| Fisetin has broad spectrum of pharmacological activity,such as anti-oxidant,anti-coagulation,anti-tumor,anti-thrombosis and treatment of diabetic renal injury.It exhibits negligible toxicity to human body and could be obtained directly from diverse sources.However,the poor aqueous solubility and low bioavailability of fisetin have limited the further development and clinical application.To address this issue,nanoparticle formulation can enhance the solubility of poorly water-soluble drugs,improve the physical and chemical properties,including stability,bioavailability and targeting of drugs.In this study,chitosan that modified with doubly fisetin and poly(lactic acid)nanoparticles were prepared to improve the solubility,bioavailability and antitumor effect of fisetin.The physical characterization,in vitro release characteristics,the in vitro anti-tumor effect as well as pharmacokinetic characteristics in vivo were investigated.This structure of this thesis was divided into the following five parts:Part I.ReviewThis chapter mainly presents a review of fisetin,nanoparticle formulations,poly(lactic acid)(PLA)nanoparticles and chitosan.At the beginning of the review,the structural properties,extraction and separation,pharmacological activity and the development of fisetin preparation were discussed.Subsequently,the properties of nanoparticles formulation were thoroughly introduced,in terms of advantages,preparation technology and characterization methods.The third part of the review mainly introduces PLA nanoparticles,regarding the structure and property of PLA,the application in medicine of PLA,and the preparation methods of PLA nanoparticles.The next part of the review summarized the physical and chemical properties of chitosan and its application.At the end of the review,we discussed the purpose and the main content of this research.Part II.Preparation of fisetin PLA nanoparticlesHerein,the fisetin PLA nanoparticles were formulated by self-emulsifying solvent diffusion method.Initially,the HPLC method for the determination of fisetin was established with high relative standard deviation(RSD)(<2%).In terms of encapsulation efficiency of fisetin,the corresponding factors were determined based on a single factor test,thus,the optimal formulation of fisetin PLA nanoparticles was subsequently identified by the orthogonal experiment.The best prescription of fisetin PLA nanoparticles was obtained at several factors,including drug/carrier ratio of 1:10,O/W ratio of 1:12,the F68 concentration of 0.5%,acetone/ethanol volume ratio of 3:3.The degree of influence factors was drug/carrier ratio > O/W ratio > emulsifier concentration > acetone/ethanol volume ratio.The variance analysis showed that the drug loading ratio had the most significant influence on the entrapment efficiency.The best prescription of fisetin PLA nanoparticles were characterized,such as the particle size and Zeta potential determined by Malvern particle size analyzer,and the entrapment efficiency of fisetin analyzed by ultrafiltration centrifugation.The optimized preparation revealed a high encapsulation efficiency(90.35%),improved drug loading efficiency(3.12%),a narrow size distribution around 226.85 nm,negatively charged surface(-15.63 mV)and acceptable RSD(<5%).These results obviously showed that the fisetin PLA nanoparticles of best prescription have good quality and reproducibility.Part III.Preparation,characterization and in vitro release of chitosanmodified fisetin PLA nanoparticlesFisetin PLA nanoparticles were modified with chitosan to improve the anti-tumor effect and decrease the release of fisetin from the nanoparticles.The optimum conjugation ratio of chitosan to fisetin PLA nanoparticles was determined as 0.2%.The chitosan modified fisetin PLA nanoparticles were found to have smooth spherical shape using transmission electron microscopy.Compared with fisetin PLA nanoparticles,the chitosan modified fisetin PLA nanoparticles demonstrated slightly larger diameters,whatever,a still narrow size distribution.Fourier transform infrared spectroscopy(FT-IR)analysis showed that hydrogen bonding might exist between the carrier and fisetin,meanwhile,the results of differential scanning calorimetry showed that fisetin was present as amorphous form in the carriers.The in vitro release property was investigated by dynamic dialysis method,and PBS buffer(pH6.8,0.5%Tween)was selected as release medium.Compared with the rapid release of free fisetin(78% in 1 h),the release of drug in chitosan modified nanoparticles were dramatically reduced,about 27% during 1 h.These results revealed that the chitosan modified fisetin PLA nanoparticles had a significant sustainable release property in comparison with free fisetin.Part IV.In vitro antitumor effect of chitosan modified fisetin PLAnanoparticlesBased on the optimum formulation,the antitumor effect of chitosan modified fisetin PLA nanoparticles was examined toward the colon cancer cell line HCT116 via MTT assay.The results revealed that the growth inhibition rate of HCT116 cells treated with free fisetin at the concentration of 200 ?g/mL over 48 h was 78.43%,while the cytotoxicity of fisetin PLA nanoparticles and chitosan modified fisetin PLA nanoparticles were 71.18% and 81.34%,respectively.Thus it's summarized that chitosan modified fisetin PLA nanoparticles exhibited the best inhibitory effect on the growth of colon cancer cells compared with other formulations.Part?.Pharmacokinetic characteristics of the chitosan modifiedfisetin PLA nanoparticles in ratsIn order to investigate the pharmacokinetic characteristics of the chitosan modified fisetin PLA nanoparticles in rats,a HPLC method for the determination of fisetin in plasma was established.The linearity of calibration curve was in the concentration range from 0.1 to 2 ?g/mL with a typical linearity line equation of A=0.4262C+0.01313 and an average correlation coefficient(R2)of 0.9962,following by low values of inter-and intra-day RSD.Thus,these results met the requirements of analysis methods in vivo.The pharmacokinetic parameters in male SD rats after tail vein administration showed that the chitosan modified fisetin PLA nanoparticles could efficiently increase the half-life of fisetin from1.84 h to 3.42 h,and decrease the clearance rate of fisetin in vivo.In addition,the average residence time of fisetin in chitosan modified fisetin PLA nanoparticles was determined as 10.67±1.81 h,compared with that of free fisetin(4.86±0.16 h).Also,the area under the drug time curve(AUC)values of free fisetin was 8.31±1.92 h?g/mL,whaterver,the AUC values of chitosan modified fisetin PLA nanoparticles was 19.28±2.15 h?g/m L,showing a 2.32 fold increase in bioavailability.These results indicatec that the chitosan modified fisetin PLA nanoparticles could significantly increase the residence time of fisetin,resulting in the improved bioavailability. |
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