Preparation And Characterization Of Folic Acid Conjugated O-Carboxymethyl Chitosan Nanoparticles Loaded With Methotrexate

Methotrexate （MTX） is a folate antimetabolite. It has been used in the treatment ofvarious malignancies including leukemia, osteosarcoma, head and neck cancer, lungcancer, and breast cancer. However, because of its nonspecific drug delivery, the clinicapplication of MTX has been limited by its undesirable side effects such asneurotoxicity. Polymeric nanoparticles have been widely used as the drug carriers forthe controlled release of the drugs, which greatly improve the treatment efficacy.However, most nanosized drug carriers can only passively accumulate in tumor sites viathe EPR （enhanced permeability and retention） effect. Folate receptors （FRs） are knownto be overexpressed on the cell membranes of many cancer cells but exhibit limitedexpression on healthy cells. Thus, folic acid （FA） can be recognized as an effectivetumor active targeting agent to conjugate with nanoparticles because of its specificbinding with the FRs.Chitosan （CS） as a natural cationic alkaline polysaccharide possesses some uniqueproperties, such as low toxicity, acceptable biocompatibility, biodegradability andantitumor activity and it has been intensively used in drug-delivery systems. However,the potential application of CS is hindered since it is only soluble in weak acidicsolution. O-Carboxymethyl chitosan （O-CMC） is a kind of water-soluble CS derivant,with the O-hydroxyl group of each monomer substituted by a carboxymethylic groupthrough the ether-bond formation, and it has been proven to have better biocompatibilityand antibacterial activity than CS. O-CMC nanoparticles can be formed via thecrosslinking of the carboxyl groups of O-CMC with calcium ions in neutral or weaklybasic conditions. An increase of the pH range is expected to improve the encapsulationefficiency of acidic drugs.In this paper, FA-O-CMC was synthesized by the reaction between the activatedFA ester and the primary amine groups of O-CMC through the formation of amide bond,and FA-CS was prepared as control. The structure of grafting FA was confirmed byultraviolet （UV） spectrophotometry, Fourier transform infrared spectrometer （FT-IR）and¹H-NMR spectroscopy. Single factor experiment shows the optimum preparationconditions of FA-O-CMC were that the mass ratio of FA ester to O-CMC was1:1andthe reaction was hold at35℃for8h.MTX/FA-CS nanoparticles and MTX/FA-O-CMC nanoparticles were prepared by ionic cross-linking with TPP and CaCl₂, respectively. The optimum conditions ofMTX/FA-CS nanoparticles were that the mass ratio of TPP/FA-CS and MTX/FA-CSwas1:5and3:20, respectively, and the pH value of the FA-CS solution （0.2%, w/v） wascontrolled as5.0. The corresponding optimum conditions of MTX/FA-O-CMCnanoparticles were that the concentration of FA-O-CMC was2mg·mL^-1, and the massratio of CaCl₂/FA-O-CMC and MTX/FA-O-CMC was1:5and1:10, respectively. Theobtained nanoparticles were spherical in shape and preserved high stability. FT-IR andX-ray diffraction confirmed the linkage between the cross-linking agent and ammoniumion of the FA-CS or FA-O-CMC. Compared to the two kinds of nanoparticles,FA-O-CMC nanoparticles showed higher drug-loading capacity （LC） than those of theFA-CS nanoparticles, and the particle size of the FA-O-CMC nanoparticles was alsosmaller. The results suggest that FA-O-CMC was more suitable for the encapsulation ofMTX than the polymeric FA-CS.In vitro release pattern in phosphate buffer saline （pH6.8） indicated that thecharacteristics of the MTX-loaded nanoparticles appeared to have an initial burst effectand followed by a slow, sustained drug release. The in vitro release profiles ofnanoparticles with different feeding drugs depict that the MTX release rate is highlyinfluenced by the amount of MTX entrapped. Polymeric nanoparticles with higher LCshowed higher drug release rates. Moreover, FA or low molecular weight FA conjugatefragments could be cleaved from the polymer and released from the blank nanoparticles,which might be utilized as an antidote opportunely and greatly reduces toxic effects ofMTX to healthy tissues within the body.