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.

Thermally reversible surface morphology transition in thin diblock copolymer films.

Many phase transitions exhibit ordering transitions at the boundary of the material that are distinct from its interior where intermolecular interactions can be significantly different. The present work considers the existence of a surface thermodynamic order-order transition between two distinct morphologies in thin block copolymer (BCP) films that are of interest in nanomanufacturing applications. Specifically, we find a thermally reversible interfacial transition between sphere-like structures and cylinders in flow-coated films of poly(styrene-block-methyl methacrylate) (PS-b-PMMA), where the BCP forms a cylinder microphase in the bulk. We present direct evidence from atomic force microscopy (AFM) of ion-etched films and grazing-incidence small-angle X-ray scattering (GISAXS) on films without etching, which shows that the order-order transition is restricted to the outer layer of the film, while the film interior remains in the cylinder state. Moreover, we find this order-order transition to be insensitive to film thickness over the range investigated (40-170 nm). This morphological transition is of importance in characterizing the thermodynamics and dynamics of thin BCP films used as templates in nanomanufacturing applications.

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.

Challenges in fabrication of mesoporous carbon films with ordered cylindrical pores via phenolic oligomer self-assembly with triblock copolymers.

Mesoporous phenol formaldehyde (PF) polymer resin and carbon films are prepared by a solution self-assembly of PF oligomers with amphiphilic triblock copolymers. After thermopolymerization of the PF to cross-link the network, the films show an ordered morphology as determined by X-ray diffraction and grazing incidence small-angle X-ray scattering (GISAXS). Our results show that the amphiphilic triblock copolymer template greatly influences the stability of the final porous mesostructures. The pyrolysis of the two-dimensional (2-D) hexagonal films with p6mm symmetry templated by Pluronic F127 yields a disordered porous structure following the template removal. Conversely, films templated by Pluronic P123 can exhibit well-ordered cylindrical pores after the template removal, but the solution composition range to yield ordered cylindrical mesopores is significantly reduced (nearly 70%) for thin films in comparison to bulk powders. We propose two dominant difficulties in fabricating well-ordered cylindrical mesopores in films: first, the stress from contraction during the pyrolysis can lead to a collapse of the mesostructure if the wall thickness is insufficient, and second, the surface wetting behavior in thin films leads to a small compositional range.

Nanoimprinted thin films of reactive, azlactone-containing polymers: combining methods for the topographic patterning of cell substrates with opportunities for facile post-fabrication chemical functionalization.

Approaches to the fabrication of surfaces that combine methods for the topographic patterning of soft materials with opportunities for facile, post-fabrication chemical functionalization could contribute significantly to advances in biotechnology and a broad range of other areas. Here, we report methods that can be used to introduce well-defined nano- and microscale topographic features to thin films of reactive polymers containing azlactone functionality using nanoimprint lithography (NIL). We demonstrate that NIL can be used to imprint topographic patterns into thin films of poly(2-vinyl-4,4-dimethylazlactone) and a copolymer of methyl methacrylate and 2-vinyl-4,4-dimethylazlactone using silicon masters having patterns of grooves and ridges ranging in width from 400 nm to 2 microm, demonstrating the potential of this method to transfer patterns to films of these reactive polymers over a range of feature sizes and densities. We demonstrate further that the azlactone functionality of these polymers survives temperatures and pressures associated with NIL, and that topographically patterned films can be readily functionalized post-fabrication by treatment of surface-accessible azlactone functionality with small molecules and polymers containing primary amines. The results of experiments in which NIH-3T3 cells were seeded onto films imprinted with lined patterns having a pitch of 4 microm demonstrated that cells attach and proliferate on these azlactone-containing films and that they align in the direction of the imprinted pattern. Finally, we demonstrate that the treatment of these materials with amine-functionalized poly(ethylene glycol) (PEG) can be used to create regions of topographically patterned films that prevent cell adhesion. The results of this study suggest approaches to the functionalization of topographically patterned surfaces with a broad range of chemical functionality (e.g., peptides, proteins, carbohydrates, etc.) of biotechnological interest. The ability to manipulate and define both the physical topography and chemical functionality of these reactive materials could provide opportunities to investigate the combined effects of substrate topography and chemical functionality on cell behavior and may also be useful in a broad range of other applications.

Apparent dewetting of ultrathin multilayered polyelectrolyte films incubated in aqueous environments.

We have investigated and characterized changes in film morphology and surface structure that occur when ultrathin multilayered polyelectrolyte films fabricated from linear poly(ethylene imine) (LPEI), sodium poly(styrene sulfonate) (SPS), and two hydrolytically degradable polyamines (polymers 1 and 2) are incubated in physiologically relevant environments. Characterization of the physical erosion profiles of films having the structure (LPEI/SPS)10(1/SPS)4(2/SPS)4 (approximately 80 nm thick) by atomic force microscopy (AFM), reflective optical microscopy, and scanning electron microscopy (SEM) demonstrated that these materials undergo large-scale changes in surface structure and morphology upon incubation in phosphate-buffered saline (PBS) at 37 degrees C. The patterns and structures generated during this transformation (e.g., nucleation and growth of holes, coalescence of holes, formation of cell-type structures, and the subsequent breakup of these features into droplets) are similar in many ways to those observed for the dewetting of thin films of conventional polymers, such as polystyrene, on nonwetting surfaces. The processes reported here are sufficiently slow (they occur over approximately 100 h) and occur under sufficiently mild conditions (e.g., incubation in PBS at 37 degrees C) to permit characterization and quantification of the structures and features that arise during the course of these transformations. The apparent dewetting of these ultrathin films upon exposure to aqueous environments creates future opportunities to investigate and characterize processes of mass transport in this class of ionically cross-linked assemblies.

Assembly of multilayered films using well-defined, end-labeled poly(acrylic acid): influence of molecular weight on exponential growth in a synthetic weak polyelectrolyte system.

We report on the influence of polyanion molecular weight on the growth and structure of multilayered thin films fabricated from poly(allylamine) (PAH) and well-defined, end-labeled poly(acrylic acid) (PAA) synthesized by atom transfer radical polymerization. We observed striking differences in the growth of PAH/PAA films fabricated using well-defined PAA compared to films fabricated using higher molecular weight, commercially available PAA. Past studies demonstrate that the thicknesses of PAH/PAA films increase as linear functions of the number of PAH and PAA layers deposited over a broad range of pH (e.g., from pH 2.5 to 4.5). We observed the thicknesses of films fabricated using solutions of PAH and PAA adjusted to pH 7.5 and 3.5, respectively, to increase in a nonlinear manner. Films fabricated using well-defined, low molecular weight samples of PAA under these conditions increased in thickness exponentially. Experiments using samples of PAA having substantially non-overlapping molecular weight distributions demonstrated a clear relationship between the molecular weight of PAA and rates of film growth. We also used confocal microscopy, in combination with fluorescently end-labeled samples of PAA, to characterize the location of PAA in these assemblies. The results of these experiments, when combined, support the general conclusion that PAA is able to penetrate or diffuse into these films over large distances during assembly. The mechanism of growth for these films thus appears similar to that recently reported for the exponential growth of films fabricated using a variety of biologically relevant polyelectrolytes. The use of living/controlled methods of polymerization to synthesize well-defined samples of PAA facilitates an interpretation of these differences in film behavior as arising largely from differences in polymer molecular weight and polydispersity. This work provides insight into the assembly and structure of a well-studied weak polyelectrolyte film system and illustrates the potential of living/controlled methods of polymerization to contribute to the characterization and understanding of the physical properties of these ionically cross-linked materials.

Nanometer-scale decomposition of ultrathin multilayered polyelectrolyte films.

We report that ultrathin multilayered films fabricated from plasmid DNA and synthetic polyamines undergo nanometer-scale transformations that resemble spinodal decomposition when incubated in aqueous media. The patterns and structures generated by this transformation are similar to those observed for the spinodal dewetting of thin films of conventional polymers. This behavior has not, however, been observed for this class of multilayered assemblies, for which long-range electrostatic interactions play significant roles in governing film structure and stability. We demonstrate that it is possible to promote this behavior, prevent it, or control it by varying polymer structure, film composition, or the conditions to which these materials are exposed. These results suggest the basis of methods that could prove useful for the generation of nanostructure on complex surfaces and contribute to methods for the localized delivery of DNA from surfaces.

Release of plasmid DNA from intravascular stents coated with ultrathin multilayered polyelectrolyte films.

Materials that permit control over the release of DNA from the surfaces of topologically complex implantable devices, such as intravascular stents, could contribute to the development of new approaches to the localized delivery of DNA. We report the fabrication of ultrathin, multilayered polyelectrolyte films that permit both the immobilization and controlled release of plasmid DNA from the surfaces of stainless steel intravascular stents. Our approach makes use of an aqueous-based, layer-by-layer method for the assembly of nanostructured thin films consisting of alternating layers of plasmid DNA and a hydrolytically degradable polyamine. Characterization of coated stents using scanning electron microscopy (SEM) demonstrated that stents were coated uniformly with an ultrathin film ca. 120 nm thick that adhered conformally to the surfaces of stent struts. These ultrathin films did not crack, peel, or delaminate substantially from the surface after exposure to a range of mechanical challenges representative of those encountered during stent deployment (e.g., balloon expansion). Stents coated with eight bilayers of degradable polyamine and a plasmid encoding enhanced green fluorescent protein (EGFP) sustained the release of DNA into solution for up to four days when incubated in phosphate buffered saline at 37 degrees C, and coated stents were capable of mediating the expression of EGFP in a mammalian cell line without the aid of additional transfection agents. The approach reported here could, with further development, contribute to the development of localized gene-based approaches to the treatment of cardiovascular diseases or related conditions.

Structure/property relationships in erodible multilayered films: influence of polycation structure on erosion profiles and the release of anionic polyelectrolytes.

We have investigated the influence of polymer structure on the erosion profiles of multilayered polyelectrolyte assemblies fabricated from sodium poly(styrene sulfonate) (SPS) and three different hydrolytically degradable polyamines. We synthesized three structurally related poly(beta-amino ester)s (polymers 1-3) having systematic variations in both charge density and hydrophobicity. These changes in structure did not influence film thickness significantly, but polymer structure was found to play an important role in defining the rates at which multilayered assemblies fabricated from these materials eroded in physiologically relevant media. Films 60 nm thick fabricated from polymer 1 and SPS eroded completely in 50 h when incubated in PBS buffer at 37 degrees C, as determined by ellipsometry. Analogous films fabricated from polymers 2 and 3 eroded and released SPS into solution over significantly longer time periods ranging from approximately 150 h (ca. 6 days) to 370 h (ca. 15 days), respectively. These differences are consistent with a systematic increase in the hydrophobicity of polymers 1-3 as well as the relative rates at which these polymers degrade hydrolytically. This work demonstrates that it is possible to tailor the rates at which thin, multilayered polyelectrolyte assemblies release incorporated anionic polyelectrolytes over a large range of time periods simply by changing the structure of the degradable polyamine used to fabricate a film. The principles reported here may therefore contribute to the design of multilayered assemblies that permit a broad range of spatial and temporal control over the release of therapeutic agents from coated surfaces.

Multilayered polyelectrolyte films promote the direct and localized delivery of DNA to cells.

Multilayered polyelectrolyte films fabricated from plasmid DNA and a hydrolytically degradable synthetic polycation can be used to direct the localized transfection of cells without the aid of a secondary transfection agent. Multilayered assemblies 100 nm thick consisting of alternating layers of synthetic polymer and plasmid DNA encoding for enhanced green fluorescent protein (EGFP) were deposited on quartz substrates using a layer-by-layer fabrication procedure. The placement of film-coated slides in contact with COS-7 cells growing in serum-containing culture medium resulted in gene expression in cells localized under the film-coated portion of the slides. The average percentage of cells expressing EGFP relative to the total number of cells ranged from 4.6% to 37.9%, with an average of 18.6%+/-8.2%, as determined by fluorescence microscopy. In addition to providing a mechanism for the immobilization of DNA at the cell/surface interface, a preliminary analysis of film topography by atomic force microscopy (AFM) demonstrated that polymer /DNA films undergo significant structural rearrangements upon incubation to present surface bound condensed plasmid DNA nanoparticles. These data suggest that the presence of the cationic polymer in these materials may also contribute to the internalization and expression of plasmid. The materials and design principles reported here present an attractive framework for the local or non-invasive delivery of DNA from the surfaces of implantable materials or biomedical devices.

Surface analysis of erodible multilayered polyelectrolyte films: nanometer-scale structure and erosion profiles.

Atomic force microscopy (AFM) and scanning electron microscopy (SEM) coupled with ellipsometry have been used to characterize the microscale and nanoscale structures of erodible multilayered films fabricated from degradable polyamine 1 and either sodium poly(styrene sulfonate) (SPS) or plasmid DNA. Striking differences were found in the topography, structures, and erosion profiles of these two materials upon incubation in PBS buffer at 37 degrees C. For films fabricated from SPS, AFM data are consistent with an erosion process that occurs uniformly without the generation of holes or pits over large, micrometer-scale areas. By contrast, films fabricated from plasmid DNA undergo structural rearrangements to present surface-bound particles ranging in size from 50 to 400 nm. Additional characterization of these particulate structures by SEM suggested that they are interpenetrated with or fused to underlying polyelectrolyte layers on the silicon surface, providing a potential mechanism to manipulate the adhesive forces with which these particles are bound to the surface. The erosion profile observed for polymer 1/SPS films suggests that it may be possible to design assemblies that release two film components with well-defined release kinetics. In the context of gene delivery, the presentation of condensed DNA as nanoparticles at these surfaces may be advantageous with respect to stimulating the internalization and processing of DNA by cells. A quantitative understanding of the factors influencing the fabrication, structure, and erosion profiles of these materials will be useful for the design of multilayered assemblies for specific applications in which controlled film erosion or the release of therapeutic materials is desired.

Dramatic reduction of the effect of nanoconfinement on the glass transition of polymer films via addition of small-molecule diluent.

The effect of nanoconfinement on the glass transition temperature T(g) in thin polymer films is studied as a function of added small-molecule diluent or plasticizer. The decrease [increase] in T(g) found in nanoconfined, neat polystyrene [poly(2-vinyl pyridine)] is suppressed by added diluent, with 13-20 nm thick polystyrene films exhibiting bulk T(g) upon addition of 9 wt % pyrene or 4 wt % dioctylphthalate [corrected]. This is explained by a connection between the size scale of the cooperative dynamics associated with T(g), which decreases with added diluent, and the size scale of the nanoconfinement effect.