Intravenous route to choroidal neovascularization by macrophage-disguised nanocarriers for mTOR modulation

Abstract

Retinal pigment epithelial (RPE) is primarily impaired in age-related macular degeneration (AMD), leading to progressive loss of photoreceptors and sometimes choroidal neovascularization (CNV). mTOR has been proposed as a promising therapeutic target, while the usage of its specific inhibitor, rapamycin, was greatly limited. To mediate the mTOR pathway in the retina by a noninvasive approach, we developed novel biomimetic nanocomplexes where rapamycin-loaded nanoparticles were coated with cell membrane derived from macrophages (termed as MRaNPs). Taking advantage of the macrophage-inherited property, intravenous injection of MRaNPs exhibited significantly enhanced accumulation in the CNV lesions, thereby increasing the local concentration of rapamycin. Consequently, MRaNPs effectively downregulated the mTOR pathway and attenuate angiogenesis in the eye. Particularly, MRaNPs also efficiently activated autophagy in the RPE, which was acknowledged to rescue RPE in response to deleterious stimuli. Overall, we design and prepare macrophage-disguised rapamycin nanocarriers and demonstrate the therapeutic advantages of employing biomimetic cell membrane materials for treatment of AMD.

Keywords: Age-related macular degeneration; Biomimetic nanoparticles; Choroidal neovascularization; Inflammation; Macrophage membrane; Rapamycin; Targeted drug delivery; mTOR signaling.

Graphical abstract

Figure 1

Schematic diagram. (A) Illustration of macrophage-disguised nanoparticles (MRaNPs) targeting choroidal neovascularization via the intravenous route. (B) Illustration of retinal homeostasis modulation by MRaNPs via the mTOR signaling pathway.

Figure 2

Characterization of MRaNPs. (A) Scheme of preparation process of MRaNP. (B) TEM images of RaNPs (left) and MRaNPs (Right). Insert: a zoomed-in view of a single MRaNP. Scale bar = 100 nm. (C to E) Hydrodynamic size, PDI and Zeta potential of M-vesicles, RaNPs and MRaNPs (n = 3). (F) Measurements of size and PDI of MRaNPs over 7 d (n = 3). (G) Drug release from RaNPs and MRaNPs in PBS at 37 °C over the course of 96 h (n = 3). (H) SDS-PAGE protein analysis of macrophage cell lysate, M-vesicles and MRaNPs. (I) WB analysis for CD49d and CD11b in macrophage cell lysate, M-vesicles and MRaNPs.

Figure 3

In vitro binding and anti-angiogenic effect of MRaNPs. (A) Cellular uptake of DiI-labeled RaNPs and MRaNPs in HUVECs analyzed by flow cytometry at different time points. (B to C) Representative CLSM images of different DiI-labeled nanoparticles (red), HUVECs (blue), and ICAM-1/VCAM-1 (green) with or without TNF-α treatment. Scale bar = 50 μm. (D) Cell proliferation detected by Ki67 immunofluorescence staining. Scale bar = 20 μm. (E) Quantitation analysis of cell proliferation (n = 3, ∗∗∗P < 0.001 and ∗∗∗∗P < 0.0001).

Figure 4

Intracellular behavior and biological effects of anti-angiogenesis and anti-inflammation of MRaNPs in ARPE-19 cells. (A) CLSM images of the intracellular behavior of MRaNPs (blue: DAPI for nucleus; green: LAMP1 for lysosome; red: DiI-labeled MRaNP). Scale bar = 10 μm. (B) WB analysis for VEGFA and the mTOR signaling pathway. (C) CLSM images of autophagy induction stained with AO (green: nucleus; red: autolysosome). Scale bar = 20 μm. (D) CLSM images of autophagic flux (blue: DAPI for nucleus; green: LAMP1 for lysosome; red: LC3 for autophagosome). Scale bar = 20 μm. (E to F) Expression of cytokine IL-6 and IL-8 measured by enzyme-linked immunosorbent assay (n = 3, ∗∗P < 0.01, ∗∗∗P < 0.001 and ∗∗∗∗P < 0.0001). (G) WB analysis for autophagy-associated proteins.

Figure 5

The In vivo CNV-targeting ability, anti-angiogenesis effect, and anti-inflammation effect of MRaNPs. (A) Representative fluorescence images of flat-mounted RCCs showing DiI-labeled nanoparticles (red) in CNV areas stained by IB4 (green). Scale bar = 100 μm. (B) Experimental workflow of drug efficacy evaluation. (C) Representative fluorescence images of in CNV areas stained by IB4 (green) after different treatments. Dashed lines delineate the lesion. Scale bar = 100 μm. (D) Quantitation analysis of CNV areas after different treatments (28–32 laser points each group, ∗∗∗P < 0.001). (E) WB analysis for proinflammatory cytokines in RCCs after different treatments.

Figure 6

In vivo angiogenesis inhibition and autophagy activation effects of MRaNPs via the mTOR signaling pathway. (A) WB analysis for VEGFA and the mTOR signaling pathway in RCCs. (B) Representative fluorescence images of immunostaining of p-mTOR (red) in CNV areas stained by IB4 (green). Scale bar = 100 μm. (C) Quantitation analysis of the p-mTOR fluorescence intensity in CNV areas (n = 3, ∗∗P < 0.01 and ∗∗∗∗P < 0.0001). (D) WB analysis for autophagy-associated proteins in RCCs. (E) Semi-quantitation analysis of the LC3-II/-I ratio (n = 3, ∗∗P < 0.01 and ∗∗∗∗P < 0.0001).

Figure 7

Biocompatibility and safety of MRaNPs. The results of AST (A), ALT (B), CREA (C), UREA (D), ALB (E) and ALP (F) examinations after different treatments for 7 days (n = 3, ns, no statistical significance). H&E staining of retina (G) and major organs (H) after different treatments indicated no tissue damage on Day 8. CHO, choroid; ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cells layer. Scale bar = 100 μm.