Tailoring Thermoreversible Hyaluronan Hydrogels by âClick

Biomacromolecules 2010, 11, 1261–1272

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Tailoring Thermoreversible Hyaluronan Hydrogels by “Click” Chemistry and RAFT Polymerization for Cell and Drug Therapy Derek Mortisen, Marianna Peroglio, Mauro Alini, and David Eglin* AO Research Institute Davos, Clavadelerstrasse 8, 7270 Davos, Switzerland Received January 18, 2010; Revised Manuscript Received March 17, 2010

Thermoreversible hydrogels are promising matrices for tissue-engineered cartilage and spine constructs. They require specific properties during all the stages of a cell therapy (e.g., cell expansion, recovery, injection, delivery). Thermoreversible hyaluronan-poly(N-isopropylacrylamide) (HA-PNIPAM) hydrogels with well-defined molecular architecture and properties were synthesized through RAFT polymerization and “click” chemistry. The effect of PNIPAM grafting length and density on HA-PNIPAM properties was evaluated by methods relevant for a cell therapy. It was found that reversibility of the PNIPAM gelling process was improved in the presence of HA. Increasing Mn of PNIPAM decreased the viscosity at 20 °C and led to high G′ at T > 30 °C; however, higher grafting density led to lower mechanical properties. Water uptake of the hydrogels was mainly dependent on PNIPAM Mn. All of the hydrogels and their degradation products were cytocompatible to hTERT-BJ1 fibroblasts. A composition with properties ideal for cell encapsulation was identified and characterized by a low viscosity at 20 °C, rapid gelling at 37 °C, absence of volume change upon gelling, and G′ of 140 Pa at 37 °C.

Introduction Tissue regeneration has made rapid progress in recent years, making possible the synthesis of constructs that closely mimic the structure and properties of native tissues. Designing functional, highly hydrated, 3D architectures on the cell scale has become the paradigm for triggering biological events via spatially and temporally controlled biological cues or drugs and the formation/regeneration of organized biological tissues.1 Among proposed solutions, injectable polymeric compositions capable of delivering cells and drugs and gelling in situ are one of the most promising. In fact, compared with large macroporous polymeric scaffolds, injectable polymeric compositions mimic more closely the hydrated 3D structure of the extracellular matrix and permit minimally invasive surgical procedures, therefore reducing potential risk of infection and damage to the surrounding tissues.2 Recently, controlled radical polymerizations such as atom transfer radical polymerization (ATRP) and reversible addition-fragmentation chain transfer polymerization (RAFT) have become popular methods because they allow the generation of highly controlled molecular weight polymers with low polydispersity (PDI). The precision and versatility offered by these methods have simplified the synthesis of hydrogels and nanogels with complex architectures.3 In particular, RAFT polymerization can occur under mild reaction conditions, in an aqueous solvent, and with high fidelity. By functionalizing the end groups of the trithiocarbonate-based chain transfer agent (CTA) used in RAFT polymerizations, it is possible to create specific polymer architectures by subsequently using “click” chemistry reactions, the most notable of which is the copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC).4 Many reports have described the preparation of polymeric hydrogel architectures (brushes, grafts, dendrimers, stars) via different “click” reactions, including new copper-free methods that can be performed under mild conditions in vivo.5 The combination of RAFT to generate specific * Corresponding author. Tel: +41-814142480. Fax: +41-814142288. E-mail: [email protected].

molecular weight polymers with the efficient coupling mechanism of CuAAC allows highly controlled formation of complex, functional architectures consisting of two or more polymers, and molecules with distinct physical properties. A polymer of choice for a cell and drug delivery platform is hyaluronic acid (HA). It is a chief component of the extracellular matrix in connective, epithelial, and neural tissues and is known to play an important role in organ development, cell proliferation, and migration.6 Additionally, HA contributes to the lubrication and maintenance of cartilage, where it is a major component of synovial fluid and forms a coating around chondrocytes.7 Ideally, hyaluronan delivery is performed using minimal invasive techniques. However, unmodified hyaluronan solutions are rapidly degraded by the body and cleared from the implant site.8 To increase the stability of hyaluronan gels sufficiently to encapsulate cells, slowly degradable cross-links need to be introduced.9 For example, injectable hyaluronan solutions based on methacrylated hyaluronan capable of gelling in situ have been proposed for tissue repair using mesenchymal stem cells.8 These cross-links can consist of chemical links between the polysaccharide chains but can also be formed by the association of hydrophobic domains synthetically introduced in the polysaccharide.10 The cross-links can be permanent (e.g., methacrylate) or reversible (e.g., disulfide bond).4,11–13 Thermoresponsive polymers, such as those based on oligo(ethylene oxide) methacrylate or poly(ethylene glycol)/poly(lactic acidco-glycolic acid), are being considered as cell encapsulation matrices because they can be injected and gelled in situ without producing toxic byproduct.14 Thermoresponsive poly(N-isopropylacrylamide) (PNIPAM) represents an attractive candidate to introduce physical cross-links via association of hydrophobic domains because it has a gelling temperature below body temperature (∼32 °C) and good biocompatibility.15 Although PNIPAM is nonbiodegradable, it has been shown that short PNIPAM chains can undergo renal excretion.16 Therefore, it is of key importance to control the PNIPAM Mn and guarantee a low polydispersity, as it has been recently reviewed by He et al.17 Pure PNIPAM hydrogels have been extensively evaluated

10.1021/bm100046n  2010 American Chemical Society Published on Web 04/06/2010

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as drug release systems but only infrequently as a matrix for cell culture.18 More recently, PNIPAM-grafted hyaluronan hydrogels for endothelial cell culture and encapsulation of human adipose-derived stem cells have been prepared by a photoradical polymerization of PNIPAM on dithiocarbamatederivatized hyaluronan and a thermoradical polymerization of PNIPAM onto the adipic dihydrazide-grafted HA.19,20 However, none of the reported synthetic procedures allows for a finetuning of the material macromolecular architecture (e.g., low PNIPAM PDI) important for the optimization of the composition properties, nor do they offer opportunities for further functionalization. Therefore, it was the goal of this work to develop a material with the following properties: (1) semisynthetic with the primary backbone being composed of hyaluronan, (2) liquid at room temperature with a sufficiently low viscosity to facilitate cell encapsulation under low shear conditions and/or mixing of additional drugs and bioactive agents, (3) injectable through a needle at room temperature, (4) with rapid thermoresponsive gelling kinetics, (5) with reversible gelling to allow retrieval of cells encapsulated and cultured in vitro, (6) cytocompatible, (7) with suitable mechanical properties when gelled at 37 °C, and (8) readily amenable to further functionalization. HA-PNIPAM copolymers with different PNIPAM Mn and grafting densities were obtained using recent advances in polymer synthesis, which allow precise tailoring of the molecular architecture: RAFT polymerization was selected for synthesizing PNIPAM chains with defined molecular weight and narrow PDI, and “click” chemistry allowed easy tuning of the PNIPAM grafting density. The effect of PNIPAM length and density on HA backbone was evaluated on properties relevant to a tissue-engineering application. First, the thermoreversibility of the gel was examined by differential scanning calorimetry (DSC) and light transmittance. Next, the viscosity of the polymer solution at 20 °C was evaluated by rheological measurements. Volume and mechanical property changes associated with temperatureinduced gelling were determined by weight and dynamic viscoelasticity measurements. Furthermore, the cytocompatibility of the gels and their degradation products was studied against a human fibroblast cell line (hTERT-BJ1). Finally, the potential of grafting drugs or other biologically relevant small molecules was demonstrated using fluorescein (FL) as a model compound. Collectively, these data establish HA-PNIPAM as a versatile hydrogel for cell encapsulation and drug delivery.

Experimental Section Materials. Hyaluronic acid sodium salt from Streptococcus equi sp. (NaHA) was purchased from Fluka. Tetrabutylammonium fluoride trihydrate (TBAF), N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), propargylamine (PPA), N-dimethylsulfoxide (DMSO), sodium chloride, sodium azide, ascorbic acid sodium salt (AANa), copper sulfate pentahydrate (CuSO4 · 5H2O), ethylenediaminetetraacetic acid (EDTA), N-ethylpiperidine hypophosphite (EPHP), N-isopropylacrylamide (NIPAM), azobisisobutyronitrile (AIBN), N-dimethylformamide (DMF), lithium bromide (LiBr), sodium nitrate (NaNO3), FL, 11-azido-3,6,9-trioxaundecan-1-amine (ATA) and Dowex 50WX8 cation exchange resin (H Type) were purchased from Sigma Aldrich and were of the purest grade. Dulbecco’s modified Eagle medium (DMEM), fetal calf serum (FCS), and penicillin-streptomycin were purchased from Gibco, Invitrogen. Synthesis of Hydrosoluble Hyaluronan Propargylamide (HApa). HApa was prepared using a procedure already established in the field,21 but to reduce deactivation of EDC/NHS by water, HA modification with PPA was performed by first solubilizing hyaluronan

Mortisen et al. in DMSO using a lipophilic cation.13 In brief, 5 g of NaHA was dissolved in 500 mL of deionized water. A Dowex 50WX8 cation exchange resin (H Type) was loaded with TBAF before addition to the NaHA solution. After ion exchange, the resin was removed by centrifugation at 5000 rpm for 2 min and HA tributylammonium salt (TBAHA) solution was frozen at -80 °C and lyophilized. TBAHA was then dissolved in 500 mL of DMSO, and the following were added to this solution: 6.5 g (0.033 mol) EDC, 7.29 g (0.063 mol) NHS, and 6.23 g (0.11 mol) PPA. The mixture was stirred at room temperature and allowed to react for 5 days protected from light. Following the reaction, the mixture was transferred to dialysis tubing (MWCO ) 12 to 14 kDa) and dialyzed first against 0.1 M NaCl in water for 24 h and then against distilled water for 5 days. After dialysis, the solution was frozen at -80 °C and lyophilized. Following the synthesis, HApa was reacted with an excess of NaN3 using CuSO4 · 5H2O as the catalyst and AANa as the reducing agent. After dialysis and lyophilization, the material was dissolved in deuterium oxide and proton nuclear magnetic resonance spectroscopy (1H NMR) was performed. The degree of substitution (DS) was determined as the ratio of the integration of the triazole proton peak (δ 7.88) to the integration of the acetyl proton peak (δ 2.00).21 Synthesis of Azido-Terminated Poly(N-isopropylacrylamide) (N3-PNIPAM). Succinctly, a CTA, S-1-dodecyl-S′-(R,R′-dimethyl-R′′acetic acid) trithiocarbonate (CTA) was first prepared.22 This CTA was then modified with an azido group according to Gondi et al.23 and used with AIBN to polymerize NIPAM.24 We prepared azido-terminated PNIPAM polymers with number-average molecular weight (Mn) equal to 10, 20, and 35 × 103 g · mol-1 by varying the polymerization time and NIPAM/CTA molar ratio. Cleavage of thiocarbonate end group was performed by radical-induced reduction using EPHP and AIBN in toluene, as reported by Vogt et al.25 We semiquantitatively checked reduction efficiency by ultraviolet-visible (UV-vis) spectroscopy analysis by monitoring absorbance at 316 nm. Mass spectroscopy (MALDI-TOF) and size exclusion chromatography (SEC) analyses were performed on a set of N3-PNIPAM polymers, and a linear correlation between MALDI-TOF and SEC values was determined.26 Molecular weights and PDI of N3-PNIPAM were measured by SEC, and corrected Mn values were calculated using the correlation determined from MALDI-TOF (Table 2). Synthesis of Hyaluronan Grafted Poly(N-isopropylacrylamide) (HA-PNIPAM). We performed the grafting of N3-PNIPAM to HApa by dissolving HApa in distilled water at 0.5% w/v and adding the desired amount of N3-PNIPAM. A catalyst solution was prepared with 0.3731 g of AANa, 0.0470 g of CuSO4 · 5H2O, and 2 mL of distilled water. The catalyst solution was added to the HApa and N3-PNIPAM solution (200 µL for 10 mL of HApa and N3-PNIPAM solution). Copper-catalyzed azide-alkyne cycloaddition (CuAAC) proceeded overnight at room temperature under stirring, at which point 0.3723 g of EDTA was added to chelate copper ions and stop the reaction. This solution was then transferred to dialysis tubing (MWCO ) 12 to 14 kDa) and dialyzed against 0.1 M NaCl in water for 24 h, followed by an additional 5 days of dialysis against distilled water. The solution was then frozen at -80 °C and lyophilized to constant weight. Six HA-PNIPAM compositions were prepared using a HApa with a degree of PPA functionalization equal to 30% of the carboxylic groups, as measured by 1H NMR. N3-PNIPAM with Mn equal to (A) 10 × 103, (B) 20 × 103, and (C) 35 × 103 g · mol-1 were grafted on HApa at a percentage corresponding to 25 and 30% of the disaccharide subunits and were named, respectively, HA-PNIPAM A25, A30, B25, B30, C25, and C30. The average amount of copper catalyst in HA-PNIPAM compositions after dialysis was measured by inductively coupled plasma optical emission spectrometry (ICP-OES) and found to be 145 ( 21 ppm. PNIPAM and HA-PNIPAM polymers were dissolved in PBS (pH 7.4) at a constant hyaluronan content of 0.8% w/v and PNIPAM concentrations of 4.1 and 5.4 mM for a DS of 25 and 30% respectively. This corresponds to HA-PNIPAM solutions with total weight polymer

Tailoring Thermoreversible Hyaluronan Hydrogels percent ranging from 4.9 to 16.8. These PNIPAM and HA-PNIPAM solutions were used for turbidity, thermal, and rheological measurements. Synthesis of HA-PNIPAM Comprising Covalently Grafted Fluorescein. An azido-modified FL was prepared by coupling FL to ATA by EDC/NHS reaction, as reported elsewhere.27 The azidomodified FL was dissolved in DMSO (4.7 mg in 125 µL of DMSO). Azido fluorescein solution (50 µL) and 0.45 g of N3-PNIPAM with a Mn,SEC of 29 × 103 g · mol-1 were then added to 3 mL of 0.5% w/v HApa solution in water. The CuAAC reaction was performed for 1 day in the absence of light. Control materials of native NaHA (lacking alkyne functionality) and HA-PNIPAM (lacking a FL label) were prepared in parallel. Solutions (1% w/v) were prepared and observed between a glass slide and coverslip under fluorescent light using an Axioplan 2 microscope (Zeiss, Go¨ttingen, Germany). For each solution, images were recorded in fluorescence with a green filter (filter set 10, excitation 450-490, beam splitter FT 510, emission BP 515-565); the exposure times were set at the beginning of the analysis to allow direct comparison of the fluorescence emission. Characterization Techniques and Instrumentation. Hyaluronan Mn and concentration in solution were measured using uronic assay and N-acetyl-D-glucosamine reducing end assay methods described in the literature.28 Reactants and products were measured by 1H NMR analysis using a Bruker Avance AV-500 NMR spectrometer and deuterated chloroform (CDCl3) for PNIPAM- and D2O for hyaluronanbased polymers as solvents. PNIPAM and hyaluronan molecular weight and polydispersity were measured by SEC. SEC analyses were carried out on a Waters (Milford, MA) modular GPC system consisting of a 510 pump, a U6K injector, and a 410 refractive index detector. Calibration was carried out with polystyrene (range 1000 to 2 × 106 g · mol-1) or pullulan (range 78.8 to 0.59 × 104 g · mol-1) molecular weight standards supplied by Shodex, Japan. PNIPAM samples were dissolved in a solution of DMF containing 0.05 M LiBr, followed by filtration through a 0.45 µm Millex SR filter prior to injection (300 µL) in Styragel HR 2 and HR 4 Waters columns. An ultrahydrogel linear Waters column blend from Waters was used to analyze hyaluronan-containing samples dissolved in the eluent (a 0.1 N NaNO3 water solution). A 0.2% w/v solution of polymer (PNIPAM, HA, or HA-PNIPAM) in the eluent was used to ensure good representation of the polymer. A flow rate of 1.0 mL/min was applied for both PNIPAM and hyaluronan samples. Data analysis was conducted using the Waters Maxima 820 software package. Lower critical solution temperature (LCST) and thermal behavior of the PNIPAM and HA-PNIPAM compositions in solution were examined using a UV-vis spectrophotometer Lambda 12 Perkin-Elmer with a Peltier temperature control system. The absorbance at 500 nm (path length ) 1 cm) was recorded stepwise (0.5 °C steps from 25 to 30 and 0.2 °C steps in the gel-transition region 30-33 °C). At each temperature, the polymer solutions were equilibrated for 20 min prior to absorbance reading. The LCST of the polymer solutions was determined as the temperature at which the solution turbidity was half of the difference between the maximum and the minimum absorbance values. After stabilization at 25 °C for 1 h, kinetics of gel formation was also recorded by monitoring the absorbance at 500 nm after an instant (Peltier controlled) temperature change from 25 to 37 °C. Ten minutes after gel formation, solubilization kinetics of the gels were also monitored with the same method by instant cooling from 37 to 25 °C. Peak onset, maximum, and area attributed to PNIPAM conformational changes were measured by DSC using a Pyris DSC-1 PerkinElmer instrument calibrated with indium. PNIPAM and HA-PNIPAM solutions (10-15 mg) were weighed in an aluminum pan. All DSC measurements were carried out with a holding time of 5 min at 15 °C, followed by a heating up to 45 °C and cooling at 15 °C at a scanning rate of 5 °C/min under a dry nitrogen atmosphere with a flow rate of 25 mL/min. The phase transition repeatability test was performed using cyclic DSC scans (up to six cycles) with the same conditions as previously mentioned with a holding step only before the first cycle.


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Evolution of temperature hysteresis (calculated as the difference between peak maximum at heating and cooling) and enthalpy could be determined. Apparent dynamic viscosity (η′), storage modulus (G′), and loss modulus (G′′) of 0.8% w/v NaHA and HApa in PBS at 25 °C were measured using a rheometer with Piezo axial vibrator (PAV) (Idm, Ulm, Germany) under a dynamic squeeze flow in the range from 0.5 Hz to 10 kHz. The Cross equation was used to determine the pseudoplasticity index and the zero-shear viscosity of NaHA and HApa (eq 1).29

η′ ) η∞ +

(

η0 + η∞ η0 + (kF)n

)

(1)

where η′ is the apparent dynamic viscosity, η0 is the zero-shear viscosity, η∞ is the viscosity at infinite share rate, F is the shear rate, k is a fitting parameter, and n is the pseudoplasticity index. Complex viscosity (η*), G′, G′′ and gel point of PNIPAM and HAPNIPAM solutions were analyzed using a CVOR-rheometer Bohlin instrument with Piezo rotary vibrator (PRV) option using a plate-plate geometry with silicon oil to avoid evaporation during measurements. The materials were subjected to a temperature-dependent analysis from 25 to 40 °C with a heating and cooling rate of 1 °C/min at several frequencies (2, 20, 200, 1000, and 2000 Hz). Gel point values were determined as the temperature for which tan δ ) 1. Water retention, or the amount of water lost or gained by the different HA-PNIPAM after 1 h in a PBS bath at 37 °C, was assessed. In brief, drops (∼40 µL) of polymer composition solutions (rehydrated in PBS at isohyaluronan concentration 0.8% w/v) were plunged in a PBS bath thermostatted at 37 °C using an autoinjector (Harvard Apparatus, 0.2 mL/min). Samples were collected after 1 h; controls consisted of 40 µL drops of polymer solutions collected in Eppendorf tubes at 22 °C. The beads and controls were weighted and, after an identical lyophilization process, weighted again (n ) 3-5). Water retention was j s), where ∆m j b is the average of the differences calculated as (∆m j b)/(∆m between wet and dry weights of the controls and ∆m j s is the average of the differences between wet and dry weights of the samples. Preparation of Degradation Products and Cytocompatibility Study. NaHA, PNIPAM (10, 20, and 35 × 103 g · mol-1), HA-PNIPAM copolymers, and their degradation products were tested for cytocompatibility to a fibroblastic cell line. Degradation of the HA backbone produced degradation products of different molecular weights, which were prepared by adding 10, 50, 100, 500, or 1000 U/mL hyaluronidase (HAse) to NaHA and HA-PNIPAM solutions at 1% w/v in PBS, followed by overnight incubation at 37 °C and HAse deactivation by heat treatment.12 The nondegraded polymers and the obtained degradation products were diluted in PBS (pH 7.4) to obtain a hyaluronan concentration of 10, 20, 100, and 200 µg/mL. This means that for each hyaluronan concentration, HA-PNIPAM copolymers were diluted, keeping hyaluronan concentration constant, and the only variable was PNIPAM length (from 10 to 35 × 103 g · mol-1). For instance, HAPNIPAM solutions with hyaluronan concentration ranging from 10 to 200 µg/mL hyaluronan contained 5-100 µM of PNIPAM. Controls with NaHA only or PNIPAM Mn,theoretical ) 10, 20, and 35 × 103 g · mol-1 were also prepared. The amount of PNIPAM in these solutions was equivalent to that in the HA-PNIPAM solutions (from 5 to 100 µM PNIPAM). Deactivated HAse (10-1000 U/mL) was also added as a control. hTERT-BJ1 cells were seeded at a density of 2000 cells per 100 µL of media (DMEM supplied with 10% FCS and 1% penicillinstreptomycin) in 96-well plates. Cells were transferred to a cell culture incubator set up at 37 °C, 5% CO2, and 95% humidity. After 14 h, deactivated HAse, PNIPAM, NaHA, PNIPAM, HA-PNIPAM, NaHA, or HA-PNIPAM degradation products or DMEM only as positive control (100 µL) were added. Cells were cultured in the presence of the polymer solutions or DMEM for 24 or 48 h. At each time point, cell viability was measured by Alamar Blue assay. Culture media was

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Scheme 1. EDC/NHS Synthesis of Hyaluronan-Propargylamide (HApa), Followed by Copper-Catalyzed Azide-Alkyne Cycloaddition of HApa with Azido-Terminated Poly(N-isopropylacrylamide) N3-PNIPAM

aspirated from the wells, and 100 µL of a 10% v/v solution of Alamar Blue reagent in DMEM supplemented with 0.5% FCS was added to each well. The plates were incubated overnight before the absorbance was read with a multilabel plate reader (Victor 3, Perkin-Elmer) at room temperature. The absorbance value was calculated to be the difference between the absorbance at 540 and 595 nm and was normalized to the positive control (cells supplemented with media only). The experiment was performed thrice with four replicas each time (n ) 12). After exclusion of the outliers, the normality of the data distribution was tested, and one-way analysis of variance (ANOVA), followed by Post Hoc test (Tukey HSD) was performed with SPSS 18 software; p values e 0.05 were considered to be statistically significant.

Results and Discussion Synthesis of PNIPAM-Grafted Hyaluronan via CuAAC. The two synthetic steps of HA-PNIPAM are depicted in Scheme 1: the EDC/NHS mediated coupling of PPA to carboxylic acid groups on the hyaluronan salt and the copper-catalyzed azide-alkyne cycloaddition of the N3-PNIPAM to the hyaluronan-propargylamide. High-molecular-weight hyaluronan macromolecules are polysaccharides that are susceptible to cleavage during ion exchange and PPA modification processing steps (e.g., by light,

Table 1. Average Molecular Weight Number (Mn) and Polydispersity (PDI) Measured by SEC and Molecular Weight Number (Mn) Measured Using Chemical Assays for the Hyaluronic Sodium Salt, the Hyaluronic Tributylammonium Salt, and the Hyaluronan-Propargylamide

NaHA TBAHA HApa

Mna

PDIa

[HA]b

[HAreducing end]c

Mn ) [HA]/ [HAreducing end]

502 600 482 100 1 116 100

1.76 2.76 1.03

20.6 19.6 22

1.74 × 10-5 1.20 × 10-4 1.36 × 10-5

1 184 900 163 500 1 617 600

a Measured by SEC (Mn in grams per mole). b Measured by uronic acid assay in grams per liter. c Measured by N-acetyl-D-glucosamine reducing end assay in grams per mole.

temperature, oxidative environment). Therefore, the average molecular weight of the hyaluronan macromolecules was measured at each step of HA preparation (Table 1). The molecular weight of NaHA as measured by SEC is relative to the pullulan standards and can be used only for comparison, whereas the Mn measured by chemical assays should give absolute values. The ion-exchange procedure from NaHA to TBAHA led to a slight decrease in the average molecular weight values and an increase in PDI values, as measured by SEC and chemical assays (Table 1). The large discrepancy between SEC and chemical assay values could be caused by the presence of

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Table 2. Average Molecular Weights (g · mol-1) and PDI of N3-PNIPAM Prepared by Reversible Addition-Fragmentation Chain Transfer (RAFT) Homopolymerization of N-Isopropylacrylamide with Azido-Functionalized Chain Transfer Agent after End-Group Reduction

A B C

Mn,theoretical

Mn,SECa

Mnb

Mwb

PDI

10 000 20 000 35 000

34 000 75 900 129 600

10 400 23 600 40 500

10 700 26 800 48 200

1.04 1.14 1.19

a Determined by SEC (grams per mole). b Calculated from MALDI-TOF and SEC analyses linear correlation (Mn ) 0.315, Mn,SEC ) 338.97, and Mw ) 0.243Mw,SEC + 364.97) (grams per mole).

very short chains that strongly affected the HA reducing end concentration, as measured by the N-acetyl-D-glucosamine reducing end assay. These data indicate that there may be cleavage of the polysaccharide chain into short oligosaccharide segments during ion exchange. An apparent increase in the HApa Mn was measured by SEC and chemical assays. In the former case, the modification of HA backbone strongly affected its elution time; therefore, the values of TBAHA and HApa could not be strictly compared. In the latter case, the final dialysis step (MWCO ) 12 to 14 kDa) used to remove EDC/NHS byproduct was probably the cause of carboxylic group and HA reducing group concentration changes. It is likely that the short oligosaccharide segments generated during ion exchange were lost during dialysis. The net result of these changes was that the HApa preparation did not strongly affect the hyaluronan average molecular weight. Three linear PNIPAM with theoretical Mn values of 10, 20, and 35 × 103 g · mol-1 and bearing an azide function on one end group were prepared by RAFT homopolymerization of NIPAM with the azido-CTA. Their experimental average molecular weights (Mn, Mw, and PDI) as measured by SEC and MALDI-TOF are reported in Table 2. Controlled molecular weight and low PDI were obtained for all azido functional N3PNIPAM synthesized by RAFT and end group reduced (Table 2). The removal of the CTA was qualitatively assessed by UV-vis spectroscopy (data not shown), as previously reported by others,25,30 and did not significantly influence the Mn and PDI values of the N3-PNIPAM. The DSC profiles of N3PNIPAM solutions also provided a good indication of the success of the end-group reduction with a temperature shift from 26 °C to around 29-30 °C for the peak maximum associated with the N3-PNIPAM conformational change, respectively, for N3-PNIPAM-CTA and N3-NIPAM end-reduced solutions.31 Several analytical techniques were employed to characterize the HA-PNIPAM macromolecular architectures prepared via “click” coupling. However, the complexity of the semisynthetic comb-grafted HA-PNIPAM architecture precluded the use of SEC; the copolymer had a molecular weight too high for MALDI-TOF, and chemical assays proved to be inadequate in the presence of PNIPAM and HA-PNIPAM. Nonetheless, the triazole proton peak (δ 7.88) was observed in all HA-PNIPAM 1 H NMR spectra, indicating the success of the CuAAC mediated grafting of PNIPAM to the HA backbone. Therefore, the combination of RAFT polymerization and “click” chemistry facilitated precise control of both graft length and density, which is essential for the fine-tuning of hydrogel behavior, both at room temperature and at 37 °C.10 The efficiency of the CuAAC reaction is anticipated to proceed at near 100% efficiency; therefore, the reported DS reflects input conditions. Reversibility of HA-PNIPAM Gelling. For regenerative medicine, expansion of autologous cells is required prior to delivery in the injured site. Therefore, it is of considerable

Figure 1. Reversibility of PNIPAM and HA-PNIPAM gelling: (A) Heating and cooling DSC thermograms of a 10.3% w/v HA-PNIPAM B30 solution (5.4 mM PNIPAM, 5.3 µM hyaluronan) and a 5.4 mM PNIPAM B30 solution in PBS buffer pH 7.4. (B) Enthalpy of a 10.3% w/v HA-PNIPAM B30 solution (5.4 mM PNIPAM, 5.3 µM hyaluronan) and a 5.4 mM PNIPAM B30 solution in PBS buffer pH 7.4 during six thermal cycles.

interest to produce a 3D matrix with reversible gelling for the encapsulation of cells. This property will allow retrieval and further manipulation of cells cultured in vitro.19 DSC is a valid technique to measure phase transition in a material during a heating or cooling scan and cycles can be easily repeated to determine the reversibility of a physical change (e.g., gelling). Hyaluronan did not show any physical change in the range of temperature studied. The grafting of PNIPAM onto the hyaluronan backbone did not significantly influence the peak maximum, as reported in Figure 1A for HAPNIPAM B30 and PNIPAM B30 solutions. All synthesized HAPNIPAM showed a coil-to-globule transition at 29 ( 1 °C, independent of their length and grafting density. This was in accordance with previous studies on HA-PNIPAM copolymers.19 Thermal cycles were performed on PNIPAM and HAPNIPAM solutions. In Figure 1B, the reversibility of the PNIPAM phase transition and its conservation up to six cycles is reported for HA-PNIPAM B30. In contrast, PNIPAM B30 enthalpy decreased over cycling. This suggests that without the HA, PNIPAM chains formed hydrophobic domains that required a greater amount of time to be disrupted. However, when they were grafted to the hydrophilic hyaluronan backbone, they changed conformation quickly.32 Therefore, all of the HA-PNIPAM solutions prepared conserved the reversibility of their thermoresponsiveness, making them suitable as an injectable carrier and matrix for in vitro expansion of cells in regenerative medicine. Viscosity of HA-PNIPAM Solutions at 25 °C. To suspend cells in the liquid matrix prior to gelling or to deliver the material via syringe to the implantation site the hydrogel should behave as a low viscosity fluid at room temperature. Therefore, at each synthesis step, the rheological behavior of HA, PNIPAM, and HA-PNIPAM was characterized. Both hyaluronan sodium salt solution and modified hyaluronan solution behaved as a non-Newtonian fluid and showed shear thinning upon frequency increase and high viscosity at low shear rate (Figure 2). HApa and NaHA had a similar n value of 0.83, indicating a shear thinning behavior. The modified hyaluronan solution behaved as a viscous fluid and showed a lower viscosity than NaHA up to 1000 Hz. (Calculated η0 values for the NaHA and HApa were 3.75 and 0.04 Pa · s, respectively.)

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Figure 2. Influence of propargylamide modification on the rheology of 0.8% w/v NaHA solutions: frequency dependence of hyaluronan sodium salt and propargylamide-modified hyaluronan moduli at 25 °C. Table 3. Viscosity (η*) of PNIPAM and HA-PNIPAM Solutions in PBS at PNIPAM Concentration of 4.1 (DS 25%) and 5.4 mM (DS 30%) at 25 °C PNIPAM η*(Pa · s), 25 °C A 25 A 30 B 25 B 30 C 25 C 30

2 Hz

3.1 5.5 8.3 10.0

HA-PNIPAM

20 Hz

2 Hz

20 Hz

0.3 0.6 2.6 2.5

23.5 30.0 4.6 13.9 10.8 16.2

3.5 4.8 1.4 2.6 2.3 2.5

To study the HA-PNIPAM gelling behavior across a wide range of temperature, another rheological setup allowing the determination of η*, G′, and G′′ was used. The η* measured at the lowest frequency is reported for PNIPAM and HA-PNIPAM solutions in Table 3. At 25 °C and low shear rate (f ) 2 Hz), the complex viscosity of PNIPAM solutions was directly proportional to PNIPAM chain length and concentration. HAPNIPAM solutions showed a complex viscosity of the same order of magnitude of PNIPAM solutions. The lowest viscosity was reached for HA-PNIPAM B25; the slight increase in viscosity measured with other compositions was due to increase in grafting density (HA-PNIPAM B30) or length (HA-PNIPAM C25 and C30). This could be explained by changes in polymer chain interactions or electrostatic interactions. A similar viscosity dependence on Mn was previously found for PNIPAM-grafted chitosan.33 At a higher shear rate (f ) 20 Hz), η* dropped for all solutions with significantly lower values for PNIPAM as compared with HA-PNIPAM solutions, except for the PNIPAM C25 and C30.2 The reasons for the high viscosity values of HAPNIPAM A25 and A30 still need to be elucidated. Therefore, at room temperature, all HA-PNIPAM solutions had an adequate viscosity for the suspension and injection of cells. Because HA-PNIPAM B25 showed the lowest viscosity, it represents a composition of choice for cell suspension preparation and injection. Gelling Kinetics of HA-PNIPAM Solutions. For a thermoresponsive solution to be suitable as an injectable cell carrier, gelling should occur below body temperature, and the gel should provide fast setting to allow good control over the localization of the hydrogel. Therefore, the kinetics of HA-PNIPAM gel formation and reversibility were assessed by UV-vis. Upon heating, PNIPAM solutions showed a similar rapid increase in turbidity for all PNIPAM molecular weights and

concentrations. However, upon cooling, an irregular decrease in absorbance was found, which indicated an inhomogeneous solubilization of PNIPAM gels. The absorbance value at the end of the experiment was higher than the initial absorbance (Figure 3A) for all PNIPAM molecular weights and concentrations. Therefore, whereas PNIPAM gelling was very quick (some seconds), its solubilization was much slower (>10 min). HA-PNIPAM C25 and C30 were too turbid at 4.1 and 5.4 mM PNIPAM concentration for a kinetics study by UV-vis. Upon heating, HA-PNIPAM A30 and B30 solutions showed a slower turbidity increase compared with that of the corresponding PNIPAM solutions (Figure 3 A). This could be related to the higher viscosity of HA-PNIPAM solutions. Upon cooling, the decrease in absorbance of HA-PNIPAM (∼2 min) was regular and faster than the corresponding PNIPAM (>10 min) (Figure 3 B). The initial absorbance of the solution was reached at the end of the experiment, indicating a more rapid and totally reversible solubilization of HA-PNIPAM solutions. The kinetics of gelling and solubilization were not significantly affected by the DS of HA-PNIPAM and were mainly governed by PNIPAM molecular weight (Figure 3C). The gelling of HA-PNIPAM solutions was also studied as a function of temperature (Figure 3D). HA-PNIPAM B30 and C30 solutions showed similar LCST (∼29 °C) and absorbance profiles. In the case of HA-PNIPAM A30, a steady and slow increase in absorbance starting from 26 to 40 °C was observed, and the LCST could not be determined. When the PNIPAM concentration of HA-PNIPAM A30 was reduced to 0.67 mM PNIPAM (which corresponds to 0.1% w/v hyaluronan), the LSCT still could not be determined. These findings are in accordance with the Ohya et al. study in which a similar dependence of the absorbance over the molecular weight of the PNIPAM graft was reported.19 Therefore, HA-PNIPAM solutions exhibited fully reversible gelling and solubilization, whereas PNIPAM alone gelled rapidly but solubilized slowly and only partially in a short time frame. HA-PNIPAM solutions seem more adequate for cell recovery after expansion and prior to delivery. Volume Changes upon Gelling. After cell expansion and injection in the defect, it is critical to assess the early swelling/ shrinkage of the gelled solutions because this would impact its utility as a cell carrier and tissue filler. It is well known that shrinkage of PNIPAM hydrogel is very high (>60%), but it can be modified by grafting to hydrophilic and charged polymers.34 The water retention values of the HA-PNIPAM gels reflect a strong swelling behavior of the HA-PNIPAM A25 and A30 (equal to half of their initial water content), which indicated a low degree of intermolecular aggregation among PNIPAM domains (Figure 4). HA-PNIPAM B25 and B30 did not show significant volume change. Interestingly, HA-PNIPAM C25 and C30 shrank to nearly half of their initial volume. The stronger shrinkage observed for HA-PNIPAM C25 as compared with that for HA-PNIPAM C30 could be due to a shielding effect of PNIPAM chains; therefore, some chains did not participate in the hydrogel formation. Because of the absence of volume changes upon gelling, HAPNIPAM B25 and B30 seem to be the best candidates for a regenerative therapy; in fact, the gelling process would be harmless to encapsulated cells, and a defect could be filled with appropriate spatial control. HA-PNIPAM of type A may be suitable for applications where the complete filling of a cavity is essential (e.g., nucleus pulposus repair). HA-PNIPAM of type C did not seem appropriate for a tissue engineering approach because its strong shrinkage would lead to inadequate filling of



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Figure 3. Influence of: (A) PNIPAM molecular weight on the gelling kinetics of PNIPAM and HA-PNIPAM, (B) PNIPAM molecular weight on the solubilization kinetics of PNIPAM and HA-PNIPAM, (C) grafting density on the gelling and solubilization kinetics of HA-PNIPAM, and (D) PNIPAM molecular weight on the absorbance of HA-PNIPAM as a function of temperature (O diluted 10 times).

Figure 4. Influence of PNIPAM molecular weight and grafting density on the water retention of HA-PNIPAM copolymers (at a hyaluronan concentration of 0.8% w/v) measured after 1 h in PBS at 37 °C.

the cavity and could be harmful for the cells encapsulated in the hydrogel. Mechanical Properties of Gel Network. Depending on the tissue to regenerate, different mechanical properties (e.g., stiffness) are demanded for the 3D matrix.35 Therefore, mechanical properties of PNIPAM and HA-PNIPAM aqueous solutions were characterized by dynamic viscoelasticity tests. In Figure 5, evolution of the storage modulus (G′) and loss modulus (G′′) as a function of the temperature and frequency are reported for PNIPAM B25 and C25 solutions. Because PNIPAM type A solutions showed inadequate gelling profiles and water retention, these hydrogels were not further characterized. Both PNIPAM B25 and C25 showed a sharp increase in moduli starting at 28 to 29 °C, and both reached similar values of G′ and G′′. However, differences between PNIPAM B25 and C25 were noticed in the evolution of moduli at higher

Figure 5. Influence of PNIPAM molecular weight on the rheology of PNIPAM solutions: G′ (solid dots) and G′′ (empty dots) of PNIPAM B25 and C25 at 4.1 mM PNIPAM in PBS solution as a function of the temperature and frequencies (9 and 0, 2 Hz; b and O 20 Hz; 4 and 2, 200 Hz; ] and [, 1000 Hz; filled left-pointing triangle and open left-pointing triangle, 2000 Hz). Represented data are the mean of four data points, and interpolation lines have been drawn for ease of interpretation.

temperatures. Whereas G′ and G′′ of PNIPAM B25 reached a maximum around 32 °C and then decreased with increasing temperatures, G′ and G′′ of PNIPAM C25 were almost constant at temperatures >30 °C (plateau). It is known that in dilute solutions PNIPAM undergo a coil-to-globule transition at around 30-32 °C.36 As the temperature increases, PNIPAM chains start to expel water and form hydrophobic domains. Depending on PNIPAM concentration and PNIPAM-solvent interactions, PNIPAM dispersions or continuous gels can be created. In semidilute solutions, intrachain foldings and interchain associations between PNIPAM chains occur at LCST. Depending on the intrachain foldings/interchain associations balance, transient or stable gels are formed.33 It is likely that PNIPAM B25 formed

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Mortisen et al.

Figure 6. Influence of PNIPAM molecular weight and grafting density on the rheology of HA-PNIPAM solutions: G′ and G′′ of HA-PNIPAM A25, A30, B25, B30, C25, and C30 as a function of the temperature and frequencies (9 and 0, 2 Hz; b and O, 20 Hz; 2 and 4, 200 Hz, [ and ], 1000 Hz; filled left-pointing triangle and open left-pointing triangle, 2000 Hz) (hyaluronan concentration is 0.8% w/v). Represented data are the mean of four data points, and interpolation lines have been drawn for ease of interpretation.

transient gels (viscosity increased at the LCST and then decreased upon heating via both aggregation/stabilization of PNIPAM hydrophobic domains and decrease in interactions with

the solvent). On the other hand, PNIPAM C25 solutions formed more stable gelled dispersions (almost constant G′ and G′′ at temperatures >30 °C), suggesting that PNIPAM interchain

Scheme 2. Schematic Representation of HA-PNIPAM Gel Formation for A, B, and C Compositions



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Table 4. PNIPAM and HA-PNIPAM Gel Point Values Measured by Rheological Tests, LCST Values Measured by UV-vis and Peak Maximum Values Measured by DSC rheology

PNIPAM HA-PNIPAM PNIPAM HA-PNIPAM PNIPAM HA-PNIPAM

a

(°C)

gel point

A A30 B B30 C C30

none none 30.1 30.6 29.9 31.0

UV-vis b

DSC peak maxc

LCST

29.2 30.2 30.6 29.5 30.5 29.8

30.5 30.2

Gel point is the temperature for which tan δ > 1 (f ) 2 Hz) during the heating ramp. b LCST corresponds to the temperature measured at half of the difference between the maximum and the minimum absorbance values. c Peak maximum measured during the first heating ramp. a

associations were more frequent for this composition. G′′ was higher than G′ over most of the frequency and temperature ranges, indicating that the PNIPAM solutions were still predominantly viscous liquids at the concentrations used. Plots of storage and loss moduli (G′ and G′′) of HA-PNIPAM solutions as a function of the temperature and frequency are reported in Figure 6. HA-PNIPAM A, B, and C showed very different rheological behaviors upon heating. In the range 24-40 °C, HA-PNIPAM A25 behaved as a viscous solution with an Arrhenius-like G′′ decrease with temperature. G′ decreased until 32 °C and then increased again because of the hydrophilic/ hydrophobic transition of the PNIPAM chains. However, PNIPAM length and density were not enough to form a gellike structure, and it is likely that mainly intrachain foldings were formed (Scheme 2A). Below LCST, HA-PNIPAM B25 had slightly lower moduli than HA-PNIPAM A25. The hydrogel formed had similar characteristics below and above 32 °C. This was in accord with the water uptake data (Scheme 2B and Figure 4), which showed no volume change upon gelling. In this case, it is likely that equilibrium between intrachain foldings and interchain associations was reached. HA-PNIPAM C25 behaved as a viscous solution in the 24-32 °C range and as a hydrogel at temperatures above 32 °C. Compared with HA-PNIPAM B25 these gels achieved much higher mechanical properties (e.g., G′ at 37 °C and 2 Hz is 0.14 kPa for HA-PNIPAM B25 and 16.1 kPa for HA-PNIPAM C25). It is likely that interchain associations between PNIPAM chains dominated (Scheme 2C) and higher entanglement density and better long-distance cohesion of the copolymer network occurred in the case of the HA-PNIPAM C25.33 All HA-PNIPAM solutions showed a frequency dependence of G′ and G′′. This was more evident for HA-PNIPAM C25. In some cases, as for HA-PNIPAM B25, the copolymer behaved as a solution (at 2 Hz) but as a gel at higher frequencies even below 32 °C. The presence of hyaluronan helped to stabilize the HA-PNIPAM hydrogels. In fact, whereas PNIPAM B25

Figure 7. Influence of PNIPAM molecular weight on the hTERT-BJ1 fibroblasts viability at 24 h (PNIPAM concentrations equal to 2.5, 5, 25, and 50 µM) as measured by Alamar Blue assay and normalized against positive control (cells with media only): viability median, quartile, maximum and minimum; *, p < 0.05 versus the two lowest PNIPAM concentrations.

formed a transient gel (Figure 5), HA-PNIPAM B25 hydrogels (Figure 6) were stable in the temperature range of 32-40 °C. In the range of grafting densities and PNIPAM molecular weights studied in this work, the effect of grafting density was less dramatic than chain length. HA-PNIPAM with a DS of 25 and 30% showed similar trends for the moduli profiles, although G′ and G′′ values were lower for HA-PNIPAM with DS ) 30% at high frequencies. This may be due to some crowding effect of the PNIPAM chains on the backbone of the hyaluronan, leading to a decrease in intermolecular associations because of steric hindrance or reduced mobility. Others have shown an increase in G′ with increasing PNIPAM grafting densities at 1 Hz.20 In our case, at lower frequencies, a higher G′ value is obtained for the lower DS, whereas the opposite is found at higher frequencies.20 It was observed that a critical value is required for the PNIPAM molecular weight to achieve gelling of the HAPNIPAM solution via aggregation of hydrophobic PNIPAM domains. When the molecular weight of PNIPAM was 80% in the whole range of PNIPAM lengths and concentration studied. Interestingly, viability was significantly higher at the two highest PNIPAM concentrations for all of the molecular weights tested without significant difference among the PNIPAM Mn values (Figure 7). This could be explained by differences in the amount of PNIPAM in contact with the cells: at 37 °C, it is likely that higher concentrations of PNIPAM formed aggregates of PNIPAM and precipitated out of solution, whereas lower concentrations of PNIPAM were unable to coalesce, thereby exposing a greater amount of PNIPAM to the cell surface. Another possible explanation is that aggregates of PNIPAM, because they are hydrophobic at 37 °C, adsorbed proteins from the media and facilitated interaction of these proteins with the cells.38 No significant differences were found between cell viability at 24 and 48 h. Therefore, it can be concluded that the RAFT polymerized N3-PNIPAM (10, 20, and 35 × 103 g · mol-1) were not cytotoxic to hTERT-BJ1 fibroblasts in the range of concentration and time frame of the study. This is in agreement with a previous study that reported no significant toxicity of high Mw PNIPAM on human carcinoma cells at 10 mg/mL (156 × 103 g · mol-1).39 After establishing the cell response of noncoupled PNIPAM, hTERT-BJ1 were exposed to NaHA, HA-PNIPAM A30, B30, and C30 and their degradation products at hyaluronan concentrations of 5, 10, 50, and 100 µg/mL. In Figure 8, cell viability at 24 h at the highest polymer concentration evaluated (100 µg HA/mL) is reported as a function of HAse concentration. From Figure 8, it is clear that increased cell numbers were associated with a decrease in the molecular weight of hyaluronan and HAPNIPAM. This trend was most apparent in the highest hyaluronan concentration evaluated (100 µg/mL). The same trend was observed at 48 h. This is in accord with previous results, which showed increased cell numbers with low-molecularweight hyaluronan.12 For HA-PNIPAM and their degradation products, the viability of hTERT-BJ1 stayed high (>90% under all performed conditions). It seems that the longer the PNIPAM chain, the better the cell response to the degradation products. This in accord with cell viability data on PNIPAM solutions and can be related to the lower exposed PNIPAM surface at 37 °C with longer chains. However, a significant increase in

Figure 9. Fluorescence image of 1% w/v solutions of (A) NaHA loaded with azido fluorescein, (B) HA-PNIPAM 29 × 103 g · mol-1 grafted with fluorescein, and (C) HA-PNIPAM 29 × 103 g · mol-1 control (without fluorescein) with an excitation of 488 nm and identical exposure time.


viability with the increase in HAse concentration was only observed for HA-PNIPAM C30 in the presence of 1000 HAse U/mL. Overall, it can be concluded that all synthesized HA-PNIPAM and their degradation products were cytocompatible to hTERTBJ1 at the concentrations evaluated in this study; therefore, the synthesized hydrogels could be used for cell encapsulation (Supporting Information). Furthermore, the biocompatibility of PNIPAM-based hydrogels has already been assessed in different animal models and tissues, and no strong inflammation or toxicity were reported.40 Although PNIPAM chains at 37 °C in biological fluid are unlikely to be significantly degraded in vivo, short PNIPAM chains below 10 × 103 g · mol-1 can undergo renal excretion.16 Therefore, in vivo the PNIPAM grafted hyaluronan hydrogels can lose their integrity because of the hyaluronan backbone degradation, after which the PNIPAM chains could diffuse and be excreted. Gels at higher molecular weight may be suitable for in vitro work; however if implanted in the body, renal clearance of the degradation products may become problematic because reduction of molecular weight will occur only on the HA backbone and PNIPAM cannot be broken down by the body. Whereas this study outlined the molecular weight requirements for these gels, future studies will focus on producing PNIPAM chains with hydrolytically degradable linkers to generate degradation products of appropriate molecular weight and solubility in the bloodstream.41 Bifunctional Hydrogel: HA-PNIPAM Grafted Fluorescein. The ability to perform multiple grafting on the polysaccharide backbone is one added feature of the CuAAC preparation of HA-PNIPAM over other synthesis methods.4 The simultaneous grafting of N3-PNIPAM and N3-fluorescein onto the HApa using CuAAC was performed to demonstrate the potential of the proposed method to create a multitude of other “active” macromolecular structures. As a proof of concept, a bifunctional hyaluronan was synthesized. On the basis of reactant weights, the FL and PNIPAM chains would substitute 10 and 8% of the disaccharide subunits, respectively. The PNIPAM-grafted HA without FL did not show autofluorescence (Figure 9A). When FL was added to a NaHA solution, punctate areas of fluorescence were observed as the result of the water insoluble azido fluorescein forming a precipitate in the native NaHA/water solution (Figure 9B). In contrast, covalent “click” binding of FL to the HA-PNIPAM backbone allowed solubilization of the FL, and a homogeneous distribution of the FL in the solution was observed (Figure 9C). These data demonstrate the feasibility of simultaneously grafting N3-PNIPAM chains and an azido-functionalized small molecule to the backbone of HA using CuAAC. The so-obtained FL-grafted thermoresponsive copolymer may be used for imaging purposes by itself or in the presence of other bioactive agents coupled to any of the additional active sites on the HA. Future studies will use this versatility to create multifunctional gels by grafting various bioactive molecules to the hyaluronan backbone.

Conclusions This work presents a semisynthetic, thermoreversible hydrogel prepared by grafting PNIPAM to HA using the CuAAC reaction. The efficiency of the “click” reaction facilitates the control of the DS of PNIPAM chains. RAFT polymerization allows the preparation of PNIPAM of controlled molecular weight and low PDI. This control of the critical parameters of PNIPAM


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molecular weight and grafting density allowed the gel to be optimized for regenerative medicine applications. A composition with properties ideal for cell encapsulation was identified as HA-PNIPAM 20 × 103 g · mol-1 (DS ) 25%) and characterized by a low viscosity at 20 °C, rapid gelling at 37 °C, absence of volume change upon gelling, and G′ of 140 Pa at 37 °C. Furthermore, coupling of FL as a model drug was presented as a proof of concept. The versatility of the CuAAC chemistry will be used in future studies to explore the effect of grafting bioactive molecules onto the HA backbone. Acknowledgment. We would like to thank Prof. M. Textor and Mrs. D. Sutter from ETH Zurich (Switzerland) for performing NMR analysis. We would also like to acknowledge the help of Dr. L. Bigler from ETH Zurich with the MALDI-TOF analysis, Dr. A. Ritter from the EMPA Du¨bendorf (Switzerland) with the ICP-EOS analysis and Prof. W. Pecchold from Idm, Ulm (Germany) for the rheology measurements, Dr. Scott Curtin for useful discussions on chemistry, and Mr. Markus Glarner for assistance with SEC. Supporting Information Available. Transmitted light image of encapsulated cells in HA-PNIPAM for 96 h and EosinHaematoxylin staining of a thin section of encapsulated cells in HA-PNIPAM for 96 h showing the homogeneous distribution of cells in the hydrogel. This material is available free of charge via the Internet at http://pubs.acs.org.

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