Pre-clinical and clinical applications of thermoreversible hydrogels in biomedical engineering: a review

Thermoreversible polymer hydrogels (TRGs) are physical aqueous networks triggered by temperature that ﬁ nd potential applications in tissue engineering (TE) and drug/gene delivery. When systems with lower critical solution temperature (LCST) behav- iour are used, the aqueous solution of polymer is mixed with cells or drugs/genes, depending on the desired application, at room temperature and then injected in vivo to form a hydrogel. This in situ forming hydrogel acts as a matrix for tissue regeneration or controlled and targeted release of the compound. This review focuses on discussing the studies in which synthetic TRGs with LCST behaviour have been applied in vivo in model animals. These studies are categorised depending on the general structure of the polymer, as follows: (i) poloxamers (also known as Pluronics®), (ii) degradable polymers, (iii) poly( N -isopropy-lacrylamide), (iv) poly(organophosphazene) and (v) poly(2-ethyl-2-oxazoline). In general, when the system is optimised, TRGs provide sustained and topical release of the drugs, thus minimising the undesired side effects. When applied in the TE ﬁ eld, tissue formation was observed. Interestingly, two polymers have reached clinical trials, namely poloxamer 407 (P407, Pluronic® F127, often combined with P188, F68) and Regel® (the formulation of Regel® with the anticancer agent paclitaxel is known as Oncogel®), indicating the challenges that need to be overcome before successful application in clinics. and (b) drug/gene delivery. In both applica- tions, the solution phase at room temperature ( T r.t. < T gel ) facilitates (i) easy mixing of the solution with cells and/or drugs and (ii) the easy loading of the mixture into a syringe. Upon injection, gelation occurs, because of the temperature increase to body temperature ( T b.t. > T gel ). In the TE application, new tissue formation takes place at the defect side, while the polymer leaves the injection side. In the drug/gene delivery concept, topical and sustained release of the molecule of interest takes place, thus increasing the bioavailability in the area of interest and minimising side effects.


INTRODUCTION
Thermoreversible polymer hydrogels (TRGs) are 3D physical polymeric networks in aqueous media. These hydrogels are formed as a result of temperature variation, via physical interactions between the different polymer chains, with the physical bonds relying on hydrogen bonding, hydrophobic junctions etc. As the nature of these bonds is physical, the gelation is reversible, with the gel returning to the solution state as the stimulus, i.e. temperature, is removed, when sufficient time is allowed. However, it should be noted that irreversibility can be caused when additional interactions are introduced, 1,2 such as stereocomplex formation within the gel structure 2 or due to other additives interfering with the physical network. 1 Depending on whether the sol-gel transition occurs as the temperature is increased or decreased, these systems are characterised by lower and upper critical solution temperature (LCST and UCST), respectively. LCST-type TRGs have received much attention due to their potential application in the tissue engineering (TE) and drug/gene delivery fields. [3][4][5] For these applications, an aqueous solution of polymer is mixed with cells (for TE) or drugs/genes (for delivery) at room temperature (r.t.); thus their structural integrity is preserved, and the mixture is easily loaded into a syringe. Upon injection into the body, a stable gel is formed, because of the increase in temperature to body temperature (b.t.), which traps the compounds of interest, i.e. cells and/or drugs/genes. In the case of TE, the cells proliferate and thus tissue formation occurs while the gel disappears, either via dissolution or degradation. In the drug/gene delivery field, controlled and local release takes place, thus increasing the bioavailability of the compound. The local release is beneficial especially in the case of drug delivery, as it minimises the side effects associated with the systemic release. This is shown schematically in Fig. 1. In drug delivery, epicutaneous application might take place for the treatment of skin disorders, such as acne 6 and psoriasis. 7 In this case, the sample is applied as a gel, while the solution phase at low temperatures facilitates the homogeneous loading of the drug.
TRGs have been widely studied during the last 50 years, with the first systematic study on TRGs with LCST behaviour dating back to 1984. 8 The progress in polymer chemistry facilitated the synthesis of thermoresponsive polymers and thus a great deal of relevant research has been published. [9][10][11][12][13][14][15][16][17][18][19][20][21][22][23][24] These studies aim to produce thermoresponsive polymers and investigate their LCST transition and thermogelation, with the main goal to design systems that show clear sol-gel transition between r.t. and b.t. Natural polymers possessing thermoresponsive properties have also been investigated as TRGs. 25,26 Synthetic polymers have received much attention due to their numerous advantages, with possibly the most important one being their tailor-ability, i.e. their properties can be tuned to match the requirements of the desired application. 27,28 Also, the synthesis is reproducible; thus they are characterised by 'batchto-batch uniformity'. In addition, depending on the polymerisation method, they can be produced in a large scale at low cost, and they have long shelf-time. 27,28 Prior to in vivo application, gelation at b.t. and in vitro biocompatibility should be confirmed. In the case of degradable systems, in vitro degradability tests should also be carried out. When the TRGs are used as drug delivery matrices, the effect of the drug on the gelation properties should also studied, since it has been shown previously that the incorporation of drugs affects the gelation. [29][30][31] In addition to the effect of any additives on the gelation, it is also well documented that the solvent, such as the presence of salts, affects the thermoresponse, i.e. LCST and/or gelation. [32][33][34] As an example, when sodium chloride is added to poloxamer solutions, the gelation point decreases as the ionic strength increases, as sodium chloride is well known to cause the 'saltingout effect', i.e. reduction of the solubility with increased ionic strength. 34 On the other hand, when sodium thiocyanate was used, a 'salting-in effect' was observed instead, with the gelation region becoming narrower and moving to higher temperatures. 34 Therefore, the gelation in the specific system of polymer-solvent of interest should be studied prior to the application.
Previously published reviews have summarised and discussed the thermodynamics of the 'hydrophobic effect', 35 the effect of the structure on the gelation, 35,36 as well as the gelation of supramolecular 37 and biodegradable hydrogels, 38,39 the application of thermoresponsive polymers in cell sheet engineering, 4 and drug delivery through micellisation. 5 The current review focuses on discussing the studies in which synthetic TRGs with LCST behaviour have been applied in vivo. These studies are categorised depending on the general structure of the polymer and/or repeated unit which provides thermoresponsive behaviour: (i) poloxamers (also known as Pluronics®), (ii) other degradable ethylene glycol (EG) based polymers besides poloxamers, (iii) poly(N-isopropylacrylamide) (PNIPAAm), (iv) poly(organophosphazene)s and (v) poly(2-ethyl-2-oxazoline) (PEtOx). These units are often combined with other species in order (i) to achieve gelation at the desired temperature, (ii) to improve the mechanical properties of the gel and/or (iii) to introduce degradability into the structure. The chemical structures of these units are shown in Fig. 2. Most of the in vivo tested injectable gels show sol-gel transitions between r.t. and b.t.; thus the sol phase is loaded into a syringe and, upon injection into a model animal, a gel is formed. Nevertheless, injection of the TRGs in the gel phase was also tested, [40][41][42] which is feasible due to the shear-thinning properties of TRGs.
The thermoresponsive systems, i.e. polymer structure, solvent and concentration, the model compounds used (if any) and the main in vivo results are summarised in Table 1. Table 1 also lists the architecture of the polymer, and the studies are shown in order of increased complexity of architecture (within the same category of TRG). Additional groups that are chemically bonded on the main thermoresponsive species are also presented. Any polymeric additives that are physically mixed in the gel precursor are listed, as they affect the gelation temperature and concentration. Figure 1. Application of thermoreversible hydrogels (TRGs) as injectable gels in (a) tissue engineering (TE) and (b) drug/gene delivery. In both applications, the solution phase at room temperature (T r.t. < T gel ) facilitates (i) easy mixing of the solution with cells and/or drugs and (ii) the easy loading of the mixture into a syringe. Upon injection, gelation occurs, because of the temperature increase to body temperature (T b.t. > T gel ). In the TE application, new tissue formation takes place at the defect side, while the polymer leaves the injection side. In the drug/gene delivery concept, topical and sustained release of the molecule of interest takes place, thus increasing the bioavailability in the area of interest and minimising side effects.
Several studies in which aqueous solutions of pure poloxamer 407, i.e. without any polymer additives that would act as gelation modifiers, were applied in vivo have been reported in the literature. 6,[43][44][45][46][47][48][49][50][51][52][53][54][55][56]195,196 Most of the studies have used solutions of poloxamer 407 at 20 w/w% and higher, due to the higher stability and strength of the gel; however, a few studies have attempted the application at 15 w/w%. In TE, the gel was used to replace the nucleus pulposus, which acts as a shock absorber, in the intervertebral disc in dogs, and it was proven to be effective in resisting the strain applied for at least a period of 3 months. 44 In another study, bone mesenchymal stromal cells (BMSC) and chondrocytes were incorporated in the gel matrix, and the TRG system led to chondrogenic differentiation of BMSC. 43 In the drug delivery concept, solutions of P407 were investigated in vivo as carriers for HIV-1 lentiviral vector, 56 quetiapine fumarate, 55 latanoprost, 196 paclitaxel (PTX), 54 lapatinib, 54 vancomycin, 53 brimonidine tartrate, 51,52 rapamycin, 50 halobetasol propionate, 49 polyphenol tannic acid, 48 dapsone, 6 soy isoflavone, 47 RNA polymerase I inhibitor 46 and simvastatin. 45 This indicates the wide variety of diseases, ranging from cancer 50,54 to chronic otitis, 53 ocularrelated diseases, 51,52 acne, 6 intimal hyperplasia 45 and wound healing, 6,47,48 for which TRGs have been in vivo tested as drug delivery carriers. It also highlights the great potential of these gel matrices in biomedical engineering, as it has been observed that incorporation of the drugs in the gel depots provides local release 52,53 and controls the drug release rate. 52,54 An approach which has been proven to be effective in improving the drug bioavailability is to incorporate the drug molecules in particles, such as liposomes and niosomes, prior to their incorporation in the gel matrix. 47,[49][50][51][52]55,196 Formulations based on mixtures of poloxamers have also been investigated as in vivo injectable systems. 59,[65][66][67][68][69][70][71][72][73] This strategy has been applied in an attempt to control the thermogelling properties of the final system as desired. In all cases, the main thermoresponsive component was P407, while most of the studies used P188 58,60-73 as polymer additive/viscosity modifier. Nevertheless, studies on mixtures of P407 with P124, 59 P123 58 and P338 57 have also been reported. These mixtures, loaded with drugs, were tested as drug delivery systems. 57,[59][60][61][62][63][64][66][67][68][69][70][71][72][73] Biocompatibility, 62 enhanced drug bioavailability, 60,61,63,64,73 as well as controlled release 59,67-73 were observed. Interestingly, in one of the studies in which PTXcontaining liposomes were injected in tumour-bearing mice, tumour growth was controlled by the local and sustained release of the drug. 70 In another interesting study, a mixture of P407 and P188 was used as drug release depot for monosialoganglioside in the spinal cord of a rat, and prolonged release as well as increased drug bioavailability were observed. 73 However, it should be noted that, depending on the ratio of the two copolymers, burst release 65,72 or short-term controlled release followed by burst release 57 might take place in vivo. As demonstrated by Al Khateb et al., when formulations based on P407 and P188 were investigated, the gelation temperature (T gel ) increased as a function of the content in P188 (Fig. 4), thus also leading to burst release rather than controlled release. 72 Therefore, optimisation of the polymer concentration and/or incorporation of additional polymers is suggested.
Other polymers, both natural and synthetic, have also been mixed with poloxamers to enhance the strength of the gels and/or provide mucoadhesive properties.  For example, Figure 2. Names, chemical structures and abbreviations of commercially available thermoresponsive polymers (top), other common thermoresponsive components (middle) and monomers typically incorporated into the structure to introduce degradability (bottom).  Applications of thermoreversible hydrogels in biomedical engineering: a review www.soci.org  a This is the concentration used for the in vivo application, and it does not necessarily correspond to the critical gelation concentration of the system. When the system consists of a mixture of poloxamers only, the concentration given is the total concentration, while for mixtures with other additives the concentration stated is the one corresponding to the main thermoresponsive component. Note that any additives, such as polymers, drugs and cells, might affect the gelation properties. b The in vivo result is system dependent, i.e. the combination of gel matrix and active compound.  83 polycarbophil, 93,99 methoxy poly(ethylene glycol)-b-poly(D,L-lactic acid) (poly(MPEG-b-PDLLA)) 95 and glycosaminoglycans, 96,102 such as hyaluronic acid (HA). 96 These systems were tested as delivery matrices for several compounds such as anticancer agents, [75][76][77]82,86,87 anti-inflammatory/anti-fungal/anti-bacterial/anti-microbial drugs, 79,81,91,100,101 cholesterol-lowering drugs, 198 antidiabetic drugs (insulin), 85 anticonvulsant agents 90,98 and vaccines. 92 Similar observations as in the previous studies were made, such as biocompatibility, 93 improved bioavailability of the compound, 88,94 controlled and prolonged release, [78][79][80][81][82][83][84][85][86][87] and control of tumour when anticancer agents were used. [75][76][77] In one of the studies, the gel loaded with vitamins was injected in a model animal suffering from skin burn, and wound healing was observed due to the antioxidant action of the vitamins. 96 In an interesting study by Luo et al., the combination of topical and controlled release of a compound from a TRG with photodynamic therapy was investigated. The gel matrix consisted of P407 and MPEG-b-PDLLA containing a two-photon absorption compound and a photosensitiser. 95 The mixture was injected in tumour-bearing mice and then irradiated with near infrared (NIR) light to initiate photodynamic therapy. Prolonged retention of compounds on the tumour and inhibition of tumour growth were observed, with the compounds showing minimal cytotoxicity when non irradiated. 95 In the only study on the TE concept, P407 was mixed with glycosaminoglycans and bone morphogenic protein (BMP-2), and upon injection in a model animal recovery of the injured cartilage was observed. 102 Another approach to modifying the properties of the poloxamers is to covalently link the polymer with other molecules, 103,104 or by further polymerising it to form pentablock terpolymers. [105][106][107] The studies in which these approaches have been applied are discussed below.
In two of the studies, P407 was covalently linked to either chitosan 104 or tetraaniline. 103 In the case of chitosan-based polymer,  wileyonlinelibrary.com/journal/pi it was concluded that the chemical bond between the polymers enhanced the stability of the gels compared to the gel formed by the physical mixture of P407 and chitosan. 104 A solution of P407-g-chitosan was loaded with poly(lactic acid-co-glycolic acid) (PLGA) nanoparticles containing vaccines, and upon in vivo injection, successful vaccine delivery was observed. 104 In another study, P407 was covalently linked to tetraaniline, which is electroactive. It was observed that the presence of tetraaniline enhanced the strength of the gel, and in vivo gelation was confirmed. 103 Ring opening polymerisation (ROP) of poloxamers facilitates the synthesis of pentablock copolymers. [105][106][107] When polymerising trimethylene carbonate (triMeCa) on P407, it was observed that the pentablock terpolymer showed enhanced gelation and improved cytotoxicity compared to P407. 105 Injection of 5 wt% copolymer solution containing mitomycin C in rabbits, which had previously undergone a glaucoma filtration surgery, improved their health. 105 In two studies by Li et al., L-lactic acid formed the outer blocks of a pentablock terpolymer based on P105. 106,107 In both studies, the gel matrix, formed by concentrated solutions (35-40 wt%), was tested as a drug delivery depot for either antimicrobial peptides or anticancer agents, such as docetaxel (DTX) and oxaliplatin. Local and controlled release of the compound was observed upon injection, while in the case of delivery of anticancer agents growth of tumour, angiogenesis and tumour cell proliferation were suppressed, which subsequently prevented metastasis. 106,107 However, it should be noted that the concentrations used in the last two studies are relatively high; thus their commercial application might be limited because of high cost.
Diblock copolymers based on EG (A block) and an ester-containing unit (B block) were investigated. [108][109][110] In two of the studies, the B block was based on either CL or both CL and p-dioxanone (DO). 109,110 Upon injection of 20 wt% solution in rats, the gels were preserved for over a month, thus sustainably releasing bovine serum albumin (BSA) or bovine insulin. Compared to poloxamer 407, the gel of P407 was destabilised after 3 days, thus limiting the drug release efficacy. It was also observed that the presence of DO increased both the T gel and the release rate, 109,110 which can be attributed to the increased hydrophilicity of DO compared to CL because of the extra oxygen in its structure. In the third study, the B block was based on LA and 1,6-bis(p-carboxyphenoxy)hexane and in vivo biocompatibility was confirmed. 108 In several studies, ABA and BAB triblock copolymers with A and B blocks based on EG and CL, respectively, were investigated. [111][112][113][114][115][116] Concentrated solutions of these copolymers (20-30 wt%) were injected in model animals, and in situ gelation and degradation of the gel were observed. 111,112 Also, these thermogels provided sustained release of lidocaine, 116 basic fibroblast growth factor 114,115 or camptothecine (CPT)-containing particles. 113 Interestingly, sustained and topical CPT release from the gel matrix both controlled the growth of tumour and prevented metastasis. 113 ABA and BAB triblock copolymers with the B block based on PLGA were also evaluated for in vivo gel formation and injectability. 30 203 ) and other groups around the world since then. 31,40,118,[123][124][125]127,204,205 The patents cover both ABA and BAB polymers with total molar mass ranging from 2000 to 4990 g mol −1 , hydrophobic content varying from 51 to 83 w/w%, and a range of hydrophobic esters which are polymerised to form a distinct B block at various percentages. [200][201][202] Amongst these, the best performing ones are BAB polymers with MM ≈ 3100-4500 g mol −1 , hydrophobic PLGA content between 51 and 83 w/w% and lactate content in the PLGA ranging from 65 to 85 mol%. The latter polymers are known as Regel®, and their T gel varies with the exact structural properties, i.e. MM and content in EG, LA and GA. 40,204 Unlike Pluronics® which form traditional core-shell micelles above their critical micellisation temperature, Regel® polymers form flower-like micelles, with the hydrophobic PLGA block forming the core of the micelle and the hydrophilic PEG block forming the 'petals' of the flower, also referred to as loops (Fig. 6). The gelation of these polymers relies on a polymer chain joining two micelles, as shown schematically in Fig. 6.
Concerning the ABA polymers, in vivo gelation, biocompatibility and degradation were confirmed. [117][118][119] Similarly to their ABA counterparts, the BAB triblock copolymers are biocompatible 122 and gel and degrade in vivo. 30,122 Their concentrated solutions (20-25 wt%) were also tested in vivo as drug delivery systems. 30,31,[121][122][123][124][125][126][127] The TRGs successfully served as matrices for sustained release of (i) interleukin-2, thus inhibiting growth of tumour, 126,127 (ii) huperzine A-phospholipid, thus reducing its toxic side effects, 121 (iii) Avastin®, which increased the half-life of the drug, 30 (iv) 5-fluorouracil, which prevented adhesion in the Achilles tendon, important to avoid pain after surgery, 122 (v) dexamethasone acetate for improved ocular drug delivery, 120 (vi) insulin or glucagon-like peptide for the treatment of diabetes [123][124][125] and (vii) irinotecan, and tumour regression was observed. 31 In three studies, end-capped PCLA-b-PEG-b-PCLA triblock copolymers were investigated, where PCLA stands for poly (ε-caprolactone-co-lactide). 42,128,129 Acetyl, 42,129 2-(2 0 ,3 0 ,5 0 -triiodobenzoyl) (TIB) 42,129 and propionyl 128 groups were used as endcapping units. TIB groups were introduced into the structure to allow visualisation, but this caused solubility issues due to the increased hydrophobicity. 42 25 wt% copolymer solution with an acetyl/TIB ratio of 25/75 was in vivo administered while in the gel phase (T gel at 10-15°C), and in vivo degradation was confirmed, 42 while the acetyl analogue promoted in vivo sustained release of celecoxib (COX). 129 The propionyl-capped polymer solutions were investigated as effective COX carriers in mice and dogs. 128 Biocompatibility was confirmed after injection in mice, while intradiscal injection in dogs suffering from intervertebral disc degeneration improved their health. 128 BAB triblock copolymers based on PEG (A block) and on a random copolymer of CL with either GA 130 or 1,4,8-trioxa [4.6]spiro-9-undecanone (TSUN) 131 (B block) were also applied in vivo. 25 wt% GA-based copolymer solution containing liraglutide was injected in mice and in vivo biocompatibility, degradation and hypoglycaemic efficacy were confirmed (Fig. 7). 130 A threecomponent system consisting of 25 wt% TSUN-based copolymer solution, doxorubicin (DOX) and iodine 131 -labelled HA was injected in tumour-bearing mice, which provided local administration of DOX and inhibition of tumour growth. 131 Degradable linear multiblock copolymers based on PEG were also tested as in vivo thermogels. 29,137 In one of the studies, a Figure 5. Acid-catalysed (top) and base-catalysed hydrolytic degradation of ester-containing polymers, with R representing an alkyl group; CH 2 , CHCH 3 and (CH 2 ) 5 groups for poly(glycolic acid), poly(lactic acid) and poly(ε-caprolactone), respectively, with glycolic acid, lactic acid and 6-hydroxyhexanoic acid being the by-products, respectively. For simplicity, the hydrolysis is shown to occur starting from the end of the polymer chain; however, the degradation can happen at any part of the polymer chain which is exposed to water molecules. During in vivo conditions, acid-catalysed hydrolytic degradation might be more favourable.
17 wt% solution of PEG-b-PCL-b-PLA-b-PCL-b-PEG, loaded with PLGA nanoparticles and vaccines, 29 was studied; PCL and PLA stand for poly(ε-caprolactone) and poly(lactic acid), respectively. The second study investigated 25 wt% solutions of multiblock copolymers based on PEG, poly(propylene glycol) and poly(polytetrahydrofuran carbonate), loaded with DOX. The TRGs were injected in tumourbearing mice, and successful inhibition of tumour growth was observed. 29,137 An interesting systematic study in which four-star copolymers were investigated as in vivo gelators was also reported. 138 The  Polym Int 2021 core of the star was based on PLGA of various LA/GA molar ratios, whereas the arms were based on methoxy PEG (MPEG). 30 wt% of the copolymer solutions were injected in rats and rapid gelation, degradation and good biocompatibility were confirmed. 138 Incorporation of carbamate bonds also introduces degradability into the system. Two studies, carried out by Wang's group, were based on ABA triblock copolymers with A and B blocks based on EG and serinol hexamethylene urethane. 132,133 In vivo biocompatibility and controlled release of bevacizumab were observed. 132,133 All the studies discussed so far are based on hydrolytically degradable systems, the degradation of which can be accelerated in an acidic environment. Chen's group investigated polymers which are based on polypeptides and PEG, and thus their degradation is enzymatically catalysed. [134][135][136] In two of the studies, L-alanine and L-phenylalanine composed the peptide part. 135,136 8 wt% of polypeptide solution was injected in rats and in vivo biocompatibility and degradation were confirmed. 136 In vivo injection of the polymer solution containing combrestatin A-4 particles and DTX showed inhibition of tumour growth. 136 When mixed with bone marrow mesenchymal stem cells and injected in rabbits with osteochondral defect, cartilage regeneration was observed. 135 In an interesting study by the group, L-methionine was used instead to protect the cells from reactive oxygen species. 134 Upon exposure to hydrogen peroxide (H 2 O 2 ), the thioether group of L-methionine is oxidised to sulfoxide and sulfone groups. Thus, the polymer becomes more hydrophilic, leading to destabilisation and degradation of the gel. Upon injection in rats, in vivo degradation was completed after 6 weeks. 134 PNIPAAm-based TRGs PNIPAAm is one of the most popular thermoresponsive polymers (Fig. 2) and it has been extensively investigated either as a homopolymer or by co-polymerising its repeated unit, i.e. N-isopropylacrylamide (NIPAAm), with other units in order to obtain the desired properties. 4 Below its LCST, NIPAAm units interact with water via hydrogen bonding; however, upon increasing the temperature, hydrogen bonding between the different NIPAAm units is favoured instead, thus leading to a thermoresponse (Fig. 8). As its cloud point (CP) is around 31°C, and independent of the MM and composition, 206 it has attracted much interest for biomedical applications. Due to its promising properties, in vivo applications of NIPAAm-based random, [139][140][141][142]144 block, [146][147][148] branch, 149-153 conjugate 163,164 and star 166 synthetic copolymers as well as NIPAAm-based polymers covalently 20,143,[154][155][156][157][158][159][160][161] or physically mixed 143,145,151,152 with natural components were reported in the literature.
Random NIPAAm-based copolymers, synthesised via freeradical polymerisation, were applied in model animals as TRGs. [139][140][141][142]144 Several units have been used as comonomers such as acrylic acid (AA), 141,142 γ-butyrolactone acrylate, 142 Nhydroxysuccinimide, 141 acryloyloxy dimethyl-γ-butyrolactone, 141 poly(ethylene glycol) methyl ether methacrylate, 140 methacrylic acid 140,144 and n-butyl acrylate (BuA). 139 Upon injection of the solutions in model animals, in situ gelation [139][140][141][142] and in vivo biocompatibility were confirmed. [139][140][141][142] Interestingly, in one of the studies, tetraaniline was incorporated into the structure to provide the electroactive and antioxidant properties needed for myocardial infarction therapy. 140 In another study, a macromonomer on which the drug was covalently linked, namely 2-hydroxyethylmethacrylate-g-poly(trimethylene carbonate)indomethacine, was copolymerised with NIPAAm, and antiinflammatory action for treatment of uveitis was confirmed. 144 In an interesting study by Bayat et al., the TRG was in vivo investigated as a possible sealant in ocular trauma. 139 The solution was injected using an in-house developed syringe, and practical workshops in which this copolymer solution was applied ex vivo in pig eyes were held. Military ophthalmologists and clinicians participated in the workshop, and they were briefly instructed on how to apply the injectable gel. On the first attempt, 43% of the participants achieved the application of the TRG, and all of them were successful on the second time. 139 This highlights the applicability of TRGs as injectable gels.
(A-co-C)-b-B-b-(A-co-C) triblock copolymer, where A, B and C units are based on NIPAAm, EG and BuA, respectively, was applied as wound dressing. 1 This copolymer solution was mixed with silver nanoparticle decorated reduced graphene oxide (GO) nanosheets, and the mixture was applied as a spray on the skin, after which gelation was observed. 1 In two studies by Gupta et al., ABC triblock copolymers were synthesised via reversible addition-fragmentation chain transfer polymerisation and were tested as in vivo drug release depots. 146,147 In both studies, B and C blocks were based on N,N-dimethylacrylamide and NIPAAm, respectively. 146,147 The A block was based on either propylene sulfide (PSu) 146,147 or CL 147 and PLGA. 147 The A block was synthesised via ROP and was incorporated into the structure to provide degradation via different mechanisms, thus controlling the drug release at a different rate: PSu provides reactive oxygen species degradation, 146,147 while PCL and PLGA provide slow and fast hydrolysis/enzymatic degradation, respectively. 147 Interestingly, in one of the studies, the copolymers formed gels at a low concentration of 2.5 wt%. 146 5 wt% of the copolymer solutions were loaded with Nile red and were injected in mice. 146,147 Sustained release from the triblock copolymer was observed over 14 days, while burst release was observed when the corresponding AB diblock copolymer was used as a control (Fig. 9). 146 In vivo degradation was controlled by the degradation mechanism and thus the PLGA-based polymer released 90% of the stain in 2 days, whereas the other two copolymers sustained the release for 12 days. 147 As the progress in polymer science facilitates the synthesis of more complicated architectures, ABCBA pentablock terpolymers with A, B and C being based on NIPAAm, CL and EG, respectively, have also been applied in vivo. 148 These polymers were synthesised via atom transfer radical polymerisation (ATRP) and ROP, and in situ gel formation was confirmed upon injection of 20 wt % polymer solution in rats. 148 Branched copolymers based on poly(PEGC-co-NIPAAm), where PEGC stands for poly(ethylene glycol) citrate, were synthesised via a combination of poly(condensation) reaction and free-radical wileyonlinelibrary.com/journal/pi polymerisation. [149][150][151][152][153] Incorporation of citrate groups in the structure gives antioxidant properties to the final copolymer. [149][150][151][152][153] In vivo studies confirmed biocompatibility, degradability, controlled release of chemokine and new tissue formation. 153 Interestingly, these copolymers were mixed with gelatin and either multipotent adipose-derived cells 152 or immortalised mouse embryonic fibroblasts, 151 and upon injection osteogenic differentiation 151,152 and vascularisation 151 were observed. When mixed with mesenchymal stem cells (MSC) and either GO 150 or strontium ions, 149 in vivo osteodifferentiation, 149 mineralisation, 149,150 osteoinduction, 150 angiogenesis 150 and bone formation 149 were observed. Nevertheless, one should bear in mind that these copolymers possess a broad molar mass distribution (MMD, final Ð of 3.41), and thus they consist of polymer chains with different chemical structures and different properties. These copolymers are also difficult to be reproduced, and therefore controlled synthetic techniques should be used for the synthesis of well-defined copolymers (MMD closer to 1).
Conjugate 163,164 and star 166 copolymers have also been applied in vivo. In the studies on conjugate polymers, poly(NIPAAm) was linked with poly(serinol hexamethylene urea) for the delivery of vascular endothelial growth factor for the treatment of myocardial infarction. 163,164 It Is worth noting that these copolymers formed a gel at concentrations as low as 1 wt%, and upon in vivo injection vascularisation and the formation of vascular endothelial cells were observed. 163,164 Star copolymers based on NIPAAm, AA, O-phosphoethanolamine, synthesised via ATRP, formed stable gels at only 0.5 wt%, 166 and in vivo mineralisation of the hydrogel was promoted. 166 The combination of poly(NIPAAm) with natural components such as HA, 20,143,154,155,160,162 gelatin, 156,157,161 chitosan, 157,158 dextran, 145 mussel adhesive protein (MAP) 159 and heparin 165 was also found in the literature. These naturally based polymers were either mixed with NIPAAm homopolymer 143,145 or covalently linked. 20,143,[154][155][156][157][158][159][160][161]165 Two studies on either a physical mixture 145 or conjugate polymer 165 were reported in the literature as drug delivery depots. In the first study, poly(NIPAAm) was mixed with conjugated polymer of dextran, CL and 2-hydroxyethyl methacrylate, 145 while in the second study heparin was conjugated with poly(NIPAAm). 165 In vivo controlled release of BSA 145 and ibuprofen 165 was observed, and the anti-inflammatory action of ibuprofen and consequent wound healing were confirmed.
In several studies, poly(NIPAAm) was combined with HA, and in vivo gelation was observed. 20,143,154,155,160,162 When a graft copolymer was synthesised, it was investigated as a drug delivery matrix by loading with either BSA, 20 bioactive microvascular fragments 160 or gentamicin. 162 The controlled drug release was shown to improve vascularisation 160 and prevent bacterial colonisation. 162 When injected in a rabbit with osteochondral defect, cartilage formation was observed, thus replacing the gel matrix 12 weeks post-injection. 155 In another study, HA methacrylate was copolymerised with NIPAAm and the random copolymer was compared to the physical mixture of HA and PNIPAAm. 143 Both the copolymer and the physical mixture were loaded with adipose derived stem cells, and they were intra-articularly injected in rabbits. It was observed that cartilage formation was enhanced when the copolymer was used instead of the mixture. 143 Grafting of NIPAAm on gelatin, 156,157,161 chitosan 157,158 and MAP 159 was also reported, primarily in the concept of TE. In two of the studies, a solution of either NIPAAm-g-gelatin 156 or NIPAAm-g-chitosan, 158 mixed with MSC, was injected in model animals, and in situ gelation and enhanced tissue formation were observed. 156,158 To combine the promising effects of both gelatin and chitosan, PNIPAAm was grafted on both natural polymers, and upon addition of biphasic calcium phosphate and subsequent in vivo injection the formation of bone tissue was observed. 157 For adipose TE, PNIPAAm was linked on MAP and decellularised adipose tissue was added in the solution, thus promoting in vivo angiogenesis. 159 In the concept of drug delivery, a NIPAAm-g-gelatin solution was loaded with pilocarpine, and controlled and topical drug release improved tissue damage in glaucomatous eyes. 161 Poly(organophosphazene)-based TRGs A different class of injectable thermogels which were tested in vivo is poly(organophosphazene)s ( Fig. 2 with R representing an organic group). [167][168][169][170][171][172][173][174][175][176][177][178][179][180] These studies were published by Song's group and their collaborators. [167][168][169][170][171][172][173][174][175][176][177][178][179][180] In the studies, the backbone was functionalised with different groups, such as the hydrophobic isoleucine ethyl ester, 167-180 a hydrophilic EG-based group, 167-180 the hydrolysable glycyl lactate ethyl ester, 167,[169][170][171][172][173][174][175] and di(glycine). 170,175,179,180 Poly(organophosphazene)s were tested as drug delivery matrices for DOX, 172,173 PTX 174 and DTX. 171 Upon injection in tumourbearing mice, local administration and control of tumour growth were observed. [171][172][173][174] When the same thermogel was injected, faster release of PTX was detected compared to DOX, 172,174 confirming the different pharmacokinetics and indicating the necessity of investigating the drug release on a defined application.
In the afore-mentioned studies, the polymer was physically mixed with the drug. [171][172][173][174] However, studies in which the drug, e.g. DOX, 180 camptothecin 179 or PTX, 178 was covalently linked to the polymer were also reported. [178][179][180] The solution was injected in tumour-bearing mice and the drug was locally released in a prolonged period via hydrolytic degradation, thus effectively inhibiting the tumour growth [178][179][180] and reducing the side effects. 180 Poly(organophosphazene)s were also used as gel depots for delivering human growth hormone (HGH) 169,175 or BMP-2. 168 In the case of HGH, the polymer was mixed with poly-L-arginine 169 or modified with protamine 175 to form complexes with HGH via electrostatic attraction, while in the case of BMP-2 the complexes were formed via both hydrophobic and electrostatic attractions. Upon injection in different model animals, controlled release was observed. 168,169,175 Interestingly, in the case of BMP-2, bone formation was observed. 168 In a similar concept, cell proliferation was observed when a mixture of poly(phosphazene), collagen and pre-osteoblasts was injected in mice. 167 In two of the latest studies of the group, polyethyleneimine (PEI) was covalently linked to the polymer to promote complex formation for gene delivery. 176,177 In the first study, controlled codelivery of gene and drug was confirmed for up to a month. 177 Interestingly, a combination of photothermal and genetic combination therapy was attempted by fabricating a triple target system. The polymer was chemically modified with (i) PEI, which electrostatically interacts with siRNA, thus protecting it from endosomal and enzymatic degradation, and (ii) folate groups, which target the folate receptor, overexpressed in cancer cells. Au-Fe 3 O 4 particles were also incorporated in the system for magnetic targeting and NIR-induced hyperthermia. Thus, the targeted therapy and a reduction in side effects were achieved via (i) an enhanced permeability and retention effect, (ii) magnetism, (iii) folate recognition and (iv) NIR-induced hyperthermia. 176 PEtOx-based TRGs Poly(2-oxazoline)s are a special class of polymers that mimic the structure of the proteins, and thus have received much attention since their discovery in the late 1960s. [207][208][209][210][211] These polymers, synthesised via cationic ROP, consist of an amide group with the nitrogen and the carbonyl groups being part of the polymer backbone and side chain, respectively. 212 The general structure is shown in Fig. 2 with R representing the side group, which is varied to modulate the final properties of the polymers, such as hydrophilicity/hydrophobicity. 212 Amongst the different poly(2-oxazoline)s, PEtOx is thermoresponsive and shows great promise as it is FDA approved to be used as an indirect food additive, and a PEtOx-based formulation has reached clinical trials (Phase 1). [212][213][214] A few studies on the in vivo application of TRGs containing PEtOx have been reported the last decade. [181][182][183] In two of the studies, PEtOx-b-PCL-b-PEtOx triblock copolymers were injected in the eyes of model animals, and biocompatibility was confirmed. 181,182 Interestingly, the oxazoline-based copolymer showed superior biocompatibility compared to Pluronic® F127 and Matrigel®, a natural protein mixture showing thermoreversible gelation. 181 In the third study, PEtOx-b-PLGA-b-PEtOx polymers were injected in mice, and in vivo biodegradation and controlled release of salmon calcitonin were confirmed, the latter successfully regulating the level of calcium in blood. 183

CLINICAL APPLICATIONS
Numerous studies have been published on the in vivo applications of TRGs in model animals, with mice and rabbits being amongst the most common. However, to the best of our knowledge, only two TRGs have reached clinical trials, Pluronic® and Regel®, showing the increased number of requirements that need to be satisfied prior to clinical studies and commercialisation. 7,41,215,216 Their applications in model animals have been discussed in the relevant sections. When PTX, an anticancer agent, is incorporated in the ReGel® solution, the system is known as Onco-Gel®, and it has been applied in clinical trials (NCT00479765, NCT00573131). However, these clinical trials have been terminated, due to sponsor decision (NCT00479765) or because the product did not show any impact on tumour regression (NCT00573131). On the other hand, several trials of Pluronic® F127 (poloxamer 407) have been completed and/or are ongoing. Pluronic® F127 has been tested as a drug delivery matrix of simvastatin (NCT03400475) and anthralin (NCT03348462) for the treatment of mastitis and psoriasis, respectively. Its formulation with Pluronic® F68 (poloxamer 188) has been applied in humans for the delivery of metronidazole (NCT02365389) for treating bacterial vaginosis, while a formulation containing P407 and P188 is currently under investigation for the delivery of timolol (NCT04139018) for the treatment of epistaxis. 7,41,215,216 Nevertheless, one should bear in mind that these formulations are in the gel state below room temperature; thus application/injection is undertaken whilst the sample is in the gel state.

CONCLUSIONS
In summary, a plethora of TRGs have been studied in model animals, and several drugs have been incorporated into the gel matrix attempting to treat many different diseases like cancer, osteoporosis and diabetes. These systems, when tested as drug delivery depots, provide controlled and local drug release, thus avoiding undesired side effects, while in TE tissue formation was observed. Despite the fact that poloxamers are the most studied type of thermoresponsive polymers, they present in vivo instability; 217,218 thus attempts to improve their gelation properties have been made by using mixtures of poloxamers and/or other additives. As their gelation temperature reduces below r.t. when the concentration increases from 15 w/w% to 20 w/w%, in some cases poloxamers are preferable for treating skin disorders, in which epicutaneous application is required. A great deal of research has been focused on the in vivo application of ester-or carbamate-containing PEG-based polymers, owing to their degradability. Interesting studies based on PNIPAAm have also been carried out, but some of the syntheses were carried out using non-controlled polymerisation techniques which might limit the reproducibility of the results. A limited number of studies on poly(organophosphazene)s and poly(2-oxazoline)s have been found in the literature and have been discussed. Finally, to the best of our knowledge, only the OncoGel® system (Regel® with PTX) and poloxamer 407 (in some cases in formulation with poloxamer 188) have successfully reached clinical applications, 40,41 indicating the difficulty of achieving the appropriate combination of structural parameters in order to obtain the desired properties for in vivo application. Nevertheless, TRGs remain very promising, due to their ease in application. Therefore, designing the TRG that meets the desired properties for the final application is highly important in the health sector, in which the TRG system will provide local and sustained drug delivery, thus minimising undesired side effects, or promote tissue regeneration.