Co-deliverying Dual Drugs Using Multifunctional Polymeric Micelles For Breast Cancer Therapy

Nowadays,a disease that seriously endangers human life on a global scale is cancer.Among numerous therapies,chemotherapy still maintains a main choice.Wherein,combination chemotherapy has been largely adopted to enhance anticancer efficacy.However,chemotherapeutics not only have poor water solubility,severe toxicity and low bioavailability,but also it still faces multidrug resistance?MDR?to free chemotherapeutics and the deadly metastasis of cancer cells.These hurdles often limit anticancer potency of treatment,leading to high mortality of cancer patients.Thus,there is an urgent need to develop ideal approaches to circumvention these puzzles for enhanced anticancer efficacy.Therefore,the drug delivery system based on polymer micelles has emerged,which improve the solubility,targeting ability and treatment potency.On the basis of predecessor's research,this paper is aiming to developing versatile polymeric nanoparticles co-delivering dual drugs,and make chemotherapy combine with photodynamic therapy or immunotherapy,to improve therapeutic efficacy of breast cancer which was drug resistant or metastatic.In the chapter 2,we consider PBA-PEG-ss-PCL and PBA-PEG-ss-PCL-hyd-DOX copolymer as basic materials.Targeting,pH-and GSH-sensitive polymer micelles were developed by solvent evaporation method.The polymeric prodrug PBA-PEG-ss-PCL-hyd-DOX with different molar ratios of DOX/HCPT was employed to harvest the DOX+HCPT-M micelles.The drug-loaded micelles still maintain a spherical shape with a uniform size distribution and a good polydispersity.The average size of these micelles was about 140 nm with weak negative charge.As the DOX/HCPT molar ratio decreases,the drug loading capacity?LC%?and drug loading efficience?EE%?of the micelles increases.The toxicity of MCF-7/ADR cells treated with free drug combination and combinatory micelles was assessed to acquire the IC₅₀ values?inhibitory concentration to produce 50%cell death?and CI₅₀ values(combination index determined by IC₅₀),which indicated that this combinatory ratio of 1:2 presented synergism.Therefore,this DOX/HCPT molar ratio was selected for the following investigations.The DOX+HCPT-M micelles remained stable in the blood at pH 7.4.Conversely,the DOX+HCPT-M micelles showed fast drug release at pH 5.0 and 10 mM GSH.As shown in the in vitro cellular uptake and subcellular localization,the micelles could be selective internalized by multidrug resistant?MDR?MCF-7/ADR cells,and escaped from lysosome to eventually locate in the nuclei,which was attributed to targeting ability of phenylboronic acid?PBA?and the responses of pH and GSH.Surprisingly,free DOX combined with HCPT could improve the cellular uptake,due to a possible existence of?–?interaction between DOX and HCPT,which was analyzed by the UV–vis absorption spectra and fluorescence emission spectrum.Furthermore,whatever in the state of drug molecule or in the micellar structure,the association of DOX and HCPT could produce?–?stacked force.This slight structural change could help free drug successfully evade the recognition of drug pumps,and increase the retained amount of DOX.As displayed in in vivo and ex vivo biodistribution,the DOX+HCPT-M micelles enhanced DOX accumulation at the tumor site owning to the targeting capacity of micelles.These micelles also could extend the circulation time in blood by pharmacokinetic assessment.In vitro and in vivo inhibition potency of MDR tumor exhibited the best treatment efficacy of the DOX+HCPT-M,which overcame the MDR to combat the growth and metastases of tumor.Further study in the 3th chapter was carried out on MCF-7/ADR breast cancer tumor with multidrug resistance.The hope is to design a nanosystem combining chemotherapy and photodynamic therapy?PDT?to reverse tumor resistance,to improve the inhibition potency of chemotherapy on drug resistant tumor.First,protoporphyrin was conjugated to the acetylated chondroitin sulfate?AC-CS?.CS targeting and light-activable ROS-sensitive micelles?AC-CS-PpIX,abbreviated as ACP?were developed through self-assembling in distilled water.The ACP micelles present spherical shape with an average size of about 100nm,and the characteristic peak at approximately 635 nm of PpIX was also dramatically found in ACP micelle.Moreover,the generation of reactive oxygen?ROS?is induced by ACP micelles upon light irradiation at 635 nm.¹H NMR spectrum of ACP copolymer shows that as light irradiation time increases,it will occur that the fracture of chondroitin sulfate skeleton resulted in the disassembly of micelleThen,DOX and Apatinib were encapsulated to ACP micelles to harvest the ACP-Dox+Apa micelles.The size changes of the drug-loaded micelles with various irradiation time further demonstrated the disassembly of the micelles.Apatinib,as a competitive inhibitor of DOX efflux,could successfully overcome the MDR and enhance DOX chemosensitivity,confirmed by Lineweaver-Burk plots.ACP micelles could selectively target the MCF-7/ADR cells,determined by cellular uptake evaluation.Meanwhile,in vitro quantitative and qualitative results displayed illuminated ACP micelles could generate a large amount of ROS in MCF-7-ADR cells,which led to the lower of MMP and the production of PDT toxicity against drug resistant cells.Confocal laser scanning microscopy?CLSM?and Pearson colocation coefficient analyses proved that the generation of ROS was induced by ACP-Dox+Apa micelles with 5-min irradiation at 635 nm,to cause the disassembly of the micelles,and eventually released DOX located in cell nucleus.Moreover,the released Apatinib could inhibit the pump function of P-gp,thus efficiently reversed drug resistance to recover the sensitivity of the MDR cells to free DOX.Further study on the inhibition efficacy of resistant tumor and apoptosis induction show that illuminated ACP-Dox+Apa micelles exerted the synergism of chemo-photodynamic therapy on the reversal of drug resistance.Additionally,in vivo antitumor activity also proves that the illuminated micelles can effectively reverse the drug resistance,to enhance the treatment potency of resistant tumor by combination of chemotherapy and PDT.Moreover,the outcomes of western blot reflect the damage of DNA and mitochondrial membrane protein induced by ROS,and the apoptotic mechanism,further implying that light-activable ROS-responsive nanomedicine delivery platform has the potential to combat the drug resistance of tumor.In the 4th chapter,we attempt to combine chemotherapy with immunotherapy,and image that the previous materials are utilized to realize the delivery of chemotherapeutic drug and immunomodulator,in order to enhance the inhibitory efficacy of chemotherapy on breast cancer by immunological approach.We have successfully prepared DOX loading phenylboric acid-polyethylene glycol-polycaprolactone micelles?abbreviated as PPP-DOX?and R837 loading acetylated chondroitin sulfate-protoporphyrin?abbreviated as ACP-R837?.Two kinds of drug loading micelles presented an average size of about 100 nm,and the micelles have stable loading ability.Furthermore,cytotoxicity measurement showed that the two blank carriers presented good cell compatibility toward respective targeted cell.In vitro CLSM and flow cytometry analyses testified that PPP-DOX micelles could specifically target the 4T1 breast cancer cells and ACP-R837 micelles could be selectively internalized by RAW264.7 macrophage cells,moreover,the ACP micelles could locate in the lysosome.We also demonstrated that R837 could activate the RAW264.7 cells and stimulate the transcription and expression of TNF-?and IL-6.The toxicity of the co-culture system of RAW264.7 and 4T1 cells was investigated in vitro,proved that the immune stimulation effect of R837 enhanced the inhibitory effect of DOX chemotherapy on 4T1 cells.Meanwhile,the results of ELISA also reflected that the ACP-R837 micelles indeed promoted the activation of RAW264.7 cells.Frozen section staining of tumor tissue testified that PPP-DOX micelles could selectively target the 4T1 tumor cells and ACP-R837 micelles could target the TAM cells,which was accordant with CLSM results in vitro.Moreover,in vivo antitumor assays reflected that PPP-DOX/ACP-R837 micelles could enhance the chemotherapeutic efficacy of breast cancer through the TAM activation mediated immune response of T cells.Meanwhile,immunohistochemistry and immunofluorescence staining demonstrated that dual-drug codelivering nanosystem could activate antitumor immune response in tumor microenvironment,and following by the cytokines secretion via associated immune cells and the activation of cytotoxic T cells and helper T cells,enventually we could realize the organic combination of chemo-immunotherapy against tumor.