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

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 1) 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 1 that could not be obtained using other methods. LSCM and flow cytometry experiments revealed that polymer 1 and DNA released from film-coated objects were both internalized extensively by cells, and that they were co-localized to a significant extent inside cells (e.g., ~58% of DNA was co-localized with polymer). Fluorescence anisotropy measurements of solutions containing partially eroded films were also consistent with the presence of aggregates of polymer 1 and DNA in solution (e.g., after release from surfaces, but prior to internalization by cells). Our results support the view that polymer 1 – which is incorporated into these materials as `layers' rather than as part of optimized, pre-formed `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.

Controllably local gene delivery mediated by polyelectrolyte multilayer films assembled from gene-loaded nanopolymersomes and hyaluronic acid

“…Our system has higher transfection efficiency than those reported substrate-mediated gene delivery systems in MSCs, whose transfection efficiency ranges from ~5% to ~40%. 3,5,12,15 But because of the different experimental conditions, including the quality of plasmid DNA, cell types, cell seeding density, and characterization methods, …”

“…In this therapeutic strategy, plasmid deoxyribonucleic acid (pDNA) is first immobilized onto a biomaterial surface and then released in a controlled and sustained manner to transfect cells surrounding materials. [1][2][3][4][5][6][7] As compared with the conventional bolus nonviral gene delivery, where cells are transfected by the suspended pDNA/vector complexes in liquid, substrate-mediated gene delivery has many advantages. The first one is its controllability, where gene transfection can be designed to meet clinical needs through controlling the amount of immobilized pDNA and its subsequent release from the substrate surfaces.…”

“…The second one is its high bioavailability of pDNA due to the high localized concentration of pDNA available to cells around substrates. It has been In PEM films, pDNA mostly acts as a polyanion layer [3][4][5][6]12,13 and partially as an adsorbed drug in a complex form (polyplex or lipoplex). 2,5,9,11 Both systems can successfully transfect cells in vitro and in vivo, but most of them have relatively low transfection efficiency (40%) due to their own defects.…”

“…The first one is that some released pDNA exists in a naked form. 3 It is well known that naked pDNA is insufficient in gene transfection due to its poor cellular uptake and rapid degradation by nuclease. 14 The second drawback is that although the gross mass of polyelectrolytes can be controlled to some extent by adjusting layer numbers in such PEM films, 5,10,11 how to precisely regulate the mass ratio of polycations to pDNA is unknown.…”

Multilayered thin films from poly(amido amine)s and DNA

“…Therapeutic agents to be released are incorporated during multilayer assembly and are subsequently released either via simple diffusion [32], biodegradation of the polyelectrolyte building block [33], or induced by internal [34] or external [35] triggers. In view of cell transfecting surfaces, Lynn and Bechler reported that the two multilayer components (i.e., hydrolysable poly(b-amino ester) and DNA) were found to co-localize within transfected cells cultured in the vicinity of the surface, indicating that either the polymer-DNA complex is released, or the separate components are released and later form a complex in cell culture medium before being internalized by cells [36].…”