(222g) 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.