(85a) DNA-Containing Polyelectrolyte Multilayers: Fluorescently Labeled Polymers Shed Light On the Roles That Cationic Polymers Play in Promoting Surface-Mediated Cell Transfection

The ability to immobilize and control the release of DNA from surfaces is important in a broad range of fundamental and applied contexts, ranging from tissue engineering and the development of gene-based therapies to the development of new tools for basic biomedical research. As one approach to the design of materials that provide such control, our group has developed methods for the layer-by-layer assembly of ultrathin polyelectrolyte-based films (or ?polyelectrolyte multilayers', PEMs) that can be used to provide control over the surface-mediated delivery of DNA. This approach is based on methods for the alternate deposition of ultrathin layers of plasmid DNA (an anionic polymer) and hydrolytically degradable poly(β-aminoester)s (cationic polymers) on surfaces. Past work by our group has demonstrated that this layer-by-layer approach can be used to design thin films and coatings that erode and release DNA gradually, and that these films can be used to promote the localized and surface-mediated transfection of cells in vitro and in vivo.

The layer-by-layer assembly of DNA-containing films offers several potential practical advantages relative to conventional methods for the encapsulation of DNA in thin films of bulk polymer, including (i) the amount of DNA incorporated into (or released from) a film can be controlled and adjusted with precision by changing the number of DNA ?layers' deposited, (ii) rates of DNA release can be tuned over a broad range of times (e.g., ranging from several hours to several days, weeks, or months) by changing the structure or nature of the cationic polymer used to fabricate the film, and (iii) these methods are entirely aqueous and thus allow films and coatings to be fabricated in the absence of organic solvents that could harm cells or the surfaces of objects on which these materials are deposited. Furthermore, layer-by-layer assembly is amenable to the fabrication of thin films and coatings on topologically complex objects typical of those used to design implants, biomedical devices, and tissue engineering scaffolds.

In the context of DNA delivery, one additional potential benefit of this layer-by-layer approach is the juxtaposition of the DNA in these materials with layers of cationic polymers (a class of materials used broadly for the delivery of DNA). The commingling of DNA with a DNA delivery agent provides opportunities to fabricate films that not only release DNA, but also release DNA in the presence of cationic materials that have the ability to interact with and thus promote the internalization of DNA by cells. Our past work demonstrates clearly that these materials can promote surface-mediated cell transfection, and studies using films fabricated using fluorescently labeled DNA have permitted the characterization of the intracellular trafficking of DNA. Unfortunately, however, these past studies have not provided direct insight into what roles the cationic polymer components of these assemblies may play in promoting cell transfection.

Here, we report an approach to the fabrication of DNA-containing films that permits characterization of both the DNA and polymer components of these materials using a range of fluorescence-based techniques. We synthesized a fluorescently end-labeled poly(β-aminoester) otherwise identical in structure and molecular weight to the polymers used in our past studies. Fabrication of polymer/DNA films using polymer and DNA labeled with two different fluorophores permitted the behavior of both of these film components to be characterized simultaneously in solution and inside cells using confocal microscopy and flow cytometry. Flow cytometry analysis of cells incubated in vitro in the presence of film-coated mesh substrates for 24 hours revealed that nearly all cells (e.g., from 95% to 99% of cells) had internalized both polymer and DNA. These studies also revealed that cells that were positive for both DNA and polymer contained higher levels of DNA than cells that were incubated in the presence of DNA alone (e.g., in control experiments for which DNA was delivered to cells as a bolus of naked DNA). Characterization of cells using confocal microscopy confirmed the internalization of both polymer and DNA by nearly all cells. Both polymer and DNA were observed to be present largely within endosomes and lysosomes containing either polymer alone, DNA alone, or, in many cases, both polymer and DNA co-localized within these vesicular bodies. Quantification of these confocal microscopy results revealed as much as 70% co-localization of labeled DNA with fluorescently labeled polymer.

Our results suggest that, in addition to serving as structural elements of these films, the cationic polymers play active roles in promoting the internalization of released DNA by cells, presumably through the formation of self-assembled polymer/DNA complexes similar to (although likely different in structure from) cationic polymer/DNA ?polyplexes' used conventionally for the delivery of DNA. The results of our fluorescence-based studies complement those of earlier studies that used light scattering, zeta potential analysis, and gel electrophoresis to characterize the presence or absence of polymer/DNA complexes released into solution during film erosion. The results of additional fluorescence anisotropy measurements suggesting the presence of complexes of fluorescently labeled polymer and DNA in solution (i.e., after surface-mediated release, but prior to internalization by cells) will also be discussed.

This work provides important insight into the roles that the cationic polymers in these DNA-containing layer-by-layer assemblies can play in promoting surface-mediated transfection. The observation of co-localized polymer and DNA inside cells suggests that these multilayered films could present a platform for the design of films that transfect cells more efficiently (e.g., through the incorporation of layers of cationic polymers that are designed to address specific intracellular barriers to transfection, such as endosomal escape, more efficiently than the model polymer used in this study). A more complete understanding of the relationships between polymer structure and surface mediated gene delivery, could facilitate the design of polymers and DNA-containing thin films tailored and optimized for applications in the areas of gene delivery, tissue engineering, and basic biomedical research.