(257g) Surface-Mediated Release of siRNA From Polyelectrolyte Multilayers Fabricated Using Degradable Polyamines | AIChE

(257g) Surface-Mediated Release of siRNA From Polyelectrolyte Multilayers Fabricated Using Degradable Polyamines


Flessner, R. M. - Presenter, University of Wisconsin - Madison
Jewell, C. M. - Presenter, Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology
Lynn, D. M. - Presenter, University of Wisconsin-Madison
Langer, R. - Presenter, Massachusetts Institute of Technology
Nguyen, D. - Presenter, Massachusetts Institute of Technology
Anderson, D. G. - Presenter, Massachusetts Institute of Technology

The incorporation of nucleic acid constructs such as DNA and RNA into ultrathin polymer films provides a basis for the surface-mediated delivery of these materials and represents a potential platform for the development of new localized gene-based therapies. We recently demonstrated that ultrathin multilayered polyelectrolyte thin films (or ?polyelectrolyte multilayers') can be fabricated by the alternating, layer-by-layer deposition of plasmid DNA and hydrolytically degradable poly(beta-amino ester)s. These ultrathin films erode gradually and release functional DNA in physiological media, and we have demonstrated that objects coated with these materials promote localized, surface-mediated transgene expression when placed in contact with cells in vitro and in vivo. These methods can be used to fabricate films that promote the surface-mediated release of DNA or other agents over periods ranging from several hours to several days, weeks, or even several months depending on the structures of the polymers used to fabricate the films.

As a platform for the incorporation and delivery of nucleic acids to cells, this layer-by-layer approach provides several potential practical advantages: (i) these fabrication methods are entirely aqueous, (ii) layer-by-layer assembly provides precise control over the amount of material incorporated and released at a surface (e.g., determined by the number of layers of polymer and nucleic acid deposited during fabrication), and (iii) the multilayered internal structures of these materials can be exploited to assemble hierarchical films that provide control over the timing and sequence of the release of multiple different nucleic acid constructs. One additional potential practical advantage of these multilayered films in the context of nucleic acid delivery is that the cationic polymer components of these materials are also useful DNA and RNA delivery agents and thus have the potential to improve levels of internalization and processing of these nucleic acid constructs by cells.

Here, we report on the fabrication and characterization of polyelectrolyte multilayers fabricated using functional siRNA constructs. Recent work on the delivery of small, double-stranded siRNA constructs to cells has generated tremendous interest as a result of the ability of these agents to specifically knockdown or silence targeted genes. However, materials and methods that have been developed or optimized for the delivery of large plasmid DNA constructs may in some cases not be directly applicable to the delivery of siRNA constructs because of large differences in the size, stability, and structure of plasmid DNA and siRNA, as well as differences in the intracellular processing and targets of these two materials. We demonstrate here that functional siRNA constructs targeted to knockdown enhanced green fluorescent protein (EGFP) can be used to fabricate ultrathin polyelectrolyte multilayers (e.g., 50 nm thick) using a model poly(beta-amino ester), similar to films fabricated from plasmid DNA. However, we observe large differences in the release behaviors of these siRNA-containing films in comparison to analogous plasmid DNA-containing films. For example, whereas films fabricated from plasmid DNA release DNA gradually into solution over a period of several days, films fabricated from siRNA release siRNA rapidly (e.g., release is generally complete over a period of 24 hours). Characterization of released siRNA by gel electrophoresis demonstrated that the siRNA that is released from these films is released in a physically intact form. Additional cell-based experiments demonstrated that the released siRNA was functional and could be used to promote the specific knockdown of EGFP in both stably transfected and transiently transfected mammalian cells. The results of additional experiments to characterize the nanometer-scale physical structures of partially eroded films will be presented, and opportunities for the use of these materials to promote the localized or surface-mediated knockdown of targeted genes in cells using a range of film-coated objects will be discussed.