| Chitosan (CS) is a material of biomacromolecule obtained by alkaline N-deacetylation of chitin, a main derivative of chitin. Chitosan has excellent biocompatibility, biodegradability, non-toxicity and low immunogenicity. Because of the free amine group and hydroxyl group in its structure, chitosan is easy to be modified to get its derivatives of different structures and properties. Micelle is a dispersion system and its particle size is generally below 200nm. Copolymer (CM) can self-assemble in aqueous phase to form copolymeric micelles with a hydrophobic inner and a hydrophilic outer shell. Micelles have several advantages as a drug delivery system, such as, solubilization of hydrophobic drugs, preservation of bioactive agents in the micellar core for long duration, passive targeting and reduction of the adverse effects and so on.In this study, a carrier of Cholesteryl Chloroformate grafted chitosan was prepared to develop an amphiphilic copolymer CF-CS, which can self-assemble in aqueous phase to form copolymeric micelles. Oridonin was packed into micelle to prepare oridonin loaded cholesteryl formate-grafted chitosan copolymer micelles (ORI-CF-CS).Formulation and preparation of synthesis of CF-CS were investigated. The IR spectra and the 13C-NMR,1H-NMR spectra of the CF-CS-were measured. In the IR spectra, the specific peak (1700cm-1) of carbonyl was clearly. And near to 3500cm-1, there were peaks of middle or high strength suggesting amied group exist. The 13C-NMR and 1H-NMR spectra showed that there were a peak of amied group (δ=155ppm) and a peak (δ=5.5ppm) of double bond respectively, demonstrating the synthetic reaction was successful.Formulation design and preparation technique of ORI-CF-CS were studied in this research. It included the mass ratio of CF-CS to oridonin, volume ratio of aqueous phase to organic phase, the minutes of stiring and the temperature of heating. The pharmaceutical properties of ORI-CF-CS were as followings:the shape of ORI-CF-CS was roughly spherical observed by used of TEM. There were drug particles of oridonin clinging on the spherical, which was an obvious difference with the blank CF-CS micelles. The mean diameter of these nanoparticles was from 68.10 to 113.8nm, Zeta potential was between -34.97 and -28.19mv. CMC was 1.15μg/mL. The encapsulation efficiency was up to 48.83%, the drug-loading rate was 9.16%. The accumulated release amount of oridonin was up to 50% in 1 day, then the release speed became slowly. Until the 11th day, the accumulated release amount was up to 90%, and nearly 100% after 15 days. Showing that oridonin had a slow-release effect in the ORI-CF-CS.The effect of the ORI-CF-CS and oridonin solution solution on the Hela cell proliferation inhibition were investigated by use of MTT method, indicating that the inhibition effect was increased with dose increment, and the IC50 of oridonin solution solution were respectively 2.75 and 2.72 times of the ORI-CF-CS, showing that the inhibition effect of ORI-CF-CS was better than the oridonin solution solution.The intacellular uptake kinetics study in Hela cells was investigated as following: after the uptake of ORI-CF-CS by Hela cells, at the time point of 30,60,120,240 and 420min, the accumulation of oridonin was remarkably higher than the Oridonin solution solution, so ORI-CF-CS can increase the uptake of oridonin by Hela cells. The elimination kinetics parameters were calculated:k and t1/2 of oridonin in ORI-CF-CS was respectively 0.39±0.005h-1 and 1.80±0.017h, suggesting that ORI-CF-CS can slow the elimination rapid of ORI comparing with oridonin solution solution.
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