This Small Business Technology Transfer (STTR) Phase I project will prove the technical and commercial feasibility of a single-use polymer microarray for electromechanical transfection of biological cells. Alternative gene transfer solutions are needed to access the potential held in biopharmaceuticals, gene therapy, and stem cell research. Although physical methods for gene transfer (e.g., electroporation) have demonstrated improved (over conventional chemical-mediated techniques) treatment outcomes in difficult-to-transfect primary and stem cells, control and uniformity of treatment remain inadequate. Further, incremental improvements in performance of cuvette-based electroporation systems have only been realized through corresponding increases in system complexity and cost. One-by-one ejection of cells through cell-sized orifices has been found to promote cell membrane poration and DNA delivery into cells by imposing an identical and carefully controlled electromechanical environment on each individual cell. Unfortunately, a relatively high material cost and limitations on achievable treatment conditions (due to constraints on the geometry of microarrays manufactured using standard silicon micromachining techniques), inhibit practical implementation of existing silicon-based microarrays. The innovative design and optimization of the polymer microarray under this STTR Phase I project will yield a low-cost system capable of generating the mechanical stress field needed to achieve improved treatment outcomes in difficult-to-transfect cells.
The broader impact/commercial potential of this project is to enable effective and economical transfection of difficult-to-transfect primary and stem cells used for a variety of research and therapeutic applications in the Life Sciences. Effective delivery of genes, drug molecules, imaging agents, peptides, antibodies, and enzymes into living cells is critical to applications ranging from the treatment of human disease through introduction of DNA to the investigation of basic cellular function through single molecule imaging; yet, intracellular delivery and transfection remain difficult tasks. While efficiencies of greater than 90% are common in basic research applications that use chemical or physical methods to transfect laboratory established and maintained ("easy") cell lines, efficiency can drop to 10% or lower for "difficult" cells. Refinements of physical methods (e.g., electroporation) have achieved incremental performance improvements; however, no system currently on the market meets all end-user requirements for efficiency, viability, functionality, and cost. The novel approach to transfection, which is the subject of this STTR Phase I project, promises to improve treatment efficacy through innovative use of multiple gene transfer techniques simultaneously, while better addressing end user needs by providing a cost-effective transfection solution for difficult cells.
Introduction of otherwise impermeable molecules into established cell line has become ubiquitous in biomedical research. Despite the substantially positive impact that extant techniques (e.g., electroporation, lipofection, and calcium phosphate-based transfection) have had on biological research, poor efficacy in difficult-to-transfect cells (e.g., primary and stem cells) provides motivation for development of novel approaches to intracellular delivery and transfection. The primary objective of this Phase I STTR project was demonstration of effective biomolecule delivery into cells using a version of our existing delivery/transfection platform that incorporated a polymer-based electrosonic acutation microarray (EAM). Polymer construction confers several advantages. First, the flexibility inherent in creating microarray freatures (either of the microarray directly or of a mold) using conventional machining provides greater diversity of achievable nozzle shapes and sizes versus the existing silicon-based microarray. Thus, nozzle geometry can be tuned to improve both hydrodynamic and cell treatment performance of the EAM. Finally, polymer-based microarrays yield a significant cost savings over equivalent silicon-based arrays. Unfortunately, these advantages are somewhat offset by the poor acoustic performance characteristic of polymers, which could be detrimental to EAM operation. Relying on our extensive experience in design and optimization of ultrasonic atomizers, and expertise in polymer manufacturing, we aimed to prove the technical and commercial feasibility of a single-use polymer microarray for electromechanical transfection of biological cells. Our specific research objectives included: (1) establishment of acoustic/hydrodynamic capabilities of the polymer EAM, (2) design/manufacture of a polymer EAM, and (3) demonstration of transfection of biological cells using polymer-based microarrays. Objective 1 was completed through development and use of a generalized two-dimensional (2D) finite element analysis (FEA) model of the complex EAM cartridge assembly. While modeling results indicated that polymer components have a negative effect on the ability of the device to eject sample at all operating conditions, results suggest that optimization of the polymer-based device should lead to performance equivalent to our current silicon-based device. Objective 2 was completed after overcoming a number of obstacles associated with conventional machining of extremely small features(in polymers and in mold materials) and difficulties in creation of consistent orifice geometries at the nozzle tips. Resultant microarrays were capable of fairly robust water ejection; however, we were unable to identify an optimal condition for effective cell treatment. A number of potential reasons for reduced capablility of the device have been identified, and potential solutions are under investigation. While we were not able to achieve our final research objective, the results of this study will aid future development of the EAM, further strengthening the commercial viablilty of the OpenCell platform.
