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HYDROGEL FROM LINUM USITATISSIMUM L.: ISOLATION,
MODIFICATION, CHARACTERIZATION AND
PHARMACEUTICAL APPLICATIONS
A DISSERTATION SUBMITTED
TO
THE UNIVERSITY OF SARGODHA, SARGODHA
IN PARTIAL FULFILLMENT OF THE REQUIREMENT
FOR
THE DEGREE OF DOCTOR OF PHILOSOPHY
IN
PHARMACEUTICS
BY
MUHAMMAD TAHIR HASEEB
COLLEGE OF PHARMACY
FACULTY OF PHARMACY
UNIVERSITY OF SARGODHA, SARGODHA
Session 2010-2015
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DEDICATED TO
MY PARENTS
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APPROVAL CERTIFICATE
It is solemnly described that the dissertation titled ―Hydrogel from Linum usitatissimum L.:
Isolation, modification, characterization and pharmaceutical applications‖ submitted by
Muhammad Tahir Haseeb in the partial fulfillment of the requirement for the award of
degree of DOCTOR OF PHILOSOPHY in Pharmaceutics is hereby approved.
Supervisor 1: __________________ Supervisor 2: _______________
Prof. Dr. Sajid Bashir Dr. Muhammad Ajaz Hussain
Dean Associate Professor
College of Pharmacy Department of Chemistry
Faculty of Pharmacy University of Sargodha, Sargodha
University of Sargodha, Sargodha
External examiner: __________
Dean: _____________________
Prof. Dr. Sajid Bashir
College of Pharmacy
Faculty of Pharmacy
University of Sargodha, Sargodha
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DECLARATION
I declare that the work described in this thesis was carried out by me under the supervision of
Prof. Dr. Sajid Bashir, Dean Faculty of Pharmacy and Dr. Muhammad Ajaz Hussain,
Associate Professor, Department of Chemistry, University of Sargodha, Sargodha, Pakistan,
in partial fulfillment of the requirement for the degree of ―Doctor of Philosophy in
Pharmaceutics‖. I certify that the main content of this thesis accounts for my own research
and has not previously been submitted for a degree at any educational institution. Further, it
is submitted that the material taken from other sources has been properly acknowledged.
MUHAMMAD TAHIR HASEEB
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ACKNOWLEDGEMENTS
All praises are for Almighty Allah, the most kind, magnificent and merciful who gave me
strength, power, knowledge and above all good health to complete this work amicably.
My deepest gratitude goes to my supervisors Prof. Dr. Sajid Bashir, Dean, Faculty of
Pharmacy and Dr. Muhammad Ajaz Hussain, Associate Professor Department of Chemistry
for their continuous support, constant encouragement and invaluable guidance throughout the
course of my research work. Particularly, rigorous, meticulous, serious and responsible
academic attitude of Dr. Muhammad Ajaz Hussain who has always been my role model. I
am also thankful to the Higher Education Commission (HEC) of Pakistan for granting
scholarship under ―Indigenous 5000 PhD Fellowship‖ and ―IRSIP‖ programs.
I would like to acknowledge Prof. Dr. Soon Hong Yuk, Dean, College of Pharmacy, Korea
University, Sejong, Republic of Korea for his cooperation and support in conducting the
research work in his laboratory under IRSIP funded by HEC for six months. I am also
thankful to him for providing research facilities and, analyses and biological and
cytotoxicity studies of some research samples. I also want to pay my gratitude to Dr. Eun
Hee Lee, Associate Prof., College of Pharmacy, Korea University, Sejong, Republic of
Korea for helpful discussion. I am grateful to Mr. Nisar Ul Khaliq, Mr. Ameeq Ul Mushtaq
and Mr. Bishal Adhikari for their cooperation and valuable scientific discussion.
I am deeply indebted to Prof. Dr. Wolfgung Tremel and Dr. Muhammad Nawaz Tahir,
Institute of Inorganic and Analytical Chemistry, Johannes Guttenberg University, Mainz,
Germany for taking TEM analyses.
I am very thankful to Dr. Muhammad Sher, Deputy Manager, Instruments Lab., University
of Sargodha, Pakistan for providing necessary facilities for the characterization of research
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samples and fruitful discussion. I also acknowledge the advice of all of my teachers in
accomplishing this research work.
I sincerely extend my gratitude to my PhD fellows: Ms Alia Erum, Mr. Muhammad Umer
Ashraf, Mr. Muhammad Amin, Mr. Khawar Abbas, Mr. Azhar Abbas, Mr. Gulzar
Muhammad and Mr. Muhammad Nauman for their cooperations and discussions.
Appreciation is due to Trison Research Laboratories (Pvt.) Ltd., Sargodha, Pakistan for
providing the facility of pharmaceutical equipment for preparation of tablet formulations.
I would also like to thank staff of all laboratories including Mr. Farhan Khan, Mr. Atta-ur-
Rehman, Mr. Laeeq, Mr. Amir Latif, Mr. Raheem, Mr. Ashfaq, Mr. Waqas, Mr. Liaqat, Mr.
Nasir, Mr. Sohail Armaghan, Mr. Muhammad Umair and Mr. Naveed for helping me in
research experimentation.
I would also like to extend my heartiest gratitude to my beloved wife Mrs. Fatima Akbar
Sheikh for her untiring efforts and moral support during my PhD studies. Furthermore, I
would also like to acknowledge the cherishing company and inspiring smiles of my beloved
son Muhammad Moeez Tahir and my sweet daughter Amna Tahir which enabled me to
overcome the bad moments during the research.
Last but not the least, special thanks to my parents who are the torch bearer for the whole of
my life, who grew me up in a way that I am able to stand before the world and face every
person and situation with a humble confidence.
MUHAMMAD TAHIR HASEEB
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ABBREVIATIONS
∆G Gibbs free energy
∆H Enthalpy
∆S Change in entropy
AFM Atomic force microscopy
Ag NPs Silver nanoparticles
ALSH Acetylated linseed hydrogel
ALT Alanine aminotransferase
ANOVA Analysis of variance
AST Aspartate aminotransferase
ATCC American type culture collection
AUC Area under curve
BS Bletilla striata
CDCl3 Deuterated chloroform
CMC Carboxymethyl cellulose
COX Cyclooxygenase
DAPI 4ʹ,6-diamidino-2-phenylindole
DLP-NPs DTX-loaded LSH Pluronic F-68 nanoparticles
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DLS Dynamic light scattering
DMAc Dimethylacetamide
DMAP 4-dimethylaminopyridine
DMF Dimethylformamide
DMSO Dimethyl sulfoxide
DMSO-d6 Dimethyl sulfoxide-deuterated
DS Diclofenac sodium
DSb Degree of substitution
DSC Differential scanning calorimetry
DTX Docetaxel
Ea Activation energy
EPR Enhanced permeability and retention
FE-SEM Field emission scanning electron microscopy
FTIR Fourier transform infrared
FWO Flynn-wall and Ozawa
GCMS Gas chromatography mass spectrometry
GIT Gastrointestinal tract
GLP Good laboratory practice
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GPC Gel permeation chromatography
HDL High density lipoprotein
HEC Hydroxyethyl cellulose
HMQC Heteronuclear multiple-quantum correlation
HPC Hydroxypropyl cellulose
HPCMC Hydroxypropyl carboxymethyl cellulose
HPMC Hydroxypropylmethyl cellulose
HSQC Heteronuclear single-quantum correlation
IPDT Integral procedural decomposition temperature
IPN Interpenetrating polymer network
ITS Index of thermal stability
KDa Kilodalton
LDL Low density lipoprotein
LiCl Lithium chloride
LSH Linseed hydrogel
MALLS Multi angle laser light scattering
MBA N,Nʹ-methylene bisacrylamide
MCF-7 Michigan cancer foundation-7
x
MCH Mean corpuscular hemoglobin
MCHC Mean corpuscular hemoglobin concentration
MCV Mean corpuscular volume
MMTS Maximum mean total score
MSC Model selection criterion
MTT 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide
NMR Nuclear magnetic resonance
NPs Nanoparticles
NSAIDs Non-steroidal anti-inflammatory drugs
OECD Organization for Economic Co-operation and Development
PDII Primary dermal irritation index
PEG Polyethylene glycol
PG Phellinus gilvus
pHEMA Poly(2-hydroxyethyl methacrylate)
PLG Polylactic-co-glycolic acid
PVA Polyvinyl alcohol
PXRD Powder X-ray diffraction
RP-HPLC Reverse-phase high performance liquid chromatography
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SEC Size exclusion chromatography
SEM Scanning electron microscopy
SGF Simulated gastric fluid
SIF Simulated intestinal fluid
SIPN Semi-interpenetrating polymer network
TBA Thiobarbituric acid
TBAF Tetrabutylammonium fluoride
TEM Transmission electron microscopy
Tg Transition temperature
TGA Thermogravimetric analysis
TOCSY Total correlation spectroscopy
USFDA United States Food and Drug Administration
USP United States Pharmacopeia
UV Ultra violet
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LIST OF FIGURES
Fig. 1.1. Structure of diclofenac sodium.
Fig. 1.2. Structure of caffeine.
Fig. 1.3. Structure of diacerein.
Fig. 1.4. Structure of docetaxel.
Fig. 3.1. FTIR spectrum of LSH.
Fig. 3.2. 1H NMR (600 MHz, ppm, 40 °C) spectrum of LSH in DMSO-d6 showing
repeating unit between 3.11-5.61.
Fig. 3.3. PXRD spectrum of LSH.
Fig. 3.4. Reaction scheme for the synthesis of acetylated LSH.
Fig. 3.5. FTIR spectra of LSH, ALSH 1, ALSH 2 and ALSH 3.
Fig. 3.6. 1H NMR (600 MHz, ppm, DMSO-d6, 40 °C) spectrum of ALSH 3 (DSb 2.91).
Fig. 3.7. 1H
1H TOCSY (600 MHz, ppm, CDCl3, 25 °C) spectrum of ALSH 3 (DSb 2.91).
Fig. 3.8. 1H
1H TOCSY (600 MHz, ppm, CDCl3, 25 °C) spectrum of ALSH 3 (DSb 2.91)
showing correlation of sugar region.
Fig. 3.9. HSQC spectrum (600 MHz, ppm, CDCl3, 25 °C) of ALSH 3.
Fig. 3.10. HSQC spectrum (600 MHz, ppm, CDCl3, 25 °C) of ALSH 3 showing
correlation of acetyl methyl region.
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Fig. 3.11. HSQC spectrum (600 MHz, ppm, CDCl3, 25 °C) of ALSH 3 showing
correlation of sugar region.
Fig. 3.12. Overlay of TG and DTG curves of LSH (a, c) and ALSH (b, d), respectively
recorded at multiple heating rates.
Fig. 3.13. Overlay of 2DTG curves of LSH (a) and ALSH 3 (b) recorded at multiple
heating rates.
Fig. 3.14. Overlay of TG (a) and DTG (b) curves of LSH and ALSH 3 recorded at 10 °C
min-1
showing stability imparted to ALSH 3.
Fig. 3.15. α vs. T graph of thermal degradation of first (a) and second (b) step of LSH at
multiple heating rates and Flynn-Wall-Ozawa (FWO) plot between log β and
1000/T (K-1
) for calculation of Ea of first degradation (c) and second degradation
(d) step at several degree of conversion for LSH.
Fig. 3.16. α vs. T graph of thermal degradation of ALSH 3 at multiple heating rates (a) and
Flynn-Wall-Ozawa (FWO) plot between log β and 1000/T (K-1
) for calculation
of Ea at several degree of conversion for ALSH 3.
Fig. 3.17. Swelling capacity (a) and second order swelling kinetics (b) of LSH in buffer of
pH 1.2, pH 6.8 and 7.4 and in deionized water (D.W).
Fig. 3.18. Swelling capacity of LSH in deionized water at different temperatures.
Fig. 3.19. Swelling capacity of LSH in different conc. of NaCl and KCl (a) and swelling-
shrinking (on-off switching) behavior of LSH; at pH 7.4 (basic) and pH 1.2
(acidic) (b), in deionized water and normal saline (0.9% NaCl solution) (c) and
in deionized water and ethanol (d), respectively.
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Fig. 3.20. SEM images of lyophilized sample of LSH showing porous and elongated
structure.
Fig. 3.21. FTIR spectra of LSH, PVP and diacerein alone, physical mixture of LSH with
diacerein and physical mixture of LSH, diacerein and PVP.
Fig. 3.22. FTIR spectra of LSH, PVP and caffeine alone, physical mixture of LSH with
caffeine and physical mixture of LSH, caffeine and PVP.
Fig. 3.23. FTIR spectra of LSH, PVP and diclofenac sodiume alone, physical mixture of
LSH with diclofenac sodiume and physical mixture of LSH, diclofenac sodiume
and PVP.
Fig. 3.24. Swelling capacity (a) and swelling kinetics (b) of LSH tablet (FH) at different
pH and in deionized water and swelling photographs (radial and axial view) of
FH formulation at pH 6.8 (c).
Fig. 3.25. Swelling capacity of FH, FC1, FC2, and FC3 at pH 1.2 (a), 6.8 (b), 7.4 (c), and
DI water (d) and swelling photographs (radial and axial view) of FC3
formulation at pH 6.8 (e).
Fig. 3.26. Swelling kinetics of LSH tablet (FH) and LSH-caffeine tablets (FC1, FC2 and
FC3) at pH 1.2 (a), 6.8 (b), 7.4 (c) and deionized water (d).
Fig. 3.27. Swelling capacity of FH, FD1, FD2, and FD3 at pH 1.2 (a), 6.8 (b), 7.4 (c) and
deionized water (d) and swelling behavior of FD3 formulation at pH 6.8
expressed in photographs (radial and axial view) (e).
Fig. 3.28. Swelling kinetics of LSH tablet (FH) and LSH-diacerein tablets (FD1, FD2 and
FD3) at pH 1.2 (a), 6.8 (b), 7.4 (c) and deionized water (d).
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Fig. 3.29. SEM images of broken surface of FH tablet (a), broken surface of FD3 tablet (b)
and cross section of swollen then freeze dried tablet formulation FD3.
Fig. 3.30. Equilibrium swelling of LSH tablet (FH), LSH-caffeine tablet (FC3) and LSH-
diacerein tablet (FD3) in different molar concentrations of salt solutions; NaCl
(a) and KCl (b).
Fig. 3.31. Stimuli responsive swelling and deswelling behavior of LSH tablet (FH), LSH-
caffeine tablet (FC3) and LSH-diacerein tablet (FD3) at basic (pH 7.4) and
acidic (pH 1.2) environment (a), in deionized water and normal saline (b) and
deionized water and ethanol (c), respectively.
Fig. 3.32. Swelling capacity of LSH based DS tablets in water (a), drug (DS) release study
from LSH matrix tablets in SGF and SIF (b) and photographs showing swelling
response (aerial and axial view) of D3 formulation in water (c).
Fig. 3.33. Caffeine release from LSH-caffeine tablet in different media; pH 6.8 (a), pH 7.4
(b), deionized water (c) and physiological pH and transit time of gastrointestinal
tract (d).
Fig. 3.34. Diacerein release from LSH-diacerein tablet in different media; pH 6.8 (a), pH
7.4 (b), deionized water (c) and physiological pH and transit time of
gastrointestinal tract (d).
Fig. 3.35. Size distribution of DLP-NPs with different drug loadings: 1% (a), 2% (b), 3%
(c); FESEM image of DLP-NPs (formulation with 1% DTX loading) (d); size
distribution of LSP (1 wt% aqueous solution) (e); size distribution calculated
from FESEM (f) and Zeta potential of 1% DLP-NPs (g).
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Fig. 3.36. PXRD (a) and FTIR (b) spectra of LSH, DTX, Pluronic F-68 and three
formualtions of DLP-NPs.
Fig. 3.37. Docetaxel release from different formulations of DLP-NPs.
Fig. 3.38. In vitro cytotoxicity of LSH, DTX and DLP-NPs at various concentrations.
Statistical significance is shown by * p < 0.05, performed by student‘s t-test for
comparison.
Fig. 3.39. Cellular uptake images of DLP-NPs (20x, a) and (40x, b).
Fig. 3.40. Schematic illustration showing synthesis of LSH mediated Ag NPs.
Fig. 3.41. Photographs of LSH-Ag+ mixture (20 mmol AgNO3) showing color change
with passage of time.
Fig. 3.42. UV/Vis spectra of LSH mediated Ag NPs: 10 mmol (a), 20 mmol (b) and 30
mmol solution of AgNO3 (c) at different reaction times and cumulative
graphical representation (d) showing increase in absorption of Ag NPs solutions
with increase in concentration and reaction time.
Fig. 3.43. TEM images of Ag NPs isolated from 10, 20 and 30 mmol LSH-Ag+
solution having size range from 10-25 nm (a), 10-30 nm (b) and 10-35 nm (c),
respectively.
Fig. 3.44. PXRD spectra: LSH (a), Ag NPs embedded LSH film (b) and isolated Ag NPs,
10 mmol (c), 20 mmol (d) and 30 mmol (e).
Fig. 3.45. UV/Vis spectra of Ag NPs synthesized from aqueous solution of AgNO3 (20
mmol) and LSH recorded after 10 h, 01, 15, 30 days and 06 months storage (a);
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PXRD spectrum of Ag NPs taken after 06 month storage of LSH-Ag NPs film
(b); Aqueous solution prepared from stored LSH-Ag NPs film (c); see through
and foldable LSH-Ag NPs film (d); TEM image of Ag NPs (10-30 nm) isolated
from LSH-Ag NPs film stored for 06 months under dark (e).
Fig. 3.46. Antimicrobial activity (a) and graphical representation of zone of inhibition of
Ag NPs (20 mmol) against different bacterial and fungal strains (b).
Fig. 3.47. Schematic illustrations of wound treatment with Ag NPs embedded LSH wound
dressing patch (a) and also showing its main parts (b).
Fig. 3.48. Collagen contents of epithelialized wound tissue of various groups after 15th
day. Statistical significance from control group is expressed by * p < 0.05.
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LIST OF TABLES
Table 2.1. Composition of different formulations to evaluate the sustained release
behavior of diclofenac sodium from LSH tablet.
Table 2.2. Tablet formulation design to evaluate the sustained release behavior of
caffeine.
Table 2.3. Constituents of various tablet formulations to study sustained release behavior
of diacerein.
Table 2.4. Group scheme for acute oral toxicity study of LSH in mice.
Table 3.1. Reaction parameters and results of the synthesis of acetylated LSH.
Table 3.2. Mean thermal decomposition temperatures, weight loss % and char yield % of
LSH at multiple heating rates.
Table 3.3. Mean thermal decomposition temperatures, weight loss % and char yield % of
ALSH 3 at various heating rates.
Table 3.4. Thermal kinetics and thermodynamic parameters of LSH.
Table 3.5. Thermal kinetics and thermodynamic parameters of ALSH 3.
Table 3.6. Physical properties of LSH.
Table 3.7. Pre-compression parameters of diclofenac sodium formulations (Mean ± SD).
Table 3.8. Pre-compression parameters of caffeine formulations (Mean ± SD).
Table 3.9. Pre-compression parameters of diacerein formulations (Mean ± SD).
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Table 3.10. Post-compression parameters of DS containing tablets (Mean ± SD).
Table 3.11. Post-compression parameters of caffeine containing tablets (Mean ± SD).
Table 3.12. Post-compression parameters of prepared tablets containing diacerein (Mean ±
SD).
Table 3.13. Mathematical data of power law.
Table 3.14. Values of drug release kinetics models for LSH-caffeine formulations at pH
6.8, 7.4 and deionized water.
Table 3.15. Values of drug release kinetics models for LSH-diacerein formulations at pH
6.8, 7.4 and deionized water.
Table 3.16. Drug loading and encapsulation efficiency of different formulations.
Table 3.17. Wound area (mm2) and wound closure (%) after selected day intervals.
Table 3.18. Scores for grading the primary eye irritation study of LSH.
Table 3.19. Clinical observations of acute oral toxicity and dermal testing of LSH.
Table 3.20. Biochemical blood analysis of control and LSH treated mice.
Table 3.21. Liver, kidney and lipid profile of mice treated with LSH.
Table 3.22. Absolute mean organ weight (g) of mice after oral administration of LSH.
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CONTENTS
ABSTRACT 1
1. INTRODUCTION 4
1.1. Polysaccharides as a biomaterial 4
1.1.1. Hydrogels 7
1.1.2. Linseed 10
1.1.3. Modification of polysaccharides 18
1.1.4. Stimuli responsive properties of polysaccharidal hydrogels 21
1.1.5. Toxicological studies of polysaccharides 25
1.2. Polysaccharides based drug delivery systems and pharmaceutical applications 27
1.2.1. Sustained release of NSAIDs from polysaccharidal materials 27
1.2.2. Polysaccharides based NPs as anticancer drug delivery system 34
1.2.3. Polysaccharides mediated synthesis and application of Ag NPs 37
1.2.4. Polysaccharides based antiseptic dressing 43
1.3. Characterization techniques 47
1.3.1. Fourier transform infrared spectroscopy 47
1.3.2. Nuclear magnetic resonance spectroscopy 47
1.3.3. Thermal analysis 48
1.3.4. Electron microscopic analysis 48
1.3.5. Powder X-ray diffraction 49
1.3.6. MTT assay 49
1.3.7. Drug release models 49
1.3.7.1. Zero order 49
1.3.7.2. First order 50
1.3.7.3. Higuchi model 51
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1.3.7.4. Hixson-Crowell model 51
1.3.7.5. Korsmeyer-Peppas model 52
1.4. Background and significance of the study 53
1.5. Aims and objectives 54
2. MATERIALS AND METHODS 56
2.1. Materials 56
2.2. Measurements 57
2.2.1. Fourier transform infrared spectroscopy 57
2.2.2. 1H NMR spectroscopy 57
2.2.3. Heteronuclear single quantum correlation spectroscopy 57
2.2.4. 1H
1H TOCSY NMR spectroscopy 57
2.2.5. UV-Vis spectrophotometry 58
2.2.6. Thermogravimetric analysis 58
2.2.7. Field emission scanning electron microscopy 58
2.2.8. Transmission electron microscopy 58
2.2.9. Powder X-ray diffraction 59
2.3. Acetylation of linseed hydrogel 59
2.3.1. Acetylation of LSH 59
2.3.2. Calculation of degree of substitution 61
2.3.3. Thermogravimetric analysis and degradation kinetics of LSH and
ALSH 61
2.4. Dynamic swelling and stimuli responsive on-off switching of LSH 62
2.4.1. Isolation of LSH 62
2.4.2. Physical properties of LSH 62
2.4.3. Preparation of buffer solutions of different pH 64
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2.4.4. Evaluation of pH responsive property of LSH 65
2.4.5. Swelling kinetics 66
2.4.6. Thermoresponsive swelling capacity of LSH in deionized water 66
2.4.7. Evaluation of salt solution-responsive properties of LSH 67
2.4.8. Evaluation of pH responsive on-off switching of LSH 67
2.4.9. Evaluation of saline responsive on-off switching of LSH 67
2.4.10. Evaluation of on-off switching of LSH in water and ethanol 68
2.5. Development of sustained drug delivery system 68
2.5.1. Formulation design 68
2.5.1.1. Drug excipient compatibility study 68
2.5.1.2. Preparation of tablets 69
2.5.1.3. Pre-compression evaluation 71
2.5.1.4. Post-compression evaluation 71
2.5.2. Dynamic swelling and stimuli responsive evaluation of LSH based
tablet formulations 73
2.5.2.1. pH responsive swelling of LSH containing tablets 73
2.5.2.2. Swelling kinetics 73
2.5.2.3. Evaluation of salt solution responsive swelling 74
2.5.2.4. Stimuli responsive swelling-deswelling (on-off) behavior 74
2.5.3. Evaluation of drug release behavior 74
2.5.3.1. In-vitro drug release studies 74
2.5.3.2. Drug release kinetics 76
2.5.3.3. Drug release mechanism 77
2.5.4. Scanning electron microscopy analysis 78
2.6. Docetaxel loaded LSH-Pluronic NPs 79
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2.6.1. Preparation of NPs 79
2.6.2. Encapsulation efficiency and drug loading 79
2.6.3. Particle size and morphology 80
2.6.4. X-ray diffraction analysis 80
2.6.5. In vitro drug release study 81
2.6.6. Cytotoxicity and cellular uptake behaviour 81
2.6.7. Statistical analysis 82
2.7. Nanobiotechnological application of LSH mediated Ag NPs 83
2.7.1. Preparation of AgNO3 solution and LSH suspension 83
2.7.2. Green synthesis of Ag NPs 83
2.7.3. Film formation 83
2.7.4. UV spectrophotometric analysis 83
2.7.5. Powder X-ray diffraction 84
2.7.6. Transmission electron microscopy 84
2.7.7. Antimicrobial activity 84
2.7.8. Wound healing studies 85
2.7.8.1. Design of wound dressing 85
2.7.8.2. Wound healing study design 85
2.7.8.3. Collagen estimation 86
2.8. Acute toxicological evaluation of LSH 87
2.8.1. Study design 87
2.8.2. Acute oral toxicity 88
2.8.3. Primary eye irritation 88
2.8.4. Acute dermal toxicity 89
2.8.5. Primary dermal irritation study 89
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2.8.6. Body weight gain study 90
2.8.7. Food and water consumption 90
2.8.8. Hematology and clinical biochemistry 90
2.8.9. Gross necropsy and histopathology 90
2.8.10. Statistical analysis 91
3. RESULTS AND DISCUSSION 92
3.1. Isolation and characterization of LSH 92
3.1.1. Isolation of LSH 92
3.1.2. FTIR spectroscopy 92
3.1.3. 1H NMR spectroscopy 93
3.1.4. PXRD 94
3.2. Synthesis and characterization of LSH-acetates 95
3.2.1. FTIR spectroscopy 96
3.2.2. 1H NMR spectroscopy 97
3.2.3. 1H
1H TOCSY spectroscopy 98
3.2.4. HSQC spectroscopy 98
3.2.5. Isoconversional thermal analysis of LSH and LSH-acetates 101
3.2.5.1. Thermal analysis 101
3.2.5.2. Degradation kinetics 104
3.2.5.3. Thermodynamic analysis 106
3.3. Dynamic swelling and stimuli responsive on-off switching of
superabsorbent LSH 107
3.3.1. Physical properties of LSH 107
3.3.2. Swelling capacity of LSH in deionized water and at different
physiological pH 108
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3.3.3. Swelling kinetics 108
3.3.4. Thermoresponsive swelling capacity of LSH 109
3.3.5. Saline responsive swelling of LSH 110
3.3.6. Responsive swelling-deswelling (on-off switching) behavior of LSH
at basic and acidic pH 110
3.3.7. Responsive swelling-deswelling (on-off switching) behavior of LSH
in deionized water and in NaCl solution (0.9%) 111
3.3.8. Responsive swelling-deswelling (on-off switching) behavior of LSH
in deionized water and ethanol 111
3.3.9. Field emission scanning electron microscopy 112
3.4. Evaluation of LSH as a novel controlled release and stimuli responsive
oral drug delivery system 113
3.4.1. Drug-excipients compatibility study 113
3.4.2. Pre-compression evaluation of tablet formulations 117
3.4.3. Post-compression evaluation of tablet formulations 118
3.4.4. Swelling response of LSH containing tablet formulations
at different pHs 120
3.4.4.1. Swelling response and swelling kinetics of LSH tablets 120
3.4.4.2. Swelling response and swelling kinetics of LSH-caffeine
tablets 121
3.4.4.3. Swelling response and swelling kinetics of LSH-diacerein
Tablets 122
3.4.5. Swelling morphology of LSH containing tablets 125
3.4.6. Morphological analysis of LSH containing tablets by SEM 127
3.4.7. Salt solution responsive swelling of LSH containing tablet
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formulations 128
3.4.8. Swelling-deswelling response of LSH tablet formulations against
external stimuli 129
3.4.8.1. Swelling-deswelling response in basic and acidic pH 129
3.4.8.2. Swelling-deswelling response in deionized water and normal
saline solution 130
3.4.8.3. Swelling-deswelling response in deionized water and ethanol 132
3.4.9. In-vitro drug release studies 132
3.4.9.1. DS release studies and release mechanism 132
3.4.9.2. Caffeine and diacerein release studies 134
3.4.9.3. Drug release kinetics and mechanism 138
3.5. Docetaxel loaded LSH-Pluronic NPs 142
3.5.1. Preparation and characterization of DLP-NPs 142
3.5.2. Particle size and morphological analysis 142
3.5.3. XRD and FTIR analysis of DLP-NPs 144
3.5.4. In vitro drug release study from DLP-NPs 145
3.5.5. Cytotoxicity and cellular uptake behaviour of DLP-NPs 146
3.6. Nanobiotechnological application of LSH 149
3.6.1. Green synthesis of Ag NPs 149
3.6.2. Characterization of Ag NPs 150
3.6.2.1. UV spectrophotometry 150
3.6.2.2. Transmission electron microscopy of isolated Ag NPs 152
3.6.2.3. Powder X-ray diffraction 153
3.6.2.4. Storage of Ag NPs in LSH thin film 154
3.6.2.5. Antimicrobial activity of Ag NPs 156
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3.6.2.6. Wound healing studies 157
3.7. Acute toxicological evaluation of LSH 160
3.7.1. Acute oral toxicity study in mice 160
3.7.2. Primary eye irritation 160
3.7.3. Acute dermal toxicity 161
3.7.4. Primary dermal irritation study 162
3.7.5. Body weight gain study 162
3.7.6. Food and water consumption 162
3.7.7. Haematology and clinical biochemistry 164
3.7.8. Gross necropsy and histopathology 164
CONCLUSIONS 166
REFERENCES 168
LIST OF PUBLICATIONS 211
1
ABSTRACT
Linseed hydrogel (LSH) was isolated from linseeds (Linum usitatissimum L.) using hot water
extraction method, characterized and used in various formulation designs. Characterization of
LSH was carried out using Fourier transform infrared (FTIR), powdered X-ray diffraction
(PXRD), nuclear magnetic resonance (NMR), scanning electron microscopy (SEM) and
thermogravimetric analysis (TGA). LSH was also modified by acetylation and structures
obtained were thoroughly characterized.
Stimuli responsive swelling of LSH was evaluated at gastrointestinal pHs (1.2, 6.8 and 7.4)
and in deionized water and also in different molar concentrations of NaCl and KCl solutions.
Swelling-deswelling (on-off) response of LSH against environmental conditions was also
observed. LSH has shown high swelling at pH 6.8, 7.4 and deionized water while negligible
swelling was seen at pH 1.2 indicating potential of LSH as intestine targeting drug delivery
system. Swelling behaviour of LSH at various pHs of gastrointestinal tract (GIT) has
followed the second order kinetics. Inverse relation between swelling of LSH and molar
concentrations (0.1, 0.2, 0.3, 0.4, 0.5, 1.0 and 2.0 M) of NaCl and KCl were observed.
Moreover, the water swollen LSH when immersed in normal saline, shrinking was observed.
A more abrupt shrinking of water swollen LSH was observed on immersing in ethanol.
Similarly, swelling-deswelling response was also observed in buffer of pH 7.4 and 1.2,
respectively. These results have revealed that LSH is a smart material and can be used to
make intelligent drug delivery systems.
High swelling and water holding capability of LSH were used to develop the sustained
release formulation of diclofenac sodium. Drug release data from LSH tablets was compared
with commercially available product (Voltral®
) and found better results. It was observed that
the release of diclofenac sodium from LSH matrix tablets was dependent on the concentration
2
of LSH and followed the anomalous transport mechanism. Therefore, LSH can be used as a
release retarding agent in sustained release formulation.
LSH-caffeine and LSH-diacerein tablets were prepared to analyze the stimuli (pH, salt
solution and ethanol) responsive swelling and swelling-deswelling (pH 7.4/1.2, water/normal
saline and water/ethanol) behaviour of LSH when used in tablet formulation. Although,
stimuli responsive properties of LSH remain the same even after compression in tablet form
but less swelling capacity was observed after compression. This might be due to the packing
arrangements of LSH and also less exposed area to the swelling medium in tablet form as
compared to powder form. LSH appeared as a novel material for stimuli responsive and pH
dependent release of NSAIDs in gastrointestinal tract.
The elongated porous structure arranged in uniformly distributed layers were seen in FE-
SEM analysis of swollen then freeze dried powder sample of LSH. Similar pattern of porous
channels was also observed even in tablet formulations of LSH. High swelling and water
holding capability of LSH are due to these porous channels.
Docetaxel loaded LSH Pluronic F-68 nanoparticles (DLP-NPs) were synthesized by core
shell formation. Drug loaded core of LSH was protected and stabilized by Pluronic F-68. Size
and morphological analysis of DLP-NPs was performed by dynamic light scattering (DLS),
PXRD and TEM. Results indicated that DLP-NPs are spherical in shape having size range of
220-335 nm. In vitro drug release study has shown a prolong release pattern for more than 4
days. Cell viability study of LSH and DLP-NPs has proved even better results when
compared with free docetaxel. Cell uptake behaviour of DLP-NPs was monitored using Nile
red and high concentration of DLP-NPs was accumulated in the cytoplasmic region of the
cell. Therefore, DLP-NPs have shown a promising anticancer drug delivery system.
LSH was used as a reducing and capping agent for the green synthesis of Ag NPs. Aqueous
suspension of LSH were mixed with silver nitrate solution and exposed to sunlight.
3
Formation of Ag NPs was monitored by noting the colour of solution and through UV
spectrophotometer. UV absorptions were observed from 410-437 nm. TEM images revealed
the formation of spherical Ag NPs in the range of 10-35 nm. Face centered cubic array of Ag
NPs was confirmed by characteristic diffraction peaks in PXRD spectrum. Significant
antimicrobial activity was observed when microbial cultures (bacteria and fungi) were
exposed to the synthesized Ag NPs. Wound healing studies revealed that Ag NPs
impregnated in LSH thin films could have potential applications as an antimicrobial dressing
in wound management procedures.
Acute toxicity study of LSH was conducted on albino mice and albino rabbits. Three groups
of mice were exposed to a single oral dose of LSH (1, 5 and 10 g/kg). For eye irritation study
and dermal toxicity study, rabbits were exposed to LSH. After day 14, the haematological
and biochemical testing were performed and the values obtained were within the normal
range. Furthermore, the histopathological evaluation of the vital organs has not shown any
abnormalities. After acute toxicity study, LSH was found safe up to the dose of 10g/kg of the
body weight of the animal.
Overall, LSH has shown itself as a highly swellable and smart biomaterial having stimuli
responsive swelling-deswelling properties both in powder form and tablet formulation.
Furthermore, the preparation of DTX loaded LSH NPs has proved its utilization in the
development of novel drug delivery system for cancer treatment. Ag NPs embedded LSH
matrix is a new biocomposite for wound dressing and wound healing. Therefore, LSH has
proved as a potential material with wide range of pharmaceutical applications.
4
1. INTRODUCTION
1.1. Polysaccharides as a biomaterial
Polysaccharides are a group of carbohydrate which is a combination of more than ten
monosaccharides attached with each other through glycosidic linkages. Depending on the
type of polysaccharides, these monosaccharides are arranged either in branched or linear
form. Nature and properties of polysaccharides are greatly dependent on the composition of
the building block, molecular weight and type of branching. Polysaccharides are abundant in
nature, e.g., starch, cellulose, glycogen, chitin, arabinoxylan, galactomannan, pectin, alginate,
guar gum and inulin, etc. Some microbes (bacteria, fungi and algae) secrete various
polysaccharides, i.e., pullulan, dextran, xanthan gum and gellan gum. Polysaccharides are
widely available, abundant in nature, inexpensive, biodegradable, biocompatible, safe and
highly stable, nontoxic and diverse in structure and properties (Hovgaard and Brondsted,
1996).
Polysaccharides are diverse in their properties. Chitin, pectin and inulin are only fermented
by different bacteria present in the intestine and colon, hence used for the site specific
delivery of different drugs (Englyst et al., 1987; Salyers et al., 1977; Tozaki et al., 1997;
Rubinstein, 1990). These polysaccharides are used in film formation for the coating of dosage
forms (Coffin and Fishman, 1993). Polysaccharides are widely used as a coating agent for
effective and site specific drug delivery system, matrix systems, prodrugs and dry coating.
Colonic bacteria secrete glycosidase enzymes that are responsible for the cleavage of
glycosidic bond present in the polysaccharide. This hydrolytic cleavage at gastric pH and
intestinal pH provided the basis to fabricate the hydrolysable (ester/amide) prodrug using
dextran as an important polysaccharide (Hovgaard and Brondsted, 1996; Hussain et al.,
2011).
5
Moreover, hydrolysable prodrugs have been formulated using a number of polysaccharides,
i.e., dextran (Larsen et al., 1991; Hussain et al., 2011), cellulose (Kumar and Negi, 2014),
pullulan (Hussain et al., 2013; Hussain and Heinze, 2008), HPMC (Hussain et al., 2009),
HEC (Amin et al., 2015; Abbas et al., 2016), HPC (Hussain 2008; Hussain et al., 2014;
Hussain et al. 2015).
HPMC based sponges containing curcumin were prepared by lyophilization with the aim to
increase its solubility and bioavailability. Curcumin was released from these sponges within 2
h and area under curve (AUC) was greater than 5-fold when compared with simple powder
formulation. In vitro drug release and bioavailability study from these sponges has proved
their utilization as an effective delivery system for water insoluble curcumin (Petchsomrit et
al., 2016).
Arabinoxylan extracted from Ispaghula seed husk was used as drug carrier for sustained
release tablet formulation (Iqbal et al., 2011a) and as a mediator for the synthesis of silver
and gold nanoparticles (Amin et al., 2013). Carboxymethylation and ethylation was carried
out to modify the swelling and solubility of arabinoxylan (Saghir, 2008; Saghir, 2009).
A polysaccharide, agarose, extracted from marine red algae has a wide range of application in
biological sciences. Monosaccharide units are attached with each other through
galactopyranose linkage and exhibited resistance against chemical and enzymatic
degradation. Agarose solution is converted into gel at 40 °C due to the formation of helices
which further shaped into bundles (Aymard et al., 2001).
Dextran is composed of linear and branched chains attached by α-1,6 glycosidic and α-1,4
glycosidic linkages and produced by different lactic acid bacteria. Depending on the
molecular weight (20-40 KDa), dextran have shown short and long term antithrombotic
effects (Qiao et al., 2009). Pullulan is a water soluble exopolysaccharide produced by fungus
6
Aureobasidium pullulans. Pullulan is odorless, tasteless, non-toxic, biodegradable, non-
antigenic, biocompatible and non-immunogenic. Due to these properties, it is widely used in
pharmaceutical, food and paper production industries (Leathers, 2003; Shingel, 2004; Singh
et al., 2008b)
Starch is a long and branched polymer mainly a combination of amylose (20-25%) and
amylopectin (75-80%). Starch being a biocompatible, biodegradable, non-toxic, non-irritant
and cheap is used in pharmaceutical, agrochemical, food, paper and packing industries.
Starch has been used as drug carrier either modified or unmodified form (Kim et al., 2003;
Abbas et al., 2015).
Carrageenan, a biopolymer, is composed of sulphated forms of 3,6-anhydro-D-galactose and
D-galactose. Six different forms of carrageenan polysaccharides are extracted and three of
them, κ, ι and λ, are important comprising 22, 32 and 38% of sulphate group. Carrageenan gel
has wide range of application in pharmaceutical and food industries due to its temperature
responsive properties, water solubility and non-toxic nature (Daniel-da-Silva et al., 2007).
Gum acacia is extracted from the stems of acacia tree and composed of acidic polysaccharide
having arabinose (27%), glucuronic acid (16%), galactose (44%), rhamnose (13%) and
peptides (2-3%) (Al-Assaf et al., 2009). Due to emulsifying properties and controlled release
behavior, gum acacia is extensively used in pharmaceutical industry (Ali et al., 2009; Nishi
and Jayakrishnan, 2007).
Polysaccharides obtained from plant origin are versatile in nature and having a wide range of
application. Locust bean gum was used to develop mucoadhesive macromolecule with the
help of sodium alginate for the delivery of aceclofenac. Optimized formulation was able to
extend the drug release up to 10 h. Particle size was found in the range from 1.328 ± 0.11 to
1.428 ± 0.13 µm (Prajapati et al., 2014).
7
1.1.1. Hydrogels
Hydrogel is a three dimensional polymeric network which can absorb and retain a large
amount of water (Buwalda et al., 2014). Hydrogels are covalently or non-covalently
crosslinked. These physically or chemically crosslinking are responsible for water
penetrability in hydrogel and water holding capacity of the hydrogel which lead to the
development of various devices for biomedical applications. Hydrogels are designed or
obtained from natural, synthetic polymers or combination of both. Natural polymers are
composed of polysaccharides, natural polyesters, nucleic acids and proteins (gelatin,
collagen, silk fibroin and elastin) (Vlierberghe et al., 2011). Therefore, hydrogel has gained
many applications in biomedical field. Two of the important applications are in tissue
engineering and sustained drug delivery from several days to months (Hoare and Kohane,
2008; Kabanov and Vinogradov, 2009).
Hydrogels of synthetic (PEG, pHEMA and PVA) and semisynthetic origins (derivatives of
cellulose) are used in biomedical fields especially in the sustained and targeted delivery of
various drugs (hydrophilic or hydrophobic drugs, protein based drugs etc.) (Ford et al., 1985).
Generally, hydrogels are biocompatible due to their water holding ability and
physicochemical resemblance with the extracellular fluid both by mechanically and
compositionally. Biodegradability of hydrogels was taken place through hydrolytic, altering
in pH or temperature and enzymatic pathways.
Hydrogels are formed by physically cross-linking of polymers chains which is triggered by
environmental factors (temperature, pH or ionic concentration) or physicochemical
interactions (hydrogen bonding, charge condensation or stereocomplexation) (Hoare and
Kohane et al., 2008).
8
Temperature and pH responsive hydrogel was fabricated using β-cyclodextrin, 2-
methylacrylic acid and N,Nʹ-methylene diacrylamide. Synthesized hydrogel was investigated
for the controlled and sustained release of atorvastatin (Samanta and Ray, 2014a). Hydrogel
was found to have high swelling at pH 8.06 while exhibited less swelling at pH ≤ 3.84 and ≥
10.34. Swelling of hydrogel is directly proportional to the temperature of the media. Drug
release from the hydrogel was high (90.5%) at pH 8.06. Solubility of atorvastatin was also
improved after incorporation in hydrogel (Yang et al., 2016). Copolymerization of sodium
alginate and acrylamide with the aid of N,Nʹ-methylene bisacrylamide (MBA) was
successfully achieved and characterization by NMR, FTIR, XRD, thermogravimetric analysis
(TGA) and SEM. Swelling of hydrogel was found to be pH dependent and drug release from
hydrogel followed the kinetic models.
Modified form of chitosan was synthesized and used to separate the heavy metal ions from
aqueous solvents by adsorption through chelation (Kandile and Nasar, 2009). Genipin, a
water soluble crosslinking agent, after reacting with chitosan produce a fluorescent hydrogel.
Chitosan/genipin hydrogel was used for sustained release formulations, cartilage substitutes
due to elasticity, encapsulating agent for the delivery of biological products and wound
healing medications (Muzzarelli, 2009). Superporous hydrogel was prepared with the help of
chitosan to deliver the insulin and other protein or peptide drugs through mucoadhesive
delivery systems (Yin et al., 2007). Synthesis of hydrogel was confirmed by FTIR, NMR,
SEM and DSC analyses. Enhanced loading capacity with more than 90% insulin release
within first hour of the delivery was achieved.
Xanthan and chitosan based hydrogel was prepared by ionic complexation method. Newly
synthesized hydrogel was used as a controlled release material for the delivery of
theophylline which was evaluated for the treatment of chronic pulmonary obstructive disease
9
(Popa et al., 2010). Chitosan based hydrogel was synthesized by copolymerization of MBA
and acrylic acid. Characterization of hydrogel was performed through FTIR, TGA, NMR,
XRD and swelling behaviour. Theophylline and tinidazole release study was carried out and
rapid release was observed at pH 7.6 than at pH 1.5 (Samanta and Ray, 2014b).
Psyllium hydrogel was synthesized through chemical method with the help of polyvinyl
alcohol and used for the controlled release of rabeprazole. Haemo-compatibility of the
synthesized hydrogel was monitored and haemolytic index was found <5%. Due to haemo-
compatibility of hydrogel and antiulcer nature of psyllium, newly synthesized hydrogel is
used as a carrier for sustained delivery of an antiulcer drug (Singh et al., 2012). Psyllium
based hydrogel was used for colon specific delivery of tetracylcline HCl. Ammonium
persulfate (initiator) and N,Nʹ-methylenebisacrylamide (crosslinker) were used in synthesis of
hydrogel. Formation of hydrogel was confirmed by FTIR. Swelling of hydrogel and drug
release study was carried out in different buffers and results have shown the drug release
followed the Fickian diffusion (Singh et al., 2008a).
Cellulose based hydrogel was develop in order to improve the mechanical strength, swelling
properties, biocompatibility and antimicrobial activity necessary for the used in disposable
diapers. Cellulose and quaternized cellulose was crosslinked in an aqueous NaOH/urea
solution to synthesize highly swellable, superabsorbent and biodegradable hydrogel.
Antimicrobial activity against Saccharomyces cerevisiae was excellent and due to the
attraction of anionic microbial membrane by cationic hydrogel leading to distraction of
microbial membrane (Peng et al., 2016).
Hydroxyethyl cellulose and gelatin were blended to prepare microspheres and evaluated as a
sustained release delivery system for theophylline. Formation of IPN was confirmed by
10
FTIR, XRD, DSC and SEM. Equilibrium and dynamic swelling of these microspheres was
determined and drug release followed the non-Fickian diffusion (Kajjari et al., 2011).
1.1.2. Linseed
Flax (Linum usitatissimum L.; Syn: Alsi) is one of the oldest crop which is known to human
being and cultivated for both seeds and fibers. Seeds of Linum usitatissimum L. (Linseed;
Syn: Flaxseeds, Alsi seeds) is a good source of edible oil which is used as a nutritional
supplement. Two varieties of linseed (yellow and brown) are cultivated to get oil, fibers and
polysaccharides. Both varieties have similar constituents including oil, carbohydrates and
proteins. Flaxseeds have shown many benefits and also enhanced the antitumor effect of
tamoxifen (Chen et al., 2007a).
In a study, flaxseed gum was extracted by using hot water and composition of
polysaccharides was determined. During extraction process, temperature of water was
maintained at 85-90 °C and pH from 6.5-7.0. Gum was separated after precipitation with
ethanol and then fractionated through ion exchange chromatography. After thorough analysis
by NMR, gel filtration chromatography and chemical treatment, presence of L-arabinose, D-
xylose, L-fucose, L-rhamnose, D-galacturonic acid and D-glalactose was confirmed.
Rheological properties of the flaxseed gum were also studied and shear thinning behavior
was observed at higher concentration (Cui et al., 1994).
Other study has reported that linseed were mixed with water and stirred at 100, 80, 25 and 4
°C for 2 h to obtain mucilage. Extracted mucilage was separated through filtration and treated
with cetyltrimethylammonium bromide to separate the low and high density polysaccharides.
These polysaccharides were analyzed through GPC, NMR, gas liquid chromatography and
acid hydrolysis. Rhamnose, galactose, arabinose, xylose and galaturonic acid are the main
components of linseed mucilage. It was also determined that the viscosity of linseed mucilage
11
is greatly affected by the pH of water and presence of electrolytes (Fedeniuk and Biliaderis,
1994).
Flaxseed mucilage is mainly composed of arabinoxylane (75%) and studied by SEC/MALLS.
After analysis, it was found that there are three distinctive proportion of arabinoxylane, i.e.,
5000000 g/mol, 200000 g/mol, and 1000000 g/mol. In depth analysis has shown that
formation of aggregates is due to the weak hydrogen bonding and can be reduced by
increasing the temperature. Rheological behavior was also investigated and presence of
hydrogen bond was also confirmed by addition of lyotropic and chaotropic salts (Warrand et
al., 2005a).
Flaxseed was evaluated for the inhibition of the breast tumors in combination with tamoxifen.
Ovariectomized mice induced with breast cancer cell line (MCF-7) were treated with
flaxseeds (5 and 10%) and tamoxifen alone (5 mg/tablet) or in combination for a period of 16
weeks (Chen et al., 2007). At the end of study, tumor was analyzed for apoptosis, cell
proliferation and expression of signal transduction and estrogen related genes. Results have
indicated that flaxseed alone has no impact on the growth of tumor and combination with
tamoxifen reduces the tumor size.
Effect of water extracted flaxseed gum was evaluated as a blood sugar and cholesterol lowing
agent in diabetic patients (Type II). Flaxseed gum was administered to 60 patients for a
period of 3 months. At the end of study, the values of fasting blood sugar, total cholesterol
and low density lipids were dropped from 154 to 136 mg/dl, 182 to 163 mg/dl and 110 to 92
mg/dl, respectively (Thakur et al., 2009).
Water extracted flaxseed gum was dried by adopting different methods (freeze drying,
ethanol precipitation then oven drying at 80 °C, vacuum drying, spray drying, drying in hot
air oven at 80 °C and 105 °C). Color of flaxseed gum was observed after drying as well as in
12
aqueous solution. Effect of drying on Zeta potential, emulsion, gelling and foaming
properties of flaxseed gum was evaluated. It was observed that the method of drying has
pronounced effect on the functional properties of flaxseed gum and appropriate method
should adopt to get potential benefits (Wang et al., 2010).
Rheological properties of flaxseed gum solution were studied after high pressure
homogenization (Wang et al., 2011b). During this process, temperature of solution was
increased and as a result viscosity was decreased. Power law was used to define the relation
of shear rate and shear stress. Conductivity of flaxseed gum solution and gelling temperature
were found to be independent to the homogenization process while clarity of solution was
significantly improved by this process.
Mucilage was extracted from flaxseed and subjected to freeze drying after precipitation with
ethanol (Mazza and Biliaderis, 1989). Composition, foamability, solubility and moisture
sorption properties were determined and compared with guar gum and locust bean gum. High
viscosity of flaxseed mucilage was observed at pH 6.0-8.0 and low in aqueous solution of
NaCl.
Gelling strength of flaxseed gum was studied along with factors effecting on the gel forming
ability (Chen et al., 2006). Gel forming temperature, pH and salt concentration was also
determined. Maximum gel strength was observed at pH 6-9. Addition of sodium ion
decreases the gel strength. Calcium chloride less than 0.3% are used to increase the gel
strength while higher concentration has negative effect. Addition of carrageenan in flaxseed
gum increased the viscosity but lowered the syneresis.
Linseed mucilage was separated by alkali extraction method. Seeds were boiled with sodium
bicarbonate and extracted mucilage was neutralized with acetic acid. Mucilage was air dried
and used to prepare mucoadhesive microspheres for effective delivery of venlafaxine using
13
spray drying method (Nerkar and Gattani, 2011). Microspheres were thoroughly
characterized by DSC, SEM and XRD and evaluated for swelling behavior, mucoadhesion, in
vitro and in vivo drug release and stability study. Results indicated that improved
bioavailability of venlafaxine was observed when compared to traditional oral route.
Flaxseed mucilage was purified by size exclusion and ion exchange chromatography and
three main polysaccharides were separated (Warrand et al., 2003). These polysaccharides
constitute the main component of the mucilage (75%). Molecular weight of these three
polysaccharides was 1.7 × 104 g/mol, 6.5 × 10
5 and 1.2 × 10
6.
Laws of extraction were applied on flaxseed mucilage extraction process (Ziolkovska, 2012).
For this purpose, extraction was carried out under different conditions of temperature (40-100
°C), duration of seed soaking in water (0-1 h), water to seed ratio (5-30 to 1) and mixer
stirring speed (0-240 rpm). Optimum temperature and water to seed ratio to get maximum
flaxseed mucilage are 80 °C and 25:1, respectively. Different mathematical equations were
used to explain the effects of different parameters on the yield of flaxseed mucilage.
Analysis of polysaccharides of flaxseed meal was performed by viscometry and light
scattering techniques. Size exclusion chromatography revealed the presence of small and
large molecular weight fraction (3.1 × 105 Da) and (1.0 × 10
6 Da), respectively (Goh et al.,
2006). SEC analysis of the yellow flaxseed mucilage confirmed three distinct types of
polymers. Molecular weight distribution of these polymers is analyzed by multi laser light
scattering and found high molecular weight (9.3 × 105 and 5.7 × 10
6 g mol
-1) and low
molecular weight (3.2 × 105 g mol
-1) fractions (Warrand et al., 2005b).
Soybean oil (10%, v/v) was emulsified using the combination of flaxseed gum (0.05-0.5%,
w/v) and soybean protein isolate (1%, w/v). Objective was to observe the emulsifying
behavior of soybean protein isolate in the presence of flaxseed gum. Formulated emulsion
14
was analyzed for stability, turbidity, surface charge, particle size and rheology (Wang et al.,
2011c). Initially, zeta-potential and turbidity of emulsion decreased as the concentration of
flaxseed gum was increased (up to 0.1%) but increased at high concentration of gum up to
0.35% (w/v). Particle size decreased with the increase of gum concentration up to 0.1% (w/v)
and increased beyond this concentration. Therefore, protein-based emulsions can be
stabilized with the addition of flaxseed gum.
Mucilage extracted from seven varieties of Italian flax was investigated for their
physiochemical, sensory and functional properties (Kaewmanee et al., 2014). Chemical
composition of polysaccharides was also determined by acid hydrolysis. Presence of
rhamnogalacturonan as a backbone of the polysaccharide was identified by NMR analysis.
Mucilage from all seven varieties was tasteless but varies with respect to viscosity, zeta-
potential, conductivity, sugar content, swelling ability, foaming capacity and emulsifying
properties.
Mucilage extracted from flaxseed hull was fractionated by ion exchange chromatography into
acidic and neutral fractions. Rhamnogalacturonans having two acidic fractions of molecular
weights 1510 kDa and 341 kDa while arabinoxylans, being a neutral fraction, having
molecular weight of 1470 kDa. Acidic and neutral fractions have shown Newtonian and
pseudoplastic flow, respectively. Physical (stability and molecular weight distribution),
chemical (composition of polysaccharide), functional (emulsify properties and surface
tension) and rheological properties (viscosity and critical concentration) of mucilage was
thoroughly examined (Qian et al., 2012b). Structure of rhamnogalacturonans was determined
after methylation and by using 1D/2D NMR spectroscopy. Rhamnogalacturonans is highly
branched structure with degree of branching 0.55 and composed of rhamnogalacturonan-1
backbone connected with homorhamnan and homogalacturonan (Qian et al., 2012a).
15
Four different methods were used to extract mucilage from flaxseed meal and optimize the
extraction process to get maximum and pleasant taste mucilage product (Singer et al., 2011).
In precipitation method, flaxseed meal was mixed with water and raised the pH up to 9 with
the addition of NaOH solution. Mixture was then treated with HCl to decrease the pH and
centrifuge to separate the precipitates. In second method, instead of HCl, ethanol was used to
get precipitates. Boiling water was used to get the mucilage and separated from the flaxseed
meal by ethanol precipitation. Hot water dispersed flaxseed meal was treated with enzymes
and then filter to obtain mucilage. After physicochemical evaluation of these four processes,
second method was considered the most appropriate to extract good quality mucilage for food
industry.
Investigation for the polysaccharides composition among 109 varieties of flaxseed from 12
geographical regions were conducted and found in a range from 3.6-8.0% (Oomah et al.,
1995). After acid hydrolysis, rhamnose, xylose, galactose, glucose, fucose and arabinose
were the main component of flaxseed mucilage. Depending on the variations in yield of
different components of mucilage, desired carbohydrates were obtained from flaxseed
mucilage.
Noodles were prepared with the addition of different concentration of flaxseed mucilage as a
replacement of wheat flour (Kishk et al., 2011). Physicochemical properties and noodle
quality were analyzed and results were compared in term of swelling index, nitrogen and
cooking loss, cooking yield, cooking time and cooking temperature. Good quality noodles
were prepared with 3% flaxseed mucilage and cooking temperature range from 68.2-70 °C.
Results were also analyzed statistically and after sensory evaluation, noodles prepared with
flaxseed mucilage have shown improved texture and pleasant in taste.
16
Effect of daily intake of flaxseed and sunflower seed on lipid profile of postmenopausal
women was studied for six weeks (Arjmandi et al., 1998). On examine the lipid profile at the
end of study, LDL level was significantly reduced in flaxseed treated group as compared to
sunflower seed treated group. Both groups have not shown any effect on HDL and
triglyceride level. LDL lowering ability of these two types of seeds is supposed to be due to
linoleic or α-linolenic acid component.
Emulsion was prepared using whey protein isolate solution in imidazole and soybean oil
under high speed mixing (Khalloufi et al., 2009). pH of emulsion was adjusted to pH 3.5 and
maintained at 4 °C. Various concentrations of flaxseed gum was prepared in imidazole at pH
7.0 and then adjusted to 3.5 before adding in the emulsion. Physicochemical properties of the
synthesized emulsion were investigated by particle size analysis, zeta potential, viscosity and
separation behavior. Results indicated that emulsion was stabilized by negatively charged
flaxseed gum.
Polysaccharide analysis of flaxseed mucilage was carried out after acid hydrolysis at various
molar concentrations and temperature (Emaga et al., 2012). Effect of chemical and enzymatic
hydrolysis was also studied. Hydrolysis with trifluoroacetic acid resulted in less damage to
the sugar components of flaxseed gum as compared to HCl and H2SO4. Moreover, enzymatic
degradation also reduced the destruction of sugars. Monosaccharide composition was
determined but quantification was found to be difficult due to strong bonding between acidic
and neutral fractions.
Flaxseed mucilage is composed of rhamnogalacturonan I and arabinoxylan and in detail study
was performed to analyze the structure of polysaccharides. After acid hydrolysis and
evaluation through GPC and SEC, presence of arabinose, xylose, galactose, glucuronic acid
17
and fucose was verified along with determination and analysis of linkages (Naran et al.,
2008).
Flaxseed mucilage was extracted by adopting three different methods and then analyzed the
mucilage for different physical parameters (Fabre et al., 2015). Ultrasonic treatment of the
hydrated seeds secreted maximum mucilage when compared with other treatments, i.e.,
microwave and magnetic stirring. Monosaccharide composition, galacturonic acid
concentration and viscosity of extracted mucilage from all three methods were evaluated. It
was found that high yield was obtained from ultrasound treatment though the viscosity and
concentration of high molecular weight fraction of mucilage was decreased.
Composite material was prepared from flax fiber and mucilage extracted after treating with
water at 20 °C and 40 °C. In order to prepare a water insoluble biocomposite material,
mucilage was treated with glutaraldehyde, as a crosslinking agent, and glycerol, as a
plasticizer. Water sorption and swelling was reduced after crosslinking while increased the
mechanical strength and rigidity (Alix et al., 2008).
Structure of polysaccharides derived from linseed mucilage was thoroughly characterized by
Muralikrishna et al. in 1987. Neutral fraction is composed of and D-galactose, L-arabinose
and D-xylose (1:3.5:6.2) while in acidic fraction, L-rhamnose, D-galacturonic acid, L-
galactose and L-fucose (2.6:1.7:1.4:1) are the main components. In neutral fraction, galactose
and arabinose are attached with the backbone of xylan. Galactose and fucose are linked as a
side chain unit with D-galactopyranosyluronic acid and rhamnopyranosyl residue
(Muralikrishna et al., 1987).
High molecular weight fractions were separated from flaxseed cake using anion exchange
chromatography. Molecular weights of arabinoxylan (arabinose to xylose ratio, 0.32) and
galactoglucan were 8.46 × 105 and 6.5 × 10
4, respectively. Furthermore, two more fractions
18
were also separated having molecular weights of 3.1 × 105 and 1.3 × 10
5. Rheological study
of the 2% solution of flaxseed gum has shown the insignificant viscosity for use as a
texturing component (Warrand et al., 2005c).
Mucilage extracted from flaxseed was crosslinked with epichlorohydrin and plasticized with
glycerol to prepare a composite material by varying the reaction conditions. Synthesized
composite material has shown very low tendency to swell. Tensile strength and other
parameters were determined and better results were found with the composite material
prepared by using glutaraldehyde as a crosslinking agent (Paynel et al., 2013).
1.1.3. Modification of polysaccharides
Naturally occurring polysaccharides are being widely used in many fields of biomedical,
pharmaceuticals, cosmetic, food industry and bioengineering. To improve some of their
properties, polysaccharides are modified to get the desired results. Therefore, the researchers
are modifying the naturally occurring polysaccharidal material by different ways, i.e.,
copolymerization, grafting, etherification, carboxymethylation, sulphonation, oxidation,
amide formation and esterification etc.
In carboxymethylation, polysaccharides are reacted with monochloroacetic acid/sodium salt
in an alkaline medium. As a result of this simple process, a variety of properties are
introduced in polysaccharide including swellability, pH responsive swelling and stimuli
responsive on-off switching etc. Cellulose (Klemm et al., 2002), chitin and chitosan (Roberts
1992), dextran (Huynh et al., 1998), pullulan (Shingel 2004) and starch (Shogrun 1998) are
successfully carboxymethylated and have number of applications. Arabinoxylan, isolated
from Ispaghula, was treated with sodium monochloroacetate in the presence of NaOH to form
carboxymethylated arabinoxylan (Saghir et al., 2008). The resulted product was verified by
19
FTIR and NMR analyses. Maximum degree of substitution of the carboxymethylated
arabinoxylan was calculated as 1.81 and product was found soluble in water.
Oxidation of polysaccharides is performed to introduce the aldehyde group which is further
used for grafting of different molecules on the polymer chain and for crosslinking reactions
with amines (Rinaudo 2010; Sarymsakov 1975). These modifications reduce the stiffness of
polysaccharides and have potential application in drug delivery. Metaperiodate was used to
oxidize the hydroxyethyl cellulose and methylcellulose to enhance the hemostatic properties
(Gibbons 1956). Flexibility and elasticity of alginate was decreased after oxidation (Gomez et
al., 2007; Andresen et al., 1977). Dextran was also oxidized with sodium periodate and
crosslinked with adipic acid dihydrazide. The resulted hydrogel was evaluated for swelling
and mechanical properties and degradation behaviour (Maia et al., 2005). Carboxyl groups
were introduced in pullulan through oxidation reaction which make it more water soluble
(Spatareanu et al., 2014).
In etherification, polysaccharidal hydrogels reacted with alkylating agents. Cellulose ethers
were prepared in homogeneous reaction condition (Takaragi et al., 1999). Cellulose was first
dissolved in a solution of dimethyl acetamide/Lithium chloride and methyl, hydroxyethyl and
hydroxypropyl ethers were synthesized using epoxide or iodomethane as alkylating agents
(McCormick and Callais, 1987). Pullulan was also transformed into propyl ether and butyl
ether with different degree of substitution when treated with sodium hydroxide and alkyl
bromide in a mixture of DMSO/H2O (Shibata et al., 2002). Carboxymethyl ether of guar
gum, xylan and konjac glucomannan were synthesized with DS value of 0.8, 1.2 and 0.3,
respectively (Lindblad and Albertsson 2004; Petzold et al., 2006). Ethylation of arabinoxylan
was performed with ethyl iodide in the presence of NaOH. Product was soluble in DMSO and
degree of substitution was calculated as 0.61 (Saghir et al., 2009).
20
Generally, esterification involved a reaction between an alcohol and an acid. There are
number of methods used for the esterification (Heinze et al., 2003). Cellulose was treated in
homogenous reaction condition with acid chloride in the presence of a base, triethylamine
(McCormick and Callais, 1987). In another method, acetylation of cellulose was carried out
in an ionic medium using acetic anhydride. Highly substituted cellulose acetate (DS up to 3)
can be prepared by simply treated with acetic anhydride or acetylchloride (Wu et al., 2004).
Starch was also esterified by acyl chloride and pyridine in a solution of DMAc/LiCl at 100 °C
(Grote and Heinze 2005). Xylan acetate was synthesized using acetic anhydride and pyridine
while DMF as a reaction medium (Belmokaddem et al., 2005). Acetylation of alginate and
chitosan was carried out in homogenous reaction conditions using DMSO/TBAF or
DMAc/LiCl as a solvent and acetic anhydride/pyridine or acid halides as acylating agent
(Pawar and Edgar 2011; Badawy et al., 2005).
Polysaccharides possess some unique properties based on the types of glycosidic linkages,
molecular weight (Li and Shah 2014), polymer chain configuration (Hattori et al., 1998),
solubility (Lu et al., 2012) and some special configurations (Wang et al., 2010). Types of
glycosidic linkages and composition of monosaccharides is the key to determine the
properties of polysaccharides. Modification at molecular level in the polysaccharides changed
or altered the properties of these polysaccharides and achieved by chemical (sulfation,
carboxymethylation, phosphorylation, selenization, acetylation, alkylation and acid/alkali
degradation), physical (ultrasonic disruption, microwave exposure and radiation treatment) or
biological (enzymatic) processing (Li et al., 2016). These methods are adopted to increase the
water solubility (Jung et al., 2011; Vasconcelos et al., 2013), reduce molecular mass (Chen et
al., 2013; Parvathy et al., 2005), reduce intrinsic viscosity (Li and Feke 2015; Yan et al.,
2015) and increase thermal stability (Carbinatto et al., 2012).
21
1.1.4. Stimuli responsive properties of polysaccharidal hydrogels
Smart hydrogels have the tendency to respond against different stimuli which may be
physical or chemical, i.e., light (Juodkazis et al., 2000; Tatsuma et al., 2007), pH (Zhang et
al., 2007; Qu et al., 2006: Kim et al., 2006), electric and magnetic field (Kwon et al., 1991;
Satarkar and Hilt, 2008; Wang et al., 2009), temperature (Yoshida et al., 1995; Ju et al., 2006)
and different species (Holtz and Asher, 1997; Chu et al., 2004). On the basis of these stimuli
responsive behaviour, hydrogels are being used in gene and controlled drug delivery systems
(Qiu and Park, 2001; Cheng et al., 2008), soft machines (Calvert, 2008), biosensors (Beebe et
al., 2000), liquid microlenses and bio or chemical separations (Ju et al., 2009; Yang et al.,
2008; Xie et al., 2009), etc.
Potato starch was modified and then used to synthesize semi-interpenetrating polymer
network (SIPN) composite with vary the degree of crosslinking (Dragan and Apopei, 2013).
Synthesized hydrogel was evaluated by FTIR and SEM. Swelling kinetics was determined in
distilled water and stimuli responsive properties of SPIN was observed in water/ethanol,
water/1 M NaCl and at pH 8/pH 1. A superfast swelling was seen in all formulations.
Swelling shrinking behaviour was found to be dependent on the incorporated functional
group and the ratio of the cross-linker.
IPN based on the derivatives of acrylamide and acrylic acid was synthesized by varying the
reaction conditions (Diez-Pena et al., 2002). Swelling response of these hydrogels was found
to be dependent on the nature of polymer, temperature and pH of the swelling media.
Formation of hydrogen bond between amide and carboxyl moiety has shown impact on the
swelling behaviour of hydrogel in acidic environment and water.
Guar gum and acrylic acid were used in different combinations to synthesize a hydrogel and
confirmed by FTIR, DSC and SEM analysis (Huang et al., 2007). Electrostatic interaction
22
between anionic group of acrylic acid and cationic group of guar gum was the main driving
force for the synthesis of hydrogel. Swelling of hydrogel is influenced by the concentration of
hydrophilic group in the composition of hydrogel. Swelling of hydrogel is increased with the
increase in the pH of the media. Hydrogel has shown swelling deswelling behaviour at pH
7.4 and 5.0, respectively. Ketoprofen was loaded in hydrogel and release was monitored at
different pHs. Release was also observed at pH 2.2 for 1 h, pH 6.8 for 4 h and pH 7.4 for 10
h.
Stimuli responsive hydrogel was prepared from poly (acrylic acid) and poly (aspartic acid)
and evaluated for pH, salt and temperature induce swelling (Zhao et al., 2006). It is noted that
the swelling ratio increased by increasing the temperature of the swelling media from 40-60
°C. pH responsive swelling deswelling was also observed and strongly dependent on the
concentration of poly (aspartic acid) in the hydrogel. An inverse relation between the
swelling capacity and concentration of poly (aspartic acid) was observed. Swelling of
hydrogel was high in deionized water as compared to other biological fluids i.e., Hank‘s
solution, glucose, urea, synthetic urine and physiological saline water.
Dynamic swelling and swelling-deswelling behaviour of an anionic hydrogel was monitored
under different conditions of pH and ionic strength (Kare and Peppas, 1995). Dynamic and
equilibrium swelling was determined in acetate buffer with a pH range from 3.2-7.6. Ionic
strength of the swelling media was maintained with the appropriate addition of NaCl. Due to
the ionization of the hydrogel at high pH, water uptake ability of the hydrogel increases
which results in the increased swelling of hydrogel. At pH 4.0, with the increase in ionic
strength from 0.0074-0.08M, dynamic swelling of the hydrogel was not affected. At pH 7.0,
due to the ionization of hydrogel, the increase in ionic strength has shown its impact on the
dynamic and equilibrium swelling.
23
Composite cryogel was synthesized in the presence of chitosan by crosslinking of acrylamide
with N,N'-methylenebisacrylamide (Dragan et al., 2012). Swelling behaviour was observed
from pH 1-12 and it was noted that the composite cryogel has not shown any swelling up to
pH 3 and then suddenly started to swell from pH 4-12 with higher rate. This swelling was due
to the presence of hydrophilic group of the gel whose ionization resulted in the repulsion.
This gel was used for the separation and removal of cationic organic dye.
Psyllium and polyacrylamide based hydrogel was prepared using N,Nʹ-methylenebis-
acrylamide as a crosslinker (Singh, 2007). Swelling trend of the polymeric network was
thoroughly studied at various temperatures, pH and different concentrations of NaCl.
Swelling was found to be dependent on the concentration of the crosslinker. Abrupt swelling
and deswelling was observed in distilled water and NaOH solution. Structural
characterization of hydrogel was investigated through SEM, TGA and FTIR.
Hydrogel having polyampholytic nature was synthesized by interaction between negatively
and positively charged polymers (Mohan and Geckeler, 2007). Tween 80 (non-ionic),
dodecylpyridinium chloride (cationic) and sodium dodecyl sulfate (anionic) were used as a
surfactant to prepare various types of interpenetrated polymer network. High swelling was
observed in hydrogel with cationic surfactant at pH 5-6. Saline responsive swelling was also
seen in various salt solutions.
Chitosan and poly(vinyl alcohol) were cross-linked by glyoxal to produce a gastro retentive
superporous hydrogel for the delivery of rosiglitazone maleate (Vishal and Shivakumar,
2010). Synthesized hydrogel has the ability to swell in acidic pH while deswell in basic pH.
A decrease in swelling was observed while increasing the ionic strength of the swelling
media. Swelling is dependent on the concentration of the chitosan and cross-linker. Release
of rosiglitazone was sustained for 6 h in acidic media.
24
Chitosan was grafted with acrylamide under the influence of a cross-linker,
methylenebisacrylamide (Pourjavadi and Mahdavinia, 2006). Chitosan grafted hydrogel was
evaluated for swelling behavior at various pHs and swelling shrinking response in acidic and
basic media, respectively. Rate of swelling was found to follow second order kinetics.
Swelling capacity was reduced with the increase in the ionic charge of the swelling media.
Superabsorbent hydrogel was prepared by mixing the aqueous solution of starch and
polyacrylonitrile under mild heating through alkaline hydrolysis (Sadeghi and Hosseinzadeh,
2008). Salt and pH responsive swelling was evaluated and found highly swellable material in
basic environment and in NaCl solution. Reversible swelling shrinking behavior at basic and
acid pH, respectively, made it a suitable material for various applications.
Hydrogel based on PEG was synthesized with prime objective for molecular recognition and
swelling deswelling response against different ionic solutions (Tominaga et al., 2013).
Swelling properties of hydrogel can be controlled by changing the composition and
concentration of reactants.
Composite hydrogel was prepared by copolymerization using sodium acrylate, HEC and
medicinal stone (Wang et al., 2011a). Formation of hydrogel and medicinal stone distribution
in the synthesized composite was analyzed by TGA, FTIR, energy dispersive spectroscopy,
FESEM, TEM and elemental map. By increasing the concentration of medicinal stone, the
swelling capacity of hydrogel was increased up to 400%. Hydrogel was evaluated for
swelling deswelling behavior at basic and acidic pH and also in water and normal saline
solution. Due to deswelling potential in surfactant solution, this hydrogel has the ability to
play an important role as absorbing agent.
25
1.1.5. Toxicological studies of polysaccharides
In order to determine the toxic characteristics (if any) of a compound, the initial screening
was performed through acute toxicity study. Toxicity of a compound is evaluated through
three types of studies, i.e., acute or sub-acute (24 h to 28 days), sub-chronic (90 days) and
chronic (6 to 12 months) toxicity studies.
Chitosan was reported as a carrier for nasal drug delivery by Aspden and coworkers and
toxicological study was carried out on different animals (guinea pig, frog, rat and mouse) as
well as on human volunteers. For the development of nasal delivery system based on
chitosan, cilia beat frequency was determined by repeatedly applying chitosan solution as a
marker of nasal toxicity on nasal tissues of guinea pigs for 28 days (Aspden et al., 1997). Rat
nasal perfusion method was employed to investigate the extent of insulin absorbance through
nasal epithelial membrane (Aspden et al., 1996).
Polysaccharide extracted from turmeric was evaluated for acute oral toxicity and
mutagenicity (Velusami et al., 2013). Oral toxicity study was conducted on Wistar rats and
results indicated the safety of turmeric polysaccharides up to the level of 5g/kg body weight.
Mutagenicity testing was assessed by chromosome aberration, bacterial reverse mutation test
and micronucleus testing. Results obtained from mutagenicity and acute oral toxicity studies
have proved the safety of turmeric polysaccharide.
α-Cyclodextrin, being a water soluble dietary fiber was investigated for oral toxicity study
over a period of 13-week at various concentrations (Lina and Bär, 2004). During this study
period, different parameters were considered as a marker of potential toxicity, i.e.,
consumption of food and water, body weights calculation, biochemical and hematological
evaluation, histopathological observations and organ to body weight ratio. Moreover, urine,
feces and ophthalmic examination were also carried out. At the end of study, treatment
26
related mortality has not seen. Mild diarrhea was observed with soft stool. Food and water
intake was slightly increased. Hematological, biochemical and histopathological evaluation
did not show any abnormality. Intake of α-cyclodextrin up to 20% of body weight of rats was
found to be safe.
Meratrim, an extract from flowers and fruits of Sphaeranthus indicus and Garciania
mangostana, respectively were used for weight management (Saiyed, et al., 2015). Safety
assessment of Meratrim intake was evaluated including acute and sub-chronic toxicity and
animal toxicological studies and was carried out on Sprague-Dawley rats. LD50 level was
found to be > 5 and 2 g/kg body weight as determined by acute and sub-chronic toxicity
study. Meratrim was non-irritating to the skin, slightly irritating to eye and non-mutagenic.
No sign of mortality, morbidity and other side effects were observed during the whole study
period. These results when combined with the human clinical trials indicated the safe use of
Meratrim.
Safety of arabinoxylan, isolated from Ispaghula husk, was investigated through acute toxicity
study carried out in mice and rabbits (Erum, et al., 2015). Different doses of arabinoxylan
(1,5 and 10 g/kg body weight) was administered in animals and kept under observation for up
to 14 days. Biochemical, histological and hematological parameters was monitored and did
not show any significant abnormalities. Moreover, effect of arabinoxylan on heart of frog was
also monitored and found safe with respect to heart rate and vascular contraction.
Partially hydrolyzed guar gum (K-13) was examined through acute and subchronic toxicity
studies (Koujitani, et al., 1997). For acute toxicity study, guar gum (6 g/kg) was given to
mice and rice. Various concentrations (0.2, 1 and 5%) of guar gum were used for subchronic
toxicity studies. Mutagenicity study was performed using bacteria by adopting reverse
mutation test. At the end, lethal dose (LD50) was more than 6 g/kg and also no death was
27
found. No significant findings were seen in subchronic toxicity study and also found
mutagenically save.
An exopolysaccharide (Bacterial Cellulose) was isolated from sugarcane molasses and
evaluated for acute toxicity study along with antigenotoxicity, cytotoxicity and genotoxicity
studies (Pinto et al., 2016). Bacterial Cellulose (2 g/kg body weight) was administered as a
single dose to Wistar rats and was observed for any sign of toxicity. Results indicated that
Bacterial Cellulose is not genotoxic, cytotoxic and acutely toxic.
1.2. Polysaccharides based drug delivery systems and pharmaceutical applications
1.2.1. Sustained release of NSAIDs from polysaccharidal materials
Non-steroidal anti-inflammatory drugs (NSAIDs) are prescribed as antipyretic, analgesic and
anti-inflammatory agent. NSAIDs act by inhibiting the enzyme, cyclooxygenase (COX),
which ultimately prevent the synthesis of prostaglandins.
Diclofenac is a phenylacetic acid derivative and its chemical name is 2-(2,6-dichloranillino)
phenylacetic acid synthesized for the first time by Alfred Sallmann and Rudolf Pfister 1973.
Diclofenac is marketed as sodium (Fig. 1.1) or potassium salt for inflammation, pain
associated with kidney or gallstones, fever, dysmenorrhea, arthritis (rheumatoid arthritis and
osteoarthritis), dental pain and endometriosis.
Diclofenac is rapidly absorbed after oral administration and absorption is dependent on dose,
salt form, composition and condition of GIT. Due to short half-life, frequent administration of
diclofenac is required for therapeutic efficiency (Davies and Anderson, 1997). Diclofenac is
also available in enteric coated, extended and sustained release formulations.
28
Controlled release of diclofenac sodium was achieved by preparing microspheres in which
sodium alginate and calcium chloride were used as a polymer and cross linking agent,
respectively (Gohel and Amin, 1998). Prolong release of diclofenac sodium was evaluated
from tablet formulation prepared with the help of polycarbophil and release profile was
compared with a commercially available product, ―Voltaren‖. In vitro and animal trials have
shown that the formulation was bioequivalent to ―Voltaren‖ (Hosny, 1996).
Drug release mechanism (either swelling or erosion of the polymer) from tablet formulation
prepared with poly(D,L-lactic acid) was investigated. It was observed that the release of
diclofenac sodium was governed both by swelling and erosion of the tablets. pH of the media,
interaction between drug and polymer, porosity and nature of the drug (acidic or basic) are
the major factors which made influence on the release of drug from the polymeric matrix
tablets. Extended release of drug is achieved at pH 7.4 as compared to pH 5.4 (Proikakis et
al., 2006).
Fig. 1.1. Structure of diclofenac sodium.
Sustained release formulation of caffeine was prepared with the aim to attain the prolong
alertness for 8-12 h. Drug and poly(ethylene oxide) were mixed to make an erodible tablet for
the sustained release of caffeine. In vitro release study and pharmacokinetic study showed the
initial burst release and then maintained a sustained systemic concentration for 8 h (Tan et al.,
2006).
29
Release retarding potential of karaya gum and xanthan gum was explored using two model
drugs, diclofenac sodium and caffeine, having different aqueous solubilities (Munday and
Cox, 2000). Gum swelling, erosion and drug release behaviour were studied. Drug release
from these two gums is dependent on the solubility of drug, agitation speed and drug to
polymer ratio. Drug release from these gums followed the zero order kinetic which is based
on erosion of the polymer matrix.
Caffeine (Fig. 1.2), being a neutral drug, is the drug choice for most of the researchers
working on the evaluation of new polymers as sustained release matrix. Talukdar and Kinget
evaluated the sustained release potential of xanthan gum using soluble neutral drug
(caffeine), soluble acidic drug (Indomethacin sodium) and insoluble acidic drug
(Indomethacin). Swelling (radial and axial) and drug release behaviour of xanthan gum
formulated tablets were observed in the media having similar physiological ionic strength.
Swelling of tablets and drug release from tablets followed Case I and Case II diffusion,
respectively (Talukdar and Kinget, 1995). Caffeine was also used to find out the release
mechanism from ethylcellulose matrix tablets. Furthermore, release kinetic models were
applied on release data and best fit model was selected (Neau et al., 1999). For the
assessment of core-in-cup drug delivery system as a sustained release matrix, caffeine and
ibuprofen were used as model drugs (Danckwerts, 1994). To develop such a system, core was
prepared with different grades of HPMC while cup shape tablet was made up of
ethylcellulose and carnauba wax. Drug release data of caffeine and ibuprofen have shown the
zero order release for 8 to 23 h depending on the drug to polymer ratio.
Stress and sleep deprivation exhibited negative impact on mood and performance of human
beings (Renner et al., 2007). Administration of different doses of caffeine (100, 200 and 300
mg) to 72 h sleep deprivation Navy trainees resulted in the improvement of visual vigilance,
30
sleepiness, reaction time, mood, memory and alertness. Caffeine (200 mg) was considered the
optimum dose to get rid of stress and problems related to sleeplessness (Lieberman et al.,
2002). Caffeine also enhanced the analgesic effect of acetaminophen when given in a
combination of 130 mg and 1000 mg, respectively. Absorption of acetaminophen was
accelerated by caffeine and relief in pain was observed for longer period of time.
Sorbitan esters containing niosomes were prepared for the effective delivery of caffeine from
dermal drug delivery systems (Khazaeli et al., 2007). Drug entrapment is dependent on the
lipophilicity of surfactants, particle size and charge of niosomes. Caffeine release was
controlled by both the erosion and diffusion mechanism. Due to high encapsulation efficiency
and stability of niosomes, this type of system is better than liposomes for topical
administration of caffeine.
Hydrolytic cleavage of poly(DL-lactic acid) (PLA50) in the presence of caffeine was
thoroughly examined in order to observe the effect of tertiary amine on breakdown of
polyester chains (Li et al., 1996). Caffeine was loaded in polymer by solvent evaporation
technique and mixture was processed into thin film and plate. Degradation of polymer is
effected by caffeine due to its catalytic action in the degradation process.
Pickering emulsion, a new form of emulsion, is prepared and compared with the conventional
w/o emulsion (Frelichowska et al., 2009). In both emulsions, caffeine was used as a
hydrophilic penetrant and effect of interfacial layer on drug release from both types of
emulsions was monitored. Due to the penetration of adsorbed caffeine on silica particles
through stratum corneum, pickering emulsion was considered the most suitable form of
emulsion.
Gastro retentive and sustained release formulation containing sodium alginate was developed
for the delivery of caffeine and chlorpheniramine (Stockwell et al., 1986). Presence of
31
sodium bicarbonate in the tablet formulation made a buoyant system. Drug release is
dependent on the nature and concentration of polymer.
For the treatment of cellulite, two topical formulations were designed containing caffeine as
an active ingredient (Hamishehkar et al., 2015). Caffeine hydrogel and caffeine loaded solid
lipid nanoparticles were prepared and evaluated through the permeation study and
histological study. Caffeine flux value through rat skin was high in case of nanoparticles as
compared to simple hydrogel. Complete breakdown of adipocytes were found in histological
study when treated with caffeine loaded solid lipid nanoparticles.
Fig. 1.2. Structure of caffeine.
Diacerein (Fig. 1.3) belongs to anthraquinone usually prescribed for the treatment of knee or
hip osteoarthritis (Nguyen et al., 1994; Pelletier et al., 2000). In vitro (Martel-Pelletier et al.,
1998) and in vivo (Moore et al., 1998) study has shown that diacerein exhibits its action by
blocking the activity and also production of a protein, interleukin-1 β, which is involved in
destruction of cartilage and commencement of inflammation (Moldovan et al., 2000; Yaron et
al., 1999) whereas, NSAIDs act by interfering with the synthesis of prostaglandin (Pelletier et
al., 1998). Diarrhea is the most common side effect of diacerein due to which daily dose of
diacerein is adjusted to half of the normal dose for first 14 to 28 days of the treatment. After
absorption, diacerein is deacetylated to rhein which is an active metabolite (Debord et al.,
1994).
32
Fig. 1.3. Structure of diacerein.
In a 16 weeks study on 484 patients of knee osteoarthritis, effective and safe dose of
diacerein was found to be 50 mg twice daily (Pelletier et al., 2000). A study was conducted to
observe the effect of diacerein on the degenerative changes in cartilage in osteoarthritis.
Results have proved that diacerein reinforced the repairing process and improved the
condition of joints (Hwa et al., 2001).
To increase the absorption of diacerein after oral administration, gastroretentive and
immediate release formulations were developed and compared with the already marketed
product (Mandawgade et al., 2016). Dissolution profile confirmed the immediate release of
diacerein and in vivo trial in healthy humans showed 1.2 fold and 1.7 fold increase in AUC0-
6h for gastroretentive and immediate release formulation, respectively.
Safety and efficacy of diacerein and piroxicam in the treatment of knee osteoarthritis was
evaluated and results are compared with each other. Piroxicam (20 mg/day) and diacereine
(100 mg/day) were administered to the patients for 16-weeks. At the end of study, diacerein
was found as effective as piroxicam in reducing pain but former has better safety profile
(Louthrenoo et al., 2007). Symptomatic efficacy of diacerein in treating osteoarthritis was
also observed by Bartels et al. during 6 month study. Diacerein was found safe and effective
against osteoarthritis and those patients can also take this medicine having problems with
NSAIDs (Bartels et al., 2010). For the treatment of osteoarthritis knee, combination of
33
diacerein and diclofenac sodium was used and evaluated the efficacy (Singh et al., 2012).
Diacerein and diclofenac sodium were given in a single dose of 50 mg and 75 mg per day,
respectively. Improvement in osteoarthritis knee was observed more rapidly when treated in
combinations of diacerein and diclofenac sodium than to the treatment with only one drug.
Niosomes based drug delivery system was developed for Diacerein to increase its dissolution
and also sustained the release the release of drug (Khan et al., 2015). Formulations were
designed with cholesterol and sorbitan monostearate with varying the ratios using reverse-
phase evaporation technique. Release studies have shown that niosomes sustained the release
of diacerein for 10 h and followed the zero order kinetics. Furthermore, non-Fickian and
anomalous transport mechanism was followed for the diacerein release.
Sustained release formulation of diacerein was developed using HPMC as a release retarding
agent (Meyyanathan et al., 2014). Drug release data was evaluated by kinetic models. Drug
release from the prepared tablets followed the first order kinetics and governed by non
Fickian diffusion. Pharmacokinetic parameters proved the sustained release of diacerein from
matrix tablet with increase in half-life.
Effectiveness of diacerein, glucosamine and NSAIDs in the treatment of osteoarthritis knee
was compared and results indicated that both glucosamine and diacerein are equally effective
but the former has fewer side effects (Kongtharvonskul et al., 2015).
Newly designed solid lipid nanoparticles were developed for the effective and sustained
delivery of diacerein with and without the loading of gold nanoparticles using solvent
emulsification-evaporation technique, hot melt encapsulation and microemulsification
method (Rehman et al., 2015). In vitro drug release study has shown the sustained release of
diacerein for three days through diffusion control mechanism. Release data followed the
Higuchi model and zero order kinetics.
34
1.2.2. Polysaccharides based NPs as anticancer drug delivery system
Nanoparticles are nanocarriers of different shapes and morphologies having the size range
between 1 to 1000 nm (Jung et al., 2000). Now a day, NPs are being used to deliver different
drugs, vaccines, genes and hormones (Pan-In et al., 2014; You and Peng, 2004; Zahoor et al.,
2005). Different materials (synthetic, semisynthetic or natural) and various methods (ionic or
covalent crosslinking, complexation or self-assembly of hydrophilic/hydrophobic groups
after modification) are being employed to synthesize NPs of desired sizes and according to
therapeutic requirements (Liu et al., 2008). Among these materials, polysaccharides have
gained the interest of researchers due to their biodegradable, biocompatible, non-toxic and
non-irritant nature (Lemarchand et al., 2004). Polysaccharides are obtained from various
sources, i.e., animal (chondroitin, chitosan), microbial (xanthan gum, dextran), plant (guar
gum, pectin) and algae (alginate) (Sinha and Kumria, 2001). Moreover, naturally occurring
polysaccharides are abundant in nature and recognized as highly stable, safe and cheap.
Mostly, naturally occurring polysaccharides are hydrophilic due to the presence of amino,
carboxyl and hydroxyl groups which play an important role in the formation of weak bonding
with biological tissues resulting in bio-adhesion and prolonged residence time (Lee et al.,
2000).
Polysaccharides are used to synthesize the drug loaded NPs for the treatment as well as
diagnostic purpose in the management of different tumours. Stable dextran based NPs with a
functional carboxylic acid group was synthesized using graft copolymerization method (Dou
et al., 2005). Reaction was carried out without any organic solvent or surfactant and initiated
from water soluble monomer. These NPs have various potential applications due to the
addition of acidic functional group.
35
Chitosan, a diverse polysaccharide, investigated extensively as a carrier for many drugs and
chemicals for diagnosis and treatment of many diseases. It has been used as a carrier for drug
delivery, cell imaging and gene delivery (Shukla et al., 2013). Chitosan NPs were synthesized
to encapsulate docetaxel and evaluated its efficiency on cancer cell line (Lozano et al., 2013).
Chitosan based NPs were prepared by solvent displacement method with 78% encapsulation
efficiency of docetaxel. Further study has proved the integrity of NPs and also the effective
intracellular delivery of docetaxel. Sustained release of doxorubicin was made possible from
chitosan NPs by Janes and coworkers (Janes et al., 2001). Similarly, paclitaxel and
doxorubicin were also delivered by chitosan NPs which made them therapeutically more
effective and less toxic (Hwang et al., 2008; Mitra et al., 2001). Nanocapsules of chitosan
were used by Lozano et al., for the intracellular transport of docetaxel in A549 and MCF-7
cell lines of human lung carcinoma and breast adenocarcinoma, respectively (Lozano et al.,
2008).
Effect of chitosan NPs were observed on the growth of hepatocellular carcinoma (Qi et al.,
2007). Chitosan NPs were evaluated by TEM, MTT assay, flow cytometry, electrophoresis,
GC/MS, spectrophotometric thiobarbituric acid (TBA) assays. After oral administration of
chitosan NPs in nude mice, size of tumour was measured periodically and morphological
changes in liver was also studied under electron microscope.
Core shell NPs was designed for doxorubicin delivery in which bovine serum albumin
conjugated chitosan act as a core and dextran conjugated chitosan as a shell (Qi et al., 2010).
Prepared NPs are highly stable having diameter of 130-230 nm. Doxorubicin is loaded in NPs
by virtue of hydrophobic and electrostatic interaction between drug, dextran and chitosan.
These NPs have shown less adverse effects of doxorubicin and increase the survival rate of
tumor bearing mice.
36
Docetaxel, an anticancer drug, belongs to taxoid family which is being used for gastric
(Cutsem, 2004), breast (Palmeri et al., 2008), pancreatic (Androulakis et al., 1999) and
urothelial carcinoma (Yafi et al., 2011). Docetaxel is an analog of paclitaxel and an inhibitor
of microtubule polymerization.
Fig. 1.4. Structure of docetaxel.
Biocompatible and biodegradable polymer, polylactic-co-glycolic acid (PLG), was used to
synthesize docetaxel loaded NPs by solvent evaporation. Small size NPs were obtained by
varying the experimental conditions. Release of docetaxel from NPs is dependent on the type
of surfactants used for the synthesis of NPs. Results indicated that PLG NPs can be used for
prolonged release of docetaxel (Keum et al., 2011). Core shell NPs was synthesized for
loading of docetaxel in which mannitol core was protected by PLG shell (Tao et al., 2013).
These NPs have proved antitumor activity against breast cancer.
In order to reduce the toxicity of docetaxel, self-assembly process was used to fabricate
docetaxel loaded albumin nanoparticles with a mean diameter of 150 nm (Tang et al., 2015).
In vivo study has shown better results as compared to Taxotere® in term of hemolysis,
tolerance, EPR effect and antitumor ability.
37
In another study, docetaxel loaded dendritic nanoparticles were prepared and used to evaluate
the in vivo antitumor activity against human breast cancer cell lines (Zhang et al., 2014). This
new type of drug delivery system has shown a promising application as a chemotherapeutic
agent. In order to evaluate the effect of NPs on pharmacokinetic parameters of docetaxel, two
different drug loading NPs were fabricated using soft-lithography technique (Chu et al.,
2013). Plasma concentration and antitumor activity of 9% docetaxel loading NPs was higher
than 20% drug loading NPs.
Poly(lactide-co-caprolactone) and poly(lactide-co-glycolide-co-caprolactone) based
docetaxel loaded NPs were prepared and evaluated for the treatment of prostate cancer
(Sanna et al., 2011). These NPs demonstrated better release properties of docetaxel with
minimum side effects as compared to pure docetaxel.
1.2.3. Polysaccharides mediated synthesis and application of Ag NPs
NPs have gained attraction of many scientists over the last two decades. NPs usually fall in
the range from 1 to 100 nm and their synthesis and application is an emerging scientific field.
There are different physical and chemical methods for the synthesis of NPs which include
photochemical reduction, electrochemical treatments, chemical reduction etc. (Frattini et al.,
2005). Now a day it is possible to synthesize NPs with high stability, longer shelf life, desired
shapes and morphology by adopting different techniques and chemicals (Knoll and
Keilmann, 1999; Sengupta et al., 2005).
Silver is used for the synthesis of ethylene oxide and formaldehyde as a catalyst (Nagy and
Mestl, 1999). Due to its good conductivity, antibacterial activity and chemical stability, it is
widely used in chemical industries (Frattini et al., 2005).
38
There are numbers of reducing agents used for the preparation of silver nanoparticles (Ag
NPs) that include ascorbate, borohydrate, elemental hydrogen and citrate (Lee and Meisel,
1982; Shirtcliffe et al., 1999; Nickel et al., 2000). A cost effective, facile and environment
friendly synthesis of Ag NPs is accomplished in three stages; solvent selection, reducing
agent selections and stability agent for Ag NPs (Raveendran et al., 2003).
Polysaccharides can serve as both capping and reducing agent for the synthesis of Ag NPs. In
starch mediated synthesis of Ag NPs, starch was used as a stabilizing agent while β-D-glucose
played its role as a reducing agent and mixture was exposed to gentle heating. A highly
spherical and monodisperse Ag NPs with diameter range of approximately 20 nm was
synthesized with environment benign material, i.e., starch (Raveendran et al., 2003).
Starch was also used as capping or reducing agent by many researchers while changing other
conditions. Ag NPs can be synthesized by keeping the solution of starch and silver nitrate in
autoclave at 121 °C and 15 psi pressure for 5 min. By this process, spherical shaped NPs in
the range of 10-34 nm was synthesized and can be stored for 3 months even in solution form
at room temperature (Vigneshwaran et al., 2006). Starch was also used to synthesize smaller
NPs with less than 10 nm in diameter (Tai et al., 2008). Solution of NaOH, starch and
glucose was used as an accelerator, capping agent and reducing agent, respectively. Mixture
of all three reagents was kept in spinning disk reactor for not more than 10 min to get these
smaller sized NPs.
Microwave irradiation was employed on the solution of silver nitrate and carboxymethyl
cellulose (CMC) sodium for the synthesis of NPs of uniformed size and with stability for at
least 2 months at ambient temperature (Chen et al., 2008). Soluble starch and basic amino
acids were used as protecting and reducing agents, respectively for the preparation of Ag NPs
under the influence of microwave radiations (Hu et al., 2008). CMC was synthesized from
39
cotton sample and used for the preparation of Ag NPs (Hebeish et al., 2010). Effect of
reaction temperature, pH of solution, concentration of CMC and silver nitrate and exposure
time on the synthesis of Ag NPs was noted. Colloidal suspension of Ag NPs was produced by
increasing the concentration of the reactants.
Oligochitosan was used as a stabilizer in the synthesis of small and biocompatible Ag NPs
when exposed to gamma radiations. Synthesized Ag NPs were in the range from 5-15 nm.
These NPs were stable between pH 1.8-9.0 and also in NaCl solution. Aggregation of Ag NPs
was observed in solution of NaNO3 and NaH2PO4 (Long et al., 2007). Solution of acetic
water, chitosan and silver nitrate were placed in front of gamma radiation to synthesize Ag
NPs. In depth analysis revealed that the size of NPs were found in the range of 4-5 nm which
were protected by chitosan chains (Chen et al., 2007b).
Arabinoxylan, isolated from seed husk of isphagula, a gel forming branched polysaccharide
were used as a reducing and stabilizing agent for the synthesis of Ag NPs. Arabinoxylan was
suspended in distilled water, mixed with silver nitrate solution and kept just below 100 °C
under continuous stirring. Change in color of the mixture was observed within 60 min
indicating the synthesis of Ag NPs. Particle size of the synthesized NPs were determined by
TEM and found in the range of 5-20 nm. Reaction mixture temperature and time,
concentration of arabinoxylan and pH of the mixture are the main determining factors for size
of NPs (Amin et al., 2013).
Gum kondagogu (Cochlospermum gossypium) was used as a reducing and stabilizing agent to
facilitate the synthesis of spherical silver nanoparticles (3 nm). Raman and FTIR
spectroscopy were used to study the mechanism of stabilization and reduction process. Ag
NPs were characterized by TEM, XRD, TGA and UV spectroscopy. These synthesized Ag
40
NPs have shown antibacterial activity against Gram positive and Gram negative bacteria
(Kora et al., 2010).
Polysaccharide (glucoxylan) was separated from the seeds of Mimosa pudica and used as a
reducing and capping agent for the green synthesis of 6 nm sized Ag NPs and 40 nm gold
NPs (Iram et al., 2014). Phyto-toxicity study of synthesized NPs was performed on the
germination of radish seed and did not found any significant effect.
A mixture of aqueous gum acacia and silver nitrate solution was exposed to gamma radiation
for the synthesis of Ag NPs. As a result, NPs of different sizes were obtained by changing the
intensity of radiation. The morphology of Ag NPs was evaluated by TEM, XRD and dynamic
light scattering (DLS). Bonding between NPs and COO─ group present on gum acacia was
confirmed by FTIR (Rao et al., 2010). Sodium alginate solution, as a reducing agent, was
used for the synthesis of Ag NPs in the presence of gamma radiation. NPs were retained their
integrity for 6 months at room temperature (Liu et al., 2009).
Nanocomposite of chitosan with silver and other metals were synthesized in the presence of
NaBH4. The synthesis of NPs was verified by UV spectroscopy and TEM analysis (Huang et
al., 2004).
A facile, cost effective and green synthesis of Ag NPs can be achieved by using gum acacia
as a reducing agent. Aqueous solution of gum acacia and silver nitrate was mixed and placed
at room temperature for 24 h. Formation of Ag NPs was confirmed by observing the change
in color and UV spectroscopy. Further, the shape and size of NPs were determined by XRD
and TEM analysis. These NPs were found stable for 5 months (Mohan et al., 2007).
Chitosan as a polymeric stabilizer was used to synthesize Ag NPs in the presence of PEG and
silver nitrate at 60 °C. Newly formed nanocomposite was characterized through UV-Vis
41
spectroscopy, TEM, FTIR and XRD. Effect of stirring time on the diameter of Ag NPs was
evaluated and it was found that the size of Ag NPs was dependent on the stirring time i.e.,
size increased with increase in stirring time (Ahmad et al., 2011).
Silver nitrate was reduced to form silver nanoparticles in the presence of chitosan. Ag NPs
bounded chitosan solution was evaporated under mild heating condition to form a thin film.
Antibacterial activity of this film was evaluated against different bacteria (Wei et al., 2009).
Dextran was used as a reducing and capping agent to synthesize Ag NPs (Bankura et al.,
2012). Aqueous solution of dextran and silver nitrate was mixed and after addition of NaOH
solution (0.001 M) at room temperature, color change of the solution was observed
considering a sign of the reduction of silver ion into metal. Formation and morphology of Ag
NPs was verified by UV-vis spectroscopy, TEM, XRD and AFM. These NPs was tested
against five different bacteria and inhibition of bacterial growth was found to be inversely
proportional to the concentration of Ag NPs.
Chitosan film was synthesized with embedded Ag NPs using thermal treatment. Dendritic
structure was observed in the chitosan-Ag NPs film (Dongwei et al., 2009). Further
investigation proved its application in surface-enhanced Raman spectroscopy.
Recently, a new approach was adopted to synthesize core-shell nanoparticles in which Ag
NPs and Au NPs acted as core and shell, respectively (El-Naggar et al., 2016). In this reaction
process, curdlan was used as a reducing and capping agent, AgNO3 and HAuCl4 as precursor
for silver and gold and microwave radiation as a source of energy. Formation and
characterization of core-shell NPs was determined by UV-vis spectroscopy, TEM, zeta
potential analysis, XRD, FTIR and atomic force microscopy (AFM). Spherical shaped core-
shell NPs with average diameter of 45 nm was obtained.
42
Ag NPs embedded chitosan film was synthesized by photochemical induce reduction of silver
nitrate in the presence of chitosan solution (Thomas et al., 2009). Film was evaluated by
TEM, TGA and XRD. Due to its antibacterial potential, it can use as wound healing dressing,
antimicrobial packaging material and biomedical implants.
Aqueous solution of hydroxypropylcellulose and silver nitrate was placed in sunlight to
induce the formation of Ag NPs (Hussain et al., 2015). Synthesized NPs were analyzed by
UV-vis spectroscopy, TEM and XRD. Ag NPs embedded HPC film was evaluated through
SEM and AFM. These Ag NPs have shown antimicrobial activity against bacteria and fungi.
The embedded Ag NPs HPC film can be stored for more than one year without any
morphological changes in NPs.
Dialysis process is used to synthesize the Ag NPs of 6 nm in diameter. HPC, ethylene glycol
and silver nitrate were used as a capping agent, solvent support and silver precursor,
respectively (Francis et al., 2010). Different concentrations of silver nitrate were used to get
optimized Ag NPs. After getting through dialysis process, the UV-visible spectrum shifted
from 410 nm to 440 nm indicating the completion of reduction process. After drying the
resulting solution at 80 °C, HPC capped Ag NPs were characterized by TEM, XRD and
FTIR. Skewed distribution of Ag NPs was observed in TEM analysis.
Hydroxypropyl cellulose was prepared by etherification reaction and used for the synthesis of
Ag NPs. Reaction mixture having HPC and silver nitrate solution was kept for 90 min at 90
°C to complete the formation of Ag NPs (Abdel-Halim and Al-Deyab, 2011).
Hydroxypropyl carboxymethyl cellulose (HPCMC) was used to prepare Ag NPs (Abdel-
Halim et al., 2015). Formation of Ag NPs is dependent on the degree of substitution of
HPCMC, concentration of silver nitrate, pH of the reaction medium, reaction time and
temperature.
43
Like other polysaccharides, dextran was used as a stabilizing and capping agent in the
synthesis of Ag NPs (Hussain et al., 2014). Spherical shaped Ag NPs of 50-70 nm in
diameter were obtained when aqueous solution of dextran and silver nitrate were placed in
sunlight. Formed particles were evaluated through TEM, PXRD, SEM, AFM and also
determined the antimicrobial activity.
1.2.4. Polysaccharides based antiseptic dressing
Wound healing is mainly a four phase process which includes inflammation, tissue
regeneration, matrix remodeling and reepithelialization (Cochrane et al., 1999). Chitosan, a
N-deacetylated polysaccharide, was used as a wound healing agent (Kojima, et al., 1998).
Hyaluronic acid, composed of N-acetyl-glucosamine and D-glucuronic acid, has known for its
lubrication, water retention capacity and cell proliferation (Bulpitt and Aeschlimann, 1999;
Hu et al., 2003). Xu et al. used chitosan and hyaluronic acid to fabricate a composite film as a
wound healing dressing and in vivo animal study also has proved its effectiveness (Xu et al.,
2007).
Aloe vera leaf gel has shown wound healing ability when applied topically as well as through
systemic administration. Wound healing ability of Aloe vera gel mainly depends on the
stability of the active constituents which is affected by gel extraction time after harvesting.
Different mechanisms of wound healing from Aloe vera gel were suggested including
migration of epithelial cells, reduction in inflammation, wound moistening and collagen
maturation (Reynolds and Dweck, 1999). Glycoprotein (5.5 kDa), isolated from Aloe vera
has shown excellent wound healing ability in hairless mice (Choi et al., 2001). Effect of Aloe
vera gel was studied on the formation of fibrous tissue, synthesis of collagen and contraction
of wound (Chithra, et al., 1998).
44
Many plants are being used in the treatment of wound. Polysaccharides extracted from
Opuntia ficus-indica L. cladodes were evaluated for their potential as a wound healing agent.
For this purpose, two polysaccharide fractions (low and high molecular weight) were isolated
and applied on the wound of rats. Results have indicated that low molecular weight fraction is
better in wound healing than high molecular weight fraction (Trombetta et al., 2005).
Wound dressing comprising carboxymethylcellulose and alginate was evaluated for their
effectiveness as engulfing and immobilization agent of two bacteria (Pseudomonas
aeruginosa and Staphylococcus aureus) present in wound. SEM examination has shown that
due to viscous nature of CMC gel, it was found a better wound healing agent than alginate gel
(Walker et al., 2003).
Alginic acid in salt form is used in wound dressing due to its high absorbency, gelling and
hydrophilic nature. Upon contact with exudate of wound, ionic interaction takes place
between the calcium ions of alginate and sodium ions present in serum. As a result, a
swellable protective film of alginate is formed (Thomas, 2000). Ionic crosslinking of alginate
makes it an ideal polymer gel for wound healing treatment and tissue engineering (Kuo et al.,
2001; Wang et al., 2003). It was also found that due to slow degradation, alginate wound
dressing is more beneficial than other hydrocolloids (Ichioka et al., 1998).
Calcium alginate has shown some pharmacological activities to speed up the healing process
including proliferation of fibroblast (Doyle et al., 1996), stimulate synthesis of tumour
necrosis factor-α (Thomas et al., 2000) and helping in clotting (Blair et al., 1990). Alginate
wound dressing is biodegradable (Gilchrist and Martin, 1983) and hence used in the
preparation of surgical sutures.
45
Hydrogel dressings are nonreactive, nonadherent and nonirritant to biological tissues. These
dressing produce the cooling effect and reduce the sensation of pain hence increase patient
compliance (Wichterle and Lim, 1960; Moody, 2006).
Aqueous solution of photocrosslinkable chitosan has the ability to convert into an elastic and
insoluble hydrogel in no time (Ishihara et al., 2002). This unique ability of chitosan gel was
used on the mouse whose skin was sliced in two pieces. After UV irradiation on the applied
crosslinked chitosan solution for 90s, the rate of wound healing and contraction was
observed. Histological evaluation also confirmed the epithelialization and tissue formation at
the wound site which make this crosslinked chitosan gel as an excellent dressing in
emergency situations.
Extracted polysaccharides from plants are used in wound healing dressings. Polysaccharides
from Bletilla striata (BS) were crosslinked and evaluated as a wound healing agent in mouse
model. For this purpose, BS hydrogel dressing is applied on the wound and healing process
was found to be fast as compared to vaseline and iodine gauze (Luo et al., 2010).
Polysaccharides were separated from the fruit of Phellinus gilvus (PG) by hot water
extraction process. Different concentrations of polysaccharide solution were applied on the
surgically induced skin wounds and Madecassol® was used for comparison. Wound
contraction diameter and rate of reepithelialization were determined periodically. At the end
of study, it was found that PG has significantly good results as compared to Madecassol®
(Bae et al., 2005).
Seed husk of psyllium was used to extract psyllium hydrogel and then in the preparation of
wound dressing film (Patil et al., 2011). Psyllium hydrogel film was loaded with Povidone
iodine and undergo for in vitro and in vivo evaluation. Psyllium dressing was found to have
antimicrobial activity against different bacteria which was usually present in wounds. After
46
applying on the wound of rat, psyllium dressing has shown better results in terms of wound
closure as compared to Band aid®.
Chitin was used to prepare an antiseptic film which was evaluated as a wound healing
material (Yusof et al., 2003). A real wound was created on the dorsal side of rat and covered
with chitin films of different concentrations. Commercially available wound dressings,
Opsite™ and Easifix™, were used as a control. A rapid healing was observed in chitin film
treated wound as compared to commercially available dressings. Moreover, chitin film
dressing was transparent, durable, biodegradable and non-fragile.
Chitosan was used as a reducing agent in the synthesis of silver nanoparticles (Li et al.,
2013). Mixture of chitosan, silver nanoparticles and poly (vinyl alcohol) was used to prepare
a fibrous mat as a wound healing dressing by electrospinning technique. This composite
material has shown antibacterial activity against E. coli and S. aureus. In vitro and in vivo
study of this composite has proved its effectiveness as wound healing material.
Dextran and poly (vinyl alcohol) was crosslinked to form a hydrogel by freezing-thawing
method (Hwang et al., 2010). Gentamicin was loaded in the hydrogel and this hydrogel film
was placed on the wound of rats and served as a wound healing dressing. Hydrogel film was
evaluated through swelling ratio, water vapor transmission test, mechanical properties,
morphology and thermal analysis. In vivo wound healing analysis confirmed gentamicin
loaded hydrogel film as an excellent wound healing dressing with improved patient
compliance.
Gamma radiation was employed for the synthesis of crosslinked chitosan/poly (vinyl alcohol)
hydrogel (El Salmawi, 2007). After successful evaluation of physical parameters of hydrogel
film, wound healing study was conducted. Hydrogel film was found a physical barrier for the
penetration of microorganisms and hence enhances wound healing.
47
Natural polymers were blended with PVA to improve the mechanical strength and
physicochemical properties of the natural hydrogel. Alginate being biocompatible and
hydrophilic in nature is used in biomedical field. Nitrofurazone was incorporated in hydrogel
composed of sodium alginate and PVA which were crosslinked by freeze-thawing process.
Size reduction of wound was seen after treating with sodium alginate/PVA hydrogel
indicating a new wound healing dressing material (Kim et al., 2008). Another method,
electrospinning, was employed for the synthesis of PVA and calcium alginate hydrogel which
was further used as wound healing dressing. In vivo study on rat has shown the formation of
epithelium without any adverse effect (Tarun and Gobi, 2012).
1.3. Characterization techniques
Polysaccharides are characterized by using various analytical, microscopic and spectroscopic
techniques.
1.3.1. Fourier transform infrared spectroscopy
Fourier transform infrared spectroscopy (FTIR) is used to characterize the different
functional groups present in a molecule. For solid material, pellet was prepared with the
mixture of KBr and the sample. Dried pellet was scanned through 4000-400 cm-1
in
transmittance or absorbance mode. Absorption peaks obtained are as a result of different
frequencies of bond‘s vibrations. Each functional group has its own characteristic frequency
of vibration. Due to advancement of technology, FTIR is used for both quantitative and
qualitative analysis (Griffiths and de Haseth, 2007).
1.3.2. Nuclear magnetic resonance spectroscopy
To determine the structure of a molecule, nuclear magnetic resonance (NMR) spectroscopy is
proved to be a power full tool. Proton NMR (1H NMR) spectroscopy is used to find the
48
structure of a molecule at the level of hydrogen-1 nuclei. Structure of polysaccharides is
evaluated by 1H NMR and more precisely by Heteronuclear single-quantum correlation
(HSQC) and total correlation spectroscopy (TOCSY). These spectroscopic techniques are
used to investigate the proton and carbon of polysaccharides (Martin and Zektzer, 1988; Lane
and Lefèvre, 1994).
1.3.3. Thermal analysis
Thermal analysis of polymer was performed to find the thermal degradation pattern, thermal
stability, glass transition temperature (Tg) and activation energy (Ea). Thermal analysis can
give the information of the stability of polymer, storage temperature and shelf life of the
polymer if used in dosage forms (Coats and Redfern, 1963). Different methods were
employed to study the kinetics of thermal degradation process. Activation energy, reaction
order (n) and frequency factor (Z) were calculated by using kinetic models. Change in
entropy (∆S), Gibbs free energy (∆G) and enthalpy (∆H) were also determined through
established methods (Evans and Polanyi, 1935).
1.3.4. Electron microscopic analysis
Analysis through scanning electron microscope (SEM) and transmission electron microscope
(TEM) is performed to observe the surface of dried form of polymer after its extraction as
well as the cross section of swollen then freeze dried sample of the polymer. Sample was
placed in front of a beam of high energy electrons whose intensity was controlled with
various mechanisms. Image was received as a result of interaction between these electrons
and the atoms on the surface of sample (Clarke and Eberhardt, 2002). Transmission electron
microscopy (TEM) was used to examine the very small specimen. TEM is very powerful and
capable to observe even a single column of atoms.
49
1.3.5. Powder X-ray diffraction
X-ray spectroscopy is widely used for the characterization of all forms of materials to get the
geometric and electric structure (Guo, 2009). For the measurement of X-ray diffraction
(XRD), sample was placed on goniometer and X-rays was bombarded on the sample. As a
result, reflections or diffraction pattern was recorded as a regularly spaced spots (Kumirska et
al., 2010). Clark and smith were the first scientists who used XRD to investigate the structure
of chitin and chitosan in 1936 (Clark and Smith, 1936).
1.3.6. MTT assay
MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) is a yellow coloured,
water soluble dye which is reduced to purple coloured formazan, a water insoluble material,
by live cells. MTT assay is a homogeneous colorimetric assay used to assess the cell
metabolic activity (Mosmann 1983). During this assay, MTT after entering into cells passes
the mitochondria and reduce to formazan which is an insoluble product. Cells are further
solubilized in an organic solvent and formazan is measured spectrophotometrically as the
reduction of MTT take place in metabolically active cells, therefore, the detection of activity
is directly related to the viability of the cells. If cell dies, the ability to reduce MTT into
formazan will also losses and is evident from the color of the cell. MTT reduction might be
due to the reducing molecule (NADH) that donates electrons to MTT (Marshall et al., 1995).
1.3.7. Drug release models
Release of drug from sustained release formulation depends mainly on the physicochemical
properties of the polymeric materials used as a release retarding agent.
1.3.7.1. Zero order
50
Slow and continuous release of drug from a controlled drug delivery system which does not
disintegrate or erode is explained by zero order release model (Eq. 1).
(Eq. 1)
where, K0 is the zero order rate constant which can be expressed as concentration per unit
time and Qt is the amount of drug released from the tablet in time t.
To calculate and study the zero order drug release kinetics, a graph is plotted between the
cumulative drug release and time (Narashimhan et al., 1999). Zero order kinetics is used to
describe mechanism of the continuous release of drug from a dosage form at a constant rate
which usually occurs in matrix system having less soluble drugs, osmotic drug delivery
systems and some transdermal systems (Yang and Fassihi, 1996; Freitas and Marchetti,
2005).
1.3.7.2. Firsst order
First order release model is used to explain the release of soluble drug from a swellable
polymeric material (Bravo et al., 2002) and expressed using Eq 2.
(Eq. 2)
where, Q is the residual concentration of drug in tablet after time t, Q0 is the initial loaded
amount of drug in tablet and K1 is the first order rate constant (Gibaldi and Feldman, 1967;
Wagner 1969).
Drug release data is plotted as a log of cumulative percentage of drug remaining in the tablet
against time which is expressed as a straight line having slop of –K/2.303. This model is
applied to explain the dissolution profile of water soluble drugs from porous matrices
(Narashimhan et al., 1999).
51
1.3.7.3. Higuchi model
In 1961 and 1961, Higuchi was the first scientist who proposed a mathematical model to
explain the release of water soluble and poorly soluble drug from semisolid and solid
pharmaceutical dosage forms. Initially, he described the drug release behaviour from a planar
system but later on from porous system and different geometrical shapes. Higuchi model was
derived to consider some assumptions that (i) in a matrix system, the drug concentration is
higher than its solubility in the media, (ii) diffusion of drug from the matrix takes place
unidirectional, (iii) particles of drug are than the thickness of system, (iv) matrix dissolution
and swelling is negligible, (v) rate of drug diffusion remains constant, (vi) perfect sink
condition of the system is maintained. Furthermore, Higuchi modified the model and covered
the system in which the concentration of drug in a matrix is less than its solubility. In this
case the release of drug occurs through the pores and channels of the matrix. Eq. 3 is the
simplest form of Higuchi model (Higuchi, 1961; Higuchi, 1963).
(Eq. 3)
where, Qt is the amount of drug released in time t and KH is the Higuchi rate constant.
In Higuchi model, cumulative drug release (%) was plotted against the square root of time.
Drug dissolution mechanism from transdermal drug delivery system and tablet formulation
containing polymeric matrix can be described by using Higuchi model (Grassi and Grassi,
2005).
1.3.7.4. Hixson-Crowell model
Hixson and Crowell (1931) described an equation showing that the particles‘ regular area is
proportional to the cube root of its volume as shown in Eq. 4.
52
(Eq. 4)
where, Q0 is the initial amount of drug in dosage form, Qt is the amount of drug released
from the dosage form in time t and KHC is the Hixson-Crowell release constant describing
surface-volume relation (Hixson and Crowell, 1931). This equation explained the release of
drug from the dosage form with respect to its surface area and diameter. Drug release data
was plotted as cube root of drug concentration remaining (%) versus time. This model
applies on the dosage form especially tablets when the dissolution process proceed in a plane
which is parallel to the surface of drug and dimensions/geometric shape of tablet remain the
same (Chen et al., 2007).
1.3.7.5. Korsmeyer-Peppas model
Korsmeyer derived an equation explaining the drug release mechanism from a polymeric
drug delivery system. For Korsmeyer-Peppas model, first 60% drug release data was
considered (Korsmeyer et al., 1983) and expressed in Eq. 5.
(Eq. 5)
where, Mt/M∞ is the fraction of drug released at time t and Kp is the Korsmeyer-Peppas
constant. n is the release exponent and its value is calculated from the slop of a graph plotted
between log Mt/M∞ and log t. The value of n indicates the drug release mechanism from a
polymeric matrix system. For a cylindrical (tablet) drug delivery system, release mechanism
is considered as Fickian diffusion (n ≤ 0.45), non-Fickian diffusion (0.45 < n < 0.89), case II
transport (n = 0.89) and super case II transport (n > 0.89) (Korsmeyer et al., 1983; Ritger and
peppas, 1987).
53
1.4. Background and significance of the study
Drug delivery systems usually require more than one ingredient/excipient for effective
delivery of therapeutic agents. These ingredients/excipients are synthetic, semisynthetic or
naturally occurring biomaterials. Due to chemical nature/composition of synthetic and
semisynthetic materials, there is always a chance that on administering, they may stimulate or
initiate the immune system of the body and may start biorejection process. Therefore, the
researchers are switching towards naturally occurring materials due to their biocompatability,
biodegradability, nonimmunogenicity and easy availability. Among naturally occurring
biomaterials, polysaccharides based materials are most important and are being used in
conventional as well as in novel and smart drug delivery systems. Furthermore, swellable
(hydrogellable) polysaccharides based materials have wide range of applications in sustained
and targeted drug delivery systems to improve the bioavailability of therapeutic agents. Due
to immense importance and unique properties of polysaccharides based material, it is utmost
important to introduce novel naturally occurring biomaterials especially water swellable and
pH sensitive polysaccharides as novel drug delivery carriers. Therefore, hydrogellable,
stimuli responsive linseed polysaccharides are one of the most important entities having great
potential for intelligent drug delivery systems.
54
1.5. Aims and objectives
Aim of this research was to evaluate a naturally occurring polysaccharide LSH for
pharmaceutical and biomedical applications. Aims were to study swelling kinetics of LSH in
deionized water and at different physiological pH. Our interests were focused on stimuli
responsive swelling-deswelling (on-off switching) in different solvents, at various ionic
concentrations and pH. Micromeritic properties of LSH will also be taken in to account.
Potential application of LSH as a sustained release oral drug delivery system for caffeine,
diclofenac sodium and diacerein are in focus. Swelling characteristics and swelling-
deswelling response of these drug containing LSH matrix tablet formulations will also be
monitored in various environments. Drug release kinetics and mechanism will be determined
using various kinetic models in order to get deeper insight into the mechanism of drug
release.
Hydrogelable polysaccharides arrange themselves into defined morphologies after swelling in
aqueous media. Therefore, our interests were to study morphological analysis of LSH in
powder form and tablet formulations using SEM to see channeling/pores in the swollen then
freeze dried LSH and its tablets.
Acetylation of LSH will be performed to check if the polysaccharides are modifiable for
esterification reactions. The obtained structures will also be characterized. As the acetylation
may enhance the stability of polysaccharides, therefore our interests were to study thermal
analysis of LSH and acetylated LSH in order to compare their thermal stability.
Aim was focused on the formation of LSH nanoparticles for the loading of docetaxel, a
chemotherapeutic agent. Docetaxel loaded LSH nanoparticles will be evaluated for
encapsulation efficiency and drug loading capability. In addition, morphology of the obtained
NPs will also be evaluated by TEM and PXRD. For an effective delivery system of
55
anticancer drug, in vitro drug release behaviour, cytotoxicity analysis and cellular uptake
studies are also the topics of interest.
Furthermore, role of LSH in the green synthesis of Ag NPs is under investigation. Factors
affecting on the synthesis of Ag NPs and potential of LSH as a storage media for Ag NPs will
be investigated. Antimicrobial activity of Ag NPs embedded LSH film as a wound healing
agent will be tested.
Due to the potential application in oral and topical drug delivery system, it was necessary to
investigate the acute toxicity study of LSH on albino mice and rabbit therefore relevant
toxicological studies will be performed.
Summarizing, it can be stated that we are interested to report a new polysaccharides based
hydrogel for sustained oral drug delivery system, wound healing applications, green synthesis
of Ag NPs and nano-particulate carrier for chemotherapeutic agents.
56
2. MATERIALS AND METHODS
2.1. Materials
Linseeds were purchased from local market, cleaned from superfluous material by passing
through different meshes and stored at ambient temperature in air tight container. Ethanol, n-
hexane, KCl, NaCl, acetone, acetic acid, sodium acetate, CHCl3, formalin, diclofenac sodium
(DS), NaOH, iso propyl alcohol, potassium dihydrogen phosphate and HCl were purchased
from Riedel-de Haën, Germany. NaOH was standardized with oxalic acid before use.
Caffeine, acetic anhydride, DMSO, 4ʹ,6-diamidino-2-phenylindole (DAPI), DMAc, AgNO3,
DMSO-d6 and CDCl3 were purchased from Sigma-Aldrich, USA. Magnesium stearate and 4-
dimethylaminopyridine (DMAP) was obtained from BDH and Alfa Aesar, England,
respectively. Microcrystalline cellulose was procured from Fluka. Simulated gastric fluid
(SGF) and simulated intestinal fluid (SIF) were prepared without enzymes as mentioned in
United States Pharmacopeia (2010). Diacerein (according to the standard of European
Pharmacopoeia) and polyvinylpyrrolidone (PVP) K30 were obtained from Consolidated
Chemical Laboratories Pvt. Ltd., Pakistan. Nylon mesh was used to separate the hydrogel
from linseeds. Pluronic F-68 (Poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene
oxide)) triblock copolymer (Mw = 8350; (EO)79(PO)28(EO)79) was purchased from BASF
Corp., Republic of Korea and used as received. Anhydrous form of docetaxel (DTX) and
Tween 80 were obtained from Parling Pharma Tech Co., Ltd. (Shanghai, China) and Sigma
(St. Louis, MO, USA), respectively. Deionized water was used during the whole research
work.
57
2.2. Measurements
2.2.1. Fourier transform infrared spectroscopy
Fourier transform infrared spectroscopy (FTIR) was used to characterize linseed hydrogel
(LSH), acetylated LSH (ALSH) and to determine the compatibility of the excipients with
active ingredient in a tablet formulation. Potassium bromide (KBr) was used to prepare the
pellets of the sample and dried at 50 °C for 2 h in vacuum oven before recording the spectra
on FTIR instrument IR Prestige-21 (Shimadzu, Japan).
2.2.2. 1H NMR spectroscopy
1H NMR spectra of LSH were recorded on Avance 600 MHz spectrometer equipped with a
triple-resonance RT probe (Bruker, Billerica, MA, USA). Deuterated solvents (DMSO-d6,
CDCl3) were used to prepare the LSH and ALSH sample (10 mg/mL). The 1H NMR spectra
were processed using TopSpin software.
2.2.3. Heteronuclear single quantum correlation spectroscopy
Heteronuclear single quantum correlation (HSQC) spectra were recorded on Bruker Avance
600 MHz spectrometer and for data analysis, TopSpin software was used. In HSQC
spectroscopy, proton and 13
C spectrum were presented along x-axis and y-axis, respectively.
1H signal is measured in direct dimension while
13C signal is recorded in indirect dimension.
2.2.4. 1H
1H TOCSY NMR spectroscopy
Total correlation spectroscopy (TOCSY) was performed on Bruker Avance 600 MHz
spectrometer and obtained data was analyzed by using TopSpin software. In TOCSY, proton
spectrum was plotted against x-axis while 13
C spectrum was shown along y-axis. 1H and
13C
signals are measured in direct and indirect dimensions, respectively.
58
2.2.5. UV-Vis spectrophotometry
UV-Vis spectra were recorded on UV-1700 PharmaSpec (Shimadzu, Japan). Formation of
Ag NPs was observed through UV-Vis spectrophotometer. Release of drugs from LSH
matrix tablets were also monitored periodically through UV-Vis spectrophotometer.
2.2.6. Thermogravimetric analysis
Thermal decomposition temperature of LSH and ALSH were recorded on SDT Q600 thermal
analyzer (TA Instruments, USA) under nitrogen flowing (100 mL/min) at heating rate of 5,
10, 15 and 20 C/min from 35-800 C. Thermal degradation data was processed by using
Universal Analysis 2000 v 4.2E software.
2.2.7. Field emission scanning electron microscopy
Dried LSH was swollen in deionized water and freeze dried. Morphology of freeze dried
sample was observed under FE-SEM (JEOL JSM-6700F, Japan). For this purpose, samples
were dispersed in deionized water to obtain a solution of 0.1 wt%. Each solution was drop
casted on a carbon mount and dried at 25 °C in a vacuum oven for 24 h prior to analysis.
Texture and cross section area of prepared tablets were investigated using scanning electron
microscope (Quanta 250, FEI, USA).
2.2.8. Transmission electron microscopy
Morphology of the LSH mediated Ag NPs was evaluated by transmission electron
microscopy (TEM). Prepared Ag NPs were first separated by centrifugation and then
examined on a Philips 420 instrument. Sample was drop casted on carbon coated copper
wired TEM grid and instrument was operated at 120 kV. Similarly, Ag NPs embedded
LSH film was air dried and stored in a dark area for six months before analyzing by TEM.
59
In another experiment, stored LSH film impregnated with Ag NPs was dissolved in
deionized water. Ag NPs were isolated by centrifugation and viewed by TEM.
Morphology and particle size determination of Docetaxel loaded LSH nanoparticles were
evaluated using TEM. Freeze dried NPs (0.1 wt%) were dispersed in deionized water.
Solution (5 µL of aqueous solution containing NPs (1 mg/1 mL in distilled water)) was
dropped on the carbon-coated grid and kept it in a vacuum oven for 24 h at 25 °C. Prepared
sample was examined with microscope (Hitachi 7600) operated at 100 kV.
2.2.9. Powder X-ray diffraction
Powder X-ray diffraction (PXRD) spectra of LSH and LSH mediated Ag NPs were recorded
on Bruker D8 Advance (Germany) diffractometer (over a range of 10-80°, 2ϴ), operated at
40 mA and 40 KV.
2.3. Acetylation of linseed hydrogel
2.3.1. Acetylation of LSH
LSH was isolated using hot water extraction method (for isolation procedure, see section
2.4.1). To study the modification ability of LSH, acetylation reaction was selected as a novel
approach (Muhammad et al., 2016). Three reactions were carried out to observe the effect of
reactants on degree of acetylation of LSH. Acetylation reaction was performed using LSH
and acetic anhydride in mole ratio of 1:6 (ALSH 1), 1:12 (ALSH 2) and 1:18 (ALSH 3),
respectively.
ALSH 1:
In a typical reaction condition, LSH (1.0 g, 6.167 mmol) was suspended in DMSO (40 mL)
followed by the addition of acetic anhydride (3.5 mL, 37.002 mmol) using DMAP (40 mg) as
60
a catalyst. The suspension was heated at 80 °C with continuous stirring for 6 h. Acetylated
LSH (ALSH 1) was dried at 50 °C after precipitation and washing with ethanol.
Yield: (1.02 g); DS: 2.11; FTIR: 1749 (COEster), 2963 (CH), 1376 (CH2), 3440 cm-1
(OH),
1042 (COC); 1H NMR (DMSO-d6; 600 MHz; NS 64): 2.05 (CH3), 3.13-5.62 (Repeating unit-
Hs).
ALSH 2:
In another reaction, LSH (1.0 g, 6.167 mmol) was allowed to suspend in DMSO (40 mL).
Acetic anhydride (6.996 mL, 74.01 mmol) and DMAP (40 mg) were added in the suspension
of LSH. Reaction mixture was kept 80 °C with continuous stirring for 6 h. Acetylated
product (ALSH 2) was separated through precipitation in ethanol. ALSH 2 was washed with
ethanol and dried at 50 °C.
Yield: (1.12 g); DS: 2.53; FTIR: 1752 (COEster), 2936 (CH), 1373 (CH2), 3425 cm-1
(OH),
1043 (COC); 1H NMR (DMSO-d6; 600 MHz; NS 64): 2.05 (CH3), 3.13-5.62 (Repeating unit-
Hs).
ALSH 3:
In this reaction, suspension of LSH (1.0 g, 6.167 mmol) was prepared in DMSO (40 mL) and
acetic anhydride (10.5 mL, 111.015 mmol) was added as an acetylating agent. DMAP (40
mg) was used as a catalyst. Reaction assembly was heated for 6 h at 80 °C with continuous
stirring. ALSH 3 was separated and purified by using ethanol. Finally, sample was dried at
50 °C and yield was calculated.
Yield: (1.33 g); DS: 2.91; FTIR: 1738 (COEster), 2926 (CH), 1376 (CH2), 3514 cm-1
(OH),
1044 (COC); 1H NMR (DMSO-d6; 600 MHz; NS 64): 2.06 (CH3), 3.14-5.62 (Repeating unit-
Hs).
61
2.3.2. Calculation of degree of substitution
For the determination of degree of substitution (DSb) of acetylation on LSH by acid-base
titration method, ALSH (100 mg) was stirred with 0.1M NaOH aqueous solution (50 mL) for
complete saponification. The solution was kept neutral at pH 7.0 with 0.01M HCl solution.
After that, 1M NaOH solution (known volume) was mixed with this neutral solution. DSb of
acetylation was calculated after neutralizing excess NaOH with 0.1M HCl solution using
following formula;
(Eq. 6)
where, n.NaOH is the number of moles of NaOH added after saponification, M (RU) is molar
mass of repeating unit of the polymer, Ms is the mass of sample taken and Mr (RCO) is
molar mass of ester functionality.
2.3.3. Thermogravimetric analysis and degradation kinetics of LSH and ALSH
Initial thermal decomposition temperature (Tdi), maximum thermal decomposition
temperature (Tdm) and final thermal decomposition temperature (Tdf) were calculated from
TG curve of LSH and ALSH. Thermal kinetics was calculated using isoconversional
methods. Flynn-wall and Ozawa (FWO) method was used to calculate the kinetic parameters
as described in Eq. 7 (Flynn, 1990; Ozawa, 1965; Sathasivam and Haris, 2012).
ln ln 5.331 1.052( )
a aAE E
Rg RT
(Eq. 7)
where, β is heating rate, A is pre-exponential factor, Ea is activation energy, R is general gas
constant and T is the temperature at the conversion rate (α). The α is calculated by Eq. 8.
o t
o f
W W
W W
(Eq. 8)
where, W0 is initial mass, Wf is final mass and Wt is the mass of sample at any temperature T.
62
FWO method is considered as a model free approach because rate of thermal degradation is
dependent on temperature for a fixed extent of conversion. To calculate fixed value of α at
different heating rate, a graph is plotted between ln β and 1000/T which results in straight line
graph. Value of Ea is calculated from the slop.
Kissinger‘s method is based on the assumption that in differential thermal analysis,
temperature required for maximum deflection is the same on which the maximum rate of
reaction is observed (Kissinger, 1957). Kissinger described the method using Eq. 9.
(Eq. 9)
Graph is plotted between ln(β/Tm2) and 1000 Tm
-2 for a constant conversion which gives Ea at
that conversion.
2.4. Dynamic swelling and stimuli responsive on-off switching of LSH
2.4.1. Isolation of LSH
Linseeds were soaked in deionized water for 48 h before heating at 80 °C for 30 min.
Mucilage was extruded from seeds, separated through nylon mesh, de-fatted with n-hexane
and washed thoroughly with deionized water. Mucilage was centrifuged at 4000 rpm for 30
min. to get LSH which was air dried for 24 h and then placed in vacuum oven at 60 °C for
another 24 h. Finally, dried LSH was ground to fine powder, passed through sieve no. 60 and
stored in an air-tight container in desiccator under vacuum. Yield: 6.5% (g/g) of dried seeds.
2.4.2. Physical properties of LSH
Physical properties of LSH were evaluated by determining moisture content, particle size,
angle of repose, bulk density, tap density, Hausner ratio and Carr‘s index (Lachman et al.,
1987). All parameters were measured in triplicate and mean value was expressed with ± SD.
63
Angle of repose
Angle of repose was calculated to determine the flow property of LSH by using fixed funnel
method (Wells, 1988). LSH was permitted to fall through a fixed funnel on a graph paper.
The height (h) and radius (r) of the heap formed was noted and angle of repose (θ) was
calculated by using Eq. 10.
an h
Tr
(Eq. 10)
Bulk and tap density
Accurately weighed LSH (1.0 g) was taken in graduated cylinder (10 mL) and bulk volume
(Vb) was noted. Tapped volume (Vt) of LSH was noted after 100 tapping. Bulk density (Db)
and tapped density (Dt) was calculated by dividing the weight of LSH with bulk volume (Vb)
and tapped volume (Vt), respectively.
Hausner ratio and Carr’s index
Hausner ratio (Wells, 1988) is the ratio of the bulk density to tap density and it was
calculated using Eq. 11. Carr‘s index (Wells, 1988) is the measure of packing arrangements
of LSH and calculated using Eq. 12.
(Eq. 11)
100 1 b
t
DCarr sindex C
D
(Eq. 12)
where, in Eq. 11 and 12, H is Hausner ratio, Db is bulk density, Dt is tap density and C is
Carr‘s index.
64
Gelation content
LSH (0.1 g) was allowed to swell in deionized water (10 mL) for 24 h at room temperature.
It was then centrifuged at 4000 rpm for 30 min. Supernatant was decanted and sediment paste
was collected and weighed. Sediment paste was oven dried at 70 ºC under vacuum until
constant weight was obtained. This dried sediment paste is the gelling content of LSH (Ring,
1985; Peerapattana et al., 2010). Percentage of gelling content was calculated using Eq. 13.
% 100f
i
WGelling content
W (Eq. 13)
where, Wf is the dried weight of the sediment paste and Wi is the weight of wet paste.
Centrifuge retention capacity
Centrifuge retention capacity is commonly known as water retention capacity. Freshly
prepared LSH (1% w/w in deionized water) was centrifuged at 4000 rpm for 30 min to
determine its water retention capacity. Sediment paste of LSH separated by decanting the
upper portion was completely dried at 70 ºC. Water retention capacity was calculated as the
ratio of weight of sediment paste to the weight of dried mass (Ring, 1985; Peerapattana et al.,
2010).
Moisture content
Moisture content of LSH was calculated by recording the weight of LSH before and after
heating at 105 ºC for 1 h in a vacuum oven.
2.4.3. Preparation of buffer solutions of different pH
Swelling study of LSH was carried out in deionized water and pH 1.2, 6.8 and 7.4. Buffer
solution of pH 1.2 was prepared by mixing KCl (250 mL, 0.2 M) and HCl solution (425 mL,
0.2 M) in 1000 mL measuring flask and final volume was adjusted to 1000 mL with
65
deionized water. To prepare buffer solution of pH 6.8, potassium dihydrogen phosphate (250
mL, 0.2 M) and NaOH solution (112 mL, 0.2 M) were taken in 1000 mL measuring flask and
volume was made up to 1000 mL with deionized water. Potassium dihydrogen phosphate
(250 mL, 0.2 M) and NaOH solution (195.5 mL, 0.2 M) were placed in 1000 mL measuring
flask and deionized water was added up to the mark to prepare buffer solution of pH 7.4.
2.4.4. Evaluation of pH responsive property of LSH
LSH was evaluated for its swelling behavior under different environmental conditions. For
this purpose, swelling of LSH was monitored at different pH mimicking the gastrointestinal
tract (GIT) pH. Furthermore, stimuli responsive swelling de-swelling capability of LSH was
also determined by altering the contact media.
Accurately weighed LSH (0.5 g) was placed in each of four cellophane bags and hung in
separate beakers (100 mL each). Buffer solutions of pH 1.2, 6.8, 7.4 and deionized water
were added in the beakers. Cellophane bags containing swollen LSH were removed after
specific time intervals. The bags were hung for some time to remove excess water, accurately
weighed and placed again in respective media for further swelling. Swelling capacity (g/g)
was calculated using Eq. 14. Experiment was performed thrice and mean of all values were
reported.
0 0 ( ) /t cSwelling capacity w w w w (Eq. 14)
where, wt is weight of wet cellophane bag containing swollen LSH, wo is weight of dry LSH
and wc is weight of wet cellophane bag.
Same experiment was performed in a single step for 24 h in order to note swelling capacity
which is another essential physical parameter for swellable inactive pharmaceutical
ingredient.
66
2.4.5. Swelling kinetics
To study the rate of absorbency of LSH, samples (0.5 g) were poured into cellophane bags
and immersed in 50 mL media (pH 1.2, 6.8, 7.4 and deionized water). At specific time
intervals, the water absorbency of the LSH was measured according to the following relation
(Krusic et al., 2006; Malana et al., 2012).
s d tt
d d
W W WQ
W W
(Eq. 15)
where, Wd is initial weight of dried powder, Ws is swollen weight of powder at time t and Wt
is weight of water penetrated into LSH at time t. The Qt is normalized degree of swelling.
The normalized equilibrium degree of swelling Qe was determined using Eq. 16.
d ee
d d
W W WQ
W W
(Eq. 16)
where, W∞ is weight of swollen gel at t∞ when swelling rate becomes constant, Wd is initial
weight of dried powder and We is amount of water penetrated into LSH at t∞.
It was possible to analyze kinetics of the swelling process by using values of normalized
degree of swelling (Qt) and normalized equilibrium degree of swelling (Qe) at time t. For
second-order kinetics following equation can be used.
2
1
t e e
t t
Q Q kQ (Eq. 17)
For second order kinetics, a plot of t/Qt vs t should be linear with the slope of 1/Qe and an
intercept of 1/kQe2.
2.4.6. Thermoresponsive swelling capacity of LSH in deionized water
Swelling response of LSH in deionized water was evaluated at different temperatures (30, 40
and 50 °C). LSH (0.5 g) was taken separately in three cellophane bags and hung in separate
beakers (50 mL). Deionized water was added up to the mark and beakers were placed in
67
water baths previously maintained at 30, 40 and 50 °C. Remaining procedure was same as
mentioned in section 2.4.4. Thermoresponsive swelling capacity of LSH was determined
three times and mean was calculated and reported.
2.4.7. Evaluation of salt solution-responsive properties of LSH
Equilibrium swelling (after 24 h) of LSH was measured in different aqueous salt solutions of
NaCl and KCl (0.1, 0.2, 0.3, 0.4, 0.5, 1, 1.5, 2 M each). LSH (0.5 g) was taken in each
cellophane bag and hung in different beakers (50 mL) having the above mentioned
concentrations of NaCl and KCl. After 24 h, cellophane bags were removed and weighed
after hanging for some time to drain out excess water. Swelling capacity (g/g) was calculated
using Eq. 14. This experiment was performed three times and mean was reported.
2.4.8. Evaluation of pH responsive on-off switching of LSH
Swelling behavior of LSH was observed at pH 7.4 and 1.2. Accurately weighed LSH (0.5 g)
was taken in cellophane bag and hung in a beaker having buffer solution (50 mL) of pH 7.4
for 15 min. Cellophane bag containing LSH was weighed after removing excess medium.
This cellophane bag was hung in another beaker having buffer solution (50 mL) of pH 1.2 for
15 min. After removing excess medium, cellophane bag was weighed and hung in buffer
solution of pH 7.4. Swelling capacity was calculated after predetermined time intervals using
Eq. 14. The on-off switching cycle were performed five times. On-off switching experiment
was repeated three times and mean was expressed graphically.
2.4.9. Evaluation of saline responsive on-off switching of LSH
Saline responsive on-off switching of LSH was determined in deionized water and NaCl
(0.9%, w/v), respectively. LSH (0.5 g) was taken in cellophane bag and hung in a beaker
containing deionized water (50 mL). Cellophane bag was removed after specific time
68
intervals, hung for some time to remove excess water and weighed to calculate the weight of
swollen LSH. After 1 h of swelling study in deionized water, swelling media was replaced
with NaCl (0.9%) and rest of the procedure was same as mentioned in section 2.4.4. This on-
off procedure was repeated four times. On-off procedure was performed three times and
mean was reported graphically.
2.4.10. Evaluation of on-off switching of LSH in water and ethanol
Accurately weighed LSH (0.5 g) was taken in cellophane bag and hung in a beaker.
Deionized water (50 mL) was added and after predetermined time intervals, cellophane bag
was removed from the beaker and weighed after removing the excess water. After 1 h,
deionized water was replaced with ethanol and swelling capacity of LSH was determined
using Eq. 14. This on-off switching as function of solvent nature was measured four times.
Whole experiment was repeated three times and mean was plotted graphically.
2.5. Development of sustained drug delivery stystem
2.5.1. Formulation design
To evaluate LSH as sustained release material, tablets were prepared and undergo for
analysis for sustained release formulation. For this purpose, diclofenac sodium (DS), caffeine
and diacerein were used to evaluate the sustained release potential of LSH. Additionally,
caffeine and diacerein containing formulations were also used to observe the stimuli
responsive behaviour of LSH in tablet formulation.
2.5.1.1. Drug-excipient compatibility study
Purity of isolated LSH and its compatibility with drugs and excipients were evaluated using
FTIR spectroscopy. Samples were mixed with KBr to prepare the pellets for FTIR analysis.
FTIR spectra were recorded on IR Prestige-21 (Shimadzu, Japan) from 4000 to 400 cm 1
.
69
2.5.1.2. Preparation of tablets
Caffeine, DS and diacerein were used to evaluate the potential of LSH as a sustained release
agent for oral drug delivery system. Tablets of LSH with DS, caffeine and diacerein were
prepared by wet granulation method according to the composition mentioned in Table 2.1,
Table 2.2 and Table 2.3, respectively. LSH, DS and microcrystalline cellulose were passed
through sieve no. 40 and mixed thoroughly in pestle and mortar. Dry mixture was kneaded
with polyvinylpyrrolidone (PVP K30) solution (5% w/v in isopropyl alcohol) to get a damp
mass. Wet mass was passed through sieve no. 12 and dried at 40 °C for 6 h. Dried granules
were finally passed through sieve no. 20, lubricated with magnesium stearate and evaluated
for pre-compression parameters. Granules (300 ± 7 mg) were compressed on a rotary press
fitted with 9 mm flat surface punch. Hardness and thickness of tablets were adjusted at 6-7
kg/cm2 and 3.45-3.65 mm, respectively.
Similarly, formulation of caffeine and diacereine were also prepared by wet granulation
method as adopted for DS containing formulation and according to the composition
mentioned in Table 2.2 and Table 2.3, respectively. Compression weight of lubricated
granules was set at 265 ± 5 mg on a rotary tablet machine fixed with flat surface punch of 9
mm diameter.
Prepared tablets of DS, caffeine and diacereine were evaluated for post-compression
parameters and stored in desiccator until further use. To observe the effect of LSH
concentration on the release of these drugs, three different formulations of each drug with
varying the concentration of LSH were prepared.
70
Table 2.1. Composition of different formulations to evaluate the sustained release behavior
of diclofenac sodium from LSH tablet.
Formulation composition (mg/tablet) D1 D2 D3
LSH 75 100 125
Diclofenac sodium 100 100 100
Microcrystalline cellulose 115 90 65
PVP K30 7 7 7
Magnesium stearate 3 3 3
Total weight 300 300 300
Table 2.2. Tablet formulation design to evaluate the sustained release behavior of caffeine.
Formulation composition (mg/tablet) FH FC FC1 FC2 FC3
LSH 100 - 50 75 100
Caffeine - 100 100 100 100
Microcrystalline cellulose 150 150 100 75 50
PVP K-30 10 10 10 10 10
Magnesium stearate 5 5 5 5 5
Total weight 265 265 265 265 265
71
Table 2.3. Constituents of various tablet formulations to study sustained release behavior of
diacerein.
Formulation composition (mg/tablet) FH FD FD1 FD2 FD3
LSH 100 - 50 75 100
Diacerein - 100 100 100 100
Microcrystalline cellulose 150 150 100 75 50
PVP K30 10 10 10 10 10
Magnesium stearate 5 5 5 5 5
Total weight 265 265 265 265 265
2.5.1.3. Pre-compression evaluation
Before feeding into tablet compression machine, lubricated granules of each formulation
(Table 2.1, 2.2 and 2.3) were evaluated for their flow properties and compressibility. For this
purpose, angle of repose, loose and tapped bulk density, Hausner ratio and compressibility
index were determined as described in section 2.4.2. All experiments were performed thrice
and mean values along with ± SD were expressed.
2.5.1.4. Post-compression evaluation
Compressed tablets were evaluated for hardness, thickness, diameter, weight variation and
friability (Lachman et al., 1987). Content uniformity of prepared tablets was also determined
to confirm the uniformly distribution of active ingredient.
Diameter, thickness and hardness test
Prepared tablets were evaluated for diameter, thickness and hardness test through hardness
tester (Pharma Test, PTB 311E, Germany). Hardness or crushing strength is the force
72
required to break the tablet diametrically. Each test was performed on randomly selected ten
tablets from each formulation and mean value along with standard deviation were reported.
Weight variation test
Compressed tablets from each formulation were tested for weight variation test. For this
purpose, twenty tablets were selected randomly and carefully weighing each tablet on
analytical balance (Shimadzu, Japan). The mean weight and standard deviation was
calculated and reported.
Friability test
Ten tablets from each formulation were randomly selected, accurately weighed, placed in a
chamber of friability tester (Pharma Test, PTF 10E, Germany) and allowed to rotate at 25
rpm for 4 min. Tablets were removed from the chamber, gently cleaned from dust particles
and accurately weighed. Friability was calculated in term of percentage weight loss using Eq.
18.
(Eq. 18)
where, wi and wf are the weight of the tablet before and after friability test, respectively.
Friability test was performed thrice and mean was calculated and reported.
Content uniformity
To determine the DS, caffeine and diacerein content in tablet formulations, ten tablets were
picked randomly from each formulation and separately crushed in pestle and mortar.
Accurately weighed crushed powder (300 mg for DS formulation and 265 mg for caffeine
and diacerein formulations) were taken in volumetric flask (50 mL), mixed vigorously with
methanol and make the volume up to the mark. Solution was filtered, properly diluted and
scan through UV spectrophotometer at 276 nm, 273 nm and 254 nm for DS, caffeine and
73
diacerein, respectively. Absorbance was compared with standard sample and content (%) of
DS, caffeine and diacerein was calculated. Mean of three values were taken and standard
deviation was calculated.
2.5.2. Dynamic swelling and stimuli responsive evaluation of LSH based tablet
formulations
Swelling and stimuli responsive behavior of LSH in tablet formulations (Table 2.2 and Table
2.3) were evaluated.
2.5.2.1. pH responsive swelling of LSH containing tablets
Swelling response of caffeine and diacerien containing formulations (Table 2.2 and Table
2.3) was evaluated in acid buffer of pH 1.2, phosphate buffer of pH 6.8 and 7.4 and also in
deionized water at 37 °C for 16 h. Buffer solutions were prepared according to United States
Pharmacopeia (USP) 37. Formulated tablets were undergo pH responsive swelling study
using cellophane bag to keep all the disintegrated fragments (if any) of swollen tablets at one
place. Four tablets of each formulation were taken separately in four cellophane bags and
placed in four beakers (100 mL) containing buffer solutions (pH 1.2, 6.8, 7.4) and deionized
water. After predetermined time intervals, these cellophane bags containing swollen tablets
were taken out of the beaker, hung for some time to drain out excess swelling media,
weighed accurately and placed them again in their respective beakers to continue the
swelling study. Swelling capacity (g/g) was calculated using Eq. 14.
2.5.2.2. Swelling kinetics
Kinetics of swelling process of tablet formulations was calculated from their swelling data in
various media and determined by the procedure as described in section 2.4.5.
74
2.5.2.3. Evaluation of salt solution responsive swelling
Prepared tablets from each formulation (Table 2.2 and Table 2.3) were evaluated for the
swelling response in different molar solutions of NaCl and KCl. Swelling response was
monitored against eight different concentrations (0.1, 0.2, 0.3, 0.4, 0.5, 1.0, 1.5 and 2.0 M) of
aqueous NaCl and KCl solution by adopting the cellophane bag method as described in
section 2.4.7. After 24 h, swelling capacity (g/g) was calculated using Eq. 14.
2.5.2.4. Stimuli responsive swelling-deswelling (on-off) behavior
Stimuli responsive behavior of the prepared tablets was observed in deionized water, normal
saline solution, ethanol and buffer solution of pH 7.4 and 1.2. For this purpose, tablets of
each formulation were first placed in the swelling media (deionized water and pH 7.4) for 1 h
and then transferred to the deswelling media (normal saline, ethanol and pH 1.2) for another
1 h. Swelling capacity was calculated after every 10 min as described in section 2.4.4. The
swelling-deswelling experiments were carried out over four cycles and repeated thrice.
2.5.3. Evaluation of drug release behavior
2.5.3.1. In-vitro drug release studies
In order to evaluate the sustained release potential of LSH, in vitro drug release study from
prepared formulations was carried out in buffer solutions of pH 1.2, 6.8, 7.4 and in deionized
water. Drug release behaviour imitating the pH and transit time through GIT was also
determined. Each experiment was performed thrice, mean was calculated and reported
graphically.
75
In-vitro release study of DS
DS release study from LSH containing tablets were carried out in USP dissolution apparatus
II (Pharma Test, PT-DT7, Germany). Drug release behavior from the prepared tablets were
studied in SGF (900 mL) for 2 h, then tablets were transferred to SIF (900 mL) and release
was monitored for 14 h at 37 °C and 50 rpm. After selected time intervals, sample (1 mL)
was taken out from respective media, filtered through 0.45 µm filter, diluted (if necessary)
and analyzed through UV spectrophotometer (Shimadzu, Japan) at 276 nm. The volume of
release media were immediately replenished with fresh SGF or SIF. This experiment was
repeated for three times and mean values were expressed in terms of cumulative percentage
of drug release. Same experiment was performed with a commercially available sustained
release formulation of diclofenac sodium (Voltral® SR 100 tablet) to compare with LSH
containing formulations.
In-vitro release study of caffeine
In vitro caffeine release from LSH containing tablets were performed by using USP
dissolution apparatus II. Four different media (buffers of pH 1.2, 6.8, 7.4; and deionized
water) were used to evaluate the release behavior of caffeine from LSH containing caffeine
formulation. Buffers of pH 1.2, 6.8 and 7.4 were prepared according to the procedure
mentioned in USP 37. Drug release study was carried out in 900 mL media kept at 37 ± 0.1
°C and 50 rpm. Aliquot (3 mL) was withdrawn from the vessels after predetermined time
intervals (0.5, 1, 1.5, 2, 3, 4, 5, 6, 8, 10, 12, 16 and 24 h) and immediately replenished with
the same media to maintain the volume at 900 mL. Samples were filtered through 0.45 µm
nylon filter, suitably diluted and analyzed through UV spectrophotometer at 273 nm. Further,
drug release pattern of each formulation mimicking the transit time and pH of the GIT was
76
also monitored. Therefore, drug release study was carried out for 2 h at pH 1.2, then 8 h at
pH 6.8 and finally for 6 h at pH 7.4.
In-vitro release study of diacerein
USP dissolution apparatus II was used to determine the release of diacerein from three
formulations. For this purpose, buffers of pH 1.2, 6.8 and 7.4, and deionized water were
selected as drug release media. Drug release study from each formulation was carried out in
900 mL media maintained at 37 ± 0.1 °C and paddle rotation speed was adjusted to 50 rpm.
Sample (3 mL) was withdrawn from the vessels after preset time intervals (0.5, 1, 1.5, 2, 3, 4,
5, 6, 8, 10, 12, 16 and 24 h) and immediately replaced with the same preheated (37 ± 0.1 °C)
media to maintain the volume at 900 mL. Samples were filtered through 0.45 µm nylon filter,
suitably diluted and analyzed through UV spectrophotometer at 254 nm. Furthermore, drug
release pattern of each formulation was also analyzed in conditions mimicking the transit
time and pH of the gastrointestinal tract. Therefore, drug release study was carried out at pH
1.2 for 2 h, then at pH 6.8 for 8 h and finally at pH 7.4 for 6 h.
2.5.3.2. Drug release kinetics
Dissolution profile of the LSH containing tablets of DS, caffeine and diacerein were analyzed
by using different kinetic models i.e., zero order (Eq. 19), first order (Eq. 20), Higuchi (Eq.
21) and Hixson-Crowell (Eq. 22). The coefficient of determination (R2) was calculated by
plotting a graph between cumulative drug release and time for zero order, log of cumulative
drug remaining and time for first order, cumulative drug release and square root of time for
Higuchi model and cube root of percentage remaining and time for Hixson-Crowell model.
The model having highest value (~1) for coefficient of determination (R2) was considered the
best fitted model.
(Eq. 19)
77
where, Qt is the amount of drug released from the tablet in time t and K0 is the zero order rate
constant.
(Eq. 20)
where, Q is the remaining amount of drug in the tablet after time t, Q0 is the initial amount of
drug in the tablet and K1 is the first order rate constant (Gibaldi and Feldman, 1967; Wagner,
1969).
(Eq. 21)
where, Qt is the amount of drug released in time t and KH is the Higuchi rate constant
(Higuchi, 1961; Higuchi, 1963).
(Eq. 22)
where, Q0 is the initial amount of drug in the tablet, Qt is the amount of drug released from
the tablet in time t and KHC is the Hixson-Crowell release constant (Hixson et al., 1931).
2.5.3.3. Drug release mechanism
The release of drug from a polymeric matrix system is a complex phenomenon and may
involve one or more mechanisms i.e., swelling and/or erosion of polymer, dissolution and/or
diffusion of drug. Drug release mechanism from a polymeric drug delivery system is best
explained by Korsmeyer-Peppas model (Korsmeyer et al., 1983; Ritger and Peppas, 1987) in
Eq. 23.
(Eq. 23)
where, Mt/M∞ is the fraction of drug release at time t, Kp is the Korsmeyer-Peppas constant
and n is the release exponent. The value of n is calculated from the slop of a graph plotted
between log Mt/M∞ and log t. The value of n indicates the release mechanism of the drug
from a polymeric matrix system. In case of cylindrical (tablet) drug delivery system, the
release mechanism is considered Fickian diffusion, non-Fickian diffusion (anomalous
78
transport), case II transport and super case II transport if n ≤ 0.45, 0.45 < n < 0.89, n = 0.89
and n > 0.89, respectively (Korsmeyer et al., 1983; Ritger and peppas, 1987).
To select a best fit model for drug release, a modified Akaike Information Criterion called
Model Selection Criterion (MSC) was used as described by Iqbal et al., 2011a and expressed
in Eq. 24.
2
- -
1
2
- -
1
. -
ln
. -
n
i i obs obs
i
n
i i obs pre
i
w y y
MSC
w y y
(Eq. 24)
where, yi_obs is the ith
observed value of y, y _obs is the mean of observed data points of y and
yi_pre is the ith
predicted value of y. wi is weighting factor and its value is usually equal to 1
for fitting the dissolution data, n is the number of all data points and p is the number of
parameters in the model.
All calculations related to drug release kinetics and mechanism was performed by using
DDSolver, an add-in program for drug dissolution modeling and comparison, as used by
Zhang et al., 2010.
2.5.4. Scanning electron microscopy analysis
Dried LSH was swollen in deionized water and freeze dried. Cross-sections of hydrogel were
obtained by using sharp blade and observed under FE-SEM to determine the morphology of
freeze-dried sample. Sample was drop casted on a carbon mount and dried at 25 °C in a
vacuum oven for 24 h prior to analysis.
Texture and cross section area of prepared tablets were also investigated using SEM. Tablets
were immersed in deionized water to swell followed by freeze drying and then observed
under SEM.
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2.6. Docetaxel loaded LSH-Pluronic NPs
2.6.1. Preparation of NPs
Docetaxel (DTX) loaded LSH-Pluronic F-68 nanoparticles (DLP-NPs) were prepared in two
steps. First, LSH (2 mg) was dispersed in distilled-deionized water (1 mL). DTX (10 mg) was
dissolved separately in ethanol (0.5 mL) and then added in LSH solution drop wise. This
mixture was kept on stirring for 6 h followed by lyophilization to prepare LSH-DTX core. In
second step, lyophilized sample was dispersed in distilled-deionized water and Pluronic F-68
(500 mg) was added with gentle stirring. After 6 h, mixture was lyophilized to complete the
core shell formation of DLP-NPs.
To get purified DLP-NPs, the prepared NPs were dispersed in deionized water and kept under
mild shaking condition at 25 °C. After 5 min, the dispersion of NPs was centrifuged at 3500
rpm for 10 min followed by freeze drying to get ultra-purified NPs.
For cellular uptake experiment, Nile red, a fluorescence dye, was loaded in DLP-NPs. For
this purpose, 100 µL of Nile red (1 mg/mL in ethanol) was mixed at drug loading phase
during the preparation of DLP-NPs (Khaliq et al., 2016).
2.6.2. Encapsulation efficiency and drug loading
Weight amount of DLP-NPs were dissolved in 4 mL distilled water and centrifuged at 3500
rpm for 5 min. The supernatant was taken, mix with methanol in 1:1 (v/v) and passed through
0.22 µm filter. Quantification of the DTX was determined by Agilent Technologies 1260
series HPLC system. The analysis were performed by reverse phase (RP-HPLC) using a Cap
cell-pack C18 column and an acetonitrile/water (60/40, v/v) as a mobile phase over 10 min at
a flow rate of 1.2 mL/min with an injection volume of 5 µL. The eluent was monitored by
UV absorption at 227 nm.
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Drug loading (DL) and encapsulation efficiency (EE) was calculated using Eq. 25 and Eq.
26, respectively.
(Eq. 25)
(Eq. 26)
where, in Eq. 25 and Eq. 26, wD, wNP and wDi are the weight of drug in nanoparticles, weight
of nanoparticles and weight of drug initially added for incorporation into nanoparticles,
respectively.
2.6.3. Particle size and morphology
Average diameter, size distribution and zeta potential of the DLP-NPs was determined by
dynamic light scattering (Zeta Sizer Nano Series, Malvern, UK) at 632.8 nm and 25 °C. For
this purpose, freeze dried DLP-NPs (1 mg) were dispersed in phosphate buffered saline
(PBS, 1 mL) of pH 7.4 and analyzed by DLS. The morphology of the prepared DLP-NPs was
examined by field emission electron microscopy (FESEM). Freeze dried DLP-NPs (0.1 wt%)
were dispersed in distilled-deionized water. Aqueous solution (5 µL) of DLP-NPs (0.1 wt%)
was dropped on the aluminum stub and kept it in a vacuum oven for 24 h at 25 °C. Prepared
sample was examined with FESEM (Nova NanoSEM 450, FEI) operated at 10 kV and
distance of 5 mm using though lens detector (TLD).
2.6.4. X-ray diffraction analysis
To evaluate the nature (amorphous or crystalline) of Pluronic F-68, LSH, DTX and DLP-
NPs, XRD pattern was determined using Bruker D8 Advance (Germany) diffractometer
operated at 40 mA and 40 kV with Cu Kα radiation. For this purpose, sample was placed in a
quartz sample holder and scanned from 5 to 40° at a scanning rate of 5°/min.
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2.6.5. In vitro drug release study
In vitro drug release study of DTX from the prepared DLP-NPs was performed in dialysis
bag (Oh et al., 2014). DLP-NPs (10 mg) were dispersed in PBS (10 mL), put in dialysis bag
(molecular weight cut off, MWCO: 12000-14000 Daltons, Spectrum®, Rancho Dominquez,
CA, USA) and completely immersed in PBS (pH 7.4, 20 mL) having Tween 80 (0.1% w/v).
Assembly was kept at 37 °C in a water bath and shaken horizontally at 100 rpm. After
predetermined time intervals, aliquot (2 mL) was withdrawn and whole medium was
replaced with fresh PBS (20 mL). Amount of released DTX was determined by reverse-phase
high performance liquid chromatography (RP-HPLC) using C18 column at detection
wavelength of 220 nm. Mobile phase (acetonitrile/water, 60/40 v/v) was run for 15 min at a
flow rate of 1.2 mL/min.
2.6.6. Cytotoxicity and cellular uptake behaviour
Murine SCC-7 (squamous cell carcinoma) cells were cultured in RPMI 1640 media (Gibco,
Grand Island, USA) containing 10% (v/v) FBS (Gibco) and 1% (w/v) penicillin-streptomycin
at 37 °C in a humidified 5% CO2-95% air atmosphere. Cytotoxicity of DLP-NPs, LSH and
free DTX (Taxotere®, a commercially available DTX formulation) was evaluated using MTT
assay. Cells were harvested in 96-well flat bottomed plates at a density of 5×103 cells/well
and allowed to adhere overnight. Cells were washed with PBS and incubated with various
concentrations of DLP-NPs, LSH and free DTX for 24 h. Cells were washed twice with fresh
PBS in order to eliminate any residual drugs. Twenty five microliters of MTT solution (5
mg/mL in PBS) was added to each well and incubated further for 2 h at 37 °C. In each well,
200 µL of DMSO was added and absorbance was measured at 570 nm with the help of
microplate reader (VERSAmax™, Molecular Devices Corp., Sunnyvale, USA). Percentage
cell viability was calculated for free DTX and empty NPs (without loading of DTX) and
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considered as equivalent DTX concentrations and NPs with same concentrations,
respectively. Cell viability was also determined for LSH and only culture medium was kept
as a negative control groups. IC50 was calculated for the drug concentration from the cell
viability data, in which cell growth was inhibited by 50%. Cell viability data was used to
calculate IC50 which is a drug concentration at which 50% inhibition of cell growth was
observed.
To confirm the cellular uptake of Nile red loaded DLP-NPs, murine squamous cell carcinoma
(SCC-7) tumor cells at a density of 1 × 105 were seeded onto a dish which was protected with
cover slip and allowed to attach for 24 h. After that, cells were treated with fresh medium (2
mL) containing Nile red loaded DLP-NPs followed by incubation for 4 h. Cells were washed
twice with PBS (pH 7.4), paraformaldehyde solution (4%) was used to fix these cells. For
nuclear staining, 4ʹ,6-diamidino-2-phenylindole (DAPI, 3 mmol) was added and incubated at
25 °C for 5 min followed by multiple washing with PBS. Intracellular localization of Nile red
loaded DLP-NPs was visualized using an IX81-ZDC focus drift compensation microscope
(Olympus, Tokyo, Japan) adjusted at 640 nm and 553 nm for emission and excitation
wavelengths, respectively.
2.6.7. Statistical analysis
All experiments were performed at least for three times and data values are expressed as the
mean ± SD. Statistical analysis was carried out using one-way ANOVA and value of p < 0.05
was considered significant.
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2.7. Nanobiotechnological application of LSH mediated Ag NPs
2.7.1. Preparation of AgNO3 solution and LSH suspension
Three different concentrations of AgNO3 solution (10, 20, 30 mmol) were prepared by
dissolving 169.87, 339.74 and 509.61 mg of AgNO3, respectively in deionized water (100
mL). LSH (0.2 g) was suspended in deionized water (10 mL) to prepare 2% (w/v) solution.
2.7.2. Green synthesis of Ag NPs
In order to synthesize silver nanoparticle (Ag NPs), freshly prepared solution of AgNO3 (10
mmol, 10 mL) was added to the suspension of LSH (10 mL). The mixture was stirred in a
dark area for 5 min at room temperature to ensure homogenous mixing of AgNO3 with LSH
suspension. This mixture was then placed in sunlight and progress of reaction was monitored
by observing the change of color. Sample (1 mL) of this reaction mixture was also analyzed
with the help of UV/Vis spectrophotometry. Same protocol was adopted for the other AgNO3
(20 and 30 mmol) solutions, respectively.
2.7.3. Film formation
A mixture of AgNO3 (10, 20 and 30 mmol) and LSH was exposed to diffused sunlight for 10
h. This mixture was air dried under dark in a petri dish to produce LSH thin films containing
the embedded Ag NPs. The thin dry films were subsequently characterized using UV/Vis
spectrophotometry, PXRD and TEM.
2.7.4. UV spectrophotometric analysis
Formation of Ag NPs with the passage of time was monitored by UV/Vis spectrophotometry.
Sample (1.0 mL) of the reaction mixture was taken at selected time intervals (0.25, 0.5, 0.75,
1, 2, 4, 6, 8 and 10 h) and analyzed in a range 200 to 800 nm on UV/Vis Spectrophotometer
84
(Optizen POP, Mecasays, Korea). Effect of various reaction conditions (conc. of AgNO3
solution and reaction time) on the progress of reaction was also studied.
2.7.5. Powder X-ray diffraction
LSH suspension containing Ag NPs and isolated Ag NPs were freeze dried and characterized
by PXRD. PXRD spectra were recorded on Bruker D8 Advance (Germany) diffractometer
(over a range of 10-80°, 2ϴ), operated at 40 mA, 40 KV.
2.7.6. Transmission electron microscopy
Morphology of Ag NPs was assessed by TEM. For this purpose, Ag NPs were isolated from
solution by centrifugation and analyzed on a Philips 420 instrument. For recording TEM
images, samples were drop casted on carbon coated copper wired TEM grid and instrument
was operated at 120 kV. The LSH thin films were air dried under dark and stored for 6
months. After the specified storage period, Ag NPs were again analyzed by TEM. In a typical
experiment, stored LSH thin film impregnated with Ag NPs was dissolved in deionized
water, Ag NPs were isolated by centrifugation and viewed by TEM.
2.7.7. Antimicrobial activity
Aqueous solution of the synthesized Ag NPs (10 mg/mL) was used to study the antimicrobial
activity against different bacterial (Gram positive and Gram negative) and fungal strains.
The tested bacterial strains include Streptococcus mutans (S. mutans) American type culture
collection (ATCC) 25175, Staphylococcus epidermidis (S. epidermidis) ATCC 12228,
Pseudomonas aeruginosa (P. aeruginosa) ATCC 27853, Escherichia coli (E. coli) ATCC
25922, Staphylococcus aureus (S. aureus) ATCC 25923, Bacillus subtilis (B. subtilis) ATCC
6633, Actinomyces odontolyticus (A. odontolyticus) ATCC 17929. The fungal strain used in
the antimicrobial assay was Aspergillus niger (A. niger). Mueller Hinton Agar Media (Oxoid
85
Ltd., England) was used as growth medium for bacterial species while fungi were grown on
Sabouraud Dextrose Agar (Hardy Diagnostics, USA). Inoculums (fungal and bacterial
culture in respective media, 10 mL) were incubated at 27-30 °C for 30-37 h for fungal strains
and at 37 °C for 24 h for bacteria. Fungal culture (7 days old) was washed, suspended in
normal saline solution and incubated at 28 °C after being filtered through aseptic glass wool.
The turbidity of inoculums was adjusted by 0.5 Mc Farland Standard.
Disk diffusion method (Bauer et al. 1966) was used to determine the antimicrobial activity of
Ag NPs. In a typical experiment, aqueous solution of the Ag NPs (20 µL) was loaded onto
filter paper discs (Whatman filter paper No. 1, 6 mm in diameter) and implanted on the
surface of the microbial culture plates. These plates were then incubated at 37 °C for 24 h for
bacterial strains and at 30 °C for 36 h for fungal strains. At the end of incubation period, zone
of inhibition was measured in millimeter. Discs loaded with pure DMSO were used as
negative control. All experiments were performed in triplicate and mean values are reported.
2.7.8. Wound healing studies
2.7.8.1. Design of wound dressing
LSH thin layer (dried) containing the embedded Ag NPs was evaluated as a wound dressing
material. Dressing patch consists of two layers. One layer is a cubic porous adhesive (USP)
backing membrane (4 × 4 cm) and the second layer is cubic sterile cotton patch (2 × 2 cm)
immersed in LSH impregnated with Ag NPs (20 mmol) which is implanted as an inner
wound dressing at the center of adhesive membrane.
2.7.8.2. Wound healing study design
Nine healthy male rabbits (2.5 ± 0.2 kg, 4 month old) were selected and divided into three
equal groups, i.e., control group, standard group and test group. All rabbits were caged under
86
standard laboratory conditions for 48 h before experiment and provided standard food and
water ad libitum throughout the whole study. Rabbits were given anesthesia and placed on
surgical table in natural position. Hair from the rear leg of the rabbit was shaved and excised
a circular wound of 6 mm in diameter using a biopsy punch. Wound of control group rabbits
were left open and untreated. A Band-aid®
dressing was applied to the wound of standard
group while the test group was applied with freshly prepared dressing patch made as
described above. The healing attributes were studied by tracing the raw wound area on
tracing paper till the wound was completely epithelialized (Lee and Tong, 1968).
Eighteen healthy male albino rats (170-220 g body weight) were selected and divided into
three groups of six animals each. The animals were given anesthesia and placed on operation
table in natural position. A round seal (3 cm diameter) was marked 3 cm away from the ears
in central dorsal thoracic region of each animal as described in literature (Morton and Malon,
1972). Skin was scratched from the marked area to induce a wound (1.0 cm diameter).
Wound of control group animals was left open and untreated. A Band-aid® dressing was
applied to the wound of standard group while the test group was applied with prepared
dressing patch. The healing attributes were studied by tracing the raw wound area on tracing
paper till the wound was completely epithelialized (Lee and Tong, 1968).
2.7.8.3. Collagen estimation
Collagen contents of the regenerated tissue were measured using a reported method (Lee and
Tong 1968). For this purpose, regenerated tissue of the wound was chopped and suspended
into 0.5 M acetic acid (10% w/v) after being washed with 0.5 M sodium acetate. It was
stirred for 48 h and then centrifuged for 2 h at 5000 × g. Collagen was precipitated by adding
sodium chloride (10% w/v) and filtered on a weighed Whatman filter paper. Amount of
87
collagen was calculated from the difference in the weight of filter paper before and after
filtration.
2.8. Acute toxicological evaluation of LSH
2.8.1. Study design
Swiss albino mice (25-31 g) and albino rabbits (1115-1225 g) of either sex bred were
obtained from the animal house of University of Sargodha, Sargodha, Pakistan and
thoroughly examined for any symptoms of sickness and anomalies. Animals were kept in
clean stainless steel cages in a 12 h photoperiod (light on at 06:00 and off at 18:00) at 22 to
25 °C and 40-70% humidity. Mice and rabbits were fed with standard laboratory diet and had
free access to ordinary tap water. All tests and procedures conformed to the good laboratory
practice (GLP) regulations as described by United States food and drug administration
(USFDA). Furthermore, acute toxicity procedures were in accordance with organization for
economic co-operation and development (OECD) test guidelines and National Institute of
Health for the care and use of laboratory animals. Animals were divided into four groups
(n=5) as described in Table 2.4. The study protocol was approved by pharmacy research
ethics committee of University of Sargodha, Sargodha, Pakistan.
Table 2.4. Group scheme for acute oral toxicity study of LSH in mice.
Group I Group II Group III Group IV
Control
Given only
standard diet
Treated with LSH
(1g/Kg bw) ground
and mixed with diet
Treated with LSH
(2g/Kg bw) ground
and mixed with diet
Treated with LSH
(5g/Kg bw) ground
and mixed with diet
88
2.8.2. Acute oral toxicity
Acute oral toxicity study of LSH was executed in Swiss albino mice following the OECD
guidelines for analysis of chemicals toxicity. At the instigation of the study, the weight
variation of animals involved was kept minimal and should be < ± 20 % of the mean weight
(Lina et al., 2004). After overnight fasting, mice were orally administered 1-5 g/Kg bw/day
of LSH mixed with food as directed in Table 2.4. The dose of LSH was according to criteria
for excipient toxicity testing and was more than estimated daily intake of excipient (George
and Shipp, 2000). The mice were observed in detail for any indication of toxic effect,
mortality, ill health and any reaction to treatment such as changes in fur, skin, mucus
membranes, eyes, behavior pattern, tremors, salivation, diarrhea, sleep and coma within the
first six hours after the treatment and daily for a further period of 14 days.
2.8.3. Primary eye irritation
This study was carried out in Albino rabbits of either sex. Animals were thoroughly
examined for any abnormalities according to Draize scale for eye lesions (Draize, 1944).
Well ground and moistened LSH (10 mg/10 mL) was placed into the conjunctival sac of the
right eye of each rabbit. Upper and lower lids were kept together with fingers for some time
in order to reduce the risk of any loss of the test material. For comparison, the left eye of
each rabbit was remained untreated and used as a control. After LSH administration, eyes
were examined after 1, 24, 48, and 72 hours for any lacrimation, redness, swelling and
irritation in cornea and pupil. Primary eye irritation score was determined with maximum
mean total score (MMTS) as described by Kay and Calandra (Kay and Calandra, 1962).
Ocular lesion was classified according to the Draize scale for scoring eye lesions (Draize et
al., 1944).
89
2.8.4. Acute dermal toxicity
Five white albino rabbits were used for acute dermal toxicity testing. Rabbits were
thoroughly examined for health and skin abnormalities before and after removal of hair from
the back of these rabbits. Thick paste of ground LSH (500 mg) in deionized water was
prepared and applied on a 4 ply gauze pad (4 in × 4 in). Prepared gauze pad was placed on
the skin of rabbit and wrapped with Micropore™ tape (3 inch wide) to avoid displacement of
the pad. Rabbits were allowed to move freely in their respective cages and observed them for
any behavioral changes for next few hours. After 24 h, the pads were carefully removed from
the skin and the site of application was observed for any color changes in skin or presence of
rashes etc. Rabbits were kept under observation for next 14 days and observed for weight
gain or loss, mortality, sign of behavioral changes, any changes in skin, fur, eyes and mucous
membrane, diarrhea, salivation, coma, convulsions and tremors (Saiyad et al., 2015).
2.8.5. Primary dermal irritation study
Five adult, young and non-pregnant female albino rabbits were selected to carry out the
primary dermal irritation study. Hair from the back of rabbits was removed by clipping the
dorsal and trunk area. Skin of rabbits was examined for any abnormalities before and after
the removal of hair and no pre-existing irritation or lesion on the skin was observed. To apply
test material on the skin, LSH (500 mg) was moistened with deionized water to make a thick
paste and applied on an area of approximately 6 cm2. Test area was covered with gauze pad
and protected from dislocation with the help of suitable semi-occlusive dressing. After 4 h,
dressing was removed and test site was gently cleaned from residual test material. Test site
on each rabbits was observed and score was recorded according to the Draize scoring system
(Draize et al., 1944) at 1, 24, 48 and 72 h after removal of dressing. Irritancy was classified
by adding the average edema and erythema scores for 1, 24, 48 and 72 h scoring intervals
90
and dividing by the number of evaluation intervals (Saiyed et al., 2015). The calculated
Primary Dermal Irritation Index (PDII) was classified according to the descriptive rating
(Sreejayan et al., 2010).
2.8.6. Body weight gain study
The body weights of mice were recorded before treatment, at day 1, 2, 3, 5, 7 and 14 of
treatment. Mean body weight was calculated for each group.
2.8.7. Food and water consumption
The amount of food and water consumed by mice before treatment, at day 1, 2, 3, 5, 7 and 14
was measured and compared with control to observe the influence of LSH intake on these
parameters.
2.8.8. Hematology and clinical biochemistry
On day 15, blood samples were collected from the overnight (about 12 h) fasted mice of each
group into lithium heparin or K2-EDTA containing tubes. Blood samples were analyzed for
hemoglobin, white blood cells count, red blood cells count, platelets, monocytes, neutrophils,
lymphocytes, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) and
mean corpuscular hemoglobin concentration (MCHC).
Blood plasma was also analyzed for alanine aminotransferase (ALT), asparate
aminotransferase (AST), creatinine, urea, uric acid, cholesterol and triglycerides.
2.8.9. Gross necropsy and histopathology
At the end of day 14, mice of each group were kept fast for next 12 h, weighed and then
sacrificed approximately at the same time. Gross necropsy of external orifices and surfaces,
thoracic, abdominal cavities, organs and tissues were performed for any lesion and
91
abnormality. Organs (heart, liver, lung, kidney, stomach and spleen) were weighed
immediately and then preserved in neutral buffered formalin (10% v/v). Preserved organs
and tissues were sliced to 4-5 µm thickness and histopathology was performed after staining
with hematoxylin-eosin.
2.8.10. Statistical analysis
The difference among the numerical values of control group and different treated groups
were analyzed through one way analysis of variance (ANOVA) followed by Student‘s t test
using statistical analyses software, Minitab 11. The level of significance was set at p < 0.05.
All values were expressed as mean ± S.D.
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3. RESULTS AND DISCUSSION
3.1. Isolation and characterization of LSH
3.1.1. Isolation of LSH
Linseed hydrogel (LSH) was isolated from linseeds using hot water extraction method.
Polysaccharides usually have hydrophilic groups (-OH, -COOH etc.) in their structure which
facilitate the penetration of water molecules in the polymer. As a result, polymer swells by
absorbing more and more water and then extrudes from the pores of the seeds. This swollen
polymer (hydrogel) was separated by filtration using nylon mesh. To purify the hydrogel
from fatty or waxy material, which may be added from the surface or inside of seeds during
heating process, n-hexane was used to wash out such impurities. Finally, hydrogel was
separated by filtration, dried, ground and stored in air tight container under vacuum for
further use.
3.1.2. FTIR spectroscopy
FTIR spectrum of LSH is shown in Fig. 3.1 and characteristic peaks glycosidic linkage (C-O-
C) and stretching vibrations of (C-O-H) pertaining to side groups of polysaccharide are
shown from 1200-1000 cm-1
(Kacurakova et al., 2000). The band observed around 1640 and
1420 cm-1
are attributed to deprotonated (COO─) carboxylic groups of uronic acid (Boulet et
al., 2007). Peaks from 3500-2500 cm-1
are assigned to C-H and O-H stretching.
93
3500 3000 2500 2000 1750 1500 1250 1000 750 500
Wavenumber, cm-1
C-O-C &
C-O-H
COO-
C-H
O-H CH2
Fig. 3.1. FTIR spectrum of LSH.
3.1.3. 1H NMR spectroscopy
Structure of LSH was characterized by 1H NMR spectroscopy (600 MHz, 40 °C) recorded in
DMSO-d6) and spectrum is shown in Fig. 3.2. Rhamnogalacturonan is a major component of
LSH which is a highly branched structure and composed of rhamnose, galacturonic acid,
galactose, fucose and xylose (Naran et al., 2008; Cui et al., 1994). Presence of these
constituents is verified through 1H NMR spectra by comparing the specific signals with
already reported results (Qian et al., 2012a; Oomah et al., 1995; Emaga et al., 2012).
94
2.503.003.504.004.505.005.50
0
500000
1000000
1500000
5.5 5.0 4.5 4.0 3.5 3.0 2.5
ppm
Fig. 3.2. 1H NMR (600 MHz, ppm, 40 °C) spectrum of LSH in DMSO-d6 showing repeating
unit between 3.11-5.61.
3.1.4. PXRD
PXRD spectrum of LSH is shown in Fig. 3.3. It was observed that the spectrum of LSH did
not have any sharp or distinct peak which is a characteristic of a crystalline material.
Therefore, the extracted LSH is free from any form of crystalline impurities and also it is a
confirmation of the amorphous nature of LSH.
95
0
100
200
300
400
500
600
700
0 10 20 30 40 50 60 70 80 90
Inte
nsi
ty
2 ϴ
Fig. 3.3. PXRD spectrum of LSH.
3.2. Synthesis and characterization of LSH-acetates
LSH was acetylated with acetic anhydride using 4-dimethylaminopyridine (DMAP) as
catalyst (Muhammad et al., 2016; Heinze et al., 2003). Acetylation reaction mechanism is
demonstrated in Fig. 3.4. Degree of substitution (DSb) of LSH was increased from 2.11-2.91,
when molar ratio of anhydroglucose unit to acetic anhydride was enhanced from 1:6 to 1:18.
The acetylated LSH (ALSH) was found soluble in dimethyl sulfoxide (DMSO), acetone,
dimethyl acetamide (DMAc) and CHCl3. The results, reaction conditions and DSb are shown
in Table 3.1. Acid-base titration after saponification was repeated thrice to obtain DSb and
mean values are discussed.
LSH + Acetic anhydride 80 C, 6 h
DMAP Acetylated LSH
Fig. 3.4. Reaction scheme for the synthesis of acetylated LSH.
96
Table 3.1. Reaction parameters and results of the synthesis of acetylated LSH.
Sample Molar ratioa Yield (g) DSb
b Solubility
ALSH 1 1:6 1.02 2.11 DMSO, acetone, DMAc, CHCl3
ALSH 2 1:12 1.12 2.53 DMSO, acetone, DMAc, CHCl3
ALSH 3 1:18 1.33 2.91 DMSO, acetone, DMAc, CHCl3
aAnhydroglucose unit of LSH:acetic anhydride
bDegree of substitution calculated from acid base titration after saponification
3.2.1. FTIR spectroscopy
FTIR spectroscopic technique was employed for the identification of ester peaks in acetylated
LSH (ALSH). FTIR spectra of ALSH are shown in Fig. 3.5. The pellets of LSH and ALSH
were prepared with KBr and characterized by FTIR spectrophotometer. A prominent peak at
1749 cm-1
, 1752 cm-1
and 1738 cm-1
in ALSH 1, ALSH 2 and ALSH 3, respectively indicates
effective acetylation of LSH. Moreover, broad peak of unreacted OH groups was still
observed at 3440 cm-1
, 3425 cm-1
and 3514 cm-1
in ALSH 1, ALSH 2 and ALSH 3,
respectively. Other peaks at 1042 cm-1
, 1043 cm-1
and 1044 cm-1
showed the presence of
COC in ALSH 1, ALSH 2 and ALSH 3, respectively. Presence of CH2 groups in ALSH 1,
ALSH 2 and ALSH 3 is evident from peaks at 1376 cm-1
, 1373 cm-1
and 1375 cm-1
,
respectively. It was also observed that as the molar concentration of acetic anhydride
increases from 1: 6 to 1:18, DSb also increased from 2.11 to 2.91.
97
Wavenumber, cm-1
4000 3600 3200 2800 2400 2000 1600 1200 800 400
C─O─C
CH2
C=O
C─H
O─H
LSH
ALSH 1
ALSH 2
ALSH 3
Fig. 3.5. FTIR spectra of LSH, ALSH 1, ALSH 2 and ALSH 3.
3.2.2. 1H NMR spectroscopy
The confirmation of successful acetylation was achieved by typical 1H NMR (DMSO-d6; 600
MHz) spectrum of ALSH 3 (Fig. 3.6.) which showed peaks at 2.06 and 3.14-5.62 ppm for
methyl group and polymer repeating units, respectively.
98
1.02.03.04.05.05.0 4.0 3.0 2.0 1.0
1.9001.9502.0002.0502.1002.1 2.0 1.9
Fig. 3.6. 1H NMR (600 MHz, ppm, DMSO-d6, 40 °C) spectrum of ALSH 3 (DSb 2.91).
3.2.3. 1H
1H TOCSY spectroscopy
1H
1H TOCSY spectra of the acetylated LSH (ALSH 3) were recorded in CDCl3 and shown in
Fig. 3.7. Correlation of sugar region present in ALSH 3 was expressed in Fig. 3.8.
3.2.4. HSQC spectroscopy
HSQC spectrum of ALSH 3 was shown in Fig. 3.9. Correlation of sugar region and acetyl
methyl region are more clearly depicted in Fig. 3.10 and Fig. 3.11, respectively.
99
Fig. 3.7. 1H
1H TOCSY (600 MHz, ppm, CDCl3, 25 °C) spectrum of ALSH 3 (DSb 2.91).
Fig. 3.8. 1H
1H TOCSY (600 MHz, ppm, CDCl3, 25 °C) spectrum of ALSH 3 (DSb 2.91)
showing correlation of sugar region.
100
Fig. 3.9. HSQC spectrum (600 MHz, ppm, CDCl3, 25 °C) of ALSH 3.
Fig. 3.10. HSQC spectrum (600 MHz, ppm, CDCl3, 25 °C) of ALSH 3 showing correlation
of acetyl methyl region.
101
Fig. 3.11. HSQC spectrum (600 MHz, ppm, CDCl3, 25 °C) of ALSH 3 showing correlation
of sugar region.
3.2.5. Isoconversional thermal analysis of LSH and LSH acetates
3.2.5.1. Thermal analysis
Thermal degradation behavior of LSH and ALSH 3 was evaluated using isoconversional
method at four different temperatures. The degradation of hydrogel and its acetate was
carried out from ambient temperature to 800 °C. The TG curves of LSH and ALSH 3
exhibited 8.492% and 4.421% loss in the mass in the temperature range of 50 to 130 °C. This
mass loss is actually the moisture present in the sample (Iqbal et al., 2011b). TGA of LSH
and ALSH 3 revealed that degradation occurs in two steps and one step, respectively. TG and
DTG curves of LSH and ALSH 3 recorded at different rates were overlaid and are shown in
Fig. 3.12. Overlay curves of 2DTG of LSH and ALSH 3 recorded at different heating rates
were also shown in Fig. 3.13. Results of thermal decomposition temperature, weight loss and
char yield of LSH and ALSH 3 are displayed in Table 3.2 and Table 3.3, respectively. For
102
first degradation step, the average of four different initial (Tdi) and final (Tdf) thermal
degradation temperatures of LSH was approximately 221 and 378 °C, respectively. For
second degradation step, average Tdi and Tdf values were 380 and 503 °C, respectively. The
average loss in mass of LSH for first and second degradation steps was approximately 46.61
and 28.91%, respectively for four different temperatures. However, average Tdm values for
first and second step was 287 and 459 °C, respectively.
0
20
40
60
80
100
45 195 345 495 645
Wei
gh
t, %
Temperature, C
5 °C/min
10 °C/min
15 °C/min
20 °C/min
0
20
40
60
80
100
40 140 240 340 440
Wei
gh
t, %
Temperature, C
5 °C/min10 °C/min15 °C/min20 °C/min
0
0.2
0.4
0.6
0.8
45 195 345 495 645
Der
iv. w
eig
ht,
%/m
in
Temperature, C
5 °C/min
10 °C/min
15 °C/min
20 °C/min
0
0.3
0.6
0.9
1.2
40 140 240 340 440
Der
iv. w
eig
ht,
%/m
in
Temperature, C
5 °C/min
10 °C/min
15 °C/min
20 °C/min
(d) (c)
(b) (a)
Fig. 3.12. Overlay of TG and DTG curves of LSH (a, c) and ALSH 3 (b, d), respectively
recorded at multiple heating rates.
The thermal degradation of ALSH 3 took place in single step and average Tdi, Tdm and Tdf
values of ALSH 3 were 139.5, 337 and 368.5 °C, respectively with mass loss of 83% at Tdf.
The maximum decomposition temperature (Tdm) of ALSH 3 (337 °C) was higher than LSH
(287 °C) indicating greater stability after acetylation (Table 3.2 and Table 3.3). TG and DTG
103
curves of LSH and ALSH 3 at a heating rate of 10 °C min-1
are compared and shown in Fig.
3.14 indicating an increased thermal stability of acetylate LSH.
-0.026
-0.011
0.004
0.019
0.034
145 225 305 385 465 545
2n
d. D
eriv
. w
eig
ht,
%/m
in2
Temperature, C
5 °C/min 10 °C/min15 °C/min 20 °C/min
-0.07
-0.05
-0.02
0.01
0.03
290 315 340 365 390
2n
d. D
eriv
. w
eig
ht,
%/m
in2
Temperature, C
5 °C/min10 °C/min15 °C/min20 °C/min
(a) (b)
Fig. 3.13. Overlay of 2DTG curves of LSH (a) and ALSH 3 (b) recorded at multiple heating
rates.
Table 3.2. Mean thermal decomposition temperatures, weight loss % and char yield % of
LSH at multiple heating rates.
Sample Step Tdi (°C) Tdm (°C) Tdf (°C) Weight loss
% at Tdf
Char yield
Wt. (%)
LSH I 221 287 378 46.61
6.81 at 700 °C II 380 459 503 28.91
Table 3.3. Mean thermal decomposition temperatures, weight loss % and char yield % of
ALSH 3 at various heating rates.
Sample Step Tdi (°C) Tdm (°C) Tdf (°C) Weight loss
% at Tdf
Char yield
Wt. (%)
ALSH 3 I 139.5 337 368.5 83 7.95 at 600 °C
104
0
20
40
60
80
100
45 195 345 495 645
Wei
gh
t, %
Temperature, C
-0.01
0.19
0.39
0.59
0.79
45 195 345 495 645
Der
iv. W
eig
ht,
%/m
in
Temperature, C
(a) (b)
LSH
—— ALSH
LSH
—— ALSH
Fig. 3.14. Overlay of TG (a) and DTG (b) curves of LSH and ALSH 3 recorded at 10 °C min-
1 showing stability imparted to ALSH 3.
3.2.5.2. Degradation kinetics
Flynn-wall, Ozawa (FWO) and Kissinger isoconversional methods were employed to
evaluate different kinetic parameters like energy of activation (Ea) and frequency factor (A).
Kissinger method was used to find the order of thermal degradation reactions (n). The Ea
values of first and second degradation steps for LSH were found to be 118.32 and 162.34 kJ
mol-1
, respectively (Table 3.4). However, the Ea value of only degradation step of LSH was
169.18 kJ mol-1
. The higher value of activation energy for ALSH 3 showed that acetylated
hydrogel is more stable than unacetylated hydrogel. Fig. 3.15 and Fig. 3.16 show the plots of
α vs. T curves of thermal degradation steps for LSH and ALSH 3, respectively at different
heating rates. FWO plots between logβ and 1000/T (K-1
) for each thermal degradation step at
several degree of conversion for LSH and ALSH 3 are shown in Fig. 3.15 and Fig. 3.16,
respectively. In Kissinger method, thermal decomposition of LSH and ALSH 3 was found to
be first order.
105
0
0.2
0.4
0.6
0.8
1
230 265 300 335 370
α
Temperature, C
5 °C/min10 °C/min15 °C/min20 °C/min
0
0.2
0.4
0.6
0.8
1
380 415 450 485 520
α
Temperature, C
5 °C/min10 °C/min15 °C/min20 °C/min
0.6
0.8
1.0
1.2
1.60 1.70 1.80 1.90
log
β
1000/T, K-1
α 0.1
α 0.2
α 0.3
α 0.4
α 0.5
α 0.6
α 0.7
α 0.8
α 0.9
0.6
0.8
1.0
1.2
1.27 1.34 1.41 1.48
log
β
1000/T, K-1
α 0.1
α 0.2
α 0.3
α 0.4
α 0.5
α 0.6
α 0.7
α 0.8
α 0.9
(a) (b)
(c) (d)
Fig. 3.15. α vs. T graph of thermal degradation of first (a) and second (b) step of LSH at
multiple heating rates and Flynn-Wall-Ozawa (FWO) plot between log β and
1000/T (K-1
) for calculation of Ea of first degradation (c) and second degradation
(d) step at several degree of conversion for LSH.
0
0.2
0.4
0.6
0.8
1
180 220 260 300 340 380
α
Temperature, C
5 °C/min
10 °C/min
15 °C/min
20 °C/min0.6
0.8
1.0
1.2
1.55 1.70 1.85 2.00 2.15
log
β
1000/T, K-1
α 0.1
α 0.2
α 0.3
α 0.4
α 0.5
α 0.6
α 0.7
α 0.8
α 0.9 (a) (b)
Fig. 3.16. α vs. T graph of thermal degradation of ALSH 3 at multiple heating rates (a) and
Flynn-Wall-Ozawa (FWO) plot between log β and 1000/T (K-1
) for calculation of
Ea at several degree of conversion for ALSH 3.
106
3.2.5.3. Thermodynamic analysis
TG data of LSH and ALSH 3 was used to calculate various thermodynamic parameters like
∆H, ∆G and ∆S (Table 3.4 and Table 3.5). Area under TG curves was used to calculate
integral procedural decomposition temperature (IPDT) and index of thermal stability (ITS)
values which are important to evaluate the thermal stability. The mean ITS values for LSH
and ALSH 3 was calculated to be 0.41 and 0.49, respectively. The mean ITS value of ALSH
3 is higher than LSH showing that ALSH 3 has greater thermal stability. Moreover, ITS
values for LSH (0.41) and ALSH 3 (0.49) are higher than other reported hydrogels such as
hydrogels from Astragalus gummifer (0.38), Acacia nilotica (0.40), Argyreia speciosa (0.35),
Acacia modesta (0.42), Ocimum basicilicum (0.41), Plantago ovata (0.39), Salvia aegyptiaca
(0.33) and P. ovata husk (0.41). It means LSH and ALSH 3 are thermally more stable than
many reported polysaccharides (Iqbal et al., 2013). Furthermore, the mean IPDT values of
LSH and ALSH 3 are 350 and 295, respectively.
Table 3.4. Thermal kinetics and thermodynamic parameters of LSH.
Sample Method Step R2 n
Ea
(kJ/mol) lnA ∆H ∆S ∆G IPDT ITS
LSH
FWO I 0.979 - 118.32 27.22 113.66 -36.58 134.14
350 0.41 Kissinger I - 0.87 - - - - -
FWO II 0.976 - 162.34 29.38 156.25 -22.67 172.85
Kissinger II - 1.17 - - - - -
Table 3.5. Thermal kinetics and thermodynamic parameters of ALSH 3.
Sample Method Step R2 n
Ea
(kJ/mol) lnA ∆H* ∆S* ∆G* IPDT ITS
ALSH 3 FWO I 0.966 - 169.18 38.97 164.11 61.97 126.30
295 0.49 Kissinger I - 1.03 - - - - -
107
3.3. Dynamic swelling, stimuli responsive on-off switching of superabsorbent LSH
3.3.1. Physical properties of LSH
Physical properties, i.e., particle size, moisture content, angle of repose, bulk density, tapped
density, swelling capacity, Carr‘s index, Hausner ratio and centrifuge retention capacity of
finely ground LSH passed through sieve no. 60 were determined. The results have indicated
that the particle size, moisture content, centrifuge retention capacity, gelling content and
swelling capacity after 24 h are 250 µm, 0.4%, 67.24%, 33.27% and 42.33, respectively
(Table 3.6). Powder flow-ability parameters, i.e., Carr‘s index, Hausner ratio and angle of
repose were noted as 41.07%, 1.70 and 59.87, respectively. These high values indicated
somewhat poor flow properties of LSH. Therefore, lubricating and gliding agents should be
used for the preparation of tablets containing LSH as an excipient.
Table 3.6. Physical properties of LSH.
Physical properties LSH
Moisture content (%) 0.4 ± 0.01
Particle size (µm) ≈ 250
Angle of repose 59.87 ± 0.32
Bulk density (g/mL) 0.33 ± 0.02
Tapped density (g/mL) 0.56 ± 0.03
Carr‘s index (%) 41.07 ± 2.87
Hausner ratio 1.70 ± 0.08
Centrifuge retention capacity (%) 67.24 ± 1.83
Swelling capacity on 24 h (g/g) 42.33 ± 1.16
Gelling content (%) 33.27 ± 0.79
108
3.3.2. Swelling capacity of LSH in deionized water and at different physiological pH
Swelling behavior of LSH at different pH of gastrointestinal tract was evaluated. Buffers
corresponding to gastric (pH 1.2), small intestine (pH 6.8) and large intestine environment
(pH 7.4) were used to study the effects of pH on swelling of LSH. Swelling of LSH was also
evaluated using deionized water. It was noted that LSH swells rapidly at pH 6.8, 7.4 and in
deionized water (Fig. 3.17.a). However, there was negligible swelling at pH 1.2. If we closely
observe the swelling behavior at pH 6.8, 7.4 and deionized water, it is noted that the
maximum swelling observed in deionized water which is a little bit higher than swelling at
pH 7.4 and 6.8. This slight less swelling at pH 7.4 than deionized water may be due to charge
screening effect of excess cations (Na+) that results in anion-anion repulsions due to shielding
of carboxylate anions (Peppas and Mikes, 1986; Pourjavadi et al., 2004).
0
10
20
30
40
50
60
0 500 1000 1500 2000 2500 3000
Sw
elli
ng
cap
acit
y, g
/g
Time, min
pH 1.2 D. W. pH 7.4 pH 6.8
0
10
20
30
40
50
60
70
0 500 1000 1500 2000 2500 3000
t/Q
t
Time, min
D. W. pH 7.4 pH 6.8
(a) (b)
Fig. 3.17. Swelling capacity (a) and second order swelling kinetics (b) of LSH in buffer of
pH 1.2, 6.8 and 7.4 and in deionized water (D.W).
3.3.3. Swelling kinetics
For practical applications of any hydrogel in formulation design, a higher swelling capacity
as well as a higher swelling rate is required. It is also academically well-known that swelling
kinetics for the hydrogels is significantly influenced by different factors such as swelling
109
capacity, size distribution of powder particles and pH (Malana et al., 2012), etc. Keeping in
view the immense importance of hydrogels, swelling kinetics of LSH was studied. Second
order kinetics appeared the best fit on swelling data acquired in water and at different
physiological pH values (Fig. 3.17.b).
3.3.4. Thermoresponsive swelling capacity of LSH
Effect of temperature on the swelling capacity of LSH in deionized water was determined at
30, 40 and 50 °C (Fig. 3.18). Swelling capacity of LSH increased significantly with the
increase in temperature of swelling medium. As the temperature of swelling medium
increased, water penetration capacity of hydrogel also increased due to relaxation of the
polymer chains. This relaxation allowed water molecules to penetrate within the polymer
chains faster and deeper. Moreover, there is a significant difference in the swelling capacity
of LSH against above mentioned temperatures for first 1000 min. As the maximum relaxation
of polymer chains was achieved at about 1500 min, therefore swelling capacity in the later
stage of swelling appeared independent of temperature.
0
10
20
30
40
50
0 500 1000 1500 2000 2500 3000
Sw
elli
ng
cap
acit
y, g
/g
Time, min
30 °C 40 °C 50 °C
Fig. 3.18. Swelling capacity of LSH in deionized water at different temperatures.
110
3.3.5. Saline responsive swelling of LSH
Swelling of naturally occurring polysaccharides depends upon the presence of
hydrophilic/hydrophobic groups, degree of crosslinking and elasticity of network. Beside
this, swelling of hydrogels is greatly affected by varying the salt concentration in swelling
medium (Pourjavadi and Mahdavinia, 2006). Therefore, effects of increase in ionic
concentration of NaCl and KCl on the swelling ratio of LSH were recorded and shown in Fig.
3.19.a. As the molar concentration of aqueous salt solution increases from 0.1-0.5 M, there is
an abrupt decrease in the equilibrium swelling. This reduced swelling in salt solutions might
be due to charge screening effect of excess cations resulting in non-perfect anion-anion
electrostatic repulsions (Peppas and Mikes, 1986). Because of this repulsion, osmotic
pressure difference between the polymer and swelling medium decreases which results in
shrinkage of swollen polymer in aqueous medium (Pass et al., 1997). It was also noted that
lesser swelling in KCl solution was observed due to higher affinity of the hydrogel towards
K+ ion.
3.3.6. Responsive swelling-deswelling (on-off switching) behavior of LSH at basic and
acidic pH
On immersing LSH in pH 7.4 solution, it rapidly swells and on transferring the swollen LSH
to the pH 1.2 solution, it rapidly deswells showing on-off switching and pH sensitive
behavior. This behavior might be due to the presence of COOH groups in the network
structure of LSH polysaccharides. Literature has indicated that LSH contains acidic sugars,
i.e., rhamanose (20.30%) and uronic acid (22.10%) in 84.25% carbohydrate portion of LSH
when isolated at ambient temperature before defatting (Barbary et al., 2009).
In pH 1.2 solution, COOH groups exist as such and make inter and intra molecular hydrogen
bonding with the polysaccharide chains which might be the driving force that allows the LSH
111
to shrink back. While at pH 7.4, the opposite process starts as the COOH groups are
converted into COONa and COO─
groups which are a cause of repulsion among the similar
groups. Due to this electrostatic repulsion property, LSH chains go away from each other and
lose hydrogen bonding amongst polymer chains and suddenly swell. Fig. 3.19b is showing
the pulsatile on-off switching of LSH at basic (7.4) and acidic pH (1.2), respectively. After
five swelling-deswelling cycles, LSH showed pH responsiveness indicating that the hydrogel
is reversibly pH sensitive.
3.3.7. Responsive swelling-deswelling (on-off switching) behavior of LSH in deionized
water and in NaCl solution (0.9%)
Swelling-deswelling behavior of LSH was examined in deionized water and 0.9% NaCl
solution, respectively (Fig. 3.19c). LSH swelled rapidly on contact with deionized water and
deswelled on contact with 0.9% NaCl solution. The reason of this swelling-deswelling
behavior in deionized water and 0.9% NaCl solution is the osmotic pressure difference
between polymer chains of LSH and the surrounding solution. When the swollen LSH comes
in contact with 0.9% NaCl solution, the presence of Na+ in the solution decreases the osmotic
pressure and as a result water molecules move out of the swollen gel and deswelling of LSH
gel occur. Similarly, when this shrunk LSH is transferred to deionized water, Na+ are washed
out and as a result the osmotic pressure is recovered and LSH again swelled.
3.3.8. Responsive swelling-deswelling (on-off switching) behavior of LSH in deionized
water and ethanol
Swelling-deswelling (on-off switching) behavior of LSH was observed in water and ethanol,
respectively as an essential parameter of swelling behavior to external stimuli (Fig. 3.19d). It
was noted that swollen LSH, abruptly deswells in ethanol. The swelling-deswelling cycles in
112
water/ethanol was repeated four times. The rapid deswelling of LSH in ethanol is because of
the fact that ethanol repels water molecules hence cause faster shrinking of hydrogel.
4
5
6
7
8
9
10
11
0 60 120 180 240 300 360 420 480
Sw
elli
ng
cap
acit
y, g
/g
Time, min
12
14
16
18
20
22
0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00
Sw
elli
ng
cap
acit
y, g
/g
Concentration, M
NaClKCl
1
2
3
4
5
6
7
0 30 60 90 120 150 180
Sw
elli
ng
cap
acit
y, g
/g
Time, min
7
7.5
8
8.5
9
9.5
10
10.5
11
0 60 120 180 240 300 360 420 480
Sw
elli
ng
cap
acit
y, g
/g
Time, min
(a) (b)
(c) (d)
Fig. 3.19. Swelling capacity of LSH in different conc. of NaCl and KCl (a) and swelling-
shrinking (on-off switching) behavior of LSH; at pH 7.4 (basic) and pH 1.2
(acidic) (b), in deionized water and normal saline (0.9% NaCl solution) (c) and in
deionized water and ethanol (d), respectively.
3.3.9. Field emission scanning electron microscopy
To observe the morphology of superabsorbent LSH, field emission scanning electron
microscopy (FE-SEM) was performed and resultant photographs are shown in Fig. 3.20. It is
obvious from the FE-SEM photographs that elongated porous structures are uniformly
distributed on the surface of LSH. This high porosity of LSH is responsible for the faster and
higher swelling of this supper-porous hydrogel.
113
20 µm 20 µm20 µm
Fig. 3.20. SEM images of lyophilized sample of LSH showing porous and elongated
structure.
3.4. Evaluation of LSH as a novel controlled release and stimuli responsive oral drug
delivery system
Isolated LSH was evaluated for the development of a sustained release formulation of
diclofenac sodium (DS) which was further compare with a commercially available product,
Voltral®. Caffeine and diacerein were selected as a neutral drug and NSAIDs, respectively
and used to explore the potential of LSH as a stimuli responsive and intelligent drug delivery
system.
3.4.1. Drug-excipients compatibility study
To find out the compatibility of LSH with active ingredient and other excipients, FTIR
spectra of different combinations were recorded using KBr pellet technique. Fig. 3.21, Fig.
3.22 and Fig. 3.23 have shown the FTIR spectra of diacerein, caffeine and diclofenac sodium
containing formulations, respectively. IR spectrum of LSH mixed with drug and excipients
indicated that LSH was compatible with the ingredients of tablets.
114
3500 3000 2500 2000 1750 1500 1250 1000 750 500
Wavenumber, cm-1
Diacerin + LSH + PVP
LSH
PVP
Diacerin
Diacerin + LSH
Fig. 3.21. FTIR spectra of LSH, PVP and diacerein alone, physical mixture of LSH with
diacerein and physical mixture of LSH, diacerein and PVP.
115
3500 3000 2500 2000 1750 1500 1250 1000 750 500
Wavenumber, cm-1
Caffeine + LSH + PVP
LSH
PVP
Caffeine
Caffeine + LSH
Fig. 3.22. FTIR spectra of LSH, PVP and caffeine alone, physical mixture of LSH with
caffeine and physical mixture of LSH, caffeine and PVP.
116
3500 3000 2500 2000 1750 1500 1250 1000 750 500
Wavenumber, cm-1
LSH
PVP
Diclofenac sodium
Diclofenac sodium + LSH
Diclofenac sodium + LSH + PVP
Fig. 3.23. FTIR spectra of LSH, PVP and diclofenac sodiume alone, physical mixture of LSH
with diclofenac sodiume and physical mixture of LSH, diclofenac sodiume and
PVP.
117
3.4.2. Pre-compression evaluation of tablet formulations
LSH was used in different concentration to prepare the tablets of diclofenac sodium, caffeine
and diacerein by wet granulation method. Lubricated granules from each formulation were
evaluated for their flow property and compressibility through the determination of angle of
repose, loose bulk density, tapped bulk density, Hausner ratio and compressibility index. The
mean of three values were taken and results are summarized for DS, caffeine and diacerein
containing formulations in Table 3.7, Table 3.8 and Table 3.9, respectively. Loose bulk
density and tapped bulk density of the lubricated granules of all formulations were in the
range of 0.502 to 0.753 g/cm3 and 0.625 to 0.833 g/cm
3, respectively. The values of Hausner
ratio, compressibility index and angle of repose were found in the range of 1.091 to 1.245,
8.358% to 17.945% and 21.11° to 31.69°, respectively. The values of pre-compression
parameters of all formulations indicated good flow and compressibility properties (Trivedi et
al., 2008, Wilson et al., 2011, El-Zahaby et al., 2014).
Table 3.7. Pre-compression parameters of diclofenac sodium formulations (Mean ± SD).
Formulation
code
Angle of
repose
n = 3
Loose bulk
density
(g/cm3)
n = 3
Tapped bulk
density
(g/cm3)
n = 3
Hausner‘s
ratio
n = 3
Compressibility
index (%)
n = 3
D1 19.33 ± 0.08 0.642 ± 0.04 0.713 ± 0.03 1.111 ± 0.03 9.958 ± 0.94
D2 23.29 ± 0.05 0.566 ± 0.03 0.633 ± 0.04 1.118 ± 0.06 10.581 ± 1.88
D3 24.78 ± 0.07 0.593 ± 0.05 0.694 ± 0.05 1.171 ± 0.06 14.561 ± 2.38
118
Table 3.8. Pre-compression parameters of caffeine formulations (Mean ± SD).
Formulation
code
Angle of
repose
n = 3
Loose bulk
density
(g/cm3)
n = 3
Tapped bulk
density
(g/cm3)
n = 3
Hausner‘s
ratio
n = 3
Compressibility
index (%)
n = 3
FH 31.69 ± 0.09 0.502 ± 0.01 0.625 ± 0.02 1.245 ± 0.01 19.680 ± 1.91
FC1 23.72 ± 0.11 0.753 ± 0.02 0.833 ± 0.01 1.106 ± 0.03 9.604 ± 0.82
FC2 28.95 ± 0.15 0.608 ± 0.01 0.714 ± 0.03 1.174 ± 0.01 14.846 ± 0.63
FC3 30.53 ± 0.13 0.535 ± 0.01 0.652 ±0.01 1.219 ± 0.01 17.945 ± 2.23
Table 3.9. Pre-compression parameters of diacerein formulations (Mean ± SD).
Formulation
code
Angle of
repose
n = 3
Loose bulk
density
(g/cm3)
n = 3
Tapped bulk
density
(g/cm3)
n = 3
Hausner‘s
ratio
n = 3
Compressibility
index (%)
n = 3
FH 31.69 ± 0.09 0.502 ± 0.01 0.625 ± 0.02 1.245 ± 0.01 19.680 ± 1.91
FD1 21.11 ± 0.05 0.625 ± 0.02 0.682 ± 0.02 1.091 ± 0.01 8.358 ± 1.11
FD2 26.67 ± 0.06 0.603 ± 0.02 0.682 ± 0.01 1.131 ± 0.02 11.584 ± 2.76
FD3 29.99 ± 0.08 0.555 ± 0.01 0.652 ± 0.01 1.175 ± 0.03 14.877 ± 3.03
3.4.3. Post-compression evaluation of tablet formulations
Compressed tablets of all formulations are analyzed through post-compression parameters
and results are tabulated in Table 3.10, Table 3.11 and Table 3.12 for DS, caffeine and
diacerein, respectively. Mean values of hardness, thickness, weight and friability of tablets
119
were in the range of 6.11 to 6.92 Kg/cm2, 3.41 to 3.95 mm, 264.5 to 266.1 mg (in case of
caffeine and diacerein formulations) and 299.5 to 301.3 (DS formulations) and 0.79% to
0.95%, respectively. Mean DS content was calculated as 99.71%, 98.45% and 97.11% for
D1, D2 and D3, respectively. Mean caffeine content in FC1, FC2 and FC3 were found to be
99.02%, 98.51% and 98.06%, respectively. In FD1, FD2 and FD3, the amount of diacerein
was 98.81%, 98.05% and 97.08%, respectively.
Table 3.10. Post-compression parameters of DS containing tablets (Mean ± SD).
Formulation
code
Hardness
(Kg/cm2)
n = 10
Thickness
(mm)
n = 10
Weight
(mg)
n = 20
Friability
(%)
n = 10
Drug content
(%)
n = 10
D1 6.33 ± 0.04 3.83 ± 0.03 301.3 ± 1.11 0.93 ± 0.06 99.71 ± 0.53
D2 6.11 ± 0.02 3.95 ± 0.04 300.8 ± 0.98 0.85 ± 0.04 98.45 ± 0.43
D3 6.64 ± 0.04 3.88 ± 0.02 299.5 ± 0.43 0.90 ± 0.04 97.11 ± 0.55
Table 3.11. Post-compression parameters of caffeine containing tablets (Mean ± SD).
Formulation
code
Hardness
(Kg/cm2)
n = 10
Thickness
(mm)
n = 10
Weight
(mg)
n = 20
Friability
(%)
n = 10
Drug content
(%)
n = 10
FH 6.13 ± 0.05 3.45 ± 0.02 264.5 ± 0.29 0.95 ± 0.01 -
FC1 6.15 ± 0.01 3.63 ± 0.02 266.1 ± 0.68 0.79 ± 0.02 99.02 ± 0.62
FC2 6.72 ± 0.03 3.41 ± 0.02 265.2 ± 0.32 0.84 ± 0.02 98.51 ± 0.91
FC3 6.45 ± 0.02 3.49 ± 0.04 265.9 ± 0.46 0.91 ± 0.01 98.06 ± 0.56
120
Table 3.12. Post-compression parameters of prepared tablets containing diacerein (Mean ±
SD).
Formulation
code
Hardness
(Kg/cm2)
n = 10
Thickness
(mm)
n = 10
Weight
(mg)
n = 20
Friability
(%)
n = 10
Drug content
(%)
n = 10
FH 6.13 ± 0.05 3.45 ± 0.02 264.5 ± 0.29 0.95 ± 0.01 -
FD1 6.92 ± 0.03 3.51 ± 0.03 264.8 ± 0.97 0.87 ± 0.01 98.81 ± 0.53
FD2 6.58 ± 0.03 3.71 ± 0.01 265.4 ± 1.12 0.91 ± 0.01 98.05 ± 0.43
FD3 6.22 ± 0.01 3.75 ± 0.03 265.1 ± 0.58 0.92 ± 0.02 97.08 ± 0.55
3.4.4. Swelling response of LSH containing tablet formulations at different pHs
Swelling response of all formulations (Table 2.2 and Table 2.3,) was observed in deionized
water and at different physiological pH, i.e., 1.2, 6.8 and 7.4. Tablets were fully immersed in
the above said media and swelling capacity was calculated periodically using Eq. 14.
3.4.4.1. Swelling response and swelling kinetics of LSH tablets
Swelling response and swelling kinetics of LSH tablet (formulation FH) is depicted in Fig.
3.24. It was noted that FH swelled more and rapidly in deionized water, at pH 7.4 and 6.8 as
compared to pH 1.2 (Fig. 3.24a). As we move from lower to higher pH, the acidic group (-
COOH) dissociates into ionic form and the ionic repulsion between similar charged ions
allow the water molecules to penetrate into the polymer chains (Amin et al., 2014; Huang et
al., 2007; Wang et al., 2011a). Hence, more swelling was observed with the increase in pH.
However, the less swelling at pH 7.4 than deionized water was due to the charge screening
effect of the more Na+ present in pH 7.4 buffer (Peppas and Mikes, 1986).
121
Furthermore, swelling kinetics of FH in different swelling media was also determined and
shown in Fig. 3.24b. The results of swelling kinetics indicated that the swelling kinetics
followed the second order kinetics.
0
2
4
6
8
10
12
0 200 400 600 800 1000
Sw
elli
ng
cap
acit
y, g
/g
Time, min
pH 1.2 pH 6.8 pH 7.4 DW
0
50
100
150
200
250
0 200 400 600 800 1000t/
Qt
Time, min
pH 1.2 pH 6.8 pH 7.4 DW
(a) (b)
0.5 h 1 h 2 h 4 h 8 h 16 h
(c)
Fig. 3.24. Swelling capacity (a) and swelling kinetics (b) of LSH tablet (FH) at different pH
and in deionized water and swelling photographs (radial and axial view) of FH
formulation at pH 6.8 (c).
3.4.4.2. Swelling response and swelling kinetics of LSH-caffeine tablets
Swelling response of LSH-caffeine formulations FC1, FC2 and FC3 were studied in
deionized water and buffer solutions of pH 1.2, 6.8, 7.4 and results are shown in Fig 3.25. It
is observed that there is very low swelling capacity for all formulations at acidic pH (Fig.
3.25a). Swelling of polymer depends on the nature and ionization of functional groups
present on the polymer chain (Huang et al., 2007). The dissociation of acidic group (-COOH)
present on the LSH polymer chain is not possible at low pH and less swelling was observed.
122
Furthermore, it is also observed that the swelling of these tablets depend on the concentration
of LSH. Formulation FH and FC3 has shown less swelling as compared to FC1 and FC2 as
former two formulations have high amount of LSH in their composition.
Swelling trend of formulations (Table 2.2) at pH 6.8, 7.4 and in deionized water is expressed
in Fig. 3.25b, Fig. 3.25c and Fig. 3.25d, respectively. The results indicated that the swelling
of tablets is increased with the increase in the concentration of LSH in each media.
Swelling kinetics was also determined for all these formulations at pH 1.2, 6.8 and 7.4 and in
deionized water and shown in Fig. 3.26a, Fig. 3.26b, Fig. 3.26c and Fig. 3.26d, respectively.
The results have shown that the data of swelling of these tablets followed the second order
kinetics.
3.4.4.3. Swelling response and swelling kinetics of LSH-diacerein tablets
Swelling response of diacerein containing LSH tablet formulations (Table 2.3) were
evaluated at pH 1.2, 6.8, 7.4 and in deionized water and shown in Fig. 3.27. It was observed
that at pH 1.2 (Fig. 3.27a), there is very less swelling for all the formulations. Moreover, the
swelling does not affect with the presence of diacerein. It is also noted that as the
concentration of LSH increases (FD3) the swelling of tablets will decrease. At pH 6.8 (Fig.
3.27b), an increase in swelling capacity was observed with the increase in the concentration
of the LSH in the tablets. It is obvious that FD1 show less swelling as compared to FD3 due
to the less amount of LSH in FD1. Same pattern of swelling was also observed for all
formulations at pH 7.4 (Fig. 3.27c) and in deionized water (Fig. 3.27d). The formulation
having less amount of LSH has shown lower tendency to swell as compared to the
formulation having high amount of LSH. The rate of swelling is determined through swelling
kinetic and it was noted that all formulations follow the second order kinetics (Fig. 3.28).
123
0
2
4
6
8
0 200 400 600 800 1000
Sw
elli
ng
cap
acit
y, g
/g
Time, min
FH FC1
FC2 FC3
0
2
4
6
8
10
0 200 400 600 800 1000
Sw
elli
ng
cap
acit
y, g
/g
Time, min
FH FC1
FC2 FC3
0
2
4
6
8
10
0 200 400 600 800 1000
Sw
elli
ng
cap
acit
y, g
/g
Time, min
FH FC1
FC2 FC3
0
2
4
6
8
0 200 400 600 800 1000
Sw
elli
ng
cap
acit
y, g
/g
Time, min
FH FC1
FC2 FC3
(a) (b)
(c) (d)
0.5 h 1 h 2 h 4 h 8 h 16 h
(e)
Fig. 3.25. Swelling capacity of FH, FC1, FC2, and FC3 at pH 1.2 (a), 6.8 (b), 7.4 (c), and DI
water (d) and swelling photographs (radial and axial view) of FC3 formulation at
pH 6.8 (e).
124
0
30
60
90
120
0 200 400 600 800 1000
t/Q
t
Time (min)
FH FC1 FC2 FC3
0
30
60
90
120
0 200 400 600 800 1000
t/Q
t
Time (min)
FH FC1 FC2 FC3
0
30
60
90
120
150
0 200 400 600 800 1000
t/Q
t
Time (min)
FH FC1 FC2 FC3
(a) (b)
(c) (d)
0
50
100
150
200
250
0 200 400 600 800 1000
t/Q
t
Time (min)
FH FC1 FC2 FC3
Fig. 3.26. Swelling kinetics of LSH tablet (FH) and LSH-caffeine tablets (FC1, FC2 and
FC3) at pH 1.2 (a), 6.8 (b), 7.4 (c) and deionized water (d).
Overall, the swelling indices of LSH containing tablets were lesser as compared to swelling
indices of powder. This lower swelling of LSH tablets as compared to LSH in powder form
can be explained due to decrease in exposed surface area of LSH particles in tablets. In the
tablets form, only the outer surfaces of the tablets are exposed to swelling medium and
swelling medium can only enters from this surface into large tablet matrix. Whereas, LSH
particles in the core of tablets are not directly exposed to media. The swelling process, that
involves the diffusion of swelling medium into the polymeric network and the relaxation of
the polymer chains, starts after the entry of medium in tablets (Razmjou et al., 2013). As the
surface area for the diffusion of medium is small in tablets therefore the swelling indices of
tablets is lower. Contrarily, in case of LSH in powder form, contact area exposed to swelling
125
medium is much greater than tablets that results in entry of more swelling medium in LSH
chains leading to higher swelling. Additionally, when the particles are compressed in tablets,
the interstitial spaces decrease that further contribute to lower swelling of tablets (Bao et al.,
2011). Although swelling indices of LSH were lesser in tablet form than in powder form,
however, LSH tablet still exhibited superabsorbent characteristics with high swelling indices.
3.4.5. Swelling morphology of LSH containing tablets
Tablets of formulations FH, FC3 and FD3 were allowed to swell at pH 6.8 and observed the
morphological changes for 16 h. It was observed that the rate of swelling of all formulations
was little bit different from each other. At the end of 16 h, more fragments of FC3 and FD3
tablets were seen as compared to FH (Fig. 3.24c, Fig. 3.25e and Fig. 3.27e, respectively).
This might be due to the release of entrapped drug which creates micropores in the tablet.
After hydration, these pores spread out and ultimately break the tablet into fragments.
126
0
2
4
6
8
0 200 400 600 800 1000
Sw
elli
ng
cap
acit
y, g
/g
Time, min
FH FD1
FD2 FD3
0
2
4
6
8
10
0 200 400 600 800 1000
Sw
elli
ng
cap
acit
y, g
/g
Time, min
FH FD1
FD2 FD3
0
2
4
6
8
10
0 200 400 600 800 1000
Sw
elli
ng
cap
acit
y, g
/g
Time, min
FH FD1
FD2 FD3
0
2
4
6
8
0 200 400 600 800 1000
Sw
elli
ng
cap
acit
y, g
/g
Time, min
FH FD1
FD2 FD3
(a)
(d) (c)
(b)
0.5 h 1 h 2 h 4 h 8 h 16 h
(e)
Fig. 3.27. Swelling capacity of FH, FD1, FD2, and FD3 at pH 1.2 (a), 6.8 (b), 7.4 (c) and
deionized water (d) and swelling behavior of FD3 formulation at pH 6.8 expressed
in photographs (radial and axial view) (e).
127
0
30
60
90
120
150
0 200 400 600 800 1000
t/Q
t
Time (min)
FH FD1 FD2 FD3
0
30
60
90
120
150
0 200 400 600 800 1000
t/Q
t
Time (min)
FH FD1 FD2 FD3
0
30
60
90
120
150
0 200 400 600 800 1000
t/Q
t
Time (min)
FH FD1 FD2 FD3
0
50
100
150
200
250
0 200 400 600 800 1000
t/Q
t
Time (min)
FH FD1 FD2 FD3
(a)
(d) (c)
(b)
Fig. 3.28. Swelling kinetics of LSH tablet (FH) and LSH-diacerein tablets (FD1, FD2 and
FD3) at pH 1.2 (a), 6.8 (b), 7.4 (c) and deionized water (d).
3.4.6. Morphological analysis of LSH containing tablets by SEM
SEM analysis of FD3 tablet formulation was conducted to observe the morphological
arrangements of LSH in tablet formulation. The SEM micrograph of tablet surface, broken
surface and cross section of swollen then freeze dried FD3 formulation are shown in Fig.
3.29. Intra-particle microscopic spaces were seen on the surface of FD3 and broken surface of
FD3 tablet as shown in Fig. 3.29a and Fig. 3.29b, respectively. Moreover, the cross section of
swollen then freeze dried FD3 tablets revealed the presence of multilayered porous structure
(Fig. 3.29c). This highly porous structure of LSH makes it an ideal material for drug loading
and control release applications.
128
a b c
Fig. 3.29. SEM images of broken surface of FH tablet (a), broken surface of FD3 tablet (b)
and cross section of swollen then freeze dried tablet formulation FD3.
3.4.7. Salt solution responsive swelling of LSH containing tablet formulations
Swelling response of all formulations was studied at various concentrations of NaCl and KCl
to observe the effects of salt solutions on swelling behavior. It was observed that in all
formulations there was an abrupt decrease in the equilibrium swelling from the molar
concentration 0.1-0.5 M but monotonous decrease from 0.5-2.0 M. Equilibrium swelling
capacity of FH, FC3 and FD3 in salt solutions of NaCl and KCl is expressed in Fig. 3.30a and
Fig. 3.30b, respectively as a typical example. Moreover, it was also monitored that the
equilibrium swelling of formulation FC3 and FD3 was less than the equilibrium swelling of
LSH. Same swelling trend was also observed in KCl salt solution. On closely observing the
equilibrium swelling of these formulations in both the media (NaCl and KCl), it was revealed
that all formulations swelled more in NaCl solution as compared to KCl solution. This less
swelling in salt solution is due to the presence of Na+ and K
+ cations. These cations decrease
the osmotic pressure difference between the polymer chains and the surrounding solutions.
As a result the water penetration ability into the tablet and swelling capability is also reduced.
The reduced swelling in the presence of salts solutions may also be explained due to the
neutralization of COO─ ions by Na
+ and K
+ ions that reduce the electrostatic repulsion
(among COO─) responsible for swelling (Pandey et al., 2013). The reason of less swelling in
129
KCl solution as compared to NaCl is due to the high charge density of the K+ as compared to
the Na+.
1
2
3
4
5
6
0 0.25 0.5 0.75 1
Sw
elli
ng
cap
acit
y, g
/g
KCl solution concentration, M
FH FC3 FD3
1
2
3
4
5
6
0 0.25 0.5 0.75 1
Sw
elli
ng
cap
acit
y, g
/g
NaCl solution concentration, M
FH FC3 FD3
(a) (b)
Fig. 3.30. Equilibrium swelling of LSH tablet (FH), LSH-caffeine tablet (FC3) and LSH-
diacerein tablet (FD3) in different molar concentrations of salt solutions; NaCl (a)
and KCl (b).
3.4.8. Swelling-deswelling response of LSH tablet formulations against external stimuli
To explore the potential of LSH as an intelligent biopolymer, swelling-deswelling response
of LSH, LSH-caffeine and LSH-diacerein tablets were observed at pH 7.4 and 1.2, in water
and normal saline solution and in water and ethanol. The evaluation of change in pH on the
swelling and deswelling of these tablets is important due to the acidic and basic environment
of gastrointestinal tract (GIT). Moreover, the presence of NaCl and ethanol in GIT can alter
the swelling capability of LSH containing tablet formulations.
3.4.8.1. Swelling-deswelling response in basic and acidic pH
Swelling-deswelling response of formulated tablets were evaluated at basic (pH 7.4) and
acidic (pH 1.2) environment (Fig. 3.31a). It was noted that all formulations were swelled at
pH 7.4 and rapidly deswelled when shifted to pH 1.2. With the increase of pH, the carboxylic
acid moiety of the polymer converts into its ionized form. The high density of these charged
130
particles creates a strong anion-anion electrostatic repulsion which results in the extension of
polymer chains and water molecules move between the free spaces of polymer chains (Wang,
et al., 2011a; Huang, et al., 2007). When these swelled tablets were placed at pH 1.2, the
protonation of the carboxylate ion (COO─) and formation of strong hydrogen bonding
regained the deswelled form of the tablets. When we compare three formulations, it was
observed that the diacerein containing formulation (FD3) has shown little bit less swelling-
deswelling response due to the presence of acidic drug (diacerein) as compare to FH and FC3
tablets. These findings indicated the swelling-deswelling potential of LSH in tablet
formulations with caffeine and diacerein and also as an oral control release drug delivery
system. The lower swelling at acidic pH (stomach) can protect the release of drug in stomach
while higher swelling at intestinal pH can be utilized to target drug release in intestinal
region. Moreover, caffeine and diacerein have negligible influence on the swelling-
deswelling capability in tablets formulation of LSH.
3.4.8.2. Swelling-deswelling response in deionized water and normal saline solution
Swelling-deswelling response of tablets in deionized water and normal saline solution were
analyzed and results are shown in Fig. 3.31b. This swelling-deswelling behavior of tablets in
water and normal saline solution was due to the difference in the osmotic pressure between
the swollen tablets and surrounding environment. The decrease in the osmotic pressure due to
the presence of sodium ion in the medium was the main force which draws the water
molecules to come out of the swollen tablets. Results have indicated the responsive swelling-
deswelling behavior of these formulations against normal saline solution.
131
0
1
2
3
4
0 60 120 180 240 300 360
Sw
elli
ng
cap
acit
y, g
/g
Time, min
FH FC3 FD3
(a)
(b)
(c)
0
1
2
3
4
0 60 120 180 240 300 360
Sw
elli
ng
cap
acit
y, g
/g
Time, min
FH FC3 FD3
0
1
2
3
4
0 60 120 180 240 300 360
Sw
elli
ng
cap
acit
y, g
/g
Time, min
FH FC3 FD3
Fig. 3.31. Stimuli responsive swelling and deswelling behavior of LSH tablet (FH), LSH-
caffeine tablet (FC3) and LSH-diacerein tablet (FD3) at basic (pH 7.4) and acidic
(pH 1.2) environment (a), in deionized water and normal saline (b) and deionized
water and ethanol (c), respectively.
132
3.4.8.3. Swelling-deswelling response in deionized water and ethanol
Swelling-deswelling response of all formulations was observed in deionized water and
ethanol (Fig. 3.31c). Tablets were allowed to swell in deionized water for 1 h and then shifted
to shrinking media i.e., ethanol for the same time period. It was observed that the rate of
shrinking is faster than the rate of swelling in all formulations. This abrupt shrinking in
ethanol was due to the fact that water molecules were quickly replaced by ethanol (Dragan
and Apopei, 2013). Therefore, the presence of ethanol will create hindrance in swelling of
LSH containing tablet formulations and as a result release of drug will be affected.
3.4.9. In-vitro drug release studies
3.4.9.1. DS release studies and release mechanism
To observe the effect of LSH on the release of DS, tablets were place in simulated gastric
fluid (SGF) and simulated intestinal fluid (SIF) for 2 h and 14 h, respectively (Fig. 3.32b).
Drug-hydrogel interaction, swelling ability of hydrogel and drug solubility in dissolution
media are the major influencing factors that control the drug release from polymeric matrix
system (Brazel and Peppas, 1999; Siepmann and Peppas, 2001). In SGF, minimum or
negligible amount of DS, 5.04%, 4.27% and 3.96%, was released from D1, D2 and D3,
respectively. Due to less swelling tendency of LSH at acidic pH (1.2) and insolubility of DS
in acidic environment, low concentration of DS was released from all three formulations.
Contrary to this, in SIF or near neutral/basic environment, both swelling of LSH and
solubility of DS was increased. After 14 h study in SIF, DS release from D1, D2 and D3 was
90.4%, 67.6% and 49.1%, respectively. Moreover, a more sustained release of DS from
tablets was observed with the increase of LSH concentration from 75-100 mg/tablet (Table
2.1). For comparison, drug release profile of DS from LSH containing formulation was
evaluated against commercially available formulation. It was observed that formulations D2
133
and D3 sustained the release of DS even better than already marketed formulation (Fig.
3.32b).
Mainly, the drug release from swellable polymeric system is controlled by swelling and
diffusion mechanism. Release mechanism was found out using Korsmeyer-Peppas model
(Eq. 23). The value of n and kp was calculated from the slop and intercept of the plot of
ln(Mt/M∞) vs ln t, respectively and values are given in Table 3.13. The value of n was found
in the range from 0.762-0.824 for all formulations. Hence, drug release from DS formulation
follows the non-Fickian diffusion (anomalous transport mechanism). Therefore, the swelling
of polymer and diffusion of drug from polymer matrix occurred simultaneously (Korsmeyer
et al., 1983; Ritger and Peppas, 1987).
Swelling capacity of all formulations (D1, D2 and D3) was studied in deionized water and
results are expressed in Fig. 3.32a. It can be seen that swelling of tablets has a direct relation
with the concentration of LSH. An increase in swelling was observed with the increase of
LSH concentration in tablets. Physical condition of the tablet during the swelling process was
noted and shown in Fig. 3.32c. It was observed that the tablet has been able to maintain its
physical state for longer period of time with minimum fragmentation.
Table 3.13. Mathematical data of power law.
Formulation n kp r2
D1 0.762 12.416 0.9277
D2 0.826 7.645 0.9488
D3 0.824 5.556 0.9566
134
0
20
40
60
80
100
0 2 4 6 8 10 12 14 16
Cu
mu
lati
ve
dru
g r
elea
se, %
Time, h
Voltral D1
D2 D3
SGF
(b)
0
2
4
6
8
10
0.25 0.5 1 2 4 8 12 16
Sw
elli
ng
cap
acit
y, g
/g
Time, h
D1 D2 D3
(a)
(c)
0 h 1 h 2 h 4 h 8 h
SIF
Fig. 3.32. Swelling capacity of LSH based DS tablets in water (a), drug (DS) release study
from LSH matrix tablets in SGF and SIF (b) and photographs showing swelling
response (aerial and axial view) of D3 formulation in water (c).
3.4.9.2. Caffeine and diacereine release studies
To explore the potential of LSH as a controlled release material, LSH containing tablets were
prepared using caffeine and diacerein. The release profile of caffeine from formulation FC1,
FC2 and FC3 at pH 6.8, 7.4 and in deionized water is shown in Fig. 3.33a, Fig. 3.33b and Fig.
3.33c, respectively. It was observed that the release of caffeine was sustained as the amount
of LSH increased from 50 mg (FC1) to 100 mg (FC3) per tablet in all the media. This
sustained release of drug from FC3 is due to the high swelling and water holding capacity of
LSH present in these tablets. Therefore, it is difficult for soluble drug to come out of the
swelled matrix of LSH. The cumulative drug release after 16 h from FC1, FC2 and FC3 was
89.09 ± 0.93, 74.11 ± 1.05 and 61.31 ± 0.62% at pH 6.8; 99.14 ± 1.25, 86.27 ± 1.11 and
135
67.33 ± 0.93% at pH 7.4 and 99.67 ± 1.71 (after 12 h), 89.52 ± 1.66 and 74.29 ± 1.01% in
deionized water, respectively. Results have indicated that release of caffeine is dependent on
the concentration of LSH in the tablet and inversely proportional relation is observed.
Furthermore, a tablet formulation (FC) was prepared without LSH and it was observed that
caffeine was completely released from the formulation within 2 h in all media.
In vitro drug release studies were performed in dissolution media mimicking the
physiological pH and maximum transit time in the GIT (Fig. 3.33d). Therefore, tablets from
formulations FC1, FC2 and FC3 were exposed to pH 1.2, 6.8 and 7.4 buffer solutions for 2, 8
and 6 h, respectively. Results have indicated that very less amount of drug was released at pH
1.2 (9.13 ± 0.08, 7.51 ± 0.05 and 6.7 ± 0.08% for FC1, FC2 and FC3, respectively), while at
pH 6.8 and 7.4, higher and sustained release behavior was observed. After 14 h, the
cumulative drug release from FC1, FC2 and FC3 was 100.25 ± 1.72, 81.23 ± 0.91 and 66.26
± 1.21%, respectively. Release of very small amount of caffeine at acidic pH is due to the
dissolution of the loosely packed caffeine from the tablet surface. From these results, it is
concluded that LSH based tablet formulations can be used for sustained and site specific
delivery of drug at small and large intestinal pH.
136
0
20
40
60
80
100
0 4 8 12 16 20 24
Cu
mu
lati
ve
dru
g r
elea
se (
%)
Time (h)
FC1
FC2
FC3
FC
0
20
40
60
80
100
0 4 8 12 16 20 24
Cu
mu
lati
ve
dru
g r
elea
se (
%)
Time (h)
FC1
FC2
FC3
FC
0
20
40
60
80
100
0 4 8 12 16 20 24
Cu
mu
lati
ve
dru
g r
elea
se (
%)
Time (h)
FC1
FC2
FC3
FC
0
20
40
60
80
100
0 2 4 6 8 10 12 14 16
Cu
mu
lati
ve
dru
g r
elea
se (
%)
Time (h)
FC1
FC2
FC3
pH 1.2 pH 6.8 pH 7.4
(c)
(a) (b)
(d)
Fig. 3.33. Caffeine release from LSH-caffeine tablet in different media; pH 6.8 (a), pH 7.4
(b), deionized water (c) and physiological pH and transit time of gastrointestinal
tract (d).
Diacerein release from formulations FD1, FD2 and FD3 was observed at GIT pHs and in
deionized water (Fig. 3.34). At pH 1.2, negligible amount of drug was released from all three
formulations (data not shown) because diacerein is insoluble at acidic pH. At pH 6.8, 7.4 and
in deionized water, a sustained release profile of the drug was observed from FD1, FD2 and
FD3. In buffer of pH 6.8, cumulative drug release (after 16 h) from FD1, FD2 and FD3 was
79.28 ± 2.11, 63.6.9 ± 4.86 and 54.31 ± 3.04%, respectively (Fig. 3.34a). At pH 7.4, diacerein
release (after 16 h) from FD1, FD2 and FD3 was 99.71 ± 0.91, 84.45 ± 2.02 and 70.04 ±
2.52%, respectively (Fig. 3.34b). Furthermore, a tablet formulation (FD) was prepared
without LSH and the release of diacerein from the formulation was completed within 3 h at
137
pH 6.8 and 7.4 whereas, in deionized water, diacerein release was completed in 4 h. This
slight delay release in deionized water was due to less solubility of diacerein in deionized
water.
0
20
40
60
80
100
0 4 8 12 16 20 24
Cu
mu
lati
ve
dru
g r
elea
se (
%)
Time (h)
FD1 FD2
FD3 FD
0
20
40
60
80
100
0 4 8 12 16 20 24
Cu
mu
lati
ve
dru
g r
elea
se (
%)
Time (h)
FD1
FD2
FD3
FD
0
20
40
60
80
100
0 4 8 12 16 20 24
Cu
mu
lati
ve
dru
g r
elea
se (
%)
Time (h)
FD1
FD2
FD3
FD
0
20
40
60
80
100
0 2 4 6 8 10 12 14 16
Cu
mu
lati
ve
dru
g r
elea
se (
%)
Time (h)
FD1
FD2
FD3
pH 1.2 pH 6.8 pH 7.4
(c)
(a) (b)
(d)
Fig. 3.34. Diacerein release from LSH-diacerein tablet in different media; pH 6.8 (a), pH 7.4
(b), deionized water (c) and physiological pH and transit time of gastrointestinal
tract (d).
After 16 h of drug release study in deionized water, the amount of diacerein released from
FD1, FD2 and FD3 was 68.03 ± 3.42, 52.84 ± 2.3 and 39.19 ± 2.84%, respectively (Fig.
3.34c).
Results have indicated that the release of diacerein from FD1, FD2 and FD3 was dependent
on the solubility of drug as well as on the concentration of LSH. Diacerein, being an acidic
138
drug is poorly soluble in deionized water while soluble in buffers of pH 6.8 and 7.4 due to the
presence of NaOH. Therefore, the cumulative drug release from FD1, FD2 and FD3 at pH 6.8
and 7.4 was high as compared to deionized water. It was also observed that the release of
diacerein was inversely proportional to the concentration of the LSH in the formulations at a
given medium.
Furthermore, to evaluate the release behavior of diacerein from the formulated tablet during
the transit through gastrointestinal tract, formulations were tested at pH 1.2 for 2 h, pH 6.8 for
8 h and pH 7.4 for 6 h (Fig. 3.34d). The results have indicated that there is negligible amount
of drug release in acidic pH which is mainly due to less swelling at such a low pH and also
due to poor solubility of the drug. When these formulations were placed in pH 6.8, a
significant and sustained release of diacerein was observed for next 8 h. In pH 7.4, a slight
increase in the drug release pattern was observed as compared to pH 6.8 due to more
solubility of diacerein at this pH. At the end of 14 h, drug released from FD1, FD2 and FD3
was 95.21 ± 2.91, 81.61 ± 1.91 and 63.91 ± 3.13%, respectively. In-vitro drug release study
suggests that LSH can be used as a potential carrier for small intestine and colon specific
drug delivery of diacerein as well as other NSAIDs. The in-vitro results indicated the
potential of LSH tablets as oral controlled release system where pH difference between the
stomach and intestine can used to trigger drug release. LSH release drug at intestinal pH
suggested that it can be further developed as site specific delivery system in intestine.
Moreover, the lower release in the stomach can also help to avoid the side effect of drugs like
NSAIDs.
3.4.9.3. Drug release kinetics and mechanism
To find out the drug release pattern and mechanism, kinetics models were applied on
dissolution data obtained at pH 6.8, 7.4 and deionized water. The value of coefficient of
139
determination (R2) were calculated from the equations of zero order, first order, Higuchi,
Korsmeyer-Peppas and Hixon-Crowell and given in Table 3.14 and Table 3.15 for caffeine
and diacerein containing LSH tablets, respectively. The value of R2 indicated that the best fit
models for both drugs are first order, Higuchi, Korsmeyer-Peppas and Hixon-Crowell. It
indicates that the release of drug depends on the swelling of polymer as well as diffusion of
the drug. Results have also indicated that caffeine and diacerein release pattern followed the
anomalous transport mechanism (non-Fickian diffusion). MSC analysis of the data indicated
that the most appropriate model in explaining the rate and mechanism of drug release was
Korsmeyer-Peppas model. The values of diffusion exponent n indicate the release mechanism
of the drug from a polymeric matrix system. In case of cylindrical geometry (tablets) drug
delivery system, the release mechanism is considered Fickian diffusion, non-Fickian
diffusion (anomalous transport), case II transport and super case II transport if n ≤ 0.45, 0.45
< n < 0.89, n = 0.89, and n > 0.89, respectively (Korsmeyer et al., 1983; Ritger and Peppas,
1987). The values of n for LSH tablets were in the range of 0.6 to 0.8 indicating that the
release of drug was governed by non-Fickian diffusion (anomalous transport), i.e., both the
swelling of polymers and diffusion of the drug occurred.
140
Table 3.14. Values of drug release kinetics models for LSH-caffeine formulations at pH 6.8,
7.4 and deionized water.
pH 6.8 pH 7.4 Deionized water
FC1 FC2 FC3 FC1 FC2 FC3 FC1 FC2 FC3
Zer
o o
rder
R2 0.7930 0.7310 0.624 0.8721 0.6834 0.6548 0.9039 0.8388 0.6746
K0 6.994 4.801 3.751 8.958 5.366 4.280 10.662 7.139 4.668
MSC 1.408 1.159 0.824 1.875 0.996 0.909 2.142 1.658 0.969
Fir
st o
rder
R2 0.9969 0.9969 0.9299 0.9924 0.9984 0.9712 0.9900 0.9947 0.9893
K1 0.136 0.096 0.065 0.168 0.120 0.080 0.194 0.138 0.093
MSC 5.601 5.617 2.504 4.693 6.287 3.394 4.406 5.080 4.386
Hig
uch
i
R2 0.9676 0.9633 0.9656 0.9557 0.9578 0.9629 0.9352 0.9490 0.9607
KH 22.05 18.067 14.287 24.89 20.302 16.242 26.82 22.36 17.681
MSC 3.262 3.152 3.215 2.935 3.011 3.140 2.536 2.809 3.082
Ko
rsm
eyer
-Pep
pas
R2 0.9995 0.9954 0.9852 0.9993 0.9977 0.9983 0.9991 0.9986 0.9954
KKP 15.42 11.61 11.47 17.05 12.49 11.39 17.23 13.718 11.603
n 0.703 0.735 0.614 0.724 0.776 0.690 0.792 0.773 0.735
MSC 7.132 4.979 3.876 6.707 5.652 5.986 6.426 6.094 4.979
Hix
son-
Cro
wel
l
R2 0.9850 0.9773 0.8700 0.9938 0.9921 0.9276 0.9987 0.9954 0.9583
KHC 0.038 0.027 0.019 0.047 0.034 0.023 0.055 0.039 0.027
MSC 4.03 3.632 1.886 4.902 4.691 2.472 6.416 5.212 3.023
141
Table 3.15. Values of drug release kinetics models for LSH-diacerein formulations at pH 6.8,
7.4 and deionized water.
pH 6.8 pH 7.4 Deionized water
FD1 FD2 FD3 FD1 FD2 FD3 FD1 FD2 FD3
Zer
o o
rder
R2 0.7833 0.8638 0.8284 0.8047 0.8329 0.7303 0.8196 0.8168 0.7286
K0 4.848 4.101 3.090 10.66 6.832 4.561 3.995 3.218 2.497
MSC 1.375 1.840 1.609 1.433 1.623 1.157 1.559 1.543 1.150
Fir
st o
rder
R2 0.9992 0.9987 0.9700 0.9991 0.9993 0.9932 0.9886 0.9687 0.8901
K1 0.095 0.069 0.045 0.204 0.129 0.088 0.067 0.048 0.034
MSC 7.022 6.523 3.353 6.841 7.168 4.830 4.318 3.309 2.055
Hig
uch
i
R2 0.9586 0.9409 0.9530 0.9590 0.9540 0.9584 0.9625 0.9618 0.9774
KH 18.12 15.123 11.47 27.16 21.43 17.16 14.86 11.98 9.403
MSC 3.029 2.675 2.903 2.995 2.911 3.027 3.131 3.110 3.636
Kors
mey
er P
eppas
R2 0.9989 0.9994 0.9894 0.9917 0.9936 0.9966 0.9952 0.9932 0.9908
KKP 10.99 8.326 8.206 19.74 13.71 10.64 10.26 8.828 7.829
n 0.753 0.776 0.640 0.738 0.752 0.754 0.664 0.627 0.577
MSC 5.634 7.093 4.237 4.226 4.612 5.275 5.014 4.678 4.376
Hix
son-
Cro
wel
l
R2 0.9889 0.9877 0.9412 0.9868 0.9906 0.9677 0.9662 0.9380 0.8479
KHC 0.027 0.020 0.014 0.057 0.036 0.025 0.020 0.014 0.010
MSC 4.349 4.246 2.681 4.126 4.499 3.279 3.234 2.627 1.729
142
3.5. Docetaxel loaded LSH-Pluronic NPs
3.5.1. Preparation and characterization of DLP-NPs
Docetaxel (DTX) loaded LSH-Pluronic F-68 NPs (DLP-NPs) was prepared by core shell
formation in which DTX loaded LSH particles act as a core and Pluronic as a shell. For the
synthesis of core, ethanolic solution of DTX was added in the aqueous suspension of LSH
before subjected to lyophilization. LSH has the tendency to shrink in ethanol (Haseeb et al.,
2016) and during this process, DTX was entangled in LSH. Moreover, DTX was also
entrapped in the polymeric network of LSH due to the crosslinking of polymeric chains
during lyophilization (Giannouli and Morris, 2003). Drug loaded LSH core was further
protected by the self-assembly of Pluronic F-68.
3.5.2. Particle size and morphological analysis
Particle size analysis of the synthesized DLP-NPs have shown the mean diameter of 163 nm,
187 nm and 223 nm for formulation with 1%, 2% and 3% DTX loading, respectively (Table
3.16). Size of DLP-NPs increased with the increase in the drug loading amount. Size
distribution of drug loaded DLP-NPs are shown in Fig. 3.35a-c. Fig. 3.35e has shown the size
distribution of aqueous LSH suspension and average diameter of the particles was found
228.6 nm. Zeta potential of 1% drug loaded NPs is shown in Fig. 3.35g and value was −31
mV indicating the formation of well dispersed NPs and having fewer tendencies to aggregate.
143
Inte
nsi
ty (
nm
)
0 200 400 600 800 1000 1200 1400 1600 1800 2000
0
5
10
15
20
Size (d.nm)
Inte
nsity
(%
)
100 200 400 0
Size (nm) 300
0
5
10
15
20
0 200 400 600 800 1000 1200 1400 1600 1800 2000
0
5
10
15
20
25
Inte
nsity
(%
)
Size (d.nm)100 200 400 0 300
Size (nm) In
ten
sity
(n
m)
Inte
nsi
ty (
nm
)
0
5
10
15
20
25
0 200 400 600 800 1000 1200 1400 1600 1800 2000
0
5
10
15
20
25
30In
tens
ity (%
)
Size (d.nm)
0 200 400 600 800 1000 1200 1400 1600 1800 2000
0
5
10
15
20
25
30
Inte
nsi
ty (
%)
Size (d.nm)
25
20
15
10
0
5
30
100 200 400 0 300
Size (nm)
Zeta potential (mV)
To
tal
cou
nts
-100 0 100 200 0
50000
100000
150000
Inte
nsi
ty (
%)
Size distribution by intensity
0.1 1 10 100 1000 10000
10
20
30
Diameter (nm)
(a) (b) (c)
(d) (e)
(f) (g)
Fig. 3.35. Size distribution of DLP-NPs with different drug loadings: 1% (a), 2% (b), 3% (c);
FESEM image of DLP-NPs (formulation with 1% DTX loading) (d); size
distribution of LSP (1 wt% aqueous solution) (e); size distribution calculated from
FESEM (f) and Zeta potential of 1% DLP-NPs (g).
Shape and size of DLP-NPs was observed through the FESEM analysis (Fig. 3.35d).
Spherical morphology of DLP-NPs was observed through FESEM and the size was found to
be 155± 44nm (Fig. 3.35f).
144
Table 3.16. Drug loading and encapsulation efficiency of different formulations.
Formulation Drug loading
(wt%)
Encapsulation efficiency
(wt%)
Average diameter
(nm)
1% (w/w) DTX loading 0.98% ± 0.19 98.12% ± 1.8 163
2% (w/w) DTX loading 1.86% ± 0.85 93.07% ± 2.1 187
3% (w/w) DTX loading 2.35% ± 1.11 78.33% ± 3.5 223
3.5.3. PXRD and FTIR analysis of DLP-NPs
PXRD spectra of DLP-NPs were recorded to find out the texture of these NPs (Fig. 3.36a)
and also the encapsulation ability of DTX and LSH by Pluronic F-68. Being amorphous in
nature, LSH has not shown any peak in the PXRD spectrum. Sharp and distinct peaks were
observed in PXRD spectra of DTX and Pluronic F-68 indicating the crystalline nature of
these samples. PXRD spectrum of DLP-NPs is somewhat similar with the spectrum of
Pluronic F-68 as represented by the characteristics peaks observed at 19° and 24° (Khaliq et
al., 2016). These results indicated that both DTX and LSH were completely surrounded by
Pluronic F-68 and no part of LSH or DTX is left out of the prepared NPs. Moreover, the
spectrum of 3% drug loaded NPs has shown less crystalline in nature as compared with other
formulations (1% and 2% DTX loading) which may be due to inefficient encapsulation of
DTX in LSH core or inability of Pluronic F-68 to completely surround the drug loaded LSH
core. Therefore, some part of LSH remained outside during the self-assembly of Pluronic F-
68.
FTIR spectra of DLP-NPs (1% DTX loading), Pluronic F-68, DTX and LSH are shown in
Fig. 3.36b. Spectrum of DLP-NPs reflecting the presence of all signals of alkyl group and
COC of Pluronic F-68, characteristic peaks of OH, COC and CH2 of LSH and specific signals
of DTX which indicated their presence in prepared DLP-NPs.
145
5 10 15 20 25 30 35 40
Inte
nsi
ty
2 ϴ
LSH Docetaxel Pluronic F-68
1% loading 2% loading 3% loading
40080012001600200024002800320036004000
40080012001600200024002800320036004000
400900140019002400290034003900
Pluronic
DLP-NPs (1% loading)
Docetaxel
LSH
40080012001600200024002800320036004000
Wave number, cm-1
(a) (b)
Fig. 3.36. PXRD (a) and FTIR (b) spectra of LSH, DTX, Pluronic F-68 and three
formualtions of DLP-NPs.
3.5.4. In vitro drug release study from DLP-NPs
Fig. 3.37 has shown the release of DTX from DLP-NPs of all three formulations with
different drug loading. Prolonged release of DTX from these NPs was observed for more than
96 h. Furthermore, in all three formulations, only 30-50% DTX was released in first 24 h and
at the end of 96 h, 42-65% drug was released indicating a good sustained release pattern.
Furthermore, it can be concluded that the integrity of NPs was also maintained for such a
long time period.
Encapsulation efficiency and drug loading was calculated and results are shown in Table
3.16. It was observed that for 1% and 2% drug loading formulations, encapsulation efficiency
was reasonably high with 98% and 93%, respectively. With the increase in drug loading
amount to 3%, encapsulation efficiency was dropped to 78.33%. This may be due to the high
146
concentration of DTX which is not being able to retain within these DLP-NPs. Total drug
loading in DLP-NPs was 0.98%, 1.86% and 2.35% for formulations with 1%, 2%, and 3%
initial loading of DTX, respectively (Table 3.16).
Drug release mechanism from the prepared DLP-NPs was analyzed using Korsmeyer
Peppas equation (Nguyen et al., 2014). Drug release followed the Fickian diffusion if the
value of n < 0.45 and for 1% drug loading formulation, it was calculated to 0.43. Therefore, it
can be concluded that the drug release from these prepared DLP-NPs (1% drug loading) was
mediated by diffusion mechanisms (Ritger and Peppas, 1987). One advantage of diffusion
controlled drug release mechanism over swelling or erosion based system is the prolong
release of drug as the integrity of the system is maintained for longer period of time.
0
20
40
60
80
100
0 12 24 36 48 60 72 84 96
Cu
mu
lati
ve
DT
X r
elea
se, %
Time, h
1 % loading 2 % loading
3 % loading DTX
Fig. 3.37. Docetaxel release from different formulations of DLP-NPs
3.5.5. Cytotoxicity and cellular uptake behaviour of DLP-NPs
MTT assay was used to observe the cytotoxicity of LSH and DLP-NPs. It was observed that
LSH have shown good cell viability (≈ 100%) up to the concentration of 250 µg/mL while at
concentration of 500 µg/mL cell viability was reduced to 82%. These values indicate that
147
LSH possess very low toxicity, hence, biocompatible to the cell culture system. DLP-NPs
have shown no effect on cell viability up to the concentration of 25 µg/mL. Cytotoxicity in
cell culture was seen at a concentration of 125 µg/mL and 52% cells were observed viable
(Fig. 3.38). IC50 values of DLP-NPs and free DTX are 116.994 ± 0.27 and 16.173 ± 0.15
µg/mL, respectively. Whereas, LSH and untreated cells did not show any IC50 value.
0
20
40
60
80
100
0 125 250 375 500
Cell
via
bil
ity, %
Concentration, µg/mL
Untreated cells LSH
DLP-NPs Free DTX *
Fig. 3.38. In vitro cytotoxicity of LSH, DTX and DLP-NPs at various concentrations.
Statistical significance is shown by * p < 0.05, performed by student‘s t-test for
comparison.
Pluronic NPs has the ability to penetrate into SCC-7 tumour cells through endocytosis
process (Rapoport et al., 2002). Therefore, cellular uptake of DLP-NPs in cell culture system
was monitored. Nile red was incorporated in DLP-NPs during drug loading phase and these
NPs were incubated in SCC-7 tumour cells. Cellular uptake of these NPs was examined using
fluorescent microscope and it was revealed that NPs are only taken up by cytoplasm of SCC-
7 tumour cells and nuclear compartment is free from such penetration (Fig. 3.39).
148
Control
0.5 h
4h
2h
1h
Phase DAPI (Nucleus) Nile Red (Cytoplasm) Merge D
LP
-NP
s w
ith
Nil
e R
ed,
20
x
Control
0.5 h
4h
2h
1h
DL
P-N
Ps
wit
h N
ile
Red
, 4
0 x
(a)
(b)
Fig. 3.39. Cellular uptake images of DLP-NPs (20x, a) and (40x, b).
149
3.6. Nanobiotechnological application of LSH
3.6.1. Green synthesis of Ag NPs
On subjecting AgNO3 solutions to diffused sunlight, hydrated electrons are generated in the
system. These electrons can be used to reduce monovalent silver cations (Ag+) to zerovalent
silver atom (Ag0). The Ag
0 atoms so generated usually
have a size ranging in nanometers.
Therefore, UV irradiation can be exploited as a green method for the synthesis of Ag NPs.
This method can also be used to avoid environmentally toxic and hazardous reducing agents.
Green synthesis of Ag NPs was carried out using LSH as a green reducing agent under
diffused sunlight. LSH was also evaluated as a storage medium for the synthesized Ag NPs.
LSH mediated synthesis of Ag NPs was carried out using different concentrations of AgNO3
solutions (10, 20 and 30 mmol). On mixing the reactants, Ag+ in the AgNO3 solution reacted
with LSH to give [Ag(LSH)]+ complex. When the mixture was subjected to diffused sunlight,
Ag+
in the complex were reduced by LSH to produce [Ag(LSH)] precursor. The positive
charge on the surface of Ag NPs was electrostatically stabilized and capped by the negative
charge on polysaccharide hydroxyls which resulted in colloidal stabilization of [Ag(LSH)].
The progress of formation of Ag NPs by irradiation of [Ag(LSH)]+ complex was followed by
noting the color change of reaction mixture with passage of time. The color of LSH and
AgNO3 mixture changed from colorless to reddish brown and finally dark brown within 10 h.
A schematic illustration of synthesis of LSH mediated synthesis of Ag NPs is shown in Fig.
3.40.
150
AgNO3
(aq)
+
LSH
Ag
+
+ +
+ +
+ +
+
O
O
O
O
O
O O
O
O
O
O
O
O
O
O
O
O
O
O O
O
Ag NPs capped by LSH
Fig. 3.40. Schematic illustration showing synthesis of LSH mediated Ag NPs.
3.6.2. Characterization of Ag NPs
3.6.2.1. UV spectrophotometry
LSH suspension (2 mL) was mixed with different conc. of AgNO3 (10, 20, 30 mmol, 2 mL
aliquot each) in dark and then placed in diffused sunlight. Reduction of Ag+ to Ag
0 was
indicated by change in color of reaction mixture and monitored by UV/Vis
spectrophotometry. The conduction electrons of Ag NPs exhibit surface plasmon resonance
(SPR) due to their collective oscillation and this SPR results in strong absorption in visible
region of spectrum (Mock et al., 2002). The wavelengths absorbed for SPR transitions of Ag
NPs are changed by change in reaction time and size of Ag NPs, so reaction mixture showed
a color change as the reaction progressed. The color changed from colorless to reddish brown
and finally dark brown with passage of time from 0.25 to 10 h. Photographs showing the
color change of reaction mixture with progress of reaction are shown in Fig. 3.41.
151
05 Min
30 Min
60 Min
90 Min
120 Min
600 Min
Fig. 3.41. Photographs of LSH-Ag+ mixture (20 mmol AgNO3) showing color change
with passage of time.
The LSH-Ag+ solutions showed UV/Vis absorptions at 410, 415, 419, 425, 426, 428, 430,
431, 436 nm for 10 mmol. Whereas, 412, 416, 422, 426, 428, 430, 431, 434, 436 nm
absorptions were recorded for 20 mmol LSH-Ag+ solution. Likewise, 30 mmol solution
showed UV absorptions at 413, 417, 424, 427, 429, 432, 433, 435 and 437 nm. The
absorption spectra were recorded at reaction time of 0.25, 0.5, 0.75, 1, 2, 4, 6, 8 and 10 h,
respectively. Similar results have been reported in literature where Ag NPs were synthesized
using exogenous reducing agents (El-Sheikh et al., 2013). The absorption spectra of Ag NPs
showed red shift indicating increase in size of Ag NPs with increase in reaction time.
Absorption intensity also showed an increase when reaction time was increased from 0.25 to
10 h which indicated continuous reduction of Ag+ by LSH with passage of time. Results of
UV/Vis analyses are summarized in Fig. 3.42.
152
280 380 480 580 680
Ab
sorb
ance
Wavelength, nm
0.25h
0.5h
0.75h
1h
2h
4h
6h
8h
10h
280 380 480 580 680
Ab
sorb
ance
Wavelength, nm
0.25h
0.5h
0.75h
1h
2h
4h
6h
8h
10h
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.2
0.4
0.6
0.8
1.0
0.2
0.4
0.6
0.8
1.0
280 380 480 580 680
Ab
sorb
ance
Wavelength, nm
0.25h
0.5h
0.75h
1h
2h
4h
6h
8h
10h
405
410
415
420
425
430
435
440
Wav
elen
gth
, n
m
Time, h
10 mmol
20 mmol
30 mmol
(a)
(c)
(b)
(d)
Fig. 3.42. UV/Vis spectra of LSH mediated Ag NPs: 10 mmol (a), 20 mmol (b) and 30 mmol
solution of AgNO3 (c) at different reaction times and cumulative graphical
representation (d) showing increase in absorption of Ag NPs solutions with
increase in concentration and reaction time.
3.6.2.2. Transmission electron microscopy of isolated Ag NPs
Size distribution and morphology of Ag NPs was assessed by TEM. Ag NPs were separated
from LSH and AgNO3 (10, 20 and 30 mmol) solutions by centrifugation and TEM image was
recorded. TEM images confirmed the formation of highly spherical Ag NPs in the size
regimen of 10-25 nm for 10 mmol, 10-30 nm for 20 mmol and 10-35 nm for 30 mmol
AgNO3 solution (Fig. 3.43).
153
500 nm 50 nm50 nm
a) b) c)
Fig. 3.43. TEM images of Ag NPs isolated from 10, 20 and 30 mmol LSH-Ag+ solution
having size range from 10-25 nm (a), 10-30 nm (b) and 10-35 nm (c),
respectively.
3.6.2.3. Powder X-ray diffraction
PXRD (Fig. 3.44) was used to confirm the crystal phase of Ag NPs isolated from AgNO3
(10, 20 and 30 mmol) solutions in the range of 10-80°, 2ϴ. The diffraction peaks centered at
(111), (200), (220) and (311) indicated that Ag NPs had face-centered cubic lattice in all
samples.
154
(e)
(d)
(c)
(b)
(a)
10 20 30 40 50 60 70 80
2θ (Degree)
(311) (220) (200)
(111)
Fig. 3.44. PXRD spectra: LSH (a), Ag NPs embedded LSH film (b) and isolated Ag NPs, 10
mmol (c), 20 mmol (d) and 30 mmol (e).
3.6.2.4. Storage of Ag NPs in LSH thin film
Potential of LSH for the storage of Ag NPs in solution as well as in the form of a thin film
was evaluated. For this purpose, Ag NPs were synthesized by reducing the AgNO3 (20
mmol) solution with LSH over 10 h. UV/Vis spectra were recorded after 10 and 24 h and
sample was stored as dry thin film under dark. Absorption spectra of the isolated Ag NPs
were acquired by dissolving the films in deionized water. Comparable absorption spectra
were obtained after the storage period (15, 30 days, and 06 months). No significant change in
the absorption wavelength and intensity was observed for the stored samples (Fig. 3.45a).
Therefore, it was inferred that the Ag NPs did not undergo agglomeration on storage in LSH
thin films. X-ray diffraction pattern was recorded for the Ag NPs isolated from stored thin
films (06 months). It was revealed that there was no change in diffraction peak of samples
155
after storage. Therefore, it was concluded that LSH could be used for the long term storage of
Ag NPs without affecting their morphology (Fig. 3.45b). Moreover, the stored film was
dissolved in deionized water and Ag NPs were isolated by centrifugation. TEM image of
isolated Ag NPs showed that the NPs retained their morphology and size over the storage
period (Fig. 3.45e). Therefore, it was concluded that the LSH thin films could be used as
storage media for Ag NPs. Results of storage experiments are illustrated in Fig. 3.45.
10 20 30 40 50 60 70 80
2θ (Degree)
(311)(220)
(200)
(111)
After 6
Months
10 h
24 h
15 days
30 days
6 months
0.2
0.4
0.6
0.8
1.0
280 380 480 580 680
Wavelength, nm
Ab
sorb
ance
50 nmc d
d
e
a b
After 6
Months
(c)
10 h
24 h
15 days
30 days
6 months
0.2
0.4
0.6
0.8
1.0
280 380 480 580 680
Wavelength, nm
Ab
sorb
ance
(a)
10 20 30 40 50 60 70 802θ (Degree)
(311) (220)
(200)
(111) (b)
(d) (e)
10 20 30 40 50 60 70 80
2θ (Degree)
(311)(220)
(200)
(111)
After 6
Months
10 h
24 h
15 days
30 days
6 months
0.2
0.4
0.6
0.8
1.0
280 380 480 580 680
Wavelength, nm
Ab
sorb
ance
50 nmc d
d
e
a b
(f)
Fig. 3.45. UV/Vis spectra of Ag NPs synthesized from aqueous solution of AgNO3 (20
mmol) and LSH recorded after 10 h, 01, 15, 30 days and 06 months storage (a);
PXRD spectrum of Ag NPs isolated after 06 month storage of LSH-Ag NPs film
(b); vial containing solution of stored LSH-Ag NPs film in water (c); foldable (d)
and see through (f) Ag NPs embedded LSH thin film; TEM image of Ag NPs
(10-30 nm) isolated from LSH-Ag NPs film stored for 06 months under dark (e).
156
3.6.2.5. Antimicrobial activity of Ag NPs
LSH based Ag NPs were tested for antimicrobial properties. Significant antibacterial and
antifungal activity was observed against S. mutans, S. epidermidis, P. aeruginosa, E. coli, S.
aureus, B. subtilis, A. odontolyticus, and A. niger strains. The cultures of above mentioned
strains showed inhibition zones of 22, 13, 18, 09, 20, 15, 16, and 10 mm on being exposed to
the aqueous solution of Ag NPs synthesized with AgNO3 (20 mmol) solution, respectively.
No antimicrobial activity was noticed for control experiments performed by using LSH
solution and sterile distilled water. However, AgNO3 (0.02 M/20 mmol) solution was found
active against the above mentioned strains. Antimicrobial properties Ag NPs prepared from
10 and 30 mmol AgNO3 solution were also tested. Results of antimicrobial activity of Ag
NPs (20 mmol) is summarized in Fig. 3.46b and average of three readings are reported. As a
typical example, antimicrobial activity of Ag NPs (20 mmol) against A. odontolyticus and S.
aureus is shown in Fig. 3.46a. It is revealed that LSH based Ag NPs show a clear zone of
inhibition against the above mentioned strains while LSH alone did not show any effect on
the microbial cultures.
157
LSH Water DMSO
AgNO3
Activity against
P. aeruginosa
0
5
10
15
20
25
Zo
ne
of
inh
ibit
ion (
mm
)
Microorganisms
(a) (b)
Fig. 3.46. Antimicrobial activity (a) and graphical representation of zone of inhibition of Ag
NPs (20 mmol) against different bacterial and fungal strains (b).
3.6.2.6. Wound healing studies
In order to evaluate wound healing properties of LSH films impregnated with Ag NPs,
excision wounds were created on rear leg of rabbit (Fig. 3.47). Average wound area was
calculated for different groups of rabbits. Wound healing was assessed after measuring the
area of wound (mm2) at regular time intervals. Control group showed a wound closure of
0.29, 1.11, 20.32, 38.76, 62.55 and 83.27 % on 1st, 3
rd, 6
th, 9
th, 12
th and 15
th day, respectively.
The animals treated with standard Band aid® dressing showed closure area of 2.17, 19.45,
63.67, 87.27, 98.49 and 100% while the test group showed 1.41, 17.73, 54.62, 93.21, 99.22
and 100% wound closure on 1st, 3
rd, 6
th, 9
th, 12
th and 15
th day, respectively. Results indicated
that tissue regeneration and percentage wound closure of LSH based wound dressing was
comparable to the standard Band aid® dressing. Results of wound healing study for various
groups are summarized in Table 3.17.
158
Fig. 3.47. Schematic illustrations of wound treatment with Ag NPs embedded LSH wound
dressing patch (a) and also showing its main parts (b).
In order to understand the mechanism of wound healing, collagen content and tensile strength
of the epithelialized wound tissue was measured. The control group showed collagen content
of 38 mg/kg while standard and test groups showed collagen content of 59 and 55 mg/kg,
respectively. Therefore, it was noticed that the standard and test group had higher collagen
content than control group which resulted rapid wound healing in the said groups in contrast
to the control. Tensile strength was also found higher for test and standard groups as
compared to control. Results of collagen content of regenerated tissue for various groups are
depicted in Fig. 3.48. It was inferred that collagen synthesis is initiated at the wound area
through formation of a polypeptide precursor. Inter and intramolecular crosslinking of this
collagen results in rapid tissue regeneration and wound closure.
159
0
10
20
30
40
50
60
70
Control Band aid®
dressing
LSH dressing
Co
llag
en c
on
ten
t (m
g/k
g)
*
Fig. 3.48. Collagen contents of epithelialized wound tissue of various groups after 15th
day.
Statistical significance from control group is expressed by * p < 0.05.
Table 3.17. Wound area (mm2) and wound closure (%) after selected day intervals.
Wound area in mm ± SD (% of wound closure) at day
1st 3
rd 6
th 9
th 12
th 15
th
Control 5.98 ± 0.06
(0.29)
5.93 ± 0.05
(1.11)
4.78 ± 0.03
(20.32)
3.67 ± 0.10
(38.76)
2.25 ± 0.06
(62.55)
1.00 ± 0.02
(83.27)
Band aid®
dressing
5.87 ± 0.04
(2.17)
4.83 ± 0.08
(19.45)
2.18 ± 0.09
(63.67)
0.76 ± 0.05
(87.27)
0.09 ± 0.08
(98.49)
(100)
LSH
dressing
5.91 ± 0.04
(1.41)
4.94 ± 0.07
(17.73)
2.72 ± 0.08
(54.62)
0.41 ± 0.06
(93.21)
0.05 ± 0.08
(99.22)
(100)
160
3.7. Acute toxicological evaluation of LSH
3.7.1. Acute oral toxicity study in mice
In acute toxicity study, a single oral dose of LSH was given to three groups (Group II, III and
IV) of male albino mice (Table 2.4). At the end of day 14, all animals were alive, active and
healthy. No other abnormalities were seen including behavioral changes, pharmacological
adverse effects and gross toxicities during the 14 day study period. Food intake was less after
day 1 as compared to remaining days. Results have indicated that the acute oral lethal dose
(LD50) of LSH is greater than 5 g/kg of body weight (bw) for male albino mice.
3.7.2. Primary eye irritation
To find out the primary eye irritation, LSH was used for a single ocular installation in the eye
of rabbit. Treated eye was not found any corneal opacity and iritis during 72 h study. Eye of
one animal was exhibited conjunctival discharge and cured within 24 h. The score was
assigned 2.0 and 1.0 after 1 h and 24 h, respectively according to Kay and Calandra eye
irritation scale (Kay and Calandra, 1962) (Table 3.18). No other adverse effects and
symptoms were seen. Therefore, the conjunctival discharge was classified as accidental and
overall LSH was found to be nonirritating to the eye.
161
Table 3.18. Scores for grading the primary eye irritation study of LSH.
Studied groups Ocular observations Time duration (h)
01 24 48 72
Group I Corneal opacity 0 0 0 0
Iritis 0 0 0 0
Conjunctivitis 0 0 0 0
Severity (MMTS) 0 0 0 0
Group II Corneal opacity 0 0 0 0
Iritis 0 0 0 0
Conjunctivitis 2 1 0 0
Severity (MMTS) 0.67 0.33 0 0
Group III Corneal opacity 0 0 0 0
Iritis 0 0 0 0
Conjunctivitis 0 0 0 0
Severity (MMTS) 0 0 0 0
Group IV Corneal opacity 0 0 0 0
Iritis 0 0 0 0
Conjunctivitis 0 0 0 0
Severity (MMTS) 0 0 0 0
MMTS (Maximum mean total scores) of 3 animals
3.7.3. Acute dermal toxicity
Acute dermal toxicity study was carried out in male albino rabbits. Single topical application
of three different doses of LSH (1, 2 and 5 g/kg bw) were applied on three groups of rabbits.
162
All animals survived, gained weight and did not found any other clinical findings or
abnormalities during the whole course of study. It is concluded that the acute dermal toxic
dose was greater than 5 g/kg body weight.
3.7.4. Primary dermal irritation study
Primary dermal irritation study was conducted to evaluate the potential of LSH irritancy on a
single topical application. No erythema or edema was found on the skin of treated animal
over the course of 14 days. Primary dermal irritation index was calculated as 0.0 indicating
the LSH as a non-irritating material to the skin.
3.7.5. Body weight gain study
Results of body weight gain study are summarized in Table 3.19. It was noted that there are
gradually increase in mean body weight of the control group during the whole study period.
A decrease in body weight of group II, group III and group IV was observed at day 1 while
mice were gained the body weight at day 7 and 14.
3.7.6. Food and water consumption
Food and water intake of each group was measured on daily basis and results have indicated
that there was not any significant difference in food and water consumption of control group
during the whole study period (Table 3.19). A significant decrease in the food and water
intake was noted for group II, group III and group IV after day 1 when compared to the
control group. At day 7 and 14, amount of food and water consumed by experimental group
had not much different when compared to the control group.
163
Table 3.19. Observations of body weight, water and food intake study of LSH.
Observations Group I Group II Group III Group IV
Signs of illness NIL NIL NIL NIL
Body weight (g)
Pretreatment 26.4 ± 2.2 28.8 ± 1.8 26.1 ± 2.2 27.7 ± 1.5
Day 1 26.5 ± 1.4 28.7 ± 1.5 26.0 ± 1.9 27.4 ± 3.5
Day 2 27.6 ± 1.2 29.1 ± 1.9 26.2 ± 1.1 27.5 ± 3.5
Day 3 28.0 ± 1.8 29.8 ± 1.1 26.5 ± 0.9 27.7 ± 3.5
Day 5 28.4* ± 2.7 30.1 ± 2.2 27.2 ± 2.0 28.8* ± 3.5
Day 7 28.5* ± 2.4 30.4* ± 1.8 27.6* ± 2.8 29.4* ± 2.5
Day 14 28.8* ± 2.0 30.8* ± 2.8 28.4* ± 2.0 29.6* ± 1.8
Water intake (mL)
Pretreatment 10.4 ± 1.0 12.2 ± 0.5 11.1 ± 2.1 11.7 ± 1.5
Day 1 10.8 ± 1.5 12.4 ± 1.5 11.6 ± 1.5 12.3 ± 1.7
Day 2 11.5 ± 1.1 12.6 ± 0.5 12.0 ± 2.1 12.6 ± 1.1
Day 3 12.8 ± 0.8 12.5 ± 1.3 12.3 ± 1.8 12.7 ± 1.1
Day 5 13.1* ± 1.8 13.3 ± 1.5 12.8* ± 1.0 12.9 ± 0.7
Day 7 13.0* ± 2.0 13.7* ± 2.0 13.0* ± 2.5 13.0 ± 2.3
Day 14 13.2* ± 1.5 13.9* ± 1.5 13.2* ± 1.5 13.2* ± 2.5
Food intake (g)
Pretreatment 3.4 ± 1.0 3.1 ± 1.5 3.6 ± 2.1 3.8 ± 1.6
Day 1 2.7 ± 1.5 2.4 ± 2.1 1.8* ± 1.1 1.1* ± 1.7
Day 2 3.2 ± 1.9 2.7 ± 2.1 2.3* ± 0.7 2.0* ± 2.1
Day 3 3.8 ± 1.0 2.9 ± 1.1 3.0 ± 1.2 2.3* ± 1.3
Day 5 3.6 ± 1.3 3.5 ± 1.4 3.2 ± 1.6 3.2 ± 0.7
Day 7 4.0 ± 1.0 4.0 ± 1.9 3.8 ± 0.5 3.6 ± 0.9
Day 14 3.8 ± 1.5 3.8 ± 1.3 4.3 ± 2.1 4.2 ± 1.1
Body weight, water and food intake are expressed as mean ± S.D. *P < 0.05 as significant
difference when compared to control.
164
3.7.7. Haematology and clinical biochemistry
Hematological parameters of each group were found to be normal and they are comparable
with the control group (Group I) as mentioned in Table 3.20. Liver, kidney and lipid profile
was also determined and results are found to be normal. All values are comparable with the
animals of control group as mentioned in Table 3.21.
3.7.8. Gross necropsy and histopathology
Macroscopic analysis mice of each group were performed and did not found any LSH-related
abnormalities (Table 3.22). All vital organs were remove and weighed separately. Results
have indicated that there were not found any significant difference in weight when compared
with the mice of control group.
Table 3.20. Biochemical blood analysis of control and LSH treated mice.
Hematology Group I Group II Group III Group IV
Hb (g/dL) 11.6 ± 0.8 12.1 ± 1.6 13.7 ± 1.6 13.5 ± 1.4
WBCs (103/µL) 4.2 ± 0.3 5.1 ± 0.4 5.9 ± 1.2 6.5 ± 0.5
RBCs (106/µL) 8.28 ± 0.23 8.39 ± 0.45 8.32 ± 0.35 8.15 ± 0.23
Platelets ( 103/µL) 549.7 ± 2.9 667.2 ± 3.9 678.6 ± 5.5 709.6 ± 4.1
Monocytes (%) 2.1 ± 0.02 2.3 ± 0.01 2.5 ± 0.01 2.2 ± 0.02
Neutrophils (%) 12.8 ± 0.5 13.5 ± 0.3 12.9 ± 0.9 13.8 ± 0.02
Lymphocytes (%) 83.3 ± 2.1 82.3 ± 1.5 84.6 ± 2.1 82.9 ± 1.3
MCV 45.8 ± 1.5 45.9 ± 2.1 51.4 ± 1.4 53.5 ± 1.4
MCH 14.5 ± 0.5 16.0 ± 0.8 15.7 ± 1.0 14.4 ± 1.2
MCHC (g/dL) 28.7 ± 0.3 30.4 ± 0.7 29.6 ± 1.5 31.6 ± 1.1
All values are expressed as mean ± S.D.
165
Table 3.21. Liver, kidney and lipid profile of mice treated with LSH.
Biochemical analysis Group I Group II Group III Group IV
ALT (IU/L) 54 ± 5.3 70 ± 3.2 58 ± 5.2 66 ± 2.1
AST (IU/L) 134 ± 8.3 145 ± 6.1 176 ± 7.1 185 ± 5.1
Creatinine (mg/dL) 0.43 ± 0.02 0.42 ± 0.01 0.35 ± 0.04 0.38 ± 0.02
Urea (mg/dL) 62 ± 2.3 53 ± 4.2 48 ± 4.2 59 ± 3.4
Uric acid (mg/dL) 5.6 ± 0.2 4.5 ± 0.05 4.6 ± 0.1 4.8 ± 0.1
Cholesterol (mg/dL) 147 ± 7.2 136 ± 2.3 108 ± 1.1 116 ± 5.2
Triglyceride (mg/dL) 132 ± 9.4 110 ± 2.3 115 ± 5.4 87 ± 4.8
All values are expressed as mean ± S.D.
Table 3.22. Absolute mean organ weight (g) of mice after oral administration of LSH.
Organ Group I Group II Group III Group IV
Heart 0.66 ± 0.05 0.58 ± 0.03 0.57 ± 0.05 0.53 ± 0.01
Liver 6.44 ± 0.37 6.15 ± 0.22 5.52 ± 0.14 5.70 ± 0.32
Lung 0.54 ± 0.12 0.63 ± 0.11 0.67 ± 0.23 0.51 ± 0.11
Kidney 0.98 ± 0.05 1.08 ± 0.07 0.88 ± 0.05 1.52 ± 0.02
Stomach 1.05 ± 0.18 1.79 ± 0.1 1.38 ± 0.4 1.6 ± 0.20
Spleen 0.63 ± 0.12 0.55 ± 0.01 0.61 ± 0.03 0.60 ± 0.12
All weights are expressed as relative organ weights and expressed the mean values ± S.D.
166
CONCLUSIONS
Polysaccharides based hydrogel was isolated from linseeds and characterized. LSH have
shown marked swelling in deionized water and at gastrointestinal tract pH (6.8 and 7.4) while
less swelling was observed at pH 1.2. Therefore, we have studied the stimuli responsive
swelling deswelling of LSH in different solvents and at basic and acid pH. Moreover, we
used said valuable properties of LSH, i.e., marked swelling, high water holding capacity and
stimuli responsible properties of LSH and successfully developed sustained release oral drug
delivery system of diclofenac sodium, caffeine and diacerein. Interestingly, the swelling
properties and swelling deswelling responses of LSH were still observed even in compressed
form, i.e., tablet formulations. Therefore, LSH can be a good candidate for the development
of sustained release formulation of short half-life drugs and also for pH dependent drug
delivery. For future prospect, LSH can be used to protect the active ingredient from the harsh
gastric environment/pH.
Highly porous structure with microscopic channeling was observed in swollen then freeze
dried sample of LSH which is considered the main reasoning of highly swellable nature of
LSH. This provided the basis to use LSH in future oral dosage forms.
LSH based nanoparticles were developed using Pluronic F68 and an anticancer drug,
docetaxel, was loaded. These docetaxel loaded LSH Pluronic (DLP) NPs has not shown any
toxicity and proved its anticancer effectiveness with minimum side effects. Formation of
DLP-NPs has opened up a new door for the delivery of other therapeutic and diagnostic
agents.
LSH appeared as a reducing and capping agent for the green synthesis of Ag NPs. The COO─
and OH groups are responsible for the synthesis of Ag NPs. Spherical shaped Ag NPs
embedded in LSH film has shown a pronounced antimicrobial and wound healing dressing.
Collagen contents and wound healing period of LSH containing wound dressing was
167
comparable with commercially available product. In future, wound healing mechanism of
these dressing can be explored to broaden the wide range of LSH application.
Additionally, LSH was proved safe after acute toxicity study without any significant
observations. Biochemical, haematological and histopathological examination has shown the
nontoxic nature of LSH. Therefore, LSH can be used as a novel drug delivery carrier for oral,
topical and parenteral administration of many drugs.
On the basis of current research work and available literature of related materials, further
research on applications of LSH in tissue engineering and tissue regeneration should be
carried out. Additionally, use of LSH as a selective chemosensor, biosensor and vaccine
adjuvant should be explored. Commercialization of LSH as an inactive pharmaceutical
ingredient should be taken into account as a future aspect of work reported here.
168
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LIST OF PUBLICATIONS
1. Haseeb, M. T., Hussain, M. A., Yuk, S. H., Bashir, S. and Nauman, M. (2016).
Polysaccharides based superabsorbent hydrogel from Linseed: Dynamic swelling, stimuli
responsive on-off switching and drug release. Carbohydr. Polym., 136:750-756.
2. Haseeb, M. T., Hussain, M. A., Bashir, S., Ashraf, M. U., Ahmad, N. (2017). Evaluation
of superabsorbent Linseed-polysaccharides as a novel stimuli-responsive oral sustained
release drug delivery system. Drug Dev. Ind. Pharm. 43:409-420.
3. Haseeb, M. T., Hussain, M. A., Abbas, K., Youssif, B. G. M., Bashir, S., Yuk, S. H.,
Bukhari, S. N. A. (2017). Linseed hydrogel mediated green synthesis of silver
nanoparticles for antimicrobial and wound dressing applications. Int. J. Nanomedicine.
12:2845-2855.
4. Haseeb, M. T., Bashir, S., Hussain, M. A., Ashraf, M. U., Erum, A., Hassan, M. N.
(2018). Acute toxicity study of a polysaccharide based hydrogel from linseed for potential
use in drug delivery system. Braz. J. Pharm. Sci. 54:e17459.
5. Haseeb, M. T., Hussain, M. A., Yuk, S. H., Amin, M., Bashir, S. Acetylation of linseed
hydrogel: Synthesis, characterization, isoconversional thermal analysis and degradation
kinetics. Cell. Chem. Technol. (Accepted on 26.06.2018).
Submitted
1. Haseeb, M. T., Khaliq, N. U., Yuk, S. H., Hussain, M. A., Bashir, S. Linseed
polysaccharides based nanoparticles for controlled delivery of docetaxel: Design, in vitro
drug release and cellular uptake. Journal of Drug Delivery Science and Technology.