Preparation Of Folate-chitosan Nanoparticles And Its Targeting Characterization As Drug Carriers

Chitosan is a kind of important natural polymer which has not been efficiently utilizated.According to the principle of electrostatic interactions, using chitosan and folic acid as raw material, folate conjugated chitosan nanoparticles and drug loaded nanoparticles were prepared by ionic crosslinking method.The chemical structure, mean diameter, polydispersity index and morphological characteristics of nanoparticles were characterized using infrared spectroscopy (IR), laser particle size analyzer, transmission electron microscopy (TEM) and zeta potential.The drug entrapment efficiency(EE), loading capacity(LC), in vitro release rate, stability, in vitro and in vivo antitumor activity, organ targeting, mean residence time of drug were determined.This research improved the preparation method of folate conjugated chitosan nanoparticles, and the drug loading range is enlarged. Drug loaded nanoparticles not only has the characteristics of high EE, high LC and good stability, but also has the obvious characteristics of good targeting and sustained release. This research could provide the foundation data and technical support for the reasonable utilization of chitosan. The main research work and some obtained novel results were performed and listed as follows:Using chitosan(CTS)as carrier, folic acid (FA)as targeting ligand, sodium tripolyphosphate (TPP)as polyanion, CTS-TPP-FA nanoparticles were prepared by the ionic crosslinking method through the electrostatic interaction between the protonated amino of chitosan in acidic conditions and the deprotonation of carboxyl of folic acid in alkaline conditions as well as trimeric sodium phosphate (TPP) in the water phase.Infrared spectroscopy (IR) confirmed that folic acid and chitosan was combined by ionic bond.With particle size (less than200nm) as index, some factors that influence the particle size were investigated.The results indicated that the optimal preparation conditions were as follows:Under the conditions of55℃water bath, ld/s of dropping speed,900～1000r/min of stirring speed (magnetic stirring), taking VmL of2.0mg.mL-1chitosan acetic acid solution (the concentration of acetic acid is1%, and the pH value is3.95)as base solution, at the first,adding V/4mL of1mg/mL-1folic acid ammonia solution in base solution, and then adding V/10mL of2.0mg/mL-1sodium tripolyphosphate solution in base solution. After finishing that, stirring was continued for10minuts to get blank folic acid-chitosan nanoparticles water dispersion system. The prepared blank folic acid chitosan nanoparticles under the best preparation process conditions were regularspherical, uniform size with nanoparticles average particle size and1.019of dispersion index.To obtain nanoparticles with high encapsulation efficiency(LE),high drug loding rate(DL) and less than200nm in averrage particle size.The adding methods and adding quantity of anticncer drug were studied on the basis of the preparation of blank nanoparticles.The results show that when HCPT was first mixed with chitosan-acetic acid solution as the ratio of3:5(HCPT:CTS solution,mg/ml),or when DOX first reacted with TPP in the ratio of2:1(DOX:TPP=2:l,molar ratio), then done as the same way of the blank nanoparticles, we could obtain uniform spherical and regular HCPT/CTS-TPP-FA nanopaticles with a size of150nm,+50.1mV surface potential,89.90%of LE,19.80%of DL and good size stability. The in vitro release profile displayed that nearly87%HCPT was released in the first24h and its release formulation fitted to Higuchi equation(PBS Buffer solution of pH=7.4). The DOX/CTS-TPP-FA nanopaticles with a size of162nm,+52.1mV surface potential,91%of LE,21.50%of DL and good size stability. The in vitro release profile displayed that nearly28.25%DOX was released in the first5d and its release formulation fitted to Higuchi equation(PBS Buffer solution of pH=7.4).Judging from the particle size and the amount of folic acid connected with it, the HCPT/CTS-TPP-FA and DOX/CTS-TPP-FA nanoparticles should have good tumor-targeting.The antitumor activity of HCPT/CTS-TPP-FA nanoparticles on human hepatocellular carcinoma Hep cells G-2, human lung cancer cell A549, and human breast cancer cell line MCF-7were investigated by in vitro cell line experiment,respectively.The results of MTT showed that HCPT/CTS-TPP-FA nanoparticles has obvious inhibiting action on all of the above cancer cells, The rate of inhibition has a time and dose dependent.It increased with the increasement of drug concentration and reaction time. Compared with the control group, the rate of inhibition of HCPT/CTS-TPP-FA had significant difference (P<0.01),and the anticancer activity of HCPT/CTS-TPP-FA was btter than that of HCPT/CTS-TPP. So HCPT/CTS-TPP-FA has good tumor targeting.The effect of antitumor rate of HCPT/CTS-TPP-FA nanoparticles on the mice bearing S180tumor cells were investigated by animal experiment in vivo. The results showed that the antitumor rate of HCPT/CTS-TPP-FA(52.91%) was better than that of HCPT/CTS-TPP (40.06%),which indicated that HCPT/CTS-TPP-FA has good tumor targeting.Organ targeting and mean residence time of HCPT/CTS-TPP/FA nanoparticles were determined in mouse blood, heart, liver, spleen, lung and kidney by HPLC method to detect the content of HCPT. Through analyzing drug targeting efficiency (Te), drug relative targeting efficiency (Re) and drug peak concentration ratio (Ce), the results showed that HCPT/CTS-TPP-FA was better than the commonly used injections in clinical on treatment of liver cancer, leukemia, and lung cancer because HCPT/CTS-TPP-FA nanoparticles have characters of targeting and controlled release.So it is expected to become a new form of HCPT in treatment of liver cancer, lung cancer and leukemia tumor targeting formulations. It has important theoretical significance in study of targeting and controlled release formulations for other anti- tumor drugs.In order to increase stability, convenient storage and use, the lyophilized powder of HCPT/FA-CTS-TPP nanoparticles for injection were prepared with glucose and mannitol as freeze-drying protectants by the freeze drying technology.With appearance and redispersibility of lyophilized powder as index, factors such as type, concentration and dosage of freeze-drying protectant influencing them were investigated.The results indicated that when adding equal volume of8%mannitol aqueous solution into the HCPT/FA-CTS-TPP water dispersion system, the lyophilized powder of the HCPT/FA-CTS-TPP with uniform color, smooth appearance, compact structure and crisp were obtained.It redispersed very soon and well in water without any precipitate.In a word, chitosan transformed into cationic polymer compound under acidic conditions, then reacted with folate acid,and then cross-linked with TPP to obtained CTS-TPP-FA namoparticles.The CTS-TPP-FA namoparticles have characters of sustained-release and good tumor targeting as well as organ targeting.It has important theoretical significance in research of targeting formulations for drugs on folate receptor mediated (such as anti-inflammatory, anti-cancer drug).