Biodegradable Hyperbranched Amphiphilic Polyurethane Multiblock

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Biodegradable Hyperbranched Amphiphilic Polyurethane Multiblock Copolymers Consisting of Poly(propylene glycol), Poly(ethylene glycol), and Polycaprolactone as in Situ Thermogels Zibiao Li,† Zhongxing Zhang,‡ Kerh Li Liu,‡ Xiping Ni,‡ and Jun Li*,†,‡ †

Department of Bioengineering, Faculty of Engineering, National University of Singapore, 7 Engineering Drive 1, Singapore 117574, Singapore ‡ Institute of Materials Research and Engineering, A*STAR (Agency for Science, Technology and Research), 3 Research Link, Singapore 117602, Singapore ABSTRACT: This paper reports the synthesis and characterization of new hyperbranched amphiphilic polyurethane multiblock copolymers consisting of poly(propylene glycol) (PPG), poly(ethylene glycol) (PEG), and polycaprolactone (PCL) segments as in situ thermogels. The hyperbranched poly(PPG/ PEG/PCL urethane)s, termed as HBPEC copolymers, were synthesized from PPG-diol, PEG-diol, and PCL-triol by using 1,6-hexamethylene diisocyanate (HMDI) as a coupling agent. The compositions and structures of HBPEC copolymers were determined by GPC and 1H NMR spectroscopy. We carried out comparative studies of the new hyperbranched copolymers with their linear counterparts, the linear poly(PPG/PEG/PCL urethane) (LPEC) copolymer and Pluronic F127 PEG-PPG-PEG block copolymer, in terms of their self-assembly and aggregation behaviors and thermoresponsive properties. HBPEC copolymers were found to show thermoresponsive micelle formation and aggregation behaviors. Particularly, the lower critical solution temperature (LCST) of the copolymers was significantly affected by the copolymer architecture. HBPEC copolymers showed much lower LCST than LPEC, the linear counterpart. Our studies revealed that the effect of hyperbranch architecture was more prominent in the gelation of the copolymers. The aqueous solutions of HBPEC copolymers exhibited thermogelling behaviors at critical gelation concentrations (CGCs) ranging from 4.3 to 7.4 wt %. These values are much lower than those reported on other PCL-contained linear thermogelling copolymers and Pluronic F127 copolymer. In addition, the CGC of HBPEC copolymers is much lower than the control LPEC copolymer. More interestingly, at high temperatures, while LPEC and other linear thermogelling copolymers formed turbid sol, HBPEC formed a dehydrated gel. Our data suggest that these phenomena are caused by the hyperbranched structure of HBPEC copolymers, which could increase the interaction of copolymer branches and enhance the chain association through synergetic hydrogen bonding effect. The thermogelling behavior of HBPEC block copolymers was further evidenced by the 1H NMR molecular dynamic study and rheological study, which further support the above hypothesis. The hydrolytic degradation study showed that the HBPEC copolymer hydrogels are biodegradable under physiological conditions. Together with the good cell biocompatibility demonstrated by the cytotoxicity study, the new thermogelling copolymers reported in this paper could potentially be used as in situ-forming hydrogels for biomedical applications.

1. INTRODUCTION Hydrogels formed by chemical cross-links or physical junctions are a special class of polymers that provide unique swelling behavior and three-dimensional structure.1 Because of its similarity to the in vivo environment, hydrogels with respect to pharmaceutical delivery and biomedical uses have been an attractive topic of extensive research in the past decades.2−4 More specifically, in situ-forming hydrogels, which undergo reversible phase transition in response to the changes in environmental conditions such as temperature and pH, have recently attracted much attention. In contrast to the permanent chemically cross-linked hydrogel networks, in situ-forming hydrogels are injectable fluids before administration but immediately turn into standing hydrogels within the desired tissue, organ, or body cavity.5 This unique characteristic of the © XXXX American Chemical Society

in situ-forming hydrogels exempts the surgical procedure for placement, and various therapeutic formulations can be incorporated by simple mixing.6 Block copolymer hydrogels that exhibit a sol−gel phase transition in response to external stimuli provide a simple and safe method of preparing in situ-forming gels. In situ-forming hydrogels formed by thermoresponsive block copolymer aqueous solutions are called temperature-sensitive hydrogels.6,7 Sensitivity to thermal environment is useful as temperature is the sole stimulus for the gelation with no other requirements for chemical or environmental treatment. Such gels are easy to Received: August 8, 2012 Revised: October 31, 2012

A

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Table 1. Molecular Characteristics and Properties of HBPEC Block Copolymersa and Control Copolymersb copolymers composition in molar ratioc

copolymer characteristics

samples

PCL

PEG

PPG

PCL (wt %)c

HBPEC1 HBPEC2 HBPEC3 LPEC F127

0.2 1.4 1.8 1.0 0.0

2.1 3.2 2.7 2.8 2.0

1.0 1.0 1.0 1.0 1.0

1.1 4.6 6.7 6.2 0.0

Mn (× 103)d

PDId

CMCe (10−2 mg/mL)

LCST (°C)f

CGC (wt%)g

15.2 13.2 12.1 11.7 12.6

1.6 1.8 1.9 1.4 1.2

33.8 31.0 28.2 30.1 700.0h

50.3 46.4 51.5 59.7 -

7.4 6.5 4.3 9.1 15.3

a

Hyperbranched poly(PPG/PEG/PCL urethane) block copolymers are denoted HBPEC, where HB represents hyperbranched, P is for PPG, E is for PEG, and C is for PCL. bLinear poly(PPG/PEG/PCL urethane) block copolymer is denoted LPEC. cCalculated from NMR results. d Determined by GPC. eCritical micellization concentration (CMC) in water determined by the dye solubilization technique at 25 °C. fDetermined from turbidimetry measurements. gDetermined by gel inversion method. hData referred from reported reference.37

and oligoester segments are arranged within a junction linkage of linear architecture. Previously, we also demonstrated the water swelling property of linear PPG/PEG/PCL multiblock copolymers based films.23 However, aqueous self-assembly property of PPG/PEG/PCL block copolymer with a nonlinear architecture is still not understood. As mentioned above, only PCL-F87-PCL and PPG/PEG/PCL copolymers coupled by diacrylchlorides in alternative/linear structure were reported and both of them showed relatively high CGC values.17,20 In this paper, we designed a series of hyperbranched PPG/ PEG/PCL multiblock copolymers by using polyurethane technique as opposed to their linear counterparts, linear PPG/PEG/PCL block copolymer and Pluronic F127 at similar molecular weight ranges. The hyperbranched structure would be expected to give an enhanced hydrogen bonding effect from increased urethane linkages and intensified hydrophobic interactions among polymer branches. Self-assembled aggregations in response to different temperatures were studied. The thermodynamic micellization and gelation behaviors showed great differences between the linear and hyperbranched block copolymers with similar polymer compositions. This study would provide a profound insight into the structure−property relationship of PPG, PEG, and PCL based thermogelling block copolymers and offer an alternative for biodegradable in situforming hydrogels with low CGCs.

control, and can be thus produced upon injection to the body when temperature is increased from ambient to physiological.8 Triblock copolymers of poly(ethylene glycol)-b-poly(propylene glycol)-b-poly(ethylene glycol) (PEG-PPG-PEG), commercially known as Pluronic or Poloxamer, have surface-active properties and are widely used in pharmaceutical systems.9,10 Some concentrated Pluronic aqueous solutions exist as a sol state at room temperature but form a gel at the physiological temperature. Therefore, Pluronic copolymers are widely used as injectable in situ-forming hydrogels in drug and gene delivery, inhibition of tissue adhesion and burn wound covering.8,11 However, Pluronic hydrogels generally have a high critical gelation concentration (CGC) (15−20 wt % or above), exhibiting inadequate mechanical integrity as well as short residence time with quick dissolution. These drawbacks have led to many limitations of their biomedical applications.6,12 More importantly, Pluronic hydrogels are nonbiodegradable and an accumulation of this copolymer has been demonstrated to induce hyperlipidemia in vivo.13 Three main strategies have been made to overcome the above-mentioned drawbacks: (i) introduction of end groups for in situ chemical cross-linking after thermal gelation, such as carbon−carbon double bonds,14 ethoxysilane groups15 and methacrylate groups;16 (ii) synthesis of PPG and PEG block copolymers using diacryl chloride as a coupling agent;17 and (iii) synthesis of PEG/PPG copolymers with incorporation of oligoester segments. The strategies (i) and (ii) have made improvement of Pluronic hydrogels in stability and mechanical strength and also show lower CGCs than unmodified ones. However, these polymers are nonbiodegradable, and the excretion from the body could be difficult. The approach (iii) could make copolymers that exhibit lower CGCs than PEGPPG-PEG triblock copolymers and, more importantly, yielded biodegradable hydrogels due to hydrolytic cleavage of the ester bonds.8 For example, Cohn et al. have synthesized reverse thermogelling multiblock copolymers based on PEG, PPG, and poly(caprolactone) (PCL).17 These biodegradable copolymers exhibited CGCs of 10 wt %. Pentablock block copolymers composed of Pluronic F87 flanked by two short polyester, that is, poly(D,L-lactide) (PLA) or PCL, showed enhanced rheological properties as compared with Pluronic F87 hydrogels.18−20 Recently, we have reported copolymers containing PEG, PPG, and poly(3-hydroxybutyrate) (PHB) with a very low CGC of 2 wt %.21 Sustained release of proteins of up to 80 days was demonstrated with this gel system.22 All these previously reported modified copolymers via the third technique have a linear structure, in which the PPG, PEG,

2. EXPERIMENTAL SECTION 2.1. Materials. Polycaprolactone triol (PCL-triol, Mn= 300), poly(ethylene glycol) (PEG, Mn = 2000), and poly(propylene glycol) (PPG, Mn = 2000) were purchased from Aldrich. They were vacuumdried at 75 °C overnight before use. Dibutyltindilaurate (95%), 1,6hexamethylene diisocyanate (HMDI) (98%), diethyl ether, methanol, 1,2-dichloroethane (99.8%), 1,6-diphenyl-1,3,5-hexatriene (DPH), and 2-propanol were also purchased from Aldrich. 1,2-Dichloroethane was distilled over CaH2 before use. Pluronic F127 with a chain composition of EG100PG65EG100 was purchased from Aldrich and used as received. 2.2. Synthesis of Hyperbranched Poly(PPG/PEG/PCL urethane) (HBPEC) Block Copolymers. Hyperbranched poly(PPG/ PEG/PCL urethane) block copolymers derived from PPG, PEG, and PCL-triol are denoted as HBPEC copolymers, where HB represents for hyperbranched, P for PPG, E represents PEG, and C for PCL. The linear counterpart of HBPEC is denoted as LPEC, where L represents linear. HBPEC block copolymers were synthesized with molar ratios of PEG/PPG fixed at 2:1 and PCL content ranging from 10 to 30 mol %. HMDI was used as a coupling reagent and the amount added was equivalent to the hydroxyl groups in the solution.21,23 Typically, 2.0 g of PPG (Mn = 2000, 1.0 × 10−3 mol), 4.0 g of PEG (Mn = 2000, 2.0 × 10−3 mol), and 0.54 g of PCL-triol (Mn = 300, 1.8 × 10−3 mol) were dried in a 250-mL three-neck flask at 75 °C under high vacuum B

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2.8. Sol−Gel Transition. Sol−gel transition was determined by a test tube inverting method with temperature increments of 2 °C per step.21,24 Each sample of a given concentration was prepared by dissolving the polymer in distilled water in a 1.5-mL vial. After equilibration at 4 °C for 24 h, the sample-containing vial was immersed in a water bath at a constant designated temperature for 15 min. The gelation temperature was characterized by the formation of a firm gel that remained intact when the tube was inverted by 180°. The critical gelation concentration (CGC) is defined as the minimum copolymer concentration in aqueous solution at which the gelation behavior could be observed. 2.9. Rheological Studies. Steady and dynamic rheological experiments were performed on an AR-G2 stress-controlled rheometer (TA Instruments, Newark, DE). The sol to gel transition of the polymer aqueous solution was investigated by dynamic rheometry. The aqueous polymer solution was placed between parallel plates of 25 mm diameter and a gap of 0.5 mm. The data were collected under a controlled stress (4.0 dyn/cm2) and a frequency of 1.0 rad/s. The heating rate was 0.5 °C/min. Critical gelation temperature (CGT) was determined at G′/G″ crossover point in temperature sweep. The storage modulus (G′) and loss modulus (G″) were obtained from the linear viscoelastic regime of dynamic stress sweep spectra recorded on each sample at 37 °C while the yield stress (τ) was defined as the applied shear stress value at G′/G″ crossover.25 2.10. Hydrolytic Degradation of HBPEC Hydrogels. HBPEC copolymers at predetermined concentrations were mixed with phosphate buffer solution in a test tube and left to equilibrate overnight at 4 °C to dissolve the polymers completely. The buffer solution had a pH of 7.4, and contained 8.0 g of NaCl, 0.2 g of KCl, 1.44 g of Na2HPO4, and 0.24 g of K2H2PO4 in 1 L of solution. In a typical preparation, 1 mL of HBPEC3 copolymer solution at concentration of 5.7 wt % was injected into a 3.5 mL sample vial and left to equilibrate at 37 °C to form a hydrogel. The hydrogel had dimensions of 1.5 cm × 0.8 cm (diameter × height). Each hydrogel sample was placed into a 37 °C oven, which was shaken at 50 rpm continuously for hydrolytic degradation study. Samples (200 μL) were taken out at predetermined time intervals and freeze-dried before performing further measurements. The molecular weight and molecular structure changes of the degraded copolymers were analyzed according to the previous methods we reported.22 Molecular weight changes were monitored by using GPC described as above. FTIR spectra recorded on Bio-Rad 165 FT-IR spectrophotometer were used to investigate molecular structure changes of the degradation products. Pressed pellets prepared by grinding the samples with KBr at 1:100 ratio were used in the measurements. Thirty-two scans were signal-averaged with a resolution of 2 cm−1 at room temperature. LPEC hydrogel at concentration of 10.7% was used as a control in the degradation study. 2.11. Cells and Media. L929 mouse fibroblasts were obtained from ATCC and cultivated in DMEM containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. Cells were grown as a monolayer and passaged upon confluence using trypsin (0.5% w/v in PBS). L929 cells were harvested from culture by incubating in trypsin solution for 5 min. The cells were centrifuged and the supernatant was discarded. Three milliliters of serum-supplemented DMEM was added to neutralize any residual trypsin. The cells were resuspended in serum-supplemented DMEM at a concentration of 2 × 104 cells mL−1. Cells were cultivated at 37 °C and 5% CO2. 2.12. Cell Viability Assay. To evaluate the biocompatibility of the HBPEC copolymers when using under biomedical conditions, in vitro cytotoxicity test was carried out using 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyl tetrazolium bromide (MTT) assay in L929 cell lines. Cells were seeded in a 96-well microtiter plate (Nunc, Wiesbaden, Germany) at densities of 6 × 104 cells/well, and cultured in complete DMEM supplemented with 10% FBS at 37 °C, 5% CO2. After 24 h, culture media were replaced with serum-supplemented culture media containing known concentrations of the polymers, and the cells were incubated for a further 48 h. Then, 10 μL of sterile-filtered MTT stock solution in PBS (5 mg/mL) was added to each well, reaching a final MTT concentration of 0.5 mg/mL. After 5 h, unreacted dye was

overnight. Then, 60 mL of anhydrous 1,2-dichloroethane was added to the flask, and any trace of water in the system was removed through azeotropic distillation with about 45 mL of 1,2-dichloroethane being left in the flask. When the flask was cooled down to 75 °C, 1.13 g of HMDI (6.7 × 10−3 mol) and two drops of dibutyltindilaurate (∼8 × 10−3 g) were added sequentially. The reaction mixture was stirred at 75 °C under a nitrogen atmosphere for 12 h. At the end of the reaction, 1 mL of 2-propanol was added and the system was allowed to age for another 2 h to prevent the allophanate reaction. The resultant polymers were precipitated from diethyl ether and further purified by redissolving in 1,2-dichloroethane followed by precipitation in a mixture of methanol and diethyl ether (5/95, v/v) to remove remaining dibutyltindilaurate. A series of products were prepared through this method. The linear counterpart LPEC copolymer was prepared according to our previously reported protocol,23 starting from PCL-diol (Mn = 530), PPG (Mn = 2000), and PEG (Mn = 2000). Polymer compositions, molecular weight and distribution are listed in Table 1. 2.3. Molecular Characterization. 1H NMR and 13C NMR spectra were recorded on a Bruker AV-400 NMR spectrometer at room temperature. Chemical shifts at 7.3 and 4.7 ppm were referred to the solvent peaks of CHCl3 and H2O, respectively. Gel permeation chromatography (GPC) analysis was carried out with a Shimadzu SCL-10A and LC-8A system equipped with a Shimadzu RID-10A refractive index detector. THF was used as the eluent at a flow rate of 0.30 mL/min at 40 °C. Monodispersed PEG standards were used to obtain a calibration curve. Copolymer compositions in molar ratios were evaluated by integrating the proton chemical shifts of −CH2− in PCL unit at 4.1 ppm, the area of −OCH− and −OCH2− in PPO units from 3.25 to 3.71 ppm, and −OCH2− in PEG unit at 3.6 ppm. Results were shown in Table 1. 2.4. Critical Micellization Concentration (CMC) Determination by UV Spectroscopy. CMC values were determined by using the dye solubilization method.21 The hydrophobic dye 1,6-diphenyl1,3,5-hexatriene (DPH) was dissolved in methanol with a concentration of 0.6 mM. Ten microliters of this solution was mixed with 1.0 mL of copolymer aqueous solution with concentrations ranging from 2.5 × 10−3 to 5.0 mg/mL and equilibrated overnight at 4 °C. A UV− Vis spectrophotometer was used to obtain the UV−vis spectra in the range of 330−430 nm at 25, 30, 35, and 40 °C for thermodynamic micellization study. The CMC value was determined by the plot of the difference in absorbance at 378 nm and at 400 nm (A378 − A400) versus logarithmic concentration. 2.5. Particle Size Measurements. Particle size and size distribution were determined by dynamic light scattering (DLS) using a Zetasizer Nano ZS (Malvern Instruments, Southborough, MA) with a laser light wavelength of 633 nm at a 173° scattering angle. Polymer solutions (600 mg/L) were passed through a 0.45 μm poresized syringe filter before measurements. Each analysis was done in triplicate at predetermined temperatures (25 or 60 °C) and the Zaverage hydrodynamic diameters of the particles were showed. For the reversible transition test, polymer aqueous solutions were equilibrated at the two above-mentioned temperatures for 30 min between each measurement run. Five parallel analyses were carried out and the average value was reported. 2.6. Transmission Electron Microscopy. Samples were imaged on a high-resolution transmission electron microscope (Philips CM300 FEGTEM) (TEM) operated at accelerating voltage of 300 kV. Samples were prepared by directly depositing one drop of the copolymer solution (0.6 mg/mL) containing 0.01 wt % phosphotungstic acid (PTA) onto 200 mesh copper grids, which were coated in advance with supportive Formvar films and carbon (Agar Scientific). The samples were kept for 24 h at 25 or 60 °C before TEM imaging. 2.7. Lower Critical Solution Temperature Determination. Lower critical solution temperature (LCST) was measured with a UV−vis spectrophotometer. It was estimated by recording the temperature point which exhibited a 50% reduction in optical transmittance at 500 nm (1 cm path length). Aqueous copolymer solution at concentration of 1 mg/mL was heated at 1 °C/min in all measurements. C

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Scheme 1. Synthesis Route of Hyperbranched Poly(PPG/PEG/PCL urethane) (HBPEC) Block Copolymers

removed by aspiration. The formazan crystals were dissolved in DMSO (100 μL/well), and the absorbance was measured using a microplate reader (Spectra Plus, TECAN) at a wavelength of 570 nm. The wells containing only the cells and the culture medium served as controls. The relative cell viability (%) related to control cells was calculated with [A]test/[A]control × 100%, where [A]test is the absorbance of the wells with polymers and [A]control is the absorbance of the control wells.26 All experiments were conducted with six repetitions and averaged.

would lead to a continuous increase in molecular weight and result in a cross-linked product. Addition of high-boiling-point 2-propanol at the end of the reaction was used to consume any unreacted −NCO groups. As such, the allophanate reaction, occurring between the −NCO and the urethane group can be eliminated.27,28 By varying PCL content from 10 to 30 mol %, a series of HBPEC block copolymers were synthesized, and its linear counterpart, linear poly(PPG/PEG/PCL urethane)(LPEC) copolymer with similar PCL weight content to HBPEC3, was prepared for comparative study. As shown in Table 1, PEG demonstrated higher hydroxyl group reactivity than PPG since the molar ratios (PEG/PPG) determined from NMR spectra showed higher values than the feed ratio of 2:1. This may have resulted from the steric hindrance of methyl group located in PPG segments. The targeted molecular weight similar to Pluronic F127 (∼12 600) was achieved at the predetermined time of polymerization. However, it should be noticed that the resultant HBPEC block copolymer showed broader polymer distribution than LPEC due to the hyperbranched structure in nature. The chemical structure of HBPEC block copolymers was verified by 1H NMR spectroscopy. Figure 1A shows the 1H NMR spectrum of HBPEC3 in CDCl3, in which all proton signals belonging to PPG, PEG, and PCL portions are confirmed. The signals at 1.1 ppm are assigned to the methyl protons of PPG, signals corresponding to methylene protons in repeated units of PEG segments are observed at 3.6 ppm, and the signals at 4.1 ppm are associated with methylene protons alpha to the ester group of PCL segments.23 The new peaks found at 4.2 ppm are attributed to the urethane linkage generated from the reaction between hydroxyl groups and isocyanate groups in HMDI (−OH + −NCO → −NHCOO−). For 13C NMR in Figure 1B, the signals corresponding to the methylene carbon alpha to the ester group of PCL segment are observed at 64.5 ppm. The signals at 17.7 ppm are assigned to the methyl carbon of PPG. And the

3. RESULTS AND DISCUSSION 3.1. Synthesis and Characterization of HBPEC Block Copolymers. Previously, we reported the synthesis and water swelling behavior of linear amphiphilic multiblock poly(ester ether urethanes) consisting of PPG, PEG and PCL blocks.23 Each segment terminated with hydroxyl group was linked with HMDI as a chain extender to form a linear structure copolymer. The obtained polymers can be cast into films with good mechanical property. However, in the current study, hyperbranched poly(PPG/PEG/PCL urethane) (HBPEC) block copolymers comprising these components were designed and synthesized. PCL-triol was used as a multifunctional branching agent and HMDI as a coupling reagent. The reaction of −OH of PPG, PEG, and PCL-triol with −NCO of HMDI in the presence of dibutyltindilaurate led to hyperbranched architecture of the final copolymers. The synthesis route is presented in Scheme 1. During the synthesis, a lower concentration of PPG, PEG, and PCL-triol prepolymers (∼15 wt %) in 1,2dichloroethane was used in comparison with previous study (∼25 wt %), since the viscosity of the polymer solution increased significantly as polymerization proceeded and it would lead to a cross-linked product at high concentration. Furthermore, molecular weight of HBPEC copolymers were carefully controlled by adjusting the polymerization time and monitored by GPC. Generally, it would achieve a similar molecular weight range with Pluronic F127 within 12 h, as listed in Table 1. Further extension in polymerization time D

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Figure 2. 1H NMR spectra of HBPEC3 at concentration of 1.0 mg/ mL in CDCl3 and D2O at 25 °C.

HBPEC3. The CMC determination was carried out for these copolymers using the dye solubilization method at 25 °C. This experiment was carried out by varying the aqueous polymer concentration in the range of 2.5 × 10−3 to 5.0 mg/mL, while keeping the concentration of DPH constant. DPH shows a higher absorption coefficient in a hydrophobic environment than in water. Thus, with increasing polymer concentration, the absorbances at 344, 358, and 378 nm increased (Figure 3A). The point where the absorbance suddenly increases corresponds to the concentration at which micelles are formed. When the micelle is formed, DPH partitions preferentially into the hydrophobic core formed in the aqueous solution.29,30 The CMC was determined by extrapolating the absorbance at 378 nm minus the absorbance at 400 nm (A378 − A400) versus logarithmic concentration (Figure 3B). The CMC values are tabulated in Table 1. At similar PPG/PEG molar ratios in all studied copolymers, the incorporation of hydrophobic PCL made a remarkable CMC decrease to around 30 × 10−2 mg/mL as compared with 7.0 mg/mL for Pluronic. This may have resulted from the enhanced driving force for self-assembly by the chain entanglement and enhanced hydrophobic−hydrophobic interaction of HBPEC copolymers.20 However, within the studied copolymers, CMC values show only a slight decrease with increasing of the incorporated PCL content, regardless of the polymer architecture. Probably at the very low polymer concentrations, the hyperbranched architecture makes limited difference in the micelle formation of the copolymers. 3.3. Thermoresponsive Behavior of Micelles. The HBPEC block copolymer were water-soluble and formed micelles with a hydrophobic PCL core and hydrophilic PEG corona, between which was interlaced by transitional PPG in hydrophilicity and hydrophobicity, depending on the temperature. When the temperature of the solution was increased, the hydrophobicity of PPG increased and PPG chains packed more tightly into the micelle core. The increased hydrophobicity of the micelles may lead to the formation of larger micellar particles or aggregates of micelles. We demonstrated the thermosensitivity of the HBPEC block copolymer micelles by observing the change in the optical absorbance of a micellar solution as a function of temperature. PPG exhibits a molecular weight and structure-dependent phase transition temperature in the range of 14−100 °C.31,32 This temperature is known as the lower critical solution temperature (LCST). The LCST values

Figure 1. 1H NMR (A) and 13C NMR (B) spectrum of HBPEC3 in CDCl3.

signals at 70.9 ppm are associated with the methylene carbon in the repeated units of PEG segments. The signals ascribed to the urethane linkages are also found in a well-split state, as labeled in Figure 1B. This observation, together with the concomitant increase in the molecular weight of the copolymers, indicates that the polymerization was successful. 3.2. Core−Shell Micelle Formation. NMR spectroscopy was used to investigate the solvent effect on the micelle structure.26,29 CDCl3 is a good nonselective solvent for PPG, PEG, and PCL, while D2O is a good selective solvent for PEG at 25 °C but poor for PCL. In CDCl3, the peaks due to PPG, PEG, and PCL segments were sharp and well-defined (Figure 2). In D2O at 25 °C, PEG is shown as sharp peak while the PCL peaks disappear, and the peaks ascribed to PPG broaden in shape. These results show that the molecular motion of PCL is slow in water, indicating a hydrophobic core structure made up of PCL with the hydrophilic PEG as the out corona structure at 25 °C, which was interlaced by transitional PPG in hydrophilicity and hydrophobicity, depending on the temperature. This study confirmed the core−corona structure of the micelle. The three HBPEC copolymers were soluble in water. So was the LPEC copolymer with similar PCL weight content to E

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Figure 3. (A) UV−vis spectra changes of DPH with increasing HBPEC3 copolymer concentration in water at 25 °C. DPH concentration was fixed at 6 μM, and the polymer concentration varied between 2.5 × 10−3 and 5 mg/mL. The increase in the absorbance band at 378 nm indicates the formation of a hydrophobic environment in water. (B) CMC determination by extrapolation of the difference in absorbance at 378 and 400 nm.

PNIPAAm behaves more freely in PNIPAAm-PCL-PNIPAAm triblock copolymer solution, so the copolymer does not make significant change in LCST.33 Interestingly, HBPEC3 micelle solution showed lagging effect in thermosensitivity at 1 °C/min temperature increasing rate. This may arise from the slow motion of PPG chains within the hyperbranched copolymer structure and this effect is proportional to the branch unit (PCL-triol) content.34 The morphology and size distributions of the HBPEC copolymer micelles in response to temperature stimulus were investigated by TEM analysis and DLS (Figure 5). The results of the DLS measurements are summarized in Table 2. From TEM micrographs in Figure 5, spherical micelles morphology were observed at 25 °C and the estimated diameters were in agreement with DLS results. These diameters of the HBPEC copolymers micelles at 0.6 mg/mL were found at around 50 nm, which is larger than 29 nm formed from the LPEC copolymer solution. However, it should be noted that PPG at this temperature behaves more favorably in hydrophilicity and the incorporated PCL provides the main driving force for selfassembly. At elevated temperature of 60 °C, the PPG units become dehydrated and collapse with each other, leading to formation of larger compact micelles with PEG as corona. This process was also confirmed by TEM and DLS (Figure 5). The diameters of the micelles at 60 °C range from 140 to 250 nm, significantly larger than those at 25 °C. After PPG dehydration at 60 °C, the driving force for self-assembly is highly enhanced, which makes the micelle aggregates pack more densely due to the increase in polymer hydrophobicity resulting from PPG blocks in the copolymer branches. This can probably explain the lower polydispersities of the particles observed at this temperature (Table 2). On the other hand, LPEC copolymer micelle solution does not show much difference in size and distribution due to its higher phase transition temperature at around 60 °C. These results illustrate that polymer chain architecture plays a crucial role in chain interaction and entanglement of the aqueous solution. This would thus further affect the self-assembled aggregations in size and distribution. The reversible transition of the micelle solution in particle size was also demonstrated by DLS. Figure 6 shows the transition of HBPEC2 copolymer solution trigged by temperature. The micelle solution was prepared for the measurement at 25 °C. The hydrodynamic diameter of the micelles increased

of the copolymers are presented in Table 1. At temperatures below LCST, PPG behaves more favorably as hydrophilic water-soluble polymer. Above this temperature, PPG becomes hydrophobic and may precipitate out of the aqueous solution. In this article, the LCST is defined as the temperature exhibiting a 50% decrease in optical transmittance of an aqueous copolymer solution (1 mg/mL) at 500 nm. The aggregation morphologies of the tested solution at temperature below and above the LCST were also presented in Figure 4. As

Figure 4. Thermoresponsive behaviors of HBPEC block copolymers in contrast to LPEC as control. Polymer concentration in aqueous solution was 1 mg/mL. Inserted graphs shows optical transmittance of copolymer aqueous solution and particle aggregation morphologies recorded on TEM.

shown, the phase transition temperatures changed to lower values with increased PCL moiety in hyperbranched series copolymers. Unlike the lack of decrease in LCST of thermosensitive poly(N-isopropylacrylamide)-PCL-poly(N-isopropylacrylamide) (PNIPAAm-PCL-PNIPAAm) copolymer micelle solutions,29 the HBPEC series copolymers could lower the LCST more dramatically (59.7 °C → 46.4 °C) when compared with LPEC copolymer (Table 1). The hyperbranched architecture of HBPEC copolymers may cause the chain entanglement and then may increase the chance of the aggregation of the hydrophobic segments (PPG and PCL) upon temperature increase, giving lower LCST.8 In contrast, F

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Figure 5. TEM micrographs (left) and particle size distribution by intensity (right) of micelles formed by HBPEC3 copolymer solution at concentration of 0.6 mg/mL. Sizes recorded on TEM graphs are in good agreement with the results obtained from DLS.

from about 50 nm at 25 °C into much larger value of about 250 nm at 60 °C. Cooling the solution at 25 °C for 30 min resulted in formation of small micelles again, indicating that the morphological changes were reversible (Figure 6). Five cycles of the transition were conducted in this experiment. The particle size changes showed very good reversibility during the cycling. This property is due to the reversible hydrophobic to hydrophilic transition of PPG segments induced by temperature.35 3.4. Thermodynamic Study of Micelle Formation. When a polymer solution is dissolved in water, there are three types of interactions that take place: between polymer molecules, polymer and water, and between water molecules. For polymer exhibiting an LCST, temperature increase results in a negative free energy of the system which makes water− polymer association unfavorable, facilitating the other two types of interactions. This negative free energy (ΔG) is attributed to the higher entropy term (ΔS) with respect to the increase in enthalpy term (ΔH) in the thermodynamic relation ΔG = ΔH − TΔS.8,36 Assuming a closed association of unimers into micelles, these thermodynamic functions for micelle formation can be extracted from the studies of the CMC dependence on temperature,30 using the following equations: the free energy of micellization can be calculated by ΔG = RT ln(XCMC) where R is the gas law constant, T is the temperature in K and XCMC is the CMC in mole fractions at temperature T. Further, the values of the standard enthalpy of micellization, ΔH, and the standard entropy of themicellization, ΔS, can be extracted from the Arrhenius plot of ln(XCMC) versus 1/T. ΔH = R(d lnXCMC/ dT−1) and ΔS = (ΔH − ΔG)/T. These thermodynamic parameters of the HBPEC copolymer solution micellization process are listed in Table 3, in which ΔH was derived from the slope of the linear plot of ln(XCMC) versus T−1 as shown in Figure 7. By referring the thermodynamic parameters

Table 2. Particle Size and PDI of Micelles Formed by HBPEC Copolymers and Control Copolymers particle diameter (nm)b samplesa

25 °C

HBPEC1 HBPEC2 HBPEC3 LPEC F127

50 43 53 29 -d

± ± ± ±

1 2 2 1

60 °C 198 250 137 24 -d

± ± ± ±

8 2 3 1

PDIc 25 °C 0.52 0.30 0.40 0.15 -d

± ± ± ±

0.02 0.01 0.01 0.02

60 °C 0.03 0.04 0.10 0.14 -d

± ± ± ±

0.02 0.01 0.04 0.01

a

Concentration of all copolymers was 0.6 mg/mL. bMeasured by dynamic light scattering. cPolydispersity index. dSample not met measurement criteria.

Figure 6. Reversible transition in thermodynamic diameter distribution of HBPEC2 copolymer solution at concentration of 0.6 mg/mL between 25 and 60 °C.

G

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Table 3. Thermodynamic Parameters of the Micellization Process of HBPEC and LPEC Copolymers samples

temperature (°C)

CMC (10−2 mg/mL)

ΔG (kJ/mol)

ΔS (kJ/(mol K))

ΔH (kJ/mol)

HBPEC1

25 30 35 40 25 30 35 40 25 30 35 40 25 30 35 40

33.8 29.6 28.8 27.7 31.0 28.3 24.1 20.3 28.2 24.8 23.2 18.5 30.1 26.9 23.4 19.9

−36.5 −37.5 −38.1 −38.9 −36.4 −37.2 −38.2 −39.3 −37.0 −38.0 −38.8 −40.0 −36.1 −37.0 −38.0 −39.0

0.207 0.207 0.206 0.205 0.196 0.196 0.196 0.196 0.193 0.193 0.192 0.193 0.193 0.193 0.193 0.193

25.3

HBPEC2

HBPEC3

LPEC

22.0

20.4

21.3

to a stronger tendency to assemble into micelles of the HBPEC aqueous solutions. 3.5. Thermo-Reversible Sol−Gel Transition. The phase diagrams of the polymers in aqueous solutions were determined by the test tube inverting method.21 The results are shown in Figure 8. Three regions are identified from the diagram, the

Figure 7. Determination of ΔHmicellization of HBPEC series block copolymers.

determined for Pluronic F127 previously, which contains similar PPG and PEG content in polymer composition but no PCL, the function values are: −27.5 kJ/mol (ΔG), 0.944 kJ/ (mol K) (ΔS) and 253 kJ/mol (ΔH).37 From Table 3, ΔG values of between −36.1 and −40.0 kJ/mol detected from all studied copolymer solutions are more negative than those for Pluronic F127. Specifically, HBPEC3 shows more negative value in ΔG than LPEC, in which the two copolymers contain similar PCL component. ΔG negative in value indicates the thermodynamically stable micelles are formed spontaneously. Thus, the negative entropy ΔS contribution must be the driving force for the micellization of the block copolymers.37 The more negative free energy signifies a greater propensity to form micelles. On the other hand, the enthalpy of the micellization, ΔH, for both HBPEC and LPEC copolymer solutions is positive, indicating that the transfer of unimers from solution to micelles is an enthalpically unfavorable endothermic process. This finding is similar to the previous report on Pluronic F127.36,37 The enthalpy values become less positive as the PCL-triol contents become higher in HBPEC copolymers. As for HBPEC3 and LPEC samples with similar PCL content but in different polymer architectures, ΔH in hyperbranched structure shows lower value than linear one, indicating more polymer chain association occurred for HBPEC3. This will lead

Figure 8. Graphics showing the gel transition of HBPEC3 (5.7 wt % in H2O) with increasing temperature (A). The transition from a clear sol to a gel and further to a dehydrated gel is observed in the graphics. LPEC (9.1 wt %) in turbid sol at 60 °C is used for comparison study. Sol−gel phase diagrams of HBPEC copolymers in comparison with LPEC and Pluronic F127 copolymers (B).

lower soluble region, gel region, and the upper dehydrated/ turbid gel region. As the temperature increased monotonically from 4 to 70 °C, the aqueous polymer solution underwent a sol−gel-dehydrated gel transition. This is different from the previously reported sol−gel−sol transition procedure resulted from the collapse of hydrogel networks.21 When the gel is H

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cooled back to 4 °C, the system reverted to the sol state. Taking HBPEC3 (5.7 wt %) as example, the polymer solution behaves a liquid-like property at 10 °C, which can flow easily in sample vial (Figure 8Aa). At raised temperature 37 °C, the above-mentioned polymer solution forms a firm gel and cannot flow even when the sample vial is inverted, confirming the gelation state (Figure 8Ab). LPEC copolymer solution at 9.1 wt % was used for comparative study. At higher temperature above 60 °C, HBPEC and LPEC solution show different morphologies which may be dominated by individual gelation mechanism. For hypebranched structure block copolymers, HBPEC, the gel deswelled and maintained its network structure, namely shrinking in size and separating from the surrounding water environment due to its strengthened intramolecular associations (Figure 8Ac), while linear block copolymer collapsed into a fluid water/polymer mixture at this temperature (Figure 8Ad). The critical gelation concentration (CGC) is defined as the minimum copolymer concentration in aqueous solution at which the gelation behavior could be observed. The CGCs of the HBPEC copolymers in this work were found to be between 4.3 and 7.4 wt % (Table 1). These values are much lower than those reported on Pluronic F127 and other PCL-contained thermogelling copolymers.38−40 For HBPEC3 copolymer with similar PCL content and molecular weight with LPEC, the CGC is lower than that of LPEC. This elucidates the importance of polymer structure in affecting selfassembly mechanism and chain association. The reasons could be, first, due to the improved urethane bond and its synergetic hydrogen bonding effect between the polymer branches. Low molecular weight of PCL-triol and its trifuctional hydroxyl groups in nature will generate more urethane linkages with isocyanate groups in HMDI during polymerization, which leads to enhanced interactions between the polymer chains in the gel assembly process. The intensively distributed urethane bonds have a strong tendency to form hydrogen bonds with each other or with water molecules and this leads to an increased degree of interaction compared with LPEC and Pluronic F127.37,41 Indeed, we noted that an original HBPEC (5.7 wt %) forms gels at 30 °C. However, upon addition of 10 wt % of ethylene diamine, the solution does not form gels at even 50 °C. This indicates that hydrogen bonding between the polymer chains is one of the factors that enhance the gel formation. Second, PPG blocks in copolymer branches in HBPEC have stronger hydrophobic association at above phase transition temperature than the individual polymer chain interaction in LPEC, which provides greater driving force to facilitate HBPEC aqueous solution to assemble into gels at certain concentrations. The intramolecular interaction in HBPEC solution was strengthened upon PPG dehydration to give a more stable network-like packing of the polymer chains, as illustrated in Figure 9. This effect can also explain the different gel morphologies of HBPEC and LPEC copolymers at above 60 °C. The molecular environment changes during the sol−gel transition of a HBPEC3 solution (6 wt %) in D2O was monitored by 1H NMR technique at different temperatures (Figure 10). The solution was equilibrated for 30 min at the setting temperature before measurement. At low temperature range between 10 and 20 °C, the peaks ascribed to PEG and PPG are sharp and well-defined because the segments interacted freely with the solvent molecules in the solution. After the gelation occurred at elevated temperatures, the peaks broaden and decrease dramatically in their intensities. Results

Figure 9. Intramolecular interaction model showing the stable network-like packing of HBPEC polymer chains.

Figure 10. 1H NMR spectrum of HBPEC3 in D2O (5.7 wt %) at different temperatures.

revealed that PEG and PPG chains underwent a decreased motion in water environment with increasing temperature.24,38,42,43 Specifically, for the methyl signals in PPG units, it decreased significantly in peak intensity and collapsed at elevated temperatures. Further, there is an upfield shift in the methyl peak, which indicated an increased hydrophobicity of PPG at high temperatures. For the changing trend of PEG peaks, the finding is different from our previously reported linear PPG/PEG/PHB based thermogels.21 In that work, the PEG peaks resorted to a sharp shape with the temperature increased up to 75 °C. This is due to the collapse of the gel into a polymer/water mixture, which again improved the PEG chain mobility at high temperature. In this paper, due to the enhanced intramolecular interaction of the network-like packing of hyperbranched polymer chains, the motion of all the components in the copolymer branches became restricted to a certain extent, as can be viewed by a continuous decline of PPG and PEG peaks with arisen temperature in Figure 10. 3.6. Thermoresponsive Rheological Properties. Rheological studies of the thermogelling copolymers were carried out to determine the physical properties of the gels. First, we determined the rheological profile of the gels as a function of temperature. In Figure 11, the rheological profile of the thermogel HBPEC3 (5.7 wt %) as a function of temperature is presented. At the start of the experiment in low temperature range, the solution is not very viscous and exists in a liquid-like state. At this stage, the loss modulus, G″ is higher than the storage modulus, G′. When the temperature is raised, both the I

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hydrogel concentration. Specifically, shear stress that leads to sample HBPEC3 hydrogel deformation can reach a comparative level to LPEC at much lower gel concentration. The enhanced H-bonding effect and intramolecular interaction of HBPEC chains in gel state may contribute to this observation. This illustrates that the hyperbranched copolymer structure can enhance the gel elasticity by dispersing the applied force uniformly.1 These results, together with the gelation temperatures shown in Table 4, in which HBPEC copolymer solutions show thermogelling transition at around or under human body temperature, show that this type of soft gels could be potentially useful for the culture of neural cells or could be used as neurite-favoring culture substrates for stem cell differentiation experiments.45 Furthermore, as suggested by Bott et al., hydrogel with higher gel stiffness (G′ > 1200 Pa) acts as a barrier for 3D-cell growth and the best performance may be achieved with G′ ranging from 10 to 1000 Pa.46 Previous study had shown cell encapsulation in F127 gels ranging from 15 to 20% (w/w) resulted in complete cell death by 5 days.47 Our results on rheological study show that by varying polymer compositions and hydrogel concentrations, the gel stiffness can be tuned in a wide range (Table 4). The improved rheological property over F127 gels may make the HBPEC copolymer hydrogels suitable biomaterials for 3D cell culture. 3.7. Hydrolytic Degradation of HBPEC Hydrogels. A firm HBPEC3 hydrogel at concentration of 5.7 wt % could be obtained after equilibrating the polymer PBS buffer solution at 37 °C. The bulk molecular weight changes at various degradation periods were monitored by GPC. As presented in Figure 12, the GPC profiles of both undegraded hyperbranched and linear copolymers were unimodal. However, after 7 days incubation, the degraded HBPEC3 showed a multimodal GPC profile with lower molecular weight (Figure 12). The degradation for 21 days resulted in a significant decrease in average molecular weight of HBPEC3 from Mn 12 100 to 8400 (31% reduction), and an increase in PDI from 1.9 to 2.6. On the other hand, the degradation of linear LPEC hydrogel was slower. The first 7 days incubation caused very limited changes of LPEC in molecular weight and molecular weight distribution. After 14 days incubation, the change in the GPC profile of LPEC was clear. The degradation for 21 days resulted in a decrease in average molecular weight of LPEC from Mn 11 700 to 9300 (21% reduction), and an increase in PDI from 1.4 to 2.2. Comparing the deductions of molecular weight of

Figure 11. Dynamic rheological analysis of HBPEC3 (5.7 wt %) aqueous solution as a function of temperature.

values of the storage modulus and the loss modulus begin to rise. At a certain temperature, the solution becomes very viscous and exists as a semisolid state, verified by the higher value of G′ than G″. The onset point of G′ over G″ is known as gelation temperature and it is dependent on the polymer concentration and polymer composition (Table 4). Thus, dynamic rheology confirms the thermogelling transition in these samples. The complete elastic modulus G′ data obtained from oscillatory stress sweep measurements, described as a function of polymer concentration for HBPEC copolymer solution based hydrogels, are also presented in Table 4. The G′ values of the tested hydrogels are lower as compared to Pluronic F127 (16.7 wt %), implying the weak stiffness of HBPEC and LPEC hydrogels under experimental conditions. On the other hand, the shear stress τ required to destroy the hydrogel network is manifested by a G′/G″ crossover into liquid-like phase.44 The τ values recorded on PPG/PEG/PCL based hydrogels are generally higher in contrast to Pluronic F127 (16.7 wt %) which yields at shear stress of only 63.1 Pa. These results reveal that the incorporation of PCL can make the hydrogel to endure a stronger gel strain. By exampling the changing trend of G′ in HBPEC copolymers, we found that we could obtain G′ in the range of 10−442 Pa, by simply changing the polymer composition and concentration in the gel. Moreover, in view of the τ values, HBPEC hydrogels show marked improvement in hydrogel strain and this improvement is proportional to the

Table 4. Rheological Characteristics of Hydrogels of HBPEC Copolymers and Control Copolymers at Various Concentrations samples

concentration (wt %)

HBPEC1

9.1 10.7 12.3 7.4 9.1 10.7 4.8 5.7 6.5 10.7 16.7

HBPEC2

HBPEC3

LPEC F127

G′ (Pa) 34 120 388 75 228 442 10 103 336 217 2354

± ± ± ± ± ± ± ± ± ± ±

1 12 81 11 50 56 1 19 68 68 176

G″ (Pa)

τ (Pa)a

CGTb

± ± ± ± ± ± ± ± ± ± ±

63.1 30.6 501.7 72.3 251.4 530.7 6.5 157.6 313.2 199.5 63.1

48.3 47.4 38.0 37.5 36.9 28.9 37.9 30.2 23.4 47.4 31.2

32 114 253 56 122 244 4 53 110 125 591

1 10 49 7 26 15 0 8 29 20 55

a

Shear stress recorded at the crossover point of G′ and G″ in stress sweep. bCritical gelation temperature (CGT) determined at the crossover point of G′ and G″ in temperature sweep. J

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Figure 13. FTIR spectra of HBPEC3 hydrogel (5.7 wt %) before and after 21 days degradation in PBS buffer at pH 7.4 and 37 °C.

corresponds to the stretching of carboxylic −CO. This confirms that the ester linkages in the copolymer were hydrolyzed to carboxylic acid groups.22 The FT-IR results indicate that the degradation of HBPEC hydrogels occurred through hydrolysis reaction of the ester linkages of PCL segments, similar to previous findings.48,49 3.8. Cytotoxicity Assay. To evaluate the biocompatibility of the HBPEC multiblock copolymers, cell viability tests were conducted by incubating the mouse fibroblast L929 cells over 48 h at 37 °C with the copolymers at various concentrations ranging from 12 to 1000 μg/mL.26,29 Quantification of the cytotoxic response was performed using the MTT assay, as shown in Figure 14. The HBPEC copolymers did not show Figure 12. GPC profiles of HBPEC3 hydrogel (5.7 wt %) and LPEC hydrogel (10.7 wt %) before and after hydrolytic degradation in PBS buffer at pH 7.4 and 37 °C in various degradation periods. One of the starting materials PEG2000 was used as a reference.

HBPEC3 and LPEC after 21 days incubation, HBPEC3 showed more significant degradation than LPEC. Although HBPEC and LPEC copolymers contain urethane and ester linkages that both are hydrolytically labile, it was reported that hydrolytic degradation of such copolymers occurs via the preferential cleavage of the ester linkages instead of the urethane ones.48 The random scission of the ester linkages of the branched PCL blocks in HBPEC may be the cause of the faster degradation of HBPEC than the linear LPEC. Previously, we reported that amphiphilic poly(PPG/PEG/PCL urethane) that is more hydrophilic than pure PCL increased the degradation rate of the PCL segments in the copolymers.49 The current study showed that hyperbranched architecture could further increase the degradation rate of poly(PPG/PEG/PCL urethane) copolymers. The molecular structures of the copolymers during hydrolytic degradation were monitored by FT-IR spectroscopy. The FT-IR spectrum of HBPEC3 after 21 days degradation in comparison with that of the undegraded copolymer is shown in Figure 13. In the spectrum of the original undegraded sample, the peak of ester −CO corresponding to the CL repeating units in PCL can be observed at 1720 cm−1, while there is only a small shoulder at 1639 cm−1.50 The degradation product showed a clear peak of −CO at 1639 cm−1, which

Figure 14. Viability assay of L929 cells incubated with HBPEC multiblock copolymers at various concentrations. LPEC and F127 were used for comparison.

significant cytotoxicity against L929 cells over a wide concentration range. Specifically, all cell viability was higher than 92% even when the copolymer concentration of HBPEC was up to 1000 μg/mL. As a comparison, LPEC and F127 were also tested, and the results showed very low cytotoxicity, which is in good agreement with previous reports.23,51 From the cytotoxicity assay results, the HBPEC copolymers, although having hyperbranched architectures, possess good biocompatibility similar to their linear counterparts. K

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4. CONCLUSIONS

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AUTHOR INFORMATION

Corresponding Author

A series of HBPEC thermogelling polyurethane block copolymers with hyperbranched structure were synthesized by using PCL-triol (Mn = 300), PPG-diol (Mn = 2000), and PEG-diol (Mn = 2000) as starting materials. The molecular weight of HBPEC copolymers was targeted at similar range with Pluronic F127 for comparative studies. A control linear LPEC copolymer was also synthesized with molecular weight and PCL content similar to HBPEC3 (PCL content 6.7 wt %). We carried out comparative studies of HBPEC copolymers with the LPEC copolymer and Pluronic F127 copolymer, in terms of their self-assembly and aggregation behaviors and thermoresponsive properties. HBPEC copolymer aqueous solutions formed thermoresponsive micelles with low CMCs, in which the micelle size became bigger and more uniform at elevated temperatures, as characterized by DLS and TEM. In addition, this change switched by temperature trigger was reversible. The hyperbranch structure showed significant effect on the LCST of the copolymers. The LCST of HBPEC copolymers became much lower than LPEC. Thermodynamic parameters such as free energy, enthalpy, and entropy were studied by temperaturedependent CMC values. Results indicated that the micellization of HBPEC aqueous solutions was a spontaneous process with a strong driving force, as verified by the more negative value of ΔG in the system than those obtained from LPEC and Pluronic F127 aqueous solutions. Our studies revealed that the effect of hyperbranch architecture was more prominent in the gelation of the copolymers. The aqueous solutions of HBPEC copolymers exhibited thermogelling behaviors at critical gelation concentrations (CGCs) ranging from 4.3 to 7.4 wt %. These values are much lower than those reported on other PCL-contained linear thermogelling copolymers and Pluronic F127 copolymer. In addition, the CGC of HBPEC copolymers is much lower than the control LPEC copolymer. More interestingly, at high temperatures, while LPEC and other linear thermogelling copolymers formed turbid sol, HBPEC formed a dehydrated gel. Our data suggest that these phenomena are caused by the hyperbranched structure of HBPEC copolymers, which could increase the interaction of copolymer branches and enhance the chain association through synergetic hydrogen bonding effect. The thermogelling behavior of HBPEC block copolymers was further demonstrated by the molecular dynamic changes during the gelation process monitored by 1H NMR spectroscopy. The HBPEC copolymer hydrogels show more elastic property than LPEC and Pluronic F127, evidenced by the great difference in storage modulus and shear stress at gel yield point. The1H NMR dynamic rheological studies further support the hypothesis that enhanced thermogelling properties are induced from the hyperbranch architecture. The hydrolytic degradation study showed that the HBPEC copolymer hydrogels are biodegradable under physiological conditions. The cytotoxicity assay results also showed that the HBPEC copolymer hydrogels have good biocompatibility. This study provides profound insights into the structure−property effect on PPG, PEG, and PCL based block polyurethane copolymers and their selfassembly behaviors.

*Address: Department of Bioengineering, Faculty of Engineering, National University of Singapore, 7 Engineering Drive 1, Singapore 117574, Singapore. Tel., +65 6516 7273; fax, +65 6872 3069. E-mail: [email protected]; [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by Agency for Science, Technology and Research (A*STAR), Singapore (SERC PSF Grant No. 102 101 0024 and JCO Grant No. 10/03/FG/06/ 05) and National University of Singapore (FSF Grant No. R397-000-136-112 and R-397-000-136-731). Z.L. thanks National University of Singapore for the Graduate Scholarship.

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