Degradable polyelectrolyte multilayers that promote the release of siRNA.

We report an approach to the design of degradable polyelectrolyte-based films for the controlled release of siRNA from surfaces. Our approach is based on stepwise, layer-by-layer assembly of multilayered polyelectrolyte films (or "polyelectrolyte multilayers", PEMs) using siRNA and a hydrolytically degradable poly(ß-amino ester) (polymer 1). Fabrication of films using siRNA sequences for green fluorescent protein (GFP) or firefly luciferase resulted in linear growth of ultrathin films (∼50 nm thick) that promoted the surface-mediated release of siRNA upon incubation in physiologically relevant media. Physicochemical characterization of these siRNA-containing films revealed large differences in film growth profiles, physical erosion profiles, and siRNA release profiles as compared to PEMs fabricated using polymer 1 and larger plasmid DNA constructs. For example, whereas films fabricated using plasmid DNA erode gradually and release DNA over a period of ∼48 h, films fabricated using siRNA released ∼65% of incorporated siRNA within the first hour of incubation, prior to the onset of any observed film erosion. This initial burst of release was followed by a second, slower phase of release (accompanied by gradual film erosion) over the next 23 h. These differences in release profiles and other behaviors likely result, at least in part, from large differences in the sizes of siRNA and plasmid DNA. Finally, we demonstrate that the siRNA in these films is released in a form that remains intact, functional, and able to silence targeted protein expression upon administration to mammalian cells in vitro. The results of this investigation provide a platform for the design of thin films and coatings that could be used to localize the release of siRNA from surfaces in a variety of fundamental and applied contexts (e.g., for development of new research tools or approaches to delivery from film-coated implants and other devices).

Fabrication of Covalently Crosslinked and Amine-Reactive Microcapsules by Reactive Layer-by-Layer Assembly of Azlactone-Containing Polymer Multilayers on Sacrificial Microparticle Templates.

We report on the fabrication of covalently crosslinked and amine-reactive hollow microcapsules using 'reactive' layer-by-layer assembly to deposit thin polymer films on sacrificial microparticle templates. Our approach is based on the alternating deposition of layers of a synthetic polyamine and a polymer containing reactive azlactone functionality. Multilayered films composed of branched poly(ethylene imine) (BPEI) and poly(2-vinyl-4,4-dimethylazlactone) (PVDMA) were fabricated layer-by-layer on the surfaces of calcium carbonate and glass microparticle templates. After fabrication, these films contained residual azlactone functionality that was accessible for reaction with amine-containing molecules. Dissolution of the calcium carbonate or glass cores using aqueous ethylenediamine tetraacetic acid (EDTA) or hydrofluoric acid (HF), respectively, led to the formation of hollow polymer microcapsules. These microcapsules were robust enough to encapsulate and retain a model macromolecule (FITC-dextran) and were stable for at least 22 hours in high ionic strength environments, in low and high pH solutions, and in several common organic solvents. Significant differences in the behaviors of capsules fabricated on CaCO(3) and glass cores were observed and characterized using scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDS). Whereas capsules fabricated on CaCO(3) templates collapsed upon drying, capsules fabricated on glass templates remained rigid and spherical. Characterization using EDS suggested that this latter behavior results, at least in part, from the presence of insoluble metal fluoride salts that are trapped or precipitate within the walls of capsules after etching of the glass cores using HF. Our results demonstrate that the assembly of BPEI/PVDMA films on sacrificial templates can be used to fabricate reactive microcapsules of potential use in a wide range of fields, including catalysis, drug and gene delivery, imaging, and biomedical research.

Characterization of pH-induced changes in the morphology of polyelectrolyte multilayers assembled from poly(allylamine) and low molecular weight poly(acrylic acid).

We report characterization of pH-dependent behavior in polyelectrolyte multilayers (PEMs) fabricated from poly(allylamine) (PAH) and low molecular weight poly(acrylic acid) (PAA) synthesized by living/controlled polymerization. Exposure of these films to solutions of low pH (e.g. pH 2.0-3.2) resulted in transformations from films that were smooth and uniform to films with porous morphologies, as characterized by scanning electron microscopy (SEM). We observed large differences in both the extent of this transformation and the sizes of the pores that resulted compared to films fabricated using higher molecular weight PAA used in past studies. Whereas transformations reported in past studies generally lead to pores with sizes in the range of 0.3-2 µm, we observed larger-scale transformations and films with cell-like internal structures comprised of networks of closed pores, interconnected pores, and through-pores with sizes as large as 10-15 µm depending on pH and the manner in which the films were incubated. Films fabricated using fluorescently end-labeled samples of PAA permitted real-time imaging of changes in internal structure using confocal microscopy (LSCM). The results of these studies also revealed large differences in the nature of these transformations when films were placed in contact with surfaces as opposed to when dipped into aqueous solutions. Our results reveal approaches that can be used to fabricate films with large pores (e.g., pores with sizes on the order of 10-15 µm) and suggest methods that could potentially be used to generate PEMs having controlled gradients in pore size.

Rapid release of plasmid DNA from polyelectrolyte multilayers: a weak poly(acid) approach.

We demonstrate an approach to the assembly of DNA-containing polyelectrolyte multilayers that can be used to promote rapid release of DNA from surfaces. The approach is based on layer-by-layer incorporation of poly(acrylic acid) to promote rapid erosion in physiologically relevant media.

Layer-by-layer assembly of DNA- and protein-containing films on microneedles for drug delivery to the skin.

Microneedle patches contain micrometer-scale needles coated with bioactive agents for minimally invasive drug delivery to the skin. In this study, we introduce layer-by-layer approaches to the fabrication of ultrathin DNA- and protein-containing polyelectrolyte films (or "polyelectrolyte multilayers", PEMs) on the surfaces of stainless steel microneedles. DNA-containing PEMs were fabricated on microneedles by the alternating deposition of plasmid DNA and a hydrolytically degradable poly(ß-amino ester). Protein-containing PEMs were fabricated using sodium poly(styrene sulfonate) (SPS) and bovine pancreatic ribonuclease A (RNase A) conjugated to a synthetic protein transduction domain. Layer-by-layer assembly resulted in ultrathin, uniform, and defect-free coatings on the surfaces of the microneedles, as characterized by fluorescence microscopy. These films eroded and thereby released DNA or protein when incubated in saline or when inserted into porcine cadaver skin and deposited DNA or protein along the edges of microneedle tracks to depths of ∼500 to 600 µm. We conclude that PEM-coated microneedles offer a novel and useful approach to the transdermal delivery of DNA- and protein-based therapeutics and could also prove useful in other applications.

Polyelectrolyte multilayers fabricated from antifungal ß-peptides: design of surfaces that exhibit antifungal activity against Candida albicans.

The fungal pathogen Candida albicans can form biofilms on the surfaces of medical devices that are resistant to drug treatment and provide a reservoir for recurrent infections. The use of fungicidal or fungistatic materials to fabricate or coat the surfaces of medical devices has the potential to reduce or eliminate the incidence of biofilm-associated infections. Here we report on (i) the fabrication of multilayered polyelectrolyte thin films (PEMs) that promote the surface-mediated release of an antifungal ß-peptide and (ii) the ability of these films to inhibit the growth of C. albicans on film-coated surfaces. We incorporated a fluorescently labeled antifungal ß-peptide into the structures of PEMs fabricated from poly-l-glutamic acid (PGA) and poly-l-lysine (PLL) using a layer-by-layer fabrication procedure. These films remained stable when incubated in culture media at 37 °C and released ß-peptide gradually into solution for up to 400 h. Surfaces coated with ß-peptide-containing films inhibited the growth of C. albicans , resulting in a 20% reduction of cell viability after 2 h and a 74% decrease in metabolic activity after 7 h when compared to cells incubated on PGA/PLL-coated surfaces without ß-peptide. In addition, ß-peptide-containing films inhibited hyphal elongation by 55%. These results, when combined, demonstrate that it is possible to fabricate ß-peptide-containing thin films that inhibit the growth and proliferation of C. albicans and provide the basis of an approach that could be used to inhibit the formation of C. albicans biofilms on film-coated surfaces. The layer-by-layer approach reported here could ultimately be used to coat the surfaces of catheters, surgical instruments, and other devices to inhibit drug-resistant C. albicans biofilm formation in clinical settings.

Characterization of nanoscale transformations in polyelectrolyte multilayers fabricated from plasmid DNA using laser scanning confocal microscopy in combination with atomic force microscopy.

Laser scanning confocal microscopy (LSCM) and atomic force microscopy (AFM) were used to characterize changes in nanoscale structure that occur when ultrathin polyelectrolyte multilayers (PEMs) are incubated in aqueous media. The PEMs investigated here were fabricated by the deposition of alternating layers of plasmid DNA and a hydrolytically degradable polyamine onto a precursor film composed of alternating layers of linear poly(ethylene imine) (LPEI) and sodium poly(styrene sulfonate) (SPS). Past studies of these materials in the context of gene delivery revealed transformations from a morphology that is smooth and uniform to one characterized by the formation of nanometer-scale particulate structures. We demonstrate that in-plane registration of LSCM and AFM images acquired from the same locations of films fabricated using fluorescently labeled polyelectrolytes allows the spatial distribution of individual polyelectrolyte species to be determined relative to the locations of topographic features that form during this transformation. Our results suggest that this physical transformation leads to a morphology consisting of a relatively less disturbed portion of film composed of polyamine and DNA juxtaposed over an array of particulate structures composed predominantly of LPEI and SPS. Characterization by scanning electron microscopy and energy-dispersive X-ray microanalysis provides additional support for this interpretation. The combination of these different microscopy techniques provides insight into the structures and dynamics of these multicomponent thin films that cannot be achieved using any one method alone, and could prove useful for the further development of these assemblies as platforms for the surface-mediated delivery of DNA.

Multilayered films fabricated from an oligoarginine-conjugated protein promote efficient surface-mediated protein transduction.

The conjugation of cationic protein transduction domains to proteins results in an increase in the extent to which proteins are internalized by cells. This investigation sought to determine whether the conjugation of a protein transduction domain to a functional protein could be used to facilitate the incorporation of the protein into multilayered polyelectrolyte films and, subsequently, whether these films could be used to promote surface-mediated protein transduction. We demonstrate that it is possible to fabricate multilayered assemblies 80 nm thick using sodium polystyrene sulfonate (SPS) and bovine pancreatic ribonuclease (RNase A) conjugated to the cationic protein transduction domain nonaarginine (R(9)) using an entirely aqueous layer-by-layer process. We demonstrate further that the conjugation of R(9) to RNase A permits the assembly of multilayered films under conditions that do not allow for the incorporation of the unmodified protein. This result suggests that R(9) functions as a cationic anchor and serves to increase the strength of electrostatic interactions with SPS and facilitate layer-by-layer assembly. We also demonstrate that RNase A-R(9)/SPS films dissolve rapidly in physiologically relevant media and that macroscopic objects coated with these materials can be used to mediate high levels of protein transduction in mammalian cells. These results suggest the basis of general methods that could contribute to the design of materials that permit spatial and temporal control over the delivery of therapeutic proteins to cells and tissues.

Pyridinium-1-yl bisphosphonates are potent inhibitors of farnesyl diphosphate synthase and bone resorption.

We report the design, synthesis and testing of a series of novel bisphosphonates, pyridinium-1-yl-hydroxy-bisphosphonates, based on the results of comparative molecular similarity indices analysis and pharmacophore modeling studies of farnesyl diphosphate synthase (FPPS) inhibition, human Vgamma2Vdelta2 T cell activation and bone resorption inhibition. The most potent molecules have high activity against an expressed FPPS from Leishmania major, in Dictyostelium discoideum growth inhibition, in gammadelta T cell activation and in an in vitro bone resorption assay. As such, they represent useful new leads for the discovery of new bone resorption, antiinfective and anticancer drugs.

Effects of bisphosphonates on the growth of Entamoeba histolytica and Plasmodium species in vitro and in vivo.

The effects of a series of 102 bisphosphonates on the inhibition of growth of Entamoeba histolytica and Plasmodium falciparum in vitro have been determined, and selected compounds were further investigated for their in vivo activity. Forty-seven compounds tested were active (IC(50) < 200 microM) versus E. histolytica growth in vitro. The most active compounds (IC(50) approximately 4-9 microM) were nitrogen-containing bisphosphonates with relatively large aromatic side chains. Simple n-alkyl-1-hydroxy-1,1-bisphosphonates, known inhibitors of the enzyme farnesylpyrophosphate (FPP) synthase, were also active, with optimal activity being found with C9-C10 side chains. However, numerous other nitrogen-containing bisphosphonates known to be potent FPP synthase inhibitors, such as risedronate or pamidronate, had little or no activity. Several pyridine-derived bisphosphonates were quite active (IC(50) approximately 10-20 microM), and this activity was shown to correlate with the basicity of the aromatic group, with activity decreasing with increasing pK(a) values. The activities of all compounds were tested versus a human nasopharyngeal carcinoma (KB) cell line to enable an estimate of the therapeutic index (TI). Five bisphosphonates were selected and then screened for their ability to delay the development of amebic liver abscess formation in an E. histolytica infected hamster model. Two compounds were found to decrease liver abscess formation at 10 mg/kg ip with little or no effect on normal liver mass. With P. falciparum, 35 compounds had IC(50) values <200 microM in an in vitro assay. The most active compounds were also simple n-alkyl-1-hydroxy-1,1-bisphosphonates, having IC(50) values around 1 microM. Five compounds were again selected for in vivo investigation in a Plasmodium berghei ANKA BALB/c mouse suppressive test. The most active compound, a C9 n-alkyl side chain containing bisphosphonate, caused an 80% reduction in parasitemia with no overt toxicity. Taken together, these results show that bisphosphonates appear to be useful lead compounds for the development of novel antiamebic and antimalarial drugs.