Study On Turmeric Derived Exosome-Like Nanoparticles Delivering Mansonones Derivative B16

Objective:In recent years,the innovation of nano-carriers has promoted the development of drug delivery system(DDS).However,the ability of nanocarriers to penetrate the biological barrier is weak,and the problems of biocompatibility and toxicity have not been effectively solved.Plant exosomelike nanoparticles(ELNs)with low immunogenicity and low toxicity are potential drug carriers.Mansonones derivative b16 has superior antitumor activity,but its cytotoxicity is strong and nonspecific.In this study,turmeric ELNs were used as b16 delivery vehicles,and curcumin in TELNs cooperated with b16 to exert antitumor activity,thereby developing a drug delivery system with high efficiency and low toxicity.Methods:(1)Separation: the ELNs of turmeric rhizome was isolated by differential centrifugation and ultracentrifugation.(2)Preliminary physical and chemical characterization: atomic force microscopy(AFM),transmission electron microscopy(TEM),dynamic light scattering(DLS),nanoparticle tracking analysis(NTA),thin layer chromatography(TLC),SDS-PAGE gel electrophoresis and agarose gel electrophoresis.(3)Component analysis and identification: lipid and protein species were analyzed by mass spectrometry,RNA was qualitatively and quantitatively analyzed by PCR and q PCR;curcumin was qualitatively and quantitatively analyzed by HPLC,and other compounds contained in TELNs were analyzed by LC-MS and GC-MS.(4)Drug loading: methodological investigation of HPLC determination of b16 content of mansonones derivatives;screening of the dominant drug loading methods and parameters of TELNs for b16 based on the drug loading rate;TELNs-b16 drug delivery system was constructed by the optimal co-incubation method;The morphology,particle size and Zeta potential of TELNs-b16 were characterized by AFM,NTA and DLS;the particle size was used as an index to investigate its stability,and the in vitro drug release characteristics were investigated by dynamic dialysis.(5)Cytology: the growth of Hela treated with b16 was monitored in real time by Incu Cyte system.The safety of TELNs,the anti-proliferative activity of b16 and TELNs-b16 against OVCAR8 and MCF-7/ADR were evaluated by MTT.The ability of tumor cells to uptake TELNs was observed by fluorescence microscope.(6)Pharmacokinetics: the distribution of TELNs and TELNs-b16 in Kunming mice(30±2 g)was investigated,and the pharmacokinetic characteristics of TELNs were reflected by blood fluorescence intensity.At the same time,the content of b16 in plasma and organs was determined by HPLC to investigate the pharmacokinetics of b16 and its distribution and accumulation in various organs.Results:(1)TELNs were extracted by ultracentrifugation.The results of AFM and TEM showed that the extract had obvious spherical nanoparticle morphology with an average particle size of 176.2±54.47 nm,and had the same teacup-like bilayer membrane structure as exosomes,and the particles were dispersed Relatively uniform and uniform in shape,singly distributed or aggregated into clusters.The results of NTA and DLS showed that the particle size of TELNs showed a normal distribution,with a concentrated distribution around 180 nm,a particle size range of 50-400 nm,and a Zeta potential of-17.6±1.19 m V.(2)The lipid components with higher content in TELNs were mainly PE(17.4%),TG(12.3%),and PI(9.82%).The proteins are mainly proteins involved in the biosynthesis of secondary metabolites such as curcumin synthase.RNA detection contained key genes for curcumin synthesis,including CURS and DCS.The chemical active components contained in TELNs are mainly curcuminoid compounds such as demethoxycurcumin and curcumin,and volatile oil substances such as curcumin and curcumene.(3)A HPLC method for the determination of b16 content in vitro was established,and a TELNs-b16 drug delivery system with a drug loading rate of48.9±1.96% was prepared by an optimized co-incubation method.AFM results showed that loading b16 did not affect the morphology of TELNs,but the particle size(221.7±5.0 nm)and potential(-22.4±3.63 Mv)were slightly increased compared with blank TELNs.The results of stability and in vitro drug release characteristics showed that the condition of 4℃ was more favorable for the preservation of TELNs and TELNs-b16,and the acidic condition of the tumor was more favorable for the release of b16.(4)In vitro,b16 inhibits the proliferation of various tumor cells and can be used as a potent chemotherapeutic drug;TELNs have a certain cytotoxicity(IC50=112 μg/m L)for a long time(72 h)at a high concentration(100 μg/m L).TELNs-b16 had stronger ability to inhibit tumor cell proliferation than free b16 at the same concentration.In vivo,the drugs mediated by TELNs are mainly distributed in the liver and kidney,and secondarily in the spleen and lung;b16loading on TELNs does not significantly affect its distribution in the body and circulation time in the blood,and its half-life in the blood is 1.72±0.08 h.Conclusion:In this study,we extracted TELNs and constructed a TELNs-b16 drug delivery system by optimizing drug loading parameters.TELNs-b16 has stronger anti-tumor cell proliferation activity in vitro,and tends to enrich in organs such as liver and kidney in vivo.Provided a theoretical basis for the application of TELNs as carriers.