Introduction Cancer drug resistance is referring to the tolerance of cancer cells when treated withchemotherapy.Once the drug resistance occurs,therapeutic effect of anticancer drugwould decrease significantly.Multi-drug resistance can be explained as a condition thatonce cancer cells became resistant to one chemotherapy drug,other drugs with differentstructures or mechanisms would also become invalid.PurposeMulti-drug resistance (MDR) is a major obstacle to successful chemotherapy ofcancer.To overcome the MDR,our research is to develop new liposome andnanomicelle delivery systems.Investigations are focusing on the aspects,includingresistant cancer cell membranes,cancer stem cells,mitochondria,apoptosis genes,vasculogenic mimicry,and heterogeneity of cancer cells.MethodsEvaluations have been performed on the cancer cells,cancer spheroids,and canceranimal models.These nano-scale formulations showed an enhanced the chemotherapyefficacy in resistant cancer and cancer stem cells in vitro and in vivo.Results1.Circumventing resistant cancer cell membraneThere are two kinds of drug resistances on the basis of the sites where the resistanceoccurs,one is due to the impaired delivery pathway of anticancer drugs to cancer cellson the membrane of cancer cell,and the other is from the changed cancer cell itselfincluding genetic and epigenetic alterations.Strategies regarding to this limitation of anticancer drug delivery have been explored.We developed a new formulation of paclitaxel.It is well known that paclitaxel has apotent efficacy against a wide spectrum of cancers in clinical treatment.However,chemotherapy with paclitaxel has been limited due to serious allergic reactions inpatients caused by cremophor EL,MDR of paclitaxel in many cancers,and restrictedpermeability across the intestinal barrier.A kind of functional paclitaxel nanomicelles,which were prepared using PEG2000-DSPE,TPGS1000 and dequalinium as thenanomicelle forming materials,was developed to overcome these obstacles.WhereinPEG2000-DSPE was used to form the micelle skeleton,TPGS1000 was added to inhibitthe drug efflux,and dequalinium was used to promote the uptake of resistant cancercells and intestinal absorption.Evaluations were performed on the breast cancer MCF-7,resistant MCF-7/Adr cells,tumor spheroids,Caco-2 cell manolayers,everted gut sacsand xenografted resistant MCF-7/Adr cancers in nude mice.Results showed that thefunctional paclitaxel nanomicelles significantly increased the intracellular uptake ofpaclitaxel,showing strong inhibitory effect on MCF-7 and MCF-7/Adr cells.Thenanomicelles were able to penetrate deeply into the central region of the tumor‐ 4 ‐spheroids,which resulted in a significant reduction of the spheroids.The intactnanomicelles were observed transporting across the Caco-2 cell manolayer or theeverted gut sac.A significant antitumor efficacy in the xenografted resistant MCF-7/Adrcancers in mice was evidenced by oral administration,which was comparable tointravenous administration.The functional paclitaxel nanomicelles provided a strategyfor oral administration of paclitaxel,increasing solubility of paclitaxel and overcomingthe multidrug resistance as well.2.Eradicating cancer stem cellsCancer stem cells (CSCs) are a small subpopulation of cancer cells with capabilitiesof self-renewal,differentiation,and tumorigenicity when transplanted into an animalhost.Most CSCs are in resting phase.In contrast,cell cycle specific agents (CCSA) areonly sensitive to cells in a certain phase of cell cycle but insensitive to the G0 phasecells.Therefore,CSCs are resistant to CCSA.We developed two types of liposomes,vinorelbine liposomes and parthenolide liposomes,to kill the cancer cells along witheliminating the cancer stem cells synchronously to prevent the potential recurrence of the breast cancer.Vinorelbine has been proven to be not a substrate of ABC transporters,which are highly expressed in cancer stem cells.Yet,the effect of vinorelbine on cancerstem cells is still limited since it is a cell-cycle specific drug which disrupts microtubuleassembly.To circumvent the problem,parthenolide (PTL) which was reported to induceapoptosis of leukemia stem cells is introduced as a co-therapy.Both drugs wereformulated in the liposomes modified with pegylated-lipid derivative which had beencharacterized for prolonged circulation and improved the accumulation of anticancerdrug in tumors through an enhanced permeability and retention (EPR) effect.Cytotoxicity and cancer stem cells identification were performed on human breastcancer cell lines MCF-7 and MDA-MB-231,and the cancer stem cells were furthersorted from MCF-7 cells.Inhibitory effect of different formulations was evaluated on the sorted CSCs and non-CSCs in vitro and in vivo.As compared to non-CSCs,inhibitory effect of vinorelbine in the CSCs was lower while a robust inhibitory effectwas observed when applying vinorelbine in combination with parthenolide.In theMCF-7 xenografts,vinorelbine liposomes plus parthenolide liposomes produced a fullinhibitory effect.This combination therapy may provide a potential strategy foreradication of breast cancer by targeting cancer together with cancer stem cells.3.Targeting mitochondriaApoptosis is a process of programmed cell death involved in cellular stress response,and most chemotherapies kill cancer cells by apoptosis.We developed the daunorubicinplus amlodipine liposomes modified with dequalinium,aiming to target cancermitochondria specifically.Evaluations were performed on the breast cancer MCF-7 andresistant MCF-7/adr cells in vitro and in vivo.The liposomes may enter into the cancercells by phagocytosis of two forms:the integrated and the ruptured liposomes.Themechanisms could be explained as follows:the integrated liposomes with dequaliniummodified on the surface can target mitochondria directly,thus daunorubicin andamlodipine synergistically inhibit Bcl-2/Bcl-XL gene encoding proteins on the outermembrane of mitochondria,resulting in apoptosis of cancer cells.Besides,the drugsmay intercalate with mitochondrial DNA and induce the permeability transition pore opening,leading to the spillage of cytochrome C.Dissimilarly,the drugs in the rupturedliposomes spread into the cytosol so that some of them can react with pro-apoptoticproteins like caspase 8,thus active pro-apoptotic gene Bid and then Bax encodingproteins,which forms large oligomers in the mitochondrial membrane and inducerelease of cytochrome C.Afterwards cytochrome C binds with Apaf-1 in the cytosol andthen pro-caspase-9 so that caspase-9 cleaves,leading to a cascade of caspase-3activation,leading to the apoptosis of cancer cells.In this study,assay with the resistantMCF/Adr cells showed that the activity of Bid was markedly increased and the TUNELassay demonstrated a higher apoptotic percentage of the mitosomal daunorubicin plusamlodipine.4.Future perspectiveCancer progress is closely related to its circulation system for nutritional supplyfrom blood or metastasize malignant cells to other tissues.Apart from the well-knowntumor angiogenesis,vasculogenic mimicry (VM),a possibly alternative circulationsystem,has been described in many malignant tumors and considered to be associatedwith a poor prognosis,early metastasis and relapse in patients with malignant tumors.Highly invasive cancer cells could form highly patterned vascular channels withoutendothelial cells and fibroblasts,namely,the vasculogenic mimicry is anangiogenesis-independent pathway of microcirculation.Moreover,cancer cells thatform the VM channels are highly malignant,poorly differentiated,and have highplasticity.Hence,routine anti-angiogenesis chemotherapy turns out to be a ratherlimited efficacy.Recently,study shows that VM-forming cancer cells exhibits part ofstem cell property and this may explain an increased therapy resistance of these cells.Under these circumstances,a chemotherapy strategy which includes bothanti-angiogenesis and elimination of VM may cut off tumor from capillary systemaround,thus resulting in a satisfactory chemotherapy.The refractoriness of cancer derive from the diversity and variability of cancer cells,and the heterogeneity of cancers render the cancer cells to be different in cellularmorphology,invasiveness,potential of differentiation and the drug resistance.Hence,comprehensive formulations will be needed in solving the drug resistance and relapse ofcancers caused by the heterogeneity.ConclusionOur research has been engaged in developing new targeting drug delivery systemsagainst MDR of cancers since 2002.Mechanisms are mainly involved in the followingaspects:(1) inhibiting or circumventing drug efflux mediated by the adenosinetriphosphate (ATP)-binding cassette (ABC) transporters; (2) eradicating cancer stemcells (CSCs) which are related to the drug-resistant relapse of cancers in the meantime;(3) inducing cancer apoptosis by targeting mitochondria which play a majorstep-limiting role in apoptosis.Apart from the three mechanisms above,there areseveral other factors which also cause MDR of cancers,such as microenvironment andheterogeneity of cancers.The approaches of targeting drug-loaded liposomes mayprovide a new strategy for chemotherapy by modulations of the mutidrug resistantcancer and cancer stem cells.