Photo-Cross-Linked Hydrogels from Thermoresponsive PEGMEMA

Biomacromolecules 2009, 10, 2895–2903

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Photo-Cross-Linked Hydrogels from Thermoresponsive PEGMEMA-PPGMA-EGDMA Copolymers Containing Multiple Methacrylate Groups: Mechanical Property, Swelling, Protein Release, and Cytotoxicity Hongyun Tai,*,†,‡,§ Daniel Howard,| Seiji Takae,‡ Wenxin Wang,*,⊥ Tina Vermonden,# Wim E. Hennink,# Patrick S. Stayton,‡ Allan S. Hoffman,‡ Andreas Endruweit,∇ Cameron Alexander,| Steven M. Howdle,§ and Kevin M. Shakesheff| School of Chemistry, Bangor University, Bangor, LL57 2UW, United Kingdom, Department of Bioengineering, University of Washington, P.O. Box 355061, Seattle, Washington 98195, School of Chemistry, School of Pharmacy, and School of Mechanical, Materials and Manufacturing Engineering, The University of Nottingham, University Park, Nottingham, NG7 2RD, United Kingdom, Network of Excellence for Functional Biomaterials, National University of Ireland, Galway, Ireland, and Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences (UIPS), Utrecht University, The Netherlands Received June 23, 2009; Revised Manuscript Received August 25, 2009

Photo-cross-linked hydrogels from thermoresponsive polymers can be used as advanced injectable biomaterials via a combination of physical interaction (in situ thermal gelation) and covalent cross-links (in situ photopolymerization). This can lead to gels with significantly enhanced mechanical properties compared to non-photo-crosslinked thermoresponsive hydrogels. Moreover, the thermally phase-separated gels have attractive advantages over non-thermoresponsive gels because thermal gelation upon injection allows easy handling and holds the shape of the gels prior to photopolymerization. In this study, water-soluble thermoresponsive copolymers containing multiple methacrylate groups were synthesized via one-step deactivation enhanced atom transfer radical polymerization (ATRP) of poly(ethylene glycol) methyl ether methacrylate (PEGMEMA, Mn ) 475), poly(propylene glycol) methacrylate (PPGMA, Mn ) 375), and ethylene glycol dimethacrylate (EGDMA) and were used to form covalent cross-linked hydrogels by photopolymerization. The cross-linking density was found to have a significant influence on the mechanical and swelling properties of the photo-cross-linked gels. Release studies using lysozyme as a model protein demonstrated a sustained release profile that varied dependent on the copolymer composition, cross-linking density, and the temperature. Mouse C2C12 myoblast cells were cultured in the presence of the copolymers at concentrations up to 1 mg/mL. It was found that the majority of the cells remained viable, as assessed by Alamar Blue, lactate dehydrogenase (LDH), and Live/Dead cell viability/cytotoxicity assays. These studies demonstrate that thermoresponsive PEGMEMA-PPGMA-EGDMA copolymers offer potential as in situ photopolymerizable materials for tissue engineering and drug delivery applications through a combination of facile synthesis, enhanced mechanical properties, tunable cross-linking density, low cytotoxicity, and accessible functionality for further structure modifications.

Introduction Hydrogels are 3D networks of physically or chemically crosslinked hydrophilic polymers and have been extensively used in various biomedical applications.1,2 The soft and hydrophilic nature of hydrogels makes them particularly suitable as protein delivery system or as a cell-entrapping scaffold in tissue engineering.3 In recent years, in situ curing gels, also called injectable scaffolds, have attracted much attention because they offer the possibility of homogeneously distributing cells and molecular signals throughout the scaffolds and can be injected directly into cavities with irregular shapes and sizes.3-14 Finding * To whom correspondence should be addressed. E-mail: h.tai@ bangor.ac.uk (H.T.); [email protected] (W.W.). † Bangor University. ‡ University of Washington. § School of Chemistry, The University of Nottingham. | School of Pharmacy, The University of Nottingham. ⊥ National University of Ireland. # Utrecht University. ∇ School of Mechanical, Materials and Manufacturing Engineering, The University of Nottingham.

suitable materials that can solidify in situ with desired mechanical and biological properties remains a challenge. The in situ gelation can be achieved through either physical or chemical cross-linking. Smart materials, which respond to external stimuli such as temperature and pH can form hydrogels via physical cross-linking.15,16 In general, gels based on these physical interactions are mechanically weak. In contrast, gelation from macromonomers via chemical cross-linking can exhibit much better mechanical performance. However, chemical crosslinking can be a harsh procedure with respect to encapsulated biological components. Photopolymerization as a cross-linking method is a relatively mild procedure and provides many benefits, including rapid polymerization, while maintaining physiological conditions and good spatial and temporal control over gelation.8,17 Therefore, photo-cross-linking has been used as an effective approach for the development of injectable systems for a number of biomedical applications including prevention of thrombosis, postoperative adhesion formation, drug delivery, coatings for biosensors, and cell transplantation.18-20 It has been shown that photo-cross-linkable materials have the

10.1021/bm900712j CCC: $40.75  2009 American Chemical Society Published on Web 09/11/2009

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potential to be used for in vitro as well as in vivo applications via minimally invasive procedures, such as laparascopic devices, catheters, or subcutaneous injection with transdermal illumination. So far, a series of polyethylene glycol (PEG) based and poly(lactic acid) (PLA) based linear multivinyl macromers,8,17,21-25 branched and star polymerizable polymers,26-29 and dendrimers with acrylate end groups12,14,30-32 have been investigated as in situ gelling materials via photopolymerization for regenerative medicine applications. It is envisioned that polymers combining both thermoresponsive and photo-cross-linkable properties should exhibit superior performance over solely thermoresponsive or photo-crosslinkable materials for tissue engineering injectable scaffolds.33,34 These polymer solutions can be rapidly confined within targeted sites after immediate administration due to in situ thermal gelation and, subsequently, form gels with the desired mechanical properties by photo-cross-linking. Recently, we have reported the successful synthesis of PEGMEMA-PPGMAEGDMA copolymers with both thermoresponsive and photocross-linkable properties via one-step deactivation enhanced atom transfer radical polymerization (ATRP).35 In this paper, the mechanical properties, swelling behavior, drug release, and cytocompatibility of the photo-cross-linked gels derived from these new smart copolymers were further investigated. The results demonstrate that thermoresponsive PEGMEMA-PPGMAEGDMA copolymers offer potential as in situ photopolymerizable materials for tissue engineering and drug delivery applications through a combination of facile synthesis, enhanced mechanical properties, tunable cross-linking density, low cytotoxicity, and accessible functionality for further structure modifications.

Experimental Section Preparation of Thermoresponsive Copolymers Containing Multiple Methacrylate Groups. The PEGMEMA-PPGMA-EGDMA copolymers were synthesized according to the previously published method.35 Briefly, the reactions were conducted in 2-butanone (99.5%, HPLC grade, Aldrich) at a volume ratio of total monomers and solvent of 1:1 with a Schlenk line system. Argon was bubbled through the solutions to eliminate oxygen and liquids were transferred under argon by means of septa and syringes or stainless steel capillaries. A round-bottom flask fitted with a three-way stopcock was charged with copper(I) chloride (CuCl, 95%, Acros), copper(II) chloride (CuCl2, 99%, Lancaster), and 2,2′-bipyridine (bpy, Aldrich) and then connected to the Schlenk line. Oxygen was removed by repeated vacuum-argon cycles. The degassed PEGMEMA (Mn ) 475, Sigma-Aldrich), PPGMA (Mn ) 375, SigmaAldrich), EGDMA (Sigma-Aldrich), and butanone were transferred into the flask. Under magnetic stirring at 500 rpm, the initiator stock solution methyl 2-chloropropionate (Aldrich) in 2-butanone was added, and the polymerization was conducted at 60 °C in an oil bath for a desired reaction time. After the polymerization, the solution was diluted with acetone and passed through a silica column to remove copper catalyst. The subsequent solutions were precipitated into a large excess of hexane to remove PPGMA and EGDMA monomers. 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 a dark environment at 4 °C to remove PEGMEMA against fresh deionized water, while the water was changed regularly. The pure polymer samples were obtained after freeze-drying, then 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

Tai et al. eluted by 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. Differential scanning calorimetry (DSC, TA 2920) was used to measure the glass transition temperatures (Tg) of the dried copolymers. Thermoresponsive Behavior of the Copolymers. The lower critical solution temperatures (LCST) of the copolymers at 0.03% w/v solutions in deionized water, phosphate buffered saline (PBS, pH 7.4), and cell culture media (Dulbecco’s modified Eagle’s medium (DMEM)), supplemented with 10% fetal bovine serum (FBS), 1% glutamine, and 2.5 mg/mL amphotericin B (antibiotic/ antimycotic solution)) were quantified by measuring their absorbance of 530 nm at temperatures from 12 to 60 °C (heating rate ) 0.5 °C/sec) with a Beckman DU-640 spectrophotometer. The data were collected every 2 s. Moreover, dynamic light scattering (DLS) was used to analyze sizes and distributions of the copolymers in water solutions on a Malvern Nano Zetasizer. Polymer solutions (0.01% w/v) were prepared in deionized water and filtered prior to measurements using a 0.45 µm disposable filter into a 12.5 × 12.5 mm polystyrene disposable cuvette. Rheological, Mechanical, and Morphological Studies of PhotoCross-Linked Gels. Real-time photo-cross-linking 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), as described elsewhere.35 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 samples were exposed to UV light for one minute after the first minute of data collection. The elastic moduli of the photopolymerized gels were obtained using a dynamic mechanical analyzer (DMA 2980, TAInstruments) 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. Photocross-linked gels with a cylindrical shape and diameter of 8 mm were prepared for DMA studies by curing PEGMEMA-PPGMA-EGDMA copolymer solutions (400 µL) with 0.1% w/v Irgacure 2959 for 5 min at 37 °C using a BluePoint lamp 4 (350-450 nm, Honle UV technology, light intensity of 450 mW/cm2). Scanning electron microscopy (SEM) was used to characterize the morphology 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 JSM-6060LV SEM machine. Preparation of Photo-Cross-Linked Gels for Swelling and Release Studies. Copolymer solutions (15% w/v) were prepared using 0.1% w/v 2-hydroxy-4′-(2-hydroxy-ethoxy)-2-methyl-propiophenone (Ciba Irgacure 2959, Sigma-Aldrich) PBS stock solution. The solutions (200 µL for swelling samples, 100 µL for release samples) were added into 1 mL flat bottom vials, preheated in an oven at 37 °C, then photocross-linked using Blak-Ray long-wave (365 nm) 100 W ultraviolet lamp with spot bulb (model B100 AP) at an intensity of 8.9 mW/cm2 for 10 min. Swelling Behavior of Photo-Cross-Linked Gels. After the exact weight of the gels was measured, 0.5 mL of PBS buffer (pH 7.4) was added to allow the gels to swell at 37 °C. At regular intervals the incubation buffer was removed and the weight of the gels was measured. Swelling ratios were calculated as below:

swelling ratio ) Wt/W0 where Wt represents the weight of the hydrogels at a certain time point and W0 represents the original weight of the hydrogels. After the swelling ratio was determined, 0.5 mL of fresh buffer was added again and the samples were further incubated at 37 °C. The swelling experiments were performed in triplicate. Drug Release from Photo-Cross-Linked Gels. The gels were saturated in carmoisine E122 (red food coloring) solution at room

Photo-Cross-Linked Hydrogels


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Scheme 1. Dendritic PEGMEMA-PPGMA-EGDMA Copolymer from One-Step ATRP Copolymerization of Poly(ethylene glycol) Methyl Ether Methacrylate (PEGMEMA), Poly(propylene glycol) Methacrylate (PPGMA), and Ethylene Glycol Dimethacrylate (EGDMA)

temperature for a week. The dye-laden gels were washed gently with deionized water to remove the dye at the surface before being transferred into deionized water at 25 and 37 °C, respectively. Digital images were taken at different time points to qualitatively demonstrate the effect of temperature on the release behavior. For a quantitative release study, lysozyme (from chicken egg white, Mw ) 14307 Da, Sigma-Aldrich) was loaded within the copolymer solutions at a concentration of 1 mg/ mL before being photo-cross-linked to form gels according to procedures described in the previous section. Then, 0.5 mL of PBS was added to each photo-cross-linked gel sample to allow protein release. At each time point, 0.35 mL of the supernatant was removed after gentle shaking and the same volume of fresh PBS was added. The concentration of lysozyme in the release samples was determined with the Micro BCA (bicinchoninic acid) assay kit (Thermo Scientific). The percentage of cumulative amounts of released lysozyme was calculated from standard calibration curve, which was generated from standard protein solutions at concentration range of 0.5-200 µg/mL. Release samples (150 µL) were pipetted into a 96-microwell plate and 150 µL of working reagent (BCA reagents A/B/C)50:48:2 v/v) was added. The plates were incubated at 37 °C for 2 h. Subsequently, the absorbance was measured at 562 nm with a TECAN Microplate Reader. Lysozyme activity was measured on Micrococcus lysodeikticus cell walls using EnzChek Lysozyme Assay Kit (Molecular Probes) following the experimental protocol recommended by the supplier. The assay is based on the hydrolysis of the outer cell membrane of Micrococcus lysodeikticus, resulting in solubilization of the affected bacteria and consequent decrease of light scattering.36 A total of 50 µL of Micrococcus lysodeikticus suspension (50 µg/mL in the buffer containing 0.1 M sodium phosphate, 0.1 M NaCl, pH 7.5 and 2 mM sodium azide) was added to each microplate well containing 50 µL of either release samples or the standard curve samples, then the loaded plate was incubated at 37 °C for 30 min while protected from light. The fluorescence intensity of reactions was measured using a TECAN fluorescence microplate reader. Digestion products have absorption maxima at 494 nm and fluorescence emission maxima at 518 nm. The release experiments were performed in triplicate. Cytotoxicity Assessments. Mouse C2C12 myoblast cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% FBS, 1% glutamine, and 2.5 mg/mL amphotericin B (antibiotic/ antimycotic solution) in a humidified incubator at 37 °C and 5% CO2. Prior to use, cells were trypsinised using 0.25% trypsin/

0.02% EDTA in PBS, centrifuged, and resuspended in DMEM. Approximately 80000 cells and 2 mL media were added into each well of a 24-well culture plates to allow cells to adhere and incubated at 37 °C for 4 h. A total of 10 µL of 15% w/v polymer solution was then added into each of the wells to make the final polymer concentration in the culture media and cultured at 37 °C for 5 days. Cell viability was assessed using the Alamar Blue assay (Biosource Europe) to measure metabolic activity of the cultured cells on days 1, 3, and 5. The experiments were performed in triplicate. At day 5, the Live/Dead viability/cytotoxicity assay (Molecular Probes, L-3224) was conducted to measure the membrane integrity of cells. Fluorescence images were taken using a Leica DMRB microscope, while viable cells fluoresce green through the reaction of calcein AM with intracellular esterase, and nonviable cells fluoresce red due to the diffusion of ethidium homodimer across damaged cell membranes and binding with nucleic acids. Light phase control microscope images were taken by Leica DMRB inverted microscope. The cytotoxicity of the copolymers was also assessed using lactate dehydrogenase (LDH) cytotoxicity detection kit (Invitrogen), which measures cell damage/death in response to chemical compounds or environmental factors using a coupled twostep reaction. Cells were seeded in 96-well plates at a density of 12000 cells/cm2 and allowed to adhere overnight. Polymer solutions at concentrations of 10-1000 µg/mL in Phenol red media were added to wells in triplicate. After cells had been incubated for 24 h with the polymer solution, 100 µL of supernatant from each well of the cultured cells was transferred to corresponding wells on a new plate. A total of 100 µL of LDH reaction solution was then added to each of the wells using a repeating pipettor. The plate was then incubated on an orbital shaker for 30 min at room temperature. The absorbance at 490 nm was measured using a microplate reader.

Results and Discussion Thermoresponsive Copolymers with Multiple Methacrylate Groups Prepared by One-Step ATRP Copolymerization. Water-soluble PEGMEMA-PPGMA-EGDMA copolymers with multiple methacrylate groups were synthesized by copolymerization of PEGMEMA (Mn ) 475), PPGMA (Mn ) 375), and EGDMA via one-step deactivation enhanced ATRP approach (Scheme 1).35 This approach was first reported for the successful

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Table 1. PEGMEMA-PPGMA-EGDMA Copolymers GPC RI a

entry

f

1 2

25/65/10 30/40/30

b

c

RT (h)

conv (%)

65 42

75 72

M

d w

GPC MALLS e

(kg/mol)

PDI

216 207

2.78 2.90

d w

M

(kg/mol)

PDIe

Ff

DBg(%)

BDh(%)

LCSTi(°C)

570 521

1.95 1.98

22:37:38 21:26:53

10 18

28 35

32 ( 0.5 33 ( 0.5

a Monomer feed mole ratio, f[PEGMEMA]/[PPGMA]/[EGDMA]. b Reaction time. c Monomer conversion, estimated using peak areas for monomers and copolymers in GPC traces. d Weight average molecular weight. e Polydispersity, Mw/Mn. f Polymer composition, F[PEGMEMA]/[PPGMA]/[EGDMA]. g Double bond content. h Branching degree. i Lower critical solution temperature. Polymerization conditions: 60 °C in butanone; total monomers/butanone (v/v) ) 1:1; [I]/[total monomers] (mol ratio) ) 1:100, [I]/[Cu+/Cu2+]/bpy ) 1:[0.375:0.125]:1. The initiator (I)/catalysts/ligand: methyl 2-chloropropionate/CuCl/CuCl2/bpy.

homopolymerization of divinyl benzene (DVB) and ethylene glycol dimethacrylate (EGDMA) to yield soluble dendritic homopolymers with multiple vinyl functional groups rather than cross-linked macrogelation.37 It was envisioned that this facile approach could be further adopted for the design and synthesis of soluble multifunctional dendritic copolymers using a high level of bifunctional vinyl monomers to achieve tunable highly branched structures. In this study, methyl 2-chloropropionate and copper(I) and (II) chloride were used as the initiator and catalyst during the reaction. The addition of Cu(II) species was used to enhance the deactivation reaction, thus, the growth rate of polymer chains was suppressed, leading to the delayed crosslinking.38 A yield of up to 75% of soluble dendritic copolymers was obtained (Table 1). The GPC with a MALLS detector was used to yield absolute molecular weight, which is found much higher than the relative molecular weight obtained by RI detector (see Supporting Information for GPC curves). The slope value (ca. 0.25) of the conformation plot for the copolymers is much lower than the typical range 0.5-0.6 for linear polymers, indicating the branched structures. Moreover, the dendritic structures of the copolymer products were also confirmed by 1 H NMR (the characteristic peaks at chemical shifts of 6.1 and 5.6 ppm are attributed to the vinyl functional groups). The composition, double bond content, and branching degree of the copolymer were calculated from the integral data of 1H NMR as described elsewhere.35 The double bond content represents the mol percentage of EGDMA with free vinyl functional groups in the copolymer and the branching degree represents the mol percentage of EGDMA as branching units (i.e., without vinyl groups) in the copolymer. The copolymers are amorphous viscous or tacky solids and their glass transition temperature (Tg) measured by DSC is about -46 °C. Thermoresponsive Properties of PEGMEMA-PPGMAEGDMA Copolymers. The resultant dendritic PEGMEMAPPGMA-EGDMA copolymers were dissolved in deionized water, phosphate buffered saline (PBS, pH 7.4), and cell culture media. The solutions reversibly became cloudy when the temperature was increased above 32 °C but were transparent below this temperature. Figure 1a shows the temperature scans of the copolymer solutions recorded by a UV spectrophotometer. The LCSTs of the copolymers were measured as about 32 °C in deionized water, suggesting this copolymer is soluble at temperatures below LCST with a random coil conformation, while above the LCST the polymer chains undergo a conformational change, collapse, and aggregate. The LCSTs of this copolymer in PBS (pH 7.4) and in cell culture media decreased slightly (LCSTPBS ) 29 °C; LCSTmedia ) 28 °C) compared to the LCST in pure water. This decrease agreed with prior data on “salting out” of thermoresponsive polymers due to the interactions and the hydrophilic/hydrophobic balance within the polymer molecules in the presence of salt additives and protein molecules.39,40 Dynamic light scattering (DLS) was also used to monitor temperature dependent changes in the conformation of the macromolecules. The results on the phase transition

Figure 1. Thermoresponsive properties of PEGMEMA-PPGMAEGDMA copolymer (2 in Table 1). (a) LCST behavior of the copolymer in 0.03% w/v deionized water, PBS buffer, and the tissue culture media, demonstrated by UV-vis spectroscopy; (b) Size distributions measured by DLS (0.01% w/v aqueous solution); (c) Polymer solution (15% w/v) became thermal gel when the temperature was raised above its LCST from 20 to 37 °C. Published with permission from ref 35. Copyright 2009 American Chemical Society.

temperature LCST agreed well with the UV measurements. Figure 1b shows a typical size distribution of the copolymers in deionized water at 20 and 37 °C. As apparent from the peak diameters increasing dramatically from 13.5 to 459 nm, the thermoresponsive property of the polymer led to aggregations at temperatures above LCST. The copolymers (100-300 mg) were dissolved in deionized water (1.00 mL) at 4 °C and then placed in 37 °C incubator to observe their gelation behavior. Gel concentration was determined as no flow within 10 s by



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Figure 2. Photo-cross-linked gels (arrow pointed) from PEGMEMA-PPGMA-EGDMA copolymer (2 in Table 1). (a) Gel sample (diameter 20 mm, thickness 0.5 mm) after realtime rheological measurements; (b) Gel sample (diameter 8 mm, thickness 8 mm) for compression test.

Figure 3. SEM images for the freeze-dried photo-cross-linked gel samples. (a) Monolith obtained from copolymer 1 (Table 1); (b) Porous structure obtained from copolymer 2 (Table 1).

visual observation (Figure 1c). It was found that gel points of these copolymers at 37 °C were 15% w/v. Rheological, Mechanical Properties, and the Morphology of Photo-Cross-Linked Gels. The thermoresponsive PEGMEMA-PPGMA-EGDMA copolymers produced by the onestep deactivation enhanced ATRP approach carry multiple methacrylate groups, which can be used to form covalent crosslinking via photopolymerization. The cross-linking of these copolymer aqueous solutions by thermally induced gelation and photopolymerization were studied by real-time rheological measurements. The copolymer solutions were found to undergo fast photo-cross-linking gelation, resulting in the crossover of loss modulus (G′) and storage modulus (G′′) within seconds of UV exposure to form elastic gels (Figure 2a) with dramatically increased moduli, that is, three orders of magnitude greater than the physically thermal gels before photo-cross-linking. The enhancement in mechanical strength was observed for gels at a high copolymer concentration, photopolymerization at a temperature above LCST, and using the copolymer with high double bond content.35 The storage modulus of 15% w/v copolymer 2 (f[PEGMEMA]/[PPGMA]/[EGDMA ) 30/40/30, Table 1) gels produced by photo-cross-linking at 37 °C is about 10 kPa, compared to about 1 kPa for photo-cross-linked gels from copolymer 1 (f[PEGMEMA]/[PPGMA]/[EGDMA ) 25/65/10, Table 1). Moreover, it was demonstrated from DMA measurements (Figure 2b) that the photo-cross-linked gels were elastic and displayed an increasing elastic modulus (E) with increasing amounts of methacrylate functional groups from 11 kPa for 15% w/v copolymer 1 gels to 22 kPa for 15% w/v copolymer 2 gels. The morphology of gels is an important factor in terms of the gel performance in tissue engineering and drug delivery applications. In this study, the freeze-dried gel samples were observed for their network structure by taking SEM images. The freeze-dried gels prepared

Figure 4. Swelling of photo-cross-linked gels in PBS buffer (pH 7.4) at 37 °C. The copolymer 1 with a lower cross-linking density demonstrated a higher swelling ratio than the copolymer 2.

from copolymer 1 formed a smooth monolith (Figure 3a), in contrast, the freeze-dried gels prepared from copolymer 2 demonstrated a porous structure with an average pore diameter about 1.63 µm (Figure 3b). The significant difference in the morphology of the photo-cross-linked gels could be the combined effect of the composition and cross-linking density on phase separation, molecular assembly and the compactness of molecules.28 The impact of copolymer composition on the morphology of photo-cross-linked gels will be further studied by preparing a series of copolymers with various compositions. It should be pointed out that the morphology of freeze-dried gels is not the same as that for the gels after swelling, especially for the gels yielded from thermoresponsive materials. A higher swelling ratio was observed for copolymer 1 gels than copolymer 2 gels. Therefore, the difference in pore size could be reduced slightly after swelling. Nevertheless, the pore sizes and porosity of the gels can be tailored by adjusting the copolymer composition and concentration, thus, to tune the cross-linking density so that to meet the specific needs for cells and/or guest molecules encapsulation and transportation.

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Figure 5. Release from photo-cross-linked gels prepared using PEGMEMA-PPGMA-EGDMA copolymer (2 in Table 1). (a) Carmoisine E122 (red food coloring) as the model molecule. The gels were releasing the carmoisine red dye at a faster rate in warm water, compared to a slow release in cold water. Images were taken immediately (left images) and after 5 min (right images) of the gels being transferred into water. (b) Cumulative release of the encapsulated lysozyme from photo-cross-linked hydrogels at a temperature above and below the LCST in 10 min.

Figure 6. Cumulative lysozyme release from photo-cross-linked gels prepared using PEGMEMA-PPGMA-EGDMA copolymers (1 and 2 in Table 1) at temperatures below and above LCST.

Swelling and Release Studies of Photo-Cross-Linked Gels. The photo-cross-linked gels were soaked in PBS for swelling at 37 °C until equilibrium to test the maximum water uptake. It was found that the weight and volume of the gels increased and the swelling reached equilibrium within 24 h (Figure 4). The copolymer 1 with a lower cross-linking density demonstrated a higher swelling ratio (about 1.2) than copolymer 2 (about 1.1). This indicates that the cross-linking density has an impact on the swelling property of the photo-cross-linked gels, thus influencing the mechanical strength of the gels and mass transport within the gels. The hydrogels prepared from thermo-responsive PEGMEMAPPGMA-EGDMA copolymers after photo-cross-linking were tested for releasing model drug with changes in temperature. The release of the red dye from the gels in the warm water

started immediately after transferring. However, in cold water the dye was released from the samples at a much slower rate (Figure 5a). Similar temperature dependent release profile was also observed from lysozyme release studies at temperatures above and below LCST of the copolymer, that is, 25 and 37 °C (Figure 5b). The higher diffusion coefficient of encapsulated compounds at a higher temperature could contribute to this apparent fast release; in addition, the gel shrinking induced by the changes in polymer chain conformation from hydrophilic to hydrophobic might squeeze the model drug dye out of the gel. Lysozyme release studies were continued up to one week using gels from copolymers 1 and 2. At 3 days, copolymer 1 exhibited ∼55% lysozyme release; while copolymer 2 showed ∼80% release (Figure 6). It is interesting that copolymer 2 showed a faster lysozyme release despite the higher cross-linking density and lower swelling ratio. The porous structure (Figure 3) might be the key factor. A crossover point was also found for the release profiles at the temperatures above and below LCST, that is, gels initially released protein faster at a temperature above LCST, then released slower at this temperature after about one day (the crossover points in Figure 6). However, owing to the experimental error bars, the differences observed for the overall release profiles at 25 and 37 °C are not considered to be significant. Statistical analysis performed using Student’s t-test with a confidence level of 0.05 (p value) on the release data shows that the difference for the release profiles of copolymers 1 and 2 is statistically significant, however, the difference for the release profiles of one copolymer at two temperatures (25 and 37 °C) is not statistically significant. It is reckoned that the release kinetics of PEGMEMA-PPGMAEGDMA photo-cross-linked gels are both diffusion and swelling controlled, and influenced by the cross-linking density, gel morphology, polymer chain conformation (from hydrophilic to hydrophobic), and the interaction of the functional groups in the copolymers with protein molecules.41-43 A higher PPGMA content in copolymer 1 provides a higher amount of OH groups



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Figure 7. Cytotoxicity assessments of PEGMEMA-PPGMA-EGDMA copolymer (1 in Table 1). (a) Alamar Blue cell metabolism assay at the copolymer concentration of 750 µg/mL. (b) LDH cell damage assay at the copolymer concentration of 10-1000 µg/mL.

in the molecular structure than in copolymer 2. This could lead to a stronger interaction for copolymer 1 with protein than copolymer 2, thus contributing to a slower and incomplete release even after 7 days (Figure 6). Moreover, the lysozyme in the release studies demonstrated more than 85% remaining activity in all cases determined by EnzChek Lysozyme Assay and normalized with released lysozyme, indicating that experimental conditions employed, including UV treatment, are not harmful to lysozyme. Cytotoxicity Assessments. Alamar Blue cell viability assays of the copolymer solution at the concentration of 750 µg/mL at days 1, 3, and 5 were performed and the results indicated that the copolymers had a slight reduction in cell activity compared to the control using cells alone (Figure 7a). Cells cultured in the copolymer/culture media solutions (750 µg/mL) showed a stellate morphology (Figure 8a), which was similar to those cultured in the media only (Figure 8b). After 5 days culture, the viable cells can be clearly seen to fluoresce green by Live/ Dead viability assay (Figure 8d). LDH cytotoxicity assay was further used to measure the cell response to the addition of the copolymers at the concentration of 10-1000 µg/mL. The results indicated that the cells showed a sign of damage at a concentration of 1 mg/mL (Figure 7b), which was lower than the concentration used as a gel in this study (15% w/v). Toxicity at higher concentration suggests interactions between copolymers and cells might exist even though these copolymers consist of solely biocompatible PEG and PPG building blocks. It would be necessary to perform further cellular interaction studies.

Possible cell interactions and penetrations of non-photo-polymerized polymers may be reduced by photopolymerization, leading to a decrease in toxicity of photopolymerized gels.34 Moreover, it was observed that with the increase in polymer concentration, there were more precipitated copolymers covering the surface of the cultured plates and cells, which led to less contact surface areas for diffusion and at higher concentration affecting the ability of cells to move and dividing cells to attach and grow. Cell seeding experiments on the PEGMEMAPPGMA-EGDMA polymer films (Figure 8c) indicated they did not adhere and spread on these materials. It is reported that structure modifications by introducing cell adhesion peptide moieties (i.e., RGD peptide) can improve cell adhesion enormously.23,44,45 Therefore, we currently conduct the ongoing research to assess cell behavior by encapsulating cells within the thermal gels and photo-cross-linked gels after modifying polymer gel network by introducing cell adhesion peptides.

Conclusions PEGMEMA-PPGMA-EGDMA copolymers with both thermoresponsive and photo-cross-linkable properties were synthesized via one-step deactivation enhanced atom transfer radical polymerization (ATRP). The photo-cross-linked gels prepared from these copolymers at a temperature above the LCST showed excellent mechanical properties. The swelling ratio and the lysozyme release rate of these gels could be controlled by simply adjusting the monomer composition within the polymers. Also,

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Figure 8. Light phase control microscope images for the cells cultured (a) in PEGMEMA-PPGMA-EGDMA copolymer (1 in Table 1) culture media solutions (750 µg/mL); (b) in the culture media without polymers, (c) on the photo-cross-linked polymer films. (d) Live/Dead viability assay for the cells cultured in the copolymer/culture media solutions after 5 days. The viable cells fluoresce green, whereas the nonviable cells fluoresce red (pointed by the arrow).

these materials were found to have low toxicity for mouse C2C12 myoblast cells as assessed with LDH, Alamar Blue, and a Live/Dead assay at concentrations less than 1 mg/mL. Further structure modifications by introducing cell-adhesion functionality are needed to improve cytocompatibility of the photo-crosslinked gels. These in situ photo-cross-linked hydrogels from thermoresponsive copolymers with multiple methacrylate groups have many advantages for the potential applications in tissue engineering and drug delivery. First, thermal gelation due to thermoresponsive properties upon injection allows easy handling and holds the shape of the gels prior to the photopolymerization during clinical practice. Second, in situ photo-cross-linked hydrogels from thermoresponsive polymers have enhanced mechanical strength and stability compared to non-photo-crosslinked gels. Third, the multiple methacrylate groups within the copolymer dendritic structure can provide a tunable high crosslinking density to allow tailoring the mechanical properties, swelling, and release profiles of the gels. Therefore, this novel thermoresponsive copolymer synthesized by one-step deactivation enhanced ATRP approach has great potential as a smart injectable system for regenerative medicine such as wound healing and tissue repair. The biodegradability of the gels can be further introduced by copolymerizing macromonomers with biodegradable building blocks, for example, PLGA based dimethacrylate, or using a biodegradable cross-linker. Acknowledgment. H.T. is supported by EPSRC with a Life Science Interface Fellowship (EP/E042619/1). The British Council and Platform Be`ta Techniek are thanked for their financial support through Partnership Programme in Science (PPS RV19).

Supporting Information Available. GPC curves of dendritic copolymers. This material is available free of charge via the Internet at http://pubs.acs.org.

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Photo-Cross-Linked Hydrogels from Thermoresponsive PEGMEMA

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