A multi-strategy liposome targeting hepatocellular carcinoma cells and stem cells enhances the chemotherapy effect of doxorubicin in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is one of the most serious malignant tumors in China and even the world, and is a global public health problem, its occurrence is insidious and has a poor prognosis. Until now, the therapeutic strategy based on doxorubicin (DOX) is not satisfactory, which may be due to their poor targeting specificity, unable to accurately identify tumor cells and tumor-like stem cells. Therefore, we aimed to established multi-strategy targeting liposomes that can improve the targeting ability and specifically recognize tumor cells and tumor-like stem cells. In this study, we prepared HCC microenvironmental targeting liposomes (PEG5000-PVGLIG-GA-DOX-Lip) by thin-film hydration and ammonium sulfate gradient method. Herein, the responsive peptide (PVGLIG) acts as the linker of the liposomes, and polyethylene glycol 5000 (PEG5000) forms a hydration layer on the surface of the liposomes, which magnifies the enhanced permeability and retention (EPR) effect while hiding the active targeting ligand glycyrrhetinic acid (GA) and reduce the uptake of DOX by normal hepatocytes, relieve toxic side effects. When liposomes were in the microenvironment overexpression of matrix metalloproteinase (MMPs), PVGLIG-responsive peptide was specifically cleaved, PEG5000 hydration layer was shed, and the targeting ligand GA was exposed, increasing the uptake of HCC cells and LCSCs through active targeting. Particle size of PEG5000-PVGLIG-GA-DOX-Lip were found to be 120.45 ± 3.58 nm with zeta potential of -(1.30 ± 0.17) mV. After incubation with MMP2, the particle size of the liposomes decreased to 107.47 ± 0.48 nm and the zeta potential was -(1.87 ± 0.06) mV. Evaluation of anti-tumor activity on HepG2 and liver cancer stem cells (LCSCs) was assessed by CCK-8 assay; IC50 values indicated around 2.2 and 1.5 folds increase in potency of PEG5000-PVGLIG-GA-DOX-Lip with MMP2 as compared to DOX-Lip, respectively. In addition, PEG5000-PVGLIG-GA-DOX-Lip with MMP2 could significantly inhibit the proliferation of HepG2 [(52.55 ± 7.42)% vs (24.26 ± 1.88)%] and LCSCs [(36.88 ± 6.64)% vs (10.98 ± 4.58)%]. Pharmacodynamic studies have shown that liposomes also have enhanced anti-HCC efficacy in vivo, which may be due to size shrinkage caused by the microenvironmental sensitivity of HCC. Thus, the unique multi-strategy targeting liposomes revealed its great potential as a promising system for the treatment of HCC.