Formation of Antifouling Brushes on Various Substrates Using a Melanin-Inspired Initiator Film.

In this study, we developed a substrate-independent initiator film that can undergo surface-initiated polymerization to form an antifouling brush. Inspired by the melanogenesis found in nature, we synthesized a tyrosine-conjugated bromide initiator (Tyr-Br) that contains phenolic amine groups as the dormant coating precursor and α-bromoisobutyryl groups as the initiator. The resultant Tyr-Br was stable under ambient air conditions and underwent melanin-like oxidation only in the presence of tyrosinase to form an initiator film on various substrates. Subsequently, an antifouling polymer brush was formed using air-tolerant activators regenerated by electron transfer for atom transfer radical polymerization (ARGET ATRP) of zwitterionic carboxybetaine. The entire surface coating procedure, including the initiator layer formation and ARGET ATRP, occurred under aqueous conditions and did not require organic solvents or chemical oxidants. Therefore, antifouling polymer brushes can be feasibly formed not only on experimentally preferred substrates (e.g., Au, SiO2, and TiO2) but also on polymeric substrates such as poly(ethylene terephthalate) (PET), cyclic olefin copolymer (COC), and nylon.

Effect of N-Methylation on Dopamine Surface Chemistry.

Dopamine (DA) surface chemistry has received significant attention because of its applicability in a wide range of research fields and the ability to graft functional molecules onto numerous solid surfaces. Various DA derivatives have been newly synthesized to identify key factors affecting the coating efficiency and to advance the coating system development. The oxidation of catechol into quinone followed by internal cyclization via the nucleophilic attack of primary amine is crucial for DA-based surface coating. Thus, it is expected that the amine group's nucleophilicity control directly affects the coating efficiency. However, it has not been systematically investigated, and most studies have been conducted with the focus on the transformation of amines into amides, despite such approaches decreasing the coating efficiency; the nitrogen in amides is less nucleophilic than that in free amines. In this study, we investigated the effect of N-alkylation on dopamine surface chemistry. N,N-Dimethyldopamine (DMDA) was newly synthesized, and the coating efficiency was systematically compared with DA and N-methyldopamine (MDA). DA N-monomethylation improved the coating rate by increasing the nitrogen nucleophilicity, whereas N,N-dimethylation dramatically decreased the DA surface coating property. In addition, MDA remained capable of universal surface coating and secondary reactions using the surface catechols. This study provides opportunities for developing coating materials with advanced functions and an improved coating rate.

Antifouling Surface Coating on Various Substrates by Inducing Tyrosinase-Mediated Oxidation of a Tyrosine-Conjugated Sulfobetaine Derivative.

Inspired by the melanogenesis occurring in nature, we report tyrosinase-mediated antifouling surface coating by synthesizing a tyrosine-conjugated sulfobetaine derivative (Tyr-SB). Synthetic Tyr-SB contains zwitterionic sulfobetaine and tyrosine, whose phenolic amine group acts as a dormant coating precursor. In contrast to catecholamine derivatives, tyrosine derivatives are stable against auto-oxidation and are enzymatically oxidized only in the presence of tyrosinase to initiate melanin-like oxidation. When the surface of interest was applied during the course of Tyr-SB oxidation, a superhydrophilic poly(Tyr-SB) film was coated on the surfaces, thereby showing antifouling performance against proteins or adherent cells. Because the oxidation of Tyr-SB occurred under mild aqueous conditions (pH 6-7) without the use of any chemical oxidants, such as sodium periodate or ammonium persulfate, we anticipate that the coating method described herein will serve as a biocompatible tool in the field of biosensors, cell surface engineering, and medical devices, whose interfaces differ in chemistry.

Development of Universal and Clickable Film by Mimicking Melanogenesis: On-Demand Oxidation of Tyrosine-Based Azido Derivative by Tyrosinase.

A tyrosine-based azido derivative (TBAD) that permits both substrate-independent surface coating and clickable film functionalization by mimicking natural melanogenesis is synthesized here. In contrast to catechol derivatives, which are generally susceptible to oxidation by air under ambient conditions, the monophenol-based TBAD remains stable under alkaline and neutral conditions and is activated to oxidized quinone in situ by tyrosinase to initiate melanin-like polymerization. The resulting poly(TBAD) film can be formed on various substrates including noble metals, metal oxides, and synthetic polymers, which can undergo click reaction with terminal alkyne moieties on the entire surface or a specific region through Cu(I)-catalyzed azide-alkyne cycloaddition. The enzyme-mediated coating can rapidly form thin films (≈10 nm) and produce a uniform film morphology, which are important aspects in surface chemistry. This on-demand, clickable coating may become a significant tool for bioconjugation, soft lithography, and labeling techniques.

Zr(IV) Coordination Chemistry for Cell-Repellent Alginate Coatings: The Effect of Surface Functional Groups.

Surface modification using alginic acid and its salt, alginate (Alg), has attracted much attention owing to its potential applications in various fields, including tissue engineering, drug delivery, antiplatelet surface preparation, and energy-storage technologies. In these applications, efficient immobilization of Alg on the solid surface is required because the delamination of the surface-bound Alg eventually leads to a significant decrease in its function. Therefore, much effort has been made to introduce Alg onto solid surfaces in a stable manner. Despite recent advances, existing methods for immobilizing Alg on surfaces have some limitations: (i) derivatization of Alg is typically also required and (ii) these methods only function under specific reaction conditions. Herein, we report a Zr(IV)-mediated strategy to immobilize Alg on solid surfaces. We demonstrate efficient Alg grafting onto carboxyl-, catechol-, polydopamine-, and tannic acid-functionalized surfaces via Zr(IV)-mediated cross-linking reactions. This strategy yields Alg multilayers that suppress fibroblast and platelet adhesion onto the solid surfaces. Furthermore, we show that the Alg multilayers can be selectively constructed on specific sites of solid surfaces. Given its ease of use and the wide selection of available carboxyl polymers, the current strategy is expected to be a useful tool for preparing functional polymer films for various applications.

Antifouling Surface Coating Using Droplet-Based SI-ARGET ATRP of Carboxybetaine under Open-Air Conditions.

The formation of a dense zwitterionic brush through surface-initiated atom transfer radical polymerization (SI-ATRP) is a typical graft-from approach used to achieve antifouling surfaces with high fidelity; however, their air-tightness may cause inconvenience to users. In this context, activator regenerated by electron transfer (ARGET) ATRP is emerging as an alternative surface-coating tool because limited amount of air is allowed to form a dense polymer brush. However, the degree of air tolerance that can ensure a thick polymer brush has not been clearly defined, limiting its practical usage under ambient-air conditions. In this study, we investigated the SI-ARGET ATRP of carboxybetaine (CB) by changing the air conditions, along with the air-related parameters, such as the concentration of the reducing agent, the volume of the polymerization solution (PS), or the solvent composition, and correlated their effects with the poly(CB) thickness. Based on the optimized reaction conditions, a poly(CB) brush with reliable thickness was feasibly formed even under open-air conditions without a degassing step. In addition, a microliter droplet (∼100 µL) of PS was sufficient to proceed with the SI-ARGET ATRP for the covering of a poly(CB) brush on the surface area of interest. By applying an optimized SI-ARGET ATRP of CB, antifouling was feasibly achieved in the surface region of interest using an array to form a large surface area under fully exposed air conditions. In other words, optimized SI-ARGET ATRP enabled the formation of a thick poly(CB) brush on the surfaces of various dimensions under open-air conditions.

Surface-Initiated ARGET ATRP of Antifouling Zwitterionic Brushes Using Versatile and Uniform Initiator Film.

In this study, we developed a uniform initiator layer that can be formed on various surfaces, and formed site-selectively, for the subsequent antifouling polymer brush formation. Initially, metal-organic films composed of tannic acid (TA) and FeIII ions (TA-FeIII) were formed on various surfaces, followed by functionalization with an aryl azide-based initiator (ABI) under photoreaction. In particular, combination with a photolithographic technique enabled the presentation of initiators only on the intended region within a single-surface platform. A resultant initiator film (TF-ABI) was formed under mild reaction conditions and meets the uniformity and transparency requirements concurrently. Subsequently, we showed that TF-ABI can be further utilized to form a polymer brush by proceeding with surface-initiated polymerization using a zwitterionic monomer, namely, sulfobetaine acrylamide (SBAA). Instead of applying a classical, yet air-sensitive atom transfer radical polymerization (ATRP) technique, we utilized an activator regenerated by electron transfer (ARGET) ATRP under air conditions without a cumbersome deoxygenation step. Overall, our initiator layer allowed the antifouling poly(SBAA) brush to be used on various surfaces, and enabled their pattern generation.

Ultralow Fouling and Functionalizable Surface Chemistry Based on Zwitterionic Carboxybetaine Random Copolymers.

Here, we report a simple yet effective surface-modification approach to imparting hydrophobic surfaces with superhydrophilicity using ultralow fouling/functionalizable carboxybetaine (CB) copolymers via a dip-coating technique. A new series of CB random copolymers with varying amphiphilicities were synthesized and coated on hydrophobic polypropylene (PP) and polystyrene (PS) surfaces. The nonfouling capability of each coating was screened by an enzyme-linked immunosorbent assay (ELISA) and further comprehensively assessed against 100% human serum by a Micro BCA protein assay kit. The random copolymer containing ∼30 mol % CB units showed superhydrophilicity with the highest air contact angle of more than 165° in DI water and the best nonfouling capability against 100% human blood serum. Surfaces of a 96-well plate coated with the optimal CB random copolymer had a significantly better nonfouling capability than those of a commercial 96-well plate with an ultralow attachment surface. The adhesion of mouse embryonic fibroblast cells (NIH3T3) was completely inhibited on surfaces coated with CB random copolymers. Furthermore, the optimal nonfouling CB copolymer surface was functionalized with an antigen via covalent bonding where its specific interactions with its antibody were verified. Thus, this CB random copolymer is capable of imparting both ultralow fouling and functionalizable capabilities to hydrophobic surfaces for blood-contacting devices.

Accelerated Development of Hippocampal Neurons and Limited Adhesion of Astrocytes on Negatively Charged Surfaces.

This work examines the development of primary neurons and astrocytes on thoroughly controlled functional groups. Negatively charged surfaces presenting carboxylate (COO-) or sulfonate (SO3-) groups prove beneficial to neuronal behavior, in spite of their supposed repulsive electrostatic interactions with cellular membranes. The adhesion and survival of primary hippocampal neurons on negatively charged surfaces are comparable to or slightly better than those on positively charged (poly-d-lysine-coated) surfaces, and neuritogenesis and neurite outgrowth are accelerated on COO- and SO3- surfaces. Moreover, such favorable influences of the negatively charged surfaces are only seen in neurons but not for astrocytes. Our results indicate that the in vitro developmental behavior of primary hippocampal neurons is sophisticatedly modulated by angstrom-sized differences in chemical structure or the charge density of the surface. We believe that this work provides new implications for understanding neuron-material interfaces as well as for establishing new ways to fabricate neuro-active surfaces.

Cell-in-Shell Hybrids: Chemical Nanoencapsulation of Individual Cells.

Nature has developed a fascinating strategy of cryptobiosis ("secret life") for counteracting the stressful, and often lethal, environmental conditions that fluctuate sporadically over time. For example, certain bacteria sporulate to transform from a metabolically active, vegetative state to an ametabolic endospore state. The bacterial endospores, encased within tough biomolecular shells, withstand the extremes of harmful stressors, such as radiation, desiccation, and malnutrition, for extended periods of time and return to a vegetative state by breaking their protective shells apart when their environment becomes hospitable for living. Certain ciliates and even higher organisms, for example, tardigrades, and others are also found to adopt a cryptobiotic strategy for survival. A common feature of cryptobiosis is the structural presence of tough sheaths on cellular structures. However, most cells and cellular assemblies are not "spore-forming" and are vulnerable to the outside threats. In particular, mammalian cells, enclosed with labile lipid bilayers, are highly susceptible to in vitro conditions in the laboratory and daily life settings, making manipulation and preservation difficult outside of specialized conditions. The instability of living cells has been a main bottleneck to the advanced development of cell-based applications, such as cell therapy and cell-based sensors. A judicious question arises: can cellular tolerance against harmful stresses be enhanced by simply forming cell-in-shell hybrid structures? Experimental results suggest that the answer is yes. A micrometer-sized "Iron Man" can be generated by chemically forming an ultrathin (<100 nm) but durable shell on a "non-spore-forming" cell. Since the report on silica nanoencapsulation of yeast cells, in which cytoprotective yeast-in-silica hybrids were formed, several synthetic strategies have been developed to encapsulate individual cells in a cytocompatible fashion, mimicking the cryptobiotic cell-in-shell structures found in nature, for example, bacterial endospores. Bioinspired silicification and phenolics-based coatings are, so far, the main approaches to the formation of cytoprotective cell-in-shell hybrids, because they ensure cell viability during encapsulations and also generate durable nanoshells on cell surfaces. The resulting cell-in-shell hybrids extrinsically possess enhanced resistance to external aggressors, and more intriguingly, the encapsulation alters their metabolic activity, exemplified by retarded or suppressed cell cycle progression. In addition, recent developments in the field have further advanced the synthetic tools available to the stage of chemical sporulation and germination of mammalian cells, where cytoprotective shells are formed on labile mammalian cells and broken apart on demand. For example, individual HeLa cells are coated with a metal-organic complex of ferric ion and tannic acid, and cellular adherence and proliferation are controlled by the programmed shell formation and degradation. Based on these demonstrations, the (degradable) cell-in-shell hybrids are anticipated to find their applications in various biomedical and bionanotechnological areas, such as cytotherapeutics, high-throughput screening, sensors, and biocatalysis, as well as providing a versatile research platform for single-cell biology.

Cytoprotective Encapsulation of Individual Jurkat T Cells within Durable TiO2 Shells for T-Cell Therapy.

Lymphocytes, such as T cells and natural killer (NK) cells, have therapeutic promise in adoptive cell transfer (ACT) therapy, where the cells are activated and expanded in vitro and then infused into a patient. However, the in vitro preservation of labile lymphocytes during transfer, manipulation, and storage has been one of the bottlenecks in the development and commercialization of therapeutic lymphocytes. Herein, we suggest a cell-in-shell (or artificial spore) strategy to enhance the cell viability in the practical settings, while maintaining biological activities for therapeutic efficacy. A durable titanium oxide (TiO2 ) shell is formed on individual Jurkat T cells, and the CD3 and other antigens on cell surfaces remain accessible to the antibodies. Interleukin-2 (IL-2) secretion is also not hampered by the shell formation. This work suggests a chemical toolbox for effectively preserving lymphocytes in vitro and developing the lymphocyte-based cancer immunotherapy.

Live cell imaging compatible immobilization of Chlamydomonas reinhardtii in microfluidic platform for biodiesel research.

This paper describes a novel surface immobilization method for live-cell imaging of Chlamydomonas reinhardtii for continuous monitoring of lipid droplet accumulation. Microfluidics allows high-throughput manipulation and analysis of single cells in precisely controlled microenvironment. Fluorescence imaging based quantitative measurement of lipid droplet accumulation in microalgae had been difficult due to their intrinsic motile behavior. We present a simple surface immobilization method using gelatin coating as the "biological glue." We take advantage of hydroxyproline (Hyp)-based non-covalent interaction between gelatin and the outer cell wall of microalgae to anchor the cells inside the microfluidic device. We have continuously monitored single microalgal cells for up to 6 days. The immobilized microalgae remain viable (viability was comparable to bulk suspension cultured controls). When exposed to wall shear stress, most of the cells remain attached up to 0.1 dyne/cm(2) . Surface immobilization allowed high-resolution, live-cell imaging of mitotic process in real time-which followed previously reported stages in mitosis of suspension cultured cells. Use of gelatin coated microfluidics devices can result in better methods for microalgae strain screening and culture condition optimization that will help microalgal biodiesel become more economically viable.

Immobilization of Antibody on a Cyclic Olefin Copolymer Surface with Functionalizable, Non-Biofouling Poly[Oligo(Ethylene Glycol) Methacrylate].

We report a perfluoroaryl azide-based photoreaction for synthesizing functionalizable and nonbiofouling poly[oligo(ethylene glycol) methacrylate] (pOEGMA) films on a chemically inert COC substrate, and an estimation of a surface coverage of the antibody immobilized onto the surface with the immuno-gold nanoparticles. The processes were confirmed by water contact angle measurement, FT-IR spectroscopy, and FE-SEM. The strategy demonstrated in this work could be applied to functionalizations of other polymeric materials and determination of the binding capacity of analytes in biosensors and microfluidic devices.

Cytoprotective silica coating of individual mammalian cells through bioinspired silicification.

The cytoprotective coating of physicochemically labile mammalian cells with a durable material has potential applications in cell-based sensors, cell therapy, and regenerative medicine, as well as providing a platform for fundamental single-cell studies in cell biology. In this work, HeLa cells in suspension were individually coated with silica in a cytocompatible fashion through bioinspired silicification. The silica coating greatly enhanced the resistance of the HeLa cells to enzymatic attack by trypsin and the toxic compound poly(allylamine hydrochloride), while suppressing cell division in a controlled fashion. This bioinspired cytocompatible strategy for single-cell coating was also applied to NIH 3T3 fibroblasts and Jurkat cells.

A cytoprotective and degradable metal-polyphenol nanoshell for single-cell encapsulation.

Single-cell encapsulation promises the cytoprotection of the encased cells against lethal stressors, reminiscent of the sporulation process in nature. However, the development of a cytocompatible method for chemically mimicking the germination process (i.e., shell degradation on-demand) has been elusive, despite the shell degradation being pivotal for the practical use of functional cells as well as for single cell-based biology. We report that an artificial shell, composed of tannic acid (TA) and Fe(III) , on individual Saccharomyces cerevisiae controllably degrades on-demand, while protecting the yeast from multiple external aggressors, including UV-C irradiation, lytic enzymes, and silver nanoparticles. Cell division is suppressed by the TA-Fe(III) shell, but restored fully upon shell degradation. The formation of a TA-Fe(III) shell would provide a versatile tool for achieving the chemical version of "sporulation and germination".

Cytoprotective alginate/polydopamine core/shell microcapsules in microbial encapsulation.

Chemical encapsulation of microbes in threedimensional polymeric microcapsules promises various applications, such as cell therapy and biosensors, and provides a basic platform for studying microbial communications. However, the cytoprotection of microbes in the microcapsules against external aggressors has been a major challenge in the field of microbial microencapsulation, because ionotropic hydrogels widely used for microencapsulation swell uncontrollably, and are physicochemically labile. Herein, we developed a simple polydopamine coating for obtaining cytoprotective capability of the alginate capsule that encapsulated Saccharomyces cerevisiae. The resulting alginate/ polydopamine core/shell capsule was mechanically tough, prevented gel swelling and cell leakage, and increased resistance against enzymatic attack and UV-C irradiation. We believe that this multifunctional core/shell structure will provide a practical tool for manipulating microorganisms inside the microcapsules.

Artificial spores: cytocompatible encapsulation of individual living cells within thin, tough artificial shells.

Cells are encapsulated individually within thin and tough shells in a cytocompatible way, by mimicking the structure of bacterial endospores that survive under hostile conditions. The 3D 'cell-in-shell' structures-coined as 'artificial spores'-enable modulation and control over cellular metabolism, such as control of cell division, resistance to external stresses, and surface-functionalizability, providing a useful platform for applications, including cell-based sensors, cell therapy, regenerative medicine, as well as for fundamental studies on cellular metabolism at the single-cell level and cell-to-cell communications. This Concept focuses on chemical approaches to single-cell encapsulation with artificial shells for creating artificial spores, including cross-linked layer-by-layer assembly, bioinspired mineralization, and mussel-inspired polymerization. The current status and future prospects of this emerging field are also discussed.

Chemical control of yeast cell division by cross-linked shells of catechol-grafted polyelectrolyte multilayers.

The chemical control of cell division has attracted much attention in the areas of single cell-based biology and high-throughput screening platforms. A mussel-inspired cytocompatible encapsulation method for achieving a "cell-division control" with cross-linked layer-by-layer (LbL) shells is developed. Catechol-grafted polyethyleneimine and hyaluronic acid are chosen as polyelectrolytes for the LbL process, and the cross-linking of polyelectrolytes is performed at pH 8.5. Cell division is controlled by the number of the LbL nanolayers and cross-linking reaction. We also suggest a new measuring unit, t-2.0 OD 600, for quantifying "cell-division timing" based on microbial growth kinetics.

Bioinspired, cytocompatible mineralization of silica-titania composites: thermoprotective nanoshell formation for individual chlorella cells.

Hard-shell case: Using a (RKK)4 D8 peptide allows mineralization to occur under cytocompatible conditions. Thus individual Chlorella cells could be encapsulated within a SiO2 -TiO2 nanoshell with high cell viability (87 %). The encapsulated Chlorella showed an almost threefold increase in their thermo-tolerance after 2 h at 45 °C.

Electrochemical release of amine molecules from carbamate-based, electroactive self-assembled monolayers.

In this paper, carbamate-based self-assembled monolayers (SAMs) of alkanethiolates on gold were suggested as a versatile platform for release of amine-bearing molecules in response to the electrical signal. The designed SAMs underwent the electrochemical oxidation on the gold surface with simultaneous release of the amine molecules. The synthesis of the thiol compounds was achieved by coupling isocyanate-containing compounds with hydroquinone. The electroactive thiol was mixed with 11-mercaptoundecanol [HS(CH(2))(11)OH] to form a mixed monolayer, and cyclic votammetry was used for the characterization of the release behaviors. The mixed SAMs showed a first oxidation peak at +540 mV (versus Ag/AgCl reference electrode), indicating the irreversible conversion from carbamate to hydroquinone groups with simultaneous release of the amine molecules. The analysis of ToF-SIMS further indicated that the electrochemical reaction on the gold surface successfully released amine molecules.