Co-reporter:Yashira M. Zayas-Gonzalez, Benjamín J. Ortiz, and David M. Lynn
Biomacromolecules May 8, 2017 Volume 18(Issue 5) pp:1499-1499
Publication Date(Web):March 23, 2017
DOI:10.1021/acs.biomac.7b00043
We report the reactive layer-by-layer assembly of amine-reactive polymer multilayers using an azlactone-functionalized polymer and small-molecule diamine linkers. This approach yields cross-linked polymer/linker-type films that can be further functionalized, after fabrication, by treatment with functional primary amines, and provides opportunities to incorporate other useful functionality that can be difficult to introduce using other polyamine building blocks. Films fabricated using poly(2-vinyl-4,4-dimethylazlactone) (PVDMA) and three model nondegradable aliphatic diamine linkers yielded reactive thin films that were stable upon incubation in physiologically relevant media. By contrast, films fabricated using PVDMA and varying amounts of the model disulfide-containing diamine linker cystamine were stable in normal physiological media, but were unstable and eroded rapidly upon exposure to chemical reducing agents. We demonstrate that this approach can be used to fabricate functionalized polymer microcapsules that degrade in reducing environments, and that rates of erosion, extents of capsule swelling, and capsule degradation can be tuned by control over the relative concentration of cystamine linker used during fabrication. The polymer/linker approach used here expands the range of properties and functions that can be designed into reactive PVDMA-based coatings, including functionality that can degrade, erode, and undergo triggered destruction in aqueous environments. We therefore anticipate that these approaches will be useful for the functionalization, patterning, and customization of coatings, membranes, capsules, and interfaces of potential utility in biotechnical or biomedical contexts and other areas where degradation and transience are desired. The proof of concept strategies reported here are likely to be general, and should prove useful for the design of amine-reactive coatings containing other functional structures by judicious control of the structures of the linkers used during assembly.
Co-reporter:Michael J. Kratochvil, Tian Yang, Helen E. Blackwell, and David M. Lynn
ACS Infectious Diseases April 14, 2017 Volume 3(Issue 4) pp:271-271
Publication Date(Web):January 24, 2017
DOI:10.1021/acsinfecdis.6b00173
We report the fabrication and biological evaluation of nonwoven polymer nanofiber coatings that inhibit quorum sensing (QS) and virulence in the human pathogen Staphylococcus aureus. Our results demonstrate that macrocyclic peptide 1, a potent and synthetic nonbactericidal quorum sensing inhibitor (QSI) in S. aureus, can be loaded into degradable polymer nanofibers by electrospinning and that this approach can deposit QSI-loaded nanofiber coatings onto model nonwoven mesh substrates. The QSI was released over ∼3 weeks when these materials were incubated in physiological buffer, retained its biological activity, and strongly inhibited agr-based QS in a GFP reporter strain of S. aureus for at least 14 days without promoting cell death. These materials also inhibited production of hemolysins, a QS-controlled virulence phenotype, and reduced the lysis of erythrocytes when placed in contact with wild-type S. aureus growing on surfaces. This approach is modular and can be used with many different polymers, active agents, and processing parameters to fabricate nanofiber coatings on surfaces important in healthcare contexts. S. aureus is one of the most common causative agents of bacterial infections in humans, and strains of this pathogen have developed significant resistance to conventional antibiotics. The QSI-based strategies reported here thus provide springboards for the development of new anti-infective materials and novel treatment strategies that target virulence as opposed to growth in S. aureus. This approach also provides porous scaffolds for cell culture that could prove useful in future studies on the influence of QS modulation on the development and structure of bacterial communities.Keywords: antivirulence; coatings; controlled release; electrospinning; nanofibers; polymers; quorum sensing;
Co-reporter:Uttam Manna;Namrata Raman;Michael A. Welsh;Yashira M. Zayas-Gonzalez;Helen E. Blackwell;Sean P. Palecek
Advanced Functional Materials 2016 Volume 26( Issue 21) pp:3599-3611
Publication Date(Web):
DOI:10.1002/adfm.201505522
Many types of slippery liquid-infused porous surfaces (‘SLIPS’) can resist adhesion and colonization by microorganisms. These ‘slippery’ materials thus offer approaches to prevent fouling on commercial and industrial surfaces. However, while SLIPS can prevent fouling on surfaces to which they are applied, they can currently do little to prevent the proliferation of non-adherent organisms. Here, multi-functional SLIPS are reported that address this issue and expand the potential utility of these materials. The approach is based on the release of antimicrobial agents from the porous matrices used to host the infused oil phases. It is demonstrated that SLIPS fabricated from nanoporous polymer multilayers can prevent colonization and biofilm formation by four common fungal and bacterial pathogens, and that the polymer and oil phases comprising these materials can be used to sustain the release of triclosan, a model antimicrobial agent, into surrounding media. This approach improves the inherent anti-fouling properties of these materials and endows them with the ability to kill non-adherent pathogens. This strategy has the potential to be general; the strategies and concepts reported here will enable the design of SLIPS with improved anti-fouling properties and open the door to new applications of slippery liquid-infused materials that host or release other active agents.
Co-reporter:Matthew C. D. Carter and David M. Lynn
Chemistry of Materials 2016 Volume 28(Issue 14) pp:5063
Publication Date(Web):June 20, 2016
DOI:10.1021/acs.chemmater.6b01897
We report approaches to the design of covalently crosslinked and physically stable surface coatings with chemically labile and dynamic surface features based on the functionalization of azlactone-containing materials with alcohol-, thiol-, and hydrazine-based nucleophiles. Past studies demonstrate that residual azlactone groups in polymer multilayers fabricated by the reactive layer-by-layer assembly of poly(2-vinyl-4,4-dimethylazlactone) and branched poly(ethylenimine) can react with amine-based nucleophiles to impart new surface and bulk properties through the creation of chemically stable amide/amide-type bonds. Here, we demonstrate that the azlactone groups in these covalently crosslinked materials can also be functionalized using less nucleophilic alcohol- or thiol-containing compounds, using an organic catalyst, or converted to reactive acylhydrazine groups by direct treatment with hydrazine. These methods (i) broaden the pool of molecules that can be used for post-fabrication functionalization to include compounds containing alcohol, thiol, or aldehyde groups and (ii) yield surface coatings with chemically labile amide/ester-, amide/thioester-, and amide/imine-type bonds that make possible the design of new dynamic and stimulus-responsive materials (e.g., surfaces that release covalently bound molecules or undergo changes in extreme wetting behaviors in response to specific chemical stimuli). Our results expand the range of functionality that can be installed in, and thus the range of new functions that can be imparted to, azlactone-containing coatings beyond those that can be accessed using primary amine-based nucleophiles. The chemical approaches demonstrated here using model polymer-based reactive multilayer coatings are general and should thus also prove useful for the design of new responsive surfaces based on other types of azlactone-functionalized materials.
Co-reporter:Namrata Raman, Myung-Ryul Lee, Angélica de L. Rodríguez López, Sean P. Palecek, David M. Lynn
Acta Biomaterialia 2016 Volume 43() pp:240-250
Publication Date(Web):1 October 2016
DOI:10.1016/j.actbio.2016.07.016
Abstract
Catheter-associated urinary tract infections (CAUTI) are the most common type of hospital-acquired infection, with more than 30 million catheters placed annually in the US and a 10–30% incidence of infection. Candida albicans forms fungal biofilms on the surfaces of urinary catheters and is the leading cause of fungal urinary tract infections. As a step toward new strategies that could prevent or reduce the occurrence of C. albicans-based CAUTI, we investigated the ability of antifungal β-peptide-based mimetics of antimicrobial peptides (AMPs) to kill C. albicans and prevent biofilm formation in synthetic urine. Many α-peptide-based AMPs exhibit antifungal activities, but are unstable in high ionic strength media and are easily degraded by proteases—features that limit their use in urinary catheter applications. Here, we demonstrate that β-peptides designed to mimic the amphiphilic helical structures of AMPs retain 100% of their structural stability and exhibit antifungal and anti-biofilm activity against C. albicans in a synthetic medium that mimics the composition of urine. We demonstrate further that these agents can be loaded into and released from polymer-based multilayer coatings applied to polyurethane, polyethylene, and silicone tubing commonly used as urinary catheters. Our results reveal catheters coated with β-peptide-loaded multilayers to kill planktonic fungal cells for up to 21 days of intermittent challenges with C. albicans and prevent biofilm formation on catheter walls for at least 48 h. These new materials and approaches could lead to advances that reduce the occurrence of fungal CAUTI.
Statement of Significance
Catheter-associated urinary tract infections are the most common type of hospital-acquired infection. The human pathogen Candida albicans is the leading cause of fungal urinary tract infections, and forms difficult to remove ‘biofilms’ on the surfaces of urinary catheters. We investigated synthetic β-peptide mimics of natural antimicrobial peptides as an approach to kill C. albicans and prevent biofilm formation in media that mimics the composition of urine. Our results reveal these mimics to retain structural stability and activity against C. albicans in synthetic urine. We also report polymer-based approaches to the local release of these agents within urinary catheter tubes. With further development, these materials-based approaches could lead to advances that reduce the occurrence of fungal urinary tract infections.
Co-reporter:Matthew C. D. Carter, James Jennings, Visham Appadoo, and David M. Lynn
Macromolecules 2016 Volume 49(Issue 15) pp:5514-5526
Publication Date(Web):July 28, 2016
DOI:10.1021/acs.macromol.6b01212
We report the design of reactive and degradable copolymers that contain both azlactone side chain functionality and hydrolyzable backbone ester groups. Copolymerization of the vinyl monomer 2-vinyl-4,4-dimethylazlactone (VDMA) and the cyclic ketene acetal 2-methylene-1,3-dioxepane (MDO) using conventional or reversible-deactivation radical polymerization techniques yielded copolymers and block copolymers that exhibit amine reactivity associated with poly(vinyl azlactone)s but also hydrolytic degradability associated with conventional polyesters. Our results demonstrate that control over monomer feed ratios and other parameters can be used to tune both copolymer composition (e.g., the number/ratio of azlactone and ester repeat units) and the physical properties of the resulting materials (e.g., glass transition temperatures and ability to self-assemble into nanoscale structures). Post-fabrication functionalization of reactive azlactone groups in MDO-co-VDMA copolymers by treatment with primary amines proceeds rapidly and quantitatively, and can be achieved without disruption or degradation of backbone ester groups. These azlactone-functionalized copolymers are thus well suited for use as templates for the design of new degradable polymers and as building blocks for the design of covalently and ionically cross-linked macromolecular thin films, capsules, and gels that degrade in aqueous environments.
Co-reporter:Matthew C. D. Carter, James Jennings, Frank W. Speetjens II, David M. Lynn, and Mahesh K. Mahanthappa
Macromolecules 2016 Volume 49(Issue 17) pp:6268-6276
Publication Date(Web):September 1, 2016
DOI:10.1021/acs.macromol.6b01268
We report a reactive polymer platform for the rapid discovery of strongly segregated diblock polymers that microphase separate into well-defined morphologies with sub-5 nm features. Our strategy employs reactive poly(styrene-block-2-vinyl-4,4-dimethylazlactone) (SV) polymers with low degrees of polymerization (N), in which the V blocks undergo selective and quantitative reactions with functional primary amines, to identify new poly(acrylamides) that are highly immiscible with poly(styrene) and induce block polymer self-assembly. Using a combination of optical birefringence and small-angle X-ray scattering (SAXS), we characterize a library of 17 block polymers produced by amine functionalization of four parent SV diblocks synthesized by sequential RAFT polymerizations. We demonstrate that V block functionalization with hydroxy- and methoxy-functional amines yields diblocks that order into lamellar mesophases with half-pitches as small as 3.8 nm. Thus, this azlactone-based reactive molecular platform enables combinatorial generation of polymer libraries with diverse side chain structures that may be rapidly screened to identify new high χ/low N systems for self-assembly at ever decreasing length scales.
Co-reporter:Jonathan W. Choi, Matthew C. D. Carter, Wei Wei, Catherine Kanimozi, Frank W. Speetjens II, Mahesh K. Mahanthappa, David M. Lynn, and Padma Gopalan
Macromolecules 2016 Volume 49(Issue 21) pp:8177-8186
Publication Date(Web):October 26, 2016
DOI:10.1021/acs.macromol.6b01734
We report on the thin film self-assembly and post-fabrication functionalization of cylinder-forming poly(styrene-block-2-vinyl-4,4-dimethylazlactone) (PS-b-PVDMA) block copolymers (BCPs). Thermal annealing of an asymmetric BCP composition, to drive microphase separation in thin films, results in high defect densities and poor long-range order. Using FTIR spectroscopy and ellipsometry, we demonstrate that thermal annealing results in reactions between the azlactone groups and the underlying substrate leading to BCP “pinning”. We demonstrate that solvent annealing circumvents these issues and drives high fidelity microphase separation of the BCP in thin films. The solvent annealing approach also enables control over domain orientation; we show that parallel and perpendicularly oriented cylinders with diameters of 11.7 ± 1.2 nm and a center-to-center distance of 25.2 ± 2.6 nm can be obtained using a BCP with Mn = 29.5 kDa and fS = 0.75. The PVDMA segments in these self-assembled thin films remain available for reaction with primary amine-functionalized nucleophiles. X-ray photoelectron spectroscopy (XPS) depth-profiling reveals that reactions between azlactone groups and incoming amines occur primarily in the top few nanometers of the reactive domains. Finally, we demonstrate that the azlactone groups in both parallel and perpendicularly oriented cylindrical thin films can selectively incorporate trimethylaluminum vapor, which can be subsequently converted into Al2O3 nanowires and nanodots with dimensions of 16 and 12 nm, respectively.
Co-reporter:Michael J. Kratochvil, Michael A. Welsh, Uttam Manna, Benjamín J. Ortiz, Helen E. Blackwell, and David M. Lynn
ACS Infectious Diseases 2016 Volume 2(Issue 7) pp:509
Publication Date(Web):May 24, 2016
DOI:10.1021/acsinfecdis.6b00065
Surfaces that can both prevent bacterial biofouling and inhibit the expression of virulence phenotypes in surrounding planktonic bacteria are of interest in a broad range of contexts. Here, we report new slippery-liquid infused porous surfaces (SLIPS) that resist bacterial colonization (owing to inherent “slippery” surface character) and also attenuate virulence phenotypes in non-adherent cells by gradually releasing small-molecule quorum sensing inhibitors (QSIs). QSIs active against Pseudomonas aeruginosa can be loaded into SLIPS without loss of their slippery and antifouling properties, and imbedded agents can be released into surrounding media over hours to days depending on the structures of the loaded agent. This controlled-release approach is useful for inhibiting virulence factor production and can also inhibit bacterial biofilm formation on nearby, non-SLIPS-coated surfaces. Finally, we demonstrate that this approach is compatible with the simultaneous release of more than one type of QSI, enabling greater control over virulence and suggesting new opportunities to tune the antifouling properties of these slippery surfaces.Keywords: anti-biofouling; antivirulence; biofilms; controlled release; quorum sensing; slippery surfaces
Co-reporter:Yashira M. Zayas-Gonzalez and David M. Lynn
Biomacromolecules 2016 Volume 17(Issue 9) pp:3067
Publication Date(Web):August 15, 2016
DOI:10.1021/acs.biomac.6b00975
We report the fabrication of reactive and degradable cross-linked polymer multilayers by the reactive/covalent layer-by-layer assembly of a non-degradable azlactone-functionalized polymer [poly(2-vinyl-4,4-dimethylazlactone), PVDMA] with hydrolytically or enzymatically degradable polyamine building blocks. Fabrication of multilayers using PVDMA and a hydrolytically degradable poly(β-amino ester) (PBAE) containing primary amine side chains yielded multilayers (∼100 nm thick) that degraded over ∼12 days in physiologically relevant media. Physicochemical characterization and studies on stable films fabricated using PVDMA and an analogous non-degradable poly(amidoamine) suggested that erosion occurred by chemical hydrolysis of backbone esters in the PBAE components of these assemblies. These degradable assemblies also contained residual amine-reactive azlactone functionality that could be used to impart new functionality to the coatings post-fabrication. Cross-linked multilayers fabricated using PVDMA and the enzymatically degradable polymer poly(l-lysine) were structurally stable for prolonged periods in physiological media, but degraded over ∼24 h when the enzyme trypsin was added. Past studies demonstrate that multilayers fabricated using PVDMA and non-degradable polyamines [e.g., poly(ethylenimine)] enable the design and patterning of useful nano/biointerfaces and other materials that are structurally stable in physiological media. The introduction of degradable functionality into PVDMA-based multilayers creates opportunities to exploit the reactivity of azlactone groups for the design of reactive materials and functional coatings that degrade or erode in environments that are relevant in biomedical, biotechnological, and environmental contexts. This “degradable building block” strategy should be general; we anticipate that this approach can also be extended to the design of amine-reactive multilayers that degrade upon exposure to specific chemical triggers, selective enzymes, or contact with cells by judicious design of the degradable polyamine building blocks used to fabricate the coatings.
Co-reporter:Uttam Manna
Advanced Materials 2015 Volume 27( Issue 19) pp:3007-3012
Publication Date(Web):
DOI:10.1002/adma.201500893
Co-reporter:Uttam Manna
Advanced Functional Materials 2015 Volume 25( Issue 11) pp:1672-1681
Publication Date(Web):
DOI:10.1002/adfm.201403735
Surfaces with extreme wetting properties are useful for the collection, manipulation, transport, and avoidance of aqueous and organic fluids of commercial and strategic importance. Two major obstacles to the deployment of synthetic non-wetting materials in practical scenarios are their lack of mechanical durability and their susceptibility to fouling in contaminated or chemically complex media. Here, crosslinked and nanoporous polymer multilayers are reported that overcome these limitations and exhibit robust and tunable “underwater superoleophobicity”, or the ability to almost completely prevent contact with oils and other organic fluids when submerged in water. These entirely organic coatings mimic key chemical and structural features found on the scales of fish and other natural anti-oil-fouling surfaces, and are remarkably tolerant to physical, chemical, and environmental insults commonly encountered in natural and synthetic aqueous environments. This approach also permits facile manipulation and patterning of surface chemistry and, thus, tunable spatial control over other important aspects of interfacial behavior, such as underwater oil adhesiveness, that extend and expand the potential utility of synthetic anti-oil-fouling surfaces in aqueous, aquatic, and marine environments.
Co-reporter:Matthew T. Holden, Matthew C. D. Carter, Cheng-Hsien Wu, Jamison Wolfer, Eric Codner, Michael R. Sussman, David M. Lynn, and Lloyd M. Smith
Analytical Chemistry 2015 Volume 87(Issue 22) pp:11420
Publication Date(Web):October 23, 2015
DOI:10.1021/acs.analchem.5b02893
The photolithographic fabrication of high-density DNA and RNA arrays on flexible and transparent plastic substrates is reported. The substrates are thin sheets of poly(ethylene terephthalate) (PET) coated with cross-linked polymer multilayers that present hydroxyl groups suitable for conventional phosphoramidite-based nucleic acid synthesis. We demonstrate that by modifying array synthesis procedures to accommodate the physical and chemical properties of these materials, it is possible to synthesize plastic-backed oligonucleotide arrays with feature sizes as small as 14 μm × 14 μm and feature densities in excess of 125 000/cm2, similar to specifications attainable using rigid substrates such as glass or glassy carbon. These plastic-backed arrays are tolerant to a wide range of hybridization temperatures, and improved synthetic procedures are described that enable the fabrication of arrays with sequences up to 50 nucleotides in length. These arrays hybridize with S/N ratios comparable to those fabricated on otherwise identical arrays prepared on glass or glassy carbon. This platform supports the enzymatic synthesis of RNA arrays and proof-of-concept experiments are presented showing that the arrays can be readily subdivided into smaller arrays (or “millichips”) using common laboratory-scale laser cutting tools. These results expand the utility of oligonucleotide arrays fabricated on plastic substrates and open the door to new applications for these important bioanalytical tools.
Co-reporter:Yan Yu, Yi Si, Shane L. Bechler, Bo Liu, and David M. Lynn
Biomacromolecules 2015 Volume 16(Issue 9) pp:
Publication Date(Web):August 19, 2015
DOI:10.1021/acs.biomac.5b00905
We report a layer-by-layer approach to the fabrication of thin polymer-based multilayers that release DNA rapidly in physiologically relevant environments. This approach exploits the properties of a weak anionic polyelectrolyte [poly(acrylic acid); PAA] to disrupt ionic interactions and promote disassembly in coatings that otherwise erode slowly. We investigated this approach using multilayers fabricated from plasmid DNA and linear poly(ethylenimine) (LPEI), a model synthetic cationic polymer used widely for DNA delivery. LPEI/DNA multilayers erode and release DNA slowly over ∼4 days when incubated in PBS buffer. In contrast, substitution of every other layer of DNA with PAA lead to thin films that released DNA rapidly, with >60% being released in the first 5 min. These quick-release coatings release bioactive DNA and can be used to fabricate uniform coatings on a variety of objects, including the tips of inflatable balloon catheters. We demonstrate that these coatings can promote high levels of cell transfection in vitro and the robust contact transfer and expression of DNA in vascular tissue in vivo using a rat model of vascular injury. These materials provide useful alternatives to multilayers and other coatings that promote the prolonged release of DNA. More broadly, approaches that depart from the use of degradable polymers to promote film erosion create opportunities to design new gene delivery coatings using a broader range of polymer-based building blocks designed for other gene delivery applications. With further development, this approach could thus provide a new and useful platform for the rapid contact transfer of DNA to cells and tissues of interest in a range of fundamental and applied contexts.
Co-reporter:Michael J. Kratochvil, Yftah Tal-Gan, Tian Yang, Helen E. Blackwell, and David M. Lynn
ACS Biomaterials Science & Engineering 2015 Volume 1(Issue 10) pp:1039
Publication Date(Web):August 26, 2015
DOI:10.1021/acsbiomaterials.5b00313
Materials and coatings that inhibit bacterial colonization are of interest in a broad range of biomedical, environmental, and industrial applications. In view of the rapid increase in bacterial resistance to conventional antibiotics, the development of new strategies that target nonessential pathways in bacterial pathogens—and that thereby limit growth and reduce virulence through nonbiocidal means—has attracted considerable attention. Bacterial quorum sensing (QS) represents one such target, and is intimately connected to virulence in many human pathogens. Here, we demonstrate that the properties of nanoporous, polymer-based superhydrophobic coatings can be exploited to host and subsequently sustain the extended release of potent and water-labile peptide-based inhibitors of QS (QSIs) in Staphylococcus aureus. Our results demonstrate that these peptidic QSIs can be released into surrounding media for periods of at least 8 months, and that they strongly inhibit agr-based QS in S. aureus for at least 40 days. These results also suggest that these extremely nonwetting coatings can confer protection against the rapid hydrolysis of these water-labile peptides, thereby extending their useful lifetimes. Finally, we demonstrate that these peptide-loaded superhydrophobic coatings can strongly modulate the QS-controlled formation of biofilm in wild-type S. aureus. These nanoporous superhydrophobic films provide a new, useful, and nonbiocidal approach to the design of coatings that attenuate bacterial virulence. This approach has the potential to be general, and could prove suitable for the encapsulation, protection, and release of other classes of water-sensitive agents. We anticipate that the materials, strategies, and concepts reported here will enable new approaches to the long-term attenuation of QS and associated bacterial phenotypes in a range of basic research and applied contexts.Keywords: anti-virulence; controlled release; polymer multilayers; quorum sensing; superhydrophobic; surface coatings
Co-reporter:Namrata Raman, Myung-Ryul Lee, Sean P. Palecek, David M. Lynn
Journal of Controlled Release 2014 Volume 191() pp:54-62
Publication Date(Web):10 October 2014
DOI:10.1016/j.jconrel.2014.05.026
Candida albicans is the most common fungal pathogen responsible for hospital-acquired infections. Most C. albicans infections are associated with the implantation of medical devices that act as points of entry for the pathogen and as substrates for the growth of fungal biofilms that are notoriously difficult to eliminate by systemic administration of conventional antifungal agents. In this study, we report a fill-and-purge approach to the layer-by-layer fabrication of biocompatible, nanoscale ‘polyelectrolyte multilayers’ (PEMs) on the luminal surfaces of flexible catheters, and an investigation of this platform for the localized, intraluminal release of a cationic β-peptide-based antifungal agent. We demonstrate that polyethylene catheter tubes with luminal surfaces coated with multilayers ~ 700 nm thick fabricated from poly-l-glutamic acid (PGA) and poly-l-lysine (PLL) can be loaded, post-fabrication, by infusion with β-peptide, and that this approach promotes extended intraluminal release of this agent (over ~ 4 months) when incubated in physiological media. The β-peptide remained potent against intraluminal inoculation of the catheters with C. albicans and substantially reduced the formation of C. albicans biofilms on the inner surfaces of film-coated catheters. Finally, we report that these β-peptide-loaded coatings exhibit antifungal activity under conditions that simulate intermittent catheter use and microbial challenge for at least three weeks. We conclude that β-peptide-loaded PEMs offer a novel and promising approach to kill C. albicans and prevent fungal biofilm formation on surfaces, with the potential to substantially reduce the incidence of device-associated infections in indwelling catheters. β-Peptides comprise a promising new class of antifungal agents that could help address problems associated with the use of conventional antifungal agents. The versatility of the layer-by-layer approach used here thus suggests additional opportunities to exploit these new agents in other biomedical and personal care applications in which fungal infections are endemic.
Co-reporter:Frank W. Speetjens II, Matthew C. D. Carter, Myungwoong Kim, Padma Gopalan, Mahesh K. Mahanthappa, and David M. Lynn
ACS Macro Letters 2014 Volume 3(Issue 11) pp:1178
Publication Date(Web):October 27, 2014
DOI:10.1021/mz500654a
We report an approach to the post-fabrication placement of chemical functionality on microphase-separated thin films of a reactive block copolymer. Our approach makes use of an azlactone-containing block copolymer that microphase separates into domains of perpendicularly-oriented lamellae. These thin films present nanoscale patterns of amine-reactive groups (reactive stripes) that serve as handles for the immobilization of primary amine-containing functionality. We demonstrate that arbitrary chemical functionality can be installed by treatment with aqueous solutions under mild conditions that do not perturb underlying microphase-separated patterns dictated by the structure of the reactive block copolymer. This post-fabrication approach provides a basis for the development of modular approaches to the design of microphase-separated block copolymer thin films and access to coatings with patterned chemical domains and surface properties that would be difficult to prepare by the self-assembly and processing of functionally complex block copolymers.
Co-reporter:Adam H. Broderick;Danielle M. Stacy;Yftah Tal-Gan;Michael J. Kratochvil;Helen E. Blackwell
Advanced Healthcare Materials 2014 Volume 3( Issue 1) pp:97-105
Publication Date(Web):
DOI:10.1002/adhm.201300119
Abstract
Staphylococcus aureus is a major human pathogen responsible for a variety of life-threatening infections. The pathogenicity of this organism is attributed to its ability to produce a range of virulence factors and toxins, including the superantigen toxic shock syndrome toxin-1 (TSST-1). While many S. aureus infections can be treated using conventional antibiotics, strains resistant to these bactericidal agents have emerged. Approaches that suppress pathogenicity through mechanisms that are nonbactericidal (i.e., antivirulence approaches) could provide new options for treating infections, including those caused by resistant strains. Here, we report a nonbactericidal approach to suppressing pathogenicity based on the release of macrocyclic peptides (1 and 2) that inhibit the agr quorum sensing (QS) circuit in group-III S. aureus. It is demonstrated that these peptides can be immobilized on planar and complex objects (on glass slides, nonwoven meshes, or within absorbent tampons) using the rapidly dissolving polymer carboxymethylcellulose (CMC). Peptide-loaded CMC films released peptide rapidly (<5 min) and promoted strong (>95%) inhibition of the agr QS circuit without inducing cell death when incubated in the presence of a group-III S. aureus gfp-reporter strain. Peptide 1 is among the most potent inhibitors of QS in S. aureus reported to date, and the group-III QS circuit regulates production of TSST-1, the primary cause of toxic shock syndrome (TSS). These results thus suggest approaches to treat the outer covers of tampons, wound dressings, or other objects to suppress toxin production and reduce the severity of TSS in clinical and personal care contexts. Because peptide 1 also inhibits QS in S. aureus groups-I, -II, and -IV, this approach could also provide a pathway for attenuation of QS and associated virulence phenotypes in a broader range of contexts.
Co-reporter:Myung-Ryul Lee, Namrata Raman, Samuel H. Gellman, David M. Lynn, and Sean P. Palecek
ACS Chemical Biology 2014 Volume 9(Issue 7) pp:1613
Publication Date(Web):May 16, 2014
DOI:10.1021/cb500203e
Candida albicans is one of the most prevalent fungal pathogens, causing both mucosal candidiasis and invasive candidemia. Antimicrobial peptides (AMPs), part of the human innate immune system, have been shown to exhibit antifungal activity but have not been effective as pharmaceuticals because of low activity and selectivity in physiologically relevant environments. Nevertheless, studies on α-peptide AMPs have revealed key features that can be designed into more stable structures, such as the 14-helix of β-peptide-based oligomers. Here, we report on the ways in which two of those features, hydrophobicity and helicity, govern the activity and selectivity of 14-helical β-peptides against C. albicans and human red blood cells. Our results reveal both antifungal activity and hemolysis to correlate to hydrophobicity, with intermediate levels of hydrophobicity leading to high antifungal activity and high selectivity toward C. albicans. Helical structure-forming propensity further influenced this window of selective antifungal activity, with more stable helical structures eliciting specificity for C. albicans over a broader range of hydrophobicity. Our findings also reveal cooperativity between hydrophobicity and helicity in regulating antifungal activity and specificity. The results of this study provide critical insight into the ways in which hydrophobicity and helicity govern the activity and specificity of AMPs and identify criteria that may be useful for the design of potent and selective antifungal agents.
Co-reporter:Rebecca J. Carlton, Yashira M. Zayas-Gonzalez, Uttam Manna, David M. Lynn, and Nicholas L. Abbott
Langmuir 2014 Volume 30(Issue 49) pp:14944-14953
Publication Date(Web):2017-2-22
DOI:10.1021/la501596b
We report a study of the wetting and ordering of thermotropic liquid crystal (LC) droplets that are trapped (or “caged”) within micrometer-sized cationic polymeric microcapsules dispersed in aqueous solutions of surfactants. When they were initially dispersed in water, we observed caged, nearly spherical droplets of E7, a nematic LC mixture, to occupy ∼40% of the interior volume of the polymeric capsules [diameter of 6.7 ± 0.3 μm, formed via covalent layer-by-layer assembly of branched polyethylenimine and poly(2-vinyl-4,4-dimethylazlactone)] and to contact the interior surface of the capsule wall at an angle of ∼157 ± 11°. The internal ordering of LC within the droplets corresponded to the so-called bipolar configuration (distorted by contact with the capsule walls). While the effects of dodecyltrimethylammonium bromide (DTAB) and sodium dodecyl sulfate (SDS) on the internal ordering of “free” LC droplets are similar, we observed the two surfactants to trigger strikingly different wetting and configurational transitions when LC droplets were caged within polymeric capsules. Specifically, upon addition of SDS to the aqueous phase, we observed the contact angles (θ) of caged LC on the interior surface of the capsule to decrease, resulting in a progression of complex droplet shapes, including lenses (θ ≈ 130 ± 10°), hemispheres (θ ≈ 89 ± 5°), and concave hemispheres (θ < 85°). The wetting transitions induced by SDS also resulted in changes in the internal ordering of the LC to yield states topologically equivalent to axial and radial configurations. Although topologically equivalent to free droplets, the contributions that surface anchoring, LC elasticity, and topological defects make to the free energy of caged LC droplets differ from those of free droplets. Overall, these results and others reported herein lead us to conclude that caged LC droplets offer a platform for new designs of LC-droplet-based responsive soft matter that cannot be realized in dispersions of free droplets.
Co-reporter:Uttam Manna;Michael J. Kratochvil
Advanced Materials 2013 Volume 25( Issue 44) pp:6405-6409
Publication Date(Web):
DOI:10.1002/adma.201302561
Co-reporter:Uttam Manna
Advanced Materials 2013 Volume 25( Issue 36) pp:5104-5108
Publication Date(Web):
DOI:10.1002/adma.201302217
Co-reporter:Uttam Manna;Matthew C. D. Carter
Advanced Materials 2013 Volume 25( Issue 22) pp:3085-3089
Publication Date(Web):
DOI:10.1002/adma.201300341
Co-reporter:Burcu S. Aytar ; John P. E. Muller ; Yukishige Kondo ; Yeshayahu Talmon ; Nicholas L. Abbott
Journal of the American Chemical Society 2013 Volume 135(Issue 24) pp:9111-9120
Publication Date(Web):May 23, 2013
DOI:10.1021/ja403546b
We report physical characterization and biological evaluation of complexes of small interfering RNA (siRNA) formed using a cationic lipid [bis(11-ferrocenylundecyl)dimethylammonium bromide (BFDMA)] containing redox-active ferrocenyl groups at the end of each hydrophobic tail. We demonstrate that control over the redox state of BFDMA can be used to influence key physical properties and control the activities of lipoplexes formed using siRNA-based constructs. Specifically, lipoplexes of siRNA and reduced BFDMA lead to high levels of sequence-specific gene silencing in cells, but lipoplexes formed using oxidized BFDMA do not. Lipoplexes of oxidized BFDMA can be activated in situ to induce high levels of silencing by addition of a chemical reducing agent, demonstrating a basis for external control over the activation/delivery of siRNA in cellular environments. Differences in activity arise from the inability of oxidized BFDMA to promote efficient internalization of siRNA; these differences also correlated to significant differences in the nanostructures of these lipoplexes (determined by cryo-TEM) and their ζ potentials as a function of oxidation state. These results are considered in view of recent studies characterizing the nanostructures, properties, and behaviors of lipoplexes formed using BFDMA and macromolecular plasmid DNA. We find that several key structural features and aspects of redox control observed for lipoplexes of plasmid DNA are maintained in complexes formed using smaller and more rigid siRNA. The ability to transform BFDMA in complex media presents opportunities to exert control over the nanostructures and behaviors of siRNA lipoplexes in ways not possible using conventional lipids. The approaches reported here could thus prove useful in both fundamental and applied contexts.
Co-reporter:Burcu S. Aytar, John P. E. Muller, Yukishige Kondo, Nicholas L. Abbott, and David M. Lynn
ACS Applied Materials & Interfaces 2013 Volume 5(Issue 17) pp:8283
Publication Date(Web):August 21, 2013
DOI:10.1021/am402594z
We report principles for active, user-defined control over the locations and timing with which DNA is expressed in cells. Our approach exploits unique properties of a ferrocenyl cationic lipid that is inactive when oxidized, but active when chemically reduced. We show that methods that exert spatial control over the administration of reducing agents can lead to local activation of lipoplexes and spatial control over gene expression. The versatility of this approach is demonstrated using both soluble and solid-phase reducing agents. These methods provide control over cell transfection, including methods for remote activation and the patterning of expression using solid-phase redox agents, that are difficult to achieve using conventional lipoplexes.Keywords: amphiphiles; chemical activation; DNA; ferrocene; redox chemistry;
Co-reporter:Uttam Manna and David M. Lynn
ACS Applied Materials & Interfaces 2013 Volume 5(Issue 16) pp:7731
Publication Date(Web):August 9, 2013
DOI:10.1021/am4026467
We report a solvent-assisted approach to the patterning and impregnation of porous superhydrophobic coatings that permits the use of entirely aqueous solutions. This approach permits immobilization of proteins and enzymes, creating opportunities to decorate superhydrophobic surfaces with hydrophilic domains and channels that can capture aliquots of aqueous media, guide and mix aqueous solutions, and chemically process streams of organic molecules. Because this approach does not require destruction of non-wetting features, it can also be used to transfer highly water-soluble polymers and small molecules without compromising superhydrophobicity, providing methods for post-fabrication loading of water-soluble agents into protective non-wetting coatings that are difficult to achieve using other approaches.Keywords: enzymes; hydrophilic; layer-by-layer; patterning; proteins; superhydrophobic;
Co-reporter:Adam H. Broderick, Matthew C. D. Carter, Matthew R. Lockett, Lloyd M. Smith, and David M. Lynn
ACS Applied Materials & Interfaces 2013 Volume 5(Issue 2) pp:351
Publication Date(Web):December 13, 2012
DOI:10.1021/am302285n
We report a top-down approach to the fabrication of oligonucleotide and protein arrays on surfaces coated with ultrathin, amine-reactive polymer multilayers fabricated by the covalent “layer-by-layer” (LbL) assembly of polyethyleneimine (PEI) and the amine-reactive, azlactone-functionalized polymer poly(2-vinyl-4,4-dimethylazlactone) (PVDMA). Manual spotting of amine-terminated oligonucleotide probe sequences on planar glass slides coated with PEI/PVDMA multilayers (∼35 nm thick) yielded arrays of immobilized probes that hybridized fluorescently labeled complementary sequences with high signal intensities, high signal-to-noise ratios, and high sequence specificity. Treatment of residual azlactone functionality with the nonfouling small-molecule amine d-glucamine resulted in regions between the features of these arrays that resisted adsorption of protein and permitted hybridization in complex media containing up to 10 mg/mL protein. The residual azlactone groups in these films were also exploited to immobilize proteins on film-coated surfaces and fabricate functional arrays of proteins and enzymes. The ability to deposit PEI/PVDMA multilayers on substrates of arbitrary size, shape, and composition permitted the fabrication of arrays of oligonucleotides on the surfaces of multilayer-coated sheets of poly(ethylene terephthalate) and heat-shrinkable polymer film. Arrays fabricated on these flexible plastic substrates can be bent, cut, resized, and manipulated physically in ways that are difficult using more conventional rigid substrates. This approach could thus contribute to the development of new assay formats and new applications of biomolecule arrays. The methods described here are straightforward to implement, do not require access to specialized equipment, and should also be compatible with automated liquid-handling methods used to fabricate higher-density arrays of oligonucleotides and proteins on more traditional surfaces.Keywords: azlactones; covalent assembly; layer-by-layer; oligonucleotide arrays; polymer multilayers; reactive surfaces;
Co-reporter:Adam H. Broderick;Anthony S. Breitbach;Reto Frei;Helen E. Blackwell
Advanced Healthcare Materials 2013 Volume 2( Issue 7) pp:993-1000
Publication Date(Web):
DOI:10.1002/adhm.201200334
Abstract
We report an approach to preventing bacterial biofilm formation that is based on the surface-mediated release of 5,6-dimethyl-2-aminobenzimidazole (DMABI), a potent and non-bactericidal small-molecule inhibitor of bacterial biofilm growth. Our results demonstrate that DMABI can be encapsulated in thin films of a model biocompatible polymer [poly(lactide-co-glycolide), PLG] and be released in quantities that inhibit the formation of Pseudomonas aeruginosa biofilms by up to 75–90% on surfaces that otherwise support robust biofilm growth. This approach enables the release of this new anti-biofilm agent for over one month, and it can be used to inhibit biofilm growth on both film-coated surfaces and other adjacent surfaces (e.g., on other uncoated surfaces and at air/water interfaces). Our results demonstrate a non-bactericidal approach to the prevention of biofilm growth and provide proof of concept using a clinically relevant human pathogen. In contrast to coatings designed to kill bacteria on contact, this approach should also permit the design of strategically placed depots that disseminate DMABI more broadly and exert inhibitory effects over larger areas. In a broader context, the non-bactericidal nature of DMABI could also provide opportunities to address concerns related to evolved resistance that currently face approaches based on the release of traditional microbicidal agents (e.g., antibiotics). Finally, the results of initial in vitro mammalian cell culture studies indicate that DMABI is not toxic to cells at concentrations required for strong anti-biofilm activity, suggesting that this new agent is well suited for further investigation in biomedical and personal care contexts.
Co-reporter:Shane L. Bechler, Yi Si, Yan Yu, Jun Ren, Bo Liu, David M. Lynn
Biomaterials 2013 34(1) pp: 226-236
Publication Date(Web):
DOI:10.1016/j.biomaterials.2012.09.010
Co-reporter:Eric M. Saurer, Christopher M. Jewell, Drew A. Roenneburg, Shane L. Bechler, Jose R. Torrealba, Timothy A. Hacker, and David M. Lynn
Biomacromolecules 2013 Volume 14(Issue 5) pp:
Publication Date(Web):April 18, 2013
DOI:10.1021/bm4005222
We report an approach to deliver DNA to vascular tissue in vivo using intravascular stents coated with degradable, DNA-containing polyelectrolyte multilayers (PEMs). Ionically cross-linked multilayers ∼120 nm thick were fabricated layer-by-layer on the surfaces of balloon-mounted stainless steel stents using plasmid DNA and a hydrolytically degradable poly(β-amino ester) (polymer 1). Characterization of stents coated using a fluorescently end-labeled analog of polymer 1 revealed film erosion to be uniform across the surfaces of the stents; differential stresses experienced upon balloon expansion did not lead to faster film erosion or dose dumping of DNA in areas near stent joints when stents were incubated in physiologically relevant media. The ability of film-coated stents to transfer DNA and transfect arterial tissue in vivo was then investigated in pigs and rabbits. Stents coated with films fabricated using fluorescently labeled DNA resulted in uniform transfer of DNA to sub-endothelial tissue in the arteries of pigs in patterns corresponding to the locations and geometries of stent struts. Stents coated with films fabricated using polymer 1 and plasmid DNA encoding EGFP resulted in expression of EGFP in the medial layers of stented tissue in both pigs and rabbits two days after implantation. The results of this study, combined with the modular and versatile nature of layer-by-layer assembly, provide a polymer-based platform that is well suited for fundamental studies of stent-mediated gene transfer. With further development, this approach could also prove useful for the design of nonviral, gene-based approaches for prevention of complications that arise from the implantation of stents and other implantable interventional devices.
Co-reporter:Uttam Manna;Adam H. Broderick
Advanced Materials 2012 Volume 24( Issue 31) pp:4291-4295
Publication Date(Web):
DOI:10.1002/adma.201200903
Co-reporter:Adam H. Broderick, Matthew R. Lockett, Maren E. Buck, Yuan Yuan, Lloyd M. Smith, and David M. Lynn
Chemistry of Materials 2012 Volume 24(Issue 5) pp:938
Publication Date(Web):November 28, 2011
DOI:10.1021/cm202720q
We report an approach to the in situ synthesis of oligonucleotide arrays on surfaces coated with crosslinked polymer multilayers. Our approach makes use of methods for the “reactive” layer-by-layer assembly of thin, amine-reactive multilayers using branched polyethyleneimine (PEI) and the azlactone-functionalized polymer poly(2-vinyl-4,4′-dimethylazlactone) (PVDMA). Postfabrication treatment of film-coated glass substrates with d-glucamine or 4-amino-1-butanol yielded hydroxyl-functionalized films suitable for the Maskless Array Synthesis (MAS) of oligonucleotide arrays. Glucamine-functionalized films yielded arrays of oligonucleotides with fluorescence intensities and signal-to-noise ratios (after hybridization with fluorescently labeled complementary strands) comparable to those of arrays fabricated on conventional silanized glass substrates. These arrays could be exposed to multiple hybridization/dehybridization cycles with only moderate loss of hybridization density. The versatility of the layer-by-layer approach also permitted synthesis directly on thin sheets of film-coated poly(ethylene terephthalate) (PET) to yield flexible oligonucleotide arrays that could be readily manipulated (e.g., bent) and cut into smaller arrays. To our knowledge, this work presents the first use of polymer multilayers as a substrate for the multistep synthesis of complex molecules. Our results demonstrate that these films are robust and able to withstand the ∼450 individual chemical processing steps associated with MAS (as well as manipulations required to hybridize, image, and dehybridize the arrays) without large-scale cracking, peeling, or delamination of the thin films. The combination of layer-by-layer assembly and MAS provides a means of fabricating functional oligonucleotide arrays on a range of different materials and substrates. This approach may also prove useful for the fabrication of supports for the solid-phase synthesis and screening of other macromolecular or small-molecule agents.Keywords: in situ synthesis; layer-by-layer; Maskless Array Synthesis; oligonucleotide arrays; polymer multilayers; reactive assembly; thin films;
Co-reporter:Burcu S. Aytar, Mark R. Prausnitz, and David M. Lynn
ACS Applied Materials & Interfaces 2012 Volume 4(Issue 5) pp:2726
Publication Date(Web):May 3, 2012
DOI:10.1021/am3003632
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(β-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.Keywords: DNA; electrochemical methods; layer-by-layer; rapid release; thin films;
Co-reporter:Burcu S. Aytar, John P.E. Muller, Sharon Golan, Yukishige Kondo, Yeshayahu Talmon, Nicholas L. Abbott, David M. Lynn
Journal of Colloid and Interface Science 2012 Volume 387(Issue 1) pp:56-64
Publication Date(Web):1 December 2012
DOI:10.1016/j.jcis.2012.07.083
We report an approach to the chemical oxidation of a ferrocene-containing cationic lipid [bis(11-ferrocenylundecyl)dimethylammonium bromide, BFDMA] that provides redox-based control over the delivery of DNA to cells. We demonstrate that BFDMA can be oxidized rapidly and quantitatively by treatment with Fe(III)sulfate. This chemical approach, while offering practical advantages compared to electrochemical methods used in past studies, was found to yield BFDMA/DNA lipoplexes that behave differently in the context of cell transfection from lipoplexes formed using electrochemically oxidized BFDMA. Specifically, while lipoplexes of the latter do not transfect cells efficiently, lipoplexes of chemically oxidized BFDMA promoted high levels of transgene expression (similar to levels promoted by reduced BFDMA). Characterization by SANS and cryo-TEM revealed lipoplexes of chemically and electrochemically oxidized BFDMA to both have amorphous nanostructures, but these lipoplexes differed significantly in size and zeta potential. Our results suggest that differences in zeta potential arise from the presence of residual Fe2+ and Fe3+ ions in samples of chemically oxidized BFDMA. Addition of the iron chelating agent EDTA to solutions of chemically oxidized BFDMA produced samples functionally similar to electrochemically oxidized BFDMA. These EDTA-treated samples could also be chemically reduced by treatment with ascorbic acid to produce samples of reduced BFDMA that do promote transfection. Our results demonstrate that entirely chemical approaches to oxidation and reduction can be used to achieve redox-based ‘on/off’ control of cell transfection similar to that achieved using electrochemical methods.Graphical abstractHighlights► The redox-active ferrocenyl lipid BFDMA can be chemically oxidized using Fe(III). ► Fe(III)-oxidized BFDMA transfects cells but electrochemically oxidized BFDMA does not. ► Characterization reveals similar nanostructures but differences in zeta potentials. ► Treatment with EDTA restores the “inactivity” of Fe(III)-oxidized BFDMA. ► Redox control of transfection can be achieved by adding chemical oxidizing and reducing agents.
Co-reporter:Elizabeth J. Tocce;Adam H. Broderick;Kaitlin C. Murphy;Sara J. Liliensiek;Christopher J. Murphy;Paul F. Nealey
Journal of Biomedical Materials Research Part A 2012 Volume 100A( Issue 1) pp:84-93
Publication Date(Web):
DOI:10.1002/jbm.a.33233
Abstract
Our study demonstrates that substrates fabricated using a “reactive” layer-by-layer approach promote well-defined cell–substrate interactions of human corneal epithelial cells. Specifically, crosslinked and amine-reactive polymer multilayers were produced by alternating “reactive” deposition of an azlactone-functionalized polymer [poly(2-vinyl-4,4-dimethylazlactone)] (PVDMA) and a primary amine-containing polymer [branched poly(ethylene imine)] (PEI). Advantages of our system include a 5- to 30-fold decrease in deposition time compared to traditional polyelectrolyte films and direct modification of the films with peptides. Our films react with mixtures of an adhesion-promoting peptide containing Arg-Gly-Asp (RGD) and the small molecule D-glucamine, a chemical motif which is nonfouling. Resulting surfaces prevent protein adsorption and promote cell attachment through specific peptide interactions. The specificity of cell attachment via immobilized RGD sequences was verified using both a scrambled RDG peptide control as well as soluble-RGD competitive assays. Films were functionalized with monotonically increasing surface densities of RGD which resulted in both increased cell attachment and the promotion of a tri-phasic proliferative response of a human corneal epithelial cell line (hTCEpi). The ability to treat PEI/PVDMA films with peptides for controlled cell–substrate interactions enables the use of these films in a wide range of biological applications. © 2011 Wiley Periodicals, Inc. J Biomed Mater Res Part A:, 2012.
Co-reporter:Shane L. Bechler and David M. Lynn
Biomacromolecules 2012 Volume 13(Issue 5) pp:
Publication Date(Web):April 2, 2012
DOI:10.1021/bm300234q
We report on conjugate addition-based approaches to the covalent layer-by-layer assembly of thin films and the post-fabrication functionalization of biointerfaces. Our approach is based on a recently reported approach to the “reactive” assembly of covalently cross-linked polymer multilayers driven by the 1,4-conjugate addition of amine functionality in poly(ethyleneimine) (PEI) to the acrylate groups in a small-molecule pentacrylate species (5-Ac). This process results in films containing degradable β-amino ester cross-links and residual acrylate and amine functionality that can be used as reactive handles for the subsequent immobilization of new functionality. Layer-by-layer growth of films fabricated on silicon substrates occurred in a supra-linear manner to yield films ∼750 nm thick after the deposition of 80 PEI/5-Ac layers. Characterization by atomic force microscopy (AFM) suggested a mechanism of growth that involves the reactive deposition of nanometer-scale aggregates of PEI and 5-Ac during assembly. Infrared (IR) spectroscopy studies revealed covalent assembly to occur by 1,4-conjugate addition without formation of amide functionality. Additional experiments demonstrated that acrylate-containing films could be postfunctionalized via conjugate addition reactions with small-molecule amines that influence important biointerfacial properties, including water contact angles and the ability of film-coated surfaces to prevent or promote the attachment of cells in vitro. For example, whereas conjugation of the hydrophobic molecule decylamine resulted in films that supported cell adhesion and growth, films treated with the carbohydrate-based motif d-glucamine resisted cell attachment and growth almost completely for up to 7 days in serum-containing media. We demonstrate that this conjugate addition-based approach also provides a means of immobilizing functionality through labile ester linkages that can be used to promote the long-term, surface-mediated release of conjugated species and promote gradual changes in interfacial properties upon incubation in physiological media (e.g., over a period of at least 1 month). These covalently cross-linked films are relatively stable in biological media for prolonged periods, but they begin to physically disintegrate after ∼30 days, suggesting opportunities to use this covalent layer-by-layer approach to design functional biointerfaces that ultimately erode or degrade to facilitate elimination.
Co-reporter:Eric M. Saurer, Ryan M. Flessner, Maren E. Buck and David M. Lynn
Journal of Materials Chemistry A 2011 vol. 21(Issue 6) pp:1736-1745
Publication Date(Web):21 Oct 2010
DOI:10.1039/C0JM02633F
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 ethylenediaminetetraacetic 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 CaCO3 and glass cores were observed and characterized using scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDS). Whereas capsules fabricated on CaCO3 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 precipitated on or within the walls of capsules after etching 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.
Co-reporter:Bin Sun, Ryan M. Flessner, Eric M. Saurer, Christopher M. Jewell, Nathaniel J. Fredin, David M. Lynn
Journal of Colloid and Interface Science 2011 Volume 355(Issue 2) pp:431-441
Publication Date(Web):15 March 2011
DOI:10.1016/j.jcis.2010.12.019
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.Graphical abstractPolyelectrolyte multilayers fabricated from poly(allylamine) and low molecular weight poly(acrylic acid) undergo large-scale transformations upon exposure to low-pH conditions to yield films with pores as large as 10–15 μm.Research highlights► The molecular weight of PAA plays a role in determining the sizes of pores that form. ► Low MW PAA leads to films with larger pores than films containing high MW PAA. ► Treatment at low pH yields films with pores up to 10–15 μm in size. ► Fabrication using fluorescently labeled PAA enables imaging of porous transformations. ► Contact with surfaces influences transformation and leads to larger pore sizes.
Co-reporter:Maren E. Buck
Advanced Engineering Materials 2011 Volume 13( Issue 10) pp:B343-B352
Publication Date(Web):
DOI:10.1002/adem.201080085
Abstract
We report a method for modulating the physicochemical properties of surfaces that is based on the reactive layer-by-layer fabrication of covalently crosslinked thin films using azlactone-functionalized copolymers. We demonstrate that copolymers containing different molar ratios of methyl methacrylate (MMA) and 2-vinyl-4,4-dimethylazlactone (VDMA) can be alternately deposited with poly(ethyleneimine) to assemble covalently crosslinked thin films. Characterization using ellipsometry demonstrates that, in general, film growth and thickness decrease as the content of reactive, azlactone functionality in the copolymer used to assemble the film decreases. Reflective infrared spectroscopy experiments demonstrate that films fabricated from MMA:VDMA copolymers contain residual azlactone functionality and that these reactive groups can be exploited to modify film-coated surfaces. Fabricating films from MMA:VDMA copolymers containing different compositions permitted modulation of the density of reactive groups within the films and, thus, the extent to which the films are functionalized by exposure to small molecule amines. For example, functionalization of MMA:VDMA copolymer films with the small molecule D-glucamine resulted in films with water contact angles that varied with the composition of the copolymer used to fabricate the film (e.g., as the azlactone content in the film increased, glucamine-modified films became more hydrophilic). We demonstrate further that treatment of copolymer-containing films with glucamine resulted in changes in the numbers of mammalian cells that grow on the surfaces of the films. Our results suggest the basis of methods that could be used to modulate or tune the density of chemical and biological functionality presented on surfaces of interest in a variety of fundamental and applied contexts.
Co-reporter:Sharon Golan, Burcu S. Aytar, John P. E. Muller, Yukishige Kondo, David M. Lynn, Nicholas L. Abbott, and Yeshayahu Talmon
Langmuir 2011 Volume 27(Issue 11) pp:6615-6621
Publication Date(Web):May 2, 2011
DOI:10.1021/la200450x
Biological media affect the physicochemical properties of cationic lipid–DNA complexes (lipoplexes) and can influence their ability to transfect cells. To develop new lipids for efficient DNA delivery, the influence of serum-containing media on the structures and properties of the resulting lipoplexes must be understood. To date, however, a clear and general picture of how serum-containing media influences the structures of lipoplexes has not been established. Some studies suggest that serum can disintegrate lipoplexes formed using certain types of cationic lipids, resulting in the inhibition of transfection. Other studies have demonstrated that lipoplexes formulated from other lipids are stable in the presence of serum and are able to transfect cells efficiently. In this article, we describe the influence of serum-containing media on lipoplexes formed using the redox-active cationic lipid bis(n-ferrocenylundecyl)dimethylammonium bromide (BFDMA). This lipoplex system promotes markedly decreased levels of transgene expression in COS-7 cells as serum concentrations are increased from 0 to 2, 5, 10, and 50% (v/v). To understand the cause of this decrease in transfection efficiency, we used cryogenic transmission electron microscopy (cryo-TEM) and measurements of zeta potential to characterize lipoplexes in cell culture media supplemented with 0, 2, 5, 10, and 50% serum. Cryo-TEM revealed that in serum-free media BFDMA lipoplexes form onionlike, multilamellar nanostructures. However, the presence of serum in the media caused disassociation of the intact multilamellar lipoplexes. At low serum concentrations (2 and 5%), DNA threads appeared to separate from the complex, leaving the nanostructure of the lipoplexes disrupted. At higher serum concentration (10%), disassociation increased and bundles of multilamellae were discharged from the main multilamellar complex. In contrast, lipoplexes characterized in serum-free aqueous salt (Li2SO4) medium and in OptiMEM cell culture medium (no serum) did not exhibit significant structural changes. The zeta potentials of lipoplexes in serum-free media (salt medium and cell culture medium) were similar (e.g., approximately −35 mV). Interestingly, the presence of serum caused the zeta potentials to become less negative (about −20 mV in OptiMEM and −10 mV in Li2SO4), even though serum contains negatively charged entities that have been demonstrated to lead to more negative zeta potentials in other lipoplex systems. The combined measurements of zeta potential and cryo-TEM are consistent with the proposition that DNA threads separate from the lipoplex in the presence of serum, resulting in a decrease in the net negative charge of the surface of the lipoplex.
Co-reporter:Eric M. Saurer, Dai Yamanouchi, Bo Liu, David M. Lynn
Biomaterials 2011 32(2) pp: 610-618
Publication Date(Web):
DOI:10.1016/j.biomaterials.2010.09.009
Co-reporter:Shane L. Bechler
Journal of Polymer Science Part A: Polymer Chemistry 2011 Volume 49( Issue 7) pp:1572-1581
Publication Date(Web):
DOI:10.1002/pola.24578
Abstract
We report the synthesis of a fluorescently end-labeled analog of a synthetic and degradable cationic poly(β-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 postpolymerization conjugate addition of amine-functionalized fluorophores. This approach enabled the synthesis of fluorescently end-labeled polymer (polymer 1FL) with molecular weights and polydispersities (Mn = 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 1FL 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. © 2011 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2011
Co-reporter:Maren E. Buck
Advanced Materials 2010 Volume 22( Issue 9) pp:994-998
Publication Date(Web):
DOI:10.1002/adma.200903054
Co-reporter:Maren E. Buck, Sarina C. Schwartz, and David M. Lynn
Chemistry of Materials 2010 Volume 22(Issue 23) pp:6319
Publication Date(Web):November 9, 2010
DOI:10.1021/cm102115e
We report an approach to the fabrication of superhydrophobic thin films that is based on the “reactive” layer-by-layer assembly of azlactone-containing polymer multilayers. We demonstrate that films fabricated from alternating layers of the azlactone functionalized polymer poly(2-vinyl-4,4-dimethylazlactone) (PVDMA) and poly(ethyleneimine) (PEI) exhibit micro- and nanoscale surface features that result in water contact angles in excess of 150°. Our results reveal that the formation of these surface features is (i) dependent upon film thickness (i.e., the number of layers of PEI and PVDMA deposited) and (ii) that it is influenced strongly by the presence (or absence) of cyclic azlactone-functionalized oligomers that can form upon storage of the 2-vinyl-4,4-dimethylazlactone (VDMA) used to synthesize PVDMA. For example, films fabricated using polymers synthesized in the presence of these oligomers exhibited rough, textured surfaces and superhydrophobic behavior (i.e., advancing contact angles in excess of 150°). In contrast, films fabricated from PVDMA polymerized in the absence of this oligomer (e.g., using freshly distilled monomer) were smooth and only moderately hydrophobic (i.e., advancing contact angles of ∼75°). The addition of authentic, independently synthesized oligomer to samples of distilled VDMA at specified and controlled concentrations permitted reproducible fabrication of superhydrophobic thin films on the surfaces of a variety of different substrates. The surfaces of these films were demonstrated to be superhydrophobic immediately after fabrication, but they became hydrophilic after exposure to water for 6 days. Additional experiments demonstrated that it was possible to stabilize and prolong the superhydrophobic properties of these films (e.g., advancing contact angles in excess of 150° even after complete submersion in water for at least 6 weeks) by exploiting the reactivity of residual azlactones to functionalize the surfaces of the films using hydrophobic amines (e.g., aliphatic or semifluorinated aliphatic amines). Our results demonstrate a straightforward and substrate-independent approach to the design of superhydrophobic and reactive polymer-based coatings of potential use in a broad range of fundamental and applied contexts.
Co-reporter:Bin Sun, David M. Lynn
Journal of Controlled Release 2010 Volume 148(Issue 1) pp:91-100
Publication Date(Web):20 November 2010
DOI:10.1016/j.jconrel.2010.07.112
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.
Co-reporter:Bin Sun, Xianghui Liu, Maren E. Buck and David M. Lynn
Chemical Communications 2010 vol. 46(Issue 12) pp:2016-2018
Publication Date(Web):19 Feb 2010
DOI:10.1039/B921664B
Azlactone-functionalized polymers are used as reactive templates for the synthesis of a library of amine-functionalized polymers of interest in the context of DNA delivery and other applications.
Co-reporter:Michael I. Kinsinger, Maren E. Buck, Maria-Victoria Meli, Nicholas L. Abbott, David M. Lynn
Journal of Colloid and Interface Science 2010 Volume 341(Issue 1) pp:124-135
Publication Date(Web):1 January 2010
DOI:10.1016/j.jcis.2009.09.026
We reported recently that amphiphilic polymers can be assembled at interfaces created between aqueous phases and thermotropic liquid crystals (LCs) in ways that: (i) couple the organization of the polymer to the order of the LC and (ii) respond to changes in the properties of aqueous phases that can be characterized as changes in the optical appearance of the LC. This investigation sought to characterize the behavior of aqueous–LC interfaces decorated with uniaxially compressed thin films of polymers transferred by Langmuir–Schaefer (LS) transfer. Here, we report physicochemical characterization of interfaces created between aqueous phases and the thermotropic LC 4-cyano-4′-pentylbiphenyl (5CB) decorated with Langmuir films of a novel amphiphilic polymer (polymer 1), synthesized by the addition of hydrophobic and hydrophilic side chains to poly(2-vinyl-4,4′-dimethylazlactone). Initial characterization of this system resulted in the unexpected observation of uniform azimuthal alignment of 5CB after LS transfer of the polymer films to aqueous–5CB interfaces. This paper describes characterization of Langmuir films of polymer 1 hosted at aqueous–5CB interfaces as well as the results of our investigations into the origins of the uniform ordering of the LC observed upon LS transfer. Our results, when combined, support the conclusion that uniform azimuthal alignment of 5CB is the result of long-range ordering of polymer chains in the Langmuir films (in a preferred direction orthogonal to the direction of compression) that is generated during uniaxial compression of the films prior to LS transfer. Although past studies of Langmuir films of polymers at aqueous–air interfaces have demonstrated that in-plane alignment of polymer backbones can be induced by uniaxial compression, these past reports have generally made use of polymers with rigid backbones. One important outcome of this current study is thus the observation of anisotropy and long-range order in Langmuir films of a novel flexible polymer. A second important outcome is the observation that the existence, extent, and dynamics of this order can be identified and characterized optically by transfer of the Langmuir film to a thin film of LC. Additional characterization of Langmuir films of two other flexible polymers [poly(methyl methacrylate) and poly(vinyl stearate)] using this method also resulted in uniform azimuthal alignment of 5CB, suggesting that the generation of long-range order in uniaxially compressed Langmuir films of polymers may also occur more generally over a broader range of polymers with flexible backbones.We report that Langmuir films of flexible polymers transferred to aqueous/liquid crystal interfaces using Langmuir–Schaefer methods induce uniform azimuthal alignment of the liquid crystal.
Co-reporter:Eric M. Saurer, Christopher M. Jewell, Jon M. Kuchenreuther, David M. Lynn
Acta Biomaterialia 2009 Volume 5(Issue 3) pp:913-924
Publication Date(Web):March 2009
DOI:10.1016/j.actbio.2008.08.022
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 (∼6 μm) 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.
Co-reporter:Maren E. Buck, Anthony S. Breitbach, Sonja K. Belgrade, Helen E. Blackwell and David M. Lynn
Biomacromolecules 2009 Volume 10(Issue 6) pp:
Publication Date(Web):May 13, 2009
DOI:10.1021/bm9001552
We report an approach to the design of reactive polymer films that can be functionalized post-fabrication to either prevent or promote the attachment and growth of cells. Our approach is based on the reactive layer-by-layer assembly of covalently crosslinked thin films using a synthetic polyamine and a polymer containing reactive azlactone functionality. Our results demonstrate (i) that the residual azlactone functionality in these films can be exploited to immobilize amine-functionalized chemical motifs similar to those that promote or prevent cell and protein adhesion when assembled as self-assembled monolayers on gold-coated surfaces and (ii) that the immobilization of these motifs changes significantly the behaviors and interactions of cells with the surfaces of these polymer films. We demonstrate that films treated with the hydrophobic molecule decylamine support the attachment and growth of mammalian cells in vitro. In contrast, films treated with the hydrophilic carbohydrate d-glucamine prevent cell adhesion and growth almost completely. The results of additional experiments suggest that these large differences in cell behavior can be understood, at least in part, in terms of differences in the abilities of these two different chemical motifs to promote or prevent the adsorption of protein onto film-coated surfaces. We demonstrate further that this approach can be used to pattern regions of these reactive films that resist the initial attachment and subsequent invasion of mammalian cells for periods of at least one month in the presence of serum-containing cell culture media. Finally, we report that films that prevent the adhesion and growth of mammalian cells also prevent the initial formation of bacterial biofilms when incubated in the presence of the clinically relevant pathogen Pseudomonas aeruginosa. The results of these studies, collectively, suggest the basis of general approaches to the fabrication and functionalization of thin films that prevent, promote, or pattern cell growth or the formation of biofilms on surfaces of interest in the contexts of both fundamental biological studies and a broad range of other practical applications.
Co-reporter:Nathaniel J. Fredin, Adam H. Broderick, Maren E. Buck and David M. Lynn
Biomacromolecules 2009 Volume 10(Issue 4) pp:
Publication Date(Web):March 16, 2009
DOI:10.1021/bm900045c
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 μm, 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 μm 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.
Co-reporter:Xianghui Liu;Jingtao Zhang
Advanced Materials 2008 Volume 20( Issue 21) pp:4148-4153
Publication Date(Web):
DOI:10.1002/adma.200800881
Co-reporter:Xianghui Liu, Jingtao Zhang and David M. Lynn
Soft Matter 2008 vol. 4(Issue 8) pp:1688-1695
Publication Date(Web):19 Jun 2008
DOI:10.1039/B804953J
We report an approach to the design of ‘charge-shifting’ anionic polymers that provides control over the disruption of ultrathin polyelectrolyte multilayers in aqueous media. We demonstrate that the addition of citraconic anhydride to poly(allylamine) yields an anionic, carboxylate-functionalized polymer (polymer 2) that can be converted readily back to cationic poly(allylamine) in acidic environments. The incorporation of polymer 2 into polyelectrolyte multilayers thus provides an approach to the fabrication of films that are stable at neutral pH but that erode over a period of several days in acidic media (e.g., pH ∼ 5). Experiments using a structural analog of polymer 2 with carboxylate side chains that do not hydrolyze readily provided support for the view that the disruption of these films occurred as a result of polymer side chain hydrolysis and a resulting change in the net charge of the polymers. Because this approach is based upon the use of anionic polymers to induce film instability, it provides a platform for the design of multilayers that can be used to provide control over the release of cationic film components. As proof of concept, we demonstrated that ultrathin films ∼100 nm thick fabricated using polymer 2 sustain the release of fluorescently labeled PAH for up to four days when incubated at pH 5.0. The synthetic approach used here is modular and tunable and can be used to introduce anionic ‘charge-shifting’ character to a broad range of other polyamines. With further development, this approach could expand significantly the range of different cationic agents (e.g., cationic proteins, peptides, polymers, nanoparticles, etc.) that can be released or delivered from surfaces using polyelectrolyte multilayers.
Co-reporter:Xianghui Liu, Jennifer W. Yang and David M. Lynn
Biomacromolecules 2008 Volume 9(Issue 7) pp:
Publication Date(Web):June 20, 2008
DOI:10.1021/bm800291v
We reported recently that the addition of ester-functionalized, “charge-shifting” side chains to linear poly(ethyleneimine) (LPEI) can be used to design polyamines that promote both self-assembly and self-disassembly with DNA in aqueous environments. This investigation sought to characterize the influence of charge-shifting side chains on the ability of LPEI to mediate cell transfection and understand the extent to which increases (or decreases) in levels of transfection could be understood in terms of time-dependent changes in the net charges of these polymers. We report that the addition of “charge-shifting” side chains to LPEI leads to significant increases in levels of LPEI-mediated transfection. In particular, polymer 1e, functionalized with 20 mol % ester-functionalized side chains, mediates levels of transgene expression in vitro up to 8-fold higher than LPEI. Experiments using an amide-functionalized analog of polymer 1e demonstrated that the esters in polymer 1e play an important role in promoting increased levels of transfection. These results, in combination with the results of additional gel electrophoresis experiments, provide support for the view that increases in transfection result from time-dependent changes in the net charge of polymer 1e and the disruption of ionic interactions in polyplexes. Additional support for this view is provided by the results of confocal microscopy experiments and measurements of fluorescence resonance energy transfer, which suggest that polymer 1e promotes the disruption of polyplexes in intracellular environments effectively. The approach reported here provides a means of addressing one important “late-stage” obstacle to polyplex-mediated transfection (polyplex unpackaging). If integrated successfully with methods that have been developed to address other important barriers to transfection, this general approach could lead to the development of multifunctional polyplexes that mimic more effectively the range of functions of viruses as agents for the delivery of DNA.
Co-reporter:David M. Lynn
Soft Matter 2006 vol. 2(Issue 4) pp:269-273
Publication Date(Web):10 Feb 2006
DOI:10.1039/B517860F
Layer-by-layer approaches to the fabrication of nanostructured polymer assemblies have contributed to the design of thin films and composites of interest in a variety of areas. Here, we highlight recent contributions from our laboratory toward the design of multilayered assemblies that erode controllably and predictably in physiological environments and permit spatial and temporal control over the administration of macromolecular therapeutics, such as DNA. Our approach makes use of assemblies fabricated from hydrolytically degradable polyamines. We place this work in the context of recent progress from other laboratories and discuss new opportunities and connections that illustrate the potential of these layered assemblies as platforms for the controlled release of one or several therapeutic agents.
Co-reporter:Jingtao Zhang;Nathaniel J. Fredin;Jingtao Zhang;Nathaniel J. Fredin
Journal of Polymer Science Part A: Polymer Chemistry 2006 Volume 44(Issue 17) pp:5161-5173
Publication Date(Web):28 JUL 2006
DOI:10.1002/pola.21613
Multilayered polyelectrolyte assemblies fabricated using hydrolytically degradable polyamines (1–3) erode gradually when incubated in physiologically relevant media. This investigation sought to characterize physically and chemically the erosion of films fabricated from these polymers and sodium poly(styrene sulfonate) (SPS) and to investigate specifically the potential role of polymer hydrolysis in governing film erosion. The characterization of erosion using reflective infrared spectroscopy revealed changes in the carbonyl region of the spectrum that were consistent with the generation of polymer hydrolysis products. To evaluate the role of the esters in these materials more directly, we also synthesized a structural analogue of polymer 2 containing amide functionality rather than ester functionality. Assemblies fabricated from this amide-containing polymer did not erode significantly or release SPS into solution when incubated in phosphate-buffered saline (PBS). Finally, we characterized the erosion of assemblies fabricated from polymer 1 in PBS buffer prepared with D2O rather than H2O. These assemblies eroded significantly more slowly in deuterated media than in buffer prepared with H2O. These results, when combined, provide support for the view that polymer hydrolysis plays an important role in governing the erosion of assemblies fabricated from these degradable polymers. © 2006 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 44: 5161–5173, 2006
Co-reporter:Marjorie O'Connell
Science 1918 Vol 48(1250) pp:588-590
Publication Date(Web):13 Dec 1918
DOI:10.1126/science.48.1250.588
Co-reporter:Melissa E. Hays, Christopher M. Jewell, Yukishige Kondo, David M. Lynn, Nicholas L. Abbott
Biophysical Journal (15 December 2007) Volume 93(Issue 12) pp:
Publication Date(Web):15 December 2007
DOI:10.1529/biophysj.107.107094
The effect of lipid oxidation state on the physical properties of complexes formed by plasmid DNA and the redox-active lipid bis-(11-ferrocenylundecyl)dimethylammonium bromide (BFDMA) is reported. With increasing concentration of BFDMA, the hydrodynamic sizes of complexes formed by BFDMA and DNA (in the presence of 1 mM Li2SO4) pass through a maximum and the ζ-potential changes monotonically from −40 mV to +40 mV. In contrast, complexes formed by oxidized BFDMA and DNA exhibit a minimum in size and maintain a negative ζ-potential with increasing concentration of BFDMA. Angle-dependent dynamic light scattering measurements also reveal the presence of relaxation processes within complexes formed by DNA and oxidized BFDMA that are absent for complexes formed by DNA and reduced BFDMA. These results, when combined, reveal that the amphiphilic nature of reduced BFDMA leads to lipoplexes with physical properties resembling those formed by classical cationic lipids, whereas the interaction of oxidized BFDMA with DNA is similar to that of nonamphiphilic cationic molecules bearing multiple charges (e.g., spermidine). In particular, the negative ζ-potential and measurable presence of DNA chain dynamics within complexes formed by oxidized BFDMA and DNA indicate that these complexes are loosely packed with excess charge due to DNA in their outer regions. These results, when combined with additional measurements performed in OptiMEM reduced-serum cell culture medium, lead to the proposition that the strong dependence of transfection efficiency on the oxidation state of BFDMA, as reported previously, is largely a reflection of the substantial change in the ζ-potentials of these complexes with changes in the oxidation state of BFDMA.
Co-reporter:Christopher M. Jewell, David M. Lynn
Advanced Drug Delivery Reviews (10 June 2008) Volume 60(Issue 9) pp:979-999
Publication Date(Web):10 June 2008
DOI:10.1016/j.addr.2008.02.010
Materials that provide spatial and temporal control over the delivery of DNA and other nucleic acid-based agents from surfaces play important roles in the development of localized gene-based therapies. This review focuses on a relatively new approach to the immobilization and release of DNA from surfaces: methods based on the layer-by-layer assembly of thin multilayered films (or polyelectrolyte multilayers, PEMs). Layer-by-layer methods provide convenient, nanometer-scale control over the incorporation of DNA, RNA, and oligonucleotide constructs into thin polyelectrolyte films. Provided that these assemblies can be designed in ways that permit controlled film disassembly under physiological conditions, this approach can contribute new methods for spatial and/or temporal control over the delivery of nucleic acid-based therapeutics in vitro and in vivo. We describe applications of layer-by-layer assembly to the fabrication of DNA-containing films that can be used to provide control over the release of plasmid DNA from the surfaces of macroscopic objects and promote surface-mediated cell transfection. We also highlight the application of these methods to the coating of colloidal substrates and the fabrication of hollow micrometer-scale capsules that can be used to encapsulate and control the release or delivery of DNA and oligonucleotides. Current challenges, gaps in knowledge, and new opportunities for the development of these methods in the general area of gene delivery are discussed.
Co-reporter:Bin Sun, Xianghui Liu, Maren E. Buck and David M. Lynn
Chemical Communications 2010 - vol. 46(Issue 12) pp:NaN2018-2018
Publication Date(Web):2010/02/19
DOI:10.1039/B921664B
Azlactone-functionalized polymers are used as reactive templates for the synthesis of a library of amine-functionalized polymers of interest in the context of DNA delivery and other applications.
Co-reporter:Eric M. Saurer, Ryan M. Flessner, Maren E. Buck and David M. Lynn
Journal of Materials Chemistry A 2011 - vol. 21(Issue 6) pp:NaN1745-1745
Publication Date(Web):2010/10/21
DOI:10.1039/C0JM02633F
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 ethylenediaminetetraacetic 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 CaCO3 and glass cores were observed and characterized using scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDS). Whereas capsules fabricated on CaCO3 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 precipitated on or within the walls of capsules after etching 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.
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