Introducing a biomimetic coating for graphene neuroelectronics: towardin-vivoapplications.

Electronic micro and nano-devices are suitable tools to monitor the activity of many individual neurons over mesoscale networks. However the inorganic materials currently used in microelectronics are barely accepted by neural cells and tissues, thus limiting both the sensor lifetime and efficiency. In particular, penetrating intracortical probes face high failure rate because of a wide immune response of cells and tissues. This adverse reaction called gliosis leads to the rejection of the implanted probe after few weeks and prevent long-lasting recordings of cortical neurons. Such acceptance issue impedes the realization of many neuro-rehabilitation projects. To overcome this, graphene and related carbon-based materials have attracted a lot of interest regarding their positive impact on the adhesion and regeneration of neurons, and their ability to provide high-sensitive electronic devices, such as graphene field effect transistor (G-FET). Such devices can also be implemented on numerous suitable substrates including soft substrates to match the mechanical compliance of cells and tissues, improving further the biocompatibility of the implants. Thus, using graphene as a coating and sensing device material could significantly enhance the acceptance of intracortical probes. However, such a thin monolayer of carbon atoms could be teared off during manipulation and insertion within the brain, and could also display degradation over time. In this work, we have investigated the ability to protect graphene with a natural, biocompatible and degradable polymeric film derivated from hyaluronic acid (HA). We demonstrate that HA-based coatings can be deposited over a wide range of substrates, including intracortical probes and graphene FET arrays without altering the underlying device material, its biocompatibility and sensitivity. Moreover, we show that this coating can be monitoredin situby quantifying the number of deposited charges with the G-FET arrays. The reported graphene functionalization offers promising alternatives for improving the acceptance of various neural interfaces.

Heparosan as a potential alternative to hyaluronic acid for the design of biopolymer-based nanovectors for anticancer therapy.

Glycosaminoglycans (GAGs) are important components of the extracellular matrix that have attracted great interest for drug delivery and pharmaceutical applications due to their diverse biological functions. Among GAGs, heparosan (Hep), a biosynthetic precursor of heparin, has recently emerged as a promising building block for the design of nanoparticles with stealth properties. Though this non-sulfated polysaccharide has a chemical structure very close to that of hyaluronic acid (HA), it distinguishes from HA in that it is biologically inert in the extracellular spaces in the body. In this study, we designed Hep- and HA-based nanogels (NGs) that differ only in the chemical nature of the hydrophilic shell. The nanogels were prepared in a very straightforward way from Hep and HA modified with a thermoresponsive copolymer properly designed to induce self-assembly below room temperature. This versatile synthetic approach also enabled further shell-crosslinking allowing an increase in the colloidal stability. After careful characterization of the un-crosslinked and crosslinked Hep and HA NGs in terms of size (Z-average diameters of un-crosslinked and crosslinked NGs ∼110 and 150 nm) and morphology, they were injected intravenously into tumor-bearing mice for biodistribution experiments. Interestingly, these show that the liver uptake of Hep nanogels is remarkably reduced and tumor accumulation significantly improved as compared to HA nanogels (intensity ratios of tumor-to-liver of 2.2 and 1.4 for the un-crosslinked and crosslinked Hep NGs versus 0.11 for the un-crosslinked and crosslinked HA ones). These results highlight the key role played by the shell-forming GAGs on the in vivo fate of nanogels, which correlates with the specific biological properties of Hep and HA.

Chitosan-lipid nanoparticles (CS-LNPs): Application to siRNA delivery.

To benefit from the biocompatibility of lipid nanoparticles associated with the transfection ability of chitosan, small chitosan lipid nanoparticles (CS-LNPs) dedicated to SiRNA delivery were formulated by an easy-to-implement one-step process. Formulations of CS-LNPs (lipid core stabilized by a shell comprising phospholipids/cationic lipids and hydrophobically modified chitosan) were optimized for their physico-chemical properties (size, zeta potential, colloidal stability) according to their shell composition. In particular, amphiphilic chitosan with various molecular weight and C12 degrees of substitution, and different phospholipids and cationic lipids (lecithin, DOTAP, DOPE) were included at the particle surface at different ratios. The ability of the particles for SiRNA complexation, NIH3T3 cell transfection, and ERK1 downregulation, were studied. Lipid nanoparticles formulated with 15,000g/mol 2% C12 substituted chitosan, DOTAP and DOPE, mediated 40% ERK1 downregulation efficiency, comparable to lipofectamine™ RNAimax, while displaying no cytotoxicity up to 500µg/mL.

Coumarin-containing thermoresponsive hyaluronic acid-based nanogels as delivery systems for anticancer chemotherapy.

Multi-stimuli responsive nanogels based on biocompatible hydrophilic polymers have emerged as promising drug delivery systems to improve anticancer therapy with hydrophobic drugs, through increase of circulating-time in the bloodstream, tumor-targeting and reduction of systemic toxicity. This paper reports on the synthesis, characterization and biological perspectives of light- and thermoresponsive hyaluronic acid (HA)-based nanogels containing coumarin as the photocleavable group. Newly synthesized nanogels exhibited interesting features: formation by a temperature-triggered self-assembly process, successful incorporation of poorly water-soluble molecules, light-responsiveness as demonstrated by a significant shift in the critical aggregation temperature after light irradiation, efficient internalization by cancer cells overexpressing the CD44 receptor of HA, ability to circulate for a prolonged period of time in the bloodstream after intravenous injection in mice and considerable detection in tumor tissues. Our findings indicate that coumarin-containing HA-based nanogels may be promising delivery systems for anticancer chemotherapy.

ß-CD-Functionalized Microdevice for Rapid Capture and Release of Bacteria.

Most procedures for detecting pathogens in liquid media require an initial concentration step. In this regard, carbohydrates have proven to be attractive affinity ligands for the solid-phase capture of bacteria that use lectins for adhesion to host cell membranes. However, the use of cyclodextrin-immobilized substrates to selectively trap bacteria has not been explored before. Here, using quartz-crystal microbalance with dissipation monitoring experiments, we demonstrate that functionalization of surfaces by ß-cyclodextrin (ß-CD) can not only allow for rapid and efficient capture of bacterial cells in liquid but also their facile elution with an aqueous solution of a selectively methylated ß-CD derivative as a competitive molecule. This capture/elution strategy, which is based on host-guest interactions between membrane components of the bacterial cell and the CD cavities, is performed in physiological conditions and can be integrated in a microchip. Indeed, proof-of-concept studies showed the potential of ß-CD-modified micropillar-integrated microfluidic devices for concentration of bacteria. The results obtained with Escherichia coli suggest that this approach could be broadly applicable among Gram-negative bacteria, which share common cell membrane structures.

Design of Hyaluronic Acid Hydrogels to Promote Neurite Outgrowth in Three Dimensions.

A hyaluronic acid (HA)-based extracellular matrix (ECM) platform with independently tunable stiffness and density of cell-adhesive peptide (RGD, arginine-glycine-aspartic acid) that mimics key biochemical and mechanical features of brain matrix has been designed. We demonstrated here its utility in elucidating ECM regulation of neural progenitor cell behavior and neurite outgrowth. The analysis of neurite outgrowth in 3-D by two-photon microscopy showed several important results in the development of these hydrogels. First, the ability of neurites to extend deeply into these soft HA-based matrices even in the absence of cell-adhesive ligand further confirms the potential of HA hydrogels for central nervous system (CNS) regeneration. Second, the behavior of hippocampal neural progenitor cells differed markedly between the hydrogels with a storage modulus of 400 Pa and those with a modulus of 800 Pa. We observed an increased outgrowth and density of neurites in the softest hydrogels (G' = 400 Pa). Interestingly, cells seeded on the surface of the hydrogels functionalized with the RGD ligand experienced an optimum in neurite outgrowth as a function of ligand density. Surprinsingly, neurites preferentially progressed inside the gels in a vertical direction, suggesting that outgrowth is directed by the hydrogel structure. This work may provide design principles for the development of hydrogels to facilitate neuronal regeneration in the adult brain.

Type, density, and presentation of grafted adhesion peptides on polysaccharide-based hydrogels control preosteoblast behavior and differentiation.

In this work, cell-responsive polysaccharide hydrogels were prepared by a simple procedure based on the sequential bioconjugation and cross-linking of the polysaccharide backbone with bioactive peptides and poly(ethylene glycol)-bis(thiol) (PEG-(SH)2), respectively. Using thiol-ene reactions, we successfully functionalized hyaluronic acid (HA) and carboxymethylcellulose (CMC) with short and long peptides (5-mer and 15-mer derivatives, respectively) derived from adhesive proteins of bone extracellular matrix. The resulting HA-peptide and CMC-peptide conjugates with varying degrees of substitution were then carefully characterized by (1)H NMR spectroscopy to precisely control the peptide density into the hydrogels cross-linked with PEG-(SH)2. Preosteoblast seeded on the hydrogels with controlled identical stiffness spread in a manner that was strongly dependent on ligand density. Surprisingly, increasing the density of the adhesive peptide anchors did not result in a plateau of initial cell spreading but rather in a bell-shaped cell response that varies with the nature of both polysaccharide backbone and functional peptide. Placing the cells under optimal conditions for cell/hydrogel interaction, we showed that in HA hydrogels, the polysaccharide moiety is not solely a passive scaffold that presents the active peptides but is an active player in cell microenvironment to control and sustain cell activity.

Thermoresponsive hyaluronic acid nanogels as hydrophobic drug carrier to macrophages.

Delivery systems for macrophages are particularly attractive since these phagocytic cells play a important role in immunological and inflammatory responses, also acting as host cells for microorganisms that are involved in deadly infectious diseases, such as leishmaniasis. Hyaluronic acid (HA) is specifically recognized by macrophages that are known to express HA receptors. Therefore, in this study, we focused on HA-based nanogels as drug carriers for these cells. The drug delivery was validated in an in vivo study on mice using intravital two-photon laser scanning microscopy. HA derivatives were modified with a biocompatible oligo(ethylene glycol)-based thermoresponsive polymer to form nanogels. These HA conjugates were readily prepared by varying the molar mass of initial HA and the degree of substitution via radical-mediated thiol-ene chemistry in aqueous solution. The derivatives were shown to self-assemble into spherical gel particles with diameters ranging from 150 to 214 nm above 37 °C. A poorly water-soluble two-photon dye was successfully loaded into the nanogels during this self-assembly process. In vitro cellular uptake tests using a RAW 264.7 murine macrophage cell line showed successful intracellular delivery of the hydrophobic dye. After intravenous injection in mice, the nanogels circulated freely in the blood but were rapidly phagocytized within 13 min by circulating macrophages and stored in the liver and spleen, as observed by two-photon microscopy. Benefit can be thus expected in using such a delivery system for the liver and spleen macrophage-associated diseases.

Thiol-ene clickable hyaluronans: from macro-to nanogels.

The fabrication of hyaluronic acid (HA) nanogels using a thiol-ene reaction has been demonstrated. HA was modified with pentenoate groups and then cross-linked with poly(ethylene glycol)-bis(thiol) by exposure to UV light. The cross-linking density and thereby the rigidity of the obtained gels were precisely controlled by the degree of substitution of pentenoate-modified HA. Their swelling properties also depended on cross-linking density. To produce hydrogels at the nanoscale, hyaluronic acid precursors were solely confined inside liposomes before cross-linking and purified after cross-linking. The size of the resulting nanogels followed their swelling properties and was also affected by their cross-linking density. Such bionanogels with tunable mechanical and swelling properties have potential in drug delivery.

Cyclodextrin-modified zeolites: host-guest surface chemistry for the construction of multifunctional nanocontainers.

The functionalization of nanoporous zeolite L crystals with ß-cyclodextrin (CD) has been demonstrated. The zeolite surface was first modified with amino groups by using two different aminoalkoxysilanes. Then, 1,4-phenylene diisothiocyanate was reacted with the amino monolayer and used to bind CD heptamine by using its remaining isothiocyanate groups. The use of the different aminoalkoxysilanes, 3-aminopropyl dimethylethoxysilane (APDMES) and 3-aminopropyl triethoxysilane (APTES), led to drastic differences in uptake and release properties. Thionine was found to be absorbed and released from amino- and CD-functionalized zeolites when APDMES was used, whereas functionalization by APTES led to complete blockage of the zeolite channels. Fluorescence microscopy showed that the CD groups covalently attached to the zeolite crystals could bind adamantyl-modified dyes in a specific and reversible manner. This strategy allowed the specific immobilization of His-tagged proteins by using combined host-guest and His-tag-Ni-nitrilotriacetic acid (NTA) coordination chemistry. Such multifunctional systems have the potential for encapsulation of drug molecules inside the zeolite pores and non-covalent attachment of other (for example, targeting) ligand molecules on its surface.