Mechanism of Polymer-Induced Hemolysis: Nanosized Pore

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Mechanism of Polymer-Induced Hemolysis: Nanosized Pore Formation and Osmotic Lysis Iva Sovadinova,† Edmund F. Palermo,‡ Rui Huang,§ Laura M. Thoma,§ and Kenichi Kuroda*,†,‡,§ Department of Biologic and Materials Sciences, School of Dentistry, Macromolecular Science and Engineering Center, and Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109, United States Received October 2, 2010; Revised Manuscript Received November 29, 2010

Hemolysis induced by antimicrobial polymers was examined to gain an understanding of the mechanism of polymer toxicity to human cells. A series of cationic amphiphilic methacrylate random copolymers containing primary ammonium groups as the cationic functionality and either butyl or methyl groups as hydrophobic side chains have been prepared by radical copolymerization. Polymers with 0-47 mol % methyl groups in the side chains, relative to the total number of monomeric units, showed antimicrobial activity but no hemolysis. The polymers with 65 mol % methyl groups or 27 mol % butyl groups displayed both antimicrobial and hemolytic activity. These polymers induced leakage of the fluorescent dye calcein trapped in human red blood cells (RBCs), exhibiting the same dose-response curves as for hemoglobin leakage. The percentage of disappeared RBCs after hemolysis increased in direct proportion to the hemolysis percentage, indicating complete release of hemoglobin from fractions of RBCs (all-or-none leakage) rather than partial release from all cells (graded leakage). An osmoprotection assay using poly(ethylene glycol)s (PEGs) as osmolytes indicated that the PEGs with MW > 600 provided protection against hemolysis while low molecular weight PEGs and sucrose had no significant effect on the hemolytic activity of polymers. Accordingly, we propose the mechanism of polymer-induced hemolysis is that the polymers produce nanosized pores in the cell membranes of RBCs, causing an influx of small solutes into the cells and leading to colloid-osmotic lysis.

Introduction We are facing a significant crisis in public health due to the emergence of antibiotic resistance in bacteria, coupled with fewer antimicrobials in the development pipeline.1–5 Creating new antibiotic agents, which can control the occurrence and spread of antibiotic-resistant bacteria, has been a major scientific challenge because it requires novel molecular designs and functions to overcome the existing resistance mechanisms. Hence, a new approach has gained considerable attention in the last several decades, which focuses on implementation of antimicrobial peptides and their synthetic analogues.6–8 In contrast to conventional antibiotics, these peptides act on the bacterial cell membranes; they display rapid bactericidal activity against a broad spectrum of bacteria including drug-resistant strains, selective toxicity to bacteria over mammalian cells, and a low level of resistance development in bacteria. While utilizing the peptides seems to be promising for the preparation of nontoxic antimicrobials, they suffer low efficacy, instability in vivo, and high manufacturing costs, which hinder their pharmaceutical and biomedical use as alternatives for antibiotics.9 On the other hand, utilizing amphiphilic polymers with cationic quaternary ammonium groups has also been a long-standing approach to prepare inexpensive disinfectants for the last several decades, which can be used in solution or on surfaces.10,11 These polymers are, however, often nonselective to cell types (or their toxicity to human cells has not been quantified), which limits * To whom correspondence should be addressed. E-mail: kkuroda@ umich.edu. † Department of Biologic and Materials Sciences. ‡ Macromolecular Science and Engineering Center. § Department of Chemistry.

their application as pharmaceuticals. Recently, this polymer approach has been further extended to include a new design strategy that mimics the structural features and biological functions of antimicrobial peptides.12–15 The peptidomimetic polymers have displayed activity profiles comparable or superior to natural peptides; however, only a few examples of polymers with potent activity but no adverse toxicity have been identified to date. The toxicity of polymers is still a major obstacle in the practical use of antimicrobial polymers. In this context, it is vital to investigate the design parameters determining polymer toxicity and underlying mechanism to assist in the rationale design of biocompatible antimicrobial polymers. To that end, we wished to study the mechanism of toxicity of polymers to human cells to gain insight into a new design strategy for nontoxic antimicrobial polymers. This report focuses on polymer-induced lysis of human red blood cells (RBCs) or hemolysis. Hemolytic activity has been widely used as an initial metric of toxicity of membrane-active antimicrobial polymers and peptides. While the mechanisms of peptide-induced hemolysis have been well-studied, the hemolysis mechanism exerted by synthetic polymers remains unexplored to date. In this study, we investigated cationic amphiphilic random methacrylate copolymers that exhibit antimicrobial and hemolytic activities but can also be controlled by fine-tuning the amphiphilic balance between cationic functionality and hydrophobicity.16,17 It has also been demonstrated that the global hydrophobicity of these polymers is the dominant factor for the hemolytic activity.18 In this report, the hemolysis induced by these amphiphilic polymethacrylates was characterized by cellular and biophysical assays. The membrane permeabilization was quantified by polymer-induced leakage of a fluorescent dye trapped in RBCs.

10.1021/bm1011739  2011 American Chemical Society Published on Web 12/17/2010

Mechanism of Polymer-Induced Hemolysis

The cell lysis was also evaluated by the relationship between the number of disappeared cells and the amount of released hemoglobin. RBCs were assayed with the polymers in the presence of poly(ethylene glycol)s (PEGs) with different molecular weights (MWs) to examine the effect of macromolecular osmolytes on the hemolysis. Based on the experimental results, we propose that the polymer-induced hemolysis is caused by formation of nanosized lesions or pores in the cell membranes of RBCs, followed by colloid-osmotic lysis of RBCs. In addition, we also propose that the mechanism of hemolysis is the complete release of hemoglobin from fractions of RBCs (all-or-none leakage) rather than the leakage of a portion of hemoglobin from individual cells (graded leakage).

Experimental Section Materials. Methyl and butyl methacrylate, methyl 3-mercaptoproprionate (MMP), 2,2′-azobisisobutyronitrile (AIBN), Triton X-100, and melittin (purity >85%) were purchased from Sigma-Aldrich. Tris(hydroxymethyl)aminomethane (Tris) and 4-(2-hydroxyethyl)piperazine1-ethanesulfonic acid (HEPES) were obtained from EMD, and calcein acetoxymethyl ester (Calcein-AM) was purchased from BD Biosciences. Human red blood cells (Red Blood Cells Leukocytes Reduced Adenine Saline Added) were obtained from the American Red Cross Blood Services Southeastern Michigan Region. PEGs and sucrose were purchased from Fisher Scientific. Egg PC was purchased from Avanti Polar Lipids (Alabaster, AL). Polymer Synthesis. Free radical polymerization of N-(tert-butoxycarbonyl)aminoethyl methacrylate (Boc-AEMA) with an alkyl methacrylate was carried out as previously described,17 with some modifications. Briefly, Boc-AEMA and alkyl methacrylates (various ratios, 0.5 mmol total), MMP (16.7 µL, 0.15 mmol) and AIBN (0.82 mg, 0.005 mmol), dissolved in acetonitrile (0.5 mL) in a sealed borosilicate glass test tube were deoxygenated with N2 bubbling for 2 min and then stirred at 60-70 °C in a mineral oil bath for 20 h. Solvent was evaporated and the crude polymer was purified by size exclusion chromatography (Sephadex LH-20 gel, methanol) monitored by thin layer chromatography (ethyl acetate/hexane ) 1:1). Fractions containing unreacted monomers and MMP were discarded. The remaining fractions were concentrated, dissolved in 1.25 M HCl in methanol (5-10 mL), and stirred at room temperature for 2 h to cleave the protecting groups. Excess acid was removed by N2 flushing, and the polymers were twice precipitated from methanol into diethylether. The precipitates were collected by centrifugation and lyophilized to afford the random copolymers bearing primary amine groups in the form of ammonium chloride salts. The polymers were characterized by 1H NMR to determine the mole percentage of alkyl groups (MPalkyl) and degree of polymerization (DP), as previously described in detail.17 The amount of unreacted monomer was found to range from 1-3%. See Supporting Information for the NMR spectra of the polymers. Antimicrobial Activity. The lowest polymer concentration required to completely inhibit growth of bacteria, defined as the minimum inhibitory concentration (MIC), was determined by a turbidity-based microdilution assay in Muller Hinton (MH) broth according to the procedure approved by The National Committee for Clinical Laboratory Standards (NCCLS), with the modifications proposed by Wiegand et al.19 and Giacometti et al.20 Each polymer was dissolved in dimethyl sulfoxide (DMSO), and 2-fold serial dilutions were prepared in 0.01% acetic acid. Midlog phase Escherichia coli ATCC 25922 were diluted to the final concentration of approximately 5 × 105 CFU/mL (OD600 ) 0.001) in MH broth based on colony counting after spreading on a MH agar plate. This stock (90 µL) was mixed with a polymer solution (10 µL) in a 96-well polypropylene microplate (Corning #3359). After incubating for 18 h at 37 °C, 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 increase was observed relative to the negative control, MH broth. As

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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 (1%). All experiments were performed three times in triplicate. The MIC values were determined below the solubility limit of the polymers in MH broth in every case. Hemolysis, Hemagglutination, and RBC Counting. Toxicity to human red blood cells (RBCs) was assessed by a hemoglobin release assay. RBCs (1 mL) were diluted into Tris-buffered saline (TBS) (9 mL; 10 mM Tris buffer, 150 mM NaCl, pH ) 7.3) and rinsed 3 times by centrifugation (5 min at 2,000 rpm). The rinsed cell suspension (10% v/v RBCs) was further diluted to give a 3% final concentration of RBC (v/v) on the plate, which corresponds to approximately 2 × 108 of cells per mL. RBC numbers for each experiment were determined after several dilutions by counting 100-200 cells using a hemocytometer. Each polymer was dissolved in DMSO, and 2-fold serial dilutions were prepared in 0.01% acetic acid. After addition of the test compounds at a 1/10 volume into a 96-well round-bottom polypropylene microplate (Corning #3359), the assay plate was incubated for 1 h at 37 °C with orbital shaking (200 rpm). The lowest concentration for hemagglutination was determined by observation and under an optical microscope. After a 1 h incubation, the plate was centrifuged at 2000 rpm for 5 min and supernatant (8 µL) from each well was diluted within TBS buffer (92 µL). The number of remaining cells in the pellet was counted after a serial dilution with TBS using a hematocytometer. Hemolysis was monitored by measuring the absorbance of the released hemoglobin at 415 nm using a microplate reader (Perkin-Elmer Lambda Reader). Hemolysis was determined relative to the positive lysis control Triton X-100 (0.2% v/v in water) for the polymers or melittin (36 µg/mL) and negative control buffer. 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 detectable hemolysis even at the highest DMSO concentration (1%). The percentage of hemolysis was calculated as the absorbance reading divided by the average of readings from positive control wells. HC50 was defined as the polymer concentration causing 50% hemolysis, which was estimated with 95% confidence intervals by a curve fitting with the following equation: H ) 100/(1 + (HC50/[polymer])n), where H is the percentage of hemolysis measured and [polymer] is the total concentration of polymer. The fitting parameters were n and HC50. The experiments were repeated at least twice. Osmoprotection Assay. To estimate the functional diameter of the pores formed by the polymers, a hemolysis assay was performed in the presence of sucrose and polyethylene glycols (PEGs) with different molecular weights (400-1500). Sucrose and PEGs were prepared in buffer and added to give a final concentration of 30 mM in the assay, according to the standard procedures for osmoprotection of RBCs against pore-forming lytic peptides and hemolytic polymers in literature.21–28 Higher concentrations of PEG were not used because of possible difficulties concerning solubility and the viscosity of the assay media, which has been reported in the similar assay procedure using bacteria.28 HEPES buffer (10 mM HEPES, 150 mM NaCl, pH 7.4) was used for the assays of PB27 and melittin. TBS buffer was used for PM63. Calcein-Leakage Assay. RBCs were washed as described in hemolysis assay and a stock solution of 6.6% v/v RBCs in TBS was prepared. This cell solution was incubated with 2 µM calcein-AM for 1 h at 37 °C to allow nonfluorescent calcein-AM to be hydrolyzed in cytosol by intracellular esterases to fluorescent calcein. Free calceinAM and calcein were removed by washing with TBS. After incubation, RBCs were counted using a hematocytometer and diluted to give approximately 2 × 107 of cells per well on a 96-well round-bottom polypropylene microplate (Corning #3359). The polymer solution was added to cells, followed by incubation for 1 h at 37 °C. The plate was centrifuged at 2000 rpm for 5 min and supernatant was used for a hemoglobin determination. RBCs were washed with TBS and 1% Triton X-100 was added to lyse the cells. Fluorescent intensity of each well

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Scheme 1. Synthesis of Amphiphilic Methacrylate Random Copolymersa

control well. Sucrose and PEGs were added to the HEPES buffer solution at a concentration of 30 mM. All experiments were performed in triplicate.

Results

a The monomers were polymerized in the presence of 3-methyl mercaptopropionate (MMP) as a chain transfer agent to obtain low molecular weight polymers. The boc-protected precursor polymers were treated by HCl in methanol to yield the polymers with primary ammonium groups. The R groups are either methyl (PMx) or butyl (PBx) and the mole percentage of R groups in the copolymer side chains (MP) is given as the subscript x. For example, PM47 is the copolymer containing an average of 47% MMA units. AIBN: 2, 2′-azobisisobutyronitrile (AIBN).

was measured on a ThermoScientific Varioskan Flash microplate reader at an excitation wavelength of 494 nm and at an emission wavelength of 517 nm. All experiments were performed three times in triplicate and the values reported are the average of the three trials. Liposome Dye Leakage. A solution of lipid (100 µL, 10 mM) in chloroform was slowly evaporated under a gentle N2 stream and subsequently dried under vacuum for 12 h. An aqueous buffer (10 mM HEPES, 50 mM sulforhodamine B (SRB), pH 7.4) was adjusted to an osmolality of 280 ( 5 mmol/kg by addition of saturated NaCl and measured using a vapor-pressure osmometer so that liposomes could be prepared without initial osmotic pressure across the membranes. The dry lipid film was resuspended in this buffer, vigorously vortexed for 5 min, and subjected to 10 freeze/thaw cycles between dry ice in acetone and a 50 °C water bath. Then it was passed 21 times through a mini-extruder equipped with two stacked polycarbonate membranes of 400 nm average pore size. Unincorporated dye was removed by size exclusion chromatography over Sepharose Cl-4B gel from Amersham Biosciences (Uppsala, Sweden) using a buffer containing no dye (10 mM HEPES, 150 mM NaCl, pH 7.4, osmolality ) 280 ( 5 mmol/kg). The concentration of lipid in the obtained suspension was determined by a colorimetric phosphorus assay.29 This solution was diluted in the same buffer to a lipid concentration of 12.5 µM. On a 96-well black microplate, the liposome suspension (160 µL) was mixed with 0.2 M stock solutions of PEG or sucrose (20 µL), and subsequently, the polymer stock solutions (20 µL) were added, to give a final lipid concentration of 10 µM in each well. The assay buffer (20 µL) and Triton X (0.1% v/v, 20 µL) were employed as the negative and positive controls instead of the polymer solutions. After a 1 h incubation at 37 °C with orbital shaking (100 rpm), the fluorescence intensity in each well was recorded using a microplate reader (Thermo Scientific Varioskan Flash) with excitation and emission wavelengths of 565 and 586 nm, respectively. The fraction of leaked SRB in each well was calculated according to the expression: L ) (F - F0)/(FTX - F0), where F is the fluorescence intensity recorded in the well, F0 is the intensity in the negative control well, and FTX is the intensity in the positive

Polymers. A series of cationic, amphiphilic methacrylate random copolymers containing primary ammonium groups as cationic functionality and either butyl (PB) or methyl groups (PM) as hydrophobic side chains have been prepared according to the procedure reported previously (Scheme 1 and Table 1).16–18 We chose to investigate the representative copolymers herein because they present a wide range of biological activity profiles in terms of toxicity to bacteria and human RBCs. The polymers have the average degree of polymerization (DP) of 14-20, which corresponds to MWs of 2300-2800, according to NMR analysis (Supporting Information). We chose this molecular weight range because low MW polymers have shown activity profiles comparable to antimicrobial peptides.17 The PM polymers have methyl side chains with 0-63% relative to the total number of monomeric units in a polymer. The mole percent of methyl methacrylate (MMA) or butyl methacrylate units in the copolymers (MP) are denoted in subscript x in PMx or PBx; for example, PM47 contains 47% MMA groups. The polymer with 27% butyl groups, PB27, was prepared also to examine the effect of longer alkyl chain length on hemolysis. Antimicrobial and Hemolytic Activities. The polymers were first tested against E. coli to evaluate their antimicrobial activity. Cationic methacrylate copolymers with alkyl side chains, including methyl and butyl groups, as hydrophobic moieties have previously shown antimicrobial activity against E. coli with minimal inhibitory concentrations (MICs) at low µg/mL or µM range.17 The MIC values of the polymers prepared here decreased with increasing the percentage of methyl groups, indicating that the hydrophobicity of polymers enhances their activity against E. coli, which is in agreement with the previous results.17 The polymers PM63 and PB27 displayed the lowest MIC values of 16 µg/mL, which is an order of magnitude lower than that of the natural antimicrobial peptide maginin-2. To assess the toxicity to human RBCs, the polymers were tested in a hemolysis assay. The series of cationic amphiphilic methacrylate copolymers previously showed moderate to high hemolytic activity, depending on their molecular weight and mole fraction of alkyl side chains.18 The P0 (homopolymer) and PM polymers displayed little to no hemolysis up to the maximum concentration tested here (2000 µg/mL) except for PM63 (Table 1 and Figure 1). However, some of these nonhemolytic polymers did induce aggregation of RBCs or hemagglutination (Figure 2). The minimal polymer concentration for the hemagglutination (Cagg) for P0 was 125 µg/mL (Table 1),

Table 1. Polymer Characterization and Biological Activity polymer or peptide P0 PM10 PM28 PM47 PM63 PB27 magainin-2 melittin

R

DPa

MPb

MWc

MICd (µg/mL)

HC50e (µg/mL)

Caggf (µg/mL)

methyl methyl methyl methyl butyl

14 14 15 20 17 16

0 10 28 47 63 27

2400 2300 2300 2800 2200 2700 2500 2800

500 ( 0 500 ( 0 500 ( 0 63 ( 0 16 ( 0 16 ( 0 125 ( 0 13 ( 0

>2000 (12% ( 1) >2000 (35% ( 6) >2000 (3% ( 1) >2000 (7% ( 2) 114 ( 7 13 ( 2 >250 (9% ( 1) 2 ( 0.2

125 ( 0 250 ( 0 250 ( 0 2000 ( 0 >2000g >2000g >250g >25g

a Degree of polymerization determined by 1H NMR. b Mole percentage of alkyl methacrylate relative to the total number of monomeric units, determined by 1H NMR. c Number averaged molecular weight calculated based on DP and MP. d Minimal inhibitory concentration of polymers for E. coli (n ) 3). e Average polymer concentration for 50% hemolysis ( standard deviation (SD; n ) 3). The hemolysis % at 2000 µg/mL is given in parentheses. f Minimal polymer concentration to induce hemagglutination (n ) 3). g Hemagglutination was not observed up to the highest polymer or peptide concentrations tested in the assays.


Figure 1. Dose-response curves in hemolysis induced by the polymers and peptide melittin. Each data point represents the average of three independent experiments ( SD.

and the Cagg increased with increasing the methyl side chains for the PM polymers. This indicates that increasing the hydrophobicity of the polymers or reducing the number of cationic groups decreases agglutination. The hemagglutination induced by the polymers is also concentration-dependent: the higher P0 concentrations induced formation of a larger cluster of cells (Figure 2). It has been reported that synthetic cationic polymers induce aggregations of RBCs;30 the cationic primary ammonium groups of the PM polymers are likely to play an important role in the agglutination mechanism. Increasing the fraction of methyl or butyl side chains causes an increase in the hydrophobicity of the polymers but, at the same time, decreases the number of cationic groups present. It has been demonstrated that the cationic charge density increases the antimicrobial activity of cationic antimicrobial peptides due to their attraction for the negatively charged bacterial cell surfaces.31–33 Net cationic charge is believed to enhance the selective binding of polymers to bacterial cell over human cells, because red blood cells display fewer anionic lipids on their membrane surface.34,35 It has been previously demonstrated that the hemolytic activity of polymers are attributed to the summation of the side chain hydrophobicities.18 It would appear that tuning the ratio of cationic to hydrophobic side chains is an effective method to optimize the polymers for maximizing antimicrobial activity as well as hemocompatibility. PM47 displayed an MIC value of 63 µg/mL, whereas the HC50 values, which are defined as the polymer concentrations for 50% hemolysis, and Cagg values were equal to or greater than 2000 µg/mL, which indicates that the polymer is selectively active against E. coli rather than RBCs. This suggests that hemocompatible antimicrobial polymers can be obtained by fine-tuning their amphiphilic balance and number of cationic charges. The polymers PM63 and PB27 displayed concentrationdependent hemolytic activity (Figure 1). The HC50 values are


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114 and 13 µg/mL for PM63 and PB27, respectively. These hemolytic polymers showed no hemagglutination up to 2000 µg/mL. The hemolysis induced by these polymers is likely a result of the high total hydrophobicity of the polymer chains.18 It has been observed that the antimicrobial and hemolytic activities of the copolymers reach a plateau when the MP is increased excessively.18 This effect has been ascribed to the tendency of the excessively hydrophobic copolymers to form aggregates in aqueous assay media, which reduces the effective concentration of polymers available to access bacterial cell membranes, limiting the maximum activity (lowest MIC) that can be achieved. Because of their potent hemolytic activity, we focused on PM63 and PB27 for this mechanistic study. The bee venom toxin melittin was also tested along with the polymers as a standard for comparison. Melittin exerts lytic effects on human and bacterial cells, which have been extensively studied.36 The assay result showed that the hemolysis induced by melittin is also concentration-dependent and that melittin is highly hemolytic compared to the tested polymers; melittin’s HC50 of 2 µg/mL is an order of magnitude lower than that of PB27 and 2 orders of magnitude lower than that of PM63. Calcein Leakage from RBCs. To determine if the membrane permeabilization of RBCs by the polymers is size-selective, the amounts of a small fluorescent dye calcein (∼1 nm)37 and hemoglobin (∼6 nm in diameter)38 remaining in RBCs after exposure to the polymers or melittin were quantified. In the experiment, nonfluorescent calcein acetoxymethyl ester (calceinAM) was incubated for loading fluorescent calcein into RBCs; calcein-AM can diffuse into the cytoplasm where it is hydrolyzed by intracellular esterases to give hydrophilic fluorescent calcein, which is membrane-impermeable and retained in the cytoplasm. Utilizing this property, calcein AM has been widely used as a fluorescent marker for spectroscopic determination of cell viability39 and dye leakage from cells.37,39,40 After removing extracellular calcein-AM by washing the RBCs, the calcein-loaded cells were incubated with the polymers or melittin. The amount of calcein remaining in the cells was determined to avoid excessive quenching of the fluorescence of released calcein by the presence of polymers. The calcein loading did not significantly affect the hemolysis as compared to the control experiment using RBCs without calcein (Supporting Information). The dose-response curves of calcein and hemoglobin remaining in RBCs are almost identical for copolymers PB27 and PM63 (Figure 3). In the same assays, melittin also showed the same dose-response curves for calcein and hemoglobin contents. This indicates the same percentages of these molecules are released from RBCs at the intermediate polymer concentrations. However, the initial amounts of calcein and hemoglobin are likely different, and the rates of calcein leakage from RBCs are expected to be larger than that of hemoglobin because of the difference in the molecular size. These could result in the different leakage percentages of dye and hemoglobin at the end

Figure 2. Hemagglutination induced by the homopolymer P0 in different polymer concentrations of 0 (A), 250 (B), and 1000 µg/mL (C) observed by optical microscopy. The scale bar represents 20 µm.

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Figure 3. Hemoglobin (closed circle) and calcein (open square) retained in human red blood cells after incubation with PB27 (A), PM63 (B), or melittin (C). The percentages of retained contents relative to the control without the polymers or melittin are presented. Each data point represents the average of three independent experiments ( SD.

point of the 1 h incubation time. We might observe differences in the dose-response curves at different incubation times, depending on the diffusion rates or lysis kinetics. Kinetic studies on isolated cells would be necessary for further clarification on the time-dependence of cell lysis and release kinetics of these molecules. Nevertheless, the same leakage curves could simply be explained by a mechanism of hemolysis involving formation of pores larger than hemoglobin or a size-independent step, which makes the diffusion rates of hemoglobin and calcein negligible at the 1 h incubation time. Considering the results presented in the following sections, the latter possibility seems more likely. Hemolysis and RBC Counting. To determine the relationship between the percent leakage of hemoglobin and the number of affected cells, the numbers of RBCs in samples treated with PB27 were counted. The supernatant of the treated samples was separated by centrifugation, and the remaining RBCs were dispersed in buffer for counting. The percentage of disappeared RBCs was calculated from the difference between the initial (before hemolysis) and final (after hemolysis) counts of RBCs and was plotted against hemolysis percentage (Figure 4A). The data points scatter about the diagonal line, indicating that the percentages of disappeared RBCs and released hemoglobin are consistent. This result suggests that the hemolysis induced by PB27 is due to the complete release of hemoglobin from fractions of RBCs (all-or-none) rather than graded release of hemoglobin from individual cells (Figure 4B). In the case of graded release, the number of remaining RBCs after hemolysis would be the same as the initial number of RBCs, regardless of hemolysis percentage. The all-or-none hemolysis mechanism is also supported by the result of the same dose-response curves for calcein and hemoglobin contents (Figure 2). It should be noted that the counting method determined only the number of visible cells under a conventional bright field microscope, which still retained some extent of hemoglobin contents; empty cells or “ghost” cells were excluded from the counting because they were not readily visible. Therefore, it is not possible to conclude at this point whether disappeared cells after hemolysis were intact but empty or completely solubilized. Osmotic Protection. It has been demonstrated that peptides,21,23,24 bacterial toxins,25–27 synthetic polymers,22,41 and a reovirus42 cause hemolysis by a mechanism of colloid-osmotic lysis of RBCs. These peptides and polymers are assumed to produce discrete lesions or pores in cell membranes. When the pores are too small for permeation of large macromolecules (such as hemoglobin) from the cytosol, the osmolarity of the cytosol becomes higher than that of outer buffer solution containing only small solutes and salt ions. This osmotic unbalance causes a hypotonic condition, which leads to an influx

Figure 4. All-or-none hemolysis induced by the polymer. (A) Relationship between cells disappeared after incubation with PB27 and percentage of hemoglobin released (hemolysis). Each data point represents the average of two independent experiments ( SD. Oneside error bars were presented for clarification. The diagonal line is presented for a guide. (B) Schematic presentation of graded and allor-none release of hemoglobin.

of small solutes into cells through the pores. This causes cell swelling and rupture of cell membranes, possibly resulting in the release of cytoplasmic contents and complete lysis of RBCs. We also hypothesized that the hemolysis induced by the polymers is caused by colloid-osmotic lysis due to the formation of pores that can allow only permeation of small solutes. To test this hypothesis, an osmoprotection assay was conducted, which has been used to determine colloid-osmotic lysis of cells as well as to estimate the size of pores formed by pore-forming peptides.21,23–28 According to the procedure reported in literature,21,23–28 sucrose (MW ) 342, 0.98 nm in diameter43) or poly(ethylene glycol) (PEGs) with different molecular weights (MW ) 400-1500, 1.36-2.4 nm in diameter43,44) was added into the assay solution with the concentration of 30 mM, which has been used as the standard assay condition (Figure 5). If the hydrodynamic size of these solutes were larger than the pores



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Figure 5. Osmoprotection against hemolysis induced by the copolymers and melittin. (A) PB27. A representative experiment of two individual experiments is shown. The data point represents the average of triplicate samples ( SD (B) PM63. Each data point represents the average of two independent experiments ( SD (C) PEG MW dependence of osmoprotection against hemolysis induced by PB27 at 31 µg/mL (closed circle) or melittin at 4.5 µg/mL (open square). The data point for melittin represents the average of triplicate samples ( SD. The final concentration of PEG in each case was 30 mM.

in the membrane, these solutes would reduce the osmolarity difference between the cytosol and the buffer solution, protecting the RBCs against hemolysis. For PB27, the lowest MW PEG 400 showed weak protection of RBCs against hemolysis (Figure 5A). The PEG 600 and sucrose displayed an intermediate effect on protection of RBCs against hemolysis. The hemolysis was increasingly inhibited as the MW of PEG was increased, and the PEGs with MWs >1000 effectively reduced the hemolysis; the hemolysis percentage at the polymer concentration of 31 µg/mL is smaller than 20%, whereas the control showed almost 100% hemolysis (Figure 5C). The protection against hemolysis started to be effective in the range of PEG MWs between 600-1000, suggesting that the hydrodynamic sizes of these PEGs are almost larger than the size of the functional diameter of pores in the cell membrane. Accordingly, because the hydrodynamic radii of PEG 600 and 1000 were estimated to be 0.8 and 1.0 nm, respectively,43 PB27 appears to produce nanosized pores with diameters of 1.6-2.0 nm at the polymer concentration of 31 µg/mL. Similarly, PEG 1500 (2.4 nm in diameter43) provided osmotic protection against hemolysis induced by PM63, while sucrose (0.92 nm in diameter43) did not affect the hemolysis (Figure 5B). Accordingly, these results support our hypothesis that the polymers produce nanosized pores in the RBC membranes, leading to an influx of small solutes and osmotic lysis of RBCs. Because the estimated pore size is 3-4 times smaller than that of hemoglobin molecule (6 nm in diameter38), we speculate that hemoglobin molecules are not able to permeate through the pores formed initially. After pore formation, the influx of small solutes into cells would lead to swelling of the cells and finally membrane damage, allowing the hemoglobin to be released subsequently. The PEGs with MW >1000 also protected RBCs against hemolysis induced by melittin (Figure 4C and Supporting Information for hemolysis curves). This also suggests that melittin causes colloid-osmotic hemolysis by forming pores with a diameter of ∼2.0 nm. Previously the pore size induced by melittin in human RBCs was found to range from 1 to 3 nm, depending on melittin concentration (∼0.1-1.5 µM) and other assay conditions.23 The PEG 600 displayed an intermediate effect on the hemolysis induced by PB27 (Figure 5C), while melittin displayed

Figure 6. Dye leakage from egg PC LUVs induced by PB27 in the presence of sucrose and PEG 1500. Each data point represents the average of triplicate samples ( SD.

a sharp transition in the effect of MWs of PEGs on osmoprotection. This may reflect a relatively broad distribution of pore sizes formed by the polymer compared to melittin. In addition, the high MW PEGs failed to inhibit hemolysis induced by the polymers and melittin at high polymer concentrations (Supporting Information), suggesting the pore size and its distribution are most likely concentration-dependent, which has also been observed in the case of the peptides melittin and gramicidin in the literature.23 Dye Leakage from Liposomes. The polymer-induced leakage of small dye molecules from lipid vesicles was further examined to quantify the membrane permeabilization by the polymers.45 The polymer PB27 induced leakage of small fluorescent dye sulforhodamine B (SRB) entrapped in egg PC large unilamellar vesicles (LUVs; Figure 6). The leakage fraction curves in the presence of PEG 1500 or sucrose are identical to that of the control (without additives), in contrast to the results of osmoprotection assays using RBCs. This indicates that the PEG itself does not protect the liposomes against permeabilization. Because liposomes do not entrap large macromolecules in their intravesicular space, induction of osmolarity differences between the intravesicular and outer buffer solutions is unlikely. The same leakage curves for the control and PEG also suggest that the protection of RBCs against osmolysis by PEGs was not due to any polymer-PEG binding, which would reduce the concentrations of polymer chains available for interacting with RBCs. Additionally, it

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also supports that any possible PEG-lipid interaction does not inhibit the formation of pores. It should be noted that there are distinctive differences between the membranes of model vesicles and red blood cells: unlike vesicles, RBCs contain a cytoskeleton, possess membrane tension, and display biomacromolecules on the membrane surfaces.46 In addition, the RBC membranes are composed of lipids with a wider variety of alkyl tail structures and are characterized by high lipid asymmetry,47 which likely affect the pore formation and lysis mechanisms. We assume that the polymer-lipid interaction is operative in the pore formation for both the model and cellular membranes. Further investigation on the effect of these structural factors in cell membranes will provide insight into the molecular mechanism of polymer-induced lysis as well as the susceptibility of cells to lysis.

Discussion The mechanism of hemolysis induced by antimicrobial polymers has been poorly understood, although hemolysis is one of the most common assessments in literature for cytotoxicity of polymers. This study provided data to support the notion that the hemolysis mechanism exerted by amphiphilic polymethacrylates involves formation of discrete nanosized pores, leading to colloid-osmotic lysis (Figure 5). The demonstrated hemolysis reflects complete release of hemoglobin from fractions of RBCs (all-or-none) rather than graded release from individual cells (Figure 4). Accordingly, we propose that the hemolysis mechanism by the polymers is that the polymers produce nanosized pores in the cell membranes, leading to an influx of small solutes into cells, which cause irreversible rupture or global destabilization of cell membranes. Such rupture rapidly releases all hemoglobin molecules and other cell contents to the aqueous milieu. The RBC counting experiment indicated that the RBC samples after hemolysis are a mixture of lysed and intact cells at the intermediate concentrations (Figure 4). We wonder which step in the hemolysis mechanism determines this heterogeneous response. We considered two possibilities: (1) the polymers form pores in only fractions of cells, followed by osmotic lysis, or (2) pores are formed in presumably all cells, but only fractions of these cells are eventually lysed. The estimated pore sizes in RBCs are larger than the size of calcein but smaller than that of hemoglobin. If pore formation were not always followed by the osmotic lysis, the leakage of calcein from RBCs would be selective over hemoglobin. The experimental result showed that the dose-response curves for calcein and hemoglobin contents are the same at the 1 h incubation time (Figure 3). This might support the notion that the leakage is not size-selective, although further kinetic study on isolated cells would be necessary to conclude the size selectivity of pores as discussed above. Accordingly, we speculate that the polymers can form pores in only fractions of cells, but once pores are formed, affected cells are lysed and release all hemoglobin, resulting in the all-ornone hemolysis. To clarify this issue on the heterogeneity in the lysis mechanism in future studies, direct observation of the leakage kinetics of hemoglobin and calcein from individual cells would provide useful information to discuss the relationship between lysis kinetics and heterogeneity of the cell response to polymer attack. We further considered why the pore formation is heterogeneous in terms of cells. One reason could be due to differing ratios of polymers bound to individual cell membranes; only

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cells with bound polymers exceeding a certain threshold concentration are lysed. Another possibility is that there is cellto-cell variation in the susceptibility to pore formation or osmotic lysis even though the polymer binding is presumably homogeneous. However, the basis of this heterogeneity remains unclear at present. Several mechanisms have been proposed to explain the membrane disruption by antimicrobial and lytic peptides, which rely on the cooperative action of multiple peptides and the formation of discrete nanosized pores in bacterial or mammalian cell membranes.8,48–50 These models suggest that the peptides form a helical structure with segregated cationic and hydrophobic phases upon the binding to cell membranes, and either multiple peptide helices insert into lipid bilayers and assemble to form trans-membrane ion channel-like pores (barrel-stave model) lined with lipid hydrophilic head groups (toroidal model), or accumulate on the cell surface to disintegrate the membranes (carpet model). In this report, amphiphilic methacrylate copolymers PB27 and PM63 displayed the formation of discrete pores with diameters estimated to be in the range of 1.6-2.0 nm, which is comparable the estimated sizes of pores generated by hemolytic toxins26,51 and peptides.23 In contrast to these natural peptides, the polymers studied here have heterogeneity in polymer structure and conformation. Although the polymers are not likely to form rigid helical conformations, the flexible polymer chains are capable of producing nanosized pores in RBCs, allowing influx of small solutes. Interestingly, a recent computational study on the action of magainin derivatives suggested that an R-helical conformation is not a prerequisite for pore formation: even when the modeled peptides were not in a stable R-helix form, they were able to form distorted toroidal pores.52 Vial et al. recently also demonstrated that hydrophobically modified poly(acrylic acid)s produce defined pores in synthetic lipid vesicles, and the pore size ranged from 0.3 to 4 nm as polymer concentration was increased.53 Poly(2-ethylacrylic acid) also displayed formation of cation-selective channels in lipid bilayers.54 These reports support the notion that a defined secondary conformation is not necessarily required for the ability of polymers to form nanosized pores. Further investigation on the pore structure will be necessary to identify what structural parameters of polymers determine the pore formation and properties. In addition, the pore formation is also likely to depend on the type and compositions of lipids in bacteria and mammalian cells.37,55–57 A detailed study on the role of lipids in the pore formation and susceptibility to lysis would provide insight into the mechanism of cell-type selectivity and specificity of polymers, enabling development of polymers that can form pores only in targeted bacteria but not in human cells. It has also been reported that cationic poly(amido amine) dendrimers form nanosized pores in mammalian cell membranes and synthetic bilayers.58,59 Computer simulation studies demonstrated that the amine functionality on the dendrimer surfaces is necessary for the insertion of dendrimers into lipid bilayers and pore formation,60 which corresponds to the experimental results for acetylated and unacetylated dendrimers.59–62 In addition, cationic synthetic polymers including poly(L-lysine)s and poly(ethylene imine)s showed membrane disruption and cytotoxicity.59,63 We have previously demonstrated that the polymethacrylates with primary or tertiary ammonium groups showed higher hemolytic activity and ability to permeabilize lipid vesicles than counterparts with quaternary ammonium groups.17 The primary ammonium functionality of PB27 might have specific


interactions with phosphate head groups of lipids, possibly by combination of electrostatic interactions and hydrogen bonding, which is expected to have an important role in the pore formation.

Conclusions This study demonstrated that the amphiphilic polymethacrylates form discrete nanosized pores, followed by colloidosmotic lysis. The diameter of membrane pores was estimated to be in the range of 1.6-2.0 nm by the osmoprotection method. The hemolysis induced by the polymers can be also characterized as an all-or-none phenomenon. We believe that the information in this study is useful to understand the mode of action in hemolysis induced by amphiphilic polymers and delineate the underlying mechanism. Further investigation, however, will be necessary to identify structural factors of polymers that determine the formation and properties of pores, and physiological relevance of the membrane permeabilization to toxicity to human cells. It would be of interest to determine if the pore formation and the following colloidosmotic lysis are also components of the antimicrobial mechanisms of amphiphilic synthetic polymers. Acknowledgment. This research was supported by the Department of Biologic and Materials Sciences, University of Michigan School of Dentistry, and NSF CAREER Award (DMR-0845592). We thank Professor Robert Davenport at the University of Michigan Hospital for supplying the red blood cells. We also thank Dr. Gregory Caputo at Rowan University for comments on the manuscript and for helpful discussions. Supporting Information Available. NMR spectra of the polymers and supplementary hemolysis dose-response curves and determination of pore sizes. This material is available free of charge via the Internet at http://pubs.acs.org.

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BM1011739

Mechanism of Polymer-Induced Hemolysis: Nanosized Pore

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