Tuning the underwater adhesiveness of antibacterial polysaccharides complex coacervates.

HYPOTHESIS: Adjusting the water content and mechanical properties of polyelectrolyte coacervates for optimal underwater adhesion requires simultaneous control of the macromolecular design and the type and concentration of the salt used. Using synthetic or bio-inspired polymers to make coacervates often involves complicated chemistries and large variations in salt concentration. The underwater adhesiveness of simple, bio-sourced coacervates can be tuned with relatively small variations in salt concentration. Bio-sourced polymers can also impart beneficial biological activities to the final material. EXPERIMENTS: We made complex coacervates from charged chitosan (CHI) and hyaluronic acid (HA) with NaCl as the salt. Their water content and viscoelastic properties were investigated to identify the formulation with optimal underwater adhesion in physiological conditions. The coacervates were also studied in antibacterial and cytotoxicity experiments. FINDINGS: As predicted by linear rheology, the CHI-HA coacervates at 0.1 and 0.2 M NaCl had the highest pull-off adhesion strengths of 44.4 and 40.3 kPa in their respective supernatants. In-situ physical hardening of the 0.2 M coacervate upon a salt switch in 0.1 M NaCl resulted in a pull-off adhesion strength of 62.9 kPa. This material maintained its adhesive properties in physiological conditions. Finally, the optimal adhesive was found to be non-cytotoxic and inherently antimicrobial through a chitosan release-killing mechanism.

Interfacial rheology of linearly growing polyelectrolyte multilayers at the water-air interface: from liquid to solid viscoelasticity.

Despite the long history of investigations of polyelectrolyte multilayer formation on solid or liquid surfaces, important questions remain open concerning the construction of the first set of layers. These are generally deposited on a first anchoring layer of different chemistry, influencing their construction and properties. We propose here an in-depth investigation of the formation of NaPSS/PAH multilayers at the air/water interface in the absence of a chemically different anchoring layer, profiting from the surface activity of NaPSS. To analyse the mechanical properties of the different layers, we combine recently established analysis techniques of an inflating/deflating bubble exploiting simultaneous shape and pressure measurement: bubble shape elastometry, general stress decomposition and capillary meniscus dynanometry. We complement these measurements by interfacial shear rheology. The obtained results allow us to confirm, first of all, the strength of the aforementioned techniques to characterize complex interfaces with non-linear viscoelastic properties. Furthermore, their sensitivity allows us to show that the multilayer properties are highly sensitive to the temporal and mechanical conditions under which they are constructed and manipulated. We nevertheless identify a robust trend showing a clear transition from a liquid-like viscoelastic membrane to a solid-like viscoelastic membrane after the deposition of 5 layers. We interpret this as the number of layers required to create a fully connected multilayer, which is consistent with previous results obtained on solid or liquid interfaces.

An increase in Wharton's jelly membrane osteocompatibility by a genipin-cross-link.

Wharton's Jelly (WJ) has attracted significant interest in the field of tissue healing thanks to its biological properties, including antibacterial activity and immunomodulation. However, due to the fast degradation and poor mechanical behavior in biological environment, its application in bone regeneration is compromised. Here, we proposed to use genipin as an efficient cross-linking agent to significantly improve the elasticity and the enzymatical stability of the WJ matrix. The degree of cross-linking, linear elastic moduli, and collagenase resistance varied over a wide range depending on genipin concentration. Furthermore, our results highlighted that an increase in genipin concentration led to a decreased surface wettability, therefore impairing cell attachment and proliferation. The genipin cross-linking prevented rapid in vitro and in vivo degradation, but led to an adverse host reaction and calcification. When implanted in the parietal bone defect, a limited parietal bone regeneration to the dura was observed. We conclude that genipin-cross-linked WJ is a versatile medical device however, a careful selection is required with regards to the genipin concentration.

Electrochemical Assembly Strategies of Polymer and Hybrid Thin Films for (Bio)sensors, Charge Storage, and Triggered Release.

In the context of functional and hierarchical materials, electrode reactions coupled with one or more chemical reactions constitute the most powerful bottom-up process for the electrosynthesis of film components and their electrodeposition, enabling the localized functionalization of conductive surfaces using an electrical stimulus. In analogy with developmental biological processes, our group introduced the concept of morphogen-driven film buildup. In this approach, the gradient of a diffusing reactive molecule or ion (called a morphogen) is controlled by an electrical stimulus to locally induce a chemical process (solubility change, hydrolysis, complexation, and covalent reaction) that induces a film assembly. One of the prominent advantages of this technique is the conformal nature of the deposits toward the electrode. This Feature Article presents the contributions made by our group and other researchers to develop strategies for the assembly of different polymer and nanoparticle/polymer hybrid films by using electrochemically generated reagents and/or catalysts. The main electrochemical-chemical approaches for conformal films are described in the case where (i) the products are noncovalent aggregates that spontaneously precipitate on the electrode (film electrodeposition) or (ii) new chemical compounds are generated, which do not necessarily spontaneously precipitate and enable the formation of covalent or noncovalent films (film electrosynthesis). The applications of those electrogenerated films will be described with a focus on charge storage/transport, (bio)sensing, and stimuli-responsive cargo delivery systems.

Physical Chemistry Study of Collagen-Based Multilayer Films.

The surface properties of a biomaterial play an important role in cell behavior, e.g., recolonization, proliferation, and migration. Collagen is known to favor wound healing. In this study, collagen (COL)-based layer-by-layer (LbL) films were built using different macromolecules as a partner, i.e., tannic acid (TA), a natural polyphenol known to establish hydrogen bonds with protein, heparin (HEP), an anionic polysaccharide, and poly(sodium 4-styrene sulfonate) (PSS), an anionic synthetic polyelectrolyte. To cover the whole surface of the substrate with a minimal number of deposition steps, several parameters of the film buildup were optimized, such as the pH value of the solutions, the dipping time, and the salt (sodium chloride) concentration. The morphology of the films was characterized by atomic force microscopy. Built at an acidic pH, the stability of COL-based LbL films was studied when in contact with a physiological medium as well as the TA release from COL/TA films. In contrast to COL/PSS and COL/HEP LbL films, COL/TA films showed a good proliferation of human fibroblasts. These results validate the choice of TA and COL as components of LbL films for biomedical coatings.

Brush-Induced Orientation of Collagen Fibers in Layer-by-Layer Nanofilms: A Simple Method for the Development of Human Muscle Fibers.

The engineering of skeletal muscle tissue, a highly organized structure of myotubes, is promising for the treatment of muscle injuries and muscle diseases, for replacement, or for pharmacology research. Muscle tissue development involves differentiation of myoblasts into myotubes with parallel orientation, to ultimately form aligned myofibers, which is challenging to achieve on flat surfaces. In this work, we designed hydrogen-bonded tannic acid/collagen layer-by-layer (TA/COL LbL) nanofilms using a simple brushing method to address this issue. In comparison to films obtained by dipping, brushed TA/COL films showed oriented COL fibers of 60 nm diameter along the brushing direction. Built at acidic pH due to COL solubility, TA/COL films released TA in physiological conditions with a minor loss of thickness. After characterization of COL fibers' orientation, human myoblasts (C25CL48) were seeded on the oriented TA/COL film, ended by COL. After 12 days in a differentiation medium without any other supplement, human myoblasts were able to align on brushed TA/COL films and to differentiate into long aligned myotubes (from hundreds of µm up to 1.7 mm length) thanks to two distinct properties: (i) the orientation of COL fibers guiding myoblasts' alignment and (ii) the TA release favoring the differentiation. This simple and potent brushing process allows the development of anisotropic tissues in vitro which can be used for studies of drug discovery and screening or the replacement of damaged tissue.

Optimization of the Rheological Properties of Self-Assembled Tripeptide/Alginate/Cellulose Hydrogels for 3D Printing.

3D printing is an emerging and powerful technique to create shape-defined three-dimensional structures for tissue engineering applications. Herein, different alginate-cellulose formulations were optimized to be used as printable inks. Alginate (Alg) was chosen as the main component of the scaffold due to its tunable mechanical properties, rapid gelation, and non-toxicity, whereas microcrystalline cellulose (MCC) was added to the hydrogel to modulate its mechanical properties for printing. Additionally, Fmoc-FFY (Fmoc: 9-fluorenylmethoxycarbonyl; F: phenylalanine; Y: tyrosine), a self-assembled peptide that promotes cell adhesion was incorporated into the ink without modifying its rheological properties and shear-thinning behavior. Then, 3D-printed scaffolds made of Alg, 40% of MCC inks and Fmoc-FFY peptide were characterized by scanning electron microscopy and infrared spectroscopy, confirming the morphological microstructure of the hydrogel scaffolds with edged particles of MCC homogeneously distributed within the alginate matrix and the self-assembly of the peptide in a ß-sheet conformation. Finally, the cytocompatibility of the scaffolds was tested in contact with the MG63 osteosarcoma cells, confirming the absence of cytotoxic components that may compromise their viability. Interestingly, MG63 cell growth was retarded in the scaffolds containing the peptide, but cells were more likely to promote adhesive interactions with the material rather than with the other cells, indicating the benefits of the peptide in promoting biological functionality to alginate-based biomaterials.

Optimization of Nanohybrid Biosensors Based on Electro-Crosslinked Tannic Acid Capped Nanoparticles/Enzyme.

Enzymes/Nanoparticles (NPs) bioconjugates are massively used nowadays to develop thin films for optical and electrochemical biosensors. Nevertheless, their full characterization as a thin coating onto electrodes remains little discussed, in particular the influence of NPs size and enzyme/NPs ratio used in the electrodeposition solution. In this study, GOx (160 kDa) and HRP (44 kDa) were used in association with tannic acid capped gold NPs (a series with sizes from 7 to 40 nm) to electrodeposit biosensor coatings, sensitive towards glucose and H2O2, respectively. The electrodeposition process was based on a mussel-inspired electro-crosslinking between gallol moieties of tannic acid (at the surface of NPs) and amine moieties of the enzymes. On one hand, the sensitivity of the GOx/NPs coatings depends strongly on the NP size and the enzyme/NPs molar ratio of the electrodeposition solution. An optimal sensitivity was obtained by electrodeposition of 11 nm NPs at a GOx/NPs molar ratio close to the theoretical value of the enzyme monolayer. On the other hand, a modest influence of the NPs size was found on the sensitivity in the case of the electrodeposited HRP/NPs coatings, reaching a plateau at the HRP/NPs molar ratio close to the value of the theoretical enzyme monolayer. In both cases, the enzyme/NPs molar ratio played a role in the sensitivity. To fully understand the parameters driving the biosensor sensitivity, a comprehensive evaluation of the colloidal state of the bioconjugates is proposed here.

Injectable Tripeptide/Polymer Nanoparticles Supramolecular Hydrogel: A Candidate for the Treatment of Inflammatory Pathologies.

Supramolecular peptide-based hydrogels attract great attention in several fields, i.e., biomedicine, catalysis, energy, and materials chemistry, due to the noncovalent nature of the self-assembly and functional tunable properties defined by the amino acid sequence. In this work, we developed an injectable hybrid supramolecular hydrogel whose formation was triggered by electrostatic interactions between a phosphorylated tripeptide, Fmoc-FFpY (F: phenylalanine, pY: phosphorylated tyrosine), and cationic polymer nanoparticles made of vinylimidazole and ketoprofen (poly(HKT-co-VI) NPs). Hydrogel formation was assessed through inverted tube tests, and its fibrillary structure, around polymer NPs, was observed by transmission electron microscopy. Interestingly, peptide self-assembly yields the formation of nontwisted and twisted fibers, which could be attributed to ß-sheets and α-helix structures, respectively, as characterized by circular dichroism and infrared spectroscopies. An increase of the elastic modulus of the Fmoc-FFpY/polymer NPs hybrid hydrogels was observed with peptide concentration as well as its injectability property, due to its shear thinning behavior and self-healing ability. After checking their stability under physiological conditions, the cytotoxicity properties of these hybrid hydrogels were evaluated in contact with human dermal fibroblasts (FBH) and murine macrophages (RAW 264.7). Finally, the Fmoc-FFpY/polymer NPs hybrid hydrogels exhibited a great nitric oxide reduction (∼67%) up to basal values of pro-inflammatory RAW 264.7 cells, thus confirming their excellent anti-inflammatory properties for the treatment of localized inflammatory pathologies.

Study of Membrane Protein Monolayers Using Surface-Enhanced Infrared Absorption Spectroscopy (SEIRAS): Critical Dependence of Nanostructured Gold Surface Morphology.

Surface-enhanced infrared absorption spectroscopy (SEIRAS) is a powerful tool that allows studying the reactivity of protein monolayers at very low concentrations and independent from the protein size. In this study, we probe the surface's morphology of electroless gold deposition for optimum enhancement using two different types of immobilization adapted to two proteins. Independently from the mode of measurement (i.e., transmission or reflection) or type of protein immobilization (i.e., through electrostatic interactions or nickel-HisTag), the enhancement and reproducibility of protein signals in the infrared spectra critically depended on the gold nanostructured surface morphology deposited on silicon. Just a few seconds deviation from the optimum time in the nanoparticle deposition led to a significantly weaker enhancement. Scanning electron microscopy and atomic force microscopy measurements revealed the evolution of the nanostructured surface when comparing different deposition times. The optimal deposition time led to isolated gold nanostructures on the silicon crystal. Importantly, in the case of the immobilization using nickel-HisTag, the surface morphology is rearranged upon immobilization of linker and the protein. A complex three-dimensional (3D) network of nanoparticles decorated with the protein could be observed leading to the optimal enhancement. The electroless deposition of gold is a simple technique, which can be adapted to flow cells and used in analytical approaches.

Localized Enzyme-Assisted Self-Assembly in the Presence of Hyaluronic Acid for Hybrid Supramolecular Hydrogel Coating.

Hydrogel coating is highly suitable in biomaterial design. It provides biocompatibility and avoids protein adsorption leading to inflammation and rejection of implants. Moreover, hydrogels can be loaded with biologically active compounds. In this field, hyaluronic acid has been largely studied as an additional component since this polysaccharide is naturally present in extracellular matrix. Strategies to direct hydrogelation processes exclusively from the surface using a fully biocompatible approach are rare. Herein we have applied the concept of localized enzyme-assisted self-assembly to direct supramolecular hydrogels in the presence of HA. Based on electronic and fluorescent confocal microscopy, rheological measurements and cell culture investigations, this work highlights the following aspects: (i) the possibility to control the thickness of peptide-based hydrogels at the micrometer scale (18-41 µm) through the proportion of HA (2, 5 or 10 mg/mL); (ii) the structure of the self-assembled peptide nanofibrous network is affected by the growing amount of HA which induces the collapse of nanofibers leading to large assembled microstructures underpinning the supramolecular hydrogel matrix; (iii) this changing internal architecture induces a decrease of the elastic modulus from 2 to 0.2 kPa when concentration of HA is increasing; (iv) concomitantly, the presence of HA in supramolecular hydrogel coatings is suitable for cell viability and adhesion of NIH 3T3 fibroblasts.

Supramolecular tripeptide self-assembly initiated at the surface of coacervates by polyelectrolyte exchange.

Spatial control of supramolecular self-assembly can yield compartmentalized structures, a key feature for the design of artificial cells. Inducing self-assembly from and on compartments is still a challenge. Polyelectrolyte complex coacervates are simple model droplet systems able to reproduce the basic features of membrane-less organelles, appearing in cells. Here, we demonstrate the supramolecular self-assembly of a phosphorylated tripeptide, Fmoc-FFpY (Fmoc: fluorenyl-methoxycarbonyl; F: phenyl alanine, pY: phosphorylated tyrosine), on the surface of poly(l-glutamic acid)/poly(allylamine hydrochloride) (PGA/PAH) complex coacervate microdroplets. The phosphorylated peptides self-assemble, without dephosphorylation, through ion pairing between the phosphate groups of Fmoc-FFpY and the amine groups of PAH. This process provides spontaneous capsules formed by an amorphous polyelectrolyte complex core surrounded by a structured peptide/PAH shell. Similar fibrillar Fmoc-FFpY self-assembled structures are obtained at the interface between the peptide solution and a PGA/PAH polyelectrolyte multilayer, a complex coacervate in the thin film or "multilayer" format. In contact with the peptide solution, PAH chains diffuse out of the coacervate or multilayer film and complex with Fmoc-FFpY at the solution interface, exchanging any PGA with which they were associated. Self-assembly of Fmoc-FFpY, now concentrated by complexation with PAH, follows quickly.

Plasmonic Resonant Nanoantennas Induce Changes in the Shape and the Intensity of Infrared Spectra of Phospholipids.

Surface enhanced infrared absorption spectroscopic studies (SEIRAS) as a technique to study biological molecules in extremely low concentrations is greatly evolving. In order to use the technique for identification of the structure and interactions of such biological molecules, it is necessary to identify the effects of the plasmonic electric-field enhancement on the spectral signature. In this study the spectral properties of 1,2-Dipalmitoyl-sn-glycero-3 phosphothioethanol (DPPTE) phospholipid immobilized on gold nanoantennas, specifically designed to enhance the vibrational fingerprints of lipid molecules were studied. An AFM study demonstrates an organization of the DPPTE phospholipid in bilayers on the nanoantenna structure. The spectral data were compared to SEIRAS active gold surfaces based on nanoparticles, plain gold and plain substrate (Si) for different temperatures. The shape of the infrared signals, the peak positions and their relative intensities were found to be sensitive to the type of surface and the presence of an enhancement. The strongest shifts in position and intensity were seen for the nanoantennas, and a smaller effect was seen for the DPPTE immobilized on gold nanoparticles. This information is crucial for interpretation of data obtained for biological molecules measured on such structures, for future application in nanodevices for biologically or medically relevant samples.

Biopolymers-calcium phosphate antibacterial coating reduces the pathogenicity of internalized bacteria by mesenchymal stromal cells.

A multifunctional material system that kills bacteria and drives bone healing is urgently sought to improve bone prosthesis. Herein, the osteoinductive coating made of calcium phosphate/chitosan/hyaluronic acid, named Hybrid, was proposed as an antibacterial substrate for stromal cell adhesion. This Hybrid coating possesses a contact-killing effect reducing by 90% the viability of Gram-positive Staphylococcus aureus (S. aureus) and Gram-negative Pseudomonas aeruginosa (P. aeruginosa) strains after 48 h of contact. In addition to the production of immunomodulatory mediators, Wharton's jelly (WJ-SCs), dental pulp (DPSCs) and bone marrow (BM-MSCs) derived stromal cells were able to release antibacterial and antibiofilm agents effective against S. aureus and P. aeruginosa strains, respectively. Studying the effect of the Hybrid coating on the internalization of S. aureus by the stromal cells, in acute-mimicking bone infection, highlighted an increase in the bacteria internalization by DPSCs and BM-MSCs when cultured on the Hybrid coating versus uncoated glass. Despite the internalization, Hybrid coating showed a beneficial effect by reducing the pathogenicity of the internalized bacteria. The formation of biofilm was reduced by at least 50% in comparison to internalized bacteria by stromal cells on uncoated glass. This work opens the route for the development of innovative antibacterial coatings by taking into account the internalization of bacteria by stromal cells.

Surface Triggered Self-Assembly of Fmoc-Tripeptide as an Antibacterial Coating.

In western countries, one patient on twenty will develop a nosocomial infection during his hospitalization at health care facilities. Classical antibiotics being less and less effective, this phenomenon is expanding year after year. Prevention of bacteria colonization of implantable medical devices constitutes a major medical and financial issue. In this study, we developed an antibacterial coating based on self-assembled Fmoc-tripeptide. Fmoc-FFpY peptides (F: phenylalanine; Y: tyrosine; p: PO4 2-) are dephosphorylated enzymatically into Fmoc-FFY by action of alkaline phosphatase functionalized silica nanoparticles (NPs@AP), previously deposited on a surface. Fmoc-FFY peptides then self-assemble through π-π stacking interactions, hydrogen bonds and hydrophobic interactions adopting ß-sheets secondary structures. The obtained hydrogel coatings show fibrillary structures observed by cryo-scanning electron microscopy with a thickness of few micrometers. At low concentration (≤0.5 mg.mL-1), self-assembled Fmoc-FFY has a superior antibacterial activity than Fmoc-FFpY peptide in solution. After 24 h of incubation, Fmoc-FFY hydrogel coatings fully inhibit the development of Gram-positive Staphylococcus aureus (S. aureus). The antibacterial effect is maintained on an in vitro model of repetitive infection in the case of S. aureus. This coating could serve in infections were Gram positive bacteria are prevalent, e.g., intravascular catheter infections. This work gives new insights toward the design of an alternative antimicrobial coating.

Effect of the Buffer on the Buildup and Stability of Tannic Acid/Collagen Multilayer Films Applied as Antibacterial Coatings.

The deposition of polyelectrolyte multilayers, obtained by the layer-by-layer (LbL) method, is a well-established technology to design biocompatible and antibacterial coatings aimed at preventing implant-associated infections. Several types of LbL films have been reported to exhibit antiadhesive and/or antibacterial (contact-killing or release-killing) properties governed not only by the incorporated compounds but also by their buildup conditions or their postbuildup treatments. Tannic acid (TA), a natural polyphenol, is known to inhibit the growth of several bacterial strains. In this work, we developed TA/collagen (TA/COL) LbL films built in acetate or citrate buffers at pH 4. Surprisingly, the used buffer impacts not only the physicochemical but also the antibacterial properties of the films. When incubated in physiological conditions, both types of TA/COL films released almost the same amount of TA depending on the last layer and showed an antibacterial effect against Staphylococcus aureus only for citrate-built films. Because of their granular topography, TA/COL citrate films exhibited an efficient release-killing effect with no cytotoxicity toward human gingival fibroblasts. Emphasis is put on a comprehensive evaluation of the physicochemical parameters driving the buildup and the antibacterial property of citrate films. Specifically, complexation strengths between TA and COL are different in the presence of the two buffers affecting the LbL deposition. This work constitutes an important step toward the use of polyphenols as an antibacterial agent when incorporated in LbL films.

Autonomous Growth of a Spatially Localized Supramolecular Hydrogel with Autocatalytic Ability.

Autocatalysis and self-assembly are key processes in developmental biology and are involved in the emergence of life. In the last decade both of these features were extensively investigated by chemists with the final goal to design synthetic living systems. Herein, we describe the autonomous growth of a self-assembled soft material, that is, a supramolecular hydrogel, able to sustain its own formation through an autocatalytic mechanism that is not based on any template effect and emerges from a peptide (hydrogelator) self-assembly. A domino sequence of events starts from an enzymatically triggered peptide generation followed by self-assembly into catalytic nanofibers that induce and amplify their production over time, resulting in a 3D hydrogel network. A cascade is initiated by traces (10-18 m) of a trigger enzyme, which can be localized allowing for a spatial resolution of this autocatalytic buildup of hydrogel growth, an essential condition on the route towards further cell-mimic designs.

Polyanionic Hydrogels as Reservoirs for Polycationic Antibiotic Substitutes Providing Prolonged Antibacterial Activity.

Implantation of biomedical devices is often followed by bacterial infections that may seriously affect implant functionalities and lead to their failure. In the context of bacterial resistance to antibiotics, which is a growing problem worldwide, new strategies that are able to overcome these problems are needed. In this work, we introduce a new formulation of hyaluronic acid (HA)-based antimicrobial material: HA hydrogels loaded with polyarginine (PAR), a polycationic antibiotic substitute. The loading is possible through electrostatic interactions between negatively charged HA and positively charged PAR. Such hydrogels absorb high quantities of PAR, which are then gradually released from the hydrogel. This original system provides a long-lasting antibacterial effect on an in vitro model of repetitive infection, thus demonstrating a strong potential to fight multiple rounds of infections that are resistant to antibiotic treatment. In addition, HA-PAR hydrogels could be deposited onto/into medical devices such as wound dressings and mesh prostheses used in clinical applications. Finally, we performed first in vivo tests of hydrogel-coated mesh materials to verify their biocompatibility in a rat model, which show no difference between control HA hydrogel and PAR-loaded hydrogel in terms of inflammation.

Enzyme assisted peptide self-assemblies trigger cell adhesion in high density oxime based host gels.

Peptide supramolecular self-assemblies are recognized as important components in responsive hydrogel based materials with applications in tissue engineering and regenerative medicine. Studying the influence of hydrogel matrices on the self-assembly behavior of peptides and interaction with cells is essential to guide the future development of engineered biomaterials. In this contribution, we present a PEG based host hydrogel material generated by oxime click chemistry that shows cellular adhesion behavior in response to enzyme assisted peptide self-assembly (EASA) within the host gel. This hydrogel prepared from poly(dimethylacrylamide-co-diacetoneacrylamide), poly(DMA-DAAM) with high molar fractions (49%) of DAAM and dialkoxyamine PEG cross-linker, was studied in the presence of embedded enzyme alkaline phosphatase (AP) and a non-adhesive cell behavior towards NIH 3T3 fibroblasts was observed. When brought into contact with a Fmoc-FFpY peptide solution (pY: phosphorylated tyrosine), the gel forms intercalated Fmoc-FFY peptide self-assemblies upon diffusion of Fmoc-FFpY into the cross-linked hydrogel network as was confirmed by circular dichroism, fluorescence emission spectroscopy and confocal microscopy. Nevertheless, the mechanical properties do not change significantly after the peptide self-assembly in the host gel. This enzyme assisted peptide self-assembly promotes fibroblast cell adhesion that can be enhanced if Fmoc-F-RGD peptides are added to the pre-gelator Fmoc-FFpY peptide solution. Cell adhesion results mainly from interactions of cells with the non-covalent peptide self-assemblies present in the gel despite the fact that the mechanical properties are very close to those of the native host gel. This result is in contrast to numerous studies which showed that the mechanical properties of a substrate are key parameters of cell adhesion. It opens up the possibility to develop a diverse set of hybrid materials to control cell fate in culture due to tailored self-assemblies of peptides responding to the environment provided by the host guest gel.

Mechanisms of Fibrinogen Adsorption on Silica Sensors at Various pHs: Experiments and Theoretical Modeling.

The adsorption kinetics of human serum fibrinogen at silica substrates was studied using optical waveguide lightmode spectroscopy (OWLS) and quartz crystal microbalance (QCM) techniques. Measurements were performed at pH 3.5, 4, and 7.4 for various ionic strengths. The experimental data were interpreted in terms of a hybrid random sequential adsorption model. This allowed the mass transfer rate coefficient for the OWLS cell and maximum coverages to be determined at various pHs. The appearance of different, pH-dependent mechanisms of fibrinogen adsorption on silica substrates was confirmed. At pH 3.5 the molecules mostly adsorb in the side-on orientation that produces a low maximum coverage of ca. 1 mg m-2. At this pH, the kinetics derived from the OWLS measurements agree with those theoretically predicted using the convective-diffusion theory. In consequence, a comparison of the OWLS and QCM results allows the water factor and the dynamic hydration of fibrinogen molecules to be determined. At pH 7.4, the OWLS method gives inaccurate kinetic data for the low coverage range. However, the maximum coverage that was equal to ca. 4 mg m-2 agrees with the QCM results and with previous literature results. It is postulated that the limited accuracy of the OWLS method for lower coverage stems from a heterogeneous structure of fibrinogen monolayers, which consist of side-on and end-on adsorbed molecules. One can expect that the results acquired in this work allow development of a robust procedure for preparing fibrinogen monolayers of well-controlled coverage and molecule orientation, which can be exploited for efficient immunosensing purposes.