1. Field of the Invention
The present invention relates to a water soluble chitosan nanoparticle (WSC-NP) for delivering an anticancer agent and a preparing method thereof, more precisely, a water soluble chitosan nanoparticle for delivering an anticancer agent which has function of targeting on a wanted area by introducing a functional group in the location of highly reactive amine group and becomes an excellent gene carrier with the use of water soluble chitosan (WSC) since the water soluble chitosan itself can combined with DNA having a negative electric charge(−) owing to the very strong positive electric charge(+) of its amine group, and a preparing method thereof.
2. Description of the Prior Art
Chitosan is formed by β-1,4 bond of pyranose monomers of glucosamine, having over 5,000 residues of glucosamine. Its molecular weight is over one million. As a biopolymer belonging to polysaccharide having polycations, chitosan is extracted from aquatic products such as Crustacea like crab or shrimp and a squid. It has a similar molecular structure to that of cellulose, a kind of polysaccharide, indicating that it has an excellent biocompatibility without rejection by immune reaction. So, chitosan has been widely used in medical industry and recently attested by FDA, U.S.A. to be safe food. Thus, chitosan is expected to be a relevant material applicable to bioindustry and biomedical industry in the 21st century.
In particular, chitosan having a molecular weight ranging from 20,000 to 100,000 is known to have very strong physiological activities, so the chitosan can be applied to the production of health food, food and beverages, cosmetics, sanitation and medical supplies.
Although the above promising characteristics of chitosan, it has not been applied successfully to the bioindustry since it is an insoluble in water owing to the strong hydrogen bond with neighboring molecules and organic acids including lactic acid, acetic acid, propionic acid, formic acid, ascorbic acid and tartaric acid, and inorganic acids including hydrochloric acid, nitric acid and sulfuric acid had to be used to dissolve chitosan. In order to overcome the problem, the present inventors have developed a water-soluble chitosan and applied for a patent (Korea Patent Application Nos. 2001-0059282 and 2001-0070052).
In the above patents, a preparing method for a pure water soluble free amine chitosan having a molecular weight ranging 1,000˜100,000 Da was reported, which includes the steps of 1) organic or inorganic acid salt solution of chitosan oligosaccharide is treated with trialkyl amine, 2) an organic solvent is added to the above solution to separate organic or inorganic acid linked with chitosan oligosaccharide which becomes trialcohol amine salt, and then, recover the chitosan oligosaccharide without organic or inorganic salt, 3) the solution of chitosan oligosaccharide without acid is treated with inorganic acid and purified by activated carbon/ion exchange resin column, resulting in a pure water soluble free amine chitosan having a molecular weight of 1,000˜100,000 Da.
Although paclitaxel, attested by FDA, U.S.A. in 1992, has a very excellent anticancer activity comparing to other conventional anticancer agents, it has a serious problem of side effects resulted from that it has to be dispersed in a mixed solution of cremophore EL (polyethoxylated oil) and ethanol (50:50) to be used as an injection because paclitaxel is hydrophobic so that it is not dispersed in water at all.
Because of insolubility in water, paclitaxel, a natural material having an anticancer activity, has limitation in use as an injection. Therefore, various systems have been proposed to make injection of paclitaxel possible. For example, human serum albumin (HSA) which is biodegradable and strongly bound to paclitaxel was combined with paclitaxel, followed by sonication, high-pressure homogenization and microfluidization according to a conventional method, resulting in an emulsion applicable as an injection.
Applicable injections prepared by combination of paclitaxel produced by VivoRx Pharmaceuticals, Inc. by taking advantage of sonication skill providing an average particle size of under 10 μm and human serum albumin particles were reported in U.S. Pat. Nos. 5,439,686, 5,498,421, 5,560,933 and WO 94/18954. However, the preparing methods reported in the above patents cannot be used on a large industrial scale. Moreover, the particles resulted from the above methods are too big to be administered to a patient.
Other reports concerning paclitaxel having an average size of under 0.2 μm obtained from dissolving freeze-dried powder in sterilized 0.9% NaCl solution and sterilized human serum albumin nano-emulsion have been made in U.S. Pat. Nos. 5,916,596, 6,096,331, WO 98/14174 and WO 99/00113 as well. According to the above reports, particles in nano-emulsion obtained through high-pressure homogenization are uniform in size and nanoparticle precipitate is not formed as time passes, indicating the stability of nano-emulsion. However, paclitaxel suspension prepared by mixing with human serum albumin is not available as an injection on industrial scale, yet.
Thus, the present inventors have studied on a method to use chitosan as a paclitaxel carrier which is applicable to medical industry successfully because chitosan has excellent biocompatibility without rejection by immune reaction as being administered, and have completed this invention by establishing the method.