MECHANISMS OF POLYMER-Ca2+ INTERACTION

AND THEIR EFFECTS ON THE

CHARACTERISTICS OF ALGINATE MICROSPHERES
AND FILMS

LEE HUEY YING

B.Sc. (Pharm.) Hons, NUS

A THESIS SUBMITTED

FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

DEPARTMENT OF PHARMACY

NATIONAL UNIVERSITY OF SINGAPORE

2006
ACKNOWLEDGEMENT

I would like to say a big Thank You to my supervisors A/P Paul Heng Wan

Sia and A/P Chan Lai Wah for their guidance and help throughout the course of my

research work. I have really learnt a lot from their scientific knowledge and advice as

well as the correct approach in problem solving.

I am grateful to the National University of Singapore for providing the

research scholarship which allowed me to do a higher degree.

I would also like to express my thanks to all my friends and laboratory mates

who have made laboratory work enjoyable and providing suggestions to my work.

Special thanks especially to Aik Jiang who has lend a listening ear with great patience

all these years and always provide me with the emotional support and encouragement

to move on.

Finally, I really appreciate the understanding of my parents and husband these

years without which all these would not be possible.

Huey Ying, 2006

ii
 Table of contents
Acknowledgement ii

Table of contents iii

Summary xi

List of tables xiii

List of figures xv

I. Introduction 1

A. Alginates 1

A1. Sources of alginates 1

A2. Composition and structure of alginates 1

A3. Properties of alginates 3

A3.1. Solubility of alginates 3

A3.2. Stability of alginates 4

A3.3. Ionic binding of alginates 5

A3.4. Interaction of alginates with other cationic 8

polymers and substances

A3.5. Stability of cross-linked alginate matrix 9

A3.6. Biocompatibility and biodegradability of 10

alginates

A4. Applications of alginates 10

B. Production of alginate microspheres by the emulsification 12

technique

B1. Microsphere production by emulsification 12

B2. Factors influencing the properties of alginate 14

iii
 microspheres produced by emulsification

B2.1. Influence of the concentration of alginate 14

B2.2. Influence of alginate composition and viscosity 16

of alginate solutions

B2.3. Influence of cross-linking method and type of 19

cross-linker used

B2.4. Influence of concentration and amount of cross- 25

linker and viscosity of cross-linking solution

B2.5. Influence of the duration of cross-linking 26

B2.6. Influence of the type, composition and 27

concentration of surfactants

B2.7. Influence of drug load on encapsulation 29

efficiency and drug release

B2.8. Influence of incorporating polymers and 31

additives

B2.9. Influence of the nature of drug encapsulated 35

C Investigation of the influence of natural polymers on the 36

properties of alginate matrix

C1. Production of alginate films 36

C2. Natural polymers that interact with calcium ions 36

C2.1. Pectins 36

C2.1.1. Chemical structure and sources of 36

pectins

C2.1.2. Physical and gelation properties of 38

pectins

iv
 C2.1.3. Applications of LM pectin 39

C2.2. Gelling carragenans: Iota carragenan and kappa 39

carrageenan

C2.2.1. Chemical structure and sources of 39

carragenans

C2.2.2. Physical and gelation properties of 40

carrageenans

C2.2.3. Applications of κ-carrageenan and ι- 41

carrageenan

C2.3. Gellan gum 42

C2.3.1. Chemical structure and source of 42

gellan gum

C2.3.2. Physical and gelation properties of 42

gellan gum

C2.3.3. Applications of gellan gum 44

C3. Blending of natural polymers with alginates 44

II Hypotheses and objectives 46

III Experimental 49

A. Materials 49

A1. Polymers 49

A2. Surfactants and hardening agents 49

A3. Continuous phase of emulsion 49

A4. Reagents and chemicals used in cross-linking 50

A5. Drugs 50

v
 A6. Reagents used in digestion of films and microspheres 51

A7. Reagents and chemicals used in media preparation 51

A8. Reagents used in laser diffractometry for sizing of 51

calcium carbonate particles

A9. Reagents and chemicals used in acid hydrolysis and 51

carbon 13 nuclear magnetic resonance (13 C-NMR)

spectroscopy

B. Methods 52

B1. Size reduction of calcium carbonate 52

B2. Sizing of calcium carbonate particles 52

B3. Acid hydrolysis of sodium alginates 54

B4. Determination of polymer composition and sequence 54

13
parameters by C nuclear magnetic resonance

spectroscopy

B5. Preparation of partially cross-linked alginate in solution 54

B6. Preparation of alginate microspheres 55

B6.1. Preparation of externally cross-linked and 55

partially cross-linked alginate microsperes

B6.2. Preparation of internally cross-linked alginate 57

microsperes

B7. Preparation of alginate micropellets 57

B8. Size analysis of microspheres 60

B9. Evaluation of aggregate size and degree of agglomeration 60

of microspheres

B10. Separation of batch of microspheres into different size 61

vi
 fractions

B11. Determination of drug content 61

B12. Determination of drug release profiles 61

B13. Preparation of sodium alginate films 62

B14. Preparation of calcium carbonate-sodium alginate films 64

B15. Preparation of films of pectin, gellan gum, carrageenan 64

and their blends with sodium alginate

B16. Preparation of cross-linked films 66

. B16.1. External and internal cross-linking of films 66

using calcium carbonate

B16.2. Cross-linking of films with calcium chloride 66

solution

B17. Determination of film thickness 66

B18. Determination of film weight before and after cross- 69

linking

B19. Determination of mechanical properties of films 69

B20. Assessment of morphology and surface topography of 70

films

B21. Determination of film opacity 70

B22. Determination of extent of water uptake and apparent 72

solubility of films

B23. Determination of percent weight increase due to moisture 72

sorption in climatic chamber

vii
 B24. Determination of film permeability 74

B25. Infra-red spectroscopy 75

B26. Atomic absorption spectrophotometry 75

B26.1. Washing of films and microspheres to remove 75

excess surface calcium

B26.2. Sample digestion and analysis 76

B27. Viscosity determination of sodium alginate solutions 77

IV Results and discussion 78

Part I. Influence of viscosity and uronic acid composition on the 78

properties of alginate films and microspheres produced by

emulsification

A Film study 78

B Microsphere study 85

Part II. Influence of different cross-linking methods on the 98

properties of alginate microspheres produced by emulsification

and alginate matrices

A. Production of alginate microspheres by the 98

emulsification/external cross-linking method

A1. Preparation of partially cross-linked alginate for 98

microsphere production

A2. Properties of microspheres produced from the partially 101

cross-linked alginate

B. Properties of alginate microspheres by the 102

emulsification/internal cross-linking method

viii
 B1. Determination of the amount of CaCO3 and volume of 102

glacial acetic acid needed

B2. Treatment of alginate microspheres produced 104

C. Mechanisms of external and internal cross-linking of the 107

alginate matrix

C1. Alginate film as a matrix model 107

C1.1. Process parameters for cross-linking 107

C1.1.1. Type of cross-linking agent used 108

C1.1.2. Amount of cross-linking agent used 108

C1.1.3. Medium employed for cross-linking of 108

alginate matrices

C1.2. FTIR of alginate films 109

C1.3. Morphology, surface topography and calcium 111

content of alginate matrices

C1.4. Thickness and weight of the alginate films 114

C1.5. Mechanical properties of alginate films 117

C1.6. Apparent solubility of alginate films 119

C1.7. Permeation study of alginate films 121

C2. Alginate micropellets 124

C2.1. Formation and morphology of calcium alginate 124

micropellets

C2.2. Drug content and drug release profiles of 125

calcium alginate micropellets

Part III. Influence of incorporating polymers on the properties of 130

ix
 the alginate matrix

A. Light transmittance, thickness and percent weight loss of films 130

and FTIR spectroscopy

B. Tensile properties of films 151

C. Apparent solubility in deionised water and extent of water 157

uptake of films

D. Apparent solubility of films in USP HCl buffer pH 1.2 and USP 161

phosphate buffer pH 6.8

E. Film permeation study 163

V Conclusion 167

VI References 170

VIII Publications and presentations arising from this study 194

x
Summary

This study involves an investigation of the effects of Ca2+-polymer interaction

on the properties of alginate matrices. Parameters investigated include different

grades of alginates, different cross-linking techniques and incorporation of natural

polymers into alginate.

Alginate microspheres were successfully produced using sodium alginate with

different viscosities and uronic acid composition. The viscosity of sodium alginate

was found to exert a greater influence on the microsphere properties than its uronic

acid composition. Generally, larger microspheres with higher drug contents and

slower drug release rates were produced from alginates of higher viscosity. The

influence of polymer composition on the above mentioned properties was variable.

The different alginate microspheres were then sieved to obtain microspheres within a

defined size range for further characterization. The viscosity of the alginates was

found to influence the microsphere properties to a greater extent than the alginate

composition. Viscosity of the alginates also affected its extent of cross-linking with

Ca2+, with higher extent of cross-linking observed in alginates of higher viscosity.

These less well cross-linked microspheres generally exhibited lower drug contents

and slower rate of drug release. The effect of microsphere size was also studied by

sieving the alginate microspheres into several size fractions. Smaller microspheres

showed greater encapsulation efficiency and faster drug release rates.

The influence of different cross-linking techniques was also studied.

Externally and internally cross-linked microspheres as well as microspheres from

partially cross-linked calcium alginate were produced. Internally cross-linked

microspheres were weak as they were adversely affected by matrix disruption due to

CO2 release. There was little difference in the drug release rates of externally cross-

xi
linked microspheres and those produced from partially cross-linked calcium alginate,

despite good drug encapsulation efficiencies. Subsequent studies were carried out to

investigate the different cross-linking mechanisms using films as a model matrix.

Films produced by both external and internal cross-linking methods had similar extent

of cross-linking but the internally cross-linked films had rougher surfaces, lower

matrix strength, stiffness and were less permeable to drugs as compared to the

externally cross-linked ones. Calcium alginate micropellets were used as a model to

examine the effects of different cross-linking mechanisms on the properties of

calcium alginate particles, including their encapsulation efficiency. The different

cross-linking kinetics gave rise to different distribution of the cross-linkages in the

matrix. This difference in structural aspect of the matrix contributed to the differences

in matrix properties observed.

The type and amount of copolymer added to alginate influenced the matrix

properties. Composite films of alginate were successfully produced with gellan gum,

carrageenans and pectin. These copolymers interacted with the alginate via Ca2+.

Pectin, ι-carargeenan and gellan gum were able to produce cross-linked films showing

high light transmittance with sodium alginate. Cross-linked composite films with

increasing amount of pectin or gellan gum showed greater change in tensile properties

after cross-linking. The stability of the alginate matrix in acidic conditions was

enhanced by the addition of these copolymers, hence these composites might be

potentially useful in enteric applications. The hydrophilicity of the alginate matrix

was also improved with the addition of carrageenans.

xii
List of Tables

Table list Table caption Page

Table 1 Median particle size of calcium carbonate batches 53

Table 2 Parameters used for production of alginate 58

microspheres by emulsification/external cross-linking
method

Table 3 Summary of conditions employed in drug assay 63

Table 4 Composition of films studied 65

Table 5 Films prepared by cross-linking with calcium carbonate 67

Table 6 Composition and sequence parameters of the different 68

types of alginate studied

Table 7 Conditions used in the determination of apparent 73

solubility and permeability of films

Table 8 Viscosity of alginate solutions, calcium ion contents of 79

cross-linked alginate films and changes in film weight
before and after the cross-linking process

Table 9 Tensile properties of alginate films 81

Table 10 Properties of alginate microspheres 87

Table 11 Release parameters of alginate microspheres in 92

different media

Table 12 Drug content and drug release parameters of MP 95

microspheres of different size fractions

Table 13 Viscosities of alginate solutions subjected to high shear 99

with/without partial cross-linking and the properties of
resultant microspheres

Table 14 Surface roughness, mechanical properties and calcium 112

content of dried calcium alginate films

Table 15 Thickness and weight of dried alginate films 115

Table 16 The hydration index (WH) and permeability coefficient 123

(P) of calcium alginate films

Table 17 Drug content of calcium alginate micropellets 127

xiii
Table 18 The influence of film thickness of SA films on light 131
transmittance

Table 19 Light transmittance of films 133

Table 20 Thickness of films before and after cross-linking 135

Table 21 Hydration indices of films 159

Table 22 Permeation coefficients of cross-linked films 164

xiv
List of figures

Figure Figure caption Page

list

Figure 1 Structural characteristics of alginates: (a) alginate 2

monomers (b) chain conformation (c) block
distribution

Figure 2 (a)The egg-box model for binding of divalent cations to 6

alginates. The cations are represented by circles.
(b) Possible chelation of the Ca2+ by the G residues

Figure 3 (a) Molecular structures of (a) pectin (b) κ- carrageenan 37

(c) ι-carrageenan; (d) gellan gum

Figure 4 Outline of methods employed in the preparation of 56

alginate microspheres. (* mixture/solution dispersed in
isooctane and cross-linked with CaCl2.2H2O. ‘
indicated the start of addition of CaCl2.2H2O solution)

Figure 5 Preparation of microspheres emulsification/internal 59

cross-linking method

Figure 6 A section of the profile of the roughness curve obtained 71

by scanning probe microscopy

Figure 7 Influence of Ca2+ content of cross-linked alginate films 82

on tensile strength and elastic modulus

Figure 8 Percentage change in tensile properties of films after 84

cross-linking with viscosity of sodium alginate solution
used

Figure 9 Size distribution of alginate microspheres produced by 86

emulsification

Figure 10 (a) Blank alginate microspheres. (b) Alginate 90

microspheres containing sulphaguanidine (indicated by
bright drug crystals present in the microspheres)

Figure 11 Changes in t25% and t50% with increased Ca2+ content of 93

alginate microspheres

Figure 12 Influence of Ca2+ content on the rate of drug release of 97

S microspheres

Figure 13 Photographs of (a) SA (b) S1 (c) S2 and (d) S3 101

microspheres

xv
Figure 14 Setup used in determination of amount of CaCO3 and 103
volume of glacial acetic acid needed

Figure 15 Photographs of freeze-dried microspheres produced 106

with (a) 0.5 g and (b) 0.8 g of CaCO3 obtained using a
light microscope under 400x magnification

Figure 16 FTIR spectra of alginic acid and alginate matrices 110

Figure 17 Scanning probe microscope images of alginate films 113

Figure 18 Apparent solubilities of externally cross-linked and 120

internally cross-linked alginate films at 37 ºC

Figure 19 Drug permeation profiles of calcium alginate films 122

Figure 20 Calcium alginate micropellets produced by extrusion 126

through a 20 gauge needle tip (S) or silicon tube with a
diameter of 2 mm (B) and cross-linked by external
(Ext) and internal (Int) gelation methods

Figure 21 Release profiles of acetaminophen from calcium 128

alginate micropellets

Figure 22 Percent change in (a) film thickness and (b) film 134
weight after the cross-linking

Figure 23 FTIR spectra of alginic acid, uncross-linked and cross- 137

linked SA films

Figure 24 Calcium contents of polymeric films after cross-linking 139

Figure 25 FTIR spectra of pectinic acid, uncross-linked and 140

cross-linked SA, PT and AP films

Figure 26 Schematic representation of possible interactions 142

between pectin and alginate with calcium ions ({)

Figure 27 FTIR spectra of uncross-linked and cross-linked SA, 145

KC and AK films

Figure 28 FTIR spectra of uncross-linked and cross-linked SA, IC 147

and AI films

Figure 29 FTIR spectra of uncross-linked and cross-linked SA, 150

GG and AG films

Figure 30 Tensile strength of uncross-linked ( ) and cross-linked 152

( ) films and the % difference ( ) in tensile strength

xvi
 of the film after cross-linking

Figure 31 Elastic moduli of uncross-linked ( ) and cross-linked 153

( ) films and the % difference ( ) in elastic moduli
of the film after cross-linking

Figure 32 Extent of moisture sorption of uncross-linked ( ) and 154

cross-linked ( ) film placed in climatic chamber.

Figure 33 Apparent solubilities of films in deionised water ( ) 158

and USP buffer pH 1.2 ( )

xvii
I. Introduction

A. Alginates

A1. Sources of alginates

Alginates are obtained from many species of brown algae such as Laminaria

hyperborea, Macrocystis pyrifera, Laminaria digitata, Ascophyllum nodosum and

Lessonia nigrescens. It is the most abundant polysaccharide in brown algae,

comprising up to 40 % of the dry matter. In the algae, the alginate component is

located in the intercellular matrix as a gel containing sodium, calcium, magnesium,

strontium and barium ions. Alginates can also be obtained from bacterial sources.

Bacteria belonging to the Azobactor and Pseudomonas species, such as Azobactor

vinelandii and Pseudomonus aeruginosa, are able to produce alginates (Wingender et

al., 2001; Saude et al., 2002).

A2. Composition and structure of the alginates

Alginates belong to a family of linear binary copolymers of (1→ 4) linked β-

D-mannuronic acid (M) and α-L-guluronic acid (G) residues in varying proportion

and sequence (Figure 1). The composition and sequential structure vary with the

seasonal and growth conditions of the algae sources (Draget, 2000). The M and G

monomers exist in the 4C1 and the 1C4 conformations respectively (Figure 1)

(Grasdalen et al., 1977). The 1C4 conformation of the guluronic acid residues results

in glycosidic linkages that are diaxial in the G blocks. This configuration hinders

rotation around the glycosidic linkage which accounts for the stiff and extended

nature of the alginate chain. Polymer chains with a high content of G residues were

found to be stiffer than polymer chains with a lower content of G residues

(Whittington, 1971). The work by Haug, Larsen and Smidrød (Haug and Smisrød,

1
 (a)
OH
OH OH
-OOC -OOC
O O OH
HO
OH
HO
OH

Mannuronic acid (M) residue Guluronic acid (G) residue

(b)

-OOC OH OH -OOC
-OOC
O HO
O OH O O O OH
O HO O
-OOC OH O
O
OH
O
OH O -OOC OH

G G M M G

(c)

MMMMGGGGGGGMMGMGMGGGGGGGGGMMMMM

M - block MG - block G -block

Figure 1. Structural characteristics of alginates: (a) alginate monomers (b) chain

conformation (c) block distribution.

2
1965; Haug et al., 1966, 1967a) provided the earliest information about the structural

sequence of the alginates. From these studies, it was concluded that alginate is a block

copolymer consisting of homopolymeric regions of M and G, also known as M blocks

and G blocks, respectively, interspersed with regions of heteropolymeric blocks of

MG (Figure 1). In a series of papers, it was shown that alginates have no regular

repeating unit (Painter et al., 1968; Smidsrød and Whittington, 1969; Larsen et al.,

1970). Detailed information of the alginate structure became available with the use of

high resolution 1H and 13

C NMR-spectroscopy (Grasdalen et al., 1979, 1981) in the

study of alginates. These techniques enabled the determination of monad frequencies

(FM and FG), the four nearest neighbouring (diad) frequencies (FGG, FMG, FGM and

FMM) and the eight next nearest neighbouring (triad) frequencies (FMMM, FMMG, FGMM,

FGMG, FGGG, FGGM, FMGG and FMGM). These frequencies allow the calculation of the

average G-block length larger than 1, which has been found to correlate well with the

gelling properties of the alginates.

The molecular weight distribution of alginates in a batch is influenced by the

processing processes of the alginates, where depolymerisation of varying degree may

occur. The molecular weight distribution is known to affect the properties of

alginates. For example, fragments of low molecular weights containing only short G

blocks may not participate in gel network formation, hence resulting in reduced gel

strength (Moe et al., 1995).

A3. Properties of alginates

A3.1. Solubility of alginates

The solubility of alginates is dependent on the pH and ionic strength of the

solvent as well as the presence of gelling ions. It was found that the pKa values of

3
mannuronic acid and guluronic acid are 3.38 and 3.65 respectively (Haug, 1964).

When alginate comes in contact with a medium of pH value lower than its pKa, it is

converted to alginic acid, which is insoluble in water (Draget et al., 1994). Alginic

acid was found to form a gel by slow and controlled release of the protons (Draget et

al., 1994; 1996). The pH value that causes precipitation of the alginates is dependent

on the composition and sequential structure of the polymer (Draget, 2000).

Any change in ionic strength of an alginate solution will influence polymer

chain extension and hence the solution viscosity. At high ionic strengths, the

solubility is affected and resulting in salting out effect. Sodium alginate may be

precipitated from a salt solution if the added salt concentration is greater than 1.4

mol/L or 0.7 mol/L of the electrolytes, KCl or NaCl respectively (Zhang et al., 1998).

The presence of gelling ions, such as Ca2+, Zn2+ and Al3+, at appropriate

concentrations will lead to the formation of an insoluble cross-linked alginate.

Alginate can form a gel (or precipitate at low polymer concentration) in the presence

of divalent ions at concentrations of > 0.1 % w/w (Yalpani, 1988). In studies on the

swelling behaviour of dry sodium alginate powder in aqueous media with Ca2+ it was

found that, almost all the alginate existed within the supernatant when the

concentration of Ca2+ was below 3 mM. In contrast, almost no alginate (1-3%) was

present in solution when the concentration of Ca2+ exceeded 3 mM (Draget, 2000).

A3.2. Stability of alginates

Sodium alginate in its dry powder form has a shelf life of up to several months

when stored in a dry, cool place without exposure to sunlight. The molecular weight

of the alginates can remain unchanged with years of storage in a deep freezer. The

viscosity of alginate solutions may be greatly reduced during storage by the action of

4
microorganisms present. Alginates are also sensitive to pH changes. Haug et al.

(1963, 1967b) reported decreased alginate stability at pH below 5 or above pH 10, due

to degradation by proton-catalyzed hydrolysis and β-alkoxy-elimination respectively.

Presence of reducing agents, such as phenolic compounds, was found to affect the

stability of alginates (Smidsrød et al., 1963). Alginates as dry powder or in solution

undergo depolymerization when subjected to sterilization by autoclaving, ethylene

oxide treatment or γ-radiation (Leo et al., 1990).

A3.3. Ionic binding of alginates

The ion binding capacity of alginate is important for its gelling property. The

selectivity of alginates for polycations is affected by their composition. It was

observed in equilibrium dialysis of alginates that the selectivity for alkaline earth

metal ions increased markedly with increasing content of G residues in the chains

(Smidsrød and Haug, 1968; Haug and Smidsrød, 1970; Smidsrød, 1974). The affinity

for these cations was found to increase in the order of Ba2+ > Sr2+ > Ca2+ >> Mg2+

(Haug, 1964). The M and MG blocks are also involved in interactions with polyvalent

cations, but to a lesser extent and are non-selective (Morris et al., 1978; Donati et al.,

2005).

The difference in selectivity for the alkaline earth metal ions suggested that the

mode of binding was unlikely to be mediated by nonspecific electrostatic interaction.

This phenomenon was eventually explained by the ‘egg-box’ model, based on the

conformation of the linkages involving the G residues (Figure 2) (Grant et al., 1973).

According to this model, the chain assembly is induced by the presence of divalent

cations, such as Ca2+, and junction zones are formed between helical chains of G

sequences that present cavities suitable for accommodating divalent cations in a

5
 (a)

(b)

O
-O
Ca2+ OH
O O
OH
O
O
O OH

O
OH
O

Figure 2. (a)The ‘egg-box’ model for binding of divalent cations to alginates. The
cations are represented by circles. (b) Chelation of the cation, Ca2+, by the G residues.

6
chelate type of binding (Figure 2). These junctions, which were described as

microcrystalline dimers (Morris et al., 1978), were found to consist of very few

laterally associated chains in circular dichronism and electron microscopic studies

(Smidsrød, 1974). The ‘egg-box’ model is widely cited as the mode of interaction

responsible for the cross-linking and gel-forming properties of the alginates.

However, the alginates produced by Pseudomonas aeruginosa were found to possess

gel forming properties despite the lack of long G blocks. This had prompted a review

of the interactions between cross-linking cations and alginate. The ability of the Ca2+

to produce a gel, with alginate that was composed strictly of alternating sequences,

had provided a new insight into the involvement of long MG blocks in the gel

network. It was proposed that junction formation can occur between long MG blocks

(Donati et al., 2005). It was also suggested that mixed GG/MG junctions were

formed, with the macroscopic outcome of a remarkable increase in Young’s modulus

for epimerized alginate samples characterized by the presence of long alternating

sequence (Donati et al., 2005). The binding of divalent cations to alginate had also

been studied by Siew et al. (2005) to further understand the gelation mechanism in

alginates. It was proposed that specific binding of a divalent cation to alginate resulted

in local charge reversal and transformation of the polyelectrolyte into a

polyampholyte, with subsequent association of these positively charged segments

with anionic groups on other chains, forming dimerss. This led to a three dimensional
13
network (Siew et al., 2005). It was observed from C-NMR studies that sol-gel

transition in connection with Cu2+, Co2+ and Mn2+ could not be explained by the egg-

box model as these cations showed low selectivity for the G residues of the alginates

(Wang et al., 1993). It was suggested that the sol-gel transition was characterized by a

complex formation in which only the carboxyl groups in both M and G residues were

7
coordinated to the cation and there was no significantly selective binding with the G

residues (Wang et al., 1993). Polarized optical microscopy revealed the magnitude of

metal ion – alginate polymer chain binding in the following order: Pb2+ > Cu2+ > Ca2+

> Zn2+ (Yokoyama et al., 1992).

For trivalent and tetravalent metal cations such as Cr3+, Al3+, Se4+ and Th4+,

the cross-linking involves more binding sites. It was suggested that the trivalent

cations coordinate with two adjacent carboxylic units and one carboxylic unit of a

different chain while the tetravalent cation chelate with two adjacent carboxylic

groups of one chain and another two groups of a different chain (Hassan, 1993).

A3.4. Interaction of alginates with other cationic polymers and substances

Alginates being anionic are able to interact with cationic polymers and

substances to form a polymer complex. The most commonly used polycations to

interact with alginates are chitosan (Gåserød et al., 1998b) and poly-L-lysine (Thu et

al., 1996). Chitosan and poly-L-lysine have been widely utilized in the encapsulation

of cells and enzymes (Stabler et al., 2001; Taqieddin and Amiji, 2004) and in coating

of alginate matrices to control drug release (Thu et al., 1996; Ribeiro et al., 2005) and

improve its bioadhesiveness (Gåserød et al., 1998a). Furthermore, chitosan-alginate

fibres have also been explored for potential use as wound dressings (Knill et al.,

2004).

Other cations that have been reported to interact with alginates include poly-L-

ornithine (Darrabie et al., 2005), poly (methylene co-guanidine) (Hearn and Neufeld,

2000), polyethyleneimine (Schneider et al., 2001; Halder et al., 2005), cetrimide

(Abletshauser et al., 1993), poly (vinylamine) and poly (allylamine) (Wang, 2000).

These polycations have been used to cross-link the alginates to improve their

8
mechanical strength (Wang, 2000) and to modulate drug release from alginate

matrices (Halder et al., 2005). They have also been utilized in coating processes

(Abletshauser et al., 1993), cell and enzyme encapsulation (Hearn and Neufeld, 2000;

Schneider et al., 2001).

A3.5. Stability of cross-linked alginate matrix

Loss of matrix integrity can occur when the cross-linking ions such as Ca2+ are

removed from the matrix in the presence of sequestering ions and chelating agents.

They include ethylenediaminetetraacetic acid (EDTA), ethylene glycol-bis(β-

aminoethyl ether) N, N, N’, N’-tetraacetic acid (EGTA), and salts of phosphate,

lactate and citrate. These compounds are able to interact with polyvalent cations and

form strong complexes with them. In solutions with a high concentration of non-

gelling ions such as Na+ and K+, the cross-linking ions could be displaced from the

cross linked matrix by these non-gelling ions resulting in reduced matrix integrity

(Draget et al., 1998; Wang and Spencer, 1998).

In water, erosion of the matrix is insignificant because the reversal of the

gelation by cation loss does not occur (Vanderbossche and Remon, 1993). Østberg et

al. (1994) suggested that the Ca2+ involved in cross-linking of alginate beads

remained in the beads during dissolution test in deionised water. This kept the beads

intact during the entire dissolution test. In simulated gastric fluid, the cross-linked

alginate was converted to alginic acid due to displacement of Ca2+ by H+ (Østberg et

al., 1994). No change in bead size was observed as alginic acid is stable below pH 2.

However, partial solubilization occurred, due to leaching of soluble alginate from the

beads at pH 4 (Draget et al., 1996). At pH 4.5 and above, solubilization increased

markedly (Draget et al., 1996). The increased instability of alginic acid with

9
increasing pH was also noted by Yotsuyanagi et al. (1991). The authors observed that

the physical integrity of alginic acid beads was maintained at pH below 2 but as the

pH of the medium was raised, the alginic acid beads swelled in a sigmoid manner

with bead disintegration occurring around pH 3.5 (Yotsuyanagi et al., 1991). In

alkaline phosphate buffer, the Ca2+ will be sequestered by phosphate ions leading to

matrix disintegration (Østberg et al., 1994).

A3.6. Biocompatibility and biodegradability of alginates

Alginates have been used in the development of many dosage forms due to

their low toxicity, biocompatibility (De Vos et al., 2003) and biodegradability (Ueng

et al., 2004). Absence of significant biological response was observed after two years

of implantation of alginate microcapsules containing pancreatic islets in rats (De Vos

et al., 2003). Alginate dressings (Suzuki et al., 1999) and beads (Ueng et al., 2004)

were also able to undergo bioresorption when implanted in vivo, eliciting only mild

host reaction.

A4. Applications of alginates

Alginates have been widely used in the food industry as additives to improve

the stability and modify the texture of a variety of foods. They are used as viscosity

enhancers, gel formers and stabilizers for aqueous mixtures, dispersions and

emulsions in the formulation of foods such as ice creams, syrups and jams (Moe et al.,

1995). Propylene glycol alginate (PGA) is used as a stabilizer for acid emulsions,

acidic fruit juice and drinks where unmodified alginate would precipitate. PGA is also

used to stabilize beer foam (Moe et al., 1995).

10
 Alginate matrices can be easily produced without the use of organic solvents

under mild room temperatures (Gombotz and Wee, 1998). Alginates are useful for

the development of encapsulating matrix and carrier vehicle due to their good gelling

properties. Alginates had been used as a carrier for a number of drugs, such as

paracetamol, theophylline, chloramphenicol (Østberg et al., 1994) and nicardipine

hydrochloride (Acartürk and Takka, 1999). They were also used in the fabrication of

an implant for the prevention or treatment of bone infections (Yenice et al., 2002).

The high porosity of the matrices makes alginates particularly suitable for

encapsulation of macromolecules. Large protein molecules such as transforming

growth factor-β (Mierisch et al., 2002), endothelial cell growth factor (Ko et al.,

1997), bovine serum albumin (BSA) and haemoglobin (Huguet and Dellacherie,

1996) were successfully encapsulated by alginates for controlled delivery. Alginates

were also successfully used to encapsulate enzymes such as dipeptidylpeptidase

(Mittal et al., 2005) and lipase (Won et al., 2005), hormones such as melatonin (Lee

and Min, 1996) and nucleic acids (Quong et al., 1998; Mittal et al., 2000)

Calcium alginate is one of the most common materials used for cell

immobilization. Entrapment within alginate matrix involves a safe, simple and

economical method without the need for organic solvents. In addition, the alginate

matrix possesses several unique properties, such as presence of a relatively inert

aqueous environment within the matrix and the dissolution and biodegradation of the

matrix under normal physiological conditions (Gombotz and Wee, 1998). The

alginate matrix also exhibits good mechanical stability (Ponce et al., 2005). Thus,

they are preferred in the encapsulation of yeast cells for use in fermentation processes

(Jamai et al., 2001), insulin secreting cells for use as artificial pancreas (De vos et al.,

11
2003), and parathyroid cells for potential parathyroid transplantation (Picariello et al.,

2001).

Alginate/antacid preparations are also useful for the treatment of reflux

oesophagitis. Gas bubbles released from the antacid upon contact with gastric acid are

trapped by the alginate gel and the resultant raft layer floats on top of the stomach

contents, thus protecting the oesophagus from acid reflux (Johnson et al., 1997a).

Alginates possess bioadhesive properties and are utilized in many bioadhesive

preparations (Choi et al., 2000; Richardson et al., 2004). Alginate fibers are readily

produced by extruding alginate solutions into cross-linking ions and dried for use in

wound dressings (Qin, 2004; Hilton et al., 2004). The wide range of uses for alginates

testifies to its great potential to be used in many innovative and exciting applications.

A thorough understanding of the properties and behaviour of the alginate matrices

under different conditions is necessary to exploit this polymer to its full potential.

B. Production of alginate microspheres by the emulsification technique

B1. Microsphere production by emulsification

One of the most common techniques used to produce alginate microspheres

involves the extrusion of alginate into a solution of cross-linking ions (Won et al.,

2005). This ionic gelation process has been used extensively to prepare alginate beads

(Bajpai and Sharma, 2004; Won et al., 2005). However, it was less suitable for the

production of small alginate microspheres (Türkoglu et al., 1997). The use of an

extrusion device with a smaller bore to produce small microspheres could be easily

clogged up by drug particles, cells or other macromolecules suspended in the alginate

solution. Therefore, the emulsification technique offers a simple and attractive method

to produce microspheres, with a particle size of less than 150 µm (Wan et al., 1992)

12
and with potential for industrial scale up (Poncelet et al., 1992b). Moreover, alginate

microspheres produced by the emulsification method have smaller pores and more

uniform surfaces compared to alginate microparticles produced by

extrusion/ionotropic gelation (Fundueanu et al., 1998). High encapsulation

efficiencies could be achieved by the emulsification technique (Wan et al., 1992;

Chan et al., 1997). However, the principal drawback of the emulsification method is

the resultant broad size distribution of the microspheres produced (Poncelet et al.,

1992a).

The emulsification process involves the dispersion of an aqueous solution of

the polymer containing the substance(s) to be encapsulated as fine globules in an

immiscible continuous phase. The microspheres are formed by interaction of the

polymer in the globules with the cross-linking agent. The cross-linking agent can be

added as an aqueous solution into the emulsion, giving rise to the external cross-

linking method which is also known as the external gelation method (Chan et al.,

2000; Chan et al., 2002). Alternatively, the cross-linking agent is released from an

insoluble form within the dispersed polymer globules giving rise to the internal cross-

linking method which is also known as internal gelation method (Poncelet et al.,

1992a). Microencapsulation by emulsification provides a rounding action to the

progressively densified microspheres through high speed stirring of the reacting

materials. Microspheres of pectin (Wong et al., 2002a; Wong et al., 2002b) and

chitosan (Thanoo et al., 1992; Shu and Zhu, 2001; Dhawan et al., 2004) can also be

easily produced by emulsification. This technique of microencapsulation using

alginates had been used for the encapsulation of drugs such as metoprolol tartrate

(Rajinikanth et al., 2003), adriamycin (Li et al., 2002), theophylline (Wan et al.,

1992), diclofenac sodium (Gürsoy and Çevik, 2000) and sulphaguanidine (Chan et al.,

13
2002), ‘short-life’ oil soluble drugs (Ribeiro et al., 1999), wheatgerm oil and evening

primrose oil (Chan et al., 2000). Large molecules such as BSA (Lemoine et al., 1998)

and haemoglobin (Ribeiro et al., 2005), as well as subtilisin for use in detergent

formulations (Chan et al., 2006) and glucose oxidase (Srivastava et al., 2005) were

also encapsulated using this technique. The emulsification technique using alginates is

also useful for entrapment of cells such as Bacillus Calmette-Guérin (Esquisabel et

al., 1997) in alginates for potential use as vaccines. The small alginate microspheres

produced by this method of encapsulation are potentially useful for nasal

administration (Lemoine et al., 1998; Rajinikanth et al., 2003) to deliver drugs with

high hepatic first pass effect (Rajinikanth et al., 2003) as well as for arterial

embolization in the treatment of haemorrhagic diseases of the kidneys and renal

cancer (Li et al., 2002).

B2. Factors influencing the properties of alginate microspheres produced by

emulsification

B2.1. Influence of the concentration of alginate

As the encapsulating matrix for microspheres, the concentration of alginate

solution used has an important influence on the properties of the microspheres

produced. Wan et al. (1990) reported that clumps of very small microspheres were

produced by emulsification with a low alginate concentration of 1 %w/w. The use of

higher concentrations of alginate solutions (2-3 %w/w) formed discrete and spherical

microspheres. However, a further increase in alginate concentration resulted in the

formation of elongated and irregular microspheres. These observations were attributed

to the effect of viscosity of the polymer solution on the efficiency of dispersion.

Therefore, viscous solutions with high concentrations of alginates would be difficult

14
to disperse and should be avoided. Similar observations were also made by Lemoine

et al. (1998) and Rajinikanth et al. (2003) for alginate-hydroxypropylmethylcellulose

(HPMC) microspheres produced by emulsification. Clumping of the small alginate-

HPMC microspheres was present with lower concentration of alginates. The

microspheres obtained were found to exhibit a higher degree of discreteness and were

larger in size when higher concentrations of alginate were used (Lemoine et al., 1998;

Rajinikanth et al., 2003). Conflicting results were obtained for beads/microspheres

produced by the extrusion/ionotropic gelation method (Aral and Akbuğa, 1998;

Pepeljnjak et al., 1994).

In addition to microsphere size and morphology, the concentration of the

alginate also exerts an influence on the encapsulation efficiency of the microspheres

produced by emulsification. The encapsulation efficiency was found to increase

proportionally with the sodium alginate concentration in alginate-HPMC

microspheres (Rajinikanth et al., 2003). The authors suggested that the instantaneous

cross-linking of Ca2+ with the alginate enhances entrapment of the drug within the

alginate matrix. Moreover, larger microspheres produced as a result of increased

alginate concentration also allowed for the entrapment of greater amount of drug.

Changes in alginate concentration have a significant impact on the release

profile of microspheres produced by emulsification. The initial burst effect from

microspheres was considerably reduced and significant decrease in the rate and extent

of drug release with increased alginate concentration was also observed (Rajinikanth

et al., 2003). Increasing the concentration of alginate also improves the mechanical

strength of the microspheres obtained. Alginate-based microspheres containing

titanium oxide and microcrystalline cellulose produced by emulsification/internal

15
gelation were found to exhibit greater mechanical strength with increased

concentrations of alginate (Chan et al., 2006).

B2.2. Influence of alginate composition and viscosity of alginate solutions

The intrinsic viscosity of alginate solutions for a given concentration is

affected by the molecular weight of the polymer and the flexibility of the polymer

chain (Johnson et al., 1997b). Chain flexibility is influenced by the alginate

composition. The decrease in chain flexibility arises from a greater restriction in

movement about the carbon-oxygen bonds joining the monomers. Parts of the chain

containing predominantly G blocks are less flexible than those containing

predominantly M blocks which are in turn stiffer than regions of alternating M and G

(Smidsrød et al., 1973). Since the cross-linker, Ca2+ , preferentially interacts with the

G blocks of the alginate, it would be expected that the alginate composition can also

affect the properties of the microspheres produced.

Gürsoy and Çevik (2000) reported a significant increase in microsphere size

when alginates of higher viscosity at a concentration of 2 % w/v were used to produce

microspheres by emulsification. The microspheres produced from higher viscosity

alginates were also found to exhibit higher drug contents and encapsulation

efficiencies (Gürsoy and Çevik, 2000). However, the alginates with a higher viscosity

used also had a higher G content. Therefore, it was not clear as to whether besides

alginate viscosity, the composition of the alginates had influenced the results

observed. Contrary to the above findings (Gürsoy and Çevik, 2000), Gürsoy et al.

(1999) using the same type of alginates did not observe significant differences in drug

contents and encapsulation efficiencies between microspheres produced from low and

high G alginates. Thus, the conflicting results obtained make it difficult to draw

16
conclusions on the exact influence of alginate viscosity and composition on the

properties of the microspheres produced by emulsification.

In another study, Poncelet and co-authors (Poncelet et al., 1992a; 1995; 1999;

Poncelet, 2001) obtained larger microspheres with high G alginates, as compared to

low G alginates, by emulsification/internal cross-linking technique. Poncelet (2001)

reported that alginate composition was an important parameter in alginate bead

formation and premature gelation occured faster with high G alginates in microsphere

production by the emulsification method. However, the high G alginates used in the

studies also had a higher viscosity than the low G alginates, with the exception of one

low G alginate that had a higher viscosity (Poncelet et al., 1992a; 1995; 1999;

Poncelet, 2001). It was also reported that alginate viscosity in the range of 100 to

>2000cp (for a 1.8 % alginate solution) had little effect on the mean size of the

microspheres produced by emulsification/internal cross-linking method (Poncelet et

al., 1995). This further suggested that the alginate composition might have

considerable effect on the microsphere size.

Liu et al. (2005) reported the production of spherical, discrete and smooth

alginate-based microparticles or granules (term used by the authors) by

emulsification/internal cross-linking method with different types of alginates.

Granules produced from lower G alginates shrunk to a smaller extent and were better

able to maintain their form during drying than those produced from high G alginates.

The more flexible polymer chains of low G alginates could swell or contract to a

larger extent than high G alginates (Amsden and Turner, 1999). In addition, the larger

number of Ca2+ cross-linking points in the high G alginates reduced matrix elasticity,

resisting shrinkage on drying (Liu et al., 2005). However, the authors did not indicate

17
the viscosities of the different alginates used and it was not known if the observations

were solely due to differences in alginate composition.

The influence of molecular weight, which is related to viscosity, on alginate-

HPMC microspheres produced by emulsification/external cross-linking was evaluated

(Lemoine et al., 1998). Alginates having low and medium viscosity but with similar

M/G ratios were used using a 5 % alginate solution. It was found that smaller

microspheres were produced from alginates with a lower viscosity. In another study

using alginates with comparable M/G ratios, those with higher molecular weight

entrapped a higher enzyme load due to lower matrix porosity (Chan et al., 2006). The

influence of viscosity and/or molecular weight on microsphere size and encapsulation

load was clearer when alginates with similar M/G ratios were used but the effects of

alginate composition were not investigated.

The alginate viscosity also influenced the release of entrapped compounds in

microspheres produced by emulsification. Lemoine et al. (1998) found that there was

a marked decrease in burst effect and release rates of bovine serum albumin in water

and phosphate buffered saline solution (PBS) from alginate-HPMC microspheres

produced with increasing viscosity or molecular weight of the alginate. This was

attributed to two reasons. Denser microsphere matrices were produced by higher

molecular weight alginates due to increased number of cross-linking points. Larger

microspheres produced by the alginates of higher viscosity also retarded bovine serum

albumin diffusion and/or microsphere erosion in PBS. The release of diclofenac

sodium from alginate microspheres in a defined size range also occurred at a slower

rate when alginates with a higher molecular weight and high G content were used

(Gürsoy and Çevik, 2000). This was attributed to the high viscosity and strong gel

network formation of high G alginate. It was not clear which factor had a greater

18
influence on drug release or if both factors had equal contribution to the outcome. The

microspheres in the above studies were produced by the emulsification/external cross-

linking method. Interestingly, differences in dissolution rates of alginate granules

produced by emulsification/internal cross-linking for detergent applications were not

noticeable between high and low G alginates for the range of alginate molecular

weights investigated (Chan et al., 2006). However, it was found that high G alginate

granules were also stronger and less susceptible to attrition. It was thus concluded that

the G content and possibly the molecular weight of the alginates did not have a

significant role in controlling the dissolution rate but played an important role in

reinforcing granule strength.

B2.3. Influence of cross-linking method and type of cross-linker used

Alginate microspheres can be produced by emulsification with either external

or internal cross-linking. In the emulsification/external cross-linking method, the

sodium alginate solution and the Ca2+-containing solution are dispersed as globules in

the reaction mixture. Upon collision of the two types of globules, Ca2+ comes in

contact with the alginate globule and diffuse inside to cross-link the macromolecules,

thus forming alginate microspheres (Wan et al., 1990; Heng et al., 2003). In the

emulsification/ internal cross-linking method, Ca2+ is released in a controlled manner

from an insoluble calcium source contained within the sodium alginate globules

(Monshipouri and Price; 1995; Poncelet et al., 1992a; 1995). The activation of the

cross-linking ions in emulsification/internal cross-linking is usually mediated by a

change in pH brought about by the addition of organic acids or lactones. Acetic acid is

the most commonly used organic acid and it reacts with the insoluble calcium source,

e.g CaCO3, to release Ca2+ for cross-linking (Poncelet et al., 1995; Poncelet, 2001).

19
Lactones such as glucono-δ-lactone had been used in the preparation of alginate gels

but not microspheres (Johansen and Flink, 1986; Draget et al., 1991). Spontaneous

breakdown of glucono-δ-lactone in water resulted in acidification of the medium,

leading to the release of Ca2+ from an insoluble salt. The Ca2+ released caused gradual

hardening of the alginate gel. This pH modifier was deemed to be unsuitable for the

emulsification method as the slow gelation would cause microsphere aggregation

(Poncelet et al., 1992a).

The different emulsification/gelation methods used in production of alginate

microspheres affect the type of cross-linking agents that can be used. In

emulsification/internal cross-linking method, many insoluble calcium salts have been

used to produce alginate microspheres. These include calcium sulphate (Monshipouri

and Price, 1995), calcium citrate (Poncelet et al., 1992a; Esquisabel et al., 1997;

2000; Li et al., 2005), calcium carbonate (Poncelet et al., 1995; 1999; Poncelet, 2000;

Chan et al 2006; Liu et al., 2005), calcium tartrate, calcium oxalate and calcium

monohydrogen phosphate (Poncelet et al., 1995; 1999; Poncelet, 2001). Calcium

oxalate and calcium tartrate were not suitable as the Ca2+ was not readily released

within the pH range (pH >5) suitable for cross-linking alginate. Calcium

monohydrogenphosphate was also found unsuitable because the salt formed

agglomerates at the centre of the beads, leading to poor gelation and bead clumping.

Only CaCO3 and calcium citrate resulted in the formation of spherical beads with

moderate size dispersities (Poncelet et al., 1995; Poncelet, 2001). However, CaCO3

formed beads that were more spherical, with a lower variability in size distribution

than those produced by calcium citrate (Poncelet et al., 1995; Poncelet, 2001).

Spherical, discrete and smooth granules were also successfully produced from

alginate using the emulsification/internal cross-linking method with CaCO3 as the

20
cross-linking agent (Liu et al., 2005). In other studies, calcium citrate was used

together with sodium alginate to bring about a gelation reaction to form a cross-linked

microsphere ‘frame’ by emulsification/internal cross-linking prior to further

interaction with other cations such as polylysine (Esquisabel et al., 1997; 2000) and

chitosan (Li et al., 2002).

In the case of calcium sulphate, its solubility is significantly higher than those

other calcium salts used in the emulsification/internal cross-linking method.

Therefore, cross-linking of alginate with Ca2+ occurred spontaneously if complexing

agents such as polyphosphates were absent to control the gelling kinetics.

Monshipouri and Price (1995) added a sequestering agent, sodium polyphosphate, to

the mixture of insoluble calcium sulphate and alginate before introduction into the oil

phase. This slowed down the rate of cross-linking due to competition between the

sequesterant and sodium alginate for the Ca2+, resulting in the successful formation of

calcium alginate microspheres. This method of producing alginate microspheres by

emulsification/internal cross-linking technique was deemed to be more suitable for

immobilization of pH sensitive cells and proteins than the addition of acids to bring

about pH change in order to release Ca2+ from insoluble calcium salts. However,

Gürsoy et al. (1998) failed to produce spherical and discrete microspheres by direct

application of the emulsification/internal cross-linking method proposed by

Monshipouri and Price (1995) and had made slight modifications to their method. In

the modified method, a mixture of sodium polyphosphate and alginate was first

dispersed into oil and the calcium sulphate suspension was then added. As mentioned

by Draget (2000), external cross-linking produces a gelling zone which moves

towards the centre of the gelling body as cross-linking ions diffuse from an ‘outer

reservoir’ into the alginate phase while internal gelation allows the gelation process to

21
start simultaneously at a large number of locations as the inert calcium source is

within the alginate solution. Therefore, the method proposed by Gürsoy et al. (1998)

could be classified as an emulsification/external cross-linking method although a

poorly soluble calcium sulphate was used. Matrix type alginate microspheres which

were generally spherical with smooth surfaces had been successfully produced using

this method (Gürsoy et al., 1998; 1999; Gürsoy and Çevіk, 2000).

The cross-linking agents used for the emulsification/external cross-linking

method include calcium chloride (Wan et al., 1990; Heng et al., 2003), calcium

acetate, calcium lactate, calcium gluconate (Heng et al., 2003), zinc chloride, zinc

sulphate, magnesium chloride, magnesium sulphate and aluminium chloride (Jin,

2001). Aldehydes such as methanal, pentanedial, octanal and octadecanal were also

added to the continuous phase of the emulsion after calcium chloride addition in an

attempt to enhance cross-linking of alginate microspheres (Chan and Heng, 2002).

Calcium chloride is one of the most common cross-linking agents used in the

successful production of alginate microspheres by emulsification/external cross-

linking. The use of other calcium salts as cross-linking agents had a significant impact

on the properties of the alginate microspheres produced (Heng et al., 2003). Calcium

lactate, calcium gluconate and calcium acetate in combination with calcium chloride

produced smaller calcium alginate microspheres as compared to microspheres

produced using calcium chloride alone (Heng et al., 2003). The cross-linking

solutions containing these salts of weak organic acids were found to have higher pH

values than the calcium chloride solution. The higher pH of the cross-linking

solutions promoted a higher degree of ionization of the alginate molecules, greater

cross-linkage and hence smaller microspheres (Heng et al., 2003).

22
 Divalent zinc ions were also used to cross-link alginate globules to form

alginate microspheres. It was reported that Zn2+ was less selective and hence able to

produce more extensive cross-linkage of the alginate (Aslani and Kennedy, 1996).

Zinc sulphate and zinc chloride had been explored to produce microspheres by

emulsification/external cross-linking (Jin, 2001; Chan et al., 2002). Zinc chloride was

found to be unsuitable to be used alone or in combination with calcium salt for

microsphere production while zinc sulphate could successfully produce microspheres

only when used in combination with calcium chloride (Jin, 2001). The higher activity

coefficient of zinc chloride as compared to zinc sulphate was responsible for the rapid

rate of reaction with the alginate globules, resulting in clump formation (Jin, 2001).

Zinc acetate in combination with calcium acetate, was successfully used to produce

alginate microspheres by emulsification/external cross-linking (Kidane et al., 2002).

When zinc sulphate was used in combination with calcium chloride, smaller

microspheres and markedly lower degree of aggregation, as compared to calcium

alginate microspheres, were obtained (Chan et al., 2002). As the proportion of zinc

sulphate increased in the cross-linking solution, the rate of drug release also

decreased, indicating that the larger proportion of zinc cations produced a more

extensively cross-linked and less permeable alginate matrix (Chan et al., 2002).

However, drug release rates of alginate microspheres cross-linked with various

proportions of zinc sulphate and calcium chloride were still rapid, with more than 90

% drug released within 60 min for a 2 % w/w alginate solution and within 150 min for

2.5 % w/w of alginate solution used (Jin, 2001).

The application of magnesium and aluminium salts as cross-linking agents for

alginates was also explored. Magnesium sulphate and magnesium chloride when used

alone were found unsuitable to produce microspheres by emulsification/external

23
cross-linking method (Jin, 2001). When used in combination with calcium chloride,

larger, more irregular and softer microspheres than those cross-linked by calcium

chloride alone were formed. These microspheres also had higher drug contents and

slightly lower release rates (Jin, 2001). A combination of calcium chloride with

aluminium chloride generally produced smaller, more compact microspheres with

rougher surfaces as a result of fast cross-linking action in the presence of large

numbers of cations. Calcium-aluminium cross-linked microspheres also exhibited

higher drug contents than calcium cross-linked microspheres (Jin, 2001). However,

the influence of calcium-aluminium combination cross-linked microspheres on drug

release was not clear as drug retardation was only observed for some formulations

(Jin, 2001). It was also reported that alginate matrices cross-linked with calcium-zinc

combinations were ‘tighter packed’ as compared to those cross-linked with a calcium-

aluminium combination (Jin, 2001).

The use of aldehydes, such as octanal and octadecanal, as cross-linking agents

produced smaller fractions (about 60%) of microspheres of size 30 µm or less as

compared to calcium alginate microspheres (Chan and Heng, 2002). In contast,

pentanedial and methanal cross-linked microspheres showed comparable size

distribution profiles as calcium alginate microspheres (Chan and Heng, 2002). It was

noted that cross-linking of microspheres with the aldehydes did not improve the drug

encapsulation efficiency. Although some of the microspheres cross-linked with

aldehydes showed a slower rate of drug release than calcium alginate microspheres,

the former was unable to significantly retard drug release as 90 % of the drug contents

were released within an hour (Chan and Heng, 2002).

24
B2.4. Influence of concentration and amount of cross-linker and viscosity of

cross-linking solution

The interaction between alginates and cross-linking cations occurs

instantaneously. The initiation of this cross-linking reaction is dependent on the

chance collision between the dispersed globules of alginate and cross-linking cations

in the emulsification/external cross-linking method (Heng et al., 2003). Higher

concentrations of cross-linking solutions were found to give rise to larger diameter

alginate microspheres (Heng et al., 2003; Rajinikanth et al., 2003). The formation of

large microspheres was attributed to the higher viscosity of the alginate when it cross-

linked with the Ca2+, resulting in less efficient dispersion (Rajinikanth et al., 2003).

Heng et al. (2003) observed an increase in tack and viscosity of higher concentrations

of cross-linking solutions and proposed that this might cause a greater propensity for

the globules to adhere, resulting in the formation of bigger microspheres. However,

there was an insignificant correlation between viscosity of the cross-linking solution

and mean size of microspheres produced (Heng et al., 2003). The morphology of

microspheres was also influenced by the amount of cross-linker added. For a given

concentration of cross-linking solution used in the emulsification method, polymer

aggregates were formed with a smaller volume of cross-linking solution while

spherical alginate microparticles were produced when a larger volume was used

(Fundueanu et al., 1998).

In their study, Heng et al. (2003) did not observe any significant change in the

drug content of alginate microspheres when the concentration of cross-linker was

increased. However, Rajinikanth et al. (2003) reported lower encapsulation efficiency

of metoprolol tartrate when higher concentrations of cross-linking solution were used.

25
 Higher concentrations of solutions of Ca2+ significantly decreased the rate and

extent of drug release from alginate microspheres (Rajinikanth et al., 2003). The

greater availability of Ca2+ led to a greater extent of cross-linking as reflected by a

higher Ca2+ content in alginate microspheres cross-linked with increased

concentration of calcium chloride solution used (Heng et al., 2003). The

bioadhesiveness of alginate microspheres was also reduced as a result of increased

extent of cross-linking (Rajinikanth et al., 2003) and this could be disadvantageous

for microspheres used for drug delivery to mucosal surfaces.

B2.5. Influence of the duration of cross-linking

Despite rapid interaction between Ca2+ and alginate, a ceratin time period is

required for adequate cross-linking of the polymer matrix to confer sufficient

mechanical strength to the microspheres for ease of collection and handling.

Cross-linking of alginate microspheres for at least 15 minutes was necessary

for the formation of sufficiently strong microspheres by emulsification/external cross-

linking (Fundueanu et al., 1998). Variation in cross-linking time was found to have

little influence on the characteristics of the microspheres or beads obtained. A longer

duration of cross-linking resulted in the formation of more rigid spherical beads

(Fundueanu et al., 1998). However, an increase in size of alginate microspheres with

increased cross-linking time was reported by Rajinikanth et al. (2003). The bulk

density of alginate microspheres also increased with longer period of cross-linking

owing to volume contraction as the polymer chains were pulled closer together by the

Ca2+. The high degree of cross-linking enabled by prolonged contact with Ca2+ during

emulsification produced microspheres that swelled less (Fundueanu et al., 1998) and

26
showed lower bioadhesiveness (Rajinikanth et al., 2003). With increased cross-

linkage, the alginate microspheres appeared hydrophobic (Fundueanu et al., 1998).

Cross-linking duration also affects the encapsulation efficiency and drug

release properties of alginate microspheres produced by the emulsification/external

cross-linking method. The encapsulation efficiency was found to decrease with

increased duration of cross-linking by Ca2+ (Rajinikanth et al., 2003). Drug loss by

diffusion from alginate globules to the immiscible continuous phase can occur during

the emulsification process (Chan et al., 1997). This phenomenon was aggravated by

longer contact time between the alginate globules and the continuous phase. The rate

and extent of drug release was markedly decreased with an increase in the duration of

cross-linking (Rajinikanth et al., 2003). Therefore, sufficient time must be allowed for

Ca2+ to diffuse into alginate globules to cross-link the microspheres adequately for

slower drug release.

The influence of varying the cross-linking time in the emulsification/internal

cross-linking method had not been well studied. However, from all the studies carried

out to date, a reaction time of 5 minutes appeared to be adequate for the successful

production of spherical microspheres by this method (Poncelet et al., 1992a; 1995;

1999; Poncelet, 2000).

B2.6. Influence of the type, composition and concentration of surfactants

The formation of microcapsules by emulsification is affected by surfactants,

which are used to serve two primary functions. Initially, surfactants reduce the

interfacial tension between the continuous and dispersed phases so that smaller

microspheres could be produced and secondly, to form a ‘film’ layer around the

globule to prevent coalescence. Poor solubility of surfactants in their respective

27
phases in an emulsion resulted in highly irregular alginate microspheres with marked

clumping (Wan et al., 1994).

The addition of surfactant(s) reduced size (Esquisabel et al., 2000; Kidane et

al., 2002; Rodrigues et al., 2006) and hydrophobicity (Kidane et al., 2002) of alginate

microspheres produced by emulsification, regardless of the method of cross-linking

used. This has important consequences for microspheres produced for use as vaccines,

where the size and surface hydrophobicity of the microspheres affect cellular uptake

by macrophages (Kidane et al., 2002). Alginate microsphere size and morphology are

affected by the type of surfactants used during emulsification (Wan et al., 1994;

Lemoine et al., 1998; Equisabel et al., 2000). Using different surfactant blends with

similar hydrophilic-lipophilic balance (HLB), Wan et al. (1994) observed that

surfactants consisting of straight chain saturated fatty acid produced smaller

microspheres than those with three fatty acid chains due to a closer and more uniform

packing at the interface of the dispersed globules. In addition, Lemoine et al. (1998)

found that Tween 85 produced less heterogeneous and smaller microspheres as

compared to sodium desoxycholate, polyvinyl alcohol or Pluronic® 68. The authors

attributed this to the variation in HLB balance as well as to the physico-chemical

properties of the surfactants.

A combination of hydrophilic and hydrophobic surfactants, rather than a

single surfactant, was found to improve the efficiency of emulsification which was

important for the formation of discrete microspheres (Wan et al., 1990). Variation in

the HLB of a surfactant blend exerted a significant impact on the shape and size

distribution of alginate microspheres (Wan et al., 1993; 1994). Surfactant blends with

HLB values of less than 3.5 were found to cause significant microsphere aggregation.

Blends with HLB values of between 4.0 and 5.0 produced smaller microspheres than

28
those produced with HLB values greater than 5.0. The microspheres were more

spherical when the HLB value was increased beyond 4.5. This was attributed to the

higher proportion of hydrophilic surfactants in the blend and hence, greater affinity

for the Ca2+ during emulsification. Thus, alginate microspheres were formed before

shape distortion occurred as a result of turbulence after cross-linking solution was

added. It was also reported that drug encapsulation was not significantly affected by

changes in the HLB but drug release rates were reduced when surfactant systems with

lower HLB values. This was attributed to adsorption of more hydrophobic surfactant

on the microspheres formed. Being hydrophobic, the surfactant was not easily

removed during the washing process. It was suggested that these surfactant molecules

impeded diffusion of water into microspheres in dissolution studies, thus showing

retarded drug release.

Smaller alginate microspheres were produced at higher concentrations of

surfactants used in the emulsification method with either internal or external cross-

linking (Wan et al., 1993; Esquisabel et al., 2000; Rodrigues et al., 2006). Using a

fixed ratio of surfactant blend, Wan et al. (1993) observed less microsphere clumping

with increasing concentrations of surfactant blends. Beyond this concentration range,

further increases were found to have negligible effect on microsphere size and the

results were similar to those obtained in another study (Esquisabel et al., 2000).

B2.7. Influence of drug load on encapsulation efficiency and drug release

The amount of drug added in the dispersed phase for encapsulation by an

emulsification method is an important variable to control the properties of the alginate

microspheres produced. In a study on encapsulation of oil, bigger alginate

microspheres were produced when more oil was added to the alginate solution (Chan

29
et al., 2000). This was attributed to less efficient dispersion of the dispersed phase by

the stirrer. A marginal increase in microsphere diameter was also observed for

alginate-HPMC microspheres with an increase in metoprolol tartrate loading

(Rajinikanth et al., 2003). However, Gürsoy and Çevik (2000) reported a lack of

influence of drug concentration on microsphere size. The microsphere morphology

was also found to be greatly affected by very high drug loads, which resulted in

aggregates of drug crystals just beneath the microsphere surfaces (Wan et al., 1992).

It was found that the drug load for encapsulation by emulsification should

exceed the solubility of the drug in the aqueous phase in order to obtain high

encapsulation efficiency in alginate microspheres (Wan et al., 1992). Several authors

reported a progressive increase in encapsulation efficiency with increasing drug

concentration (Li et al., 2002; Rajinikanth et al., 2003) or ratio of drug to

encapsulating material (Wan et al., 1992). The rate of increase in encapsulation

efficiency declined when a high ratio of drug to encapsulating material was used

(Wan et al., 1992). In addition, Gürsoy and Çevik (2000) reported that increasing

amount of diclofenac sodium added into the alginate solution resulted in a linear

increase in drug content of the alginate microspheres produced by emulsification but

no change in the encapsulation efficiency.

Drug load was found to have an insignificant effect on the rate and extent of

drug release for alginate microspheres produced by emulsification/external cross-

linking (Rajinikanth et al., 2003). Alginate beads produced by an extrusion/gelation

method were found to exhibit higher rates of drug release with a higher drug load

(Arıca et al., 2002) but an opposite trend was observed for alginate microparticles

(Acartürk and Takka, 1999).

30
B2.8. Influence of incorporating polymers and additives

The matrix of alginate microspheres can be modified by adding polymers and

additives to the aqueous alginate phase prior to dispersion in the immiscible

continuous phase. The use of synthetic polymers such as acrylate and methacrylate

based polymers (Eudragit®) (Gürsoy et al., 1998; 1999; Gürsoy and Çevіk, 2000),

semi-synthetic polymers such as cellulose derivatives (Chan et al., 1997; Gürsoy et

al., 1998; Gürsoy and Çevіk, 2000) and natural polymers such as starch (Chan et al.,

2000), tragacanth and pectin (Gürsoy et al., 1999) have been explored.

Spherical alginate microspheres could be produced with the addition of certain

acrylate-based polymers (Eudragit®) to the alginate solution. Microspheres with

smooth surfaces were obtained for Eudragit® RS30D but not Eudragit® S-100, where

white particles were seen on the composite microspheres (Gürsoy et al., 1998; Gürsoy

et al., 1999; Gürsoy and Çevіk, 2000). Eudragit® S-100 and Eudragit L100-55 are

both anionic but only Eudragit® L100-55 was able to form spherical particles with

alginate (Gürsoy et al., 1998; Gürsoy et al., 1999; Gürsoy and Çevіk, 2000).

Bodmeier and Wang (1993) reported the unsuccessful formation of microparticles by

an extrusion/gelation method using a combination of Eudragit® RS30D and alginate.

This was attributed to immediate flocculation and precipitation when the oppositely

charged polymers were added together. However, other workers were able to produce

spherical microspheres by emulsification with this composite mixture (Gürsoy et al.,

1998; Gürsoy and Çevіk, 2000). The addition of Eudragit® polymers had little

influence on the size of the alginate microspheres and had insignificant effects on the

drug contents. The addition of Eudragit® NE30D to alginate retarded drug release to a

greater extent than Eudragit® RS30D at pH 5, 6 and 6.8. However, both Eudragit®

polymers had no effect on the release rate under acidic conditions, with drug release

31
as rapid as that of plain alginate microspheres (Gürsoy et al., 1998; Gürsoy et al.,

1999). In fact, 100 % of the drug contents was released within 30 mins for Eudragit®

NE30D-alginate composite microspheres produced by the emulsification/external

cross-linking (Gürsoy et al., 1998).

Ethylcellulose (EC) is a neutral polymer commonly used for controlling drug

release and taste masking (Rhodes and Porter, 1998). The use of equal amounts of

alginate and EC resulted in hard clumps of microspheres when produced by

emulsification/external cross-linking method (Chan and Heng, 1998). These clumps

were more easily powdered when a lower proportion of EC was used although size

analysis revealed the presence of mostly aggregated microspheres. The degree of

aggregation was also found to increase when a higher viscosity of EC was used.

However, spherical and smooth surfaced microspheres were obtained with

Aquacoat®, an aqueous dispersion of ethycellulose at alginate: EC ratio of 1:1.5

(Gürsoy et al., 1998; Gürsoy and Çevіk, 2000). Gürsoy and Çevіk (2000) reported

that the addition of Aquacoat® to alginate did not influence the size and drug contents

of the alginate microspheres. However, Chan and Heng (1998) observed a lower drug

content for alginate-EC microspheres than control calcium alginate microspheres. The

different findings could probably be attributed to the different EC formulations used

or variation in the cross-linking technique. Aquacoat® dispersion contained

surfactant(s) and other additives in addition to ethylcellulose and these might affect

microsphere formation by altering emulsification efficiency. Calcium chloride, which

liberated Ca2+ readily, was used to cross-link alginate-EC (Chan and Heng, 1998)

while CaSO4, which liberates Ca2+ gradually with the aid of a sequestering agent, was

used to cross-link alginate-Aquacoat® (Gürsoy and Çevіk, 2000). In both studies, it

was noted that the addition of EC or Aquacoat did not retard drug release to a great

32
extent as 80 % of the drug contents were released within 20-40 min in distilled water

and in media with pH 5-6.8 (Chan and Heng, 1998).

The influence of the hydrophilic HPMC on alginate-based microspheres

produced by the emulsification/external cross-linking method was also studied by

different reaserchers (Wan et al., 1992; Chan et al., 1997; Lemoine et al., 1998;

Kidane et al., 2002; Rajinikanth et al., 2003). Microspheres prepared from a mixture

of sodium alginate and HPMC were more spherical with smoother surfaces than those

prepared from sodium alginate alone (Wan et al., 1990). However, in another study,

alginate-HPMC microspheres produced were observed to have a higher degree of

surface indentation and agglomeration than calcium alginate microspheres (Chan et

al., 1997). This suggested that the HPMC decreased the rigidity of the alginate matrix,

which resulted in greater deformation as immature microspheres collided. The

differences observed in the different studies could be due to the different grades and

proportions of HPMC used. Kidane et al. (2000) observed that alginate-HPMC

microspheres were smaller in size than alginate microspheres. HPMC was described

as ‘surface active molecules’ that formed a multimolecular film around the dispersed

alginate globules, thereby preventing aggregation (Kidane et al., 2002). The influence

of surface active properties of substances was previously discussed (section B2.6) and

it could be responsible for the formation of smaller, more spherical and smoother

surface alginate-HPMC microspheres. These composite microspheres were also more

hydrophobic due to the more hydrophobic HPMC as compared to alginate and are

favoured for cellular uptake (Kidane et al., 2002). Alginate microspheres showed

higher encapsulation efficiency with the addition of HPMC and the effectiveness of

alginate-HPMC as an encapsulating matrix increased with increasing viscosity of the

HPMC used (Chan et al., 1997). The higher viscosity HPMC impeded drug diffusion

33
from the aqueous polymer phase to the continuous phase during emulsification and

more drug was entrapped. However, the composite microspheres obtained

demonstrated faster drug release than the control calcium alginate microspheres in

distilled water (Chan et al., 1997). Other cellulose derivatives such as methylcellulose

(Chan et al., 1997; Kidane et al., 2002), hydroxypropylcellulose and sodium

carboxymethylcellulose (Chan et al., 1997) had also been explored to modify the

matrix properties of alginate microspheres produced by the emulsification/external

cross-linking method (Chan et al., 1997). The microspheres produced were generally

spherical with a high encapsulation efficiency but the composite microsphere matrix

remained permeable, with 90 % of the encapsulated drug released within 20 min.

Attempts to incorporate poly(vinylpyrrolidone), achieved reliable rates of drug

dissolution when used in sustained release formulations (Robinson et al., 1990),

however alginate matrices failed to retard drug release from the microspheres

although spherical and discrete microspheres with good flow properties were

produced (Chan et al., 1997). Blends of alginate and the natural gum, tracaganth, were

also used to produce alginate microspheres by the emulsification/external cross-

linking method. The incorporation of tracaganth had no effect on the microsphere size

although greater microsphere uniformity was observed (Gürsoy et al., 1999). In

addition, higher encapsulation efficiencies and lower drug release rates were observed

(Gürsoy et al., 1999).

Alginate granules produced by the emulsification/internal cross-linking

method were incorporated with different amounts of additives such as

microcrystalline cellulose, polyvinyl alcohol, sucrose and titanium dioxide for use in

detergent formulations (Chan et al., 2006). Granules without additives appeared to be

smoother and were more resistant to attrition, with the exception of sucrose. The

34
additives occupied void spaces in the alginate matrix resulting in a decrease in

alginate chain interaction, subsequently leading to the formation of weaker granules

(Chan et al., 2006).

B2.9. Influence of the nature of drug encapsulated

The nature of drug encapsulated can have a significant influence on the

properties of alginate microspheres produced by emulsification. The physical

properties of the encapsulated drug, such as solubility, can influence the morphology,

encapsulation efficiency and drug release rate from alginate microspheres. Using the

emulsification/external cross-linking technique and a constant alginate:drug ratio,

dipyridamole and diclofenac sodium were encapsulated using alginate, producing

spherical matrix type microspheres (Gürsoy et al., 1999; Gürsoy and Çevіk, 2000).

However, the encapsulation efficiency for diclofenac sodium (about 40 %) was much

lower than that for dipyridamole (>60 %). This was attributed to the lower aqueous

solubility of dipyradamole and hence less drug lost during washing of the

microspheres with water. Diclofenac sodium with its higher solubility in water, was

more easily removed during the washing process, resulting in a smaller amount of

drug being encapsulated. Rapid dipyridamole release was observed in 0.1 N HCl, with

90 % of the drug release within 15 min, compared to 40 % for diclofenac sodium. The

higher solubility of dypyridamole in the acidic medium accounted for the higher rate

of drug release observed. This further illustrates the importance of the physical

properties of the encapsulated drug on drug release from alginate microspheres.

35
C. Investigation of the influence of natural polymers on the properties of alginate

matrix

C1. Production of alginate films

Alginate films could be easily produced by dissolving sodium alginate in

water and drying in a suitable mold to form films. Cross-linked films can be produced

by immersing sodium alginate film in a cross-linking solution. This simple method of

film production by a solvent evaporation technique allows alginate films to be

prepared with ease. For studies involving the alginate matrix, films are preferred to

other models such as microspheres and beads as film shape is constant and does not

influence the results obtained.

C2. Natural polymers that interact with calcium ions

C2.1. Pectin

C2.1.1. Chemical structure and sources of pectins

Pectin is present in the cell wall of most plants and the dominant feature of

pectin is a linear chain of α-(1→ 4) linked D-galacturonic acid units with varying

proportion of acidic groups present as methoxyl esters (Figure 3a). Pectins can be

classified into high methoxyl (HM) pectin and low methoxyl (LM) pectin (depending

on the percentage of carboxyl groups that are esterified with methanol (degree of

methylation). If more than 50 % of the carboxyl groups are methylated, the pectins are

classified as HM pectin; otherwise, they are classified as LM pectin. Amidated pectins

(mostly of the low methoxyl type) can be produced by reaction of a suitable HM

pectin with ammonia. The major sources of pectins are citrus peel and apple pomace

(May, 2000) in addition to sugar beet, carrot and potato (Voragen et al., 1995).

36
(a)
O O O
CO2H CO2CH3 CO2CH3
O O O
OH OH OH
HO HO HO

(b)
OSO3-
HOH2C O
O
O
O O
OH
OH

(c)
OSO3-
HOH2C O
O
O
O O
OH
OSO3-

(d)
O

O C CH3 OH
H2C O HO OH HOH2C O HO
O O
O O
HO O HO OH H3C
O O HOOC
O C
CHOH n

CH2OH

Figure 3. Molecular structures of (a) pectin (b) κ- carrageenan (c) ι-carrageenan (d)
gellan gum.

37
C2.1.2. Physical and gelation properties of pectins

Pectins are generally soluble in water and insoluble in most organic solvents,

with decreased solubility as ionic strength increases. HM pectins will only gel in the

presence of sugars or other co-solutes and at sufficiently low pH, so that the acid

groups in the polymer are not completely ionized (May, 2000). Of interest are the LM

pectins that can gel in the presence of divalent cations such as Ca2+. Calcium ions

interact with pectin to form water-insoluble calcium pectinate. Gelation is due to the

formation of intermolecular junction zones between homogalacturonic regions of

different polymer chains (Voragen et al., 1995). It had been proposed that pectins

form junctions with Ca2+ through an ‘egg box’ binding process similar to that in

alginates (Grant et al., 1973). The gel-forming ability of pectins increases with

decreasing degree of methylation and amidation but is decreased by acetylation. The

gelling process also depends on external factors such as temperature, pH, ionic

strength and amount of cross-linking calcium added (Axelos and Thibault, 1991). At

pH around 3.5, more calcium was needed for gelation of sodium pectinates than under

neutral or very low pH conditions. The charge at the junction-zone cavities was

neutralized by hydrogen ions at very low pH and aggregation or precipitation

occurred (Axelos and Thibault, 1991). The affinity of the pectin macromolecules for

Ca2+ was decreased when ionic strength increased with addition of NaCl, regardless of

the degree of methylation or polymer concentration (Garnier et al., 1994). Sol-gel

transition occurred at highly reduced polyme and calcium concentrations when the

setting temperature was increased (Garnier et al., 1993).The binding of Ca2+ to pectin

was probably less stable at high temperature and therefore more Ca2+ was required to

form an elastically active junction zone (Garnier et al., 1993). Pectins can undergo

38
degradation by oxidation (Abdel-Hamid et al., 2003) and γ-irradiation as radiation

induces attack of glycosidic linkages by free radicals (Jo et al., 2005)

C2.1.3 Applications of LM pectin

Pectins have been widely used in food industries for preparing jams, jellies,

milk gels and desserts (May, 2000). The conversion of pectin to pectinic acid in acidic

conditions was exploited for use as an anti-reflux agent to treat gastro-oesophageal

reflux (Waterhouse et al., 2000). Pectin tablets had also been explored for drug

delivery to the colon (Ahrabi et al., 2000). LM pectins can interact with Ca2+ to form

calcium pectinate. Calcium pectinate was shown to possess good mechanical stability

with a greater potential for cell encapsulation (Sriamornsak, 1998). Moreover, it

meets the requirements for a matrix suitable for drug delivery applications and these

include suitable mechanical properties, biodegradability and ease of processing

(Sriamornsak, 1999). Indomethacin was efficiently entrapped in calcium pectinate gel

beads and the cross-linked barrier was able to control its release from the beads

(Sriamornsak and Nunthanid, 1998). Calcium pectinate had also been suggested as a

suitable agent in delayed release preparations for site-specific delivery of drugs to the

colon (Rubinstein et al., 1993). Besides drug delivery, calcium pectinate also

exhibited great potential in oral controlled delivery of proteins (Sriamornsak, 1999).

C2.2. Gelling carrageenans: Iota carrageenan and kappa carrageenan

C2.2.1. Chemical structures and sources of carrageenans

Carrageenan is a high molecular weight linear polysaccharide extracted from

various species of red seaweeds (Rhodophycae). It comprises repeating galactose

units and 3, 6-anhydrogalactose, both sulphated and non sulphated, joined by

39
alternating α-(1, 3) and β-(1, 4) glycosidic links (Figure 3b and 3c) (Imeson, 2000).

The main carrageenan types include κ-carrageenan, ι-carrageenan and λ-carrageenan.

κ-carrageenan and ι-carrageenan have similar disaccharide repeating units but the 2-

sulphate substituent on the anhydrogalactose residues are absent in κ-carrageenan

(Figure 3b).

C2.2.2. Physical and gelation properties of carrageenans

All carrageenans are soluble in hot water. However, only λ-carrageenan,

sodium salts of κ-carrgeenan and ι-carrageenan are soluble in cold water (Imeson,

2000). Ordered helices are not formed by the highly sulphated λ-carrageenan and

therefore it does not gel. Hot solutions of κ- and ι-carrageenans set to form a range of

gels when cooled to between 40°C and 60°C displaying a range of textures which

depend on the cations present. The carrageenan gels are thermoreversible and they are

stable at room temperature but can be converted to a sol by heating to temperatures

above its gelling point. On cooling again, the system will gel. Viscosity of

carrageenan solutions and carrageenan gel strength will decrease at pH values below

4.3. This was attributed to autohydrolysis which occurrs at low pH conditions where

carrageenan in the acid form is cleaved at the 3, 6-anhydrogalactose linkages

(Hoffman et al., 1996). The rate of hydrolysis increases at elevated temperatures and

low cation levels.

The properties of these gelling carrageenans are sensitive to the ionic

environment such as the type and amount of ions, and their valencies. It has been

suggested that gelation of κ-carrgeenan is preceded by a transition from a disordered

state or random coil to an ordered conformation or helix and gel formation occurs in

the presence of helix aggregation. For κ-carrgeenan, cations such as Cs+, K+, Rb+ and

40
NH4+ have been reported to have greater helix stabilization effects than other

monovalent (Na+, Li+) or divalent (Ca2+, Ba2+, Co2+, Zn2+) cations in promoting the

aggregation and gelation of κ-carrgeenan (Rochas and Rinaudo, 1980). The former

are collectively known as specific cations while the latter monovalent and divalent

cations are known as non-specific cations. Aggregation of the charged carrageenan

helices is generally promoted by increasing the salt concentration but at similar

conditions of ionic strength, the specific cations are more effective than the non-

specific cations in promoting helix aggregation. However, some rheological studies

suggested that Ca2+ was more efficient for gelling κ-carrgeenan than Na+ and K+

(Morris and Chilvers, 1983). κ-carrgeenan formed firm, brittle gels with K+ but it also

formed gels with divalent ions such as Ca2+and Ba2+ (Morris and Chilvers, 1983;

Watase and Nishinari, 1986).

Weak gel formation occurred for ι-carrageenan as a result of conformational

transition, with increased gel strength as helical content increased despite the absence

of helix-helix aggregation (Piculell et al., 1992). Divalent cations such as Ca2+ exerted

a very strong helix stabilizing effect in ι-carrageenan and they interact with ι-

carrageenan to form soft gels (Michel et al., 1997).

C2.2.3. Applications of κ-carrageenan and ι- carrageenan

The carrageenans are widely used for preparing food gels and cake glazes.

They are also used in milk-based products such as whipped cream, milkshakes and

yoghurt (Piculell, 1995). For pharmaceutical applications, carrageenans had been

tabletted to produce matrices with near zero-order release rates for theophylline

monohydrate but showed faster drug release rates when tabletted with calcium salt

(Picker, 1999). κ-carrageenan and ι-carrageenan beads and spheres cross-linked with

41
Ca2+ were investigated as controlled release systems for drugs such as acetaminophen,

verapamil hydrochloride and ibuprofen (Garcia and Ghaly, 1996; Sipahigil and

Dortunc, 2001). Viable yeast cells have also been entrapped within κ-carrageenan

beads produced by ionotropic gelation with Ca2+ (Navrátil et al., 2000).

C2.3. Gellan gum

C2.3.1. Chemical structure and source of gellan gum

Gellan gum is an anionic extracellular polysaccharide secreted by the

microorganism Sphingomonas elodea, which is also known as Pseudomonas elodea

(Sworn, 2000). The primary structure of this linear polysaccharide is composed of

repeating tetrasaccharide units (β-D-glucose, β-D-glucuronic acid, β-D-glucose, α-L-

rhamnose), with each unit consisting of one carboxyl group (glucuronic acid) per

repeating unit (Figure 3d). Gellan gum is produced with two acyl substituents, acetate

and glycerate, present on the 3-linked glucose sub-unit. This is also known as the high

acyl form. The removal of the acyl groups in commercial samples such as Gelrite and

Kelcogel gives rise to the low acyl forms of gellan gum.

C2.3.2. Physical and gelation properties of gellan gum

Gellan gum is insoluble in cold water and is readily dispersed in deionised

water by stirring and adding the gum slowly to the vortex. Heating of high acyl gellan

gum to 85-95°C is sufficient to fully hydrate the gum in both water and milk systems.

Hydration can also occur below pH 4 but can be inhibited by the presence of sugars.

The temperature at which low acyl gellan gum hydrates is dependent on the type and

concentration of ions in solution. The presence of ions such as Na+ and Ca2+ will

42
inhibit hydration of low acyl gellan gum (Sworn, 2000). However, low acyl gellan

gum will not fully hydrate below pH 3.9.

Gels of high acyl gellan gum can be formed by cooling hot solutions and

cation addition is not necessary for the formation of these gels. Their properties are

much less dependent on the concentration of ions in solution. Of special interest is the

low acyl gellan gum which is able to form gels with a variety of cations, notably Na+,

K+, Li+ , Ca2+, Mg2+ and Ba2+ (Grasdalen and Smidsrød, 1987). Divalent cations are

more efficient at promoting gelation of gellan gum than monovalent cations (Giavasis

et al., 2000). Gel strength was found to increase with increasing cation concentration

until a maximum was reached and further addition of the cations resulted in a

reduction in gel strength (Tang et al., 1995). Gellan gum also formed gels in the

presence of hydrogen ions (Grasdalen and Smidsrød, 1987; Moritaka et al., 1995). It

was suggested that at high temperatures, gellan gum exists as a disordered coil

(Robinson et al., 1991). Cooling under non-gelling conditions resulted in reversible

double helix formation and association of these helices by weak interactions gave rise

to ‘weak gel’ formation. Gelation in the presence of gel-promoting cations resulted in

the formation of stronger ‘cation-mediated’ aggregates of the double helices

(Robinson et al., 1991). It was also proposed that gellan gum showed a reversible

helix-coil transition in the absence of gel-promoting cations and this helix formation

led to formation of filamentous aggregates at moderately high polymer concentration.

Branching of these structures resulted in a weak network structure. Formation of

strong gels in the presence of gel-promoting cations was attributed to a localized

lateral association or crystallization of regions of these filaments (Morris, 1991;

1992).

43
C2.3.3. Applications of gellan gum

Gellan gum is mainly used in the manufacture of dessert jellies, dairy

products, sugar confectioneries, fruit preparations (Sworn, 2000) and encapsulated

flavours (Chalupa, 1996). Gellan gum had been used in the formulation of buccal

preparations (Remuñán-López et al., 1998) and ophthalmic preparations for the

delivery of timolol maleate (Dickstein et al., 2001; Shedden et al., 2001; Mundorf et

al., 2004). Calcium cross-linked gellan gum had also been explored for drug

encapsulation (Kedzierewicz et al., 2003) and was shown to have potential for

sustained oral delivery of drugs such as paracetamol (Kubo et al., 2003), cimetidine

(Miyazaki et al., 2001) and theophylline (Alhaique et al., 1996; Santucci et al., 1996).

C3. Blending of natural polymers with alginates

Natural polymers had been blended with alginate for various purposes.

Alginate-carrageenan hydrogel gel films had been produced for use as a coat for

hydrophobic membranes used in membrane distillation and osmotic distillation for

protection against wet out by surface active agents (Xu et al., 2003). The alginate-

carrageenan gels were also capable of good entrapment of the enzyme β-galactosidase

(Mammarella and Rubiolo, 2005). Alginate-pectin microbeads used to encapsulate

fibroblasts also showed good mechanical stability against the host’s immune system

when implanted in vivo (Ponce et al., 2005).

In addition, some studies reported better performance of these cross-linked

polymer composities as compared to cross-linked alginates. Alginate-pectin matrix

cross-linked with Ca2+ was found to have a higher encapsulation efficiency for folic

acid and reduced leakage when compared to that consisting of alginate alone

(Madziva et al., 2005). Liu and Krishnan (1999) also reported a significantly lower

44
rate of release of theophylline from alginate-pectin-polylysine particulates as

compared to alginate-polylysine particulates in both acidic and alkaline media. The

addition of pectin to alginate and cross-linking of the composite matrix with Ca2+

reduce the matrix permeability, enabling greater potential for use in sustained drug

delivery systems. Calcium cross-linked gellan gum produced gels of higher strength

than calcium alginate (Kubo et al., 2003). It is clear that the properties of the alginate

matrix may be modified by the addition of natural polymers, and this may serve as a

potentially useful strategy for the development of an alginate matrix for drug delivery

applications.

45
II. Hypotheses and objectives

Calcium alginate microspheres can be produced easily by the emulsification

method. Previous studies had shown that the formation and properties of the

microspheres were significantly affected by the emulsification process parameters (As

discussed in Introduction). Different types of sodium alginate, as well as different

methods of cross-linking, had been used by different researchers in the study of

alginate microspheres, beads and films, making comparison of the results difficult.

Therefore, this study was undertaken to prove the following hypotheses:

1. The viscosity and composition of sodium alginate exert a significant influence

on the properties of alginate microspheres produced by the emulsification

method. These two parameters act by different mechanisms and affect the

microsphere properties to different extents.

2. The properties of the alginate microsphere matrix, particularly mechanical

strength, encapsulation efficiency and permeability, are significantly affected

by the method of cross-linking.

3. The properties of the alginate matrix are modified by the incorporation of

natural gums through polymer-polymer interaction, mediated by the cross-

linking cation.

This study was therefore divided into 3 parts with specific objectives to prove the

above hypotheses.

Objectives of Part I

i) To produce calcium alginate microspheres by the emulsification/external

cross-linking method using different grades of sodium alginate and evaluate

the morphology, drug content and drug release profiles of the microspheres

produced.

46
ii) To determine the viscosity and composition of the different grades of sodium

alginate and relate these parameters to the properties of the microspheres

produced.

The influence of sodium alginate composition was studied by using a specific

size fraction of microspheres produced from different grades of sodium

alginate.

The influence of sodium alginate viscosity was studied by using different size

fractions of a particular grade of sodium alginate.

iii) To use alginate films as a model to evaluate the mechanical properties of the

alginate matrices formed from different grades of sodium alginate.

Objectives of Part II

i) To produce calcium alginate microspheres by the emulsification method, using

external and internal cross-linking.

ii) To study the effect of using partially cross-linked sodium alginate in the

production of alginate microspheres by emulsification.

iii) To evaluate the size, morphology, encapsulation efficiency and drug release

properties of the microspheres produced.

iv) To elucidate the mechanism of cross-linking using alginate films and

micropellets as a model.

Objectives of Part III

i) To prepare films consisting of sodium alginate, other natural gums and a

combination of both for evaluation of their properties, such as light

transmittance, thickness, mechanical strength, extent of hydration, extent of

moisture sorption, apparent solubility and permeability.

47
ii) To investigate the interactions of sodium alginate with the different types of

natural gums.

iii) To study the effects of different types of natural gums on the properties of

alginate matrices.

The findings of this study would provide a better understanding of the influence and

mechanisms by which the test parameters affect the properties of alginate

microspheres produced by the emulsification method. The proper choice of these

parameters would enable the formation of microspheres with the desired properties.

48
III. Experimental

A. Materials

A1. Polymers

Different types of sodium alginate were used in this study. The types used

include Manucol® DH, Nigrescens (acid precipitated), Flavicans (acid precipitated)

and Macro (acid precipitated), supplied by International Speciality Products (USA). In

addition, two other alginates derived from Macrocystis pyrifera (Sigma Chemicals,

USA), Laminaria hyperborea (BDH Chemicals, UK) were used. Other natural

polymers used include low methoxylated pectin (Citrus pectin, LM-208), κ-

carrageenan (Satiagum® type CSM 37) and ι-carrgeenan (Satiagel® type CT-52)

which were obtained from Natural Colloids (Singapore) and gellan gum (Kelcogel®

F) from CP Kelco (USA).

A2. Surfactants and hardening agents

The non-ionic surfactants, sorbitan trioleate (Sigma Chemicals, USA) and

polyoxyethylene (20) sorbitan trioleate (Merck, Germany) were used to facilitate the

emulsification process. Sorbitan trioleate and polyoxyethylene (20) sorbitan trioleate

have HLB values of 1.8 and 11.0 respectively (Kruglyakov, 2000).

Isopropyl alcohol and glutaraldehyde (Merck, Germany) were used to harden

the alginate microspheres.

A3. Continuous phase of emulsion

A water-immiscible solvent, isooctane (analytical grade, Merck, Germany)

was used as the continuous phase.

49
A4. Reagents and chemicals used in cross-linking

Calcium chloride dihydrate, calcium carbonate (Merck, Germany) and

nanoparticulate calcium carbonate (NanoMaterials Technology, Singapore) were used

as cross-linking agents. Glacial acetic acid (Merck, Germany) and sodium acetate

anhydrous (AnalaR, BDH Laboratories Supplies, England) were used in the

preparation of the cross-linking solution.

Calcium chloride dihydrate consists of hygroscopic granules with a molecular

weight of 147.02 and is freely soluble in water. Calcium carbonate is practically

insoluble in water and reaction with acids results in the release of carbon dioxide.

A5. Drugs

The drugs used included sulphathiazole, sulphaguanidine and acetaminophen.

All the drugs were of B.P. grade.

Acetaminophen has a solubility of 1 in 70 parts of water at 20°C (Clarke,

1969). The solubility of sulphaguanidine is about 1 in 1000 parts of water at 25°C and

that of sulphathiazole is about 1 in 1700 parts of water at 25°C (Budavari et al.,

1989). Sulphaguanidine and sulphathiazole are freely soluble in dilute acids (Budavari

et al., 1989).

Sulphathiazole was milled (Retsch ZM 1000, Germany) to give drug particles

with mean size of 5.80 µm (Olympus, BH2, Japan) before use. Sulphaguanidine was

milled using a centrifugal ball mill (Retsch, Germany) and passed through a 32 µm

aperture sieve (Micron Air jet Sieve, Hosokawa Micron, New Jersey, USA) before

use.

50
A6. Reagents used in the digestion of films and microspheres

Nitric acid (65% or 70%), perchloric acid (60%) and hydrogen peroxide (30%)

(Merck, Germany) of analytical grade were used in sample digestion prior to atomic

absorption spectrophotometric analysis.

A7. Reagents and chemicals used in media preparation

Potassium dihydrogen phosphate, potassium chloride, sodium hydroxide and

concentrated hydrochloric acid (37%, fuming) from Merck, Germany were used for

the preparation of media used in the determination of the apparent solubility of films

and drug release studies from microspheres. The 0.1 N HCl solution prepared had a

pH of about 1.2.

A8. Reagents used in laser diffractometry for sizing of calcium carbonate

particles

Alcohol (analytical grade) was obtained from Merck, Germany.

A9. Reagents and chemicals used in acid hydrolysis and carbon 13 nuclear

magnetic resonance (13 C-NMR) spectroscopy

Sodium hydroxide and concentrated hydrochloric acid (37%, fuming) (Merck,

Germany) were used in the hydrolysis of alginate. Deuterated water, D2O, (Aldrich,

USA) was used to dissolve the hydrolyzed alginate samples and sodium 3-

(trimethylsilyl) propionate (Merck, Germany) was used as the internal standard in

13
C-NMR spectroscopy.

51
B. Methods

B1. Size reduction of calcium carbonate

Calcium carbonate (CaCO3-S) was comminuted using an air jet mill

(Hosokawa Alpine AG, 100 AFG, Germany) with a classifier wheel rotational speed

of 18 000 rpm at a milling pressure of 0.5 MPa to give milled calcium carbonate

(CaCO3-M) (Table 1).

B2. Sizing of calcium carbonate particles

The particle sizes of different calcium carbonate batches: unmilled calcium

carbonate (CaCO3-S), milled calcium carbonate (CaCO3-M) and nanoparticulate

calcium carbonate (CaCO3-N) were determined (Table 1). Laser diffractometry

(Coulter Particle Sizer LS 230, Coulter, USA) using the small volume module was

used for the determination of the size and size distribution of CaCO3-S and CaCO3-M

particles. The calcium carbonate was dispersed in 70 %w/w ethanol and a suitable

amount of the suspension was added to the diffractometer’s liquid sample cell

containing ethanol. The ethanol used in this study was prefiltered through a 0.45 µm

membrane filter (Sartorius, Germany). The size measurements were carried out in

triplicate and the mean size was expressed as the volume median diameter (VMD),

which is the diameter below which 50 % by volume of the particles reside.

Nanoparticulate calcium carbonate (CaCO3-N) particles were coated with

Osmium and sized by a scanning electron microscope (Shimadzu, SS-550, Japan). At

least 20 particles were measured.

52
 Table 1. Median particle size of calcium carbonate batches.

Type of calcium
carbonate used Code Median diameter (µm)

Unmilled calcium
carbonate CaCO3 -S 5.663 ± 0.18

Milled calcium carbonate CaCO3 -M 4.123 ± 0.061

Nanoparticulate calcium
CaCO3 -N 0.071 ± 0.013
carbonate

53
B3. Acid hydrolysis of sodium alginate

Partial hydrolysis of sodium alginate was carried out prior to structural

13
characterization by C-NMR spectroscopy. The hydrolysis procedure used was

adapted from the method described by Schürks et al. (2002). A 1 % w/v sodium

alginate solution was hydrolyzed with 1M hydrochloric acid at pH 3.0 and 100°C for

1 h. After hydrolysis, the solution was cooled and neutralized with sodium hydroxide

to a pH 7.0. The neutral solution was dialysed against deionized water for 24 h and

then dried by evaporation under reduced pressure.

13
B4. Determination of polymer composition and sequence parameters by C

nuclear magnetic resonance spectroscopy

The samples were made up with D2O to a concentration of between 80-100

mg/ml and kept at about 60°C for 5 h prior to spectra acquisition. Sodium 3-

(trimethylsilyl) propionate was used as the internal reference. The spectra were

recorded on a Bruker DPX 300 spectrometer using 32K data points at a resonance

frequency of 75 MHz and spectral width of 20 kHz. The spectra were accumulated

from 5000-10000 scans using a 45° pulse with a pulse repetition time of 0.8 s under

complete proton decoupling. The temperature was kept at 75°C throughout spectra

acquisition. The spectra were analyzed with the aid of commercial software (‘1D-

WIN NMR’, Bruker). The monomer composition and the sequence parameters were

determined from the NMR spectra as described by Grasdalen et al. (1981).

B5. Preparation of partially cross-linked alginate in solution

Various amounts of calcium chloride dihydrate solution (0.5 %w/v) were

added to the sodium alginate (Manucol® DH) solution under high speed shearing

54
using a homogenizer (Ultra-Turrax T25 with dispersing element S25 KV-25F, IKA

Labortechnik, Germany) to produce mixtures S2 and S3 (Figure 4). The calcium

chloride dihydrate solution was delivered at a rate of 4 ml/min using a peristaltic

pump (Minipuls 3 Model M312, Gilson, France) to give a final alginate concentration

of 2 % w/w solution. Solution S1 was obtained by subjecting the sodium alginate

solution to high speed shearing without calcium chloride addition. The total

concentrations of calcium chloride dihydrate used to obtain S2 and S3 mixtures are

given in Figure 4.

B6. Preparation of alginate microspheres

B6.1. Preparation of externally cross-linked microspheres from sodium alginate

and partially cross-linked alginate

Fifty grams of a solution consisting of 2 %w/w sodium alginate or partially

cross-linked alginate (Manucol® DH), with 0.5 g of drug, were dispersed in 75 g of

isooctane containing sorbitan trioleate by a mechanical stirrer (IKA-WERK RW 20

DZM, Germany) at 1000 rpm for 10 min. Five grams of solution containing

polyoxyethylene (20) sorbitan trioleate was then added. The dispersion was stirred for

another 5 min, after which a 25 % w/w solution of calcium chloride dihydrate was

added and allowed to react with the aqueous alginate-drug globules. In all, 5 g of

calcium chloride were added for cross-linking to form microspheres. The total

amounts of sodium alginate, drug and calcium chloride used were kept constant. The

dispersion was then allowed to stand for 3 h and the microspheres formed were

harvested by in vacuo filtration and washed with 30 ml of distilled water. The

microspheres were oven dried at 40°C until constant weight. The microspheres were

then passed through a mesh (0.8 mm) and the amount of microspheres obtained was

55
 2 % w/w sodium Sodium alginate
alginate solution. solution.
(SA solution)

15 000 rpm
(1 min)

CaCl2
added
* 15 000 rpm
(1 min)
16 000 rpm
SA 16 000 rpm (1 min)
(1 min)
microspheres
16 500 rpm
16 500 rpm (1 min)
(1 min)
17 500 rpm
(1 min)
17 500 rpm
(1 min)
18 000 rpm
(2 min)
18 000 rpm
(2 min) 19 000 rpm
(1 min)
19 000 rpm
(1 min) 20 000 rpm
(1 min)
20 000 rpm
(1 min)

S2 mixture S3 mixture
S1 solution (0.04 %w/w (0.08 %w/w
CaCl2.2H2O) CaCl2.2H2O)

*
* *

S1 S2 microspheres S3
microspheres microspheres

Figure 4. Outline of methods used in the preparation of alginate microspheres.

(* mixture/solution dispersed in isooctane and cross-linked with CaCl2.2H2O. ‘ indicates
the start of addition of CaCl2.2H2O solution).

56
reported as the yield. The different types of microspheres produced are shown in

Table 2. The amounts of surfactants used, type of drug added for encapsulation and

duration of stirring after the addition of calcium chloride dihydrate (CaCl2.2H2O)

solution for each type of calcium alginate microspheres are also listed in Table 2.

B6.2. Preparation of internally cross-linked alginate microsperes

Various amounts of calcium carbonate (CaCO3-M) (0.5, 0.8 and 1.0 g),

dispersed in water, were incorporated into the sodium alginate (Manucol® DH)

solution to give a final alginate concentration of 2 % w/w. The sodium alginate

solution (50 g) containing calcium carbonate was dispersed in isooctane containing

2.54 g of sorbitan trioleate using a mechanical stirrer (IKA-WERK RW 20 DZM,

Germany) at 1000 rpm. Five grams of solution containing 1.36 g of polyoxyethylene

sorbitan trioleate were added after 10 min of stirring. Glacial acetic acid (1.1, 1.6 and

1.8 ml respectively) was then added to react with the CaCO3 to release Ca2+ for cross-

linking with the alginate to produce microspheres. The alginate microspheres

produced were subjected to different treatments, washed and collected by filtration

(Figure 5). The microspheres were then oven-dried at 45°C or freeze-dried.

B7. Preparation of alginate micropellets

Acetaminophen was added to 30 g of 2 % w/w sodium alginate (Manucol®

DH) solution to give a drug to polymer ratio of 1:2. Externally cross-linked

micropellets were produced by dropwise extrusion of drug-polymer mixture into 50

ml of Solution A, to which 0.25 g of calcium carbonate (CaCO3-M) was added as

source of Ca2+. In the preparation of internally cross-linked micropellets, 0.25 g of

CaCO3-M was added to the drug-polymer mixture instead and the mixture extruded

57
 Table 2. Parameters used for production of alginate microspheres by emulsification/external cross-linking method

Formulation conditions Microsphere Amount of Amount of Drug used Duration of

code sorbitan polyoxyethylene stirring after
trioleate (20) sorbitan addition of
added (g) trioleate added CaCl2.2H2O
(g) solution (min)

Using sodium alginate

Manucol® DH

(Without high shear stirring) SA 3.71 0.84 Sulphathiazole 10

(With high shear stirring) S1

Laminaria hyperborea LH
Macrocystis pyrifera MP
Macro (acid precipitated) MA
Nigrescens (acid precipitated) NI 2.93 1.57 Sulphaguanidine 25
Flavicans (acid precipitated) FL

Using partially cross-linked alginate

Manucol® DH with 0.04 % w/w CaCl2 S2

3.71 0.84 Sulphathiazole 10
Manucol® DH with 0.08 % w/w CaCl2 S3
58

58
 Dispersion of
aqueous phase
(sodium alginate and
calcium carbonate) in
Glacial acetic
organic phase at
acid.
1000 rpm
Organic phase removed by
decantation

30 ml of 50 %w/v
calcium chloride
solution added.
50 ml of 30 %w/v
calcium chloride
solution added.

Stirred for
15 min at 10 ml of
Stirred for 15 or 20 isopropyl
min at 500 rpm 500 rpm
alcohol added.
Microspheres
collected by in
vacuo filtration
and dried in the
oven. (M3)
Microspheres
collected by in Microspheres collected
vacuo filtration by gravitational filtration
and redispersed in 30 ml 20 ml of
and dried in the
of distilled water before glutaraldehyde
oven.
freeze-drying. added.
(M1) Microspheres
(M5)
collected by in
Microspheres vacuo filtration
further hardened Microspheres collected by and dried in the
with calcium gravitational filtration, and oven. (M4)
chloride, redispersed in 30 ml of
collected by in distilled water and further
vacuo filtration hardened with 50 ml of 30
and dried in %w/v calcium chloride
oven. (M2) solution for 20 min.

Microspheres collected and redispersed in 30 ml of

distilled water before freeze-drying. (M6)

Figure 5. Preparation of microspheres by emulsification/internal cross-linking

method.

59
into Solution A. Extrusion through a syringe needle (20 gauge) or a silicon tubing

with an internal diameter of 2 mm at a flow rate of 0.7 g/min was carried out using

peristaltic pump (Gilson, Minipuls 3 Model M312, France). The alginate micropellets

were allowed to cure overnight in the cross-linking solution. Micropellets were then

harvested, washed three times with 50 ml of deionised water and oven-dried at 40°C

to constant weight.

B8. Size analysis of microspheres

The diameter and shape of the microspheres were determined using a

microscope (Olympus BX61-TRF, Japan) connected to an image analyzer. The

microspheres were mounted in water and the microscope was calibrated using a stage

micrometer. Sphericity is defined by Equation 1:

Sphericity = 4π(area)/ (perimeter)2 (1)

Sphericity is an indication of the roundness of the microspheres with a value of unity

corresponding to a perfect circle. As sphericity increases the particle departs from a

perfect circle. Each mean value reported was obtained by averaging the measured

values obtained from at least 200 microspheres.

B9. Evaluation of aggregate size and degree of agglomeration of microspheres

The size of the aggregates, as well as number of single and aggregated

microspheres, were determined using a microscope (Olympus BX61-TRF, Japan)

connected to an image analyzer. The degree of agglomeration is expressed as

Equation 2:

60
 Degree of Number of aggregates
agglomeration = x 100 % (2)
Total number of single and
(%) aggregated microspheres

B10. Separation of batch of microspheres into different size fractions

The alginate microspheres were separated into different size fractions by air

jet sieving (Hosokawa Micron, NJ, USA) with sieves of aperture sizes 32, 53, 75, 100

and 125 µm.

B11. Determination of drug content

An accurately weighed amount of alginate microspheres or micropellets was

diluted to 50 ml with 0.1N HCl. The sample was placed in an ultrasonic water bath for

3 consecutive periods of 20 min each for each batch of microspheres and 40 min each

for micropellets, with a rest period of 20 min and 40 min in between for microspheres

and micropellets respectively. It was then allowed to stand overnight at room

temperature. Aliquots were removed via filtration through a 0.45 µm filter and

appropriately diluted with 0.1N HCl. The drug content was assayed

spectrophotometrically (Shimadzu UV-1201, Japan) at a suitable wavelength (Table

3). For each sample, at least 3 determinations were carried out. The mean amount of

drug in 50 mg of dried alginate microspheres or micropellets or the percent drug

content was reported.

B12. Determination of drug release profiles

Dissolution testing was carried out by suspending an accurately weighed

amount of microspheres or micropellets in 900 ml of dissolution medium at 37 ±

61
0.5°C using USP Apparatus 1 or 2 (Table 3) (Optimal DT-1, USA). The paddle or

basket was rotated at 50 rpm. For alginate micropellets, SA, S1, S2 and S3

micropsheres, 5 ml of samples were collected at specified time intervals, passed

through a 0.45 µm filter and the drug present assayed spectrophotometrically

(Shimadzu UV-1201, Japan) at the appropriate wavelength (Table 3). The samples

were collected at 5, 15, 30, 60, 90, 120, 180, 240, 300 and 360 min for drug release

from alginate micropellets. For drug release from SA, S1, S2 and S3 microspheres,

samples were collected at 2, 5, 10, 15, 20, 30, 60, 90, 120, 180 and 240 min. For

LH, MA, MP, FL and NI microspheres, a flow cell coupled to a diode array

spectrophotometer (Hewlett Packard 8452A, USA) was used to determine the amount

of sulphaguanidine released at specified time intervals. The amount of drug released

from the microspheres was determined every 20 sec. At least 3 dissolution runs were

carried out for each type of microspheres or micropellets and the results averaged.

The model-independent time at which 25 % (t25%), 50 % (t50%) or 75 % (t75%)

of drug was released from the microspheres were determined.

B13. Preparation of sodium alginate films

A 2 %w/w solution of sodium alginate was prepared by dissolving an

appropriate amount of sodium alginate in deionised water. The solution was left to

stand overnight to remove air bubbles. The films were produced by a solvent

evaporation technique. Thirty grams of the sodium alginate solution was transferred

into respective glass petri dishes of 10 cm diameter and oven-dried on a leveled

surface at 40°C to constant weight. The dried films were then removed from the petri

dishes and stored in a desiccator at 24 ± 1°C for at least 72 hours prior to

characterization and cross-linking.

62
Table 3. Summary of conditions used in drug assay.

Type of Wavelength Type of Medium Wavelength

microspheres used for drug dissolution used in used in drug
and alginate assay (nm) apparatus drug release
micropellets used release studies (nm)
studies

SA
S1 279.5 USP Deionized 284
S2 apparatus 2 water
S3

LH
MP 265 USP Deionized 259
MA apparatus 1 water
NI
FL

0.1 N HCl 265

Alginate
micropellets 245 USP Deionized 242
apparatus 1 water

63
B14. Preparation of calcium carbonate-sodium alginate films

A specific amount of calcium carbonate (CaCO3-S, CaCO3-M or CaCO3-N)

was suspended in an appropriate amount of deionised water, ultrasonicated for 30 min

and further mixed using a magnetic stirrer. The required amount of suspension was

added to the sodium alginate solution which was thoroughly mixed to give a final

alginate concentration of 2 %w/w. Films were prepared and stored as described in

Section B13.

B15. Preparation of films of pectin, gellan gum, carrageenan and their blends

with sodium alginate.

An appropriate amount of pectin, gellan gum, ι-carrageenan or κ-carrageenan

was dissolved in distilled water to produce a 2% w/w solution. Heating was required

to dissolve the carrageenan and gellan gums. The respective gums were combined

with sodium alginate to produce a 2 % w/w polymer solution for the preparation of

composite films. Different alginate:gum ratios were used: 90:10, 70:30 and 50:50.

Appropriate amounts of the above solutions were placed in levelled plastic or

glass petri dishes and oven-dried at 40 oC for 48 h. Pectin was cast in glass petri

dishes. Due to difficulties in removing films of carrageenans and gellan gum from

glass petri dishes, these polymers were cast in plastic petri dishes instead. The dried

films were removed from the petri dishes and stored as described in Section B13. The

different types of films studied are listed in Table 4.

64
 Table 4. Composition of films studied

Film composition Film Code

sodium alginatea SA

sodium alginate: pectin

90:10 AP90/10
70:30 AP70/30
50:50 AP50/50

sodium alginate:κ-carrageenan

AK90/10
90:10
70:30 AK70/30
50:50 AK50/50

sodium alginate: ι-carrageenan

90:10 AI90/10
70:30 AI70/30
50:50 AI50/50

sodium alginate: gellan gum

90:10 AG90/10
70:30 AG70/30
50:50 AG50/50

pectin PT
gellan gum GG
κ-carrageenan KC
ι-carrageenan IC

a
The type of sodium alginate used was Mannucol® DH.

65
B16. Preparation of cross-linked films

B16.1. External and internal cross-linking of films using calcium carbonate

Externally cross-linked films were prepared by immersing each sodium

alginate film for about 24 h in 50 ml of 1% sodium acetate with an appropriate

amount of glacial acetic acid (Solution A), to which the required amount of calcium

carbonate was added (0.15 g, 0.25 g or 0.35 g of CaCO3-M). Glacial acetic acid was

added to liberate Ca2+ from the insoluble salt. Internally cross-linked films were

produced by immersing each calcium carbonate-sodium alginate film for about 24 h

in 50 ml of Solution A. The different types of alginate films prepared are shown in

Table 5.

B16.2. Cross-linking of films with calcium chloride solution

The films were immersed in a specific volume of calcium chloride solution for

1 hr. The amount of Ca2+ available for cross-linking each gram of alginate polymer

(Manucol® DH) was 4.2 x 10-3 moles while films made from the following types of

sodium alginate: MP, LH, MA, NI and FL (Table 6), were cross-linked with 50 ml of

0.15M of calcium chloride solution (0.0125 moles of Ca2+ per gram of sodium

alginate). All cross-linked films were washed with 50 ml of deionised water three

times and oven-dried at 40°C to constant weight. The dried films were stored in a

desiccator at 24 ± 1°C for at least 72 h prior to further test.

B17. Determination of film thickness

The thickness of the films was measured by using a micrometer screw gauge

66
 Table 5. Films prepared by cross-linking with calcium carbonate

Type of film Code

Sodium alginatea SA

Calcium alginate externally

cross-linked with different
amounts of CaCO3 –M

0.15 g CAE 0.15

0.25 g CAE 0.25
0.35 g CAE 0.35

Calcium alginate internally

cross-linked with different
amounts CaCO3 –M

0.15 g CAI 0.15

0.25 g CAI 0.25
0.35 g CAI 0.35

Calcium alginate internally

cross-linked with 0.25 g of
calcium carbonate batch of
different particle size

CaCO3 -M CAI 0.25

CaCO3 -S CAI 0.25S
CaCO3 -N CAI 0.25N

a
The type of sodium alginate used was Mannucol® DH.

67
 Table 6: Composition and sequence parameters of the different types of alginate studied

Type of Alginate Monad frequencies Doublet frequencies

alginate code M/G
FM FG FMM FGG FGM ratio
(FMG)

Nigrescens NI 0.62 0.38 0.41 0.19 0.20 1.65

(acid precipitated)

Macro MA 0.58 0.42 0.37 0.28 0.17 1.40

(acid precipitated)

Flavicans FL 0.41 0.59 0.28 0.48 0.12 0.70

(acid precipitated)

Macrocystis MP 0.64 0.36 0.43 0.15 0.21 1.75

pyrifera

Laminaria LH 0.43 0.57 0.28 0.44 0.14 0.74

hyperborea
68

68
(Mitutoyo, 293-721-30 CE, Japan). At least 5 thickness readings from different

regions of each piece of film were taken and the results averaged to give the thickness

of that piece of film. The thickness of a given type of film reported is the average of

the thickness values of at least 5 film samples.

B18. Determination of film weight before and after cross-linking

The weights of alginate film before (Wb) and after (Wc) cross-linking were

determined. For films containing CaCO3, the corresponding weight of the calcium salt

was deducted from Wb. For each type of film, the individual weights of 5 pieces of

film samples were determined.

B19. Determination of mechanical properties of films

Films were cut into strips of 1 cm by 7 cm for tensile measurements. The

thickness of each film strip was measured at 5 random points (Mitutoyo, 293-721-30

CE, Japan) and mean thickness calculated. Only film samples with deviation of

thickness within 10% from the mean were used. The tensile strength was determined

(Shimadzu, EZ tester, Japan) with an initial grip separation of 5 cm and a test speed of

10 mm/min. Tensile strength was calculated by dividing the maximum load with the

original cross-sectional area of the test film. Elastic modulus, which describes the

rigidity or stiffness of a film, was obtained from the gradient of the initial linear

portion of the load-deformation curve. The percent elongation at break was also

determined. For each type of film, at least 5 determinations were made and the results

average.

69
B20. Assessment of morphology and surface topography of films

The morphology of the films was examined under a light microscope

(Olympus, BX16TRF, Japan). Quantitative surface measurement was carried out by

scanning probe microscopy (Shimadzu, SPM 9500, Japan) in the dynamic mode, at a

scan rate of 1 Hz. A scan area of 25 µm by 25 µm with Z range (depth of scan) set at

1.5 µm was used in the acquisition of surface images. At least 30 scans were carried

out for each type of film and the mean roughness values (Ra) calculated (Heng et al.,

2000).

Ra was defined as the average absolute deviation of the roughness

irregularities from the mean line over one sampling length (L) that was obtained from

the roughness curve (Gadelmawla et al., 2002)(Figure 6). Ra was computed from

equation 3 where the roughness curve is expressed as y = f(x) where y is the length, x

is the deviation of roughness irregularities from the mean line.

L
Ra = 1/L ∫{f(x)} dx (3)
0

B21. Determination of film opacity

A strip of film (20 mm x 35 mm) was mounted onto a cell holder of a UV

spectrophotometer (UV-1201, Shimadzu, Japan). Only strips with a thickness in the

range of 0.041-0.058 mm were used. The amount of light transmitted through the film

at 600 nm, with air as reference was measured. All experiments were performed in

triplicate and the results averaged. The film opacity was indicated by % transmittance,

with a higher value corresponding to a less opaque film.

70
 y (nm)

Mean line

x (µm)

L L

Figure 6. A section of the profile of the roughness curve obtained by scanning probe
microscopy.

71
B22. Determination of extent of water uptake and apparent solubility of films

Rectangular pieces of films, 1 cm by 7 cm, were cut and stored in a desiccator

for at least 72 h before testing. Each piece of film was weighed (W1), immersed in 40

ml of medium in a boiling tube, which was then placed in a shaker water bath and

shaken at 52 oscillations/min at 37°C. The different media used are listed in Table 7.

At the end of the study, the film was removed, carefully placed between two pieces of

filter paper to remove surface water and weighed (W0). It was then spread out on a

clean petri dish and oven-dried at 40°C to constant weight. The dried film was kept in

a desiccator for at least 72 h before reweighing (W2). The apparent solubility of the

films (WA) and the hydration index (WH) was calculated using Equations 4 and 5

respectively.

W1 –W2
WA = x 100 %
W1 (4)

Wo (5)
WH = x 100 %
W1

For each type of film, 5 determinations were made and the results averaged.

B23. Determination of percent weight increase due to moisture sorption in

climatic chamber

At least 3 samples of each uncross-linked and cross-linked films were weighed

before being placed in a climatic chamber (WTB Binder Labortechnik GmbH,

Germany). The relative humidity in the climatic chamber was kept at 90 + 3% and the

72
Table 7. Conditions used in the determination of apparent solubility and permeability
of films.

Medium used in Duration of Medium Concentration

Type of film determination of apparent used in film of
apparent film film permeation acetaminophen
solubility solubility studies solution used
study (hr) (mg/ml)

Cross-linked
alginate films Deionised water 6 Deionised 0.48
using CaCO3
as cross- water
linking agent

Deionised water 6
Cross-linked
USP HCl buffer 2 Deionised 0.72
alginate films
pH 1.2 water
using CaCl2
as the cross-
USP phosphate
linking agent
buffer 4
pH 6.8

73
temperature was maintained at 25 + 1°C. The weight of each sample was taken at

intervals of 2, 5, 10, 25, 30, 35, 48, 72 and 100 h. The average percent weight increase

for each type of film was reported.

B24. Determination of film permeability

A two-compartment horizontal diffusion cell was used. The film was securely

mounted between the compartments. The donor compartment contained 250 ml of

acetaminophen solution (Table 7) while the receptor compartment was filled with an

equal volume of deionised water. The available area for permeation was 8.042 cm2.

The diffusion cell was placed in a shaker water bath shaken at 52 oscillations/min at

37°C. At 15, 30, 60, 90, 120, 180, 240, 300 and 360 min, 5 ml samples were

withdrawn from the receptor compartment with replacement of deionised water to

maintain constant volume. Sink conditions were maintained throughout the

permeation study as no more than 5 % of the initial drug quantity in the donor

compartment entered the receptor compartment (Aslani and Kennedy, 1996). The

samples were analyzed spectrophotometrically (Shimadzu, UV-1201, Japan) at 242

nm. All experiments were repeated at least 3 times and the results averaged.

The total amount of drug, Qt, which penetrated through the film in time, t,

from the donor compartment at constant concentration (C) to the receptor

compartment at sink condition is given by:

∞
t _ 1 _ 2 (- 1)n - Dn2 π2t
Qt = AKhC [D h2 6 π2
Σ
n=1
n2
exp ( h2 )] (6)

where A is the actual surface area for diffusion, K is the partition coefficient of the

drug between the film and the donor solution, h is the film thickness, D is the drug

74
diffusion coefficient through the film. The product of Kh and D/h2, which is equal to

the permeability coefficient (P) and was calculated by curve fitting the permeation

data to Equation 6 (Crank, 1975) using a statistical program (GraphPad Prism 3). At

least 3 determinations were carried out for each type of film and the mean

permeability coefficient through films calculated.

B25. Infra-red spectroscopy

Absorption spectra were obtained using a Fourier Transform Infra-red (FTIR)

spectrometer (Jasco, FTIR-430, Japan) equipped with a triglycin sulphate (TGS)

detector. The alginic acid and pectinic acid used was obtained by precipitation of

sodium alginate and sodium pectinate using 0.1N HCl. Potassium bromide discs

containing 1 %w/w of film material was scanned at 4 mm/s at a resolution of 4 cm-1

over a wavenumber range of 400 to 4000 cm-1, averaging over 128 scans for each type

of film.

B26. Atomic absorption spectrophotometry

B26.1. Washing of films and microspheres to remove excess surface calcium

Films cross-linked using CaCO3 as the cross-linking agent were washed in 50

ml of deionised water for 5 min. The washing process was repeated three times. The

films were then oven-dried at 100 ± 1 °C for 3 h and allowed to cool overnight in

desiccator before use.

Films cross-linked by immersion in calcium chloride solution were washed in

50 ml of deionised water for 1 min under agitation using a magnetic stirrer. The

75
washing process was repeated and films were then oven-dried at 65 ± 1 °C until

constant weight and allowed to cool overnight in a desiccator before use.

The microspheres were suspended in 50 ml of deionised water for 2 min under

agitation using a magnetic stirrer. The microspheres were then collected by in vacuo

filtration and resuspended in 50 ml of deionised water under agitation for 5 min. The

latter procedure was repeated before the microspheres were collected by in vacuo

filtration, oven-dried at 105 ± 1 °C for 3 h and allowed to cool overnight in a

desiccator before use.

B26.2. Sample digestion and analysis

For CaCO3 cross-linked films, approximately 50 mg of dried film material was

accurately weighed. Twenty millilitres of concentrated nitric acid (HNO3) was added

to the film material in a conical flask and heated for 1 h, keeping the solution boiling

throughout. On cooling, 10 ml of perchloric acid was added and heated at a slightly

boiling state for 1.5 h. It was then cooled and transfered to a 100 ml volumetric flask

and made up to volume with deionised water.

For alginate microspheres and films cross-linked with calcium chloride

solution, approximately 50 mg of dried sample was accurately weighed into a

carousel (AC-10 Advanced Carousel, Milestone, Italy). Five ml of concentrated nitric

acid and 1 ml of hydrogen peroxide were added and the samples were digested by

microwave digestion (Ethos 900, Milestone, Italy), cooled, transfered to a 100 ml

volumetric flask and made up to volume with deionised water.

Appropriate dilutions were carried out for all samples and the Ca2+ content was

determined by atomic absorption spectrophotometry (Shimadzu, AA6800, Japan) at

422.7 nm. Three determinations were carried out for each type of film and

76
microspheres and the results averaged. The Ca2+ content of the microspheres or films

was defined as the amount of Ca2+ present in 1 g of the sample.

B27. Viscosity determination of sodium alginate solutions

Viscosities of the alginate solutions were measured in triplicates using 3

individually prepared solutions with a rheometer (RheoStress 1, ThermoHaake,

Germany) at a shear rate of 600 s-1 at 20°C for solutions of partially cross-linked

alginate mixtures and 25 °C for solutions of sodium alginate.

77