Research On Preparation And Antitumor Activity Of Mitochondria Targeted Nanoparticles In Tumor Cells

ObjectiveDoxorubicin(Dox)is a commonly used broad-spectrum anticancer drug,but its application is severely limited due to its cardiotoxicity and high resistance rate.Dox in addition to the role of nuclear DNA,mitochondria is also its main target,As an important cell structure of eukaryotic cells,mitochondria play an important role in the process of apoptosis signal transduction,and mitochondria also contain functional DNA,which is very sensitive to anti-tumor drugs.Therefore,in order to reduce the damage of normal cells by anti-tumor drugs,it is very important to prepare a targeting vector that can effectively deliver Dox to the mitochondria of tumor cells.In this study,based on the high mitochondrial transmembrane potential of tumor cells,a triphosphoric lipophilic triphenyl phosphine(TPP)with a high mitochondrial binding specificity was attached to a good drug carrier chitosan nanoparticles(CS-NPs),The lung cancer A549 cells and cervical cancer Hela cells were used as tumor cell models to investigate the targeting effects of doxorubicin-triphenylphosphine chitosan nanoparticles(Dox-TPP-NPs)on tumor cell mitochondria,and its anti-tumor activity.MethodsDoxorubicin,chitosan,sodium polyphosphate and triphenyl phosphine were used as raw materials to prepare CS-NPs,TPP-NPs,Dox-CS-NPs and Dox-TPP-NPs by ion exchange method.The structures of the nanoparticleswere characterized by infrared spectroscopy.The structure of the nanoparticles was identified by infrared spectroscopy.First,The prepared Dox-TPP-NPs were performed by prescription optimization,quality evaluation and characterization analysis.A preliminary study was performed on its targeting.A549 cells and Hela cells were cultured in vitro.The uptake of free Dox and Dox-TPP-NPs by tumor cells and the presence of Dox in tumor cells were observed by confocal laser scanning microscopy.And the intracellular uptake of Dox was quantitatively analyzed.Finally,the anti-tumor activity of Dox-TPP-NPs was assayed.Free Dox and Dox-TPP-NPs were detected by MTT assay in vitro.Confocal laser scanning microscopy was used to observe the fluorescence intensity changes of JC-1 dyes.The changes of mitochondrial membrane potential after free Dox and Dox-TPP-NPs treatment were studied.The generation of reactive oxygen species(ROS)in cells was quantitatively analyzed.The regulation of mitochondrial apoptosis pathway-related proteins by Free Dox and Dox-TPP-NPs was investigated by Western Blot.ResultsAfter IR spectroscopy,we successfully connected the TPP to the surface of CS-NPs.In the characterization of the nanoparticles,TEM images showed that the morphology of Dox-TPP-NPs was spherical with an average diameter of100.1 ± 4.3 nm The dispersion index was 0.016,indicating good dispersibility of nanoparticles.The Zeta potential was positive with a size of 15.6 ± 2.3 m V.The encapsulation efficiency and drug loading of Dox-TPP-NPs were 91.3% and13.2%,respectively.In vitro release experiments showed that Dox sustained release from Dox-TPP-NPs,and as the pH value of the medium decreased,the cumulative drug release increased significantly,promoting the rapid release of the drug.A549 cells and Hela cells were cultured in vitro.Laser confocal microscopy showed that uptake of the nanoparticles by the tumor cells was basically completed within 6 hours.When co-incubated with Free Dox,Dox gradually accumulated in the nucleus of the tumor,co-incubated with Dox-TPP-NPs,Dox evenly distributed in the cytoplasm,with the help ofmitochondrial indicator that our nanocarriers effectively and specifically deliver Dox into the tumor cell mitochondria.The cytotoxicity of Free Dox and Dox-TPP-NPs on tumor cells was determined by MTT assay.Both of them could inhibit the growth of tumor cells in a concentration-dependent manner,and Dox-TPP-NPs could induce higher cytostatic effect.The change of fluorescence intensity of JC-1 dye was observed by laser confocal microscopy.The results showed that Dox-TPP-NPs could induce the targeted delivery of Dox to mitochondria and significantly enhance the mitochondrial damage of tumor cells by decreasing the mitochondrial membrane potential.Fluorescence quantitative analysis of ROS production in tumor cells mitochondria showed that there was a significant increase of ROS in cells treated with Free Dox and Dox-TPP-NPs.Compared with Free Dox,the Dox-TPP-NPs treated cells Produced more amounts of ROS,indicating that Dox-TPP-NPs are more cytotoxic.Finally,we examined the regulation of mitochondrial apoptotic pathway-related proteins by Free Dox and Dox-TPP-NPs through the Western Blot.The results showed that compared with Free Dox,more cytochrome C was released from the mitochondria to the cytoplasm of Dox-TPP-NPs treated with the same concentration of Dox,and apoptosis-related proteins Bax,Caspase-9 and Caspase-3 were significantly increased,indicating that Dox-TPP-NPs induce apoptosis of tumor cells through the mitochondrial pathway.ConclusionsIn this study,we successfully prepared a new type of tumor cell mitochondria-targeting nanoparticles.Dox-TPP-NPs is a spherical or similar spherical solid microspheres of a complete structure,uniformsize and stability,particle size distribution,positive surface charge,dissolution in vitro has a significant sustained-release effect,good stability.The targeting and antitumor activity of the NPs were studied by laser confocal microscopy,MTT and Western Blotting.The results show that nanoparticles have mitochondrial targeting,can specifically deliver doxorubicin to the mitochondria of tumor cells,increase its accumulation in tumor cells mitochondria,and interfere with the mitochondrialrespiratory chain,resulting in high cytotoxicity,inducing significant apoptosis effect in the mitochondrial pathway,enhancing the antitumor activity of Dox,Reversible tumor resistance,there is value for further research.