Construction And Absorption Mechanism Of Oral Curcumin Nanomicelles Targeting Transporter Proteins

Oral administration is the most widely used method because of its convenience,rapidity,and strong patient adaptability.However,many fat-soluble drugs,due to their low solubility,poor stability and low transmembrane transport efficiency,which seriously affect the oral absorption and reducing the efficacy of drugs.Nanoparticles can effectively improve the solubility and stability of drugs,increase the transmembrane transport efficiency of drugs,and then increase the oral bioavailability of drugs.However,transmembrane transport efficiency of nanoparticles is still limited.Currently,transporter mediated nanometer preparations can further improve drug transmembrane transport efficiency by targeting drugs to transporters.Intestinal epithelial cell oligopeptide transporter 1(PepTl)is an uptake transporter that promotes the body’s absorption of drugs.P-glycoprotein(P-glycoprotein,P-gp)is an efflux transporter that reduces the body’s absorption of drugs.Therefore,in order to further improve the transmembrane transport efficiency of micelles and better increase the oral absorption of drugs dual functional micelles with PepTl targeting and P-gp inhibitory action were constructed and studied.This transporter mediated micelles,targeting PepTl and inhibiting P-gp,could further increase the transmembrane transfer efficiency of the nanomicelles,thereby improving the oral bioavailability and clinical efficacy of the drugs.First,the amphipathic carrier materials of Val-TPGS and Phe-TPGS were synthesized with polyethylene glycol vitamin E succinate(TPGS)and benzyloxyproline(Cbz-valine)or benzyloxyphenyl phenylalanine(Cbz-phenylalanine)after esterification reaction and palladium carbon catalytic hydrogenation reaction.The structure of the newly synthesized polymers was determined by 1H-NMR,13C-NMR and IR.The critical micelle concentration(CMC)of the polymer was measured using a fluorene fluorescent probe method.The CMC values of TPGS,Val-TPGS,and Phe-TPGS were 15.01,8.55,and 9.55μg·mL-1.Val-TPGS and Phe-TPGS with lower CMC could exert better dilution stability in comparison withcommercially available TPGS.The curcumin loaded micelles(ie,TP-PMs,Val-PMs and Phe-PMs)were prepared with curcumin as a model drug,commercially available TPGS and self-synthesized Val-TPGS,Phe-TPGS as nanomaterials.Based on the particle size and entrapment efficiency,the preparation process and formulation of nanomicelles were optimized and screened,final determination of preparation nanomicelles by membrane hydration,and the nanomicelles properties were investigated.The results showed that the three drug-loaded nanomicelles were small(15 nm)in size and spherical in shape;the Zeta potential of TP-PMs was-5 mV,but due to the surface modification of amino acids,the surface of Val-PMs and Phe-PMs was positively charged(potential is about 5 mV);differential scanning calorimetry(DSC)measurement results show that the drug exists in the nanomicelles in an amorphous form;three drug-loaded micelles stored at room temperature and 4℃ for 15 days,the nanomicelles was no significant change in the particle size and content,with good storage stability.In vitro gastrointestinal fluid release results showed that cumulative release of TP-PMs,Val-PMs,and Phe-PMs for 24 h was 52%,42%,and 41%,it shows that the three micelles release was slowly and have good gastrointestinal stability.Using Caco-2 cells as a model,cell uptake,uptake mechanism,and cell trafficking of drug-loaded nanomicelles were investigated in vitro cell level.First,the cytotoxicity results show that the concentration of curcumin loaded micelles of 20 μg·mL-1 and cells incubated for 2 h had no toxic or side effects on cells,it could be used for subsequent uptake studies.RT-PCR results showed that after one week of induction of Caco-2 cells with leptin,the expression of PepTl in the cells was 3.5 times that of normal Caco-2 cells,and the cells with high expression of PepTl were successfully induced.After the co-incubation of TPGS,Val-TPGS and Phe-TPGS with high expression of PepTl Caco-2,the expression of PepTl in TPGS group did not change significantly,but the expression of PepTl in Val-TPGS and Phe-TPGS groups decreased significantly(P<0.01),the PepTl targeting of self-synthesized materials Val-TPGS and Phe-TPGS was demonstrated.Cell uptake results showed that the cell uptake of the Cur-Sol and drug-loaded micelles was time-dependent and concentration-dependent,drug-loaded micelles significantly increased the uptake of the drug by the cells(P<0.01),in Caco-2 cells with high expression of PepTl,the uptake of Val-PMs and Phe-PMs was 1.34-5.05 times higher than TP-PMs(P<0.05).Gly-Sar significantly inhibited the uptake of Val-PMs and Phe-PMs in PepTl high-expressing cells,confirming that Val-PMs and Phe-PMs mediate increased cellular uptake of micelles through PepTl-mediated.At the same time,laser scanning confocal microscope results show that the fluorescence intensity of C6-Val and C6-Phe in PepTl high expression cells is stronger than C6-TP,the results of qualitatively proves that Val-PMs and Phe-PMs can further promote the uptake of nanomicelles by transporters of Caco-2 cells.In addition,the uptake of drug-loaded micelles into cells is via energy-dependent,endocytic pathways.The transport results showed that the apparent permeability coefficients(Papp)of Val-PMs and Phe-PMs were 1.29 and 1.22 times higher than TP-PMs,indicating that the targeted micelles of PepTl could be significantly increased(P<0.05)transmembrane transport efficiency.At the same time,the distribution of different micelles in different intestinal tissues of rats was qualitatively and quantitatively investigated from the in vivo level by rats.The results showed that the contents of Val-PMs,Phe-PMs and TP-PMs in the duodenum,jejunum and ileum of rats were significantly higher than Cur-Sol(P<0.01),confirming that the nanomicelles can increase the drug intestinal absorption;the intestinal absorption of the three drug-loaded micelles was different,and the absorption of Val-PMs and Phe-PMs was 1.34 and 1.54 times of TP-PMs in the duodenum;2.48 and 2.63 times of TP-PMs in the jejunum;3.24 and 3.41 times of TP-PMs in the ileum,was significantly increased(P<0.05).The qualitative results further confirmed that the fluorescence intensity of C6-Val and C6-Phe in different intestinal tissues was significantly stronger than C6-TP,demonstrating that PepTl targeted micelles can further promote the absorption of micelles into small intestine of rats.The pharmacokinetic study in rats further investigated the oral absorption of PepTl targeted micelles.The results showed that the AUC0-t of TP-PMs,Val-PMs,and Phe-PMs were 3,9,and 10 times of Cur-Sol,and t1/2 were 2.24,4.02,and 3,15 times of Cur-Sol.The AUC0-t of Val-PMs and Phe-PMs was 3.41 and 3.57 times t of TP-PMs,t1/2 were 1.79 and 1.40 times of TP-PMs,indicating that PepTl targeted micelles of Val-PMs and Phe-PMs can significantly increase the oral absorption compared with TP-PMs,so PepTl as a target can significantly increase the oral absorption of micelles.