Thermoresponsive Hydrogels from Phosphorylated ABA Triblock

Jun 13, 2013 - Moreira Teixeira , L. S.; Bijl , S.; Pully , V. V.; Otto , C.; Jin , R.; Feijen , J.; van Blitterswijk , C. A.; Dijkstra , P. J.; Karpe...
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Thermoresponsive Hydrogels from Phosphorylated ABA Triblock Copolymers: A Potential Scaffold for Bone Tissue Engineering Zaifu Lin, Shuqin Cao, Xingyu Chen, Wei Wu, and Jianshu Li* College of Polymer Science and Engineering, Sichuan University, Chengdu 610065, China S Supporting Information *

ABSTRACT: A series of thermoresponsive and biocompatible ABA triblock copolymers in which the outer A blocks comprise poly(Nisopropylacrylamide) and the central B block consists of Ophosphoethanolamine (PEA) grafted poly(acrylic acid) (PAA(PEA)) are achieved by atom transfer radical polymerization (ATRP) and subsequent modification. At a relatively low concentration (2 w/v% in phosphate buffered saline), the triblock copolymers can form freestanding gels at 37 °C. Using a combination of variable-temperature 1 H NMR, dynamic light scattering, and rheological measurements, it is demonstrated that the gelation behavior is highly dependent on both the length of A blocks and the substitution degree of phosphate group. To examine the potential application as scaffold for bone tissue engineering, the physical gels are incubated in the simulated body fluid (SBF) for 2 weeks. Obvious nucleation and growth of hydroxyapatite are found in the gels, as indicated by the scanning electron microscope, energy dispersive spectroscopy, and X-ray diffraction measurements. The triblock copolymers also exhibit low cytotoxicity in cell viability test. Thus the triblock copolymers have great potential for bone tissue engineering.

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INTRODUCTION Stimuli-responsive polymeric hydrogels that can switch between free-flowing liquid and free-standing gel states have aroused great interest in the past few decades, especially in the biomedical field.1−6 Their water-based sol−gel transition triggered by the stimulus under physiological condition enables so-called “minimally invasive therapy”, i.e., entrapping pharmaceutical agents, biomacromolecules, or cells to form a sustained delivery system or a cell-growing matrix by solution mixing and injection at the target site.7,8 Meanwhile, the in situ transition from soluble molecules to a physical cross-linking network means a perfect adaption to irregular substrate surface, by which it can improve the interface adhesion and benefit the practical applications, such as bone tissue regeneration.9−13 For example, Cao et al. described a novel application of PEO− PPO−PEO (PEO: poly(ethylene oxide); PPO: poly(propylene oxide)) triblock copolymers in the formation of cartilage on the host bone: isolated chondrocytes were suspended in the polymer solution and painted on a viable osseous surface. Then, they formed a semisolid sticky gel within a few minutes and then transformed into a cartilage layer after 8 weeks. It was noted that the newly formed cartilage could infiltrate the underlying osseous substrate and generate a good bonecartilage interface.14 Therefore, reversible hydrogels based on stimuli-responsive polymers show great potential as tissue engineering scaffolds for bone tissue regeneration, owing to their tunable chemical structures and gelation property.15,16 Meanwhile, the rational molecular design may be inspired by the biomimetic strategy. It © 2013 American Chemical Society

is found that in the formation of natural bone, the acidic extracellular matrix proteins are attached to the collagen scaffolds and can induce the transformation of amorphous calcium phosphates into stable mature bone apatite with increased crystallinity. The acidic groups can serve as the binding sites for calcium ion and align them in an orientation that matches the apatite crystal lattice.17,18 Hence, it is desirable to design a multifunctional polymeric scaffold with sol−gel transition to realize “minimally invasive therapy” and adapt irregular substrate surfaces, as well as with the capability of inducing in situ mineralization to promote bone regeneration. It is well-known that ABA triblock copolymers with a permanently water-soluble B block and stimuli-responsive A blocks can form either flower micelles or free-standing gels, depending on the copolymer compositions and concentration.7,19 Their interesting self-assembly structures and gelation properties are based on three possible conformations: “loops”, “bridges”, and “dangling chain ends”.20,21 In dilute solution, the application of external stimuli triggers the aggregation of A blocks to create discrete flowerlike micelles with central B blocks forming loops in the corona layer.22 When the polymer concentration is efficiently high, i.e., above the critical gelation concentration, a three-dimensional micellar network is formed, in which the central B blocks forming bridges among the Received: March 6, 2013 Revised: May 29, 2013 Published: June 13, 2013 2206

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neighboring micelles. In the above condition, the free-flowing solution is transformed into a free-standing micellar gel.7 Numerous works have been carried out to investigate the structure−property relationship of those stimuli-responsive ABA triblock copolymers. In a previous report, it was indicated that a critical copolymer volume fraction of 0.05−0.10 is required for gelation, depending on the molecular weight of the copolymers.13 Other theoretical studies consider the molecular weights of both blocks and the hydrophobic character of the outer A blocks as the main reason for the gelation.22 By atom transfer radical polymerization (ATRP), Armes et al. synthesized a series of ABA triblock copolymers with sol−gel transition in aqueous solution in response to pH or temperature changes.19,24−29 For instance, ABA triblock copolymers with poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC) as the B blocks and poly(N-isopropylacrylamide) (PNIPAM) as the outer blocks gelled at 37 °C in phosphate buffered saline (PBS; pH = 7.4) and showed sufficient biocompatibility for cell culture.27 In another case, the ABA triblock copolymers in which the outer A blocks comprise poly(2-hydroxypropyl methacrylate) (PHPMA) and the central B block is poly(2-(dimethylamino)ethyl methacrylate) (PDMA) is achieved via ATRP.19 Gel rheology measurements were carried out and demonstrated the high dependence of critical gelation temperature and mechanical properties on copolymer composition and concentration. Kirkland and his co-workers prepared PNIPAM−PDMA−PNIPAM by reversible addition−fragmentation chain transfer (RAFT) polymerization.20 Reversible physical gels were formed above the phase transition temperature of PNIPAM at concentrations as low as 7.5 wt % and showed similar mechanical properties to that of collagen. Herein, we report the synthesis of a kind of thermoresponsive ABA triblock copolymer with potential application for bone tissue regeneration. PNIPAM is chosen as the stimuliresponsive outer blocks due to its phase transition temperature (32 °C) between room and body temperature. In the beginning, poly(acrylic acid) (PAA) was designed as the B block of copolymer due to its affinity for calcium ion, which will benefit the biomineralization process. However, our preliminary experiment confirmed that there is undesirable strong interpolymer complexation between PNIPAM and PAA by forming hydrogen bonds, resulted in precipitation in aqueous solution. Thus, we designed phosphorylated PAA as the B block, since the phosphate group has a higher affinity for calcium ions than PAA and does not form hydrogen bonds with PNIPAM.28,29 Syntheses involved two-step ATRP of tert-butyl acrylate (tBA) and N-isopropylacrylamide (NIPAM), subsequent hydrolysis of the PtBA segment into the PAA, and final modification of the carboxyl group with O-phosphorylethanolamine (PEA). The obtained ABA triblock copolymers with PNIPAM as the A blocks and PEA-grafted PAA (PAA(PEA)) as the B block were tested with rheological measurement, variable-temperature 1H NMR, and MTT assay. Physical gels from PNIPAM−PAA(PEA)−PNIPAM were incubated in simulated body fluid (SBF) for 2 weeks to investigate the capability of inducing hydroxyapatite under physiological conditions. The mineralized gel is analyzed with SEM, energy dispersive spectroscopy (EDS) and X-ray powder diffraction (XRD).

Article

EXPERIMENTAL SECTION

Materials. NIPAM (97%, Aldrich) was purified by recrystallization two times from n-hexane, dried in vacuum, and then stored at 0 °C before use. tBA (98%, TCI) was rinsed with 5% NaOH aqueous solution and dried over CaCl2. It was then distilled under reduced pressure and stored at 0 °C. N,N,N′,N″,N″-Pentamethyldiethylenetriamine (PMDETA, 98%, Aldrich), tris(2-(dimethylamino)ethyl)amine (Me6TREN, 99%, Alfa Aesar), diethyl-meso-2,5-dibromo adipate (DEDBA, 98%, Alfa Aesar), O-phosphorylethanolamine (98%, TCI), dicyclohexyl carbodiimide (DCC, 97%, TCI), N-hydroxysuccinimide (NHS, 97%, TCI) and trifluoroacetic acid (TFA, 99%, Kelong Reagent Co. Ltd.) were used as received. CuBr and CuCl (Kelong Reagent Co. Ltd.) were washed with acetic acid and ethanol in turn and dried in vacuum before use. A dialysis membrane (molecular weight cutoff (MWCO) = 8000−14000, Cellu SepH1, USA) was used. All solvents used in the syntheses were freshly distilled. Synthesis of Br−PtBA−Br ATRP Macroinitiator. Br−PtBA−Br was synthesized according to previous reports.30 Typically, Br− PtBA303−Br was prepared as follows. DEDBA (36 mg, 0.1 mmol), PMDETA (46 μL, 0.22 mmol), anisole (1.45 mL, 20 vol% with respect to monomer) and tBA (7.24 mL, 50 mmol) were added into a flask with a three-way valve. After dissolution, the flask was kept at −20 °C and deoxygenated by three inflate-pump cycles with nitrogen and bubbling for 20 min. Finally, fresh CuBr (29 mg, 0.2 mmol) was added under nitrogen flow, and then the flask was sealed and placed in an oil bath at 60 °C. Once CuBr was added, the systems turned light-green and gradually deepened with the reaction. After 16 h, excess tetrahydrofuran (THF) was poured in to stop the polymerization. The resulting solution was then stirred overnight and passed through an alumina column to remove the catalyst. After removal of solvent, the concentrated solution was dropped into a 10-fold-excess water/ methanol (1:1) mixture. The macroinitiator was obtained by filtration and drying under vacuum. The polymer was 3.565 g, and the yield was 55.77%. Synthesis of PNIPAM−PtBA−PNIPAM Triblock Copolymer. Typically, PNIPAM117−PtBA303−PNIPAM117 was prepared as follows. Difunctional macroinitiator Br−PtBA303−Br (0.5 g, 0.0141 mmol), NIPAM (0.799g, 7.07 mmol), Me6TREN (35 μL, 0.143 mmol), and 1.33 mL of dimethylformamide (DMF) were added into a flask with a three-way valve. Three inflate−pump cycles and bubbling for 20 min with nitrogen at −20 °C were performed to remove oxygen from the polymerization solution. Then, fresh CuCl (14 mg, 0.143 mmol) was added under the nitrogen flow, and the sealed flask was placed in an oil bath at 35 °C for 48 h. Finally, the polymerization was terminated by pouring DMF and stirred overnight. The resulting solution was transferred to dialysis tubes (MWCO = 8000−14000) and dialyzed against distilled water for 3 days at room temperature to remove the unreacted chemical reagents and catalyst. After dialysis, the sample was obtained by freeze-drying process. The resulting copolymer was 0.77 g with a yield of 59.276%. Synthesis of PNIPAM−PAA(PEA)−PNIPAM. Typically, PNIPAM117−PtBA303−PNIPAM117 (0.6 g, 0.00813 mmol) was dissolved in 20 mL of dry dichloromethane in a 50 mL round-bottom flask before the addition of triflouroacetic acid (1.83 mL, 24.63 mmol). The mixture was stirred at 25 °C for 48 h. Then, most of the volatiles and solvent were removed by a rotavapor. The obtained oily mixture was dropped into 10-fold-excess distilled water. After filtration, the hydrolysate was dried under vacuum. The obtained PNIPAM117− PAA303−PNIPAM117 was 0.417 g with 94.0% yield. Then, a mixture of PNIPAM117−PAA303−PNIPAM117 (350 mg, 2.36 mmol) and NHS (325 mg, 2.83 mmol) was dissolved in 10 mL of DMF. DCC (971 mg, 4.71 mmol) dissolved in another 10 mL of DMF was then added dropwise at 0 °C. The reaction mixture was stirred at 25 °C for 48 h, followed by filtration and concentration. Then, NaOH (565 mg, 6 mmol) was gradually added into the mixture of 25 mL methanol and PEA (997 mg, 7.07 mmol) to form a clear solution, which was poured into the above reaction mixture, and stirred at 25 °C for 48 h. Then, the solvent was evaporated, and a proper quantity of water was added to dissolve the product. Further separation and purification involves 2207

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Scheme 1. Synthetic Route of PNIPAM−PAA(PEA)−PNIPAM via ATRP and Subsequent Modification

Figure 1. 1H NMR spectra of (a) Br−PtBA303−Br (in CDCl3), (b) PNIPAM117−PtBA303−PNIPAM117 (in CDCl3), (c) PNIPAM117−PAA303− PNIPAM117 (in DMSO-d6) and (d) PNIPAM117−PAA(PEA)303−PNIPAM117 (in D2O). Peak-identification: a: −C(CH3)3; b: −CH(CH3)2; c: −CH(CH3)2; d: −CO−NH−CH(CH3)2; e: −NH−CH2−CH2−OPO3X2; f: −NH−CH2−CH2−OPO3X2. centrifugation (8000 rpm, 10 min), filtration (0.45 μm), dialysis (MWCO = 8000−14000), and freeze-drying. The 0.332 g of endproduct was obtained with a yield of 43.6%. Characterization. Nuclear Magnetic Resonance (NMR) Spectroscopy. 1H NMR spectra from a Bruker 400 MHz spectrometer provided the main structure information of polymer. Meanwhile, variabletemperature 1H NMR spectra were obtained by a Bruker 600 MHz spectrometer. Sample was dissolved in D2O to form a 2.0 w/v% solution (TMSP-2,2,3,3-d4 as the internal standard). At each

temperature point, the solution was equilibrated for 20 min before acquiring the data. Fourier Transform Infrared (FT-IR) Spectroscopy. FT-IR spectra were recorded on a Nicolet 6700 FT-IR spectrometer using dispersion of polymer powders in potassium bromide (KBr) pellets. Characterization of Absolute Molecular Weight. Tandem gel permeation chromatography/light scattering (GPC/LLS) was performed using an SSI pump connected to Wyatt Optilab DSP and Wyatt DAWN EOS light scattering detectors at 50 °C. DMF with 0.02 mg/mL LiBr was used as the eluent, and the flow rate was kept at 1.0 2208

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Table 1. Summary of the Syntheses and Absolute Molecular Weight Data for Br−PtBA−Br and PNIPAM−PtBA−PNIPAM via ATRP samples

initiator

[M]0/[I]0/[Cu]0/[L]0

Mn (kDa) (GPC/LLS)

PDI

dn/dc

Br−PtBA303−Br Br−PtBA377−Br PNIPAM64−PtBA303−PNIPAM64 PNIPAM117−PtBA303−PNIPAM117 PNIPAM155−PtBA303−PNIPAM155 PNIPAM48−PtBA377−PNIPAM48 PNIPAM326−PtBA377−PNIPAM326

DEDBA DEDBA Br−PtBA303−Br

500/1/2/2 800/1/2/2 300/1/10/10 500/1/10/10 700/1/10/10 500/1/10/10 1000/1/10/10

38.8 48.3 53.2 62.2 73.8 59.2 122.0

1.03 1.14 1.14 1.15 1.14 1.16 1.29

0.0360 0.0380 0.0599 0.0598 0.0604 0.0640 0.0609

Br−PtBA377−Br

μL minimum essential medium alpha medium (α-MEM), supplemented with 10% heat-inactivated fetal bovine serum (FBS) for 24 h. The culture media was replaced with 100 μL of fresh culture media containing serial dilutions of copolymers, and cells were incubated for another 24 h. Then, MTT reagent (in 10 μL of PBS, 5 mg/mL) was added to each well. After 5 h, the media was drawn off carefully, and 100 μL DMSO was added into each well. The absorbance values were measured at a wavelength of 492 nm using a KHB ST-360 microplate reader (Shanghai Kehua).

mL/min. The dn/dc value of each polymer was determined at 532 nm in DMF at 50 °C using a BI-DNDC differential refractometer. Transmission Electron Microscopy (TEM). TEM measurements were carried out by a Hitachi H-600 instrument (JEOL Ltd., Japan) at an accelerating voltage of 75 kV. Samples were prepared as follows: the aqueous solution of the triblock copolymer (0.5 mg/mL), phosphotungstic acid (PTA) and Formvar-coated copper grids were placed in an oven with temperature of 38 °C for 30 min to reach temperature equilibrium. A droplet of sample solution was then dripped onto the copper grids, which were dried in the oven for another 30 min. Finally, the samples were negatively stained with PTA and dried in the oven before observation. Dynamic Light Scattering (DLS). Copolymer solutions for light scattering studies were prepared with PBS (0.5 mg/mL) and filtered through a 0.45 μm filter prior to use. DLS measurement was conducted with a BI-200SM (Brookhaven Instruments). Scattering angle and He−Ne laser operating were set at 90° and 532 nm. During the measurement, the polymer solution was equilibrated for 10 min at each temperature point. The results were analyzed by the regularized CONTIN method. Rheometric Studies. The copolymers were dissolved in PBS solution. The pH of the solution was tested by pH meter. Rheometric measurements were performed with a Bohlin Gemini 200 rheometer (Malvern Instruments) with a 40 mm parallel plate and gap of 200 μm. Precautions were taken to minimize water evaporation. The linear viscoelasticity regimes were determined by the strain tests from the stain of 0.001 to 0.5 at several frequencies at 20 and 50 °C respectively. A optimum frequency of 0.8 Hz was chosen, and a appropriate strain of 0.4 was determined within the corporate linear viscoelasticity regimes of all copolymers and concentrations. The measuring temperatures range from 20 to 50 °C with a heating rate of 2 °C/min. Mineralization in SBF. A dialysis bag with MWCO 8000−14000 was loaded with PNIPAM−PAA(PEA)−PNIPAM aqueous solution (2.0 w/v% in PBS, 2 mL) and immersed in 100 mL of SBF in a polyethylene beaker (100 mL, diameter 5 cm, height 5.6 cm, supplier: Haihong, Chengdu) at 37 °C. SBF was replaced by preheated fresh solution every day. The conventional SBF was used:31,32 NaCl (7.996 g/L), NaHCO3 (0.350 g/L), KCl (0.224 g/L), K2HPO4·3H2O (0.228 g/L), MgCl2·6H2O (0.305 g/L), HCl (1 mol/L, 40 mL), CaCl2 (0.278 g/L), Na2SO4 (0.071 g/L), and tris(Hydroxymethyl)aminomethane (Tris, 6.057 g/L). The concentration and pH of SBF were Na+ 142, Ca2+ 5.0, Mg2+ 1.5, Ca2+ 2.5, Cl− 147.8, HCO3− 4.2, HPO42− 1.0, SO42− 0.5 mmol/L and 7.25, which was almost equal to those of human blood plasma. After incubation for 2 weeks, the dialysis bag was soaked in 1 L of distilled water (37 °C) for 12 h to remove the free ion. After that, liquid nitrogen was used to freeze the sample, which was then freeze-dried to obtain the mineralized gels. SEM (Hitachi S-450, 20 kV, Japan) was used to examine the surface of the mineralized gels. The samples were sputter-coated with gold before observation. Energy disperse spectra (EDS) (INC350, Oxford, UK) was applied to measure the Ca/P ratio. X-ray diffraction analysis (Dmax 1400, 40 kV, 110 mA, Japan) was performed to examine the formed crystal Cell Viability Test. The cytotoxicity of PNIPAM−PAA(PEA)− PNIPAM was evaluated via MTT assay. Bone marrow stromal cells (BMSCs) were cultured to the third generation. Cells were seeded in a 96-well plate at an initial density of 104 cells/well and cultured in 100

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RESULTS AND DISCUSSION Copolymer Synthesis. The PNIPAM−PAA(PEA)−PNIPAM triblock copolymers were synthesized by two-step ATRP Table 2. Summary of the Composition and Gelation Behavior of PNIPAM−PAA(PEA)−PNIPAM samples

PEA% in B blocka

Tgelb(oC)

PNIPAM64−PAA(PEA)303−PNIPAM64 PNIPAM117− PAA(PEA)303−PNIPAM117 PNIPAM155− PAA(PEA)303−PNIPAM155 PNIPAM48− PAA(PEA)377−PNIPAM48 PNIPAM326− PAA(PEA)377−PNIPAM326

27.2 38.8 6.8 26.9 39.1

 32.0 45.3 37.8 32.9

a Calculated by 1H NMR data. bThe temperature at which G′ equals G″ is designated the critical gelation temperature, Tgel.

and subsequent modification with phosphate groups. As shown in Scheme 1, the difunctional initiator DEDBA and the CuBr/ PMDETA catalytic system are used to initiate the ATRP of tBA to produce the bromo-terminated macroinitiator, i.e., Br− PtBA−Br.33 After that, the copolymerization with NIPAM was carried out in DMF using a CuCl/Me6TREN catalytic system. DMF was chosen as the reaction solvent due to the different solubilities of PtBA and NIPAM. In this reaction, the halogen exchange between Br of the macroinitiator and Cl of the catalyst could increase the initiation efficiency.30 The successful copolymerization of NIPAM onto the PtBA homopolymers was confirmed by both 1H NMR and FT-IR spectra. Figure 1a,b displays the signals at 1.44 ppm assigned to the tert-butyl ester group of PtBA, and also signals at 1.19, 4.0, and 6.3 ppm, which are ascribed to the isopropyl groups and amide bond of PNIPAM. Meanwhile, the FT-IR spectra (Figure S1a,b) exhibit strong absorption bands at 1730 cm−1 assigned to the stretching of CO, a scissor band at 1470−1380 cm−1 due to the spin−spin splitting of the methyl of the tert-butyl ester groups, which indicates the existence of PtBA block. Characteristic peaks at 1650 cm−1 and 1550 cm−1 are ascribed to the amide I and amide II of PNIPAM blocks, respectively. The absolute molecular weight and molecular weight distribution of Br−PtBA−Br and PNIPAM−PtBA−PNIPAM were characterized by GPC/LLS analysis and shown in Table 1. The absolute molecular weight of PNIPAM−PtBA−PNIPAM 2209

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Figure 3. TEM image of the dried flower-like micelles obtained by drying the 0.5 mg/mL aqueous solution of PNIPAM117−PAA(PEA)303−PNIPAM117 and then staining with phosphotungstic acid at 38 °C.

were formed between O-phosphoethanolamine and polycarboxylic acid, resulting in the singlets at 3.8 and 3.4 ppm in Figure 1d, which were ascribed to the methylenes of Ophosphoethanolamine. Further evidence of the successful conjunction is shown in the FT-IR spectrum (Figure S1d), as there are characteristic absorption bands at 905 cm−1 (the symmetric vibration of P−O), 970 cm−1 (the asymmetric vibration of P−O), and 1200 cm−1 (the phosphonyl group (PO), indicating the existence of phosphate groups.35 The PNIPAM−PAA−PNIPAM samples are grafted with different amounts of PEA for comparison purposes, as shown in Table 2. Aqueous Solution Properties. As mentioned above, the ABA triblock copolymer with soluble B block and stimuliresponsive A blocks can form either flower-like micelles or micellar gels in concentrated or dilute solution, following the similar stimuli-induced self-assembly process.19,21 Thus the dilute solution properties from the variable-temperature DLS and TEM may shed light on the nature of the sol−gel transition. Figure 2a shows the hydrodynamic diameter (Dh) distribution of PNIPAM117−PAA(PEA)303−PNIPAM117 in PBS at the concentration of 0.5 mg/mL. Both PNIPAM and

Figure 2. (a) Distribution of the hydrodynamic diameter (Dh) measured by DLS for PNIPAM117 −PAA(PEA) 303−PNIPAM 117 aqueous solution (0.5 mg/mL, PBS) at different temperatures. (b) Changes of the Dh of PNIPAM117−PAA(PEA)303−PNIPAM117 aqueous solution (0.5 mg/mL) with the increase of temperature.

measured by GPC/LLS is quite close to the value calculated from the 1H NMR peak area ratio at 1.19 ppm (isopropyl, 6 H) to 1.44 ppm (tert-butyl, 9 H), indicating the reliability of the GPC data. Meanwhile, the GPC traces shown in Figure S2 intuitively exhibit the molecular weight distribution of the triblock copolymer and confirm the successful copolymerization by the shift of the elution peak. The triblock copolymer PNIPAM−PtBA−PNIPAM was then hydrolyzed by triflouroacetic acid to obtain PNIPAM− PAA−PNIPAM. The signal at 1.44 ppm attributed to the resonance of the methyl protons of tert-butyl moieties disappeared in Figure 1c, suggesting the complete hydrolysis of esters. After that, phosphate groups were grafted to the obtained hydrolysate, i.e., PNIPAM−PAA−PNIPAM, by the classical NHS/DCC active ester method.34 New amide bonds

Scheme 2. Aqueous Solution Properties of PNIPAM−PAA(PEA)−PNIPAMa

a (a) At low concentration, loose aggregates due to the hydrogen bonds will turn into flower-like micelles and shrink with the increase of temperature. (b) At high concentration, loose aggregates will turn into micellar network and shrink with the increase of temperature, to form a template for mineralization.

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Figure 4. (a) Variable-temperature 1H NMR spectra of PNIPAM117−PAA(PEA)303−PNIPAM117 recorded at different temperatures (2.0 w/v%, in D2O). (b) Signal changes as a function of temperature (the data are normalized according to the intensity of TMSP internal standard). (c) Photos of the free-flowing liquid and free-standing gel of PNIPAM117−PAA(PEA)303−PNIPAM117 in PBS (2.0 w/v%) at 26 and 37 °C, respectively.

PAA(PEA) blocks are believed to be water-soluble at 26 °C, but the Dh at 400 nm is observed in Figure 2a. The DLS data strongly indicates the existence of aggregation at low temperature, which should be the result of hydrogen bonds between PNIPAM and residual carboxyl groups. With the increase of temperature, the Dh of main aggregates declines sharply from 400 nm to reach an almost constant value of 180 nm in Figure 2b (the discrete DLS data at each point are provided in Figure S3). It is reasonable since the dehydration of PNIPAM with the increase of temperature may break the hydrogen bonds between NIPAM and AA units,28 and change the disordered and loose aggregates into flower-like micelles. The micellization and shrinkage of the micellar core should be responsible for the sharp decline of Dh, as shown in Scheme 2. This hypothesis is also proved by the TEM image of flower-like micelles, which was obtained by drying a 0.5 mg/mL aqueous solution of PNIPAM117−PAA(PEA)303−PNIPAM117 and staining with PTA at 38 °C (Figure 3). As can be seen, individual flower-like micelles without bridging were formed at low concentration as reported in lots of related experimental,19 theoretical,22 and simulation studies,23,26 which suggests that the effect of hydrogen bonds is limited, especially at elevated temperature. On the other hand, when the concentration is sufficiently high, a transition from loose aggregates to micellar network is preferred, and the shrinkage of the micellar cores (or hydrophobic domains) may create irregular channels or pores in the gel (see Scheme 2b). Further evidence can be found in mineralization results of the thermoreponsive gel, as shown in a later section. The variable-temperature 1H NMR study of concentrated copolymer solution was carried out to reveal more detail for the thermo-induced sol−gel transition. PNIPAM 117 −PAA(PEA)303−PNIPAM117 was dissolved in D2O to create a solution that can switch between free-flowing liquid and freestanding gel. As shown in Figure 4a,b, the intensity of signals “b” and “c” ascribed to the methyl and methylene protons of the isopropyl group decrease gradually over the range of 26 to 41 °C, suggesting reduced solvation and mobility of the PNIPAM blocks. Meanwhile, the signals labeled “e” and “f” due

to the methylene protons of the PAA(PEA) block remain as they were, except for slight changes in chemical shift. The reduction in intensity of signals assigned to the PNIPAM blocks provides the positive evidence for the increase in the incompatibility between polymers and solvent. As the result, the enthalpic penalty increases, leading to the incorporation of dangling PNIPAM chain ends into hydrophobic domains and forming bridges and loops. It is noteworthy that the signals of isopropyl groups do not disappear in spectra even if the temperature is raised to 41 °C, which implies that the PNIPAM blocks still possess a certain level of mobility beyond the LCST of PNIPAM. It may be due to the dangling of the copolymer chain ends in the solution. In Figure 4c, the free-flowing liquid at 26 °C and free-standing gel at 37 °C of PNIPAM117− PAA(PEA)303−PNIPAM117 in PBS are somewhat transparent in appearance, indicating a delicate but stable balance between hydrophilicity and hydrophobicity over the temperature range. The Rheological Properties. Considering the potential practical applications of those copolymers, the rheological measurements were conducted to reveal more about the thermo-induced sol−gel transitions and their influencing factors, such as the length of the blocks and copolymer concentration. During the oscillatory shear measurement of the PNIPAM−PAA(PEA)−PNIPAM in PBS aqueous solution, the linear viscoelasticity regime was first determined (Figure S4 and Figure S5), by which the appropriate frequency and shear strain were selected for all of the subsequent temperature sweep measurements. Under that condition, rheological data from different copolymer solutions can be discussed, in terms of copolymer composition and concentration. For most of the solutions, the same trend is observed as shown in Figure 5. At low temperature, both the storage modulus (G′) and loss modulus (G″) are low. Additionally, the storage modulus G′ is always lower than the loss modulus G″, suggesting a freeflowing liquid. With the increase of temperature, both G′ and G″ increase by 1−3 orders of magnitude to reach an almost constant high level. Since the increasing rate of G′ is larger than that of G″, a crossover is reached, and the G′ is larger than G″ 2211

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blocks, the modulus curves of PNIPAM117−PAA(PEA)303− PNIPAM117 start to increase at 30 °C and approximately reach a high constant value (10 Pa or higher) at around 40 °C. A similar phenomenon has been observed in Figure 5b. With much longer PNIPAM blocks, the PNIPAM 326 −PAA(PEA)377−PNIPAM326 has a lower onset temperature, lower Tgel, and higher moduli than that of PNIPAM48− PAA(PEA)377−PNIPAM48. It is reasonable since the size of PNIPAM blocks could contribute to the incompatibility between copolymers and solvent at elevated temperature, leading to a strong aggregation tendency and stable junctions or micelle cores, which are related to the Tgel and value of modulus, respectively. Besides the outer PNIPAM block, the middle PAA(PEA) block also plays an interesting role on the gelation behavior of the copolymers. In Figure 5a,b, the PNIPAM64−PAA(PEA)303−PNIPAM64 and PNIPAM48−PAA(PEA)377−PNIPAM48 have similar size of outer blocks, but their rheological behaviors are quite different. According to the Mattice’s simulation, the middle block size doses not significantly affect the overall gelation behavior; but can affect the distribution of chain conformation.23 It has been proven that the increase in middle block size leads to the decrease of loops and the increase of bridges and dangling chain ends, due to a corresponding increase in the entropy loss from the backfolding of the middle block.28 In a word, a longer inner block means larger possibility of bridges and therefore more obvious gelation. However, attention should be paid to the unexpected curves of PNIPAM155− PAA(PEA)303−PNIPAM155 in Figure 5a, as the one with longer PNIPAM blocks does not exhibit obvious sol−gel transition. It could be due to the low phosphorylation degree of PNIPAM155−PAA(PEA)303−PNIPAM155 (Table 2), which means there is lower electrostatic repulsion from the phosphate group located at the inner blocks and less stretched chain conformation. At this condition, intramolecular aggregates are more appropriate, which leads to the loops conformation instead of bridges. Figure 5c shows the modulus curves of PNIPAM117− PAA(PEA)303−PNIPAM117 at different concentrations. At 0.5 w/v%, the G′ and G″ curves show no significant changes. While the concentration is increased to 1 w/v%, the sol−gel transition phenomenon is observed and gradually shifts to the low temperature range. Meanwhile, the G′ and G″ undergo a notable increase as expected. The effect of concentration may be explained as follows. Bridge conformation does not emerge until the critical concentration point is reached, where the average intermicellar distance is small enough to allow bridging without significant chain stretching. A higher concentration indicates shorter intermicellar distance, which could lead to more bridges conformation. It is also noted that the triblock copolymers can form free-standing gels below 37 °C at a relatively low concentration (2 w/v% in PBS, Tgel = 32 °C). Mineralization Properties. To examine its capability of inducing in situ mineralization, the physical gel formed by the PBS solution of PNIPAM117−PAA(PEA)303−PNIPAM117 (2 w/v%, in dialysis bag) was incubated in the SBF for 2 weeks.37 Figure 6a,c shows SEM photos of the original gel before mineralization. As shown in Figure 6b, the mineralized gel exhibits porous structure, which can benefit its potential application for bone tissue engineering. The porous structure is proved by diffusion experiment as discussed in Figure S6. These mineralized gels possess three-dimensional structure with fine details and seem to be the hybrid composites of micellar

Figure 5. Plots of dynamic storage modules (G′) and dynamic loss modulus (G″) versus temperature for (a) PNIPAM64−PAA(PEA)303− PNIPAM64, PNIPAM117−PAA(PEA)303−PNIPAM117, and PNIPAM155−PAA(PEA)303−PNIPAM155 (2.0 w/v%, in PBS); (b) PNIPAM 48 −PAA(PEA) 377 −PNIPAM 48 and PNIPAM 64 −PAA(PEA)303−PNIPAM64 (2.0 w/v%, in PBS); and (c) PNIPAM117− PAA(PEA)303−PNIPAM117 in PBS aqueous solution at concentrations of 0.5, 1.0, 1.5, and 2.0 w/v%.

at high temperatures, which indicates the gelation of the triblock copolymers. The above trends can be partially explained by Mattice’s Monte Carlo simulation for ABA triblock copolymers with solvent-incompatible A blocks.23,36 Loops, bridges, and dangling chain ends suffer a certain level of enthalpic penalty because of folding, stretching, and dangling in an incompatible solvent, respectively. At low temperature (above the LCST of PNIPAM), enthalpic penalty due to the poor solubility is relatively low, and the dangling chain end conformations take the majority. While the temperature is elevated, the increase in enthalpic penalty will trigger the incorporation of dangling PNIPAM chain ends into hydrophobic domains, forming loops or bridges. If the concentration is sufficiently high, or in other words, the micellar distance is small enough to allow bridging without significant chain stretching, the bridges confirmation is more favorable. As a result, a three-dimensional network is formed, which can be indicated by the changes of G′ and G″. In Figure 5a, the G′ and G″ curves of PNIPAM64− PAA(PEA)303−PNIPAM64 exhibit a very slight increase over the range of 30−40 °C and show no Tgel. With longer outer 2212

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Biomacromolecules

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Figure 6. SEM images of PNIPAM117−PAA(PEA)303−PNIPAM117 gel before (a,c) and after (b,d) mineralization. The area within the white box was selected for EDS measurement, as shown in the same image.

Figure 8. Cytotoxicity assay of PNIPAM117−PAA(PEA)303−PNIPAM117 at different concentrations in BMSC cells by MTT assay.

Figure 7. XRD patterns of mineralized PNIPAM117−PAA(PEA)303− PNIPAM117, original PNIPAM117−PAA(PEA)303−PNIPAM117 gel, and HA.

[310]) of crystalline HA, which differs from that of the original gel (Figure 7). The cytotoxicity of PNIPAM117−PAA(PEA)303−PNIPAM117 at a series of concentrations from 7.81 to 500 μg/mL was evaluated by MTT assay using BMSCs. As shown in Figure 8, the triblock copolymer displays superior cell viability in the range of 84.57 to 95.83%, which indicates that it has very low cytotoxicity at a wide range of concentration and is biocompatible for potential medical applications.

network and inorganic particles (Figure 6d). The strong affinity between acid groups of the gels and calcium ion in the solution will result in so-called template-driven nucleation and mineral growth, especially on the surface.17 Additionally, the micellar network of the thermoresponsive gel served as the template during the mineralization and seems to be “frozen” by the inorganic phase in that way (as shown in Scheme 2b). The calibrated EDS (inset of Figure 6d) area analysis performed on the surface of the mineralized gels revealed a Ca/P ratio of 1.61, which is similar to that of the hydroxyapatite (HA) in natural bones. Also, the XRD pattern of the composite exhibits three broad peaks that match the typical reflection ([002], [211] and

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CONCLUSIONS In this study, a type of thermoresponsive ABA triblock copolymers, i.e., PNIPAM−PAA(PEA)−PNIPAM, was successfully synthesized via ATRP and subsequent modification. 1 H NMR, FT-IR, and GPC/LLS spectra have been used to 2213

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Protocols; Morgan, J. R., Yarmush, M. L., Eds.; Humana Press: Totowa, NJ, 1999; pp 75−83. (15) Taguchi, T.; Xu, L.; Kobayashi, H.; Taniguchi, K.; Kataoka, K.; Tanaka, J. Biomaterials 2005, 26 (11), 1247−1252. (16) Ohya, S.; Kidoaki, S.; Matsuda, T. Biomaterials 2005, 26 (16), 3105−3111. (17) Song, J.; Saiz, E.; Bertozzi, C. R. J. Am. Chem. Soc. 2003, 125 (5), 1236−1243. (18) Addadi, L.; Weiner, S. Pro. Natl. Acad. Sci. U.S.A. 1985, 82 (12), 4110−4114. (19) Madsen, J.; Armes, S. P.; Bertal, K.; Lomas, H.; Macneil, S.; Lewis, A. L. Biomacromolecules 2008, 9 (8), 2265−2275. (20) Kirkland, S. E.; Hensarling, R. M.; McCormick, S. D.; Guo, Y.; Jarrett, W. L.; MoCormick, C. L. Biomacromolecules 2008, 9 (2), 491− 486. (21) Zhou, C.; Hillmyer, M. A.; Lodge, T. P. J. Am. Chem. Soc. 2012, 134 (25), 10365−10368. (22) Balsara, N. P.; Tirrell, M.; Lodge, T. P. Macromolecules 1991, 24 (8), 1975−1986. (23) Nguyen-Misra, M.; Mattice, W. L. Macromolecules 1995, 28 (5), 1444−1457. (24) Ma, Y.; Tang, Y.; Billingham, N. C.; Armes, S. P.; Lewis, A. L. Biomacromolecules 2003, 4 (4), 864−868. (25) Li, C.; Buurma, N. J.; Haq, I.; Turner, C.; Armes, S. P.; Castelletto, V.; Hamley, I. W.; Lewis, A. L. Langmuir 2005, 21 (24), 11026−11033. (26) Madsen, J.; Armes, S. P.; Lewis, A. L. Macromolecules 2006, 39 (22), 7455−7457. (27) Li, C.; Tang, Y.; Armes, S. P.; Morris, C. J.; Rose, S. F.; Lloyd, A. W.; Lewis, A. L. Biomacromolecules 2005, 6 (2), 994−999. (28) Staikos, G.; Bokias, G.; Karayann, K. Polym. Int. 1996, 41 (3), 345−350. (29) Morozowich, N. L.; Modzelewski, T.; Allcock, H. R. Macromolecules 2012, 45 (19), 7684−7691. (30) Ravi, P.; Wang, C.; Dai, S.; Tam, K. C. Langmuir 2006, 22 (17), 7167−7174. (31) Oyane, A.; Kim, H. M.; Furuya, T.; Kokubo, T.; Miyazaki, T.; Nakamura, T. J. Biomed. Mater. Res., Part A 2003, 64A (2), 188−195. (32) Kokubo, T.; Hushitani, H.; Sakka, S.; Kitsugi, T.; Yamamuro, T. J. Biomed. Mater. Res 1990, 24 (6), 721−734. (33) Shipp, D. A.; Wang, J. L.; Matyjaszewski, K. Macromolecules 1998, 31 (23), 8005−8008. (34) Ko, Y. G.; Ma, P. X. J. Colloid Interface Sci. 2009, 330 (1), 77− 83. (35) Kim, S. Y.; Lee, S. C. J. Appl. Polym. Sci. 2009, 113 (6), 3460− 3469. (36) Nguyen-Misra, M.; Mattice, W. L. Macromolecules 1995, 28 (20), 6976−6985. (37) Kokubo, T. Acta Mater. 1998, 46 (7), 2519−2527. (38) Sohier, J.; Hamann, D.; Koenders, M.; Cucchiarini, M.; Madry, C.; Blitterswijk, V.; Groot, K.; Bezemer, J. M. Int. J. Pharm. 2007, 332 (1−2), 80−89.

confirm the syntheses. The triblock copolymers can form freestanding gels at 37 °C even at a quite low concentration (2 w/v % in PBS). The results from variable-temperature 1H NMR, dynamic light scattering, and rheological measurements indicate that the gelation behavior is based on the hydrophobic interactions among the PNIPAM blocks and is highly dependent on both the length of PNIPAM bocks and substitution degree of PEA content. The gels could induce in situ mineralization of hydroxyapatite after being incubated in the SBF for 2 weeks. PNIPAM−PAA(PEA)−PNIPAM also exhibits low cytotoxicity in cell viability tests, thus it shows great potential for bone tissue engineering.

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ASSOCIATED CONTENT

S Supporting Information *

Additional characterizations of polymers, including FT-IR spectra, GPC traces, distribution of hydrodynamic diameter by DLS, shear strain sweep, and the diffusion test. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Financial support from the National Natural Science Foundation of China (51073102), Fok Ying Tung Education Foundation (122034), Program for New Century Excellent Talents in University (NCET-10-0592), Program for Changjiang Scholars and Innovative Research Team in University (IRT1163), Foundations of Sichuan Province (2012JQ0009), Fundamental Research Funds for the Central Universities (2010SCU22001, 2011SCU04A04) and Natural Science Foundation of Jiangsu Province (BK2010248, BK2011340) are gratefully acknowledged.

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