Antibacterial Efficacy of Gold and Silver Nanoparticles Functionalized with the Ubiquicidin ( 29 – 41 ) Antimicrobial Peptide

Recent studies have demonstrated that drug antimicrobial activity is enhanced when metallic nanoparticles are used as an inorganic support, obtaining synergic effects against microorganisms. The cationic antimicrobial peptide ubiquicidin 29–41 (UBI) has demonstrated high affinity and sensitivity towards fungal and bacterial infections. The aim of this research was to prepare and evaluate the antimicrobial efficacy of engineered multivalent nanoparticle systems based on silver or gold nanoparticles functionalized with UBI. Spectroscopy techniques demonstrated that NPs were functionalized with UBI mainly through interactions with the -NH2 groups. A significant increase in the antibacterial activity against Escherichia coli and Pseudomonas aeruginosawas obtained with the conjugate AgNP-UBI with regard to that of AgNP. No inhibition of bacterial growth was observedwith AuNP andAuNP-UBI using a nanoparticle concentration of up to 182μgmL. Nonetheless, silver nanoparticles conjugated to the UBI antimicrobial peptide may provide an alternative therapy for topical infections.

Metallic NPs such as silver (AgNP) and gold (AuNP) show unique and considerably distinct physical, chemical, and biological properties due to their high surface-to-volume ratio, with which surfaces can be modified with ligands containing functional groups, providing an electrostatic or steric stabilization [8].
Recent studies demonstrated that AuNPs also have antimicrobial activity [14].Different techniques for NP surface synthesis and functionalization with antimicrobial drugs through covalent or noncovalent interactions have been described, such as amoxicillin-coated AuNPs [15], vancomycin-capped AuNPs [16], and ampicillin-, streptomycin-, and kanamycin-conjugated AuNPs [17], as well as cefaclor- [18] and aminoglycoside-conjugated AuNPs [19].The aforementioned studies are in agreement that the use of nanoparticles combined with other antimicrobial effects can help reduce intrinsic toxicity of nanoparticles for mammalian cells, enhance the microbicidal effect by increasing damage mechanisms, and diminish the probability development of resistance.
Antimicrobial peptides (AMP) represent ancient host defence effector molecules.The basis of antimicrobial activity is the interaction of the peptide's cationic domains (positively charged) with the surface of microorganisms (negatively charged).The membranes of the latter expose negatively charged lipoteichoic acid and phospholipids while, in normal mammalian cells, negatively charged lipids face the cytoplasm.These properties explain the poor binding of cationic peptides to mammalian cells and the selective binding to bacteria in physiological conditions [20].
The aim of this research was to evaluate the antimicrobial efficacy of engineered multivalent nanoparticle systems based on silver or gold nanoparticles functionalized with the antimicrobial peptide ubiquicidin 29-41 (UBI).), sodium dodecyl sulfate, and solvents were purchased from Sigma-Aldrich Chemical Co. and used as received.All water was deionized and sterile, obtained from a MilliQ5 purified water system.

Synthesis and Stabilization of Silver and Gold Nanoparticles.
Gold nanoparticles were prepared by citrate reduction of tetrachloroauric acid according to the method described by Kimling et al. [26].Silver nanoparticles were prepared according to Dong et al. 's procedure [27].Nanoparticles were purified by dialysis against injectable-grade water during 24 h and sterilized by membrane filtration with a 0.22 m filter (Millipore, Bedford, Massachusetts, USA).Posteriorly, NPs were stabilized with the sodium dodecyl sulfate (SDS) surfactant in order to prevent NP aggregation and establish a suitable surface for peptide adsorption.SDS (1 mL, 0.05% w/w ) was added to 100 mL of the nanoparticle suspension (AgNP or AuNP) and stirred for 10 min.The mixture was then purified by size-exclusion chromatography (PD-10 column) using injectable-grade water as eluent.The peak obtained (3.0-4.0 mL) corresponded to the void volume of the column, which contained AuNP-SDS or AgNP-SDS.The volume mean diameter measured by DLS was not significantly different from nonstabilized nanoparticles.

Determination of Gold and Silver Content. Gold content was quantified through potentiometric titration of gold (III).
A constant volume of gold nanoparticles was oxidized by a HF/aqua regia mixture and determined through Robles et al. 's methodology [28].Silver content was determined using a back titration, following Volhard's method described by Xu et al. [29].

Conjugation and Standardization of Stabilized
Nanoparticles to the UBI Peptide.In order to determine the stability of the NP-UBI colloidal suspension, different volumes (from 20 L to 100 L) of 20 M UBI were added to 1 mL of stabilized SDS-NPs and  were then stirred for 15 minutes at room temperature.Z-potential (Microtrac, USA) was then measured to determine the peptide's effect on colloidal stability.
Once the most stable suspension was selected, it was scaled as follows: 750 L of UBI (29-41) 20 M (1.5 × 10 −8 mol) was added to 25 mL of stabilized SDS-NPs.The peptide/NP radio was calculated and adjusted to obtain 5-20 peptides per nanoparticle.Finally, functionalized NPs were purified and concentrated using ultracentrifugation (22,000 rpm for 20 minutes at −4 ∘ C).The final suspension of AuNP-UBI or AgNP-UBI was spectrophotometrically adjusted to an absorbance of 1 u.a. and stored (in darkness at 4 ∘ C) for further use.

Infrared Spectroscopy.
The FT-IR spectra of lyophilized samples were acquired on a Perkin Elmer System 2000 spectrometer with an ATR platform (Pike Technologies) by applying Attenuated Total Reflection Fourier Transform Infrared (ATR-FTIR) spectroscopy from 570 to 4400 cm −1 .

Raman Spectroscopy.
Raman spectra of the samples were performed on a MicroRaman OLYMPUS BX 41 spectrometer with a wavelength of 632.817 nm, a D0.6 filter, and a 100 microhole filter/array.Ten scans of 60 s were acquired on a pretreated glass cover (washed in three steps): 1HNO 3 /3HCl (v/v) solution, bidistilled water, and injectable water.10 L aliquots of the sample were deposited and dried under nitrogen atmosphere at room temperature in a laminar flow hood.2.5.5.XPS Spectroscopy.X-ray photoelectron spectra were acquired on a Jeol JPS 9200 spectrometer equipped with a MgKR X-ray source operated at 10 kV/20 mA and calibrated using Au 4f 7/2 (84.0 eV) and Ag 3d 5/2 (368.2 eV) from foil samples.The collection was made using 20 scans for Au 4f and Ag 3d with energy step size of 0.1 eV.The binding energies were referenced to the C 1 s peak at 284.3 eV.From all spectra, a Shirley background was subtracted to perform the peak fitting with a symmetric Gauss-Lorentz sum function (SpecSurf software).

Microbiological Evaluation.
To determine the antibacterial efficacy of the conjugates, Escherichia coli (ATCC 8739) and Pseudomonas aeruginosa (ATCC 27853) were used as Gram-negative models because of their epidemiologic and clinical importance.Antimicrobial activity of the NPs with or without conjugation was expressed in terms of minimum inhibitory concentration (MIC).Evaluated NPs and conjugates were AuNP, AuNP-SDS, AuNP-UBI, AgNP, AgNP-SDS, AgNP-UBI, and UBI (free peptide).NaCl 0.9% solution was employed as a growth control.Due to the high difficulty in determining the exact nanoparticle concentration, all nanoparticle suspensions and conjugates were standardized spectrophotometrically to 1 a.u. of absorbance and expressed as mass silver or gold concentrations.Titration showed that the standardized solutions contained 637 ± 74 g mL −1 of Au and 588 ± 19 g mL −1 of Ag.To obtain a range in nanoparticle concentrations, 1, 2, 3, 4, and 5 mL aliquots of standardized NPs were placed in test tubes and completed to 6 mL with distilled water, representing 106, 424, 955, 1698, and 2654 g mL −1 of Au and 98, 392, 883, 1569, and 2452 g mL −1 of Ag.
Microbiological evaluation was performed using a modified agar dilution method.Briefly, Muller-Hinton agar was hydrated (77%), sterilized, and mixed with 1 mL of nanoparticle solution.The final volume was completed to 20 mL with sterilized medium.The modified agar was slowly mixed, poured into petri dishes for gelation, and posteriorly incubated 24 h for microbiological control.Final concentrations for modified Agar-NP media were 5, 20, 46, 81, 126, and 182 g mL −1 of Au and 5, 19, 42, 75, 117, and 168 g mL −1 for Ag.
To determine the effect of the UBI peptide, different concentrations, ranging from 20 M (0.33 g mL −1 ) to 0.06 M (0.001 g mL −1 ), were evaluated.Each experiment was carried out in triplicate.
Inoculation was conducted in accordance with the CLSI M07-A9 method [30], using a suspension equivalent to a 0.5 McFarland standard of E. coli and P. aeruginosa (1 to 2 × 10 8 colony-forming units; CFU).Appropriate dilutions were made from 10 −1 to 10 −6 .Inoculation was performed with 2 L of the 10 −1 dilution.Five inoculums were placed in the previously prepared agar to obtain approximately 10 4 CFU per spot.Aliquots of 10 −6 were cultured in nanoparticle-free agar as a growth control.Inoculated plates were inverted and incubated at 35 ± 2 ∘ C for 16-20 hours as an end point.Growth of colonies was posteriorly evaluated.

Chemical Characterization
3.1.1.UV-Vis Spectroscopy.The citrate-stabilized AuNP and AgNP spectra showed a surface plasmon resonance band at 520 nm and 426 nm (Figure 1), respectively.It is wellknown that size, shape, and chemical environment modify the dielectric constant at the nanoparticle surface [31].A redshift was observed in the functionalized systems of AuNP-SDS (3 nm), AuNP-UBI (6 nm), and AuNP (4 nm), which is associated with an increase in the local refractive index at the nanoparticle surface [32].A blue-shift was observed for AgNP-SDS (2 nm), which could be related to stabilization by the coating surfactant, promoting the dispersion of small aggregates.
The stability of functionalized NPs in water was monitored by means of an SPR band for 6 months.There were not significant modifications in intensity and width of the SPR band.

Particle Size and Zeta
Potential.TEM images of AgNP, AgNP-UBI, AuNP, and AuNP-UBI showed monodispersed solutions.Additionally, an increase in the volume mean diameter was observed as an effect of peptide conjugation.The interaction of stabilizer/peptide and peptide conjugation was observed as a low electronic density around the gold nanoparticle cores (Figure 2(b)).
The mean hydrodynamic diameters, determined by DLS, showed no significant changes in stabilized SDSnanoparticles.Molero  formation at concentrations below 8 mM has no significant increase on micellar diameter [33].Measured diameters were 12.3 ± 3.9 nm and 14.4 ± 4.3 nm for AgNP and AgNP-UBI and 14.5 ± 5.7 and 16.5 ± 6.2 nm for AuNP and AuNP-UBI, respectively (Figure 2(a)).Nanoparticles did not show a significant change in dispersity; the slight increase in diameters was attributed to adsorbed peptide and its significant contribution to molecular weight.The Z-potential of AgNP and AuNP was 96.5 mV and 63.0 mV, respectively, and 113.9 mV and 82 mV for the AgNP-UBI and AuNP-UBI, correspondingly, indicating that the peptide interaction conferred a high colloidal stability to the nanosystems.

Infrared Spectroscopy.
The main vibrational frequencies of the primary amine of Lys (3281 cm −1 ) and the amide I (at 1619 cm −1 , C=O stretch) of the secondary amide observed in the UBI spectrum (Figure 3 This supports the argument that the main bond in NP-UBI is through the amine NP-NH 2 -Lys-UBI (Figure 3).
The AgNPs spectra are characterized by weak and broad band centred at 3369 cm −1 from (COO-H) st vibrations, a broad and intense band from (C-H)  vibrational modes from citrate skeletal are localized at 1390 cm −1 , and additional vibrational modes (832-1100 cm −1 ) from citrate are observed.AuNP shows strong bands between 3000-2700 cm −1 assigned to (>C-H 2 ) st sy ,  as (>C-H 2 ) st as vibrational modes and 1118-1258 cm −1 assigned to (COO-) st sy .In both cases, predominant vibrational modes correspond to functional groups of the citrate employed as a reducer agent and stabilizer (see Supplemental Material Figure 1 in Supplementary Material available online at https://doi.org/10.1155/2017/5831959).Characteristic spectra of dodecyl sulphate were obtained.SDSstabilized NPs spectra were characterized by strong, multiple, and well defined bands attributed principally to (-CH 3 ) as between 1470 and 1430 cm −1 , (S=O) st as at 1225-980 cm −1 , (S=O) st sy at 1200-1000 cm −1 , (C-O) st at 1200-1080 cm −1 , (S-O) st at 870-690 cm −1 , and (C-H 2 ) g between 770 and 720 cm −1 (see Supplemental Material Figure 2).The spectra of UBI show a broad band between 3100 and 3500, corresponding to (N-H) st , H-bonded (O-H) st , and (N-H) st free vibrational modes.Strong bands corresponding to amide I (C=O) st and amide II (N-C=O) st sy can be observed between 1655-1630 cm −1 and 1560-1510 cm −1 , respectively (Figure 3  3.1.4.Raman Spectroscopy.Significant spectral differences were observed in functionalized silver and gold NPs with respect to citrate-stabilized suspensions.Absence of Raman signalling was observed for AuNP-citrate and AgNP-citrate (Figures 4(a) and 4(c)), whereas peptide-functionalized NPs (Figures 4(b) and 4(d)) showed broad bands in the region of 1800 to 800 cm −1 .In both systems, significant bands attributable to amide I (C=O) st , amide II (N-H) st , and amide III (C-N) st were observed near 1600, 1550, and 1231 cm −1 , respectively.Additionally, bands with a significant contribution in intensity were observed at 1574 and 1542 cm −1 , which could be attributable to (C-N 3 ) st from guanidine groups of arginine, present in high concentration in the peptide.Skeletal chains of aliphatic (-CH 2 -) and methylene groups (-CH 3 ) could contribute to frequencies near 1410 and 1422 cm −1 .Additional vibrational bands of Tyr could be assigned at 685 and 820 cm −1 .The latter is caused by characteristic Fermi resonance between the in-plane breathing mode of the phenol ring and an overtone of the out-of-plane deformation mode.FT-IR and Raman spectroscopy were used to confirm the surface modification of silver or gold nanoparticles with the UBI peptide.
The vibrational modes from functionalized nanoparticles are hardly sensitive to conformational changes and electrostatic interactions; shifts in vibrational modes from the amide II region are correlated with the increase of strength of van der Waal interactions with NP surfaces.The C-H stretching band appears as superimposed bands under the prominent O-H stretching in functionalized nanoparticles, with respect to SDS or citrate-stabilized nanoparticles.This gives good evidence that the molecules increase their intermolecular hydrogen bonds and suggests that electrostatic interactions play a determinant role in peptides bound to NP surfaces.
TEM and spectroscopy techniques demonstrated that AgNPs and AuNPs were functionalized with the UBI peptide.The IR spectrum of nanoparticles showed vibrational modes from citrate, SDS, or peptides adsorbed onto the nanoparticle surface.
3.1.5.XP Spectroscopy.The AuNP spectrum (Figure 5(a)) showed two main peaks corresponding to the binding energies (BE, eV) of electrons in Au 4f orbitals at 87.2 eV (Au 4f 5/2 ) and 83.3 eV (Au 4f 7/2 ) and the deconvoluted spectrum for Au 4f 7/2 showed peaks at 83.9 and 83.2 eV, the first probably due to weak bond with oxygen by electrostatic forces to metallic gold core and phosphate groups of surfactant.For Ag spectrum (Figure 5(b)) 3d 3/2-5/2 core level binding energies appear at 374.1 and 368.1 eV, respectively, in good agreement with bulk silver metallic values, corresponding to the presence of fcc metallic silver structures found in nanoparticles.Deconvoluted Ag 3d 5/2 spectra can be fitted in terms of two different chemically species with binding energies at 368.5 and 367.8 eV, assigned to metallic silver (Ag0) and silver ions in Ag 2 O (Ag+), respectively, in agreement with previous reports [34,35]. 1 shows the minimum NP concentrations required to inhibit growth of E. coli and P. aeruginosa.A statistically significant difference was observed between noncapped gold and silver nanoparticles, whereas AuNP and gold-functionalized nanoparticles seem to need high NP concentration to produce a significant deleterious effect on microbial growth.AgNPs showed an important effect over both evaluated microorganisms.Significant activity was observed with functionalized NPs when compared with citrate or SDS-stabilized NPs.The free peptide did not show any inhibitory effect at the evaluated concentrations.

Microbiological Assay. Table
The mechanism of nanoparticle interaction with ionic surfactants has not been completely elucidated yet.However, [36] suggests a possible mechanism of SDS distribution on the NP surface, in which the hydrophilic groups of the surfactant molecules are adsorbed onto the nanoparticle surface and the hydrophobic tails are directed outward to form the first layer.Consequently, a counter-layer is oriented the opposite way, resulting in interpenetration of the surfactant hydrophobic tails between the two layers with hydrophilic groups headed outward [13].The addition of cationic surfactant is justified by at least two reasons.The first is an increase of colloidal stability of zero valent nanoparticles, enhanced by steric repulsion from adsorbed surfactants and the balance in electrostatic forces, following a possible mechanism in which surfactant molecules form a compact micellar layer with hydrophilic groups oriented to the nanoparticle surface.The second justification is related to the increase of peptide interaction with the nanoparticle surface through the use of the surfactant bilayer's capability to adsorb and stabilize the peptide by electrostatic interactions between anionic, cationic, and nonpolar domains of the peptide.Additionally, it has been reported that ionic surfactants are adsorbed in a less-compact mode at the nanoparticle surface compared to nonionic surfactants [37].Established mechanisms have demonstrated that silver nanoparticles have a higher antimicrobial activity compared to free silver ions.Additionally, the Ag + ions delivered by nanoparticles located within the cell increase the antibacterial activity.Reported bactericidal effects from gold NPs are still controversial.A great variability in MIC exists, attributable mainly to microorganism type, specific conditions of particular procedures, as well as diffusion mechanisms in growth media, support materials (applicable in disc procedures), biofilm formation, and so forth.It is important to note the differences in analytical procedures for microbiological evaluation; in most cases, analysis was carried out by disk diffusion assay on Müller-Hinton agar plates.However, in our experience, there are significant disadvantages in such procedure.We have observed that the interaction of nanoparticles with adsorbent materials does not demonstrate the same behaviour as with the desorption process.Interaction with fibres could modify nanoparticle nature observed by significant alteration in nanoparticle-support colour.In this sense, it is difficult to establish a relationship between previous reports based on exposure concentrations.The NP microbicidal effect has been associated with bacteria class.In E. coli, complete growth was impeded with AgNP concentrations above 75 g mL −1 .However, evidence demonstrates that the shape of AgNPs determines their deleterious effects.The most effective antibacterial activity has been observed with triangular nanoparticles [38,39].In this research, synthetized silver nanoparticles showed a variable distribution in nanoparticle shape, with a predominant spheroidal form.These and other factors associated with the particular development of the methodology could be responsible for the increase of the MIC in noncapped AgNPs.For example, Rai et al. reported a weak bactericidal activity (<10% of inhibition) on S. aureus exposed for 6 h to 500 g mL −1 of AgNPs of 23-to 52 nm diameter [18].The absence of inhibition was most likely attributable to the presence of a peptidoglycan layer on the cell walls of S. aureus, which does not allow for the penetration of AgNPs.Umamaheswari et al. reported MCI 80 on E. coli, S. typhi, P. aeruginosa, and K. pneumoniae, which ranged from 20 to 40 g mL −1 with a damage mechanism mediated by ROS and loss of membrane integrity [40].
The diversity in results showed that microbicidal effects are limited to certain types of microorganisms and the interaction with the cellular microenvironment, such as cell wall, glycocalyx, slime layer, or biofilm formation, since differences in peptidoglycan composition, variations in the peptide stem, and particular cross-linking determine nanoparticle diffusion.Additionally, certain variations were observed between members of the same species and the species themselves, depending on growth conditions such as growth phase, media composition, and presence of antibiotics [41].
In one particular case, a concentration of 182 g mL −1 of gold nanoparticles was insufficient to produce cellular damage in Gram-negative microorganisms (Figure 6   but a significant increase in inhibition was observed with silver nanoparticles.Specifically, a decrease of 31.6% for E. coli and 38% for P. aeruginosa was observed when the peptide was conjugated to silver (Figure 6(b)).We propose that the enhanced mechanisms of the antimicrobial agent could be attributable to at least two effects.The first is the local concentration increase of peptide surrounding the nanoparticle and the intrinsic effect of the nanoparticle, including the release of silver ions from the nanoparticle core.As with silver nanoparticles, the gold cores increase the local concentration of peptide.However, the damage mechanisms differ significantly with respect to AgNP.The nature of the damage produced by silver nanoparticles has been widely studied, even though the specific mechanisms have not yet been elucidated.
We demonstrated that the antimicrobial activity produced by silver nanoparticles was modified when antimicrobial peptides were bound to the nanoparticle surface.The effect was determined using two microorganisms representative of the Gram-negative group, E. coli and P. aeruginosa.However, in order to prove their therapeutic potential, the AgNP-UBI conjugate needs to be tested on in vivo models.Silver nanoparticles conjugated to antimicrobial peptides increase the antibacterial activity against Gram-negative bacteria providing a promising alternative therapy for topical infections.

Conclusions
Currently, an intense investigation in nanotechnology and its applications in infectious or contagious diseases has led to the development of antimicrobial formulations from nanoparticles that act as an effective bactericidal agent.In this study, it was demonstrated that an increase in the antibacterial activity against Gram-negative bacteria appears when silver nanoparticles are capped with the UBI antimicrobial peptide, probably due to the multimeric or polyvalent arrangement of ligands distributed on the nanoparticle surface.Additional studies are required to understand the structure of multivalent nanoparticles and stabilization mechanisms not yet elucidated.

Figure 2 :
Figure 2: Particle size analysis determined by DLS (a) and TEM (b).
(a)) were no longer seen as defined bands in the AuNP-UBI and AgNP-UBI spectra (Figures 3(b) and 3(c)).Instead, broad bands centered at 3294 cm −1 (Figure 3(b)) and 3257 cm −1 (Figure 3(c)) corresponding to the secondary amide and bands at 1719 cm −1 , 1589 cm −1 (Figure 3(b)), 1656 cm −1 , and 1573 cm −1 (Figure 3(c)), related to primary amide I or II, are observed (in the solid state, amides I and II may overlap).The interaction of the NH 2 -lysine with the NP surface is also revealed in the CH 2 -NH 2 band position, which changed from 1179 cm −1 (CH 2 /NH 2 twisting vibration) to 1394 cm −1 (Figure 3(b)) or 1389 cm −1 (Figure 3(c)) (CH 2 wagging vibration) as a result of a significant change of symmetry in the peptide due to the NP-Lys-UBI interaction.
(a)).Peptide-functionalized silver and gold NPs (Figures 3(b) and 3(c)) show vibrational modes associated with UBI.Vibrational modes of (C-H) st near 2925 cm −1 and (-CH 2 -O-)st, between 2880 and 2830 cm −1 , are attributable to the hydrocarbon chain and ester bond from the SDS employed as a stabilizing agent.

Figure 6 :
Figure 6: Effect on bacterial growth of Escherichia coli and Pseudomonas aeruginosa exposed to UBI peptide, silver and gold nanoparticles, and conjugate AuNP-UBI or AgNP-UBI.
Evaluation. Results were expressed as mean ± standard deviation (SD).Statistical significance was verified through Student's t-test, considering a significance of  < 0.05, unless otherwise indicated.
Figure 1: UV-Vis spectra of silver and gold nanoparticles stabilized with SDS and functionalized with the UBI peptide.

Table 1 :
Minimum concentrations needed to inhibit the growth of bacterial cells.