Side Chain Degradable CationicâAmphiphilic Polymers with Tunable

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Side Chain Degradable Cationic-amphiphilic Polymers with Tunable Hydrophobicity Show In-vivo Activity Divakara S S M Uppu, Sandip Samaddar, Jiaul Hoque, Mohini Mohan Konai, Krishnamoorthy Paramanandham, Bibek R Shome, and Jayanta Haldar Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.6b01057 • Publication Date (Web): 21 Jul 2016 Downloaded from http://pubs.acs.org on July 27, 2016

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Side Chain Degradable Cationic-amphiphilic Polymers with Tunable Hydrophobicity Show In-vivo Activity Divakara S.S.M. Uppua, Sandip Samaddara, Jiaul Hoquea, Mohini M. Konaia, Krishnamoorthy Paramanandhamb, Bibek R. Shomeb and Jayanta Haldara,* a

Chemical Biology & Medicinal Chemistry Laboratory, New Chemistry Unit, Jawaharlal Nehru Centre for Advanced Scientific Research (JNCASR), Jakkur, Bangalore 560064, India; bICARNational Institute of Veterinary Epidemiology and Disease Informatics (NIVEDI), Ramagondanahalli, Yelahanka, Bengaluru 560064, India.

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ABSTRACT Cationic-amphiphilic antibacterial polymers with optimum amphiphilicity generally target the bacterial membranes instead of mammalian membranes. To date, this balance has been achieved either by varying the cationic charge or side chain hydrophobicity in a variety of cationicamphiphilic polymers. Optimum hydrophobicity of cationic-amphiphilic polymers has been considered as the governing factor for potent antibacterial activity yet minimal mammalian cell toxicity. However, the concomitant role of hydrogen bonding and hydrophobicity keeping constant cationic charge in the interactions of antibacterial polymers with bacterial membranes has not been understood. Also, degradable polymers that result in non-toxic degradation byproducts offer promise as safe antibacterial agents. Here we show that amide and ester (degradable) bearing cationic-amphiphilic polymers with tunable side chain hydrophobicity can modulate antibacterial activity and cytotoxicity. Our results suggest that an amide polymer can be potent antibacterial agent with lower hydrophobicity whereas the corresponding ester polymer needs relatively higher hydrophobicity to be as effective as its amide counterpart. Our studies reveal that at higher hydrophobicity both amide and ester polymers have similar profiles of membrane-active antibacterial activity and mammalian cell toxicity. On contrary, at lower hydrophobicity amide and ester polymers are less cytotoxic but the former have potent antibacterial and membrane activity than the latter. Incorporation of amide and ester moieties made these polymers side chain degradable with amide polymers being more stable than the ester polymers. Further, the polymers are less toxic and their degradation-by products are non-toxic to mice. More importantly, the optimized amide polymer reduces the bacterial burden of burn wound infections in mice models. Our design introduces a new strategy of interplay between the hydrophobic and hydrogen bonding interactions keeping constant cationic charge density for


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developing potent membrane-active antibacterial polymers with minimal toxicity to mammalian cells.

INTRODUCTION Natural antimicrobial peptides (AMPs) are part of the innate immune defense against pathogens in eukaryotes targeting primarily the microbial cell membranes1-3. Taking inspiration from membrane-active nature of AMPs, antibacterial polymers have been developed by tuning the key structural features of AMPs such as cationic charge and hydrophobicity required for their interaction with the bacterial membranes4. Cationic charge and hydrophobicity are responsible for electrostatic and hydrophobic interactions respectively with the negatively charged phospholipids of bacterial membranes whereas the mammalian membranes contain zwitterionic phospholipids. Amphiphilicity (ratio of cationic charge hydrophobicity) is important for driving the interactions of AMPs to bacterial instead of mammalian membranes1-3. Cationic and amphiphilic polymers as well as small molecules have been developed that mimic AMPs, to address the problems such as high cost of manufacture, mammalian toxicity and low stability invivo that have prevented the AMPs from reaching the clinics5-7. Tuning the amphiphilicity is required even in their mimics to achieve selective antibacterial activity without causing toxicity to mammalian cells5-7. Two key structural features of AMPs such as cationic charge and hydrophobicity were reported to be important till now in literature for membrane interactions 4. To that end, a plethora of

cationic

and

amphiphilic

polymers

including

polyamides8,

9

,

polynorbornenes6,

polymethacrylates7, 10-12, pyridinium copolymers13, polycarbonates14, 15, peptidopolysaccharides16 and others17-47 have been synthesized over the last decade as synthetic antimicrobial polymers. 3 ACS Paragon Plus Environment

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Optimum amphiphilicity has been achieved either by varying cationic charge or side chain hydrophobicity in a variety of cationic-amphiphilic polymers. Optimum amphiphilicity of cationic-amphiphilic polymers has been considered as the governing factor for good antibacterial activity yet minimal cytotoxicity. We have recently shown that hydrogen bonding plays a key role in the membrane interactions of antibacterial polymers with bacteria48. However, the balance of hydrogen bonding and hydrophobicity in the interactions of antibacterial polymers with bacterial membranes has remained elusive. In this report, we have envisaged identifying a subtle balance between hydrophobicity and hydrogen bonding keeping constant cationic charge density in antibacterial polymers for membrane interactions. Reported antibacterial polymers nondegradable and a very few are degradable4. Toxicity due to by-products from degradation of polymers is another safety issue that has not yet been addressed. Moreover, some of the conventional antibiotics are released into the environment in their active form contributing to the development of bacterial resistance.4 However, generation of non-active degradation by-products might have fewer tendencies for the development of bacterial resistance. Also, it is important to determine the potential of antibacterial polymers in-vivo for clinical applications. Here, we demonstrate the side chain degradable poly(isobutylene-alt-N-alkyl maleimide) antibacterial polymers with tunable side chain hydrophobicity. We found that optimum side hydrophobicity in amide and ester polymers is required for potent antibacterial activity with minimal mammalian cell toxicity. Mechanistic investigations on bacterial membranes and model lipid bilayers were used to understand the molecular interactions. Degradation studies showed that amide polymers were more stable than ester polymers. Toxicity profiles of the polymers and their degradation by-products were assessed in mice models. In-vivo efficacy against Acinetobacter baumannii burn wound infections in mice models is also reported.


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MATERIALS AND METHODS Synthesis of amide and ester based alkylating agents was performed as described previously48-52. Synthesis of the polymeric derivatives was performed as described previously48-52. Chemical degradation of polymers. The hydrolysis of the EC3P/AC3P was done using 8 M HCl at 50 °C for 72 h to give the zwitterionic derivative QZwiP. Treatment of either the EC3P or QZwiP with 1 M NaOH at 50 °C for 24 h degraded the succinimide ring yielding the corresponding open ring by-product with net anionic charge. All these by-products were obtained after dialyzing against DI water at room temperature using a dialysis membrane (Mol. wt. cut off =10 KDa) followed by freeze-drying. The complete conversion from the reactant to the product was confirmed quantitatively by FT-IR. Antibacterial, hemolytic, depolarization and permeabilization studies were performed as described previously.48, 49, 52, 53 In-vivo studies in mice models.41,52 Animal studies were performed as described previously. In-vitro degradation of the polymers. The degradation study (chemical hydrolysis) was performed for both amide and ester-containing polymers in the presence of acid (0.2 M DCl in D2O), base (0.2 M NaOD in D2O) and enzyme lipase (from Candida antarctica) (5 U/mL in D2O). 500 µL of polymer solution (4 mg/mL) in the above mentioned acid, base or enzyme solution was added to a 5 mm NMR tube and proton NMR spectra were recorded immediately. Proton NMR spectra was taken by incubating the tubes at 37 °C. Degradation of these polymers will generate 1-aminopropane or 1-propanol. Thus the degree of hydrolysis at different times was calculated from the relative integrals originating from β−CH2− protons (1.5 ppm) of the


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hydrolysed product HOCH2CH2CH3 for ester containing polymer or from α−CH2− protons (2.5 ppm) of the hydrolysed product NH2CH2CH2CH3 for the amide containing polymer and from β−CH2− protons (δ 2.0 ppm) of the NCH2CH2CH2N+(Me2)− for the polymer. The appearance of the peaks at 1.48-1.51 and 3.3-3.4 ppm that correspond to the β−CH2− and α−CH2− groups of 1propanol confirmed the hydrolysis of the ester-containing polymer. On the other hand, the appearance of the peaks at 1.49-1.52 and 2.4-2.5 ppm that correspond to the β−CH2− and α−CH2− groups of 1-aminopropane confirmed the hydrolysis of the amide-containing polymer. Laurdan fluorescence and Isothermal titration calorimetry (ITC) were performed as described previously48.

RESULTS AND DISCUSSION Rational design. Polymers were prepared as per the post-functionalization approach (Scheme 1).48-52 The characterization has been provided in the supporting information. The calculation of degree of quaternization (90-95 %) and determination of molecular weight have been done as described previosuly48-52. As reported earlier48-52 the polydispersity index (PDI) is 1.2 and the range of molecular weight is 15-18 KDa. Polymers in Scheme are represented according to the functional group (amide (A) or ester (E)) in the side chain of different lengths (Cm, for number (m) of carbon (C) atoms in the alkyl chain). The hydrophobicity of the polymers is modulated by changing the hydrophobicity from hexyl to ethyl in both amide and ester polymers keeping nearly constant cationic charge density.


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Scheme 1: Synthesis of amide and ester containing cationic-amphiphilic polymers of varying hydrophobicity.

Antibacterial activity and cytotoxicity. Both the amide and ester polymers with appended butyl (AC4P and EC4P) and hexyl (AC6P and EC6P) chains were potent antibacterial agents (MIC = 4-31 µg mL-1) against both E. coli and S. aureus (Table 1). However, even at these longer alkyl


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chains, the ester polymers (EC6P and EC4P) were also hemolytic (HC50 = 30 µg mL-1 and 124 µg mL-1) but less than their amide counterparts (HC50 = 12 µg mL-1 and 83 µg mL-1). On the other hand, the amide polymers with propyl and ethyl chains (AC3P and AC2P) have MIC of 31 µg mL-1 and 62 µg mL-1 respectively, whereas their corresponding ester polymers (EC3P and EC2P) were less antibacterial (MIC = 125-500 µg mL-1) against both bacteria (Table 1). Higher alkyl chain amide polymers (C6 & C4) were found to be more toxic to human red blood cells (hRBCs) than the corresponding ester polymers (Table 1). However, all the lower alkyl chain (C3 and C2) containing amide and ester polymers had HC50 > 1000 µg mL-1 due to lower hydrophobicity. Hence, the higher alkyl chain amide and ester polymers showed low selectivity (0.4 - 4) to bacteria (Table 1). AC3P and AC2P showed more selectivity than EC3P and EC2P (Table 1). The amide polymer, AC3P with lower hydrophobicity (propyl chain) and the ester polymer, EC4P with higher hydrophobicity (butyl chain) showed similar antibacterial activity of 31 µg mL-1 against E. coli. However, the EC4P was found to be more hemolytic (HC50 = 124 µg mL-1) than AC3P (HC50 >1000 µg mL-1). These results suggest that an amide containing polymer can be potent antibacterial agent with lower hydrophobicity whereas an ester containing polymer needs relatively higher hydrophobicity to be as effective as its amide counterpart. Thus, optimization of hydrophobicity is required to tune the amphiphilicity. We have previously shown that amide polymers possess strong hydrogen bonding interactions with the bacterial lipid bilayers compared to the ester polymers48. Hence, higher hydrophobic amide polymers (AC4P & AC6P) were highly antibacterial but were more toxic to mammalian cells than their corresponding ester polymers (EC6P & EC4P). This is due to stronger hydrogen bonding interactions in amide polymers than ester polymers though both have same hydrophobic alkyl chains. Thus, at higher hydrophobicity 8 ACS Paragon Plus Environment

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Table 1. Biological activity profiles of cationic-amphiphilic polymers. Polymer

MICa (µg mL-1)

HC50b (µg mL-1)

Selectivityc

S. aureus S. aureus E. coli E. coli AC6P 31 16 12 0.4 0.8 EC6P 16 8 30 2 4 AC4P 31 4 83 3 21 EC4P 31 16 124 4 8 AC3P 31 31 >1000 >32 >32 EC3P 125 250 >1000 >8 >4 AC2P 62 62 >1000 >16 >16 EC2P >500 >500 >1000 >2 >2 a MIC, minimum inhibitory concentration in cation adjusted Mueller-Hinton broth (CAMHB); b HC50, concentration required to cause 50% hemolysis; cSelectivity, is defined as HC50/MIC.

both amide and ester polymers were potent antibacterial but more hemolytic. The differences between the amide and ester polymers were clearer at lower hydrophobicity. Hence, the lower hydrophobic amide polymer (AC3P) was potent antibacterial than the corresponding ester polymer (EC3P) due to the strong hydrogen bonding interactions. Due to lower hydrophobicity, both AC3P and EC3P were non-toxic to human erythrocytes. However, even at C2 chains, the amide polymer, AC2P was more potent in antibacterial activity than the EC2P supporting the importance of amide than the ester moieties. Amide polymers showed more selectivity of killing bacteria over mammalian cell compared to their ester counterparts and this depends on optimum hydrophobicity. With respect to the MIC values, these polymers fare well with the polymers reported in literature. The advantages our approach include ease of functionalization, simple two step, inexpensive and readily available commercial maleic anhydride copolymers. Similar class of these copolymers are Food and Drug Administration (FDA) approved for use in humans54. More importantly, the presence of degradable bonds in the side chains make them switch their antibacterial activity and toxicity as shown later in this work.


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Polymers cause cytoplasmic membrane and permeabilization. Membrane depolarization was studied using DiSC3(5) dye against E. coli and S. aureus at 25 µg mL-1. At longer appended alkyl chains (hexyl, C6) the ester polymers showed similar dissipation of membrane potential than its amide counterparts (Fig. 1A & Fig. 1B). At all the other appended alkyl chains (C2-C4), the amide polymers were always found to possess higher ability to dissipate the membrane potential than their ester counterparts. The optimum alkyl chain amide polymer, AC3P has more depolarization than EC3P that correlates with the latter’s low antibacterial activity. EC2P (MIC > 500 µg mL-1) did not have any appreciable effect on membrane potential of E. coli and S. aureus. A plot of MIC against membrane potential for E. coli as shown in Fig. S2 indicated correlation between them. Membrane permeabilization was monitored using propidium iodide against E. coli and S. aureus at 25 µg mL-1. All the amide containing polymers have greater membrane permeabilization than their ester counterparts at all the appended alkyl chain lengths (Fig. 1C & Fig. 1D). Lower alkyl chain amide and ester polymers that showed membrane depolarization were devoid of membrane permeabilization.

Mechanistic investigations with lipid bilayers Polymers affect membrane hydration. To study membrane fluidity, model lipid bilayers (liposomes) of bacteria DPPG:DPPE (88:12) and human erythrocytes DPPC were prepared by with Laurdan dye (6-Dodecanoyl-2-dimethylaminonaphthalene) encapsulated in them. Polymer interactions with the model lipid bilayers cause changes in Laurdan dye fluorescence that can be quantified by calculating general polarization (GP =(I440-I490)/(I440+I490)). Lower GP shows increased fluidity of the bilayer.55 This experiment has been performed as described previously48 and the data for AC3P and EC3P has been taken from our published paper48. Laurdan GP for the


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Figure 1. Membrane-disrupting properties of isosteric ester and amide polymers. Membrane depolarization against S. aureus (A) and E. coli (B); permeabilization against E. coli (C) and S. aureus (D). Concentration of the polymers used was 25 µg mL-1.

interaction of DPPG: DPPE (88:12) and DPPC AC6P, AC3P,EC6P, EC3P (lipid: polymer = 7.4:1) was calculated (Fig. 2A). The longer alkyl chain amide polymer, AC6P showed the lowest GP followed by similar GP for both AC3P and EC6P for DPPG:DPPE lipid system compared to the untreated system. Hence AC3P is as effective as EC6P indicating that lower hydrophobicity is sufficient for amide polymers. On the other hand, EC3P had no change on GP after treatment with DPPG:DPPE. For DPPC bilayer, only the longer alkyl chain amide polymer, AC6P but not EC6P showed low GP (Fig. 2A). This suggested that at higher hydrophobicity amide polymer (AC6P) is highly antibacterial but more cytotoxic compared to the lower hydrophobic amide polymer (AC3P) whereas EC3P was less antibacterial and non-toxic. 11 ACS Paragon Plus Environment

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Thermodynamics of membrane interactions. Isothermal titration calorimetry (ITC) was used to understand the thermodynamics of interactions of DPPG:DPPE (88:12) or DPPC model lipid bilayers with polymers (lipid:polymer = 30:1) as described by us previously48. The longer alkyl chain amide polymer, AC6P had a very fast and complete interaction (within 10 injections) with the DPPG:DPPE (88:12) (Fig. 2B). The reaction was found to be very spontaneous with ∆G = 10.96 kcal mol-1 (Gibbs free energy change) and was entropy-driven with a positive entropy change, ∆S = 63.7 cal mol-1 K-1 (positive enthalpy change, ∆H = 8.8 kcal mol-1). The shorter alkyl chain amide polymer AC3P as shown earlier48 had complete interaction with nearly the same ∆G = -11.03 kcal mol-1 but with a lower positive ∆S = 43.9 cal mol-1 K-1 (∆H = 2.57 kcal mol-1). EC6P although had ∆G = -9.3 kcal mol-1, ∆H = 8.94 kcal mol-1 and ∆S = 58.8 cal mol-1 K-1 but still did not have complete interaction by the end of 40 injections. These results suggested that AC3P had stronger interaction with DPPG:DPPE compared to EC6P due to amide functionality. EC3P as reported previously48 has weak and incomplete interactions. With DPPC polymer had negligible interactions under same conditions (Fig. S3). AMPs such as mellitin56 and PGLa57 like these polymers were reported to possess membrane interactions

through

positive entropy processes like hydrophobic effect and membrane pore formation.58,59 Hence, the higher entropy change in the interaction of higher hydrophobic AC6P compared to lower hydrophobic AC3P with DPPG:DPPE model lipid bilayer is justified. These results also explain the stronger membrane interactions and good antibacterial activity of amide polymers compared to ester polymers.


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Figure 2. Interactions of amide and ester polymers with model lipid bilayers. (A) Membrane fluidity of DPPG: DPPE (88:12) and DPPC 48; (B) Isothermal titration calorimetry (ITC) studies with DPPG:DPPE (88:12)48.


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Degradation of amide and ester polymers. The degradation properties of both the amide and ester-bearing polymers were studied under acidic, alkaline and enzymatic hydrolytic conditions using 1H NMR at 37 °C for 10 days. Amide-containing polymer (AC3P) was found to hydrolyze only under alkaline conditions whereas the ester-containing polymer (EC3P) hydrolyzed under all the three conditions (Fig. 3). Amide-group being chemically more stable did not get hydrolyzed in presence of acid even till day 10. On the other hand, hydrolysis of ester-containing polymer started at day 1 and increased gradually with time. The degree of hydrolysis of estercontaining polymer was 20-22% after day 10 in presence of acidic conditions under the experimental conditions. However, amide-containing polymer was found to hydrolyze in the presence of the base. Hydrolysis of amide-containing polymer started at day 1 and increased with time. The degree of hydrolysis of was 71-76% after day 10 in presence of base under the experimental conditions. On the other hand, ester-containing polymer degraded completely (100% hydrolysis) just after day 1 which again proves that the polymers having ester groups are more susceptible towards both acid and base treatment than the amide containing polymers. When tested the susceptibility of these polymer towards enzyme lipase (particularly known to hydrolyze ester and in some cases amide groups), the ester containing polymer was found to hydrolyze in presence of lipase whereas the amide-containing polymer remained un-hydrolyzed till day 10 under the experimental conditions. Hydrolysis of ester-containing polymer started at day 1 and increased gradually with time in presence of lipase. The degree of hydrolysis of estercontaining polymer was 25-28% after day 10 under the experimental conditions (Fig. 3). These preliminary in-vitro degradation results suggest that the amide polymers are relatively more stable than their ester counterparts under given experimental conditions that is required for their long lasting antibacterial activity but certainly need further investigations in detail.


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Figure 3. In-vitro degradation profiles of isosteric amide and ester polymers. Rate of hydrolysis of amide and ester-containing polymers in presence of acidic, basic and enzymatic conditions. % of hydrolysis of AC3P and EC3P in presence of 0.2 M DCl in D2O (A), 0.2 M NaOD in D2O (B) and 5 U/mL of lipase (C).

In-vivo toxicity of polymers and their degradation by-products. AC3P and EC3P resulted in intravenous LD50 values of 20 mg kg-1 and 37 mg kg-1 respectively (Table 2). This compares favorably with clinically approved antibiotics such as polymixins, which work by a comparable cell-lytic mechanism as AMPs, and have lower LD50 levels of approximately 8-10 mg kg-1 with reported neuro- and nephro-toxicity.60

Also, AC3P had LD50 more than17.5 mg kg-1

(intraperitoneal (i.p.)) and 55 mg kg-1 (subcutaneous (s.c.)) respectively.

We have also

chemically synthesized the possible polymeric degradation by-products (Fig. 4). These degradation by-products have zwitterionic, anionic and neutral character unlike their parent cationic polymers. Hence, the cationic polymers were more toxic to human erythrocytes compared to their degradation by-products (Table 2). We investigated the in-vivo systemic toxicity of the polymeric by-products ZwiP and OprP of AC3P or EC3P and found that they did not induce any mortality in mice up to 400 mg kg-1, the highest tested dose whereas their parent cationic polymers had LD50 in the rage of 20-37 mg/kg in mice (Table 2). The molecular weight of cationic polymers and negative or zwitterionic polymeric by- products was in the range of 620 KDa, which is below the limit for

clearance through kidneys (< 50 KDa).61 More

importantly, the degradation-by products were found to devoid of antibacterial activity even up 15 ACS Paragon Plus Environment

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Figure 4. Synthesis of degradation by-products of the polymers.

Table 2. Antibacterial activity and toxicity of polymers and their degradation by-products. MICa (µg mL-1) HC50b LD50c -1 (µg mL ) (mg/kg) E. coli S. aureus AC3P (Cationic) 31 31 >1000 20 EC3P (Cationic) 125 250 > 1000 37 QZwiP (Zwitterionic) > 1000 > 1000 > 40000 > 400 QOprP (Anionic) > 1000 > 1000 > 40000 > 400 PIBMA (Neutral) > 1000 > 1000 > 40000 > 400 a MIC, minimum inhibitory concentration in cation adjusted Mueller-Hinton broth (CAMHB); b HC50, concentration required to cause 50% hemolysis; cLD50 (lethal dose required to kill 50% of mice). Polymer


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to 1000 µg mL-1 (Table 2). Some of the conventional antibiotics are released into the environment in their active form contributing to the development of bacterial resistance.4 However, these non-active degradation by-products might have fewer tendencies for the development of bacterial resistance. These results indicate the good safety profiles of the polymers for use in-vivo. The effect of amide and ester replacement has also been found to play a role in restoring the efficacy of antibiotics against drug-resistant Gram-negative bacteria.53 Enhancing the efficacy of antibiotics is a promising approach to target antibiotic resistant bacteria.62 We have reported that AC3P can restore the efficacy of tetracycline antibiotics at lower concentrations than EC3P towards Gram-negative clinical isolates.53

In-vivo activity. The in-vitro antibacterial activity of AC3P against A. baumannii has been found to be 4 µg mL-1 as reported previously52. The in-vivo antibacterial efficacy studies of these polymer antibiotics were studied in a mouse burn wound model of A. baumannii.63 Burn wounds were infected with 107 cfu of A. baumannii (Fig. 5). Mice were treated with topical application of 40 µL of AC3P (50 mg kg-1), minocycline (50 mg kg-1) and AC3P + minocycline (50 mg kg1

+ 50 mg kg-1) every 24 h for 5 days. After six days of infection, untreated mice had an increased

bacterial burden of 10 log(cfu/g). Mice treated with AC3P showed reduction of 3 log(cfu/g) of bacterial burden. Mice receiving minocycline, a known bacteriostatic antibiotic, showed 3 log(cfu/g) reduction in bacterial burden. However, mice that had received AC3P + minocycline had a high reduction of 6 log(cfu/g). These results prove the potential of these polymers alone or in combination with antibiotics for the topical treatment of bacterial infections.


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Figure 5. In-vivo antibacterial efficacy. In-vivo A. baumannii burn wound infection. Mice were treated with Minocycline (Mino, 50 mg kg-1), AC3P (50 mg kg-1) and AC3P + Mino (50 mg kg-1 + 50 mg kg-1) . The data are expressed as mean ± standard deviation based on values obtained from four mice (n = 4). P value was calculated using Student’s t test (2 tailed one sample, unpaired distribution).

Overall, these findings suggest that the subtle balance of hydrophobicity and hydrogen bonding interactions can be used to drive the selectivity of cationic-amphiphilic antibacterial polymers to selectively kill bacteria sparing the mammalian cells. Till now in literature4, the amphiphilicity (hydrophobic/hydrophilic balance) has been optimized that account for the electrostatic and hydrophobic interactions of antibacterial polymers with the bacterial and mammalian cell membranes. The numerous ways achieve this include the optimization using macromolecular architectures (polymeric sequence or backbone), alkyl spacers and tails as well as cycles and fused cycles4. To date, the key structural parameters exploited in designing highly selective cationic antimicrobial polymers are: amphiphilicity (hydrophilic/hydrophobic balance) and backbone structure/sequence (random, block and alternating copolymers or homo polymers)4,

5, 64

. Amphiphilicity has been optimized by different approaches such as the

“segregated monomer approach”, the “facially amphiphilic approach” and the “same centered approach” in literature. It has been shown that a polymer synthesized using the “segregated 18 ACS Paragon Plus Environment

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monomer approach” was highly hemolytic compared to that of the “same centered approach” having the same alkyl chain length13, 64. When it comes to the polymeric backbone structure, Sampson and co-workers have demonstrated that random copolymers were found to be 2-6 fold less active than alternating copolymers towards different bacteria18. Kuroda and co-workers have shown that the random copolymers are more hemolytic compared to block copolymers but both have similar antibacterial activity profiles7. Yang and co-workers reported that self-assembling nanoparticles of amphiphilic polycarbonate based triblock copolymers were active towards Gram-positive bacteria but were ineffective towards Gram-negative bacteria64. This could be due to the fact that the hydrophobic region of these nanoparticles buried inside their core could not interact with the outermost lipopolysaccharide (LPS) layer of the Gram-negative bacteria. Overall, these reports suggest the importance of structural strategies like the “same centered approach” and an ordered backbone conformation such as alternating/block copolymer structure to achieve optimum selectivity towards bacteria over mammalian cells compared to random copolymers. The others parameters include studying the effect of molecular weight and ionic/polar groups (ammonium, phosphonium, sulfonium, cationic metals, uncharged polar or neutral groups, charge density and position, and counter ions) for improving the selectivity to bacteria over mammalian cells4. Polymethacrylates with ammonium moieties might possess hydrogen bonding interactions with the phosphate lipid head groups7. Also, a variety of antimicrobial polymers and AMPs might possess hydrogen bonding interactions with the bacterial membranes. Literature reports of both antimicrobial polymers and AMPs highlight electrostatic and hydrophobic interactions with the bacterial membranes but the role or importance of hydrogen bonding in this regard has not been studied or reported before1-6. However, this report for the first time shows the balance between hydrophobic and hydrogen


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bonding interactions with constant cationic charge density for the optimum interactions with bacterial and mammalian cell membranes. Our approach has the advantage of the involvement of three major non-covalent interactions such as electrostatic, hydrophobic and hydrogen bonding interactions with the bacterial and mammalian cell membranes. More importantly, these polymers showed good in-vivo antibacterial activity in combination with antibiotics for topical treatment of Gram-negative infections. Also, the need for degradable antimicrobials is important for ecology and to evade bacterial resistance development. Hence, our side-chain degradable cationic-amphiphilic antibacterial polymers will be able to address such issues of long-term toxicity and bacterial resistance.

CONCLUSIONS In conclusion, we showed the importance of delicate balance between hydrophobicity and hydrogen bonding in bacterial membrane interactions using polymers containing amide and ester moieties in their side chains. Till now in literature, cationic charge and hydrophobicity have been optimized to obtain selective antibacterial polymers. The present work demonstrated the concept of interplay between the hydrogen bonding and hydrophobic interactions in amphiphilic polymers. We believe that this understanding will guide the future design of selective membraneactive molecules.

Moreover, the polymers were less toxic and their degradation by-products

were non-toxic in mice. More importantly, anti-infective studies and good safety profiles of polymers in animal models support their potential for possible in-vivo applications.


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ASSOCIATED CONTENT Supporting Information. Synthesis, characterization and ITC figures of DPPC bilayers are provided in supporting information (PDF). This material is available free of charge via the Internet at http://pubs.acs.org.

This material is available free of charge via the Internet at http://pubs.acs.org.

Corresponding Author *E-mail: [email protected]; Fax: +91-80-2208-2627; Telephone: +91-80-2208-2565.

ACKNOWLEDGEMENTS We thank Prof. C.N.R. Rao, FRS (JNCASR) for his constant support and encouragement.

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Side Chain Degradable CationicâAmphiphilic Polymers with Tunable

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