylene oxide block copolymer

(Pluronic F68) as modifiers. In vivo studies in rats were carried out and the drug

treatment to the rats was provided by using Dry Powder Insufflators (Penn-Century,

DP-4). Reductions in the inflammatory biochemical parameters were observed which

indicated that the drug was efficiently delivered in the pulmonary tissues by this

technique [238-240].

Salama et al prepared a series of co-spray dried microparticles containing the anti-

asthmatic drug di-sodium cromoglycate and different concentrations of polyvinyl

alcohol as controlled release vehicles for drug delivery to the respiratory tract. The in

vitro performance was tested using a 5-stage Marple Miller Impactor and


49

Cyclohaler® DPI device and resulted in the FPF of 16.71±0.73% to 30.76±1.5%

from increasing concentrations of PVA (0% to 90%) [241].

The literature indicates that polymer matrices have been used in pulmonary drug

delivery to control or sustain the release of drugs of which PLGA and chitosan have

been widely investigated. PLGA microspheres incorporating SS have been reported

but they have been used for achieving sustained drug release into the lungs and not

for the purpose of carrier in DPIs. Composites of SS and chitosan have also been

reported but they were formulated with the perspective of achieving controlled drug

release. Thus there have been no reports of the use of polymers (PCL or PLGA) as

carriers for SS in DPIs per se and our work revolves round the use of PCL and PLGA

polymers as carriers for dry powder inhalers.

2.11. Hypothesis that polymers can fill in the gap

The use of polymeric systems to achieve controlled drug delivery in the pulmonary

system is still in its infancy and their use as carriers in DPIs is still an unexplored

arena. Hence it is important to evaluate potential polymer candidates as alternative

carriers in DPI products. Controlling the particle size, shape and surface roughness of

polymers is much easier [50] as compared to sugars. Thus it will be worthwhile to

research biodegradable polymers with controlled surface functionality which can be

used as carriers for the pulmonary delivery of drugs from powder formulation.

Thus our work involved producing microparticles of PCL and PLGA polymers with

controlled size and shape and extensively studying the surface properties of carrier

particles. It would involve thorough investigation of the surface roughness of the

polymeric carrier particle and the measurement of adhesional forces between the

drug and the carrier particles. Adhesion force measurements between the polymer

film and silica sphere, polymer film and the drug coated silica sphere, polymer

particle and silica sphere and finally polymer particle and drug coated silica sphere

will be measured. These data would largely help us to understand the nature of the

surface of the polymer carrier and its role in influencing the adhesion to the drug and

thereby finally influencing the dispersion of the drug from the carrier.

Chapter 3 General Methods

51

CChhaapptteerr 33

GGeenneerraall MMeetthhooddss



53

3.1. Materials

3.1.1. Model drug

Micronized Salbutamol Sulfate (SS) of inhalation grade [Volume Median Diameter

(VMD) 5 µm] (GlaxoSmithKline, Australia) was used to prepare the formulations.

The compound is a β2 adrenoreceptor agonist and is used as a bronchodilator. The

chemical name of SS is (R, S)-4-[2-(tert-butylamino)-1-hydroxyethyl]-2-

(hydroxymethyl) phenol, sulfuric acid salt can also be named as bis[(1RS)-2-[(1,1-

dimethylethyl)amino]-1-[4-hydroxy-3-hydroxymethyl)phenyl]ethanol]sulfate (Figure

3.1). The molecular weight of SS is 576.7 and the empirical formula is

(C13H21NO3)2H2SO4. SS is white crystalline powder freely soluble in water (1 part in

4 parts of water), practically insoluble or very slightly soluble in 96% ethanol and in

methylene chloride.

HO

OH

N

OH

H

H

CH3

CH3

CH3

2

H2SO4

Figure 3.1 Chemical structure of Salbutamol Sulfate (SS)


54

3.1.2. Carrier

3.1.2.1. Polycaprolactone (PCL)

H

O

OH

O

n

Figure 3.2 Structure of Polycaprolactone (PCL)

Polycaprolactone (MW 80,000) (Sigma Aldrich) was used for the microsphere

preparation. Polycaprolactone (PCL) is a biodegradable polyester (Figure 3.2) with a

low melting point of 60 °C and a glass transition temperature of about −60 °C. PCL

is a Food and Drug Administration (FDA) approved material that is used in the

human body as a drug delivery device. PCL is degraded by hydrolysis of its ester

linkages in physiological conditions. It is a semicrystalline polymer with longer

hydrophobic segments and slower hydrolytic degradation [242].

3.1.2.2. Poly (DL-lactide-co- glycolide) (PLGA)

Poly (DL-lactide-co-glycolide) 50:50 (MW 65 kDa) (SurModics Pharmaceuticals,

Lakeshore Biomaterials) was used for the microsphere preparation. PLGA is a FDA

approved polymer composed of glycolic acid and lactic acid (Figure 3.3). It is widely

used in drug delivery owing to its biodegradability and biocompatibility. Depending

on the ratio of lactide to glycolide used for the polymerization, different forms of

PLGA can be obtained. e.g. PLGA 50:50 is a copolymer whose composition is 50%

lactic acid and 50% glycolic acid. All PLGAs are amorphous rather than crystalline

and show a glass transition temperature in the range of 40-60 °C [243-244]. PLGA

degrades by hydrolysis of its ester linkages in the presence of water. The time

required for degradation of PLGA is related to the monomers' ratio used in

production; the higher the content of glycolide units, the lower the time required for

http://en.wikipedia.org/wiki/Biodegradable

http://en.wikipedia.org/wiki/Polyester

http://en.wikipedia.org/wiki/Melting_point

http://en.wikipedia.org/wiki/Glass_transition_temperature

http://en.wikipedia.org/wiki/Food_and_Drug_Administration

http://en.wikipedia.org/wiki/Drug_delivery

http://en.wikipedia.org/wiki/Hydrolysis

http://en.wikipedia.org/wiki/Ester

http://en.wikipedia.org/wiki/Glycolic_acid

http://en.wikipedia.org/wiki/Lactic_acid

http://en.wikipedia.org/wiki/Biodegradation

http://en.wikipedia.org/wiki/Biocompatibility

http://en.wikipedia.org/wiki/Amorphous_solid

http://en.wikipedia.org/wiki/Crystallinity

http://en.wikipedia.org/wiki/Glass_transition_temperature

http://en.wikipedia.org/wiki/Hydrolysis

http://en.wikipedia.org/wiki/Water_(molecule)


55

degradation. An exception is the copolymer with 50:50 monomers' ratio which

exhibits the faster degradation (about two months).

HO

O

O

H

O

Om n

Figure 3.3 Structure of Poly (DL-lactide-co- glycolide) (PLGA)

Two biodegradable polymers: PCL and PLGA were selected to act as carriers in DPI

formulations because of the difference in the hydrophobicity and degradation rates.

PCL is more hydrophobic and degrades at a slower rate than PLGA.

3.1.2.3. Lactose

O

HO

OH

OH

OH

O

O

HO

OH

OH

OH

Figure 3.4 Structure of Lactose

Lactose was used as a control in our experiments. Samples of lactose monohydrate

Aeroflo-95 was obtained from Foremost Farm, USA. It is a disaccharide sugar that is

formed from galactose and glucose. It has a formula of C12H22O11 and its systematic

name is β-D-galactopyranosyl-(1→4)-D-glucose (Figure 3.4). It exists in two optic

isomeric forms α-lactose and β-lactose.


56

3.1.3. Ternary Components

Magnesium stearate and L-Leucine were used as ternary components for the coating

of the microspheres that have been fabricated. L-Leucine was procured from Sigma

Aldrich and Magnesium stearate was obtained from PCCA, Australia.

3.1.4. Solvents and Chemicals

Tween 80 (Ajax, Australia) was used in particle sizing experiments. HPLC grade

methanol (LiChrosolv®) (Merck, Germany), Ammonium acetate (AR reagent, Ajax

Chemicals, Australia) and Milli-Q grade water were used in analytical method

development. Polyvinyl alcohol (87-89% hydrolysed, MW 85,000-124,000) (Sigma

Aldrich) was used as the surfactant in the microsphere preparation. Solvents like

Ethanol, Methanol, Acetone, Isopropyl alcohol and Dichloromethane were supplied

by Merck Pty Ltd, Australia and were of analytical grade.

3.2. General methods

3.2.1. Microparticle preparation

3.2.1.1. Oil in water solvent evaporation technique

3.2.1.1.1. Using an overhead stirrer

The microparticles were prepared by oil in water (o/w) solvent evaporation method.

The polymer PCL was dissolved in dichloromethane (DCM) at 10% and 15%

concentration. This polymer solution was added dropwise into 1% w/v aqueous

Polyvinyl Alcohol (PVA) solution with the aid of a dropping funnel. The emulsion

was stirred with an overhead stirrer (IKA® RW 20 digital Labtek, Model RW20D) at

a constant speed of 2000 rpm continuously for 40 minutes under ambient pressure

and then stirred for another 20 minutes under reduced pressure on a rotary evaporator

(Rotavapor R-210, BUCHI, Switzerland). Finally the microspheres were collected by

filtration, washed with deionized water and dried in a vacuum desiccator at room

temperature.

For PLGA microsphere preparation, the polymer PLGA was dissolved in

concentrations of 10%, 15% and 20% in DCM. This polymer solution was added

dropwise into 1% w/v aqueous PVA solution with the aid of a dropping funnel. The

emulsion was stirred with an overhead stirrer (IKA® RW 20 digital Labtek, Model

RW20D or IKA® Eurostar 6000, Labtek) at a constant speed of 2000 rpm or 6000


57

rpm continuously for a period of 4 hours. The microspheres formed were collected

by filtration, washed with deionized water and dried in a vacuum desiccator at room

temperature.

3.2.1.1.2. Using a homogenizer

The same emulsion technique was employed to prepare the microspheres as

described above. The PCL polymer solution at 10% concentration was dissolved in

DCM and this solution was added completely in the aqueous phase (1% PVA

solution). This emulsion was stirred initially at a very high speed [Speed 1 (≈8000

rpm) or Speed 3 (≈15000 rpm)] with the help of homogenizer (Heidolph DIAX 900,

Germany) for 5 minutes. Later the stirring was continued with the help of an

overhead stirrer (IKA® RW 20 digital Labtek, Model RW20D) at 2000 rpm for 4

hours until all the DCM was evaporated from the emulsion. The hardened

microspheres were later collected by filtration, washed with deionized water and

dried in desiccator at room temperature.

3.2.1.2. Electrospraying

The electrospraying apparatus consists of a 2.5 mL glass syringe (Hamilton, USA)

fitted with a 23 gauge stainless steel nozzle (Terumo, Japan and Becton Dickinson,

USA). PCL solution (10% v/v) was filled in the syringe and extruded through the

nozzle at a constant rate of 1.0 mL/hour by using a syringe pump (WPI, USA). An

aluminium foil collector (20x20 cm2) was placed opposite to the syringe as a counter

electrode. The distance from the steel nozzle to the collecting plate was maintained at

25 cm. A strong electric field was applied between the nozzle and the collector

ranging from 8 to 13 kV. Temperature and relative humidity ranged from 22-23 °C

and 44-50% respectively.

The solution contained in the syringe was supplied to the nozzle at a constant flow

rate forming a droplet. The electric field induces charges on the droplet surface. A

liquid jet occurs that breaks up in droplets with a narrow size distribution. Solid

particles formed by solvent evaporation from the droplets travel through the electric

field and were collected on the aluminium collector. After electrospraying, the

collectors were placed under vacuum for 72 hours to remove the solvent residue from

the microspheres. The microspheres were later transferred into glass vials and further

evacuated for storage.


58

3.2.2. Coating of microspheres

3.2.2.1. Dry powder coating

The coating of the PCL or PLGA microspheres with MgSt or leucine was done by a

hand mixing method [245-246]. Mixtures of MgSt (1% and 2%) or leucine (1% and

2%) and PCL or PLGA microspheres were prepared in 5.0 g batches. The powder

was placed between two layers of microspheres in a glass test tube along with three

ceramic beads of approximately 10 mm in diameter. The test tube was vigorously

shaken by hand for 5 minutes to ensure proper mixing. During this process ceramic

beads provided a ball milling effect for breaking up the agglomerates formed during

mixing. This same technique was used to prepare mixture of SS (2.5%) with various

carriers i.e. PCL or PLGA microspheres or lactose monohydrate (Aeroflo-95). These

mixtures were finally used for homogeneity test and in vitro aerosol deposition tests

as mentioned in Sections 3.4.2 and 3.4.3

3.2.2.2. Solution coating

The solution of MgSt was prepared in ethanol (1% and 2%) with the aid of heat and

the aqueous solution of leucine was prepared in milliQ water (1% and 2%).

Approximately 4.0 g of PCL or PLGA microspheres was coated with 10 mL of either

concentrations of MgSt solution or leucine solution by stirring for a period of 24

hours. Finally the coated microspheres were collected by filtration and dried in a

vacuum desiccator at room temperature.

3.2.3. Particle size measurement

3.2.3.1. For microspheres prepared by emulsion technique

The particle size of the carrier particles was measured by laser diffraction (Malvern

Mastersizer, Malvern Instruments Ltd, UK) using the small volume dispersion unit

(capacity 150 mL). The carrier particles were dispersed in water containing Tween

80 with the aid of sonication in a water bath for 5 minutes. This sonicated sample

was added dropwise to the sample cell containing 100 ml of distilled water until an

obscuration between 15-30% was obtained. The average particle size distribution

was measured from five replicates of each sample. The VMD is the parameter used

to characterize the size distributions of the polymeric carrier.

Particle size of SS was determined by dispersing SS in Isopropyl alcohol (IPA) with

the aid of Tween 80. A suspension of the above solution was sonicated for 5 minutes.


59

This sonicated sample was added dropwise to the sample cell containing 100 mL of

IPA until an obscuration between 15-30% was obtained. The VMD was measured

from five replicates of each sample. For lactose carrier, ethanol was used as a

solvent.

3.2.3.2. For microspheres prepared by electrospraying

In order to determine the particle size of the electrosprayed microspheres a

microscope glass slide was introduced in the electrospraying box and held in contact

with the collector in the centre of the spraying zone for 5 minutes. The slide was then

removed and analyzed by light microscopy (AxioVision, Carl Zeiss MicroImaging,

Germany). The particle size was determined from three replicates and the size was

assessed using Image J analysis software.

The laser diffraction technique for particle sizing requires approximately 100-200 mg

of the carrier particles for size measurements. As electrospraying technique is a low

throughput technique of producing microspheres, the microscopy technique was used

to measure the size as it does not require large quantities of the sample for size

determination.

3.2.4. Scanning electron microscopy (SEM)

For surface morphological studies of the carrier particles and the drug-carrier

mixture, samples were glued onto aluminium stubs using double-sided carbon sticky

tape. Particles were then sputtered with gold (thickness ~ 15-20 nm) with a sputter

coater (BIORAD SC-500) using an electrical current of 15 mA for 3 minutes.

Several photomicrographs of the samples were taken at different magnifications

using a Scanning Electron Microscope (FEI Quanta 200 SEM).

3.2.5. Energy Dispersive X-ray Analysis (EDX)

Elemental composition analysis of the carrier particles mixed with the drug was

carried out using Energy Dispersive X-ray Analysis in an SEM (Quanta 3D FIB

instrument). Samples were transferred onto aluminium stubs with the help of double-

sided carbon sticky tape. Particles were then carbon coated with a Cressington

Carbon coater using an electrical current of 100 A for 2 minutes and 10-4

mbar

pressure. Samples were analyzed for sulfur that was present as the salt of the

Salbutamol powder and magnesium for MgSt coated microspheres.


60

3.2.6. X-Ray Photoelectron Spectroscopy (XPS)

The surface composition of the uncoated and coated PCL or PLGA microspheres

was analyzed by XPS. Samples were mounted onto stainless steel sample holders

using double-sided adhesive tape. XPS analysis was performed with a Kratos Axis

Ultra spectrometer (Kratos Analytical, Manchester, UK) equipped with

monochromatized aluminium X-ray source (powered at 10 mA and 15 kV) and an

eight-channeltron detector. The analyzed area was 800 x 200 µm. The constant pass

energy was set at 160 eV for the survey spectrum and 20 eV for the narrow spectrum.

The following sequence of spectra was recorded: Survey spectrum, O1s, N1s, C1s, and

Mg2p multiplex spectra. The elemental compositions of each material were

determined using Casa XPS software Version 2.3.14.

3.2.7. Adhesion force

The adhesion forces of the polymer microspheres and the films with the uncoated

and coated silica sphere were determined by Atomic Force Microscopy (AFM) using

a colloid probe technique [127, 247]. The silicon nitride cantilever (225 ± 10 µm

long, 28 ± 7.5 µm wide and 3 ± 1 µm thick) used for the determination of adhesion

forces had a single silica particle (sphere of diameter 3.5 ± 0.1 µm) attached on the

tip of the cantilever (CP-FM-SiO-B-5, NanoAndMore, Germany). The cantilevers

have resonance frequencies ranging from 45-115 kHz and force constants ranging

from 0.5-9.5 N/m.

3.2.7.1. Measurement of spring constant

The spring constant (k) for each cantilever used was determined using the thermal

noise method [248-249]. The spring constant was found to vary from 1.12 - 2.55

N/m.

3.2.7.2. Sample preparation

3.2.7.2.1. Plasma cleaning of the cover slips

Cover slips of the size 22 x 22 mm were used (Menzel Glaser, Germany) for the

deposition of films. Harrick Plasma Cleaner (Model: PDC-002, Ithaca, NY) was used

to eliminate possible contamination from the coverslips and to make them

hydrophilic. The vacuum was attained with the help of dry oxygen service pump

(Model XDS5, Edward, USA). Oxygen flow was kept at 0.1 SCFH (Standard Cubic

Feet per Hour) using a plasma flow controller. The cleaning step consisted of 30


61

seconds oxygen plasma clean at 380 mTorr pressure and 29.6 W of discharge power

at high RF level.

3.2.7.2.2. Cleaning of the glass slides

Microscope glass slides 25.4 mm x 76.2 mm x 1 mm (Sail brand, China) were used.

The glass slides were first washed with acetone and then with ethanol to remove the

impurities and contaminants from the surface. Later the slides were blown with

nitrogen to ensure complete drying.

3.2.7.2.3. Polymer microspheres

The polymer microspheres were glued on a clean glass slide with Araldite five-

minute epoxy resin glue (Selleys Chemical Company, Australia). A uniform thin

smear of the glue was made in the centre of the clean glass slide. The microspheres

were sprinkled on the resin glue and allowed to dry for five minutes. The excess

microsphere particles were removed from the slide by blowing with nitrogen.

3.2.7.2.4. Polymer films (Spin coating)

The cover slips were used as a substrate and Spin coater (Model PWM-32, Headway

Research, Inc., Texas) was used for the deposition of films on the cover slip.

Approximately 1 mL of the sample solution was placed on the centre of the cover

slip and was spun at a constant speed until all the solvent was evaporated and a thin

film was deposited on it. These samples were stored in a desiccator and later used for

adhesion force measurements and imaging.

The PCL film was formed by spinning the 10% PCL solution in DCM at 1000 rpm

for 20 seconds. The PCL + PVA film was formed by depositing the layer of PCL

film as described above and allowing it to dry for 30 minutes. Later the PCL layer

was covered with 1% PVA solution and spun at 2000 rpm for 60 seconds. The 3

layer film of PCL + PVA + MgSt was prepared by depositing the first 2 layers in the

similar manner as described above and later 1% MgSt solution was deposited and

spun for 1000 rpm for 60 seconds. Similarly the 3 layered film of PCL + PVA +

Leucine was prepared by depositing the first 2 layers as described above and later

spinning 1% leucine solution at 5000 rpm for 60 seconds. The PLGA film was

formed by spinning the 10% PLGA solution in DCM at 500 rpm for 120 seconds.

The deposition of PVA, MgSt and leucine layer on PLGA film were done at similar


62

speed and time intervals as described for PCL. A summary of the speed and the time

interval used for each sample is provided in Table 3.1.

Table 3.1 Speed of the spin coater and time intervals of spinning of each sample

SAMPLE SPEED

(rpm)

TIME

(seconds)

PCL 1000 20

PVA 2000 60

MgSt 1000 60

Leucine 5000 60

PLGA 500 120

3.2.7.2.5. Functionalization of silica probe with SS

The silica sphere on the cantilever was functionalized with SS solution to act as SS

probe. The drug was coated on the silica sphere of the cantilever with the aid of

approach/retract cycle of AFM scanner. This procedure was first developed in the lab

and was validated. A small drop of supersaturated solution of SS was placed on a

clean glass slide and the slide was placed on the scanner of the AFM. The silica

probe to be functionalized was secured on the cantilever holder and positioned

exactly above the SS solution. The cantilever was made to approach to the solution

on the surface using the microscope‟s feedback loop with a controlled motion, was

kept immersed in the SS solution for 10 minutes; retracted from the solution after 10

minutes and allowed to dry for a period of 30 minutes. This resulted in the formation

of multilayered coating of SS on the silica sphere. This method ensured a SS layer on

the spherical silica probe, which is perfect for adhesion force measurement.

3.2.7.3. Force Measurement

All force measurements were performed using AFM MFP-3D-BIO (Asylum

Research, Technical Manufacturing Corporation, USA) and IGOR Pro 6.21 software

(Wavemetrics, USA) in air and ambient humidity. Measurements in force volume

mode were performed between an uncoated silica sphere and the film and

microsphere, or an SS coated silica sphere and the film and microsphere. In the force

volume mode, the AFM raster scans the substrate under the colloidal probe to

produce a series of force curves, each from a well-defined location in the x and y


63

directions. The results were produced as a force map that shows the variation in the

forces of interaction in the defined area. For microspheres, the individual force

curves were measured over a 10 µm x10 µm area, at a scan rate of 1 Hz and a total of

32 x 32 (n=1024) force points. The adhesion force determination was performed on 5

different microspheres for each sample (n=5) and a total number of 5120 force

curves were measured for each sample. For the films, the individual force curves

were measured over a 10 µm x 10 µm area, at a scan rate of 1 Hz and a total of 8 x 8

(n=64) force points. The adhesion force determination was performed at 5 different

spots on each film (n=5) and a total number of 320 force curves were measured for

each film.

3.2.8. Surface Roughness

The imaging and the determination of surface roughness of the samples were done in

air and ambient humidity and were performed using AFM MFP-3D-BIO (Asylum

Research, Technical Manufacturing Corporation, USA) and IGOR Pro 6.21 software

(Wavemetrics, USA).

3.2.8.1. Imaging of polymer particles and films

Polymer microspheres were glued on the glass slide as described before in Section

3.2.7.2.3. The films were prepared in a similar manner as described in the Section

3.2.7.2.4. Tapping mode surface scans were performed so that the sample is not

damaged during imaging. Tapping mode AFM tips with force constants of 40 N/m

and resonant frequencies of 300 kHz were used for imaging (Tap 300 DLC Silicon

AFM probes, Budget Sensors, USA). A scan rate of 1.00 Hz, a defined scan size of

10 x 10 µm2 with 512 x 512 points in each image was used.

3.2.8.2. Roughness measurement

After capturing an image, it was flattened to remove the effect of piezo motion. The

root mean square (RMS) roughness was determined using the IGOR Pro software

available with AFM. The software used the following equation for the calculation of

surface roughness.

RMS of Y values = 2

_

1iY

npntsV 3.1


64

Where: V_npnts: number of points= 512 x 512 and Yi is the height of the Y values.

3.2.9. Surface energy determination

Surface energies were determined using Inverse Gas Chromatography (IGC 2000,

Surface Measurement Systems Ltd, London, UK) with a flame ionization detector.

The sample (PCL or PLGA microspheres) was packed into pre-silanised glass

columns of 300 mm length and 3 mm internal diameter. The columns were filled

with approximately 0.6 g of the samples by tapping until no cracks, voids or channels

were visible in the powder bed. Silanised glass wool was used to plug at both the

ends of the column to prevent the powder from falling out. The columns were

conditioned at 30 oC for 2 hours before each measurement to remove impurities

adsorbed onto the surface. Probes were carried into the column at a flow rate of 10

sccm (standard cubic centimeter per minute) by helium carrier gas. The dead volume

calculations were based on the elution time of methane which was run at a

concentration of 0.1 p/p0 (where p denotes the partial pressure and p

0 the vapour

pressure). Triplicate measurements were performed for each sample.

Non-polar surface energy distributions (NP

profile)

The non-polar surface energy distributions or the dispersive energies (NP

profiles) of

the samples were determined by running five alkane probes (hexane, heptane, octane,

nonane and decane) at concentrations of 0.03, 0.10, 0.25, 0.35, 0.55, 0.70, 0.80, 0.90

and 0.95 p/p0

[250-252]. The retention time of the alkane probes were used to

calculate the dispersive surface energy of the powder in the column. Peak maximum

method was used to create the adsorption isotherms for each alkane [253]. The

Brunauer-Emmet-Teller (BET) surface area was calculated from heptane adsorption

isotherms. The surface coverage (n/nm) was calculated by dividing the adsorbed

amount (n) by the monolayer capacity (nm, the number of moles of the probe

adsorbed for monolayer coverage). The net retention volume (VN) was then

calculated for each probe at each surface coverage. The non-polar surface energy

(NP

) was calculated from the slope (2 NA √NP

) of a plot of RTlnVN against a √NP

of

alkanes [254] where, NP

is the non-polar surface energy, NA is Avogadro‟s number,

R is the gas constant, VN is net retention volume, and T is the column temperature.


65

3.3. Analytical methods

3.3.1. UV spectrophotometric assay

A spectrophotometric (UV) assay was used for the determination of SS in the

homogeneity studies. The UV spectrum of SS in water was analyzed over a

wavelength range of 190-400 nm by a UV spectrophotometer (U-2800

spectrophotometer, Hitachi) using 10 mm quartz cells to measure the wavelength of

maximum absorbance (λmax). The Beer-Lambert calibration curve was prepared at a

wavelength of 276 nm using concentrations ranging from 0-500 µg/mL. Three

replicates of the measurements were done. The accuracy and the precision of the

assay were determined at three concentrations (high, medium and low) using three

replicates of each concentration. The Limit of Detection (LOD) and Limit of

Quantification (LOQ) were determined.

3.3.2. HPLC assay

SS was analyzed by HPLC (Agilent HP1100) using a C18 column (µBondapak®, 3.9

x 300 mm, Waters) and an UV Diode Array detector (Agilent) at a wavelength of

276 nm. A mixture of methanol and 0.2% w/v ammonium acetate solution (45:55)

was used as a mobile phase at a flow rate of 1.0 mL/minute by a HPLC pump (Quat

pump, Agilent). An injection volume of 100 µL was used. The peak area was

recorded by integration. The retention time of SS was found to be 4.3 minutes. A

HPLC calibration curve was prepared with concentrations ranging between 0 - 100

µg/mL. Five replicates of the measurements were done. The accuracy and the

precision of the assay were determined at three concentrations (high, medium and

low) using five replicates of each concentration. The LOD and LOQ were

determined.

3.4. Powder formulation

3.4.1. Powder mixing

The drug-carrier mixture was prepared by a hand mixing method [245-246] in a

similar manner as described in Section 3.2.2.1

3.4.2. Homogeneity tests

The homogeneity of each drug-carrier mixture prepared in the Section 3.4.1 was

determined by sampling 20 x 20 mg samples from the mixtures and assaying for SS

content. The samples were dissolved in 10 mL of water and the amount of SS was


66

quantified based on UV absorbance in triplicates. An acceptable degree of

homogeneity is achieved when the mean drug content is 95%-105% of the theoretical

value and the Coefficient of variation (CV) is less than 5% which indicates that 95%

of the samples would fall within 10% of the mean [255].

3.4.3. In vitro aerosol deposition

The in vitro aerosol deposition of the powder formulations was determined by a

Twin Stage Impinger (TSI apparatus, Apparatus A, Glass Impinger, British

Pharmacopoeia, 2009) (Copley, UK) (Figure 3.5). Rotahaler® (Glaxo Wellcome)

was the DPI device used and the collection liquid used for the study was water. 7 and

30 mL of water was placed in stage one (S1) and stage two (S2) of the TSI,

respectively. The air flow was drawn through the TSI using a vacuum pump (D-

63150, Erweka, Germany) and the air flow rate was adjusted to 60 ± 5 L/min at the

mouthpiece, prior to each measurement (Fisher and Porter, Model 10A3567SAX,

UK).

The powder formulations were loaded (about 20.0 mg) into hard gelatin capsules

(size 3, Fawns and McAllan Pty Ltd.; Australia). The filled capsule was inserted into

the Rotahaler® which was placed into a moulded mouthpiece attached to the TSI.

The Rotahaler® was twisted to release the powder into the body of the device and an

air volume of 5 litres (5 seconds at 60 L/min) was drawn for each measurement

which produces a cut-off mass median aerodynamic diameter of 6.4 µm between the

two stages [256]. Each section (Inhaler, S1 and S2) was rinsed with water and the

liquid was collected and volume was adjusted to 100 mL. The SS content was

measured by HPLC analysis. Five replicates of each mixture were performed for TSI

measurement.

The recovered dose (RD) was the total amount of drug collected from the inhaler, S1

and S2. The emitted dose (ED) was the fraction of the RD delivered from the inhaler

expressed as a percentage.

10021

RD

SSED 3.2


67

The fine particle fraction (FPF) was defined as the fraction of the recovered dose

deposited in the lower stage of TSI expressed as a percentage of RD.

1002

RD

SFPF 3.3

Figure 3.5 Twin Stage Impinger (TSI) apparatus

3.4.4. Statistical Analysis

Comparison between different groups of FPF was performed by one-way analysis of

variance (ANOVA) to ascertain statistical significance; p<0.05 was considered to be

statistically significant.

Mouth piece + Rotahaler®

Stage one (S1)

Stage two

(S2)

Chapter 4 Method Validation

69

CChhaapptteerr 44

MMeetthhoodd VVaalliiddaattiioonn



71

4.1. Summary

This chapter discusses the validation for the following experiments:

UV assay for the determination of SS in the homogeneity studies

HPLC assay for the determination of SS in the in vitro aerosol studies

Homogeneity of the powder mixtures

The inter-batch and intra-batch variability of in vitro drug dispersion

Functionalization of silica probe with SS

The adhesion force measurements by AFM using a colloid probe

4.2. Analytical validation

4.2.1. Spectrophotometric assay

The UV spectrum of SS in water was determined and the wavelength of maximum

absorption (λmax) was found to be 276 nm (Figure 4.1). The Beer-Lambert calibration

curve at 276 nm in the concentration range of 0-150 µg/mL using three replicates is

presented below (Figure 4.2). A linear line was obtained with the regression

coefficient (r2) of 1.000. The accuracy and precision of the assay was examined using

high, medium and low concentrations (Table 4.1). The accuracy was in the range of

99%-100.1% and the coefficient of variation (CV) for precision ranged from

0.007%-0.03%. The LOD and LOQ were found to be 4 µg/mL and 12 µg/mL,

respectively.

Figure 4.1 Ultraviolet scan of SS in water over the range of 190-400 nm for the

determination of the wavelength of maximum absorbance (λmax= 276 nm)


72

Figure 4.2 Beer’s Law calibration curve of SS (n=3)

Table 4.1 Accuracy and precision of the UV assay for SS (n=3)

Concentration (µg/ml) Mean Accuracy (%) Variation (% CV)

12 99.0 0.03

50 100.1 0.007

150 99.6 0.01

4.2.2. HPLC

The HPLC method was validated and a representative HPLC chromatogram of SS of

lowest and highest standards is presented in Figure 4.3 and Figure 4.4 respectively

which showed the retention time of 4.2-4.3 minutes. The calibration curve of HPLC

for determining SS in the concentration range of 0-100 µg/mL is presented below

(Figure 4.5). A linear line was obtained with the regression coefficient (r2) of 1.000.

The accuracy and precision of the assay was examined using high, medium and low

concentrations and is presented in Table 4.2. The accuracy ranged from 99.9%-100.8

% and the coefficient of variation (CV) for precision ranged from 0.3%-2.6%. The

LOD and LOQ were found to be 0.2 µg/mL and 0.6 µg/mL, respectively.

y = 0.0055x + 0.0052

R² = 1

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

0 50 100 150 200

Ab

sorb

an

ce

Concentration (µg/ml)


73

Figure 4.3 A representative HPLC chromatogram of Salbutamol Sulfate (0.6

µg/ml) showing retention time at 4.224 minutes

Figure 4.4 A representative HPLC chromatogram of Salbutamol Sulfate (100

µg/ml) showing retention time at 4.314 minutes


74

Figure 4.5 HPLC calibration curve of SS (n=5)

Table 4.2 Accuracy and precision of the HPLC assay for SS (n=5)

Concentration (µg/ml) Mean Accuracy (%) Variation (% CV)

0.6 100.8 2.6

50 100.3 0.3

100 99.9 0.3

4.3. Powder Formulations

Five batches (A, B, C, D and E) of powder formulation containing 2.5% of SS and

PCL carrier coated with 1% magnesium stearate solution were prepared as

previously described (Section 3.2.2.1, Chapter 3). The homogeneity and in vitro TSI

deposition of each batch were examined in order to confirm that the mixing

technique gave reproducible homogenous powder mixtures.

4.3.1. Homogeneity test by UV

The accuracy and variation of the mean drug content for each mixture is shown in

Table 4.3. The accuracy ranged from 99.0%-100.8% and the CV was found to be

0.6%-1.3%.

y = 15.567x - 0.2574

R² = 1

0

200

400

600

800

1000

1200

1400

1600

1800

0 20 40 60 80 100 120

Are

a

Concentration (µg/ml)


75

Table 4.3 Homogeneity tests on five batches of 2.5% Drug-Carrier mixture (n=20)

Batch Mean Accuracy (%) Variation (% CV)

A 99.3 0.9

B 99.0 1.3

C 100.8 0.7

D 99.8 0.6

E 99.6 0.7

4.3.2. In vitro aerosol deposition

4.3.2.1. Inter-batch and intra-batch variability

The results of the in vitro aerosol deposition of five different batches (Inter-batch) of

2.5% SS- PCL carrier coated with 1% magnesium stearate solution mixtures are

presented in Figure 4.6. No significant differences (p<0.05) were observed in the

deposition of drug which confirmed that the mixing technique produced reproducible

in vitro aerosol deposition and no batch to batch variability was observed with

respect to deposition. The in vitro aerosol deposition of the five replicates from one

batch (Intra-batch) is shown in Figure 4.7. No significant differences occurred in

terms of emitted dose and the drug deposition in S1 and S2 of the TSI apparatus

which implies that similar in vitro aerosol deposition was obtained from replicates of

the same powder formulation.

Thus it implied that the overall deposition, washing and analytical procedures were

reliable and reproducible.


76

Figure 4.6 Inter-batch comparison of TSI deposition of SS for 2.5% mixture of SS

and PCL microspheres coated with 1% magnesium stearate solution (n=5)

Figure 4.7 Intra-batch comparison of TSI deposition of SS for 2.5% mixture of SS

and PCL microspheres coated with 1% magnesium stearate solution (n=5)

4.4. Adhesion Force measurements

4.4.1. Functionalization of silica probe with SS

In order to determine the adhesion forces between the SS and the polymer samples,

the silica sphere on the cantilever was functionalized with SS solution to act as SS

0

10

20

30

40

50

60

70

Rotahaler Stage 1 Stage 2

% D

rug

Batch A

Batch B

Batch C

Batch D

Batch E

0

10

20

30

40

50

60

70

Rotahaler Stage 1 Stage 2

% D

rug

Replicate 1

Replicate 2

Replicate 3

Replicate 4

Replicate 5


77

probe. This experiment validated the time required by the silica sphere to get

completely coated by the SS solution. For this purpose, silica spheres were coated

with SS solution for a period of 5, 10 and 30 minutes. Later these samples were

analyzed by XPS to detect the presence of sulfur (as SS is present as a sulfate salt)

and silica. The XPS analysis revealed that the silica spheres coated for the time

interval of 5 minutes showed the presence of sulfur in addition to the signal of silica

(Figure 4.8B). This indicated that the spheres were coated with SS but some traces of

silica were still present. However the spheres coated for the time interval of 10 and

30 minutes had a very strong signal of sulfur and silica was not found in the

spectrum (Figure 4.8C and D) indicating that the spheres coated for 10 minute time

interval was completely coated by SS. Hence the time period of 10 minutes was

selected to functionalize the silica probe with SS.

Figure 4.8 XPS survey scan of (A) Silica spheres (B) Silica spheres coated with SS

for 5 minutes (C) Silica spheres coated with SS for 10 minutes and (D) Silica

spheres coated with SS for 30 minutes

Figure 4.9 below gives the SEM images of the uncoated silica sphere coated with SS

at various time intervals. Figure 4.9C and D reveals that the silica sphere has been

well-covered with SS as compared with Figure 4.9B which was coated for just 5

minutes.


78

(A) (B)

(C) (D)

Figure 4.9 SEM images of (A)Silica sphere at 6250×, (B)Silica sphere coated with

SS for 5 minutes at 10,000×, (C) Silica sphere coated with SS for 10 minutes at

10,000× and (D) Silica sphere coated with SS for 30 minutes at 9375×

The cantilevers were coated with SS according to the procedure described in Chapter

3, Section 3.2.7.2.5. The silica probe was functionalized with SS by coating it for the

time interval of 10 minutes. The SEM images of the uncoated and coated cantilevers

were done using SEM (NeoScope Benchtop SEM [JCM-5000, JEOL, USA]). An

SEM image of the uncoated cantilever is presented in Figure 4.10. and of the

cantilever functionalized with SS is shown in Figure 4.11.


79

Figure 4.10 SEM image of the uncoated cantilever

Figure 4.11 SEM image of the cantilever coated with SS

4.4.2. Individual adhesion forces by silica probe

The adhesion forces between silica probe and the PCL microspheres were

measured on three different microspheres at the time interval of 5 minutes

each over a period of 30 minutes. The reproducibility of the adhesion forces

between the silica probe and three different PCL microspheres is shown in

Figure 4.12. The mean adhesion force and CV obtained for the three different

PCL microspheres over a period of 30 minutes were 93.2 nN (0.7%), 135.4

nN (1.5%) and 167.3 nN (1.2%). The low variability in the adhesion forces at

each adhesion site confirmed that the adhesion forces were reproducible and

remained stable over a period of time.


80


microspheres over a period of 30 minutes determined by AFM using 3.5 µm silica

probe

4.4.3. Individual adhesion forces by SS probe

The adhesion forces between SS probe and the PCL microspheres were

measured on three different microspheres at the time interval of 5 minutes

each over a period of 30 minutes. The reproducibility of the adhesion forces

between the SS probe and three different PCL microspheres is shown in

Figure 4.13. The mean adhesion force and CV obtained for the three different

PCL microspheres over a period of 30 minutes were 267.2 nN (0.1%), 298.8

nN (0.6%) and 326.4 nN (0.4%). The low variability in the adhesion forces at

each adhesion site confirmed that the adhesion forces were reproducible and

remained stable over a period of time.

0

20

40

60

80

100

120

140

160

180

0 5 10 15 20 25 30

Ad

hes

ion

forc

es (

nN

)

Time (minutes)

Microsphere 1

Microsphere 2

Microsphere 3


81


microspheres over a period of 30 minutes determined by AFM using 3.5 µm silica

probe functionalized with SS

0

50

100

150

200

250

300

350

0 5 10 15 20 25 30

Ad

hes

ion

Forc

es (

nN

)

Time (minutes)

Microsphere 1

Microsphere 2

Microsphere 3

Chapter 5 Drug Dispersion from PCL and PLGA Microspheres

83

CChhaapptteerr 55

DDrruugg DDiissppeerrssiioonn ffrroomm PPCCLL aanndd PPLLGGAA

MMiiccrroosspphheerreess

Chapter 5 Drug Dispersion from PCL and PLGA

Microspheres


85

5.1. Introduction

The traditional carrier lactose has been widely discussed in the literature. The

purpose of this present study is to explore the use of the biodegradable polymers with

spherical shape and reproducible surface as an alternative carrier to lactose for the

purpose of drug dispersion from DPI formulations.

This chapter discusses the fabrication of microspheres of two different biodegradable

polymers: PCL and PLGA, their characterization and eventually the role of these

microspheres in the drug dispersion from DPI formulations.

5.2. Results and Discussion

5.2.1. Particle sizing and drug dispersion from lactose

Lactose is used as a control in the experiments because it is the most widely used

carrier in all the DPI products currently available in the market. Inhalation grade

Aeroflo-95 lactose was used as a control because it has a VMD of 112.9 ± 2.5 µm

which is comparable with the particle size of the PCL and PLGA microspheres that

have been fabricated (VMD: 104 ± 0.4 µm and 90.2 ± 1.0 µm for PCL and PLGA

microspheres, respectively. Particle sizes have been measured by laser diffraction

according to the procedure described in Chapter 3, Section 3.2.3). One important

point to be considered here is that the morphologies of lactose and polymer particles

are quite different. It is noted that the lactose carriers used as control showed a wider

particle size distribution (Figure 5.1). This was due to the presence of a significant

amount of associated fine particles of lactose attached on the surface of large

carriers; whereas, PCL and PLGA microspheres showed comparatively narrow size

distribution (Figure 5.1). The large lactose particles have a significant amount of

associated fine lactose (less than 10 μm) and the effect of these fine particles on the

particle size distribution as well as the SS dispersion has not been taken into account

in this study.


86

Figure 5.1 Particle size distribution of Lactose particles, PCL microspheres and

PLGA microspheres

The lactose carrier, drug (SS) and the mixture of SS and lactose were characterized

by SEM imaging as shown in Figure 5.2.

0

2

4

6

8

10

12

14

16

1 10 100 1000

% V

olu

me

Particle Size (µm)

Lactose

PCL

PLGA


87

(A) (B)

(C)

Figure 5.2 SEM images of (A) Lactose (Aeroflo-95) at 500X , (B) SS at 10,000X

and (C) SS-Lactose mixture at 4000X

It can be seen that the lactose particles are highly irregular in shape and have a

significant amount of associated fine particles of lactose on their surface (Figure

5.2A). The drug particles SS are flat, plate-like and exist as a very cohesive powder

(Figure 5.2B). Figure 5.2C showed the DPI formulation of SS and lactose. The fine

particles of lactose attached on the surface of large lactose made it very difficult to

differentiate them from the particles of SS in the mixture of SS and lactose. Hence an

EDX analysis of the lactose-SS mixture according to the procedure described in

Chapter 3, Section 3.2.5 was carried out to distinguish between the two and confirm

the presence of drug adhered onto the carrier surface. Since the drug is present as a

sulfate salt, the detection of the sulfur in the EDX spectrum confirms the presence of

the SS on the lactose carriers (Figure 5.3).


88

Figure 5.3 EDX spectrum of SS-Lactose mixture

This drug-carrier mixture was found to be homogenous (Table 5.1). The

homogeneity of the drug-carrier mixture is determined according to procedure

described in Chapter 3, Section 3.4.2.


(n=20)

Batch Accuracy (%) Variation (%)

SS and Lactose (Aeroflo-95) 99.8 0.4

SS and PCL 99.8 0.5

SS and PCL coated with 1% MgSt powder 99.4 0.7

SS and PCL coated with 2% MgSt powder 99.6 0.5

SS and PCL coated with 1% MgSt solution 99.3 0.9

SS and PCL coated with 2% MgSt solution 99.9 0.2

SS and PCL coated with 1% leucine powder 99.0 0.5

SS and PCL coated with 2% leucine powder 98.7 0.3

SS and PCL coated with 1% leucine solution 98.8 0.8

SS and PCL coated with 2% leucine solution 99.2 0.8

SS and PLGA 99.3 1.0

SS and PLGA coated with 1% MgSt solution 99.8 0.5

SS and PLGA coated with 1% leucine solution 99.8 0.5


89

When the SS-lactose mixture was subjected to in vitro deposition tests using TSI

(procedure described in Chapter 3, Section 3.4.3), FPF of 13.5 ± 2.5% was obtained

as shown in Table 5.2. This indicated that with the traditional carrier lactose; 13.5%

of the SS was obtained in the S2 of the TSI. The amount of drug deposited in the DPI

device and S1 of TSI was found to be 45.4 ± 8.4% and 41.0 ± 8.5% respectively

(Table 5.3).


90

Table 5.2 Fine Particle Fraction (FPF) of SS

Sample FPF (%)

Emitted Dose

(ED) (%)

Recovered

Dose

(RD) (%)

SS and Lactose (Aeroflo-95) 13.5 ± 2.5 54.5 ± 8.4 98.0 ± 2.0

SS and PCL (Prepared by o/w solvent evaporation method)

0 63.6 ± 7.2 92.4 ± 5.0

SS and PCL (Prepared by Electrospraying)

0 77.7 ± 4.8 95.5 ± 3.8

SS and PCL coated with 1% MgSt powder

12.0 ± 1.3 60.3 ± 5.0 87.0 ± 5.2

SS and PCL coated with 2% MgSt powder

15.8 ± 1.5 50.3 ± 7.1 96.9 ± 3.9

SS and PCL coated with 1% MgSt

solution

11.4 ± 1.0 40.3 ± 2.4 86.2 ± 5.0


solution

15.4 ± 1.6 64.4 ± 5.6 95.0 ± 5.7

SS and PCL coated with 1% leucine

powder

6.0 ± 0.5 67.1 ± 5.4 90.6 ± 5.0


powder

10.2 ± 1.2 65.3± 3.0 97.4 ± 6.2


solution

11.3 ± 1.1 56.1 ± 9.3 85.0 ± 3.5


solution

11.3 ± 0.8 67.0 ± 4.1 84.4 ± 3.3

SS and PLGA 16.6 ± 1.6 63.7 ± 2.7 88.2.0 ± 3.0

SS and PLGA coated with 1% MgSt

powder

15.8 ± 1.0 63.0 ± 4.5 93.7 ± 5.7


solution

15.3 ± 1.0 62.8 ± 4.7 93.5 ± 6.0

SS and PLGA coated with 1% leucine

powder

15.3 ± 1.0 64.5 ± 2.0 90.2 ± 3.7


solution

15.4 ± 0.4 65.0 ± 4.0 87.2 ± 2.2


91

Table 5.3 TSI data of SS from different carriers

Sample DPI device

(%)

Stage 1

(%)

FPF

(Stage 2)

SS and Lactose (Aeroflo-95) 45.4 ± 8.4 41.0 ± 8.5 13.5 ± 2.5

SS and PCL (Prepared by o/w solvent

evaporation method)

36.3 ± 7.3 63.4 ± 7.3 0

SS and PCL (Prepared by

Electrospraying)

22.3 ± 4.8 77.4 ± 4.8 0


powder

39.6 ± 5.0 48.4 ± 4.0 12.0 ± 1.3


powder

50.0 ± 7.1 34.2 ± 6.0 15.8 ± 1.5


solution

59.7 ± 2.4 28.8 ± 3.0 11.4 ± 1.0


solution

35.5 ± 5.7 48.9 ± 6.5 15.4 ± 1.6


powder

32.8 ± 5.4 61.1 ± 5.6 6.0 ± 0.5


powder

34.6 ± 3.0 55.0 ± 3.8 10.2 ± 1.2


solution

49.4 ± 2.0 39.2 ± 2.8 11.3 ± 1.1

SS and PCL coated with 2% leucine solution

33.0 ± 4.1 55.7 ± 3.4 11.3 ± 0.8

SS and PLGA 36.2 ± 2.8 47.1 ± 3.8 16.6 ± 1.6


powder

37.0 ± 4.5 47.2 ± 4.5 15.8 ± 1.0


solution

37.1 ± 4.7 47.5 ± 4.3 15.3 ± 1.0


powder

35.1 ± 4.0 50.0 ± 4.4 15.3 ± 1.0


solution

35.0 ± 4.0 49.5 ± 4.1 15.4 ± 0.4


92

5.2.2. Characterization of microspheres and drug dispersion

5.2.2.1. PCL microspheres

The microspheres were fabricated using the o/w solvent evaporation method as

described in Chapter 3, Section 3.2.1. SEM images revealed that the PCL

microspheres are spherical and had a rough surface morphology (Figure 5.4A). These

PCL microspheres were dry-mixed with the drug particles according to the procedure

described in Chapter 3, Section 3.4.1. The SEM images confirm that the drug

particles had adhered onto the entire surface of the PCL carrier (Figure 5.4B). The

drug-polymer mixture was found to be homogenous (Table 5.1). The drug

dispersion was determined by TSI using the procedure as discussed in Chapter 3,

Section 3.4.3. The TSI experiment indicated that the drug had been emitted

effectively from the DPI device (% ED: 63.6 ± 7.2) (Table 5.2) but no FPF of SS was

obtained, which means a significant amount of powder was retained in the DPI

device and S1 of TSI (36.3 ± 7.3% and 63.4 ± 7.3%, respectively) (Table 5.3). It

seems that the drug was strongly adhered on the surface of the PCL microspheres

and could not be detached by the airflow in the TSI. As evident from the SEM image

(Figure 5.4B), the micronized SS particles formed a close-packed array on the

surface of the PCL carrier which is smooth and hence difficult to detach from the

surface. Therefore, it was vital to determine the surface composition of the PCL

microspheres to understand the reason behind the strong SS-PCL adhesion. The

surface composition was determined using XPS analysis which is described in the

following section 5.2.3.

(A) (B)

Figure 5.4 SEM images of (A) PCL microspheres at 2188X and (B) SS-PCL

mixture at 2187X. Note the high surface coverage of SS on the PCL particle

compared to the lactose particle in Figure 5.2C


93

5.2.2.2. PLGA microspheres

The PLGA microspheres were fabricated in a similar manner as PCL microspheres

using o/w solvent evaporation method described in Chapter 3, Section 3.2.1. SEM

images revealed that the microspheres are spherical and had a smooth surface

morphology in contrast to PCL microspheres which were comparatively rougher

(Figure 5.5A and Figure 5.4A). These microspheres were mixed with the drug

particles and a homogenous mixture was formed (Table 5.1). The SEM images

confirm that the drug has adhered onto the surface of the PLGA carrier (Figure

5.5B). In this case, the micronized SS particles did not form a close-packed array on

the surface of the PLGA carrier when compared with the PCL carrier (Figure 5.4B).

It seems the drug was loosely bound to the PLGA surface which was not much

difficult to detach from the surface. The drug dispersion from the TSI experiment

indicated that the FPF of SS obtained was 16.6 ± 1.6% (Table 5.2) and the remaining

drug obtained in the DPI device and S1 of TSI was 36.2 ± 2.8% and 47.1 ± 3.8%,

respectively (Table 5.3). Thus in contrast to the PCL microspheres, the drug particles

were capable of detaching from the PLGA surface and FPF of the drug was obtained.

However to get an idea about the chemical composition of the PLGA surface, the

microspheres were subjected to XPS analysis.

(A) (B)

Figure 5.5 SEM images of (A) PLGA microspheres at 2344X and (B) SS- PLGA

mixture at 2813X. Note that the surface coverage of SS on the PLGA particle

which is to a lesser extent than the PCL particle in Figure 5.4C

5.2.3. XPS analysis of microspheres

To understand the surface composition of the microspheres, XPS analysis was

carried out in accordance with the procedure described in Chapter 3, Section 3.2.6.


94

XPS scan of the pure polymer was also carried out simultaneously to understand the

differences between the pure polymer and the microspheres that have been fabricated

from the respective polymer.


XPS analysis shows that the spectrum of PCL microspheres was different from pure

PCL powder (Figure 5.6). A higher C-O signal in Figure 5.6B suggests that that the

initially hydrophobic surface of the PCL microspheres was covered with PVA which

was used as a surfactant in the manufacture of the microspheres. The analysis is

complicated by the fact that the PVA used for the microsphere preparation was 87-

89% hydrolysed so it contained 11-13% of residual vinyl acetate groups. However,

comparison with the spectrum of the PVA used in the microparticle preparation

shows that the surface of the PCL microspheres contains a layer of PVA along with

some residual acetate groups. Thus the hydrophobic surface of PCL was rendered

more hydrophilic due to the presence of PVA.

Figure 5.6 XPS scan of (A) PCL powder and (B) PCL microspheres


95

Earlier it was speculated that the presence of hygroscopic PVA on the surface might

have led to the strong adhesion between the PCL microspheres and the hydrophilic

drug particles. The hypothesis was that the strong adhesion might have occurred due

to the formation of a hydrogen bonds between hydroxyl groups of PVA on the

surface of PCL (which has a low glass transition temperature of -60 oC and is thus

rubbery at room temperature) and either hydroxyl groups or nitrogen group of SS. In

order to test our hypothesis we analyzed pure PCL, pure SS, PCL microspheres and

SS-PCL mixture using infrared spectroscopy. When the spectrum of SS was

subtracted from that of the SS-PCL mixture the spectrum obtained was similar to the

spectra of PCL microspheres indicating that there was no detectable hydrogen bond

formation between SS and PCL. The more likely reason for the strong adhesion

could be due to the formation of capillary bridges between the hygroscopic PVA,

which is present on the surface of PCL, and the hydrophilic SS. The hygroscopic

PVA might have led to the absorption of moisture from the atmosphere. This

atmospheric moisture around the surface contact sites usually exerts strong force

between the adjacent solid bodies and this force would be dominant compared to the

other surface forces such as electrostatic forces and van der Waals forces. Therefore,

there was no detachment of the drug particles from the PCL surface resulting in no

FPF of SS.

However in order to further confirm whether PVA was the true cause for the strong

adhesion, the author fabricated PCL microspheres which were devoid of PVA. This

was done by using the electrospraying technique described in Chapter 3, Section

3.2.1.2. The particle size of these microspheres was determined by light microscopy

using the procedure described in Chapter 3, Section 3.2.3.2. Malvern Mastersizer

was not used to determine the size of the electrosprayed microspheres.

Electrospraying is a low throughput technique, hence the yield of the microspheres is

less and the Mastersizer technique needs a large amount of microspheres to

determine their size based on laser diffraction. Hence light microscopy was used to

determine the size of the electrosprayed batch.


96

Figure 5.7 Particle size distribution of PCL microspheres fabricated by

electrospraying

These microspheres had the median particle diameter of 19.9 ± 4.2 µm (Figure 5.7)

and were spherical in shape. The SEM images showed that the surface of these

microspheres was covered with small indentations or dimples and the surface

appeared quite rough (Figure 5.8A). The microspheres were mixed with SS to form a

homogenous mixture. The SEM images reveal that the SS has adhered on the surface

of microspheres (Figure 5.8B). However the coverage of the SS particles on the

surface of PVA-free PCL microparticles was more scattered and isolated when

compared with the close-packing arrangement on the PVA-coated PCL

microparticles (Figure 5.4B). This difference in the packing of the SS particles on the

surface of the microspheres could be attributed to the difference in the morphology

of the microspheres. The drug-carrier mixture was analyzed for drug dispersion by

TSI. The TSI results from the PCL microspheres devoid of PVA yielded no FPF of

the drug as no drug was detected in the S2 of the TSI. With respect to device

retention, only 22.3 ± 4.8% of the drug was found in the DPI device and the

remaining 77.4 ± 4.8% of the drug was detected in the S1 of the TSI (Table 5.3).

This suggested that the drug was very strongly adhering on the surface of the PCL

microspheres.

0

5

10

15

20

25

30

35

40

45

50

5 12 18 24 31 37

Nu

mb

er o

f P

art

icle

s (%

)

Particle Diameter (µm)


97

(A) (B)

Figure 5.8 SEM images of (A) PCL microspheres prepared by electrospraying at

15,994X and (B) SS- PCL mixture at 8000X.

The results of TSI from PVA-free microspheres gave a clear idea about the true

reason of the strong attachment of the drug particles on the PCL surface. It was clear

from this experiment that PVA was not the causative factor for the strong adhesion

of the drug particles as the drug dispersion from both PVA-coated and PVA-free

PCL microspheres was nil.

Although presence of PVA was detected on the surface of the microspheres which

were prepared by o/w solvent evaporation method, but the extent of the coverage of

PVA on the surface is unknown. It can be possible that the coating of the PVA on the

surface of the microspheres is not uniform as the microspheres after fabrication were

thoroughly washed with water. It would have stripped off some amount of PVA but

left some residual PVA behind on the surface of the microspheres which was

detected by XPS. Hence it could be that the PCL polymer by itself was the actual

reason for the strong adhesive forces as it was established that the presence of PVA

was not the contributing factor to the strong adhesion forces between PCL

microspheres and the SS particles. This is explained in detail in Chapter 7, Section

7.2.1.1 where the adhesion forces are determined between the PCL film and SS

particles.

Thus to overcome the problem of strong adhesion between SS and PCL carrier, PCL

surface was modified with a suitable excipient to decrease the forces of adhesion

with an objective to achieve better detachment of drug particles from the PCL

surface. This was achieved by coating the surface of the microspheres with a ternary

agent, which exhibits anti-adherent properties. Based on a consideration of the need


98

for excipients that would reduce the bonding to SS, MgSt and leucine were chosen.

Both these agents are hydrophobic and may act as lubricants between surfaces thus

improving the dispersibility of the powders.

Solution coating and dry mixing were both used for coating the microspheres. The

coating of the microspheres was carried out according to the procedure described in

Chapter 3, Section 3.2.2. The coated polymer microspheres were further subjected to

XPS analysis to confirm the presence of the MgSt and leucine on the PCL

microspheres. The XPS data (Table 5.4) confirmed the presence of magnesium and

nitrogen elements on the PCL microspheres which indicated that the PCL particles

were successfully coated with these two agents. Also this technique was used to

determine the thickness of the coating on the surface of the PCL microspheres [257].

If a uniform layer is formed, the thickness of the MgSt or leucine coating obtained

from solution may be determined using the following equation:

I

Il

dn 1 5.1

Where d is the thickness of the layer on the substrate, λ is the photoelectron mean

free path in the overlayer, I is the signal intensity of the overlayer (intensity of

magnesium or nitrogen obtained from coated PCL microspheres) and I∞ is the signal

intensity from a homogenous infinitely thick sample of the overlayer (intensity of

magnesium or nitrogen obtained from pure MgSt and leucine powders respectively).

According to Table 5.4, I∞= 2.1 and 11.17 for MgSt and leucine powder samples,

respectively. The mean free path (λ) for a magnesium photoelectron was unavailable

and Si-2p was taken as a reference element because both photoelectrons have similar

kinetic energies. λ was taken to be 40 Å on average for a silicon layer (Si-2p) on

polyester or polystyrene material. λ was taken to be 32 Å for nitrogen which is the

reference value of N-1s on polyester or polystyrene material [258].Using this

equation and the I values in Table 5.4, the thickness of MgSt coating on the surface

of the PCL microspheres was determined to be 10 Å and 38 Å when deposited from

1% and 2% MgSt solutions, respectively. In contrast, it was found by the same

method of analysis that 1% and 2% leucine solution coating provided 5 Å and 8 Å

layers on the surface of PCL microspheres. It should be noted that these calculations


99

assume that a uniform layer has been deposited on the surface, which as noted later is

only the case for leucine.

Table 5.4 XPS results of PCL and PLGA microspheres coated with MgSt and

Leucine solution

Sample Atomic Percentage of Elements

C O Mg N

MgSt powder 89.10 8.85 2.10 -

PCL microspheres coated with 1% MgSt

solution 77.86 21.67 0.47 -

PCL microspheres coated with 2% MgSt

solution 86.39 12.3 1.31 -

Leucine powder 68.77 20.06 - 11.17

PCL microspheres coated with 1% leucine

solution 72.20 26.25 - 1.55

PCL microspheres coated with 2% leucine

solution 70.98 26.57 - 2.45

PLGA microspheres coated with 1% MgSt

solution 86.98 11.21 1.80 -

PLGA microspheres coated with 1% leucine

solution 71.19 26.60 - 2.21


XPS analysis shows that the spectrum of PLGA microspheres was different from

PLGA powder (Figure 5.9). The presence of C*-COO signal in Figure 5.9B

suggested that the surface of the PLGA microspheres was rendered hydrophilic due

to the presence of PVA on the surface. Comparing the spectrum of the PLGA

microspheres with the spectrum of the PVA used in the microparticle preparation

shows that the surface of the PLGA microspheres contains a layer of PVA along with

some residual acetate groups. Thus the hydrophobic surface of PLGA is rendered

more hydrophilic due to the presence of PVA similar to the case of PCL

microspheres.

The presence of PVA on the surface of PLGA nanoparticles has been reported in the

literature. The fraction of PVA remains on the surface of the nanoparticles despite

repeated washing with water as PVA is capable of forming an interconnected

network with the polymer at the interface [259].


100

Figure 5.9 XPS scan of (A) PLGA powder and (B) PLGA microspheres

But in the case of PLGA, the presence of PVA on the surface of the microspheres did

not hamper the dispersion of the drugs unlike PCL microspheres. When the TSI

experiments were conducted with the PLGA microspheres, the drug readily detached

from the PLGA surface and approximately 16% FPF of the drug was obtained. This

could be because the adhesion forces between the drug and the PLGA microspheres

were not that strong and hence it facilitated easy detachment of the SS from the

surface. This is explained in detail in Chapter 7, Section 7.2.1.2.

In order to further improve the drug dispersion from PLGA microspheres it was

decided to coat the microspheres with the hydrophobic glidants. MgSt and leucine

which had been used for coating the PCL microspheres were selected. PLGA

microspheres were coated with MgSt and leucine according to the procedure

described in Chapter 3, Section 3.2.2. The coated polymer microspheres were further

subjected to XPS analysis to confirm the presence of the MgSt and leucine on the


101

surface of the PLGA microspheres. The XPS data (Table 5.4) confirmed the presence

of magnesium and nitrogen elements on the PLGA microspheres which indicated

that the PLGA particles were successfully coated with these two agents. Also the

XPS data was used to calculate the thickness of the coat formed by using the same

overlayer equations as described for PCL microspheres (Section 5.2.3.1). The

thickness of the coating on the surface of the PLGA microspheres was determined to

be 78Å when deposited from 1% MgSt solutions and 26Å when deposited from 1%

leucine solutions. As stated earlier these calculations are applicable only when a

uniform layer has been deposited on the surface.

5.2.4. Characterization of microspheres after surface-coating and drug

dispersion


The coated PCL microspheres were mixed with SS and homogenous mixtures were

obtained (Table 5.1). The coated PCL microspheres and the SS-PCL mixture were

characterized using SEM imaging. It was expected that MgSt solution coating would

form a thin layer on the surface of PCL microspheres but in the SEM images Figure

5.10A and B, it was observed that MgSt had deposited in the form of crystals on the

surface of the PCL microspheres. This could be due to crystallization of MgSt on

evaporation of the ethanol solvent. Usually MgSt is used in DPI formulations to

increase the flow property of the powder mixture and the mixing is done either by

ball milling or by mechanofusion [40] to make a solid coat around the carrier surface.

In our study, MgSt was mixed by ball milling. Additionally, MgSt solution was used

by solvent evaporation technique to form a thin coat on the polymer carrier surfaces.

The SEM images reveal that since the MgSt coat was not formed in a continuous

film but was found as discontinuous patches of crystals on the surface of

microspheres, then the calculations for the determination of its thickness via XPS

analysis were inappropriate for this particular sample. In contrast, leucine formed a

uniform coating on the surface of the microspheres and no deposition of the crystals

was found on the surface (Figure 5.10C and D). The powder coated samples were

also imaged with SEM. The MgSt powder coated samples looked similar to the

solution coated microspheres (Figure 5.10 E and F). However the leucine powder

coated microspheres showed the tiny specs of leucine powder adhered on their

surface (Figure 5.10G and H) in contrast to leucine solution coated microspheres


102

which had a uniform coating. The SEM images further confirm that the drug has

adhered onto the surface of the coated PCL carrier but not to the same extent as

compared with the uncoated PCL microspheres. It is noted that in case of coated

microspheres the attachment of the SS particles on the carrier is scattered and more

isolated and the surface corona is rougher as compared to the uncoated PCL carrier

(Figure 5.11 and Figure 5.4B).

(A) (B)

(C) (D)

(E) (F)


103

(G) (H)

Figure 5.10 SEM images of (A) 1% MgSt solution coated PCL microspheres at

1852X, (B) 2% MgSt solution coated PCL microspheres at 1250X, (C) 1% Leucine

solution coated PCL microspheres at 1250X, (D) 2% Leucine solution coated PCL

microspheres at 938X, (E) 1% MgSt powder coated PCL microspheres at 1000X

(F) 2% MgSt powder coated PCL microspheres at 700X, (G) 1% Leucine powder

coated PCL microspheres at 1099X and (H) 2% Leucine powder coated PCL

microspheres at 1583X

(A) (B)


104

(C) (D)

(E) (F)

(G) (H)


PCL microspheres at 1500X, (B) 2% MgSt solution coated PCL microspheres at

1875X, (C) 1% Leucine solution coated PCL microspheres at 1327X, (D) 2%

Leucine solution coated PCL microspheres at 2400X, (E) 1% MgSt powder coated

PCL microspheres at 1500X, (F) 2% MgSt powder coated PCL microspheres at

1828X, (G) 1% Leucine powder coated PCL microspheres at 938X and (H) 2%

Leucine powder coated PCL microspheres at 847X


105

As may be seen from the SEM images (Figure 5.11A and B), in case of 1% and 2%

MgSt coated PCL microspheres, it was difficult to distinguish between the particles

of MgSt and SS; therefore, it was necessary to determine elemental composition to

confirm the presence of drug and MgSt on the surface of PCL. The presence of

elemental magnesium and sulfur in the EDX spectrum confirms that the PCL

microspheres have been coated with MgSt and the drug particles are adhered onto it

(Figure 5.12).

Figure 5.12 EDX spectrum of interactive mixture of 2.5% SS and PCL

microspheres coated with 1% MgSt solution

The FPF values obtained from these surface-modified samples are shown in Table

5.2 and Figure 5.13. The FPF obtained with MgSt and leucine coating was found to

be promising. It was found that 1% MgSt powder coating yielded an FPF of 12 ±

1.3%; however no significant improvement in the FPF of the drug was obtained from

PCL carriers coated with 1% MgSt solution (FPF: 11.4 ± 1.0%) (p>0.05, n=5). The

DPI device retention for 1% MgSt powder coated and solution coated carrier was

found to be 39.6 ± 5.0% and 59.7 ± 2.4%, respectively and the amount of SS

deposited in S1 of the TSI with these two carriers was observed to be 48.4 ± 4.0%

and 28.8 ± 3.0%, respectively (Table 5.3). Similarly, for the 2% MgSt powder coated

and solution coated carriers, the amount of SS recovered from Rotahaler® device

was found to be 50.0 ± 7.1% and 35.5 ± 5.7%, respectively and the amount of SS


106

found in S1 of TSI was found to be 34.2 ± 6.0% and 48.9 ± 6.5%, respectively

(Table 5.3). It is evident from the SEM images that even in the case of solution

coating the MgSt had precipitated in the form of crystals on the surface of the PCL

microspheres. 2% MgSt powder coated and solution coated PCL microspheres

showed a significant improvement in the FPF of SS (15.8 ± 1.5% and 15.4 ± 1.6%)

as compared with 1% MgSt powder coated and solution coated PCL microspheres

(p<0.05, n=5). However again there was no significant improvement in the FPF

when MgSt was used to coat the microspheres from solution compared to powder

coating. This is explained since in both cases, SEM shows crystals of MgSt were

deposited on the PCL surface. SEM shows that the coating was formed as

discontinuous patches (Figure 5.10A and B). As the concentration of the MgSt

increased there was more crystallization of the MgSt on the PCL surface resulting in

increased surface coating of the PCL microspheres with increased FPF of SS.

For the leucine excipient, the dry powder coating yielded a lower FPF (6.0 ± 0.5%

and 10.2 ± 1.2% for 1% and 2% concentrations, respectively) compared to the

solution coating process. The amount of SS retained in the DPI device was found to

be 32.8 ± 5.4% and 34.6 ± 3.0%, respectively and the amount recovered in the S1 of

TSI was 61.1 ± 5.6% and 55.0 ± 3.8%, respectively (Table 5.3). Coating the PCL

particles with either 1% or 2% leucine gave the same FPF (11.3 ± 1.1% and 11.3 ±

0.8%, respectively) (p>0.05, n=5). The device retention of the SS with 1% and 2%

leucine solution coated carriers was 49.4 ± 2.0% and 33.0 ± 4.1%, respectively and

the amount recovered in the S1 of TSI was found to be 39.2 ± 2.8% and 55.7 ± 3.4%,

respectively (Table 5.3).

In contrast for the earlier values quoted for dry powder coating the FPF obtained

with 2% leucine was statistically different from that obtained with 1% leucine

(p<0.05, n=5). In dry powder leucine coating, as the concentration of leucine

increased on the surface of PCL, it caused improved surface coating of the PCL

microspheres and increased the FPF by masking the PCL surface with leucine. In

contrast, the lack of an effect of leucine concentration in the solution coating results

could be due to the fact that 1% concentration of leucine was sufficient to completely

mask the surface of PCL. This may have caused easy detachment of SS and hence

increased FPF. There was no evidence for crystallization of leucine on the surface of

the PCL in contrast to the results for MgSt. Consequently, the overlayer calculation


107

is applicable and a thickness of only 5 Å of leucine is required to mask the PVA

surface layer on PCL.

Thus these anti-adherent agents helped in improving the FPF of SS from the coated

PCL microspheres probably by the reduction of the adhesion forces which is

explained in details in Chapter 7, Section 7.2.1.1.

Figure 5.13 FPF of coated PCL microspheres


The coated PLGA microspheres were mixed with SS and homogenous mixtures were

formed (Table 5.1). The coated PLGA microspheres and the SS-PLGA mixture were

characterized using SEM imaging. Similar to PCL microspheres, in case of solution

coating it was found that MgSt had crystallized on the surface of the microspheres

(Figure 5.14A). Thus the MgSt coating was non-uniform and hence the thickness of

the coat determined for this sample with XPS analysis was not valid. In concurrence

with the previous leucine results, again leucine formed a uniform coat from the

solution (Figure 5.14B) and hence the overlayer calculation of the determination of

the thickness of the coat is valid for this sample. The MgSt and leucine powder

coated samples were also imaged by SEM and the microspheres showed the presence

of MgSt and leucine powder respectively on their surfaces (Figure 5.14C and D). The

0 2 4 6 8

10 12 14 16 18 20

% F

PF


108

SEM images further confirm that the drug has adhered onto the surface of the coated

PLGA carrier (Figure 5.15). The attachment of the SS particles on both uncoated and

coated PLGA carrier did not form a close-packed array and the particles were more

isolated on the surface of the carrier (Figure 5.15A and Figure 5.5B).

(A) (B)

(C) (D)

Figure 5.14 SEM images of (A) 1% MgSt solution coated PLGA microspheres at

3906X, (B) 1% Leucine solution coated PLGA microspheres at 8000X, (C) 1%

MgSt powder coated PLGA microspheres at 4405X and (D) 1% Leucine powder

coated PLGA microspheres at 2400X


109

(A) (B)

(C) (D)


PLGA microspheres at 4501X, (B) 1% Leucine solution coated PLGA

microspheres at 3750X, (C) ) 1% MgSt powder coated PLGA microspheres at

5244X and (D) 1% Leucine powder coated PLGA microspheres at 5859X

The FPFs obtained from these surface-modified samples are shown in Table 5.2 and

Figure 5.16. It was found that 1% MgSt solution coated PLGA microspheres

provided an FPF of 15.3 ± 1.0% and 1% leucine solution coated PLGA microspheres

provided an FPF of 15.4 ± 0.4%. The FPFs obtained with the 1% MgSt and leucine

powder coated samples were found to be 15.8 ± 1.0% and 15.3 ± 1.0% respectively.

The DPI device retention of SS with all the four coated PLGA carrier samples was

found to be in the range of 35-37% and 47-50% of the drug was found to be in the S1

of TSI (Table 5.3).

The FPF data indicates that both the coated microspheres provided no significant

improvement in the FPF when compared with the uncoated PLGA microspheres

which yielded an FPF of 16.6 ± 1.6% (p>0.05, n=5). Also there were no significant


110

differences in the FPF of SS from the dry powder coatings when compared with the

solution coatings (p>0.05, n=5). Thus these results indicated that the coatings of

MgSt and leucine on the surface of PLGA microspheres did improve the FPF of the

SS unlike PCL microspheres. In fact the FPFs obtained in the case of uncoated and

coated PLGA microspheres were similar in either case of powder and solution

coatings. It could be because there was no significant reduction in the adhesion

forces between the drug and the surface of PLGA even when the coating agents were

used which is explained in detail in Chapter 7, Section 7.2.1.2.

Figure 5.16 FPF of uncoated and coated PLGA microspheres

In summation, when the uncoated PCL microspheres were used as a carrier, the drug

did not detach from its surface which could be due to the strong adhesion force

between SS and PCL carriers. However upon coating the carrier with the anti-

adherent agents like MgSt or leucine, the drug detached from the surface, which may

be due to the reduction of adhesion force and significant improvement in the FPF of

drug was obtained. This was further examined by AFM adhesion measurements in

Chapter 7, Section 7.2.1.1.

For PLGA microspheres, the drug readily detached from the surface; however, the

anti-adherent coating agents were not capable of further improving the drug

dispersion. This could be because these hydrophobic agents were not capable of

0

2

4

6

8

10

12

14

16

18

20

PLGA PLGA+1%

MgSt

powder

PLGA+1%

MgSt

solution

PLGA+1%

Leucine

powder

PLGA+1%

Leucine

solution

% F

PF


111

reducing further the adhesion forces existing between the PLGA polymer and the

drug which is discussed in Chapter 7, Section 7.2.1.2.

5.3. Conclusion

The work presented in this chapter highlighted the potential of surface coated

biodegradable PCL microparticles and PLGA microspheres as carriers for pulmonary

drug delivery system. The surface coating of PCL microparticles carriers helped in

significantly increasing the aerosolization of SS from the carrier based DPI

formulation. Without surface coating, the drug adhering on the PCL carrier has been

efficiently emitted from the device but showed no FPF. The solution coatings of

MgSt and leucine facilitated the easy detachment of the drug particles from the PCL

surface with a concomitant increase in drug deposition. On the other hand, PLGA

microspheres readily allowed the detachment of the drug from the surface and hence

no surface coating of the PLGA microspheres was essential. These results suggested

that the surface coated biodegradable PCL microspheres and the uncoated PLGA

microspheres can act as potential carriers in DPI formulations.


113

CChhaapptteerr 66

EEffffeecctt ooff tthhee SSiizzee ooff tthhee CCaarrrriieerr oonn tthhee

DDiissppeerrssiioonn ooff SSSS

Chapter 6 Effect of the Size of the Carrier on the

Dispersion of SS


115

6.1. Introduction

The size of the carrier plays a very important role in the dispersion of the drug from

the DPI devices as it affects the detachment of the drugs and eventually the FPF of

the drug reaching the lungs. The effect of the size of the lactose carrier on the FPF of

the drug has been discussed in detail in Chapter 2. Contrary reports exist in the

literature regarding the effect of the size of the carrier on the FPF of the drug. Some

studies suggest that the small size of the carrier is favourable in improving the

dispersion of the drug from the surface of the carrier [21, 48, 89-90] while other

studies contradict that the larger size of the carrier is beneficial [100-101].

Hence, it was the aim of the present study to examine the influence of the particle

size of the polymeric carrier on the aerosol performance of the binary drug-polymer

powder mixture. This chapter discusses the effect of the size of the carrier on the FPF

of the SS from the interactive mixtures of SS and the polymer (PCL and PLGA)

carriers. The relationship between the size and FPF was determined by fabricating

microspheres of four different sizes each of PCL (25 µm, 48 µm, 104 µm and 150

µm) and PLGA (20 µm, 45 µm, 90 µm and 150 µm) and then determining the

dispersion of SS from them using the TSI.


6.2.1. Particle size and Morphology

6.2.1.1. Salbutamol Sulfate (SS)

The average VMD of the SS powder was found to be 4.5 ± 0.04 µm and the Mass

Median Aerodynamic Diameter (MMAD) was found to be 3.8 ± 0.06 µm from the

distribution shown in Figure 6.1. This distribution showed that about 50% of the

particles are below the size of MMAD; hence capable of reaching deep into the

lungs. The SEM images showed that SS particles are flat and elongated and exists as

cohesive powder (Figure 6.2).


116

Figure 6.1 Particle size distribution of SS, n=5

Figure 6.2 SEM images of SS powder at 2000× and 10,000× respectively

6.2.1.2. Carriers

6.2.1.2.1. PCL microspheres

PCL microspheres were fabricated as discussed in Chapter 3 (Section 3.2.1). The

PCL microspheres were prepared in four different sizes: 25 µm, 48 µm, 100 µm and

150 µm by varying the concentration of the polymer and the speed of the stirring.

The coating of the PCL microspheres with MgSt and leucine were carried out using

the procedure described in Chapter 3 (Section 3.2.2). The particle size distribution of

the PCL microspheres is presented in Figure 6.3. The average VMD of the PCL

microspheres were found to 25.5 ± 0.2 µm, 48.2 ± 0.1 µm, 104.4 ± 0.4 µm and 150.3

0

1

2

3

4

5

6

7

8

9

0.01 0.1 1 10 100 1000

% V

olu

me

Particle Size (µm)


117

± 0.4 µm. The surface morphology of the PCL microspheres coated with MgSt and

leucine was studied using SEM. The SEM images confirmed that the PCL

microspheres are spherical in shape and had irregular surface morphology (Figure

6.4 and Figure 6.5). Also contrary to the expectation that MgSt would form a

continuous thin layer on the surface of the microspheres, actually it was found that

MgSt had deposited in the form of crystals (Figure 6.4). This could be due to

crystallization of MgSt on the surface of microspheres on evaporation of the ethanol

solvent which was used to dissolve MgSt as explained earlier in Chapter 5. However

with leucine, the coatings were found to be uniform as opposed to the MgSt coatings

(Figure 6.5).

Figure 6.3 Particle size distribution of four different batches of PCL microspheres,

n=5

0

2

4

6

8

10

12

14

0.01 0.1 1 10 100 1000

% V

olu

me

Particle Size (µm)

25 µm

48 µm

104 µm

150 µm


118

(A) (B)

(C) (D)

Figure 6.4 SEM images of PCL microspheres coated with MgSt of various sizes:

(A) 25 µm (4687×), (B) 48 µm (4442×), (C) 104 µm (1852×) and (D) 150 µm

(946×)


119

(A) (B)

(C) (D)

Figure 6.5 SEM images of PCL microspheres coated with leucine of various sizes:

(A) 25 µm (6392×), (B) 48 µm (10,535×), (C) 104 µm (1250×) and (D) 150 µm

(1200×)

6.2.1.2.2. PLGA microspheres

PLGA microspheres were fabricated in four different sizes: 20 µm, 45 µm, 90 µm

and 150 µm by varying the concentration of the polymer and the speed of the stirring

as discussed in Chapter 3 (Section 3.2.1). The particle size distribution of the PLGA

microspheres is shown in Figure 6.6. The average VMD of the PCL microspheres

were found to 20.1 ± 0.07 µm, 45.6 ± 0.04 µm, 90.2 ± 1.0 µm and 150.2 ± 0.8 µm.

The surface morphology of the PLGA microspheres was obtained using SEM. The

SEM images confirmed that the PLGA microspheres are spherical in shape and had a

smooth surface morphology especially when compared to PCL microspheres (Figure

6.7).


120

Figure 6.6 Particle size distribution of four different batches of PLGA

microspheres, n=5

(A) (B)

(C) (D)

Figure 6.7 SEM images of PLGA microspheres of various sizes: (A) 20 µm

(10,000×), (B) 45 µm (3662×), (C) 90 µm (2344×) and (D) 150 µm (1500×)

0

2

4

6

8

10

12

14

16

0.01 0.1 1 10 100 1000

% V

olu

me

Particle Size (µm)

20 µm

45 µm

90 µm

150 µm


121

6.2.1.3. Interactive mixtures of SS and Carriers

6.2.1.3.1. Interactive mixtures of SS and PCL carrier

The coated PCL microspheres were later dry-mixed with SS and homogenous

mixtures were formed as discussed in Chapter 3 (Section 3.2.1). The homogeneity of

the mixtures (Table 6.1) is confirmed using the procedure described in Chapter 3

(Section 3.2.2). The drug-coated PCL microspheres were characterized using SEM

imaging. The SEM images further confirm that the drug has adhered onto the surface

of the coated PCL carrier (Figure 6.8 and Figure 6.9). As can be seen in the SEM

images, as the carrier size increased, there was increased deposition of the SS

particles on the surface of the PCL carrier. This can be explained by the concept of

particle number (N) which gives the number of particles per unit weight of the

sample and is expressed by the following equation.

3

6

dN 6.1

Where, N is the particle number, d is the diameter of the spherical particle and ρ is

the density.

It can be seen in Table 6.2 and Table 6.3, as the size of the carrier particle increases,

the particle number decreases. For each sizes of the microspheres, the drug-carrier

ratio was maintained constant (2.5%). Hence for a fixed mass of drug, the mass of SS

particles per unit area of carrier particles is more in larger size as compared with the

small sized microspheres due to the reduction in the carrier particle number. Thus as

the carrier size increases the mass of SS particles per unit area also increases.


122

(A) (B)

(C) (D)


coated with MgSt of various sizes: (A) 25 µm (3418×), (B) 48 µm (4194×), (C) 104

µm (1500×) and (D) 150 µm (800×)


123

(A) (B)

(C) (D)


coated with leucine of various sizes: (A) 25 µm (7786×), (B) 48 µm (6662×), (C)

104 µm (1327×) and (D) 150 µm (1131×)

6.2.1.3.2. Interactive mixtures of SS and PLGA carriers

The PLGA microspheres were later dry-mixed with SS and homogenous mixtures

were formed as discussed in Chapter 3 (Section 3.2.1). The homogeneity of the

mixtures (Table 6.1) is confirmed using the procedure described in Chapter 3

(Section 3.2.2). The drug- coated PLGA microspheres were characterized using SEM

imaging. The SEM images further confirm that the drug has adhered onto the surface

of the PLGA carrier (Figure 6.10). As the size of the carrier increased, there was

increased deposition of the SS particles on the surface of the PLGA carrier (Figure

6.10). For a given formulation mass with a fixed formulation ratio (constant drug to

carrier blend ratio), the mass of SS particles per carrier is increased in the larger

carriers due to the reduction in the carrier particle number (Table 6.4). Thus when the

carrier size is increased, the number of carrier particles is decreased, the specific

surface area of the particles is decreased and there is an increase in the number of


124

drug particles per carrier. It is pointed out that in contrast to the study of the PCL

microparticles there was no surface coating of PLGA microparticles prior to the

loading of the drug.

(A) (B)

(C) (D)

Figure 6.10 SEM images of interactive mixtures of 2.5% SS and PLGA

microspheres of various sizes: (A) 20 µm (7500×),(B) 45 µm (4000×), (C) 90 µm

(2813×) and (D) 150 µm (1200×)


125


(n=20)

Batch Accuracy

(%)

Variation

(%)

SS and PCL coated with 1% MgSt solution (25 µm) 99.9 0.6






SS and PCL coated with 2% MgSt solution (100µm) 99.9 0.2


SS and PCL coated with 1% leucine solution (25 µm) 99.7 0.5


SS and PCL coated with 1% leucine solution (100µm) 98.8 0.8






SS and PLGA 99.3 1.0


126

Table 6.2 The % FPF of SS from 1% and 2% MgSt coated PCL microspheres of

four different sizes having different surface areas

VMD of PCL

microspheres

(µm)

Surface

Area

(cm2)

Particle

number

(N)

Mass of SS

particles per

unit area of

PCL particles

(mg)

% FPF

1% MgSt

coated

2% MgSt

coated

25 0.00002 1.2 x 108 0.011 3.6 ± 0.3 5.3 ± 0.9

48 0.00007 1.5 x 10

7

0.023 4.9 ± 0.3 7.2 ± 2.1

104 0.00034 1.4 x 10

6

0.050 11.4 ± 1.0 15.4 ± 1.6

150 0.00071 4.9 x 10

5

0.072 13.1 ± 0.8 20.4 ± 2.7

Table 6.3 The % FPF of SS from 1% and 2% Leucine coated PCL microspheres of

four different sizes having different surface areas

VMD of PCL

microspheres

(µm)

Surface

Area

(cm2)

Particle

number

(N)

Mass of SS

particles per

unit area of

PCL particles

(mg)

% FPF

1% Leucine

coated

2%

Leucine

coated

25 0.00002 1.2 x 108 0.011 2.9 ± 0.3 3.2 ± 0.3

48 0.00007 1.5 x 107 0.023 4.7 ± 0.8 4.6 ± 0.3

104 0.00034 1.4 x 106 0.050 11.3 ± 1.1 11.3 ± 0.8

150 0.00071 4.9 x 105 0.072 13.4 ± 0.5 13.9 ± 1.4


127

Table 6.4 The % FPF of SS from PLGA microspheres of four different sizes

having different surface areas

VMD of PLGA

microspheres

(µm)

Surface Area

(cm2)

Particle

number

(N)

Mass of SS

particles per unit

area of PLGA

particles (mg)

% FPF

20 0.00001 1.9 x 10

8

0.01 5.6 ± 0.7

45 0.00006 1.6 x 10

7

0.023 11.0 ± 1.0

90 0.00025 2.0 x 10

6

0.047 16.6 ± 1.6

150 0.00071 4.5 x 10

5

0.078 21.3 ± 1.2

6.2.2. Effect of carrier size on drug dispersion

6.2.2.1. In vitro TSI deposition of SS from the carrier

6.2.2.1.1. Drug dispersion from PCL carrier

Four different sizes of the MgSt coated and leucine coated PCL microspheres were

used as carriers to prepare homogenous mixtures with 2.5% SS. The microspheres

had different particle size distributions with VMD ranging from 25 to 150 µm. The

dispersion of SS from these mixtures was measured by TSI. The FPF from these

interactive mixtures was found to be increasing as the carrier size increased. As the

VMD increased from 25 to 150 µm, the FPF values increased from 3.6% to 13.1%

for 1% MgSt coated PCL microspheres and from 5.3% to 20.4% for 2% MgSt coated

PCL microspheres. Similarly the FPF of the SS from leucine coated microspheres

also increased ranging from 2.8% to 13.4% for 1% leucine coated microspheres and

3.2% to 13.9% for 2% leucine coated microspheres. The amounts of drug recovered

in the Rotahaler® device and S1 were in the range of 25-50% and 28-70%,

respectively (Table 6.5). The recovered dose of SS in the TSI was found to be 85%

to 98% and the emitted dose was found to be ranging from 65% to 75%. Thus the

FPF of SS was found to be greater with the highest particle size of the carrier.

The relationship between the FPF of SS from 1% and 2% MgSt coated PCL

microspheres and the size of the microspheres is depicted in Figure 6.11. It can be

seen very clearly that as the size of the carrier increased there is an increase in the


128

FPF of the drug from both 1% and 2% MgSt coated PCL microspheres. There is also

significant difference in the FPF of SS amongst the two concentrations of MgSt

coatings. Similarly the relationship between the FPF of SS from 1% and 2% leucine

coated PCL microspheres and the size of the microspheres is depicted in Figure 6.12.

As in the case of MgSt coatings, the FPF of the drug from both 1% and 2% leucine

coated PCL microspheres increased with the increase in the carrier size; however

there was no significant difference in the FPF of SS amongst the two concentrations

of leucine coatings suggesting again that the FPF of SS is independent of the

concentration of the leucine coatings (over the values studied of 1% and 2%).


129

Table 6.5 TSI data of SS from different carriers

Sample

DPI

device

(%)

Stage 1

(%)

FPF

(Stage 2)

SS and PCL (25 µm) coated with 1% MgSt solution 48.6 ± 4.5 47.7 ± 4.5 3.6 ± 0.3


SS and PCL (25 µm) coated with 1% leucine solution 28.2 ± 3.6 68.8 ± 3.6 2.9 ± 0.3








SS and PCL (104 µm) coated with 1% leucine solution

49.4 ± 2.0 39.2 ± 2.8 11.3 ± 1.1

SS and PCL (104 µm) coated with 2% leucine

solution

33.0 ± 4.1 55.7 ± 3.4 11.3 ± 0.8




solution

24.4 ± 5.6 62.5 ± .7 13.4 ± 0.5


solution

28.0 ± 3.3 58.0 ± 4.8 13.9 ± 1.4

SS and PLGA (20 µm) 20.1 ± 2.7 73.5 ± 3.1 5.6 ± 0.7

SS and PLGA (45 µm) 30.1 ± 3.7 59.0 ± 3.6 11.0 ± 1.0

SS and PLGA (90 µm) 36.2 ± 2.8 47.1 ± 3.8 16.6 ± 1.6

SS and PLGA (150 µm) 30.7 ± 6.5 48.0 ± 7.0 21.3 ± 1.2


130


microspheres coated with 1% and 2% MgSt solution respectively, n=5


microspheres coated with 1% and 2% leucine solution respectively, n=5

6.2.2.1.2. Drug dispersion from PLGA carrier

Four different sizes of the PLGA microspheres were used as carriers to prepare

homogenous mixtures with 2.5% SS. The microspheres had different particle size

distributions with VMD ranging from 20 to 150 µm. The dispersion of SS from these

mixtures was measured by TSI. The FPF from these interactive mixtures was found

0

5

10

15

20

25

0 50 100 150 200

% F

PF

Particle size (µm)

1%

2%

0

2

4

6

8

10

12

14

16

18

0 50 100 150 200

% F

PF

Particle Size (µm)

1%

2%


131

to be increasing from 5.6% to 21.3% as the carrier size increased. The DPI device

retention with the PLGA carrier in the different size range was found to be 20-36%

and in S1 was found to be 47-73% (Table 6.5). The recovered dose of SS in the TSI

was ranging between 85% to 98% and the emitted dose was found to be 65% to 75%.

The FPF was found to be greater with the highest particle size of the carrier. The

relationship between the FPF of SS from PLGA microspheres and the size of the

microspheres is depicted in Figure 6.13. It can be seen very clearly that as the size of

the PLGA microspheres increased there is a concurrent increase in the FPF of SS.

Figure 6.13 The relationship between the FPF of SS and the size of the PLGA

microspheres, n=5

6.2.2.2. Influence of inherent carrier size on dispersion

It was observed that as the VMD of the carrier increased, there was a concurrent

increase in the FPF of the drug. This occurs because there is a difference in the

mechanism in the way the drug detaches from the larger carriers as compared to the

small sized carriers which is discussed in detail in Chapter 2, Section 2.3.3.

There are two major mechanisms which govern the detachment of drug from the

carrier surface i.e. detachment by the flow stream (fluid forces) and detachment by

impaction (mechanical forces) [80, 82, 260]. When the flow stream has unobstructed

path to access and remove the drug from the carrier surface, then the mechanism is

called detachment by the flow stream (fluid forces). This mechanism occurs mostly

0

5

10

15

20

25

0 50 100 150 200

% F

PF

Particle Size (µm)


132

on a relatively flat carrier surface with minimal asperities. Detachment by

mechanical forces occurs when the collisions occurs between the carrier particles and

the inhaler wall and in between the carrier particles. This generally occurs in those

carrier surfaces which have increased amount of surface roughness [80, 260]. Due to

the collisions, there is a transfer of momentum which leads to the detachment of the

drug from the surfaces. The momentum of any particle is dependent on the mass and

the velocity of the carrier. The larger particles will have greater mass. The force of

mechanical detachment would be directly proportional to the cube of carrier particle

diameter (Force = Mass*Acceleration, Mass = Density*Volume and Volume =

4/3πr3). Thus larger particles will have greater mass which in turn will lead to

generation of greater detachment forces due to impaction. Hence as the magnitude of

the detachment forces increases with the carrier size, there is more detachment of

drugs from the carrier surface and consequently increase in the FPF of the drug.

Therefore the larger carriers will increase the mechanical detachment forces due to

the stronger particle-inhaler and particle-particle collisions [260]. This was further

confirmed in a study by Concession et al [261]. The study determined the

relationship between the impact forces and particle detachment. They found that as

the diameters of the lactose or maltodextrin carrier particles decreased, greater

magnitude of forces were required to separate the SS particles from the surface. In

addition the greater drag forces encountered by the larger carriers in the turbulent air

stream will contribute to a greater detachment force [48]. Hence the larger carrier

demonstrates better performance in terms of FPF as compared to their smaller

counterparts.

It is also known that as the size of the carrier decreases, there in an increase in the

specific surface area of the carrier particles with subsequent increase in the overall

surface energy. This increase in surface energy results in higher adhesive forces

between drug and the lactose carrier particles [96]. As the particle size decreases,

cohesive and frictional resistance increases due to more points of contact arising

from the increase in surface area-to-volume ratio [262]. In addition, a large surface

area with decreased particle size renders the particles subject to greater potential for

charging and moisture uptake. Also the size of the particles renders them more

susceptible to the influence of van der Waals forces [65].


133

Hence, all these factors might have contributed to the decreased detachment of the

drug from the smaller carrier as compared to the larger carrier. Therefore, as the

mechanical forces increased with the larger carrier particles, the role of the increased

size of the carrier particles was confirmed in improving the dispersion performance

of the drug from the carrier.

The author found that increasing the size of both PCL and PLGA carrier, increased

the aerosol performance of the drug. However, these observations were inverse to the

observations by Ooi et al who used spherical polystyrene as a carrier for SS [48].

Contrary to their expectation, they found that increasing the size of the carrier

particle in fact, decreases the FPF of the drug which was attributed to the increased

press-on forces when the drug-carrier blend was prepared. Futher the glass carrier

was used to confirm the contribution of press-on forces to the aerosolization

performance of the carrier. As no significant difference in the FPF of SS was

obtained with the glass carrier, it was concluded that the press-on forces were not a

governing factor for the aerosolization performance of the drug. It should be noted

that the materials employed in these studies are totally different. The previous study

used polystyrene and glass as spherical carrier and the present study utilized PCL and

PLGA as the carrier. These materials will vary widely in their surface chemistry and

Young‟s modulus and this explains the difference obtained in the FPF of the drug.

The maximum particle size employed in the present study was 150 µm. It is not

known whether the particle size higher than 150 µm will further improve the drug

delivery. As the carrier mass continues to increase it will encounter reduced velocity

due to its increasing mass which can hamper the drug dispersion. As a consequence,

it is speculated that further increase in the size of the microspheres may inhibit the

dispersion of drug from the carrier.

6.3. Conclusion

The work presented in this chapter emphasized the importance of the size of the

carrier in the dispersion and the detachment of the drug from the carrier surface in

DPI formulations. It was observed that the smaller size of the carrier resulted in

lower FPF of the SS. As the carrier size increased, there was an increased mechanical

force experienced by larger carriers which resulted in more detachment of the drug

from the surfaces and eventually increased FPF of the drug. These results suggested


134

that the larger size of the PCL and PLGA carriers were effective in improving the

dispersion of the drug from the carrier surfaces.

Chapter 7 Characterization of PCL and PLGA Surfaces and their Relationship with SS dispersion

135

CChhaapptteerr 77

CChhaarraacctteerriizzaattiioonn ooff PPCCLL aanndd PPLLGGAA

SSuurrffaacceess aanndd tthheeiirr RReellaattiioonnsshhiipp wwiitthh

SSSS DDiissppeerrssiioonn

Chapter 7 Characterization of PCL and PLGA Surfaces

and their Relationship with SS dispersion


137

7.1. Introduction

The surface characteristics (adhesional forces, surface roughness and surface free

energy) of the carrier play a crucial role in the dispersion of the drug from the DPI

devices and thereby affect the efficiency of DPI systems. The adhesion forces

between the drug and the carrier have to be overcome by the concomitant efforts of

the patient‟s inhalation and the mechanical forces from the device so that the drug is

detached from the surface and is available to the lungs. The various factors affecting

the adhesion forces and the relationship between the surface properties and drug

dispersion have been discussed in Chapter 2. Researchers have carried out various

studies with the lactose carrier to investigate the effect of its surface roughness on the

FPF of the drug and have also measured the adhesion forces. All these studies have

been discussed earlier in Chapter 2.

It was the aim of the present study to investigate the influence of the surface

roughness of the polymeric carrier, surface free energy and the adhesion forces

between the drug and the polymeric carrier on the aerosol performance of the drug.

This chapter will discuss the correlation of the adhesional properties, surface

roughness and surface free energies of the PCL and PLGA carrier with the dispersion

of the SS from the interactive mixtures of SS and carriers.


The adhesion forces between the silica probe and microspheres and the drug probe

(silica probe coated with SS) and microspheres were measured by AFM. As

discussed in Chapter 2, the surface roughness of the sample is a predominant factor

affecting the adhesion forces. The adhesion forces experienced by the drug particle at

the top of the peak on the surface of the carrier would be certainly less as compared

with the drug particle which is situated deep in the valley of the carrier surface

(Chapter 2, Figure 2.5). This occurred because the particles had different areas of

contact [166]. Hence in order to eliminate the effect of surface roughness on the

determination of adhesion forces, spin-coated films were prepared in this study.

Similar sets of adhesion force experiments were done with films instead of the

microspheres as the films are flat, smooth and have minimized surface roughness.

The surface roughness of both the films and microspheres were determined by AFM.


138

7.2.1. Determination of adhesion forces

The adhesion forces of the microspheres and the films were determined with the

uncoated silica sphere and then with the silica sphere functionalized with the drug

(SS) according to the procedure described in Chapter 3, Section 3.2.7. The typical

force distribution map in a 10x10µm area with 8x8 force points or 32x32 force points

which is obtained from the AFM is shown below in the Figure 7.1. The dark pixels

represent the lower force values whereas the light pixels represent the higher force

values.

(A) (B)

Figure 7.1 Force distribution map of the adhesion forces from AFM with (A) 8X8

force points for films and (B) 32X32 force points for microspheres

A typical force-distance curve obtained from the AFM is shown in Figure 7.2. Red

line represents the approach curve of cantilever and blue line represents the retraction

curve.

Figure 7.2 An example of force-distance curve obtained from AFM.


139

7.2.1.1. Adhesion forces with the PCL carrier

The adhesion forces were determined between the uncoated silica sphere and PCL

microspheres and films and between the silica sphere coated with SS and PCL

microspheres and films.

(A) (B)

(C) (D)

Figure 7.3 Adhesion force measurements of (A) Uncoated silica sphere and PCL

microspheres, (B) Silica sphere coated with SS and PCL microspheres, (C)

Uncoated silica sphere and PCL films and (D) Silica sphere coated with SS and

PCL films, n=5

The adhesion force between the uncoated silica sphere and PCL microspheres was

found to be 150.1 ± 15.5 nN. When the adhesion forces were determined between the

uncoated silica sphere and 1% or 2% MgSt coated PCL microspheres, the forces of

interaction were found to decrease from 150.1 ± 15.5 nN to 73.3 ± 16.3 nN and 75.6

± 16.9 nN, respectively (p<0.05, n=5). Similarly the adhesion forces between the

uncoated silica sphere and 1% or 2% leucine coated PCL microspheres were found to

reduce from 150.1 ± 15.5 nN to 81.6 ± 16.6 nN and 80.3 ± 11.7 nN, respectively

(p<0.05, n=5) (Table 7.1). However there were no significant differences in the

0

20

40

60

80

100

120

140

160

180

PCL PCL-MgSt PCL-Leucine

Ad

hes

ion

Fo

rces

(n

N)

Uncoated silica sphere and microspheres

1% 2%

0

50

100

150

200

250

300

350

PCL PCL-MgSt PCL-Leucine

Ad

hes

ion

Fo

rces

(n

N)

SS-coated silica sphere and microspheres

1% 2%

0 20 40 60 80

100 120 140 160 180

Ad

hes

ion

Fo

rces

(n

N)

Uncoated silica sphere and films

1% 2%

0 100 200 300 400 500 600 700 800 900

Ad

hes

ion

Fo

rces

(n

N)

SS-coated silica sphere and films

1% 2%


140

adhesion forces between the two concentrations (1% or 2%) in either cases of MgSt

or leucine (p>0.05, n=5) (Figure 7.3A). The reduction in the adhesion forces between

the drug and the lactose carrier upon coating with MgSt and leucine have often been

reported in the literature [31-32, 181-183, 185, 191]. It was found that coating the

lactose particles with MgSt or leucine helped in modifying the interaction between

the drug and carrier by formation of the hydrophobic coating. Some studies propose

that MgSt has high affinity for the active sites on lactose carrier and forms a layer to

cover the depressions and thereby aids in drug separation [189]. The role of MgSt

and leucine in reducing the drug-carrier interactions has been explained in details in

Chapter 2.

The adhesion force between the silica sphere functionalized with SS (drug probe)

and PCL microspheres were found to be 301.4 ± 21.7 nN (Table 7.1). The adhesion

forces measured with functionalized silica were much higher as compared to the

uncoated silica sphere. This can be attributed to the size and geometry of the probes.

In case of silica probe, the silica particle is spherical in shape and the diameter of the

particle is 3.5 ± 0.1 µm. For the drug probe (silica sphere functionalized with SS),

the SS particles are flat and plate-like and are adhered on the surface of the silica

sphere. Thus as the SS probe is plate-like, this probe might possess greater contact

with the valleys of the rough carrier surface as compared to the spherical silica probe.

Hence the adhesion forces were greater with the drug probe as compared to the silica

probe. When the adhesion forces were determined between SS and 1% or 2% MgSt

coated PCL microspheres, the forces of interaction were found to decrease drastically

to 110.9 ± 30.5 nN and 121.8 ± 24.6 nN, respectively (p<0.05, n=5). Similarly the

adhesion forces between SS and 1% or 2% leucine coated PCL microspheres were

found to be reduced to 148.1 ± 21.0 nN and 150.2 ± 18.1 nN, respectively (p<0.05,

n=5) (Table 7.1). However, there were no significant differences in the adhesion

forces between the two concentrations (1% or 2%) in either cases of MgSt or leucine

(p>0.05, n=5) (Figure 7.3B).

In the present study, MgSt and leucine coating decreased significantly the interaction

forces between the carrier and the drug. Summarizing the results for both uncoated

and coated silica sphere, it was found that the adhesion forces were high with the

PCL microspheres but these forces were reduced to nearly half with the MgSt or


141

leucine coatings on PCL microspheres. The concentration of the coating solution

(1% or 2% of MgSt or leucine) had no significant effect on the adhesion forces.

In case of PCL microspheres, the adhesion forces were measured for PVA-coated

PCL microspheres and not for PVA-free microspheres. The particle size of the PVA-

free microspheres which were prepared by electrospraying was very small (19.9 ±

4.2 µm); such small sized spheres were very difficult to be used for adhesion force

measurements due to instrumental limitations. For a scan size of 10x10 µm, it was

quite uncertain that the cantilever will land on the top of the microsphere. The

cantilever could easily go off the sphere. To add to this, the adhesion forces from

PVA-coated PCL microspheres and PVA-free PCL microspheres cannot be

compared as both the microspheres have drastic difference in the surface

morphologies. The PVA-coated PCL microspheres are smoother (Chapter 5, Figure

5.4A) as compared to dimpled surface morphology of PVA-free PCL microspheres

(Chapter 5, Figure 5.8A). These variations in the surface morphology and roughness

will in turn lead to the variabilites in the adhesion forces. Hence the adhesion force

data from these two sets of microspheres is incomparable.

As mentioned earlier in order to eliminate the effect of surface roughness, adhesion

force measurements were also conducted on films. The adhesion force measurements

from the films gave an idea of the interaction forces between the drug and pure PCL

polymer and the combination of PCL and PVA film. It was found that with the PCL

film the adhesion force was high (153.7 ± 7.0 nN) and it drastically reduced to 56.9 ±

6.0 nN and 60.3 ± 8.5 nN for 1% and 2% MgSt coatings respectively and to 73.7 ±

5.1 nN and 77.8 ± 4.8 nN for 1% and 2% leucine coatings respectively (Table 7.2).

Thus the uncoated PCL film exhibited high adhesion forces with the silica sphere and

this force was reduced in the presence of MgSt and leucine coatings on the PCL

surface (Figure 7.3C). It is evident that the distribution of forces still existed in case

of films but the distribution was relatively narrow when compared with the

microspheres (Figure 7.1A and B). The distribution of forces could be attributed to

the intrinsic adhesion of the spin-coated surface of the sample. The adhesion force

between the SS-functionalized silica sphere and the uncoated PCL film was found to

be 776.1 ± 26.9 nN and with the PCL+PVA film was found to be 295.6 ± 26.2 nN. In

contrast, when the adhesion forces were determined between SS and three-layered

film of PCL+PVA+1% or 2% MgSt, the forces of interaction declined to 131.3 ±


142

14.7 nN and 139.1 ± 25.3 nN, respectively (p<0.05, n=5). Similarly the adhesion

forces between SS and 1% or 2% leucine coated PCL+PVA films were found to be

reduced to 137.1 ± 24.5 nN and 153.1 ± 23.8 nN, respectively (p<0.05, n=5) (Table

7.2). Again, there were no significant differences (p>0.05, n=5) in the adhesion

forces between the two concentrations (1% or 2%) in either cases of MgSt or leucine

(Figure 7.3D).

For the films, the adhesion forces reduction were found in the order of PCL >

PCL+PVA > PCL+PVA+MgSt ≈ PCL+PVA+Leucine. The adhesion forces of SS

with PCL polymer were very high and upon deposition of PVA on the surface of

PCL, the forces of interaction were decreased between PCL+PVA and SS. This

could be due to the presence of certain functional groups on the surface of the

polymer which could have led to strong interaction between the drug and the

polymer. The surface chemistry of the polymer could be responsible for strong

adhesion which needs detailed investigation. Further deposition of MgSt and leucine

on the surface of PCL+PVA decreased the adhesion forces with SS on account of its

anti-adherent and lubricant property by forming a hydrophobic film on the surface.

This data indicated the interaction force between PCL polymer and SS was higher

than PCL+PVA and PCL+PVA+MgSt or PCL+PVA+Leucine. Thus PCL exhibited

the highest adhesion forces and the addition of MgSt and leucine which acts as anti-

adherent aided in the reduction of adhesion forces.


143

Table 7.1 Adhesion force measurements of PCL and coated PCL microspheres

with uncoated and coated silica sphere, n=5

Sample Adhesion forces (nN)

Uncoated Silica sphere Silica sphere coated

with SS

PCL microspheres 150.1 ± 15.5 301.4 ± 21.7

PCL microspheres coated with

1% MgSt solution

73.3 ± 16.3 110.9 ± 30.5


2% MgSt solution

75.6 ± 16.9 121.8 ± 24.6


1% leucine solution

81.6 ± 16.6 148.1 ± 21.0


2% leucine solution

80.3 ± 11.7 150.2 ± 18.1

Table 7.2 Adhesion force measurements of PCL and coated PCL films with

uncoated and coated silica sphere, n=5



with SS

PCL film 153.7 ± 7.0 776.1 ± 26.9

PCL+ PVA film 103.7 ± 8.4 295.6 ± 26.2

PCL+ PVA+ MgSt (1%) film 56.9 ± 6.0 131.3 ± 14.7

PCL+ PVA+ MgSt (2%) film 60.3 ± 8.5 139.1 ± 25.3

PCL+ PVA+ leucine (1%) film 73.7 ± 5.1 137.1 ± 24.5

PCL+ PVA+ leucine (2%) film 77.8 ± 4.8 153.1 ± 23.8

7.2.1.2. Adhesion forces with the PLGA carrier

The adhesion forces were investigated between the uncoated silica sphere and PLGA

microspheres and films and between the silica sphere coated with SS and PLGA

microspheres and films.


144

(A) (B)

(C) (D)

Figure 7.4 Adhesion force measurements of (A) Uncoated silica sphere and PLGA

microspheres, (B) Silica sphere coated with SS and PLGA microspheres, (C)

Uncoated silica sphere and PLGA films and (D) Silica sphere coated with SS and

PLGA films, n=5

The adhesion force between the uncoated silica sphere and PLGA microspheres were

found to be 95.0 ± 9.7 nN (Table 7.3). The adhesion forces were also determined

between the uncoated silica sphere and 1% or 2% MgSt coated PLGA microspheres.

In this case the forces of interaction were found to be 81.2 ± 9.6 nN and 88.1 ± 7.1

nN, respectively. The adhesion forces between the uncoated silica sphere and 1% or

2% leucine coated PLGA microspheres was found to be 94.5 ± 13.5 nN and 97.4 ±

9.1 nN, respectively (Table 7.3) which was in the similar range as uncoated and

MgSt coated PLGA microspheres. This indicated that there was no significant

difference (p>0.05, n=5) in the adhesion forces of the MgSt and leucine coated

PLGA microspheres as compared to the uncoated PLGA microspheres. There was

also no significant differences in the adhesion forces between the two concentrations

(1% or 2%) in either cases of MgSt or leucine (p>0.05, n=5) (Figure 7.4A).

0

20

40

60

80

100

120

PLGA PLGA-MgSt PLGA-Leucine

Ad

hes

ion

Fo

rces

(n

N)

Uncoated silica sphere and microspheres

1% 2%

0

50

100

150

200

250

PLGA PLGA-MgSt PLGA-Leucine

Ad

hes

ion

Fo

rces

(n

N)

SS-coated silica sphere and microspheres

1% 2%

0

20

40

60

80

100

120

Ad

hes

ion

Fo

rces

(n

N)

Uncoated silica sphere and films

1% 2%

0

50

100

150

200

250

300

Ad

hes

ion

Fo

rces

(n

N)

SS-coated silica sphere and films

1% 2%


145

In case of PLGA, MgSt or leucine were not capable of reducing the adhesion forces

which was contrary to PCL. There are some reports in the literature that addition of

powdered MgSt after a certain extent, does not improve the dispersibility of the drug;

infact it decreases the FPF of the drug [40, 188]. Begat et al found that the energy of

interaction between the drug SS and lactose coated with MgSt, leucine or lecithin

reduced to such an extent that the adhesive drug-carrier system shifted to cohesive

system. This led to the segregation of the drug from the carrier particles and

decreased the aerosolization performance of the drug [40]. It was proposed by Islam

et al that a minimum amount of MgSt is required to lower the threshold level of

interaction forces between the drug and the carrier particles [188]. In the case of

PLGA, the interaction forces were as such low and further addition of 1% or 2%

MgSt or leucine was not capable of altering the interaction forces between the

particles.

Similar trend of the interaction forces was observed in case of silica sphere

functionalized with SS and PLGA microspheres. The adhesion force between the SS

probe and PLGA microspheres were found to be 157.5 ± 26.8 nN (Table 7.3). When

the adhesion forces were determined between SS and 1% or 2% MgSt coated PLGA

microspheres, the interaction forces were found to be similar to the uncoated PLGA

microspheres. Adhesion forces were found to be 134.6 ± 26.6 nN and 145.0 ± 18.6

nN for 1% and 2% MgSt coatings, respectively. Similarly the adhesion forces

between SS and 1% or 2% leucine coated PLGA microspheres were found to be

181.5 ± 31.1 nN and 169.7 ± 30.8 nN, respectively (Table 7.3). In concurrence with

the previous results there were no significant differences in the adhesion forces

between the two concentrations (1% or 2%) in either cases of MgSt or leucine

(p>0.05, n=5) (Figure 7.4B).

Analogous results for the adhesion forces were found in the case of films. For both

the uncoated and SS coated silica sphere, the extent of interaction forces was same

for the uncoated PLGA film and the MgSt and leucine coated PLGA films (Table

7.4) (Figure 7.4 C and D).

Summarizing the above results for uncoated and coated silica spheres, it was found

that the adhesion forces were unchanged for both uncoated and coated PLGA

microspheres. Thus the adhesion forces between the drug and the PLGA were found


146

to be in the following order: PLGA ≈ PLGA+PVA ≈ PLGA +PVA+MgSt ≈ PLGA

+PVA+Leucine. The surface chemistry of the PLGA polymer could be a parameter

influencing the adhesion forces between the polymer and the drug which needs to be

investigated in detail in future.

Table 7.3 Adhesion force measurements of PLGA and coated PLGA microspheres

with uncoated and coated silica sphere, n=5



with SS

PLGA microspheres 95.0 ± 9.7 157.5 ± 26.8

PLGA microspheres coated with

1% MgSt solution

81.2 ± 9.6 134.6 ± 26.6


2% MgSt solution

88.1 ± 7.1 145.0 ± 18.6


1% leucine solution

94.5 ± 13.5 177.6 ± 29.2


2% leucine solution

97.4 ± 9.1 169.7 ± 30.8


147

Table 7.4 Adhesion force measurements of PLGA and coated PLGA films with

uncoated and coated silica sphere, n=5



with SS

PLGA film 81.4 ± 9.8 253.8 ± 19.0

PLGA +PVA film 90.2 ± 7.7 234.2 ± 23.0

PLGA + PVA+ MgSt (1%) film 80.0 ± 16.5 243.1 ± 13.2

PLGA + PVA+ MgSt (2%) film 78.2 ± 13.4 234.8 ± 21.2

PLGA + PVA+ leucine (1%) film 91.1 ± 8.2 254.3 ± 23.4

PLGA + PVA+ leucine (2%) film 97.0 ± 11.0 254.7 ± 20.3

7.2.2. XPS analysis of the films

XPS analysis of the films was carried out to detect the presence of coating materials

(MgSt or leucine) on the surface of the polymer.

7.2.2.1. XPS analysis of coated PCL films

The three layered coated PCL films were analyzed using XPS for confirming the

presence of MgSt and leucine on their surface. The presence of the characteristic

peak of magnesium (Mg2p) at 49.5eV (Figure 7.5A) and nitrogen (N1s) at 398eV

(Figure 7.5B) confirmed that the films had been successfully coated with MgSt and

leucine solutions respectively.


148

Figure 7.5 Survey spectra of (A) three layered film of PCL, PVA and MgSt and (B)

three layered film of PCL, PVA and Leucine

7.2.2.2. XPS analysis of coated PLGA films

The coated PLGA films were also analyzed using XPS for the confirming the

presence of MgSt and leucine on their surfaces. The presence of the characteristic

peak of magnesium (Mg2p) at 50.5eV (Figure 7.6A) and nitrogen (N1s) at 401.2eV

(Figure 7.6B) confirmed that the films had been effectively coated with MgSt and

leucine solutions respectively.


149

Figure 7.6 Survey spectra of (A) three layered film of PLGA, PVA and MgSt and

(B) three layered film of PLGA, PVA and Leucine

7.2.3. Relationship between adhesion force and FPF

7.2.3.1. PCL carriers

The TSI results which were discussed in Chapter 5 indicated that there was no FPF

of the drug from the formulation containing uncoated PCL microspheres and SS;

however, from the interactive mixture of surface coated microspheres and SS,

significant improvement in the FPF of the drug was obtained. This indicated that the

coatings enhanced the detachment of the drug from the carrier surface.

The AFM results indicated that the adhesion forces between SS and uncoated PCL

microspheres was 301.4 ± 21.7 nN which resulted in no FPF of the SS from the

carrier surface. However, upon coating the PCL microspheres with MgSt and

leucine, the adhesion forces were reduced nearly by half. For MgSt coated PCL


150

microspheres (104 µm) the adhesion forces were reduced from 301.4 ± 21.7 nN to

110.9 ± 30.5 nN and 121.8 ± 24.6 nN for 1% and 2% coatings, respectively which

increased the FPF from zero to 12 ± 1.3% and 15.8 ± 1.5% with the powder coatings

(1% and 2% respectively) and to 11.4 ± 0.9% and 15.4 ± 1.6% with the solution

coatings (1% and 2% respectively). For leucine coated PCL microspheres, the

adhesion forces were reduced from 301.4 ± 21.7 nN to 148.1 ± 21.0 nN and 150.2±

18.1 nN for 1% and 2% coatings, respectively, which increased the FPF from zero to

6.0 ± 0.5% and 10.2 ± 1.2% (1% and 2% respectively) with the powder coatings and

to 11.3 ± 1.1% and 11.3 ± 0.8% with the solution coatings (1% and 2% respectively).

The AFM results indicated that the adhesion forces were reduced nearly by half in

case of MgSt and leucine coated microspheres when compared with the uncoated

PCL microspheres. Thus this reduction in the adhesion forces must have contributed

to the increasing FPF of SS with the coated PCL microspheres.

Although adhesion forces reduction between 1% MgSt coated PCL and SS was

similar to that of 2% MgSt coated PCL carriers, the latter carrier provided a

significant improvement in the FPF over 1% MgSt coating. This would have

occurred due to the fact that when 2% MgSt solution was used, a significant amount

of MgSt had crystallized on the surface of the PCL microspheres (discussed earlier in

Chapter 5, Section 5.2.4.1), which led to increased coating on the surface resulting in

easy detachment of the SS from the surface of microspheres. In addition, MgSt is a

highly hydrophobic glidant, which contributed to reduced adhesion between

hydrophilic SS and coated carriers, resulting in increasing FPF of SS. In contrast,

leucine coated PCL microspheres, although showed to reduce adhesion forces and

significantly increased the FPF of SS; there was no difference in the FPF of SS in

both concentrations (1% or 2%) of leucine. It was explained previously that the

solution coating was uniform in case of leucine and 1% concentration was enough to

coat the entire surface of PCL microspheres (Chapter 5, Section 5.2.4.1). In addition,

leucine is relatively hydrophobic and hence reduced adhesion forces between

hydrophilic SS and coated carriers; however, the reduction of adhesion force is less

than that of the forces between hydrophilic SS and hydrophobic MgSt coated PCL

carriers. In addition, leucine plays an important role as an aerosolization enhancer

and thus leucine coating contributed to the enhanced aerosolization leading to

increased FPF of SS. Thus both the MgSt and the leucine coating may be


151

contributing to the SS detachment by reducing the adhesion forces between SS and

PCL particles, as well as increased aerosolization, resulting in increased dispersion of

SS.

7.2.3.2. PLGA carriers

For PLGA microspheres, strikingly different results were obtained in comparison to

PCL microspheres. The TSI results which were discussed in Chapter 5 indicated that

similar FPF of the drug was obtained from both the uncoated and surface coated

PLGA microspheres. The surface coating of MgSt and leucine on the surface of the

PLGA microspheres did not facilitate in significant improvement of the FPF of the

drug. It was observed from the AFM results that the adhesion forces between the SS

and the coated PLGA microspheres were similar as compared with the uncoated

PLGA microspheres.

The AFM results indicated that the adhesion forces between SS and uncoated PLGA

microspheres (90 µm) was 157.5 ± 26.8 nN which resulted in 16.6 ± 1.6% FPF of the

SS from the PLGA carrier surface. However upon coating the PLGA microspheres

with MgSt and leucine, the adhesion forces were same as in the case of uncoated

microspheres. In case of MgSt coated PLGA microspheres the adhesion forces was

134.6 ± 26.6 nN for 1% coatings which gave the FPF of 15.8 ± 1.0% with the

powder coatings and 15.3 ± 1.0% with the solution coatings. Similarly for leucine

coated PLGA microspheres, the adhesion forces was 177.6 ± 29.2 nN for 1% coating

which gave the FPF of 15.3 ± 1.0% with the powder coatings and 15.4 ± 0.4% with

the solution coatings.

These data indicate that the coating of the surface of the PLGA microspheres did not

result in any alterations or further reduction of the adhesion forces. These results

were also in agreement with the TSI results of the PLGA microspheres. This

suggests that as similar extent of interaction forces existed between SS and the

uncoated and coated PLGA microspheres, hence any coatings on the surface of

PLGA microspheres did not result in any significant improvement in the FPF of the

SS.

7.2.4. Surface Roughness

The morphology of the PCL and PLGA films or microspheres has been investigated

by AFM. Studies on the crystallization behaviour of PCL (MW 22,000 Da) thin films


152

have been investigated extensively [263]. PCL-toluene solutions have been spin-

casted on mica substrates and films were made of varied thickness (4-120 nm). The

morphology of films was studied by AFM and it was found that PCL (MW 76,598

Da) can crystallize into spherulites or dendrites or islands like structure can be

obtained which is dependent on the thickness of the film formed. The substrate also

affects the morphology of the polymer. The ring banded spherulites have been

reported on the surface of PCL which is solution casted in tetrahydrofuran (THF)

solvent over glass slides [264]. Banded spherulites have also been reported in PCL-

Polyvinyl chloride (PVC) blends [265]. PCL films have been prepared at various

concentrations, spinning speed and time to yield films of various thickness and

roughness and have been investigated by AFM. It was found that the surface

roughness and the thickness of the film was dependent on the spinning speed and the

concentration of PCL solution [266]. The significant changes in the surface

roughness of ovalbumin loaded PLGA microspheres after γ-irradiation have been

characterized by AFM [267]. The surface properties of PLGA in chloroform film

before and after oxygen plasma treatment casted in poly(tetrafluoroethylene) mould

have been investigated by AFM [268]. It was observed that the roughness of the

PLGA surface increased after treatment with oxygen plasma.

In the present study, surface roughness of the PCL and PLGA microspheres were

determined by AFM according to the procedure described in Chapter 3, Section

3.2.8. The films were also analyzed for surface roughness as they are supposed to

have minimal surface roughness when compared to the microspheres. For the present

study, the PCL and PLGA films were prepared in DCM solvent and on a glass

substrate.

7.2.4.1. Surface roughness of PCL microspheres

The PCL microspheres were found to be rough with the root mean square (RMS)

value of 73.0 ± 16.8 nm. The surface topography of the PCL microspheres in 2D and

3D view is shown in Figure 7.7A and B. Figure 7.7C is a section view of the surface

topography of the PCL microspheres which gives an idea of the distribution of the

peaks and valleys on the surface of the microspheres. The deposition of MgSt and

leucine on the surface of the PCL microspheres did not make the surface more rough

(Table 7.5). The RMS values of the MgSt and leucine coated PCL microspheres were

found to be 76.8 ± 15.2 and 70.7 ± 11.7 nm, respectively.


153

(A) (B)

(C)

Figure 7.7 Surface topography of PCL microspheres in (A) 2D view, (B) 3D view

and (C) Section view

Table 7.5 Surface roughness measurements of the PCL and coated PCL

microspheres, n=5

Sample RMS (nm)

PCL microspheres 73.0 ± 16.8

PCL microspheres coated with 1% MgSt solution 76.8 ± 15.2

PCL microspheres coated with 1% leucine solution 70.7 ± 11.7

7.2.4.2. Surface roughness of PCL films

The films exhibited less roughness as compared to their microsphere counterparts.

The surface roughness of PCL+PVA films and three-layered film of PCL+ PVA+

MgSt and PCL+ PVA+ Leucine yielded similar RMS values and the trend was

similar to microspheres (Table 7.6).


154

Table 7.6 Surface roughness measurements of the PCL and coated PCL films, n=5

Sample RMS (nm)

PCL + PVA film 12.7 ± 1.2

PCL + PVA+ MgSt film 13.0 ± 1.6

PCL + PVA+ leucine film 14.0 ± 0.5

Thus it can be seen that the coatings of MgSt and leucine on the surface of PCL did

not significantly increase the roughness of the surface as compared with their

uncoated counterparts in case of both the microspheres and the films (p>0.05, n=5)

(Figure 7.8).

Figure 7.8 Surface roughness measurements of coated PCL films and

microspheres, n=5

7.2.4.3. Surface roughness of PLGA microspheres

In contrast to PCL microspheres, the PLGA microspheres were found to be very

smooth. This is also proved by the SEM images which visually confirm that the

surface of PLGA microspheres is very smooth as compared to the PCL microspheres

(Chapter 5, Section 5.2.2.2). However the surface of the PLGA microspheres showed

a very distinct flower-like pattern which appeared as specs on the surface (Figure

7.9). This could be due to the phase separation of the polymer during the evaporation

of solvent which could have led to the formation of these patterns on the surface of

0 10 20 30 40 50 60 70 80 90

100

RM

S (

nm

)

Films

Microspheres


155

the microspheres. It has been reported in literature that the blend of PLGA 50:50 and

polyphosphazene in dichloromethane solvent resulted in the formation of microphase

separation structure on the surface [269]. The close view of this flower-like pattern is

shown in Figure 7.9D. The effect of these patterns on the dispersion of drug from the

surface is unknown. The surface topography of the PLGA microspheres in 2D view,

3D view and section view is shown in Figure 7.9A, B and C. The PLGA

microspheres yielded the RMS values of 3.8 ± 1.5 nm. The deposition of MgSt and

leucine on the surface of the PLGA microspheres made the surface quite rough

(Table 7.7). The RMS values were markedly increased in case of MgSt coating

which is due to the deposition of the MgSt crystals on the surface. In case of leucine,

the surface became rougher than PLGA microspheres but not to the same extent as

MgSt coated PLGA because leucine forms a comparatively uniform coating on the

surface of the microspheres as compared to MgSt coating (Chapter 5, Section

5.2.4.2).

Table 7.7 Surface roughness measurements of the PLGA and coated PLGA

microspheres, n=5

Sample RMS (nm)

PLGA microspheres 3.8 ± 1.5

PLGA microspheres coated with 1% MgSt solution 50.1 ± 20.0

PLGA microspheres coated with 1% leucine solution 7.7 ± 1.0


156

(A) (B)

(C) (D)

Figure 7.9 Surface topography of PLGA microspheres in (A) 2D view, (B) 3D

view, (C) Section view and (D) Close view of the specs on the surface of the PLGA

microspheres

7.2.4.4. Surface roughness of PLGA films

The roughness determination of the PLGA films gave results similar to the

microspheres. Although the films exhibited less surface roughness as compared to

the microspheres but the deposition of MgSt and leucine layers on the PLGA+PVA

film increased the RMS values significantly (Table 7.8).

10

8

6

4

2

0

µm

1086420

µm

-30

-20

-10

0

10

20

30

nm

2.0

1.5

1.0

0.5

µm

9.59.08.58.0

µm

-20

-10

0

10

20

nm


157

Table 7.8 Surface roughness measurements of the PLGA and coated PLGA films,

n=5

Sample RMS (nm)

PLGA + PVA film 1.4 ± 0.5

PLGA + PVA+ MgSt film 14.3 ± 0.5

PLGA + PVA+ leucine film 5.4 ± 1.5

Thus it was observed that the coatings of MgSt and leucine significantly increased

the roughness of the PLGA surface in comparison with the uncoated microspheres

and films (p<0.05, n=5) (Figure 7.10)

Figure 7.10 Surface roughness measurements of coated PLGA films and

microspheres, n=5

7.2.5. Relationship between FPF and RMS

7.2.5.1. PCL carriers

It was found that the PCL microspheres by themselves did not allow any detachment

of the drug due to the strong adhesion forces between the two and hence no FPF of

the drug was obtained. However the coatings of MgSt and leucine on the surface of

the PCL microspheres reduced the forces of interaction between the two, which

caused easy detachment of the drug and hence FPF of the drug was obtained. In

terms of surface roughness, both the uncoated and coated PCL microspheres

0

10

20

30

40

50

60

70

80

RM

S (

nm

)

Films

Microspheres


158

exhibited similar surface roughness but the coated microspheres yielded better FPF

of SS as compared to no FPF from the uncoated microspheres.

7.2.5.2. PLGA carriers

In case of PLGA microspheres it was found that the drug readily detached from the

surface and FPF of the drug was obtained. But the coating of the surface of the

PLGA microspheres with MgSt and leucine was not capable of improving the FPF of

the drug. The FPF of the SS remained similar in uncoated and coated PLGA

microspheres due to the same extent of adhesion forces prevailing between them. In

terms of surface roughness, the PLGA surface was found to be very smooth but the

MgSt and leucine coatings made the surface of the microspheres very rough. Thus

for PLGA microspheres there was an increase in the surface roughness of the

microspheres but there was no improvement in the FPF of the drug.

Thus it was found that both uncoated and coated PCL microspheres had similar kind

of surface roughness but improvement in the FPF of the SS was observed with the

coatings. However, in case of PLGA, there was an increase in the roughness of the

microspheres with the deposition of coatings but no significant improvement in the

FPF of the drug was obtained. Thus this data provided no evidence that a correlation

exists between the surface roughness of the microspheres and the FPF of the drug.

7.2.6. Surface free energy determination

Surface energy is defined as the amount of energy required to create a unit area of

solid surface. It can also be defined as the excess energy at the surface of the material

compared to the bulk. The surface free energy of the PCL and PLGA microspheres

were determined in accordance with the procedure described in Chapter 3, Section

3.2.9. It was measured for approximately the same size of PCL and PLGA

microspheres (104 µm and 90 µm respectively). Surface free energy consists of two

components: Dispersive component or non-polar energy and acid-base component or

polar energy. The magnitude of the dispersive component of the surface free energies

are based on the interaction of the alkane probes with the sample; the stronger the

interaction, the higher the energy values.

Researchers have utilized IGC technique to measure the surface free energies of PCL

and PLGA. Dispersive component of the surface free energy of the pellets of PCL

biopolymer has been measured and is reported to be 40 mJ/m2 [270]. It should be


159

noted that this magnitude of the surface free energy is for the pure PCL polymer and

not for the microspheres. Dispersive surface free energies of the various blends of

Amylopectin-PCL have been investigated using IGC and it was found that the

addition of PCL increased the dispersive component of the surface free energies of

Amylopectin [271]. The dispersive surface free energy of PLGA microparticles

prepared by spray drying (3.3 µm) was measured by IGC at 25°C and was found to

be 31.65 mJ/m2 [235].

In the present study, it should be noted that the measurements were done on PCL

microspheres and not pellets. For PLGA, the measurements were done at 30°C on

microspheres which had VMD of 104 µm and were prepared by o/w solvent

evaporation method. The dispersive component of the surface free energies of the

PCL and PLGA microspheres were 99.2 ± 5.2 mJ/m2 and that of PLGA microspheres

was 38.0 ± 0.3 mJ/m2, respectively (Figure 7.11).

Figure 7.11 Dispersive surface free energies of PCL and PLGA microspheres

Thus the dispersive contribution to the surface energy was significantly higher for

PCL microspheres in comparison to PLGA microspheres. These values indicated that

the surface of PCL microspheres was more energetic than PLGA microspheres. The

difference in the surface free energies of PCL and PLGA microspheres can be

attributed to the surface roughness of the microspheres. The variation in the surface

roughness of a solid surface changes the free energies of the surface by influencing

the contact angle [272]. From the surface roughness measurements, it is clear that the

PCL microspheres exhibited a rough surface of 73.0 ± 16.8 nm (Table 7.5) in

0

20

40

60

80

100

120

PCL PLGA

Dis

per

sive

fre

e e

ner

gy

(mJ/m

2)


160

comparison to PLGA microspheres which were very smooth (3.8 ± 1.5 nm) (Table

7.7). Thus these variations in the roughness of the surface of the microspheres have

contributed to the difference in the surface free energies of the two which further

affected the detachment of the drug from the surface.

The surface free energy differences detected by IGC are dependent on the size [273],

crystal morphology [146], optical forms [274], preparation route [275]and relative

humidity [276]. Even on the same crystal form, every crystal face and edge could

experience different forces pulling from bulk and hence have different surface

energy. Even different physical forms of the same drug have different surface

energies. For amorphous forms, the molecules at the surface have greater freedom to

move and reorientate than the molecules in the crystal surface, so the amorphous

surfaces can have changes in the surface energy with time [277]. All these factors

can contribute to the variations in the free energies of the two surfaces.

7.2.7. Difference between the PCL and PLGA polymers

The above discussion indicate that there is a difference in the adhesion forces,

surface roughness, surface free energies and the drug dispersion from the two

biodegradable polymers: PCL and PLGA. The uncoated PCL surface had high

surface roughness (73.0 ± 16.8 nm), high dispersive component of surface free

energy (99.2 ± 5.2 mJ/m2) and exhibited high adhesion forces (301.4 ± 21.7 nN) with

the drug which resulted in no FPF of the drug. The PCL microspheres were coated

with antiadherent agents, MgSt and leucine to reduce the adhesion forces with the

drug; although they exhibited similar surface roughness to uncoated PCL

microspheres but led to easy detachment of the drug from their surface. On the other

hand, in case of PLGA microspheres, the uncoated PLGA microparticles exhibited

minimal surface roughness (3.8 ± 1.5 nm), less dispersive component of surface free

energy (38.0 ± 0.3 mJ/m2) and less adhesion forces (157.5 ± 26.8 nN) in comparison

to PCL microspheres and they easily allowed detachment of the drug from their

surface. The FPF of the drug obtained from PLGA was 16.6 ± 1.6%. Coating of the

microspheres with MgSt and leucine increased the surface roughness of the polymer

but did not lead to significant improvement of the drug delivery because similar kind

of adhesion forces existed between the uncoated and coated polymers and the drug.


161

It should be noted that the dispersion of the drug from the surface is a complex

process. This difference in the dispersion of drug from the surface of PCL and PLGA

polymers can be attributed to the surface, thermal and mechanical properties of the

polymers which are described below.

7.2.7.1. Surface free energy

The interparticular interactions are influenced by surface energies of the individual

components and hence they are capable of affecting the dispersibility of the drug

from the carrier surface [278]. The relationship between surface free energy and the

detachment or the dispersibilty of the drug from the carrier surface was established in

a number of studies [148, 152-153, 155, 279]. In these investigations, a negative

correlation was established between the dispersibility of the aerosol and the surface

free energy which indicates that the decrease in surface energy led to an increase in

the FPF of the drug. This is discussed in details in Section 2.4.4.2.2, Chapter 2. As

measured the dispersive component of surface free energy of PCL microspheres was

much higher than the PLGA microspheres. This indicated that the surface of PCL

microspheres were more energetic than PLGA microspheres. This energetic surface

led to increased interaction of the PCL surface with SS and hence it contributed to no

detachment of the drug from the surface. In contrast, the surface of PLGA

microspheres was less energetic than the PCL microspheres, hence SS readily

detached from the surface.

7.2.7.2. Glass transition temperature (Tg)

The glass transition temperature (Tg) is the critical temperature at which the material

changes its behavior from being „glassy‟ to being „rubbery‟. Glassy in this context

means hard and brittle and therefore relatively easy to break, while „rubbery‟ means

elastic and flexible. Hence at temperatures below Tg, the polymers are hard, stiff and

glassy and at temperatures well above the Tg, the polymers are rubbery and they

might flow.

As mentioned earlier in Chapter 3, Section 3.1.2.1 and Section 3.1.2.2, the Tg of PCL

is -60 °C and that of PLGA is 40-60 °C. This indicates that at room temperature, the

PCL polymer is rubbery and elastic while the PLGA polymer is hard, stiff and

glassy. This difference in the Tg of PCL and PLGA polymers can lead to the

variability in the way the drug detaches from the rubbery surface in comparison to


162

the glassy surface. For a rubbery surface, the particle-particle impact will be

characterised by large elastic deformations which will lead to increased contact area

between the drug and the carrier particles and higher drug-carrier forces of

interaction and lower FPF of the drug. On the other hand, the elastic deformations in

the case of glassy surface will be less which will lead to less contact between the two

particles and lowered interaction forces and higher FPF of the drug. The particle-

particle impact, the elastic deformations and eventually the adhesion forces will

affect the detachment potential of the drug from the surface and eventually the FPF

of the drug.

7.2.7.3. Elasticity of the polymer and the inhaler wall

The elastic modulus of the polymer and the wall of the DPI device will have a major

influence on the carrier-carrier and carrier-inhaler collisions which in turn will

influence the impact based detachment from the surface of the carrier. This mode of

detachment is explained in details in Chapter 2, Section 2.3.3.1 which will eventually

influence the FPF of the drug. The effect of the wall material on the collision process

has been analyzed by Sommerfeld et al. They found that the roughness of the wall

material is capable of altering the rebound behaviour of the particles and causes the

reduction of the gravitational settling of the particles which keeps the particles

redispersed [280].

Thus these surfaces, thermal and mechanical properties of the PCL and PLGA

polymer might have led to the differences in the way the drug detaches from their

surface.

7.3. Conclusion

A good correlation was observed with AFM force measurements and the TSI studies.

The extent of the interaction forces existing between the drug and the polymeric

carrier was a major controlling factor in affecting the detachment of the drug from

the surface in TSI studies. No direct relationship was observed between the surface

roughness of the microspheres and the FPF of the drug. An inverse correlation

between dispersive component of surface free energy and the FPF of the drug was

established. Thus it can be concluded that the adhesion forces between the drug and

the polymer surfaces played a very important role in the detachment of the drugs

from the carrier surface and the subsequent FPF of the drugs.

Chapter 8 Overall Conclusions and Further Directions

163

CChhaapptteerr 88

OOvveerraallll CCoonncclluussiioonnss aanndd FFuurrtthheerr

DDiirreeccttiioonnss



165

8.1. Summary and Conclusions

The overall objective of this research project was to study the surface properties of

the biodegradable polymer as a carrier in DPI formulations with an aim to achieve

efficient drug dispersion goals. Hence, this project focused on the formulation

development of DPIs.

The currently used traditional carrier in DPI formulations, lactose, poses many

challenges with respect to dispersion of drug from its surface. The lactose carrier

allows only 20-30% of the total dose to reach the lungs. This has been attributed

primarily to the irregular shape and morphology of lactose. There are reports in the

literature which suggests that the spherical carriers can be of help in improving the

dispersion of the drug from the surface. Hence, this research project proposed the use

of biodegradable polymers as an alternative carrier to lactose for DPI formulation as

they can be easily fabricated as microspheres. For this purpose, we selected PCL and

PLGA, two biodegradable polymers, to be used as carriers for delivery of the anti-

asthmatic drug, SS. Characterization strategies for the formulations were undertaken.

In order to gain a better insight into drug dispersion from DPI formulations, an

understanding of surface chemistry was essential. Hence, thorough investigations on

the surface properties of the DPI formulations were made by analysing adhesion

forces and surface roughness.

The first step in the project was to fabricate microspheres from the polymer, and then

later characterize them with various surface analytical techniques and eventually

determine drug dispersion from the formulation. When the PCL microspheres were

used for drug dispersion, no drug detached from the surface. This was because of the

high adhesion forces prevailing between PCL and SS which was determined using

AFM. In order to improve drug dispersion, the microspheres were coated with anti-

adherent agents: MgSt and leucine. These anti-adherent agents helped in reducing the

strong adhesion forces between PCL and SS. This helped SS to readily detach from

the surface due to reduced adhesion between them and helped in improving drug

dispersion.

In contrast, in the case of PLGA microspheres, the drug detached from the surface of

the uncoated microspheres and the FPF of the drug was obtained. To test any further

improvement in drug delivery, the PLGA microspheres were coated with MgSt and


166

leucine, but there was no significant improvement in the FPF of SS obtained when

compared with uncoated microspheres. This was because the adhesion forces

existing between PLGA and SS were present to the same extent as the interaction

forces existing between MgSt or leucine coated PLGA microspheres and SS. Hence

no further improvement in the FPF of the drug was obtained with the coated PLGA

microspheres.

Secondly, this project established the effect of the size of the spherical carrier in the

aerosolization and dispersion of the drug from the carrier surface. The FPF of the

drug was obtained from four different sizes of the carrier. It was found that as the

size of the PCL and PLGA microspheres increased, there was a subsequent increase

in the FPF of the drug. Thus, it was concluded that the larger size of the PCL and

PLGA carrier provided efficient drug delivery when compared with their smaller

counterparts due to a higher magnitude of impaction detachment as experienced by

the larger carriers.

Thirdly, a comprehensive investigation into the surface properties of the polymeric

carriers was done using the AFM. Determining the adhesion properties and surface

roughness of the polymeric carrier helped to correlate the aerosolization results with

the surface properties. With respect to interaction forces, a very good correlation was

established. It was found that as the adhesion forces decreased between the PCL

carrier and the drug due to the coating with anti-adherent agents, there was an

increased detachment of the drug from the surface and, subsequently a high FPF of

the drug was obtained in the TSI experiments. In the case of the PLGA microspheres,

there were similar kind of adhesion forces between the SS and the uncoated and

coated PLGA microspheres; therefore the drug detached to a similar extent in the

cases of both the uncoated and coated PLGA. On the contrary, no correlation was

observed between the aerosolization of SS and the surface roughness of PCL or the

PLGA carrier. Thus, it was concluded that the adhesion forces between the carrier

and the drug played a major role in controlling the dispersion of the drug from the

surface.

This study successfully proved that coated PCL microspheres and PLGA

microspheres can be used as alternative carriers to lactose in DPI formulations. The

author agrees that there has been no significant enhancement in the FPF of SS from


167

PCL and PLGA microspheres as compared with lactose. In fact, the FPF obtained

from the lactose and polymeric microspheres are comparable. However, the author

would like to emphasize that the production of microspheres with a particular surface

morphology is reproducible as compared with lactose, which has highly irregular

surfaces. There are various grades and brands of lactose currently available in the

market which have different surface morphologies and variable amounts of fine

lactose associated with it. All these, in turn, lead to variability in the dispersion of the

drug from the lactose surface. In the case of polymeric microspheres, reproducible

surfaces are achieved and this subsequently leads to reproducible drug delivery.

8.2. Future Directions

The author has been successful in matching drug delivery from polymeric

microspheres to the current gold standard, lactose; however there are still a few

challenges which can be addressed.

Further investigations are recommended to explore novel hydrophobic anti-adherent

agents other than MgSt or leucine. Hydrophobic agents should be such that they can

help in reducing the adhesion forces to such an extent that more detachment of the

drug is achieved from the carrier surfaces and, eventually, better drug dispersion is

obtained.

The PCL microspheres can be used for delivery of hydrophobic drugs rather than the

hydrophilic drug SS, with which it exhibited highly interactive forces. It could be

possible that these PCL microspheres are a much more suitable candidate for

aerosolizing the hydrophobic drug as they will exhibit less adhesive interactions

between them when compared with hydrophilic drugs. An example of an anti-

asthmatic hydrophobic drug is SX.

Future research should be conducted to obtain a better understanding of the influence

of the drug to carrier ratio on aerosolization performance. This can be done by

mixing the carrier with varying concentrations of drug and conducting in vitro

aerosolization experiments. In the current study, the author has used one fixed drug

to carrier ratio (2.5% SS) for the increasing sizes of the carrier microspheres. Thus,

varying ratios of drug and carrier can be used for different sizes of carrier

microspheres to study its effect on the FPF of the drug.


168

Also the surface free energies of various sizes of the microspheres can be measured

using the IGC technique. This will provide an idea of the free energy of the

microspheres which can be correlated to the in vitro dispersion of the drug from their

surfaces.

In order to understand whether the presence of PVA on PCL microspheres

contributes to the highly adhesion forces between the microspheres and the drug and,

eventually, the dispersion of drug from the polymer surface, PVA-free microspheres

were fabricated in the present study. This was done by electrospraying PCL and

evaluating the drug dispersion from these PCL microspheres which were devoid of

PVA. One of the limitations of this study was that the morphology from these two

sets of microspheres was quite different. PVA containing PCL microspheres were

smoother when compared with the „dimpled‟ surface morphology of the PCL

microspheres, which were prepared by electrospraying. This posed the challenge that

the adhesion force data from these microspheres of varied morphologies is not

comparable. In order to obtain PCL microspheres with the same morphology but

devoid of PVA, the microspheres could be fabricated using the same o/w emulsion

technique but with a different emulsifier other than PVA. One possibility is to use

Hydroxypropyl methyl cellulose (HPMC) to make PVA-free PCL microspheres

using the emulsion technique. These microspheres can be further used to determine

drug dispersion.

Other polymers can be screened which do not exhibit higher adhesion forces with the

drug. These polymers can be used as effective carriers for DPI formulations.

The electrosprayed PCL microspheres which are PVA-free could be coated with

MgSt and leucine and could be tested for drug dispersion. It would be interesting to

compare and contrast the FPFs of the SS from the PVA-free and PVA bound PCL

microspheres as the morphologies of both microspheres are quite different.

The molecular modelling of the PCL and PLGA polymer could be conducted to gain

an understanding of the functional groups that are present on the surface of the

polymer, which leads to the difference in the way both the polymers interact with the

drug.

Bibliography

169

BBiibblliiooggrraapphhyy

Bibliography

Bibliography

171

1. Byron, P.R. and J.S. Patton, Drug delivery via the respiratory tract. J Aerosol

Med, 1994. 7(1): p. 49-75.

2. Daniher, D.I. and J. Zhu, Dry powder platform for pulmonary drug delivery.

Particuology, 2008. 6(4): p. 225-238.

3. Haynes, A., M.S. Shaik, A. Chatterjee, and M. Singh, Evaluation of an

aerosolized selective COX-2 inhibitor as a potentiator of doxorubicin in a

non-small-cell lung cancer cell line. Pharm Res, 2003. 20(9): p. 1485-95.

4. Tian, Y., M.E. Klegerman, and A.J. Hickey, Evaluation of microparticles

containing doxorubicin suitable for aerosol delivery to the lungs. PDA J

Pharm Sci Technol, 2004. 58(5): p. 266-75.

5. Chougule, M.B., B.K. Padhi, and A. Misra, Nano-liposomal dry powder

inhaler of Amiloride Hydrochloride. J Nanosci Nanotechnol, 2006. 6(9-10):

p. 3001-9.

6. Thomas, S.H., M.J. O'Doherty, A. Graham, C.J. Page, P. Blower, D.M.

Geddes, and T.O. Nunan, Pulmonary deposition of nebulised amiloride in

cystic fibrosis: comparison of two nebulisers. Thorax, 1991. 46(10): p. 717-

21.

7. Laube, B.L., Treating diabetes with aerosolized insulin. Chest, 2001. 120(3

Suppl): p. 99S-106S.

8. Laube, B.L., Advances in treating diabetes with aerosolized insulin. Diabetes

Technol Ther, 2002. 4(4): p. 515-8.

9. Yamamoto, H., Y. Kuno, S. Sugimoto, H. Takeuchi, and Y. Kawashima,

Surface-modified PLGA nanosphere with chitosan improved pulmonary

delivery of calcitonin by mucoadhesion and opening of the intercellular tight

junctions. Journal of Control Release, 2005. 102(2): p. 373-81.

10. Yang, T., F. Mustafa, S. Bai, and F. Ahsan, Pulmonary delivery of low

molecular weight heparins. Pharm Res, 2004. 21(11): p. 2009-16.

Bibliography

172

11. Islam, N. and R. Shafiqur, Pulmonary drug delivery: Implication for new

strategy for pharmacotherapy for neurodegenerative disorders. Drug

Discoveries and Therapeutics, 2008a. 2(5): p. 264-276.

12. Groneberg, D.A., C. Witt, U. Wagner, K.F. Chung, and A. Fischer,

Fundamentals of pulmonary drug delivery. Respir Med, 2003. 97(4): p. 382-

7.

13. Brocklebank, D., F. Ram, J. Wright, P. Barry, C. Cates, L. Davies, G.

Douglas, M. Muers, D. Smith, and J. White, Comparison of the effectiveness

of inhaler devices in asthma and chronic obstructive airways disease: a

systematic review of the literature. Health Technol Assess, 2001. 5(26): p. 1-

149.

14. Siddiqui, M.A. and G.L. Plosker, The Novolizer: a multidose dry powder

inhaler. Treat Respir Med, 2005. 4(1): p. 63-9.

15. Bunnag, C., R. Fuangtong, C. Pothirat, and P. Punyaratabandhu, A

comparative study of patients' preferences and sensory perceptions of three

forms of inhalers among Thai asthma and COPD patients. Asian Pac J

Allergy Immunol, 2007. 25(2-3): p. 99-109.

16. Prime, D., P.J. Atkins, A. Slater, and B. Sumby, Review of dry powder

inhalers. Advanced Drug Delivery Reviews, 1997. 26(1): p. 51-58.

17. Hersey, J.A., Letter: Powder mixing by frictional pressure: specific example

of use of ordered mixing. J Pharm Sci, 1974. 63(12): p. 1960-1.

18. Islam, N., P. Stewart, I. Larson, and P. Hartley, Effect of carrier size on the

dispersion of salmeterol xinafoate from interactive mixtures. J Pharm Sci,

2004a. 93(4): p. 1030-8.

19. Frijlink, H.W. and A.H. de Boer, Trends in the technology-driven

development of new inhalation devices. Drug Discovery Today:

Technologies, 2005. 2(1): p. 47-57.

Bibliography

173

20. Chougule, M.B., B.K. Padhi, K.A. Jinturkar, and A. Misra, Development of

Dry Powder Inhalers. Recent Patents on Drug Delivery & Formulation,

2007(1): p. 11-21.

21. Steckel, H. and B.W. Muller, In vitro evaluation of dry powder inhalers I:

drug deposition of commonly used devices. International Journal of

Pharmaceutics, 1997. 154(1): p. 19-29.

22. Smith, I.J. and M. Parry-Billings, The inhalers of the future? A review of dry

powder devices on the market today. Pulmonary Pharmacology &

Therapeutics, 2003. 16(2): p. 79-95.

23. Islam, N., P. Stewart, I. Larson, and P. Hartley, Lactose surface modification

by decantation: are drug-fine lactose ratios the key to better dispersion of

salmeterol xinafoate from lactose-interactive mixtures? Pharm Res, 2004b.

21(3): p. 492-9.

24. Zeng, X.M., G.P. Martin, C. Marriott, and J. Pritchard, The influence of

carrier morphology on drug delivery by dry powder inhalers. International

Journal of Pharmaceutics, 2000. 200(1): p. 93-106.

25. French, D.L., D.A. Edwards, and R.W. Niven, The influence of formulation

on emission, deaggregation and deposition of dry powders for inhalation.

Journal of Aerosol Science, 1996. 27(5): p. 769-783.

26. Steckel, H. and B.W. Müller, In vitro evaluation of dry powder inhalers II:

influence of carrier particle size and concentration on in vitro deposition.

International Journal of Pharmaceutics, 1997. 154(1): p. 31-37.

27. Larhrib, H., X.M. Zeng, G.P. Martin, C. Marriott, and J. Pritchard, The use of

different grades of lactose as a carrier for aerosolised salbutamol sulphate.


28. Steckel, H. and B.W. Muller, In vitro evaluation of dry powder inhalers II:

influence of carrier particle size and concentration on in vitro deposition.


Bibliography

174

29. Karhu, M., J. Kuikka, T. Kauppinen, K. Bergström, and M. Vidgren,

Pulmonary deposition of lactose carriers used in inhalation powders.


30. Kawashima, Y., T. Serigano, T. Hino, H. Yamamoto, and H. Takeuchi, Effect

of surface morphology of carrier lactose on dry powder inhalation property

of pranlukast hydrate. International Journal of Pharmaceutics, 1998. 172(1-

2): p. 179-188.

31. Ganderton, D., D.A.V. Morton, and P. Lucas, Improvements in or relating to

powders. International patent WO/ 00/33811, 2000.

32. Lucas, P., K. Anderson, U.J. Potter, and J.N. Staniforth, Enhancement of

Small Particle Size Dry Powder Aerosol Formulations using an Ultra Low

Density Additive. Pharmaceutical Research, 1999. 16(10): p. 1643-1647.

33. Staniforth, J.N., Performance-Modifying Influences in Dry Powder Inhalation

Systems. Aerosol Science and Technology, 1995. 22(4): p. 346 - 353.

34. Staniforth, J.N., Carrier particles for use in dry powder inhalers.

International patent WO/ 96/23485, 1996.

35. Staniforth, J.N., Improvements in or relating to powders for use in dry

powder inhalers. International patent WO/ 97/03649, 1997.

36. Chew, N.Y., B.Y. Shekunov, H.H. Tong, A.H. Chow, C. Savage, J. Wu, and

H.K. Chan, Effect of amino acids on the dispersion of disodium cromoglycate

powders. Journal of Pharmaceutical Sciences, 2005. 94(10): p. 2289-2300.

37. Yue, B., J. Yang, Y. Wang, C.-Y. Huang, R. Dave, and R. Pfeffer, Particle

encapsulation with polymers via in situ polymerization in supercritical CO2.

Powder Technology, 2004. 146(1-2): p. 32-45.

38. Lahde, A., J. Raula, and E. Kauppinen, I. , Production of L-leucine

nanoparticles under various conditions using an aerosol flow reactor method.

J. Nanomaterials, 2008. 2008: p. 1-9.

Bibliography

175

39. Raula, J., A. Kuivanen, A. Lahde, and E.I. Kauppinen, Gas-phase synthesis of

l-leucine-coated micrometer-sized salbutamol sulphate and sodium chloride

particles. Powder Technology, 2008. 187(3): p. 289-297.

40. Begat, P., R. Price, H. Harris, D. Morton, and J. Staniforth, The Influence of

Force Control Agents on the Cohesive-Adhesive Balance in Dry Powder

Inhaler Formulations. KONA, 2005. 23: p. 109-121.

41. Tee, S.K., C. Marriott, X.M. Zeng, and G.P. Martin, The use of different

sugars as fine and coarse carriers for aerosolised salbutamol sulphate.

International Journal of Pharmaceutics, 2000. 208(1-2): p. 111-123.

42. Jaspart, S., P. Bertholet, G. Piel, J.-M. Dogné, L. Delattre, and B. Evrard,

Solid lipid microparticles as a sustained release system for pulmonary drug

delivery. European Journal of Pharmaceutics and Biopharmaceutics, 2007.

65(1): p. 47-56.

43. Sanna, V., N. Kirschvink, P. Gustin, E. Gavini, I. Roland, L. Delattre, and B.

Evrard, Preparation and in vivo toxicity study of solid lipid microparticles as

carrier for pulmonary administration. AAPS PharmSciTech, 2004. 5(2): p.

27.

44. Steckel, H. and N. Bolzen, Alternative sugars as potential carriers for dry

powder inhalations. International Journal of Pharmaceutics, 2004. 270(1-2):

p. 297-306.

45. Katainen, J., M. Paajanen, E. Ahtola, V. Pore, and J. Lahtinen, Adhesion as

an interplay between particle size and surface roughness. Journal of Colloid

and Interface Science, 2006. 304(2): p. 524-529.

46. Li, Q., V. Rudolph, and W. Peukert, London-van der Waals adhesiveness of

rough particles. Powder Technology, 2006. 161(3): p. 248-255.

47. Podczeck, F., The Influence of Particle Size Distribution and Surface

Roughness of Carrier Particles on the in vitro Properties of Dry Powder

Inhalations. Aerosol Science and Technology, 1999. 31(4): p. 301 - 321.

Bibliography

176

48. Ooi, J., D. Traini, S. Hoe, W. Wong, and P.M. Young, Does carrier size

matter? A fundamental study of drug aerosolisation from carrier based dry

powder inhalation systems. International Journal of Pharmaceutics, 2011.

413(1-2): p. 1-9.

49. Liechty, W.B., D.R. Kryscio, B.V. Slaughter, and N.A. Peppas, Polymers for

Drug Delivery Systems. Annual Review of Chemical and Biomolecular

Engineering, 2010. 1(1): p. 149-173.

50. Emami, J., H. Hamishehkar, A.R. Najafabadi, K. Gilani, M. Minaiyan, H.

Mahdavi, H. Mirzadeh, A. Fakhari, and A. Nokhodchi, Particle size design of

PLGA microspheres for potential pulmonary drug delivery using response

surface methodology. Journal of Microencapsulation, 2009. 26(1): p. 1-8.

51. Arnold, M.M., E.M. Gorman, L.J. Schieber, E.J. Munson, and C. Berkland,

NanoCipro encapsulation in monodisperse large porous PLGA

microparticles. Journal of Controlled Release, 2007. 121(1-2): p. 100-109.

52. Celebi, N., N. Erden, and A. Türkyilmaz, The preparation and evaluation of

salbutamol sulphate containing poly(lactic acid-co-glycolic acid)

microspheres with factorial design-based studies. International Journal of

Pharmaceutics, 1996. 136(1-2): p. 89-100.

53. Coowanitwong, I., V. Arya, G. Patel, W.S. Kim, V. Craciun, J.R. Rocca, R.

Singh, and G. Hochhaus, Laser-ablated nanofunctional polymers for the

formulation of slow-release powders for dry powder inhalers:

physicochemical characterization and slow-release characteristics. J Pharm

Pharmacol, 2007. 59(11): p. 1473-84.

54. Erden, N. and N. Celebi, Factors influencing release of salbutamol sulphate

from poly(lactide-co-glycolide) microspheres prepared by water-in-oil-in-

water emulsion technique. International Journal of Pharmaceutics, 1996.

137(1): p. 57-66.

55. Singh, R.K., W.S. Kim, M. Ollinger, V. Craciun, I. Coowantwong, G.

Hochhaus, and N. Koshizaki, Laser based synthesis of nanofunctionalized

Bibliography

177

particulates for pulmonary based controlled drug delivery applications.

Applied Surface Science, 2002. 197-198: p. 610-614.

56. Scheuch, G., M.J. Kohlhaeufl, P. Brand, and R. Siekmeier, Clinical

perspectives on pulmonary systemic and macromolecular delivery. Advanced

Drug Delivery Reviews, 2006. 58(9-10): p. 996-1008.

57. Byron, P.R., Prediction of drug residence times in regions of the human

respiratory tract following aerosol inhalation. J Pharm Sci, 1986. 75(5): p.

433-8.

58. Martini, F.H., Ober W.C., Garrison, C.W., Hutchings, R.T., Fundamentals of

anatomy and physiology. 7th edition ed. 2006, San Francisco: Pearson

Education Inc, Benjamin Cummings. 814-815.

59. Tortora, G., Principles of Human Anatomy. 5th edition ed. 1989, New York:

Harper Collins. 636.

60. Rang, H.P., M.M. Dale, and J.M. Ritter, Pharmacology. 4th edition ed. The

Respiratory System. 1999, Edinburgh: Harcourt Publishers Ltd. 338-350.

61. Sweetman, S.C., Martindale-The Complete Drug reference. 35th ed, London:

Pharmaceutical Press. 997.

62. Darquenne, C., Particle deposition in the lung, in Encyclopedia of

Respiratory Medicine, J.L. Geoffrey and D.S. Steven, Editors. 2006,

Academic Press: Oxford. p. 300-304.

63. Gonda, I., Aerosols for delivery of therapeutic and diagnostic agents to the

respiratory tract. Critical Reviews in Therapeutic Drug Carrier Systems,

1990. 6(4): p. 273-313.

64. Stuart, B.O., Deposition and clearance of inhaled particles. Environmental

Health Perspectives, 1984. 55: p. 369-90.

65. Telko, M.J. and A.J. Hickey, Dry powder inhaler formulation. Respir Care,

2005. 50(9): p. 1209-27.

Bibliography

178

66. Srichana, T., G.P. Martin, and C. Marriott, Dry powder inhalers: The

influence of device resistance and powder formulation on drug and lactose

deposition in vitro. European Journal of Pharmaceutical Sciences, 1998. 7(1):

p. 73-80.

67. Freedman, T., Medihaler therapy for bronchial asthma; a new type of aerosol

therapy. Postgrad Med, 1956. 20(6): p. 667-73.

68. Altounyan, R.E.C., Inhibition of experimental asthma by a new compound-

disodium cromoglycate. Intal. Acta Allergol 1967(22): p. 487-489.

69. Geller, D.E., Comparing clinical features of the nebulizer, metered-dose

inhaler, and dry powder inhaler. Respir Care, 2005. 50(10): p. 1313-21;

discussion 1321-2.

70. Islam, N. and E. Gladki, Dry powder inhalers (DPIs)--A review of device

reliability and innovation. International Journal of Pharmaceutics, 2008b.

360(1-2): p. 1-11.

71. Hickey, A.J. and N.M. Concessio, Descriptors of irregular particle

morphology and powder properties. Advanced Drug Delivery Reviews, 1997.

26(1): p. 29-40.

72. Zeng, X.M., G.P. Martin, S.-K. Tee, and C. Marriott, The role of fine particle

lactose on the dispersion and deaggregation of salbutamol sulphate in an air

stream in vitro. International Journal of Pharmaceutics, 1998. 176(1): p. 99-

110.

73. Lucas, P., K. Anderson, and J.N. Staniforth, Protein Deposition from Dry

Powder Inhalers: Fine Particle Multiplets as Performance Modifiers.

Pharmaceutical Research, 1998. 15(4): p. 562-569.

74. Timsina, M.P., G.P. Martin, C. Marriott, D. Ganderton, and M. Yianneskis,

Drug delivery to the respiratory tract using dry powder inhalers.


Bibliography

179

75. Bérard, V., E. Lesniewska, C. Andrès, D. Pertuy, C. Laroche, and Y.

Pourcelot, Dry powder inhaler: influence of humidity on topology and

adhesion studied by AFM. International Journal of Pharmaceutics, 2002a.

232(1-2): p. 213-224.

76. Ferron, G.A., Aerosol properties and lung deposition. Eur Respir J, 1994.

7(8): p. 1392-4.

77. Aiache, J.M., Les préparations pour inhalation. S. T. P. Pharma, 1990. 6(10):

p. 753-761.

78. Saint-Lorant, G., P. Leterme, A. Gayot, and M.P. Flament, Influence of

carrier on the performance of dry powder inhalers. International Journal of

Pharmaceutics, 2007. 334(1-2): p. 85-91.

79. Wynn, E. and S. Nichols, Importance of Wall Collisions for Particle-Particle

Detachment in Dry Powder Inhalers and Advanced Wall Collision Models, in

Drug Delivery to the lungs 19. 2008: Scotland, UK.

80. de Boer, A.H., P. Hagedoorn, D. Gjaltema, J. Goede, and H.W. Frijlink, Air

classifier technology (ACT) in dry powder inhalation: Part 1. Introduction of

a novel force distribution concept (FDC) explaining the performance of a

basic air classifier on adhesive mixtures. International Journal of

Pharmaceutics, 2003a. 260(2): p. 187-200.

81. Donovan, M.J., S.H. Kim, V. Raman, and H.D. Smyth, Dry powder inhaler

device influence on carrier particle performance. Journal of Pharmaceutical

Sciences, 2011.

82. Voss, A. and W.H. Finlay, Deagglomeration of dry powder pharmaceutical

aerosols. International Journal of Pharmaceutics, 2002. 248(1-2): p. 39-50.

83. Zanen, P., L.T. Go, and J.-W.J. Lammers, The optimal particle size for

parasympathicolytic aerosols in mild asthmatics. International Journal of

Pharmaceutics, 1995. 114(1): p. 111-115.

Bibliography

180

84. Zanen, P., L.T. Go, and J.W. Lammers, Optimal particle size for beta 2

agonist and anticholinergic aerosols in patients with severe airflow

obstruction. Thorax, 1996. 51(10): p. 977-80.

85. Usmani, O.S., M.F. Biddiscombe, J.A. Nightingale, S.R. Underwood, and P.J.

Barnes, Effects of bronchodilator particle size in asthmatic patients using

monodisperse aerosols. J Appl Physiol, 2003. 95(5): p. 2106-2112.

86. Zanen, P., L.T. Go, and J.-W.J. Lammers, The optimal particle size for

[beta]-adrenergic aerosols in mild asthmatics. International Journal of

Pharmaceutics, 1994. 107(3): p. 211-217.

87. Bates, D.V., B.R. Fish, T.F. Hatch, T.T. Mercer, and P.E. Morrow,

Deposition and retention models for internal dosimetry of the human

respiratory tract. Task group on lung dynamics. Health Physics, 1966. 12(2):

p. 173-207.

88. Carvalho, T.C., J.I. Peters, and R.O. Williams Iii, Influence of particle size on

regional lung deposition - What evidence is there? International Journal of

Pharmaceutics, 2011. 406(1-2): p. 1-10.

89. Louey, M.D., S. Razia, and P.J. Stewart, Influence of physico-chemical

carrier properties on the in vitro aerosol deposition from interactive

mixtures. International Journal of Pharmaceutics, 2003. 252(1-2): p. 87-98.

90. Podczeck, F., The relationship between physical properties of lactose

monohydrate and the aerodynamic behaviour of adhered drug particles.


91. Srichana, T., G.P. Martin, and C. Marriott, On the relationship between drug

and carrier deposition from dry powder inhalers in vitro. International

Journal of Pharmaceutics, 1998. 167(1-2): p. 13-23.

92. Corn, M., The adhesion of solid particles to solid surfaces. I. A review. J Air

Pollut Control Assoc, 1961a. 11: p. 523-8.

Bibliography

181

93. Kendall, K., Adhesion: Molecules and Mechanics. Science, 1994. 263(5154):

p. 1720-1725.

94. Bell, J.H., P.S. Hartley, and J.S.G. Cox, Dry powder aerosols I: A new

powder inhalation device. Journal of Pharmaceutical Sciences, 1971. 60(10):

p. 1559-1564.

95. Ganderton, D., The generation of respirable clouds from coarse powder

aggregates. Journal of Biopharmaceutical Sciences, 1992. 3: p. 101-105.

96. Gilani, K., A.R. Najafabadi, M. Darabi, M. Barghi, and M. Rafiee-Tehrani,

Influence of formulation variables and inhalation device on the deposition

profiles of cromolyn sodium dry powder aerosols DARU 2004. 12(3): p. 123-

130.

97. Kassem, N.M., K.K.L. Ho, and D. Ganderton, The effect of air flow and

carrier size on the characteristics of an inspirable cloud. J. Pharm.

Pharmacol. , 1989. 41: p. 14P.

98. Cline, D. and R. Dalby, Predicting the Quality of Powders for Inhalation

from Surface Energy and Area. Pharmaceutical Research, 2002. 19(9): p.

1274-1277.

99. de Boer, A.H., P. Hagedoorn, D. Gjaltema, J. Goede, K.D. Kussendrager, and

H.W. Frijlink, Air classifier technology (ACT) in dry powder inhalation Part

2. The effect of lactose carrier surface properties on the drug-to-carrier

interaction in adhesive mixtures for inhalation. International Journal of

Pharmaceutics, 2003b. 260(2): p. 201-216.

100. Byron, P., Jashnani, R. and Germain, S., Efficiency of aerosolization from dry

powder blends of terbutaline sulfate and lactose NF with different particle

size distribution. Pharm. Res, 1980. 7: p. S81.

101. Hamishehkar, H., J. Emami, A.R. Najafabadi, K. Gilani, M. Minaiyan, H.

Mahdavi, and A. Nokhodchi, Influence of carrier particle size, carrier ratio

and addition of fine ternary particles on the dry powder inhalation

Bibliography

182

performance of insulin-loaded PLGA microcapsules. Powder Technology,

2010. 201(3): p. 289-295.

102. Chew, N.Y. and H.K. Chan, Effect of powder polydispersity on aerosol

generation. Journal of Pharmacy and Pharmaceutical Sciences, 2002. 5(2): p.

162-8.

103. Fults, K.A., I.F. Miller, and A.J. Hickey, Effect of Particle Morphology on

Emitted Dose of Fatty Acid-Treated Disodium Cromoglycate Powder

Aerosols. Pharmaceutical Development and Technology, 1997. 2(1): p. 67-

79.

104. Rumpf, H., Particle Technology. 1990, London: Chapman and hall. 162-163.

105. Dunbar, C.A.H., A. J.; Holzner, P., Dispersion and Characterization of

Pharmaceutical Dry Powder Aerosols. Kona, 1998. 16: p. 7-45.

106. Braun, M.A., R. Oschmann, and P.C. Schmidt, Influence of excipients and

storage humidity on the deposition of disodium cromoglycate (DSCG) in the

Twin Impinger. International Journal of Pharmaceutics, 1996. 135(1-2): p. 53-

62.

107. Maggi, L., R. Bruni, and U. Conte, Influence of the moisture on the

performance of a new dry powder inhaler. International Journal of

Pharmaceutics, 1999. 177(1): p. 83-91.

108. Shoyele, S.A. and S. Cawthorne, Particle engineering techniques for inhaled

biopharmaceuticals. Advanced Drug Delivery Reviews, 2006. 58(9-10): p.

1009-1029.

109. Chan, H.-K., What is the role of particle morphology in pharmaceutical

powder aerosols? Expert Opinion on Drug Delivery, 2008. 5(8): p. 909-914.

110. Heng, P.W., L.W. Chan, and L.T. Lim, Quantification of the surface

morphologies of lactose carriers and their effect on the in vitro deposition of

salbutamol sulphate. Chemical and Pharmaceutical Bulletin, 2000. 48(3): p.

393-8.

Bibliography

183

111. Larhrib, H., G.P. Martin, C. Marriott, and D. Prime, The influence of carrier

and drug morphology on drug delivery from dry powder formulations.


112. Young, P.M., D. Cocconi, P. Colombo, R. Bettini, R. Price, D.F. Steele, and

M.J. Tobyn, Characterization of a surface modified dry powder inhalation

carrier prepared by "particle smoothing". Journal of Pharmacy and

Pharmacology, 2002. 54: p. 1339-1344.

113. Chan, L.W., L.T. Lim, and P.W. Heng, Immobilization of fine particles on

lactose carrier by precision coating and its effect on the performance of dry

powder formulations. Journal of Pharmaceutical Sciences, 2003. 92(5): p.

975-84.

114. Adi, H., D. Traini, H.-K. Chan, and P. M.Young, The influence of drug

morphology on aerosolisation efficiency of dry powder inhaler formulations.

Journal of Pharmaceutical Sciences, 2008. 97(7): p. 2780-2788.

115. Hickey, A.J., K.A. Fults, and R.S. Pillai, Use of particle morphology to

influence the delivery of drugs from dry powder aerosols. Journal of

Biopharmaceutical Sciences, 1992. 3: p. 107-113.

116. Chew, N.Y.K. and H.-K. Chan, Use of Solid Corrugated Particles to

Enhance Powder Aerosol Performance. Pharmaceutical Research, 2001.

18(11): p. 1570-1577.

117. Chew, N.Y., P. Tang, H.K. Chan, and J.A. Raper, How much particle surface

corrugation is sufficient to improve aerosol performance of powders?


118. Kawashima, Y., T. Serigano, T. Hino, H. Yamamoto, and H. Takeuchi, A

New Powder Design Method to Improve Inhalation Efficiency of Pranlukast

Hydrate Dry Powder Aerosols by Surface Modification with

Hydroxypropylmethylcellulose Phthalate Nanospheres. Pharmaceutical

Research, 1998. 15(11): p. 1748-1752.

Bibliography

184

119. Louey, M.D., P. Mulvaney, and P.J. Stewart, Characterisation of adhesional

properties of lactose carriers using atomic force microscopy. Journal of

Pharmaceutical and Biomedical Analysis, 2001. 25(3-4): p. 559-567.

120. Stewart, P.J., Particle interaction in pharmaceutical systems. Pharmacy

International, 1986. 7(6): p. 146-149.

121. Finot, E.L., J.-C Mutin, S.H. Hosain and J.-P. Goudonnet, Contact force

dependence on relative humidity: investigation using atomic force Scanning

Microscopy, 1996. 10: p. 697-708.

122. Ripperger, S. and K. Hein, Measurement of adhesion forces between particles

and rough substrates in air with the vibration method. KONA, 2004. 22: p.

121-133.

123. Okada, J., Y. Matsuda, and Y. Fukumori, Measurement of the adhesive force

of pharmaceutical powders by the centrifugal method. I. Yakugaku Zasshi,

1969. 89(11): p. 1539-44.

124. Okada, J., Y. Matsuda, and Y. Fukumori, Measurement of the adhesive force

of pharmaceutical powders by the centrifugal method. II. Relation between

the surface condition of substrates and the separation force-particle residue

curves. Yakugaku Zasshi, 1971. 91(11): p. 1207-10.

125. Otsuka, A., Iida, K. , Danjo, K. , Sunada, H., Measurements of the adhesive

force between particles of powdered organic substances and a glass substrate

by means of the impact separation method. II. Effect of addition of light

anhydrous silicic acid on the adhesive force of potato starch. Chemical

pharmaceutical bulletin, 1985. 33(9): p. 4054-4056.

126. Corn, M., The adhesion of solid particles to solid surfaces. II. Journal of the

Air Pollution Control Association, 1961a. 11: p. 566-75.

127. Ducker, W.A., T.J. Senden, and R.M. Pashley, Direct measurement of

colloidal forces using an atomic force microscope. Nature, 1991. 353(6341):

p. 239-241.

Bibliography

185

128. Binnig, G., C.F. Quate, and C. Gerber, Atomic force microscope. Physical

Review Letters, 1986. 56(9): p. 930-933.

129. Butt, H.-J., B. Cappella, and M. Kappl, Force measurements with the atomic

force microscope: Technique, interpretation and applications. Surface

Science Reports, 2005. 59(1-6): p. 1-152.

130. Nanda, K.K., S.N. Sarangi, and S.N. Sahu, Measurement of surface

roughness by atomic force microscopy and Rutherford backscattering

spectrometry of CdS nanocrystalline films. Applied Surface Science, 1998.

133(4): p. 293-297.

131. Bunker, M., M. Davies, and C. Roberts, Towards screening of inhalation

formulations: measuring interactions with atomic force microscopy. Expert

Opinion on Drug Delivery, 2005. 2(4): p. 613-624.

132. Begat, P., P. Young, M. , S. Edge, J.S. Kaerger, and R. Price The effect of

mechanical processing on surface stability of pharmaceutical powders:

Visualization by atomic force microscopy. Journal of Pharmaceutical

Sciences, 2003. 92(3): p. 611-620.

133. Islam, N., P. Stewart, I. Larson, and P. Hartley, Surface roughness

contribution to the adhesion force distribution of salmeterol xinafoate on

lactose carriers by atomic force microscopy. Journal of Pharmaceutical

Sciences, 2005. 94(7): p. 1500-1511.

134. Hooton, J.C., M.D. Jones, and R. Price, Predicting the behavior of novel

sugar carriers for dry powder inhaler formulations via the use of a cohesive–

adhesive force balance approach. Journal of Pharmaceutical Sciences, 2006.

95(6): p. 1288-1297.

135. Traini, D., P.M. Young, M. Jones, S. Edge, and R. Price, Comparative study

of erythritol and lactose monohydrate as carriers for inhalation: Atomic force

microscopy and in vitro correlation. European Journal of Pharmaceutical

Sciences, 2006. 27(2-3): p. 243-251.

Bibliography

186

136. Strathmann, S.C., M.A. Murphy, B.A. Goeckner, P.W. Carter, and J.-B.D.

Green, Forces between insulin microspheres and polymers surfaces for a dry

powder inhaler. International Journal of Pharmaceutics, 2009. 372(1-2): p.

147-153.

137. Lam, K.K. and J.M. Newton, Influence of particle size on the adhesion

behaviour of powders, after application of an initial press-on force. Powder

technology 1992a. 73(2): p. 117-125.

138. Mullins , M.E., L.P. Michaels, V. Menon, B. Locke, and M.B. Ranade, Effect

of Geometry on Particle Adhesion Aerosol Science and Technology, 1992.

17(2): p. 105 - 118

139. Rowley, G., Quantifying electrostatic interactions in pharmaceutical solid

systems. International Journal of Pharmaceutics, 2001. 227(1-2): p. 47-55.

140. Lam, K.K. and J.M. Newton Effect of temperature on particulate solid

adhesion to a substrate surface. Powder Technology 1992b. 73(3): p. 267-

274.

141. Lam, K.K. and J.M. Newton, The influence of the time of application of

contact pressure on particle adhesion to a substrate surface

Powder Technology, 1993. 76(2): p. 149-154.

142. Attard, P. and J.L. Parker, Deformation and adhesion of elastic bodies in

contact. Physical Review A, 1992. 46(12): p. 7959.

143. Corn, M., The adhesion of solid particles to solid surfaces. I. A review.

Journal of the Air Pollution Control Association, 1961b. 11: p. 523-8.

144. Kulvanich, P. and P.J. Stewart, The effect of particle size and concentration

on the adhesive characteristics of a model drug-carrier interactive system.

Journal of Pharmacy and Pharmacology, 1987. 39(9): p. 673-678.

145. Podczeck, F., The relationship between particulate properties of carrier

materials and the adhesion force of drug particles in interactive powder

Bibliography

187

mixtures. Journal of Adhesion Science and Technology, 1997. 11: p. 1089-

1104.

146. Grimsey, I.M., J.C. Feeley, and P. York, Analysis of the surface energy of

pharmaceutical powders by inverse gas chromatography. Journal of

Pharmaceutical Sciences, 2002. 91(2): p. 571-583.

147. James, J., B. Crean, M. Davies, R. Toon, P. Jinks, and C.J. Roberts, The

surface characterisation and comparison of two potential sub-micron, sugar

bulking excipients for use in low-dose, suspension formulations in metered

dose inhalers. International Journal of Pharmaceutics, 2008. 361(1-2): p. 209-

221.

148. Sethuraman, V.V. and A.J. Hickey, Powder properties and their influence on

dry powder inhaler delivery of an antitubercular drug. AAPS PharmSciTech,

2002. 3(4): p. E28.

149. Shekunov, B., P. Chattopadhyay, H. Tong, and A. Chow, Particle Size

Analysis in Pharmaceutics: Principles, Methods and Applications.


150. Shekunov, B.Y., J.C. Feeley, A.H.L. Chow, H.H.Y. Tong, and P. York,

Physical properties of supercritically-processed and micronised powders for

respiratory drug delivery. KONA, 2002. 20: p. 178-187.

151. Traini, D., P.M. Young, F. Thielmann, and M. Acharya, The Influence of

Lactose Pseudopolymorphic Form on Salbutamol Sulfate-Lactose

Interactions in DPI Formulations. Drug Development and Industrial

Pharmacy, 2008. 34(9): p. 992 - 1001.

152. Schiavone, H., S. Palakodaty, A. Clark, P. York, and S.T. Tzannis,

Evaluation of SCF-engineered particle-based lactose blends in passive dry

powder inhalers. International Journal of Pharmaceutics, 2004. 281(1-2): p.

55-66.

153. Tong, H.H., B.Y. Shekunov, P. York, and A.H. Chow, Predicting the aerosol

performance of dry powder inhalation formulations by interparticulate

Bibliography

188

interaction analysis using inverse gas chromatography. Journal of

Pharmaceutical Sciences, 2006. 95(1): p. 228-33.

154. Jiang, R.G., P.W. Zhang, L.Q. Wang, H. Liu, W.S. Pan, and C.L. Wang,

Effect of surface modification on surface energy of lactose and performance

of dry powder inhalations. Yao Xue Xue Bao (Acta Pharmaceutica Sinica),

2005. 40(4): p. 373-6.

155. Saleem, I., H. Smyth, and M. Telko, Prediction of Dry Powder Inhaler

Formulation Performance From Surface Energetics and Blending Dynamics.

Drug Development and Industrial Pharmacy, 2008. 34(9): p. 1002-1010.

156. Das, S., I. Larson, P. Young, and P. Stewart, Surface energy changes and

their relationship with the dispersibility of salmeterol xinafoate powders for

inhalation after storage at high RH. European Journal of Pharmaceutical

Sciences, 2009. 38(4): p. 347-354.

157. Bérard, V., E. Lesniewska, C. Andrès, D. Pertuy, C. Laroche, and Y.

Pourcelot, Affinity scale between a carrier and a drug in DPI studied by

atomic force microscopy. International Journal of Pharmaceutics, 2002b.

247(1-2): p. 127-137.

158. Tsukada, M., R. Irie, Y. Yonemochi, R. Noda, H. Kamiya, W. Watanabe, and

E.I. Kauppinen, Adhesion force measurement of a DPI size pharmaceutical

particle by colloid probe atomic force microscopy. Powder Technology,

2004. 141(3): p. 262-269.

159. Price, R., P.M. Young, S. Edge, and J.N. Staniforth, The influence of relative

humidity on particulate interactions in carrier-based dry powder inhaler

formulations. International Journal of Pharmaceutics, 2002. 246(1-2): p. 47-

59.

160. Dey, F.K., J.A.S. Cleaver, and P.A. Zhdan, Atomic force microscopy study of

adsorbed moisture on lactose particles. Advanced Powder Technology, 2000.

11(4): p. 401-413.

Bibliography

189

161. Podczeck, F., J.M. Newton, and M.B. James, The influence of constant and

changing relative humidity of the air on the autoadhesion force between

pharmaceutical powder particles. International Journal of Pharmaceutics,

1996. 145(1-2): p. 221-229.

162. Hooton, J., C. German, S. Allen, M. Davies, C. Roberts, S. Tendler, and P.

Williams, An Atomic Force Microscopy Study of the Effect of Nanoscale

Contact Geometry and Surface Chemistry on the Adhesion of Pharmaceutical

Particles. Pharmaceutical Research, 2004. 21(6): p. 953-961.

163. Young, P.M., R. Price, M.J. Tobyn, M. Buttrum, and F. Dey, The influence of

relative humidity on the cohesion properties of micronized drugs used in

inhalation therapy. Journal of Pharmaceutical Sciences, 2004. 93(3): p. 753-

761.

164. Young, P.M., R. Price, M.J. Tobyn, M. Buttrum, and F. Dey, Investigation

into the effect of humidity on drug–drug interactions using the atomic force

microscope. Journal of Pharmaceutical Sciences, 2003. 92(4): p. 815-822.

165. Young, P.M., M.J. Tobyn, R. Price, M. Buttrum, and F. Dey, The use of

colloid probe microscopy to predict aerosolization performance in dry

powder inhalers: AFM and in vitro correlation. Journal of Pharmaceutical

Sciences, 2006. 95(8): p. 1800-1809.

166. Beach, E.R., G.W. Tormoen, J. Drelich, and R. Han, Pull-off Force

Measurements between Rough Surfaces by Atomic Force Microscopy. Journal

of Colloid and Interface Science, 2002. 247(1): p. 84-99.

167. Meine, K., K. Kloß, T. Schneider, and D. Spaltmann, The influence of surface

roughness on the adhesion force. Surface and Interface Analysis, 2004. 36(8):

p. 694-697.

168. Mizes, H.A., Surface roughness and particle adhesion. Journal of Adhesion

1995. 51: p. 155-165.

Bibliography

190

169. Adi, S., H. Adi, P. Tang, D. Traini, H.-k. Chan, and P.M. Young, Micro-

particle corrugation, adhesion and inhalation aerosol efficiency. European

Journal of Pharmaceutical Sciences, 2008. 35(1-2): p. 12-18.

170. Eve, J.K., N. Patel, S.Y. Luk, S.J. Ebbens, and C.J. Roberts, A study of single

drug particle adhesion interactions using atomic force microscopy.


171. Rabinovich, Y.I., J.J. Adler, A. Ata, R.K. Singh, and B.M. Moudgil, Adhesion

between Nanoscale Rough Surfaces: I. Role of Asperity Geometry. Journal of

Colloid and Interface Science, 2000a. 232(1): p. 10-16.

172. Rabinovich, Y.I., J.J. Adler, A. Ata, R.K. Singh, and B.M. Moudgil, Adhesion

between Nanoscale Rough Surfaces: II. Measurement and Comparison with

Theory. Journal of Colloid and Interface Science, 2000b. 232(1): p. 17-24.

173. Quon, R.A., A. Ulman, and T.K. Vanderlick, Impact of Humidity on Adhesion

between Rough Surfaces. Langmuir 2000. 16: p. 8912-8916.

174. Podczeck, F., J.M. Newton, and M.B. James, The estimation of the true area

of contact between microscopic particles and a flat surface in adhesion

contact. Journal of Applied Physics, 1996. 79(3): p. 1458-1463.

175. Iida, K., Y. Hayakawa, H. Okamoto, K. Danjo, and H. Leuenberger,

Preparation of dry powder inhalation by surface treatment of lactose carrier

particles. Chem Pharm Bull (Tokyo), 2003a. 51(1): p. 1-5.

176. Flament, M.-P., P. Leterme, and A. Gayot, The influence of carrier roughness

on adhesion, content uniformity and the in vitro deposition of terbutaline

sulphate from dry powder inhalers. International Journal of Pharmaceutics,

2004. 275(1-2): p. 201-209.

177. Podczeck, F., J. Newton, and M. James, Assessment of adhesion and

autoadhesion forces between particles and surfaces: I. The investigation of

autoadhesion phenomena of salmeterol xinafoate and lactose monohydrate

particles using compacted powder surfaces. Journal of Adhesion Science and

Technology, 1994. 8(12): p. 1459-1472.

Bibliography

191

178. Podczeck, F., J.M. Newton, and M.B. James, Assessment of adhesion and

autoadhesion forces between particles and surfaces. Part II. The

investigation of adhesion phenomena of Salmeterol Xinafoate and lactose

monohydrate particles in particle-on-particle and particle-on-surface

contact. Journal of Adhesion Science and Technology, 1995b. 9: p. 475-486.

179. Podczeck, F., Adhesion forces in interactive powder mixtures of a micronized

drug and carrier particles of various particle size distributions. Journal of

Adhesion Science and Technology, 1998. 12: p. 1323-1339.

180. Zeng, M.X., G.P. Martin, C. Marriott, and J. Pritchard, The use of lactose

recrystallised from carbopol gels as a carrier for aerosolised salbutamol

sulphate. European Journal of Pharmaceutics and Biopharmaceutics, 2001.

51(1): p. 55-62.

181. Stewart, P., Infleunce of magnesium stearate on the homogeneity of a

prednisolone-granule ordered mix. Drug Development and Pharmacy, 1981.

7(5): p. 485-495.

182. Bolhuis, G.K., C.F. Lerk, H.T. Zijlstra, and A.H. De Boer, Fil formation by

magnesium stearate during mixing and its effect on tabletting. Pharm.

Weekbl., 1975: p. 110.

183. Gold, G., R.N. Duvall, B.T. Palermo, and J.G. Slater, Powder flow studies III.

Factors affecting the flow of lactose granules. Journal of Pharmaceutical

Sciences, 1968. 57(4): p. 667-671.

184. Guchardi, R., M. Frei, E. John, and J.S. Kaerger, Influence of fine lactose and

magnesium stearate on low dose dry powder inhaler formulations.


185. DesRosiers Lachiver, E., N. Abatzoglou, L. Cartilier, and J.-S. Simard,

Insights into the Role of Electrostatic Forces on the Behavior of Dry

Pharmaceutical Particulate Systems. Pharmaceutical Research, 2006. 23(5):

p. 997-1007.

Bibliography

192

186. Weber, D., Y. Pu, and C.L. Cooney, Quantification of Lubricant Activity of

Magnesium Stearate by Atomic Force Microscopy. Drug Development and

Industrial Pharmacy, 2008. 34(10): p. 1097 - 1099.

187. Begat, P., D.A.V. Morton, J. Shur, P. Kippax, J.N. Staniforth, and R. Price,

The role of force control agents in high-dose dry powder inhaler

formulations. Journal of Pharmaceutical Sciences, 2009. 98(8): p. 2770-2783.

188. Islam, N., A. Rashid, and G. Camm, Effects of magnesium stearate on the

efficient dispersion of Salbutamol Sulfate from carrier-based dry powder

inhaler formulations, in Respiratory Drug Delivery, Europe 2011. 2011.

189. Iida, K., Y. Hayakawa, H. Okamoto, K. Danjo, and H. Luenberger, Effect of

surface layering time of lactose carrier particles on dry powder inhalation

properties of salbutamol sulfate. Chem Pharm Bull (Tokyo), 2004a. 52(3): p.

350-3.

190. Tay, T., S. Das, and P. Stewart, Magnesium stearate increases salbutamol

sulphate dispersion: What is the mechanism? International Journal of

Pharmaceutics, 2010. 383(1-2): p. 62-69.

191. Lechuga-Ballesteros, D. and M.-C. Kuok, Dry powder compositions having

improved dispersivity. International patent WO/ 01/32144, 2001.

192. Staniforth, J.N., S. Cryer, H.A. Ahmed, and S.P. Davies, Aspects of

pharmaceutical tribology. Drug Development and Industrial Pharmacy, 1989.

15: p. 2265-2294.

193. Najafabadi, A.R., K. Gilani, M. Barghi, and M. Rafiee-Tehrani, The effect of

vehicle on physical properties and aerosolisation behaviour of disodium

cromoglycate microparticles spray dried alone or with L-leucine.


194. You, Y., M. Zhao, G. Liu, and X. Tang, Physical characteristics and

aerosolization performance of insulin dry powders for inhalation prepared by

a spray drying method. Journal of Pharmacy and Pharmacology, 2007. 59(7):

p. 927-934.

Bibliography

193

195. Ren, Y., C. Yu, K. Meng, and X. Tang, Influence of formulation and

preparation process on ambroxol hydrochloride dry powder inhalation

characteristics and aerosolization properties. Drug Development and

Industrial Pharmacy, 2008. 34(9): p. 984-91.

196. Prota, L., A. Santoro, M. Bifulco, R.P. Aquino, T. Mencherini, and P. Russo,

Leucine enhances aerosol performance of Naringin dry powder and its

activity on cystic fibrosis airway epithelial cells. International Journal of

Pharmaceutics, 2011. 412(1-2): p. 8-19.

197. Paajanen, M., J. Katainen, J. Raula, E.I. Kauppinen, and J. Lahtinen, Direct

evidence on reduced adhesion of Salbutamol sulphate particles due to L-

leucine coating. Powder Technology, 2009. 192(1): p. 6-11.

198. Raula, J., J.A. Kurkela, D.P. Brown, and E.I. Kauppinen, Study of the

dispersion behaviour of l-leucine containing microparticles synthesized with

an aerosol flow reactor method. Powder Technology, 2007. 177(3): p. 125-

132.

199. Raula, J., A. Lähde, and E.I. Kauppinen, Aerosolization behavior of carrier-

free l-leucine coated salbutamol sulphate powders. International Journal of

Pharmaceutics, 2009. 365(1-2): p. 18-25.

200. Raula, J., F. Thielmann, M. Naderi, V.-P. Lehto, and E.I. Kauppinen,

Investigations on particle surface characteristics vs. dispersion behaviour of

l-leucine coated carrier-free inhalable powders. International Journal of

Pharmaceutics, 2010. 385(1-2): p. 79-85.

201. Louey, M.D. and P.J. Stewart, Particle Interactions Involved in Aerosol

Dispersion of Ternary Interactive Mixtures. Pharmaceutical Research, 2002.

19(10): p. 1524-1531.

202. Jones, M.D., R. Price, J.C. Hooton, M.L. Dawson, and A.R. Ferrie, Using

AFM to investigate how fines improve DPI performance. Proceedings of

Drug Delivery to Lungs, The Aerosol Society, Edinburgh, UK, 2005. 16: p.

11-14.

Bibliography

194

203. Iida, K., Y. Hayakawa, H. Okamoto, K. Danjo, and H. Luenbergerb, Effect of

surface covering of lactose carrier particles on dry powder inhalation

properties of salbutamol sulfate. Chem Pharm Bull (Tokyo), 2003b. 51(12):

p. 1455-7.

204. Iida, K., Y. Inagaki, H. Todo, H. Okamoto, K. Danjo, and H. Luenberger,

Effects of surface processing of lactose carrier particles on dry powder

inhalation properties of salbutamol sulfate. Chem Pharm Bull (Tokyo),

2004b. 52(8): p. 938-42.

205. El-Sabawi, D., S. Edge, R. Price, and P.M. Young, Continued investigation

into the influence of loaded dose on the performance of dry powder inhalers:

surface smoothing effects. Drug Dev Ind Pharm, 2006. 32(10): p. 1135-8.

206. Young, P., P. Kwok, H. Adi, H.-K. Chan, and D. Traini, Lactose Composite

Carriers for Respiratory Delivery. Pharmaceutical Research, 2009. 26(4): p.

802-810.

207. Zeng, X.M., G.P. Martin, C. Marriott, and J. Pritchard, Crystallization of

Lactose from Carbopol Gels. Pharmaceutical Research, 2000a. 17(7): p. 879-

886.

208. Dhumal, R., S. Biradar, A. Paradkar, and P. York, Ultrasound Assisted

Engineering of Lactose Crystals. Pharmaceutical Research, 2008. 25(12): p.

2835-2844.

209. Zeng, X.-M., G.P. Martin, C. Marriott, and J. Pritchard, Lactose as a carrier

in dry powder formulations: The influence of surface characteristics on drug

delivery. Journal of Pharmaceutical Sciences, 2001a. 90(9): p. 1424-1434.

210. Malcolmson, R.J. and J.K. Embleton, Dry powder formulations for

pulmonary delivery. Pharmaceutical Science & Technology Today, 1998.

1(9): p. 394-398.

211. Momin, M.-N., A. Hedayati, and A. Nokhodchi, Investigation into

Alternative Sugars as Potential Carriers for Dry Powder Formulation of

Budesonide. BioImpacts, 2011. 1(2): p. 105-111.

Bibliography

195

212. Sebti, T. and K. Amighi, Preparation and in vitro evaluation of lipidic

carriers and fillers for inhalation. European Journal of Pharmaceutics and

Biopharmaceutics, 2006. 63(1): p. 51-58.

213. Ringsdorf, H., Hermann Staudinger and the Future of Polymer Research

Jubilees - Beloved Occasions for Cultural Piety. Angewandte Chemie

International Edition, 2004. 43(9): p. 1064-1076.

214. Martin del Valle, E.M., M.A. Galan, and R.G. Carbonell, Drug Delivery

Technologies: The Way Forward in the New Decade. Industrial &

Engineering Chemistry Research, 2009. 48(5): p. 2475-2486.

215. Pillai, O. and R. Panchagnula, Polymers in drug delivery. Current Opinion in

Chemical Biology, 2001. 5(4): p. 447-451.

216. Upton, C.E.K., Catherine A; Shakesheff, Kevin M and Howdle, Steven M,

One dose or two? The use of polymers in drug delivery. Polymer

International, 2007. 56: p. 1457-1460.

217. Fiegel, J., J. Fu, and J. Hanes, Poly(ether-anhydride) dry powder aerosols for

sustained drug delivery in the lungs. Journal of Controlled Release, 2004.

96(3): p. 411-423.

218. Fu, J., J. Fiegel, and J. Hanes, Synthesis and Characterization of PEG-Based

Ether Anhydride Terpolymers: Novel Polymers for Controlled Drug Delivery.

Macromolecules, 2004. 37(19): p. 7174-7180.

219. Fu, J., J. Fiegel, E. Krauland, and J. Hanes, New polymeric carriers for

controlled drug delivery following inhalation or injection. Biomaterials,

2002. 23(22): p. 4425-4433.

220. Zhang, Q., Z. Shen, and T. Nagai, Prolonged hypoglycemic effect of insulin-

loaded polybutylcyanoacrylate nanoparticles after pulmonary administration

to normal rats. International Journal of Pharmaceutics, 2001. 218(1-2): p. 75-

80.

Bibliography

196

221. Rytting, E., J. Nguyen, X. Wang, and T. Kissel, Biodegradable polymeric

nanocarriers for pulmonary drug delivery. Expert Opin Drug Deliv, 2008.

5(6): p. 629-39.

222. Edwards, D.A., A. Ben-Jebria, and R. Langer, Recent advances in pulmonary

drug delivery using large, porous inhaled particles. J Appl Physiol, 1998.

85(2): p. 379-85.

223. Shenoy, D.B. and M.M. Amiji, Poly(ethylene oxide)-modified poly([var

epsilon]-caprolactone) nanoparticles for targeted delivery of tamoxifen in

breast cancer. International Journal of Pharmaceutics, 2005. 293(1-2): p.

261-270.

224. Kim, S.Y. and Y.M. Lee, Taxol-loaded block copolymer nanospheres

composed of methoxy poly(ethylene glycol) and poly([var epsilon]-

caprolactone) as novel anticancer drug carriers. Biomaterials, 2001. 22(13):

p. 1697-1704.

225. Prabu, P., A.A. Chaudhari, N. Dharmaraj, M.S. Khil, S.Y. Park, and H.Y.

Kim, Preparation, characterization, in-vitro drug release and cellular uptake

of poly(caprolactone) grafted dextran copolymeric nanoparticles loaded with

anticancer drug. Journal of Biomedical Materials Research Part A, 2009.

90A(4): p. 1128-1136.

226. Zheng, D., X. Li, H. Xu, X. Lu, Y. Hu, and W. Fan, Study on docetaxel-

loaded nanoparticles with high antitumor efficacy against malignant

melanoma. Acta Biochimica et Biophysica Sinica, 2009. 41(7): p. 578-587.

227. Damgé, C., P. Maincent, and N. Ubrich, Oral delivery of insulin associated to

polymeric nanoparticles in diabetic rats. Journal of Controlled Release, 2007.

117(2): p. 163-170.

228. Shah, L.K. and M.M. Amiji, Intracellular delivery of saquinavir in

biodegradable polymeric nanoparticles for HIV/AIDS. Pharmaceutical

Research, 2006. 23(11): p. 2638-45.

Bibliography

197

229. Espuelas, M.S., P. Legrand, P.M. Loiseau, C. Bories, G. Barratt, and J.M.

Irache, In Vitro Antileishmanial Activity of Amphotericin B Loaded in Poly(ε-

Caprolactone) Nanospheres. Journal of Drug Targeting, 2002. 10(8): p. 593-

599.

230. Kho, K., W.S. Cheow, R.H. Lie, and K. Hadinoto, Aqueous re-dispersibility

of spray-dried antibiotic-loaded polycaprolactone nanoparticle aggregates

for inhaled anti-biofilm therapy. Powder Technology, 2010. 203(3): p. 432-

439.

231. Rawat, A., Q.H. Majumder, and F. Ahsan, Inhalable large porous

microspheres of low molecular weight heparin: In vitro and in vivo

evaluation. Journal of Controlled Release, 2008. 128(3): p. 224-232.

232. Kawashima, Y., H. Yamamoto, H. Takeuchi, S. Fujioka, and T. Hino,

Pulmonary delivery of insulin with nebulized -lactide/glycolide copolymer

(PLGA) nanospheres to prolong hypoglycemic effect. Journal of Controlled

Release, 1999. 62(1-2): p. 279-287.

233. Philip, V.A., R.C. Mehta, P.P. Deluca, and M.K. Mazumder, E-SPART

Analysis of Poly (D, L-lactide-co-glycolide) Microspheres Formulated for

Dry Powder Aerosols. Particulate Science and Technology, 1997a. 15(3-4): p.

303-316.

234. Philip, V.A., R.C. Mehta, M.K. Mazumder, and P.P. DeLuca, Effect of

surface treatment on the respirable fractions of PLGA microspheres

formulated for dry powder inhalers. International Journal of Pharmaceutics,

1997b. 151(2): p. 165-174.

235. Shi, S. and A. Hickey, PLGA Microparticles in Respirable Sizes Enhance an

In Vitro T Cell Response to Recombinant Mycobacterium Tuberculosis

Antigen TB10.4-Ag85B. Pharmaceutical Research, 2010. 27(2): p. 350-360.

236. Learoyd, T.P., J.L. Burrows, E. French, and P.C. Seville, Modified release of

beclometasone dipropionate from chitosan-based spray-dried respirable

powders. Powder Technology, 2008. 187(3): p. 231-238.

Bibliography

198

237. Corrigan, D.O., A.M. Healy, and O.I. Corrigan, Preparation and release of

salbutamol from chitosan and chitosan co-spray dried compacts and

multiparticulates. European Journal of Pharmaceutics and Biopharmaceutics,

2006. 62(3): p. 295-305.

238. Huang, Y.C., A. Vieira, M.K. Yeh, and C.H. Chiang, Pulmonary Anti-

inflammatory Effects of Chitosan Microparticles Containing Betamethasone.

Journal of Bioactive and Compatible Polymers, 2007. 22(1): p. 30-41.

239. Huang, Y.C., M.K. Yeh, S.N. Cheng, and C.H. Chiang, The characteristics of

betamethasone-loaded chitosan microparticles by spray-drying method.

Journal of Microencapsulation, 2003. 20(4): p. 459-472.

240. Huang, Y.C., M.K. Yeh, and C.H. Chiang, Formulation factors in preparing

BTM-chitosan microspheres by spray drying method. International Journal of

Pharmaceutics, 2002. 242(1-2): p. 239-242.

241. Salama, R., S. Hoe, H.-K. Chan, D. Traini, and P.M. Young, Preparation and

characterisation of controlled release co-spray dried drug-polymer

microparticles for inhalation 1: Influence of polymer concentration on

physical and in vitro characteristics. European Journal of Pharmaceutics and

Biopharmaceutics, 2008. 69(2): p. 486-495.

242. Sinha, V.R., K. Bansal, R. Kaushik, R. Kumria, and A. Trehan, Poly-

[epsilon]-caprolactone microspheres and nanospheres: an overview.


243. Engelberg, I. and J. Kohn, Physico-mechanical properties of degradable

polymers used in medical applications: A comparative study. Biomaterials,

1991. 12(3): p. 292-304.

244. Miller, R.A., J.M. Brady, and D.E. Cutright, Degradation rates of oral

resorbable implants (polylactates and polyglycolates): rate modification with

changes in PLA/PGA copolymer ratios. Journal of Biomedical Materials

Research, 1977. 11(5): p. 711-9.

Bibliography

199

245. Alway, B., R. Sangchantra, and P.J. Stewart, Modelling the dissolution of

diazepam in lactose interactive mixtures. International Journal of

Pharmaceutics, 1996. 130(2): p. 213-224.

246. Liu, J. and P.J. Stewart, Deaggregation during the dissolution of

benzodiazepines in interactive mixtures. Journal of Pharmaceutical Sciences,

1998. 87(12): p. 1632-1638.

247. Larson, I., C.J. Drummond, D.Y.C. Chan, and F. Grieser, Direct force

measurements between titanium dioxide surfaces. Journal of the American

Chemical Society, 1993. 115(25): p. 11885-11890.

248. Hutter, J. and J. Bechhoefer, Calibration of atomic force microscope tips

Review of Scientific Instruments, 1993. 64: p. 1868-1873.

249. Senden, T. and W. Ducker, Experimental Determination of Spring Constants

in Atomic Force Microscopy. Langmuir, 1994. 10(4): p. 1003-1004.

250. Ho, R., S.J. Hinder, J.F. Watts, S.E. Dilworth, D.R. Williams, and J.Y. Heng,

Determination of surface heterogeneity of D-mannitol by sessile drop contact

angle and finite concentration inverse gas chromatography. International

Journal of Pharmaceutics, 2010. 387(1-2): p. 79-86.

251. Thielmann, F., D.J. Burnett, and J.Y.Y. Heng, Determination of the Surface

Energy Distributions of Different Processed Lactose. Drug Development and

Industrial Pharmacy, 2007. 33(11): p. 1240-1253.

252. Yla-Maihaniemi, P.P., J.Y. Heng, F. Thielmann, and D.R. Williams, Inverse

gas chromatographic method for measuring the dispersive surface energy

distribution for particulates. Langmuir, 2008. 24(17): p. 9551-7.

253. Thielmann, F. and D. Pearse, Determination of surface heterogeneity profiles

on graphite by finite concentration inverse gas chromatography. Journal of

Chromatography A, 2002. 969(1-2): p. 323-7.

254. Schultz, J., L. Lavielle, and C. Martin, The Role of the Interface in Carbon

Fibre-Epoxy Composites. The Journal of Adhesion, 1987. 23(1): p. 45-60.

Bibliography

200

255. Crooks, M.J. and R. Ho, Ordered mixing in direct compression of tablets.

Powder Technology, 1976. 14(1): p. 161-167.

256. Hallworth, G.W. and D.G. Westmoreland, The twin impinger: a simple device

for assessing the delivery of drugs from metered dose pressurized aerosol

inhalers. J Pharm Pharmacol, 1987. 39(12): p. 966-72.

257. Atanasoska, L., V. Cammarata, B.J. Stallman, W.S.V. Kwan, and L.L. Miller,

Langmuir-Blodgett films of rigid rod oligoimides on gold: An x-ray

photoelectron spectroscopy study. Surface and Interface Analysis, 1992.

18(2): p. 163-172.

258. Andrade, J.D., X-Ray Photoelectron Spectroscopy (XPS), in Surface and

Interfacial aspects of Biomedical polymers, J.D. Andrade, Editor. 1985,

Plenum Publishing Corporation: New York. p. 181.

259. Sahoo, S.K., J. Panyam, S. Prabha, and V. Labhasetwar, Residual polyvinyl

alcohol associated with poly (D,L-lactide-co-glycolide) nanoparticles affects

their physical properties and cellular uptake. Journal of Controlled Release,

2002. 82(1): p. 105-14.

260. Donovan, M.J. and H.D.C. Smyth, Influence of size and surface roughness of

large lactose carrier particles in dry powder inhaler formulations.


261. Concessio, N.M., M.M. VanOort, M.R. Knowles, and A.J. Hickey,

Pharmaceutical Dry Powder Aerosols: Correlation of Powder Properties

with Dose Delivery and Implications for Pharmacodynamic Effect.


262. Hickey, A.J., H.M. Mansour, M.J. Telko, Z. Xu, H.D.C. Smyth, T. Mulder,

R. McLean, J. Langridge, and D. Papadopoulos, Physical characterization of

component particles included in dry powder inhalers. II. Dynamic

characteristics. Journal of Pharmaceutical Sciences, 2007. 96(5): p. 1302-

1319.

Bibliography

201

263. Qiao, C., S. Jiang, X. Ji, L. An, and B. Jiang, Studies on confined

crystallization behavior of polycaprolactone thin films. Frontiers of

Chemistry in China, 2007. 2(4): p. 343-348.

264. Hua, C., Z. Chen, Q. Xu, and L. He, Ring-banded spherulites in PCL and

PCL/MWCNT solution-casting films and effect of compressed CO2 on them.

Journal of Polymer Science Part B: Polymer Physics, 2009. 47(8): p. 784-

792.

265. Yong, J., L. Yanhong, F. Zefu, W. Xiayu, X. Jun, G. Baohua, and L. Lin,

Obsevation of banded spherulites and lamellar structures by atomic force

microscopy. Science in China (Series B), 2003. 46(2): p. 153-159.

266. Simon, D., A. Holland, and R. Shanks, Poly(caprolactone) thin film

preparation, morphology, and surface texture. Journal of Applied Polymer

Science, 2007. 103(2): p. 1287-1294.

267. Dorati, R., M. Patrini, P. Perugini, F. Pavanetto, A. Stella, T. Modena, I.

Genta, and B. Conti, Surface characterization by atomic force microscopy of

sterilized PLGA microspheres. Journal of Microencapsulation, 2006. 23(2): p.

123-133.

268. Wan, Y., X. Qu, J. Lu, C. Zhu, L. Wan, J. Yang, J. Bei, and S. Wang,

Characterization of surface property of poly(lactide-co-glycolide) after

oxygen plasma treatment. Biomaterials, 2004. 25(19): p. 4777-4783.

269. Study on the phase separation of poly(lactide-co-glycolide)/polyphosphazene

blend film.

270. Cava, D., R. Gavara, J.M. Lagarón, and A. Voelkel, Surface characterization

of poly(lactic acid) and polycaprolactone by inverse gas chromatography.

Journal of Chromatography A, 2007. 1148(1): p. 86-91.

271. Al-Ghamdi, A., M. Melibari, and Z.Y. Al-Saigh, Characterization of

biodegradable polymers by inverse gas chromatography. II. Blends of

amylopectin and poly(ε-caprolactone). Journal of Applied Polymer Science,

2006. 101(5): p. 3076-3089.

Bibliography

202

272. Ahn, H.B., S.J. Ahn, S.J. Lee, T.W. Kim, and D.S. Nahm, Analysis of surface

roughness and surface free energy characteristics of various orthodontic

materials. American Journal of Orthodontics and Dentofacial Orthopedics,

2009. 136(5): p. 668-74.

273. York, P., M.D. Ticehurst, J.C. Osborn, R.J. Roberts, and R.C. Rowe,

Characterisation of the surface energetics of milled dl-propranolol

hydrochloride using inverse gas chromatography and molecular modelling.


274. Grimsey, I.M., M. Sunkersett, J.C. Osborn, P. York, and R.C. Rowe,

Interpretation of the differences in the surface energetics of two optical forms

of mannitol by inverse gas chromatography and molecular modelling.


275. Grimsey, J., A. Edwards, B. Shekunov, R. Forbes, and P. York. The effect of

processing on the surface energetics of poly(l-lactide) microparticles as

determined by inverse gas chromatography (IGC), . in Proceedings of the

Annual Conference of American Association of Pharmaceutical Scientists,

PharmSci. 1999.

276. Ticehurst, M.D., P. York, R.C. Rowe, and S.K. Dwivedi, Characterisation of

the surface properties of [alpha]-lactose monohydrate with inverse gas

chromatography, used to detect batch variation. International Journal of

Pharmaceutics, 1996. 141(1-2): p. 93-99.

277. Aulton, M., Aulton's Pharmaceutics: The design and manufacture of

medicines. 3rd ed, ed. K. Taylor. 2007, United Kingdom. 736.

278. Mukhopadhyay, P. and H.P. Schreiber, Aspects of acid-base interactions and

use of inverse gas chromatography. Colloids and surfaces. A,

Physicochemical and engineering aspects 1995. 100(1-3): p. 47-71.

279. Feeley, J.C., P. York, B.S. Sumby, and H. Dicks, Processing effects on the

surface properties of α-lactose monohydrate assessed by inverse gas

chromatography (IGC). Journal of Materials Science, 2002. 37(1): p. 217-

222.

Bibliography

203

280. Sommerfeld, M. and N. Huber, Experimental analysis and modelling of

particle-wall collisions. International Journal of Multiphase Flow, 1999.

25(6-7): p. 1457-1489.

Documents

Studies on the Surface Properties of Biodegradable Polymer ... · Rinku Tuli B.Pharm, M.Pharm This thesis is submitted in fulfilment of the requirements for the degree of Doctor of