Studies On Construction And Evaluation Of Redox-Responsive Glycolipid-Like Drug Delivery System

Nano-scale drug delivery system has made considerable progress in cancer therapy. However, it should be noted that the current nano drug delivery system is far from optimal due to the drug leakage profiles, low drug loading capacity and lacking of targeting ability. Thus, much effort has been concentrated on smart drug delivery system to maximize the therapeutic potential of drug molecules at target tissue and minimize their effects in healthy ones. Smart drug delivery system (smart DDS) can be modified in response to environmental stimuli based on the biological differences between tumor and normal tissues, and selectively enriched the encapsulated drug in tumor tissues. When triggered by the stimuli, the properties of DDS change, which makes it quickly and efficiently drug release at the target site. This specificity allows the Smart DDS to significantly enhance the therapeutic efficacy and to substantially reduce the side effects. By responding to the endogenous occurring difference between the extra-and intra-cellular redox environments (100-1000 folds) and the elevated production of reducing substances (7-10 folds) in tumor cells than normal cells, this study aims to establish a selective redox-response smart DDS which holds the minimal cleavage rate in circulation and sufficient drug release in tumor cells. The main content of the thesis is as follows:The selective redox-response smart DDS (CSO-ss-SA) was synthesized by a two-step reaction with chitosan as the hydrophilic segment and stearylamine as the hydrophobic segment. Amphiphilic CSO-ss-SA could self-aggregate into micelles in an aqueous medium, which emerged a uniform spherical shape and under 100nm average diameter. The CSO-ss-SA showed an improved redox sensitivity which only fast degraded and released drug in lOmM levels of glutathione (GSH). With a hydrophobic inner core and a particular spatial structure, model drug paclitaxel (PTX) was easily encapsulated into the CSO-ss-SA using a dialysis method to form CSO-ss-SA/PTX. The release of PTX from CSO-ss-SA in lOmM GSH showed a significant increase compared with micelles with low concentrations of GSH. The glycolipid-like CSO-ss-SA had an effective internalization capacity into both of tumor cells and normal cells, and can be utilized for intracellular drug delivery. Utilizing normal cells and tumor cells as the model cells, and nile red as the model drug, CSO-ss-SA released drug in response to the high reducing milieus in tumor cells and only few red fluorescent bodies were found in normal cells in 12h. The cytotoxicity of CSO-ss-SA/PTX was 13.2-fold and 7.55-fold higher compared with free Taxol and PTX-loaded non-responsive CSO-SA, respectively. By regulating the intracellular GSH concentration in tumor cells, it indicated that the cellular inhibition of the PTX-loaded CSO-ss-SA showed a positive correlation with the GSH concentration. Using BALB/C+/nu nude mice bearing SKOV-3 xenografts as model, near infrared imaging was applied to calculate the fluorescent intensity of tumor tissue, which evidenced the passive tumor targeting ability. We used NR loaded CSO-ss-SA to observe the drug release behaviors in vivo. Triggered NR release from CSO-ss-SA occurred in 12h in the tumor, which was resulted from selective redox sensitivity to the higher GSH concentration. While in the liver and spleen, only a few NR fluorescence signals were released and dispersed at the tissue in 72h. The inhibition rates of tumor growth of CSO-ss-SA/PTX, which were 88.4%(25mg/kg), and were significantly better than Taxol (59.4%) at the same doses.Based on selective redox-response ability of CSO-ss-SA, we built a co-delivery system for the delivery of PTX and small interfering RNA (siRNA). With the cationic characteristics, CSO-ss-SA could compact siRNA effectively, and release compacted siRNA fast in 10mM GSH conditions.The CSO-ss-SA could transport siRNA fast into the tumor cells with certain endosomal escape capacity and induce the silencing of target gene expression efficiently (85.8%). Fluorescence resonance energy transfer (FRET) and molecular beacons (MB) were used to qualitatively and quantitatively determine the intracellular siRNA release, which indicated that fast siRNA release only occured in high GSH concentration in tumor cells. The cell cycle arrest and cell viability experiments confirmed that co-delivery of PTX and siRNA’s targeting anti-apoptosis genes (siBcl-2) possessed the advantages to simultaneously overcome the drug resistance.To further improve the anti-tumor efficacy and avoid the macrophage phagocytosis, we constructed the PEGylation CSO-ss-SA (PEG-CSO-ss-SA) as a vector for pDNA delivery. PEG-CSO-ss-SA could compact pDNA efficiently, protect pDNA from the degradation of DNase I and release the compacted pDNA fast in response to 10mM GSH conditions. The modification of PEG shell on the surface of micelle significantly reduced the macrophage phagocytosis, and slowed down the uptake rate of micelles on the tumor cells slightly. PEG-CSO-ss-SA/pDNA complexes showed effective transfection on the tumor cells, and the transfection efficiency was similar to the positive control. Wild-type p53 was selected as the target gene for the success of gene transfection. PEG-CSO-ss-SA/pDNA complexes could effectively up-regulate the p53 expression and induce apoptosis in tumor cells, and the inducing-apoptosis ability of the complexes had the correlation with the intracellular GSH concentration. In vivo studies showed that PEGylation of the CSO-ss-SA extended the circulation time and increased the accumulation of micelles in the tumor site. After i.v. injection of PEG-CSO-ss-SA/p53 complexes for 48h, its expression could be detected in the tumor and the tumor sections further confirmed inducing-apoptosis of the tumor cells by the complexes.