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Objective Combination of chemotherapy with gene therapy is a promising strategy for cancer treatment.However traditional combination therapy is just simultaneously delivering two or more chemotherapeutic drugs or genes to the same tumor cells for efficient anti-tumor effect.Here, a modified combination therapy was accomplished to stimulate lasting tumor-specific immunity by the co-delivery of doxorubicin (DOX) and DNA.Doxorubicin, an anthracyclines, had capacity to drive calreticulin (CRT) translocation to the tumor cell surface to induce immunogenic cell death.We hypothesized that the polymer could self-assembled into micelles loaded with drugs and pcDNA-CRT, and the micelles reached to the tumors via EPR effect, rapidly release pcDNA-CRT to guide CRT protein synthesis once into the tumor cells.At the same time, the nano-carriers could slowly release DOX to exert cytotoxicity and transfer CRT to tumor cell surface, causing cancer calls "eaten" by immune system.Thus, the anti-tumor effect was improved and the side-effect was reduced significantly.Methods An innovative cationic polymer poly monomethoxy (ethylene glycol)-liner-poly(ethyleneimine)-poly(e-caprolactone) (mPEG-LPEI-PCL) was systhesized via living radical polymerization.Complex mPEG-LPEI-PCL/DNA/DOX was prepared with DOX loaded in the hydrophobic core of the micelles and DNA electrostatic band to the cationic shell.The cytotoxicity of the polymer was evaluated by MTT experiment, and the transfection efficiency of the polymer was qualitative and quantitative confirmed using green fluorescent protein plasmid EGFP-C2 and luciferase plasmid pGL4.74, respectively.With B16F10 cell gene as template, primers were designed with double digestion sites EcoR Ⅰ and HindⅢ and pcDNA3.1/CRT recombinant plasmid was built as the therapy gene.The cellular uptake of the complex was evaluated using scanning confocal microscopy, and the anti-tumor mechanism was researched by Western blotting, phagocytosis and immunohistochemistry experiments.Finally, the in vivo efficacy of the complex was assessed by building H22 tumor-bearing mice.Results mPEG-LPEI-PCL could self-assembled into micelles loaded with DOX and DNA.The drug loading of the micelles was 15.74%, and the encapsulation efficiency was as high as 94.41%.DOX/DNA/mPEG-LPEI-PCL complex were easily formed in aqueous medium with an average size of 150nm and positive surface charge, which were suitable for cellular uptake.The release of DOX from the micelles was pH-dependent.It released much faster in the simulated acidic environment such as tumor tissues than in pH 7.4 physiological conditions.At 48h, the releases of DOX from the micelles at pH of 5.0, 6.8 and 7.4 were 85.3%, 74.8% and 62.82% respectively.IC50 of mPEG-LPEI-PCL was 329.13μg/mL, less cytotoxicity than PEI.The novel polymer enhanced green fluorescent protein expression in cells, and optimal transfection efficiency was observed at N/P of 20, when the luciferase gene pGL4.74 expression level was (1.05±0.07) × l06 RLU/mg protein.Sequencing results revealed a synonymous mutation, namely, amino acid sequence was identical with that in gene bank.pcDNA3.1/CRT recombinant plasmid could be expressed in mammalian cells.In the cell uptake experiment, green fluorescence (DNA) could be observed in the whole cell, while red fluorescence (DOX) was observed only in the cytoplasm rather than in the cell nuclei after 24 h incubation.With another 12 h culture of the transfected cells, DOX fluorescence in nucleus increased and green fluorescence was still significant.The results suggested that the complex was internalized through endocytosis and released DOX slowly.Western blot proved that mPEG-LPEI-PCL increased CRT plasmid entering into cells, thereby increasing the protein expression.Primary cultured macrophages and tumor cells were both stained with fluorescent dyes.Calreticulin overexpressed and doxorubicin induced tumor cells tend to be phagocytized.The complex mPEG-LPEI-PCL/DNA/DOX causing more infiltration of macrophages in the tumor tissue, with CD68 as macrophage marker.Western blotting, phagocytosis and immunohistochemistry experiments demonstrated that the activation of tumor immune response seen in these mice was a result of overexpression of CRT protein, which is enhanced by co-delivered of DOX.Thus, co-delivered DOX and CRT could work together to trigger more macrophages infiltration in tumor tissue and enhance tumor cell phagocytosis, resulting in asynergistic tumor immunity and following tumor cell disruption.The systemic administration of the complex induced a profound tumor suppression in BI6F10 cell xenograft murine model.mPEG-LPEI-PCL/CRT/DOX could suppress tumor growth and prolong their life remarkably, with a tumor volume of 259.10 ± 26.03 mm3 and an inhibition rate of 79.66% after three weeks treatment.Conclusion mPEG-LPEI-PCL is a promising carrier for the co-delivery of gene and chemotherapeutic drugs to achieve a synergistic effect.Combination therapy based on immunogenicity of cancer cells is a pioneer in gene therapy and chemotherapy.