Thermoresponsive Block Copolymers of Poly(ethylene glycol) and

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Biomacromolecules 2009, 10, 66–73

Thermoresponsive Block Copolymers of Poly(ethylene glycol) and Polyphosphoester: Thermo-Induced Self-Assembly, Biocompatibility, and Hydrolytic Degradation Yu-Cai Wang,† Ling-Yan Tang,‡ Yang Li,‡ and Jun Wang*,‡ Department of Polymer Science and Engineering, University of Science and Technology of China, Hefei, Anhui 230026, People’s Republic of China, and Hefei National Laboratory for Physical Sciences at Microscale and School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230027, People’s Republic of China Received July 19, 2008; Revised Manuscript Received October 29, 2008

Novel thermoresponsive block copolymers of poly(ethylene glycol) and polyphosphoester were synthesized, and the thermo-induced self-assembly, biocompatibility, and hydrolytic degradation behavior were studied. The block copolymers with various molecular weights and compositions were synthesized through ring-opening polymerization of 2-ethoxy-2-oxo-1,3,2-dioxaphospholane (EEP) and 2-isopropoxy-2-oxo-1,3,2-dioxaphospholane (PEP) using poly(ethylene glycol) monomethyl ether (mPEG) as the initiator and stannous octoate as the catalyst. The obtained block polymers exhibited thermo-induced self-assembly behavior, demonstrated by dynamic light scattering and UV-vis measurements using 1,6-diphenyl-1,3,5-hexatriene as the probe. It was found that the critical aggregation temperature (CAT) of the block copolymers shifted to higher temperature with increased molecular weight of mPEG, while copolymerization with more hydrophobic monomer PEP led to lower transition temperature; thus, the CAT can be conveniently adjusted. The block copolymers did not induce significant hemolysis and plasma protein precipitation. In vitro MTT and live/dead staining assays indicated they are biocompatible, and the biocompatibility was further demonstrated in vivo by the absence of local acute inflammatory response in mouse muscle following intramuscular injection. Unlike most frequently studied thermoresponsive poly(N-isopropylacrylamide), polyphosphoesters were hydrolytically degradable in aqueous solution that was proven by gel permeation chromatography and NMR analyses, and the degradation products were proven to be nontoxic to HEK293 cells. Therefore, with good biocompatibility and thermoresponsiveness, these biodegradable block copolymers of mPEG and polyphosphoesters are promising as stimuli-responsive materials for biomedical applications.

Introduction Stimuli-responsive polymers that exhibit unique property changes in response to environmental stimuli, for example, temperature, pH, electric fields, and light, are promising for many biomedical applications, including smart drug/gene delivery systems, injectable tissue engineering scaffolds, cell culture, and separation sheets.1-7 Polymers that undergo a transition between water-soluble and water-insoluble states have been particularly attractive and intensively investigated in recent years, since such a reversible transition generally does not require additional chemical reagents to induce the switch though it may depend on the structure of stimuli responsive polymers. Most frequently studied poly(N-substituted acrylamide)s, including poly(N-isopropylacrylamide) (PNIPAAm),8 poly(N,N′-diethylacrylamide),9 and poly(2-carboxyisopropylacrylamide),10 are representatives of temperature-responsive polymers, all showing lower critical solution temperature (LCST). In aqueous solution, such thermoresponsive polymers undergo phase transition from a soluble to an insoluble state or reversely. In addition, many other thermoresponsive polymers have also been reported. For example, poly(N-vinylcaprolactam) is hydrophilic at room temperature and gradually becomes hydrophobic from 25 to * To whom correspondence should be addressed. Tel.: (+86) 5513600402. Fax: (+86) 551-3600335. E-mail: [email protected]. † Department of Polymer Science and Engineering. ‡ Hefei National Laboratory for Physical Sciences at Microscale and School of Life Sciences.

35 °C.11 Copolymers of 2-(2-methoxyethoxy)ethyl methacrylate and oligo(ethylene glycol) methacrylate recently reported by Lutz and colleagues are another type of thermoresponsive copolymers with tunable LCST.12 Commercially available block copolymers of poly(ethylene oxide) (PEO) and poly(propylene oxide) (PPO), known as poloxamers (Pluronic) and poloxamines (Tetronic), also exhibit phase transitions at various temperatures, which are of great interest for their reverse thermal gelation behaviors.13 However, for in vivo biomedical applications, the main limitation of above thermoresponsive polymers lays on the nondegradability of carbon-carbon or ether-based backbone and the limitation on biocompatibility associated with themselves or residual monomers,14 although it may be partially overcome by advanced polymer synthesis techniques. For example, Lutz and colleagues introduced labile linkages to the backbone of copolymers of 2-(2-methoxyethoxy)ethyl methacrylate and oligo(ethylene glycol) methacrylate, retaining stimuli-responsiveness.15 Another example is thermo-responsive PEO-PPOPEO based poly(ether ester urethane) developed by Cohn and colleagues.16 Such polymer is thermosensitive and degradable through hydrolysis of ester based bonds. Block copolymer of poly(lactic acid) and poly(ethylene glycol) (PEG) has been successfully used as thermosensitive biomaterial, which exhibits reverse phase transition property and is degradable and resorbable.17,18 Many other analogues based on PEG and biodegradable aliphatic polyesters have also been developed.19-21

10.1021/bm800808q CCC: $40.75  2009 American Chemical Society Published on Web 12/02/2008

Thermoresponsive and Biodegradable Block Copolymers

Biomacromolecules, Vol. 10, No. 1, 2009

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Scheme 1. Syntheses Pathway of Block Copolymers of mPEG and Polyphosphoestera

a

The x, y, and z represent the degrees of polymerization of ethylene glycol, EEP, and PEP, respectively.

Table 1. Composition and Molecular Weight Distribution of Block Copolymer of mPEG and Polyphosphoester code

Mn of mPEG

feeding molar ratio (mPEG/EEP/PEP)

yield (%)

DP of EG/EEP/PEP

Mna

Mnb

PDIb

CAT (°C)

mPEG17-b-PEEP181 mPEG45-b-PEEP172 mPEG45-b-P(EEP177-co-PEP37) mPEG45-b-P(EEP149-co-PEP67) mPEG114-b-PEEP196

750 2000 2000 2000 5000

1:250:0 1:250:0 1:200:50 1:170:80 1:250:0

71.2 78.1 82.0 84.4 80.8

17:181:0 45:172:0 45:177:37 45:149:67 114:196:0

28260 28140 35040 35770 34800

37300 39800 49490 52010 42320

1.53 1.48 1.42 1.38 1.48

35 40 32 27 50

a

Determined by 1H NMR.

b

Determined by GPC.

It has been recently reported that a kind of polyphosphoester, namely, poly(ethyl ethylene phosphate) and its copolymers with poly(isopropyl ethylene phosphate) are thermosensitive, exhibiting varied LCSTs depending on the compositions.22 Due to their favorable biocompatibility and biodegradability, polyphosphoesters have shown potential in drug and gene delivery23,24 and tissue engineering applications.25 Polyphosphoesters were traditionally synthesized by polycondensation, transesterfication, polyaddition, enzyme-catalyzed, or tri-isobutyl aluminum initiated ring-opening polymerization.26-29 However, such methods failed to produce polyphosphoesters with well-defined structure and adjustable physiochemical properties controlled by the composition or molecular weights.30,31 Such problems have recently been overcome through well-controlled ring-opening polymerization of cyclic phosphoester monomers initiated by aluminum isoproxide or alcohol/stannous octoate.24,32 In this study, aiming at the development of biodegradable and biocompatible polymers with thermosensitivity for potential biomedical applications, we synthesized thermoresponsive block copolymers of PEG and polyphosphoesters with various molecular weights and compositions through controlled ringopening polymerization. We studied the thermo-induced assembly behavior of the block polymers in aqueous solution and investigated the effect of polymer composition on the phase transition behavior. We further demonstrated the biocompatibility of these thermosensitive block copolymers both in vitro and in vivo and studied the hydrolytic properties in vitro.

Experimental Section Materials. Ethyl ethylene phosphate (EEP, 2-ethoxy-2-oxo-1,3,2dioxaphospholane) and isopropyl ethylene phosphate (PEP, 2-isopropoxy-2-oxo-1,3,2-dioxaphospholane) were synthesized, as previously described in literature,33 and purified with two consecutive vacuum distillations. Monomethyl ethers of poly(ethylene glycol) (mPEG) with molecular weights of 750, 2000, and 5000 were purchased from Fluka Chemical Co. and dried by azeotropic distillation with toluene. Stannous octoate (Sn(Oct)2) was purchased from Shanghai Chemical Co. and purified as previously described.24 1,6-Diphenyl-1,3,5-hexatriene (DPH), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), poly(ethylene glycol) (PEG20K, MW 20000), and branched poly(eth-

Figure 1. GPC chromatograms of monomethyl poly(ethylene glycol) with molecular weight of 750 (mPEG17) (a), 2000 (mPEG45) (b), 5000 (mPEG114) (c), and of block copolymers mPEG17-b-PEEP181 (d), mPEG45-b-PEEP172 (e), and mPEG114-b-PEEP196 (f).

yleneimine) (PEI, Mn 25000) were obtained from Sigma Chemical Co. All other solvents and reagents, including tetrahydrofuran (THF), N,Ndimethylformamide (DMF), ethyl ether, triethylamine (TEA), Triton X-100, and sodium dodecyl sulfate (SDS), were used as received. Live/ dead viability/cytotoxicity kit was obtained from Molecular Probes, Inc. Synthesis of Block Copolymers of mPEG and Polyphosphoester. Block copolymers of mPEG and poly(ethyl ethylene phosphate) (mPEG-b-PEEP) were synthesized through ring-opening polymerization of EEP using mPEG as an initiator and Sn(Oct)2 as the catalyst, as shown in Scheme 1. Typically, EEP (6.2 g, 40.7 mmol) and mPEG (MW 5000, 0.8 g, 0.16 mmol) were added into a fresh flamed and nitrogen-purged round-bottomed flask, and the mixture was melted at 90 °C in a glovebox with H2O and O2 contents less than 0.1 ppm. Sn(Oct)2 (0.012 g, 0.03 mmol) was added and the mixture was maintained at 90 °C for 2 h. The product was dissolved in THF and precipitated in cold ethyl ether twice. The precipitate was dried under vacuum at room temperature (yield 80%). Block copolymers of mPEG and random polyphosphoesters, composed of poly(ethyl ethylene phosphate) and poly(isopropyl ethylene phosphate) (mPEG-b-P(EEPco-PEP)), were synthesized in a similar procedure, where PEP and EEP were mixed before polymerization. Gel Permeation Chromatography Measurement. Number and weight average molecular weights (Mn and Mw) and molecular weight distributions (polydispersity index, PDI ) Mw/Mn) were determined by gel permeation chromatography (GPC) measurements on a Waters GPC system, which was equipped with a Waters 1515 HPLC solvent pump, a Waters 2414 refractive index detector, and three Waters

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Figure 2. 1H NMR spectrum of mPEG45-b-P(EEP177-co-PEP37) in CDCl3 (ppm).

Figure 3. Absorbance at 365 nm of DPH measured at 45 and 25 °C (inset) with mPEG17-b-PEEP181 at different concentrations.

Styragel High Resolution columns (HR4, HR2, HR1, effective molecular-weight ranges of 5000-500000, 500-20000, and 100-5000, respectively). HPLC grade chloroform was purchased from J.T. Baker and used as the eluent at 40 °C, delivered at a flow rate of 1.0 mL min-1. Monodispersed polystyrene standards, obtained from Waters Co. with a molecular weight range 1310-5.51 × 104, were used to generate the calibration curve. 1 H NMR. Bruker AV300 NMR spectrometer (300 MHz) was used to collect 1H NMR spectra for the determination of the structure and composition of the block copolymers. Deuterated chloroform (CDCl3) containing 0.03% tetramethylsilane (TMS) was used as the solvent for NMR measurements. Dynamic Light Scattering (DLS). The size and size distribution of particles in aqueous solution were measured by dynamic light scattering carried out on a Malvern Zetasizer Nano ZS90 with a He-Ne laser (633 nm) and 90° collecting optics. All samples were prepared in aqueous solution at a concentration of 10 mg mL-1 and filtered through Millipore 0.45 µm filter prior to measurements. The aqueous solution was kept in the thermostat of the apparatus at various temperatures for 20 min to reach the equilibrium prior to the measurements. The data were analyzed by Malvern Dispersion Technology Software 4.20. UV-vis Determination. The assembly behavior of block copolymer was determined according to the following method based on the solubilization of probe molecules within self-assembled polymer aggregates. To 4 mL of aqueous solution of the copolymer (10 mg mL-1), 40 µL of methanol containing 0.4 mmol L-1 DPH was added. The mixture was left in the dark for 2 h. The UV-vis absorption spectra of the mixture from 300 to 550 nm were recorded at 25 or 45 °C using a UV-2802 PC (UNICO, China) spectrophotometer. MTT Assay. The relative cytotoxicity of block polymers was assessed with a methyl tetrazolium (MTT) viability assay against HEK293 cells. The cells were seeded in 96-well plates at 5000 cells

Figure 4. Temperature dependence of the hydrodynamic diameter (Dh) of block polymers in aqueous solution (10 mg mL-1): (A) effect of mPEG molecule weight and (B) effect of composition of polyphosphoester.

per well in 100 µL of complete DMEM containing 10% fetal bovine serum, supplemented with 50 units mL-1 penicillin and 50 units mL-1 streptomycin, and incubated at 37 °C in 5% CO2 atmosphere for 24 h, followed by removing culture medium and adding block polymer solution (100 µL in complete DMEM medium) at different concentrations. After a 72 h incubation, the medium was removed and replaced with 100 µL of fresh medium. MTT stock solution (25 µL, 5 g L-1 in phosphate-buffered saline (PBS)) was added to each well, with the exception of the wells as blank, to which 25 µL of PBS was added. After incubation for an additional 2 h, the extraction buffer (100 µL, 20% SDS in 50% DMF, pH 4.7, prepared at 37 °C) was added to the wells and incubated overnight at 37 °C. The solution was mixed gently, and the absorbance of the solution was measured at 570 nm using a Bio-Rad 680 microplate reader. The cell viability was normalized to that of HEK293 cells without polymer treatment. SDS with the same concentrations was incubated for 24 h as the positive control. Live/Dead Staining. HEK293 cells were seeded in 24-well plates at 60000 cells per well in 1 mL of complete DMEM and cultured for 24 h, followed by removing culture medium and adding polymer solutions (1 mL in complete DMEM medium) at concentration of 10 mg mL-1. After a 24 h incubation, cells were rinsed by PBS twice and incubated in PBS containing calcein acetoxymethyl ester (calcein-AM, 1 µM) and ethidium homodimer-1 (4 µM) at 37 °C in 5% CO2 for 10 min. Live and dead cells were imaged with a Nikon TE 2000-U fluorescence microscope. Samples were excited with light of 488 nm (green emission) to show viable cells and excited with light of 532 nm (red emission) to show the dead cells. Hemolysis. The experiments related to blood compatibility were performed under the supervision of Blood Administration of Hefei of China. Human blood was obtained from Central Blood Bank of Hefei in evacuated packs containing 3.2% sodium citrate (nine parts blood



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Figure 6. Hemolysis of RBCs when incubated with mPEG17-bPEEP181 and mPEG45-b-P(EEP177-co-PEP37) for 0.5 and 1.5 h (A); hydrodynamic diameter of plasma affected by incubation with mPEG45b-PEEP172 or polyethylene glycol (Mw ) 20000; B).

Figure 5. Cell viability of HEK293 cells incubated with block copolymers of mPEG and polyphosphoester for 72 h (A); overlaid fluorescence images of HEK293 cells after incubation with mPEG17b-PEEP181 (10 mg mL-1) for 24 h (B); and Triton X-100 (10 mg mL-1; C).

to one part anticoagulant) or EDTA. To obtain red blood cells (RBCs), 30 µL of human blood was suspended in 10 mL of PBS (pH ) 7.4, 0.01 M) and centrifuged at 1750 g min-1 for 5 min. Supernatant was removed and RBCs were resuspended and rinsed three times. Block copolymers dissolved in PBS (0.5 mL) were mixed with 0.5 mL of RBC (108 per mL) suspended in PBS in 1.5 mL eppendorf tubes. The final polymer concentration was from 0.078 to 10 mg mL-1. RBCs were then incubated at 37 °C in a thermostatted water bath for 0.5 and 1.5 h and centrifuged at 12000 g min-1 for 1 min. Free hemoglobin in the supernatant was measured with a UV-vis spectrophotometer at 414 nm. Triton X-100 (10 mg mL-1), a surfactant known to lyse RBCs, was used as the positive control. Samples treated with Triton X-100 (10 mg mL-1) were used to determine the absorbance with 100% hemolysis. RBC-PBS solution was the negative control. All hemolysis experiments were carried out in triplicates. Stability of Plasma Incubated with Polymer. Plasma was obtained from Central Blood Bank of Hefei of China in evacuated packs. The stability of plasma treated with block copolymer was evaluated by dynamic light scattering measurements and PEG20K was used as the control. Briefly, polymer was dissolved in plasma to final concentration of 10 mg mL-1 with NaN3 (0.09% w/v). The mixture was incubated at

37 °C and the Z-average diameters of solutions were determined by DLS at predetermined intervals. Tissue Response. Five-week-old female C57BL/6J mice were obtained and housed in University of Science and Technology of China Animal Holding Unit. Mice were maintained on ad libitum rodent diet and water at room temperature, 40% humidity. The animal procedures were approved by the University of Science and Technology of China School of Life Sciences Animal Care and Use Committee. Polymer dissolved in saline (10 mg mL-1, 30 µL) was injected into the tibialis anterior muscle in C57BL/6J mice. The muscles that received the polymer injections were isolated at days 3 and 7, fixed in phosphatebuffered formalin containing 4% (w/v) formaldehyde, washed, and embedded in paraffin. Tissue sections were cut into 8 µm in thick slices, placed on gelatin-coated slides, and stained with hematoxylin and eosin (H&E) for histological examination. Mice receiving intramuscular injection of 30 µL of saline and PEI in saline (10 mg mL-1, 30 µL) were used as negative and positive controls.34 In Vitro Hydrolytic Degradation. The in vitro hydrolytic degradation was performed at 37 °C in phosphate buffer (0.05 mol L-1, pH 7.4) with NaN3 (0.09% w/v). The concentration of polymer was set at 10 mg mL-1. At predetermined time intervals, samples were taken out and freeze-dried for GPC and 1H NMR analyses.

Results and Discussion Synthesis and Characterization of Block Copolymers. We have previously demonstrated that block copolymers of poly(εcaprolactone) and polyphosphoester can be synthesized by sequential polymerization of ε-caprolactone and cyclic phosphoester monomer using aluminum isopropoxide as the initiator.32 In another way, we have also reported that ring-opening

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Figure 7. Tissue response after intramuscular injection of PEG17-b-PEEP181, PEG45-b-P(EEP177-co-PEP37) as compared with saline and PEI injection. Tissue sections were analyzed by H&E staining after 3 and 7 days following injection (scale ) 100 µm).

Figure 8. (A) GPC chromatograms of mPEG114-b-PEEP196 after degradation in PBS (pH 7.4); (B) molecular weight change of degraded mPEG114-b-PEEP196 as a function of degradation time.

polymerization of cyclic phosphoester monomer can be wellcontrolled using alcohol and stannous octoate initiation system.35 The latter allows convenient synthesis of block copolymer of polyphosphoester using hydroxyl end-capped macromer as initiator. According to such a procedure, we have prepared a series of block copolymers of poly(ε-caprolactone) and poly(ethyl ethylene phosphate) and developed micellar system for targeted drug delivery, where poly(ethyl ethylene phosphate) worked as the hydrophilic component.36 In this study, aiming at the synthesis of block copolymers consisting of polyphosphoester, we selected mPEG as the

initiator due to its good solubility and compatibility. mPEG with molecular weights of 750, 2000, or 5000 were used. The polymerization was performed in bulk with Sn(Oct)2 at 90 °C instead of in THF solution, as previously reported.24 The synthesis pathway is depicted in Scheme 1. We expected that higher molecular weights could be achieved because we observed that it was difficult to obtain a polyphosphoester with a molecular weight above 20000 when the reaction was performed in THF. According to GPC analyses, results summarized in Table 1, the molecular weights of synthesized polymers are around 40000-50000 and the monomer conversions are all above 70%. The molecular weight distributions of the block copolymers are around 1.50 and are only slightly higher than those obtained in solution polymerization. Representative GPC chromatograms of block copolymers with unimodal peaks are shown in Figure 1 and compared with that of the initiators. To further demonstrate the chemical structure of obtained copolymers, the polymers were analyzed by NMR. A typical 1 H NMR of mPEG45-b-P(EEP177-co-PEP37) is shown in Figure 2. Resonance at 4.26 ppm (b) is a characteristic signal of protons of polyphosphoester backbone (-POCH2CH2O-, 4H). Resonance at 4.18 ppm (c) is assigned to methylene protons of PEEP (-OCH2CH3, 2H), while resonance at 4.67 ppm (e) is assigned to methine protons (-OCH(CH3)2, 1H) of PPEP. Methylene protons of PEG block give a signal at 3.65 ppm. The degree of polymerization (DP) of PEP was calculated from the integration of peak (e) by that of the triplets at 3.65 ppm (a), while the DP of EEP was calculated based on the integration of peak (d + f) after subtraction of six times the integration of peak (e). Temperature-Induced Assembly of Block Copolymers. Block copolymers consisting of one block with thermoresponsive hydrophilic-to-hydrophobic transition property are expected to undergo reversible self-assembly as temperature changes.37,38 A typical example is poly(ethylene oxide)-block-poly(N-isopropylacrylamide), which self-assembles into vesicles in water above the LCST and shows potential as thermosensitive drug carrier.39 It is assumed that such block copolymers are highly soluble in aqueous solution below the LCST of the thermosensitive block, while above the LCST, the thermosensitive block becomes hydrophobic, therefore triggering the self-assembly of the block copolymers. Such self-assemblies will disassociate when the temperature is decreased below the LCST due to hydrophobic-to-hydrophilic transition of the core material. As reported by Iwasaki’s group, PEEP homopolymer or copolymer



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Figure 9. 1H NMR analyses of mPEG114-b-PEEP196 (A) and its degradation products after hydrolytic degradation for 3 months (B) and 8 months (C; CDCl3, ppm).

Figure 10. Cytotoxicity of the hydrolytic degradation products of mPEG114-b-PEEP196 to HEK293 cells.

with PPEP exhibited phase transition behavior in water, showing LCST around 38 °C or lower.22 Therefore, the aqueous solution property of PEEP and hydrophilic PEG block copolymer is expected to respond to temperature change. To demonstrate the self-assembly induced by the increased temperature, we used DPH, which is a hydrophobic dye that partitions into the hydrophobic environment within self-assembled polymer aggregates as a probe.40 DPH is stable within our experimental temperature range (25-50 °C) as was proven by the constant absorbance at 356 nm (the maximum absorption wavelength from 300 to 600 nm; data not shown). We monitored the absorption spectra of block copolymer solutions (from 5 × 10-4 to 10 mg mL-1) in the presence of DPH (0.4 mmol L-1) at 25 and 45 °C. The absorbance intensities at 356 nm against concentrations of mPEG17-b-PEEP181 are plotted and shown in Figure 3. At 25 °C (inset of Figure 3), the absorbance intensities at 356 nm remained almost constant, independent of polymer concentration, revealing the solubility of DPH was not affected

by increased polymer concentration and indicating no hydrophobic domain formation. However, at 45 °C, following the relatively constant values at lower polymer concentrations, a rapid absorption intensity enhancement at around 1 mg mL-1 of mPEG17-b-PEEP181 was observed. This can be attributed to preferential partition of DPH probe from the aqueous into hydrophobic environment, which is a proof of hydrophobic domain formation. Such difference on absorption at 25 and 45 °C demonstrated the temperature-dependent assembly of mPEG17-b-PEEP181 into particles. Effect of mPEG Molecular Weight and Polyphosphoester Composition on Critical Aggregation Temperature (CAT). The response of the block copolymers to temperature change is also detectable by measuring the particle size changes using DLS. The CAT, which is an important indicator of the thermosensitivity of polymers in aqueous solution, can be defined as the temperature when the radius increases sharply in measurements of particle size versus temperature.41 Figure 4 shows the dependence of Z-average diameter on temperature for 10 mg mL-1 block copolymer solutions. As summarized in Table 1, it is obvious that CAT of block copolymers shifts to higher temperature when the molecular weight of mPEG increases while the molecular weight of PEEP block is roughly constant. This should be due to the hydrophilicity of mPEG segment. A similar effect has been reported previously for copolymers of PEG-b-PNIPAAm.37 On the other hand, CAT can also be tuned by adjusting the composition of the polyphosphoester block. Typically, CAT of mPEG45-bPEEP172 (without PEP copolymerization) was 40 °C from DLS measurements, while copolymerization of PEP into polyphosphoester block resulted in significantly lower CAT. It is possible that at temperatures below the LCST, the hydrogen bonding between PEEP backbone (-OP(O)O-) and water molecules is dominant. An increase of temperature would cause the disruption of hydrogen bonding and dehydration, making the PEEP more hydrophobic. Therefore, the balance of the hydrophilicity and hydrophobicity would shift, resulting in a transition of the PEEP

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chains into a compact and collapsed conformation and in insolubility in water. It is assumed that dehydration of the polymer preferably occurred with the addition of the more hydrophobic PPEP unit, similar to the effect to PNIPAAm with copolymerization of the hydrophobic component.42 Thus, CAT of mPEG and polyphosphoester block copolymers can be tuned conveniently to meet the need for physiological conditions and the polymers are potentially applicable in biomedical fields. In Vitro Cytotoxicity of the Block Copolymers. To evaluate the biocompatibility of the block copolymers, the in Vitro cytotoxicity to HEK293 cells was determined by MTT assay. As shown in Figure 5A, at all tested concentrations up to 10 mg mL-1, the viabilities of the HEK293 cells were around 100% after 72 h incubation, while cells did not tolerate the treatment with same dose of SDS for only 24 h. Correspondingly, live/ dead staining results were more direct to demonstrate the cell compatibility of the block copolymers. As an example, almost all of HEK293 cells incubated with mPEG17-b-PEEP181 (10 mg mL-1) for 24 h exhibited green fluorescence after both “live” staining with 1 µM calcein-AM and “dead” staining with 4 µM ethidium homodimer (EthD-1), indicating that the cells were viable (Figure 5B). On the contrary, to show the success of live-dead staining, all of HEK293 cells were dead with treatment with Triton X-100 (10 mg mL-1; Figure 5C). The blood compatibility of these block copolymers was assessed by a hemolysis assay. The hemolysis ratio (HR) represents the extent of the membranes of RBC broken by the sample in contact with blood. The greater the value of HR is, the more broken RBCs there are. A smaller HR value represents better blood compatibility of a biomaterial. RBCs and block copolymers were coincubated at different polymer concentrations for 0.5 and 1.5 h, and then HR values were determined with a UV-vis spectrophotometer. As shown in Figure 6A, the representative mPEG17-b-PEEP181 did not show conspicuous hemolytic activity on RBC even at a very high concentration of 10 mg mL-1. Incubation of RBCs with mPEG45-b-P(EEP177co-PEP37) gave similar results, indicating that copolymers with PPEP are also hemocompatible for potential biomedical application.43 Plasma proteins like serum proteins can bind to polymers and may lead to an alteration in physicochemical properties of polymers.44 For this consideration, the interaction between the block copolymers and plasma protein was investigated by incubating polymer solution with human plasma at 37 °C for 72 h to test if they would cause protein precipitation.45 PEG20K was used as control since it is known to cause plasma protein precipitation.46 At 37 °C, with incubation with block copolymer mPEG45-b-PEEP172, no visual evidence of precipitation was observed even when the concentration of mPEG45-b-PEEP172 was 20 mg mL-1. The Z-average diameters of plasma were determined by DLS and the results are shown in Figure 6. It was compared with direct incubation of plasma or incubation with PEG20K. The diameter remained constant after incubation for 24 h in all cases. However, increased diameter from 100 to 720 nm was observed when plasma was incubated with PEG20K for 72 h. Only a minor increase of diameter was observed when plasma was incubated with 10 mg mL-1 of mPEG45-b-PEEP172, indicating block copolymer of mPEG and polyphosphoester was more tolerated by plasma. Tissue Response to Block Copolymers. The acute inflammatory response to block copolymers was evaluated in muscle in C57BL/6J mice using saline and branched PEI (Mn 25000) injections as controls. In our previous study, we have shown that PEI induced severe acute inflammatory response in muscle

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tissue following intramuscular injection,34 therefore, it was used as a control in this study. Similarly, severe necrosis and accumulation of macrophages, histiocytes, and neutrophils at the injection site were observed in muscle samples after 3 and 7 days following PEI injection (Figure 7). However, histological analysis revealed no obvious inflammatory reaction at injection site with the same mass dose injection of mPEG17-b-PEEP181 or mPEG45-b-P(EEP177-co-PEP37) as PEI, which was comparable to muscle samples receiving saline injection, indicating the good biocompatibility of block copolymers. In Vitro Hydrolytic Degradation. An important advantage of polyphosphoester for biomedical applications over conventional thermoresponsive polymers (e.g., PNIPAAm) is that phosphoester bonds are biodegradable in contrast to nondegradable carbon-carbon bonds in aqueous solution at neutral pH. Moreover, the degradation rate of polyphosphoester may be adjusted by controlling the chemical structure of the backbone and pendent groups. For example, it has been previously demonstrated that polyphosphoester with pendent amino groups, namely, poly(2-aminoethyl propylene phosphate) degraded rapidly in aqueous solution, with 15 and 83% of Mw losses in 24 h and 7 days at 37 °C and pH 7.4, respectively.47 On the other hand, by choosing biocompatible building blocks of polyphosphoester, degradation products can have minimal toxic effects and good biocompatibility. The in vitro degradation behavior of block copolymer mPEG114-b-PEEP196 was evaluated as a representative at 37 °C and pH 7.4 in PBS. The degradation products were freeze-dried and molecular weights (Mn) were analyzed by GPC measurements. As shown in Figure 8, the molecular weights decreased gradually with increased incubation time up to 8 months in this study, which is a reflection of the hydrolytic cleavage of the phosphoester bonds in the backbone. The Mn dropped to 39350 in 2 months, corresponding to 7% Mn loss of PEEP block. The degradation was accelerated after the fourth month, and Mn declined gradually to 14000 after 8 months according to GPC analyses. To better understand the change during hydrolysis, the degradation products after 3 and 8 months degradation were freeze-dried and analyzed by 1H NMR, which was compared with the 1H NMR spectrum of the starting polymer. The results shown in Figure 9 revealed that signals assigned to protons of PEEP block declined, while signals assigned to protons of mPEG remained prominent, demonstrating that PEEP block was hydrolytically degraded. Several newly appeared resonances that correspond to novel proton chemical environments due to the degradation have been carefully assigned. The cytotoxicity of the degradation products is important in the evaluation of the biological safety of the materials. For example, it is reported that poly(lactide) and poly(lactide-coglycolide) have satisfactory biocompatibility, while high concentrations of the degradation products have a toxic influence.48 The degradation products of mPEG114-b-PEEP196 over 8 months incubation were freeze-dried and the cytotoxicity was evaluated using MTT method. Figure 10 shows the cell viability after 72 h incubation with 8 month degradation products at different concentrations. It was demonstrated that cells remained viable even when the concentration of degraded products was up to 0.5 mg mL-1, suggesting their good biocompatibility to HEK293 cells.

Conclusion Block copolymers of poly(ethylene glycol) and polyphosphoester with various molecular weights and compositions have


been synthesized through ring-opening polymerization of cyclic phosphate monomers. Dynamic light scattering and UV-vis studies revealed that the aqueous solution of these block copolymers underwent thermo-induced phase transitions. By copolymerization of different phosphate monomers, the critical aggregation temperature can be tuned in a wide range. The in vitro studies, including hemolysis, plasma stability, and cytotoxicity through MTT and live/dead assay, revealed that these block copolymers are biocompatible. The in vivo tissue response to the block copolymers also demonstrated their good tissue compatibility. This type of block copolymers is hydrolytically degraded at neutral condition, generating degradation products without cytotoxicity. These biodegradable, biocompatible, and thermoresponsive block copolymers may be promising for biomedical applications. Acknowledgment. This work was financially supported by the National Natural Science Foundation of China (50733003, 20774089), the Ministry of Science and Technology of the People’s Republic of China (2006CB933300, 2009CB930300), and Chinese Academy of Sciences (“Bairen” Program).

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BM800808Q

Thermoresponsive Block Copolymers of Poly(ethylene glycol) and

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