Small Synthetic Peptides Bioconjugated to Hybrid Gold Nanoparticles

Subscriber access provided by University of South Dakota

Article

Small Synthetic Peptides Bioconjugated to Hybrid Gold Nanoparticles Destroy Potentially Deadly Bacteria in Submicromolar Concentration Gianna Palmieri, Rosarita Tatè, Marta Gogliettino, Marco Balestrieri, Ilaria Rea, Monica Terracciano, Yolande Therese Proroga, Federico Capuano, Aniello Anastasio, and Luca De Stefano Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.8b00706 • Publication Date (Web): 23 Oct 2018 Downloaded from http://pubs.acs.org on October 25, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Bioconjugate Chemistry

Small Synthetic Peptides Bioconjugated to Hybrid Gold Nanoparticles Destroy Potentially Deadly Bacteria in Submicromolar Concentration Gianna Palmieri†°, Rosarita Tatè‡, Marta Gogliettino†, Marco Balestrieri†, Ilaria Rea§, Monica Terracciano§°, Yolande Therese Prorogaç, Federico Capuanoç, Aniello Anastasio^ and Luca De Stefano§°* †Institute

of Biosciences and BioResources, UOS Na, National Research Council, Via Pietro Castellino

111, 80131 Naples Italy ‡Institute

of Genetics and Biophysics, National Research Council, Via Pietro Castellino 111, 80131

Naples Italy çDepartment

of Food Microbiology, Istituto Zooprofilattico Sperimentale del Mezzogiorno, via della

Salute, 2, 80055 Portici (NA) Italy §Institute

for Microelectronics and Microsystems, National Research Council, Via Pietro Castellino

111, 80131 Naples Italy °Materias

S.r.l., Corso N. Protopisani n. 50, 80146 Naples, Italy

^Department

of Veterinary Medicine and Animal Production, University of Naples Federico II, Via

Federico Delpino 1, 80137 Naples Italy Correspondent author: Dr. L. De Stefano, Email: [email protected]

Abstract Synthetic antibacterial peptides are advanced weapons that scientists design and produce facing current treats due to harmful and mortal pathogens, which could affect humans in everyday life. Recently, many small amino acid sequences, greatly efficient in the antibacterial action, have been reported in the literature. To date, only few synthetic peptides, acting in micromolar or even tenths of micromolar concentration, are on the market as commercial products, mainly due to the high cost of production. In

ACS Paragon Plus Environment

Bioconjugate Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 26

this context, material science can give a fundamental help by engineering small synthetic peptides, powered by hybrid gold nanoparticles, which have been found to strongly enhance the antimicrobial activity against bacterial infections. Submicromolar concentrations of the 1018K6 peptide, bioconjugated to hybrid polymer-gold nanoparticles, kill almost 100% of pathogen bacteria, such as Listeria and Salmonella genera, paving the way to economically sustainable commercial products based on this synthetic nanocomplex.

Introduction One of the scariest hazards that applied science still faces concerns the potentially deadly bacteria, for which there are no antimicrobial agents. Moreover, some microorganisms causing serious diseases became multidrug-resistant pathogens due to the long-term use of conventional antibiotics. Actually, there are twelve listed families of bacteria, for which a cocktail of antibiotic drugs able to destroy efficiently them after human infections1, is not still available. Moreover, other bacteria are less difficult to fight but much more widespread. Beyond new chemical compounds with proven antimicrobial and/or antibiofilm activity, so costly that only few can be found on the market2,3, material science proposes two alternative antibacterial approaches. One is based on the use of metallic (gold and silver) or semiconducting (titanium oxide and tin oxide, for example) nanoparticles4. The other one involves the design of short peptide sequences, which specifically fold themselves near the cell membrane. The antibacterial ability of metal and metal oxide nanoparticles can be ascribed to the release of free metal ions resulting in cell membrane damage, DNA interactions, and free radical generation5. Naturally occurring antimicrobial peptides (AMPs) are a useful source in fighting bacteria since they can penetrate cell membranes and also inhibit the biosynthesis of the cell wall, nucleic acid, and proteins of these microorganisms. There is a great structural variety among almost three thousand AMPs that have been investigated until today6 together with their mechanisms of action7-9. The


Page 3 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


capability of these small molecules in disrupting and permeating plasma cell membranes depends on a number of biophysical properties, such as amphipathicity, hydrophobicity, structural folding (including secondary structure, orientation, and dynamics), overall net charge, size and balance between hydrophobic and polar regions10. Many recent papers report on nature-derived AMP bound to metallic nanoparticles, mainly gold or silver11,12, or loaded into porous nano-carriers13, showing antibacterial activity against human pathogens or bacteria model (i.e., mainly Escherichia coli). Even if some of these nano-complexes are effective at tenths of micromolar concentration, the peptides used are mostly composed of long sequences of amino acids (more than 20). Synthesis costs of these long peptides prevent their economic sustainability. Recently, Hancock’s group has projected a peptide, called innate defense regulator (IDR)-1018, which showed a strong antibiofilm activity14-16. IDR-1018 is a 12-amino acids (VRLIVAVRIWRR) peptide derivative of bactenecin, a bovine host-defense peptide (HDP), which belongs to the cathelicidins family. The peptide is effective toward a large panel of Gram-negative and Gram-positive bacteria.

Moreover,

the

new

1018-derivative

peptide,

the

single-point

mutated

1018K6

(VRLIVKVRIWRR), properly designed by G. Palmieri and its group17, is even more active as compared to the parent IDR-1018, specifically against Listeria monocytogenes, one of the most common food pathogen, also potentially dangerous for humans. Even if 1018K6 has been demonstrated to have both bactericidal and antibiofilm properties at micromolar concentration, synthesis costs are still too high for the formulation of sanitizing products, which are strongly required by the food industry. In this work, we have engineered hybrid polymer-gold nanoparticles (AuNPs) by covalent conjugation of 1018K6 to AuNPs surface, by using carbodiimide chemistry. The resulting nanobiocomplex shows enhanced antibacterial activity at submicromolar concentration, which could allow the industrial production at feasible costs.



Page 4 of 26

Results and discussion In our previous papers18-20, we proved that the one-pot synthesized polyethylene glycol (PEG)stabilized AuNPs were really hybrid polymer-metal nano-aggregates forming a stable colloidal solution of about 8-10 nm in size, which could be effectively bioconjugated with the enzymes or proteins. Following this experience, we covalently grafted the 1018K6 peptide to AuNPs by using standard 1ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC)/N-hydroxysuccinimide (NHS) reaction (details are reported in the Materials and Methods section), which is schematically shown in Fig. 1A.


Page 5 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Fig. 1. Conjugation scheme of 1018K6 peptide to AuNPs (A); UV-Vis spectra of AuNPs before and after peptide conjugation (in the inset an optical view of the two solutions is reported) (B); hydrodynamic diameters (by DLS measurement) and surface charge before and after bioconjugation (C); TEM imaging and counting, before (D) and after bioconjugation (E).



Page 6 of 26

The modification of AuNPs by 1018K6 conjugation has been characterized by UV-Vis spectroscopy. This measurement highlighted a 7 nm red-shift of the local surface plasmon resonance (LSPR) of the absorbance spectrum, due to the grafting of 1018K6 on the AuNPs surface. It quantitatively confirmed the change of colour from red to violet of the two stock solutions before and after 1018K6 peptide coupling (Fig. 1B). Moreover, the tryptophan (Trp) band in the UV spectrum at 280 nm appeared after peptide bioconjugation. Size and surface charge of nanoparticles have been estimated by Dynamic Light Scattering (DLS) and -potential measurements, respectively (Fig. 1C). DLS quantified the hydrodynamic diameter of AuNps in 8±2 nm, while 1018K6-AuNPs were 16±4, suggesting dimerization of the nanoparticles. The surface charge was -26±10 mV, before peptide conjugation, mainly due to the exposure of PEG –COOH groups, and +27±7 mV after peptide coupling, due to positive amino acids of 1018K6, which had 4 positive net charges. The AuNPs and 1018K6-AuNPs nanoparticles were characterized also by Transmission Electron Microscopy (TEM) imaging. Bare AuNPs were well dispersed and showed an average size of 13±5 nm (Fig. 1D); after 1018K6 grafting, the nanoparticles were arranged in a cluster and showed an average size of 14±7 nm (Fig. 1E). These data were in accord to that of DLS within their experimental errors. Despite the quite high positive surface charge and the high stability in solution, the 1018K6-AuNPs revealed some tendency to aggregate after drying. This effect was probably due to peptide clustering, as showed by TEM images after fixing the 1018K6-AuNPs with glutaraldehyde (Fig. S1A) since AuNPs, fixed in the same way, were always well dispersed (Fig. S1B). Antibacterial peptides that well work in solution often become inactive when bound to a support surface. In order to quantitatively compare the biological activity of the tethered peptide with that of the free molecule, the conjugation yield of 1018K6 immobilized on the gold nanoparticles was determined to find out the amount of peptide present in each 1018K6-AuNPs solution. Since there was ACS Paragon Plus Environment

Page 7 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


not any straightforward protocol described in the literature, we designed direct and indirect quantification methods to overcome this limitation. Briefly, after conjugation and centrifugation of the 1018K6-AuNPs to remove the excess of the peptide, the nano-complexes were treated with trifluoroacetic acid (TFA) 50% for 24 h at room temperature in order to hydrolyze the covalent bond between the peptides and the gold particles surface. Then, the amount of tethered peptides was directly estimated by reverse phase-high precision liquid chromatography (RP-HPLC) analysis, evaluating the peptide peak area after the immobilization procedure. Therefore, by knowing the initial peptide concentration that was used for the conjugation, it was possible to correlate the quantity of peptide attached to the AuNPs surface. The results of the direct quantification of yield conjugation demonstrated that no more than 9.0±1.0 % of the peptide was covalently grafted to AuNPs surface (Fig. S2). Moreover, we verified that no acidolysis event occurred on the peptide bonds within the amino acidic chain, by incubating the free peptide under the same experimental conditions used in the direct method, as revealed by RP-HPLC analysis (Fig. S2). In order to further validate coupling yield, it was also quantified the unbound peptide in solution after incubation with AuNPs, that is an indirect method to estimate the immobilized peptides amount (Fig. S3A). In this second experiment, once the reaction was completed, the supernatant solution was recovered after centrifugation of the nanoparticles and analysed by RP-HPLC. In this procedure, complementary to the direct approach, the peak area quantified the amount of the peptide not bound to nanoparticles surface. The data showed similar results to those obtained by direct quantification method since the coupling yield resulted equal to 11.0±1.0 %. The same amount of bound peptide was also determined via interpolation of a calibration curve that has been constructed by spiking known 1018K6 concentrations (Fig. S3B). Therefore, on average, we assumed that 10% of the initial peptide concentration (125 M) was covalently bound on the gold nanoparticles, resulting in approximately 12.5 M peptide concentration in the 1018K6-AuNP batch solution. ACS Paragon Plus Environment


Page 8 of 26

The immobilization yield, which is one of the most critical parameters to achieving the formation of stable and active nanoparticles, could also depend on the peptide concentration that is incubated with the AuNPs. The effect of the 1018K6 concentration was tested by using different amounts of the peptide in order to optimize the coupling reaction conditions. As shown in Fig. S4, it was evident that the most suitable coupling conditions for improving the immobilization yield were obtained with a peptide concentration of 125 M. Indeed, when a smaller amount of 1018K6 was added to the mixture, the coupling reaction resulted less effective as compared to 125 M concentration of peptide and it was also accompanied by precipitation of nanoparticles. The aggregation was probably due to the fact that AuNP surfaces were not completely covered by the peptide. Similarly, the further increase in 1018K6 concentration did not determine significant changes in the conjugation yield of the peptide to gold nanoparticles. Hence, 125 M was considered the saturation concentration and the most suitable for all the experiments. Finally, it was also possible to calculate the approximate number of peptide bound to each AuNP by taking into account the number of hybrid nanoparticles in the reaction volume (details can be found in SI): 12.5 nmol of peptides were bound to 7.8x1010 AuNPs, resulting in about 9.6x104 molecules of peptide per each AuNP. The antimicrobial power of AMPs strongly depends on their structure, but also on their microenvironment since they must have the conformational freedom to fold into their functional form. Therefore, to find out whether the peptide 1018K6 could still assume this active conformation when tethered to the gold surface, CD and fluorescence spectroscopy data relative to 1018K6-AuNPs were compared to the free peptides. These analyses gave deep insight into the possible structural changes of the peptide, which could promote inactive states. Specifically, if the charged Lys residues resulted to be bonded to the gold core, they could not be able to contribute to the right conformation, which was the one active against the bacteria.


Page 9 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


As reported in Fig. 2A, the free peptide 1018K6, which was unfolded in aqueous buffers17, folded as amphiphilic α-helices, in order to be biologically active, in presence of SDS micellar solutions (3 mM), which could be considered a bacterial membrane-mimicking model. After nanoparticles conjugation, there were no significant changes in the peptide secondary structure in presence of SDS, highlighted by its CD spectrum (Fig. 2B). It was not possible to acquire CD spectra of the free and the conjugated form of 1018K6 at the same concentration, due to AuNPs interference. Therefore, CD analysis of the unbounded 1018K6 was set up choosing the minimal concentration at which a good quality of spectra was recorded in case of 1018K6-AuNPs. The obtained results suggested that coupling strategy was successful in tethering the peptides, leaving enough conformational freedom to peptides that could switch their secondary structures, and keeping the cationic side chains freely available for interactions with bacterial membranes.



Page 10 of 26

Fig. 2. Conformational changes of 1018K6 and 1018K6-AuNPs by CD and Fluorescence spectroscopy. CD spectra of the 1018K6 free at 10 µM (A) or tethered to the gold nanoparticles (B) at 2 µM concentration in water, were recorded in the 195 nm–250 nm range at 25 °C, after addition of 3 mM SDS. Overlay of fluorescence emission spectra (ex = 280 nm) of free 1018K6 (C) and 1018K6 bound Au-NPs (D) in the presence or absence of SDS (3 mM) at different pHs.

Fluorescence spectroscopy studies, exploiting the tryptophan residues of the peptide sequence, checked the formation of tertiary structure conjugates. The increase in fluorescence emission intensity of 1018K6-AuNPs (Fig. 2D) compared to the free 1018K6 (Fig. 2C) suggested that a high number of ACS Paragon Plus Environment

Page 11 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


molecules bound to the nanoparticles contributed to the strengthening of the 1018K6 emission intensity. On exposure to SDS micellar solution, the fluorescence spectra of 1018K6-AuNPs revealed a small “blue shift” of the emission maximum towards shorter wavelengths (from 353 nm to 340 nm), similar to that observed for free 1018K6 (from 353 nm to 337 nm). This behavior could be attributed to the migration of the Trp residues in largely buried and non-polar environment during the folding process in micellar solutions. On the other hand, the contemporary large decrease in peak fluorescence intensity shown by 1018K6-AuNPs in presence of SDS could be due to the densely packed layer of peptides on the AuNPs surface, which resulted in a fluorescence quenching effect. The same behavior was observed when samples were incubated for 24 h at extreme pH values, thus demonstrating a high stability of the nanosystem under these conditions. Starting from the conformational results, the in vitro antimicrobial activity of 1018K6-AuNPs was evaluated against two of the major foodborne dangerous pathogens, the Gram-positive Listeria monocytogenes, and the Gram-negative Salmonella Typhimurium bacteria. Since L. monocytogenes strains in different food-production environments can remarkably modify their virulence and pathogenicity20, the antimicrobial activity had been tested on a strain directly selected from seafood samples, the isolates LM2, previously characterized as a 4b serotype17. Preliminary experiments were carried out culturing increased Listeria cell density (approximately 102, 104, 105 CFU/ml) with 1018K6-AuNPs at 0.16 M concentration, calculated on the basis of the immobilization yield. Plates with only the gold nanoparticles, which are recognized to have antimicrobial properties, were used as the control and the number of the viable bacterial colonies was counted in order to assess the bactericidal activity of the nanosystems. Interestingly, as shown in Fig. 3A, the 1018K6-AuNPs exerted a strong bactericidal activity against the food-isolate strain LM2 of L. monocytogenes, causing a significant reduction in the number of CFU, as compared to bare AuNPs.



Page 12 of 26

Fig. 3. Bactericidal activity of gold nanoparticles functionalized with the antimicrobial peptide 1018K6 (1018K6-AuNPs) against L. monocytogenes. Killing activity of (A) 1018K6-AuNPs at 0.16 M towards the reported bacterial cell concentrations (CFU/ml). Data were determined by counting the surviving colony forming units (CFU) on plates (ALOA) seeded with the pathogen incubated with 1018K6-AuNPs for 6 hours and expressed as bactericidal activity (percent CFU respect to the colony counted in the control plates); (B) increasing amounts of 1018K6-AuNPs against a fixed concentration of L. monocytogenes. The error bars represent the standard deviation (SD) from the mean for a triplicate experiment (n=3).

Specifically, even if AuNPs demonstrated a 96.2% inhibition in growth of gram-positive bacteria, due to the release of gold ions from the nanoparticle core, when the inoculum was equal to 1.5x103 CFU/ml, 1018K6-AuNPs increased the effectiveness up to 100%. Much more impressive was the


Page 13 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


inhibition in bacterial growth, up to 99.8% and 99.9%, observed in plates inoculated with 1.5x105 and 1.5x106 bacterial concentrations, on exposure to 1018K6-AuNPs, whereas AuNPs were really not active. Based on these results, a starting inoculum of approximately 1.5 x 104 CFU/ml, at which no lethal activity was displayed by bare AuNPs, was used for the testing dose-dependent 1018K6-AuNPs antibacterial activity. After exposing this inoculum with increasing 1018K6-AuNPs concentrations, the number of viable bacteria was compared to that obtained with both AuNPs and free 1018K6 (Fig. 3B). A dose-dependent bacterial cells death behavior was detected on exposure to the free peptide 1018K6 at concentrations ranging from 0.10 µM to 0.58 µM (Fig. 3B), and the minimal bactericidal concentration able to cause at least 50% decrease in the number of CFU (MBC50) was 0.58±0.03 µM. In case of 1018K6-AuNPs, it was found not only a concentration-dependent effect but also a significantly higher killing activity, which led to a MBC50 value equal to 0.14±0.01 µM, 4-fold lower than that of the free 1018K6. This result was greatly important, since L. monocytogenes strains, isolated from food products or human listeriosis cases, often displayed strong adaptation or resistance phenomena to antibiotics and disinfectants, possibly due to the presence of mobile genetic elements carrying resistance genes or an altered permeability of the bacterial cell wall, such as structural modifications or a consequence of the action of active pumps which detoxify the cell21-23. Moreover, the conjugation of peptide to gold nanoparticles clearly potentiated its antimicrobial activity, due to a local concentration increase of the peptide surrounding each nanoparticle that in the proximity of bacterial membrane resulted more effective. In further experiments, the antibacterial properties of the 1018K6-AuNPs nano-system were tested against S. Typhimurium, one of the most dangerous foodborne pathogens, chosen as a model of Gram-negative bacteria. Preliminary experimental conditions were set up in order to evaluate the killing ability of 1018K6-AuNPs in presence of different concentrations of bacteria. As shown in Fig. S5, it is worth noting that only the hybrid peptidenanoparticles caused a pronounced reduction in the number of viable bacterial cells (up to 99.9 %) of S.



Page 14 of 26

Typhimurium, when 0.16 µM concentration of 1018K6-AuNPs was added to 1.5 x 103 CFU/ml; while at lower cell density (15 x 101 CFU/ml) also the uncoated gold nanoparticles exhibited 30% of antimicrobial activity. These results demonstrated that the gold tethered 1018K6 peptide not only preserved the activity of the free 1018K6 peptide, but also showed an enhanced ability in killing both Gram-negative and Gram-positive bacteria after the conjugation to the AuNPs surface. Moreover, the value of foodborne pathogens infectious doses used in the experiments was of the same order of that found in human outbreaks, thus suggesting a potential application of the 1018K6-AuNPs nanosystems in some industrial sectors. In recent years, many AMPs have been chemically immobilized on different types of surfaces; however, it is known that only a limited fraction of AMPs is still active after surface immobilization. This effect strongly depends on several factors including peptide composition, length of peptide spacer, peptide orientation and concentration23. Many studies have shown that an immobilized AMP could keep the antibacterial activity, but with very low efficiency and at extremely high concentration24. The gold tethered 1018K6 peptide had an excellent long-term stability as antibacterial colloidal solution, after storing (pH=7.4; room temperature; deionized water) and reuse (1018K6-AuNPs complex was stable for at least 7 months). This result was confirmed by CD analysis (Fig. S6A) and anti-bacterial tests (Fig. S6B). The high stability was mainly due to two factors: the ability of gold nanoparticles to keep active the peptide on their surface and the steric repulsion between the peptides immobilized on the surface, which prevented aggregation and precipitation of nanoparticles. TEM imaging provided a direct picture of the effects induced on Listeria cells after treatment with 1018K6-AuNPs with spatial resolution in the nanometer range. In general, TEM data analysis gave deep insight into particles-membrane interactions, such as uptake and/or aggregation outside and/or inside the cells and finally the intracellular fate of hybrid NPs in biological specimens. In TEM microphotos, both untreated Listeria cells (Fig. 4A-A’ and Fig. S7A) and those incubated with bare AuNPs


Page 15 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(Fig. 4B-B’ and Fig. S7B) presented cellular membranes integrity, distinctly visible nucleoids (highlighted by white asterisks) and high electron density cytoplasm.

Fig. 4. TEM micrographs of L. monocytogenes cells untreated (A-A’), incubated for 6 h with Au nanoparticles (B-B’) or with modified nanoparticles (C-C’) at 0.16 M concentration. A’ was a magnified view of the blue line surrounded rectangular area of A; B’ was a magnified view of the red ACS Paragon Plus Environment


Page 16 of 26

line surrounded rectangular area of B; C’ was a magnified view of the green line surrounded rectangular area of C. Red arrows show AuNPs located outside of the Listeria cells. Green arrows indicate hybrid NPs mixed with cellular debris. White lightning in C’ clearly demonstrated the alterations in the cell membrane. The bacterial nucleoids were visible only in untreated and treated with AuNPs Listeria cells (Asterisk). Blue star points out the DNA exit from the cell. The bar was equal to 1 m in A, B and C; in A’, B’ and C’ it was equal to 500 nm.

High-intensity cytosol (i.e. dark region inside the bacteria cell) represented healthy cells with strong absorption of electrons by biomolecules present in the cytosol. In samples of Listeria cells treated with bare AuNPs, the nanoparticles aggregated outside the cells (see red arrows in Fig. 4B-B’ and Fig. S7B). This result suggested that there was not any internalization of AuNPs. Listeria cell images treated by 1018K6-AuNPs revealed a significant decrease in the number of the cells present in each field observed and a diffuse presence of cellular debris mixed with gold nanoparticles (see green arrow in Fig. 4C-C’ and Fig. S7C-C’-C’’-C’’’). Moreover, the cytosol of Listeria cells treated by 1018K6AuNPs showed very low electron-density, and the nucleoids inside the cell could not be observed anymore. In many cases, cell membranes were almost disaggregated (see white lightning in Fig. 4C’ and Fig. S7C’-C’’-C’’’). The alterations of cell membranes caused the release of bacterial nucleoids (blue star in Fig. S7C’’’) and cytosol through macroscopic channels in the cell envelopes. In other images, Listeria cells appeared as empty cells or vacant cell envelopes of bacteria, usually defined as bacterial ghosts (BG in Fig. S7C’)25. All these results suggested that 1018K6-AuNPs killed the Listeria bacteria through permeabilization and solubilization of cytoplasmic membranes. Charge mediated interactions between 1018K6-AuNPs and the cell lipid bilayers destabilized the membrane barrier causing bacteria destruction. These findings were also consistent with the proposed mechanism of deactivation of microorganisms by antimicrobial peptides through pore formation26. ACS Paragon Plus Environment

Page 17 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Conclusions In conclusions, the small synthetic 1018K6 peptide tethered to the AuNPs nanoparticles was able to strongly retain its antimicrobial activity by folding into a functionally relevant -helix structure when in presence of a membranous environment. In addition, the AuNPs enhanced the peptide bacteriakilling ability, against both Gram-positive and Gram-negative bacteria. Due to proper covalent chemistry, which assured strong binding of 1018K6 on the gold nanoparticle and good surface coverage, the antimicrobial complex was fully effective in submicromolar concentration, and for this reason, it has been patented (PCT/EP2018/069304)27. The effective concentration of 1018K6 used in the experiment was found of the order of less than 100 nM. Such low concentration of this small peptide, bioconjugated to AuNPs, would allow its large-scale production and use at sustainable costs. Moreover, the 1018K6-AuNPs solution in deionized water, stored at room temperature, resulted in a high chemically stable bio-complex, also re-usable after seven months. The results obtained paved the way for an economically sustainable product useful for different purposes such as the development of antibacterial coatings of materials active against lethal foodborne pathogens, innovative sanitizing bioformulations and for the production of antimicrobial surfaces in the medical devices field. Materials and methods Synthesis of PEG-stabilized Au nanospheres Gold nanoparticles (AuNPs) were synthesized from chloroauric acid (0.25 mM, 25 mL; HAuCl4 3H2O, Sigma-Aldrich, USA) by mixing poly(ethylene glycol) diacid (0.25 mM, 0.25 mL, PEG-diacid, Sigma-Aldrich, USA) and sodium tetrahydroborate (0.01 M, 20 mL, NaBH4, Sigma-Aldrich, USA) as surfactant and reducing agents, respectively28. The formation of the PEG-stabilized AuNPs was observed as an instantaneous color change of the solution from pale yellow to bright red after addition of the reducing agent. The as-prepared AuNPs solution was centrifuged at 15 000 rpm for 30 min for ACS Paragon Plus Environment


Page 18 of 26

three times and then the supernatant discarded. The resulting pellet was re-suspended in an equivalent amount of MilliQ-water. AuNPs covalent peptide conjugation The carboxyl groups of PEG-stabilized AuNPs were covalently conjugated to the N-amine groups of 1018K6 (0.125 or 0.030 mM) by 1-ethyl-3-[3-dimethylaminopropyl]carbodiimidehydrochloride/Nhydroxysuccinimide (EDC 5 mM/NHS 2.5 mM, Sigma-Aldrich, USA), under stirring overnight (ON) at room temperature following the reaction scheme reported in Fig. 129. The site-specific addition of the carboxyl group of PEG at the N-terminus of the peptide has been controlled by lowering the pH at which the PEGylation reaction takes place. Indeed, the lower pH takes advantages of the differences in pKa values of the -amino group versus the -amino group on the side chain of a lysine residue, whose amino groups are an attractive target for conjugation chemistry30. After the bioconjugation with 1018K6 peptide, the NPs (1018K6-AuNPs) were washed twice with MilliQ-water and re-suspended in MilliQ-water. Characterization of bare and 1018K6-modified AuNPs Transmission electron microscopy (TEM) was used to study NPs morphology before and after 1018K6 conjugation. To this aim, 1 mL of the sample was centrifuged for 30 min at 15 000 rpm and the supernatant discarded. The solid portion was re-dispersed in 1 ml of MilliQ-water and 10 μL of NPs dispersion were placed on a formvar carbon film 400 Mesh TEM copper grid, dried at room temperature and then observed by a FEI Tecnai G2 Spirit BT TEM at an accelerating voltage of 100KV. Hydrodynamic diameter (size) and surface charge (ζ potential) of NPs dispersed in water (pH 7) were performed by dynamic light scattering (DLS) using a Zetasizer Nano ZS (Malvern Instruments, Malvern, UK) equipped with a He–Ne laser (633 nm, fixed scattering angle of 173°, room temperature 25°C). 1 mL of NPs solution was centrifuged for 30 min at 15000 rpm and re-dispersed in MilliQwater before each measurement. Absorption spectra of bare and modified-AuNPs (1 mL) were ACS Paragon Plus Environment

Page 19 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


recorded using a V-570 UV/ VIS/ NIR Cary 100 (Agilent) spectrophotometer from Jasco Int. Co. Ltd, Tokyo, Japan in the range between 300 and 900 nm. AuNPs functionalization yield analysis Functionalization yield analysis of 1018K6-modified AuNPs was performed by using the Reversephase High-performance liquid chromatography (RP-HPLC) analysis. In the direct method, the peptide was cleaved from NPs surface by acidolysis of the covalent bond with 50 % (v/v) trifluoroacetic acid (TFA, Sigma-Aldrich, USA), under stirring at room temperature31 for 24 h. Then, the supernatant was recovered by centrifugation at 15000 rpm for 30 min at 10 °C, in order to remove any AuNPs that could interfere and analyzed to quantify the amount of the bound peptide by HPLC chromatography. For the analyses, 200 μl of sample were injected over a μBondapak C18 reverse-phase column (3.9×300 mm, Waters Corporation) connected to a HPLC system (Shimadzu) using a linear gradient of 0.1% TFA in acetonitrile from 5% to 95%. A reference solution was prepared with the initial peptide concentration used for the functionalization at the same reaction conditions and run in parallel. Therefore, by knowing the added peptide (reference solution) it was possible to correlate the amount of peptide bound (expressed as percentage) to the nanoparticles by comparing the peak area. As a control, the same experimental conditions and analyses were performed by using the free peptide. In the indirect method, once functionalization was completed the supernatant solution was recovered after centrifugation of the nanoparticles to remove the coupled peptides and the peak area of the peptide not attached to the AuNPs was calculated. All measurements were carried out in triplicate. Circular dichroism (CD) spectroscopy The secondary structure of the peptides bound to gold nanoparticles was investigated by circular dichroism spectroscopy (CD) using a Jasco J-810 spectropolarimeter. CD spectra were recorded at 25°C using quartz cuvettes with 0.1 cm optical path length (Hellma Analytics). Spectra were acquired after sonication in the 194-250 nm measurement range at 20 nm/min scanning speed, 1 nm bandwidth,



Page 20 of 26

4 s response time, 1.0 data pitch, by averaging 5 scans. 1018K6-AuNPs concentration for CD measurement was 2 µM in water using 3 mM SDS as membrane-mimicking environment. Fluorescence spectroscopy Fluorescence emission intensity of 1018K6 bound to AuNPs was monitored by using fluorescence spectrophotometer (Jasco FP-8200 spectrofluorometer). The samples (12.5 µM) were excited at 280 nm (fluorescence emission from tryptophan residue), setting the slit widths at 2.5 nm. The emission spectra recorded at the range 300-400 nm in phosphate buffer (10 mM) at pH 7.0, glycine-HCl buffer (100 mM) at pH 2.0, glycine-NaOH buffer (100 mM) at pH 10.0, following the addition of SDS (3 mM final concentration) to samples. The fluorescent signals were recorded at 25 °C after 24 h incubation. Bacterial growth Listeria and Salmonella cultures were stored in deep-freezer at -80°C. Before to start the experiments, the frozen stocks of Listeria and Salmonella were plated on Half Fraser (Biorad- Italia) and BPW (Biomerieux- Italia) agar plates respectively and incubated at 37°C for 16 h to obtain single colonies. Antimicrobial activity of 1018K6-AuNPs The antimicrobial efficacy of the peptide bound to the gold nanoparticles were determined against L. monocytogenes (LM2 food-isolated strain) and S. Typhimurium (ATCC 13311) bacteria, which allowed the evaluation of the Minimal Bactericidal Concentration (MBC50), corresponding to the lowest peptide concentration able to cause at least 50% reduction in the number of viable bacteria on agar plates. L. monocytogenes and S. Typhimurium were grown at 37 °C in Half Fraser (Biorad- Italia) and BPW (Biomerieux- Italia) broths, respectively. The antibacterial activity of 1018K6-AuNPs was determined to incubate 15 x 104 CFU/mL (colony forming units/mL) of Listeria with increasing concentrations of 1018K6-AuNPs for 6 h at 37 °C. Each serial dilution included control plates with only the free peptide (1018K6). In all the experimental conditions explored, the plate counting method was used to estimate the minimal bactericidal activity


Page 21 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


of 1018K6-Au-NPs. Specifically, the number of colonies grown on agar plates in the presence of the different dilutions of 1018K6 peptide bound to the gold nanoparticles were counted and compared with those of the free peptide, whose MBC was reported in Palmieri et al. 201817. In another set of experiments, a fixed concentration of 1018K6-Au-NPs (0.16 µM) was incubated with different dilutions of Listeria (ranging from 15 x 102 to 15 x 105) or S. Typhimurium (ranging from 15 x 101 to 15 x 103) cultures. Transmission electron microscopy (TEM) L. monocytogenes (LM2 food-isolated strain) cells untreated (as control sample) and treated for 6 h with bare AuNPs or modified 1018K6-AuNPs were fixed with glutaraldehyde, post-fixed with osmium tetroxide, dehydrated by passing through a graded ethanol series and embedded in Poly/Bed 812 resin (Polysciences, Warrington, PA, U.S.A.). The embedded samples were cut by Leica UCT ultramicrotome (Leica Microsystems, Germany) into thin sections (50 nm thickness), attached to the formvar /carbon copper grids, and, to increase the contrast of the samples, an additional staining with uranyl acetate was used. Finally, the samples on the grids were observed under a Jeol JEM-1011 (JEOL, Tokyo, Japan) transmission electron microscope using an accelerating voltage of 100 kV. Low and high magnification images were captured by using the iTEM software (Olympus Soft Imaging System, Münster, Germany). At least ten different microscopic fields on a high number of thickness sections from different samples (untreated and treated) were observed and captured to obtain a good confidence in the data. Supporting Information Available TEM images of stained 1018K6-Au-NPs; HPLC analysis of 1018K6 on exposure to TFA 50%; HPLC calibration curve; immobilization yield; Bactericidal activity of 1018K6-AuNPs against S. Typhimurium; long term stability characterization; TEM micrographs of L. monocytogenes cells on exposure to AuNPs and 1018K6-Au-NPs.



Page 22 of 26

References (1) World Health Organization, Global priority list of antibiotic-resistant bacteria to guide research, discovery,

and

development

of

new

antibiotics;

http://www.who.int/medicines/publications/global-priority-list-antibiotic-resistantbacteria/en/, accessed: 10 April 2018. (2) Mishra, B., Reiling, S., Zarena, D., and Wang, G. (2017), Curr. Opin. Chem. Biol. 38, 87-96. (3) O’Neill, J. (2015), The Review on Antimicrobial Resistance. http://amr-review.org/, accessed: 22 March 2018. (4) Palmieri, V., Bugli, F., Lauriola, M. C., Cacaci, M., Torelli, R., Ciasca, G., Conti, C., Sanguinetti, M., Papi, M., and De Spirito, M. (2017), Bacteria meet graphene: modulation of graphene oxide nanosheet interaction with human pathogens for effective antimicrobial therapy, ACS Biomater. Sci. Eng. 3 (4), 619-627. (5) Hemeg, H.A. (2017), Nanomaterials for alternative antibacterial therapy, Int. J. Nanomed. 12, 8211-8225. (6) Wang, G., Li, X., and Wang, Z. (2016), APD3: the antimicrobial peptide database as a tool for research and education, Nucleic Acids Res. 44, D1087–D1093. (7) Brogden N.K., and Brogden K.A. (2011), Will new generations of modified antimicrobial peptides improve their potential as pharmaceuticals?, Int. J. Antimicrob. Agents 38, 217-225. (8) Mika, J. T., Moiset, G., Cirac, A. D., Feliu, L., Bardají, E., Planas, M., and Sengupta, D. (2011), Structural basis for the enhanced activity of cyclic antimicrobial peptides: the case of BPC194, Biochim. Biophys. Act. 1808, 2197-2205.


Page 23 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(9) Nguyen, U. T., Wenderska, I. B., Chong, M. A., Koteva, K., Wright, G. D., and Burrows, L.L. (2011), Small-molecule modulators of Listeria monocytogenes biofilm development, App. Environ. Microbiol. 78, 1454-1465. (10) Teixeira, V., Feio, M. J., and Bastos, M. (2012), Role of lipids in the interaction of antimicrobial peptides with membranes, Prog. Lipid Res. 51, 149-177. (11) Morales-Avila, E., Ferro-Flores, G., Ocampo-García, B.E., López-Téllez, G., López-Ortega, J., Rogel-Ayala, D.G., and Sánchez-Padilla, D. (2017), Antibacterial efficacy of gold and silver nanoparticles functionalized with the ubiquicidin (29–41) antimicrobial peptide, J. Nanomater. 2, 10. (12) Li, X., Robinson, S.M., Gupta, A., Saha, K., Jiang, Z., Moyano, D.F., Sahar, A., Riley, M.A., and Rotello, V.M. (2014), Functional gold nanoparticles as potent antimicrobial agents against multi-drug-resistant bacteria, ACS Nano 8, 10682-10686. (13) Kwon, E. J., Skalak, M., Bertucci, A., Braun, G., Ricci, F., Ruoslahti, E., Sailor, M. J., and Bhatia, S.N. (2017), Porous Silicon Nanoparticle Delivery of Tandem Peptide Anti‐Infectives for the Treatment of Pseudomonas aeruginosa Lung Infections, Adv. Mater. 29, doi: 10.1002/adma.201701527. (14) de la Fuente-Núñez, C., Korolik, V., Bains, M., Nguyen, U., Breidenstein, E.B., Horsman, S., Lewenza, S., Burrows, L., and Hancock, R.E. (2012), Inhibition of bacterial biofilm formation and 332 swarming motility by a small synthetic cationic Peptide, Antimicrob. Agents Chemother. 56, 2696-2704. (15) de la Fuente-Núñez, C., Reffuveille, F., Haney, E.F., Straus, S.K., and Hancock, R.E. (2014), Broad-spectrum anti-biofilm peptide that targets a cellular stress response, PLoS Pathog. 10, e1004152.



Page 24 of 26

(16) Reffuveille, F., de la Fuente-Núñez, C., Mansour, S., and Hancock, R.E. (2014), A broadspectrum anti-biofilm peptide enhances antibiotic action against bacterial biofilms, Antimicrob. Agents Chemother. 58, 5363-5371. (17) Palmieri, G., Balestrieri, M., Capuano, F., Proroga, Y.T.R., Pomilio, F., Centorame, P., Riccio, A., Marrone, R., and Anastasio, A. (2018), Bactericidal and antibiofilm activity of bactenecin-derivative peptides against the food-pathogen Listeria monocytogenes: New perspectives for food processing industry, Int J. Food Microbiol. 279, 33-42. (18) Politi, J., Spadavecchia, J., Iodice, M., and De Stefano, L. (2015), Oligopeptide–heavy metal interaction monitoring by hybrid gold nanoparticle based assay, Analyst 140, 149-155. (19) Politi, J., Spadavecchia, J., Fiorentino, G., Antonucci, I., and De Stefano, L. (2016), Arsenate reductase from Thermus thermophilus conjugated to polyethylene glycol-stabilized gold nanospheres allow trace sensing and speciation of arsenic ions, J. R. Soc. Interface 13, 20160629. (20) Beuchat, L.R., and Brackett, R.E. (1990), Survival and growth of Listeria monocytogenes on lettuce as influenced by shredding, chlorine treatment, modified atmosphere packaging and temperature, J. F. S. 55, 755-758. (21) Charpentier, E., and Courvalin P. (1999), Antibiotic Resistance in Listeria spp, Antimicrob. Agents Chemother. 43, 2103-2108. (22) Soumet, C., Ragimbeau, C., and Maris (2005), P., Screening of benzalkonium chloride resistance in Listeria monocytogenes strains isolated during cold smoked fish production, Lett. Appl. Microbiol. 41, 291-296. (23) Costa F., Carvalho I.F., Montelaro R.C., Gomes P., and Martins M.C. (2011), Covalent immobilization of antimicrobial peptides (AMPs) onto biomaterial surfaces, Acta Biomater. 7, 1431-1440.


Page 25 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(24) Rai, A., Pinto, S., Evangelista, M.B., Gil, H., Kallip, S., Ferreira, M.G., and Ferreira, L. (2016), High-density antimicrobial peptide coating with broad activity and low cytotoxicity against human cells, Acta Biomater. 33, 64-77. (25) Wu, X., Singh, A.K., Lyu, Y., Bhunia, A.K., and Narsimhan (2016), G., Characterization of antimicrobial activity against Listeria and cytotoxicity of native melittin and its mutant variants, Colloids and Surfaces B: Biointerfaces 143, 194-205. (26) Zhou, L., Narsimhan, G., Wu, X., and Du, F. (2014), Pore formation in 1, 2-dimyristoyl-snglycero-3-phosphocholine/cholesterol mixed bilayers by low concentrations of antimicrobial peptide melittin, Colloids Surf. B Biointerfaces 123, 419-428. (27) Balestrieri, M., Palmieri, G., Neglia, G., Anastasio, A., Capuano, F., De Stefano, L., and Nicolais, L., Antimicrobial Peptides, 16 July 2018, PCT/EP2018/069304. (28) Spadavecchia, J., Perumal, R., Barras, A., Lyskawa, J., Woisel, P., Laure, W., Pradier, C.M., Boukherroub, R., and Szunerits, S. (2014), Amplified plasmonic detection of DNA hybridization using doxorubicin-capped gold particles, Analyst 139, 157-164. (29) Terracciano, M., Shahbazi, M.A., Correia, A., Rea, I., Lamberti, A., De Stefano, L., and Santos, H.A. (2015), Surface bioengineering of diatomite based nanovectors for efficient intracellular uptake and drug delivery, Nanoscale 7, 20063-20074. (30) Kinstler, O.B., Brems, D.N., Lauren, S.L., Paige, A.G., Hamburger, J.B., and Treuheit, M.J. (1996), Characterization and Stability of N-terminally PEGylated rhG-CSF, Pharm. Res. 13, 996-1002. (31) Merrifield, R.B. (2006), in Advances in Enzymology and Related Areas of Molecular Biology, Vol. 32 (Ed: F.F. Nord), John Wiley & Sons, Inc., Weinheim, Germany, Ch. 6.



TOC graphic


Page 26 of 26

Small Synthetic Peptides Bioconjugated to Hybrid Gold Nanoparticles

Recommend Documents