Browsing by Subject "Electroporation"

Electroporation is a biological cell's natural reaction to strong electric fields, where transient pores are created in the cell membrane. While electroporation holds promise of being a safe and effective tool for enhancing molecular delivery in numerous medical applications, it remains largely confined to preclinical research and clinical trials due to an incomplete understanding of the exact mechanisms involved. Muscle fibers are an important delivery target, but traditional theoretical studies of electroporation ignore the individual fiber geometry, making it impossible to study the unique transverse and longitudinal effects from the pulse stimulus. In these long, thin muscle fibers, the total reaction of the fiber to the electric field is due to fundamentally different effects from the constituent longitudinal and transverse components of the electric field generated by the pulse stimulus. While effects from the transverse component have been studied to some degree, the effects from the longitudinal component have not been considered.

This study develops a model of electroporation and delivery of small molecules in muscle tissue that includes effects from both the transverse and longitudinal components of the electric field. First, an asymptotic model of electric potential in an individual muscle fiber is derived that separates the full 3D boundary value problem into transverse and a longitudinal problems. The transverse and longitudinal problems each have their own respective source functions: the new "transverse activating function" and the well known longitudinal activating function (AF). This separation enhances analysis of the different effects from these two AFs and drastically reduces computational intensity. Electroporation is added to the asymptotic fiber model, and simplified two-compartment mass transport equations are derived from the full 3D conservation of mass equations to allow simulation of molecular uptake due to diffusion and the electric field. Special emphasis is placed on choosing model geometry, electrical, and pulsing parameters that are in accordance with experiments that study electroporation-mediated delivery of small molecules in the skeletal muscle of small mammals.

Simulations reveal that for fibers close to the electrodes the transverse AF dominates, but for fibers far from the electrodes the longitudinal AF enhances uptake by as much as 2000%. However, on the macroscopic tissue level, the increase in uptake from the longitudinal AF is no more than 10%, given that fibers far from the electrodes contribute so little to the total uptake in the tissue. The mechanism underlying the smaller effect from the longitudinal AF is found to be unique to the process of electroporation itself. Electroporation occurs on the short time scale of polarization via the transverse AF, drastically increases membrane conductance, and effectively precludes further creation of pores from charging of the membrane via the longitudinal AF. The exact value of enhancement in uptake from the longitudinal AF is shown to depend on pulsing, membrane, and tissue parameters. Finally, simulation results reproduce qualitative, and in some cases quantitative, behavior of uptake observed in experiments.

Overall, percent increase in total tissue uptake from the longitudinal AF is on the order of experimental variability, and this study corroborates previous theoretical models that neglect the effects from the longitudinal AF. However, previous models neglect the longitudinal AF without explanation, while the asymptotic fiber model is able to detail the mechanisms involved. Mechanisms revealed by the model offer insight into interpreting experimental results and increasing efficiency of delivery protocols. The model also rigorously derives a new transverse AF based on individual fiber geometry, which affects the spatial distribution of uptake in tissue differently than predicting uptake based on the magnitude of the electric field, as used in many published models. Results of this study are strictly valid for transport of small molecules through small non-growing pores. For gene therapy applications the model must be extended to transport of large DNA molecules through large pores, which may alter the importance of the longitudinal AF. In broader terms, the asymptotic model also provides a new, computationally efficient tool that may be used in studying the effect of transverse and longitudinal components of the field for other types of membrane dynamics in muscle and nerves.

Successful transfection of genetically active materials is essential to gene delivery and genome editing. Electrotransfection, also known as electroporation, is a fast, safe, and convenient non-viral method for introducing materials such as proteins and nucleic acids into cells and tissues. It has been widely used in academic research, industrial manufacturing, and clinical therapeutics. Particularly, electrotransfection is one of the most commonly used method in gene delivery into mammalian cells. However, despite its many advantages comparing to other gene delivery methods, the application of electrotransfection is limited by inconsistent transfection efficiency, which is caused by the poor understanding of the mechanism of electrotransfection.

The goal of my research is to understand the fundamental biological mechanisms of electrotransfection and to develop novel strategies that can improve the transfection efficiency of gene delivery and genome editing. To this end, this study is divided into two phases. Phase 1 aims at understanding the key cellular components involved in the transport process. Phase 2 focuses on the development of strategies to enhance electrotransfection by controlling the biological pathways that are involved in electrotransfection.

In the first phase of my study, we investigated the dependence of electrotransfection efficiency on endocytosis. Data from this study demonstrated that macropinocytosis is involved in electrotransfection. The results revealed that electric pulses induced cell membrane ruffling and actin cytoskeleton remodeling. Using fluorescently labeled pDNA and a macropinocytosis marker (i.e., dextran), the study showed that electrotransfected pDNA co-localized with dextran in intracellular vesicles formed from macropinocytosis. Furthermore, electrotransfection efficiency was reduced significantly by lowering temperature or treatment of cells with a pharmacological inhibitor of Rac1 and could be altered by changing Rac1 activity. Taken together, the findings suggested that electrotransfection of pDNA involved Rac1-dependent macropinocytosis.

Second phase of this study focuses on the intracellular transport of plasmid DNA, especially the transport of DNA molecules towards degradative compartments. Our data elucidated that components in both endocytic and autophagic pathways are responsible for intracellular trafficking and processing of transfected materials such as pDNA. In addition, we also characterized a new type of vesicle named amphisome-like vesicle (ALB) and revealed its involvement in electrotransfection. Based on these findings, we propose a novel strategy to enhance electrotransfection by blocking degradative routes within the endocytic pathways, which led to the development of a new technique called transfection by redirection of endocytic and autophagic traffic (TREAT). Transfection of plasmid DNA (pDNA), messenger RNA (mRNA), sleeping beauty transposon system (SB), and different forms of CRISPR/Cas9 system by TREAT achieved superior efficiency in various cell lines including difficult-to-transfect human primary cells. In addition, we successfully applied TREAT method to improve clinically relevant applications including SB-based gene integration and CRISPR/Cas9-based editing of T cell receptor alpha constant (TRAC). In summary, we studied the biological mechanism of electrotransfection and developed a general, flexible, and reliable technique to enable highly efficient non-viral gene delivery and genome editing. Furthermore, the insights gained on the mechanism of electrotransfection provide better understanding of cellular response to exogenous materials. In the future, our study could potentially pave new paths for a wide range of research and therapeutic applications such as CRISPR/Cas9 mediated high-throughput loss-of-function gene screening analysis, correction of disease-related mutations, as well as genetic engineering of immune cells and stem cells for transplantation.

Electrowetting-on-dielectric (EWD) digital microfluidics is a droplet-based fluid handling technology capable of radically accelerating the pace of genome engineering research. EWD-based laboratory-on-chip (LoC) platforms demonstrate excellent performance in automating labor-intensive laboratory protocols at ever smaller scales. Until now, there has not been an effective means of gene transfer demonstrated in EWD microfluidic platforms. This thesis describes the theoretical and experimental approaches developed in the demonstration of an EWD-enabled electrotransfer device. Standard microfabrication methods were employed in the integration of electroporation (EP) and EWD device architectures. These devices enabled the droplet-based bulk transformation of E. coli with plasmid and oligo DNA. Peak on-chip transformation efficiencies for the EP/EWD device rivaled that of comparable benchtop protocols. Additionally, ultrasound induced in-droplet microstreaming was developed as a means of improving on-chip electroporation. The advent of electroporation in an EWD platform offers synthetic biologists a reconfigurable, programmable, and scalable fluid handling platform capable of automating next-generation genome engineering methods. This capability will drive the discovery and production of exotic biomaterials by providing the instrumentation necessary for rapidly generating ultra-rich genomic diversity at arbitrary volumetric scales.