Zein As a Natural Material for the Controlled Delivery of Non-Viral Gene Delivery Vectors: Zein Films and Nanoparticle Coatings

Zein as a Natural Material for the Controlled Delivery of Non-Viral Gene Delivery Vectors: Zein Films and Nanoparticle Coatings

Eric Farris¹, Jessica Taylor¹, Christopher Davidson¹, and Angela K. Pannier¹

¹Department of Biological Systems Engineering, University of Nebraska-Lincoln, Lincoln NE, 68588

Nonviral strategies for the delivery of nucleic acids are considered a promising alternative to viral vectors due to their increased safety and lower immunogenicity. However, nonviral vectors still lack the efficiency needed to reach clinical relevance. The inefficiency, which can be attributed to many factors, such as degradation of the DNA by nucleases and other enzymes, and instability of the vector in physiological conditions, can potentially be overcome through use of particulates that physically incorporate plasmid DNA. The use of particulates incorporating DNA are promising vehicles for gene delivery as they have the ability to protect DNA from degradation and allow for sustained and controlled release of DNA for increased transfection.

Another strategy at increasing the efficiency of nonviral systems involves immobilizing delivery vectors to a substrate prior to cell seeding. This strategy, termed substrate-mediated gene delivery, allows for an increased concentration of DNA, which is localized at the cell surface for increased uptake and transfection. Zein, the major prolamine, or storage protein, in corn, is biocompatible and biodegradable and has been shown to have unique properties that allow it to form particulates, films, and coatings through coacervation or evaporation induced self-assembly. Moreover, zein has very low aqueous solubility and has been shown to be stable in acidic environments. Due to these properties zein has the ability to be tailored for applications in substrate-mediated gene delivery as well as in nanoparticulate coatings for oral DNA delivery applications.

For this work, our objective was to investigate zein's ability to form films for substrate-mediated gene delivery, as well as investigate zein as a material for particulate DNA delivery, in particular as a protective coating material for non-viral gene delivery complexes. For substrate-mediated gene delivery applications, zein was found to be able to form uniform films on a variety of substrates, including glass, titanium, and gold. Observation of the films by scanning electron microscopy indicated the films were composed of nanospherical particles, the size of which could be modulated by altering the pH of the initial zein solutions to alter the surface roughness and resulting hydrophobicity of the films. Zein films were shown to adsorb both poly(ethylene imine)/DNA (PEI/DNA) complexes and Lipofectamine 2000/DNA complexes and once cells were seeded on these immobilized complexes, transfection in HEK 293T cells was demonstrated.  Furthermore, zein films were shown to increase cellular proliferation, which is often critical to gene delivery efficacy.

Due to the enzymatic and pH resistant properties of zein, we next investigated the use of zein as a protective coating for DNA delivery systems for use in oral gene delivery and DNA vaccination applications. Zein was used to encapsulate chitosan/DNA (CS/DNA) complexes via a water-in-oil emulsion method to generate Zein/CS/DNA micro/nanoparticles. The resulting microparticles ranged in size from 5 to 10 µm and DNA loading in the particles was found to increase from 1.68 to 7.72 mg DNA/g of zein by increasing the concentration of the zein solution from 3 to 7% (w/v).   Zein particles are capable of protecting DNA cargo within the stomach for up to two hours, and after incubation in simulated intestinal fluid (SIF), zein degrades rapidly, releasing CS/DNA particles that were shown to mediate transfection in HEK 293T cells. A significant increase in transgene expression mediated by the ZN/CS/DNA particles after incubation in SIF was observed with increasing ZN concentration, demonstrating the ability of this system to release intact and transfection competent CS/DNA NPs in a site specific manner for targeted gene delivery in the intestinal environment. Overall, these studies demonstrate the versatility of zein as a natural material for controlled delivery systems for non-viral gene delivery.