Integrated endotoxin-adsorption and antibacterial properties of platelet-membrane-coated copper silicate hollow microspheres for wound healing

Abstract

Serious infection caused by drug-resistant gram-negative bacteria and their secreted toxins (e.g., lipopolysaccharide) is a serious threat to human health. Thus, treatment strategies that efficiently kill bacteria and reducing the impact of their toxins simultaneously are urgently required. Herein, a novel antibacterial platform composed of a mesoporous copper silicate microsphere (CSO) core and a platelet membrane (PM) shell was prepared (CSO@PM). CSO@PM specifically targets bacteria owing to formyl peptide receptors on the PM and, combined with photothermal therapy (PTT), exhibits highly effective bacter icidal activity. Importantly, CSO@PM can adsorb lipopolysaccharide secreted by gram-negative bacteria, resulting in inflammation reduction. Thus, CSO@PM stimulates re-epithelialization and granulation-tissue formation, promoting wound healing. Moreover, this antibacterial platform exhibits no obvious toxicity at all the test concentrations in vitro and in vivo. Thus, CSO@PM exhibits a robust antibacterial effect and a strong toxin-adsorption capacity, facilitating the clinical treatment of many bacterial infections and the development of next-generation antibacterial nanoagents.

Keywords: Bacterial infection; Mesoporous copper silicate microspheres; Photothermal therapy; Toxin adsorption; Wound healing.

Fig. 1

Schematic showing the preparation of CSO@PM NPs and the treatment of wounds infected with multi-drug-resistant Gram-negative bacteria. CSO@PM NPs specifically target bacteria, adsorbing their LPS and, in combination with NIR laser irradiation, effectively kill them, reducing inflammatory reactions and ultimately promoting wound healing

Fig. 2

Characterization of CSO@PM. Representative TEM images showing the morphologies of CSO A before and B after PM coating. C Zeta potential values for CSO, PM, and CSO@PM. Data are mean ± SD (n = 3). D Hydrodynamic diameters of CSO and CSO@PM. Data are mean ± SD (n = 3). E Preservation of the PM proteins and E intracellular proteins detected in platelets, PM, and CSO@PM using proteomic analysis. F Western blot analysis of TLR4, FRP1, GPVI, GLEC-2, and GAPDH in platelets, PM, and CSO@PM. G Corresponding thermographic images and H temperature evolution curves of CSO suspensions with different concentrations in PBS (pH 7.4) under 808 nm NIR laser irradiation at 1.5 W cm⁻². I Thermal response of CSO under repeated laser irradiation (n = 5; 808 nm, 1.5 W cm^–2)

Fig. 3

In vitro bactericidal activity of CSO@PM. A SEM images for the control (PBS treated), CSO, CSO@PM, CSO + NIR, CSO@PM + NIR groups after incubation with P. aeruginosa. Blue indicates NPs. B Mass of NPs bound to P. aeruginosa after co-incubation with CSO or CSO@PM. Values are mean ± SD (n = 3) and ** indicates P < 0.001. C Representative images and D quantitative analysis of bacterial colonies formed by P. aeruginosa after exposure to CSO and CSO@PM with or without 808 nm NIR irradiation. The values are shown as mean ± SD (n = 3) and ** indicates P < 0.001 compared with the corresponding control group. E Representative images and F quantitative analysis of bacterial colonies formed by P. aeruginosa after exposure to CFP, CSO, or CSO@PM with or without 808 nm NIR irradiation. The values are shown as mean ± SD (n = 3) and ** indicates P < 0.001 compared with the control group

Fig. 4

Anti-inflammatory activity of CSO@PM in vitro. A LPS adsorption capacity of CSO@PM. The initial concentration of LPS was 1 ng mL⁻¹. B LPS adsorption for the PBS, LPS (100 ng mL⁻¹), CSO (50 μg mL⁻¹), and CSO@PM (50 μg mL⁻¹) groups. C Schematic of the co-culture system composed of RAW246.7 cells, LPS, and CSO@PM. D mRNA and E protein expression of IL-1β and IL-6 in RAW264.7 macrophages. The values are shown as mean ± SD (n = 3) and ** indicates P < 0.001 compared with the corresponding control group

Fig. 5

Effects of CSO@PM on the healing of P. aeruginosa-infected wounds. A Photographs of P. aeruginosa-infected wounds under different treatments. The round blue card with a 5 mm diameter indicates initial wound size. B Fractions of the wounds healed by the different treatments on days 5, 7, and 9. The values are shown as mean ± SD (n = 3). C Quantitative analysis of wound area for each group. The values are shown as mean ± SD (n = 3). * P < 0.1 and ** P < 0.001 compared to the control group. D Photographs and E quantitative counts of bacterial colonies formed by P. aeruginosa obtained from wound tissues. The values were shown as mean ± SD (n = 3). * P < 0.1 and ** P < 0.001 compared to the corresponding control group. F H&E staining images of mouse wound tissue from all groups at days 7. Black arrows indicate the length of newly regenerated epidermis. Yellow arrows indicate thickness of granulation tissue. Scale bar: 200 μm. G Neo-epidermis length and H thickness of granulation tissue data. The values are show as mean ± SD (n = 3). * P < 0.1 and ** P < 0.001 compared to the corresponding control group. I Masson images of mouse wound tissue from all groups on day 7. Scale bar: 100 μm

Fig. 6

Effect of CSO@PM on LPS-infected wound healing. A Photographs of LPS-infected wounds under different treatments. The round blue card with a 5 mm diameter indicates initial wound size. B Fractions of the wounds healed by the different treatments on days 5, 7, and 9 (n = 3). C Quantitative wound area analysis for each group (n = 3). * P < 0.1 and ** P < 0.001 compared to the corresponding control group. D H&E images of mouse wound tissue from all groups at day 7. Black arrows indicate the length of newly regenerated epidermis. Yellow arrows indicate thickness of granulation tissue. Scale bar: 200 μm E Neo-epidermis length and F granulation tissue thickness data. G Real-time quantitative PCR detection results for IL-1β and IL-6 mRNA and H protein expression of IL-1β and IL-6 in LPS-infected wounds at day 7. The values are shown as mean ± SD (n = 3). * P < 0.1 and ** P < 0.001 compared to the corresponding control group. I Masson images of mouse wound tissue from all groups on day 7. Scale bar: 100 μm

Fig. 7

In vitro and in vivo biocompatibility of CSO@PM. A Cell viabilities of 3T3 fibroblasts treated with different concentrations of CSO@PM. B–H Hematology and blood biochemistry analysis results for healthy Balb/c mice sacrificed 21 days after intravenous injection with CSO@PM at a concentration of 500 mL⁻¹ (n = 3). PBS-treated mice were used as controls. B White blood cells, C red blood cells, D platelets, E aspartate aminotransferase (AST), F creatinine (Cr), G blood urea nitrogen (BUN), and H alanine aminotransferase (ALT). I H&E staining images of the major organs (heart, liver, spleen, lung, and kidney) from mice 21 days after intravenous injection with CSO@PM (500 μg mL⁻¹). Scale bar: 100 μm