| Objective: Based on chitosan-stearic acid(CSO-SA)polymers and modified with aldehyde-terminated polyethylene glycol(m PEG-CHO)and folic acid(FA),FA/PEG-CSO-SA nanomicelles were prepared,and then a poorly soluble drug(Osthole,OST)were encapsulated to obtain Osthole nanomicelles with active targeting of tumor cells,and the preparation process,particle size,Zeta potential,encapsulation efficiency,drug loading,stability,in vitro release,and in vitro antitumor activity of the nanomicelles were also investigated in order to obtain a novel and safe and effective Osthole nanomicelles with active antitumor effect.Methods:1?The structures of CSO-SA,PEG-CSO-SA and FA/PEG-CSO-SA were synthesized and characterized by infrared spectroscopy and NMR.2?The synthesis conditions of CSO-SA,PEG-CSO-SA and FA/PEG-CSO-SA blank micelles were investigated by dialysis.The micelles was observed by a transmission electron microscope Microscopic morphology;and the amino substitution of the micelles was determined by the TNBS method.3?Methodological investigation of osthole(OST)by HPLC,on the basis of single-factor investigation,the factors that are important for the preparation of FA/PEG-CSO-SA/OST were screened out,and the encapsulation efficiency,drug loading and particle size were used as comprehensive indicators,and orthogonal tests of three factors and three levels were designed to optimize the best preparation process.4?Ultrasonic method was used to prepare osthole nanomicelles,ultrafiltration centrifugation method was used to determine the encapsulation efficiency and drug loading of drug-loaded micelles,X-ray diffraction analysis of the structure and crystal morphology of drug-loaded micelles;the particle size analyzer measures the particle size and Zeta potential of drug-loaded micelles,determine the optimal process of drug-loaded micelles and study its stability,In vitro release of osthole nanomicelles and free osthole at different p H(5.0,6.8 and 7.4)was investigated by dialysis.5? The MTT method was used to study the cytotoxicity and cell uptake of CSO-SA,PEG-CSO-SA,and FA/PEG-CSO-SA using Hep G2 cells as a cell model,and evaluates the in vitro antitumor effects of free osthole and carrier osthole nanomicelles.Results:1?Infrared spectroscopy showed that the amino group of CSO reacted with the carboxyl group of SA to form an amide bond,indicating that CSO-SA were successfully synthesized;and m PEG-CHO reacted with the amino group on chitosan in CSO-SA by Schiff base reaction the resulting Schiff base bond indicates that PEG has chemically reacted with CSO-SA to obtain PEG-CSO-SA;the carboxyl group in FA reacted with the residual amino group on the CSO in m PEG-CSO-SA micelles,forming the amide bond,indicating that FA has been successfully attached to m PEG-CSO-SA and the FA/PEG-CSO-SA have been obtained.The structures of CSO-SA,PEG-CSO-SA and FA/PEG-CSO-SA were confirmed by H-NMR.PEG and FA were successfully grafted onto CSO-SA respectively.2?The particle size of the blank carrier prepared by the optimized process(96.01±5.99),(112.93±1.06)and(216.01±4.76)nm(n=3),the Zeta potentials are(39.30±1.75),(38.03±2.91)and(15.17±2.10)m V(n=3);transmission electron microscopy(TEM)images showed that the blank micelles were elliptical,with uniform size and good dispersion.The amino substitution degrees of CSO-SA,PEG-CSO-SA and FA/PEG-CSO-SA determined by the trinitrobenzenesulfonic acid method(TNBS)were 17.78%,19.05%,and 24.13%,respectively.3?The results of orthogonal experiments show that the optimal preparation conditions for Osthole nanomicelles are: carrier concentration of 4 mg·m L-1,drug loading ratio of 5:1,and the number of ultrasounds 200 times/min.4?The encapsulation efficiency and drug loading of the osthole nanomicelles prepared by the optimal process were(86.5±1.48)% and(24.7±0.49)%;particle size(316.6±2.47)nm,Zeta potential(41.7±1.01)MV(n=3).The results of in vitro drug release tests showed that the drug-loaded micelles were p H-sensitive and had a relatively slow release compared to free Osthole.5?The cytotoxicity test results of FA/PEG-CSO-SA blank micelles showed their inhibition rate<20% for Hep G2 cells,which could be used as a safe carrier materials for further study of nano-drug delivery system.The IC50 of OST solution and FA/PEG-CSO-SA / OST on human liver cancer Hep G2 cells were(62.08±5.21)and(27.49±0.50)?g·m L-1(n=3).In vitro antitumor experiments have shown that FA/PEG-CSO-SA/OST can improve the inhibitory effect on Hep G2 cells compared with OST,and it is expected to become a new drug delivery system for antitumor applications.Conclusions: In this study,a new type of nanomicelles was prepared with simple preparation method,good stability,high encapsulation efficiency and drug loading capacity,in vitro drug release showed sustained release and p H sensitivity of drug-loaded micelles.The prepared nanomicelles can significantly increase the inhibitory effect of OST on Hep G2 cells,such nanomicelles are expected to become a new type of antitumor drug carrier. |
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