Characterization of Degradable
Polyelectrolyte Multilayers
Fabricated Using DNA and a Fluorescently-Labeled Poly(β-amino
ester): Shedding Light on the Role of the Cationic Polymer in Promoting
Surface-Mediated Gene Delivery
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Abstract
Polyelectrolyte multilayers (PEMs) fabricated from cationic
polymers
and DNA have been investigated broadly as materials for surface-mediated
DNA delivery. One attractive aspect of this “multilayered”
approach is the potential to exploit the presence of cationic polymer
“layers” in these films to deliver DNA to cells more
effectively. Past studies demonstrate that these films can promote
transgene expression in vitro and in vivo, but significant questions
remain regarding roles that the cationic polymers could play in promoting
the internalization and processing of DNA. Here, we report physicochemical
and in vitro cell-based characterization of DNA-containing PEMs fabricated
using fluorescently end-labeled derivatives of a degradable polycation
(polymer <b>1</b>) used in past studies of surface-mediated
transfection. This approach permitted simultaneous characterization
of polymer and DNA in solution and in cells using fluorescence-based
techniques, and provided information about the locations and behaviors
of polymer <b>1</b> that could not be obtained using other methods.
LSCM and flow cytometry experiments revealed that polymer <b>1</b> and DNA released from film-coated objects were both internalized
extensively by cells and that they were colocalized to a significant
extent inside cells (e.g., ∼58% of DNA was colocalized with
polymer). Fluorescence anisotropy measurements of solutions containing
partially eroded films were also consistent with the presence of aggregates
of polymer <b>1</b> and DNA in solution (e.g., after release
from surfaces, but prior to internalization by cells). Our results
support the view that polymer <b>1</b>, which is incorporated
into these materials as “layers” rather than as part
of optimized, preformed “polyplexes”, can act to promote
or enhance surface-mediated DNA delivery. More broadly, our results
suggest opportunities to improve the delivery properties of DNA-containing
PEMs by incorporation of additional “layers” of other
conventional cationic polymers designed to address specific intracellular
barriers to transfection, such as endosomal escape, more effectively