Title：Biomimetic Bacteriophage-Like Particles Formed from Probiotic Extracts and NO Donors for Eradicating Multidrug-Resistant Staphylococcus aureus

Journal：《Advanced Materials》

IF：29.4

Original link：https://doi.org/10.1002/adma.202206134

Reporter：Yunyan Bai, Master of Grade 2022

Effectively clearing multidrug-resistant bacteria through nonantibiotic treatments is crucial for the recovery of infected tissues in favorable biological environments. Herein, a thermally responsive donor of cell-messenger nitric oxide (NO) is combined with extracts of food-grade Lactobacillus casei to form biomimetic phage-like microparticles with a tailspike structure. These particles can invade bacterial membranes and release NO to disrupt nitrogen and respiratory metabolisms, which initiates the programmed death of multidrug-resistant Staphylococcus aureus (MRSA) for inducing lysis, like the bacterial virus. Experiments suggest that these microparticles can also weaken bacterial toxicity and provide favorable conditions for cell proliferation because of the continuously released NO. By encapsulating these microparticles into graphene-oxide-doped polymers, a dual-mode antibacterial hydrogel (DMAH) can be constructed. In vivo results reveal that the DMAH achieves a long-time sterilization of MRSA with 99.84 ± 0.13% antibacterial rate in the dark because of the phage-like performance of the biomimetic microparticles. In its other antibacterial mode, DMAH subjected to 20 min of near-infrared irradiation release NO, which, together with the photothermal effect, synergistically damages bacterial cell membranes to achieve very fast disinfection (97.13 ± 0.41% bactericidal rate). This multifunctional hydrogel can also significantly accelerate wound healing due to the phage-like particles.

As the largest human organ, the skin undertakes plenty of metabolic activities and protects the body from the outside. This means that, once wounds have formed, the soft tissues face direct threats of pathogens during the long reconstruction process, and risks of infection are particularly elevated in chronic wounds, such as burns and diabetic ulcers. Although traditional antibiotic therapy has been the mainstream clinical method for preventing infection for several decades, the rapid development of bacterial drug resistance has demonstrated its limitation. It should also be noted that bacterial infection, rather than the virus itself, has become the leading cause of COVID-19-related death, and more challenges to the healing of infected wounds should be expected in the struggle against coronavirus. This highlights the significance and urgency of finding nonantibiotic therapies for avoiding drug resistance.

In recent years, inspired by nature and life, physical effects have been increasingly involved in constructing efficient sterilization methods. For example, biomimetic surfaces based on the microstructures of plants or insect wings can destroy bacterial membranes through mechanical forces, and light has been introduced and combined with photothermal conversion materials (e.g., graphene oxide (GO), MoS2) to denature the proteins on bacterial membranes through high temperatures over a short time.Furthermore, some gas molecules (e.g., O2, NO, H2S) are not only closely related to physiological activities, but also able to damage bacteria through chemical reactions; for instance, inspired by macrophage-secreted nitric oxide (NO) molecules that can oxidize protein and DNA, NO-led strategies have been used to treat various infectious bacterial diseases and to achieve tissue repair. However, there remain many limitations to these attempts at the therapeutic application of NO—endogenous NO is often insufficient to remove bacteria, and it is difficult for exogenous NO to achieve a decent therapeutic effect because of its uncontrollable release kinetics and short half-life.Therefore, to disinfect pathogens thoroughly, NO gas often needs to be combined with other strategies, such as phototherapy or drug therapy.

Apart from the NO molecules secreted by immune cells, the biological effects of probiotics or bacteria that are symbiotic with humans can also provide references for exploring nonantibiotic strategies, and some studies have found excellent therapeutic effects. For instance, the competition between probiotic flora and pathogenic bacteria—so-called living bacteria therapy—can reduce inflammation and promote tissue reconstruction and can even involve engineered or modified bacteria with specific biological characteristics to eradicate lesions.but although it has been reported that the introduction of living microorganisms can have an excellent antibacterial effect, controllability and biosafety still need to be considered.

Based previous work, Lactobacillus casei, which has been approved by the Food and Drug Administration (FDA) for food industries because it can produce antibacterial agents (H2O2, lactic acid, and bacteriocin), was selected as one of the raw materials, and the biocompatible and thermoresponsive S-nitrosothiols (RSNO) were selected as NO donor; in combination, they would be used to construct an antibacterial material to efficiently inhibit bacteria. Considering the temperature sensitivity of RSNO, if it could be further combined with photothermal materials and loaded by hydrogel materials, it may not only integrate the advantages of probiotics and NO for efficient bacteriostasis, but also deliver to construct another rapid photothermal/NO disinfection.

1. A mimetic particle (MP) with a structure similar to a phage tail spike was prepared by hydrogen bonding interactions between probiotic extract (PES) and RSNO.

We The MP was labelled as PES-RSNO, and both theoretical and experimental results confirmed its excellent antimicrobial properties. In addition, the PES-RSNO MPs were encapsulated in a hydrogel consisting of a polyvinyl alcohol (PVA)-polyacrylic acid (PAA) interpenetrating network (GO@PVA-PAA). The hydrogel was endowed with two different antimicrobial therapeutic modes under different conditions in terms of thermal responsiveness and phage-like antimicrobial properties of the bionic MPs. In vivo treatment showed that this dual-mode antimicrobial hydrogel (DMAH) not only killed MRSA by phage-like therapy in the dark (99.84 ± 0.13%). Moreover, the hydrogel was able to achieve rapid sterilisation (97.13±0.41%) by synergistic photothermal/NO therapy under near-infrared light (808 nm) irradiation for 20 min and both in vitro and in vivo experiments have shown that the DMAH accelerated the reconstruction of infected wounds.

Figure 1. Characterization of DMAH and its in vitro sterilization in the dark. a) Schematic diagram illustrating bacteriophage-like PES-RSNO MPs being generated by the formation of hydrogen bonds between the temperature-sensitive NO donors (RSNO) and the PES, with a structure similar to the tailspike of a phage. This PES-RSNO can attack MRSA and disrupt bacterial nitrogen and respiratory metabolisms, leading to programmed death and lysis. With this phage-like disinfection, a DMAH can be constructed and endowed with outstanding antibacterial performance in the dark by encapsulating these MPs. b) SEM images of the GO@PVA–PAA hydrogel and DMAH. All scale bars are 20 µm. c) UV–vis spectra of GO, PES, RSNO, PVA–PAA, GO@PVA–PAA, and DMAH. d) Antibacterial results of DMAH against MRSA at 37 °C in 12 h and photographs of the changes in the different groups’ MRSA solutions. e) Growth of MRSA treated with different samples in 12 h. Data were evaluated as mean values ± standard deviation (SD) from a representative experiment (n = 3 independent samples per group) and error bars meant the standard deviations. p-Values were calculated using a one-way analysis of variance (ANOVA) program with Tukey's multiple comparisons post hoc test (*p < 0.05, **p < 0.01, ***p < 0.001, and ns = no significant difference).

2. The antimicrobial agent in PES-RSNO was further analyzed to explore its antimicrobial mechanism.

Results It was inferred that MRSA was removed by bacterial proteins in PES and NO released continuously by PES-RSNO.

Figure 2. Analysis of antibacterial substances in PES-RSNO. a) H2O2 detections from deionized water and PES solution. b) Hydrogen ion concentrations detected in deionized water, dispersion of L. casei, and PES solution. c) Lactic acid detection from deionized water and PES liquid. d) Photographs of Tyndall test in different liquids and Tricine-SDS-PAGE detection from PES liquid. e) Protein leakage assay of MRSA treated with different treatments. f) Photographs of the caramelized PES and its antibacterial test. g) Concentrations of NO released by RSNO and PES-RSNO in the dark for 12 h at 37 °C. Data were evaluated as mean values ± standard deviation (SD) from a representative experiment (n = 3 independent samples per group) and error bars meant the standard deviations. p-Values were calculated using a one-way ANOVA program with Tukey's multiple comparisons post hoc test (*p < 0.05, **p < 0.01, ***p < 0.001, and ns = no significant difference)..

3. Through a series of experiments and theoretical simulations to find the antibacterial mechanism.

The results show that the sterilization of PES-RSNO is divided into three stages. First, PES builds phage-like PES-RSNO MPs by forming hydrogen bonds to load RSNOs. Secondly, PES-RSNO's tail-prick structure invades the bacterial membrane, injecting NO gas released by RSNO into MRSA. Third, bacterial nitrogen metabolism is destroyed, causing nitrous/oxidative stress to initiate PCD, causing bacterial cell cleavage, just like phages.

Figure 3. Antibacterial mechanism of PES-RSNO. a) Tertiary structure prediction of the PES protein performed by I-TASSER and its structure-based functions. b) Simulation of polar contacts (hydrogen bonds), the PES protein, and RSNO. c) Venn diagram of differentially expressed genes in the control (Ctrl) and PES-RSNO groups. d) Downregulated gene ontology enrichment analysis in PES-RSNO compared with Ctrl. e) Upregulated geneontology enrichment analysis in PES-RSNO compared with Ctrl. f) KEGG pathway analysis of the differentially expressed genes in PES-RSNO compared with Ctrl. g) Expression changes of the nitrogen metabolism, programmed cell death, amino acid biosynthesis, IMP biosynthetic process, protein binding, and obsolete pathogenesis of Ctrl and PES-RSNO. h) Schematic diagram of the bacteriophage-like disinfection mechanism of PES-RSNO.

4. Rapid antimicrobial treatment of DMAH in near-infrared light and in vitro cell migration test show that DMAH is expected to promote the repair of infection wound in vivo by various mechanisms.

Figure 4. Rapid antibacterial therapy of DMAH under NIR light and cell migration test in vitro. a) Schematic diagram illustrating the rapid release of NO gas from donors due to the thermosensitivity of RSNO. DMAH containing photothermal GO can be used to achieve photothermal/NO-guided antibacterial therapy. b) Temperature changes of PVA–PAA, GO@PVA–PAA, and DMAH under 808 nm NIR light (0.5 W cm−2, 20 min). c) Antibacterial results of PVA–PAA (as control group), GO@PVA–PAA, and DMAH under 808 nm NIR light (0.5 W cm−2, 20 min). d) Fluorescence images of NIH-3T3 cells treated with or without DMAH for 24 h in vitro. All scale bars are 100 µm. e) Fluorescence images of cell scratch test and quantitative data. All scale bars are 400 µm. Data were evaluated as mean values ± SD from a representative experiment (n = 3 independent samples per group) and error bars meant the standard deviations. p-Values were calculated using a one-way ANOVA program with Tukey's multiple comparisons post hoc test (*p < 0.05, **p < 0.01, ***p < 0.001, and ns = no significant difference).

5. Two antibacterial therapies directed by DMAH in vivo and the experiment to promote wound healing.

This demonstrates that DMAH can achieve different antimicrobial treatment patterns in the body. DMAH-guided antimicrobial therapy not only effectively removes MRSA, but also reduces inflammation and provides favorable conditions for wound repair..

Figure 5. Two antibacterial therapies and the promotion of wound healing guided by DMAH in vivo. a) Schematic diagram of the in vivo experimental process. b) Antibacterial results of the MRSA's CFU in the wounds of the different groups on day 2. c) Photographs of wounds in the differently treated groups on days 0, 2, 5, 10, and 15. d) Quantitative data on wound areas and final healing rates in (c). e) H&E staining images of wound tissues in the different groups on days 2, 5, and 15. The green arrows and circles represent the neutrophils. All scale bars are 20 µm. Data were evaluated as mean values ± SD from a representative experiment (n = 3 independent samples per group) and error bars meant the standard deviations. p-Values were calculated using a one-way ANOVA program with Tukey's multiple comparisons post hoc test (*p < 0.05, **p < 0.01, ***p < 0.001, and ns = no significant difference).

The authors prepared PES-RSNO MPs by simple method. A multifunctional hydrogel is built in the light-thermal GO @ PVA-PAA interconnection network. It can guide a variety of sterilization methods in different practical situations, thus promoting the healing of MRSA infection wounds. While this design strategy is currently only proven in the case of the combined use of Lactobacillus caseii and RSNO, metabolism-induced bacterial mechanisms may be prevalent in other bacteria. Therefore, there is some significance in trying to use phage-like MPs to eliminate other bacteria. At the same time, hydrogen bonding between PES and RSNO is the most common material combination method, meaning that the strategy can attempt to use probiotics with different biological properties (e.g. Bacillus subtilis, Bacillus bifidus, C. butyrate) or change NO donors (e.g. organic nitrates, metal-NO complexes, oz) Response to different external energies (e.g. light, ultrasound, microwave) To meet the individual and diverse needs of various diseases. We believe this novel design concept, which combines probiotics with NO therapy, will provide additional references and options for other biomedical material designs.