In Vitro And In Vivo Study Of Raddeanin A Long-acting Liposome In The Treatment Of Ovarian Clear Cell Carcinoma

Ovarian clear cell carcinoma（OCCC）is the second most common histologic subtype of ovarian cancer,following high-grade plasmacytoma.It is considered one of the most challenging cancers due to its unique molecular biology,insensitivity to first-line platinum-based chemotherapy,and poorer prognosis compared to other subtypes of ovarian cancer.There is currently no expert consensus on the treatment guidelines for OCCC.Raddeanin A（RA）is an oleanolane triterpenoid saponin extracted from the rhizomes of the anemone raddeana regel.RA has been shown to inhibit the growth and promote the apoptosis of various types of cancer cells,including cervical cancer,breast cancer,hepatocellular carcinoma,gastric cancer,and non-small cell lung cancer.Furthermore,RA can enhance the sensitivity of cholangiocarcinoma and prostate cancer to chemotherapy drugs,and reverse the drug resistance of drug-resistant choriocarcinoma.Therefore,we venture to speculate that RA could be a potential drug for the treatment of OCCC.Unfortunately,RA has a short half-life and low bioavailability in vivo,and saponin drugs have been shown to have potential biological toxicity,such as weight loss,hair loss,and liver damage.Liposomes,one of the earliest developed nanomaterials,can effectively prolong the half-life of drugs in the body,improve drug stability,reduce non-specific organ toxicity,and achieve passive targeting of drugs by utilizing the enhanced permeability and retention effect of tumor tissues.Polyethylene glycol（PEG）is non-toxic,non-antigenic,and non-immunogenic,which can make the liposome structure more stable and less likely to be captured by the reticuloendothelial system.Therefore,PEGylated liposomes are also known as long-acting or stealth liposomes.Currently,dozens of PEGylated liposomal drugs have been approved to use in clinical treatment.Therefore,this study chose to utilize PEGylated liposomes to encapsulate RA to improve its application deficiencies.The present study utilized the thin-film dispersion method to construct long-acting liposomal formulations of Raddeanin A long-acting liposome（RA-PEG-LPs）and characterized their physicochemical properties.Subsequently,human OCCC cell line ES-2-based subcutaneous xenograft models in nude mice were established to investigate the antitumor effects of RA and RA-PEG-LPs in vitro and in vivo,as well as to evaluate their biosafety.The findings indicate that:1.Preparation and Characterization of RA-PEG-LPsRA-PEG-LPs were synthesized using the thin-film dispersion method with RA as the model drug,hydrogenated soybean phospholipid and cholesterol as the membrane materials,and DSPE-PEG₂₀₀₀ as the modifier.The hydrated particle size of RA-PEG-LPs was 173.5±15.3 nm,and it remained stable in p H 7.4 PBS.The particles also exhibited sustained release in PBS at different p H values（p H 7.4 and 6.8）,with a release rate of 59.2±5.5%after 72 hours in p H 6.8 buffer.2.Evaluation of antitumor activity of RA and RA-PEG-LPs in vitroES-2 cells were selected as model cells,and quantitative experiment of endocytosis demonstrated that ES-2 cells continuously uptake RA-PEG-LPs over time.Cytotoxicity experiments showed that RA and RA-PEG-LPs inhibited the growth of ES-2 cells in a time-and dose-dependent manner.Apoptosis experiments demonstrated that RA and RA-PEG-LPs promoted ES-2 cell apoptosis in a time-and dose-dependent manner.Western blotting results indicated that RA and RA-PEG-LPs upregulated the expression of Bax and Grim19 in ES-2 cells,while downregulating the expression of Bcl-2 and Stat3.3.Evaluation of antitumor activity of RA and RA-PEG-LPs in vivoFemale BALB/c nude mice were used to construct ES-2 subcutaneous transplantation tumor model.Compared with the control group,both RA and RA-PEG-LPs were able to inhibit tumor growth,with a more pronounced antitumor effect observed from RA-PEG-LPs.Histological examination of tumor slices stained with HE and TUNEL staining demonstrated that both RA and RA-PEG-LPs promoted apoptosis in tumor tissues,with a more significant effect observed from RA-PEG-LPs.Western blot analysis indicated that RA and RA-PEG-LPs upregulated the expression of Bax and Grim19 in tumor tissues,while downregulating the expression of Bcl-2 and Stat3.4.Biosafety evaluation of RA and RA-PEG-LPsThe body weight of nude mice in the RA group decreased significantly compared with the control group and the RA-PEG-LPTs group.The serum liver function indexes（including alanine aminotransferase、aspartate aminotransferase、alkaline phosphatase、γ-glutamyl transpeptidase）were significantly elevated in the RA group,suggesting hepatotoxicity of RA.HE staining of RA liver tissue showed significant cell swelling,nuclear condensation and dissolution,and Masson staining showed significant liver fibrosis,while no significant pathological changes were observed in the RA-PEG-LPs group.These indicated that RA-PEG-LPs could effectively alleviate the drug toxicity of RA.In conclusion,the RA model drugs selected in this study were effective in treating OCCC both in vitro and in vivo.The prepared RA-PEG-LPs were of appropriate particle size and stable properties,and showed better antitumor effects than RA alone in vivo.More importantly,RA-PEG-LPs could effectively alleviate the toxic side effects of weight loss and liver damage caused by RA to the organism.