Design And Evaluation Of Targeting Nanoparticulate Drug Delivery Systems Based On Amino Acid Transporter LAT1

More and more attentions are focused on developing new targets for tumor treatment because the types of receptors expressed in tumor cells are limited and the ligands are poorly stable in vivo. In comparison, simple amino acids modifications are enough to satisfy amino acid transporters targeting, and the amino acids have no steric hindrance and good stability in vivo. Therefore, large amino acid transporter 1 highly expressed on tumors is selected as the target. Then glutamate modified PLGA nanoparticles were developed for LAT1 targeting. The LAT1 targeting efficiency, the roles of LAT1 in nanoparticles binding and endocytosis, and in vivo anticancer effects were investigated.Glutamate was conjugated to polyoxyethylene stearate via the reaction between hydroxyl group of polyoxyethylene stearate and carboxyl group of glutamate. The chemical structures of the synthesized targeting copolymers (glutamate-polyoxy-ethylene stearate, SPGn) were characterized by H-NMR. The molecular and molecular distribution of SPGn were investigated. Results showed that glutamate was successfully conjugated to polyoxyethylene stearate with different PEG chains. Besides, the targeting copolymers had a narrow molecular distribution, indicating that the copolymers had a high purity.Paclitaxel (PTX) was encapsulated into LAT1 targeting glutamate-polyoxy-ethylene stearate modified PLGA nanoparticles by emulsion solvent evaporation method. The LAT1 targeting nanoparticles were modified with different intensities of SPGn, named as 5% PTX SPGn NPs and 10% PTX SPGn NPs. The size of PTX SPGn NPs ranged from 152 nm to 181 nm with a narrow size distribution. TEM results showed that nanoparticles were spherical. The release profiles of PTX SPGn NPs were investigated by dialysis in pH 7.4 PBS. PTX SPGn NPs demonstrated sustained release profiles of PTX. With an increase in the PEG chain length, the drug release was reduced. The in vitro colloidal stability of different PTX SPGn NPs was evaluated in pH 7.4 PBS containing 10% FBS at 37℃. The changes in particle size were determined by DLS at predetermined time points. With an increase in PEG chain length, the physical stability of PTX SPGn NPs was increased, indicating that a longer PEG chain length produced better steric protection to a harsh environment.The expression of LAT1 in MCF-7, HeLa and NIH 3T3 cells was characterized by immunofluorescence analysis. Results showed that LAT1 was overexpressed in MCF-7 and HeLa cells, and was lowexpressed in NIH 3T3 cells. The cytotoxicity, LAT1 targeting efficiency, subcellular distribution and endocytosis mechanisms of glutamate modified nanoparticles and unmodified nanoparticles were investigated. The results of cytotoxicity indicated that glutamate modification could improve the inhibition rate of nanoparticles on MCF-7 and HeLa cells. The cellular internalization of glutamate-modified NPs was significantly higher than that of unmodified NPs. Finally,10% SPG25 NPs were selected as the optimum LAT1 targeting nanoparticles. Subcellular distribution found that the targeting NPs could escape from endosomal/ly-sosomal compartments. The main internalization pathway for 10% SPG25 NPs was energy-dependent clathrin-mediated endocytosis. It was found that 10% SPG25 NPs could bind with LAT1 both at 4℃ and at 37℃, and the process was inhibited by LAT1 substrates.The effects of 10% SPG25 NPs on LAT1 were studied by immunofluorescence and western blot analysis. The results indicated that LAT1 was internalized simultaneously with 10% SPG25NPS into the cytoplasm, leading to a reduced LAT1 protein level on the cell membrane at 1 h. The recycling of LAT1 to the cell membrane from the cytoplasm contributed to the increased LAT1 level on the cell membrane.To investigate the LAT1 targeting ability of 10% SPG25 NPs in tumor-bearing mice, the in vivo distribution of the NPs was observed using an FX Pro in vivo imaging system.10% SPG25 NPs reduced the liver and spleen phagocytosis and enhanced the tumor accumulation. From the in vivo antitumor effects, groups treated with Taxol and PTX-loaded NPs showed apparent antitumor effects compared with the saline group. And the 10% SPG25 PTX NPs-treated group exhibited a better tumor inhibition.The therapeutic outcome of glioma treatment is rigorously limited by blood-brain barrier (BBB) and infiltrating growth of glioma. To tackle the dilemma, more and more attentions were focused on developing nutrient transporters-mediated dual-targeted drug delivery system, in one side for BBB penetration, another for intracranial glioma targeting. Herein, Large amino acid transporter 1 (LAT1), overexpressed both on BBB and glioma cells, was selected as a target. Docetaxel-loaded glutamate-D-a-tocopherol polyethylene glycol 1000 succinate copolymer (Glu-TPGS) functionalized LAT1-targeting liposomes (DTX-TGL) were applied to enhance the BBB penetration and glioma therapy. The in vivo results of the fluorescent image indicated that TGL possessed an effective BBB penetration than that of unmodified ones in mice. Compared with the unmodified liposomes, a significant higher cellular uptake and cell cytotoxicity was found in TGL treated group. Our results indicated that LAT1-targeting docetaxel-loaded liposome paves up a new direction using LAT1 transporter as a good target in designing brain glioma-targeting nanosystems.