Peptide-Based PolymerâPolyoxometalate Supramolecular Structure

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Peptide Based Polymer- Polyoxometalate Supramolecular Structure with Differed Antimicrobial Mechanism Lakshmi Priya Datta, Riya Mukherjee, Subharanjan Biswas, and Tapan Kumar Das Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.7b02916 • Publication Date (Web): 14 Nov 2017 Downloaded from http://pubs.acs.org on November 15, 2017

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Peptide based Polymer- Polyoxometalate Supramolecular Structure with Differed Antimicrobial Mechanism Lakshmi Priya Datta,† Riya Mukherjee, †Subharanjan Biswas,‡ Tapan Kumar Das*,† †

Department of Biochemistry & Biophysics, University of Kalyani, Kalyani, Nadia - 741235,

Nadia, West Bengal, India ‡

Department of Chemical Sciences, Indian Institute of Science Education and Research Kolkata,

Mohanpur-741246, Nadia, West Bengal, India

1 ACS Paragon Plus Environment

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ABSTRACT. Because of the increasing prevalence of multi-drug resistance feature, several investigations have been so far reported regarding the antibiotic alternative supramolecular bioactive agents made of hybrid assemblies. In this regard, it is well established that combinational therapy inherited by assembled supramolecular structures can improve the bioactivity to some extent but their mode of actions have not been studied in details. We provide the first direct evidence that the improved mechanism of action of antimicrobial supraamphiphilic nanocomposites differ largely from their parent antimicrobial peptide based polymers. For the construction of hybrid combinational system, we have synthesized side chain peptide based antimicrobial polymers via RAFT polymerization and exploited their cationic nature

to

decorate

supra-amphiphilic

nanocomposites

via

interaction

with

anionic

polyoxometalates. Due to cooperative antimicrobial properties of both polymer and polyoxometalates, the nanocomposites shows enhanced antimicrobial activity with different antimicrobial mechanism. The cationic stimuli responsive peptide based polymers attacks bacteria via membrane disruption mechanism whereas free radical mediated cell damage is the likely mechanism of polymer-polyoxometalate based supra-amphiphilic nanocomposites. Thus our study highlights the different antimicrobial mechanism of combinational systems in details which improves our understanding of enhanced antimicrobial efficacy.

INTRODUCTION. Supramolecular self-assembly driven electrostatic interaction plays a pivotal role in several biological events by creating hierarchical structural features with diverse functionality.1-6 By exploiting the supramolecular interaction it is possible to make a plethora of macromolecular hybrids having distinct bioactive properties along with enhanced biocompatible feature.7, 8 Thus utilization of supramolecular self-assembly becomes an attractive way to modulate microbial 2 ACS Paragon Plus Environment

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killing feature. The tuning of bacterial killing ability by reversible supramolecular assemblydisassembly processes have recently explored by several groups.3, 9-11 For example, Diacon et al. have reported hybrid sandwich material consists of polymer colloids, silver nanoparticles and multiwall carbon nanotubes with potential bioactivity.12 Similar observation has been addressed by T. Pradeep group where the antimicrobial activity enhances due to combined action of both nanoparticles and the polymer.13 Very recently the Lee group has reported supramolecular selfassembly driven multivalent nanofibers with improved bioactivity.14 The yang group has reported self-assembled core–shell nano-micelles bearing a hydrophobic cholesterol core and a hydrophilic cationic peptide shell with TAT peptide molecules bearing enhanced antimicrobial activity. The self-assembled nano-micelles are expected to increase local charge density and mass thereby promoting increased bioactivity.15 Apart from improved bioactive feature the selfassembly topology provides excellent biomimetic extracellular surfaces with differential actions that may improve the cell-cell interaction in a differential manner.16 These observations suggest that supramolecular assemblies possess improved bioactivity however the pattern of work might be substantially different from their parent components. To date antimicrobial polymers with cationic and amphiphilic features are of increasing interest due to their reduced cytotoxicity, protease inactivity, higher blood circulation time and enhanced antimicrobial efficacy.17-20 Because of the absence of specific receptor-ligand interaction, the development of mutation associated drug resistance pattern is limited here rather than the conventional antibiotics. The physicochemical properties of the polymers can be modified via adjustment of their hydrophobicity, ion functionality and amphiphilicity.21,

22

Incorporation of cationic charges increases the possibility of binding to the anionic bacterial cell membrane via electrostatic interactions followed by membrane disruption and leakage of cellular


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constituents.23-25 The increasing interest in antimicrobial polymers stimulated us to design new peptide based cationic antimicrobial polymethacrylates with potential bioactivity. Incorporation of peptide sequences within the side chain of the polymers is significantly appealing due to formation of ordered secondary structures along with biocompatible feature.26-28 Although there are few reports regarding the polymerization of side-chain peptide based monomers by several techniques,29, 30 to the best of our knowledge, there are no reports of antimicrobial activity study of side chain peptide based cationic polymers synthesized via RAFT polymerization. Since the antimicrobial activity depends on cationic-hydrophobic balance of the polymers, we have chosen phenylalanine-alanine dipeptide sequence for designing of amphiphilic peptide based pH responsive polymers via RAFT polymerization procedure. Though the incorporation of peptides within the polymeric architecture makes them highly biocompatible but the highly cationic nature of the polymer may interfere forming colloidal aggregation when it come in contact with some anionic moieties during disinfection procedure. Notably during oral uptake the cationic moieties may interact with anionic mucin of salivary gland leading to astringency or with the anionic moieties of the residual digestive components followed by compromised bioactivity.31, 32 It is therefore important to improve the quality of bioactivity profile along with maintaining the biocompatible feature. As mentioned earlier, the supramolecular self-assembly driven conformation can be able to improve the microbial killing activity; therefore the cationic feature of the polymer has been exploited to form stimuli responsive polymeric supra-amphiphiles. The major advantages offered by the supra-amphiphiles is the possibility to decorate hybrid assemblies derived from wide range of functional materials with properties predominate over their parent components. Realizing that the advantageous feature of both organic and inorganic based polymeric materials


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is much broader in scope, herein, we have attempted to develop inorganic-organic polymeric hybrids via exploiting supramolecular chemistry. Polyoxometalates are chosen as the anionic counterparts with remarkable ability of self-assembly driven functionalization via ionic interactions.33-36 The rigid ordered framework and bioactivity also make them unique in terms of potential bioapplication.37, 38 Apart from their anticancer, antibacterial and antiviral properties, polyoxometalates are potentially reported for amyloid inhibition via preventing Aβ aggregation.39 The main driving force for such inhibition was simple electrostatic interaction between cationic peptide and anionic polyoxometalates. Ionic interaction mediated fabrication of higher order assemblies are also particularly appealing for multifaceted applications along with biocompatible feature. The enlargements of applications of polyoxometalates are significant from biological perspective since their cytotoxic feature and physiological instability restricts their clinical applications.40, 41 Therefore in order to achieve the essential bioactive feature along with construction of higher order assemblies it is important to investigate self-assembly feature for a wide range of substances. Herein, the ionic self-assembly between side chain peptide based cationic polymers and anionic polyoxometalates leads to the formation of well-defined multivalent nanocomposites with enhanced antimicrobial activity. The polyoxometalates also have the ability to transform the β-sheet polymers to cationic multivalent nanorods upon interaction. Noteworthy is that the antimicrobial mechanism of the rod like nanocomposites is totally different from their parent systems that explain their advanced efficacy over their counterparts. In addition to enhanced bioactive feature, the co-assembly between polymer and polyoxometalate leads to formation of highly biocompatible nanocomposites thereby expanding their applicability.


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EXPERIMENTAL SECTION Methods Monomer Synthesis The alanine based vinyl monomer, Boc-L-Ala-HEMA, was synthesized by esterification reaction between HEMA and Boc-L-Ala-OH, according to the previous procedure reported for the synthesis of amino acid based vinyl monomers.42, 43 Herein, ethyl acetate has been used as coupling solvent for the esterification reaction. The condensation reaction was carried out in presence of DCC as a coupling agent and DMAP as catalyst. At first, 12 g (63.4 mmol) of BocL-Ala-OH was added in ethyl acetate (50 mL) and purged with argon gas followed by constant stirring. DCC (13.081 g, 63.4 mmol) and DMAP (0.77 g, 6.34 mmol) were eventually dissolved within the reaction system followed by dropwise addition of HEMA (8.244 g, 63.4 mmol) under ice-bath condition. The reaction mixture was allowed to keep under room temperature for 24 h. After completion of the required time period, insoluble N,N′-dicyclohexylurea (DCU) was removed via suction filtration from the reaction environment. The organic layer was then thoroughly washed with 2 × 60 mL 1.0 N HCl solution, 2 × 60 mL saturated NaHCO3 solution, and finally with 2 × 60 mL brine solution. Then, the collected organic layer was dried over anhydrous Na2SO4, concentrated by a rotary evaporator and purified by column chromatography using 7% ethyl acetate in hexanes to a get white solid, gravimetric yield = 92%. 1H NMR (Figure S1, CDCl3, δ, ppm):

6.12 and 5.59 (C=CH2, 2H, s), 5.05 (NHCOO, 1H, s), 4.49-4.29

(OCH2CH2O and CH3CHCOO, 5H, m), 1.94 (C=CCH3, 3H, s), 1.42 (C(CH3)3, 9H, s), 1.26 (CH3CHCOO, 3H, m). To eliminate the Boc group, the purified Boc-Ala-HEMA (7.0 g, 23.33 mmol) was then dissolved in 40 mL of ethyl acetate and 15 mL of TFA was added to this solution in an ice-water


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bath followed by overnight stirring at room temperature. The reaction mixture was thoroughly washed with saturated NaHCO3 solution and the organic layer was dried over anhydrous sodium sulfate. After removing the excess organic solvent by rotary evaporation, the resultant product was further purified by silica gel column chromatography using 4% methanol/DCM, resulting in pure colorless liquid compound H2N-Ala-HEMA, with a yield of 87%. Boc-Phe-OH (4 g, 10.6 mmol) and NH2-Ala-HEMA (3.2 g, 10.6 mmol) were then dissolved in dry DCM (30 mL) and the solution was kept under argon atmosphere. DCC (2.18 g, 10.6 mmol) and DMAP (0.12 g, 1mmol) in 10 mL of dry DCM was subsequently added to the reaction mixture in an ice-water bath under stirring and was allowed to keep at room temperature for 24 h. After 24 h, insoluble N, N´-dicyclohexylurea (DCU) was removed from the reaction system via suction filtration. The organic layer was then washed with 1 N HCl, saturated NaHCO3andbrine solution and dried over anhydrous Na2SO4. After removing the excess organic solvent rotary evaporator, the crude product was purified by silica gel column chromatography using 4% methanol/DCM as mobile phase), to get a white solid compound Boc-Phe-Ala-oxyethylmathacrylate (Boc-FA-HEMA), with a yield of 85%. 1H NMR (Figure S2 in the ESI,† CDCl3, d, ppm): 7.32–7.16 (C6H5, 5H,m), 6.43–6.39 (CONHCH, 1H, d), 6.13 and 5.57 (C=CH2, 2H, s),4.87–4.78 (C6H5CH2CH, 1H, m), 4.55–4.29 (OCH2CH2O and COCHNH, 5H, m), 1.23 (NHCCH3, 3H, s), 1.39 (C(CH3)3, 9H, s). ESI-MS (Figure S3 in the ESI†): [M + Na+]= 471.209 m/z. General method of RAFT polymerization For the RAFT polymerization process, Boc-FA-HEMA (0.20 g, 0.446 mol), CDP (7.20 mg, 0.017 mol), AIBN (0.558 mg, 0.0034 mmol) and DMF (1 mL) were added in a 20 mL septa sealed glass vial. In a preheated reaction chamber of 70 0C the reaction vial was placed under constant stirring and argon gas has been passed within the reaction mixture for 20 minute to


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create inert atmosphere. The reaction kinetics was monitored by withdrawing around 0.1 mL of reaction mixture from the reaction system. After a predetermined time, the polymerization reaction has been quenched by keeping the reaction vial under ice in presence of air. The polymer has been purified by acetone-hexane purification method by dissolving in minimum amount of acetone and re-precipitated several times in cold hexane to get yellow powdered polymer. Deprotection of Boc group The water soluble polymers have been obtained by the elimination of Boc group from the pendant amine moieties using TFA in DCM at room temperature. 0.02 g polymer was dissolved in 0.5 mL DCM in a 15 mL vial followed by addition of 0.5 mL of TFA under constant stirring. The reaction environment was kept at room temperature under stirring for 2 h. Finally, the Boc deprotected polymer was isolated by precipitation from cold diethyl ether. Preparation of polymeric supra-amphiphiles with polyoxometalate For the preparation of polymeric supra-amphiphiles, at first the polymer aqueous solution (1 mg/mL) was prepared by the addition of powdered polymer in deionized water (Millipore 0.22 µm filter, 18 Ω) to obtain homogeneous and optically transparent solutions. The supramolecular self-assembled polymer-polyoxometalate conjugates were prepared by adding different amount of powdered polyoxometalate to the polymer solution under constant stirring to obtain different molar ratios of polymer and polyoxometalate with the concentration of polymer kept constant at 1 mg/mL. Appearance of initial turbidity as initiated by Tyndall effect indicates towards formation of stable supramolecular aggregates. The resulting supramolecular complexes were then isolated from the aqueous suspension via centrifugation at 5,000 rpm for 15 min followed by lyophilization.


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RESULTS AND DISCUSSION The synthesis of small dipeptide derived Boc-Phe-Ala-oxyethyl methacrylate (Boc-FAHEMA) monomer has shown in Scheme 1 depicting simple coupling reaction between H2N-AlaHEMA with Boc-L-Phe-OH results in the formation of Boc-FA-HEMA monomer. Firstly, BocAla-HEMA has been synthesized (Figure S1) via simple coupling reaction between Boc-Ala-OH and HEMA. The modification efficiency was determined by 1H NMR spectroscopy by comparing the integration of the vinyl protons of Boc-Ala-HEMA monomer with the DMF protons at 8.02 ppm. DMF solvent has been taken as an external standard and the conversion efficiency as determined is 85%. Formation of Boc-Ala-HEMA has been further confirmed via FTIR spectroscopic analysis. Figure S2 represents the FTIR spectra of Boc-Ala-HEMA depicting characteristic absorption band at 1515 cm-1 for N-H deformation and at 1730 cm-1 for C=O stretching. The resulting compound is then treated with TFA in chloroform at room temperature for subsequent deprotection of the Boc groups.43, 44 The deprotected vinyl monomer NH2-AlaHEMA was then introduced for amide coupling reaction with Boc-Phe-OH in presence of DCC and DMAP followed by synthesis of Boc-Phe-Ala-HEMA or Boc-FA-HEMA. The appearance of two vinyl proton peaks at 5.60 and 6.11 ppm followed by the presence of amide peak of the dipeptide linkage at 6.47 ppm confirms the successful amide coupling reaction via peptide bond formation (Figure S3). Characteristic resonance signals for the phenyl group protons at 7.18– 7.30 ppm, 4.34–4.53 (backbone methylene protons in HEMA), 3.04 (benzyl peak), 1.23 (alanine side chain methyl protons), 1.39 (methyl protons of the Boc group) also assures the formation of coupling product. The experimental molecular mass ([M + Na]+ = 471.209 m/z) as obtained from the ESI-MS analysis matched nicely with the theoretical mass confirming successful synthesis of the Boc-FA-HEMA monomer (Figure S4).


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The structural evidence of the formation of Boc-FA-HEMA was further confirmed by the FTIR spectra (Figure S5); where the characteristic absorbance peaks for the amide groups appear at 3326 and 1533 cm-1. Presence of strong amide-I bond at 1660 cm-1further suggests towards peptide bond formation. The C-O stretching band appears at 1168 cm-1 whereas the two sharp band at 1727 and 1757 cm-1 are assigned to the stretching vibrations of carbonyl (C=O) groups, confirming the presence of C=O (ester) linkages.

Scheme 1. Schematic representation of formation of side-chain peptide based polymers and their self-assembly with polyoxometalate.


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Taking into account that CDP can efficiently polymerize the dipeptide based polymers via RAFT polymerization method as reported elsewhere,29 herein we have used CDP as chain transfer agent for the efficient polymerization of Boc-FA-HEMA at 70 0C in DMF using AIBN as initiator. The polymerization have been performed at different [monomer]/[CDP]/[AIBN] ratio. The yield and molecular weight distributions have been summarized in table 1. The initial characterizations of the homopolymers have been done by GPC and 1H NMR spectroscopy technique. In all the GPC measurements with increasing conversion unimodal refractive index traces shifted towards lower elution volume suggesting the controlled feature of RAFT polymerization. Absence of any bimodal peak along with the presence of narrow poly dispersity index (PDI) in Figure 1a implicates that no bimolecular termination takes place during polymerization. The appearance of different resonance signals from the repeating unit protons and chain transfer agents in the 1H NMR spectrum confirms the efficient polymerization reaction (Figure 2a). Comparison of the integration areas from the terminal CH2–CH2– protons (from the HOOC–CH2–CH2–C(CN)(CH3)– chain end) at 2.3 ppm and Boc group protons at 1.44 ppm allowed calculation of the number-average molecular weight (Mn,NMR) from the 1H NMR spectroscopy. Table 1 clearly indicates nice agreement between the theoretical molecular weight, molecular weight from GPC and molecular weight from NMR. The pseudo first order kinetic plot as depicted in Figure 1b along with a constant number of propagating chain ends confirms the controlled radical polymerization process. The solubility of the resulting homopolymers has been analyzed in different solvents. Except water, hexane and pet ether, the homopolymers were found to be soluble in most organic solvents.


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Table 1. Results from the synthesis of P(Boc-FA-HEMA) homopolymers in DMF at 70 °C. Expt. No. 1 2 3

[M]/[CTA]/[AIBN] Time (h) 15:1:0.2 5 25:1:0.2 5 50:1:0.2 5

Conv.a (%) 52 58 62

Mn, GPCb (g/mol) 4580 6700 18200

Ð 1.15 1.23 1.34

Mn, NMRc (g/mol) 5100 7200 NDi

Mn,theod (g/mol) 3400 7820 13700

Figure 1. GPC RI traces of a) P(Boc-FA-HEMA) synthesized at different [Monomer]/[CDP] ratio, b) pseudo first order kinetic plot of P(Boc-FA-HEMA) homopolymers.

Aqueous solubility and charge balance maintenance are two crucial parameters for bioactivity applications. Thus to make the polymer water soluble and to incorporate cationic charge within the macromolecular framework, the pendant Boc groups have been eliminated by treating the polymer with TFA/DCM at room temperature. Successful expulsion of the Boc group from the polymer has been confirmed by the absence of Boc proton signals at 1.44 ppm in the 1H NMR spectroscopy (Figure 2b). After successful removal of the Boc group, the resulting polymer


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Figure 2. 1H NMR spectra of a) P(Boc-Phe-Ala-HEMA) in CDCl3, and b) P(H3N+-Phe-AlaHEMA) in D2O. becomes soluble in aqueous media. The FT-IR spectra in Figure S6 also confirms the Boc group elimination where the amide II peak for N-H deformation at 1527 cm-1 present in the monomer and Boc protected polymer certainly disappear in the deprotected polymer. However appearance of a new peak at 1587 cm-1 may be due to the presence of N–H (amide II band) band from pendant amine moiety after Boc group deprotection. Since the free amine pendant groups in the Boc deprotected polymer can reversibly protonated and deprotonated by modulating the solution pH, thus the pH responsive feature of the polymers were evaluated by turbidimetric measurement as a function of pH at 25 0C (Figure S7). The reversible protonation-deprotonation can result the transparent solution to opaque gradually and above a particular pH, initial turbidity


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appears within the solution due to complete deprotonation of amine groups, thus indicating the pH responsive feature. For our polymer the pH responsiveness have been observed at pH 7.14. Since the phenylalanine containing dipeptide segments are already reported to acquire beta sheet conformation,45,

46

thus the possibility of formation of secondary structures have been

evaluated in water by FESEM analysis. The FESEM images in Figure S8a confirmed the formation of extended sheet like structures in aqueous solution. Formation of sheet like secondary conformation is evoked by the several factors like amphiphilic feature of the polymer, alkyl groups of alanine and the aromatic phenyl groups group in phenylalanine. Presence of phenylalanine segment is important since aromatic stacking actually dominates the higher order secondary orientation. Apart from the aromatic stacking, the hydrophobicity, more specifically hydrophilic-hydrophobic balance plays a crucial role during formation of secondary structures. Noteworthy that ile-phe dipeptide is reported to form secondary structure while val-phe inhibits any secondary orientation might be due to lower hydrophobicity of valine unit.46 Deprotection of pendant amine moieties is a significant feature regarding secondary orientation since the monomer is devoid of any higher order structure formation (Figure S8b). To confirm that the secondary conformation is not an artifact of experimental processes specially drying process, the CD analysis has been performed. The CD spectrums of the deprotected peptide polymer and the monomer have been recorded in methanol. The spectra of Boc-deprotected polymer represents characteristic peaks correspond to β-sheet conformation as compared to the monomer (Figure S9). The presence of strong positive maxima 195 nm along with a negative minimum at 213 nm corresponds to the n–π* and π-π* transitions demonstrating the presence of β-sheet orientation. The H-bonding type interaction between ester oxygen and


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free pendant N-H proton of amine units however contributes to some extent towards formation of ordered structure. The FT-IR spectra also support the above conformational organization (Figure S6). The βsheet structures in proteins and synthetic biomacromolecules are mainly associated with amide I band in the region of 1600 to 1700 cm-1. Appearance of characteristic amide I band at 1677 cm-1 represents the presence β-sheet structure within our polymer. Apart from the amide I band existence of peaks at amide II and amide V region at 1587 cm-1 and at 700 cm-1along with the absence of peak at 620 cm-1 (corresponds to α-helix) confirms the β-pleated structure. Another strong intense band at 3422 cm-1 suggests presence of intermolecular hydrogen-bonded network of NH groups thereby complementing the FE-SEM analysis and CD spectra. With the anionic feature of polyoxometalate in mind, the self-assembled supra-amphiphilic nanocomposites have been prepared by interaction of polyoxometalates with the cationic peptide polymer. The self-assembly process have been initiated by mixing different molar ratios of polymer and polyoxometalate in tris-HCl buffer solution of pH 7 with the concentration of polymer fixed at 1 mg/mL. In order to obtain stable nano-dispersions, the formation of supraamphiphiles was monitored by DLS, SEM and turbidimetric measurement procedure. At a polymer: polyoxometalate ratio of 1:0.2 and 1:0.5, transparent solutions were observed with the DLS count rate negligibly small. This may be due to the increased amount of cationic charges of polymer with respect to the anionic charges, thus the yield of supraamphiphiles become low. At a ratio of 1:1 non agglomerated nanorods with stable dispersion have been observed. The increase in DLS count rate (Figure S10) further confirms the beginning of formation of selfassembled supra-amphiphilic architecture. This electrostatically driven supra-amphiphilic interaction thought to originate from the coacervation triggered weak interactions, where two


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oppositely charged biomolecules results in complexation via multi-phase separation.47 The FESEM images in Figure 3 depict formation of rod like morphology. The polyoxometalate mediated charge neutralization of the cationic side chains of the polymers along with assemblage of the anions around the secondary structure of the peptide polymer could affect the morphological switching. The evidence of morphological switching was obtained by analyzing the SEM images (Figure 3) of time dependent complexation. Herein the peptide based cationic polymers are attracted by the anionic polyoxometalates via electrostatic interaction which leads to the breakage of integrity of the extended sheet like structure of the polymer. Figure 3a is the low magnification image depicting the assembly of β-sheet peptides while Figure 3e is the higher magnification image corresponding to Figure 3a. Figure 3e indicates the presence of extended sheet like structure (marked with yellow arrow) of the polymers. Figure 3b features a transition state image prior formation of nanocomposite after 45 min of addition of polyoxometalate in polymer system. Corresponding higher magnification FESEM image (Figure 3f) emphasizes the initial breakage of extended sheet like morphology, as evidenced from the lowering of the diameter; breakage of sheets are marked by white arrows. Figure 3c is the image after 90 min of addition of polyoxometalate. The disintegration and fracture along the sheet can be visualized in Figure 3c. The corresponding higher magnification image more clearly indicates the dissolution of compact sheets (yellow arrows) and evolution of rod like structures (red arrows). Figure 3d implicates completion of nanorods formation where there is no evidence of presence of any sheet like structures instead presence of individual rods are visualized. Figure 4f is the higher magnification image of Figure 4d precisely featuring a bundle of rod like nanocomposites (red arrows).


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The conformation evolution is typically a thermodynamically favorable process as evidenced by the isothermal titration calorimetry (Figure S11) measurement. The measurement proves that the composite formation induced via electrostatic interaction is a highly exothermic process with ∆H= -100 KJ mol-1 and ∆S= -213 J mol-1K-1. The high binding constant (K= 2. 2 X 106 M-1) implies that the composite formation is largely facilitated via ionic interactions. Breakage of ordered structure is entropy driven favorable phenomenon and here the highly ordered extended sheet has been cleaved via anionic polyoxometalates therefore providing energetic stabilization for the rod like nanocomposites. From the observations it can be proposed that the electron-rich polyoxometalates can insert within the densely packed H-bonding network of β-pleated sheet forcing the network to break up into individual rods. At a ratio of 1:2, turbidity of the reaction system increases initially along with rise of agglomeration as depicted in SEM. However the DLS count rate did not change indicating no further advancement in complexation by changing the molar ratio. The rise in turbidity though limits their applicability. Further rise in molar ratio to 1:5 results in immediate precipitation.

Figure 3. Time dependent morphological studies of the mechanistic pathway of formation of supramolecular nanocomposites after addition of polyoxometalates within polymer P(H2N-FAHEMA) solution, upper panel (lower magnification) and lower panel (higher magnification). a) 17 ACS Paragon Plus Environment

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& e) FE-SEM images of P(H2N-FA-HEMA) before addition of polyoxometalate, which indicate sheet like morphology of the polymer; individual sheets are marked by yellow arrows. b) & f) after 45 min of addition of polyoxometalate; breakage of sheets are indicated by white arrows. c) & g) after 90 min of addition; along with dissolution of sheets (yellow arrow), appearance of nanorods observed (red arrows). d) & h) after 120 min of addition; complete disappearance of sheets, only nanorods are observed (red arrows).

The composite formation was further confirmed via FT-IR spectra in Figure S12a. The feature associated with the characteristic groups mainly O-P stretching frequency at 1077 cm-1, the W-O terminal frequency at 977 cm-1, W-O-W stretching frequency at 893 cm-1 and the edge sharing octahedral frequency of W-O-W at 798 cm-1 are all present within the composite material. However they are slightly shifted towards shorter wavelength which is an expected feature of composite formation. The composite formation has been further verified by Raman spectroscopic analysis. Raman spectrum of polyoxometalate is shown in Figure S12b. A characteristic doublet is observed at 218 cm-1 (δ(O-W-Ot), δ(W-O-W)), and 238 cm-1 (νs(W-O), δ(W-O-W)). Band at 518 cm-1 is due to (δ(W-O-W), δ(W-O-W), δ(O-P-O)). At higher energy region bands observed at 952 cm-1 (νas(W-Ot)) and 1004 cm-1 (νs(W-Ot)). All of these are peaks are characteristic bands of phosphotungstate Keggin. In the Raman spectrum of the composite, all of these bands are present with a little bit of shift for some cases indicating intactness of the Keggin network and composite formation, respectively. Some bands of the polyoxometalate are coupled with fingerprint bands of the polymeric network in the composite; such as the doublet at 218 and 238 cm-1 and the peaks at 518 and 1004 cm-1 get broadened due to intermolecular interactions. Band


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at 611 cm-1 is due to methacrylate moiety. Thus Raman spectra confirm the composite formation between polyoxometalate and the polymeric network. Since both the cationic polymers and polyoxometalates are already individually reported for their antimicrobial activity,5, 14, 37, 48, 49 we have evaluated the antimicrobial activity of cationic peptide based polymer, polyoxometalate and the polymer-polyoxometalate nanocomposites separately. Generally the cationic amphiphilic polymers are renowned for their electrostatically derived membrane disrupting affect. For that purpose, the gram negative bacteria consisting of negatively charged outer membranes are seems to be more sensitive towards actively designed cationic polymers. Keeping this feature in mind, we have first explored the antimicrobial activity over the representative gram negative bacteria Escherichia coli or E. coli. Selecting the gram negative bacteria is also challenging from the standpoint of antimicrobial study because of their wide capability to inhibit the drug mediated actions as inherited by their cell wall structure and responsibility towards formation of more than half of the infections worldwide. Few recent studies have reported that the bioactivity of cationic peptides significantly enhances via formation of self-assembled structure.14, 50 However the mechanism of action is likely to be the same as peptides. We herein introduced an alternative possibility that the supramolecular nanocomposites not only enhance the antimicrobial activity at significantly low doses but their antimicrobial mechanism solely different from their parent molecules (vide infra). The general growth inhibitory effect of the polymers, composites and polyoxometalates has been measured by the traditional spectrophotometric measurement. Evaluation of the minimum inhibitory concentration (MIC) value is the primary determinants of cell killing feature by the external agents. The MIC value of our peptide polymer is 70 µg/mL and the MIC value of our nanocomposite is 25 µg/mL, whereas the MIC value of the polyoxometalate is greater than 330


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µg/mL. The MIC value of polyoxometalates is accountable with previous data where it has been observed that the keggin type phosphate containing polyoxomolybdates shows lowest antimicrobial activity (MIC> 256 µg/mL) rather than other polyoxometalates.51 The MBC value of the polymer-polyoxometalate nanocomposites has been estimated as 32.5 µg/mL. The colloidal stability and surface charge of the components were measured by zeta potential measurement. The surface charges of the deprotected peptide polymers were +48 mv. The cationic primary amine groups are basically responsive for the positive zeta potential value. Whereas the zeta potential value of polymeric nanocomposites are found to be +17 mv, indicating the outer surface of the composites are covered by the cationic polymer segment though most of the cationic charges have been balanced via anionic polyoxometalate. The physicochemical properties specifically the surface charge distribution plays a supreme role regarding the cellular uptake of external agents followed by activity differentiation. Generally the uptake of external agents by the bacterial cells occurs via two consecutive steps: first is the binding interaction and the next step is cell permeabilization. Since the bacterial cell surfaces are dominated by negative charges thus the most possible hypothesis is cellular uptake mediated interactions facilitated by highly cationic charged moieties. Indeed we have observed increased


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Figure 4. FESEM images of E. coli cells: a) control cells, where the bacterial cell membrane have been maintained, b) treated with P(H2N-FA-HEMA) at their MIC value, corrugated surface have been observed, c) treated with P(H2N-FA-HEMA) at their 2X MIC value, fractured cell debris have been observed, d) treated with nanocomposites at their sub-MIC value, cell filamentation have been observed, e) treated with nanocomposites at their MIC value, e) treated with nanocomposites at their 2X MIC value.

bioactivity from less cationic nanocomposites suggesting the active insertion of the composites within bacterial system. The active insertion within the bacterial system however be facilitated by the hydrophobic effect assimilated by the two hydrophobic amino acids along with the long hydrophobic tail of CDP predominates over the cationic charge due to reduced charge density. In case of polymethacrylates, Kuroda et al. have observed a gain in biocidal activity along with increasing hydrophobicity.52 In this context, it can be said that cationic charges within the 21 ACS Paragon Plus Environment

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polymers are important for selectivity, whereas masking of cationic charges increases the hydrophobicity which ensures the activity parameter. In the next stage, we have attempted to investigate the mechanism of action of both polymer and polymeric composites over bacterial cell membrane. We have not proceeded further with only polyoxometalates due to its extremely high MIC value that restricts its significance as a potential antimicrobial agent. The bacterial cell suspensions were incubated with both the samples at their different MIC value for 2 h. After that specific time, FESEM images were taken to observe any type of morphological changes in the bacteria. From the FESEM images in Figure 4 it can be seen that the cells treated with polymers exhibit distorted and corrugated cell surface which in turn forms fractured cell debris after further incubation. This phenomenon suggests that the biocidal activity happens through membrane disruption mechanism. It is already hypothesized that the biocidal activity of cationic beta sheet polymers occurs through electrostatic interactions.49,

53

This hypothesis actually obeys three consecutive steps: i)

interaction followed by binding of the cationic polymers with anionic bacterial cell membrane ii) insertion of the polymer within cell interior iii) cell wall disruption and leakage of the cell constituents. At a certain local concentration, the hydrophobicity of the polymeric architecture facilitates the entry of the polymer through the membrane lipid bilayer. Rather than α-helix, βsheet structured peptides are more potent towards cytoplasmic insertion and membrane leakage while maintaining their biocompatible feature.54 Surprisingly, in case of polymerpolyoxometalate nanocomposites, the bacterial cell morphology appear to be filamentous. Formation of filamentous morphology leads us to think about alternative cell killing mechanism apart from the membrane disrupting mechanism. The cell filament formation is primary determinant of free radical generation.


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Figure 5. Phase contrast microscopic images of E. coli cells: a) control cells, b) treated with nanocomposites at their MIC value, c) treated with nanocomposites at their 2 X MIC value.

Cell filamentation feature in bacteria has been recommended as an alteration in cell size and shape under oxidative stress. This is a type of SOS response in which cell defends themselves from external atmospheres by growing without any cell division.40 Adaptation of filamentous structure is a distinct mechanism adhered by bacterial cells in response to oxidative stress where living cells continue to elongate with multiple chromosomal copies without undergoing any cell division .The cell filamentation upon treatment with nanocomposites have been visualized via phase contrast microscopy. Figure 5 shows that in presence of composite cell filamentation occur prior to cell death as compared to the control cell. From the Figure it can be further evidence that the bacterial cell size increases from about 2 to 20 µm upon treatment of the cells at the MIC value of the nanocomposites (Figure 5b). However above the MIC value, the filament size of the cells becomes decreased due to cell death. This phenomenon signifies that the killing of the bacterial cells occur through filament formation. This observation is consistent with the FESEM images where upon treatment with nanocomposites the bacterial cells exhibit filamentous morphology instead of cell membrane disruption. To further explore whether the antimicrobial mechanism of polymer composites are free radical mediated, we have assessed the free radical generation via flow cytometric method. The 23 ACS Paragon Plus Environment

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Figure 6. Flow cytometric analysis of free radical generation in intact E. coli cells: a) unlabeled control cells; b) cells treated with nanocomposites at their MIC value; c) cells treated with nanocomposites at their MBC value.

chemical

compound

2´,7´dichlorodihydrofluoresceindiacetate

(DCFH-DA)

have

been

exploited for the estimation of free radical generation. This is a highly authentic method for the detection of intracellular ROS generation by using the same chemical DCFH-DA. Instead of other physico-chemical methods like electron spin resonance and spin trapping, flow cytometry is advantageous due to its highly sensitive, accurate and rapid cellular assay that measure the quantitative responses of individual cells within total cell population. Furthermore simultaneous characterization of many related cellular parameters like cell size, cell granularity promotes identification of specific subsets within a particular time. The measurement in Figure 6 depicts the percentage distributions of cells in different quadrants. Increase in ROS generation will results in a shift of the cell populations to a different quadrant. The cells were treated at their


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MIC and MBC value to check whether any intracellularly generated reactive oxygen species can hydrolyze the DCFH to highly fluorescent DCF. Nearly 100% of the control labeled cells was represented by the dots in the left lower quadrant (Figure 6a). When the cells were treated with polymer composite at their MIC value (25 µg/mL) for 30 min (Figure 6b), about 36.88% of cells acquired considerable fluorescence label and the remaining 62.89% cells did not. Treatment of cells with polymer composite at their MBC value (32.5 µg/mL), (Figure 6c) corresponds to the labeling of 41.23% of cells at right lower quadrant and 18.64% of cells at right upper quadrant. Considering that cell membrane potential plays a significant role in the free radical mediated cell killing mechanism, we have determined the cell membrane potential of the polymer composite treated cells. The alternation in membrane potential also a primary determinant for the filamentous feature of cells since several protonophores blocks the cell division pathway by altering the transmembrane potential of cells.55, 56 Subsequent depolarization of the membranes inactivate the ‘Min’ site selection system thereby preventing localization of one of the conserved cell division protein FtsZ followed by hindering cell division. The membrane potential has been determined by treating the cells at their MIC (25 µg/mL) and MBC (32.5 µg/mL) value. The flow cytometric measurements were initiated to confirm the membrane depolarization feature. The dye 3,3'-diphenylthiocarbocyanineiodide have been used as a marker for determination of alteration in membrane potential value. Since the binding of the dye is membrane potential dependent; more negative the membrane potential more positive the binding mode, thus measuring the fluorescence quenching it is possible to determine the alteration in membrane potential value.57 In Figure 7 the cell populations represented by dot in the lower left quadrant signifies the unlabeled control cells. That means nearly 95% of the unlabeled cells are limited in the lower left quadrant. Whereas, when the cells were labeled with the fluorescent dye 3,3´-


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diphenylthiocarbocyanine iodide for 2 h about 76.55% of cells acquired considerable fluorescence label followed by shifting towards lower right quadrant.

Figure 7. Flow cytometric analysis of membrane potential of intact E. coli cells: a) unlabeled control cells; b) labeled control cells; c) cells treated with nanocomposites at their MIC value; d) cells treated with nanocomposites at their MBC value.

When the cells treated at their MIC and MBC values, only about 36.56 and 21.89% of cells were labeled. The decrease in the amount of labeling in the treated cells with respect to the labeled control cells implied that the nanocomposites caused dissipation of the bacterial plasma membrane potential. In case of treated samples, it was observed that maximum number of cells with depolarized membranes were populated in the lower left quadrant compared to that in the case of control labeled cells. The increase in membrane potential results in fluorescence quenching. Thus, the flow cytometric results clearly signified that the exposure of the bacterial cells to the nanocomposites at their MIC and MBC value caused cell membrane depolarization subsequently causing dissipation of membrane potential.


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Figure 8. Flow cytometric histograms of PI uptake by E. coli; a) control, b) cells treated at subMIC value, c) cells treated at MIC value and d) cells treated at MBC value.

The membrane disintegration or permeabilization feature of the nanocomposite treated bacterial cells has been further confirmed via flow cytometric analysis using the fluorescent dye propidium iodide (PI). According to the principle the compound cannot penetrate the live cell membranes, while it can easily permeate the dead cell membranes followed by intercalating with DNA. Intercalation with DNA leads to excitation at the longer wavelength 488 nm of which the intensity can be collected using the FL3 channel. Figure 8 represents the fluorescence histogram plots designating the H-1 and H-2 regions which indicate the position of live (non-fluorescent) and dead (fluorescent) cells respectively. Based on the fluorescence intensity distribution, the histogram in Figure 8a indicates that almost 82% of the normal living cells occupied the H1 region. Figure 8b represents the cells treated with nanocomposites at their sub-MIC value where about 72% of living cells are occupied within H1 region. Compared to Figure 8a and 8b, 8c and 8d implicates localization of about 57 % and 90% cells in the H2 region suggesting


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disintegration of membranes followed by uptakes of PI within cell interior. These two histograms are particularly for the cells treated with nanocomposites at their MIC and MBC value. At the MBC value, most of the bacteria presumed to be dead thereby uptake of dye followed by DNA intercalation results in increased fluorescence. This feature indicates the predominance of dead cells with considerable fluorescence property is inheriting by the uptake of PI through disintegrated membranes.

Figure 9. a) Agarose gel electrophoretic pattern of the chromosomal DNA isolated from the E. coli K12 cells treated with polymer. b) Agarose gel electrophoretic pattern of the chromosomal DNA isolated from the E. coli K12 cells treated with polymer-polyoxometalate nanocomposites.

The major consequences of free radical generation are cleavage of the chromosomal DNA by attacking the base and sugar moieties present in DNA. Cleavage of the DNA results in single and double strand break. To back proof the free radical formation we have performed the DNA degradation experiment in presence of both polymer and polymer composites at their MIC value. We have investigated the fate of DNA by gel electrophoresis assay. The lane 1 in Figure 9a corresponds to the control sample where the intact band of DNA demonstrates that no degradation occurs at the control cell. The lane 2 and 3 corresponds to the cells treated with 28 ACS Paragon Plus Environment

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polymer at their sub-MIC and MIC value respectively for 2 hours. The band pattern in lane 2 and 3 also remain intact may be due to formation of stable polyplexes between the cationic peptide polymer and anionic DNA followed by inhibition of migration. We have then performed the UVVisible spectrometric measurement to ascertain the interaction of DNA with cationic polymer. The complexation with DNA mainly follows three specific binding modes; one is intercalation, second is groove binding and third is electrostatic binding.58, 59 The hyperchromic characteristic is attributed towards external electrostatic binding whereas hypochromism usually indicative of intercalation or groove binding. We have observed the hyperchromic shift in the absorbance spectra with polymer at their sub-MIC and MIC value (Figure S13). The hyperchromic shifts observed is typically ascribed to the electrostatic interaction between cationic polymer and anionic DNA followed by the appearance of non-covalent complexation. In Figure 9b we observe different phenomenon, where incubation of cells with polymer-polyoxometalate nanocomposites at their sub-MIC and MIC value for 2 hour results in fragmentation of the chromosomal DNA due to free radical induced extensive damage w.r.t. control in lane 1. Thereby they left from the groove within a specific time during electrophoresis w.r.t. the control. Free radical induced DNA damage typically leads to formation of very small lesions followed by formation of very small DNA fragments. As discussed earlier, in Figure 9b presence of intact DNA band in lane 1 signifies that no degradation of genomic DNA occurred in untreated cells under normal condition. On the other hand, the smear like band pattern in lane 2 and 3 implies chromosomal degradation in E. coli cells when treated with composites at their sub-MIC and MIC value. The smear like band pattern results from an extensive cleavage where many short DNA fragments of nearly same molecular weights are formed and gathered within a particular zone during the running time of the gel. This phenomenon may lead us to conclude that in vivo


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chromosomal DNA degradation in E. coli starts within 2 hour of treatment with the polymer composite resulting complete killing of bacterial cells.

Scheme 2. Schematic representation of mechanism of action of a) polymer, b) nanocomposite over bacterial cell membrane.

From the above analysis, it is evident that the impact of two antimicrobial agents over bacterial cells is different in nature. The binding of cationic peptide based polymers to anionic bacterial cell membrane typically mediated through a combination of weak interactions. More specifically ionic interaction between cationic polymer and anionic constituents of cell membrane facilitates the entry of polymer within cell interior. Once the polymer enters within cell interior formation of a leakage in the bacterial outer cell membrane may facilitate the entrapment of external agent within cell interior, also termed as self-promoted uptake.60 The entry into cellular cytosol leads to osmotic imbalance followed by release of cellular constituents. In case of polymerpolyoxometalate nanocomposites, they do not appear to interact directly with bacterial cell membrane. The action of nanocomposites typically resembles the mechanism of action induced by metal nanoparticles.6,

61

After entering into the bacterial cell interior, the nanocomposites


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attack the DNA resulting damage of bacterial DNA. The DNA damage induces production intracellular reactive oxygen species (ROS) accelerating the cellular apoptosis. We have introduced the diverse mechanism of action schematically (Scheme 2) where the cationic polymers in section a attacks via electrostatic interaction followed by membrane disruption while the nanocomposites in section b attacks via free radical formation. In the next stage, we have investigated the antimicrobial activity of our components over gram positive bacteria B. subtilis. In general the activity of antimicrobial cationic polymers relies on electrostatic interaction mediated cellular internalization followed by cell disruption. Henceforth the adaptability of our components as potential antimicrobial agents should not be selective towards only gram negative bacteria. However the extent of killing and mechanism of action are important determinants to examine. The MIC and MBC measurements established that both the polymer and the nanocomposites are less effective towards gram positive bacteria rather gram negative ones. The MIC for the polymer was found to be 120 µg/mL while it was 76.5 µg/mL for the composite. The MBC value as determined was 62 µg/mL for the composite. We did not get any MIC value for the polyoxometalates upto 360 µg/mL. In case of cationic polymers, the higher bactericidal activity towards gram negative bacteria over gram positive bacteria has been well documented. The reason behind higher selectivity might be conditional on their cellular structures that inhibit the bactericidal action to some extent.62, 63 The gram positive bacteria B. subtilis consists of very thick peptidoglycan layer (20-90 nm thick) while in case of E. coli the peptidoglycan layer is very thin (8-10 nm thick). Thus the thick peptidoglycan layer may present a hurdle for the external agents to penetrate the cell wall thus hindering the membrane disruption ability. To probe the mechanism of action of our compounds over gram positive bacteria, we have performed mechanistic investigation of morphological changes by FESEM analysis. To


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visualize the impact on bacterial cell membrane the images was taken from bacterial suspensions grown with and without the polymers and composites separately at their MIC. The control B. subtilis cells without any treatment showed the expected cellular morphology with normal membrane structure indicating the healthiness of the cells. The polymer treated B. subtilis cells showed fractured, divided cellular fractions strongly suggesting their compromised situations (Figure 10). However their smooth surfaces have not been fully corrugated as observed in case of gram negative E. coli bacteria. On the basis of MIC results, a possible explanation for this observation would be less penetration ability of the polymer within gram positive bacterial cell interior that might hinders the cell disruption ability. In case of treatment with nanocomposites we observed filamentous morphology instead of divided cellular fractions suggesting an altered mechanism based on free radical mediated interaction of nanocomposites within bacterial cell interior. These observations strongly stimulate the possibility of diverse mechanism of action of polymers and polymer-polyoxometalate nanocomposites.

Figure 10. FESEM images of B. subtilis cells: a) control cells, where the smoothness of bacterial cell membrane have been maintained, b) treated with P(H2N-FA-HEMA) at their MIC value, corrugated surface with fractured morphology have been observed, c) treated with nanocomposites at their MIC value, cell stacking and filamentation as observed.


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The free radical mediated cell damage mechanism has been further confirmed via flow cytometric analysis of free radical generation and membrane depolarization. In case of free radical formation analysis, it has been observed that nearly 100% of the control labeled cells is gated in the left lower quadrant (Figure S14a), while among the MIC dosage treated cells about 50% of cells acquired considerable fluorescence label and the remaining 43% cells did not (Figure S14b). About 72% of the MBC dosage treated cells (Figure 14c) have been labeled. The membrane depolarization of treated cells results in considerable fluorescence quenching in the MIC and MBC treated cells w.r.t. the control labeled cells therefore is evidencing the plausibility of formation of free radicals within cell interior (Figure S15). To observe the fate of the cells, the propidium iodide has been used as a death stain to compare with live cells. The histogram plot of control cells (Figure S16a) indicates that most of the cells are situated in the H1 region. Upon treatment with nanocomposite at their sub-MIC value, about 25% cells have been shifted towards H2 region (Figure S16b). Treatment with nanocomposite at MIC and MBC value designates the shifting of about 72% and 80% cells towards H2 region (Figure S16c and S16d). These features strongly corroborated by other results suggesting the loss of membrane integrity at corresponding MIC and MBC values of the nanocomposites. The non-cytotoxic features of our peptide polymer and nanocomposites have been evaluated over HeLa (human cervical cancer) cell line. The MTT assay has been chosen for in vitro assessment of mitochondrial activity. In presence of mitochondrial dehydrogenase enzyme the terazolium dye reduced to purple colored chromogenic formazan salts. The colorimetric determination thus enables to quantify number of viable cells present within the system. To analyze the cytotoxic feature, HeLa cells were incubated separately with both polymer and nanocomposites at different concentrations starting from 20 µg/mL to 100 µg/mL including the


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untreated controls. The histogram in Figure S17a depicts that both the compounds do not exhibit any potential cytotoxicity over HeLa cell lines suggesting the plausibility of both polymer and nanocomposites as potential bioagents. This observed selectivity over mammalian cell lines may be due to the fact that the anionic surface charge of bacterial cell membrane facilitates the entry of the components via electrostatic interaction followed by cell disruption as compared to mammalian cells. We have further extended our study to visualize the cell morphologies after treating the cells with our samples (Figure 17b). The intact cell morphology of the HeLa cell lines has been visualized by confocal microscopy. The DAPI stained fluorescence images of cells treated with polymer and nanocomposites showed no particular changes in nuclear structure compared to the control. The morphological integrity of the cells was further confirmed by overlaying the DAPI images on bright field phase contrast images. Since the mammalian cell cytotoxicity assessment is a key index for antimicrobial applications thus the biocompatible feature of both polymer and polymer-polyoxometalate nanocomposites hold great potential as novel antimicrobial agents.

CONCLUSION In conclusion, we have employed the facile RAFT polymerization approach for the incorporation of dipeptide sequence within the side chain of polymethacrylates. The pH responsive cationic β-sheet polymers show efficient antimicrobial activity against both gram positive and gram negative bacteria cells via membrane disruption mechanism as evident by the switching of bacterial cell morphology. The cationic feature of the peptide based polymers has been

further

utilized

to

construct

supramolecular

self-assembly

between

anionic

polyoxometalates. Simple electrostatic interaction results in the formation of nanorods from the


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sheet type polymers upon destruction of secondary interactions. The supra-amphiphilic nanocomposites lead to enhanced antimicrobial efficacy with altered mechanism of action. The origin of this altered antimicrobial mechanism is most likely inherited by the polyoxometalates that is enhanced in presence of another potent antimicrobial agent, the polymer. This study has provided an improved understanding of the structural alterations of the nanocomposites with respect to the polymers followed by the consequences of these alterations on the bioactivity profile. Both the peptide based polymer and the nanocomposites exhibit non-cytotoxic feature as evident by the MTT assay against HeLa cell line The DNA binding activity of the cationic polymers also opens up their possibility as gene vector. Finally, we expect that the detailed understanding of altered antimicrobial mechanism will provide fundamental guidelines to design antibiotic mimic smart nanostructured materials.

ASSOCIATED CONTENT Supporting Information. Materials, method, detail description of experiments, additional figures containing FTIR spectra, DLS data, UV-Vis spectrum and pH responsiveness feature are included in the SI. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *[email protected] ACKNOWLEDGMENT Financial support from University of Kalyani is gratefully acknowledged.


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REFERENCES 1.

Wang, C.; Chen, Q.; Wang, Z.; Zhang, X. An Enzyme-Responsive Polymeric

Supramphiphile. Angew. Chem. Int. Ed. 2010, 49 (46), 8612-8615. 2.

Biswas, S.; Mani, E.; Mondal, A.; Tiwari, A.; Roy, S. Supramolecular Polyelectrolyte

Complex (SPEC): pH Dependent Phase Transition and Exploitation of its Carrier Properties. Soft Matter 2015, 12 (7), 1989-1997. 3.

Chang, Y.; McLandsborough, L.; McClements, D. J. Cationic Antimicrobial (ε-

Polylysine)–Anionic Polysaccharide (Pectin) Interactions: Influence of Polymer Charge on Physical Stability and Antimicrobial Efficacy. J. Agric. Food. Chem. 2012, 60 (7), 1837-1844. 4.

Bai, H.; Zhang, H.; Hu, R.; Chen, H.; Lv, F.; Liu, L.; Wang, S. Supramolecular

Conjugated Polymer Systems with Controlled Antibacterial Activity. Langmuir 2017, 33 (4), 1116-1120. 5.

Zhou, C.; Wang, F.; Chen, H.; Li, M.; Qiao, F.; Liu, Z.; Hou, Y.; Wu, C.; Fan, Y.; Liu, L.

Selective Antimicrobial Activities and Action Mechanism of Micelles Self-Assembled by Cationic Oligomeric Surfactants. ACS Appl. Mater. Interfaces 2016, 8 (6), 4242-4249. 6.

Datta, L. P.; Chatterjee, A.; Acharya, K.; De, P.; Das, M. Enzyme Responsive Nucleotide

Functionalized Silver Nanoparticles with Effective Antimicrobial and Anticancer Activity. New J. Chem. 2017, 41 (4), 1538-1548. 7.

Chu, H.; Pazgier, M.; Jung, G.; Nuccio, S.-P.; Castillo, P. A.; de Jong, M. F.; Winter, M.

G.; Winter, S. E.; Wehkamp, J.; Shen, B.; Salzman, N. H.; Underwood, M. A.; Tsolis, R. M.; Young, G. M.; Lu, W.; Lehrer, R. I.; Bäumler, A. J.; Bevins, C. L. Human α-Defensin 6 36 ACS Paragon Plus Environment

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Promotes Mucosal Innate Immunity Through Self-Assembled Peptide Nanonets. Science 2012, 337 (6093), 477. 8.

Castelletto, V.; de Santis, E.; Alkassem, H.; Lamarre, B.; Noble, J. E.; Ray, S.; Bella, A.;

Burns, J. R.; Hoogenboom, B. W.; Ryadnov, M. G. Structurally Plastic Peptide Capsules for Synthetic Antimicrobial Viruses. Chem. Sci. 2016, 7 (3), 1707-1711. 9.

Ignatova, M.; Labaye, D.; Lenoir, S.; Strivay, D.; Jérôme, R.; Jérôme, C. Immobilization

of Silver in Polypyrrole/Polyanion Composite Coatings: Preparation, Characterization, and Antibacterial Activity. Langmuir 2003, 19 (21), 8971-8979. 10. Li, L.-L.; Xu, J.-H.; Qi, G.-B.; Zhao, X.; Yu, F.; Wang, H. Core–shell Supramolecular Gelatin Nanoparticles for Adaptive and “On-Demand” Antibiotic Delivery. ACS Nano 2014, 8 (5), 4975-4983. 11. Giano, M. C.; Ibrahim, Z.; Medina, S. H.; Sarhane, K. A.; Christensen, J. M.; Yamada, Y.; Brandacher, G.; Schneider, J. P. Injectable Bioadhesive Hydrogels with Innate Antibacterial Properties. Nat. Commun. 2014, 5, 4095-4095. 12. Rusen, E.; Mocanu, A.; Nistor, L. C.; Dinescu, A.; Călinescu, I.; Mustăţea, G.; Voicu, Ş. I.; Andronescu, C.; Diacon, A. Design of Antimicrobial Membrane Based on Polymer Colloids/Multiwall Carbon Nanotubes Hybrid Material with Silver Nanoparticles. ACS Appl. Mater. Interfaces 2014, 6 (20), 17384–17393. 13. Nair, A. S.; Binoy, N. P.; Ramakrishna, S.; Kurup, T. R. R.; Chan, L. W.; Goh, C. H.; Islam,

M.

R.;

Utschig,

T.;

Pradeep,

T.

Organic-Soluble

Antimicrobial

Silver


Langmuir

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 38 of 45

Nanoparticle−Polymer Composites in Gram Scale by One-Pot Synthesis. ACS Appl. Mater. Interfaces 2009, 1 (11), 2413-2419. 14. Li, J.; Chen, Z.; Zhou, M.; Jing, J.; Li, W.; Wang, Y.; Wu, L.; Wang, L.; Wang, Y.; Lee, M. Polyoxometalate‐Driven Self‐Assembly of Short Peptides into Multivalent Nanofibers with Enhanced Antibacterial Activity. Angew. Chem. Int. Ed. 2016, 55 (7), 2592-2595. 15. Liu, L.; Xu, K.; Wang, H.; Jeremy Tan, P. K.; Fan, W.; Venkatraman, S. S.; Li, L.; Yang, Y.-Y. Self-Assembled Cationic Peptide Nanoparticles as an Efficient Antimicrobial Agent. 2009, 4, 457. 16. Faruqui, N.; Bella, A.; Ravi, J.; Ray, S.; Lamarre, B.; Ryadnov, M. G. Differentially Instructive Extracellular Protein Micro-nets. J. Am. Chem. Soc. 2014, 136 (22), 7889-7898. 17. Kuroda, K.; DeGrado, W. F. Amphiphilic Polymethacrylate Derivatives as Antimicrobial Agents. J. Am. Chem. Soc. 2005, 127 (12), 4128-4129. 18. Punia, A.; Mancuso, A.; Banerjee, P.; Yang, N.-L. Nonhemolytic and Antibacterial Acrylic copolymers with Hexamethyleneamine and Poly (Ethylene Glycol) Side Chains. ACS Macro Lett. 2015, 4 (4), 426-430. 19. Guo, J.; Xu, Q.; Zheng, Z.; Zhou, S.; Mao, H.; Wang, B.; Yan, F. Intrinsically Antibacterial Poly (Ionic Liquid) Membranes: The Synergistic Effect of Anions. ACS Macro Lett. 2015, 4 (10), 1094-1098. 20. Engler, A. C.; Wiradharma, N.; Ong, Z. Y.; Coady, D. J.; Hedrick, J. L.; Yang, Y.-Y. Emerging Trends in Macromolecular Antimicrobials to Fight Multi-Drug-Resistant Infections. Nano Today 2012, 7 (3), 201-222. 38 ACS Paragon Plus Environment

Page 39 of 45

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

Langmuir

21. Palermo, E. F.; Kuroda, K. Chemical Structure of Cationic Groups in Amphiphilic Polymethacrylates Modulates the Antimicrobial and Hemolytic Activities. Biomacromolecules 2009, 10 (6), 1416-1428. 22. Uppu, D.; Bhowmik, M.; Samaddar, S.; Haldar, J. Cyclization and Unsaturation Rather than Isomerisation of Side Chains Govern the Selective Antibacterial Activity of CationicAmphiphilic Polymers. Chem. Commun. 2016, 52 (25), 4644-4647. 23. Kenawy, E.-R.; Worley, S.; Broughton, R. The Chemistry and Applications of Antimicrobial Polymers: a State-of-the-art Review. Biomacromolecules 2007, 8 (5), 1359-1384. 24. Palermo, E. F.; Kuroda, K. Structural Determinants of Antimicrobial Activity In Polymers which Mimic Host Defense Peptides. Appl. Microbiol. Biotechnol. 2010, 87 (5), 16051615. 25. Mizerska, U.; Fortuniak, W.; Chojnowski, J.; Hałasa, R.; Konopacka, A.; Werel, W. Polysiloxane Cationic Biocides with Imidazolium Salt (ImS) Groups, Synthesis and Antibacterial Properties. Eur. Polym. J. 2009, 45 (3), 779-787. 26. Murata, H.; Sanda, F.; Endo, T. Highly radical-Polymerizable Methacrylamide Having Dipeptide Structure. Synthesis and Radical polymerization of N-methacryloyl-L-leucyl-LAlanine Methyl Ester. Macromolecules 1996, 29 (17), 5535-5538. 27. Ayres, L.; Adams, P. H. H.; Löwik, D. W.; van Hest, J. C. β-Sheet Side Chain Polymers Synthesized by Atom-Transfer Radical Polymerization. Biomacromolecules 2005, 6 (2), 825831.


Langmuir

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 40 of 45

28. Börner, H. G.; Smarsly, B. M.; Hentschel, J.; Rank, A.; Schubert, R.; Geng, Y.; Discher, D. E.; Hellweg, T.; Brandt, A. Organization of Self-Assembled Peptide− Polymer Nanofibers in Solution. Macromolecules 2008, 41 (4), 1430-1437. 29. Kumar, S.; Bheemireddy, V.; De, P. Aβ17–20 Peptide‐Guided Structuring of Polymeric Conjugates and Their pH‐Triggered Dynamic Response. Macromol. Biosci. 2015, 15 (10), 14471456. 30. Kumar, S.; Acharya, R.; Chatterji, U.; De, P. Controlled Synthesis of β-Sheet Polymers Based on Side-Chain Amyloidogenic Short Peptide Segments via RAFT Polymerization. Polym. Chem. 2014, 5 (20), 6039-6050. 31. Chang, Y.; McLandsborough, L.; McClements, D. J. Physicochemical Properties and Antimicrobial Efficacy of Electrostatic Complexes Based on Cationic ε-Polylysine and Anionic Pectin. J. Agric. Food. Chem. 2011, 59 (12), 6776-6782. 32. Chang, Y.; McLandsborough, L.; McClements, D. J. Interactions of a Cationic Antimicrobial (ε-Polylysine) with an Anionic Biopolymer (Pectin): an Isothermal Titration Calorimetry, Microelectrophoresis, and Turbidity Study. J. Agric. Food. Chem. 2011, 59 (10), 5579-5588. 33. Rieger, J.; Antoun, T.; Lee, S. H.; Chenal, M.; Pembouong, G.; Lesage de la Haye, J.; Azcarate, I.; Hasenknopf, B.; Lacôte, E. Synthesis and Characterization of a Thermoresponsive Polyoxometalate–Polymer Hybrid. Chem. Eur. J. 2012, 18 (11), 3355-3361.


Page 41 of 45

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

Langmuir

34. Gong, Y.; Hu, Q.; Wang, C.; Zang, L.; Yu, L. Stimuli-Responsive Polyoxometalate/Ionic Liquid Supramolecular Spheres: Fabrication, Characterization, and Biological Applications. Langmuir 2016, 32 (2), 421-427. 35. Li, W.; Yin, S.; Wang, J.; Wu, L. Tuning Mesophase of Ammonium AmphiphileEncapsulated Polyoxometalate Complexes through Changing Component Structure. Chem. Mater. 2008, 20 (2), 514-522. 36. Gao, P.; Wu, Y.; Wu, L. Co-assembly of Polyoxometalates and Peptides towards Biological Applications. Soft Matter 2016, 12 (41), 8464-8479. 37. Rhule, J. T.; Hill, C. L.; Judd, D. A.; Schinazi, R. F. Polyoxometalates in Medicine. Chem. Rev. 1998, 98 (1), 327-358. 38. Judd, D. A.; Nettles, J. H.; Nevins, N.; Snyder, J. P.; Liotta, D. C.; Tang, J.; Ermolieff, J.; Schinazi, R. F.; Hill, C. L. Polyoxometalate HIV-1 Protease Inhibitors. A New Mode of Protease Inhibition. J. Am. Chem. Soc. 2001, 123 (5), 886-897. 39. Geng, J.; Li, M.; Ren, J.; Wang, E.; Qu, X. Polyoxometalates as Inhibitors of the Aggregation of Amyloid β Peptides Associated with Alzheimer’s Disease. Angew. Chem. 2011, 123 (18), 4270-4274. 40. Yu, H.; Li, M.; Liu, G.; Geng, J.; Wang, J.; Ren, J.; Zhao, C.; Qu, X. Metallosupramolecular Complex Targeting an [Small Alpha]/[Small Beta] Discordant Stretch of Amyloid [Small Beta] Peptide. Chem. Sci. 2012, 3 (11), 3145-3153. 41. Song, Y.-F.; Tsunashima, R. Recent Advances on Polyoxometalate-Based Molecular and Composite Materials. Chem. Soc. Rev. 2012, 41 (22), 7384-7402. 41 ACS Paragon Plus Environment

Langmuir

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 42 of 45

42. Kumar, S.; Roy, S. G.; De, P. Cationic Methacrylate Polymers Containing Chiral Amino Acid Moieties: Controlled Synthesis via RAFT Polymerization. Polym. Chem. 2012, 3 (5), 12391248. 43. Datta, L. P.; Maiti, B.; De, P. Synthetic Polymeric Variant of S-Adenosyl Methionine Synthetase. Polym. Chem. 2015, 6 (45), 7796-7800. 44. Kumar, S.; Acharya, R.; Chatterji, U.; De, P. Controlled Synthesis of pH Responsive Cationic Polymers Containing Side-Chain Peptide Moieties via RAFT Polymerization and Their Self-assembly. J. Mater. Chem. B 2013, 1 (7), 946-957. 45. Maity, S.; Kumar, P.; Haldar, D. An Amyloid-like Fibril-Forming Supramolecular Crossβ-Structure of a Model Peptide: a Crystallographic Insight. Org. Biomol. Chem. 2011, 9 (10), 3787-3791. 46. de Groot, N. S.; Parella, T.; Aviles, F. X.; Vendrell, J.; Ventura, S. Ile-Phe Dipeptide Self-Assembly: Clues to Amyloid Formation. Biophys. J. 2007, 92 (5), 1732-1741. 47. Dutta, L. P.; Das, M. Coacervation—A Method for Drug Delivery. In Advancements of Medical Electronics; Springer, 2015, pp 379-386. 48. Carmona-Ribeiro, A. M.; de Melo Carrasco, L. D. Cationic Antimicrobial Polymers and Their Assemblies. Int. J. Mol. Sci. 2013, 14 (5), 9906-9946. 49. Timofeeva, L.; Kleshcheva, N. Antimicrobial Polymers: Mechanism of Action, Factors of Activity, and Applications. Appl. Microbiol. Biotechnol. 2011, 89 (3), 475-492.


Page 43 of 45

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

Langmuir

50. Tian, Y. F.; Hudalla, G. A.; Han, H.; Collier, J. H. Controllably Degradable β-Sheet Nanofibers and Gels from Self-Assembling Depsipeptides. Biomater. Sci. 2013, 1 (10), 10371045. 51. Inoue, M.; Segawa, K.; Matsunaga, S.; Matsumoto, N.; Oda, M.; Yamase, T. Antibacterial Activity of Highly Negative Charged Polyoxotungstates, K27[KAs4W40O140] and K18[KSb9W21O86], and Keggin-Structural Polyoxotungstates against Helicobacter Pylori. J. Inorg. Biochem. 2005, 99 (5), 1023-1031. 52. Kuroda, K.; Caputo, G. A.; DeGrado, W. F. The Role of Hydrophobicity in the Antimicrobial and Hemolytic Activities of Polymethacrylate Derivatives. Chem. Eur. J. 2009, 15 (5), 1123-1133. 53. 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 (33), 12225-12234. 54. Blazyk, J.; Wiegand, R.; Klein, J.; Hammer, J.; Epand, R. M.; Epand, R. F.; Maloy, W. L.; Kari, U. P. A Novel Linear Amphipathic β-Sheet Cationic Antimicrobial Peptide with Enhanced Selectivity for Bacterial Lipids. J. Biol. Chem. 2001, 276 (30), 27899-27906. 55. Strahl, H.; Hamoen, L. W. Membrane Potential is Important for Bacterial Cell Division. Proc. Natl. Acad. Sci. 2010, 107 (27), 12281-12286. 56. Chimerel, C.; Field, C. M.; Piñero-Fernandez, S.; Keyser, U. F.; Summers, D. K. Indole Prevents Escherichia Coli Cell Division by Modulating Membrane Potential. Biochim. Biophys. Acta 2012, 1818 (7), 1590-1594.


Langmuir

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 44 of 45

57. Hiraoka, S.; Nukui, K.; Uetake, N.; Ohta, A.; Shibuya, I. Amplification and Substantial Purification of Cardiolipin Synthase of Escherichia Coli. J. Biochem. 1991, 110 (3), 443-449. 58. Khare, D.; Pande, R. Experimental and Molecular Docking Study on DNA Binding Interaction of N-Phenylbenzohydroxamic Acid. Der. Pharma. Chemica. 2012, 4 (1), 66-75. 59. Shahabadi, N.; Fili, S. M.; Kheirdoosh, F. Study on the Interaction of the Drug Mesalamine with Calf Thymus DNA using Molecular Docking and Spectroscopic Techniques. J. Photochem. Photobiol., B 2013, 128, 20-26. 60. Hancock, R. E. Peptide Antibiotics. The Lancet 1997, 349 (9049), 418-422. 61. Raffi, M.; Hussain, F.; Bhatti, T.; Akhter, J.; Hameed, A.; Hasan, M. Antibacterial Characterization of Silver Nanoparticles against E. coli ATCC-15224. J. Mater. Sci. Technol. 2008, 24 (2), 192-196. 62. Muñoz-Bonilla, A.; Fernández-García, M. Polymeric Materials with Antimicrobial Activity. Prog. Polym. Sci. 2012, 37 (2), 281-339. 63. Yuan, H.; Liu, Z.; Liu, L.; Lv, F.; Wang, Y.; Wang, S. Cationic Conjugated Polymers for Discrimination of Microbial Pathogens. Adv. Mater. (Weinheim, Ger.) 2014, 26 (25), 4333-4338.


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Table of Content Graphic:

Title: Peptide Based Polymer-Polyoxometalate Supramolecular Structure with Differed Antimicrobial Mechanism Authors: Lakshmi Priya Datta, Riya Mukherjee, Subharanjan Biswas, Tapan Kumar Das*, Synopsis: Schematic illustration of antimicrobial activity of peptide base cationic antimicrobial polymers and their supramolecular hybridization with metal inorganic frameworks.


Peptide-Based PolymerâPolyoxometalate Supramolecular Structure

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