Engineered Biomimetic Platelet Membrane-Coated Nanoparticles Block Staphylococcus aureus Cytotoxicity and Protect Against Lethal Systemic Infection

Abstract

Staphylococcus aureus is a leading human pathogen capable of producing severe invasive infections such as bacteremia, sepsis and endocarditis with high morbidity and mortality, exacerbated by expanding antibiotic-resistance exemplified by methicillin-resistant strains (MRSA). S. aureus pathogenesis is fueled by secretion of secreted toxins including the membrane damaging pore-forming α-toxin that have diverse cellular targets including epithelium, endothelium, leukocytes and platelets. Here we examine human platelet membrane-coated nanoparticles (PNPs) as a biomimetic decoy strategy to neutralize S. aureus toxins and preserve host cell defense functions. PNPs blocked platelet damage induced by S. aureus secreted toxins, supporting platelet activation and bactericidal activity. Likewise, PNPs blocked macrophage damage induced by S. aureus secreted toxins, supporting macrophage oxidative burst and nitric oxide production and bactericidal activity, and diminishing MRSA-induced neutrophil extracellular trap release. In a mouse model of MRSA systemic infection, PNP administration reduced bacterial counts in the blood and protected against mortality. Taken together, the present work provides proof-of-principle of therapeutic benefit of PNPs in toxin neutralization, cytoprotection and increased host resistance to invasive S. aureus infection.

Keywords: Nanotherapeutics; Staphylococcus aureus; bacterial toxins; nanosponge; platelet; sepsis.

Fig. 1.. Formulation and analysis of platelet membrane-coated nanoparticles (PNPs).

(a) Model showeing our rationale of applying PNPs to modulate immune cells, pathogen binding, and control in vivo therapeutic efficacy. (b, c) Dynamic light scattering to evaluate hydrodynamic size (diameter, nanometers) of the PLGA polymeric cores before and after platelet membrane coating. (d) Transmission electron microscopy images of derived PNPs using uranyl acetate counterstain.

Fig. 2.

PNPs prevented human platelet damage and dysfunction induced by MRSA supernatants. (a) PNP reduced MRSA supernatant-induced platelet cytotoxicity as measured by LDH release; 0.52 ± 0.13 in MRSA supernatant alone, 0.27±0.08 with PNP treatment, p = 0.002 (b, c) P-selectin expression on platelets exposed to MRSA supernatant in the presence or absence of PNPs. PNP treatment led to a striking increase of P-selectin expression beginning at the early time point (10 min). (d, e) Increased platelet viability upon PNP treatment is accompanied by improved MRSA killing. **P < 0.01; ***P < 0.001.

Fig. 3.

PNPs prevented macrophage damage and dysfunction induced by MRSA supernatants (A) LDH cytotoxicity test and (B) MTT assay showed that PNP treatment showed about 75% reduction of THP-1 monocyte cell cytotoxicity. (C) PNP also improved the viability of THP-1 differentiated macrophages after MRSA supernatant treatment. (D, E) Pre-treatment of macrophages with PNP showed higher bactericidal efficiency regardless of bacterial load and incubation time. *P < 0.05, **P < 0.01 and ***P < 0.001.

Fig. 4.. The effect of PNPs on activation of macrophage and neutrophil bactericidal mechanisms.

(a) PNP treatment increases macrophage oxidative burst as measured by DCFH-DA assay for superoxide production (b) Increased nitrite production reflecting nitric oxide production was observed in PNP treated macrophages. (c) Reduced cytokine IL-1β production at the earliest (4h) time point in PNP-treated macrophages. (d) Immunostaining of human neutrophil extracellular traps (NETs) elicited by MRSA in the presence or absence of PNP treatment; PMA serves as a positive control. (e) Quantification of NETs by PICO-green assay. *P < 0.05, **P < 0.01 and ***P < 0.001.

Fig. 5.. PNPs improve survival in a murine MRSA systemic infection model.

(a) Schematic diagram of setup of in vivo experiments. (b) Survival rates of mice over 144 h following an intraperitoneal injection of MRSA (3×10⁸ bacteria per mouse). 100 μg PNP at 5.0 mg/ml was injected twice, at 0 h and 3 h after bacterial inoculation (n = 8 in each group). Treatment with PNPs provided a significant survival benefit. (c) Enhanced bacterial clearance in blood during upon PNP treatment.