Effect Of Tanshinone ⅡA On Transplacental Transport Of Digoxin

Objective To address the problem of placental barrier in fetal treatment,the natural Chinese medicine component tanshinone ⅡA which contain inhibitory effect on P-gp transporter protein in placenta and P-gp substrate digoxin,were loaded into core-shell lipid-polymer nanoparticles(CSLP-NP)with poly(lactic-co-glycolic acid)(PLGA)contained digoxin,the shell is a phospholipid layer containing tanshinone ⅡA.CSLP-NP allow step-wise release of tanshinone ⅡA and digoxin.Tanshinone ⅡA was first released to inhibit placental ABC transporter proteins,and it can improve the transplacental amount of digoxin which was released from CSLP-NP after tanshinoneⅡA.Finally,it can improve fetal treatment and also reduce maternal adverse reactions.Methods1.Using Be Wo cells to construct an in vitro placental model,the effect and mechanism of tanshinone ⅡA on the expression of ABC transporter proteins in BeWo cells were observed by Rho123 celluar accumulation test and Be Wo tight cell monolayers transport test,ATP assay test,RT-PCR and Western-blotting method.2.Subsequently,the effect and mechanism of tanshinone ⅡA on the function of ABC transporter proteins was studied using both MCF-7/ADR and MDCKⅡ-MDR1 cells by Rho123 celluar accumulation test and MDCKⅡ-MDR1 tight cell monolayers transport test,ATP assay test,doxorubicin hydrochloride(DOX)uptake test,RT-PCR and Western-blotting method.3.Digoxin-loaded PLGA nanoparticles were prepared by a modified solvent displacement method.Statistical modeling and factorial design was applied to investigate the influence of process parameters on the following nanoparticle characteristics:particle size,polydispersity index,zeta potential,and drug encapsulation efficiency.4.Dual drug delivery CSLP-NP was prepared by film dispersion method.The effects of different concentrations of PLGA nanoparticles,different concentrations of lipid component,different content of drug and other process parameters on CSLP-NP characteristics was observed.5.The cytotoxicity of blank nanoparticles and drug-loaded nanoparticles was investigated by MTT assay.The effects of tanshinone ⅡA and nanoencapsulation on the transport of digoxin were investigated in Be Wo and MDCKⅡ-MDR1 tight cell monolayers.6.Establishing a human placental in vitro perfusion model to provide a less ethical approach to research drug transport across placenta barrier.Results1.The expression of the BCRP protein was higher in BeWo cells.Cell lines also expressed the MRP1 protein.The expression of P-gp and MRP2 was very low or non-detectable in all immunoblots.Tanshinone ⅡA could promote the celluar accumulation of Rho123,decrease the intracellular ATP depletion as well as the expression of MRP 1 and BCRP.BeWo cells were seeded at a density of 105cells/well onto polycarbonate Transwell? inserts(pore size 3.0 μm,1.12 cm2 growth area)coated with rat tail collagen.The formation of tight junctions for barrier integrity and the presence of a single monolayer on the filters were confirmed by measuring the transepitihelial electrical resistance(TEER).Only the Transwell? inserts with cell monolayers displaying TEER values of ≈60Ω cm2 were used in transport studies.2.Tanshinone ⅡA can inhibit the function and expression of P-gp transporter protein in both MDCKⅡ-MDR1 and MCF-7/ADR cell,it also has inhibitory effect on the expression of BCRP and MRP1 protein in MCF-7/ADR cells.3.The mean particle size of PLGA digoxin nanoparticles prepared by the optimized process parameters is about 120 nm,the Zeta potential is about-40 mV,and the entrapment efficiency is about 30%.Tansmission electron microscopy showed that it is spherical and has a smooth surface,and is a rigid nanostructure.Several factors affect the physical characteristic of CSLP-NP:methods applied to produce lipid vesicles(direct hydration,sonication,or extrusion),type and concentration of lipids,ionic strength of buffers used,surface charge of lipid vesicles,the content of tanshinone ⅡA and the concentration of PLGA nanoparticles,etc.The mean particle size of CSLP-NP prepared by the optimized process parameters is approximately 200 nm,the Zeta potential is approximately 30 mV,and it is spherical.Tansmission electron microscopy confirmed the expected structure,with lipid bilayers adhering to the surface of CSLP-NP and subsequently spreading,leading to a lipid shell formation around the nanoparticle surface.4.The encapsulation of phospholipid layer can further improve the cytotoxicity of PLGA nanoparticles,so CSLP-NP has good biocompatibility.Loading digoxin into PLGA decreases its cytotoxicity,and dual drug delivery CSLP-NP does not increase toxicity compared to digoxin.Tanshinone ⅡA reduces the efflux ratio for the permeation of digoxin across both Be Wo and MDCKⅡ-MDR1 cell monolayer.Loading digoxin into PLGA did not increase digoxin transport rate across both BeWo and MDCKⅡ-MDRlcell monolayers,but the transport rate of digoxin was greatly enhanced after PLGA nanoparticles were coated with anshinone ⅡA-loaded cationic phospholipid outer layer.5.The establishment of human placenta in vitro perfusion model can make the placenta for a certain period of time to maintain good activity.The model can be used for drug placental transport research.Conclusion Tanshinone ⅡA can inhibit the function and expression of P-gp,BCRP and MRP1 transporter proteins in placental cells.The efflux ratio for the permeation of digoxin across both BeWo and MDCKⅡ-MDR1 cell monolayer was decreased.The tanshinone ⅡA and digoxig loaed core-shell lipid nanoparticles can increase the transplacental transfer of digoxig in in vitro placental cell models.