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Kaiyu Wang,1,* Yan Gao,1,* Shan Wu,1,* Jin Zhang,1 Manfang Zhu,2 Xiayu Chen,1 Xizi Fu,1 Xingmei Duan,2 Ke Men1 1State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University, Chengdu, 610041, People’s Republic of China; 2Department of Pharmacy, Personalized Drug Therapy Key Laboratory of Sichuan Province, Sichuan Academy of Medical Sciences & Sichuan Provincial People’s Hospital, School of Medicine, University of Electronic Science and Technology of China, Chengdu, 610072, People’s Republic of China*These authors contributed equally to this workCorrespondence: Xingmei Duan, Department of Pharmacy, Personalized Drug Therapy Key Laboratory of Sichuan Province, Sichuan Academy of Medical Sciences & Sichuan Provincial People’s Hospital, School of Medicine, University of Electronic Science and Technology of China, Chengdu, 610072, People’s Republic of China, Email duanxingmei2003@163.com Ke Men, State Key Laboratory of Biotherapy and Cancer Center, West China Hospital of Sichuan University, Chengdu, 610041, People’s Republic of China, Email mendingbob@hotmail.comBackground: Messenger RNA (mRNA)-based immunogene therapy holds significant promise as an emerging tumor therapy approach. However, the delivery efficiency of existing mRNA methods and their effectiveness in stimulating anti-tumor immune responses require further enhancement. Tumor cell lysates containing tumor-specific antigens and biomarkers can trigger a stronger immune response to tumors. In addition, strategies involving multiple gene therapies offer potential optimization paths for tumor gene treatments.Methods: Based on the previously developed ideal mRNA delivery system called DOTAP-mPEG-PCL (DMP), which was formed through the self-assembly of 1.2-dioleoyl-3-trimethylammonium-propane (DOTAP) and methoxypoly (ethylene glycol)-b-poly (ϵ-caprolactone) (mPEG-PCL), we introduced a fused cell-penetrating peptide (fCPP) into the framework and encapsulated tumor cell lysates to form a novel nanovector, termed CLSV system (CLS: CT26 tumor cell lysate, V: nanovector). This system served a dual purpose of facilitating the delivery of two mRNAs and enhancing tumor immunogene therapy through tumor cell lysates.Results: The synthesized CLSV system had an average size of 241.17 nm and a potential of 39.53 mV. The CLSV system could not only encapsulate tumor cell lysates, but also deliver two mRNAs to tumor cells simultaneously, with a transfection efficiency of up to 60%. The CLSV system effectively activated the immune system such as dendritic cells to mature and activate, leading to an anti-tumor immune response. By loading Bim-encoded mRNA and IL-23A-encoded mRNA, CLSV/Bim and CLSV/IL-23A complexes were formed, respectively, to further induce apoptosis and anti-tumor immunity. The prepared CLSV/dual-mRNA complex showed significant anti-cancer effects in multiple CT26 mouse models.Conclusion: Our results suggest that the prepared CLSV system is an ideal delivery system for dual-mRNA immunogene therapy.Keywords: mRNA gene therapy, dual-mRNA delivery, tumor cell lysates, Bim and IL-23A mRNAs, immune response

Jing Huang,1,* Kaiyu Wang,1,* Xizi Fu,1,* Manfang Zhu,2 Xiaohua Chen,1 Yan Gao,1 Pingchuan Ma,3 Xingmei Duan,2 Ke Men1 1State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University, Chengdu, 610041, People’s Republic of China; 2Department of Pharmacy, Personalized Drug Therapy Key Laboratory of Sichuan Province Sichuan Academy of Medical Sciences & Sichuan Provincial People’s Hospital, School of Medicine, University of Electronic Science and Technology of China, Chengdu, 610072, People’s Republic of China; 3State Key Laboratory of Oral Diseases & National Center for Stomatology & National Clinical Research Center for Oral Diseases & Department of Head and Neck Oncology, West China Hospital of Stomatology, Sichuan University, Chengdu, Sichuan, 610041, People’s Republic of China*These authors contributed equally to this workCorrespondence: Ke Men, State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University, Chengdu, 610041, People’s Republic of China, Email mendingbob@hotmail.com Xingmei Duan, Department of Pharmacy, Personalized Drug Therapy Key Laboratory of Sichuan Province Sichuan Academy of Medical Sciences & Sichuan Provincial People’s Hospital, School of Medicine, University of Electronic Science and Technology of China, Chengdu, 610072, People’s Republic of China, Email duanxingmei2003@163.comBackground: Messenger ribonucleic acid (mRNA)-based gene therapy has great potential in cancer treatment. However, the application of mRNA-based cancer treatment could be further developed. Elevated delivery ability and enhanced immune response are advantages for expanding the application of mRNA-based cancer therapy. It is crucial that the prepared carrier can cause an immune reaction based on the efficient delivery of mRNA.Methods: We reported DMP nanoparticle previously, which was obtained by the self-assembly of 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) and (ethylene glycol)-b-poly (ϵ-caprolactone) (mPEG-PCL). Research demonstrated that DMP can deliver mRNA, siRNA, and plasmid. And it is applied to various tumor types. In our work, the tumor cell lysate was introduced to the internal DMP chain, fusing cell-penetrating peptides (CPPs) modification on the surface forming the CLSV system. And then mixed encoded IL-22BP (interleukin-22 binding protein) mRNA and CLSV to form CLSV/IL-22BP complex.Results: The size of the CLSV system was 213.2 nm, and the potential was 45.7 mV. The transfection efficiency of the CLSV system is up to 76.45% in C26 cells via the micropinocytosis pathway. The CLSV system also could induce an immune response and significantly elevate the expression of CD80, CD86, and MHC-II in vivo. Then, by binding with IL-22BP (Interleukin-22 binding protein) mRNA, the CLSV/IL-22BP complex inhibited tumor cell growth, with an inhibition rate of up to 82.3% in vitro. The CLSV/IL-22BP complex also inhibited tumor growth in vivo, the tumor cell growth inhibition up to 75.0% in the subcutaneous tumor model, and 84.9% in the abdominal cavity metastasis tumor model.Conclusion: Our work demonstrates that the CLSV system represents a potent potential for mRNA delivery.Keywords: mRNA, cell-penetrating peptide, gene therapy, tumor cell lysate, immune response

Jingmei Li,1,* Jin Zhang,1,* Yan Gao,1 Sibei Lei,1 Jieping Wu,1 Xiaohua Chen,1 Kaiyu Wang,1 Xingmei Duan,2 Ke Men1 1Department of Biotherapy, Cancer Center and State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu, 610041, People’s Republic of China; 2Department of Pharmacy, Personalized Drug Therapy Key Laboratory of Sichuan Province Sichuan Academy of Medical Sciences & Sichuan Provincial People’s Hospital, School of Medicine, University of Electronic Science and Technology of China, Chengdu, 610072, People’s Republic of China*These authors contributed equally to this workCorrespondence: Ke Men, State Key Laboratory of Biotherapy and Cancer Center, National Clinical Research Center for Geriatrics, West China Hospital of Sichuan University, Chengdu, 610041, People’s Republic of China, Email mendingbob@hotmail.com Xingmei Duan, Department of Pharmacy, Personalized Drug Therapy Key Laboratory of Sichuan Province, Sichuan Academy of Medical Sciences & Sichuan Provincial People’s Hospital, School of Medicine, University of Electronic Science and Technology of China, Chengdu, 610072, People’s Republic of China, Email duanxingmei2003@163.comIntroduction: Cell-membrane nanocarriers are usually constructed by modifying the nanoparticle surface with cell membrane extracts, which has a direct benefit in endowing targeting capacity to nanocarriers based on their original cell types. However, delivering nucleic acid cargos by cell membrane–based nanoparticles is difficult owing to the strong negative charge of the cell membrane fraction. In this study, we developed a cancer cell membrane–based drug delivery system, the cMDS, for efficient siRNA delivery. Meanwhile, the cancer-specific immune response stimulated by the gene vector itself could offer synergistic anti-cancer ability.Methods: The cMDS was prepared by ultrasound, and its transfection efficiency and anti-cancer ability were examined using cultures of CT26 cells. MTT and red blood cell hemolysis tests were performed to assess the safety of cMDS, while its targeted gene delivery and strong immune stimulation were investigated in a subcutaneous tumor model. Moreover, the detailed anti-cancer immune stimulation mechanisms of cMDS are uncovered by protein chip analysis.Results: The cMDS was spherical core-shell structure. It showed high transfection efficiency and anti-cancer ability in vitro. In animal experiments, intravenously administered cMDS/siStat3 complex efficiently suppress the growth of colon cancer. Moreover, the result of protein chip analysis suggested that cMDS affect the migration and chemotaxis of immune cells.Conclusion: The cMDS shows obvious tumor tissue-specific accumulation properties and strong immune stimulation ability. It is an advanced targeted gene delivery system with potent immunotherapeutic properties.Keywords: bioinspired material, cancer cell membrane, targeted siRNA delivery, immunotherapeutic, colon cancer

Jing Huang,1,* Mengmeng Dai,1,* Mingxia He,1,* Weicheng Bu,1 Liwen Cao,1 Jing Jing,1 Run Cao,1 Hailong Zhang,1 Ke Men2 1Joint National Laboratory for Antibody Drug Engineering, The First Affiliated Hospital, Henan University, Kaifeng, Henan Province, 475004, People’s Republic of China; 2State Key Laboratory of Biotherapy and Cancer Center, West China Hospital of Sichuan University, Chengdu, Sichuan Province, 610044, People’s Republic of China*These authors contributed equally to this workCorrespondence: Hailong Zhang, Joint National Laboratory for Antibody Drug Engineering, The First Affiliated Hospital, Henan University, Kaifeng, Henan Province, 475004, People’s Republic of China, Email hailong6891@163.com Ke Men, State Key Laboratory of Biotherapy and Cancer Center, West China Hospital of Sichuan University, Chengdu, Sichuan Province, 610044, People’s Republic of China, Email mendingbob@hotmail.comPurpose: The abnormal activation of NLRP3 inflammasome is related to the occurrence and development of ulcerative colitis (UC). However, the ideal drug and delivery system remain important factors limiting the targeting of NLRP3 inflammasome in UC therapy. Gene therapy by delivering siRNA is effective in treating various diseases. Therefore, delivering siNLRP3 using an ideal vector for UC treatment is necessary.Materials and Methods: Nanoparticles delivering siNLRP3 were developed based on cationic liposome (CLP/siNLRP3). Their ability to inhibit NLRP3 inflammasome activation was monitored using Western blot (WB) and Enzyme-linked Immunosorbent Assay (ELISA). The ASC oligomerization in LPS-primed peritoneal macrophages (PMs) was detected by WB and immunofluorescence. Moreover, we assessed the role of CLP/siNLRP3 on dextran sodium sulfate (DSS)-induced UC by examining NLRP3 levels, pro-inflammatory cytokines expression, and disease-associated index (DAI). Flow cytometry (FCM) was used to detect the contents of macrophages and T cells. Finally, we assessed the safety of CLP/siNLRP3.Results: The prepared CLP was spherical, with a small particle size (94 nm) and low permeability. The CLP could efficiently protect siNLRP3 from degradation and then deliver siNLRP3 into PMs, inhibiting NLRP3 inflammasome activation. Also, the CLP/siNLRP3 could inhibit the secretion of mature IL-1β and IL-18 from PMs, thereby achieving a favorable anti-inflammation effect. In vivo, CLP/siNLRP3 could effectively alleviate intestinal injury in UC mice, which was attributed to down-regulating levels of IL-1β and IL-18, inhibiting infiltration of macrophages and other immune cells, and the polarization of M1 macrophages. Finally, pathological testing of tissue sections and blood biochemical tests showed no significant toxic effects of CLP/siNLRP3.Conclusion: We introduced a prospective approach for the efficient delivery of siRNA in vitro and in vivo with high safety and stability, which was found to have great potential in treating NLRP3-driven diseases in an RNA-silencing manner.Keywords: cationic liposome, NLRP3, siRNA, macrophage, ulcerative colitis

Pingchuan Ma,1 Jingmei Li,2 Yan Gao,2 Jieping Wu,2 Ke Men,2 Chunjie Li,1 Yi Men,1 Xingmei Duan3 1State Key Laboratory of Oral Diseases, National Clinical Research Center for Oral Diseases, Department of Head and Neck Oncology, West China Hospital of Stomatology, Sichuan University, Chengdu, Sichuan Province, 610041, People’s Republic of China; 2State Key Laboratory of Biotherapy and Cancer Center, West China Hospital of Sichuan University, Chengdu, Sichuan Province, 610041, People’s Republic of China; 3Department of Pharmacy, Personalized Drug Therapy Key Laboratory of Sichuan Province, Sichuan Academy of Medical Sciences & Sichuan Provincial People’s Hospital, School of Medicine, University of Electronic Science and Technology of China, Chengdu, Sichuan Province, 610072, People’s Republic of ChinaCorrespondence: Yi Men, State Key Laboratory of Oral Diseases, National Clinical Research Center for Oral Diseases, Department of Head and Neck Oncology, West China Hospital of Stomatology, Sichuan University, Chengdu, 610041, Sichuan Province, People’s Republic of China, Email yurimen@163.com Xingmei Duan, Department of Pharmacy Personalized Drug Therapy Key Laboratory of Sichuan Province, Sichuan Academy of Medical Sciences & Sichuan Provincial People’s Hospital, School of Medicine, University of Electronic Science and Technology of China, Chengdu, 610072, People’s Republic of China, Email duanxingmei2003@163.comPurpose: Oral squamous cell carcinoma (OSCC) is the most common type of oral cancer, with more than 300,000 new cases annually. Despite advances in existing treatments, including surgery, radiation, chemotherapy, and immunotherapy, the overall survival and prognosis have remained poor. However, gene therapy based on non-viral vectors provides new ideas for the treatment of OSCC. Here, we aimed to prepare and describe the synthesis, biosafety, and preclinical efficacy of DOTAP-mPEG-PCL (DMP) in OSCC gene therapy.Methods: We prepared a nano-sized hybrid cationic micelle DMP. DMP micelles were prepared by self-assembling cationic lipid DOTAP and mPEG-PCL polymer. We evaluated the characteristics of this cationic micelle in vitro. Combined with encoding the apoptosis-inducing BimS gene, we established the DMP/phBimS complex and evaluated its anti-tumor effect in vitro. We also established a mouse tongue xenograft model to evaluate the antitumor effect of the DMP/phBimS complex in vivo through local and systemic administration prospectively.Results: The DMP cationic micelle is spherical in shape, with an average diameter of 28.32 ± 3.56 nm and an average zeta potential of 43.43 ± 0.82 mV. By activation of lipid raft-mediated endocytosis caveolin-mediated endocytosis, DMP could efficiently deliver plasmid into SCC15 cells (efficiency: 52.07% ± 1.63%), with an ideal biosecurity. When loaded by plasmid encoding the apoptosis-inducing BimS gene, the DMP/phBimS complex exhibited an obvious anti-proliferation effect of SCC15 in vitro through the apoptosis pathway (33.9% ± 2.62% apoptosis rate). By local administration, the DMP/phBimS complex showed ideal anti-tumor properties in the nude mouse tongue xenograft model, with an average tumor inhibition rate of 65.66%. Furthermore, through systematic administration, the DMP/phBimS complex obviously inhibited OSCC growth, with an average inhibition rate of 45.63% (DMP/phBimS) and an appropriate biocompatibility.Conclusion: The DMP/phBimS complex is an optional effective option for suicide gene therapy for OSCC.Keywords: gene therapy, Bim, micelle, oral squamous cell carcinoma, non-viral delivery

Yan Gao,1,* Ke Men,1,* Congbin Pan,1 Jingmei Li,1 Jieping Wu,1 Xiaohua Chen,2 Sibei Lei,1 Xiang Gao,1 Xingmei Duan2 1State Key Laboratory of Biotherapy and Cancer Center, West China Hospital of Sichuan University, Chengdu, Sichuan Province, People’s Republic of China; 2Department of Pharmacy, Sichuan Academy of Medical Sciences & Sichuan Provincial People’s Hospital, Personalized Drug Therapy Key Laboratory of Sichuan Province, School of Medicine, University of Electronic Science and Technology of China, Chengdu, 610072, People’s Republic of China*These authors contributed equally to this workCorrespondence: Ke MenState Key Laboratory of Biotherapy and Cancer Center, West China Hospital of Sichuan University, Chengdu, 610041, People’s Republic of ChinaEmail mendingbob@hotmail.comXingmei DuanDepartment of Pharmacy, Sichuan Academy of Medical Sciences & Sichuan Provincial People’s Hospital, Personalized Drug Therapy Key Laboratory of Sichuan Province, School of Medicine, University of Electronic Science and Technology of China, Chengdu, 610072, People’s Republic of ChinaEmail duanxingmei2003@163.comBackground: Gene therapy has emerged as a new strategy for cancer therapy. As an alternative nucleic acid material, messenger ribonucleic acid (mRNA) is being increasingly utilized in cancer gene therapy. However, unfulfilled requirements and a lack of ideal mRNA delivery vectors persist.Methods: We developed an advanced mRNA delivery system, DMP-039, by fusing a cell-penetrating peptide, cRGD-R9, and a cationic nano-sized DMP backbone together. The DMP gene vector backbone was synthesized by the self-assembly of DOTAP lipid and mPEG-PCL polymer. Introduction of the cRGD-R9 peptide onto the DMP backbone was performed to elevate the mRNA delivery capacity, which resulted in a peptide-functionalized hybrid delivery system.Results: The average size of the synthesized DMP-039 was 268.9 ± 12.4 nm (PDI = 0.382), with a potential of 17.4 ± 0.5 mV. The synthesized DMP-039 hybrid nanoparticles exhibited high mRNA delivery efficiency through multiple mechanisms during transmembrane transportation. By loading the encoding mRNA from the suicide gene Bim, a locally administered mBim/DMP-039 complex strongly inhibited growth in two colon cancer models. Moreover, intravenous administration of the mBim/DMP-039 complex efficiently suppressed C26 pulmonary metastatic tumor progression with high safety. The in vivo distribution, degradation, and excretion were also investigated in detail.Conclusion: Our results suggest that the DMP-039 peptide-functionalized hybrid nanoparticle is an advanced candidate for mRNA-based suicide gene therapy.Keywords: mRNA, Bim, cell-penetrating peptide, gene therapy

Hongjia Zhang,1,* Ke Men,1,* Congbin Pan,1 Yan Gao,1 Jingmei Li,1 Sibei Lei,1 Guonian Zhu,1 Rui Li,1 Yuquan Wei,1 Xingmei Duan2 1State Key Laboratory of Biotherapy and Cancer Center, National Clinical Research Center for Geriatrics, West China Hospital of Sichuan University, Chengdu, Sichuan Province, People’s Republic of China; 2Department of Pharmacy, Sichuan Academy of Medical Sciences & Sichuan Provincial People’s Hospital, Personalized Drug Therapy Key Laboratory of Sichuan Province, School of Medicine, University of Electronic Science and Technology of China, Chengdu 610072, People’s Republic of China*These authors contributed equally to this workCorrespondence: Ke Men StateKey Laboratory of Biotherapy and Cancer Center, National Clinical Research Center for Geriatrics, West China Hospital of Sichuan University, Chengdu 610041, People’s Republic of ChinaEmail mendingbob@hotmail.comXingmei DuanDepartment of Pharmacy, Sichuan Academy of Medical Sciences & Sichuan Provincial People’s Hospital, Personalized Drug Therapy Key Laboratory of Sichuan Province, School of Medicine, University of Electronic Science and Technology of China, Chengdu 610072, People’s Republic of ChinaEmail duanxingmei2003@163.comBackground: Drugs that work based on the mechanism of RNA interference have shown strong potential in cancer gene therapy. Although significant progress has been made in small interfering RNA (siRNA) design and manufacturing, ideal delivery system remains a limitation for the development of siRNA-based drugs. Particularly, it is necessary to focus on parameters including delivery efficiency, stability, and safety when developing siRNA formulations for cancer therapy.Methods: In this work, a novel degradable siRNA delivery system cRGD-R9-PEG-PEI-Cholesterol (rrPPC) was synthesized based on low molecular weight polyethyleneimine (PEI). Functional groups including cholesterol, cell penetrating peptides (CPPs), and poly(ethylene oxide) were introduced to PEI backbone to attain enhanced transfection efficiency and biocompatibility.Results: The synthesized rrPPC was dispersed as nanoparticles in water with an average size of 195 nm and 41.9 mV in potential. rrPPC nanoparticles could efficiently deliver siRNA into C26 clone cancer cells and trigger caveolae-mediated pathway during transmembrane transportation. By loading the signal transducer and activator of transcription 3 (STAT3) targeting siRNA, rrPPC/STAT3 siRNA (rrPPC/siSTAT3) complex demonstrated strong anti-cancer effects in multiple colon cancer models following local delivery. In addition, intravenous (IV) injection of rrPPC/siSTAT3 complex efficiently suppressed lung metastasis tumor progression with ideal in vivo safety.Conclusion: Our results provide evidence that rrPPC nanoparticles constitute a potential candidate vector for siRNA-based colon cancer gene therapy.Keywords: RNA interfering, nanoparticle, STAT3, colon cancer

Xueyan Zhang,1 Ke Men,1 Yuanfa Zhang,1 Rui Zhang,1 Li Yang,1 Xingmei Duan21State Key Laboratory of Biotherapy and Cancer Center/Collaborative Innovation Center for Biotherapy, West China Hospital, Sichuan University, Chengdu 610041, People’s Republic of China; 2Department of Pharmacy and Personalized Drug Therapy Key Laboratory of Sichuan Province, Sichuan Academy of Medical Sciences & Sichuan Provincial People’s Hospital, Chengdu 610072, People’s Republic of ChinaBackground: In vitro transcribed (IVT) mRNA has been applied as an alternative therapeutic molecule to plasmid DNA in the field of cancer therapy and biomedical research studies. mRNA-based therapy has demonstrated several advantages over its DNA counterparts. However, its further therapeutic application is largely restricted by delivery method.Methods: In this work, a liposome-protamine lipoplex (CLPP) was prepared to deliver IVT mRNA encoding survivin-T34A gene, forming a novel core-shell structured nanoparticle formulation (CLPP/mSur-T34A).Results: The prepared CLPP/mSur-T34A particle had an average size of 186.1±3.1 nm, displaying high mRNA transfecting and expression efficiency on C26 tumor cells through lipid rafts-mediated endocytosis. CLPP/mSur-T34A mRNA formulation demonstrated obvious therapeutic effects on various models of C26 colon cancer both in vitro and in vivo. Particularly, local and systemic administration of CLPP/mSur-T34A particle exhibited superior antitumor effect regarding its DNA plasmid counterpart with high safety.Conclusion: Our results indicated the high delivery capacity of liposome-protamine lipoplex and further suggested CLPP/mSur-T34A mRNA formulation to be a potential candidate for colon cancer therapy.Keywords: mRNA gene therapy, colon cancer, nonviral vector, systemic delivery

Luo L, Du T, Zhang J, et al. Int J Nanomedicine. 2016;11:501—513. The authors have advised due to an error at the time of figure assembly, Figure 11 on page 510 is incorrect. The correct Figure 11 is shown below. The authors apologize for this error and advise it does not affect the results of the paper. Read the original article

Xiaowei Liu,1,* Zhan Li,1,* Xiaodong Wang,2,3 Yujuan Chen,2,3 Fengbo Wu,2,3 Ke Men,1 Ting Xu,2,3 Yan Luo,1 Li Yang1 1State Key Laboratory of Biotherapy/Collaborative Innovation Center for Biotherapy, 2Department of Thyroid and Breast Surgery, 3Department of Pharmacy, West China Hospital, Sichuan University, Chengdu, People’s Republic of China *These authors contributed equally to this work Abstract: Infections caused by multidrug-resistant bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA), have become a public threat; therefore, development of new antimicrobial drugs or strategies is urgently required. In this study, a new antibacterial peptide DP7-C (Chol-suc-VQWRIRVAVIRK-NH2) and DP7-C-modified azithromycin (AZT)-loaded liposomes (LPs) are developed for the treatment of MRSA infection, and it was found that DP7-C inserted into the LP lipid bilayer not only functioned as a carrier to encapsulate the antibiotic AZT but also synergized the antibacterial effect of the encapsulated AZT. In vitro assays showed that DP7-C-modified LPs possessed sustained drug release profile and immune regulatory effect and did not show obvious cytotoxicity in mammal cells, but they did not possess direct antibacterial activity in vitro. In vivo studies revealed that DP7-C-modified LPs did not exhibit obvious side effects or toxicity in mice but were able to significantly reduce the bacterial counts in an MRSA-infectious mouse model and possessed high antibacterial activity. In particular, DP7-C-modified AZT-loaded LPs showed more positive therapeutic effects than either DP7-C-modified blank LPs or nonmodified AZT-loaded LPs treatment alone. Molecular mechanism studies demonstrated that DP7-C formulations effectively upregulated the production of anti-inflammatory cytokines and chemokines without inducing harmful immune response, suggesting that DP7-C was synergistic with AZT against the bacterial infection by activating the innate immune response. Most importantly, although DP7-C activated the innate immune response, it did not possess direct antibacterial activity in vitro, indicating that DP7-C did not possess the potential to induce bacteria resistance. The findings indicate that DP7-C-modified AZT-loaded LPs developed in this study have a great potential required for the clinical treatment of MRSA infections. Keywords: DP7-C, azithromycin, antimicrobial resistance, MRSA infections, immune-regulation

Li Luo,1,* Ting Du,1,* Jiumeng Zhang,1 Wei Zhao,2 Hao Cheng,1 Yuping Yang,1 Yujiao Wu,1 Chunmei Wang,1 Ke Men,1 Maling Gou1 1State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University and Collaborative Innovation Center for Biotherapy, 2Department of Thoracic Oncology, Cancer Center, West China Hospital, Medical School, Sichuan University, Chengdu, People’s Republic of China *These authors contributed equally to this work Abstract: Gene therapy has promising applications in ovarian cancer therapy. Blocking the function of the survivin protein could lead to the growth inhibition of cancer cells. Herein, we used degradable heparin–polyethyleneimine (HPEI) nanoparticles to deliver a dominant-negative human survivin T34A (hs-T34A) gene to treat ovarian cancer. HPEI nanoparticles were characterized and were found to have a dynamic diameter of 66±4.5 nm and a zeta potential of 27.1±1.87 mV. The constructed hs-T34A gene expression plasmid could be effectively delivered into SKOV3 ovarian carcinoma cells by HPEI nanoparticles with low cytotoxicity. Intraperitoneal administration of HPEI/hs-T34A complexes could markedly inhibit tumor growth in a mouse xenograft model of SKOV3 human ovarian cancer. Moreover, according to our results, apparent apoptosis of cancer cells was observed both in vitro and in vivo. Taken together, the prepared HPEI/hs-T34A formulation showed potential applications in ovarian cancer gene therapy. Keywords: human survivin T34A, heparin–polyethyleneimine nanoparticles, gene therapy, ovarian cancer

Lin Zhou,1,2,* Xingmei Duan,1,2,* Shi Zeng,1 Ke Men,1 Xueyan Zhang,1 Li Yang,1 Xiang Li1 1State Key Laboratory of Biotherapy, Cancer Center and Department of Urology, West China Hospital, Sichuan University, Chengdu, People’s Republic of China; 2Sichuan Food and Drug Safety Monitoring and Review of Certification, Adverse Reaction Monitoring Center, Drug Abuse Monitoring Center, Chengdu, People’s Republic of China *These authors contributed equally to this work Abstract: Natural product curcumin (Cur) and H2S-releasing prodrug SH-aspirin (SH-ASA) are potential anticancer agents with diverse mechanisms, but their clinical application prospects are restricted by hydrophobicity and limited efficiency. In this work, we coencapsulated SH-ASA and Cur into methoxy poly(ethylene glycol)-poly (lactide-coglycolide) (mPEG-PLGA) nanoparticles through a modified oil-in-water single-emulsion solvent evaporation process. The prepared SH-ASA/Cur-coloaded mPEG-PLGA nanoparticles had a mean particle size of 122.3±6.8 nm and were monodispersed (polydispersity index =0.179±0.016) in water, with high drug-loading capacity and stability. Intriguingly, by treating with SH-ASA/Cur-coloaded mPEG-PLGA nanoparticles, obvious synergistic anticancer effects on ES-2 and SKOV3 human ovarian carcinoma cells were observed in vitro, and activation of the mitochondrial apoptosis pathway was indicated. Our results demonstrated that SH-ASA/Cur-coloaded mPEG-PLGA nanoparticles could have potential clinical advantages for the treatment of ovarian cancer. Keywords: drug delivery, cancer therapy, ovarian cancer, synergistic effect

Jin-Feng Qiu,1 Xiang Gao,1,2 Bi-Lan Wang,1 Xia-Wei Wei,1 Ma-Ling Gou,1 Ke Men,1 Xing-Yu Liu,1 Gang Guo,1 Zhi-Yong Qian,1 Mei-Juan Huang1 1Department of Thoracic Oncology, State Key Laboratory of Biotherapy and Cancer Center, West China Hospital and Medical School, Sichuan University, Chengdu, People’s Republic of China; 2Medical School and Department of Pathophysiology, College of Preclinical and Forensic Medical Sciences, Sichuan University, Chengdu, People’s Republic of China Abstract: Luteolin (Lu) is one of the flavonoids with anticancer activity, but its poor water solubility limits its use clinically. In this work, we used monomethoxy poly(ethylene glycol)-poly(ε-caprolactone) (MPEG-PCL) micelles to encapsulate Lu by a self-assembly method, creating a water-soluble Lu/MPEG-PCL micelle. These micelles had a mean particle size of 38.6 ± 0.6 nm (polydispersity index = 0.16 ± 0.02), encapsulation efficiency of 98.32% ± 1.12%, and drug loading of 3.93% ± 0.25%. Lu/MPEG-PCL micelles could slowly release Lu in vitro. Encapsulation of Lu in MPEG-PCL micelles improved the half-life (t½; 152.25 ± 49.92 versus [vs] 7.16 ± 1.23 minutes, P = 0.007), area under the curve (0–t) (2914.05 ± 445.17 vs 502.65 ± 140.12 mg/L/minute, P = 0.001), area under the curve (0–∞) (2989.03 ± 433.22 vs 503.81 ± 141.41 mg/L/minute, P = 0.001), and peak concentration (92.70 ± 11.61 vs 38.98 ± 7.73 mg/L, P = 0.003) of Lu when the drug was intravenously administered at a dose of 30 mg/kg in rats. Also, Lu/MPEG-PCL micelles maintained the cytotoxicity of Lu on 4T1 breast cancer cells (IC50 = 6.4 ± 2.30 µg/mL) and C-26 colon carcinoma cells (IC50 = 12.62 ± 2.17 µg/mL) in vitro. These data suggested that encapsulation of Lu into MPEG-PCL micelles created an aqueous formulation of Lu with potential anticancer effect. Keywords: luteolin, micelle, MPEG-PCL, cancer therapy

Xiang Gao,1 BiLan Wang,1 XiaWei Wei,1 Wang Rao,2 Fang Ai,2 Fen Zhao,2 Ke Men,1 Bowen Yang,1 Xingyu Liu,1 Meijuan Huang,1 Maling Gou,1 ZhiYong Qian,1 Ning Huang,1 Yuquan Wei11Department of Pathophysiology, College of Preclinical and Forensic Medical Sciences, and State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, West China Medical School, Sichuan University, Chengdu, People's Republic of China; 2Department of Surgery, First Affiliated Hospital, Xinxiang Medical School, Xinxiang, People's Republic of ChinaAbstract: Star-shaped polymer micelles have good stability against dilution with water, showing promising application in drug delivery. In this work, biodegradable micelles made from star-shaped poly(ε-caprolactone)/poly(ethylene glycol) (PCL/PEG) copolymer were prepared and used to deliver doxorubicin (Dox) in vitro and in vivo. First, an acrylated monomethoxy poly (ethylene glycol)-poly(ε-caprolactone) (MPEG-PCL) diblock copolymer was synthesized, which then self-assembled into micelles, with a core-shell structure, in water. Then, the double bonds at the end of the PCL blocks were conjugated together by radical polymerization, forming star-shaped MPEG-PCL (SSMPEG-PCL) micelles. These SSMPEG-PCL micelles were monodispersed (polydispersity index = 0.11), with mean diameter of ≈25 nm, in water. Blank SSMPEG-PCL micelles had little cytotoxicity and did not induce obvious hemolysis in vitro. The critical micelle concentration of the SSMPEG-PCL micelles was five times lower than that of the MPEG-PCL micelles. Dox was directly loaded into SSMPEG-PCL micelles by a pH-induced self-assembly method. Dox loading did not significantly affect the particle size of SSMPEG-PCL micelles. Dox-loaded SSMPEG-PCL (Dox/SSMPEG-PCL) micelles slowly released Dox in vitro, and the Dox release at pH 5.5 was faster than that at pH 7.0. Also, encapsulation of Dox in SSMPEG-PCL micelles enhanced the anticancer activity of Dox in vitro. Furthermore, the therapeutic efficiency of Dox/SSMPEG-PCL on colon cancer mouse model was evaluated. Dox/SSMPEG-PCL caused a more significant inhibitory effect on tumor growth than did free Dox or controls (P < 0.05), which indicated that Dox/SSMPEG-PCL had enhanced anticolon cancer activity in vivo. Analysis with terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) showed that Dox/SSMPEG-PCL induced more tumor cell apoptosis than free Dox or controls. These results suggested that SSMPEG-PCL micelles have promising application in doxorubicin delivery for the enhancement of anticancer effect.Keywords: drug delivery, star-shaped polymer, MPEG-PCL, CMC

Bi Lan Wang1, Xiang Gao1,2, Ke Men1, Jinfeng Qiu1, Bowen Yang3, Ma Ling Gou1, Mei Juan Huang1, Ning Huang2, Zhi Yong Qian1, Xia Zhao1, Yu Quan Wei11State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, West China Medical School, 2Department of Pathophysiology, College of Preclinical and Forensic Medical Sciences, 3College of Life Science, Sichuan University, Chengdu, People’s Republic of ChinaAbstract: Intravesical application of an anti-inflammatory drug is an efficient strategy for acute cystitis therapy. Quercetin (QU) is a potent anti-inflammatory agent; however, its poor water solubility restricts its clinical application. In an attempt to improve water solubility of QU, biodegradable monomethoxy poly(ethylene glycol)-poly(ε-caprolactone) (MPEG-PCL) micelles were used to encapsulate QU by self-assembly methods, creating QU/MPEG-PCL micelles. These QU/MPEG-PCL micelles with DL of 7% had a mean particle size of ~34 nm, and could release QU for an extended period in vitro. The in vivo study indicated that intravesical application of MPEG-PCL micelles did not induce any toxicity to the bladder, and could efficiently deliver cargo to the bladder. Moreover, the therapeutic efficiency of intravesical administration of QU/MPEG-PCL micelles on acute cystitis was evaluated in vivo. Results indicated that QU/MPEG-PCL micelle treatment efficiently reduced the edema and inflammatory cell infiltration of the bladder in an Escherichia coli-induced acute cystitis model. These data suggested that MPEG-PCL micelle was a candidate intravesical drug carrier, and QU/MPEG-PCL micelles may have potential application in acute cystitis therapy.Keywords: nanomedicine, MPEG-PCL, self-assembly

Ling Zhang1,*, Xiang Gao1,2,*, Ke Men1, BiLan Wang1, Shuang Zhang1, Jinfeng Qiu1, Meijuan Huang1, MaLing Gou1, Ning Huang2, ZhiYong Qian1, Xia Zhao1, YuQuan Wei11State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, West China Medical School, 2Department of Pathophysiology, College of Preclinical and Forensic Medical Sciences, Sichuan University, Chengdu, People’s Republic of China*These authors contributed equally to this workBackground: Gene therapy provides a novel method for the prevention and treatment of cancer, but the clinical application of gene therapy is restricted, mainly because of the absence of an efficient and safe gene delivery system. Recently, we developed a novel nonviral gene carrier, ie, heparin-polyethyleneimine (HPEI) nanoparticles for this purpose.Methods and results: HPEI nanoparticles were used to deliver plasmid-expressing mouse survivin-T34A (ms-T34A) to treat C-26 carcinoma in vitro and in vivo. According to the in vitro studies, HPEI nanoparticles could efficiently transfect the pGFP report gene into C-26 cells, with a transfection efficiency of 30.5% ± 2%. Moreover, HPEI nanoparticle-mediated ms-T34A could efficiently inhibit the proliferation of C-26 cells by induction of apoptosis in vitro. Based on the in vivo studies, HPEI nanoparticles could transfect the Lac-Z report gene into C-26 cells in vivo. Intratumoral injection of HPEI nanoparticle-mediated ms-T34A significantly inhibited growth of subcutaneous C-26 carcinoma in vivo by induction of apoptosis and inhibition of angiogenesis.Conclusion: This research suggests that HPEI nanoparticle-mediated ms-T34A may have a promising role in C-26 colon carcinoma therapy.Keywords: gene therapy, mouse survivin-T34A, colon cancer, polyethyleneimine, nanoparticles, cancer therapy

Non-coplanar volumetric modulated arc therapy (ncVMAT) is proposed to reduce toxicity in heart and lungs for locoregional radiotherapy of left-sided breast cancer, including internal mammary nodes (IMN).