Synthesis, Characterization and Application of Thermoresponsive

Jun 28, 2016 - Molecular weights and polydispersity index of samples were measured by a Shimadazu LC-20A instrument equipped with a RID-10A refractive...
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Synthesis, Characterization and Application of Thermoresponsive Polyhydroxyalkanoate-graf t-Poly(N‑isopropylacrylamide) Yi-Ming Ma,†,‡ Dai-Xu Wei,† Hui Yao,† Lin-Ping Wu,§ and Guo-Qiang Chen*,†,‡ †

Center of Synthetic and Systems Biology, School of Life Science, Tsinghua-Peking Center for Life Sciences, Tsinghua University, Beijing 100084, China ‡ Center for Nano and Micro Mechanics, Tsinghua University, Beijing 100084, China § Department of Pharmacy, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen 2100, Denmark ABSTRACT: A thermoresponsive graft copolymer polyhydroxyalkanoate-g-poly(N-isopropylacrylamide) or short as PHA-gPNIPAm, was successfully synthesized via a three-step reaction. First, PNIPAm oligomer with a trithiocarbonate-based chain transfer agent (CTA), short as PNIPAm-CTA, with designed polymerization degree was synthesized via reversible addition− fragmentation chain transfer (RAFT) polymerization. Subsequently, the PNIPAm-CTA was treated with n-butylamine for aminolysis in order to obtain a pendant thiol group at the end of the chain (PNIPAm-SH). Finally, the PNIPAm-SH was grafted onto unsaturated P(3HDD-co-3H10U), a random copolymer of 3hydroxydodecanoate (3HDD) and 3-hydroxy-10-undecylenate (3H10U), via a thiol−ene click reaction. Enhanced hydrophilicity and thermoresponsive property of the resulted PHA-g-PNIPAm were confirmed by water contact angle studies. The biocompatibility of PHA-g-PNIPAm was comparable to poly-3hydroxybutyrate (PHB). The graft copolymer PHA-g-PNIPAm based on biopolyester PHA could be a promising material for biomedical applications.

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INTRODUCTION Polyhydroxyalkanoates (PHA) are a family of hydrophobic, biodegradable, and biocompatible polyesters synthesized by many microorganisms. They are generally considered to be candidates for various biomedical applications including tissue engineering and drug delivery carrier.1−6 The above properties allow PHA to be studied in medical areas. Although PHA has been developed as an environmentally friendly Bioplastic with tunable thermal and mechanical properties for a long time, it is still facing problems of high production costs as well as poor mechanical properties compared to many petroleum based plastics.7 In order to improve its properties and increase its values, PHA is usually physically or chemically modified.8 However, PHA with saturated main/side chains is relatively inert, thus the efficiency of traditional modifications is generally low, while the modification effects are usually limited.9 In some cases, the modification processes are so violent that many favorable properties of PHA are even compromised, let alone the toxic residues after many modifications.10 Therefore, it is necessary to introduce functional groups directly to PHA for possible high value-added applications.11−13 It has already been demonstrated to be feasible to biosynthesize PHA containing functional groups such as double/triple bonds, epoxy, carbonyl, cyano, phenyl, halogen et al., respectively;14−18 the resulting PHA with functional groups in the side chains can thus allow further modifications.8 Due to the chemical activity © XXXX American Chemical Society

of carbon−carbon double bonds, unsaturated PHA have been chosen in some studies. For example, poly(3-hydroxy-10undecenoate) (PHU) and other unsaturated PHA were reported to be synthesized by various Pseudomonas strains grown on various unsaturated carbon sources.19−21 In a previous study, we reported successful biosynthesis of structurally controlled unsaturated poly(3-hydroxydodecanoate-co-3-hydroxy-9-decenoate) or P(3HDD-co-3H9D) for the first time using Pseudomonas entomophila LAC23 defected in its β-oxidation pathway.7 The double bonds in the side chains can thus be transformed into various functional groups such as hydroxyl, carboxyl, epoxy, chlorine, amide, etc.8,22 These functional groups with different physical and chemical properties can then be further conjugated with oligomers, bioactive compounds or targeting molecules. Thiol−ene click reaction is a simple, specific and efficient method to perform functionalization of the unsaturated bonds and has attracted increasing interests in recent years due to its valuable properties including: mild reaction conditions, compatible with various functional groups (carboxyl, hydroxyl, amine, etc.), biocompatible (no involvement of metallic catalysts compared with copper-catalyzed azide−alkyne cycloaddition (CuAAc) reacReceived: May 18, 2016 Revised: June 16, 2016

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DOI: 10.1021/acs.biomac.6b00724 Biomacromolecules XXXX, XXX, XXX−XXX

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Scheme 1. (a) Chemical Structure of Unsaturated P(3HDD-co-3H10U); (b) Synthesis of Thermoresponsive PHA-g-PNIPAm via a Three-Step Reaction; n = 10, 20

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tion, thus avoiding biological toxicity) and environmentfriendly.23−27 For example, Le Fer et al.10,28 prepared PHA-gPEG and PHA-g-Jeffamine [Jeffamine is short for α-amino-ωmethoxy poly(oxyethylene-co-oxypropylene)] amphiphilic copolymers, respectively, via thiol−ene click reactions. However, functionalization of unsaturated PHA through thiol−ene click reactions is still rarely reported so far. It has not yet reported any feasibility to obtain functional PHA for “smart” applications, such as thermoresponsive PHA. Interest in thermoresponsive biopolymers has increased due to its promising potential in the field of biomedical applications, ranging from functional bioactive surfaces, thermo-reversible separators, biosensor, and tissue engineering to intelligent drug/gene delivery.29−33 Poly(N-isopropylacrylamide) (PNIPAm) as a “gold standard”, represents one of the most intensively studied thermoresponsive polymers. The phase transition of PNIPAm is accompanied by the formation of inter- and/or intrachain hydrogen bonds above its lower critical solution temperature (LCST) in water;34 it is soluble in water below around 32.8 °C, yet insoluble above this cloud point (CP).35,36 The LCST of PNIPAm is close to physiological temperature and is relatively insensitive to environmental conditions. Yet it can be tuned by copolymerization with other monomers; 37 for example, the addition of hydrophilic monomers typically increases the LCST, while the incorporation of more hydrophobic units has the opposite effect.38 The modification of the LCST of PNIPAm is of primary interest,34,39−42 for example, Toraman et al. prepared PHAbased amphiphilic graft copolymers with PNIPAm via bromination, substitution, and RAFT polymerization.43 In this study, we aimed to synthesize thermoresponsive PHA graft copolymers for possible medical applications.

EXPERIMENTAL SECTION

Materials. Unsaturated P(3HDD-co-28.8% 3H10U) (Scheme 1a (Mw ≈ 85 600, Mn ≈ 48 762, PDI ≈ 1.76) was microbially synthesized by Pseudomonas entomophila LAC23 with chromosomal β-oxidation related genes fadB, fadA, and PSEEN 0664 deleted when fed with dodecanoic acid (66.7%) and 10-undecenol (33.3%). PHB (98%, Tianan Biotech Co. Ltd., Ningbo/China), polylactide (PLA, Daigang Biotech Co. Ltd./China, Mw = 10 000), 2-(Dodecylthiocarbonothioylthio)-2-methylpropionic acid (98%, Sigma-Aldrich), dimethoxy2-phenylacetophenone (DMPA, 98%, Tci), n-butylamine (99%, Tci), chloroform (Analytical reagent, Beijing Chemical works), tetrahydrofuran (THF, analytical reagent, Beijing Chemical Works), toluene (Analytical reagent, Beijing Chemical Works) and n-hexane (Analytical reagent, Beijing Chemical Works) were used as received. NIsopropylacrylamide (NIPAm, 98%, Sigma-Aldrich) was recrystallized in n-hexane before use. 2,2′-Azobis (2-methylpropionitrile) (AIBN, 98%, Tci) was recrystallized in methanol before use. Synthesis of PNIPAm-CTA Oligomer via RAFT Polymerization. PNIPAm-CTA oligomer was synthesized by reversible addition−fragmentation chain transfer (RAFT) polymerization, using a trithiocarbonate-based chain transfer agent (CTA). For example, if the polymerization degree of the PNIPAm oligomer is designed to be n, the CTA of 1/n molar ratio of the NIPAm monomer will be used, while AIBN of 20 mol % of the CTA will be used as an initiator. The reaction was carried out at 75 °C in argon atmosphere for 12 h, using toluene as the solvent (the concentration of NIPAm was about 0.5 mmol/mL). After the reaction, the solution was precipitated in cold nhexane. The product PNIPAm-CTA was collected after centrifugation at 10,000 rpm for 20 min (CR22GIII, Hitachi, Japan, equipped with a R12 rotor) and vacuum-dried at room temperature for 24 h before further characterization and reactions. Synthesis of PNIPAm-SH Oligomer via Aminolysis. The PNIPAm-CTA was redissolved in THF (the concentration of NIPAm was about 0.5 mmol/mL) and then treated with n-butylamine (6 mol equiv of CTA) to obtain thiol terminated PNIPAm-SH. The reaction was accomplished at room temperature in less than 3 h, with a very obvious color change from dark yellow to colorless. After the reaction, the product PNIPAm-SH was also precipitated in cold n-hexane, B

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Figure 1. 1H NMR (C4D8O, 600 MHz) spectrum (left) and photos (right) of PNIPAm-CTA (red) and PNIPAm-SH (green). centrifuged and vacuum-dried at room temperature for 24 h before further characterization and reactions. Synthesis of PHA-g-PNIPAm via Thiol−ene Click Reaction. Unsaturated P(3HDD-co-3H10U) was grafted with the PNIPAm-SH via a thiol−ene click reaction. Both 75 °C heating and 365 nm UV light were used to initiate reactions, the latter was proven to be more efficient, and thus was chosen in this study. The molar ratio of thiol groups to double bonds was 1.2:1 to achieve a higher reaction yield, as steric hindrance of this reaction is high. The reaction was carried out under 365 nm UV light in argon atmosphere for 12 h, using 1 wt % of DMPA as the photo initiator and THF as the solvent (the concentration of NIPAm was about 0.5 mmol/mL). After the reactions, the solution was precipitated in cold n-hexane. Subsequently, the final product was collected by centrifugation at 10 000 rpm for 20 min (CR22GIII, Hitachi, Japan, equipped with a R12 rotor) and vacuum-dried at room temperature for 24 h. Also, polymer films were solvent-casted at room temperature in Corning glass Petri dishes using THF as the solvent (at a concentration of 50 mg/mL) before further characterization. The thickness of the films was around 0.15−0.2 mm. Nuclear Magnetic Resonance (NMR) Characterization. 1H NMR spectra were recorded on a JEOL JNM-ECA600 nuclear magnetic resonance instrument using deuterated chloroform (CDCl3) or THF (C4D8O) as a solvent to determine the structure of the above intermediates and products. The chemical shifts were calibrated against the solvent signals or 0.03% v/v tetramethylsilane (TMS) as the internal reference. Matrix-Assisted Laser Desorption/Ionization Time-of-flight (MALDI-TOF) Analysis. All mass spectra were measured by a Shimadzu AXIMA-Performance MA mass spectrometer equipped with a nitrogen laser. 2,5-Dihydroxybenzoic acid was used as the matrix, 1 μL matrix solution in ethyl acetate (20 mg/mL) was applied to the target plate, and 1 μL saturated methanolic NaBF4 was added. After evaporation, a smooth layer of crystals was formed. Then 1 μL sample solution (5 mg/mL, chloroform) was applied and evaporated. The laser power was adjusted for good ion yield to improve the S/N ratio.44 Gel Permeation Chromatography (GPC) Characterization. Molecular weights and polydispersity index of samples were measured

by a Shimadazu LC-20A instrument equipped with a RID-10A refractive index detector. The measurements were carried out with a Shimadzu GPC-804C column. Chromatographically pure chloroform was used as an elution liquid at a flow rate of 1 mL/min at 40 °C, and the sample volume was set to be 30 μL. Sample solutions were prepared at a concentration of 1 mg/mL and filtered by 2.5 μm nylonmicroporous membrane (ALLPURE NY 0.02 μm, Membrane Solution, USA). A standard calibration curve was prepared by polystyrene standards with different number-average molecular weights (3 × 103, 5 × 103, 3 × 104, 5 × 104, 1.5 × 105, and 3 × 105; Sigma-Aldrich, USA), and was utilized to calculate the molecular weights and polydispersity index of the samples. Thermal Gravity Analysis (TGA). TGA measurements were carried out using a Shimadazu TGA-50 at a heating rate of 10 °C/min from room temperature to 500 °C, with a nitrogen flow of 50 mL/min. The typical sample weight was between 5 and 10 mg. Light Absorbance Test. The LCST of the sample in aqueous solutions was determined by measuring their UV/vis spectroscopy at 650 nm from 25 to 55 °C on a WPA Biowave UV/vis spectrophotometer equipped with a Peltier 1 × 1 cell holder. The sample in aqueous solution with a concentration of 10 mg/mL was filtered into a dust-free quartz cell through a 0.45 μm Millipore filter, and was then equilibrated in thermostatic water bath for at least 10 min to ensure a stable temperature before measurement. Each data point was measured for three times. The average light absorbance at 55 °C was defined as 100% absorbance, while the temperature corresponding to the middle point of absorbance between 35 and 40 °C was defined as the LCST. Water Contact Angle Measurement. A DataPhysics OCA20 contact angle system was used to measure the water contact angle of the sample films. Droplets of 3 μL deionized water were dropped carefully onto the sample surface, and the average contact angle was obtained by measuring at least three different positions of the same sample. Cell Proliferation Assays. NIH 3T3-E1 mouse embryo fibroblast cells (purchased from Cell Bank of Chinese Academy of Sciences, Shanghai, China) were employed to investigate the cell proliferation on the PHA-g-PNIPAm film. Cells were cultured in DMEM medium C

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Figure 2. Molecular weights of PNIPAm-SH determined by MALDI-TOF-MS.

Figure 3. 1H NMR (CDCl3 with 0.03 v/v TMS, 600 MHz) spectrum of neat PHA (red) and PHA-g-PNIPAm (green). (Gibco, USA) with 10% fetal bovine serum (FBS, Gibco, USA) in an incubator supplied with 5% CO2 at 37 °C. The PHA-g-PNIPAm films were successively placed into 96-well tissue culture plates (TCPs), sterilized by immersion in 75% (v/v) ethanol for 6 h, washed three times with 10 mM PBS to remove ethanol, and then immersed in DMEM medium with 10% FBS for 2 h. NIH 3T3-E1 cells (10 000 cells per well) were seeded on each film and cultured for 3 and 6 days, respectively. Then the media were removed, and the CCK-8 assays were performed.45 Briefly, 180 μL DMEM medium containing 10% FBS and 20 μL CCK-8 were added to each well, incubating at 37 °C for 1.5 h, and then 100 μL of the above

solution was transferred to a 96-well plate. The absorbance at 450 nm was measured by a microplate reader (Varioskan Flash 3001−2016, Thermo, USA) and corrected by subtracting the absorbance of the mixture of 90 μL DMEM containing 10% FBS with 10 μL CCK-8. Neat PHA, PHB, and PLA films and TCPs were used as controls in this study. All the above films were prepared by solvent evaporation:46 the polymer was dissolved in THF at a concentration of 50 mg/mL, placed into Corning glass Petri dishes, dried at room temperature for 48 h, and then vacuum-dried for 12 h. The thickness of the films was around 0.15−0.2 mm. At least three replicates were prepared for each sample. Statistical comparisons were performed using Student’s t test. D

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Figure 4. TGA studies on neat PHA, PNIPAm-CTA, PNIPAm-SH and PHA-g-PNIPAm.

Figure 5. Thermoresponsive property of PNIPAm-SH. (a) Photos of PNIPAm-SH aqueous solution at 20 and 55 °C. (b) Light absorbance of PNIPAm-SH aqueous solution at different temperatures. (c) Thermal reversibility of PNIPAm-SH aqueous solution between 35 and 40 °C.

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Fluorescence Microscopy. The prepared films were placed into 96-well tissue culture plates. DiO membrane dye was used for staining NIH 3T3-E1 cells, and 35 000 cells per well were seeded on each film. Then living cell fluorescence images were captured at different times by a Nikon Eclipse Ti−S inverted microscope under 488 nm excitation light. Scanning Electron Microscopy (SEM). For SEM characterization, NIH 3T3-E1 cells on each film were fixed by 4 wt % of paraformaldehyde solution, and dehydrated with gradient ethanol of 30%, 50%, 60%, 70%, 80%, 90%, 95% and 100% (v/v), respectively. The samples were vacuum-coated with a layer of Pd/Pt alloy and observed with scanning electron microscope (Quanta 200, FEI, USA) at an accelerating voltage of 15 kV.

RESULTS AND DISCUSSION Synthesis of PHA-g-PNIPAm via a Three-step Reaction and Its Characterization. PNIPAm oligomers with a trithiocarbonate-based Chain Transfer Agent (CTA), short as PNIPAm-CTA, were synthesized via RAFT polymerization in the presence of AIBN as an initiator (Scheme 1b). A precalculated amount of CTA was utilized to design a polymerization degree of 10 or 20. The yield of PNIPAmCTA was around 90% in 24 h. After purification, the PNIPAm-CTA was redissolved in THF and treated using n-butylamine to obtain pendant thiol-groups via aminolysis (Scheme 1b). In less than 3 h, the solution gradually turned from dark yellow to colorless, indicating E

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Figure 6. Water contact angles of PHA-g-PNIPAm with different grafting rates at 30 and 37 °C, respectively. (a) PHA-g-7.1% PNIPAm, (b) PHA-g11.7% PNIPAm, (c) PHA-g-15.7% PNIPAm.

spectrum. The methylene protons (2H, −CH2−S−, 2) of PNIPAm-CTA appeared at 3.30 ppm, while the methylene protons (2H, HS−CH−CH2−, 3′) of PNIPAm-SH were visible at 2.78 ppm. The degree of polymerization and molecular weights of PNIPAm oligomers can be approximately estimated by integrations of the above three peaks. Precise molecular weights of PNIPAm-SH ranging from 800 to 1400 were determined by MALDI-TOF-MS (Figure 2). The middle value of molecular weight was found around 1250, which includes 10 NIPAm monomers as designed (MW: 1130) together with a CTA group after aminolysis (MW: 120). Finally, purified PNIPAm-SH was reacted with unsaturated PHA via a thiol−ene click reaction initiated by heating or UV light (Scheme 1b). Reaction rate of this step could be controlled by different initiating times. The molecular weights and polydispersity of the PHA-g-PNIPAm were measured by GPC (Mw ≈ 84 357, Mn ≈ 51 994, PDI ≈ 1.62), and found no significant change compared with the neat PHA (Mw ≈ 85 600, Mn ≈ 48 762, PDI ≈ 1.76) within the error range of the GPC. This also shows that the three-step reaction was performed without obvious PHA degradation or chain scission. The purified final product was characterized by 1H NMR in deuterated chloroform taken neat P(3HDD-co-3H10U) as a comparison (Figure 3).

Table 1. Water Contact Angles of PHA-g-PNIPAm, Neat PHA, and TCPS at 30 and 37°Ca

a

Sample

30 °C

37 °C

PHA-g-7.1% PNIPAm PHA-g-11.7% PNIPAm PHA-g-15.7% PNIPAm neat PHA TCPS

96° ± 1.39° 77.96° ± 1.06° 64.34° ± 1.68° 103.62° ± 2.98° 104.54° ± 2.95°

106.08° ± 2.21° 98.45° ± 3.07° 85.48° ± 1.82° 101.84° ± 4.92° 101.6° ± 1.06°

Each data was measured from at least three parallel studies.

successful aminolysis of the trithiocarbonate-based CTA (Figure 1). The purified PNIPAm-CTA and PNIPAm-SH were characterized using 1H NMR in deuterated THF (Figure 1): δ −0.85 (3H, −CH3, 1), 1.10 (12H, −CH3, 6, 9, 6′ and 8′), 1.26 (2H, −CH2−CH2−, 2), 1.61 (2H, −CH−CH2−, 7 and 3′), 2.13 (2H, −CH−CH2−, 3 and 2′), 2.78 (2H, HS−CH− CH2−, 3′), 3.30 (2H, −CH2−S−, 2), 3.49−3.50 (1H, HS− CH−, 2′), 3.96 (1H, −CH−NH−, 5 and 5′). Aminolysis can be visually confirmed via the color change of the solution (from dark yellow to colorless), which was attributed to cleavage of the trithiocarbonate group. The characteristic signals of NIPAm (1H, −CH−NH−, 5 and 5′) were observed at 3.96 ppm in the F

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Figure 7. Biocompatibility studies of PHA-g-PNIPAm. (a) CCK-8 assays of cells proliferated on PHA-g-PNIPAm, neat PHA, PHB, PLA films and tissue culture plates (TCPs) respectively, *P < 0.05, **P < 0.01, n = 4. (b,c) SEM images of NIH 3T3-E1 cell proliferated on the PHA-g-PNIPAm film after 3 and 6 days, respectively.

In this 1H NMR spectrum, signals at 0.85−0.87 (3H, −CH3, 12 and 12‴), 1.24−1.27 (2H, −CH2−, 5a−11a, 5b−8b, 5′−8′, and 5‴−11‴), 1.56 (2H, −CH−CH2− CH2−, 4, 4′, and 4‴), 2.45−2.57 (2H, −CH2−CO, 2, 2′, and 2‴) and 5.14−5.18 (1H, −CH−O−, 3, 3′, and 3‴) were typical PHA chemical shifts. Signals at 4.90−4.98 (2H, −CHCH2−, 11b and 11′b) and 5.75−5.80 (1H, −CHCH2−, 10b and 10′b) were assigned to protons of the double bonds, while signals at 2.01− 2.02 (2H, −CH2−CHCH2, 9b and 9′b) represented the methylene protons adjacent to the double bonds. The characteristic signals of NIPAm (1H, −CH−NH−, 2″) were observed at 4.00 ppm in this spectrum. By calculating integrations of the above signals, the neat PHA contained 28.8 mol % 3H10U ((1.00 + 1.87)/(3.15 + 6.80) × 100% = 28.8%), while the PHA-g-PNIPAm contained 26.7 mol % 3H10U ((1.00 + 2.26)/(3.87 + 8.34) × 100% = 26.7%). Thus, the reaction ratio of the unsaturated bonds is calculated to be 7.3% ((28.8 − 26.7)/28.8 × 100% = 7.3%). Considering the signals at 4.00 ppm, each reacted unsaturated bond was grafted with approximately 10 NIPAm monomers (24.6%/ (28.8%− 26.7%) = 11.7) on average. The total grafting rate of NIPAm monomers to PHA was estimated to be 15.7% (28.8%−26.7%) × (11.7*113g/mol + 120g/mol)/(193g/mol) × 100%= 15.7%) (Here, we define PHA-g-PNIPAm short for PHA-g-15.7% PNIPAm, if not specially mentioned below). TGA curves of neat PHA, PNIPAm-CTA, PNIPAm-SH, and PHA-g-PNIPAm were compared (Figure 4). In the TGA curve of PNIPAm-SH, there were two weight loss stages similar to

PNIPAm-CTA. The neat PHA had only one weight loss stage. After the thiol−ene click reaction, PHA-g-PNIPAm showed three weight loss stages associated with overlays of the two PNIAPAm-SH stages and one PHA stage; the result indicated the successful grafting of PNIPAm oligomers on unsaturated PHA. Characterization of Thermoresponsive Property of PNIPAm-SH. An obvious solubility change of PNIPAm-SH in aqueous solution was observed visually under 20 and 55 °C (Figure 5a), and concentrations of the PNIPAm-SH aqueous solution were all 10 mg/mL. Figure 5b is a typical light absorbance-temperature curve of PNIPAm oligomer aqueous solution, all light absorbance data were measured by UV/vis spectroscopy at 650 nm, and each data point was measured three times. The average light absorbance at 55 °C was defined here as 100% absorbance. A sharp increase of light absorbance from 35 to 40 °C was exhibited in the curve. The LCST of PNIPAm oligomer was generally reported to be around 32.8 °C.35,36 Here the middle point 37.5 °C is defined as the LCST of PNIPAm-SH, which lies in the range of physiological temperature. The reason for higher LCST of this oligomer could be attributed to the hydrophilic thiol-group at the chain end of PNIPAm-SH. Further thermal reversibility test of PNIPAm-SH between 35 and 40 °C confirmed its good reproducibility of hydrophilicity/hydrophobicity transformation, which undergoes a reversible coil-to-globule transition in aqueous solution around the LCST (Figure 5c). G

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Figure 8. Cell attachment and detachment studies of PHA-g-PNIPAm. (a) CCK-8 assays of cells proliferated on PHA-g-PNIPAm, neat PHA, PHB, and PLA films and tissue culture plates (TCPs), at different temperatures. Inoculum cell density: 35 000 cells/well. (b) Cell detachment ratio of PHA-g-PNIPAm, neat PHA, PHB, PLA films and tissue culture plates (TCPs).

Characterization of Thermoresponsive Property of PHA-g-PNIPAm. Water contact angles of PHA-g-PNIPAm with different grafting rates at 30 and 37 °C are shown (Figure 6). There are reversible water contact angle changes between 30 and 37 °C due to the thermoresponsive property of PHA-gPNIPAm. Water contact angles of PHA-g-PNIPAm, neat PHA and TCPS (Tissue Culture Petri Dish, Corning) are also compared (Table 1). The neat PHA samples and TCPS systems are both considered to be hydrophobic polymers. It was obviously observable that the water contact angles decreased at both 30 and 37 °C after the thiol−ene click reaction, which can be attributed to the successful grafting of hydrophilic PNIPAm oligomers. While there are reversible water contact angle changes for PHA-g-PNIPAm between 30 and 37 °C, no obvious change could be found for the neat PHA and TCPS samples. Also, the water contact angles decreased obviously at both 30 and 37 °C, with an increasing grafting rate of PNIPAm oligomers on PHA. Biocompatibility Studies of PHA-g-PNIPAm. Activities of NIH 3T3-E1 mouse embryo fibroblast cells grown on different films were studied using CCK-8 assay after 3 and 6

days of incubation, respectively (Figure 7a): inoculum cell density was 10 000 cells/well. After 3 days of incubation, the cell growth on PHA-g-PNIPAm, neat PHA, PHB, and PLA films had no significant difference. After 6 days of incubation, the activity of NIH 3T3-E1 cells grown on the neat PHA and PLA films was slightly better than that of cells grown on the PHA-g-PNIPAm and PHB films. During the 6 days of incubation, the increase of the cell activity on all the samples were similar (although the best was tissue culture plate), indicating that PHA-g-PNIPAm was comparable with PHB for cell growth. The viable NIH 3T3-E1 cells grown on PHA-g-PNIPAm films were observed using scanning electron microscope (Figure 7). In the first 3 days of incubation, only a few cells adhered on the surface of PHA-g-PNIPAm films (Figure 7b). The viable cells grown on the film surface increased with increasing incubation time. After 6 days of incubation, cells grew to a higher density on the film surface (Figure 7 c). In this study, NIH 3T3-E1 cells proliferated well on the surface of PHA-g-PNIPAm films, demonstrating the good biocompatibility of PHA-g-PNIPAm. H

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Figure 9. Fluorescence microscopy images of NIH 3T3-E1 cells on different films at different culture temperatures. Films: (a) PHA-g-PNIPAm, (b) neat PHA, (c) PHB, (d) PLA; Temperatures: (1) 25 °C, 12 h, (2) 37 °C, 12 h and then 25 °C, 12 h, (3) 37 °C, 12 h.

Figure 10. Scanning electron microscopy images of NIH 3T3-E1 cells on different films at different culture temperatures. Films: (a) PHA-gPNIPAm, (b) neat PHA, (c) PHB, (d) PLA; Temperatures: (1) 25 °C, 12 h, (2) 37 °C, 12 h and then 25 °C, 12 h, (3) 37 °C, 12 h.

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Thermoresponsive Attachment and Detachment Studies of Cells Grown on PHA-g-PNIPAm. Viabilities of NIH 3T3-E1 mouse embryo fibroblast cells grown on different films were studied using CCK-8 assay, under different culture conditions (Details: (1) culture at 25 °C for 12 h; (2) culture at 37 °C for 12 h, and then culture at 25 °C for another 12 h; (3) culture at 37 °C for 12 h) (Figure 8a). After the incubations, the activity of NIH 3T3-E1 cells grown on different films showed similar trend: the 37 °C, 12 h groups had the best cell viability; the 25 °C, 12 h groups the worst. This may be due to a better growth at 37 °C for the cells compared with that at 25 °C. In order to evaluate thermoresponsive cell detachment ability of the different films, we defined a “cell detachment ratio” here as

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AUTHOR INFORMATION

Corresponding Author

* Tel: +86-10-62783844; Fax: +86-10-62794217; E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Dr. Lei Tao and Dr. Lei Liu from Department of Chemistry, Tsinghua University provided technical support, and Mr Longwei Cai helped us produce unsaturated PHA via microbial fermentation. We are grateful to the Center of Biomedical Analysis, Tsinghua University for the SEM studies. This research was supported by the Dutch Polymer Institute (DPI), the State Basic Science Foundation 973 (Grant no. 2012CB725201) and National Natural Science Foundation of China (Grant Nos. 31430003 and 31270146).

Cell Detachment Ratio = 1 − Cell Viability (37− 25°C)/Cell Viability (37°C)

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Under the culture conditions (short as 25 °C, 12 h; 37 °C, 12 h and then 25 °C, 12 h; 37 °C, 12 h), the PHA-g-PNIPAm films showed the highest cell detachment ratio, demonstrating PHA-g-PNIPAm could induce thermoresponsive cell attachment and detachment (Figure 8b). The viable NIH 3T3-E1 cells grown on PHA-g-PNIPAm films under different culture conditions were also studied using fluorescence microscope and scanning electron microscope (Figures 9 and 10). Obviously, both results showed similar biocompatibility for the PHA-g-PNIPAm films with that of the other films based on CCK-8 assay results (Figure 7a). Viability of NIH 3T3-E1 cells grown on different films agreed well with the CCK-8 assay results (Figure 8a): Studies conducted under 37 °C for 12 h were revealed with better cell viability than that of the proliferation groups at 37 °C for 12 h and then at 25 °C for 12 h. While the ones conducted at 25 °C for 12 h had the poorest results. Similarly, the PHA-g-PNIPAm films also showed the most observable cell detachment phenomenon (Figure 8b), indicating the strongest thermoresponsive property among the different films mentioned in this study. It is therefore confirmed that NIH 3T3-E1 cells proliferated on the surface of PHA-g-PNIPAm films demonstrated the best thermoresponsive cell detachment property.

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CONCLUSIONS A three-step reaction to prepare PHA-based graft copolymer for high value-added applications has been developed. Designed side chain PNIPAm oligomers were synthesized via a living radical RAFT polymerization with a trithiocarbonate-based chain transfer agent. The PNIPAm oligomers were then grafted onto unsaturated PHA via aminolysis and thiol−ene click reaction to obtain graft copolymer PHA-g-PNIPAm. Structures of the above intermediates and products were characterized and confirmed by 1HNMR, MALDI-TOF and GPC. The graft copolymer films showed surface hydrophilicity improvement, obvious thermoresponsive property at different temperatures, good biocompatibility for cell growth as well as thermoresponsive cell detachment ability. The reported synthesis pathway provides a new vision for PHA modifications and produced a promising material for potential biomedical applications, such as temperature-controlled drug release, thermoresponsive filter and functional textile. J

DOI: 10.1021/acs.biomac.6b00724 Biomacromolecules XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.biomac.6b00724 Biomacromolecules XXXX, XXX, XXX−XXX