Preparation and characterization of [email protected]
As shown in Fig. 1, the preparation of [email protected] mainly included the following three steps: 1) synthesis of Ag-MOF with 2-methylimidazole and AgNO3 in a high temperature reactor; 2) loading vancomycin to Ag-MOF to synthesize Ag-MOF-Vanc; 3) adding platelet membrane vesicles to encapsulate Ag-MOF-Vanc to form the final product [email protected] Transmission electron microscopy (TEM) (Fig. 2a) showed that the synthesized Ag-MOF particles had uniform size (130–150 nm) and shape of radiating corolla. After the fusion with PLTm vesicles, Ag-MOF was observed to be encapsulated into PLTm vesicles. The protein composition of PLT membrane vesicles and [email protected] was detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE). The results showed that [email protected] and platelet membrane vesicles had similar protein profiles, which also confirmed the successful transfer of platelet membrane proteins to [email protected] (Fig. 2b). Dynamic light scattering (DLS) data showed that the particle size of Ag-MOF was about 133 nm. After the encapsulation by PLT membrane vesicles, the particle size of [email protected] was about 148 nm and was close to the size of PLT membrane vesicles (146 nm, Fig. 2c), which was comparable with the TEM results. Zeta potential of Ag-MOF, PLTm, and [email protected] were − 19.0 mV, − 18.6 mV, and − 25.6 mV, respectively (Fig. 2d).
UV-vis spectrum analysis showed that [email protected] had three characteristic absorption peaks, located at 199 nm, 213 nm, and 281 nm, which were consistent with the characteristic absorption peaks of PLT membrane vesicles, Ag-MOF, and vancomycin, respectively (Fig. 2e). FTIR results also showed that the infrared spectrum of Ag-MOF-Vanc contained the characteristic peaks of Ag-MOF and vancomycin (Fig. 2f).
MOF is an ideal drug carrier due to its high porosity and specific surface area. In this study, vancomycin was loaded in Ag-MOF, and the encapsulation efficiency (EE) and loading efficiency (LE) of vancomycin were 81.0% and 64.7%, respectively (Fig. 3a). The ideal drug carrier should efficiently load the drug but also reach specific sites to achieve responsive release. The pH values of the infected site and intracellular environment were lower than those of healthy tissue and extracellular environment, respectively8, 22. Therefore, pH-sensitive nanoparticles could have a better inhibitory effect on the infection of Staphylococcus aureus. In order to verify the pH-responsive release of [email protected] in infected microenvironment, pH 7.4 and pH 5.0 were used in this study to simulate neutral blood circulation environment and the acidic infection microenvironment, respectively. As shown in Fig. 3b, Vanc was more easily released at pH 5.0 than at pH 7.4. It is beneficial for Vanc in [email protected] to be released at the infected site rather than in neutral circulation, indicating that [email protected] can be used for drug delivery, especially at the infected site. Similar to Vanc, the release rate of Ag+ in Ag-MOF increased with the decrease in pH value (Fig. 3c). In summary, Ag+ and Vanc in [email protected] can be released rapidly in the weak acidic environment of the infected area. In addition, the cumulative release rate of Ag+ and Vanc in [email protected] was lower than that in Ag-MOF-Vanc, indicating that PLT membrane inhibited the rapid release of the drug to some extent and played a role in continuous release. These results indicate that [email protected] is an effective drug carrier.
To evaluate whether [email protected] was endowed with good blood compatibility after PLT membrane encapsulation, hemolysis test was performed. Namely, 5% erythrocytes were incubated with different concentrations of Ag-MOF and [email protected] (0, 5, 10, 20, 40, 80, and 160 µg/mL) for 2 h. As shown in Fig. 4a, Ag-MOF and [email protected] did not cause significant hemolysis during 2 h (less than 3% for both). In addition, the hemolysis rate induced by [email protected] was significantly lower than that of Ag-MOF, indicating that the encapsulation by PLT membrane increased [email protected] blood compatibility.
To demonstrate the immune escape ability of PLT membrane-camouflaged [email protected], the phagocytosis of RAW264.7 macrophages was evaluated by laser confocal fluorescence microscopy (LCFM). The green fluorescence of Ag-MOF was used for cell imaging. As shown in Fig. 4b, 24 hours after injection of Ag-MOF-Vanc, a large number of Ag-MOF without red PLT was engulfed by RAW264.7 cells. Meanwhile, under the same conditions, the green fluorescence in RAW264.7 cells treated with [email protected] was significantly reduced; these data indicated that after being encapsulated by PLT membrane vesicles, the immunogenicity of [email protected] decreased and was not recognized as a non-self-component by macrophages, so the phagocytosis was effectively inhibited. These above characteristics endow [email protected] with prolonged circulatory half-life by reducing recognition and clearance by phagocytes from the reticuloendothelial system in vivo.
To further evaluate the cytotoxicity of the material, HeLa cells and HUVECs were treated with different concentrations (0, 5, 10, 20, 40, 80, and 160 ug/mL) of Ag-MOF and [email protected], and the cell vitality was detected by CCK-8. As shown in Fig. 4c, after 48 h of mixed culture with the added material, cell vitality did not significantly decrease; therefore, Ag-MOF and [email protected] have no obvious cytotoxicity. In this study, the effects of the materials on cell apoptosis and reactive oxygen species (ROS) production were further detected by flow cytometry. Compared with the control group, the apoptosis rate and ROS production were not increased in Ag-MOF, Ag-MOF-Vanc, and [email protected] groups (Fig. 4d and 4e). Apoptosis is a mechanism of cell death, and ROS is a key molecule in cell apoptosis and autophagy23. These results indicate that the synthesized material in this study does not lead to cell death by promoting apoptosis.
In vitro antibacterial effect of Ag-MOF-Vanc
In this study, the antibacterial effect of the newly synthesized Ag-MOF on three common clinical strains (Escherichia coli ATCC25922, Pseudomonas aeruginosa ATCC27853, and Staphylococcus aureus ATCC25923) was investigated by disc method. The results of antibacterial zone showed that Ag-MOF had a good inhibitory effect on the growth of the three strains (Fig. 5a and 5b). For MRSA (ATCC25923), the MIC of Ag-MOF was 8 µg/mL (Fig. 5c). The antibacterial effects of vancomycin alone and Ag-MOF-Vanc on MRSA were compared; the results showed that the antibacterial zone of Ag-MOF-Vanc was larger than that of vancomycin alone at different concentrations (Fig. 5d). The MIC of vancomycin alone was 2 µg/mL; in contrast, the MIC of Ag-MOF-Vanc was 0.5 µg/mL (Fig. 5e), which was only about one fourth of that of vancomycin alone. The above results indicate that Ag-MOF could enhance the antibacterial effect of Vanc against MRSA and reduce the dosage of vancomycin.
When MRSA was exposed to different concentrations (0, 10, 20, 40 µg/mL) of Vanc and Ag-MOF-Vanc for 1 h, the number of dead bacteria increased in a dose-dependent manner, suggesting that the antibacterial activity of Ag-MOF-Vanc was concentration-dependent (Fig. 5f). MRSA was treated with 5 µg/ mL Vanc and Ag-MOF-Vanc. The permeability of the bacteria increased with prolonged time, suggesting the time dependence of Ag-MOF-Vanc (Fig. 5g).
A series of studies were conducted to explore the antibacterial mechanism of [email protected] The first step for [email protected] to exert its antibacterial effect is to target MRSA with the assistance of PLT membrane. In order to clarify the interaction between [email protected] and MRSA, Ag-MOF-Vanc and [email protected] at a certain concentration were co-incubated with MRSA for 3 h and observed by Scanning electron microscopy (SEM). Figure 6a shows that the surface of MRSA was relatively smooth when exposed to Ag-MOF-Vanc, while a large number of nanoparticles were attached to the surface after the exposure to [email protected] This indicates that PLT membrane promotes the binding of [email protected] to MRSA and has a certain targeting effect.
To evaluate the effect of [email protected] on bacterial metabolism, intracellular ATP levels were measured. The results showed that [email protected] led to a significant decrease in ATP level, which was significantly greater than that caused by Ag-MOF and Vanc (Fig. 6b). The decrease in ATP levels may be attributed to the inactivation of F-type ATP synthase(F-ATPase) (Fig. 6c). The functions of F-ATPase include catalyzing the synthesis of ATP in the last step of oxidative phosphorylation, working in reverse as an ATPase to produce the transmembrane proton electrochemical gradient required for molecular transport24. [email protected] can significantly decrease the activity of F-ATPase.
The death of bacteria exposed to nanoparticles can be attributed to the disruption of energy production caused by the decoupling of oxidized phosphate in the cellular respiratory chain, the interference in membrane permeability, and the loss of enzyme activity involved in key metabolic pathways; among them, the excessive ROS production by cells is the most effective component for triggering bacterial cell death25. Therefore, it is very important to study the effect of nanoparticles on the formation of ROS in bacterial cells. We used 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) method to quantitatively detect ROS. Figure 6d shows the ROS production level of MRSA after the treatment with Ag-MOF, vancomycin alone, Ag-MOF-Vanc, and [email protected] Compared with the control group and the vancomycin alone group, Ag-MOF treatment significantly increased ROS levels. The variation was more obvious in the bacteria treated with Ag-MOF-Vanc and [email protected] Ag-MOF-Vanc. High ROS levels were observed in [email protected] bacteria, indicating that [email protected] effectively bound to the bacterial surface, thus releasing a high proportion of silver ions in the target cells.
One of the main consequences of intracellular ROS accumulation is the damage to the membrane integrity caused by the gradual establishment of oxidative stress. In addition, nanoparticles can also cause physical damage to the cell membrane. Therefore, we continued to use malondialdehyde (MDA) method to detect cell lipid peroxidation to determine the degree of membrane damage. There were significant differences in MDA content among bacteria in different treatment groups (Fig. 6e). Compared with vancomycin alone, the content of MDA in [email protected] cells increased significantly. These results suggest that the interaction between Ag-MOF and bacterial surface increases the degree of bacterial damage.
One of the reasons for drug resistance and poor therapeutic effect of antibiotics is the generation of biofilms. Because of their high permeability, nanoparticles can penetrate thick biofilms. We speculated that Ag-MOF-Vanc may have a good inhibitory and scavenging effect on Staphylococcus aureus biofilm. During the early- and mid-stages during biofilm formation, two assays, crystal violet staining and the 2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carbox-anilide (XTT) assay, are regarded as crucial experimental tools. The results of crystal violet staining and XTT staining after treating the MRSA biofilm with different drugs showed that [email protected] effectively destroyed the biofilm formed by MRSA, and the effect was obviously better than that of Ag-MOF and vancomycin alone (Fig. 4f and 4h). The results obtained by confocal laser scanning microscopy were identical to those obtained by crystal violet and XTT analysis (Fig. 4g).
In conclusion, [email protected] can kill MRSA through a comprehensive physical and chemical mechanism, including targeting MRSA via PLT membranes; interfering with the intracellular metabolism of bacteria; catalytic production of ROS; damage to cell membrane integrity; and inhibiting the formation of biofilm.
Distribution of Intravenously Injected [email protected] Ag-MOF-Vanc
To demonstrate that [email protected] can target the MRSA-infected sites in vivo, biodistribution in MRSA pneumonia model mice was evaluated by Small Animal In Vivo Imaging at 6, 24, and 48 h after Ag-MOF-Vanc and [email protected] tail vein injection. As shown in Fig. 7a, Ag-MOF-Vanc rarely aggregated at the infected site within 48 h after injection, while [email protected] mostly accumulated at the infected site. Then, 48 h after injection, the mice were killed by cervical dislocation, and fluorescence imaging of the heart, liver, spleen, lung, and kidney was performed in vitro (Fig. 7b). There was a small amount of Ag-MOF-Vanc aggregation in the lung, liver, and spleen; the accumulation of [email protected] in the lung was much higher than that of Ag-MOF-Vanc. The above results indicate that [email protected] has a good targeting effect on the MRSA-infected sites in vivo.
The present study further evaluated the anti-infection effect of [email protected] in vivo in the MRSA pneumonia model of Kunming mice. The infected mice were divided into the following five groups: normal saline group, Ag-MOF group, Vanc group, Ag-MOF-Vanc group, and [email protected] group. After establishing the model, the corresponding drugs were injected daily, and every day one mouse was taken from each group for Hematoxylin-eosin(HE) staining so as to observe the alveolar structure and integrity of ciliated endothelial cells, inflammation, necrosis, and infiltration by inflammatory cells (macrophages) in the alveoli. The results showed significantly better improvement rate of the lung condition in [email protected] group compared with other groups, and the alveoli recovered from the third day of the treatment, with no obvious inflammatory cell infiltration (Fig. 8a). Four days after the treatment, the levels of inflammatory cytokines IL-6 and TNF-α in the lung tissue of mice were examined by immunohistochemical staining; the results showed that the expression levels of IL-6 and TNF-α in the normal saline group, Ag-MOF group, Vanc group, and Ag-MOF-Vanc group were still significantly higher compared with the normal control mice, while those in [email protected] group almost returned to normal control level (Fig. 8b). In addition, blood was taken for hematological tests; the results showed that the WBC and neutrophil (NEU) count and inflammatory marker CRP level were significantly reduced in the [email protected] group (Fig. 8c). The levels of IL-6 and TNF-α in blood were examined by ELISA; it was shown that the level of inflammatory cytokines significantly decreased in the [email protected] group (Fig. 8c). The bacterial count in alveolar lavage fluid of different treatment groups also showed that the number of residual bacteria was the lowest after the treatment with [email protected] (Fig. 8d). After 5 days of treatment, the survival rate of mice in the [email protected] group was 100%, while death occurred in other groups (Fig. 8e). All the above results show that [email protected] has a good anti-infective effect, which is significantly superior to Vanc alone and Ag-MOF or uncoated Ag-MOF-Vanc, indicating that Ag-MOF and Vanc have a synergistic anti-infective effect. Meanwhile, after encapsulation with platelet membrane, Ag-MOF-Vanc could be targeted and transported to the MRSA-infected site, thus further strengthening the anti-infective effect of [email protected]
In order to evaluate the potential toxicity of [email protected], hematological indicators of normal mice were measured 1 week after the tail vein drug injection. We measured complete blood counts(red blood cell (RBC), white blood cell (WBC), platelet (PLT)), liver function indicators (alanine transaminase (ALT), aspartate aminotransferase (AST)), and renal function indicators (blood urea nitrogen (BUN), and creatinine (CREA)). There was no significant difference in any of these indicators among different groups (Fig. 9a), indicating that [email protected] had no significant effect on the production of red blood cells, white blood cells, and platelets in blood, and it had no obvious hepatorenal toxicity.
To further clarify the in vivo toxicity of [email protected], we used HE staining to evaluate the pathological changes in major organs (heart, liver, spleen, lung, and kidney). The results showed that [email protected] did not cause significant damage to the heart, liver, spleen, lung, and kidney; it had low toxicity in vivo and good biocompatibility (Fig. 9b).