prr.hec.gov.pkprr.hec.gov.pk/jspui/bitstream/123456789/9237/1... · iii Development and Evaluation...

Development and Evaluation of Self-assembled

Stabilized Supramolecular Hydrogels of

Polyoxometalates

By

Aziz Ullah

CIIT/FA13-R60-004/ATD

PhD Thesis

In

Pharmacy

COMSATS Institute of Information Technology

Abbottabad-Pakistan

Spring, 2017

ii

COMSATS Institute of Information Technology



Polyoxometalates

A Thesis Presented to

COMSATS Institute of Information Technology, Abbottabad

In partial fulfillment

of the requirement for the degree of

PhD (Pharmacy)

By

Aziz Ullah


Spring, 2017

iii



Polyoxometalates

A Post Graduate Thesis submitted to the Department of Pharmacy as partial

fulfillment of the requirement for the award of Degree of PhD in Pharmacy.

Name Registration Number

Aziz Ullah CIIT/FA13-R60-004/ATD

Supervisor

Dr. Jamshed Iqbal (T.I.)

Head CADR

Professor, Department of Pharmacy

Abbottabad Campus.

COMSATS Institute of Information Technology (CIIT),

Abbottabad.

Co–Supervisor

Dr. Nisar-ur-Rehman

Professor, Department of Pharmacy

Abbottabad Campus

COMSATS Institute of Information Technology (CIIT),

Abbottabad.

iv

v

vi

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DEDICATION

Dedicated to my beloved parents, wife, siblings and

my son Muhammad Saud Khan for their never-

ending love, provision and encouragement

ix

ACKNOWLEDGEMENTS

In particular, I would like to express gratitude to Allah Almighty, the most

Gracious the most Merciful whose infinite blessings lend me to accomplish my final

dissertation. Subsequently, Salawat and Salam to Muhammad PBUH who brings us

from darkness to the brightness.

I would like to thank my beloved parents and family for their encouragement who

are so supportive to me throughout my life.

I wish to express my extreme gratitude and appreciation to my supervisor, Prof.

Dr. Jamshed Iqbal for his guidance and patience in providing priceless ideas that led

to the success of my research. I am also grateful to my co–supervisor, Prof. Dr. Nisar–

ur–Rehman for his guidance in my work.

I am very thankful to one of our research collaborators Prof. Dr. Ulrich Kortz,

and his research students especially Wenjing Liu, Sachin A. Joshi and Dr. Ali Haider

from Jacobs University Bremen Germany, who helped in the polyoxometalate

synthesis.

I want to extend my gratitude to Prof. Peter Langer, Department of Organic

Chemistry, University of Rostock, Germany, for giving me an opportunity and built

my research skills throughout my stay. I am extremely thankful to the Dr. Mariya Al-

Rasheeda, department of Chemistry, Forman Christian College University, Lahore,

Pakistan for providing me support in terms of samples analyses.

My special thanks to all my colleagues, especially Dr. Muhammad Sohail Khan,

Dr. Saifullah Afridi, Dr. Syed Hamid Saeed Shah, Dr. Sumera Zaib, Syeda Abida

Ejaz, Syed Mubashar Ali Abid, Kaleem Ullah Khan, Shafiullah Khan and Syed Jawad

Ali Shah who supported me during the most painstaking phase of my research with

their knowledge, perception, and companionship.

My special thanks to Higher Education Commission (HEC), Pakistan for their

support in terms of fee wave off under prime mister fee reimbursement program

throughout my PhD tenure.

Aziz Ullah


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ABSTRACT

Development and Evaluation of Self–assembled Stabilized

Supramolecular Hydrogels of Polyoxometalates

Polyoxometalates (POMs) are discrete anions and have got immense attraction in

biomedical research due to their structural diversity rendering them highly active

against viral, cancer, bacterial, and HIV infections. In this study 36 different hydrogel

formulations of Polyanion (P2W15) were prepared with four different cationic

polymers including gelatin, polyethyleneimine (PEI) and two water soluble chitosan

derivatives i.e. carboxymethyl chitosan (CMCh) and chitosan hydrochloride (ChCl).

For induction of pH responsiveness in prepared hydrogel formulations methacrylic

acid (MAA) and acrylic acid (AA) were grafted on polymer backbone. The swelling

and in–vitro dissolution experiments of prepared formulations were conducted at pH

1.2 and pH 7.4 showing maximum swelling and cumulative % in–vitro release at pH

7.4. Out of all 36 developed formulations, the maximum swelling and cumulative %

in–vitro release (92%) was observed for MAA–ChCl–POM hydrogel formulation

with sample ID CHCP6. Prepared hydrogels were physically cross–linked using

electrostatic interactions between POM and polycations that were further

characterized by scanning electron microscopy (SEM), fourier transform infrared

spectroscopy (FTIR), thermal analysis (thermogravimetric analysis, (TGA),

differential scanning calorimetry (DSC) and X–ray diffraction (XRD). FTIR

spectroscopy confirmed the interaction among different functional groups of polymer,

monomer and POM. Thermal properties of the prepared hydrogels were higher than

their individual components as suggested by TGA/DSC curves. The hydrogel

possessed glassy smooth surface morphology supporting an equal distribution of

POM throughout hydrogel network. Safety evaluation of the POM solution and oral

acute toxicity/tolerability of the hydrogel system (MAA–ChCl–POM) were also

conducted in rabbits using both male and female species. For oral tolerability/ acute

toxicity of hydrogel dispersion, maximal tolerated dose method was used suggesting

4000 mg/kg body weight dose as safe maximal tolerated dose (MTD).

Histopthological examinations of the rabbit‘s vital organs like heart, liver, kidney,

spleen and lungs showed no gross significant signs of toxicity. The findings of serum

xi

chemistry and haematological investigations confirmed that blood, liver and kidney

functions for both oral POM solution and hydrogel dispersion were normal except

reduction in blood glucose level which is due to inhibition of glucose–6–phosphatase

enzyme by POM. In-vitro cytotoxicity studies were also performed to check the

cytotoxic potential of the free as well as encapsulated POM (hydrogel) showing the

dose dependent cytotoxicity against two different cancer cell lines (MCF–7 and HeLa

cells) with minimal effects on normal cells (Vero). The highest in-vitro release

hydrogel formulation (sample ID CHCP6) was further selected for in–vivo evaluation

of POM for pharmacokinetic parameters through a validated and sensitive ion pair

HPLC method in rabbits. Kinetica 5.1 was used to determine the pharmacokinetic

profile of the POM. After oral administration of hydrogel disc having 20 mg POM,

relatively longer plasma concentration profiles were observed. Area under the curve

(AUC) and mean retention time (MRT) obtained for hydrogel formulation were 2.87

times and 1.91 times greater respectively than oral POM solution. Time for maximum

plasma concentration (Tmax) obtained for oral solution was 9.6 hours while for

hydrogel disc it was 14.6 hours. Half–life (t1/2) recorded for oral POM solution was 2

hours while in the case of hydrogel formulation, extended t1/2 of 4.87 hours was

observed. Apparent volume of distribution (Vz) obtained for oral POM solution was

0.003L while for hydrogel system that was 0.0038L. Clearance of oral POM solution

occurred at the rate of 0.0012 L/h while for hydrogel it was 0.00056 L/h. Elimination

half–life (Lz) observed for oral solution was 0.345 (hr) and that for the hydrogel

enclosed POM was 0.147 (hr). Cmax values calculated for oral POM solution and

hydrogel disc were lying in close proximity. The findings of the current study

indicated that prepared hydrogel system showed controlled encapsulated POM

release. The hydrogel system and concentration of POM used were safe from in–vivo

point of view. The cytototoxity data of normal Vero cells suggest that toxicity issue of

POMs can be addressed by closing it inside polymeric network and controlling its

release pattern.

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List of Publication/s

1. Azizullah, Nisar-ur-Rehman, Haider. A., Kortz, U., Afridi S. U., Sohail M., Joshi,

S.A., Iqbal, J. (2017). Novel pH Responsive Supramolecular Hydrogels of

Chitosan hydrochloride and Polyoxometalate: In-Vitro, In-Vivo and Preliminary

Safety Evaluation. Internal Journal of Pharmaceutics, (533) 125-137.

2. Azizullah, Nisar–ur–Rehman, Liu, W., Haider, A., Kortz, U., Sohail, M. Iqbal, J.

(2016). Novel gelatin–polyoxometalate based self–assembled pH responsive

hydrogels: formulation and in vitro characterization. Designed Monomers and

Polymers, 19(8), 697–705.

3. Azizullah, Haider,

A., Kortz,

U., Joshi. S.A.

and Iqbal, J. (2017).

Polyethyleneimine–Polyoxometalate-Based Supramolecular Self– Assembled pH

Responsive Hydrogels: Formulation and in vitro Evaluation. ChemistrySelect,

21(2), 5905-5912.

4. Azizullah, Ali Haider, Nisar-ur-Rehman, Ulrich Kortz, Sachin A. Joshi, Jamshed

Iqbal. Development and In-vitro Anticancer Evaluation of Self Assembled

Supramolecular pH Responsive Hydrogels of Carboxymethyl Chitosan and

Polyoxometalate. (Accepted in ChemistrySelect).

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TABLE OF CONTENTS

1 Introduction ............................................................................................ 1

Hydrogels ......................................................................................... 2 1.1

1.1.1 Natural and synthetic hydrogels ....................................................... 3

1.1.2 Biodegradability ............................................................................... 3

1.1.3 pH responsive hydrogels .................................................................. 4

1.1.4 Temperature responsive hydrogels .................................................. 5

1.1.5 Thermo–reversible hydrogels .......................................................... 5

1.1.6 According to cross–linking in hydrogel networks ........................... 6

1.1.7 Physically cross–linked or supramolecular hydrogels ..................... 8

1.1.8 Biomedical Applications of supramolecular hydrogels ................. 11

Cationic polymers .......................................................................... 15 1.2

1.2.1 Chitin and chitosan......................................................................... 16

1.2.2 Chemical modifications in chitosan ............................................... 18

1.2.3 Carboxymethyl chitosan (CMCh) .................................................. 18

1.2.4 Chitosan hydrochloride (ChCl) ...................................................... 20

1.2.5 Gelatin ............................................................................................ 22

1.2.6 Polyethyleneimine .......................................................................... 25

Polyoxometalates ........................................................................... 28 1.3

1.3.1 History of POMs ............................................................................ 28

1.3.2 Classification of POMs .................................................................. 29

1.3.3 General properties of POMs .......................................................... 30

1.3.4 Synthesis mechanism of POMs ..................................................... 31

1.3.5 Structural isomerism ...................................................................... 31

1.3.6 Applications of POMs .................................................................... 32

Problem statement .......................................................................... 34 1.4

Aims and objectives ....................................................................... 35 1.5

2 Materials and Methods ........................................................................ 36

Instruments ..................................................................................... 36 2.1

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Materials ......................................................................................... 37 2.2

Buffers and working solutions ....................................................... 39 2.3

2.3.1 Phosphate buffered saline .............................................................. 39

2.3.2 Swelling and dissolution buffers .................................................... 39

2.3.3 0.001 M tetra–n–butyl ammonium phosphate buffer .................... 39

Synthesis of pH responsive cationic polymer–POM complexes ... 39 2.4

Swelling studies of POM loaded hydrogels ................................... 42 2.5

In–vitro POM release ..................................................................... 43 2.6

POM release kinetics from hydrogel system ................................. 43 2.7

Fourier transform infrared spectroscopy (FTIR) ........................... 44 2.8

Thermogravimetric analysis (TGA) ............................................... 44 2.9

Differential Scanning calorimetry (DSC) ...................................... 44 2.10

Scanning electron microscopy (SEM) ........................................... 44 2.11

Powder X–ray diffraction............................................................... 45 2.12

Pharmacokinetic study of POM in rabbits ..................................... 45 2.13

2.13.1 High performance liquid chromatography (HPLC) analysis ......... 45

2.13.2 Validation of HPLC method .......................................................... 45

2.13.3 Animal models and drug administration ........................................ 47

2.13.4 Polyanion plasma concentration quantification and

pharmacokinetic profiling .............................................................. 47

Statistical analysis .......................................................................... 48 2.14

Acute toxicity evaluation and safety evaluation of the MAA–2.15

ChCl–POM hydrogel and POM oral solution ................................ 48

2.15.1 Histopathology studies ................................................................... 49

2.15.2 Serum chemistry and hematological investigations ....................... 49

Cytotoxicity assay .......................................................................... 50 2.16

2.16.1 Cell culturing .................................................................................. 51

3 Results.................................................................................................... 53

Synthesis of pH responsive gelatin–POM complexes ................... 54 3.1

3.1.1 Physical appearance ....................................................................... 54

3.1.2 Effect of gelatin content on swelling and P2W15 release ............... 54

3.1.3 Effect of acrylic acid content on swelling and P2W15 release ........ 55

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3.1.4 Effect of POM ratio on swelling and P2W15 release ...................... 56

3.1.5 POM release kinetics from pH responsive gelatin-POM complexes

56

3.1.6 FTIR Spectroscopy analysis .......................................................... 57

3.1.7 Scanning electron microscopy (SEM) ........................................... 58

3.1.8 Thermogravimetric analysis (TGA) ............................................... 59

3.1.9 Differential scanning calorimetry (DSC) ....................................... 60

3.1.10 X–ray diffraction ............................................................................ 61

3.1.11 Cytotoxicity assay .......................................................................... 62

Synthesis of pH responsive PEI–POM supramolecular complexes3.2

........................................................................................................ 63


3.2.2 Effect of Polyethyleneimine content on swelling and in–vitro

P2W15 release .................................................................................. 64

3.2.3 Effect of acrylic acid content on swelling and P2W15 release ........ 65


3.2.5 POM release kinetics ..................................................................... 66

3.2.6 FTIR spectroscopy ......................................................................... 67


3.2.8 Thermal analysis ............................................................................ 69

3.2.9 Powder X–ray diffraction............................................................... 70

3.2.10 Cytotoxicity assay of pH responsive PEI-POM complexes .......... 71

Synthesis of pH responsive carboxymethyl chitosan– POM 3.3

supramolecular complexes ............................................................. 71


3.3.2 Effect of CMCh concentration on swelling and P2W15 release ..... 72

3.3.3 Effect of methacrylic acid content on swelling and P2W15 release 73


3.3.5 POM release kinetics of pH responsive carboxymethyl chitosan-

POM supramolecular complexes ................................................... 74

3.3.6 FTIR Spectroscopy analysis .......................................................... 75


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3.3.8 Thermogravimetric analysis ........................................................... 77

3.3.9 Differential scanning calorimetry .................................................. 78

3.3.10 X–ray diffraction ............................................................................ 79

3.3.11 Cytotoxicity assay of pH responsive carboxymethyl chitosan-POM

supramolecular complexes ............................................................. 80

Synthesis of pH responsive chitosan hydrochloride–POM 3.4

complexes ....................................................................................... 80


3.4.2 Effect of ChCl concentration on swelling and P2W15 release ........ 81

3.4.3 Effect of methacrylic acid content on swelling and P2W15 release 82

3.4.4 Effect of POM concentration on swelling and in–vitro release ..... 83

3.4.5 POM release kinetics of pH responsive chitosan hydrochloride-

POM complexes ............................................................................. 83

3.4.6 FTIR spectroscopy analysis ........................................................... 84

3.4.7 Scanning electron microscopy ....................................................... 85

3.4.8 Thermogravimetric analysis ........................................................... 86

3.4.9 Differential scanning calorimetry .................................................. 87

3.4.10 X–ray diffraction analysis .............................................................. 88

3.4.11 Cytotoxicity assay .......................................................................... 89

Pharmacokinetic study of POM in rabbits ..................................... 89 3.5

3.5.1 HPLC method validation ............................................................... 90

3.5.2 Limit of detection and limit of quantification ................................ 91

3.5.3 Accuracy and precision .................................................................. 91

3.5.4 Pharmacokinetics of POM ............................................................. 93

Oral acute toxicity and safety evaluation of MAA–ChCl–POM 3.6

hydrogel and POM solution ........................................................... 96

3.6.1 Maximal tolerated dose of hydrogel dispersion ............................. 96

3.6.2 General conditions ......................................................................... 96

3.6.3 Hematological investigations and serum chemistry profiling ....... 97

3.6.4 Histopathologic study .................................................................. 100

4 Discussion ............................................................................................ 103

Effect of gelatin content on swelling and P2W15 release ............. 104 4.1

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Effect of polyethyleneimine content on swelling and P2W15 release4.2

...................................................................................................... 104

Effect of CMCh content on swelling and P2W15 release ............. 105 4.3

Effect of ChCl content on swelling and P2W15 release ................ 106 4.4

Effect of acrylic acid content on swelling and P2W15 release ...... 106 4.5

Effect of methacrylic acid content on swelling and P2W15 release4.6

...................................................................................................... 108

Effect of POM content on swelling and in–vitro release ............. 109 4.7

Polyanion release Kinetics ........................................................... 111 4.8

Characterization ........................................................................... 112 4.9

4.9.1 FTIR spectroscopy ....................................................................... 112

4.9.2 Scanning electron microscopy ..................................................... 115

4.9.3 Thermal analysis .......................................................................... 115

4.9.4 Powder X–ray diffraction............................................................. 119

Pharmacokinetic parameters ........................................................ 120 4.10

Acute oral toxicity evaluation and safety evaluation of the MAA–4.11

ChCl–POM hydrogel and POM oral solution .............................. 120

Cytotoxicity profiling of the developed hydrogels ...................... 121 4.12

Conclusion ................................................................................... 121 4.13

5 References ........................................................................................... 123

6 Appendices .......................................................................................... 156

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LIST OF FIGURES

Fig 1.1 Chemical structure of chitin and chitosan ....................................................... 17

Fig 1.2 Structural unit of carboxymethyl chitosan....................................................... 19

Fig 1.3 Formation of gelatin from collagen ................................................................. 24

Fig 1.4 Schematic of polyethyleneimine structural unit .............................................. 26

Fig 1.5 Structure of Lindqvist ion [M6O19]n–

............................................................... 29

Fig 1.6 Keggin ion [XM12O40] n–

and Wells–Dawson ion [X2W18O62]n–

............ 30

Fig 3.1 Sole form and B) developed hydrogel disc ..................................................... 54

Fig 3.2 A) Effect of gelatin concentration on swelling and B) cumulative % P2W15

release. Data represents mean value ± SEM (n=3) ...................................................... 55

Fig 3.3 A) Effect of acrylic acid concentration on swelling and B) cumulative %

P2W15 release. Data represents mean value ± SEM (n=3). .......................................... 55

Fig 3.4 A) Effect of P2W15 concentration on swelling and B) cumulative % release.

Data represents mean value ± SEM (n=3). .................................................................. 56

Fig 3.5 A) FTIR spectra of gelatin B) acrylic acid C) polyanion salt and D) hydrogel.

...................................................................................................................................... 58

Fig 3.6 Scanning electron microscope images of powdered hydrogel (a, b, c) and (d)

intact hydrogel disc. ..................................................................................................... 59

Fig 3.7 TGA plots of gelatin, POM salt and hydrogel. ................................................ 60

Fig 3.8 DSC curves of POM salt, gelatin and hydrogel............................................... 61

Fig 3.9 A) XRD pattern of gelatin (B) POM and C) hydrogel. .................................. 62

Fig 3.10 Cytotoxicity profiling gelatin–POM Hydrogel (encapsulated POM), free

POM (polyoxometalate, P2W15, normal control), doxorubicin (positive control) and

blank wells (Negative control): (A) Cytotoxicity against MCF–7 cells (B)

Cytotoxicity against HeLa cells (C) Cytotoxity against normal Vero cells shown as %

cell viability. ................................................................................................................ 63

Fig 3.11 A) Sole form and B) slightly dried developed hydrogel disc. ...................... 64

Fig 3.12 A) Effect of polyethyleneimine concentration on swelling and B)

cumulative % P2W15 release at of hydrogels at pH 1.2 and pH 7.4. Each data point

represents the mean ± SEM, n=3. ................................................................................ 65

Fig 3.13 A) Effect of acrylic acid concentration on swelling and B) cumulative %

P2W15 release of hydrogels at pH 1.2 and pH 7.4. Each data point represents the mean

± SEM, n=3. ................................................................................................................. 65

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Fig 3.14 A) Effect of POM concentration on swelling and B) cumulative % in–vitro

release of hydrogels at pH 1.2 and pH 7.4. Each data point represents the mean ±

SEM, n=3. .................................................................................................................... 66

Fig 3.15 A) FTIR spectra of polyethyleneimine B) acrylic acid C) POM D) hydrogel.

...................................................................................................................................... 68

Fig 3.16 a, b, c) Scanning electron microscope images of powdered hydrogel and d)

intact hydrogel. ............................................................................................................ 69

Fig 3.17 A) Thermograms and differential scanning calorimetry curves of POM and

B) hydrogel. ................................................................................................................. 70

Fig 3.18 A) X–ray diffraction pattern of POM and B) developed hydrogel. .............. 70

Fig 3.19 Anticancer and Cytotoxicity profiling of AA–PEI–POM hydrogel

(encapsulated POM), free POM polyoxometalate, P2W15 (normal control),

doxorubicin (positive control) and blank wells (Negative control). Anticancer activity

shown as % cell viability against A) MCF-7 cells B) HeLa cells and C) normal Vero

cells shown as % viability. ........................................................................................... 71

Fig 3.20 A) Sole form of hydrogel components and B) developed supramolecular

hydrogel disc. ............................................................................................................... 72

Fig 3.21 A) Effect of CMCh concentration on swelling and B) cumulative % release

of hydrogels. Data are shown as mean ± SEM, n=3. ................................................... 73

Fig 3.22 A) Effect of MAA concentration on swelling and B) cumulative % POM

release of hydrogels. Data are shown as mean ± SEM, n=3. ....................................... 73

Fig 3.23 (A) Effect of POM concentration on swelling and (B) cumulative % in–vitro

release of hydrogels. Data are shown as mean ± SEM, n=3. ....................................... 74

Fig 3.24 A) FTIR spectra of CMCh B) MAA C) polyanion salt and D) hydrogel ..... 76

Fig 3.25 Scanning electron microscope images of powdered hydrogel (A, B, C) and

(D) intact hydrogel disc. .............................................................................................. 77

Fig 3.26 TGA plots of CMCh, POM and hydrogel. .................................................... 78

Fig 3.27 DSC curves of CMCh, POM and hydrogel. .................................................. 79

Fig 3.28 A) X–ray diffraction patterns of CMCh B) POM and C) developed hydrogel.

...................................................................................................................................... 79

Fig 3.29 Cytotoxicity profiling of MAA–CMCh–POM Hydrogel (encapsulated

POM), free POM (polyoxometalate, P2W15, normal control), doxorubicin (positive

control) and blank wells (Negative control): (A) Cytotoxicity against MCF–7 cells

shown as % cell viability (B) HeLa cells C) normal Vero cells shown as % viability.

...................................................................................................................................... 80

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Fig 3.30 A) Sole form and B) developed MAA–ChCl–POM supramolecular hydrogel

disc. .............................................................................................................................. 81

Fig 3.31 (A) Effect of ChCl concentration on swelling and (B) cumulative % POM

release of hydrogels. Each data points represents average value ± SEM, (n=3). ........ 82

Fig 3.32 A) Effect of MAA concentration on swelling and (B) cumulative % POM

release of hydrogels. Each data points shows average value ± SEM, (n=3). .............. 82

Fig 3.33 A) Effect of POM concentration on swelling and B) cumulative % release of

hydrogels. Each data points shows average value ± SEM, (n=3). ............................... 83

Fig 3.34 A) FTIR spectrum of ChCl B) MAA C) polyanion salt and D) hydrogel. .... 85

Fig 3.35 A, B, C) Scanning electron microscope images of powdered hydrogel and D)

intact hydrogel disc. ..................................................................................................... 86

Fig 3.36 TGA Plots of ChCl, POM and hydrogel........................................................ 87

Fig 3.37 DSC curves of ChCl, POM and hydrogel..................................................... 88

Fig 3.38 A) X–ray diffraction patterns of ChCl B) POM and C) hydrogel. ................ 89

Fig 3.39 A–B) Cytotoxicity profiling of ChCl–POM Hydrogel (encapsulated POM),

free POM (Polyoxometalate, P2W15, normal control), doxorubicin (positive control)

and blank wells (negative control) Cytotoxicity against MCF–7, HeLa cells and (C)

cytotoxicity against normal Vero cells shown as % viability. ..................................... 89

Fig 3.40 Calibration curve of polyanion spiked in rabbit plasma. .............................. 90

Fig 3.41 Representative chromatograms of blank and polyanion spiked rabbit plasma.

...................................................................................................................................... 92

Fig 3.42 Plasma profile of POM after oral solution, oral hydrogel and combined

plasma profile after oral solution and oral hydrogel. ................................................... 94

Fig 3.43 Individual Plasma Profiles of POM obtained for rabbits control group in

comparison with mean plasma profile. ........................................................................ 95


comparison with mean plasma profile. ........................................................................ 95

Fig 3.45 A) Histopathalogy photographs of heart muscle of control group rabbit B)

hydrogel dispersion treated rabbit and C) POM treated rabbits. ............................... 100

Fig 3.46 A) Histopathology photographs of liver of control group rabbit B) hydrogel

dispersion treated rabbit and C) POM treated rabbit. ................................................ 101

Fig 3.47 A) Histopathalogy photographs of spleen of control group rabbit B) hydrogel

dispersion treated rabbit and C) POM treated rabbits. ............................................... 101

Fig 3.48 A) Histopathalogy photographs of lung of control group rabbit B) hydrogel

dispersion treated rabbit and C) POM treated rabbits. ............................................... 102

xxi

Fig 3.49 A) Histopathology photographs of kidney of control group rabbit B)

hydrogel dispersion treated rabbit and C) POM treated rabbits. ............................... 102

xxii

LIST OF TABLES

Table 2.1 Composition of the various AA–gelatin–POM hydrogel formulations. ...... 40

Table 2.2 Composition of the various AA–PEI–POM hydrogel formulations. ........... 41

Table 2.3 Composition ratios of the various MAA–CMCh–POM hydrogel

formulations. ................................................................................................................ 41

Table 2.4 Composition ratios of various MAA–ChCl–POM hydrogel formulations. . 42

Table 3.1 Mechanism of encapsulated POM release in dissolution experiments of

AA–gelatin–POM hydrogels. ...................................................................................... 57

Table 3.2 Mechanism of POM release in dissolution experiments of AA–PEI–POM

hydrogels. ..................................................................................................................... 67

Table 3.3 Mechanism of POM release in dissolution experiments from MAA–CMCh–

POM hydrogels. ........................................................................................................... 75

Table 3.4 Mechanism of POM release during dissolution experiments from MAA–

ChCl–POM hydrogels. ................................................................................................. 84

Table 3.5 Linearity summary of the method ................................................................ 90

Table 3.6 Determination of LOD and LOQ by signal to noise ratio method (n=6). ... 91

Table 3.7 Precision and accuracy of the developed HPLC method in rabbit plasma. . 92

Table 3.8 Mean values ± SEM of pharmacokinetic parameters of POM following

administration of oral solution and oral hydrogel in rabbits (n=12) ............................ 93

Table 3.9 Toxicity signs not observed in hydrogel dispersion (4000 mg/kg) and POM

treated groups. .............................................................................................................. 96

Table 3.10 Mean values ± SEM of rabbit Serum levels of albumin, total protein,

globulin, alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase,

cholesterol, glucose, bilirubin, creatinine, urea, magnesium, potassium and sodium.

Control, (n=3 male, n=3 female), hydrogel dispersion (n=3 male, n=3 female), POM

(n=3 male, n=3 female) ................................................................................................ 98

Table 3.11 Mean values ± SEM of rabbit blood count of WBCs, lymphocytes,

eosinophils, basophils, neutrophils, platelets, RBCs, hemoglobin level, packed cell

volume and mean cell volume. (Control, n=3 Male, n=3 female, hydrogel dispersion

n=3 Male, n=3 female, POM, n=3 male, n=3 female) ................................................. 99

xxiii

LIST OF ABBREVIATIONS

AA Acrylic acid

AAM Acrylamide

Au Gold

AUC Area under the curve

aq Aqueous solution

AsPC–1 Pancreatic carcinoma cells

β–CD β–cyclodextrin

BPEI Branched polyethyleneimine

Ca2+

Calcium ion

Cl Clearance

cm2 Centimeter square

Cmax Maximum plasma concentration

CMCh Carboxymethyl chitosan

ChCl Chitosan hydrochloride

Cu Copper

Cd Cadmium

Co Cobalt

CaCo–2 Colorectal adenocarcinoma cell line

CLSM Confocal laser scanning microscopy

Da Dalton

DLS Dynamic light scattering

DSC Differential scanning calorimetry

DMEM Dulbecco's Modified Eagle's medium

DNA Deoxyribonucleic acid

DPBS Dulbecco's phosphate–buffered saline

EGDMA Ethylene glycol dimethacrylate

E.coli Escherichia coli

FBS Fetal bovine serum

FTIR Fourier transform infrared spectroscopy

g Gram

GIT Gastrointestinal tract

H Hour

xxiv

H157 Human squamous cell carcinoma cell line

HeLa Human cervical adenocarcinoma cell line

HER2 Human epidermal growth factor receptor 2

Hg Mercury

HmGOD Hydrophobically modified glucose oxidase

HIV Human immunodeficiency virus

HPA Heteropoly acid

HPAs Hetero polyanions

HPV Human Papilloma virus

HPLC High performance liquid chromatography

IARC International Agency for Research on Cancer

IPAs Iso polyanions

IPN Interpenetrating polymer network

IUPAC International Union of Pure and Applied Chemistry

KCl Potassium chloride

KDa Kilo Dalton

KH2PO4 Potassium dihydrogen phosphate

kV Kilo–volt

LPEI Linear polyethyleneimine

Lz Elimination half life

MCF–7 Human breast cancer cell line

MEC Minimum effective concentration

mg Milligram

Mg2+

Magnesium ion

MgCl2 Magnesium chloride

MRSA Methicillin resistant staphylococcus aureus

min Minute

mL Milliliter

M Molar

mM Millimolar

MRI Magnetic resonance imaging

MRT Mean retention time

MTT (3–(4, 5–dimethylthiazolyl–2)–2,5–diphenyltetrazolium

bromide)

MTD Maximal tolerance dose

https://en.wikipedia.org/wiki/Mercury_(element)

xxv

Na2HPO4 Di–sodium hydrogen phosphate

nm Nanometer

nM Nano molar

NMR Nuclear magnetic resonance

Pb Lead

PC–3 Human prostate cancer cell line

PEI Polyethyleneimine

PEG Polyethylene glycol

PCL Poly(ɛ–caprolactone)

PLA Poly (lactic acid)

PLGA Poly lactic–co–glycolic acid

PLGA–b–PEG Poly (D,L–lactic–co–glycolic acid)–b–poly(ethylene glycol)

PM–17 [Me3NH]6[H2Mo(V)12O28(OH)12(Mo(VI)O3)4]∙2H2O

PM–8 [NH3Pri]6[Mo7O24]·3H2O

Po Polonium

POMs Polyoxometalates

P2W15 Na12[P2W15O56].12H2O

PVP Polyvinyl pryrolidone

PMAA Poly methacrylic acid

PAA Poly acrylic acid

PNIPAAM Poly (N–isopropyl acrylamide)

PEO–PPO–PEO Polyethylene oxide–polypropyleneoxide–polyethylene oxide

PLGA Poly–lactic acid–co–glycolic acid

PVA Polyvinyl alcohol

R2 Regression coefficient

ROS Reactive oxygen species

rpm Revolution per minute

S.E.M. Standard error of mean

SEM Scanning electron microscopy

SODN antisense phosphorothioate oligonucleotides

SGC–7901 Human gastric cancer cell line

SRB Sulphorhodamine B

SiRNA Small interfering ribonucleic acid

TCA Trichloroacetic acid

TGA Thermogravimetric analysis

xxvi

TNBS Trinitrobenzene sulfonate

Tmax Maximum plasma time concentration

Trizma® 2–Amino–2–(hydroxymethyl)–1,3 propanediol

Trypan blue (3Z,3'Z)–3,3'–[(3,3'–dimethylbiphenyl–4,4'diyl)di(1Z)hydrazine

–2–yl–1–ylidene]bis(5–amino–4–oxo–3,4dihydronaphthalene–

2,7–disulfonic acid)

U Unit

UV Ultraviolet radiation

USP United States Pharmacopeia

Vero African green monkey kidney cell line

Vd Volume of distribution

XRD X–ray diffraction

ZP Zeta potential

µg Microgram

µL Microliter

µM Micromolar

Å Armstrong

α Alpha

β Beta

γ Gamma

δ Delta

λ Lambda

1

Chapter 1

1 Introduction

2

Hydrogels 1.1

Hydrogels represent three dimensional, polymeric, hydrophilic networks having

ability to absorb large amounts of aqueous biological fluids (Annabi et al., 2014; Caló &

Khutoryanskiy, 2015). The hydrogels structure in aqueous environment is created by the

hydration of the hydrophilic groups, which are present inside the polymeric network

structure. Some sorts of crosslinks have to be necessarily present in hydrogels for

avoiding dissolution of hydrophilic segments. Aqueous solutions of hydrogels show

Newtonian behavior at low concentration so they can be described rheologically. The

Newtonian behavior of hydrogels gets converted to elastic or viscoelastic behavior when

cross linker is introduced in between the polymer chains. Hydrophilic groups present in

the hydrogel skeleton absorb large amount of water, so they have wide variety of

applications in tissue engineering scaffolds, contact lenses, artificial heart linings,

artificial skin materials and biosensors (Faghihi et al., 2014; D. Kim & Park, 2004).

Hydrophilicity of hydrogels can be attributed to various functional groups present in the

polymeric strands .These functional groups include hydroxyl, amide, amino and carboxyl

groups. Based on cross–linking methods, hydrogels are of two types; chemically cross–

linked and physically cross–linked hydrogels. In chemically cross–linked hydrogels, the

cross–linking is mediated through covalent interactions while in other type the hydrogels

are fabricated as a result of non–covalent interactions. Physical gels are reversible gels as

the polymeric network is held together either through molecular entanglements or

secondary forces like hydrogen bonding, ionic bonding or hydrophobic forces

(Campoccia et al., 1998; Prestwich et al., 1998).

Hydrogel made up of poly(2–hydroxyethyle methacrylate) was first synthesized

by (Wichterle & Lim, 1960) for application in contact lenses. Lim and Sun (1980),

successfully prepared and applied the microcapsules of calcium alginate for cell

encapsulation. In 1980s, natural polymers like shark cartilage and collagen were

incorporated into hydrogels by Yannas et al., 1998 for preparation of artificial burn

setting the grounds for use of hydrogels in various fields like wound dressing, drug

delivery, ophthalmic materials and ―tissue engineering‖ for regenerating and repairing the

organs and tissues (Cao et al., 1998; Chow & Khor, 2000; Clapper et al., 2000; Cruise et

3

al., 1998; Hubbell, 1998; Hunter et al., 2000; Ichi et al., 2000; Jeong et al., 2000; Kim et

al.,1999; Kim et al., 2000; Lutolf et al., 2000; Marcolongo et al., 2000; Marler et al.,

2000; Ohya et al., 2000; Woerly, 1997; Yamamoto et al., 1999).

Swelling of the hydrogels is directly related to the extent of osmotic driving force

encouraging water or biological fluids entrance and the cohesive forces between polymer

strands. The cohesive forces among polymer strands resists the expansion of hydrogels

during swelling. The magnitude of these cohesive forces is directly dependent on cross–

linking density (Guenet, 1992; Markey, Bowman, & Bergamini, 1989). In other words

more is the polymer hydrophilicity, high will be the total water content taken up by

hydrogel. Whereas higher the cross–linking density, lower will be the rate of gel

hydration. In dried form hydrogels are termed as xerogels but if some drying technique is

involved in the preparation of hydrogels like solvent extraction based drying or freeze

drying then the resulting hydrogels are called aerogels (Guenet, 1992).

Hydrogels are classified on numerous bases depending on its origin, preparation

method, physical properties, ionic charge, biodegradation and nature of cross–linking

(Qiu & Park, 2001).

1.1.1 Natural and synthetic hydrogels

Based on source of origin, hydrogels can be classified into three classes, natural,

synthetic and semi synthetic. Natural hydrogels are fabricated from polymers obtained

from natural sources like polypeptides, polysaccharides and polynucleotides. Such

polymers are obtained from natural sources like chitin and chitosan from shellfish of

exoskeleton and collagen from mammals. Vinyl activated monomers polymerized

through traditional methods are mainly used as fabricating agents for the preparation of

synthetic hydrogels. Monomer hydrophilicity and extent of cross–linking are the main

factors defining the swelling index of prepared hydrogels.

1.1.2 Biodegradability

Hydrogels can be either durable or biodegradable depending on their stability and

integrity in physiological environment. In the current era, significant research is focused

4

on the preparation and utilization of biodegradable hydrogels which are employed in

many areas covering both biomedical and non–medical applications. Biodegradable

hydrogels are degraded into oligomers chains scission process which either eliminated

directly from the body or subjected to further degradation process (W. Zhu & Ding,

2006). According to hydrogel response or stimuli responsive hydrogels

In the recent past a unique class of hydrogels has emerged which has drawn much

interest of research scientists. This unique class of the hydrogels is called smart hydrogels

or intelligent hydrogels. These intelligent hydrogels are also termed as stimuli responsive

hydrogels, responding smartly to surrounding environment (Gil & Hudson, 2004; N.

Peppas, Bures, Leobandung, & Ichikawa, 2000). Physical stimuli include temperature,

light, electric field, pressure etc. while chemical stimuli include chemical agents, ionic

factors and pH. Another class of hydrogels known is called dual responsive hydrogels

which responds to two different stimuli in one hydrogel system. Example of the dual

responsive hydrogel is polyacrylic acid–co–polyvinyl sulfonic acid hydrogel system

(Kang & Bae, 2003). Stimuli responsive biomaterials are attractive agents regarding

biomedical, pharmaceutical and biotechnological applications (Kashyap et al., 2005).

1.1.3 pH responsive hydrogels

This class of hydrogels lies in the class of smart hydrogels. pH responsive

hydrogels are very much investigated for stimuli based responsive drug delivery. This

category of hydrogels got immense attraction because of pH variations across

gastrointestinal tract (Hall, 2015). These hydrogels are made from polymers having

pendant ionic groups present along the polymers backbone. These polymers include

polymethacrylic acid (PMAA), polyacrylic acid (PAA) and poly(diethyl–aminoethyle

methacrylate) (PDEAEMA) (Lowman et al., 1999). These pendant ionic groups play a

key role in pH sensitivity of these hydrogels. At specific pH values the pendant ionic

groups are ionized and generate charges on polymeric network resulting in electrostatic

repulsive forces creating voids inside polymer network leading to swelling of hydrogels

(Kofinas, et al., 1996).These hydrogels are of two types, cationic and anionic hydrogels.

In cationic hydrogels the pendant groups are amine type undergo ionization below pKb

resulting in swelling due to electrostatic repulsive forces (Baker et al., 1992; Gupta et al.,

2002). In anionic hydrogels the pendant ionic groups are like sulfonic acid or carboxylic

5

acid which become ionize when pH is greater than pKa leading to ionization and results

into swelling of hydrogels (Jabbari & Nozari, 1999; Jianqi & Lixia, 2002; Li et al.,

2006).

1.1.4 Temperature responsive hydrogels

Temperature responsive hydrogels responds to the changes in surrounding fluid

temperature and they swell and deswell accordingly (Kuckling et al., 2009). These

hydrogels are furthermore classified as positive temperature responsive hydrogels and

negative temperature responsive hydrogels. Positive hydrogels have upper critical

solution temperature (UCST) value (Serra et al.,2006) and these hydrogels undergo

collapsing phenomenon because of hydrogel bonds formation at temperature below

UCST and the fluids are released form the hydrogel matrix. When the temperature

exceeds the UCST, hydrogels starts swelling. There are many polymers and copolymers

which are dependent on temperature, i.e. poly (AAm–co–BMA and poly (AA–co–AAm–

co–BMA) (Gong et al., 2013). Negatively temperature sensitive hydrogels are

characterized by lower critical solution temperature (LCST). These hydrogels are

shrinked when the temperature exceeds the LCST and swells when the temperature value

is below LCST. Lower critical solution temperature is one of the important parameter of

the negatively temperature sensitive hydrogels which can also be altered by either

changing the solvent composition or by adding ionic copolymer in small amount. Lower

critical solution temperature of hydrogels can also be changed by changing the

hydrophilic to hydrophobic content ratio in hydrogel structure. At temperatures below

LCST fluids interact by forming hydrogen bonds with the hydrophilic portion and as a

result swelling and dissolution of these gels improves. When temperature exceeds the

LCST, interaction with the hydrophobic part becomes stronger resulting in reduced

swelling. Example of the negatively temperature sensitive hydrogel is PVP/PNIPAAM

hydrogel system (Geever et al., 2008).

1.1.5 Thermo–reversible hydrogels

Structurally and content wise thermo–reversible hydrogels are same like negative

and positive temperature responsive hydrogels with only difference in network bond

types. There is no covalent cross–linking in thermo–reversible hydrogels and instead of

6

swelling–deswelling there is only phase transition from sol to gel. The thermosenstive

hydrogels undergoing volume changes have covalent cross–linking and they can‘t

undergo sol–gel transitions. Example of the thermo–reversible hydrogel is the

Poly(ethylene oxide)-poly(propylene oxide)- Poly(ethylene oxide) (PEO–PPO–PEO)

based hydrogels. Parenteral preparation contains biodegradable Thermo–reversible

hydrogel system. Therefore to make the PEO–PPO–PEO based hydrogel biodegradable,

PPO segment is replaced with poly(L–Lactic acid). Tectonics and pleuronics are the

vastly used Thermo–reversible polymers recommended by EPA and FDA for

pharmaceuticals, food additives and agricultural purposes (Li et al., 2011).

1.1.6 According to cross–linking in hydrogel networks

Based on cross–linking hydrogels can be divided into two classes, chemically

cross–linked also called synthetic hydrogels and physically cross–linked also called

supramolecular hydrogels (Hennink & Van Nostrum, 2012).

1.1.6.1 Chemically cross–linked or synthetic hydrogels

In chemically cross–linked or synthetic hydrogels permanent bonds like covalent

bonds are present and they can be prepared through different methods. Some of which are

listed below (Hennink & Van Nostrum, 2012).

1.1.6.2 Cross–linking by radical polymerization

Several hydrogels are prepared by this method (Langer & Peppas, 1981) in which

gels are prepared by cross–linking of the monomers having low molecular weight with

various crosslinkers. The first hydrogels prepared by this method was prepared by

Wichterle and Lim as mentioned earlier. Swelling of the so prepared hydrogels can also

be modulated as this characteristic of the hydrogels is proportionally dependent on the

amount of crosslinker used. Moreover through this route of hydrogel preparation various

sophisticated environment responsive hydrogel systems can also be prepared, for

example pH responsive hydrogels from monomers like acrylic acid, methacrylic acid

(Bettini et al., 1995) etc. and temperature sensitive hydrogels using N–

isopropylacrylamide (Cicek & Tuncel, 1998).

7

1.1.6.3 Cross–linking by complementary groups chemical reaction

In water soluble polymers certain functional groups are present like (NH2, OH,

and COOH) which are responsible for their water solubility and these functional groups

are in turn involved in hydrogel formation through the development of covalent bonds

between polymer strands bearing complementary functional groups responsible for its

complementary reactivity. Examples of such complementary reactions include Schiff

base reactions, amine–carboxylic acid reactivity and isocyanate–NH2/OH reactions.

1.1.6.4 Cross–linking by addition reactions

Water soluble polymers make hydrogels by cross–linking via addition reactions.

The network properties of the so developed hydrogels can be easily tailored by the

concentration of cross–linking agent and amount of dissolved polymer. Cross–linking is

done in the presence of organic solvents but the gels are then extracted very extensively

before application as the crosslinkers are highly toxic and the unreacted contents have to

be removed. Such type of matrices are loaded with drugs after successful gel formation

and extraction (Coviello et al., 1999; Gehrke et al., 1998).

1.1.6.5 Cross–linking by condensation reaction

Various types of polyamides and polyesters are produced by the condensation

reactions between amines with carboxylic acid or hydroxyl groups. Hydrogels can also be

prepared by these reactions. Example of hydrogel system prepared by this method is the

gelatin hydrogel cross–linked by ,N–(3–dimethylaminopropyl)–N–ethyl carbodiimide

(EDC) (Kuijpers et al., 2000). Alginate and polyethyleneglycol based gels were also

produced via same mechanism using same crosslinking agent (EDC) (Eiselt et al.,1999).

1.1.6.6 Cross–linking using enzymes

Hydrogels can also be produced using enzyme based cross–linking phenomenon.

Example of such enzyme based developed hydrogels is the PEG based hydrogels

prepared by sperinde et al. In this study glutaminyl groups were used for the

functionalization of the tetrahydroxy PEG (PEG–Qa). Networks of PEG were then

fabricated by transglutaminase enzyme between poly(lysine–co–phenylalanine) and

PEG–Qa. Amide bonds were produced as a result of reaction between PEG–Qa γ–

carboxamide group and lysine ε–amine group (Sperinde & Griffith, 1997).

8

1.1.6.7 Cross–linking using high energy irradiation

For the polymerization of the unsaturated compounds high energy radiations

particularly electron beam and gamma radiations are used. Similarly water soluble

polymers bearing vinyl groups can also be converted into gels using high energy

radiations (Giammona et al.,1999). Hydrogels of monofunctional acrylate and suitable

crosslinker can be developed as a result of polymerization induced by high energy

irradiation (Peppas & Mikos, 1986). The swelling characteristics of the developed

hydrogels depends on polymer load and radiation dose as the cross–linking density

depends on these two variable parameters. Some polymers which can be cross–linked

with high energy radiations include polyacrylic acid, polyethylene glycol and polyvinyl

alcohol (Jabbari & Nozari, 2000; Kofinas et al., 1996; Stringer & Peppas, 1996 ; Peppas

& Merrill, 1977).

1.1.7 Physically cross–linked or supramolecular hydrogels

Practically no commercial products are available in the market in spite of

extensive efforts made regarding fabrication of hydrogels through various cross–linkers

(Bromberg & Ron, 1998). Hydrogels prepared as a result of chemical reactions should be

extremely pure to meet specifications of FDA, as some of the cross linkers used are not

only highly toxic, teratogenic and carcinogenic (Qui et al., 2012) but they also disturb the

entrapped substance integrity as well, particularly in case of proteins and cells. So such

substances either need removal/extraction from gels before application or the hydrogels

should be developed through physical cross–linking for which various methods are

applied as discussed below.

In physically cross–linked or supramolecular hydrogels non covalent crosslinks

are present avoiding the use of crosslinkers. They are also called self–assembled

hydrogels. Self–assembly is evident in nature and by definition is the natural and

spontaneous association or organization of different molecules into balanced and rational

structures or arrangements without external involvement (Kopeček & Yang, 2012; Kyle,

et al., 2009) through various non–covalent interactions (Zhang, 2002). Self–assembly is

also called supramolecular assembly in which various physically interactive forces are

involved such as hydrogen bonding, electrostatic interactions, hydrophilic or hydrophobic

9

interactions, van der Waals forces and ԉ–ԉ interactions (Chung & Park, 2009; Yang et

al., 2015; Zhao et al., 2015). These physical interactions directly impact the gel properties

and integrity of the superstructure (Nagarajan, 2001). Supramolecular interactions are

being known for long but the ground breaking work of Lehn (Lehn, 1988) , Cram (Cram,

1988) and Pederson(Pedersen, 1988) (Nobel Prize awarded in 1987) caused the rapid

expansion of this field. The capacity of molecular species for reversible and spontaneous

bonding is a driving force for developing self–healed (Kakuta et al., 2013), dynamic

(Seiffert & Sprakel, 2012) and structured materials (Fyfe & Stoddart, 1997; Hoeben et

al., 2005; Percec et al., 2004) as well as its applications in pharmaceuticals (Brewster &

Loftsson, 2007; Ma & Zhao, 2014; Zhou & Ritter, 2010).

Synthetic hydrogels are widely used for various biomedical applications but there

are certain limitations of synthetic hydrogels like they are brittle and opaque, unable to

heal when network structure is broken. Incorporation of therapeutic agents in synthetic

hydrogels is through sorption process which is also a time taking process and only limited

content can be loaded. Moreover, sometimes drugs also undergo conjugation process

with hydrogel during cross–linking disturbing the integrity of incorporated drugs and the

hydrogel system may by itself become non–biodegradable by adopting ill–defined

composition ( Li, 2010). Therefore an advanced drug delivery system is keenly

anticipated in which drug loading and gelation in aqueous media can be simultaneously

achieved.

Supramolecular hydrogels presents a novel class of polymeric materials which are non–

covalently cross–linked (Appel et al., 2012; Babu et al., 2014; Zhang & Ma, 2013; Zhao

et al., 2009) but at the same time they possess all the advantages of synthetic hydrogels as

well (Aida et al., 2012; Dong et al., 2015; Wang et al., 2014). In supramolecular hydrogel

system the cross–linking is achieved through electrostatic interaction, host–guest

recognition, metal–ligand coordination or hydrogen bonding. These interactions results in

remarkable reduction in structural flexibility leading to the formation of three

dimensional networks. These supramolecular hydrogels possess the moderate mechanical

properties and undergo sol–gel behavior in response to external stimuli like redox agents,

pH, bioactive molecules and enzymes. Supramolecular hydrogels are used as a carrier for

variety of therapeutics like proteins, genes and drug molecules etc. and are promising

10

tools in tissue engineering as well for regenerating and repairing human tissues and

organs ( Dong et al., 2015).

1.1.7.1 Size of the supramolecular hydrogels

Supramolecular hydrogels adopt different sizes depending on cross–linked

networks. On size basis supramolecular hydrogels are divided into three types, macro–

hydrogel, micro–hydrogel and nano–hydrogel. In macro–hydrogels infinite cross–linked

networks are present leading to uncontrollable growth. In majority cases the interactions

are through multivalent non–covalent interactions. Macrohydrogels are the good

candidates for tissue engineering as well as delivery of therapeutic agents because of

presence of three dimensional dynamic networks and in situ gelation behavior in intimate

contact with the tissue.

Internally micro and nanohydrogels are similar to macrohydrogels with the

difference in responsiveness and size. Size of microhydrogels is comparable with human

cell having 1–100µm size while that of nanohydrogels is less than 200 nm.

Nanohydrogels are especially engineered gels which are transportable inside cell by the

process of endocytosytosis. Therefore nanohydrogels provide a very suitable platform for

efficient and safe delivery of therapeutic agents like proteins, drugs and genes in the

targeted regions of the human body (Yallapu et al., 2011). Size of nanohydrogels is

important in various aspects like cytotoxicity, therapeutic efficacy and internalization

pathways (W. Jiang, Kim, Rutka, & Chan, 2008). Therefore there was a need of

developing simple and fast approach for controlling the nano size. The size issue of

nano–hydrogels can also be controlled by simply altering the molar ratios of the contents

providing the size controlled nano–hydrogels for effective application in cancer therapy

and diagnosis (Dong et al., 2012; W. Jiang et al., 2008).

1.1.7.2 Building blocks of the supramolecular hydrogels

Based on building blocks, supramolecular hydrogels are classified into three

levels. 1) Molecular hydrogels 2) Supramolecular polymeric hydrogels and 3)

supramolecular hybrid hydrogels. Preparation of molecular hydrogels occurs in two steps.

In first step fiber like structures are developed through non–covalent interactions from

low molecular weight building blocks and then supramolecular structure is developed

through non–covalent entanglements between nano fibers. These hydrogels undergo

11

smooth biodegradation because of low molecular weight building blocks and they

respond very smartly to the external stimuli. Molecular hydrogels can also be prepared

from multitopic building blocks possessing low molecular weight by direct multivalent

non–covalent interactions (Castellano et al., 2000; Zhang et al., 2013).

Supramolecular polymeric hydrogels (Cao et al., 2013; Ma et al., 2014; Zhang et

al., 2013) are prepared from multitopic polymer chains bearing complementary

supramolecular motifs. In supramolecular polymeric hydrogels, host–guest

supramolecular polymeric hydrogels are more stable and flexible and they have the

ability to change the macroscopic properties as well as microscopic structure in response

to external stimuli. Supramolecular hybrid gels possess inorganic components in their

networks. These inorganic components may be either graphenes (Han & Yan, 2014),

clays (Tamesue et al., 2013), quantum dots (Wu et al., 2013) or metal nanostructures

(Jung et al., 2014). These gels shows good mechanical properties and they are

comparatively stable and can be used for studying electrical conductivity, florescence

properties and antibacterial activities.

1.1.8 Biomedical Applications of supramolecular hydrogels

Beside exclusive physicochemical properties supramolecular hydrogels can be

used for specific functions and they can respond very smartly to external stimuli. All

these characteristics prove supramolecular hydrogels ideal for variety of biomedical

applications like drug delivery, biodetection, bioimaging, tissue engineering, protein

delivery and gene transfection (Dong et al., 2015).

1.1.8.1 Biodetection

The advancement in biodetection techniques for disease diagnosis and

environmental analysis of biological agents is one of the crucial issue. Biosensors have

been developed as a result of combination of signal conversion and biological recognition

elements which are now used for variety of applications. Many biological agents are

significantly involved in various biological events happening in diseased condition. So

the detection of pathological condition biomarkers is extremely important not only from

basic science point of view but also for diagnostic purposes. Nano supramolecular

hydrogels are used for the biodetection purposes because of their rational size, good

12

biocompatibility and smart responsive behavior to the external stimuli like biological

molecules and enzymatic reactions (Siegel et al., 2010).

Recently, large number of supramolecular hydrogels based sensors have been

developed for the detection of various biological molecules like POMs, glycoconjugates,

alkaline phosphatase, cancer cells, polyamines and glycosidase etc. Hamachi in recent

past developed a supramolecular hydrogel based detection system for exploring the

prostate specific antigen and selective targeting, sensing of the prostate cancer cells

(Ikedaet al., 2010).

1.1.8.2 Bioimaging

The aim of bioimaging is to visualize in–vivo disease related molecular pathways

by using sophisticated probes. Bioimaging facilitates the scientists in visualization of the

disease processes at molecular level, hence can be used in diagnosis and therapy of

cancer (Zhu et al., 2013). Now a day‘s various bioimaging tools and techniques are in use

for clinical diagnosis of cancer. Various techniques used for bioimaging include magnetic

resonance imaging involving paramagnetic agents, optical imaging using fluorescent

probes, nuclear imaging through radio–labelled probes and ultrasound imaging using

nanostructures which are acoustically active. On the basis of molecular structures

bioimaging probes have three classes, supramolecular polymers, conventional polymers

and small molecule compounds. Supramolecular bioimaging probes are infact the

supramolecular hydrogels, offering several advantages in this regard like excellent

biocompatibility, biodegradability, defined three dimensional structure and smart

responsiveness to external physiological stimuli (Dong et al., 2015). Various bioimaging

agents like radioactive isotopes, quantum dots, fluorescent dyes and contrast agents can

be easily carried on nanoscale supramolecular hydrogels because they are easily taken by

cells through the process of endocytosis. Supramolecular Cu labelled nanoparticles were

fabricated by Tseng through host– guest interaction mechanism for the purpose of

microPET/CT imaging (Wang et al., 2009). Amphiphilic peptide based fluorescent

supramolecular hydrogels were developed by Xu and coworkers for the purpose of cancer

cell diagnosis and imaging (Li et al., 2013). Supramolecular nanoparticles containing

Gd3+

represents novel class probing agent which will be used in future for the cancer

metastasis diagnosis in future (Chen et al., 2011). In the field of nanomedicine

13

supramolecular nanoparticles are providing the most exciting platform for both diagnostic

as well as therapeutic purposes because of its rational design, size and multifunctional

applications (Wu et al., 2013). Supramolecular hybrid multifunctional nanoparticles were

used by Li for the co–delivery of the anticancer drug paclitaxel and DNA into the tumor

cells. The nanoparticles were composed of quantum dot red fluorescence core and

cationic polymer shell. Therefore additionally they were also used for cellular imaging as

well and due to imaging ability it was possible to track co–delivery system inside

untransfected and transfected cells (Wu et al., 2013).

1.1.8.3 Tissue Engineering

Tissue engineering can be defined as ―development and understanding of the life

sciences and engineering principles and methods for understanding the relationship of

structure–function of the normal and pathological tissues and synthesis of the substitutes

for regeneration or repairing of organ or tissue function‖. In tissue engineering the main

goal is achieving local regeneration of the malfunctioning or lost organ, tissue by

culturing victims own body cells on a suitable polymeric matrix. To achieve this goal two

main factors play a key role, interaction of the biomaterial with the cell and physiological

environment of the body. Supramolecular hydrogels can be used for sustained tissue

growth because on one side they are biodegradable possessing tunable mechanical

properties and on another side they can excellently afford physiological environment.

Moreover, biologically active agents like cells and growth factors can be efficiently

encapsulated and delivered using supramolecular hydrogels as delivery carriers. In recent

past supramolecular hydrogels are used as efficient biomaterials in issue engineering

prepared from bioactive building blocks through host–guest interaction or hydrogen

bonding. Oligopeptides having naphthalene moiety can be converted into supramolecular

hydrogels –in vivo using phosphatase/Kinase switch which showed good biocompatibility

profile in mice (Yang et al., 2006). Similarly yang and coworkers successfully

demonstrated the self–assembled hydrogelation capability of naphthalene containing

tripeptide on platelets surface satisfactorily inhibiting platelet aggregation (Zheng et al.,

2012).

14

1.1.8.4 Therapeutics delivery via supramolecular hydrogels

Most of the supramolecular hydrogels are prepared from natural sources so they

are highly compatible and can be used for relevant biomedical purposes. Supramolecular

gels are providing fertile platform for controlled release of various molecules especially

drugs. They can also be used for external stimuli based programmable drug delivery.

Various studies are conducted in this regard to investigate the therapeutics delivery

potential of the supramolecular hydrogels (Dong et al., 2013; Dong et al., 2011). Sorbiton

monosteartae multicomponent organogels were investigated for their drug in–vivo

delivery ability (Murdan et al., 1999). Peptide based supramolecular hydrogels are used

for drug delivery purposes and at low pH undergo sol–gel phase transition, hence used

for controlled delivery of anti–HIV prodrugs ( Li et al., 2013), anti–inflammatory agents

(Li et al., 2012), Taxol (Yang et al., 2014) and kanamycin (Zheng et al., 2012).

Supramolecular hydrogels which are prodrug based are more significant in inhibiting

cancer growth (Jiayang Li et al., 2013; J. Li et al., 2012). Cyclodextrin and its derivatives

form supramolecular injectable hydrogels through host–guest interaction and offer

several advantages like weak immunogenicity and good biocompatibility. Therefore they

are used as promising candidates for treating cancer (Ha et al., 2014; Lin & Dufresne,

2013). Cyclodextrin based supramolecular hydrogels exhibit thermoreversible thixotropic

behavior, therefore they are used for long term controlled and sustained drug delivery

purposes in the form of injectable hydrogels. Cationic supramolecular hydrogels are

efficiently used for the in–vivo and in–vitro gene therapy. They have the ability to

condense nuclei acids and transfer it to the specific cancer cells or tissues. Recently a lot

of attempts have been made to accomplish gene delivery through supramolecular

hydrogels and the results obtained were equivalent or even higher as compare to the

covalently cross–linked hydrogels. Nano sized supramolecular hydrogels are considered

as efficient and safe gene transfecting agents because of easy surface functionalization,

controlled size and switchable morphological characteristics (Yallapu et al., 2011). A

photo sensitive supramolecular hydrogel was prepared through host guest interaction

mechanism by Kros between dextran bearing azobenzenes and β–cyclodextrin modified

dextran for protein release in response to light (Peng, Tomatsu, & Kros, 2010). Similarly

Tseng and colleagues prepared a nano supramolecular hydrogel system for the delivery of

15

the intact transcription factor possessing high transduction efficiency. Very recently a

novel supramolecular hydrogel system was designed by Meijer and Dankers for the

efficient delivery of the growth factor. This system was prepared from supramolecular

polymers linked through hydrogen bonding (Bastings et al., 2014; Dankers et al., 2012).

Cationic polymers 1.2

Polymeric systems bearing positive charges in their backbone or polymer skeleton

are called cationic polymers. Cationic moieties are incorporated on the backbone of such

polymers during synthesis process. Cationic polymers possess exclusive physicochemical

properties and capacity for further modification, therefore they are used for biological

purposes. In the current era, various cationic polymers and its derivatives are employed in

research and some has reached into clinical trials as well (Li et al., 2011; McLachlan et

al., 2007; Zhou et al., 2012). Both anionic and cationic polymers have significant

applications in their respective area and are investigated for relevant therapeutic

purposes. Anionic polymers form complexes with cationic molecules like basic peptides

and cationic drugs resulting in therapeutic effects. Cationic polymers form

polyelectrolyte complexes through electrostatic interactions with anionic biological

molecules like proteins and nucleic acids. Moreover, cationic polymers possess various

other important bioactive properties like anticancer, antioxidant, antimicrobial, anti–

inflammatory and stimuli responsiveness, further enhancing their therapeutic potential

(Govende et al., 1999; Kobayashi et al.,, 2003).

Cationic polymers got interest of the scientists because of their complexation

ability with nucleic acids, forming electrostatically interacted complexes with biological

molecules. These are used as excellent gene delivery vehicles due to their property to

condense nucleic acids, enhance the cellular uptake and give protection from enzyme

based degradation. However the use of cationic polymers is now extended to drug

delivery, drug conjugation, various therapeutic applications and tissue engineering. Some

examples of cationic polymers are poly(amidoamines), poly(ethyleneimine), poly–L–

(lysine), chitosan, poly[2–(N,N–dimethylamino) ethyl methacrylate] (PDMAEMA),

dextrans and cationic cyclodextrin. Cationic polymers possess protonable amino groups

varying from polymer to polymer. Cationic polymers exists in different architectures

including dendrimers, branched, linear and hyper branched form. Some polymers like

16

poly–L–(lysine) are in linear forms and polyethyleneimine exists in both branched and

linear structures. In some polymers like poly–L–(lysine) the cationic functionality is

attributed to the side chain groups attached while in others like polyethyleneimine the

positive charges are present in polymer backbone. Block and comb type graft copolymers

have also been developed having polycationic backbone. Example of the block

copolymer is polyethylene glycol–poly–L–lysine (PEG–PLL) copolymer while that of

comb type copolymer is PLL–g–dextran (Samal et al., 2012).

1.2.1 Chitin and chitosan

Chitin is one of the most abundant nitrogen containing compound and second

most existing polysaccharide after cellulose (Jolláes, 1999). Chitin is a linear polymer

having β(1,4)–linked N–acetyl glucosamine (NAG) units, stabilized by hydrogen

bonding, presenting α–helical three dimensional configuration (Kas, 1997; Singla &

Chawla, 2001). The main source of the chitin include; crustaceans cuticles, crab/shrimp

shells, cephalopods exoskeletons and food industry bye products (Domard et al., 2002).

Extraction of chitin from crustacean shells is performed through the process of

deproteinization and demineralization using sodium hydroxide concentrated solution at

high temperature. Chitin and chitosan are extensively investigated for various purposes.

They are mainly used for enzyme immobilization, tissue engineering, gene and drug

delivery.

Chitosan is amino polysaccharide copolymer composed of repeated units of N–

acetyl glucosamine and 1,4 D–glucosamine as shown in Figure 1.1 (Chung et al., 2003).

Chitosan is obtained from chitin through the process of enzymatic or alkaline

deacetylation (Araki & Ito, 1974) .Therefore chitosan is same like chitin with the

difference in degree of deacetylation (DD). Term chitosan is used if degree of

deacetylation is more than 40%. Chitin and chitosan are not found in mammals but they

can be hydrolyzed by lysozyme found in specific concentration in tears and serum

(Nordtveit et al., 1996). Biodegradation of chitosan depends on two factors; Distribution

pattern of N–acetyl glucosamine units and DD (Lee et al., 1995). In–vivo degradation rate

of chitosan is not same like in–vitro degradation and it is reported that after 50 hours of

lysozyme exposure 10% chitosan film was degraded and 80% film chitosan was

remaining in Wistar rates even after 12 weeks of implantation (Tomihata & Ikada, 1997).

17

The physicochemical properties and potential applications of chitosan depends on

molecular weight (MW) and degree of deacetylation (DD). In chitosan chain, the

percentage of deacetylation affects biological, physical and chemical properties like

immunoadjuvant activity, metal ions chelation and tensile strength. DD also affects

chitosan intrinsic pKa which in turn affects its solubility in dilute acid solutions. Chitosan

will be soluble in dilute acid solution if DD is ≥ 40%. DD also affects gene delivery

properties of chitosan as well. The effect of chitosan DD on gene transfection efficiency

was investigated by Kiang et al and found that with the decrease in DD, DNA binding

efficacy also decreases (Kiang et al., 2004).

Chitin

Chitosan

Fig 1.1 Chemical structure of chitin and chitosan

Effect of DD on cell adhesion has also been investigated on different cancer cell

lines (Prasitsilp et al., 2000) and it is reported that cells attachment properties of the

chitosan are significantly changed if there is only 10% difference in degree of acetylation.

Large number amino groups are free and can be protonated if percent DD is higher,

imparting cationic functionality. The increase in cationic nature of chitosan increases the

interaction with negatively charged moieties (cells) hence facilitating cell adhesion

(Prasitsilp et al., 2000). However some cell lines like fibroblasts greatly adhere to

chitosan films having high DD, affecting cell proliferation (Chellat et al., 2000). So DD

selection should be done carefully in order to achieve optimum results.

18

1.2.2 Chemical modifications in chitosan

Chitosan is the only biomass derived cationic polysaccharide found in nature

possessing exclusive biological and physicochemical properties making it worthy

regarding biomedical and pharmaceutical applications. However some of its properties

like solubility can be modified chemically or physically. The main objective of chemical

modification is the production of water soluble chitosan derivatives. Additionally

hydrophobicity, anionic and cationic properties can also be incorporated and controlled

with the attachment of various ligands and functional groups. Fortunately, chitosan can

be chemically modified because of the presence of amine, acetamido and hydroxyl

functional groups (Mourya & Inamdar, 2008). Chemical modifications do not change the

basic chitosan skeleton and preserve the original biochemical, physico–chemical

properties while adding up improved properties.

1.2.3 Carboxymethyl chitosan (CMCh)

Carboxymethyl chitosan (CMCh) is one of the water soluble chitosan

derivative which is widely used in several fields like food technology (Khanjari et al.,

2013), gene therapy (Dong et al., 2012), biosensors (Zhang et al., 2010), in–vitro

diagnostics (Hawary et al., 2011; Liu et al., 2011), bio–imaging (Xie et al., 2005) with the

greatest impact in tissue engineering and drug delivery. Chemically CMCh is a versatile

compatible material possessing both –COOH and –NH2 groups and is available in various

molecular weights. As compared to chitosan, its exclusive features like water solubility,

moisture retention capability, excellent biocompatibility and biodegradability (Chen et

al., 2002), non–toxicity, enhanced antibacterial (Fei Liu et al.,2001), antifungal (Seyfarth

et al., 2008) and antioxidant (Zhao et al., 2011) properties has attracted researchers to use

it for developing scaffolds and formulations for the purpose of tissue engineering and

drug delivery.

19

Fig 1.2 Structural unit of carboxymethyl chitosan

1.2.3.1 Applications of carboxymethyl chitosan

Carboxymethyl chitosan has been used by many researchers for fabricating

hydrogels and other drug delivery systems (Chen et al., 2007; Chen et al., 2004a; Chen et

al., 2004b; Y. Chen, Liu, Tan, & Jiang, 2009; Dolatabadi-Farahani et al.,2006; El-

Sherbiny & Smyth, 2010; Guo & Gao, 2007; Hiroki et al.,2009; Kumar Singh Yadav &

Shivakumar, 2012; X. Li et al., 2012; Ma et al., 2007; Mohamed et al.,, 2012; Poon et

al., 2007; C. Yang et al., 2010a; Yang et al., 2010b; Yin et al., 2007; Zhu et al., 2011) . In

coming paragraphs some of the reported studies are given in detail.

Vaghani et al., in 2012 prepared the pH responsive hydrogels of carboxymethyl

chitosan cross–linked through glutaryldehyde encapsulating ornidazole. Carboxymethyl

chitosan was prepared from chitosan. The entrapment of drug, its interaction with the

hydrogel network and carboxymethylation of chitosan were evaluated through NMR,

FTIR, XRD and DSC studies. The swelling behavior of the developed hydrogels was

evaluated at different pH values like 1.2, 6.8 and 7.4. Hydrogels exhibited very less

swelling at pH 1.2, quick swelling at 6.8 and linear at 7.4 with a minor increase. The drug

release pattern was dependent on swelling (Vaghani et al., 2012).

Li et al., in 2012 prepared the Insitu nano–composite injectable hydrogels of

oxidized alginate and carboxymethyl chitosan loaded with the nano–curcumin for the

purpose of wound healing. Nano curcumin was developed by using copolymer of

poly(ethylene glycol)–b–poly(–caprolactone). In-vitro release profile was evaluated for

the curcumin and invivo analysis were performed on dorsal wounds of rats. Histological

20

results showed that the nano curcumin loaded alginate/carboxymethyl chitosan hydrogel

markedly enhance the collagen deposition and epidermis re–epithelialization in wounded

tissue (Li, et al., 2012).

Mohamed et al., in 2012 fabricated the novel hydrogels consisting of

carboxymethyl chitosan and terephthaloyl thiourea in various concentrations. The

developed hydrogels were characterized through NMR, XRD, FTIR ,SEM, elemental

analysis and swellability studies in different solvents. Hydrogels were also evaluated for

its antimicrobial potential against bacteria (Escherichia coli, Staphylococcus aureus,

Bacillus subtilis) and fungi (Candida albicans, Geotrichum candidum, Aspergillus

fumigatus). Hydrogels showed enhanced antifungal and antibacterial potential as compare

to parent carboxymethyl chitosan ( Mohamed et al., 2012).

Luo et al in 2013 prepared hydrogel beads of carboxymethyl chitosan and alginate

through a novel method in aqueous–alcohol binary solvent system. For attaining the

structural integrity glutaryldehyde was used as crosslinker. Spherical hydrogel beads

were obtained at 30% alcohol and 3% calcium concentration. Vitamin D3 was loaded as

a model drug and 96.9% encapsulation efficiency was achieved. Effects of room

temperature and freeze drying on release characteristics and swelling were investigated in

conditions simulated with the GIT. (Luo et al., 2013).

Wei et al., 2015 studied the pH responsive hydrogels of carboxymethyl chitosan

and poly acrylic acid. The synthesized hydrogels were evaluated by FT–IR for hydrogel

confirmation and SEM for Surface morphologies. Swelling studies were conducted in

respective buffer solutions and it was concluded that swelling index of the prepared

hydrogels was dependent on buffer solution pH and compositional feed ratio. Swelling of

the hydrogels was increasing with the increase in carboxymethyl chitosan content (Wei et

al., 2015).

1.2.4 Chitosan hydrochloride (ChCl)

Likewise carboxymethyl chitosan, chitosan hydrochloride (ChCl) is also one of

the water soluble derivative of the chitosan. It exists in the form of white fine powder.

Chitosan hydrochloride is prepared from normal solution of the chitosan treated with 1N

21

HCl at pH 4.7. The resulting solution is then filtered, undissolved traces are removed and

evaporated.

1.2.4.1 Applications of Chitosan hydrochloride

Chitosan hydrochloride have also been studied in forming gels and fabrication

of various drug delivery systems (Dergunov et al., 2005; Colo et al., 2002; Gamzazade &

Nasibov, 2002; Harris et al., 2010; J. Liang et al., 2011; Maestrelli et al., 2004; Rai et al.,

2005; Rezaei Mokarram & Alonso, 2016; Ruel-Gariepy et al., 2000; Seyfarth et al.,

2008). Some of the reported studies are given here under in detail.

Chen et al., in 2012 conducted a study to check the effects of chitosan

hydrochloride and chitosan on enhancing the absorption of berberin from rat intestine.

The results revealed that chitosan has dose dependent enhancement in berberin

absorption. Formulations having 0.5%, 1.5% and 3% chitosan results in improved AUC0–

36 h at the rate of 1.9, 2.2 and 2.5 times respectively. The increase in the absorption rate of

chitosan–berberin formulation is due to improvement in intestinal paracellular pathway of

berberin due to chitosan. The impact of chitosan hydrochloride on berberin intestinal

absorption was not much more significant because of the less solubility of berberin

chloride in solution of chitosan hydrochloride. Therefore the optimum formulation

resulting in highest berberin absorption was the berberin solution having 3% chitosan

concentration (Chen et al., 2012).

Cai et al., in 2013 conducted a study to prepare, characterize and evaluate a new

chitosan derivative for its antibacterial potential. The prepared novel chitosan derivative

was ortho–biguanidinyl benzoyl chitosan hydrochloride and was characterized by NMR

and FTIR data analysis. Molecular weight of the prepared derivative was determined

through gel permeation chromatography technique while degree of substitution was

evaluated through elemental analysis technique. The antibacterial potential of the ortho–

biguanidinyl benzoyl chitosan hydrochloride was evaluated against Staphylococcus

aureus and Escherichia coli using agar plate method. The antimicrobial potential of the

prepared derivative was higher than chitosan hydrochloride and can be attributed to

guanidinylation substitution (Cai et al., 2013).

22

Zhang et al., in 2014 conducted an experimental attempt to prepare, characterize

and evaluate the antimicrobial potential of the zein nanoparticles loaded with thymol and

stabilized by double layer of sodium caseinate and chitosan hydrochloride. Nanoparticles

stabilized by sodium caseinate were having negatively charged surface and well defined

size. Sodium caseinate coated nanoparticles exhibited isoelectric shift to 5.05 from 6.18

and were redispersible after lyophilization at neutral pH in water. Sodium caseinate

coated zein nanoparticles were again coated with chitosan hydrochloride resulting in

reversal of negative zeta potential to positive, enhanced encapsulation efficiency and

increased particle size. The surface of the double coated nanoparticles was rough

presenting some clumps as well. As compare to un–encapsulated thymol, the

encapsulated thymol shown the long lasting antimicrobial activity against gram–positive

bacterium (Zhang et al., 2014).

Straccia et al., in 2015 prepared the alginate hydrogels which were coated with

chitosan hydrochloride for the purpose of imparting the antimicrobial potential and

hydrophilic molecules delayed release as well. Alginate hydrogels were prepared by

internal setting method with different calcium concentrations and coated with the

chitosan hydrochloride by immersing hydrogels in solution of chitosan hydrochloride.

The alterations in surface morphology due to chitosan hydrochloride coating were

evaluated through cryo–scanning electron microscopy. The Stability, swellability and

release characteristics in physiological solution were evaluated using rhodamine B as a

model hydrophilic drug and were compared with the uncoated alginate hydrogels. The

results revealed that chitosan coated alginate hydrogels can be used for wound dressing

possessing intrinsic antimicrobial activity and controlled drug release characteristics

(Straccia et al., 2015).

1.2.5 Gelatin

Gelatin is a natural polymer obtained from collagen hydrolysis which is one of the

abundant protein and structural mainstay of the kingdom Animalia (Bailey & Paul, 1998).

Gelatin is biodegradable and biocompatible at physiological environment having the

ability to load charged biomolecules and is used as plasma expander and drug

formulation ingredient. (Santoro et al., 2014; Young et al., 2005). Gelatin is one of the

23

important hydrocolloid and high molecular weight polypeptide possessing proven

popularity largely because of its thickening and gelling properties. It differs from

polysaccharides and possess all essential amino acids in its structure except tryptophan.

The amino acid composition of gelatin may vary from specie to specie especially from

hydroxyprolin and prolin point of view because of some environmental exposures like

temperature (Kasankala et al., 2007). Some of the main sources of gelatin are fish, pig

skin, hides cattle bones and insects.

During the hydrolysis of collagen the triple helix structure of collagen is broken

down to generate the gelatin coils. Inspite of the collagen basic structure destruction

during hydrolysis the chemical structure of the gelatin is maintained (Harris, 2012).

Basically there are two processes involved in the denaturation of collagen to yield gelatin

i.e. thermal treatment and breakage of covalent bonds due to hydrolysis. Collagen is

thermally treated at 40oC for the destruction of the electrostatic interactions and hydrogen

bonds. Hydrolysis of the collagen either takes place in acidic or basic conditions resulting

in the production of gelatin type A and B respectively. For the production of gelatin A the

collagen is treated with dilute acid and then extraction at pH 4 but at this pH the other

non–collagenous proteins present as impurities are less soluble (Dreesmann et al., 2007).

However in alkaline conditions these impurities are highly soluble so type B gelatin is

more pure then type A gelatin.

During the hydrolysis process the resulting gelatin molecular weight, number of

amino acids present and number of present polypeptide chains depends on the position of

the collagen bond broken down. There is no specific bond to be broken, instead every

bond is susceptible depending on the pH and temperature. Therefore the heterogeneity in

gelatin molecule is due to random hydrolysis of the collagen bonds. Moreover this

molecular heterogeneity in gelatin also depends on the source of raw material. As the sex,

age, tissue and specie variations are there leading to variable amino acid composition in

obtained collagen. After collagen hydrolysis the gelatin coils are present in solution form

at temperature of 40–50oC. At temperature below 35

oC there is conformational change in

gelatin chains called coil to helix transition.

24

Fig 1.3 Formation of gelatin from collagen

1.2.5.1 Applications of gelatin

Gelatin has widespread applications regarding drug delivery (Abruzzo et al.,

2012; Buhus et al., 2009; Camci-Unal et al., 2013; Dong et al., 2006; Frutos et al., 2010;

Jiang & Zhu, 2006; Kajjari et al., 2011; Liu et al., 2006; Mallick et al., 2012; Nagahama

et al., 2009; Raafat, 2010; Rathna, 2008; Sadeghi & Hosseinzadeh, 2011; Salamon et al.,

2014) . In coming paragraphs some of the detailed reported studies are given.

Anirudhan & Mohan et al., 2014 prepared a novel pH responsive hydrogel

composed of the gelatin and β–cyclodextrin (β–CD) cross–linked through oxidized

dextran using 5–flourouracil (5–FU) as a model drug. This hydrogel system was

developed for the colonic delivery of 5–FU. The developed hydrogel system was

characterized through FTIR for the confirmation of grafting, drug encapsulation and

cross–linking. Powder X–ray diffraction showed the amorphous nature of the hydrogel.

Developed hydrogels were evaluated for swelling characteristics. Results showed that

drug encapsulation efficiency get enhanced with the grafting of β–CD. Release models

and mathematical equations were applied for the determination of the 5–FU release

pattern revealing that it follows the Higuchi model. Release characteristics of the

hydrogel system were evaluated both at pH 1.2 and pH 7.4 giving minimum release at pH

1.2 and maximum at pH 7.4. Swelling and degradation were the primary factors involved

in release of the 5–FU. The results of –in vitro cytotoxicity studies showed that

developed hydrogel system caused the enhanced inhibition as compare to the drug alone

(Anirudhan & Mohan, 2014).

Bukhari et al., 2015 fabricated the pH responsive hydrogels of gelatin and acrylic

acid using ethylene glycol dimethylacrylate (EGDMA) as crosslinker. Pheniramine

maleate was loaded as model drug. The hydrogels were prepared in different monomer,

25

crosslinker and polymer feed ratios to evaluate their respective effects on swelling and

release characteristics of the developed hydrogel system. The swelling and in–vitro

release characteristics were evaluated at pH 1.2, 5.5 and 7.5. 0.05M phosphate buffer was

used as dissolution medium at different pH values. Hydrogels were characterized by

FTIR and SEM for the confirmation of the hydrogel system and analysis of surface

morphology (Bukhari et al., 2015).

Li et al., 2015 prepared the nanocomposite hydrogels of the gelatin,

polyacrylamide and laponite and evaluated the effect of gelatin on hemocompatible and

physicochemical properties of the developed nanocomposite hydrogel. The developed

nanocomposite hydrogel was evaluated for mechanical properties, thermal stability,

structure and compatibility parameters. Results showed that the developed hydrogels

exhibited good mechanical properties and thermal stability. Incorporation of gelatin

impart the pH responsive property inside hydrogel system and did not affect the

homogenecity and transparency of the hydrogel system. Incorporated gelatin enhanced

the clotting time, degree of hemolysis and resisted the adsorption of nonspecific protein

revealing that hemocompatibility of the hydrogels get enhanced considerably with

gelatin. Therefore such hydrogels can be potentially applied for various biomedical

applications (Li et al., 2015).

1.2.6 Polyethyleneimine

Polyethyleneimine (PEI) is one of the most important and widely used cationic

polymer bearing primary, secondary and tertiary amino groups in its skeleton. It exists in

both linear as well as branched form having different molecular weights. Linear PEI

(LPEI) exists in solid form while branched PEI (BPEI) exists in viscous liquid form at

room temperature. Linear polyethyleneimine is obtained as a result of 2–ethyl–2–

oxazoline ring opening polymerization followed by hydrolysis while branched

polyethyleneimine is obtained through aziridine acid catalyzed polymerization (Brissault

et al., 2003). In polyethyleneimine skeleton chemically reactive cationic nature imparting

amino groups are present which react with the negatively charged molecules like nucleic

acids and drugs making polyelectrolyte complexes (Ulasov et al., 2011).

The most important and prominent property of the PEI is the presence of cationic

charge density on its skeleton. In PEI skeleton almost every third atom is nitrogen

26

capable of protonation leading to the extraordinary cationic charge density. The cationic

charge density in biological environment is due to the protonation of amino groups

leading to a close relation between cationic charge density and biological environment

pH. PEI possess the effective buffer capacity and all this can be attributed to apparent

pKa value of PEI which changes from environment to environment bearing different pH

values.

PEI is one of the non–biodegradable polymer limiting its wide therapeutic

applications (Koo et al., 2010). This shortcoming of the PEI is fixed by the incorporation

of the short PEI chains in association with biodegradable linkers into longer chains

leading to the development of biodegradable derivatives of PEI possessing low toxicity

and high transfection efficiency ( Liang et al., 2009). Lee et al. in 2007 synthesized the

reducible derivatives of LPEI from bismercapto–ethyleneimine oligomers through the

oxidative poly condensation process. The molecular weight of the resulting poly

(ethylenimine sulfide) (LPEIS) was ranging between 10 to 20 kDa (Lee et al., 2007).

Several research groups have also synthesized the biodegradable derivatives of PEI by

the attachment of acid labile ester linkages to PEI skeleton (Kim et al., 2005).

Fig 1.4 Schematic of polyethyleneimine structural unit

1.2.6.1 Applications of polyethyleneimine

In literature, numerous studies have been reported regarding use of

polyethyleneimine for the purpose of delivery of therapeutic agents (Chen et al., 2014;

27

El-Din & El-Naggar, 2012; Fleischer & Schmuck, 2014; Giano et al., 2014; Han et al.,

2009; Kie Shim et al., 2008; J. Kim et al., 2011; Mallikarjuna Setty et al., 2005; Mimi et

al., 2012; Sutton et al., 2006; Vinogradov et al., 1999; Yang & Kim, 2011; Yang et al.,

2012; Zhang et al., 2008). Some of the detailed conducted studies are given below.

Paciello & Santonicola (2015) developed the highly swellable supramolecular

polycationic hydrogels of polyethyleneimine through its methacrylation process. The

generated macromolecules were able of self-assembling in water leading to the formation

of highly swollen hydrogels having tunable microstructure and hydrophilicity.

Methacrylation of polyethyleneimine was performed under appropriated conditions and

the resultant molecules were then evaluated by NMR and FTIR to understand the

correlation between the methacrylate moieties in modified polymer and reaction

conditions. The surface morphology and the dynamic structure of the developed hydrogel

network was evaluated by fluorescence microscopy and small X–ray scattering technique

showing the interconnected micro-cavities in the superstructure (Paciello & Santonicola,

2015).

Zhao et al., 2015 prepared the aerogel of cellulose nanofibril–grafted–

polyethyleneimine and evaluated its use as a novel drug delivery system by loading

sodium salicylate as a model drug. The structure and morphology of the cellulose

nanofibrils before and after chemical modification were evaluated through various

analytical techniques like X–ray diffraction, FTIR, SEM and X–ray photoelectron

spectroscopy (XPS). The developed aerogels showed excellent drug loading capability.

The drug release form the developed aerogels was in controlled manner and temperature,

pH dependent. The release of sodium salicylate was following pseudo–second order

release pattern. It was concluded that due to unique pH and temperature responsive

properties the aerogel can be used as a good alternative to the conventional drug delivery

system made from synthetic polymers (Zhao et al., 2015).

Tian et al., 2016 conducted a study regarding synthesis of the silica modified

nanogel of polyethyleneimine and chitosan for the gene carrying purpose. The nanogel

was developed due to supramolecular interactions between chitosan, polyethyleneimine

and modified silica resulting in stable and rigid gene carrier material. Comparison of the

physically developed nanogel was also done with the chemically developed nanogel

28

involving chemical cross–linking and it was concluded that physically developed nanogel

were easy to prepare and its components can be easily adjusted resulting in enhanced

gene carrying ability. The results showed that combination of the modified silica and

polyethyleneimine with chitosan hydrogel results in the factual increase of its stability,

strength and gene carrying ability (Tian et al., 2016).

Polyoxometalates 1.3

Polyoxometalates (POMs) are macro anionic metal oxide clusters of early d–

block metal ions in high oxidation states like molybdenum, tungsten and vanadium linked

by shared oxygen atoms with a large compositional and structural variety, resulting in

manifold associated properties. They are soluble in water and are produced as a result of

condensation reactions of metalate groups in acidic conditions. POMs have been studied

for potential applications in many diverse areas such as catalysis (Hill, 2007),

electrochemistry (Yang et al., 2013), material science (Long et al., 2007), agriculture,

nanotechnology and medicine (Izarova et al., 2012; Kortz et al., 2009; Pope & Müller,

1991).

1.3.1 History of POMs

History of POMs began since 1826 when Berzelius reported the very first yellow

colored hetero polyanion formed as a result of reaction between Mo As, or P (Berzelius,

1826). After that Marignac in 1862 discovered and characterized the hetropolytungstates.

Then, in the next 70 years hundreds of POMs were synthesized and systematically

investigated. In 1929 Pauling put a significant contribution in the field of POM chemistry

by proposing the structure of POMs having central XO4 unit enclosed by twelve corners

having MO6 (Pauling, 1929). Later on Keggin in 1933 elucidated the factual structure of

heteroPOM using Powder X–ray diffraction technique (Keggin, 1933). In 1948 Evans

interpreted and analyzed the structural arrangement of [TeMo6O24]6–

using single crystal

X–ray analysis presenting the self–assembled structure called Anderson–Evans structure

(Evans Jr, 1948). Next to the elucidation of Anderson–Evans structure, Dawson in 1953

reported the heteroPOM [P2W18O62]6–

called Wells–Dawson ion (Dawson, 1953).

29

1.3.2 Classification of POMs

POMs are classified into two main classes depending on the homogenecity or

heterogenecity of addenda atoms.

1.3.2.1 Iso polyanions (IPAs)

Iso polyanions are composed of only addenda (metal) atoms including Mo, W, V

and oxygen ligands. The well–known example of IPAs is the Lindqvist ion also called

hexametallate [M6O19]2–

, where M is either Mo or W. Some of the other examples of iso

polyanions are [W6O19]2–

(Fuchs et al.,1978), [Mo6O19]2–

(Nagano & Sasaki, 1979) and

[V6O19]8–

(Chae et al., 1989). Iso polyanions are highly basic, hence more unstable than

hetero polyanions (HPAs). IPAs are highly charged bearing moieties due to which they

are able to form huge structures (Pope & Müller, 1991).

Fig 1.5 Structure of Lindqvist ion [M6O19]n–

1.3.2.2 Hetero polyanions (HPAs)

HetroPOMs possess additional hetero elements mainly from d–block of the

periodic table. The most common examples of the HPAs are the Wells–Dawson and

Keggin anions. Wells–Dawson anions are represented by general formula [X2M18O62]n–

while Keggin ions are represented as [XM12O40]n–

(where M=Mo or W and X= Si or P).

HPAs are stable as compare to IPAs and are extensively used in the field of catalysis.

Keggin ions are highly symmetrical having central XO4 tetrahedral hetero unit enclosed

by 12 MO6 octahedra while Wells–Dawson ions possess 18 metal atoms and two XO4

hetero units (Dawson, 1953).

30

Fig 1.6 Keggin ion [XM12O40] n–

and Wells–Dawson ion [X2W18O62]n–

1.3.3 General properties of POMs

Polyoxometalates chemistry is one of the fast growing field of inorganic

chemistry because of the structural versatility of POMs regarding size, structure, charge

distribution, photochemical properties and redox chemistry. Generally POMs are stable in

air and water with admirable stability over entire pH range (1–14). The stability of POMs

also depends on type of POM involved. The acids and salts of POMs are highly hydrated

having up to 50 molecules of water per anion. POMs are soluble in both polar and non–

polar solvents supplied with appropriate counterions. POMs have excellent thermal

stability and the decomposition process starts at 400oC. POMs possess large sizes ranging

from 6–25Å and high ionic masses (1000 –10,000 g/mol) (Pope, 1983).

According to the Pope classification, Type I POMs are those which have single

terminal oxygen and can reversibly exchange electrons without ensuring any structural

change in respective POM. Reduction of type II POMs is a difficult and irreversible

phenomenon (Casan-Pastor et al., 1991). The reduction of hetropolymolybdates is easier

than polyoxotungstates. Reduction of POMs depends on POM charge and pH of the

medium. In order to achieve electro neutrality, the protonation effect of the respective

solvents balance the negative charge of the POMs (Maestre et al., 2001).

The reduction of the Keggin ions derived from type I octahedra produces the

intensively colored mixed valence species called ―heteropolyblues‖ (Launay et al., 1980).

HeteroPOMs having extra proton (H+) behaves like acids called heteropolyacid (HPA).

Heteropolyacids are stronger than classic inorganic acids like HBr, HNO3, HCl and even

HClO4, hence can be used for acid based catalysis (Pope & Müller, 1991). The strong

acidic behavior of HPA is due to its huge structure and high negative charge. The

strength of the HPA depends on the type of hetero and addenda atoms present in the

31

POM structure. For example, polyoxotungstates are highly acidic than

polyoxomolybdates. H3[PW12O40] represents the highly acidic Keggin type structure

(Kozhevnikov, 1998).

1.3.4 Synthesis mechanism of POMs

The synthesis of POMS is a simple and easy process involving lesser number of

steps or sometimes just one step called one–pot syntheses. Both hetero and isoPOMs are

synthesized and then isolated from aqueous solution in which the reaction is carried out.

Acidified precursor metal salts and respective counterions are mixed in solution form

resulting in the formation of polyhedral units (MOx). These polyhedral units are the

building blocks, basically formed as a result of edge and corner sharing. These small

building blocks then self–assemble to form various POMs. The self–assembling of

building blocks is dependent on the appropriate reaction conditions like concentration of

reaction starting material, pH, counterions, temperature, reaction time and ionic strength

etc.

After synthesis another important step is the preparation of respective POM crystals so

that its structure can be determined. The produced POMs are crystallized in the presence

of suitable countercation like organic cation and filtered (Johnson et al., 2002). The

synthesis of POMs can be represented by following reactions schemes.

12[WO4]2–

+ [HPO4]2–

+ 23H [PW12O40]3–

+ 12H2O

7[MoO4]2–

+ 8H [Mo7O24]6–

+ 4H2O

For the determination of POMs structure, X–ray crystallography and NMR spectroscopy

is used.

1.3.5 Structural isomerism

Isomers are the two or more compounds having same chemical formula with

different arrangement of atoms and different properties. Isomerization is the process

concerned with the changing of one molecule into another form having same number of

atoms but arranged in different fashion. The resultant molecules are termed as isomers.

POMs also exhibit isomerism and the typical examples are provided by both Wells–

Dawson and kegging ions. There are five proposed structural isomers of Keggin ion

32

denoted as α, β, γ, δ and ε isomers. Among all these, α– and β–Keggin isomers are more

stable while others are less stable.

1.3.6 Applications of POMs

The applications and significance of POMs is largely dependent on their various

chemical and physical properties including pH, thermal stability, mass, size, redox

potential, electron transfer ability and proton storage ability (Ammam, 2013). POMs

have advantage that their shape, size, composition, charge, solubility, redox property etc.

can be tuned with respect to the desired reactivity with the targeted molecules. Some of

the fascinating applications of the POMs are discussed below.

1.3.6.1 Catalysis

POMs and Hetropoly compounds have good role in catalytic synthesis. HPAs

possess strong Bronsted acidic properties. POMs are good oxidants with rigid electron

reservoir resulting in multielectron oxidation and reduction reversible transformation in

mild conditions. Many POMs are thermally stable. These are anionic in nature and can

exchange cations in metathesis catalytic reactions resulting in the formation of organic

and inorganic salts. Catalysis can occur in hetro or homogeneous phase. HPAs can be

used as HPAred or HPAox presenting oxidized and reduced forms.

[HPA]ox + substrate + nH+ Hn[HPA]red + oxidized substrate

Hn[HPA]red + n/4 O2 [HPA]ox + n/2H2O

1.3.6.2 Biological applications of POMs

1.3.6.2.1 Antibacterial potential

Various studies are conducted to explore the biological potential of the various

types of POMs. Regarding antibacterial potential of POMs it has been demonstrated that

they show cidal effect on various biologically important bacterial strains like methicillin

resistant staphylococcus aureus (MRSA). In a study designed by Yamase et al., 1996 the

synergistic antibacterial effect of POMs K7[PTi2W10O40].6H2O and K7[BVW11O40].7H2O

was observed when used in combination with oxacillin. The cidal effect was exerted

33

through the inhibition of the penicillin binding protein 2 enzyme resulting in cell wall

destruction (Inoue et al., 2006).

The conjugation of the POMs (H3PMo12O40 and H3PW12O40) with metallic

nanoparticles resulted in significant antibacterial activity against both gram negative and

gram positive bacteria (Daima et al., 2014). Similarly the formation of H5PV2Mo10O40

nanoparticles significantly enhanced the antibacterial potential against resistant bacterial

strains like staphylococcus aureus (S.aureus), pseudomonas aeruginosa (P. aeruginosa)

and Escherichia coli (E.coli). The underlying mechanism regarding antibacterial effect of

POMs like K18[KSb9W12O86] and K27[KAs4W40O140] can be attributed to the presence of

high negative charge causing the bacterial transformation from bacillus to cocci making

them susceptible to POM action (Inoue et al., 2005). POMs have also been used in

wound dressings for antibacterial purposes when immobilized on suitable biocompatible

material like chitosan (Chen et al., 2006). In comparison with standard antibiotics like

penicillin, Silicon containing POMs like and K5[SiW11VO40] have shown higher

antibacterial action against fruit spoiling microorganisms like yeast, aspergillus niger,

bacillus subtilis, and E.coli (Chen et al., 2011a).

1.3.6.2.2 Anticancer potential

POMs possess promising anticancer potential as revealed by various studies

conducted to evaluate its anticancer potential. The main mechanism involved in

anticancer effect is the production of reactive oxygen species (ROS) like superoxides

causing DNA fragmentation of the cancer cells and subsequent cell death. Mukherjee

(1965), administered intramuscularly an exceptional combination of molybdenum and

tungsten bearing POMs with caffeine in the form of sodium bicarbonate aqueous solution

for mitigation of adenocarcinoma. The combination used was having phosphotungstic

acid H3[PW12O40] and phosphomolybdic acid (H3[PMo12O40]) administered for 6 days.

The results demonstrated the complete eradication of cancer after 2–4 weeks with no

remaining‘s (Mukherjee, 1965). Yanagie et al., (2006) evaluated in vitro cytotoxic

potential of the [NH3Pri]6[Mo7O24].3H2O abbreviated as PM–8 exhibiting a significant

anticancer potential greater than 5–flourouracil against Meth–A sarcoma in mice

(Yanagie et al., 2006). The cytotoxic mechanism of POMs is ascribed to relapse of redox

34

phases and inhibition of ATP production in mitochondria resulting in prevention of

electron shift from nicotinamide adenine dinucleotide to coenzyme Q (Yamase, 1993).

PM–8 have also shown significant anti–cancer potential against gastric carcinoma in

nude mice given intraperitoneally for 14 days and a 50% reduction was observed in

gastric carcinoma in 10 weeks (Ogata et al., 2005). Similarly other POM candidates like

[Me3NH]6[H2Mo(V)12O28(OH)12(Mo(VI)O3)4].2H2O showed promising anticancer

potential against two different cancer cell lines including gastric cancer cell line

(MKN45) and pancreatic carcinoma cell line (AsPC–1) (Ogata et al., 2008). A POM

molecule N16[(O3POPP3)4W12O36].38H2O have shown higher antipoliferative activity

than standard vincristine against squamous cell carcinoma cell line (H157) (Raza et al.,

2012).

The anticancer activity of POM

([(CH3)4N]2Na65(NH4)2[SnII

1.5(WO2(OH))0.5(WO2)2(SbW9O33)2].32H2O) showing higher

anticancer potential than cisplatin against KCN cancer cell line using MTT assay (Gerthet

al., 2005). Similarly in another study bismuth and tungsten bearing POMs were tested

against gastric cancer cell line (SGC–7901). For apoptosis observation ethidium bromide

or acridine orange dye was used (Wang et al., 2013).

Due to such tremendous applications there was a need to develop proper dosage form for

the controlled delivery of POM, stepping forward towards its use in treating pathological

conditions.

Problem statement 1.4

POMs have shown marked activity against various cancerous cell lines, bacterial

and viral strains. The excessive availability of POM concentration at cellular level causes

various toxic effects like production of reactive oxygen species. The targeted and

controlled delivery of POMs using various biodegradable and biocompatible polymers

might result in reduced POMs toxicity by controlling the release pattern and subsequent

higher cellular uptake. The blocking and sealing of POMs inside cationic polymer based

hydrogels may also result in reducing toxicity by governing its release patterns from

hydrogel network.

35

Aims and objectives 1.5

To develop the pH–responsive supramolecular hydrogels for the controlled

release of encapsulated POMs.

To determine the dynamic swelling index, in–vitro release profile and

characterization of the developed hydrogels through different characterization

tools like FTIR, XRD, SEM and thermal analysis (TGA/DSC).

Preliminary safety evaluation of the POM and hydrogel formulation in rabbit‘s

selected for in–vivo analysis.

In–vitro cytotoxicity evaluation of the developed hydrogels against two different

cancer cell lines and a normal cell line.

In–vivo analysis of the enclosed POM in rabbits through an optimized HPLC

method.

36

Chapter 2

2 Materials and Methods

Instruments 2.1

Analytical balance Schimadzu, ATY244

Autoclave WiseClaveTM

Korea

Centrifuge C2 Series United Kingdom

CO2 Incubator SANYO, MCO-19AIC Japan

Filter Whatman™ GF 51, Germany

Glass test tubes Pyrex, Germany

FTIR spectrophotometer Tensor II, Bruker USA

Thermal analyzer SDT Q600, USA

SEM Hitachi S–2460N

Incubator Contherm Scientific Ltd. Europe

Laminar safety cabinets NUNC® BIOFLOW

Magnetic stirrer VELP Italy

37

Microplate reader Bio–Tek ELx 800TM

, USA

Microplates Corning USA

Oven Memmert E07086

pH meter WTW, Germany

Pipettes Eppendorf Research, Germany

Software‘s Chemdraw, Kinetica 5.1,

Sonicator WiseClean Germany, Daihan WUC-AU01H

Sterile filters 0.2 micron Germany

Vortex mixer WiseMix® VM–10 Korea

HPLC SPD 10A, Japan

HPLC Column C18, bondapak

HPLC Detection system LC10

Hematology analyzer SYSMEX 100, Germany

Serum chemistry analyzers Roche cobas C111, Roche cobas E 411, Germany

Dissolution apparatus Galvano Pak

UV spectrophotometer IRMECO U2020, Germany

Water Bath Memmert, WNB 45, Germany

X-ray Diffractometer Bruker D8 Discover, Germany

Materials 2.2

O–Phosphoric acid (H3PO4) 7664–38–2 Sigma

Hydrochloric acid (HCl) 7647–01–0 Sigma

Ethanol 64–17–5 Sigma

Gelatin 9000–70–8 DAEJUNG

Polyethyleneimine 9002–98–6 Sigma

Carboxymethyl chitosan 83512–85–0 Santa Cruz Biotechnology

Chitosan hydrochloride 70694–72–3 Sigma

Acrylic acid 79–10–7 DAEJUNG

Methacrylic acid 79–41–4 DAEJUNG

Benzoyl peroxide 94–36–0 DAEJUNG

Sodium hydrogen sulfite 7631–905–5 DAEJUNG

Ammonium persulphate 7727–54–0 DAEJUNG

38

Sodium phosphate monobasic 7558–80–7 Sigma

Roswell park memorial institute 2240–089 Invitrogen

(RPMI) medium

Human cervical adenocarcinoma ATCC USA

cell line (HeLa)

Human breast cancer cell line ATCC USA

(MCF–7)

African green monkey kidney RIKEN Japan

cell line (Vero)

L–Glutamine (200 mM) Thermo Scientific

Fetal bovine serum Thermo Scientific

Penicillin–Streptomycin Thermo Scientific

(10,000 U/mL, 10 mg/mL)

Trypsin/EDTA (TE) solution Thermo Scientific

Glacial acetic acid 64–19–7 Sigma

Trichloroacetic acid (TCA) 76–03–9 Sigma

Trizma® base (Tris base) 77–86–1 Sigma

Sodium hydroxide pellets 1310–73–2 Sigma

Sulphorhodamine B dye S2902 Sigma

0.4 % solution in 1 % acetic acid

Trypan blue solution (0.4 %) 72–57–1 Sigma

10% neutral buffered formalin HT501128 Sigma

Tetra–n–butyl ammonium phosphate 5574–97–0 Alfa Aesar

Acetonitrile 75–05–8 Sigma

The POM, Na12[α–P2W15O56]·24H2O having IUPAC name 15–tungsto–2–phosphate with

code P2W15 was synthesized in Prof. Dr. Ulrich Kortz Laboratory, Department of Life

Sciences and Chemistry, Jacobs University Bremen–Germany.

39

Buffers and working solutions 2.3

2.3.1 Phosphate buffered saline

Phosphate buffered saline (PBS) of pH 7.4 was prepared by dissolving 8.0 g of

NaCl (137 mM), 0.3 g of KCl (2.7 mM), 0.2 g of KH2PO4 (1.47 mM) and 0.609 g of

Na2HPO4 (4.3 mM) in one liter of purified water.

2.3.2 Swelling and dissolution buffers

2.3.2.1 0.2 M phosphate buffer

A 50 mL of 0.2 M potassium di–hydrogen phosphate was placed in 200 mL

volumetric flask and 39.1 mL of 0.2 M NaOH was added. Both were mixed, water added

to make up volume and mixed.

For the preparation of 0.2 M Potassium dihydrogen phosphate, 27.21 g potassium

dihydrogen phosphate was dissolved in distilled water and diluted with distilled water to

make up 1000 mL of 0.2 M KH2PO4.

For 0.2 M Sodium Hydroxide, 8.0 g of sodium hydroxide weighed and dissolved

in distilled water to make 1000 mL of 0.2 M NaOH.

After preparation of phosphate buffer, pH was checked on pH meter and was 7.4 + 5%.

2.3.2.2 0.1 M HCl buffer

For 0.1 M HCl preparation, a 9.86 mL of 37% HCl solution was mixed in some

quantity of distilled water and volume made up to 1000 mL with distilled water. After

proper mixing, pH was checked on pH meter. pH was 1.2 + 5%.

2.3.3 0.001 M tetra–n–butyl ammonium phosphate buffer

For the preparation of 0.001 M tetra–n–butyl ammonium phosphate buffer 339

mg tetra–n–butyl ammonium phosphate was dissolved in 1000 mL of HPLC grade

deionized water.

Synthesis of pH responsive cationic polymer–POM complexes 2.4

Cationic polymer–POM complexes (hydrogels) were prepared using self–

assembling phenomenon through electrostatic interactions in polymer, monomer and

40

polyoxometalate in various ratios as shown in Tables 2.1, 2.2, 2.3 & 2.4. Specific amount

of polymer/s were dissolved in weighed amount of water. For gelatin water was pre–

warmed at 50oC. Nitrogen gas was purged through polymer solution in order to remove

the dissolved oxygen. Weighed amount of initiators, sodium hydrogen sulfite and

ammonium persulfate were dissolved in specific amount of water. For benzoyl peroxide

ethanol was used as solvent. Monomer (acrylic acid, methacrylic acid) and initiators

solutions were added gradually to the polymer solution while continuous stirring. At the

end respective amount of POM sodium salt (20, 25, 30, 35 mg) was added with constant

stirring to small amount of the final solution sufficient to make hydrogel disc and placed

in water bath. Temperature schedule was 55oC for 4 hours and 60

oC for 4 hours. After

successful formulation development it was air dried and placed in oven at 35oC.

Table 2.1 Composition of the various AA–gelatin–POM hydrogel formulations. Formulation

code

Gelatin/100g AA/100g SHS/APS/100g POM salt (mg)

per hydrogel

disc

GP1 1 10 0.3/0.3 20

GP2 2 10 0.3/0.3 20

GP3 3 10 0.3/0.3 20

GP4 1 08 0.3/0.3 20

GP5 1 12 0.3/0.3 20

GP6 1 16 0.3/0.3 20

GP7 1 10 0.3/0.3 25

GP8 1 10 0.3/0.3 30

GP9 1 10 0.3/0.3 35

*AA for acrylic acid, * SHS/APS for sodium hydrogen sulfite/ ammonium persulfate, * POM for

polyoxometalate.

41

Table 2.2 Composition of the various AA–PEI–POM hydrogel formulations. Formulation

Code

PEI/100 g AA/100 g BPO/100 g POM salt

(mg) per

hydrogel disc

PEP1 0.4 20 0.4 20

PEP2 0.6 20 0.4 20

PEP3 0.8 20 0.4 20

PEP4 0.6 18 0.4 20

PEP5 0.6 20 0.4 20

PEP6 0.6 22 0.4 20

PEP7 0.6 20 0.4 25

PEP8 0.6 20 0.4 30

PEP9 0.6 20 0.4 35

* PEI for polyethyleneimine, * AA for acrylic acid, * BPO for benzoyl peroxide, * POM for

polyoxometalate.

Table 2.3 Composition ratios of the various MAA–CMCh–POM hydrogel formulations. S. No Formulation

Code

CMCh

g/100g

MAA

g/100g

BPO g/100g POM salt (mg)

per hydrogel

disc

1 CMCP1 0.4 18 0.4 20

2 CMCP2 0.6 18 0.4 20

3 CMCP3 0.8 18 0.4 20

4 CMCP4 0.6 16 0.4 20

5 CMCP5 0.6 20 0.4 20

6 CMCP6 0.6 24 0.4 20

7 CMCP7 0.6 18 0.4 25

8 CMCP8 0.6 18 0.4 30

9 CMCP9 0.6 18 0.4 35

* CMCh for carboxymethyl chitosan, * MAA for methacrylic acid, * BPO for benzoyl peroxide,* POM for

polyoxometalate.

42

Table 2.4 Composition ratios of various MAA–ChCl–POM hydrogel formulations. S. No Formulation

Code

ChCl

g/100g

MAA

g/100g

BPO g/100g POM salt (mg)

per hydrogel

disc

1 CHCP1 0.5 15 0.4 20

2 CHCP2 0.7 15 0.4 20

3 CHCP3 0.9 15 0.4 20

4 CHCP4 0.7 13 0.4 20

5 CHCP5 0.7 16 0.4 20

6 CHCP6 0.7 19 0.4 20

7 CHCP7 0.7 15 0.4 25

8 CHCP8 0.7 15 0.4 30

9 CHCP9 0.7 15 0.4 35

* ChCl for chitosan hydrochloride, * MAA for methacrylic acid, * BPO for benzoyl peroxide,* POM for

polyoxometalate.

Swelling studies of POM loaded hydrogels 2.5

Swelling studies of the developed hydrogels were conducted to evaluate their pH

sensitive behavior. For the provision of the gastric simulated pH conditions swelling

studies were performed in pH 1.2 (0.1 M HCl buffer) and 7.4 (0.2 M Phosphate buffer)

buffer solutions. Hydrogel discs (dried) were accurately weighed and placed at room

temperature in respective 100 mL buffer solution of each pH 1.2 and 7.4. Swollen

hydrogel discs after regular time intervals were removed from respective buffer solutions

and weighed using analytical balance (SHIMADZU, model ATY224) after blotting the

excess water present on the surface of hydrogel discs. Discs were again placed in

respective buffer solutions after weighing and the process was carried out until the

constant weight was achieved by hydrogel discs. Following formula was used to calculate

the swelling index (Q).

( )

⁄

Where ―Wh‖ is the weight of disc in swollen state at specific time while ―Wd‖ represents

the dry weight of hydrogel disc.

43

In–vitro POM release 2.6

To confirm the drug release behavior and controlled delivery characteristics from

the developed hydrogels, in vitro drug release studies were performed both at pH (1.2) as

well as pH (7.4) conditions in 900 mL dissolution medium at 37 ±0.5oC using USP

dissolution apparatus–II (Galvano Pak/ GDT_ 7TV3) After specific time interval, a 5 mL

dissolution medium sample was collected and analyzed by UV–Spectrophotometer

(IRMECO U2020, Germany) at 210 nm detection wavelengths following an already

reported method (Lis & But, 2000).

POM release kinetics from hydrogel system 2.7

In order to investigate POM release mechanism from supramolecular hydrogels

different mathematical models (zero order, first order, Higuchi and Peppas models) were

applied. The equations for these models are given below.

Zero order Kinetics Equation …………….. (1)

Where Qo denotes the amount of released POM at time ―to‖, Qt is the amount of released

POM at specific time ―t‖ while Ko represents zero order rate constant.

First order Kinetics Equation ………. (2)

Mt represents the remaining POM amount at time ―t‖, Mo denotes initial POM amount, k1

is the first order rate constant.

Higuchi equation ………………. (3)

M represents cumulative amount of released POM at time‗t‘, t represents time in hours

while kH is the Higuchi constant.

Peppas model (

) ……. (4)

(Mt /M∞) shows the fraction of POM released at time‗t‘ and n shows slope defining the

type of polymer matrix diffusion process.

44

Fourier transform infrared spectroscopy (FTIR) 2.8

To confirm the formation of the pH responsive self–assembled hydrogels, Fourier

transform infrared spectroscopy was used. FTIR analysis was performed for all

components like polymers, monomers, POM and developed hydrogel formulations. All

solid samples were properly crushed before analysis. For FTIR analysis TENSOR II,

Bruker, USA, instrument was used obtaining spectra in the range of 4000–500 cm–1

.

Thermogravimetric analysis (TGA) 2.9

Thermogravimetric analysis for POM, polymers and developed hydrogel

formulations were performed using TA instrument SDT Q600 series thermal analysis

system. Powdered samples of 0.5 to 5 mg were used for analysis in the sample pan at a

temperature ranging from 0 to 800oC. Analysis was performed under nitrogen gas

purging at a flow rate of 100 mL/min with the adjusted heating rate at 20oC/min.

Measurements for all samples were recorded in triplicate.

Differential Scanning calorimetry (DSC) 2.10

In order to determine samples glass transition temperature TA instrument SDT

Q600 series thermal analysis system was used. Calibration was performed with indium

99% at 156.6oC followed by confirmation with zinc standard at 419.5

oC. In standard

aluminum pan samples were sealed keeping the temperature range from 0 to 800oC and

purging the nitrogen at 100 mL/min, maintaining the heating rate at 20oC/min.

Scanning electron microscopy (SEM) 2.11

For surface morphological characteristics of the hydrogels Scanning Electron

Microscope (Hitachi S–2460N) was used. The accelerating voltage was 3KV. With

double adhesive tape powdered hydrogel and intact hydrogel disc samples were fixed on

aluminum stub and coated with gold under argon atmosphere with gold sputter. For

analysis of surface morphology random photomicrographs were taken at different

magnification levels.

45

Powder X–ray diffraction 2.12

X–ray diffraction (XRD) patterns were recorded for POM, Polymers and prepared

hydrogels, in reflection with a Bruker D8 Discover (Germany) instrument at 25oC

temperature, using the nickel filtered CuKa radiation (k = 1.54050oA) and operating at

voltage (40 kV), and current (35 mA). Eva software was used for the data processing

(Evaluation Package Bruker, Germany). The range of diffraction angle was 10o–80

o at

2h.

Pharmacokinetic study of POM in rabbits 2.13

2.13.1 High performance liquid chromatography (HPLC) analysis

A simple, accurate, reproducible and sensitive HPLC method was developed and

validated for polyanion analyses in rabbit‘s plasma. In-vivo profiling of the selected

hydrogel system was studied using an optimized HPLC method. The separation was

carried out using HPLC system (Schimadzu SPD 10A, Japan) supplied with C18 bonda

pak column, LC 10 detection system and the quaternary Pump with the detection

wavelength at 210 nm at a flow rate of 1.0 mL/min. The mobile phase was composed of

90:10 V/V tetra-n- butylammonium phosphate buffer (0.001M) and acetonitrile. O-

phosphoric acid solution was used to adjust the pH of the mobile phase at 7.0. Polyanion

concentration in µg/mL was plotted against time (hours) as a result of HPLC analyses.

2.13.2 Validation of HPLC method

2.13.2.1 Sensitivity and specificity

Sensitivity in terms of limit of detection (LOD) and specificity in terms of limit

of quantification (LOQ) of the HPLC method were calculated through signal to noise

ratio method by injecting the lowest amount of rabbit plasma spiked with analyte. Total

46

of 06 signal to noise ratio readings were taken from the integration system of HPLC

software. Following equations were used to calculate the LOD and LOQ.

Limit of detection (LOD)

× amount found ……….……… (5)

Limit of quantification (LOQ)

× amount found…………..... (6)

2.13.2.2 Linearity

Polyanion stock solution of 10mg/ml or 10µg/µl was prepared from which

25µg/ml, 50µg/ml, 100µg/ml, 250µg/ml, 500µg/ml, 1000µg/ml and 2000µg/ml plasma

spiked dilutions were prepared. The linearity of the HPLC method was evaluated by

constructing 5 seven pointed calibration curves from the above dilutions and linearity of

each was calculated through correlation coefficient (R2) using Microsoft excel 2007

(Barrett et al., 2007).

2.13.2.3 Accuracy and precision

To check the accuracy, precision and intraday, interday assay variations in the

developed HPLC method, three plasma dilutions 25µg/ml, 250 µg/ml and 1000 µg/ml

were selected. Accuracy and precision were determined by intermediate precision and

repeatability. Dilutions were prepared using stock solution (10mg/ml) of polyanion in

water. For interday precision (intermediate precision), five samples of three different

concentration were analyzed for three days while for intraday precision (repeatability)

five replicates of three different concentrations were analyzed in a single day. Precision

was calculated as percentage of relative standard deviation (% RSD) and accuracy was

determined as percentage of relative error (% RE) using following equations (Tan et al.,

2013).

( )

………….. (7)

( )

…………. (8)

Where SD is the standard deviation, Co is the concentration of polyanion spiked or added

and C1 is the concentration of polyanion found.

47

2.13.3 Animal models and drug administration

To avoid the effect of physiological changes, healthy rabbits (n=12) having

average weight of 2.2 Kg were obtained from Veterinary Hospital and Research Centre,

Abbottabad Pakistan. The study protocols were reviewed and approved by research ethics

committee, department of pharmacy COMSATS Abbottabad, Pakistan (Voucher/

notification number, PHM.ETHICS/FA13-08-17-01.N.24). Rabbits were randomly

divided into two groups, control and hydrogel group each having 6 rabbits and

quarantined in separate wooden boxes one month before the start of the experiment for

the purpose of acclimatization. Rabbits of each group were properly tagged for easy

handling in dosing and sampling process. Prior to the dose administration, the rabbits

were fasted for 12 h but were given free access to water. Parallel study design was

selected for this study. To the hydrogel group, hydrogel discs (4mm) were administered

having 20mg POM flushed by 5ml distilled water. Each rabbit of control group was also

given 20mg/2ml of the aqueous solution of POM. After regular time intervals i.e. at 0,

0.5, 1, 2, 3, 4, 6, 10, 14, 18, 22, 26, 30 and 48 hours blood samples (1 mL) were collected

from marginal ear vein of the and stored at -20oC in heparinized tubes. The obtained

samples were centrifuged at 5000 rpm for 25 min. the thawed plasma of rabbit was used

to perform the plasma spiking. Then rabbit plasma was vortexed for 5 min having POM.

For the precipitation of the protein, 1mL of the acetonitrile was added into spiked plasma

and vortexed for 5min.

2.13.4 Polyanion plasma concentration quantification and pharmacokinetic

profiling

Plasma concentrations of polyanion in obtained rabbit plasma samples were

determined after the administration of oral polyanion solution in water (20mg/2mL) and

hydrogel disc having 20mg of preloaded POM via constructed calibration curves.

Polyanion concentration from both receiving groups was calculated by using Microsoft

48

excel 2013. For the non-compartmental model based pharmacokinetic parameters like

time for maximum plasma concentration (Tmax), maximum plasma concentration

(Cmax), clearance (Cl), elimination half-life (Lz), volume of distribution (Vz) and area

under the curve (AUC0-t) etc. was performed through scientific application package

Kinetica® 5.1 version (Thermo Electron Corporation).

Statistical analysis 2.14

A software program (MedCalc®

Belgium) was used for statistical analysis of data.

Unpaired sample T-test was used for comparison of the results and determination of the

significant/ non-significant interpretation at 95% confidence interval, p-value less than

0.05 was considered as significant difference in results.

Acute toxicity evaluation and safety evaluation of the MAA–ChCl–2.15

POM hydrogel and POM oral solution

The hydrogel formulation selected for in–vivo study was evaluated for acute oral

toxicity studies in rabbits. As no median lethal dose (LD50) or lethal dose could be

determined, so maximal tolerance dose (MTD) method was adopted for oral safety

evaluation of the hydrogel (Tan et al., 2013; Wang et al., 2012b). Initially four rabbits (02

male, 02 female) were taken for determining the MTD of hydrogel dispersion and then

total of 18 rabbits (09 male, 09 female) having average body weight of 2.2 kg were

purchased and properly housed in separate cages. The rabbits were divided into 03

groups, hydrogel group (n = 06, 03 male, 03 female), POM group (n = 06, 03 male, 03

female) and control group (n = 06, 03 male, 03 female). All rabbits were kept on fasting

15 hour before dosing except free excess to water. Hydrogel group was given hydrogel

dispersion twice at an interval of 4 hours at maximal tolerable dose of 4000 per kg body

weight. The control group was given normal saline. To the POM group, 20 mg/2mL

solution was administered to each rabbit. All animals were continuously observed twice

daily for seven days. The daily observations include general condition, mortality, activity,

abnormal behavior, feces etc. On day 7th

animals were sacrificed and organs like heart,

spleen, liver, kidney and lung were properly preserved in 10% buffered formalin and

were subjected to histopthological examination at Ayub medical and teaching hospital,

49

Abbottabad, Pakistan. The blood of each animal was divided into two parts. One part was

used for serum chemistry and other for routine blood analysis following the published

procedure (Wang et al., 2012).

2.15.1 Histopathology studies

For histopathology studies rabbit organs were preserved in a sufficient quantity of

10% buffered formalin composed of pure formalin (10 mL), sodium dihydrogen

phosphate (0.4 g), disodium hydrogen phosphate (0.65 g) and normal saline for volume

make up to 100 mL, and transported to the laboratory. The sections of the buffered

formalin fixed organs were cut and placed in properly labeled histopathology cassettes.

The tissues (cut sections) were then dehydrated with agitation using absolute alcohol for

2 hours followed by clearing alcohol with xylene. The tissues were then impregnated

with paraffin wax at 50–60oC (2–3 times) to make it harder, consistent and xylene free.

Molten wax was again poured on the tissue samples and rapidly cooled with water and

placed in embedding station after cutting and trimming individual blocks which were

fixed on glass slides. The glass slides were placed in xylol and then absolute alcohol each

for 3 minutes. Slides were then placed in methylated spirit for 2 minutes and washed for a

minute in running water and treated with Harris hematoxylin for 3–5 minutes. Slides

were then washed for 30 seconds in running water and treated with 1% acid alcohol for

15 seconds to clear the excess dye. After that slides were washed in flowing water for 30

seconds and dipped 2–3 times in ammonia water solution until the tissue get blue colored.

The slides were then dipped in water 2–3 times and then treated with counter stain eosin

for 2–3 minutes and washed in flowing water for 30 seconds and then dehydrated by

treating with the increasing alcohol concentration i.e. 70% alcohol, 95% and absolute

alcohol, cleared in xylol and mounted with Canada balsam.

2.15.2 Serum chemistry and hematological investigations

Serum chemistry was performed for the determination of serum levels of albumin,

total protein, alkaline phosphatase, aspartate transferase, alanine aminotransferase,

amylase, cholesterol, glucose, bilirubin, creatinine, urea, magnesium, phosphorous,

potassium and sodium were determined through fully automated Roche cobas C111 and

50

Roche cobas e 411 chemistry analyzers, Germany. Globulin level was determined

through the difference of total protein and albumin level. Hematological investigations

like RBC, WBC, Platelets, eosinophils, basophils count, mean cell volume, packed cell

volume and hemoglobin levels were determined through fully automated hematology

analyzer (SYSMEX 100, Germany).

Cytotoxicity assay 2.16

Cytotoxicity assay for the prepared hydrogels was performed using SRB

(Sulphorhodamine B) assay (Skehan et al., 1990) using MCF–7 breast cancer cells, HeLa

cells and normal Vero cells. In All of the four developed different hydrogel formulations

only best release formulation were selected in each series and different concentration of

POM were incorporated for this study. SRB assay is one of the highly sensitive

colorimetric assays used for the determination of the total cells protein content after

treatment with the anticancer agents. Sulphorhodamine B is one the fluorescent, water

soluble and negatively charged dye which binds electrostatically with cells protein at

decreased pH value after cells fixing with trichloroacetic acid (TCA). Cell suspension of

1× 105 cells/mL in Roswell Park Memorial Institute (RPMI) medium were seeded in 24

well cell culture plate and incubated at 95 ± 5% relative humidity, 5.0 ± 0.1 % CO2 and

37 ± 1oC to achieve confluent cells monolayer. Prior to use hydrogels were sterilized at

121oC for 20 min. 20, 25, 30 and 35 mg/mL doxorubicin solution were used as positive

controls and blank cell wells as negative control. After incubation, cells were treated with

100 µL 40% ice cold TCA and kept for 90 minutes. Halogen atom present in TCA

produces the chloride ions (Cl–) which causes the disruption of the electrostatic bonds

ensuring the stability of cellular proteins. The Plate was washed 05 times with distilled

water and then treated with 100 µL of 0.4% sulphorhodamine B (SRB) dye prepared in

1% acetic acid and placed for 30 minutes. Cells were then washed with 1% acetic acid 5

times and then 100 µL of 10 mM tris base was added in each well and kept for 10

minutes at room temperature. The absorbance was measured at 540 nm using microplate

reader.

51

2.16.1 Cell culturing

2.16.1.1 Preparation of culture medium

RPMI media was used for cell culture and RPMI (500 mL) was prepared by

adding heat inactivated 10% FBS, streptomycin 1mL (10 mg/mL), penicillin 1mL

(10000U/mL) and 1 mL of 200 mM L–glutamine. The prepared media was stored in

refrigerator at 4oC.

2.16.1.2 Thawing

Initially the cells were taken from the cryopreserved vial kept at –80oC and was

placed for a minute in water bath at 37oC. The cryovial contents were then immediately

transferred to falcon tube (15 mL) having 10 mL cell culture medium preheated at 37oC.

The cell suspension was then subjected to centrifugation process at room temperature for

5 minutes at 5000 rpm and the obtained pellet was then again re–suspended in cell culture

medium and transferred to cell culture flask (75cm2) having preheated cell growth

medium.

2.16.1.3 Cells splitting

When 80–90% of the cells confluency was achieved, the media was removed and

washed 03 times with PBS for restoration of the physiological pH of cultured cells. After

washing, cells were trypsinized with 3 mL EDTA/trypsin solution (0.05%) and were

incubated for 3–5 minutes. The flasks were gently tapped to detach the remaining

adherent cells. For stopping reaction, 10 mL of the fresh culture medium was added and

the cell suspension was transferred to 15 mL falcon tube and centrifuged for 5 minutes at

5000 rpm. Pellet obtained was uniformly re–dispersed in growth medium by pipetting it

several times. This cell suspension was either then used for cell culturing or

cryopreservation.

2.16.1.4 Cells Cryopreservation

The medium used for vials cryopreservation was composed of 50% FBS (v/v),

10% DMSO (v/v), and 40% RPMI (v/v). On cells counting chamber the cells were

manually counted and approximately 3× 106/mL were cryopreserved per vial at low

temperature (–80oC).

52

2.16.1.5 Cell viability

Trypan blue dye exclusion method was adopted for counting the viable cells in

cell suspension using naubaeur chamber. Trypan blue can easily enter in cells presenting

with poor cell membrane integrity imparting dark dull blue stain in dead cells while live

cells do not capture any stain. For preparing the cells counting solution, 10µL of the 0.4%

trypan blue solution was added into the 10 µL of cell suspension and incubated at room

temperature for 3 min. A particular volume was taken from the suspension and put in the

naubaeur chamber for manual counting under inverted microscope. The dull blue colored

cells were accounted as dead while glowing brighter cells as live ones. Following formula

was used for counting per mL live cells.

…………. (9)

Where n represents average cell count, df denotes dilution factor while 104 is the counting

chamber actual depth (0.1mm3

= 1× 10–4

).

Dilution factor df was calculated as per following formula;

………………..(10)

Where A represents volume of cell suspension (10 µL) and B shows trypan blue dye

volume (10 µL).

53

Chapter 3

3 Results

54

Synthesis of pH responsive gelatin–POM complexes 3.1

Nine formulations having changed concentration of the gelatin, acrylic acid and

POM were prepared as shown in Table 2.1.

3.1.1 Physical appearance

Stable acrylic acid–gelatin–Polyoxometalate hydrogels were prepared after

successful physical cross–linking through electrostatic interactions. Generally hydrogels

were light yellowish in color when freshly prepared but turns into yellow color on drying

and become transparent on swelling. Freshly prepared hydrogels color was largely

dependent on gelatin content ratio. Hydrogels having high gelatin ratio were creamy in

color (Figure 3.1) which turned also transparent on swelling. The mechanical strength of

the hydrogels was largely dependent on POM content.

Fig 3.1 Sole form and B) developed hydrogel disc

3.1.2 Effect of gelatin content on swelling and P2W15 release

Three formulations with different concentrations of gelatin (GP1, GP2, and GP3)

were prepared keeping ratios of acrylic acid, POM and initiator constant. Swelling and

release studies were conducted at pH 1.2 and pH 7.4 using 0.1M HCl buffer and 0.2M

phosphate buffers respectively. Effect of gelatin ratio on release and swelling of the

prepared formulations is shown in Figure 3.2. At pH 1.2 the swelling and P2W15 release

55

increases (GP1, 15%, GP2, 17% and GP3, 19% release) while at pH 7.4 the swelling and

P2W15 decreases (69%, 67% and 64% release).

Fig 3.2 A) Effect of gelatin concentration on swelling and B) cumulative % P2W15

release. Data represents mean value ± SEM (n=3)

3.1.3 Effect of acrylic acid content on swelling and P2W15 release

To check the effect of acrylic acid ratio on swelling and release of the

encapsulated POM, three formulations GP4, GP5, and GP6 were prepared. Figure 3.3

shows the effect of acrylic acid content on P2W15 release and swelling of the hydrogels.

At pH 1.2 there is decrease in the swelling while there was no significant effect on P2W15

release. At pH 7.4 swelling and P2W15 release increases with the respective increase in

acrylic acid concentration (GP4, 69%, GP5, 77% and GP6 83% release).

Fig 3.3 A) Effect of acrylic acid concentration on swelling and B) cumulative % P2W15

release. Data represents mean value ± SEM (n=3).

56

3.1.4 Effect of POM ratio on swelling and P2W15 release

In a series of developed formulations, three formulations (GP7, GP8, and GP9)

were prepared having different concentrations of P2W15. With the increasing

concentration of P2W15 swelling and release decreases accordingly at both pH values.

The effect of P2W15 concentration on swelling and release are shown in Figure 3.4. At pH

1.2 release profile was (GP7 15%, GP8 13% and GP9 12%). Similarly at pH 7.4 observed

release was GP7 64%, GP8 59% and GP9 56%).

Fig 3.4 A) Effect of P2W15 concentration on swelling and B) cumulative % release. Data

represents mean value ± SEM (n=3).

3.1.5 POM release kinetics from pH responsive gelatin-POM complexes

The regression coefficient (R2) values of the different mathematical models are

listed in Table 3.1. As value of n is greater than 1 so it follows super case transport–II

release mechanism followed by zero order release kinetics.

57

Table 3.1 Mechanism of encapsulated POM release in dissolution experiments of AA–

gelatin–POM hydrogels.

Formulation

Code

Zero

order R2

First

order R2

Higuchi

model R2

Peppas

model R2

n

GP1 0.978 0.913 0.745 0.996 1.235

GP 2 0.983 0.981 0.856 0.990 0.892

GP 3 0.992 0.945 0.785 0.996 1.103

GP 4 0.992 0.939 0.790 0.995 1.081

GP 5 0.991 0.939 0.805 0.991 1.027

GP 6 0.993 0.920 0.804 0.994 1.036

GP 7 0.994 0.948 0.784 0.999 1.111

GP 8 0.999 0.951 0.781 0.998 1.121

GP 9 0.992 0.954 0.778 0.999 1.126

*n represents slope of Peppas model

Formulation with sample ID GP6 showed best swelling index and in-vitro release profile.

Therefore was selected for evaluation through different characterization techniques.

3.1.6 FTIR Spectroscopy analysis

FTIR spectrum of the gelatin, acrylic acid, POM and developed hydrogel (GP6)

were recorded as shown in Figure 3.5 for the determination of functional group

interactions. Gelatin spectra showed bands at 3279 cm–1

, 1626 cm–1

, 1520 cm–1

, 1230

cm–1

, 3066 cm–1

and 2865 cm–1

. In acrylic acid FTIR spectra, absorption bands were

recorded at 2988cm–1

, 1696 cm–1

, 1294 cm–1

and 1183 cm–1

. POM FTIR spectra showed

absorption peaks at 1083 cm–1

, 977 cm–1

, 861 cm–1

and 805 cm–1

. Hydrogel FTIR spectra

showed typical peaks at 2930 cm–1

and 500–1000 cm–1

region.

58

*SV for stretching vibration * TS for terminal stretch

Fig 3.5 A) FTIR spectra of gelatin B) acrylic acid C) polyanion salt and D) hydrogel.

3.1.7 Scanning electron microscopy (SEM)

The surface morphology of the intact hydrogel and powdered hydrogel were

evaluated at magnification level of 130X, 210X, 420X and 450X using 3.0 kV electrical

voltage showing the glassy smooth surface morphology. Photomicrographs obtained are

shown in Figure 3.6.

59

Fig 3.6 Scanning electron microscope images of powdered hydrogel (a, b, c) and (d)

intact hydrogel disc.

3.1.8 Thermogravimetric analysis (TGA)

Thermogravimetric analysis of the hydrogel and its components were performed

to evaluate the change in mass with increasing temperature because of water loss. TGA

plots of the hydrogel, gelatin and POM are shown in Figure 3.7. In gelatin TGA Plot it is

evident that there is 8% water loss upto 80oC. In TGA plot of the POM, 4% water loss

occurs up to 100oC and after 100 to 300

oC the crystallized water is lost gradually by the

POM salt which contributes to 6% of the total weight. In case of hydrogel the water loss

of 6% occurred at 170oC.

60

Fig 3.7 TGA plots of gelatin, POM salt and hydrogel.

3.1.9 Differential scanning calorimetry (DSC)

DSC analysis was also performed for the purpose of identification of any thermal

transitions in hydrogel network and its components. DSC spectra of the hydrogel, gelatin

and POM are shown in Figure 3.8. Two endothermic peaks in gelatin DSC spectra are

present in the range of 50 to 90oC and 250 to 300

oC. POM DSC spectra revealed first

endothermic peak at 100oC and second at 410

oC while in case of hydrogel DSC spectra,

two endothermic peaks were observed, first at 220 oC and second at 420

oC.

61

Fig 3.8 DSC curves of POM salt, gelatin and hydrogel.

3.1.10 X–ray diffraction

To evaluate the crystallinity of the prepared hydrogel formulation and its contents

X–ray diffraction was performed. The X–ray diffraction patterns of the gelatin, POM and

developed hydrogel network are shown in Figure 3.9. In case of POM XRD pattern there

is a characteristic peak at 2θ equals to 25.91o in association with other small peaks at 2θ ~

28.73o and 31

o.The XRD pattern of the gelatin showed no intense peak. In case of

hydrogel XRD pattern there is one broad peak recorded at 2θ ~ 72.64o.

62

Fig 3.9 A) XRD pattern of gelatin (B) POM and C) hydrogel.

3.1.11 Cytotoxicity assay

The anticancer potential of the AA–gelatin–POM (encapsulated POM) and free

POM was evaluated against MCF–7, HeLa cells and normal cells (Vero). Both

encapsulated as well as free POM showed dose dependent toxicity against cancer cells.

Doxorubicin was used as positive control. The anticancer potential of the free POM was

higher than doxorubicin. Results of the study are shown in Figure 3.10 expressed as %

viability against concentration (mg/mL). The cytotoxic effect of free POM was higher

than doxorubicin on normal cells but was considerably less than that of MCF–7 and HeLa

cells.

63

Fig 3.10 Cytotoxicity profiling gelatin–POM Hydrogel (encapsulated POM), free POM

(polyoxometalate, P2W15, normal control), doxorubicin (positive control) and blank wells

(Negative control): (A) Cytotoxicity against MCF–7 cells (B) Cytotoxicity against HeLa

cells (C) Cytotoxity against normal Vero cells shown as % cell viability.

Synthesis of pH responsive PEI–POM supramolecular complexes 3.2

Total of nine formulations were prepared having changed concentrations of PEI,

acrylic acid and POM as shown in Table 2.2.


Freshly prepared hydrogels were sea green in color which becomes transparent

after swelling. The color intensity of the hydrogel was largely dependent on

polyethyleneimine content. Hydrogels having low polyethyleneimine content were

transparent in color (Figure 3.11). The prepared hydrogels were having little bit low

mechanical strength and uniform structure.

64

Fig 3.11 A) Sole form and B) slightly dried developed hydrogel disc.

3.2.2 Effect of Polyethyleneimine content on swelling and in–vitro P2W15

release

To check the effect of PEI content on swelling and release characteristics of the

hydrogel system three formulations with sample codes PEP1, PEP2, PEP3 were prepared.

The effect of PEI content on swelling and release at pH 1.2 and pH 7.4 are shown in

Figure 3.12. At pH 1.2 with increasing PEI concentration, the swelling as well as

encapsulated P2W15 release increases (PEP1 24%, PEP2 33% and PEP3 41%). At pH 7.4

the swelling and P2W15 release decreases (PEP1 70%, PEP2 64% and PEP3 59%) as we

increase the PEI content in the respective three formulations.

65

Fig 3.12 A) Effect of polyethyleneimine concentration on swelling and B) cumulative %

P2W15 release at of hydrogels at pH 1.2 and pH 7.4. Each data point represents the mean

± SEM, n=3.

3.2.3 Effect of acrylic acid content on swelling and P2W15 release

For evaluation of the acrylic acid content on swelling and release parameters three

formulations with sample codes PEP4, PEP5 and PEP6 were prepared. The effect of

acrylic acid content on swelling and release at pH 1.2 and pH 7.4 are shown in Figure

3.13. At pH 1.2 there was no significant swelling and P2W15 release decreases as well

(PEP4 25%, PEP5 21%, PEP6 18%). At pH 7.4 with the increasing concentration of the

acrylic acid the swelling and P2W15 release increases accordingly (PEP4 61%, PEP5

68%, PEP6 77%).

Fig 3.13 A) Effect of acrylic acid concentration on swelling and B) cumulative % P2W15

release of hydrogels at pH 1.2 and pH 7.4. Each data point represents the mean ± SEM,

n=3.

66


For evaluation of the POM content on swelling and release parameters, three

formulations with sample codes PEP7, PEP8 and PEP9 were prepared. The effect of

POM content on swelling and release at pH 1.2 and pH 7.4 are shown in Figure 3.14. At

both pH 1.2 and 7.4 with the increase in POM ratio swelling and P2W15 release decreases.

For pH 1.2 release profile was (PEP7 27%, PEP8 21%, PEP9 18%) while for pH 7.4

release profile of (PEP7 67%, PEP8 61% and PEP9 55%) was observed.

Fig 3.14 A) Effect of POM concentration on swelling and B) cumulative % in–vitro

release of hydrogels at pH 1.2 and pH 7.4. Each data point represents the mean ± SEM,

n=3.

3.2.5 POM release kinetics

Regression coefficient (R2) was used to analyze the model which can best fit to

the release data and suitable model was selected having regression coefficient (R2) value

closer to 1. The release profile of the P2W15 from hydrogels showed that it follows zero

order kinetics. In Table 3.2 regression coefficient values are shown. The value of n is

greater than 1 suggesting the super case transport II mechanism. Regression values of

zero order are closer to 1 hence the release of POM follows zero order kinetics.

67

Table 3.2 Mechanism of POM release in dissolution experiments of AA–PEI–POM

hydrogels.

Formulation

code

Zero order

R2

First order

R2

Higuchi

model R2

Peppas

model R2

n

PEP 1 0.9949 0.9515 0.8898 0.9395 1.3081

PEP2 0.9941 0.9578 0.8875 0.9548 1.3195

PEP3 0.9960 0.9670 0.8926 0.9703 1.3502

PEP4 0.9987 0.9820 0.9134 0.9510 1.3141

PEP5 0.9991 0.9804 0.9286 0.9197 1.2839

PEP6 0.9977 0.9743 0.9322 0.8958 1.2789

PEP7 0.9963 0.9659 0.8954 0.9478 1.3198

PEP8 0.9923 0.9586 0.8779 0.9750 1.3653

PEP9 0.9911 0.9615 0.8761 0.9798 1.3470


Formulation with sample ID PEP6 showed best swelling and in-vitro release profile out

of all developed nine formulations of this class. Therefore this formulation was selected

for evaluation through different characterization techniques

3.2.6 FTIR spectroscopy

FTIR spectra of polyethyleneimine, POM, acrylic acid and hydrogel are shown in

Figure 3.15. The absorption spectrum of PEI showed some typical peaks at 1109 cm–1

,

1458 cm–1

and 1647 cm–1

. In acrylic acid FTIR spectra peaks were recorded at 2988cm–1

,

1696 cm–1

, 1586 cm–1

, 1294 cm–1

and 1183 cm–1

. POM FTIR spectra showed absorption

peaks at 1083 cm–1

, 977 cm–1

, 861 cm–1

and 805 cm–1

. In hydrogel FTIR spectra peaks

were recorded at 1697 cm–1

, 1449 cm–1

, 1162 cm–1

, 1115 cm–1

and some modified

specific absorption peaks in the 1000–500 cm–1

region.

68

*BV for bending vibration * FP for finger print region

Fig 3.15 A) FTIR spectra of polyethyleneimine B) acrylic acid C) POM D) hydrogel.


Surface morphology of the powered and intact hydrogel disc were evaluated at the

magnification level of X120, X270, X420 and X950 at the electrical voltage of 3.0 kV.

Photomicrographs are shown in Figure 3.16 revealing the glassy smooth surface

morphology.

69

Fig 3.16 a, b, c) Scanning electron microscope images of powdered hydrogel and d)

intact hydrogel.

3.2.8 Thermal analysis

TGA and DSC analysis were performed for both POM and developed self–

assembled hydrogel and the thermograms are shown in Figure 3.17. In the thermogram of

the POM there is continuous loss of crystal waters from room temperature to about

200oC. Thermogram of the hydrogel shows initial weight loss at 180

oC. Major loss in

mass occurs between 300oC to 500

oC.

70

Fig 3.17 A) Thermograms and differential scanning calorimetry curves of POM and B)

hydrogel.

3.2.9 Powder X–ray diffraction

To determine the degree of crystallinity in prepared hydrogel samples and it

components X–ray diffraction patterns were obtained. The XRD patterns of prepared

hydrogel formulation and POM are shown in Figure 3.18. In case of POM XRD pattern

there is a characteristic peak at 2θ equals to 25.91o in association with other small peaks

at 2θ ~ 28.73o

and 31o. Hydrogel XRD pattern depicts other small peaks present at 2θ ~

30o and 35

o there is one intense peak at 2θ equals to72.52

o.

Fig 3.18 A) X–ray diffraction pattern of POM and B) developed hydrogel.

71

3.2.10 Cytotoxicity assay of pH responsive PEI-POM complexes

The cytotoxicity potential of the encapsulated POM (hydrogel) against MCF–7,

HeLa cells and Vero cells was determined through sulphorhodamine B assay. The

obtained results showed that free POM exhibited dose dependent toxicity on both

cancerous cell lines (MCF–7, HeLa) while there was a minimal effect on normal Vero

cells. In comparison with doxorubicin concentrations POM showed more cytotoxic

potential on cancerous as well as on normal Vero cells. In case of hydrogel graphs it can

be seen that it has also shown dose dependent toxicity on both cancer cell lines but the

viable cell count is high as compare to unbound POM as shown in Figure 3.19.

Fig 3.19 Anticancer and Cytotoxicity profiling of AA–PEI–POM hydrogel (encapsulated

POM), free POM polyoxometalate, P2W15 (normal control), doxorubicin (positive

control) and blank wells (Negative control). Anticancer activity shown as % cell viability

against A) MCF-7 cells B) HeLa cells and C) normal Vero cells shown as % viability.

Synthesis of pH responsive carboxymethyl chitosan– POM 3.3

supramolecular complexes

Electrostatically driven total nine formulations were prepared having changed

concentration of carboxymethyl chitosan, methacrylic acid and POM as shown in Table

2.3.

72


The freshly prepared hydrogels were transparent in colure (Figure 3.20). The

hydrogels prepared with highest amount of POM were having more mechanical strength

because of hydrogel overall increased cross–linking density.

Fig 3.20 A) Sole form of hydrogel components and B) developed supramolecular

hydrogel disc.

3.3.2 Effect of CMCh concentration on swelling and P2W15 release

In a series of formulations three formulations (CMCP1, CMCP2, and CMCP3)

were prepared having changed concentration of the CMCh keeping other content ratios

constant as shown in Table 2.3. The effect of CMCh concentration on both swelling and

release was significant on both respective pH values releasing maximum of 39% P2W15 at

pH 1.2 and maximum of 71% at pH 7.4. The effect of CMCh concentration on swelling

and P2W15 release are shown in Figure 3.21.

73

Fig 3.21 A) Effect of CMCh concentration on swelling and B) cumulative % release of

hydrogels. Data are shown as mean ± SEM, n=3.

3.3.3 Effect of methacrylic acid content on swelling and P2W15 release

To evaluate the effect of methacrylic acid concentration on swelling and POM

release three formulations (CMCP4, CMCP5 and CMCP6) were prepared. The effect of

methacrylic acid concentration on swelling and POM release is shown in Figure 3.22. At

pH 1.2 there was minimum swelling and less P2W15 release of 26% while at pH 7.4 there

was maximum swelling and P2W15 release of 86%.

Fig 3.22 A) Effect of MAA concentration on swelling and B) cumulative % POM release

of hydrogels. Data are shown as mean ± SEM, n=3.


To check the effect of POM content on swelling and itself POM release of

hydrogel network, three different formulations (CMCP7, CMCP8 and CMCP9) were

74

prepared having changed concentration of POM. The effect of POM content on swelling

and POM release is shown in Figure 3.23. At pH 1.2 there was minimum swelling and

P2W15 release of 30% while at pH 7.4 there was maximum swelling and P2W15 release of

74%.

Fig 3.23 (A) Effect of POM concentration on swelling and (B) cumulative % in–vitro

release of hydrogels. Data are shown as mean ± SEM, n=3.

3.3.5 POM release kinetics of pH responsive carboxymethyl chitosan-POM


Regression coefficient (R2) was used to analyze the model which can best fit to the

release data and suitable model was selected having regression coefficient (R2) value

closer to 1. In Table 3.3 regression coefficient values are shown. The value of n is greater

than 1 suggesting the super case transport II mechanism followed by zero order kinetics.

75

Table 3.3 Mechanism of POM release in dissolution experiments from MAA–CMCh–

POM hydrogels.

Formulation

code

Zero

order R2

First order

R2

Higuchi

model R2

Peppas

model R2

n

CMCP1 0.9972 0.9765 0.9019 0.9494 1.2926

CMCP2 0.9980 0.9716 0.9027 0.9489 1.3173

CMCP3 0.9961 0.9515 0.8916 0.9384 1.3090

CMCP4 0.9982 0.9553 0.9047 0.9246 1.2981

CMCP5 0.9963 0.9319 0.8983 0.9188 1.3132

CMCP6 0.9991 0.9304 0.9147 0.9141 1.3358

CMCP7 0.9953 0.9407 0.8893 0.9525 1.3606

CMCP8 0.9955 0.9516 0.8909 0.9505 1.3352

CMCP9 0.9961 0.9730 0.8963 0.9594 1.3269


Formulation with sample ID CMCP6 showed best swelling and in-vitro release profile

out of all developed nine formulations of this class as well. Hence, selected for evaluation

through different characterization techniques.

3.3.6 FTIR Spectroscopy analysis

FTIR spectrums were obtained for CMCh, MAA, POM and developed hydrogel

as shown in Figure 3.24. FTIR spectrum of CMCh exhibited typical peaks at 1407 cm–1

,

1585 cm–1

, 1046 cm–1

, 1307 cm–1

, 895 cm–1

, 3362 cm–1

and 2871 cm–1

. MAA FTIR

spectra showed peaks at 1633 cm–1, 2962 cm

–1, 1557 cm

–1, 1201

cm

–1 and 1685 cm

–1.

POM FTIR spectra showed absorption peaks at 1083 cm–1

, 977 cm–1

, 861 cm–1

and 805

cm–1

. In hydrogel FTIR spectra the absorption bands were recorded at 1481 cm–1

, 1388

cm–1

, 1256 cm–1, 1537 cm

–1 and distinctive bands present in the region of the 1000–500

cm–1

.

76

*S for stretch *SV for bending vibration * FP for finger print region

Fig 3.24 A) FTIR spectra of CMCh B) MAA C) polyanion salt and D) hydrogel


Surface morphology of the powered and intact hydrogel disc were evaluated at the

magnification level of X120, X270, X420 and X950 at the electrical voltage of 3.0 kV

and the obtained Photomicrographs of the powdered and intact hydrogel disc are shown

in Figure 3.25. Images obtained shows that hydrogel network possesses the smooth

glassy morphology.

77

Fig 3.25 Scanning electron microscope images of powdered hydrogel (A, B, C) and (D)


3.3.8 Thermogravimetric analysis

Thermogravimetric analysis were performed exhibiting the change in mass and

material decomposition with the respective increase in temperature. Thermogravimetric

plots of carboxymethyl chitosan, POM salt and hydrogel are shown in Figure 3.26. In the

first stage carboxymethyl chitosan weight loss starts at 60 oC contributing about 7%

weight loss due to loss of water. In second stage carboxymethyl chitosan undergo weight

loss process in the range of 240–290oC contributing 35% weight loss. In TGA plot of the

POM salt 4% water loss occurs up to 100oC and after 100 to 300

oC there is gradual loss

of mass contributing 6% of the total weight and there is a heat change at 450oC. In

hydrogel the first stage of weight loss occurs upto 200oC contributing 16% weight loss

because of evaporation of water. The onset of second phase of weight loss occurs at

270oC upto 390

oC contributing 30% loss in weight.

78

Fig 3.26 TGA plots of CMCh, POM and hydrogel.

3.3.9 Differential scanning calorimetry

For the identification of the thermal transitions in the developed hydrogel network

differential scanning calorimetry was performed in association with thermogravimetric

analysis. DSC curves of the carboxymethyl chitosan, POM salt and hydrogel are shown

in Figure 3.27. In case of CMCh, endothermic peak is present at 70oC and exothermic

peak at 290oC. POM DSC spectra reveals first endothermic peak at 100

oC and second at

410oC. In hydrogel DSC curve first endothermic peak is evident at 240

oC and second at

410o C.

79

Fig 3.27 DSC curves of CMCh, POM and hydrogel.

3.3.10 X–ray diffraction

To check the degree of crystallinity of the samples, XRD pattern of the CMCh,

POM and developed hydrogels were recorded as shown in Figure 3.28. The X–ray

diffraction pattern of the CMCh exhibited a broad peak at 2θ of 26.51o. POM XRD

pattern showed characteristic peak at 2θ equals to 25.91o in association with other peaks

at 2θ ~ 28.73o and 31

o. Hydrogel XRD pattern showed less intense peak recorded at 2θ ~

17.61o.

Fig 3.28 A) X–ray diffraction patterns of CMCh B) POM and C) developed hydrogel.

80

3.3.11 Cytotoxicity assay of pH responsive carboxymethyl chitosan-POM


The anticancer potential of the MAA–CMCh–POM hydrogel (encapsulated POM)

and free POM was evaluated against MCF–7, HeLa cells and normal cells (Vero). The

anticancer effect of encapsulated POM was less than free POM as compare to free POM

as well as doxorubicin (positive control). On HeLa cells the hydrogel showed anticancer

effect expressed as % cell viability upto 32% while in case of POM at 35mg

concentration, % cell viability was 15%. On MCF–7 cells the hydrogel exhibited

anticancer affect up to 30% in terms of % cell viability as shown in Figure 3.29.

Fig 3.29 Cytotoxicity profiling of MAA–CMCh–POM Hydrogel (encapsulated POM),

free POM (polyoxometalate, P2W15, normal control), doxorubicin (positive control) and

blank wells (Negative control): (A) Cytotoxicity against MCF–7 cells shown as % cell

viability (B) HeLa cells C) normal Vero cells shown as % viability.

Synthesis of pH responsive chitosan hydrochloride–POM 3.4

complexes

Total of nine formulations were prepared having changed concentration of

chitosan hydrochloride (ChCl), methacrylic acid (MAA) and POM as shown in Table 2.4.

81


The freshly prepared hydrogel formulations were transparent in colure possessing

good mechanical strength and uniform structure as shown in figure 3.30.

Fig 3.30 A) Sole form and B) developed MAA–ChCl–POM supramolecular hydrogel

disc.

3.4.2 Effect of ChCl concentration on swelling and P2W15 release

Three formulations with sample codes as CHCP1, CHCP2 and CHCP3 were

prepared with changed ChCl concentration. The effect of ChCl on swelling parameter

and release profile of the hydrogel formulations is shown in Figure 3.31. The hydrogels

exhibited less swelling and release at pH 1.2 and high swelling and release profile at pH

7.4. Maximum of 28% P2W15 release was observed at pH 1.2 while at pH 7.4 74% P2W15

release was observed.

82

Fig 3.31 (A) Effect of ChCl concentration on swelling and (B) cumulative % POM

release of hydrogels. Each data points represents average value ± SEM, (n=3).

3.4.3 Effect of methacrylic acid content on swelling and P2W15 release

In a series of developed formulations three formulations coded as CHCP4,

CHCP5 and CHCP6 were prepared having changed concentration of MAA. The effect

MAA on swelling and release parameters of the hydrogels are shown in Figure 3.32. At

pH 1.2 with increasing concentration of MAA both swelling and release decreases while

at pH 7.4 there is significant increase in swelling and release of encapsulated P2W15 with

maximum release of 92%.

Fig 3.32 A) Effect of MAA concentration on swelling and (B) cumulative % POM

release of hydrogels. Each data points shows average value ± SEM, (n=3).

83

3.4.4 Effect of POM concentration on swelling and in–vitro release

To check the effect of POM concentration on swelling and POM release itself

three formulations were prepared with changed POM concentration bearing sample codes

CHCP7, CHCP8 and CHCP9. Figure 3.33 contains the effect of POM concentration on

swelling and release parameters. Both at pH 1.2 and pH 7.4 the swelling and POM

release decreases with the increasing POM concentration.

Fig 3.33 A) Effect of POM concentration on swelling and B) cumulative % release of

hydrogels. Each data points shows average value ± SEM, (n=3).

3.4.5 POM release kinetics of pH responsive chitosan hydrochloride-POM

complexes

Regression coefficient (R2) was used to analyze the model which can best fit to

the release data and suitable model was selected having regression coefficient (R) value

closer to 1. In Table 3.4 regression coefficient values of different mathematical models

are shown. The value of n (slope of Peppas model) is greater than 1 suggesting the super

case transport II mechanism of P2W15 release. Regression values of zero order are closer

to 1 hence the release of POM follows zero order kinetics.

84

Table 3.4 Mechanism of POM release during dissolution experiments from MAA–ChCl–

POM hydrogels.

Formulation

code

Zero order

R2

First order

R2

Higuchi

model R2

Peppas

model R2

n

CHCP 1 0.9982 0.9804 0.9153 0.9328 1.2996

CHCP 2 0.9961 0.9822 0.9192 0.9289 1.3015

CHCP 3 0.9961 0.9756 0.9292 0.9406 1.2791

CHCP 4 0.9979 0.9730 0.9162 0.8998 1.3060

CHCP 5 0.9970 0.9465 0.9104 0.9047 1.3069

CHCP 6 0.9989 0.9127 0.9325 0.8676 1.2772

CHCP 7 0.9992 0.9581 0.9130 0.9258 1.3216

CHCP 8 0.9993 0.9683 0.9201 0.9215 1.2818

CHCP 9 0.9983 0.9813 0.9139 0.9502 1.3322


Formulation with sample ID CHCP6 showed best swelling and in-vitro release profile

among all different formulation of this class, hence selected for evaluation through

different characterization techniques.

3.4.6 FTIR spectroscopy analysis

FTIR spectra were recorded for ChCl, MAA, POM and developed hydrogel as

shown in Figure 3.34. FTIR spectrum of ChCl showed peaks at 1614, 1582, 1316, 1393,

1138, 1092 and 3283 cm–1

. FTIR spectrum of MAA showed absorption peaks at 1633

cm–1, 2962 cm

–1 , 1557 cm

–1, 1201

cm

–1 and 1685 cm

–1. POM FTIR spectra showed

absorption peaks at 1083 cm–1

, 977 cm–1

, 861 cm–1

and 805 cm–1

. The hydrogel FTIR

spectrum exhibited absorption bands at 1254, 1387, 1685, 3126, 1541 and modified

absorption peaks in the range of 1000–500 cm–1

and 3500–2500 cm–1

.

85

*S for stretch *SV for bending vibration * TS for terminal stretch.

Fig 3.34 A) FTIR spectrum of ChCl B) MAA C) polyanion salt and D) hydrogel.

3.4.7 Scanning electron microscopy

Scanning electron microscopy was performed for the evaluation of the cross

sectional and intact hydrogel disc morphology at magnification level of X120, X420,

X270, X950 and electrical voltage of 3.0 KV. Photomicrographs obtained are shown in

Figure 3.35 showing the smooth and glassy surface morphology.

86

Fig 3.35 A, B, C) Scanning electron microscope images of powdered hydrogel and D)


3.4.8 Thermogravimetric analysis

Thermogravimetric analysis were performed for ChCl, POM and the developed

hydrogel system and the plots obtained are shown in Figure 3.36. ChCl TGA plot

exhibited no significant weight loss up to 180oC. At 180

oC there is a heat change up to

250oC accountable for 45% loss in mass and after this there is gradual decomposition of

polymer backbone up to 450oC. In TGA plot of the POM, 4% water loss occurs up to

100oC and after 100 to 300

oC the crystallized water is lost gradually by the POM which

contributes to 6% of the total weight. In hydrogel TGA plot first stage of weight loss

starts at 200oC contributing 12% mass loss, second at 260

oC accounting for 21% loss in

mass and the third heat change was observed at 370oC extending upto 470

oC.

87

Fig 3.36 TGA Plots of ChCl, POM and hydrogel.

3.4.9 Differential scanning calorimetry

DSC curves were also obtained for evaluation of thermal transitions in hydrogel

system and its components. DSC curves of the ChCl, POM and hydrogel are shown in

Figure 3.37. DSC curve of ChCl exhibited sharp endothermic peak at 220oC and full

decomposition at 500oC. POM DSC spectra reveals first endothermic peak at 100

oC and

second at 410oC. DSC curve of hydrogel network exhibited multi–stage thermal

transitions. First minute endothermic peak was observed at 40oC, second at 250

oC, third

at 380oC and fourth endothermic peak at 430

oC.

88

Fig 3.37 DSC curves of ChCl, POM and hydrogel.

3.4.10 X–ray diffraction analysis

To evaluate the crystallinity of the prepared hydrogels, XRD patterns were

obtained and compared with its components. Figure 3.38 shows the XRD patterns of

ChCl, hydrogel and POM. ChCl XRD pattern gives major peaks at 2θ ~ 10.161, 15.352,

and 20.538o in association with other small peaks. POM XRD pattern showed

characteristic peak at 2θ equals to 10.91o. Hydrogel XRD pattern showed a broad peak

recorded at 2θ ~ 19.51o.

89

Fig 3.38 A) X–ray diffraction patterns of ChCl B) POM and C) hydrogel.

3.4.11 Cytotoxicity assay

The anticancer potential of the ChCl–POM hydrogel (encapsulated POM) and

free POM was evaluated against MCF–7, HeLa cells and normal cells (Vero). The

anticancer effect of encapsulated POM was less than free POM as compare to free POM

as well as doxorubicin (positive control). On HeLa cells the hydrogel showed anticancer

effect expressed as % cell viability upto 30% while in case of POM at 35mg

concentration, % cell viability was 15%. On MCF–7 cells the hydrogel exhibited

anticancer affect up to 25% in terms of % cell viability as shown in Figure 3.39.

Fig 3.39 A–B) Cytotoxicity profiling of ChCl–POM Hydrogel (encapsulated POM), free

POM (Polyoxometalate, P2W15, normal control), doxorubicin (positive control) and blank

wells (negative control) Cytotoxicity against MCF–7, HeLa cells and (C) cytotoxicity

against normal Vero cells shown as % viability.

Pharmacokinetic study of POM in rabbits 3.5

Hydrogel formulation (Sample ID CHCP6) Showed highest in-vitro release profile.

Therefore was selected for in-vivo evaluation of POM pharmacokinetic behavior in

rabbits.

90

3.5.1 HPLC method validation

3.5.1.1 Linearity of the method

The seven pointed calibration curve showed good linearity over the whole

concentration range (25–2000µg/mL) covering the plasma concentration of POM found

after administration of 20mg/2mL oral POM solution and hydrogel disc having 20mg

POM. Correlation coefficient (R2) was 0.9999 for all five constructed calibration curves

which was highly significant (Figure 3.40 and Table 3.5).

Table 3.5 Linearity summary of the method

Run Number Equation form: Y=Ax + B

A B

Correlation

Coefficient (R2)

1 1022.5 1224.8 0.9999

2 1022.4 1185.7 0.9999

3 1022.4 1157.0 0.9999

4 1023.2 974.91 0.9999

5 1022.7 656.99 0.9999

Mean 1022.64 1039.88 0.9999

SD 0.336155 234.5218 0

*SD for standard deviation

Fig 3.40 Calibration curve of polyanion spiked in rabbit plasma.

91

3.5.2 Limit of detection and limit of quantification

Limit of detection (LOD) was 7.50 µg/mL and limit of quantification (LOQ) was

25µg/mL calculated through signal to noise (S/N) ratio method according to FDA

guidelines. LOD and LOQ calculations are given in Table 3.6.

Table 3.6 Determination of LOD and LOQ by signal to noise ratio method (n=6).

S.No

Conc.

spiked

(µg/mL)

Noise/Signal

ratio

amount found

(µg/mL)

LOD

(µg/mL)

LOQ

(µg/mL)

1 8 0.3466 7.34 7.63 25.44

2 8 0.3509 7.25 7.63 25.44

3 8 0.3599 7.3 7.88 26.27

4 8 0.3365 7.2 7.27 24.22

5 8 0.3469 7.1 7.39 24.62

6 8 0.329 7.3 7.21 24.01

Mean LOD, LOQ values 7.50 25.00 *LOD for limit of detection *LOQ for limit of quantification

3.5.3 Accuracy and precision

For intraday precision (% relative standard deviation, % RSD) and accuracy (%

relative error, % RE) were calculated and found between 0.01– 2.95% and –0.31–14%

respectively. For interday precision and accuracy were 1.87–2.62% and 3.27–13.46%

lying within allowed limits according to FDA guidelines (Health & Services, 2001).

92

Table 3.7 Precision and accuracy of the developed HPLC method in rabbit plasma.

Conc. added

(µg/mL)

Mean conc.

found

(µg/mL)

SD* Precision

(RSD, %)

Accuracy

(RE, %)

Intraday (n=5)

25 µg/mL

28.60

0.84

2.95

14.43

250 µg/mL

244.21

0.11

0.04

–2.31

1000 µg/mL

996.89

0.14

0.01

–0.31

Interday (n=15)

25 µg/mL 28.36 0.30

2.62

13.46

250µg/mL 244.23 0.05

1.87

3.27

1000 µg/mL 996.99

0.06

1.93

4.61

*SD for standard deviation *RSD for relative standard deviation * RE for relative error

Representative blank and spiked plasma chromatograms are shown in Figure 3.41.

Fig 3.41 Representative chromatograms of blank and polyanion spiked rabbit plasma.

93

3.5.4 Pharmacokinetics of POM

Mean plasma concentrations obtained after oral Polyoxometalate solution and

hydrogel plotted against time are shown in Table 3.8. Time wise a clear–cut difference

was observed between plasma concentrations obtained after oral POM solution and

hydrogel disc. After administration of hydrogel disc having equivalent 20mg POM,

relatively longer plasma concentration profiles were obtained. AUC and MRT obtained

for hydrogel formulation were greater 2.87 and 1.91 times respectively than oral POM

solution. Tmax obtained for oral solution was 9.6 hours while for hydrogel disc it got

extended to 14.6 hours. Half–life (t1/2) recorded for oral POM solution was 2 hours while

in case of hydrogel formulation t1/2 of 4.87 hours was observed. Apparent volume of

distribution (Vz) obtained for oral POM solution was 0.0034L while for hydrogel system

was 0.0038L. Clearance of oral POM solution occurred at the rate of 0.0012 L/h while for

hydrogel it was 0.00056 L/h. Elimination half–life (Lz) observed for oral solution was

0.345 (hr) while for hydrogel encapsulated POM was 0.147 (hr). Interestingly Cmax

values calculated for oral POM solution and hydrogel disc were lying in close proximity.

Table 3.8 Mean values ± SEM of pharmacokinetic parameters of POM following

administration of oral solution and oral hydrogel in rabbits (n=12)

S.No Pharmacokinetic

Parameter

Oral solution ± SEM Hydrogel ± SEM

1 Cmax(µg/mL) 1810 ± 53.4 1801 ±32.3

2 Tmax (hour) 9.6 ± 0.61 14.6 ± 0.67

3 t1/2 (hour) 2.0 ± 0.08 4.8 ± 0.48

4 AUCtot (µg/mL*hour) 13807.6± 251 39628.9 ± 665

5 MRT (hour) 10 ± 0.06 19 ± 0.28

6 Vz (L) 0.0034 ± 0 0.0038 ± 0

7 Cl (L/hour) 0.0012 ± 0 0.00050 ± 0

8 Lz (hour) 0.345 ± 0.02 0.147 ± 0.02

94

Combined as well as individual rabbits plasma profiles of POM obtained after oral

solution and hydrogel formulation are shown in Figure 3.42, Figure 3.43 and Figure 3.44.

Fig 3.42 Plasma profile of POM after oral solution, oral hydrogel and combined plasma

profile after oral solution and oral hydrogel.

95


comparison with mean plasma profile.


comparison with mean plasma profile.

96

Oral acute toxicity and safety evaluation of MAA–ChCl–POM 3.6

hydrogel and POM solution

3.6.1 Maximal tolerated dose of hydrogel dispersion

The dosing of hydrogel dispersion was administered in the range of 50–4300

mg/kg body weight with the increasing rate of 100 mg/kg. During dosing, rabbits were

critically monitored for vital signs and symptoms. At a dose of 4300 mg/kg body weight,

pupil dilation and gesture abnormalities were observed for average time period of 3–5

hours probably due to limited stomach capacity of rabbits. Therefore, maximum tolerable

dose of hydrogel dispersion was decided as 4000 mg/kg body weight for further study.

3.6.2 General conditions

Throughout all oral acute toxicity, safety profiling no rabbit death occurred and

no toxic response was observed in hydrogel dispersion and polyoxometalate oral solution

treated rabbits. The conditions of the hair, eye slits, eyes, teeth and oral cavity were

normal. Other activities like reflection, breathing, response to light and sound were

normal. There was no edema, hyper salivation and abnormal eye secretions thorough out

the safety profiling. The colure and frequency of the rabbit‘s feces were normal and free

of blood, pus and mucus. Regarding food consumption, there was no difference between

the treatment and control groups throughout the test.

Table 3.9 Toxicity signs not observed in hydrogel dispersion (4000 mg/kg) and POM

treated groups.

Motor activities Hyperactivity, restlessness and phonation.

Symptoms of nervous system Twitching, tail erection, movement disorder,

abnormal gesture

Symptoms of autonomic nervous

system

Excessive urination, hypersalivation, eyeballs

protrusion, hair discoloration and bristling up,

dyspnea.

Mortality Death

97

3.6.3 Hematological investigations and serum chemistry profiling

After administration of oral polyoxometalate solution and hydrogel dispersion,

serum chemistry profiling (Table 3.10) and complete blood count (CBC) were performed

on 7th

day. CBC was performed to sort out any abnormalities inside blood system caused

by polyoxometalate or hydrogel dispersion in comparison with the control group. Serum

chemistry was performed to evaluate the kidneys and liver functions mainly. In Table

3.11 red blood cells, platelets, white blood cells count, hemoglobin level, mean cell

volume, packed cell volume of the polyoxometalate and hydrogel dispersion group are

shown. The serum chemistry profile and CBC of both treatment groups revealed no

significant difference as compared to control group except reduction of blood glucose

level in both hydrogel dispersion as well as POM solution treated rabbits groups.

Metabolite concentrations of the both treatment groups were normal as compare to

control group as shown in Figures 3.43–3.47. Results of both the treatment groups exhibit

that kidney, liver functions and blood system were fairly normal.

98

Table 3.10 Mean values ± SEM of rabbit Serum levels of albumin, total protein, globulin, alkaline phosphatase, aspartate

aminotransferase, alanine aminotransferase, cholesterol, glucose, bilirubin, creatinine, urea, magnesium, potassium and

sodium. Control, (n=3 male, n=3 female), hydrogel dispersion (n=3 male, n=3 female), POM (n=3 male, n=3 female)

Parameter

Male

control

group

Male

treatment

group (HS)

Male treatment

group (POM)

Female control

group

Female

treatment

group (HS)

Female treatment

group (POM)

Total protein (g/L) 71.04 ± 0.77 68.49 ± 0.28 67.85 ± 1.28 71.81 ± 0.24 69.21 ± 0.45 70.56 ± 0.42

Albumin (g/L) 51.3 ± 0.9 49.0 ± 0.6 49.7 ± 1.3 54.7 ± 1.0 53.7 ± 1.0 53.7 ± 1.0

Globulin (g/L) 20.04 ± 0.20 19.49 ± 0.30 18.19 ± 0.61 17.15 ± 0.48 16.73 ± 0.82 16.89 ± 0.26

ALP (U/L) 120 ± 0.20 116 ± 0.30 118 ± 0.61 137 ± 0.48 127 ± 0.82 132 ± 0.26

AST (U/L) 64.83 ± 1.35 63.59 ± 1.24 63.96 ± 1.52 68.29 ± 0.66 66.67 ± 0.88 65.03 ± 1.56

ALT (U/L) 81.83 ± 1.35 79.59 ± 1.24 80.96 ±1.52 79.29 ± 0.66 76.67 ± 0.88 77.03 ± 1.56

Amylase (U/L) 491 ± 3.93 485 ± 2.96 482 ± 5.9 504.57 ± 1.23 499 ± 1.73 513.67±5.61

Cholesterol (mmol/L) 107.04±0.61 102±0.58 102.2±2.15 108.23±0.48 104.81±0.45 105.86±0.94

Glucose (mmol/L) 7.10±0.06 3.96±0.035 4.26±0.032 7.19±0.065 3.87±0.022 4.05±0.074

Bilirubin (µmol/L) 10.17±0.03 10.05±0.03 10.05±0.06 10.21±0.03 10.09±0.02 9.99±0.06

Creatinine (µmol/L) 156.78±3.12 151.45±2.71 154±3.21 152.51±2.25 141.89±1.80 143.55±4.12

Urea (mmol/L) 156.78±3.12 151.45±2.71 154±3.21 6.52±0.12 6.37±0.10 6.22±0.07

Magnesium (mmol/L) 0.81±0.01 0.78±0.01 0.80±0.03 0.83±0.03 0.82±0.03 0.81±0.03

Phosphorous(mmol/L) 1.98±0.05 1.9 ± 0.02 1.93±0.03 2.28±0.04 2.21±0.01 2.20±0.03

Potassium (mmol/L) 6.11±0.07 5.94±0.03 6.03±0.09 6.1±0.06 5.91±0.03 5.96±0.07

Sodium (mmol/L) 141.27±0.84 140.67±0.68 140.5±0.81 141.79±0.84 141±0.58 141.17±0.56

*HS for hydrogel dispersion *POM for polyoxometalate

99

Table 3.11 Mean values ± SEM of rabbit blood count of WBCs, lymphocytes, eosinophils, basophils, neutrophils, platelets,

RBCs, hemoglobin level, packed cell volume and mean cell volume. (Control, n=3 Male, n=3 female, hydrogel dispersion n=3

Male, n=3 female, POM, n=3 male, n=3 female)

Parameter Male control

group

Male

treatment

group (HS)

Male

treatment

group (POM)

Female

control group

Female

treatment group

(HS)

Female

treatment

group (POM)

WBC ×109/L 8.213±0.09 8.12±0.015 8 ± 0.01 8.24 ± 0.023 8.1 ± 0.012 7.98 ± 0.018

Lymphocytes ×109/L 2.29 ± 0.02 2.16 ± 0.029 2.20 ± 0.020 2.3 ± 0.02 2.23 ± 0.01 2.51 ± 0.02

Eosinophils ×109/L 0.028 ±0.06 0.026±0.0003 0.027±0.0003 0.0913±0.0009 0.0907±0.0003 0.0893±0.0015

Basophils ×109/L 0.21 ± 0.012 0.323 ± 0.003 0.323 ± 0.013 0.223 ± 0.007 0.227 ± 0.009 0.217 ± 0.003

Neutrophils ×109/L 4.27 ± 0.15 4.28±0.09 4.25±0.16 4.23 ± 0.009 4.18 ± 0.009 4.19±0.003

Platelets ×109/L 335.8 ± 2.28 332.36±1.60 331.87±1.80 329.06±0.59 327.08±1.69 328.33±0.33

RBC ×1012

/L 5.82±0.021 5.8±0.01 5.82±0.015 5.34±0.015 5.30±0.009 5.32±0.021

Hemoglobin (g/L) 115.28±2.06 112.19±1.29 113.43±1.46 106.91±1.04 104.26±0.98 105.48±0.86

Packed cell volume

(L/L) 0.374±0.002 0.37±0.003 0.368±0.002 0.349±0.002 0.343±0.003 0.343±0.003

Mean cell volume L/L 60.99±1.072 58.29±0.756 57.773±1.349 64.733±0.635 62.52±0.764 62.793±0.589

*HS for hydrogel dispersion *POM for polyoxometalate

100

3.6.4 Histopathologic study

The representative histopathology slides photographs of the different rabbit

organs treated with hydrogel dispersion, polyoxometalate oral solution and distilled water

(control) including heart, lung, kidney, spleen, liver are shown in figure 3.45–3.49. Both

the hydrogel dispersion and POM solution had no significant histopathologic effect on

heart, liver, kidney, lung, spleen and liver of the rabbit model in comparison with the

control group.

Figure 3.45 describes the microscopic image of the hydrogel dispersion treated group,

polyoxometalate treated group heart muscle as compared to the control group showing

normally ordered cardiac myocytes. There was no effect on pericardium, myocardium

and endocardium. No hypertrophy of the cardiac muscle was observed.

Fig 3.45 A) Histopathalogy photographs of heart muscle of control group rabbit B)

hydrogel dispersion treated rabbit and C) POM treated rabbits.

Figure 3.46 exhibits the microscopic images of the rabbit liver treated with hydrogel

dispersion and polyoxometalate oral solution compared with the control group. There was

no apparent liver necrosis and degeneration in both treatment groups. There was no

neutrophils, macrophages, lymphocytes infiltration and hyperemia, hypertrophy on

hepatic sinusoid. The hepatic lobules were in arranged form and the hepatic cord was

observed in good order.

101

Fig 3.46 A) Histopathology photographs of liver of control group rabbit B) hydrogel

dispersion treated rabbit and C) POM treated rabbit.

Figure 3.47 shows the normal spleen corpuscle structure revealing no gross pathologic

changes in red pulp, white pulp and spleen sinus.

Fig 3.47 A) Histopathalogy photographs of spleen of control group rabbit B) hydrogel

dispersion treated rabbit and C) POM treated rabbits.

Figure 3.48 shows the morphological structures of the lung tissue after receiving

hydrogel dispersion treatment and polyoxometalate oral solution treatment compared

with the normal control group. There was no significant abnormality in the treatment

group of rabbits. Both the treatment groups showed no alveolar or bronchioles collapse

and there was no infiltration of the inflammatory cells surrounding the bronchus. The

cilia of the airways were seen normal.

102

Fig 3.48 A) Histopathalogy photographs of lung of control group rabbit B) hydrogel


The microscopic images of the rabbit kidneys of the control group and both treatment

groups are shown in Figure 3.49. The nephron shape was normal revealing defined space

around glomerulus. There was no bleeding, necrosis and degeneration in various kidney

tubes and glomerulus. Shape of the adrenal cortex and adrenal medulla were normal.

Fig 3.49 A) Histopathology photographs of kidney of control group rabbit B) hydrogel


103

Chapter 4

4 Discussion

104

Effect of gelatin content on swelling and P2W15 release 4.1

In a series of physically cross–linked acrylic acid–gelatin–POM supramolecular

hydrogels, three formulations with different concentrations of gelatin (GP1, GP2, and

GP3) were prepared keeping ratios of acrylic acid, POM and initiator constant. Swelling

and release studies were conducted at pH 1.2 and pH 7.4 using 0.1M HCl buffer and 0.2

M phosphate buffers respectively. Effect of gelatin ratio on swelling behavior of

formulations is opposite of the acrylic acid ratio. Dynamic swelling coefficient of

formulations having increased gelatin ratio decreases at pH 7.4 while it increases at pH

1.2. Isoelectric point of gelatin plays important roles both in swelling as well as in release

of drug from gelatin based polymeric formulations (Samal et al., 2012). Gelatin chains

stays protonated at pH below isoelectric point possessing NH3+ ions responsible for

cationic repulsions and creation of intermolecular spaces leading to high swelling, same

results were also revealed by other hydrogels. (Curcio et al., 2013) Extent of swelling has

direct impact on percent release of drug. Increased concentration of gelatin increases the

release of (P2W15) at low pH because of the increased swelling and decreases at high pH

because of decreased swelling at this pH, corresponding with the results obtained by

Burugapalli et al. 2010 (Burugapalli et al., 2001) while studying the thermal and

swelling behaviors of the gelatin and polyacrylic acid interpenetrating polymeric

networks.

Effect of polyethyleneimine content on swelling and P2W15 release 4.2

Out of all different formulations three formulations with sample codes PEP1,

PEP2 and PEP3 were formulated with different polyethyleneimine content ratios keeping

other contents ratios constant. Swellability index and release properties of the prepared

formulations were evaluated at low pH 1.2 and high pH 7.4 for the provision of the

respective gastric fluid and intestinal fluid simulation. At low pH with increasing

concentration of the polyethyleneimine the swelling as well as encapsulated P2W15

release increases (El-Din & El-Naggar, 2012). At low pH the amino groups present in the

polymer backbone are protonated resulting in repulsive forces creating voids and hence

increase swelling as well as P2W15 release. At high pH the swelling and P2W15 release

decreases as we increase the polyethyleneimine content in the respective three

105

formulations. The network formed as a result of interaction of acrylic acid with

polyethyleneimine behave like a polyampholyte as both are pH responsive so it must

have some isoelectric point. Therefore the pH value has a great effect on swelling and

subsequent release of encapsulated drug from polyethyleneimine and acrylic acid

hydrogels. The P2W15 release from polyethyleneimine and acrylic acid based

supramolecular hydrogels was markedly pH dependent with the highest amount of drug

released at pH 7.4, simulated with intestinal fluid and less drug released at simulated

gastric fluid pH 1.2. Results of the study were similar to the findings of various other

studies regarding controlled release behavior of the polyethyleneimine based systems

(Zhao et al., 2015).

Effect of CMCh content on swelling and P2W15 release 4.3

In a series of formulations, three formulations (CMCP1, CMCP2, and CMCP3)

were prepared having changed concentration of the CMCh keeping other content ratios

constant. CMCh possessed both carboxylic and amino groups in its structure so it

responds to both low and high pH conditions. The network formed as a result of a CMCh

interaction process, both oppositely charged groups get mobilized on exposure to

respective pH conditions. Their swelling and release at two different pH (low and high)

was directly dependent on the amount of CMCh incorporation. Swelling and P2W15

release was increased as the amount of CMCh was increased. At low pH, high amount of

amino groups is protonated resulting in expansion of the gel network leading to high

swelling as well as release. While, at high pH a high amount of unprotonated carboxyl

groups are present in the gel network causing the repulsion forces and increased osmotic

pressure (Yadav & Shivakumar, 2012). These repulsive forces are in turn responsible for

high swelling and the release of the encapsulated P2W15. Therefore, as we increase the

amount of CMCh, respective ionic groups get dominant both at low and high pH values

exhibiting subsequent high swelling and release. Our obtained results were in accordance

with the study of Zhou et al., 2012 designed for the ornidazole targeted delivery to colon

previously and other conducted studies as well. (Yadav & Shivakumar, 2012; Vaghani et

al., 2012; Y. Zhou et al., 2012). The maximum amount of POM released at pH 1.2 was

39%, while at pH 7.4, around 71% of the POM was released.

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Effect of ChCl content on swelling and P2W15 release 4.4

Chitosan hydrochloride (ChCl) is the water soluble derivative of chitosan

possessing amino groups in its skeleton. Three formulations with sample codes CHCP1,

CHCP2 and CHCP3 were prepared having changed concentration of the ChCl. As the

concentration of the ChCl increases, concentration of amino groups also increases

responding very smartly to the respective variations in surrounding pH values. With the

increase in concentration of ChCl in developed hydrogel formulations, the swelling and

P2W15 release increases accordingly at low pH because of the chain relaxation

phenomenon due to repulsions of the amino groups leading to enhanced solvent diffusion

inside hydrogel network. At high pH 7.4 although the swelling and release is much

significant but with the increase in polymer concentration the hydrogel undergoes

collapsing phenomenon, hence comparatively less solvent diffusion inside and gradually

decreased swelling and P2W15 release. Same response of the chitosan content in both high

and low pH values media is also reported by (Paloma et al., 2003) while studying the

chitosan and polyacrylic acid interpolymer complexes. Dergunov 2005 and Rao et al.,

2006 also reported the same results while investigating the chitosan based pH sensitive

networks for the controlled delivery of the encapsulated agents like cefadroxil.

Effect of acrylic acid content on swelling and P2W15 release 4.5

Two hydrogel formulations i.e. acrylic acid–gelatin–POM and acrylic acid–PEI–

POM were prepared. In both of these formulations acrylic acid content has played vital

role regarding swelling and release properties of the hydrogel systems. In the

forthcoming paragraphs effect of acrylic acid content on swelling and release parameters

of both formulations is discussed jointly.

To check the effect of acrylic acid ratio on swelling and release of the

encapsulated POM from acrylic acid-gelatin-POM hydrogels, three formulations GP4,

GP5, and GP6 were prepared. The sensitive response of hydrogels to pH variation is

likely because of the carboxylic group hydrophilicity present in the hydrogel structure.

(Amin et al., 2012). When pH increases, carboxylic group is deprotonated to carboxylate

ion bearing negative charge (da Silva & de Oliveira, 2007), resulting in electrostatic

repulsions causing the swelling of hydrogels (Akala et al., 1998). At higher pH, the

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restricted interaction of the acid molecules with water molecules and carboxylic group

non–ionized condition results in less swelling ratios. (Chauhan & Chauhan, 2008; Halib

et al., 2010) Cumulative percent release of P2W15 was less at pH 1.2 than the pH 7.4

which was due to the less swelling of hydrogel. Formulation GP6 having highest amount

of acrylic acid was showing highest swelling and highest P2W15 release at pH 7.4

showing greater pH sensitivity. In a study designed by (Elliot et al., 2004) it is reported

in the same way that as we increase the concentration of acrylic acid in polymerization

reaction subsequently swelling also increases and same results were also produced by

(Changez et al., 2003). Highest swelling and P2W15 release at pH 7.4 can be attributed to

the deprotonation phenomenon of the carboxyl group (COOH) to (COO)– resulting in the

generation of repulsive forces creating spaces. Voids produced then result in high

swelling as well as high POM release from the hydrogel.

Three formulations PEP4, PEP5 and PEP6 having varied acrylic acid content

were evaluated to observe the effect of acrylic acid on release behavior of the

encapsulated POM and swelling properties of the AA-PEI-POM hydrogel system. In the

current study as the nature of monomer (acrylic acid) is ionic, responding very smartly to

the respective variations in pH so it will influence the capacity of hydrogel swelling as

well as release of the POM. The responsive behavior of the hydrogels to variations in pH

is because of the hydrophilic response of the various functional groups like carboxylic

group present inside hydrogel structure (Amin et al., 2012). With the increase in pH,

negatively charged carboxylate ions are produced due to deprotonation of the carboxylic

group (da Silva & de Oliveira, 2007) resulting in hydrogel swelling due to electrostatic

repulsions (Akala et al., 1998). The limited interaction of water molecules with the acid

molecules at low pH and non–ionized condition of the carboxylic group results in

significantly less swelling ratios (Chauhan & Chauhan, 2008; Halib et al., 2010) . At

simulated gastric fluid pH 1.2 the percent P2W15 release was less due to less swelling and

at simulated intestinal fluid pH 7.4 the swelling and release were high because the pKa of

the carboxylic acid is about 4.5 and so the respective dissociation occurs at high pH value

of intestinal fluid (Nesrinne & Djamel, 2013). Increase in acrylic acid ratio during

polymerization reaction increases the subsequent swelling and release of the encapsulated

drug candidates as reported by (Changez et al., 2003) and (Elliott et al., 2004) .Therefore

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with the increase in the acrylic acid content more carboxylic groups will be deprotonated

creating voids due to electrostatic repulsive forces between the negatively charged

carboxylate ions at higher pH resulting in enhanced swelling and subsequent

encapsulated P2W15 release from the polymeric networks in hydrogel. At pH 1.2 there

was no significant swelling and POM release because of the hydrogel collapsing

phenomenon while at pH 7.4 as the voids are produced due to electrostatic repulsive

forces so there was significant swelling as well as encapsulated POM release (78%).

Effect of methacrylic acid content on swelling and P2W15 release 4.6

Two hydrogel formulations i.e. methacrylic acid–CMCh–POM and methacrylic

acid–ChCl–POM were prepared. In both of these formulations methacrylic acid content

has played vital role regarding swelling and release properties of the hydrogel network. In

the forthcoming paragraphs effect of methacrylic acid content on swelling and release

parameters of both formulations is discussed jointly.

To evaluate the effect of methacrylic acid concentration on swelling and POM

release, three formulations (CHCP4, CHCP5 and CHCP6) were prepared. With increase

in MAA concentration in formulations, grafting percentage on backbone of chitosan

derivatives also increases due to the presence of higher number of MAA molecules in the

vicinity of immobilized CMCh macro–radicals (El-Tahlawy, 2006). The incorporation of

increased concentration of MAA in the reaction mixture potentially increased the density,

degree of cross–linking and number of effective elastic polymer chains of resulting

xerogels (Panic et al., 2010). Due to the ionic nature of MAA it responds efficiently to

the respective variations in surrounding pH value. At low pH hydrogels exhibit less

swelling because of the collapsing phenomenon and protonation of carboxylic groups

resulting in the generation of less osmotic pressure, hence less swelling and subsequent

P2W15 release. As the pH of the medium exceeds the pKa value of the MAA, the

equilibrium water content inside hydrogel network increases turning the polymeric

network into more hydrophilic one. At high pH the carboxylic groups are present in more

ionized form because of the deprotonation phenomenon. Meanwhile, electrostatic

repulsive forces between negatively charged carboxylic groups leading to the generation

of increased osmotic pressure and voids inside the polymer network. Therefore, most

water molecules go inside the polymer network resulting in enhanced swelling and

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encapsulated P2W15 release. The hydrogels having changed concentration of MAA

showed minimum swelling and POM release at pH 1.2 while the maximum at pH 7.4.

Garcia et al., 2004 reported the same swelling behavior of the polymethacrylic acid

hydrogels designed for the delivery of metoclopramide (Garcıa et al., 2004). Similarly in

other studies it is also reported in the same way that polyelectrolyte complexes exhibit

significant swelling and drug release in weak basic conditions ( Yao et al., 1996;

Milosavljević et al., 2011).

Effect of POM content on swelling and in–vitro release 4.7

The mechanism of hydrogel formation between POM and cationic polymer is

through electrostatic interaction between the oppositely charged moieties. Cationic

polymers like PEI, ChCl, CMCh and gelatin bears primary, secondary and tertiary amino

groups in its skeleton possessing charged nitrogen atoms imparting the cationic

functionality (Samal et al., 2012). POMs, are negatively charged and hence strongly

bonded to cationic polymer skeleton (Kim & Park, 2004). In 2015 Pandeya et al. and in

our previous papers same mechanism of hydrogel formation between cationic

carboxymethyl chitosan, gelatin and POMs through electrostatic interactions is also

reported (Pandya et al., 2015).

In a series of acrylic acid–gelatin–POM based developed hydrogel formulations,

three formulations (GP7, GP8, and GP9) were having different concentrations of (P2W15).

Encapsulated (P2W15) here acts as a cross linking density enhancer because of the

electrostatic interaction between the polycationic polymer and the POM (P2W15). The

formulation got optimized at specific concentration of the (P2W15) which was safe from

the toxicity point of view, as the dose administered to the rats while studying the

pharmacokinetics of antiviral POMs (Ni et al., 1996). In last formulation (GP9) there is

the highest concentration of (P2W15) so consequently there was minimum swelling and

release from the formulation. From the GP9 formulation 57%, 59% from the GP8 and

63% POM release was observed from GP7.

To evaluate the effect of POM concentration on acrylic acid–PEI–POM hydrogel

network swelling and release profile, three formulations (PEP7, PEP8, and PEP9) were

prepared having changed concentration of the POM content. In a formulation having

highest amount of POM there was a strong electrostatic interaction. The effect of POM

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content variation on swelling and release was significant supporting the statement that

cross–linking density play a major role in swellability and release characteristics of the

hydrogels (Han et al., 2009). With the increasing concentration of the POM content the

swelling and P2W15 release decreases because of the overall increase in cross–linking

density of the developed hydrogel formulations. Again the toxicity of the POM was

considered while formulating the hydrogels on specific concentration of POM which on

one side was addressed by stabilizing POM inside polyethyleneimine through organic

functionalization (Boglio et al., 2008; Dolbecq et al., 2010; Hu et al., 2012; Meißner et

al., 2006) and on another side, safe concentration of the POM was used as the dose

administered by Ni et al., 1996 to the rates for investigating the pharmacokinetic

parameters of antiviral polyoxometalates.

In MAA–CMCh–POM based hydrogel network, to check the effect of POM

content on swelling and itself POM release of hydrogel network, again three different

formulations (CMCP7, CMCP8 and CMCP9) were prepared having changed

concentration of POM. The POMs and polycations are linked through electrostatic

interactions between the oppositely charged moieties. Therefore as we increase the

concentration of POM in formulation. The electrostatic interaction increases and as a

result cross–linking density increases. Increase in cross–linking density further effects the

swellability index and release profile of the hydrogel network supporting the statement of

Han et al., 2009 that cross–linking density play a major role in the swelling and release

characteristics of the hydrogel systems (Han et al., 2009). Same mechanism of hydrogel

formation is also reported by Pandya et al., 2015 between the carboxymethyl chitosan

and POM with the only addition of the methacrylic acid grafting on polymer backbone

for inducing pH responsiveness as evident from FTIR data. The swelling and release of

the developed hydrogel networks decreases both and low and high pH values with the

increasing POM concentration. Maximum of 30% POM was released at pH 1.2 by

formulation having comparatively less POM content while at pH 7.4 74% of the POM

release was observed. A worth mentioning point here is the concentration of the POM

which was kept less than its toxic level as suggested by a study designed for the

evaluation of the pharmacokinetic parameters of antiviral polyoxometalates in rates (Ni et

al., 1996) .

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As already mentioned, the developed hydrogel system was prepared by using

non–covalent interactions between the different moieties forming a superstructure

through self–healing phenomenon. As POM is highly anionic specie, hence strongly

bounded to the cationic polymer backbone. The bonding of the POM with the polymer

backbone results in increase in the compactness and cross–linking density of the

superstructure. Therefore to assess the effect of POM concentration on the swelling and

POM release itself, three formulations (CHCP7, CHCP8 and CHCP9) were developed

having changed concentration of the POM. With the increase in POM concentration the

swelling and release decreases both at low and high pH values suggesting that increases

in cross–linking density results in decreased swelling as well as release. Cross–linking

density plays a major role in keeping the polymer chains in either relaxed or compact

form. In case of low cross–linking density the polymer chains are in relaxed form

resulting in enhanced penetration of the solvent or dissolution medium causing the

expansion of the gel network, hence increased swelling and POM release. However if the

cross–linking density is high then there is limited interaction of the medium molecules,

hence less solvent penetration and subsequent less swelling and release profile.

Maximum of 16% release was observed at low pH and 77% at high pH for CHCP7

formulation having less concentration of the POM.

Polyanion release Kinetics 4.8

While formulating a delivery system, some factors ought to be considered like

drug diffusion, aqueous solubility of drug molecule and polymer matrix erosion

capabilities. The drug release from biodegradable polymers usually results from diffusion

and later on erosion of polymer matrix (von Burkersroda et al., 2002). Different

mathematical models like zero order, first order, Higuchi and Peppas models were

applied to find the best fit regression (R2) value closer to 1. As value of n (slope of

Peppas model) in all of the formulations is > 1, hence it follows super case transport–II

mechanism of release involving polymer erosion and diffusion processes (Costa & Lobo,

2001). The R2

value of zero order in all formulations was closer to 1, hence POM release

follows zero order kinetics.

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Characterization 4.9

4.9.1 FTIR spectroscopy

To confirm the functional group interactions inside the developed hydrogel

network, FTIR spectroscopy was performed giving the particular absorption bands

produced as result of stretching and bending etc. vibrations and were compared with the

individual hydrogel components i.e. polymer, monomer and POM. In the coming

paragraphs the FTIR spectroscopy analysis results for all developed hydrogel

formulations are discussed.

4.9.1.1 FTIR Spectroscopy analysis of pH responsive gelatin–POM hydrogels

In gelatin spectra, wide absorption band at 3279 cm–1

is due to stretching of N–H

bond connected to O–H bond. (Yu & Xiao, 2008) Absorption Peaks at 1626 cm–1

and

1520 cm–1

are because of amide–I and amide–II of gelatin respectively. (Zhu & Ding,

2006) Peak present at 1230 cm–1

is due to stretching vibration of C–N bond while peaks

at 3066 cm–1

and 2865 cm–1

are due to stretching of C–H bond in CH3 and CH2 groups. In

FTIR spectra of acrylic acid the absorption peak at 2988cm–1

refers to O–H stretching

and absorption band recorded at 1696 cm–1

is attributed to C=O stretching.(Tang et

al.,2009) Peak recorded at 1568cm–1

represents bending of C=O of COOH group. Peak

observed at 1294 cm–1

shows C–C stretching while C–O stretching is shown by peak

present at 1183 cm–1

. Appearance of new modified absorption peak in hydrogel FTIR

spectra at 2930 cm–1

corresponds to the successful development of interaction between

gelatin and acrylic acid. In POM salt FTIR spectra, the characteristic region (1000–500

cm–1

) is called ―finger print‖ because the absorptions occur here due to oxygen–metal

stretching vibrations. As compare to the simple FTIR spectra of the POM, there are some

characteristic bands appeared in the region of 1000–500 cm–1

showing the successful

development of the hydrogel between gelatin and POM P2W15. (Shah et al., 2014)

4.9.1.2 FTIR Spectroscopy analysis of pH responsive PEI–POM hydrogels

The absorption peak at 1109 cm–1

refers to the secondary amines present in the

PEI skeleton (Paciello & Santonicola, 2015a). Absorbance band at 1458 cm–1

indicates

N–H bending of secondary amines and –CH2 vibrations (Pang et al., 2011; Wang & Li,

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2015). Absorption peak at 1647 cm–1

shows N–H bending vibrations of primary amines

(Paciello & Santonicola, 2015b; Tan et al., 2007). In acrylic acid FTIR spectra the

absorption band at 2988 cm–1

is attributed to O–H stretching and absorption peak at 1696

cm–1

refers to C=O stretching (Tang et al., 2009). Band recorded at 1568cm–1

shows

bending of acrylic acid (C=O) carboxyl group. Peak recorded at 1294 cm–1

present C–C

stretching while peak observed at 1183 cm–1

shows C–O stretching. In FTIR spectra of

hydrogel the peak observed at 1697 cm–1

is attributed to N–H bending vibrations. Peak

recorded at 1449 cm–1

refers to deformation of C–H group (El-Din & El-Naggar, 2012;

Paciello & Santonicola, 2015a). In comparison with the normal polyethyleneimine

spectra the peaks recorded at 1162 cm–1

and 1115 cm–1

shows successful interaction of

polyethyleneimine (Paciello & Santonicola, 2015a). Appearance of modified absorbance

band at 2925 cm–1

up to 3400 cm–1

shows cross–linking between polyethyleneimine and

acrylic acid. Absorptions in the POM FTIR characteristic region of (1000–500 cm–1

) is

the finger print of the polyoxometalates and is because of the stretching vibrations of the

oxygen and metal atoms. In comparison with the normal FTIR spectra of the POM,

appearance of some new specific absorption peaks in the 1000–500 cm–1

region shows

successful interaction between cationic polyethyleneimine and POM P2W15 (Shah et al.,

2014)

4.9.1.3 FTIR Spectroscopy analysis of pH responsive CMCh–POM hydrogels

In CMCh FTIR spectra the Peak recorded at 1407 cm–1

can be attributed to

symmetrical –COO–

stretching vibrations (Sun et al., 2003). The characteristic absorption

band present at 1585 cm–1

shows anti–symmetrical stretch of –COO– associated

carboxylic acid salt, suggesting the presence of carboxymethyl groups. C–O stretch of

primary alcohols is shown by the absorption band at 1046 cm–1

(Chen et al., 2004).

Absorption at 1307 cm–1

can be attributed to the extension vibration of the C–O group.

Out of plane N–H bending vibration present in primary amino group is shown by an

absorption band recorded at 895 cm–1

( Chen et al., 2009; Y. Chen & Tan, 2006). Peak

observed at 3362 cm–1

shows –O–H stretching while peak observed at 2871 cm–1

shows –

C–H stretch. In case of MAA, FTIR spectra the absorption band recorded at 1633 cm–1

shows specific peak of vinyl monomers (Bai et al.,2007). Wider band at 2962 cm–1

is due

114

to intermolecular hydrogen bonding. Presence of bands at 1557–1201 cm

–1 can be

attributed to –C–O and –C–H vibrations (De Vasconcelos et al., 2006; Sun et al., 2003)

while absorption band at 1685 cm–1

is due to carboxylic group C=O stretching vibration.

In POM FTIR spectra, the characteristic region (1000–500 cm–1

) is termed as ―finger

print‖ as the absorptions occurring here are due to oxygen–metal atom stretching

vibrations. Following IR characteristics were observed for POM in association with the

finger print region. Peak recorded at 1083 cm–1

shows P–O stretching while W–O

terminal stretching is shown by absorption band at 977 cm–1

. Absorption band recorded at

861 cm–1

can be attributed to W–O–W inter–bridges stretching while peak present at 805

cm–1

is due to W–O–W intra–bridges stretching (Abia & Ozer, 2013). In hydrogel FT–IR

spectra there is characteristic absorption band at 1161 cm–1

. Other specific peaks at 1481

cm–1

, 1388 cm–1

and 1256 cm–1

are from MAA (Sun et al., 2003). The band recorded at

1537 cm–1

can be assigned to the ionic interaction between methacrylic acid and CMCh

(Milosavljević et al., 2010). In comparison with normal POM FTIR spectra the

distinctive bands present in the region of the 1000–500 cm–1

displays the successful ionic

interaction between POM and CMCh (Shah et al., 2014).

4.9.1.4 FTIR Spectroscopy analysis of pH responsive ChCl–POM hydrogels

In ChCl FTIR spectra the absorption band recorded at 1614 cm–1

can be attributed

to primary amides and the peaks present at 1582 and 1316 cm–1

can be assigned to the

secondary and tertiary amides present in the ChCl skeleton (Cai et al., 2013). Absorption

peak present at 1393, 1138 cm–1

represents the –CH3 group bending vibration and –CH

group stretching vibrations respectively and the peak recorded at 1092 cm–1

is due to the

C–O group vibrations (Zhao‐Sheng et al., 2012).N–H and O–H groups stretching

vibrations are denoted at the absorption band recorded at 3283 cm–1

(Zhao‐Sheng et al.,

2012). In case of methacrylic acid FTIR spectra the absorption band recorded at 1633

cm–1

shows specific peak of the vinyl monomers (Bai et al., 2007). Wider band at 2962

cm–1

is due to intermolecular hydrogen bonding. Presence of bands at 1557 cm–1

to 1201

cm–1

can be attributed to –C–O and –C–H vibrations (Sun et al., 2003), while absorption

band at 1685 cm–1

is due to carboxylic group C=O stretching vibration. In POM salt FT–

IR spectra, the characteristic region (1000–500 cm–1

) is termed as ―finger print‖ as the

115

absorptions occurring here are due to oxygen–metal atom stretching vibrations.

Following IR characteristics were observed for POM salt in association with finger print

region. Peak recorded at 1083 cm–1

shows P–O stretching while W–O terminal stretching

is shown by absorption band at 977 cm–1

. Absorption band recorded at 861 cm–1

can be

attributed to W–O–W inter–bridges stretching while peak present at 805 cm–1

is due to

W–O–W intra–bridges stretching (Abia & Ozer, 2013). In hydrogel FTIR spectra in

association with other peaks the peaks recorded at 1541 cm–1

is due to the ionic

interaction between MAA and ChCl (Milosavljević et al., 2010). The modified absorption

bands in the region of 1000–500 cm–1

represents the successful interaction of the ChCl

with the POM as here unlike ChCl and POM FTIR spectra‘s some modified bands are

present (Shah et al., 2014).

4.9.2 Scanning electron microscopy

Morphological evaluation of the micro structured hydrogels as well as in intact

form is one of the important property of hydrogel to be considered and evaluated [Sadegi

& Hosseinzadeh, 2010]. The SEM results of the different pH responsive formulations

prepared for the controlled delivery of the encapsulated POM showed glassy smooth

surface morphology of both intact hydrogel discs as well as cross–sectional view

confirming the uniform distribution of the POM inside hydrogel network.

4.9.3 Thermal analysis

Thermal analysis including Thermogravimetric analysis and differential scanning

calorimetry were performed to evaluate the mass in loss due to water molecules

evaporation, decomposition of the material with the increasing temperature and the

expected thermal transitions inside hydrogel network and its components.

Thermogravimetric and differential scanning calorimetry results of the prepared pH

responsive hydrogel formulations are discussed below.

4.9.3.1 Thermal analysis of the gelatin–POM pH responsive supramolecular

hydrogels

In gelatin TGA plot normally there are three stages i.e. water loss at 25 to 100oC,

decomposition of gelatin from 250 to 450°C and combustion of the residual material at

450oC to 700

oC. (Bertoldo et al., 2007; Panzavolta et al., 2009) TGA plot of gelatin is

116

compared with the one of cross–linked gelatin (hydrogel) in a temperature range starting

from 0oC to 700

oC. Both the samples exhibited the thermal phenomenon like loss of

water and material decomposition. Gelatin TGA plot shows the 8% water loss up to 80oC

while in case of hydrogel the water loss of 6% occurred at 170oC showing that the

hydrogel system impedes the water content removal. In TGA plot of the POM salt 4%

water loss occurs up to 100oC and after 100 to 300

oC the crystallized water is lost

gradually by the POM salt which contributes to 6% of the total weight. After 300oC there

may be the process of phase change from α to β as there is heat change at 450oC. In case

of the hydrogel spectra it can be seen that the water loss occurs up to 170oC and the

degradation of the hydrogel occurs up to 600oC showing the thermal stability of hydrogel.

DSC analysis were also performed for the purpose of identification of thermal

transitions in hydrogel network. In comparison with the other polymers, the biopolymers

including gelatin thermal characterization are not always straightforward and depend

largely on the polymer source and thermal history of the material. Therefore no sole

value is reported in literature (Bigi et al., 2001; Chiou et al., 2008; Rahman et al., 2008) ,

but the non–cross–linked gelatin DSC spectrum displays generally an endothermic peak

which is associated with the helix–coil transition. Two endothermic peaks in gelatin DSC

spectra are present in the range of 50 to 90oC and 250 to 300

oC indicating water loss and

decomposition respectively. From POM salt DSC spectra it is evident that the first

endothermic peak at 100oC shows water loss while second at 410

oC shows the

decomposition or the phase inversion phenomena from α to β. In case of hydrogel the

first endothermic peak at 220oC shows water loss and second at 420

oC dictates

decomposition showing that thermal stability of the hydrogel get increased as a result of

cross–linking between gelatin– P2W15 complexes.

4.9.3.2 Thermal analysis of the PEI–POM pH responsive supramolecular

hydrogels

The thermal behavior and stability of the developed hydrogel formulation were

checked by performing thermogravimetric analysis and differential scanning calorimetry

(DSC). In case of polyethyleneimine the glass transition temperature increases with the

degree of substitution. Polyethyleneimine presents two stage weight loss in the range of

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50oC to 600

oC with the first stage at 50

oC to 161.5

oC with the complete decomposition up

to 400oC (Wang et al., 2012a). In the thermogram of the POM it is evident 4% water loss

occurs up to 100oC and after 100 to 300

oC the crystallized water is lost gradually by the

POM salt contributing 6% of the total weight. After 300oC there may be the process of

phase change from α to β as there is heat change at 450oC. From the thermogram of the

hydrogel it is evident that there is multi–stage decomposition process with initial weight

loss at 180oC because of water evaporation and major loss in mass occurs between 300

oC

to 500oC which could be attributed to the degradation of polymer backbone (Arabi et al.,

2016) showing that developed hydrogel have enhanced thermal stability than its

components.

4.9.3.3 Thermal analysis of the CMCh–POM pH responsive supramolecular

hydrogels

The TGA plot of CMCh exhibits two stage weight loss. At the first stage about

7% CMCh weight were lost that was started at 60 °C (Milosavljević et al., 2010). In the

second stage about 35% CMCh weight were lost that was started in the range of 240–290

°C, involving saccharide ring dehydration and breakage of glycosidic bond (C–O–C) in

main polysaccharide chain (Chen & Tan, 2006; Neto et al., 2005). In TGA plot of the

POM salt 4% water loss occurs up to 100°C and after 100–300°C the crystallized water is

lost gradually by the POM salt which contributes to 6% of the total weight. After 300°C

there may be the process of phase change from α to β as there is heat change at 450°C. In

hydrogel the first stage of weight loss occurs up to 200 °C contributing 16% weight loss.

While, second phase of weight loss occurs at 270–390 °

C contributing 30% loss in

weight.

In DSC curve of CMCh, endothermic peak present at 70°C shows water loss

(Guinesi & Cavalheiro, 2006). Glucosamine units present in the backbone of the polymer

undergo decomposition giving an exothermic peak at 290°C (Kittur et al., 2002). From

POM salt DSC spectra it is evident that the first endothermic peak at 100°C shows water

loss while second at 410°C shows decomposition or phase inversion phenomena from α

to β. In hydrogel DSC curve first endothermic peak is evident at 240°C and second at

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410°C showing the decomposition of the hydrogel network with the complete

decomposition up to 500 °C showing the enhanced thermal stability of the hydrogel

network.

4.9.3.4 Thermal analysis of the ChCl–POM pH responsive supramolecular

hydrogels

Thermal analysis of the ChCl, MAA, hydrogel and encapsulated POM were

performed to evaluate the mass in loss and thermal transitions in developed hydrogel

network and its components with the increasing temperature. The TGA plot of ChCl

shows a major loss in mass at about 180oC contributing 45% loss in mass due to the

evaporation of water bound molecules. After this the main skeleton of the polymer

undergoes gradual degradation process with the cleavage of the glycosidic bonds up to its

final conversion into residues and the findings were similar to the analysis of (Neto et al.,

2005). In TGA plot of the POM 4% water loss occurs up to 100oC and after 100 to 300

oC

the crystallized water is lost gradually by the POM which contributes to 6% of the total

weight. After 300oC there may be the process of phase change from α to β as there is heat

change at 450oC. In case of hydrogel or cross–linked chitosan skeleton showed three

stages of weight loss. The first stage occurring at 200oC is due to the evaporation of

bound water molecules. The second stage present at 260oC can be assigned to the

degradation of saccharide units attached through glycoside bonds. Third stage at 370oC is

due to the complete decomposition of the chitosan basic structural nucleus and its

conversion into residues.

Thermal transitions were also evaluated for the MAA–ChCl–POM hydrogel and

its components by the technique of differential scanning calorimetry. Pure sample of

ChCl exhibited a sharp endothermic peak at 220oC due to evaporation of the water

molecules bounded to –NH2 and –OH groups of the chitosan structure and the finding is

in exact accordance with the results of Sohail et al., 2015 while studying the controlled

delivery of valsartan using chitosan matrices (Sohail et al., 2015). From POM salt DSC

spectra it is evident that the first endothermic peak at 100oC shows water loss while

second at 410oC shows the decomposition or the phase inversion phenomena from α to β.

As compare to non–cross–linked chitosan cross–linked chitosan exhibits three stages of

119

thermal transitions. The first thermal transition recorded at 260oC is due to the

evaporation of the bound water molecules present inside hydrogel network. Second and

thermal shifts recorded at 390oC and 440

oC are due to the decomposition of the

saccharide structural units and glycosidic bonds cleavage with the complete

decomposition of the hydrogel network up to 600oC. The so observed thermal transition

pattern of the cross–linked chitosan (MAA–ChCl–POM hydrogel) was in exact

accordance with the (Neto et al., 2005) .

4.9.4 Powder X–ray diffraction

To evaluate the crystallinity of the prepared hydrogel formulation and its contents

X–ray diffraction was performed. In case of POM XRD pattern there is a characteristic

peak at 2θ equals to 25.91o in association with other small peaks at 2θ ~ 28.73

o and 31

o

showing its crystalline structure. The XRD pattern of the gelatin shows a broad peak at

2θ ~ 20o suggesting its amorphous nature. Developed hydrogel showed a peak at 2θ ~ 70

o

exhibiting its slight crystalline structure after the incorporation of the POM and

successful cross–linking.

In XRD pattern of the pH responsive PEI–POM hydrogel, it can be seen that

beside other small peaks present at 2θ ~ 30o and 35

o there is one intense peak at 2θ equals

to72.52o suggesting that prepared hydrogels exhibit crystalline structure.

The X–ray diffraction pattern of CMCh exhibited a strong peak at 2θ of 19.8°

revealing its semi–crystalline structure (Khan & Ranjha, 2014). In case of POM XRD

pattern there is a characteristic peak at 2θ equals to 25.91° in association with other peaks

at 2θ ~ 28.73° and 31

° showing its crystalline structure. Regarding developed hydrogel

XRD pattern, it can be seen that there is an intense peak recorded at 2θ ~72.61o showing

the crystallinity of developed hydrogel formulation.

ChCl XRD pattern gives major peaks at 2θ ~ 10.161, 5.352, 5.151, 3.697 and

3.538o in association with other small peaks suggesting its crystalline structure. In case of

hydrogel developed as a result of successful physical cross–linking of MAA, ChCl and

POM, a broad peak was recorded at 2θ ~ 15.921o revealing comparatively less crystalline

nature of the prepared hydrogel.

120

Pharmacokinetic parameters 4.10

POMs are the ionic compounds representing non–nucleoside analogs possessing

versatile biological activities and altered pharmacokinetic profiles. Wang et al., (2014)

studied pharmacokinetic behavior of Cs2K4Na[SiW9Nb3O40].H2O in rats and found that

Tmax of the administered POM was ranging from 0.1 to 3 hour and half–life (t1/2) 20–30

hours (Wang et al., 2014). Similarly in another study Ni et al.1996 studied the

pharmacokinetics of antiviral POMs in rats and found that POMs can be given at the

single dose of 50mg/Kg without any toxicity reaching the maximum plasma

concentration in five days (Ni et al., 1996). In the current study the maximum plasma

concentration of POM was achieved in 8 hours for oral POM solution while in case of

hydrogel it was obtained in 18 hours with the complete elimination in two days (48

hours). Maximum plasma concentration (Cmax) calculated for oral POM solution was

1810 µg/ml while in case of hydrogel 1801 µg/ml was observed. Mean retention time

(MRT) recorded for oral solution was 10 hours while for hydrogel it was recorded as 19

hours. Statistical significant difference at 95% confidence interval (p ˂ 0.05) was found

between all pharmacokinetic parameters except Cmax and Vz for which the p-values

were found 0.827 and 0.491 respectively. The comparison of pharmacokinetic data of the

oral POM solution and hydrogel formulation clearly indicates that POM release from

hydrogel formulation followed controlled release pattern.

Acute oral toxicity evaluation and safety evaluation of the MAA–4.11

ChCl–POM hydrogel and POM oral solution

Generally acute toxicity is defined as, the adverse effects occurring in a short time

after the oral administration of single or multiple doses of a substance in 24 hours (Chen

et al., 2008). An important aspect of toxicology studies is the identification of a dose at

which the target article is continuously observed but the dose is not so high that study is

affected by morbidity or mortality. This dose is called maximal tolerated dose (MTD)

because the animal will not then tolerate adverse effects that may occur at higher doses.

By definition MTD is the highest dose that can be tolerated for specific study duration.

Typically MTD is affected by the duration of dosing because the animal might not be

tolerating the consecutive high doses for a long duration. Shorter the duration of a study

121

higher will be the MTD and vice versa (Robinson et al., 2009). All of the serum

chemistry findings were fairly normal except reduction in glucose levels in both hydrogel

dispersion as well as POM groups because of the inhibition of glucose–6–phosphatase

enzyme by POM which is responsible for the hydrolysis of glucose–6–phosphate

resulting in production of free glucose and phosphate group (Stephan et al.,2013). The

hematology data and histopathology profiling findings indicated that hydrogel dispersion

and the concentration of the polyoxometalate used in developing hydrogel systems were

safe from –in vivo point of view.

Cytotoxicity profiling of the developed hydrogels 4.12

Cytotoxicity potential of the encapsulated P2W15 (hydrogel) and free P2W15 was

evaluated through sulphorhodamine B dye (SRB) assay. SRB assay is one the highly

sensitive colorimetric assay determining protein content in subjected cells (Skehan et al.,

1990). Blank wells were kept as negative control while doxorubicin (20, 25, 30 and

35mg/ml) was kept as standard positive control. The cytotoxicity results exhibited that

both encapsulated as well as free P2W15 showed the dose dependent toxicity against

MCF–7 and HeLa cells. The cytotoxic potential of the free P2W15 was higher than

doxorubicin on both cancer as well as normal cells supporting the results of Wang et al.,

2000 & 2003 that POMs have better efficacy than some established anticancer drugs like

cisplatin (Wang et al., 2000; Wang et al., 2003). Encapsulated P2W15 (hydrogel) showed

reduced cytotoxic activity as compare to free POM because of controlled release of POM

from polymeric network. Encapsulated POM showed less toxicity supporting the

statement that the problem of toxicity and lack of selectivity regarding POMs can be

addressed by enclosing and immobilizing it inside polymeric network through organic

functionalization (Boglio et al., 2008; Hu et al., 2012; Meißner et al., 2006).

Conclusion 4.13

From the current study it was concluded that stabilized supramolecular hydrogels

of polyoxometalate were developed through physical cross–linking. The hydrogel

formulations showed maximum swelling and release at pH 7.4 and less at pH 1.2.

Characterization of hydrogel samples through different analytical techniques like FTIR,

122

XRD, thermal analyses (TGA/DSC) and SEM confirmed the successful hydrogel

formation in terms of successful functional group interactions, crystallinity, improved

thermal stability and glassy smooth surface morphology. The prepared hydrogels were

completely safe from in–vivo point of view at maximal tolerable dose of 4000mg/kg body

weight as depicted from histopathological data of different rabbit‘s organs like heart,

kidney, lungs, spleen and liver. Serum chemistry findings suggested that the prepared

hydrogels can be used for treating diabetic conditions as it has shown marked reduction

effect on blood glucose levels of rabbits. Hematological investigations exhibited that both

hydrogel dispersion as well as oral POM solution were completely safe. The

pharmacokinetic profiling of hydrogel encapsulated POM and oral POM solution

indicated that the prepared hydrogels can be used for controlled and targeted delivery of

encapsulated POM. Both free as swell as hydrogel encapsulated POM showed dose

dependent cytotoxicity against both cancerous cell lines (HeLa & MCF-7). Free POM

showed more cytotoxic potential against normal Vero cells as compared to standard

reference anticancer drug doxorubicin which get reduced when POM was sealed and

blocked inside polymeric network (hydrogel) showing that the toxicity issue of POM can

be reduced through organic functionalization in polymer network.

123

Chapter 5

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124

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6 Appendices

Table A1. Swelling data index of pH responsive gelatin-POM formulations at pH 1.2

Time

(h) GP1 GP2 GP3 GP4 GP5 GP6 GP7 GP8 GP9

0 1 1 1 1 1 1 1 1 1

157

0.25 1.24 1.31 1.06 1.23 1.22 1.21 1.09 1.08 1.00

0.5 1.31 1.39 1.23 1.33 1.28 1.24 1.19 1.12 1.09

1 1.39 1.47 1.45 1.40 1.34 1.29 1.25 1.21 1.16

1.5 1.54 1.58 1.62 1.49 1.40 1.34 1.33 1.29 1.20

2 1.65 1.63 1.75 1.61 1.45 1.39 1.48 1.42 1.26

3 1.74 1.72 1.91 1.72 1.50 1.43 1.56 1.51 1.34

4 1.83 1.74 1.94 1.82 1.54 1.49 1.59 1.55 1.39

5 1.90 1.81 2.04 1.92 1.59 1.54 1.69 1.61 1.44

6 1.96 1.87 2.14 1.96 1.64 1.60 1.75 1.66 1.49

7 2.04 2.01 2.25 2.03 1.71 1.65 1.79 1.69 1.55

8 2.07 2.11 2.30 2.09 1.80 1.70 1.80 1.73 1.59

10 2.19 2.34 2.36 2.12 1.91 1.76 1.84 1.79 1.65

12 2.26 2.49 2.45 2.17 1.99 1.84 1.94 1.85 1.71

14 2.33 2.61 2.77 2.20 2.10 1.90 1.99 1.90 1.79

Table A2. Swelling data index of pH responsive gelatin-POM formulations at pH 7.4

Time (h) GP1 GP2 GP3 GP4 GP5 GP6 GP7 GP8 GP9

0 1 1 1 1 1 1 1 1 1

158

0.25 1.24 1.22 1.05 1.24 1.24 1.28 1.24 1.07 1.04

0.5 1.38 1.34 1.31 1.28 1.34 1.42 1.38 1.20 1.14

1 1.55 1.48 1.46 1.37 1.50 1.64 1.53 1.40 1.29

1.5 1.75 1.67 1.69 1.50 1.60 1.91 1.63 1.54 1.42

2 1.94 1.90 1.92 1.60 1.69 2.21 1.90 1.74 1.66

3 2.16 2.16 2.21 1.75 1.91 2.52 2.18 1.95 1.84

4 2.40 2.44 2.54 1.91 2.24 2.86 2.48 2.20 1.98

5 2.58 2.64 2.79 2.10 2.42 3.15 2.57 2.40 2.14

6 2.77 2.90 3.05 2.30 2.63 3.48 2.86 2.60 2.37

7 2.97 3.16 3.32 2.60 2.96 3.76 3.02 2.77 2.51

8 3.13 3.39 3.58 3.00 3.35 4.05 3.29 2.98 2.76

10 3.54 3.87 4.12 3.50 3.97 4.49 3.65 3.36 3.05

12 3.77 4.14 4.43 4.10 4.57 5.19 3.99 3.61 3.29

14 4.12 4.60 4.91 4.70 5.21 5.76 4.35 3.86 3.53

Table A3. Percent in-vitro release data of pH responsive gelatin-POM formulations

at pH 1.2

Time GP1 GP2 GP3 GP4 GP5 GP6 GP7 GP8 GP9

159

(h)

1 0.72 0.80 0.88 0.40 0.32 0.24 0.48 0.37 0.37

2 1.20 1.28 1.92 0.72 0.64 0.48 0.86 0.96 0.96

3 2.00 2.80 2.96 1.28 0.96 1.28 0.96 1.93 1.71

4 2.80 3.99 4.15 2.88 2.24 2.24 1.28 2.99 2.94

5 4.39 5.59 5.19 4.07 3.83 3.99 2.67 4.28 4.39

6 5.19 6.95 6.79 5.27 4.95 5.75 3.32 5.72 5.29

7 6.39 8.15 8.79 6.47 6.31 7.75 4.28 7.06 6.95

8 8.23 9.35 9.98 7.91 8.31 9.03 5.35 9.04 8.77

9 10.22 10.86 11.82 9.35 9.74 10.54 6.68 10.48 9.95

10 11.90 12.54 13.66 10.38 10.70 12.30 8.56 11.87 11.07

11 13.58 14.70 14.94 11.74 11.90 13.82 10.00 12.94 11.34

12 14.54 16.21 15.97 13.42 13.74 14.62 11.18 13.74 12.14

13 15.97 17.09 17.17 15.10 14.54 15.65 12.41 13.85 12.78

14 16.77 17.41 19.17 15.42 15.58 15.89 14.28 13.90 13.37

Table A4 Percent in-vitro release data of pH responsive gelatin-POM formulations

at pH 7.4

Time

(h) GP1 GP2 GP3 GP4 GP5 GP6 GP7 GP8 GP9

160

1 3.75 4.71 3.99 4.79 4.87 5.27 4.06 3.10 2.51

2 6.87 10.86 8.55 9.11 10.22 10.70 7.54 6.63 5.51

3 11.98 15.50 12.54 13.58 15.50 16.21 11.12 10.86 9.73

4 16.13 21.81 16.05 17.17 21.49 22.68 15.51 14.97 13.96

5 19.65 27.16 21.49 22.84 27.32 29.71 20.05 19.73 18.72

6 23.48 32.91 24.92 28.99 32.75 37.54 25.56 23.21 22.99

7 26.60 39.62 29.31 35.70 40.89 44.09 30.27 27.06 26.68

8 32.03 44.17 33.71 40.34 46.65 50.88 35.03 31.55 31.12

9 39.14 48.56 39.54 45.69 54.31 58.31 40.37 35.24 35.72

10 45.37 54.95 45.13 51.92 61.02 64.22 45.19 39.73 39.52

11 51.04 58.31 51.36 58.39 68.53 72.68 50.27 44.22 44.39

12 56.31 61.74 57.19 64.22 72.76 79.31 55.13 50.32 49.47

13 63.02 64.78 60.86 69.09 74.04 82.51 60.21 56.31 53.80

14 68.21 65.73 63.02 69.65 77.88 84.35 63.85 59.04 57.33

Table A5. Swelling data index of pH responsive PEI-POM formulations at pH 1.2

Time

(h) PEP1 PEP2 PEP3 PEP4 PEP5 PEP6 PEP7 PEP8 PEP9

161

0 1 1 1 1 1 1 1 1 1

0.25 1.11 1.20 1.14 1.14 1.09 1.12 1.10 1.08 1.07

0.5 1.29 1.30 1.28 1.31 1.24 1.26 1.23 1.23 1.20

1 1.41 1.42 1.38 1.46 1.35 1.36 1.37 1.32 1.29

1.5 1.56 1.54 1.62 1.55 1.42 1.44 1.49 1.45 1.40

2 1.72 1.64 1.76 1.77 1.53 1.54 1.61 1.53 1.51

3 1.89 1.74 1.91 2.01 1.63 1.66 1.70 1.64 1.62

4 2.00 1.87 2.05 2.21 1.74 1.78 1.82 1.75 1.74

5 2.07 1.99 2.19 2.38 1.83 1.88 1.91 1.91 1.85

6 2.19 2.14 2.31 2.53 1.94 2.01 2.02 2.01 1.90

7 2.33 2.24 2.43 2.65 2.06 2.13 2.08 2.12 2.01

8 2.44 2.35 2.53 2.80 2.15 2.23 2.20 2.23 2.10

10 2.53 2.56 2.68 2.94 2.34 2.37 2.36 2.32 2.20

12 2.66 2.72 2.82 3.47 2.50 2.49 2.49 2.45 2.31

14 2.80 2.84 3.20 3.69 2.62 2.62 2.69 2.62 2.39

16 2.86 3.30 3.58 3.84 2.84 2.80 3.11 2.77 2.50

18 2.93 3.56 4.08 3.99 3.35 3.29 3.37 2.99 2.61

20 2.99 4.01 4.71 4.16 3.68 3.52 3.52 3.27 2.69

Table A6. Swelling data index of pH responsive PEI-POM formulations at pH 7.4

Time


0 1 1 1 1 1 1 1 1 1

162

0.25 1.32 1.24 1.18 1.18 1.20 1.17 1.13 1.09 1.12

0.5 1.51 1.60 1.35 1.38 1.43 1.56 1.31 1.30 1.28

1 1.77 1.93 1.53 1.53 1.69 1.87 1.65 1.66 1.59

1.5 2.08 2.28 1.73 1.68 2.04 2.30 2.08 2.02 1.83

2 2.41 2.68 1.87 1.92 2.47 2.64 2.55 2.21 2.14

3 2.70 3.08 2.08 2.13 3.03 3.59 2.95 2.98 2.48

4 3.90 3.41 2.33 2.43 3.65 4.45 4.12 3.55 2.71

5 5.03 3.57 2.51 2.81 4.38 5.34 4.58 3.99 2.91

6 5.71 3.76 2.65 3.15 4.89 6.21 5.05 4.43 3.18

7 6.42 4.08 2.77 3.34 5.51 6.93 5.50 4.93 3.31

8 6.91 4.35 2.94 3.63 6.04 7.73 5.92 5.50 3.67

10 7.43 4.89 3.22 3.90 6.84 8.72 6.93 6.25 4.11

12 8.42 5.46 3.69 4.45 7.73 10.42 7.87 7.01 4.64

14 9.01 6.34 4.09 5.10 8.57 11.52 8.75 7.71 5.09

16 9.92 6.81 4.70 5.72 9.65 13.14 10.66 8.57 5.55

18 11.30 7.44 5.54 6.58 10.48 14.58 11.56 9.15 6.08

20 11.75 8.07 6.14 7.22 11.52 16.40 13.22 9.83 6.63

Table A7. Percent in-vitro release data of pH responsive PEI-POM formulations at

pH 1.2

Time


1 1.17 1.49 1.49 0.59 0.91 0.59 0.89 0.71 0.46

163

2 2.40 3.67 4.74 1.33 1.97 1.01 1.68 1.39 1.14

3 3.09 6.66 7.19 2.61 3.35 1.54 2.89 2.75 2.21

4 4.53 9.05 10.17 4.31 4.42 2.50 3.78 3.89 2.96

5 6.76 11.18 13.79 5.64 5.11 3.67 6.10 4.96 3.74

6 9.21 13.21 16.29 7.40 6.28 4.53 7.74 6.60 4.71

7 11.24 15.50 18.90 8.63 7.29 5.80 10.16 7.24 5.49

8 13.74 17.15 21.99 9.80 8.09 6.71 13.05 8.52 6.63

9 16.93 18.53 24.39 11.29 9.05 8.04 14.58 9.84 8.45

10 18.48 20.29 27.85 12.94 10.44 9.85 16.29 12.51 9.95

11 20.29 22.47 30.30 14.64 12.09 10.81 17.50 14.69 11.44

12 21.25 25.19 32.32 16.51 12.99 11.93 19.29 15.69 12.05

13 22.42 27.42 34.24 18.90 14.96 12.89 20.86 16.61 13.12

14 23.80 29.07 36.32 20.18 15.97 14.38 22.50 17.79 14.37

16 24.28 29.87 38.07 22.20 17.41 14.96 24.21 18.57 15.26

18 24.49 31.47 40.04 23.70 19.33 16.72 25.49 19.64 17.08

20 24.97 33.01 41.91 25.03 21.09 18.21 27.52 21.35 18.11

Table A8. Percent in-vitro release data of pH responsive PEI-POM formulations at

pH 7.4

Time


164

1 4.31 3.41 2.40 3.30 4.05 4.69 3.53 2.39 2.10

2 7.45 6.44 5.32 6.66 7.83 8.15 7.09 4.92 4.71

3 12.09 10.60 8.09 9.90 11.66 14.43 10.09 9.16 8.16

4 15.28 14.96 12.25 13.90 16.83 19.33 14.65 12.48 11.27

5 21.67 18.58 15.18 16.35 21.62 24.55 18.11 15.15 14.97

6 28.27 23.75 18.32 20.82 26.04 29.66 21.85 18.68 18.15

7 33.60 28.22 21.51 26.36 30.09 35.36 25.99 21.46 21.03

8 38.45 31.95 25.77 30.51 34.56 40.63 29.34 26.06 25.78

9 44.14 38.13 30.72 34.13 38.87 45.31 33.80 29.63 30.20

10 48.94 43.02 35.84 39.56 44.73 50.00 38.54 32.23 34.97

11 54.47 45.79 40.31 45.26 49.31 55.06 41.89 36.40 39.57

12 59.21 50.16 45.37 50.32 54.15 61.29 46.06 40.96 42.42

13 62.78 53.51 49.04 52.13 59.69 66.24 50.30 45.63 46.24

14 64.70 56.44 52.45 54.90 62.25 70.34 55.58 51.02 49.38

16 66.93 60.86 55.96 57.14 64.91 72.79 60.18 54.08 51.52

18 68.53 62.25 58.63 59.27 66.08 75.99 64.56 58.07 53.80

20 70.18 64.54 59.90 61.29 69.38 78.75 67.06 61.35 55.15

Table A9. Swelling data index of pH responsive CMCh-POM formulations at pH 1.2

Time

(h) CMCP1 CMCP2 CMCP3 CMCP4 CMCP5 CMCP6 CMCP7 CMCP8 CMCP9

165

0 1 1 1 1 1 1 1 1 1

0.25 1.27 1.14 1.24 1.30 1.19 1.19 1.20 1.14 1.08

0.5 1.47 1.17 1.35 1.51 1.39 1.35 1.28 1.25 1.16

1 1.64 1.24 1.55 1.73 1.54 1.48 1.47 1.35 1.25

1.5 1.75 1.33 1.74 2.01 1.73 1.68 1.69 1.43 1.39

2 1.87 1.49 1.93 2.27 1.96 1.87 1.80 1.56 1.48

3 1.93 1.68 2.25 2.51 2.16 2.05 1.91 1.75 1.58

4 2.03 1.85 2.51 2.76 2.50 2.27 2.05 1.88 1.72

5 2.12 2.09 2.83 2.97 2.76 2.56 2.23 1.98 1.80

6 2.19 2.31 3.16 3.34 2.92 2.63 2.45 2.09 1.88

7 2.26 2.56 3.36 3.46 3.07 2.67 2.51 2.19 1.96

8 2.31 2.77 3.60 3.69 3.13 2.70 2.57 2.35 2.03

10 2.35 3.05 3.75 3.77 3.16 2.74 2.64 2.48 2.12

12 2.42 3.28 3.93 3.85 3.22 2.76 2.97 2.62 2.18

14 2.47 3.40 4.07 3.93 3.27 2.84 3.21 2.78 2.22

16 2.50 3.49 4.13 3.98 3.30 3.11 3.36 2.92 2.26

18 2.51 3.50 4.21 4.05 3.43 3.24 3.54 3.05 2.27

Table A10. Swelling data index of pH responsive CMCh-POM formulations at pH

7.4

Time CM CM CM CM CM CM CM CM CM

166

(h) CP1 CP2 CP3 CP4 CP5 CP6 CP7 CP8 CP9

0 1 1 1 1 1 1 1 1 1

0.25 1.12 1.08 1.50 1.20 1.24 1.39 1.24 1.12 1.05

0.5 1.26 1.35 2.43 1.44 1.87 2.59 1.79 1.25 1.15

1 1.54 1.77 3.65 2.00 2.08 3.36 2.95 1.55 1.35

1.5 1.80 2.25 4.68 2.76 2.86 3.85 3.36 2.13 1.67

2 2.04 2.64 6.10 3.08 3.31 4.83 4.13 2.67 1.96

3 2.53 2.96 6.66 3.52 3.96 5.53 4.66 3.05 2.55

4 2.87 3.45 7.14 4.05 4.30 5.65 4.93 3.52 2.84

5 3.14 3.95 8.21 4.63 4.79 6.83 5.25 4.35 3.18

6 3.56 4.17 8.90 5.10 5.39 8.35 5.82 4.61 3.40

7 3.76 4.93 9.25 5.48 6.11 9.07 6.40 5.09 3.85

8 4.07 6.13 9.80 5.88 6.84 9.89 6.96 5.55 4.02

10 4.27 6.72 10.65 6.00 7.43 11.00 7.85 6.11 4.31

12 4.51 7.33 11.64 6.17 8.26 11.83 8.44 6.46 4.56

14 4.64 8.39 12.68 6.72 9.20 12.87 9.78 6.99 4.81

16 4.94 9.38 13.88 7.33 10.58 13.84 10.77 8.34 5.27

18 5.60 9.90 14.17 8.07 11.59 15.01 11.57 8.52 5.57

Table A11. Percent in-vitro release data of pH responsive CMCh-POM formulations

at pH 1.2

167

Time

(h)

CMCP

1

CMCP

2

CMCP

3

CMCP

4

CMCP

5

CMCP

6

CMCP

7

CMCP

8

CMCP

9

0 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

1 0.96 1.01 2.02 0.96 1.01 0.37 0.96 0.78 0.43

2 1.81 2.50 3.35 2.08 2.02 1.22 2.21 1.93 0.75

3 2.88 3.83 4.63 2.93 3.14 2.24 4.06 3.21 1.25

4 4.37 4.95 6.71 4.63 3.67 2.77 6.10 4.74 1.60

5 5.17 6.55 8.04 5.48 4.26 3.57 8.27 6.81 2.60

6 6.44 8.20 9.64 6.87 5.11 4.69 10.05 8.88 4.24

7 7.67 9.85 11.77 8.79 6.39 5.43 12.69 10.70 5.56

8 9.00 11.77 13.42 10.12 7.77 6.23 14.90 12.34 7.20

9 10.54 13.21 15.18 11.93 8.89 7.19 16.83 13.69 8.48

10 12.19 14.96 17.36 13.10 10.12 8.31 18.68 15.51 9.91

11 13.68 16.45 19.49 13.63 11.08 9.27 20.57 17.90 10.80

12 15.12 17.94 21.41 14.96 12.30 9.90 22.67 19.25 12.19

13 15.87 19.17 22.90 15.97 12.78 10.38 25.13 20.36 13.16

14 17.04 20.18 24.71 16.77 13.53 10.92 27.09 21.43 14.55

16 17.73 21.73 25.99 17.25 13.79 11.45 29.23 21.78 15.61

18 18.58 22.52 26.41 17.41 14.22 11.24 30.05 22.03 16.58

Table A12. Percent in-vitro release data of pH responsive CMCh-POM formulations

at pH 7.4

Time CMCP CMCP CMCP CMCP CMCP CMCP CMCP CMCP CMCP

168

(h) 1 2 3 4 5 6 7 8 9

0 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

1 3.30 3.78 4.10 4.74 4.85 5.48 4.10 3.32 2.46

2 7.51 6.44 8.68 9.16 9.96 10.38 8.88 6.95 4.96

3 11.13 10.38 12.73 14.38 15.50 16.03 13.58 10.37 7.99

4 13.79 13.58 16.45 19.17 20.61 22.52 18.29 14.26 12.37

5 17.63 18.42 20.82 23.70 25.24 29.55 23.53 19.57 16.76

6 22.04 22.10 24.49 29.55 30.14 36.58 28.16 23.71 20.29

7 25.72 27.42 28.27 34.66 36.90 43.61 32.48 27.42 24.31

8 28.91 32.06 34.35 40.10 43.66 49.79 36.11 30.84 28.34

9 34.88 37.75 40.10 43.08 50.21 54.37 41.60 34.97 31.37

10 40.10 41.80 45.85 47.76 56.39 60.06 45.95 39.75 35.65

11 45.85 47.82 50.48 53.04 63.74 66.29 50.09 43.81 40.82

12 49.73 53.83 57.03 58.31 69.22 72.47 55.69 48.74 44.88

13 52.98 57.99 62.30 62.46 74.17 77.53 60.50 53.83 50.59

14 56.23 61.98 66.72 67.63 77.16 81.79 65.95 59.71 56.01

16 59.16 64.48 70.13 71.09 79.93 83.65 70.62 64.99 58.15

18 59.96 65.18 71.62 74.01 81.20 86.58 74.51 69.70 60.61

Table A13. Swelling data of pH responsive ChCl-POM formulations at pH 1.2

Time CHCP CHCP CHCP CHCP CHCP CHCP CHCP CHCP CHCP

169

(h) 1 2 3 4 5 6 7 8 9

0 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00

0.25 1.30 1.12 1.34 1.30 1.23 1.12 1.13 1.32 1.14

0.5 1.45 1.15 1.55 1.44 1.36 1.22 1.32 1.45 1.26

1 1.65 1.21 1.75 1.60 1.52 1.35 1.48 1.54 1.37

1.5 1.75 1.30 1.94 1.78 1.72 1.52 1.63 1.70 1.52

2 1.87 1.37 2.15 1.97 1.89 1.65 1.82 1.83 1.57

3 1.96 1.53 2.38 2.14 2.07 1.75 2.01 2.03 1.70

4 2.03 1.77 2.52 2.35 2.27 1.90 2.19 2.18 1.84

5 2.12 2.00 2.73 2.52 2.45 2.02 2.35 2.27 2.01

6 2.19 2.21 2.84 2.67 2.60 2.15 2.47 2.42 2.12

7 2.26 2.26 2.99 2.82 2.81 2.24 2.73 2.52 2.13

8 2.31 2.39 3.18 3.21 2.92 2.39 2.88 2.75 2.21

10 2.38 2.48 3.31 3.08 2.99 2.50 3.05 2.85 2.27

12 2.45 2.57 3.43 3.22 3.10 2.62 3.16 2.98 2.33

14 2.47 2.65 3.63 3.32 3.15 2.71 3.32 3.08 2.42

16 2.53 2.77 3.78 3.50 3.21 2.82 3.48 3.19 2.48

18 2.57 2.92 3.91 3.50 3.22 2.92 3.65 3.24 2.54

Table A14. Swelling data index of pH responsive ChCl-POM formulations at pH 7.4

Time CHCP1 CHCP2 CHCP3 CHCP4 CHCP5 CHCP6 CHCP7 CHCP8 CHCP9

170

(h)

0 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00

0.25 1.36 1.07 1.13 1.10 1.16 1.15 1.08 1.07 1.05

0.5 1.60 1.25 1.33 1.33 1.42 2.06 1.23 1.13 1.10

1 1.77 1.78 1.76 1.62 1.80 2.68 1.54 1.25 1.20

1.5 1.99 2.09 2.36 1.90 2.19 3.56 1.98 1.48 1.38

2 2.22 2.31 2.99 2.22 2.77 4.55 2.37 1.65 1.54

3 2.59 2.73 3.77 2.61 3.24 5.17 3.45 1.82 1.71

4 2.87 3.48 4.41 3.32 4.25 5.74 4.40 2.05 1.91

5 3.24 4.36 4.95 3.56 4.96 6.16 5.47 2.31 2.14

6 3.51 4.82 6.01 3.89 5.67 7.65 6.21 2.73 2.30

7 3.72 5.14 6.83 4.59 6.65 9.11 7.36 3.45 3.04

8 3.97 5.87 7.63 5.12 7.46 10.75 8.02 4.36 3.84

10 4.29 6.26 8.28 5.95 8.63 12.32 8.51 5.37 4.42

12 4.50 7.26 9.12 6.39 10.02 14.08 8.93 6.44 4.73

14 4.75 7.95 9.99 7.40 11.27 15.75 9.35 6.95 5.15

16 4.90 8.31 10.83 8.34 12.13 16.68 9.77 7.07 5.55

18 5.04 8.49 11.87 9.06 12.99 17.05 10.15 7.19 5.79

Table A15. In-vivo plasma concentrations (µg/mL) data of control group rabbits

Time (h) Rabbit 1 Rabbit 2 Rabbit 3 Rabbit 4 Rabbit 5 Rabbit 6 Mean

171

0.5 60.33 61.28 56.15 54.54 63.18 62.04 59.59

1 205.87 123.63 134.53 179.81 182.27 160.20 164.39

2 236.11 158.76 161.43 201.96 241.82 202.54 200.44

3 442.88 397.77 412.37 420.86 495.38 455.34 437.43

4 589.92 702.22 745.00 639.22 618.63 519.79 635.80

6 799.12 775.52 765.68 756.45 763.76 913.46 795.67

8 1975.11 1937.57 1739.69 1647.51 1702.48 1711.61 1785.66

10 1939.27 1879.14 1617.74 1681.27 1666.47 1668.64 1742.09

12 1898.48 1408.33 1474.78 1343.71 1423.42 1114.47 1443.86

14 1433.57 838.68 657.28 736.76 776.74 770.66 868.95

16 444.34 475.19 421.47 350.62 421.36 346.12 409.85

20 105.58 95.39 101.91 96.97 107.60 105.58 102.17

24 0 0 0 0 0 0 0.00

Table A16. In-vivo plasma concentrations (µg/mL) data of hydrogel group rabbits

Time

(h) Rabbit 1 Rabbit 2 Rabbit 3 Rabbit 4 Rabbit 5 Rabbit 6 Mean

1 131.0 139.6 151.2 161.9 147.8 148.9 146.7

2 492.3 296.5 313.2 187.6 205.4 201.4 282.7

3 611.5 377.5 375.2 310.0 347.3 297.0 386.4

4 625.0 465.6 473.9 472.9 480.9 484.4 500.4

6 802.9 595.6 603.3 589.9 584.2 586.8 627.1

8 1006.9 1115.9 910.6 950.9 984.8 988.2 992.9

10 1378.7 1413.3 1282.2 1285.3 1220.8 1230.3 1301.8

14 1642.9 1647.0 1409.4 1466.4 1476.4 1315.5 1492.9

18 1937.6 1751.3 1676.7 1598.7 1623.8 1729.0 1719.5

22 1543.2 1583.2 1784.1 1734.8 1758.0 1608.8 1668.7

26 1202.0 1146.3 1315.0 1276.6 1319.4 1337.1 1266.1

30 763.8 771.3 825.6 783.7 617.2 533.7 715.9

36 296.0 257.3 186.4 185.5 270.1 184.7 230.0

48 35.0 35.0 35.0 100.8 38.0 36.0 46.6

Table A17. Cytotoxicity data of hydrogel formulations on MCF-7 cancer cell line

172

Gelatin-POM Hydrogels (MCF-7)

Conc.

mg/mL

Negative

control

Polyanion

Doxorubicin Gelatin-POM Hydrogel

20 99.21125 72.49657 76.33745 88.95748

25 97.25652 54.86968 57.9561 69.44444

30 96.94787 37.41427 36.24829 51.20027

35 99.10837 21.98217 25.34294 31.31001

PEI-POM Hydrogels (MCF-7)

Conc.

mg/mL

Negative

control

Polyanion

Doxorubicin PEI-POM Hydrogel

20 99.21125 72.49657 76.33745 87.68861

25 97.25652 54.86968 57.9561 64.54047

30 96.94787 37.41427 36.24829 49.41701

35 99.10837 21.98217 25.34294 30.07545

CMCh-POM Hydrogels (MCF-7)

Conc.

mg/mL

Negative

control

Polyanion

Doxorubicin CMCh-POM Hydrogel

20 99.21125 72.49657 76.33745 87.68861

25 97.25652 54.86968 57.9561 64.54047

30 96.94787 37.41427 36.24829 49.41701

35 99.10837 21.98217 25.34294 30.07545

ChCl-POM Hydrogels (MCF-7)

Conc.

mg/mL

Negative

control

Polyanion

Doxorubicin ChCl-POM Hydrogel

20 99.21125 72.49657 76.33745 82.92181

25 97.25652 54.86968 57.9561 57.23594

30 96.94787 37.41427 36.24829 41.04938

35 99.10837 21.98217 25.34294 25.61728

Table A18. Cytotoxicity data of hydrogel formulations on HeLa cancer cell line

173

Gelatin-POM Hydrogels (Hela Cells)

Conc.

mg/mL

Negative

control

Polyanion

Doxorubicin Gelatin-POM Hydrogel

20 99.85994 87.5 81.33754 86.27451

25 97.11934 54.97199 59.52381 68.10224

30 98.11385 27.90616 35.39916 47.47899

35 97.56516 15.86134 23.3882 34.41877

PEI-POM Hydrogels (Hela Cells)

Conc.

mg/mL

Negative

control

Polyanion

Doxorubicin PEI-POM Hydrogel

20 99.85994 87.5 81.33754 86.27451

25 97.11934 54.97199 59.52381 68.10224

30 98.11385 27.90616 35.39916 47.47899

35 97.56516 15.86134 23.3882 34.41877

CMCh-POM Hydrogels (Hela Cells)

Conc.

mg/mL

Negative

control

Polyanion

Doxorubicin CMCh-POM Hydrogel

20 99.85994 87.5 81.33754 80.91737

25 97.11934 54.97199 59.52381 60.15406

30 98.11385 27.90616 35.39916 40.82633

35 97.56516 15.86134 23.3882 32.52801

ChCl-POM Hydrogels (Hela Cells)

Conc.

mg/mL

Negative

control

Polyanion

Doxorubicin ChCl-POM Hydrogel

20 99.85994 87.5 81.33754 80.04552

25 97.11934 54.97199 59.52381 64.70588

30 98.11385 27.90616 35.39916 44.85294

35 97.56516 15.86134 23.3882 30.91737

Table A19. Cytotoxicity data of hydrogel formulations on normal Vero cell line

174

Gelatin-POM Hydrogels

Conc.

mg/mL

Negative

control

Polyanion

Doxorubicin

Gelatin-POM

Hydrogel

20 99.46422 83.42901 95.40758 97.85687

25 99.31114 78.45388 89.24608 93.53234

30 98.69881 70.37887 82.9315 89.43743

35 98.43092 63.18408 77.42059 85.76349

PEI-POM Hydrogels

Conc.

mg/mL

Negative

control

Polyanion

Doxorubicin

PEI-POM

Hydrogel

20 99.46422 83.42901 94.64217 97.85687

25 99.31114 78.45388 89.16954 93.53234

30 98.69881 70.37887 82.70188 89.43743

35 98.43092 63.18408 78.03291 85.76349

CMCh-POM hydrogels

Conc.

mg/mL

Negative

control

Polyanion

Doxorubicin

CMCh-POM

Hydrogel

20 99.46422 83.42901 94.64217 98.43092

25 99.31114 78.45388 89.16954 95.13969

30 98.69881 70.37887 82.70188 92.46077

35 98.43092 63.18408 78.03291 88.74856

ChCl-POM Hydrogels

Conc.

mg/mL

Negative

control

Polyanion

Doxorubicin

ChCl-POM

Hydrogel

20 99.46422 83.42901 94.64217 95.94336

25 99.31114 78.45388 89.16954 89.36089

30 98.69881 70.37887 82.70188 84.27095

35 98.43092 63.18408 78.03291 80.71183

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