Scavenger receptor A-mediated nanoparticles target M1 macrophages for acute liver injury

Abstract

Acute liver injury (ALI) has an elevated fatality rate due to untimely and ineffective treatment. Although, schisandrin B (SchB) has been extensively used to treat diverse liver diseases, its therapeutic efficacy on ALI was limited due to its high hydrophobicity. Palmitic acid-modified serum albumin (PSA) is not only an effective carrier for hydrophobic drugs, but also has a superb targeting effect via scavenger receptor-A (SR-A) on the M1 macrophages, which are potential therapeutic targets for ALI. Compared with the common macrophage-targeted delivery systems, PSA enables site-specific drug delivery to reduce off-target toxicity. Herein, we prepared SchB-PSA nanoparticles and further assessed their therapeutic effect on ALI. In vitro, compared with human serum albumin encapsulated SchB nanoparticles (SchB-HSA NPs), the SchB-PSA NPs exhibited more potent cytotoxicity on lipopolysaccharide (LPS) stimulated Raw264.7 (LAR) cells, and LAR cells took up PSA NPs 8.79 times more than HSA NPs. As expected, the PSA NPs also accumulated more in the liver. Moreover, SchB-PSA NPs dramatically reduced the activation of NF-κB signaling, and significantly relieved inflammatory response and hepatic necrosis. Notably, the high dose of SchB-PSA NPs improved the survival rate in 72 h of ALI mice to 75%. Hence, SchB-PSA NPs are promising to treat ALI.

Keywords: Acute liver injury; M1 macrophages; Palmitic acid-modified human serum albumin; Schisandrin B.

Graphical abstract

Scheme 1

Schematic representation of the M1 macrophage targeting effect of SchB-PSA NPs on ALI. SchB-PSA NPs can specifically target SR-A overexpressed on M1 macrophages so that SchB can be released at sites of inflammation in the liver and inhibit the production of pro-inflammatory mediators to reduce liver injury.

Fig. 1

Schematic illustration of the formation of SchB-PSA NPs. SchB chemical structure (A), and the preparation of PSA and SchB-PSA NPs (B).

Fig. 2

Physicochemical properties of the SchB-PSA/HSA NPs (n = 3). PDI and TEM of SchB-PSA (A) or SchB-HSA NPs (B). Storage stability of SchB-PSA/HSA NPs at 4 °C (C) or 37 °C (D). The dilution stability of SchB-PSA/HSA NPs in water (E) or PBS (F). The stability of SchB-PSA/HSA NPs in plasma (G) or serum (H) for 48 h.

Fig. 3

The cellular uptake of DiD-PSA/HSA NPs on Raw264.7 and LPS-activated macrophages for 2 h (A) (n = 3); Cytotoxicity of SchB -PSA NPs, SchB -HSA and SchB solution on Raw264.7 (B) and LAR (C) (n = 6); Confocal images of DiD-PSA/HSA NPs on Raw264.7cells (D) and LAR (E) (n = 3). Red: DiD; Blue: DAPI. Scale bar = 50 µm; Effects of SchB-PSA NPs, SchB-HSA NPs and SchB solution on the generation of TNF-α (F), IL-1β (G) and IL-6 (H) on LAR cells. The concentration of these cytokines in the negative control group is below the standard curve. *P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001. NS: no significance.

Fig. 4

The M1 macrophages targeting effect of PSA NPs in vivo (n = 5). (A) The co-localization of SR-A (red) and M1 macrophages (purple). Scale bar: 100 µm. (B) Confocal micrographs of DiD-PSA NPs, DiD-PSA NPs and free DiD in the liver of ALI mice. Green: SR-A, Red: DiD, Blue: DAPI. Sclar bar = 50 µm. (C) Cellular uptake of DiD-PSA NPs, DiD-PSA NPs and free DiD by M1 macrophages. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. NS: no significance.

Fig. 5

In vivo experiments of SchB-PSA/HSA NPs. The drug concentration-time curve (A) and parameters (B) of SchB-PSA NPs, SchB-HSA NPs and free SchB (n = 6); The ex vivo imaging in the liver after the administration of DiD-PSA NPs, DiD-HSA NPs and free DiD during different time intervals (C). The fluorescence analysis of the liver at 2 h (D), 6 h (E) and 9 h (F) in different groups. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. NS: no significance.

Fig. 6

The effects of different formulations against ALI. The diagrammatic figure of the LPS/D-GalN-induced ALI model (A). The gross examination of the liver (B), H&E staining of liver sections (C), histological scores (D) and hepatic index (E) under different treatment conditions, Scale bar: 100 µm. Serum TNF-α (F), IL-1β (G) and IL-6 (H), AST (I) and ALT (J) levels of ALI mice were analyzed. Survival curves of ALI mice in 72 h (K). *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. NS: no significance.

Fig. 7

The mechanisms of SchB-PSA NPs in ALI mice. Total protein in liver tissues was analyzed by Western blotting (A). The densitometric analysis was used to quantify the expression of p-p65, p65, p-IκBα and IκBα (B-C) (n = 3). The rate of M1 macrophages was measured by flow cytometry (D) (n = 6) *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. NS: no significance.