One-Pot Synthesis of Bioinspired Peptide-Decorated Apatite Nanoparticles for Nanomedicine.

Hybrid organic-inorganic bio-inspired apatite nanoparticles (NPs) are attractive for biomedical applications and especially in nanomedicine. Unfortunately, their applications in nanomedicine are limited by their broad particle size distributions and uncontrolled drug loading due to their multistep synthesis process.  Besides, very few attempts at exposing bioactive peptides on apatite NPs are made. In this work, an original one-pot synthesis of well-defined bioactive hybrid NPs composed of a mineral core of bioinspired apatite surrounded by an organic corona of bioactive peptides is reported. Dual stabilizing-bioactive agents, phosphonated polyethylene glycol-peptide conjugates, are prepared and directly used during apatite precipitation i) to form the organic corona during apatite precipitation, driving the size and shape of resulting hybrid NPs with colloidal stabilization and ii) to expose peptide moieties (RGD or YIGSR sequences) at the NPs periphery in view of conferring additional surface properties to enhance their interaction with cells. Here, the success of this approach is demonstrated, the functionalized NPs are fully characterized by Fourier-transform infrared, Raman, X-ray diffraction, solid and liquid state NMR, transmission electron microscopy, and dynamic light scattering, and their interaction with fibroblast cells is followed, unveiling a synergistic proliferative effect.

A bioinspired approach for the modulation of electroosmotic flow and protein-surface interactions in capillary electrophoresis using silylated amino-amides blocks and covalent grafting.

We explore a bioinspired approach to design tailored functionalized capillary electrophoresis (CE) surfaces based on covalent grafting for biomolecules analysis. First, the approach aims to overcome well-known common obstacles in CE protein analysis affecting considerably the CE performance (asymmetry, resolution, and repeatability) such as the unspecific adsorption on fused silica surface and the lack of control of electroosmotic flow (EOF). Then, our approach, which relies on new amino-amide mimic hybrid precursors synthesized by silylation of amino-amides (Si-AA) derivatives with 3-isocyanatopropyltriethoxysilane, aims to recapitulate the diversity of protein-protein interactions (π-π stacking, ionic, Van der Waals…) found in physiological condition (bioinspired approach) to improve the performance of CE protein analysis (electrochromatography). As a proof of concept, these silylated Si-AA (tyrosinamide silylation, serinamide silylation, argininamide silylation, leucinamide silylation, and isoglutamine silylation acid) have been covalently grafted in physiological conditions in different amount on bare fused silica capillary giving rise to a biomimetic coating and allowing both the modulation of EOF and protein-surface interactions. The analytical performances of amino-amide functionalized capillaries were assessed using lysozyme, cytochrome C and ribonuclease A and compared to traditional capillary coatings poly(ethylene oxide), poly(diallyldimethylammonium chloride), and sodium poly(styrenesulfonate). EOF, protein adsorption rate, protein retention factor k, and selectivity were determined for each coating. All results obtained showed this approach allowed to modulate the EOF, reduce unspecific adsorption, and generate specific interactions with proteins by varying the nature and the amount of Si-AA in the functionalization mixture.

Superiority of the Triple-Acting 5-HT6R/5-HT3R Antagonist and MAO-B Reversible Inhibitor PZ-1922 over 5-HT6R Antagonist Intepirdine in Alleviation of Cognitive Deficits in Rats.

The multifactorial origin and neurochemistry of Alzheimer's disease (AD) call for the development of multitarget treatment strategies. We report a first-in-class triple acting compound that targets serotonin type 6 and 3 receptors (5-HT-Rs) and monoamine oxidase type B (MAO-B) as an approach for treating AD. The key structural features required for MAO-B inhibition and 5-HT6R antagonism and interaction with 5-HT3R were determined using molecular dynamic simulations and cryo-electron microscopy, respectively. Bioavailable PZ-1922 reversed scopolamine-induced cognitive deficits in the novel object recognition test. Furthermore, it displayed superior pro-cognitive properties compared to intepirdine (a 5-HT6R antagonist) in the AD model, which involved intracerebroventricular injection of an oligomeric solution of amyloid-ß peptide (oAß) in the T-maze test in rats. PZ-1922, but not intepirdine, restored levels of biomarkers characteristic of the debilitating effects of oAß. These data support the potential of a multitarget approach involving the joint modulation of 5-HT6R/5-HT3R/MAO-B in AD.

Development of hybrid bioactive nanofibers composed of star Poly(lactic acid) and gelatin by sol-gel crosslinking during the electrospinning process.

The design of a biomimetic scaffold is a major challenge in tissue engineering to promote tissue reconstruction. The use of synthetic polymer nanofibers is widely described as they provide biocompatible matrices whose topography mimics natural extracellular matrix (ECM). To closely match the biochemical composition of the ECM, bioactive molecules such as gelatin are added to the nanofibers to enhance cell adhesion and proliferation. To overcome the rapid solubilization of gelatin in biological fluids and to allow a lasting biological effect, the covalent crosslinking of this macromolecule in the network is crucial. The sol-gel route offers the possibility of gentle crosslinking during shaping but is rarely combined with electrospinning. In this study, we present the creation of Poly(lactic acid)/Gelatin hybrid nanofibers by sol-gel route during electrospinning. To enable sol-gel crosslinking, we synthesized star-shaped PLA and functionalized it with silane groups; then we functionalized gelatin with the same groups for their subsequent reaction with the polymer and thus the creation of the hybrid nanonetwork. We evaluated the impact of the presence of gelatin in Poly(lactic acid)/Gelatin hybrid nanofibers at different percentages on the mechanical properties, nanonetwork crosslinking, degradation and biological properties of the hybrid nanofibers. The addition of gelatin modulated nanonetwork crosslinking that impacted the stiffness of the nanofibers, resulting in softer materials for the cells. Moreover, these hybrid nanofibers also showed a significant improvement in fibroblast proliferation and present a degradation rate suitable for tissue reconstruction. Finally, the bioactive hybrid nanofibers possess versatile properties, interesting for various potential applications in tissue reconstruction.

3D high-precision melt electro written polycaprolactone modified with yeast derived peptides for wound healing.

In this study melt electro written (MEW) scaffolds of poly(ε-caprolactone) PCL are decorated with anti-inflammatory yeast-derived peptide for skin wound healing. Initially, 13 different yeast-derived peptides were screened and analyzed using both in vitro and in vivo assays. The MEW scaffolds are functionalized with the selected peptide VLSTSFPPW (VW-9) with the highest activity in reducing pro-inflammatory cytokines and stimulating fibroblast proliferation, migration, and collagen production. The peptide was conjugated to the MEW scaffolds using carbodiimide (CDI) and thiol chemistry, with and without plasma treatment, as well as by directly mixing the peptide with the polymer before printing. The MEW scaffolds modified using CDI and thiol chemistry with plasma treatment showed improved fibroblast and macrophage penetration and adhesion, as well as increased cell proliferation and superior anti-inflammatory properties, compared to the other groups. When applied to full-thickness excisional wounds in rats, the peptide-modified MEW scaffold significantly enhanced the healing process compared to controls (p < 0.05). This study provides proof of concept for using yeast-derived peptides to functionalize biomaterials for skin wound healing.

Amelogenin-Derived Peptide (ADP-5) Hydrogel for Periodontal Regeneration: An In Vitro Study on Periodontal Cells Cytocompatibility, Remineralization and Inflammatory Profile.

A relevant alternative to enamel matrix derivatives from animal origin could be the use of synthetic amelogenin-derived peptides. This study aimed to assess the effect of a synthetic amelogenin-derived peptide (ADP-5), alone or included in an experimental gellan-xanthan hydrogel, on periodontal cell behavior (gingival fibroblasts, periodontal ligament cells, osteoblasts and cementoblasts). The effect of ADP-5 (50, 100, and 200 µg/mL) on cell metabolic activity was examined using Alamar blue assay, and cell morphology was assessed by confocal imaging. An experimental gellan-xanthan hydrogel was then designed as carrier for ADP-5 and compared to the commercial gel Emdogain®. Alizarin Red was used to determine the periodontal ligament and cementoblasts cell mineralization. The inflammatory profile of these two cells was also quantified using ELISA (vascular endothelial growth factor A, tumor necrosis factor α, and interleukin 11) mediators. ADP-5 enhanced cell proliferation and remineralization; the 100 µg/mL concentration was more efficient than 50 and 200 µg/mL. The ADP-5 experimental hydrogel exhibited equivalent good biological behavior compared to Emdogain® in terms of cell colonization, mineralization, and inflammatory profile. These findings revealed relevant insights regarding the ADP-5 biological behavior. From a clinical perspective, these outcomes could instigate the development of novel functionalized scaffold for periodontal regeneration.

Gelatin modified with alkoxysilanes (GelmSi) forms hybrid hydrogels for bioengineering applications.

Biopolymers are ideal candidates for the development of hydrogels for tissue engineering applications. However, chemical modifications are required to further improve their mechanical properties, in particular to cross-link them for long-lasting applications or biofabrication. Herein, we developed a novel gelatin-based hydrogel precursor, "GelmSi" which consist on modified gelatin with triethoxysilyl groups. Gelatin was chosen as starting material because of its biocompatibility and bioactivity, favouring cell adhesion and migration. Alkoxysilane moieties were introduced in a controlled manner on the lysine side chains of gelatin to obtain a hybrid precursor which reacts in physiological conditions, forming covalent siloxane bonds and allowing the formation of a three-dimensional chemical network. On the contrary to unmodified gelatin, siloxane covalent network dramatically increases the stiffness and the thermal stability of the resulting gelatin-based hydrogel, making it suitable for cell encapsulation and cell culture. The biorthogonality and versatility of the GelmSi hybrid hydrogel unlock a broad range of gelatin-based bioengineering applications.

Silylated biomolecules: Versatile components for bioinks.

Physical hydrogels prepared from natural biopolymers are the most popular components for bioinks. However, to improve the mechanical properties of the network, in particular its durability for long-lasting tissue engineering applications or its stiffness for bone/cartilage applications, covalent chemical hydrogels have to be considered. For that purpose, biorthogonal reactions are required to allow the inclusion of living cells within the bioink reservoir before the 3D printing procedure. Interestingly, such reactions also unlock the possibility to further multifunctionalize the network, adding bioactive moieties to tune the biological properties of the resulting printed biomaterial. Surprisingly, compared to the huge number of studies disclosing novel bioink compositions, no extensive efforts have been made by the scientific community to develop new chemical reactions meeting the requirements of both cell encapsulation, chemical orthogonality and versatile enough to be applied to a wide range of molecular components, including fragile biomolecules. That could be explained by the domination of acrylate photocrosslinking in the bioprinting field. On the other hand, proceeding chemoselectively and allowing the polymerization of any type of silylated molecules, the sol-gel inorganic polymerization was used as a crosslinking reaction to prepare hydrogels. Recent development of this strategy includes the optimization of biocompatible catalytic conditions and the silylation of highly attractive biomolecules such as amino acids, bioactive peptides, proteins and oligosaccharides. When one combines the simplicity and the versatility of the process, with the ease of functionalization of any type of relevant silylated molecules that can be combined in an infinite manner, it was obvious that a family of bioinks could emerge quickly. This review presents the sol-gel process in biocompatible conditions and the various classes of relevant silylated molecules that can be used as bioink components. The preparation of hydrogels and the kinetic considerations of the sol-gel chemistry which at least allowed cell encapsulation and extrusion-based bioprinting are discussed.

Development of Amino Acids Functionalized SBA-15 for the Improvement of Protein Adsorption.

Ordered mesoporous materials and their modification with multiple functional groups are of wide scientific interest for many applications involving interaction with biological systems and biomolecules (e.g., catalysis, separation, sensor design, nano-science or drug delivery). In particular, the immobilization of enzymes onto solid supports is highly attractive for industry and synthetic chemistry, as it allows the development of stable and cheap biocatalysts. In this context, we developed novel silylated amino acid derivatives (Si-AA-NH2) that have been immobilized onto SBA-15 materials in biocompatible conditions avoiding the use of toxic catalyst, solvents or reagents. The resulting amino acid-functionalized materials (SBA-15@AA) were characterized by XRD, TGA, EA, Zeta potential, nitrogen sorption and FT-IR. Differences of the physical properties (e.g., charges) were observed while the structural ones remained unchanged. The adsorption of the enzyme lysozyme (Lyz) onto the resulting functionalized SBA-15@AA materials was evaluated at different pHs. The presence of different functional groups compared with bare SBA-15 showed better adsorption results, for example, 79.6 nmol of Lyz adsorbed per m2 of SBA-15@Tyr compared with the 44.9 nmol/m2 of the bare SBA-15.

Structure-Based Design and Optimization of FPPQ, a Dual-Acting 5-HT3 and 5-HT6 Receptor Antagonist with Antipsychotic and Procognitive Properties.

In line with recent clinical trials demonstrating that ondansetron, a 5-HT3 receptor (5-HT3R) antagonist, ameliorates cognitive deficits of schizophrenia and the known procognitive effects of 5-HT6 receptor (5-HT6R) antagonists, we applied the hybridization strategy to design dual-acting 5-HT3/5-HT6R antagonists. We identified the first-in-class compound FPPQ, which behaves as a 5-HT3R antagonist and a neutral antagonist 5-HT6R of the Gs pathway. FPPQ shows selectivity over 87 targets and decent brain penetration. Likewise, FPPQ inhibits phencyclidine (PCP)-induced hyperactivity and displays procognitive properties in the novel object recognition task. In contrast to FPPQ, neither 5-HT6R inverse agonist SB399885 nor neutral 5-HT6R antagonist CPPQ reversed (PCP)-induced hyperactivity. Thus, combination of 5-HT3R antagonism and 5-HT6R antagonism, exemplified by FPPQ, contributes to alleviating the positive-like symptoms. Present findings reveal critical structural features useful in a rational polypharmacological approach to target 5-HT3/5-HT6 receptors and encourage further studies on dual-acting 5-HT3/5-HT6R antagonists for the treatment of psychiatric disorders.

Neuropathic pain-alleviating activity of novel 5-HT6 receptor inverse agonists derived from 2-aryl-1H-pyrrole-3-carboxamide.

The diverse signaling pathways engaged by serotonin type 6 receptor (5-HT6R) together with its high constitutive activity suggests different types of pharmacological interventions for the treatment of CNS disorders. Non-physiological activation of mTOR kinase by constitutively active 5-HT6R under neuropathic pain conditions focused our attention on the possible repurposing of 5-HT6R inverse agonists as a strategy to treat painful symptoms associated with neuropathies of different etiologies. Herein, we report the identification of compound 33 derived from the library of 2-aryl-1H-pyrrole-3-carboxamides as a potential analgesic agent. Compound 33 behaves as a potent 5-HT6R inverse agonist at Gs, Cdk5, and mTOR signaling. Preliminary ADME/Tox studies revealed preferential distribution of 33 to the CNS and placed it in the low-risk safety space. Finally, compound 33 dose-dependently reduced tactile allodynia in spinal nerve ligation (SNL)-induced neuropathic rats.

A Collagen-Mimetic Organic-Inorganic Hydrogel for Cartilage Engineering.

Promising strategies for cartilage regeneration rely on the encapsulation of mesenchymal stromal cells (MSCs) in a hydrogel followed by an injection into the injured joint. Preclinical and clinical data using MSCs embedded in a collagen gel have demonstrated improvements in patients with focal lesions and osteoarthritis. However, an improvement is often observed in the short or medium term due to the loss of the chondrocyte capacity to produce the correct extracellular matrix and to respond to mechanical stimulation. Developing novel biomimetic materials with better chondroconductive and mechanical properties is still a challenge for cartilage engineering. Herein, we have designed a biomimetic chemical hydrogel based on silylated collagen-mimetic synthetic peptides having the ability to encapsulate MSCs using a biorthogonal sol-gel cross-linking reaction. By tuning the hydrogel composition using both mono- and bi-functional peptides, we succeeded in improving its mechanical properties, yielding a more elastic scaffold and achieving the survival of embedded MSCs for 21 days as well as the up-regulation of chondrocyte markers. This biomimetic long-standing hybrid hydrogel is of interest as a synthetic and modular scaffold for cartilage tissue engineering.

2-Phenyl-1H-pyrrole-3-carboxamide as a New Scaffold for Developing 5-HT6 Receptor Inverse Agonists with Cognition-Enhancing Activity.

Serotonin type 6 receptor (5-HT6R) has gained particular interest as a promising target for treating cognitive deficits, given the positive effects of its antagonists in a wide range of memory impairment paradigms. Herein, we report on degradation of the 1H-pyrrolo[3,2-c]quinoline scaffold to provide the 2-phenyl-1H-pyrrole-3-carboxamide, which is devoid of canonical indole-like skeleton and retains recognition of 5-HT6R. This modification has changed the compound's activity at 5-HT6R-operated signaling pathways from neutral antagonism to inverse agonism. The study identified compound 27 that behaves as an inverse agonist of the 5-HT6R at the Gs and Cdk5 signaling pathways. Compound 27 showed high selectivity and metabolic stability and was brain penetrant. Finally, 27 reversed scopolamine-induced memory decline in the novel object recognition test and exhibited procognitive properties in the attentional set-shifting task in rats. In light of these findings, 27 might be considered for further evaluation as a new cognition-enhancing agent, while 2-phenyl-1H-pyrrole-3-carboxamide might be used as a template for designing 5-HT6R inverse agonists.

Sol-gel process: the inorganic approach in protein imprinting.

Proteins play a central role in the signal transmission in living systems since they are able to recognize specific biomolecules acting as cellular receptors, antibodies or enzymes, being themselves recognized by other proteins in protein/protein interactions, or displaying epitopes suitable for antibody binding. In this context, the specific recognition of a given protein unlocks a range of interesting applications in diagnosis and in targeted therapies. Obviously, this role is already fulfilled by antibodies with unquestionable success. However, the design of synthetic artificial systems able to endorse this role is still challenging with a special interest to overcome limitations of antibodies, in particular their production and their stability. Molecular Imprinted Polymers (MIPs) are attractive recognition systems which could be an alternative for the specific capture of proteins in complex biological fluids. MIPs can be considered as biomimetic receptors or antibody mimics displaying artificial paratopes. However, MIPs of proteins remains a challenge due to their large size and conformational flexibility, their complex chemical nature with multiple recognition sites and their low solubility in most organic solvents. Classical MIP synthesis conditions result in large polymeric cavities and unspecific binding sites on the surface. In this review, the potential of the sol-gel process as inorganic polymerization strategy to overcome the drawbacks of protein imprinting is highlighted. Thanks to the mild and biocompatible experimental conditions required and the use of water as a solvent, the inorganic polymerization approach better suited to proteins than organic polymerization. Through numerous examples and applications of MIPs, we proposed a critical evaluation of the parameters that must be carefully controlled to achieve sol-gel protein imprinting (SGPI), including the choice of the monomers taking part in the polymerization.

Design of PEGylated Three Ligands Silica Nanoparticles for Multi-Receptor Targeting.

The synthesis of silica nanoparticles (SiNPs) decorated on their surface with a range of various elements (e.g., ligands, drugs, fluorophores, vectors, etc.) in a controlled ratio remains a big challenge. We have previously developed an efficient strategy to obtain in one-step, well-defined multifunctional fluorescent SiNPs displaying fluorophores and two peptides ligands as targeting elements, allowing selective detection of cancer cells. In this paper, we demonstrate that additional level of controlled multifunctionality can be achieved, getting even closer to the original concept of "magic bullet", using solely sol-gel chemistry to achieve conjugation of PEG chains for stealth, along with three different ligands. In addition, we have answered the recurrent question of the surface ungrafting by investigating the stability of different siloxane linkages with the ERETIC Method (Electronic Reference to Access In Vivo Concentrations) by 19F NMR quantification. We also compared the efficiency of the hybrid silylated fluorophore covalent linkage in the core of the SiNP to conventional methods. Finally, the tumor-cell-targeting efficiency of these multi-ligand NPs on human endothelial cells (HUVEC or HDMEC) and mixed spheroids of human melanoma cells and HUVEC displaying different types of receptors were evaluated in vitro.

Epimerization-Free C-Term Activation of Peptide Fragments by Ball Milling.

Peptides were produced in high yields and, if any, very low epimerization, by mechanochemical coupling of peptide fragments containing highly epimerization-prone and/or highly hindered amino acids at C-term. Ball milling was clearly identified as the key element enabling one to obtain such results.

Encapsulation of BSA in hybrid PEG hydrogels: stability and controlled release.

Hybrid hydrogels based on silylated polyethylene glycol, Si-PEG, were evaluated as hybrid matrices able to trap, stabilize and release bovine serum albumin (BSA) in a controlled manner. Parameters of the inorganic condensation reaction leading to a siloxane (Si-O-Si) three dimensional network were carefully investigated, in particular the temperature, the surrounding hygrometry and the Si-PEG concentration. The resulting hydrogel structural features affected the stability, swelling, and mechanical properties of the network, leading to different protein release profiles. Elongated polymer assemblies were observed, the length of which ranged from 150 nm to over 5 µm. The length could be correlated to the Si-O-Si condensation rate from 60% (hydrogels obtained at 24 °C) to about 90% (xerogels obtained at 24 °C), respectively. Consequently, the controlled release of BSA could be achieved from hours to several weeks, with respect to the fibers' length and the condensation rate. The protein stability was evaluated by means of a thermal study. The main results gave insight into the biomolecule structure preservation during polymerisation, with ΔG < 0 for encapsulated BSA in any conditions, below the melting temperature (65 °C).

Hydrocarbon-Stapled Peptide Based-Nanoparticles for siRNA Delivery.

Small interfering RNAs (siRNAs) are promising molecules for developing new therapies based on gene silencing; however, their delivery into cells remains an issue. In this study, we took advantage of stapled peptide technology that has emerged as a valuable strategy to render natural peptides more structured, resistant to protease degradation and more bioavailable, to develop short carriers for siRNA delivery. From the pool of stapled peptides that we have designed and synthesized, we identified non-toxic vectors that were able to efficiently encapsulate siRNA, transport them into the cell and induce gene silencing. Remarkably, the most efficient stapled peptide (JMV6582), is composed of only eight amino-acids and contains only two cationic charges.

Direct Synthesis of Peptide-Containing Silicones: A New Way to Bioactive Materials.

A simple and efficient way to synthesize peptide-containing silicone materials is described. Silicone oils containing a chosen ratio of bioactive peptide sequences were prepared by acid-catalyzed copolymerization of dichlorodimethylsilane, hybrid dichloromethyl peptidosilane, and Si(vinyl)- or SiH-functionalized monomers. Functionalized silicone oils were first obtained and then, after hydrosilylation cross-linking, bioactive polydimethylsiloxane (PDMS)-based materials were straightforwardly obtained. The introduction of an antibacterial peptide yielded PDMS materials showing activity against Staphylococcus aureus. PDMS containing RGD ligands showed improved cell-adhesion properties. This generic method was fully compatible with the stability of peptides and thus opened the way to the synthesis of a wide range of biologically active silicones.

Correction to: Real-time shear wave ultrasound elastography: a new tool for the evaluation of diaphragm and limb muscle stiffness in critically ill patients.

In the publication of this article [1], there was an error in the Family Name of one of the authors. This has now been updated in the original article.