| Popis: |
Extracellular vesicles (EVs) are naturally secreted by cells to the extracellular environment. EVs contain the genetic information of their donor cells and play important roles in intercellular communications. Because of their nanoscale-size and high biocompatibility, EVs would not trigger the immune response and could efficiently penetrate the physiological barriers. Therefore, EVs have a great potential as a drug carrier for therapeutic use. However, several major barriers of the mainstream technologies for producing therapeutic EVs have greatly limited the therapeutic use of EVs. First is the low production yield of EVs from the donor cells. Second is the low loading efficiency of therapeutic RNAs (large mRNA in particular). Asymmetric Cell Electroporation (ACE), which is designed for controllable and non-invasive cell transfection, could efficiently stimulate cells to secrete a large amount of EVs. Also, by delivering a specific type of DNA plasmid into the cell by ACE, the secreted EVs would contain a high copy of the mRNA of interest transcribed from the delivered plasmid. However, the transition of this ACE platform from good cell transfection to enhanced EV production has not been well understood because the mechanism of the ACE-induced EV production remains obscure today. In this thesis, we first clearly define several important stages of ACE by using a Si-based ACE chip with a well-arranged array of nanopores. Among these stages, the transfection stage and the EV production stage are the most important ones. The ‘Transfection stage’ is defined as the transient Transmembrane potential (TMP) is slightly higher than the threshold TMP for porating the cell membrane. At this stage, cells can be transfected well with minimal cell damage (high viability) and a high cellular protein expression. However, each cell can only secrete a very limited number of EVs. The ‘EV production stage’ is defined as the transient TMP is far beyond the threshold TMP for cell membrane poration. At this stage, each cell can secrete a large quantity of EVs encapsulating a high copy number of a mRNA transcribed from the delivered plasmid DNA. The cellular RNA profiling results reveal strong changes of both lysosome and reactive oxygen species (ROS) related genes after ACE, the two important mechanisms related to ACE-induced EV production. Lysosome exocytosis, which is closely correlated to the cell membrane damage and resealing during and after ACE, is another factor for the enhanced EV production. The asymmetric electric field induces asymmetric membrane damage. We defined cell membrane facing the channel as ‘bottom membrane’ and the rest of the membrane as ‘top membrane’. To stimulate the cell for EV production, ‘large pores’ (with a size larger than the radius of gyration of an 18-mer oligonucleotide) are formed on the ‘bottom membrane’ and might take up to an hour to reseal to a small size that propidium iodide (PI) can not penetrate. However, only a large number of ‘small pores’ (with a size smaller than the radius of gyration of an 18-mer oligonucleotide) are generated on the top membrane and take around 10 minutes to reseal to a size small enough that PI dye would not penetrate. The membrane damage triggers the Ca2+ activated lysosome exocytosis, which greatly consumes the lysosome and reduces the lysosome content inside the cell. Therefore, late endosomes or multivesicular bodies (MVB) would have less chances to be fused with lysosomes for degradation, and instead tend to be secreted out as EVs. ACE-induced mitochondria depolarization and heat shock are the two main reasons of ROS accumulation after ACE, that led to the enhanced EV production. ACE induced mitochondrial potential decrease lasted for hours and recovered gradually, which led to the ROS-release from mitochondria into cytosol. Joule heating during ACE led to an intense local temperature oscillation but the heat dissipated to the surroundings rapidly within seconds. However, the heat shock triggered long-term cellular response also contributed to the ROS accumulation. The ROS accumulation led to an increase of endosome formation which is essential for EV production.A major challenge of the current ACE platform is the DNA aggregation caused by an off-balance of counter ions induced by the extremely high electric field inside the submicron-sized channel, which significantly reduced the plasmid delivery efficiency and accordingly the loading efficiency of therapeutic RNAs into EVs. We found that ACE chips with a smaller pore size and a higher voltage would lead to more severe DNA aggregation. DNA aggregates were formed inside the submicron-sized channel and the large DNA aggregates could not be delivered into the cell easily. Even being forced into the cell cytosol by the electric field, the DNA aggregation was not functional for hours after ACE till some individual DNA molecules were dissociated from the aggregate by counter ions. We conducted s systematic study and proposed some ideas for resolving the DNA aggregation problem during ACE. |
