Thermoresponsive and Photocrosslinkable PEGMEMA-PPGMA

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Biomacromolecules 2009, 10, 822–828

Thermoresponsive and Photocrosslinkable PEGMEMA-PPGMA-EGDMA Copolymers from a One-Step ATRP Synthesis Hongyun Tai,*,† Wenxin Wang,‡ Tina Vermonden,§ Felicity Heath,‡ Wim E. Hennink,§ Cameron Alexander,‡ Kevin M. Shakesheff,‡ and Steven M. Howdle† School of Chemistry and School of Pharmacy, The University of Nottingham, University Park, Nottingham, NG7 2RD, United Kingdom, and Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences (UIPS), P.O. Box 80082, 3508 TB Utrecht, Utrecht University, The Netherlands Received November 13, 2008; Revised Manuscript Received January 3, 2009

Thermoresponsive and photocrosslinkable polymers can be used as injectable scaffolds in tissue engineering to yield gels in situ with enhanced mechanical properties and stability. They allow easy handling and hold their shapes prior to photopolymerization for clinical practice. Here we report a novel copolymer with both thermoresponsive and photocrosslinkable properties via a facile one-step deactivation enhanced atom transfer radical polymerization (ATRP) using poly(ethylene glycol) methyl ether methylacrylate (PEGMEMA, Mn ) 475) and poly(propylene glycol) methacrylate (PPGMA, Mn ) 375) as monofunctional vinyl monomers and up to 30% of ethylene glycol dimethacrylate (EGDMA) as multifunctional vinyl monomer. The resultant PEGMEMAPPGMA-EGDMA copolymers have been characterized by gel permeation chromatography (GPC) and 1H NMR analysis, which demonstrate their multivinyl functionality and hyperbranched structures. These water-soluble copolymers show lower critical solution temperature (LCST) behavior at 32 °C, which is comparable to poly(Nisopropylacrylamide) (PNIPAM). The copolymers can also be cross-linked by photopolymerization through their multivinyl functional groups. Rheological studies clearly demonstrate that the photocrosslinked gels formed at a temperature above the LCST have higher storage moduli than those prepared at a temperature below the LCST. Moreover, the cross-linking density of the gels can be tuned to tailor their porous structures and mechanical properties by adjusting the composition and concentration of the copolymers. Hydrogels with a broad range of storage moduli from 10 to 400 kPa have been produced.

Introduction In tissue engineering, injectable scaffolds offer the possibility of homogeneously distributing cells and molecular signals throughout the scaffolds, and can be injected directly into tissue defects with irregular shapes and sizes.1 The use of injectable scaffolds can minimize patient discomfort, risk of infection, scar formation, and the cost of treatment. Most studies on the development of injectable scaffolds have focused on bone and cartilage tissue repair,2-8 as well as corneal wound healing.9-11 Finding suitable materials that can solidify in situ to form 3-D structures with desired biological and mechanical properties is one of the challenging issues for tissue engineering, where in situ gelation can be achieved through either physical or chemical cross-linking.12 Smart polymers that change in response to external stimuli such as temperature and pH have attracted much attention.13,14 Thermoresponsive polymers exhibit a reversible, temperaturedependent phase transition, which is defined as the cloud point or lower critical solution temperature (LCST). Examples of these thermoresponsive polymers include poly(N-isopropylacrylamide) (PNIPAM), poly(ethylene oxide)-co-poly(propylene oxide) (PEOPPO) copolymers, 2-(2-methoxyethoxy)ethyl methacrylate (MEO2MA), and oligo(ethylene glycol) methacrylate (OEGMA) * To whom correspondence should be addressed. Current address: Bioengineering Department, University of Washington, Box 355061, Seattle, WA 98105. E-mail: [email protected]. † School of Chemistry, The University of Nottingham. ‡ School of Pharmacy, The University of Nottingham. § Utrecht University.

copolymers, poly(caprolactone) (PCL) and poly(lactide) (PLA) block copolymers, and PEO-PCL-PEO and PLGA-PEG-PLGA triblock copolymers.15-20 However, gels formed by these polymers via physical cross-linking are normally too mechanically unstable to be used for cell scaffolds/delivery. Gelation from macromonomers via chemical cross-linking seems promising because the network structures are controllable by designing constitutional units, resulting in gels with the desired mechanical properties. Cross-linking via photopolymerization of macromonomers provides many benefits, including rapid polymerization while maintaining physiological conditions and good spatial and temporal control over polymerizations.5,21,22 Photopolymerized hydrogels have been investigated for a number of biomedical applications including prevention of thrombosis, postoperative adhesion formation, drug delivery, coatings for biosensors, and cell transplantation.22-24 These studies have shown that photocrosslinkable materials have the potential to be used for in vitro as well as in vivo applications via a minimally invasive manner, for example laparascopic devices, catheters, or subcutaneous injection with transdermal illumination. Therefore, photocrosslinking is an effective approach for injectable systems in regenerative medicine applications. A series of linear vinyl (macro)monomers have been used, including poly(ethylene oxide)-dimethacrylate and PEG semiinterpenetrating network,21 PEG-diacrylate derivatives,25-27 poly(propylene glycol) (PPG) diacrylate,26 PEG-PLA diacrylate,28 and PLA-b-PEG-b-PLA macromers.5 To increase the cross-linking density and functionality, branched and star polymers have been investigated.29-32 Furthermore, dendrimers,

10.1021/bm801308q CCC: $40.75  2009 American Chemical Society Published on Web 02/18/2009

PEGMEMA-PPGMA-EGDMA Copolymers

Biomacromolecules, Vol. 10, No. 4, 2009

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Scheme 1. Copolymerization of Poly(ethylene glycol) Methyl Ether Methacrylate (PEGMEMA), Poly(propylene glycol) Methacrylate (PPGMA), and Ethylene Glycol Dimethacrylate (EGDMA) via Deactivation Enhanced ATRP (DE-ATRP)a

a

Note: the end functional groups introduced from ATRP initiators have not been illustrated in the structure.

highly branched macromolecules with well-defined molecular weight and chain structure, have generated much interest as advanced materials for a variety of applications ranging from drug and gene delivery, tissue engineering, and biological imaging to nanobuilding blocks.9,33-36 Grinstaff et al. synthesized a photocrosslinkable dendrimer for corneal wound healing sealant and cartilage repair.9,11,37-39 However, the difficulties of preparation of dendrimers, which normally involve solventintensive and multistep synthetic routes, have severely impeded their practical applications. Most importantly, it is difficult to tailor the composition and structure of dendrimers for a wide variety of applications. Recently, materials with both thermoresponsive and photocrosslinkable properties have attracted attention for tissue engineering applications.40 These materials enable the formation of gels with desired mechanical properties in situ to encapsulate bioactive agents and cells for the controlled drug release and to support cell growth. Such polymers are also easy to work with clinically because the polymer solutions can be localized within targeted sites after administration due to thermal gelation, then form gels with the desired mechanical properties by photocrosslinking. In this paper, we report the synthesis of water-soluble copolymers, consisting of biocompatible building blocks and having both thermoresponsive and photocrosslinkable properties, via one-step atom transfer radical polymerizations (ATRP) of monofunctional vinyl macromonomers poly(ethylene glycol) methyl ether methylacrylate (PEGMEMA) and poly(propylene glycol) methacrylate (PPGMA) with multifunctional vinyl monomer ethylene glycol dimethacrylate (EGDMA) Scheme 1).

Experimental Section Materials. The (macro)monomers PEGMEMA (Mn ) 475), PPGMA (Mn ) 375), and EGDMA were purchased from Sigma-Aldrich. The methyl 2-bromopropionate and methyl 2-chloropropionate (Aldrich) were used as the initiators and their stock solutions were prepared at 0.4075 mol/L in 2-butanone (99.5%, HPLC grade, Aldrich) for use. Azobisisobutyronitrile (AIBN, analytical grade, Aldrich), copper(I) bromide (CuBr, 98%, Aldrich), copper(II) bromide (CuBr2, 99%, Lancaster), copper(I) chloride (CuCl, 95%, Acros), copper(II) chloride (CuCl2, 99%, Lancaster), and 2,2-bipyridine (bpy, Aldrich) were used as received. Photoinitiator 2-hydroxy-4′-(2-hydroxy-ethoxy)-2-methylpropiophenone (Ciba Irgacure 2959) was purchased from SigmaAldrich. Synthesis and Purification of PEGMEMA-PPGMA-EGDMA Copolymers. The copolymers were prepared in butanone (the volume ratio of total monomers and solvent ) 1:1) at 60 °C with a Schlenk

line system, where argon was bubbled through the solutions to eliminate oxygen. Liquids were transferred under argon by means of septa and syringes or stainless steel capillaries. A typical reaction procedure is described: A round-bottom flask fitted with a three-way stopcock was charged with CuCl (18.7 mg, 0.189 mmol), CuCl2 (10.7 mg, 0.063 mmol), and bpy (78.6 mg, 0.504 mmol) and then connected to the Schlenk line. Oxygen was removed by repeated vacuum-argon cycles. The degassed PEGMEMA (6.0 g, 12.6 mmol), PPGMA (12.3 g, 32.8 mmol), EGDMA (1.0 g, 5.04 mmol), and butanone (20 mL) were transferred into the flask. The solution was stirred at 500 rpm, the initiator stock solution (1.24 mL) was added, and the polymerization was conducted at 60 °C in an oil bath for a desired reaction time. Samples were withdrawn periodically for GPC analysis to monitor the monomer conversions by comparing the peak areas for monomers and copolymers. After polymerization, the solution was diluted with acetone and passed through a silica column to remove the copper catalyst. The obtained polymers were precipitated by dropping the solution into a large excess of hexane to remove PPGMA and EGDMA. The precipitated mixture of the polymer and PEGMEMA was dissolved in deionized water and purified by dialysis (Spectrum dialysis membrane, molecular weight cut off 3500) for 72 h in the dark at 4 °C to remove PEGMEMA using deionized water, which was changed regularly. Polymer samples were obtained after freeze-drying and weighed to obtain the final yields. Characterizations of PEGMEMA-PPGMA-EGDMA Copolymers. The resultant copolymers were characterized by gel permeation chromatography (GPC) and 1H NMR. Number average molecular weight (Mn), weight average molecular weight (Mw), and polydispersity (Mw/Mn) were obtained by GPC (PL-120, Polymer Laboratories) with an RI detector and multiangle laser light scattering (MALLS) detector (mini-Dawn) supplied by Wyatt Technology. The columns (30 cm PLgel Mixed-C, two in series) were eluted using THF and calibrated with polystyrene standards. All calibrations and analyses were performed at 40 °C and a flow rate of 1 mL/min. 1H NMR was carried out on a 300 MHz Bruker NMR with MestReC processing software. The chemical shifts were referenced to the lock CDCl3. Lower critical solution temperatures (LCST) of the copolymers in deionized water (0.03% w/v solutions) were quantified by measuring their absorbance of 530 nm at temperatures from 12 to 60 °C (heating rate ) 0.5 °C/s) with a Beckman DU-640 spectrophotometer. The data were collected every 2 s. Rheological Studies of Thermally Induced and Photocrosslinked Gelation of PEGMEMA-PPGMA-EGDMA Copolymers. Rheological studies on the thermally induced gelation were performed using an Anton Paar Physica 301 rheometer equipped with parallel plate geometry (20 mm diameter). This instrument was fitted with a Peltier element and a water-bath for temperature control. Oscillatory temperature sweeps were recorded, while a frequency of 10 Hz, a strain of 0.5%, and a heating rate of 1 °C/min were employed, and the gap

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Table 1. Copolymerizations of Poly(ethylene glycol) Methyl Ether Methacrylate (PEGMEMA), Poly(propylene glycol) Methacrylate (PPGMA), and Ethylene Glycol Dimethacrylate (EGDMA) via Deactivation Enhanced ATRP GPC RI

GPC MALLS

entry

monomer feed mole ratio f[PEGMEMA]/[PPGMA]/[EGDMA]

RTah

monomer conversionb %

Mwc kg/mol

PDId

Mwc kg/mol

PDId

1 2 3 4 5 6 7 8

25/65/10 25/65/10 25/65/10 25/65/10 25/65/10 25/65/10 25/65/10 30/40/30

27 16 24 31 40 51 65 42

40 5 10 17 30 54 75 72

167 12 17 29 52 126 216 207

2.13 1.19 1.24 1.29 1.39 2.28 2.78 2.90

432 14 20 35 63 376 570 521

1.77 1.15 1.20 1.25 1.32 1.89 1.95 1.98

a Reaction time. b Monomer conversion, estimated using peak areas for monomers and copolymers in GPC traces. c Weight average molecular weight. Polydispersity, Mw/Mn; polymerization conditions: 60 °C in butanone; total monomers/butanone (v/v) ) 1:1; [I]/[total monomers] (mole ratio) ) 1:100, [I]/[Cu+/Cu2+]/bpy ) 1:[0.375:0.125]:1, [Cu+]/[Cu2+] ) 3:1. The initiator (I)/catalysts/ligand are methyl 2-bromipropionate/CuBr/CuBr2/bpy for entry 1, and methyl 2-chloropropionate/CuCl/CuCl2/bpy for the rest of the experiments. d

distance between two plates was set as 0.5 mm. Real-time photocrosslinking rheological studies were performed on an AR1000-N (TA Instruments) using parallel-plate geometry (20 mm diameter) equipped with a UV light source (BluePoint lamp 4, 350-450 nm, Honle UV technology, light intensity of 50 mW/cm2). The bottom plate was made of glass through which the samples were exposed to UV light. The oscillatory measurements were performed at 37 and 20 °C, respectively, for 5 min, with a frequency of 10 Hz, a strain of 0.5%, and a gap of 0.5 mm. The strain was within the linear viscoelastic region. The samples were exposed to UV light for 1 min after the first minute of data collection. Photocrosslinked Gels: Preparation, Mechanical Property, and Morphology. The PEGMEMA-PPGMA-EGDMA copolymers were dissolved in 0.1% w/v Irgacure 2959 water solution to prepare 15 and 30% (w/v) copolymer solutions. These solutions were preheated in an oven at 37 °C before photopolymerization. A BluePoint lamp 4 (350-450 nm, Honle UV technology, light intensity of 450 mW/cm2) was used for the preparation of photocrosslinked hydrogels. Gels with a cylindrical shape were prepared with a typical diameter of 8 mm and a volume of 400 µL by UV exposure of 5 min. The elastic moduli of the photopolymerized gels were obtained using a dynamic mechanical analyzer (DMA 2980, TA-Instruments) in the controlled force mode, where a force ramp from 0.001 to 1.0 N at a rate of 0.1 N/min was applied at 25 °C. Scanning electron microscopy (SEM) was used to characterize the porous structure of freeze-dried gels. The samples were mounted on an aluminum stub using an adhesive carbon tab and sputter coated with gold before images were obtained using a JEOL JSM6060LV SEM machine.

Results and Discussion One-Step ATRP Synthesis of PEGMEMA-PPGMAEGDMA Copolymers. Recently, Wang and Howdle41 reported the successful homopolymerization of divinyl benzene (DVB) and ethylene glycol dimethacrylate (EGDMA) to yield soluble hyperbranched polymers with multitude vinyl functional groups via deactivation enhanced ATRP. Here, we demonstrate the successful copolymerizations of PEGMEMA (Mn ) 475), PPGMA (Mn ) 375), and EGDMA via this one-step ATRP approach for the preparation of thermoresponsive and photocrosslinkable copolymers (Scheme 1 and Table 1). Methyl 2-bromopropionate was first adopted as the initiator and copper bromide/bpy ligand was used as the catalyst. During the reactions, the addition of Cu(II) species was used to enhance deactivation. It was found that no macrogelation occurred until 27 h reaction time (monomer conversion ca. 40%, entry 1 in Table 1). A parallel conventional ATRP copolymerization of PEGMEMA, PPGMA, and EGDMA without the addition of Cu(II) species was also carried out for comparison, and the mixture gelled after only 30 min reaction time. To further

Figure 1. GPC results of entries 2-7 in Table 1. (a) GPC traces from RI detector; (b) GPC traces from MALLS detector. Note: increases in molecular weight and polydispersity with monomer conversion are observed.

decrease the polymerization rate, methyl 2-chloropropionate and copper(I) and (II) chloride were adopted instead of the bromide compounds, and a yield of up to 75% of soluble copolymer was obtained with a controlled molecular weight (entry 7 in Table 1). These results demonstrated that the addition of Cu(II) species delayed the occurrence of the gelation. To study the kinetics of this copolymerization, the reaction was monitored by GPC analysis (entries 2-7 in Table 1). Clear shifts to the short retention time in GPC elution traces were observed with the increase in reaction time (Figure 1a and b), indicating an increase of molecular weight with the monomer conversion. In



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r ) SL ) 1

(1)

n ) (2Sc - 3Sb) ⁄ 30

(2)

m ) (Sa - Sb - 13n) ⁄ 33

(3)

p ) [3Sb - 6(m + n)] ⁄ 12

(4)

The double bond content and degree of branching of the copolymers were calculated from eqs 5 and 6, respectively.

Figure 2. Typical conformation plot for the PEGMEMA-PPGMAEGDMA copolymers (7 in Table 1). The slope of the conformation plot is 0.25, indicating the hyperbranched structure. (dn/dc ) 0.076, measured by a differential refractometer). Note: the slope of conformation plot for linear polymers is in the range 0.5-0.6.

Figure 3. 1H NMR for the PEGMEMA-PPGMA-EGDMA copolymer in CDCl3 (7 in Table 1). Note: the spectrum shows clearly the double bonds within the structure at the chemical shifts of 6.1 and 5.6 ppm. The copolymer composition (m, n, r, p) can be calculated from the integral data Sa, Sb, Sc, and SL.

addition, the molecular weight distribution became broader, which is commonly observed in the synthesis of hyperbranched polymers.41 The GPC with a MALLS detector can yield absolute molecular weight, root-mean-square radius (rms) and branching parameters.42 It is known that for the polymers at a constant molar mass, the rms decreases with increasing degree of branching. A typical conformation plot of rms versus molar mass for the resultant hyperbranched copolymers is given in Figure 2. The slope of the line (0.25) is lower than the typical value for linear random coils (in the range 0.5-0.6),43 confirming the hyperbranched structures. These hyperbranched structures are also confirmed by 1H NMR (Figure 3). The characteristic peaks at chemical shifts of 6.1 and 5.6 ppm are attributed to the vinyl functional groups in the copolymer and the others are assigned as indicated in Figure 3. The reactivity of the monomers influences the final composition of the copolymer, which commonly differs from the initial feed composition of the monomers. The composition of the copolymer, represented by m, n, r, and p values in the macromolecule structure (in Figure 3), was calculated from the integral data Sa, Sb, Sc, and SL, respectively, according to eqs 1-4. These equations were derived from the four equations established for the proton integral regions of Sa, Sb, Sc, and SL, respectively.

double bond content % ) r ⁄ (m + n + r + p) × 100 (5) branching degree % ) p ⁄ (m + n + r + p) × 100 (6) The double bond content represents the mole percentage of EGDMA with free vinyl functional groups in the copolymer and the degree of branching represents the mole percentage of EGDMA as branching units (i.e., without vinyl groups) in the copolymer. Table 2 shows the composition, double bond content and degree of branching of the hyperbranched copolymers prepared at feed monomer ratio f[PEGMEMA]/[PPGMA]/[EGDMA] of 25/ 65/10 and 30/40/30. These copolymer composition results indicate that EGDMA has a higher reactivity in the copolymerizations. In recent years, some research groups, such as Sherrington,44-47 Guan,48 Armes,49-52 and Perrier,53 have reported the preparation of soluble hyperbranched copolymers via copolymerizations of vinyl and divinyl monomers (see Supporting Information). In our copolymerization system, a much higher concentration of EGDMA was successfully employed to achieve the copolymers with high levels of branching degree and multivinyl functionality. We realize that defects could exist in our hyperbranched structures due to possible internal cyclization and there are also debates on whether these copolymers are (micro)nanogels or hyperbranched macromolecules. Therefore, further theoretical and experimental studies are required and currently undergoing to clarify the polymerization mechanism. However, we hypothesize that the slow growth of each independent and complex hyperbranched molecule due to the addition of Cu(II) species might have played the key role to avoid cross-linking.41 The active radicals with a very short lifetime due to the presence of excess Cu(II) species should favor the addition of small molecular species, such as monomers and low molecular weight oligomeric polymer chains rather than large polymer species because of the diffusion effect. Moreover, the two methacrylate groups of EGDMA should have the same reactivity; however, after one of them has polymerized, the other one could have less chance to react than the methacrylate groups in the free EGDMA molecules due to concentration, steric and diffusion effects. All these can result in delayed gelation and incomplete methacrylate conversion of EGDMA during copolymerization, therefore, leading to the resultant copolymers with mutivinyl functional groups and hyperbranched structures. Thermoresponsive Properties of PEGMEMA-PPGMAEGDMA Copolymers. UV-vis spectrophotometer was used to measure the LCST of the PEGMEMA-PPGMA-EGDMA copolymers. The results (Figure 4) demonstrate that the phase transition temperature (LCST) of the copolymer solution is around 32 °C, that is, the copolymer is soluble below this temperature. To study the thermal gelation behavior, the copolymers (100-300 mg) were dissolved in 1 mL of deionized water at 4 °C and then placed at 37 °C for 5 min. Gel concentration was determined as no flow upon inversion of the vial within 10 s (Figure 5a). It was found that gel points of copolymers with Mw above 52 kg/mol (entries 5-8 in Table 1) at 37 °C were about 15%. The copolymers with a low molecular weight showed precipitation, and no gels formed up to 30%

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Table 2. Properties of PEGMEMA-PPGMA-EGDMA Copolymersa entry

monomer feed ratio f[PEGMEMA]/[PPGMA]/[EGDMA]

polymer composition f[PEGMEMA]/[PPGMA]/[EGDMA]b

double bond contentb

branching degreeb

7 8

25/65/10 30/40/30

22/37/38 21/26/53

10 18

28 35

LCST °C

c

32 33

a The reaction conditions to prepare these copolymers were detailed in Table 1 (entries 7 and 8). b Determined by 1H NMR. c Determined by UV-vis spectrophotometer.

Figure 4. LCST behavior of the PEGMEMA-PPGMA-EGDMA copolymer (7 in Table 1) in 0.03% w/v deionized water, measured by UV-vis spectroscopy.

Figure 5. Thermal gelation of the PEGMEMA-PPGMA-EGDMA copolymer aqueous solution (7 in Tables 1 and 2, concentration 15% w/v). (a) digital images; (b) oscillation rheological temperature sweep.

concentration. The thermal gelation behavior of the copolymer solutions was also studied by oscillation rheology experiments. Figure 5b demonstrates their rheological properties (storage modulus, G′; loss modulus, G′′; and complex viscosity, η*) and indicates that both the loss and the storage moduli are low at a low temperature. With increasing the temperature, both moduli increase by 2-5 orders of magnitude. However, the increase in G′ is stronger than that of G′′, which demonstrates that this aqueous solution undergoes thermally induced gelation. However, there is no crossover of G′ and G′′ occurring, which indicates that the thermally induced gels are mechanically weak

Figure 6. Real-time photocrosslinking rheological measurements of the PEGMEMA-PPGMA-EGDMA copolymers (7 and 8 in Table 2). (a) effect of temperature (8 at a concentration of 30% w/v); (b) effect of copolymer concentration (solutions of 8 cured at 37 °C); (c) effect of copolymer composition (7 and 8, at a concentration of 30% w/v, cured at 37 °C). The square symbols represent the storage modulus G′ and the triangles represent the loss modulus G′′.

and injectable through a syringe when a force is applied. Their injectability has been confirmed by compression tests on Stable Micro TA Texture Analyzer (experimental data not shown).



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Figure 7. Photocrosslinked gels: (a) elastic modulus of 15 and 30% (w/v) copolymer gels after photopolymerizations of copolymers 7 and 8 (in Table 1) using 0.1% photoinitiator. Data are shown as average (n ) 3). (b,c) SEM images of freeze-dried photocrosslinked gels prepared from 15 and 30% (w/v) copolymer 8 solutions using 0.1% photoinitiator. Note: the mean values of pore diameters measured by the average of 100 pores are 1.63 µm (SD ) 0.5) and 2.83 µm (SD ) 0.82), respectively.

Photocrosslinkable Properties of PEGMEMA-PPGMAEGDMA Copolymers. The thermoresponsive PEGMEMAPPGMA-EGDMA copolymers have photocrosslinkable properties due to a multitude of vinyl functional groups within their structures. The mechanical properties of the photocrosslinked gels from these copolymers were studied by in situ photocrosslinking rheology experiments in the presence of photoinitiator Irgacure 2959, which was selected for its known biocompatibility.4 The results showed that a plateau value in the hydrogel storage modulus was reached and the crossover of G′ and G′′ occurred within seconds of UV exposure, leading to photocrosslinked elastic gels with moduli 3 orders of magnitude greater than the thermally induced gels (Figure 6). A prolonged UV exposure time of 900 s did not lead to further increases in moduli. These results suggest that the PEGMEMA-PPGMAEGDMA copolymer solutions underwent fast photopolymerizations and reached almost complete methacrylate conversion within seconds. The photocrosslinked gels formed at a temperature (37 °C) above the LCST demonstrated higher moduli (ca. 400 kPa) than those gels photocrosslinked at a temperature below the LCST (20 °C; ca. 160 kPa; Figure 6a). This could be related to the conformations of macromolecule chains of the thermoresponsive copolymers, which are more compact at a temperature above LCST due to increased hydrophobic interactions. This may lead to higher photocrosslinking density and therefore higher mechanical properties. These results convincingly demonstrate that the enhanced mechanical properties of the photocrosslinked gels have been achieved by adopting these copolymer architectures with both thermoresponsive and photocrosslinkable properties. Furthermore, Figure 6b shows that the storage modulus plateau value of the photocrosslinked gels from copolymer 8 increases from 10 to 400 kPa when the concentration increases from 15 to 30%. Figure 6c shows that

the gels yielded from copolymer 8 (with high double bond content) have higher storage moduli than the gels from copolymer 7. These results are attributed to higher cross-linking densities of the gels when employing the PEGMEMA-PPGMAEGDMA copolymers at a high concentration and with high double bond content. The compression modulus of UV-cured gels was tested using DMA measurements. The gels were photopolymerized upon UV exposure for 5 min at 37 °C in the presence of photoinitiator Irgacure 2959. After photopolymerizations, the gels were elastic and displayed an increasing elastic modulus (E) with increasing number of methacrylate functional groups and polymer concentration as shown in Figure 7a. Hydrogels with elastic moduli from 10 to 250 kPa have been produced. The sol content of the gels was undetectable by GPC, indicating the photopolymerization reaction was rapid and almost complete due to multivinyl functional groups within the macromolecular structure. SEM images (Figure 7) for the freeze-dried gels from copolymer 8 demonstrated that the pores for the gels prepared at 30% (w/v) polymer concentration have larger diameter (ca. 2.83 µm) compared to those prepared at 15% (w/v) (ca. 1.63 µm), while the walls of the pores were thicker due to higher cross-linking densities. Therefore, the pore sizes and porosity of the photocrosslinked gels can be tailored by adjusting the copolymer concentration to meet the requirements for controlled drug release and cell encapsulation.

Conclusions This study shows that thermoresponsive water-soluble PEGMEMA-PPGMA-EGDMA copolymers were prepared via a facile one-step deactivation enhanced ATRP copolymerization of monofunctional and multifunctional vinyl monomers, where

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up to 30% of ethylene glycol dimethacrylate (EGDMA) was successfully used as the multifunctional vinyl monomer to achieve high levels of branching and multivinyl functionality. These copolymers have demonstrated lower critical solution temperature (LCST) behavior and photocrosslinkable properties. The photocrosslinked gels produced from the thermally induced gels, where photopolymerization was performed at a temperature above the LCST, have demonstrated higher storage moduli than those gels prepared from polymer solutions, where photopolymerization was performed at a temperature below the LCST. Hydrogels with storage moduli from 10 to 400 kPa have been produced. These hyperbranched copolymers consist of biocompatible building blocks and have many advantages, including one-step facile synthesis, tuneable high density of multivinyl functionality for photocrosslinking, as well as dendritic topology to provide multifunctionality for further biomolecule conjugations. Therefore, these materials are promising for smart injectable systems in regenerative medicine applications such as wound healing and tissue repair. Biodegradability can be introduced by copolymerizing macromonomers with degradable building blocks, for example, using macromonomers with lactide and glycolide units. Follow-up studies on the gel swelling, protein drug release, and cytotoxicity assessments of these smart copolymers are undergoing and will be reported shortly. Acknowledgment. EPSRC and British Council are gratefully acknowledged for the financial supports. H.T. also thanks to Prof. Allan Hoffman, Prof. Patrick Stayton, Dr. D. Benoit, Dr. C. Duvall, and Dr. S. Takae for the scientific discussions and constructive comments. Dr. Andreas Endruweit is thanked for helping with access to UV equipment. The helpful advice and suggestions from the reviewers are also highly appreciated. Supporting Information Available. Information about the state-of-the-art studies on the synthesis of hyperbranched polymers from copolymerizations of vinyl and divinyl functional monomers. This material is available free of charge via the Internet at http://pubs.acs.org.

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Thermoresponsive and Photocrosslinkable PEGMEMA-PPGMA

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