Biocompatible Lipopeptide-Based Antibacterial Hydrogel

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Biocompatible Lipopeptide-Based Antibacterial Hydrogel Anindyasundar Adak, Subhajit Ghosh, Varsha Gupta, and Surajit Ghosh Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b01836 • Publication Date (Web): 12 Apr 2019 Downloaded from http://pubs.acs.org on April 14, 2019

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Biocompatible Lipopeptide-Based Antibacterial Hydrogel Anindyasundar Adak,1 Subhajit Ghosh,1 Varsha Gupta, Surajit Ghosh*1,2 1. Organic & Medicinal Chemistry Division, CSIR-Indian Institute of Chemical Biology, 4 Raja S. C. Mullick Road, Jadavpur, Kolkata-700032, West Bengal, India. Fax: +91-33-24735197/0284; Tel: +91-33-2499-5872 2. Academy of Scientific and Innovative Research (AcSIR), CSIR-Indian Institute of Chemical Biology Campus, 4 Raja S. C. Mullick Road, Kolkata 700 032, India. CORRESPONDING AUTHOR INFORMATION: Fax: +91-33-2473-5197/0284; Tel: +91-33-2499-5872; E-mail: [email protected] KEYWORDS: Self-assembly, Peptide hydrogel, Anti-bacterial gel, Live-dead cell assay, Co-culture assay, Hemolytic Activity. ABSTRACT: A biocompatible hydrogel containing hexa-peptide as key unit has been designed and fabricated. Our design construct comprises a β-sheet rich short hexa-peptide in the centre with a hydrophobic long chain and hydrophilic triple lysine unit attached at the Nand C- terminals respectively. Thus, it is this amphiphilic nature of the molecule that facilitates the gelation. It can capture solvent molecules in the three dimensional cross-linked fibrillary networks. The amphiphilic character of the construct has been modulated to produce an excellent biocompatible soft-material for the inhibition of bacterial growth by rupturing the bacterial cell membrane. This hydrogel is also stable against the enzymatic degradation (Proteinase K) and most importantly offers a bio-compatible environment for the

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growth of normal mammalian cells due to its non-cytotoxic nature as observed through the cell viability assay. From the haemolytic assay, the morphology of the hRBCs is found to be almost intact, which suggests that the hydrogel can be used in biomedical applications. Thus, this newly designed anti-bacterial hydrogel can be used both as antibacterial biomaterials and biocompatible scaffolds for mammalian cell culture. INTRODUCTION Peptide self-assembly displays immense application in 3D biomaterial scaffolds,1,2 in tissue regenerative technique,3,4 different kinds of drug delivery,5-7 and wound healing.8 It leads to different nano-structures governed by variable factors like secondary structure, hydrophobic effects,9-13 π−π interactions,14,15 hydrogen bonding16 etc. Now, these interactions enforce the formation of three dimensional (3D) dense cross-linked nano-fibers, which ultimately facilitates the formation of hydrogel. Peptide based hydrogels have recently gained huge potential in various biotechnological applications due to their excellent biocompatibility and non-cytotoxic property.17,18 The hydrogel also contains plenty of vacant sites in their three dimensional cross-linked networks. For that reason, they can capture a lot of water molecules under appropriate conditions, which is the key factor in providing necessary fluids for cell survival and delivery of drugs like cyanocobalamin, curcumin, doxorubicin, morpholine at the desired sites.19-22 This added facility of the hydrogel microenvironment provides an optimum humid condition which not only facilitates tissue regeneration but also prohibits shrinking of wound. There are some added advantages of this ideal environment as it can offer a large supply of cytokines, necessary fluids, growth factors and blood cells.23 However, the applications of these hydrogels should be handled carefully since the moist environment provides ideal conditions for bacterial growth.24 Over the year, this has always been a great problem that causes trouble during the implant surgery, tissue engineering and cell culture. To address these issues, more hydrogels with antibacterial property can be designed and


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developed based on its application in biomedical field. One of the potent approaches towards development of anti-bacterial hydrogels includes silver nanoparticle based antibacterial hydrogels which are able to combat against the bacterial contamination and infection.25-29 But, considering the several side effects of hydrogels like inflammation, oxidative DNA damage and pigmentation of the skin or eyes,30 their designing and development should be carefully monitored. The reported methods often suffers from several drawbacks such as their uncontrolled release, toxic side effects and lack of selectivity.31 In that respect, peptides offer distinct advantages like their easy modulation as it contains multiple functional groups in their side chain, natural biocompatibility, antimicrobial activity, non-cytotoxicity and biodegradable nature. This makes them promising biomaterials for different purposes with versatile applications. Lysine and arginine amino acids possesses positive charge in their side chains, which are often incorporated in different peptides and polymers to generate or enhance antibacterial property.32-36 The negatively charged membrane often interact with cationic biomaterials through electrostatic interactions followed by attachment of the hydrophobic counter parts into the lipid layer of membrane. This could lead to the formation of pores in the bacterial cell membrane thereby killing the bacteria. Now hydrophobic moiety and positively charged moiety can be combined in the different spacing and orientation to generate various building blocks to facilitate the formation of different antibacterial biomaterials. But considering their cell membrane perturbation character, their high cost in synthesis, limited stability, and unknown toxicology and pharmacokinetics, one has to choose ideal sequence to overcome these drawbacks. Inspired by the research and development in the amphiphilic lipopeptides37 we have tried to design and develop an antibacterial lipopeptide by modulating its hydrophobic and hydrophilic character. The amphiphilic character of bacterial walls can be targeted by designing different amphiphilic peptides for killing bacteria by rupturing their cell walls. Another interesting fact is that the peptide with extensive β-sheet



secondary structure can rupture bacterial walls.38 Considering the above concepts, we have designed a new antibacterial molecule, which forms stable injectable hydrogel, are capable of encapsulating solvent molecules in its void spaces, display non-cytotoxic nature and shows biocompatibility for the culture of normal mammalian cells. EXPERIMENTAL SECTION Materials Chemicals. Rink Amide resin and Fmoc-amino acids, Diisopropylethylamine (DIPEA) HBTU were bought from Novabiochem. Pyridine, Methanol, Trifluoroacetic acid (TFA) Dimethylsulfoxide (DMSO), Pipyridine, Ethanedithiol (EDT), and Ether were purchased from Spectrochem. Phenol, Dichloromethane (DCM), TritonX-100, Hydrogen peroxide (30% solution), Acetone, Dichloromethane and N, N′-Dimethylformamide (DMF) were purchased from Merck. Tubulin (EP1332Y) antibody and Goat polyclonal anti-Rabbit IgG H&L (Cy3.5 ®) were purchased from Abcam N, N′- Diisopropylcarbodiimide (DIC), 5(6)Carboxyfluorescence (FITC), 5-diphenyltetrazolium bromide (MTT), Proteinase K enzyme, DMSO for cell culture and formaldehyde solution (molecular biology grade) were purchased from Sigma Aldrich. Penicillin-Streptomycin, and fetal bovine serum (FBS) were purchased from Invitrogen. Lipopeptides have been purified by Simadzu HPLC with C-18 (Waters) semi preparative column in the reverse phase. Cell Culture WI-38 cells (fibroblasts derived from lung cell line) have been cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% heal inactivated Fetal Bovine Serum (FBS), 100 units/mL penicillin, and 100 µg/mL streptomycin in a humidified 5% CO2 atmosphere at 37 ºC.


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Peptide Amphiphile (PA-NV) Synthesis Solid phase peptide synthesis technique has been applied to synthesize the nonapeptide (NH2NAVSIQKKK-CONH2) by CEM-LIBERTY1 automated microwave peptide synthesizer taking resin (Rink Amide) as solid support. 20% v/v piperidine in DMF was used for the deprotection of Fmoc group along with DIPEA (12 equiv.) and HBTU (5 equiv.) were used as base and coupling agent. The free carboxylic group of palmitic acid has been attached at the N-terminal of synthesized nonapeptide using HBTU and DIPEA in anhydrous DMF solvent for overnight. The lipopeptide has been cleaved from the resin with a cocktail mixture containing TFA, EDT, H2O, phenol 94:2.0:2.0:2.0 (v/v/v/v) at room temperature for 3 h. Next TFA has been evaporated by nitrogen gas to minimum volume and ice cold diethyl ether has been added into it to get white precipitate. The precipitate has been centrifuged, lyophilized and purified by reverse phase HPLC (Simadzu). The synthesized final molecule has been characterized by MALDI Mass spectroscopy and 1H-NMR. Preparation of Peptide Hydrogel 1 mL Phosphate-Buffered Saline (PBS, 10 mM) has been added to PA-NV (20 mg) to get 2 wt% solutions at pH 7.4. The dissolved mixture was heated gently for 5-10 minutes. After that the solution was kept at room temperature to get well defined homogeneous hydrogel in half an hour, which is physically confirmed by inverting the vials. Characterization of the PA-NV Hydrogel Fourier Transforms Infrared Spectroscopy (FT-IR) FT-IR analysis of the PA-NV hydrogel (1 wt%) has been performed on a Perkin-Elmer Spectrum 100 FT-IR spectrometer, mixing PA-NV hydrogel with KBr pellets in a CaF2 cell. Next, FT-IR spectrum has been recorded in Perkin-Elmer Spectrum 100 series spectrometer



by the LiTaO3 detector. To remove errors from the experiment, background correction has been performed each time. Atomic Force Microscopy (AFM) Study Inner structure of the PA-NV hydrogel has been examined by a Nanosurf C3000 Controller AFM. The sample has been prepared by depositing 2 wt % PA-NV hydrogel sample over a microscopic glass slide. Glass slide was allowed to dry under vacuum for overnight. The experiment was performed in dynamic mode. Transmission Electron Microscopy (TEM) Study To analyse the nanostructure of PA-NV hydrogel in more details, TEM has been performed. Three carbon coated TEM grids (Quantifoil, Jena, Germany) were prepared by depositing 1 wt%, 2 wt% hydrogel and PA-NV solution before gelation. The excess sample solution has been removed by blotting paper. Next, 2% uranyl acetate has been added over the grids followed by washing with Milli-Q water. The EM has been performed at an accelerating voltage of 120 kV. Rheology Study To check the gelation property of PA-NV hydrogel dynamic frequency sweep and time dependent step-strain rheological experiment has been performed. 2 wt% and 4 wt% PA-NV hydrogel has been prepared and the experiment is carried out in TA Instrument, USA rheometer with a cone-plate (diameter 40 mm and angle 4º). Similarly Thixotropy experiment has been carried out at room temperature over 800 ns with alternation of sudden change in strain in two cycles. In vitro Proteolytic Degradation by Proteinase K


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Proteolytic degradation study of PA-NV compound has been performed by dissolving it in HEPES (100 mM) buffer to obtain 1 mM solution. After that, Proteinase K enzyme has been added to the PA-NV solution in HEPES buffer (3.2 units/ mL) followed by time to time analysis with the help of MALDI-TOF mass spectrometry. Cell Viability Assay of PA-NV Hydrogel MTT assay has been performed to analyse the biocompatibility of the hydrogel in WI-38 cells. Approximately 6000 cells have been seeded per well in a 96-well plate (with a medium volume of 100 mL). After 24 h, the medium has been exchanged with solutions of different concentration of hydrogel to each well (each concentration repeated in five wells). Next, MTT assay have been carried out after 24 h of treatment. Cells without treatment were taken as control. Antibacterial Assays For the antibacterial assays, 96-well plates were taken and 2 wt% hydrogel has been prepared and coated at the bottom surface of the individual wells, followed by the addition of equal volume of PBS buffer. The buffer due to its ionic strength strengthens the gelation of the hydrogel. The resulting 2 wt% PA-NV hydrogels has been allowed to incubate at 37 ºC for 2 hours. Pure cultures of bacterial strains was kept as glycerol stocks and stored in -80 oC which were further quadrant streaked on a TrypticaseTM Soy Agar plate with 5% sheep blood, followed by incubation at 37 ºC overnight. After that, colonies from the fourth quadrant were transferred to a fresh agar plate and quadrant streaked followed by incubation for 24 hours. For each bacterial strain, one colony of bacteria from the fourth quadrant of the second agar plate was suspended in 1 mL of TSB in a 5 mL eppendorf tube and incubated at 37°C until the optical density of this suspension was reached to OD625nm = 0.1 AU, resulting in a 108 colony forming units (CFU)/mL bacterial stock solution. For each assay, 100 μL of



bacteria-free TSB was introduced onto the surface of the hydrogels. Next, 100 μL of the 108 CFU/mL bacterial stock solution was introduced to the surface of a given hydrogel and serial 1:10 dilutions were performed across the plate, resulting in final bacterial concentrations of 2 x 103, 2 x 104, 2 x 105, 2 x 106, 2 x 107, 2 x 108 and 2 x 109 CFU/dm2 respectively, for each of seven wells. Controls experiments were carried out on only tissue culture-treated polystyrene (TCTP) surfaces. Bacteria were incubated for 48 h on control and PA-NV hydrogel surfaces at 37 ºC. Next bacterial growth was monitored by measuring OD625nm of the media above the gel by adding 100 μL of bacteria-free TSB to the surfaces, gently mixing and transferring the supernatant to a cuvette. Control OD625nm was calculated to correct for dilution and to normalize for scattering that occurs for each individual bacterial strain. Susceptibility Assay of Bacteria using Colony Count Assay The activity of PA-NV towards reduction of viable bacteria was checked using previously reported colony counting method.36 Briefly E. coli and S. aureus were sub-cultured for around 6 hours at 37 °C in TSB and NB respectively. When the optical density was around 0.6 at 625 nm (1×106 CFU/ml), 100 µL of this culture was added into each well of microtiter plate containing 100 µL of different concentration of PA-NV (2, 1 and 0.5 mM). In control wells 100 µL PBS was added in place of hydrogel. Inoculated microtiter plates were incubated for 24 hours at 37 °C in a BOD incubator. After 24 hours, 20 µL of sample was taken out from each well and serially diluted in PBS and spread either on TSA or NA plates for E coli or S aureus respectively. Plates were incubated at 37 °C in a BOD incubator for overnight. Colonies were counted using Miles and Misra method. Results reported here are the mean (Log10 CFU/mL) of four replicates. Outer Membrane Permeabilization Assay


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Outer membrane permeabilization of our compound PA-NV was determined by Nphenylnapthylamine (NPN) as described previously14 with some modifications. Briefly midlog phase of both the bacteria (E. coli and S.aureus) was harvested at 3500 rpm for 3 min. The pellet was once washed and resuspended in 1:1 mixture of 5 mM glucose and 5 mM HEPES buffer (pH 7.2). 150 μL of bacterial suspension containing around 108 colony forming unit per mL was transferred into the wells of a black 96 well flat bottom plate. Then NPN dye (10 µM, 50 μL) was added to the bacterial suspension containing wells and incubated for around 30 min. On ending of pre-incubation period, fluorescence intensity of the wells was monitored for next 7 min at every 1 min interval at an excitation wavelength of 350 nm and emission wavelength of 420 nm. Then different concentrations (500 µg/mL and 250 μg/mL) of PANV were added into each well from a very high concentration of PA-NV solution. One well was left as control and into another well valinomycin from very high concentration was added as a final concentration 100 μg/mL. Remaining very small volume was adjusted with 1:1 mixture of 5 mM glucose and 5 mM HEPES buffer. Further fluorescence intensity was measured immediately for another 20 min at every 1 min interval keeping the parameters same. Results reported here is the average intensity of triplicate wells. Cytoplasmic Membrane Permeabilization Assay This assay of PA-NV was performed as described with some modifications.14 Briefly, midlog phase of both the bacteria (E. coli and S.aureus) were harvested as mentioned earlier. It was then washed and resuspended in 1:1 mixture of 5 mM glucose and 5 mM HEPES buffer (pH 7.4). 200 μL of bacterial suspension containing around 108 colony forming unit per mL was transferred into the wells of a black 96 well flat bottom plate. 10 μM of Propidium iodide (PI) was added to each well containing bacterial suspension. Immediately after adding fluorescence intensity was measured for 7 min with 1 min interval at excitation wavelength of 535 nm and emission wavelength of 617 nm. Next different concentration (500 µg/mL and



250 μg/mL) of PANV was added into each well from very high concentration of PANV solution. One well was left as control and into another well valinomycin from very high concentration was added at a final concentration of 100 μg/mL. Remaining very small volume was adjusted with 1:1 mixture of 5 mM glucose and 5 mM HEPES buffer. Further fluorescence intensity was measured immediately for another 20 min. at every 1 min interval keeping the parameters same. As a measure of the extent of cytoplasmic membrane permeabilization increase in fluorescence intensity was observed. Results reported here is the average intensity of triplicate wells. Confocal Microscopy Mid log phase (6 h grown) culture of E. coli and S. aureus were grown to approximately 106 CFU/mL in a TSA broth (for E. coli) or in nutrient broth (for S. aureus). 2 wt % PA-NV gel was prepared on sterile 35 mm cover glass bottom dishes. 1 mL of this bacterial suspension with 1 mL fresh respective culture medium was then added into either on empty cover bottom dish plates for control experiment or on gel surface. The plates were then incubated under stationary conditions for 24h. After that, the medium was removed and planktonic bacteria were carefully washed out with 1 × PBS (pH = 7.4). Bacteria adhering on the surface of cover glasses were stained with SYTO-9 (3 μM) and 30 μM propidium iodide. Following incubation for 15 min, the cells were imaged using 10 X magnification on fluorescence (Olympus IX83) Microscope. Bacteria with intact membranes display green fluorescence and (Em = 500 nm) and bacteria with comprised membranes fluoresce red (Em = 635 nm). Co-culturing Assay Co-culture experiments were performed on 35 mm tissue culture treated glass cover bottom dishes with mammalian and bacterial cells. PA-NV hydrogel was first prepared on the 35mm glass cover bottom dishes. PA-NV hydrogels has been equilibrated for 12 h with DMEM.


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Then the medium is removed and WI38 (Human Lung Fibroblast) cells in DMEM (1 × 105 cells/mL, 1000 μL) has been plated over PA-NV hydrogel surface. WI38 cells have been incubated for 2 days at 37 °C under 5% CO2, which leads to the spreading of WI-38 cells over the gel surface. After that, over the plate 40 µL of aliquots containing E. coli and S. aureus (about 106 CFU/mL) (mid log phase culture after 6 h incubation) have been spread to get a final concentration of 1 × 103 CFU/mL. After that they have been co-cultured for 24 hrs followed by addition of an aliquots (60 μL) containing solution of SYTO 9, PI, and calceinAM. Then the system has been incubated for 1 h at 37 °C. After that, images of WI-38 cells and bacteria have been captured with a fluorescence microscope (Olympus IX83). The excitation wavelength for both the viable bacteria and WI38 cells has been fixed at 488 nm which gives green fluorescence, while at 568 nm red fluorescence has been observed due to dead bacteria. The 35mm glass cover bottom dish without the peptide PA-NV hydrogel are considered as control. Haemolytic Assays Human blood has been used for this experiment. Blood was collected in a BD Vacutainer™, coated with heparin as anti-coagulating agent. To remove the white blood cells and serum, the blood has been centrifuged for twelve minutes at 3000 rpm. The resulting pelleted human red blood cells (hRBCs) has been collected and mixed in 12 mL of PBS buffer (10 mM, pH 7.4). The resulting solution has been centrifuged again at 3000 rpm for twelve minutes and again the pellet was dissolved in 12 mL of PBS buffer. 200 μL hRBC stock solutions was taken in duplicate wells and marked as control in the 96 well plate. 200 μL hRBC stock solutions was taken in another set of duplicate wells and and 1 percent of Triton-X-100 added in these wells and marked as positive control. Further 200 μL hRBC stock solutions were taken in another set of wells and 2 wt % of PA-NV hydrogel was added in it and marked as test. Red blood cell count has been performed using a Neubauer counting chamber and found



to be 5.2 x 1012 hRBCs/L. Additional PBS buffer was added to each well to adjust the final buffer volume to 300 μL to ensure even distribution of hRBCs with respect to the surface being assayed. Samples were incubated at 37 ºC under dynamic conditions using a rocker platform for 1 hour. Next, the supernatant was collected and optical density of each sample was measured at 450nm after centrifugation at 4000 rpm for 5 min. The release of Hemoglobin, as a result of hRBC lysis, was assessed. RESULTS AND DISCUSSION Amphiphilic peptides are able to form β-sheet secondary structures. They are well-known for exhibiting antimicrobial properties. According to Shai-Matsuzaki-Huang model, β-sheet secondary structure rich peptide leads to the perturbation of membrane, which results in loss of cellular substances. Due to formation of β-sheet secondary structure, they can amass as an oligomeric transmembrane pore, which further helps to permeabilize the membrane.39 Keeping this fact in mind, we have chosen an amphiphilic hexapeptide NAVSIQ, which is amphiphilic in nature and are able to form β-sheet secondary structure.40 To further accelerate the β-sheet secondary structure, the triple lysine unit was added at the C-terminal to get NAVSIQKKK nonapeptide. MD-simulation of two NAVSIQKKK peptides has been performed for 40 ns using Gromos 9653a6 force field 41 to check whether they adopt β-sheet structure or not. Initially, V7, S6 and I5 residues of both the peptides started to interact by Hbonding at 9 ns of simulation. In addition, K1 residue of one peptide started to interact with each other after 10 ns of simulation and results in β-sheet conformation (Figure 1). This study motivated us to further modulate this sequence by inserting palmitic acid at the N-terminal, so that the resulting amphiphilic character leads to an efficient gelator. So, we have synthesized the NAVSIQKKK by solid phase peptide synthesis procedure and palmitic acid has been attached to the N-terminal of this peptide by amide coupling to get the desired molecule (PA-NV) (Figure 2A), followed by HPLC purification to get the pure compound


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(Figure S1) and characterization by MALDI mass spectrometry (Figure S2) and 1H-NMR spectroscopy (Figure S3, the characteristic NMR peaks are assigned in ESI both for Mass and 1H-NMR).

The gelation property of PA-NV molecule has been examined by preparing 2 wt%

PA-NV hydrogel (Figure 2C), and from the image, we have found that stable homogeneous hydrogel is formed at room temperature. Self-assembly property of the hydrogel has been confirmed by FT-IR study with 2 wt% hydrogel in KBr pellet. From this experiment we have observed the amide - I peak at 1635 cm-1, which indicates the formation of the β-sheet secondary structure (Figure S4). To further validate the secondary structure, circular dichroism (CD) experiment has been carried out with 2 wt% hydrogel and observed the characteristic peak at 216 nm with negative ellipticity of β-sheet secondary structure, which is one of the key factors for the formation of the hydrogel (Figure S5). Next, to understand the ultrastructural details of the hydrogel and its gelation mechanism, we have performed Atomic Force Microscopy (AFM) of PA-NV 2 wt% hydrogel. AFM images reveal that three dimensional cross-linked networks of nano-fiber are the main backbones of the PA-NV hydrogel (Figure 2D). To further explore the inner structure of hydrogel, the TEM experiments have been performed with different concentrations, such as 1 wt%, 2 wt% PANV hydrogel and another with a solution of PA-NV before gelation. From the experiment intense cross-linked nano-fiber for both 1 wt% and 2 wt% hydrogel were observed (Figure. S6). But very little amount of nano-fiber was observed for PA-NV before gelation, which were fragile, weak, random and without any cross-linkage between them (Figure. S6A). This clearly indicates that with increasing concentration of the hydrogel, the extent of three dimensional cross-linked nano-fiber structures enriched, which significantly facilitates the gelation property of hydrogel. To check that, we have further studied the gelation property by performing the rheological study with 2 wt% and 4 wt% PA-NV hydrogel. Firstly, a frequency sweep experiment of both 2 wt% and 4 wt% hydrogel has been performed, which



shows that the storage modulus is more than the loss modulus and they have maintained a significant difference. (Figure 3A and S7) Since we have observed in frequency sweep rheology study that with 4 wt% concentration of the hydrogel, the storage modulus significantly enhanced (around 800 Pa) compared to the 2 wt% hydrogel (around 100 Pa), so we can conclude that the enhancement in three dimensional cross-linked nano-fibers with increasing concentration accelerates the gelation property of PA-NV, which leads to the formation of stable homogeneous hydrogel. Next, a time dependent step-strain rheological experiment has been studied to check the injectable nature of this hydrogel. First low constant strain of 0.1% has been applied to 2 wt% (w/v) hydrogel and monitored for first 400 s, and then suddenly a higher strain of 40% was applied for 200 ns. This leads to increase the loss modulus values (G″) above the storage modulus values (G′). Afterward, the strain has been suddenly reduced to a constant low strain of 0.1% and again monitored for 400 ns followed by repetition of same cycle again. From the data restoration of hydrogel has been observed since storage modulus showed (G′) higher value than loss modulus (G″) (Figure 3B). This experiment indicated that PA-NV hydrogel is injectable in nature. The thixotropic property of the hydrogel was further investigated by taking the 10 mg of PA-NV in glass vial and dissolved it in 1 mL of PBS buffer. Then the resulting solution was allowed to form the hydrogel. After the formation of the hydrogel, it was shaken vigorously, which leads to the formation of clear solution. Now this solution can be taken in syringe and kept at room temperature for few minutes in that a glass vial and we obtained our desired hydrogel (Figure 3C). Next, the proteolytic stability of the peptide hydrogel PA-NV has been studied. Now, since long aliphatic chain i.e. palmitic acid has been attached to the N-terminal of the peptide molecule and C-terminal has been modified to amide functional group so it can be assumed that it will show some proteolytic stability. For that we have performed proteolytic


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degradation experiment taking 2 wt% the peptide hydrogel PA-NV in HEPES buffer (pH 7.46) followed by addition of proteinase K enzyme at 37°C. The degradation of the PA-NV molecule has been examined at successive time intervals using HPLC. From the Figure 4A, we have found that PA-NV molecule shows significant resistance against the degradation by Proteinase – K enzyme. At 24 hours, about 60% of the peptide molecule remains intact. So, it can be concluded that these PA-NV peptide hydrogel are moderately proteolytically stable and have bio-stability. Cell morphologies and non-cytotoxicity study of PA-NV hydrogel has been studied using WI-38 lung fibroblast cells. To determine the cytotoxicity, cell viability assays with WI-38 cells (normal lung cell line) and HeLa cells (cancer cell line) have been performed up to 1 mM (0.12 wt%) concentrations of PA-NV hydrogel. No significant cytotoxicity was observed in case of PA-NV hydrogel as we have found almost 100% viable cells in both the cases (Figure 4B and 4C respectively). Next, cellular morphologies of these cultured WI38 cells on hydrogel were observed under the inverted microscope (Olympus IX83 fluorescence microscope, at 40× objective) in DIC mode. Cells at a density of 5,000 per dish were seeded on a confocal dish for 24 h prior to hydrogel treatment. Then, DMEM media containing different concentrations (2 wt% hydrogel) of the compound has been added and kept for 24 more hours in a 37 °C incubator. One confocal dish was kept untreated for the control study. The morphology of the cells was examined and images were captured in DIC mode and found them to be healthy and unaffected along with control (Figure 4D and S8 respectively). So, from the above two experiments, we found that our hydrogel is non-cytotoxic in nature and provides healthy environment for culturing mammalian cells. Next, for the investigation of anti-bacterial activity of PA-NV hydrogel, hydrogel surface (Figure S9) was treated with the various concentrations of colony forming units (CFU) of both Gram positive (S. aureus) and Gram negative (E. coli) bacteria. A varying number of



CFUs were inoculated independently either on the surface of 2 wt% hydrogel or polystyrene treated tissue culture plate’s surface (control) and incubated for 24 hours at 37 °C. In this time period cells were able to proliferate in the solution above the hydrogels’ surface. If the gel is permissive, the cells coming in contact with the hydrogel will grow otherwise their growth will be inhibited. The growth of bacteria was monitored by recording absorption spectra at 625 nm. To avoid error pre mixed bacterial solution was taken from above of the each well. From the data (Figure 5A) we found that the PA-NV hydrogel surface able to inhibit E. coli proliferation when up to 2 × 106 CFUs/dm2 inoculum was treated with PANV surface. When 2 × 108 CFUs/dm2 or higher concentration of inoculum was presented, PA-NV surface was not able to show its inhibitory effect and significant bacterial growth was observed over the PA-NV surface. Moreover, when very lower CFU of bacteria was treated with control surface vast bacterial growth over the control surface was found (Figure S10). The data shows that the inhibitory activity of PA-NV hydrogel surface seemingly decreases since the PA-NV hydrogel was active against up to 2 × 106 CFUs/dm2 treated initially. Similarly, PA-NV hydrogel surface was treated with the Gram-positive bacteria, S. aureus (Figure 5B), which showed that the hydrogel’s surface was able to inhibit this bacterial growth effectively. It was found that when the PA-NV surface was treated with 2 × 105 CFUs/dm2 (S. aureus) for 24 h, bacterial proliferation was inhibited. However, in case of the control surface massive growth of bacteria was found even inoculating with very small numbers of bacteria (Figure S10). We have shown that PA-NV hydrogel could effectively inhibit bacterial growth by its surface. To further confirm it, viability of bacteria has been performed in the solution above of the hydrogel surface and of those at the hydrogel/liquid interface after 24 h of incubation. The bacteria (E. coli and S. aureus) were introduced at a concentration of 1 × 106 CFU/mL. As shown in Figure 5C and 5D, there was insignificant growth of bacteria in the solution


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above the hydrogel from the two strains, in sharp contrast to the control in which bacterial growth in solution was substantial. Further, different concentrations of gel solutions have been incubated with E. coli and S. aureus bacterial solution suspended in their respective broths for 24 hrs at 37 °C. After that the absorbance at 625 nm has been recorded and from the data Figure 5C and 5D, we have found that there is appreciable amount of inhibition in bacterial growth. Antibacterial activity of PA-NV was further checked using a viable colony count assay. The result shows a significant reduction in viable bacterial load with increasing concentration of PA-NV (Figure S11). The mechanism of action of PA-NV is likely to be the cationic charge enabling interaction with bacterial membrane. Further, impact of PA-NV on cellular death pathway was analysed by flow cytometry using annexin-V and propidium iodide (PI). E. coli was treated with different concentrations of PA-NV. The results indicated that with increase in concentration, a sharp increase in the population of necrotic ∼24.2% (Q1) cells and late apoptotic ∼1.8% (Q2) in case of 500 μM PA-NV treatment were observed (Figure 5E). This may be due to the rupture of cell membrane, which leads to the uncontrolled release of products of cell death into the extracellular space. Necrosis is caused by factors external to the cell and in necrosis various receptors are activated. Next, outer membrane permeabilization assay was performed to show the impact of PA-NV on bacterial cell membrane. Generally NPN is a hydrophobic dye which is excluded from the outer membrane (OM) of gram negative bacteria. When the OM is ruptured or damaged, NPN screens into the perturbed OM, as a result an increment in fluorescence was observed. On treatment with our compound PA-NV an increase in fluorescence was observed, (Figure 6A) which mean outer membrane of the bacteria was damaged by our compound. The reason behind such observation may be due to the cationic nature of the compound.



On the other hand, permeabilization of the cytoplasmic membrane of the bacteria was checked using PI. PI generally enters inside bacteria only through compromised membrane and only fluoresces upon binding to the cellular DNA. Upon treatment with PA-NV, an enhancement in the fluorescence intensity was observed in both E. coli and S aureus bacteria (Figure 6B and 6C). It proves that PA-NV is competent enough to permeabilize the cytoplasmic membrane of both Gram-positive (S. aureus) and Gram-negative (E. coli) bacteria. As we have already shown that our compound PA-NV is permeable to the outer membrane and interacts with cytoplasmic membrane, indicates that PA-NV is involved in cell injury, which results in the premature death of cells. Furthermore, to know the mechanistic aspect of the bacterial death we have performed Live/Dead assay using 2 wt% hydrogel. Firstly, the bottom surface of the 35 mm glass cover bottom dish was coated with 2 wt% PA-NV hydrogel, followed by addition of E. coli at a concentration of 106 CFUs and incubated it for 24 hrs. From figure 6, we have found that bacteria when incubated over the PA-NV hydrogel, gives red signal due to the staining of PI rather than green, while another batch without PA-NV hydrogel (considered as control) bacteria showed green fluorescence. From figure 7, we found higher extent of bacterial adhesion under static culture conditions rather than substrate irrespective of the presence of the peptide hydrogel. On the contrary, the bacteria over the PA-NV hydrogel displayed red signal rather than green signal, while in case of control green signal was observed. From these experiment and data, we concluded that bacterial growth above the PA-NV hydrogel surface are significantly inhibited, in spite of a high inoculum, suggesting its high antibacterial activity. PA-NV hydrogel can efficiently kill bacteria via a mechanism of membrane permeabilization. It is well known that PI only penetrates damaged cell membranes and interacts with DNA and RNA. Since bacteria have been stained by PI in presence of PA-NV hydrogel, this indicates that that the peptide hydrogel prompted bacterial


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death through cell membrane destabilization mechanism. From figure 7, it was further observed that dead bacteria were susceptible to aggregation, signifying membrane permeabilization mechanism. Co-culturing experiments were carried out to check the selectivity of the PA-NV hydrogel following previously reported procedure.42 After the incubation of E. coli along with WI-38 human normal cells without the peptide hydrogel PA-NV, the morphology of WI-38 cells was observed to be distorted with spherical like shape with the red signal due to the staining of nuclei by PI. But, green signal has been observed for bacteria due to the staining of SYTO 9 (Figure 8A). It is expected due to the co-culture of both bacteria and WI-38 cells without any antibiotics. In another set of experiment, where WI-38 cells have been cultured along with bacteria in presence of PA-NV hydrogel, the WI-38 cell morphology has been observed healthy. From the Figure 8B we have found that the morphology of WI-38 cells are intact on the gel surface which has been stained by calcein-AM (green), while the bacteria gives red signal (PI). This suggests that PA-NV hydrogel offers high cellular selectivity in case of bacterial contamination. Since our hydrogel provide good biocompatibility, non-cytotoxicity in nature with significant antibacterial activity with this added facility of exceptional cellular selectivity, it becomes a great scaffold material for biomedical applications. To apply it in clinical purpose, we are motivated to focus in future about its further enhancement of antibacterial activities under dynamic culture conditions, along with biofilm formation on the hydrogel surface. Finally, hemolytic activity of the PA-NV hydrogel has been examined by addition of 2 wt% PA-NV hydrogel on hRBC’s and rocking the system in an orbital shaker. After that, the absorbance of the cell lysis at 450 nm has been recorded. The absorbance at 450 nm is due to the release of hemoglobin from hRBC’s in the lysis process (Figure 7C). The solutions containing 6.9 × 105 hRBC’s considered as control showed minimal absorbance at 450 nm,



revealing that the cells healthy (Figure 8C). With another batch, 1% Triton-X-100 has been introduced to hRBC’s, which is considered as positive control. Due to the surfactant nature of Triton-X-100 rapid cell lysis occurs, which shows maximum absorbance at 450 nm. In a another set, 2 wt % PA-NV hydrogel has been added with hRBC’s and incubated under shaking conditions for 1 h. From the Figure 7C, the cells were observed to be healthy and intact, which suggested the non-hemolytic nature of PA-NV hydrogel. Figure 8D indicates the effect of PA-NV hydrogel on hRBC’s after incubation for 5 h. Cells display its inherent spherical morphologies which suggests that hRBC’s are healthy. Thus from this experiment we can say that the gel’s surface is non-hemolytic. CONCLUSIONS PA-NV hydrogel surfaces exhibited substantial antibacterial activity against E. coli, S. aureus. It was found to be non-cytotoxic towards mammalian cells, biocompatible and stable against enzyme degradation. It is hypothesized that PA-NV compound binds to bacterial cell membrane, which leads to cell membrane lysis and necrotic death of bacteria. From coculture experiments, we found that when WI-38 human normal cells and E. coli were cultured onto the PA-NV hydrogel, its surface is able to prohibit bacterial growth whereas mammalian cells remain intact with healthy morphology. This shows the potential application of selective nature of our hydrogel. Moreover, haemolysis experiments confirmed that the hydrogel surfaces are non-hemolytic toward hRBC’s. Overall, our newly developed hydrogel shows a lot of promises for future and opens up opportunities to further design new generation antibacterial gel for clinical applications. ASSOCIATED CONTENT Supporting Information


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The Supporting Information is available free of charge on the ACS Publications website at DOI: 8b01681. http://pubs.acs.org. Experimental section; characterization of peptide (HPLC, MALDI-Mass, NMR), peptide hydrogel formation (FTIR, CD, TEM, rheology study); cell morphology, surface coating, colony inhibition study, colony count assay (PDF). AUTHOR INFORMATION Corresponding Author E-mail: [email protected]. Notes The authors declare no competing financial interest. ACKNOWLEDGMENT Authors wish to thank Prof. A. K. Nandi for accessing his laboratory and NCCS-Pune for cell lines. SG thanks DST (EMR/2015/002230) and CSIR-Network project (HCP-0012) for financial assistance. ABBREVIATIONS NAVSIQKKK, NV. Palmitic acid conjugated NAVSIQKKK peptide, PA-NV. REFERENCES 1.

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Figure 1. MD-simulation of two NVK3 peptides for 40 ns applying Gromacs force field.


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Figure 2. (A) Schematic representation of the PA-NV molecule with the modulation of amphiphilicity. (B) MD-Simulation of NVK3 peptide for 40 ns. (C) Optical image of inverted PA-NV hydrogel. (D) AFM images of PA-NV hydrogel showing three dimensional nanofiber network. (E) TEM image of PA-NV hydrogel shows the nano-fiber structure. Scale bars correspond to the 200 µm for (D) and 400 for µm (E).

Figure 3. (A) Frequency sweep rheology study of PA-NV hydrogel. (B) Rheology study shows the injectibility nature of the hydrogel. (C) Demonstration of the injectable nature of the hydrogel, which shows the thixotropy property of PA-NV hydrogel.


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Figure 4. (A) Proteolytic degradation assay of PA-NV molecule in presence of Proteinase K enzyme in HEPES buffer up to 24 hrs. (B) Cell viability assay of WI-38 cells treated with PA-NV hydrogel at different concentrations. Error bar corresponds to standard deviation of value from mean (n = 3, *p< 0.05, performing two tailed student’s t test). (C) Cell viability assay of HeLa cells treated with PA-NV hydrogel at different concentrations. Error bar



corresponds to standard deviation of value from mean (n = 3, *p< 0.05, performing two tailed student’s t test). (D) Images of cell morphology of WI-38 cells treated with 2 wt % PA-NV hydrogel, indicates healthy morphology.

Figure 5. (A) E. coli coloni formation inhibition by PA-NV hydrogel surface. (B) Inhibition of S. areus coloni formation by PA-NV hydrogel. (C) E. coli bacteria viability assay by PA-


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NV hydrogel. Error bar corresponds to standard deviation of value from mean (n = 3, **p< 0.01, performing two tailed student’s t test). (D) S. aureus bacteria viability assay by PA-NV hydrogel at λmax = 625 nm. Error bar corresponds to standard deviation of value from mean (n = 3, **p< 0.01, performing two tailed student’s t test). (E) Apoptosis study using flow cytometry in E. coli bacteria in different concentration of PA-NV.

Figure 6. Outer membrane permeabilization assay of E. coli (A), cytoplasmic membrane permeabilization assay of E. coli (B) S. aureus (C) at different concentration of PA-NV and valinomycin as positive control.



Figure 7. Live-dead cell assay by SYTO9 and PI staining: (A) Control image of E. coli in green channel. (B) E. coli in red channel. (C) Merge image of both the channels. (D) E. coli


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in presence of PA-NV hydrogel in green channel. (E) E. coli in presence of PA-NV hydrogel in red channel. (F) Merge image of both the channels. Scale bars correspond to the 25 µm.



Figure 8. Co-culture Assay: (A) WI-38 cells in presence of bacteria upon the control surface. (B) WI-38 cells in presence of bacteria upon the PA-NV hydrogel surface. (C) Haemolytic assay of hRBC in presence of 2 wt% PA-NV hydrogel. Error bar corresponds to standard deviation of value from mean (n = 3, **p< 0.01, performing one way ANOVA) (D) Morphology of hRBC after 5 hr. incubation in presence of 2 wt% PA-NV hydrogel. Scale bars correspond to the 25 µm for (A), (B) and 10 µm for (D).


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New antibacterial hydrogel has been designed based on a lipopeptide template, which shows high antibacterial effect with significant biocompatibility.


Biocompatible Lipopeptide-Based Antibacterial Hydrogel

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