Polyvinylpyrrolidone-Modified Taxifolin Liposomes Promote Liver Repair by Modulating Autophagy to Inhibit Activation of the TLR4/NF-κB Signaling Pathway

Abstract

Taxifolin (TAX) is a hepatoprotective flavanol compound, which is severely limited by poor solubility and low bioavailability. Liposomes (Lips) are used as well-recognized drug carrier systems that improve the water solubility and bioavailability of drugs, but are easily damaged by gastric juice after oral administration, resulting in the release of drugs in the gastric juice. Therefore, it is important to find materials that modify liposomes and avoid the destruction of the liposomal phospholipid bilayer structure by the gastrointestinal environment. Taxifolin liposomes (TAX-Lips) were modified by polyvinylpyrrolidone-k30 (PVP-TAX-Lips) and manufactured using a thin-film hydration technique. Particle size (109.27 ± 0.50 nm), zeta potential (-51.12 ± 3.79 mV), polydispersity coefficient (PDI) (0.189 ± 0.007), and EE (84.7 ± 0.2%) of PVP-TAX-Lips were studied. In addition, the results of in vitro release experiments indicated that the cumulative release rates of TAX-Lips and PVP-TAX-Lips were 89.73 ± 5.18% and 65.66 ± 4.86% in the simulated gastric fluid after 24 h, respectively, while the cumulative release rates were 68.20 ± 4.98% and 55.66 ± 3.92% in the simulated intestinal fluid after 24 h, respectively. Moreover, PVP-TAX-Lips were able to reverse lipopolysaccharide and D-galactosamine (LPS/D-GalN)-induced acute liver injury (ALI) by inducing autophagy to inhibit the expression levels of the TLR4/NF-κB signaling pathway and inflammatory factors, which suggested that PVP-TAX-Lips played an important role in the prevention of ALI and also provided a promising drug delivery system for the application of TAX.

Keywords: TLR4/NF-κB; acute liver injury; autophagy; liposomes; polyvinylpyrrolidone-K30; taxifolin.

FIGURE 1

The schematic diagram of the structure of PVP-TAX-Lips.

FIGURE 2

Characterization of TAX-Lips and PVP-TAX-Lips. (A) The HPLC chromatogram of TAX standard. (B) The HPLC chromatogram of TAX-Lips. (C) The HPLC chromatogram of PVP-TAX-Lips. (D) Particle size of TAX-Lips. (E) Zeta potential of TAX-Lips. (F) Particle size of PVP-TAX-Lips. (G) Zeta potential of PVP-TAX-Lips. (H) TEM image TAX-Lips. (I) TEM image of PVP-TAX-Lips. (J) FTIR spectra of TAX-Lips, PVP-TAX-Lips, and pure TAX, and a physical mixture of PVP-K30, TAX, and SPC.

FIGURE 3

(A) In vitro release profiles of free TAX, TAX-Lips, and PVP-TAX-Lips in simulated gastric juice (pH = 1.2). (B) In vitro release profiles of free TAX, TAX-Lips, and PVP-TAX-Lips in simulated intestinal fluids juice (pH = 6.8) (mean ± SD, n = 3).

FIGURE 4

Acute liver injury in mice. (A) Effects of TAX-Lips and PVP-TAX-Lips on the appearance of the liver in mice. (B) H&E-stained liver tissue sections, with magnifications of ×100 and 200X, and the necrosis score of the liver. (C,D) ALT and AST levels in mice serum. (E–G) GSH, MDA, and SOD content in the mice liver tissue. As compared with the control group, ##p < 0.01,###p < 0.001; As compared with the model group, *p < 0.05, **p < 0.01, ***p < 0.001. The data are presented as the mean ± SD (n = 8).

FIGURE 5

Inflammatory factors in mice with ALI induced by LPS/D-GalN. (A) Protein levels of iNOS, IL-1β, TNF-α, and β-actin analyzed by Western blotting. (B–D) Relative band intensity analyzed by ImageJ Analysis System, and β-actin was used as a control for equal loading. As compared with the control group, #p < 0.05. As compared with the model group, *p < 0.05. The data are presented as the mean ± SD (n = 3).

FIGURE 6

Expression of TLR4/NF-κB signaling pathway induced by LPS/D-GalN. (A) Protein levels of TLR4, MyD88, IκBα, p-IκBα, NF-κB, and β-actin analyzed by Western blotting. (B–E) Relative band intensity analyzed by ImageJ Analysis System, and β-actin was used as a control for equal loading. As compared with the control group, ##p < 0.01, ###p < 0.001; As compared with the model group, **p < 0.01, ***p < 0.001. The data are presented as the mean ± SD (n = 3).

FIGURE 7

Expression of the autophagy proteins induced by LPS/D-GalN. (A) Protein levels of LC3, P62, ATG5, ATG7, and β-actin analyzed by Western blotting. (B–E) Relative band intensity analyzed by ImageJ Analysis System, and β-actin was used as a control for equal loading. As compared with the control group, #p < 0.05. As compared with the model group, *p < 0.05, **p < 0.01. The data are presented as the mean ± SD (n = 3).

FIGURE 8

The diagram of PVP-TAX-Lips oral administration into the body circulation.