A Comparative Study On The Antitumour Effects And Underlying Mechanisms Of Doxorubicin And Its Nano-drug Delivery Systems In Parental Sensitive And Resistant Human Breast Tumors

Doxorubicin is one of the widely used chemotherapeutic drugs in clinics.However,due to the defects of low tumor targeting ability and poor specificity,doxorubicin may cause significant toxicities to normal tissues.In addition,doxorubicin may induce tumor cell multidrug resistance,which further restricts its clinical application.Macromolecular drug delivery systems provide new pathways to solve the defects of doxorubicin.Liposomal doxorubicin Doxil(?)is the first liposomal anticancer drug.Despite Doxil shows good biocompatibility,good biodegradability,and reduced toxicity to certain organs,it still has obvious limitations including low drug-loading,poor efficacy and significant side effects.The main purpose of this thesis is to study the characteristics of antitumor activity in vitro and in vivo,and the underlying mechanisms between doxorubicin and the doxorubicin-loaded pegylated liposomes delivery system in parental sensitive and resistant breast tumors.Meanwhile,the influence of the tumor-associated macrophages(TAM)on the anticancer activity of the above two formulations of doxorubicin was also evaluated.In addition,we preliminarily determined the in vitro antitumor activity,and the underlying mechanisms of the self-assembled doxorubicin prodrug PEGx-DOX in parental sensitive and resistant breast tumors,in order to obtain useful information for preparation of better doxorubicin-loaded nano-drug delivery systems.In this thesis,we chose our patented sensitive human breast cancer lines BCap37(no P-gp expression)and its resistant cell lines Bats-72(expressing low level of P-gp),Bads-200(expressing high level of P-gp)(Chapters 2-4),and the classical MCF-7(no P-gp expression),MCF-7/ADR(expressing high level of P-gp)cells(Chapter 5)as the biological models.Our data demonstrated that the in vitro cytotoxicity of doxorubicin in resistant tumor cell lines was lower than that in parental sensitive cell lines.Meanwhile,the cytotoxicity of doxorubicin-loaded pegylated liposomes were lower than doxorubicin in all tested cell lines,which might be related to its endocytosis pathway to enter cells and the slow release profile of doxorubicin.In tumor clonal ball(similar to cancer stem cell spheres)model,the sensitivity of cancer cells to both doxorubicin and doxorubicin-loaded pegylated liposomes was further reduced.In xenograft model,doxorubicin showed gradually decreased antitumor activity with increased expression levels of P-gp,while doxorubicin-loaded pegylated liposomes exhibited obviously increased antitumor activity in BCap37,Bats-72 and Bads-200 xenograft models.These findings demonstrated that doxorubicin-loaded pegylated liposomes have advantages for the treatment of both sensitive and resistant tumors,which may be related to their improved drug biodistribution,the increase of tumor targeting ability and the bypass of P-gp-mediated drug efflux.The determination of ATP levels demonstrated that when administered with doxorubicin and doxorubicin-loaded pegylated liposomes for 5h,the cellular ATP levels did not change in BCap37,while they were obviously increased in Bats-72 and Bads-200 cell lines,especially in Bads-200.We hypothesized that these differences were caused by energy-consuming P-gp-mediated drug efflux.Further determination of ROS levels demonstrated that when administered with doxorubicin,the increase of ROS levels was negatively related with the tumor resistance(BCap37>Bats-72>Bads-200).When administered with doxorubicin-loaded pegylated liposomes,the ROS levels did not show significant difference in three cell lines.This data suggest that doxorubicin-loaded pegylated liposomes could bypass the P-gp mediated tumor resistance effectively,leading to effective doxorubicin accumulation in both sensitive and resistant tumor cells.Moreover,the measurement of MMP demonstrated that when administered with doxorubicin and doxorubicin-loaded pegylated liposomes,both drugs decreased MMP(reduction level:BCap37>Bats-72>Bads-200).In addition,we successfully induced and obtained tumor associated macrophages(TAM)in vitro,which showed elevated expression level of CD68.When co-cultured with TAM,Bads-200 cells showed obviously decreased sensitivity to both doxorubicin and doxorubicin-loaded pegylated liposomes,while was accompanied with the increased P-gp protein level..Finally,we utilized the hydrophilic PEG chains of different molecular weights(MW:550,2K)as carriers to react with the hydrophobic doxorubicin and synthesized amphiphilic PEGx-DOX prodrug,which could self-assemble into stable PEGx-DOX nanoparticles with high drug loading.The in vitro experiments demonstrated that PEGx-DOX showed good antitumor activity in both sensitive MCF-7 and resistant MCF-7/ADR cells.In particular the IC50 values of PEG2K-DOX in MCF-7/ADR cells was obviously lower than that of doxorubicin.Further studies confirmed that in resistant MCF-7/ADR cells,doxorubicin increased the ATP levels while PEGx-DOX did not affect the intracellular ATP levels,which might be related to the effective bypass of P-gp by PEGx-DOX.Moreover,in MCF-7/ADR cell line,PEG2K-DOX obviously increased the intracellular ROS levels and decreased MMP.These data indicate that PEG2K-DOX nanoparticles may hold promise for the treamten of resistant tumors and deserve further studies.