Studies On The Targeted Hepatocyte Mitochondrial Nano Drug Delivery System

Preventing and curing the tumors is always a big obstacle in the field of life science research. According to the physiological environment of the tumor and the requirement of mitochondrial targeting drug delivery, two novel polymer materials N-glycyrrhetinic acid(GA)-polyethylene glycol(PEG)-chitosan derivatives(NGPC) and N-quaternary ammonium-chitosan derivatives(NQC) are designed and then we choose them as basic construction unit to construct multifunctional nanoparticles(MNPs) encapsulating brucine as a novel mitochondrial-targeted nanocarrier system for cancer therapy. In this system, PEG has a longer hydrophilic chain and can form a hydration layer around in nanoparticles, thus reduce the phagocytosis of macrophage in the blood circulation. At the same time, this hydrophilic barrier can shield the positive ion of quaternary ammonium, which reducing its toxicity in physiological environments. The GA of the hydration layer surface has the function of active targeting to liver parenchymal cells, when the MNPs endocytosis into endosomes/lysosomes by liver cells, due to the reduction of pH value of the microenvironment, hydration layer the surface of the nanoparticles with GA-PEG abscission and disappeared, bare the positively charged chitosan nanoparticles with quaternary ammonium cations, meanwhile the imine group produces the sponge effect for endo-lysosomal escape. As escaping from endo-lysosomes to cytoplasm, MNPs are able to accumulate at the mitochondria by electrostatic interaction for mitochondrial targeting, owing to the transmembrane electric potential of mitochondria, and release anticancer drugs for enhancing anti-proliferation and apoptosis of tumor cells.Based on our preliminary research, we prepared three kinds of blank chitosan nanoparticles(NGPC-NPs, NQC-NPs, MNPs) and drug loaded chitosan nanoparticles(Brucine/ NGPC-NPs, Brucine/NQC-NPs, Brucine/MNPs) by ionic crosslinking method, respectively. In vitro physicochemical properties including the morphology, particle size distribution, zeta potential, drug entrapment efficiency(EE), drug loading content(LC), in vitro release and proton sponge effect were investigated.The results showed that the optimal formulation of Brucine/MNPs with EE of 68.53 ± 2.42%, LC of 6.01 ± 0.27% and particle size of 185.3 ± 6.9nm, and remained charge conversion similar to bare MNPs. Brucine/MNPs is spherical under TEM observation, and the size is quite basic with the size determined by dynamic light scattering(DLC). There was no remarkable difference between in vitro release of Brucine/MNPs at pH 7.4 and pH 4.5. Compared with Bucine solution, brucine released from three kinds of drug loaded chitosan nanoparticles slowly, which has sustained release effect.To verify the targeting efficiency of Brucine/MNPs in vivo, the biodistributions of different brucine formulations were estimated in mice. The results showed that the Brucine/NGPC-NPs, Brucine/NQC-NPs and Brucine/MNPs were subjected to phagocytosis in the reticuloendothelial system(RES) after intravenous administration. However, compared with Brucine/NQC-NPs, the GA of Brucine/NGPC-NPs and Brucine/MNPs, via receptor-mediated endocytosis, contributed to increased brucine accumulation and prolonged residence time in the liver tissue. Meanwhile, we observed that Brucine/NGPC-NPs and Brucine/MNPs significantly targeted the liver, as exhibited by the high relative ratio(Re) and the targeting efficiency(Te) parameter.Subsequently, the influence of time, concentration, temperature and GA on cellular uptake and endocytosis pathway of Brucine/NGPC-NPs, Brucine/NQC-NPs and Brucine/MNPs were investigated in human hepatocarcinoma(HepG2) cells. The Brucine-CTS/NPs presented concentration- and time-dependent cellular uptake. Nevertheless, the uptake of Brucine/NGPC-NPs and Brucine/MNPs was significantly higher than that of Brucine/NQC-NPs and Brucine solution. In order to further study that whether Brucine/NGPC-NPs and Brucine/MNPs are combined via the surface of glycyrrhetinic acid ligand and hepatocyte surface glycyrrhetinic acid receptor way to improve the concentration of nano drug carriers in liver cells, and to explore its mechanism internalized by cells, we especially studied the effects of glycyrrhetinic acid and various endocytic inhibitors on cellular uptake. The results showed that transporter proteins are involved in the uptake processes of HepG2 cells and the enhanced uptake of Brucine/MNPs in tumor tissue was partially attributable to receptor-mediated endocytosis. The presence of chlorpromazine and sucrose, inhibitors of clathrin-mediated endocytosis, and sodium azide, an inhibitor of energy metabolism, significantly decreased the cellular uptake of Brucine/CTS-NPs compared with inhibitors of other endocytotic processes, implying that Brucine/CTS-NPs encountered the primary barrier, endolysosomes, during intracellular delivery. Besides, the intracellular delivery efficiency of C6-CTS/NPs on HepG2 cells was evaluated by using confocal laser scanning microscopy(CLSM), and the brucine content in the mitochondrial fraction of HepG2 cells was further quantified by HPLC for quantitatively assessing the role of MNPs in mitochondrial targeting. It was confirmed that MNPs played a prominent role in endo-lysosome escape and mitochondrial targeting in comparison with NGPC-NPs and NQC-NPs.Then we investigated the in vitro antitumor efficacy of four brucine formulations and evaluated the apoptosis mechanism preliminary. The results showed that Brucine-CTS/NPs had a strong capability of killing tumor cells, and Brucine/MNPs was stronger than that of other preparations, the total cell apoptosis rate increased, and the mitochondria appeared obvious swelling and deformation, accompanied by decreased mitochondrial membrane potential and release of cytochrome C, the cell cycle was arrested at G2 phase. It was suggested that the novel MNPs facilitated the cellular uptake,realized the efficient intracellular drug delivery, and thus enhanced the tumor cells apoptosis and cytotoxicity.To further verify the feasibility of Brucine/MNPs for cancer therapy in vivo, the antitumor efficacies of different formulations were estimated in murine Heps cell xenograft models. The tumor-bearing mice received an tail vein injection of various formulations of brucine once every other day at a brucine dose of 5 mg/kg. Tumor growth was inhibited to a large extent when the mice were treated with smart Brucine/MNPs, thus resulting in significantly prolonged animal survival time. Pathological sections revealed that after Brucine/MNPs treatment, tumor necrosis area is the largest, accounting for the total number of apoptotic cells increased. The Cleaved caspase-3 activation detection results showed that compared with other groups, the Brucine/MNPs group has abundant expression of Cleaved caspase-3 protein, thus activated a series of enzyme related apoptosis, resulting in more tumor cells apoptosis, play stronger antitumor effect.In the work reported here, we designed and evaluated a novel mitochondrial-targeted multifunctional nanoparticles(MNPs) based on chitosan derivatives according to the physiological environment of the tumor and the requirement of mitochondrial targeting drug delivery. The intelligent chitosan nanoparticles possess various functions such as stealth, hepatocyte targeting, multistage pH-response, lysosomal escape and mitochondrial targeting, which lead to targeted drug release after the progressively shedding of functional groups, thus realize the efficient intracellular delivery and mitochondrial localization, inhibit the growth of tumor, elevate the antitumor efficacy, and reduce the toxicity of anticancer drugs. It provides a safe and efficient nanocarrier platform for mitochondria targeting anticancer drug delivery.