Apelin-13-Loaded Macrophage Membrane-Encapsulated Nanoparticles for Targeted Ischemic Stroke Therapy via Inhibiting NLRP3 Inflammasome-Mediated Pyroptosis

Abstract

Purpose: Ischemic stroke is a refractory disease wherein the reperfusion injury caused by sudden restoration of blood supply is the main cause of increased mortality and disability. However, current therapeutic strategies for the inflammatory response induced by cerebral ischemia-reperfusion (I/R) injury are unsatisfactory. This study aimed to develop a functional nanoparticle (MM/ANPs) comprising apelin-13 (APNs) encapsulated in macrophage membranes (MM) modified with distearoyl phosphatidylethanolamine-polyethylene glycol-RVG29 (DSPE-PEG-RVG29) to achieve targeted therapy against ischemic stroke.

Methods: MM were extracted from RAW264.7. PLGA was dissolved in dichloromethane, while Apelin-13 was dissolved in water, and CY5.5 was dissolved in dichloromethane. The precipitate was washed twice with ultrapure water and then resuspended in 10 mL to obtain an aqueous solution of PLGA nanoparticles. Subsequently, the cell membrane was evenly dispersed homogeneously and mixed with PLGA-COOH at a mass ratio of 1:1 for the hybrid ultrasound. DSPE-PEG-RVG29 was added and incubated for 1 h to obtain MM/ANPs.

Results: In this study, we developed a functional nanoparticle delivery system (MM/ANPs) that utilizes macrophage membranes coated with DSPE-PEG-RVG29 peptide to efficiently deliver Apelin-13 to inflammatory areas using ischemic stroke therapy. MM/ANPs effectively cross the blood-brain barrier and selectively accumulate in ischemic and inflamed areas. In a mouse I/R injury model, these nanoparticles significantly improved neurological scores and reduced infarct volume. Apelin-13 is gradually released from the MM/ANPs, inhibiting NLRP3 inflammasome assembly by enhancing sirtuin 3 (SIRT3) activity, which suppresses the inflammatory response and pyroptosis. The positive regulation of SIRT3 further inhibits the NLRP3-mediated inflammation, showing the clinical potential of these nanoparticles for ischemic stroke treatment. The biocompatibility and safety of MM/ANPs were confirmed through in vitro cytotoxicity tests, blood-brain barrier permeability tests, biosafety evaluations, and blood compatibility studies.

Conclusion: MM/ANPs offer a highly promising approach to achieve ischemic stroke-targeted therapy inhibiting NLRP3 inflammasome-mediated pyroptosis.

Keywords: apelin-13; cerebral ischemia-reperfusion injury; ischemic stroke therapy; macrophage membrane; pyroptosis.

Graphical abstract

Figure 1

Preparation of MM/ANPs. (A) MM/ANPs are synthesized using an APN-PLGA-COOH polymer and DSPE-PEG-RVG29 targeting peptide-modified macrophage membranes under hybrid ultrasound. (B) Drug LE and EE of MM/ANPs (n = 3). (C) Representative transmission electron microscopy images of MM/ANPs. (D) Hydrodynamic size, PDI, and zeta potentials of MM/ANPs (n = 3). (E) In vitro drug release profile of MM/ANPs in phosphate-buffered saline at 37°C (n = 6). (F) UV-Vis spectra of MM/ANPs. (G) Fluorescence measurement curves of MM/ANPs.

Figure 2

In vitro cytotoxicity and blood–brain barrier penetration. (A-C) No significant change was observed in Bend.3, BV2, or HT22 cell viability after 24-hour incubation with different concentrations of MM/ANPs. (D) Schematic of the Transwell model for assessing the permeability of MM-coated nanoparticles through endothelial cells. (E) Representative images showing the fluorescence content of different concentrations of MM/ANPs in the lower chamber culture solution using an IVIS. (F) IVIS images of the brains of normal C57BL/6 mice at different timepoints after tail vein injection of MM/ANPs. Values are expressed as the mean ± standard error of the mean (n = 3). There was no statistical difference between the control group and any of the other groups in graphs A, B, and C, as determined by Tukey’s post hoc test.

Figure 3

Brain in vivo imaging after tail vein injection of MM/ANPs in MCAO/R model mice. (A) Representative in vivo images of mice in sham, MCAO, and MM/ANPs groups were recorded at the indicated timepoints after immediate tail vein injection of MM/ANPs in MCAO, showing significant accumulation of MM/ANPs in the ischemic region (n = 3). (B) Quantitative analysis of fluorescence intensity in the cerebral ischemic region at different timepoints in each group (n = 3). (C) Fluorescence intensity ratio of MCAO/contralateral hemisphere in each group of mice. MM/ANPs, macrophage membrane-encapsulated apelin-13-bound nanoparticle; MCAO/R, middle cerebral artery occlusion/reperfusion. Statistical significance was determined by one-way analysis of variance (Tukey’s multiple comparison test), ****p < 0.0001.

Figure 4

In vivo evaluation of the therapeutic effect of MM/ANPs in MCAO/R mice. (A) Laser speckle contrast imaging of cerebral blood flow in mice of different treatment groups (n = 3). (B) TTC staining and (C) infarct volume of mouse brain in different treatment groups (n = 6). (D) Representative micrographs of H&E staining and Nissl staining of brain tissues of different treatment groups. (E) Cerebral edema content based on the ratio of dry weight to wet weight 24 hours after MCAO. (F) Neurological deficit score (n = 6). (G) Trajectory line graph and trajectory heat map of the Y maze. (H) The percentage of spontaneous exploration of novelty in each group of mice was measured using a Y-maze test (n = 6). (I) The bodyweight changes of mice 3 days after different treatments (n = 5). (J) Representative images and (K) Quantification of TUNEL staining in different groups. Values are expressed as the mean ± standard error of the mean. Statistical significance was determined by one-way analysis of variance (Tukey’s multiple comparison test), with **p < 0.01, and ***p < 0.001, ****p < 0.0001.

Figure 5

Changes in brain tissue protein levels in the NLRP3 pathway and APJ in mice from different treatment groups. Representative bands of the Western blot (A-B) and quantitative analysis of NLRP3 (C), GSDMD-N (D), caspase-1 (E), cleaved caspase-1 (F), pro-IL-1β (G), IL-1β (H), IL-18 (I), ASC (J), and APJ (K) normalized to tubulin. Values are expressed as the mean ± standard error of the mean. Statistical analyses were performed using one-way analysis of variance (Tukey’s multiple comparison test). Statistical significance is indicated by *p < 0.05, **p < 0.01, and ***p < 0.001, ****p < 0.0001.

Figure 6

MM/ANPs reduced the number of pyroptosis cells. (A) The spatial patterns between pyroptosis and apoptosis are shown by TUNEL staining (red) and caspase-1 double staining (green). (B) Detection of caspase-1 activity in HT22 cells after oxygen–glucose deprivation injury by colorimetric assay. (C-F) Immunofluorescence staining for NeuN/caspase-1, NeuN/GSDMD, NeuN/IL-1β, and NeuN/ASC (NeuN-green and GSDMD/caspase-1/IL-1β/ASC-red) colocalization showed the neuronal cell pyroptosis in the infarcted area. Scale bar = 50 µm. Values are expressed as the mean ± standard error of the mean. Statistical significance was determined by one-way analysis of variance (Tukey’s multiple comparison test), with ****p < 0.0001.

Figure 7

Treatment with MM/ANPs reduces caspase-1 and microglia activation and inflammatory factors production in the brain tissue of MCAO/R in mice. (A) Representative immunofluorescence images of microglia showed a decrease in the number of microglia in the MM/ANPs group compared to the MCAO/R model. (B-E) ELISA shows that MM/ANPs significantly reduced the release of inflammatory factors relative to the MCAO/R group. (F) Ultrastructural changes were examined using transmission electron microscopy. Representative microphotographs showing mitochondria in different groups. High magnification images from the cropped rectangle are shown in the lower panel. Arrows indicate varying mitochondrial sizes, whereas arrowheads show mitochondrial swelling and disarrayed cristae. Scale bar = 1 µm. Values are expressed as the mean ± standard error of the mean. Statistical analyses were performed using one-way analysis of variance (Tukey’s multiple comparison test). Statistical significance is indicated by ****p < 0.0001.

Figure 8

Knockdown of SIRT3 reverses the protective effect of MM/ANPs. (A) Effect of MCC950, an inhibitor of NLRP3, on the salvage effect of MM/ANPs. (B-C) Quantitative analysis of SIRT3 and NLRP3. (D-H) Western blot and quantitative analyses of the salvage effect of MM/ANPs after MCAO/R injury in SIRT3 knockout mice. (I) Number of SIRT3- and Iba-1-positive cells in immunofluorescence experiments. Statistical analyses were performed using one-way analysis of variance (Tukey’s multiple comparison test). Statistical significance is indicated by *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Figure 9

In vivo safety and blood compatibility studies of MM/ANPs. (A) Hemolysis tests using MM/ANPs. (B) The absorbance of MM/ANPs measured at 540 nm (n = 6). (C) Representative images of H&E staining of vital organs from different treatment groups. All micrographs are acquired at 200× magnification. (D-G) Biochemical markers relevant to hepatic and kidney function (n = 6). MM/ANP, macrophage membrane-encapsulated apelin-13-bound nanoparticle. Values are expressed as the mean ± standard error of the mean. Statistical significance was determined by one-way analysis of variance (Tukey’s multiple comparison test), with ****p < 0.0001.