Study On The Drug Delivery Of L-carnitine-conjugated Nanoparticles Via Targeting Transporter

Oral administration is the most preferred route for therapeutic drugs for most patients.But this route has proven not to be efficacious for all drugs due to the poor oral bioavailability.The application of nanotechnology could improve the druggability of these pharmacological agents for oral administration.Furthermore,nanoparticles could effectively improve the absorption after functionalized with ligands.OCTN2?organic cation/carnitine transporter 2?,mainly expressed at intestine,is responsible for the absorption of L-carnitine from the dietary and the transport of some organic cations.OCTN2 can be used as an ideal target for oral drug delivery systems.In our preliminary study,L-carnitine was covalently linked with stearic acid to make stearyl-L-carnitine,which was used to modify poly?lactic-co-glycolic acid??PLGA?nanoparticles to make L-carnitine-conjugated nanoparticles.The prepared L-carnitine-conjugated nanoparticles could effectively improve the oral bioavailability of loaded drug.Based on the preliminary study,using paclitaxel as model drug,a series of L-carnitine-conjugated nanoparticles with different ligand density?5%LC-PLGA NPs,10%LC-PLGA NPs,20%LC-PLGA NPs,40%LC-PLGA NPs?were prepared by emulsion-solvent evaporation method and characterized on shape,size distribution,zeta potential,encapsulated efficiency,drug load,in vitro drug release,stability assay,et al.The uptake of L-carnitine-conjugated nanoparticles in Caco-2 cells was conducted.Of them,10%LC-PLGA NPs got the highest uptake efficiency,indicating that there is an optimal ligand density on the surface of the NPs that is needed for maximal uptake.The uptake mechanism of L-carnitine-conjugated nanoparticles were conducted.The results showed that the uptake and binding process of L-carnitine-conjugated nanoparticles was Na+-dependent,inhibitable by excessive free L-carnitine,and multiple endocytosis pathways involved,suggesting that the uptake mechanism was OCTN2-dependent endocytosis.The Na+ dependence of L-carnitine-conjugated nanoparticles in uptake process was further investigated by molecular dynamic simulation.The results showed that the binding energy was decreased in the presence of Na+,indicating that Na+ played a key role in the uptake of L-carnitine-conjugated nanoparticles.In addition,it was shown that a portion of OCTN2 proteins was degraded in the uptake process of L-carnitine-conjugated nanoparticles.However,this effect would not change the normal function of cells,because the proteins could be reproduced in the following hours.The uptake mechanism of L-camitine-conjugated nanoparticles in tissue level was further investigated.L-carnitine conjugation could increase the uptake of nanoparticle in intestine,and the uptake process was Na+-dependent,inhibitable by free L-carnitine,and multiple endocytosis pathways involved,indicating OCTN2-dependent endocytosis was responsible for the uptake of L-carnitine-conjugated nanoparticles in tissue level.In vivo pharmacokinetics of L-camitine-conjugated nanoparticles with various ligand density in rats was conducted.The results were consistent with those in cellular level.10%LC-PLGA NPs achieved the highest oral bioavailability?3.22-fold increased compared to PLGA NPs?,and the oral absorption could be inhibited by free L-carnitine,indicating that OCTN2-mediated absorption contributed to the increased oral bioavailability of L-carnitine-conjugated nanoparticles.The mesenteric lymph duct ligation rat model was used to investigate the contribution of lymphatic transport in oral absorption of L-carnitine-conjugated nanoparticles.The results showed that the oral bioavailability of 10%LC-PLGA NPs was decreased 85.1%,and that of unmodified PLGA NPs was decreased 37.2%in mesenteric lymph duct ligation rats.Based on the results,it was suggested that compared to unmodified PLGA NPs,L-carnitine-conjugated nanoparticles could keep intact after absorption by enterocyte and enter into systemic circulation via lymphatic pathway.L-carnitine can be transported by OCTN2 and ATB^0,+ with different affinity.By uptake assay under different situation and colocalization assay,it was suggested that L-carnitine-conjugated nanoparticles dual-target to both transporters.ATB^0,+ is overexpressed in colon cancer,and we found that OCTN2 is also highly expressed in colon cancer cells.Considering that L-carnitine-conjugated nanoparticles could dual-target to both OCTN2 and ATB^0,+,normal colon cells and colon cancer cells were used to study the potential of L-carnitine-conjugated nanoparticles for targeted drug delivery to colon cancer.The results showed that L-carnitine-conjugation significantly increased the uptake of NPs in colon cancer cells,and enhanced the cytotoxicity against colon cancer cells,and improve the inhibition of nanoparticles to 3D tumor spheroids.