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Miao Zou, Xirui Yin, Xuan Zhou, Xinhui Niu, Yi Wang, Manman Su Department of Regenerative Medicine, School of Pharmaceutical Sciences, Jilin University, ChangChun, People’s Republic of ChinaCorrespondence: Manman Su; Yi Wang, Department of Regenerative Medicine, School of Pharmaceutical Sciences, Jilin University, 1266 Fujin Road, Changchun, 130021, People’s Republic of China, Tel/Fax +86 431 85619252, Email summ@jlu.edu.cn; wangyi@jlu.edu.cnBackground: Effective treatments for ovarian cancer remain elusive, and survival rates have long been considered grim. Ovarian cancer stem cells (OCSCs) and epithelial–mesenchymal transition (EMT) are associated with cancer progression and metastasis, as well as drug resistance and eventual treatment failure. Salinomycin (Sal) has an extensive effect on a variety of cancer stem cells (CSCs); however, its poor water solubility and toxicity to healthy tissues at high doses limit further research into its potential as an anti-cancer drug. We proposed a therapeutic strategy by constructing a tumor-targeting carrier that mimics high-density lipoprotein (HDL) to synthesize salinomycin-loaded high-density lipoprotein (S-HDL). This strategy helps reduce the side effects of salinomycin, thereby improving its clinical benefits.Methods: OCSCs were isolated from ovarian cancer cells (OCCs) and the uptake of HDL nanoparticles was observed using laser confocal microscopes. After the cell viability analysis revealed the inhibitory effect of S-HDL on OCCs and OCSCs, the main biological processes influenced by S-HDL were predicted with a transcriptome sequencing analysis and verified in vitro and in vivo.Results: Cellular uptake analysis showed that the HDL delivery system was able to significantly enhance the uptake of Sal by OCCs, tentatively validating the targeting role of recombinant HDL, so that S-HDL could reduce the toxicity of Sal and increase its anti-ovarian cancer effects. Conversely, S-HDL could exert anti-ovarian cancer effects by inhibiting the proliferation of OCCs and OCSCs, promoting apoptosis, blocking EMT, and suppressing stemness and angiogenesis-related protein expression in vitro and in vivo.Conclusion: S-HDL had stronger anti-ovarian cancer effects than unencapsulated Sal. Thus, it may be a potential agent for ovarian cancer treatment in the future.Keywords: ovarian cancer, ovarian cancer stem cells, epithelial–mesenchymal transition, salinomycin, high-density lipoprotein

Xirui Yin,1 Yuhui Lu,1 Miao Zou,1 Liuli Wang,1 Xuan Zhou,1 Yingyu Zhang,2 Manman Su1 1Department of Regenerative Medicine, School of Pharmaceutical Sciences, Jilin University, Changchun, People’s Republic of China; 2Department of Medical Science, Chang Chun Medical College, Changchun, People’s Republic of ChinaCorrespondence: Manman SuDepartment of Regenerative Medicine, School of Pharmaceutical Sciences, Jilin University, 1266 Fujin Road, Changchun, 130021, People’s Republic of ChinaTel +86 13843037881Fax +86 431 85619252Email summ@jlu.edu.cnBackground: Cervical cancer stem cells (CCSCs), a small part of tumor population, are one of the important reasons for metastasis and recurrence of cervical cancer. Targeting CCSCs may be an effective way to eliminate tumors. Salinomycin (Sal) has been proved to be an effective anticancer drug in many studies, especially for cancer stem cells (CSCs). However, the cytotoxicity of salinomycin limits its further research as an anticancer drug. High-density lipoprotein (HDL) nanoparticles are an excellent drug carrier, which can reduce the toxicity of Sal, have a certain targeting effect and improve the clinical benefit of Sal.Methods: Salinomycin-loaded high-density lipoprotein (S-HDL) was synthesized and characterized by various analytical techniques. CD44highCD24low CCSCs were isolated from HeLa cells by magnetic separation. The uptake of HDL nanoparticles was observed by laser confocal microscopy, and the effect of S-HDL on the proliferation of CCCs and CCSCs was detected by cell viability analysis. Genome-wide analysis was used to analyze the effects of S-HDL on the biological processes of CCCs and then cell apoptosis, cell cycle and cell migration were selected for verification.Results: S-HDL had a particle size of 38.98 ± 1.78 nm and an encapsulation efficiency of 50.73 ± 4.29%. Cell uptake analysis showed that HDL nanoparticles could enhance the drug uptake of CCCs and CCSCs and may target CCCs and CCSCs. In cell viability analysis, CCCs and CCSCs showed high sensitivity to S-HDL. S-HDL can more efficiently prevent CCSCs from developing tumorspheres than Sal in tumorsphere formation study. S-HDL had stronger ability to induce cell cycle arrest, promote cell apoptosis and inhibit cell migration compared with free Sal, which was consistent with the results of Genome Wide analysis.Conclusion: S-HDL can effectively target and eliminate CCCs and CCSCs, which is a potential drug for the treatment of cervical cancer.Keywords: HDL, salinomycin, cervical cancer stem cells, cellular uptake, LPR1

Jianguo Wang,1,* Jianyong Zhuo,2,* Yaoye Tao,2 Shengjun Xu,2 Zun Chen,2 Fan Yang,2 Qinghong Ke,2,3 Haiyang Xie,2,3 Shusen Zheng,2– 4 Hangxiang Wang,2,3 Xiao Xu1 1Department of Hepatobiliary and Pancreatic Surgery, Affiliated Hangzhou First People’s Hospital, Zhejiang University School of Medicine, Hangzhou 310006, People’s Republic of China; 2NHC Key Laboratory of Combined Multi-Organ Transplantation, Hangzhou 310003, People’s Republic of China; 3Department of Hepatobiliary and Pancreatic Surgery, First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310003, People’s Republic of China; 4Department of Hepatobiliary and Pancreatic Surgery, Shulan (Hangzhou) Hospital, Hangzhou 310003, People’s Republic of China*These authors contributed equally to this workCorrespondence: Xiao XuDepartment of Hepatobiliary and Pancreatic Surgery, Affiliated Hangzhou First People’s Hospital, Zhejiang University School of Medicine, Hangzhou 310006, People’s Republic of ChinaTel/Fax +86-571-87236567Email zjxu@zju.edu.cnBackground: There is currently no effective treatment for advanced hepatocellular carcinoma (HCC), and chemotherapy has little effect on long-term survival of HCC patients, largely due to the cancer stem cell (CSC) chemoresistance of HCC.Methods: We constructed a small-molecule nanometer-sized prodrug (nanoprodrug) loaded with salinomycin (SAL) for the treatment of HCC. SAL was encapsulated by the prodrug LA-SN38 (linoleic acid modified 7-ethyl-10-hydroxycamptothecin) to construct a self-assembled nanoprodrug further PEGylated with DSPE-PEG2000. We characterized this codelivered nanoprodrug and its antitumor activity both in vitro in human HCC cell lines and in vivo in mice.Results: Delivery of the SAL- and LA-SN38-based nanoprodrugs effectively promoted apoptosis of HCC cells, exerted inhibition of HCC tumor-sphere formation as well as HCC cell motility and invasion, and reduced the proportion of CD133+ HCC-CSC cells. In nude mice, the nanoprodrug suppressed growth of tumor xenografts derived from human cell lines and patient.Conclusion: Our results show that SAL-based nanoprodrugs are a promising platform for treating patients with HCC and a novel strategy for combination therapy of cancers.Keywords: small-molecule prodrugs, salinomycin, self-assemble, cancer stem cells, hepatocellular carcinoma

Qin Wang,1,* Fangcen Liu,2,* Lifeng Wang,1 Chen Xie,3 Puyuan Wu,1 Shiyao Du,1 Shujuan Zhou,1 Zhichen Sun,1 Qin Liu,1 Lixia Yu,1 Baorui Liu,1 Rutian Li1 1The Comprehensive Cancer Centre of Drum Tower Hospital, Medical School of Nanjing University, Clinical Cancer Institute of Nanjing University, Nanjing 210008, People’s Republic of China; 2The Comprehensive Cancer Centre, Nanjing Drum Tower Hospital, Clinical College of Nanjing Medical University, Nanjing, People’s Republic of China; 3Key Laboratory for Organic Electronics and Information Displays, Institute of Advanced Materials (IAM), Jiangsu National Synergetic Innovation Center for Advanced Materials (SICAM), Nanjing University of Posts & Telecommunications, Nanjing 210023, People’s Republic of China*These authors contributed equally to this workCorrespondence: Rutian Li; Baorui LiuThe Comprehensive Cancer Centre of Drum Tower Hospital, Medical School of Nanjing University, Clinical Cancer Institute of Nanjing University, 321 Zhongshan Road, Nanjing 210008, People’s Republic of ChinaTel +86-25-83107081Fax +86-25-83317016Email rutianli@nju.edu.cn; baoruiliu@nju.edu.cnBackground: Cervical cancer stem cells (CCSCs) represent a subpopulation of tumor cells that possess self-renewal capacity and numerous intrinsic mechanisms of resistance to conventional chemotherapy and radiotherapy. These cells play a crucial role in relapse and metastasis of cervical cancer. Therefore, eradication of CCSCs is the primary objective in cervical cancer therapy. Salinomycin (Sal) is an agent used for the elimination of cancer stem cells (CSCs); however, the occurrence of several side effects hinders its application. Nanoscale drug-delivery systems offer great promise for the diagnosis and treatment of tumors. These systems can be used to reduce the side effects of Sal and improve clinical benefit.Methods: Sal-loaded polyethylene glycol-peptide-polycaprolactone nanoparticles (Sal NPs) were fabricated under mild and non-toxic conditions. The real-time biodistribution of Sal NPs was investigated through non-invasive near-infrared fluorescent imaging. The efficacy of tumor growth inhibition by Sal NPs was evaluated using tumor xenografts in nude mice. Flow cytometry, immunohistochemistry, and Western blotting were used to detect the apoptosis of CSCs after treatment with Sal NPs. Immunohistochemistry and Western blotting were used to examine epithelial–mesenchymal transition (epithelial interstitial transformation) signal-related molecules.Results: Sal NPs exhibited antitumor efficacy against cervical cancers by inducing apoptosis of CCSCs and inhibiting the epithelial–mesenchymal transition pathway. Besides, tumor pieces resected from Sal NP-treated mice showed decreased reseeding ability and growth speed, further demonstrating the significant inhibitory ability of Sal NPs against CSCs. Moreover, owing to targeted delivery based on the gelatinase-responsive strategy, Sal NPs was more effective and tolerable than free Sal.Conclusion: To the best of our knowledge, this is the first study to show that CCSC-targeted Sal NPs provide a potential approach to selectively target and efficiently eradicate CCSCs. This renders them a promising strategy to improve the therapeutic effect against cervical cancer.Keywords: nanoparticles, salinomycin, tumor-targeted delivery, cancer stem cells, epithelial interstitial transformation

Jie Gao,1,2,* Junjie Liu,1,3,* Fangyuan Xie,4,* Ying Lu,3 Chuan Yin,5 Xian Shen1 1Department of Gastrointestinal Surgery, The Second Affiliated Hospital and Yuying Children’s Hospital of Wenzhou Medical University, Zhejiang, People’s Republic of China; 2Scientific Research Center, The Second Affiliated Hospital and Yuying Children’s Hospital of Wenzhou Medical University, Zhejiang, People’s Republic of China; 3Department of Pharmaceutical Sciences, Second Military Medical University, Shanghai, People’s Republic of China; 4Department of Pharmacy, Shanghai Eastern Hepatobiliary Surgery Hospital, Shanghai, People’s Republic of China; 5Department of Gastroenterology, Changzheng Hospital, Second Military Medical University, Shanghai, People’s Republic of China*These authors contributed equally to this workCorrespondence: Jie Gao; Xian ShenDepartment of Gastrointestinal Surgery, The Second Affiliated Hospital and Yuying Children’s Hospital of Wenzhou Medical University, 109 Xueyuan W Road, Lucheng, Wenzhou, Zhejiang 325000, People’s Republic of ChinaTel +86-0577-88816381; 86-0577-88002709Fax +86-0577-88816381Email gaojiehighclea@163.com; shenxian1963@sina.comPurpose: Conventional chemotherapy is hampered by the presence of breast cancer stem cells (BCSCs). It is crucial to eradicating both the bulky breast cancer cells and BCSCs, using a combination of conventional chemotherapy and anti-CSCs drugs. However, the synergistic ratio of drug combinations cannot be easily maintained in vivo. In our previous studies, we demonstrated that the simultaneous delivery of two drugs via nanoliposomes could maintain the synergistic drug ratio for 12 h in vivo. However, nanoliposomes have the disadvantage of quick drug release, which makes it difficult to maintain the synergistic drug ratio for a long time. Herein, we developed a co-delivery system for docetaxel (DTX)—a first-line chemotherapy drug for breast cancer—and salinomycin (SAL)—an anti-BCSCs drug—in rigid nanoparticles constituted of polylactide-co-glycolide/D-alpha-tocopherol polyethylene glycol 1000 succinate (PLGA/TPGS).Methods: Nanoparticles loaded with SAL and DTX at the optimized ratio (NSD) were prepared by the nanoprecipitation method. The characterization, cellular uptake, and cytotoxicity of nanoparticles were investigated in vitro, and the pharmacokinetics, tissue distribution, antitumor and anti-CSCs activity of nanoparticles were evaluated in vivo.Results: We demonstrated that a SAL/DTX molar ratio of 1:1 was synergistic in MCF-7 cells and MCF-7-MS. Moreover, the enhanced internalization of nanoparticles was observed in MCF-7 cells and MCF-7-MS. Furthermore, the cytotoxicity of NSD against both MCF-7 cells and MCF-7-MS was stronger than the cytotoxicity of any single treatment in vitro. Significantly, NSD could prolong the circulation time and maintain the synergistic ratio of SAL to DTX in vivo for 24 h, thus exhibiting superior tumor targeting and anti-tumor activity compared to other treatments.Conclusion: Co-encapsulation of SAL and DTX in PLGA/TPGS nanoparticles could maintain the synergistic ratio of drugs in vivo in a better manner; thus, providing a promising strategy for synergistic inhibition of breast cancer.Keywords: combined therapy, breast cancer stem cells, docetaxel, salinomycin, nanoparticles

Puyuan Wu, Qin Liu, Qin Wang, Hanqing Qian, Lixia Yu, Baorui Liu, Rutian Li The Comprehensive Cancer Center of Drum Tower Hospital, Medical School of Nanjing University, Clinical Cancer Institute of Nanjing University, Nanjing, People’s Republic of China Background: A multi-drug delivery platform is needed as the intra-tumoral heterogeneity of cancer leads to different drug susceptibility. Cancer stem cells (CSCs), a small population of tumor cells responsible for tumor seeding and recurrence, are considered chemotherapy-resistant and have been reported to be sensitive to salinomycin (Sal) instead of paclitaxel (Ptx). Here we report a novel silk fibroin (SF) hydrogel-loading Sal and Ptx by incorporating drug-loaded silk fibroin nanoparticles (SF-NPs) to simultaneously kill CSCs and non-CSCs.Methods: Using the method we have previously reported to prepare Ptx-loaded SF-NPs (Ptx-SF-NPs), Sal-loaded SF-NPs (Sal-SF-NPs) were fabricated under mild and non-toxic conditions. The drug-loaded SF-NPs were dispersed in the ultrasound processed SF solution prior to gelation.Results: The resulting SF hydrogel (Sal-Ptx-NP-Gel) retained its injectable properties, exhibited bio-degradability and demonstrated homogeneous drug distribution compared to the non-NP incorporated hydrogel. Sal-Ptx-NP-Gel showed superior inhibition of tumor growth compared to single drug-loaded hydrogel and systemic dual drug administration in the murine hepatic carcinoma H22 subcutaneous tumor model. Sal-Ptx-NP-Gel also significantly reduced CD44+CD133+ tumor cells and demonstrated the least tumor formation in the in vivo tumor seeding experiment, indicating superior inhibition of cancer stem cells.Conclusion: These results suggest that SF-NPs incorporated SF hydrogel is a promising drug delivery platform, and Sal-Ptx-NP-Gel could be a novel and powerful locoregional tumor treatment regimen in the future. Keywords: silk fibroin, nanoparticle, hydrogel, salinomycin, cancer stem cells

Jun Li,1,* Wenqing Xu,2,* Xiaoli Yuan,3,* Huaiwen Chen,3 Hao Song,1,4 Bingquan Wang,5 Jun Han5 1College of Pharmacy, Liaocheng University, Liaocheng, Shandong, 2Railway Police College, Zhengzhou, 3Department of Cadre Health Care, Nanjing General Hospital of Nanjing Military Command, Nanjing, Jiangsu, 4Centre for Stem Cell & Regenerative Medicine, Liaocheng People’s Hospital, 5Institute of Biopharmaceutical Research, Liaocheng University, Liaocheng, Shandong, China *These authors contributed equally to this work Purpose: Breast cancer stem cells (CSCs) are responsible for the initiation, recurrence, and metastasis of breast cancer. Sufficient evidence has established that breast cancer cells can spontaneously turn into breast CSCs. Thus, it is essential to simultaneously target breast CSCs and cancer cells to maximize the efficacy of breast cancer therapy. HER2 has been found to be overexpressed in both breast CSCs and cancer cells. We developed salinomycin-loaded polymer–lipid hybrid anti-HER2 nanoparticles (Sali-NP-HER2) to target both HER2-positive breast CSCs and cancer cells.Methods: The antitumor activity of Sali-NP-HER2 constructed by conjugating anti-HER2 antibodies to polymer–lipid salinomycin nanoparticles was evaluated in vitro and in vivo.Results: Sali-NP-HER2 efficiently bound to HER2-positive breast CSCs and cancer cells, resulting in enhanced cytotoxic effects compared with non-targeted nanoparticles or salinomycin. In mice bearing breast cancer xenografts, administration of Sali-NP-HER2 exhibited superior efficacy in inhibiting tumor growth. Sali-NP-HER2 reduced the breast tumorsphere formation rate and the proportion of breast CSCs more effectively than non-targeted nanoparticles or salinomycin alone.Conclusion: Sali-NP-HER2 represents a promising approach in treating HER2-positive breast cancer by targeting both breast CSCs and cancer cells. Keywords: nanoparticles, breast cancer, cancer stem cells, salinomycin, HER2

Xi-Feng Zhang,1 Sangiliyandi Gurunathan2 1College of Biological and Pharmaceutical Engineering, Wuhan Polytechnic University, Wuhan, People’s Republic of China; 2Department of Stem Cell and Regenerative Biology, Konkuk University, Seoul, South Korea Abstract: Ovarian cancer is one of the most important malignancies, and the origin, detection, and pathogenesis of epithelial ovarian cancer remain elusive. Although many cancer drugs have been developed to dramatically reduce the size of tumors, most cancers eventually relapse, posing a critical problem to overcome. Hence, it is necessary to identify possible alternative therapeutic approaches to reduce the mortality rate of this devastating disease. To identify alternative approaches, we first synthesized silver nanoparticles (AgNPs) using a novel bacterium called Bacillus clausii. The synthesized AgNPs were homogenous and spherical in shape, with an average size of 16–20 nm, which are known to cause cytotoxicity in various types of human cancer cells, whereas salinomycin (Sal) is able to kill cancer stem cells. Therefore, we selected both Sal and AgNPs to study their combined effect on apoptosis and autophagy in ovarian cancer cells. The cells treated with either Sal or AgNPs showed a dose-dependent effect with inhibitory concentration (IC)-50 values of 6.0 µM and 8 µg/mL for Sal and AgNPs, respectively. To determine the combination effect, we measured the IC25 values of both Sal and AgNPs (3.0 µM and 4 µg/mL), which showed a more dramatic inhibitory effect on cell viability and cell morphology than either Sal or AgNPs alone. The combination of Sal and AgNPs had more pronounced effect on cytotoxicity and expression of apoptotic genes and also significantly induced the accumulation of autophagolysosomes, which was associated with mitochondrial dysfunction and loss of cell viability. Our data show a strong synergistic interaction between Sal and AgNPs in tested cancer cells. The combination treatment increased the therapeutic potential and demonstrated the relevant targeted therapy for the treatment of ovarian cancer. Furthermore, we provide, for the first time, a mode of action for Sal and AgNPs in ovarian cancer cells: enhanced apoptosis and autophagy. Keywords: apoptosis, autophagy, cell viability, caspase activity, ovarian cancer, salinomycin, silver nanoparticles