Assembly of erodible, DNA-containing thin films on the surfaces of polymer microparticles: toward a layer-by-layer approach to the delivery of DNA to antigen-presenting cells

Abstract

We report a layer-by-layer approach to the assembly of ultrathin and erodible DNA-containing films on the surfaces of polymer microparticles. DNA-containing multilayered films were fabricated layer-by-layer on the surfaces of polystyrene microspheres (approximately 6 microm) by iterative and alternating cycles of particle suspension, centrifugation and resuspension in solutions of plasmid DNA and a hydrolytically degradable polyamine. Film growth occurred in a stepwise manner, as demonstrated by characterization of the zeta potentials and fluorescence intensities of film-coated particles during film assembly. Characterization of film-coated particles by confocal fluorescence microscopy and scanning electron microscopy revealed the multilayered particle coatings to be smooth, uniform and free of large-scale physical defects. Film-coated microparticles sustained the release of transcriptionally active DNA into solution for approximately three days when incubated in physiologically relevant media. Previous studies have demonstrated that the adsorption of DNA onto the surfaces of cationic microparticles can be used to target the delivery of DNA to antigen-presenting cells. As a first step toward the application of this layer-by-layer approach to the development of methods for the delivery of DNA to antigen-presenting cells, we demonstrated that film-coated microparticles could be used to transport DNA into macrophage cells in vitro using a model mouse macrophage cell line. Our results suggest the basis of a general approach that could, with further development, prove useful for the delivery of DNA-encoded antigens to macrophages, or other antigen-presenting cells, and provide new materials-based methods for the formulation and delivery of DNA vaccines.

Figure 1

A) Representative confocal microscopy image of particles coated with a film 2.5 bilayers thick fabricated from polymer 1 and Cy3-labeled DNA. Scale bar = 20 μm for image, 2μm for inset. B) SEM image of a particle coated with 2.5 bilayers of polymer 1 and DNA. Scale bar = 1 μm.

Figure 2

A) Zeta potentials of particles as a function of the number of alternating polymer 1 and DNA layers adsorbed. Particles were suspended in sodium acetate buffer (100 mM; pH = 5) prior to measurement. B) Average grayscale intensities of coated particles as a function of the number of alternating polymer 1 and Cy3-labeled DNA layers adsorbed.

Figure 3

Plot of DNA released into solution versus time for particles coated with a polymer 1/DNA film 2.5 bilayers thick incubated in PBS at 37 °C.

Figure 4

A) Agarose gel electrophoresis data characterizing release of DNA from film-coated particles. Lane 1 shows control DNA at a concentration of 2 μg/mL. Lanes 2–7 are solutions from DNA release experiments (corresponding to solutions collected after 3, 6, 14, 24, 48, and 72 hours, respectively). B–C) Representative fluorescence microscopy images showing EGFP expression in COS-7 cells transfected with DNA released over 0–3 hours (B) and 24–48 hours (C) using Lipofectamine 2000 (see text). Scale bars = 100 μm.

Figure 5

A) Representative confocal microscopy image of p388d1 cells treated with film-coated microparticles fabricated using polymer 1 and Cy3-labeled DNA. Scale bar = 10 μm. B) Higher magnification image of the cell in panel A that has internalized a coated particle. Scale bar = 5μm. C) Representative image of p388d1 cells treated with microparticles coated with 2 bilayers of a polymer 1/Cy3-labeled DNA film capped with an outer layer of LPEI. Scale bar = 10 μm. D) Enlarged image of the cell in panel C that has internalized a coated particle. Scale bar = 5 μm. The red, green and blue fluorescence in these images correspond to Cy3, WGA-Alexa 488, and Hoechst fluorescent probes, respectively.

Scheme 1

Assembly of multilayered polyelectrolyte films on polymer microspheres is accomplished by successive cycles of centrifugation and resuspension in solutions of positively and negatively charged polymers. Passing the particles through a narrow-gauge syringe needle facilitates resuspension and reduces particle aggregation (see text).