Engineering of self-assembled silver-peptide colloidal nanohybrids with enhanced biocompatibility and antibacterial activity | Scientific Reports

Nov 02, 2024

Scientific Reports volume 14, Article number: 26398 (2024) Cite this article

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Several bacterial strains have developed resistance against commercial antibiotics, and interestingly, supramolecular nanomaterials have shown considerable advantages for antibacterial applications. However, the main challenges in adopting nanotechnology for antibacterial studies are random aggregation, compromised toxicity, multi-step preparation approaches, and unclear structure-function properties. Herein, we designed the amphiphilic tripeptide that acts as a reducing and capping agent for silver metal to form silver-peptide colloidal nanohybrids with the mild assistance of UV light (254 nm) through the photochemical reduction method. The nanohybrids are characterized by different spectroscopic and microscopic techniques, and non-covalent molecular interactions between metal and peptide building blocks confirm their central role in the formation of nanohybrids. The tripeptide is biocompatible and can reduce the toxicity of silver ions (Ag+) by reducing to Ag0. These colloidal nanohybrids showed antibacterial activity against gram-negative and gram-positive bacterial strains, and the possible mechanism of killing bacterial cells could be membrane disruption. This synthetic strategy is facile and green, which helps avoid using toxic chemicals or reagents and complicated methods for colloidal nanohybrid preparation for biomedical applications.

Over the last decade, several bacterial strains have developed resistance against the marketed antibiotics due to their unnecessary use, and microbial infections have significantly increased morbidity and mortality rates, prolonged treatment time, and patient economic loss1,2,3,4,5. Intelligent materials such as polymeric nanostructures6,7, inorganic nanomaterials8,9, carbon-based nanomaterials10, and self-assembling peptides11can treat bacterial and cancerous infectious and prevent biofilm formation. Among inorganic nanomaterials, metal nanoparticles (MNPs), particularly silver and gold, have inspired researchers to utilize them in different applications because of their unique physical properties like tunable size, shape, and surface plasmonic resonance12,13. In particular, silver nanoparticles (AgNPs) have shown promising biological and physicochemical properties like high surface area to volume ratio and lower toxicity than other metals14. These properties have made AgNPs an exceptional material in biomedical applications, including biosensing, imaging and drug delivery, and antibacterial activities15,16. However, the selectivity of individual AgNPs is a significant drawback that hinders the replacement of antibiotic drugs because AgNPs can mainly accumulate in healthy tissues. Besides this, silver ions (Ag+) have also shown side effects like allergic reactions, skin staining, and cellular toxicity when used at high dosages or for a long time as a treatment remedy17. Consequently, the need of the hour is to develop broad-spectrum antibacterial agents with enhanced biosafety and biocompatibility properties that exhibit a low propensity for drug-resistance development.

Typical synthesis methods can influence the biological properties of AgNPs and, thereby, affect the antibacterial activity and expected biomedical outcomes. For example, synthesizing AgNPs using microbes, plant extracts, and hazardous chemicals, which brings the toxicity concerns, and several complicated steps are involved in solvothermal and hydrothermal that can compromise their safety and reproducibility, needed for biomedical applications13,18,19,20. Moreover, these synthesis methods can fabricate less stable nanocomposites, which resultantly show random aggregation or gelations to make hydrogel, decreasing their surface area-to-volume ratio. Indeed, AgNPs have the potential to be used at the clinical level and replace antibiotic drugs, which is far beyond the concept because they may accumulate in normal tissues and organs. The most important aspect is that unreacted Ag+may also cause local or systemic side effects when used for longer time or high dosages, for example, skin staining, allergic reactions, and cellular toxicity21.

Interestingly, self-assembling and phase-separating amino acids, peptides, proteins, and polymeric nano-, micro- materials have shown fascinating properties for drug/protein delivery, artificial cells, and biocatalytic applications22,23,24,25. Therefore, using peptides that can act as reducing and capping agents to develop green and sustainable silver-peptide colloidal nanohybrids could be a promising strategy while maintaining biosafety and biocompatibility26. Recently, many efforts have been made to utilize a variety of biological compounds like natural polymers, nucleotides, amino acids, peptides, and proteins as effective templates and reducing agents for the synthesis of inorganic-organic nanohybrids27,28,29,30. Metal composites with proteins/peptides that act as capping, reducing, and stabilizing agents have been developed to tune the physicochemical properties further to solve the stability issues31,32. For example, Seferji et al. explored the photochemical synthesis of size-controlled biocompatible AgNPs using tetramer peptides without using chemicals for reducing, capping, and stabilizing agents for antibacterial applications33. Besides this, we reported colloidal composites prepared using silver metal and phase-separating peptide through a photochemical reduction method and used for bacterial wound healing28.

In this article, we synthesized metal-peptide colloidal nanohybrids using two chemical components: (1) amphiphilic tripeptide building block (IFH) and (2) silver nitrate (AgNO3), which has reduced the intrinsic limitations of each compound for nanomaterial formation and antibacterial activity. A self-assembling peptide containing three amino acids, namely, H2N-IFH-COOH, as a reducing and capping agent to synthesize Ag-IFH nanohybrids via a photochemical reduction approach with the assistance of mild UV light and non-covalent intermolecular interactions play a vital role in the self-assembly. These nanohybrids show antibacterial activity against Escherichia coli and Staphylococcus aureus strains. The Ag-IFH nanohybrids kill bacteria more effectively than their constitutive components, reducing the toxicity stemming from silver ions. Reactive oxygen species (ROS), release of silver ions from nanohybrids, and membrane disruption could be possible mechanisms that result in the death of bacterial cells. The present approach is simple and green in its synthetic aspects, and interestingly, these nanohybrids have shown promising biocompatibility. The interactions between metal and peptide could minimize aggregation and improve the antibacterial efficacy of colloidal nanohybrids.

All exmethods were carried out in accordance with relevant guidelines and regulations.

The ethics committee of the School of Biological Sciences, University of Punjab, reviewed and approved the research experiments involving human subjects with reference number PU/D-No/97/DFENS.

Informed consent was obtained from donors before taking the blood samples.

Dulbecco’s modified eagle medium (DMEM), Fetal Bovine Serum, and Penicillin/Streptomycin (Pen/Strep) 100x were purchased from Life Technologies, GibcoTM, USA. Phosphate Buffered Saline (Tablets) was obtained from BioWorld, Canada - Cedarlane Laboratories. Trypsin was purchased from ATCC PCS-999-003, USA. Yeast Extract and Tryptone were purchased from BIO BASIC INC., Canada. Agar was purchased from Neogen, Michigan, United States. All solvents and metal salts were purchased from Sigma Aldrich. IFH tripeptide was purchased from customized peptide company PEPMIC, China.

2 mg lyophilized powder of tripeptide H2N-IFH-COOH was dissolved in 500 µL Milli-Q water, and then 500 µL HEPES buffer (1 M) with pH 7.4 or PBS (1 M) of pH 7.2 was added, and subsequently, 250 µL AgNO3 (200 mM) was added to the reaction mixture. Then, the mixture was transferred to a quartz cuvette and irradiated with UV light (254 nm) for 10 min. The transparent color of the mixture was changed to reddish, indicating the formation of silver-peptide nanohybrids with the final concentration of silver 40 mM and peptide 2 mM.

The surface potential and size of silver peptide nanohybrids (silver 40 mM and peptide 2 mM) were measured using the Malvern ZS Nano instrument at room temperature, and measurements were recorded in triplicate.

Lyophilized powder of IFH (2 mM) and Ag-IFH nanohybrids (silver 40 mM and peptide 2 mM) were used for the XRD pattern using an X-ray diffractometer PANalytical Empyrean instrument. The spectra were recorded by Cu Kα radiation (\(\:\lambda\:\) = 0.154 Ao) and a nickel monochromator filtering wave at 40 kV and 30 mA. The diffraction pattern was obtained at diffraction angles 2θ between 5o and 80o, with a scanning speed of 0.4o/min at room temperature. The crystallinity of the lyophilized Ag-IFH nanohybrids was calculated using Scherrer’s relationship.

FTIR spectrometer (Thermo Scientific, Nicolet iS10, Smart iTR) was used at room temperature to identify the molecular structure of the prepared IFH powder and lyophilized Ag-IFH nanohybrids (silver 40 mM and peptide 2 mM) in ATR mode from 500 to 4000 cm−1 with the resolution of 4 cm−1 over 128 scans.

The UV-Vis spectra of IFH peptide, silver nitrate, and Ag-IFH nanohybrids (silver 40 mM and peptide 2 mM) were recorded using a Shimadzu UV-1900 UV-Vis spectrophotometer. The optical absorption spectra of IFH peptide and Ag-IFH nanohybrids were recorded using 1 cm quartz cuvettes in the 200–800 nm spectral range at a 200 nm/min scan speed. And data were plotted using the Origin 2023b.

Ag-IFH nanohybrids (silver 40 mM and peptide 2 mM) were freeze-dried to obtain the dry powder, which was coated on carbon tape and placed on the sample holder. The XPS experiment was performed using a Thermo-Fisher Scientific Escalab Xi + instrument equipped with a monochromatic Al KαX-ray source (hν = 1486.6 eV) operated at a power of 300 W under UHV conditions with ∼10 − 10 mbar. The XPS spectra were recorded in standard mode through magnetic lenses (120 μm to 1 mm range) at resolutions up to 1 μm. The survey and high-resolution spectra were acquired at fixed analyzer pass energies of 100 and 20 eV (unless otherwise specified). To avoid differential charging, this system combines a flood gun and an ion gun (argon) to compensate for the potential surface charges. The peak fitting was performed using Originpro2023b with/without background subtraction and the standard 100% Gaussian for curve fitting. No preliminary smoothing was conducted during the analysis.

The sample of freshly prepared nanohybrids (silver 40 mM and peptide 2 mM) (~ 2 µL) was directly drop-casted onto a holey carbon-coated copper grid (mesh size 300 nm) (purchased from Electron Microscopy Sciences) for 45 s, and the excess sample was blotted with a filter paper. Afterward, the sample containing grids was negatively stained with a reagent phosphotungstic acid (~ 2 µL) applied, and the excess reagent was also blotted with a filter paper after 30 s. The TEM grid was then dried at room temperature before imaging the morphology of Ag-IFH nanohybrids using ThermoFisher Scientific’s Talos 200 transmission electron microscope (TEM), equipped with a field emission gun. The experiments were performed by operating the microscope at 200 kV accelerating voltage. Several bright-field TEM (BF-TEM) images were obtained by setting the microscope at different magnifications to understand the nanohybrids’ morphology and nanostructure. The average diameter of nanohybrids was calculated by processing the acquired BF-TEM images with ImageJ and Originpro2023b software packages.

We confirm that Punjab University approved all in-vitro experimental protocols. For this experiment, we used two bacterial strains: Staphylococcus aureus (ATCC # 28923) as the gram-positive model and Escherichia coli (ATCC # 25922) as the gram-negative model. The bacterial cells were kept at 4˚C on an agar slant, subcultured overnight at 37 C, and maintained in Luria Burtani (LB) medium. These freshly prepared cultures of both species of bacteria were incubated overnight and diluted to give the desired concentration before any susceptibility test. We used a Well Diffusion Method and minimum inhibitory concentration for antimicrobial activity for three samples, as demonstrated below.

Antibacterial activity was evaluated against Escherichia coli (ATCC # 25922) and Staphylococcus aureus (ATCC # 28923) by well diffusion method. The working samples of IFH solution, AgNO3 solution, and Ag-IFH nanohybrids were prepared, and the entire agar plate surface was inoculated by spreading the 200 µL microbial culture of 0.01 OD. Then, holes were punched with sterile yellow and blue tips. After that, different concentrations of IFH, AgNO3, and Ag-IFH nanohybrids were introduced into the wells, and the zone of inhibitions was measured using ImageJ. The marketed antibiotic ampicillin (100 µg/mL) was used as a positive control, and Tris buffer was used as a negative control. The samples containing Petri dishes were incubated for 16 h at 37 °C.

In microbiology, the minimum inhibitory concentration (MIC) is the lowest concentration of an antimicrobial agent, preventing visible in vitro growth of bacteria strains. The MIC of Ag-IFH nanohybrids was determined by the microdilution method in Luria-Bertani (LB) broth. Various concentrations of Ag-IFH nanohybrids (Ag: IFH, 0.0424 mg/mL:0.00606 mg/mL, 0.0849 mg/mL :0.0121 mg/mL, 0.1698 mg/mL :0.0242 mg/mL, 0.33975 mg/mL : 0.0485 mg/mL, 0.6795 mg/mL: 0.097 mg/mL, 1.359 mg/mL : 0.194 mg/mL respectively, were tested against Escherichia coli (ATCC# 25922) and Staphylococcus aureus (ATCC # 28923). The starting inoculum was approximately 0.5 × 106 cells/mL, and bacterial colonies growing on agar plates were inoculated in LB medium overnight at 37 °C with agitation at 200 rpm to achieve an approximate concentration of one million cells (106)/mL. Various dilutions of nanohybrids were prepared in 96-well plates for testing with each bacterial species. Each dilution of Ag-IFH nanohybrids had an equal number of bacteria and was allowed to incubate for 18 h. A well without nanohybrids inoculated with the tested bacteria species was a positive control, whereas a negative control had only the liquid broth. The well showing no visible growth corresponds to the MIC of nanohybrids in this experiment to prevent bacterial growth. To enhance the validity of the findings, all the antibacterial assays for evaluating MIC were conducted in triplicate.

To check hemocompatibility, 3 mL of blood from the human donor (with prior permission from the donor and the ethics committee of Punjab University) was taken into vacutainer tubes coated internally with K2-EDTA to prevent coagulation. The blood sample was washed with 0.9% saline solution and PBS, diluted with a 1:4 ratio of PBS, and split into five vials. Different concentrations of Ag-IFH nanohybrids 0.0849 mg/mL : 0.0121 mg/mL, 0.1698 mg/mL : 0.0242 mg/mL, 0.33975 mg/mL : 0.0485 mg/mL, 0.6795 mg/mL : 0.097 mg/mL, respectively were added to it and incubated at 37 OĊ for 24 h in constant stirring. The nanohybrids incubated samples were centrifuged to collect the supernatant, and OD was measured at 540 nm. For positive control, we used 1 mL of diluted blood, and 1 mL of 0.01 N HCl was added, while for negative control, 1 mL of diluted blood was added with 1 mL of 0.9% saline water. The percentage hemolysis was calculated by using the following equation:

To assess the cytotoxicity, we used a mice fibroblast cell line (NIH3T3) with a density of 3 × 104 cells per well, cultured in a cell culture medium (DMEM) in a 48-well plate. The plate was incubated at 37 °C with 5% CO2 for 24 h. After that, 0.0424 mg/mL : 0.00606 mg/mL, 0.0849 mg/mL : 0.0121 mg/mL, 0.1698 mg/mL : 0.0242 mg/mL, 0.33975 mg/mL : 0.0485 mg/mL ,0.6795 mg/mL : 0.097 mg/mL, 1.359 mg/mL : 0.194 mg/mL of Ag-IFH nanohybrid were added (treated group) and blank well with just cells (non-treated group) and incubated for another three days. After three days, 75 µM of resazurin dye was added to all wells, and the optical density was recorded using a microplate reader at a wavelength of 570 nm (reduced). Cell viability was calculated based on the degree of reduction in Alamar blue with reference to control cells.

The Escherichia coli and Staphylococcus aureus bacterial strains were cultured until the optical density (OD) reached 0.1, measured by a spectrophotometer at 600 nm. Then, the bacterial cultures were treated with the sub-MIC dosage of nanohybrids and incubated for 12 h, followed by centrifugation at 4000 rpm for 5 to 10 min at 4 °C to get the bacterial pellets. These bacterial pellets were fixed in 2.5% glutaraldehyde for 4 h and gradient ethanol dehydration for 15 min. The bacteria samples were placed on silicon wafers to observe the morphology by SEM.

Several biological and photophysical methods have been reported to synthesize silver nanohybrids, and oligopeptides have received enormous attention for in-situ preparation of metal nanoparticles for antibacterial and catalytic applications to overcome the drawbacks of existing reducing and capping agents28,31. Therefore, we designed the amphiphilic tripeptide H2N-IFH-COOH (IFH) composed of three amino acids, isoleucine, phenylalanine, and histidine, through amide bonds that act as reducing and capping agents to synthesize the nanohybrids through photochemical reduction approach. The C-terminal of IFH contains carboxylic acid, and the N-terminal includes an amine group (for building block characterization, see supplementary Fig. 1) that plays a central role in reducing the silver ions (Ag+) to make nanohybrids. To prepare these nanohybrids, 2 mg/mL IFH peptide was dissolved in MQ water, HEPES/Tris- buffer with pH 7.4 was used to increase the pH, and 200 µL silver nitrate solution (200 mM) was added. However, no particle formation was observed, and the solution remained transparent until the sample was irradiated by a UV light source (254 nm) for 10 min. The color was changed to reddish (Fig. 1a-b), and the characteristic plasmonic peak of silver nanoparticles at 430 nm34 was observed in the absorption spectra, confirming the presence of silver nanoparticles in colloidal nanohybrids (Fig. 1c). The absorption peak is not as sharp as we expect from the hydrothermal and other reducing methods of silver nanoparticles35,36, and relatively broadening plasmonic peaks show that peptide somehow surrounds the nanoparticles instantly through biomolecular interactions. The emission spectra of nanohybrids also indicate the presence of silver nanoparticles embedded with peptide, as fluorescence intensity is not increased in the case of only peptide and silver nitrate when excited at the same wavelength of 430 nm (Fig. 1d). Interstingly, irradiation time plays a significant role, and the intensity of a particular peak increases with time, indicating the density of silver nanoparticles formed (Supplementary Fig. 2). Hence, these results collectively elucidate that silver nanoparticles are capped with the peptide building block, forming colloidal nanohybrids.

Structural information, schematic illustration, and spectroscopic characterization, (a) chemical structure and schematic illustration for preparation of nanohybrids for antibacterial activity, (b) photographs to show nanohybrids formation, (c) UV-Vis absorption spectra of pure peptide and nanohybrids, (d) fluorescence spectra of silver nitrate, pure peptide, and nanohybrids.

Self-assembly of peptides alone or with other components is driven by weak molecular interactions, including electrostatic, cation-pi, metal coordination, and hydrogen bonding, to create nano-to-microscale materials37. Interestingly, peptide and polymeric materials have been considered multivalent materials and could potentially mitigate infectious diseases, and these molecular interacting sites are crucial in the multivalent role of materials38. Thus, the functional groups and molecular interactions between metal silver and tripeptide play a pivotal role in reducing and capping to make the Ag-IFH nanohybrids through self-assembly in aqueous media. The tripeptide IFH contains carboxylic acid and amine functional groups at the terminal, and upon increasing the pH, the compound becomes the zwitterionic species as it includes one (+) and one (-) charge. Notably, the surface potential was positive ~ +2.20 mV at 5 min aged Ag-IFH colloidal nanohybrids (Supplementary Fig. 3), which was due to the dissociation of silver salt into Ag+ ions and nitrate ions, and interestingly, the surface potential was shifted to negative ~ – 9.0 mV after aging the sample for four days. This shift in surface potential demonstrates that silver ions (Ag+) have been converted to (Ag0). The size of nanohybrids is approximately 5–6 nm, calculated from the TEM image (Sect. 3.3), and it was not so convenient to observe in the dynamic light scattering (DLS) technique and appeared around ~ 1 μm (Supplementary Fig. 4). That could be due to light scattering from peptide building blocks presumably attached to silver metal in nanohybrids, and the polydispersity index (PDI) was between 0.266 and 0.294, suggesting particle distribution of the colloidal nanohybrids is not well uniform. Furthermore, we used Fourier Transform Infra-Red (FT-IR) to show the shifts in peaks of functional groups in IFH peptide and Ag-IFH nanohybrids, confirming the molecular interactions between the two components. Amino acids present in the tripeptide can bind AgNPs through the C=O from carboxyl groups, which prevents their agglomeration and possibly makes nanohybrids stable39. In detail, the 1658 cm−1 peak indicates the C=O stretching bond (amide) in the IFH tripeptide, and this peak has been broadening in Ag-IFH nanohybrids, revealing the interactions between silver and carbonyl oxygen, while the peak at 1464 cm−1 represents the C-H bending, and the 1168 cm−1 peak represents C-O stretching (Fig. 2a). The peaks at 2840 and 2990 cm−1 indicate the C-H stretching of the molecules, and the peak at 3414.77 cm−1 represents N-H stretching (Supplementary Fig. 5). Thus, the difference in bending and stretching peaks of different functional groups confirms the molecular interactions between the IFH peptide and silver metal in colloidal nanohybrids. Other characteristic peaks remain unchanged, which could be due to the low concentration of respective components in nanohybrids.

To further elucidate and validate the hypothesis of molecular interactions involved in colloidal nanohybrid formation, we performed the X-ray photoelectron spectroscopy (XPS) analysis for the binding energy of two components and elements present in the composition of colloidal nanohybrids. For elemental analysis, the peaks in the survey profile of colloidal nanohybrids at 283.35, 367.53, 397.17, and 529.66 eV demonstrate the presence of carbon, silver, nitrogen, and oxygen, respectively, while silver is missing in the survey profile of the control sample of pure peptide IFH (Supplementary Fig. 6a-b). The XPS spectrum of Ag 3d showed two peaks of Ag 3d5/2 and Ag3d3/2, at 367.07 and 373.12 eV, which confirms the reduction of silver ions (Fig. 2b). For c1s spectra, the binding energy shifted from 283.55 to 284.63 eV (Supplementary Fig. 6c-d), and the N1s spectrum suggests the binding energy shift from 397.7 to 400.54 eV from N1 to N3 (Fig. 2c-d). However, the O1s spectrum revealed that the O1 peak shifted toward the O2 peak from 530.11 to 530.86 eV, and a new peak emerged, presumably the carbonyl oxygen bonded with silver (Fig. 2e-f).

Fourier Transform Infra-Red (FTIR), experimental and deconvoluted X-ray photoelectron spectroscopy (XPS) spectra for the binding energy of molecular interaction, (a) FTIR spectra of pure peptide and colloidal nanohybrids, (b) Ag3d in nanohybrids, (c) N1s in IFH, (d) N1s in nanohybrids, (e) O1s in IFH, (f) O1s in nanohybrids.

We then further investigated the size and crystallinity-related information of silver-peptide colloidal nanohybrids by transmission electron microscope (TEM). Firstly, we calculated the size of colloidal nanohybrids, around 6 nm (Fig. 3a), and the inset histogram shows the size profile extracted from the same TEM image. This ultrasmall size of colloidal nanohybrids is well aligned with the silver nanoparticles synthesized by different methods40,41. After confirming the presence of silver metal in nanohybrids in comparatively bulk material using the XPS, we performed the energy dispersive X-ray spectroscopy (EDS), and the spectrum confirms the presence of silver along with other elements in the nanohybrids (Figure 3b). Figure 3c shows the selected area electron diffraction (SAED) pattern, which demonstrates the lattices of silver metal in nanohybrids. The crystallinity of metal in nanohybrids and the pure peptide was analyzed by powder X-ray diffraction (Figure 3d). The crystalline features of the nanohybrids are evident from the PXRD profiles, which display seven peaks at the 2θ position of ≈ 23.4°, ≈ 22.3°, ≈ 21.2°, ≈ 20.6°, ≈ 19.6°, ≈ 16.4°. ≈13.5°. The poly-crystalline patterns of the nanohybrid’s 69.40% crystallinity calculated using the Scherrer equation could be due to the nano-level nucleation of the silver nanoparticles. Moreover, the absence of any pure peptide crystalline peaks indicates the amorphous nature of the peptide building block.

Microscopic analysis of Ag-IFH nanohybrids, (a) TEM micrograph and inset histogram calculated from the same TEM image, (b) EDS spectrum confirming the presence of silver element in the nanohybrids, (c) selected area electron diffraction (SAED) pattern to represent the lattices of silver in nanohybrids, (d) powder x-ray diffraction graph of pure peptide and colloidal nanohybrids.

To assess the antibacterial activity of nanohybrids against gram-negative and gram-positive bacterial cells, we used bacterial strains of Escherichia coli and Staphylococcus aureus. We first utilized the Well Diffusion Method described in the material and method section, and we prepared three working samples such as Ag-IFH nanohybrids, silver nitrate, and pure peptide (IFH). In sample preparation, the same concentration of silver nitrate was used alone and in Ag-IFH nanohybrids. Similarly, the same peptide concentration was used alone and in Ag-IFH nanohybrids. For evaluating the antibacterial activity, in the case of Escherichia coli, the diameter was calculated for three concentrations such as 2.718 mg/mL-0.388 mg/mL,2.19 mg/mL-0.322 mg/mL,1.359 mg/mL-0.194 mg/mL from Ag-IFH nanohybrids respectively, which were 0.9 mm,1.12 mm, and 1.16 mm, respectively. Similarly, for Staphylococcus aureus, the diameter of the zone of inhibition was calculated for three different concentrations such as 2.718 mg/mL : 0.388 mg/mL, 2.19 mg/mL : 0.322 mg/mL, 1.359 mg/mL : 0.194 mg/mL from Ag-IFH nanohybrids respectively, and that was 2.84 mm, 2.16 mm, 1.8 mm, respectively (Fig. 4a-b). Thus, nanohybrids showed higher in-vitro antibacterial activity against Staphylococcus aureus because the diameter of the zone of inhibition was significantly higher than that of Escherichia coli. Furthermore, We checked the antibacterial activity of silver nitrate to see which component of nanohybrid has an antibacterial effect, for which we used silver nitrate (67.96 mg/mL) in three different concentrations. The average diameter for the zone of inhibition was calculated as 2.24 mm for Escherichia coli and 1.02 mm for Staphylococcus aureus (Supplementary Fig. 7). This shows the significantly higher antibacterial activity of silver ions, and that is why we hypothesized that silver metal could be utilized as a biological building block for bactericidal properties by decreasing its inherent toxicity of metal ions. On the other hand, the pure peptide (IFH) did not show antibacterial activity against both strains because no significant zone of inhibition was observed in agar plates (Supplementary Fig. 8). Furthermore, we used the commercially available antibiotic ampicillin (100 µg/mL) as a positive control, and ZOI was 3.14 mm for Escherichia coli and 4.62 mm for Staphylococcus aureus. The results showed a significantly higher zone of inhibition due to its higher concentration and well-tested antibiotic. At the same time, we used 20 mM Tris buffer (40 µL) pH 7.4 as negative control against Escherichia coli and Staphylococcus aureus (Supplementary Fig. 9).

To validate the Well Diffusion Method results, we decided to calculate the Minimum Inhibitory Concentration (MIC) using the same bacterial strains, Escherichia coli and Staphylococcus aureus, and the optical density (OD) of broth cultures when treated with different concentrations of nanohybrids and silver nitrate were measured. Interestingly, with increasing concentrations of nanohybrids, the OD of bacterial cultures decreased significantly compared to the control sample, where no nanohybrids were incubated, and the growth of bacterial culture significantly reduced at 1.359 mg-0.194 mg per ml of Ag-IFH concentration (Fig. 4c-d). However, the MIC of silver nitrate solution against Escherichia coli and Staphylococcus aureusis higher than nanohybrids with the same concentration of silver nitrate used (Supplementary Fig. 10). This result envisions that silver ions are more toxic than silver elementary (nanoparticle form) and are unsafe for biological application42. To get further insights into the antimicrobial mechanism of nanohybrids, treated bacterial strains (Fig. 4a-b) were fixated on the scanning electron microscope wafers, and we observed the distorted morphology of both bacterial strains (Fig. 4e-f), which potentially confirms the membrane disruption.

Evaluation of the antibacterial activity of Ag-IFH nanohybrids by Well Diffusion Method and MIC, (a) Zone inhibition in Escherichia coli growth, (b) Zone inhibition in Staphylococcus aureus growth, (c,d) In-vitro antimicrobial activity of nanohybrids against Escherichia coli and Staphylococcus aureus, there is a decrease in the growth of bacterial cultures with increasing concentration of nanohybrids, (e, f) SEM images of treated Escherichia coli and Staphylococcus aureus. The scale bar is 1 μm.

Biocompatibility of any nanomaterial is crucial to deciding whether the formulation is helpful for the next phase of testing and is factually essential for the clinical levels of that formulation43. Therefore, we decided to test the biocompatibility using two essays: (1) hemocompatibility and (2) fibroblast cells. We followed the American Society for Testing and Materials (ASTM) standard protocols for the biocompatibility of nanohybrids, and the hemocompatibility of biomaterials should be less than 5%. In positive control, we used 0.01 N HCl, and due to its highly acidic nature, the membranes of red blood cells were damaged, which caused hemolysis. The nanohybrid’s shape, size, and surface charge can affect its compatibility with blood OD at 540 nm, which is for free plasma hemoglobin44. Nanohybrids should be compatible with blood and non-toxic or could not damage the membrane of red blood cells; otherwise, hemoglobin will be released. Therefore, Ag-IFH nanohybrids with 0.0849 mg/mL: 0.0121 mg/mL, 0.1698 mg/mL : 0.0242 mg/mL, 0.33975 mg/mL − 0.0485 mg/mL, 0.6795 mg/mL-0.097 mg/mL respectively have hemocompatibility of 3.3%, 3.5%, 4.2%, and 5%, respectively, which demonstrate that the nanohybrids Ag-IFH are biocompatible with blood up to the concentration of 0.6795 mg/mL: 0.097 mg/mL respectively.

To validate the hemocompatibility results, we tested the cytotoxicity of nanohybrids against fibroblast cell lines with resazurin dye using a UV spectrophotometer45. Interestingly, metabolically active fibroblast cells have reduced the blue-colored nonfluorescent resazurin dye into pink-colored fluorescent resorufin within 4 h of incubation. For this study, we used a range of concentrations of Ag-IFH nanohybrids between 0.0424 mg/mL : 0.00606 mg/mL, 0.0849 mg/mL : 0.0121 mg/mL, 0.1698 mg/mL : 0.0242 mg/mL, 0.33975 mg/mL : 0.0485 mg/mL, 0.6795 mg/mL : 0.097 mg/mL, 1.359 mg/mL : 0.194 mg/mL respectively, calculated the percentage viability, which was between 94.23% and 82.54%, respectively. However, relatively higher cytotoxicity was noticed when a higher concentration of 1.359 mg-0.194 mg per ml of Ag-IFH nanohybrid used, as shown in Supplementary Fig. 11. These results show that the cytotoxicity of nanohybrids against fibroblast cell lines is not high, and cells were found compatible against nanohybrids to a specific concentration.

In summary, we designed the silver-peptide nanohybrids using amphiphilic tripeptide that acts as a reducing and capping agent with the mild assistance of UV light. Silver nitrate dissociates into silver ions Ag+, which is toxic and can damage healthy cells and tissues. At the same time, its nanoparticle form (Ag0) has been considered more bio-friendly and could be used for antibacterial applications. Therefore, we used biological building blocks to avoid the inherent toxicity of metal ions and toxicity stemming from solvents or reagents used to reduce metal ions. This in-situ facile method is based on a photochemical reduction approach and interactions between reduced silver and peptide, which result in nanohybrids. The nanohybrids are characterized by different spectroscopic and microscopic techniques, and non-covalent molecular interactions between silver and peptide building blocks confirm their role in capping the nanohybrids. Nanohybrids showed antibacterial performance against gram-negative and gram-positive bacterial strains with the possible killing mechanism due to reactive oxygen species (ROS) and membrane disruption. This synthetic strategy provides a green approach for synthesizing nanohybrid and could provide insight into the design of novel materials for biomedical applications.

All data generated or analyzed during this study are included in this present article and are available from the corresponding author upon reasonable request.

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MA thanks the financial support from Khalifa University (Project No. 8474000462). MA thanks the support of the KU research laboratories department (Core laboratories) and Thomas Delclos for XPS measurements. The authors thank the support from research facilities available in the Core Technology Platform (CTP) of New York University, Abu Dhabi (NYUAD).

Institute of Physics, The Islamia University of Bahawalpur, Bahawalpur, Pakistan

Nyla Saeed & Maria Atiq

Division of Science and Technology, Department of Physics, University of Education, Lahore, Pakistan

Atia Atiq & Zahid Usman

School of Biological Sciences, University of the Punjab, Lahore, Pakistan

Farhat Rafiq & Muhammad Saleem

Department of Chemistry, Khalifa University of Science and Technology, P.O. Box 127788, Abu Dhabi, United Arab Emirates

Iliyas Khan & Manzar Abbas

Department of Physics, Khalifa University of Science and Technology, P.O. Box 127788, Abu Dhabi, United Arab Emirates

Dalaver H. Anjum

Functional Biomaterial Group, Khalifa University of Science and Technology, P.O. Box 127788, Abu Dhabi, United Arab Emirates

Manzar Abbas

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MA and MA conceived the project and designed the experiments. NS, AA, ZU, and IK performed the material characterization and analysis. NS and FR performed the antibacterial, haemocompatibility, and cytotoxicity experiments. DHA performed the TEM of nanohybrids. MS, MA, and MA analyzed the results and wrote the manuscript with the help of all authors. All authors were involved in the discussion and approved the submission.

Correspondence to Maria Atiq or Manzar Abbas.

The authors declare no competing interests.

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Saeed, N., Atiq, A., Rafiq, F. et al. Engineering of self-assembled silver-peptide colloidal nanohybrids with enhanced biocompatibility and antibacterial activity. Sci Rep 14, 26398 (2024). https://doi.org/10.1038/s41598-024-78320-7

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Received: 01 August 2024

Accepted: 30 October 2024

Published: 02 November 2024

DOI: https://doi.org/10.1038/s41598-024-78320-7

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