Surface functionalized silver nanoparticles augment macrophage activation followed by intracellular killing of pathogenic bacteria

Surface functionalized silver nanoparticles augment macrophage activation followed by intracellular killing of pathogenic bacteria | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Surface functionalized silver nanoparticles augment macrophage activation followed by intracellular killing of pathogenic bacteria Balaram Das, Jaydeep Adhikary, Sandeep Kumar Dash, Kankan Kumar Maity This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7036578/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Nanoparticles interact with these immune cells and modulate its function, leading to immunosuppression or immunostimulation. The immunostimulatory activity of modified metal nanoparticles is based on the activation of immune system against pathogenic bacteria. In this study, we evaluate the efficacy of modified metal nanoparticles to activate immune cells (Macrophage) and also to examine the efficacy of those activated macrophages towards intracellular killing of pathogenic S. aureus and P. aeruginosa bacterial strains. Synthesized Ag-NPs were modified by polyethylene glycol (PEG). The modified nanoparticles successfully activated macrophage (MФ) evident by the increasing the serum TNF-α level and intracellular NO generation. To check the effective contribution of TNF-α and COX-2 pathway underlying the Ag@PEG NPs pulsed macrophage induced bacterial killing addition of POF and ASA in co-culture setup was done. PEG functionalized nanoparticles enhanced the intracellular killing of pathogenic bacteria in macrophage. We also found that, Ag@PEG NPs pulsed RAW 264.7 secreted elevated level of proinflammatory cytokines. Pulse macrophages were also successfully killed intracellular pathogenic S. aureus and P. aeruginosa bacteria in in vitro setup at 1:1 ratio for 24 h. The use of TNF-α inhibitor and NO blocker confirmed the association between Ag@PEG NPs with TNF-α and NO function. These findings will enrich the biomedical applications of Ag@PEG NPs as a potent immune stimulating agent and this macrophage stimulating efficacy might be an effective way in the bacterial immunotherapy. Such a metal nanoparticles offers versatility in that it can simultaneously activate the primary Immune cells as well as kill the ingested microbes. PEG functionalized Ag-NPs Immunostimulation Macrophage activation intracellular killing of bacteria Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 1. Introduction Studies relating to the immunomodulatory activity of Ag-NPs are relatively limited. Immunotherapeutic approaches are the alternative strategies to treat microbial infections as the conventional antibiotics were resistant to different bacterial species. However, the aim of our study is to evaluate the immunomodulatory activity of Ag@PEG-NPs in antibacterial therapy. Multifunctional role of metal nanoparticles enhances its wide applicability in various biomedical application systems as well as in many industrial processes [ 1 ]. Silver has long been recognized as effective antimicrobial agent and thus it is widely used in topical ointments and various creams in its nano forms or ionization state to prevent infection of burns and wounds [ 1 , 2 ]. This use of biological elements for the development of nanoparticles has been designated as “green synthesis” and is highly considered to be far more beneficial than chemical methods which require toxic chemicals, more complex and expensive chemical reactions. The absence of toxic by-products and consequential decrease in degradation of the particles proved this technique more preferable over physical and classical chemical methods [ 3 ]. In spite of presence of various bioactive plant materials over the surface of green synthesized Ag-NPs as capping agent it is still shows significant toxicity towards health cells. Thus, more effective surface functionalization using PEG, PLGA, BSA reduces the toxicity of the particles upto considerable limit and increases solubility in aqueous solutions [ 4 ]. Macrophage plays crucial role in innate and cell mediated immunity in response to killing and eliminations of pathogens [ 5 ]. Being potent phagocytic cells, it can eliminate intracellular bacteria through generation of phagosomes using germline-encoded pattern recognition receptors specific for microbial products [ 6 ]. AgNPs has been found to activate macrophage through inflammatory stimuli which helps for successive elimination of intracellular microbes [ 7 ]. Nishanth and co-workers reported that AgNP treatment with macrophages induced various inflammatory response mainly increased IL-6 production, increased ROS concentration, nuclear translocation of NF-κB, induction of cyclooxygenase-2 (COX-2), and increased tumor necrosis factor-alpha (TNF-α) mediated by NF-κB [ 8 ]. Those inflammatory stimuli are responsible for M1 polarization of macrophage which promotes the antimicrobial killing [ 5 ]. But the toxicity of bare Ag NPs towards macrophages limits these functions and decreases the viability of macrophage itself without promoting microbicidal effects [ 9 ]. Though it was found that biologically prepared Ag NPs and chitosan coating produces less toxicity to murine RAW 264.7 macrophages [ 10 , 11 ], but their effective contribution in intracellular killing was not examined in detail. However, there are few instances on the comparative study of surface functionalized green synthesized AgNps mediated macrophage activation and its ability to kill intracellular pathogenic bacteria (both gram positive and negative). Based on the above background this study was conducted to investigate the ability of PEG coated Ag NPs (Ag@PEG NPs) towards activation of RAW 264.7 cells and also to examine the efficacy of those activated RAW 264.7 towards intracellular killing of clinically isolated pathogenic S. aureus and P. aeruginosa strains. 2. Materials and Methods 2.1. Chemicals and reagents Silver nitrate (AgNO 3 ), polyethylene glycol (PEG), Histopaque 1077, sodium acetate, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT reagent), Acetylsalicylic acid (ASA), Sulfasalazine, indomethacin, pentoxifylline (POF) were procured from Sigma (St. Louis, MO, USA). Minimum Essential Medium (MEM), RPMI 1640, fetal bovine serum (FBS), penicillin, streptomycin, sodium chloride (NaCl), sodium carbonate (Na 2 CO 3 ), sucrose, Hanks balanced salt solution, ethylene diamine tetra acetate (EDTA), dimethyl sulfoxide (DMSO), NaOH Nutrient agar and Luria broth were purchased from Himedia, Mumbai, India. Tris–HCl, Tris buffer, KH 2 PO 4 , K 2 HPO 4 , HCl, formaldehyde, alcohol and other chemicals were procured from Merck Ltd., Mumbai, India. All other chemicals of the highest purity grade (MB and cell culture grade) were purchased from Merck Ltd., SRL Pvt., Ltd., Mumbai, India. 2.2. Green synthesis purification and Surface functionalization of silver nanoparticles Silver nanoparticles were synthesized using Ocimum gratissimum leaves extract according to our previously reported method. The purification of Ag-NPs was done by sucrose density gradient centrifugation [ 12 ]. Polyethylene glycol (PEG) loading on green synthesised Ag nanoparticles has been achieved by adopting slightly modified procedure as reported previously [ 12 , 13 ]. 2.2.1. Fourier transform infrared spectroscopy. Ag-NPs and Ag@PEG were investigated by Fourier transform IR spectroscopy with a PerkinElmer Spectrum RX I Fourier transform IR system with a frequency ranging from 500 to 4,000 cm − 1 and a resolution of 4 cm − 1 . Potassium bromide (KBr) was used to prepare the pellet for analysis of samples for this study [ 14 ]. 2.2.2. Dynamic light scattering (DLS) and Zeta potential. The hydrodynamic diameter of the sample was analyzed by DLS technique using Zetasizer Nano ZS (Malvern, Malvern Hills, U.K.). At first, 100µg/mL concentration of the nanoparticles were prepared and sonicated for 2 min, then dynamic particle sizes were measured by suspending two drops of an aqueous suspension of NPs in 10 mL of Millipore water. When the NPs had absolutely dispersed in water, the samples were analyzed with a DLS analyzer. This measurement was repeated for several times to achieve the average size of the NPs. The zeta potential of the nanoparticles were measured by using a Zetasizer-Nano ZS (Malvern, Malvern Hills, U.K.) [ 14 ]. 2.3. Bacterial Strains used in this study Multidrug resistant and virulent Pseudomonas aeruginosa strains were isolated from urine samples of urinary tract infected patients [ 15 ] and Staphylococcus aureus strains were isolated from pus samples [ 16 ] in our laboratory. We used Multidrug resistant Pseudomonas aeruginosa (PA-7) and Staphylococcus aureus (SA-20) bacterial strains for this study. The strains were subculture and used throughout the study. 2.4. Cell culture The Raw 264.7 cell line (macrophage-like, Abelson leukemia virus transformed cell line derived from BALB/c mice) was procure from NCCS, Pune, India. This cell line was cultured in RPMI 1640 medium with 10% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin, 4 mM L-glutamine under 5% CO 2 , and 95% humidified atmosphere at 37 0 C for in vitro experiments. 2.5. Macrophage Pulsing RAW 264.7 cells (1x10 6 cells per milliliter) were pulsed with 1, 5, 10, 25, 50µg/ml Ag@PEG NPs in complete RPMI 1640 medium for different time interval at 37°C. 2.6. The dose dependent cytotoxicity of Ag@PEG NPs toward RAW 264.7 cells. RAW 264.7 cells were seeded into 96 wells of tissue culture plates containing 180 µl of complete medium and were incubated for 48 h. Ag@PEG NPs were added to the cells at different concentrations (1, 5, 10, 25 and 50 µg/ml), and the mixtures were incubated for 48 h at 37°C in a humidified incubator (NBS) maintained at 5% CO 2 . The cell viability was estimated by MTT assay according to the method described elsewhere [ 17 ]. The plates were read on a microplate reader (model 550, Bio-Rad, Tokyo, Japan) at a wavelength of 570 nm. 2.7. The duration dependent cytotoxicity of Ag@PEG NPs toward RAW 264.7 cells. RAW 264.7 cells were treated as same as the dose (in terms of concentration) dependant study and were incubated for 24h, 48h and 72h at 10 µg/ml concentrations (selected dose) at 37°C in a humidified incubator maintained at 5% CO 2 . The cell viability was estimated by MTT assay. 2.8. Bacterial cell co-culture with pulsed RAW 264.7 cells. The pulsed RAW 264.7 cells were collected by trypsinization and were centrifuged at 1,500 rpm for 5 min. The pellet cells were washed with PBS at room temperature. After they had been washed three times, the cells were resuspended in RPMI 1640 medium with 5% FBS. Bacterial strains were cultured in liquid media [ 12 ]. After the culture of bacterial cells, pulsed RAW 264.7 cells and the bacterial cells were co-cultured in a 96 well plate in a ratio of 1:1 (RAW 264.7 cells to bacterial cells) at the density of each cultured cells was 1x10 6 cells per well and were then incubated for 24 hrs at 37°C [ 18 ]. 2.9. Bacterial cell viability assay After co-culture, each well was washed with sterile saline to remove the dead macrophages and residual bacterial cells and lysed using 200 µl of 1% saponin in sterile water. Cell lysates were plated on solid medium and incubated at 37°C. After 24 hrs of incubation CFU were counted, and the results were expressed graphically. 2.10. Intracellular localization of nanoparticles and bacteria The intracellular localization of Ag@PEG NPs and both bacterial strains (i.e S. aureus and P. aeruginosa strains) were visualized under fluorescence microscopy. For this assay, 100 µl Fluorescein isothiocyanate (FITC) solution (100 µg/ml, dissolved in 0.1 M carbonate–bicarbonate buffer and vortexed for 1 minute) was added to the Ag@PEG NPs solution (1mg/ml, dissolved in 0.1 M carbonate–bicarbonate buffer). The mixture was incubated for 2 hrs at 37°C wrapped in aluminium foil to prevent photo bleaching. The FITC conjugated Ag@PEG NPs solution (FITC-Ag@PEG NPs) was washed thrice using ice cold PBS by centrifugation at 8000g to remove excess FITC. The pallet was again resuspended using 1 ml PBS and 10µl of the FITC-Ag@PEG NPs solution was treated with RAW 264.7 cells (1X10 6 /ml) for 8 hrs at 37°C. The both bacterial cells were labelled with Rhodamine B (Rh-B) (10 µg/ml solution of Rh-B incubated with 1ml of each bacterial strain for 1 hr followed by washing at 5000g using PBS). The pulsed RAW 264.7 cells were co-cultured with Rh-B labelled both bacterial cells (1:1) for 4 hrs followed by washing with PBS and layered on to glass slide for fluorescent microscopic imaging (NIKON ECLIPSE LV100POL). 2.11. NO release assay The NO concentration was measured by a microplate assay method with Griess reagent (1% sulfanilamide, 0.3% naphthylethylene diamine dihydrochloride, 7.5% H 3 PO 4 ). Briefly, culture supernatants (100 µl) were mixed with 100 µl of Griess reagent. The nitrite concentration in the culture supernatant was measured at a wavelength of 550 nm after 10 min of mixing [ 19 ]. 2.12. Cytokine analysis To investigate the effect of Ag@PEG NPs on cytokine production before and after co-cultured with bacteria, an ELISA technique was used for the determination of interferon-γ (IFN-γ), TNF-α, and IL-12 production. RAW 264.7 cells were cultured at 1x10 6 cells per millilitre and were treated with 10µg/ml concentrations of Ag@PEG for 24 h. another set was prepared where RAW 264.7 cells were cultured with bacterial cells for 24 hrs at 37°C. After treatment, cell-free supernatants were harvested via successive 10-min centrifugations (2,000, 7,000, and 13,000 rpm) and were stored at -80°C until analysis. ELISA was performed by the protocol supplied by the manufacturer company (Invitrogen™ eBioscience™ ELISA Ready-SET-Go!™ Kits). 2.13. Incubation with POF RAW 264.7 cells (macrophages) were co-cultured with bacterial cells in a ratio of 1:1 for 24 hrs in RPMI medium alone, and in the presence of 1 mM POF [ 20 ]. After incubation, cell viability was measured by MTT assay [ 20 ]. 2.14. Incubation with ASA and indomethacin In another experiment, ASA and indomethacin at final concentrations of 1 mM and 50 µM respectively, were applied together in the reaction mixture of pulsed RAW 264.7 cells and bacterial cells. After the treatment schedule cell viability was measured by MTT assay [ 20 ]. 2.15. Statistical Analysis Each of the above assays was performed in triplicate. The data were expressed as mean ± SEM, n = 6. Comparisons between the means of control and treated group were made by two-way ANOVA test with multiple comparison t-tests, p < 0.05 as a limit of significance. 3. Results 3.1. Fourier transform infrared spectroscopy: FTIR spectra of Ag and Ag@PEG are presented in Figure-1. The FTIR measurements of Ag-NPs, PEG functionalized Ag-NPs were carried out to identify the possible interaction between coating agents with Ag-NPs. Results of FTIR study showed sharp absorption peaks at about 1635.16cm -1 , 1451.34cm -1 , 1018.11cm -1 3433.57 cm -1 for Ag-NPs (Figure-1). The FTIR spectra of Ag@PEG consists several new peaks at 1589.44 cm -1 , 1385.40cm -1 , 1350.64 cm -1 and shifting of peaks from 1451.34cm -1 to 1458.03cm -1 and decrease the intensity of the peak at 1818.11cm -1 , in addition to the characteristic peaks of Ag-NPs suggests the successful loading of PEG on the surface of Ag-NPs. 3.2. Dynamic light scattering (DLS) and Zeta potential: Average particle size, distribution and polydispersity index (PDI) of synthesized silver nanoparticles in solutions were evaluated by DLS technique, which is shown in Figure-2. The DLS pattern revealed that Ag-NPs synthesized by green method had a Z average diameter of 38.33 ± 10 nm according to the size distributions by volume with PDI of 0.479. The average particle size of Ag@PEG had a Z average diameter of 58.89 ± 16 nm with PDI of 0.569. The zeta potential value of synthesized silver nanoparticles was − 21.8 mV and PEG functionalized Ag-NPs showed the zeta potential was − 4.08 mV which is shown in Figure-3. The stability of Ag@PEG NPs also checked for 5 moth old NPs solution (stored at RT) using DLS and Zeta potential techniques and found no significant detonation of the stability of NPs (Figure-S1). 3.3. Effect of Ag@PEG NPs on RAW 264.7 cells. The toxicity of the Ag@PEG NPs toward normal RAW 264.7 cells in vitro was checked. It was found that there was no significant difference in cell viability between the cells treated with Ag@PEG NPs. The cell viability assay showed that Ag@PEG NPs upto 10µg/ml concentration has no major toxicity. This nanoparticle shows significant toxicity after the concentration of 25mg/ml. It was found that Ag@PEG NPs kills RAW 264.7 cells by 3.79%, 6.883%, 8.92%, 19.23% and 33.72% after 24 h incubation at 1, 5, 10, 25, 50 µg/ml concentrations respectively compared with the negative control. (Figure-4A). On the other hand, in duration dependant study the cell viability assay showed that Ag@PEG NPs was effective in 24hrs after treatment in selected concentration or dose have no toxicity. It was found that Ag@PEG NPs kills RAW 264.7 cells by 9.43%, 14.28%, and 16.32% after 1 day, 2 days and 3 days incubation respectively at 10µg/ml concentration compared with the control (Figure-4B). Hence, these NPs are safe in 10µg/ml concentration and 24 hrs treatment for biomedical applications. 3.4. Effect of Ag@PEG NP pulsed RAW 264.7 cells on bacterial cells The antibacterial activity of Ag@PEG pulsed RAW 264.7 cells towards both types of bacterial cells was significant. The present result suggested that Ag@PEG NPs pulsed RAW 264.7 cells have greater killing activity toward S. aureus cells in comparison with P. aeruginosa . As shown in Figure-5, during the co-culture of pulsed RAW 264.7 cells with both types of bacterial cells in a ratio of 1:1. The intracellular killing of S. aureus and P. aeruginosa were substantially higher after 16 h of treatment period. At 24h treatment period the killing percentage of S. aureus and P. aeruginosa were slightly reduced. As shown in Figure-5, during the co-culture of pulsed macrophage with bacterial cells, a significant amount of S. aureus and P. aeruginosa cell killing induced by pulsed macrophage was observed on 16 hours (85.287% and 91.631%) and 24 hours (92.284% and 88.802%). All the results show significant difference at the level of p < 0.05. 3.5. Intracellular localization of nanoparticles and bacteria The intracellular localization of both Ag@PEG NPs and bacterial strains within RAW 264.7 cells were visualized under fluorescence microscopy. During treatment of S. aureus and P. aeruginosa infected RAW 264.7 cells by Ag@PEG nanoparticles, large number of intracellular bacteria were seen in fluorescence images. All the bacteria appeared in the process of destruction within the macrophage as images shows defragmented red florescence. The fluorescence of Ag@PEG NPs was localized in the cytoplasm, with which the bacteria undergoing degradation were often associated (Figure-6). From these results it was clear that both Ag@PEG NPs and bacterial cells are distributed throughout the RAW 264.7 cells or macrophages indicating successful internalization through endocytosis process. 3.6. NO release In the present study, presence of the Ag@PEG stimulates RAW 264.7 cells to generate a significant amount (84.59%) of NO (p < 0.05) in the medium after 16 hrs of incubation period (Figure-7A). Nitric oxide level was also measured in duration dependent manner when pulsed RAW 264.7 cells were co-cultured with both bacterial cells (Figure-7B). Results shows that the NO generation were increased by 1.062-fold, 1.189 fold, 1.266 fold, 1.246 fold, 1.311 fold in pulsed macrophage infected with S. aureus at 4hrs, 12 hrs, 16 hrs, 24 hrs and 48 hrs respectively compared with the control. In case of P. aeruginosa infection in pulsed macrophage increases the NO generation by 1.001 fold, 1.117 fold, 1.215 fold, 1.208 fold and 1.333 fold at 4hrs, 12 hrs, 16 hrs, 24 hrs and 48 hrs respectively compared with the control. 3.7. Cytokine estimation from Ag@PEG NP pulsed RAW 264.7 cells. The contributing effects of cytokine production after stimulation of Ag@PEG NPs on RAW 264.7 cells were measured by ELISA assay. After treatment both bacterial cells with Ag@PEG-NPs pulsed RAW 264.7 cells, cell-free supernatants were used to quantify cytokine levels. The results demonstrated significant increases in the levels of pro inflammatory cytokines such as IL-12, TNF-α and decrease anti-inflammatory cytokine IL-10, TGF-β in NPs treated group then control group. Concentrations of TNF-a, IL-12, and TGF-b, IL-10 were quite low in culture supernatants of unpulsed RAW 264.7 cells. The results showed that pulsed RAW 264.7 cells increased the production of TNF-α by 1.68-fold, IL-12 by 1.45-fold compared with controls at 10 µg/ml NPs concentration (Figure-8) and decreased the production of TGF-β by 0.74-fold, and IL-10 by 0.76-fold compared with controls at same dose or concentration (Figure-8). Thus Ag@PEG pulsation elevated of Th1 cytokines and decrease in Th2 cytokines which contributed better immunostimulatory activity as well as better bacterial killing. 3.8. Incubation with POF To check the effective contribution of TNF-α underlying the Ag@PEG NPs pulsed RAW 264.7 cells induced bacterial killing addition of POF in co-culture setup was done. The present study showed that use of POF in culture medium increased the bacterial cell viability more than 90% after co-culture with Ag@PEG NPs pulsed RAW 264.7 cells (Figure-9) 3.9. Incubation with ASA Effect of NO towards pulsed macrophage mediated bacterial cell death was monitored in the presence of ASA (Figure-10). ASA acts as an inhibitor of the COX-2 pathway [ 21 ]. It was noted that presence of ASA results both types bacterial strains were not killed by the pulsed RAW 264.7 cells. But the protection ability was less than POF co-treatment group. 4. Discussion The surface charge and chemical properties of functionalized nanoparticles were determined by various physio-chemical measurements. Among various coating materials, polyethylene glycol (PEG) has been widely used because of their nontoxic, biodegradable, biocompatible properties [22, 23, 24]. In general, PEG functionalization improves drug solubility, increases drug stability, the retention time of the conjugates in blood, and reduces proteolysis and renal excretion, thereby allowing a reduced dosing frequency. This functionalization changes the physical and chemical properties of the biomedical molecule, electrostatic binding, and hydrophobicity results in an improvement in the pharmacokinetic behaviour of the drug [25, 26]. Fourier transforms infrared spectroscopy (FT-IR) study exhibits characteristic peaks for Ag-NPs at 1635.16cm -1 , 1451.34cm -1 , 1018.11cm -1 3433.57 cm -1 . The FTIR spectra of Ag@PEG have several new peaks at 1589.44 cm -1 , 1385.40cm -1 , 1350.64 cm -1 and shifting of peaks from 1451.34cm -1 to 1458.03cm -1 and decrease the intensity of the peak at 1818.11cm -1 , in addition to the characteristic peaks of Ag-NPs suggests the successful loading of PEG on the surface of Ag-NPs (Figure-1), DLS analysis confirmed that synthesized silver nanoparticles and functionalized silver nanoparticles were in nanometer size range having particle sizes of 38.33± 10 nm with PDI of 0.479 for Ag-NPs and 58.89 ±16 nm with PDI of 0.569 for Ag@PEG (Figure-2). After attachment of PEG onto the surface of Ag-NPs the average sizes were enhanced. The zeta potential value of synthesized silver nanoparticles was −21.8 mV and PEG functionalized Ag-NPs showed the zeta potential was −4.08 mV (Figure-3). The enhancement of particle size, surface charge and alteration in PDI clearly suggest the successful surface modification of Ag-NPs. The microbial pathogenesis is an event that results of a continuous battle between host and the microbial pathogens. Last few decades, new insights were opened in host immune response to bacterial pathogens, which has been underlying the development of new strategies to modulate immune system against those infecting microbes. Several approaches have been taken up to improve the immune responses against microbial pathogens [27, 28]. Particulate adjuvants, nanoparticles (NPs) have generated a lot of interest due to their unique features. Important pathogens such as Staphylococcus aureus, Pseudomonas aeruginosa, Listeria monocytogenes, Shigella flexneri etc adopted different strategies to avoid lysosomal degradation, including escaping phagocytosis, release of toxins that are lethal to phagocytes. Staphylococcus aureus can avoid destruction by releasing toxin leukocidin AB [29, 30] which form octameric pores in the plasma membrane of the target cells [31] results osmotic imbalance and cell death [29, 30]. Pseudomonas aeruginosa eliminate phagocytes as a strategy to escape the immune defense by producing toxins, such as phenazines [32] which induce the reactive oxygen species (ROS) generation [33] causes rapid and overwhelming apoptosis [32]. PEG modification reduces the toxicity of Ag-NPs by decreasing excess release of Ag ion in the medium. Moreover, it was also reported that in acidic environment (micro environment of cancer cells) the PEG coated Ag NPs showed more toxicity; while in physiological pH (pH of normal cell environment) it lowers the toxicity significantly [34]. The microbial infected area is also acidic in nature. So, Ag@PEG nanoparticles release more amount of ion whereas in normal cellular environment it release less amounts of ion results this nanocomposites is effective in infected area and kill the microbes but cannot toxic to normal cells. Thus, in the present study Ag@PEG showed lower toxicity towards RAW 264.7 cells and it was more toxic when macrophage cells (RAW 264.7) are present with bacterial cells. This may be due to differences in ion release. Previous report revealed that the drastic reduction of bacterial cell counts by activating immune functions of activated macrophages [35]. In the case of unpulsed macrophage the killing percentage for both bacterial cells were much lower than nanoparticle pulsed macrophage. This may be due to inactivation of macrophages which lowers the phagocytic effects. Activated macrophage secretes various inflammatory mediators such as TNF-α and NO [17]. These inflammatory mediators have various effects in our body. They activate immune competent cells to recruit at site of infection or directly kill the pathogens and thereby help to cure from infections. In the present study, presence of the Ag@PEG stimulates macrophages to generate a significant amount (84.59%) of NO (p<0.05) in the medium after 16 hrs of incubation period (Figure-7A). This result indicated the presence of a high concentration of NO in the co-culture medium from pulsed macrophages which may attribute to bacterial killing during treatment of pulsed macrophages with bacterial cells. Nitric oxide level was also measured in duration dependent manner when pulsed macrophages were co-cultured with both bacterial cells (Figure-7B). Hence, the amount of this NO was correlated with the cytotoxicity toward bacterial cells. Several studies have evaluated the relationship between inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 [36, 37]. The contributing effects of cytokine production after stimulation of Ag@PEG NPs on macrophage was measured by ELISA assay. After treatment both bacterial cells with Ag@PEG pulsed RAW 264.7 cells, cell-free supernatants were used to quantify cytokine levels. The results demonstrated significant increases in the levels of pro inflammatory cytokines such as IL-12, TNF-α and decrease anti-inflammatory cytokine IL-10, TGF-β in NP treated group then control group. Concentrations of TNF-a, IL-12, and TGF-b, IL-10 were quite low in culture supernatants of unpulsed macrophages. The results showed that pulsed macrophages increased the production of TNF-α and decreased the production of TGF-β. Thus Ag@PEG pulsation elevated of Th1 cytokines and decrease in Th2 cytokines which contributed better immunostimulatory activity as well as better bacterial killing. Nanomaterials have been shown to modulate expression of cytokines, which are soluble biological protein messengers that regulate the immune system [38]. Published studies have demonstrated the ability of certain nanomaterials to induce cytokine production, although this appears heavily dependent on a variety of factors, including material composition, size, and method of delivery [39]. In the present study elevated levels of IL-12, and TNF-α represent critical pathways that are involved in the inflammatory response and differentiation processes as well as confirmed the activation process of macrophages by pulsing with Ag@PEG NPs. Thus the findings indicate that careful titration of Ag@PEG NPs based therapeutic interventions may be successful in elevating the levels of a group of cytokines important for eliciting a Th1-mediated immune response with effective antibacterial actions (Scheme-1). To check the effective contribution of TNF-α underlying the Ag@PEG NPs pulsed macrophage induced bacterial killing addition of POF in co-culture setup was done. Recent studies reported the ability of theophylline and POF, both phosphodiesterase inhibitors, to suppress monocyte/macrophage TNF-α production by increasing the intracellular accumulation of cAMP [40]. The present study showed that use of POF in culture medium increased the bacterial cell viability more than 90% after co-culture with Ag@PEG NPs pulsed macrophages (Fig. 9). Thus this findings indicated that TNF-α is main causative agent responsible for Ag@PEG NPs mediated antibacterial therapy. The production of TNF-α by mononuclear phagocytes is regulated by the intracellular levels of cyclic adenosine monophosphate (cAMP) [41]. Exogenous cAMP analogues and substances such as prostaglandin E, which are capable of increasing the intracellular level of cAMP, reduce the release of bioactive TNF-α by downregulating the expression of the TNF-α gene [20]. There is increasing evidence that POF may also play a therapeutic role in the inhibition of inflammatory processes. POF has been shown to improve resistance against sepsis or endotoxin challenge in mice, rats, and humans [19, 20], most likely by decreasing circulating TNF-α levels. POF is able to inhibit the synthesis of messenger RNA for TNF-α in mouse murine macrophages at the transcriptional level. Also in humans, POF is able to reduce the release of TNF-α by peripheral blood macrophages. In contrast to ASA and indomethacin, which totally blocks the synthesis of cyclo-oxygenase intermediate endoperoxides (PGG2, PGH2) [21]. Effect of NO towards pulsed macrophage mediated bacterial cell death was monitored in the presence of ASA along with indomethacin (Figure-10). ASA and indomethacin acts as an inhibitor of the COX-2 pathway [21]. It was noted that presence of ASA and indomethacin protected both bacterial cells from pulsed macrophage mediated killing. But the protection ability was less than POF co-treatment group. Thus, it may be suggested that TNF-α was the most abundant contributing factor responsible for macrophage activation and intracellular killing of pathogenic bacteria. Moreover, studies have reported that ASA decreases the intracellular internalization of microorganism within activated macropahage [42]. Thus it was manifest that COX pathway plays prevotal role in the process of microbial invasion. In addition, this previous study also showed that iNOS expression in macrophages was increased with ASA treatment, suggesting that iNOS-dependent production is responsible for ASA effects. 5. Conclusions This study shows that Ag@PEG NPs efficiently killed bacterial cells after activation of macrophages. The pulsed macrophages show good antibacterial activity. Treatment of Ag@PEG NPs results increased the generation of NO and increase the concentration Th1 type cytokines results the activation of macrophage. This activated macrophage then killed the engulfed bacterial cells. Such a metal nanoparticles offers versatility in that it can simultaneously activate the primary Immune cells as well as kill the ingested microbes. Abbreviations Ag-NPs: Silver nanoparticles MTT: 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide PBS: Phosphate buffer saline Rh-B: Rhodamine B PEG: polyethylene glycol Ag@PEG NPs: modified metal nanoparticles () MФ: Macrophage TNF-a: Tumor necrosis factor alpha IL-6: Interleukine-6 COX-2: cyclooxygenase-2 iNOS: inducible nitric oxide synthase ROS: Reactive oxygen species NF-κB: Nuclear factor –κB POF: pentoxifylline ASA: Acetylsalicylic acid cAMP: cyclic adenosine monophosphate TGF-β: Transforming growth factor Declarations Conflict of interests The authors declare there are no conflicts of interest. Acknowledgement The authors express gratefulness to the Belda College, Belda and USIC, Vidyasagar University, Midnapore, University for providing the facilities to execute these studies. Authors Contribution: BD: Conceptualization, Data Curation, Analysis of Data, Investigation, Methodology, Validation and visualization, manuscript writing, editing. JA: Methodology , manuscript writing, editing. SKD: Methodology, manuscript writing, editing. KKM: Methodology, manuscript writing, editing. Funding Declaration : No funding received for the entire work. Ethics and Consent: Bacterial strains were isolated previously prior to ethical permission and consent to participate and publication. References Murphy C-J (2008) Sustainability as an emerging design criterion in nanoparticle synthesis and applications, J Mater Chem. 18: 2173-2176. Schultz S, Smith D-R, Mock J-J, Schultz D-A (2000) Single-target molecule detection with non bleaching multicolor optical immunolabels, Proce Nat Aca Sci. 97: 996-1001. Mandal D, Dash S-K, Das B, Chattopadhyay S, Ghosh T, Das D, Roy S (2016) Bio-fabricated silver nanoparticles preferentially targets Gram positive depending on cell surface charge, Biomed & Pharmacother. 83: 548-558. 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Zolnik B-S, Gonzalez-Fernandez A, Sadrieh N, Dobrovolskaia M-A (2010) Minireview: nanoparticles and the immune system. Endocrinology. 151:458-65. Hanley C, Thurber A, Hanna C, Punnoose A, Zhang J, Wingett D-G (2009) The Influences of Cell Type and ZnO Nanoparticle Size on Immune Cell Cytotoxicity and Cytokine Induction. Nanoscale Res Lett, 4: 1409–1420. Endres S, Fülle H-J, Sinha B, Stoll D, Dinarello C-A, Gerzer R, Weber P-C (1991) Cyclic nucleotides differentially regulate the synthesis of tumour necrosis factor-alpha and interleukin-1 beta by human mononuclear cells. Immunology. 72: 56–60. Katakami Y, Nakao Y, Koizumi T, Katakami N, Ogawa R, Fujita T (1988) Regulation of tumour necrosis factor production by mouse peritoneal macrophages: the role of cellular cyclic AMP. Immunology. 64: 719–724. Malvezi A-D, da Silva R-V, Panis C, Yamauchi L-M, Lovo-Martins M-I, Zanluqui N-G, Tatakihara V-L, Rizzo L-V, Verri W-A, Martins-Pinge M-C, Yamada-Ogatta S-F (2014) Aspirin modulates innate inflammatory response and inhibits the entry of Trypanosoma cruzi in mouse peritoneal macrophages. Mediators of inflammation. 19: 2014. Scheme Scheme 1 is available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files supportingfile.doc FigureS1.jpg Scheme1.jpg Scheme-1:Schematic representation of the macrophage activation and mechanisms of intracellular killing of bacteria in Ag@PEG nanoparticles treatment. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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08:27:57","extension":"html","order_by":39,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":134755,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7036578/v1/f3b41d29afafbf28d1b02df9.html"},{"id":95802012,"identity":"92756da9-bc3d-4c5e-95a4-7b577865554f","added_by":"auto","created_at":"2025-11-13 08:26:37","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":166743,"visible":true,"origin":"","legend":"\u003cp\u003eFourier transform infrared (FTIR) spectroscopic images of Ag-NPs and PEG functionalized Ag-NPs. Here, The FTIR spectroscopic images of Ag-NPs shows the characteristic peak of 1635.16cm\u003csup\u003e-1\u003c/sup\u003e, 1451.34cm\u003csup\u003e-1\u003c/sup\u003e, 1018.11cm\u003csup\u003e-1\u003c/sup\u003e etc. whereas the FTIR spectroscopic images of PEG functionalized Ag-NPs consists several new peaks at 1589.44 cm\u003csup\u003e-1\u003c/sup\u003e, 1385.40cm\u003csup\u003e-1\u003c/sup\u003e, 1350.64 cm\u003csup\u003e-1\u003c/sup\u003e and shifting of peaks from 1451.34cm\u003csup\u003e-1 \u003c/sup\u003eto 1458.03cm\u003csup\u003e-1\u003c/sup\u003e\u0026nbsp; and decrease the intensity of the peak at 1818.11cm\u003csup\u003e-1\u003c/sup\u003e, in addition to the characteristic peaks of Ag-NPs suggests the successful loading of PEG on the surface of Ag-NPs. KBr was used to prepare pallet for FTIR spectroscopy.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7036578/v1/e4dc8629866aa4f3f9546736.jpg"},{"id":95802593,"identity":"17944d62-6f6c-42b8-bb69-ee00912fdc98","added_by":"auto","created_at":"2025-11-13 08:28:03","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":153835,"visible":true,"origin":"","legend":"\u003cp\u003eThe hydro-dynamic size determination of Ag-NPs and Ag@ PEG-NPs by dynamic light scattering (DLS). Here, \u003cstrong\u003eA:\u003c/strong\u003e The average particle size of Ag-NPs had a Z average diameter of 38.33 ±10 nm with PDI of 0.479; \u003cstrong\u003eB:\u003c/strong\u003e The average particle size of Ag@PEG had a Z average diameter of 58.89± 16 nm with PDI of 0.569.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7036578/v1/730bf9f43427a3ba4b099587.jpg"},{"id":95802197,"identity":"50327c8d-61a2-46c9-b7e6-e2a82997e9b0","added_by":"auto","created_at":"2025-11-13 08:27:07","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":175447,"visible":true,"origin":"","legend":"\u003cp\u003eThe zeta potential of Ag-NPs and PEG functionalized Ag-NPs. was. Here, \u003cstrong\u003eA:\u003c/strong\u003e Ag-NPs showed the zeta potential was −21.8 mV; \u003cstrong\u003eB:\u003c/strong\u003e PEG functionalized Ag-NPs showed the zeta potential was −4.08 mV.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7036578/v1/b892844aca9f293f3c381f50.jpg"},{"id":95802001,"identity":"2b094e19-fd7f-432b-b977-a98d3ca625e2","added_by":"auto","created_at":"2025-11-13 08:26:35","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":41476,"visible":true,"origin":"","legend":"\u003cp\u003eCellular toxicity analysis of Ag@PEG toward macrophage by MTT assay (A), Dose dependent study (B), Duration dependent study. All the measurements were performed in triplicate. Values are expressed as mean ±SEM.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7036578/v1/940113664422c6d35e0031ec.jpg"},{"id":95762760,"identity":"6484012b-7afc-4faf-9093-81767448eef3","added_by":"auto","created_at":"2025-11-12 18:28:48","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":66910,"visible":true,"origin":"","legend":"\u003cp\u003eBacterial cells killing by Ag@PEG NPs pulsed macrophages co-cultured with \u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eP. aeruginosa\u003c/em\u003e cells at ratios of macrophages to bacterial cells of 1:1. All values are expressed as the mean ± the standard error of the mean. * indicate a significant difference as compared with the control only bacteria group.\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7036578/v1/17fee77521aa5794d863e19c.jpg"},{"id":95762755,"identity":"b6102d26-66c6-4854-91f9-06293172e351","added_by":"auto","created_at":"2025-11-12 18:28:48","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":420519,"visible":true,"origin":"","legend":"\u003cp\u003eThe intracellular localization of both Ag@PEG NPs and bacterial strains within RAW 264.7 cells were visualized under fluorescence microscopy. Here, \u003cstrong\u003eA:\u003c/strong\u003e Gray scale images of \u003cem\u003eS. aureus\u003c/em\u003e infected macrophages treated with Ag@PEG NPs. \u003cstrong\u003eB:\u003c/strong\u003e Green fluorescence image of \u003cem\u003eS. aureus\u003c/em\u003einfected macrophages treated with Ag@PEG NPs. \u003cstrong\u003eC:\u003c/strong\u003e Merge A and B images. \u003cstrong\u003eD:\u003c/strong\u003eRed fluorescence images of \u003cem\u003eS. aureus\u003c/em\u003einfected macrophages treated with Ag@PEG NPs. \u003cstrong\u003eE:\u003c/strong\u003e Merge A and D images. \u003cstrong\u003eF:\u003c/strong\u003eMerge C and E images. \u003cstrong\u003eG: \u003c/strong\u003eGray scale image of \u003cem\u003eP. aeruginosa\u003c/em\u003e infected macrophages treated with Ag@PEG NPs. \u003cstrong\u003eH:\u003c/strong\u003eGreen fluorescence image of \u003cem\u003eP. aeruginosa\u003c/em\u003einfected macrophages treated with Ag@PEG NPs. \u003cstrong\u003eI:\u003c/strong\u003e Merge G and H images. \u003cstrong\u003eJ:\u003c/strong\u003eRed fluorescence images of \u003cem\u003eP. aeruginosa\u003c/em\u003einfected macrophages treated with Ag@PEG NPs. \u003cstrong\u003eK:\u003c/strong\u003e Merge G and J images. \u003cstrong\u003eL:\u003c/strong\u003eMerge I and J images.\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7036578/v1/d581156c1808c85441650a13.jpg"},{"id":95802598,"identity":"532badb9-f69d-48a9-9684-042b65e348a0","added_by":"auto","created_at":"2025-11-13 08:28:05","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":61148,"visible":true,"origin":"","legend":"\u003cp\u003eRelease of NO after pulsation of Ag@PEG NP exposed macrophages (A). NO release after co-culture of Ag@PEG NP pulsed macrophages with \u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eP. aeruginosa\u003c/em\u003ecells at ratios of macrophages to bacterial cells of 1:1 (B). n = 6; values are expressed as the mean ± the standard error of the mean. Asterisks indicate a significant difference as compared with the control group\u003c/p\u003e","description":"","filename":"Figure7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7036578/v1/258de492d734e9ee3f3cc711.jpg"},{"id":95762761,"identity":"213f6e95-07ed-4996-8966-d543c3b21040","added_by":"auto","created_at":"2025-11-12 18:28:48","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":45137,"visible":true,"origin":"","legend":"\u003cp\u003eMeasurement of pro- inflammatory and anti-inflammatory Cytokine released from Ag@PEG pulsed Macrophages at 10µg/ml concentration. Here, (A), Pro-inflammatory Cytokine levels (B) anti-inflammatory Cytokine levels All the measurements were performed in triplicate. Values are expressed as mean ±SEM, * (asterisks) indicates the significant difference as compared with control group.\u003c/p\u003e","description":"","filename":"Figure8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7036578/v1/d5d2e82717df738377e51dba.jpg"},{"id":95802135,"identity":"61b204e7-16be-4150-810f-003f55fef187","added_by":"auto","created_at":"2025-11-13 08:26:59","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":86443,"visible":true,"origin":"","legend":"\u003cp\u003eViability of bacterial cells after co-culture with Ag@PEG NPs pulsed macrophage in the presence of a pentoxifylline (POF); n = 6; values are expressed as the mean ± the standard error of the mean.\u003c/p\u003e","description":"","filename":"Figure9.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7036578/v1/d5d5f829182fabd92c3e93c9.jpg"},{"id":95762764,"identity":"8283c368-a768-461a-a185-d3d62023c036","added_by":"auto","created_at":"2025-11-12 18:28:48","extension":"jpg","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":88053,"visible":true,"origin":"","legend":"\u003cp\u003eViability of bacterial cells after co-culture with Ag@PEG NPs pulsed macrophage in the presence of Acetylsalicylic acid (ASA); n = 6; values are expressed as the mean ± the standard error of the mean.\u003c/p\u003e","description":"","filename":"Figure10.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7036578/v1/1cd87dc4f9d84833091f4837.jpg"},{"id":104769246,"identity":"d023cb4f-1175-4822-99a0-7d8682270371","added_by":"auto","created_at":"2026-03-17 04:40:45","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2370208,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7036578/v1/1cb3c82c-c7d6-4081-99a2-eef1fbfb7600.pdf"},{"id":95762749,"identity":"185060fa-f747-4d3b-9919-f3f16cc3dae9","added_by":"auto","created_at":"2025-11-12 18:28:47","extension":"doc","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":129536,"visible":true,"origin":"","legend":"","description":"","filename":"supportingfile.doc","url":"https://assets-eu.researchsquare.com/files/rs-7036578/v1/eef506c24148c36339347fce.doc"},{"id":95762750,"identity":"6dce0b66-3097-44dc-b99b-87036bf8909a","added_by":"auto","created_at":"2025-11-12 18:28:48","extension":"jpg","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":209680,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7036578/v1/354e4fcdc467d14e1c8510ed.jpg"},{"id":95802616,"identity":"83704ebb-ce43-42c8-88bc-36ed5c64b12e","added_by":"auto","created_at":"2025-11-13 08:28:08","extension":"jpg","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":62116,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScheme-1:\u003c/strong\u003eSchematic representation of the macrophage activation and mechanisms of intracellular killing of bacteria in Ag@PEG nanoparticles treatment.\u003c/p\u003e","description":"","filename":"Scheme1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7036578/v1/2dce8406b5428e323eee8b90.jpg"}],"financialInterests":"No competing interests reported.","formattedTitle":"Surface functionalized silver nanoparticles augment macrophage activation followed by intracellular killing of pathogenic bacteria","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eStudies relating to the immunomodulatory activity of Ag-NPs are relatively limited. Immunotherapeutic approaches are the alternative strategies to treat microbial infections as the conventional antibiotics were resistant to different bacterial species. However, the aim of our study is to evaluate the immunomodulatory activity of Ag@PEG-NPs in antibacterial therapy. Multifunctional role of metal nanoparticles enhances its wide applicability in various biomedical application systems as well as in many industrial processes [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Silver has long been recognized as effective antimicrobial agent and thus it is widely used in topical ointments and various creams in its nano forms or ionization state to prevent infection of burns and wounds [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. This use of biological elements for the development of nanoparticles has been designated as \u0026ldquo;green synthesis\u0026rdquo; and is highly considered to be far more beneficial than chemical methods which require toxic chemicals, more complex and expensive chemical reactions. The absence of toxic by-products and consequential decrease in degradation of the particles proved this technique more preferable over physical and classical chemical methods [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. In spite of presence of various bioactive plant materials over the surface of green synthesized Ag-NPs as capping agent it is still shows significant toxicity towards health cells. Thus, more effective surface functionalization using PEG, PLGA, BSA reduces the toxicity of the particles upto considerable limit and increases solubility in aqueous solutions [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Macrophage plays crucial role in innate and cell mediated immunity in response to killing and eliminations of pathogens [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Being potent phagocytic cells, it can eliminate intracellular bacteria through generation of phagosomes using germline-encoded pattern recognition receptors specific for microbial products [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. AgNPs has been found to activate macrophage through inflammatory stimuli which helps for successive elimination of intracellular microbes [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Nishanth and co-workers reported that AgNP treatment with macrophages induced various inflammatory response mainly increased IL-6 production, increased ROS concentration, nuclear translocation of NF-κB, induction of cyclooxygenase-2 (COX-2), and increased tumor necrosis factor-alpha (TNF-α) mediated by NF-κB [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Those inflammatory stimuli are responsible for M1 polarization of macrophage which promotes the antimicrobial killing [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. But the toxicity of bare Ag NPs towards macrophages limits these functions and decreases the viability of macrophage itself without promoting microbicidal effects [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Though it was found that biologically prepared Ag NPs and chitosan coating produces less toxicity to murine RAW 264.7 macrophages [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], but their effective contribution in intracellular killing was not examined in detail. However, there are few instances on the comparative study of surface functionalized green synthesized AgNps mediated macrophage activation and its ability to kill intracellular pathogenic bacteria (both gram positive and negative). Based on the above background this study was conducted to investigate the ability of PEG coated Ag NPs (Ag@PEG NPs) towards activation of RAW 264.7 cells and also to examine the efficacy of those activated RAW 264.7 towards intracellular killing of clinically isolated pathogenic \u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eP. aeruginosa strains.\u003c/em\u003e\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1. Chemicals and reagents\u003c/h2\u003e\u003cp\u003eSilver nitrate (AgNO\u003csub\u003e3\u003c/sub\u003e), polyethylene glycol (PEG), Histopaque 1077, sodium acetate, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT reagent), Acetylsalicylic acid (ASA), Sulfasalazine, indomethacin, pentoxifylline (POF) were procured from Sigma (St. Louis, MO, USA). Minimum Essential Medium (MEM), RPMI 1640, fetal bovine serum (FBS), penicillin, streptomycin, sodium chloride (NaCl), sodium carbonate (Na\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e), sucrose, Hanks balanced salt solution, ethylene diamine tetra acetate (EDTA), dimethyl sulfoxide (DMSO), NaOH Nutrient agar and Luria broth were purchased from Himedia, Mumbai, India. Tris\u0026ndash;HCl, Tris buffer, KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, K\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e, HCl, formaldehyde, alcohol and other chemicals were procured from Merck Ltd., Mumbai, India. All other chemicals of the highest purity grade (MB and cell culture grade) were purchased from Merck Ltd., SRL Pvt., Ltd., Mumbai, India.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2. Green synthesis purification and Surface functionalization of silver nanoparticles\u003c/h2\u003e\u003cp\u003eSilver nanoparticles were synthesized using \u003cem\u003eOcimum gratissimum\u003c/em\u003e leaves extract according to our previously reported method. The purification of Ag-NPs was done by sucrose density gradient centrifugation [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Polyethylene glycol (PEG) loading on green synthesised Ag nanoparticles has been achieved by adopting slightly modified procedure as reported previously [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e\u003cdiv id=\"Sec5\" class=\"Section3\"\u003e\u003ch2\u003e2.2.1. \u003cb\u003eFourier transform infrared spectroscopy.\u003c/b\u003e\u003c/h2\u003e\u003cp\u003eAg-NPs and Ag@PEG were investigated by Fourier transform IR spectroscopy with a PerkinElmer Spectrum RX I Fourier transform IR system with a frequency ranging from 500 to 4,000 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and a resolution of 4 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Potassium bromide (KBr) was used to prepare the pellet for analysis of samples for this study [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section3\"\u003e\u003ch2\u003e2.2.2. \u003cb\u003eDynamic light scattering (DLS) and Zeta potential.\u003c/b\u003e\u003c/h2\u003e\u003cp\u003eThe hydrodynamic diameter of the sample was analyzed by DLS technique using Zetasizer Nano ZS (Malvern, Malvern Hills, U.K.). At first, 100\u0026micro;g/mL concentration of the nanoparticles were prepared and sonicated for 2 min, then dynamic particle sizes were measured by suspending two drops of an aqueous suspension of NPs in 10 mL of Millipore water. When the NPs had absolutely dispersed in water, the samples were analyzed with a DLS analyzer. This measurement was repeated for several times to achieve the average size of the NPs. The zeta potential of the nanoparticles were measured by using a Zetasizer-Nano ZS (Malvern, Malvern Hills, U.K.) [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.3. Bacterial Strains used in this study\u003c/h2\u003e\u003cp\u003eMultidrug resistant and virulent \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e strains were isolated from urine samples of urinary tract infected patients [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] and \u003cem\u003eStaphylococcus aureus\u003c/em\u003e strains were isolated from pus samples [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] in our laboratory. We used Multidrug resistant \u003cem\u003ePseudomonas aeruginosa (PA-7) and Staphylococcus aureus (SA-20)\u003c/em\u003e bacterial strains for this study. The strains were subculture and used throughout the study.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.4. Cell culture\u003c/h2\u003e\u003cp\u003eThe Raw 264.7 cell line (macrophage-like, Abelson leukemia virus transformed cell line derived from BALB/c mice) was procure from NCCS, Pune, India. This cell line was cultured in RPMI 1640 medium with 10% FBS, 100 U/ml penicillin, and 100 \u0026micro;g/ml streptomycin, 4 mM L-glutamine under 5% CO\u003csub\u003e2\u003c/sub\u003e, and 95% humidified atmosphere at 37\u003csup\u003e0\u003c/sup\u003eC for in vitro experiments.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.5. Macrophage Pulsing\u003c/h2\u003e\u003cp\u003eRAW 264.7 cells (1x10\u003csup\u003e6\u003c/sup\u003e cells per milliliter) were pulsed with 1, 5, 10, 25, 50\u0026micro;g/ml Ag@PEG NPs in complete RPMI 1640 medium for different time interval at 37\u0026deg;C.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.6. The dose dependent cytotoxicity of Ag@PEG NPs toward RAW 264.7 cells.\u003c/h2\u003e\u003cp\u003eRAW 264.7 cells were seeded into 96 wells of tissue culture plates containing 180 \u0026micro;l of complete medium and were incubated for 48 h. Ag@PEG NPs were added to the cells at different concentrations (1, 5, 10, 25 and 50 \u0026micro;g/ml), and the mixtures were incubated for 48 h at 37\u0026deg;C in a humidified incubator (NBS) maintained at 5% CO\u003csub\u003e2\u003c/sub\u003e. The cell viability was estimated by MTT assay according to the method described elsewhere [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The plates were read on a microplate reader (model 550, Bio-Rad, Tokyo, Japan) at a wavelength of 570 nm.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e2.7. The duration dependent cytotoxicity of Ag@PEG NPs toward RAW 264.7 cells.\u003c/h2\u003e\u003cp\u003eRAW 264.7 cells were treated as same as the dose (in terms of concentration) dependant study and were incubated for 24h, 48h and 72h at 10 \u0026micro;g/ml concentrations (selected dose) at 37\u0026deg;C in a humidified incubator maintained at 5% CO\u003csub\u003e2\u003c/sub\u003e. The cell viability was estimated by MTT assay.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e2.8. Bacterial cell co-culture with pulsed RAW 264.7 cells.\u003c/h2\u003e\u003cp\u003eThe pulsed RAW 264.7 cells were collected by trypsinization and were centrifuged at 1,500 rpm for 5 min. The pellet cells were washed with PBS at room temperature. After they had been washed three times, the cells were resuspended in RPMI 1640 medium with 5% FBS. Bacterial strains were cultured in liquid media [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. After the culture of bacterial cells, pulsed RAW 264.7 cells and the bacterial cells were co-cultured in a 96 well plate in a ratio of 1:1 (RAW 264.7 cells to bacterial cells) at the density of each cultured cells was 1x10\u003csup\u003e6\u003c/sup\u003e cells per well and were then incubated for 24 hrs at 37\u0026deg;C [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e2.9. Bacterial cell viability assay\u003c/h2\u003e\u003cp\u003eAfter co-culture, each well was washed with sterile saline to remove the dead macrophages and residual bacterial cells and lysed using 200 \u0026micro;l of 1% saponin in sterile water. Cell lysates were plated on solid medium and incubated at 37\u0026deg;C. After 24 hrs of incubation CFU were counted, and the results were expressed graphically.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e2.10. Intracellular localization of nanoparticles and bacteria\u003c/h2\u003e\u003cp\u003eThe intracellular localization of Ag@PEG NPs and both bacterial strains (i.e \u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eP. aeruginosa strains)\u003c/em\u003e were visualized under fluorescence microscopy. For this assay, 100 \u0026micro;l Fluorescein isothiocyanate (FITC) solution (100 \u0026micro;g/ml, dissolved in 0.1 M carbonate\u0026ndash;bicarbonate buffer and vortexed for 1 minute) was added to the Ag@PEG NPs solution (1mg/ml, dissolved in 0.1 M carbonate\u0026ndash;bicarbonate buffer). The mixture was incubated for 2 hrs at 37\u0026deg;C wrapped in aluminium foil to prevent photo bleaching. The FITC conjugated Ag@PEG NPs solution (FITC-Ag@PEG NPs) was washed thrice using ice cold PBS by centrifugation at 8000g to remove excess FITC. The pallet was again resuspended using 1 ml PBS and 10\u0026micro;l of the FITC-Ag@PEG NPs solution was treated with RAW 264.7 cells (1X10\u003csup\u003e6\u003c/sup\u003e/ml) for 8 hrs at 37\u0026deg;C. The both bacterial cells were labelled with Rhodamine B (Rh-B) (10 \u0026micro;g/ml solution of Rh-B incubated with 1ml of each bacterial strain for 1 hr followed by washing at 5000g using PBS). The pulsed RAW 264.7 cells were co-cultured with Rh-B labelled both bacterial cells (1:1) for 4 hrs followed by washing with PBS and layered on to glass slide for fluorescent microscopic imaging (NIKON ECLIPSE LV100POL).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e2.11. NO release assay\u003c/h2\u003e\u003cp\u003eThe NO concentration was measured by a microplate assay method with Griess reagent (1% sulfanilamide, 0.3% naphthylethylene diamine dihydrochloride, 7.5% H\u003csub\u003e3\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e). Briefly, culture supernatants (100 \u0026micro;l) were mixed with 100 \u0026micro;l of Griess reagent. The nitrite concentration in the culture supernatant was measured at a wavelength of 550 nm after 10 min of mixing [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e2.12. Cytokine analysis\u003c/h2\u003e\u003cp\u003eTo investigate the effect of Ag@PEG NPs on cytokine production before and after co-cultured with bacteria, an ELISA technique was used for the determination of interferon-γ (IFN-γ), TNF-α, and IL-12 production. RAW 264.7 cells were cultured at 1x10\u003csup\u003e6\u003c/sup\u003e cells per millilitre and were treated with 10\u0026micro;g/ml concentrations of Ag@PEG for 24 h. another set was prepared where RAW 264.7 cells were cultured with bacterial cells for 24 hrs at 37\u0026deg;C. After treatment, cell-free supernatants were harvested via successive 10-min centrifugations (2,000, 7,000, and 13,000 rpm) and were stored at -80\u0026deg;C until analysis. ELISA was performed by the protocol supplied by the manufacturer company (Invitrogen\u0026trade; eBioscience\u0026trade; ELISA Ready-SET-Go!\u0026trade; Kits).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e2.13. Incubation with POF\u003c/h2\u003e\u003cp\u003eRAW 264.7 cells (macrophages) were co-cultured with bacterial cells in a ratio of 1:1 for 24 hrs in RPMI medium alone, and in the presence of 1 mM POF [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. After incubation, cell viability was measured by MTT assay [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003e2.14. Incubation with ASA and indomethacin\u003c/h2\u003e\u003cp\u003eIn another experiment, ASA and indomethacin at final concentrations of 1 mM and 50 \u0026micro;M respectively, were applied together in the reaction mixture of pulsed RAW 264.7 cells and bacterial cells. After the treatment schedule cell viability was measured by MTT assay [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003e2.15. Statistical Analysis\u003c/h2\u003e\u003cp\u003eEach of the above assays was performed in triplicate. The data were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM, n\u0026thinsp;=\u0026thinsp;6. Comparisons between the means of control and treated group were made by two-way ANOVA test with multiple comparison t-tests, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 as a limit of significance.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003e3.1. Fourier transform infrared spectroscopy:\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eFTIR spectra of Ag and Ag@PEG are presented in Figure-1. The FTIR measurements of Ag-NPs, PEG functionalized Ag-NPs were carried out to identify the possible interaction between coating agents with Ag-NPs. Results of FTIR study showed sharp absorption peaks at about 1635.16cm\u003csup\u003e-1\u003c/sup\u003e, 1451.34cm\u003csup\u003e-1\u003c/sup\u003e, 1018.11cm\u003csup\u003e-1\u003c/sup\u003e 3433.57 cm\u003csup\u003e-1\u003c/sup\u003e for Ag-NPs (Figure-1). The FTIR spectra of Ag@PEG consists several new peaks at 1589.44 cm\u003csup\u003e-1\u003c/sup\u003e, 1385.40cm\u003csup\u003e-1\u003c/sup\u003e, 1350.64 cm\u003csup\u003e-1\u003c/sup\u003e and shifting of peaks from 1451.34cm\u003csup\u003e-1\u003c/sup\u003e to 1458.03cm\u003csup\u003e-1\u003c/sup\u003e and decrease the intensity of the peak at 1818.11cm\u003csup\u003e-1\u003c/sup\u003e, in addition to the characteristic peaks of Ag-NPs suggests the successful loading of PEG on the surface of Ag-NPs.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003e3.2. Dynamic light scattering (DLS) and Zeta potential:\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eAverage particle size, distribution and polydispersity index (PDI) of synthesized silver nanoparticles in solutions were evaluated by DLS technique, which is shown in Figure-2. The DLS pattern revealed that Ag-NPs synthesized by green method had a Z average diameter of 38.33\u0026thinsp;\u0026plusmn;\u0026thinsp;10 nm according to the size distributions by volume with PDI of 0.479. The average particle size of Ag@PEG had a Z average diameter of 58.89\u0026thinsp;\u0026plusmn;\u0026thinsp;16 nm with PDI of 0.569. The zeta potential value of synthesized silver nanoparticles was \u0026minus;\u0026thinsp;21.8 mV and PEG functionalized Ag-NPs showed the zeta potential was \u0026minus;\u0026thinsp;4.08 mV which is shown in Figure-3. The stability of Ag@PEG NPs also checked for 5 moth old NPs solution (stored at RT) using DLS and Zeta potential techniques and found no significant detonation of the stability of NPs (Figure-S1).\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec23\" class=\"Section2\"\u003e\u003ch2\u003e3.3. Effect of Ag@PEG NPs on RAW 264.7 cells.\u003c/h2\u003e\u003cp\u003eThe toxicity of the Ag@PEG NPs toward normal RAW 264.7 cells \u003cem\u003ein vitro\u003c/em\u003e was checked. It was found that there was no significant difference in cell viability between the cells treated with Ag@PEG NPs. The cell viability assay showed that Ag@PEG NPs upto 10\u0026micro;g/ml concentration has no major toxicity. This nanoparticle shows significant toxicity after the concentration of 25mg/ml. It was found that Ag@PEG NPs kills RAW 264.7 cells by 3.79%, 6.883%, 8.92%, 19.23% and 33.72% after 24 h incubation at 1, 5, 10, 25, 50 \u0026micro;g/ml concentrations respectively compared with the negative control. (Figure-4A). On the other hand, in duration dependant study the cell viability assay showed that Ag@PEG NPs was effective in 24hrs after treatment in selected concentration or dose have no toxicity. It was found that Ag@PEG NPs kills RAW 264.7 cells by 9.43%, 14.28%, and 16.32% after 1 day, 2 days and 3 days incubation respectively at 10\u0026micro;g/ml concentration compared with the control (Figure-4B). Hence, these NPs are safe in 10\u0026micro;g/ml concentration and 24 hrs treatment for biomedical applications.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\u003ch2\u003e3.4. Effect of Ag@PEG NP pulsed RAW 264.7 cells on bacterial cells\u003c/h2\u003e\u003cp\u003eThe antibacterial activity of Ag@PEG pulsed RAW 264.7 cells towards both types of bacterial cells was significant. The present result suggested that Ag@PEG NPs pulsed RAW 264.7 cells have greater killing activity toward \u003cem\u003eS. aureus\u003c/em\u003e cells in comparison with \u003cem\u003eP. aeruginosa\u003c/em\u003e. As shown in Figure-5, during the co-culture of pulsed RAW 264.7 cells with both types of bacterial cells in a ratio of 1:1. The intracellular killing of \u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eP. aeruginosa\u003c/em\u003e were substantially higher after 16 h of treatment period. At 24h treatment period the killing percentage of \u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eP. aeruginosa\u003c/em\u003e were slightly reduced. As shown in Figure-5, during the co-culture of pulsed macrophage with bacterial cells, a significant amount of \u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eP. aeruginosa\u003c/em\u003e cell killing induced by pulsed macrophage was observed on 16 hours (85.287% and 91.631%) and 24 hours (92.284% and 88.802%). All the results show significant difference at the level of p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec25\" class=\"Section2\"\u003e\u003ch2\u003e3.5. Intracellular localization of nanoparticles and bacteria\u003c/h2\u003e\u003cp\u003eThe intracellular localization of both Ag@PEG NPs and bacterial strains within RAW 264.7 cells were visualized under fluorescence microscopy. During treatment of \u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eP. aeruginosa\u003c/em\u003e infected RAW 264.7 cells by Ag@PEG nanoparticles, large number of intracellular bacteria were seen in fluorescence images. All the bacteria appeared in the process of destruction within the macrophage as images shows defragmented red florescence. The fluorescence of Ag@PEG NPs was localized in the cytoplasm, with which the bacteria undergoing degradation were often associated (Figure-6). From these results it was clear that both Ag@PEG NPs and bacterial cells are distributed throughout the RAW 264.7 cells or macrophages indicating successful internalization through endocytosis process.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec26\" class=\"Section2\"\u003e\u003ch2\u003e3.6. NO release\u003c/h2\u003e\u003cp\u003eIn the present study, presence of the Ag@PEG stimulates RAW 264.7 cells to generate a significant amount (84.59%) of NO (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in the medium after 16 hrs of incubation period (Figure-7A). Nitric oxide level was also measured in duration dependent manner when pulsed RAW 264.7 cells were co-cultured with both bacterial cells (Figure-7B). Results shows that the NO generation were increased by 1.062-fold, 1.189 fold, 1.266 fold, 1.246 fold, 1.311 fold in pulsed macrophage infected with \u003cem\u003eS. aureus\u003c/em\u003e at 4hrs, 12 hrs, 16 hrs, 24 hrs and 48 hrs respectively compared with the control. In case of \u003cem\u003eP. aeruginosa\u003c/em\u003e infection in pulsed macrophage increases the NO generation by 1.001 fold, 1.117 fold, 1.215 fold, 1.208 fold and 1.333 fold at 4hrs, 12 hrs, 16 hrs, 24 hrs and 48 hrs respectively compared with the control.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec27\" class=\"Section2\"\u003e\u003ch2\u003e3.7. Cytokine estimation from Ag@PEG NP pulsed RAW 264.7 cells.\u003c/h2\u003e\u003cp\u003eThe contributing effects of cytokine production after stimulation of Ag@PEG NPs on RAW 264.7 cells were measured by ELISA assay. After treatment both bacterial cells with Ag@PEG-NPs pulsed RAW 264.7 cells, cell-free supernatants were used to quantify cytokine levels. The results demonstrated significant increases in the levels of pro inflammatory cytokines such as IL-12, TNF-α and decrease anti-inflammatory cytokine IL-10, TGF-β in NPs treated group then control group. Concentrations of TNF-a, IL-12, and TGF-b, IL-10 were quite low in culture supernatants of unpulsed RAW 264.7 cells. The results showed that pulsed RAW 264.7 cells increased the production of TNF-α by 1.68-fold, IL-12 by 1.45-fold compared with controls at 10 \u0026micro;g/ml NPs concentration (Figure-8) and decreased the production of TGF-β by 0.74-fold, and IL-10 by 0.76-fold compared with controls at same dose or concentration (Figure-8). Thus Ag@PEG pulsation elevated of Th1 cytokines and decrease in Th2 cytokines which contributed better immunostimulatory activity as well as better bacterial killing.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec28\" class=\"Section2\"\u003e\u003ch2\u003e3.8. Incubation with POF\u003c/h2\u003e\u003cp\u003eTo check the effective contribution of TNF-α underlying the Ag@PEG NPs pulsed RAW 264.7 cells induced bacterial killing addition of POF in co-culture setup was done. The present study showed that use of POF in culture medium increased the bacterial cell viability more than 90% after co-culture with Ag@PEG NPs pulsed RAW 264.7 cells (Figure-9)\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec29\" class=\"Section2\"\u003e\u003ch2\u003e3.9. Incubation with ASA\u003c/h2\u003e\u003cp\u003eEffect of NO towards pulsed macrophage mediated bacterial cell death was monitored in the presence of ASA (Figure-10). ASA acts as an inhibitor of the COX-2 pathway [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. It was noted that presence of ASA results both types bacterial strains were not killed by the pulsed RAW 264.7 cells. But the protection ability was less than POF co-treatment group.\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThe surface charge and chemical properties of functionalized nanoparticles were determined by various physio-chemical measurements. Among various coating materials, polyethylene glycol (PEG) has been widely used because of their nontoxic, biodegradable, biocompatible properties [22, 23, 24]. In general, PEG functionalization improves drug solubility, increases drug stability, the retention time of the conjugates in blood, and reduces proteolysis and renal excretion, thereby allowing a reduced dosing frequency. This functionalization changes the physical and chemical properties of the biomedical molecule, electrostatic binding, and hydrophobicity results in an improvement in the pharmacokinetic behaviour of the drug [25, 26]. Fourier transforms infrared spectroscopy (FT-IR) study exhibits characteristic peaks for Ag-NPs at 1635.16cm\u003csup\u003e-1\u003c/sup\u003e, 1451.34cm\u003csup\u003e-1\u003c/sup\u003e, 1018.11cm\u003csup\u003e-1\u003c/sup\u003e 3433.57 cm\u003csup\u003e-1\u003c/sup\u003e. The FTIR spectra of Ag@PEG have several new peaks at 1589.44 cm\u003csup\u003e-1\u003c/sup\u003e, 1385.40cm\u003csup\u003e-1\u003c/sup\u003e, 1350.64 cm\u003csup\u003e-1\u003c/sup\u003e and shifting of peaks from 1451.34cm\u003csup\u003e-1\u0026nbsp;\u003c/sup\u003eto 1458.03cm\u003csup\u003e-1\u003c/sup\u003e and decrease the intensity of the peak at 1818.11cm\u003csup\u003e-1\u003c/sup\u003e, in addition to the characteristic peaks of Ag-NPs suggests the successful loading of PEG on the surface of Ag-NPs (Figure-1), DLS analysis confirmed that synthesized silver nanoparticles and functionalized silver nanoparticles were in nanometer size range having particle sizes of 38.33\u0026plusmn; 10 nm with PDI of 0.479 for Ag-NPs and 58.89 \u0026plusmn;16 nm with PDI of 0.569 for Ag@PEG (Figure-2). After attachment of PEG onto the surface of Ag-NPs the average sizes were enhanced. The zeta potential value of synthesized silver nanoparticles was \u0026minus;21.8 mV and PEG functionalized Ag-NPs showed the zeta potential was \u0026minus;4.08 mV (Figure-3). The enhancement of particle size, surface charge and alteration in PDI clearly suggest the successful surface modification of Ag-NPs.\u0026nbsp;\u003c/p\u003e\n\u003cp skip=\"true\"\u003eThe microbial pathogenesis is an event that results of a continuous battle between host and the microbial pathogens. Last few decades, new insights were opened in host immune response to bacterial pathogens, which has been underlying the development of new strategies to modulate immune system against those infecting microbes. Several approaches have been taken up to improve the immune responses against microbial pathogens [27, 28]. \u0026nbsp;Particulate adjuvants, nanoparticles (NPs) have generated a lot of interest due to their unique features. Important pathogens such as \u003cem\u003eStaphylococcus aureus, Pseudomonas aeruginosa, Listeria monocytogenes, Shigella flexneri\u0026nbsp;\u003c/em\u003eetc\u003cem\u003e\u0026nbsp;\u003c/em\u003eadopted different strategies to avoid lysosomal degradation, including escaping phagocytosis, release of toxins that are lethal to phagocytes. \u003cem\u003eStaphylococcus aureus\u003c/em\u003e can avoid destruction by releasing toxin leukocidin AB [29, 30] which form octameric pores in the plasma membrane of the target cells [31] results osmotic imbalance and cell death [29, 30]. \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e eliminate phagocytes as a strategy to escape the immune defense by producing toxins, such as phenazines [32] which induce the reactive oxygen species (ROS) generation [33] causes rapid and overwhelming apoptosis [32].\u003c/p\u003e\n\u003cp\u003ePEG modification reduces the toxicity of Ag-NPs by decreasing excess release of Ag ion in the medium. Moreover, it was also reported that in acidic environment (micro environment of cancer cells) the PEG coated Ag NPs showed more toxicity; while in physiological pH (pH of normal cell environment) it lowers the toxicity significantly [34]. The microbial infected area is also acidic in nature. So, Ag@PEG nanoparticles release more amount of ion whereas in normal cellular environment it release less amounts of ion results this nanocomposites is effective in infected area and kill the microbes but cannot toxic to normal cells. Thus, in the present study Ag@PEG showed lower toxicity towards RAW 264.7 cells and it was more toxic when macrophage cells (RAW 264.7) are present with bacterial cells. This may be due to differences in ion release. Previous report revealed that the drastic reduction of bacterial cell counts by activating immune functions of activated macrophages [35]. \u0026nbsp;In the case of unpulsed macrophage the killing percentage for both bacterial cells were much lower than nanoparticle pulsed macrophage. This may be due to inactivation of macrophages which lowers the phagocytic effects. Activated macrophage secretes various inflammatory mediators such as TNF-\u0026alpha; and NO [17]. These inflammatory mediators have various effects in our body. They activate immune competent cells to recruit at site of infection or directly kill the pathogens and thereby help to cure from infections. \u0026nbsp;In the present study, presence of the Ag@PEG stimulates macrophages to generate a significant amount (84.59%) of NO (p\u0026lt;0.05) in the medium after 16 hrs of incubation period (Figure-7A). This result indicated the presence of a high concentration of NO in the co-culture medium from pulsed macrophages which may attribute to bacterial killing during treatment of pulsed macrophages with bacterial cells. \u0026nbsp;Nitric oxide level was also measured in duration dependent manner when pulsed macrophages were co-cultured with both bacterial cells (Figure-7B). Hence, the amount of this NO was correlated with the cytotoxicity toward bacterial cells. Several studies have evaluated the relationship between inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 [36, 37].\u003c/p\u003e\n\u003cp\u003eThe contributing effects of cytokine production after stimulation of Ag@PEG NPs on macrophage was measured by ELISA assay. After treatment both bacterial cells with Ag@PEG pulsed RAW 264.7 cells, cell-free supernatants were used to quantify cytokine levels. The results demonstrated significant increases in the levels of pro inflammatory cytokines such as IL-12, TNF-\u0026alpha; and decrease anti-inflammatory cytokine IL-10, TGF-\u0026beta; in NP treated group then control group. Concentrations of TNF-a, IL-12, and TGF-b, IL-10 were quite low in culture supernatants of unpulsed macrophages. The results showed that pulsed macrophages increased the production of TNF-\u0026alpha; and decreased the production of TGF-\u0026beta;. Thus Ag@PEG pulsation elevated of Th1 cytokines and decrease in Th2 cytokines which contributed better immunostimulatory activity as well as better bacterial killing. Nanomaterials have been shown to modulate expression of cytokines, which are soluble biological protein messengers that regulate the immune system\u0026nbsp;[38]. Published studies have demonstrated the ability of certain nanomaterials to induce cytokine production, although this appears heavily dependent on a variety of factors, including material composition, size, and method of delivery\u0026nbsp;[39]. In the present study elevated levels of IL-12, and TNF-\u0026alpha; represent critical pathways that are involved in the inflammatory response and differentiation processes as well as confirmed the activation process of macrophages by pulsing with Ag@PEG NPs. Thus the findings indicate that careful titration of Ag@PEG NPs based therapeutic interventions may be successful in elevating the levels of a group of cytokines important for eliciting a Th1-mediated immune response with effective antibacterial actions (Scheme-1).\u003c/p\u003e\n\u003cp\u003eTo check the effective contribution of TNF-\u0026alpha; underlying the Ag@PEG NPs pulsed macrophage induced bacterial killing addition of POF in co-culture setup was done. Recent studies reported the ability of theophylline and POF, both phosphodiesterase inhibitors, to suppress monocyte/macrophage TNF-\u0026alpha; production by increasing the intracellular accumulation of cAMP\u0026nbsp;[40]. The present study showed that use of POF in culture medium increased the bacterial cell viability more than 90% after co-culture with Ag@PEG NPs pulsed macrophages (Fig. 9). Thus this findings indicated that TNF-\u0026alpha; is main causative agent responsible for Ag@PEG NPs mediated antibacterial therapy. The production of TNF-\u0026alpha; by mononuclear phagocytes is regulated by the intracellular levels of cyclic adenosine monophosphate (cAMP)\u0026nbsp;[41]. Exogenous cAMP analogues and substances such as prostaglandin E, which are capable of increasing the intracellular level of cAMP, reduce the release of bioactive TNF-\u0026alpha; by downregulating the expression of the TNF-\u0026alpha; gene\u0026nbsp;[20]. There is increasing evidence that POF may also play a therapeutic role in the inhibition of inflammatory processes. POF has been shown to improve resistance against sepsis or endotoxin challenge in mice, rats, and humans\u0026nbsp;[19, 20], most likely by decreasing circulating TNF-\u0026alpha; levels. POF is able to inhibit the synthesis of messenger RNA for TNF-\u0026alpha; in mouse murine macrophages at the transcriptional level. Also in humans, POF is able to reduce the release of TNF-\u0026alpha; by peripheral blood macrophages.\u003c/p\u003e\n\u003cp\u003eIn contrast to ASA and indomethacin, which totally blocks the synthesis of cyclo-oxygenase intermediate endoperoxides (PGG2, PGH2) [21]. Effect of NO towards pulsed macrophage mediated bacterial cell death was monitored in the presence of ASA along with indomethacin (Figure-10). ASA and indomethacin acts as an inhibitor of the COX-2 pathway [21]. It was noted that presence of ASA and indomethacin protected both bacterial cells from pulsed macrophage mediated killing. But the protection ability was less than POF co-treatment group. Thus, it may be suggested that TNF-\u0026alpha; was the most abundant contributing factor responsible for macrophage activation and intracellular killing of pathogenic bacteria. Moreover, studies have reported that ASA decreases the intracellular internalization of microorganism within activated macropahage [42]. Thus it was manifest that COX pathway plays prevotal role in the process of microbial invasion. In addition, this previous study also showed that iNOS expression in macrophages was increased with ASA treatment, suggesting that iNOS-dependent production is responsible for ASA effects.\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eThis study shows that Ag@PEG NPs efficiently killed bacterial cells after activation of macrophages. The pulsed macrophages show good antibacterial activity. Treatment of Ag@PEG NPs results increased the generation of NO and increase the concentration Th1 type cytokines results the activation of macrophage. This activated macrophage then killed the engulfed bacterial cells. Such a metal nanoparticles offers versatility in that it can simultaneously activate the primary Immune cells as well as kill the ingested microbes.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eAg-NPs: Silver nanoparticles\u003c/p\u003e\n\u003cp\u003eMTT: 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide\u003c/p\u003e\n\u003cp\u003ePBS: Phosphate buffer saline\u003c/p\u003e\n\u003cp\u003eRh-B: Rhodamine B\u003c/p\u003e\n\u003cp\u003ePEG: polyethylene glycol\u003c/p\u003e\n\u003cp\u003eAg@PEG NPs: \u0026nbsp;modified metal nanoparticles ()\u003c/p\u003e\n\u003cp\u003eMФ: Macrophage\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTNF-a:\u0026nbsp;Tumor\u0026nbsp;necrosis factor alpha\u003c/p\u003e\n\u003cp\u003eIL-6: Interleukine-6\u003c/p\u003e\n\u003cp\u003eCOX-2: cyclooxygenase-2\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eiNOS: \u0026nbsp;inducible nitric oxide synthase\u003c/p\u003e\n\u003cp\u003eROS: Reactive oxygen species\u003c/p\u003e\n\u003cp\u003eNF-\u0026kappa;B: Nuclear factor \u0026ndash;\u0026kappa;B\u003c/p\u003e\n\u003cp\u003ePOF: \u0026nbsp;pentoxifylline\u003c/p\u003e\n\u003cp\u003eASA: \u0026nbsp;Acetylsalicylic acid\u003c/p\u003e\n\u003cp\u003ecAMP: \u0026nbsp;cyclic adenosine monophosphate\u003c/p\u003e\n\u003cp\u003eTGF-\u0026beta;: Transforming growth factor\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eConflict of interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare there are no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors express gratefulness to the Belda College, Belda and USIC, Vidyasagar University, Midnapore, University for providing the facilities to execute these studies.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors Contribution:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBD: Conceptualization, Data Curation, Analysis of Data, Investigation, Methodology, Validation and visualization, manuscript writing, editing. JA: Methodology , manuscript writing, editing. SKD: Methodology, manuscript writing, editing. KKM: \u0026nbsp; Methodology, manuscript writing, editing.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eFunding Declaration\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e: \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo funding received for the entire work.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eEthics and Consent:\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBacterial strains were isolated previously prior to ethical permission and consent to participate and publication.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eMurphy C-J (2008) Sustainability as an emerging design criterion in nanoparticle synthesis and applications, J Mater Chem. 18: 2173-2176.\u003c/li\u003e\n\u003cli\u003eSchultz S, Smith D-R, Mock J-J, Schultz D-A (2000) Single-target molecule detection with non bleaching multicolor optical immunolabels, Proce Nat Aca Sci. 97: 996-1001.\u003c/li\u003e\n\u003cli\u003eMandal D, Dash S-K, Das B, Chattopadhyay S, Ghosh T, Das D, Roy S (2016) Bio-fabricated silver nanoparticles preferentially targets Gram positive depending on cell surface charge, Biomed \u0026amp; Pharmacother. 83: 548-558.\u003c/li\u003e\n\u003cli\u003eHabiba K, Bracho-Rincon D-P, Gonzalez-Feliciano J-A, Villalobos-Santos J-C, Makarov V-I, Ortiz D, Avalos J-A, Gonzalez C-I, Weiner B-R, Morell G (2015) Synergistic antibacterial activity of PEGylated silver\u0026ndash;graphene quantum dots nanocomposites. 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Nanoscale Res Lett, 4: 1409\u0026ndash;1420.\u003c/li\u003e\n\u003cli\u003eEndres S, F\u0026uuml;lle H-J, Sinha B, Stoll D, Dinarello C-A, Gerzer R, Weber P-C (1991) Cyclic nucleotides differentially regulate the synthesis of tumour necrosis factor-alpha and interleukin-1 beta by human mononuclear cells. Immunology. 72: 56\u0026ndash;60.\u003c/li\u003e\n\u003cli\u003eKatakami Y, Nakao Y, Koizumi T, Katakami N, Ogawa R, Fujita T (1988) Regulation of tumour necrosis factor production by mouse peritoneal macrophages: the role of cellular cyclic AMP. Immunology. 64: 719\u0026ndash;724.\u003c/li\u003e\n\u003cli\u003eMalvezi A-D, da Silva R-V, Panis C, Yamauchi L-M, Lovo-Martins M-I, Zanluqui N-G, Tatakihara V-L, Rizzo L-V, Verri W-A, Martins-Pinge M-C, Yamada-Ogatta S-F (2014) Aspirin modulates innate inflammatory response and inhibits the entry of Trypanosoma cruzi in mouse peritoneal macrophages. Mediators of inflammation. 19: 2014.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Scheme ","content":"\u003cp\u003eScheme 1 is available in the Supplementary Files section.\u003c/p\u003e\n"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"PEG functionalized Ag-NPs, Immunostimulation, Macrophage activation, intracellular killing of bacteria","lastPublishedDoi":"10.21203/rs.3.rs-7036578/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7036578/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eNanoparticles interact with these immune cells and modulate its function, leading to immunosuppression or immunostimulation. The immunostimulatory activity of modified metal nanoparticles is based on the activation of immune system against pathogenic bacteria. In this study, we evaluate the efficacy of modified metal nanoparticles to activate immune cells (Macrophage) and also to examine the efficacy of those activated macrophages towards intracellular killing of pathogenic \u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eP. aeruginosa\u003c/em\u003e bacterial strains. Synthesized Ag-NPs were modified by polyethylene glycol (PEG). The modified nanoparticles successfully activated macrophage (MФ) evident by the increasing the serum TNF-α level and intracellular NO generation. To check the effective contribution of TNF-α and COX-2 pathway underlying the Ag@PEG NPs pulsed macrophage induced bacterial killing addition of POF and ASA in co-culture setup was done. PEG functionalized nanoparticles enhanced the intracellular killing of pathogenic bacteria in macrophage. We also found that, Ag@PEG NPs pulsed RAW 264.7 secreted elevated level of proinflammatory cytokines. Pulse macrophages were also successfully killed intracellular pathogenic \u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eP. aeruginosa\u003c/em\u003e bacteria in \u003cem\u003ein vitro\u003c/em\u003e setup at 1:1 ratio for 24 h. The use of TNF-α inhibitor and NO blocker confirmed the association between Ag@PEG NPs with TNF-α and NO function. These findings will enrich the biomedical applications of Ag@PEG NPs as a potent immune stimulating agent and this macrophage stimulating efficacy might be an effective way in the bacterial immunotherapy. Such a metal nanoparticles offers versatility in that it can simultaneously activate the primary Immune cells as well as kill the ingested microbes.\u003c/p\u003e","manuscriptTitle":"Surface functionalized silver nanoparticles augment macrophage activation followed by intracellular killing of pathogenic bacteria","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-12 18:28:43","doi":"10.21203/rs.3.rs-7036578/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"8540bec8-818c-462a-99aa-fe0a8aa33756","owner":[],"postedDate":"November 12th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-03-17T04:40:05+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-12 18:28:43","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7036578","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7036578","identity":"rs-7036578","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}