Study On The Preparation And The Liver Cancer Targeting Of CD147Antibody-mediated α-Hederin Chitosan Nanoparticle

Objective: Taking chitosan (CS) as the basic carrier,-Hederin (-Hed, a waterinsoluble drug) as the model drug, to prepare-Hed-CS-NP (HCS); taking CD147monoclonal antibody (mAb) as the active target, and to modify CD147to the surface of thenanoparticles to prepare-Hed-CS-CD147-NP (αHCD), so as to decrease the toxicity andto improve the efficacy of-Hed with the slow-release character of nanoparticles and theCD147-mediated active targeting.Methods：(1) Taking particle size, polydisperse index(P.I.) and entrapment efficiency(EE) as comprehensive evaluation indexes, αHCSwere prepared by the emulsion-solventdiffusion technique, and prescription and preparation processes were investigated throughthe single factor and the orthogonal tests. The in vitro drug release and the pharmaceuticalproperties of nanoparticles were studied with FT-IR, DSC, XRD.(2) αHCD were preparedby EDC/NHS amidation reaction, and then were evaluated through TEM, FT-IR, XRD.The concentration and activity reservation of CD147monoclonal antibody wererespectively determined by BCA and ELISA method. The proper amount of modifiedCD147was determined by mensurating the uptake rate of nanoparticles modified withdifferent amount of antibody.(3) The cytoxicty of CD147and Void CS-CD147-NP to livercancer cells were determined with MTT method. The cytotoxity of nanopaticles to HepG2,SMMC-7721was confimed through the same method, and taking HSC cell as control. Theapoptosis and cell cycle tests of HepG2were carried out.(4) Taking HSC as control cell,HepG2cell uptake of nanoparticles was determined, and by pretreating with saturatedCD147, the active targeting was further validated. By preconditioning with different kindsof endocytosis inhibitors, the endocytosis mechanism of nanoparticles was studied, thusunderstanding the related pathways. And the in vivo uptake of nanoparticles was observedthrough inverted fluorescence microscope.(5) Taking normal saline as the negative controlgroup, cyclophosphamide as the positive control, the nude mice bearing HepG2as tumor model, the pharmacodynamics was studied by intraperitoneal injection of nanoparticles.Using the in vivo imaging technology in small animal, the in vivo absorption anddistribution of nanoparticles to tissues and the targeting to tumor were investigated.Results:(1) The properties of good drug entrapment, sustained release, averageparticle size (92.7±2.77) nm, low P.I.(0.144±0.037) and high EE (75.63±4.06)%werereflected in the prepared HCS. And the fact that-Hed had been dispersed into thenanoparticle was approved by series characterizations.(2) CD147could be modified toααHCSvia amide coupling reaction to prepare αHCD with the particle size of little change,and the CD147saturation modification quantity was (6.55±0.234) μg/mgNP. Accordingto the results of cell uptake rate,1.5μg/mg NP was finally chosen, with the activityretention rate (57.07±0.60)%.(3) The VCD and CD147antibody had no cytotoxic effecton liver cancer cells by MTT experiments, and the antiproliferation of nanoparticles toHepG2and SMMC-7721was more formidable, with the IC50of αHCD being the lowest.By Annexin V-FITC/PI double staining, the apoptosis results were consistent with MTT;and with the addition of drugs, the cell percentage in S phase was increased.(4) The uptakeof αHCD on HepG2was significantly higher than that of HCS, and there was nosignificant difference between αHCSand αHCD on HSC cell, and the uptake of αHCDcould be extremely reduced by adding the supersaturation of CD147. With the seriesresults above, nanoparticles modified with antibody was inferred to have better affinity forliver tumor cell. With the addition of different endocytosis inhibitors, the endocytic uptakeof nanoparticles is an energy-dependent process, which needed to form the coated pits toget into cells, and was probably related to the clathrin-mediated pathways and the stabilityand mobility of cell membrane. Through the inverted fluorescence microscope, thenanoparticles were observed gathering on the surface of cell membrane in one hour andingested more into cell in three hours.(5) Through the pharmacodynamics experiments, thetumor inhibition rate of αHCS(79.02%,1mg/kg) and αHCD (88.43%,1mg/kg) werediscovered much higher than that of-Hed (53.83%,1mg/kg), and the tumor inhibitionrate of nanoparticles was dose-dependent. Through the near infrared imaging of smallanimals, the fluorescence was detected more concentrated to liver and tumor in one hourafter the tail vein, and was found gradually weakening in tumor in six hours and thenstarting to disappear in twelve hours. Conclusion: The αHCSprepared through the emulsion solvent diffusion method haduniform size distribution, with its roundness, high entrapment efficiency, slow-releasecharacteristics, the in vitro and in vivo pharmacodynamics experiments showed that theblank nanoparticles were biocompatible with cells, and the antiproliferation of αHCD onliver tumor cells were enhanced, and the CD147mAb mediated active targeting wasvalidated by a series of uptake tests. In summary, αHCD had the application potential withthe prospect of becoming a novel carrier for water-poorly-soluble drugs.