Human neutrophil membrane-derived nanovesicles as a drug delivery platform for improved therapy of infectious diseases

Abstract

Resolvins are a group of specialized proresolving lipid mediators (SPMs) enzymatically produced from omega-3 fatty acids during acute inflammation response to infections or tissue injury. Resolvin D1 (RvD1) is one of resolvins and is well studied in resolution of inflammation to treat inflammatory diseases. Resolution of inflammation includes the inhibition of polymorphonuclear leukocyte recruitment and reduced cytokine production. However, effective delivery of RvD1 to inflammatory tissues is challenging because of its lack of tissue targeting and poor physicochemical properties. Here, we proposed nanovesicles made from human neutrophil membrane which can specifically target inflamed tissues, and we loaded RvD1 on the surface of nanovesicles and antibiotic (ceftazidime, CEF) inside nanovesicles for improved treatment of bacterial infections. In a mouse model of bacterium-induced peritonitis, we demonstrated that human neutrophil cell membrane-formed vesicles (NMVs) enhanced inflammation resolution and bacterial killing after co-delivery of RvD1 and CEF. Our studies reveal that neutrophil nanovesicles may be critical for enhanced therapy to infectious diseases.

Keywords: Ceftazidime; Neutrophil-membrane formed vesicles; Peritonitis; Pseudomonas aeruginosa; Resolvin D1.

Figure 1. Schematic shows that human neutrophil membrane-derived nanovesicles can specifically bind to inflamed vasculature in bacterial infections.

Bacterial infections cause the inflammatory response. During inflammation response, activated neutrophils bind to endothelium via several intercellular adhesion molecules, such as ICAM-1 expressed on endothelium and integrin β₂ expressed on neutrophils. We proposed that neutrophil membrane-derived nanovesicles may bind to activated endothelium like their parent neutrophils, thus delivering therapeutics to infectious tissues for improved anti-infection. In this study, we proposed to co-load RvD1 (a new inflammation resolution agent) and antibiotic (ceftazidime) in nanovesicles to treat a mouse model of bacterium-induced peritonitis.

Figure 2. Generation of NMVs and their physical and biological properties.

(A) Illustration of generation of human neutrophil membrane nanovesicles including human blood collection, isolation of neutrophils and generation of NMVs. (B) Cryo-TEM image of NMVs. (C) Size distribution of NMVs measured using dynamic light scattering (DLS). (D) Zeta potentials of NMVs and their parent neutrophils. (E) Western blots of NMVs and neutrophils at the same amount loading of proteins.

Figure 3. NMVs bind to inflamed endothelial cells.

(A) In vitro binding of NMVs to activated endothelial cells characterized by confocal microscopy. HUVECs were treated with TNF-α at 25 ng/ml for 4 h or without TNF-α before the nanoparticle uptake experiments were performed. Then, DiD-NMVs (5 μg) were incubated with HUVECs at 37°C for 30 min. Anti-ICAM-1 antibody or its isotype antibody was used to block the binding of NMVs to endothelial cells. Scale bar, 50 μm. (B) Flow cytometry used to quantitatively measure the binding of DiD-NMVs to activated endothelial cells and (C) their quantification. Data are presented as the means ± SD, n=3. *P < 0.05; **P < 0.05.

Figure 4. Co-loading of RvD1 and CEF in NMVs.

(A) Schematic shows the co-loading of CEF-RvD1-NMVs. CEF was trapped inside NMVs and RvD1 was incorporated to a lipid bilayer of NMVs. Loading efficiencies of CEF (B) and of RvD1 (C) in NMVs (n=3). (D) The size distribution of CEF-RvD1-NMVs determined by DLS. (E) Release profiles of CEF and RvD1 in CEF-RvD1-NMVs at 37°C (n=3).

Figure 5. Co-delivery of CEF and RvD1 using NMVs enhances inflammation resolution and bacterial killing in a mouse model of bacterium-induced peritonitis.

(A) Animal protocol for bacterium-induced peritonitis. Neutrophil numbers (B) and CFU (C) in the peritoneal cavity. CFUs were measured using LB plates. (D-F) Cytokines of TNF-α, IL-1β and IL-6 in the peritoneal cavity. (G) CFUs of P. aeruginosa in blood. Data are presented as the mean ± SD, n=3, 4. The differences were assessed by one-way ANOVA and *P < 0.05, **P < 0.01, *** P < 0.001 compared to control (HBSS) unless specified otherwise. Copyright of the animal image was obtained from Encapsula NanoScience.

Figure 6. RvD1 and CEF inhibits ICAM-1 expression of endothelial cells and bacterial growth, respectively.

(A) HUVECs were activated by TNF-α at 50 ng/ml in the presence of CEF (5 μg/ml), RvD1 (33ng/ml) and CEF+RvD1 (5 μg/ml and 33 ng/ml, respectively) for 4 h. The ICAM-1 expression was quantified based on western blots as shown in Fig. S7. (B) P aeruginosa was incubated with or without RvD1 (33 ng/ml), CEF (5 μg/ml) CEF+RvD1 (5 μg/ml and 33 ng/ml) for 24 h. The inhibition of bacterial growth was quantified based on the data in Fig. S8. The data were expressed as means ± SD, n=3. One-way ANOVA was used for statistical analysis. NS, not significance.