| Metastatic breast cancer seriously affect women’s life and health.Immunotherapy based on the cytokine interleukin-12(IL-12)is considered to be one of the effective ways to inhibit the growth and metastasis of breast cancer.However,the severe adverse effects after systemic administration and the limited IL-12 mediated immune response under tumor immunosuppressive microenvironment hinder its application.The first-line chemotherapeutic drug doxorubicin(DOX),can not only inhibit the proliferation and induce the apoptosis of tumor cells directly but also generate immunogenic cell death of tumor cells and neutralize the immune-tumor suppressive microenvironment.Therefore,the combination of DOX and IL-12,generating synergistic chemo-immune action,is a practical strategy for metastatic breast cancer treatment.In order to further improve the therapeutic efficacy of antitumor therapy and reduce the side effects of IL-12,a tumor microenvironment charge reversal polymetformin(PMet)/thiolated hyaluronic acid(HA-SH)nanosystem co-delivering DOX and plasmid encoding IL-12 gene(pIL-12)was developed for chemo-gene combination therapy on metastatic breast cancer.The cationic PMet can self-assembled into micelles for DOX physical encapsulation and pIL-12 complexation,formed pIL-12/DOX-PMet micelleplexes.Moreover,a hyaluronidase-sensitive HA-SH was then collaboratively assembled to the pIL-12/DOX-PMet micelleplexes,abbreviated as HA/pIL-12/DOX-PMet co-drug loaded micelleplexes.This micelleplexes has the characteristics of enzyme response,which can realize the charge reversal based on the tumor tissue-specific overexpressed hyaluronidase,complete the co-release of drugs,and generating synergistic chemoimmunotherapy action to fight against growth and metastasis of breast cancer.The contents are as follows:The cationic polymer PMet with micellar self-assembling property was synthesized by reversible addition-fragmentation transfer(RAFT)polymerization.The chemical structure confirmation of PMet was performed by ~1H NMR,the content of MET in PMet was 30.4%.The pIL-12/DOX-PMet micelleplexes were prepared by solvent-evaporation method combined with electrostatic interactions,the pIL-12/DOX-PMet micelleplexes exhibited satisfying characteristics for drug delivery with excellent DOX encapsulation performance.In addition,the gel retardation assay indicated that the cationic polymer PMet had a high condensing capacity for pIL-12.The HA-SH was synthesized by chemical coupled reaction,and was collaborative assembly on pIL-12/DOX-PMet based on electrostatic interaction and thiol cross-link to form HA/pIL-12/DOX-PMet micelleplexes to shield higher positive charge and enhance its blood circulation stability in vivo.In response to the hyaluronidase,the charge of HA/pIL-12/DOX-PMet micelleplexes is reversible,and re-exposing the cationic PMet groups on the surface of micelleplexes,favor pIL-12/DOX release.The cytotoxicity of HA/IL-12/DOX-PMet in 4T1 breast cancer cells was investigated by MTT assay,the results showed that the micelleplexes could effectively inhibit the proliferation of tumor cells.Apoptosis experiment showed the higher pro-apoptotic effect of HA/IL-12/DOX-PMet.The intracellular co-delivery efficacy of DOX and FITC-pIL-12 in HA/PMet micelleplexes was analyzed by flow cytometry,to make sure the two drugs could be co-delivered simultaneously into 4T1 tumor cells for anti-tumor efficacy.Meanwhile,the HA receptor competitive inhibition assay showed that HA/IL-12/DOX-PMet could effectively target 4T1 cells by CD44-mediated endocytosis pathway.The transfection efficiency of HA/PMet nanosystem in 4T1 cells was further investigated.The results showed that the HA/PMet nanosystem had excellent p EGFP and pIL-12 transfection performance,and the following in vivo studies were conducted at N/P of 20/1.In vivo pharmacokinetic studies showed that HA/IL-12/DOX-PMet significantly prolonged the retention time of DOX in blood compared with DOX solution.The 4T1 breast cancer mice mode was established,and the distribution of HA/IL-12/DOX-PMet in various tissues of tumor mice was determined by UPLC-FIR.The results showed that the micelleplexes significantly increased the content of DOX in tumor tissues,indicating that HA/IL-12/DOX-PMet can be effectively targeted to the tumor tissues to reduce the toxic and side effects of free drugs.On the other hand,in vivo gene transfection results showed that the HA/PMet had good transfection efficiency of p EGFP and pIL-12.Therefore,the HA/PMet micelleplexes would be a superior DOX delivery and pIL-12 transfection carrier for DOX and cytokine IL-12 mediated combination therapy.The In vivo anti-tumor and anti-metastasis efficacy of HA/pIL-12/DOX-PMet were studied in mice bearing 4T1 tumors.The highest antitumor activity was achieved in the mice with HA/pIL-12/DOX-PMet micelleplexes treatment,and the survival period was prolonged significantly.The TUNEL and H&E experiments further demonstrated that DOX and pIL-12co-delivered by the micelleplexes induced an improved therapeutic effect on proliferation inhibition and apoptosis induction.The body weight and H&E staining showed that HA/pIL-12/DOX-PMet had good biocompatibility,and could significantly reduced the toxicity of DOX to the heart and kidneys of mice.In addition,HA/pIL-12/DOX-PMet led to the highest antimetastasis activity with the fewest number of tumor nodules in the lungs.The above results indicated that the synergistic anti-tumor effect of DOX and IL-12 was significantly improved by targeting co-delivery of two drugs,which proved the important role of co-loaded and co-delivery of drugs in the nano drug delivery system in improving the efficacy of the combination therapy.In order to further explore the molecular mechanism of the HA/pIL-12/DOX-PMet micelleplexes chemoimmunotherpy,the T lymphocytes(CD4,CD8),NK cells and immune negative regulating Treg cells proliferation,and M2-type macrophages to M1-type macrophages repolarization levels were detected by flow cytometry.The levels of cytokines IL-12,IFN-γand TNF-αwere detected by ELISA kit.The results showed that HA/IL-12/DOX-PMet increased CD4~+T,CD8~+T and NK cells,decreased regulatory T cells and upregulated levels of IL-12,IFN-γand TNF-α.In addition,HA/pIL-12/DOX-PMet significantly increased the number of M1-type macrophages while decreasing the number of M2-type macrophages.The immunofluorescence staining analysis was further employed to verify the infiltration of CD8~+T and NK cells in tumors.The results showed that HA/pIL-12/DOX-PMet treatment group had the strongest immunofluorescence signal of NK cells and CD8~+T cells.These results suggest that the HA/IL-12/DOX-PMet can promote the proliferation of relevant immune effector cells and the secretion of cytokines in the tumor microenvironment,thus producing the best chemo-immuno-synergistic anti-tumor effect.In conclusion,the tumor microenvironment charge reversal HA/PMet nanosystem holds great promise for DOX/pIL-12 co-delivery and exploitation in chemo-gene combination therapy on metastatic breast cancer. |
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