The Biocompatibility and Toxin-Neutralization Capacity of EM@MoS2
The CCK-8 assay on RAW 264.7 cells was applied to assess the cytotoxicity of MoS2 NDs and EM@MoS2. As shown in Fig. S5, when the concentrations of two nanomaterials were increased to 200 mg/L, relative cell viabilities still maintained at a high level (approximately up to 90%), and there were no significant differences between MoS2 NDs group and EM@MoS2 group. However, the cell survival rate decreased to 56.77%, after treatment with 400 mg/L of MoS2 NDs, which was notably lower than that of cells treated with 400 mg/L EM@MoS2 (78.10%). These data indicated that both MoS2 NDs and EM@MoS2 were not cytotoxic to cells even at a concentration of 200 mg/L, consistent with previous studies. Of note, EM@MoS2 exhibited higher cytocompatibility than MoS2 NDs at a high concentration (400 mg/L), which might be attributed to the toxicity isolation effect of erythrocyte membranes.
The capacity of EM@MoS2 to neutralize toxins and further avoid hemolysis was tested using α-hemolysin derived from staphylococcus aureus. As presented in Fig. 2a and 2c, after pretreated with toxin + EM@MoS2 or toxin + anti-α-toxin, supernatants in samples were clear and corresponding absorbance at 540 nm was relatively low, demonstrating that EM@MoS2 had an α-hemolysin neutralizing ability comparable to that of anti-α-toxin antibody. On the contrary, severe hemolysis was observed in toxin and toxin + MoS2 NDs group, implying MoS2 NDs alone is ineffective to prevent α-haemolysin-induced erythrocyte lysis. Subsequently, the efficiency of EM@MoS2 to absorb α-hemolysin was determined. As presented in Fig. 2b and 2d, after pretreated with EM@MoS2 (200 mg/L, 500 µL), toxin with an amount up to 5 µg caused no apparent hemolysis, indicating 100 µg of EM@MoS2 was able to neutralize at least 5 µg of α-haemolysin. However, when toxin amount increased to 10 µg, partial hemolysis was observed, indicating that the toxins cannot be completely captured and some unbound toxins attacked the erythrocyte membrane. Furthermore, to investigate the effect of EM@MoS2-detained toxins on cell adhesion and proliferation, cell growth was assessed by cytoskeleton staining (Fig. 2e). In control group, cells with spindle-like morphology spread flat and packed tightly. In contrast, in α-toxin group, the number of adhering cells decreased to a large extent, and the cytoplasm became crumpled and even cracked (white circles), suggesting severe impairment caused by the toxin. As expected, there were still a large number of cells attaching and spreading well on the plates, with only minor injuries, after treatment with toxin-bound EM@MoS2. Overall, EM@MoS2 was able to efficiently absorb pore-forming toxins and keep them away from normal cells, thus contributing to reducing the virulence of biofilms.
In Vitro Antibiofilm Activity of MoS 2 NDs and EM@MoS2.
With the bacterial metabolic pathway spontaneously altering towards anaerobic glycolysis, biofilm microenvironment is featured by a low pH (4.5-6.5) and high H2O2 content (100-300 µmol/L), which would promote the generation of ROS. Based on good photothermal capacities and peroxidase-like properties, the in vitro anti-biofilm abilities of MoS2 NDs and EM@MoS2 were evaluated. Standard plate counting tests in vitro (Fig S8a and S8c) showed that MoS2 NDs and EM@MoS2 reduced MRSA viability by 0.28 lg CFU/mL and 0.23 lg CFU/mL, respectively, which is accordingly equivalent to a moderate antibiofilm efficiency of 46.72% and 41.27%, respectively. Hyperthermia therapy, wherein the treating temperature was maintained slightly above 50 ℃ for 5 min, had a higher antibiofilm efficiency of 70.61% comparing with MoS2 NDs and EM@MoS2 groups. Particularly, the bacterial viability in EM@MoS2 + NIR group decreased by 2.47 lg CFU/mL (antibacterial rates of 99.66%).
To visualize the role of nanomaterials in disrupting the stacking biofilms, SEM was applied to further observe the changes in bacterial morphology and density after treatment. As shown in Fig. 3a, compared with control group, bacteria cells were distributed, to a moderate extent, more sparsely and some bacterial membranes became rough even ruptured (red arrows) following treated with MoS2 NDs (or EM@MoS2), indicating that a certain degree of impairment had occurred. This trend was more noticeable in the NIR group. When subjected to EM@MoS2 and NIR simultaneously, the amount of adherent bacteria decreased considerably and a substantial fraction of bacteria had suffered irreversible damage, including deformation, perforation, and fragmentation. Additionally, although sub-10 nm MoS2 NDs were hardly visible in MoS2 NDs group, EM@MoS2 or its aggregates (denoted as red points) could be found both on the bacterial surface and within the biofilms in EM@MoS2 and EM@MoS2 + NIR groups. Thus, these nanomaterials were able to penetrate into the tightly packed biofilm structure to cause hyperthermia and generate ROS in situ. In summary, these findings revealed that MoS2 NDs or EM@MoS2 have the potential to physically disintegrate the biofilm, particularly when combined with hyperthermia therapy. Subsequently, the residual biofilm biomass of all samples was qualitatively and quantitatively assessed using confocal laser scanning microscopy (CLSM). As shown in Fig. 3b and 3d, the control group was completely covered with a dense green biofilm structure, with nearly no red signals. While in MoS2 NDs EM@MoS2 groups, a slight redshift occurred and quite a few irregular voids of varying size appeared, suggesting that a small proportion of the biofilm fragments were depolymerized from the substrates. Consequently, there were 61.05% and 69.14% biomass left respectively. In the NIR group, the red fluorescence signals were more obvious and the biofilm biomass amounted to 42.54%. Remarkably, for the EM@MoS2 + NIR group, only 15.53% of the biofilm debris was attached to the implant surface, implying that the biofilm was totally dissociated from the implant. Subsequently, crystal violet staining assays (Fig S8b and S8d) revealed comparable experimental data (100%, 61.75%, 71.10%, and 35.66% for control, NIR, MoS2 NDs, EM@MoS2, and EM@MoS2 + NIR group, respectively). These results suggested that MoS2 NDs itself possesses a certain degree of bactericidal ability in the biofilm environment, which, mechanically speaking, was mediated by toxic ROS. Obviously, this ability was barely affected by the cell membrane coating of EM@MoS2. And it could be speculated that the antibiofilm effect of ROS could be enhanced by hyperthermia. hyperthermia and ROS.
Therefore, the synergistic effect between hyperthermia and ROS was investigated by detecting the ROS concentrations in each group of biofilms using2',7'-Dichlorodihydrofluorescein diacetate (DCFH-DA), which would be converted into green fluorescent 2′,7′-dichlorofluorescein (DCF) in the presence of ROS. As presented in Fig. 3c and 3e, there was hardly any green fluorescence signal in control group and it was slightly enhanced in NIR group, reflecting low levels of ROS. Whereas, this signal was moderately strengthened following pretreated with MoS2 NDs or EM@MoS2, implying intermediate levels of ROS and it was suitable for MoS2 NDs (or EM@MoS2) to induce ROS generation in acidic and hydrogen peroxide-containing microenvironment of biofilms. Notably, the EM@MoS2 + NIR group emitted a robustly high green fluorescence, indicating that ROS production was strongly accelerated by hyperthermia. On the other hand, the standard plate counting tests and biofilm biomass analysis concurred that the antibiofilm effect of the EM@MoS2 + NIR treatment was more superior than that of the NIR treatment. This suggested that ROS-induced oxidative damage to cell membranes rendered bacteria more sensitive to heat. Overall, we have confirmed that MoS2 NDs or EM@MoS2 acted as a synergistic therapeutic platform for hyperthermia- and ROS-based therapy.
It is well established that drug resistance of biofilms has been a persistent and non-negligible issue[37, 38]. Thus, it seemed indispensable to investigate whether drug resistance developed in response to EM@MoS2-mediated synergistic therapy (Fig. 3f). Our results indicated that MRSA viabilities had been steadily maintained at approximately 0.5% for 8 consecutive generations of screening. This confirmed our hypothesis that the synergistic therapy dose prevented the emergence of drug resistance and remains highly effective.
Consequently, these results indicated that both MoS2 NDs and fabricated EM@MoS2 were effective synergistic antibiofilm agents with superior photothermal and peroxidase-like performance. This is partly because MoS2 NDs and EM@MoS2 met the requirements to permeate biofilms, corroborating with previous reports that stated that ideal diameter for nanoparticles to penetrate the biofilms must not exceed 200 nm.[39, 40] Moreover, an ultra-small particle size increases the specific surface-area and active sites, which improves the catalytic activity of nanoparticles. Thus, the characteristics above account for the effectiveness and efficiency of MoS2 NDs and EM@MoS2.
In Vitro Immunomodulatory Properties of MoS 2 NDs and EM@MoS2.
Despite a relatively high efficiency of MoS2 NDs-based synergistic therapy in destroying biofilms, inevitably, a small number of released bacteria can survive and reconstruct biofilms using cracked EPSs in an immunosuppressive microenvironment. Therefore, more attention needs to be paid to reverse this unfavorable immune state to avoid an infection recurrence. Macrophages have a powerful phagocytic ability and possess dynamic plasticity, conferring them as the most promising target to enhance immune therapy. Macrophages can differentiate into the M1 phenotype, facilitating bacterial elimination, or into the M2 phenotype, that is correlated with immune suppression and infection recurrence. To this end, we treated the macrophages with MoS2 NDs and first-hand discovered its superior properties to reverse macrophage phenotype from M2 to M1.
Firstly, the results of immunofluorescence staining (Fig. 4a and 4c) revealed that red fluorescence corresponding to the expression of CCR7 (an M1 marker) was gradually enhanced with increasing MoS2 NDs concentrations and ultimately reached a maximum when the concentration of MoS2 NDs or EM@MoS2 increased to 200 mg/L. In contrast, the expression of Arg-1 (an M2 marker) was marginally inhibited by MoS2 NDs or EM@MoS2in a concentration-dependent manner. It’s worth noting that there seemed no obvious differences in CCR7 and Arg-1 expression levels between MoS2 NDs (200 mg/L) and EM@MoS2 (200 mg/L) groups according to semi-quantitative analysis. Similarly, flow cytometry (Fig. 4b) was subsequently conducted to verify the immunomodulatory effect of MoS2 NDs and EM@MoS2 using another pair of markers, CD86 (M1 marker) and CD206 (M2 marker). The proportion of CD86-positive cells increased remarkably and reached a maximum of 56.99% when treated with 200 mg/L MoS2 NDs, while that of the control group is 4.89%. On the other hand, the proportion of CD206-positive cells remained low, and displayed a slight downward trend (11.49-6.89%) with increasing concentrations of MoS2 NDs. Meanwhile, enzyme-linked immunosorbent assay (ELISA) (Fig. 4d) showed that the secretion levels of pro-inflammatory factors, TNF-α, IL-1β, MCP-1, were elevated in a concentrate-dependent manner, whereas that of anti-inflammatory factor, IL-10, was decreased in the same way. An identical tendency was observed at gene level using RT-PCR tests (Fig. 4e).
Therefore, we explored the intrinsic mechanisms underlying MoS2 NDs-induced macrophage polarization using transcriptome sequencing. Initially, the Pearson correlation analysis was applied to evaluate the reproducibility of the samples within the groups. As shown in Fig S9a, the Pearson coefficients within groups were very close to 1, significantly greater than that between groups, indicating a high gene expression stability. The volcano plot was chosen to present the gene expression profile of macrophages (Fig. 5a). Comparing with control group, there were 1274 genes up-regulated and 830 genes down-regulated in MoS2 NDs group, which were together defined as differential expressed genes (DEGs). Next, some DEGs correlated with macrophage polarization were selected and shown in the heatmap (Fig. 5b). In MoS2 NDs group, the expression levels of M1 phenotype-related genes, including that coding for surface markers (CD86, CCR7, CD80), cytokines (IL-23, IL-1a, IL-1b, IL-6, TNF-α), and upstream regulators (NF-κB) were considerably up-regulated. Conversely, the expression levels of M2-related genes, like that coding for CD206, CD301, CD115, IL-10, PPARG, and KLF4 were substantially inhibited.[44, 47, 48] These findings supported and complemented the RT-PCR results. Furthermore, to classify all DEGs into various biological functions, Gene Ontology (GO) analysis was performed and top 30 enriched terms are shown in Fig S9b. Obviously, multiple DEGs were implicated in macrophage polarization-related terms (i.e., inflammatory response, immune response, cell migration, and response to lipopolysaccharide). Subsequently, the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis was used to identify the signaling pathways underlying macrophage polarization. As depicted in bubble plots (Fig. 5c and 5d), signaling pathways mediating M1 macrophage activation, including TNF, NOD-like receptor, NF-κB, and MAPK signaling pathways, had comparably high q values. This implied an explicit pro-inflammatory effect of MoS2 NDs on macrophages. On the other hand, signaling pathways related to M2 polarization like Jak-STAT and PPAR were significantly down-regulated[43, 49]. Thus, MoS2 NDs were well substantiated to be an intrinsic immunomodulator, which can promote the differentiation of macrophages to M1 phenotype to establish a pro-inflammatory microenvironment. This finding broadens the biological applications of MoS2 NDs and sheds light on the mechanisms underlying immuno-antibacterial therapy mediated by MoS2 NDs.
Immuno-Antibiofilm Therapy Mediated by EM@MoS2.
The ability of macrophages to penetrate EPS and engulf bacteria largely determines the host defense response against biofilm infection. Inspired by MoS2 NDs’ newfound property to activate macrophages, we initially explored the ability of activated macrophages (AM) to invade biofilms using CellTraker Red CMTPX dyes to trace macrophages and CFDA-SE to trace bacteria. As shown in Fig. 5e and 5f, after two hours of coculture, the number of macrophages infiltrating either an intact or a cracked biofilm increased when stimulated by EM@MoS2. This indicated that AM possessed stronger chemotactic ability than unactivated counterparts (UM). Simultaneously, we also found that both AM and UM could easily enter the broken biofilm. These results illustrated that in the process of macrophage attacking biofilms, the characteristics of the biofilm itself, that is, the degree of fragmentation, play a decisive role, while the states of macrophages play a supporting role. Subsequently, we compared the capacities of AM and UM to phagocytose and kill biofilm fragments. As presented in Fig. 5e, macrophages possessed an intrinsic trait to phagocytose biofilm fragments (green dots) and, obviously, this ability was enhanced by EM@MoS2. After phagocytosis, bacteria survival rates were evaluated by plate counting test (Fig. S10 and Fig. 5g), which suggested that AM have a stronger bacteria-killing efficiency than UM. Thus, it could be concluded that EM@MoS2 NDs could act as an immunological adjuvant to endow macrophages with potent bactericidal ability. In summary, it seems to be a more compelling antibiofilm strategy to initially destroy the biofilm structure and subsequently drive the macrophages to eliminate biofilm debris in comparison with monotherapy.
Combinatory Antibiofilm Effect of EM@MoS2 in Vivo.
In order to assess the ability of EM@MoS2 to synergistically eliminate biofilms on implants in vivo, we constructed a mouse subcutaneous implant-related infection model. Firstly, we took general pictures of all mice on 1st, 3rd, 7th, 10th, and 13th day to dynamically monitor skin lesions (Fig S11). Evidently, varying degrees of skin impairments, including skin edema, subcutaneous purulent exudates, and skin ulceration and necrosis, had occurred in all groups except for EM@MoS2 + NIR group over the course of thirteen-day observation. Thereafter, magnetic resonance imaging (MRI) was employed to accurately evaluate the severity of the subcutaneous infections. As presented in Fig. 6a and 6g, the high signal oval regions around the PEEK discs on T2-weighted image represented inflammatory exudates, pus, or liquefactive tissues. The average abscess volume in MoS2 NDs group (173.2 mm3) was smaller than that in the control and NIR groups (393.3 mm3 and 307.1 mm3, respectively). Strikingly, the extent of infection was substantially reduced in mice treated with EM@MoS2 and was almost negligent in those belonging to EM@MoS2 + NIR group. After the mice were sacrificed, the infection status of peri-implant soft tissues was further assessed. As shown in Fig. S12, the PEEK discs of control and NIR groups were completely covered with pus and necrotic tissues. And there was hardly any visible bleeding in control group, implying an impaired blood supply. However, white pus was remarkably decreased in MoS2 NDs group. In comparison, no purulent exudates were visible and the implants were encapsulated by a layer of vascularized fibrous tissues in EM@MoS2 and EM@MoS2 + NIR groups. Lastly, H&E staining was used to evaluate the degree of edema and inflammatory reaction (Fig. 6b and 6h). The thicknesses of skin connective tissues were decreased obviously after various treatment, especially in EM@MoS2 and EM@MoS2 + NIR groups. Similarly, a downward trend could be observed as for inflammatory cell infiltration. These results implied that MoS2 NDs could appreciably relieve the infection symptoms, which was probably achieved through a combination of direct antibiofilm effect and immunomodulatory mechanisms. Moreover, EM@MoS2 provided better protection on peri-implant soft tissues, indicating that the pore-forming toxins play a critical role in spreading of infection, and erythrocyte membrane coating of EM@MoS2 exhibits a superb protective effect against bacterial toxins.
We further estimated the antibiofilm ability of MoS2 NDs and EM@MoS2 in vivo. Plate counting tests (Fig. 6c, d for implant, and i, j for peri-implant soft tissues) revealed that the bacterial burden after treatment with MoS2 NDs decreased by 0.82 lg CFU/ml (implant) and 0.60 lg CFU/ml (peri-implant tissue). This indicated a more remarkable antibiofilm effect in vivo than that in vitro, possibly attributable to the immunomodulatory role of MoS2 NDs in polarizing resident macrophages. Noteworthily, we also observed that the number of live bacteria in EM@MoS2 group was significantly reduced whether in implant (p<0.01) or in peri-implant tissues (p < 0.05) compared with that in MoS2 NDs group. This was distinctly different from our in vitro analysis results wherein MoS2 NDs and EM@MoS2 showed similar antibiofilm effect. This difference may be attributed to the adsorption of a large amount of toxins by EM@MoS2 that could, otherwise, impair macrophage functions. Additionally, the EM@MoS2 + NIR group had only sporadic bacterial colonies on blood agar plates, suggesting a relatively powerful antibiofilm efficacy. Afterwards, residual biofilms were observed through SEM (Fig. 6e) and Giemsa staining (Fig S13). In control and NIR groups, a large number of dense bacterial communities were stacked on the implant surfaces or embedded in surrounding soft tissues (marked as red arrows). And the bacterial loads in other groups were correspondingly reduced in the same magnitude as plate counting tests. In general, MoS2 NDs or EM@MoS2 possess excellent in vivo antibiofilm abilities, especially combined with hyperthermia therapy.
Subsequently, immunofluorescence staining was performed to further evaluate the in vivo immunoregulatory properties of MoS2 NDs or EM@MoS2. As shown in Fig. 6f, Arg-1-positive M2 macrophages accounted for a higher proportion than CD86-positive M1 macrophages in control and NIR groups, which illustrated that the biofilm itself would skew macrophages from M1 to M2 phenotype. However, in other three groups treated with MoS2 NDs or EM@MoS2, the proportion was just the opposite, that is, M1 macrophages occupied the dominant position. Thus, the above results indicated that even in a complex in vivo biofilm microenvironment, the immunoregulatory mechanisms of MoS2 NDs still worked, which would contribute to reverse immunosuppressive microenvironment and prevent the recurrence of infection. Lastly, according to histologic examination of the vital organs (the heart, liver, spleen, lung, and kidney; Fig S14) of the mice, there was no visual damage among all the groups, implying that these nanomaterials were nontoxic and safe for in vivo application.