Purpose(s)
According to data estimated by the WHO, primary liver cancer is currently the fourth most common malignant tumor and the second leading cause of death around the world. Hepatocellular carcinoma (HCC) is one of the most common primary liver malignancies, so effective therapy is highly desired for HCC.
Herein we presented a new idea that co-administration of poly(L-Aspartic acid)-poly(ethylene glycol) /combretastatin A4 nanoparticles (CA4-NPs) and aPD-L1 possessed notable anti-tumor efficacy for HCC treatment.
Method(s)
1. Design, synthesis and characterization CA4-NPs, including determining the structure of nano-medicine by 1H NMR, detecting nano-drug particle by DLS and TEM, etc.
2. In vitro cytotoxicity and uptake of the CA4-NPs experimental section. Cytotoxicity was evaluated by MTT assay. Then FITC was labeled to investigate the uptake effect by using CLSM and FACS.
3. Pharmacokinetics and biodistribution of CA4-NPs. To verify the behaviors in vivo, Cy5.5 was used to monitor imaging method in vivo.
4. Mechanism of combination aPD-L1 with CA4-NPs. The level of HIF-1α and PD-L1 in tumor tissues under immunofluorescence staining was compared.
5. In vivo anti-tumor efficacy. A Hepa1-6 mouse subcutaneous liver cancer model was constructed. After administration, the tumor volume, size and weight were observed, and experiment of survival percentages of mice was performed. Then immunofluorescence and Elisa were used to detect Ki67, TUNEL, CD31, IFN-γ/IL-4, CD4/CD8, LAG-3/TIM-3 and other indicators to evaluate the anti-tumor effect of each group of drugs.
Result(s)
CA4-NPs arrest the tumor by disrupting the established central tumor vasculature, which can induce severe hypoxic conditions and high expression of HIF-1α in tumor tissues. The hypoxic condition can increase the expression of PD-L1 in tumor microenvironment, so the PD-L1 inhibitors can release the ‘immune brake’ to reactivate the T-cell immune response to the tumor effect in the peripheral area.
Conclusion(s)
In this study, a combination of CA4-NPs with aPD-L1 was developed for systemic treatment of HCC. The use of CA4-NPs was aimed to cause extensive tumor necrosis mainly due to the triple target effects - EPR effect, acid-sensitive (pH = 5.5) effect to TME and good selectivity of CA4 for tumor blood vessel. Also, the circulation time of CA4-NPs in vivo was about 10 times that of CA4P. However, CA4-NPs also induced overexpression of PD-L1 that was confirmed by immunofluorescence staining in the treated tumors. Therefore, the aPD-L1 was applied to efficiently up-regulate the growth and proliferation of T cells and activate their attack and killing functions. The combination of PEG-b-PAsp-g-CA4 at 40 mg kg-1 with aPD-L1 (50 µL) showed a nearly 90% tumor suppression rate in a Hepa1-6 subcutaneous tumor model with low systemic toxicity. These findings indicated that the two-pronged attack of aPD-L1 with a strategy of VDAs, CA4-NPs, was really a potential therapeutic approach for HCC treatment.