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Biomacromolecules 2011, 12, 50–56
Effect of Steric Hindrance on the Properties of Antibacterial and Biocompatible Copolymers Thomas R. Stratton,† Bruce M. Applegate,‡ and Jeffrey P. Youngblood*,† School of Materials Engineering, Purdue University, 701 West Stadium Avenue, West Lafayette, Indiana 47907, United States, and Departments of Food Science and Biological Sciences, Purdue University, 745 Agriculture Mall Drive, West Lafayette, Indiana 47907, United States Received August 17, 2010; Revised Manuscript Received November 11, 2010
The development of polymers that are both bactericidal and biocompatible would have many applications and are currently of substantial research interest. It is well known that polymers of alkyl-quaternized poly(4-vinylpyridine) are known to be effective against a wide range of microbes: when copolymerized with monomers that form biocompatible materials, they has also been shown to possess biocompatible properties. However, the relationship of the various physical and chemical properties of these polymers and copolymers with the antibacterial and biocompatible properties remains poorly understood: maximizing the selectivity and performance of these materials is absolutely needed before they have the potential for commercial applications. Maximizing the performance will require a complete understanding of the effect of physical and chemical adjustments on these quaternized polymer bactericides. This article seeks to explore and characterize the effect of one specific property, steric hindrance, on the copolymers’ antibacterial and biocompatible properties. We have thus synthesized and characterized a new class of copolymers from 2-vinylpyridine and poly(ethylene glycol) methyl ether methacrylate, measured its bactericidal and biocompatible properties, and compared its performance to chemically similar but sterically different polymer bactericides. This work thereby enables both a greater understanding of the properties of the 2-vinylpyridine copolymers and an improved understanding of the material properties that are vital for the development of antibacterial polymers.
Introduction Many recent events have brought increased interest to the development of new microbicides. As an example, the cost of hospital-acquired infections has become a major burden on health care providers because it amounts to ∼5% of hospital admissions in the United States alone, a percentage that has remained constant over the last 25 years1 despite the fact that a third of these infections are deemed preventable.2,3 Current alternatives to traditional antibiotics, such as triclosan and silver nanoparticles, suffer from serious drawbacks, for example, short active lifetimes or high production costs.4-6 Compared with the drawbacks of the alternatives, polymer microbicides have been increasingly seen as a viable alternative for microbicidal applications.7-10 Polymer approaches offer novel methods of action, straightforward synthesis, and low costs. One example of such a microbicidal polymer is quaternized poly(4-vinylpyridine) (4VP); however, this polymer has been shown to have poor biocompatibility.11 Previous work has shown improvements in the biocompatibility and bactericidal activity through the copolymerization of 4VP with monomers that form biocompatible polymers like poly(ethylene glycol) methyl ether methacrylate (PEGMA).12-16 Poly(ethylene glycol) (PEG) is an FDA-approved, widely used, hydrophilic, water-soluble, biocompatible polymer.17 PEGMA was chosen as a copolymer unit in place of PEG because of the ease of polymerizing and copolymerizing PEGMA. This approach led to a surprising result: quaternized copolymers that included a small amount of the biocompatible monomer exhibited much stronger antibacterial properties than the quaternized P4VP homopolymer * Corresponding author. E-mail:
[email protected]. † School of Materials Engineering. ‡ Departments of Food Science and Biological Sciences.
as well as improved biocompatibility.14,15 This fascinating result begs an additional question: what chemical or physical properties mediate these results and how can these properties be manipulated to improve the antimicrobial and biocompatible characteristics? The copolymerization approach toward the development of safe, effective, microbicidal materials can be further expanded and explored. Substantial research has already been completed on the effects of various configurations and chemistries on the performance of this and similar antibactericidal polymers. It is theorized for similar systems that the antibacterial behavior of these polymers is related to the amphipathic balance18-22 and the spatial relationship of the cation to the alkyl chain.23 Further research has explored the effects of the chemical structure of the amphilicity of the polymer chain by controlling the separation of the differently charged groups and found that the spatial relationship of the various polymer groups was crucial in determining the bactericidal and biocompatible properties.18 In this context, the performance of a bactericidal polymer is measured by its ability to strike a balance between microbicidal and biocompatible properties, maximizing the “selectivity” of the material.9,18,24 The term selectivity typically refers to the ratio of the concentration at which a material is found to be biocompatible according to a specified standard or assay, over the minimum bactericidal concentration (MBC), the lowest concentration where the material will kill a specified population of a specified species of bacteria. A high value in the numerator indicates a low toxicity to humans, whereas a low value in the denominator indicates a strongly microbicidal material. A high selectivity thus indicates a material that can kill bacteria readily without harming human cells. These two values are not absolutes: the concentration of microbicide needed varies depending on the assay selected and testing procedure, com-
10.1021/bm1009624 2011 American Chemical Society Published on Web 12/15/2010
Antibacterial and Biocompatible Copolymers
plicating the comparison and interpretation of results. Also, the selectivity value of a material is by no means the final or complete evaluation of the performance or future potential of a material. For example, a bactericide featuring antibacterial activity in the nanogram concentration range (nanograms per milliliter) would be of profound interest because of its substantially improved performance compared with currently researched polymers. In addition, the applicability of a particular in vitro or even in vivo test can always be called into question because of the complexity of biocompatibility and the human body. That said, the selectivity ratio provides a valuable figure of merit for materials comparison and study, as long as one remains apprised of the advantages and disadvantages of such a calculation. Following previous work,12,13,16,18 we chose to synthesize and quaternize copolymers of a relative of 4VP, 2-vinylpyridine (2VP), to observe its antibacterial potential as well as biocompatibility and to compare these properties to the related quaternized homopolymer and copolymers of 4VP. 2VP differs from 4VP in the relative orientation of the nitrogen atom and vinyl functional group and has been relatively ignored in its potential to form an antibacterial polymer. The change in sterics and orientation will affect the distribution of the cation charge as well as the nature of the interaction between the alkyl chain and bacteria surface because the nitrogen cationic center and the alkyl lipophile are sterically shielded by the polymer and may interact differently with cells. For our antibacterial testing, we made use of a previously described high-throughput method utilizing a bioluminescent reporter strain of pathogenic Escherichia coli O157:H7 and a multiwell plate reader.14 E. coli is an important bacteria because of its ubiquity in the human digestive track and the dangerous and widespread infections caused by pathogenic strains like O157:H7.25 Biocompatibility is an equally important property to study to improve the development of useful polymer bactericides, and to measure this property, we completed the red blood cell (RBC) hemolysis assay.9,26 RBCs are an early site of interaction between a biomaterial and bodily humors. In hemolysis testing, as erythrocytes are removed from the protective plasma, proteins and bodily humors that mediate the interactions of RBCs are exposed to our polymers as well as to positive and negative controls.27,28 RBC testing is by no means the final or definitive measurement of biocompatibility but is a simple and effective window to understanding a material’s biocompatible potential. Previous work has shown that the results of in vitro cell viability experiments on similar polymers yielded largely identical results.15 We were able to synthesize and quaternize these polymers successfully and discovered that they exhibited antibacterial and biocompatible properties that substantially differed from their 4VP analogues.
Experimental Section Materials. 2VP, PEGMA of Mn 300 and 1100 g/mol, 2,2′azobisisobutyronitrile (AIBN), 1-bromohexane, methanol, chloroform, hexane, tetrahydrofuran (THF), tris(hydroxymethyl)aminomethane buffered saline (Tris), Triton-X 100, and lactose were purchased from Sigma-Aldrich (St. Louis, MO). 2VP was purified by vacuum trap-totrap distillation, whereas PEGMA monomers were purified by column chromatography through 70-240 µm silica particles (Sorbent Technologies, Atlanta, GA) in chloroform. Tris-buffered saline powder was dissolved in deionized water as instructed (pH 8.0). All other materials were used as received. Agar plates were purchased from Cole-Parmer (Vernon Hills, IL). Kanamycin was purchased from Boston Bioproducts (Ashland, MA). Polypropylene 96-well plates were purchased from Axygen Scientific (Union City, CA). Minimal salts media (MSM) culture solution was prepared by the addition of 200 µL of 50 000 ppm lactose solution dissolved in deionized water, 100 µL of kanamycin
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Figure 1. Synthesis of 2VP-based copolymers.
dissolved at 50 mg/mL in deionized water, and 10 µL of trace elements solution to 100 mL of MSM. The trace elements solution consisted of 2 g of magnesium oxide, 0.4 g of calcium carbonate, 1.08 g of iron(III) chloride hexahydrate, 0.288 g of zinc sulfate heptahydrate, 0.05 g of copper sulfate pentahydrate, 5.6 mg of calcium sulfate heptahydrate, 0.0124 g of boric acid, and 0.098 g of sodium molybdate dihydrate dissolved in 190 mL of deionized water and a 10 mL solution of 40% hydrochloric acid in water (all of which were purchased from SigmaAldrich). The bacteria strain utilized was Escherichia coli O157:H7 modified with the luxCDABE gene cassette to luminesce while metabolically active.29-31 All other materials were used as received. Copolymer Synthesis. Copolymers of 2VP and PEGMA n were synthesized through a free radical polymerization technique adapted from Sellenet et al.13 A total of 3.5 g of monomer was mixed with 50 mg of AIBN as an initiator and 7.0 mL of chloroform as a solvent, evacuated, and backfilled with nitrogen to remove oxygen, and propagated by heating to 70 °C for 48 h. Molar ratios of 2VP to PEGMA n were varied across the composition spectrum and, after polymerization, were precipitated in hexane. Two sets of 2VP copolymers were produced, a copolymer of 2VP and PEGMA 1100 (P(2VP-co-PEGMA 1100)) and a copolymer of 2VP and PEGMA 300 (P(2VP-co-PEGMA 300)) under identical reaction conditions. A general synthetic scheme is displayed in Figure 1. The copolymer composition was confirmed by 1H nuclear magnetic resonance (NMR) spectroscopy32 on a Varian 300 MHz spectrometer (Varian, Palo Alto, CA) and follows from the results of previous publications.13 The results of NMR performed on the 2VP copolymers are available in the Supporting Information. Gel permeation chromatography (GPC) was used to determine the molecular weight of the polymers using a Polymer Laboratories PL-GPC 20 (Varian) with a Waters Styragel HR column (Milford, MA) using HPLC-grade THF as a solvent at a flow rate of 1 mL/min. Calibration was carried out using polystyrene standards (Polymer Laboratories, Palo Alto, CA) ranging from 4000 to 205 000 g/mol. The complete results of the GPC analysis are available in the Supporting Information. The copolymers of 2VP and PEGMA 1100, P(2VP-co-PEGMA 1100), showed somewhat low molecular weights (Mn 6000 to 15 000 g/mol), especially for those copolymers with high PEGMA content. Because of these lower molecular weights, some of these copolymers (those high in PEGMA content) were liquids in their quaternized state, even after evacuating and heating the samples to 70 °C overnight to remove any residual solvent. These copolymers also displayed remarkably low PDIs, below 1.15, in all cases except for the homopolymer of 2VP. This is probably due to a shielding effect caused by the long PEG groups of PEGMA 1100, decreasing the potential for further polymerization. This type of steric hindrance has been used in the past to create low PDI copolymers using macromonomers of Mn similar to that of PEGMA 1100.33 Because of the low molecular weights and PDIs displayed in the P(2VP-coPEGMA 1100) copolymer, we chose to synthesize the P(2VP-coPEGMA 300) copolymers as well to observe the reaction without the hindrance to polymerization presented by the longer PEG chain of the PEGMA 1100. These polymers showed higher molecular weights, ranging from 10 000 to 20 000 g/mol, were consistently solid and had PDIs keeping in line with polymers made through free radical polymerization (∼1.4). Previous work showed relatively similar
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Figure 2. Quaternization procedure of 2VP-based copolymers.
bactericidal activity in the range of Mn 8600-16 000 g/mol with some decreased effect beyond this point,34 whereas another work showed similar activity in the Mn range of 16 000-36 900 g/mol,35 which is similar to the Mn reported for the polymers in this and related publications.12,14,16 Copolymers were redissolved in the appropriate solvent, and 1-bromohexane was added to quaternize the pyridine ring, as shown in Figure 2. Three times the amount of 1-bromohexane necessary to quaternize fully the vinylpyridine molecules of the polymer were added to ensure total quaternization in a sealed reaction vessel, stirred at 70 °C for at least 72 h. Copolymers were then precipitated again in hexane and then dried under vacuum at 70 °C for 12 h to remove excess 1-bromohexane (abbreviated as HB for hexyl bromide, an alternative name for the chemical). The set of P(2VP-co-PEGMA 1100)-HB copolymers appeared almost uniformly green and ranged from a brittle solid at 100 mol % 2VP content to a liquid structure for a copolymer of high PEGMA 1100 monomer content. The copolymers of 2VP and PEGMA 300 appeared brown-green at low 2VP compositions and green at higher concentrations and ranged in consistency from a highly viscous solid at low 2VP compositions to a brittle solid at higher 2VP compositions. Characterization was confirmed using 1H NMR, as discussed in the Supporting Information. All quaternized copolymers of 2VP and PEGMA n, including the homopolymer P2VP-HB, were soluble in water, in contrast with quaternized homopolymer and some copolymers of 4VP, which were not. This surprising result may be due to the increased separation of the cation and anion in solution due to the additional steric hindrance of the 2VP monomer. It should be noted that the unquaternized homopolymer, P2VP, was insoluble in water. Bactericidal Activity Assay. Antibacterial assays were completed using a previously described high-throughput method utilizing a bioluminescent reporter strain of pathogenic Escherichia coli O157: H7 and a multiwell plate reader, as reported in a previous publication.14 Polymers were dissolved in deionized water and diluted to the desired testing concentrations (from as large as 10 mg/mL to as small as 1 µg/mL), of which 200 µL of each dissolved polymer was added to the individual wells of a polypropylene 96-well plate (Axygen Scientific, Union City, CA). Bacteria were grown in culture flasks in MSM culture solution and diluted in the same solution to achieve a concentration of 106 colony forming units per mL (CFU/mL), and 200 µL of this diluted solution was added to the polymer-containing wells of the testing plate. The bacteria were previously determined to be in the logarithmic growth phase through regular sampling, and the bacteria was added to the wells immediately following addition of the polymer to prevent the possibility of solvent evaporation. A Wallac multiwell photomultiplier plate reader (PerkinElmer, Waltham, MA) was then used to measure the increase or decrease in luminescence from the bacteria over time, in accordance with the increase or decrease in bacterial population in each well. This technique enabled the testing of up to 27 samples in each experimental run, which were completed in triplicate for rigorous results. The results of these experiments generated time-sensitive plots of the growth and elimination of the bacteria over time. With the data thus generated, it is possible to determine quantitative MBCs for the antibacterial efficacy of these copolymers. The results of the novel high-throughput bioluminescence technique were confirmed with traditional agar plate testing. The bacterial and
Figure 3. Bacterial luminescence results for P(2VP-co-PEGMA 300)-HB copolymers in aqueous testing with a polymer concentration of 5000 µg/mL. Percentages labeled in the legend refer to the 2VP composition of the polymer. Some copolymer compositions are not shown; these copolymers displayed results identical to the control.
polymer suspensions from the plate testing were afterward applied to agar plates under a series of six serial dilutions (106 total dilution), enabling a manual count of the bacteria on the final plate all the way up to the 106 CFU/mL count of the control sample and were also completed in triplicate for rigorous results. There is a potential for only an inhibitory effect due to the continuing presence of polymer in the solution, but it is possible to conject that these materials are bactericidalbased on their strong chemical similarity to insoluble bactericidal polymer.14,36 The linear relationship between bacteria density and luminescence was also verified through the traditional agar plate colony counting technique. Hemolysis Assay. The experimental procedure was identical to that of Allison et al.,12 which was modified from previous work by Ilker et al.37 Human blood (30 µL) was suspended in 10 mL of Tris and centrifuged at 1500 rpm for 5 min to clean and separate the RBCs from the other components of blood plasma. This process was repeated three times to ensure the removal of other compounds. The polymers were dissolved in Tris to an initial concentration of 10 mg/mL. A solution comprising 50% polymer solution and 50% RBC solution was mixed in a centrifuge tube, yielding a final polymer concentration of 5 mg/mL. The tubes were then placed in a rotating hybridization oven at 37 °C for 30 min, after which they were centrifuged at 3000 rpm for 10 min to separate the RBCs from other contents. Hemoglobin was then detected in this liquid through the use of a DU800 ultraviolet-visible (UV-vis) spectrophotometer at 414 nm (Beckman Coulter, Brea, CA). Triton-X was employed as a positive control, and the RBC-Tris and PEG solutions were employed as negative controls.38 Serial dilutions of the polymer concentration were performed until hemolysis was not observed. Each test was completed in triplicate for rigorous results.
Results Bactericidal Activity Assay. With this technique, we were able to determine accurately the antibacterial activity and MBCs of the copolymers of P(2VP-co-PEGMA 300)-HB and P(2VPco-PEGMA 1100)-HB as compared with the copolymers of 4VP.14 Figure 3 shows the bacteria growth results for the experimentally interesting copolymers of 2VP and PEGMA 300 at 5000 µg/mL; this high concentration was chosen to illustrate clearly the antibacterial power of the copolymers. (Some copolymer compositions not shown; these copolymers produced results identical to the control.) All plots include the P2VP-HB
Antibacterial and Biocompatible Copolymers
Figure 4. Bacterial luminescence results for P(2VP-co-PEGMA 1100)-HB copolymers in aqueous testing with a polymer concentration of 5000 µg/mL. Percentages labeled in the legend refer to the 2VP composition of the polymer. Some copolymer compositions are not shown; these copolymers displayed results identical to the control.
homopolymer in addition to an empty control well. For both plotting and modeling, bacteria concentrations are relative to the initial value (t ) 0) of the control well as one with the background signal as zero. As opposed to the 4VP-based copolymers, where a small amount of a PEGMA n monomer in the copolymer composition improved antibacterial properties,14 the 2VP copolymers exhibiting increasing bactericidal capabilities as the 2VP composition increased: a major difference from previous results. This effect arises perhaps from the solubility of these copolymers in water, which increases the likelihood of polymer-bacteria interaction. Figure 4 shows the bacteria growth results of the copolymers of 2VP and PEGMA 1100, which follow the same trend as the 2VP and PEGMA 300 copolymers. The homopolymer P2VP-HB and several of the copolymers show an extremely fast response, completely eliminating the bacteria before the sample can even be loaded into the sample chamber (∼15 s), showing even swifter bactericidal effects than the copolymers of 4VP and PEGMA n previously reported in Stratton et al.;14 this effect could be useful in the synthesis and testing of improved polymer biocides. The dependence of polymer concentration on antibacterial activity, as shown in Figures 5-8, was also measured because this concentration dependence is used as a metric for activity by determining the MBC for a biocide. To specify the MBC without an overly large number of assays, we used the model previously developed14 to determine approximate MBC values quickly. Some polymers show an extreme dependence on the concentration, either killing all bacteria quickly or scarcely hindering growth (as seen in Figures 6 and 8). Others do not show such extreme switching behavior between active and inactive concentrations. (See Figures 5 and 7.) This effect may be related to the hydrophilic/hydrophobic balance that affects the bactericidal power of these materials.39 We used the quantitative results from the model to predict the MBC for each bactericide by fitting to a line representing the hypothetical shape of bacterial growth at the bacteriostatic point, which would be the ultimate limit of bactericidal capability. These results are displayed in Table 1, along with the results for the 4VP copolymers, as reported in previous
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Figure 5. Bacterial luminescence results for P(2VP-co-PEGMA 300)-HB with a monomer composition of 80 mol % (61 wt %) 2VP and 20 mol % (39 wt %) PEGMA 300 at varying concentrations in units of micrograms per milliliter.
Figure 6. Bacterial luminescence results for P(2VP-co-PEGMA 300)-HB with a monomer composition of 90 mol % (81 wt %) 2VP and 10 mol % (19 wt %) PEGMA 300 at varying concentrations in units of micrograms per milliliter.
work.14 For the 2VP copolymers, several results that are distinct from those of the 4VP copolymers are present. First, the 2VP homopolymer (P2VP-HB) showed a much lower MBC (greater antibacterial effect) than the related 4VP homopolymer (P4VP-HB), which may be due to its water solubility or the effect of moving the cationic center and reconfiguring the position of the alkyl tail. The copolymers with a 2VP content of 60 wt % 2VP and above (the copolymers of 2VP and PEGMA 300) show very low MBCs below 10 µg/mL, whereas the copolymer with a 2VP content of 57 wt % (95 mol %) 2VPco-PEGMA 1100 shows a much lower MBC of ∼100 µg/mL, whereas the 42 wt % 2VP and PEGMA 300 copolymer shows almost no antibacterial activity (MBC greater than 5000 µg/ mL), thereby illustrating that the 60 wt % area may indicate the threshold between the active and inactive copolymer compositions. Several of these copolymers such as P(2VP-coPEGMA 300)-HB with 90 mol % 2VP display very low MBCs that are comparable to or even lower than those found with the
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Figure 9. Human red blood cell (RBC) hemolysis of water-soluble P(2VP-co-PEGMA)-HB at a concentration of 1 mg/mL as a function of 2VP mol %, relative to the hemolysis induced by a 10 mg/mL Triton-X solution (Hemolysis of Triton-X ) 1). Figure 7. Bacterial luminescence results for P(2VP-co-PEGMA 1100)-HB with a monomer composition of 95 mol % (57 wt %) 2VP and 5 mol % (43 wt %) PEGMA 1100 at varying concentrations in units of micrograms per milliliter.
Figure 10. Human red blood cell (RBC) hemolysis of water-soluble P(2VP-co-PEGMA)-HB at a concentration of 1 mg/mL as a function of 2VP wt % relative to the hemolysis induced by a 10 mg/mL Triton-X solution (Hemolysis of Triton-X ) 1). The transition region from hemocompatible to noncompatible was ∼60 wt % for both sets of copolymers.
Figure 8. Bacterial luminescence results for P(2VP)-HB at varying concentrations in units of micrograms per milliliter. Table 1. MBCs for the 2VP and 4VP Copolymers As Determined by the Model and the Data Introduced in Stratton et al.14 for 4VP Copolymers, As Compared with the Upper Limit As Determined through Traditional Agar Plate Countinga material P(4VP-co-PEGMA P(4VP-co-PEGMA P4VP-HB P(2VP-co-PEGMA P(2VP-co-PEGMA P(2VP-co-PEGMA P2VP-HB PHMB Triclosan a
1100)-HB 50 mol % 4VP 1100)-HB 90 mol % 4VP 300)-HB 80 mol % 2VP 300)-HB 90 mol % 2VP 1100)-HB 95 mol % 2VP
model MBC
plated MBC
70 5 100 6.0 1.4 120 6.0 0.6 0.2
250 15 100 10 10 100 10 5 0.5
MBC units are in micrograms per milliliter.
copolymers of 4VP and PEGMA n. It is possible that incorporating steric hindrance into a bactericidal polymer could provide improved bactericidal properties.
Hemolysis Assay. The results of the hemolysis assays completed on the copolymers of 2VP and PEGMA n at a concentration of 1 mg/mL are displayed in Figures 9 and 10 based on the mol % and wt % of 2VP, respectively. In general, the results of these plots are similar to those of Allison et al.;12 copolymers with a low VP content show low hemolysis consistent with the saline control sample (0 on the relative hemolysis scale), whereas those with a high VP concentration show broad hemolysis on a level equaling or even exceeding that of the Triton-X control (1 on the relative hemolysis scale). The results of assays done at the higher concentration of 5 mg/ mL are displayed in the Supporting Information; as for some copolymers, the measured hemolysis is in fact greater than that of the Triton-X control: this is due to heavy absorption of light by the polymer. The key feature of these plots is the transition region, where the material shifts from being biocompatible to noncompatible. As with the copolymers of 4VP, the location of this transition region was nearly identical for the various comonomers of PEGMA and 2VP as compared with the weight percentage composition of the polymer components. (See Figure 10.) In the copolymers of 4VP and PEGMA n, this transition was found to be quite abrupt and to occur at ∼18 wt % 4VP.12 In these copolymers, the transition appears to be somewhat more
Antibacterial and Biocompatible Copolymers
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Table 3. Comparison of Antibacterial Efficacy and Biocompatibility of P(2VP-co-PEGMA 1100)-HB Copolymersa mol % 2VP
wt % 2VP
MBC
HC50
selectivity
55 70 80 95 100
10 17 28 57 100
ineffective ineffective ineffective 120 6.0
5000 5000 5000 1000 1
N/A N/A N/A 8.33 0.17
a Selectivity measurements are calculated by dividing the HC50 by the MBC as per Gabriel et al.9
Table 4. Comparison of Antibacterial Efficacy and Biocompatibility of P(2VP-co-PEGMA 300)-HB Copolymersa
Figure 11. Comparison of the minimum bactericidal concentrations and HC50 concentrations for the copolymers of 4VP and PEGMA n with 2VP and PEGMA n. The results for the 4VP copolymers are from in Stratton et al.14 and Allison et al.12 The lines indicate the general trend of each characteristic of the copolymers. The polymer compositions where no data points exist for the MBC value indicate that the copolymer composition was ineffective at killing bacteria at the maximum concentration of 5000 µg/mL. Table 2. Hemolytic Concentrations (HC50) for the Quaternized Copolymers of 2VP material P(2VP-co-PEGMA P(2VP-co-PEGMA P(2VP-co-PEGMA P(2VP-co-PEGMA P(2VP-co-PEGMA P(2VP-co-PEGMA P(2VP-co-PEGMA P(2VP-co-PEGMA P(2VP-co-PEGMA P(2VP-co-PEGMA P(2VP-co-PEGMA P(2VP-co-PEGMA P(2VP-co-PEGMA
300)-HB 300)-HB 300)-HB 300)-HB 300)-HB 300)-HB 300)-HB 300)-HB 1100)-HB 1100)-HB 1100)-HB 1100)-HB 1100)-HB
mol % 2VP
wt % 2VP
HC50 (µg/mL)
0 20 30 40 65 80 90 100 55 70 85 90 100
0 9 14 19 42 61 81 100 10 17 28 57 100
5000 5000 5000 5000 5000 1 1 1 5000 5000 5000 1000 1
gradual and occurs instead at the much greater 2VP composition of 57 wt %. (See Figure 11.) For the samples that were found to be hemolytic, serial dilutions of the polymer concentration were performed until hemolysis was no longer observed. The highest concentration where 57 wt %, as compared with only 9 wt % for the 4VP copolymers. We can plot and observe the differences between the two sets of copolymers graphically, as shown in Figure 11. In this Figure, it is clear to see the shift in the minimum VP composition, which exhibits antibacterial properties, and the shift in the maximum VP composition, which exhibits biocompatibility. It is conceivable that there exists a region of highly selective overlap between biocompatibility and bactericidal function in the 2VP copolymers, as has been observed in the 4VP copolymers, lying somewhere in the range of 42 and 57 wt % 2VP composition. Previous research has shown that the region of composition overlap between bactericidal and bicompatible properties in the 4VP copolymers is quite small,12 and a conceivable related region could exist for these materials, but given the results observed thus far, such a region would remain quite small (in composition terms), and, based on the results of the P(2VP-co-PEGMA 1100)-HB 57 wt % 2VP copolymers (Table 4), such a material might not likely exhibit greater selectivity than the P(4VP-co-PEGMA 1100)-HB 9 wt % copolymer. (See Table 5.) These effects may very well be due to the shielding of the cation by the steric hindrance caused by moving the nitrogen group from the ortho position relative to
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Table 5. Comparison of Antibacterial Efficacy and Biocompatibility of P(4VP-co-PEGMA 1100)-HB Copolymers Including Results from Allison et al.12 and Stratton et al.14 a mol % 4VP
wt % 4VP
MBC
HC50
selectivity
0 10 50 75 90
0 1 9 21 47
ineffective ineffective 70 ∼40 5
10000 10000 10000 1
