department of pharmacy faculty of pharmacy & alternative...
TRANSCRIPT
i
Formulation and Evaluation of Smart Polymeric
Systems for Controlled Release of Antihypertensive
Drugs
A Thesis
Submitted in partial fulfillment of requirement for the
Degree of
Doctor of Philosophy
(Pharmaceutics)
By
Jawad Ahmad Khan
B.Pharm., M.Phil.
DEPARTMENT OF PHARMACY
Faculty of Pharmacy & Alternative Medicine
The Islamia University of Bahawalpur
PAKISTAN
2010-2016
ii
In the name of Allah, the Most Merciful, the Most Kind
iii
DEDICATION
To
My Father & Mother
who have supported me all the way since the beginning of my studies, experienced the tension, doubt, and frustration accompanying, consciously or unconsciously, assent to
"investigative judgment" doctrine
iv
DECLARATION
I, Jawad Ahmad Khan, Ph.D. Scholar of Department of Pharmacy, the Islamia
University of Bahawalpur hereby declares that this research work entitled:
―Formulation and Evaluation of Smart Polymeric Systems for Controlled
Release of Antihypertensive Drugs” has completed successfully. I also certify that
nothing has been incorporated in this dissertation without acknowledgment and that to
the best of my knowledge and belief it does not contain any material previously
published or any material previously submitted for a degree in any University; where
due reference is not made in the text.
Jawad Ahmad Khan
v
CERTIFICATE
It is hereby certified that work presented by Jawad Ahmad Khan S/O Umer Daraz Ali
in the dissertation entitled ―Formulation and Evaluation of Smart Polymeric
Systems for Controlled Release of Antihypertensive Drugs” has been successfully
carried out in partial fulfillment of the requirements for the degree of Doctor of
Philosophy (Pharmaceutics) under my supervision in the Department of Pharmacy,
Faculty of Pharmacy and Alternative Medicine, The Islamia University of
Bahawalpur.
Dr. Fahad Pervaiz
Supervisor, Assistant Professor
Faculty of Pharmacy and Alternative Medicine,
The Islamia University of Bahawalpur
vi
Acknowledgments
I glorify with the depth of my heart to Almighty Allah. He is Alone and Magnificent.
All praises for Prophet Hazrat Muhammad (PBUH) who is the real embodiment of
human morality. He showed the humanity its actual supreme status that it really
deserves.
From the formative stages of this thesis, to the final draft, I owe an immense debt of
gratitude to my supervisor, Dr. Fahad Pervaiz, Assistant Professor, Department of
Pharmacy, Faculty of Pharmacy and Alternative Medicine, The Islamia University of
Bahawalpur, for his hard work, guidance, unfailing supportive attitude and sound
advice throughout this entire thesis process and gently leading me in the proper
direction. Thank you so much for a great experience.
I am also thankful to Prof. Dr. Mahmood Ahmad, Dean, Faculty of Pharmacy and
Alternative Medicine, the Islamia University of Bahawalpur for providing best
research facilities. I am also thankful to Prof. Dr. Naveed Akhtar, Chairman
Department of Pharmacy, for carrying out in time documentation for conductance of
research project.
I am also thankful to Prof. Dr. Nazar Muhammad Ranjha, Chairman Department
of Pharmacy, Bahaudin Zakariya University Multan for helping me in my research
project.
I am also indebted to Mr. Muhammad Naeem, Liaqat ali, Nayab Khalid and
Muhammad Yousuf for co-ordination and co-operation during the whole time by
sparing their valuable time. I am also thankful to Mr Shafeeq, store Incharge and Lab
attendants who supported me during my lab work.
I am highly grateful to Higher Education Commission of Pakistan for financial
support in the form of Indigenous scholarship.
vii
Manuscripts Published, Accepted/Submitted in HEC
Approved / Impact Factor Journals
Jawad Ahmad Khan, Fahad Pervaiz, Nazar Mohammad Ranjha, Muhammad
Naeem, Nayab Khalid, and Zeeshan Javaid, 'Design and Characterization of Pva–
Methacrylic Acid Based Smart Polymeric System for Controlled Release of
Metoprolol', Journal of Polymers and the Environment (2016), 1-13 (Published,
Impact factor 1.969).
Jawad Ahmad Khan, Fahad Pervaiz, Nazar Muhammad Ranjha, Muhammad
Naeem, Muhammad Yousaf, Fabrication and characterization of carrageenan-
methacrylic acid hydrogel for controlled release of anti-hypertensive perindopril
(Submitted in Colloid and Polymer Science Impact factor 1.890)
Jawad Ahmad Khan, Fahad Pervaiz, Muhammad Naeem, Design and in vivo study
of robust carrageenan-methacrylic acid hydrogel for controlled release of perindopril
(Submitted in International Journal of Pharmaceutics Impact factor 3.994)
viii
LIST OF CONTENTS
Serial no Title Page No
1 Title Page i
2 Bismillah ii
3 Dedication iii
4 Declaration iv
5 Certificate v
6 Acknowledgement vi
7 Manuscripts Published, Accepted/Submitted in
HEC Approved / Impact Factor Journals
vii
8 List of Contents viii
9 List of Tables xv
10 List of Figures xvii
11 Abstract xix
CHAPTER NO. 1.
1. INTRODUCTION 1
CHAPTER NO. 2.
2. LITERATURE REVIEW 6
2.1. Oral delivery systems 6
2.2. Biodegradable systems 7
2.3. Controlled drug delivery system 8
ix
2.3.1. Controlled release systems of biologically active
compounds
8
2.3.2. Mechanisms of Drug Release 9
2.3.3. Materials utilized for controlled release systems 10
2.3.3.1 Drug-Polymer complexes 10
2.3.3.2. Polymers utilization in controlled drug delivery
system
11
2.3.3.2.1. Biocompatible polymers 11
2.3.3.2.2. Recombinant polymers 12
2.3.3.2.3. Stimuli sensitive polymers for drug delivery 13
2.3.3.2.3.1. Applications and examples 13
2.3.3.2.4. Stimuli sensitive systems based on temperature 14
2.3.3.2.5. Responsive systems based on pH 16
2.3.3.2.6. Responsive systems based on redox potential 17
2.3.3.2.7. Supremacy of polymer therapeutics for drug
delivery
17
2.4. Hydrogels 18
2.4.1. Classifications of hydrogels 20
2.4.1.1. Types of hydrogels on the basis of crosslinking 20
2.4.1.1.1. Physically and chemically crosslinked hydrogels 21
2.4.1.1.2. Crosslinking through radical polymerization 22
2.4.1.1.3. Crosslinking via chemical reactions 22
2.4.1.1.4. Crosslinking via ionic reactions 22
2.4.1.1.5. Crosslinking by crystallization 22
x
2.4.1.2 Interpenetrating polymer network gels 23
2.4.1.2.1. Interpenetrating and semi-interpenetrating
networks
23
2.4.1.2.2. Homo-polymeric hydrogel 25
2.4.1.2.3. Co-polymeric hydrogel 25
2.4.1.3. Stimuli responsive hydrogels 25
2.4.1.4. pH responsive hydrogels 26
2.4.1.4.1. Salient features of pH responsive hydrogel 27
2.5. Hypertension and its remedy 28
2.5.1. Perindopril erbumine 29
2.5.1.1. Overview 29
2.5.1.2. Pharmacodynamics 30
2.5.1.3. Pharmacokinetic Profile 31
2.5.1.4. Therapeutic Efficacy 32
2.5.1.5. Tolerability 33
2.5.1.6. Dosage and Administration 33
2.5.2. Metoprolol 33
2.5.2.1. Pharmacodynamic features 34
2.5.2.2. Pharmacokinetic features 34
2.5.2.3. Therapeutic Use 35
2.5.2.4. Dosage and administration 36
CHAPTER NO. 3.
3. MATERIALS AND METHODS 37
xi
3.1. Materials 37
3.2. Synthesis of covalently crosslinked pH
sensitive hydrogels
37
3.3. Characterization of synthesized hydrogels 42
3.3.1. Swelling investigations 42
3.3.1.1. Swelling parameters studies 42
3.3.2. Water diffusion coefficient of the formulations 42
3.3.3. Networking parameters of smart hydrogels 43
3.3.3.1. Molecular weight between crosslinks (Mb) 43
3.3.3.2. Solvent interaction parameters (χp) 43
3.3.3.3. Crosslinkeddensity (qh) 44
3.3.4. Sol-gel and porosity studies 44
3.3.4.1. Sol gel calculation 44
3.3.4.2. Porosity 44
3.3.5. Structural spectroscopy 45
3.3.6. Thermal studies 45
3.3.6.1. Differential scanning calorimetry 45
3.3.6.2. Thermogravimetric analysis 45
3.3.7. X-ray diffraction 46
3.3.8. Scanning electron microscopy 46
3.4. In vitro studies 46
3.4.1. Loading of drug 46
3.4.2. Determination of drug loaded in disks 46
3.4.3. Drug release studies 47
xii
3.4.4. Analysis of drug release pattern 47
3.5. In Vivo study 47
3.5.1. Selection of Animals 47
3.5.2. HPLC Conditions and mobile phase 48
3.5.3. Stock Solution Preparation 48
3.5.4. Blank plasma sample 49
3.5.5. Plasma spiking 49
3.5.6. Sampling 49
3.5.7. Statistical evaluation 50
CHAPTER NO. 4.
4. RESULTS AND DISCUSSIONS 51
4.1 Swelling and drug release pattern 51
4.1.1 Effect of pH on swelling and on drug release of poly
vinyl alcohol/methacrylic acid hydrogels
51
4.1.2. Implications of tuned pH on swelling of CA-g-MA
hydrogels
52
4.1.3. Implications of concentrations of composites on
swelling of CA-g-MA hydrogels
52
4.1.4. Effect of monomer concentration on swelling and on
drug release of PVA/MA hydrogel
56
4.1.5. Effect of polymer concentration on swelling of
PVA/MA hydrogel
59
4.1.6. Effect of degree of crosslinking on swelling and on
drug release of PVA/MA hydrogel
61
4.1.7. Implications of concentrations of composites on
drug release of CA-g-MA hydrogel
66
4.2. Diffusion coefficient investigations 69
4.2.1. Diffusion coefficient of polymers (Dw) of PVA/MA
hydrogel
69
xiii
4.2.2. Diffusion coefficient study of CA-g-MA
formulations
69
4.3. Networking structural analysis 69
4.3.1. Molecular weight between crosslinks (Mb) and
solvent interaction parameters (χp) in PVA/MA
hydrogel
69
4.3.2. Networking structural analysis of CA-g-MA
formulations
70
4.4. Sol-gel and porosity analysis 71
4.4.1. Sol-gel analysis of PVA/MA hydrogel 71
4.4.2. Porosity measurement of PVA/MA hydrogel 72
4.5. Spectral confirmation 76
4.6. Insights into thermal behavior 79
4.6.1. Differential scanning calorimetry 79
4.6.2. Thermogravimetric analysis 82
4.7. X-Ray diffraction 85
4.8. Interpretation of morphological
characteristics
87
4.9. In vitro evaluation 91
4.9.1. Drug release mechanism 91
4.10. In-vivo Investigations 96
4.10.1. HPLC Insights 96
4.10.2. Calibration curve of Perindopril erbumine 96
4.10.3. In vivo findings 99
CHAPTER NO. 5.
xiv
5. CONCLUSION 104
CHAPTER NO. 6.
6. REFERENCES 105
xv
LIST OF TABLES
Table no Title Page No
3.1 Formulation compendia of polyvinyl alcohol/methacrylic
acid hydrogel.
38
3.2. Feed module of CA-g-MA hydrogel formulations. 39
4.1. Equilibrium swelling ratios of PVA/MA hydrogel. 64
4.2. Percent metoprolol tartrate released in different
formulations of PVA/MA hydrogel.
65
4.3. Drug loading through weight and extraction method of
PVA/MA hydrogel
65
4.4. Swelling measurements of CA-g-MA hydrogel. 66
4.5. Drug release study of optimum formulations of CA-g-MA/MA
hydrogel.
67
4.6. Drug loading through weight and extraction method of CA-g-MA
MA hydrogel
67
4.7. Flory-Huggins network parameters of PVA/MA hydrogel. 70
4.8. Flory-Huggins network parameters of tuned CA-g-MA
formulations.
71
4.9. Gel fraction and porosity measurement in different
formulations of PVA/MA hydrogel.
72
4.10. Effect of different concentrations of methacrylic acid on
drug release kinetics of PVA/MA hydrogel in solution of
different pH using glyoxal as crosslinking agent.
92
4.11. Effect of degree of crosslinking on drug release kinetics of
PVA/MA hydrogel in solution of different pH.
92
4.12. Effect of tuned concentrations of methacrylic acid on drug
release kinetics of CA-g-MA hydrogel in solution of
different pH using glyoxal as crosslinking agent.
93
4.13. Effect of degree of crosslinking on drug release kinetics of
CA-g-MA hydrogel in solution of different pH.
93
xvi
4.14. Effect of different concentrations of methacrylic acid on
drug release mechanism of PVA/MA hydrogel in solution of
different pH using glyoxal as crosslinking agent.
94
4.15. Effect of degree of crosslinking on drug release mechanism
of PVA/MA hydrogel in solution of different pH.
94
4.16 Effect of tuned concentrations of methacrylic acid on drug
release mechanism of CA-g-MA hydrogel in solution of
different pH using glyoxal as crosslinking agent.
95
4.17 Effect of degree of crosslinking on drug release mechanism
of CA-g-MA hydrogel in solution of different pH.
95
4.18. Major parameters regarding regression analysis for
calibration line.
97
4.19. Pharmacokinetic data of antihypertensive perindopril
erbumine.
99
xvii
LIST OF FIGURES
Figure No Title Page No
2.1. Internal structural parts of a hydrogel. 19
2.2. Major divisions of hydrogel on the basis of different
characteristics.
21
2.3. pH dependent ionization of polyelectrolyte. 27
2.4. Structural formula of perindopril erbumine. 31
2.5. Structural formula of metoprolol tartrate. 35
3.1. Presumptive structure of PVA/MA hydrogel. 40
3.2. Proposed structure of CA-g-MA hydrogel. 41
4.1. Dynamic swelling coefficient of CA-g-MA hydrogels
with different amounts of (a) glyoxal (b) MA and (c)
CA in solutions of tuned pH values: pH 2 ( ), pH 4.7 (
), pH 6.4 ( ) and pH 7.4 ( ).
55
4.2. Dynamic swelling coefficient of PVA/MA hydrogels
with different concentrations of MA (38, 46 & 54 g)
using Glyoxal as crosslinking agent (0.75 %) in
solutions of different pH in 0.05M USP phosphate
buffer at 37 0C. The pH values are: pH 1.2 ( ), pH 6.5
( ) and pH 7.5 ( ).
57
4.3. Cumulative release % of metoprolol after 48 h (a)
using different concentrations of methacrylic acid (38,
46 & 54 g) and (b) complete release profile of J7.
58
4.4. Dynamic swelling coefficient of PVA/MA hydrogels
with different concentrations of PVA (0.30, 0.60 & 1.2
g) using Glyoxal as crosslinking agent (0.75 %) in
solutions of different pH in 0.05M USP phosphate
buffer at 37 0C.The pH values are: pH 1.2 ( ), pH 6.5
( ) and pH 7.5 ( ).
60
4.5. Dynamic swelling coefficient of PVA/MA hydrogels
with different concentrations of Glyoxal (0.30, 0.50
and 1 %) in solutions of different pH in 0.05 M USP
phosphate buffer at 37 0C. The pH values are: pH 1.2 (
), pH 6.5( ) and pH 7.5 ( ).
62
4.6. Cumulative release % of metoprolol after 48 h (a)
using different concentrations of Glyoxal as
63
xviii
crosslinking agent (0.30 %, 0.50 % and 1 %) and (b)
complete release profile of J8.
4.7. Release of perindopril from CA-g-MA hydrogels
using different amounts of (a) glyoxal and (b) MA at
tuned pH values: pH 2 ( ), pH 4.7 ( ) and pH 7.4 ( ).
68
4.8. Effect of different concentrations of PVA on gel
fraction.
73
4.9. Effect of different concentrations of Glyoxal on gel
fraction.
74
4.10. Effect of different concentrations of MA acid on gel
fraction.
75
4.11. FTIR spectra of (a) PVA (b) PVA/MA hydrogel
without drug (c) PVA/MA hydrogel with drug.
77
4.12. FTIR spectra of (a) CA (b) Blank CA-g-MA hydrogel
(c) Drug loaded CA/MA hydrogel.
78
4.13 DSC of MA, PVA and PVA/MA hydrogel. 80
4.14. DSC of MA, CA and CA-g-MA hydrogel. 81
4.15. TGA of MA, PVA and PVA/MA hydrogel. 83
4.16. TGA of MA, CA and CA-g-MA hydrogel. 84
4.17. XRD spectra of (a) PVA (b) PVA/MA hydrogel. 86
4.18. Scanning electron micrographs of PVA/MA hydrogel
without drug.
88
4.19. Scanning electron micrographs of PVA/MA hydrogel
with drug.
89
4.20. Scanning electron micrographs of (a) blank and (b)
loaded CA-g-MA hydrogel.
90
4.21. Calibration curve of Perindopril erbumine in the
spiked plasma.
98
4.22. Chromatogram of blank plasma. 101
4.23. Chromatogram of plasma spiked perindopril (16
ng/mL) and internal standard perindoprilat (8
ng/mL).
102
4.24. Mean serum data of Perindopril erbumine in the
rabbits, after delivery of oral hydrogel and tablet.
103
xix
Abstract
In the first investigation covalently crosslinked smart polymeric system of hydrogel
based on poly vinyl alcohol (PVA) and methacrylic acid (MA) was designed by free
radical polymerization with different compositions using glyoxal (40 % water
solution) as crosslinker. It was observed that swelling of hydrogel had a pronounced
enhancing effect on increasing the concentration of MA due to availability of more
ionized carboxylic groups of MA but produced an opposite effect on increasing the
concentration of glyoxal owing to less porous structure. As far as PVA is concerned,
swelling did not show significant effect on increasing the concentration of PVA.
Hydrophilic polymer PVA rich in hydroxyl group pertained to be highly interactive
with water. It was examined that the release of metoprolol tartrate decreased with
increased concentration of glyoxal, but increased with increase in concentration of
MA. PVA/MA hydrogel was characterized by Fourier transform infrared
spectroscopy (FTIR) and X-ray diffraction (XRD) to study the structure and
crystallinity of hydrogel respectively. Morphology was studied through scanning
electron microscopy (SEM). Furthermore differential scanning calorimetry (DSC) and
thermogravimetric analysis (TGA) were also performed to characterize thermal
stability. It may be concluded that the mechanism of drug release was mainly non-
Fickian diffusion.
The second investigation enlightened a robust design to develop biodegradable pH
responsive hydrogel of carrageenan (CA) / methacrylic acid (MA) and to evaluate the
potential of formulations to sustain the release of perindopril. Glyoxal crosslinked
carrageenan graft methacrylic acid (CA-g-MA) macromolecule formulations were
fabricated in the presence of potassium persulphate as free radical initiator system.
Crosslinker composition was varied in JA1, JA2 and JA3 formulations, while JA4, JA5
xx
and JA6 had variable MA concentrations respectively. JA7, JA8 and JA9 had variable
CA concentrations. Preliminary experiments on swelling ratio, drug loading and drug
release were carried out to investigate the ability of CA-g-MA formulations as
controlled drug delivery system. Release of drug was dependent on monomeric
composition, crosslinker and pH of release media. The morphology, structure,
swelling capacity, interpenetrating polymeric networking parameters and thermal
stability were evaluated. The results obtained indicated that the modulated
formulations have considerable potential as drug-carrier materials for controlled
delivery system.
1
1. INTRODUCTION
The current dissertation enlightened robust designs to develop and characterize
biodegradable smart polymeric systems and to evaluate the potential of formulations
to sustain the release of antihypertensive drugs.
Polymeric systems have been promisingly employed in every field of our life from
nano-scale to mega-scale. In current scenario the study of physicochemical properties
of polymeric systems is the evolution in designing and fabrication of mature
polymeric systems. Their implications are largely seen in pharmaceutical industries as
controlled drug delivery systems. Controlled drug delivery designing is the glorious
field of science in which scientists are playing their vital role in health care
disciplines. This delivery system leads over conventional system due to its fascinating
features of low toxicity, ease of access, enhanced patient compliance and suitability.
Mostly synthetic polymers are utilized for these types of systems.
Drug delivery system focuses mainly on the betterment of drug therapy. This
betterment can be achieved by minimizing side effects and frequent dosing.
Controlled release of drugs can be explained through temporal and distribution
controlled methods. Temporal method involves the import of drugs for prolonged
time or for appropriate period through treatment phase whereas distribution controlled
involves the delivery of drugs at right position of activity inside body. Mechanism
behind temporal controlled release can be better understood through the release of
drugs from polymeric devices. Temporal release of drugs is carried out through three
types of mechanisms which are (i) delayed dissolution (ii) diffusion controlled and
(iii) drug solution flow control [1, 2].
2
Polymers are gaining attention in biomedical era owing to their controlled, localized
and sustained release behavior. Polymer hydrogels are now being extensively
employed in bioactive factor and therapeutic drug delivery systems. Hydrophilic
novel polymers are the core of modern research in biotechnological developments.
Recent developments in biotechnology evolve the engineering of sequences of the
components of biomacromolecules. Hydrogels can be conveniently prepared from
polymers [3, 4]. Hydrogels are three dimensional (3D) macromolecular networks
capable of imbibing an immense amount of water which remain insoluble in water
due to the presence of crosslinks. These crosslinks maintain the polymeric network
and physical configuration in hydrogels [5]. Hydrogels are stimuli responsive smart
materials exhibiting unique characteristics owing to modulation in temperature, pH,
ionic strength or type of swelling agent used and irradiation. Network permeability
and swelling ability of hydrogels are the most fascinating parameters which play a
vital role in drug release [6]. In this modern era strategies have been developed to
invade cell delivery in 3D microenvironment through hydrogel. Hydrogels have a
versatile characteristic of cell encapsulation which makes them a strong candidate for
cell delivery. The other salient features of hydrogels like biocompatibility,
hydrophilicity, low toxicity and stimuli responsive behaviours hold a promise to
deploy them in tissue engineering scaffolds, protein therapeutics, dental materials,
stem cell culture, regenerative medicine, cancer immunotherapy and as biomimetic
materials [7, 8]. Hydrogels can be designed mainly through physical and chemical
crosslinking methods. Hydrogels prepared through safe crosslinkers have fascinating
features of biocompatibility, cytocompatiblity and facile cell encapsulation. In this
modern era, new emerging techniques are now being utilized to get nanocomposite,
3
triblock copolymer, macromolecule microsphere composite, biodegradable,
injectable, superporous and double network hydrogels [9-12].
Radical polymerization is one of the techniques utilized in chemical crosslinking of
hydrogels [13]. Release of drug from hydrogels mainly depends on the diffusion rates
of solute molecules in the polymeric matrix [14]. Improved mechanical properties of
hydrogels can be tuned through chemical crosslinking methods. Hydrogels exhibit
visco-elastic and pure elastic behaviour due to the presence of crosslinks present in
polymeric chains. Chemically crosslinked gels have covalent bonds in polymeric
chains and physically crosslinked gels have physical interactions in different
polymeric chains [15, 13]. Crosslinking through enzymes, high energy radiation and
radical polymerization fall under the category of chemical crosslinking. Radical
polymerization boosted the utilization of hydrophilic polymers to develop hydrogels
[16, 17]. Broadly speaking stimuli responsive hydrogels are classified as light,
electrical signal, glucose, temperature, pH and other stimuli sensitive hydrogels.
Among which pH sensitive hydrogels have fascinating features of swelling in
response to change in pH caused by polyelectrolytes that make them a strong
candidate for oral controlled release formulations. They have a number of imperative
applications in developing permeation switches, biosensors and drug carrier systems
[18].
Interpenetrating polymer networks (IPNs) are described as the combination of two
crosslinked polymers. In semi IPN structure one of the polymers is linear and the
other one is crosslinked. IPNs can be designed through physical and chemical
crosslinking methods which are followed by either spontaneous or sequential
pathway. These IPN structures exhibited wonderful physico-chemical features as
compared to its constituents. Vast implications of IPN have been observed in tissue
4
engineering and biomedical era owing to their fascinating mechanical and biophysical
features [19, 20]
IPN hydrogels are being extensively employed in sustained release formulations. IPN
hydrogels have complex skeleton with better features. IPN hydrogels can be
categorized into nonionic and ionic IPN hydrogels on the basis of the nature of
polymers. Non-ionic IPN hydrogels contain non ionic polymers whereas ionic IPN
hydrogel contain mostly anionic, cationic and cationic/anionic. These ionic IPN
hydrogels may contain both cationic and anionic groups connected to various chains
in the structure are bound mainly through covalent and ionic bonds, which in turn
improve them mechanically and enhance their sensitivity to external stimuli such as
pH, ionic strength and temperature. IPNs physical and mechanical features can be
tuned by modulating the concentrations of polymers and crosslinking agent. A new
and emerging type of ionic IPN hydrogel having polyion complexes is fabricated by
keeping a controlled proportion of cationic and anionic groups in the formulation.
These polyelectrolyte gels contain ionizable moieties. These moieties will regulate the
electrochemical balance between the hydrogel and its external environment. Swelling
of these gels depends on the pH of external medium. Gels with acidic groups swell at
higher pH. These salient features make them strong candidate for controlled release
system [21, 22].
In current investigation covalently crosslinked polymeric systems of polyvinyl
alcohol (PVA) and carrageenan (CA) hydrogels are formulated by modulating
amounts of crosslinking agent glyoxal and monomer methacrylic acid (MA). Free
radical solution copolymerization technique was implied to prepare PVA/MA and
CA-g-MA hydrogels. In this regard, optimized formulations with sustained release of
hydrophilic drug over prolonged period of time are prepared by studying the impact
5
of copolymer composition on mass absorption. Metoprolol tartrate and perindopril
erbumin are loaded and release study of drugs was conducted in different buffer
solutions. Swelling characterization and the potential of formulation composites to
control the release of freely water soluble drugs is evaluated. Network parameters are
calculated. Finally, hydrogel characterization is carried out by fourier transform
infrared spectroscopy (FTIR), differential scanning calorimetry (DSC),
thermogravimetric analysis (TGA) and scanning electron microscopy (SEM) to
investigate structure, thermal stability and surface morphology of hydrogels. In vivo
study was carried out on the rabbits by using CA-g-MA hydrogels with loaded
perindopril erbumine and compared the pharmacokinetic data with the conventional
dosage form (tablet).
6
2. LITERATURE REVIEW
2.1. Oral delivery systems
There are number of new systems for oral drug delivery studies. One important
outcome is prolonged constant release for drug when it crosses across the
gastrointestinal tract (GIT).
A second outcome is to decrease pills one requires and to capacitate the system for
prolonged released. Another new approach for getting prolonged constant release
includes drugs binding to the ion-exchange resins which could be coated using semi-
permeable membranes. As the drug crosses across GIT, salts in the bodily fluids could
relocate the drug; enable it for movement through membrane at comparatively
constant rate. This strategy is fruitful for charged drugs.
Another strategy includes with erodible polymers. For instance, hydroxyl-propy1-
methyl-cellulose is utilized to fabricate tablets whose outer layer formulates gel which
act as diffusion barrier after exposure to water; with time the network wholly
dissolves.
Other strategies for oral administration include the micro-encapsulation of drug, with
or without its combination with non-encapsulated drug; a suitable successfully
fabricated for continuous release theophylline for curing patients of asthma. Another
main challenge for oral delivery is fabrication of bio-adhesive systems that can be
employed to alter transit time of drug moving across GIT. Several strategies including
cellulose-derivatives and polymers are studied to avail bio-adhesion. Instead of active
research under study for oral delivery the controlled release study might not be
required in few cases. For instance, if greater quantity of drug is needed, it can be
hard for all drugs to encapsulate in single tablet. Likewise, if drug has prolonged in
vivo half-life like digoxin then controlled release is not required. Additionally, when
7
there are greater first pass effects including metabolism of drug in liver, controlled
release cannot be fruitful. Finally, wide range difference between minimal effective
dose (MED) and maximum toxic dose (MTD) are unlikely to be worthwhile for
controlled release systems. Instead of such drawbacks, the fabrication of systems
which would improve bioavailability of oral drugs is found to be major challenges for
drug delivery research [23].
2.2. Biodegradable systems
Biodegradable polymers delineate an important group of materials for drug delivery
systems. Mostly applied alternately, degradation and erosion differentiate in that
covalent bond breakage through chemical reactions took place during degradation.
Erosion takes place by the dissolution of chain segments in non-crosslinked networks
without chemical modifications to the molecular entity. For dissolution to take place,
the polymer must absorb the around aqueous solvent and must collaborate with water
through charge interaction or hydrogen bonding. Degradation and erosion can take
place like surface or bulk procedures. During surface degradation, polymer matrix is
gradually removed from surface, yet the volume fraction of polymer remains clearly
same. Contrarily, during bulk degradation, no distinct change takes place in the
physical dimension of the polymer transporter till it is nearly fully diminished or
eroded however polymer fraction present in the transporter falls with time. The
predominant process is calculated through relative rates of solvent entrance in the
polymer, diffusion of the degraded product and dissolution or degradation of the
macro-molecular entity [24]. Such rate evaluations are particularly significant in
fabricating biodegradable hydrogels as they are usually polymerized in aqueous
solvent. Hydrogels chemically degraded by the breakage of bonds present in the
polymer backbone or crosslinks. Most of the biodegradable synthetic materials based
8
on hydrolytic breakage of ester derivatives or ester bonds like poly-ε- caprolactone
and polylactic/glycolic acid. Besides derivatives of ester, hydrolysis also reacts on a
number of other derivatives such as polyphosphazenes, polycyanoacrylate,
polyphosphoesters, polyorthoesters, and polyanhydrides [25, 26].
A renowned problem with biodegradable polymers is ambiguity regarding the safety
of degradation outcomes as degradation usually results in dissemination in small
particle sizes, toxicity is tough to calculate experimentally. Theoretically,
intravenously administered polymers would break in small, metabolic materials which
are seemed to be nontoxic and are short enough to clear natural mechanisms.
2.3. Controlled drug delivery system
Controlled drug delivery system is able to deliver an active biologically substance for
prolonged time period in a biological system from a polymer network.
2.3.1. Controlled release systems of biologically active compounds
These systems are capable to integrate and deliver a particular quantity of active
substance to human body with a purpose to achieve greater therapeutic efficacy by
monitoring the rate of release, time for release and place to show mechanism of
action. The main objective of this system is to keep steady amount of active substance
which was between minimum effective concentration (MEC) and minimum toxic
concentration (MTC) for prolonged time period [27-29]. The mechanism for
controlling release rate for biologically active substance are distinguished into two
levels, owing to mechanism of release of biological active substance.
Chemical mechanisms: It involves the enzymatic degradation which resulted
in breaking or constructing bonds of active compound with matrix or external
environment.
9
Physical mechanisms: It involves ion exchange, matrix dissolution, diffusion
of biologically active substance and osmotic pressure)
Release kinetics was modulated using number of factors by encapsulating biologically
active substance in controlled release carrier including immobilized molecule
chemical structure, nature of polymer which described controlled release carrier,
admixtures employed to get controlled release pattern and feasible chemical
interactions [27, 30-32]. Owing to the technique employed in preparing procedure
along with its dependence on chemical, physical and biochemical characteristics,
there are various kinds of controlled release carriers: nanospheres [33], polymeric
micelles, nanocapsules, dendrimers and liposomes [34-37].
Various studies have been employed on materials which are responsible for achieving
control release pattern due to greater clinical requirements. Controlled released
carriers have to fulfill the requirements of bioavailability, biodegradability and
selectivity to show its viability.
2.3.2. Mechanisms of Drug Release
Controlled release delivery can be defined as system which administer drug at pre-
determined rate for a prolonged time period. These systems should be manufactured
using pumps or polymers. Due to its little size and smaller cost, polymers are
considered the most frequently used vehicles for controlled release. There are three
basic techniques for releasing drug from polymers.
Diffusion: reservoir systems, the drug are encircled with polymer membrane
like microcapsule or capsule.
Matrix systems: the drug is uniformly and equally distributed throughout the
system.
10
From above both cases the drug diffusion from backbone of polymer or pores
in polymer membrane is found to be rate-limiting technique. Release rates are
measured from membranes using steady-state Fick’s Law diffusion equation.
2.3.3. Materials utilized for controlled release systems
There are various momentous demands for all bio-materials employed for modulating
controlled released. Additionally, specific criteria are intended for controlled release
delivery which contains the characteristics of polymer permeability to many
substances along with degradability extent.
2.3.3.1. Drug-Polymer complexes
Therapeutic entities can be covalently combined with polymer backbone. In these
kinds of drug-polymer complexes, the aim is to rectify the procedure of cellular
internalization and cell particular to attain ideal release of the drug at the defined
target.
On the other hand, the systems may contain drugs enclosed or encapsulated in the
polymer inquire to increase the distribution and serum stability and to get a fall in
drug immunogenicity. Polysaccharides for example, comprised of hydroxyl groups
that permit direct reaction of drugs with carboxylic acid functions, hence forming
ester linkages which are biodegradable and so assist the release of the drug in the
system.
Drug polymer complexes are important when the drugs are attached to the polymer
with linkers that behave in a peculiar manner to some digestive enzymes or acidic pH.
Such kind of release is applied in nanoparticles that have good mobility in capillaries,
permitting for effective usage and selective drug aggregation on target sites. This kind
of nanoparticle is regularly employed in cancer therapy [38].
11
2.3.3.2. Polymers utilization in controlled drug delivery system
Polymers employed in controlled drug delivery system are usually named as
therapeutic polymers. Even though, these materials are being employed as excipients
in different formulation but they may not have any particular therapeutic effect. The
major insight in polymers for drug delivery is in designing controlled drug delivery
system. In these formulations, drugs are absorbed or encapsulated in polymer
matrices. According to the polymer chemistry point of view, it should be kept in mind
that prominent mechanisms of controlled delivery system demanded polymers with
different physicochemical characteristics [39]. So the fabrication of drug delivery
contains various architectural patterns in terms of shape and size. Different kinds of
polymers have been used as efficient delivery systems, containing nano- and micro-
particles, capsosomes and micelles, micro- and nano-spheres. The major
discrimination in nano- and micro-formulation is of size. The smaller size of the nano-
formulation draws attention towards the delivery of drugs into the cells. Nano-
formulations can be given through different routes, such as enteral, pulmonary
intravenous and intraocular: though, few routes cannot be implied in
microformulations, for example the intravenous route, due to the blockage of veins.
2.3.3.2.1. Biocompatible polymers
The polymers frequently employed for achieving nanocarriers are biocompatible
materials that control the release for an active substance. A polymer frequently
utilized in acquiring controlled release is co-polymer poly-lactic-co-glycolic acid and
poly-L-lactic acid. The release of drug is primarily induced by using different ratios of
two materials (polymers) employed in fabricated system [40]. Various studies showed
enhancement in pharmacological efficacy with association of at most one polymer to
achieve the controlled release pattern. The term can be defined as Inter-penetrating
12
Polymer Network (IPN) [41]. Owing to the kind of bond they encircle, various IPNs
defined as non-covalent semi-IPN, covalent semi-IPN or non-covalent full IPN [42,
43]. IPNs particular characteristic contains: enhanced polymeric structures elasticity,
raised thermal stability, ameliorate aspects of the dielectric characteristics, optical
clarity and permeability of nutrient, [41, 44]. Researchers studied formation of IPNs
from the poly-(vinyl alcohol) (PVA) and locust bean gum (LBG) for buflomedil
hydrochloride (BH) release. Improved release pattern was beheld of the biologically
active substance for oral administration [43].
2.3.3.2.2. Recombinant polymers
Another kind of system for controlled release is achieved from polymers obtained by
recombinant technology. These are ordinarily engineered from the natural proteins.
The most general amino acid sequences employed in procuring such materials are:
silk-elastin-like protein, silk-like protein and elastin-like polypeptide polymers [45].
Systems for controlled release emanated from the recombinant technology polymers
are a contemporary field in pharmaceutical industry which constrains further studies.
Controlled release systems thoroughly studied now-a-days because they have unique
properties. System for controlled release has following characteristics: cell
recognition, bioavailability, reduced toxicity, biodegradability, low costs and
enhanced bio-activity capability. Additionally, their controlled action proves them
exemplar candidates in fabrication of many pharmaceutical formulations capable to
fulfill clinical requirements. Research in field of controlled release systems has
proved efficiently because it shows benefits of safety, patient convenience and
efficacy. Moreover, various new drugs (peptides) which are being delivered by using
these polymers.
13
Systems for controlled release are represented in various forms containing
particularly developed tablets which should be taken orally, transdermal patches,
implants or injectable microspheres.
2.3.3.2.3. Stimuli sensitive polymers for drug delivery
Stimuli sensitive polymers are a group of materials composed of different branched
and linear copolymers or networks of crosslinked polymer. Fascinating features of
responsive polymers is their property to go through a distinct chemical or physical
change in reply to an external stimulus. Modulation in pH and temperature are usually
implied to initiate behavioral changes, whereas different stimuli, like radiation,
ultrasound, redox potential, electromagnetic, chemical or biochemical agents and
ionic strength can be utilized. Such stimuli can be categorized into separate groups on
the basis of chemical or physical nature.
Physical stimuli such as magnetic field, electrical field, light, temperature and
ultrasound are used. These stimuli directly transform the energy level of
polymer/solvent system and induct polymer reaction at specific energy level.
Chemical stimuli such as redox potential, ionic strength, chemical agents and pH
initiate responses on modulating molecular interactions within polymer/solvent or
within polymer chains [46]. Different types of behavioral changes may contain
modifications in conformation, hydrophilic-hydrophobic balance and solubility [47].
Such modifications are described in different ways, for example sol-gel modification
in physically crosslinked hydrogels [48], the modulation of coil-globule in polymeric
chains [49] and swelling/deswelling of crosslinked hydrogels [50].
2.3.3.2.3.1. Applications and examples
Stimuli responsive polymers can be classified as conjugated drug-polymer conjugates,
polyplexes, hydrogels or micelles that are explained and elaborated in detail.
14
Hydrogels are demystified as hydrophilic polymeric systems ability to absorb
immense quantity of water or biological fluids [51]. Crosslinks present in networks
restrict hydrogels dissolution in water. Hydrogels can be fabricated in such a way that
they quickly respond to different stimuli and express distinct value in pharmaceutical
and medical fields [52]. Scientists have engineered such pH-responsive hydrogels
which can be employed in oral protein delivery like insulin. Such formulations
successfully designed to absorb, protect, and release the delivery of proteins like
interferon β [53], calcitonin [54] and insulin [55]. Polymers implied in micelles
include polymers of polyuronics, polypropylene oxide, and polyethylene oxide, have
been utilized in drug delivery systems [56]. Such polymers represent temperature-
sensitive micellization [57], like the polymers of polyethylene glycol combined with
poly N-isopropylacrylamide (PNIPAAm) [58]. Polyplexes formulated through
electrostatic interactions of DNA and polyethyleneimine (PEI) are investigated for
delivery of gene. Many authors have elaborated about the delivery of genes in detail
and also discussed about the safety and importance of the fabricated formulations [59-
61].
2.3.3.2.4. Stimuli sensitive systems based on temperature
Temperature is an important stimulant playing a key role in the applications of
controlled drug delivery system. [46]. Scientists explored about the impact of
temperature on swelling of polymeric systems [62].
One of the most promising thermo-sensitive polymers, PNIPAAm, has been studied
due to its property to induce an inverse, reversible temperature-dependent phase
modulation [52]. Below its, PNIPAAm maintains itself as a hydrophilic coil at a
temperature less than its lower critical solution temperature (LCST) about 32°C ,
while above this temperature i.e. higher LCST hydrophobic globule is formed on the
15
cleavage of the chains [63]. This modification takes place as a result of the
hydrophilic/hydrophobic balance of polymeric chains [64], which is modified through
continual formation and destabilization in hydrophobic and intra-intermolecular
interactions.
Progress in polymer therapeutics has remained successful since the last few decades
in designing effective and better delivery of bioactive materials to overcome a large
number of clinical and medical conditions. Advancement in investigations has
revealed a great promise in promoting drug delivery systems that drugs will be
reached at target site in therapeutically required quantities and will decrease the
frequent dosing of patient. Looking forward, investigation efforts should explore a
comprehensive understanding about how polymer products and polymers interact
with biological networks. New advancement in this modern era have investigated on
smart chemical roots to design advanced controlled release systems, but most of the
time incomplete or no biocompatibility investigations are carried out in designing
formulations. It resulted in the failure of a large number of formulations at the end of
their design. In vivo investigations in the beginning of device designing will aid to
make sure that polymer-related outcomes and laboratory trials resulted in fair and
refined platforms for advanced drug delivery systems [65].
It is suitable to change the required temperature to a particular temperature range of
phase transition for volume during drug delivery. This is achieved through the
incorporation of hydrophilic (water loving) or hydrophobic (water hating) fractions in
chain of polymer. A polymer with enlarged hydration area (hydrophobic) occupies
greater interactive forces and bears collision at low temperature [49, 66]. Contrarily,
LCST increased with increasing the content (hydrophilic) of polymer chain.
Polymers which show transition in globule-to-coil after increasing temperature keep
16
an upper-critical-solution-temperature (UCST). Substantially, cross-linked gels show
phase transition from sol to gel about its LCST [56]. These substances are suitable
components for in-situ implants that facilitate the implantation of solid drug depot
preparations by exploiting thermo-reversible gelation [65]. In such systems, a liquid
solution of drug and polymer is injected at optimal temperature to the targeted site.
Drug entraps in polymers gel matrix is diffused and released at sustained pattern as
temperature increased to body temperature. This technique was employed in a study
demonstrating release profile of bovine serum albumin (model protein) from chitosan
grafted with PEG (PEG-g-chitosan) [67]. Novel polymerization method, for example
click chemistry reactions and controlled radical polymerizations present better control
over substance network and offer the open door to make novel materials exceedingly
custom fitted for particular responsive pattern.
2.3.3.2.5. Responsive systems based on pH
pH (Physiological) changes systematically along GIT, where enzymatic conditions
and brutal pH in stomach (pH 2) metabolize the macromolecules. Small intestine is
generally extra alkaline (pH 6.2-7.5). Physiological pH will likewise vary amid
cellular compartments. Such as, endosomes and lysosomes specifically show pH of
5.0-6.8 and 4.5-5.5, respectively [68, 69].
Besides, it is notable that ailing or aroused tissues show diverse pH profiles than
typical tissue [70]. Tumors are broadly reported to give acidic conditions (pH 6.5) for
extracellular milieu [71]. Subsequently, it is nothing unexpected that engineers and
researchers have committed impressive exertion toward the polymers rational design
fit for exhibiting these pH varieties to specifically convey important therapeutics to
particular intracellular or extracellular targeted destinations of activity. By prudent
17
materials choice and cautious atomic design building, pH responsive polymer
conveyance systems are produced to present controlled drug release and pH.
Appropriate characteristics, for example biocompatibility, swelling ratio, surface
charge and critical swelling pH were customized using tuning polymer crosslinking
density, composition, and addition of hydrophobic fractions across hydrogel core.
2.3.3.2.6. Responsive systems based on redox potential
Polymers including labile linkages display an appealing chance to fabricate
bioerodible or biodegradable delivery appliances. A significant part of initial study in
this area concentrated on acid labile linkages of poly (lactic/glycolic acid) [72],
polyanhydrides [73, 74], and further lately poly (β-amino esters) [75, 76]. Although,
intracellular signals are presently being researched as a way to trigger cytoplasmic
metabolism of polymer carriers adding advanced therapeutics, for example, anticancer
medications. Disulfide linkages are notable to be precarious for reductive
environment because disulfide bond is promptly divided for relating thiol bunches.
2.3.3.2.7. Supremacy of polymer therapeutics for drug delivery
Polymer therapeutics is used to portray an undeniably critical area of bio-
pharmaceutics whither a branched or straight polymer chain carries on either as the
bioactive polymeric drug or, all the more ordinarily, as the inactive carrier to which a
restorative is covalently connected, as on account of multicomponent polyplexes,
polymer-medication conjugates, polymeric micelles and polymer protein conjugates
[77]
Therapeutic conjugation to the polymer enhances the pharmacodynamic and
pharmacokinetic characteristics of bio-pharmaceuticals through an assortment of
measures, comprising diminishment in immmunogenicity, therapeutic molecule safety
from proteolytic catalysts, expanded plasma half-life (which enhances patient
18
compliance in light of the fact that less incessant dosages are required), improved
steadiness of proteins, increased solubility of little molecular weight drugs and
targeted delivery potential [78, 79].
For conjugation of drug and polymer to be both effective and practical various
characteristics are required: (a) non-immunogenic and non-toxic polymer carrier, (c)
sufficient carrying and loading capability with regard to drug potency (b) high
molecular weight assure prolonged circulation times (e) active and passive means for
target desired tissue and (d) stable linkage for transport that easily broke after arrival
at target for optimum delivery [78]. The conventional strategy to amalgamating
conjugation of protein polymer entails the modification in post-polymerization for
polymeric carrier. There are three common prerequisites for useful conjugated
protein-polymer system: a polymer bearing single reactive group on one terminal end
for preventing cross-linking of protein, a non immunogenic and non toxic linker
comprising of intermediate by-products, and a technique that will produce
conjugation at specific site [77]. Besides, various novel techniques are evaluated to
evade protein-polymer coupling reactions and post-polymerization modifications.
There is a powerful impetus currently present for these techniques that permit the
fabrication directly from protein-reactive initiators of polymer, indebted the approach
of controlled polymerization techniques, for example ATRP (atom transfer radical
polymerization) and RAFT (reversible addition-fragmentation chain transfer) because
these are less time rigorous, straightforward and about assuring each polymer chain
comprises one reactive end group [80-82].
2.4. Hydrogels
Hydrogels are three dimensional polymeric materials which have tendency to absorb
an enormous amount of water. Hydrogels have been widely employed in cell
19
therapies, tissue regeneration, drug delivery carriers and therapies of wound.
Hydrogels prepared through safe crosslinkers have fascinating qualities of
biocompatibility, cytocompatiblity and facile cell encapsulation. In this modern era,
new emerging techniques are now being utilized to get nanocomposite, triblock
copolymer, macromolecule microsphere composite, biodegradable, injectable,
superporous and double network hydrogels [83-86]. Major parts of internal structure
of hydrogel are given in the Figure 2.1.
Hydrogels are a class of materials which can be designed through physical and
chemical crosslinking methods. Improved mechanical properties of hydrogels can be
tuned through chemical crosslinking methods. Crosslinking through enzymes, high
energy radiation and radical polymerization fall under the category of chemical
crosslinking. Radical polymerization boosted the utilization of hydrophilic polymers
to develop hydrogels [87, 88]. Broadly speaking stimuli responsive hydrogels are
classified as light, electrical signal, glucose, temperature, pH and other stimuli
sensitive hydrogels. Among which pH sensitive hydrogels have fascinating features of
swelling in response to change in pH caused by polyelectrolytes that make them a
strong candidate for oral controlled release formulations. They have a number of
imperative applications in developing permeation switches, biosensors and drug
carrier systems.
Figure 2.1. Internal structural parts of a hydrogel.
20
2.4.1. Classifications of hydrogels
Hydrogels can be classified on the basis of their physical characteristics, kind of
swelling, method of formulation, source, ionic charges, degree of bio-degradation and
type of crosslinking [89]. It is clearly manifested from the Figure 2.2 that
classification covers almost each type, however few of the smart hydrogels, which
potentially drew attention of the researchers are described.
Physical gels are distinguished on the basis of type of crosslinking which is physical.
These gels are fabricated through different physical processes like hydrogen bonding,
polymer chain complexion, chain aggregation, crystallization and hydrophobic
association. However, chemically crosslinked hydrogels are designed through
chemical processes i.e. covalent crosslinking. Physical hydrogels can be reversible
owing to the conformational modifications while chemical hydrogels remain
irreversible. One more important class is the dual-network hydrogel, fabricated as a
result of electrostatic interaction on combining physical and chemical crosslinked
smart hydrogels.
It has been implied to minimize the drawbacks of individually utilizing chemical or
physical hydrogels with great liquid uptake capability for a broad range of pH and an
increased sensitivity on modulating the pH as matched with chemical hydrogels. A
new smart dual-network comprising grapheme polymer hydrogel exhibited wonderful
mechanical characteristics and a better self-healing capability was recently prepared
[90, 91].
2.4.1.1. Types of hydrogels on the basis of crosslinking
Crosslinks present in hydrogels play a key role in maintaining its elastic integrity
which may be pure elastic or visco-elastic.
21
2.4.1.1.1. Physically and chemically crosslinked hydrogels
Scientific researches regarding crosslinking have categorized it into two major
divisions which are physical and chemical crosslinking. Current scenario is different
from the old one as it mainly focuses on the preparation of physically crosslinked
formulations due to the toxicity of most of the crosslinkers which are present in
chemically crosslinked gels. Physically crosslinked formulations do not contain
crosslinkers in their composition and the absence of crosslinkers has no impact on
their integrity [92].
Figure 2.2. Major divisions of hydrogel on the basis of different
characteristics.
22
Physically crosslinked networks can be prepared through different methods.
2.4.1.1.2. Crosslinking through radical polymerization
Swelling property of hydrogels can be tuned through the modulation of crosslinker
concentration. Furthermore, stimuli responsive networks are designed with the
addition of a crosslinker. In addition to the radical polymerization of the combination
of vinylmonomers, chemically crosslinked gels can also be formulated through radical
polymerization of hydrophilic polymers obtained from polymerizable entities. A
variety of hydrophilic natural, synthetic and semi-synthetic polymers have been
applied for the fabrication of gels.
2.4.1.1.3. Crosslinking via chemical reactions
Hydrophilic polymers employed in the fabrication of hydrogels, manifest their
solubility characteristics due to the functional groups (-OH, -NH2, -COOH) present in
their structure. Covalent links can be created in polymeric structures by combining
complementary reactivity with functional groups. Moreover various techniques have
been employed in chemical crosslinked hydrogels such as crosslinking through
enzymes, high energy irradiation condensation and addition reactions.
2.4.1.1.4. Crosslinking via ionic reactions
Crosslinking of some polymeric materials like alginate can be done at physiological
pH and room temperature that is why alginate is mostly implied in matrix to release
proteins [93] and to encapsulate cells [94]. Most of the ionotropic gels deteriorate on
exposure to physiological environment.
2.4.1.1.5. Crosslinking by crystallization
Mechanically weak hydrogels can be formulated on preparing aqueous solutions of
hydrophilic polymers at room temperature. Their mechanical strength can be
enhanced through freeze-thaw cycles [95]. Gels prepared through this technique
23
exhibited different characteristics which mainly depend on the number of freeze-thaw
cycles, amounts of water and polymer. Prepared gels manifested a better stability at
38 °C for more than 5 months [17].
Physically crosslinked networks are usually prepared with grafting or multi-block
polymers. Different other techniques implied in crosslinking are irradiation,
suspension polymerization, hydrogen bonding and protein crosslinking [90, 96-98].
Hydrogels prepared through crosslinking agent may have some issues regarding
toxicity. Fascinating features of hydrogels attract scientists to explore new ideas
regarding their ability to deploy in contact lenses, controlled release formulations,
nutrient carrier for soil and cosmetics.
One of the hydrogel divisions is based on the type of side group attached to the
structure. Such types of hydrogels are cationic, anionic and neutral, whereas the
mechanism behind swelling in neutral hydrogels is the polymer- water
thermodynamic combination [17].
2.4.1.2. Interpenetrating polymer network gels
Hydrogels can be categorized into following types on the basis of formulation.
Interpenetrating
Semi-interpenetrating
Copolymeric
Homo-polymeric
2.4.1.2.1. Interpenetrating and semi-interpenetrating networks
Interpenetrating polymer networks (IPNs) are described as the combination of two
crosslinked polymers. In semi IPN structure one of the polymers is linear and the
other one is crosslinked. IPNs can be designed through physical and chemical
crosslinking methods which are followed by either spontaneous or sequential
24
pathway. These IPN structures exhibited wonderful physico-chemical features as
compared to its constituents. Vast implications of IPN have been observed in tissue
engineering and biomedical era owing to their fascinating mechanical and biophysical
features [19, 20]
IPN hydrogels are being extensively employed in sustained release formulations. IPN
hydrogels have complex skeleton with better features. IPN hydrogels can be
categorized into nonionic and ionic IPN hydrogels on the basis of the nature of
polymers. Non-ionic IPN hydrogels contain non ionic polymers whereas ionic IPN
hydrogel contain mostly anionic, cationic and cationic/anionic. These ionic IPN
hydrogels may contain both cationic and anionic groups connected to various chains
in the structure are bound mainly through covalent and ionic bonds, which in turn
improve them mechanically and enhance their sensitivity to external stimuli such as
pH, ionic strength and temperature. IPNs physical and mechanical features can be
tuned by modulating the concentrations of polymers and crosslinking agent. A new
and emerging type of ionic IPN hydrogel having polyion complexes is fabricated by
keeping a controlled proportion of cationic and anionic groups in the formulation.
These polyelectrolyte gels contain ionizable moieties. These moieties will regulate the
electrochemical balance between the hydrogel and its external environment. Swelling
of these gels depends on the pH of external medium. Gels with acidic groups swell at
higher pH. These salient features make them strong candidate for controlled release
system [21, 22].
25
2.4.1.2.2. Homo-polymeric hydrogel
Polymer networks obtained from the same species of monomers are called as
homopolymeric hydrogels which is the simplest structural entity of polymer network
[99]. Homopolymeric gels are crosslinked structures containing similar monomer
hydrophilic units, while copolymeric gels are designed through crosslinking of two
co-monomer units and their swelling mostly depends on the hydrophilic monomer.
Major factors influencing the homopolymer network shapes are the type of monomer
and polymerization method implied. Polyethyleneglycol (PEG) gels have stimuli
responsive networks which respond to external stimuli and widely applied in
controlled delivery systems. Some of the other applications of PEG hydrogels are
seen in tissue engineering, pharmaceutical industries and scaffold for the protein
recombination technology [100].
2.4.1.2.3. Co-polymeric hydrogel
Co-polymeric networks are formulated by combining two kinds of monomer, one of
which may be hydrophilic in nature. Researchers fabricated different biodegradable
triblock hydrogels for the development of controlled release systems. During the
triblock preparation, different polymers, initiators and catalysts were employed. Such
co polymeric blocks have ability to design hydrogels for in-situ purposes [101].
2.4.1.3. Stimuli responsive hydrogels
Such type of hydrogels which quickly react on exposure to external environmental
and exhibited unusual modifications in mechanical strength, permeability growth
actions and network structure are stimuli responsive networks [51, 102]. These can be
further categorized mainly in three types which are physical, chemical and
biochemical responsive hydrogels. External stimuli which play a key role in their
modifications are intensity of different energy sources, pressure, mechanical stress,
26
temperature, electric field, magnetic field, and light, which alters the molecular
interactions on crucial onset areas. Some other stimuli are chemical agents, ionic
strength and pH.
Dual responsive structures are designed by joining two stimuli responsive networks in
a single hydrogel entity. One of the best examples is the combination of Polyacrylic
acid-co-polyvinyl sulfonic acid [103]. A number of implications of stimuli responsive
gels are seen in biotechnological, biomedical and pharmaceutical arena [104].
2.4.1.4. pH responsive hydrogels
Polymeric networks containing ionic groups which can donate or accept protons on
exposure to different pH media are named as pH responsive hydrogels. pH responsive
Structures exhibited a dramatic change in their degree of ionization at some critical
pH. This rapid modification in the whole charge of ionized entity produces a change
in volume of the structure due to the presence of electrostatic repulsion in the ionized
entities that produces a strong osmotic swelling capacity. pH responsive hydrogels
are divided into two types
Anionic hydrogels
Cationic hydrogels
Most of the anionic hydrogels contain active pendant groups like sulfonic or
carboxylic acids, which are responsible for their swelling in different pH media [36–
39], whereas cationic hydrogels have amine groups that are responsible for their
swelling in different pH media owing to the greater electrostatic repulsive forces [5,
105].
27
2.4.1.4.1. Salient features of pH responsive hydrogel
Swelling of ionic structures mainly depends on the two major factors. First major
factor is the features regarding polymers which are given as
pKa or pKb of the ionizable groups
Degree of ionization
Ionic charge,
Hydrophilicity or hydrophobicity
Concentration
Following Figure 2.3 clearly indicated the ionization of polydiethyl-aminoethyl-
methacrylate (PDEAEMA) as well as their copolymer at basic pH [5, 106-108].
Figure 2.3. pH dependent ionization of polyelectrolyte.
Second factor includes the features regarding swelling medium like
Counter-ion
28
Ionic strength
pH
Most of the hydrogel preparations exhibits swelling at alkaline pH and release drugs
in colon whereas some hydrogels are specific to release the drug at acidic pH. Many
scientists explored new horizons in the fabrication of pH responsive networks.
Swelling features of acrylic polymers were found due to the presence of carboxylic
groups in the structures.
pH responsive superabsorbent hydrogel were investigated by the scientists and their
drug release study was performed in different pH media. Drug release was found
maximum at basic pH when compared to acidic pH. Non-biodegradable behaviour is
not an issue in some applications, like oral drug delivery, but it becomes a necessary
limitation in some other applications such as in case of implantable biosensor. So the
efforts have been made to design and fabricate biodegradable pH responsive
hydrogels [109].
2.5. Hypertension and its remedy
Hypertension i.e. high blood pressure is the most common and distinct issue,
influencing approximately 20 % of the young generation throughout the world. High
blood pressure is an alarming sign regarding cerebro and cardiovascular
complications, which records almost 15 million causalities per annum. Cardiac
disease is the major reason for human casualties worldwide. Human deaths are
increasing day by day due to hypertension and this rise in deaths may lead to an
alarming figure of 23.3 million by 2030 and current scenario also depicts an increase
in or keeps static cardiac diseases and strokes [110].
In most of the developing countries a rise in disability and mortality rate is seen [111].
Patients are said to be hypertensive whenever their diastolic blood pressure ≥ 90mm
29
Hg and/or with systolic blood pressure ≥ 140 mm Hg. To treat hypertensive patients
few important risk factors must be kept in mind such as previous disease record
(history), severity of hypertension and presence of any other risk factor. After
investigating these factors, few modifications in life style of hypertensive patients are
required which are proper diet, exercise and to avoid from hectic routine work, while
proper treatment should be started in patients with high or very high risk category.
Most of the drugs employed in the treatment are angiotensin converting enzyme
(ACE) inhibitors angiotensin II antagonists, β-blockers and calcium channel blockers
etc [112].
2.5.1. Perindopril erbumine
2.5.1.1. Overview
Perindopril erbumine is an antihypertensive drug which presides in angiotensin
converting enzyme inhibitor (ACE) class. It belongs to pro-drug an ester of the
perindoprilat with a daily dose of 4 to 8 mg. To overcome this problem of
hypertension, controlled release formulations of perindopril erbumine were prepared.
Perindopril falls in the category of angiotensin converting enzyme inhibitor (ACE)
concerned to enalapril and acts by blocking the conversion of angiotensin I to
angiotensin II. It is employed to cure congestive heart failure, acute myocardial
infarction and in different hypertension conditions by decreasing systolic and diastolic
pressure. Poor bioavailability and potent action of perindopril erbumine makes it a
strong candidate for the controlled release medication [113].
30
2.5.1.2. Pharmacodynamics
On oral prescribing perindopril lowers the plasma ACE activity; the amount of
perindoprilat implied to lower 50 % of the ACE activity (IC50) is approximately 3.60
nmol/L. ACE activity diminishes within 3 to 4 hours after administering perindopril 2,
4 or 8 mg and plasma ACE activity inhibited > 70 % one day after giving a dose of
perindopril 4, 8 or 16 mg. Ethnicity or age did not have any impact on ACE
inhibition, however it depicted a distinct rise on single dose with subsequent doses in
patients with renal complications. Structural formula of perindopril erbumine is given
in the Figure 2.4.
For hypertensive patients, perindopril is considered effective in maintaining normal
blood pressure. Complications regarding hypertension like haemodynamic and arterial
complications are treated through perindopril; carotid-femoral aortic pulse wave
velocity (PWV) gave a downfall from baseline (–1.1 m/sec with p < 0.001) in
noncomparative half year study (where n = 1703). Downfalls in aortic PWV within
antihypertensive treatment (71 % patients need proper medication, 46 % of them need
perindopril 4 to 8 mg for 48 hours alone or combined with other antihypertensive)
were linked with a clear downfall in both cardiovascular and mortality rate during
cohort study with 150 patients of renal complications. Survival chances of the patients
increased with the use of perindopril instead nitrendipine or atenolol. Perindopril
produces a distinct rise in the compliance, blood flow and arterial diameter whereas a
clear fall in the values of systemic vascular resistance. In hypertensive patients, blood
pressure brought to the normal values by administering perindoprilat intravenously
which also produced dilation effect in coronary arteries. Perindopril had no distinct
impact on cerebral blood flow in hypertensive patients whereas a distinct impact on
reduction of systolic and diastolic blood pressure was observed.
31
N
NH
O
OH
O
CH3
H
H
O
CH3
CH3
O CH3
CH3
H3C NH2.
Figure 2.4. Structural formula of perindopril erbumine.
In many clinical trials a distinct impact on reduction of left ventricular mass index
(LVMI) was also observed. LVMI fall below the baseline by 13.55 g/m2 (p < 0.001),
when treatment was ongoing with single dose of perindopril 4 to 8 mg for
approximately 3 months in stable hypertensive patients and moderate LV
hypertrophy. Perindopril also depicted a better impact on echocardiographic
parameters as well as a fall in intraventricular septal and thickness diastolic wall but
produced a rise in relaxation of left ventricle. Perindopril had an advantage that it had
less or no severe impact on lipid profiles or glycaemic control.
2.5.1.3. Pharmacokinetic Profile
Orally given perindopril extensively metabolized into perindoprilat after hydrolysis.
Its bioavailability limits in between 65 and 95 % whereas it remained 16.8 % in
plasma in the form of perindoprilat. Plasma protein binding of this drug is
approximately 74 % at a steady-state concentration and perindoprilat gives 15 %
binding in plasma protein. Perindopril quickly absorbed and depicted peak plasma
32
concentration (Cmax) 64.2 μg/L reached in 0.7 to 0.9 hours with single dose of 4 mg.
Perindoprilat gave Cmax 4.7 μg/L in 3.5 hours with post-dose. Perindopril and
perindoprilat depicted linear pharmacokinetics with therapeutic dosing whereas
volume of distribution of perindopril is 0.22 L/kg and distributes into tissues with
high ACE activity. On passing through liver, perindopril is metabolized into two
major products perindoprilat and perindoprilat glucuronide. After 8 hours plasma
concentration of the drug almost diminished whereas elimination half-life of
perindoprilat glucuronide, perindoprilat and perindopril were found to be 1.7, 10.9
and 2.9 hours, respectively.
The data obtained from healthy volunteers and hypertensive patients gave a similar
trend regarding pharmacokinetics of the drug. In elderly people a rise in perindoprilat
bioavailability was seen. Pharmacokinetic parameters study in hepatic impaired
patients gave no distinct impact whereas these parameters like area under the plasma
concentration-time curve, half-life and Cmax were distinctly rise in renally impaired
patients.
2.5.1.4. Therapeutic Efficacy
Systolic and diastolic blood pressure can be maintained to the normal values by using
perindopril with a single dose of ≤ 8 mg. In comparative study with other drugs like
fosinopril, enalapril, lisinopril and ramipril a similar pattern in lowering the blood
pressure was observed whereas a distinct reduction in diastolic pressure was seen as
compared metoprolol. Perindopril exhibited a better impact on response rate as
compared to atenolol.
Mostly perindopril is implied as combination with other drugs like indapamide or
hydrochlorothiazide to get better responses regarding blood pressure. In most of the
33
cases when perindopril monotherapy failed to reduce the blood pressure, then the
combined therapy of perindopril with nifedipine and hydrochlorothiazide are implied.
Perindopril is considered to be a better drug at doses ≥ 4 mg as compared to other
antihypertensive drugs though a limited data is present yet it is recommended by the
Food and Drug Administration authority to administer daily dose.
2.5.1.5. Tolerability
Tolerance profile of perindopril goes side by side with the other members of ACE
inhibitors. Most probably the adverse effects observed through the treatment with
daily dose of 2 to 8 mg in the period of almost one year were asthenia, GI
upset/dyspepsia and cough in a post marketing surveillance investigation with about
fifty thousand hypertensive patients.
2.5.1.6. Dosage and Administration
Initially recommended dose of 2 mg is given to hypertensive patients which can be
increased upto 8 mg depending upon the severity of disease provided in the European
guidelines. Renally impaired patients require special attention because perindoprilat is
excreted renally whereas hepatic impaired patients don’t require special monitoring. It
is contraindicated in patients with angioneurotic edema and also in pregnancy.
Combination therapy with potassium-sparing diuretics and potassium supplements is
not suitable [114].
2.5.2. Metoprolol
Beta-blockers are mostly employed to cure cardiovascular diseases like arrhythmias
and hypertension. Beta-blockers are usually better tolerated though have been
attached with some side effects such as psychosis and delirium. Other major factors
34
which enhance a greater risk for side effects are impaired hepatic function and old age
[115, 116].
Metoprolol falls under the category of β1-blocker employed in the treatment of angina
pectoris, hypertension and myocardial infarction. Metoprolol has poor membrane
stabilizing performance with no intrinsic sympathomimetic activity.
2.5.2.1. Pharmacodynamic features
Metoprolol treatment in hypertensive patients may cause a fall in haemodynamic
activity. Drug therapy for almost three months of metoprolol with a dose of 100 to
150 mg/day may cause a controlled systolic function and heart rate which is validated
by increased stroke work and volume index. Metoprolol treatment improved
myocardial efficiency and is also helpful in maintaining improved systolic and
diastolic activities. Left ventricular geometry is improved and mass is reduced on
therapy for one and half year.
2.5.2.2. Pharmacokinetic features
Metoprolol tartrate is completely absorbed when taken orally and gives 50%
bioavailability due to first pass effect. Volume of distribution of drug is found to be
5.6 L/kg. It is mainly metabolized by cytochrome P4502D6 with an elimination half-
life of almost 3 to 4 hours. Major metabolites of metoprolol are O-
demethylmetoprolol and α-hydroxymetoprolol which account for the 5%
pharmacological activity of the metoprolol. Pharmacokinetic parameters are not
affected by the kidney malfunction and age; whereas liver cirrhosis may cause a fall
in total body clearance and enhanced elimination half-life and bioavailability. Patients
with hyperthyroidism suffer a fall in area under the plasma concentration-time curve
35
and peak plasma amount. Metoprolol tartrate salt is more soluble as compared to
succinate salt. Structural formula of metoprolol tartrate is given in the Figure 2.5.
H3CO
O
OH
NH CH3
CH3
.HO
O
HOH
OH
O
HO H
Figure 2.5. Structural formula of metoprolol tartrate.
2.5.2.3. Therapeutic Use
Metoprolol is widely implied in a number of complications like congestive heart
failure ventricular tachycardia, myocardial infarction, hypertension (by lowering high
blood pressure different complications such as heart attack, renal failure and strokes
can be prevented), acute supraventricular tachycardia, treatment of heart failure,
vasovagal syncope, adjunct in hyperthyroidism treatment. It can also be used to treat
long QT syndrome, mostly for asthmatic patients owing to the β1 selectivity of
metoprolol [117, 118]. β1 selectivity of metoprolol makes it a strong candidate
for off-label prescription use in different complications like social anxiety disorder,
performance anxiety and for other anxiety disorders.
36
2.5.2.4. Dosage and administration
Metoprolol initial dose initiated was in range of 6.25 to 12.5 mg/day (divided doses)
for immediate release. It was then increased up to 100 to 200 mg dose in BID.
Metoprolol CR/XL was initiated at a dosage of 12.5 or 25 mg once daily and
increased at 2-weekly intervals up to a target dosage of 200mg once daily. Metoprolol
CR/XL initial dose was 12.5 or 25 mg once daily (OD). It was then increased up to
200 mg OD for two weeks. The metoprolol dose is reduced in hepatic impairment
patients. Drug was utilized cautiously in diabetes mellitus patients, bronchospastic
disease, thyrotoxicosis, and in anaesthesia surgery. The metoprolol usage is found to
be contraindicated in severe bradycardia patients, decompensated cardiac failure,
cardiogenic shock, first degree greater heart block, and in right ventricular failure
secondary to pulmonary hypertension. Conventional preparations are commonly
administered twice in day (BID). Drug has capability to diminish its selectivity at
greater plasma concentrations. Metoprolol is marketed for the first time in Europe
during mid-1970. Afterwards, it is utilized vastly for treatment of angina and
hypertension [119].
37
3. MATERIALS & METHODS
3.1. Materials
For the preparation of covalently crosslinked pH sensitive hydrogels, methacrylic acid
(MA) (Merck, Germany) was used as monomer. Poly vinyl alcohol (PVA) (Fluka,
Buchs, Swizerland), carrageenan (CA) (Fluka, Buchs, Switzerland) and glyoxal
(Merck, Germany) were used as polymers and crosslinking agent, respectively.
Potassium per sulfate (Fluka, Buchs, Swizerland) was used as an initiator. Deionized
water and distilled water were used as solvents. Potassium dihydrogen phosphate,
sodium chloride, sodium hydroxide, and hydrochloric acid (Merck, Germany) were
used.
3.2. Synthesis of covalently crosslinked pH sensitive hydrogels
Smart polymeric systems of hydrogels were designed by the slight modification of the
previous method [120, 121]. First of all the aqueous polymer solution was prepared
by mixing polymer with water while the other solution was prepared by dissolving
potassium per sulfate in MA. Now both solutions were combined and tuned
concentrations of glyoxal added. After that the weight of the each formulation
prepared was made equivalent to 100 g with the help of ethanol and mix it until clear
solution obtained. After mixing thoroughly the final solution was poured into glass
tubes (Pyrex). Nitrogen bubbling was implied for about 7-8 minutes to remove
oxygen present in the test tubes. Then these tubes were further moved for heating
process. Heating procedure was completed by placing test tubes in water bath and
gradually heated from 40 0C up to 70
0C. Test tubes were allowed to cool and then cut
into same sizes. After washing the disks of same sizes were dried at 44 0C to constant
weight and kept in air tight jars for further studies. PVA/MA hydrogels were prepared
by using PVA as hydrophilic polymer, while CA/MA hydrogels were prepared by
38
incorporating CA as hydrophilic polymer. Formulation compendia of PVA/MA and
CA/MA hydrogels are given in Tables 3.1 and 3.2, respectively. The presumptive
structures of hydrogels are described in Figures 3.1 and 3.2.
Table 3.1. Formulation compendia of polyvinyl alcohol/methacrylic acid
hydrogel.
Sample code PVA/100g
solution
MA/100g
solution
Glyoxal/100g
solution
J1 0.30 59.70 0.44
J2 0.60 59.70 0.44
J3 1.20 59.70 0.44
J4 1.20 38.00 0.28
J5 1.20 46.00 0.34
J6 1.20 54.00 0.40
J7 1.20 60.00 0.18
J8 1.20 60.00 0.30
J9 1.20 60.00 0.60
39
Table 3.2. Feed module of CA-g-MA hydrogel formulations.
Sample code CA/100g
solution
MA/100g
solution
Glyoxal/100g
solution
JA1 1.00 50.00 0.05
JA2 1.00 50.00 0.15
JA3 1.00 50.00 0.50
JA4 1.00 31.66 0.15
JA5 1.00 38.33 0.19
JA6 1.00 45.00 0.22
JA7 0.25 49.75 0.24
JA8 0.50 49.75 0.24
JA9 1.00 49.75 0.24
40
O
O
O
OH
OH
O O O
O
OH
O O
n
n
n
Poly vinyl alcohol Glyoxal Methacrylic acid
++
Figure 3.1. Presumptive structure of PVA/MA hydrogel.
41
Figure 3.2. Proposed structure of CA-g-MA hydrogel.
42
3.3. Characterization of synthesized hydrogels
3.3.1. Swelling investigations
3.3.1.1. Swelling parameters studies
Swelling investigations were carried out in different buffer solutions from acidic to
alkaline pH to simulate the pH of GIT of human body. Swelling study of washed
dried hydrogel was conducted at room temperature in different buffer solutions (pH
(1.2-7.5)). Gel disks begin to swell in different pH media. The swelled disks were
taken out from the solution media after specific time duration, and kept in the same
bath media after weighing. The swelling parameters like dynamic and equilibrium
swellings were calculated by dividing the swollen weight of each disk with its initial
dried weight. [122]:
3.3.2. Water diffusion coefficient of the formulations
Drug release from the formulations usually takes place through diffusion process.
Water diffusion coefficient of the prepared optimized formulations was given by the
equation [123]:
D ( t.
4Sgeq ) (1)
Here Dw represents diffusion coefficient of prepared formulations, t is the initial
thickness of the gel before swelling, gives the value of slope of linear part of
swelling curves and Sgeq gives the swelling of gel at equilibrium.
43
3.3.3. Networking parameters of smart hydrogels
3.3.3.1. Molecular weight between crosslinks (Mb)
Molecular weight between crosslinks was investigated by impling Flory-Rehner
theory. This theory states that Mb has a direct relation with swelling ratio of hydrogels
which can be better investigated by the following equation [124]:
Mb - dhVs(Vf,s
- Vf,s/2)
ln (1- Vf,s ) Vf,s V f,s (2)
Volume fraction of the polymer Vf,s is calculated by the following equation [125]:
V f,s *1 d
ds M
M -1 )+
-1
(3)
where p is the Flory-Huggins polymer solvent interaction parameters. Ms and Md are
the masses (g) of the swollen and dry hydrogels, respectively. Vf,s (mL/mol) is
volume fraction of the swollen hydrogel in the equilibrium state and dh and ds are
densities (g/mL) of the hydrogel and solvent, respectively.
3.3.3.2. Solvent interaction parameters (χp)
Flory-Huggins theory was used to determine ( p) from the following equation [123]:
ln (1- Vf,s ) Vf,s
V f,s (4)
44
3.3.3.3. Crosslinkeddensity (qh)
Crosslinked density was measured by the following formula [121]:
q
Mb
M (5)
where Mu is the molar mass of the repeating unit and is given as:
Mu mpolymerMpolymer mmonomerMmonomer mcrosslinkerMcrosslinker
mpolymer mmonomer mcrosslinker(6)
where mpolymer, mmonomer and mcrosslinker are the masses of polymer, monomer and
crosslinker respectively, while Mpolymer, Mmonomer and Mcrosslinker are the molar masses
of polymer, monomer and crosslinker respectively.
3.3.4. Sol-gel and porosity studies
3.3.4.1. Sol gel calculation
Uncrosslinked polymers present in the prepared structures can be determined through
sol-gel calculation. Sol fraction was determined by dividing the difference in the
weights of initial and extracted gel disks with initial weight whereas gel fraction can
be obtained by subtracting the sol fraction value from100 [126]:
3.3.4.2. Porosity
Porosity of the prepared structures can be determined by dipping the prepared disks in
pure ethanol solution overnight and then the weight of the dipped disks were
measured and is given by the following method [127]:
45
[ –
] (7)
where pe is the density of pure ethanol and Vg is the volume of gel. W1 and W2 are
weights of hydrogel before and after immersion in absolute ethanol, respectively.
3.3.5. Structural spectroscopy
Prepared dried formulations were grounded to fine powdered and combined with KBr
(potassium bromide) (Merck IR spectroscopy level) in the ratio of 1:100 and then
dried at 42 0C. The mixture was compressed for the specified time. FTIR
Spectrometer (FT-IR 8400 S, Shimadzu) was utilized to record spectrum over the
wavelength range 4000-500 cm-1
[128, 129].
3.3.6. Thermal studies
3.3.6.1. Differential scanning calorimetry
Thermal stability of PVA, CA, MA, CA-g-MA and PVA/MA hydrogels were
determined by DSC with DSC822 Mettler Toledo (Switzerland). Nitrogen flow rate
was kept at 70 mL/min whereas 4 mg samples were placed on aluminum pans with
heating at 10 °C min
-1 from 0
°C to 600
°C for PVA/MA hydrogel whereas 0
°C to 500
°C for CA-g-MA hydrogel [130-133].
3.3.6.2. Thermogravimetric analysis
Thermal analysis of samples was performed on a SDTA 85 Mettler Toledo
(Switzerland) TGA system at the rate of 10 0C min
−1 and a temperature range of 20–
600 0C for 60 min for PVA/MA hydrogel and a temperature range of 20–800
0C
[131-133].
46
3.3.7. X-ray diffraction
X-ray diffraction (XRD) patterns were recorded, in reflection, with a Bruker D8
Discover (Germany) instrument, at 25 0C temperature, using the nickel filtered Cu Kα
radiation (λ 1.54Å) and operating at 40 kV and 35 mA, step scan 0.1° of 2 and 1s of
counting time. The range of diffraction angle was 10° -70° 2 [134, 135].
3.3.8. Scanning electron microscopy
The surface morphology of PVA/MA and CA-g-MA hydrogels was determined
through a scanning electron microscope (Hitachi, S 3000H, Japan) [136].
3.4. In vitro studies
3.4.1. Loading of drug
Formulations exhibiting maximum swelling were selected for drug loading and
release studies.. The weighed dried gel samples of CA-g-MA were placed in 1 %
(w/v) drug, whereas PVA/MA hydrogel disks were placed in 5 % (w/v) drug solution
upto equilibrium swelling. Drug solutions were prepared by dissolving hydrophilic
drugs in deionized water and the quantity of ethanol to deionized water was added to
get drug solution of 50 % (v/v) ethanol/deionized water. After equilibrium swelling of
drug loaded hydrogels were removed from drug solution, first dried at room
temperature and then in oven at 45 0C to constant weight [137].
3.4.2. Determination of drug loaded in disks
Concentration of drug loaded in a disk was determined through two methods. One of
the methods through which the amount of drug loaded in hydrogels can be calculated
by difference in the weights of after and before dipping in drug solution, while the
other method involves the determination of percent drug loading calculated by
extracting the drug loaded discs in alkaline buffer solution with pH 7.5 till the loaded
drug completely wear out and the amount of extracted drug was measured through
47
spectrophotometer. Amount of extracted metoprolol tartarate was measured at
λmax274 nm, whereas perindopril erbumine was evaluated at λmax215 nm. Total
amount of drug present in all portions was considered as the amount of drug
entrapped or loaded [138, 139].
3.4.3. Drug release studies
For the measurement of drug release, the dissolution apparatus (Phamatest; type PT-
DT 7, Germany) and UV-Visible spectrophotometer (IRMECO, UV-Vis. U2020)
were used. The weighed disks were placed in dissolution medium with 0.05 M USP
phosphate buffer solutions of tuned pH (1.2, 6.5 and 7.5) for PVA/MA hydrogel,
whereas CA-g-MA hydrogel disks were placed in buffer solutions of pH (2, 4.7 and
7.4) and stirred to keep a uniform drug concentration in the medium. Drugs assay
during release studies were conducted at λmax215 nm and λmax 274nm for perindopril
erbumine and metoprolol tartarate, respectively up to 72 hrs [140-142].
3.4.4. Analysis of drug release pattern
Various models were utilized to clarify the release patterns of drug. Zero-order [143],
First-order [144], Higuchi [145] and Korsmeyer-Peppas [146] models were used. To
interpret the solute release mechanism, the release pattern was sketched through
Peppas equation with the help of semi-empirical power.
3.5. In Vivo study
3.5.1. Selection of Animals
In vivo study was conducted on 10-12 weeks old healthy male rabbits of weighing
approximately 1-1.25 kg. Rabbits were placed in well ventilated environment with
suitable temperature (28 ºC) and humid (55 ± 10 %) conditions. Rabbits were fed with
water and food. In vivo study was carried out after getting an approval from the
pharmacy research ethics committee of The Islamia University of Bahawalpur.
48
Rabbits were placed in two groups for in vivo evaluation each group containing 8
rabbits. Single dose crossover design with a wash out period of two weeks was
implied. Commercially available tablets were administered to each rabbit of 1st group
in solution form through syringe whereas hydrogel disks were given to the 2nd
group
through silicone rubber gastric intubation tube with gavage. Rabbits were placed in a
suitable environment with proper feeding.
3.5.2. HPLC Conditions and mobile phase
HPLC equipment containing binary pump solvent transporting system, reverse phase
C-18 (Discovery ® HS, 15 cm 4.6 mm, 5 μm) stainless steel analytical column.
Samples were analyzed at a wavelength 230 nm. Mobile phase was prepared by
mixing 0.1 % (v/v) aqueous ammonia solution with methanol in the ratio of 30:70
(v/v). The mobile phase was filtered by using 0.45 μm diameter membranes
(Sartorius, Germany) and degassed through ultra-sonication. Pump was handled at a
flow rate of 500 µL/min.
3.5.3. Stock Solution Preparation
Perindopril (Drug) and perindoprilat’s (internal standard) stock solutions were
prepared individually with water. 1 mg/mL solution was prepared by mixing the 100
mg drugs in water to make final volume 100 mL. This solution is further diluted with
water to make final stock solution of 100 ng/ml and after that mobile phase was
implied to prepare the dilutions of perindopril in the limits of 4-128 ng/mL. Internal
standard with a concentration of 8 ng/ml was prepared and further utilized in each
dilution of the drug. Further findings of peak area and height were observed on
injecting 10 µL of injection from each dilution.
49
3.5.4. Blank plasma sample
Plasma was taken into tubes and mixed with 10 % per-chloric acid solution to settle
down the proteins. Then this mixture was vortexed for 3 minutes and then centrifuged
for 5 minutes at 3500 rpm. The supernatant was collected in Eppendorf 2 mL micro-
centrifuge tube. This supernatant was concentrated through nitrogen flux and after
that mobile phase was introduced to it. Final solution was filtered with Millipore filter
0.45 μm and then 10 μL of this solution was injected to determine the peak height and
area.
3.5.5. Plasma spiking
Serum extraction was done through the above given procedure. Required dilutions
were prepared in serum with perindopril in the range of 4-128 ng/mL and keeping
perindoprilat concentration constant at 8 ng/mL. Peak area and height were
determined after injecting 10 μL of each dilution.
3.5.6. Sampling
Blood sampling was carried out after variable intervals 0, 45, 90, 150, 240, 480, 720,
960, 1200, 1560, 2160, 2880 min. Blood samples were collected in heparinized tubes
and then centrifuged at 3000 rpm (revolutions per minute) for 20 minutes. After
collecting plasma from the sample, it was stored at -20 °C. Serum extraction was
carried out from the above given procedure.
An earlier reported HPLC method with slight modification was employed to analyze
the unknown concentration of drug in rabbit serum [113]. Different mathematical
calculations were solved through MS Excel 2013 whereas non-compartmental model
based pharmacokinetic parameters like maximum plasma concentrations (Cmax), time
50
for achieving maximum plasma concentrations (tmax) and area under the plasma
concentration time curve (AUCTotal) were determined through Kinetica software
(version 4.4).
3.5.7. Statistical evaluation
One way analysis of variance (ANOVA) and paired sample t-test were employed to
investigate statistical evaluation with P < 0.05 as the minimal level of significance.
Data was explained with the mean value + S.D.
51
4. RESULTS AND DISCUSSION
4.1. Swelling and drug release pattern
4.1.1. Effect of pH on swelling and on drug release of poly vinyl
alcohol/methacrylic acid hydrogels
The ionization of carboxyl varies with pH of the immersion medium that exhibit great
diversity in swelling behavior of hydrogel. Effect of pH on swelling and drug release
was investigated in solutions of pH (1.2, 5.5, 6.5 and 7.5) as shown in Table 4.1 and
4.2, respectively. Dried hydrogels were immersed in 0.05 M USP phosphate buffer
solution of varying pH. The pH values in the physiological medium change from
highly acidic condition in the stomach (pH 1-3) to almost neutral values in the small
(6.37-7.04) and large intestine (6.63-7.49). MA containing gels have a low degree of
swelling in the acidic pH (gastric pH), but as they passed into the gastrointestinal tract
(GI), the degree of swelling increase due to increase in pH. MA has carboxyl group,
swelling of gel is greatly affected by localization of charges on the polymer chains.
Ionization of the carboxyl groups of MA varies with pH of the immersion medium
that results in different swelling behavior of gel at different pH. At higher pH,
electrostatic repulsion along the chain takes place that causes an expansion of the
originally coiled molecules while at lower pH carboxylic groups of MA show no
swelling behavior due to formation of protonated groups. The ionization in turn
stretches the molecules to an extent which depends on the percent ionization of the
carboxylic group. Water soluble drug metoprolol tartrate is selected as model drug. It
is observed that drug release and swelling of gel increased by increasing the pH of
medium. Drug loading of the samples with maximum swelling is given in the Table
4.3. Osmotic pressure inside the gel also causes maximum drug to release at higher
pH (7.5) due to more swelling as compared to lower pH (1.2).
52
4.1.2. Implications of tuned pH on swelling of CA-g-MA hydrogels
pH sensitive hydrogels have polymers which accept or donate protons in modulated
pH environment through acidic or basic groups. As it is known that a great variation
in pH values is observed in the human physiological medium from highly acidic (pH
1-3 in the stomach) to almost neutral values (6.37-7.04 in the small intestine) and
shifted to basic values (6.63-7.49 in large intestine). Presently prepared CA-g-MA
hydrogels were utilized for swelling in tuned pH (2, 4.7, 6.4 and 7.4) as shown in
Table 4.4. Dried disks were kept in 0.05 M USP phosphate buffer solutions of tuned
pH. Dynamic and equilibrium swelling is found to be fine in solution of pH 7.4 as
compared to pH 2, 4.7 and 6.4. At lower pH protonation of carboxylic groups of MA
show no significant swelling whereas in basic pH a forceful electrostatic repulsion
induces which causes a distinct increase in the size of the originally coiled molecules.
So it is clear that swelling of gel increased in alkaline pH.
4.1.3. Implications of concentrations of composites on swelling of CA-g-MA
hydrogels
Degree of crosslinking on swelling was observed by using tuned crosslinking agent
concentration (0.1 %, 0.3 % and 1%) as shown in Table 4.4. Swelling of gel falls on
raising glyoxal concentration (Figure 4.1a) owing to rich physical entanglements in
the structure which triggers elastic restrain forces to hold the network structure from
expanding. The enhanced concentration of glyoxal crosslinker results in more thick
and closely packed structures which in turn gives poor swelling behavior. The rise in
crosslinking concentration had a significant impact on the swelling mechanism
because it causes the polymer networks to come close to each other and retard the
pore formation by reducing the chain flexibility [147].
53
Variable concentrations of MA (31.66 g, 38.33 g and 45 g per 100 g of solution) were
implied in the formulations with glyoxal as crosslinking agent (0.75 %) to get insight
the impact of MA contents on the swelling as shown in Table 4.4. Swelling of
formulations improves on increasing the concentrations of MA owing to the
ionization of rich carboxylic groups of MA which caused the network chain to move
apart leading to enhanced swelling as depicted in Figure 4.1b. At lower pH values, no
distinct swelling was seen due to the complex formation. These complexes are
influenced by the hydrogen bonding that raises the constraints in the network structure
and forms the structure hydrophobic as the carboxylic groups are captured in the
complexes. So, at lower pH values the formulations did not give a sound swelling
whereas at higher pH values the case is reverse in the sense that complexes broke and
carboxylic groups start ionizing. This ionization of carboxylic groups produced an
electrostatic repulsion which in turn moves the network chains far from each other
that resulted in higher swelling [148].
CA-g-MA hydrogels of different polymeric compositions (0.25, 0.50 and 1.0 g per
100 g of solution) were fabricated to evaluate the impact of polymeric composition on
swelling pattern depicted in Table 4.4. At acidic pH, the hydroxyl groups present in
the carrrageenan forms hydrogen bonding with carboxylic groups. These hydrogen
bonds within carboxylic groups form a dense structure which retards the contraction
and relaxation of internal chains. It also affects the flexibility of structure that results
in less swelling whereas in alkaline media, carboxylic groups get ionized. An
electrostatic repulsion is created within polymer networks due to the ionized
carboxylic groups. This repulsion within chains cause more expansion of network and
resulted in distinct swelling. CA contains sulfate and hydroxyl groups which makes it
a strongly anionic polymer owing to greater ionic strength inside. It depicted high
54
impact on swelling at higher pH values as given in Figure 4.1c. Electrostatic
repulsion is created due to negatively charged sulfate groups present in the structure at
different chains which in turn enhances distance between chains. This gap between
chains allows it to swell in alkaline pH. It was found that by increasing the
carrageenan content in the hydrogel formulation swelling ratio increases [149].
55
Figure 4.1. Dynamic swelling coefficient of CA-g-MA hydrogels with different
amounts of (a) glyoxal (b) MA and (c) CA in solutions of tuned pH
values: pH 2 ( ), pH 4.7 ( ), pH 6.4 ( ) and pH 7.4 ( ).
0
2
4
6
8
10
0.1 0.3 1
Dy
na
mic
sw
elli
ng
co
effi
cien
t
Glyoxal (w/w%)
pH 2
pH 4.7
pH 6.4
pH 7.4
0
2
4
6
8
10
31.66 38.33 45Dy
na
mic
sw
elli
ng
co
effi
cien
t
Methacrylic acid (w/w%)
pH 2
pH 4.7
pH 6.4
pH 7.4
0
2
4
6
8
10
0.25 0.5 1
Dy
na
mic
sw
elli
ng
co
effi
cien
t
Carrageenan (w/w%)
pH 2
pH 4.7
pH 6.4
pH 7.4
(c)
)
(a)
(b)
56
4.1.4. Effect of monomer concentration on swelling and on drug release of
PVA/MA hydrogel
Amount of MA was varied from 38 g, 46 g and 54 g per 100 g of solution in
PVA/MA hydrogels using glyoxal as crosslinking agent (0.75 %) were prepared to
investigate the effect of MA contents on the swelling and on drug release as shown in
Table 4.1 and 4.2. The hydrophilic parts of the PVA/MA hydrogels like carboxylic
groups make hydrogen bonds with the water molecules. These bonds serve as a strong
environment of hydration which surrounds the hydrophobic portions that result in
higher swelling and more water intake [150]. At alkaline pH carboxylic groups
present in the structure become highly ionized and resulted in the cleavage of
hydrogen bond The electrostatic repulsion caused the network to expand, producing a
larger swelling ratio when the hydrogel was at pH 7.2 [151] whereas at acidic pH
these hydrogel structures depicted poor swelling due to the formation of hydrogen
bonds in the structure [152-154].
It is observed in Figures 4.2 and 4.3(a) that swelling and drug release of gel
increases with increasing the concentration of MA due to the availability of more
carboxylic groups of MA for ionization and this ionization caused the network chain
to move apart which brought about enhanced swelling. Drug release studies were
carried out for 72 hrs. Samples J7 and J8 gave maximum drug loading and release. As
shown in Figure 4.3(a) cumulative drug release was observed 84.12 %, 85.77 % and
89.56 % at pH 7.5, 78.91 %, 79.44 % and 79.9 % at pH 6.5 and 30.93 %, 31.77 % and
31.92 % at pH 1.2 with respect to the compositions of PVA/MA 1.2/38, 1.2/46 and
1.2/54 per 100 g, respectively after 48 hrs, while Figure 4.3(b) provided the complete
release profile of sample J7 as a function of time.
57
Figure 4.2. Dynamic swelling coefficient of PVA/MA hydrogels with different
concentrations of MA (38, 46 & 54 g) using Glyoxal as crosslinking
agent (0.75 %) in solutions of different pH in 0.05M USP
phosphate buffer at 37 0C. The pH values are: pH 1.2 ( ), pH 6.5 (
) and pH 7.5 ( ).
0
2
4
6
8
10
38 46 54
Dy
na
mic
sw
elli
ng
co
effi
cien
t
Methacrylic acid concentration (w/w%)
pH1.2
pH6.5
pH7.5
58
Figure 4.3. Cumulative release % of metoprolol after 48 h (a) using different
concentrations of methacrylic acid (38, 46 & 54 g) and (b) complete
release profile of J7.
0
10
20
30
40
50
60
70
80
90
100
38 46 54
Cu
mu
lati
ve
met
op
rolo
l r
ele
ase
%
Methacrylic acid concentration (w/w%)
pH 1.2
pH 6.5
pH 7.5
0
10
20
30
40
50
60
70
80
90
100
0 8 16 24 32 40 48
Cu
mu
lati
ve
met
op
rolo
l re
lea
se %
Time (h)
pH 1.2
pH 6.5
pH 7.5
(a)
(b)
59
4.1.5. Effect of polymer concentration on swelling of PVA/MA hydrogel
PVA/MA hydrogels of different polymeric composition (0.3, 0.6 and 1.2 g per 100 g
of solution) were prepared to investigate the effect of polymeric composition on
swelling of PVA/MA hydrogels as shown in the Table 4.1 PVA contain high
hydroxyl groups that make this polymer highly interactive with water. It is observed
in Figure 4.4 that swelling of gel increased on increasing PVA concentration due to
the increased hydrophilic properties of PVA/MA hydrogels which leads to increased
swelling. It is also seen that PVA does not show significant difference in swelling
behavior as compared to the tuned concentrations of crosslinker and monomer. At
alkaline pH of the swelling environment (5.5–7.5), an enhanced swelling is observed
in the structure. This rise in swelling may be due to the increase of pH of the outer
environment which is usually due to the hydroxyl (–OH) groups present in PVA
[155].
It was also found that at much larger amounts of PVA, equilibrium swelling is
attained earlier. The reason behind is that the hydrophilicity of PVA make the
structure highly hydrated due to the high proportion of PVA [156-158].
60
Figure 4.4. Dynamic swelling coefficient of PVA/MA hydrogels with different
concentrations of PVA (0.30, 0.60 & 1.2 g) using Glyoxal as
crosslinking agent (0.75 %) in solutions of different pH in 0.05M
USP phosphate buffer at 37 0C.The pH values are: pH 1.2 ( ), pH
6.5 ( ) and pH 7.5 ( ).
0
2
4
6
8
10
0.3 0.6 1.2
Dy
na
mic
sw
elli
ng
co
effi
cien
t
Polyvinyl alcohol concentration (w/w%)
pH1.2
pH6.5
pH7.5
61
4.1.6. Effect of degree of crosslinking on swelling and on drug release of
PVA/MA hydrogel
A series of PVA/MA hydrogels of different crosslinking agent concentration (0.3 %,
0.50 % and 1%) were designed to study the impact of crosslinking on swelling and on
drug release as shown in Table 4.1 and 4.2. It was observed in Figure 4.5 that
swelling of gel decreased with increase of glyoxal concentration due to presence of
more physical entanglements between hydrogels. This provides elastic restrain forces
to retard the expanding of the network. Formulation with higher glyoxal
concentrations like J9 is tightly packed and less flexible as compared to the
formulations with low concentrations of glyoxal such as J7 and J8 [159].
As shown in the Figure 4.6 (a) that cumulative drug release was 94.12 %, 90.21 %
and 85.13 % at pH 7.5, 84.95 %, 82.94 % and 76.26 % at pH 6.5 and 34.65 %, 31.26
% and 28.59 % at pH 1.2 with respect to concentration of glyoxal (0.3 %, 0.5 % and 1
%) as crosslinking agent respectively after 48 hrs, while Figure 4.6 (b) provided the
complete release profile of sample J8 as a function of time. So it is clear from the
above findings that formulations containing higher concentrations of glyoxal depicted
low amount of drug release as compared to the formulations with low concentrations
of glyoxal therefore it can be predicted that drug release behavior has an indirect
relation with the glyoxal concentration used in the formulations [160].
62
Figure 4.5. Dynamic swelling coefficient of PVA/MA hydrogels with different
concentrations of Glyoxal (0.30, 0.50 and 1 %) in solutions of
different pH in 0.05 M USP phosphate buffer at 37 0C. The pH
values are: pH 1.2 ( ), pH 6.5( ) and pH 7.5 ( ).
0
2
4
6
8
10
0.3 0.5 1
Dy
na
mic
sw
elli
ng
co
effi
cien
t
Glyoxal concentration (w/w%)
pH1.2
pH6.5
pH7.5
63
Figure 4.6. Cumulative release % of metoprolol after 48 h (a) using different
concentrations of Glyoxal as crosslinking agent (0.30 %, 0.50 %
and 1 %) and (b) complete release profile of J8.
0
10
20
30
40
50
60
70
80
90
100
0.3 0.5 1
Cu
mu
lati
ve
met
op
rolo
l re
lea
se %
Glyoxal concentration (w/w%)
pH1.2
pH6.5
pH7.5
0
10
20
30
40
50
60
70
80
90
100
0 8 16 24 32 40 48
Cu
mu
lati
ve
met
op
rolo
l re
lea
se %
Time (h)
pH 1.2
pH 6.5
pH 7.5
(a)
(b)
64
Table 4.1. Equilibrium swelling ratios of PVA/MA hydrogel.
Samples
No
Equilibrium swelling coefficient in solution of various pH
pH 1.2 pH 5.5 pH 6.5 pH 7.5
J1 1.05 5.38 12.32 b
J2 1.23 5.13 14.34 b
J3 1.30 5.25 15.67 b
J4 4.21 6.24 25.62 b
J5 4.43 6.46 28.90 b
J6 4.58 6.64 47.06 b
J7 4.61 7.01 54.07 b
J8 4.40 6.99 44.66 b
J9 4.17 5.67 24.81 b
“b” stand for samples broken
65
Table 4.2. Percent metoprolol tartrate released in different formulations
of PVA/MA hydrogel.
Samples
No
Amount of metoprolol tartrate released (%) up to 48hrs
pH 1.2 pH 6.5 pH 7.5
J4 30.93 78.91 84.12
J5 31.77 79.44 85.77
J6 31.92 79.9 89.56
J7 34.65 84.95 94.12
J8 31.26 82.94 90.21
J9 28.59 76.26 85.13
Table 4.3. Drug loading through weight and extraction method of PVA/MA
hydrogel.
Samples
No
Concentration of metoprolol tartrate loaded (g/g) of dry gel
Through weight Through extraction
J4 0.1067 0.1028
J5 0.1302 0.1297
J6 0.1431 0.1423
J7 0.1683 0.1676
J8 0.1568 0.1554
J9 0.1274 0.1271
66
Table 4.4. Swelling measurements of CA-g-MA hydrogel.
Samples No Equilibrium swelling coefficient in solutions of tuned pH
pH 2 pH 4.7 pH 6.4 pH 7.4
JA1 2.1355 2.7675 5.632 b
JA2 1.9037 2.3076 5.101 b
JA3 1.2964 1.9645 4.432 b
JA4 2.4083 2.9123 4.456 b
JA5 2.5081 2.8429 4.723 b
JA6 2.753 3.183 5.098 b
JA7 2.457 2.694 3.925 b
JA8 2.516 2.814 4.193 b
JA9 2.817 2.989 4.835 b
“b” stand for samples broken
4.1.7. Implications of concentrations of composites on drug release of CA-g-MA
hydrogel
Consequences of drug release profile of CA/MA hydrogels were studied in the
solutions of tuned pH (2, 4.7 and 7.4) as shown in Table 4.5, whereas drug loading
through weight and extraction method is given in Table 4.6. Presently hydrophilic
drug perindopril is utilized as model drug. Release profile of formulations correlates
with the swelling characteristic in the sense that it also enhances in alkaline pH as
compared to acidic pH (Figure 4.7). Mechanism behind is the same as discussed in
the swelling earlier.
67
Table 4.5. Drug release study of optimum formulations of CA-g-MA hydrogel.
Samples
No
Amount of perindopril released (%) upto 48 hrs
pH 2 pH 4.7 pH 7.4
JA1 38.52 44.98 96.62
JA2 32.82 41.69 92.52
JA3 22.45 31.26 75.01
JA4 23.75 39.66 81.75
JA5 29.27 49.44 88.60
JA6 33.17 58.15 91.81
Table 4.6. Drug loading through weight and extraction method of CA-g-MA
hydrogel.
Samples
No
Concentration of perindopril erbumine loaded (g/g) of dry gel
Through weight Through extraction
J4 0.0107 0.0106
J5 0.0104 0.0103
J6 0.0089 0.0088
J7 0.0092 0.0091
J8 0.0098 0.0097
J9 0.0101 0.0100
68
Figure 4.7. Release of perindopril from CA-g-MA hydrogels using different
amounts of (a) glyoxal and (b) MA at tuned pH values: pH 2 ( ),
pH 4.7 ( ) and pH 7.4 ( ).
0
10
20
30
40
50
60
70
80
90
100
0.1 0.3 1
Per
ind
op
ril
rele
ase
%
Glyoxal (w/w%)
pH 2
pH 4.7
pH 7.4
0
10
20
30
40
50
60
70
80
90
100
31.66 38.33 45
Per
ind
op
ril
rele
ase
%
Methacrylic acid (w/w%)
pH 2
pH 4.7
pH 7.4
(a)
(b)
69
4.2. Diffusion coefficient investigations
4.2.1. Diffusion coefficient of polymers (Dw) of PVA/MA hydrogel
Diffusion coefficient is indirectly measure of solute diffusion into hydrogel. Fick’s
law of diffusion was applied during membrane permeation method or sorption and
desorption phenomenon. It was investigated that diffusion coefficient decreased with
the increasing of MA concentration because swelling of hydrogel increased on
increasing the concentration of MA as shown in Table 4.7 [161,162].
4.2.2. Diffusion coefficient study of CA-g-MA formulations
Diffusion coefficient is indirectly measure of solute diffusion into hydrogel. Fick’s
law of diffusion was applied during membrane permeation method or sorption and
desorption phenomenon. It was investigated that diffusion coefficient decreased with
the increasing of MA concentration because swelling of hydrogel increased on
increasing the concentration of MA as shown in Table 4.8 [163].
4.3. Networking structural analysis
4.3.1. Molecular weight between crosslinks (Mb) and solvent interaction
parameters (χp) in PVA/MA hydrogel
Increased in values of molecular weight between crosslinks (Mb) was observed by
increasing the concentration of MA as shown in Table 4.7. Higher swelling of
polymer was reported due to impart of MA carboxylic groups into polymer chain.
Solvent interaction parameters ( p) were studied to check the effect of solvent
interaction between polymer and solvent. It was reported that greater the values ( p)
weaker the values of interaction between polymer and solvent. It is observed in Table
4.7 that on increasing the concentration of MA, the value of p decreased which
exhibit the hydrophilic nature of the chains and strong interaction with solvent.
70
4.3.2. Networking structural analysis of CA-g-MA formulations
Molecular weight between crosslinks (Mb) and solvent interaction parameters ( p)
were calculated to get insight structural network. Values of Mb boosted on enhancing
the concentration of MA as given in Table 4.8. Presence of carboxylic groups in MA
caused a climb up in the values of swelling of polymer. Implications of interaction
between solvent and polymer were evaluated through p. An enhanced interaction
with solvent was found on leveling up the concentration of MA which in turn declined
the values of p. These boosted Mb and p values give a clue that a rise in hydrophilic
behavior is observed due to the ionization of carboxylic groups which are being added
in the chains. On addition of these moieties Mb values increased which in turn
enhanced the swelling of the hydrogels [164].
Table 4.7. Flory-Huggins network parameters of PVA/MA hydrogel.
Samples
No
Vf,s χp Mb Mu qh (Mb/Mu) Dw
10ˉ6 (cm²/sec)
J4 0.4414 0.7233 32.6635 83.7297 0.3901 0.8537
J5 0.3807 0.6794 59.1009 84.1026 0.7027 0.4458
J6 0.3599 0.6657 60.8333 84.3703 0.7210 0.2862
Vfs: Volume fraction of the polymer at swelling equilibrium in phosphate buffer solution. Mb: Average molecular
weight between crosslinks. Mu: Molar mass of the repeating unit. χp: Solvent interaction parameter. qh:
Crosslinking density. Dw: Diffusion coefficient.
71
Table 4.8. Flory-Huggins network parameters of tuned CA-g-MA
formulations.
Samples
No
Vf,s χp Mb Mu qh (Mb/Mu) Dw
10ˉ6 (cm²/sec)
JA4 0.555877 0.82776 22.884 76.5240 0.2990 1.5894
JA5 0.285595 0.62172 109.371 77.2769 1.4153 0.8060
JA6 0.24921 0.60251 145.041 78.3304 1.8516 0.6358
Vfs: Volume fraction of the polymer at swelling equilibrium in phosphate buffer solution. Mb: Average molecular
weight between crosslinks. Mu: Molar mass of the repeating unit. χp: Solvent interaction parameter. qh:
Crosslinking density. Dw: Diffusion coefficient.
4.4. Sol-gel and porosity analysis
4.4.1. Sol-gel analysis of PVA/MA hydrogel
Sol-gel fraction analysis of hydrogel was conducted to calculate the uncrosslinked
fraction of polymer. PVA/MA hydrogels of different crosslinker concentrations (0.3
%, 0.5 %, and 1 %), different polymeric composition (0.3, 0.6 & 1.2 g/100 g) and
different monomeric composition (38, 46 & 54 g/100 g) were prepared. It is observed
that on increasing the concentration of the MA in PVA/MA hydrogels there is a
gradual decrease in the sol fraction. As the concentration of glyoxal increased, sol
fraction also decreased. In the same manner on increasing the concentration of PVA,
sol fraction also decreased. It is concluded that sol fraction is inversely proportional to
the concentration of the MA, glyoxal and PVA. Gel fraction of hydrogels is calculated
by subtracting sol fraction from the value 100 [165]. It is observed from the results in
the Table 4.9 that gel fraction is directly proportional to the concentration of the PVA
(shown in Figure 4.8), glyoxal (shown in Figure 4.9) and MA (shown in Figure
4.10).
72
4.4.2. Porosity measurement of PVA/MA hydrogel
It is observed from the results in Table 4.9 that porosity increased by enhancing the
concentration of PVA and MA due to the improved viscosity of the hydrogel solution.
Viscous solution efficiently prevents the bubbles escaping from the solution that
result in increased porosity due to formation of interconnected channels, while on
increasing the glyoxal concentration porosity decreased due to increased physical
entanglement between PVA/MA that results in more compressed and thick network
structure. These dense structures retard the pore formation so the prepared hydrogels
represented a fall in porosity values [166, 167].
Table 4.9. Gel fraction and porosity measurement in different formulations
of PVA/MA hydrogel.
Samples No Gel fraction
(%)
Porosity
(%)
J1 87.24 17.12
J2 90.87 18.36
J3 93.74 22.84
J4 90.18 12.36
J5 92.67 19.76
J6 94.51 22.78
J7 89.16 37.98
J8 91.46 28.79
J9 93.86 12.69
73
Figure 4.8. Effect of different concentrations of PVA on gel fraction.
82
84
86
88
90
92
94
96
0.3 0.6 1.2
Gel
fra
ctio
n
PVA concentration (w/w%)
74
Figure 4.9. Effect of different concentrations of Glyoxal on gel fraction.
82
84
86
88
90
92
94
96
0.3 0.5 1
Gel
fra
ctio
n
Glyoxal concentration (w/w%)
75
Figure 4.10. Effect of different concentrations of MA on gel fraction.
82
84
86
88
90
92
94
96
38 46 54
Gel
fra
ctio
n
Methacrylic acid concentration (w/w%)
76
4.5. Spectral confirmation of hydrogel structures
Figure 4.11 depicts the absorption spectra of PVA, PVA/MA hydrogel without drug
and PVA/MA with drug in the region of 500-4000 cm ־ ¹. The characteristic peaks of
pure PVA obtained at 3286 cm ־ ¹, 2942 cm ־ ¹ and 1090 cm ־ ¹ exhibiting O-H, C-H and
-C-O- stretching vibrations respectively. The broader band appeared in the range of
2900–3500 cm ־ ¹ may be due to the asymmetric stretching of the O-H groups present
in the PVA and the sharp band appeared at 2942 cm ־ ¹ as a result of C-H asymmetric
stretching. The stretching vibration at 1715 cm ־ ¹ denotes ester configuration which
confirms the graft polymerization of MA onto PVA [168, 169]. FTIR analysis
confirmed that hydrogels network has chemical linkages which formed during the free
radical polymerization reaction and as there is no distinct shift in peculiar peaks, so
we can elucidate that there is no interaction between polymer and drug.
Absorption spectra of CA, CA-g-MA blank hydrogel and drug loaded CA-g-MA is
observed in the region of 500-4000 cm ־ ¹ (Figure 4.12). The peculiar peaks of
carrageenan polymer were found to be at 1224 cm-1
, 1035 cm-1
, 925 cm-1
and 844 cm-1
representing stretching of ester sulfate, glycosidic linkage, 3,6-anhydro-D-galactose
and D-galactose-4-sulfate respectively while extended peak band at 3389 cm-1
exhibiting stretching vibration of –OH group. Spectrum band of blank CA-g-MA gave
two new characteristic peaks at 1691 cm-1
and 1483 cm-1
. These peaks confirmed the
carbonyl stretching of carboxylic group and symmetric stretching of carboxylate
group respectively. Peak found at 1693 cm-1
of drug loaded CA-g-MA gel gave the
indication of no major shift of peak owing to no interaction between polymer and
drug [170].
77
Figure 4.11. FTIR spectra of (a) PVA (b) PVA/MA hydrogel without drug (c)
PVA/MA hydrogel with drug.
78
Figure 4.12. FTIR spectra of (a) CA (b) Blank CA-g-MA hydrogel (c) Drug
loaded CA-g-MA hydrogel.
79
4.6. Insights into thermal behavior
4.6.1. Differential scanning calorimetry
DSC thermograms of PVA/MA hydrogel depicts the thermal degradation which starts
with the formation of anhydride structures between pairs of carboxyl group with the
elimination of water at 100 0C, while the other peak at 260
0C represents the
destruction of crosslinked structure. In case of PVA, the first endothermic peak of
PVA is shown at 83 0C and the second peak explains the Tm (melting temperature)
which is found to be around 205-215 0C and MA melts very early just at 30
0C as
shown in Figure 4.13. As PVA/MA exhibited enhanced thermal stability pattern, it
indicates that cross-linking between the PVA and MA raises the thermal stability of
PVA. This determined that the prepared formulation could be handled at enhanced
temperature as compared to its individual components (PVA and MA).
Transition temperature is evaluated by comparing DSC thermogram profiles of CA-g-
MA hydrogel, CA and MA as given in the Figure 4.14. Transition temperature can be
correlated to the swelling of hydrogel owing to the strength of polymer chain.
Carrageenan gave a peak at 106.4 0C depicting its transition temperature while this
peak shifted towards higher temperature at 119.9 0C in CA-g-MA hydrogel. This
shifted peak gives a clue of rise in transition temperature due to the strong crosslinked
structure of CA-g-MA hydrogel. MA with a low transition temperature started
degrading at 30 °C.
80
Figure 4.13. DSC of MA, PVA and PVA/MA hydrogel.
-4
-3.5
-3
-2.5
-2
-1.5
-1
-0.5
0
0.5
1
-100 0 100 200 300 400 500
Hea
t F
low
(w
/g)
Temperature (◦c)
MA
PVA
PVA/MA hydrogel
81
Figure 4.14. DSC of MA, CA and CA-g-MA hydrogel.
-7
-6
-5
-4
-3
-2
-1
0
1
-100 0 100 200 300 400 500
Hea
t F
low
(w
/g)
Temperature (◦c)
MA
Carrageenan
CA-g-MA hydrogel
82
4.6.2. Thermogravimetric analysis
The crosslinked hydrogel formulation exhibits better thermal stability as compared to
individual polymer and monomer as shown in Figure 4.15. Thermogram of PVA/MA
hydrogel formulation could be explained into two divisions. First division depicts the
slow weight loss from 220 0C to 310
0C, while second division exhibits the
decomposition curve from 410 0C to 490
0C. PVA exhibits less thermal stability than
PVA/MA hydrogel formulation. Thermal degradation of PVA occurs at about 270 0C
while MA has the least thermal stability as weight loss abruptly above 100 0C.
TGA was performed to study thermal stability of formulation and its major
composites. Thermal degradation of crosslinked CA-g-MA structure takes place in
two phases. First phase determined (9.36 %) weight loss in the range of 36 - 190 0C.
This weight loss is attributable to escape of moisture. Weight loss of (52.8 %) was
observed in the second phase from 190 0C to 710
0C. Weight loss at this stage
prompts the destruction of crosslinked structure. Thermogram pattern of CA polymer
explained the weight loss in two major portions. First portion suggests the water loss
from 20 – 180 0C while the second portion sounds the destruction of internal structure
in the range of 180 – 400 0C. MA vanished quickly above 100
°C which turns out poor
thermal stability Overall findings suggest that the formulation of crosslinked compact
hydrogel structure gives improved thermal stability than its composites (Figure 4.16).
From this overall discussion, it can be elucidated that glyoxal crosslinked structures of
PVA/MA and CA-g-MA hydrogels have strong thermally stable structures which
require a higher thermal treatment as compared to their constituents present in the
formulations [171, 172].
83
Figure 4.15. TGA of MA, PVA and PVA/MA hydrogel.
0
20
40
60
80
100
120
0 100 200 300 400 500 600
Wei
gh
t (
%)
Temperature (◦c)
MA
PVA
PVA/MA hydrogel
84
Figure 4.16. TGA of MA, CA and CA-g-MA hydrogel.
0
20
40
60
80
100
120
0 100 200 300 400 500 600 700 800
Wei
gh
t (%
)
Temperature (°C)
MA
CA
CA-g-MA hydrogel
85
4.7. X-Ray diffraction
Typical peaks of PVA appeared at 2Ө 20.10. PVA/MA hydrogel indicated diverse
XRD design from the pure PVA that affirmed the establishment of another polymer.
The peak at 2 20.1° is weekend fundamentally and also exhibited a sharply
decreased wide peak at 2Ө 500. It is cleared from the Figure 4.17 that an amorphous
structure of PVA/MA hydrogel is obtained. Hence during hydrogel formation of
PVA/MA, there is decrease in crystallinity due to the crosslinking.
86
Figure 4.17. XRD spectra of (a) PVA (b) PVA/MA hydrogel.
87
4.8. Interpretation of morphological characteristics
The hydrogels formed by PVA/MA are highly porous. SEM analysis of PVA/MA
hydrogel with and without drug was performed. Figure 4.18 (a, b) depicts scanning
electron micrographs of PVA/MA hydrogel without drug which clearly shows a
highly porous rough surface with thick cracks, while micrographs of PVA/MA
hydrogel with drug as shown in Figure 4.19 (a, b) shows a different morphology
which is less porous and possessed a scaly structure [173, 174]. Compact masses and
sponge like structure appeared in loaded PVA/MA hydrogel which seemed to be the
aggregate of drug particles. The porous morphology of PVA/MA hydrogel without
drug could have a prominent effect on the rate and extent of hydrogel swelling and
drug loading.
Swelling and drug loading characteristics correlates with the openings present in the
structure of CA-g-MA hydrogels. Sound difference is observed in the scanning
electron microscopic images of blank (Figure 4.20 (a)) and loaded formulations
(Figure 4.20 (b)). Numerous cracks and openings observed on the surface of blank
disks, whereas loaded disks have few apertures with packed masses and ragged
surface could be the identification of drug particles aggregates.
The pores present in the hydrogel structures may be inducted as a result of removal of
water molecules present in the formulation solution when heat is applied. The
findings are relevant to our prospect that a porous morphology can be obtained by free
radical polymerization technique. Swelling of the prepared structures can be found
due to the intake of water molecules into the pores, while the drug is loaded in these
pores of prepared hydrogel matrix. The porous structure of blank disk can have
plausible relation with the extent of swelling and drug loading. [175].
88
Figure 4.18. Scanning electron micrographs of PVA/MA hydrogel without
drug.
89
Figure 4.19. Scanning electron micrographs of PVA/MA hydrogel with drug.
90
Figure 4.20. Scanning electron micrographs of (a) blank and (b) loaded CA-g-
MA hydrogel.
91
4.9. In vitro evaluation
4.9.1. Drug release mechanism
The method that best fits the release data was evaluated by the regression coefficient
(r). Criteria for selecting the most appropriate model were based on the ideal fit
indicated by the values of regression coefficient (r) near to 1.
Regression coefficient (r) values obtained from PVA/MA at varying content of MA
and degree of crosslinking are given in the Table 4.10 and 4.11, respectively whereas
the values of ‘r’ concerning CA-g-MA hydrogel with tuned concentrations of MA and
crosslinker are provided in the Table 4.12 and 4.13, respectively. The value of ‘r’
obtained from zero order release rate constants were found higher with those of first
order rates which indicates that drug release from the samples of varying monomeric
compositions and degree of crosslinking are according to zero order release.
The R values of Higuchi model at different monomeric composition and at different
degree of crosslinking indicated that the drug release mechanism was diffusion
controlled. Effects of monomer concentration and degree of crosslinking on ‘n’ values
of PVA/MA hydrogels are given in Table 4.14 and 4.15, respectively while ‘n’
values of CA-g-MA hydrogels with tuned concentrations of MA and crosslinker are
depicted in Table 4.16 and 4.17, respectively.
As it is clear from the obtained data that the ‘n’ values of all formulations found
between 0.45 and 0.89 so it can be predicted that release mechanism is non-fickian.
All samples of PVA/MA hydrogel represent Non-fickian behavior at pH 1.2, 6.5 and
7.5 while CA-g-MA formulations at variable pH 2, 4.7 and 7.4 also show Non-fickian
behavior. This means that swelling and relaxation of polymer is involved in drug
release mechanism [176, 177].
92
Table 4.10. Effect of different concentrations of methacrylic acid on drug
release kinetics of PVA/MA hydrogel in solution of different pH
using glyoxal as crosslinking agent.
Samples
No
MA contents
(%)
pH Zero order
kinetics
First order
kinetics
Higuchi
Model
Ko (h¹־) r K1 (h¹־) r K2 (h¹־) R
J4 38 1.2 2.375 0.982 0.028 0.861 0.099 0.947
6.5 5.607 0.950 0.095 0.653 0.232 0.900
7.5 6.378 0.958 0.143 0.895 0.277 0.996
J5 46 1.2 2.462 0.954 0.030 0.765 0.106 0.976
6.5 6.2 0.993 0.126 0.872 0.262 0.979
7.5 6.143 0.994 0.146 0.857 0.26 0.978
J6 54 1.2 2.459 0.977 0.030 0.688 0.105 0.990
6.5 6.527 0.992 0.129 0.782 0.277 0.983
7.5 6.423 0.994 0.168 0.852 0.272 0.980
Table 4.11. Effect of degree of crosslinking on drug release kinetics of
PVA/MA hydrogel in solution of different pH.
Samples
No
Glyoxal
(%)
pH Zero order
kinetics
First order
kinetics
Higuchi Model
Ko (h¹־) r K1 (h¹־) r K2 (h¹־) R
J7 0.3 1.2 2.509 0.949 0.032 0.736 0.111 0.981
6.5 7.350 0.996 0.167 0.795 0.319 0.985
7.5 7.208 0.989 0.229 0.886 0.134 0.984
J8 0.5 1.2 2.515 0.981 0.031 0.674 0.109 0.989
6.5 6.673 0.996 0.143 0.794 0.288 0.993
7.5 6.639 0.987 0.178 0.881 0.285 0.993
J9 1 1.2 2.048 0.972 0.025 0.666 0.089 0.989
6.5 5.782 0.996 0.110 0.692 0.247 0.991
7.5 5.803 0.989 0.135 0.785 0.248 0.993
93
Table 4.12. Effect of tuned concentrations of methacrylic acid on drug release
kinetics of CA-g-MA hydrogel in solution of different pH using
glyoxal as crosslinking agent.
Samples
No
MA contents
(%)
pH Zero order
kinetics
First order
kinetics
Higuchi
Model
Ko (h¹־) r K1 (h¹־) r K2 (h¹־) R
J4 31.66 2 1.946 0.986 0.018 0.786 0.076 0.932
4.7 3.702 0.963 0.084 0.845 0.156 0.975
7.4 6.134 0.998 0.267 0.932 0.365 0.991
J5 38.33 2 1.436 0.989 0.025 0.698 0.135 0.981
4.7 2.991 0.992 0.135 0.863 0.245 0.984
7.4 5.934 0.996 0.253 0.937 0.46 0.979
J6 45 2 1.342 0.979 0.029 0.769 0.115 0.994
4.7 3.425 0.995 0.249 0.782 0.197 0.981
7.4 5.893 0.989 0.154 0.882 0.372 0.980
Table 4.13. Effect of degree of crosslinking on drug release kinetics of CA-g-
MA hydrogel in solution of different pH.
Samples
No
Glyoxal
(%)
pH Zero order
kinetics
First order
kinetics
Higuchi Model
Ko (h¹־) r K1 (h¹־) r K2 (h¹־) R
J7 0.05 2 1.432 0.984 0.064 0.765 0.151 0.967
4.7 5.241 0.991 0.187 0.895 0.239 0.985
7.4 6.981 0.949 0.318 0.872 0.498 0.974
J8 0.15 2 1.245 0.990 0.069 0.765 0.128 0.988
4.7 4.652 0.993 0.272 0.872 0.276 0.992
7.4 6.534 0.997 0.189 0.897 0.296 0.995
J9 0.5 2 1.652 0.968 0.029 0.766 0.567 0.986
4.7 4.725 0.994 0.210 0.699 0.238 0.981
7.4 6.845 0.995 0.245 0.654 0.296 0.991
94
Table 4.14. Effect of different concentrations of methacrylic acid on drug
release mechanism of PVA/MA hydrogel in solution of different
pH using glyoxal as crosslinking agent.
Samples
No
MA contents
(%)
pH Release
exponent (n)
r Order of
release
J4 38 1.2 0.672 0.979 Non-fickian
6.5 0.838 0.979 Non-fickian
7.5 0.672 0.994 Non-fickian
J5 46 1.2 0.660 0.965 Non-fickian
6.5 0.706 0.979 Non-fickian
7.5 0.561 0.968 Non-fickian
J6 54 1.2 0.623 0.984 Non-fickian
6.5 0.825 0.984 Non-fickian
7.5 0.557 0.969 Non-fickian
Table 4.15. Effect of degree of crosslinking on drug release mechanism of
PVA/MA hydrogel in solution of different pH.
Samples
No
Glyoxal
(%)
pH Release
exponent (n)
r Order of release
J7 0.3 1.2 0.608 0.977 Non-fickian
6.5 0.803 0.971 Non-fickian
7.5 0.625 0.969 Non-fickian
J8 0.5 1.2 0.699 0.983 Non-fickian
6.5 0.779 0.989 Non-fickian
7.5 0.592 0.987 Non-fickian
J9 1 1.2 0.544 0.987 Non-fickian
6.5 0.694 0.995 Non-fickian
7.5 0.531 0.988 Non-fickian
95
Table 4.16. Effect of tuned concentrations of methacrylic acid on drug release
mechanism of CA-g-MA hydrogel in solution of different pH using
glyoxal as crosslinking agent.
Samples
No
MA contents
(%)
pH Release
exponent (n)
r Order of release
J4 31.66 2 0.653 0.983 Non-fickian
4.7 0.745 0.989 Non-fickian
7.4 0.697 0.991 Non-fickian
J5 38.33 2 0.654 0.976 Non-fickian
4.7 0.706 0.987 Non-fickian
7.4 0.662 0.969 Non-fickian
J6 45 2 0.613 0.974 Non-fickian
4.7 0.742 0.984 Non-fickian
7.4 0.657 0.978 Non-fickian
Table 4.17. Effect of degree of crosslinking on drug release mechanism of CA-
g-MA hydrogel in solution of different pH.
Samples
No
Glyoxal
(%)
pH Release
exponent (n)
r Order of release
J7 0.05 2 0.508 0.967 Non-fickian
4.7 0.763 0.974 Non-fickian
7.4 0.805 0.989 Non-fickian
J8 0.15 2 0.619 0.981 Non-fickian
4.7 0.767 0.986 Non-fickian
7.4 0.587 0.992 Non-fickian
J9 0.5 2 0.534 0.985 Non-fickian
4.7 0.624 0.991 Non-fickian
7.4 0.580 0.978 Non-fickian
96
4.10. In-vivo Investigations
In-vivo investigations were implied in rabbits by administering oral hydrogels and
conventional tablets containing about 0.5 mg drug. Male healthy rabbits were selected
in place of humans or other animals owing to different benefits such as easy to handle
and small size of animal group can be handy in estimating drug in plasma. Rabbits are
being frequently implied in bioavailability investigations of various drug delivery
systems.
4.10.1. HPLC Insights
Concentration of drug in different rabbit plasma samples were quantified through high
performance liquid chromatography (HPLC). An earlier reported method with slight
modification was used to quantify drug in plasma [113]. Perindopril erbumine and
perindoprilat depicted retention time of 0.85 minutes and 2.2 minutes, respectively.
This variation in retention time of the drugs minimizes the possibilities of merging of
peaks. Run time of every sample was adjusted at 5 min.
4.10.2. Calibration curve of Perindopril erbumine
Perindopril erbumine was estimated in ng/ml of rabbit plasma through a highly
sophisticated method of better sensitivity as very low concentrations of drug
penetrated into the system at different time spans. Calibration curve was plotted by
drawing the drug concentration against peak height which is given in the Figure 4.21.
Drug concentration was calculated through the calibration curve by implying the
regression equation (R2
= 0.994)
y = 0.091x + 0.203
97
Where y represents the response ratio, x gives the slope of regression line, 0.091 is a
slope and 0.203 is the intercept of the regression line. The main parameters are given
in the following table (Table 4.18).
Table 4.18. Major parameters regarding regression analysis for calibration
line.
Serial No Parameters
Perindopril
erbumine
1 Number of samples 6
2 Concentration range (ng/ml) 4-128
3 Regression equation y = ax + b
4 Slope (a) 0.091
5 Intercept (b) 0.203
6 Regression coefficient (r²) 0.994
98
Figure 4.21. Calibration curve of Perindopril erbumine in the spiked
plasma.
y = 0.0912x - 0.2038 R² = 0.9947
0
2
4
6
8
10
12
14
0 20 40 60 80 100 120 140
Pea
k h
eig
ht
rati
o
Concentration (ng/ml)
99
4.10.3. In vivo findings
In vivo studies of hydrogel and conventional tablet were carried out on rabbits. An
HPLC method with slight modification was used to quantify the perindopril in
plasma. Perindopril was quantified in the concentration range of ng/mL. Limit of
detection (LOD) and Limit of quantification (LOQ) were observed at 2 ng/mL and 4
ng/mL respectively. Chromatogram of blank plasma is depicted in Figure 4.22
whereas chromatogram of plasma spiked perindopril (16 ng/mL) and internal standard
perindoprilat (8 ng/mL) is represented in Figure 4.23.
The pharmacokinetic parameters like Cmax, Tmax, AUC and MRT (mean residence
time) are given in Table 4.19. A distinct difference was observed between
pharmacokinetic parameters of conventional tablet and oral hydrogel with a 95%
confidence interval (P<0.05).
Table 4.19. Pharmacokinetic data of antihypertensive perindopril erbumine.
No Pharmacokinetic
parameters
Cmax
(ng/mL)
t1/2
(min)
Tmax
(min)
AUCtotal
(ng/mL/min)
AUClast
(ng/mL/min)
Kel MRT
(min)
1 Oral tablet
(Immediate
release)
29.113 302.4 120 9576.57 7847.87
0.00229 429.56
2 Oral hydrogel
(Controlled
release)
31.8317 974.4 720 66568.2 55713.2
0.000711
1687.02
The result is significant at p<0.05
100
Mean plasma concentrations of perindopril achieved after administration of
conventional tablet and CA/MA hydrogel, was plotted against time as given in the
Figure 4.24.
The absorption of perindopril upon administration of conventional tablet oral solution
was immediate as compared to hydrogel disks. Cmax from oral solution was found at
29.11ng/mL in 120 min whereas Cmax of oral hydrogel disks was achieved at 31.83
in 720 min.
Statistical data provides a significant difference with the F ratio value 5.1. The p-
value is 0.034 which clearly represents that the result is significant at p<0.05.
Hydrogel disks provided drug concentration for an enhanced period of time as
compared to oral conventional tablet. It was clarified from the values of MRT (mean
residence time) of perindopril found in hydrogels and oral conventional tablet that
MRT of perindopril in hydrogel formulations was observed almost 4 times greater
than the MRT of perindopril in oral conventional tablet.
It was revealed that the peak plasma concentration of the hydrogel was found greater
than the oral tablet while the values of Kel for hydrogel formulation were less than the
conventional oral tablet. Distinct difference in AUC values of hydrogel formulation
and oral conventional tablet was seen. It clearly gives a clue about the improved
bioavailability of perindopril erbumine from oral hydrogel owing to the deletion of
first pass effect as compared to oral conventional tablet. So it can be elucidated that
oral hydrogel represented better therapy for the prolonged period of time to manage
hypertension.
101
Figure 4.22. Chromatogram of blank plasma.
102
Figure 4.23. Chromatogram of plasma spiked perindopril (16 ng/mL) and
internal standard perindoprilat (8 ng/mL).
103
Figure 4.24. Mean serum data of Perindopril erbumine in the rabbits, after
delivery of oral hydrogel and tablet.
0
5
10
15
20
25
30
35
0 500 1000 1500 2000 2500 3000 3500
Ser
um
con
cen
trati
on
(n
g/m
l)
Time (minutes)
In vivo bioavailability
Hydrogel
Tablet
104
5. CONCLUSION
Crosslinked pH sensitive PVA/MA and CA-g-MA hydrogels were developed by
blending tuned concentrations of PVA, CA, MA and glyoxal (40% water solution).
An appreciable swelling was observed in the CA-g-MA formulations containing
higher amounts of MA and lower amounts of crosslinker. In alkaline media
formulations exhibited maximum swelling due to enhanced force of repulsion in
chains and presence of increased ionized carboxylic groups which created spaces in
the structure. Drug release behavior in CA-g-MA formulations was also found to be
dependent on the concentrations of formulation composites. Developed formulations
of PVA/MA hydrogel exhibited an enhanced impact on swelling, porosity, sol-gel and
drug release behaviour by increasing the concentration of MA, while a declined
impact on swelling and drug release was observed on increasing the concentration of
crosslinker. FTIR of CA-g-MA hydrogel confirms the hydrogel structure while DSC
and TGA findings gave an idea of thermally stable hydrogel structure. Surface gaps
in the structure of blank and drug loaded CA-g-MA formulations were clearly
exposed through SEM. PVA/MA hydrogel was also characterized by FTIR, TGA,
DSC and additional feature of XRD to study the characteristics changes in hydrogels.
The SEM of PVA/MA hydrogel determined the porous structure of hydrogel. It was
also evident that crosslinked hydrogel exhibited better thermal stability. The results
suggested that PVA/MA and CA-g-MA hydrogels can be deployed in pH sensitive
controlled release vehicle for drug delivery owing to swelling and prolonged drug
release characteristics in alkaline pH.
105
6. REFERENCES
1. Majid Moosavi, and Nima Soltani, 'Prediction of the Specific Volume of
Polymeric Systems Using the Artificial Neural Network-Group Contribution
Method', Fluid Phase Equilibria, 356 (2013), 176-184.
2. Kathryn E Uhrich, Scott M Cannizzaro, Robert S Langer, and Kevin M
Shakesheff, 'Polymeric Systems for Controlled Drug Release', Chemical Reviews,
99 (1999), 3181-3198.
3. Carmen Alvarez-Lorenzo, Barbara Blanco-Fernandez, Ana M Puga, and Angel
Concheiro, 'Crosslinked Ionic Polysaccharides for Stimuli-Sensitive Drug
Delivery', Advanced drug delivery reviews, 65 (2013), 1148-1171.
4. Minh Khanh Nguyen, and Eben Alsberg, 'Bioactive Factor Delivery Strategies
from Engineered Polymer Hydrogels for Therapeutic Medicine', Progress in
Polymer Science, 39 (2014), 1235-1265.
5. Piyush Gupta, Kavita Vermani, and Sanjay Garg, 'Hydrogels: From Controlled
Release to pH-Responsive Drug Delivery', Drug discovery today, 7 (2002), 569-
579.
6. Yury E Shapiro, 'Structure and Dynamics of Hydrogels and Organogels: An Nmr
Spectroscopy Approach', Progress in Polymer Science, 36 (2011), 1184-1253.
7. Chunming Wang, Rohan R Varshney, and Dong-An Wang, 'Therapeutic Cell
Delivery and Fate Control in Hydrogels and Hydrogel Hybrids', Advanced drug
delivery reviews, 62 (2010), 699-710.
8. Tina Vermonden, Roberta Censi, and Wim E Hennink, 'Hydrogels for Protein
Delivery', Chemical Reviews, 112 (2012), 2853-2888.
9. Qiang Chen, Lin Zhu, Chao Zhao, Qiuming Wang, and Jie Zheng, 'A Robust,
One‐Pot Synthesis of Highly Mechanical and Recoverable Double Network
106
Hydrogels Using Thermoreversible Sol‐Gel Polysaccharide', Advanced Materials,
25 (2013), 4171-4176.
10. Adam J Singer, and Alexander B Dagum, 'Current Management of Acute
Cutaneous Wounds', New England Journal of Medicine, 359 (2008), 1037-1046.
11. Jeong-A Yang, Junseok Yeom, Byung Woo Hwang, Allan S Hoffman, and Sei
Kwang Hahn, 'In Situ-Forming Injectable Hydrogels for Regenerative Medicine',
Progress in Polymer Science, 39 (2014), 1973-1986.
12. Nermin Seda Kehr, Eko Adi Prasetyanto, Kathrin Benson, Bahar Ergün, Anzhela
Galstyan, and Hans‐Joachim Galla, 'Periodic Mesoporous Organosilica‐Based
Nanocomposite Hydrogels as Three‐Dimensional Scaffolds', Angewandte Chemie,
125 (2013), 1194-1198.
13. Mehrdad Hamidi, Amir Azadi, and Pedram Rafiei, 'Hydrogel Nanoparticles in
Drug Delivery', Advanced drug delivery reviews, 60 (2008), 1638-1649.
14. B Falk, S Garramone, and S Shivkumar, 'Diffusion Coefficient of Paracetamol in
a Chitosan Hydrogel', Materials Letters, 58 (2004), 3261-3265.
15. WE Hennink, and CF van Nostrum, 'Novel Crosslinking Methods to Design
Hydrogels', Advanced drug delivery reviews, 54 (2002), 13-36.
16. Yanjiao Jiang, Jing Chen, Chao Deng, Erik J Suuronen, and Zhiyuan Zhong,
'Click Hydrogels, Microgels and Nanogels: Emerging Platforms for Drug
Delivery and Tissue Engineering', Biomaterials, 35 (2014), 4969-4985.
17. WE Hennink, and C_F Van Nostrum, 'Novel Crosslinking Methods to Design
Hydrogels', Advanced drug delivery reviews, 64 (2012), 223-236.
18. Yong Qiu, and Kinam Park, 'Environment-Sensitive Hydrogels for Drug
Delivery', Advanced drug delivery reviews, 53 (2001), 321-339.
107
19. Mehlika Pulat, and Hediye İrem Özgündüz, 'Swelling Behavior and
Morphological Properties of Semi-Ipn Hydrogels Based on Ionic and Non-Ionic
Components', Bio-medical materials and engineering, 24 (2014), 1725-1733.
20. Mehlika Pulat, Anıl Sera Kahraman, Nur Tan, and Menemşe Gümüşderelioğlu,
'Sequential Antibiotic and Growth Factor Releasing Chitosan-Paam Semi-Ipn
Hydrogel as a Novel Wound Dressing', Journal of Biomaterials Science, Polymer
Edition, 24 (2013), 807-819.
21. Subham Banerjee, Lubna Siddiqui, Shiv Sankar Bhattacharya, Santanu Kaity,
Animesh Ghosh, Pronobesh Chattopadhyay, Anurag Pandey, and Lokendra Singh,
'Interpenetrating Polymer Network (Ipn) Hydrogel Microspheres for Oral
Controlled Release Application', International journal of biological
macromolecules, 50 (2012), 198-206.
22. Minh Khanh Nguyen, and Eben Alsberg, 'Bioactive Factor Delivery Strategies
from Engineered Polymer Hydrogels for Therapeutic Medicine', Progress in
Polymer Science, 39 (2014), 1235-1265.
23. ER Edelman, J Kost, H Bobeck, and R Langer, 'Regulation of Drug Release from
Polymer Matrices by Oscillating Magnetic Fields', Journal of biomedical
materials research, 19 (1985), 67-83.
24. JA Tamada, and R Langer, 'Erosion Kinetics of Hydrolytically Degradable
Polymers', Proceedings of the National Academy of Sciences, 90 (1993), 552-556.
25. Lakshmi S Nair, and Cato T Laurencin, 'Biodegradable Polymers as Biomaterials',
Progress in Polymer Science, 32 (2007), 762-798.
26. Haesun Park, Kinam Park, and Waleed SW Shalaby, Biodegradable Hydrogels
for Drug DeliveryCRC Press, 2011).
108
27. Jinhyun Hannah Lee, and Yoon Yeo, 'Controlled Drug Release from
Pharmaceutical Nanocarriers', Chemical engineering science, 125 (2015), 75-84.
28. Katsuhiko Ariga, Yuri M Lvov, Kohsaku Kawakami, Qingmin Ji, and Jonathan P
Hill, 'Layer-by-Layer Self-Assembled Shells for Drug Delivery', Advanced drug
delivery reviews, 63 (2011), 762-771
29. Fredy Munoz, Gursel Alici, and Weihua Li, 'A Review of Drug Delivery Systems
for Capsule Endoscopy', Advanced drug delivery reviews, 71 (2014), 77-85.
30. Iyabo Oladunni Babasola, Wei Zhang, and Brian G Amsden, 'Osmotic Pressure
Driven Protein Release from Viscous Liquid, Hydrophobic Polymers Based on 5-
Ethylene Ketal Ε-Caprolactone: Potential and Mechanism', European journal of
pharmaceutics and biopharmaceutics, 85 (2013), 765-772.
31. Rubén Machín, José Ramón Isasi, and Itziar Vélaz, 'Hydrogel Matrices
Containing Single and Mixed Natural Cyclodextrins. Mechanisms of Drug
Release', European polymer journal, 49 (2013), 3912-3920.
32. Anil Kumar, Xu Zhang, and Xing-Jie Liang, 'Gold Nanoparticles: Emerging
Paradigm for Targeted Drug Delivery System', Biotechnology advances, 31
(2013), 593-606.
33. Nadiah Zafar, Hatem Fessi, and Abdelhamid Elaissari, 'Cyclodextrin Containing
Biodegradable Particles: From Preparation to Drug Delivery Applications',
International journal of pharmaceutics, 461 (2014), 351-366.
34. Liangrong Yang, Chen Guo, Lianwei Jia, Xiangfeng Liang, Chunzhao Liu, and
Huizhou Liu, 'Dual Responsive Copolymer Micelles for Drug Controlled Release',
Journal of colloid and interface science, 350 (2010), 22-29.
35. CE Mora-Huertas, H Fessi, and A Elaissari, 'Polymer-Based Nanocapsules for
Drug Delivery', International journal of pharmaceutics, 385 (2010), 113-142.
109
36. David M Webster, Padma Sundaram, and Mark E Byrne, 'Injectable
Nanomaterials for Drug Delivery: Carriers, Targeting Moieties, and Therapeutics',
European journal of pharmaceutics and biopharmaceutics, 84 (2013), 1-20.
37. Theresa M Allen, and Pieter R Cullis, 'Liposomal Drug Delivery Systems: From
Concept to Clinical Applications', Advanced drug delivery reviews, 65 (2013), 36-
48.
38. Jayanth Panyam, Sanjeeb K Sahoo, Swayam Prabha, Tom Bargar, and Vinod
Labhasetwar, 'Fluorescence and Electron Microscopy Probes for Cellular and
Tissue Uptake of Poly (D, L-Lactide-Co-Glycolide) Nanoparticles', International
journal of pharmaceutics, 262 (2003), 1-11.
39. Gemma Vilar, Judit Tulla-Puche, and Fernando Albericio, 'Polymers and Drug
Delivery Systems', Current Drug Delivery, 9 (2012), 367-94.
40. Takashi Sasaki, Hiroaki Matsuura, and Kazuki Tanaka, 'Preparation and Drug-
Release Kinetics of Porous Poly (L-Lactic Acid)/Rifampicin Blend Particles',
ISRN Polymer Science, 2014 (2014).
41. Subham Banerjee, Lubna Siddiqui, Shiv Sankar Bhattacharya, Santanu Kaity,
Animesh Ghosh, Pronobesh Chattopadhyay, Anurag Pandey, and Lokendra Singh,
'Interpenetrating Polymer Network (Ipn) Hydrogel Microspheres for Oral
Controlled Release Application', International journal of biological
macromolecules, 50 (2012), 198-206.
42. Alka Lohani, Garima Singh, Shiv Sankar Bhattacharya, and Anurag Verma,
'Interpenetrating Polymer Networks as Innovative Drug Delivery Systems',
Journal of drug delivery, 2014 (2014).
110
43. Santanu Kaity, Jinu Isaac, and Animesh Ghosh, 'Interpenetrating Polymer
Network of Locust Bean Gum-Poly (Vinyl Alcohol) for Controlled Release Drug
Delivery', Carbohydrate polymers, 94 (2013), 456-467.
44. Murugesh Shivashankar, and Badal Kumar Mandal, 'A Review on
Interpenetrating Polymer Network', Int. J. Phram. Phram. Sci, 4 (2012), 1-7.
45. Robert Price, Azadeh Poursaid, and Hamidreza Ghandehari, 'Controlled Release
from Recombinant Polymers', Journal of controlled release, 190 (2014), 304-313.
46. Eun Seok Gil, and Samuel M Hudson, 'Stimuli-Reponsive Polymers and Their
Bioconjugates', Progress in Polymer Science, 29 (2004), 1173-1222.
47. Dirk Schmaljohann, 'Thermo-and Ph-Responsive Polymers in Drug Delivery',
Advanced drug delivery reviews, 58 (2006), 1655-1670.
48. Martin Malmsten, and Bjoern Lindman, 'Self-Assembly in Aqueous Block
Copolymer Solutions', Macromolecules, 25 (1992), 5440-5445.
49. Howard G Schild, 'Poly (N-Isopropylacrylamide): Experiment, Theory and
Application', Progress in Polymer Science, 17 (1992), 163-249.
50. Atul R Khare, and Nikolaos A Peppas, 'Swelling/Deswelling of Anionic
Copolymer Gels', Biomaterials, 16 (1995), 559-567.
51. NA Peppas, P Bures, W Leobandung, and H Ichikawa, 'Hydrogels in
Pharmaceutical Formulations', European journal of pharmaceutics and
biopharmaceutics, 50 (2000), 27-46.
52. Yong Qiu, and Kinam Park, 'Environment-Sensitive Hydrogels for Drug
Delivery', Advanced drug delivery reviews, 53 (2001), 321-339.
53. Noriyasu Kamei, Mariko Morishita, Hitomi Chiba, Nikhil J Kavimandan,
Nicholas A Peppas, and Kozo Takayama, 'Complexation Hydrogels for Intestinal
111
Delivery of Interferon Β and Calcitonin', Journal of controlled release, 134
(2009), 98-102.
54. Madeline Torres-Lugo, and Nikolaos A Peppas, 'Molecular Design and in Vitro
Studies of Novel Ph-Sensitive Hydrogels for the Oral Delivery of Calcitonin',
Macromolecules, 32 (1999), 6646-6651.
55. AM Lowman, M Morishita, M Kajita, T Nagai, and NA Peppas, 'Oral Delivery of
Insulin Using Ph‐Responsive Complexation Gels', Journal of pharmaceutical
sciences, 88 (1999), 933-937.
56. Eve Ruel-Gariepy, and Jean-Christophe Leroux, 'In Situ-Forming Hydrogels
Review of Temperature-Sensitive Systems', European journal of pharmaceutics
and biopharmaceutics, 58 (2004), 409-426.
57. Paschalis Alexandridis, Josef F Holzwarth, and T Alan Hatton, 'Micellization of
Poly (Ethylene Oxide)-Poly (Propylene Oxide)-Poly (Ethylene Oxide) Triblock
Copolymers in Aqueous Solutions: Thermodynamics of Copolymer Association',
Macromolecules, 27 (1994), 2414-2425.
58. MDC Topp, PJ Dijkstra, H Talsma, and J Feijen, 'Thermosensitive Micelle-
Forming Block Copolymers of Poly (Ethylene Glycol) and Poly (N-
Isopropylacrylamide)', Macromolecules, 30 (1997), 8518-8520.
59. Hyejung Mok, and Tae Gwan Park, 'Functional Polymers for Targeted Delivery of
Nucleic Acid Drugs', Macromolecular bioscience, 9 (2009), 731-743.
60. Michael A Gosselin, Wenjin Guo, and Robert J Lee, 'Efficient Gene Transfer
Using Reversibly Cross-Linked Low Molecular Weight Polyethylenimine',
Bioconjugate chemistry, 12 (2001), 989-994.
112
61. WT Godbey, Kenneth K Wu, and Antonios G Mikos, 'Size Matters: Molecular
Weight Affects the Efficiency of Poly (Ethyleneimine) as a Gene Delivery
Vehicle', Journal of biomedical materials research, 45 (1999), 268-275.
62. Toyoichi Tanaka, 'Collapse of Gels and the Critical Endpoint', Physical Review
Letters, 40 (1978), 820.
63. Michael Heskins, and James E Guillet, 'Solution Properties of Poly (N-
Isopropylacrylamide)', Journal of Macromolecular Science—Chemistry, 2 (1968),
1441-1455.
64. You Han Bae, Teruo Okano, and Sung Wan Kim, 'Temperature Dependence of
Swelling of Crosslinked Poly (N, N′‐Alkyl Substituted Acrylamides) in Water',
Journal of Polymer Science Part B: Polymer Physics, 28 (1990), 923-936.
65. Leda Klouda, and Antonios G Mikos, 'Thermoresponsive Hydrogels in
Biomedical Applications', European journal of pharmaceutics and
biopharmaceutics, 68 (2008), 34-45.
66. Hiroshi Inomata, Shuichi Goto, and Shozaburo Saito, 'Phase Transition of N-
Substituted Acrylamide Gels', Macromolecules, 23 (1990), 4887-4888.
67. Narayan Bhattarai, Hassna R Ramay, Jonathan Gunn, Frederick A Matsen, and
Miqin Zhang, 'Peg-Grafted Chitosan as an Injectable Thermosensitive Hydrogel
for Sustained Protein Release', Journal of controlled release, 103 (2005), 609-
624.
68. Sushmita Mukherjee, Richik N Ghosh, and Frederick R Maxfield, 'Endocytosis',
Physiological reviews, 77 (1997), 759-803.
69. Ira Mellman, 'Endocytosis and Molecular Sorting', Annual review of cell and
developmental biology, 12 (1996), 575-625.
113
70. Srinivas Ganta, Harikrishna Devalapally, Aliasgar Shahiwala, and Mansoor Amiji,
'A Review of Stimuli-Responsive Nanocarriers for Drug and Gene Delivery',
Journal of controlled release, 126 (2008), 187-204.
71. Peter Vaupel, Friedrich Kallinowski, and Paul Okunieff, 'Blood Flow, Oxygen
and Nutrient Supply, and Metabolic Microenvironment of Human Tumors: A
Review', Cancer research, 49 (1989), 6449-6465.
72. Smadar Cohen, Toshio Yoshioka, Melissa Lucarelli, Lena H Hwang, and Robert
Langer, 'Controlled Delivery Systems for Proteins Based on Poly (Lactic/Glycolic
Acid) Microspheres', Pharmaceutical research, 8 (1991), 713-720.
73. Edith Mathiowitz, Jules S Jacob, Yong S Jong, Gerardo P Carino, Donald E
Chickering, Pravin Chaturvedi, Camilla A Santos, Kavita Vijayaraghavan, Sean
Montgomery, and Michael Bassett, 'Biologically Erodable Microspheres as
Potential Oral Drug Delivery Systems', Nature, 386 (1997), 410-414.
74. KW Leong, BC Brott, and R Langer, 'Bioerodible Polyanhydrides as Drug‐Carrier
Matrices. I: Characterization, Degradation, and Release Characteristics', Journal
of biomedical materials research, 19 (1985), 941-955.
75. Dinesh Shenoy, Steven Little, Robert Langer, and Mansoor Amiji, 'Poly (Ethylene
Oxide)-Modified Poly (Β-Amino Ester) Nanoparticles as a Ph-Sensitive System
for Tumor-Targeted Delivery of Hydrophobic Drugs. 1. In Vitro Evaluations',
Molecular pharmaceutics, 2 (2005), 357-366.
76. Dinesh Shenoy, Steven Little, Robert Langer, and Mansoor Amiji, 'Poly (Ethylene
Oxide)-Modified Poly (Β-Amino Ester) Nanoparticles as a Ph-Sensitive System
for Tumor-Targeted Delivery of Hydrophobic Drugs: Part 2. In Vivo Distribution
and Tumor Localization Studies', Pharmaceutical research, 22 (2005), 2107-
2114.
114
77. Ruth Duncan, 'The Dawning Era of Polymer Therapeutics', Nature reviews Drug
discovery, 2 (2003), 347-360.
78. Ruth Duncan, 'Polymer Conjugates as Anticancer Nanomedicines', Nature
Reviews Cancer, 6 (2006), 688-701.
79. MJ Roberts, MD Bentley, and JM Harris, 'Chemistry for Peptide and Protein
Pegylation', Advanced drug delivery reviews, 54 (2002), 459-476.
80. Karina L Heredia, and Heather D Maynard, 'Synthesis of Protein–Polymer
Conjugates', Organic & biomolecular chemistry, 5 (2006), 45-53.
81. Debora Bontempo, and Heather D Maynard, 'Streptavidin as a Macroinitiator for
Polymerization: In Situ Protein-Polymer Conjugate Formation', Journal of the
American Chemical Society, 127 (2005), 6508-6509.
82. Cyrille Boyer, Volga Bulmus, Jingquan Liu, Thomas P Davis, Martina H Stenzel,
and Christopher Barner-Kowollik, 'Well-Defined Protein-Polymer Conjugates Via
in Situ Raft Polymerization', Journal of the American Chemical Society, 129
(2007), 7145-7154.
83. Todd R Hoare, and Daniel S Kohane, 'Hydrogels in Drug Delivery: Progress and
Challenges', Polymer, 49 (2008), 1993-2007.
84. Enrica Calo, and Vitaliy V Khutoryanskiy, 'Biomedical Applications of
Hydrogels: A Review of Patents and Commercial Products', European polymer
journal, 65 (2015), 252-267.
85. Jindrich Kopecek, 'Hydrogel Biomaterials: A Smart Future?', Biomaterials, 28
(2007), 5185-5192.
86. Jennifer Elisseeff, 'Hydrogels: Structure Starts to Gel', Nature Materials, 7 (2008),
271-273.
115
87. Gwi-Taek Jeong, Kyoung-Min Lee, Hee-Seung Yang, Seok-Hwan Park, Jae-Hee
Park, Changshin Sunwoo, Hwa-Won Ryu, Doman Kim, Woo-Tae Lee, and Hae-
Sung Kim, 'Synthesis of Poly (Sorbitan Methacrylate) Hydrogel by Free-Radical
Polymerization', Applied biochemistry and biotechnology, 137 (2007), 935-946.
88. Miguel AD Gonçalves, Virgínia D Pinto, Rita AS Costa, Rolando Dias, Julio C
Hernándes‐Ortiz, and Mário Rui PFN Costa, 'Stimuli‐Responsive Hydrogels
Synthesis Using Free Radical and Raft Polymerization', in Macromolecular
SymposiaWiley Online Library, 2013), 41-54.
89. Faheem Ullah, Muhammad Bisyrul Hafi Othman, Fatima Javed, Zulkifli Ahmad,
and Hazizan Md Akil, 'Classification, Processing and Application of Hydrogels: A
Review', Materials Science and Engineering: C, 57 (2015), 414-433.
90. Huai-Ping Cong, Ping Wang, and Shu-Hong Yu, 'Stretchable and Self-Healing
Graphene Oxide–Polymer Composite Hydrogels: A Dual-Network Design',
Chemistry of materials, 25 (2013), 3357-3362.
91. Manssur Yalpani, Polysaccharides: Syntheses, Modifications and
Structure/Property Relations. Vol. 36Elsevier, 2013).
92. Iwona Gibas, and Helena Janik, 'Review: Synthetic Polymer Hydrogels for
Biomedical Applications', 4 (2010), 297–304.
93. Giuliana Gorrasi, Valeria Bugatti, and Vittoria Vittoria, 'Pectins Filled with Ldh-
Antimicrobial Molecules: Preparation, Characterization and Physical Properties',
Carbohydrate polymers, 89 (2012), 132-137.
94. B Thu, P Bruheim, T Espevik, O Smidsrød, P Soon-Shiong, and G Skjåk-Bræk,
'Alginate Polycation Microcapsules: I. Interaction between Alginate and
Polycation', Biomaterials, 17 (1996), 1031-1040.
116
95. Nasim Annabi, Kelly Tsang, Suzanne M Mithieux, Mehdi Nikkhah, Afshin
Ameri, Ali Khademhosseini, and Anthony S Weiss, 'Highly Elastic
Micropatterned Hydrogel for Engineering Functional Cardiac Tissue', Advanced
Functional Materials, 23 (2013), 4950-4959.
96. YuXi Zhang, FeiPeng Wu, MiaoZhen Li, and ErJian Wang, 'Ph Switching on-Off
Semi-Ipn Hydrogel Based on Cross-Linked Poly (Acrylamide-Co-Acrylic Acid)
and Linear Polyallyamine', Polymer, 46 (2005), 7695-7700.
97. Biancamaria Baroli, 'Photopolymerization of Biomaterials: Issues and
Potentialities in Drug Delivery, Tissue Engineering, and Cell Encapsulation
Applications', Journal of Chemical Technology and Biotechnology, 81 (2006),
491-499.
98. Yea Garcia, Russell Collighan, Martin Griffin, and A Pandit, 'Assessment of Cell
Viability in a Three-Dimensional Enzymatically Cross-Linked Collagen Scaffold',
Journal of Materials Science: Materials in Medicine, 18 (2007), 1991-2001.
99. Takashi Iizawa, Hatsumi Taketa, Makoto Maruta, Takashi Ishido, Takehiko
Gotoh, and Shuji Sakohara, 'Synthesis of Porous Poly (N‐Isopropylacrylamide)
Gel Beads by Sedimentation Polymerization and Their Morphology', Journal of
applied polymer science, 104 (2007), 842-850.
100. Jongdoo Lim, Abdellatif Chouai, Su-Tang Lo, Wei Liu, Xiankai Sun, and Eric E
Simanek, 'Design, Synthesis, Characterization, and Biological Evaluation of
Triazine Dendrimers Bearing Paclitaxel Using Ester and Ester/Disulfide
Linkages', Bioconjugate chemistry, 20 (2009), 2154-2161.
101. ChangYang Gong, Shuai Shi, PengWei Dong, Bing Kan, MaLing Gou, XianHuo
Wang, XingYi Li, Feng Luo, Xia Zhao, and YuQuan Wei, 'Synthesis and
117
Characterization of Peg-Pcl-Peg Thermosensitive Hydrogel', International journal
of pharmaceutics, 365 (2009), 89-99.
102. Mario Casolaro, Ilaria Casolaro, Severino Bottari, Barbara Del Bello, Emilia
Maellaro, and Konstantinos D Demadis, 'Long-Term Doxorubicin Release from
Multiple Stimuli-Responsive Hydrogels Based on Α-Amino-Acid Residues',
European journal of pharmaceutics and biopharmaceutics, 88 (2014), 424-433.
103. Seong Il Kang, and You Han Bae, 'A Sulfonamide Based Glucose-Responsive
Hydrogel with Covalently Immobilized Glucose Oxidase and Catalase', Journal of
controlled release, 86 (2003), 115-121.
104. N Kashyap, N Kumar, and MNV Ravi Kumar, 'Hydrogels for Pharmaceutical and
Biomedical Applications', Critical Reviews™ in Therapeutic Drug Carrier
Systems, 22 (2005), 107-150.
105. John P Baker, David R Stephens, Harvey W Blanch, and John M Prausnitz,
'Swelling Equilibria for Acrylamide-Based Polyampholyte Hydrogels',
Macromolecules, 25 (1992), 1955-1958.
106. Young-Chang Nho, Sung-Eun Park, Hyung-Il Kim, and Taek-Sung Hwang,
'Retracted: Oral Delivery of Insulin Using Ph-Sensitive Hydrogels Based on
Polyvinyl Alcohol Grafted with Acrylic Acid/Methacrylic Acid by Radiation',
Nuclear Instruments and Methods in Physics Research Section B: Beam
Interactions with Materials and Atoms, 236 (2005), 283-288.
107. A Ariffin, MS Musa, MBH Othman, MAA Razali, and F Yunus, 'Effects of
Various Fillers on Anionic Polyacrylamide Systems for Treating Kaolin
Suspensions', Colloids and Surfaces A: Physicochemical and Engineering
Aspects, 441 (2014), 306-311.
118
108. Frédéric Bossard, Thierry Aubry, Georgios Gotzamanis, and Constantinos
Tsitsilianis, 'Ph-Tunable Rheological Properties of a Telechelic Cationic
Polyelectrolyte Reversible Hydrogel', Soft Matter, 2 (2006), 510-516.
109. Mohammad Sadeghi, and Hossein Hosseinzadeh, 'Synthesis of Starch—Poly
(Sodium Acrylate-Co-Acrylamide) Superabsorbent Hydrogel with Salt and Ph-
Responsiveness Properties as a Drug Delivery System', Journal of bioactive and
compatible polymers, 23 (2008), 381-404.
110. Dena Dorniani, Aminu Umar Kura, Mohd Zobir Bin Hussein, Sharida Fakurazi,
Abdul Halim Shaari, and Zalinah Ahmad, 'Controlled-Release Formulation of
Perindopril Erbumine Loaded Peg-Coated Magnetite Nanoparticles for
Biomedical Applications', Journal of Materials Science, 49 (2014), 8487-8497.
111. Edmund H Sonnenblick, 'Perindopril Treatment for Congestive Heart Failure', The
American journal of cardiology, 88 (2001), 19-27.
112. Suzanne Oparil, 'Efficacy of Perindopril in the Treatment of Systemic
Hypertension', The American journal of cardiology, 88 (2001), 3-12.
113. Deepak S Jain, Gunta Subbaiah, Mallika Sanyal, Umesh C Pande, and Pranav
Shrivastav, 'First Lc–Ms/Ms Electrospray Ionization Validated Method for the
Quantification of Perindopril and Its Metabolite Perindoprilat in Human Plasma
and Its Application to Bioequivalence Study', Journal of Chromatography B, 837
(2006), 92-100.
114. Miriam Hurst, and Blair Jarvis, 'Perindopril', Drugs, 61 (2001), 867-896.
115. Merit-HF Study Group, 'Effect of Metoprolol Cr/Xl in Chronic Heart Failure:
Metoprolol Cr/Xl Randomised Intervention Trial in-Congestive Heart Failure
(Merit-Hf)', The Lancet, 353 (1999), 2001-2007.
119
116. Martin John Kendall, Simon RJ Maxwell, Anders Sandberg, and Gudrun
Westergren, 'Controlled Release Metoprolol', Clinical pharmacokinetics, 21
(1991), 319-330.
117. David L Geffner, and Jerome M Hershman, 'Β-Adrenergic Blockade for the
Treatment of Hyperthyroidism', The American journal of medicine, 93 (1992), 61-
68.
118. Mauro Biffi, Giuseppe Boriani, Paolo Sabbatani, Gabriele Bronzetti, Lorenzo
Frabetti, Romano Zannoli, Angelo Branzi, and Bruno Magnani, 'Malignant
Vasovagal Syncope: A Randomised Trial of Metoprolol and Clonidine', Heart, 77
(1997), 268-272.
119. Amitabh Prakash, and Anthony Markham, 'Metoprolol', Drugs, 60 (2000), 647-
678.
120. NM Ranjha, 'Synthesis and Characterization of Noncrosslinked and Crosslinked
Poly (Vinyl Alcohol-Co-Crotonic Acid) Hydrogels', Saudi Pharmaceutical
Journal, 7 (1999), 130-136.
121. Xiuyu Li, Wenhui Wu, Jianquan Wang, and Yufeng Duan, 'The Swelling
Behavior and Network Parameters of Guar Gum/Poly (Acrylic Acid) Semi-
Interpenetrating Polymer Network Hydrogels', Carbohydrate polymers, 66 (2006),
473-479.
122. Liesbeth Vervoort, Guy Van den Mooter, Patrick Augustijns, and Renaat Kinget,
'Inulin Hydrogels. I. Dynamic and Equilibrium Swelling Properties', International
journal of pharmaceutics, 172 (1998), 127-135.
123. Crank J (1975) The mathematics of diffusion, 2nd edn. Clarendon, Great Britain.
124. Flory PJ (1953) Principles of polymer chemistry, Cornell University, Ithaca New
York.
120
125. NA Peppas, Y Huang, M Torres-Lugo, JH Ward, and J Zhang, 'Physicochemical
Foundations and Structural Design of Hydrogels in Medicine and Biology',
Annual review of biomedical engineering, 2 (2000), 9-29.
126. Safaa G Abd Alla, Horia M Nizam El-Din, and Abdel Wahab M El-Naggar,
'Structure and Swelling-Release Behaviour of Poly (Vinyl Pyrrolidone)(Pvp) and
Acrylic Acid (Aac) Copolymer Hydrogels Prepared by Gamma Irradiation',
European polymer journal, 43 (2007), 2987-2998.
127. Ashwani Goyal, Manju Nagpal, Shikha Bhalla, and Gitika Arora Dhingra,
'Superporous Hydrogel Composites of 2 Acrylamide Using Starch-Silicone
Dioxide 3 Coprecipitate as Composite Agent 4', British Journal of
Pharmaceutical Research, 4 (2014), 338-351.
128. Nazar M Ranjha, Gohar Ayub, Shahzad Naseem, and Muhammad Tayyab Ansari,
'Preparation and Characterization of Hybrid Ph-Sensitive Hydrogels of Chitosan-
Co-Acrylic Acid for Controlled Release of Verapamil', Journal of Materials
Science: Materials in Medicine, 21 (2010), 2805-2816.
129. Zhi‐Lan Liu, Han Hu, and Ren‐Xi Zhuo, 'Konjac Glucomannan‐Graft‐Acrylic
Acid Hydrogels Containing Azo Crosslinker for Colon‐Specific Delivery', Journal
of Polymer Science Part A: Polymer Chemistry, 42 (2004), 4370-4378.
130. Jan Golebiewski, and Andrzej Galeski, 'Thermal Stability of Nanoclay
Polypropylene Composites by Simultaneous Dsc and Tga', Composites Science
and Technology, 67 (2007), 3442-3447.
131. José S Torrecilla, Ester Rojo, Juan C Domínguez, and Francisco Rodríguez,
'Chaotic Parameters and Their Role in Quantifying Noise in the Output Signals
from Uv, Tga and Dsc Apparatus', Talanta, 79 (2009), 665-668.
121
132. Ramón Artiaga, Salvador Naya, A Garcia, F Barbadillo, and Laura García,
'Subtracting the Water Effect from Dsc Curves by Using Simultaneous Tga Data',
Thermochimica acta, 428 (2005), 137-139.
133. Bo Li, Gang Chen, Hui Zhang, and Changdong Sheng, 'Development of Non-
Isothermal Tga–Dsc for Kinetics Analysis of Low Temperature Coal Oxidation
Prior to Ignition', Fuel, 118 (2014), 385-391.
134. Vittorio Berbenni, and Amedeo Marini, 'Oxidation Behaviour of Mechanically
Activated Mn 3 O 4 by Tga/Dsc/Xrpd', Materials research bulletin, 38 (2003),
1859-1866.
135. Pei Sun, Z Zak Fang, Mark Koopman, James Paramore, KS Ravi Chandran, Yang
Ren, and Jun Lu, 'An Experimental Study of the (Ti–6al–4v)–Xh Phase Diagram
Using in Situ Synchrotron Xrd and Tga/Dsc Techniques', Acta Materialia, 84
(2015), 29-41.
136. CaiBing Liu, ChangYang Gong, YiFeng Pan, YangDe Zhang, JiWei Wang,
MeiJuan Huang, YongSheng Wang, Ke Wang, MaLing Gou, and MingJing Tu,
'Synthesis and Characterization of a Thermosensitive Hydrogel Based on
Biodegradable Amphiphilic Pcl-Pluronic (L35)-Pcl Block Copolymers', Colloids
and Surfaces A: Physicochemical and Engineering Aspects, 302 (2007), 430-438.
137. Jahanzeb Mudassir, and Nazar Mohammad Ranjha, 'Dynamic and Equilibrium
Swelling Studies: Crosslinked Ph Sensitive Methyl Methacrylate-Co-Itaconic
Acid (Mma-Co-Ia) Hydrogels', Journal of polymer research, 15 (2008), 195-203.
138. Laura Serra, Joseph Doménech, and Nicholas A Peppas, 'Drug Transport
Mechanisms and Release Kinetics from Molecularly Designed Poly (Acrylic
Acid-G-Ethylene Glycol) Hydrogels', Biomaterials, 27 (2006), 5440-5451.
122
139. Ulf Siemoneit, Christoph Schmitt, Carmen Alvarez-Lorenzo, Asteria Luzardo,
Francisco Otero-Espinar, Angel Concheiro, and José Blanco-Méndez,
'Acrylic/Cyclodextrin Hydrogels with Enhanced Drug Loading and Sustained
Release Capability', International journal of pharmaceutics, 312 (2006), 66-74.
140. KSV Krishna Rao, B Vijaya Kumar Naidu, MCS Subha, M Sairam, and TM
Aminabhavi, 'Novel Chitosan-Based Ph-Sensitive Interpenetrating Network
Microgels for the Controlled Release of Cefadroxil', Carbohydrate polymers, 66
(2006), 333-344.
141. AFR Pimenta, J Ascenso, JCS Fernandes, R Colaço, AP Serro, and B Saramago,
'Controlled Drug Release from Hydrogels for Contact Lenses: Drug Partitioning
and Diffusion', International journal of pharmaceutics, 515 (2016), 467-475.
142. N Najib, and MS Suleiman, 'The Kinetics of Drug Release from Ethylcellulose
Solid Dispersions', Drug Development and Industrial Pharmacy, 11 (1985), 2169-
2181.
143. Mallinath S Birajdar, and Jonghwi Lee, 'Sonication-Triggered Zero-Order Release
by Uncorking Core–Shell Nanofibers', Chemical Engineering Journal, 288
(2016), 1-8.
144. SJ Desai, P Singh, AP Simonelli, and WI Higuchi, 'Investigation of Factors
Influencing Release of Solid Drug Dispersed in Inert Matrices. 3. Quantitative
Studies Involving the Polyethylene Plastic Matrix', Journal of pharmaceutical
sciences, 55 (1966), 1230-1234.
145. T Huguchi, 'Mechanism of Sustained-Action Medication', J. Pharm. Sci., 52
(1963), 1145-49.
146. NA Peppas, 'Analysis of Fickian and Non-Fickian Drug Release from Polymers',
Pharmaceutica Acta Helvetiae, 60 (1985), 110-111.
123
147. N Vishal Gupta, and HG Shivakumar, 'Preparation and Characterization of
Superporous Hydrogels as Gastroretentive Drug Delivery System for
Rosiglitazone Maleate', Daru, 18 (2010), 200-210.
148. Cristi L Bell, and Nikolaos A Peppas, 'Water, Solute and Protein Diffusion in
Physiologically Responsive Hydrogels of Poly (Methacrylic Acid-G-Ethylene
Glycol)', Biomaterials, 17 (1996), 1203-1218.
149. Hadi Hezaveh, and Ida Idayu Muhamad, 'Controlled Drug Release Via
Minimization of Burst Release in Ph-Response Kappa-Carrageenan/Polyvinyl
Alcohol Hydrogels', Chemical Engineering Research and Design, 91 (2013), 508-
519.
150. Jeannine E Elliott, Mara Macdonald, Jun Nie, and Christopher N Bowman,
'Structure and Swelling of Poly (Acrylic Acid) Hydrogels: Effect of Ph, Ionic
Strength, and Dilution on the Crosslinked Polymer Structure', Polymer, 45 (2004),
1503-10.
151. Shilan Chen, Mingzhu Liu, Shuping Jin, and Bin Wang, 'Preparation of Ionic-
Crosslinked Chitosan-Based Gel Beads and Effect of Reaction Conditions on
Drug Release Behaviors', International journal of pharmaceutics, 349 (2008),
180-187.
152. Ke Wang, Shao Zhi Fu, Ying Chun Gu, Xu Xu, Peng Wei Dong, Gang Guo, Xia
Zhao, Yu Quan Wei, and Zhi Yong Qian, 'Synthesis and Characterization of
Biodegradable Ph-Sensitive Hydrogels Based on Poly (Ɛ-Caprolactone),
Methacrylic Acid, and Poly (Ethylene Glycol)', Polymer Degradation and
Stability, 94 (2009), 730-737.
124
153. Yu-Yang Liu, Wei-Qing Liu, Wei-Xing Chen, Le Sun, and Guo-Bin Zhang,
'Investigation of Swelling and Controlled-Release Behaviors of Hydrophobically
Modified Poly (Methacrylic Acid) Hydrogels', Polymer, 48 (2007), 2665-2671.
154. T Riley, S Stolnik, CR Heald, CD Xiong, MC Garnett, L Illum, SS Davis, SC
Purkiss, RJ Barlow, and PR Gellert, 'Physicochemical Evaluation of Nanoparticles
Assembled from Poly (Lactic Acid)-Poly (Ethylene Glycol)(Pla-Peg) Block
Copolymers as Drug Delivery Vehicles', Langmuir, 17 (2001), 3168-3174.
155. S Bahram Bahrami, Soheila S Kordestani, Hamid Mirzadeh, and Parvin
Mansoori, 'Poly (Vinyl Alcohol)-Chitosan Blends: Preparation, Mechanical and
Physical Properties', Iranian Polymer Journal, 12 (2003), 139-146.
156. Sandeep Shukla, AK Bajpai, and RA Kulkarni, 'Preparation, Characterization,
and Water‐Sorption Study of Polyvinyl Alcohol Based Hydrogels with Grafted
Hydrophilic and Hydrophobic Segments', Journal of applied polymer science, 95
(2005), 1129-1142.
157. Gergely Kali, Theoni K Georgiou, Béla Iván, Costas S Patrickios, Elena Loizou,
Yi Thomann, and Joerg C Tiller, 'Synthesis and Characterization of Anionic
Amphiphilic Model Conetworks of 2-Butyl-1-Octyl-Methacrylate and
Methacrylic Acid: Effects of Polymer Composition and Architecture', Langmuir,
23 (2007), 10746-10755.
158. Csaba Fodor, Gergely Kali, and Béla Iván, 'Poly (N-Vinylimidazole)-L-Poly
(Tetrahydrofuran) Amphiphilic Conetworks and Gels: Synthesis, Characterization,
Thermal and Swelling Behavior', Macromolecules, 44 (2011), 4496-4502.
159. Limin Wang, and Jan P Stegemann, 'Glyoxal Crosslinking of Cell-Seeded
Chitosan/Collagen Hydrogels for Bone Regeneration', Acta biomaterialia, 7
(2011), 2410-2417.
125
160. JV Cauich-Rodriguez, S Deb, and R Smith, 'Effect of Cross-Linking Agents on
the Dynamic Mechanical Properties of Hydrogel Blends of Poly (Acrylic Acid)-
Poly (Vinyl Alcohol-Vinyl Acetate)', Biomaterials, 17 (1996), 2259-2264.
161. Z Abdeen, 'Swelling and Reswelling Characteristics of Cross-Linked Poly (Vinyl
Alcohol)/Chitosan Hydrogel Film', Journal of Dispersion Science and
Technology, 32 (2011), 1337-1344.
162. Meera George, and T Emilia Abraham, 'Polyionic Hydrocolloids for the Intestinal
Delivery of Protein Drugs: Alginate and Chitosan—a Review', Journal of
controlled release, 114 (2006), 1-14.
163. Kang De Yao, Tao Peng, Han Bao Feng, and Yu Ying He, 'Swelling Kinetics and
Release Characteristic of Crosslinked Chitosan: Polyether Polymer Network
(Semi‐Ipn) Hydrogels', Journal of Polymer Science Part A: Polymer Chemistry,
32 (1994), 1213-1223.
164. Paul J Flory, Principles of Polymer ChemistryCornell University Press, 1953.
165. Yi Lyn Lam, Saravanan Muniyandy, Hashim Kamaruddin, Ahmad Mansor, and
Pushpamalar Janarthanan, 'Radiation Cross-Linked Carboxymethyl Sago Pulp
Hydrogels Loaded with Ciprofloxacin: Influence of Irradiation on Gel Fraction,
Entrapped Drug and in Vitro Release', Radiation Physics and Chemistry, 106
(2015), 213-22.
166. Nasim Annabi, Jason W Nichol, Xia Zhong, Chengdong Ji, Sandeep Koshy, Ali
Khademhosseini, and Fariba Dehghani, 'Controlling the Porosity and
Microarchitecture of Hydrogels for Tissue Engineering', Tissue Engineering Part
B: Reviews, 16 (2010), 371-83.
167. Nasim Annabi, Jason W Nichol, Xia Zhong, Chengdong Ji, Sandeep Koshy, Ali
Khademhosseini, and Fariba Dehghani, 'Controlling the Porosity and
126
Microarchitecture of Hydrogels for Tissue Engineering', Tissue Engineering Part
B: Reviews, 16 (2010), 371-83.
168. F Kartal, A Akkaya, and A Kilinc, 'Immobilization of Porcine Pancreatic Lipase
on Glycidyl Methacrylate Grafted Poly Vinyl Alcohol', Journal of Molecular
Catalysis B: Enzymatic, 57 (2009), 55-61.
169. Sang-Man Ahn, Jong-Wook Ha, Jeong-Hoon Kim, Yong-Taek Lee, and Soo-Bok
Lee, 'Pervaporation of Fluoroethanol/Water and Methacrylic Acid/Water Mixtures
through Pva Composite Membranes', Journal of membrane science, 247 (2005),
51-57.
170. A Pourjavadi, AM Harzandi, and H Hosseinzadeh, 'Modified Carrageenan. 6.
Crosslinked Graft Copolymer of Methacrylic Acid and Kappa-Carrageenan as a
Novel Superabsorbent Hydrogel with Low Salt-and High pH-Sensitivity',
Macromolecular research, 13 (2005), 483-490.
171. Alina Gabriela Rusu, Marcel Ionel Popa, Gabriela Lisa, and Liliana Vereştiuc,
'Thermal Behavior of Hydrophobically Modified Hydrogels Using Tga/Ftir/Ms
Analysis Technique', Thermochimica acta, 613 (2015), 28-40.
172. Mohammad Sadeghi, and Hossein Hosseinzadeh, 'Synthesis and Super-Swelling
Behavior of a Novel Low Salt-Sensitive Protein-Based Superabsorbent Hydrogel:
Collagen-G-Poly (Amps)', Turkish Journal of Chemistry, 34 (2010), 739-752.
173. Ying Guan, Jing Bian, Feng Peng, Xue-Ming Zhang, and Run-Cang Sun, 'High
Strength of Hemicelluloses Based Hydrogels by Freeze/Thaw Technique',
Carbohydrate polymers, 101 (2014), 272-280.
174. Ruixia Hou, Guohua Zhang, Gaolai Du, Danxia Zhan, Yang Cong, Yajun Cheng,
and Jun Fu, 'Magnetic Nanohydroxyapatite/Pva Composite Hydrogels for
127
Promoted Osteoblast Adhesion and Proliferation', Colloids and Surfaces B:
Biointerfaces, 103 (2013), 318-325.
175. Jingyi Qiu, Ling Xu, Jing Peng, Maolin Zhai, Long Zhao, Jiuqiang Li, and
Genshuan Wei, 'Effect of Activated Carbon on the Properties of
Carboxymethylcellulose/Activated Carbon Hybrid Hydrogels Synthesized by Γ-
Radiation Technique', Carbohydrate polymers, 70 (2007), 236-242.
176. Hui Zhao, Jun Gao, Ruina Liu, and Sanping Zhao, 'Stimulus-Responsiveness and
Methyl Violet Release Behaviors of Poly (Nipaam-Co-Aa) Hydrogels Chemically
Crosslinked with Β-Cyclodextrin Polymer Bearing Methacrylates', Carbohydrate
research, 428 (2016), 79-86.
177. Heung Soo Shin, So Yeon Kim, and Young Moo Lee, 'Indomethacin Release
Behaviors from pH and Thermoresponsive Poly (Vinyl Alcohol) and Poly
(Acrylic Acid) Ipn Hydrogels for Site‐Specific Drug Delivery', Journal of applied
polymer science, 65 (1997), 685-693.