Research On Novel Curcumin-Oleic Acid Derivative Nanoparticle Preparation,Characteristic And Anti-tumor Activity Evaluation In Vitro

Objective:In order to improve the stability,water dispersibility,enhangce its bioavailability and antitumor activite of Curcumin(Cur),prodrug modification method and nano-preparation technology was proposed.Meanwhile,the stability,hydrolytic release characteristics and antitumor activity in vitro of the novel nanoparticle was evaluated so as to lay the foundation of developing a stable and injectable novel Cur preparation.Methods:(1)Using optimized prodrug synthesis technology,Curcumin-(Oleic acid)2[Cur(OA)2]prodrug was obtained and characterized by thin layer chromatography(TLC),ultraviolet spectrum(UV),high performance liquid chromatography(HPLC)and nuclear magnetic resonance spectroscopy(NMR).Take mPEG5000-PLGA as carrier,nano particle of Cur(OA)2 was prepared by modified solvent evaporation method[mPEG5000-PLGA-Cur(OA)2-NP,mPPCO-NP].The best preparation conditions of the nanoparticle was determined through orthogonal experiment which taken morphology,particle size and entrapment efficiency rate as indicators.Then the particle size,zeta potential and morphology of the nanoparticle was characteristiced by laser particle size analyzer(Nano-ZS90)and transmission electron microscopy(TEM).Furthermore,the drug loading amount(Drug Loading,DL)and drug encapsulation efficiency(Drug Entrapment Efficiency,DEE)was determined as well.(2)Free Cur loaded nanoparticle(mPPC-NP)was prepared in the same way,and the stability,photosensitivity and drug release property(in PBS solution,health healthy human plasma and inactivated human plasma)between mPPC-NP and mPPCO-NP was compared by UV-spectrophotometry.(3)The cell growth inhibition activity of mPPCO-NP against HepG2 cells was evaluated by MTT method;meanwhile,the phagocytosis of HepG2 cell to the mPPCO-NP marked with DiI by the fluorescent staining was observed using the Confocal Laser Scanning Microscope(CLSM)FV1000.Results:(1)The Oleic Acid-modified prodrug of Curcumin:Cur(OA)2 was synthesized and confirmed.The optimized mPPCO-NP preparation conditions was screened out through orthogonal design:amount ratio of Cur(OA)2 and mPEG5000-PLGA(m/m)was 1:4,the volume ratio of organic solvent and aqueous solvent(V/V)was 1:2,the stirring speed was 1200 r·min-1,and the content of poloxamer 188(Pluronic F68)was 0.30%.The nanoparticles were uncovered as spherical particles with uniform distribution,and the average particle diameter of mPPCO-NP was 86.23nm with Zeta potential-23.8mV.Entrapment eEfficiency rate and drug loading capacity were(91.25±0.10)%and(18.25 ± 0.02%)respectively.(2)In the condition of 37?,mPPCO-NP and mPPC-NP was released slowly in the normal saline.And mPPCO-NP was released 16.68%in the first 1d,while 60.85%of mPPC-NP was released;moreover,only about 29.81%of mPPCO-NP was released after 9d comparing to 92.42%of that mPPC-NP.Comparing with mPPC-NP,the stability of mPPCO-NP had been greatly improved,while the photosensitivity of mPPCO-NP was reduced,hence the mPPCO-NP had good storage stability in the dark.Results of drug release test in vitro showed that the drug release rate of the mPPCO-NP was significantly lower(P<0.01)than mPPC-NP,which can persistent release drug in the flowing 100h,attributed to Oleic acid modification.(3)In vitro anti-tumor activity results indicated that mPPCO-NP still with inhibition activity against HepG2 cells with IC50 at 40.61 ?M after the treatment for 48h,but it's active was declined slightly comparing with free curcumin(IC50 at 15.76 ?M),which can be explained as the attenuated toxic effect.CLSM revealed that mPPCO-DiI-NP can be intaken by HepG2 in the way of endocytosis and the intake was increased over time.Conclusion:mPPCO-NP was uniform,spherical,with high drug loading capacity.The stability of Cur modified by OA and prepared into mPPCO-NP was significantly improved;The naoparticle enter into HepG2 cells mainly through phagocytosis and exert its antitumor proliferation activity in vitro.The research on mPPCO-NP set a solid foundation for further exploring the liver targeting of the nanoparticle and uncovering the relationship between the Cur concentration and its pharmacodynamic in vivo.