Substrate-Mediated Gene Delivery from Glycol-Chitosan/Hyaluronic Acid Polyelectrolyte Multilayer Films Christina A. Holmes † and Maryam Tabrizian* ,†,‡ † Department of Biomedical Engineering and ‡ Faculty of Dentistry, Duff Medical Science Building, 3775 University Street, McGill University, Montreal, H3A 2B4, Canada ABSTRACT: Substrate-mediated transfection is one of the key strategies for localized gene delivery. Layer-by-layer (LbL) polyelectrolyte deposition is a promising technique which enables controlled delivery of a number of biofactors, including nucleic acids. Here, we embed lipoplexes containing plasmid DNA within polyelectrolyte multilayers composed of glycol- chitosan (Glyc-CHI) and hyaluronic acid (HA) in order to produce a film system that enables localized, surface-based transfection. The topography and morphology of the resulting multilayers were characterized after lipoplex absorption and during subsequent film build-up via atomic force microscopy (AFM) and scanning electron microscopy (SEM), respectively. DNA embedding efficiency and release were then examined. Lipoplex- containing Glyc-CHI/HA films were found to successfully transfect NIH3T3 fibroblasts and HEK293 kidney cells in vitro, maintaining transfection levels of approximately 20% for a period of at least 7 days. KEYWORDS: gene delivery, lipoplex, substrate-mediated, layer-by-layer deposition, glycol-chitosan ■ INTRODUCTION Spatial control of gene delivery is an essential feature of many biomedical applications, including inductive tissue engineering, medical implant coatings, and cellular transfection microarrays. One main approach to localized gene transfer, termed “substrate-mediated gene delivery” 1 or “reverse-transfection”, 2 involves the immobilization of DNA and carrier vectors onto a biomaterial surface, as opposed to the traditional “bolus” transfection method of adding the DNA and vector to an aqueous media. A variety of strategies can be employed to surface-immobilize gene transfection vectors, including non- specific absorption, 3 surface attachment via biotin-streptavidin 1 and antibody-antigen conjugate systems, 4 or encapsulation within a thin polymeric or hydrogel film. 2 Among these many methods, layer-by-layer polyelectrolyte deposition has emerged as a simple yet versatile technique which can be utilized with biomaterials of nearly any type, shape, or size. Layer-by-layer (LbL) deposition involves the sequential surface assembly of alternating layers of oppositely charged polyelectrolytes (PEs) 5 and has been widely used for the controlled release of drugs, bioactive proteins, and genes (reviewed in ref 6). Naked plasmid DNA, PEI- and cyclo- dextran-complexed plasmids, and adenoviral vectors have been incorporated into a variety of polyelectrolyte multilayer designs (reviewed in ref 7) and have been successfully used to transfect cells in vitro and in vivo. 8,9 Careful selection of the PEs used and the layer architecture and chemistry employed enables both the tailoring of release kinetics and sequential delivery of several different genes. 10,11 Plasmid DNA itself is often directly used as the anionic PE for LbL assembly, alongside degradable cationic polymers. For example, Lynn and colleagues built PE multilayers from a synthetic hydrolytically degradable cationic polyamine (“poly- mer 1”) and naked plasmid DNA encoding EGFP or RFP, 12 which, used alone or as a stent-coating, could transfect cells in vitro 13 and in vivo. 9 Atomic force microscopy (AFM) analysis suggested that the DNA/polymer layers rearranged themselves to present surface-bound condensed DNA nanoparticles. 13 Naked plasmid DNA-based LbL multilayers have also been constructed using chitosan, 14 galactosylated chitosan, 15 poly(2- aminoethyl propylene phosphate), 16 poly(ethylimine), 17 and reducible hyperbranched poly(amido amine) 8 as the cationic PEs, with similar film surface rearrangements into nanoparticle complexes observed in most cases. As these plasmid-cationic polymer films degrade, these complexes are released, as verified via electrophoresis and transmission electron microscopy (TEM) 15,16 and are thought to act like other typical cationic gene delivery vectors. Alternatively, plasmids precomplexed with a viral or nonviral gene carrier vector can also be incorporated within multilayer films for controlled, substrate-mediated transfection. The Voegel and Jessel groups, for example, have done extensive work using PLL/PGA, chitosan (CHI)/hyaluronic acid (HA), PAH/PSS, and PLL/HA multilayer films to deliver PEI- condensed plasmids, 18 pyridylamino cyclodextrin complexed plasmids, 11 or adenoviral vectors 19 to several different cell lines as well as primary cells. While results varied greatly between systems, generally, lower transfection levels were observed Received: May 30, 2012 Accepted: January 2, 2013 Published: January 2, 2013 Research Article www.acsami.org © 2013 American Chemical Society 524 dx.doi.org/10.1021/am303029k | ACS Appl. Mater. Interfaces 2013, 5, 524-531