Efficient gene delivery is a fundamental goal of biotechnology and has numerous applications in both basic and applied science. Substrate-mediated delivery and reverse transfection enhance gene transfer by increasing the concentration of DNA in the cellular microenvironment through immobilizing a plasmid to a cell culture substrate prior to cell seeding. In this report, we examine gene delivery of plasmids that were complexed with cationic polymers (polyplexes) or lipids (lipoplexes) and subsequently immobilized to cell culture or biomaterial substrates by adsorption. Polyplexes and lipoplexes were adsorbed to either tissue culture polystyrene or serumadsorbed tissue culture polystyrene. The quantity of DNA immobilized increased with time of exposure, and the deposition rate and final amount deposited depended upon the properties of the substrate and complex. For polyplexes, serum modification enhanced reporter gene expression up to 1500-fold relative to unmodified substrates and yielded equivalent or greater expression compared to bolus delivery. For lipoplexes, serum modification significantly increased the number of transfected cells relative to unmodified substrates yet provided similar levels of expression. Immobilized complexes transfect primary cells with improved cellular viability relative to bolus delivery. Finally, this substrate-mediated delivery approach was extended to a widely used biomaterial, poly(lactide-coglycolide). Immobilization of DNA complexes to tissue culture polystyrene substrates can be a useful tool for enhancing gene delivery for in vitro studies. Additionally, adapting this system to biomaterials may facilitate application to fields such as tissue engineering.

Gene transfer has many potential applications in basic and applied sciences. In vitro, DNA delivery can be enhanced by increasing the concentration of DNA in the cellular microenvironment through immobilization of DNA to a substrate that supports cell adhesion. Substrate-mediated delivery describes the immobilization of DNA, complexed with cationic lipids or polymers, to a biomaterial or substrate. As surface properties are critical to the efficiency of the surface delivery approach, selfassembled monolayers (SAMs) of alkanethiols on gold were used to correlate surface chemistry of the substrate to binding, release, and transfection of non-specifically immobilized complexes. Surface hydrophobicity and ionization were found to mediate both DNA complex immobilization and transfection, but had no effect on complex release. Additionally, SAMs were used in conjunction with soft lithographic techniques to imprint substrates with specific patterns, resulting in patterned DNA complex deposition and transfection, with transfection efficiencies in the patterns nearing 40%. Controlling the interactions between complexes and substrates, with the potential for patterned delivery, can be used to locally enhance or regulate gene transfer, with applications to tissue engineering scaffolds and transfected cell arrays.

Adapting controlled release technologies to the delivery of DNA has the potential to overcome extracellular barriers that limit gene therapy. Controlled release systems can enhance gene delivery and increase the extent and duration of transgene expression relative to more traditional delivery methods (e.g., injection). These systems typically deliver vectors locally, which can avoid distribution to distant tissues, decrease toxicity to nontarget cells, and reduce the immune response to the vector. Delivery vehicles for controlled release are fabricated from natural and synthetic polymers, which function either by releasing the vector into the local tissue environment or by maintaining the vector at the polymer surface. Vector release or binding is regulated by the effective affinity of the vector for the polymer, which depends upon the strength of molecular interactions. These interactions occur through nonspecific binding based on vector and polymer composition or through the incorporation of complementary binding sites (e.g., biotin -avidin). This review examines the delivery of nonviral and viral vectors from natural and synthetic polymers and presents opportunities for continuing developments to increase their applicability.

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Human mesenchymal stem cells (hMSCs) are one of the most widely researched stem cell types with broad applications from basic research to therapeutics, the majority of which require introduction of exogenous DNA. However, safety and scalability issues hinder viral delivery, while poor efficiency hinders nonviral gene delivery, particularly to hMSCs. Here, we present the use of a pharmacologic agent (glucocorticoid) to overcome barriers to hMSC DNA transfer to enhance transfection using three common nonviral vectors. Glucocorticoid priming significantly enhances transfection in hMSCs, demonstrated by a 3-fold increase in efficiency, 4-15-fold increase in transgene expression, and prolonged transgene expression when compared to transfection without glucocorticoids. These effects are dependent on glucocorticoid receptor binding and caused in part by maintenance of normal metabolic function and increased cellular (5-fold) and nuclear (6-10-fold) DNA uptake over hMSCs transfected without glucocorticoids. Results were consistent across five human donors and in cells up to passage five. Glucocorticoid cell priming is a simple and effective technique to significantly enhance nonviral transfection of hMSCs that should enhance their clinical use, accelerate new research, and decrease reliance on early passage cells.

BackgroundParticulates incorporating DNA are promising vehicles for gene delivery, with the ability to protect DNA and provide for controlled, localized, and sustained release and transfection. Zein, a hydrophobic protein from corn, is biocompatible and has properties that make it a promising candidate material for particulate delivery, including its ability to form nanospheres through coacervation and its insolubility under physiological conditions, making it capable of sustained release of encapsulated compounds. Due to the promise of this natural biomaterial for drug delivery, the objective of this study was to formulate zein nanospheres encapsulating DNA as the therapeutic compound, and to characterize size, charge, sustained release, cell cytotoxicity and cellular internalization of these particles.ResultsZein nanospheres encapsulating DNA were fabricated using a coacervation technique, without the use of harsh solvents or temperatures, resulting in the preservation of DNA integrity and particles with diameters that ranged from 157.8 ± 3.9 nm to 396.8 ± 16.1 nm, depending on zein to DNA ratio. DNA encapsulation efficiencies were maximized to 65.3 ± 1.9% with a maximum loading of 6.1 ± 0.2 mg DNA/g zein. The spheres protected encapsulated DNA from DNase I degradation and exhibited sustained plasmid release for at least 7 days, with minimal burst during the initial phase of release. Zein/DNA nanospheres demonstrated robust biocompatibility, cellular association, and internalization.ConclusionsThis study represents the first report on the formation of zein particles encapsulating plasmid DNA, using simple fabrication techniques resulting in preservation of plasmid integrity and tunable sizes. DNA encapsulation efficiencies were maximized to acceptable levels at higher zein to DNA ratios, while loading was comparable to that of other hydrophilic compounds encapsulated in zein and that of DNA incorporated into PLGA nano- and microspheres. The hydrophobic nature of zein resulted in spheres capable of sustained release of plasmid DNA. Zein particles may be an excellent potential tool for the delivery of DNA with the ability to be fine-tuned for specific applications including oral gene delivery, intramuscular delivery, and in the fabrication of tissue engineering scaffolds.

Substrate-mediated gene delivery describes the immobilization of gene therapy vectors to a biomaterial, which enhances gene transfer by exposing adhered cells to elevated DNA concentrations within the local microenvironment. Surface chemistry has been shown to affect transfection by nonspecifically immobilized complexes using self-assembled monolayers (SAMs) of alkanethiols on gold. In this report, SAMs were again used to provide a controlled surface to investigate whether the presence of oligo(ethylene glycol) (EG) groups in a SAM could affect complex morphology and enhance transfection. EG groups were included at percentages that did not affect cell adhesion. Nonspecific complex immobilization to SAMs containing combinations of EG-and carboxylic acidterminated alkanethiols resulted in substantially greater transfection than surfaces containing no EG groups or SAMs composed of EG groups combined with other functional groups. Enhancement in transfection levels could not be attributed to complex binding densities or release profiles. Atomic force microscopy imaging of immobilized complexes revealed that EG groups within SAMs affected complex size and appearance and could indicate the ability of these surfaces to preserve complex morphology upon binding. The ability to control the morphology of the immobilized complexes and influence transfection levels through surface chemistry could be translated to scaffolds for gene delivery in tissue engineering and diagnostic applications.

BackgroundMesenchymal stem cells (MSCs) are multipotent stem cells that can be isolated and expanded from many tissues, and are being investigated for use in cell therapies. Though MSC therapies have demonstrated some success, none have been FDA approved for clinical use. MSCs lose stemness ex vivo, decreasing therapeutic potential, and face additional barriers in vivo, decreasing therapeutic efficacy. Culture optimization and genetic modification of MSCs can overcome these barriers. Viral transduction is efficient, but limited by safety concerns related to mutagenicity of integrating viral vectors and potential immunogenicity of viral antigens. Nonviral delivery methods are safer, though limited by inefficiency and toxicity, and are flexible and scalable, making them attractive for engineering MSC therapies.Main textTransfection method and nucleic acid determine efficiency and expression profile in transfection of MSCs. Transfection methods include microinjection, electroporation, and nanocarrier delivery. Microinjection and electroporation are efficient, but are limited by throughput and toxicity. In contrast, a variety of nanocarriers have been demonstrated to transfer nucleic acids into cells, however nanocarrier delivery to MSCs has traditionally been inefficient. To improve efficiency, plasmid sequences can be optimized by choice of promoter, inclusion of DNA targeting sequences, and removal of bacterial elements. Instead of DNA, RNA can be delivered for rapid protein expression or regulation of endogenous gene expression. Beyond choice of nanocarrier and nucleic acid, transfection can be optimized by priming cells with media additives and cell culture surface modifications to modulate barriers of transfection. Media additives known to enhance MSC transfection include glucocorticoids and histone deacetylase inhibitors. Culture surface properties known to modulate MSC transfection include substrate stiffness and specific protein coating. If nonviral gene delivery to MSCs can be sufficiently improved, MSC therapies could be enhanced by transfection for guided differentiation and reprogramming, transplantation survival and directed homing, and secretion of therapeutics. We discuss utilized delivery methods and nucleic acids, and resulting efficiency and outcomes, in transfection of MSCs reported for such applications.ConclusionRecent developments in transfection methods, including nanocarrier and nucleic acid technologies, combined with chemical and physical priming of MSCs, may sufficiently improve transfection efficiency, enabling scalable genetic engineering of MSCs, potentially bringing effective MSC therapies to patients.