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Article Cite This: Biomacromolecules 2019, 20, 2743−2753

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Synthesis and Characterization of Peptide−Chitosan Conjugates (PepChis) with Lipid Bilayer Affinity and Antibacterial Activity Thais H. Costa Petrin,† Valmir Fadel,‡ Danubia B. Martins,‡ Susana A. Dias,§ Ana Cruz,§ Luciana Marciano Sergio,∥ Manoel Arcisio-Miranda,∥ Miguel A. R. B. Castanho,§ and Marcia P. dos Santos Cabrera*,†,‡

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†

Departamento de Química e Ciências Ambientais and ‡Departamento de Física, Instituto de Biociências Letras e Ciências Exatas (Ibilce), Universidade Estadual Paulista (Unesp), Câmpus São José do Rio Preto, Sao Jose do Rio Preto 15054-000, São Paulo, Brazil § Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Lisboa 1649-028, Portugal ∥ Laboratório de Neurobiologia Estrutural e Funcional (LaNEF), Departamento de Biofísica, Universidade Federal de São Paulo, São Paulo 04023-062, Brazil S Supporting Information *

ABSTRACT: Antimicrobial peptides appear among innovative biopolymers with potential therapeutic interest. Nevertheless, issues concerning efficiency, production costs, and toxicity persist. Herein, we show that conjugation of peptides with chitosans can represent an alternative in the search for these needs. To increase solubility, deacetylated and degraded chitosans were prepared. Then, they were functionalized via N-succinimidyl-S-acetylthiopropionate or via glutathione (GSH), an endogenous peptide linker. To the best of our knowledge, it is the first time that GSH is used as a thiolating agent for the conjugation of peptides. Next, thiolated chitosans were conjugated through a disulfide bond with designed shortchain peptides, one of them derived from the antimicrobial peptide Jelleine-I. Conjugates and respective reaction intermediates were characterized by absorciometry, attenuated total reflectance−Fourier transform infrared, and 1H NMR. Zeta potential measurements showed the cationic nature of these biomacromolecules and their preferential partitioning to Gram-positive bacterial-like model membranes. In vitro investigation using representative Gram-positive and -negative bacteria (Staphylococcus aureus and Escherichia coli, respectively) showed that the conjugation strategies lead to enhanced activity in relation to the unconjugated peptide and to the unconjugated chitosan. The obtained products showed selectivity toward S. aureus at low cytotoxicity as determined in NIH/3T3 cells. Overall, our study demonstrates that an appropriate choice of antimicrobial peptide and chitosan characteristics leads to increased antimicrobial activity of the conjugated product and represents a strategy to modulate the activity and selectivity of antimicrobials resorting to low-cost chemicals. The present proposal starts from less expensive raw materials (chitosan and short-chain peptide), is based on aqueous solvents, and minimizes the use of reactants with a higher environmental impact. The final biopolymer contains the backbone of chitosan, just 3−6% peptide derived from royal jelly and GSH, all of them considered safe for human use or as a physiological molecule.

1. INTRODUCTION Antimicrobial peptides (AMPs) have been largely studied as alternatives to the current antibiotic therapies.1,2 These peptides are highly effective, selective toward bacterial targets, and are considered less likely to induce resistance.1,3 The hallmarks of AMPs are the presence of cationic groups and structural amphipathicity, which are, respectively, determinants of the specificity in the binding to the bacterial cell membrane and of the subsequent membrane permeablization.3,4 An appropriate balance of hydrophobicity and cationicity is a requisite for selectivity and efficient antimicrobial activity.1,3 Despite the accumulation of convincing examples that encourage further efforts,5,6 a rapid development of AMP application has been limited by difficulties in achieving high © 2019 American Chemical Society

efficiency/toxicity ratios, proteolytic stability, and desirable pharmacokinetics at fair manufacturing costs.1,7 To overcome these obstacles to improving AMPs’ drugability, several structural modifications have been proposed,5−7 among them the peptide conjugation with polymers.8 Chitosan, a natural copolymer of glucosamine and Nacetylglucosamine, exhibits cationic character at pH below 6.5. This characteristic endows chitosans with antimicrobial activity, which is not seen with other abundant natural polymers. Moreover, besides being not toxic, biocompatible, Received: April 12, 2019 Revised: June 7, 2019 Published: June 11, 2019 2743

DOI: 10.1021/acs.biomac.9b00501 Biomacromolecules 2019, 20, 2743−2753

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Biomacromolecules

yl-S-acetylthiopropionate) and via GSHand, afterwards, conjugated with different peptides. The antimicrobial activity of these conjugates was evaluated and further insights into their interaction with bacterial model membranes are presented. Given the recognized importance of electrostatic interaction on the mechanism of action of peptides and chitosan,3,9 zeta potential measurements with phospholipid vesicles were performed to evaluate the partitioning of conjugates and potential antimicrobial activity. We are interested in the investigation and development of peptide conjugation with chitosan, aiming to improve chitosan antimicrobial activity and reduce the minimal inhibitory concentration (MIC) of the peptide. This would represent a way to reduce costs of peptide-based drugs, starting from less expensive raw materials (chitosan and short-chain peptides), using aqueous solvents and minimizing the use of reactants with higher environmental impact. Moreover, our approach has a view to avoid the development of mechanisms of resistance toward antimicrobial peptides and to circumvent problems related to bioavailability and potential toxicity.3,23

and biodegradable, chitosan was preferred in relation to other polymers because it also acts as a bioadhesive, has wound healing properties, and its reactive amino group enables the synthesis of several different derivatives.9−12 The antimicrobial activity of chitosan was demonstrated to depend on the species of microorganisms used in the evaluation of the antimicrobial activity, but also on chitosan physical−chemical features, such as the molecular weight (Mw), the degree of deacetylation, and the polycationic character, which may contribute to the selective association of chitosan with the anionic groups on the surface of bacteria and fungi membranes.9,10 There is parallelism between the mechanisms of action attributed to AMPs and chitosan derivatives, which depend on their physicochemical features and on the lipid composition of bacterial membranes.3,9 Both, antimicrobial peptides and chitosan derivatives, are of a cationic nature, target bacterial membranes, and may accumulate and/or permeabilize them. Accumulation of AMPs or chitosan derivatives compromises the vital functions and/or barrier properties of membranes. Comprehensive reviews on the mechanisms of action of peptides and chitosan can be found elsewhere.3,5,9,10 For longer than a decade, conjugated molecules obtained with modified amino acids or with peptides have been studied in an attempt to obtain antimicrobials with increased efficiency and selectivity. Besides the increased efficiency, the main advantages searched for with peptide/chitosan conjugates in relation to the unconjugated biopolymers are the combination with the bioadhesiveness and tissue regeneration contributions of chitosan.13−24 The enhanced efficiency has been attributed to the increase in the local peptide concentration, to the stability toward proteolytic degradation, and to the reduced cytotoxicity.22,25 Additional contribution to the efficiency may be brought about by increasing hydrophobicity, but this is not so clearly established.10 Whereas some polylysine peptides (K6) exhibited improved antimicrobial activity compared to peptides with higher hydrophobicity as poly(lysine-ran-phenylalanine),17,22 nonpeptide derivatization showed that the presence of a dodecyl substituent was important for increased activity.26 Moreover, according to Li et al.17 and Su et al.,22 conjugates of chitosan and peptides hold a close similarity with the peptidoglycan layer of bacterial cell walls and this structural affinity favors the primary interaction with antimicrobial agents. Thus, depending on the nature of its derivatization, chitosan conjugates can provide a permanent cationic character, increased solubility at a physiological pH, structural affinity with bacterial cell walls, which would enhance the antimicrobial activity and improve the selectivity of Pep− Chitosan conjugates. One of the possible strategies to conjugate amino acids or peptides to the chitosan backbone is its thiolation followed by conjugating a peptide that contains a cysteine residue.22,24 An interesting thiolating agent, unexplored in the conjugation of peptides until now, is glutathione (GSH; γGlu-Cys-Gly), an ubiquitous cellular peptide found in all organisms that exerts several functions including the maintenance of biological membranes. GSH also contributes to chitosan properties of mucoadhesiveness and tissue permeation combined with a convenient toxicological profile and is endowed with high conformational flexibility.21,27−31 Here, we report the preparation of conjugates that consist of a thiolated chitosan backbone with grafted peptides (PepChis). They were obtained by two different strategies of chitosans’ functionalization with thiol groupsvia SATP (N-succinimid-

2. EXPERIMENTAL SECTION 2.1. Materials. Chitosans were obtained from two sources, reagent grade from Sigma-Aldrich (SP, Brazil, named CS) and commercial grade from Polymar (Polymar Ind. Com. Exp. Ltda. CE, Brazil, named, CP) and were used in parallel. Deuterium chloride, 1-ethyl-3(3-dimethylaminopropyl)carbodiimide hydrochloride (EDAC), hydroxylamine, L-cysteine, L-glycine, L-glutathione reduced form (GSH), and N-hydroxysuccinimide (NHS) were purchased from SigmaAldrich (Brazil). SATP and Ellman’s reagent were from Thermo Fisher Scientific (Waltham, MA USA). Deuterium oxide was from Cambridge Isotopes Labs. Inc. (USA). Egg L-α-phosphatidylcholine (PC) and L-α-phosphatidylglycerol sodium salt (PG) were supplied by Avanti Polar Lipids (Alabaster, AL). Water was deionized and materials were of analytical grade quality or superior and used as received unless otherwise stated. 2.2. Synthesis. 2.2.1. Peptides. Peptides for conjugation reaction were supplied by GenScript USA Inc. (Piscataway, NJ, USA) at minimum 98% purity. 2.2.2. Chitosan Deacetylation and Degradation. Chitosan samples (from both suppliers) were solubilized in glacial acetic acid and deacetylated by the use of sodium hydroxide (pellets) as described by Tiera et al.32 Next, they were depolymerized with NaNO2 solution (NaNO2/NH2 = 0.25 molar ratio), followed by treatment with NaBH4 at pH 5.5 (NaBH4/NH2 = 0.75) for 24 h at room temperature, to convert reactive mannose endings to lessreactive mannitol endings.33,34 Finally, they were precipitated, washed, dialyzed, and freeze-dried according to Huang et al.35 for further use. Right before the functionalization steps, chitosan samples were gently dried at 50 °C for 4 h in Petri dishes. 2.2.3. SATP-Functionalized Chitosan. (Scheme 1) This procedure was inspired by Lee et al.16 and adapted from the SATP manufacturer’s instructions. Under magnetic stirring, 1.7 mg of deacetylated and depolymerized chitosan (approx. 10 μmol NH2 groups) were dissolved in 300 μL of HCl 0.1 mol L−1, then 750 μL 12 mmol L−1 phosphate buffer (3.15 mmol L−1 KH2PO4, 8.70 mmol L−1 Na2HPO4·7H2O, 5 mmol L−1 EDTA), pH 7.4, was added. When solubilization completes, 200 μL of SATP solution (2.5 mg in 200 μL of dimethyl sulfoxide (DMSO); −NH2/SATP mol ratio = 1) freshly prepared was added, kept under stirring for 90 min, and then incubated for 30 min. The reaction mixture was dialyzed twice against 1 mmol L−1 phosphate buffer (0.26 mmol L−1 KH2PO4, 0.73 mmol L−1 Na2HPO4·7H2O, 2.5 mmol L−1 EDTA), pH 7.4, using an MWCO 500−1000 Da membrane (Float-A-LyzerG2, Spectrum Laboratories Inc.). 2.2.3.1. Determination of the Content of the Thiol Group. This quantification was carried out using Ellman’s reagent (2 mg dissolved 2744


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Biomacromolecules Scheme 1. Synthesis of Functionalized Chitosan by SATPa

determined at 412 nm in the Shimadzu UV-2600 spectrophotometer (Shimadzu, Japan). The total thiol content was calculated from the molar extinction coefficient of 14 150 M−1 cm−1 using a 1 cm path length quartz cuvette. 2.2.5. Peptide Conjugation. 2.2.5.1. Peptide Conjugation with SATP-Functionalized Chitosan. (Scheme 3) As SATP introduces

Scheme 3. Conjugation of peptidesa

a

Chitosan is shown as the deacetylated monomer only; SATP in red.

in 250 μL of ethanol, then in 250 μL phosphate buffer, pH 7.4). Around 1 mg of the thiolated chitosan sample (lyophilized) was diluted in 470 μL of phosphate buffer, pH 7.4, and reacted with 50 mM hydroxylamine solution [10 mM EDTA in phosphate-buffered saline (PBS)] to generate free thiol groups. A 50 μL aliquot of Ellman’s solution was added, homogenized, and incubated for 15 min. The absorbance was determined at 412 nm in the Shimadzu UV-2600 spectrophotometer (Shimadzu, Japan). The total thiol content was calculated from the molar extinction coefficient of 14 150 M−1 cm−1. 2.2.4. Glutathione (GSH)-Functionalized Chitosan. (Scheme 2) This procedure was carried out according to Kafedjiiski et al.28

a

Chitosan is shown as the deacetylated monomer only; SATP in red, GSH in green, and the peptide highlighted in gray.

Scheme 2. Synthesis of Functionalized Chitosan by GSH (Chitosan is Shown as the Deacetylated Monomer Only; GSH in Green)

Depolymerized chitosan (1.7 mg) was hydrated with 12.8 μL of HCl 0.1 mol L−1, then diluted in deionized water to make a 1% m/v solution. The pH was adjusted to 6.0 with 5 mol L−1 NaOH. Under magnetic stirring, GSH aqueous solution (8 mg/160 μL) was added. Next, an EDAC aqueous solution was added to a 200 mmol L−1 final concentration, and then an NHS aqueous solution was added also to a 200 mmol L−1 final concentration, under vigorous stirring. The pH was adjusted to 6.0 with 5 mol L−1 NaOH. The reaction mixture was kept under magnetic stirring for 7 h at room temperature, then dialyzed once against 5 mmol L−1 HCl, twice against 5 mmol L−1 HCl containing 1% NaCl, and twice against 1 mmol L−1 HCl, using the same membrane as for the SATP functionalization. GHS-functionalized chitosans were freeze-dried. 2.2.4.1. Determination of the Content of the Thiol Group. This quantification was carried out as for the SATP-functionalized chitosans with the following modifications. The sample (around 1 mg) was diluted in 500 μL of Tris−Glycine buffer (10 × 10−3 M EDTA), pH 8.0, and reacted with 500 μL of 2% NaBH4 solution for 30 min at 40 °C, with occasional stirring to generate free thiol groups. The excess NaBH4 was neutralized with 300 μL of 1 M HCl under stirring for 2 min. Next, 1 mL of acetone was added and stirred for 3 min. The pH was adjusted to 7.5−8.5 with 1 mL Tris-HCl buffer (10 mM EDTA), pH 8.5. A 100 μL aliquot of Ellman’s solution was added, homogenized, and incubated for 15 min. The absorbance was 2745

protected sulfhydryl groups by forming a stable covalent amide bond with primary amines, deprotection is carried out with hydroxylamine to generate the free SH group. Samples (approx. 10 μmol NH2 groups) were dissolved in 1 mL of 1 mmol L−1 phosphate buffer pH 7.4, and 100 μL of 50 mmol L−1 hydroxylamine solution (in phosphate buffer pH 7.4 containing 10 mmol L−1 EDTA) was added under stirring. After 90 min of stirring followed by 30 min of resting, the pH was adjusted to 5.5 and the peptide solution (1 mg in 200 μL of deionized water) was added. The mixture was incubated for 5 h, then kept at 4 °C overnight, dialyzed five times against 1 mmol L−1 phosphate buffer, pH 7.4, and freeze-dried. 2.2.5.2. Peptide Conjugation with GSH-Functionalized Chitosan. Samples (approx. 10 μmols NH2 groups) were dissolved in 1 mL of 1 mmol L−1 phosphate buffer, pH 8.0, and 200 μL of aqueous sodium borohydride solution (2%) was added to free any disulfide bond that could be formed. The reaction mixture was incubated for 30 min at 40 °C, with occasional stirring. The reaction was stopped by adding HCl 1 mol L−1 (100 μL/mg chitosan) under stirring for 2 min. Next, 1 mmol L−1 phosphate buffer (500 μL/mg chitosan), pH 7.4, was added under stirring for 3 min. The following steps, from peptide solution addition until getting the freeze-dried PepChis conjugates, were the same as for the chitosan functionalized with SATP. 2.3. Characterization. 2.3.1. 1H NMR. Deacetylated, depolymerized, and functionalized chitosan samples were analyzed by 1H NMR. From the previously dried samples, 10−15 mg was hydrated with 10 μL of deuterium chloride and solubilized in 700 μL of deuterium oxide under magnetic stirring. All 1D 1H NMR spectra were acquired with solvent presaturation, 32k points in 5.2 s of acquisition time and 3 s of relaxation delay at 70 °C,36 in an Agilent Technologies 500 MHz spectrometer. With the formula ÄÅ I ÉÑ ÅÅ ÑÑ H3 ÅÅ ÑÑ 3 Ñ the deacetylation degree was calculated for DDA = 1 − ÅÅÅ IH3 Ñ ÅÅ (IH1 + 3 ) ÑÑÑ ÅÇ ÑÖ the deacetylated and depolymerized samples. It considers the integration of the areas IH3 and IH1 under peaks H3 and H1, which respectively correspond to H of the methyl groups of acetylated monomers with a chemical shift of 2.37 ppm and are referred to H of the anomeric carbon with a chemical shift of 5.22 ppm. The content of GSH-functionalized chitosan could be calculated from the ratio between the integral of the areas IG1 and IG2 under peaks corresponding to the resonances of the methylene G1 and G2 hydrogens of GSH and the respective integral of the peak of the H1 ÄÅ I É ÅÅi G1 y i IG2 yÑÑÑ ÅÅjj 2 zz jj 2 zzÑÑ by the formula FF = 1/2ÅÅjj I zz + jj I zzÑÑ. ÅÅÅÇjk H1 z{ jk H1 z{ÑÑÑÖ DOI: 10.1021/acs.biomac.9b00501 Biomacromolecules 2019, 20, 2743−2753

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Biomacromolecules 2.3.2. Molecular Weight. The samples (10 mg) were solubilized in 1 mL of acetate buffer (0.2 mol L−1 acetic acid, 0.15 mol L−1 sodium acetate), pH 4.5,37,38 and filtered on 0.45 μm pore size membranes (Millipore) before injection. A Viscotek TDAmax system (Malvern Instruments, Worcestershire, U.K.) equipped with detector TDA305 and with a set of GPC columns of 300 × 7.8 mm ID (Water Co., Milford, MA) was used. Analyses were carried out in the buffer solution as the mobile phase, at a flow rate of 0.5 mL/min and sample injection volume of 1000 μL. 2.3.3. Attenuated Total Reflectance-Fourier Transform Infrared. Depolymerized, functionalized, and peptide-conjugated chitosan samples were analyzed with an attenuated total reflectance−Fourier transform infrared spectrometer, Spectrum Two (PerkinElmer, Beaconsfield, UK). Depending on the signal to noise ratio, 1.25− 1.50 mg of samples were dissolved in 25 μL of deuterium oxide and kept under stirring for 1 h. Approx. 7 μL of solutions are applied on the detector equipped with a sapphire crystal. For each spectrum, 32 interferograms were collected at 2 cm−1 resolution from 4000 to 400 cm−1. Solvent contribution was subtracted and spectra were analyzed with Spectrum 10 software (PerkinElmer, Beaconsfield, UK) 2.3.4. Spectrophotometric Determination of Peptide Content in the Conjugate. Peptide sequences contain a tryptophan residue, which strongly absorbs at 280 nm. PepChis samples of 0.7 mg were solubilized in 1 mL of deionized water and analyzed in the Shimadzu UV-2600 spectrophotometer (Shimadzu, Japan). After correcting for the solvent and base line absorption, the absorbance observed at 280 nm was used to calculate the content of peptide, considering the molar absorptivity of 5580 M−1 cm−1 for the Trp residue in aqueous medium. 2.4. PepChis Interaction with Model Membranes. 2.4.1. Large Unilamellar Vesicles’ Preparation. Lipid large unilamellar vesicles (LUVs) were composed of PC/PG 70:30 molar ratio and 100% PG. Films in these compositions were obtained by mixing aliquots of stock solutions of phospholipids in chloroform, evaporating the solvent under a gentle nitrogen flow, and drying under vacuum for at least 3 h. To prepare LUVs’ suspension at 7−10 mmol L−1 lipid concentration, films were hydrated with citrate/ phosphate buffer (10 mmol L−1 C6H 807 ·H 2O, 20 mmol L−1 Na2HPO4·2H2O, 150 mmol L−1 NaF) pH 5.5, and vortex-mixed at room temperature. Using an Avanti (Alabaster, AL) mini-extruder, LUVs were obtained within a diameter range of 105−130 nm [confirmed by dynamic light scattering (DLS), with a Zetasizer Nano ZS, Malvern Instruments, Worcestershire, U.K.] after extrusion, at room temperature, through polycarbonate membranes (Nuclepore Track-etch Membrane, Whatman, USA), six times through 400 nm followed by 11 times through 100 nm pore size membranes. LUVs were used within 24 h of preparation, kept under refrigeration (8 °C), and protected from light. 2.4.2. Particle Size Measurements and Zeta Potential Determination. The Zetasizer Nano ZS (Malvern Instruments, Worcestershire, U.K.) was used to determine changes in the size of LUVs induced by PepChis in comparison to the correspondent depolymerized chitosans, through its size mode. Also, zeta potential changes were obtained to verify the binding of the same samples to the LUVs. PepChis solutions in the concentration range of (44−352) × 10−3 g L−1 were prepared in citrate/phosphate buffer, pH 5.5, in plastic vials and an aliquot LUVs’ suspension was added to a final concentration of 100 μmol L−1. Before measurement, each preparation was left to equilibrate for 15 min at 25 °C and then transferred to a disposable cuvette for the size evaluation (Sarstedt 67.754/Malvern DTS0012). Next, the same preparation was transferred to a DTS1070 cell (Malvern) for zeta potential measurement. 2.5. Antibacterial Activity. The antibacterial activity of PepChis conjugates was evaluated by a standard broth microdilution procedure to determine the growth inhibition and the MIC values.39,40 Bacterial suspensions of Gram-positive Staphylococcus aureus (ATCC 25923) and Gram-negative Escherichia coli (ATCC 25922) were prepared by direct suspension of morphologically similar colonies in Mueller Hinton broth (BD, Franklin Lakes, NJ) to a final concentration of 1 × 10 6 CFU mL−1 and added to a sterile 96-well microtiter

polypropylene plate (Corning, NY, USA) containing twofold dilutions of each PepChis sample. The final bacterial concentration was 5 × 105 CFU mL−1, whereas PepChis concentration ranged from (800 to 12.5) × 10−3 g L−1 and Jelleine-I from (96 to 1.5) × 10−3 g L−1. A triplicate was prepared. The plates were incubated at 37 °C for 18 h. The negative control was the broth in the absence of inoculum and the positive controls were 20 μg mL−1 vancomycin for S. aureus and 10 μg mL−1 ampicillin for E. coli. MIC was defined as the lowest PepChis or Jelleine-I concentration, which visibly inhibited bacterial growth. 2.6. Cytotoxicity. The fibroblast cell line NIH/3T3 (ATCC CRL1658) was cultured in Dulbecco’s modified Eagle’s medium (GibcoThermo Fisher Scientific Inc., MA, USA), supplemented with 10% fetal bovine serum (Gibco-Thermo Fisher Scientific Inc., MA, USA), 100 U·mL−1 penicillin, and 100 mg mL−1 streptomycin (GibcoThermo Fisher Scientific Inc., MA, USA). The cells were maintained in a humidified incubator with 5% CO2 at 37 °C. Cell viability was assessed by 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma-Aldrich Co., MO, USA) assay as previously described.41 Briefly, cells were plated in 96-well plates at a density of 5 × 103 cells/well. Twenty-four hours after cell plating, the medium was replaced by medium without penicillin/streptomycin supplemented with 5% fetal bovine serum. PepChis conjugates were tested at (200 and 800) × 10−3 g L−1. After 18 h, 0.1 × 10−3 g mL−1 of MTT was added into each well and the plates were further incubated at 37 °C for 3 h. The supernatants were carefully removed and 100 μL of DMSO was added into each well to solubilize the formazan crystals. Finally, the optical density was measured at 570 nm with a microplate reader (PowerWave XS, BioTek Instruments, VT, USA). The results are presented as mean ± standard error mean (SEM) of at least three independent experiments. In order to access the different PepChis effects on NIH/3T3 viability, ANOVA one-way followed by Tukey multi comparison test was performed in GraphPad Prism 6 (GraphPad Software, San Diego, CA, USA). Results with probability values of p < 0.05 were considered statistically significant.

3. RESULTS AND DISCUSSION 3.1. Design of Peptides for Conjugation. The aim of having innovative biopolymers consisting of peptides is to take advantage of peptides’ specificity and optimal in vitro activity and, simultaneously, circumvent their shortcomings through the properties of conjugated chitosan. We started by designing two model pentapeptides to work as minimalistic probes (PChis-1 and PChis-2, Table 1) of the conjugation reaction Table 1. Designed Unconjugated Peptidesa peptides

sequences

net charge

⟨H⟩

PChis-1 PChis-2 JIChis-1

RWAAC-NH2 CAAWR-NH2 PWKISIHLAAC-NH2

+2 +2 +2.5 (+3b)

−0.017 −0.017 0.097

Net charge considered at physiological pH; ⟨H⟩, mean hydrophobicity calculated according to Eisenberg et al.44 bAt pH below 6.0, His residues will be mostly protonated. a

and of the bilayer binding activity. These peptides consisted of a permanent positive charge, conferred by an Arg residue, an intrinsically absorbing probe, the Trp residue, the spacer, two Ala residues,14 and the Cys residue, to enable the disulfide bridge with the functionalized chitosan. The presence of a spacer is expected to maintain the activity of the coupled peptide.19 A sequence of three Gly residues could be used as a spacer;16 however, we preferred the two-Ala which confer additional hydrophobicity. In a previous work,26 we found out that hydrophobicity is an important factor to increase the antimicrobial activity of chitosan derivatives. 2746


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sample exemplifies the difference between the two procedures (Table 2). Mw and DDA characteristics of CS and CP following DA and DG show that before functionalization steps both chitosans are similar. These unique chitosan samples (CS-DADG and CP-DADG) were employed in the next reactions. For their Mw of around 5.0 kDa, our DADG chitosans can be classified as oligo-chitosans.9 3.3. SATP Functionalization of CS- and CP-DADG. SATP brings some advantages to thiolation reactions. It reacts specifically with primary amine groups, under mild and nondenaturing conditions, in the pH range of 7−9, temperatures from 4 to 37 °C, and relatively short incubation times. Also, sulfhydryl groups are introduced in a protected form, allowing longer storage of the intermediate product. Thiolation of chitosan with the aim to conjugate peptides was not often used and detailed results of the obtained products are not supplied.16 This reaction was repeated at least three times for each chitosan sample and the content of sulfhydryl groups ranged from 1.0 to 2.4 × 10−7 mol SH groups/mg chitosan as determined by Ellman’s reagent method. In average, this is roughly equivalent to 3% SH groups (1 mg of chitosan contains approx. 6 μmol NH2 groups). 3.4. GSH Functionalization of CS- and CP-DADG. GSH is a tripeptide with a gamma peptide bond between the carboxyl group of the Glu-residue side chain and the Nterminal of a Cys-residue, which is normally bound to a Glyresidue. The reduced cysteine is covalently attached to chitosan via a carbodiimide-mediated coupling reaction forming amide bonds between the carboxylic group at the Cterminal of GSH and amine groups in chitosan.28,45 This reaction occurs preferentially with the carboxylic groups of the Gly-residue. It was attributed to a much higher mobility of this group than the corresponding groups in the Glu-residue under physiological conditions.31 Additionally, there is a small difference between the pKa values of the α-carboxylic groups in Gly- and Glu-residues, which also carries a protonated Nterminal, both facts favoring the Gly-residue interaction with the chitosan amino groups. Carboxylic groups are activated by EDAC that catalyzes the amide bond formation with enhanced yield by the presence of NHS.28,45 To the best of our knowledge it is the first time that GSH is used as a thiolating agent for the conjugation of peptides. This reaction was repeated three times for each, CS- and CP-DADG, and the content of sulfhydryl groups were 7.9 and 14.4 × 10−7 mol SH groups/mg chitosan, respectively, as determined by Ellman’s reagent method. This is, respectively, equivalent to 13 and 24% SH groups (1 mg of chitosan contains approx. 6 μmol NH2 groups). This thiolation range is compatible with results obtained for an 18 kDa, 96% DDA chitosan using the same procedure.45 Liu et al.30 reported ca. 6 × 10−7 mol total SH groups/mg chitosan when thiolation was carried out with Nacetyl cysteine. Figure 1 exemplifies the 1H NMR spectra of the GSH-functionalized CP, which allowed to identify the resonances of the methylene (G1) and (G2) hydrogens at 2.91 and 2.56 ppm, respectively.46 These spectra showed that the yield of the GSH functionalization reaction was 20% for CP. The content of −SH groups may differ when determined by Ellman’s test or by NMR. In the former test, the total thiol content is determined immediately after reduction (with hydroxylamine or with NaBH4 solutions for the SATP or GSH thiolation) whereas in the latter, samples are prepared in advance, and some oxidation may occur. In this case, the result obtained reports the free thiol content.

Two peptides were designed with reversed sequences (PChis-1 and PChis-2), which allow us to check the effect of having the disulfide bridge either at the N- or at the Cterminal. Additionally, an analog of the well-studied antimicrobial octapeptide found in royal jelly, Jelleine-I, was included in the study. This analogue was modified to contain a Trp residue, to include the same spacer of two Ala residues, and the Cys residue (Table 1). Jelleine-I was chosen because of its amenable source, shown to be neither cytolytic nor directly involved with inflammatory processes associated with a good antimicrobial profile.42 Moreover, it is a soluble, short-chain peptide of cationic character endowed with some hydrophobic character. These physicochemical features reduce production costs and contribute to a membranolytic mechanism of interaction with microbial membranes. Nevertheless, previous work pointed out to a detrimental aggregation tendency,43 which is limiting for biotechnological and pharma applications but can in principle be avoided if the peptide is conjugated. This set of peptides allows us to study the importance of cationic charges being located closer to the backbone and of the peptide being conjugated through its N-terminal instead of the C-terminal in relation to its binding potential.10,23 3.2. Chitosan Preparation for Functionalization. CS and CP showed Mw of 422 and 338 kDa, respectively, with the same Mn/Mw = 4.40 (considering pullulan samples as references) and deacetylation degree (DDA) of 80 and 73%, respectively. CS was deacetylated once and CP twice. Depolymerization of deacetylated CS and CP leads to CSDADG and CP-DADG of equivalent Mw (Table 2). The Table 2. Chitosans’ Characteristics before Functionalizationa samplesb

% DDA

Mw (kDa)

Mw/Mn

DPW

CS CS DA CS-DADG CP DA CP-DADG

80 96 93 97 94

169 73 5.3 163 5.0

1.2 1.6 1.6 1.4 1.6

996 448 32 1003 31

a Deacetylation degree (% DDA) obtained by 1H NMR (see Figure SI1); weight-average molecular weight, Mw, obtained by GPC (RALS), and the deduced weight-average degrees of polymerization, DPw. bCS, CS DA, and CS-DADG refer, respectively, to chitosan obtained from Sigma, then deacetylated (DA), and further depolymerized (DG); CP refers to commercial chitosan, obtained from Polymar.

purpose of these preparation steps was to take advantage of the tendency of chitosans with higher DDAs and lower Mw, having lower MIC values against bacterial strains.9,10 DDAs were determined based on the integral values from the 1H NMR spectra, using the ratios between the N-acetyl signal (H3 at 2.37 ppm) and the anomeric carbon (H1 at 5.22 ppm). 1H NMR spectra do not differ significantly in relation to the linewidth. Linewidth may be qualitatively related to the molecular weight and the spectra show that after degradation there are no substantial differences in the Mw between CS and CP (Figure SI1). Peaks referring to the mannose monomer were not detected. After deacetylation (DA) and depolymerization (DG), Mw was characterized by a system based on right angle light scattering (RALS), which determines the absolute molecular weight for small polymers. The result obtained for the CS 2747


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Figure 1. 1H NMR spectra of DADG chitosan samples’ functionalization with GSH, CP-GSH, and CS-GSH. The indicated H1, G1, and G2 peaks correspond, respectively, to 1H resonances of the anomeric carbon of the deacetylated monomer (5.22 ppm), and of the methylene G1 and G2 carbon of GSH (shown in the inset).

Table 3. Peptide Content in PepChis Conjugates As Determined by Trp Characteristic UV-Absorption in Several Reactions CS-/CP-DADGSATP CS-PChis-1 CP-PChis-1 CS-PChis-2 CP-PChis-2 CS-JIChis-1 CP-JIChis-1

SATP thiolation no. 1/9 1/2 to 7/9 2 to 5 2 to 7 8 2 to 7

% peptide (m/m) 1.8/1.9 2.0/1.4/4.1 1.7 1.5 2.2 1.4

CS-/CP-DADGGSH CS-PChis-1 CP-PChis-1 CS-PChis-2 CP-PChis-2 CS-JIChis-1 CP-JIChis-1

3.5. Peptide Conjugation. The formation of the conjugate PepChis was evidenced by the FTIR spectra and further quantified by the spectrophotometric determination of peptide through the Trp content (Table 3). The set of FTIR spectra following the steps of chitosan modification are shown in Figure 2. Figure 2A,B (DADG), either for CS or CP chitosans, shows the characteristic bands of chitosan as previously described:14,23,47 an intense broad band around 3400 cm−1, attributed to stretching of −NH and of hydrogen bonded −OH, at 2900 cm−1, a low-intensity band ascribed to the stretching of −CH, at 1630, 1540, and 1454 cm−1, respectively, the regions of amide I (CO stretching), of amide II (low-intensity NH bending) and of amide III (C−N stretching and NH in plane deformation). Additionally, we observed bands at 1070 cm−1 (C−O stretching) and at 1030 cm−1 (C−O−C glucopyranose ring stretching). Spectra obtained for the functionalized chitosan either with SATP (Figure 2A) or GHS (Figure 2B) exhibit an increase in the intensity of the peak in the amide I region, consistent with the coupling of both reactants, which in the PepChis conjugates is further increased. The PepChis conjugates (PChis-1, PChis-2, and JIChis-1) exhibit some distinct changes in relation to the spectra of −DADG chitosans. Although the characteristic bands of chitosan (at 3400, 1630, 1540, 1454, and 1070 cm−1) are present, there are significant contributions at vibrations related to CO and C−N, respectively, the regions of amide I and III. The amide I band is frequently used to determine the secondary structure of peptides because of its higher intensity48 and has been taken as indicative of successful coupling of peptide and chitosan.14,23,45 The coupling of the three different peptides is evidenced by the same important contributions in the amide I and III regions either functionalized with SATP or GHS.

GSH thiolation no. 3 3 1, 2 1, 2 1, 2 1, 2

% peptide (m/m) 3.7 4.3 0.4 1.1 3.0 3.0

Figure 2. FTIR spectra of chitosans (CS-derivatives, thick lines; CPderivatives, thin lines), functionalized derivatives (red lines) and PepChis conjugates. (A) SATP-functionalized derivatives. (B) GSHfunctionalized derivatives. Conjugates: PChis-1 (blue); PChis-2 (gray); JIChis-1 (green). Spectral regions’ characteristics of chitosan and of the peptide conjugation are shown as vertical dotted lines.

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Figure 3. Comparing peptides, PChis-1 (black/dark gray symbols) vs PChis-2 (red symbols) vs JIChis-1 (light grey symbols). Zeta potential isotherms for PepChis conjugates in the presence of 100 μM LUVs, at pH 5.5 and 25 °C. (A,C) For conjugates obtained via SATP in the presence of PG and PC/PG 70:30 vesicles, respectively. (B,D) for conjugates obtained via GSH, in the presence of PG and PC/PG 70:30 LUVs, respectively. Closed symbols for CS- and open symbols for CP-derivatives. Error bars calculated from triplicates are about the size of symbols and were hidden for clarity. (E) Zeta potential change (Δζ = ζ − ζ0) induced by PepChis conjugates at 176 × 10−3 g L−1 in the zeta potential of PG LUVs (ζ0). (F) In the zeta potential of PC/PG 70:30 LUVs.

sponding to the thiolation with SATP or GSH show similar profiles independently of the PepChis conjugate tested. Nevertheless, stronger interactions are observed with PG LUVs, emphasizing the importance of electrostatic interactions. For example, considering all PChis-1 conjugates independently of the starting chitosan and functionalization reactant, the Δζ (ζ − ζ0) at 176 × 10−3 g L−1 ranges from 50.1 to 40.0 mV in PG, and ranges from 28.9 to 21.5 mV in PC/PG 70:30. Fully charged PG vesicles better discriminate differences between PepChis conjugates. However, both vesicles’ compositions show that JIChis-1 tends to impart weaker interactions (Figure 3E,F). Comparing the two thiolation processes, in PG LUVS, charge neutralization occurs for half of the samples at 176 × 10−3 g L−1 when prepared via SATP and for 1/3 of those prepared via GSH. In PC/PG 70:30 LUVs, charge neutralization occurs for 1/3 of the samples at doubled concentration either when prepared with SATP or GSH (Figure SI2). Although the characterization of the PepChis conjugates showed some differences among them (Tables 1 and 3), the interaction with model membranes just suggest a tendency of weaker binding of the products made of the longer chain length peptide JIChis-1, better evidenced in PG LUVs. In these products, the permanent positive charges are located farther from the chitosan backbone than in those made of Chis-1 and Chis-2. Quaternized amino groups located closer to the chitosan backbone have been correlated with improved antimicrobial activity.20 In relation to the coupling of peptides through their N- or C-terminal, PChis-2 showed slightly lower binding than PChis-1, but differences are not systematically significant. Peptides richer in cationic residues coupled (by different processes with higher Mw chitosans) through their Nterminal have shown mostly enhanced antibacterial activ-

The quantification of peptide molecules bound to functionalized chitosans through the content of Trp residue is shown in Table 3, as obtained in several reactions either when functionalization was made with SATP or GSH. We found that functionalization with GSH generally brought about higher contents of bound peptides, with exceptions made for CS- and CP-PChis-2. Nevertheless, both functionalization procedures lead to very limited coupling of peptides. Most frequently, higher amino acid/peptide concentrations were reported for different functionalization reactants: with EDAC/ NHS 8.7−28.4% for arginine coupling;45 through coppercatalyzed alkyne-azide coupling chemistry, from 6 to 23% for Anoplin coupling20 but 0.2% for Dhvar-5 conjugation.23 3.6. PepChis Interacts with Lipid Bilayers As Model Membranes. The interaction of PepChis conjugates with model membranes was evaluated by carrying out zeta potential measurements of anionic LUVs made of pure PG or 70:30 PC/PG, which approximately mimic the membranes of Grampositive and Gram-negative bacteria, respectively.49 Electrostatic attraction is an important feature when antimicrobials are expected to target membranes as is the case with peptides and chitosan derivatives.3,27 We employed zeta potential to compare PepChis conjugates in relation to the thiolation process (SATP × GSH) and in relation to the different peptides PChis-1 × PChis-2 × JIChis-1 (Figure 3). At pH 5.5, chitosan amino groups and Arg or Lys and His residues in the peptide sequences are mostly protonated and biopolymers have cationic character. We observed a steady increase of the initial zeta potential (ζ0) of the vesicles (for PG, ζ0 ≈ −40 mV; for PC/PG 70:30, ζ0 ≈ −30 mV) tending to a plateau as a consequence of the increase in the concentration of PepChis conjugates at the membrane, and concomitant neutralization of charges by cationic conjugated peptides. Isotherms corre2749


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Biomacromolecules Table 4. Physicochemical Parameters and Antibacterial Activity of Chitosan and Derivativesa Δζ (mV) at 176 × 10−3 g L−1 biopolymer

% peptide (m/m)

CS-DADG CP-DADG CSSATPPChis-1 CPSATPPChis-1 CSSATPJIChis-1 CPSATPJIChis-1 CSGSHJIChis-1 CPGSHJIChis-1 Jelleine-I

0 0 1.8 1.4 2.2 1.4 3.0 3.0 100

peptide net charge (at pH 7.0)

+2 +2 +2.5 +2.5 +2.5 +2.5 +2.5

PG

50.1 45.6 28.5 29.7 36.4 25.2

PC/PG 70:30

28.9 27.0 27.5 12.8 27.7 17.8

MIC (×10−3 g L−1) ATCC S. aureus 25923

E. coli 25922

>800 >800 200 (3.6) 200 (2.8) 200 (4.4) 200 (2.8) 200 (6.0) 200 (6.0) 96

>800 >800 800 (24) 800 (24) 800 (24) 800 (24) 600 (18) 800 (24) 12

a % peptide is the content of peptide in each conjugate. Net charge is the free peptide charge at physiological pH. Δζ (ζ − ζ0) is the LUV zeta potential variation induced by 176 × 10−3 g L−1 of each conjugate in the zeta potential of vesicles in the absence of conjugates (ζ0). In parentheses, equivalent peptide concentration.

ity.20,23 However, with PepChis conjugates the N-terminal coupling results in a lower cationic contribution from the PChis-2 peptide. The tendency to a plateau observed in the isotherms suggests that PepChis conjugates are inducing aggregation, either in PG or in PC/PG 70:30 (figure A−D). This is supported by vesicle size measurements, which ranged from around 2000−6000 nm at the concentration of 88 × 10−3 g L−1 of PepChis conjugates (Table SI1). Membrane disturbance capacity was observed in pure PG monolayers [1,2dipalmitoyl-sn-glycero-3-phospho-(1′-rac-glycerol)] induced by thiolated low Mw chitosans, which exhibited significant expansion and decreased elasticity.27 3.7. PepChis Shows Enhanced Antibacterial Activity Against S. aureus Coupled with Low Cytotoxicity. The antibacterial activity data of PepChis conjugates and their parent biopolymers, CS- and CP-DADG, against Grampositive (S. aureus) and Gram-negative (E. coli), which are representative of the most clinically important bacteria, are shown in Table 4. The MIC values are in general agreement with reported results for chitosans of higher Mw and higher acetylation degree.9,10 PepChis conjugates exhibited higher efficiency toward S. aureus than toward E. coli. Interestingly, these results diverge from those obtained by Fernandes et al.,27 in which a thiolated chitosan of 15 kDa conjugated with Nacetyl-cysteine was used. They found equivalent MIC values for S. aureus and E. coli. It appears that the higher Mw is counteracted by the N-acetyl-cysteine residues, which increase the overall hydrophobicity of the product (by lowering the content of available amino groups for protonation), and could favor interactions with less anionic Gram-negative lipid membranes.49 PepChis conjugates made of JIChis-1 showed improved antibacterial activity compared to Jelleine-I, especially against S. aureus but also toward E. coli (considering that the conjugates contain an average of 2% peptide that means approx. 2 μg mL−1). This observation suggests that increasing polarity is a way to improve selectivity against Gram-positive bacteria. Importantly, in the range of the MIC values for S. aureus the PepChis conjugates show low cytotoxicity toward NIH/3T3 cells and they are relatively cytotoxic in the MIC range for E. coli (Figure 4). Chitosan peptidopolysaccharide conjugates synthetized using different strategies showed diverse antibacterial profiles. The data in references Pranantyo et al.,24 Li et al.,17 and Su et al.22 do not show a preferential activity toward S. aureus or E. coli. The first study shows an MIC value of 128 × 10−3 g L−1

Figure 4. Effects of PepChis on NIH/3T3 cell viability. The cell viability was assessed by the MTT method after 18 h of incubation with PepChis at the MIC concentrations for S. aureus (white bars) and E. coli (blac). Data are expressed as the mean ± SEM of at least three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 vs control (C) (conjugates-untreated bacteria) by one-way ANOVA followed by Tukey multicomparison test. ns is non-significant.

(corresponding to 58 × 10−3 g L−1 of peptide) for a peptidopolysaccharide obtained from a decapeptide with a positive net charge of +5, which originally displayed MIC values of 32 × 10−3 g L−1. Employing peptides, either practically deprived of antimicrobial activity (polylysine K16 and K25), or a very active one (EPL with MIC of 4 × 10−3 g L−1) in their conjugates, the latter studies show, respectively, MIC values of 10 × 10−3 or 8 × 10−3 g L−1. However, Sahariah et al.20 used a decapeptide with a positive net charge of +3 and found a preferential activity toward E. coli with MIC values as low as 4 × 10−3 g L−1 (corresponding to 2.3 × 10−3 g L−1 of peptide). These results show that different conjugation strategies lead to a broad range of MIC values and that tuning the selectivity toward Gram-positive or -negative strains will rely on further efforts. Moreover, the peptide contents in PepChis at the MIC for S. aureus and E. coli are, respectively, 1 order of magnitude lower but 1 order of magnitude higher than those contents observed for the conjugation with anoplin, whose conjugates displayed selectivity toward Gram-negative strains.20 The dependence of the antimicrobial activity of chitosan and derivatives like PepChis in relation to the bacterial strains has been attributed mainly to the differences in the composition of the Gram-positive and Gram-negative cell wall.10 Despite the differences between these cell walls, their constituents 2750


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concentrations, bacterial growth was eliminated, but the antibacterial activity of the parent chitosans could not be detected until the highest tested concentration of 800 × 10−3 g L−1. PepChis conjugates made of JIChis-1 showed improved antibacterial activity compared to Jelleine-I, especially against S. aureus but also toward E. coli. Additionally, in the range of the MIC values for S. aureus the PepChis conjugates show low cytotoxicity toward NIH/3T3 cells and they are relatively cytotoxic in the MIC range for E. coli (Figure 4). Our results show that the conjugation strategies, via SATP or GSH, lead to equivalent MIC values, but cytotoxicity levels are increased in the GSH-functionalized products. We believe that the present approach may constitute a general strategy for enhancing the activity and selectivity of AMPs. Our proposal starts from less-expensive raw materials (chitosan and short-chain peptide), is based on aqueous solvents, and minimizes the use of reactants with higher environmental impact. The final biopolymer contains a backbone of chitosan, just 3−6% peptide derived from royal jelly and GSH, all of them considered safe for human use or as a physiological molecule. Future efforts will be directed toward optimizing the degree of polymerization of the conjugates, increasing the fraction of conjugated peptide as well as investigating the scope and mechanism of these findings by examining alternative AMP−chitosan conjugates. Additional efforts are also required to analyze the viability of developing these conjugates for therapeutic or surface coating purposes.

lipoteichoic acids (LTA) in Gram-positive and lipopolysaccharides (LPS) in Gram-negative cell wallsplay similar roles.3 As cationic peptidopolysaccharides bind to and aggregate on the cell wall, the structural disturbance increases permeability and the structural resemblance with LTA and LPS helps the interaction and crossing through the peptidoglycan layer and the access to the cytoplasmic membrane.24 This structural similarity is greater with the cell wall of Gramnegative bacteria.17 In this sense, our designed PepChis conjugates obtained via GSH as the coupling agent is a closer peptidoglycan mimetic than the conjugates obtained via SATP (Schemes 1 and 2). The thickness of the peptidoglycan layer may not impair peptides or low Mw PepChis conjugates from crossing it,9,10 but strong peptide binding with LPS or LTA may reduce the required concentration at the cytoplasmic membrane.3 Nevertheless, our work and recent reports indicate that peptidopolysaccharides may reach higher antibacterial activity with greater selectivity than just peptides or chitosan.17,20

4. CONCLUSIONS In the present work, peptides were designed to study two conjugation strategies based on the formation of a disulfide bond with chitosan, which was previously functionalized via SATP or via GSH. Both processes are carried out under mild conditions, but the conjugation with SATP rendered a lower thiolation ratio (3%) than GSH (18% in average) did, with lower cost of chemicals. Over the SATP, the GSH thiolation has the advantages of introducing an endogenous peptide as a linker, which may also contribute to chitosan properties of mucoadhesiveness and tissue permeation.21,27−30 To the best of our knowledge it is the first time that GSH is used as a thiolating agent for the conjugation of peptides. Nevertheless, the formation of the conjugate PepChis, as evidenced by FTIR spectra and further quantified by the Trp content, did not reach the same level as the thiolation ratio would enable. However, the functionalization with GSH generally reached higher contents of bound peptides, with exceptions made for CS- and CP-PChis-2. PChis-2 peptide is linked by a Cys residue in its N-terminal, which is protonated because of peptide amidation. This positive charge may hinder the oxidation process of forming the disulfide bond. Zeta potential measurements showed the binding of conjugates to bacterial mimetic membranes, besides confirming the PepChis cationic nature because net charge variation occurred. Although the characterization of the PepChis conjugates showed some differences among them (Tables 1 and 3), we just found a tendency of weaker binding of the products made of the longer chain length peptide JIChis-1. In these conjugates, the permanent positive charges are located farther from the chitosan backbone than in those made of Chis-1 and Chis-2. In relation to the coupling of peptides through their N- or C-terminal, PChis-2 (coupled through its N-terminal) showed slightly lower binding than PChis-1, but differences are not systematically significant. Besides electrostatic interactions, we found that vesicles’ aggregation is also involved in the mechanism of interaction of PepChis conjugates as denoted by the increasing vesicle size measurements (Table SI1). The more pronounced interaction observed in PG in relation to the less anionic charged PC/PG 70:30 vesicles is in agreement with the superior antibacterial activity observed with Gram-positive S. aureus (Table 4). At the indicated

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biomac.9b00501. NMR characterization spectra of chitosan, charge neutralization determination through zeta potential isotherms, and DLS determination of vesicles’ size (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Susana A. Dias: 0000-0001-8910-5404 Miguel A. R. B. Castanho: 0000-0001-7891-7562 Marcia P. dos Santos Cabrera: 0000-0001-7443-2883 Author Contributions

The article was written through contributions of all the authors. All the authors have given approval to the final version of the article. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by Fundaçaõ de Amparo à Pesquisa do Estado de São PauloFAPESP nos. 2012/24259-0, 2012/ 02065-0, 2014/08372-7, 2015/07548-7, 2016/13368-4, 2016/ 50178-8, FCT (Fundaçaõ para a Ciência e a Tecnologia, Portugal) grant PTDC/QEQ-MED/4412/2014, and European Commission, Marie S. Curie action RISE, H2020-MSCARISE-2014, grant 644167. DBM was a recipient of the CAPES scholarships, LMS is a recipient of the CNPq scholarship, and 2751


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AC and SAD are recipients of scholarships PD/BD/136866/ 2018 and PD/BD/114425/2016 from FCT, respectively. In addition to funders, we acknowledge Prof. Dr. Márcia Bisinoti ́ Boscolo, Prof. and Prof. Dr. Altair Moreira, Prof. Dr. Mauricio Dr. Márcio Tiera and Prof. Dr. Vera Tiera for the use of the spectrophotometer, the IR spectrometer, and the lyophilizer, respectively. We are thankful to Dr. Maicon Petrônio for fruitful discussions.

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ABBREVIATIONS PepChis, peptide−chitosan conjugate; SATP, N-succinimidylS-acetylthiopropionate; GHS, glutathione; JI, Jelleine-I; LUV, large unilamellar vesicle

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REFERENCES

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