This journal is c The Royal Society of Chemistry 2013 Chem. Commun.

Cite this: DOI: 10.1039/c3cc43751e

Polymers with tunable side-chain amphiphilicity asnon-hemolytic antibacterial agents†

Divakara S. S. M. Uppu, Padma Akkapeddi, Goutham B. Manjunath,Venkateswarlu Yarlagadda, Jiaul Hoque and Jayanta Haldar*

Quaternized polymers mimicking the antimicrobial peptides were

created by tuning the side-chain amphiphilicity using a first-time

approach of post-functionalization. They displayed excellent efficacy

against pathogenic bacteria even in human plasma and membrane

disruptive mode of action. The optimized polymers and degraded

products were non-hemolytic.

The ongoing explosion of resistant superbugs coupled with thediminishing antibiotic pipeline creates an urgent need for thedevelopment of new antimicrobial agents which exhibit novelmechanisms of action.1 Antimicrobial peptides (AMPs) have gainedincreased attention to address such an unmet need. The develop-ment of synthetic mimics by incorporating cationic charge andamphiphilicity that can overcome the limitations of AMPs has beenthe Holy Grail of the field.2 Over the years, a great deal of effort hasgone into the design and development of synthetic mimics ofAMPs, including oligomers,3 polymers,4 and cationic amphiphiles,5

etc. Despite the development of a plethora of cationic and amphi-philic antimicrobial polymers, most of them still face seriouschallenges including the use of tedious and expensive syntheticmethodologies, high toxicity to mammalian cells (for e.g. hemo-lysis) that leads to low selectivity towards bacteria over mammaliancells. Also, achieving antibacterial efficacy under human serum/plasma conditions is still an unsolved issue.

Herein, we report the design and development of a novel seriesof synthetic cationic and amphiphilic polymers as mimics of AMPsbased on quaternized poly(isobutylene-alt-N-(N0,N0-dimethyl N0-alkylaminopropyl)-maleimide) (QAlk_PIBMI) derivatives starting from acheap and commercially available synthetic polymeric precursorpoly(isobutylene-alt-maleic anhydride) (PIBMA, average Mw B6000 g mol�1). Most of the cationic and amphiphilic antimicrobial

polymers reported in the literature are synthesized in a mannerwhere the entire polymeric backbone needs to be constructedresulting in tedious and expensive methodologies. Whereas, wedescribe an approach for post-functionalization of a syntheticpolymer wherein the amphiphilicity was optimized by merelyvarying the chemical nature of the side chain alkylating agent usedto quaternize the polymer (Scheme 1). Simple functionalization ofthe polymer backbone based on the highly reactive anhydride ringwith 3-aminopropyldimethylamine followed by quaternization withdifferent alkylating agents gave the corresponding water solubleQAlk_PIBMI derivatives in a simple two-step process with nearlysimilar degree of quaternization of 93–98% (Scheme 1, Fig. S1 andTable S1. See ESI† for experimental details and characterization).

The need for quaternization with different alkylating agents camefrom the observation that the protonated derivative, QPro_PIBMI, didnot have optimum antibacterial efficacy (minimum inhibitory concen-tration, MIC of 105 and 91 mg mL�1 against E. coli and S. aureusrespectively) (Fig. S1, ESI† data not shown). To increase the hydro-phobicity, with alternating sequence of isobutylene and N-alkylmale-imide as the backbone structure, we varied the amphiphilicity of thederivatives by quaternization with hydrophobic alkyl chain lengthsranging from ethyl (C2) to decyl (C10) (Fig. S1, ESI†). The MIC values ofC2–C10 derivatives against E. coli and S. aureus varied from >1000 to3–20 mg mL�1 (from C2–C8) and again increased to 100–120 mg mL�1

(for C10) (Fig. S4, ESI†). Overall, a parabolic relationship was observedbetween the antibacterial activities of these C2–C10 derivatives (againstE. coli and S. aureus) and their hydrophobic chain lengths (Fig. S4,ESI†). Also, their toxicity towards human erythrocytes increased (con-centration at which 50% hemolysis occurs, HC50 values decreasedfrom >1000 to 4 mg mL�1) as the hydrophobicity increased fromC2–C10 (Fig. S4, ESI†). Apart from the hydrophobicity that affected theoverall amphiphilicity of these C2–C10 derivatives, their molecularweight varied in the range of 16 000–20 000 g mol�1 which might beanother factor that affected the antibacterial activity and hemolysis(Table S1, ESI†). Amongst all, QPen_PIBMI (C5) with optimumamphiphilicity showed high selectivity towards bacteria over humanerythrocytes (selectivity = HC50/MIC) compared to the antimicrobialpeptide MSI-78,3a,6 which is in phase-III clinical trials as a topical anti-bacterial agent (Table 1). QPen_PIBMI demonstrated a selectivity of

Bioorganic and Medicinal Chemistry Laboratory, New Chemistry Unit,

Jawaharlal Nehru Centre for Advanced Scientific Research, Jakkur, Bengaluru,

560064, India. E-mail: jayanta@jncasr.ac.in; Fax: +91-80-2208-2627;

Tel: +91-80-2208-2565

† Electronic supplementary information (ESI) available: Experimental details ofsynthesis, characterization and degree of quaternization of the derivatives, bio-assays of the polymeric derivatives and supplementary figures. See DOI: 10.1039/c3cc43751e

Received 18th May 2013,Accepted 28th June 2013

DOI: 10.1039/c3cc43751e

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114 whereas MSI-78 had a maximum selectivity of 15 against S. aureus(Table 1). QPen_PIBMI also showed comparable or better selectivityin the case of E. faecium (43), E. coli (25), K. pneumoniae (6) andP. aeruginosa (4) compared to the AMP (Table 1).

For further optimization of selectivity, we varied the amphiphili-city by quaternizing with alkylating agents containing hydrophilicmoieties like ester (QEst_PIBMI), amide (QAmi_PIBMI) and ether(QOEG_PIBMI) moieties by keeping the chain length almost constant(Scheme 1). Apart from this, the goal is to increase biocompatibilityand to impart biodegradability into the polymers. Interestingly,these three derivatives did not show 50% hemolysis even up to1000 mg mL�1, the highest tested concentration (i.e. HC50 =>1000 mg mL�1) (Table 1). Among these, QAmi_PIBMI had the highestselectivity of more than 91, 67 and 45 against S. aureus, E. faecium andE. coli respectively compared to MSI-78 and its alkyl chain counterparts QHex_PIBMI (C6) and QPen_PIBMI (C5) (Table 1). QEst_PIBMIalso had the selectivity of more than 15 and 91 towards E. coli andS. aureus compared to its alkyl chain counterparts QHex_PIBMI (C6)and QPen_PIBMI (C5) as well as the AMP (Table 1). Interestingly, thederivative quaternized with the highly hydrophilic alkylating agentOEG (oligoethyleneglycol), QOEG_PIBMI, showed ‘‘double selectivity’’towards S. aureus over other bacteria and human erythrocytes.

These three derivatives also showed comparable or better selectivitytowards K. pneumoniae and P. aeruginosa as well (Table 1).

QHex_PIBMI, QPen_PIBMI and QAmi_PIBMI displayed excellentMICs of 4, 4, and 6 mg mL�1 respectively against vancomycin resistantEnterococcus faecium (VRE), whereas vancomycin was found to beineffective up to the highest tested concentration of 156 mg mL�1

(Table 1 and Fig. S5, ESI†). In literature reports, however it has beenshown that vancomycin is inactive against VRE even at concentra-tions above 1000 mg mL�1.7 Qpen_PIBMI and QAmi_PIBMI had thehighest selectivity of 86 and >166 against VRE, whereas it has beenshown in the literature that MSI-78 has a selectivity of only 15(Table 1).3a,6 The derivatives QHex_PIBMI, QPen_PIBMI andQAmi_PIBMI had MICs of 113, 104, 224 mg mL�1 respectively againstMRSA, whereas QEst_PIBMI and QOEG_PIBMI were found to beineffective up to 1000 mg mL�1 (Table 1). This 5–50 fold increase inMICs of QAlk_PIBMIs against MRSA might be due to that fact thatS. aureus has been shown to have modified its cell surface andanionic membranes by increasing the net positive charge resulting inthe repulsion of AMPs.2b However, detailed structural investigationsabout the bacteria are required to fully understand the variation inactivity among different bacterial strains. Additionally, QAmi_PIBMIshowed a rapid 5 log10 of reduction of bacteria within 2 h and 1 h at a

Scheme 1 Design and synthesis of QAlk_PIBMI derivatives.

Table 1 In vitro antibacterial and haemolytic activities and selectivities of the QAlk_PIBMI derivatives

Polymer MIC (mg mL�1) HC50 (mg mL�1)

E. coil K. pneumoniae P. aeruginosa S. aureus E. faecium MRSAa VREb

QHex_PIBMI 7 (4.3) 36 (0.8) 62 (0.5) 20 (1.5) 4 (7.5) 113 (0.3) 4 (7.5) 30QPen_PIBMI 14 (24.5) 57 (6) 94 (4) 3 (114) 8 (43) 104 (3.3) 4 (86) 343QAmi_PIBMI 22 (>45) 59 (>17) 101 (>10) 11 (>91) 15 (>67) 224 (>4.5) 6 (>166) >1000QEst_PIBMI 65 (>15.4) 46 (>22) 208 (>4.8) 11 (>91) 510 (>2) >1000 123 (>8) >1000QOEG_PIBMI >1000 150 (>6.6) >1000 20 (>50) >1000 >1000 >1000 >1000MSI-78 16–32c (4–8) 8–16c (8–15) 8–16c (8–15) 8–16c (8–15) 64e,c (2) 16–32c (4–8) 8c (15) 120d

a Methicillin resistant Staphylococcus aureus. b Vancomycin resistant Enterococcus faecium. c Literature values.3a,6 d Literature values.3a e Literaturevalues for E. faecalis,6 selectivity (HC50/MIC) is given in parentheses.

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concentration of 6 and 12 times the MIC respectively whereas at MICit rather showed bacteriostatic activity (Fig. S6, ESI†). So, by merelyvarying the side chain amphiphilicity, the overall amphiphilicity ofthe polymeric mimics of AMPs is varied that resulted in thederivatives with maximum selective toxicity towards bacteria overhuman erythrocytes. Supporting our hypothesis, these observa-tions suggest the role of optimization of amphiphilicity in selec-tive toxicity to bacteria.

One of the major disadvantages of natural AMPs is the loss ofantibacterial efficacy because of their susceptibility to proteases inblood plasma. For the first time in the field of polymeric mimics ofAMPs, to assess the compound’s stability, we performed MIC experi-ment of QAmi-PIBMI against S. aureus as a model bacterium in 50%of human plasma. The MIC of QAmi_PIBMI remained constant evenin the presence of 50% of human plasma (12 � 1 mg mL�1) than inthe absence of the plasma (100% media, 11� 1 mg mL�1) (Fig. 1(A)).It has been reported in the literature that the MIC of MSI-78 againstS. aureus increases to two fold in 50% of human serum itself than inthe absence of serum (100% media).6 We have also performed theexperiment with pre-incubation of the derivatives in 50% of humanplasma for 3 h. The MIC values were nearly unchanged even after 3 hof preincubation in human plasma (20� 5 mg mL�1) (Fig. 1(A)). Thissuggests that these synthetic macromolecular mimics can be used assystemic therapeutics as they resist the plasma proteases and otherplasma components that reduce the efficacy of AMPs.

Toxicity caused by the degradation by-products of the anti-microbial polymers is another issue that needs to be addressed toassess their safety profile as antimicrobial drugs. As a preliminarystudy, we investigated the toxicity of the chemically degraded(pH) polymeric by-products of the derivatives QPen_PIBMI,

QESt_PIBMI/QAmi_PIBMI against human erythrocytes (Fig. S2and Scheme S2, ESI†). All the polymeric by-products were foundto be significantly non-hemolytic up to 40 000 mg mL�1, thehighest tested concentration (Fig. S7, ESI†). Moreover, the mole-cular weight of all the derivatives and the polymeric by-products isin the range of 6000–20 000 g mol�1 (Table S1, ESI†), which ismuch below the threshold for renal clearance (o50 000 g mol�1).8

Membrane permeabilization/disruption causing loss of mem-brane integrity of the bacteria is the main mechanism of action ofAMPs. QAmi_PIBMI at 50 mg mL�1 showed cytoplasmic membranedepolarization and permeabilization when tested against bothE. coli and S. aureus (Fig. S8, ESI†). Field emission scanning electronmicroscopy (FESEM) images of both S. aureus and E. coli treatedwith QAmi_PIBMI (6 � MIC) for 2 h (Fig. 1(B)) give visual insightsinto the morphological membrane disruption. The untreatedbacteria showed intact morphology whereas the irregularly shapedmorphological membrane was disrupted and thus probably thedead bacteria were found upon treatment with QAmi_PIBMI(Fig. 1(B)). The cationic polymers therefore possibly interactwith the mostly negatively charged bacterial cell membrane anddisrupt the membrane, which might lead to cell death.5

In conclusion, these mimics, unlike AMPs, can be produced atvery low cost, are stable in the presence of human plasma and arehighly toxic to pathogenic bacteria including the multi-drug resistantbacteria like VRE but less or non-toxic to human erythrocytes. Thisstructure–activity relationship, demonstrated by varying the chemicalnature of the alkyl side chains appended to quaternized polymers,emphasizes the role of optimum amphiphilicity in developing non-hemolytic yet potent membrane active antibacterial agents.

We thank Prof. C. N. R. Rao for his constant support andencouragement. Haldar acknowledges the Department ofScience and Technology (DST), Govt. of India, for Ramanujanfellowship [SR/S2/RJN-43/2009]. Uppu thanks UGC for JuniorResearch Fellowship (JRF).

Notes and references1 G. Taubes, Science, 2008, 321, 356.2 (a) M. Zasloff, Nature, 2002, 415, 389; (b) K. A. Brogden, Nat. Rev.

Microbiol., 2005, 3, 238; (c) R. E. W. Hancock and H. G. Sahl, Nat.Biotechnol., 2006, 24, 1551.

3 (a) D. Liu, S. Choi, B. Chen, R. J. Doerksen, D. J. Clements, J. D. Winkler,M. L. Klein and W. F. DeGrado, Angew. Chem., Int. Ed., 2004, 43, 1158;(b) I. S. Radzishevsky, S. Rotem, D. Bourdetsky, S. Navon-Venezia, Y. Carmeliand A. Mor, Nat. Biotechnol., 2007, 25, 657; (c) L. Motiei, S. Rahimipour, D. A.Thayer, C. H. Wong and M. R. Ghadiri, Chem. Commun., 2009, 3693;(d) S. Padhee, Y. Hu, Y. Niu, G. Bai, H. Wu, F. Costanza, L. West,L. Harrington, L. N. Shaw, C. Cao and J. Cai, Chem. Commun., 2011, 47, 9729.

4 (a) K. Kuroda and W. F. DeGrado, J. Am. Chem. Soc., 2005, 127, 4128;(b) J. Haldar, D. An, L. Alvarez de Cienfuegos, J. Chen and A. M. Klibanov,Proc. Natl. Acad. Sci. U. S. A., 2006, 103, 17667; (c) V. Sambhy, B. R. Petersonand A. Sen, Angew. Chem., Int. Ed., 2008, 47, 1250; (d) K. Lienkamp,A. E. Madkour, A. Musante, C. F. Nelson, K. Nusslein and G. N. Tew, J. Am.Chem. Soc., 2008, 130, 9836; (e) F. Nederberg, Y. Zhang, J. P. Tan, K. Xu,H. Wang, C. Yang, S. Gao, X. D. Guo, K. Fukushima, L. Li, J. L. Hedrickand Y. Y. Yang, Nat. Chem., 2011, 3, 409.

5 J. Hoque, P. Akkapeddi, V. Yarlagadda, D. S. Uppu, P. Kumar andJ. Haldar, Langmuir, 2012, 28, 12225.

6 Y. Ge, D. L. MacDonald, K. J. Holroyd, C. Thornsberry, H. Wexler andM. Zasloff, Antimicrob. Agents Chemother., 1999, 43, 782.

7 G. A. McKay, S. Beaulieu, F. F. Arhin, A. Belley, I. Sarmiento, T. Parr,Jr. and G. Moeck, J. Antimicrob. Chemother., 2009, 63, 1191.

8 H. S. Choi, W. Liu, P. Misra, E. Tanaka, J. P. Zimmer, B. Itty Ipe,M. G. Bawendi and J. V. Frangioni, Nat. Biotechnol., 2007, 25, 1165.

Fig. 1 (A) Antibacterial efficacy in 50% of human plasma (the numbers representthe MIC values) of QAmi_PIBMI against S. aureus and (B) FESEM images showingthe morphological disruption of the bacteria by QAmi_PIBMI. Scale bar is 1 mm(S. aureus) and 2 mm (E. coli).

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