---- .LIBRARY -INTERNATIONAL tSLAMIC UNIVERSITY MALAYSIA

MIC.ROENCAPSULATION AND CHARACTERIZATION OF GENTAMICIN-PLGA

MICROSPHERE INTENDED FOR ORTHOPAEDIC INFECTION

BY

AHMAD FAHMI BIN HARUN @ ISMAIL

A dissertation in fulfilment of the requirement for the degree of Master in Pharmaceutical Science

(Pharmaceutical Technology)

Kulliyyah of Pharmacy International Islamic University

Malaysia

AUGUST2012

ABSTRACT

The study was done for the purpose of developing biodegradable gentamicin-loaded

microspheres fabricated using poly(D.L-lactic-co-glycolic acid) (PLGA). The

microspheres were fabricated by manipulating several variables i.e. molecular weight

of PLGA, types of surfactant/emulsifier, different concentrations of polyvinyl alcohol

(PVA) as well as the oil phase and different HLB values of surfactants in order to

achieve the best formulation for W /0/W emulsion during the fabrication process.

Antibiotic treatment of orthopaedic infection is complicated by systemic toxicity and

the need of effective therapeutic concentration necessary to ensure optimum killing of

bacteria. To overcome the problem of systemic toxicity and to achieve a high initial

release followed by sustained release of antibiotics, a new method of delivering

gentamicin is attempted by encapsulating gentamicin into PLGA using multiple

emulsion, solvent-evaporation method. Gentamicin was first extracted from the

microspheres and quantified using Ninhydrin assay before the concentration was

measured using UV spectrophotometer. Gentamicin loading after encapsulation was

preserved when CTAB (83.51 ± 1.42%) and low molecular weight (LMW) PLGA

(82.38 ± 9.08%) were used as indicated by drug loading of more than 80% in the disc­

diffusion assay. LMW PLGA enabled high burst release (-90%) of gentamicin within

the first 10 hours corresponding to zone inhibition of 13.78 ± 0.86 mm, only 30%

smaller than the positive control (10 mg/ml gentamicin). The effects of Tg and

molecular weight rather than surfactant types influence the initial burst release. The in

vitro release profile suggests that by having a mixture of various PLGA microspheres

in one dosage implant system, the high burst release can be sustained within

therapeutic concentration for a prolonged period (> 1 months). This biodegradable

delivery system does not entail another surgery to remove the implant hence reducing

the high treatment cost usually associated with the non-bidegradable proprietary

gentamicin-polymethyl-methacrylate (PMMA) beads currently in use.

11

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04 J:!Al4 ~I~ 04 ~I~~ ~W:,..JI u1:bi,,Loll ~~ ul~..9_;S;-JI

''F~'il J.oWl/~I <>- ~WI J.,WI e.~ ,~;.Jl ~~I wJJll ~.J ,ul~I

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.JI t'l~4 4t.l,.o.o.ll ul~..9.~ 14'i~hi ~ 0/o80CTAB ~I c>, ~\.9 J.ola.S

~ .u.,:illJ 14000 ~~' wJJll (jj ~.J-='31 t'1~4 4t.l..o.o.11 ul~..9_;S;-JI ~i.J

.lb_,I J!..9 o/020 ~ J:!j:i ~ ~ld ~ ~I ul~.J~ _;~I [ j~ _;~I

· t-..1.:. u .. Ji · 1.A~I • o/ 80 • i ~ • I.Al.:wJJ .Jj · • t1 ·~1 ·- -:t1 ~ _)~ ~ • (.)A /0 U .. ~ • ~-..>- '-r • _)J-1"'

u.,:illJ 14000 ~.J-='31 t'l~4 o~I ul~..9.A!,,oll 04 c)..9~1 ~\.wJI PLGA ~ •

.. I ui...... .( ··'I • ul • .. · · ·· • .:. t La 30 .. • 1.Al.:wJJ J.a.\.o.11 ·- -:t1 · t ·- · .. -: '-,I ..n--.JJ'*:l"'""' (.)A ~ u-- ~ ~ ~ . .. _)J-1"' ~

t'l~4 4t.l..o.o.11CTAB .J TRITON X-100 °/06.J 0/o5 ,~I c>, ~tj J.ol.Jl,S

..1l J:!SJi c)! U~} ~jll ~I J:!Sljll ~/6J.JPVA ul~.J~ .~/6J.J 0/ol

.JIPLGA lAJJ~.J lf:l,..,al_;J c) ~jill ~ ~ o~ ~ti.a! u~i ~I.A~4 Uu.oll

.9'1..9.lll ~~I 9'U:i~I ~ J'+4 c) _;~~I¥ 0$.l..9 ~JAS

11l

ABSTRAK

Kajian itu dilakukan b&gi tujuan membangunkan gentamicin yang dimuatkan ke

dalam mikrosfera terbiodegradasi menggunakan poli (D.L-laktik-co Glycolik asid)

(PLGA). Mikrosfera telah direka oleh memanipulasi beberapa pembolehubah iaitu

berat molekul PLGA, jenis surfaktan / pengemulsi yang berbeza kepekatan polivinil

alkohol (PVA) serta fasa minyak dan nilai-nilai yang berbeza HLB surfaktan untuk

mencapai rumusan terbaik untuk W / 0 / W emulsi ketika proses fabrikasi. Rawatan

ortopedik bagi jangkitan antibiotik rumit oleh ketoksikan sistemik dan memerlukan

kepekatan terapeutik yang berkesan diperlukan untuk memastikan pembasmian

bacteria yang optimum. Untuk mengatasi masalah ketoksikan sistemik dan untuk

mencapai kadar antibiotik awal yang tinggi yang diikuti oleh kadar antibiotic yang

berterusan, satu kaedah baru telah digunakan dengan memberikan gentamicin

menggunakan emulsi berganda, pelarut-kaedah penyejatan. Gentamicin diekstrak dari

mikrosfera dan dengan menggunakan ninhydrin kepekatan gentamicin diukur

menggunakan spektrofotometer UV. Muatan gentamicin selepas pengkapsulan telah

dikekalkan apabila CTAB (83,51 ± 1.42%) dan berat molekul yang rendah (LMW)

PLGA (82,38 ± 9,08%) telah digunakan seperti yang ditunjukkan oleh jumlah

antibiotik lebih daripada 80% dalam cerakin cakera-resapan. LMW PLGA

membolehkan pembebasan gentamicin yang tinggi (- 90%) dalam tempoh 10 jam

pertama sepadan dengan zon perencatan daripada 13, 78 ± 0,86 mm, hanya 30% lebih

kecil daripada kawalan positif (10 mg / ml gentamicin). Kesan Tg dan berat molekul

dan bukannya jenis surfaktan mempengaruhi pelepasan pecah awal. Dalam profil

pelepasan vitro mencadangkan bahawa dengan mempunyai campuran pelbagai PLGA

mikrosfera dalam satu sistem dos implan, pelepasan pecah yang tinggi boleh

dikekalkan dalam kepekatan terapeutik untuk tempoh yang lama(> 1 bulan). Sistem

penyampaian terbiodegradasi ini tidak melibatkan pembedahan untuk membuang

implan seterusnya mengurangkan kos rawatan yang tinggi yang biasanya dikaitkan

dengan mamc bukan-biodegradasi proprietari gentamicin-polimetil-metakrilat

(PMMA) yang kini dalam penggunaan di hospital-hospital.

iv

APPROVAL PAGE

I certify that I have supervised and read this study and that in my opinion, it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a thesis for the degree of Master in Pharmaceutical Science

(Pharmaceutical Technology). . ... ~~ ............................... . F~4~d~h

1Mohamed

Supervisor

I certify that I have read this thesis and that in my opinion, it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a thesis for the degree of Master in Pharmaceutical Science (Pharmaceutical

Technology). . .... k ........................................ . Kausar Ahmad Internal Examiner

Wong Tin Wui External Examiner

This dissertation was submitted to the Department of Pharmaceutical Technology and is accepted as a partial fulfilment of the requirements for the degree of Master in Pharmaceutical Science (Pharmaceutical Technology) .

. . .. ·:;;;i;;;;;~ Md.j:ii~ ...................... . Head Department of Pharmaceutical Technology

This dissertation was submitted to the Kulliyah of Pharmacy and is accepted as a partial fulfilment of the requirements for the degree aster in Pharmaceutical Science (Pharmaceutical Technology).

Dean, Kulliyyah of Pharmacy

V

DECLARATION PAGE

I hereby declare that this dissertation is the result of my own investigations, except

where otherwise stated. I also declare that it has not been previously or concurrently

submitted as a whole for any other degrees at IIUM or other institutions.

Ahmad Fahrni Bin Harun@Ismail

Signature ......................................... . Date ......................... .

VI

INTERNATIONAL ISLAMIC UNIVERSITY MALAYSIA

DECLARATION OF COPYRIGHT AND AFFIRMATION OF FAIR USE OF UNPUBLISHED RESEARCH

Copyright ©2011 by International Islamic University Malaysia. All rights reserved.

MICROENCAPSULATION AND CHARACTERIZATION OF GENTAMICIN-PLGA MICROSPHERE INTENDED FOR ORTHOPAEDIC

INFECTION

I hereby affirm that The International Islamic University Malaysia (IIUM) hold all

rights in the copyright of this Work and henceforth any reproduction or use in any

form or by means whatsoever is prohibited without the written consent of IIUM. No

part of this unpublished research may be reproduced, stored in a retrieval system, or

transmitted, in any form or by means, electronic, mechanical, photocopying,

recording or otherwise without prior written permission of the copyright holder.

Affirmed by Ahmad Fahrni Bin Harun @ Ismail

Signature Date

Vll

.. To My Beloved Wife and Daughter .. .. The Apple of My Eye ..

Vlll

ACKNOWLEDGEMENTS

Alhamdulillah. As all praises are belong to Almighty God. Peace and blessing be upon our beloved Prophet SAW. First and foremost, I would like to thank to ALLAH as by HIS permission and will, I have completed my research and this thesis successfully. It was indeed a great experience to complete such an important task in order for me to graduate. May ALLAH provide benefits from this research to those who seek knowledge and make this research as a reason for others to increase their Iman towards HIM.

Here I would like to thank my parents, Hj. Harun @ Ismail bin Yusof and Hjh. Juwariah bt Mustafa and also all my siblings for supporting me during my studies here and also for giving me many ideas on how to improve my research. I appreciated all the ideas and suggestions.

And also a very big thank to my supervisor, Asst. Prof. Dr. Farahidah Mohamed for helping me and giving me guidance throughout the research and giving me endless advises in completing this thesis.

I would like to dedicate this thesis to my beloved wife, Nurul Aziyana Kamaruddin for her supports and for being with me through thick and thin, looked after our lovely daughter Nurhusna when I had to do my lab work. May ALLAH shower us with happiness and blessing till the end. InsyaALLAH.

Next, I want to express my gratitude to all lab assistants that were involved in this research especially Sis. Haryanti, Bro. Dzadil, and our science officer of Pharmaceutical Technology Department, Sis. Zaililah for their assistances during the lab works.

Not to forget, the important persons throughout the experiment, my project colleagues; AbdAlmonem, Aimen and Mulham. They had been helping me a lot during the research and providing me with many ideas to make our research into reality. Special gratitude also goes to Bro. Saadi for being my SPSS expert, and also to my good friend, Usop, for all those lame jokes when I needed one. Thank you.

To all my friends, Bro. Suhaib, Bro. Anung, Pak Dedi, Bro. Firdaus, Bro. Muhammad and Bro. Abdurrahman, I thank all of you and may ALLAH repay you with HIS blessing and HIS mercy, here and hereafter. JnsyaALLAH

Thank you, Wassalam.

IX

TABLE OF CONTENTS

Abstract ......................................................................................................................... ii Abstract in Arabic .............................................................................. iii Abstract in Malay ........................................................................................................ iv Approval Page .............................................................................................................. v Declaration Page .......................................................................................................... vi Copyright Page ........................................................................................................... vii Dedication ................................................................................................................. viii Acknowledgements ............................................................................. .ix List of Tables .................................................................................. xiii List of Figures ............................................................................................................ xvi List of Abbreviations ................................................................................................. xix

CHAPTER 1: .............................. ............................................. .......... 1 1.1 Introduction .............................................................................................. 1 1 .2 Research Background .............................................................................. 2 1.3 Literature Review .................................................................................... 6

1.3.1 Poly (D,L-Lactic-Co-Glycolic Acid) (PLGA) ............................... 6 1.3.1.1 The Overview ............................................................... 6 1.3.1.2 Glass Transition Temperature (T g) ............................. l 0 1.3.1.3 Related Work .............................................................. 12

1.3.2 Gentamicin ............................... '. ................................................... 14 1.3.2.1 An Overview .............................................................. 14 1.3.2.2 Gentamicin Toxicity ................................................... 15 1.3.2.3 Pharmacokinetic ......................................................... 17 1.3.2.4 PMMA Beads ............................................................. 18

1.3.3 Double Emulsion Solvent Evaporation Method of Microsphere Fabrication ................................................................................... 20

1.3 .3 .1 The Overview ............................................................. 20 1.3.3.2 Surfactants .................................................................. 2 I 1.3 .3 .3 Chitosan Microspheres ............................................... 24 1.3.3.4 Drug Release Mechanism ........................................... 25

1.4 Objectives and Scope of Study .............................................................. 29 1.5 Hypotheses ............................................................................................. 30

CHAPTER 2: DEVELOPMENT OF GENTAMICIN-LOADED PLGA MICROSPEHERES ................................................................................................. 31

2.1 Introduction ............................................................................................ 31 2.2 Materials and Chemicals ........................................................................ 32 2.3 Methodology .......................................................................................... 33

2.3.1 Fabrication OfGentamicin-Loaded Microspheres ...................... 33 2.4 Characterization of Microspheres .......................................................... 41

2.4.1 Particle Morphology .................................................................... 41

X

2.4.2 Particle Size Analysis ................................................................. .41 2.4.3 Drug Loading ............................................................................... 42 2.4.4 Gentamicin Stock Preparation .................................................... .43 2.4.5 Construction Of Gentamicin Standard Curve ............................. .44 2.4.6 Ninhydrin Assay .......................................................................... 44 2.4.7 HLB Preparation .......................................................................... 45 2.4.8 Statistical Analysis ....................................................................... 46

2.5 Result and Discussion ............................................................................. 47 2.5.1 Particle Size Distribution, Morphology and Drug Loading

Efficiency ..................................................................................... 4 7 2.5.1.1 Surfactant Study ........................................................ .47 2.5.1.2 HLB Value ................................................................. 55 2.5.1.3 Molecular Weight of PLGA ....................................... 60 2.5 .1.4 PV A Concentration .................................................... 63 2.5.1.5 PLGA Concentration in Oil Phase ............................. 68

CHAPTER 3: IN-VITRO RELEASE AND ANTIMICROBIAL STUDY .......... 74 3.1 Introduction ............................................................................................ 74 3 .2 Material .................................................................................................. 7 5 3.3 Method ................................................................................................... 75

3.3.1 Fabrication ofMicrospheres ........................................................ 75 3.3.2 In-Vitro Release Profile ............................................................... 76 3.3.3 Antimicrobial Study ..................................................................... 77

3.3.3.1 Nutrient Agar Preparation .......................................... 77 3.3.3.2 Nutrient Broth Preparation ......................................... 77 3.3.3.3 Preparation of Bacterial Culture ................................. 77 3.3.3.4 McFarland Standard for Bacterial Culture ................. 78 3.3.3.5 Extraction of Gentamicin From Microspheres ........... 78 3.3.3.6 Disk Diffusion Assay ................................................. 79

3.4 Result & Discussion .............................................................................. 80 3.4.1 In-Vitro Release Profile ............................................................... 80 3 .4.2 Antimicrobial Study ..................................................................... 86

CHAPTER 4: GENTAMICIN-LOADED CHITOSAN/PLGA MICROSPHERES .................................................................................................................................... 91

4.1 Introduction ............................................................................................ 91 4 .2 Material ......................................................................... , ........................ 92 4.3 Methodology .......................................................................................... 93

4.3.1 Fabrication of Gentamicin-Loaded Chitosan/PLGA Microspheres ..................................................................................................... 93

4.3.2 Microspheres Characterization .................................................... 95 4.3.2.1 Surface Morphology ................................................... 95 4.3.2.2 Particle Size Distribution ........................................... 95 4.3.2.3 Drug Loading Efficiency ............................................ 95

4.4 Results & Discussion ............................................................................. 96 4.4.1 Morphology ................................................................................. 96 4.4.2 Particle Size ................................................................................. 96 4.4.3 Drug Loading ............................................................................... 98

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CHAPTER 5: GLASS TRANSITION OF PLGA MICROSPHERES .............. 102 5 .1 Introduction .......................................................................................... 102 5.2 Material ................................................................................................ 103 5.3 Method ................................................................................................. 103 5.4 Results & Discussion ........................................................................... 104

5.4.1 Effect of PLGA Molecular Weight on Tg .................................. 106 5.4.2 Effect of Surfactant Types on Tg of the Gentamicin-Loaded

Microspheres .............................................................................. 108 5.4.3 Effect of Different PVA Concentrations Used In Aqueous 2 and

Hardening Tank on Tg···· ............................................................ 110 5.4.4 Effect of Concentration of PLGA in Oil Phase on Tg ................ 112 5.4.5 Effect ofHLB Values on Tg .................................... : .................. 113

CHAPTER 6: GENERAL DISCUSSION ............................................................ 115

CHAPTER 7: CONCLUSION .............................................................. 122 7.1 Future Study ......................................................................................... 122

BIBLIOGRAPHY ............................................................................. 124

PUBLICATIONS AND PRESENTATIONS ............................................ 135

APPENDIX A .................................................................................... 136

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Table No.

1.1

2.1

2.2

2.3

2.4

2.5

2.6

2.7

2.8

2.9

LIST OF TABLES

Decreasing order of nephrotoxic aminoglycosides used systemically. 1 is the highly toxic, 5 is the least toxic (Decker & Molitoris, 2008).

Composition of microsphere fabrication process for the various types of surfactant.

Composition of microspheres fabrication process for different concentration of PLGA.

Composition of microsphere fabrication process with different HLB values of surfactants used.

Composition of microsphere fabrication process to study the effect of molecular weight of PLGA employed to synthesize the microspheres.

Composition of microsphere fabrication process with different concentrations of PV A for aqueous 2 and hardening tank.

The HLB value for the nonionic surfactants used provided by the (Manual Safety Data Sheet) MSDS from the supplier.

Particle size distribution of gentamicin-loaded microspheres synthesized with selected surfactants. Data were expressed as mean± standard deviation (S.D); n=3.

Particle size distribution of gentamicin-loaded microspheres synthesized with selected HLB values of surfactant blends. Data are expressed as the mean ± standard deviation (S.D); n=3.

Particle size distribution of gentamicin-loaded microspheres synthesized with selected molecular weight of PLGA. Data are expressed as the mean ± standard deviation (S.D); n=3 and analyzed by ANOV A Tukey multiple comparison test.

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Page No.

16

35

36

38

39

40

45

47

57

61

Table No.

2.10

2.11

3.1

4.1

4.2

5.1

5.2

5.3

5.4

Particle size distribution of gentamicin-loaded microspheres with selected PV A concentration for aqueous 2 and hardening tank. Data are expressed as the mean± standard _deviation (S.D); n=3.

Particle size distribution of gentamicin-loaded microspheres synthesized with selected PLGA concentration. Data are expressed as the mean ± standard deviation (S.D); n=3

The ingredients of four formulations out of six formulations of gentamicin-loaded PLGA microspheres that achieved more than 20% drug loading efficiency and more. I% w/v aqueous PV A were employed in aqueous 2 and in hardening tank for all formulations.

The chitosan concentration used m formulation of gentamicin-loaded chitosan/PLGA microspheres.

Particle size distribution of gentamicin-loaded microspheres synthesized with selected chitosan concentration. Data expressed as the mean ± standard deviation (S.D); n=3.

The onset, midpoint and endpoint of glass transition for the gentamicin-loaded microspheres fabricated using different PLGA molecular weight. The data tabulated was collected post quench cool.

The onset, midpoint and endpoint of glass transition for the gentamicin-loaded microspheres (100 kDa PLGA) fabricated using different types of surfactant. The data tabulated was collected post quench-cooled.

The onset, midpoint and endpoint of glass transition for the gentamicin-loaded microspheres (100 kDa PLGA) fabricated using different concentrations of PV A m Aquoues 2 and hardening tank. The data tabulated was collected post quench cool.

The onset, midpoint and endpoint of glass transition for the gentamicin-loaded microspheres (100 kDa PLGA) fabricated using different concentrations of oil phase. The data tabulated was collected post quench cool

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Page No.

65

70

75

94

97

106

108

111

112

Table No.

5.5 The onset, midpoint and endpoint of glass transition for the gentamicin-loaded microspheres (100 kDa PLGA) fabricated using different concentrations of oil phase. The data tabulated was collected post quench cool.

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Page No.

113

Figure No.

1.1

1.2

1.3

2.1

2.2

2.3

2.4

2.5

LIST OF FIGURES

Examples of hydrolytic biodegradable polymers (Hoffman, 2006).

(a) Formation of PLGA polymer from lactic acid and glycolic acid and (b) degradation of PLGA chain into its monomers via hydrolysis (Bouissou & Walle, 2006).

The schematic diagram drawn shows how the drug released from the microenvironment of small particle (S. Yang & Washington, 2006).

Schematic diagram to prepare gentam1cm PLGA Microspheres. Oil Phase (#), Aqueous I (€), Primary Emulsion (Q) and Secondary Emulsion (~).

Surfaces morphologies of the microspheres fabricated using PV A in the secondary emulsion and the following surfactants in the primary emulsion: (A) PVA; (B) Tween 20; (C) Tween 80; (D) Span 80; (E) Span 85; (F) SDS; (G) CTAB and (H) Triton X-100.

The bar chart illustrates the data for drug loading efficiency based on types of surfactant used. The group marked with asterisks showed significantly higher with 95% confidence level (P<0.05) when compared with the control group (PV A).

Surfaces morphologies of the microspheres fabricated using PV A in the secondary emulsion and surfactant blends with the following HLBs in the primary emulsion: (A) HLB 4; (B) HLB 10; (C) HLB 13.5 and (D) HLB 16.

Drug loading of gentamicin microspheres with difference HLB of surfactant. All formulations showed statistically no significant different compared to control (PV A). The HLB consisted of the surfactant blends mentioned in Table 2.7.

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Page No.

7

8

26

34

50

52

56

58

Figure No.

2.6

2.7

2.8

2.9

2.10

2.11

2.12

Surfaces morphologies of the microspheres fabricated using Tween 80 in primary emulsion, PV A in the secondary emulsion and the following PLGA molecular weight in the primary emulsion: (A) PLGA 14 kDa; (B) PLGA 34 kDa and (C) PLGA 100 kDa.

Drug loading of gentamicin in microspheres fabricated based on different molecular weight of PLGA. Data was analyzed using multiple comparison Tukey Test.

Surfaces morphologies of the microspheres fabricated using the following PV A concentration for Aquoues 2 and hardening tank: (A) 1 % w/v PV A; (B) 3% w/v PV A and (C) 5% w/v PV A. All microspheres were fabricated employing PLGA 100 kDa, Triton X-100 as surfactant in primary emulsion, 1 % PV A in aqueous 2 and in the hardening tank

Drug loading of gentamicin in microspheres fabricated based on different concentrations of PV A. Asterisk indicates the drug loading is significantly higher than control (PVA) based on One-way ANOVA (Dunnett's Test).

Surfaces morphologies of the microspheres fabricated using PV A in the secondary emulsion and the following oil phase concentrations in the primary emulsion: (A) 5% w/v; (B) 10% w/v and (C) 20% w/v

Drug loading of gentamicin in microspheres fabricated based on different concentrations of PLGA in oil phase. Asterisks showed drug loading significantly higher than the control group (microspheres fabricated with PV A).

The bar chart illustrates the 6 formulations of gentamicin microspheres with more than 20% drug loading efficiency and a control group. The figures were the average taken from triplicate measurements (n=3). All 6 formulations showed significantly higher drug loading efficiency (marked with asterisk) with 95% confidence level (P<0.05) when compared against microspheres fabricated using PV A as surfactant in the primary emulsion ( control group)

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Page No.

61

62

64

66

68

71

73

Figure No .:.

3.1

3.2

3.3

3.4

4.1

4.2

4.3

5.1

Configuration of the four points (1 - 4) for measurement of the diameters of inhibition zone. The centre point (C) corresponds to the paper disc centre.

The cumulative data (%) for the release profile of gentamicin microspheres plotted as mean values with error bar; n=3.

The zone of inhibition of microspheres fabricated using (A) Fl formulation, (B) F2 formulation, (C) F3 formulation, (D) F4 formulation, (E) naked gentamicin at 10 mg/ml and (F) positive control marked with ([+ve] ctrl) and negative control marked with ([-ve] ctrl) and PBS.

The average zone of inhibition (mm); n=3, based on the four formulations (Fl-F4) and 'naked' gentamicin (10 mg/ml) as positive control. The negative control was not included as the value for zone of inhibition was zero. Data for four formulations were analyzed using multiple comparison Tukey test.

The SEM images of gentamicin microspheres fabricated by using three different concentrations of chitosan added in aqueous 2 and hardening tank labeled with (A) Formulation 1, (B) Formulation 2 and (C) Formulation 3.

The bar charts represent mean data for drug loading with error bar for triplicate samples of gentamicin microspheres for chitosan concentration study. One-way ANOVA (Dunnett's Test) showed significantly higher (P<0.05) with 95% confidence level (marked with asterisks) compared with control group.

The chemical structure of (A) acetylated chitin and (B) chitosan (Aranaz et al., 2009).

Simplified schematic diagram illustrates the process of determining glass transition temperature (Tg). The quench cool step marked with *.

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Page No .

79

83

87

88

96

99

101

104

LIST OF ABBREVIATIONS

ATCC American Type Culture Collection

BSA Bovine Serum Albumin

CTAB Cetyl trimethylammonium bromide

CFU Colony Forming Unit

DSC Differential Scanning Calorimeter

DCM Dichloromethane

DMSO Dimethylsulfoxide

HLB Hydrophile-Lyphophile Balance

LE Drug Loading Efficiency

MAA Methacrylic acid

MSDS Material safety data sheet

MIC Minimum Inhibition Concentration

NIPAM N-isopropylacrylamide

O.D. Optical density

PBS Phosphate Buffer Saline

PDI Potential determining ions

PGA polyglycolic acid

PLA poly(D,L-lactic) acid

PVA Poly vinyl alcohol

PLGA Poly (D,L-lactide-co-glycolide) acid

PZC Potential of zero charge

S.D Standard Deviation

SDS Sodium dodecyl sulfate

SEM Scanning Electron Microscopy

Tg Glass Transition Temperature

USFDA United State Food Drug Administration

MW Molecular Weight

xix

CHAPTER ONE

1.1 INTRODUCTION

The science of drug delivery can be defined as the control of the in vivo temporal and

spatial location of drug molecules for clinical purposes by the biological and chemical

applications (Uchegbu, 2006). As a drug is being administered, only small fraction of

the dosage will reach the intended site and give the pharmaceutical effect. The rest

will be wasted in a few ways either being taken up by the wrong tissue, being

removed by the tissue too fast before the drug can exhibit the effect or being

metabolized on its way to the target site by many possible enzymatic processes

(Uchegbu, 2006). There are two main reasons for researchers to persistently continue

explore in the field of drug delivery which are to maximize the drug activities and to

minimize the side effects (Allen & Cullis, 2004).

Nowadays research on advanced drug delivery has become more and more

demanding in the scientific world as there are several reasons for this booming

phenomenon. This is due to increase use of biological materials with poorly

understood physical properties or questionable shelf life, emergence of more

challenging low-molecular-weight molecules and biomacromolecules with either poor

aqueous solubility or poor tissue permeation and realization of having to control the

release of some portion of drug on the target sites to reduce the toxicity and to give

higher therapeutic index of that drug itself (Uchegbu, 2006). In addition, some clinical

advancements have also been the driving force for such researches to take place,

especially those treatment which are better using the implant system rather than using

the conventional way.

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1.2 RESEARCH BACKGROUND

Clinical problems arise from infection of the bone due to fractures or post surgery

complications are amongst the problems faced by the general practitioners nowadays

and yet the best possible solutions are still under debate amongst the scholars. A

common, debilitating infection in orthopaedics setting is osteomyelitis, mainly caused

by Staphylococcal aureus (Balmayor et al., 2011). It is an inflammatory bone disease

with deep bone involvement infiltrating the medullary cavity, cortex and periosteum

(Chung, 2001). Osteomyelitis normally arises as a nosocomial infection due to post­

operative orthopaedic surgeries, introduced during implantation of a prosthesis or

carried to the biomaterial surface by a temporary bacteraemia where they adhere and

grow to form a biofilm (Neut et al., 2001). Osteomyelitis is especially complicated if

patients are immunocompromised (Brin et al., 2008; Chung, 2001 ).

In addition, osteomyelitis is one of the inflammatory bone diseases caused by

infectious bacteria which can induce microbial infection of the medullary cavity of the

bone, the cortex as well as the periosteum (Chung, 2001). The infections are either

introduced during implantation of a prosthesis or are carried to the biomaterial surface

by a temporary bacteraemia, where they adhere and grow to form a biofilm (Neut et

al., 2001). But the most common cause of this bone disease is the post-operative

sepsis following orthopaedic procedures which has become a major concern amongst

the orthopedic surgeons (Chung, 2001).

In the case of osteomyelitis, the treatment is sometimes quite complicated

since the optimum concentration level of the antibiotic at the site of infection is

difficult to be achieved via systemic administration. Many contributing factors have

been identified which lead to this particular problem in treating such infection.

Successful treatment is difficult to achieve without causing irreversible side effects to

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the patient i.e. the systemic toxicity of the antibiotic which in return prohibits a high

systemic dose of the drug administrated (Stephens et al., 2000). Besides, inherent

problems of the drug such as short half-life of the antibiotic and poor circulation to the

infected area can slow down the rate of therapeutic outcome. However, the problems

associated with the drug itself can be mitigated by re-formulating the drug or creating

a carrier system that can directly deliver the drug to the site of infection.

One of the conventional treatments for osteomyelitis which is still being

practiced clinically is by local administration of antibiotic either by spraying or by

injecting to the infected site followed by oral administration to optimize the

therapeutic effect of the drug given (Xue & Shi, 2004). Although injection

(intravenous) can give adequate blood level of gentamicin, the long-term indwelling

catheter as well as the daily dose of administration remain the drawbacks of the

conventional method (Brin et al., 2008). This method will give rise to the typical drug

concentration in the blood that shows peaks and valleys wherein below the therapeutic

level the clinical effect of the drug is ineffective while above the minimum toxic

concentration (MTC), the side effects can be greatly manifested clinically.

Hence, the toxicity and the minimum effective levels give a very narrow

window for any mistakes to happen in administrating any drugs. The toxicity of

gentamicin to the patient which can cause ototoxic and nephrotoxic effects is the main

concern whenever the patient is administered with gentamicin (Chaisri, Ghassemi,

Hennink & Okonogi, 2011). The toxicity of gentamicin in treating bone infections for

instance, researchers have been considering alternative ways of administrating the

drug by using polymers to get a controlled release effect. Besides, it has been proven

that by using an implant system to administer gentamicin, the risk of ototoxicity and

nephrotoxicity can be minimized (Chaisri et al., 2011; Chang, Perrie & Coombes,

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2006). That is why amongst the treatment options for osteomylistis is by using a local

implant containing polymethyl-methacrylate (PMMA) beads or calcium phosphate

bone cements. PMMA by itself delivers several advantages as vehicle for gentamicin

(Brin et al., 2008). However, a major problem associated with this PMMA beads; is its

non-biodegradability which requires second surgery to remove them.

The progression of scientific findings nowadays have further shown a few

biodegradable polymers can be used in medical aspects due to their hydrolysable

backbone linkages such as orthoester, anhydride, ester, carbonate, urea and urethane.

These polymers have undergone many scientific tests and to date, two of them seem to

get the spotlight in medical field. These two biodegradable and biocompatible

polymers namely poly(glycolic acid) or PGA and poly(o,L-lactic acid) or PLA have

been showing promising biological natures and have received approval by the US

FDA to be used on human (Di Toro, Betti & Spampinato, 2004)

One of the most commonly employed co-polymers in drug delivery is Poly(o.L­

lactic-co-glycolic acid) (PLGA) a combination of PLA and PGA. Having the property

to degrade naturally in the human body without creating any harmful by products

systemically, PLGA is a promising candidate to be used as micro carrier system for

gentamicin for several reasons which are (1) to protect the drug from being

metabolised prematurely; (2) to deliver the drug safely to the infection site; (3) to

provide a controlled release of the gentamicin which can be extended to a significant

period oftime depending on the PLGA molecular weight used (Fischer, Foerg, Merkle

& Gander, 2004 ). Hence, gentamicin-loaded PLGA microsphere is envisaged to solve

the problem of having multiple administrations via the conventional way as it can

replace it with a single dose which can give a sustained-release over several weeks to

months.

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In this study, gentamicin-loaded PLGA microspheres were fabricated by using

water-in-oil-in-water (w/o/w) double emulsion solvent evaporation method. Even

though there were some extensive studies on gentamicin microspheres, a lot more

variables and parameters are waiting to be manipulated in order to fabricate the best

microspheres with the best formulation. The background of this study is to manipulate

several parameters such as different molecular weight of PLGA, different

concentrations of poly-vinyl alcohol (PV A), different Hydrophile Lipophile balance

(HLB) values of the surfactants, different types of surfactant or emulsifier used such

as PV A, Tween 20, Span 80, Tween 80, Span 85, Triton XI 00 and combinations of

the surfactants and last but not least, the concentration of oil phase used ranging from

5% up to 20%. The influences of different concentrations of chitosan in fabricating the

microspheres were also studied. 0.1 %, 0.3% and 0.5% of chitosan concentrations were

used during the fabrication process.

The fabricated microspheres were characterized based on the particle size

distributions, the external morphologies, the encapsulation efficiency of gentamicin

inside the microspheres and the profiling of the release patterns of gentamicin-loaded

PLGA microspheres over time. The outcome of the study is intended to broaden the

knowledge under this field apart from giving an alternative way of administrating

small molecule, water-soluble drugs in the future.

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