Non-viral delivery of plasmid DNA has traditionally relied upon physical forces applied directly to target tissues. These physical methods typically involve contact between an applicator and the target tissue and often cause transient patient discomfort. To overcome the contact-dependent limitations of such delivery methodologies, an atmospheric direct current plasma source was developed to deposit ionized gas molecules onto localized treatment sites. The deposition of charged species onto a treatment site can lead to the establishment of an electric field with strengths similar to those used for traditional electroporation. In vitro experiments proved that this technology could transiently permeabilize cell membranes and that membrane restabilization followed first order kinetics. Optimum delivery of tracer molecules to cell suspensions occurred after 10 minutes of plasma exposure and was attained without adversely effecting cell viability. In vivo testing of the plasma discharge demonstrated the capability of this system to deliver plasmid DNA to murine skin. Initial experiments involved the injection of plasmid DNA encoding luciferase into the dermis of C57BL/6J mice and then exposing the tissue to plasma discharge for 10 mintues. Delivery by this method resulted in increased luminescence that was as much as 19-fold greater than DNA injection alone. Follow-up optimization experiments demonstrated it was possible to obtain luminescence results that were similar in magnitude to those obtained using electroporation, which under optimum conditions resulted in about a 40-fold increase in peak luminescence. Finally, optimum conditions were used to deliver a plasmid DNA encoding for the 120 kilodalton glycoprotein present on the surface of a macrophage tropic HIV. Results from this vaccination experiment indicated this method was capable of producing antigen specific humoral immune responses at similar levels as when electroporation was utilized as the delivery method.

Delivery of biologically relevant ions such as drugs, neurotransmitters and hormones have been recognized as powerful a tool to control physiology of animals and plants for research purposes and practical applications. In the plant research community, ions are most commonly delivered as part of a solvent by soaking, spraying, pipetting or by adding to the soil. These methods have low control of the delivery dynamics and quantity of ion uptake. These issues motivated the development of the Organic Electronic Ion Pump (OEIP), which delivers only ions of interest by applying an external electric field through a polyelectrolyte membrane of high fixed charge concentration. A miniaturized, implantable version of the OEIP based on capillary fibres (c-OEIP), where the polyelectrolyte is enclosed in a capillary, enabled even higher precision of the delivery. In this master thesis, c-OEIP has been applied in the tropical plant Kalanchoe Blossfeldiana, chosen due to its characteristic skill to gradually learn to save water: while maturing it shifts to night time photosynthesis and transpiration, called Constitutive Crassulacean Acid Metabolism. A better understanding of this metabolism and water saving ability could guide engineering of enhanced drought tolerance in crop plants, which is motivated by the increasing global warming. One of the biologically relevant ions that is potentially involved in this water-saving learning process is the malate ions. The aim of this thesis is to test the hypothesis that c-OEIP is able to deliver malate ions to cause a reduction in stomatal conductance and transpiration of intact leaves of Kalanchoe Blossfeldiana. To test this hypothesis, firstly, the capillary-based OEIP were fabricated using polyimide coated glass capillaries filled with AETMAC polyelectrolyte. The ability of these devices to deliver malic acid (MA) was verified by using current-voltage characterisation during loading and delivery of MA. Secondly, the setup for MA delivery with c-OEIP to intact kalanchoe leaf was developed, optimising the insertion method to minimize the wounding of the plant and increase assay reproducibility. Finally, the MA was delivered to intact kalanchoe leaves via c-OEIP, where the plant transpiration response was evaluated using standard gas exchange porometer and also novel infrared camera, as plant temperature can be correlated with plant transpiration status. The results indicate that c-OEIP can deliver MA and trigger reduction of transpiration of young kalanchoe leaves, supporting the hypothesis that malate ions act to reduce stomatal conductance, potentially conveying a feedback message from the mesophyll to the guard cells. / <p>Examensarbetet är utfört vid Institutionen för teknik och naturvetenskap (ITN) vid Tekniska fakulteten, Linköpings universitet</p>