Polyphosphoester/DNA Multilayer Thin Films Via Layer-by-Layer Technique For Controlled Gene Delivery

Tissue engineering for disease and trauma treatments requires signaling to promote cell proliferation or control cell fate. The potential signals include immobilized ligands that promote cell adhesion and cell-cell communication, and soluble factors such as growth factors that can signal over various distances and initiate cellular events such as cell differentiation, migration and proliferation. A number of systems exist to deliver soluble growth factors to cells both in vitro and in vivo. However, proteins have very short half-lives in vivo when delivered directly, therefore, it may not provide the therapeutic benefit of natural proteins.Gene therapy approaches utilize DNA vectors to transfect cells, and use the cellular machinery to produce therapeutic proteins. Viruses provide high transfection efficiency and have been used for systemic applications in diseases treatment, but viral immunogenicity has raised safety concerns, which has motivated the development of synthetic systems capable of delivering DNA to cells. Synthetic vector can significantly decrease toxicity, but their limitations include constrained duration and area of expression, and deactivation by serum proteins. One approach to control the duration and location of gene expression with non-viral vectors is to immobilize DNA constructs in localized depots with polymeric scaffolds that provide a platform for localized DNA delivery.Sustained release of functional plasmid DNA from the surfaces of materials which support cell adhesion for tissue formation could have a significant impact on gene therapy and tissue engineering. We report here layer-by-layer assembled multilayer film from a degradable cationic poly (2-aminoethyl propylene phosphate) and plasmid DNA encoding for enhanced green fluorescent protein (EGFP) for mouse osteoblast cell adhesion and prolonged gene delivery. Multilayer film growth was monitored by UV spectrophotometry and intensity of absorbance at 260 nm related to incorporated DNA increased in an exponential manner with increase the number of deposited polymer and plasmid layers. It degraded upon incubation in phosphate-buffered saline (PBS) at 37℃and sustained the release of bioactive plasmid DNA up to 2 months. The multilayer film facilitated initial mouse osteoblast cell adhesion onto the surface and enhanced cellular alkaline phosphatase activity and calcium accumulation. It sustained delivering transcriptional active DNA to mouse osteoblast cells cultured on the film, and directly prolonged gene expression in the presence of serum without any exogenous transfection agent. Besides, we do some changes to the synthesis of cationic polyphosphoester. N-(t-butoxy carbonyl) ethanolamine (EABoc) was reacted with 2-cholo-2-oxo-1,3,2-dioxaphospholane (COP) to synthesize amine-protected monomer PEEABoc. The ring-opening polymerization of PEEABoc with initiation of stannous octoate yields cationic polyphosphoester PPEEA. The synthetic route of polymer PPEEA was confirmed by ¹H NMR, ¹³C NMR and GPC. By controlling the synthetic conditions, different molecular weight of cationic polyphosphoester like PPE-EA may be achieved in the synthesis route.Above all, this biodegradable multilayer assembly is promising for the local and sustained delivery of plasmid DNA and such a layer-by-layer system suggests an alternative method for plasmid DNA incorporation which may be useful for surface modification of implanted materials or scaffold for gene therapy and tissue regeneration.