Research On Tissue Distribution And Pharmacodynamics Of Liver-targeting Cur-（OA）₂ Loaded MPEG-PLGA Nanoparticles In Vivo

Objective:To prepare the novel liver-targeting Cur（OA）₂ nanoparticles（Cur（OA）₂-NPs）and research on tissue distribution and pharmacodynamics of the nanoparticles.Methods:1.Synthesis and evalution of Cur（OA）₂-NPs:Cur（OA）₂ loaded mPEG5000-PLGA nanoparticles were prepared by the emulsion solvent evaporation technique according to the optimized preparation conditions obtained in preliminary work:the 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,and the content of poloxamer 188（Pluronic F68）was 0.3%.The drug loading amount（Drug Loading,DL）and drug encapsulation efficiency（Drug Entrapment Efficiency,DEE）were determined by UV-spectrophotometry.Then the particle size and morphology of the nanoparticle were characteristiced by laser particle size analyzer（Nano-ZS90）and transmission electron microscopy（TEM）.2.Study on the tissue distribution and drug release in vivo:Male KM mice（20±2.0g）were obtained from the Laboratory Animal Center Zhejing Chinese Medical university.Then 25mg/kg of Cur（OA）₂-NPs dissolved in normal saline solution was intravenously injected with 1mL.A 200μL blood sample was obtained by eyeball extirpating with a heparinized centrifuge tube at 0.05,0.25,0.75,2,4,6,8,12 h after injection.The organs（liver,heart,spleen,lung,kidney and brain）were harvested for the measurment of Cur（OA）₂ and curcumin with HPLC method after extracting the drug by liquid-liquid extraction method,then analysis the tissue distribution and drug release in vivo.3.Monitor the targeting of nanoparticles by the in vivo NIR fluorescence imaging:DiR and Cur（OA）₂ were loaded into nanoparticles according to the method described in the preparation of Cur（OA）₂-loaded nanoparticles（by replacing Cur（OA）₂ with DiR and Cur（OA）₂）.Normal and H22 cancer model mice were injected intravenously.At different time intervals,the mice were anesthetized and scanned using an in-vivo imaging system with an excitation bandpass filter at 785 nm and an emission filter at 820 nm（LI-COR Odyssey Infrared Imaging System）.4.Tumor inhibition effect of the Cur（OA）₂-NPs in vivo:40 male KM mice were randomly divided into 4 groups and each of 10:A（control group,blank nanoparticles,0.2mL/animal,iv）,B（cyclophosphamide group,25mg/kg,ip）,C（low dose of nanoparticles,15mg/kg,ⅳ）and D（high dose of nanoparticles,300mg/kg,ⅳ）,and was administered intravenously once a day during the treatment cycle.During the program,mice weight was measured by every two days and tumor growing was observed.Results:The average particle size and encapsulation efficiency of nanoparticles was（93.39±1.71）nm,（92.32±3.13）%,respectively.The record from TEM suggested that nanoparticles were spherical and the particle size（below 100nm）was similar to the data determined by dynamic light scattering.After intravenous injection,the Cur（OA）₂ in the plasma reduced from 310.33μg·mL^-1 to 28.94μg·mL^-1 within 4h,and 90%of Cur（OA）₂ was cleared from blood vessel rapidly,indicating that the nanoparticles have distributed in mice.The results in the tissue distribution show that Cur（OA）₂ was found in spleen,heart,lung and kidney but not brain,Cur（OA）₂ reached the highest level of 368.93μg·g^-1.Observed from curcumin release in vivo,curcumin was only detected in the liver,spleen and kidney,the level was 125.72μg·g^-1,33.60μg·g^-1,16.81μg·g^-1 respectively,but not detected in plasma,lung and brain.In genaral,Cur（OA）₂-NPs distributed and released mainly in liver,spleen and kidney,the peak value was found at the 2th hour after intravenous injection.We found that Cur（OA）₂ in the liver was greater than the other organs significantly,and the level of Cur（OA）₂-NPs was expected to exhibit the anti-tumor activity.In Vivo NIR optical imaging provided evidence that Cur（OA）₂-NPs were accumulated in the liver and tumor,the NIR signal reached the climax at the second hour and descreased slowly as time went by,and we observed a strong NIR signal in the tumor mass at the 8th hour after the administration of nanoparticles.The signal intensity reached the climax in the tumor mass at the 12th hour and decreased follow,and it was stronger than liver significantly.Therefore,the Cur（OA）₂-NPs was demonstrated to target the tumor mass.Tumor inhibition effect of the Cur（OA）₂-NPs in vivo revealed furtherly that the anticancer effect of curcumin was maintained when modified by oleic acid and incorporated into mPEG5000-PLGA nanoparticles.As shown in the liver cancer model,tumor inhibition ratio of high dose was reached 48.98%,and was equivalent to 45.33%of CTX group.Thus,Cur（OA）₂-NPs was worth to develop and apply.Conclusion:This resrarch on issue distribution and pharmacodynamics in vivo completed the evaluation of Cur（OA）₂-NPs in vivo.Then study on the tissue distribution and drug release in vivo displayed that the Cur（OA）₂-NPs resolved the problem on the curcumin’s Iow oral bioavailability and rapid tissue metabolism,and the concentration of curcumin was the highest in the research reports nowadays and reached 125.72μg·g^-1,as the expected tissue concentration on playing the efficacy.Therefore,the Cur（OA）₂,NPs were suitable probablly to treat for the liver-disorders Iik liver cancer and hepatic fibrosis.In addtion,this study has laid the foundation for research on molecular mechanisms of anti-tumor of Cur（OA）₂-NPs in future.Meanwhile,It can offer some clue for developing a new drug which is insoluble,easily metabolic or toxic on the human body.