Editing Chitosan Nanoparticles

http://www.google.com/patents/US7883723<br>

EXAMPLE 1 Preparation of a Water-soluble Chitosan Nanoparticle 1

A water-soluble chitosan with molecular weight of 18 kDa and degree of deacetylation of 87% was supplied from KITTOLIFE Co. Ltd. Methoxy poly (ethylene glycol) p-nitrophenyl carbonate (MPEG p-NP)), hydrophilic moiety, was purchased from Sigma Co. and cholesteryl chloroformate, hydrophobic group, was purchased from Aldrich Co. Dialysis tubing (MWCO 12,000) was commercially obtained from Spectrum Co. All other chemicals were reagent grade and used as received.

Preparation of a water-soluble chitosan nanoparticle was carried out in the following steps. In step 1, 0.1 g of water-soluble chitosan was dissolved in 15 ml of PBS (pH 7.0˜8.0), followed by stirring for one hour. 0.5 g of Methoxy poly (ethylene glycol) p-nitropheyl carbonate (MPEG p-NP) was dissolved in PBS (pH 8.0), which was dropped slowly to the above chitosan reaction solution while the solution was stirring. Two hours later, the solution was dialyzed using a molecular cut-off 12,000 g/mol dialysis tube against cold distilled water for 48 hours, resulting in the formation of a clear product in supernatant and by-product in precipitation layer. Those products were separated and a clear supernatant layer was freeze-dried.

A white product obtained through freeze-drying in the above step 1 was dissolved in 15 ml of PBS (pH 7.0˜8.0), followed by stirring for one hour. Cholesteryl chloroformate was dissolved in 1˜2 ml of anhydrous DMF by 1.0 per 10 chitosan glucose amine monomers. The cholesteryl chloroformate solution was slowly dropped into the above reaction solution while the reaction solution was stirring for 2 hours. Upon completing the reaction, dialysis was performed with distilled water for 24 hours. Centrifugation was then performed at 10,000 rpm for 10 minutes, followed by filtering with 0.45 μm syringe filter and freeze-drying. A final product, a white water-soluble chitosan nanoparticle, was obtained.<br>EXAMPLE 2 Preparation of a Water-soluble Chitosan Nanoparticle 2

A water-soluble chitosan nanoparticle was prepared by the same method as described in the above Example 1 except that 1.5 cholesteryl chloroformate per 10 chitosan glucose amine monomers were used.<br>EXAMPLE 3 Preparation of a Water-soluble Chitosan Nanoparticle 3

A water-soluble chitosan nanoparticle was prepared by the same method as described in the above Example 1 except that 1.8 cholesteryl chloroformate per 10 chitosan glucose amine monomers were used.<br>EXAMPLE 4 Preparation of a Water-soluble Chitosan Nanopaclitaxel 1

25 mg of water-soluble chitosan nanoparticle prepared in the above Example 1 was dispersed in 10 ml of PBS (pH 7.4), followed by stirring at 40□ for 30 minutes. 4 mg of paclitaxel was dissolved in 2 ml of ethanol, which was slowly dropped into the above solution, during which sonication was performed using a water type sonicator. Sonication with the above solution was performed by using a bar type sonicator again for 2 seconds, which was repeated 10 times. Dialysis was performed in PBS (pH 7.4). On the next day, dialysis was performed again in distilled water for 12 hours, followed by centrifugation. Then, the product was filtered by 0.45 μm syringe filter and freeze-dried, resulting in a water-soluble chitosan nanopaclitaxel containing paclitaxel therein.<br>EXAMPLE 5 Preparation of a Water-soluble Chitosan Nanopaclitaxel 2

Water-soluble chitosan nanopaclitaxel was prepared by the same method as described in the above Example 4 except that WSC-NP prepared in the above Example 2 was used.<br>EXAMPLE 6 Preparation of a Water-soluble Chitosan Nanopaclitaxel 3

Water-soluble chitosan nanopaclitaxel was prepared by the same method as described in the above Example 4 except that WSC-NP prepared in the above Example 3 was used.<br>





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