Chitosan Derivatives Active against Multidrug-Resistant Bacteria and

Subscriber access provided by CORNELL UNIVERSITY LIBRARY

Article

Chitosan Derivatives Active against Multi-Drug-Resistant Bacteria and Pathogenic Fungi: In Vivo Evaluation as Topical Antimicrobials Jiaul Hoque, Utsarga Adhikary, Vikas Yadav, Sandip Samaddar, Mohini Mohan Konai, Relekar Gnaneshwar Prakash, Krishnamoorthy Paramanandham, Bibek R. Shome, Kaustuv Sanyal, and Jayanta Haldar Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.6b00764 • Publication Date (Web): 02 Sep 2016 Downloaded from http://pubs.acs.org on September 3, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Molecular Pharmaceutics is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 36

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Molecular Pharmaceutics

Chitosan

Derivatives

Active

against

Multi-Drug-Resistant

Bacteria and Pathogenic Fungi: In Vivo Evaluation as Topical Antimicrobials

Jiaul Hoque†, Utsarga Adhikary†, Vikas Yadav‡, Sandip Samaddar†, Mohini Mohan Konai†, Relekar Gnaneshwar Prakash†, Krishnamoorthy Paramanandham§, Bibek R. Shome§, Kaustuv Sanyal‡ and Jayanta Haldar†*

†

Chemical Biology and Medicinal Chemistry Laboratory, New Chemistry Unit, Jawaharlal

Nehru Centre for Advanced Scientific Research, Jakkur, Bengaluru 560064, India E-mail: [email protected]

‡

Molecular Mycology Laboratory, Molecular Biology and Genetics Unit, Jawaharlal Nehru

Centre for Advanced Scientific Research, Jakkur, Bengaluru 560064, India §

National Institute of Veterinary Epidemiology and Disease Informatics (NIVEDI)

Ramagondanahalli, Yelahanka, Bengaluru 560064, India

ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abstract: The continuous rise of antimicrobial resistance and the dearth of new antibiotics in the clinical pipeline raise an urgent call for the development of potent antimicrobial agents. Cationic chitosan derivatives, N-(2-hydroxypropyl)-3-trimethylammonium chitosan chlorides (HTCC), have been widely studied as potent antibacterial agents. However, their systemic structure-activity relationship, activity towards drug-resistant bacteria and fungi and mode of action are very rare. Moreover, toxicity and efficacy of these polymers under in vivo conditions are yet to be established. Herein, we investigated antibacterial and antifungal efficacies of the HTCC polymers against multi-drug resistant bacteria including clinical isolates and pathogenic fungi, studied their mechanism of action and evaluated cytotoxic and microbial activities in vitro and in vivo. The polymers were found to be active against both bacteria and fungi (MIC = 125-250 µg/mL) and displayed rapid microbicidal kinetics, killing pathogens within 60-120 min. Moreover, the polymers were shown to target both bacterial and fungal cell membrane leading to membrane disruption and found to be effective in hindering bacterial resistance development. Importantly, very low toxicity toward human erythrocytes (HC50 = >10000 µg/mL) and embryo kidney cells were observed for the cationic polymers in-vitro. Further, no inflammation towards skin tissue was observed in vivo for the most active polymer even at 200 mg/kg when applied on the mice skin. In a murine model of superficial skin infection, the polymer showed significant reduction of methicillin-resistant Staphylococcus aureus (MRSA) burden (3.2 log MRSA reduction at 100 mg/kg) with no to minimal inflammation. Taken together, these selectively active polymers show promise to be used as potent antimicrobial agents in topical and other infections.

Keywords: Antimicrobial polymer, cationic chitosan derivatives, drug-sensitive and drugresistant bacteria, pathogenic fungi, topical infection, antimicrobial resistance


Page 2 of 36

Page 3 of 36

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1. INTRODUCTION The persistent threat to human health posed by resistance development in bacteria has been further augmented by the steady decline in the approval of new antibiotics.1, 2 Rising levels of multi-drug resistance is another serious hazard to public health and is a primary hurdle for the introduction of new antibiotics in the clinical settings.3,

4

In addition, opportunistic fungal

infections are becoming a major problem globally.5 The high mortality rate currently associated with fungal infections is a cause of concern and reflects the low efficacy and multiple side effects of current antifungal drugs.5, 6 Additionally, emerging fungal populations resistant to conventional antifungal therapy are especially alarming and demand immediate attention.7,

8

Identifying agents that specifically inhibit fungal growth without deleterious

effects is challenging because both fungi and humans are eukaryotes. Nevertheless, developing agents that are selectively active towards fungal populations would be advantageous.9, 10 Of even greater therapeutic applicability would be molecules that possess potent activity against both bacterial and fungal populations in general while sparing mammalian cells. The aim of the study was therefore to evaluate the potential of the HTCC polymers as broad spectrum antimicrobial agents that can be used for healthcare such as topical applications. Naturally occurring cationic antimicrobial peptides (AMPs) have drawn attention due to their broad-spectrum of activity and ability to combat multi-drug-resistant microbes.11, 12 However, high cost of manufacture, low in-vivo stability and high toxicity of AMPs have reduced their use as alternatives to antibiotics.13,

14

In order to overcome the limitation

associated with AMPs, synthetic mimics of AMPs (SMAMPs) have been put forward to curtail infections caused by multi-drug-resistant pathogens. Among various SMAMPs, cationic antimicrobial polymers are well studied due to ease in chemical tailoring and broadspectrum activity.15-23 In recent years chitosan, a derivative of naturally occurring polymer



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

chitin, has attracted special attention because of its excellent biocompatibility, biodegradability and its inherent antibacterial activity.24-29 However, insolubility in aqueous solvent and low antibacterial activity limits its use as an effective antimicrobial agent. To increase the solubility and enhance the activity, various cationic chitosan derivatives have been developed as effective antimicrobials.30-32 To this end, N-(2-hydroxypropyl)-3trimethylammonium chitosan chloride (HTCC) is one of the efficient polymers in terms of ease in synthesis and potent microbicidal properties and is therefore considered as an excellent material for application in various fields such as in food, medical, textile and papermaking industries as potent antimicrobial agent.33-36 However, despite the evidence of antibacterial activity there are no comprehensive reports on antimicrobial activity of HTCC polymers. Ko, S.-K. et al. have shown that the antibacterial activity of the polyacrylonitrile (PAN) fiber can be achieved by adding only a small amount of cationic HTCC (degree of quaternization, DQ = 92%).37 Jang, J. et al. have shown that HTCC (DQ = 96%) has a lower minimum inhibition concentration (MIC) against bacteria compared to that of chitosan and demonstrated its use as an antibacterial agent in cotton fabrics.38 Polyelectrolyte multilayer microcapsules were developed using HTCC and hyaluronic acid and the antibacterial activity of the capsules was studied.39 To the best of our knowledge, no systematic activity and toxicology profiles of these polymers are reported. Moreover, though the antimicrobial activity of these polymers has been reported against bacteria, any exploration into their potency towards fungal infections has been left unattempted. This is of particular relevance considering the fact that the fungal proliferation has been shown to induce an increased frequency or severity of diseases upon association with bacterial infections.40 To this end, an investigation into the potency of these quaternary chitosan polymers against both bacteria and fungi is therefore deemed important. An exploration of this kind will provide key insights to a number of unanswered questions


Page 4 of 36

Page 5 of 36

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


pertaining to HTCC polymers such as their in-vitro and in-vivo efficacy against pathogenic bacteria and fungi, their mode of action, ability to delay the onset of microbial resistance and selective potency to microbial cells over mammalian cells both in-vitro and in-vivo. These inquiries will lead to collective knowledge crucial to the translation of these polymers into biomedical applications. Herein, we investigated the antimicrobial efficacy of the HTCC polymers against drug-resistant bacteria and pathogenic fungi. Further, these polymers have also been assessed against clinically isolated multi drug-resistant bacterial strains. We also determined the rate at which these polymers killed bacteria and fungi, looked into the mechanism of action and studied the proclivity of the microbes to grow resistant towards these polymers. The selectivity of the polymers was assessed by comparison of their antimicrobial and cytotoxic activity. For in vivo assessment of the safety profile of these polymers, toxicity studies aimed at evaluating the lethal dose and tissue inflammation of the lead polymer administered superficially were performed in mice. Finally, a superficial murine model of skin infection was used to evaluate the potential of these polymers as topical anti-infective agents. 2. EXPERIMENTAL SECTION 2.1. Materials and Instruments. Chitosan with molecular weight 15 kDa and a degree of acetylation (DA) of 0.15 was purchased from Polysciences, USA. Chitosan with molecular weight 50-190 kDa and DA of 0.21, glycidyltrimethyl ammonium chloride (GTMAC) and silver nitrate (AgNO3) were purchased from Sigma-Aldrich, USA. Glacial acetic acid (AcOH), acetone and all other solvents were purchased from SD Fine, India and were of analytical grade. The water used in all experiments was Millipore water with a resistivity of 18.2 MΩ cm. Bacterial strains Staphylococcus aureus (MTCC 737), Escherichia coli (MTCC 443) and Acinetobacter baumannii (MTCC 1425) were purchased from MTCC (Chandigarh, India). Vancomycin-resistant Enterococcus faecium (VRE) (ATCC 51559), beta-lactam



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

resistant Klebsiella pneumoniae (ATCC 700603), methicillin-resistant Staphylococcus aureus (MRSA) (ATCC 33591) were obtained from ATCC (Rockville, MD). All the clinical isolates were obtained from National Institute of Mental Health and Neuro Sciences (NIMHANS), India. The fungal strains are Candida albicans (SC5314), Candida dubliniensis (CD36), Candida tropicalis (MYA3404), Cryptococcus neoformans var. grubii (serotype A) (H99), Cryptococcus gattii (serotype B) (WM276) and Cryptococcus neoformans var. neoformans (serotype D) (JEC21). Nuclear magnetic resonance spectra (1H-NMR, 400 MHz) spectra were recorded on a Bruker AMX-400 instrument. 13C NMR was recorded by Bruker Avance III HD (AscendTM 400) solid state NMR instrument using 13C cross-polarization magic-angle spinning (CP-MAS) with 4 mm MAS probe at 100 MHz. FT-IR spectra of the chitosan derivatives were recorded on Bruker IFS66 V/s spectrometer using KBr pellets. Centrifugation was performed by Eppendorf 5810R centrifuge. TECAN (Infinite series, M200 pro) Plate Reader performed optical density and fluorescence measurements. Bacterial and fungal growth media and agar were obtained from HIMEDIA, India. Studies on animals were carried out in accordance with protocols provided by the Institutional Animal Ethics Committee (IAEC) at the Jawaharlal Nehru Centre for Advanced Scientific Research (JNCASR). 2.2. Synthesis of polymers. The quaternary chitosan derivatives were synthesized according to earlier reports with slight modifications.39 A total of six different polymers with different degrees of quaternization and molecular weights were synthesized (Scheme 1). See Supporting Information for the synthesis of polymers. HTCC 1: FT-IR (ATR): ν = 3450-3100 cm-1 (−OH and −NH2 or −NH−),1686 cm−1 (amide I, C=O str.), 1559 cm−1 (amide II, NH ben.), 1477 cm−1 (−N+(CH3)3 ben.); 1H NMR (400 MHz, CDCl3, 25 ˚C): δ = 1.908 (s, −CH3COO−), 2.068 (s, −CH3CO−), 2.568-2.939 (m, −NHCH2CH(OH)CH2− and Cell C2H), 3.227 (s, −CH(OH)CH2N+(CH3)3), 3.408-3.993 (m,


Page 6 of 36

Page 7 of 36

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Cell C3H-C5H, −CH(OH)CH2N+(CH3)3), 4.290 (s, −CH2CH(OH)CH2−), 4.542 (s, Cell C1H); 13C NMR (CP-MAS, 100 MHz): δ = 24.161, 55.297, 557.574, 61.925, 63.812, 64.170, 64.917, 75.225, 84.513, 105.657, 106.061, 174.107. HTCC 2: FT-IR (ATR): ν = 3440-3160 cm-1 (−OH and −NH2 or −NH−),1684 cm−1 (amide I, C=O str.), 1560 cm−1 (amide II, NH ben.), 1480 cm−1 (−N+(CH3)3 ben.); 1H NMR (400 MHz, CDCl3, 25 ˚C): δ = 1.908 (s, −CH3COO−), 2.067 (s, −CH3CO−), 2.565-2.935 (m, −NHCH2CH(OH)CH2− and Cell C2H), 3.225 (s, −CH(OH)CH2N+(CH3)3), 3.409-3.969 (m, Cell C3H-C5H, −CH(OH)CH2N+(CH3)3), 4.289 (s, −CH2CH(OH)CH2−), 4.504 (s, Cell C1H); 13C NMR (CP-MAS, 100 MHz): δ = 24.345, 55.308, 62.022, 62.719, 62.983, 63.723, 64.310, 64.660, 64.982, 75.123, 84.570, 104.617, 105.770, 174.068. HTCC 3: FT-IR (ATR): ν = 3445-3162 cm-1 (−OH and −NH2 or −NH−),1682 cm−1 (amide I, C=O str.), 1555 cm−1 (amide II, NH ben.), 1478 cm−1 (−N+(CH3)3 ben.); 1H NMR (400 MHz, CDCl3, 25 ˚C): δ = 1.910 (s, −CH3COO−), 2.068 (s, −CH3CO−), 2.568-2.939 (m, −NHCH2CH(OH)CH2− and Cell C2H), 3.228 (s, −CH(OH)CH2N+(CH3)3), 3.412-3.991 (m, Cell C3H-C5H, −CH(OH)CH2N+(CH3)3), 4.292 (s, −CH2CH(OH)CH2−), 4.546 (s, Cell C1H); 13C NMR (CP-MAS, 100 MHz): δ = 24.064, 55.265, 61.417, 61.715, 62.023, 63.922, 64.293, 64.953, 75.050, 84.552, 104.858, 105.633, 174.047. HTCC 4: FT-IR (ATR): ν = 3455-3160 cm-1 (−OH and −NH2 or −NH−),1679 cm−1 (amide I, C=O str.), 1559 cm−1 (amide II, NH ben.), 1472 cm−1 (−N+(CH3)3 ben.); 1H NMR (400 MHz, CDCl3, 25 ˚C): δ = 1.915 (s, −CH3COO−), 2.069 (s, −CH3CO−), 2.567-2.966 (m, −NHCH2CH(OH)CH2− and Cell C2H), 3.227 (s, −CH(OH)CH2N+(CH3)3), 3.571-3.969 (m, Cell C3H-C5H, −CH(OH)CH2N+(CH3)3), 4.281 (s, −CH2CH(OH)CH2−), 4.537 (s, Cell C1H); 13C NMR (CP-MAS, 100 MHz): δ = 24.048, 55.225, 61.326, 61.720, 62.041, 62.337, 63.047, 63.716, 64.079, 64.799, 74.838, 84.402, 104.864, 106.168, 173.521.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 36

HTCC 5: FT-IR (ATR): ν = 3475-3140 cm-1 (−OH and −NH2 or −NH−),1680 cm−1 (amide I, C=O str.), 1560 cm−1 (amide II, NH ben.), 1481 cm−1 (−N+(CH3)3 ben.); 1H NMR (400 MHz, CDCl3, 25 ˚C): δ = 1.911 (s, −CH3COO−), 2.071 (s, −CH3CO−), 2.570-2.943 (m, −NHCH2CH(OH)CH2− and Cell C2H), 3.231 (s, −CH(OH)CH2N+(CH3)3), 3.414-3.974 (m, Cell C3H-C5H, −CH(OH)CH2N+(CH3)3), 4.287 (s, −CH2CH(OH)CH2−), 4.541 (s, Cell C1H); 13C NMR (CP-MAS, 100 MHz): δ = 24.073, 55.115, 58.124, 61.817, 62.151, 64.014, 64.213, 64.873, 75.225, 84.256, 105.504, 105.890, 173.939. HTCC 6: FT-IR (ATR): ν = 3462-3158 cm-1 (−OH and −NH2 or −NH−),1688 cm−1 (amide I, C=O str.), 1551 cm−1 (amide II, NH ben.), 1474 cm−1 (−N+(CH3)3 ben.); 1H NMR (400 MHz, CDCl3, 25 ˚C): δ = 1.909 (s, −CH3COO−), 2.059 (s, −CH3CO−), 2.588-2.929 (m, −NHCH2CH(OH)CH2− and Cell C2H), 3.224 (s, −CH(OH)CH2N+(CH3)3), 3.409-3.989 (m, Cell C3H-C5H, −CH(OH)CH2N+(CH3)3), 4.278 (s, −CH2CH(OH)CH2−), 4.541 (s, Cell C1H); 13C NMR (CP-MAS, 100 MHz): δ = 24.148, 55.029, 60.921, 61.421, 63.183, 63.817, 64.171, 64.623, 74.847, 84.414, 105.108, 105.363, 174.729. 2.3. Antibacterial assays. Antibacterial activities of the HTCC polymers were determined by a micro-dilution broth method.41 Details of antibacterial activity, kinetics of bactericidal action, mechanism of antibacterial activity and propensity of bacterial resistance development were provided in the Supporting Information. 2.4.

Antifungal assays.

Minimum

inhibitory concentration, minimum

fungicidal

concentration and mechanism of fungicidal action were evaluated following earlier published protocol.42, 43 2.5. In-vivo toxicity. The details of in-vivo skin toxicity procedure was provided in the Supporting Information.44 2.6. In-vivo activity. The experiment was performed in accordance with the earlier protocols with slight modifications.45 BALB/c mice (female, 6 to 8 weeks old, 18-21g) were used for


Page 9 of 36

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


the experiments. First, the mice were rendered neutropenic (∼100 neutrophils/mL) by injecting two doses of cyclophosphamide i.p. (150 mg/kg first dose and 100 mg/kg after 3 days of the first dose). After 24 h of the second dose, mice were anesthetized by ketaminexylazine (40 mg/kg ketamine and 2 mg/kg) mixture i.p.. The fur of each mouse was clipped and then shaved using a razor. While shaving, a wound (reddening and glistening of the skin without bleeding) was introduced on the dorsal midline of each mouse. To the wound site, MRSA was added drop wise (∼109 cells/mL, 20 µL) and allowed to dry to ensure that the bacteria remained within the shaved area. Mice (n = 5 in each group) were treated after 4 h of the infection with 80 µL of HTCC 3 (concentration of 12.5 mg/mL and 25 mg/mL) at the site of infection (i.e., 50 and 100 mg/kg). The polymer solution was carefully added and spread on the entire wound surface to avoid any loss of solution. Another group of mice (n = 5) were treated with fusidic acid (50 mg/kg). The dosages were continued for three days. One group of mice (n = 5) were left untreated and used as a control. Mice were sacrificed 18 h after the last dose using isofluorane and the infected skin was collected, homogenized, and enumerated for cell counting. The bacterial count was finally expressed as log CFU/g of the tissue collected and expressed as mean ± standard error of mean. In order to visualize the effect of the polymer treatment, some skin tissue samples were fixed in formalin for 24 h, subsequently dried using 30, 50, 70, 90 and 100% ethanol. The tissue samples were then sputter coated with gold and imaged using Quanta 3D FEG, FEI field emission scanning electron microscope. 3. RESULTS AND DISCUSSION 3.1. Synthesis of the Polymers. The quaternary chitosan derivatives were prepared by reacting chitosan with glycidyltrimethylammonium chloride (GTMAC) under aqueous acidic conditions (Scheme 1).36 The homogenous conditions favored the substitution of the sugar units of chitosan with selective grafting only onto the primary amine groups (as amines are



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

more nucleophilic than the hydroxyl groups). The introduction of the quaternary ammonium groups onto chitosan and the selective substitution at the primary amine groups were confirmed by 1H and

13

C CP-MAS NMR spectroscopy (Figures S1 and S2, Supporting

Information) as the spectra showed the presence of the −N+Me3 group in the polymer chain (the presence of −N+Me3 group was confirmed by an intense peak at 3.2 ppm in 1H NMR spectrum and ∼55.3 ppm in

13

C CP-MAS spectrum). However, due to overlapping of the

signals from the protons of substituent group and the polymeric backbone in 1H NMR, the degree of substitution (DS) or degree of quaternization (DQ) was not calculated from the proton spectra. The DS of the polymers was determined by conductometric titration of Cl− ions with AgNO3 solution (Figure S3, Supporting Information).39 Six derivatives were prepared all under identical reaction temperature, time, and concentration of polymer and only by varying the mole ratio of GTMAC to sugar unit of chitosan (Table S1, Supporting Information). Mole ratio of GTMAC to sugar unit was used as 4:1, 6:1 and 8:1, which led to three quaternary polymers from chitosan of a particular molecular weight. Two different molecular weights of chitosan were used (relatively lower molecular weight chitosan of 15 kDa and of higher molecular weight chitosan of 50-190 kDa). The DS values ranged from 0.35 to 0.55. (Table S1, Supporting Information). All the derivatives were found to be soluble in water till 40 mg/mL. However, polymers with lower DS were found to form gel at higher concentrations (> 40 mg/mL) and thus heated to obtain clear aqueous solution when needed. 3.2. Antibacterial Activity. Though the antibacterial activity of quaternary chitosan derivatives has been studied earlier, no systematic structure-activity relationship (SAR) studies of the HTCC polymers are reported. To evaluate the antibacterial efficacy of these polymers with different DS or DQ and to evaluate the role of molecular weight, the polymers were first challenged against a wide spectrum of drug-sensitive bacteria such as S. aureus, E. coli and A. baumannii and drug-resistant bacteria such as methicillin-resistant Staphylococcus


Page 10 of 36

Page 11 of 36

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


aureus (MRSA), vancomycin-resistant Enterococcus faecium (VRE), β-lactam-resistant K. pneumoniae. The antibacterial efficacy for all the bacteria was determined in nutrient broth except VRE which was performed in brain-heart infusion broth, and represented as minimum inhibitory concentration (MIC), i.e., the minimum concentration of the polymers required to inhibit the growth of bacteria. In general, the polymers showed activity against all the bacteria tested (Table 1). The MIC values of the polymers varied from 125-1000 µg/mL. Variation of DS and molecular weight of chitosan derivatives were found to have slight effect on the antibacterial activity. For example, HTCC 1 (with DS 31% obtained from chitosan of 15 kDa) showed MIC values of 250 µg/mL and 500 µg/mL against S. aureus and E. coli respectively whereas HTCC 3 (with DS 58% obtained from chitosan of 15 kDa) showed MIC values of 125 µg/mL and 250 µg/mL against S. aureus and E. coli respectively. On the other hand, HTCC 3 (with DS 58% obtained from chitosan of 15 kDa) and HTCC 6 (with DS 54% obtained from chitosan of 50-190 kDa) showed similar MIC values against both S. aureus and E. coli (MIC = 125 µg/mL and 250 µg/mL against S. aureus and E. coli respectively). In general, the polymers were found to be more active towards Gram-positive bacteria than Gram-negative bacteria. For example, the range of MIC values for the polymers were 125-250 µg/mL against Gram-positive bacteria whereas the range of MIC values was 250-1000 µg/mL against Gram-negative bacteria for both low and high molecular weight HTCC polymers (Table 1). Like drug-sensitive bacteria, the polymers showed similar activity against drug-resistant bacteria such as MRSA, VRE and K. pneumoniae. The two most active polymers (HTCC 3 and HTCC 6) displayed MIC values of 125 µg/mL each against MRSA and VRE, and 500 µg/mL each against K. pneumoniae respectively (Table 1). The relatively low antibacterial activity of chitosan derivatives could be attributed to the lack of sufficient hydrophobicity.46



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Antibacterial activity of the chitosan derivatives was also evaluated against various multi-drug resistant (MDR) clinical isolates. Clinical isolates of carbapenem-resistant bacteria, New Delhi Metallo-beta-lactamase-1 (NDM-1) with bla-NDM-1 gene were previously characterized using polymerase chain reaction and gel electrophoresis by our group.44 All the isolates mentioned in this report were highly resistant to common antibiotics such as ampicillin, kanamycin, meropenem, ciprofloxacin, erythromycin, tetracycline, doxycycline and minocycline (MIC values of most of the antibiotics were >250 µg/mL) and were sensitive to only tigecycline and colistin (MIC = 0.5-1.0 µg/mL).44 A total of 12 such isolates were used in this study. Like the drug-sensitive and drug-resistant laboratory strain, HTCC polymers were found to be active against the multi-drug resistant clinical strains (Table 2). One of the most active polymers, HTCC 3 displayed MIC value of 125 µg/mL each against Gram-positive MDR MRSA R3545 and MRSA R3889 strains. The polymer also showed activity against Gram-negative clinical isolates except P. aeruginosa (e.g., MIC values of HTCC 3 were 125-250 µg/mL against clinical isolates of K. pneumoniae, E. coli, E. cloacae and A. baumannii) (Table 2). The above results thus emphasize the broadspectrum nature of the cationic chitosan derivatives and show their potency as antibacterial agents. The polymers were found to be not only bacteriostatic but also bactericidal against both Gram-positive and Gram-negative bacteria (Table S2, Supporting Information). The most active polymer HTCC 3 displayed MBC values of 250 µg/mL and 500 µg/mL against both S. aureus and E. coli respectively (Table S2, Supporting Information). The rate of bactericidal action is important in clinical settings as the first 6 h of post-infection is critical.47 The rate of bacterial killing was therefore evaluated towards both S. aureus and E. coli using two most active polymers HTCC 3 and HTCC 6 at two different concentrations (MIC and 6 × MIC). HTCC 6 killed S. aureus (∼5 log reduction) at 30 min whereas HTCC 3 killed


Page 12 of 36

Page 13 of 36

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


within 60 min at 6 × MIC. On the other hand, HTCC 6 killed E. coli (∼5-log reduction) at 60 min whereas HTCC 3 killed at 90 min at 6 × MIC (Figure S4, Supporting Information). Thus, both the polymers showed rapid bactericidal activity against both classes of bacteria. However, the polymers showed bacteriostatic effect at their respective MIC values against S. aureus whereas killed E. coli at 120 min and 240 min at their MIC values, which are also the respective MBC values (Figure S4, Supporting Information). 3.3. Mechanism of Antibacterial Action. Since the bacterial cell membrane is anionic due to the presence of negatively charged phospholipids, the cationic polymers are known to interact with the anionic cell membrane and cause membrane damage.48, 49 To confirm that these cationic polymers act by disrupting the bacterial membrane integrity, the molecular mechanism of action against both S. aureus and E. coli was studied using various spectroscopic and microscopic methods. Maintaining cell membrane potential is very important for the survival of the unicellular bacteria. To determine whether the polymers act by depolarizing the cell membrane, potential sensitive dye 3,3'-dipropylthiadicarbocyanine iodide (DiSC35) was used to monitor the changes in the potential across the membrane using fluorescence spectroscopy.50 Due to the presence of potential gradient across the membrane DiSC35 is taken up by the bacteria and accumulated in the membrane which leads to a decrease in the fluorescence intensity of the dye because of self-quenching. However, when the membrane-potential is dissipated upon interaction with an antibacterial agents, an increase in the fluorescence intensity is observed due to the displacement of DiSC3(5) dye into the solution. When tested, all the polymers showed an enhancement in the fluorescence intensity of the DiSC35 dye therefore indicated that these cationic polymers indeed dissipated the membrane potential of the both Gram-positive and Gram-negative bacteria (Figure 1a and 1b). Intracellular K+ ion is an essential ion for the survival of bacteria. To evaluate whether the polymers also cause leakage of intracellular K+ ion, K+ ion sensitive dye PBFI-AM was



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

used and fluorescence intensity was monitored. PBFI-AM is known to bind to K+ ion selectively and to show an increase in the fluorescence intensity.51 Interestingly, when both Gram-positive and Gram-negative bacteria were treated with the polymers, the dye showed an increase in fluorescence intensity compared to non-treated bacteria thus indicated that the cationic polymers caused significant leakage of intracellular K+ ions of bacteria (Figure 1c and 1d). Gram-negative bacteria are protected by an additional membrane outside the cell wall known as outer membrane (OM). The OM plays a vital role as barrier to unwanted molecules such as drug molecules. The OM of the Gram-negative bacteria mostly consists of lipopolysaccharides (LPS) which are generally negatively charged. Cationic polymers are therefore expected to bind to the negatively charged LPS of Gram-negative bacteria, thus disrupting the integrity of OM and resulting in loss of the additional barrier function. The OM permeabilization was studied using a hydrophobic dye N-phenyl naphthylamine (NPN). NPN is generally known to get excluded from the OM of the bacteria. However, when OM is damaged, NPN distributes into the perturbed OM thereby exhibits an increase in fluorescence intensity. Upon addition of the cationic polymers to E. coli suspensions in presence of preequilibrated NPN, increased fluorescence was observed for all the polymers.52 Thus, the above result indicated that the cationic polymers interacted with the E. coli outer membrane thus disrupting its integrity (Figure S5, Supporting Information). The ability of the polymers to permeabilize the cytoplasmic membrane of bacteria was also studied using a membrane impermeable dye propidium iodide (PI). PI enters inside the cell only through compromised cell membrane and fluoresces strongly upon binding to the nucleic acid. When bacteria were treated with the polymers, an increase in the fluorescence intensity was found to observe against both S. aureus and E. coli (Figure 1e and 1f). Thus, the cationic polymers were efficient in permeabilizing the cytoplasmic membrane of both Gram-positive and Gramnegative bacteria. Notably, all the polymers were found to be membrane-active against both


Page 14 of 36

Page 15 of 36

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


types of bacteria. In summary, all the above studies indicated that these cationic polymers interacted with the anionic bacterial cell membrane and disrupted the membrane integrity thus causing cell death. In order to further consolidate the mechanistic action of these polymers, both treated and untreated bacteria were imaged by confocal laser scanning microscope (CLSM).53, 54 Bacteria were imaged using a mixture of two nucleic acid binding dyes, membrane-permeable green fluorescent dye SYTO 9, and membrane-impermeable red fluorescent dye PI. CLSM images of bacteria treated with the polymer HTCC 3 and without any treatment (as control) were captured and shown in Figure 2. Cell viability was clearly seen from green fluorescence observed in case of non-treated bacteria (Figure 2a and Figure 2c for S. aureus and E. coli respectively). In contrast, bright red fluorescence was observed for both bacteria upon treatment with the polymer which indicated membrane permeabilization in both bacterial species (Figure 2b and Figure 2d for S. aureus and E. coli respectively). 3.4. Propensity of bacterial resistance development. Bacterial resistance development against most of the clinically approved drugs is one of the major threats of current times.55 Thus, it would be of significant importance to evaluate the propensity of developing resistance by bacteria against these polymeric biocides. The ability of these polymers to suppress the development of resistance was evaluated by taking the most active polymer (HTCC 3) against a Gram-positive S. aureus and a Gram-negative A. baumannii. Norfloxacin, an antibiotic commonly used to treat Gram-positive bacterial infections, was used as a positive control for S. aureus, whereas colistin, a lipopeptide antibiotic active against Gram-negative bacteria, was used in case of A. baumannii. The polymer was repeatedly challenged against these bacteria at their sub-MIC values. Interestingly, no change in MIC of HTCC 3 was observed for both S. aureus and A. baumanii even after 14 passages whereas around 280-fold and 350-fold increase in MIC was observed in case of norfloxacin



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

and colistin respectively (Figure 2e and 2f). Thus, the above results indicated that bacteria find it difficult to develop resistance against the polymer within the experimental time period. 3.5. Antifungal Activity. Cationic polymers have previously been shown to possess potent antifungal activity.56 However, to the best of our knowledge, no systematic antifungal study of HTCC polymers has been reported. In order to find the efficacy of the cationic chitosan derivatives as antifungal agents, activity of all the polymers were evaluated against different pathogenic Candida spp. (C. albicans SC5314, C. dubliniensis CD36, C. tropicalis MYA3404) and Cryptococcus spp. (C. neoformans var. grubii (serotype A) H99, C. gattii (serotype B) WM276 and C. neoformans var. neoformans (serotype D) JEC21). The polymers were found to inhibit the growth of all the human pathogenic fungi except C. dubliniensis at the tested concentrations. HTCC 3 and HTCC 6 showed maximum antifungal activity therefore suggested pan-antimicrobial features of these polymers (Table 1). The MIC values for HTCC 3 and HTCC 6 were 250 µg/mL each against Candida spp. and 125-250 µg/mL against Cryptococcus spp. indicating Cryptococcus spp. are more susceptible to these polymers. Furthermore, these cationic polymers were not only fungistatic but also fungicidal in nature. For example, minimum fungicidal concentration (MFC) of HTCC 3 was found to be 250 µg/mL against both C. tropicalis and C. neoformans Ser. D (Figure 3a and Figure S6, Supporting Information). To evaluate how fast these polymers kill fungi, the rate of action was determined against C. albicans using HTCC 3. HTCC 3 killed C. albicans within 2 h at 8 × MIC (Figure 3b) and in 8 h at its MIC which is also its MFC. The cationic polymers interacted with the cellular membrane of bacteria and thereby disrupted the membrane integrity of the pathogen leading to cell death as observed earlier. To verify whether these polymers also interact with the fungal cell membrane, we performed cell viability assay by fluorescence microscopy. C. albicans was used as a model fungal pathogen and treated with HTCC 3 at two different concentrations. The microscopic images showed


Page 16 of 36

Page 17 of 36

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


the presence of viable cells in case of control samples (non-treated) as observed by green fluorescence of SYTO 9 (live stain) and absence red fluorescence of PI (dead stain). However, the cells treated with the polymer HTCC 3 showed red color fluorescence of PI indicating that the membrane was compromised (Figure 3c). These results signify that the cationic chitosan derivatives interact with the fungal cell membrane and disrupt the integrity of the membrane presumably leading to cell death.57 3.6. In-vitro toxicity. One of the major concerns in the development of clinically useful antibacterial agents for health care applications is the toxicity towards mammalian cells. In order to be used in clinical settings, the biocides should be active towards microbial cells and non-active towards mammalian cells. To evaluate the cytotoxic effects of the polymers, hemolytic activity against human red blood (hRBC) cells as well as cytotoxicity against human embryo kidney (HEK) cells were performed. The ability of the polymers to lyse the human red blood cells (hRBC) was expressed as HC50 (µg/mL), i.e., the concentration of the polymers at which 50% RBC gets lysed. In general, HC50 of all the polymers was found to be more than 10000 µg/mL (Table 1). This showed that these cationic polymers were 40-80 times more selective (selectivity = HC50/MIC) towards bacteria over mammalian cells. Notably, all the polymers showed negligible hemolysis even at 8000 µg/mL (32-64 times more than their MIC values). The above results therefore indicated that these polymers are non-toxic towards human erythrocytes. To strengthen our inquiry into the selectivity of the polymers, cytotoxicity was also performed with HEK 293 cell lines. Cells were treated with HTCC 3 at 125, 250 and 500 µg/mL and were imaged by fluorescence microscopy using green fluorescent dye calcein AM and red fluorescent dye PI (Figure 4).58 The polymer treated cells showed green fluorescence for all tested concentrations and were almost similar to the untreated cells, which was indicative of healthy cells. Cells treated with triton-X, on the other hand, showed complete red fluorescence thereby indicated the presence of completely



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

dead cells (Figure S7, Supporting Information). These results thus indicated that these polymers are indeed non-toxic towards mammalian cells. 3.7. In-vivo toxicity. In-vivo toxicity of cationic polymers remains a major concern that prevents them from being used in therapeutic applications. The acute dermal toxicity of the polymers was evaluated by applying HTCC 3 at the back of mice. After 14 days, almost complete regeneration of fur was observed in all the test animals. No morbidity was observed among the test animals at 200 mg/kg and it was therefore concluded that the 50% lethal dose (LD50) in the acute dermal toxicity test was more than 200 mg/kg. Moreover, the skin tissue samples did not show any inflammatory responses in hematoxylin and eosin staining and showed like untreated skin tissue at 200 mg/kg dosage of HTCC 3 (Figure 4). Taken together, the above studies demonstrate that the chitosan derivatives possess an impressive safety profile which has potential to be translated into therapeutic applications. 3.8. In-vivo activity. For any antibacterial agents to be used in actual clinical disinfection, the agents should be active under in-vivo conditions.59 In order to show the efficacy of the polymer under in-vivo conditions and to evaluate its potential in alleviating topical infections, murine model of superficial skin wound infection was used (Figure 5a).45 First, a wound was created while shaving the back of each neutropenic mouse until skin tissue was red and glistening. The wounds were then inoculated with approximately 20 µL of 109 CFU/mL of MRSA. Treatment of the polymer was initiated 4 h after the application of bacteria. Infection site was treated with HTCC 3 for three days by adding 80 µL of the polymer on the infected site to give 50 mg/kg and 100 mg/kg (once a day). Fusidic acid (50 mg/kg) was also used as a control drug to compare the efficacy of the polymers. The polymer was able to reduce bacterial count significantly in a course of three days (almost 2.1 log reduction at 50 mg/kg and 3.2 log reduction at 100 mg/kg as compared to untreated mice sample) (Figure 5b). In fact, the effect of the polymer was comparable to that of the approved drug fusidic acid at the


Page 18 of 36

Page 19 of 36

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


same dose (fusidic acid showed 2.0 log reduction of MRSA at the same dose). While development of resistance to fusidic acid is major limitation to its efficacy as an antibacterial agent, cationic chitosan derivatives with less likelihood of inducing microbial resistance might thus be more promising in treating microbial infection. The skin tissue was also imaged by scanning electron microscopy to have visual effect. As can be seen from Figure 5, the skin tissue sample showed a higher density of bacterial cells on the skin after 4 h of infection (Figure 5c). The treated sample, in contrast, showed lower bacterial amounts on the tissue surface along with white blood cells (Figure 5d). The skin tissue samples were also imaged by hematoxylin and eosin staining. It was observed that the infected but non-treated skin tissue showed inflammation (infiltration of inflammatory cells mainly neutrophils in dermis layer) whereas treated tissue samples showed minimal to negligible inflammation (Figure 5e-5h). The above results therefore indicated that the chitosan derivative was able to reduce the skin infection. 4. CONCLUSIONS In summary, the cationic chitosan derivatives were shown to be active against various drugsensitive and drug-resistant bacteria. The polymers were also shown to be active against clinically isolated multi drug-resistant bacteria and human pathogenic fungi. Mechanistic studies suggested that the polymers inactivated both bacteria and fungi primarily by disruption of membrane integrity. Bacteria were shown to have negligible tendency towards development of resistance against the polymers up to 14 passages. Low cytotoxic activity against human erythrocytes and mammalian cells confirmed their in-vitro non-toxic behaviour. Further, the polymers were found to have high lethal doses in acute dermal toxicity determination in-vivo and showed negligible skin tissue inflammation. Interestingly, the most active polymer showed activity against MRSA in a mouse model of superficial skin infection at a dose that exerted no cytotoxic effects. The findings reported herein thus stress



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

the importance of these cationic chitosan derivatives to be used as safe antimicrobial agents in topical and other healthcare applications.

Supporting Information Synthetic procedure of polymers, protocols for antibacterial activity, protocols for in-vitro and in-vivo toxicity, 1H and

13

C CP-MAS NMR spectra of the HTCC polymers, tables

showing reaction parameters and bactericidal activity, figures of antibacterial and antifungal activity, figures of in-vivo toxicity.

Acknowledgements We thank Prof. C. N. R. Rao, FRS (JNCASR) for his constant support and encouragement. J. Hoque thanks JNCASR for senior research fellowship (SRF). This work was partially funded by the DST-Fast Track project (SR/FT/Cs-097/2009), Department of Science and Technology, Government of India and Indo-Portugal Joint Collaborative Project under IndoPortuguese Program for Co-operation in Science and Technology (INT/PORTUGAL/P12/2013) between Department of Science and Technology (DST), India, and FCT, Portugal.

References 1. Alekshun, M. N.; Levy, S. B. Molecular mechanisms of antibacterial multidrug resistance. Cell 2007, 128, 1037-1050. 2. Wright, G. D. Molecular mechanisms of antibiotic resistance. Chem. Commun. 2011, 47, 4055-4061. 3. Walsh, C. Molecular mechanisms that confer antibacterial drug resistance. Nature 2000, 406, 775-781. 4. Taubes, G. The bacteria fight back. Science 2008, 321, 356-361.


Page 20 of 36

Page 21 of 36

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


5. Kontoyiannis, D. P.; Lewis, R. E. Antifungal drug resistance of pathogenic fungi. Lancet 2002, 359, 1135-1144. 6. Mintzer, M. A.; Dane, E. L.; O’Toole, G. A.; Grinstaff, M. W. Exploiting dendrimer multivalency to combat emerging and re-emerging infectious diseases. Mol. Pharmaceutics 2012, 9, 342-354. 7. Chandra, J.; Kuhn, D. M.; Mukherjee, P. K.; Hoyer, L. L.; McCormick, T.; Ghannoum, M. A. Biofilm formation by the fungal pathogen Candida albicans: development, architecture, and drug resistance. J. Bacteriol. 2001, 183, 5385-5394. 8. Pfaller, M. A. Antifungal drug resistance: mechanisms, epidemiology, and consequences for treatment. Am. J. Med. 2012, 125, S3-13. 9. Pushpanathan, M.; Pooja, S.; Gunasekaran, P.; Rajendhran, P. Critical evaluation and compilation of physicochemical determinants and membrane interactions of MMGP1 antifungal peptide. Mol. Pharmaceutics 2016, 13, 1656-1667. 10. Liu, R.; Chen, X.; Hayouka, Z.; Chakraborty, S.; Falk, S. P.; Weisblum, B.; Masters, K. S.; Gellman, S. H. Nylon-3 polymers with selective antifungal activity. J. Am. Chem. Soc. 2013, 135, 5270-5273. 11. Oh, D.; Sun, J.; Shirazi, A. M.; LaPlante, K. L.; Rowley, D. C.; Parang, K. Antibacterial activities of amphiphilic cyclic cell-penetrating peptides against multidrug-resistant pathogens. Mol. Pharmaceutics 2014, 11, 3528-3536. 12. Zasloff, M. Antimicrobial peptides of multicellular organisms. Nature 2002, 415 (6870), 389-95. 13. Uppu, D. S.; Samaddar, S.; Ghosh, C.; Paramanandham, K.; Shome, B. R.; Haldar, J. Amide side chain amphiphilic polymers disrupt surface established bacterial bio-films and protect mice from chronic Acinetobacter baumannii infection. Biomaterials 2016, 74, 131143.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 36

14. Kuroda, K.; DeGrado, W. F. Amphiphilic polymethacrylate derivatives as antimicrobial agents. J. Am. Chem. Soc. 2005, 127, 4128-4129. 15. Worley, B. V.; Schilly, K. M.; Schoenfisch, M. H. Anti-biofilm efficacy of dual-action nitric

oxide-releasing

alkyl

chain

modified

poly(amidoamine)

dendrimers.

Mol.

Pharmaceutics 2015, 12, 1573-1583. 16. Lienkamp, K.; Madkour, A. E.; Musante, A.; Nelson, C. F.; Nusslein, K.; Tew, G. N. Antimicrobial polymers prepared by ROMP with unprecedented selectivity: a molecular construction kit approach. J. Am. Chem. Soc. 2008, 130, 9836-9843. 17. Yang, C.; Krishnamurthy, S.; Liu, J.; Liu, S.; Lu, X.; Coady, D. J.; Cheng, W.; De Libero, G.; Singhal, A.; Hedrick, J. L.; Yang, Y. Y. Broad-spectrum antimicrobial star polycarbonates functionalized with mannose for targeting bacteria residing inside immune cells. Adv. Healthc. Mater. 2016, 5, 1272-1281. 18. Li, P.; Zhou, C.; Rayatpisheh, S.; Ye, K.; Poon, Y. F.; Hammond, P. T.; Duan, H.; ChanPark, M. B. Cationic peptidopolysaccharides show excellent broad-spectrum antimicrobial activities and high selectivity. Adv. Mater. 2012, 24, 4130-4137. 19. Svenson, J.; Karstad, R.; Flaten, G. E.; Brandsdal, B.-O.; Brandl, M.; Svendsen, J. S. Altered activity and physicochemical properties of short cationic antimicrobial peptides by incorporation of arginine analogues. Mol. Pharmaceutics 2015, 12, 996-1005. 20. Radzishevsky, I. S.; Rotem, S.; Bourdetsky, D.; Navon-Venezia, S.; Carmeli, Y.; Mor, A. Improved antimicrobial peptides based on acyl-lysine oligomers. Nat. Biotechnol. 2007, 25, 657-659. 21. Padhee, S.; Hu, Y.; Niu, Y.; Bai, G.; Wu, H.; Costanza, F.; West, L.; Harrington, L.; Shaw, L. N.; Cao, C.; Cai, J. Non-hemolytic alpha-AApeptides as antimicrobial peptidomimetics. Chem. Commun. 2011, 47, 9729-9731.


Page 23 of 36

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


22. Chakraborty, S.; Liu, R.; Hayouka, Z.; Chen, X.; Ehrhardt, J.; Lu, Q.; Burke, E.; Yang, Y.; Weisblum, B.; Wong, G. C.; Masters, K. S.; Gellman, S. H. Ternary nylon-3 copolymers as host-defense peptide mimics: beyond hydrophobic and cationic subunits. J. Am. Chem. Soc. 2014, 136, 14530-14535. 23. Chen, H.; Liu, C.; Chen, D.; Madrid, K.; Peng, S.; Dong, X.; Zhang, M.; Gu, Y. Bacteriatargeting conjugates based on antimicrobial peptide for bacteria diagnosis and therapy. Mol. Pharmaceutics 2015, 12, 2505-2516. 24. Kumar, M. N.; Muzzarelli, R. A.; Muzzarelli, C.; Sashiwa, H.; Domb, A. J. Chitosan chemistry and pharmaceutical perspectives. Chem. Rev. 2004, 104, 6017-6084. 25. Shi, Z. L.; Neoh, K. G.; Kang, E. T.; Wang, W. Antibacterial and mechanical properties of bone cement impregnated with chitosan nanoparticles. Biomaterials 2006, 27, 2440-2449. 26. Ueno, H.; Mori, T.; Fujinaga, T. Topical formulations and wound healing applications of chitosan. Adv. Drug Deliv. Rev. 2001, 52, 105-115. 27. Diez-Pascual, A. M.; Diez-Vicente, A. L. Wound healing bionanocomposites based on castor oil polymeric films reinforced with chitosan-modified ZnO nanoparticles. Biomacromolecules 2015, 16, 2631-2644. 28. Jin, R.; Moreira Teixeira, L. S.; Dijkstra, P. J.; Karperien, M.; van Blitterswijk, C. A.; Zhong, Z. Y.; Feijen, J. Injectable chitosan-based hydrogels for cartilage tissue engineering. Biomaterials 2009, 30, 2544-2551. 29. Freier, T.; Montenegro, R.; Shan Koh, H.; Shoichet, M. S. Chitin-based tubes for tissue engineering in the nervous system. Biomaterials 2005, 26, 4624-4632. 30. Li, P.; Poon, Y. F.; Li, W.; Zhu, H. Y.; Yeap, S. H.; Cao, Y.; Qi, X.; Zhou, C.; Lamrani, M.; Beuerman, R. W.; Kang, E. T.; Mu, Y.; Li, C. M.; Chang, M. W.; Leong, S. S.; ChanPark, M. B. A polycationic antimicrobial and biocompatible hydrogel with microbe membrane suctioning ability. Nat. Mater. 2011, 10, 149-156.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

31. Belalia, R.; Grelier, S.; Benaissa, M.; Coma, V. New bioactive biomaterials based on quaternized chitosan. J. Agric. Food Chem. 2008, 56, 1582-1588. 32. Badawy, M. E. I. Structure and antimicrobial activity relationship of quaternary N-alkyl chitosan derivatives against some plant pathogens. J. Appl. Polym. Sci. 2010, 117, 960-969. 33. Lapitsky, Y.; Zahir, T.; Shoichet, M. S. Modular biodegradable biomaterials from surfactant and polyelectrolyte mixtures. Biomacromolecules 2008, 9, 166-174. 34. Xu, Y. M.; Du, Y. M.; Huang, R. H.; Gao, L. P. Preparation and modification of N-(2hydroxyl) propyl-3-trimethyl ammonium chitosan chloride nanoparticle as a protein carrier. Biomaterials 2003, 24, 5015-5022. 35. Xiao, B.; Wan, Y.; Wang, X. Y.; Zha, Q. C.; Liu, H. M.; Qiu, Z. Y.; Zhang, S. M. Synthesis and characterization of N-(2-hydroxy)propyl-3-trimethyl ammonium chitosan chloride for potential application in gene delivery. Colloids Surf. B 2012, 91, 168-174. 36. Kaminski, K.; Szczubialka, K.; Zazakowny, K.; Lach, R.; Nowakowska, M. Chitosan derivatives as novel potential heparin reversal agents. J. Med. Chem. 2010, 53, 4141-4147. 37. Nam, C. W.; Kim, Y. H.; Ko, S. W. Modification of polyacrylonitrile (PAN) fiber by blending with N-(2-hydroxy)propyl-3-trimethylammonium chitosan chloride. J. Appl. Polym. Sci. 1999, 74, 2258-2265. 38. Kim, Y. H.; Nam, C. W.; Choi, J. W.; Jang, J. H. Durable antimicrobial treatment of cotton fabrics using N-(2-hydroxy)propyl-3-trimethylammonium chitosan chloride and polycarboxylic acids. J. Appl. Polym. Sci. 2003, 88, 1567-1572. 39. Cui, D.; Szarpak, A.; Pignot-Paintrand, I.; Varrot, A.; Boudou, T.; Detrembleur, C.; Jerome, C.; Picart, C.; Auzely-Velty, R. Contact-killing polyelectrolyte microcapsules based on chitosan derivatives. Adv. Funct. Mater. 2010, 20, 3303-3312. 40. Lynch, A. S.; Robertson, G. T. Bacterial and fungal biofilm infections. Annu. Rev. Med. 2008, 59, 415-428.


Page 24 of 36

Page 25 of 36

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


41. Wiegand, I.; Hilpert, K.; Hancock, R. E. W. Agar and broth dilution methods to determine the minimal inhibitory concentration (MIC) of antimicrobial substances. Nat. Protoc. 2008, 3, 163-175. 42. Hoque, J.; Akkapeddi, P.; Yadav, V.; Manjunath, G. B.; Uppu, D. S.; Konai, M. M.; Yarlagadda, V.; Sanyal, K.; Haldar, J. Broad spectrum antibacterial and antifungal polymeric paint materials: synthesis, structure-activity relationship, and membrane-active mode of action. ACS Appl. Mater. Interfaces 2015, 7, 1804-1815. 43. Lee, M. R.; Raman, N.; Gellman, S. H.; Lynn, D. M.; Palecek, S. P. Hydrophobicity and helicity regulate the antifungal activity of 14-helical beta-peptides. ACS Chem. Biol. 2014, 9, 1613-1621. 44. Uppu, D. S.; Manjunath, G. B.; Yarlagadda, V.; Kaviyil, J. E.; Ravikumar, R.; Paramanandham, K.; Shome, B. R.; Haldar, J. Membrane-active macromolecules resensitize NDM-1 gram-negative clinical isolates to tetracycline antibiotics. PLoS One 2015, 10, e0119422. 45. Ghosh, C.; Manjunath, G. B.; Konai, M. M.; Uppu, D. S.; Hoque, J.; Paramanandham, K.; Shome, B. R.; Haldar, J. Aryl-alkyl-lysines: agents that kill planktonic cells, persister cells, biofilms of MRSA and protect mice from skin-infection. PLoS One 2015, 10, e0144094. 46. Sahariah, P.; Benediktssdottir, B. E.; Hjalmarsdottir, M. A.; Sigurjonsson, O. E.; Sorensen, K. K.; Thygesen, M. B.; Jensen, K. J.; Masson, M. Impact of chain length on antibacterial activity and hemocompatibility of quaternary N-alkyl and N,N-dialkyl chitosan derivatives. Biomacromolecules 2015, 16, 1449-1460. 47. Hetrick, E. M.; Schoenfisch, M. H. Reducing implant-related infections: active release strategies. Chem. Soc. Rev. 2006, 35, 780-789.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

48. Yang, X.; Hu, K.; Hu, G.; Shi, D.; Jiang, Y.; Hui, L.; Zhu, R.; Xie, Y.; Yang, L. Long hydrophilic-and-cationic polymers: a different pathway toward preferential activity against bacterial over mammalian membranes. Biomacromolecules 2014, 15, 3267-3277. 49. Mosca, S.; Keller, J.; Azzouz, N.; Wagner, S.; Titz, A.; Seeberger, P. H.; Brezesinski, G.; Hartmann, L. Amphiphilic cationic beta(3R3)-peptides: membrane active peptidomimetics and their potential as antimicrobial agents. Biomacromolecules 2014, 15, 1687-1695. 50. Hoque, J.; Konai, M. M.; Gonuguntla, S.; Manjunath, G. B.; Samaddar, S.; Yarlagadda, V.; Haldar, J. Membrane active small molecules show selective broad spectrum antibacterial activity with no detectable resistance and eradicate biofilms. J. Med. Chem. 2015, 58, 54865500. 51. Yarlagadda, V.; Samaddar, S.; Paramanandham, K.; Shome, B. R.; Haldar, J. Membrane disruption and enhanced inhibition of cell-wall biosynthesis: a synergistic approach to tackle vancomycin-resistant bacteria. Angew. Chem. Int. Ed. 2015, 54, 13644-13649. 52. Hoque, J.; Konai, M. M.; Samaddar, S.; Gonuguntala, S.; Manjunath, G. B.; Ghosh, C.; Haldar, J. Selective and broad spectrum amphiphilic small molecules to combat bacterial resistance and eradicate biofilms. Chem. Commun. 2015, 51, 13670-13673. 53. Abd-El-Aziz, A. S.; Agatemor, C.; Etkin, N.; Overy, D. P.; Lanteigne, M.; McQuillan, K.; Kerr, R. G. Antimicrobial organometallic dendrimers with tunable activity against multidrug-resistant bacteria. Biomacromolecules 2015, 16, 3694-3703. 54. Parthasarathy, A.; Pappas, H. C.; Hill, E. H.; Huang, Y.; Whitten, D. G.; Schanze, K. S. Conjugated polyelectrolytes with imidazolium solubilizing groups. properties and application to photodynamic inactivation of bacteria. ACS Appl. Mater. Interfaces 2015, 7, 28027-28034. 55. Jennings, M. C.; Minbiole, K. P. C.; Wuest, W. M. Quaternary ammonium compounds: an antimicrobial mainstay and platform for innovation to address bacterial resistance. Acs Infect. Dis. 2015, 1, 288-303.


Page 26 of 36

Page 27 of 36

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


56. Liu, R. H.; Chen, X. Y.; Falk, S. P.; Mowery, B. P.; Karlsson, A. J.; Weisblum, B.; Palecek, S. P.; Masters, K. S.; Gellman, S. H. Structure-activity relationships among antifungal nylon-3 polymers: identification of materials active against drug-resistant strains of Candida albicans. J. Am. Chem. Soc. 2014, 136, 4333-4342. 57. Ravikumar, T.; Murata, H.; Koepsel, R. R.; Russell, A. J. Surface-active antifungal polyquaternary amine. Biomacromolecules 2006, 7, 2762-2769. 58. Hoque, J.; Akkapeddi, P.; Yarlagadda, V.; Uppu, D. S.; Kumar, P.; Haldar, J. Cleavable cationic antibacterial amphiphiles: synthesis, mechanism of action, and cytotoxicities. Langmuir 2012, 28, 12225-12234. 59. Thoma, L. M.; Boles, B. R.; Kuroda, K. Cationic methacrylate polymers as topical antimicrobial agents against Staphylococcus aureus nasal colonization. Biomacromolecules 2014, 15, 2933-2943.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Scheme 1. Synthesis of quaternary chitosan derivatives by reacting chitosan with glycidyl trimethylammonium chloride (GTMAC) led to different degrees of substitution.


Page 28 of 36

Page 29 of 36

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 1. Mechanism of antibacterial action of quaternary chitosan derivatives. Membrane depolarization of bacteria by HTCC polymers against (a) S. aureus and (b) E. coli respectively; Intracellular K+ ion leakage of bacteria by HTCC polymers against (c) S. aureus and (d) E. coli respectively; Cytoplasmic membrane permeabilization of bacteria by HTCC polymers against (e) S. aureus and (f) E. coli respectively.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 2. Mechanism of action by confocal laser scanning microscopy (CLSM) and propensity of bacterial resistance development. CLSM images of (a and c) untreated S. aureus and E. coli, and (b and d) polymer treated S. aureus and E. coli respectively. Fold increase in MIC against (e) S. aureus and (f) E. coli respectively.


Page 30 of 36

Page 31 of 36

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 3. Antifungal activity of the chitosan derivatives. (a) Minimum fungicidal concentration of the polymers against C. albicans was determined by the spot plate method; (b) kinetics of antifungal killing; (c) fluorescence microscopic images of non-treated and polymer (HTCC 3)-treated C. albicans cells.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 4. Mice skin histopathology via haematoxylin and eosin staining in different groups of mice. (a) Control mice showing normal architecture of epidermis, dermis, subcutaneous tissues and with sweat glands, sebaceous glands (arrow); (b) mice skin treated with 50 mg/kg of HTCC 3 showing normal dermis layer of skin with sweat and sebaceous gland along with normal adipose tissue (arrow); (c) mice skin treated with 100 mg/kg of HTCC 3 showed normal skin epidermis and dermis layer with sebaceous and sweat glands (arrow); (d) mice skin treated with 200 mg/kg of HTCC 3 showing normal skin epidermis and dermis layer with sebaceous and sweat glands and subcutaneous tissues (arrow).


Page 32 of 36

Page 33 of 36

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 5. In vivo antibacterial efficacy of quaternary chitosan derivative. (a) Experimental plan of murine model of superficial MRSA skin infection after mice were rendered neutropenic; (b) bacterial count of the treated and non-treated skin tissue samples from mice; (c) scanning electron microscopy images of the skin tissue sample representing bacteria after 4 h of infection and (d) after 3 days of treatment with HTCC 3. Color arrow indicates MRSA cells or macrophages present on the skin tissue samples. Mice skin histopathology: (e) MRSA-infected mice skin showing infiltration of inflammatory cells mainly neutrophils in dermis layer (arrow); (f) skin treated with 50 mg/kg of HTCC 3 showing normal appearance of skin with thin layer of epidermis and hair follicles (arrow); (g) skin treated with 100 mg/kg of HTCC 3 showing normal epidermis layer with different types of cells and sweat, sebaceous glands (arrow); (h) skin treated with 50 mg/kg of fusidic acid showing normal epidermis and dermis layer with mild inflammation (arrow).



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Table 1: Antimicrobial and hemolytic activity of cationic chitosan derivatives Microorganism MIC or HC50 (µg/mL) HTCC 1 HTCC 2 HTCC 3 HTCC 4 HTCC 5 HTCC 6 or mammalian cell S. aureus 250 250 125 250 250 125 E. coli 500 500 250 500 250 250 Bacteria A. baumannii 1000 500 250 500 500 250 MRSA 250 250 125 250 250 125 VRE 250 250 125 250 250 125 K. pneumoniae 1000 500 500 1000 1000 250 C. albicans 250 250 250 250 250 500 C. dubliniensis >250 >250 >250 >250 >250 >250 Fungi C. tropicalis 500 250 250 250 250 250 C. neoformans Ser A 250 250 250 250 250 250 C. neoformans Ser B 250 125 125 250 250 125 C. neoformans Ser D 500 500 250 500 250 250 Human red blood cell (hRBC) >10000 >10000 >10000 >10000 >10000 >10000 MIC = Minimum inhibitory concentration, MRSA = methicillin-resistant S. aureus, VRE = vancomycin-resistant E. faecium, HC50 = hemolytic concentration at which 50% hemolysis occurs against hRBC


Page 34 of 36

Page 35 of 36

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Table 2. Antibacterial activity of chitosan derivatives against clinical isolates Bacterial clinical isolates

MIC (µg/mL) HTCC 1

HTCC 2

HTCC 3

HTCC 4

HTCC 5

HTCC 6

MRSA R3545 MDR 250 125 125 125 125 125 MRSA R3889 MDR >250 250 125 >250 250 250 E. coli R3597 MDR 250 125 125 250 125 125 E. coli R250 MDR >250 250 250 250 250 250 A. baumannii R676 NDM-1 250 125 125 250 125 125 A. baumannii R674 >250 >250 250 >250 >250 250 E. cloacae R3921 NDM-1 250 125 125 250 250 125 E. cloacae R2928 >250 250 125 >250 250 125 K. pneumoniae R3421 MDR 500 250 125 250 250 125 K. pneumoniae R3949 NDM1 >250 >250 250 >250 250 250 P. aeruginosa R596 MDR >250 >250 >250 >250 >250 >250 P. aeruginosa R590 MDR >250 250 250 >250 250 250 MIC = Minimum inhibitory concentration, MRSA = methicillin-resistant S. aureus, MDR = multi drug-resistance, NDM = New Delhi Metallo-beta lactamase



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Table of Contents Graphic:


Page 36 of 36

Chitosan Derivatives Active against Multidrug-Resistant Bacteria and

Recommend Documents