Functionalized nanoparticles with monocyte membranes and rapamycin achieve synergistic chemoimmunotherapy for reperfusion-induced injury in ischemic stroke

Abstract

Background: Ischemic stroke is an acute and severe neurological disease, and reperfusion is an effective way to reverse brain damage after stroke. However, reperfusion causes secondary tissue damage induced by inflammatory responses, called ischemia/reperfusion (I/R) injury. Current therapeutic strategies that control inflammation to treat I/R are less than satisfactory.

Results: We report a kind of shield and sword nano-soldier functionalized nanoparticles (monocyte membranes-coated rapamycin nanoparticles, McM/RNPs) that can reduce inflammation and relieve I/R injury by blocking monocyte infiltration and inhibiting microglia proliferation. The fabricated McM/RNPs can actively target and bind to inflammatory endothelial cells, which inhibit the adhesion of monocytes to the endothelium, thus acting as a shield. Subsequently, McM/RNPs can penetrate the endothelium to reach the injury site, similar to a sword, and release the RAP drug to inhibit the proliferation of inflammatory cells. In a rat I/R injury model, McM/RNPs exhibited improved active homing to I/R injury areas and greatly ameliorated neuroscores and infarct volume. Importantly, in vivo animal studies revealed good safety for McM/RNPs treatment.

Conclusion: The results demonstrated that the developed McM/RNPs may serve as an effective and safe nanovehicles for I/R injury therapy.

Keywords: Ischemia/reperfusion injury; Monocyte membrane; Nanoparticles; Rapamycin; Stroke.

Scheme 1.

Schematic of McM/RNPs fabrication and its treatment for I/R

Fig. 1

Characterization of the McM/RNPs. a The sizes and b zeta potentials of McM, RNPs and McM/RNPs (n = 3, mean ± SD). c Drug loading efficiency (LE) and encapsulating efficiency (EE) of RNPs. d TEM images of RNPs and McM/RNPs (scale bar = 100 nm). e Western blotting results of integrin α4 (150 kDa /140 kDa), integrin β1 (135 kDa), CD 47 (35 kDa) and β-actin (42 kDa). f Proteins in cell lysis, McM, McM/RNPs, and RNPs by SDS-PAGE. Coomassie brilliant blue staining was used to detect the protein content. g In vitro release profiles of RNPs and McM/RNPs (n = 6)

Fig. 2

Cellular uptake nanoparticles by macrophages. a Confocal laser microscope images of DiDNPs and McM/DiDNPs internalized by macrophages (scale bar = 10 μm). b Cellular uptake of DiDNPs and McM/DiDNPs in macrophages by flow cytometry at different time points. c Quantification of the cellular uptake of DiDNPs and McM/DiDNPs in macrophages at different times points (n = 3). ****, p < 0.0001. ns no significance

Fig. 3

The adhesion of monocytes on inflammatory endothelial cells and the transmigration capability of McM-coated nanoparticles. a Images of monocyte adhesion on HUVECs after different treatments. b Quantification of monocyte adhesion (n = 5). c Schematic illustration of the Transwell model to evaluate the penetration capability of McM-coated nanoparticles across the inflamed endothelium. d Quantification of the fluorescence intensity of McM/DiDNPs in the bottom chamber at different time points. **, p < 0.01. ***, p < 0.001. ****, p < 0.0001. ns, no significance

Fig. 4

a Ex vivo fluorescent images of DiDNPs and McM/DiDNPs in the major organs at 24 h after i.v. injection. b Quantitative fluorescence intensity of DiDNPs and McM/DiDNPs in major organs (n = 3). ****, p < 0.0001. ns no significance

Fig. 5

I/R treatment with different nanoparticles 7 days after tMCAO. a Representative TTC staining images of coronal sections (n = 6). b Quantified infarct ratio (n = 6) and c neurological scores (n = 6). *p < 0.05. **p < 0.01. ***p < 0.001. ****p < 0.0001

Fig. 6

Representative immunofluorescent staining of NeuN and GFAP at 7 days after tMCAO treatment. a Immunofluorescence staining of NeuN and GFAP in the injury side after different treatments in tMCAO rats (scale bar = 50 μm). b Statistical analysis of NeuN in the injury side (n = 6). c Statistical analysis of GFAP in ipsilateral zone (n = 6). *p < 0.05. **p < 0.01. ***p < 0.001. ****p < 0.0001. ns, no significance

Fig. 7

Representative inflammatory immunofluorescence staining of CD11B and CD68 at 1 day after tMCAO treatment. a Immunofluorescence staining of CD11B (scale bar = 50 μm. Enlarged, scale bar = 20 μm). b Immunofluorescence staining of CD68 (scale bar = 50 μm. Enlarged, scale bar = 20 μm). c Statistical analysis of the number of microglia in ischemic zone (n = 6). d Statistical analysis of the number macrophages in the ischemic zone (n = 6). *, p < 0.05. ***, p < 0.001. ****, p < 0.0001. ns, no significance

Fig. 8

Preliminary safety evaluation. a H&E staining images of main organs from rats after different treatments. All micrographs were acquired at ×200 magnification. b–e Biochemical markers relevant to hepatic and kidney function (n = 3). f The percentage of inflammatory cells in the blood (n = 3). g, h Typical hematological parameters (n = 3)