A Versatile Protein and Cell Patterning Method Suitable for Long-Term Neural Cultures.

Herein, we present an easy-to-use protein and cell patterning method relying solely on pipetting, rinsing steps and illumination with a desktop lamp, which does not require any expensive laboratory equipment, custom-built hardware or delicate chemistry. This method is based on the adhesion promoter poly(allylamine)-grafted perfluorophenyl azide, which allows UV-induced cross-linking with proteins and the antifouling molecule poly(vinylpyrrolidone). Versatility is demonstrated by creating patterns with two different proteins and a polysaccharide directly on plastic well plates and on glass slides, and by subsequently seeding primary neurons and C2C12 myoblasts on the patterns to form islands and mini-networks. Patterning characterization is done via immunohistochemistry, Congo red staining, ellipsometry, and infrared spectroscopy. Using a pragmatic setup, patterning contrasts down to 5 µm and statistically significant long-term stability superior to the gold standard poly(l-lysine)-grafted poly(ethylene glycol) could be obtained. This simple method can be used in any laboratory or even in classrooms and its outstanding stability is especially interesting for long-term cell experiments, e.g., for bottom-up neuroscience, where well-defined microislands and microcircuits of primary neurons are studied over weeks.

Easy to Apply Polyoxazoline-Based Coating for Precise and Long-Term Control of Neural Patterns.

Arranging cultured cells in patterns via surface modification is a tool used by biologists to answer questions in a specific and controlled manner. In the past decade, bottom-up neuroscience emerged as a new application, which aims to get a better understanding of the brain via reverse engineering and analyzing elementary circuitry in vitro. Building well-defined neural networks is the ultimate goal. Antifouling coatings are often used to control neurite outgrowth. Because erroneous connectivity alters the entire topology and functionality of minicircuits, the requirements are demanding. Current state-of-the-art coating solutions such as widely used poly(l-lysine)-g-poly(ethylene glycol) (PLL-g-PEG) fail to prevent primary neurons from making undesired connections in long-term cultures. In this study, a new copolymer with greatly enhanced antifouling properties is developed, characterized, and evaluated for its reliability, stability, and versatility. To this end, the following components are grafted to a poly(acrylamide) (PAcrAm) backbone: hexaneamine, to support spontaneous electrostatic adsorption in buffered aqueous solutions, and propyldimethylethoxysilane, to increase the durability via covalent bonding to hydroxylated culture surfaces and antifouling polymer poly(2-methyl-2-oxazoline) (PMOXA). In an assay for neural connectivity control, the new copolymer's ability to effectively prevent unwanted neurite outgrowth is compared to the gold standard, PLL-g-PEG. Additionally, its versatility is evaluated on polystyrene, glass, and poly(dimethylsiloxane) using primary hippocampal and cortical rat neurons as well as C2C12 myoblasts, and human fibroblasts. PAcrAm-g-(PMOXA, NH2, Si) consistently outperforms PLL-g-PEG with all tested culture surfaces and cell types, and it is the first surface coating which reliably prevents arranged nodes of primary neurons from forming undesired connections over the long term. Whereas the presented work focuses on the proof of concept for the new antifouling coating to successfully and sustainably prevent unwanted connectivity, it is an important milestone for in vitro neuroscience, enabling follow-up studies to engineer neurologically relevant networks. Furthermore, because PAcrAm-g-(PMOXA, NH2, Si) can be quickly applied and used with various surfaces and cell types, it is an attractive extension to the toolbox for in vitro biology and biomedical engineering.

Imparting Nonfouling Properties to Chemically Distinct Surfaces with a Single Adsorbing Polymer: A Multimodal Binding Approach.

Surface-active polymers that display nonfouling properties and carry binding groups that can adsorb onto different substrates are highly desirable. We present a postmodification protocol of an active-ester-containing polymer that allows the creation of such a versatile platform. Poly(pentafluorophenyl acrylate) has been postmodified with a fixed grafting ratio of a nonfouling function (mPEG) and various combinations of functional groups, such as amine, silane and catechol, which can provide strong affinity to two model substrates: SiO2 and TiO2 . Adsorption, stability and resistance to nonspecific protein adsorption of the polymer films were studied. A polymer was obtained that maintained its surface functionality under a variety of harsh conditions. EG surface-density calculations show that this strategy generates a denser packing when both negatively and positively charged groups are present within the backbone, and readily allows the fabrication of a broad combinatorial matrix.

General in vitro method to analyze the interactions of synthetic polymers with human antibody repertoires.

Recent reports on the hitherto underestimated antigenicity of poly(ethylene glycol) (PEG), which is widely used for pharmaceutical applications, highlight the need for efficient testing of polymer antigenicity and for a better understanding of its molecular origins. With this goal in mind, we have used the phage-display technique to screen large, recombinant antibody repertoires of human origin in vitro for antibodies that bind poly(vinylpyrrolidone) (PVP). PVP is a neutral synthetic polymer of industrial and clinical interest that is also a well-known model antigen in animal studies, thus allowing the comparison of in vitro and in vivo responses. We have identified 44 distinct antibodies that bind specifically to PVP. Competitive binding assays show that the PVP-antibody binding constant is proportional to the polymerization degree of PVP and that specific binding is detected down to the vinylpyrrolidone (VP) monomer level. Statistical analysis of anti-PVP antibody sequences identifies an amino-acid motif that is shared by many phage-display-selected anti-PVP antibodies that are similar to a previously described natural anti-PVP antibody. This suggests a role for this motif in specific antibody/PVP interactions. Interestingly, sequence analysis also suggests that only a single antibody chain containing this shared motif is responsible for antibody binding to PVP, as confirmed upon systematic deletion of either antibody chain for 90% of selected anti-PVP antibodies. Overall, a large number of antibodies in the human repertoires we have screened bind specifically to PVP through a small number of shared amino acid motifs, and preliminary comparison points to significant correlations between the sequences of phage-display-selected anti-PVP antibodies and their natural counterparts isolated from immunized mice in previous studies. This study pioneers the use of antibody phage-display to explore the antigenicity of biotechnologically relevant polymers. It also paves the way for a fast, cost-effective, and systematic in vitro analysis, thus reducing the need for animal immunization experiments. Moreover, identifying the encoding DNA sequence of polymer-binding antibodies via phage-display enables future applications of a molecular biology approach to protein-polymer conjugation, based on protein-antibody fusion.

Photochemically prepared, two-component polymer-concentration gradients.

A versatile, photochemical surface-modification approach using nitrene-insertion reactions has been employed to develop an ultrathin, two-component, polymer-gradient coating. Perfluorophenyl azide (PFPA) acted as the photosensitive moiety, forming a nitrene radical upon 254 nm UV exposure. Cationic poly(allyl amine) was grafted with PFPA and surface-anchored onto silicon wafers by means of electrostatic self-assembly. After spin-coating of polystyrene (PS), the substrate was illuminated from behind a moving shutter, thereby controlling the azide-to-nitrene conversion degree across the substrate, and leading to a gradually varying PS density after rinsing. Backfilling with poly(vinyl pyrrolidone) (PVP) and re-exposing to UV light formed a two-component polymer-density gradient. The composition varied linearly following exposure to a linear UV exposure profile, as determined with spectroscopic ellipsometry (ELM) and X-ray photoelectron spectroscopy (XPS). High-spatial-resolution, time-of-flight secondary ion mass spectrometry (ToF-SIMS) revealed a high degree of mixing between the two incompatible polymers on the micrometer scale. The dynamic water-contact angle (dCA) was found to depend strongly on the sample history, suggesting adaptive properties of the coating, which was further confirmed by angle-resolved XPS (ARXPS). To confirm the applicability of the system for biological investigations, gradients were exposed to zoospores of the macrofouling alga Ulva linza , and a critical PS composition of 70% was identified, above which settlement started to increase. It has been shown that a two-component polymer-density gradient can provide a high-throughput platform for determining critical surface properties of polymer blend materials.

Poly(ethylene glycol) and hydroxy functionalized alkane phosphate mixed self-assembled monolayers to control nonspecific adsorption of proteins on titanium oxide surfaces.

The spontaneous formation of alkane phosphate self-assembled monolayers (SAMs) on titanium oxide was chosen as a tool to tailor the surface physicochemical properties in terms of nonspecific adsorption of proteins. For this aim, poly(ethylene glycol)-modified (PEG) alkane phosphate was codeposited with OH-terminated alkane phosphates. X-ray photoelectron spectroscopy and ellipsometry of the resulting mixed SAMs indicate that the PEG density can be controlled by varying the mole fraction of PEG-terminated phosphates in the solutions used during the deposition process, leading to surfaces with different degrees of protein resistance.

Self-assembly of poly(ethylene glycol)-poly(alkyl phosphonate) terpolymers on titanium oxide surfaces: synthesis, interface characterization, investigation of nonfouling properties, and long-term stability.

This contribution deals with the self-assembling of a terpolymer on titanium oxide (TiO(2)) surface. The polymer structure was obtained by polymerization of different methacrylates, i.e., alkyl-phosphonated, butyl and PEG methacrylate, in the presence of a chain transfer agent. The resulting PEG-poly(alkyl phosphonate) material, characterized mainly by SEC and NMR, self-organized at the interface of TiO(2). AR-XPS demonstrated the binding of phosphonate groups to TiO(2) substrate and the formation of a PEG-brush layer at the outermost part of the system. The stability of this terpolymer adlayer, after exposure to solutions of pH 2, 7.4, and 9 up to 3 weeks, was evaluated quantitatively by XPS and ellipsometry. We demonstrated an overall stability improvements of this coating against desorption in contact with aqueous solutions in comparison with reference self-assembly systems. Finally, the PEG-terpolymer adlayer proved to impart to TiO(2) substrate antifouling properties when exposed to full blood serum.

Fabricating chemical gradients on oxide surfaces by means of fluorinated, catechol-based, self-assembled monolayers.

Catechols bind strongly to several metal oxides and can thus be used as a binding group for generating self-assembled monolayers. Furthermore, their derivatives can be used to produce well-defined, centimeter-scale surface-chemical gradients on technologically relevant surfaces, such as titanium dioxide (TiO(2)). A simple dip-and-rinse gradient-preparation technique was utilized to produce surface-hydrophobicity gradients from perfluoro-alkyl catechols and nitrodopamine (ND). Chemical composition, quality, and properties of the functionalized surfaces were determined by means of X-ray photoelectron spectroscopy (XPS), variable-angle spectroscopic ellipsometry (VASE), and static water contact angle (sCA) measurements. Contact angles were found to be in the range of 30°-95°, correlating well with the determined surface chemical composition and adlayer thickness.

Protein-resistant surfaces through mild dopamine surface functionalization.

The synthesis and evaluation of new dopamine-based catechol anchors coupled to poly(ethylene glycol) (PEG) for surface modification of TiO(2) are reported. Dopamine is modified by dimethylamine-methylene (7) or trimethylammonium-methylene (8) groups, and the preparation of mPEG-Glu didopamine polymer 11 is presented. All these PEG polymers allow stable adlayers on TiO(2) to be generated through mild dip-and-rinse procedures, as evaluated both by variable angle spectroscopic ellipsometry and X-ray photoelectron spectroscopy. The resulting surfaces substantially reduced protein adsorption upon exposure to full human serum.

Delineating fibronectin bioadhesive micropatterns by photochemical immobilization of polystyrene and poly(vinylpyrrolidone).

Bioadhesive micropatterns, capable of laterally confining cells to a 2D lattice, have proven effective in simulating the in vivo tissue environment. They reveal fundamental aspects of the role of adhesion in cell mechanics, proliferation, and differentiation. Here we present an approach based on photochemistry for the fabrication of synthetic polymer micropatterns. Perfluorophenyl azide (PFPA), upon deep-UV exposure, forms a reactive nitrene capable of covalently linking to a molecule that is in close proximity. PFPA has been grafted onto a backbone of poly(allyl amine), which readily forms a self-assembled monolayer on silicon wafers or glass. A film of polystyrene was applied by spin-coating, and by laterally confining the UV exposure through a chromium-on-quartz photomask, monolayers of polymers could be immobilized in circular microdomains. Poly(vinylpyrrolidone) (PVP) was attached to the background to form a barrier to nonspecific protein adsorption and cell adhesion. Micropatterns were characterized with high-lateral-resolution time-of-flight secondary ion mass spectrometry (TOF-SIMS), which confirmed the formation of polystyrene domains within a PVP background. Fluorescence-microscopy adsorption assays with rhodamine-labeled bovine serum albumin demonstrated the nonfouling efficiency of PVP and, combined with TOF-SIMS, allowed for a comprehensive characterization of the pattern geometry. The applicability of the micropatterned platform in single-cell assays was tested by culturing two cell types, WM 239 melanoma cells and SaOs-2 osteoblasts, on micropatterned glass, either with or without backfilling of the patterns with fibronectin. It was demonstrated that the platform was efficient in confining cells to the fibronectin-backfilled micropatterns for at least 48 h. PVP is thus proposed as a viable, highly stable alternative to poly(ethylene glycol) for nonfouling applications. Due to the versatility of the nitrene-insertion reaction, the platform could be extended to other polymer pairs or proteins and the surface chemistry adapted to specific applications.

Adsorption and lubricating properties of poly(l-lysine)-graft-poly(ethylene glycol) on human-hair surfaces.

We have characterized the adsorption and lubricating properties of the polycation-PEG graft copolymer poly(l-lysine)-graft-poly(ethylene glycol) (PLL-g-PEG) on human-hair surfaces by means of X-ray photoelectron spectroscopy (XPS), fluorescence microscopy, and atomic force microscopy (AFM). XPS measurements indicated that PLL-g-PEG copolymers spontaneously adsorbed onto the surface of bleached-hair samples (a good model of a weathered, damaged hair surface for cosmetic care applications) from an aqueous solution. Further treatment with cationic surfactants present in common shampoo formulations removed the adsorbed PLL-g-PEG from the hair samples. Fluorescence microscopy showed that the adsorption of PLL-g-PEG onto the hair samples from an aqueous polymer solution occurred inhomogeneously. Nanotribological studies with AFM (friction vs load plots) revealed that the relationship between load and friction was approximately linear for all hair samples, while the slopes of the plots varied considerably along the hair sample surface. Under ambient, "dry" conditions, the frictional properties of the bleached, bleached + PLL-g-PEG-treated, and bleached + PLL-g-PEG-treated and subsequently surfactant-treated hair samples did not reveal a clear difference. In distilled water, however, the bleached + PLL-g-PEG-treated hair samples showed statistically lower frictional properties than simply bleached or bleached + PLL-g-PEG-treated and subsequently surfactant-treated hair samples. Overall, the three instrumental techniques have consistently shown that the adsorption of PLL-g-PEG onto the hair sample surface occurs unevenly, which can be ascribed to the intrinsically heterogeneous properties of the human-hair surface. A control experiment, involving an injection of concentrated PLL-g-PEG solution into a liquid cell where an AFM tip was already scanning over a specific area (line scan mode), revealed an immediate and apparent reduction in the frictional force. Despite the inhomogeneity of the hair surface, the adsorption of the polymer seems to be extremely effective in promoting lubrication of the fiber. This suggests that the adsorbed graft copolymers act as a boundary lubricant on the hair surface. The presence of a more organized, brushlike layer of polymers contrasts with the usual random adsorption of chains that is believed to be present in the case of linear polyelectrolytes that are nowadays applied for shampoos and conditioners in the cosmetic or textile industries.

Biomimetic surface modifications based on the cyanobacterial iron chelator anachelin.

Siderophores are natural iron chelators that have been evolutionarily selected to bind to Fe ions with very high binding constants. We utilize these unique properties to bind to metal oxide surfaces using a fragment of the cyanobacterial siderophore anachelin. The resulting poly(ethylene glycol) conjugate forms stable adlayers on TiO2 as has been shown by variable angle spectroscopic ellipsometry and X-ray photoelectron spectroscopy. Moreover, these coated surfaces are highly protein-resistant against the adsorption of full human serum.