The development of thin films and coatings that control the release of DNA from the surfaces of materials could have a significant impact on localized approaches to gene therapy. Here, we report multilayered polyelectrolyte assemblies that sustain the release of functional plasmid DNA from the surfaces of model substrates under physiological conditions. Multilayered assemblies consisting of alternating layers of plasmid DNA encoding for enhanced green fluorescent protein (EGFP) and a synthetic degradable polyamine were deposited on planar silicon and quartz substrates using a layer-by-layer fabrication process. Film growth was monitored by ellipsometry and UV spectrophotometry and correlated linearly with the number of polymer and plasmid layers deposited. In general, the thickness of deposited layers was found to be a function of both the pH and the ionic strength of the polyelectrolyte solutions used. Films up to 100 nm thick were investigated in this study. These assemblies erode gradually upon incubation in phosphate-buffered saline at 37 degrees C, as determined by ellipsometry and UV spectrophotometry, and sustain the release of incorporated plasmid into the incubation medium for a period of up to 30 h. Characterization of the released plasmid by agarose gel electrophoresis revealed that the DNA was released in a relaxed, open circular, rather than supercoiled, topology; subsequent cell transfection experiments demonstrated that the released plasmid is transcriptionally viable and promotes the expression of EGFP in the COS-7 cell line. These layered materials could represent an approach to the controlled administration of one or more functional DNA constructs from the surfaces of biomedical materials and devices.

The combinatorial, automated high-throughput synthesis and evaluation of small molecules has revolutionized modern drug discovery. Here we describe the first high-throughput, semi-automated methodology for the synthesis and screening

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We recently reported the parallel synthesis of 140 degradable poly(beta-amino esters) via the conjugate addition of 20 primary or secondary amine monomers to seven different diacrylate monomers. To explore possible structure/function relationships and further characterize this class of materials, we investigated the ability of each DNA-complexing polymer to overcome important cellular barriers to gene transfer. The majority of vectors were found to be uptake-limited, but complexes formed from polymers B14 and G5 displayed high levels of internalization relative to "naked" DNA (18x and 32x, respectively). Effective diameter and zeta potential measurements indicated that, in general, small particle size and positive surface charge led to higher internalization rates. Of the 10 DNA/polymer complexes with the highest uptake levels, all had effective diameters less than 250 nm and nine had positive zeta potentials. Lysosomal trafficking was investigated by measuring the pH environment of delivered DNA. Complexes prepared with polymers G5, G10, A13, B13, A14, and B14 were found to have near neutral pH measurements, suggesting that they were able to successfully avoid trafficking to acidic lysosomes. This work highlights the value of parallel synthesis and screening approaches for the discovery of new polymers for gene delivery and the elucidation of structure/function relationships for this important class of materials.

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Methods that permit the deposition or assembly of reactive polymer films on topologically complex substrates are useful for the patterning or chemical modification of surfaces of interest in a broad range of applications. [1][2][3][4][5] Here, we report a layer-by-layer approach to the assembly of covalently-crosslinked ultrathin films that makes use of fast and efficient 'click'-type interfacial reactions between poly(2-alkenyl azlactone)s and appropriately functionalized polyamines. In contrast to conventional, aqueous methods for the layer-by-layer fabrication of multilayered assemblies composed of polyelectrolytes, [6][7][8][9] fabrication of these materials occurs in organic solvents and is driven by rapid interfacial formation of covalent bonds during assembly. These methods permit precise, nanometer-scale control over the thicknesses and compositions of covalently crosslinked thin films. In addition, we demonstrate that it is possible to chemically tailor the properties of surfaces coated with these ultrathin films post-fabrication by exploiting the accessibility and reactivity of residual 'spring-loaded' azlactone functionality. These results suggest the basis of methods for the post-fabrication modification of curved or topologically complex surfaces coated with multilayered films and the patterning or passivation of surfaces with chemical or biological functionality of interest in the contexts of catalysis, medicine, and other areas of biotechnology. Aqueous methods for the layer-by-layer deposition of oppositely charged polyelectrolytes on surfaces are used widely for the bottom-up assembly of nanostructured polymer films. [6][7][8][9] These methods generally take advantage of multivalent weak interactions (e.g., electrostatic or hydrogen bonding interactions) between polyelectrolytes and oppositely charged surfaces and allow precise control over the thicknesses, compositions, and morphologies of thin films fabricated from a broad range of water-soluble polymers.

We present the fabrication of conformal, hydrolytically degradable thin films capable of administering sustained, multiagent release profiles. Films are constructed one molecular layer at a time by using the layer-by-layer, directed-deposition technique; the subsequent hydrolytic surface erosion of these systems results in the release of incorporated materials in a sequence that reflects their relative positions in the film. The position of each species is determined by its ability to diffuse throughout the film architecture, and, as such, the major focus of this work is to define strategies that physically block interlayer diffusion during assembly to create multicomponent, stratified films. By using a series of radiolabeled polyelectrolytes as experimental probes, we show that covalently crosslinked barriers can effectively block interlayer diffusion, leading to compartmentalized structures, although even very large numbers of ionically crosslinked (degradable or nondegradable) barrier layers cannot block interlayer diffusion. By using these principles, we designed degradable films capable of extended release as well as both parallel and serial multiagent release. The ability to fabricate multicomponent thin films with nanoscale resolution may lead to a host of new materials and applications.T he ability to engineer surfaces that present multiple functionalities when and where they are needed could lead to important advances in electrooptical devices, separations, and biomaterials (1, 2). For example, in the area of drug delivery, there is a need for low-cost ''smart'' coatings that balance the ability to release complex drug profiles with the flexibility of incorporation into a range of biomaterials, including those with large area sizes or nonplanar geometries, such as pins, sutures, prosthetic bones, devices, and microparticles. The layer-by-layer (LbL) electrostatic assembly technique is ideally suited for such applications because it allows for absolute control over the order in which multiple functional elements are incorporated into a growing film (3). However, the development of truly stratified, multicompartment LbL films has been largely unsuccessful with many biomacromolecules because of the phenomenon of interlayer diffusion, which results in blended structures lacking regular, controlled order (4). In this work, we systematically probe a range of strategies designed to solve this problem by placing physical barriers between various components within a single film to control interlayer diffusion. We measure the effect of each type of barrier with a system consisting of a hydrolytically degradable polymer (Fig. 1, polymer 1) alternately deposited with a series of radiolabeled polyelectrolytes. Top-down film degradation results in the release of components in a sequence that reflects their relative positions in the film; thus, we can quantify the effects of various barrier strategies aimed at limiting diffusion behavior. With this approach, we uncover a set of strategies that allow for the production...

Materials that provide spatial and temporal control over the delivery of DNA and other nucleic acid-based agents from surfaces play important roles in the development of localized gene-based therapies. This review focuses on a relatively new approach to the immobilization and release of DNA from surfaces: methods based on the layer-by-layer assembly of thin multilayered films (or polyelectrolyte multilayers, PEMs). Layer-by-layer methods provide convenient, nanometer-scale control over the incorporation of DNA, RNA, and oligonucleotide constructs into thin polyelectrolyte films. Provided that these assemblies can be designed in ways that permit controlled film disassembly under physiological conditions, this approach can contribute new methods for spatial and/or temporal control over the delivery of nucleic acid-based therapeutics in vitro and in vivo. We describe applications of layer-by-layer assembly to the fabrication of DNA-containing films that can be used to provide control over the release of plasmid DNA from the surfaces of macroscopic objects and promote surface-mediated cell transfection. We also highlight the application of these methods to the coating of colloidal substrates and the fabrication of hollow micrometer-scale capsules that can be used to encapsulate and control the release or delivery of DNA and oligonucleotides. Current challenges, gaps in knowledge, and new opportunities for the development of these methods in the general area of gene delivery are discussed.