Patterning poly(organophosphazenes) for selective cell adhesion applications

Five polyphosphazenes with different hydrophilicites were synthesized and screened in vitro. The purpose was to identify unique types of polymeric substrates that distinctly favored or markedly prevented cellular adhesion. The SK-N-BE(2c) human neuroblastoma cell line, utilized for its electrogenic responses, was used to test this differential adhesion. In particular, the objective was to specifically culture this cell line in a highly selective pattern. Each candidate polymer was cast into films and plated with neuroblastoma cells for 3 days. The polyphosphazene materials which showed negative cellular adhesive properties (-CAPs) were poly[bis(trifluoroethoxy)phosphazene] (TFE) and poly[bis(methoxyethoxyethoxy)phosphazene] (MEEP). The polyphosphazenes which showed positive cellular adhesive properties (+CAPs) were poly[(methoxyethoxyethoxy)(1.0)(carboxylatophenoxy)(1.0)phosphazene] (PMCPP), poly[(methoxyethoxyethoxy)(1.0)(cinnamyloxy)(1.0)phosphazene] (PMCP), and poly[(methoxyethoxyethoxy)(1.0)(p-methylphenoxy)(1.0)phosphazene] (PMMP). To test cellular selectivity, films of -CAP and +CAP were copatterned onto glass substrates. The micropatterned films were plated with SK-N-BE(2c) neuroblastoma cells for one week. The results showed that neuroblastoma cells adhere selectively (over 60%) to the +CAP microfeatures. We also showed that multiple properties can be achieved with a single material and that we can use TFE as both a -CAP and an insulation layer and PMCP as a conductive +CAP layer.     

Hepatocyte culture on biodegradable polymeric substrates

The interactions of primary rat liver cells with biodegradable polymeric substrates were investigated in vitro to assess the suitability of the polymer materials for use in cell transplantation devices. The kinetics of cell adhesion to, and the growth and biochemical function of cells maintained on, films formed from poly (D,L-lactic-co-glycolic acid, 88: 12) (PLGA) or from a 50/50 (w/w) blend of PLGA and poly (L-lactic acid) (PLLA) were evaluated in comparison to two control substrates, matrigel coated or collagen-coated polystyrene petri dishes. The rate of cell adhesion to both types of polymeric substrates was similar to the rate of adhesion to the collagen control substrate, but of the two polymers, only the blend was suitable for extended culture. Hepatocytes maintained on the polymer blend films showed retention of differentiated cell function as measured by the rate of albumin secretion-the rate of albumin secretion by cells on the films was the same as the rate for cells on matrigel and reached a level in the range of reported in vivo levels (140-160 mug/10(6) cells/24 h). In contrast, albumin secretion by hepatocytes maintained on collagen-coated polystyrene culture dishes declined over five days to a level one third that of the initial level and one fifth that of cells maintained on the polymer blend films on day five. Such retention of differentiated cell function by hepatocytes in culture has previously been observed only when hepatocytes were cultured in the presence of exogenous extracellular matrix proteins or were cocultured with another cell type. In addition to retention of differentiated function, the cells maintained on the polymer blend films also displayed rates of DNA synthesis similar to controls maintained on collagen-coated polystyrene, a substrate optimal for DNA synthesis.     

Surface modification with poly(sulfobetaine methacrylate-co-acrylic acid) to reduce fibrinogen adsorption, platelet adhesion, and plasma coagulation

Zwitterionic sulfobetaine methacrylate (SBMA) polymers were known to possess excellent antifouling properties due to high hydration capacity and neutral charge surface. In this study, copolymers of SBMA and acrylic acid (AA) with a variety of compositions were synthesized and were immobilized onto polymeric substrates with layer-by-layer polyelectrolyte films via electrostatic interaction. The amounts of platelet adhesion and fibrinogen adsorption were determined to evaluate hemocompatibility of poly(SBMA-co-AA)-modified substrates. Among various deposition conditions by modulating SBMA ratio in the copolymers and pH of the deposition solution, poly(SBMA(56)-co-AA(44)) deposited at pH 3.0 possessed the best hemocompatibility. This work demonstrated that poly(SBMA-co-AA) copolymers adsorbed on polyelectrolyte-base films via electrostatic interaction improve hemocompatibility effectively and are applicable for various substrates including TCPS, PU, and PDMS. Furthermore, poly(SBMA-co-AA)-coated substrate possesses great durability under rigorous conditions. The preliminary hemocompatibility tests regarding platelet adhesion, fibrinogen adsorption, and plasma coagulation suggest the potential of this technique for the application to blood-contacting biomedical devices.     

Addition of biological functionality to poly(epsilon-caprolactone) films

Biodegradable polyesters such as poly(epsilon-caprolactone) (PCL) have a number of biomedical applications; however, their usage is often limited by a lack of biological functionality. In this paper, a PCL-based polymer containing pendent groups activated by 4-nitrophenyl chloroformate (NPC) and reactive toward primary amines has been cast into thin films. The reactivity of the films toward poly(l-lysine) and the cell adhesion peptide, GRGDS, was assessed, and their cell adhesive capabilities were characterized. ATR-FTIR analysis found that NPC functional groups were present on the surface of the cast film, and the synthesis, conjugation, and visualization of a fluorescent molecule on these films further demonstrated the success of this functionalization methodology. The immersion of these films into a solution of either poly(l-lysine) (PLL) or GRGDS in PBS (pH 7.4) and subsequent 3T3 fibroblast adhesion studies demonstrated significant improvement in cell adhesion and spreading over films cast from unmodified PCL. This investigation has shown that this novel NPC-containing polymer can be utilized in many applications where increased cellular adhesion is required, or the coupling of specific molecules to polymer surfaces is of interest.     

Stable biopassive insulation synthesized by initiated chemical vapor deposition of poly(1,3,5-trivinyltrimethylcyclotrisiloxane)

The permanent implantation of electronic probes capable of recording neural activity patterns requires long-term electrical insulation of these devices by biopassive coatings. In this work, the material properties and neural cell compatibility of a novel polymeric material, poly(trivinyltrimethylcyclotrisiloxane) (poly(V3D3)), are demonstrated to be suitable for application as permanently bioimplanted electrically insulating films. The poly(V3D3) polymeric films are synthesized by initiated chemical vapor deposition (iCVD), allowing for conformal and flexible encapsulation of fine wires. The poly(V3D3) also exhibits high adhesive strength to silicon substrates, a common material of manufacture for neural probes. The poly(V3D3) films were found to be insoluble in both polar and nonpolar solvents, consistent with their highly cross-linked structure. The films are pinhole-free and extremely smooth, having a root-mean-square (rms) roughness of 0.4 nm. The material possesses a bulk resistivity of 4 x 1015 Ohm-cm exceeding that of Parylene-C, the material currently used to insulate neurally implanted devices. The iCVD poly(V3D3) films are hydrolytically stable and are demonstrated to maintain their electrical properties under physiological soak conditions, and constant electrical bias, for more than 2 years. In addition, biocompatibility studies with PC12 neurons demonstrate that this material is noncytotoxic and does not influence cell proliferation.     

Poly(2-oxazoline) hydrogels as next generation three-dimensional cell supports

Synthetic hydrogels selectively decorated with cell adhesion motifs are rapidly emerging as promising substrates for 3D cell culture. When cells are grown in 3D they experience potentially more physiologically relevant cell–cell interactions and physical cues compared with traditional 2D cell culture on stiff surfaces. A newly developed polymer based on poly(2-oxazoline)s has been used for the first time to control attachment of fibroblast cells and is discussed here for its potential use in 3D cell culture with particular focus on cancer cells toward the ultimate aim of high-throughput screening of anticancer therapies. Advantages and limitations of using poly(2-oxazoline) hydrogels are discussed and compared with more established polymers, especially polyethylene glycol (PEG).     

Poly(2-oxazoline) hydrogels as next generation three-dimensional cell supports

Synthetic hydrogels selectively decorated with cell adhesion motifs are rapidly emerging as promising substrates for 3D cell culture. When cells are grown in 3D they experience potentially more physiologically relevant cell–cell interactions and physical cues compared with traditional 2D cell culture on stiff surfaces. A newly developed polymer based on poly(2-oxazoline)s has been used for the first time to control attachment of fibroblast cells and is discussed here for its potential use in 3D cell culture with particular focus on cancer cells toward the ultimate aim of high-throughput screening of anticancer therapies. Advantages and limitations of using poly(2-oxazoline) hydrogels are discussed and compared with more established polymers, especially polyethylene glycol (PEG).     

Modulation of spreading, proliferation, and differentiation of human mesenchymal stem cells on gelatin-immobilized poly(L-lactide-co--caprolactone) substrates

Controlled adhesion and continuous growth of human mesenchymal stem cells (hMSCs) are essential for scaffold-based delivery of hMSCs in tissue engineering applications. The main goal of this study is to develop biofunctionalized synthetic substrates to actively control adhesion, spreading, and proliferation of hMSCs. gamma-Ray irradiation was employed to graft acrylic acid (AAc) to biodegeradable poly(L-lactide-co--caprolactone) (PLCL) films. Gelatin, a natural polymer, was then immobilized on the AAc grafted PLCL film (AAc-PLCL) to induce biomimetic interactions with the cells. The graft yield of AAc increased as the irradiation dose and AAc concentration increased, and the presence of gelatin (gelatin-AAc-PLCL) following immobilization was confirmed using ESCA. To investigate cell responses, hMSCs isolated from a human mandible were cultured on the various substrates and their adhesion, spreading, and proliferation were examined. After three days of culture, the DNA concentration from the cells cultured on gelatin-AAc-PLCL film was 2.9-fold greater than that on the PLCL film. Immunofluorescent staining of hMSCs cultured on the gelatin-AAc-PLCL films demonstrated homogeneous localization of F-Actin and vinculin in their cytoplasm, while mature adhesive structure was not observed from the cells cultured on other substrates. Furthermore, the ratio of projected area of adherent single cells on gelatin-AAc-PLCL films was significantly larger (116.80 +/- 12.78%) than that on the PLCL films (30.11 +/- 5.07%). Our results suggest that gelatin-immobilized PLCL substrates may be potentially used in tissue engineering, particularly as a stem cell delivery carrier for the regeneration of target tissue.     

Rational design of cytophilic and cytophobic polyelectrolyte multilayer thin films

Nanostructured polyelectrolyte multilayer thin films electrostatically assembled alternately from such polymers as poly(allylamine hydrochloride) (PAH) and poly(acrylic acid) (PAA) were investigated for their in vitro cell interactions. Not surprisingly, NR6WT cells, a highly adhesive murine fibroblast cell line, attached to many different multilayer combinations tested. However, PAH/PAA multilayers constructed at pH deposition conditions of 2.0/2.0 were completely bioinert. Analogous cell interactions were observed with PAH/poly(methacrylic acid) (PAH/PMA), PAH/sulfonated poly(styrene) (PAH/SPS), and poly(diallyldimethylammonium chloride)/SPS (PDAC/SPS) systems, thereby suggesting a general trend in the fibroblasts' response to multilayers. Specifically, highly ionically stitched films attracted cells, whereas weakly ionically cross-linked multilayers, which swell substantially in physiological conditions to present richly hydrated surfaces, resisted fibroblast attachment. Thus, by manipulating the multilayer pH or ionic strength assembly conditions or both, which in turn dictate the molecular architecture of the thin films, one may powerfully direct a single multilayer combination to be either cell adhesive or cell resistant.     

Ricin toxicity to microglial and monocytic cells.

Microglial cells, like macrophages, are very sensitive to ricin, a galactose-specific toxic lectin belonging to the family of ribosome-inactivating proteins. This toxin can be taken up by most cells through the binding of its B chain to galactose-containing molecules on the cell membrane. In macrophagic cell types it can be internalised also by mannose receptors which are present on the surface of these cells. Endocytosis of the toxin by either pathway was evaluated by ricin toxicity to primary cultures of rat microglial cells and to a microglial N11 cell line in the presence or absence of lactose and mannan, which compete for the endocytosis via the ricin lectin chain or cellular mannose receptors, respectively. Results were compared with those obtained in cultures of mouse macrophages, human monocytes, and a monocytic JM cell line. All cultures were protected from ricin toxicity more by lactose than by mannan, indicating that ricin endocytosis via its lectin B chain is prevalent over that mediated by cellular mannose receptors. However, a partial protection by mannan was observed in all cases but not-stimulated N11 cells, either in the form of direct protection or of significant additional protection over that afforded by lactose. Mannose receptor expression by N11 cells was negative before, and positive after, treatment with endotoxin, as assessed by the specific binding of 125I-mannose-bovine serum albumin. Moreover, a partial protection from ricin toxicity by mannan was induced in the N11 microglial line after stimulation, consistently with an inducible expression of the mannose receptor by activated cells switched towards a microglial phenotype.     

Characterization of porous PLGA/PLA microparticles as a scaffold for three dimensional growth of breast cancer cells

We have designed and evaluated biodegradable porous polymeric microparticles as a scaffold for cell growth. The hypothesis was that microparticles with optimized composition and properties would have better cell adhesion and hence cell growth into a tissue-like structure. Solvent-evaporation method was modified using sucrose as an additive to form large porous microparticles of poly(D,L-lactic-co-glycolic) (PLGA) and polylactide (PLA) polymers. Microparticles containing hydrophilic polymers (poly(vinyl alcohol) and chitosan) incorporated in their internal matrix structure were also formulated. Different formulations of microparticles were evaluated for physical properties, cell adhesion, and cell growth in culture. PLA microparticles containing poly(vinyl alcohol) (PVA) in the matrix structure (PLA-PVA) and treated with serum prior to cell seeding demonstrated better cell adhesion and cell growth than other formulations of microparticles. Cells were seen to grow into clumps, engulfing microparticles completely with time, and forming a 3-D tissue-like structure. Cell density of 1.5 x 10(6) cells per mg of microparticles was achieved in 9 days of culture, which was a 7-fold increase from the initial seeding cell density. The mechanism of better cell growth on PLA-PVA microparticles appears to be due to the PVA associated with the internal matrix structure of microparticles. These microparticles demonstrated better wetting in culture and also cell adhesion. In addition to tissue engineering applications, microparticles with cancer cells grown into a tissue-like structure in vitro can be potentially used as a model system for preclinical evaluation of the cytotoxic effect of anticancer agents.     

SOFT‐MI: A novel microfabrication technique integrating soft‐lithography and molecular imprinting for tissue engineering applications

An innovative approach has been employed for the realization of bioactive scaffolds able to mimic the in vivo cellular microenvironment for tissue engineering applications. This method is based on the combination of molecular imprinting and soft‐lithography technology to enhance cellular adhesion and to guide cell growth and proliferation due to presence of highly specific recognition sites of selected biomolecules on a well‐defined polymeric microstructure. In this article polymethylmethacrylate (PMMA) scaffolds have been realized by using poly(dimethylsiloxane) (PDMS) microstructured molds imprinted with FITC‐albumin and TRITC‐lectin. In addition gelatin, an adhesion protein, was employed for the molecular imprinting of polymeric scaffolds for cellular tests. The most innovative aspect of this research was the molecular imprinting of whole cells for the development of substrates able to enhance the cell adhesion processes. Biotechnol. Bioeng. 2010;106: 804–817. © 2010 Wiley Periodicals, Inc.     

Imaging cell interactions with native and crosslinked polyelectrolyte multilayers

The adhesion of primary chondrocytes to polyelectrolyte multilayer films, made of poly(l-lysine) (PLL) and hyaluronan (HA), was investigated for native and crosslinked films, either ending by PLL or HA. Crosslinking the film was achieved by means of a water-soluble carbodiimide in combination with N-hydroxysulfosuccinimide. The adhesion of macrophages and primary chondrocytes was investigated by microscopical techniques (optical, confocal, and atomic), providing useful information on the cell/film interface. Native films were found to be nonadhesive for the, primary chondrocytes, but could be degraded by macrophages, as could be visualized by confocal laser scanning microscopy after film labeling. Confocal microscopy images show that these films can be deformed by the condrocytes and that PLL diffuses at the chondrocyte membrane. In contrast, the cells adhered and proliferated well on the crosslinked films, which were not degraded by the macrophages. These results were confirmed by a MTT test over a 6-d period and by atomic force microscopy observations. We thus prove that chemical crosslinking can dramatically change cell adhesion properties, the cells being more stably anchored on the crosslinked films. Both authors kcontributed equally.     

Glycated collagen and altered glucose increase endothelial cell adhesion strength

Cell adhesion strength is important to cell survival, proliferation, migration, and mechanotransduction, yet changes in endothelial cell adhesion strength have not yet been examined in diseases such as diabetes with high rates of cardiovascular complications. We therefore investigated porcine aortic endothelial cell adhesion strength on native and glycated collagen‐coated substrates and in low, normal, and high glucose culture using a spinning disc apparatus. Adhesion strength increased by 30 dynes/cm² in cells on glycated collagen as compared to native collagen. Attachment studies revealed that cells use higher adhesion strength α_vβ₃ integrins to bind to glycated collagen instead of the typical α₂β₁ integrins used to bind to native collagen. Similarly, endothelial cells cultured in low and high glucose had 15 dynes/cm² higher adhesion strength than cells in normal glucose after 2 days. Increased adhesion strength was due to elevated VEGF release and intracellular PKC in low and high glucose cells, respectively. Thus glucose increased endothelial cell adhesion strength via different underlying mechanisms. These adhesion strength changes could contribute to diabetic vascular disease, including accelerated atherosclerosis and disordered angiogenesis. J. Cell. Physiol. 228: 1727–1736, 2013. © 2013 Wiley Periodicals, Inc.     

Myocilin promotes substrate adhesion,spreading and formation of focal contacts in podocytes and mesangial cells

Myocilin, a secreted glycoprotein of the olfactomedin family, is constitutively expressed in podocytes of the rat kidney and induced in mesangial cells during mesangioproliferative glomerulonephritis. As myocilin has been found to be associated with fibrillar components of the extracellular matrix, and adhesive properties have been shown for other members of the olfactomedin family, we hypothesized that myocilin might play a role in cell-matrix interactions in the glomerulus. To elucidate functional properties of myocilin, recombinant myocilin was expressed in 293 EBNA cells and purified by Ni-chelate and heparin chromatography. Culture plates were coated with myocilin, and primary rat mesangial cells and cells from an immortal murine podocyte cell line were seeded onto the plates in serum free conditions. Both cell types showed concentration-dependant attachment to myocilin, an effect that was statistically significant and could be blocked with specific antibodies. When compared to equal amounts of fibronectin or collagen 1, myocilin was less effective in promoting substrate adhesion. Synergistic effects in substrate adhesion were observed when myocilin was added to low concentrations of fibronectin. Twenty-five percent of cells that had attached to myocilin substrates showed spreading and expressed focal contacts which were labeled by vinculin/phalloidin staining. Comparable findings were observed when human or murine trabecular meshwork cells were seeded on myocilin substrates. Adhesive properties of myocilin required multimer formation, and were not observed when culture plates were coated with a C-terminal fragment of myocilin, containing the olfactomedin domain. We conclude that myocilin promotes substrate adhesion of podocytes and mesangial cells, and might contribute to cell-matrix adhesion of both cell types in vivo.     

Surface immobilization of galactose onto aliphatic biodegradable polymers for hepatocyte culture

A novel surface modification method of biodegradable polymers was investigated for inducing the attachment of specific cells onto the polymer surface via ligand-receptor interactions. Galactose, a targeting ligand specific to asialoglycoprotein receptors present on cell membrane of hepatocytes, was introduced on the surface of poly(D,L-lactic-co-glycolic acid) (PLGA) films. A terminal end group of carboxylic acid in PLGA was activated by dicyclohexylcarbodiimide and N-hydroxysuccinimide for the direct conjugation of lactose by reductive amination reaction. Di-block copolymers of PLGA-b-poly(ethylene glycol) (PEG) having a free terminal amine group were also synthesized and used for the conjugation of galactose for the introduction of a PEG spacer between PLGA and galactose. The presence of galactose moieties on the blend film surface was characterized by measuring water contact angle and X-ray photon spectroscopy, and the amount of galactose was indirectly determined by a specific lectin-binding assay. With increasing the galactose concentration on the blend film surface, the initial attachment as well as the cell viability of hepatocyates concomitantly increased. The introduction of PEG spacer reduced the cell attachment and viability. Albumin secretion rate from hepatocytes was enhanced for galactose modified surfaces, whereas it was reduced for the surfaces not having galactose moieties.     

Human Neutrophil Flow Chamber Adhesion Assay

Neutrophil firm adhesion to endothelial cells plays a critical role in inflammation in both health and disease. The process of neutrophil firm adhesion involves many different adhesion molecules including members of the β₂ integrin family and their counter-receptors of the ICAM family. Recently, naturally occurring genetic variants in both β₂ integrins and ICAMs are reported to be associated with autoimmune disease. Thus, the quantitative adhesive capacity of neutrophils from individuals with varying allelic forms of these adhesion molecules is important to study in relation to mechanisms underlying development of autoimmunity. Adhesion studies in flow chamber systems can create an environment with fluid shear stress similar to that observed in the blood vessel environment in vivo. Here, we present a method using a flow chamber assay system to study the quantitative adhesive properties of human peripheral blood neutrophils to human umbilical vein endothelial cell (HUVEC) and to purified ligand substrates. With this method, the neutrophil adhesive capacities from donors with different allelic variants in adhesion receptors can be assessed and compared. This method can also be modified to assess adhesion of other primary cell types or cell lines.     

Tunable cell-surface mimetics as engineered cell substrates

Most recent breakthroughs in understanding cell adhesion, cell migration, and cellular mechanosensitivity have been made possible by the development of engineered cell substrates of well-defined surface properties. Traditionally, these substrates mimic the extracellular matrix (ECM) environment by the use of ligand-functionalized polymeric gels of adjustable stiffness. However, such ECM mimetics are limited in their ability to replicate the rich dynamics found at cell-cell contacts. This review focuses on the application of cell surface mimetics, which are better suited for the analysis of cell adhesion, cell migration, and cellular mechanosensitivity across cell-cell interfaces. Functionalized supported lipid bilayer systems were first introduced as biomembrane-mimicking substrates to study processes of adhesion maturation during adhesion of functionalized vesicles (cell-free assay) and plated cells. However, while able to capture adhesion processes, the fluid lipid bilayer of such a relatively simple planar model membrane prevents adhering cells from transducing contractile forces to the underlying solid, making studies of cell migration and cellular mechanosensitivity largely impractical. Therefore, the main focus of this review is on polymer-tethered lipid bilayer architectures as biomembrane-mimicking cell substrate. Unlike supported lipid bilayers, these polymer-lipid composite materials enable the free assembly of linkers into linker clusters at cellular contacts without hindering cell spreading and migration and allow the controlled regulation of mechanical properties, enabling studies of cellular mechanosensitivity. The various polymer-tethered lipid bilayer architectures and their complementary properties as cell substrates are discussed.     

Thrombospondin modulates focal adhesions in endothelial cells

We examined the effects of thrombospondin (TSP) in the substrate adhesion of bovine aortic endothelial cells. The protein was tested both as a substrate for cell adhesion and as a modulator of the later stages of the cell adhesive process. TSP substrates supported the attachment of some BAE cells, but not cell spreading or the formation of focal adhesion plaques. In contrast, cells seeded on fibrinogen or fibronectin substrates were able to complete the adhesive process, as indicated by the formation of focal adhesion plaques. Incubation of cells in suspension with soluble TSP before or at the time of seeding onto fibronectin substrates resulted in an inhibition of focal adhesion formation. Furthermore, the addition of TSP to fully adherent cells in situ or prespread on fibronectin substrates caused a reduction in the number of cells, which were positive for focal adhesions, although there was no significant effect on cell spreading. In a dose-dependent manner, TSP reduced the number of cells with adhesion plaques to approximately 60% of control levels. The distribution of remaining adhesion plaques in TSP-treated cells was also altered: plaques were primarily limited to the periphery of cells and were not present in the central cell body, as in control cells treated with BSA. The observed effects were specific for TSP and were not observed with platelet factor 4, beta-thromboglobulin, or fibronectin. The TSP-mediated loss of adhesion plaques was neutralized by the addition of heparin, fucoidan, other heparin-binding proteins, and by a monoclonal antibody to the heparin binding domain of TSP, but not by antibodies to the core or carboxy-terminal regions of TSP. The interaction of the heparin- binding domain of TSP with cell-associated heparan sulfate appears to be an important mechanistic component for this activity of TSP. These data indicate that TSP may have a role in destabilizing cell adhesion through prevention of focal adhesion formation and by loss of preformed focal adhesions.     

Enhanced adhesion of dopamine methacrylamide elastomers via viscoelasticity tuning

We present a study on the effects of cross-linking on the adhesive properties of bio-inspired 3,4-dihydroxyphenylalanine (DOPA). DOPA has a unique catechol moiety found in adhesive proteins in marine organisms, such as mussels and polychaete, which results in strong adhesion in aquatic conditions. Incorporation of this functional group in synthetic polymers provides the basis for pressure-sensitive adhesives for use in a broad range of environments. A series of cross-linked DOPA-containing polymers were prepared by adding divinyl cross-linking agent ethylene glycol dimethacrylate (EGDMA) to monomer mixtures of dopamine methacrylamide (DMA) and 2-methoxyethyl acrylate (MEA). Samples were prepared using a solvent-free microwave-assisted polymerization reaction and compared to a similar series of cross-linked MEA materials. Cross-linking with EGDMA tunes the viscoelastic properties of the adhesive material and has the advantage of not reacting with the catechol group that is responsible for the excellent adhesive performance of this material. Adhesion strength was measured by uniaxial indentation tests, which indicated that 0.001 mol % of EGDMA-cross-linked copolymer showed the highest work of adhesion in dry conditions, but non-cross-linked DMA was the highest in wet conditions. The results suggest that there is an optimal cross-linking degree that displays the highest adhesion by balancing viscous and elastic behaviors of the polymer but this appears to depend on the conditions. This concentration of cross-linker is well below the theoretical percolation threshold, and we propose that subtle changes in polymer viscoelastic properties can result in significant improvements in adhesion of DOPA-based materials. The properties of lightly cross-linked poly(DMA-co-MEA) were investigated by measurement of the frequency dependence of the storage modulus (G') and loss modulus (G'). The frequency-dependence of G' and magnitude of G' showed gradual decreases with the fraction of EGDMA. Loosely cross-linked DMA copolymers, containing 0% and 0.001 mol % of EGDMA-cross-linked copolymers, displayed rheological behavior appropriate for pressure-sensitive adhesives characterized by a higher G' at high frequencies and lower G' at low frequencies. Our results indicate that dimethacrylate cross-linking of DMA copolymers can be used to enhance the adhesive properties of this unique material.