Preparation Of Doxorubicin And Metformin Co-Loaded Liposomes To Overcome Drug Resistant Tumors

Tumors recurrence represents one of the major challenges after the conventional cancer radio/chemo therapy,which is always accompanied with enhanced tumor malignancy,including drug resistance and cell metastasis.This phenomenon accounts for the failures in chemotherapies and decreases patient survival rate.Recent studies indicate that many first-line chemodrugs are effective in inhibiting tumor growth,yet may otherwise enhance tumor malignancy.These unexpected adverse effects might lead to tumor relapse.Liposomes,one of the most explored drug delivery carriers,can change the cellular drug uptake by prolonging drug release,significantly reduce the side effect of chemodrugs.Taking doxorubicin(DOX)as the model,we first evaluated the effect of liposomal DOX on transcriptional pattern of MCF7 breast cancer cells,and revealed the relationship between the formulation and tumor malignancy.Two types of DOX liposomal formulations were prepared by thin film method:the HSPC DOXlipo(Hydrogenated soy phosphatidylcholine doxorubicin liposomes)with slow drug release profile,and the DPPC DOXlipo(1,2-dipalmitoyl-sn-glycero-3-phosphocholine doxorubicin liposomes)with faster drug release.Parts of HSPC DOXlipos entered the cells through clathrin-mediated endocytosis,while the cellular uptake of DPPC DOXlipo were more similar with free DOX.Based on the cellular uptake results,transcriptome-wide elucidation of different DOX formulations on MCF7 cells was performed by microarray.Genes regulated by DOX focusing on DNA damage and repair in the early stage.DOX liposomes slowed the drug release kinetics,which delay the development of tumor malignancy.Compared with free DOX,liposomal DOX reduced the expression of drug resistant genes(e.g.,ABCB1)and significantly inhibited the cell migration behavior.According to the transcriptional analysis and previous studies,GPR87 is closely related to tumor cell proliferation,metastasis and survival under stress,which might involved in DOX induced malignant phenotypes.Therefore,siRNA targeting GPR87 was chosen to co-deliver with DOX using cationic DOX liposomes.The siGPR87/DOX liposomes could inhibit the up-regulation of ABCB1,Twistl,Nanog induced by DOX.Since changing drug release kinetics alone couldn't reverse chemodrug-induced tumor malignancy,we combined metformin(MET)with DOX so as to enhance tumor repression and prevent the development of drug resistance.Recently,epidemiological studies have shown that MET is associated with lower incidence and mortality of numerous cancers.We first found that MET could inhibit cell proliferation,induce cell apoptosis and cell cycle arrest in DOX-resistant breast cancer cells MCF7/ADR.Mechanism studies showed that MET induced mitochondrial toxicity,by reducing mitochondrial membrane potential,stimulating ROS production,repressing ATP level and oxygen consumption.Further,tumor microenvironment,especially glucose starvation significantly enhanced toxicity of MET.In vivo study showed that MET suppressed tumor growth of MCF7/ADR xenograft models,and eliminated Ki67-positive cancer cells.In addition,MET exerted synergistical effect with DOX against MCF7/ADR by increasing nuclear doxorubicin accumulation.Importantly,MET overcame drug resistance by down-regulating drug-resistant genes such as ABCB1,ABCC1,ABCC2 and ABCG2 on mRNA level.MET also inhibited P-glycoprotein(Pgp)expression on protein level through AMPK/mTOR/HIF1? cell signal pathway.MET enhanced anti-tumor effect of DOX in vivo and decreased Pgp expression in MCF7/ADR xenografts.Based on the mechanism studies of DOX and MET co-application,we further investigated how to improve the delivery efficiency of the two drugs.MET is highly hydrophilic and difficult to cross the cell membrane,and easily eliminated in vivo.Then liposomes were used as carriers to co-deliver both drugs into tumor site more efficiently.First,optimized cationic liposomal formulation(HSPC-DOTAP Lipo)were used to promote the cellular uptake of MET.Then DOX was hydrophobized and co-encapsulated with MET to form DOX/MET liposomes.In vitro drug release profile showed that HSPC-DOTAP Lipos had sustained release for both DOX and MET.In addition,DOX/MET liposomes had higher cytotoxicity than free DOX or DOX-only liposomes.Our previous studies indicated that MET had obvious inhibition on oxygen consumption,and therefore might improve the hypoxia tumor microenvironment.Hypoxia is considered as a negative factor for tumor malignancy and drug resistance.Our studies showed that DOX/MET liposomes significantly inhibited oxygen consumption,and suppress HIFla and Pgp expression through AMPK/mTOR activation,which benefits the reduction of hypoxia induced drug resistance.In vivo distribution results showed that liposomes had EPR effect,which enhanced tumor targeting.Furthermore,DOX/MET liposomes relieved hypoxia in vivo,by increasing oxygen concentration in tumor vessels.Finally,the DOX/MET liposomes exhibited higher in vivo tumor inhibition against MCF7/ADR than free DOX or DOX-only liposomes as well.In general,our study demonstrates that liposome can delay the development of malignant tumor phenotype by slowering cellular uptake of DOX.Combination DOX and MET can reverse the drug resistance phenotype on the other side.Further,DOX/MET co-encapsulated liposomes can enhance cytotoxicity against MCF7/ADR in vitro.By reducing cellular oxygen consumption,the DOX/MET liposomes can improve the hypoxia tumor microenvironment,which enhance anti-tumor effect and prevent the development of tumor malignancy.This study provides novel theory and policy for treatment of drug resistant tumors and prevent chemodrug induced tumor malignancy.