| Background:Colorectal cancer (CRC) is a prevalent lethal malignancy in our country. Only a small number of patients can be early detected and surgical radical resection, the majority of patients with advanced disease require chemotherapy. Clinical studies have demonstrated that combined use of OXA with5-fluorouracil and leucovorin (FOLFOX) for metastatic CRC has achieved response rates31～56%and median survival approaching2years, but nearly all responding CRCs eventually develop OXA resistance and progress within8months. These suggest that chemotherapy fails to current cancer treatments. Therefore, it is of great importance to identify these residual cancer cells and find suitable agents for the treatment of patients.Recent evidences have identified a small subset of cancer-initiating cells within tumors that drive tumor growth and recurrence, so-called cancer stem cells (CSCs). CSCs possess both self-renewal capabilities and the ability to generate tumor bulk. CSCs are demonstrated to exist in various solid tumours, including brain, lung, breast, pancreatic, liver and colorectal carcinoma. In addition, CSCs are proven to be more resistant than other cancer cells to drug treatment, and chemotherapy significantly results in the enrichment of these CSCs populations. It suggests that CSCs are the main reason for the failure of chemotherapy drugs.To explain why chemotherapies ultimately fail to cure cancer, the CSC hypothesis has highlighted the therapeutic resistance of CSCs in tumors as a possible mechanism. Such characteristics of CSCs may result from several causes, including high expression levels of ABC transporters, resistance to apoptosis, and active DNA-repair capacity. Studies of cancer cells have revealed that CSCs commonly express high levels drug efflux pumps, such as ABCB1, ABCC1, and ABCG2protein. The drug pumps are responsible for protecting cancer cells from cytotoxic damage by efflux pumping mechanisms. However, many chemotherapy drugs inhibit cancer cells through entering tumor cell nucleus to interacte DNA or related enzymes. Therefore, as a result of these biophysical and biological properties, CSCs render resistance to chemotherapeutic agent treatment.In addition, some traditional Chinese medicine has been reported to eliminate CSCs. such as curcumin (Cur). It has been applied such as anti-oxidant, anti-bacteria, anti-inflammatory, and anti-cancer. Despite all these extraordinary anti-cancer properties, its application for cancer treatment is limited by the high hydrophobicity, instability, poor pharmacokinetics, which greatly hampers its in vivo efficacy.Nanotechnology offers one of innovative approaches to overcome these problems. Polymeric micelles with core-shell structure, formed by self-aggregation of amphiphilic graft or block copolymers, provide a significant advantage for delivering antitumor drugs to solid tumors.Moreover, polymeric micelles are largely accumulated in tumor tissue via the passive "enhanced permeability and retention (EPR) effect". Among the natural polysaccharides, the stearic acid-g-chitosan oligosaccharide (CSO-SA) micelles have been widely utilized as polymeric drug carrier due to its biocompatibility and biodegradabiliry. CSO is non-toxic, low immunogenic and biodegradable, obtained from the enzymatic degradation of chitosan. And SA also has low cytotoxicity as it is mainly composed of fat. CSO-SA micelle is thus used as a carrier for physically or chemically loading antitumor drug with excellent tumor cellular uptakes. Importantly, the drug-loaded micelles can enhance the antitumor activity and reverse multi-drug resistance (MDR) of tumors.Therefore, in order to improve the drug concentration in tumor cells and effectively kill CSCs, we use chemotherapy drugs such as oxaliplatin and Chinese medicine such as curcumin as model drug, and chitooligosaccharides-stearic micelles as drug delivery system, to papere composite nano micelles.Objectives:The mature platform of chitooligosaccharides-stearic (CSO-SA) micelles delivery (Laboratory Fu-Qiang Hu, College of Pharmaceutical Science, Zhejiang University), and CSCs research technology has been established. Polymeric micelles with core-shell structure, formed by self-aggregation of amphiphilic graft or block copolymers, provide a significant advantage for delivering antitumor drugs to solid tumors. Moreover, polymeric micelles are largely accumulated in tumor tissue via the passive "enhanced permeability and retention (EPR) effect". Drug effects are limited to the tumor tissue, and reduce damage to normal tissue. Utilizing these advantages, the objective of our study is to develop new agents loaded with chemotherapy drugs such as oxaliplatin (OXA) as a model, or Chinese medicine such as curcumin (Cur) as a model, for providing beneficial pharmacological properties to eliminate colorectal CSCs. In the study, we evaluated antitumor activity and CSCs eradication of CSO-SA/OXA and CSO-SA/Cur micelles respectively. CSO-SA/OXA micelles were synthesized for anti-CSCs pharmacodynamic studyMethods:1) Synthesis and characteristics of CSO-SA:CSO-SA was synthesized by esterification reaction between amine groups of CSO (18kDa) and carboxyl group of stearic acid (SA, C18H36O2) in the presence of carbodiimide (EDC). The amino substituted degree was tested as trinitrobenzene sulfonic acid method. The critical micelle concentration (CMC) of the synthesized CSO-SA was determined by fluorescence methods.(Laboratory Fu-Qiang Hu, Zhejiang University, China)2) Synthesis and characteristics of CSO-SA/OXA micelles:CSO-SA/OXA micelles were prepared through thin film dispersed steps. The size and its distribution of CSO-SA/OXA micelles were measured by dynamic light scattering using a Zetasizer. The OXA content in the drug loaded micelles was detected using inductively coupled plasma mass spectrometer (ICP-MS).3) Viability assay in vitro:The cytotoxicities of CSO-SA micelles, OXA and CSO-SA/OXA micelles were assessed by MTS. The formation of spheroids was evaluated under DMEM/F12basal serum free medium.4) In vivo tumorigenicity assay and mouse treatment:After the tumor formed in subcutaneously of nude mice, mice were treated with CSO-SA/OXA micells and evaluated the effects:tumor inhibitory rate, percentage of CSCs subpopulation, cancer cells apoptotic events and drug toxicity.Results:1) Preparation and Characteristics of CSO-SA micelles and CSO-SA/OXA micelles The size of CSO-SA micelles was25.4nm and zeta potential was29.9mV in pure water. After the OXA was loaded, the size increased (90.0nm) and the zeta potential decreased (24.4mV). The encapsulation efficiency of CSO-SA/OXA micelles could reach to about 60wt%by present preparation method. TEM images showed CSO-SA/OXA micelles was spherical and well dispersed in aqueous medium. The drug released profiles of CSO-SA/OXA micelles showed that about90%total OXA was released from micelles for48hr.2) OXA-loaded CSO-SA micelles effectively suppressing both CSCs and the bulk cancer cellsDose response curves revealed that CSO-SA/OXA micelles reversed free OXA resistance of CSCs and increased cytotoxicity to both CSCs and the bulk cancer cells in vitro. Moreover, CSO-SA/OXA micelles were able to decrease the spheroid formation of cancer cells.3) CSO-SA/OXA micelles enhancing antitumor efficacy and reducing CSCs enrichment in vivoCSO-SA/OXA micelles could improve cytotoxicity and significantly reduced CSCs enrichment in xenografts, and did not increase cytotoxic to mice compared free OXA as determined by body weight. As a control, free OXA resulted in the enrichment of CSCs populations.4) The reasons of CSO-SA/OXA micelles effects were that the effective accumulation and sustained release of the OXA from CSO-SA/OXA into the tumor tissues, causing suppression of the tumor growth and increasing of apoptosis in tumor cells. However, the details of mechanism need further studies.Conclusions:OXA loaded CSO-SA micelles were prepared, which had excellent internalization ability that increased OXA concentration in colorectal cancer cells and tissues. It was effective for suppressing both CSCs and the bulk cancer cell populations. The assay of anticancer activity in xenograft tumors indicated that CSO-SA/OXA micelles could reduce CSCs enrichment and effectively suppress the tumor growth comparing with free OXA. The reasons that might explain the results were that the effective accumulation into the tumor tissues and the sustained release of the drug from CSO-SA/OXA. Moreover, micelles could overcome ATP-binding cassette (ABC) transporters in CSCs and deliver drug to cell nucleus, which facilitated the antitumor efficacy.Novel CSO-SA/Cur micelles were synthesized for anti-CSCs pharmacodynamic studyMethods:1) CSO-SA micelles were prepared as the above work.2) Preparation and physicochemical properties of CSO-SA/Cur micelles. CSO-SA/Cur micelles were obtained by complexation method. The encapsulation efficiency (EE) of CSO-SA/Cur micelles was determined using a fluorescence spectrophotometer. The surface appearance and shape of CSO-SA/Cur micelles were observed by transmission electron microscopy. In vitro stability and water-soluble of CSO-SA/Cur micelles was evaluated.3) Cytotoxicity assay. The cytotoxicities of free curcumin, empty vector, and CSO-SA/Cur micelles were investigated using colorectal cancer cells by MTS assay. The CSC surface marker was assessed after treatment with differet drugs.4) In vivo antitumor activities of curcumin-loaded CSO-SA micelles. The antitumor efficacy of CSO-SA/Cur micelles was assessed in a nude mouse bearing orthotopic colorectal tumors. At the end of drug treatment, we observed curcumin biodistribution analysis and CSC surface marker expression.Results:1) Preparation and Characteristics of CSO-SA and CSO-SA/Cur micelles. The mean size of CSO-SA/OXA micelles was114.7nm and zeta potential was18.5mV in pure water. TEM images showed the CSO-SA/OXA micelles were regular spherical appearance. CSO-SA/Cur micelles could easily disperse in aqueous medium and encapsulated curcumin was relatively stable in neutral pH.2) In vitro antitumor activity. The dose response curves showed that the cytotoxicity of CSO-SA/Cur micelles was about6-fold higher than that treated with free curcumin solution at the equivalent concentration of curcumin. CSO-SA/Cur micelles could greatly decrease the spheroid formations of colorectal cancer cells and inhibited CSCs.3) In vivo antitumor activity. After intravenous injection for14days, only CSO-SA/Cur micelles marginally inhibited colorectal tumor growth. It was found that CSO-SA/Cur micelles could significantly decrease CSCs population compared with PBS and empty vehicle group. In contrast, empty vehicle had no effect. Moreover, the fact that no obvious body weight declined in all groups was suggestive of the chosen doses within the safe range.Conclusions:CSO-SA/Cur micelles were prepared successfully in the present work. The synthesized CSO-SA/Cur copolymer could self-assemble to form nanosized micelles in aqueous medium and had excellent internalization ability. The results clearly demonstrated that CSO-SA/Cur micelles could increase antitumor efficacy and eliminate colorectal CSCs in vitro. Moreover, intravenous administration of CSO-SA/Cur micelles marginally suppressed the tumor growth and inhibited CSCs enrichment. In addition, CSO-SA/Cur micelles were low toxicity and well tolerated in vivo. |
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