34
6.0 Preparation and Evaluation of Tenofovir nanoparticles Page 53 6.1 Introduction Nanoparticle technology has evolved over the years in pharmaceutical drug delivery. This technology has been used to targeting drug to different sites of the body as per requirements of the disease. Well documented and extensive work has been done in cancer therapy using nanoparticle technology. The major problem involved in treatment of complex diseases is penetration of drug through blood brain barrier. Nanoparticles have overcome this problem to a great extent. Over the past decade nanotechnology has evolved for anti- retroviral therapy. Published literature from Christy Anthony pillai (2006), Scott Letendre (2008) and P.P. Koopmans (2009) to name a few have reported on the need and effectiveness of nanoparticle technology in HIV therapy. In the following chapter we have developed and optimised Tenofovir nanoparticle formulation and established all parameters required for its optimisation. Drug interaction has been studied using differential scanning calorimetry and infrared spectroscopy as a part of Preformulation studies. Attempt was made to prepare tenofovir nanoparticle formulation by various techniques like nanoprecipitaion, double emulsion method and the steps were taken to optimise formulation. Physio chemical characterisation of optimised formulation like the size of the nanoparticle, zeta potential (which establishes the surface charge), drug entrapment studies for establishing the amount of drug present inside the nanoparticle, Surface morphology was done by scanning electron microscopy. Stability studies were carried out to check the stability as per standard guidelines. The topics are discussed under various subtopics below. 6.2 Materials and Methods During the development and optimisation of Tenofovir nanoparticle formulation various materials, reagents, instruments, chemicals are used and they are mentioned in detail.

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Page 1: 6.0 Preparation and Evaluation of Tenofovir nanoparticlesshodhganga.inflibnet.ac.in/bitstream/10603/17103/15/15... · 2018. 7. 9. · 6.3 Experimental methods 6.3.1 Pre formulation

6.0 Preparation and Evaluation of Tenofovir nanoparticles

Page 53

6.1 Introduction

Nanoparticle technology has evolved over the years in pharmaceutical drug

delivery. This technology has been used to targeting drug to different sites of the

body as per requirements of the disease. Well documented and extensive work

has been done in cancer therapy using nanoparticle technology. The major

problem involved in treatment of complex diseases is penetration of drug

through blood brain barrier. Nanoparticles have overcome this problem to a

great extent. Over the past decade nanotechnology has evolved for anti-

retroviral therapy. Published literature from Christy Anthony pillai (2006), Scott

Letendre (2008) and P.P. Koopmans (2009) to name a few have reported on the

need and effectiveness of nanoparticle technology in HIV therapy.

In the following chapter we have developed and optimised Tenofovir

nanoparticle formulation and established all parameters required for its

optimisation. Drug interaction has been studied using differential scanning

calorimetry and infrared spectroscopy as a part of Preformulation studies.

Attempt was made to prepare tenofovir nanoparticle formulation by various

techniques like nanoprecipitaion, double emulsion method and the steps were

taken to optimise formulation. Physio chemical characterisation of optimised

formulation like the size of the nanoparticle, zeta potential (which establishes

the surface charge), drug entrapment studies for establishing the amount of drug

present inside the nanoparticle, Surface morphology was done by scanning

electron microscopy. Stability studies were carried out to check the stability as

per standard guidelines. The topics are discussed under various subtopics below.

6.2 Materials and Methods

During the development and optimisation of Tenofovir nanoparticle formulation

various materials, reagents, instruments, chemicals are used and they are

mentioned in detail.

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6.0 Preparation and Evaluation of Tenofovir nanoparticles

Page 54

6.2.1 Drug

Tenofovir disproxyl fumarate gift sample supplied by Matrix pharmaceuticals

limited Hyderabad.

6.2.2 Chemicals and reagents

Acetonitrile and Methanol (HPLC grade) supplied by Merck India, Water by

Millipore waters system, Eudragit RS 100 and RL 100 gift sample from Evonic

Mumbai, India, PLGA (50:50) polymer purchased from Boehringer Ingelheim

Germany, Polyvinyl alcohol (30000 to 70000) molecular weight from Sigma-

Aldrich, Mannitol is supplied by Himedia.

6.2.3 Instruments used

HPLC system (Waters 2695 separation module with millennium software),

Lichrocart analytical column 250 x 4.6mm, 5µ, pH meter (Systronics make),

UV spectrophotometer (2450 Shimadzu Kyoto, Japan), Infrared

spectrophotometer (Shimadzu FT-IR 8300), Differential scanning calorimeter

(DSC 60 Shimadzu Japan), ultra centrifuge (Sigma Aldrich 3k30), Analytical

balance (Sartorius), deep freezer (Sanyo), waters purification system (Milli-Q),

vortexer (Spinix USA), micropipette (eppendorf), particle size analyser(), high

speed homogeniser(Polytron PT 3100 Germany),lyophiliser().

6.3 Experimental methods

6.3.1 Pre formulation studies

Drug excipient compatibility studies performed using DSC-60 instrument. Alumina

was taken as reference. Temperature range is set from 350c to 800

0c. Samples were

analysed with the above mentioned parameters and data collected using the collection

monitor software. Fourier transform infrared spectroscopy (FT-IR), the samples are

mixed with potassium bromate, triturated with help of mortar and pestal to ensure

uniform mixing of the sample. Pellets are prepared by taking the mixture into IR

holder and using a hydraulic press. It is ensured that thickness of pellet is uniform and

thin. Spectrum was recorded by Shimadzu IR solution 1.30 software. The FT-IR

range for preformulation studies were carried out at 4000cm-1

to 400 cm-1

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6.0 Preparation and Evaluation of Tenofovir nanoparticles

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Differential Scanning Calorimetry (DSC) were performed for pure drug alone

and drug with excipients in the ratio of 1:1

Table 11: Samples analysed for FT-IR and DSC

S.No Sample

1. Tenofovir

2. Tenofovir+ PLGA 50:50 + Mannitol

3. Tenofovir+ Eudragit RL+ Mannitol

4. Tenofovir+ Eudragit RS+ Mannitol

6.3.2 Preparation of Nanoparticles

Preparation of nanoparticle was done by simple double emulsion method.

[Lamprecht et al (2000), Cristina et al (2002)] Tenofovir was dissolved in 0.5%

poly vinyl alcohol in a beaker and sonicated using ultra sonicator to ensure

complete dissolution of drug and vortexed to ensure uniform distribution in

solution. Accurately weighed amount of polymer (weights of polymer taken are

given in the table 12,13&14) was dissolved in two ml of dichloromethane in a

beaker and the solution obtained was clear with no particulate suspension

visible to naked eye. Primary emulsion was prepared in the following way.

Drug solution was added to polymer solution drop by drop and homogenised by

high speed homogeniser at 10000 RPM for 15 minutes. The time is counted

after complete addition of drug to polymer solution. The solution is probe

sonicated for 8 minutes (80w). Once the primary emulsion was formed,

secondary emulsion is prepared.

In another beaker, 0.5% Poly vinyl alcohol was homogenised with addition of

primary emulsion drop by drop at 10000 rpm for 15 minutes. The above

emulsion was probe sonicated 8 minutes (80w). The secondary emulsion was

kept for DCM evaporation for 4 hours at 370c and checked for odour of solvent

at regular intervals. The same procedure was followed for Eudragit-RS and

Eudragit-RL polymers.

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6.0 Preparation and Evaluation of Tenofovir nanoparticles

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6.3.3 Separation of nanoparticles

After complete evaporation of DCM, the nanoparticles were centrifuged at 5000

rpm at 50c for 5 minutes to remove un entrapped drug and micro particles if

any. Sigma centrifuge (Germany) was used and further centrifugation was

carried out at 30000g for 30 minutes at 50c. The nanoparticles were dispersed in

Mannitol solution in 1:1 ratio (mannitol & Polymer). The sample solution was

kept at 00c for 2 hours and latter freezed at -70

0c overnight before proceeding

for lyophilisation.

6.3.4 Lyophilisation of Nanoparticles

Freeze drying of nanoparticles is method of choice to remove the water from

nanoparticles. Freeze drying of nanoparticles will help in overcoming stability

issues like aggregation of nanoparticles, drug leakage etc. After lyophilisation

of nanoparticles further steps like characterization of nanoparticles, particle

size, zeta potential, poly dispersity index and drug entrapment were carried out.

Table 12: Composition for TNF formulation and PLGA (50:50)

nanoparticles

Ingredient TNF-

04

TNF-

05

TNF-

06

TNF-

25A

TNF-

25B

TNF-

25C

Tenofovir

(mg)

8.0 8.0 8.0 8.0 8.0 8.0

Poly lactic

glycolic acid

(PLGA) 50:50

(mg)

10 20 40 60 60 60

Poly vinyl

alcohol

(0.25%) (ml)

-- -- -- 2.0 -- --

Poly vinyl

alcohol (0.5%)

(ml)

2.0 2.0 2.0 -- 2.0 --

Poly vinyl

alcohol (1.0%)

(ml)

-- -- -- -- -- 2.0

Dichlorometha

ne (ml)

2.0 2.0 2.0 2.0 2.0 2.0

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Table 13: Composition for TNF formulation and Eudragit RL-100

nanoparticles

Ingredient TNF-

15

TNF-

19

TNF-

21

TNF-

30A

TNF-

30B

TNF-

30C

Tenofovir

(mg)

8.0 8.0 8.0 8.0 8.0 8.0

Eudragit-RL

100 (mg)

10 20 40 60 60 60

Poly vinyl

alcohol

(0.25%) (ml)

-- -- -- 2.0 -- --

Poly vinyl

alcohol

(0.5%) (ml)

2.0 2.0 2.0 -- 2.0 --

Poly vinyl

alcohol

(1.0%) (ml)

-- -- -- -- -- 2.0

Dichlorometh

ane (ml)

2.0 2.0 2.0 2.0 2.0 2.0

Table 14: Composition for TNF formulation and Eudragit RS-100

nanoparticles

Ingredient TNF-

18

TNF-

20

TNF-

29

TNF-

31A

TNF-

31B

TNF-

31C

Tenofovir

(mg)

8.0 8.0 8.0 8.0 8.0 8.0

Eudragit-RS

100 (mg)

10 20 40 60 60 60

Poly vinyl

alcohol

(0.25%) (ml)

-- -- -- 2.0 -- --

Poly vinyl

alcohol

(0.5%) (ml)

2.0 2.0 2.0 -- 2.0 --

Poly vinyl

alcohol

(1.0%) (ml)

-- -- -- -- -- 2.0

Dichlorometh

ane (ml)

2.0 2.0 2.0 2.0 2.0 2.0

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6.0 Preparation and Evaluation of Tenofovir nanoparticles

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6.3.5 Nanoparticle characterization

Particle size analysis & Polydispersity index, Zeta potential, Drug entrapment,

Percentage yield, FE-SEM (Field Emission Scanning Electron Microscope),

FT-IR (Fourier transform infrared spectroscopy) and Differential Scanning

Calorimetry (DSC) were carried out. Zambaux et al (1998)

6.3.6 Particle size Analysis

The nanoparticle size was determined by dynamic light scattering (DLS)

technique. using Zeta sizer nano instrument from Malvern. The technique

measures the time dependent fluctuations in the intensity of scattered light

which occurs due to the particles in constant Brownian motion. This technique

provides the information about whole particulate system. Particle size of nano

formulation is very important. Parameters like biological fate, distribution in the

body and physiochemical parameters depend on particle size. Polydispersity

index (PDI) is the measure for width of size distribution. The value of PDI close

to zero indicates homogenous distribution of Nano particles and values close to

one indicates heterogeneous distribution. The Nano formulation is dispersed in

water as medium and temperature at 250c. Clear disposable zeta cells are used

for measurements were taken in triplicates and mean particle size reported.

Thirumala Govender et al (1999).

6.3.7 Zeta potential

It is described as charge of electric double layer created by ions of the liquid

which exists around each particle. (Malvern Webinar on particle size and zeta

potential available over the web) suggests this particle will attract positive ions

to the surface, they form a layer and this layer is called as “Stern layer”. If we

move further from the particles ions diffuse more freely around the particle.

This layer is called as the “Diffuse layer”. Somewhere within this diffuse layer

there is a band ring called as “Hydrodynamic plane of shear” also called as

“Slipping plane”. Ions outside the slipping plane will not move along with the

particle whereas ions within the plane move with it.

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There are 3 types of potential. First one is Surface potential (Surface of

particle), second one Stern potential (potential of stern layer) and third Zeta

potential (potential of slipping plane). So zeta potential is the potential at hydro

dynamic shear or also called as slipping plane. It depends not only on the

particle surface but also on the dispersant, it may be unrelated to the surface

potential, it can be affected by the small change in the pH or ionic charge.

Particles interact according to the magnitude of zeta potential not the surface

charge, Therefore it helps in knowing dispersion stability.

If all the particles have higher negative or positive zeta potential then there will

be dispersion stability due to repulsion. The dividing line between an aqueous

particle dispersion being stable and un stable is considered to be +30mV and -

30mV. Particles outside the above stated range are stable. The Nano

formulation is dispersed in water as medium and temperature at 250c. Clear

disposable zeta cells are used for measurements were taken in triplicates and

mean particle size reported.

6.3.8 Percentage yield

The percentage yield of the formulation of PLGA (50:50), Eudragit-RL 100 and

Eudragit-RS 100 formulation are done by using the formula

Percentage yield = Weight of freeze dried nanoparticles (mg) X 100

Weight of drug (mg) + Polymer (mg) + Mannitol (mg)

6.3.9 Percentage Entrapment Efficiency

It is defined as the amount of drug entrapped inside the nano formulation and is

assayed by HPLC assay method developed and validated as indicated in early

chapter. The sample was prepared in the following way. Amount equivalent to

10 mg of Tenofovir from its salt form was weighed and dissolved in 10 ml

volumetric flask. The solution was sonicated for 10 minutes and volume made

up to mark with mobile phase. The clear supernatant solution was centrifuged

for 5000 rpm for 5 minutes and injected into the system. Percentage entrapment

efficiency is calculated by using the formula, Jin-Wook Yoo et al (2011)

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6.0 Preparation and Evaluation of Tenofovir nanoparticles

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EE (%) = Amount of drug in the prepared nanoparticle x 100

Amount of drug loaded in nanoparticle

6.3.10 Fourier Transform Infrared Spectroscopy (FT-IR) and Differential

Scanning Calorimetry (DSC)

Fourier Transform Spectroscopy (FT-IR) and DSC analysis of optimised batch

was carried out for Tenofovir formulation involving PLGA (50:50), Eudragit-

RS 100 and Eudragit-RL 100 polymers. The above mentioned parameters help

us in understanding the physical characteristic of drug and polymer interactions.

6.3.11 Field Emission Scanning Electron Spectroscopy (FE-SEM)

The surface morphology of optimised formulations was studied using Field

Emission Scanning Electron Spectroscopy (FE-SEM Hitachi SU 6600 Japan).

Dry nano formulation powder was taken on a dye with double side sticky tape

attached to it. The formulation is finely dispersed over the tape and gold coated

for 20 seconds. The die along with sample was loaded into the system and

photographed for its surface morphology.

6.3.12 Invitro Drug Release

Invitro release studies were carried out to nanoparticle formulation by Diffusion

method [T. Niwa, H. Takeuchi, T (1992), Einat et al (2009)] .0.1N HCl was

taken as dissolution media. Due to very high solubility of drug (13.9 mg/ml). 40

ml media was taken. Sigma membrane immersed in the dissolution media is

kept for overnight. This differentiates the donor and acceptor compartment.

Amount equivalent to one dose of drug was taken and placed in donor

compartment. A magnetic needle with 50 RPM was used and temperature is

maintained at 370c. Time points were 5, 10, 15, 30, 45, 60, 120, 240, 360, 480,

720, 960, 1200 and 1440 minutes. Sample were taken at the designated time and

replaced with fresh disso media maintained at the same temperature. Detection

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6.0 Preparation and Evaluation of Tenofovir nanoparticles

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of drug release was done at UV wavelength of 260 nm. The experiments are

performed in duplicate and the drug concentration is converted into cumulative

drug release and results reported.

6.3.13 Release Kinetics

Release Kinetics is an important parameter for pharmaceutical dosage forms

and determining the release kinetics of our nano formulation is a proof of

adequacy of its design. To fulfil these criteria, cumulative release profiles of

optimised nano formulation are described using mathematical equation. These

models illustrate the drug release from nano formulation. In the present study

the best describing kinetic models like Krosmeyers-Peppas model (Mt/M∞ = kt

n), „n‟ is the diffusional exponent, „k‟ is kinetic constant, „Mt‟ is the amount

drug released at time t, „M∞‟ is amount released at time infinity. Higuchi (Q =

Kt1/2) model, Zero order model (percent release against time), First order (log

percent release against time). The criteria to choose the best model was

determined by coefficient of determination R2 to assess the best fit model.

Srinivas et al (2008).

6.3.14 Stability Studies

Stability is a very important criterion for formulation development. Stability

helps in assessment of shelf life. Different regulatory bodies like ICH have

guidelines and stringent norms of real time stability data that needs to be

established. This stability data gives information about shipping and storage of

samples. Stability studies were conducted for the optimised formulation as per

ICH guidelines. The optimised formulations were stored in glass vials at 40c +

20c. all three batches TNF-25, TNF-30 and TNF-31 were stored under the above

stated condition as well as at accelerated stability condition of 400 + 2

0c / 75% +

5% RH for a period of 15, 30,60,90 and 180 days. The samples were analysed

for change in physical appearance, entrapment efficiency, particle size and zeta

potential.

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6.0 Preparation and Evaluation of Tenofovir nanoparticles

Page 62

6.4 Results and Discussion

6.4.1 Preformulation studies

As mentioned earlier Preformulation studies were carried out of Drug +PLGA

(50:50) polymer ( TNF-25 batch), Drug + Eudragit-RL 100 (TNF-30) and Drug

+ Eudragit-RS 100( TNF-31). Drug excipient reaction was studied by FT-IR

and DSC.

6.4.2 IR interpretation:

Presence of following functional groups in Tenofovir drug was confirmed by

the following functional groups, Carbonyl group at 1759 cm-1

and OH group at

3227 cm-1

. PLGA polymer contain the following group OH at 3516 cm-1

and

carbonyl group at 1757 cm-1

. In TNF-25 (PLGA 50:50) the spectrum of

polymer with drug showed that there is a physical interaction between carbonyl

group of drug and OH group of polymer due to hydrogen bond formation by

weak van der Waal‟s forces. Further release of drug invitro indicates only

physical interaction not chemical interaction.

Eudragit RL (TNF-30) the following functional group are found, Alkyl groups

below 3000cm-1

, bend of alkyl CH3 and CH2 at 2359 cm-1

and 2332cm-1

.

Carbonyl group at 1735cm-1

and 1726cm-1

.In TNF-30 and TNF-31 IR

chromatograms indicates that drug entrapment is within the polymer by weak

forces of attraction like hydrogen bonding and van der Waal‟s forces.

6.4.3 DSC interpretation:

Thermogram of Tenofovir disproxil fumarate showed a sharp melting transition

endotherm ranging from 114.64°c to 120.52°c with a peak at 117.69°c. The

DSC Thermogram of formulation TNF 25 (drug + PLGA polymer) showed

small melting endotherm at 157.21°c followed by a sharp endotherm at

158.58°c indicating decomposition of drug. Thermogram of formulation TNF

30 (drug + Eudragit RS) showed a small melting endotherm at 157.29°c

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6.0 Preparation and Evaluation of Tenofovir nanoparticles

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followed by an endotherm at 169.49°c indicating decomposition of drug. A

similar type of DSC Thermogram was observed for formulation TNF 31 with a

slight change in the peak position, 157.84°c followed by an endotherm at

168.25°c, with increased intensity. The absence of endothermic peak of the drug

at 117.69°c in the DSC Thermogram of the formulations i.e. TNF 25, TNF 30

and TNF 31 suggests that the drug existed in an amorphous or crystalline phase

as a molecular dispersion in the polymeric matrix.

Figure 17: FT-IR Spectrum of TNF-25 (Tenofovir + PLGA 50:50)

Figure 18: FT-IR Spectrum of TNF-31 (Tenofovir + Eudragit-RS 100)

75015002250300037501/cm

25

50

75

%T

32

71

.38

29

33

.83

28

75

.96

27

66

.01

23

59

.02

23

32

.02 1

75

5.2

8

16

37

.62

14

58

.23

13

71

.43

13

30

.93

12

55

.70

11

97

.83

10

89

.82

10

22

.31

96

8.3

09

31

.65

88

1.5

0

71

7.5

4 68

2.8

26

28

.81

51

1.1

5

TNF-25

75

0 150

0 225

0 300

0 375

0 1/c

m

0

2

5

5

0

7

5

10

0 %

T

3730.4

5

3273.3

1

2993.6

2

2935.7

6

2767.9

4

2677.2

9 2

561.5

5

2359.0

2

2332.0

2

1732.1

3

1458.2

3 1

375.2

9

1329.0

0

1253.7

7

1192.0

5

1149.6

1

1087.8

9

1022.3

1

964.4

4

929.7

2

879.5

7

715.6

1 6

26.8

9

509.2

2

476.4

3

412.7

8

TNF-

31

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Figure 19: FT-IR Spectrum of TNF-30 (Tenofovir + Eudragit-RL100)

DSC Spectrum

Figure 20: DSC of TNF-25 (Tenofovir + PLGA 50:50)

100.00 200.00 300.00

Temp [C]

-20.00

-10.00

0.00

10.00

mW

DSC

117.69 x100C

157.21 x100C

168.58 x100C

TNF + TNF 25

75

0 150

0 225

0 300

0 375

0 1/c

m

0

2

5

5

0

7

5 %

T 328

2

.95

327

3

.31

298

9

.76

293

5

.76

276

7

.94 26

77

.29

235

9

.02 233

2

.02

173

2

.13

145

8

.23

137

5

.29

133

2

.86 1

25

1

.84

118

8

.19

115

1

.54

108

7

.89

102

2

.31

966

.

37

931

.

65

881

.

50

715

.

61

628

.

81

509

.

22

TNF-

30

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Figure 21: DSC of TNF-30 (Tenofovir + Eudragit-RL100)

100.00 200.00 300.00

Temp [C]

-20.00

-10.00

0.00

10.00

mW

DSC

117.69 x100C

157.29 x100C

169.49 x100C

TNF + TNF 30

Figure 22: DSC of TNF-31 (Tenofovir + Eudragit-RS100)

100.00 200.00 300.00

Temp [C]

-10.00

0.00

10.00

mW

DSC

117.69 x100C

157.84 x100C

168.25 x100C

TNF + TNF 31

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6.4.4 Formulation Development

In the field of nano formulations PLGA (50:50) is most widely used bio

degradable polymer. The polymer is widely accepted by regulatory bodies

around the world for its safety was well known, no known major toxic effects

till now. Eudragit-RL 100 and Eudragit-RS 100 are selected for their stability at

varying pH. pH plays an important role in Tenofovir nano formulation

preparation by nano precipitation method Thirumala Govender et al (1999).

The problem with Tenofovir formulation was its high aqueous solubility of drug

which makes drug entrapment difficult as most of the drug remains in the

aqueous phase in this method both the drug and polymer is dissolved in organic

phase and then this was added to aqueous phase with constant stirring. The

organic layer was evaporated and then further steps of centrifugation at 20,000

rpm 50c were carried out. For aqueous phase we have chosen 2-[4-(2-Hydroxy

ethyl)1-piperazinyl] ethane sulphonic acid (HEPES buffer) 1mM strength and

pH adjusted to 7.0 by NaOH. The idea was to prevent the ionization of TNF

into aqueous solution so that more amount of drug is entrapped to the polymer.

pka of TNF is 3.75, so the pH 7.0 was chosen as trial. The particle size analysis

of the nanoparticles obtained by this method was high (884 nm), so double

emulsion method was tried out. The procedure for double emulsion method was

as mentioned earlier.

Most commonly used Poly vinyl alcohol was chosen as stabiliser. The ratio of

polymer with respect to drug was increased and this resulted in increase of drug

entrapment and drug yield. Varying concentrations of stabiliser did not make a

significant difference in nano particle characteristics like size, zeta potential or

drug entrapment etc. Homogenisation speed was optimised at 10000 rpm after

trials at lower and higher speeds. Probe sonication for reducing the particle size

was tried at varying amplitudes and the final optimised condition was kept at

80w/8 amps/ 8 mts.

Centrifugation speed was optimised at 30000g for 30 minutes at 40C. Higher

speeds made the re dispersion of nano formulation difficult and in few batches

re dispersion was not possible resulting in lump formation in the centrifuge

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tube. Lyophilisation to remove aqueous phase was done and it did not

contribute in any significant change in particle size or poly dispersity index and

zeta potential. The above method was good enough to have batch to batch

reproducibility and minimum variability with respect to final formulation

characterisation. Invitro release studies were carried out by widely reported

diffusion cell method with minor modifications to suit the developed

formulation and drug release was established for a period of 24 hours.

6.4.5 Characterisation of Nanoparticles

6.5 Physiochemical characterisation

6.5.1 Percentage yield

The percentage yield of Tenofovir nano formulation increased with increase in

polymer. Increase in polymer was necessary for increase in drug entrapment.

The final yield of all the three polymers (PLGA 50:50, Eudragit-RL, Eudragit-

RS) was comparable and reproducible. The final yield consists of drug, polymer

and cryoprotectant Mannitol.

Table 15: Percentage yield

Batch Number TNF-25 TNF-30 TNF-31

Polymer used in

nano

formulation

PLGA (50:50)

Polymer

Eudragit-RL 100

Polymer

Eudragit-Rs 100

Polymer

Total yield 87.14% + 1.54 86.45% + 1.72 87.00% + 1.34

6.5.2 Percentage Encapsulation Efficiency

It is the measure of drug entrapped with polymer and is of great importance in

the final optimised formulation for drug delivery. Entrapment of drug is

dependent on factors like nature and types of polymers used. The encapsulation

efficiency for TNF-25 optimised formulation is found to be 13.45% + 1.20. In

case of TNF-30, Eudragit RL 100 the entrapment was established at 11.55% +

1.24. For TNF-31, Eudragit RS 100 the entrapment was established at 11.12% +

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1.32. There is no significant change in entrapment with change in Poly vinyl

alcohol which is used as stabiliser. The drug entrapment was increased with

change in drug polymer ratio from 1:1.25 to 1:7.5. This was attributed to

increase in surface area available to drug with increase in polymer.

Table 16: Tenofovir + PLGA (50:50) Polymer + Mannitol

Drug:

Polymer PVA

Batch

No

Particle

Size PDI

Zeta

Potential % EE

1:1.25 0.50% TNF-4 250.8 0.117 -21.3 1.72

1:2.5 0.50% TNF-5 226.2 0.143 -23.6 3.85

1:50 0.50% TNF-6 738 0.596 -28 11.9

1:7.5 0.50% TNF-25 231 0.348 -8.46 13.45

Table 17: Tenofovir + Eudragit-RL 100 + Mannitol

Drug:

Polymer PVA Batch No

Particle

Size PDI

Zeta

Potential % EE

1:1.25 0.50% TNF-15 845 1.000 30.4 0.99

1:2.5 0.50% TNF-26 290 0.252 49.6 9.45

1:50 0.50% TNF-28 284 0.314 51.4 10.26

1:7.5 0.50% TNF-30 325 0.351 47.7 11.55

0

5

10

15

20

1 2 3 4

0

5

10

15

20

1 2 3 4

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Table 18: Tenofovir + Eudragit-RS 100 + Mannitol

Drug:

Polymer PVA Batch No

Particle

Size PDI

Zeta

Potential % EE

1:1.25 0.50% TNF-18 291 0.418 34.5 1.75

1:2.5 0.50% TNF-27 290 0.316 49.4 8.87

1:50 0.50% TNF-29 333 0.352 40.5 9.98

1:7.5 0.50% TNF-31 187 0.209 30.9 11.12

6.5.3 Particle Size Analysis

Mean particle size and poly dispersity index is the measure of quality of nano

formulation. Particle size was determined by using dynamic light scattering

technique of Malvern zeta sizer. The average particle size of TNF-25

(Tenofovir + PLGA 50:50) batch is found to be 232 nm and poly dispersity

index was found to be 0.348. For Eudragit-RL 100 nano formulation the size

was found to be 326 nm and poly dispersity index 0.351. Eudragit-RS 100

particle size was found to be 187 nm and poly dispersity index a healthy 0.209.

0

2

4

6

8

10

12

14

1 2 3 4

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Figure 23: Particle size of TNF-25 Tenofovir + PLGA (50:50) Polymer

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Figure 24: Particle size of TNF-30 Tenofovir + Eudragit-RL 100 Polymer

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Figure 25: Particle size of TNF-31 Tenofovir + Eudragit-RS 100 Polymer

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6.5.4 Zeta potential

It is one of the important parameters for stability of nano formulation and the

negative charge indicates repulsion of particles thus preventing aggregation of

formulation and more stable during stability of formulation on storage. The

results show that zeta potential depends on particle size. The TNF-25 batch

containing PLGA (50:50) polymer has zeta potential -8.46 + 1.42 mV and TNF-

30 batch containing Eudragit-RL 100 polymer‟s has a zeta potential +47.7 +

1.34 mV, TNF-31 batch containing Eudragit-RS 100 polymer‟s zeta potential is

established at + 30.9 + 1.45 mV. TNF-25 negative charge is due to surfactants

adhering to the nanoparticle surface covering the carboxylic groups in the

polymer and presence of terminal carboxylic group in PLGA 50:50 polymer.

Eudragit polymer formulations have a very high value of zeta potential which is

well above the specified value of + 30 mV. This value of zeta potential

indicates the stability of formulation.

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Figure 26: Zeta Potential of TNF-25 Tenofovir + PLGA (50:50) Polymer

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Figure 27: Zeta Potential of TNF-30 Tenofovir + Eudragit-RL 100 Polymer

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Figure 28: Zeta Potential of TNF-31 Tenofovir + Eudragit-RS 100 Polymer

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6.5.5 Field Emission Scanning Electron Microscope

The surface morphology of optimised formulations was studied and the SEM

images indicate that the nanoparticles were spherical and no agglomeration of

particles is seen which supports the zeta potential data. The particles are

spherical in nature and seen against Mannitol in background.

Figure 29: SEM image of TNF-25 Tenofovir + PLGA (50:50) Polymer

Figure 30: SEM image of TNF-30 Tenofovir + Eudragit-RL 100 Polymer

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Figure 31: SEM image of TNF-31 Tenofovir + Eudragit-RS 100 Polymer

6.5.6 Fourier Transform Infrared (FT-IR) Spectroscopy

FT-IR and DSC analytical techniques give us the complete picture of drug

polymer interactions and also the influence it has on nanoparticles. Tenofovir

disproxyl fumarate displays the aliphatic CH stretching at 2985 cm-1

and

aromatic CH stretching at 3051 cm-1

. C=O stretching is spotted at 1759 cm-1

.

The predominant group of NH stretching is seen at 3227 cm-1

and 3271 cm-

1.C=O stretching is also seen at 1269 cm

-1. The major group (NH) has not

interacted with polymer indicating the stability of the nano formulation as

evident in the spectrum.

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Figure 32: TNF-25 Tenofovir Drug + Formulation + PLGA (50:50) Polymer

Figure 33: TNF-30 Tenofovir Drug + Formulation + Eudragit RL 100

Polymer

75015002250300037501/cm

0

25

50

75

100

%T

3227.02

3099.71 305

1.49298

5.91293

5.76

2748.65

2683.07

2532.62 247

6.68

1759.14 168

1.98162

6.05

1506.46

1467.88

1421.58

1381.08

1269.20

1180.47 115

5.40110

1.39103

3.88987

.59 950.94

895.00

829.42 788

.91729

.12 698.25

653.89

599.88

567.09

478.36

TNF

75015002250300037501/cm

0

25

50

75

%T

3282.9

5327

3.31

2989.7

6293

5.76

2767.9

4 2677.2

9

2359.0

2 2332.0

2

1732.1

3

1458.2

3137

5.29

1332.8

6 1251.8

4118

8.19

1151.5

4108

7.89

1022.3

1966

.37931

.65881

.50

715.61

628.81

509.22

TNF-28

75015002250300037501/cm

60

70

80

90

%T

3591.57

3554.93

3510.56

3437.26 338

1.33

2359.02

2332.02

1735.99

1726.35

1637.62

1446.66 138

4.94

1274.99

1242.20

1151.54

1024.24

989.52 958

.65850

.64

EUDRAGIT

75015002250300037501/cm

0

25

50

75

100

%T

3227.02

3099.71 305

1.49298

5.91293

5.76

2748.65

2683.07

2532.62 247

6.68

1759.14 168

1.98162

6.05

1506.46

1467.88

1421.58

1381.08

1269.20

1180.47 115

5.40110

1.39103

3.88987

.59 950.94

895.00

829.42 788

.91729

.12 698.25

653.89

599.88

567.09

478.36

TNF

75015002250300037501/cm

25

50

75

%T

3271.38

2933.83

2875.96

2766.01

2359.02 233

2.02

1755.28

1637.62

1458.23

1371.43

1330.93

1255.70 119

7.83

1089.82

1022.31

968.30

931.65

881.50

717.54 682

.82 628.81

511.15

TNF-25

75015002250300037501/cm

0

25

50

75

100

%T

3936.84 3880.91

3863.55 3850.04 3811.47

3797.96

3730.45

3649.44

3516.35

3230.87

2999.41

2955.04

2887.53

2748.65

2661.85

2613.63 2546.12

2503.69

2409.17

2357.09

2258.72

2114.05

2079.33

1757.21

1620.26

1454.38

1429.30

1392.65

1278.85

1174.69 112

6.47108

9.82

956.72 862

.21

750.33 711

.76

570.95

PLGA

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Figure 34: TNF-31 Tenofovir Drug + Formulation + Eudragit RS 100

Polymer

Figure 35: TNF-25 Tenofovir Drug + Formulation + PLGA (50:50)

Polymer

100.00 200.00 300.00

Temp [C]

-10.00

0.00

10.00

mW

DSC

117.69 x100C

157.21 x100C

168.58 x100C

45.38 x100C

TNF + TNF 25+PLGA

75015002250300037501/cm

0

25

50

75

100

%T

3227.0

2

3099.7

1305

1.49

2985.9

1 2935.7

6

2748.6

5268

3.07

2532.6

2247

6.68

1759.1

4 1681.9

8162

6.05

1506.4

6 1467.8

8142

1.58

1381.0

8126

9.20

1180.4

7115

5.40

1101.3

9103

3.88

987.59 950

.94895

.00829

.42 788.91

729.12 698

.25653

.89599

.88567

.09478

.36

TNF

75015002250300037501/cm

60

75

90

%T

3585.7

9354

9.14

3437.2

6339

0.97

2987.8

4295

1.19

2359.0

2 2330.0

9

1734.0

6

1637.6

2

1479.4

5144

6.66

1386.8

6

1271.1

3124

2.20

1149.6

1

1026.1

6987

.59

848.71 756

.12

671.25

480.29

EUDRAGIT RS

75015002250300037501/cm

0

25

50

75

%T

3282.95

3273.31

2989.76

2935.76

2767.94 267

7.29

2359.02 233

2.02

1732.13

1458.23

1375.29

1332.86 125

1.84118

8.19115

1.54108

7.89 1022.31

966.37

931.65

881.50

715.61

628.81

509.22

TNF-28

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Figure 36: TNF-30 Tenofovir Drug + Formulation + Eudragit RL 100

Polymer

100.00 200.00 300.00

Temp [C]

-30.00

-20.00

-10.00

0.00

10.00

mW

DSC

117.69 x100C

157.29 x100C

169.49 x100C

TNF + TNF 30 + RS 100

Figure 37: TNF-31 Tenofovir Drug + Formulation + Eudragit RS 100

Polymer

100.00 200.00 300.00

Temp [C]

-20.00

-10.00

0.00

10.00

mW

DSC

117.69 x100C

157.84 x100C

168.25 x100C

TNF + TNF 31 + RL 100

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DSC is useful in the investigation of solid-state interactions. Thermograms were

generated for pure drug and different formulations. The thermal curve of

tenofovir showed an endothermic peak at 117.69°c corresponding to its melting

point/transition temperature displaying crystalline nature of the pure compound.

In the freeze dried nanoparticles the peak of tenofovir was disappeared

indicating possibility of conversion of tenofovir in to amorphous form. The

drug molecules might have entirely dispersed in the polymer matrix. As per

literature, decrease in crystallinity and amorphization of the drug exhibits more

solubility (Usha et al., 2008; Mutalik et al., 2008). However appearance of the

endothermic peaks at 168-169°c is due to the presence of cryoprotectant,

mannitol. This was confirmed by the native thermogram of mannitol.

6.5.7 Invitro Release Study

Invitro release studies give an insight about the formulation behaviour invivo.

The release pattern shows that the release is erosion kind of release. TNF-25

(PLGA 50:50 polymer) has faster release than TNF-30 & 31 (Eudragit RL 100

& RS 100). Tenofovir has a very high solubility in water (13.46 mg/ml), and it

goes into the solution at a rapid rate. In TNF-25 the drug release rate was high

from initial time point and at 60 minutes it was 85% + 2.7. TNF 30 the drug

release rate was 64% + 3.42 .TNF 31 batch the drug release rate was 59% +

3.29. This is due to the affinity of drug to the polymer which was due to

hydrogen bonding by van der Waals forces between the polymer and drug.

However it was observed that at TNF-25 drug released was 101% + 1.97 in four

hours and the concentration remained constant throughout indicating its

complete release from the polymer. In case of TNF 30 batches the drug release

was 100% + 1.48 in 8 hours more than double the time taken for PLGA

polymer formulation. TNF-31 batch drug release was 100% + 1.89 in 12 hours.

All the batches were tested for 24 hours and more and found out the drug

release was constant after 16 hours.

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Figure 38: Invitro drug release profile of TNF-25 (PLGA 50:50) polymer

Figure 39: Invitro drug release profile of TNF-30 (Eudragit RS 100)

polymer

Figure 40: Invitro drug release profile of TNF-30 (Eudragit RS 100)

polymer

0

20

40

60

80

100

120

0 500 1000 1500

D

r

u

g

R

e

l

a

s

e

Time in Minutes

0

20

40

60

80

100

120

0 500 1000 1500

D

r

u

g

R

e

l

e

a

s

e

Time in Minutes

0

20

40

60

80

100

120

0 500 1000 1500

D

r

u

g

R

e

l

a

s

e

Time in Minutes

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6.0 Preparation and Evaluation of Tenofovir nanoparticles

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6.5.8 Release Kinetics

The non curvilinear shape of the graph shows that the optimised batches have

followed Higuchi release kinetics. The obtained data was also tried out in other

mathematical models. The data suggest that drug entrapped outside the nano

particle released immediately followed by polymer breaking down and

releasing the entrapped drug.

Table 19: Release Kinetics data of optimised formulation

Nano particle Batch TNF-25 TNF-30 TNF-31

Zero order (R2) 0.2359 0.5241 0.7400

First order (R2) 0.7879 0.6280 0.6656

Higuchi model (R2) 0.8035 0.8713 0.8635

Krosmeyers-Peppas (n) 0.7951 0.7835 0.7889

Model fit Higuchi Higuchi Higuchi

6.5.9 Stability Studies

Stability studies were conducted as per the ICH guidelines. The results of

optimised nano formulation of TNF-25, TNF-30 and TNF-31 are performed at

accelerated stability conditions 400 + 2

0c / 75% + 5% RH and the results are

given in the following table no 21,22&23. Nano formulations of the above

batches did not show any agglomeration or flocculation of dry powder. The

percentage encapsulation efficiency was reduced in all the formulations with

time. Particle size was constant but slight fluctuation of zeta potential in TNF-

25 batch. Poly dispersity index was well within 0.5 value so the optimised

formulation was homogenous. In TNF-30 batch there was fluctuation in particle

size but it did not cross more than 400 nm. Zeta potential was well outside the

+30mV value which indicates the stability of formulation. Poly dispersity index

was well within the limit but showed a slight increase in 06 months data. In

TNF-31 batch, there was steady increase in particle size but till the last 06

months it has not crossed 400 nm which is good for oral formulation. Poly

dispersity index was also well within the limit with one value going above 0.5.

Zeta potential was outside+30mV but in 06 months the zeta potential has come

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down to + 19mv. which was a good value which indicates the stability of

formulation.

Table 20: Stability data of TNF-25 Tenofovir + PLGA (50:50) Polymer at

400 + 2

0c / 75% + 5% RH (n = 3)

Time

(Months)

% Encapsulation

Efficiency

Particle

Size (nm)

PDI Zeta

Potential

0.0 13.45 231.0 0.348 -08.46

0.5 13.44 224.2 0.120 -10.62

01 13.40 226.2 0.143 -23.60

02 13.39 250.8 0.117 -21.30

03 13.24 265.1 0.239 -18.20

06 13.19 267.9 0.244 -22.80

Figure 41: Log % Drug remaining against time plot of TNF-25 at 400 + 2

0c

/ 75% + 5% RH

Table 21: Stability data of TNF-30 Tenofovir + Eudragit-RL 100 Polymer

at 400 + 2

0c / 75% + 5% RH (n = 3)

y = -0.0015x + 2 R² = 0.8903

1.99

1.992

1.994

1.996

1.998

2

2.002

0 2 4 6 8

L

o

g

%

R

e

m

a

i

n

i

n

g

Time in Months

Time

(Months)

% Encapsulation

Efficiency

Particle

Size (nm)

PDI Zeta

Potential

0.0 11.55 325 0.351 +47.70

0.5 11.52 284 0.314 +51.40

01 11.48 290 0.252 +49.46

02 11.47 275 0.396 +37.00

03 11.36 291 0.418 +34.50

06 11.35 371 0.613 +19.50

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Figure 42: Log % Drug remaining against time plot of TNF-30 at 400 + 2

0c

/ 75% + 5% RH

Table 22: Stability data of TNF-31 Tenofovir + Eudragit-RS 100 Polymer

at 400 + 2

0c / 75% + 5% RH (n = 3)

Time

(Months)

% Encapsulation

Efficiency

Particle

Size (nm)

PDI Zeta

Potential

0.0 11.22 187 0.209 +30.90

0.5 11.22 241 0.130 +38.40

01 11.18 290 0.316 +49.40

02 11.10 291 0.418 +34.50

03 11.09 333 0.352 +40.50

06 11.05 371 0.613 +19.50

Figure 43: Log % Drug remaining against time plot of TNF-31 at 400 + 2

0c

/ 75% + 5% RH

y = -0.0013x + 1.9991 R² = 0.8287

1.99

1.992

1.994

1.996

1.998

2

2.002

0 2 4 6 8

L

o

g

%

R

e

m

a

i

n

i

n

g

Time in Months

y = -0.0012x + 1.9995 R² = 0.8345

1.992

1.994

1.996

1.998

2

2.002

0 2 4 6 8

L

o

g

%

R

e

m

a

i

n

i

n

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Time in Months