The Interaction Between Nanoparticle Carriers And Pulmonary Epithelium Cell A549 And The Pharmacodynamics Of Chitosan Nanoparticles Loaded Insulin In Vivo

In this study,we chose chitosan (CS) as delivery system material for macromolecules. Chitosan with different molecular weights were prepared using enzymatic degradation. Their molecular weights were determined by viscometry and gel permeation chromatography (GPC). Chitosan nanoparticles were prepared by solvent diffusion technique. Several characteristics of chitosan nanoparticles such as morphology,particle size,Zeta potential and drug entrapment efficiency were evaluated. The compound of chitosan grafted on fluorescein isothiocyanate (FITC) by chemical bond was prepared and called FITC-labeled CS (FITC-cs). Using pulmonary epithelium cell A549 as model cell,the cytotoxicity of chitosan nanopartilces was estimated. The interaction of chitosan nanopartilces and A549 cell was investigated. The intracellular fluorescence intensities were observed under inverted fluorescent microscope and were detected using flow cytometer. When the chitosan nanoparticles loaded insulin (INS),the pharmacodynamics after pulmonary administration in vivo was evaluted.Chitosan with different molecular weights was obtained after controlled thecondition of enzymatic degradation and ultrafiltration separation. The molecular weights of chitosan determined by gel permeation chromatography (GPC) were 10289,41500,101870Da,respectively. The chitosan nanoparticles prepared by solvent diffusion technique showed a spheric shape and exhibited a narrow particle size distribution,and became smaller after enhanced the proportion of anhydrous alcohol in the system or increased the treatment sonication number of times. The particle size distribution also turned to small when using lower molecular weight chitosan.The nanoparticle system belongs to thermadynamics unstable system. Due to the higher Zeta potentials of chitosan nanoparticle systems (over +30 mV),these systems were relatively stable. The average entrapment efficiency of insulin was 95.54+ 1 . 14% (n=3 ) by treatment of high speed centrifugation and filtration.The investigation of cytotoxicity made it clear that chitosan gained by enzymatic degradation,FITC-cs and chitosan nanoparticles showed low toxicity. With the concentration of 32 ng/ml of chitosan nanoparticles (10289Da and 41500Da),their cell suvival rate were 87.8% and 82.9%,respectively. The investigation of the interaction of chitosan nanopartilces and A549 cell showed the different fluorescence intensity in cells when incubated with FITC-cs and their nanoparticles. It also suggested that the fluorescence intensity were in relation with chitosan molecular weight and the incubation time. There was no intracellular fluorescence after incubation with FITC solution or the complex of fluorescein isothiocyanate and chitosan.The result determined by flow cytometer following the incubation of carriers and A549 cell indicated that the intracellular fluorescence intensities were in relation with chitosan molecular weight when using the same concentration of FITC-cs. With the same molecular weight of chitosan,the fluorescence intensityof FTIC-cs was obviously higher than that of its nanoparticles. In comparison with the insulin solution,five times relative bioavailability was observed with the same dosage of insulin chitosan nanoparticles after pulmonary administration.