Influence of Surface Chemistry on Host/Guest Interactions: A Model Study on Redox-Sensitive ß-Cyclodextrin/Ferrocene Complexes.

While host/guest interactions are widely used to control molecular assembly on surfaces, quantitative information on the effect of surface chemistry on their efficiency is lacking. To address this question, we combined electrochemical characterization with quartz crystal microbalance with dissipation monitoring to study host/guest interactions between surface-attached ferrocene (Fc) guests and soluble ß-cyclodextrin (ß-CD) hosts. We identified several parameters that influence the redox response, ß-CD complexation ability, and repellent properties of Fc monolayers, including the method of Fc grafting, the linker connecting Fc with the surface, and the diluting molecule used to tune Fc surface density. The study on monovalent ß-CD/Fc complexation was completed by the characterization of multivalent interactions between Fc monolayers and ß-CD-functionalized polymers, with new insights being obtained on the interplay between the surface chemistry, binding efficiency, and reversibility under electrochemical stimulus. These results should facilitate the design of well-defined functional interfaces and their implementation in stimuli-responsive materials and sensing devices.

Hydrogel-Colloid Composite Bioinks for Targeted Tissue-Printing.

The development of extrusion-based bioprinting for tissue engineering is conditioned by the design of bioinks displaying adequate printability, shape stability, and postprinting bioactivity. In this context, simple bioink formulations, made of cells supported by a polymer matrix, often lack the necessary versatility. To address this issue, intense research work has been focused on introducing colloidal particles into the ink formulation. By creating weak cross-links between polymer chains, added particles modify the rheology and mechanical behavior of bioinks to improve their printability and structural integrity. Additionally, nano- and microscopic particles display composition- and structure-specific properties that can affect the cellular behavior and enhance the formation of tissue within the printed material. This Review offers a comprehensive picture of the role of colloids in bioprinting from a physicochemical and biological perspective. As such, it provides guidance on devising adaptable bioinks for the fabrication of biomimetic tissues.

Injectable Self-Healing Hydrogels Based on Boronate Ester Formation between Hyaluronic Acid Partners Modified with Benzoxaborin Derivatives and Saccharides.

We demonstrate here, for the first time, formation of injectable dynamic covalent hydrogels at physiological pH using benzoxaborin-saccharide complexation as a reversible cross-linking method. The gels were prepared by simply mixing hyaluronic acid modified with an original boronic acid derivative, 3,4-dihydro-2H-benzo[e][1,2]oxaborinin-2-ol (1,2-ABORIN), and HA functionalized with 1-amino-1-deoxy-d-fructose. Dynamic rheological experiments confirmed the gel-like behavior (storage modulus (G') > loss modulus (G″) in the frequency window explored) for the designed HA-1,2-ABORIN/HA-fructose network. Furthermore, this hydrogel exhibited excellent self-healing and injectability behaviors in aqueous conditions and was found to be responsive to pH. Additionally, fibroblast cells encapsulated in the HA network showed high viability (>80% after 7 days of cell culture), as monitored by Live/Dead staining. Taken together, this new class of boronate ester cross-linked hydrogel provides promising future for diverse biomedical applications.

Boronic acid and diol-containing polymers: how to choose the correct couple to form "strong" hydrogels at physiological pH.

Dynamic covalent hydrogels crosslinked by boronate ester bonds are promising materials for biomedical applications. However, little is known about the impact of the crosslink structure on the mechanical behaviour of the resulting network. Herein, we provide a mechanistic study on boronate ester crosslinking upon mixing hyaluronic acid (HA) backbones modified, on the one hand, with two different arylboronic acids, and on the other hand, with three different saccharide units. Combining rheology, NMR and computational analysis, we demonstrate that carefully selecting the arylboronic-polyol couple allows for tuning the thermodynamics and molecular exchange kinetics of the boronate ester bond, thereby controlling the rheological properties of the gel. In particular, we report the formation of "strong" gels (i.e. featuring slow relaxation dynamics) through the formation of original complex structures (tridentate or bidentate complexes). These findings offer new prospects for the rational design of hydrogel scaffolds with tailored mechanical response.

Dynamic Covalent Chemistry Enables Reconfigurable All-Polysaccharide Nanogels.

Dynamic covalent bonds are established upon molecular recognition of sugar derivatives by boronic acid molecules. These reversible links can be used in a cross-linking method to fabricate polymer-based responsive nanosystems. Herein, the design of the first dynamic nanogels made entirely of polysaccharides (PS) is reported. Based on PS chains alternately modified with phenyl boronic acid groups and sugar moieties, these colloids self-assemble in physiological conditions and combine the biocompatible nature of their PS backbone with the reconfiguration capacities of their cross-linking chemistry. These dynamic nanogels are easily prepared, stable for a long time, pH responsive, and efficiently internalized by cancer cells.

Designing multivalent probes for tunable superselective targeting.

Specific targeting is common in biology and is a key challenge in nanomedicine. It was recently demonstrated that multivalent probes can selectively target surfaces with a defined density of surface binding sites. Here we show, using a combination of experiments and simulations on multivalent polymers, that such "superselective" binding can be tuned through the design of the multivalent probe, to target a desired density of binding sites. We develop an analytical model that provides simple yet quantitative predictions to tune the polymer's superselective binding properties by its molecular characteristics such as size, valency, and affinity. This work opens up a route toward the rational design of multivalent probes with defined superselective targeting properties for practical applications, and provides mechanistic insight into the regulation of multivalent interactions in biology. To illustrate this, we show how the superselective targeting of the extracellular matrix polysaccharide hyaluronan to its main cell surface receptor CD44 is controlled by the affinity of individual CD44-hyaluronan interactions.

A3K2A3-induced apoptotic cell death of Leishmania amazonensis occurs through caspase- and ATP-dependent mitochondrial dysfunction.

Leishmaniasis is a neglected tropical disease that affects millions of people worldwide. Current therapies mainly rely on antimonial drugs that are inadequate because of their high toxicity and increased drug resistance. An urgent need exists to discover new, more effective, more affordable, and more target-specific drugs. Pathways that are associated with apoptosis-like cell death have been identified in unicellular eukaryotes, including protozoan parasites. In the present study, we studied the mechanism of cell death that is induced by A3K2A3 against L. amazonensis. A3K2A3 is a dibenzylideneacetone that has an acyclic dienone that is attached to aryl groups in both ß-positions, which is similar to curcuminoids and chalcone structures. This compound was previously shown to be safe with regard to cytotoxicity and active against the parasite. Biochemical and morphological approaches were used in the present study. The results suggested that A3K2A3 caused mitochondrial dysfunction in L. amazonensis promastigotes, leading to mechanisms of cell death that share some common phenotypic features with metazoan apoptosis, such as an increase in reactive oxygen species production, a decrease in the adenosine triphosphate ratio, phosphatidylserine exposure, a decrease in cell volume, caspase production, and DNA fragmentation. Altogether, these findings indicate that apoptosis can indeed be triggered by chemotherapeutic agents.

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.

Superselective targeting using multivalent polymers.

Despite their importance for material and life sciences, multivalent interactions between polymers and surfaces remain poorly understood. Combining recent achievements of synthetic chemistry and surface characterization, we have developed a well-defined and highly specific model system based on host/guest interactions. We use this model to study the binding of hyaluronic acid functionalized with host molecules to tunable surfaces displaying different densities of guest molecules. Remarkably, we find that the surface density of bound polymer increases faster than linearly with the surface density of binding sites. Based on predictions from a simple analytical model, we propose that this superselective behavior arises from a combination of enthalpic and entropic effects upon binding of nanoobjects to surfaces, accentuated by the ability of polymer chains to interpenetrate.

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.

Readily prepared dynamic hydrogels by combining phenyl boronic acid- and maltose-modified anionic polysaccharides at neutral pH.

Dynamic covalent hydrogels are facilely prepared from biocompatible polysaccharides in physiological conditions by the formation of phenylboronate ester cross-links. This is based on the simple mixing of carboxylate-containing polysaccharides (i.e., hyaluronic acid or carboxymethylcellulose) modified with phenylboronic acid and maltose moieties according to mild coupling reactions performed in aqueous solution. The formation of dynamic networks based on reversible boronic-ester cross-links is demonstrated by analyzing their rheological behavior. This study shows that these gels can adapt their structure in response to chemical stimuli such as variations in pH or addition of glucose and self-heal.

Hyaluronic acid single-network hydrogel with high stretchable and elastic properties.

Stretchable materials have demonstrated great interest in wearable or implantable applications. Most of the existing hydrogels with high stretchability characteristics are based on double networks, exhibit large hysteresis loops, and cannot recover after deformation due to permanent rupture of network. Elastic, biodegradable, and biocompatible hydrogels are desirable for wound dressing of joints with frequent motions or post-surgical healing of mobile tissues. Here, we show a simple strategy for the preparation of a hyaluronic acid (HA) single-network hydrogel that can be stretchable and highly elastic without the addition of other components/partners or complicated processes of preparation. Our strategy relies on the use of high Mw HA to create a chemical hydrogel in which densely entangled HA chains are tied together by a small number of covalent bonds. While the presence of covalent cross-links can prevent disintegration of the HA network, entanglements endow the hydrogel with high stretchability through transmission of tension along the length of the long HA chains. The stretching-relaxation cycles show negligible hysteresis and perfect recovery of material after the release of force. The diminution of Mw together with increasing the concentration or cross-linker amount leads to brittle hydrogels.

Design of hyaluronan-based dopant for conductive and resorbable PEDOT ink.

Conformable biocompatible conductive materials are increasingly sought for the development of bioelectronics. If additionally resorbable, they could serve for the design of transient implantable electronic devices, opening the way to new healthcare applications. Hyaluronan (HA) derivatives including sulfate and aminophenylboronic acid (PBA) groups (HAS-PBA) were therefore designed to serve as dopants of poly(3,4-ethylenedioxy)thiophene (PEDOT). The optimized HA sulfation protocol allowed good control on polymer sulfation degree while minimizing polymer chain degradation. Sulfated HA was shown to be degradable in physiological conditions. A synergy was observed between the sulfate negative charges and the PBA aromatic groups promoting hydrophobic interactions and π-stacking between PEDOT and HAS-PBA, to boost the material conductivity that reached 1.6 ± 0.2 S/cm in physiological conditions. Moreover the PEDOT:HAS-PBA material was not cytotoxic and could be formulated for easy processing by inkjet printing, appearing as promising candidate for the design of soft transient electronics for in vivo applications.

A novel injectable radiopaque hydrogel with potent properties for multicolor CT imaging in the context of brain and cartilage regenerative therapy.

Cell therapy is promising to treat many conditions, including neurological and osteoarticular diseases. Encapsulation of cells within hydrogels facilitates cell delivery and can improve therapeutic effects. However, much work remains to be done to align treatment strategies with specific diseases. The development of imaging tools that enable monitoring cells and hydrogel independently is key to achieving this goal. Our objective herein is to longitudinally study an iodine-labeled hydrogel, incorporating gold-labeled stem cells, by bicolor CT imaging after in vivo injection in rodent brains or knees. To this aim, an injectable self-healing hyaluronic acid (HA) hydrogel with long-persistent radiopacity was formed by the covalent grafting of a clinical contrast agent on HA. The labeling conditions were tuned to achieve sufficient X-ray signal and to maintain the mechanical and self-healing properties as well as injectability of the original HA scaffold. The efficient delivery of both cells and hydrogel at the targeted sites was demonstrated by synchrotron K-edge subtraction-CT. The iodine labeling enabled to monitor the hydrogel biodistribution in vivo up to 3 days post-administration, which represents a technological first in the field of molecular CT imaging agents. This tool may foster the translation of combined cell-hydrogel therapies into the clinics.

α-Acetal, ω-alkyne poly(ethylene oxide) as a versatile building block for the synthesis of glycoconjugated graft-copolymers suited for targeted drug delivery.

α-Acetal, ω-alkyne poly(ethylene oxide) was synthesized as building block of glycoconjugated poly(ε-caprolactone)-graft-poly(ethylene oxide) (PCL-g-PEO) copolymers. The alkyne group is indeed instrumental for the PEGylation of a poly(α-azido-ε-caprolactone-co-ε-caprolactone) copolymer by the Huisgen's 1,3 dipolar cycloaddition, i.e., a click reaction. Moreover, deprotection of the acetal end-group of the hydrophilic PEO grafts followed by reductive amination of the accordingly formed aldehyde with an aminated sugar is a valuable strategy of glycoconjugation of the graft copolymer, whose micelles are then potential. A model molecule (fluoresceinamine) was first considered in order to optimize the experimental conditions for the reductive amination. These conditions were then extended to the decoration of the graft copolymer micelles with mannose, which is a targeting agent of dendritic cells and macrophages. The bioavailability of the sugar units at the surface of micelles was investigated by surface plasmon resonance (SPR). The same question was addressed to nanoparticles stabilized by the graft copolymer. Enzyme linked lectin assay (ELLA) confirmed the availability of mannose at the nanoparticle surface.

Synthesis of poly(lactide-co-glycolide-co-ε-caprolactone)-graft-mannosylated poly(ethylene oxide) copolymers by combination of "clip" and "click" chemistries.

Poly(lactide-co-glycolide) (PLGA) is extensively used in pharmaceutical applications, for example, in targeted drug delivery, because of biocompatibility and degradation rate, which is easily tuned by the copolymer composition. Nevertheless, synthesis of sugar-labeled amphiphilic copolymers with a PLGA backbone is quite a challenge because of high sensitivity to hydrolytic degradation. This Article reports on the synthesis of a new amphiphilic copolymer of PLGA grafted by mannosylated poly(ethylene oxide) (PEO). A novel building block, that is, α-methoxy-ω-alkyne PEO-clip-N-hydroxysuccinimide (NHS) ester, was prepared on purpose by photoreaction of a diazirine containing molecular clip. This PEO block was mannosylated by reaction of the NHS ester groups with an aminated sugar, that is, 2-aminoethyl-α-d-mannopyroside. Then, the alkyne ω-end-group of PEO was involved in a copper alkyne- azide coupling (CuAAC) with the pendent azides of the aliphatic copolyester. The targeted mannose-labeled poly(lactide-co-glycolide-co-ε-caprolactone)-graft-poly(ethylene oxide) copolymer was accordingly formed. Copolymerization of d,l-lactide and glycolide with α-chloro-ε-caprolactone, followed by substitution of chlorides by azides provided the azido-functional PLGA backbone. Finally, micelles of the amphiphilic mannosylated graft copolymer were prepared in water, and their interaction with Concanavalin A (ConA), a glyco-receptor protein, was studied by quartz crystal microbalance. This study concluded to the prospect of using this novel bioconjugate in targeted drug delivery.

Design of biomimetic cell-interactive substrates using hyaluronic acid hydrogels with tunable mechanical properties.

Hyaluronic acid (HA) is a natural polysaccharide abundant in biological tissues with excellent potential for constructing synthetic extracellular matrix analogues. In this work, we established a simple and dependable approach to prepare hyaluronic acid-based hydrogels with controlled stiffness and cell recognition properties for use as cell-interactive substrates. This approach relied on a new procedure for the synthesis of methacrylate-modified HA macromers (HA-MA) and, on photorheometry allowing real time monitoring of gelation during photopolymerization. We showed in this way the ability to obtain gels that encompass the range of physiologically relevant elastic moduli while still maintaining the recognition properties of HA by specific cell surface receptors. These hydrogels were prepared from HA macromers having a degree of methacrylation <0.5, which allows to minimize compromising effects on the binding affinity of HA to its cell receptors due to high substitution on the one hand, and to achieve nearly 100% conversion of the methacrylate groups on the other. When the HA hydrogels were immobilized on glass substrates, it was observed that the attachment and the spreading of a variety of mammalian cells rely on CD44 and its coreceptor RHAMM. The attachment and spreading were also shown to be modulated by the elastic properties of the HA matrix. All together, these results highlight the biological potential of these HA hydrogel systems and the needs of controlling their chemical and physical properties for applications in cell culture and tissue engineering.

Tailored hyaluronic acid-based nanogels as theranostic boron delivery systems for boron neutron cancer therapy.

Boron-rich nanocarriers possess great potential for advanced boron neutron capture therapy (BNCT) as an effective radiation treatment for invasive malignant tumors. If additionally, they can be imaged in a non-invasive and real-time manner allowing the assessment of local boron concentration, they could serve for dose calculation and image-guided BNCT to enhance tumor treatment efficacy. To meet this challenge, this study describes the design of a theranostic nanogel, enriched in 10B and fluorescent dye, to achieve selective imaging, and sufficient accumulation of boron at the tumor site. The boron-rich and fluorescent nanogels can be easily obtained via temperature triggered-assembly of hyaluronic acid (HA) modified with a thermoresponsive terpolymer. The latter was specifically designed to enable the efficient encapsulation of the fluorescent dye - an aza­boron-dipyrromethene (aza-BODIPY) - linked to 10B-enriched sodium borocaptate (BSH), in addition to induce nanogel formation below room temperature, and to enable their core-crosslinking by hydrazone bond formation. The HA nanogel considerably concentrates aza-BODIPY-BSH into the hydrophobic nanodomains made of the terpolymer chains. Here, we present the detailed synthesis of the HA-terpolymer conjugate, nanogel formation, and characterization in terms of size, morphology, and stability upon storage, as well as the biological behavior of the boron nanocarrier using real-time fluorescence imaging in cells and in vivo. This work suggested the potential of the theranostic HA nanogel as a boron delivery system for the implementation of BNCT in brain cancer and sarcoma.

Polysaccharide-blend multilayers containing hyaluronan and heparin as a delivery system for rhBMP-2.

It is shown that blend multilayers of hyaluronan (HA) and heparin (HEP) as polyanions and poly(L-lysine) (PLL) as a polycation can be used to prepare films with different thicknesses and chemical compositions. The amounts of recombinant human BMP-2 (rhBMP-2) loaded and the fraction initially released from the films depend on the film's chemical composition. The amounts of rhBMP-2 loaded in the films are much higher for HA mass fractions of more than 0.4. The bioactivity of the rhBMP-2-loaded films is investigated on C2C12 myoblasts, which differentiates into osteoblasts in contact with the films. The alkaline phosphatase expression for cells grown on nanoblend films of various compositions falls over a unique curve. This suggests that the cells "sensing" the rhBMP-2 are not influenced by the film's chemistry. The rhBMP-2 can sustain at least three successive culture sequences while remaining bioactive, thus confirming the important and protective effect of rhBMP-2. Altogether, these results indicate that crosslinked PLL/HA films have superior properties for the incorporation of rhBMP-2 and on its long-lasting bioactivity.

Electrochemically controlled adsorption of Fc-functionalized polymers on beta-CD-modified self-assembled monolayers.

This work presents an in situ study of the adsorption/desorption behavior of ferrocene(Fc)-functionalized linear polymers on a gold surface covered with beta-cyclodextrin(beta-CD)-modified self-assembled monolayers (SAMs). The characterization of binary SAMs obtained with HS-(CH(2))(11)-EG(6)-N(3) and HS-(CH(2))(11)-EG(4)-OH (EG, ethylene glycol) was performed using a quartz crystal microbalance with dissipation monitoring (QCM-D), cyclic voltammetry, and contact angle measurements. The functionalization of SAMs with beta-CD was made via the "click" reaction between the beta-CD monoalkyne derivative and azide groups exhibited by SAMs. The formation of the host-guest complex between SAM-beta-CD and Fc-derivatized polymers (chitosan (CHI) and poly(allylamine hydrochloride) (PAH)) was studied by QCM-D. The viscoelastic model of Voinova was used to fit QCM-D curves recorded during the adsorption and electrochemically controlled desorption of CHI-Fc and PAH-Fc on SAM-beta-CD. Using QCM-D coupled to cyclic voltammetry, we demonstrated that CHI-Fc and PAH-Fc can be successfully deposited on a SAM-beta-CD-coated gold surface forming a stable multivalent inclusion complex between Fc moieties of polymer and beta-CD cavities of SAM. We also showed that all specifically attached polymer chains can be detached from the SAM-beta-CD-coated gold surface by applying an electric field.