Effect Of TPGS Sub - 2k / PLGA Nanoparticles On Multidrug Resistance In Lung Adenocarcinoma Cells And Its Mechanism

BackgroundD-a-tocopheryl polyethylene glycol succinate (Vitamin E TPGS, or simply TPGS) is a highly efficient emulsifiers, solubilizer, absorption promoter, not only can increase the rate of envelopment, but also can control drug release. TPGS that modified to the surface of nanoparticles can bring out steric hindrance which cat as the stabilizer of the nanoparticles. Also, the chain length of PEG2k in new TPGS (TPGS2k) is long enough to maintain efficient emulsification, facilitate the release of drug and ensure the nanoparticles to extend blood circulation times to reduce liver accumulation. It has also been found that, vitamin E TPGS with the lipophilic alkyl tail was often used as inhibitor in P-glycoprotein mediated multidrug resistance.Experimental purposesEsterification reaction between mPEG2k and a-TOS was employed to Synthesize TPGS2k which was used as the emulgator in TPGS2k/PLGA NPs and TPGS2k/PLGA/SN-38 NPs preparation. Nanoparticles were characterized for necessary physical and chemical characteristics, and explored its therapy effect on MDR cell lines. Nevertheless, intracellular accumulation, efflux inhibition and Oxygen consumption rate were tested to explore the mechanisms of MDR reversal.MethodsMaterial preparation:TPGS2k was synthesized referred to published methods and confirmed by FT-IR and 1HNMR. The synthesized TPGS2k was used as emulgator in nanoparticles preparation. SEM, DLS, XRD and DSC etc. were employed to explore physicochemical characterization. The loading efficiency, encapsulation efficiency and release index were researched by ultraviolet spectroscopy.Safety evaluation:WST-1 and AnnexinV-FITC/PI were used to confirm security of NPs and demonstrate the cell death were induced by necrosis or apoptosis.Mechanism study:To investigate uptake and efflux mechanisms, we also prepared fluorescent-labeled nanoparticles for tracing their intracellular distribution. They were coumarin-6 labeled nanoparticles (TPGS2k/PLGA/C6 NPs) for uptake mechanism research and R123 fluorescentlabeled nanoparticles (TPGS2k/PLGA/R123 NPs) for efflux mechanism study. Mitochondrial Ultrastructure Morphology and Respiration Rate of cells were confirmed by TEM and XF96 cell mito-stress test kit.Result1. The successful synthesis of TPGS2k was confirmed by FT-IR and 1H NMR spectra. According to SEM, the NPs emulsified by TPGS2k was global particles with smooth surface and an average diameter about 100 nm. XRD and DSC were employed to settle the existing condition of NPMs and model drug in the NPs. The EE% and LE% of SN-38 in TPGS2k/PLGA/SN-38 NPs were 83.6 and 7.85%, respectively. TPGS2k/PLGA/SN-38 NPs were stable at physiological condition, implying the nanoparticles may have lower toxicity and fewer side effects during anticancer treatment in vivo.2. Both TPGS2k and TPGS2k/PLGA NPs exhibit slight toxicity at higher concentration or for a longer incubation time. The results of AnnexinV-FITC/PI apoptosis assay were consistent with the viability detection experiments and further revealed that the tested cell viability decreased after the cells were treated with TPGS21/PLGA NPs, mainly owing to cells apoptosis.3. TPGS21/PLGA NPs prolonged intracellular residence time and enhanced the therapeutic effect of the anticancer drug that ws substrate of P-gp by more effective uptake and fainter efflux in MDR cells. Further study showd that TPGS2k/PLGA NPs could overcome Pgp mediated MDR maybe through interfering the generation of energy.ConclusionWe successfully synthesized TPGS2k and prepared TPGS2k/PLGA/SN-38 NPs. The nanoparticles had with higher encapsulation efficiency and favorable drug released characteristic. Further studies on the mechanism for the nanoparticles in increasing the death of MDR cells showed that TPGS2k/PLGA NPs might interfere with the microenvironment of P-gp efflux pump, particularly the structure and function of mitochondria.The results indicated that TPGS2k-emulsified PLGA NPs could become the functional NPMs to have the ability to overcome Pgp mediated MDR and improve anticancer effect of some chemotherapy drugs as P-gp substrates.