Design and Synthesis of Self-Degradable Antibacterial Polymers by

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Design and Synthesis of Self-Degradable Antibacterial Polymers by Simultaneous Chain- and Step-Growth Radical Copolymerization Masato Mizutani,† Edmund F. Palermo,‡ Laura M. Thoma,§ Kotaro Satoh,† Masami Kamigaito,*,† and Kenichi Kuroda*,‡,§,∥ †

Department of Applied Chemistry, Graduate School of Engineering, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8603, Japan ‡ Macromolecular Science and Engineering Center, §Department of Chemistry, and ∥Department of Biologic and Materials Sciences, School of Dentistry, University of Michigan, Ann Arbor, Michigan 48109, United States S Supporting Information *

ABSTRACT: Self-degradable antimicrobial copolymers bearing cationic side chains and main-chain ester linkages were synthesized using the simultaneous chain- and step-growth radical polymerization of t-butyl acrylate and 3-butenyl 2chloropropionate, followed by the transformation of t-butyl groups into primary ammonium salts. We prepared a series of copolymers with different structural features in terms of molecular weight, monomer composition, amine functionality, and side chain structures to examine the effect of polymer properties on their antimicrobial and hemolytic activities. The acrylate copolymers containing primary amine side chains displayed moderate antimicrobial activity against E. coli but were relatively hemolytic. The acrylate copolymer with quaternary ammonium groups and the acrylamide copolymers showed low or no antimicrobial and hemolytic activities. An acrylate copolymer with primary amine side chains degraded to lower molecular weight oligomers with lower antimicrobial activity in aqueous solution. This degradation was due to amidation of the ester groups of the polymer chains by the nucleophilic addition of primary amine groups in the side chains resulting in cleavage of the polymer main chain. The degradation mechanism was studied in detail by model reactions between amine compounds and precursor copolymers.

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INTRODUCTION Cationic amphiphilic copolymers have long attracted scientific and commercial interest due to their ability to control bacterial growth in solution and on surfaces by a mechanism involving disruption of bacterial cytoplasmic membranes.1 Alkyl quaternary ammonium groups have been widely used as cationic groups, which are likely responsible for polymer-binding to bacteria and membrane disruption. Recently, the design of antimicrobial polymers has been extended to use primary ammonium groups to mimic the amphiphilic property and cationic functionality of natural antimicrobial peptides.2−5 In this new approach, the random copolymers based on norbornene,6 methacrylates,7 methacrylamides,8 and β-lactams5 displayed potent antimicrobial activity and hemocompatibility, which can be controlled by tuning the balance between hydrophobic and cationic properties as well as the polymer length. These synthetic polymers are chemically stable and resistant to proteolysis in physiological conditions, which could provide potent activity and extended lifetime at infection sites. However, for biomedical applications it would be of interest to develop antimicrobial polymers that degrade to inactive oligomers within a controlled time frame to prevent undesired long-term toxicity. © 2012 American Chemical Society

Synthetic and naturally occurring polymers that degrade in physiological conditions to give nontoxic low molecular weight compounds have been utilized in biomedical applications.9 In this field, polymers with ester groups in the main chain, including polylactate, polyglycolide, and polycaprolactone, have been used as biodegradable sutures, fracture fixations, oral implants, and drug delivery microspheres.10−12 The degradation process of these polymers involves hydrolysis of mainchain ester linkages in water or by enzymes,11 resulting in scission of polymer chains. Although these polymers have been useful to produce particles and scaffolds, the inherent low water solubility of polyesters limits the applications of the materials in aqueous conditions. In this study, we demonstrate the design and synthesis of water-soluble degradable amphiphilic copolymers with antimicrobial activity, which contain ester linkages in the main chains and primary amine groups in the side chains. In our polymer design, polyester-based polymers with cationic functionality are expected to display amphiphilic structures necessary for antimicrobial activity, and the ester linkage of the Received: February 17, 2012 Revised: April 10, 2012 Published: April 13, 2012 1554

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the solvents. The products were purified by repeated precipitation from CHCl3 into hexane and dried under vacuum.27 Deprotection of Boc-Protected Copolymer. The Boc-protected copolymer (0.10 g) was treated with TFA (30.0 mL) or hydrochloric acid in 1,4-dioxane (4.0 M; 3.8 mL) at room temperature. Within 1 h, 1H NMR indicated disappearance of the Boc group. The product was evaporated to remove the solvents to result in the deprotected products. Synthesis of Copolymers Containing Quaternary Ammonium Salts. 2-(Dimethylamino)ethanol (0.45 mL) was added with vigorous stirring to a solution of poly(acrylic acid-co-1) (35.1 mg), DCC (0.34 g), and DMAP (0.15 g) in CH2Cl2 (2.06 mL) at 0 °C. The mixture was stirred for 5 min at 0 °C and then for 3 h at room temperature. After the dilution with CHCl3, the mixture was washed with distilled water and was evaporated to remove the solvents. The products were purified by repeated precipitation from CHCl3 into hexane and dried under vacuum. The obtained product (2.0 mg) was dissolved in methanol (0.05 mL). Ethyl iodide (0.01 mL) was added to the solution. After 2 h, the product was evaporated to remove the solvents, resulting in the products containing quaternary ammonium salts. Amidation of Poly(acrylic acid-co-1). N-Boc-ethylenediamine (0.27 mL) was added with vigorous stirring to a solution of poly(acrylic acid-co-1) (50.0 mg), DCC (0.13 g), and DMAP (0.06 g) in CH2Cl2 (0.69 mL) at 0 °C. The mixture was stirred for 5 min at 0 °C, then for 3 h at room temperature, and was evaporated to remove the solvents. The products were purified by repeated precipitation from CHCl3 into hexane and dried under vacuum. Antimicrobial Activity. The lowest polymer concentration required to completely inhibit growth of bacteria, defined as the minimum inhibitory concentration (MIC), was determined by a standard microbroth dilution assay according to the Clinical and Laboratory Standards Institute guidelines (CLSI28) with suggested modifications by REW Hancock Laboratory (University of British Columbia, Vancouver, British Columbia, Canada29) and Giacometti et al.30 for testing cationic agents. Each polymer was dissolved in dimethyl sulfoxide (DMSO), and eight 2-fold serial dilutions of the stock were prepared in 0.01% acetic acid. Escherichia coli ATCC 25922 in the midlogarithmic growth phase were diluted to OD600 = 0.001 in MH broth. This stock suspension of bacteria (90 mL) was mixed with serial dilutions of a polymer stock solution (10 mL) in each well of a 96-well polypropylene microplate, which was not treated for tissue culture (Coming #3359). After incubating at 37 °C for 18 h, the OD600 in each well was recorded using a microplate reader (Perkin-Elmer Lambda Reader). The MIC was defined as the lowest polymer concentration at which no turbidity was observed relative to the negative growth control, sterile MH broth. As an additional negative control, 2-fold serial dilutions of the DMSO in 0.01% acetic acid, without polymer, were tested in the same conditions and showed no inhibitory effects, even at the highest DMSO concentration (5%). All experiments were performed three times in triplicate and MIC values reported are the average of the three trials. The MIC values were determined below the solubility limit of the polymers in MH broth in every case. Hemolytic Activity. Toxicity to human red blood cells (RBCs) was assessed by a hemoglobin release assay. RBCs (1 mL) were diluted into HEPES buffered saline (9 mL; HBS = 10 mM HEPES, 150 mM NaCl, pH 7) and then centrifuged at 1000 rpm for 5 min. The supernatant was carefully removed using a pipet. The RBCs were then washed with HEPES two additional times. The resulting stock (10% v/ v RBC) was diluted 3-fold in HEPES to give the assay stock (3.3% v/v RBC). The assay stock (90 μL) was then mixed with each of the polymer serial dilutions (10 μL) on a sterile 96-well round-bottom polypropylene microplate to give a final solution of 3% v/v RBC, which corresponds to approximately 108 red blood cells per mL based on counting in a hemacytometer. HEPES (10 μL) or Triton X-100 (10 μL, 1% v/v) was added instead of polymer solution as negative and positive hemolysis controls, respectively. The microplate was secured in an orbital shaker at 37 °C and 180 rpm for 60 min. The plate was then centrifuged at 1000 rpm for 10 min. The supernatant (10 μL)

polymer backbone would be a potential site of polymer chain scission for degradation to inactive low molecular weight oligomers. For polymer synthesis, we utilized a polymerization method based on the metal-catalyzed simultaneous chain- and step-growth radical polymerization to prepare cationic amphiphilic copolymers, which was quite recently developed.13−17 This polymerization method involves evolution of the conventional radical addition reaction18−24 into simultaneous chain- and step-growth propagation, enabling incorporation of ester units into the polymer backbone as well as functional groups of vinyl monomers into the side chain in one polymerization procedure.13,15 Because the polyester units are relatively hydrophobic, the amphiphilic properties of polymers could be tuned by altering the ratios between amine and ester units for optimal antibacterial activity. Recently, antimicrobial activity of random copolymers with a biodegradable polycarbonate backbone and quaternary ammonium groups in the side chains have been reported.25 Compared to previously reported syntheses of biodegradable monomers and polymers with carbonate and ester polymer chains, we prepared copolymers by a more facile radical polymerization method to incorporate vinyl monomer functionality and ester linkage into polymer chains. This radical polymerization method is also compatible to a wide range of functional monomers. We herein report the antimicrobial and hemolytic activities of these polymers and their polymer degradations. We also investigated the degradation mechanism of the polymers and model compounds in detail by matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry (MALDI-TOFMS).

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MATERIALS AND METHODS

Materials. tBA (TCI, >98%) was distilled from calcium hydride under reduced pressure before use. 3-Butenyl 2-chloropropionate (1) was synthesized according to the literature.14 CuCl (Aldrich, 99.99%) was used as received and was handled in a glovebox (VAC Nexus) under a moisture- and oxygen-free argon atmosphere (O2 < 1 ppm). Toluene and CH2Cl2 (Both from Kanto, > 99.5%, H2O < 10 ppm) were further dried and deoxygenized by passage through columns of Glasscontour solvent system before use. HMTETA (Aldrich, 97%) was distilled from calcium hydride before use. Polymerizations. Polymerization was carried out under dry nitrogen in baked glass tubes equipped with a three-way stopcock. A typical example for the polymerization procedure is given below. To a suspension of CuCl (99.0 mg, 1.0 mmol) in toluene (3.64 mL) was added HMTETA (0.27 mL, 1.0 mmol), and the mixture was stirred for 12 h at 80 °C to give a heterogeneous solution of CuCl/HMTETA complex. After the solution was cooled to room temperature, tBA (2.93 mL, 20.0 mmol) and 1 (3.16 mL, 20.0 mmol) were added. The tube was immersed in a thermostatic oil bath at 80 °C. The polymerization was terminated by cooling the reaction mixtures to room temperature. Monomer conversions were determined from the concentration of residual monomers by 1H NMR spectroscopy with toluene as an internal standard. Deprotection of the tert-Butyl Group. The poly(tBA-co-1) (2.77 g) in CH2Cl2 (9.0 mL) was treated with TFA (9.0 mL) at room temperature. Within 30 min, 1H NMR indicated disappearance of the tert-butyl group. The product was evaporated to remove the solvents to result in the poly(acrylic acid-co-1).26 Esterification of Poly(acrylic acid-co-1). The 2-Boc-ethanolamine (9.6 g) was added with vigorous stirring to a solution of poly(acrylic acid-co-1) (1.5 g), DCC (4.5 g), and DMAP (3.3 g) in CH2Cl2 (22.6 mL) at 0 °C. The mixture was stirred for 5 min at 0 °C and then for 3 h at room temperature. After dilution with CHCl3, the mixture was washed with distilled water and was evaporated to remove 1555

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was diluted into HEPES (90 μL) and the absorbance at 415 nm was recorded using a microplate reader (Perkin-Elmer Lambda Reader). The fraction of hemolysis was defined as H = (A − A0)/(ATX − A0), where A is the absorbance reading of the sample well, A0 is the negative hemolysis control (buffer), and ATX is the positive hemolysis control (Triton X-100). Hemolysis was plotted as a function of polymer concentration and the HC50 was defined as the polymer concentration that causes 50% hemolysis relative to the positive control. All experiments were performed three times in triplicate. Degradation of Polymer in Buffer Solution. A portion of the polymer (10 mg) was dissolved in ethanol (1.0 mL) and diluted into phosphate-buffered saline of pH varying from 7.0 to 12.0 (20 mL; 10 mM 4-(2-hydroxyethyl)piperazine 1-ethanesulfonic acid, tris(hydroxymethyl)aminomethane, NaHCO3, or K2CO3, 150 mM NaCl) in a vial. The solution was reacted at 37 °C. The products were evaporated to remove the solvents to result in the degraded products. Degradation of Poly(acrylic acid-co-1) with 2-Ethanolamine Hydrochloride in Buffer Solution. A portion of the polymer (5 mg) and 2-ethanolamine hydrochloride (2.1 mg) was dissolved in ethanol (0.5 mL) and diluted into buffered saline (10 mL; 10 mM 4(2-hydroxyethyl)piperazine 1-ethanesulfonic acid, tris(hydroxymethyl)aminomethane, NaHCO3, or K2CO3, 150 mM NaCl) in a vial. The solution was reacted for 18 h at 37 °C. The products were evaporated to remove the solvents to result in the degraded products. Measurements. Monomer conversions were determined from the residual monomers by 1H NMR spectroscopy with toluene as an internal standard. 1H NMR spectra were recorded in CDCl3 at 25 °C on a JEOL ECS-400 or a Varian Gemini 2000 spectrometer, operating at 400 MHz. The number-average molecular weight (Mn), the weightaverage molecular weight (Mw), and the molecular weight distribution (Mw/Mn) of the product polymers were determined by size-exclusion chromatography (SEC) in THF at 40 °C on two polystyrene gel columns [Shodex K-805 L (pore size: 20−1000 Å; 8.0 mm i.d. 30 cm); flow rate 1.0 mL/min] connected to Jasco PU-980 precision pump and a Jasco 930-RI refractive index detector. The columns were calibrated against eight standard poly(MMA) samples (Shodex; Mp = 202− 1950000; Mw/Mn = 1.02−1.09). MALDI-TOF-MS spectra were measured on a Shimadzu AXIMA-CFR Plus mass spectrometer (linear mode) with dithranol (1,8,9-anthracenetriol) as the ionizing matrix and sodium trifluoroacetate as the ion source. ESI-MS spectra were recorded on a JEOL JMS-T100CS spectrometer.

Scheme 1. Synthesis of Self-Degradable Antimicrobial Polymer

The precursor copolymers were first prepared by simultaneous polymerization with various monomer feed ratios of tBA and 1 ([tBA]0/[1]0 = 1/1, 3/1, and 1/3) in toluene at 80 °C using CuCl/1,1,4,7,10,10-hexamethyltriethylenetetramine (HMTETA) as the catalyst. Under the same conditions, methyl acrylate (MA) and 1 have been successfully polymerized simultaneously.15 In all cases, both monomers were quantitatively consumed in 120 h (>98% total monomer conversion), in which the consumption of tBA was slightly faster than that of 1 (Figure 1A). The molecular weight of polymers significantly increased as the total conversion approached 100% (Figure 1B,C), which is characteristic to simultaneous polymerization.15 The copolymers P1−P3 possessed molecular weights in the range of 1200−2500 with similar molecular weight distributions (Mw/Mn ∼ 2.5; Table 1). The low molecular weights of the polymers were intentional as previous reports showed that amphiphilic polymethacrylates with such low molecular weights displayed better hemocompatibility than high molecular weight polymers.31 The mole fractions of the main-chain ester groups relative to the total number of units in a polymer chain, fester, ranged from 0.25 to 0.74, and are expected to control the antimicrobial activity and degradability of the copolymers. The 1 H NMR spectrum of the obtained products showed the combined signals from the both homopolymers of tBA and 1, which were prepared separately (Figure S1). The MALDITOF-MS spectrum also showed that both monomers were randomly copolymerized (Figure S2). This result is in agreement with the previous report on the copolymerization

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RESULTS AND DISCUSSION Polymer Synthesis. The copolymers were prepared by metal-catalyzed simultaneous chain- and step-growth radical polymerization (Scheme 1). We used t-butyl acrylate (tBA) and 3-butenyl 2-chloropropionate (1) for the copolymerizations to give the precursor polymers. In this method, the monomer 1 undergoes step-growth polymerization through radical formation and deactivation by a metal catalyst. On the other hand, the acrylate monomer is also polymerized by chain-growth radical polymerization catalyzed by the same metal catalyst, that is, metal-catalyzed living radical polymerization or atom transfer radical polymerization (ATRP). These polymerizations progress simultaneously in the mixture of monomers by metal catalysis, and cross-reaction of these monomers yields random copolymers with acrylate and ester repeat units in the polymer chains. This polymerization method enables incorporation of ester units into the polymer backbones as well as functional groups of vinyl monomers into the side chain through one polymerization procedure.13,15 The t-butyl groups were further converted to t-butyloxycarbonyl (Boc)-protected amine groups, and the following deprotection yielded the targeted polymer with ammonium salts in the side chains. 1556

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Figure 1. Simultaneous chain- and step-growth radical polymerization of tBA and 1 with CuCl/HMTETA in toluene at 80 °C: [tBA]0 = 2.0 M; [1]0 = 2.0 M; [CuCl]0 = 100 mM; [HMTETA]0 = 100 mM. (A) Consumption of tBA measured by 1H NMR and 1 measured by gas chromatography. (B) Mw and Mw/Mn values of the obtained copolymers vs total monomer conversion of tBA and 1. (C) Size-exclusion chromatograms of the obtained copolymers.

Table 1. Simultaneous Chain- and Step-Growth Radical Polymerization of tBA and 1a entry

[tBA/1]0, (M)

time (h)

conversion (tBA/1; %)b

Mnb

Mwb

Mw/Mnb

yield (%)

tBA/1c

P1 P2 P3

2.0/2.0 3.0/1.0 1.0/3.0

140 140 500

>99/97 >99/99 >99/97

1500 2500 1200

4100 6700 3200

2.83 2.64 2.72

84 83 78

51/49 75/25 26/74

a [CuCl]0 = 100 mM, [1,1,4,7,10,10-hexamethyltriethylenetetramine (HMTETA)]0 = 100 mM, in toluene at 80 °C. bThe number-average molecular weight (Mn), the weight-average molecular weight (Mw), and distribution (Mw/Mn) were determined by size-exclusion chromatography. c Determined by 1H NMR.

copolymers, we prepared a series of copolymers (P4−P14) with different structural features in terms of molecular weight, monomer composition, amine functionality, and side chain structure to examine the effect of polymer properties on their antimicrobial and hemolytic activities. These parameters of copolymers have been reported to determine their biological activity and ability to disrupt bacterial cell membranes.2 We evaluated the antimicrobial activity of the copolymers P4−P14 against Escherichia coli in a turbidity-based assay and determined the minimal inhibitory concentration of the copolymers (MIC) in which the bacterial growth is completely inhibited (Table 2). The acrylate copolymers containing primary amine groups P4, P6, P7, and P8 displayed antimicrobial activity against E. coli (MIC = 16−104 μg/mL). Under the same assay conditions, the natural antimicrobial peptide magainin-2 displayed an MIC of 125 μg/mL for comparison.7 When copolymers with the similar fester of 0.22− 0.28 (P6−P8) are compared, higher molecular weight polymers displayed lower MIC values (higher activity) against E. coli. Comparing copolymers of similar molecular weights (4000− 5500; P4, P6, P7, and P9), P4 displayed the lowest MIC value of 16 μg/mL, while other polymers with both higher or lower fester have larger MIC values. This indicates that there is an optimal fester for high activity. It has been reported that amphiphilic polymers show higher activity as the hydrophobicity of polymers is increased.31 However, high hydrophobicity of polymers in the high fester range may cause strong aggregation of polymer chains in aqueous solution. This may reduce the number of polymers available to interact with the bacteria cells, resulting in low antibacterial activity.31 The acrylate copolymer with quaternary ammonium groups, P13, showed no activity (MIC > 500 μg/mL) although the copolymer of similar molecular weight and fester with primary amine groups, P6, displayed an order of magnitude lower MIC value (MIC = 31 μg/mL). This result is in agreement with the previous report on the antimicrobial activity of polymethacry-

of MA and 1, in which each monomer unit was randomly distributed in the main chain.15 These results suggest that the copolymerization proceeded by the random simultaneous cross-propagation of monomers. The t-butyl ester groups of the copolymers were acidolyzed using trifluoroacetic acid (TFA) to give carboxylic acids, which were coupled with Boc-protected ethanolamine using N,N′dicyclohexylcarbodiimide (DCC) in the presence of N,N′dimethyl-4-aminopyridine (DMAP). The quantitative acidolysis of the t-butyl ester groups was confirmed by 1H NMR spectra (Figure 2A,B), where the broad peak of the carboxylic acid (7.6−8.0 ppm) appeared along with the disappearance of the tbutyl signal (1.4−1.5 ppm; P14 in Figure 3). The Bocprotected aminoethyl group was successfully introduced into the side chains, which was supported by the 1H NMR spectrum indicating that the peak of the carboxylic acid disappeared and that the large sharp peak of Boc groups appeared (1.4−1.5 ppm; Figure 2C). Finally, the Boc-protected amine groups were deprotected with TFA or HCl to give the desired cationic copolymers with primary ammonium groups as trifluoroacetate (Figure 2D) or chloride salts (P4−9; Table 2). With similar procedures, the acrylamide copolymer bearing primary ammonium groups was prepared using mono-Boc protected ethylenediamine instead of Boc-protected ethanolamine, followed by acid deprotection (P10−12). The acrylate copolymers with quaternary ammonium salts were prepared using N,N-dimethylaminoethanol, followed by alkylation of amine groups by ethyl iodide (P13). The ratios of amine to ester units closely matched with the feed ratios, which supports the notion that the polymerization and following modification of side chains were quantitative. Antibacterial and Hemolytic Activities. Cationic, amphiphilic random copolymers with cationic groups in the side chains have previously displayed antimicrobial effects against a broad spectrum of bacteria.1−3 As our first approach to the investigation on the structure−activity relationship of 1557

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Figure 2. 1H NMR spectra of (A) poly(tBA-co-1) (Mn = 1200, n = 4.9, m = 3.5), (B) poly(acrylic acid-co-1) (Mn = 890, n = 4.4, m = 3.5), (C) Bocprotected copolymer (Mn = 1500, n = 4.2, m = 3.4), and (D) cationic copolymer (Mn = 1400, n = 4.7, m = 3.6; A−C: CDCl3, r.t.; D: DMSO-d6, r.t.).

late derivatives with primary or quaternary ammonium groups.32 Acrylamide copolymers P10−12 showed low activity (MIC ≥ 500 μg/mL). This might be due to the hydrophilic nature of acrylamide, which could discourage insertion of

polymers into the hydrophobic regions of bacterial cell membranes, reducing the ability of polymers to permeabilize the membranes and resulting in low antimicrobial activity. It has been reported that polymethacrylamide derivatives are less active than their polymethacrylate counterparts and need more hydrophobic groups to be effective in membrane disruption.8 The acrylic acid copolymer P14 was inactive, which might suggest that cationic functionality of polymers is essential for antibacterial activity. Despite limited testing of polymer parameters, these results seem to indicate that the acrylate copolymers with primary amine side chains may be a platform for further optimization in order to achieve potent antimicrobial activity. To assess hemocompatibility, we examined the lytic activity of the polymers against human red blood cells (RBCs) (hemolysis) and determined the polymer concentration necessary for 50% hemolysis (HC50; Table 2; see Figure S3, Supporting Information for hemolysis dose−response curves.). The hemolytic activity of the copolymers depended on the monomer identity and the comonomer ratio, likely because the

Figure 3. Copolymers possessing main-chain ester linkages and pendent ammonium salts. 1558

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Table 2. Characterization and Biological Activities of Polymers entry

precursor

P4 P5 P6 P7 P8 P9 P10 P11 P12 P13 P14

P1 P1 P2 P2 P2 P3 P1 P2 P3 P2 P1

vinyl monomer acrylate

ammonium

festera

ammonium (m)/ester (n)b

DPc

Mnd

MICe (μg/mL)

HC50f (μg/mL)

HC50/MIC

primary

0.48 0.50 0.22 0.26 0.28 0.73 0.52 0.26 0.72 0.26 0.47

14.5/13.5 5.4/5.4 27.7/7.9 23.2/8.1 12.7/4.9 7.0/18.6 12.3/13.3 27.0/9.3 7.1/17.9 16.9/5.8 18.6/16.7

28.1 10.8 35.5 31.3 17.6 25.5 25.6 36.3 25.0 22.7 35.3

4400 2100 5500 4800 2700 4100 4000 5600 4000 6000 4100

16 500 31 104 >500 63 >500 >500 500 >500 >500

1000 (9%) >500 (0%) >250 (36%) >500 (14%) >1000 (1%)

0.50

a Average mole fraction of vinyl ester units relative to the total number of monomer units, calculated from analysis of the peak integration of the 1H NMR spectra. bAmmonium (m)/ester (n) units in polymer. cDegree of polymerization calculated by NMR. dNumber average molecular weight calculated by NMR. eMinimum inhibitory concentration of polymers for E. coli. fPolymer concentration for 50% hemolysis of human red blood cells. Number following in parentheses is the percent hemolysis at the highest or lowest polymer concentration tested. See Supporting Information for hemolysis curves. gNot determined.

Figure 4. Mp of degraded products after incubation at pH 7.0 in buffer solution for 0.5−360 h (A) and at pH 7.0−12.0 in buffer solution for 18 h at 37 °C (B), and MALDI-TOF-MS spectra of (C) cationic copolymer (P6; Mn = 5500, n = 27.7, m = 7.9) and the degraded products at pH 7.0 in buffer solution at (D) 0.5, (E) 3, (F) 18, or (G) 360 h at 37 °C: [cationic polymer (P6) or poly(acrylic acid-co-1) (P14)]0 = 500 mg/mL, [HEPES]0 = 10 mM, [NaCl]0 = 150 mM.

copolymers with increasing fester may be attributed to the polymers’ increased hydrophobicity due to the hydrophobic properties of ester units.31 Interestingly, P4 showed higher hemolytic activity than more hydrophobic P9. It is possible that the polymer chains of P9 strongly aggregate in aqueous assay media due to high hydrophobicity, reducing the number of polymers active against the red blood cell membranes.31 The acrylamide copolymers, P10−12, the acrylate copolymer with quaternary ammonium groups, P13, and the acrylic acid copolymer, P14, displayed no hemolytic activity. Such stark

hydrophobicity of the polymers is the main determinant of hemolytic activity.31 When the hemolytic activity of acrylate copolymers with similar MW (∼4000−5000; P4, P7, and P9) are compared, the copolymer P7, having the lowest fester of 0.26 in the series, did not show significant hemolytic activity (HC50 > 500 μg/mL). P4 ( fester = 0.48) and P9 (fester = 0.73), which have higher fester, displayed moderate to high hemolytic activity (HC50 < 3.9 and 87 μg/mL, respectively). For comparison, in this assay, the HC50 of the toxic bee venom peptide melittin is 2 μg/mL.33 The increased hemolytic activity of acrylate 1559

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Figure 5. Antimicrobial and hemolytic activities of degraded products at (A) pH 7.0 or (B) pH 10.0.

copolymer. The Mp rapidly decreased for the first 18 h and eventually reached 1700 at 360 h (Figure 4). However, this still appears to be higher than the theoretical value of degradation products for the completion (Mp = 617, n = 3, m = 0) assuming all ester groups are cleaved. We further examined the effect of pH on polymer degradation. As pH was increased, the degradation products of P6 after 18 h have lower molecular weights than those at pH 7 (Figures 4 and S4) and the Mp appears to reach to ∼1000 at pH 12. Interestingly, the precursor copolymer with acid side chains, P14, also degraded in aqueous solutions, which is likely due to hydrolysis of ester linkages (Figures 4B and S5). However, the molecular weight of degradation products of P14 showed Mp = 2600 at pH 12 although the copolymer with primary amine groups P6 degraded to lower MW oligomers (Mp = ∼1000) under the same condition. This may suggest that the primary amine groups in the side chains may facilitate the degradation process of P6. The degradation mechanism of copolymers will be discussed in detail below. We further examined the effects of the polymer degradation on the antimicrobial and hemolytic activities of the copolymer P6. The MIC and HC50 values of P6 after degradation at pH 7 or 10 were determined (Figure 5 and Table S1). At pH 7, the molecular weight of the polymer decreased from 6000 to ∼2000 after 200−300 h. The MIC value increased from 31 to 63 μg/mL after 120 h, and the HC50 is increased more significantly from 500 μg/mL, HC50 > 500 μg/mL). The molecular weight of degradation products at pH 10 reached Mp ∼ 1000 after 2−3 h, and these low molecular weight oligomers are evidently inactive against both bacteria and red blood cells. The degradation rate at pH 10 is significantly higher than at pH 7, and the oligomers produced at pH 10 have lower molecular weight relative to the degradation products obtained at pH 7. The small reduction in antimicrobial activity at pH 7 may reflect slower degradation of polymer chains. These results indicate that polyester-based polymers with primary amine groups in the side chains

differences in hemolysis likely arise from the properties of amine side chains as well as the hydrophilic nature of the polymers as previously shown for polymethacrylates containing quaternary ammonium groups32 or the polymethacrylamides.8 The copolymers P4 and P6 showed highest antimicrobial activity against E. coli (MIC of 16−32 μg/mL) among the polymers that were prepared in this study. However, these polymers showed high hemolytic activity (HC50 < 3.9−7.8 μg/ mL) and a low selective index (HC50/MIC) of 500 μg/mL), giving a selective index of >4.8. Although the quantitative comparison of MIC and HC50 values in literature may be challenging because these values strongly depend on the assay conditions, the polymers reported here seem to show moderate antimicrobial activity and be relatively hemolytic (low selectivity) as compared to other polycations.5−7,34,35 The selective index of these polymers needs to be improved for them to be good antimicrobial candidates. In previous reports on antimicrobial polymethacrylate derivatives, the antimicrobial activity and selectivity of these polymers were improved by reducing molecular weight to 2000−3000 and balancing the ratio of cationic and hydrophobic side chains.31 In addition, monodisperse nylon copolymers displayed lower hemolytic activity compared to polydisperse samples as high molecular weight components increase hemolytic activity.36 The copolymers prepared in this report have relatively high molecular weight (4000−5500) and broad molecular weight distribution (Mw/Mn = ∼2.7−2.8). Synthesis of lower molecular weight polymers, followed by column chromatography or dialysis to remove high molecular weight polymer components will give more monodisperse oligomers, which may display higher antimicrobial activity and low hemolysis. Polymer Degradation and Activity. To assess the degradability of the copolymers studied here, the copolymer P6 was incubated in aqueous buffer solution in the pH range of 7.0−12.0 at 37 °C for 0.5−360 h. P6 was chosen as a model polymer because of high water solubility, which would allow a homogeneous reaction of the polymer in aqueous solutions. In the MALDI-TOF-MS spectra, the original polymer before the degradation showed a broad peak centered on ∼5800 molecular weight represented as an Mp (the most probable peak molecular weight determined from the highest peak intensity in the MALDI spectrum; Figure 4). The spectra showed that the Mp of the products after incubation at pH 7.0 decreased to 3500 at 18 h, indicating the degradation of the 1560

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Figure 6. ESI-MS spectra of (A) poly(acrylic acid-co-1) (P15; Mn = 1300, n = 5.5, m = 5.4), (B) the degraded products at pH 7.0, (C) 8.0, (D) 9.0, (E) 10.0, (F) 11.0, and (G) 12.0 in buffer solution for 18 h at 37 °C: [poly(acrylic acid-co-1) (P15)]0 = 500 mg/mL, [2-ethanolamine hydrochloride]0 = 2.2 mM, [HEPES, Tris, NaHCO3, or K2CO3]0 = 10 mM, [NaCl]0 = 150 mM.

ethanolamine at the terminal (opened circles), while the minor series is likely from residual nondegraded polymer chains (filled circles). This result indicates that the copolymer degraded mainly by nucleophilic addition of amine groups of ethanolamine to polymer ester groups (amidation), followed by the cleavage of polymer chains rather than ester hydrolysis. In addition, no significant amount of degradation products by hydrolysis was found after incubation of P15 with 2(dimethylamino)ethanol at pH 7 (Figure S6), suggesting that ester hydrolysis is not a primary cause of polymer degradation. These results suggest that the copolymer P6 with primary amine side chains could possibly degrade by intramolecular amidation of polymer ester linkage by the amine groups in the side chains. We have previously reported that the apparent pKa of primary ammonium side chains of polymethacrylate derivatives is 8.3, as estimated by extrapolation of the titration curve,37 which is smaller than that of free ethanolamine in solution (pKa = 9.5 in literature38) because of electrostatic repulsion between neighboring ammonium groups in the polymer chains. Although the pKa of the copolymer was not determined because of polymer degradation, we speculate that the pKa would be similar to these values, and a fraction of the amine groups of the copolymer are nonprotonated at pH 7, which is likely responsible for the degradation process (Scheme 2). Because the pKa of the copolymer could be smaller than that of ethanolamine in solution, the fraction of nonprotonated amine groups of the copolymer would be greater than that of ethanolamine at pH 7, which may facilitate the copolymer

degraded into nontoxic oligomers, and the rate of polymer inactivation by degradation can be controlled by solution pH. Degradation Mechanism. The pH dependence of the degradation of copolymer P6 and poly(acrylic acid-co-1) P14 (Figure 4) suggested that the primary amine groups may play an important role in the degradation mechanism. To probe the effect of amine groups on the degradation process, we examined the degradation of the precursor copolymer with acid side chains, poly(acrylic acid-co-1), in the presence of a free small molecule with a primary amine group, 2-ethanolamine. The reaction of the copolymer with 2-ethanolamine hydrochloride at different pHs after 18 h was monitored by electrospray ionization mass spectrometry (ESI-MS) to identify the degradation products (Figure 6). Poly(acrylic acid-co-1) P15 with a low molecular weight (Mn = 1300) was used to obtain distinctive peaks in MS spectra to identify the products, because the higher MW polymer P14 (Mn = 4100) showed broad peaks (Figure S5). As a control, a series of peaks were observed in the spectrum of P15 in the absence of ethanolamine, which was separated by the formula weights of each monomer unit (72.0 for acrylic acid and 162.0 for 1). However, the degradation products of the copolymer P6 with amine groups showed broad peaks in the spectrum (Figure 4), which is likely due to the cationic groups in the side chains. The degradation products of the polymer in the presence of ethanolamine at pH 7.0 consisted of two main series of peaks. Based on the molecular weight analysis, the major series of the peaks was assignable to the degraded products possessing 21561

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We further tested the polymer for degradation at higher pHs of 9−12 (Figure 6C−G). Although the signals of the control poly(acrylic acid-co-1) remain at pH 8 after 18 h incubation, they disappeared for the samples at pH > 9.0, and more signals of lower molecular weight compounds appeared as the pH is increased. We speculate that increasing the pH enhanced the degradation, which could be due to accelerated ester amidation as the number of deprotonated basic amine groups increases at high pH. Poly(acrylic acid-co-1) incubated with 2(dimethylamino)ethanol showed little degradation by hydrolysis at pH 11 (Figure S6) and a relatively smaller change in polymer degradation at high pH compared to the copolymer P6 (Figure 4). These results may indicate that intramolecular amidation followed by chain scission is a primary mechanism for degradation, even at high pH.

Scheme 2. Degradation Mechanism of Polymer via Intramolecular Amidation

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CONCLUSIONS We have synthesized cationic random copolymers bearing pendent amine groups and degradable main-chain ester linkages using simultaneous chain- and step-growth radical polymerization. The copolymers exhibited antimicrobial activity against E. coli by varying the molecular weight, monomer structure, and monomer composition. The model polymer with primary amine groups in the side chains (P6) degraded to low molecular weight oligomers in aqueous solution by the intramolecular nucleophilic addition of primary amine groups in the side chains to the main chain ester linkages, followed by amidation and scission of the main chain. The antibacterial and hemolytic activities decreased as the copolymer degraded to oligomers. This self-degradation property of copolymers and their subsequent deactivation would be useful for antimicrobial agents to avoid undesired toxicity. Altering monomer compositions and amine functionality will enable tuning of the polymer degradation rate, leading to precise control of antibacterial lifetime. The polymers tested in this study showed relatively moderate antimicrobial activity against E. coli and low selectivity to bacteria over RBCs as compared to several classes of other polycations reported in literature. The polymer structures in terms of molecular weight, distribution, and ratio of the cationic groups to the hydrophobic ester unit require further optimization for potent antimicrobial efficacy with low hemolysis to be antimicrobial candidates with high efficacy. In addition, more investigation would be necessary to elucidate the effect of physiological conditions such as ionic strength and serum proteins on the antimicrobial activity of these polymers and their degradation process for use as antimicrobials, as these factors are known to inhibit the activity of antimicrobial peptides and polymers.7,42 Although the structural parameters and assay conditions of copolymers explored in this study were limited and only one model polymer was demonstrated for degradation, the presented results provide a proof of concept for the design and synthesis of self-degradable antimicrobial polymers. Through this research, we show the first application of the novel simultaneous chain- and step-growth polymerization technique to prepare biologically active polymers. The advantage of this approach is that biodegradable units can be directly copolymerized with a wide variety of functionalized vinyl monomers. In this report, we demonstrated the antimicrobial activity of cationic biodegradable polymers. In the future, this new class of biodegradable materials may also be useful as drug/gene delivery carriers as well as tissue engineering scaffolds.

degradation by amidation as compared to free amines in solution. As the peaks of the MALDI-MS spectra of P6 at pH 7 shifted to lower molecular weights with time, the spectra become narrower (Figure 4). This indicates that the degradation proceeded without significant intermolecular amidation between the polymers, which would otherwise lead to cross-linking of the polymer chains, resulting in the production of higher molecular weight polymers. The “selfdegradation” mechanism by intramolecular amidation likely facilitated disintegration of polymer structures at neutral pH in a relatively short period of time compared to hydrolysis. A similar self-degradation mechanism has been previously reported for polyester platforms containing primary amine groups in the side chains, which were synthesized based on polycondensation of hydroxyacids.39−41 Compared to these approaches, the simultaneous chain- and step-growth radical copolymerization enables the preparation of copolymers with ester groups in polymer backbones by a one-pot synthesis as well as the incorporation of vinyl monomer functionalities. Recently, antimicrobial random copolymers with a biodegradable polycarbonate backbone have also been synthesized.25 Compared to these biodegradable polymers, the copolymers reported here likely degrade in a relatively short period of time, even in physiological conditions. The self-degradation strategy using primary amine side chains would be useful for quick disintegration of antimicrobial effects to circumvent potential toxic side effects by polymers. 1562

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ASSOCIATED CONTENT

S Supporting Information *

1

H NMR spectra, MALDI-TOF-MS spectra, characterization of polymer degradation, hemolysis curves, and antimicrobial activity of degradation products. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] (K.K.); kamigait@apchem. nagoya-u.ac.jp (M.K.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported in part by JSPS Research Fellowships for Young Scientists (No. 22-8341 to M.M.), a Grant-in-Aid for Young Scientists (S) (No. 19675003 to M.K.), Institutional Program for Young Researcher Overseas Visits by the Japan Society for the Promotion of Science, and the Global COE Program “Elucidation and Design of Materials and Molecular Functions.” This work was also supported by NSF CAREER Award (DMR-0845592 to K.K.) and the Department of Biologic and Materials Sciences at the University Michigan School of Dentistry. We thank Dr. Robertson Davenport of the University of Michigan Hospital for providing a unit of human red blood cells for this work and Dr. Eric Krukonis, University Michigan School of Dentistry, for use of the microplate reader.

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