Hybrid Membrane-Coated Nanoparticles for Precise Targeting and Synergistic Therapy in Alzheimer's Disease

Abstract

The blood brain barrier (BBB) limits the application of most therapeutic drugs for neurological diseases (NDs). Hybrid cell membrane-coated nanoparticles derived from different cell types can mimic the surface properties and functionalities of the source cells, further enhancing their targeting precision and therapeutic efficacy. Neuroinflammation has been increasingly recognized as a critical factor in the pathogenesis of various NDs, especially Alzheimer's disease (AD). In this study, a novel cell membrane coating is designed by hybridizing the membrane from platelets and chemokine (C-C motif) receptor 2 (CCR2) cells are overexpressed to cross the BBB and target neuroinflammatory lesions. Past unsuccessful endeavors in AD drug development underscore the challenge of achieving favorable outcomes when utilizing single-mechanism drugs.Two drugs with different mechanisms of actions into liposomes are successfully loaded to realize multitargeting treatment. In a transgenic mouse model for familial AD (5xFAD), the administration of these drug-loaded hybrid cell membrane liposomes results in a significant reduction in amyloid plaque deposition, neuroinflammation, and cognitive impairments. Collectively, the hybrid cell membrane-coated nanomaterials offer new opportunities for precise drug delivery and disease-specific targeting, which represent a versatile platform for targeted therapy in AD.

Keywords: Alzheimer's disease; hybrid cell membrane‐coated liposomes; inflammatory targeting; synergistic therapy.

Figure 1

Scheme of the preparation of drug‐loaded hybrid cell membrane liposomes and their application for AD therapy. Platelet membranes were obtained from retro‐orbital blood collected from mice, and CCR2‐RFP overexpressing membranes were extracted from stable CCR2‐RFP‐transfected HEK293T cells. Two types of cell membranes were fused and loaded with rapamycin and TPPU. The drug‐loaded hybrid cell membrane liposomes migrated to inflammatory lesions in the CNS via intravenous injection. The drugs were released to enhance autophagy, alleviate neuroinflammation and ultimately treat AD.

Figure 2

Expression, sublocalization and chemotaxis of CCR2‐RFP. A) Western blotting and B) immunofluorescence (scale bar: 20 µm) of HEK293T cells transfected with the CCR2‐RFP plasmid or mock vector. C) Western blotting of HEK293T cells transfected with the CCL2 plasmid. D) Schematic of the Transwell assay and representative images of migrated cells under different conditions (scale bar: 50 µm). E) Number of migrated cells in different groups (n = 5, ***p < 0.001).

Figure 3

Extraction and hybridization of platelet membranes and CCR2‐RFP HEK293T cell membranes. A) Marker proteins (CD41, CD61, and CD62P) located in the membrane in different cell components of platelets. WC: whole cell; CM: cell membrane. B) Marker proteins (CCR2‐RFP and Na‐K‐ATPase) located in the membrane in different cell components of HEK293T cells. WC: whole cell; CM: cell membrane; SN: supernatant. C) Expression of marker proteins in the single cell membrane and hybrid cell membrane. D) Colocalization analysis of the hybrid cell membrane using Pearson's R ² (n = 3, ***p < 0.001). E) Fluorescence images of colocalization in hybrid cell membranes with different fusion ratio (red: CCR2‐RFP HEK293T cell membrane; green: platelet cell membrane; scale bar: 200 µm).

Figure 4

Characteristics of drug‐loaded hybrid cell membrane liposomes. TEM images of A) the hybrid cell membrane and B) the drug‐loaded hybrid cell membrane liposomes (scale bar: 20 nm). C) The particle sizes of different groups. D) The zeta potential of the different groups (n = 3). E) Loading efficiency and F) entrapment efficiency of cell membrane liposomes coloaded with rapamycin and TPPU (n = 3). Cumulative release of H) rapamycin and I) TPPU within 72 hours (n = 3).

Figure 5

Biodistribution and targeting effect of hybrid cell membrane liposomes. A) Representative in vivo fluorescence images of 5xFAD mice treated with different ratios of hybrid cell membrane liposomes at various time‐points. B) Curve of the Δaverage radiant efficiency in the brain at various time‐points (n = 3, blue: G1, green: G2, red: G3; G1 versus G3: ^# p < 0.05, ^## p < 0.001, ^### p < 0.001; G2 versus G3: *p < 0.05, ***p < 0.001). C) Fluorescence biodistribution of different organs in G3 at various time‐points (n = 3). D) Immunofluorescence showing the migration of liposomes to microglia (green: Iba‐1; red: liposomes; scale bar: 50 µm). E) Number of liposomes around the microglia (n = 3, *p < 0.05). F) Immunofluorescence showing the migration of liposomes to astrocytes (green: GFAP; red: liposomes; scale bar: 50 µm). G) Number of liposomes around the astrocytes (n = 3, *p < 0.05).

Figure 6

Behavioral tests of 5xFAD mice treated with different liposomes. A) Timeline of the animal experiment. B) Average velocity, C) distance and D) time spent in the center area by 5xFAD mice in the open field test (n = 6–8). E) Representative tracks of mice from seven groups in the NOR test. F) Recognition indices of mice in the NOR test (n = 6–8, *p < 0.05, ***p < 0.001, ns p > 0.05). G) Curve of average latency during the acquisition phase. H) Average latency of mice on Day 5 (n = 6‐8, **p < 0.01, ***p < 0.001, ns p > 0.05). I) Time spent in the platform quadrant (%) by the mice in the probe trial. J) Representative tracks of the mice in the probe trial. (n = 6–8, FAD versus liposome treatment: **p < 0.01, ***p < 0.001; TR@CPL versus the other three groups with liposome treatment: ^# p < 0.05).

Figure 7

Drug‐loaded hybrid cell membrane liposomes reduced amyloid plaques and alleviated neuroinflammation. A) Western blot analysis of amyloid, tau, and autophagy related pathological proteins. Relative protein expression of B) APP, C) phosphorylated tau, D) LC3 (n = 5, FAD versus liposome treatment: *p < 0.05). E) Immunofluorescence of amyloid plaques in different treatment groups. The number of amyloid plaques in the F) cortex and G) hippocampus (n = 4‐5, FAD versus liposome treatment: *p < 0.05, **p < 0.01, ***p < 0.001; TR@CPL versus the other three groups with liposome treatment: ^# p < 0.05; scale bar: 200 µm). The density of H) microglia and I) astrocytes in the different treatment groups (n = 4‐5, FAD vs liposome treatment: *p < 0.05, **p < 0.01, ***p < 0.001; TR@CPL vs the other three groups with liposomes treatment: ^# p < 0.05, ^## p < 0.05). J) Immunofluorescence of glial cells in different treatment groups (scale bar: 100 µm).