(645d) Transdermal Delivery of DNA and Protein Using Stainless Steel Microneedle Arrays Coated with Ultrathin Polyelectrolyte Multilayers

Transdermal delivery of macromolecular drugs using microneedles presents an attractive platform for the administration of protein- or DNA-based vaccines. By creating small channels in the stratum corneum, microneedles substantially improve the transdermal transport of macromolecules. In addition, the insertion of microneedles is generally painless, and arrays of needles can be applied in the form of patches for simple self-administration. Although a variety of methods have been reported for the fabrication of microneedle arrays and the loading and release of a range of small-molecule and macromolecular agents, the development of new materials and methods for the fabrication of DNA- and protein-containing thin films on the surfaces of microneedles will be critical to further advancing this approach.

Here, we report (i) the fabrication and characterization of ultrathin multilayered polyelectrolyte assemblies (or ?polyelectrolyte multilayers') containing either plasmid DNA or a cationic protein to coat the surfaces of arrays of stainless steel microneedles (e.g., ~750 micrometers in length) and (ii) the ability of film-coated needles to deliver DNA or protein across porcine cadaver skin. Ultrathin films (e.g., ~100 to 200 nm thick) were fabricated layer-by-layer directly on the surfaces of microneedle arrays using entirely aqueous assembly methods adapted from those used previously for the assembly of DNA- and protein-containing films on the surfaces of macroscopic objects. DNA-containing multilayers were fabricated by the alternating deposition of layers of fluorescently labeled plasmid DNA encoding enhanced green fluorescent protein (EGFP) and a synthetic, hydrolytically degradable poly(beta-amino ester). Protein-containing multilayers were fabricated by the alternating deposition of layers of sodium poly(styrene sulfonate) and fluorescently labeled Ribonuclease A (RNase A) modified with a cationic nonaarginine (R₉) sequence.

Assembly of DNA- and protein-containing multilayers on microneedle arrays resulted in ultrathin, uniform, and defect-free coatings on the surfaces of the microneedles, as characterized by fluorescence microscopy. Incubation of microneedles coated with DNA-containing films in physiologically relevant media (e.g., PBS, pH = 7.4, 37 °C) resulted in the release of DNA into solution over a period of ~2 days. Subsequent characterization of released DNA by agarose gel electrophoresis demonstrated that the DNA was released in a physically intact form. Additional cell-based experiments demonstrated that the released DNA was functional and capable of promoting the expression of EGFP in a mammalian cell line. Incubation of microneedle arrays coated with protein-containing films in physiologically relevant media resulted in rapid release of protein (e.g., release of protein from the surfaces of the microneedles was typically complete in ~1 hour). These results are similar to the results of past studies on the release of DNA and protein from these films fabricated on the surfaces of macroscopic objects.

We also performed a series of experiments to determine whether these ultrathin films could withstand the range of physical forces associated with the penetration of microneedles into skin, and, subsequently, whether film-coated microneedle arrays could be used to promote the transdermal delivery of fluorescently labeled DNA and protein. The results of experiments using porcine cadaver skin as a model demonstrated that coated microneedle arrays could deliver significant levels of DNA and protein into the epidermal and dermal layers of the skin when inserted for a period of up to two hours. Characterization of histological sections of samples of skin after removal of the needle arrays using fluorescence microscopy demonstrated that DNA and protein were released and delivered into the skin along the surfaces of needle tracks to depths of ~500 to 600 nm into the skin, suggesting that these ultrathin films are mechanically robust and able to withstand shear forces associated with the insertion of the needles into porcine cadaver skin (i.e., the films do not peel or crack and are not otherwise scraped off of the surfaces of the microneedles during insertion). Characterization of the surfaces of the microneedles after removal from skin demonstrated that nearly all of the DNA and protein were released from the needles during the two-hour incubation period.

When combined, the results above suggest that layer-by-layer coatings may be suitable for promoting the transdermal delivery of therapeutic macromolecules or protein- or DNA-based vaccines. The results of additional experiments to characterize and tune the release profiles of these films both in solution and when inserted into skin will be presented, and opportunities for the development of new approaches to the transdermal delivery of therapeutically relevant amounts of DNA-based vaccines will be discussed.