Polyethylene glycol (PEG)–Poly(N-isopropylacrylamide) (PNIPAAm) based thermosensitive injectable hydrogels for biomedical applications

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

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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 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 potential and benefits of PNIPAAm based thermoresponsive hydrogels for the biomedical application. Ó 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 [1–3]. The extensive research had evolved the invention of long acting injections and implants [4–7] 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: [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.

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) triblock copolymers comprising of poloxamer and PEG.5 Characteristically, 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], 4 5

Hydrogels. Polyethylene glycol.

http://dx.doi.org/10.1016/j.ejpb.2014.07.005 0939-6411/Ó 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

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

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 specifically, 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 Waal’s interactions, crystal formation 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 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 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 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 fluorescent probe as well as an anti-cancer drug too. The study showed that PEO-bPNIPAAm-BAC nanoparticles sequester Dox. The outcome of this modification 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 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 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 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 suitable 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 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 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]. 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) xxx–xxx

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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

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 30–35 25.0–98.5 13–65

[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 [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-biodegradable, 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 (monomer) and acrylate terminated PLEL16 (biodegradable macromonomer 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 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

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the poly(NIPAAm-co-PEG) and poly(NIPAAm-coPEG)-acrylate copolymers form gel above their LCSTs. Poly(NIPAAm-co-PEG) demonstrates 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 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– 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 thermoresponsive and biodegradable copolymers. Copolymer PNIPAM– PLLA–PEG–PLLA–PNIPAM exhibited thermoresponsive properties which shows more biocompatibility with probably partial biodegradability [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 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. 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 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 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 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 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 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 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 immediately but in subsequent weeks, a significant 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.

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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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. PNIPAAm–PCL–PEG–PCL–PNIPAAm, thermosensitive pentablock copolymer injectable carriers for sustained drug delivery systems Thermosensitive PLGA copolymers required several hours to solubilize in water, making it a difficult and time-consuming process. 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 hydrophobic polyester, such as PCL–PEG–PCL (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 PCL–PEG–PCL 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 sol–gel system based on copolymers that contain both PCL–PEG– 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) (PNIPAAm–PCL–PEG–PCL–PNIPAAm), which was synthesized by a combination of controlled ROP27 and ATRP28 (Fig. 4). This pentablock 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–PNIPAAm penta-block copolymer while keeping its thermogelling property 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.

27 28

Ring-opening polymerization. Atom transfer radical polymerization.

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

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, 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 [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 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 sufficient 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 (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 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. 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 occurrence of a thermoreversible change between the polymer-enriched phase

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) xxx–xxx

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.)

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. 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) (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 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 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. 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 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 efficient 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 significant finding 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 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 viscosity 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 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.

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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

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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 [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 by graft polymerization of NIPAAm on to chitosan using ceric ammonium 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 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 films 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, 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 biodegradable 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 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 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 34

Polymeric nanoparticles.

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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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 cellular uptake 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 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 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 multiple 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 preferred 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 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.

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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 PEG–PNIPAAm 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 PEG–PNIPAAm 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 sol–gel 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. PEG–PNIPAAm 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 PEG–PNIPAAm based hydrogels specifically for the parenteral route, increase the efficacy of various proteins and peptides along with other bioactives, leading 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 various mustard derivatives used for the DNA alkylation can be delivered using the PEG–PNIPAAm 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 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 hydrogels can be prepared, but the injectable hydrogels stimuli triggered by temperature are still a more effective delivery system. Some PEG based copolymers are PEG–PNIPAM; PEG/PLGA; PEG–PCL.

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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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