Water soluble chitosan nanoparticle for delivering an anticancer agent and preparing method thereof
View Patent ↗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 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. Therefore, a water-soluble chitosan nanoparticle for delivering an anticancer agent of the present invention can effectively envelope paclitaxel by introducing hydrophilic and hydrophobic groups in the position of highly reactive amine group of the water-soluble chitosan. A water soluble chitosan nanopaclitaxel prepared as the above has an excellent re-dispersion force, after freeze-drying, in distilled water and has an outstanding anticancer effect with its accumulation in tumor cells greater than that of the other anticancer agent carriers.
1. A chitosan nanoparticle-paclitaxel complex, comprising: a water-soluble chitosan nanoparticle (WSC-NP) and paclitaxel,
wherein the water-soluble chitosan nanoparticle is spherical and having a hydrophobic core and paclitaxel is enveloped in the water-soluble chitosan nanoparticle to be present in the hydrophobic core,
wherein the water-soluble chitosan nanoparticle comprises chitosan derivatives with hydrophobic cholesterol groups attached to amino groups of the water soluble chitosan and hydrophilic methoxy poly (ethylene glycol) groups attached to other amino groups of the water soluble chitosan, and
wherein the water soluble chitosan is prepared by the method comprising:
a) enzymatically hydrolyzing an organic or inorganic acid salt of a chitosan oligosaccharide;
b) reacting the hydrolysis product of step (a) with trialkylamine;
c) adding an organic solvent to the product of step (b) to remove a trialkylamine salt at the C2-position of the chitosan oligosaccharide;
d) adding an inorganic acid to the product of step (c) to remove a trialkylamine salt at the C-6 position of the chitosan oligosaccharide; and
e) purifying the chitosan from the product of step (d) to obtain a chitosan with free amine groups and having a molecular weight ranging from 10,000 to 50,000 Da,
wherein the number ratio of glucose amine monomers of water-soluble chitosan:methoxy poly (ethylene glycol):cholesterol is 10:2:1.8,
wherein the size of the water-soluble chitosan nanoparticle ranges from 30 to 150 nm, and
wherein the chitosan nanoparticle-paclitaxel complex is prepared by the method comprising:
1) adding MPEG to the water-soluble chitosan, thereby forming an amide bond;
2) eliminating the p-nitrophenyl groups, by-products of step 1), by dialysis with ice water, and completely eliminating the remaining by-products by purifying with anhydrous ethanol;
3) adding cholesteryl chloroformate to the above reaction solution to form another amide bond between free amine group of the water-soluble chitosan and cholesteryl chloroformate; and
4) adding paclitaxel to the product of step 3) while sonicating,
wherein the steps 1)-4) are performed in Phosphate Buffered Saline (PBS) solution.
2. A method of making the chitosan nanoparticle-paclitaxel complex of claim 1 comprising:
1) adding MPEG to a water-soluble chitosan, thereby forming an amide bond; wherein the water soluble chitosan is prepared by a method comprising:
a) enzymatically hydrolyzing an organic or inorganic acid salt of a chitosan oligosaccharide;
b) reacting the hydrolysis product of step (a) with a trialkylamine;
c) adding an organic solvent to the product of step (b) to remove a trialkylamine salt at the C2-position of the chitosan oligosaccharide;
d) adding an inorganic acid to the product of step (c) to remove a trialkylamine salt at the C-6 position of the chitosan oligosaccharide; and
e) purifying the chitosan from the product of step (d) to obtain a chitosan with free amine groups;
2) eliminating the p-nitrophenyl groups, by-products of step 1), by dialysis with ice water, and completely eliminating the remaining by-products by purifying with anhydrous ethanol;
3) adding cholesteryl chloroformate to the above reaction solution to form another amide bond between free amine group of the water-soluble chitosan and cholesteryl chloroformate; and
4) adding paclitaxel to the product of step 3) while sonicating;
wherein steps 1)-4) are performed in Phosphate Buffered Saline (PBS) solution.