Studies On Administration Of Micelles Based On α-tocopherol Succinate-Modified Chitosan

Paclitaxel (PTX) has been successfully used in the clinical treatment of several cancer types,especially breast and ovarian cancer. Due to its poor water solubility, PTX is currently solubilized in a 50:50 mixture of Cremophor EL and dehydrated ethanol as Taxol(?). Unfortunately,Cremophor EL was shown to induce severe side effects. Therefore, many attempts have been made to find less toxic and better tolerated carriers for PTX delivery.Based on the research achievements at home and abroad, the polymeric micelle system ofα-tocopherol succinate-modified chitosan (CS-TOS) for paclitaxel delivery has been developed in this study. Polymeric micelles have unique core-shell architecture, which is composed of hydrophobic segments as the inner core and hydrophilic segments as the outer shell in aqueous medium. Poorly water-soluble drugs can be solubilized within the core by hydrophobic interactions without any surfactant added.First, the amphiphilic chitosan derivative CS-TOS was synthesized by the coupling reaction of carboxyl group of a-tocopherol succinate (TOS) with amino group of chitosan in the presence of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS). The formation of CS-TOS was confirmed by 1H-NMR, FT-IR, DSC and XRD analysis. Furthermore, a series of CS-TOS polymer with different degree of substitution were prepared by adjusting the molar ratio of sugar units of chitosan to a-tocopherol succinate. The degree of the substitution was 5.2%, 11.5%, and 17.4%, respectively, which was determined by 2,4,6-trimitrobenzene sulfonic acid (TNBS) method. In aqueous medium, the polymer could self-aggregate to form micelles. The critical aggregation concentrations (CAC) of these three different CS-TOS were determined to be 1.5×10-2 mg/ml,9.8×10-3 mg/ml, and 5.8×10-3 mg/ml by pyrene fluorescent probe method. The low CAC value indicated that the polymer would be stable upon dilution.Based on the preliminary study, the polymer with optimized degree of substitution 17.4% was chosen to prepare blank and drug-loaded micelles by probe sonication. The mean particle size and zeta potential of drug-loaded micelles was about 78 nm and +25.7 mV, respectively. Transmission electron microscopy (TEM) observation revealed that both bare and paclitaxel-loaded micelles were near spherical in shape with narrow distribution. The drug encapsulation efficiency and drug loading of the PTX-loaded micelles was 90.3% and 3.8%, respectively. The results of DSC and XRD analysis indicated that paclitaxel was entrapped in the micelles in molecular or amorphous state. The in vitro release profile of the micelles in PBS 7.4 was studied by dialysis method. It was revealed that drug-loaded micelles showed prolonged release.Furthermore, in vitro cytotoxicity studies revealed that compared to Cremophor EL-based formulation, drug-loaded micelles have equivalent cytotoxicity against MCF-7 tumor cells. Cellular uptake studies showed that with the longer incubation time, the uptake amount of CS-TOS micelles by MCF-7 cells was enhanced, which was equal to Cremophor EL-based formulation. From fluorescent images of MCF-7 cells incubated with FITC-labled CS and CS-TOS micelles, it was revealed that the micelles could entry the cells through cell membrane,while the CS control could only adhere to the outside surface of cell wall, but not into the cells. Hemolysis study revealed the safety of this micellar delivery system.For the determination of PTX in biosamples, an effective HPLC method was established. The pharmacokinetics study in rats showed that the micelles had prolonged the action time of PTX in Cremophor EL-based formulation. In vivo biodistribution experiment in mice revealed that the micelles showed liver and spleen-targeting and low toxicity to heart and kidney.Moreover, antitumor activity assessed in U14 tumor-bearing mice showed that the micelles significantly reduced the tumor size. The tumor inhibition of the micelles and Cremophor EL-based formulation was 68.0% and 66.6%, respectively. The systemic toxicity of micelles was much lower than Cremophor EL-based formulation at the dose of 20 mg/kg by determination of body weight change after i.v. administration. It can prolong the life span of tumor-bearing mice. It can be concluded that CS-TOS may be a promising micellar carrier for paclitaxel.