EXPLORING THE POTENTIAL OF OPTIMISED STARCH...

EXPLORING THE POTENTIAL OF OPTIMISED STARCH

PARTICULATES AS PLATFORM FOR ORAL DELIVERY

OF A MODEL GASTROLABILE DRUG CEFOTAXIME

by

NADEEM IRFAN BUKHARI

Thesis submitted in fulfilment of the

requirements for the degree

of Doctor of Philosophy

2009

ii

ACKNOWLEDGEMENT

I would like to express my sincere gratitude to my supervisor, Professor Dr. Saringat

Haji. Bai, School of Pharmacy, Universiti Sains Malaysia (USM), Penang for his scientific

guidance, insightful discussion, for allowing me to develop my work in higher tier research

approaches and for giving me the freedom to develop my own ideas.

I am indebted to my co-supervisor Professor Dr. Yuen Kah Hay, School of

Pharmacy, USM, Penang for his incessant support, guidance, encouragement and attention to

detail rigor for helping me throughout my PhD. Through his supervision, I have gained a lot

more than scientific development.

My sincere gratitude is for my Field Supervisor, Professor Dr. Abu Bakar Abdul

Majeed, Dean Faculty of pharmacy, Universiti Technologi Mara (UiTM), Kuala Selangor,

Malaysia for his invaluable logistic input, intellectual support and constant interest in the

PhD work and making UiTM research facilities available.

I am also indebted to Professor Dr. Peter Pook, Dean School of Pharmacy and Allied

Sciences, International Medical University (IMU), Kuala Lumpur for grant of the protected

time, suggestion and cooperation during my research work. Part of the investigations for this

study has been undertaken within the scope of research projects funded by International

Medical University (vide grants, IMU 069/04 and IMU 108/06). The above fundings are

gratefully acknowledged.

I am thankful to Professor Dr. Oya Alpar, School of Pharmacy, University of

London, UK for providing me opportunity to work with her research group on new drug

delivery systems and for her generous support during research work at School of Pharmacy,

iii

University of London, UK. I am thankful to Dr. Gabriele Anisah Froemming, Head Institute

of Medical Molecular Biotechnology (IMMB), UiTM for permission for Scanning Electron

Microscopy (SEM) and Mr. Abdul Karim, at IMMB, UITM and David McCarthy at Electron

Microscopy Unit, School of Pharmacy, University of London for carrying out SEM.

The start of this work is much owing to the cooperation of my friends, Dr. Nisar ur

Rehman, Associate Professor, Islamia University, Bhawalpur Pakistan and Waseem Sarwar,

Manager Research and Development, Highnoon Laboratories, Lahore-Pakistan. I am also

thankful to Khalid Ahmad Sheikh, Lecturer, IMU for his encouragement and interesting

discussions. I pay thanks to Dr Rosa Elizabeth Pereira, then PhD scholar at UiTM and Mr.

Tommy Julianto, Lecturer, UiTM for their informal discussions. I am also thankful to Dr.

Rosa for her help in HPLC method validation, Dr. Adhiyaman Rajendran, Lecturer

Pharmaceutics, IMU for help in kinetic analysis of drug release and Khalid Hussain, PhD

scholar, USM for his help during pharmacokinetic studies.

Special thanks go to Shari Kraber, Wayne F. Adams and Mark J. Anderson, Stat

Ease Inc., Minneapolis, USA and Colbourn Elizabeth, Intelligensys, Ltd., UK for their email

discussions on the predictive model development and data analysis and data visualization. I

also appreciate Pharsight Corporation, Mountain View, CA, USA for providing WinNonlin®

Ver 5.2.1 under the Pharsight Academic Licence programme.

I am also deeply thankful to my siblings who are always there for me and for

reminding me that my research should serve good purposes for all humankind. My spouse

has been very supportive for this work. Her understanding and moral support during my

work has been very much appreciated. I am also thankful to her for weathering through and

forgiving my bouts of moans and whines and thank to her for hanging in there with me

during the hard times. This work is dedicated to her and to our sons, Muhammad Haris

Bukhari, Muhammad Usama Bukhari and particularly, to Muhammad Qasim Bukhari.

iv

Last, but not the least, I thank my heavenly parents, for their efforts to give me the

best training, educating me and for unconditional support and encouragement to pursue my

interests. They never leave me aside and never once allowed my foot to slip. Their prayers

have always been with me.

Nadeem Irfan Bukhari, B.Pharm., M.Phil Pharm. (Pak)

v

TABLE OF CONTENTS

Page ACKNOWLEDGEMENT ii

TABLE OF CONTENTS v

LIST OF TABLES xx

LIST OF FIGURES xxii

LIST OF PLATES xxviii

LIST OF SYMBOLS xxix

LIST OF ABBREVIATION xxx

ABSTRAK xxxiii

ABSTRACT xxxv

CHAPTER 1 – GENERAL INTRODUCTION AND AIMS OF STUDY

1

1.1 THE ORAL ROUTE OF ADMINISTRATION 1

1.1.1 Drug absorption after oral route of administration 1

1.1.2 Barriers to drug absorption after oral administration 2

1.1.2(a) Gastrointestinal tract milieu 2

1.1.2(b) Mucus 3

1.1.2(c) Epithelial cells – the mechanical barrier on lumen side 3

1.1.2(d) Epithelial permeation, transport pathways and mechanisms 3

1.1.2(e) Efflux transporters and pumps 4

1.1.2(f) Endothelial cells – the mechanical barrier on apical side 5

1.1.2(g) Presystemic cellular metabolism 5

1.1.2(h) Presystemic hepatic metabolism 5

1.1.2(i) Enterohepatic shunt 6

1.2 STRATEGIES TO OVERCOME BARRIERS FOR ORAL DELIVERY 6

1.2.1 Approaches addressing gastric liability 6

1.2.1(a) Chemical modification – prodrug and drug analogue 7

1.2.1(b) Lipidisation strategies 7

1.2.1(c) Polymeric coating, encapsulation or entrapment 8

1.2.1(d) Drug targeting to lymphatics 8

1.2.1(e) Drug targeting to the Peyer’s patches 9

1.2.1(f) Targeted drug delivery to colon 10

1.2.1(g) General use of particulate drug carriers 11

1.3 PARTICULATE DELIVERY SYSTEMS 11

vi

1.3.1 Liposomes 12

1.3.2 Solid lipid nanoparticles 13

1.4 GENERAL METHODS FOR PREPARATION OF PARTICULATES 13

1.4.1 Bottom-up procedures 13

1.4.1(a) Emulsion/dispersion polymerization 13

1.4.1(b) Preparation from preformed polymers 14

1.4.1(c) Solvent deposition/displacement (nanoprecipitation) 15

1.4.1(d) Emulsification-cross-linking 16

1.4.1(e) Desolvation of macromolecules 16

1.4.2 Supercritical fluid technology 16

1.4.3 Top-down procedure 17

1.4.3(a) Extrusion 17

1.4.3(b) Wet-grinding (nanomilling) 17

1.4.4 Materials of fabrication of particulates 17

1.5 GENERAL APPLICATIONS OF PARTICULATES 18

1.5.1 Oral delivery of peptides, gastrolabile drugs and vaccines 18

1.5.2 Targeting infected and inflamed cells 18

1.5.3 Targeting cytostatics to tumours 19

1.5.4 Ophthalmic delivery 19

1.5.5 Vaginal drug delivery 19

1.5.6 Enhancing drug safety 19

1.5.7 Adjuvants for vaccines 20

1.6 STARCH AS THE MATERIAL OF PARTICULATE FABRICATION 20

1.6.1 Starch composition and molecular structure 21

1.6.1(a) Amylose 21

1.6.1(b) Amylopectin 21

1.6.1(c) Molecular arrangement of amylose and amylopectin 21

1.6.1(d) Other minor components 22

1.6.2 Types of starches 22

1.6.2(a) Modified starches 22

1.6.3 Functionality of starch 23

1.6.3(a) Gelatinisation 23

1.6.3(b) Hydrocolloidal functionality 23

1.6.3(c) Thickening agent 24

1.6.3(d) Gel and film formation 24

1.6.3(e) Retrogradation 24

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1.6.3(f) Water binding capacity 25

1.6.4 Features of starch usable in new and novel drug delivery systems 25

1.6.5 New and novel starch-based delivery systems 26

1.6.6 Applications of new and novel starch-based systems 26

1.6.6(a) Enhanced pharmaceutical excipient 26

1.6.6(b) Delivery carrier 26

1.6.6(c) Control release 27

1.6.6(d) Bioadhesive drug delivery system 27

1.6.6(e) Regional chemotherapy 28

1.6.6(f) Miscellaneous 28

1.6.7 Methods for the fabrication of starch particulates 28

1.7 MODEL DRUG AND THE MATERIAL OF FABRICATION 28

1.7.1 Cefotaxime sodium as model drug 28

1.7.2 Starch as the material of fabrication 32

1.8 MULTIVARIABLE OPTIMISATION 33

1.8.1 Statistical optimisation algorithms 33

1.8.2 Artificial intelligent optimisation algorithms 35

1.9 Problem statement 36

1.10 Experimental work and the scope of study 37

CHAPTER 2 – HPLC METHOD VALIDATION AND STABILITY

STUDIES OF CEFOTAXIME SODIUM 38

2.1

INTRODUCTION

38

2.1.1 Validation of HPLC method 38

2.1.1(a) Specificity 38

2.1.1(b) Linearity 39

2.1.1(c) Accuracy 39

2.1.1(d) Precision 39

2.1.1(e) Sensitivity 40

2.1.1(f) Robustness 40

2.1.2 Stability studies of cefotaxime sodium 40

2.1.3 Objectives of the study 41

2.2 EXPERIMENTAL FOR HPLC 41

2.2.1 Materials 41

2.2.2 HPLC instrumentation 42

2.2.2(a) Mobile phase 42

viii

2.2.2(b) HPLC conditions 42

2.2.2(c) Preparation of cefotaxime sodium standard solution 42

2.2.2(d) Preparation of drug-spiked plasma 43

2.2.2(e) Processing of the plasma-spiked samples 43

2.2.3 Method validation 44

2.2.3(a) Assessment of specificity 44

2.2.3(b) Determination of linearity and range 44

2.2.3(c) Evaluation of accuracy and precision 44

2.2.3(d) Estimation of recovery 44

2.2.3(e) Sensitivity 45

2.3 ASSESSMENT OF DRUG STABILITY 45

2.3.1 Chemicals 45

2.3.2 Software tool 45

2.3.3 Assessment of stability 46

2.3.3(a) Preparation of drug aqueous solutions with different pH values 46

2.3.3(b) Preparation of glutaraldehyde and amylase solutions 46

2.3.3(c) Sample incubation, collection and processing 47

2.3.3(d) Analysis of cefotaxime sodium concentration in samples 47

2.3.3(e) Data analysis 47

2.3.3(f) Selection of the predictive model 48

2.4 RESULTS 48

2.4.1 HPLC method validation 48

2.4.1(a) Specificity 48

2.4.1(b) Linearity 49

2.4.1(c) Recovery, accuracy and precision 49

2.4.1(d) Sensitivity 50

2.4.2 Stability of cefotaxime sodium 50

2.4.2(a) Drug stability in aqueous solutions at different pH values 51

2.4.2(b) Stability of drug in different preparatory media 51

2.5 DISCUSSION 53

2.5.1 HPLC 53

2.5.2 Stability study of cefotaxime sodium 54

2.6 CONCLUSION 56

ix

CHAPTER 3 – FABRICATION OF STARCH PARTICULATES BY

SPRAY DRYING 57

3.1

INTRODUCTION

57

3.1.1 Advantages of spray-drying 58

3.1.2 Applications of spray-drying in developing starch particulates 59


3.2 EXPERIMENTAL 60

3.2.1 Materials 60


3.2.3 Methods 60

3.2.3(a) Experimental design 60

3.2.3(b) Preparation of starch premixes 61

3.2.3(c) Spray-drying of starch premixes 61

3.2.3(d) Determination of particle size 61

3.2.3(e) Determination of size distribution (dispersion in uniformity) 62

3.2.3(f) Measurement zeta potential 62

3.2.3(g) Determination of particle yield 62

3.2.3(h) Data analysis and selection of the predictive model 62

3.2.3(i) Scanning electron microscopy of starch particles 63

3.2.3(j) Optimisation of responses and selection criteria 63

3.2.3(k) Validation of predicted formulation 63

3.3 RESULTS 64

3.3.1 Particle size 65

3.3.2 Dispersion in uniformity of particle size 66

3.3.3 Zeta potential 68

3.3.4 Powder yield 68

3.3.5 Simultaneous optimisation and validation 70

3.3.6 Scanning electron micrographs of corn starch particles 71

3.4 DISCUSSION 71

3.4.1 Experimental design 71

3.4.2 Factors and experimental conditions 71

3.4.3 Data analysis and model selection 73

3.4.3(a) Particle size 74

3.4.3(b) Uniformity of size distribution 75

3.4.3(c) Zeta potential 75

3.4.3(d) Powder yield 75

x

3.4.4 Optimisation and validation 76

3.4.5 Scanning electron micrographs of corn starch particles 76

3.5 CONCLUSION 77

CHAPTER 4 – FABRICATION OF STARCH PARTICULATES BY

ULTRASONICATION 78

4.1

INTRODUCTION

78

4.1.1 Ultrasonication 79

4.1.1(a) Possibility of size reduction with ultrasonication 80


4.2 EXPERIMENTAL 81

4.2.1 Materials 81

4.2.2 Instruments 81

4.2.2(a) Homogenizer 81

4.2.2(b) Probe sonicator 81

4.2.2(c) Software tool 81

4.2.3 Methods 81


4.2.3(b) Construction of design layout for optimisation 82

4.2.3(c) Preparation of starch premixes 82

4.2.3(d) Ultrasonication of starch premixes 83

4.2.3(e) Determination of particle size and polydispersity index (PDI) 83

4.2.3(f) Measurement of zeta potential 84

4.2.3(g) Data analysis 84

4.2.3(h) Predictive model selection in optimisation study 85

4.2.3(i) Optimisation criteria 85

4.2.3(j) Assessment of model validation and output reproducibility 86

4.3 RESULTS 86

4.3.1 Pre-optimisation study 86

4.3.1(a) Pulsed ultrasonication 86

4.3.1(b) Continuous ultrasonication 88

4.3.1(c) Effect of phospholipid 90

4.3.1(d) Effect of ultrasonication time and phospholipid concentration 91

4.3.1(e) Effect of surfactants 92

4.3.1(f) Effect of stabilizers 94

4.3.2 Optimisation study 94

xi

4.3.2(a) Optimisation of factors for particle size 95

4.3.2(b) Optimisation of factors for polydispersity index (PDI) 100

4.3.2(c) Optimisation of factors for zeta potential 104

4.3.2(d) Simultaneous optimisation of all responses and validation of prediction

105

4.4 DISCUSSION 106

4.4.1 Pre-optimisation study 106

4.4.1(a) Factors in study 106

4.4.1(b) Effect of pulse ultrasonication 107

4.4.1(c) Effect of continuous ultrasonication 107

4.4.1(d) Effect of phospholipid 108

4.4.1(e) Effect of phospholipid with continuous ultrasonication 108

4.4.1(f) Effect of surfactants 109

4.4.1(g) Effect of stabilisers 109


4.4.2(a) Experimental design – D-optimal 110

4.4.2(b) Data analysis in optimisation study 110

4.4.2(c) Predictability of model 111

4.4.2(d) Particle size 112

4.4.2(e) Polydispersity index 113

4.4.2(f) Zeta potential 113

4.4.2(g) Simultaneous optimisation 113

4.4.2(h) Validation of predicted formulation 114

4.4.3 Comment on ultrasonication as method of size reduction 114

4.4.3(a) Mechanisms for size reduction by ultrasonication 114

4.5 CONCLUSION 115

CHAPTER 5 – STARCH PARTICULATES BY NANOMILLING:

A PRE-EXPERIMENTAL PILOT STUDY 117

5.1

INTRODUCTION

117


5.2 EXPERIMENTAL 120

5.2.1 Materials 120

5.2.2 Grinding media 120

5.2.3 Instrumentation 120

5.2.4 Methods 120

xii

5.2.4(a) Preparation of starch premixes 120

5.2.4(b) Nanomilling of starch premixes 121

5.2.4(c) Determination of size and polydispersity index (PDI) 124

5.2.4(d) Measurement of zeta potential 124

5.2.4(e) Data analysis 125

5.2.4(f) Selection of appropriate formulation parameters for optimisation study

125

5.3 RESULTS 125

5.3.1 Milling time 126

5.3.2 Heating the starch premixes 126

5.3.3 Addition of surfactants 128

5.3.3(a) Pluronic-F68 128

5.3.3(b) Addition of Spans and Tweens at different concentrations 128

5.3.4 Addition of cross-linker and plasticizer at varied cross-linking reaction times

131

5.3.5 Change of grinding media (beads) size 132

5.3.6 Addition of amylase 133

5.3.7 Change of starch content 134

5.3.8 Variation in pH 135

5.3.9 Omission of glycerol at pH 5.5 136

5.3.10 Addition of surfactants and other dispersant 137

5.3.10(a) Addition of PEG as dispersant 137

5.3.10(b) Addition of different surfactants with different concentrations at pH 5.0

138

5.3.11 Selection of suitable formulation parameters and process conditions

140

5.4 DISCUSSION 141

5.4.1 Effect of milling time 142

5.4.2 Effect of heating starch premixes 143

5.4.3 Effect of addition of surfactants 144

5.4.4 Effect of addition of cross-linker and plasticizer 145

5.4.5 Effect of changing grinding media 147

5.4.6 Effect of addition of amylase 148

5.4.7 Effect of changing the starch contents 148

5.4.8 Effect of variation of pH 148

5.4.9 Effect of omission of glycerol at pH 5.5 149

5.4.10 Effect of dispersant, PEG and other surfactants 149

5.4.11 Identification and selection of critical factors 150

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5.5 CONCLUSION 151

CHAPTER 6 – STARCH PARTICULATES BY NANOMILLING –

OPTIMISATION STUDY 153

6.1

INTRODUCTION

153



6.2.1 Materials 154

6.2.2 Grinding media 155

6.2.3 Instrumentation 155


6.2.5 Methods 155


6.2.5(b) Preparation of starch premixes 156

6.2.5(c) Nanomilling of the premixes 156

6.2.5(d) Determination of particle size, PDI and zeta potential 157

6.2.5(e) Data analysis and model selection 157

6.2.5(f) Optimisation criteria 157

6.2.5(g) Validation and assessment of reproducibility 157

6.3 RESULTS 157

6.3.1 Pre-optimisation screening study 158

6.3.1(a) Factors for particle size 158

6.3.1(b) Factors for polydispersity index 163

6.3.1(c) Factors for zeta potential 166


6.3.2(a) Optimisation of factors for particle size 169

6.3.2(b) Optimisation of factors for polydispersity index 170

6.3.2(c) Optimisation of factors for zeta potential 173

6.3.3 Simultaneous optimisation of particle size, PDI and zeta potential 174

6.4 DISCUSSION 175

6.4.1 Factors and their levels in pre-optimisation and optimisation study 175

6.4.2 Experimental designs 176

6.4.3 Data analysis 177

6.4.4 Model selection and testing predictability 177

6.4.5 Particle size 178

6.4.6 Polydispersity index 179

xiv

6.4.7 Zeta potential 180

6.4.8 Simultaneous prediction of optimum levels 181

6.4.9 Validation and reproducibility 181

6.5 CONCLUSION 182

CHAPTER 7 – IN VITRO CHARACTERISATION OF STARCH

PARTICULATES AND DRUG RELEASE STUDY 183

7.1

INTRODUCTION

183

7.1.1 Particle size, distribution and surface charges 183

7.1.2 Drug loading analysis 183

7.1.3 In vitro drug release 184

7.1.4 Biological and other characterisation methodologies 185



7.2.1 Materials 186

7.2.2 Methods 186

7.2.2(a) Preparation and drug loading on optimised starch formulations 186

7.2.2(b) Particle size, PDI/dispersion in uniformity and zeta potential 187

7.2.2(c) Particle yield and drug loading analysis 187

7.2.2(d) Quantification of drug in samples 188

7.2.2(e) Scanning electron microscopy 188

7.2.2(f) Swelling index 188

7.2.2(g) Assessment of physical stability of optimised particles 189

7.2.2(h) In vitro drug release study 189

7.2.2(i) Kinetic analysis of release data 190

7.2.2(J) Statistical data analysis 190

7.3 RESULTS 191

7.3.1 Particle size, PDI and zeta potential 192

7.3.2 Particulate recovery (yield) and loading efficiency 192

7.3.3 Scanning electron micrographs of starch particulates 193

7.3.4 Degree of swelling 193

7.3.5 Physical stability 195

7.3.6 Release kinetics 198

7.4 DISCUSSION 200

7.4.1 Particle size, PDI and zeta potential of blank and loaded particles 200

7.4.2 Drug loading 201

xv

7.4.3 Scanning electron micrographs of starch particles 202

7.4.4 Swelling index 202


7.4.6 Kinetics of in vitro drug release 203

7.5 CONCLUSION 204

CHAPTER 8 – BIOAVAILABILITY AND PHARMACOKINETIC

STUDY OF CEFOTAXIME SODIUM LOADED STARCH

PARTICULATE CARRIERS

205

8.1

INTRODUCTION

205

8.1.1 Pharmacokinetic properties of and issues for cefotaxime sodium 206

8.1.2 Approaches for improving of bioavailability of cefotaxime sodium 206

8.2 Objectives of the study 207


8.3.1 Materials 207

8.3.2 Experimental animals and their housing prior to experimentation 207

8.3.3 Methods 208


8.3.3(b) Preparation of aqueous drug solution and starch vehicles 209

8.3.3(c) Preparation and characterisation of starch particulate formulations

209

8.3.3(d) Administration of dose 210

8.3.3(e) Blood sampling and sample processing 210

8.3.3(f) Determination of drug in biological samples 210

8.3.3(g) Pharmacokinetic data analysis 211

8.3.3(h) Statistical data analysis 211

8.4 RESULTS 211

8.4.1 Particle size and their characterisation 211

8.4.2 Plasma level time curves 212

8.4.3 Pharmacokinetic parameters 213

8.5 DISCUSSION 219

8.5.1 Study design 219

8.5.2 Pharmacokinetics computation approach 220

8.5.3 Characteristics of the particulate formulations 221

8.5.4 Plasma level time curves and derived parameters 221

8.5.4(a) Appearance of double peak phenomenon 222

xvi

8.5.4(b) Pharmacokinetics parameters 223

8.5.5 Enhanced oral bioavailability of cefotaxime sodium 224

8.5.6 Possible reasons for enhanced oral bioavailability 225

8.5.6(a) Protective effects of starch 226

8.5.6(b) Macromolecular/drug endocytosis 226

8.5.6(c) Starch absorption and transport 226

8.5.6(d) Particulate characteristics 227

8.5.6(e) Mucoadhesion 228

8.5.6(f) Presence of surfactant (composition) 229

8.5.6(g) Co-grinding (processing) 229

8.5.7 In vivo release mechanisms from particulates 229

8.5.7 (a) Digestibility of starch– a basis for drug release from starch 230

8.6 CONCLUSION 231

CHAPTER 9 – GENERAL DISCUSSION, CONCLUSION AND

SUGGESTIONS FOR IMPROVEMENT AND FURTHER

DEVELOPMENT

232

9.1

GENERAL DISCUSSION

232

9.1.1 HPLC method validation and drug stability assessment 232

9.1.2 Production methods for starch particulates 233

9.1.3 Use of computer-aided optimisation algorithms 234

9.1.4 Important features of starch particles 234

9.1.5 In vitro characterisation, drug loading and particle shape 235

9.1.6 Bioavailability and pharmacokinetics 235

9.2 ENVISIONED BIOMEDICAL APPLICATIONS SUITABLE FOR THIS SYSTEM

236

9.2.1 Lymph targeting 236

9.2.2 Application for other drugs 236

9.2.3 Tumour therapy 236

9.2.4 Local periodontal delivery carrier 236

9.2.5 Pharmaceutical excipient 237

9.2.6 Future of starch particulates 237

9.3 SUGGESTIONS FOR IMPROVEMENTS AND FURTHER DEVELOPMENT

237

9.3.1 Spray-drying 238

9.3.2 Ultrasonication 238

9.3.3 Nanomilling 238

xvii

9.3.4 Polydispersity 239

9.3.5 Drug loading 239


9.3.7 In vitro drug release 240

9.3.8 Microscopic, radiopharmaceutical and pharmacokinetic evaluation 240

9.3.9 Assessment of therapeutic potential 240

9.3.10 Miscellaneous 241

BIBLIOGRAPHY 242

APPENDICES

A 2.1 Data on the stability of cefotaxime sodium in different experimental conditions

273

A 3.1 Data for particle size, polydispersity index, zeta potential of corn starch based on DX® generated template

274

A 3.2 Predicted levels of factors to prepare optimised corn starch particles using spray-drying

275

A 4.1 Effect of pulsed ultrasonication on the particle size and polydispersity index of potato starch, soluble starch and corn starch

276

A 4.2 Effect of continuous ultrasonication on the particle size and polydispersity index of potato starch, soluble starch and corn starch

277

A 4.3 Effect of different concentrations of phospholipid on the particle size of potato starch. (SD is given in parentheses)

278

A 4.4 Effect of different concentrations of phospholipid on the particle size of soluble starch particles. (SD is given in parentheses)

279

A 4.5 Effect of different concentrations of phospholipid on the particle size of corn starch particles. (SD is given in parentheses)

280

A 4.6 Effect of different concentrations of phospholipid on the polydispersity index of potato starch particles. (SD is given in parentheses)

281

A 4.7 Effect of different concentrations of phospholipid on the polydispersity index of soluble starch particles. (SD is given in parentheses)

282

A 4.8 Effect of different concentrations of phospholipid on the polydispersity index (PDI) of corn starch. (SD is given in parentheses)

283

A 4.9 Effect of surfactants at different concentrations on the particle size and polydispersity index of potato starch, soluble starch and corn starch

284

A 4.10 Effect of stabilizers on the particle size, polydispersity index and zeta charge of potato starch, soluble starch and corn starch

285

A 4.11 Particle size, polydispersity index and zeta charge of particles prepared following DX®- generated combinations for factors in optimisation study

286

A 4.12 Predicted levels and responses generated by DX®7.1.5 for ultrasonication

287

xviii

A 5.1 Effect of milling time and heat on the particle size and polydispersity index of potato starch, soluble starch and corn starch. Mean ± SD, N = 3.

288

A 5.2 Effect of surfactants at different concentrations on the particle size and polydispersity index of potato starch, soluble starch and corn starch at pH 10.0. Mean ± SD, N = 3.

289

A 5.3 Effect of the time of cross-linking on the particle size and polydispersity index of potato starch, soluble starch and corn starch. Mean ± SD, N = 3.

290

A 5.4 Effect of the size of media (beads) on the particle size and polydispersity index of potato starch, soluble starch and corn starch. Mean ± SD, N = 3

290

A 5.5 Effect of addition of amylase on the particle size and polydispersity index of potato starch, soluble starch and corn starch. Mean ± SD, N = 3.

290

A 5.6 Effect of different starch content on the particle size and polydispersity index of potato starch, soluble starch and corn starch. Mean ± SD, N = 3.

290

A 5.7 Effect of pH of the starch premixes on the size and polydispersity index of potato starch, soluble starch and corn starch. Mean ± SD, N = 3.

291

A 5.8 Effect of omission of glycerol at pH 5.5 on the size and polydispersity index of potato starch, soluble starch and corn starch. Mean ± SD, N = 3.

291

A 5.9 Effect of addition of PEG on the size and polydispersity index of potato starch, soluble starch and corn starch. Mean ± SD, N = 3.

291

A 5.10 Effect of surfactants with different concentrations at pH 5.5 on the particle size and polydispersity index of potato starch, soluble starch and corn starch. Mean ± SD, N = 3.

292

A 6.1 Size, polydispersity index and zeta potential of the formulations prepared using the pre-optimisation screening design

293

A 6.2 Predicted levels of the factors and responses for optimized potato starch particles formulations

295

A 6.3 Particle size, polydispersity index and zeta potential of the validation formulations of potato starch

295

A 7.1 Effect of drug loading and storage on the particle size, polydispersity index and zeta potential of potato starch particulates. Mean ± SD, N = 4.

296

A 7.2 Effect of drug loading and storage on the particle size, polydispersity index and zeta potential of soluble starch particulates. Mean ± SD, N = 4.

297

A 7.3 Effect of drug loading on the size of corn starch particulates 298

A 7.4 The p-value for the particle size, polydispersity index (PDI) and zeta potential when blank and drug loaded starch particulate formulations compared at zero to day 7

298

A 7.5 Loading efficiency of different starch particles 299

A 7.6 The p-value for the yield, loading and loading efficiency when blank and loaded starch particulate formulations were compared

299

A 7.7 Zeta potential of the different processing ingredients used in this study 299

A 8.1 Approval letter from Animal Ethics Committee to carry out bioavailability and pharmacokinetic study

300

xix

A 8.2 Pharmacokinetic parameters of cefotaxime sodium after single oral dose of CAQUEOUS, as free drug (control) to male Sprague-Dawley rats

301

A 8.3 Pharmacokinetic parameters of cefotaxime sodium after single oral dose of drug containing potato starch vehicle (CVEHICLE-PS) to male Sprague-Dawley rats

301

A 8.4 Pharmacokinetic parameters of cefotaxime sodium after single oral dose of drug loaded cross-linked potato starch (FPS-CL) to male Sprague-Dawley rats

302

A 8.5 Pharmacokinetic parameters of cefotaxime sodium after single oral dose of drug loaded uncross-linked potato starch (FPS-UCL) to male Sprague-Dawley rats

302

A 8.6 Pharmacokinetic parameters of cefotaxime sodium after single oral dose of drug containing soluble starch vehicle (CVEHICLE-SS) to male Sprague-Dawley rats

303

A 8.7 Pharmacokinetic parameters of cefotaxime sodium after single oral dose of drug loaded uncross-linked cationic soluble starch particulates (FSS-

UCL+) to male Sprague-Dawley rats

303

A 8.8 Pharmacokinetic parameters of cefotaxime sodium after single oral dose of drug loaded uncross-linked anionic soluble starch particulates (FSS-

UCL–) to male Sprague-Dawley rats

304

A 8.9 Pharmacokinetic parameters of cefotaxime sodium after single oral dose of drug loaded uncross-linked neutral soluble starch particulates (FSS-

UCL0) to male Sprague-Dawley rats

304

A 8.10 Pharmacokinetic parameters of cefotaxime sodium after single oral dose of drug loaded spray-dried cross-linked corn starch (FCS-SpD) to male Sprague-Dawley rats

305

A 8.11 The p-values for the pharmacokinetic parameters of potato starch controls and the potato starch particulate formulations

305

A 8.12 The p-values for the pharmacokinetic parameters of soluble starch controls and the soluble starch particulate formulations

306

A 8.13 The p-values for the pharmacokinetic parameters of corn starch particles (FCS-SpD) and aqueous drug control (CAQUEOS) and, between particles of potato starch (FPS-UCL) and soluble starch (FSS-UCL

+)

306

12 LIST OF PUBLICATIONS AND AWARDS 307

xx

LIST OF TABLES

Table Page

1.1 Review of the methods used for development of starch particulates 29

2.1 Physicochemical properties of cefotaxime sodium 41

2.2 Multiple response optimisation criteria for the stability of cefotaxime sodium

47

2.3 The recovery, within- and between-day precision and accuracy for cefotaxime sodium

50

2.4 The values of R2, LOD and LOQ cefotaxime sodium 50

3.1 The fixed and varied experimental conditions 61

3.2 Exerprimental design matrix for 4 factors each at 3 levels with response variables

64

3.3 ANOVA statistics of screening design for particle size, dispersion in uniformity, zeta potential and yield

65

3.4 Numerical goodness of fit statistical criteria for response data of corn starch

66

3.5 Comparison of validation formulations with the predicted formulations

71

4.1 Particle size, polydispersity index (PDI) and zeta potential on the starch particlulates of potato starch, soluble starch and corn starch. Mean ± SD, N = 3.

96

4.2 ANOVA statistics of screening design for particle size, polydispersity index and zeta potential of starches under study

97

4.3 Comparison of the validation formulations with the predicted formulation

106

5.1 Formulation variables and the processing conditions for premixes of potato starch, soluble starch and corn starch

122

5.2 Parameters and conditions found to be favourable for potato and soluble starch

141

5.3 Particle size, polydispersity index and zeta potential for soluble starch particles. Mean ± SD, N = 4.

141

6.1 The fixed and varied experimental conditions 156

6.2 Experimental design matrix for 7 factors, each at 2 levels (27) along with the data for particle size, polydispersity index (PDI and zeta potential. Mean ± SD, N = 3.

159

6.3 ANOVA of screening design for particle size, polydispersity index and zeta potential

160

6.4 Complementary array of the low, middle and high levels for 7 factors along with the data for particle size, polydispersity index and zeta potential. Mean ± SD, N = 3.

169

6.5 ANOVA statistics of the optimisation design for particle size, polydispersity index and zeta charge of potato starch

170

xxi

Table Page

6.6 Comparison of validation formulations with the predicted formulation

173

7.1 Composition of the starch particulate formulations selected for in

vivo study 187

7.2 Description of the mathematical models used to describe drug release from potato starch particulate formulations

191

7.3 Particle size, polydispersity index and zeta potential of blank and drug loaded starch particulate formulations. Mean ± SD, N = 4.

192

7.4 The swelling index of three starch and the selected starch particulates 195

7.5 Correlation coefficient (R2) values in various kinetic models tested to describe drug release from different starch particulate formulations

200

7.6 Mean dissolution time of free drug in aqueous solution and that of the starch particulate formulations. Mean ± SD, N = 6.

200

8.1 Detail on the comparison of bioavailability of starch particulate to that of controls

209

8.2 Statistical tests for comparing the control and starch particulate formulations

211

8.3 Particle size, polydispersity index and zeta potential for different starch particles under investigation. Mean ± SD, N = 4.

212

8.4 Pharmacokinetic parameters of cefotaxime sodium after single oral dose of different drug loaded starch particles and controls to the male Sprague-Dawley rats. Mean ± SD (in parentheses), N = 6.

216

8.5 The reported pharmacokinetic parameters of cefotaxime sodium in rats

225

xxii

LIST OF FIGURES

Figure Page

1.1 Morphological and functional barriers for drug absorption after oral administration

2

1.2 Microvilli showing epithelial cells and underneath two circulatory systems, blood and lymphatic stream. A special transport mechanism, persorption is also shown

9

1.3 A suggested exprimentation strategy for optimum design of experiment (DoE) (Adapted and modified from Anderson and Whitcomb, 2005)

34

1.4 Schematics for the architect of a neural network for the solution of complex problems

35

2.1 Chromatograms for blank (I) and spiked (II) rat plasma 49

2.2 Calibration curve of cefotaxime sodium 50

2.3 Response surface plot for effect of pH and time on percentage of drug remaining

52

2.4 Overlay plot with sweet spot showing optimised conditions for maximum drug stability (I). Interaction plot showing combined effect of pH and time on drug stability (II)

52

2.5 Artificial neural network (ANN)-generated response surface plot for the effect of pH and time on percentage of drug remaining

52

2.6 Drug remaining at pH 1.0, 4.0, 7.0 and 5.5 in aqueous solution. Mean ± SD, N = 3.

53

2.7 Drug remaining in amylase and glutaraldehyde solution and at pH 10.0. Mean ± SD, N = 3

53

3.1 A simplified diagram of the Buchi Mini Spray-Drier (Buchi Laboratotiums – Technik AG, Flawil, Switzerland) (Adapted and modified from Maa et al., 1998)

58

3.2 Half-normal plot (I) and Pareto chart (II) indicating the critical factors for particle size of corn starch

66

3.3 Interaction plot showing combined effect of corn starch content and surfactant on particle size

67

3.4 Half-normal plot (I) and Pareto chart (II) indicating the critical factors for the dispersion in uniformity of particle size of corn starch

67

3.5 One factor plot showing the effect of the volume of DMSO on the dispersion in uniformity of particle size of corn starch

67

3.6 Half-normal plot (I) and Pareto chart (II) indicating the critical factors for the zeta potential of corn starch

68

3.7 One factor plot showing the effect of the volume of DMSO on the zeta potential of corn starch

69

3.8 Half-normal plot (I) and Pareto chart (II) indicating the critical factors for particle yield of corn starch

69

3.9 One factor indicating the effect of surfactant on the yield corn starch 70

xxiii

Figure Page

3.10 Interaction plot showing combined effect of starch content and DMSO on yield of corn starch

70

3.11 A simplified illustration showing drying mechanism for droplet drying and the types of different particles as outputs

77

4.1 Working of High Intensity Sonic Vibra Cell Probe Sonicator®, Autotune series (Sonic and Materials Inc., USA)

79

4.2 Peaks showing size distribution of particles as monomodal (I), skewed (II), skewed and bi-modal (III) and multi modal (IV)

85

4.3 Effect of pulsed ultrasonication on the particle size of potato starch, soluble starch and corn starch. Mean ± SD, N = 3.

87

4.4 Effect of pulsed ultrasonication on the polydispersity index (PDI) of potato starch, soluble starch and corn starch. Mean ± SD, N = 3.

88

4.5 Effect of continuous ultrasonication on the particle size of potato starch, soluble starch and corn starch. Mean ± SD, N = 3.

89

4.6 Effect of continuous ultrasonication on the polydispersity index (PDI) of potato starch, soluble starch and corn starch. Mean ± SD, N = 3.

89

4.7 Effect of different concentrations of phospholipid on the particle size of potato starch, soluble starch and corn starch. Mean ± SD, N = 3.

91

4.8 Effect of different concentrations of phospholipid on the polydispersity index (PDI) of potato starch, soluble starch and corn starch. Mean ± SD, N = 3.

91

4.9 Effect of different surfactants on the particle size of potato starch, soluble starch and corn starch. Mean ± SD, N = 3.

93

4.10 Effect of different surfactants on the polydispersity index (PDI) of potato starch, soluble starch and corn starch. Mean ± SD, N = 3.

93

4.11 Effect of trehalose and glucose on the particle size (I), polydispersity index (PDI) (II) and zeta charge (III) of potato starch, soluble starch and corn starch. Mean ± SD, N = 3.

95

4.12 Effect of the types of starch on the particle size 98

4.13 Perturbation plot showing the relative effect of factors on particle size of potato starch (I), soluble starch (II) and corn starch (III)

98

4.14 Counter and response surface plots showing the effect of homogenisation time and ultrasonication amplitude on the particle size of potato starch (1-II), soluble starch (III-IV) and corn starch (V-VI)

99

4.15 Counter and response surface plots showing the effect of homogenisation speed and ultrasonication amplitude on the particle size of potato starch (I-II), soluble starch (III –IV) and corn starch (V-VI)

101

4.16 Interaction plots showing combined effect of starch type and each of homogenisation time (I) and homogenisation speed (II) on particle size of potato starch, soluble starch and corn starch

102

xxiv

Figure Page

4.17 Perturbation plots showing the relative effect of factors on polydispersity index (PDI) of potato starch (I), soluble starch (II) and corn starch (III)

103

4.18 Effect of types starch on the polydispersity index (PDI) of potato starch, soluble starch and corn starch

103

4.19 Response surface plots showing the effect of homogenisation speed and homogenisation time on polydispersity index (PDI) of potato starch (I), soluble starch (II) and corn starch (III) and of homogenisation time and ultrasonication amplitude on PDI of potato starch (IV), soluble starch (V) and corn starch (VI)

104

4.20 Interaction plots showing combined effect of types of starch and each of homogenisation time, speed and ultrasonication amplitude on polydispersity index (PDI) of potato starch (I), soluble starch (II) and corn starch (III)

105

4.21 Effect of starch type on the zeta potential of potato starch, soluble starch and corn starch

106

4.22 Mechanism of size reduction by ultrasonication 115

5.1 Mechanism of size reduction (I) Schematic illustration (II) and Photograph (III) of Netzsch MiniCer® laboratory circulation machine

118

5.2 Effect of milling time on the particle size and polydispersity index (PDI) of potato starch, soluble starch and corn starch Mean ± SD, N = 3.

126

5.3 Effect of heating on the particle size and polydispersity index (PDI) of potato starch, soluble starch and corn starch. Mean ± SD, N = 3.

127

5.4 Effect of Pluronic-F68 on the particle size and polydispersity index (PDI) of potato starch, soluble starch and corn starch. Mean ± SD, N = 3.

128

5.5 Effect of different surfactants and milling time on the particle size of potato starch (PS), soluble starch (SS) and corn starch (CS). Mean ± SD, N = 3.

129

5.6 Effect of different surfactants on the polydispersity (PDI) index of potato starch (PS), soluble starch (SS) and corn starch (CS). Mean ± SD, N = 3.

130

5.7 Three-D surface plot of starch type and surfactants for particle size and polydispersity index (PDI) at the mid levels of milling time and surfactant concentration

131

5.8 Effect of cross-linking reaction time and milling time on the particle size and polydispersity index (PDI) of potato starch (PS), soluble starch (SS) and corn starch (CS). Mean ± SD, N = 3.

132

5.9 Effect of the size of grinding media on the particle size of potato starch (PS), soluble starch (SS) and corn starch (CS). Mean ± SD, N = 3.

133

xxv

Figure Page

5.10 Effect of amylase on the particle size and polydispersity index (PDI) of potato starch (PS), soluble starch (SS) and corn starch (CS). Mean ± SD, N = 3.

134

5.11 Effect of starch contents on the particle size and polydispersity index (PDI) of potato starch (PS), soluble starch (SS) and corn starch (CS). Mean ± SD, N = 3.

135

5.12 Effect of pH on the particle size and polydispersity index (PDI) of potato starch (PS), soluble starch (SS) and corn starch (CS). Mean ± SD, N = 3.

136

5.13 Effect of omission of glycerol on the particle size and polydispersity index (PDI) of potato starch (PS), soluble starch (SS) and corn starch (CS). Mean ± SD, N = 3.

137

5.14 Effect of addition of PEG on the particle size and polydispersity index (PDI) of potato starch (PS), soluble starch (SS) and corn starch (CS). Mean ± SD, N = 3.

138

5.15 Effect of different surfactants and their concentrations on the particle size of potato starch (PS), soluble starch (SS) and corn starch (CS). Mean ± SD, N = 3.

139

5.16 Effect of different surfactants and their concentrations on polydispersity index of potato starch (PS), soluble starch (SS) and corn starch (CS). Mean ± SD, N = 3.

140

6.1 Half-normal (I) and Pareto chart (II) showing effects of 7 variables on particle size of potato starch

161

6.2 Interaction plot showing combined effect of citric acid and surfactant on particle size of potato starch

161

6.3 Perturbation plot showing the relative effect of factors on the particle size

162

6.4 Response surface plot showing the efect of surfactnat and citric acid on the particl size of potato starch when all other parameters are set at their mid levels

162

6.5 Cube plot showing combined effect of citric acid, surfactant and speed together

163

6.6 Half-normal plot (I) and Pareto chart (II) indicating critical factors for polydispersity index of potato starch

163

6.7 The perturbation plot showing the relative effect of factors on the polydispersity index (PDI) of potato starch nanoparticles

164

6.8 Response surface plot for the effect of phospholipid and gycerol (I), speed and glycerol (II) and surfactant and speed (III) on the polydispersity index of potato starch

165

6.9 Cube plot, showing simultaneoulsy effect of three parameters, phospholipid, glycerol and surfactant concentration (I) and glycerol, surfactant and speed (II) on the polydispersity index of potato starch

165

6.10 Half-normal plot (I) and Pareto chart (II) indicating critical factors for zeta potential of potato starch

166

xxvi

Figure Page

6.11 The perturbation plot showing the relative effect of factors on the zeta potential of potato starch nanoparticles

167

6.12 Response surfaces, respectively for the effect of D and F (I), D and E (II), A and G (III) and A and E (IV) on zeta potential of potato starch

168

6.13 Cube plot showing effect of glycerol, cirtic acidd and surfactant concentration (I) and citric acid, grinding time and speed (II) on zeta potential of potato starch

168

6.14 Effect of increasing concentration of citric acid (I) and processing time (II) on the particle size of potato starch

171

6.15 Perturbation plot showing the relative effect of factors on polydispersity index (PDI) of potato starch

171

6.16 Response surfaces, respectively for the effect of glycerol and phospholipid (I-II), glycero and speed (III-IV), phospholipid and processing time (V-VI) and citric acid and surfactant (VII-VIII) on the PDI of potato starch

172

6.17 Perturbation plot showing the relative effect of factors on zeta potential of potato starch

173

6.18 One factor plot (I to III) and cube plot (IV) showing the individual effect of glycerol (I), citric acid (II), surfactant concentration (III) and milling speed and collectively (IV) on the polydispersity index of potato starch

174

7.1 Side-by-side diffusion cells, clamped (left) and unclamped (right) 190

7.2 Particle yield (%) and drug loading efficiency (%) of starch particulate formulations. Mean ± SD, N = 4.

193

7.3 Particle size of the blank and drug loaded potato starch particulates. Mean ± SD, N = 4.

196

7.4 Polydispersity index of the blank and drug loaded potato starch particulates. Mean ± SD, N = 4.

196

7.5 Zeta potential of the blank and drug loaded potato starch particulates. Mean ± SD, N = 4.

197

7.6 Particle size of the blank and drug loaded soluble starch particulates. Mean ± SD, N = 4.

197

7.7 Polydispersity index of the blank and drug loaded soluble starch particulates. Mean ± SD, N = 4.

198

7.8 Zeta potential of the blank and drug loaded soluble starch particulates. Mean ± SD, N = 4.

199

7.9 Average drug release (%) for free drug and the starch particulates in phosphate buffer, pH 5.5. Mean ± SD, N = 6.

199

8.1 Experimental design for the controls and starch micro- and nano-particles

208

xxvii

Figure Page

8.2 Plasma concentrations of cefotaxime sodium after single oral administration as free drug (CAQUEOUS), potato starch vehicle (CVEHICLE-PS) and loaded with cross- and uncross-linked potato starch particulates (FPS-CL and FPS-UCL, respectively) to male Sprague-Dawley rats. Mean ± SD, N = 6. (The lower error bar has been removed from the last two points of CAQUEOUS)

213

8.3 Plasma concentrations of cefotaxime sodium after single oral administration as free drug (CAQUEOUS) and soluble starch vehicle (CVEHICLE-SS) and loaded with cationic, anionic and neutral soluble starch particulates (FSS-UCL

+, FSS-UCL– and FSS-UCL

0, respectively) to male Sprague-Dawley rats. Mean ± SD, N = 6. (The lower error bar has been removed from the last two points of CAQUEOUS)

214

8.4 Plasma concentrations of cefotaxime sodium after single oral administration as free drug (CAQUEOUS) and as drug loaded spray-dried corn starch particulates (FCS-SpD) to male Sprague-Dawley rats. Mean ± SD, N = 6. (The lower error bar has been removed)

214

8.5 Plasma concentrations of cefotaxime sodium after single oral administration as loaded uncross-linked potato starch (FPS-UCL), soluble starch (FSS-UCL

+) and spray-dried corn starch particulates (FCS-

SpD) to male Sprague-Dawley rats. Mean ± SD, N = 6)

215

8.6 Illustration to propose release of drug from starch particulates by swelling of starch in the presence of water and subsequent digestion

231

xxviii

LIST OF PLATES

Plate Page

3.1 Plate 3.1: Starch particles by using DMSO in PVA 0.1 % aqueous solution (I), in water containing acacia 0.5 % and magnetic stirring at 40°C (II), in trichloroethylene added in ethyl acetate (III) and in DMSO added in hot water (IV)

72

7.1 Scanning electron micrographs of potato starch, soluble starch and corn starch

194

xxix

LIST OF SYMBOLS

Symbol Description

∞ Infinity

µ micro

σ standard deviation of intercept

xxx

LIST OF ABBREVIATIONS

Abbreviation Description

Adeq Pre adequate precision

Adj R2 adjusted correlation coefficient

ANN artificial neural network

ANOVA analysis of variance

AOT aerosol OT (1,4-bis(2-ethylhexyl) sodium sulfosuccinate)

ATR-AOT combination of surfactants AOT and ATR

ATR aerosol TR (sodium 1,2-bis (tridecoxycarbonyl) ethanesulfonate)

AUC0-∞ area under the curve from zero to infinity

AUC0-∞, norm dose and weight normalised area under the curve

AUC0-t area under the curve from zero to time t

AUFS absorbance units full scale

BBD box Behnken Design

BCS biopharmaceutical classification system

BOS based on starch

°C Degree Celsius

CAQUEOUS control containing cefotaxime sodium in aqueous solution

CCD central composite design

CI confidence interval

Cmax peak concentration

Cmax, norm dose and weight normalised peak concentration

CS corn starch

CTAB cetyl trimethyl ammonium bromide

CVEHICLE-PS control containing vehicle for potato starch

CVEHICLE-SS control containing vehicle for soluble starch

CYP 450 cytochrome P 450

DDS drug delivery system

DMSO dimethyl sulfoxide

DoE Design of experiment

DX® Design Expert®

EDTA ethyl diamine tetra acetate

FCD face-centre composite design

xxxi


FCS-SpD microparticles of cross-linked, spray-dried corn starch

FDA food and drug regulation authority

FEG-ESEM field-emission gun environmental scanning electron microscopy

FI factor interaction

FPS-CL nanoparticles of cross-linked potato starch

FPS-UCL nanoparticles of uncross-linked potato starch

FSS-UCL– nanoparticles of uncross-linked anionic soluble

starch

FSS-UCL+ nanoparticles of uncross-linked cationic soluble

starch

FSS-UCL0 nanoparticles of uncross-linked neutral soluble

starch

F value Fisher’s statistics value

g/mol gram per mole

GIT gastro intestinal tract

HLB hydrophilic-lipophilic balance

HPLC high performance liquid chromatography

Hr hour

Hz hertz

ICH International committee on harmonisation

Kelim elimination rate constant

Kg Kilogram

kPA kilopascal

LOD Limit of detection

LOQ Limit of quantification

M molar

M-cells modified epithelial cells

MDT mean dissolution time

mg milligram

min. minute

ml millilitre

mm Millimetre

MRPs Multidrug resistance proteins

mV milli electron volt

xxxii


MW molecular weight

n release exponent

N normal; number of replicate/samples

nER number of experimental runs

nF number of factors

nL number of levels of the factors

nm nanometre

O/W oil in water

OFAT one factor at a time

p probability

PAS publicly available standard

PDI polydispersity index

PE processing element

PEG polyethylene glycol

P-gp p-glycoprotein

pH power of hydrogen ion concentration

PI prediction index

Pred R2 predicted coefficient of correlation

PS Potato starch

PSA polar surface area

R2 correlation coefficient

RES reticuloendothelial system

RPM revolution per minute

RSD relative standard deviation

RSM response surface methodology

SEM scanning electron microscopy

SLNs solid lipid nanoparticles

SS soluble starch

Tg transition temperature

Tmax time for peak concentration

VIF variation inflation factor

v/v volume per volume

W Watt

W/OW water in oil in water

w/w weight by weight

xxxiii

MENGKAJI POTENSI ZARAH KANJI TEROPTIMUM SEBAGAI

PELANTAR UNTUK PENYAMPAIAN ORAL SATU MODEL DRUG

GASTROLABILE SEFOTAKSIM NATRIUM

ABSTRAK

Penggunaan zarah adalah pendekatan utama untuk sistem penghantaran kejuruteraan

bagi mengatasi rintangan-rintangan di gastrousus. Kanji mempunyai ciri-ciri yang sesuai

diperlukan sebagai satu zarah bahan pembuatan. Kanji mudah dibioteruraikan, selamat,

murah dan banyak walaubagaimanapun, kurang digunakan sebagai bahan penyelidikan,

berbanding biopolymer yang lain. Kerja ini melaporkan kaedah-kaedah yang mudah dan

berinovasi termasuk pengeringan sembur, ultrasonikasi dan pengisaran nano untuk produksi

saiz micron dan submicron zarah kanji. Zarah kanji teroptimum direka melalui kaedah

pemprosesan dan formulasi menggunakan algoritma dengan bantuan komputer.

Ultrasonifikasi gagal menghasilkan zarah kanji secara berulang dengan

menggunakan bantuan pemprosesan. Pengeringan sembur menghasilkan partikel dengan saiz

21.64 ± 1.36 µm (keseragaman 0.56 ± 0.135) bagi kanji jagung. Kaedah pengisaran nano

kelihatan menjanjikan untuk penghasilan partikel submikron secara berulang dengan

kawalan mengenai ciri-ciri zarah melalui modulasiparameter proses dan formulatif . Kanji

kentang menghasilkan partikel bersilang dengan saiz dan indeks serakan berganda (PDI)

masing-masing 200 nm dan 0.25. Potensi zeta untuk kanji kentang adalah > –21 mV. Kanji

terlarutkan yang tidak bersilang menghasilkan partikel yang bersifat kation, anion dan

neutral dengan potensi masing-masing +8.59 ± 0.45, –10.5 ± 0.20 dan 2.00 ± 0.30 mV, saiz

partikel tersebut < 200 nm dan PDI < 0.25. Kanji jagung pula menghasilkan partikel tidak

bersilang dengan saiz sekitar 500 nm dengan PDI > 0.40, iaitu satu nilai yang sedikit tinggi

daripada nilai PDI yang boleh diterima, oleh itu partikel kanji jagung tidak dioptimakan.

Persilangan kanji kentang dan jagung berciri hidrofobik.

xxxiv

Semburan kering kanji jagung, pengisaran nano silang- dan tidak bersilang kanji

kentang dan beranion, kation dan neutral kanji terlarut dipilih untuk kajian in vitro dan in

vivo. Kentang dan kanji terlarut menunjukkan keberkesanan muatan > 9.55%. Kanji jagung

menunjukkan keberkesanan muatan yang rendah iaitu 2.30%, mungkin disebabkan hasil

rendah kanji jagung (27.77 ± 1.48%) berbanding kanji kentang dan kanji jagung (> 84%).

Pertumbuhan saiz zarah telah diperhatikan selepas tempoh waktu tertentu, dalam zarah kanji

terlarut. Pembebasan in vitro zarah mematuhi model tertib sifar dengan eksponen resapan

<1, menggambarkan sifat pembebasan oleh resapan bengkak. Perbandingan bagi sefotaksim

natrium bebas, kanji kentang tidak bersilang dan kanji terlarut kation menunjukkan 4.82 dan

2.59 lipatan peningkatan dalam ketersediaan biohayati, masing-masing. Oleh itu, kanji

mungkin mempunyai potensi bagi menyampaikan gastrolabile drug, sefotaksim natrium dan

dengan itu, untuk meningkatkan keterbiosediaan oral.

Sebagai kesimpulan, partikel kanji bersaiz mikron dan submikron yang

teroptimumkan boleh disediakan menggunakan kaedah fizikal melalui gabungan sains

farmaseutikal dengan kaedah pengoptimuman dengan bantuan komputer. Zarah adalah

mampu untuk meningkatkan keterbiosediaan sefotaksim natrium. Walaubagaimanapun,

kestabilan fizikal zarah perlu terus untuk diperbaiki.

xxxv

EXPLORING THE POTENTIAL OF OPTIMISED STARCH

PARTICULATES AS PLATFORM FOR ORAL DELIVERY OF A MODEL

GASTROLABILE DRUG CEFOTAXIME SODIUM

ABSTRACT

Use of particulates is a prominent approach for engineering delivery systems to

overcome gastrointestinal barriers. Starch has desired properties necessary for a particulate

fabrication material. It is biodegradable, safe, cheap and abundant however, has attracted less

research attention, comparative to other biopolymers. This work reports simple and

innovative top-down physical methods including spray-drying, ultrasonication and

nanomilling for production of micron and submicron size starch particles. The optimised

starch particulates were fabricated with use of processing and formulative aids employing

computer-aided algorithm.

Ultrasonication was unsuccessful in generating reproducible starch particles with

processing aids used. Spray-drying produced particle size of 21.64 ± 1.36 µm (dispersion in

uniformity of particle size 0.56 ± 0.135) for corn starch. Nanomilling seemed promising for

production of reproducible submicron particles with control over particle characteristics by

modulation of process and formulative parameters. Potato starch produced cross-linked

particles with size and polydispersity (PDI) of 200 nm and 0.25, respectively. The zeta

potential of potato starch was > –21 mV. Uncross-linked soluble starch yielded cationic,

anionic and neutral particles with charges +8.59 ± 0.45, –10.50 ± 0.20 and 2.00 ± 0.30 mV,

respectively with particle size < 200 nm and PDI < 0.25. Corn starch uncross-linked particle

size was around 500 nm with PDI > 0.40 which was slightly higher than generally accepted

PDI values, thus corn starch particles were not optimised. Cross-linked potato and corn

starch were somewhat hydrophobic.

The spray-dried corn starch, nanomilled cross- and uncross-linked potato starch and

anionic, cationic and neutral soluble starch were selected for in vitro and in vivo studies.

xxxvi

Potato and soluble starch demonstrated loading efficiency > 9.55 %. Corn starch showed low

loading efficiency of 2.30 %., probably due to its lower yield (27.77 ± 1.48 %) as compared

to that of potato and corn starch (> 84 %). Particle size growth was observed over time

prominently, in soluble starch particulates. The in vitro release of particulates followed zero

order model with diffusion exponent < 1, characterising release by swelling-diffusion.

Comparative to free cefotaxime sodium, the uncross-linked potato starch and cationic

soluble starch showed 5.28 and 3.11 folds increase in bioavailability, respectively. Thus,

starch may have potential to deliver gastrolabile drug, cefotaxime sodium and thereby, to

enhance its oral bioavailability.

In conclusion, the micron and submicron size optimised starch particles can be

prepared using physical methods by blending pharmaceutical science with computer-aided

optimisation. The particulates are capable to enhance bioavailability of cefotaxime sodium.

However, physical stability of particulates is needed to be improved further.

1

CHAPTER 1

GENERAL INTRODUCTION AND AIMS OF STUDY 1.1 THE ORAL ROUTE OF ADMINISTRATION

Among the routes of drug administration, per oral administration is the most

dominant, convenient and preferred portal by which drugs are presented for systemic effects

(Davenport, 1982; Lee and Yang, 2001). It is relatively safe and affords high acceptability,

convenience and maximum compliance, in combination with the relative low cost, simple

and cost-efficient manufacturing process (Lee and Yang, 2001). Nevertheless, the route is

not without limitations, which prohibit the accomplishment of the full benefits of the route

(Mahato et al., 2003). Substantial number of existing and new pharmaceuticals moieties

exhibits poor oral absorption (Amidon et al., 1995; Hoerter and Dressman, 1997; Devane,

1998). Despite of the above limitation, the design of an oral dosage form is usually the first

choice for the new medicinal entities and aims to reformulate and/or optimise dosage forms

of the existing drugs. The objective of the current oral formulation design is to develop a

dosage form capable of absorption into blood in right time and amount after administration

(Macheras et al., 1995). The drug absorption after oral administration is a complex process,

requiring series of events (Ungell, 1997).

1.1.1 Drug absorption after oral route of administration

The small intestine usually is the most favourable site for drug absorption due to its

large surface area endowed by microvilli and, therefore, delivery of drug to this region is

usually a prerequisite for absorption (Davis et al., 1986). The microvilli consist of loose

connective tissue and are supplied with blood and lymphatic system at their base (DeMarco

and Levine, 1969). If absorbed into blood stream, the drug passes through the liver before its

entry into the systemic circulation. However, the drug taken up by lymph accesses the

systemic circulation without passing through liver (O’Driscoll, 1992; Ungell, 1997; Martinez

and Amidon, 2002). Drug absorption after oral administration depends on physicochemical,

pharmaceutical and physiological factors and on the efficiency with which a drug crosses the

2

several barriers prior to reaching into the systemic circulation (Kinget et al., 1998; Ashford,

2002a; Ashford, 2002b; Mahato et al., 2003; Barrett, 2004). In line with the scope of the

present study, only the physiological factors will be discussed under barriers to drug

absorption after oral administration (Section 1.1.2).

1.1.2 Barriers to drug absorption after oral administration

GIT provides a variety of morphological (spatial) and physiological (functional)

barriers to the arrival of a drug to systemic circulation (Ashford, 2002b). Morphological

barriers are mucus and epithelial and endothelial cells while the physiological factors are pH,

enzymes, presystemic biotransformation, enterohepatic circulation and counter absorption

mechanisms (Shargel et al., 2004). The above barriers are given in Figure 1.1.

Figure 1.1: Morphological and functional barriers for drug absorption after oral administration

1.1.2(a) Gastrointestinal tract milieu

The pH, enzymes, secretions and foods in gastrointestinal tract (GIT), together make

the GIT milieu. The pH varies along the length of GIT, from very slightly acidic mouth

region to very acidic region of stomach followed by another change to the neutral pH in the

proximal small intestine (Davenport, 1982). Thus, the drug is exposed to fluctuating pH

3

while it passes along the GIT. The GIT pH influences or completely halts the absorption of

drugs by affecting their chemical stability and ionisation or by acid- or base-mediated drug

inactivation (Lee and Yang, 2001). Pepsin is the primary enzyme in the stomach while

lipase, amylases and proteases are secreted from pancreas into the small intestine

(Davenport, 1982). Drugs, particularly peptides, nucleotides and fatty acids are susceptible to

enzymatic degradation (Lee, 1991; Wilson and Washington, 2001). The presence of food

interferes with the drug absorption by retarding the gastric emptying time, provision of

viscous environment and/or provoking the enzyme and acid secretions (Toothaker and

Welling, 1980; Lee and Yang, 2001).

1.1.2(b) Mucus

Mucus on the lining of GIT epithelial cells is secreted by the mucosal cells of GIT

epithelia. It consists of glycoprotein and glycocalyx (sulphated mucopolysaccharides) and is

hydrophilic which is attributed to 95 % of water it contains (Csaky, 1984). Its most important

property is the viscoelasticity, enabling it to act as a physical diffusion barrier for drug or to

make complexes to some drugs thus, leading to impaired absorption (Gu et al., 1988; Norris

et al., 1998; Ashford, 2002b).

1.1.2(c) Epithelial cells – the mechanical barrier on the lumen side

The small intestine is lined with a single layer of tightly joined columnar epithelial

cells, called enterocytes (Squier, 1992; Lennernas, 1998). Higher molecular weight, lack of

lipophilicity, presence of charge or epithelial binding prohibits translocation of certain drugs

across intestinal epithelium (Gu et al., 1988; Tenhoor and Dressman, 1992; Squier, 1992;

Amidon et al., 1995; Hoerter and Dressman, 1997).

1.1.2(d) Epithelial permeation, transport pathways and mechanisms

The uptake pathways, permeation mechanisms, transporters and efflux pumps have

been regarded as biophysical and biochemical barriers. In the intestinal epithelium, the

4

possible mechanisms for the permeation of drugs are passive diffusion, carrier-mediated and

endocytosis through the paracellular and transcellular pathways (Squier, 1992; Tsuji and

Tamai, 1996). Paracellular drug flux is limited to small molecules and is of limited relevance

in the most cases (Carino and Mathiowitz, 1999). The transcellular route is available for

diffusion of small lipophilic molecules. The hydrophilic compounds are transported by

carrier-mediated transport or by endocytosis (Carino and Mathiowitz, 1999; Ashford,

2002b).

In endocytosis, the particles are internalised by phagocytosis or pinocytosis and this

occurs at specialised regions of membrane. Thus, drug absorption depends upon the region

where they are present (Russell-Jones, 2004; Belting et al., 2005). After internalisation, the

molecule may be acted upon by a range of destructive processes or substances including

proteases and nucleases or is extruded out by efflux pumps. Distribution, affinity, capacity,

specificity and direction of epithelial transporters and pumps play an important role in drug

absorption (Ayrton and Morgan, 2001; Steffansen et al., 2004). Absorption of the following

drugs is particularly affected; a) drugs having affinity to the intestinal transporters and b)

drugs for which the passive diffusion is of no relevance due to higher molecular weight or

low partition coefficient (Tsuji and Tamai, 1996; Ayrton and Morgan, 2001; Daniel, 2004;

Steffansen et al., 2004).

1.1.2(e) Efflux transporters and pumps

The epithelial or endothelial permeability of a drug depends on the membrane

carriers, known as “efflux transporters” which recognize substrate drugs and restrict their

transport. The most documented efflux transporters belong to ATP-binding cassette, super

family of transporters, including P-glycoprotein (P-gp) and multidrug resistance proteins

(MRPs) (Kleinzeller, 1999; Westpahal et al., 2000; Walgren et al., 2000). The P-gp acts as a

barrier by preventing drugs from crossing GIT epithelium into blood or extruding them from

blood into bile. The transporters include a broad structural diversity of substrates and thus,

5

affect several therapeutic classes (Leonard et al., 2002; Chan et al., 2004). The MRPs confer

resistance to substrate lipophilic anions. Many structurally diverse drugs are the substrates

for MRPs (Borst et al., 1999; Bakos et al., 2000; Miller, 2001; Kim, 2003; Kruh and

Belinsky, 2003).

1.1.2(f) Endothelial cells – the mechanical barriers on apical side

The endothelium, a cellular lining of the blood vessels underlying the GIT mucosa

has fenestrations with pores of 80-100 nm size that allow paracellular transport. However,

the presence of P-gp efflux in endothelium is highly restrictive for the transport of drugs

using the transcellular route (Firth, 2002; Feng et al., 2002).

1.1.2(g) Presystemic cellular metabolism

The enterocytes contain phase I drug metabolizing enzymes, dominantly

Cytochrome P450 (Wacher et al., 1996; Watkins, 1997) and hydrolytic and phase II drug

metabolizing enzyme such as, acetyltransferases and sulfotransferases (Krishna and Klotz,

1994; Wacher et al., 1998). The substrate drugs for the Cytochrome P450 (CYP450) enzyme

undergo extensive metabolism after uptake in the intestinal wall (Kolars et al., 1991; Wacher

et al., 1996; Benet et al., 1996; Wacher et al., 1998). The co-localisation of efflux

transporters and metabolizing enzymes in the enterocytes, together with the substantial

overlap in their substrate specificity, seems to indicate the existence of interplay between

drug transporters and metabolizing enzymes suspected to synergize recycling of the substrate

over the apical membrane. This increases the exposure of substrate drugs to the metabolizing

enzymes (Cummins et al., 2002; Benet et al., 2004; Chan et al., 2004).

1.1.2(h) Presystemic hepatic metabolism

The heptoportal blood exposes the drugs to liver on its way to systemic circulation

and the substrate drugs are extensively metabolised via oxidation by CYP450 enzyme or

phase II reactions arising from conjugation with glutathione, sulphate or glucuronic acid.

6

Only CYP3A4, a predominant member of the CYP450 mixed-function oxidase enzyme,

metabolizes 50 % of all the drugs currently prescribed (Evans and Relling, 1999; Clarke and

Jones, 2002). Thus, liver extraction and thereby premetabolism substantially reduces the

systemic availability of susceptible drugs (Arimori and Nakano, 1998).

1.1.2(i) Enterohepatic shunt

The enterohepatic shunt is a cycle of events after drug absorption from intestine,

where a drug secreted into the bile is presented to the intestine for absorption again and then

secretion into bile. This cycle entails the increased persistence of drug in the body or

inactivation of drug (Shargel et al., 2004).

1.2 STRATEGIES TO OVERCOME BARRIERS FOR ORAL DELIVERY

To be able to permeate through intestinal membrane, a drug must have following

biopharmaceutical properties; ability to withstand the insults with appropriate stability in the

hostile GIT environment, appropriate hydrophilicity to dissolve in the gastric fluids and

sufficiently lipophilic to permeate through the cellular barrier (Thompson, 1997).

Furthermore, it should not be a substrate for presystemic cellular or hepatic inactivation of

efflux pumps (Ashford, 2002b). Several strategies have been proposed to generate or

enhance the above biopharmaceutical features in the dosage form for oral delivery. Among

many absorption barriers, the gastric liability has been addressed as one of the most

important barriers in drug absorption (Amidon et al., 1995; Hoerter and Dressman, 1997;

Macheras and Iliadis, 2005). In line with the scope of the present work, only the strategies to

overcome insufficient gastric stability will be discussed.

1.2.1 Approaches addressing gastric liability

The drugs with adverse biopharmaceutical properties are usually delivered through

parenteral route or are enteric-coated (Moes, 1993; Hillery et al., 2001). The parenteral route

is invasive, usually with brief effects and impossible to terminate drug action, if required

7

(Robinson and Lee, 1987; Evers, 1997). The enteric coatings contain indigestible solids and

make product often of considerable size, prohibitive for patients with gastric hypomotility or

pyloric channel narrowing. The location where an enteric coating starts dissolving is

uncertain and release may occur within the small intestine or deep in the colon. The large

inter- and intra-patient variations leads to a varied drug release (Lee and Yang, 2001). To

avoid the above limitations the recent research has been directed towards the use of

convenient alternatives to parenteral route, such as sublingual, pulmonary and vaginal route

for gastric liable drugs (MacGregor and Graziani, 1997) and preferably, to develop

successful oral delivery systems (Lee, 1991; Zhou, 1994; Hillery et al., 2001).

1.2.1(a) Chemical modification – prodrug and drug analogue

A drug can be made enzymatically stable by presenting it as a prodrug with the

substitution of chemical group(s) in the drug molecule (Vilhardt, 1990; Lloyd and Smith,

1998). Recently PEGylisation, a chemical modification approach using conjugation or cross-

linking of drug with polyethylene glycol (PEG) has gained significant interest (Hinds, 2005).

The approach prevents the degradation of PEGylised drug from proteolytic enzymes, reduces

drug clearance rate and offers a shield to uptake by the reticuloendothelial system (RES)

(Roberts et al., 2002). Nevertheless, prodrug strategy might not be adequate because a

prodrug is considered as new chemical entity, thus requires the preclinical and clinical

investigations. Therefore, approaches involving the change of formulation or engineering of

the drug absorption are feasible (Hillery et al., 2001).

1.2.1(b) Lipidisation strategies

Lipidisation involves the formation of drug in appropriate pre-solubilised lipid

phase, which by the metabolism of the lipids in GIT facilitates drug absorption

(Humberstone and Charman, 1997). It may be achieved by incorporation of drug into lipid

vehicle such as oils, surfactants, dispersions, micelles of mixed bile salt, liposomes, or

emulsifying systems (Gershanik and Benita, 2000; Frokjaer et al., 2005). Such systems have

8

reported to improve bioavailability due to impeded gastric emptying time, increased

permeability and facilitated lymphatic uptake mediated by special carriers, chylomicrons and

very low density lipoproteins in epithelial cells (Sernka et al., 1979; Schilling and Mitra,

1990; Macheras et al., 1995).

1.2.1(c) Polymeric coating, encapsulation or entrapment

Coating with, encapsulation and entrapment of drug in polymer is used to protect the

gastric labile drugs. Depending upon polymeric materials used, they may form coating film,

hydrogels and matrices (Grahan and McNeil, 1984; Kopecek et al., 1992; Brondsted and

Kopeck, 1992). Several pH-dependent polymeric materials are available which are stable at

acidic pH and dissolve in the intestinal or colonic pH. Thus, coating with such polymer

protects the gastric unstable drugs (Touitou and Rubinstein, 1986; Saffran, 1991). Hydrogels

are the cross-linked hydrophilic polymers able to swell in aqueous environment. This

swellability is controllable by pH, degree of cross-linking and temperature. Hydrogels

encapsulated drugs can be targeted to specific pH and thus, are attractive as drug delivery

systems able to protect drugs at acidic pH (Frokjaer et al., 2005).

1.2.1(d) Drug targeting to the lymphatics

Oral route has proven feasible for the lymphatic uptake of lipophilic drugs and their

subsequent access to the systemic circulation without passing through liver. The total flow

rate of the lymph in the thoracic duct is approximately 100-200 ml/hr, 500 times less than the

blood flow in portal vein (O’Driscoll, 1992; Porter and Charman, 2001). Thus, the lymph

portal may be used to achieve sustained drug release and to avoid first pass metabolism. The

endothelial layer of lymphatic capillaries is discontinuous or without basement membrane

thus, allowing a free permeation, independent of the molecular size (Hastewell et al., 1991).

A drug with molecular weight greater than 16,000 reaches systemic circulation

through the lymph (Porter and Charman, 2001; Swartz, 2001; Feng et al., 2002). Delivery

9

systems can selectively redirect drug absorption into the lymphatics. Drugs in lipid-based

particles or oil enhances lymphatic uptake, while macromolecules and colloidal particles,

after endocytosis or persorption (Figure 1.2) may enter the lymphatic system through clefts

in the terminal vessels (Muranishi, 1997; Nishioka and Yoshino, 2001; Wassan, 2002).

1.2.1(e) Drug targeting to the Peyer's patches

Peyer's patches, concentrated in the ileum are the collection of large lymph tissues in

the intestinal mucus-secreting lining and appear as the elongated thickened areas lacking villi

(Figure 1.2). The patches are associated with the immune system and contain large number

of lymphocytes. Specialised antigen-presenting epithelial cells, called modified epithelia (M)

cells cover the patches. Features of M-cells, important in connection with drug absorption

can be discriminated from that of the enterocytes. The M-cells have apical microfolds and

lack or if have, under-developed microvilli and the mucus covering is assumed to be

attenuated to allow antigenic uptake perhaps by adsorptive and/or receptor-mediated process.

Thus, M-cells are capable of relatively extensive macromolecular endocytosis (Eldridge et

al., 1990; Walgren et al., 2000). The phagocytotic potential of lymphocytes provides a

concentration gradient for drug permeation.

Figure 1.2: Microvilli showing epithelial cells and underneath two circulatory systems, blood and lymphatic stream. A special transport mechanism, persorption is also shown

10

Targeting the Peyer’s patches helps an enhanced drug uptake into lymphatics that

depends on particles size, surface hydrophobicity and availability of specific targeting

ligands (Eldridge et al., 1990; Muranishi and Yamamoto, 1994). Particle with size range of

50-3,000 nm are taken up by the Peyer's patches and through the lymphatics, subsequently

translocated to blood. The particles of size 0.5–1.0 µm are phagocytised by lymphocytes,

which migrate to the draining lymph nodes (Jani et al., 1992; Randolph et al., 1999; Jung et

al., 2000). The particles of size 3-10 µm are often retained within the Peyer's patches.

Particles of size 2-3 µm exhibit the maximal phagocytosis (Champion et al., 2008). A higher

uptake is generally observed for more hydrophobic microparticles. Monoclonal antibodies

which bind to the specific M-cells-bound carbohydrate residues may increase

microparticulates uptake (Eldridge et al., 1990).

1.2.1(f) Targeted drug delivery to the colon

A couple of strategies have been explored for targeting drug to the colon. One of the

colonic drug delivery strategies is based on the design of a prodrug which is metabolised by

enzymes found exclusively in the colon (Lee and Yang, 2001; Chourasia and Jain, 2003).

Azoreduction of polymeric coating is a further important approach used for targeted drug

delivery to the colon. The colonic anaerobic bacteria reductively cleave azo bonds leading to

the disruption of the polymer network in the coating to release drugs (Van Den-Mooter et al.,

1992). Coating of azo polymeric systems onto pellets and solid unit dosage forms is another

novel approach and shown to promote the oral administration of insulin and desmopressin

(Saffran, 1991; Kopecek et al., 1992).

Hydrogels are reported to have excellent persistence properties in the lower part of

the rectum and hence demonstrate a potential of enhancing the colonic absorption of drugs

(Grahan and McNeil, 1984; Brondsted and Kopecek 1992; Wilson et al., 1997). Dosage

forms using pH-sensitive polymers and hydrogels have also been used for the drug targeting

to desirable pH (Saffran et al., 1986; Chiu et al., 1999; Khan et al., 1999). Some other

11

approaches such as conjugation with glycoside, glucuronide, cyclodextrins, dextrans, amino

acid and time-release system and bioadhesive systems have also been reported for the

colonic drug delivery by Chourasia and Jain (2003). Impermeability of the colonic epithelia

for drugs and long transit time of the colon are the major limitations for colonic drug

delivery (Sangalli et al., 1999; Lee and Yang, 2001; Washington et al., 2001).

1.2.1(g) General use of particulate drug carriers

The colloidal particulates are becoming increasingly prominent in the novel drug

delivery systems because of their potential for drug delivery, targeting, protecting gastric

labile drugs and in parallel, protecting the GIT from drug toxicity and capacity for

permeation into membranes (Ferrari, 2005). They are translocated in enterocytes, Peyer’s

patches and M-cells simultaneously by para- and intra-cellular pathways and subsequently,

gain access to lymphatics (Walgren et al., 2000; Feng et al., 2002). Several particulate

carrier systems have been described including liposomes, nanoparticles, microparticles,

microspheres, dendrimers and Ferro fluids (Artursson et al., 1984; Levy and Andry, 1990;

Fournier et al., 1994; Jameela and Jayakrishnan, 1995). In accordance with the scope of the

present study, the particulates will be emphasised more in Section 1.3.

1.3 PARTICULATE DELIVERY SYSTEMS

There is no agreed international definition of a nanoparticle, though Kreuter (1983a),

Couvreur et al. (1986) and Allemann et al. (1993) have defined nanoparticles as the particles

ranging in size from 10-1000 nm. A review of the internet (http://www.sciencedirect.com

accessed on 27 May, 2006), revealed that the majority of the researchers are partial towards

the above definition. However, according to the document PAS71 by Publicly Available

Standard in collaboration with British Standards Institution, a nanoparticle is a particle

having one or more dimensions of the order of 100 nm or less with the properties different

from that of the bulk material (http://www.malvern.co.uk; accessed on 27 May, 2006). The

nanoparticles are typically made of a single material in which a drug is entrapped,

12

encapsulated or adsorbed onto the surface (Kreuter, 1993; Kreuter, 1994). Generally, the

spherical formulation with a particle size above 1 µm, with a distinguishable coating and

core regions are referred to as microcapsules while matrix type structures are the

microspheres or microparticles. Micelles are self-assembled block copolymers that form a

hydrophilic layer and an inner hydrophobic core or vice versa. Dendrimers are

monodispersed symmetric macromolecules with a large number of reactive end groups

(Passirani and Benoit, 2005). The terms used to describe particulate carriers vary among the

authors but herein the “particulate” will collectively be used for the systems of colloidal

particulate intended for drug delivery. However, the references sustain the original

terminology used by the authors.

1.3.1 Liposomes

Liposomes are microscopic vesicles composed of one or more lipid bilayers

surrounding an aqueous space (Gregoriadis, 1991). Phospholipids are the most commonly

used materials of fabrication for liposomes (Knight, 1981; Barratt, 2000). Both water and

lipid soluble drugs can be entrapped within the aqueous compartment or lipid bilayers,

respectively (Betageri el al., 1993a; Sharma and Sharma, 1997). Surface modification has

improved the stability of liposomes in the gut and has facilitated permeation of drugs from

gastrointestinal mucosa (Morgan and Williams 1980).

Liposomes protect drug against enzymatic degradation and improve drug

hydrophilicity (Al-Meshal et al., 1998; Bayomi et al., 1998; Weiner and Chiang, 1988; Kisel

et al., 2001). Liposome-entrapped cefotaxime sodium has been reported to be a potential

delivery system for oral route as indicated by its enhanced bioavailability (Betageri et al.,

1993b; Rojers and Habib, 1999; Ling et al., 2006). The phospholipids are readily hydrolysed

or oxidised, thus liposomes require storage under nitrogen atmosphere. The liposomes are

not compatible with several coatings of surfactants and are instable against the bile salts

(Betageri et al., 1993c; Sharma and Sharma, 1997).

13

1.3.2 Solid lipid nanoparticles

Solid lipid nanoparticles (SLNs) are the colloidal carrier for lipophilic drugs and

composed of a high melting point lipid as a solid core coated with surfactants (Gassco, 1993;

Muller et al., 1995; Chen et al., 2001). SLNs, the promising alternative delivery system to

emulsions, liposomes and polymeric nanoparticles, can be administered through parenteral,

oral or dermal route (Siekman and Westesen, 1992; Gassco, 1993) and share all the

advantages of above in one carrier (Schwarz and Mehnert, 1999; Muller et al., 2000).

1.4 GENERAL METHODS FOR PREPARATION OF PARTICULATES

Nano- and microparticulates are formed by bottom-up or top-down procedures, the

former procedure being used more frequently. In bottom-up procedure, the particulates are

produced by polymerisation of monomers (dispersion polymerisation) or by shaping or

condensation of macromolecules (coacervation, spray-drying, or solvent evaporation).

1.4.1 Bottom-up procedures

1.4.1(a) Emulsion/dispersion polymerisation

In emulsion-polymerisation, particles are formed by chemically induced

polymerisation of monomers dispersed in a continuous phase (Kreuter, 1983a). The

polymerisation media may be aqueous or organic, employed for water-insoluble and soluble

monomers, respectively (Gassco and Trotta, 1986; Krause et al., 1986). The polymer, on

reaching certain molecular weight, form particles and becomes insoluble, leading to phase

separation (Kreuter, 1982). The chemical initiator may be ammonium or potassium

peroxodisulphate, bases or basic drugs (Kreuter, 1990; Kreuter 1992).

The surfactants solubilise or disperse the insoluble monomers in continuous phase

and stabilize the particulates while the cross-linker links the monomers (Schwarts and

Rembaum, 1985). Emulsifiers are not mandatory for a hydrophilic macromolecule since it

acts as dispersant (Kreuter, 1983a; Al-Khouri Fallouh et al., 1986). The drugs can either be

14

incorporated in particles during polymerisation process or adsorbed onto the formed

nanoparticles (Allemann et al., 1993). The limitations of the applications are the conditions

and solvents with respect to the stability of incorporated drug, drug-polymer reaction and the

practically difficult removal of contaminants, un-reacted monomers or reaction initiator

residues from the product (Fessi et al., 1989; Quintanar-Guerrero et al., 1996).

1.4.1(b) Preparation from preformed polymers

Use of the preformed and biodegradable macromolecules to prepare particulate

carriers may prevent the problems associated with emulsion-polymerisation method. The

principal methods for production of particulates from preformed polymers are

emulsification-solvent evaporation, precipitation, or denaturation (Tice and Gilley, 1985;

Fessi et al., 1989). Solvent evaporation is usually referred to as the phase separation when a

water insoluble polymer is in question, while the term coacervation is used for separation of

water-soluble macromolecules (Fessi et al., 1989; Jeffery et al., 1991; Jeffery et al., 1993).

I Emulsification-solvent evaporation

In the emulsification-solvent evaporation, a polymer dissolved in a water-immiscible

volatile organic solvent is emulsified in the aqueous medium to form oil-in-water emulsion.

The subsequent evaporation of the organic solvent, by heating or reduced pressure solidifies

the organic phase droplets to particles. Drug is incorporated during the production as

dissolved or suspended in the polymer solution in the organic solvent (Tice and Gilley, 1985;

Al-Khouri Fallouh, 1986). The final product may require centrifugation, washing and

lyophilisation (Krause et al., 1985; Jeffery et al., 1991).

Double emulsion solvent-evaporation, a modification of the emulsification solvent

technique improves the loading efficiency of hydrophilic drugs but yields relatively larger

particle size. In this approach, an aqueous drug solution is first emulsified in an organic

solution of polymer to yield primary emulsion, which then is poured to the larger volume of

15

aqueous phase (Blanco-Prieto et al., 1994; Rafati et al., 1997; Delie et al., 2001). Limitations

of the method are the same as those mentioned for emulsion polymerisation (Verrecchia et

al., 1995).

II Coacervation (phase separation) method

Coacervation involves a separation of a polymer from its solution and deposition

around a drug to be coated. In this process, a synthetic polymer is dissolved in a water-

immiscible organic solvent and the separation is induced by the addition of another organic

solvent or oil (Ruiz et al., 1989; Sampath et al., 1992). The polymer may also be dissolved in

a water miscible solvent and then poured into a non-solvent (usually water with surfactant).

The polymer separation is also induced by a change of surface charge by ionic gelation,

temperature, or pH and addition of salt (salting out) or incompatible polymer (Calvo et al.,

1997). Addition of desolvating agents causing the so-called salting out phenomenon can also

be used to form coacervate. The organic solvent is evaporated under a reduced pressure

(Oppenhiem et al., 1984). Hardening is achieved by addition of a cross-linker, thus,

purification from hardening agent is a disadvantage of this method (Fessi et al., 1989).

Recently, a complex coacervation or polyelectrostatic complexation method has been

reported for the preparation of nanoparticles in which the polymer-polymer or polymer-drug

complex is formed by electrostatic interactions (Liu et al., 2007; Bayat et al., 2008a; Bayat et

al., 2008b).

1.4.1(c) Solvent deposition/displacement (nanoprecipitation)

Nanoprecipitation is a simple, one-step and straightforward technique for

preparation of nanoparticles. It is carried out with two miscible solvents, the first one must

be the solvent ideally for both polymer and drug while the second is a non-solvent for both.

Addition of polymer solution to the non-solvent leads to nanoprecipitation by a rapid

diffusion of polymer-containing solvent into the dispersing medium (also called desolvation

of polymer). The drug is encapsulated in the polymer (Fessi et al., 1989; Bilati et al., 2005).

16

1.4.1(d) Emulsification-cross-linking

In this method, the aqueous solutions of polymer and of the drug to be incorporated

into the particles are emulsified in oil, which result in the formation of droplets. The droplets

can then be hardened by addition of cross-linkers or by denaturation of the molecules at high

temperatures (Gupta el al., 1986; Kreuter, 1992). The addition of surfactants stabilizes the

emulsion and helps re-suspension of the final dry nanoparticles in water (Widder et al.,

1979; Gupta et al., 1986).

1.4.1(e) Desolvation of macromolecules

Particles can be produced after dissolution of relatively hydrophilic copolymers or

macromolecules in water-miscible solvents. The solutions of the polymer and of the drug to

be entrapped in the above solvents are then poured into water, resulting in the spontaneous

formation of particles due to desolvation. The particles produced by this method however,

are not redispersible in water after spray- or freeze-drying (Bodmeir et al., 1991). Addition

of desolvating agents results in precipitation of macromolecules or coacervation as a new

phase. The polymer may be gelatine, human serum albumin, bovine serum albumin, casein

and ethyl cellulose. In some cases, surfactants are required in order to solubilise certain

drugs or to facilitate the redispersion of the final freeze-dried product (Marty et al., 1978).

1.4.2 Supercritical fluid technology

Use of organic solvents/or some chemical reaction stabilizing the colloid structure

and thus, the need of extensive purification can be avoided by use of supercritical fluid

technology. The supercritical fluid technology yields organic solvent-free and pure particles

thus, is highly environment-friendly. In this technology, the solution of material of interest in

a supercritical fluid is expanded through a nozzle. An abrupt decrease in the solvent power

on expansion results in precipitation of solute (Tom and Debenedetti, 1991). Nevertheless,

insolubility of polymers (MW >10,000 D) in supercritical fluid had made this technique

impractical (Tom et al., 1994).

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1.4.3 Top-down procedure

Top-down procedures employ the physical energy or stress to reduce the particle

size of a coarse polymer. Extrusion and wet-grinding (nanomilling) are the two common

physical methods and require the coarse polymer to be grinded in a dispersion medium

containing surfactants, stabilizers and other adjunctive materials. Size reduction is achieved

by shearing forces (tearing apart), influencing forces (crushing and impinging) and attrition

(tearing-impacting combined) (Yilmaz et al., 2001).

1.4.3(a) Extrusion

Extrusion is employed by an instrument, called extruder that has two counter

rotating screws through which the material is passed to reduce the size (Patent No WO

00/40617, 2000). Extrusion is cost effective, environment-friendly technology and thus, is an

attractive method to encapsulate a great variety of bioactive substances in starch matrices.

Yilmaz et al. (2001) has comprehensively reviewed the use of extrusion for this purpose.

Perhaps, use of a higher volume is a dominating reason for its limited use in the current

research.

1.4.3(b) Wet-grinding (nanomilling)

Wet-grinding or nanomilling, using media-mills requires small volume, this coupled

with efficiency in reducing the particle size and use of wet-grinding is gaining attraction in

size reduction. So far, application of the wet-grinding has been confined only to the material

research and there are a few reports on its use for pharmaceutically applicable nanomaterials

(Merisko-Liversidge et al., 1996; Merisko-Liversidge et al., 2003).

1.4.4 Materials of fabrication of particulates

Natural polymeric materials or their derivatives such as dextran, maltodextrin,

mannan, or other starch derivatives, albumin and gelatine have been studied for developing

particulate delivery systems (Schroder et al., 1985; Artursson et al., 1987; Artursson et al.,

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1988; Fahlvik et al., 1990). However, the synthetic polymeric materials are usually preferred

due to their consistent properties, biodegradability, biocompatibility and compatibility with

drugs. Copolymers such as poly (lactic-co-glycolic acid) or poly(lactic acid)-polyethylene

oxide, have been well-documented (Vert, 1986; Kissel and Koneberg, 1996). Majeti and

Kumar (2000) reviewed materials currently used in preparation of particulate materials.

Some of the synthetic materials are costly while others have some degree of toxicity

(Kreuter, 1992; Kreuter, 1993; Kreuter, 1994). Several features of starch, stated in Section

1.6.4 have motivated the use of starch as the fabrication material.

1.5 GENERAL APPLICATIONS OF PARTICULATES

1.5.1 Oral delivery of peptides, gastrolabile drugs and vaccines

Presenting the gastric instable drugs in particulates offers gastric stability, improves

cellular uptake and due to their access to lymphatics, avoids first pass effects (Gautier et al.,

1992; Bayat et al., 2008a Bayat et al., 2008b). Particulate-bound gastric labile and water

insoluble drugs exhibit enhanced bioavailability (Eldridge et al., 1990; Jani et al., 1992).

Recently, Pereira (2008) demonstrated an enhanced absorption of Eudragit-loaded

cefotaxime sodium in rats. A drug in nanoparticles may have a long-term effect due, in part

to the formation of a depot in the gut wall, Peyer's patches, lymphatic system and/or other

body compartments (Grangier et al., 1991).

1.5.2 Targeting infected and inflamed cell

The nanoparticles are readily taken up by phagocytic cells, Kupffer cells and

monocytes (Lam and Mathison, 1982), making targeting possible for nanoparticle-bound

drug to these cells and the alveoli with an improved drug efficacy (Eltahawy, 1983). The

infected reticuloendothelial system (RES) cells with human immunodeficiency virus or

leishmaniasis and inflamed cells show preferentially higher phagocytosis of nanoparticles-

bound drugs (Mizushima, 1985; Fouarge et al., 1989; Meltzer et al., 1990; Losa et al., 1991).

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1.5.3 Targeting cytostatics to tumours

The particulates accumulate in a number of tumours, may be due to their increased

bioadhesiveness towards the tumours (Gipps et al., 1986; Alpar et al., 1989; Illum et al.,

1989). It may also be attributed to endocytosis of particles by endothelial cells lining the

tumour vasculature or escape of particles through leaky or open blood vessels (Couvreur et

al., 1982; Widder et al., 1983; Gupta et al., 1989). Nanoparticles have prolonged persistence

time in a variety of tumours and metastases with enhanced efficacy and decreased toxicity of

a number of cytostatics (Brasseur et al., 1980; Couvreur et al., 1986; Ferrari, 2005).

1.5.4 Ophthalmic delivery

Ocular drug in a particulate delivery system increases the residence time of drug as

compared to the conventional ocular delivery systems that have fast washout due to rapid

tear turnover, lachrymal drainage and dilution of tears (Lee and Robinson, 1979; Losa et al.,

1993; Zimmer et al., 1995). The persistence time of drug can be further enhanced by using a

strong bioadhesive substance for nanoparticle fabrication (Gurny et al., 1987).

1.5.5 Vaginal drug delivery

Microparticulate systems have been investigated for drug and vaccine delivery

through vaginal mucosa (Okada, 1991; Hagan et al., 1993). Starch microspheres, 40 µm in

diameter, were shown to be capable of enhancing the vaginal absorption of insulin (Okada,

1991).

1.5.6 Enhancing drug safety

Due to the selective localisation in the infected cell and tumours, the particulate drug

delivery systems reduce drug delivery to the non-targeted tissues (Lam and Mathison, 1982;

Eltahawy, 1983). Thus, such systems have potentials to improve efficacy, safety and reduce

the chances of drug resistance (Fouarge et al., 1989; Schafer et al., 1992).

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1.5.7 Adjuvants for vaccines

Nano- and microparticles claim most of the desired features that the vaccine carriers

must have (Wikingsson and Sjoholm, 2002), thus are promising for use as adjuvant for

vaccines (Chen et al., 1998; Singh et al., 2000; Briones et al., 2001). Their slow degradation

offers long persistence in body, prolonged contact of antigen with immunocompetent cells

and thus able to maintain a long immunity (Kreuter et al., 1986; Hedley et al., 1999;

Herrman et al., 1999; Cui and Mumper, 2003).

1.6 STARCH AS THE MATERIAL OF PARTICULATE FABRICATION

Starch, the material of fabrication for particulate carrier in this study is the major

carbohydrate reserve in plant. It is a semi-crystalline material extracted from a variety of

sources, maize being the predominant followed by wheat potato and rice barley (Buléon et

al., 1998; De-Baere, 1999; Gordon, 1999; Wesslén and Wesslén, 2002). Chemically, starch

is a polysaccharide that consists of repeating glucose units (Li and Yeh, 2001; Singh et al.,

2003). Starch is abundantly available, cheap and is widely used polymer. The properties of

native starch, being dependent upon the source are inconsistent but the commercially

available starch is with consistently uniform quality (Gordon, 1999) as freely flowing white

coloured powder.

Starch has found diverse and wide range of applications in pharmaceutical and non-

pharmaceutical fields. It is used as staple food, raw material (Aime et al., 2001), source of

energy (calories), thickener, gelling agent (Aime et al., 2001; Tester and Karkalas, 2002) and

as a dietary fibre (www.Isbu.ac.uk/water/hysta.html; Accessed on 7 December, 2005). In

pharmaceutical field, starch is used conventionally as a thickener, tablet binder, diluent,

disintegrant, stabilizer, gelling agent and as a drug carrier. In topical preparations, it is used

as dusting-powder for its absorbency , as a protective covering in ointment formulations and

as an emollient (Bayazeed et al., 1989; Whaley et al., 1999; Schmidt et al., 2001; Jeffcoat et

al., 2002; Lind et al., 2002).

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1.6.1 Starch composition and molecular structure

Though varied in structure and composition, each starch granule primarily contains

blends of two polyglucans, amylose and amylopectin, both being associated through

hydrogen bonding and arranged in layers to form granules (Hovenkamp-Hermelink et al.,

1987; Tomlinson et al., 1997; Li and Yeh, 2001; Vorwerg et al., 2002).

1.6.1(a) Amylose

Amylose is mostly linear fraction consisting of about 500 – 20000 α-D-glucose units

linked through α (1→ 4) linkage and accounting for 20-30 % of the total starch (Li and Yeh,

2001). It has lower molecular weight (105–106 g/mol) but has a relatively extended shape

with radius of 7-22 nm and generally tends to wind up into single or double helical junction

zones. Hydrogen bonding is also present between the aligned chains (Morrison et al., 1986;

Morrison et al., 1993a; Hoover, 2001).

1.6.1(b) Amylopectin

Amylopectins are branched fractions containing short chains linking linear chains

via α (1→ 6) linkages, forming branching point at one residue in every twenty and some

cases, every thirty residues, thus making it a huge but compact molecule with molecular

weights between 106 and 107 g/mol (Hoover, 2001; Li and Yeh, 2001). Each amylopectin

molecule contains up to two million glucose residues and has a hydrodynamic radius of 21-

75 nm (Parker and Ring, 2001). It accounts for 70-80 % of total starch. Hydrogen bonding

between sequential residues encourages a helical conformation in amylopectin (French et al.,

1984; Li and Yeh, 2001; Parker and Ring, 2001).

1.6.1(c) Molecular arrangement of amylose and amylopectin

Amylose and amylopectin molecules are oriented as alternating semi-crystalline

(dark) and amorphous layers, forming concentric regions in granules. In semi-crystalline

layer, the ordered regions compose of double helices of short amylopectin branches. The

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amorphous regions of the semi-crystalline layers and the amorphous layers are composed of

amylose and non-ordered amylopectin branches (Yamaguchi et al., 1979; Jenkins et al.,

1993; Donald et al., 1997; Gallant et al., 1997; Baldwin et al., 1998; Baker et al., 2001;

Parker and Ring, 2001).

1.6.1(d) Other minor components

Starch may have some minor components such as lipids, fatty acids, phosphorus and

proteins (Morrison, 1993a; Lim et al., 1994; Hizukuri et al., 1996; Jane et al., 1996; Ellis et

al., 1998; Tester and Karkalas, 2002).

1.6.2 Types of starches

Based on source, the starches are grouped as corn (maize), potato, rice, tapioca and

wheat. Depending on contents, starches are waxy when the amylose to amylopectin ratio is

low (≈ 15 %), normal when amylose represents ≈ 16-35 % and high-amylose when amylose

content exceed ≈36 % (Morrison, 1993b). The common corn starch has 25 % amylose while

the waxy corn starch is almost totally made up of amylopectin. The granules of high-amylose

starch tend to be smooth and have a narrower size range. Potato starch has about 20 % of

amylose (Jane et al., 1994).

1.6.2(a) Modified starches

Starch is modified to enhance and expand its functionality and is achieved

chemically, enzymatically, physically, or by biotechnological approach due to rupture of all

or part of starch granules (Jobling, 2004). Physical modification can be achieved by milling

to produce damaged starch granules. Pregelatinised starch is obtained using a physical

method by the simultaneous gelatinisation and drying of aqueous starch dispersions. In this

process, heating of starch in excess water induces many physical changes such as loss of

crystallinity, swelling and rupturing of starch granules (Blanshard, 1987; Tester, 1997).

Gelatinised starch is easily dispersed in cold water. Extrusion of starch is another physical

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method (Giezen et al., 2004). Other methods include enzymatic modification by α- β-

amylase and amyloglucosidase are used to hydrolyse starch. The cross-linked starches are

produced by chemical modification (Tester and Karkallas, 2002).

1.6.3 Functionality of starch

1.6.3(a) Gelatinisation

On heating starch in excess water, starch granules absorb water and while hydrogen

bonding between amylose and amylopectin maintains granule integrity, it begins to swell

from hilum (centre). Granules lose organised structure, crystallinity and become amorphous.

Ultimately, granule structure is completely lost resulting into a thin paste (<4 %) or gel (>4

%). Typically, this process begins at 45°C, peaks at 60°C and completes at 75°C.

Gelatinisation renders starch easy deformation and complete digestibility. Degree of swelling

dictates the extent of starch water binding capacity (Jane et al., 1996; Li and Yeh, 2001;

Rosa, 2004; Tester et al., 2004). After gelatinisation starch, which is otherwise water

insoluble, becomes water soluble (Dziechciarek et al., 1998; Jane et al., 1999; Tester and

Sommervillie, 2003). The common solvents for starch are sodium or potassium hydroxide

and dimethyl sulfoxide (Radosta et al., 2001). Higher amylose content, owing to the

extensive hydrogen bonding, requires more energy to gelatinise the starch. Thus, high

amylose starch have less swelling power and higher gelatinisation temperatures (Li and Yeh,

2001; Singh et al., 2003). The presence of phosphate groups in starch increases its swelling

power. Certain salts such as calcium chloride and lithium chloride promote gelatinisation of

starch at room temperature (Jane, 1994; Jane et al., 1999; Pan and Jane, 2000).

1.6.3(b) Hydrocolloidal functionality

Starch functions as hydrocolloid due to the presence of amylose and amylopectin.

The extended and loose helical amylose chains have a relatively hydrophobic inner surface.

Similarly, helical structure in amylopectin may present continuous hydrophobic surface

(King, 1995; Whaley, 1999).

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1.6.3(c) Thickening agent

Viscosity is purely a function of molecular weight of substances. The branched

structure of amylopectin with all attached chains yields much larger molecule than amylose.

Consequently, it builds higher viscosity thus the waxy starches have higher viscosity.

Amylopectin interferes with the interaction between amylose chains and its solution that can

lead to an initial loss in viscosity followed by a more slimy consistency. After gelatinised,

the swollen granules increase the viscosity of dispersion (Bayazeed et al., 1989).

1.6.3(d) Gel and film formation

In a solution, amylose molecules easily align themselves with one another and

associate through hydrogen bonding to form gels or films. This property is useful for several

applications such as coating and for the inclusion of fats and flavours in starch (Shinsato et

al., 1999). In general, higher degree of polymerisation (length of amylose in starch) gives

greater gel strength due to its increased ability to associate through hydrogen bonding. High

amylose starches show better film forming properties. Gelatinisation creates more tendencies

for association of granules to form gels and films (Roper and Koch, 1990; Zeller et al., 1999;

Haugaard et al., 2001).

1.6.3(e) Retrogradation

Tendency of re-association of the gelatinised starch molecules with one another on

cooling and thereby, forcing water out of the molecule, causing starch to recrystallise is

called retrogradation. This unique property of starch is due to the presence of amylose.

Branched amylopectin impedes retrogradation but the long branched amylopectin are

susceptible to retrogradation. Mixing with carrageenan, alginate and xanthan gum and low

molecular weight sugars can reduce retrogradation. On release of water, the aligned chains

may form double stranded crystallites that are resistant to amylase and due to extensive inter-

and intra-strand hydrogen bonding makes hydrophobic structure of low solubility (Shogren

et al., 1992; www.lsbu.ac.uk/water/hysta.html: Accessed on 7 December, 2005).

EXPLORING THE POTENTIAL OF OPTIMISED STARCH...

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