Preparation Of Self-Assembled Dihydroartemisinin Nanodrugs And Its Anti-Liver Cancer Effect

Background: Liver cancer is one of the common malignant tumors in China,which has the characteristies of difficult early diagnosis,rapid disease progression,poor prognosis and high mortality.Traditional anti-liver drugs have problems such as poor efficacy,strong toxic and side effects,easy to produce drug resistance.Therefore,it has important clinical significance to explore high-efficiency and low toxicity anti hepatoma drugs.Dihydroartemisinin(DHA)is the main active product of artemisinin(Art),which belongs to sesquiterpenoids and has significant antimalarial activity.Recent researches have shown that DHA can inhibit the proliferation of tumor cells,showing potential clinical application value.However,DHA has problems such as poor water solubility,fast metabolism in the body,and unclear action targets.Therefore,the development of new formulations of DHA is of great significance for its application in anti-tumor.Nano drug delivery system is a new drug delivery strategy,which can not only improve the drug efficacy and reduce the toxic and side effects of chemotherapy,but also realize the controlled release of drugs and the effective accumulation of targets in vivo.Self-assembled nano drug delivery system has high drug loading and avoids the systemic toxicity caused by the factors of traditional nano carriers,which has potential application value in drug delivery system.Therefore,the use of self-assembled nano drug delivery system is expected to solve the bottleneck of DHA clinical application.Objective: By preparing self-assembled Dihydroartemisinin Nanoparticles(DHA NPs),the purpose of improving the water solubility,half-life and antitumor activity of DHA,achieving to target delivery of DHA to tumor cells and reduce the effect of nanocarriers on cells were achieved.The liver cancer cells were used as the object to preliminarily explore the anti-hepatocellular carcinoma effect and mechanism of DHA NPs in vitro.Methods: Self-assembled DHA NPs were prepared by precipitation method.The morphology of nanoparticles was determined by transmission electron microscopy(TEM).The particle size and particle size distribution of DHA NPs were investigated by dynamic light scattering(DLS).Zeta potential was used to measure the surface potential of DHA and DHA NPs.The stability of DHA NPs was investigated by measuring the changes of particle size and polymer dispersion index(PDI)in aqueous solution for 7 days and in phosphate buffer saline(PBS)containing 10% serum for 48 hours by DLS.Thiazole blue(MTT)test was used to detect the proliferation inhibition of different concentrations(10,20,40,60 and 80 μM)DHA NPs on human hepatoma HepG2 cells.DHA NPs loaded with Nile red(NR)fluorescent dye was prepared and the uptake of NR-DHA NPs by HepG2 cells was observed by useing laser confocal microscope.The changes of cell apoptosis after DHA NPs treatment were detected by flow cytometry.High throughput transcriptome sequencing(RNA-seq)was used to analyze the gene expression changes in HepG2 cells after DHA NPs treatment.The differentially expressed genes(DEGs)were enriched and analyzed by Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG).Nextly,the related protein expression of p53 pathway was detected by Western blot.Results: DHA NPs were prepared successfully.The morphology of DHA NPs was uniform and regular.The average particle size was 93.47 nm,and the distribution was normal.Zeta potential measurement showed that the surface potential of DHA NPs decreased compared with that of DHA,which proved that DHA NPs were assembled successfully.The stability test results showed that the particle size and PDI of DHA NPs did not change significantly after being placed in aqueous solution for 7 days and in PBS containing 10% serum for 48 hours,which proved that DHA NPs had good stability.The proliferation inhibition experiment result of DHA NPs HepG2 cells showed that compared with control group,DHA and DHA NPs could significantly inhibit HepG2 cell proliferation in a dose-dependent manner.Under the same concentration,the inhibition rate of cell proliferation in DHA NPs group was higher than that in DHA group.That indicated DHA NPs can enhance the antitumor activity of DHA.The results of cell uptake experiment showed that the nucleus showed red fluorescence in NR-DHA NPs treated group and the intensity of red fluorescence signal increased with the extension of action time and the increase of dosage,indicating that DHA NPs can improve the DHA uptake by cells.The results of apoptosis detected by flow cytometry showed that the early apoptosis rate were increased significantly in DHA NPs group compared with the control group.RNA-seq analysis result showed that 6546 DEGs were detected in DHA NPs treated group compared with control group.The GO analysis results showed that DEGs were mainly enriched in nucleoplasm,protein kinase binding and cell cycle.The results of KEGG pathway enrichment analysis result showed that oxidative phosphorylation,p53 and other signal pathways were involved in DHA NPs treated group.Western blot result showed that compared with the control group,the expression of apoptosis related Bcl-2associated X protein(Bax)increased,the expression of B-cell lymphoma-2 gene(Bcl-2)decreased,the ratio of Bax/Bcl-2 increased in DHA NPs treated group.The expressions of p53 protein,Cytochrome c(Cyt c),Cleaved Caspase-3 and Cleaved Caspase-9 also increased.Conclusion: DHA NPs with regular morphology,uniform size and good stability were prepared successfully.DHA NPs can enhance cellular uptake,inhibit the proliferation of HepG2 cells and induce their apoptosis.The possible mechanism may be related to regulate the p53 cell signal transduction pathway.The results provide new technical support for the clinical application of DHA.