Characterization of degradable polyelectrolyte multilayers fabricated using DNA and a fluorescently-labeled poly(?-amino ester): shedding light on the role of the cationic polymer in promoting surface-mediated gene delivery.
ABSTRACT: Polyelectrolyte multilayers (PEMs) fabricated from cationic polymers and DNA have been investigated broadly as materials for surface-mediated DNA delivery. One attractive aspect of this "multilayered" approach is the potential to exploit the presence of cationic polymer "layers" in these films to deliver DNA to cells more effectively. Past studies demonstrate that these films can promote transgene expression in vitro and in vivo, but significant questions remain regarding roles that the cationic polymers could play in promoting the internalization and processing of DNA. Here, we report physicochemical and in vitro cell-based characterization of DNA-containing PEMs fabricated using fluorescently end-labeled derivatives of a degradable polycation (polymer 1) used in past studies of surface-mediated transfection. This approach permitted simultaneous characterization of polymer and DNA in solution and in cells using fluorescence-based techniques, and provided information about the locations and behaviors of polymer 1 that could not be obtained using other methods. LSCM and flow cytometry experiments revealed that polymer 1 and DNA released from film-coated objects were both internalized extensively by cells and that they were colocalized to a significant extent inside cells (e.g., ~58% of DNA was colocalized with polymer). Fluorescence anisotropy measurements of solutions containing partially eroded films were also consistent with the presence of aggregates of polymer 1 and DNA in solution (e.g., after release from surfaces, but prior to internalization by cells). Our results support the view that polymer 1, which is incorporated into these materials as "layers" rather than as part of optimized, preformed "polyplexes", can act to promote or enhance surface-mediated DNA delivery. More broadly, our results suggest opportunities to improve the delivery properties of DNA-containing PEMs by incorporation of additional "layers" of other conventional cationic polymers designed to address specific intracellular barriers to transfection, such as endosomal escape, more effectively.
Project description:We report the synthesis of a fluorescently end-labeled analog of a synthetic and degradable cationic poly(?-amino ester) (PBAE; polymer 1) used in past studies for the delivery of DNA and the layer-by-layer assembly of erodible polyelectrolyte multilayers (PEMs). The synthesis of an analog of polymer 1 having acrylate functionalized end groups provided a platform for the introduction of fluorescent labels by post-polymerization conjugate addition of amine-functionalized fluorophores. This approach enabled the synthesis of fluorescently end-labeled polymer (polymer 1(FL)) with molecular weights and polydispersities (M(n) = 18,000; PDI ~1.8) similar to those used in past studies for the fabrication of PEMs using polymer 1. Layer-by-layer assembly of PEMs using polymer 1(FL) and poly(styrene sulfonate) enabled characterization of film erosion and, for the first time, direct observation of the release of cationic polymer from these assemblies using fluorescence microscopy and fluorometry. Our results shed new light on the behaviors of the cationic components of these PEMs and could prove useful for the design of thin films for a range of different controlled release applications. Our results also provide new fluorescent cationic polymer probes that could be useful for characterization of the behaviors of PBAEs in other fundamental or applied biotechnological contexts.
Project description:We report an approach to the design of multilayered polyelectrolyte thin films (or 'polyelectrolyte multilayers', PEMs) that can be used to provide tunable control over the release of plasmid DNA (or multiple different DNA constructs) from film-coated surfaces. Our approach is based upon methods for the layer-by-layer assembly of DNA-containing thin films, and exploits the properties of a new class of cationic 'charge-shifting' polymers (amine functionalized polymers that undergo gradual changes in net charge upon side chain ester hydrolysis) to provide control over the rates at which these films erode and release DNA. We synthesized two 'charge-shifting' polymers (polymers 1 and 2) containing different side chain structures by ring-opening reactions of poly(2-alkenyl azlactone)s with two different tertiary amine functionalized alcohols (3-dimethylamino-1-propanol and 2-dimethylaminoethanol, respectively). Subsequent characterization revealed large changes in the rates of side chain ester hydrolysis for these two polymers; whereas the half-life for the hydrolysis of the esters in polymer 1 was ~200 days, the half-life for polymer 2 was ~6 days. We demonstrate that these large differences in side chain hydrolysis make possible the design of PEMs that erode and promote the surface-mediated release of DNA either rapidly (e.g., over ~3 days for films fabricated using polymer 2) or slowly (e.g., over ~1 month for films fabricated using polymer 1). We demonstrate further that it is possible to design films with release profiles that are intermediate to these two extremes by fabricating films using solutions containing different mixtures of these two polymers. This approach can thus expand the usefulness of these two polymers and achieve a broader range of DNA release profiles without the need to synthesize polymers with new structures or properties. Finally, we demonstrate that polymers 1 and 2 can be used to fabricate multilayered films with hierarchical structures that promote the sequential release of two different DNA constructs with separate and distinct release profiles (e.g., the release of a first construct over a period of ~3 days, followed by the sustained release of a second for a period of ~70 days). With further development, this approach could contribute to the design of functional thin films and surface coatings that provide sophisticated control over the timing and the order of the release of two or more DNA constructs (or other agents) of interest in a range of biomedical contexts.
Project description:We report the design of erodible 'mixed multilayer' coatings fabricated using plasmid DNA and combinations of both hydrolytically degradable and charge-shifting cationic polymer building blocks. Films fabricated layer-by-layer using combinations of a model poly(?-amino ester) (polymer 1) and a model charge-shifting polymer (polymer 2) exhibited DNA release profiles that were substantially different than those assembled using DNA and either polymer 1 or polymer 2 alone. In addition, the order in which layers of these two cationic polymers were deposited during assembly had a profound impact on DNA release profiles when these materials were incubated in physiological buffer. Mixed multilayers ?225 nm thick fabricated by depositing layers of polymer 1/DNA onto films composed of polymer 2/DNA released DNA into solution over ?60 days, with multi-phase release profiles intermediate to and exhibiting some general features of polymer 1/DNA or polymer 2/DNA films (e.g., a period of rapid release, followed by a more extended phase). In sharp contrast, 'inverted' mixed multilayers fabricated by depositing layers of polymer 2/DNA onto films composed of polymer 1/DNA exhibited release profiles that were almost completely linear over ?60-80 days. These and other results are consistent with substantial interdiffusion and commingling (or mixing) among the individual components of these compound materials. Our results reveal this mixing to lead to new, unanticipated, and useful release profiles and provide guidance for the design of polymer-based coatings for the local, surface-mediated delivery of DNA from the surfaces of topologically complex interventional devices, such as intravascular stents, with predictable long-term release profiles.
Project description:New therapeutic approaches that eliminate or reduce the occurrence of intimal hyperplasia following balloon angioplasty could improve the efficacy of vascular interventions and improve the quality of life of patients suffering from vascular diseases. Here, we report that treatment of arteries using catheter balloons coated with thin polyelectrolyte-based films ('polyelectrolyte multilayers', PEMs) can substantially reduce intimal hyperplasia in an in vivo rat model of vascular injury. We used a layer-by-layer (LbL) process to coat the surfaces of inflatable catheter balloons with PEMs composed of nanolayers of a cationic poly(?-amino ester) (polymer 1) and plasmid DNA (pPKC?) encoding the ? isoform of protein kinase C (PKC?), a regulator of apoptosis and other cell processes that has been demonstrated to reduce intimal hyperplasia in injured arterial tissue when administered via perfusion using viral vectors. Insertion of balloons coated with polymer 1/pPKC? multilayers into injured arteries for 20 min resulted in local transfer of DNA and elevated levels of PKC? expression in the media of treated tissue three days after delivery. IFC and IHC analysis revealed these levels of expression to promote downstream cellular processes associated with up-regulation of apoptosis. Analysis of arterial tissue 14 days after treatment revealed polymer 1/pPKC?-coated balloons to reduce the occurrence of intimal hyperplasia by ~60% compared to balloons coated with films containing empty plasmid vectors. Our results demonstrate the potential therapeutic value of this nanotechnology-based approach to local gene delivery in the clinically important context of balloon-mediated vascular interventions. These PEM-based methods could also prove useful for other in vivo applications that require short-term, surface-mediated transfer of plasmid DNA.
Project description:Polycations have been successfully used as gene transfer vehicles both in vitro and in vivo; however, their cytotoxicity has been associated with increasing molecular weight. Polymers that can be rapidly degraded after internalization are typically better tolerated by mammalian cells compared to their non-degradable counterparts. Here, we report the use of a dibromomaleimide-alkyne (DBM-alkyne) linking agent to reversibly bridge cationic polymer segments for gene delivery and to provide site-specific functionalization by azide-alkyne cycloaddition chemistry. A panel of reducible and non-reducible, statistical copolymers of (2-dimethylamino)ethyl methacrylate (DMAEMA) and oligo(ethylene glycol)methyl ether methacrylate (OEGMA) were synthesized and evaluated. When complexed with plasmid DNA, the reducible and non-reducible polymers had comparable DNA condensation properties, sizes, and transfection efficiencies. When comparing cytotoxicity, the DBM-linked, reducible polymers were significantly less toxic than the non-reducible polymers. To demonstrate polymer functionalization by click chemistry, the DBM-linked polymers were tagged with an azide-fluorophore and were used to monitor cellular uptake. Overall, this polymer system introduces the use of a reversible linker, DBM-alkyne, to the area of gene delivery and allows for facile, orthogonal, and site-specific functionalization of gene delivery vehicles.
Project description:Soft polymer films, such as polyelectrolyte multilayers (PEMs), are useful coatings in materials science. The properties of PEMs often rely on the degree of hydration, and therefore the study of these films in a hydrated state is critical to allow links to be drawn between their characteristics and performance in a particular application. In this work, we detail the development of a novel soft contact cell for studying hydrated PEMs (poly(sodium 4-styrenesulfonate)/poly(allylamine hydrochloride)) using FTIR microspectroscopy. FTIR spectroscopy can interrogate the nature of the polymer film and the hydration water contained therein. In addition to reporting spectra obtained for hydrated films confined at the solid-solid interface, we also report traditional ATR FTIR spectra of the multilayer. The spectra (microspectroscopy and ATR FTIR) reveal that the PEM film build-up proceeds as expected based on the layer-by-layer assembly methodology, with increasing signals from the polymer FTIR peaks with increasing bilayer number. In addition, the spectra obtained using the soft contact cell indicate that the PEM film hydration water has an environment/degree of hydrogen bonding that is affected by the chemistry of the multilayer polymers, based on differences in the spectra obtained for the hydration water within the film compared to that of bulk electrolyte.
Project description: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(?-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).
Project description:Glioblastoma multiforme is a devastating disease that has attracted enormous attention due to poor prognosis and high recurrence. Small interfering RNA (siRNA) in principle offers a promising therapeutic approach by the downregulation of disease-related genes via RNA interference. For efficient siRNA delivery to target sites, cationic polymers are often used in preclinical studies for the protection of siRNA and complex formation based on electrostatic interactions. In an effort to develop biocompatible and efficient nanocarriers with a translational outlook for optimal gene silencing at reduced toxicity, we synthesized two sets of nylon-3 copolymers with variable cationic content (DM or NM monomer) and hydrophobic subunits (CP monomer) and evaluated their suitability for in vitro siRNA delivery into glioblastoma cells. DM0.4/CP0.6 and NM0.4/CP0.6 polymers with similar subunit ratios were synthesized to compare the effect of different cationic subunits. Additionally, we utilized NM0.2/CP0.8 polymers to evaluate the impact of the different hydrophobic content in the polymer chain. The siRNA condensation ability and polymer-siRNA complex stability was evaluated by unmodified and modified SYBR gold assays, respectively. Further physicochemical characteristics, e.g., particle size and surface charge, were evaluated by dynamic light scattering and laser Doppler anemometry, whereas a relatively new method for polyplex size distribution analysis-tunable resistive pulse sensing-was additionally developed and compared to DLS measurements. Transfection efficiencies, the route of cell internalization, and protein knockdown abilities in glioblastoma cells were investigated by flow cytometry. Furthermore, cellular tolerability was evaluated by MTT and LDH assays. All the polymers efficiently condensed siRNA at N/P ratios of three, whereas polymers with NM cationic subunits demonstrated smaller particle size and lower polyplex stability. Furthermore, NM0.2/CP0.8 polyplexes with the highest hydrophobic content displayed significantly higher cellular internalization in comparison to more cationic formulations and successful knockdown capabilities. Detailed investigations of the cellular uptake route demonstrated that these polyplexes mainly follow clathrin-mediated endocytotic uptake mechanisms, implying high interaction capacity with cellular membranes. Taken together with conducive toxicity profiles, highly hydrophobic nylon-3 polymers provide an appropriate siRNA delivery agent for the potential treatment of glioblastoma.
Project description:Polymer composition and morphology can affect the way polymers interact with biomolecules, cell membranes, and intracellular components. Herein, diblock, triblock, and statistical polymers that varied in charge center type (primary and/or tertiary amines) were synthesized to elucidate the role of polymer composition on plasmid DNA complexation, delivery, and cellular toxicity of the resultant polyplexes. The polymers were synthesized via RAFT polymerization and were composed of a carbohydrate moiety, 2-deoxy-2-methacrylamido glucopyranose (MAG), a primary amine group, N-(2-aminoethyl) methacrylamide (AEMA), and/or a tertiary amine moiety, N,N-(2-dimethylamino)ethyl methacrylamide (DMAEMA). The lengths of both the carbohydrate and cationic blocks were kept constant while the primary amine to tertiary amine ratio was varied within the polymers. The polymers were characterized via nuclear magnetic resonance (NMR) and size exclusion chromatography (SEC), and the polyplex formulations with pDNA were characterized in various media using dynamic light scattering (DLS). Polyplexes formed with the block copolymers were found to be more colloidally stable than statistical copolymers with similar composition, which rapidly aggregated to micrometer sized particles. Also, polymers composed of a higher primary amine content were more colloidally stable than polymers consisting of the tertiary amine charge centers. Plasmid DNA internalization, transgene expression, and toxicity were examined with each polymer. As the amount of tertiary amine in the triblock copolymers increased, both gene expression and toxicity were found to increase. Moreover, it was found that increasing the content of tertiary amines imparted higher membrane disruption/destabilization. While both block and statistical copolymers had high transfection efficiencies, some of the statistical systems exhibited both higher transfection and toxicity than the analogous block polymers, potentially due to the lack of a hydrophilic block to screen membrane interaction/disruption. Overall, the triblock terpolymers offer an attractive composition profile that exhibited interesting properties as pDNA delivery vehicles.
Project description:We report an approach to the rapid release of DNA based on the application of electrochemical potentials to surfaces coated with polyelectrolyte-based thin films. We fabricated multilayered polyelectrolyte films (or "polyelectrolyte multilayers", PEMs) using plasmid DNA and a model hydrolytically degradable cationic poly(?-amino ester) (polymer 1) on stainless steel substrates using a layer-by-layer approach. The application of continuous reduction potentials in the range of -1.1 to -0.7 V (vs a Ag/AgCl electrode) to film-coated electrodes in PBS at 37 °C resulted in the complete release of DNA over a period of 1-2 min. Film-coated electrodes incubated under identical conditions in the absence of applied potentials required 1-2 days for complete release. Control over the magnitude of the applied potential provided control over the rate at which DNA was released. The results of these and additional physical characterization experiments are consistent with a mechanism of film disruption that is promoted by local increases in pH at the film/electrode interface (resulting from electrochemical reduction of water or dissolved oxygen) that disrupt ionic interactions in these materials. The results of cell-based experiments demonstrated that DNA was released in a form that remains intact and able to promote transgene expression in mammalian cells. Finally, we demonstrate that short-term (i.e., non-continuous) electrochemical treatments can also be used to promote faster film erosion (e.g., over 1-2 h) once the potential is removed. Past studies demonstrate that PEMs fabricated using polymer 1 can promote surface-mediated transfection of cells and tissues in vitro and in vivo. With further development, the electrochemical approaches reported here could thus provide new methods for the rapid, triggered, or spatially patterned transfer of DNA (or other agents) from surfaces of interest in a variety of fundamental and applied contexts.