Upregulation of endothelial gene markers in Wharton's jelly mesenchymal stem cells cultured on polyelectrolyte multilayers.

Designing convenient substrates is a pertinent parameter that can guide stem cell differentiation. Current research is directed toward differentiating mesenchymal stem cells (MSCs) into endothelial cells (ECs). It is generally accepted that MSCs cannot be easily differentiated into ECs without high concentrations of proangiogenic factors. To guide either bone marrow-derived mesenchymal stem cells (BM-MSCs) and Wharton's jelly-derived mesenchymal stem cells (WJ-MSCs) into ECs-like phenotype, poly(allylamine-hydrochloride)/poly(styrene-sulfonate) multilayers film (PAH/PSS) was used as culture coating and compared to type I collagen (as control coating). After 2 weeks of culture and in absence of angiogenic growth factors, PAH/PSS upregulated KDR, PECAM-1, and CDH5 genes, whereas combining PAH/PSS with endothelial growth media (EGM-2® ) led to the production of respective proteins by WJ-MSCs. In contrast, not fully EC-like phenotype is obtained from the differentiation of BM- or WJ-MSCs cultured on type I collagen. Thus, using PAH/PSS coating in synergy with EGM-2® appears as an ideal condition promoting WJ-MSCs differentiation into ECs-like. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 292-300, 2017.

Immunomodulation with Self-Crosslinked Polyelectrolyte Multilayer-Based Coatings.

This study aims to design an optimal polyelectrolyte multilayer film of poly-l-lysine (PLL) and hyaluronic acid (HA) as an anti-inflammatory cytokine release system in order to decrease the implant failure due to any immune reactions. The chemical modification of the HA with aldehyde moieties allows self-cross-linking of the film and an improvement in the mechanical properties of the film. The cross-linking of the film and the release of immunomodulatory cytokine (IL-4) stimulate the differentiation of primary human monocytes seeded on the films into pro-healing macrophages phenotype. This induces the production of anti-inflammatory cytokines (IL1-RA and CCL18) and the decrease of pro-inflammatory cytokines secreted (IL-12, TNF-α, and IL-1ß). Moreover, we demonstrate that cross-linking PLL/HA film using HA-aldehyde is already effective by itself to limit inflammatory processes. Finally, this functionalized self-cross-linked PLL/HA-aldehyde films constitutes an innovative and efficient candidate for immunomodulation of any kind of implants of various architecture and properties.

Cell Alignment Driven by Mechanically Induced Collagen Fiber Alignment in Collagen/Alginate Coatings.

For many years it has been a major challenge to regenerate damaged tissues using synthetic or natural materials. To favor the healing processes after tendon, cornea, muscle, or brain injuries, aligned collagen-based architectures are of utmost interest. In this study, we define a novel aligned coating based on a collagen/alginate (COL/ALG) multilayer film. The coating exhibiting a nanofibrillar structure is cross-linked with genipin for stability in physiological conditions. By stretching COL/ALG-coated polydimethylsiloxane substrates, we developed a versatile method to align the collagen fibrils of the polymeric coating. Assays on cell morphology and alignment were performed to investigate the properties of these films. Microscopic assessments revealed that cells align with the stretched collagen fibrils of the coating. The degree of alignment is tuned by the stretching rate (i.e., the strain) of the COL/ALG-coated elastic substrate. Such coatings are of great interest for strategies that require aligned nanofibrillar biological material as a substrate for tissue engineering.

Cell guidance into quiescent state through chromatin remodeling induced by elastic modulus of substrate.

Substrate stiffness is known to strongly influence the fate of adhering cells. Yet, little is known about the influence of the substrate stiffness on chromatin. Chromatin integrates a multitude of biochemical signals interpreted by activation or gene silencing. Here we investigate for the first time the organization of chromatin of epithelial cells on substrate with various mechanical properties. On stiff substrates (100-200 kPa), where cells preferentially adhere, chromatin is mainly found in its euchromatin form. Decreasing the Young modulus to 50 kPa is correlated with a partial shift from euchromatin to heterochromatin. On very soft substrates (≪10 kPa) this is accompanied by cell lysis. On these very soft substrates, histone deacetylase inhibition by adding a drug preserves acetylated histone and thus maintains the euchromatin form, thereby keeping intact the nuclear envelope as well as a residual intermediate filament network around the nucleus. This allows cells to survive in a non-adherent state without undergoing proliferation. When transfer on a stiff substrate these cells retain their capacity to adhere, to spread and to enter a novel mitotic cycle. A similar effect is observed on soft substrates (50 kPa) without need of histone deacetylase inhibition. These new results suggest that on soft substrates cells might enter in a quiescence state. Cell quiescence may thus be triggered by the Young modulus of a substrate, a major result for strategies focusing on the design of scaffold in tissue engineering.

Templated assembly of albumin-based nanoparticles for simultaneous gene silencing and magnetic resonance imaging.

In this article, we address the design of innovative human serum albumin (HSA)-based nanoparticles loaded with silencing RNA and grafted with gadolinium complexes having average sizes ranging from ca. 50 to 150 nm according to the siRNA/HSA composition. The non-covalent siRNA/HSA assembly is formed on isobutyramide-modified mesoporous silica and the self-supported HSA-based nanoparticles are obtained following the silica template dissolution. These original protein particles provide simultaneous magnetic resonance imaging contrast enhancement and cellular in vitro gene silencing.

Influence of the interaction strength between supramolecular complexes on the topography of neutral polymer multilayer films.

Step-by-step polymer film buildup processes lead to polymer coatings, e.g., polyelectrolyte multilayers, of various structures ranging from continuous smooth films to droplet like discontinuous coatings. Yet, the origin of these different behaviors depending upon the system is not yet known. This study is a first attempt to rationalize the evolution of the coating structure as a function of the strength of the interactions between the polymers constituting the film. We investigated the influence of the strength of noncovalent host-guest interactions between cyclodextrin (CD) and pyrene (Py), ferrocene (Fc) or adamantane (Ad) on the structure of neutral poly(N-hydroxypropylmethacrylamide) (PHPMA) multilayers films formed in a step-by-step manner. In solution, the strength of the inclusion complex (measured by log K where K is the complex association constant) is increasing in the order Py/ß-CD < Fc/ß-CD < Ad/ß-CD and can be further varied in the presence of different sodium salts at different ionic strengths. Depending upon this strength, the buildup process is limited to the formation of isolated aggregates for PHPMA-CD/PHPMA-Py, leading to smooth continuous films for PHPMA-CD/PHPMA-Fc and to droplet-like films, not entirely covering the substrate, for PHPMA-CD/PHPMA-Ad. To study the influence of the strength of the host-guest interactions on the film topography, PHPMA-CD/PHPMA-Fc films were built in the presence of different sodium salts at different ionic strengths. For low host-guest interactions, only isolated aggregates are formed on the substrate. As the strength of the host-guest interactions increases (increase of log K), the formed films go through a droplet-like structure, before becoming continuous but rough for stronger interactions. When the interaction strength is further increased, the roughness of the films decreases, leading to a smooth continuous film before becoming rough again at still higher interaction strength. Smooth continuous multilayers seem thus to be obtained for an optimal range of the interaction strength.

Nanosized films based on multicharged small molecules and oppositely charged polyelectrolytes obtained by simultaneous spray coating of interacting species.

Simultaneous spraying of polyelectrolytes and small multicharged molecules of opposite charges onto a vertical substrate leads to continuous buildups of organic films. Here, we investigate the rules governing the buildup of two such systems: poly(allylamine hydrochloride)/sodium citrate (PAH/citrate) and PAH/sulfated α-cyclodextrin (PAH/CD-S). Special attention is paid to the film growth rate as a function of the spraying rate ratio of the two constituents. This parameter was varied by increasing the spraying rate of one of the constituents while maintaining constant that of the other. For PAH/CD-S systems, whatever the constituent (PAH or CD-S) whose spraying rate was kept fixed, the film growth rate first increases and passes through a maximum before decreasing when the spraying rate of the other constituent is increased. For PAH/citrate, the film growth rate reaches a plateau value when the spraying rate of citrate is increased while that of PAH is maintained constant, whereas when the spraying rate of citrate is maintained constant and that of PAH is increased, a behavior similar to that of PAH/CD-S is observed. The composition of PAH/CD-S sprayed films determined by X-ray photoelectron spectroscopy is independent of the spraying rate ratio of the two constituents and corresponds to one allylamine for one sulfate group. For PAH/citrate, by increasing the PAH/citrate spraying rate ratio, the carboxylic/nitrogen ratio in the film increases and tends to 1. There is thus always a deficit of carboxylic groups (COO(-) + COOH) with respect to amines (NH2 + NH3(+)). Yet, the ratio (COO(-)/NH3(+)) is always close to 1, ensuring exact charge compensation. The film morphology determined by atomic force microscopy is granular for PAH/CD-S and is smooth and liquid-like for PAH/citrate. A model based on strong (respectively weak) interactions between PAH and CD-S (respectively citrate) is proposed to explain these features.

Contribution of soft substrates to malignancy and tumor suppression during colon cancer cell division.

In colon cancer, a highly aggressive disease, progression through the malignant sequence is accompanied by increasingly numerous chromosomal rearrangements. To colonize target organs, invasive cells cross several tissues of various elastic moduli. Whether soft tissue increases malignancy or in contrast limits invasive colon cell spreading remains an open question. Using polyelectrolyte multilayer films mimicking microenvironments of various elastic moduli, we revealed that human SW480 colon cancer cells displayed increasing frequency in chromosomal segregation abnormalities when cultured on substrates with decreasing stiffness. Our results show that, although decreasing stiffness correlates with increased cell lethality, a significant proportion of SW480 cancer cells did escape from the very soft substrates, even when bearing abnormal chromosome segregation, achieve mitosis and undergo a new cycle of replication in contrast to human colonic HCoEpiC cells which died on soft substrates. This observation opens the possibility that the ability of cancer cells to overcome defects in chromosome segregation on very soft substrates could contribute to increasing chromosomal rearrangements and tumor cell aggressiveness.

Interspecies differences with in vitro and in vivo models of vascular tissue engineering.

In arterial replacement there is a clear clinical need for a functional substitute possessing appropriate haemocompatible properties to be implanted as small diameter artery. Endothelial cell seeding constitutes an appreciated method to improve blood compatibility on the condition that cells firmly adhere to the support. Along this way, an innovative technique based on multilayered polyelectrolyte films (PEM) as cell adhesive substrate was previously validated in vitro and in vivo in a small-animal model. In this study, we extended the work on a larger animal (sheep) to validate furthermore the paradigm of PEM functionalization for vascular substitutes. We tested in vitro: the efficiency of PEM to induce endothelial progenitor differentiation in sheep endothelial cells; the ability of PEM to sustain cell proliferation and allow resistance to shear stress; the fate of PEM-coated de-endothelialized human saphenous veins under flow conditions, a prerequisite step before in vivo experiments. Despite in vitro differences we were encouraged by testing in vivo PEM-coated prosthesis as carotid replacement in sheep, but without success. In order to explain the implantation failure, an in vitro haemocompatibility evaluation was performed that highlighted interspecies differences able to explain, at least in part, the graft failure obtained.

Self-construction of supramolecular polyrotaxane films by an electrotriggered morphogen-driven process.

The design of films using a one-pot process has recently attracted increasing interest in the field of polymer thin film formation. Herein we describe the preparation of one-pot supramolecular polyrotaxane (PRX) films using the morphogen-driven self-construction process. This one-pot buildup strategy where the film growth is triggered by the electrochemical formation and diffusion of a catalyst in close vicinity of the substrate has recently been introduced by our group. A one-pot mixture was used that contained (i) poly(acrylic acid) (PAA) functionalized by azide groups grafted on the polymer chain through oligo(ethylene glycol) (EG) arms, leading to PAA-EG13-N3, (ii) cyclodextrins (α and ß CD), as macrocycles that can be threaded along EG arms, (iii) alkyne-functionalized stoppers (ferrocene or adamantane), to cap the PRX assembly by click chemistry, and (iv) copper sulfate. The one-pot mixture solution was brought into contact with a gold electrode. Cu(I), the morphogen, was generated electrochemically from Cu(II) at the electrode/one-pot solution interface. This electrotriggered click reaction leads to the capping of polypseudorotaxane yielding to PRXs. The PRXs can self-assemble through lateral supramolecular interactions to form aggregates and ensure the cohesion of the film. The film buildup was investigated using different types of CD and alkyne functionalized stoppers. Supramolecular PRX aggregates were characterized by X-ray diffraction measurements. The film topographies were imaged by atomic force microscopy. The influence of the concentration in CD and the presence of a competitor were studied as well. The stability of the resulting film was tested in contact with 8 M urea and during the electrochemical oxidation of ferrocene.

Endothelialized and preconditioned natural umbilical arteries with long term patency open the route for future human uses.

The major challenge of vascular tissue engineering is to develop a small calibre vascular graft with a high patency rate. In native vessels, the thrombosis is prevented by the endothelium located at the luminal site of the vessel. The aim of this study was to develop a resistant endothelial lining on the inner surface of vascular graft using a polyelectrolyte multilayers (PEM) film. Umbilical arteries were de-endothelialized, coated with 3.5 bilayers of poly(styrene sulfonate) (PSS)/poly(allylamine hydrochloride) (PAH) and then cellularized with endothelial cells. The grafts were cultured for a week in static condition and preconditioned by exposure to a shear stress of at 1 Pa for three hours before implantation on the rabbit carotid site. Histological and confocal microscopy in vitro investigations showed that PEMs films improve cell adhesion and retention on the luminal surface after shear stress preconditioning. In vivo Doppler data showed that graft preconditioning is a crucial factor for graft patency. Indeed, preconditioned grafts remained over the whole experimental period, whereas unpreconditioned grafts were obstructed after only one week of implantation. These results open the route toward the development of a new generation of vascular substitutes having a long term patency.

Bioaffinity sensor based on nanoarchitectonic films: control of the specific adsorption of proteins through the dual role of an ethylene oxide spacer.

The identification and quantification of biomarkers or proteins is a real challenge in allowing the early detection of diseases. The functionalization of the biosensor surface has to be properly designed to prevent nonspecific interactions and to detect the biomolecule of interest specifically. A multilayered nanoarchitecture, based on polyelectrolyte multilayers (PEM) and the sequential immobilization of streptavidin and a biotinylated antibody, was elaborated as a promising platform for the label-free sensing of targeted proteins. We choose ovalbumin as an example. Thanks to the versatility of PEM films, the platform was built on two types of sensor surface and was evaluated using both optical- and viscoelastic-based techniques, namely, optical waveguide lightmode spectroscopy and the quartz crystal microbalance, respectively. A library of biotinylated poly(acrylic acids) (PAAs) was synthesized by grafting biotin moieties at different grafting ratios (GR). The biotin moieties were linked to the PAA chains through ethylene oxide (EO) spacers of different lengths. The adsorption of the PAA-EOn-biotin (GR) layer on a PEM precursor film allows tuning the surface density in biotin and thus the streptavidin adsorption mainly through the grafting ratio. The nonspecific adsorption of serum was reduced and even suppressed depending on the length of the EO arms. We showed that to obtain an antifouling polyelectrolyte the grafting of EO9 or EO19 chains at 25% in GR is sufficient. Thus, the spacer has a dual role: ensuring the antifouling property and allowing the accessibility of biotin moieties. Finally, an optimized platform based on the PAA-EO9-biotin (25%)/streptavidin/biotinylated-antibody architecture was built and demonstrated promising performance as interface architecture for bioaffinity sensing of a targeted protein, in our case, ovalbumin.

Potent calcium phosphate nanoparticle surface coating for in vitro and in vivo siRNA delivery: a step toward multifunctional nanovectors.

The present study describes hybrid nanoparticles, built by alternate deposition of siRNA and modified polyethyleneimine (tyrosine-grafted PEI or tyrosine/galactose-grafted PEI) on calcium phosphate nanoparticles. These "easy to produce" nanoparticles (NPs) present an efficient gene silencing effect demonstrated in vitro in a luciferase expressing cell culture model and in vivo in a tumour xenograft mouse model. The luciferase gene silencing percentage reached up to 95% in vitro with biocompatible doses of siRNA. Interestingly, we show by SPECT imaging of radiolabeled particles that without modifying the size, stability and in vitro efficiency, the grafting of a sugar moiety on PEI can modify the in vivo biodistribution of the particles. The proof of concept that galactose-grafting on PEI could change biodistribution without changing the gene silencing efficiency makes them versatile tools for specific delivery of small interfering RNA. As they have been designed so far, biodistribution is mainly located in the liver and thus these innovative nanoparticles open a realistic and feasible strategy for siRNA delivery into the liver in vivo.

Protein capsules assembled via isobutyramide grafts: sequential growth, biofunctionalization, and cellular uptake.

We report the sequential assembly of proteins via the alternating physical adsorption of human serum albumin (HSA) and chemical grafting with isobutyramide (IBAM) or bromoisobutyramide (BrIBAM) groups. This approach, performed on silica template particles, leads to the formation of noncovalent protein films with controlled growth at the nanometer scale. Further, after template removal, hollow protein capsules with tunable wall thicknesses and high mechanical stability are obtained. The use of BrIBAM, compared to IBAM grafts, leads to significantly thicker capsule walls, highlighting the influence of the bromine atoms in the assembly process, which is discussed in terms of a theoretical model of noncovalent interactions. Another feature of the process is the possibility to functionalize the HSA capsules with other biologically active macromolecules, including enzymes, polysaccharides, or DNA plasmids, demonstrating the versatility of this approach. We also report that BrIBAM-HSA and IBAM-HSA capsules display negligible cytotoxicity in vitro with HeLa cells and that their cellular uptake is dependent on the thickness of the capsule walls. These findings support the potential use of these protein capsules in tailored biological applications such as drug delivery.

Stretch-induced biodegradation of polyelectrolyte multilayer films for drug release.

The design of stimuli-responsive polymer assemblies for the controlled release of bioactive molecules has raised considerable interest these two last decades. Herein, we report the design of mechanically responsive drug-releasing films made of polyelectrolyte multilayers. A layer-by-layer (LbL) reservoir containing biodegradable polyelectrolytes is capped with a mechanosensitive LbL barrier and responds to stretching by a total enzymatic degradation of the film. This strategy is successfully applied for the release in solution of an anticancer drug initially loaded within the architecture.

Collagen-based fibrillar multilayer films cross-linked by a natural agent.

Surface functionalization plays an important role in the design of biomedical implants, especially when layer forming cells, such as endothelial or epithelial cells, are needed. In this study, we define a novel nanoscale surface coating composed of collagen/alginate polyelectrolyte multilayers and cross-linked for stability with genipin. This buildup follows an exponential growth regime versus the number of deposition cycles with a distinct nanofibrillar structure that is not damaged by the cross-linking step. Stability and cell compatibility of the cross-linked coatings were studied with human umbilical vein endothelial cells. The surface coating can be covered by a monolayer of vascular endothelial cells within 5 days. Genipin cross-linking renders the surface more suitable for cell attachment and proliferation compared to glutaraldehyde (more conventional cross-linker) cross-linked surfaces, where cell clumps in dispersed areas were observed. In summary, it is possible with the defined system to build fibrillar structures with a nanoscale control of film thickness, which would be useful for in vivo applications such as inner lining of lumens for vascular and tracheal implants.

Mobility of proteins in highly hydrated polyelectrolyte multilayer films.

The lateral diffusion of a protein (human serum albumin labeled with fluorescein isothiocyanate) within a highly hydrated polyelectrolyte film is studied. The film is built up with poly(L-lysine) as polycation and hyaluronate as polyanion. Fluorescence recovery after photobleaching is used to evaluate the mobility of the labeled protein. Spatial Fourier transformation is applied to the fluorescence intensity recorded at various times after bleaching of a narrow rectangular area within an image representative of the film. This approach necessitates no hypothesis on the intensity distribution at the end of the bleaching provided that the bleach has not appreciably changed the concentration ratios of the different diffusing species. Furthermore, under the hypothesis that molecules move according to Fick's law, we represent the Fourier transform by a weighted sum of exponentials each containing another diffusion coefficient and evaluate the proportion attached to each term of this sequence using the simulated annealing method. A criterion, combining goodness-of-fit and the entropy characterizing the diffusion coefficient spectrum, is proposed to avoid overinterpretation of the experimental data. The optimum spectrum of the diffusion coefficient is then extracted from the time evolution of the light intensity at various albumin concentrations within the films. It appears that the mobility, quantified by the amount of tracer molecules having a diffusion coefficient smaller than, e.g., 0.1 µm(2)/s, undergoes a transition between 20 and 2000 µg/mL of internal concentration. This suggests that the mutual interactions of the albumin molecules and the interactions between fluorescently labeled albumin and the film network become increasingly important in the reduction of the albumin mobility as the albumin concentration increases.

Spray-assisted polyelectrolyte multilayer buildup: from step-by-step to single-step polyelectrolyte film constructions.

The alternate deposition of polyanions and polycations on a solid substrate leads to the formation of nanometer to micrometer films called Polyelectrolyte Multilayers. This step-by-step construction of organic films constitutes a method of choice to functionalize surfaces with applications ranging from optical to bioactive coatings. The method was originally developed by dipping the substrate in the different polyelectrolyte solutions. Recent advances show that spraying the polyelectrolyte solutions onto the substrate represents an appealing alternative to dipping because it is much faster and easier to adapt at an industrial level. Multilayer deposition by spraying is thus greatly gaining in interest. Here we review the current literature on this deposition method. After a brief history of polyelectrolyte multilayers to place the spraying method in its context, we review the fundamental issues that have been addresses so far. We then give an overview the different fields where the method has been applied.

Cyto-mechanoresponsive polyelectrolyte multilayer films.

Cell adhesion processes take place through mechanotransduction mechanisms where stretching of proteins results in biological responses. In this work, we present the first cyto-mechanoresponsive surface that mimics such behavior by becoming cell-adhesive through exhibition of arginine-glycine-aspartic acid (RGD) adhesion peptides under stretching. This mechanoresponsive surface is based on polyelectrolyte multilayer films built on a silicone sheet and where RGD-grafted polyelectrolytes are embedded under antifouling phosphorylcholine-grafted polyelectrolytes. The stretching of this film induces an increase in fibroblast cell viability and adhesion.

Layer-by-Layer Enzymatic Platform for Stretched-Induced Reactive Release.

An original "all-in-one" platform combining polymers, enzymes, and enzymatic substrates in a unique film is designed. A polymeric barrier stratum prevents any contact between enzymes adsorbed on top of the film and substrates loaded in an underlying reservoir. Upon stretching of the film, a continuous diffusion of substrates through the barrier is triggered, followed by a catalytic reaction. This leads to the formation of products that are released from the film. This new platform acts as a stretch-induced reactive release system and emerges as an innovative concept in mechano-responsive materials.