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8/19/2019 Alexander, Amit; Ajazuddin, ; Khan, Junaid; Saraf, Swarnlata; Sa -- Polyethylene Glycol

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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 University Institute of Pharmacy, Pt. Ravishankar Shukla University, Raipur, Indiab 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(ethyl-

ene glycol) (PEG) based copolymers play a crucial role as a biomedical material for biomedical applica-

tions, 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 specifically, PEG/Poly(N-isopropylacrylamide) (PNIPAAm) based

thermoresponsive injectable hydrogels, recently utilized for biomedical applications. PEG–PNIPAAm

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 sol–gel 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 potentialand benefits 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 paren-

teral 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 [1–3]. Theextensive research had evolved the invention of long acting injec-

tions and implants [4–7] to prolong the release of proteins and

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 fluid 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) tri-

block copolymers comprising of poloxamer and PEG.5 Characteristi-cally, poloxamer shows reversible gelification upon repeated cooling

and warming [10], hence best suited for biomedical applications

[11–13]. 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 efficacy.

Out of various stimuli-triggered external factors such as,

temperature [14–16], pH, electric and photofields [17–19],

http://dx.doi.org/10.1016/j.ejpb.2014.07.005

0939-6411/ 2014 Elsevier B.V. All rights reserved.

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

University, Raipur, Chhattisgarh 492010, India. Tel.: +91 788 2262832.

E-mail addresses: [email protected] (A. Alexander), write2ajaz@gmail.

com ( Ajazuddin), [email protected] (J. Khan), swarnlata_saraf@rediffmail.

com (S. Saraf), [email protected] (S. Saraf).1 Mobile: +91 990733846.2 Mobile: +91 9827199441.3 Mobile: +91 9826141303.

4 Hydrogels.5 Polyethylene glycol.

European Journal of Pharmaceutics and Biopharmaceutics xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

European Journal of Pharmaceutics and Biopharmaceutics

j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / e j p b

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


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temperature stimuli triggered hydrogel remain the most studied

and preferred one for the controlled drug delivery [20]. These hydro-

gels have proven to play a vital role for the delivery of bioactives.

More specifically, PEG based hydrogel comprising from the blocks

of hydrophobic polyesters such as PLGA6 and PCL 7 has gain more

responsiveness in the recent past, because of its good biodegradabil-

ity and biocompatibility properties in contrast with those of

Poloxamers [21]. Among the above-mentioned polymers, PNIPAAm

8

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 Waal’s interactions, crystal forma-

tion and/or physical entanglements are most appropriate [23–26].

2. Reason to develop PNIPAAm–PEG hydrogels over simple

PNIPAAm hydrogels

Crosslinking design improves the inherent properties of hydro-

gels. Crosslinking prevents the molecules of the hydrogels from

being dissolved in a swelling medium by holding the entire mole-

cule together. The advantage of physical crosslinked hydrogel

includes no use of crosslinking agents or initiators. Physical

crosslinking includes hydrogen or ionic bonds, van der Waal’s

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 biocompati-

ble and biodegradable polysaccharide, chitosan by Sun et al. [27].

However, the systems formed were brittle and showed poor phys-

ical and mechanical properties. Thus, to improve this, author had

incorporated PEG, to improve the mechanical properties of the

hydrogels. Chitosan/PNIPAAms hydrogels exhibit lower crystallin-

ity than each individual component, which got higher after the

introduction of PEG i.e., chitosan/PEG/PNIPAAm gels. The introduc-

tion of PEG activated the crystals as crosslinker and affect the prop-

erties 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 under-

stood that the PEG crosslinked PNIPAAm can improve the physicaland chemical properties of the hydrogel up to a great extent [27].

Some of the works patented on the above-related work are summa-

rized in Table 1.

3. Biodegradability and biocompatibility of PNIPAAm-based

hydrogels

Biodegradability and non-toxicity are the basic desired proper-

ties, when working with the thermogelling block copolymers

hydrogels for parenteral delivery. To make PNIPAAm biodegrad-

able 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 cross-

linked BAC, cores of micelles is copolymerized with the NIPAAm. The

model drug used for the study (Dox

12

) acts like a fluorescent probeas well as an anti-cancer drug too. The study showed that PEO-b-

PNIPAAm-BAC nanoparticles sequester Dox. The outcome of this

modification had made it stable up to two weeks even at room tem-

perature and at the same time biodegradable too so that they do not

build up the body. Likewise, the PEG-based triblock copolymers are

also fulfilling 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 poly-

mer is best suited to be applied as an injectable in-situ forming gel-

ling 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 parame-

ters of these PEG-based copolymer hydrogels utilized for biomedical

applications.

4. Biomedical applications of thermogelling PEG–PNIPAM

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 suit-

able polymer for in situ hydrogel [30]. At room temperature it is a

free-flowing 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 sol–gel phase-transitions at critical tempera-

tures, 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 increas-

ing 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 mono-mers 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

sol–gel transition state is rapid and reversible too. With such fast

transition to temperature stimuli, drugs can be quickly released from

the hydrogel, exhibiting on–off switching release system [35].

6 Poly(lactic-co-glycolic) acid.7 Polycaprolactone.8 Poly(N-isopropylacrylamide).9

Lower critical solution temperature.10 Molecular Weight.

11 N,N-bis(acryloyl)cystamine.12 Doxorubicin.13

Food and Drug Administration.14 Upper critical solution temperatures.

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







PNIPAAm along with its copolymers is extensively used for biomed-

ical 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

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

[41–44]. 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-biodegrad-

able, resulting in the formation of a non-biodegradable hydrogel.

Thus, incorporation of PEG significantly 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. PNIPAM–PLLA–PEG–PLLA–PNIPAM, 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 (mono-

mer) and acrylate terminated PLEL 16 (biodegradable macromono-

mer crosslinker, PLA–PEG–PLA terminated with diacrylate) to get

PNIPAM thermosensitive formulation. PEG and PLA were employed

as polymeric micelles for the investigation as both comprise of

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 modified

temperature responsive PNIPAAm/PU

hydrogel and the use thereof in

preparation of facial mask

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. Themechanical strength also get boosted by the Grafting

of PNIPAAm and PU onto the surface of cellulose

fabrics

WO 01/68768

A1

September

20, 2001

CHENG, Yu-Ling; LIN, Hai-Hui Environment responsive gelling

copolymer

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

WO9828364

(A1)

July 02,

1998

WU CHI; JIANG SUHONG Novel polymer gel composition and uses

therefor

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

20040258727 December

23, 2004

Liu, Lina; (Hamilton, CA);

Sheardown, Heather D.;

(Nobleton, CA)

Ophthalmic biomaterials and preparation

thereof

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 IPNsrespectively) were successfully synthesized to

enhance the mechanical strength of the hydrogel

US 6,238,688 B1 May 29,

2001

Chi Wu, Yeung Long; Suhong J

iang, Shatin

Method for repairing blood vessel System 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, andin

temperature-dependent drug delivery systems

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

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

WO2004060429

A1

July 22,

2004

Howard Allen Ketelson Compositions comprising

n-isopropylacrylamide and methods for

inhibiting protein adsorption on surfaces

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 LCST (C) References

PNIPAMa 32 [48,49]

DEAMb 25 [50]

PNEMAMc 58 [50]

PMVEd 34 [51]

PEOVEe 20 [52]

PNVIBAMf 39 [53]

PNVCag 30–35 [54]

Poly(organophosphazenes) 25.0–98.5 [55]

PHPMAM-mono/di lactateh 13–65 [56]

a Poly(N-isopropylacrylamide).b Poly(N,N-diethylacrylamide).c Poly(N-ethylmethacrylamide).

d Poly(methyl vinyl ether).e Poly(2-ethoxyethyl vinyl ether).f Poly(N-vinylisobutyramide).

g Poly(N-vinylcaprolactam).h Poly(N-(2-hydroxypropyl) methacrylamide mono/di lactate).

15 Poly(ethylene glycol) diacrylate. 16 Poly(L -lactic acid)-b-poly(ethylene glycol)-poly(L -lactic acid).

A. Alexander et al. / European Journal of Pharmaceutics and Biopharmaceutics xxx (2014) xxx–xxx 3





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hydrophilic and hydrophobic segments, respectively. Solubility of

copolymer in water can be increased when PEG concentration

becomes higher; increasing the molecular weight of PEG; keepingconstant 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 cross-

linking 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–

PEG–PLA.17 For such preparations, calculated amount of LA18 and

PEG were introduced into a dried 100 ml three-necked flask

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 ther-

moresponsive and biodegradable copolymers. Copolymer PNIPAM–

PLLA–PEG–PLLA–PNIPAM exhibited thermoresponsive properties

which shows more biocompatibility with probably partial biode-

gradability [62,63]. In this study, to validate the delivery system,

ofloxacin 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 field [62].

4.1.2. NIPAAm-co-PEG, thermally gelling injectable biomaterial

hydrogel for arteriovenous malformationVicki Cheng and colleague reported the synthesis of poly(NIP-

AAm-co-PEG) by free radical polymerization with acrylate termi-

nated 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

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

copolymers form gel above their LCSTs. Poly(NIPAAm-co-PEG) dem-

onstrates LCST property at 27–28 C and was confirmed 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 confirms the probable use of this

copolymer with enhanced mechanical strength and biocompatibil-

ity, for aneurysm or AVM

22

occlusion as a thermally gelling inject-able biomaterial.

4.1.3. PNIPAAm–PEG-DA, hydrogels intravitreal injection for ocular

drug delivery

Thermoresponsive PNIPAAm–PEG-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 [67–69]. An ideal

hydrogel must retain its thermoresponsive characteristic and should

retain homogeneous pores throughout. For achieving the said prop-erty, 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 addi-

tion, 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 intravi-

treal route via small-gauge needles. Hydrogels formed by PNIPAAm–

PEG-DA exhibited a significant 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 hydro-

gel collapse. Pure PNIPAAm hydrogel altered its phase (LCST) at

31 C while PNIPAAmPEG-DA hydrogel altered its phase at

32

C, due to the increased hydrophilicity [70]. Moreover, thishydrogel system shows ideal syringeability and injectability. Rodent

model was used to study the injectability of the hydrogel for the vit-

reous chamber. The PNIPAAm–PEG-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 PNI-

PAAm 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 confirming 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 significant change was observed in the IOP26 immedi-

ately but in subsequent weeks, a significant change was observed

when compared to control IOP value. The PEG-DA crosslinked PNI-

PAAm hydrogel for intravitreal injection thus had minimal impact

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

17 Diacrylate of polyethylene glycol and polylactides.18 Lactic acid.19

N-isopropylacrylamide.20 Pentaerythritol tetrakis 3-mercaptopropionate.

21 Differential scanning calorimetry.22 Arteriovenous malformation.23 Bovine serum albumin.24 Volume phase transition temperature.25

Optical coherent tomography.26 Intraocular pressure.








on IOP. PEG-DA crosslinked PNIPAAm hydrogels prove to be a poten-

tial drug delivery system for the posterior segment of the eye [39].

4.1.4. PNIPAAm–PCL–PEG–PCL–PNIPAAm, thermosensitive penta-

block copolymer injectable carriers for sustained drug delivery systems

Thermosensitive PLGA copolymers required several hours tosolubilize in water, making it a difficult and time-consuming pro-

cess. Also, these copolymers having PLGA segment exhibit sticky

paste morphology, resulting in difficulty to transfer or weigh

[73]. Substitution of PLGA with the PCL in the backbone of hydro-

phobic polyester, such as PCL–PEG–PCL (PCEC) can be an alterna-

tive 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 multi-

block copolymer synthesized from coupling of PCL–PEG–PCL tri-

block 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 sol–gel system based on copolymers that contain both PCL–PEG–

PCL tri-block copolymer and N-isopropylacrylamide in the samecontext. Working on the same concept recently, Hamid Sadeghi

Abandansari et al., grafted a new biocompatible, biodegradable

and thermosensitive penta-block copolymer poly(N-isopropylac-

rylamide)-b-poly(e-caprolactone)-b-poly ethylene glycole

b-poly(e-caprolactone)-b-poly(N-iso-propylacrylamide) (PNI-

PAAm–PCL–PEG–PCL–PNIPAAm), which was synthesized by a

combination of controlled ROP27

and ATRP28

(Fig. 4). This penta-block copolymer undergoes reversible sol–gel transitions between

room temperature (22 C) and human body temperature (37 C).

Amalgamation of poly(N-isopropylacrylamide) (PNIPAAm) block at

the end of PCL–PEG–PCL (PCEC) triblock copolymer improves the

mechanical strength and high sol stability of PNIPAAm–PCEC–PNI-

PAAm penta-block copolymer while keeping its thermogelling prop-

erty in the range of physiological temperatures 20–50 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.

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 whenpoly(NIPAAm-coPEG)-acrylate is allowed to react with QT. Reprinted with

permission from Cheng et al. [64].

27

Ring-opening polymerization.28 Atom transfer radical polymerization.






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4.2. Tissue engineering

4.2.1. PNIPAAm–PEG, hydrogels injection for load-bearing soft tissue

applications

Amalgamation of PEG to the PNIPAAm polymer system, utiliz-

ing branching and grafting technology improves the swelling abil-

ity of the copolymer (Fig. 3). PNIPAAm-based hydrogels are usually

of low compressive modulus with poor elastic recovery after load-

ing when used in its pure form [41–44]. 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

PNIPAAm–polyethylene glycol (PEG) hydrogels crosslinked with

MPS29 had shown a remarkable value above 600 kPa [78]. LCST val-

ues 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

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

branched polymers (%PEGP 7%) show better gel-like reaction mix-

ture with 25% aqueous solutions due to sufficient network like struc-

tures 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

(%PEGP 7%) show LCSTs that fell within the temperature range suit-

able for an injectable (25–37 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 net-

work) in raising the water content of PNIPAAm hydrogels, that can

hold onto and entrap water. PEG grafts were not effective in improv-

ing the elastic recovery of the PNIPAAm hydrogels, as PEG branches

were effective in increasing the water content of PNIPAAm hydro-

gels. In totality, a care must be taken to balance the PNIPAAm/PEG

ratio to get better resulting material for implantation.

4.2.2. PNIPAAm–PEG, impregnated microgel injection for possible

applications in biomedical and biotechnology fields

The hydrogen-bonding efficiency becomes weaker to solubilize

PNIPAAm, at a temperature above its LCST. Due to the occurrenceof a thermoreversible change between the polymer-enriched phase

Fig. 3. Synthetic scheme of PNIPAAm–PCEC–PNIPAAm penta-block copolymer [76].

29 3(methacryloxy) propyltrimethoxysilane.








and aqueous phase, PNIPAAm hydrogel is applied for various bio-

medical applications such as, controlled drug release, protein–

ligand recognition including immobilization of enzyme [79,80].

To improve the thermoresponsive properties of PNIPAAm hydro-gels, 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 aus-

picious 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 matri-

ces. The prepared microgel-impregnated PNIPAAm hydrogels signif-

icantly showed tighter and array porous network in comparison with

the pure form of the PNIPAAm. As the concentration of the impreg-

nated-microgel increases, the pore size reduces. Although there is no

difference in the LCST of the impregnated-microgel, PNIPAAm andthe 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. Chitosan–PEG–PNIPAAm, hydrogels influenced by PEG

(molecular weight)

It was observed that molecular weight of PEG (MW 2000–

20,000) significantly improves the physical and mechanical

properties of the chitosan-PEG-poly(N-isopropylacrylamide) (PNI-

PAAm) hydrogels. Increased molecular weight of PEG reduces the

crystallinity of the physical hydrogel, subsequently, improving its

polymer-to-polymer interactions. Similarly, an increase in themolecular 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. Chemi-

cally cross-linked hydrogel polymer such as the PNIPAAm has its

own limitation of being non-biodegradable. Thus, the hydrogels

formed specifically 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 MWPEG 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 chainitself. The crystallinity peak of the PEG dramatically

increases as the MWof the PEG increases from 2000 to 20,000. Low

MW PEG (2000) weakens the physical crosslinking with the chito-

san 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 inter-

action and the presence of the free PEG chain segments, exhibiting

the higher level of crystallinity with reduction in the crystallization

temperature, T c of PEG. Chitosan–PEG–PNIPAAm 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 swell-

ing. 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 pre-

pared, 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 applica-

tions. Previously, gelatin had been used as an efficient carrier for

the delivery of pilocarpine in the form of a device known as Gel-

foam sponge [83]. In addition to prolong the residence of pilocar-

pine at the eye surface bioadhesive gelatin nanoparticles were

used for topical applications. Another significant finding includes

sustained release of epidermal growth factor from cationized gela-

tin 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-cappedPN31 through a carbodiimide-mediated coupling reaction. Phase

transition occurs due to alteration in the external temperature,

resulting in the modification 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 vis-

cosity binding effect of gelatin the GN32 gels possess remarkable

adherence properties. As the concentration of polymer increases

solution flow 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 poly-

mer chains. Moreover, when dissolved in deionized water, the PNand GN had LCST of 31.3 ± 0.1 and 32.2 ± 0.1 C, respectively. To val-

idate the prepared biodegradable in situ forming GN gels, animal

model was selected and the formulation was administered parente-

raly 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 pharma-

cological 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.

Fig. 4. The schematic image of sol–gel transition of PNIPAAm–PCEC–PNIPAAm

penta-block copolymer. (For interpretation of the references to color in this figure

legend, the reader is referred to the web version of this article.)

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

31

Poly(N-isopropylacrylamide.32 Graft copolymer.






http://-/?-

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

‘Smart’ polymers undergo reversible phase transition once trig-

gered 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 PNI-

PAAm, the temperature-dependent interactions are a result of bal-

ance between hydrogen bonding of hydrophilic segment of the

polymer chain and hydrophobic interaction among polymer chainsand hydrophobilc interaction among isopropyl domains. The

limitation of PNIPPAm polymer is associated with its non-

biodegradability [86], which is overcome by combining the bio-

polymers. When small chains of NIPAAm were grafted onto

chitosan polymer backbone forms a material showing both tem-

perature and pH dependence. An applied and controllable

approach for the attachment of polymer to surface is the use of

LbL 33 technique. In this method, the formation of polyelectrolyte

multilayer is sequentially treated with a charged surface solution

comprising oppositely charged polyelectrolytes [87–90]. Martins

and colleague [93] demonstrated the formation of in situ hydrogel

of a new thermoresponsive thin film build by electrostatic assembly.

They utilized the LbL approach for cell sheet purpose by traditional

grafting techniques. Chitosan-graft-NIPAAm was synthesized bygraft polymerization of NIPAAm on to chitosan using ceric ammo-

nium nitrate (CAN) as an initiator. In the wet state, final 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 PNI-

PAAm 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 ‘salt-

ing 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

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

successfully attached and proliferate at 37 C followed by detach-

ment of cell sheets with deposited extracellular matrix triggered

by temperature. This technique can be better used in cell sheet engi-

neering. LbL technique in addition is a suitable candidate for drug

delivery and controlled release systems, sensory devices, filters

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 biode-

gradable poly(lactide)–poly(ethylene glycol) (PLA–PEG) and

poly(lactide-co-glycolide)–poly(ethylene glycol) (PLGA–PEG)

copolymers [95–98]. PLA–PEG and PLGA–PEG 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 identified 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 graft-

ing 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-phos-

phate (BP)-encapsulated NPs. The NPs were formed from a blend

of PLA homopolymers and PNIPAAm–PLA 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/PNIPAAm–PLA 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

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

33 Layer-by-layer. 34 Polymeric nanoparticles.








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/PEG–PLA NPs. Whereas, PLA/PNIPAAm–PLA

NPs showed a f potential of 20 mV at low temperature. Above

the LCST, the release of the BP from the NPs accelerated. The cellularuptake of the PLA/PNIPAAm–PLA NP was not noticed below the

LCST. On the other hand, above LCST the PLA/PNIPAAm–PLA NP

was noticed inside the cells around the cell nuclei. These results

indicated that PLA/PNIPAAm–PLA NP could allow controllable drug

release and cellular uptake by changing the temperature [101].

5. Author’s perspective

The present article highlights the significance 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 proper-

ties 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

modification 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 multi-

ple routes of delivery systems such as oral, ocular, rectal, vaginal,

and parenteral routes make this system more versatile. These

PEG–PNIPAAm based copolymers are considered to be the pre-

ferred copolymer for the delivery of proteins and peptides over

PEG–PLGA based copolymers, when prolong action of drug release

is concerned. This is due to the ease in the grafting procedure andwith 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 stimuli-

induced physical interactions, such as micellar ordered-packing,

phase-separation, hydrophobic association, crystallization, stereo-

complexation and electrostatic interactions are the basic principles

leading to the success of the various PEG based triblock copoly-

mers. Among various stimuli factors, temperature is most conve-

nient 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 maintain-

ing 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 visco-

elastic and pure elastic behavior. Crosslinked polymers prevent

dissolution of the hydrophilic polymer chains in an aqueous med-

ium. 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 biocompati-

bility and the degradation products formed should have a low

toxicity. The degraded products formed thereof can be tailoredby 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 poly-

mer exhibits thermogelling copolymer solution, which below the

LCST is solution and form gel when temperature increases above

theLCST. Compared to the Poloxamers, injectable hydrogels formed

by PEG–PNIPAAm based triblock copolymers are more biocompati-

ble andbiodegradable. Thehydrogel formed by theuse of thePolox-

amer 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, Polox-

amer with high concentration (>16%, w/w), exhibit toxicity when

administered intraperitoneally. These limitations can be bypassed

by designing biodegradable thermogelling copolymers. On consid-

ering these facts thePEG–PNIPAAm appearsas a suitablecopolymer

for the preparation of thermoresponsive in situ forming hydrogels.

Triblock copolymers composed of PEG/PLGA, PEG/poly(caprolac-

tone), PEG/poly(propylene fumarate), PEG/poly(propylene glycol)/

polyester, PEG/peptide, chitosan/glycerolphosphate, and

poly(phosphazenes) exhibits sol–gel transition in water as the tem-

perature rises. Amongthem 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. PEG–PNIPAAm based hydrogels are biode-

gradable and deliver the drug at the target site for several hrs. PEG–

PLGA based hydrogels are also biodegradableandnon-toxicandcan

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 compo-

nent of a multiple block copolymer for the injectable preparation,

some issues must be cleared. NIPPAM homopolymer and copoly-

mers are of potential for its application in various biomedical

sectors; however, its clinical applications are still a major chal-lenge. PNIPAAM and its copolymers are not biodegradable, until

grafted with PEG. Thus, the use of PEG/PNIPAAm based thermore-

sponsive hydrogel will be more compatible, as usingonly the acryl-

amide-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 PEG–PNIPAAm based

hydrogels specifically for the parenteral route, increase the efficacy

of various proteins and peptides along with other bioactives, lead-

ing to an increase in patient compliance and will definitely

improve the host acceptance. Anticancer drugs could be loaded

to such copolymers and delivered to the specific site, providing a

local and a prolong action over the injection/tumor site. The vari-ous mustard derivatives used for the DNA alkylation can be deliv-

ered using the PEG–PNIPAAm based hydrogel as these will reduce

the initial burst of the molecule and thus will reduce the host tox-

icity. 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

PEG–PNIPAAm 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 hydro-

gels can be prepared, but the injectable hydrogels stimuli triggered

by temperature are still a more effective delivery system. SomePEG based copolymers are PEG–PNIPAM; PEG/PLGA; PEG–PCL.






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Moreover, PEG–PNIPAAm 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 conflict

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 financial assistance under DST-FIST

scheme as well as the Maulana Azad National Fellowship (MANF)

UGC, New Delhi, India for providing financial 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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Please cite this article in press as: A Alexander et al Polyethylene glycol (PEG) Poly(N-isopropylacrylamide) (PNIPAAm) based thermosensitive injectable























































































































































































































































































































































































alexander, amit; ajazuddin, ; khan, junaid; saraf, swarnlata; sa -- polyethylene glycol

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