Self-Activated Cascade-Responsive Sorafenib And USP22 ShRNA Co-Delivery System For Synergetic Hepatocellular Carcinoma Therapy

Background:Hepatocellular carcinoma(HCC)is one of the most common malignant tumors in China.Owing to the lack of specific early clinical symptoms and efficient screening methods,most HCC patients are diagnosed at advanced stages and miss the opportunity for surgical resection and liver transplantation.The patients with unresected HCC could only choose locoregional therapy or systemic therapy,which resulted in the poor prognosis.Sorafenib is the first-line therapy for unresectable HCC.However,only about 30% to 40% of HCC patients can benefit from sorafenib.Resistance to sorafenib severely hinders its effectiveness against HCC and remains one of largest challenges in HCC therapy.Cancer stemness is closely connected with resistance to sorafenib.Methods for reversing the cancer stemness remains one of the largest concerns in research and the lack of such methods obstructs current HCC therapeutics.Ubiquitin-specific protease 22(USP22),one of markers in cancer stem cells(CSCs),has been reported to play a pivotal role in multidrug resistance(MDR)and promote HCC stemness by a HIF-1α/USP22 positive feedback loop.Therefore,USP22-specific gene therapy seems to be a promising method for reversing the cancer stemness and sensitizing HCC cells to sorafenib.Aim:We aimed to analyze the USP22 expression in HCC and explore whether the USP22 expression in HCC cells would influence the sensitivity to sorafenib.Reactive oxygen species(ROS)-responsive charge-reversal polymer B-PDEAEA was synthesized and utilized for condensing USP22(sh USP22)to obtain polyplexes with various N/P ratios.The most optimal N/P ratio was screened based on the gene transfection efficiency of polyplexes with different N/P ratios.Polyplexes were coated with a sorafenib-loaded and galactose-decorated lipid layer to afford Gal-SLP.The synergetic effect of sorafenib and USP22-specific gene therapy was assessed.A sorafenib-insensitive HCC patient-derived xenograft(PDX)model was established and utilized for antitumor efficiency and biosafety evaluation.Methods:Section 1: TCGA and KM-plotter databases were used for analyzing the USP22 expression in HCC and the relationship between USP22 expression and overall survival of HCC patients.USP22-knockdown(SH)and USP22-overexpressing(OE)HCC cells were established via lentiviral infection to explore the role of USP22 in sorafenib sensitivity and glycolysis.Section 2: ROS-responsive charge-reversal polymer B-PDEAEA was synthesized and utilized for condensing sh USP22 to obtain polyplexes with various N/P ratios.Gene transfection efficiency assessment was conducted to choose the most optimal N/P ratio for further experiments.Polyplexes were coated with a sorafenib-loaded and galactose-decorated lipid layer to afford Gal-SLP.The size,zeta potential,morphology and sorafenib release kinetics of Gal-SLP were further explored.Section 3: The ability of Gal-SLP to in vitro inhibit growth and proliferation of HCC was assessed.The synergetic effect of sorafenib and USP22-specific gene therapy was analyzed.DHE staining was executed to figure out whether sorafenib was able to induce intracellular ROS elevation in HCC cells.Cellular uptake and intracellular trafficking study and ROS scavenger assay were conducted to confirm the role of sorafenib-induced ROS in sh USP22 rapid releasing.The USP22 knockdown efficiency and influence in the AKT/GSK-3β/MRP1 of Gal-SLP was assessed and the intracellular sorafenib accumulation was detected.Change of glycolysis rate in Huh-7 cells treated with sorafenib,Gal-LP and Gal-SLP was evaluated and the potential of combinational therapy of glycolysis inhibitors and sorafenib against HCC was explored.A sorafenib-insensitive HCC PDX model was established and utilized for antitumor efficiency and biosafety evaluation.Results:Section 1: According to the TCGA database,the expression of USP22 in HCC was higher than that in normal liver tissue.HCC patients with high USP22 expression have a poorer prognosis than patients with low USP22 expression.HCC cells with different USP22 expression were constructed through lentiviral infection.It was found that suppressing the expression of USP22 would sensitize HCC cells to sorafenib and HCC cells with up-regulated USP22 showed resistant to sorafenib.HCC cells with overexpressed USP22 exhibited higher glycolysis rates;whereas,HCC cells with suppressed USP22 exhibited lower glycolysis rates,which indicated that the glycolysis rate has a positive correlation with USP22 expression in HCC cells.Section 2: ROS-responsive charge-reversal polymer B-PDEAEA condensed sh USP22 and formed spherical polyplexes with uniform sizes of ～ 50 nm and zeta potentials from +20 to +25 m V.Polyplexes exhibited excellent ROS responsiveness and high gene transfection efficiency.According to gene transduction efficiency,N/P=17X was chosen as the most optimal N/P ratio for further experiments.After cloaking the surface of polyplexes with a sorafenib-loaded galactose-decorated lipid layer,the zeta potential of Gal-SLs decreased to-5.6 ± 0.8 m V and the size increased to 95.6 ± 5.2 nm.Sorafenib in Gal-SLP showed a retained release and a favorable faster release in the acidic environment.Section 3: Combination indexes(CI)of sorafenib and sh USP22 gene therapy against Huh-7 and BEL-7402 cells were 0.759 and 0.713,respectively,which indicated a synergetic effect of sorafenib and sh USP22 combinational therapy(Gal-SLP).Gal-SLP showed potent in vitro antitumor efficiency.DHE staining,cellular uptake and intracellular trafficking study and ROS scavenger assay together verified that both sorafenib and sorafenib entrapped in Gal-SLP would induce an elevation of intracellular ROS and a rapid release of sh USP22,thus suppressing USP22 efficiently.The downregulation of USP22 suppressed the expression of MRP1 and led to an enhanced intracellular sorafenib accumulation,further generating an ROS-responsive positive feedback loop.Gal-LP could suppress the glycolysis of Huh-7 and sorafenib would enhance the glycolysis.Furthermore,Gal-SLP would offset the increase of glycolysis rate triggered by sorafenib.And the potential of combination therapy of glycolysis inhibitor and sorafenib against HCC was shown.A sorafenib-insensitive HCC PDX model was established and utilized for antitumor efficiency evaluation.Gal-SLP suppressed USP22 expression in tumors and inhibited the growth of tumors,resulting in a tumor-growth inhibition rate(TIR %)of 82.7 ± 4.9% in terms of tumor weight at the end of experiment,significantly higher than that of sorafenib(53.4 ± 15.5%)and Gal-LPs(55.0 ± 7.8%).Meanwhile,Gal-SLP was biosafe and did no harm to major organs of mice.Conclusions:In this study,we developed a self-activated cascade-responsive sorafenib and sh USP22 co-delivery system(Gal-SLP)for synergetic HCC therapeutics.Gal-SLPs exhibited potent antitumor efficiency via a trio synergetic effect:(i)sorafenib elevated intracellular levels of ROS,which oxidize B-PDEAEA to trigger rapid sh USP22 release for efficient gene downregulation;(ii)the downregulation of USP22 led to downregulation of MRP1 and inhibition of glycolysis,dramatically impairing MDR and achieving higher intracellular sorafenib accumulation,thus generating an ROS-responsive positive feedback loop;(iii)the downregulation of USP22 suppressed the cell metabolism of cancer cells and further influenced cancer stemness.Gal-SLPs exhibited potent antitumor efficiency against a sorafenib-insensitive HCC PDX model and biosafety.Therefore,Gal-SLPs are expected to have great potential in the clinical treatment of HCC.