Hierarchical protein patterning by meso to molecular scale self-assembly.

Numerous protein patterning methodologies are used extensively for biomedical research and development. We have developed a novel bottom-up protein patterning method using a combination of self-assembly processes in the meso to molecular scale range to allow hierarchical protein patterns to be straightforwardly fabricated with low cost over large areas. As a proof of principle, we patterned vitronectin in various dimensional hierarchies using meso to nanoscale colloids and self-assembled monolayers.

Spatial mapping and quantification of soft and hard protein coronas at silver nanocubes.

Protein coronas around silver nanocubes were quantified in serum-containing media using localized surface plasmon resonances. Both soft and hard coronas showed exposure-time and concentration-dependent changes in protein surface density with time-dependent hardening. We observed spatially dependent kinetics of the corona-formation at cube edges/corners versus facets at short incubation times, where the polymer stabilization agent delayed corona hardening. The soft corona contained more protein than the hard corona at all time-points (8-fold difference with 10% serum conditions).

Biofunctional surface patterns retaining activity after exposure to whole blood.

Biofunctional surface patterns capable of resisting nonspecific bioadsorption while retaining bioactivity play crucial roles in the advancement of life science and biomedical technologies. The currently available functional surface coatings suffer from a high level of nonspecific surface adsorption of proteins under biologically challenging conditions, leading to a loss of activity in functional moieties over time. In this study, the recently discovered facile method of temperature-induced polyelectrolyte (TIP) grafting has been used to graft two biofunctional variants (biotin and nitrilotriacetic acid, NTA) of poly(l-lysine)-grafted PEG (PLL-g-PEG) onto a titanium surface. A significant increase in the polymer adsorption was observed from the TIP-grafted surfaces assembled at 80 °C, compared to the polymer surfaces assembled at ambient temperature (20 °C). These functional PLL-g-PEG surfaces were subsequently incubated in whole human blood continuously for up to 7 days, and the TIP-grafted surfaces achieved close-to-zero nonspecific protein adsorption, as confirmed by ultrasensitive time-of-flight secondary ion mass spectrometry (ToF-SIMS). To test the maintenance of the bioactivity of the biotin and NTA moieties, submicrometer-scale mono- (biotin) and bi- (biotin/NTA) functional surface chemical patterns were fabricated via two-step TIP grafting using colloidal lithography (CL), preincubated in blood for up to 7 days and sequentially exposed to streptavidin and Ni(2+)-histidine-tagged calmodulin. The fluorescence microscopy studies revealed that the PLL-g-PEG-NTA and -biotin surfaces grafted from the TIP method were still capable of recognizing the corresponding affinity proteins for up to 1 and 7 days of preincubation in blood, respectively. These results highlight the bioresistant robustness realized by the facile TIP grafting method, which in turn preserves the activities of biofunctional moieties over a prolonged period in whole blood.

Onset of bonding plasmon hybridization preceded by gap modes in dielectric splitting of metal disks.

Dielectric splitting of nanoscale disks was studied experimentally and via finite-difference time-domain (FDTD) simulations through systematic introduction of multiple ultrathin dielectric layers. Tunable, hybridized dark bonding modes were seen with first-order gap modes preceding the appearance of bonding dipole-dipole disk modes. The observed bright dipolar mode did not show the energy shift expected from plasmon hybridization but activated dark higher order gap modes. Introducing lateral asymmetry was shown to remodel the field distribution resulting in 3D asymmetry that reoriented the dipole orientation away from the dipole of the elementary disk modes.

Assembly of poly(dopamine)/poly(N-isopropylacrylamide) mixed films and their temperature-dependent interaction with proteins, liposomes, and cells.

Many biomedical applications benefit from responsive polymer coatings. The properties of poly(dopamine) (PDA) films can be affected by codepositing dopamine (DA) with the temperature-responsive polymer poly(N-isopropylacrylamide) (pNiPAAm). We characterize the film assembly at 24 and 39 °C using DA and aminated or carboxylated pNiPAAm by a quartz crystal microbalance with dissipation monitoring (QCM-D), X-ray photoelectron spectroscopy, UV-vis, ellipsometry, and atomic force microscopy. It was found that pNiPAAm with both types of end groups are incorporated into the films. We then identified a temperature-dependent adsorption behavior of proteins and liposomes to these PDA and pNiPAAm containing coatings by QCM-D and optical microscopy. Finally, a difference in myoblast cell response was found when these cells were allowed to adhere to these coatings. Taken together, these fundamental findings considerably broaden the potential biomedical applications of PDA films due to the added temperature responsiveness.

Dopamine-assisted rapid fabrication of nanoscale protein arrays by colloidal lithography.

The development of cost-effective methodologies for the precise nanometer-scale positioning of biomolecules permits the low-cost production of various biofunctional devices for a range of biomedical and nanotechnological applications. By combining colloidal lithography and the mussel-inspired multifunctional polydopamine coating, we present a novel parallel benchtop method that allows rapid nanoscale patterning of proteins without the need for electrically powered equipment in the fabrication process. The PDA-immobilized binary nanopattern consisting of BSA surrounded by PLL-g-PEG is fabricated over a large area, and the integrity of the pattern is confirmed using AFM and FM.

Assembly of poly(dopamine) films mixed with a nonionic polymer.

Poly(dopamine) (PDA) coatings have recently attracted considerable interest for a variety of applications. Here, we investigate the film deposition of dopamine mixed with a nonionic polymer (i.e., poly(ethylene glycol) (PEG), poly(vinyl alcohol) (PVA), and poly(N-vinyl pyrrolidone) (PVP)) onto silica substrates using X-ray photoelectron spectroscopy and quartz crystal microbalance. Furthermore, we assess the possibility of coating silica colloids to yield polymer capsules and liposomes with these mixtures. We found that mixed PDA/PEG and PDA/PVA films are deposited without the need for a covalent linker such as an amine or thiol. We also discovered the first material, namely, PVP, that can suppress PDA film assembly. These fundamental findings give further insight into PDA film properties and contribute to establish PDA as a widely applicable coating.

Temperature-induced ultradense PEG polyelectrolyte surface grafting provides effective long-term bioresistance against mammalian cells, serum, and whole blood.

We report a facile method of generating ultradense poly(l-lysine)-graft-poly(ethylene glycol) (PLL-g-PEG) surface by using high temperature alone, which in turn provides dramatic improvement in resisting nonspecific bioadsorption. X-ray photoelectron spectroscopy (XPS) revealed that the surface graft density increased ~4 times higher on the surface prepared at 80 °C compared to 20 °C. The studies from small-angle X-ray scattering (SAXS) and the effect of varying ionic strength during/post assemblies at 20 and 80 °C indicated that the "cloud point grafting effect" is not the cause for obtaining high density grafting. Stringent long-term bioresistance tests have been conducted and the temperature-induced PLL-g-PEG surfaces have achieved (1) zero mammalian cell adsorption/migration for up to 36 days and (2) extremely close-to-zero protein adsorptions have been observed even after 36 days in 10% serum media and 24 h in whole blood within the ultrasensitive detection limit of time-of-flight secondary ion mass spectrometry (ToF-SIMS).

Surface mass spectrometry of two component drug-polymer systems: novel chromatographic separation method using gentle-secondary ion mass spectrometry (G-SIMS).

In recent years, there has been an increase in the use of time-of-flight secondary ion mass spectrometry (TOF-SIMS) for characterizing material surfaces. A great advantage of SIMS is that the analysis is direct and has excellent spatial resolution approaching a few hundred nanometers. However, the lack of the usual separation methods in mass spectrometry such as chromatography or ion mobility combined with the complexity of the heavily fragmented ions in the spectra means that the interpretation of multicomponent spectra in SIMS is very challenging indeed. The requirements for high-definition imaging, with say 256 × 256 pixels, in around 10 min analysis time places significant constraints on the instrument design so that separation using methods such as ion mobility with flight times of milliseconds are incompatible. Clearly, traditional liquid and gas chromatographies are not at all possible. Previously, we developed a method known as Gentle-SIMS (G-SIMS) that simplifies SIMS spectra so that the dominant ions are simply related to the structure of the substances analyzed. The method uses a measurement of the fragmentation behavior under two different primary ion source conditions and a control parameter known as the g-index. Here, we show that this method may be used "chromatographically" to separate the mass spectra of a drug molecule from the matrix polymer. The method may be used in real-time and is directly compatible with the majority of TOF-SIMS instruments. The applicability to other imaging mass spectrometeries is discussed.

Tuning the hydrophobicity of mica surfaces by hyperthermal Ar ion irradiation.

The hydrophobicity of surfaces has a strong influence on their interactions with biomolecules such as proteins. Therefore, for in vitro studies of bio-surface interactions model surfaces with tailored hydrophobicity are of utmost importance. Here, we present a method for tuning the hydrophobicity of atomically flat mica surfaces by hyperthermal Ar ion irradiation. Due to the sub-100 eV energies, only negligible roughening of the surface is observed at low ion fluences and also the chemical composition of the mica crystal remains almost undisturbed. However, the ion irradiation induces the preferential removal of the outermost layer of K(+) ions from the surface, leading to the exposure of the underlying aluminosilicate sheets which feature a large number of centers for C adsorption. The irradiated surface thus exhibits an enhanced chemical reactivity toward hydrocarbons, resulting in the adsorption of a thin hydrocarbon film from the environment. Aging these surfaces under ambient conditions leads to a continuous increase of their contact angle until a fully hydrophobic surface with a contact angle >80° is obtained after a period of about 3 months. This method thus enables the fabrication of ultrasmooth biological model surfaces with precisely tailored hydrophobicity.

High-resolution surface chemical analysis of a trifunctional pattern made by sequential colloidal shadowing.

We present a new method for creating surface chemical patterns where three chemistries can be periodically arranged at alternate positions on a single substrate without the use of top-down approaches. High-resolution chemical imaging by time-of-flight secondary ion mass spectrometry (ToF-SIMS), with nanometer spatial resolution, is used to prove the success of the patterning and subsequent chemical modification steps. We use a combination of colloidal self-assembly, plasma etching, self-assembled monolayers (SAMs) and physical vapour deposition (PVD). The method utilizes a double colloid assembly process in which a first layer of close-packed colloids is created, followed by plasma etching, coating with gold and deposition of a first SAM layer. A second particle layer is deposited on top of the first layer masking the interstitial spaces containing the first SAM. A second gold layer is deposited followed by a second SAM. After particle removal the surface consists of the pattern containing two different SAMs and a SiO(2) layer that can be readily functionalized with silanes. The possibility in the replacement of the two different thiols is investigated by X-ray photoelectron spectroscopy (XPS) and it was found that no replacement is taking place. ToF-SIMS imaging is used to show the periodicity of the chemical patterns by tracking unique fragment ions from the different surface regions. The patterning method is adaptable to create smaller or larger chemical patterns by appropriate choice of particle sizes. The patterns are useful for immobilizing biomolecules for cell studies or as multiplexed biosensors.

Organic depth profiling of a binary system: the compositional effect on secondary ion yield and a model for charge transfer during secondary ion emission.

In recent years, it has been demonstrated that cluster ion beams may be used to sputter some materials, particularly organic materials, without the significant accumulation of damage. It is therefore possible to use cluster ion beam sputtering in conjunction with a surface analytical technique, such as SIMS, to obtain depth profiles and three-dimensional images of the distribution of organic species in the near-surface region. For SIMS organic depth profiling to be useful as an analytical tool, it is important that it is able to measure physically meaningful quantities, such as the local concentration of a species within a blend. In this paper, we investigate a model system of a miscible binary mixture of codeine and poly(lactide). We show that there is a strong surface enrichment of poly(lactide), which provides a reference signal and permits the direct comparison of different samples in terms of secondary ion yield behavior. We demonstrate that it is possible to relate secondary ion intensities to local concentrations for a binary system and that there is a direct correspondence between the yield enhancement of one component and the yield suppression of the other. The dependence of secondary ion yield on composition is described using a model of the kinetically limited transfer of charge between secondary ions and secondary neutrals. Application of the model to pure materials under the assumption that only highly fragmented secondary ions are initially produced and interact with unfragmented secondary neutrals leads to the prediction that high molecular mass quasi-molecular ions have intensities proportional to the square of the total secondary ion yield. This relationship has been independently observed in other work (Seah, M. P. Surf. Interface Anal. 2007, 39, 634.).

Ultra-dense polymer brush coating reduces Staphylococcus epidermidis biofilms on medical implants and improves antibiotic treatment outcome.

Staphylococcal biofilm formation is a severe complication of medical implants, leading to high antibiotic tolerance and treatment failure. Ultra-dense poly(ethylene glycol) (udPEG) coating resists adsorption of proteins, polysaccharides and extracellular DNA. It is therefore uniquely resistant to attachment by Staphylococcus epidermidis, which remains loosely adhered to the surface. Our aim was to determine if S. epidermidis remains susceptible to antibiotics when adhering to udPEG, and if udPEG coatings can improve the treatment outcome for implant-associated infections. We tested the in vitro efficacy of vancomycin treatment on recently adhered S. epidermidis AUH4567 on udPEG, conventional PEG or titanium surfaces using live/dead staining and microscopy. udPEG was then applied to titanium implants and inserted subcutaneously in mice and inoculated with S. epidermidis to induce infection. Mice were given antibiotic prophylaxis or a short antibiotic treatment. One group was given immunosuppressive therapy. After five days, implants and surrounding tissue were harvested for CFU enumeration. Only few S. epidermidis cells adhered to udPEG compared to conventional PEG and uncoated titanium, and a much lower fraction of cells on udPEG survived antibiotic treatment in vitro. In vivo, the bacterial load on implants in mice receiving vancomycin treatment was significantly lower on udPEG-coated compared to uncoated implants, also in neutropenic mice. Our results suggest that the improved outcome results from the coating's anti-adhesive properties that leads to less biofilm and increased efficacy of antibiotic treatment. Thus, the combination of udPEG with antibiotics is a promising strategy to prevent acute implant-associated infections that arise due to perioperative contaminations. STATEMENT OF SIGNIFICANCE: Infections of medical implants is an ever-present danger. Here, bacteria develop biofilms that cannot be eradicated with antibiotics. By using an ultra-dense polymer-brush coating (udPEG), bacterial attachment and the subsequent biofilm formation can be reduced, resulting in increased antibiotic susceptibility of bacteria surrounding the implant. udPEG combined with antibiotics proved to significantly reduce bacteria on implants inserted into mice, in our animal model. As the coating is not antibacterial per se, it does not induce antimicrobial resistance and its effect is independent of the bacterial species. Our results are encouraging for the prospect of preventing and treating implant-associated infections that arise due to perioperative contaminations.

Combinatorial Biomolecular Nanopatterning for High-Throughput Screening of Stem-Cell Behavior.

A novel combinatorial biomolecular nanopatterning method is reported, in which multiple biomolecular ligands can be patterned in multiple nanoscale dimensions on a single surface. The applicability of the combinatorial platform toward cell-biology applications is demonstrated by screening the adhesion behavior of a population of human dental pulp stem cell (hDPSC) on 64 combinations of nanopatterned extracellular matrix (ECM) proteins in parallel.

Non-proteinaceous bacterial adhesins challenge the antifouling properties of polymer brush coatings.

Polymer brushes of poly(ethylene glycol) have long been considered the gold standard for antifouling surfaces that resist adsorption of biomolecules and attachment of microorganisms. However, despite displaying excellent resistance to protein adsorption, the polymer brush coatings cannot entirely avoid colonization by bacteria. Here we investigate and identify which non-proteinaceous bacterial adhesins challenge the antifouling properties of polymer brush coatings and how these challenges might be overcome. We quantified biofilm formation on a well-known polymer brush coating of poly(l-lysine)-graft-poly(ethylene glycol) (PLL-g-PEG) on titanium. The coating successfully resisted colonization by Staphylococcus aureus and Pseudomonas aeruginosa, but not Staphylococcus epidermidis. This colonization pattern was also reflected on the adhesion forces measured on single bacterial cells. The biofilm produced from S. epidermidis on PLL-g-PEG were found to be rich in polysaccharides and extracellular DNA, and quantification of DNA, polysaccharides and proteins on PLL-g-PEG surfaces revealed that although the coating almost fully resisted protein adsorption, polysaccharides could adsorb, and exposure to DNA led to desorption of the polymer from the titanium surface. We hypothesized that this problem could be overcome by increasing the polymer brush density to better resist the penetration of DNA and polysaccharides into the polymer layer. Indeed, high density PLL-g-PEG brushes prepared by the recently discovered temperature-induced polyelectrolyte (TIP) grafting method resisted the interaction with DNA and polysaccharides, and therefore also the colonization by S. epidermidis. The TIP grafting is a simple improvement of PLL-g-PEG brush formation, and our results suggest that it provides an important advancement to the bacterial resistance by polymer brush coatings. STATEMENT OF SIGNIFICANCE: The antifouling properties of poly(ethylene glycol) brush coatings against protein adsorption are well documented, but it is not well understood why these coatings do not perform as well against bacterial colonization when tested against a wide range of species and over periods of days. Here we investigated bacterial colonization on poly(l-lysine)-graft-poly(ethylene glycol) (PLL-g-PEG) grafted on Ti, and revealed that bacteria relying mostly on polysaccharides and extracellular DNA for adhesion and biofilm formation could successfully colonize PLL-g-PEG coated surfaces. The coatings could not resist adsorption of polysaccharides, and DNA could even desorb the coatings from the Ti surface. Fortunately, the shortcomings of conventional PLL-g-PEG could be overcome by increasing the graft density, using the recently discovered and very simple grafting method, 'temperature-induced polyelectrolyte (TIP) grafting'. Our study highlights that it is of utmost importance to develop coatings which resist adsoprtion of non-proteinaceous bacterial adhesins such as polysaccharides and DNA, and we demonstrated that TIP grafted high density PLL-g-PEG coatings are promising materials to achieve diverse bacterial resistance.

Mixed poly(dopamine)/poly(L-lysine) (composite) coatings: from assembly to interaction with endothelial cells.

Engineered polymer films are of significant importance in the field of biomedicine. Poly(dopamine) (PDA) is becoming more and more a key player in this context. Herein, we deposited mixed films consisting of PDA and poly(L-lysine) (PLL) of different molecular weights. The coatings were characterized by quartz crystal microbalance with dissipation monitoring, atomic force microscopy, and X-ray photoelectron spectroscopy. The protein adsorption to the mixed films was found to decrease with increasing amounts of PLL. PDA/PLL capsules were also successfully assembled. Higher PLL content in the membranes reduced their thickness while the ζ-potential increased. Further, endothelial cell adhesion and proliferation over 96 h were found to be independent of the type of coating. Using PDA/PLL in liposome-containing composite coatings showed that sequential deposition of the layers yielded higher liposome trapping compared to one-step adsorption except for negatively charged liposomes. Association/uptake of fluorescent cargo by adherent endothelial cells was found to be different for PDA and PDA/PLL films. Taken together, our findings illustrate the potential of PDA/PLL mixed films as coatings for biomedical applications.

Ultrastable green fluorescence carbon dots with a high quantum yield for bioimaging and use as theranostic carriers.

Carbon dots (Cdots) have recently emerged as a novel platform of fluorescent nanomaterials. These carbon nanoparticles have great potential in biomedical applications such as bioimaging as they exhibit excellent photoluminescence properties, chemical inertness and low cytotoxicity in comparison to widely used semiconductor quantum dots. However, it remains a great challenge to prepare highly stable, water-soluble green luminescent Cdots with a high quantum yield. Herein we report a new synthesis route for green luminescent Cdots imbuing these desirable properties and demonstrate their potential in biomedical applications. Oligoethylenimine (OEI)-ß-cyclodextrin (ßCD) Cdots were synthesised using a simple and fast heating method in phosphoric acid. The synthesised Cdots showed strong green fluorescence under UV excitation with a 30% quantum yield and exhibited superior stability over a wide pH range. We further assembled the Cdots into nanocomplexes with hyaluronic acid for potential use as theranostic carriers. After confirming that the Cdot nanocomplexes exhibited negligible cytotoxicity with H1299 lung cancer cells, in vitro bioimaging of the Cdots and nanocomplexes was carried out. Doxorubicin (Dox), an anticancer drug, was also loaded into the nanocomplexes and the cytotoxicity effect of Dox loaded nanocomplexes with H1299 lung cancer cells was evaluated. Thus, this work demonstrates the great potential of the novel OEI-ßCD Cdots in bioimaging and as theranostic carriers.

Routing of individual polymers in designed patterns.

Synthetic polymers are ubiquitous in the modern world, but our ability to exert control over the molecular conformation of individual polymers is very limited. In particular, although the programmable self-assembly of oligonucleotides and proteins into artificial nanostructures has been demonstrated, we currently lack the tools to handle other types of synthetic polymers individually and thus the ability to utilize and study their single-molecule properties. Here we show that synthetic polymer wires containing short oligonucleotides that extend from each repeat can be made to assemble into arbitrary routings. The wires, which can be more than 200 nm in length, are soft and bendable, and the DNA strands allow individual polymers to self-assemble into predesigned routings on both two- and three-dimensional DNA origami templates. The polymers are conjugated and potentially conducting, and could therefore be used to create molecular-scale electronic or optical wires in arbitrary geometries.

Highly-branched poly(N-isopropylacrylamide) as a component in poly(dopamine) films.

Mixed one-step poly(dopamine) (PDA)/highly branched poly(N-isopropylacrylamide) (pNiPAAm) coatings have been assembled and characterized by X-ray photoelectron spectroscopy (XPS), UV-vis spectroscopy, atomic force microscopy, and quartz crystal microbalance with dissipation monitoring (QCM-D) depending on the deposition temperature below and above the lower critical solution temperature (LCST) of the pNiPAAm. Mixed films were confirmed. The protein adsorption at 24 °C was found to be reduced with increasing amount of pNiPAAm in the mixed coatings, while there was no difference observed for proteins deposition at 39 °C. Further, the ability of these mixed coatings in comparison to the pure PDA and pNiPAAm films to serve as capping layer for surface-immobilized zwitterionic or positively charged liposomes has been assessed by QCM-D. The adhesion of hepatocytes, macrophages, and myoblast to these liposomes-containing hybrid coatings and the uptake of fluorescent lipids from the surface by the adhering cells depending on the capping layers were compared. The latter aspect was found to be dependent on the used capping layer and the type of liposome as carrier for the fluorescent lipid, with the highest uptake found for positive liposomes and pure pNiPAAm as capping layer. Taken together, the assembled hybrid coatings have the potential to be used as functional coatings toward surface-mediated drug delivery.

Complex protein nanopatterns over large areas via colloidal lithography.

The patterning of biomolecules at the nanoscale provides a powerful method to investigate cellular adhesion processes. A novel method for patterning is presented that is based on colloidal monolayer templating combined with multiple and angled deposition steps. Patterns of gold and SiO2 layers are used to generate complex protein nanopatterns over large areas. Simple circular patches or more complex ring structures are produced in addition to hierarchical patterns of smaller patches. The gold regions are modified through alkanethiol chemistry, which enables the preparation of extracellular matrix proteins (vitronectin) or cellular ligands (the extracellular domain of E-cadherin) in the nanopatterns, whereas the selective poly(l-lysine)-poly(ethylene glycol) functionalization of the SiO2 matrix renders it protein repellent. Cell studies, as a proof of principle, demonstrate the potential for using sets of systematically varied samples with simpler or more complex patterns for studies of cellular adhesive behavior and reveal that the local distribution of proteins within a simple patch critically influences cell adhesion.