| Traditional chemotherapeutic drugs remain the major treatment for advanced colorectal cancer. Yet, due to the lack of tumor specificity that induces unacceptable systemic toxicity, low solubility and/or low stability has led to the failure of tumor treatment and and mortality. In addition, the development of multidrug resistant (MDR) phenotypes classified into cellular and physiological factors ones is also an important reason for the failure of the traditional chemotherapy. The MDR is concerned with the over-expressed drug efflux transporters, mutated molecular targets and defective apoptotic mechanisms along with the tumor microenvironment complications as hypoxia, poor vasculature and low extracellular pH. Targeted drug delivery systems, especially by nanoparticles, have been the focus of cancer therapy research during the last decade to improve bioavailability and specific delivery of anticancer drugs to tumor sites, thereby reducing toxicity and side effects to normal tissues. However, the positive antitumor effects of these nanocarriers observed in conventional monolayer cultures frequently fail during in vivo due to the lack of resembling physical and biological barriers seen in the actual body. Therefore, in this study, we first developed a collagen based 3-D multicellular culture system for new drug nanocarriers screening to obtain a more adequate and better predict therapeutic outcomes in preclinical experiments. This 3-D culture model was successfully established using optimized density of cells. The result showed that 3-D cell colonies were successfully developed from 95-D, U87 and HCT116 cell lines respectively after seven day culture in the collagen matrix. The coumarin-conjugated nanoparticles were able to penetrate into the matrix gel to reach tumor cells. The model is supposedly more accurate to reflect/predict the dynamics and therapeutic outcomes of drug transporting candidates in vivo and/or investigation of tumor biology, thus pacing up the discovery of novel drug delivery systems for cancer therapy.In the second place, we synthesized a copolymer by graft epsilon-caprolactone into chitosan via ring-opening polymerization. The efficiency of sustained drug delivery system based on chitosan and epsilon-caprolactone to overcome multidrug resistance in monolayer and drug resistance associated with the 3-D tumor microenvironment was performed in 2-D models and in our established 3-D models. The 5-FU loaded nanoparticles were characterized by transmission electron microscope, dynamic light scattering and released property was determined at different pH values.5-FU/NPs exhibited well sustained release properties and markedly enhanced the cytotoxicity of 5-FU against HCT116/L-OHP or HCT8/VCR MDR cells in 2-D and its parental cells in 3-D collagen gel culture with 2-to 3-fold decrease in the IC50 values, as demonstrated by MTT assay, Hoechst/PI staining and flow cytomertry analysis. Furthermore, the possible mechanism was explored by HPLC and Rh-123 accumulation experiment. These results demonstrated that 5-FU/NPs increase intracellular concentration of 5-FU and enhance it anticancer efficiency by inducing apoptosis. And more, it was suggest this novel nanoparticles are a promising carrier to decrease toxic of 5-FU and has the potential to reverse forms of both intrinsic and acquired drug resistance in 2-D and 3-D cultures.Folate receptor (FR) is a highly selective tumor marker overexpressed in malignant tumors. Folic acid (FA) as one of the most common ligands retains a high affinity for FR. Therefore, folate-based drug delivery systems present an attractive target for tumor-selective drug delivery. Folic acids conjugates combine with FR situated at the surface of tumor cells and are internalized to intracellular compartments to form endosomes. Then folate conjugates are degraded by lysosome and released drug into the cytosol. In following, we has introduce a chemical active group, carbamic acid benzyl easter (CAB), to PEG-b-PCL based material using mPEG as an initiator to to make FA conjugated poly(ε-caprolactone) derivatives and increase the 5-FU loading. FA conjugated mPEG-b-P(CABCL-co-ACL) diblock copolymers were synthesized and characterized by TEM and NMR. Drug loaded nanoparticles were prepared using dialysis method and was obtained with a mean diameter of 45.2 nm with sustained in vitro release profile. In vitro cytotoxicity assay indicated that the cytotoxicity of folate modified nanoparticles were significantly increased compared to free drug and non-folate nanoparticles. In addition, results of hemolytic and histopathologic study suggested that the non-loaded nanoparticle (NL/NP) was non-toxic and biocompatible at the testing concentration. Moreover, in vivo results showed that FA/5-FU/NP effectively inhibited growth of HCT-8 cell-based xenograft tumors in BALB/c mice and revealed stronger antitumor efficacy than other treated groups. Thus, both in vitro and in vivo results exhibited that the folate conjugated mPEG-b-P(CABCL-co-ACL) copolymers have great potential to be used as sustainable and specific colon cancer targeting delivery system for anticancer agents.In the current research, extensive knowledge has been gathered and significant advances have been made in the field of nanocarrier delivery systems. Nanocarriers have also provided a great platform for the delivery of chemotherapeutic agents and imaging compounds, or both. Nevertheless, there are many challenges involved with the nanocarrier systems, such as biodegradation, immune responses, large scale manufacturing, and batch to batch variability issues. In respect to testing of efficient delivery systems, more efforts are required to utilize 3-D culture for high-throughput screening for potential nanosystems due to the advantages it offers. Briefly, this study has developed a novel poly(ε-caprolactone) based nanodelivery system that carry 5-FU to to the tumor site while limiting exposure to non-target tissues. Still, this study has documented the nanocarrier could improve efficiency of chemotherapy drugs and partly reveal how the nanoparticle reverse drug resistance in cancer cells. Finally, these findings contribute to better understanding for the possible therapeutic effects of 5-FU nanoparticles in further clinical research. Moreover, these findings may be beneficial for further research of utilizing nanopreparations for cancer therapy, especially in the multidrug resistance case. |
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