Biocatalytic Fabrication of Fast-Degradable, Water-Soluble

2550

Biomacromolecules 2010, 11, 2550–2557

Biocatalytic Fabrication of Fast-Degradable, Water-Soluble Polycarbonate Functionalized with Tertiary Amine Groups in Backbone Hua-Fen Wang,† Wei Su,† Chao Zhang,‡ Xiao-hua Luo,† and Jun Feng*,† Key Laboratory of Biomedical Polymers (The Ministry of Education), Department of Chemistry, Wuhan University, Wuhan, Hubei 430072, China, and Division of Biomedical Engineering, School of Engineering, Sun Yat-Sen University, Guangzhou, Guangdong 510275, China Received February 7, 2010; Revised Manuscript Received August 29, 2010

Degradable polymers with specifically designed functionality have wide applications in biomedical fields. We reported herein the synthesis and characterization of a water-soluble and fast-degradable polycarbonate, functionalized with tertiary amine groups in the backbone. A novel cyclic carbonate monomer, namely, 6,14dimethyl-1,3,9,11-tetraoxa-6,14-diaza-cyclohexadecane-2,10-dione (ADMC)2, was synthesized and polymerized to provide the title polycarbonate [poly(ADMC)] via Novozym-435 lipase or tin(II) 2-ethylheaxanoate [Sn(Oct)2] catalyzed ring-opening polymerization (ROP). Novozym-435 lipase exhibited high activity toward the ROP in terms of molecular weight (Mn) and monomer conversion, whereas the attempt with Sn(Oct)2 failed. In the presence of molecular sieves-4 Å, the highest Mn value of 1.2 × 104 g/mol was obtained in toluene with an initial monomer concentration of 0.58 M at 75 °C in the presence of 10 wt % of Novozym-435 to the monomer. Parameters that influence the polymerization, including reaction temperature, enzyme concentration, monomer concentration, and solvent composition, were investigated systematically. The resultant data suggested “living” characteristics for this enzyme-catalyzed polymerization, and the “living” feature seemed independent of the lipase concentration. The polymerization conducted in mixed solvents (toluene/isooctane) showed that product Mns were heavily dependent on the solvent composition. Poly(ADMC) was demonstrated to be amorphous by DSC technique. The obtained poly(ADMC) was found to be soluble in most of the organic solvents and interestingly in H2O as well. In vitro hydrolytic degradation of poly(ADMC) as monitored by GPC indicated the degradation was a relatively fast process. HPLC-ESI/MS and 1H NMR analyses demonstrated that N-methyl diethanolamine was the main product after degradation. Poly(ADMC) presented low cytotoxicity toward human cervix carcinoma (HeLa) cells and hepatoblastoma cells (Hep G2), as demonstrated by MTT assay.

Introduction Synthetic degradable materials represented by aliphatic polyesters have found their numerous applications in the vast field of biomedical and pharmaceutical sciences. Among those polymers, aliphatic polycarbonates (APCs) have been paid increasing attention due to their excellent physicochemical and mechanical properties.1–3 Relative to polyester, the application of APCs in vivo avoids the creation of acidic microenvironments, which was reported to result from the accumulation of released acid components during polyester degradation and usually causes the local aseptic inflammation as well as deactivation/denaturation of loaded drugs such as proteins or plasmid DNA.4–6 Some APCs have been commercialized and applied to clinical use. Traditional APCs, however, lack functionality and compatibility with cell/organs, and their medical applications are eventually limited. One main impetus for the research in this field currently focuses on the preparation of specifically designed functional APCs by introducing functional groups to polymer chains. Functional groups, properly located on a polymer, are usually responsible for material’s biocompatibility and may tailor its macroscopic properties such as hydrophilicity/ * To whom correspondence should be addressed. Tel.: + 86 27 6875 4509. Fax: + 86 27 6875 4509. E-mail: [email protected]. † Wuhan University. ‡ Sun Yat-Sen University.

hydrophobicity, membrane permeability, bioadhesive ability, and biodegradability. For example, cell and protein binding reactions and growth may be strongly affected by the functional groups of an implanted polymer. The presence of pendent hydroxyl and sugar groups can effectively enhance the biodegradability and biocompatibility of polycarbonates, thus, favoring their applications as controlled drug release matrices and tissue engineering scaffolds.7–9 Meanwhile, the presence of functional groups also offers a great possibility for extensive postmodification, including sequential binding of appropriate biochemical cues, which in turn allow specific interactions between material and cells for drug targeting and tissue engineering purpose. Currently, one of the main strategies to achieve APCs bearing functional groups is based on the innovative design and polymerization of cyclic monomers substituted by a functional group. Functional groups including hydroxyl, carboxyl, sugar groups, and so on can be attached to the cyclic carbonate monomers.7,8,10–16 By means of ring-opening polymerizations (ROP) in the presence of selected initiators, the precise control over functional APCs with respect to molecular weight (Mn) and polymer architecture can be readily realized. Additionally, the preparation of functional monomers facilitates the manufacture of a variety of new materials with a well-defined arrangement of functional groups along the backbone through copolymerization with other available unsubstituted monomers such as trimethylene carbonate (TMC), lactide (LA), ε-caprolactone (ε-CL), and so on.17–20 Nevertheless, the number of

10.1021/bm1001476  2010 American Chemical Society Published on Web 09/13/2010

Biocatalytic Fabrication of Polycarbonate Scheme 1. Preparation of (ADMC)2 and Poly(ADMC)

reported functional cyclic carbonates are very limited due to elaborate synthetic procedures. For the first time, we report here the controlled synthesis and characterization of a novel functional polycarbonate poly(ADMC) containing tertiary amine groups in the backbone (Scheme 1). A biosynthetic pathway like metal-free enzymatic ROP has attracted great attention as a new approach for biomaterial synthesis due to its nontoxicity and mild reaction conditions. Enzymatic ROP was herein employed in poly(ADMC) synthesis from a novel cyclocarbonate, 6,14-dimethyl1,3,9,11-tetraoxa-6,14-diaza-cyclohexadecane-2,10-dione [(ADMC)2] (Scheme 1), in the presence of Novozym-435 lipase. Due to the presence of tertiary amine groups, poly(ADMC) demonstrated attractive properties such as water-solubility and fast degradability; whereas one knows that in addition to natural polymers such as proteins and polysaccharides, few synthetic cationic polymers with both water-solubility and degradability are reported. It is expected that poly(ADMC) should share many features of polyelectrolyte and be offered a variety of potentials in use for medical purposes. For example, it is potentially useful as a polycationic vehicle21,22 to electrovalently bind protein drug and therapeutic DNA, aiming at sustained drug delivery and gene transfection. It can also be utilized as building blocks of smart soft materials to “intelligently” respond to external stimuli (pH or ionic strength), resulting from the protonation/deprotonation behavior of tertiary amine groups accompanied with variation in aquatic environment.23,24 Furthermore, the combination of high degradability and water solubility of poly(ADMC) would significantly expand its possible applications in the future. It should also be pointed out that there’s no elaborate protection-deprotection process in the preparation of this novel polycarbonate, which is usually involved in functional polymer synthesis due to the antagonism of functional groups toward the active site of the catalysts.7,8,10–16 This approach is attractive in view of the easy manipulation of functional polycarbonate.

Experimental Section Materials. Lipase acrylic resin from Candida antarctica (Novozym435) was purchased from Sigma. Diethyl carbonate, N-methyl diethanolamine, tetrahydrofuran, and dichloromethane (Shanghai Chemical Co. China) were of analytical grade and used as received. Toluene was dried over sodium-potassium alloy and distilled before use. Tin(II) 2-ethyl hexanoate [Sn(Oct)2] (Shanghai Chemical Co. China) was purified by distillation under reduced pressure and dissolved in fresh anhydrous toluene. All other reagents were used without further purification. Fetal bovine serum (FBS), Dulbecco’s modified Eagle’s medium (DMEM), trypsin, Dubelcco’s phosphate buffered saline (PBS),

Biomacromolecules, Vol. 11, No. 10, 2010

2551

and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Invitrogen Corp. Instrumentation. Melting point was measured on an X-4 microscopic melting point apparatus (Beijing Keyi Company) without calibration. Mass spectrum was recorded on a JEOL JMS-700 mass spectrometer (MStation). Fourier-transformation infrared (FT-IR) spectra were recorded on a Perkin-Elmer-2 spectrophotometer. Samples were film-cast in chloroform onto sodium chloride (NaCl) plates. Proton nuclear resonance spectroscopy (1H NMR) and carbon-13 nuclear resonance spectroscopy (13C NMR) were performed on a Varian Mercury-VX 300 spectrometer using CDCl3 or D2O as solvent. Differential scanning calorimetry (DSC) measurements were carried on using a Perkin-Elmer DSC 7 thermal analyzer. Samples of 8 ( 0.1 mg were used in test and sealed in aluminum pans. The heating/cooling rate was 10 °C/min. Gel permeation chromatography (GPC) was carried out on a Waters HPLC system equipped with a Model 2690D separation module, a Module 2410 differential refractive index detector, and a Shodex K803 column. THF was used as eluent at a flow rate of 1.0 mL/min. Waters MILLIENIUM32 module software was used to calculate molecular weights based on a calibration curve generated by narrow molecular weight distribution polystyrene standards. The sample concentration and injection volume was 0.3% (wt/v) and 50 µL, respectively. Crystallography data of (ADMC)2 single crystal were collected on a SMART APEX II CCD diffractometer at room temperature. HPLC with electrospray ionization mass spectrometry (HPLC-ESI/ MS) consisted of a Binary HPLC pump (Waters1525) and quadruple detector (MicromassZQ4000). Data acquisition and analysis was performed using the MassLynx software package (MassLynx 4.0, Micromass). The column was equilibrated for at least 30 min with methanol as the mobile phase at a flow rate of 0.2 mL/min. Positive ion monitor mode was adopted and mass parameters for HPLC-ESI/ MS were set as follows: capillary voltage was 3.5 kV, cone voltage was 40 V, source and desolvation temperature were 105 and 450 °C, and desolvation gas flow was 750 mL/h. Synthesis of 1,3,9,11-Tetraoxa-cyclohexadecane-2,10-dione [(ADMC)2]. N-Methyl diethanolamine (59.5 g, 0.5 mol), diethyl carbonate (61 g, 0.51 mol), and potassium carbonate (0.2 g, 1.45 mmol) were charged into a 250 mL round-bottom flask equipped with a fractioning column. The mixture was stirred at 120 °C for 15 h, whereby most of the produced ethanol was distilled off. Then excessive diethyl carbonate was removed under vacuum (15 mmHg). The residue was dissolved in 20 mL of dichloromethane and washed twice with 50 mL of saturated brines. The organic layer was dried over anhydrous Na2SO4 overnight and then filtered. The filtrate was concentrated to provide the oligomers in vacuo. The oligomers were depolymerized at 220-250 °C/60 Pa and the product was collected by distillation. The crude product was recrystallized from ethyl acetate for three times to isolate a white solid (yield 25%), mp 134-136 °C. Mass spectroscopy revealed a peak at 291 g/mol. IR: ν ) 1744 cm-1 (CdO). 1H NMR (CDCl3, ppm): 4.23 (t, O-CH2-CH2-N(CH3) 4H), 2.68 (t, O-CH2-CH2-N(CH3) 4H), 2.37 (s, O-CH2-CH2-N(CH3) 3H). 13C NMR (CDCl3, ppm): 155.4 (CO), 65.1 (O-CH2-CH2-N(CH3)), 55.9 (O-CH2-CH2-N(CH3)), 42.8 (OCH2-CH2-N(CH3)). Novozym-435 Catalyzed Ring-Opening Polymerization of (ADMC)2 in Toluene. Enzymatic ROP of (ADMC)2 was carried out in toluene or a mixed solvent (toluene/isooctane) using Novozyme435 as the catalyst. Both monomer and enzyme were dried (40 Pa, room temperature, 24 h) over phosphorus pentoxide before polymerization. Typically, at 10 wt % of enzyme to monomer, the mixture of (ADMC)2 (0.1 g) and Novozym-435 (10 mg) were introduced to a dried flask. The flask was degassed under vacuum and purged with argon for several cycles. To the flask was added 0.6 mL of fresh anhydrous toluene by syringe and the ratio of toluene to (ADMC)2 was 6:1 (v/w, mL/g). The flask was then closed with a glass stopper and immersed in an oil bath at a determined temperature. After a period of time, the resulting product was dissolved in 5 mL of dichloromethane and the

2552 Biomacromolecules, Vol. 11, No. 10, 2010 solution was filtered to remove the insoluble enzymes. One part of the filtrate was concentrated and precipitated in 25 mL of ethyl ether two times. Pale yellow viscous products were isolated and then dried in vacuo to a constant weight for IR and NMR analysis. IR: ν ) 1743 cm-1(CdO). 1H NMR (CDCl3, ppm): 4.20 (t, O-CH2-CH2-N(CH3), 4H), 3.65 (t, O-CH2CH2N(CH3)CH2CH2OH), 2.74 (t, O-CH2-CH2-N(CH3), 4H), 2.35 (s, O-CH2-CH2-N(CH3), 3H). 13C NMR (CDCl3, ppm): 155.4 (CO), 65.7 (O-CH2-CH2-N(CH3)), 56.03 (O-CH2-CH2-N(CH3)), 42.93 (O-CH2-CH2-N(CH3)). For the remaining filtrate, dichloromethane was removed in vacuo, and the crude product without purification treatment was dissolved in CDCl3 to make a 0.3% (w/v) solution for 1H NMR measurement. The monomer conversion was calculated by comparing the area integral (Amonomer) of the signal at 2.68 ppm assigned to methylene proton O-CH2-CH2-N(CH3) connected to nitrogen atom of monomer with that of polymer at 2.74 ppm (Apolymer).

monomer conversion(%) )

Apolymer × 100 Amonomer + Apolymer

Number-average molecular weight (Mn) of the polymer was determined by comparing the Apolymer mentioned above with that at 3.65 ppm (Aterminal), which was assigned to the methylene group -CH2OH adjacent to the two terminal hydroxyl groups.

Mn ) 145 ×

Apolymer

/Aterminal

Ring-Opening Bulk Polymerization of (ADMC)2 under Sn(Oct)2 Catalysis. For the polymerizations, different molar ratios of (ADMC)2 versus Sn(Oct)2 were applied in the range of 100-500. (ADMC)2 was introduced to a dried glass vessel that was pretreated with Me3SiCl solution in toluene. A predetermined amount of Sn(Oct)2 in toluene was introduced. The vessel was vacuumed (60 Pa) for 6 h and then sealed under vacuum and immersed in an oil bath at 140 °C for 12-24 h. In all the cases, brown viscous liquids were obtained. One part of the product was dissolved in CDCl3 with a concentration of 0.3% (w/ v) for 1H NMR analysis. The remaining part was dissolved in CH2Cl2 and poured in excessive ethyl ether, but no precipitate was observed. Bulk polymerization under an argon atmosphere afforded the similar results. Ring-Opening Solution Polymerization of (ADMC)2 in Toluene under Sn(Oct)2 Catalysis. The molar ratios of (ADMC)2 versus Sn(Oct)2 was 100:1 in this study. Polymerizations were carried out under an argon atmosphere by adjusting the ratios of toluene versus (ADMC)2 in the range of 5:1-10:1 (v/w, mL/g). The reactions were immersed in an oil bath for 48 h at 75 and 100 °C, respectively. After cooling to room temperature, no polymer products were detected and a large amount of crystal was observed that was proved to be (ADMC)2 by means of NMR analysis and mp measurement. Cell Culture. Human cervix carcinoma (HeLa) cells and human hepatoblastoma cells (Hep G2) were incubated in 24-well tissue culture plates in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS) and 1% antibiotics (penicillin-streptomycin, 10000 U/mL) at 37 °C in a humidified atmosphere containing 5% CO2. In Vitro Biocompatibility Assay. The cytotoxicity of poly(ADMC) was evaluated by MTT assay. The Hep G2 cell (6000 cells/well) and HeLa cell (6000 cells/well) were seeded in a 96-well plate, respectively. After polymer solutions with a particular concentration were added, the cells were cultured for 24 and 48 h in 100 µL DMEM containing 10% FBS. Then the medium was replaced with 200 µL of fresh medium and 20 µL of MTT (3-(4,5-dimethylthiazol2-yl)-2,5-diphenyl tetrazolium bromide) solution (5 mg/mL) was added and the solution was incubated for an additional 4 h. After that, the medium was removed and 150 µL of DMSO was added to dissolve the formazan crystals. The optical density (OD) was

Wang et al.

Figure 1. IR spectra of (ADMC)2 and poly(ADMC) purified by precipitation in ether.

measured using a microplate reader (model 550, Bio-Rad) at 570 nm. The cell viability was calculated as

cell viability(%) ) (OD570(samples)/OD570(control)) × 100 where OD570(control) was obtained in the absence of polymers and OD570(samples) was obtained in the presence of polymers. In Vitro Degradation Test. A total of 6 mg of the polymer was incubated in 4 mL of 0.1 M PBS (pH 7.4) at 37 °C for studying the degradation behavior in vitro. After a determined time, a portion of the solutions were taken and lyophilized. The obtained mixture was extracted with a total amount of 10 mL of distilled THF three times. The THF solution was concentrated and then dried in vacuum to a constant weight for GPC analysis.

Results and Discussion Polymer chemistry has made a lasting impact on materials science through innovative monomer design as well as improvements in catalysts, and the strategies for the controlled polymerization of monomers.25 In this paper, a novel functional monomer (ADMC)2 containing tertiary amine group was designed and its enzymatic ROP was attempted in a controlled manner. It is well-known that the general route to synthesize aliphatic cyclocarbonates involves (1) direct cyclization via reaction of R, ω-alkanediol with phosgene analogues in the presence of acid-binding agent and (2) thermo-decomposition of the corresponding oligomers in vacuo. As for the direct cyclization method, 6- or 7-membered monomeric cyclocarbonates can be easily synthesized in high yields.26,27 In contrast, no cyclic monomer could be obtained when reacting N-methyl diethanolamine with triphosgene or ethyl chloroformate by the same method. The synthesis of low-molecular-weight oligomers and subsequent thermal depolymerization of corresponding oligomers appears to be a more realistic approach to obtain the cyclic monomer compared with the direct cyclization. In the case of thermal depolymerization, IR (Figure 1), NMR (Figure 2), MS (Figure 3), and single crystal X-ray diffraction analyses (Figure 4) distinctly demonstrated that dimeric cyclocarbonate 6,14dimethyl-1,3,9,11-tetraoxa-6,14-diaza-cyclohexadecane-2,10-dione [(ADMC)2] other than monomeric ADMC was synthesized. It is well-known that Sn(Oct)2 is a highly efficient catalyst for ROP of cyclic carbonates.27–30 Whereas no polymer can be isolated via a precipitation method for Sn(Oct)2-catalyzed bulk polymerizations of (ADMC)2. The obtained viscid product before precipitation was subjected to 1H NMR measurement (Figure 5). Compared to 1H NMR spectrum of (ADMC)2 (Figure

Biocatalytic Fabrication of Polycarbonate


2553

Figure 5. 1H NMR spectrum of the obtained products in the Sn(Oct)2catalyzed bulk polymerization without any treatment. The polymerization was carried out at 120 °C for 12 h, while the molar ratio of (ADMC)2 vs Sn(Oct)2 was fixed at 100:1.

Figure 2. 1H and 13C NMR spectra of the monomer (ADMC)2 in CDCl3.

Figure 3. MS spectrum of the monomer (ADMC)2.

Figure 6. 1H and 13C NMR spectra of poly(ADMC) (Mn ) 0.7 × 104 g/mol) purified by precipitation in ethyl ether.

Figure 4. Illustration of the single crystal structure of (ADMC)2.

2), a peak at 3.45 ppm with high intensity was found, which is the characteristic resonance of ether groups (-CH2-O-CH2-). It can be suggested that a possible decarboxylation reaction, which is a common phenomenon for TMC polymerization in the presence of anionic catalyst3 but not Sn(Oct)2,27–32 occurred during the process. The control experiment in the absence of Sn(Oct)2 afforded the same result. Thus an explanation to this extraordinary phenomenon would be more possibly the relatively lower thermal stability of (ADMC)2 under high temperature. However, the possibility of the reaction between tertiary amine groups and Sn(Oct)2 can not be excluded yet. To avoid the destabilization of (ADMC)2 in bulk polymerization, an attempt for Sn(Oct)2-catalyzed polymerization in toluene at lower temperature of 70 or 100 °C was performed. Nevertheless, no polymerization was observed. From the results, traditional Sn(Oct)2-catalysis is not a suitable way toward polymerization of functionalized (ADMC)2, although it has shown high efficiency for nonfunctionlized cyclic carbonates.

The outstanding ability of the Novozym-435 lipase to catalyze ROP of nonfunctionlized cyclic carbonates has been demonstrated. It is still challenging to employ enzymatic catalysis for polymerization of (ADMC)2, because the influence of the functional tertiary amine groups toward the enzyme activity is unclear. In this work, Novozym-435 catalyzed polymerization of (ADMC)2 was investigated in toluene rather than in bulk due to the high melting point of (ADMC)2. Interestingly, the resultant data demonstrated that Novozym-435 is an effective biocatalyst toward (ADMC)2 polymerization, whereas no polymerization occurred in the absence of catalysts. The polymer structure was confirmed by IR and NMR analyses. For obtained polymers, IR spectra displayed a “CdO” band at 1743.2 cm-1 that was almost identical with that of (ADMC)2 at 1744.1 cm-1 (Figure 1). 1H NMR spectra showed that the characteristic signal of (ADMC)2 at 2.68 ppm (signal b in Figure 2), belonging to methylene group adjacent to nitrogen atom (O-CH2-CH2-N(CH3)-), shifted to 2.74 ppm after polymerization (Figure 6). No evidence was seen in the NMR spectra for decarboxylation of polymer backbone during the polymerization.33–35 Besides, a triplex signal (signal d in Figure 6) was found at 3.65 ppm in 1H NMR spectra of poly(ADMC)s, which can be ascribed to the methylene group (-CH2CH2OH) connected

2554


Figure 7. Plots of monomer conversion (a) and Ln[M0/Mt] (b) as a function of reaction time and plot of Mn vs monomer conversion (c). The ratio of toluene to (ADMC)2 was fixed at 6:1 [vol (mL)/wt (g)]. -Oweight ratio of lipase vs (ADMC)2 ) 1:5, reaction temperature ) 75 °C; -0- weight ratio of lipase vs (ADMC)2 ) 1:10, reaction temperature ) 75 °C;. -2- weight ratio of lipase vs (ADMC)2 ) 1:10, reaction temperature ) 60 °C.

to the terminal hydroxyl groups and has been well-documented in enzymatic ROP of TMC33–35 and lactones.25 Compared with IR spectra of (ADMC)2, a broad peak appeared at around 3400 cm-1 in polymer IR spectrum, further confirming the presence of terminal hydroxyl groups (Figure 2). This agreed well with the finding by Gross et al. in their study of enzymatic polymerization of TMC, where trace water may act as the initiators during the polymerization and result in the formation of terminal hydroxyl groups on both ends of polymer chain.33 For comparison purposes, a reaction product with Mn,NMR of 0.7 × 104 g/mol determined from NMR analysis was purified by precipitation in ethyl ether and subjected to GPC measurement. A unimodal GPC trace was observed, and the Mn,GPC was found to be 0.75 × 104 g/mol with a PDI of 1.34. The influence of Novozym-435 concentration on polymerization of (ADMC)2 was explored at 75 °C with lipase/(ADMC)2 ratios at 1:5 and 1:10 (w/w). Seen from Figure 7a, the monomer conversion increased as a function of reaction time and then leveled off. From Figure 7b, the data showed that higher lipase concentration led to faster monomer consumption. Up to monomer conversion of around 90%, there was a good linear relationship between Ln[M0/Mt] and reaction time (Figure 7b),

Wang et al.

indicating monomer consumption might follow a first-order law. Further analysis of the polymerization showed that Mns increased linearly with monomer conversion in both cases (Figure 7c). Those results suggested that few chain transfer reaction or termination took place up to 90% monomer conversion under the specified condition. This polymerization catalyzed by Novozym-435 presented many features of an immortal polymerization. Moreover, it appeared that variation in the lipase concentration did not affect this “living” characteristic, which has also been reported in the enzymatic polymerization of ε-CL.25 Further prolonging the reaction time resulted in a deviation from linearity of Ln[M0/Mt] versus reaction time (Figure 7b) as well as the deviation from linearity of Mn versus monomer conversion (Figure 7c). Mns of the polymers thereafter decreased, although the monomer conversion has no significant change. Therefore, the deviation from linearity in the later stage could be an indication of the occurrence of side reactions such as the polymer degradation or chain transfer at higher monomer conversion/prolonged reaction time. In Figure 7c, an inverse relationship between Mn and Novozym-435 concentration was found. At the same monomer conversion, higher lipase concentration led to lower polymer molecular weight. This can be explained by the amount of water inherently contained in the enzyme, which is regarded as the initiator and engender more polymer chains in the case of higher enzyme concentration.33 In that sense, decreasing the water content in the reaction system might produce polymers with higher Mns. This is demonstrated by a significant increase in Mn when introducing 4 Å molecular sieves to decrease the water content in the system. For instance, the highest Mn of 8000 g/mol was obtained for the polymerization case with an enzyme/ monomer ratio of 1:10 (series -0- in Figure 7), while a much higher Mn up to 1.2 × 104 g/mol was provided in the presence of 4 Å molecular sieves at the identical condition. The polymerization kinetics at different temperatures was also investigated at 60 and 75 °C, respectively (Figure 7). It was shown that the temperature had no obvious influence on the monomer consumption rate, although it is anticipated that a higher temperature should accelerate the reaction. It was possible due to the partial deactivation of Novozym-435 at higher temperature. The results also indicated that slightly higher molecular weight could be obtained at higher temperature of 75 °C. Kobayashi and co-workers previously also found large increases in Mn by increasing the reaction temperature in the enzymatic polymerization of PDL.36 It was explained as a result from increasing the mobility of the monomer and the polymer components in viscous reaction mixtures at higher temperature. Another series of experiments at 75 °C for 12 h were performed to investigate the effect of the monomer concentration on the polymerization. The polymerizations were conducted with a constant weight ratio of lipase versus (ADMC)2 and varied toluene/monomer ratio. It was found that a further increase in the toluene/monomer ratio would retard the monomer consumption (Figure 8), which could be due to the excessively diluted reaction system. The nature of organic solvents was reported to play an important role in the enzymatic polymerization. Although the explicit mechanism is not clear, it is believed that solvent polarity would exert marked influences on the enzymatic polymerization.25,37,38 One main explanation may lie in the enzymatic conformational changes induced by the interaction between polar solvent and enzyme, thus, affecting the catalytic activity of enzyme. Generally speaking, lower solvent polarity



2555

Figure 8. Plot of monomer conversion vs ratio of toluene to (ADMC)2 for 12 h polymerization at 75 °C. Weight ratio of the lipase vs (ADMC)2 was 1:10.

Figure 9. GPC curves of poly(ADMC) in PBS (0.1M) at 37.4 °C at marked degradation time.

Table 1. Enzymatic Polymerization of (ADMC)2 in Mixed Solvents Composed of Isooctane and Toluenea

insoluble in both methanol and H2O. Obviously the introduction of the tertiary amine groups into the polymer main chain greatly improved its hydrophilicity. For DSC analyses from -60 to 100 °C, poly(ADMC) was revealed to be amorphous, as evidenced by the absence of a endothermic melting peak in its DSC curve. Biodegradable materials provide the significant advantage that the polymer will ultimately disappear in vivo, obviating longterm biological toxicity and negative tissue responses. It is known that nonfunctional aliphatic polycarbonates are even more resistant to hydrolysis than polyesters due to its low hydrophilicity and the lack of autocatalysis effect.39 The rate of hydrolytic chain scission of PTMC in phosphate-buffered saline at 37 °C, pH 7.4, is 20 times lower than that of poly(ε-caprolactone).40 The degradation of purified poly(ADMC) sample (Mn,NMR ) 0.7 × 104 g/mol) in PBS at 37 °C was monitored by GPC measurements, and the results are shown in Figure 9. Marked changes in Mns and molecular weight distributions (MWD) of the polymer during the degradation process were observed. The initial polymer (degradation time zero) exhibited a unimodal MWD (PDI ) 1.34) peaked at 8000 g/mol. After degradation of 3 weeks, bimodal peaks at 6140 and 575 g/mol were observed. The MWD of the peak at 6140 g/mol were much broader compared with that before hydrolysis. After degradation for 7.5 weeks, bimodal peaks at 575 and 170 g/mol were detected, while the peak at 6140 g/mol almost disappeared. The resulting data demonstrated that degradation of poly(ADMC) might be a relatively fast process due to the enhanced hydrophilicity and hydrolytic catalysis caused by the presence of tertiary amine groups. After a prolonged degradation test to 3 months, the degraded product was subjected to 1H NMR and HPLC/ESI-MS analyses. In contrast to the poly(ADMC) spectrum, the signal at 4.23 ppm, belonging to methylene group adjacent to carbonate of ADMC unit, dispeared and a signal at 3.63 ppm with strong intensity emerged in the spectrum of degradation product (Figure 10). The spectrum showed a fairly good agreement with that of N-methyl diethanolamine (MDEA). It indicated that the signal at 3.63 ppm should be ascribed to the methylene connected to the hydroxyl groups in MDEA, and poly(ADMC) ultimately degraded into MDEA. The HPLC/ESI-MS graph distinctly exhibited the production of [M + H]+ and [M + Na]+ molecular ions, further confirming that MDEA was the main product of degradation (Figure 11). The cytotoxicity of water-soluble polymers appears more important to their potential biomedical applications. Therefore, the cytotoxicity of poly(ADMC) was evaluated via MTT assay toward human cervix carcinoma (HeLa) cells and human hepatoblastoma (Hep G2) cells, respectively. Poly(ADMC)

entry

isooctane/toluene ratio (v/v)

monomer conversion (%)

Mn

1 2 3 4 5

3/0 2/1 1/1 1/2 0/3

94.6 95.5 95.4 93.6 94.5

3710 4010 4030 5500 8000

a The polymerizations were conducted at 75 °C for 24 h with the weight ratio of lipase to monomer fixed at 1:10 and the ratio of total solvent volume vs monomer at 6:1 (v/w).

may contribute to the enhancement in polymer Mns. As an example, polymerization of lactones in polar solvents such as 1,4-dioxane, acetonitrile, acetone, and 2-butanone hardly took place, while less polar hydrocarbon solvents (toluene, heptane, cyclooctane, and isooctane) are more suitable for the polymerization with respect to monomer conversion and Mns.25 Therefore, mixed solvents with different isooctane portions were employed herein for the purpose of tailoring solvent polarity while keeping the ratio of total solvent volume versus monomer constant (6:1; Table 1). Mns of polymerization products were found to be heavily dependent on the solvent composition. In opposition to our expectation, compared with isooctane-free case (Mn ) 8000 g/mol), lowered solvent polarity by adding isooctane did not result in increase in Mn. Instead, the increase in the isooctane/toluene ratio led to a decrease in Mns. Similar results have been reported when using single solvents with different polarity,25,37,38 which revealed that more factors such as solvent dipole moment, substrate, or polymer solubility, and enzyme specificity in addition to solvent polarity may be systematically considered to understand the complicated polymerization behavior in solvents. One fact should be noted that, when the polymerization was conducted in pure isooctane (ratio of 3/0), the produced polymer product (Mn ) 3710 g/mol) was readily isolated from reaction system in the form of precipitates. Thus, the decrease in Mn values in the case of much higher isooctane portion may be more possibly attributed to the lower solubility of polymers in the mixed solvents, which affect the conformation of the polymer in solution as well as the polymer diffusivity. The obtained poly(ADMC) was found to be soluble in most solvents such as toluene, THF, methanol, DMF, and ethyl acetate, and is attractively soluble in water. From the 1H NMR spectra in D2O (Figure 6), all the characteristic signals belonging to poly(ADMC) can be clearly detected. In contrast, nonfunctional aliphatic polycarbonates such as poly(trimethylene carbonate) (PTMC) are known to be highly hydrophobic and

2556


Figure 10. 1H NMR in CDCl3 of the product obtained after 3 months degradation in PBS (0.1M) at 37.4 °C of poly(ADMC).

Figure 11. HPLC/ESI-MS graph of the product obtained after 3 months degradation in PBS (0.1M) at 37.4 °C of poly(ADMC).

Figure 12. In vitro cytotoxicity of poly(ADMC) (Mn ) 7000) at different concentrations.

exhibited low cytotoxicity to both cell types. For HeLa cells, the cell viability was above 90% when the polymer concentration was below 200 mg/L (Figure 12). When the polymer concentration was increased to a higher level at about 1200 mg/ L, the viabilities of HeLa cells was still kept above 75%. In comparison, the incubation of Hep G2 cells with the polymers resulted in much higher cell viabilities. Within the range of 1300 mg/L, the cell viabilities remained above 90% (Figure 12). To sum up, the development of new functional groups for polymers is an active area of investigation in polymer science. A novel functional polycarbonate of poly(ADMC) with welldefined structure were designed and fabricated via enzymatic ROP in a controlled manner. The monomer (ADMC)2 was synthesized in two steps starting from N-methyl diethanolamine (MDEA), which is a commercially available and low toxic material with cheap price. It is also noteworthy that the polymer

Wang et al.

synthesis from this novel cyclic monomer avoids elaborate protection-deprotection process,10,11,13–19 which was usually involved in functional polymer syntheses. Enzymatic polymerization has proved advantageous as opposed to traditional preparative routes by Sn(Oct)2 catalysis in terms of higher catalytic efficiency. Different from traditional APCs, poly(ADMC) with attractive water-solubility should share many features of polyelectrolyte due to the presence of a great number of tertiary amine groups in the backbone. Meanwhile, poly(ADMC) exhibited much higher degradability demonstrated in the study. Those unique properties are advantageous in the application where high hydrophilicity, cell affinity, and relatively fast degradation rate is desired. For instance, poly(ADMC) or its quarternized derivatives are potentially useful as cationic polymeric vectors to allow the highly efficient loading of therapeutic DNA through counterion condensation. The relatively higher degradability may find its application in the effective release of loaded DNA into nucleus and fast eradication of the toxicity caused by a high charge density of polycationic materials, resulting in a high expression of therapeutic DNA and promoted material biocompatibility.41 It can also be utilized to build the hydrophilic blocks of amphiphilic polymeric micelles for controlled drug release to “intelligently” respond to external stimuli of pH and ionic strength. Meanwhile, the availability of functional (ADMC)2 favors the design of new materials by means of well-established copolymerization strategy with other common monomers including TMC, LA, or CL. By careful design of copolymer’s architecture, biomaterials with tunable properties such as hydrophilicity/hydrophobicity, degradability, and charge density could be synthesized and meet other specified requirements such as in use as matrix for cell proliferation and tissue engineering. On the other hand, we should keep in mind that poly(ADMC) with acceptable biocompatibility ultimately degraded into MDEA with low toxicity characterized by the LD50/oral/rat value of 4780 mg/kg (according to EC Directive 2001/58/EC). MDEA can be substantially metabolized in vivo and eliminated through urinary excretion.42,43 It has been utilized as the building block of polymeric vectors specifically designed for enhancing DNA vaccine efficacy in vivo as well as controlled insulin release.44,45 Given those considerations, the degradation into MDEA is advantageous for application in the medical field from the perspective of stringent safety requests. With regard to the unique function together with the low toxicity, it is expected that, with further study, this novel aliphatic polycarbonate and its monomer shall have promising potential in biomedical applications.

Conclusions A novel cyclocarbonate, 6,14-dimethyl-3,9,11-tetraoxa-6,14diaza- cyclohexadecane-2,10-dione (ADMC)2, was synthesized and characterized. Starting from (ADMC)2, we developed a lipase-catalyzed ring-opening polymerization process to synthesize the functional polycarbonate of poly(ADMC), containing tertiary amine groups in the backbone. Novozyme-435 lipase exhibited high activity toward the polymerization in terms of high Mns and monomer conversions. The resultant data suggested a “living” characteristic for this enzyme-catalyzed polymerization, and the “living” feature seemed independent of the lipase concentration. It was found that, under the identical reaction condition, higher lipase concentration led to a polymer with lower Mns. Addition of molecular sieves (4 Å) into the polymerization system would markedly enhance the Mns of the


polymer. In the presence of molecular sieves (4 Å), the highest Mn value of 1.2 × 104 g/mol was obtained in toluene with monomer concentration of 0.58 M at 75 °C in the presence of 10 wt % Novozym-435 to the monomer. No marked difference in monomer consumption rate was observed for different reaction temperature of 60 and 75 °C, whereas relatively higher molecular weight could be obtained at 75 °C. The resultant data of the polymerization in mixed solvents (toluene/isooctane) indicated that polymer solubility may exert significant influences on the polymerization with respect to polymer Mns. The obtained poly(ADMC) was water-soluble and displayed low toxicity toward HeLa and Hep G2 cells by MTT assay. The degradation test in vitro indicated that poly(ADMC) was a fast degradable material and would ultimately degrade into low toxic N-methyl diethanolamine. Acknowledgment. This work was financially supported by the National Natural Science Foundation of China (Grant No. 20874075), National Key Basic Research Program of China (2005CB623903 and 2009CB930301), and Ministry of Education of China (Cultivation Fund of Key Scientific and Technical Innovation Project 707043).

References and Notes (1) William, C.; Ray, I.; Grinstaff, M. W. Macromolecules 2003, 36, 3557– 3562. (2) Albertson, A.-C.; Sjoling, M. J. Macromol. Sci., Part A 1992, 29, 43–54. (3) Feng, J.; He, F.; Zhuo, R. X. Macromolecules 2002, 35, 7175–7177. (4) Fu, K.; Pack, D. W.; Klibanov, A. M.; Langer, R. Pharm. Res. 2000, 17, 100–106. (5) Tinsley-Bown, A. M.; Fretwell, R.; Dowsett, A. B.; Davis, S. L.; Farrar, G. H. J. Controlled Release 2000, 66, 229–241. (6) Walter, E.; Moelling, K.; Pavlovic, J.; Merkle, H. P. J. Controlled Release 1999, 61, 361–374. (7) Kricheldorf, H. R.; Weegen-Schulz, B. J. Polym. Sci., Part A: Polym. Chem. 1995, 33, 2193–2201. (8) Shen, Y.; Chen, X.; Gross, R. A. Macromolecules 1999, 32, 3891– 3897. (9) Mei, H. J.; Zhong, Z. L.; Long, F. F.; Zhuo, R. X. Macromol. Rapid Commun. 2006, 27, 1894–1899. (10) Feng, J.; Wang, X. L.; He, F.; Zhuo, R. X. Macromol. Rapid Commun. 2007, 28, 754–758. (11) Chen, X.; McCarthy, S. P.; Gross, R. A. Macromolecules 1997, 30, 3470–3476. (12) Kalbe, M.; Keul, H.; Ho¨cker, H. Macromol. Chem. Phys. 1995, 196, 3305–3321. (13) Wang, L.-S.; Cheng, S.-X.; Zhuo, R.-X. Macromol. Rapid Commun. 2004, 25, 959–963. (14) Wang, X.-L.; Zhuo, R.-X.; Liu, L.-J.; He, F.; Liu, G. J. Polym. Sci., Part A: Polym. Chem. 2001, 40, 70–75. (15) Sanda, F.; Kamatani, J.; Endo, T. Macromolecules 2001, 34, 1564– 1569.


2557

(16) Al-Azemi, T. F.; Bisht, K. S. J. Polym. Sci., Part A: Polym. Chem. 2002, 40, 1267–1274. (17) Chen, X.; Gross, R. A. Macromolecules 1999, 32, 308–314. (18) Chen, X.; McCarthy, S. P.; Gross, R. A. J. Appl. Polym. Sci. 1997, 67, 547–557. (19) Cheng, S.-X.; Miao, Z.-M.; Wang, L.-S.; Zhuo, R.-X. Macromol. Rapid Commun. 2003, 24, 1066–1069. (20) Wang, X.-L.; Zhuo, R.-X.; Huang, S.-W.; Liu, L.-J.; He, F. Macromol. Chem. Phys. 2002, 203, 985–990. (21) Zhu, J. L.; Cheng, H.; Jin, Y.; Cheng, S. X.; Zhang, X. Z.; Zhuo, R. X. J. Mater. Chem. 2008, 18, 4433–4441. (22) Kundu, P. P.; Sharma, V. Curr. Opin. Solid State Mater. Sci. 2009. (23) Lee, A. S.; Bu¨tu¨n, V.; Vamvakaki, M.; Armes, S. P.; Pople, J. A.; Gast, A. P. Macromolecules 2002, 35, 8540–8551. (24) Li, Y. Y.; Zhang, X. Z.; Cheng, H.; Zhu, J. L.; Li, U. N.; Cheng, S. X.; Zhuo, R. X. Nanotechnology 2007, 18, 215605–215612. (25) Kumar, A.; Gross, R. A. Biomacromolecules 2000, 1, 133–138. (26) Kricheldorf, H. R.; Lee, S.-R.; Weegen-Schulz, B. Macromol. Chem. Phys. 1996, 197, 1043–1054. (27) Kricheldorf, H. R.; Mahler, A. J. Polym. Sci., Part A: Polym. Chem. 1996, 34, 2399–2406. (28) Kricheldoef, H. R.; Malhle, A.; Lee, S.-R. New Polym. Mater. 1996, 5, 25–34. (29) Kricheldorf, H. R.; Mahler, A. Polymer 1996, 37, 4383–4388. (30) Kricheldorf, H. R.; Mahler, A.; Lee, S.-R. J. Macromol. Sci., Part A 1997, 34, 417–428. (31) Kricheldorf, H. R.; Stricker, A. Polymer 2000, 41, 7311–7320. (32) Kricheldorf, H. R.; Stricker, A. Macromol. Chem. Phys. 2000, 201, 2557–2565. (33) Bisht, K. S.; Svirkin, Y. Y.; Henderson, L. A.; Gross, R. A. Macromolecules 1997, 30, 7735–7742. (34) Matsumura, S.; Tsukada, K.; Toshima, K. Macromolecules 1997, 30, 3122–3124. (35) Kobayashi, S.; Kikuchi, H.; Uyarna, H. Macromol. Rapid Commun. 1997, 18, 575–579. (36) Uyama, H.; Kikuchi, H.; Takeya, K.; Kabayashi, S. Acta Polym. 2003, 47, 4. (37) Albertsson, A. C.; Srivastava, R. K. AdV. Drug DeliVery ReV. 2008, 60, 1077–1093. (38) Gross, R. A.; Kumar, A.; Kalra, B. Chem. ReV. 2001, 101, 2097– 2124. (39) Kawaguchi, T.; Nakano, M.; Juni, K.; Inoue, S.; Yoshida, Y. Chem. Pharm. Bull. 1983, 31, 1400–1403. (40) Zhu, K. J.; Hendren, R. W.; Jensen, K.; Pitt, C. G. Macromolecules 1991, 24, 1736–1740. (41) Wang, C.-F.; Lin, Y.-X.; Jiang, T.; He, F.; Zhuo, R.-X. Biomaterials 2009, 30, 4824–4832. (42) Werley, M. S.; Ballantyne, B.; Leung, H.; Hermansky, S. J.; Fowler, E. H. J. Toxicol. Cutaneous Ocul. Toxicol. 1997, 16, 157–171. (43) Leung, H.; Ballantyne, B.; Frantz, S. W. J. Toxicol. Cutaneous Ocul. Toxicol. 1996, 15, 343–353. (44) Wang, C.; Ge, Q.; Ting, D.; Nguyen, D.; Shen, H.; Chen, J.; Eisen, H. N.; Heller, J.; Langer, R.; Putnam, D. Nat. Mater. 2004, 3, 190– 196. (45) Heller, J.; Chang, A. C.; Rood, G.; Grodsky, G. M. J. Controlled Release 1990, 13 (2-3), 295–302.

BM1001476

Biocatalytic Fabrication of Fast-Degradable, Water-Soluble

Recommend Documents