Studies On Codelivery Of Doxorubicin And Elacridar Loaded PLGA/TPGS Nanoparticles For Targeting Both Liver Cancer Cells And Cancer Stem Cells

Hepatocellular carcinoma(HCC)is one of the most common tumors,which is a serious threat to human health.At present,the treatment of liver cancer is still based on traditional treatment,but the treatment effect is poor.Liver cancer stem cells(LCSCs)are a kind of tumor cells with a similar subpopulation of normal stem cells,with the ability of self-renewal,unlimited proliferation,which is considered to be the main cause of tumor recurrence.Therefore,it is very crucial to enhance the targeted killing ability of drugs to LCSCs to improve the therapeutic effect of tumor.It is difficult to inhibit LCSCs of the traditional antitumor drugs,which is due to the characteristics of LCSCs with multidrug resistance,the main reason is the over expression of ABC protein,which can discharge the intracellular drug and reduce drug effects.Elacridar(ELC)is the third generation of P-glycoprotein(P-gp)inhibitor that inhibits ABC transporter ABCB1 and ABCG2.Studies have shown that the combination of ELC and chemotherapy drugs can improve the sensitivity of tumor cells to drugs and enhance the inhibition of drugs.In this study,we constructed a co-delivery drug system,and PLGA was used as the carrier to carry the antitumor drug doxorubicin(DOX)and ELC,in order to kill HCC cells and LCSCs.In the first part,HCC cell line HepG2 cells were cultured and Hep G2 microspheres(HepG2-TS)were obtained by serum-free suspension culture.The results of cytotoxicity test showed that the IC50 values of free DOX on HepG2 cells and HepG2-TS were 0.230 ± 0.0221 ?M and 0.477 ± 0.0514 ?M,respectively.The IC50 values of free ELC on HepG2 cells and Hep G2-TS were 9.266 ± 2.609 ?M and 13.740 ± 4.271 ?M,respectively.The results showed that Hep G2 are more sensitive to the DOX,ELC low cytotoxicity;the study of DOX and ELC to kill HepG2 cells and HepG2-TS in different ratios exhibited the optimum synergistic ratio of DOX and ELC combined on HepG2 cells and HepG2-TS was 1:1(mol/mol).In the second part,the determination methods of DOX and ELC were established.The experimental results showed that: the linear regression equation of DOX was A=0.021C+0.002,R2=0.999 in the concentration range of 1 ~ 40?g / m L,with good linearity,high specificity,and intra and inter day precision were less than 2%,the recovery rate was more than 98%;linear regression equation of ELC was A=120742.4626C+1974.5704,and R2=1.0000 in the concentration range of 0.5~100?g / mL.The linearity was good,the specificity was high,and intra and inter day precision were less than 2%,the recovery rate is l more than 98%.These coincided with experimental requirements.In the third part,the blank nanoparticles(NB),DOX loaded nanoparticles(ND),ELC loaded nanoparticles(NE)and DOX and ELC co-loaded nanoparticles(NDE)were prepared according to the nanoparticle precipitation method by using PLGA as carrier and TPGS as surfactant.After the prescription optimization,the particle size of NDE was about 50 nm,which was spherical shape and uniform distribution,and Zeta potentialwas about-15.6mV,drug encapsulation efficiency and drug loading of two drugs was high and the molar ratio was 1:1,and achieved synergy ratio;nanoparticles preservation is more stable under the condition of 4 ?;in vitro release assay showed that NDE had a good sustained-release effect,and the drug release was faster and the cumulative release rate was higher under the condition of pH5.5.In the fourth part,we investigated the antitumor activity including HepG2 cells and HepG2-TS of nanoparticles in vitro.Using DOX as a fluorescence probe on the uptake of nanoparticles on cells,the experimental results showed that nanoparticles could promote the uptake of drugs on HepG2 cells and HepG2-TS and cause higher accumulation;using cytotoxicity experiment,plate cloning experiment and mammosphere formation rate to study antitumor activity in vitro,the results showed that the combination of the two drugs enhanced the cytotoxicity on HepG2 cells and HepG2-TS,and inhibit the proliferation of cells and the formation of microspheres;nanoparticles could enhance the anti-tumor effect of drugs,among which the ND and NE group and NDE group had the strongest inhibitory effect on HepG2 cells and the HepG2-TS.In the fifth part,we investigated the antitumor activity of nanoparticles in vivo.DiR as fluorescent probe to investigate in vivo distribution of nanoparticles in a tumor-burdened nude mice,the experimental results showed that the nanoparticles group was targeted to tumor and could quickly reach the tumor site and accumulate more in the tumor;the results of pharmacokinetic experiments showed that nanoparticles could prolong the half-life of drugs and improve the circulating time of the drug in the body,significantly to improve the drug area under curve;pharmacodynamics experimental results indicated that the blank nanoparticles group system with low toxicity,free DOX+ELC group although also had good anti hepatoma effect,but the system was more toxic and was not conducive to the combined administration;in addition,NDE group had the strongest effect on anti-hepatoma in vivo,and the tumor inhibition rate was 89.99 ± 1.66%.According to the review,compared with the free DOX,free ELC,the combination of the two drugs,the loaded-DOX nanoparticles,the loaded-ELC nanoparticles and the combination of the two nanoparticles,the co-delivery drug delivery system in this study had a good antitumor effect in vivo and in vitro,and provided a new strategy for the treatment of hepatoma.