European Journal of Pharmaceutics and Biopharmaceutics xxx (2014) xxxxxx

Contents lists available at ScienceDirect

European Journal of Pharmaceutics and Biopharmaceutics

journal homepage: www.elsevier.com/locate/ejpb

Review article

Polyethylene glycol (PEG)Poly(N-isopropylacrylamide) (PNIPAAm)

based thermosensitive injectable hydrogels for biomedical applications
Amit Alexander a,1, Ajazuddin b,2, Junaid Khan a,3, Swarnlata Saraf a, Shailendra Saraf a,
a
b

University Institute of Pharmacy, Pt. Ravishankar Shukla University, Raipur, India

Rungta College of Pharmaceutical Sciences and Research, Bhilai, India

a r t i c l e

i n f o

Article history:
Received 14 January 2014
Accepted in revised form 8 July 2014
Available online xxxx
Keywords:
Hydrogel
Injectable
In situ thermo responsive
Poly ethylene glycol
Poly(N-isopropylacrylamide) (PNIPAAm)
Novel

a b s t r a c t
Protein and peptide delivery by the use of stimuli triggered polymers remains to be the area of interest
among the scientist and innovators. In-situ forming gel for the parenteral route in the form of hydrogel
and implants are being utilized for various biomedical applications. The formulation of gel depends upon
factors such as temperature modulation, pH changes, the presence of ions and ultra-violet irradiation,
from which drug is released in a sustained and controlled manner. Among various stimuli triggered
factors, thermoresponsive is the most potential one for the delivery of protein and peptides. Poly(ethylene glycol) (PEG) based copolymers play a crucial role as a biomedical material for biomedical applications, because of its biocompatibility, biodegradability, thermosensitivity and easy controlled
characters. This review, stresses on the physicochemical property, stability and compositions prospects
of smart thermoresponsive polymer specically, PEG/Poly(N-isopropylacrylamide) (PNIPAAm) based
thermoresponsive injectable hydrogels, recently utilized for biomedical applications. PEGPNIPAAm
based hydrogel exhibits good gelling mechanical strength and minimizes the initial burst effect of the
drug. In addition, upon changing the composition and proportion of the copolymer molecular weight
and ratio, the gelling time can be reduced to a great extent providing better solgel transition. The
hydrogel formed by the same is able to release the drug over a long duration of time, meanwhile is also
biocompatible and biodegradable. Manuscript will give the new researchers an idea about the potential
and benets of PNIPAAm based thermoresponsive hydrogels for the biomedical application.
2014 Elsevier B.V. All rights reserved.

1. Introduction
The administration of the proteins and peptide through parenteral routes is the most preferred one since a long time. However,
frequent administration had led to poor patient compliance due to
pain and irritation. Even though, there are various other routes for
the delivery of protein and peptides such as transdermal; vaginal;
intranasal and intra-pulmonary routes, among them is parenteral
route always designated as the main area of interest [13]. The
extensive research had evolved the invention of long acting injections and implants [47] to prolong the release of proteins and

Corresponding author. University Institute of Pharmacy, Pt. Ravishankar Shukla

University, Raipur, Chhattisgarh 492010, India. Tel.: +91 788 2262832.
E-mail addresses: itsmeamitalex@gmail.com (A. Alexander), write2ajaz@gmail.
com ( Ajazuddin), junaid1010@gmail.com (J. Khan), swarnlata_saraf@rediffmail.
com (S. Saraf), drssaraf@yahoo.in (S. Saraf).
1
Mobile: +91 990733846.
2
Mobile: +91 9827199441.
3
Mobile: +91 9826141303.

peptides for extended duration of time. HG,4 due to their insoluble

polymers network help to retain shape and therefore, suitable for the
loading of the bioactive [8]. Injectable hydrogels are triggered by
temperature, which remain uid at room temperature and transform
to viscous gel, as the temperature rises [9]. These gelling systems
sustain the drug release to larger extent and subsequently increase
the bioavailability by providing local effect. Injectable hydrogels
were prepared by a series of thermoresponsive (or reversible) triblock copolymers comprising of poloxamer and PEG.5 Characteristically, poloxamer shows reversible gelication upon repeated cooling
and warming [10], hence best suited for biomedical applications
[1113]. However, the hydrogels prepared with Poloxamers have
its own limitation regarding its biodegradability. Thus, there is a
need for an alternative biomaterial required to prepare the hydrogel,
which must be biocompatible along with safety and efcacy.
Out of various stimuli-triggered external factors such as,
temperature [1416], pH, electric and photoelds [1719],
4
5

Hydrogels.
Polyethylene glycol.

http://dx.doi.org/10.1016/j.ejpb.2014.07.005
0939-6411/ 2014 Elsevier B.V. All rights reserved.

Please cite this article in press as: A. Alexander et al., Polyethylene glycol (PEG)Poly(N-isopropylacrylamide) (PNIPAAm) based thermosensitive injectable
hydrogels for biomedical applications, Eur. J. Pharm. Biopharm. (2014), http://dx.doi.org/10.1016/j.ejpb.2014.07.005

A. Alexander et al. / European Journal of Pharmaceutics and Biopharmaceutics xxx (2014) xxxxxx

temperature stimuli triggered hydrogel remain the most studied

and preferred one for the controlled drug delivery [20]. These hydrogels have proven to play a vital role for the delivery of bioactives.
More specically, PEG based hydrogel comprising from the blocks
of hydrophobic polyesters such as PLGA6 and PCL7 has gain more
responsiveness in the recent past, because of its good biodegradability and biocompatibility properties in contrast with those of
Poloxamers [21]. Among the above-mentioned polymers, PNIPAAm8
because of its LCST9 of 32 C, remains to be the most suitable
temperature sensitive polymer. PNIPAAm based hydrogels can be
prepared by either chemical or physical crosslinking method. Among
these two methods, chemical crosslinking method is preferred
because of its ease in manufacturing by tuning/altering the initiator
ratio; crosslinking agents; precursor ratio and concentration. Some
crosslinking agents and initiator show toxicity, which need to be
removed further [22]. In addition, hydrogels formed by chemical
crosslinking method are generally nonbiodegradable. To overcome
such limitation, hydrogels formed via physical method like through
hydrogen or ionic bonds, van der Waals interactions, crystal formation and/or physical entanglements are most appropriate [2326].
2. Reason to develop PNIPAAmPEG hydrogels over simple
PNIPAAm hydrogels
Crosslinking design improves the inherent properties of hydrogels. Crosslinking prevents the molecules of the hydrogels from
being dissolved in a swelling medium by holding the entire molecule together. The advantage of physical crosslinked hydrogel
includes no use of crosslinking agents or initiators. Physical
crosslinking includes hydrogen or ionic bonds, van der Waals
interactions, crystal formation and/or physical entanglements
[25]. Physically crosslinked hydrogels fail to show strength and
at the same time are not stable as covalent crosslinked systems.
To improve the same, PNIPAAm is crosslinked with a biocompatible and biodegradable polysaccharide, chitosan by Sun et al. [27].
However, the systems formed were brittle and showed poor physical and mechanical properties. Thus, to improve this, author had
incorporated PEG, to improve the mechanical properties of the
hydrogels. Chitosan/PNIPAAms hydrogels exhibit lower crystallinity than each individual component, which got higher after the
introduction of PEG i.e., chitosan/PEG/PNIPAAm gels. The introduction of PEG activated the crystals as crosslinker and affect the properties of the physically crosslinked hydrogels thereof. According to
the results, PEG with 2000 MW10 showed limited swelling, very few
pores were formed because of its high crystalline regions; with 6000
bigger, and more pores were formed because of lower crystallinity of
the physical hydrogel. When PEG with MW 10,000 and 20,000 was
incorporated into the system, very few pores were formed because
of the increased MW of PEG which limits the mobility of PNIPAAm
molecules and made it harder even at LCST. Thus, it can be understood that the PEG crosslinked PNIPAAm can improve the physical
and chemical properties of the hydrogel up to a great extent [27].
Some of the works patented on the above-related work are summarized in Table 1.
3. Biodegradability and biocompatibility of PNIPAAm-based
hydrogels
Biodegradability and non-toxicity are the basic desired properties, when working with the thermogelling block copolymers
6
7
8
9
10

Poly(lactic-co-glycolic) acid.
Polycaprolactone.
Poly(N-isopropylacrylamide).
Lower critical solution temperature.
Molecular Weight.

hydrogels for parenteral delivery. To make PNIPAAm biodegradable and biocompatible the researchers adopted various synthetic
approaches. Among them crosslinked cores of the poly(ethylene
oxide)-b-poly(N-isopropylacrylamide) (PEO-b-PNIPAAm) micelle
with a biodegradable crosslinker BAC11 forms a stable micelle like
nanoparticles. Due to the hydrophobicity of the biodegradable crosslinked BAC, cores of micelles is copolymerized with the NIPAAm. The
model drug used for the study (Dox12) acts like a uorescent probe
as well as an anti-cancer drug too. The study showed that PEO-bPNIPAAm-BAC nanoparticles sequester Dox. The outcome of this
modication had made it stable up to two weeks even at room temperature and at the same time biodegradable too so that they do not
build up the body. Likewise, the PEG-based triblock copolymers are
also fullling the same, with desired and tunable control over the
delivery system. Some of the investigated PEG-based copolymers
are discussed here, highlighting the innovators idea behind the
development of these copolymers. In addition, PEG is approved by
the FDA13 for the use in pharmacological applications [28]. This polymer is best suited to be applied as an injectable in-situ forming gelling biomaterial whose mechanical properties go beyond those of
purely physical gels, however still allows a temperature-triggered
gelation. The section includes the synthesis and evaluation parameters of these PEG-based copolymer hydrogels utilized for biomedical
applications.
4. Biomedical applications of thermogelling PEGPNIPAM
blocks copolymers
A PNIPAAm-based system due to its phase transition between
ambient and body temperature and copolymerization of PNIPAAm
with different types of monomers, remains to be one of the most
commonly used thermosensitive materials to formulate hydrogels
[29]. PNIPAAm exhibits an LCST around 32 C, making it most suitable polymer for in situ hydrogel [30]. At room temperature it is a
free-owing solution, once the temperature is raised (body
temperature) it solidify into an elastomeric hydrogel. Moreover,
crosslinked PNIPAAm, owing to its highly swollen nature allows
injectability even through small gauge needles [31,32]. PNIPAAm
is water-soluble at a temperature below its LCST; though, at a LCST
temperature or higher, weak hydrogen bond interaction between
PNIPAAm and water tend to release the water from the system.
At this stage, PNIPAAm undergoes a coil to globule transition and
become insoluble. Thermo-sensitive hydrogels exhibit volume
phase transitions or solgel phase-transitions at critical temperatures, i.e., LCST or UCST.14 Some of the LCSTs among several typical
thermosensitive polymers are shown in Table 2. The LCST polymers
exhibit swelling-to-shrinking (or sol-to-gels) transition with increasing temperature, whereas the UCST systems undergo the opposite
transitions. This LCST can be altered by incorporation of various
comonomers. In addition, conjugation of hydrophobic monomers
leads to a decrease in LCST whereas, addition of hydrophilic monomers will give the reverse result. Poly(NIPAAm) undergoes gelation
by physical cross-linking. As already discussed, at temperatures
below its LCST, the polymer chains are hydrophilic and thus soluble
in the aqueous environment. Gradual increase in hydrophobicity is/
was observed as the temperature of the polymer chain is increased
above its LCST. Shrinkage of the chains is due to the dispersion of
the water present between chains to form a gel [33,34]. Here, the
solgel transition state is rapid and reversible too. With such fast
transition to temperature stimuli, drugs can be quickly released from
the hydrogel, exhibiting onoff switching release system [35].
11
12
13
14

N,N-bis(acryloyl)cystamine.
Doxorubicin.
Food and Drug Administration.
Upper critical solution temperatures.

Please cite this article in press as: A. Alexander et al., Polyethylene glycol (PEG)Poly(N-isopropylacrylamide) (PNIPAAm) based thermosensitive injectable
hydrogels for biomedical applications, Eur. J. Pharm. Biopharm. (2014), http://dx.doi.org/10.1016/j.ejpb.2014.07.005

A. Alexander et al. / European Journal of Pharmaceutics and Biopharmaceutics xxx (2014) xxxxxx

Table 1
Patents on PNIPAM based and temperature sensitive hydrogels.
Patent/
application no.

Pub. date

Inventors

Title

Work description

20060286152

December
21, 2006

Hu; Jinlian; (Hong Kong, CN);

Liu; We nguang; (Hong Kong,
CN); Liu; Baohua; (Hong Kong,
CN)

Fabric-supported chitosan modied

temperature responsive PNIPAAm/PU
hydrogel and the use thereof in
preparation of facial mask

WO 01/68768
A1

September
20, 2001

CHENG, Yu-Ling; LIN, Hai-Hui

Environment responsive gelling

copolymer

WO9828364
(A1)

July 02,
1998

WU CHI; JIANG SUHONG

Novel polymer gel composition and uses

therefor

20040258727

December
23, 2004

Liu, Lina; (Hamilton, CA);

Sheardown, Heather D.;
(Nobleton, CA)

Ophthalmic biomaterials and preparation

thereof

US 6,238,688 B1

May 29,
2001

Chi Wu, Yeung Long; Suhong J

iang, Shatin

Method for repairing blood vessel System

US005997961A

December
7, 1999

Xiangdong Feng; Jun Liu, both

of West Richland; Liang Liang,
Richland, all of Wash

Method of Bonding Functional Surface

Materials To Substrates And Applications
In Microtechnology And Antifouling

WO2004060429
A1

July 22,
2004

Howard Allen Ketelson

Compositions comprising
n-isopropylacrylamide and methods for
inhibiting protein adsorption on surfaces

For the preparation of facial mask, PNIPAAm/PU

hydrogel including fabric-supported chitosan
triggered by temperature stimuli is utilized. The
advantage of this technique is reversibly swelling and
deswelling of hydrogel near body temperature. The
mechanical strength also get boosted by the Grafting
of PNIPAAm and PU onto the surface of cellulose
fabrics
This work relates with composition of comprising
copolymer of PEG and PNIPAAM, having a liquid form
at room temperature and gel at body temperature.
This makes it suitable for the in situ implants
This work highlights the application of hydrogel for
the repair of damage tissues. The inventor used the
preparation of hydrophobic polymer matrix PNIPAAM
and the interpenetrating polymer network, supplied
by incorporation of an amount of protein, typically
gelatin, with in the PNIPAAM
The work highlights, interpenetrating network (IPN) of
polydimethyl siloxane (PDMS) and PNIPAAM.
Transparent vinyl and hydroxyl terminated PDMS/
PNIPAAM IPNs (PDMS-V and PDMS-OH IPNs
respectively) were successfully synthesized to
enhance the mechanical strength of the hydrogel
The compositions of the invention and particular use
in surgical applications for the repair of damaged
tissues, e.g., blood vessels, neurons, and the like, and in
temperature-dependent drug delivery systems
Innovator proposed a simple and effective method to
bond a thin coating of poly(N-isopropylacylamide)
(NIPAAIII) on a glass surface by UV
photopolymerization, and the use of such a coated
surface in nano and micro technology applications
This work in particular directed to reduction of the
adsorption of proteins on surfaces of contact lenses
and other medical prosthetics

Table 2
LCSTs of several typical thermosensitive polymers [47].
Polymer
a

PNIPAM
DEAMb
PNEMAMc
PMVEd
PEOVEe
PNVIBAMf
PNVCag
Poly(organophosphazenes)
PHPMAM-mono/di lactateh
a
b
c
d
e
f
g
h

LCST (C)

References

32
25
58
34
20
39
3035
25.098.5
1365

[48,49]
[50]
[50]
[51]
[52]
[53]
[54]
[55]
[56]

Poly(N-isopropylacrylamide).
Poly(N,N-diethylacrylamide).
Poly(N-ethylmethacrylamide).
Poly(methyl vinyl ether).
Poly(2-ethoxyethyl vinyl ether).
Poly(N-vinylisobutyramide).
Poly(N-vinylcaprolactam).
Poly(N-(2-hydroxypropyl) methacrylamide mono/di lactate).

PNIPAAm along with its copolymers is extensively used for biomedical applications, including as embolic agents [36], for drug delivery
[37], as a nucleus pulposus replacement [30], as an injectable
multifunctional scaffold for tissue engineering applications [38]
and for the treatment of ocular diseases [39]. Previously, it had been
shown that crosslinked PNIPAAm hydrogel with PEG-DA15 exhibited
a thermoresponsive and sustained release and can be used for the

15

Poly(ethylene glycol) diacrylate.

ocular drug delivery system [35,40]. PNIPAAm, due to its structural

function, preferably is not in its pure form and due to its poor
mechanical behavior, and PNIPAAm based hydrogels exhibit low
compressive modulus with poor elastic recovery after drug loading
[4144]. In addition, PNIPAAm based hydrogel suffers from limited
amount of drug released with respect to change in temperature
[45]. The crosslinked bond in PNIPAAm hydrogel is non-biodegradable, resulting in the formation of a non-biodegradable hydrogel.
Thus, incorporation of PEG signicantly enhanced the mechanical
and other properties of the hydrogels. As the concentration of the
PEG increases, shrinking increases for other diffusants, e.g. salts or
ethanol [46]. Moreover, PEG is known for its inert behavior toward
biosystems in general and to protein adsorption in particular.

4.1. Drug delivery

4.1.1. PNIPAMPLLAPEGPLLAPNIPAM, hydrogel for sustained
release of hydrophilic drugs
The non-biocompatible property of PNIPAM hydrogels restricts
its utility in many biomedical applications [57]. Thus, attempts
were made to introduce a biodegradable, biocompatible linker into
PNIPAM backbone [58,59]. Saibo Chen and colleague investigated a
unique study based on in situ gelling system on PNIPAM (monomer) and acrylate terminated PLEL16 (biodegradable macromonomer crosslinker, PLAPEGPLA terminated with diacrylate) to get
PNIPAM thermosensitive formulation. PEG and PLA were employed
as polymeric micelles for the investigation as both comprise of
16

Poly(L-lactic acid)-b-poly(ethylene glycol)-poly(L-lactic acid).

Please cite this article in press as: A. Alexander et al., Polyethylene glycol (PEG)Poly(N-isopropylacrylamide) (PNIPAAm) based thermosensitive injectable
hydrogels for biomedical applications, Eur. J. Pharm. Biopharm. (2014), http://dx.doi.org/10.1016/j.ejpb.2014.07.005

A. Alexander et al. / European Journal of Pharmaceutics and Biopharmaceutics xxx (2014) xxxxxx

the poly(NIPAAm-co-PEG) and poly(NIPAAm-coPEG)-acrylate

copolymers form gel above their LCSTs. Poly(NIPAAm-co-PEG) demonstrates LCST property at 2728 C and was conrmed by the
results obtained from DSC21 and rheology. In addition, this system
shows good gelation behavior and temperature induced physical
cross-linking [64,65]. The study conrms the probable use of this
copolymer with enhanced mechanical strength and biocompatibility, for aneurysm or AVM22 occlusion as a thermally gelling injectable biomaterial.

Fig. 1. Synthesis of biodegradable crosslinking agents [62].

hydrophilic and hydrophobic segments, respectively. Solubility of

copolymer in water can be increased when PEG concentration
becomes higher; increasing the molecular weight of PEG; keeping
constant the LA/PEG block ratio. Thus, it is correlated with the fact
that higher the PEG block length or molecular weight, better will
be the solubility of the copolymer [19,21]. The biodegradable crosslinking agent PNIPAM hydrogels were prepared from enhanced
macromonomer [60,61]. The PNIPAM with high molecular weight
is generally not biodegradable or soluble when tested in preclinical
studies as compared to the PNIPAM having low molecular weight,
exhibiting better solubility and excretion. For the synthesis, the
author had used biodegradable crosslinking agents such as PLA
PEGPLA.17 For such preparations, calculated amount of LA18 and
PEG were introduced into a dried 100 ml three-necked ask
equipped with a magnetic stirrer, under a nitrogen atmosphere. A
catalyst, stannous octoate (Sn (Oct)2) was added. The reaction
system was kept at 150 C for 6 h to produce PLEL (Fig. 1). Further,
various amounts of PLEL and PNIPAM were added to produce thermoresponsive and biodegradable copolymers. Copolymer PNIPAM
PLLAPEGPLLAPNIPAM exhibited thermoresponsive properties
which shows more biocompatibility with probably partial biodegradability [62,63]. In this study, to validate the delivery system,
ooxacin was used as a hydrophilic model drug to understand the
drug release behavior. The initial drug release of the hydrogel was
observed very rapidly and further the release rate was slowed down
due to the diffusion and degradation of the hydrogel. In totality, it
was well understood that the PNIPAM copolymer hydrogel plays a
vital role as injectable drug delivery system in biomedical eld [62].
4.1.2. NIPAAm-co-PEG, thermally gelling injectable biomaterial
hydrogel for arteriovenous malformation
Vicki Cheng and colleague reported the synthesis of poly(NIPAAm-co-PEG) by free radical polymerization with acrylate terminated pendant groups by copolymerizing NIPAAm19 with
poly(ethylene glycol)-monoacrylate (PEG-monoacrylate) followed
by the alteration of hydroxyl terminus of the PEG. Further, it forms
a chemical gel with the help of Michael-type addition reaction when
it is mixed with a multi-thiol compound such as QT20 in phosphate
buffer saline solution of pH 7.4. Poly(NIPAAm-co-PEG)-acrylate was
synthesized by permitting terminal OH groups of PEG to react with
acryloyl chloride as shown in Fig. 2. The physical gels prepared by

4.1.3. PNIPAAmPEG-DA, hydrogels intravitreal injection for ocular

drug delivery
Thermoresponsive PNIPAAmPEG-DA hydrogel was applied for
the extended release of the drug delivery to the posterior segment.
Proteins (bevacizumab and ranibizumab) were encapsulated into
the hydrogels, including BSA,23 immunoglobulin G (IgG). PEG is
cross-linked with PNIPAAm to get a hydrogel having a homogenous
structure [66]. PEG, because of its pore-forming property remains to
be the matter of choice for the above synthesis [6769]. An ideal
hydrogel must retain its thermoresponsive characteristic and should
retain homogeneous pores throughout. For achieving the said property, PEG-DA is hosted to PNIPAAm. Here, PEG-DA (cross-linker) was
used as a tuner for controlling the pore size of the hydrogel. In addition, altering the degree of cross-linker density, the protein release
rate can be regulated. Thermoresponsive hydrogels formed by such
crosslinking have shown faster and reversible phase transition with
altered temperature. Hydrogels with lesser cross-linking agents
exhibit fast release and better syringeability, when injected intravitreal route via small-gauge needles. Hydrogels formed by PNIPAAm
PEG-DA exhibited a signicant improved mechanical strength. Use of
PEG-DA as a cross-linker did not alter the LCST, it was observed that
below the LCST, the hydrogel swells and above the LCST, the hydrogel collapse. Pure PNIPAAm hydrogel altered its phase (LCST) at
31 C while PNIPAAm PEG-DA hydrogel altered its phase at
32 C, due to the increased hydrophilicity [70]. Moreover, this
hydrogel system shows ideal syringeability and injectability. Rodent
model was used to study the injectability of the hydrogel for the vitreous chamber. The PNIPAAmPEG-DA hydrogel is biocompatible
and has a unique polymerization characterization, as acrylates are
used as end groups due to rapid polymerization [71]. Extending
the same work, authors developed another intravitreal injection of
a PEG Poly(ethylene glycol) diacrylate (PEG-DA) crosslinked PNIPAAm hydrogel for injectable drug delivery on retinal function.
Crosslinked PNIPAAms showed the thermoresponse behavior at
approximately 32 C exhibiting a VPTT24 [35,72], above which the
swelling behavior decreases with subsequent burst release. One of
the advantages associated with the PNIPAAm was seen with its
highly swollen nature of crosslinked PNIPAAm. At this stage (room
temperature), the crosslinked PNIPAAm shows better syringeability
[32]. Thermoresponsive hydrogels were prepared by dissolving
PEG-DA solution followed by N-isopropylacrylamide. OCT25 was
used for measuring the retinal thickness conrming a small decrease
in retinal thickness after one week post-injection, which was
returned to initial levels in later weeks. As soon as the injection is
applied, no signicant change was observed in the IOP26 immediately but in subsequent weeks, a signicant change was observed
when compared to control IOP value. The PEG-DA crosslinked PNIPAAm hydrogel for intravitreal injection thus had minimal impact
21
22

17
18
19
20

Diacrylate of polyethylene glycol and polylactides.

Lactic acid.
N-isopropylacrylamide.
Pentaerythritol tetrakis 3-mercaptopropionate.

23
24
25
26

Differential scanning calorimetry.

Arteriovenous malformation.
Bovine serum albumin.
Volume phase transition temperature.
Optical coherent tomography.
Intraocular pressure.

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A. Alexander et al. / European Journal of Pharmaceutics and Biopharmaceutics xxx (2014) xxxxxx

Fig. 2. (A) Scheme showing Poly(NIPAAm-co-PEG) prepared by free radical polymerization. (B) Nucleophilic thiol attacking the double bond adjacent to the carbonyl forming
a covalent bond between the two entities. (C) Chemical cross-linking network that is formed when poly(NIPAAm-coPEG)-acrylate is allowed to react with QT. Reprinted with
permission from Cheng et al. [64].

on IOP. PEG-DA crosslinked PNIPAAm hydrogels prove to be a potential drug delivery system for the posterior segment of the eye [39].
4.1.4. PNIPAAmPCLPEGPCLPNIPAAm, thermosensitive pentablock copolymer injectable carriers for sustained drug delivery systems
Thermosensitive PLGA copolymers required several hours to
solubilize in water, making it a difcult and time-consuming process. Also, these copolymers having PLGA segment exhibit sticky
paste morphology, resulting in difculty to transfer or weigh
[73]. Substitution of PLGA with the PCL in the backbone of hydrophobic polyester, such as PCLPEGPCL (PCEC) can be an alternative approach to alter the morphology to powder state, which
can be transfer or weighed easily. Increase in the molecular weight
of PCL, decreases its crystallinity [74]. Therefore, PEG/PCL multiblock copolymer synthesized from coupling of PCLPEGPCL triblock copolymer exhibited lesser crystallinity and apart from this,
because of the high molecular weight of polycaprolactone, the sol
stability gets improved [75]. Therefore, it is also feasible to develop
a solgel system based on copolymers that contain both PCLPEG
PCL tri-block copolymer and N-isopropylacrylamide in the same
context. Working on the same concept recently, Hamid Sadeghi

Abandansari et al., grafted a new biocompatible, biodegradable

and thermosensitive penta-block copolymer poly(N-isopropylacrylamide)-b-poly(e-caprolactone)-b-poly
ethylene
glycole
b-poly(e-caprolactone)-b-poly(N-iso-propylacrylamide)
(PNIPAAmPCLPEGPCLPNIPAAm), which was synthesized by a
combination of controlled ROP27 and ATRP28 (Fig. 4). This pentablock copolymer undergoes reversible solgel transitions between
room temperature (22 C) and human body temperature (37 C).
Amalgamation of poly(N-isopropylacrylamide) (PNIPAAm) block at
the end of PCLPEGPCL (PCEC) triblock copolymer improves the
mechanical strength and high sol stability of PNIPAAmPCECPNIPAAm penta-block copolymer while keeping its thermogelling property in the range of physiological temperatures 2050 C (Fig. 5) [76].
The resulting good mechanical strength of the copolymer hydrogel,
with storage modulus up to 60,000 Pa makes it the most suitable
candidate as a thermogelling injectable for sustained drug release.

27
28

Ring-opening polymerization.
Atom transfer radical polymerization.

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A. Alexander et al. / European Journal of Pharmaceutics and Biopharmaceutics xxx (2014) xxxxxx

Fig. 3. Synthetic scheme of PNIPAAmPCECPNIPAAm penta-block copolymer [76].

4.2. Tissue engineering

4.2.1. PNIPAAmPEG, hydrogels injection for load-bearing soft tissue
applications
Amalgamation of PEG to the PNIPAAm polymer system, utilizing branching and grafting technology improves the swelling ability of the copolymer (Fig. 3). PNIPAAm-based hydrogels are usually
of low compressive modulus with poor elastic recovery after loading when used in its pure form [4144]. The PNIPAAm self-ability
to show thermal transitioning makes it a suitable candidate for the
development of an injectable in situ forming biomaterial for the
use in soft tissue restoration or replacement. Earlier reported study
related to PNIPAAm-based hydrogels for load-bearing applications
is characterized for mechanical properties [30,77]. PNIPAAm-based
hydrogels exhibited compression modulus values in the range
from 0.7 to 600 kPa [41,44]. Compressive modulus of injectable
PNIPAAmpolyethylene glycol (PEG) hydrogels crosslinked with
MPS29 had shown a remarkable value above 600 kPa [78]. LCST values for such hydrogels usually fall within ranges (LCST for PEG grafts
and branches ranged from 33.17 0.10 C at 2.2% PEG to
29

3(methacryloxy) propyltrimethoxysilane.

37.65 0.43 C at 31.3% PEG). Author concluded that the heavily

branched polymers (%PEG P 7%) show better gel-like reaction mixture with 25% aqueous solutions due to sufcient network like structures created by PEG branching. In addition, 31% PEG-branched
polymer exhibited too many PEG branches to form a cohesive gel
in water. Moderate concentrations of PEG grafts or branches
(%PEG P 7%) show LCSTs that fell within the temperature range suitable for an injectable (2537 C) in contrast to 31% PEG which shows
LCSTs that were too high for the injectable application. Grafted PEGs
are not as effective as compared to branches (forms a porous network) in raising the water content of PNIPAAm hydrogels, that can
hold onto and entrap water. PEG grafts were not effective in improving the elastic recovery of the PNIPAAm hydrogels, as PEG branches
were effective in increasing the water content of PNIPAAm hydrogels. In totality, a care must be taken to balance the PNIPAAm/PEG
ratio to get better resulting material for implantation.
4.2.2. PNIPAAmPEG, impregnated microgel injection for possible
applications in biomedical and biotechnology elds
The hydrogen-bonding efciency becomes weaker to solubilize
PNIPAAm, at a temperature above its LCST. Due to the occurrence
of a thermoreversible change between the polymer-enriched phase

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A. Alexander et al. / European Journal of Pharmaceutics and Biopharmaceutics xxx (2014) xxxxxx

Fig. 4. The schematic image of solgel transition of PNIPAAmPCECPNIPAAm

penta-block copolymer. (For interpretation of the references to color in this gure
legend, the reader is referred to the web version of this article.)

and aqueous phase, PNIPAAm hydrogel is applied for various biomedical applications such as, controlled drug release, protein
ligand recognition including immobilization of enzyme [79,80].
To improve the thermoresponsive properties of PNIPAAm hydrogels, PNIPAAm/PEG-DA30 microgels were exploited during the
polymerization and/or crosslinking. The impregnation of PNIPAAm/
PEG-DA microgels to PNIPAAm hydrogel improves its mechanical
property. PEG, due to its spherical shape [81] is being used as a
promising pore-forming agent to get a macroporous PNIPAAm
hydrogel. This is the reason that PEG was extensively used as an auspicious pore-forming agent to obtain a macroporous PNIPAAm
hydrogel [67]. Impregnated PNIPAAm/PEG-DA microgel additive
too has the thermo-responsive capability in the surrounding matrices. The prepared microgel-impregnated PNIPAAm hydrogels significantly showed tighter and array porous network in comparison with
the pure form of the PNIPAAm. As the concentration of the impregnated-microgel increases, the pore size reduces. Although there is no
difference in the LCST of the impregnated-microgel, PNIPAAm and
the pure PNIPAAm, due to the similar chemical nature between
the microgel and its surrounding PNIPAAm matrix. Thus, a novel
microgel consisting of a copolymer of PNIPAAm and (PEG-DA) could
be used as novel pore-forming additive to develop a quick response
PNIPAAm hydrogels with enhanced mechanical property [82].
4.2.3. ChitosanPEGPNIPAAm, hydrogels inuenced by PEG
(molecular weight)
It was observed that molecular weight of PEG (MW 2000
20,000) signicantly improves the physical and mechanical
properties of the chitosan-PEG-poly(N-isopropylacrylamide) (PNIPAAm) hydrogels. Increased molecular weight of PEG reduces the
crystallinity of the physical hydrogel, subsequently, improving its
polymer-to-polymer interactions. Similarly, an increase in the
molecular weight of the PEG increases the water uptake capacity
of the physical hydrogel. However, it was observed that increase
in molecular weight of PEG increases the mechanical strength of
physical hydrogel up to a remarkable level, which get deteriorate
with further increase in the molecular weight of the PEG. Chemically cross-linked hydrogel polymer such as the PNIPAAm has its
own limitation of being non-biodegradable. Thus, the hydrogels
formed specically with such polymer remain non-biodegradable.
Thus, for the release of the macromolecules from a hydrogel, its
degradability factor always remains a main concern in biomedical
applications. Low MW PEGs due to a higher number of the polar
hydroxyl end group exhibits higher degree of plasticization, on
the other hand higher MW PEG plasticizers gets involved in various

types of interactions with chitosan and PNIPAAm. This higher MW

PEG interacts not only with the chitosan and PNIPAAm but also
with PEG chain itself. The crystallinity peak of the PEG dramatically
increases as the MW of the PEG increases from 2000 to 20,000. Low
MW PEG (2000) weakens the physical crosslinking with the chitosan and PNIPAAm, improving the properties of the hydrogels with
slow chain mobility of PEG molecules, exhibiting the lower level of
crystallinity. Higher MW PEG (20,000) shows lesser extent of interaction and the presence of the free PEG chain segments, exhibiting
the higher level of crystallinity with reduction in the crystallization
temperature, Tc of PEG. ChitosanPEGPNIPAAm hydrogel having
the PEG 2000 shows very few pores because of high crystalline
region and higher crosslinking level with limited swelling. When
PEG 6000 is used, bigger and more pores were formed because of
reduced crystallinity of the physical hydrogel and increased swelling. In case of 10,000 and 20,000 MW, very few pores were formed
because of increase in the MW of PEG, which limits its mobility of
PNIPAAm molecules. Thus, when PEG based hydrogels are prepared, choice of appropriate MW of PEG is an important step [27].
4.3. Other synthetic approaches for improving PNIPAAm thermogelling
properties
4.3.1. Gelatin-g-poly(N-isopropylacrylamide) for the intracameral
administration
Gelatin carriers for intraocular delivery of cell/tissue sheets are
used since a long time as a good candidate for ophthalmic applications. Previously, gelatin had been used as an efcient carrier for
the delivery of pilocarpine in the form of a device known as Gelfoam sponge [83]. In addition to prolong the residence of pilocarpine at the eye surface bioadhesive gelatin nanoparticles were
used for topical applications. Another signicant nding includes
sustained release of epidermal growth factor from cationized gelatin hydrogels placed over the rabbit corneal epithelial defect for
enhanced ocular surface wound healing [84]. Recently, Lai et al.
have developed a biodegradable in situ forming delivery systems
utilizing aminated gelatin grafted with carboxylic end-capped
PN31 through a carbodiimide-mediated coupling reaction. Phase
transition occurs due to alteration in the external temperature,
resulting in the modication of the hydrophilic hydrophobic balance.
Previously, it was proved that an aminated gelatin does not undergo
morphological changes as the temperature was raised from 25 C to
34 C. At low polymer concentration fragiled PN unable to adhere at
the bottom of the vial upon inversion. In contrast, owing to the viscosity binding effect of gelatin the GN32 gels possess remarkable
adherence properties. As the concentration of polymer increases
solution ow gets decreased, exhibiting improved thermal gelation
ability. Hydrophobic interactions at temperature above LCST are
responsible for the aggregation of macromolecules in solution due
to thermal dissociation of hydrating water molecules from the polymer chains. Moreover, when dissolved in deionized water, the PN
and GN had LCST of 31.3 0.1 and 32.2 0.1 C, respectively. To validate the prepared biodegradable in situ forming GN gels, animal
model was selected and the formulation was administered parenteraly using 30-guage needle directly into anterior chamber. Upon
injection, the drug-polymer solution exhibited an instantaneous
phase transitions from liquid to solid. In totality, it was found that
to improve the ocular bioavailability and achieve sustained pharmacological responses of pilocarpine, intracameral administration using
GN was found to be more effective. Thus, the biodefradable and
thermo-responsive gelatin-g-PNIPAAm is an effective carrier for
the biomedical application including an injectable in situ depot
forming hydrogel for intraocular drug delivery.
31

30

Poly(N-isopropylacrylamide)/poly(ethylene glycol) diacrylate.

32

Poly(N-isopropylacrylamide.
Graft copolymer.

Please cite this article in press as: A. Alexander et al., Polyethylene glycol (PEG)Poly(N-isopropylacrylamide) (PNIPAAm) based thermosensitive injectable
hydrogels for biomedical applications, Eur. J. Pharm. Biopharm. (2014), http://dx.doi.org/10.1016/j.ejpb.2014.07.005

A. Alexander et al. / European Journal of Pharmaceutics and Biopharmaceutics xxx (2014) xxxxxx

Fig. 5. Chemical structure of PNIPAAm with graft and branches.

4.3.2. Chitosan-graft-NIPAAm and alginate for biomedical applications

Smart polymers undergo reversible phase transition once triggered by temperature. These thermoresponsive hydrogels are used
in various biomedical applications including self-regulated drug
delivery systems as injectable hydrogels for local wound healing.
In addition these are also used in cell sheet engineering for tissue
reconstruction. This emerging technique of cell sheet engineering
is based on the control of cellular adhesion [85]. In case of PNIPAAm, the temperature-dependent interactions are a result of balance between hydrogen bonding of hydrophilic segment of the
polymer chain and hydrophobic interaction among polymer chains
and hydrophobilc interaction among isopropyl domains. The
limitation of PNIPPAm polymer is associated with its nonbiodegradability [86], which is overcome by combining the biopolymers. When small chains of NIPAAm were grafted onto
chitosan polymer backbone forms a material showing both temperature and pH dependence. An applied and controllable
approach for the attachment of polymer to surface is the use of
LbL33 technique. In this method, the formation of polyelectrolyte
multilayer is sequentially treated with a charged surface solution
comprising oppositely charged polyelectrolytes [8790]. Martins
and colleague [93] demonstrated the formation of in situ hydrogel
of a new thermoresponsive thin lm build by electrostatic assembly.
They utilized the LbL approach for cell sheet purpose by traditional
grafting techniques. Chitosan-graft-NIPAAm was synthesized by
graft polymerization of NIPAAm on to chitosan using ceric ammonium nitrate (CAN) as an initiator. In the wet state, nal thickness
for the graft polymer was found to be around 50 nm. As the number
of layer was increased the thickness increases too. The pendant PNIPAAm chains are responsible for the increase of molecular weight,
leading to thicker multilayer. The homopolymer PNIPAAm solution
exhibited a phase transition around 33 C in aqueous condition. In
contrast, grafted polymer showed the respective transition at
34 C. The LCST of thermoresponsive graft polymer was found to
be 2 C lower compared with the respective cloud points. With the
addition of salts such as NaCl, LCST may be decreased known as salting out [91,92]. In the present study, there is a least effect of NaCl on
to the LCST of graft polymer though it successfully reduced the phase
33

Layer-by-layer.

transition of PNIPAAm. Thus, (chitosan-graft-NIPAAm)/alginate lms

successfully attached and proliferate at 37 C followed by detachment of cell sheets with deposited extracellular matrix triggered
by temperature. This technique can be better used in cell sheet engineering. LbL technique in addition is a suitable candidate for drug
delivery and controlled release systems, sensory devices, lters
and controllable membrane [93].
4.3.3. Temperature-controllable drug release and intracellular uptake
Polymeric nanoparticles (NPs34) and micelles are novel drug
therapeutic agents and are promising carriers for the drug delivery.
Poor water soluble drugs are the best candidate for the NPs as the
outer core of the NPs comprises of the hydrophilic shell and the
inner one consists of the hydrophobic core [94]. These core shell is
made compatible by the application of biocompatible and biodegradable poly(lactide)poly(ethylene glycol) (PLAPEG) and
poly(lactide-co-glycolide)poly(ethylene
glycol)
(PLGAPEG)
copolymers [9598]. PLAPEG and PLGAPEG nanoparticles are
investigated for their ability to form a controlled and targeted drug
delivery system. In the lieu of development, a new temperature
responsive polymer, PNIPAAm is identied as an intelligent material.
PNIPAAm has a LCST of 32 C, allowing a broad gelation window
facilitating ease in formulation. Alteration in this polymer by grafting can induce a reversible alteration in the surface hydrophilic or
hydrophobic properties by hydration/dehydration changes of
polymer side-chain isopropyl groups [99,100]. Recently, Ayano
et al. formulated hydrophilic betamethasone disodium 21-phosphate (BP)-encapsulated NPs. The NPs were formed from a blend
of PLA homopolymers and PNIPAAmPLA block copolymers. Block
copolymers were obtained by the ring-opening polymerization of
DL-lactide using the terminal hydroxyl group of the NIPAAm. BP
loaded NPs were prepared in zinc. During their experimentation,
they found that the LCST of this polymer increased due to the
hydration of the polar terminal hydrophilic hydroxyl group on the
polymer. Increase in temperature does not affect the diameter of
the NPs. PLA/PNIPAAmPLA NP diameter was found to be 140 nm,
which remain constant as the temperature increases. At higher
concentration the particles aggregate due to the hydrophobic
34

Polymeric nanoparticles.

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A. Alexander et al. / European Journal of Pharmaceutics and Biopharmaceutics xxx (2014) xxxxxx

interactions. The f potential of PLA NPs was found to be 50 mV

mainly due to the ionization of the PLA due to ionization of the
PLA carboxylic end-groups at the particle surface in the presence
of water. The presence of the PEG chains at the particle surface
masks the carboxylate group of PLA chains showing the f potential
of 15 mV in case of PLA/PEGPLA NPs. Whereas, PLA/PNIPAAmPLA
NPs showed a f potential of 20 mV at low temperature. Above
the LCST, the release of the BP from the NPs accelerated. The cellular
uptake of the PLA/PNIPAAmPLA NP was not noticed below the
LCST. On the other hand, above LCST the PLA/PNIPAAmPLA NP
was noticed inside the cells around the cell nuclei. These results
indicated that PLA/PNIPAAmPLA NP could allow controllable drug
release and cellular uptake by changing the temperature [101].

5. Authors perspective
The present article highlights the signicance of using PEG
based injectable hydrogel, especially those formed with PNIPAAm
copolymers for biomedical applications. Modifying the synthesis
method (change in molar ratio) can alter the thermogelling properties of these triblock copolymers up to a great extent. We have
underlined the thermoresponsive in situ forming hydrogels owing
to its simple manufacturing procedure and biocompatibility. The
modication can be achieved by a simple grafting procedure and
with a proportional change in the composition and the molecular
weight of the initiator (PEG), changing the physicochemical
property of the copolymer. Preparation of the hydrogel via these
biocompatible copolymers involves a very simple mixing method
and therefore, remains a matter of great interest and concern
among the scientists and the innovators. Apart from this, its multiple routes of delivery systems such as oral, ocular, rectal, vaginal,
and parenteral routes make this system more versatile. These
PEGPNIPAAm based copolymers are considered to be the preferred copolymer for the delivery of proteins and peptides over
PEGPLGA based copolymers, when prolong action of drug release
is concerned. This is due to the ease in the grafting procedure and
with the use of PNIPAAm; the release of the drug can be prolonged
largely as compared to that of PLGA. Ability to self-materialize
transient (or reversible) polymer network caused by the stimuliinduced physical interactions, such as micellar ordered-packing,
phase-separation, hydrophobic association, crystallization, stereocomplexation and electrostatic interactions are the basic principles
leading to the success of the various PEG based triblock copolymers. Among various stimuli factors, temperature is most convenient and effective for loading of the bioactive for the desired
effect. In situ forming hydrogels are three-dimensional crosslinked
polymeric networks that can swell in the presence of an aqueous
medium and retain large amounts of the medium while maintaining their structures. These highly hydrated hydrogels are having
identical structure with natural tissue and are biocompatible too.
At low or moderate aqueous concentrations, hydrophilic polymer
shows Newtonian behavior as no substantial entanglement of
chains occurs. In addition, once crosslinks between the different
polymers chains are introduced, obtained networks show viscoelastic and pure elastic behavior. Crosslinked polymers prevent
dissolution of the hydrophilic polymer chains in an aqueous medium. There are many approaches by which cross-linking has been
used to prepare hydrogels. Since, it is used in various biomedical
applications, the hydrogels are biodegradable and therefore labile
bonds are frequently introduced in the gels. These bonds either
are present in the polymer backbone or in the crosslinks used to
prepare the gel. Implanted hydrogels must have good biocompatibility and the degradation products formed should have a low
toxicity. The degraded products formed thereof can be tailored
by proper selection of the hydrogel building blocks.

Poly(N-isopropylacrylamide) is extensively used as an excellent

thermosensitive segment for in vivo drug delivery applications due
to its lower critical solution temperature (LCST) (32 C) which can
alter volume and shape and show transition around physiological
temperature. Copolymerization of PNIPAAm with hydrophilic polymer exhibits thermogelling copolymer solution, which below the
LCST is solution and form gel when temperature increases above
the LCST. Compared to the Poloxamers, injectable hydrogels formed
by PEGPNIPAAm based triblock copolymers are more biocompatible and biodegradable. The hydrogel formed by the use of the Poloxamer are non-biodegradable and dissolve at the injection site
within a few days, limiting their applicability for the sustained
delivery of bioactives for longer duration of time. In addition, Poloxamer with high concentration (>16%, w/w), exhibit toxicity when
administered intraperitoneally. These limitations can be bypassed
by designing biodegradable thermogelling copolymers. On considering these facts the PEGPNIPAAm appears as a suitable copolymer
for the preparation of thermoresponsive in situ forming hydrogels.
Triblock copolymers composed of PEG/PLGA, PEG/poly(caprolactone), PEG/poly(propylene fumarate), PEG/poly(propylene glycol)/
polyester,
PEG/peptide,
chitosan/glycerolphosphate,
and
poly(phosphazenes) exhibits solgel transition in water as the temperature rises. Among them PEG/polyester copolymer hydrogels are
more studied in various sectors of biomedical applications such as
drug delivery, cell therapy, tissue regeneration, and wound healing
due to their biocompatibility and long persistence in the gel form
under in vivo conditions. PEGPNIPAAm based hydrogels are biodegradable and deliver the drug at the target site for several hrs. PEG
PLGA based hydrogels are also biodegradable and non-toxic and can
deliver both lipophilic and hydrophilic drugs for several days. In
addition, PEG-PC based hydrogels are having the advantage of ease
in handling, as it remains in solid state at ambient temperature.
Apart from all these advantages of using PNIPAAm as a component of a multiple block copolymer for the injectable preparation,
some issues must be cleared. NIPPAM homopolymer and copolymers are of potential for its application in various biomedical
sectors; however, its clinical applications are still a major challenge. PNIPAAM and its copolymers are not biodegradable, until
grafted with PEG. Thus, the use of PEG/PNIPAAm based thermoresponsive hydrogel will be more compatible, as using only the acrylamide-based polymers can activate platelets, upon contact with
blood.
These grafted blends of PEG/PNIPAAm will surely reveal the
metabolism of PNIPAAm, making them more ease in procurement
of FDA approvals. The application of the PEGPNIPAAm based
hydrogels specically for the parenteral route, increase the efcacy
of various proteins and peptides along with other bioactives, leading to an increase in patient compliance and will denitely
improve the host acceptance. Anticancer drugs could be loaded
to such copolymers and delivered to the specic site, providing a
local and a prolong action over the injection/tumor site. The various mustard derivatives used for the DNA alkylation can be delivered using the PEGPNIPAAm based hydrogel as these will reduce
the initial burst of the molecule and thus will reduce the host toxicity. In situ thermoresponsive injectable will then be a fruitful dart
for the local and prolong action of the drug to the tumor site.
6. Conclusion
Current review highlights the biomedical applications of the
PEGPNIPAAm based in situ injectable hydrogels. The article
highlights the emerging works carried out in recent years with
PNIPAAm. There are many types of copolymers with which hydrogels can be prepared, but the injectable hydrogels stimuli triggered
by temperature are still a more effective delivery system. Some
PEG based copolymers are PEGPNIPAM; PEG/PLGA; PEGPCL.

Please cite this article in press as: A. Alexander et al., Polyethylene glycol (PEG)Poly(N-isopropylacrylamide) (PNIPAAm) based thermosensitive injectable
hydrogels for biomedical applications, Eur. J. Pharm. Biopharm. (2014), http://dx.doi.org/10.1016/j.ejpb.2014.07.005

10

A. Alexander et al. / European Journal of Pharmaceutics and Biopharmaceutics xxx (2014) xxxxxx

Moreover, PEGPNIPAAm among them is gaining more attention

and can be better utilized for the delivery of anticancer drug,
specially, with nitrogen mustard. Although, the extensive use of
these hydrogels are the matter of concern among the scientists
but still to procure the FDA approvals, a strong investigation in
the clinical studies is necessary.
Declaration of conict
Declared none.
Acknowledgements
The authors acknowledge Department of Science and
Technology (No. SR/FST/LSI-434/2010), New Delhi (SERC Division),
India and UGC-SAP F.No.3-54/2011 (SAP II) dated March 2011, New
Delhi, India for providing nancial assistance under DST-FIST
scheme as well as the Maulana Azad National Fellowship (MANF)
UGC, New Delhi, India for providing nancial assistance. The
authors are also grateful to the e-library of Pt. Ravishankar Shukla
University, Raipur, Chhattisgarh, India, 490001 for providing
UGC-INFLIBNET facility.
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