Structure–property relationships of antibacterial amphiphilic polymers derived from 2-aminoethyl acrylate

Abstract

The findings from the investigation of an ensemble of amphiphilic polymers derived from 2-aminoethyl acrylate establish significant effects of variation in the topographical position of the cationic center and hydrophobic segments on their biological activities. For example, the isomeric polymer pair of poly(6-aminohexylacrylate) and poly(2-(butylamino)ethyl acrylate) show striking differences in their biological activities, with the former having biological activities orders of magnitude higher. The trend of the activities of alkyl tails attached to the charge center shows an abrupt increase in biological activity at butyl length in the series of methyl to butyl tail. The distribution and interaction of the charge center in the chain domain is one of the main parameters in influencing polymer activities. Within the 2-aminoethyl acrylate system of homo- and copolymer, the homopolymer has its cationic centers closely distributed along the amphiphilic macromolecular chain with proximity to the backbone leading to rigid conformations not conducive to the attachment of the polymer to the cell surface. In copolymers, the incorporation of uncharged counits increases the distance between the cationic centers, resulting in significant reduction of charge repulsion and thus enhancing the flexibility of the chain conformation. This is conducive for polymer-cell association, leading to a remarkable surge in orders of magnitude of biological activity but with low selectivity against bacteria over red blood cells.

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Introduction

The steady increase in infections involving multi-drug resistant bacteria (superbugs) have now threatened to upend a century of medical advances in antibiotics.¹ A recent report has projected 10 million deaths due to antimicrobial resistance annually by 2050, which will surpass the number of deaths caused by cancer.² Biopolymer antimicrobial peptides (AMPs) are considered capable of destroying bacteria with very low likelihood of bacterial resistance development.³ Large scale therapeutic applications of AMPs have been impeded by their costly synthesis and challenging drug administration due to proteolysis.⁴ On the other hand, synthetic amphiphilic polymers, mimicking the fundamental principles of AMPs, have emerged as promising antibacterial candidates because of their facile synthesis and ease of structural tunability.⁵ We report here our findings from our ongoing investigation into structure–property relationships for antibacterial synthetic amphiphilic polymers. The polymers described here are based on a 2-aminoethyl acrylate monomer leading to their cationic centers being only 2 carbons away from the chain backbone.

For therapeutic applications, it is highly desirable for synthetic amphiphilic polymers to selectively attack bacteria while exhibiting minimal toxicity towards mammalian cells. To this end, a number of studies have been focused on various structural determinants of the antibacterial and hemolytic activity of amphiphilic polymers including the effects of amphiphilic balance,⁶ identity of amine functionality,⁷ block versus random copolymer architecture,⁸ charge density,⁹ counter-ion effect,¹⁰ and inclusion of poly(ethylene glycol) (PEG) side groups.¹¹ The hemolytic activity of polymers towards red blood cells (RBCs) has been widely used as a benchmark to assess the toxicity of polymers towards mammalian cells.

Our investigation in the area of synthetic antibacterial polymers has been focused on polyacrylate systems with the goal to further our understanding of the effect of controlled amphiphilicity of the polymer. Recently we reported our findings on systems based on aminoethyl and n-aminohexyl acrylates.¹² The two counits led to copolymer side chains having 2- and 6-carbon spacer arms (distance from polymer backbone to cationic center), facilitating the study of the effect of controlled topography of cationic charge distribution in connection with the polyacrylate polymer main chain backbone.

A 2-carbon spacer arm homopolymer displayed low activity against Escherichia coli (E. coli) and low hemolytic activity while a 6-carbon spacer arm homopolymer displayed high antibacterial activity but with very high hemolytic activity.^12a,13 We found polymers with high antibacterial and low hemolytic activity by utilizing the high antibacterial activity of the 6-carbon spacer arm component while mitigating its very high hemolytic effect through incorporation of counits with designed characteristics. In one approach, we tuned the polymer amphiphilicity through copolymerizing the 6-carbon spacer arm monomer with non-ionic poly(ethylene glycol) methyl ether methacrylate monomers.^12b Through control of the polymer composition and length of PEG side groups, a copolymer was synthesized with >100 times selectivity (hemolytic activity/antibacterial activity) towards E. coli over RBCs. In another strategy, the control of spacer arm lengths through various combinations of 6-carbon spacer arms and 2-carbon spacer arms led to a high antibacterial activity concomitant with a low hemolytic activity.^12a Copolymerization of just 10 mol% of smaller 2-carbon spacer arm counits led to a drastic reduction in the hemolytic activity while retaining the high antibacterial activity, leading to >200 times selectivity towards E. coli over RBCs. These results illustrate that the incorporation of 2-carbon spacer arm acrylate comonomer units can be an effective tool to obtain antibacterial polymers with dramatically reduced hemolytic activity.

Thus, the comonomer 2-aminoethyl acrylate is of keen interest as a comonomer with a short spacer arm from the polymer backbone to the cationic center. The focus of the present study is on this acrylate monomer and its derivatives as cationic building units for antibacterial homo- and copolymers. The amphiphilic balance of 2-carbon spacer arm homopolymers was varied by controlling the length of the alkyl side tail attached to the cationic center. These polymers were synthesized at two molecular weight levels. For copolymers of 2-aminoethyl acrylate with alkyl acrylate, the number of cationic and hydrophobic groups on each polymer chain can affect the initial interactions of the polymers with bacterial and mammalian cell surfaces and further steps during cell membrane penetration. Random copolymers with butyl side chains and cationic groups on separate repeating units were synthesized to investigate their biological activity for comparison with similar homopolymers having cationic groups and hydrophobic groups on the same repeating units. Furthermore, the effect of the shape of 6-carbon alkyl isomers on the biological activity of random copolymers was explored. The antibacterial activities of the polymers were evaluated against E. coli and Staphylococcus aureus (S. aureus), and the toxicities of the polymers against mammalian cells were evaluated in terms of the hemolytic activity of the polymer against mouse RBCs.

Experimental

Materials and methods

(Methylamino)ethanol, 2-(ethylamino)ethanol, 2-(propylamino)ethanol, 2-(butylamino)ethanol, dichloromethane (anhydrous), N,N-diisopropylethylamine, acetonitrile (anhydrous), 2,2′-azobis(2-methylpropionitrile) (AIBN), methyl 3-mercaptopropionate (MMP), 1-hexanol, cyclohexanol, 3,3-dimethyl-2-butanol, hexane, and diethyl ether were purchased from Sigma-Aldrich and used without further purification. Acryloyl chloride from Sigma-Aldrich was distilled prior to use. Butyl acrylate was stirred with inhibitor remover for 20 minutes and filtered before use. Di-tert-butyl dicarbonate and trifluoroacetic acid were purchased from VWR. ¹H NMR spectra were obtained with 300 MHz and 600 MHz Varian NMR spectrometers using CDCl₃ or DMSO-d₆ as solvents. Molecular weights (M_w and M_n) of Boc protected polymers, and their molecular weight distributions (M_w/M_n, PDI) were obtained with Waters alliance GPCV 2000 using linear polystyrene as standard. Tetrahydrofuran (HPLC grade) was used as an eluent at a flow rate of 1 mL min⁻¹. OD₆₀₀ was obtained to measure bacterial growth with an Agilent 8453 spectrophotometer (using 1 cm path length plastic cuvette). OD₅₉₅ and OD₄₁₄ were obtained using a SpectraMax 340 PC microplate reader (molecular devices).

N-Boc protection of alkylamino alcohols^12a,14

Di-tert-butyl dicarbonate (26 g, 119 mmol) was added to a 250 mL single neck round bottom flask, already containing 2-(methylamino)ethanol (8.6 mL, 108 mmol) and H₂O (110 mL). The reaction mixture was then stirred at 34 °C for 6 hours. After 6 hours, the reaction mixture was extracted with ethyl acetate (3 × 125 mL) and dried with sodium sulfate. After filtration, the ethyl acetate was evaporated using a rotary evaporator to yield a pure product (90% yield). ¹H NMR (300 MHz, CDCl₃): δ 1.45 (s, 9H), 2.92 (s, 3H), 3.38 (s, 2H), 3.73 (s, 2H). N-Boc protection of 2-(ethylamino)ethanol, 2-(propylamino)ethanol, and 2-(butylamino)ethanol was carried out following a modified work-up procedure (ESI†).

Synthesis of amine functionalized monomers^6c,12a

A representative monomer synthesis is as follows. 2-(N-Boc-butylamino)ethanol (12.84 g, 60 mmol) was added into a 500 mL, 3-neck round bottom flask, loaded with N,N-diisopropylethylamine (17.4 mL, 100 mmol) and dichloromethane (100 mL). Acryloyl chloride (5.52 mL, 68 mmol) was then added drop-wise at 0 °C, under a nitrogen atmosphere. The reaction mixture was allowed to warm to room temperature, and stirred overnight. After 18 hours, the reaction mixture was washed with distilled water (3 times), 10% citric acid (2 times), 10% potassium carbonate (2 times), and saturated sodium bicarbonate solution (3 times). The organic layer was separated and dried over sodium sulfate, followed by the removal of excess solvent using a rotary evaporator. The resultant liquid was purified by silica gel column chromatography, using hexane/ethyl acetate (1 : 1) as an eluent. 60% yield (monomer 4). ¹H NMR (300 MHz, CDCl₃): δ 0.92 (t, 3H), 1.30 (m, 2H), 1.48 (bs, 11H), 3.23 (s, 2H), 3.48 (s, 2H), 4.3 (s, 2H), 5.86 (d, 1H), 6.14 (m, 1H), 6.43 (d, 1H). A similar procedure was followed for the synthesis of all other monomers.

Synthesis of polymers^6c,12a

A representative synthesis procedure is as follows. A 3 neck 100 mL flask was filled with monomer 2 (2.25 g, 9.25 mmol, R = ethyl (Scheme 1)), AIBN (15.2 mg), methyl-3-mercaptopropionate (MMP, 0.205 mL), and acetonitrile (10 mL). The reaction mixture was degassed with dry nitrogen for 20 minutes and stirred at 65 °C for 18 hours. Acetonitrile was then evaporated using a rotary evaporator, and a small amount (1 mL) of dichloromethane was added into reaction mixture, followed by precipitation into hexane (3 times). The resultant polymer was dried under reduced pressure. By varying the mole ratios of MMP and the monomer, two series of molecular weights were obtained (M_w approximately 6k g mol⁻¹ and 1.6k g mol⁻¹).

Removal of Boc protecting groups

For the deprotection of the Boc protecting group, polybutyl was dissolved in a minimum quantity of dichloromethane, and an excess quantity of trifluoroacetic acid (TFA) was added. The mixture was stirred for 4 hours at room temperature. TFA was removed under reduced pressure using a rotary evaporator. A small quantity of acetonitrile (2 mL) was added and the mixture was repeatedly precipitated into diethyl ether to obtain an amphiphilic polymer. The polymer was dried under reduced pressure and lyophilized. All other cationic amphiphilic polymers were similarly obtained.

Preparation of polymer dilutions for antibacterial and hemolytic activity determination

Each polymer was dissolved in DMSO to prepare a stock solution with 20 mg mL⁻¹ concentration. Further two-fold dilutions were prepared by adding deionized water. Control solutions (without polymers) were prepared in a similar way. As described below, a ten-fold dilution of the polymer concentrations would take place in the assays for antibacterial and hemolytic activity determination.

Determination of antibacterial activity^6c,12

The antibacterial activities of the polymers were studied by following a slightly modified literature protocol. To assess the antibacterial activities of the polymers against Gram negative bacteria, E. coli TOP 10 (ampicillin resistant) was incubated overnight at 37 °C in Luria Bertani (LB) broth (containing ampicillin, 100 μg mL⁻¹). OD₆₀₀ was obtained with an Agilent 8453 spectrophotometer using plastic disposable cuvettes (1 cm path length) to measure bacterial cell growth. This cell suspension was diluted to obtain OD₆₀₀ = 0.1, by adding fresh LB broth. The cell suspension was allowed to grow at 37 °C (under shaking) for approximately 1.5 hours, and OD₆₀₀ increased to around 0.5 (log phase growth). A final stock cell suspension with OD₆₀₀ = 0.001 was obtained through a final dilution with fresh LB broth. To each well of a 96 well sterile tissue culture plate (REF 353916, BD falcon, flat bottom), 90 μL of the cell stock suspension (with ampicillin) was added followed by the addition of 10 μL of the polymer solutions, or control solutions. Each polymer concentration was added in triplicate, and the assay plates were incubated at 37 °C for 18 hours. OD₅₉₅ values were then obtained using a SpectraMax 340 PC micro plate reader, and the minimum inhibitory concentration (MIC) is defined as the lowest polymer concentration required to completely inhibit the bacterial cell growth. The antibacterial activities of the polymers against S. aureus ATCC 25923 were examined by following a similar protocol to that described above for E. coli, except Mueller-Hinton (MH) broth was used in place of Luria-Bertani (LB) broth. The MIC values reported in this study are the averages of three independent MIC values obtained on different days under similar conditions.

Determination of hemolytic activity^6c,12a

Freshly drawn RBCs were obtained by centrifuging mice blood, and discarding the white blood cells and plasma (as supernatant). 4.5 mL of TBS (Tris buffer, 10 mM, pH = 7, 150 mM NaCl) was added to 0.5 mL RBCs. To 250 μL of this cell suspension, 10 mL of TBS was further added to obtain a final stock cell suspension (40 fold dilution, 0.25% RBCs). 130 μL of this stock solution was added to a 600 μL centrifugation tube containing 15 μL of the polymer solution (or control solution) and TBS (15 μL). Centrifugation tubes were incubated at 37 °C for 1 h, and then centrifuged for 4 minutes at 4000 rpm. The supernatant (30 μL) was obtained and diluted with TBS (70 μL) in a 96 well sterile assay plate (in triplicate). Hemoglobin concentration as optical density at OD₄₁₄ was obtained with a microplate reader (SpectraMax 340 PC). 1% triton X-100 was used as a reference for 100% hemolysis (positive control), and the control solutions were used as a reference for 0% hemolysis (negative control). The percent hemolysis corresponding to each polymer concentration was obtained by using the following formula:
The HC₅₀ values reported here are the averages of 3 independent experiments conducted on different days.

Results

Synthesis of polymers

The strategy employed for the synthesis of the monomers and amphiphilic polymers is as shown in Scheme 1. The homopolymers were synthesized by free radical polymerization of N-Boc protected monomers (Scheme 1b). Likewise, N-(tert-butoxycarbonyl)aminoethyl acrylate was copolymerized with butyl acrylate (1 : 1, feed mole ratio, Scheme 1c) to synthesize a random copolymer in order to compare its antimicrobial activity with a similar homopolymer. Two series of molecular weights (M_w ∼ 6k g mol⁻¹, degree of polymerization (DP ∼ 22) and 1.6 g mol⁻¹ (DP ∼ 6)) were synthesized for each homopolymer and copolybutyl. Data from ¹H NMR (600 MHz or 300 MHz) was used to estimate the DP of products and to ascertain the copolymer composition. Gel permeation chromatography (linear polystyrene standards) was employed to estimate the molecular weights and polydispersities (PDI) of the Boc protected polymers. Poly(N-methyl)-6k represents the homopolymer with a methyl chain attached to the amine group and with a molecular weight of approximately 6k g mol⁻¹. Scheme 1c represents the synthesis of the copolymers designed to evaluate the shape effect of the 6-carbon hydrophobic side chain on the antibacterial and hemolytic activity of the amphiphilic copolymers. The mole percentage feed of the hydrophobic comonomer was kept at 40%.

Scheme 1 Synthesis of monomers and polymers (X⁻ is CH₃COO⁻).

Antibacterial activity

The antibacterial activities of the polymers were determined in terms of minimum inhibitory concentration (MIC) against E. coli TOP 10 (ampicillin resistant) and S. aureus ATCC 25923 (Fig. 1 and Table 1). Homopolymers with one to three carbon tails in the 6k g mol⁻¹ series displayed high and similar activities against Gram positive S. aureus (Fig. 1a, MIC = 62 μg mL⁻¹). However, this homopolymer series displayed much lower antibacterial activity towards E. coli than with S. aureus (Fig. 1a). With the addition of one more carbon to the three carbon tail, the homopolymer poly(N-butyl) showed a marked increase in biological activity towards both bacteria and RBCs. A conspicuous surge in orders of magnitude of the biological activities was observed in going from homopolymers to copolymers. Random copolymer copoly(butyl)-6k displayed high antibacterial activity against both S. aureus (MIC = 34 μg mL⁻¹) and E. coli (MIC = 13 μg mL⁻¹), as shown in Fig. 1c and Table 1. Similarly, the three copolymers with six-carbon alkane groups attached to the polymer backbone: copoly(linear)-6k, copoly(cyclo)-6k, and copoly(branched)-6k, displayed high activity against both S. aureus and E. coli (Fig. 1c and Table 1). The role of the molecular weight of the homopolymers in their biological activity is apparent from Fig. 1 and Table 1. Smaller molecular weight homopolymers were inactive against E. coli (Fig. 1b), in contrast to the moderately active 6k g mol⁻¹ series homopolymers. Similarly, the 1.6k g mol⁻¹ series homopolymers, except poly(N-butyl)-1.6k, did not show activity against S. aureus, whereas the 6k g mol⁻¹ series homopolymers demonstrated high activity against S. aureus. The 1.6k g mol⁻¹ series with low DP of ∼6 has a much lower likelihood of association with the cell surface at initial contact than the 6k g mol⁻¹ series. In contrast to homopolymers, the effect of molecular weight on the antibacterial activity of the random copolymer was not observed as both copoly(butyl)-6k and copoly(butyl)-1.6k manifested very high and similar activities against E. coli and S. aureus.

Fig. 1 Antibacterial and hemolytic activities of (a) 6k g mol⁻¹ series homopolymers; (b) 1.6k g mol⁻¹ homopolymers; and (c) random copolymers. Error bars represent standard deviation.

Table 1 Characterization and biological activities of homopolymers and copolymers

Polymer	[MMP]/[monomer]	Mw (GPC)	PDI	DP^a	MIC, μg mL^−1 (E. coli)	MIC, μg mL^−1 (S. aureus)	HC50 (RBCs)	Selectivity (HC50/MIC)
								E. coli	S. aureus
a Degree of polymerization (DP) is calculated from ^1H NMR (ESI).
Poly(N-methyl)-1.6k	0.20	1646	1.54	6.9	>2000	>2000	>2000	1	1
Poly(N-ethyl)-1.6k	0.20	1730	1.5	5.7	>2000	>2000	>2000	1	1
Poly(N-propyl)-1.6k	0.20	1723	1.5	6.0	>2000	809	>2000	1	>2.5
Poly(N-butyl)-1.6k	0.20	1637	1.8	6.6	2000	62	42	0.02	0.68
Poly(N-methyl)-6k	0.05	7359	1.33	19	1048	62	>2000	>2	>32
Poly(N-ethyl)-6k	0.05	6299	1.31	23	905	62	>2000	>2.2	>32
Poly(N-propyl)-6k	0.05	5314	1.37	23	810	62	>2000	>2.5	>32
Poly(N-butyl)-6k	0.05	6380	1.47	24	417	62	253	0.61	4.1
Copoly(butyl)-1.6k	0.20	1420	1.57	4.1	13	18	16	1.2	0.9
Copoly(butyl)-6k	0.05	6493	1.5	24	13	34	<7	<0.5	<0.2
Copoly(linear)-6k	0.05	6316	1.26	—	16	18	26	1.6	1.4
Copoly(cyclo)-6k	0.05	5478	1.48	16	21	18	<7	<0.3	<0.4
Copoly(branched)-6k	0.05	5350	1.34	22	16	18	<7	<0.4	<0.4

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Hemolytic activity

To ascertain the toxicity of the polymers towards mammalian cells, the hemolytic activity (HC₅₀) of the polymers was measured against mouse RBCs. HC₅₀ is defined as the minimum concentration of a polymer solution required to cause lyses in 50% of RBCs within an incubation period of 1 h at 37 °C. Our study found that all of our homopolymers, except poly(N-butyl), are non-hemolytic (HC₅₀ > 2000 μg mL⁻¹), whereas copoly(butyl), the random copolymer, was highly hemolytic at both 6k g mol⁻¹ and 1.6k g mol⁻¹ molecular weight levels (Fig. 1 & Table 1). Copoly(linear)-6k, copoly(cyclo)-6k, and copoly(branched)-6k displayed similar and high hemolytic activities despite having different shapes of hydrophobic side groups.

Selectivity of polymers

Selectivity is defined as the HC₅₀/MIC ratio of polymers. Polymers with selective activity against bacteria over RBCs are highly desired. Poly(N-methyl)-6k, poly(N-ethyl)-6k, and poly(N-propyl)-6k demonstrated a selectivity of greater than 32 times for S. aureus over RBCs. Furthermore, the 6k series homopolymers displayed selectively higher activity against S. aureus over E. coli. Thus, these homopolymers are doubly selective: S. aureus over RBCs and S. aureus over E. coli. In contrast to the homopolymers, the random copolymers in this study did not demonstrate selective activity against bacteria over RBCs, and showed similar activity against both S. aureus and E. coli.

Discussion

Antibacterial activity of polymers

All the homopolymers in the 6k series displayed high activity against S. aureus but lacked high activity against E. coli. S. aureus has a single plasma membrane surrounded by a negatively charged peptidoglycan layer (∼15–80 nm), whereas E. coli has a double membrane structure in which the negatively charged peptidoglycan layer (∼8 nm) is sandwiched between an outer membrane and an inner membrane.¹⁵ Thus, the double membrane structure of the E. coli cell surface can be considered more challenging to lyse than the single membrane structure of S. aureus. In contrast to the homopolymers, the copolymers did not demonstrate selectivity between the types of bacteria and showed high activity against both E. coli and S. aureus. The ability of polymers to selectively attack S. aureus over E. coli can be advantageous in treating S. aureus and potentially other Gram positive infections without negatively affecting the gut microbiome consisting of Gram negative bacteria.¹⁶

The significantly different levels of antibacterial activity between the homopolymers and the copolymers mainly arise from the topographical variation of the locations of the hydrophobic side segments and cationic centers, and the chain backbone conformation. The homopolymer macromolecule carries a positive charge on each repeat unit while the copolymer molecule has positive center on every other counit. The distance between the positive centers in poly(N-butyl)-6k is about half of that in the copoly(butyl)-6k; its activity against E. coli is 32 times lower than that of copoly(butyl)-6k. The high cationic charge density of the homopolymers leads to a rigid conformation due to charge repulsion and thus may hinder the initial process of polymer–cell surface association and their permeabilization through the hydrophobic core of the lipid bilayer, especially with E. coli due to its double bilayer structure. Furthermore, the short 2-carbon spacer arm of these polymers can also be a deterring factor behind their low activity against E. coli. Poly(N-butyl)-6k and PM6-100% (cationic poly(6-aminohexylacrylate) homopolymer) are isomeric in that both contain six hydrophobic alkane carbons on their side chains (Fig. 2). The structural difference is that the former polymer has a 4-carbon alkane tail attached as a tail to the charge center while the latter has all six carbons on the spacer arm. Both polymers have similar values of cationic charges and hydrophobicity, but PM6-100%, with a 6-carbon long spacer arm, is highly antibacterial (MIC_{E. coli} = 5.8 μg mL⁻¹; MIC_{S. aureus} = 16 μg mL⁻¹) and hemolytic (HC₅₀ < 1.9 μg mL⁻¹) as compared with poly(N-butyl)-6k (MIC_{E. coli} = 417 μg mL⁻¹; MIC_{S. aureus} = 62 μg mL⁻¹; and HC₅₀ = 253 μg mL⁻¹).^12a The striking difference in biological activity can be attributed to the topology of their amphiphilicity arrangement.

Fig. 2 A structural isomeric polymer pair: poly(2-(butylamino)ethyl acrylate), i.e. poly(N-butyl) and poly(6-aminohexylacrylate), i.e. (PM6-100%). The effect of spacer arm, the locations of hydrophobic segments, and proximity of the cationic center to the chain backbone on the antibacterial and hemolytic activities of polyacrylate homopolymers.

In poly(N-butyl), a hydrophobic butyl tail is attached to the positive center, which may hamper the ionic interaction with the cell surface but can also assist with hydrophobic interactions within the double layer of the cell membrane. PM6 100% has a longer spacer arm with four more carbons. A long spacer arm enhances the snorkel effect in which the cationic center can attach to the cell surface and the polymer backbone can then permeabilize through the cell membrane core.¹³ On the other hand, the shorter 2-carbon spacer arm hinders the permeabilization of the polymer backbone through the hydrophobic core of the cell membrane. Molecular weight has a substantial effect on the antibacterial activity of these homopolymers. As compared with higher molecular weight polymers, the lower molecular weight polymers (DP ∼ 6) will have a lower number of attached points per chain to the surface of bacterial cells leading to a lower ability to bind to the cell surface of bacteria. This effect may be especially exacerbated in the case of homopolymers with cationic groups that are sterically hindered due to the presence of a hydrophobic tail on the cationic center. Copoly(linear)-6k, copoly(cyclo)-6k, and copoly(branched)-6k displayed similar activities against both E. coli and S. aureus. Thus the shape of the hydrophobic alkyl tail of 6 carbons in these polymers has no significant effect on their antibacterial activity, indicating the spatial resolution of the recognition of biological agents by the cell surface not sensitive down to the 6-carbon structural level.

Hemolytic activity of polymers

The toxicity of polymers towards mammalian cells has been a major concern hindering the therapeutic applications of amphiphilic polymers. All our homopolymers, except poly(N-butyl), demonstrated non-hemolytic activity, as opposed to the random copolymers with very high hemolytic activity. The outer surface of RBCs’ cell membrane lacks net negative charge and the hemolytic activity of amphiphilic polymers is believed to primarily result from the insertion of the hydrophobic alkyl tail of the polymer into the hydrophobic domain of the lipid bilayer.⁴ The presence of a cationic group along with every alkyl side tail in the homopolymers and the high cationic charge density would not favor the hydrophobic interactions of the homopolymers with the lipid bilayer of RBCs. However, in the copolymers cationic charges and hydrophobic alkyl side groups are placed on separate repeat units, thus the hydrophobic side groups can more readily insert into the lipid membrane of RBCs. Copoly(linear)-6k, copoly(cyclo)-6k, and copoly(branched)-6k showed similar hemolytic activity, suggesting that the shape of the hydrophobic alkyl tail on a six carbon level in these polymers does not substantially affect their hemolytic activity.

Conclusions

The findings here on the biological activities of a series of amphiphilic copolymers and homopolymers derived from a 2-aminoethyl acrylate comonomer can serve as significant references for further development of structure–property relationships in the subject of synthetic antibacterial polymers. The biological activities of the structural isomeric pair (Fig. 2): poly(6-aminohexylacrylate) and poly(2-(butylamino)ethyl acrylate), clearly show the strong impact of the spatial arrangement of the hydrophobic segments and the charge center. An increase in the spacer length and keeping the cationic center unhindered for ionic interactions leads to a dramatic rise in antibacterial and hemolytic activities. The distribution and interaction of the charge center in the chain domain is one of the main parameters. In the homopolymers, the cationic centers are closely distributed along the amphiphilic macromolecular chain with proximity to the backbone leading to rigid conformations not conducive to the attachment of the polymer to the cell surface. In the copolymers, the incorporation of non-charged counits doubles the distance between the cationic centers, resulting in a significant reduction of the charge repulsion and thus enhancing the flexibility of the chain conformation. The position and properties of the hydrophobic alkyl side group with respect to the cationic center has a substantial effect on the biological activity. The acrylate homopolymers with hydrophobic groups directly attached to the cationic center showed low antibacterial activity against E. coli and low hemolytic activity against RBCs. High charge density and a short cationic spacer arm can hinder the permeability of homopolymers through the hydrophobic core of the outer and inner cell membranes in E. coli and the lipid bilayer of RBCs.

In contrast to doubly selective homopolymers (S. aureus over RBCs and S. aureus over E. coli), the random copolymers which had hydrophobic segments and cationic groups on separate repeating units, displayed very high but non-selective activity against bacteria and RBCs.

Acknowledgements

We acknowledge the support from the Center for Engineered Polymeric Materials, Department of Chemistry at the College of Staten Island of CUNY; Ph.D. Program in Chemistry at the Graduate Center of CUNY and CUNY RF 68464-00 46.

Notes and references

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Footnote

† Electronic supplementary information (ESI) available: NMR spectra, calculation of DP. See DOI: 10.1039/c5ra17875d

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