A potential strategy against clinical carbapenem-resistant Enterobacteriaceae: antimicrobial activity study of sweetener-decorated gold nanoparticles in vitro and in vivo | Journal of Nanobiotechnology


Synthesis and characterization of different Au NPs

The synthesis of Au NPs through a green one-pot method is a widely accepted and frequently employed approach known for its environmentally friendly, uncomplicated, and cost-effective characteristics [37]. This method principally involves combining tetrachloroauric acid (HAuCl4) with a reducing agent within the same container to produce organic or inorganic Au NPs [38]. The standard approach for generating Au NPs employs sodium borohydride (NaBH4) as the reducing agent [39]. Previous investigations have highlighted that reducible functional groups like hydroxyl, amino, and amide groups in organic compounds can effectuate the reduction of HAuCl4, yielding decorated Au NPs exhibiting varied functionalities [40]. In our study, we observed that NAS share similar chemical structures.

In this study, we synthesized SAC-decorated Au NPs (SAC_Au NPs), SUC-decorated Au NPs (SUC_Au NPs), ACE-decorated Au NPs (ACE_Au NPs), and ASP_Au NPs by combining SAC (0.05 mmol), SUC (0.05 mmol), ACE (0.05 mmol), ASP (0.05 mmol) (Fig. 1A), and HAuCl4 (0.05 mmol). Due to the distinctive reducibility of NAS, the Au3+ ions in HAuCl4 undergo reduction to Au0 through Au-O or Au–N bonds, facilitating the formation of the aforementioned decorated Au NPs. Residual NAS within the system underwent removal through dialysis. Dynamic light scattering (DLS) measurements revealed that SAC_Au NPs, SUC_Au NPs, ACE_Au NPs, and ASP_Au NPs exhibited average sizes of 52.52, 51.34, 42.56, and 27.18 nm, respectively, with polydispersity indices (PDIs) of 0.511, 0.270, 0.258, and 0.199 (Fig. 1B). This suggests the presence of small and uniformly dispersed sizes among the four Au NPs. As depicted in Fig. 1C, SAC_Au NPs, SUC_Au NPs, ACE_Au NPs, and ASP_Au NPs displayed average zeta potentials of − 10.2, − 24.9, − 28, and − 24.4 mV, respectively, indicative of negatively charged nanoparticles with commendable stability. The UV–visible spectra exhibited absorption peaks within the 500–600 nm range, corresponding to the distinctive surface plasmon resonance of Au NPs (Fig. 1D). The UV–visible spectra of the Au NPs reduced by NaBH4 are shown inAdditional file 1: Figure S1.The noticeable redshift in the absorption peaks of the four NAS-decorated Au NPs (NAS_Au NPs) compared to those of the NaBH4-reduced Au NPs, which confirms the successful decoration of NAS on the gold nanoparticles [41]. The size, PDI, and zeta potential of NaBH4-reduced Au NPs are shown inAdditional file 1: Figure S2. Transmission electron microscopy (TEM) images (Fig. 1E) unveiled uniform morphology and reduced particle sizes for SAC_Au NPs, SUC_Au NPs, ACE_Au NPs, and ASP_Au NPs, approximately measuring 30, 28, 35, and 12 nm, respectively. The TEM sizes proved smaller than the DLS sizes, as DLS reflects hydrodynamic size while TEM evaluates the size of dry particles [42]. Notably, smaller-sized Au NPs have demonstrated enhanced antimicrobial activity [43], and ASP_Au NPs emerging as the standout among the four Au NPs due to their stability, well-defined circular shape, minimal size, and uniform dispersion. Further, the extent of NAS bound to the AuNPs was measured using the ortho-pthaldehyde (OPA) fluorescence assay [44]. As shown in Additional file 1: Table S1, it appears that approximately half of the NAS have bound to the Au NPs. OPA reacts with primary amines to produce fluorescence, and the lower binding efficiency observed may be due to some NAS participating in the reduction process, while others are involved in conjugation. This could result in alterations to the amino nitrogen groups, preventing their interaction with OPA. Additionally, we prepared standard Au NPs utilizing the NaBH4 reduction method for subsequent experimental control.

Fig. 1
figure 1

Characterization of different Au NPs. A Molecular structures of SAC, SUC, ACE, and ASP. B Particle size and dispersity of SAC_Au NPs, SUC_Au NPs, ACE_Au NPs, and ASP_Au NPs. C Zeta potential of SAC_Au NPs, SUC_Au NPs, ACE_Au NPs, and ASP_Au NPs. D UV–Vis spectra of SAC_Au NPs, SUC_Au NPs, ACE_Au NPs, and ASP_Au NPs. E TEM images of SAC_Au NPs, SUC_Au NPs, ACE_Au NPs, and ASP_Au NPs

For in vitro antimicrobial activity, antibiofilm effects, biocompatibility, and in vivo model studies, the concentrations of SAC_Au NPs, SUC_Au NPs, ACE_Au NPs, and ASP_Au NPs were kept consistent with those of the Au NPs control group. Considering the challenges in determining post-dialysis concentrations of SAC, SUC, ACE, and ASP, their concentrations were maintained at levels similar to those of the pre-dialysis SAC_Au NPs, SUC_Au NPs, ACE_Au NPs, and ASP_Au NPs groups. In all experiments, the Au NPs control group refers to the group of NaBH4-reduced Au NPs.

Antimicrobial activity of four NAS_Au NPs

As depicted in Additional file 1: Table S2, we selected 30 unique clinical CRE strains and 2 standard strains assessed their drug resistance profiles using the microbroth dilution technique. Polymerase chain reaction (PCR) and sequencing methodologies were employed to elucidate their resistance mechanisms, certain mechanism data were drawn from our prior studies [45, 46]. The chosen strains exhibited resistance mechanisms spanning all four classes of carbapenemases [47], encompassing Ambler class A (KPC, SHV, TEM, CTX-M, etc.), class B (NDM, IMP, etc.), class C (AmpC, CMY, etc.), and class D (OXA). Additional resistance mechanisms, such as efflux pumps, OmpK37 mutations, and downregulation of OmpC and OmpF, were also implicated. Overall, these strains displayed pronounced resistance to ertapenem (ETP) (≥ 64 µg/mL). Accordingly, we selected ETP as the antibiotic control for subsequent experiments, maintaining its concentration consistent with that of the ASP_Au NPs group to underscore the clinical applicability of our formulated Au NPs in addressing CRE-related challenges.

As delineated in Table 1, the MIC values of SAC_Au NPs, SUC_Au NPs, and ACE_Au NPs against the 32 tested bacterial strains all exceeded ≥ 256 µg/mL, signifying a lack of notable antimicrobial activity. Strikingly, ASP_Au NPs demonstrated exceptional antimicrobial potency against all strains, displaying MIC values ranging from 8 to 16 µg/mL, except for CG1330, which exhibited an MIC value of 4 µg/mL. The remarkable antimicrobial efficacy of ASP_Au NPs vis-à-vis the other three types of Au NPs may be attributed to their uniform particle shape, diminutive size, and the presence of ester, methyl, and phenyl moieties, which confer increased hydrophobicity. Enhanced hydrophobicity facilitates the internalization of particles into bacterial cells [48]. Considering ASP_Au NPs as a member of the NAS_Au NPs group with exceptional antimicrobial effects, we designated them for further characterization and subsequent exploration encompassing antimicrobial activity, antimicrobial mechanisms, antibiofilm activity, biocompatibility, anti-inflammatory properties, and in vivo antimicrobial assessments.

Table 1 Antimicrobial susceptibility of the SAC_Au NPs, SUC_Au NPs, ACE_Au NPs and ASP_Au NPs against the 32 strains used in this study

Further characterization of ASP_Au NPs

Given the potential of ASP_Au NPs in addressing clinical CRE challenges, we conducted supplementary characterization through X-ray photoelectron spectroscopy (XPS) and Fourier-transform infrared spectroscopy (FTIR) to gain deeper insights into ASP_Au NPs. As demonstrated in the XPS survey spectrum (Fig. 2A), ASP exhibited three discernible absorption peaks at approximately 530 eV, 400 eV, and 288 eV, correlating with the O1s, N1s, and C1s spectra, respectively. In contrast, ASP_Au NPs manifested three prominent absorption peaks at approximately 530 eV, 288 eV, and 87 eV, corresponding to the O, C, and Au elements, respectively. A comparison of the two spectra unveiled the disappearance of the N element peak and the amplification of the Au element peak in ASP_Au NPs, suggesting the plausible interaction between ASP’s amino and amide groups with Au, leading to the establishment of Au–N bond connections and consequently causing the obliteration of the absorption peaks that were initially attributed to the amino and amide groups in ASP. To substantiate this hypothesis, we subsequently executed fitting of the Au 4f, N1s, and O1s spectra to scrutinize specific electron transfers during the chemical reaction (Additional file 1: Figure S3). The Au 4f spectra (Additional file 1: Figure S3A, B) exhibited two distinct absorption peaks for both ASP_Au NPs and standard Au NPs reduced by NaBH4, denoting Au 4f5/2 and Au 4f7/2. In view of the database and binding energy positions, these peaks indicated the presence of Au0 formation. Additionally, ASP_Au NPs displayed a feeble absorption peak at around 85.78 eV, suggesting marginal Au1+ formation, possibly attributable to electron transfer with -OH or nitrogen-containing groups. The N1s spectra (Additional file 1: Figure S3C, D) displayed two prominent absorption peaks for ASP located at 400.14 eV and 401.57 eV, attributed to –NH2 and –NH–, respectively. In ASP_Au NPs, two absorption peaks emerged at 397.06 eV and 399.78 eV, also corresponding to –NH2 and –NH–, implying a shift in the nitrogen-containing group’s peak pattern after loading Au onto ASP, signifying electron transfer between Au and N, leading to the establishment of an Au–N bond. The O1s spectra (Additional file 1: Figure S3E, F) unveiled conspicuous absorption peaks for ASP at 531.12 eV, 532.12 eV, and 533.88 eV, aligning with C = O, O = C–O, and C–OH, respectively. In comparison, ASP_Au NPs exhibited absorption peaks at 531.88 eV, 532.48 eV, and 532.92 eV, ascribed to C = O, O = C–O, and C–OH, respectively. The only notable shift was observed in the C–OH peak, suggesting electron transfer between C–OH and Au after Au loading, likely due to the emergence of a small quantity of Au1+.

Fig. 2
figure 2

Further characterization of ASP_Au NPs. A XPS spectra of ASP and ASP_Au NPs. B FTIR spectra of ASP, Au NPs, and ASP_Au NPs

The findings from FTIR analyses (Fig. 2B) indicated that the peak patterns of ASP_Au NPs resembled those of Au NPs. ASP displayed multiple absorption peaks, with a distinct peak at 1666.28 nm being the characteristic peak of ASP [49]. Following Au loading, ASP_Au NPs exhibited conspicuous shifts in absorption peaks at wavelengths of 611 nm, 1666.28 nm, and 3339.16 nm, attributed to N–H, C = O–N, and –NH2 [50]. The disappearance of these three peaks implied the reaction between ASP’s amide structure (C = O–N–H) and amino group (–NH2) with Au, whereby the hydrogen on the nitrogen atom underwent substitution by Au to establish an Au–N bond, consistent with the XPS analysis. The distinct characteristic peaks of Au NPs, ASP_Au NPs, and ASP are presented in Additional file 1: Figure S4. FTIR of the remaining three nano-cargos are available in the Additional file 1: Figure S5.

In vitro antimicrobial activity study of ASP_Au NPs

To investigate the antimicrobial activity of the raw materials, we conducted a microbroth dilution assay using the 32 bacterial strains. We evaluated the antimicrobial effects of ASP, Au NPs, and ASP_Au NPs separately. The results, as shown in Table 2, revealed that ASP and Au NPs alone had MIC values ≥ 256 µg/mL, indicating minimal antimicrobial activity as anticipated. However, ASP_Au NPs demonstrated remarkable antimicrobial potency. Although reports suggest that NAS shows some antimicrobial activity at concentrations of 1500 µg/mL and above [51], we believe that this concentration is much greater than the clinically used antibiotic doses and might cause certain side effects [52], making them unsuitable for use as antimicrobial agents.

Table 2 Antimicrobial susceptibility of the ASP, Au NPs, and ASP_Au NPs against the 32 strains used in this study

For further insights into the effects of ASP_Au NPs on CRE strain growth kinetics, selected strains underwent growth curve analysis. As depicted in Fig. 3A, strains treated with phosphate-buffered saline (PBS), ETP, ASP, and Au NPs displayed normal growth, while ASP_Au NPs treatment led to effective antimicrobial action, preventing bacterial growth even after 24 h. The concentrations for each group were previously specified. Furthermore, to directly observe the morphological changes of bacteria after different treatments, a scanning electron microscopy (SEM) analysis was performed, and DC8647, FK3006, and CG1381 were randomly selected as the experimental strains. The SEM images (Fig. 3B) revealed that bacteria treated with ASP_Au NPs at the MIC concentration exhibited flattened and wrinkled morphology, along with distorted and ruptured cell bodies. In contrast, the bacteria in the other treatment groups maintained regular and intact cellular structures. Elevated ROS levels and membrane permeability changes are potential antimicrobial mechanisms of nanomaterials [53]. This aligns with our hypothesis, prompting us to conduct related experiments. The ROS detection assay demonstrated that ASP_Au NPs significantly raised ROS levels in strains DC8647, FK3006, and CG1381. The ROS increase displayed a dose-dependent pattern and even induced substantial ROS elevation in bacteria below the MIC concentration (Fig. 3C). To confirm that ROS is the core antimicrobial mechanism of ASP_Au NPs, we further performed ROS clearance experiments to investigate whether ASP_Au NPs still have significant antimicrobial activity after clearing ROS. In this study, quercetin, a plant-derived natural flavonoid known for its powerful antioxidant activity and ROS scavenging properties [54, 55], was used as the ROS scavenger. After quenching ROS, the MIC values of ASP_Au NPs for the same bacterial strains increased from 4–16 µg/mL to ≥ 256 µg/mL, an MIC escalation of at least 16–64 fold (Additional file 1: Table S3). This confirmed that elevated ROS levels are the primary antimicrobial mechanism of ASP_Au NPs. Additionally, we investigated the impact on inner membrane permeability. In the propidium iodide (PI) membrane permeability assay (Additional file 1: Figure S6), ASP_Au NPs at various concentrations significantly increased membrane permeability compared to the PBS control group (represented as 0 µg/mL), as evidenced by the corresponding rise in fluorescence intensity. Furthermore, ASP_Au NPs at the MIC concentration (8 µg/mL) exhibited a sharp increase in fluorescence intensity, albeit to a lesser extent at sub-inhibitory concentrations, but still with significant statistical differences. This suggests that ASP_Au NPs can interfere with bacterial morphology and normal physiological functions. Conversely, Au NPs and ASP at different concentrations did not exhibit a notable increase in fluorescence intensity. This finding aligns with the observed changes in bacterial morphology under scanning electron microscopy. Therefore, membrane permeability could potentially serve as a secondary antimicrobial mechanism of ASP_Au NPs.

Fig. 3
figure 3

The antimicrobial ability of ASP_Au NPs in vitro and its core mechanism. A Growth curves of clinical CRE strains after treatment with different formulations. B Representative SEM images of clinical CRE strains after treatment with different formulations. C ROS levels in clinical CRE strains after treatment with different concentrations of formulations

Moreover, to exclude potential synergistic effects between ASP and Au NPs that might impact the study, a checkerboard assay was conducted for validation. As depicted inAdditional file 1: Table S4, within the randomly chosen experimental strains, both the combination treatment group and the monotherapy group exhibited MIC values of ≥ 256 µg/mL. This suggests the absence of a significant synergistic effect over a broad range. This implies that the antimicrobial potency of ASP_Au NPs originated from the “decoration” of ASP onto Au NPs, rather than a mere “synergistic” effect.

In vitro biofilm inhibition study of ASP_Au NPs

Bacterial biofilms are communities of bacteria attached to surfaces, providing internal bacteria with significantly greater antibiotic resistance, up to thousands of times more [56]. In the context of hospital infection control, biofilms formed on medical devices and wound surfaces greatly impact patient health [57]. Furthermore, within the food industry, microbial biofilms often develop on food packaging, posing risks to food safety [58, 59]. Thankfully, numerous studies have reported the antimicrobial biofilm activity of various nanoparticles [60]. Hence, our focus was to investigate whether the ASP_Au NPs we synthesized possess biofilm inhibition properties, using a biofilm inhibition assay with crystal violet staining. Simultaneously, we employed confocal live/dead staining to directly observe the viability of bacteria within the biofilm.

In the biofilm inhibition assay, we selected six strains randomly as experimental subjects, and the concentration of ASP_Au NPs was determined based on 1/2 of the MIC. The concentrations of ETP, ASP, and Au NPs were specified earlier. Figure 4A outlines the general process of the biofilm inhibition assay. As depicted in Fig. 4B, the group treated with ASP_Au NPs significantly impeded biofilm formation, whereas the ETP and Au NPs groups did not exhibit this effect. Intriguingly, we also observed a slight inhibitory effect on biofilm formation by ASP. Regarding this observation, we found supporting evidence in the relevant literature that ASP has potential ability to inhibit biofilm formation [61, 62]. This implies that the potential ability of ASP_Au NPs to inhibit biofilm formation might stem from the decoration of ASP. Furthermore, confocal live/dead staining substantiated this observation. As depicted in Additional file 1: Figure S7, green fluorescence represents live bacteria, while red fluorescence indicates dead bacteria. At sub-inhibitory concentrations, ASP causes a slight reduction in bacterial density within the biofilm, while ASP_Au NPs are able to kill nearly half of the bacteria. Consequently, We contend that ASP_Au NPs exhibit superior antibiofilm activity compared to ASP and Au NPs because they can better penetrate bacterial biofilms and kill more bacteria, resulting in the inhibition of biofilm growth within the same timeframe.

Fig. 4
figure 4

Inhibition of biofilm formation by ASP_Au NPs in vitro. A Schematic illustration of the crystal violet staining method to assess biofilm formation inhibition. B Crystal violet staining results at OD595 after treatment with different formulations in clinical CRE strains

Safety assessment of ASP_Au NPs

In order to simulate in vivo safety and further explore the therapeutic effects, we conducted hemolysis and cytotoxicity assays. The hemolysis assay, depicted in Fig. 5A, demonstrated that ASP_Au NPs at concentrations 2–8 times the MIC (≤ 32 µg/mL) did not manifest any hemolytic activity, remaining below the defined cutoff of hemolysis rates ≤ 5%. For the cytotoxicity assay, the CCK-8 assay method was employed. As illustrated in Fig. 5B, exposure to ASP_Au NPs at concentrations 2–8 times the MIC (≤ 32 µg/mL) had no impact on cell viability. Collectively, ASP_Au NPs exhibited antimicrobial and antibiofilm effects without eliciting corresponding toxicity.

Fig. 5
figure 5

Safety evaluation of ASP_Au NPs. A Hemolytic effects of ASP_Au NPs at various concentrations. B Cell toxicity of ASP_Au NPs at different concentrations using the CCK-8 assay. Statistical differences in OD450 readings indicate cytotoxicity

In vivo antimicrobial performance study

Building upon the remarkable in vitro antimicrobial efficacy and robust safety profile of ASP_Au NPs, we proceeded to investigate their effects within a Galleria mellonella infection model and a mouse acute intra-abdominal infection model. The Galleria mellonella infection model was chosen to validate the in vivo antimicrobial performance of ASP_Au NPs. Multiple authoritative studies have highlighted Galleria mellonella‘s role as a stable in vivo model, characterized by its cost-effectiveness, user-friendliness, and absence of ethical limitations. This model produces results similar to those achieved through vertebrate in vivo experiments [63,64,65]. In our initial step, we introduced various concentrations of ASP_Au NPs into healthy Galleria mellonella to gauge in vivo safety and dismiss the possibility of drug-induced mortality. Illustrated in Additional file 1: Figure S8, all Galleria mellonella (10 per group) injected with ASP_Au NPs survived across the wide concentration range tested. Consequently, DC5113 and FK7513 were selected as the experimental strains to establish a CRE infection model. Adjustments were made to the methods according to prior studies [66]. The general experimental procedure is outlined in Fig. 6A. Succinctly, healthy Galleria mellonella weighing between 200–300 mg were distributed into five groups: PBS, ETP, ASP, Au NPs, and ASP_Au NPs, with 10 larvae per group. Bacterial suspensions (DC5113, 10 µL, 1.5 × 108 CFU/mL; FK7513, 10 µL, 7.5 × 106 CFU/mL) were microsyringe-injected into the penultimate left limb under consistent conditions. After a 2 h bacterial exposure, ASP_Au NPs were injected into the penultimate right limb at a concentration of 16 µg/mL, as determined from the MIC in the in vitro antimicrobial assay. The concentrations of ETP, ASP, and Au NPs remained as previously indicated. Larval survival was documented daily, with mortality ascertained in the absence of response to physical stimuli. As demonstrated in Fig. 6B, Galleria mellonella infected with DC5113 and treated with ASP_Au NPs exhibited a 100% survival rate, while those infected with FK7513 and treated with ASP_Au NPs showed a 70% survival rate. Conversely, larvae in the PBS, ETP, ASP, and Au NPs groups predominantly perished within 7 days. These findings underscored the notable enhancement in the survival rate of CRE-infected Galleria mellonella through ASP_Au NPs, aligning with mouse model results. Furthermore, bacterial colony counting was conducted to reflect the in vivo antimicrobial effects of ASP_Au NPs, with slight decorations based on bacterial strain characteristics [67]. Briefly, bacteria resistant to CRE strains were selectively isolated from Galleria mellonella a day after standard treatment. Following the euthanasia of four larvae per group, their bodies were processed, yielding a suspension in PBS. A 10 µL aliquot of the suspension was placed onto an Luria Bertani (LB) agar plate containing ETP, and visible colonies were tallied the following day. As depicted in Fig. 6C, the bacterial load in Galleria mellonella treated with ASP_Au NPs was markedly lower than that in the PBS, ETP, ASP, and Au NPs groups, for both DC5113 and FK7513 infections. The ASP_Au NPs treatment group witnessed nearly a 2 log10 CFU/g reduction in bacterial load, whereas no significant differences were noted in the other groups. These results affirm the remarkable in vivo antimicrobial properties of ASP_Au NPs.

Fig. 6
figure 6

In vivo antimicrobial performance of ASP_Au NPs. A General experimental procedure of the Galleria mellonella bacterial infection model. B Survival of Galleria mellonella larvae infected with clinical CRE and treated with different formulations at different time points. C Bacterial load in Galleria mellonella larvae infected with clinical CRE and treated with different formulations after 24 h. D General experimental procedure of the acute intraperitoneal infection model in mice. E Survival of mice infected with clinical CRE and treated with different formulations at different time points

Regarding the mouse acute intra-abdominal infection model, Fig. 6D outlines the comprehensive procedure for in vivo mouse experiments. We employed immunosuppressant cyclophosphamide (150 mg/kg) to induce immune-deficient mice, simulating individuals with compromised immune function. DC8647 was selected as the experimental strain. Following three consecutive intraperitoneal immunosuppressant injections, mice were inoculated with a bacterial suspension (200 µL, 1.5 × 108 CFU/mL) supplemented with 5% yeast extract into the peritoneal cavity [68]. Given the frequent clinical application of carbapenems like ETP, an ETP control group was incorporated. The mice were divided into five groups—PBS, ETP, ASP, Au NPs, and ASP_Au NPs—each consisting of 10 mice. Previous reports informed the methods employed to construct mouse models in vivo [69]. After a 2 h bacterial exposure, the corresponding drugs were administered at 0 h, 12 h, and 24 h. ASP_Au NPs were administered at a concentration of 10 mg/kg, as previously described, through a 200 µL injection. Mouse survival was monitored every 12 h, alongside measurement of the mice’s body weight. As revealed in Fig. 6E, mice treated with ASP_Au NPs showed a 70% survival rate, while those in the PBS, ETP, ASP, and Au NPs groups succumbed within 48 h. These results highlight the potential of ASP_Au NPs in countering CRE infections in vivo and significantly improving the survival rates of infected mice. Moreover, changes in mouse body weight were tracked as an indirect indicator of the drug’s effects. As depicted in Additional file 1: Figure S9, mice treated with ASP_Au NPs exhibited a significantly slower decrease in body weight compared to those in the PBS, ETP, ASP, and Au NPs groups, underscoring the in vivo anti-infective effects of ASP_Au NPs. To conclude, both the mouse model and Galleria mellonella model corroborate the potential of ASP_Au NPs as a promising option against clinical CRE infections.

In vivo biocompatibility assessment

In light of the demonstrated efficacy of ASP_Au NPs treatment in mice, an in-depth exploration of the inflammatory response and physiological alterations in mice was conducted to assess the physiological ramifications of ASP_Au NPs in vivo. The mouse model methodology was replicated, except for the administration of immunosuppressants and bacterial inoculation, with the objective of observing the impact of ASP_Au NPs on mouse immune response and physiological functions. Initially, histopathological sections were prepared from the heart, liver, spleen, lung, and kidney of mice that were sacrificed 48 h post-treatment. Hematoxylin and eosin (H&E) staining was carried out to evaluate the infiltration of inflammation in various organ tissues. As illustrated in Additional file 1: Figure S10, the organ tissue morphology in mice treated with ASP_Au NPs appeared normal and similar to the PBS group, with no marked indications of inflammation infiltration or tissue congestion. This suggests that ASP_Au NPs are nonhazardous to mouse organ tissues and do not elicit excessive inflammatory reactions.

Subsequently, blood samples were obtained via retro-orbital bleeding for comprehensive blood count analysis in order to monitor the differential leukocyte count. The outcomes, as shown in Additional file 1: Figure S11, indicated that the counts of white blood cells (WBC) and neutrophils (NEUT) in mice treated with ASP_Au NPs were comparable to those in the PBS group, with all values falling within the normal reference range (WBC: 0.8–10.6 109/L; NEUT: 0.23–3.6 109/L). This suggests that ASP_Au NPs did not lead to a significant rise in white blood cell count that could trigger an undue immune response. Additionally, an analysis of biochemical markers was performed by subjecting the blood samples to centrifugation and serum testing. This analysis focused on eight liver function markers, four cardiac enzyme markers, and three kidney function markers, all in line with clinical practice. As depicted in Additional file 1: Figure S12, all the biochemical markers in mice treated with ASP_Au NPs showed no notable disparities in comparison to the PBS group, with the exception of alkaline phosphatase (ALP) and creatinine (CREA) levels, which were below the reference range. Meanwhile, the other biochemical markers remained within the normal reference range, which is outlined in Additional file 1: Table S5. It is important to note that elevated ALP and CREA levels might imply impaired liver or kidney function. However, it usually holds no clinical significance when these markers fall below the reference range and might be attributed to methodological factors. In conclusion, ASP_Au NPs have demonstrated outstanding biocompatibility in vivo, positioning them as promising candidates for potential in vivo applications.

Evaluation of anti-inflammatory effects

Commercially available NAS has shown specific anti-inflammatory effects [70]. As a NAS member, ASP also exhibits analgesic properties similar to nonsteroidal anti-inflammatory drugs, and it can diminish the levels of the inflammatory cytokine IL-6 [71]. Hence, the hypothesis was formulated that ASP_Au NPs could possess potential anti-inflammatory effects. To validate this hypothesis, real time quantitative PCR (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA) were conducted. For the RT-qPCR experiment, an inflammatory response was triggered in RAW264.7 mouse macrophage cells in vitro utilizing lipopolysaccharide (LPS) [72]. The LPS-stimulated cells were divided into two groups: the PBS group and the ASP_Au NPs group. The ASP_Au NPs concentration was determined based on the MIC from the antimicrobial assay, which was 16 µg/mL. Following 4 h of drug treatment, cellular RNA was extracted for RT-qPCR analysis to quantify the relative expression levels of inflammatory cytokines. As depicted in Fig. 7A, the levels of IL-1β and TNF-α in the ASP_Au NPs group were significantly downregulated compared to the PBS group, indicating the potential anti-inflammatory effect of ASP_Au NPs. For the ELISA experiment, a similar approach to the RT-qPCR experiment was adopted, wherein IL-1β and TNF-α protein detection kits were utilized to measure the cytokine levels in the cell supernatant obtained after centrifugation. As shown in Fig. 7B, the protein levels of IL-1β and TNF-α in the ASP_Au NPs group were significantly diminished in comparison to the PBS group, aligning with the outcomes of the RT-qPCR analysis. Thus, our findings suggest that ASP_Au NPs may hold potential anti-inflammatory effects.

Fig. 7
figure 7

Evaluation of the anti-inflammatory performance of ASP_Au NPs. A Gene expression levels of IL-1β and TNF-α mRNA in cells treated with PBS and ASP_Au NPs measured by RT-qPCR. B Protein levels of IL-1β and TNF-α in cells treated with PBS and ASP_Au NPs detected using an ELISA assay


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