Skip to main content
NCBI home page
Nanomedicine logoLink to Nanomedicine
. 2024 Sep 3;19(23):1913–1929. doi: 10.1080/17435889.2024.2382073

Lipopolysaccharide targeting-peptide-capped chitosan gold nanoparticles for laser-induced antibacterial activity

Samraggi Choudhury a, Aakrati Mehra a, Swapnil Srivastava a, Manju Sharma a, Manish Singh a,**, Jiban Jyoti Panda a,*
PMCID: PMC11457656  PMID: 39225175

Abstract

Aim: We present the synthesis of anti-bacterial gold nanoparticles using chitosan as a dual-functional agent. The resulting ChAuNPs were further modified with a lipopolysaccharide-targeting antibacterial peptide to aid in biocompatibility and specificity.

Materials & methods: The nanoparticles' antibacterial activity against Escherichia coli was tested in the presence of a 450 nm laser.

Results: Our data suggested that the peptide and laser emissions had a synergistic impact on the gold nanoparticles, resulting in strong antibacterial effects. The study shows that advanced nanomaterials, including chitosan, gold nanoparticles and lipopolysaccharide targeting peptides, can boost antibacterial functions at a low concentration of 250 μg/ml.

Conclusion: The findings highlight ChAuNPs' potential as strong antibacterial agents, with targeted alterations critical for maximizing their utilization.

Keywords: : antibacterial activity, E. coli, gold nanoparticles, LPS targeting peptide, ROS production

Plain language summary

Article highlights.

  • Use of chitosan as a dual-functional i.e. reducing and stabilizing agent for synthesizing the gold nanoparticles.

  • Lipopolysaccharide-targeting antibacterial peptide decorated chitosan-coated gold nanoparticles (ChAuNPs) with improved biocompatibility and specificity.

  • Advanced nanomaterials featuring chitosan, gold nanoparticles and lipopolysaccharide-targeting peptides were developed.

  • The antibacterial effectiveness of the nanoparticles was tested against E. coli with 450 nm laser exposure.

  • The study showed significant antimicrobial effects due to the combined action of the peptide and laser.

  • The results highlight the potential of ChAuNPs as potent antibacterial and ROS producing agents due to its tailored modifications.

  • The nanomaterials demonstrated enhanced antimicrobial properties at a low concentration of 250 μg/ml.

  • The study contributes to advanced nanomaterials for antimicrobial applications, emphasizing the potential of LPS targeting ChAuNPs in fighting bacterial infections.

1. Background

Metal-based nanoparticles are a common class of inorganic nanoparticles that present a viable platform for fighting antibiotic resistance [1]. These nanoparticles function in different ways than standard antibiotics, making them effective against germs that have developed resistance. They prevent resistant strains from forming by targeting several biomolecules [2]. The electrostatic attraction of negatively charged bacterial cell walls and positively charged nanoparticles promotes their interaction, resulting in membrane rupture and enhanced permeability [3]. Furthermore, metal-based nanoparticles can release metal ions into cells, causing the generation of reactive oxygen species (ROS) and suppressing bacterial antioxidant defenses by oxidizing glutathione. Metal ions interact with proteins, membranes and DNA, causing oxidative stress that affects cellular functioning. Metal ions can create strong coordination bonds with bacteria by interacting with specific metal sensor proteins that form unique coordination complexes [4,5].

Colloidal gold nanoparticles with polymer coating comprise of a gold (Au) core and polymeric shell like structure [6]. These polymer coated gold nanoparticles (AuNPs) possess plasmonic and photothermal properties of AuNPs combined with biocompatibility, stability and robustness provided by the polymers [7]. The surface plasmon resonance (SPR) characteristics of gold nanoparticles aid in exhibiting a photothermal effect when exposed to visible light laser irradiation [8].

Chitosan is a biopolymer derived from chitin through the deacetylation process, and is primarily sourced from crustacean waste in nature. This biopolymer exhibits cationic properties due to its β (1–4) linkages, featuring varying amounts of N-acetyl glucosamine and glucosamine residues. The amine group's presence imparts a positive charge to the compound, enhancing its affinity for Gram-negative bacteria's negatively charged lipopolysaccharide membrane [9].

In the recent past antimicrobial peptides (AMPs) have gained attention as biological alternatives due to the increased cases of antibiotic resistance. AMPs mainly consist of non-polar residues and have an amphipathic nature. Their amphipathic nature allows them to interact with both the outer and inner membranes, leading to the membrane disintegration of bacteria [10]. Studies have reported that AMPs can be conjugated to diverse metal nanoparticles, as they are nontoxic in nature, and the chemistry between AMPs and nanoparticles is tunable, leading to an enhanced anti-microbial effect [11].

Antimicrobial peptides called lipopolysaccharides (LPS) targeting peptides specifically target the LPS of Gram-negative bacteria's surface [12]. These peptide when interact with LPS, it damage the bacterial membranes and eventually kills the bacteria. Coating LPS targeting peptides on gold nanoparticles can increase their antibacterial efficacy [13]. As a result, the peptides may become more stable and bioavailable, improving their capacity to interact with the bacterial membrane. LPS targeting peptides are a potential family of antimicrobial peptides that can be utilized to target particular bacterial membrane areas and cause membrane disruption, which will eventually cause the bacterium to die.

The LPS targeting peptide-coated gold nanoparticles for antibacterial applications is a rapidly evolving field with significant advancements. Covalent binding of antimicrobial peptides (AMPs) to gold nanoparticles (AuNPs) has been shown to enhance their antimicrobial activity against various bacterial strains [13]. Utilizing EDC-NHS chemistry to covalently bind AMPs to AuNPs is an effective strategy for increasing antimicrobial efficacy. Targeting peptides are employed to direct AuNPs to specific bacterial targets, further improving their antimicrobial activity [11]. The surface chemistry of AuNPs also affects their antimicrobial properties, with specific coatings like chitosan, enhancing activity against particular bacteria.

In this study, we utilized chitosan capped gold nanoparticles, to engineer functional nanoparticles with a superior bactericidal effect compared to plain gold nanoparticles. Chitosan targets the crosslinked murein multilayers within bacteria, penetrating the plasma membrane. The resultant particles exhibited an overall positive charge, which interacted electrostatically with the negatively charged bacterial membrane, contributing to the bactericidal effect and the accumulation of deceased bacterial cells as shown in Figure 1 [14,15]. ChAuNPs conjugated with LPS-targeting peptides are an optimal choice for antibacterial applications due to their unique properties. The novelty of this work lies in the development of LPS targeting peptide sequence which was further conjugated onto gold nanoparticles (ChAuNPs) for antibacterial applications. The positive surface charge of the chitosan coating facilitates strong electrostatic interactions with the negatively charged bacterial cell wall, particularly the LPS layer in gram-negative bacteria. The targeted peptide conjugation enhances the binding efficiency of the nanoparticles to the bacterial cell wall along with 450 nm laser irradiation, leading to synergistic antibacterial effects. This approach offers a promising solution for combating multidrug-resistant bacteria, as it exploits the unique properties of chitosan and gold nanoparticles to create a targeted and efficient antibacterial agent. Additionally, the mechanism of action, which involves physical disruption of the bacterial cell membrane, makes it less likely for bacteria to develop resistance compared with traditional antibiotics [16].

Figure 1.

Figure 1.

Open in a new tab

Illustration of antibacterial efficacy showcased by ChAuNPs modified with LPS-targeting peptides coactivated in the presence of laser source. (Schematic representation was developed with the help of Biorender.com).

2. Materials & experimental methods

2.1. Materials

Sigma Aldrich supplied the Fmoc amino acids, propidium iodide (PI) and HAuCl4 along with dimethylformamide (DMF), diisopropyl carbodiimide (DIC), oxyma, dichloromethane (DCM), methanol, trifluoroacetic acid (TFA), thioanisole, ethanedithiol and anisole. Sisco Research Laboratories (SRL) provided acetic acid. Loba Chemie provided deacetylated chitosan from shrimp shells, and Luria Bertani (LB) broth and agar were provided by Hi Media. All materials were handled in their original condition, and their respective aqueous solutions were prepared in deionized water (DI).

2.2. Chitosan capped AuNP (ChAuNPs) synthesis

ChAuNPs were synthesized by dispersing deacetylated chitosan in 1% acetic acid solution [17]. The mixture was then probe-sonicated at 25A and centrifuged at 3000 rpm. The supernatant was then filtered using a 0.2 μm syringe filter and boiled with constant stirring, on to which 1 mM of HAuCl4 was added dropwise. The solution was kept on stirring for 1 hour at room temperature until stable-sized purplish colour particles were formed. The nanoparticles were further centrifuged at 15,000 rpm for 30 min at 4°C, which allowed the separation of particles based on their size. Following centrifugation, the particles were washed thrice to remove any unbound reactants and impurities. The resulting pellet was then resuspended in double-distilled water to ensure the removal of any residual contaminants and to facilitate further characterization of the ChAuNPs [18].

2.3. Peptide functionalization of chitosan AuNPs (ChAuNPs+P)

The sequence of amino acids referred to as “KNKSR” functions as an anchor to the LPS found in the outer layer of bacterial cell walls, ultimately leading to the disruption of the bacterial cell membrane [10]. In the experiment, 1 ml of ChAuNPs was mixed with 2 mM of EDC and NHS respectively, which were activated to facilitate a chemical reaction. Then, 1 mg of the peptide was carefully measured and added to the mixture, subsequently placed on a rotary shaker overnight. The peptide molecules were bound to the surface of the nanoparticles through the formation of amide bonds (Supplementary Figure S1). The protocol was adapted from Ho et al., where they chemically conjugated RGDS peptide with chitosan [19].

2.4. Characterization of the chitosan AuNPs

After synthesis, these nanoparticles were characterized by measuring UV-visible spectra using a dual-beam monochromator from SHIMADZU (UV-2600) using a quartz cuvette with a resolution of 1 nm between the wavelength 300 to 800 nm with a pathlength of 1 cm. The average hydrodynamic diameter based on the intensity and the polydispersity index (PDI) along with zeta potential were obtained from Malvern Zetasizer Nano ZSP. The average hydrodynamic diameter and PDI for the particles were obtained from the dynamic light scattering based study. Performed at a backscattering angle of 173° in a 12 mm disposable cuvette. Zeta potential was measured using disposable folded capillary cells. The experimentation was carried out at room temperature (RT ∼ 27°C) [20].

For High Resolution Transmission Electron Microscopy (HRTEM) and Selected Area Electron Diffraction (SAED) analysis, the diluted sample was drop cast onto a carbon-coated copper grid and then dried using a desiccator. It was then observed and imaged using a JEOL JEM-2100 transmission electron microscope operating at an acceleration voltage of 200 KV.

The chemical similarity between the ChAuNPs and ChAuNPs+P were determined using x-ray photoelectron spectroscopy (XPS). For this study, samples were drop cast on silicon wafers and examined using the x-ray photoelectron spectrophotometer Kα, Thermo Fisher Scientific, USA.

Fourier transformed infrared spectra (FTIR) of the nanoparticles were obtained between 4000 to 400 cm-1 using an ATR-FTIR system, Equinox 55 IR Spectrophotometer.

For x-ray diffraction (XRD) pattern of the nanoparticles, which was recorded via Bruker D8 Advance Eco diffractometer with CoKα radiation source with an operating voltage of 40 kV and a filament current of 25 mA. The concentrated samples were drop cast on silicon wafers for the analysis.

2.5. Internalization of chitosan AuNPs in bacteria as analyzed by a confocal laser scanning microscope

Gram-negative Escherichia coli DH5α bacteria were incubated with Rhodamine B (red fluorescent dye) loaded nanoparticles and peptide-functionalized nanoparticles in Luria Bertani (LB) broth and incubated for 4 h at 37°C in darkness. Subsequently, the bacterial sample was centrifuged at 5000 rpm and resuspended in phosphate buffer saline (PBS). The samples were then placed on a glass dish and examined using a confocal laser scanning microscope (Zeiss-LSM 880 with Aryscan) for imaging and analysis [21].

2.6. Determination of the antibacterial properties of the chitosan AuNPs

To promote the growth of E. coli bacteria, LB broth was inoculated with the bacteria and subsequently incubated at 37°C, at 180 revolutions per minute (rpm). After that, the bacterial culture was stripped of the culture medium by centrifugation, followed by at least three washes in a solution consisting of 0.85% sodium chloride. After determining the bacterial concentration at 600 nm (using an UV-Visible spectrophotometer), the sample was diluted in phosphate-buffered saline (PBS) to a final concentration of 1 × 107 CFU (Colony forming units)/ml. After preparing two different sets of bacterium samples, each one was treated with nanoparticles at a concentration ranging from 25 μg/ml to 250 μg/ml. One batch of these treatments was subjected to a blue laser source for five min. Both the samples were kept in the incubator for 18 h. Counting the CFUs in each treatment allowed us to determine the effect of ChAuNPs and ChAuNPs+P nanoparticles when they were exposed to the laser and when they were not. The study was carried out three-times, on three separate days and in triplicate each time.

2.7. SEM analysis of bacteria

Bacterial cultures at a concentration of 1 × 107 CFU/ml were inoculated into LB broth, followed by treatment with ChAuNPs and ChAuNPs+P nanoparticles in the presence and absence of laser irradiation. Subsequently, the bacterial culture was centrifuged at 5000 rpm. The resulting bacterial cells were subjected to triple washing with PBS and then fixed using 4% paraformaldehyde. The fixed bacterial cells underwent a dehydration process by sequential immersion in ethanol solutions at concentrations of 30, 50, 70, 80 and 90%, with each stage of incubation lasting for 10 min. Finally, the cells were immersed in 100% ethanol for 1 h. Following this, the samples were pelleted and drop-casted onto a substrate for scanning electron microscopy.

2.8. PI staining assay

Bacteria at 1 × 107 CFU/ml were maintained in LB broth along with 50 μg/ml of ChAuNPs and ChAuNPs+P nanoparticles followed by laser irradiation for 5 min to one set of treatment groups. The samples were then kept at 37°C and at 180 rpm for approximately the whole day (18 h). After being incubated, the samples were centrifuged at 5000 rpm. The pellet that resulted was then collected and resuspended in fresh LB broth. The bacterial sample was incubated with propidium iodide (PI) at 37°C for 20 min. The protocol was adapted [22] and modifications were made. These samples were then analyzed under a confocal laser scanning microscope (Zeiss-LSM 880 with Aryscan).

2.9. Nitro blue tetrazolium reduction method

E. coli cells were suspended in PBS at a 1 × 106 CFU/ml density. Different concentrations of ChAuNPs and ChAuNPs+P nanoparticles (250, 150, 50 and 25 μg/ml) were then introduced into the culture. One set of bacterial cells were then exposed to 450 nm laser irradiation. The bacterial culture was subsequently left to incubate overnight for 18 h at 37°C. Following this, 1 mg/ml solution of NBT was prepared and introduced to the bacterial cells treated with nanoparticles. Afterward, they were incubated with NBT for 30 min. The bacterial cells were centrifuged at a speed of 5000 rpm, after which the supernatant was extracted. The pellet was treated with DMSO and finally the absorbance of the samples was measured at 575 nm wavelength. This method was adapted from the work of Banerjee et al. [23].

2.10. Detection of intracellular reactive oxygen species

Intracellular reactive oxygen species (ROS) levels within bacterial cells were assessed utilizing dichlorofluorescein diacetate (DCFH-DA), following the method outlined by Lyon et al. [24] with slight modifications. Bacterial cultures (1 × 106 CFU/ml) were centrifuged at 5000 rpm to form pellets. These pellets were then suspended for 4 h at 37°C containing ChAuNPs and ChAuNPs+P nanoparticle treatment at different concentrations in PBS. After incubation, the cells were supplemented with 30 μg/ml DCFH-DA dye and incubated at 37°C for 30 min. Subsequently, the treated cultures were pelleted again and resuspended in PBS, and the fluorescence intensity produced by the cells at an excitation wavelength of 485 nm and an emission wavelength of 528 nm was captured under a confocal laser scanning microscope (Zeiss-LSM 880 with Aryscan).

3. Results

3.1. Synthesis & morphological studies of ChAuNPs

The synthesis of gold nanoparticles involved utilizing a chitosan solution performing two roles, functioning as both a reducing and stabilizing agent [25]. This dual role is crucial in ensuring the synthesis of uniformly dispersed nanoparticles, preventing aggregation attributed to van der Waal's interactions and Ostwald ripening. The interaction between AuCl4- ions and chitosan lead to a reduction of Au3+ to Au0 through a three-electron transfer mechanism [26]. This process is triggered by the oxidation of protonated amine groups on glucosamine units within chitosan. The observable transformation of the solution from yellow to colorless and finally to a majestic purple signified the successful synthesis of chitosan-coated gold nanoparticles. The yield percentage of ChAuNPs was observed to be 45.38%.

One of the fundamental analytical techniques used to characterize metal-based nanoparticles is UV-visible spectrophotometry. This method provides valuable insights into particle size and their overall stability [27,28]. In our study, we harnessed UV-visible spectrophotometry to assess the chitosan-gold structures with and without peptide coating. The acquired spectra revealed distinct surface plasmon resonance (SPR) peaks of around 530 nm, The red shift in the UV peak after conjugation of a peptide on chitosan nanoparticles is attributed to the interaction between the peptide and the chitosan backbone. This interaction caused a change in the electronic structure of the peptide, resulting in a shift of its absorption peak toward longer wavelengths (red shift) consistent with previous findings [29,30] shown in Figure 2A. Notably, the SPR peak revealed the presence of monodispersity in the synthesized particles following peptide conjugation. Specifically, the ChAuNPs sample exhibited an absorption maximum at 534 nm, while the ChAuNPs+P displayed a 4 nm redshift, with an absorption maximum at 538 nm. Due to gold nanoparticle's SPR properties, a photothermal effect is observed when exposed to light in the visible to infrared region [31]. When these nanoparticles are irradiated with a laser source, they induce a localized increase in temperature due to the conversion of light energy into heat. In the case of ChAuNPs sample exposed to a 450 nm laser source, a temperature rise of approximately 32°C was observed after 15 min of laser exposure (shown in Supplementary Figure S2).

Figure 2.

Figure 2.

Open in a new tab

Characterization studies of ChAuNPs and ChAuNPs+P. (A) UV-visible spectra of ChAuNPs and ChAuNPs+P. (B) The average hydrodynamic diameter of the ChAuNPs and ChAuNPs+P was determined using DLS. ChAuNPs and ChAuNPs+P showed a hydrodynamic diameter of 227 nm and 258 nm, respectively. (C) (i) TEM images of ChAuNPs demonstrating an average size of 11 nm. (ii) Size distribution graph based on the TEM particle size analysis of ChAuNPs (D) (i) TEM images of ChAuNPs+P demonstrating an average size of 22 nm and (ii) Size distribution graph based on the TEM particle size analysis of ChAuNPs+P (E) (i) High-resolution C1s XPS spectra of ChAuNPs and ChAuNPs+ P obtained in the carbon region (ii) High-resolution N1s XPS spectra of ChAuNPs and ChAuNPs+ P obtained in the nitrogen region. (F) Fourier transform infrared (FTIR) spectra of ChAuNPs and ChAuNPs+ P. (G) XRD and SAED patterns of ChAuNPs and ChAuNP+P. (i) The crystalline nature of the ChAuNPs is evident from the diffraction patterns observed at the (111), (200) and (220) planes. (ii) After peptide conjugation a decrease in the availability of the crystal planes due to structural rearrangements, resulted in only a prominent peak at the (111) plane. These characterization techniques confirmed the formation of ChAuNPs and peptide conjugation on them, respectively.

Furthermore, we employed DLS to determine the particles' average hydrodynamic diameters (Z Avg), which revealed that the particles were uniformly distributed in the solution, indicating a state of minimal agglomeration. as illustrated in Figure 2B. This analysis revealed the formation of stable particles with an average size of 227 nm and a polydispersity index of 0.33, indicating a favorable dispersion of the nanoparticles. The zeta potential of these structures was found to be approximately +42 mV (Supplementary Figure S3). Following peptide conjugation, the average particle size increased to 258 nm, with a polydispersity index of 0.3.

TEM images of the chitosan nanoparticles depicted the formation of spherical particles with an average size of 11 nm (Figure 2C (i) and (ii)). Remarkably, after peptide conjugation, the average particle size increased to 22 nm. Average particle size of each particle was calculated from the TEM images as shown in Figure 2D (i) and (ii).

XPS analysis shown in Figure 2E (i) and (ii) was used to depict and confirm the peptide functionalization on the chitosan nanoparticles, as well as to look into the chemical composition and identify similarities between the two nanostructures. Three C1s peaks, those were characteristic to chitosan, were found at 284, 286 and 288 eV. These peaks correspond to the C-C, C-OH and C=O groups. Peaks for N1s were found at 402 eV and 398 eV, which correspond to N-H and C-N, respectively. The establishment of an amide bond between the amine group of chitosan and the carboxyl group of the peptide was indicated by the positive shift in the C1s and N1s peaks, and the rise in C-N binding energy was observed between the amine group of chitosan and the carboxyl group of the peptide [32].

The FTIR spectroscopic analysis of ChAuNPs and ChAuNPs+P nanoparticles depicted in Figure 2F revealed significant distinctions between the ChAuNPs and ChAuNPs+P nanoparticles while also sharing essential characteristics. A distinct peak at 3332 cm-1 in ChAuNPs, indicating NH stretching from the inherent secondary amines in chitosan, was notably absent in the ChAuNPs+P spectra. Conversely, the spectra of ChAuNPs+P nanoparticles displayed an intensified peak at 1696 cm-1, suggesting notable C=O stretching due to the presence of carboxylic acid, confirming the chemical modification of the nanoparticle surface through peptide attachment. Additionally, an additional peak at 1625 cm-1 was observed, indicating C=C stretching vibrations, providing insights into the structural changes induced by the peptide on the gold nanoparticle surface.

Furthermore, similar bands at 1378 cm-1 and 1272 cm-1 were observed in both ChAuNPs and ChAuNPs+P samples, suggesting shared underlying structural features preserved in both nanoparticle types as illustrated in Figure 2F. The observed variations in the FTIR spectra may indicate the successful modification of ChAuNPs with peptides, leading to alterations in the surface chemistry of the nanoparticles. The absence of specific peaks in ChAuNPs+P and the emergence of new peaks suggest specific changes in the functional groups present on the nanoparticle surface. It's important to note that the presented results align with the work of Mohan et al., demonstrating the reproducibility and consistency of the observed spectral characteristics in the context of ChAuNPs and ChAuNP+P counterparts [30].

For XRD analysis, the ChAuNPs and peptide-functionalized ChAuNPs samples were drop-cast on a silicon wafer. The crystalline nature of the ChAuNPs was confirmed by the presence of similar diffraction patterns at the (111), (200) and (220) planes corelating with JCPDS card no 04–0784 and SAED analysis [33], as shown in Figure 2G (i) [34]. However, after the peptide was latched onto the surface of the ChAuNPs, the capping by the peptide decreased the availability of the crystal planes of the nanoparticles. As a result, only a prominent peak at the (111) plane was observed in the XRD pattern, as depicted in Figure 2G (ii). This change in the XRD profile suggests that the peptide functionalization altered the crystalline structure of the system and exposed crystal planes of the ChAuNPs, potentially due to the surface interactions between the peptide and the nanoparticles. The observations made from the XRD were further corroborated by the SAED analysis of the nanoparticles. The HRTEM images revealed a lattice fringe spacing of 0.24 nm (Supplementary Figure S4), corresponding to the spacing in the (111) plane of the face-centered cubic (fcc) lattice of gold. The SAED analysis, collected from a single nanoparticle, showed diffraction rings that could be assigned to the (111), (200) and (220) planes, indicating the crystalline nature of the nanoparticles. However, after the attachment of the peptide to these nanoparticles, only the (111) crystalline plane was observed, suggesting structural change or rearrangement upon peptide functionalization [35,36].

3.2. Uptake & internalization of ChAuNPs in bacteria

Rhodamine loaded ChAuNPs (Rb-ChAuNPs) and ChAuNPs +P (Rb-ChAuNPs+P) interacted with E. coli cells through specific mechanisms based on their design and surface functionalization. The LPS targeting ChAuNP+P nanoparticles interacted with E. coli through the peptide attached on their surface, facilitating higher internalization. The peptide can target the LPS layer on the bacterial surface, leading to enhanced nanoparticle-bacterial cell interactions that may disrupt membrane integrity, interfere with cellular processes, or trigger specific responses within the bacterial cell. Overall, the interactions of these ChAuNPs+P nanoparticles with bacteria are multifaceted and can involve various pathways, including surface binding, internalization using LPS targeting peptides and potential modulation of cellular functions, ultimately influencing the bacteria's behavior and viability. As a result of its distinctive affinity for the bacterial membrane, an increased uptake of Rb-ChAuNPs+P was observed when compared with Rb-ChAuNPs, as illustrated in Figure 3.

Figure 3.

Figure 3.

Open in a new tab

Confocal laser scanning microscopic data showing the nanoparticle uptake in E. coli DH5α cells after they were incubated with the red-fluorescent, Rhodamine B tagged ChAuNPs (Rb-ChAuNPs) and ChAuNPs+P NPs (Rb-ChAuNPs+P). (Scale bar indicates 2 μm). (Schematic representation was developed with the help of Biorender.com).

3.3. Determination of the anti-microbial effect of the nanoparticles

To evaluate the bactericidal efficacy of the NPs, we subjected the bacterial suspension to a series of dilutions and spread it onto LB agar plates as illustrated in Figure 4. Subsequently, the colony-forming units (CFUs) were counted and compared with those from the control group. Notably, both the ChAuNPs and ChAuNPs+P groups exhibited a pronounced reduction in CFUs count. Furthermore, when both sets were exposed to a 450 nm laser, a further decrease in CFU count was evident due to localized heat production in the presence of the nanoparticles, which target and degrade bacterial membranes, leading to cellular damage

Figure 4.

Figure 4.

Open in a new tab

Graphical representation of the spread plate assay performed in E. Coli DH5α cells treated with ChAuNPs and ChAuNPs+P at varying concentrations (25, 50, 150 and 250 μg/ml) in the presence and absence of a 450 nm laser (Schematic representation was developed with the help of Biorender.com).

As shown in Figure 5 A & B. At the lowest concentration of 25 μg/ml, the average CFU values for ChAuNPs, both in the absence and presence of laser irradiation, were quantified as 136 ± 13 and 103 ± 6, respectively. Similarly, for ChAuNPs+P, the average CFU values, in the absence and presence of laser irradiation, were found to be 81 ± 3 and 69 ± 4, respectively.

Figure 5.

Figure 5.

Open in a new tab

Spread plate assay results demonstrating the antibacterial efficacy of ChAuNPs and ChAuNPs+P nanoparticles. (A) Spread plate images of E. coli DH5α cells treated with ChAuNPs and ChAuNPs+P at varying concentrations (25, 50, 150 and 250 μg/ml). The spread plate method detected a notable decrease in the CFUs after the bacterial cells were treated with ChAuNPs+P. This reduction in CFUs was observed both in the presence and absence of a 450 nm laser, with an enhanced reduction in the laser group indicating the antibacterial efficacy of these nanoparticles against E. coli DH5α cells across different concentrations and treatment conditions. (B) The results were further visualized in a bar graph representing the mean values of the counted colonies. A two-way ANOVA analysis followed by Dunnett's test was employed to assess the levels of significance between control groups and other treated samples, with the results presented as mean ± SD. The p values were considered significant at *p < 0.05 and **p < 0.01, while a non-significant (ns) result was indicated by no difference in the growth inhibition between the control and treated samples.

3.4. SEM analysis of treated bacteria

The antibacterial effects of ChAuNPs and ChAuNPs+P nanoparticles were further investigated using the spread plate method and SEM analysis.

The results of SEM analysis also showed a significant reduction in the bacterial count after treatment with ChAuNPs and ChAuNPs+P, both with and without laser exposure (Figure 6). The laser irradiation further enhanced the antibacterial effect, indicating a synergistic impact of the nanoparticles combined with laser sources on the bacteria. The reduction in bacterial count was consistent with the observations from SEM analysis, which showed elongated structures in bacteria and a simultaneous decrease in bacterial count following nanoparticle treatment with laser exposure.

Figure 6.

Figure 6.

Open in a new tab

Scanning electron microscopic (SEM) images illustrate the interaction between bacteria and ChAuNPs and ChAuNPs+P. The presence of elongated structures, coupled with a concurrent reduction in bacterial count, suggests the combined effect of these nanoparticles with the laser source on E. coli DH5α cells. (Scale bars indicate 1 μm).

3.5. Estimation of bacterial cell death through PI staining assay in nanoparticle treated bacteria

Live/dead assay was performed using propidium iodide (PI) to evaluate the antibacterial effectiveness of ChAuNPs and ChAuNPs+P nanoparticles at a concentration of 50 μg/ml, under conditions with or without a 450 nm laser source. Notably, bacterial cells showed even dispersion across the surface in the control groups and demonstrated motility.

When exposed to the laser, some cells in the control samples demonstrated cell death by picking up the PI stain, showing the presence of more live cells than dead cells, whereas the treated samples revealed evidence of more dead cells and less viable cells.

Upon introducing ChAuNPs to the bacterial cells, a reduction in motility was observed, paralleled with an increase in the red fluorescence intensity, signifying the ChAuNPs' bactericidal properties (Supplementary Video). The most substantial increase in the red fluorescence intensity was observed in the ChAuNPs+P nanoparticles, which additionally displayed larger aggregates, as shown in Figure 7A & B. It can be inferred here that the conjugation of a lipopolysaccharide targeting peptide sequence onto the nanoparticles enabled heightened interaction of the particles with the bacterial surface and simultaneously amplified the bactericidal attributes of the ChAuNPs. Furthermore, it was noted that bacterial cells exhibited heightened aggregation in the presence of the laser source, particularly in the context of ChAuNPs+P nanoparticles, which would further enhance their antibacterial activity.

Figure 7.

Figure 7.

Open in a new tab

The propidium iodide (PI) assay outcomes, as demonstrated through confocal laser scanning microscopy images. The results reveal the cytotoxic effects on E. coli cells following their exposure to ChAuNPs and ChAuNPs+P nanoparticles under two distinct conditions: (A) in the absence of laser irradiation and (B) under 450 nm laser exposure. (Scale bars indicate 10 μm). The cells treated with nanoparticles and laser irradiation exhibited a more intense red fluorescence, suggesting a greater degree of cell death compared with control groups.

3.6. Assessment of intracellular ROS production ability of the NPs

All aerobic organisms in limited quantities produce ROS to facilitate fundamental cellular processes, but in presence of ChAuNPs any enhancement in the degree of ROS generation in the E. coli cells was examined using DCFH-DA staining. Cellular esterases hydrolyze the diacetate group, forming the DCFH group, which is subsequently oxidized to fluorescent DCF in the presence of intracellular ROS. The intracellular ROS production efficacy of ChAuNPs and ChAuNPs+P at a concentration of 50 μg/ml, was assessed both in the presence and absence of a 450 nm laser source. The cellular imaging of treated bacterial cells was performed as shown in Figure 8A.

Figure 8.

Figure 8.

Open in a new tab

Representation of intracellular ROS produced by chitosan nanoparticles after interaction with E. coli DH5α. (A) DCFH-DA staining represented via confocal laser scanning microscopic images. Results showing enhanced green fluorescence in the nanoparticle and laser-treated cells (ChAuNPs and ChAuNPs) confirming significant ROS production (Scale bars indicate 5 μm) (B) Graphical representation of the heat map generated from the quantified fluorescence intensity of the confocal images of the DCFH-DA stained bacteria. Results display the relative levels of the ROS produced within the bacteria. (C) NBT assay results showcase the influence of ChAuNPs and ChAuNPs+P nanoparticles on bacterial intracellular ROS production, along with the presence and absence of laser sources at various concentrations and treatment conditions. Both assays demonstrated an increased production of ROS when bacterial cells were exposed to a combination of nanoparticle and laser treatment in comparison to control groups. A two-way ANOVA analysis followed by Dunnett's test was employed to assess the levels of significance between control groups and other treated samples, with the results presented as mean ± SD. The p values were considered significant at *p < 0.05 and **p < 0.01, while a non-significant (ns).

In the laser-irradiated groups, particularly an elevated fluorescence intensity was observed compared with the non-irradiated group as shown in Figure 8A & B. Additionally, following nanoparticle treatment, the formation of cellular aggregates was observed. Especially, the highest fluorescence intensity was detected in the laser-irradiated group treated with ChAuNPs+P nanoparticles.

The NBT assay was carried out to assess the generation of free oxygen radicals by the nanoparticles, both in the presence and absence of a 450 nm laser source. Concentrations ranging from 25 to 250 μg/ml were tested. Results revealed a heightened concentration-dependent absorbance intensity of the sample at 575 nm in the presence of the laser source. Especially, ChAuNPs+P exhibited a greater NBT dye reduction, leading to an increased absorbance intensity compared with ChAuNPs alone, as shown in Figure 8C.

4. Discussion

Chitosan based nanoparticles are made up of the biopolymer coating making them suitable for various biomedical applications due to their biocompatibility, biodegradability and ability to encapsulate and deliver bioactive molecules. These nanoparticles can be created utilizing a variety of processes, including ionic gelation, emulsion crosslinking and solvent evaporation [37], to produce particles of specified sizes and surface qualities Chitosan based nanoparticles are reported to have the potential biological applications like gene delivery, tissue engineering, cancer therapy, immunomodulation, etc. [37] Chitosan based nanoparticles can attach to DNA and prevent it from being degraded by nucleases, increasing DNA's residence time in the gastrointestinal system [38]. They have been exploited as gene delivery vehicles for a variety of medicinal purposes Chitosan based nanoparticles were demonstrated to have antiviral capabilities against a variety of viruses, including HIV and herpes simplex virus. They have been utilized in the creation of antiviral medications for the treatment of viral illnesses [38]. They have served as immunoadjuvants in vaccine development [39]. Our current work involves the synthesis bacterial LPS targeting peptide and chitosan capped antibacterial gold nanoparticles as potential anti-microbial agent. The gold nanoparticles were synthesized using chitosan serving dual functions: reduction and overall stabilization of the particles. This dual role is critical for assuring the creation of uniformly dispersed nanoparticles. The visible transition of the solution from yellow to colorless and then to a majestic purple color indicated the effective synthesis of ChAuNPs. The synthesized ChAuNPs, were further functionalized with a lipopolysaccharide (LPS) targeting peptide sequence through a covalent bond formation. This was achieved via EDC-NHS chemistry, where the carboxyl group of the peptide sequence reacted with the amine group of chitosan present on the ChAuNPs, resulting in the formation of an amide bond. This conjugation process allowed the peptide sequence to be specifically attached to the surface of the ChAuNPs, enabling targeted interactions with LPS on the bacterial surface.

The synthesised nanoparticles were then characterized using various characterization techniques which pronounced about various properties of these nanoparticles. Like the UV-visible spectrophotometry was used to assess the probable shape and size of ChAuNPs after peptide coating. The acquired spectra revealed a distinct SPR peak of around 530 nm. The interaction between the peptide and the chitosan backbone was responsible for the red shift in the UV peak that occurred following peptide conjugation to the ChAuNPs. Several implications can be derived from the red shift of the UV-Vis peak observed following peptide conjugation to the gold nanoparticles. The red shift implies good conjugation, conjugate stability and binding efficiency. It also indicates surface modification on the nanoparticles and colloidal stability. The magnitude of the red shift indicates binding efficiency, whereas the distinct UV-Vis spectra reveal conjugate stability.

Dynamic light scattering was used to determine the particles' average hydrodynamic diameters, agglomeration state as well as the surface charge of the nanoparticles, which revealed that the particles were uniformly distributed in the solution, indicating a state of minimal agglomeration. The surface zeta potential of these structures measured approximately +42 mV. Following peptide conjugation, the average particle size increased to 258 nm, with a polydispersity index of 0.3. Peptide conjugation to ChAuNPs lead to some changes in the nanoparticles' hydrodynamic size The hydrodynamic diameter of the nanoparticles showed an upward trend, indicating an increase in the overall particle size. This increase in size was most likely caused by the peptide's conjugation on the nanoparticles' surfaces,

TEM images of the chitosan nanoparticles depicted the formation of spherical particles with an average size of 11 nm. Remarkably, after peptide conjugation, the average particle size increased to 22 nm.

XPS analysis was utilized to confirm the peptide functionalization on the chitosan capped nanoparticles, as well as to investigate the chemical composition and detect commonalities between the two nanostructures. The change in binding energy of the nanoparticles following peptide conjugation, as determined by XPS, suggests that the peptide modification had a considerable impact on the nanoparticles' binding characteristics. The XPS study demonstrated a change in binding energy, showing that the binding energy the ChAuNPs shifted during peptide conjugation. This shift in binding energy peaks indicated that the peptide molecules interacted with the nanoparticles, modifying their surface characteristics and increasing their binding affinity to target bacterial molecules or surfaces.

The FTIR spectroscopic investigation of ChAuNPs and ChAuNPs+P nanoparticles revealed considerable differences between the nanoparticles while sharing important particle properties. The spectrum variations and comparison of the FTIR spectra confirmed the peptide's attachment to the ChAuNPs via an amide bond. This bonding resulted in a conjugate complex that was stable and was unaffected by other biomolecules. The observed modifications in the FTIR spectra may imply that ChAuNPs were successfully modified with the peptide, resulting in changes to the nanoparticles' surface chemistry. The lack of specific peaks in ChAuNPs+P and the appearance of new peaks indicate specific alterations in the functional groups on the nanoparticle surface.

The XRD and SAED analysis of the ChAuNPs revealed similar diffraction planes as previously reported by our group and [33] K. Saravanakumar et al., for gold nanostuctures [34]. After peptide conjugation dimimishing of prominent XRD peaks as well reduction in the electron diffraction intensity was obseved. Several variables contribute to the absence of strong XRD peaks following peptide coating on ChAuNPs. The peptide coating on the surface of ChAuNPs can reduce the availability of crystal planes, resulting in a decrease or absence in the intensity of XRD peaks. This is because peptide molecules can bond to the gold surface, decreasing the crystal planes' exposure to x-ray radiation. Furthermore, peptide coating might alter the crystal structure of the gold nanoparticles, causing peak positions to shift or broaden, resulting in a drop in XRD peak intensity.

After confirming thepeptide conjugation with the ChAuNPs these nanoparticles were investigated for their uptake, antibacterial and ROS generating abilities. These ChAuNPs and ChAuNPs+P nanoparticles were loaded with a fluorescent dye named Rhodamine B to aid in their cellular detection. The results displayed a higher intensity of fluorescence from the bacterial cells when incubated with ChAuNPs+P nanoparticles in comparison to only ChAuNPs. This proved that the positively charged ChAuNPs+P nanoparticles demonstrated a higher surface interaction with the gram-negative bacterium, E. coli when compared with only ChAuNPs. The peptide sequence can target the LPS layer on the bacterial surface, leading to nanoparticle interactions that may disrupt membrane integrity, interfere with cellular processes, or trigger specific responses within the bacterial cells.

The 450 nm laser also played an important role in determining the antibacterial effect of these nanoparticles. The precise control of the photothermal effect allowed targeted antibacterial applications, where the heat generated by the nanoparticles can selectively damage bacterial cells while minimizing harm to surrounding healthy tissues. Liu et al. performed experiments involving the irradiation of diverse metallic nanoparticles using visible light laser sources. They observed that when AuNPs were exposed to a 450 nm laser source, there was an increase in temperature of 11.4°C which was higher than silver nanoparticles (AgNPs) and silver nanowires (AgNWs) tested in the study [8]. De Sousa et al., demonstrated the antibacterial effect of blue laser sources, they analyzed the results on three bacterial strains such as Pseudomonas aeruginosa, Escherichia coli and Staphylococcus aureus. Their research findings indicated that blue laser light sources can impede bacterial growth consistently, unaffected by time variations [16]. In case of ChAuNPs, after 15 min of exposure to the 450 nm laser, ChAuNPs showed a temperature increase of around 32°C. To be precise the temperature of the nanoparticles reached from 27°C to 60°C.

These nanoparticles were then checked for their antibacterial effects via colony forming assay, SEM analysis and PI staining assay. E. coli cells were treated with varied doses ChAuNPs and ChAuNPs+P nanoparticles, ranging from 25 μg/ml to 250 μg/ml, in the presence and absence of a 450 nm laser source. When exposed to the laser source, the ChAuNPs+P treated bacterial group formed the fewest or no colonies, aiding to our hypothesis. The addition of the LPS-targeting peptide, which aided the ChAuNPs internalization, accounts for the increased binding effectiveness of the ChAuNPs+P group to bacterial cells. The combination of the 450 nm laser source and the ChAuNPs+P nanoparticles instigated photothermal ablation of the bacterial cells, which resulted in a considerable reduction in colony development. Similar results were also observed in the SEM analysis which revealed a substantial reduction in bacterial count after treatment with Chitosan- ChAuNPs and ChAuNPs+P, both in the presence and absence of laser irradiation. The laser exposure significantly enhanced the antibacterial effect of the particles as mentioned earlier, indicating a synergistic effect of the nanoparticles and laser on the bacteria. This synergistic effect was consistent with the SEM observations, which showed elongated structures within the bacteria and a concurrent decrease in bacterial count following nanoparticle treatment with laser exposure.

The synergistic effect of ChAuNPs+P nanoparticles and laser exposure has been observed to enhance antibacterial activity against E. coli. This synergistic effect is comparable to other nanoparticle-laser combinations that have been studied in this article for their antimicrobial properties. Additionally, the influence of photothermal therapy (PTT) using nanoparticles has also been demonstrated in PI staining assay. Where the bacterial cells were stained with PI dye which is a widely used stain of detecting dead cells as they permeate through the dead cell membrane and intercalate to DNA/RNA leading them to fluoresce in the red region [40]. Our findings demonstrated significant changes in bacterial cells after treatment ChAuNPs and ChAuNPs+P in the presence and absence of a 450 nm laser. Specifically, we found a significant increase in the red fluorescence and a consistent decrease in bacterial motility when they were treated with ChAuNPs+P compared with ChAuNPs. This substantiates our hypothesis that the conjugation of the peptide moiety to the ChAuNPs, boosted their antibacterial efficacy while inducing cellular damage, as indicated by increased red fluorescence and decreased motility. The presence of the 450 nm laser amplified these effects, implying a synergistic interaction between the nanoparticles and laser source which can lead to enhanced antimicrobial and therapeutic effects.

As per literature sources, metallic nanoparticles also exhibit a heightened tendency to generate elevated levels of ROS [41]. Owing to their robust oxidation potential, the additional ROS generated by the nanoparticles can impair biomolecules and organelle constituents [42]. This can result in oxidative carbonylation of proteins, peroxidation of fats, DNA/RNA fragmentation and disruption of membrane structures, ultimately contributing to increased necrosis and apoptosis [43].Thus we determined the ROS generation capabilities of the ChAuNPs ChAuNPs+P nanoparticles under laser exposure using DCFH-DA a fluorescent probe and NBT a dark blue dye. In DCFH-DA assay, the intracellular ROS production causes the bacterial cells to fluoresce in the green region, similar observation was recorded, the ChAuNPs+P laser treated bacterial cells fluoresced brighter and intense compared with all other groups. NBT assay also concluded similar results where these nanoparticles were externally checked for ROS production. A concentration-dependent increase in absorbance intensity at 575 nm was further observed when the bacterial cells were exposed to the nanoparticles in the presence of a laser source. Notably, the ChAuNPs+P exhibited a more pronounced reduction of the NBT dye, resulting in a significantly higher absorbance intensity compared with the ChAuNPs alone. This enhanced NBT dye reduction by the ChAuNPs+P nanoparticles suggest a greater generation of superoxide radicals, which can lead to oxidative stress and ultimately contribute to the observed antibacterial effects.

These above findings concluded that the synergistic combination of the peptide functionalization and laser exposure appears to amplify the generation of reactive oxygen species, potentiating the antibacterial activity of the ChAuNPs in a concentration-dependent manner.

5. Conclusion

In the current study, we synthesized gold nanoparticles using chitosan as a reducing and stabilizing agent. Chitosan, a positively charged biopolymer, played a crucial role in the environment friendly synthesis of these nanoparticles, imparting an overall positive charge that facilitated their binding to gram-negative bacteria. Various characterization techniques such as DLS, TEM, UV-visible spectroscopy, FTIR, XPS and XRD confirmed the formation of these nanoparticles.

The ChAuNPs exhibited notable antibacterial properties against E. Coli. Furthermore, a peptide that targeted the bacterial lipopolysaccharide (LPS) layer was chemically conjugated with the ChAuNPs. The LPS targeting sequence enhanced the cellular internalization of these nanoparticles.

These nanoparticles have been shown to exert multifaceted effects on bacteria, contributing to their antibacterial properties. The positive charge of the nanoparticles enables charge-based interactions with gram-negative bacteria, while the addition of an LPS-targeting peptide further enhances the binding affinity of the nanoparticles toward the bacteria. The metallic component of the nanoparticles plays a dual role in generating ROS and inducing a photothermal effect under laser irradiation. The combination of these effects, including charge-based interactions, increased binding affinity, ROS production and photothermal effect, lead to a heightened antibacterial response and increased cell death observed in the case of both ChAuNPs and peptide-functionalized ChAuNPs, with enhanced photothermal-based cell killing effect witnessed under exposure to a 450 nm laser source.

The results indicated that the combination of a positively charged nanoparticle and a peptide targeting LPS, along with laser activation, can give rise to a prospective framework with antibacterial activity, highlighting the potential of this approach for generating potential antibacterial agents.

Supplementary Material

Supplementary Figures S1-S4
INNM_A_2382073_SM0001.docx (2.1MB, docx)
Supplementary Video
INNM_A_2382073_SM0002.zip (1.2GB, zip)

Acknowledgments

We would like to thank BioRender.com for generously offering their exceptional templates, enabling the creation of professional and visually appealing scientific schematics.

Funding Statement

The authors express gratitude for the support provided by the ICMR extramural grant (IIRPIG-2023-0000790), the SERB-POWER grant from the Department of Science and Technology (DST) India (SPG/2021/002910), the Council of Scientific and Industrial Research (CSIR) India (37/1755/23/EMR-II), the Department of Biotechnology (DBT), India (BT/PR36632/NNT/28/1694/2020; BT/13/IYBA/2020/08).

Supplemental material

Supplemental data for this article can be accessed at https://doi.org/10.1080/17435889.2024.2382073

Author contributions

S Choudhury: conceptualization, methodology, software, validation, formal analysis, investigation, resources, data curation, writing - original draft, writing - review and editing. A Mehra: conceptualization, methodology, writing - review and editing. S Srivastava: formal analysis, investigation, resources, data curation, writing - review and editing. M Sharma: conceptualization, formal analysis, writing - review and editing. M Singh: conceptualization, methodology, investigation, supervision, writing - original draft, writing - review and editing. JJ Panda: conceptualization, methodology, software, validation, formal analysis, investigation, resources, data curation, writing - original draft, writing - review and editing, visualization, supervision, project administration, funding acquisition.

Financial disclosure

The authors express gratitude for the support provided by the ICMR extramural grant (IIRPIG-2023-0000790), the SERB-POWER grant from the Department of Science and Technology (DST) India (SPG/2021/002910), the Council of Scientific and Industrial Research (CSIR) India (37/1755/23/EMR-II), the Department of Biotechnology (DBT), India (BT/PR36632/NNT/28/1694/2020; BT/13/IYBA/2020/08). The authors have no financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Competing interests disclosure

The authors have no competing interests or relevant affiliations with any organization or entity with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Writing disclosure

No writing assistance was utilized in the production of this manuscript.

References

Papers of special note have been highlighted as: • of interest; •• of considerable interest

  • 1.Slavin YN, Asnis J, Häfeli UO, et al. Metal nanoparticles: understanding the mechanisms behind antibacterial activity. J Nanobiotechnology BioMed Central Ltd. 2017;15:1–20. doi: 10.1186/s12951-017-0308-z [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Lee NY, Ko WC, Hsueh PR. Nanoparticles in the treatment of infections caused by multidrug-resistant organisms. Front Pharmacol Frontiers Media SA. 2019;10:1153. doi: 10.3389/fphar.2019.01153 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Stensberg MC, Wei Q, McLamore ES, et al. Toxicological studies on silver nanoparticles: challenges and opportunities in assessment, monitoring and imaging. Nanomedicine. 2011;6(5):879–898. doi: 10.2217/nnm.11.78 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Frei A, Verderosa AD, Elliott AG, et al. Metals to combat antimicrobial resistance. Nat Rev Chem Nature Research. 2023;7(3):202–244. doi: 10.1038/s41570-023-00463-4 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Yuan P, Ding X, Yang YY, et al. Metal nanoparticles for diagnosis and therapy of bacterial infection. Adv Healthc Mater Wiley-VCH Verlag. 2018;7(13):1701392. doi: 10.1002/adhm.201701392 [DOI] [PubMed] [Google Scholar]
  • 6.Pereira SO, Barros-Timmons A, Trindade T. Polymer@gold nanoparticles prepared via RAFT polymerization for opto-biodetection. Polymers MDPI AG. 2018;10(2):189. doi: 10.3390/polym10020189 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Shan J, Tenhu H. Recent advances in polymer protected gold nanoparticles: synthesis, properties and applications. Chem Commun Royal Soc Chem. 2007;4580–4598. doi: 10.1039/b707740h [DOI] [PubMed] [Google Scholar]
  • 8.Liu X, Shan G, Yu J, et al. Laser heating of metallic nanoparticles for photothermal ablation applications. AIP Adv. 2017;7:7. doi: 10.1063/1.4977554 [DOI] [Google Scholar]
  • 9.Wei D, Ye Y, Jia X, et al. Chitosan as an active support for assembly of metal nanoparticles and application of the resultant bioconjugates in catalysis. Carbohydr Res. 2010;345:74–81. doi: 10.1016/j.carres.2009.10.008 [DOI] [PubMed] [Google Scholar]; • This study is of interest as it discusses the use of chitosan as a reducing agent in the synthesis of metal nanoparticles, which is a key component of our manuscript.
  • 10.Ghosh A, Datta A, Jana J, et al. Sequence context induced antimicrobial activity: insight into lipopolysaccharide permeabilization. Mol Biosyst. 2014;1596–1612. doi: 10.1039/C4MB00111G [DOI] [PubMed] [Google Scholar]; •• This study is of considerable interest as it explores the LPS targeting properties of the peptide sequence, which is a key aspect of our research.
  • 11.Rajchakit U, Sarojini V. Recent developments in antimicrobial-peptide-conjugated gold nanoparticles. Bioconjug Chem American Chemical Society. 2017;28(11):2673–2686. doi: 10.1021/acs.bioconjchem.7b00368 [DOI] [PubMed] [Google Scholar]; •• This study is of considerable interest as it highlights the potential of gold nanoparticles in antimicrobial applications, which is a key area of research in our manuscript.
  • 12.Barreto-Santamaría A, Arévalo-Pinzón G, Patarroyo MA, et al. How to combat gram-negative bacteria using antimicrobial peptides: a challenge or an unattainable goal? Antibiotics MDPI. 2021;10(12):1499. doi: 10.3390/antibiotics10121499 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Rai A, Pinto S, Velho TR, et al. One-step synthesis of high-density peptide-conjugated gold nanoparticles with antimicrobial efficacy in a systemic infection model. Biomaterials. 2016;85:99–110. doi: 10.1016/j.biomaterials.2016.01.051 [DOI] [PubMed] [Google Scholar]
  • 14.Sokary R, Abu el-naga MN, Bekhit M, et al. A potential antibiofilm, antimicrobial and anticancer activities of chitosan capped gold nanoparticles prepared by γ–irradiation. Mater Technol. 2020;37:493–502. doi: 10.1080/10667857.2020.1863555 [DOI] [Google Scholar]; • This study is of interest as it discusses the antibiofilm properties of chitosan capped gold nanoparticles which is a key component of our manuscript.
  • 15.Yan D, Li Y, Liu Y, et al. Antimicrobial properties of chitosan and chitosan derivatives in the treatment of enteric infections. Molecules MDPI. 2021;7136. doi: 10.3390/molecules26237136 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Teixeira N, De Sousa A, Santos MF, et al. Blue laser inhibits bacterial growth of Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa. Photomed Laser Surg. 2015;33(5):278–282. doi: 10.1089/pho.2014.3854 [DOI] [PMC free article] [PubMed] [Google Scholar]; •• This study is of considerable interest as it highlights the potential of 450 nm laser source on various bacterial strains, which is a key area of research in our manuscript.
  • 17.Thanayutsiri T, Patrojanasophon P, Opanasopit P, et al. Rapid synthesis of chitosan-capped gold nanoparticles for analytical application and facile recovery of gold from laboratory waste. Carbohydr Polym. 2020;250:116983. doi: 10.1016/j.carbpol.2020.116983 [DOI] [PubMed] [Google Scholar]
  • 18.Shaabani E, Sharifiaghdam M, De Keersmaecker H, et al. Layer by layer assembled chitosan-coated gold nanoparticles for enhanced siRNA delivery and silencing. Int J Mol Sci. 2021;22:1–23. doi: 10.3390/ijms22020831 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Ho MH, Wang DM, Hsieh HJ, et al. Preparation and characterization of RGD-immobilized chitosan scaffolds. Biomaterials. 2005;26:3197–3206. doi: 10.1016/j.biomaterials.2004.08.032 [DOI] [PubMed] [Google Scholar]
  • 20.Dube T, Kumar N, Bishnoi M, et al. Dual blood-brain barrier-glioma targeting peptide-poly(levodopamine) hybrid nanoplatforms as potential near infrared phototheranostic agents in glioblastoma. Bioconjug Chem. 2021;9:2014–2031. doi: 10.1021/acs.bioconjchem.1c00321 [DOI] [PubMed] [Google Scholar]; • This study is of interest as it discusses the manual synthesis of peptide sequence and its conjugation with metal nanoparticles which is a key component of our manuscript.
  • 21.Białas N, Sokolova V, van der Meer SB, et al. Bacteria (E. coli) take up ultrasmall gold nanoparticles (2 nm) as shown by different optical microscopic techniques (CLSM, SIM, STORM). Nano Sel. 2022;3:1407–1420. doi: 10.1002/nano.202200049 [DOI] [Google Scholar]
  • 22.Tong C, Li L, Xiao F, et al. Daptomycin and AgNP co-loaded rGO nanocomposites for specific treatment of Gram-positive bacterial infection in vitro and in vivo. Biomater Sci. 2019;7:5097–5111. doi: 10.1039/C9BM01229J [DOI] [PubMed] [Google Scholar]
  • 23.Banerjee M, Mallick S, Paul A, et al. Heightened reactive oxygen species generation in the antimicrobial activity of a three component iodinated chitosan?silver nanoparticle composite. Langmuir. 2010;26:5901–5908. doi: 10.1021/la9038528 [DOI] [PubMed] [Google Scholar]; • This study is of considerable interest as it explores the ROS producing properties of chitosan-coated nanoparticles.
  • 24.Lyon DY, Brunet L, Hinkal GW, et al. Antibacterial activity of fullerene water suspensions (nC 60) is not due to ROS-mediated damage. Nano Lett. 2008;8:1539–1543. doi: 10.1021/nl0726398 [DOI] [PubMed] [Google Scholar]
  • 25.Li S-T, Xi L, Shi W-K, et al. The synthesis of biocompatible and SERS-active gold nanoparticles using chitosan. Nanotechnology. 2009;20:315602. doi: 10.1088/0957-4484/20/31/315602 [DOI] [PubMed] [Google Scholar]
  • 26.Simeonova S, Georgiev P, Exner KS, et al. Kinetic study of gold nanoparticles synthesized in the presence of chitosan and citric acid. Colloids Surfaces A Physicochem Eng Asp. 2018;557:106–115. doi: 10.1016/j.colsurfa.2018.02.045 [DOI] [Google Scholar]
  • 27.Qian LH, Yan XQ, Fujita T, et al. Surface enhanced Raman scattering of nanoporous gold: smaller pore sizes stronger enhancements. Appl Phys Lett. 2007;90. doi: 10.1063/1.2722199 [DOI] [Google Scholar]
  • 28.Noginov MA, Zhu G, Bahoura M, et al. The effect of gain and absorption on surface plasmons in metal nanoparticles. Appl Phys B Lasers Opt. 2007;86:455–460. doi: 10.1007/s00340-006-2401-0 [DOI] [Google Scholar]
  • 29.De Matteis V, Rizzello L, Cascione M, et al. Sustainable synthesis of FITC chitosan-capped gold nanoparticles for biomedical applications. Clean Technol. 2022;4:942–953. doi: 10.3390/cleantechnol4040058 [DOI] [Google Scholar]
  • 30.Mohan CO, Gunasekaran S, Ravishankar CN. Chitosan-capped gold nanoparticles for indicating temperature abuse in frozen stored products. NPJ Sci Food. 2019;3(1):2. doi: 10.1038/s41538-019-0034-z [DOI] [PMC free article] [PubMed] [Google Scholar]; • Focuses on the biological application of chitosan coated gold nanoparticles which helps us to determine its biocompatibility, which is a key component of our manuscript.
  • 31.Yang T, Wang D, Liu X. Assembled gold nanorods for the photothermal killing of bacteria. Colloids Surf. B Biointerfaces. 2019;173:833–841. doi: 10.1016/j.colsurfb.2018.10.060 [DOI] [PubMed] [Google Scholar]
  • 32.Das SK, Dickinson C, Lafir F, et al. Synthesis, characterization and catalytic activity of gold nanoparticles biosynthesized with Rhizopus oryzae protein extract. Green Chem. 2012;14:1322–1334. doi: 10.1039/c2gc16676c [DOI] [Google Scholar]
  • 33.Dube T, Kumar N, Kour A, et al. Gold nano-/microroses on levodopa microtubes for SERS-based sensing of gliomas. ACS Appl Nano Mater. 2019;2:2663–2678. doi: 10.1021/acsanm.9b00155 [DOI] [Google Scholar]
  • 34.Saravanakumar K, Mariadoss AVA, Sathiyaseelan A, et al. Synthesis and characterization of nano-chitosan capped gold nanoparticles with multifunctional bioactive properties. Int J Biol Macromol. 2020;165:747–757. doi: 10.1016/j.ijbiomac.2020.09.177 [DOI] [PubMed] [Google Scholar]
  • 35.Goswami AM, Ghosh S. Biological synthesis of colloidal gold nanoprisms using Penicillium citrinum MTCC9999. J Biomater Nanobiotechnol. 2013;04:20–27. doi: 10.4236/jbnb.2013.42A003 [DOI] [Google Scholar]
  • 36.Sun D, Zhang G, Huang J, et al. Plant-mediated fabrication and surface enhanced raman property of flower-like Au@Pd nanoparticles. Materials. 2014;7:1360–1369. doi: 10.3390/ma7021360 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Reshad RAI, Jishan TA, Chowdhury NN. Chitosan and its broad applications: a brief review. J Clin Exp Investig. 2021;12:em00779. doi: 10.29333/jcei/11268 [DOI] [Google Scholar]
  • 38.Xia Y, Wang D, Liu D, et al. Applications of chitosan and its derivatives in skin and soft tissue diseases. Front Bioeng Biotechnol Frontiers Media SA. 2022;10:894667. doi: 10.3389/fbioe.2022.1082945 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Gong X, Gao Y, Shu J, et al. Chitosan-based nanomaterial as immune adjuvant and delivery carrier for vaccines. Vaccines MDPI. 2022. doi: 10.3390/vaccines10111906 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Atale N, Gupta S, Yadav UCS, et al. Cell-death assessment by fluorescent and nonfluorescent cytosolic and nuclear staining techniques. J Microsc. 2014;255:7–19. doi: 10.1111/jmi.12133 [DOI] [PubMed] [Google Scholar]
  • 41.El-Naggar NEA, Shiha AM, Mahrous H, et al. Green synthesis of chitosan nanoparticles, optimization, characterization and antibacterial efficacy against multi drug resistant biofilm-forming Acinetobacter baumannii. Sci Rep. 2022;12(1):19869. doi: 10.1038/s41598-022-24303-5 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Quan Z, Luo C, Zhu B, et al. Synthesis and antimicrobial activities of chitosan/polypropylene carbonate-based nanoparticles. RSC Adv. 2021;11:10121–10129. doi: 10.1039/D0RA09257F [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Yu Z, Li Q, Wang J, et al. Reactive oxygen species-related nanoparticle toxicity in the biomedical field. Nanoscale Res Lett Springer. 2020;15(1):115. doi: 10.1186/s11671-020-03344-7 [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Figures S1-S4
INNM_A_2382073_SM0001.docx (2.1MB, docx)
Supplementary Video
INNM_A_2382073_SM0002.zip (1.2GB, zip)

Articles from Nanomedicine are provided here courtesy of Taylor & Francis

RESOURCES