Synthesis, structural characterization and antimicrobial evaluation

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Peptide functionalized metallic nanoconstructs: Synthesis, structural characterization and antimicrobial evaluation Manish Bajaj, Satish Pandey, Nishima Wangoo, and Rohit Kumar Sharma ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.7b00729 • Publication Date (Web): 22 Jan 2018 Downloaded from http://pubs.acs.org on January 24, 2018

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ACS Biomaterials Science & Engineering

Peptide functionalized metallic nanoconstructs: Synthesis,

structural

characterization

and

antimicrobial evaluation Manish Bajaj,a Satish K. Pandey,b Nishima Wangoo*c and Rohit K. Sharma*a a

Department of Chemistry and Centre for Advanced Studies in Chemistry, Panjab University,

Chandigarh-160014, India b

Ubiquitous Analytical Techniques, Central Scientific Instruments Organisation (CSIR-

CSIO), Sector 30 C, Chandigarh 160030, India c

Department of Applied Sciences. University Institute of Engineering and Technology,

Panjab University, Sector 25, Chandigarh-160014, India * Corresponding authors.

E-mail address: [email protected] (R. K. Sharma), [email protected] (N. Wangoo) Tel.: +91 1722534409

1 ACS Paragon Plus Environment

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ABSTRACT Biomolecule based conjugation of metallic nanoparticles is an important area of research lately especially considering the role of such nanoconjugates in diverse applications. Herein, synthesis of cationic dipeptide conjugated gold/silver nanoparticles through covalent approach using carbodiimide crosslinker chemistry has been reported. Owing to the exceptional

optoelectronic

properties

of

metallic

nanoparticles,

peptide-gold/silver

nanoparticle conjugates were synthesized employing 2-mercaptopropanoic acid as a crosslinker. The conjugates were further compared with their non-covalently synthesized counterparts using ultraviolet-visible spectroscopy. It was generally observed that the conjugates synthesized using covalent approach were more stable than conventional noncovalent approach. The synthesized conjugates were further evaluated for their antimicrobial efficacy. The experimental findings demonstrated that peptide capped silver nanoparticles possessed relatively better antimicrobial activity than peptide capped gold nanoparticles, native peptides as well as unconjugated gold/silver nanoparticles which was also evident in time kill assay studies. The morphological effects of active compounds on Escherichia coli and Candida albicans exhibited complete disruption of the cell wall. Thus, this study is an important step towards opening up of avenues for the applicability of covalent approach for functionalization of metallic nanoparticles with not only short peptide based systems but also for other biomolecules in areas such as anti-infectives, drug delivery and bio-imaging.

KEYWORDS metallic nanoparticles, cationic peptides, covalent approach, nano-peptide conjugates, antimicrobials, FE-SEM analysis


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INTRODUCTION Over the last decade, bioconjugation of the metallic nanoparticles has become an important area of research due to its various biological applications.1 Metallic nanoparticles, especially gold and silver, owing to their extraordinary optoelectronic properties can be easily functionalized using variety of organic ligands, polymers and biomolecules etc.2 In particular, the surface modulation of nanoparticles is critical for obtaining stable peptide nanoconjugates. There are different types of interactions involved between nanoparticles and biomolecules which have been mainly categorized into non-covalent and covalent interactions.3 The non-covalent bonding involves electrostatic interactions between two oppositely charged ions (e.g. between negatively charged metallic nanoparticles and positively charged biomolecules) while covalent bonding is based on the interaction between metallic nanoparticles and biomolecule containing specific groups such as thiols. Since, gold/silver nanoparticles (AuNPs/AgNPs) have a strong binding affinity to thiols, these can be exploited to assist their conjugation with biomolecules such as aptamers, proteins, antibodies and peptides.4,5 Thus, the commonly used ligands for the preparation of AuNPs/AgNPs possess thiol group with generally a terminal amine or carboxylic moiety for further functionalization with biomolecules.6,7 The bioconjugation of metallic nanoparticles using covalent approach is quite a challenging job as it requires the presence of specific functionalities at both ends.8 It is in sharp contrast to the non-covalent approach which simply involves adsorption of ligands. Among different methods available for covalent binding, the most commonly used is the EDC/NHS coupling chemistry which catalyzes the formation of amide bonds.9 This method has been used for the synthesis of AuNP-antibody conjugates which were found to be highly stable for imaging of a tumour cell marking its applicability in a number of clinical and surgical applications.10 Further, applications of biomolecule conjugated nanoparticle involves



a report where spherical/rod shaped AuNPs have been modified using oligoethylene glycol as a spacer and conjugated with octapeptide (KPQPRPLS) prompting its crucial role in activating angiogenic genes.11 The role of a spacer molecule between biomolecule and nanoparticle has also been exploited where it was shown that enhancement in the loading of the oligonucleotides onto the AuNPs takes place by the use of thiolated PEG spacer.12 A comparative toxicological analysis of glutathione coated AuNPs modified with a non-polar hydrophobic and polar basic amino acid showed high biocompatibility in biological systems and hence used for imaging and metal chelation.13 The silver/polymer nano-composites were synthesized using an amine capped AgNPs and acid terminated polymers possessed strong bactericidal and X-ray contrast properties which make them potentially applicable in various biomedical applications.14 Apart from above discussed applications, biomolecule conjugated nanoparticles have considerable effects on the biological properties of both individual biomolecule and nanoparticle. One such report showed the effect of such a conjugation on the antimicrobial activity where enhancement in the antimicrobial activity of the food protecting peptide nisin has been observed when conjugated with AgNPs.15 Similarly, ceftriaxone, a well-known β-lactam antibiotic has showed a better efficacy after conjugation with AgNPs.16 Recently, the synthesis of short cationic dipeptide labeled AuNPs/AgNPs based nanohybrid materials using non-covalent approach was reported which displayed good antimicrobial activity against tested strains.17 The stability of nanoparticles was an issue in non-covalent approach as the prepared nanoconjugates were aggregated at higher peptide concentration. To overcome this, more stable nanoconjugates are needed to be synthesized. For this purpose, the present approach focuses on conjugation of short cationic peptides with AuNPs/AgNPs using covalent approach. The biomolecule conjugation with metallic nanostructures has been a relatively recent phenomenon; however, the rapid pace at which its application is being reported compels the need for understanding the properties of


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conjugated systems in an elaborated manner. Considering these facts and increasing importance of short peptide based nanoconjugates, herein, metallic short peptide-nano conjugates have been synthesized using EDC/NHS chemistry and characterized using various techniques such as UV-visible (UV-vis) spectroscopy, transmission electron microscopy (TEM), Fourier transform infrared (FT-IR) spectroscopy, differential scanning calorimetry (DSC), Field emission scanning electron microscopy (FE-SEM) and energy dispersive X-ray spectroscopy (EDS). Further, these nanoconjugates were subjected to different bacterial and fungal strains followed by time kill studies, FE-SEM and cytotoxicity analysis. Although reports regarding covalent conjugation of metallic nanoparticles with long chain peptides are available in literature, but, to the best of our knowledge, this is the first report where the conjugation of short cationic peptide with AuNPs/AgNPs has been thoroughly investigated via covalent approach using EDC/NHS chemistry. MATERIALS AND METHODS Materials Tetrachloroauric acid trihydrate (HAuCl4.3H2O), silver nitrate (AgNO3), tri-sodium citrate (TSC), sodium borohydride (NaBH4), 2-mercaptopropanoic acid (MPA), 1-ethyl-3-(3 dimethylaminopropyl)carbodiimide (EDC), N-hydroxy succinimide (NHS) and boric acid were obtained from Sigma, India and used as received. The Milli Q water having a resistivity of 18.2 MΩ-cm was used for the synthesis. All glasswares used for the synthesis were cleaned with aqua-regia. Synthesis of MPA capped AuNPs Synthesis of AuNPs was carried out using Turkevich method.18 In this method, 0.1 ml of 0.25 M HAuCl4.3H2O was dissolved in 100 ml of water. The solution was heated to boil and 2 ml, 34 mM TSC was added and kept at boiling temperature until a deep ruby red color appeared. Afterwards, the colloidal suspension of AuNPs was cooled to room temperature and used as



substrate to prepare MPA capped AuNPs. In a typical protocol, AuNPs (3 nM) were mixed with a solution of MPA (100 µM, 10:1 v/v) in water and incubated for 24 h at room temperature.4 The resulting colloidal suspension was purified using centrifuging at 9000 rpm for 30 min and characterized using UV-vis spectroscopy, TEM, FT-IR and EDS. Synthesis of MPA capped AgNPs Synthesis of AgNPs was carried out by using sodium borohydride reduction method.19 In this method, 1 ml of 100 mM AgNO3 was mixed with 1 ml of 34 mM TSC in 100 ml water. To this solution 0.1 ml, 2 mM NaBH4 was added under vigorous stirring. The mixture was stirred for 1 h and as prepared particles were used as substrate to prepare MPA capped AgNPs. In a typical protocol, AgNPs (3 nM) were mixed with a solution of MPA (100 µM, 10:1 v/v) in water and incubated for 24 h at room temperature.4 The resulting colloidal suspension was purified using centrifuging at 10000 rpm for 30 min and characterized using UV-vis spectroscopy, TEM, FT-IR and EDS. EDC/NHS coupling reactions In a typical protocol, 100 µl, 1 mM of earlier synthesized cationic dipeptides L-Arg-L-ArgOMe (4a), L-His-L-Arg-OMe (4b) and L-His-L-His-OMe (4c) (Figure S1) was dissolved in borate buffer (pH 8.5) and added to MPA capped AuNPs/AgNPs and mixed. To this solution, EDC (50 µl, 250 µM) and NHS (100 µl, 250 µM) were introduced simultaneously and reaction mixture was incubated for 24 h at room temperature.11 The particles were purified with repeated cycle of centrifuging at 9000 rpm for 20 min and characterized using UV-vis spectroscopy, TEM, FT-IR spectroscopy, DSC and EDS. Characterization of peptide capped AuNPs/AgNPs UV-vis spectroscopy


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UV-visible spectra were obtained in the range of 200-800 nm using JASCO V 530 model spectrophotometer with a precision of ± 0.2 nm using quartz cells having a path length of 1 cm. TEM analysis A drop of AuNPs/AgNPs suspension was applied to 300 mesh copper grid, left undisturbed for 1 min and finally excess water was removed by absorbing on a filter paper. The grid was analyzed using Hitachi (H-7500) 120 kV equipped with CCD camera with a resolution of 0.36 nm and 40-120 kV operating voltage. FT-IR spectroscopy The solid state FT-IR spectra of peptide and peptide capped AuNPs/AgNPs were obtained on a Thermoscientific, Nicolet iS50 FTIR spectrophotometer. DSC analysis It was carried using differential scanning calorimetry (DSCQ20 calorimeter, TA Instruments) in the temperature range 25-400 oC at a heating rate of 20 oC/min under nitrogen atmosphere. EDS analysis EDS spectrum was collected with the help of Bruker (XFlash 6130) analyzer attached to FESEM (Hitachi, SU8010). Antimicrobial studies Microbial strains The bacterial and fungal strains used in the this study were received from National Collection of Pathogenic Fungi (NCPF), Post-Graduate Institute of Medical Education and Research (PGIMER) and Microbial Culture Collection Centre (MTCC), Institute of Microbial Technology (IMTECH), Chandigarh, India. The bacterial strains Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) were cultured in Mueller Hinton broth (HiMedia); while fungal strains Candida albicans (C. albicans) and Candida glabrata (C. glabrata) in



yeast extract-peptone-dextrose (YEPD broth, HiMedia) and RPMI 1640 media (HiMedia). All strains with 15% glycerol were stored at -80 °C as frozen stocks. In each experiment, microbial cells were freshly revived on respective agar plates. Antimicrobial studies The two bacterial strains (E. coli and S. aureus) were grown overnight and diluted in Mueller-Hinton broth (MHB) to a cell density of 105 colony forming unit (CFU)/mL into 96well flat-bottomed microtiter plate. The plate was incubated at 37 ºC without shaking for 24 h. The visual and optical density at 600 nm was measured using microplate reader (BioRed, Model 680). The antifungal activities against two fungal species (C. albicans and C. glabrata) were performed according to the Clinical and Laboratory Standards Institute (CLSI) guidelines by broth microdilution methods. The minimum inhibitory concentration (MIC) was determined by a broth micro dilution method based on Clinical and Laboratory Standards Institute (CLSI) and kanamycin, a well-known standard antibacterial drug and Amphotericin B, a well-known standard antifungal drug was used as a positive control. The microtiter plates were incubated at 30 oC for 48 h. The visual and optical density at 492 nm was measured using microplate reader (BioRed, Model 680). The concentrations of MPAAuNPs/MPA-AgNPs and peptides capped MPA-AuNPs/MPA-AgNPs ranged 1-1000 µM was used to check the antibacterial and antifungal activity. Time kill assay analysis The E. coli and S. aureus cells (~105 CFUs/mL) were inoculated in MHB medium containing peptides, MPA-AuNPs/MPA-AgNPs and peptide capped MPA-AuNPs/MPA-AgNPs. The tube was incubated (37 °C, 200 rpm) and 100 µl of aliquots were removed at different time intervals (0, 4, 8, 12, 16, 20 and 24 h). Similarly, C. albicans and C. glabrata cells (~1 x 105 CFU/ml) were inoculated in YEPD medium containing MPA-AuNPs/MPA-AgNPs and peptide capped MPA-AuNPs/MPA-AgNPs. The tube was incubated (37 °C, 200 rpm) and


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100 µl of aliquots were removed at pre-determined time points (0, 4, 8, 12, 16, 20 and 24 h). The numbers of colonies were counted after incubating the plates at 30 °C for 24 h. FE-SEM analysis of microbial strains The cell suspension of E. coli and C. albicans was prepared in MHB and RPMI-1640 medium (pH 7) respectively. MPA-AgNPs, peptide capped MPA-AgNPs was added to the E. coli and C. albicans (~1 x 105 CFU/ml) and incubated at 30 oC for 2 h. Cytotoxicity analysis Cytotoxicity analysis of MPA-AuNPs/MPA-AgNPs, peptides and their conjugates against HaCaT cells was done using MTT (3-(4, 5)-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide) protocol.20 Briefly, HaCaT cells (9×104/well) were cultured in a RPMI-1640 supplemented with 10 % fetal bovin serum in 96-well microtiter plate at 37 °C for overnight. On the subsequent day, all compounds were added to cells in a separate well and incubated at 37 ºC for 24 h. The cells were further treated with 20 µl of MTT solution (2.5 mg/ml) in phosphate buffer saline (PBS) and incubated at 37 ºC for 4 h. The supernatant was removed and 100 µl DMSO was added to dissolve formazan crystals. The percentage viability of cells was calculated by the ratio of OD570 of treated cells to the OD570 of untreated cells. Untreated cells and 10 % DMSO were taken as negative and positive control respectively. RESULTS AND DISCUSSION Synthesis and characterization of MPA capped AuNPs/AgNPs Among various metals, gold and silver nanoparticles were proposed to be synthesized, since their remarkable optical properties and strong affinity towards sulfur atom render the nanoparticle surface effective for further functionalization with biomolecules such as peptides and proteins.2 In order to prepare a carboxyl terminated AuNPs/AgNPs, MPA was chosen as a linker. MPA is a bifunctional compound and has both a carboxylic acid (COOH) and thiol (SH) group.21 The SH group binds with the metal surface of AuNPs/AgNPs and enables the



COOH group for further functionalization. For preparation of MPA stabilized AuNPs/AgNPs, citrate capped AuNPs/AgNPs were modified with MPA using ligand exchange method.22 Initially, MPA was mixed with AuNPs/AgNPs and incubated for 24 h. Afterwards, the particles were purified and analyzed using UV-vis spectroscopy (Figure S2). A shift in the absorption maxima was observed for MPA-AuNPs and MPA-AgNPs (Figure S2) with respect to citrate capped AuNPs/AgNPs which indicated the formation of MPA stabilized AuNPs/AgNPs. The morphology of MPA-AuNPs and MPA-AgNPs was determined using TEM which demonstrated that particles were spherical in shape with a diameter of 12 ± 2 nm (Figure S3). Further, in order to confirm the binding interactions of MPA with AuNPs/AgNPs, FT-IR studies were carried out (Figure S4). The FT-IR spectrum of MPA displayed a broad peak in the 2550-2600 cm-1 corresponding to the characteristic S-H stretching vibration (Figure S4). However, when MPA was conjugated to AuNPs and AgNPs, the peak at 2550-2600 cm-1 corresponding to S-H stretching vibration disappeared which confirmed the binding of MPA to the nanoparticle surface through sulfur.23 Also, the carbonyl stretching frequency observed at 1694 cm-1 in MPA was shifted to 1613 cm-1 and 1573 cm-1 in MPA-AuNPs and MPAAgNPs respectively. These observations attributed to the fact that MPA bind to AuNPs/AgNPs through sulfur group possibly via complete exchange of citrate ion.22 EDC/NHS coupling reactions The nanoparticle-peptide conjugates were synthesized using EDC/NHS coupling chemistry.11 The advantage of using EDC is derived from its high solubility in water and NHS is used to enhance the stability of active intermediates involved during the coupling reaction. Initially, EDC was used to activate the carboxyl group present on the surface of AuNPs by forming Oacylisourea intermediate which was further replaced with reagent NHS and finally made to undergo coupling with peptide to form amide bond (Figure 1). In order to synthesize these


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conjugates, the concentration of peptide (4a-c), EDC and NHS was optimized using UV-vis spectroscopy (Figure 2). It was observed that interactions between MPA-AuNPs/MPAAgNPs and EDC/NHS proceeded with a change in the absorption maxima with respect to their SPR band. In the first set of experiments, concentration of peptide was kept constant and EDC/NHS was varied. To achieve this, varying concentrations of EDC (0.001-10 mM) and NHS (0.002-20 mM) were added to the 1 ml colloidal solution of both MPA-AuNPs and MPA-AgNPs containing 0.1 ml of 1 mM peptide (4a) and incubated for 24 h at room temperature. In case of MPA-AuNPs, new absorption band was observed in the region 650750 nm at higher concentrations of EDC (0.5-10 mM) and NHS (1-20 mM) which is probably due to formation of larger sized particles as depicted from color change (Figure 2A) as MPA-AuNPs displayed red color, prior to the addition of coupling reagents. However, absorption maxima of MPA-AuNPs was retained at 518 nm for all other tested concentrations of EDC (0.001-0.5 mM) and NHS (0.002-1 mM) clearly indicating that particles are quite stable (Figure 2A). So, the concentration of 0.1 mM of EDC and 0.2 mM of NHS was taken for further functionalization. Similarly, broadening of absorption band was observed for MPA-AgNPs at concentration of EDC (0.1-10 mM) and NHS (0.2-20 mM), indicated the destabilization of MPA-AgNPs as depicted from color change (Figure 2B). The absorption maxima of MPA-AgNPs observed at 394 nm for concentration of EDC (0.001-0.5 mM) and NHS (0.002-1 mM) indicated the formation of stable nanoconjugates (Figure 2B). Hence, the concentration of 0.01 mM of EDC and 0.02 mM of NHS was marked to be taken for further functionalization. To understand the chemistry of metal peptide conjugation, peptide (4a-c) capped AuNPs/AgNPs were also synthesized using non-covalent approach. Nano conjugates synthesized from both the approaches were compared for their stability. It was observed that dipeptide L-Arg-L-Arg-OMe (4a) capped AuNPs synthesized at 1 mM concentration of



peptide using both a covalent and electrostatic approach does not show any change in the characteristic absorption maxima of AuNPs (Figure 3A). It is probably due to the strong affinity of arginine towards the gold surface through a side chain guanidinium group.24 Further, the morphology of peptide 4a capped MPA-AuNPs showed the formation of stable spherical particles with diameter of 12 ± 2 nm (Figure 3B). However, peptide L-His-L-ArgOMe (4b) and L-His-L-His-OMe (4c) capped MPA-AuNPs synthesized at 1 mM concentration of peptide using covalent approach were observed to be more stable than noncovalent approach as depicted from absorption spectra (Figure 3C and 3E). The particles synthesized using non-covalent approach exhibited aggregation for peptide 4b as no absorption in UV-vis spectra while peptide 4c showed absorption maxima in the range 650750 nm for larger sized particles (Figure 3C and 3E). This peak can be attributed to the extra stability provided by interaction between gold and sulphur in covalent approach. The morphology of peptide 4b and peptide 4c capped MPA-AuNPs determined using TEM showed the formation of mono-dispersed spherical nanoparticles with a diameter of 12 ± 2 nm (Figure 3D and 3F). Interestingly, particles were stable at 1mM concentration of peptide (4a-c) in covalent approach, while in non-covalent approach it showed aggregation which proved that particles were covalently attached. Thus, it can be safely concluded that choice of synthetic approach for nano-conjugation process has a large impact on nanoparticle stability. In a similar manner, the conjugation of peptide (4a-c) with MPA-AgNPs was done using covalent approach and compared with non-covalent approach. The observations made in case of AgNPs were quite similar to AuNPs. It was observed that dipeptide L-Arg-L-Arg-OMe (4a) capped AgNPs does not show any change in the surface plasmon of AgNPs synthesized using both covalent and non-covalent approach (Figure 4A). The morphology of peptide 4a capped MPA-AgNPs formed spherical structures with diameter of 12 ± 2 nm (Figure 4B). In contrast, silver nanoconjugates of peptide L-His-L-Arg-OMe (4b) and L-His-L-His-OMe (4c)


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synthesized using covalent approach were more stable than non-covalent approach as depicted from absorption spectra (Figure 4C and 4E). It was observed that peptide 4b conjugates synthesized using non-covalent approach does not show any absorption in UV-vis spectra due to aggregation while peptide 4c shows absorption maxima in the range 600-700 nm for larger sized particles (Figure 4C and 4E). It was probably due to the stability provided by interaction between silver and sulphur. The morphology of peptide 4b and peptide 4c capped MPA-AgNPs determined by TEM showed spherical particles with a diameter of 12 ± 2 nm (Figure 4D and 4F). Further to confirm the binding interactions between peptide and MPA-AuNPs/MPA-AgNPs, FT-IR analysis was carried out. FT-IR spectrum of peptide L-Arg-L-Arg-OMe (4a) displayed two peaks at 3356.68 and 3128.49 cm-1 which corresponded to primary amine N-H stretching vibration (Figure 5A). However, when peptide 4a was conjugated with MPA-AuNPs, the NH stretching peak broadened and one hump appeared at ~3232.27 cm-1 indicating that interaction between MPA-AuNPs and peptide 4a occurs through N-terminal peptide amino group.23 Also, the carbonyl stretching vibration observed at 1636.95 cm-1 in peptide 4a was broadened and shifted to 1616.32 cm-1 in peptide 4a capped MPA-AuNPs, indicating that carbonyl bond is playing a part in conjugation through electronegative interaction with carbonyl oxygen. Similar trends were obtained for peptide 4b and 4c capped MPA-AuNPs therby confirming nanoparticle-peptide interactions (Figure 5B-C). Similarly, peptide 4a capped MPA-AgNPs showed shift in the N-H stretching region at 3429.19 and 3234.93 cm-1 and carbonyl bond stretching vibration shifted to 1555.78 cm-1. The shift in the amine and carbonyl stretching vibration confirmed the capping of peptide 4a on MPA-AgNPs (Figure 5D). Similar results were obtained for peptide 4b and peptide 4c capped MPA-AgNPs (Figure 5E-F). The FT-IR peak values (in cm-1) of all synthesized peptides and MPAAuNPs/MPA-AgNPs peptide conjugates have been summarized in Table S1.



To ascertain the surface chemistry of MPA-AuNPs/MPA-AgNPs and peptide capped MPAAuNPs/MPA-AgNPs, elemental analysis was carried out using EDS which gives percentage chemical composition of different elements present on the surface of nanoparticles (Figure 6). The EDS spectrum of MPA-AuNPs (Figure 6A) showed peaks for Au (3.16 %) and sulphur (0.05 %) while EDS of peptide (4b) capped MPA-AuNPs (Figure 6B) showed peaks for Au (3.05 %), sulphur (0.05 %) and nitrogen (0.35 %). The EDS spectrum of peptide (4b) capped MPA-AuNPs displayed relative decrease in percentage composition of Au whereas that of sulphur was intact and appearance of nitrogen peak indicated the labeling of peptide (4b) on MPA-AuNPs.25 In case of AgNPs, EDS spectra of MPA-AgNPs (Figure 6C) showed peaks of Ag (3.92 %) and sulphur (0.37 %) whereas peptide capped MPA-AgNPs showed (Figure 6D) peaks of Ag (1.54 %), sulphur (0.16 %) and nitrogen (0.70 %). Similarly, on comparing the EDS spectra of MPA-AgNPs and peptide (4b) capped MPA-AgNPs, percentage composition of Ag and sulphur was decreased and appearing of nitrogen peak in nanoconjugates indicated the labeling of peptide (4b) on MPA-AgNPs. In order to understand the interaction of peptide with MPA-AuNPs/MPA-AgNPs, differential scanning calorimetric studies were performed. The exothermic characteristic peaks were observed in thermogram of peptide (4b) and MPA-AuNPs at ~326.72 oC and ~77.88 oC (Figure S5). Correspondingly, these two peaks were identified in thermogram of peptide (4b) conjugate at ~ 342.27 oC and ~ 63.81 oC which suggested capping of peptide (4b) on MPAAuNPs (Figure S5A).26 In case of MPA-AgNPs, peak appeared at ~161.31 oC and peptide (4b) conjugate showed two peaks at ~311.18 oC and ~161.01 oC, indicating the formation of peptide (4b) capped MPA-AgNPs (Figure S6). Antimicrobial activity All the synthesized compounds including MPA-AuNPs, MPA-AgNPs and their peptide conjugates were evaluated against bacterial strains such as E. coli and S. aureus and fungi


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species C. albicans and C. glabrata (Table 1). The antimicrobial activity of dipeptides (4a-c) exhibited MIC values of 50.83, 42.28 and 24.16 µM respectively against Gram positive bacteria S. aureus. The conjugates MPA-AgNPs_4a, MPA-AgNPs_4b and MPA-AgNPs_4c were observed to exhibit MIC of 8, 8 and 8 µM respectively and found to be more potent than MPA-AgNPs which exhibited MIC value of 24 µM against Gram positive bacteria S. aureus indicated the additive effect of peptides and MPA-AgNPs (Table 1). Further, the conjugates MPA-AgNPs_4a, MPA-AgNPs_4b and MPA-AgNPs_4c exhibited MIC of 4, 4 and 8 µM respectively against Gram negative bacteria E. Coli which was better than MIC of peptide molecules (29.05, 30.75 and 32.66 µM ) and comparable with MPA-AgNPs (8 µM) (Table 1). Thus, it can be safely interpreted that peptide conjugates possessed potent antimicrobial activity against tested bacterial strains and opens a new class of nanoparticle peptide conjugate based compounds as effective antibacterial agents. As already described, dipeptides did not show any significant activity as such but when conjugated to MPA-AgNPs, exhibited better efficacy against both Gram-positive and Gram-negative bacteria. These results justified the coupling of peptides with nanoparticles.15 Furthermore, the MICs of MPA-AgNPs_4a, MPA-AgNPs_4b, MPA-AgNPs_4c and MPAAgNPs were found to be 24, 24, 16 and 32 µM respectively against fungi C. albicans and 24, 16, 16 and 40 µM against C. glabrata respectively (Table 1). It was noted that peptides (4a-c) alone exhibited better activity against C. albicans as compared to MPA-AgNPs and peptide conjugates (Table 1). On the other hand, peptide conjugates provide potent activity against C. glabrata as compared to MPA-AgNPs and dipeptides (Table 1). These observations suggested that MPA-AgNPs are more potent antimicrobials than antifungal agents. Interestingly, peptide (4a-c) capped MPA-AgNPs exhibited greater efficacy against both bacteria and fungi strains as compared to native peptides and bare MPA-AgNPs. In a similar manner, peptide capped MPA-AuNPs were evaluated against bacterial and fungal strains



(Table 1). It was noted that neither MPA-AuNPs nor peptide conjugates show any activity against all the tested strains. These observations were in agreement with antimicrobial properties of AgNPs as it can be interpreted that peptide conjugated MPA-AgNPs possessed a potent antimicrobial activity as compared to peptide conjugated MPA-AuNPs. Time Kill assay In order to account for the efficacy of active compounds, the time kill kinetic studies were performed in which colony forming units (CFUs) of the E. coli and S. aureus were rapidly reduced after treatment with MPA-AgNPs (at MIC 24 µM), MPA-AgNPs_4a (at MIC 4 µM), MPA-AgNPs_4b (at MIC 4 µM) and MPA-AgNPs_4c (at MIC 8 µM) respectively (Figure S7A-B). It was observed that the maximum killing of E. coli and S. aureus cells with MPAAgNPs and peptide conjugates were observed after 10 h in comparison to the used control. Therefore, it can be safely interpreted that the MPA-AgNPs and peptide conjugates successfully inhibited the growth of Gram positive and Gram negative bacterial strains. In a similar manner, the antifungal action of MPA-AgNPs (at MIC 24 µM), MPA-AgNPs_4a (at MIC 4 µM), MPA-AgNPs_4b (at MIC 4 µM) and MPA-AgNPs_4c (at MIC 8 µM) was determined separately against C. albicans and C. glabrata (Figure S7C-D). It was observed that the maximum killing of C. albicans and C. glabrata cells with AgNPs and peptide conjugates were observed after 10 h in comparison to the used control. FE-SEM analysis of microbial strains FE-SEM was used to investigate the morphological effects of active compounds on the E. coli and C. albicans (Figure 7). It was observed that when E. coli and C. albicans were treated with MPA-AgNPs_4b, an increased roughness and cell disruption was noted after 2 h of incubation (Figure 7C & 7F respectively) as compared to untreated E. coli (Figure 7A) and C. albicans (Figure 7D). Notably, the effect in terms of disruption of the cell wall by MPAAgNPs_4b was similar to that of well-known antibiotics i.e. Kanamycin and Amphotericin B


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which were used as positive control (Figure 7B & 7D respectively). However, on the other hand, treatment against MPA-AuNPs_4b did not show such morphological changes; thereby further asserting that peptide conjugated MPA-AgNPs are more potent antimicrobial agents as compared to peptide conjugated MPA-AuNPs (Figure S8). Cytotoxicity analysis The toxicity effect of all synthesized dipeptides and their conjugates on mammalian cells was evaluated to determine their safety profile against HaCaT cells at their MIC values. The purpose of choosing HaCaT cell line is to evaluate the skin cell line for C. albicans related infections.27 It was observed that all the compounds exhibited less than 20 % toxicity against HaCaT cells (Figure S9). The cytotoxicity data has been reported as mean ± SD of three independent experiments performed in quadruplicate. From MTT assay, it is safely concluded that peptide capped metallic nanoparticles are safe and non-toxic drug alternative and can be used against various microbial pathogens. CONCLUSIONS To best of our knowledge, this is the first report where short cationic peptide capped AuNPs/AgNPs have been synthesized via covalent approach using EDC/NHS chemistry. AuNPs and AgNPs were modified with MPA owing to their strong affinity towards sulfur and coupled with peptide using EDC/NHS coupling chemistry. The concentration of EDC/NHS used for conjugation was optimized using UV-vis spectroscopy. It was interesting to note that conjugates synthesized using covalent approach were much more stable than noncovalent approach. Further, the size of the peptide conjugates were found to be around 12 ± 2 nm analyzed using TEM. The capping of peptide onto the surface of nanoparticles was confirmed using FT-IR spectroscopy and EDS described the percentage composition of elements on nanoparticle surface. The capping of peptide on nanoparticles was further confirmed using DSC by virtue of exothermic peaks corresponding to the respective



ingredients. The synthesized peptides and their conjugates were evaluated for antimicrobial activity against host of bacterial and fungal strains. It was observed that peptide conjugated MPA-AgNPs demonstrated higher activity against two bacterial strains and one fungal strain as compared to MPA-AgNPs and native peptide. In particular, peptide L-Arg-L-Arg-OMe (4a) capped MPA-AgNPs exhibited MIC of 4 and 8 µM respectively against E. coli and S. aureus respectively. Further, L-His-L-His-OMe (4c) capped MPA-AgNPs exhibited MIC of 16 µM against both C. albicans and C. glabrata respectively. To the contrary, peptide conjugated MPA-AuNPs did not display any remarkable activity. Further, the time kill studies were performed for active compounds and confirmed that a very low dosage is required to kill the infected cells. FE-SEM analysis of peptide capped MPA-AgNPs against E. coli and C. albicans confirmed the morphological dissociation of bacterial and fungal cell membrane. Cell cytotoxicity studies against HaCaT cells revealed that tested compounds exhibited acceptable cytotoxicity. Particularly, long chain peptides have been used for covalent conjugation since they provide stabilization to the nanoparticle surface owing to capability of different orientations. Notably, here for the first time the stable metallic short peptide-nano conjugates was synthesized using covalent approach. Moreover, one-pot EDC/NHS coupling chemistry utilized for nanopeptide conjugation process has a large impact on nanoparticle stability and can lead towards the design of short peptide labeled nanostructures as novel antimicrobials. The peptide conjugates synthesized using present approach can be exploited to various applications such as drug delivery, sensing, bio-imaging etc. ASSOCIATED CONTENT Supporting information UV-vis spectra, TEM, FT-IR, 1H-NMR, Mass spectrum, FTIR values, DSC thermogram, Time kill assay, Cytotoxicity assay (PDF) is provided in the supporting information.


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AUTHOR INFORMATION * Corresponding authors: Tel.: +91 172 2534409 E-mail address: [email protected] (R.K. Sharma), [email protected] (N. Wangoo) Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Funding Sources This work was supported by Department of Science and Technology (DST-INSPIRE), India grant no. IFA12-CH-52, Science Engineering & Research Board (SERB), India grant no. SB/SO/BB/0040/2013 and DST-PURSE II grant. ACKNOWLEDGEMENTS MB thanks the University Grants Commission (UGC), New Delhi, India for research fellowship. REFERENCES (1) Yang, W.; Guo, W.; Chang, J.; Zhang, B. Protein/Peptide-Templated Biomimetic Synthesis of Inorganic Nanoparticles for Biomedical Applications. J. Mater. Chem. B 2017, 5, 401-417 DOI: 10.1039/c6tb02308h. (2) Boken, J.; Khurana, P.; Thatai, S.; Kumar, D.; Prasad, S. Plasmonic Nanoparticles and their Analytical Applications: A Review. APPL SPECTROSC REV 2017, 1-47 DOI: 10.1080/05704928.2017.1312427. (3) Biju, V.; Chemical Modifications and Bioconjugate Reactions of Nanomaterials for Sensing, Imaging, Drug Delivery and Therapy. Chem. Soc. Rev. 2014, 43, 744-764 DOI: 10.1039/c3cs60273g.



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Sedighimoghaddamf, B. Various Methods of Gold Nanoparticles (GNPs) Conjugation to Antibodies. Sens. Bio-sensing Res. 2016, 9, 17–22 DOI: 10.1016/j.sbsr.2016.04.002. (6) Wangoo, N.; Bhasin, K. K.; Mehta, S. K.; Suri, C. R. Synthesis and Capping of WaterDispersed Gold Nanoparticles by an Amino Acid: Bioconjugation and Binding Studies. J. Colloid Interface Sci. 2008, 323, 247-254 DOI: 10.1016/j.jcis.2008.04.043. (7) Wangoo, N.; Kaur, S.; Bajaj, M.; Jain, D. V. S.; Sharma, R. K. One Pot, Rapid and Efficient Synthesis of Water Dispersible Gold Nanoparticles Using Alpha-Amino Acids. Nanotechnology 2014, 25, DOI: 10.1088/0957-4484/25/43/435608. (8) Lin, S-Y.; Tsai, Y-T.; Chen, C-C.; Lin, C-M.; Chen, C-h. Two-Step Functionalization of Neutral and Positively Charged Thiols onto Citrate-Stabilized Au Nanoparticles. J. Phys. Chem. B 2004, 108, 2134-2139 DOI: 10.1021/jp036310w. (9) Totaro, K. A.; Liao, X.; Bhattacharya, K.; Finneman, J. I.; Sperry, J. B.; Massa, M. A.; Thorn, J.; Ho, S. V.; Pentelute, B. L. Systematic Investigation of EDC/sNHS-Mediated Bioconjugation Reactions for Carboxylated Peptide Substrates. Bioconjug. Chem. 2016, 27, 994-1004 DOI: 10.1021/acs.bioconjchem.6b00043. (10) Eck, W.; Craig, G.; Sigdel, A.; Ritter, G.; Old, L. J.; Tang, L.; Brennan, M. F.; Allen, P. J.; Mason, M. D. PEGylated Gold Nanoparticles Conjugated to Monoclonal F19 Antibodies as Targeted Labeling Agents for Human Pancreatic Carcinoma Tissue. ACS Nano 2008, 2, 2263-2272 DOI: 10.1021/nn800429d.


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Figure 1. Schematic representation of amide bond formation between dipeptide (4a-c) and MPA NPs using EDC/NHS coupling.


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Figure 2. UV-vis absorption spectra for EDC/NHS optimization of peptide 4a conjugated MPA-AuNPs (A) and MPA-AgNPs (B).



Figure 3. UV-vis absorption spectra and TEM images of peptide 4a conjugated AuNPs (A, B); peptide 4b conjugated AuNPs (C, D); and peptide 4c conjugated AuNPs (E, F), respectively (scale bar = 20 nm).


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Figure 4. UV-vis absorption spectra and TEM images of peptide 4a conjugated AgNPs (A, B); peptide 4b conjugated AgNPs (C, D); and peptide 4c conjugated AgNPs (E, F), respectively (scale bar = 20 nm).



Figure 5. FT-IR spectra of peptide 4a capped MPA-AuNPs (A), peptide 4b capped MPAAuNPs (B), peptide 4c capped MPA-AuNPs (C), peptide 4a capped MPA-AgNPs (D), peptide 4b capped MPA-AgNPs (E) and peptide 4c capped MPA-AgNPs (F).


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Figure 6. EDS of MPA-AuNPs (A), peptide 4b capped MPA-AuNPs (B), MPA-AgNPs (C) and peptide 4b capped MPA-AgNPs (D).



Figure 7. FE-SEM images of (A) Control cells (E. coli); (B) MPA-AgNPs_4b treated E. coli; (C) Kanamycin treated E. coli; (D) Control cells (C. albicans); (E) MPA-AgNPs_4b treated C. albicans; (F) Amphotericin B treated C. albicans.


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Table 1. MIC of peptide (4a-c) and peptide capped MPA-AgNPs/MPA-AuNPs against various bacterial and fungal strains. Bacterial Strains

a

Fungal Strains

Compound

E. colia,b

S. aureusa,b

C. albicansa,b

C. glabrataa,b

4a

29.05

50.83

3.00

80.00

4b

30.75

42.28

3.00

80.00

4c

32.66

24.16

2.40

60.00

MPA-AgNPs

8

24

32

40

MPA-AgNPs_4a

4

8

24

24

MPA-AgNPs_4b

4

8

24

16

MPA-AgNPs_4c

8

8

16

16

MPA-AuNPs

no inhibition

no inhibition

no inhibition

no inhibition

MPA-AuNPs_4a

no inhibition

no inhibition

no inhibition

no inhibition

MPA-AuNPs_4b

32

64

no inhibition

no inhibition

MPA-AuNPs_4c

64

64

no inhibition

no inhibition

Standard used: Kanamycin (9.29 µM for E. coli and 10.33 µM for S. aureus); Amphotericin B

(8.65 µM for C. albicans and 10.82 µM for C. glabrata). Concentration [in µM].


b

MIC: Minimum Inhibitory


For Table of Contents Use Only

Peptide functionalized metallic nanoconstructs: Synthesis, structural characterization and antimicrobial evaluation Manish Bajaj,a Satish K. Pandey,b Nishima Wangoo*c and Rohit K. Sharma*a


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Graphical Abstract

EDC/NHS Peptide

Metallic NPs MPA capped NPs

Peptide

ACS Paragon Plus Environment

Peptide conjugated NPs

Synthesis, structural characterization and antimicrobial evaluation

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