Construction Of Mitochondrial Targeting Drug Delivery System And Its Application In Tumor Treatment

Objective:In order to overcome the multidrug resistance,enhance the anti-tumor activity and reduce the systemic toxicity of DOX,pH-sensitive nanoparticles HA-hydra-DOX-TPP were set up to specifically deliver DOX to mitochondria of the tumor cells by modifying triphenylphosphine to doxorubicin(DOX-TPP),and GSH-sensitive non-viral vector was constructed to delivery DOX-TPP and siRNA.Methods:1.Synthesize mitochondrial targeting conjugate DOX-TPP,HA-hydra-DOX-TPP nanoparticles and HSTC non-viral vector.The structure of conjugates were identified by 1H-NMR,FTIR and LC-MASS.The particle sizes of them were measured by zetasizer,and the morphology was observed under transmission electron microscopy(TEM).2.The cytotoxicity of HA-hydra-DOX-TPP nanoparticles and HSTC/siTwist complexes were measured by MTT method.Intracellular distribution of DOX-TPP,HA-hydra-DOX-TPP and HSTC/siRNA complexes were observed by Confocal Laser Microscopy.The effect of HSTC/siRNA on cellular uptake and HA-hydra-DOX-TPP on mitochondrial membrane potential,reactive oxygen species(ROS)production,caspase 3 activity and apoptosis rate were measured by Flow Analysis Cytometry.3.The ability of compressing siRNA,the ratio of incubation,and the release of siRNA in different concentration of DTT were determined by agarose gel retardation assay.The RNAi efficiency of HSTC/siTwist complexes were determined by PCR assay.4.Nude mice bearing MCF-7/ADR breast tumor models were established.The accumulation of HA-hydra-DOX-TPP in tumor tissue was observed under Confocal Laser Microscopy.The expression of cytochrome C protein in tumor and DNA fragmentation of nucleus in late apoptotic cells were assayed by immunohistochemical staining.The cytotoxicity of heart,liver,spleen,lung,kidney and tumor were observed by hematoxylin and eosin(H&E)staining.Results:The results of 1H-NMR,FTIR and LC-MASS showed that mitochondrial targeting conjugate DOX-TPP,HA-hydra-DOX-TPP and HSTC were successfully synthesized.TPP-DOX had excellent ability to achieve mitochondrial targeting and improved cytotoxicity of DOX.The particle size of HA-hydra-DOX-TPP was 192nm and the zeta potential was-24mV,which were beneficial to the biodistribution in tumor by enhanced permeability and retention(EPR)effect.Drug release of HA-hydra-DOX-TPP showed pH-dependent manner and DOX-TPP released from HA-hydra-DOX-TPP aggregated in mitochondria.It also could enhance Caspase 3 activity,produce more ROS,induce more apoptosis,and increase the uptake of MCF-7/ADR cells.HA-hydra-DOX-TPP showed better anti-tumor effect and good security in vivo.siRNA could completely be retared when the mass ratio of HSTC and siRNA was 100:1.The particle size of HSTC/siRNA complexs was 80nm and positive zeta potential was 19mV.When HSTC/siRNA complexs exposing to high concentration of glutathione,siRNA was triggered to release.It also could achieve endosomal escape reliant on cationic excipient,release DOX-TPP targeting mitochondria,and release siRNA in cytoplasm.HSTC/siRNA complexs were also confirmed to increase drug accumulation and inhibit Twist mRNA expression in vitro.Conclusion:In this study,we successfully constructed pH-sensitive nanoparticle HA-hydra-DOX-TPP,which could deliver DOX to mitochondria,increase the accumulation of tumor tissue and enhance the antitumor activity of DOX.Meanwhile,non-viral gene vector HSTC,simultaneously delivered siTwist and mitochondrial targeting drugs,was constructed to effectively inhibit the expression of Twist mRNA.