Enhancing the Activity of Surface Immobilized Antimicrobial Peptides Using Thiol-Mediated Tethering to Poly(ethylene glycol).

Considering the need for versatile surface coatings that can display multiple bioactive signals and chemistries, the use of more novel surface modification methods is starting to emerge. Thiol-mediated conjugation of biomolecules is shown to be quite advantageous for such purposes due to the reactivity and chemoselectivity of thiol functional groups. Herein, the immobilization of poly(ethylene glycol) (PEG) and antimicrobial peptides (AMPs) to silica colloidal particles based on thiol-mediated conjugation techniques, along with an assessment of the antimicrobial potential of the functionalized particles against Pseudomonas aeruginosa and Staphylococcus aureus is investigated. Immobilization of PEG to thiolated Si particles is performed by either a two-step thiol-ene "photo-click" reaction or a "one-pot" thiol-maleimide type conjugation using terminal acrylate or maleimide functional groups, respectively. It is demonstrated that both immobilization methods result in a significant reduction in the number of viable bacterial cells compared to unmodified samples after the designated incubation periods with the PEG-AMP-modified colloidal suspensions. These findings provide a promising outlook for the fabrication of multifunctional surfaces based upon the tethering of PEG and AMPs to colloidal particles through thiol-mediated biocompatible chemistry, which has potential for use as implant coatings or as antibacterial formulations that can be incorporated into wound dressings to prevent or control bacterial infections.

Chitosan-Based Biomaterials: Insights into Chemistry, Properties, Devices, and Their Biomedical Applications.

Chitosan is a marine-origin polysaccharide obtained from the deacetylation of chitin, the main component of crustaceans' exoskeleton, and the second most abundant in nature. Although this biopolymer has received limited attention for several decades right after its discovery, since the new millennium chitosan has emerged owing to its physicochemical, structural and biological properties, multifunctionalities and applications in several sectors. This review aims at providing an overview of chitosan properties, chemical functionalization, and the innovative biomaterials obtained thereof. Firstly, the chemical functionalization of chitosan backbone in the amino and hydroxyl groups will be addressed. Then, the review will focus on the bottom-up strategies to process a wide array of chitosan-based biomaterials. In particular, the preparation of chitosan-based hydrogels, organic-inorganic hybrids, layer-by-layer assemblies, (bio)inks and their use in the biomedical field will be covered aiming to elucidate and inspire the community to keep on exploring the unique features and properties imparted by chitosan to develop advanced biomedical devices. Given the wide body of literature that has appeared in past years, this review is far from being exhaustive. Selected works in the last 10 years will be considered.

Synthesis and Characterization of Alkoxysilane-Bearing Photoreversible Cinnamic Side Groups: A Promising Building-Block for the Design of Multifunctional Silica Nanoparticles.

The present study reports on the synthesis of a new alkoxysilane-bearing light-responsive cinnamyl group and its application as a surface functionalization agent for the development of SiO2 nanoparticles (NPs) with photoreversible tails. In detail, cinnamic acid (CINN) was activated with N-hydroxysuccinimide (NHS) to obtain the corresponding NHS-ester (CINN-NHS). Subsequently, the amine group of 3-aminopropyltriethoxysilane (APTES) was acylated with CINN-NHS leading to the generation of a novel organosilane, CINN-APTES, which was then exploited for decorating SiO2 NPs. The covalent bond to the silica surface was confirmed by solid state NMR, whereas thermogravimetric analysis unveiled a functionalization degree much higher compared to that achieved by a conventional double-step post-grafting procedure. In light of these intriguing results, the strategy was successfully extended to naturally occurring sepiolite fibers, widely employed as fillers in technological applications. Finally, a preliminary proof of concept of the photoreversibility of the obtained SiO2@CINN-APTES system has been carried out through UV diffuse reflectance. The overall outcomes prove the consistency and the versatility of the methodological protocol adopted, which appears promising for the design of hybrid NPs to be employed as building blocks for photoresponsive materials with the ability to change their molecular structure and subsequent properties when exposed to different light stimuli.

Fishery waste valorization: Sulfated ZrO2 as a heterogeneous catalyst for chitin and chitosan depolymerization.

Chitin and chitosan are abundant unique sources of biologically-fixed nitrogen mainly derived from residues of the fishery productive chain. Their high potential as nitrogen-based highly added-value platform molecules is still largely unexploited and a catalytic way for their valorization would be strongly desirable within a biorefinery concept. Here we report our results obtained with a series of heterogeneous catalysts in the depolymerization of chitosan and chitin to acetylglucosamine. Copper catalysts supported on SiO2, SiO2-Al2O3, SiO2-ZrO2, ZrO2 and the corresponding bare oxides/mixed oxides were tested, together with a sulfated zirconia system (ZrO2-SO3H) that revealed to be extremely selective towards glucosamine, both for chitosan and chitin, thus giving pretty high yields with respect to the values reported so far (44% and 21%, respectively). The use of a heterogeneous catalyst alone, without the need of any additives or the combination with a mineral acid, makes these results remarkable.

Fabrication Strategies Towards Hydrogels for Biomedical Application: Chemical and Mechanical Insights.

This review aims at giving selected chemical and mechanical insights on design criteria that should be taken into account in hydrogel production for biomedical applications. Particular emphasis will be given to the chemical aspects involved in hydrogel design: macromer chemical composition, cross-linking strategies and chemistry towards "conventional" and smart/stimuli responsive hydrogels. Mechanical properties of hydrogels in view of regenerative medicine applications will also be considered.

Galactose Tethered Decellularized Liver Matrix: Toward a Biomimetic and Biofunctional Matrix for Liver Tissue Engineering.

The major challenge in liver tissue engineering is the replication of the microenvironment and microarchitecture of the liver tissue at the nanoscale. Decellularized liver matrix (DLM) provides an ideal material for scaffold preparation, as it retains the relevant structural and biochemical composition. However, the loss of bioactive factors during decellularization needs to be taken into account when using DLM and should be supplemented accordingly for an expected outcome. This study reports on the modification of DLM by the addition of galactose residues using a two-step thiol-ene-mediated photoclick chemistry for the coupling of galactose moieties to the DLM. Modification with galactose enhanced the function of hepatocytes and provides many advantages over currently used DLM and DLM-based materials. The galactose modified DLM enhanced the initial HepG2 cell adhesion to the substrate with changes in dynamics over time such as spheroid formation and further migration on the matrix. Our observation is that the galactose ligand decoration can also enhance the liver-specific metabolism of HepG2 compared to unmodified DLM. Galactosylated DLM also showed a better establishment of cellular polarity which also contributes to the function of HepG2 cells. Together our results demonstrate the advantages of adding galactose residues to currently available biomaterials, which makes this approach an attractive method for ECM-based liver tissue engineering.

Squarate Cross-Linked Gelatin Hydrogels as Three-Dimensional Scaffolds for Biomedical Applications.

Hydrogels are useful platforms as three-dimensional (3D) scaffolds for cell culture, drug-release systems, and regenerative medicine applications. Here, we propose a novel chemical cross-linking approach by the use of 3,4-diethoxy-3-cyclobutene-1,2-dione or diethyl squarate for the preparation of 5 and 10% w/v gelatin-based hydrogels. Hydrogels showed good swelling properties, and the 5% gelatin-based hydrogel proved suitable as a 3D cell culture scaffold for the chondrocyte cell line C28/I2. In addition, diffusion properties of different sized molecules inside the hydrogel were determined.

Photoinduced Porcine Gelatin Cross-Linking by Homobi- and Homotrifunctional Tetrazoles.

Gelatin is a costless polypeptide material of natural origin, able to form hydrogels that are potentially useful in biomaterial scaffold design for drug delivery, cell cultures, and tissue engineering. However, gelatin hydrogels are unstable at physiological conditions, losing their features only after a few minutes at 37 °C. Accordingly, treatments to address this issue are of great interest. In the present work, we propose for the first time the use of bi- and trifunctional tetrazoles, most of them unknown to date, for photoinduced gelatin cross-linking towards the production of physiologically stable hydrogels. Indeed, after UV-B irradiation, aryl tetrazoles generate a nitrilimine intermediate that is reactive towards different functionalities, some of them constitutively present in the amino acid side chains of gelatin. The efficacy of the treatment strictly depends on the structure of the cross-linking agent used, and substantial improved stability was observed by switching from bifunctional to trifunctional cross-linkers.

Differential glycosylation of collagen modulates lung cancer stem cell subsets through ß1 integrin-mediated interactions.

In lung cancer, CD133+ cells represent the subset of cancer stem cells (CSC) able to sustain tumor growth and metastatic dissemination. CSC function is tightly regulated by specialized niches composed of both stromal cells and extracellular matrix (ECM) proteins, mainly represented by collagen. The relevance of collagen glycosylation, a fundamental post-translational modification controlling several biological processes, in regulating tumor cell phenotype remains, however, largely unexplored. To investigate the bioactive effects of differential ECM glycosylation on lung cancer cells, we prepared collagen films functionalized with glucose (Glc-collagen) and galactose (Gal-collagen) exploiting a neoglycosylation approach based on a reductive amination of maltose and lactose with the amino residues of collagen lysines. We demonstrate that culturing of tumor cells on collagen determines a glycosylation-dependent positive selection of CSC and triggers their expansion/generation. The functional relevance of CD133+ CSC increase was validated in vivo, proving an augmented tumorigenic and metastatic potential. High expression of integrin ß1 in its active form is associated with an increased proficiency of tumor cells to sense signaling from glycosylated matrices (glyco-collagen) and to acquire stemness features. Accordingly, inhibition of integrin ß1 in tumor cells prevents CSC enrichment, suggesting that binding of integrin ß1 to Glc-collagen subtends CSC expansion/generation. We provide evidence suggesting that collagen glycosylation could play an essential role in modulating the creation of a niche favorable for the generation and selection/survival of lung CSC. Interfering with this crosstalk may represent an innovative therapeutic strategy for lung cancer treatment.

Synthesis, Molecular Modeling and Biological Evaluation of Metabolically Stable Analogues of the Endogenous Fatty Acid Amide Palmitoylethanolamide.

Palmitoylethanolamide (PEA) belongs to the class of N-acylethanolamine and is an endogenous lipid potentially useful in a wide range of therapeutic areas; products containing PEA are licensed for use in humans as a nutraceutical, a food supplement, or food for medical purposes for its analgesic and anti-inflammatory properties demonstrating efficacy and tolerability. However, the exogenously administered PEA is rapidly inactivated; in this process, fatty acid amide hydrolase (FAAH) plays a key role both in hepatic metabolism and in intracellular degradation. So, the aim of the present study was the design and synthesis of PEA analogues that are more resistant to FAAH-mediated hydrolysis. A small library of PEA analogues was designed and tested by molecular docking and density functional theory calculations to find the more stable analogue. The computational investigation identified RePEA as the best candidate in terms of both synthetic accessibility and metabolic stability to FAAH-mediated hydrolysis. The selected compound was synthesized and assayed ex vivo to monitor FAAH-mediated hydrolysis and to confirm its anti-inflammatory properties. 1H-NMR spectroscopy performed on membrane samples containing FAAH in integral membrane protein demonstrated that RePEA is not processed by FAAH, in contrast with PEA. Moreover, RePEA retains PEA's ability to inhibit LPS-induced cytokine release in both murine N9 microglial cells and human PMA-THP-1 cells.

Neoglycosylated Collagen: Effect on Neuroblastoma F-11 Cell Lines.

The regeneration of the nervous system is a challenging task. Currently, regenerative medicine approaches that exploit nature-inspired cues are being studied and hold great promise. The possibility to use protein-based matrices functionalized with small oligo- and monosaccharides is of interest since these can be finely tuned to better mimic the native environment. Collagen has been selected as a promising material that has the potential to be further tailored to incorporate carbohydrates in order to drive cell behavior towards neuroregeneration. Indeed, the grafting of carbohydrates to collagen 2D matrices is proved to enhance its biological significance. In the present study, collagen 2D matrices were grafted with different carbohydrate epitopes, and their potential to drive F-11 neuroblastoma cells towards neuronal differentiation was evaluated. Collagen functionalized with α-glucosides was able to differentiate neuroblastoma cells into functional neurons, while sialyl α-(2→6)-galactosides stimulated cell proliferation.

Histological validation of adipogenic differentiation potential of ASC on collagen-based 2D scaffolds.

Determination of the adipogenic potential and behavior of adipose-derived mesenchymal stem/stromal cells (ASCs) is particularly relevant for their potential clinical application in regenerative medicine, especially when regeneration is supported by biomaterials or scaffolds. Scaffolds need to be able to induce tissue repair and limit undesired adipogenic differentiation. Depending on the scaffold employed, determination of cell behavior may be hindered by material interference with staining, which will limit either cells identification or dye quantification. Collagen is a promising biomaterial in regenerative medicine, however, histological analysis of cells cultured on collagen-based scaffolds is challenging. Here we describe a new histological method based on iron hematoxylin combined with Oil red O (ORO) staining, for the determination of the adipogenic differentiation of ASCs cultivated on a collagen-based 2D scaffold. ASCs were seeded on collagen films or plastic, differentiated into adipocytes for 14 days, and then stained with either ORO or iron hematoxylin and ORO combined. The collagen films avidly absorbed the ORO dye; conventional staining and quantification by dye extraction failed to discriminate between differentiated and undifferentiated cells on the films. On the contrary, the iron hematoxylin-ORO combination provided a quantitative and more reliable determination of adipocytes based on single cell count. This method is particularly recommended for determining the adipogenic differentiation potential of ASCs and other cell types grown on highly absorptive materials that need to be validated for their potential use in bioengineering and regenerative medicine.

Gelatin-Based Hydrogels for the Controlled Release of 5,6-Dihydroxyindole-2-Carboxylic Acid, a Melanin-Related Metabolite with Potent Antioxidant Activity.

The ability of gelatin-based hydrogels of incorporating and releasing under controlled conditions 5,6-dihydroxyindole-2-carboxylic acid (DHICA), a melanin-related metabolite endowed with marked antioxidant properties was investigated. The methyl ester of DHICA, MeDHICA, was also tested in view of its higher stability, and different solubility profile. Three types of gelatin-based hydrogels were prepared: pristine porcine skin type A gelatin (HGel-A), a pristine gelatin cross-linked by amide coupling of lysines and glutamic/aspartic acids (HGel-B), and a gelatin/chitosan blend (HGel-C). HGel-B and HGel-C differed in the swelling behavior, showed satisfactorily high mechanical strength at physiological temperatures and well-defined morphology. The extent of incorporation into all the gelatins tested using a 10% w/w indole to gelatin ratio was very satisfactory ranging from 60 to 90% for either indoles. The kinetics of indole release under conditions of physiological relevance was evaluated up to 72 h. The highest values were obtained with HGel-B and HGel-C for MeDHICA (90% after 6 h), and an appreciable release was observed for DHICA reaching 30% and 40% at 6 h for HGel-B and HGel-C, respectively. At 72 h, DHICA and MeDHICA were released at around 30% from HGel-A at pH 7.4, with an increase up to 40% at pH 5.5 in the case of DHICA. DHICA incorporated into HGel-B proved fairly stable over 6 h whereas the free compound at the same concentration was almost completely oxidized. The antioxidant power of the indole loaded gelatins was monitored by chemical assays and proved unaltered even after prolonged storage in air, suggesting that the materials could be prepared in advance with respect to their use without alteration of their efficacy.

Glycan Carriers As Glycotools for Medicinal Chemistry Applications.

Carbohydrates are one of the most powerful and versatile classes of biomolecules that nature uses to regulate organisms' biochemistry, modulating plenty of signaling events within cells, triggering a plethora of physiological and pathological cellular behaviors. In this framework, glycan carrier systems or carbohydrate-decorated materials constitute interesting and relevant tools for medicinal chemistry applications. In the last few decades, efforts have been focused, among others, on the development of multivalent glycoconjugates, biosensors, glycoarrays, carbohydrate-decorated biomaterials for regenerative medicine, and glyconanoparticles. This review aims to provide the reader with a general overview of the different carbohydrate carrier systems that have been developed as tools in different medicinal chemistry approaches relying on carbohydrate-protein interactions. Given the extent of this topic, the present review will focus on selected examples that highlight the advancements and potentialities offered by this specific area of research, rather than being an exhaustive literature survey of any specific glyco-functionalized system.

Gelatin-Based Hydrogels through Homobifunctional Triazolinediones Targeting Tyrosine Residues.

Gelatin is a biopolymer with interesting properties that can be useful for biomaterial design for different applications such as drug delivery systems, or 3D scaffolds for tissue engineering. However, gelatin suffers from poor mechanical stability at physiological temperature, hence methods for improving its properties are highly desirable. In the present work, a new chemical cross-linking strategy based on triazolinedione ene-type chemistry towards stable hydrogel is proposed. Two different homobifunctional 1,2,4-triazoline-3,5(4H)-diones, namely 4,4'-hexane-1,6-diylbis(3H-1,2,4-triazoline-3,5(4H)-dione) 1 and 4,4'-[methylenebis(4,1-phenylene)]bis(3H-1,2,4-triazoline-3,5(4H)-dione) 2 were used as cross-linkers in different ratio to tyrosine residues in gelatin. The reaction was proved effective in all experimented conditions and hydrogels featured with different thermal stability were obtained. In general, the higher the cross-linker/tyrosine ratio, the more thermostable the hydrogel. The swelling properties are strictly dependent upon the chemical nature of the cross-linker.

Towards hydrophobic carminic acid derivatives and their incorporation in polyacrylates.

Carminic acid, a natural hydrophilic dye extensively used in the food and cosmetic industries, is converted in hydrophobic dyes by acetylation or pivaloylation. These derivatives are successfully used as biocolourants for rubber objects. In this paper, spectroscopic properties of the carminic acid derivatives in dimethyl sulfoxide and in polybutylacrylate are studied by means of photoluminescence and time-resolved photoluminescence decays, revealing a hypsochromic effect due to the presence of bulky substituents as the acetyl or pivaloyl groups. Molecular mechanics and density functional theory calculations confirm the disruption of planarity between the sugar ring and the anthraquinoid system determined by the esterification.

Glycomics: New Challenges and Opportunities in Regenerative Medicine.

Tissue engineering relies on the possibility to engineer cell microenvironments by means of bioactive materials, biochemical and physical stimuli in order to guide cell behaviour and to regenerate damaged tissue. Despite the relevance of glycan epitopes as signaling molecules, and the recent advances in glycomics, their use as biomolecular cues at the interface between materials and cells for the controlled stimulation of adhesion and differentiation processes for regenerative medicine applications is still limited. In this concept article we will briefly outline the basis and the impact on health and economics of regenerative medicine, together with the recent applications of the glycocode in tissue regeneration approaches.

Synthetic sulfoglycolipids targeting the serine-threonine protein kinase Akt.

The serine-threonine protein kinase Akt, also known as protein kinase B, is a key component of the phosphoinositide 3-kinase (PI3K)-Akt-mTOR axis. Deregulated activation of this pathway is frequent in human tumors and Akt-dependent signaling appears to be critical in cell survival. PI3K activation generates 3-phosphorylated phosphatidylinositols that bind Akt pleckstrin homology (PH) domain. The blockage of Akt PH domain/phosphoinositides interaction represents a promising approach to interfere with the oncogenic potential of over-activated Akt. In the present study, phosphatidyl inositol mimics based on a ß-glucoside scaffold have been synthesized as Akt inhibitors. The compounds possessed one or two lipophilic moieties of different length at the anomeric position of glucose, and an acidic or basic group at C-6. Docking studies, ELISA Akt inhibition assays, and cellular assays on different cell models highlighted 1-O-octadecanoyl-2-O-ß-d-sulfoquinovopyranosyl-sn-glycerol as the best Akt inhibitor among the synthesized compounds, which could be considered as a lead for further optimization in the design of Akt inhibitors.

Multivalent ligand mimetics of LecA from P. aeruginosa: synthesis and NMR studies.

Molecular recognition of glycans plays an important role in glycomic and glycobiology studies. For example, pathogens have a number of different types of lectin for targeting host sugars. In bacteria, lectins exist sometimes as domains of bacterial toxins and exploit adhesion to glycoconjugates as a means of entering host cells. Herein, we describe the synthesis of three glycodendrons with the aim to dissect the fine structural details involved in the multivalent carbohydrate-protein interactions. LecA, from the pathogen Pseudomonas aeruginosa, has been used to characterize galactose dendrons interaction using one of the most widespread NMR technique for the elucidation of receptor-ligand binding in solution, the saturation transfer difference (STD) NMR. Furthermore, the effective hydrodynamic radius of each dendrimer recognized by LecA was estimated from the diffusion coefficients determined by pulsed-field-gradient stimulated echo (PFG-STE) NMR experiments.

The collaggrecan: Synthesis and visualization of an artificial proteoglycan.

An artificial aggrecan-like proteoglycan has been designed and synthesized in vitro. At variance with natural proteoglycans, whose glycosaminoglycan chains are always O-linked via a tetrasaccharide bridge to the serine residues of a specific protein core, the present structure consists of chondroitin-6-sulfate chains directly bound to the lysine and hydroxylysine residues of a collagen molecule backbone. The resulting macromolecule has been characterized by histochemistry, atomic force microscopy and FTIR. The number of variables involved (e.g., length and type of the collagen backbone, glycosaminoglycan species, sulfation type and pattern, molecular weight, number and length of side chains, etc.) makes possible to conceive an almost endless variety of artificial proteoglycans, each precisely tailored to a specific functional role. In addition to their use as biomaterials, glycated collagens interact with cells in complex ways and a previous study has already shown the ability of a glycated collagen to redirect fibroblastoma cells from proliferation to differentiation. The research is still underway.