Study On Cell Membrane Transport And Intracellular Delivery Of RNA Nano-drugs

RNA nanotechnology is developing into a promising platform for therapeutical application,which has certainly sparked interest for applications in nanomedicine.Multifunctional RNA nanoparticles（RNPs）designed and constructed based on RNA nanotechnology have controllable shapes,sizes,therapeutic effects and drug delivery systems,showing great application prospects in nanomedicine.This thesis will contribute to understand the rapid dynamic transmembrane process of RNPs,which is conducive to our further understanding of RNA nano-drugs and provide theoretical guidance for the development,optimization and clinical translation of multifunctional RNA nanodrugs and m RNA vaccines.In this thesis Antimir21-RNP-Apt was selected as a model to study the process of RNPs cell membrane transport and intracellular delivery.The dynamic parameters of Antimir21-RNP-Apt and Antimir21-RNP-MUT entry different cell lines were evaluated using high spatio-temporal resolution force tracing technique based on atomic force microscopy（AFM）at single-particle level.Furthermore,the intracellular delivery of Antimir21-RNP-Apt was tracked through the real-time fluorescence tracking based on spinning-disk confocal microscopy with high resolution.The main research contents of this thesis are as follows:1.Using force tracing technique with high temporal and spatial resolution,monitored the transmembrane dynamic mechanism of single targeted RNA nano-drug Antimir21-RNP-Apt entry triple-negative breast cancer cell lines（MDA-MB-231 cells with EGFR overexpression on cell membrane）under physiological conditions.Measured the cell membrane transport parameters such as force,duration,displacement,average velocity,and endocytosis efficiency required by Antimir21-RNP-APT.The Antimir21-RNP-Apt transmembrane force ranged from 22 to 77 p N,with a mean value of 46±12 p N,the duration ranged from 27.1 to 189.5 ms with a mean value of 81.9±39.8 ms,the mean displacement was 26.0±1.6 nm,the average velocity was 0.40±0.19μm·s^-1,and the probability of observed force signal was 18.3%.2.Studied transmembrane transport parameters of Antimir21-RNP-MUT（the mutated sequence MUT replaces the aptamer sequence targeting EGFR,with no target specificity）entry MDA-MB-231 cells（cell membrane EGFR overexpression）and Antimir21-RNP-Apt entry normal human kidney cell line（HEK-293 cells,barely EGFR expressed on the cell membrane）.We found that the duration of Antimir21-RNP-MUT entry into MDA-MB-231cells was shortened to 69.5±47.2 ms,and the endocytic force was almost unchanged,the duration of Antimir21-RNP-Apt entry HEK-293 cells was shortened to 50.8±20.8 ms,and there was no significant difference in endocytic force.The results showed that the specific interaction of EGFR aptamer（Apt）carried by Antimir21-RNP-Apt with the overexpressed EGFR on the surface of the cell membrane could prolong the time for Antimir21-RNP-Apt entry cell.By combining the force tracing technique with confocal fluorescence imaging,different specific inhibitors were used to block the cell endocytosis pathway mediated by different transmembrane proteins respectively to study the transmembrane transport mechanism of Antimir21-RNP-Apt entry MDA-MB-231 cells,the results found that antimir21-RNP-Apt entry cells mainly through clathrin-mediated endocytic pathway.3.The dynamic transport process of Antimir21-RNP-Apt in MDA-MB-231 cells was investigated using single particle tracking technique based on spinning-disk confocal microscopy.Different organelles were labeled with different fluorescence molecules respectively,the intracellular delivery pathway of Cy3-labeled Antimir21-RNP-Apt was analyzed by confocal fluorescence imaging.As the incubation time increases,Antimir21-RNP-Apt colocalizes with different organelles,such a time-evolution reflects the sequence of intracellular traffic of Antimir21-RNP-Apt from early endosomes to late endosomes and eventually in lysosomes.The dynamic analysis indicates the low motility of Antimir21-RNP-Apt in early endosomes and the high mobility in late endosomes.