Plasma-treated collagen functionalized with chondroitin sulfate as bioactive and nanostructured extracellular matrix mimics.

Aim: Cell microenvironment contains a plethora of information that influences cell modulation. Indeed, the extracellular matrix plays a central role in tissue development. Reproducing the cell-extracellular matrix crosstalk able to recapitulate both physical and biochemical signals is crucial to obtain functional tissue models or regenerative strategies. Materials & methods: Here, a combined method is proposed to easily functionalize collagen surface films, tailoring morphological properties. Oxygen nonthermal plasma treatment and glyco-conjugation with chondroitin sulfate are used to modify surface properties. Results: It results in higher adhesion, proliferation and morphological organization of U87 glioblastoma cells. Conclusion: Our finding suggests new promising strategies for the development of collagen-based biomaterials, which can be employed for advanced in vitro models.

Tyrosine glucosylation of collagen films exploiting Horseradish Peroxidase (HRP).

The development of human tissue models for regenerative medicine and animal-free drug screening requires glycosylated biomaterials such as collagen. An easy and fast biomaterial glycosylation method exploiting Horseradish Peroxidase (HRP) phenol coupling reaction is proposed. The protocol is adaptable to any polymer functionalized with phenol residues or tyrosine containing proteins. As a model the tyrosine residues on collagen films were functionalized with salidroside, a natural ß-glucoside with a phenol in the aglycone. Scanning Electron Microscope (SEM) and contact angle analysis revealed the influence of glycosylation on the sample's morphology and wettability. Preliminary biological evaluation showed the cytocompatibility of the glucosylated collagen films.

Alginate-Gelatin Self-Healing Hydrogel Produced via Static-Dynamic Crosslinking.

Alginate-gelatin hydrogels mimicking extracellular matrix (ECM) of soft tissues have been generated by static-dynamic double crosslinking, allowing fine control over the physical and chemical properties. Dynamic crosslinking provides self-healing and injectability attributes to the hydrogel and promotes cell migration and proliferation, while the static network improves stability. The static crosslinking was performed by enzymatic coupling of the tyrosine residues of gelatin with tyramine residues inserted in the alginate backbone, catalyzed by horseradish peroxidase (HRP). The dynamic crosslinking was obtained by functionalizing alginate with 3-aminophenylboronic acid which generates a reversible bond with the vicinal hydroxyl groups of the alginate chains. Varying the ratio of alginate and gelatin, hydrogels with different properties were obtained, and the most suitable for 3D soft tissue model development with a 2.5:1 alginate:gelatin molar ratio was selected. The selected hydrogel was characterized with a swelling test, rheology test, self-healing test and by cytotoxicity, and the formulation resulted in transparent, reproducible, varying biomaterial batch, with a fast gelation time and cell biocompatibility. It is able to modulate the loss of the inner structure stability for a longer time with respect to the formulation made with only covalent enzymatic crosslinking, and shows self-healing properties.

3D printed tissue models: From hydrogels to biomedical applications.

The development of new advanced constructs resembling structural and functional properties of human organs and tissues requires a deep knowledge of the morphological and biochemical properties of the extracellular matrices (ECM), and the capacity to reproduce them. Manufacturing technologies like 3D printing and bioprinting represent valuable tools for this purpose. This review will describe how morphological and biochemical properties of ECM change in different tissues, organs, healthy and pathological states, and how ECM mimics with the required properties can be generated by 3D printing and bioprinting. The review describes and classifies the polymeric materials of natural and synthetic origin exploited to generate the hydrogels acting as "inks" in the 3D printing process, with particular emphasis on their functionalization allowing crosslinking and conjugation with signaling molecules to develop bio-responsive and bio-instructive ECM mimics.

3D bioprinted colorectal cancer models based on hyaluronic acid and signalling glycans.

In cancer microenvironment, aberrant glycosylation events of ECM proteins and cell surface receptors occur. We developed a protocol to generate 3D bioprinted models of colorectal cancer (CRC) crosslinking hyaluronic acid and gelatin functionalized with three signalling glycans characterized in CRC, 3'-Sialylgalactose, 6'-Sialylgalactose and 2'-Fucosylgalactose. The crosslinking, performed exploiting azide functionalized gelatin and hyaluronic acid and 4arm-PEG-dibenzocyclooctyne, resulted in biocompatible hydrogels that were 3D bioprinted with commercial CRC cells HT-29 and patient derived CRC tumoroids. The glycosylated hydrogels showed good 3D printability, biocompatibility and stability over the time. SEM and synchrotron radiation SAXS/WAXS analysis revealed the influence of glycosylation in the construct morphology, whereas MALDI-MS imaging showed that protein profiles of tumoroid cells vary with glycosylation, indicating that sialylation and fucosylation of ECM proteins induce diverse alterations to the proteome of the tumoroid and surrounding cells.

Preventing extrinsic mechanisms of bioprosthetic degeneration using polyphenols.

OBJECTIVES: The purpose of this study was to evaluate the impact of a polyphenols-based treatment on the extrinsic mechanisms responsible for early bioprosthetic heart valve (BHV) degeneration. Structural degeneration can be driven by both extrinsic and intrinsic mechanisms. While intrinsic mechanisms have been associated with inherent biocompatibility characteristics of the BHV, the extrinsic ones have been reported to involve external causes, such as chemical, mechanical and hydrodynamic, responsible to facilitate graft damage. METHODS: The chemical interaction and the stability degree between polyphenols and pericardial tissue were carefully evaluated. The detoxification of glutaraldehyde in commercial BHVs models and the protective effect from in vivo calcification were taken into relevant consideration. Finally, the hydrodynamic and biomechanical features of the polyphenols-treated pericardial tissue were deeply investigated by pulse duplicator and stress-strain analysis. RESULTS: The study demonstrated the durability of the polyphenols-based treatment on pericardial tissue and the stability of the bound polyphenols. The treatment improves glutaraldehyde stabilization's current degree, demonstrating a surprising in vivo anti-calcific effect. It is able to make the pericardial tissue more pliable while maintaining the correct hydrodynamic characteristics. CONCLUSIONS: The polyphenols treatment has proved to be a promising approach capable of acting simultaneously on several factors related to the premature degeneration of cardiac valve substitutes by extrinsic mechanisms.

Multivalent γ-PGA-Exendin-4 Conjugates to Target Pancreatic ß-Cells.

Targeting of glucagon-like peptide 1 receptor (GLP-1R), expressed on the surface of pancreatic ß-cells, is of great interest for the development of advanced therapies for diabetes and diagnostics for insulinoma. We report the conjugation of exendin-4 (Ex-4), an approved drug to treat type 2 diabetes, to poly-γ-glutamic acid (γ-PGA) to obtain more stable and effective GLP-1R ligands. Exendin-4 modified at Lysine-27 with PEG4-maleimide was conjugated to γ-PGA functionalized with furan, in different molar ratios, exploiting a chemoselective Diels-Alder cycloaddition. The γ-PGA presenting the highest number of conjugated Ex-4 molecules (average 120 per polymeric chain) showed a double affinity towards GLP-1R with respect to exendin per se, paving the way to improved therapeutic and diagnostic applications.

Combined Analytical Approaches to Standardize and Characterize Biomaterials Formulations: Application to Chitosan-Gelatin Cross-Linked Hydrogels.

A protocol based on the combination of different analytical methodologies is proposed to standardize the experimental conditions for reproducible formulations of hybrid hydrogels. The final hybrid material, based on the combination of gelatin and chitosan functionalized with methylfuran and cross-linked with 4-arm-PEG-maleimide, is able to mimic role, dynamism, and structural complexity of the extracellular matrix. Physical-chemical properties of starting polymers and finals constructs were characterized exploiting the combination of HP-SEC-TDA, UV, FT-IR, NMR, and TGA.

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.

Design and Synthesis of Chitosan-Gelatin Hybrid Hydrogels for 3D Printable in vitro Models.

The development of 3D printable hydrogels based on the crosslinking between chitosan and gelatin is proposed. Chitosan and gelatin were both functionalized with methyl furan groups. Chemical modification was performed by reductive amination with methyl furfural involving the lysine residues of gelatin and the amino groups of chitosan to generate hydrogels with tailored properties. The methyl furan residues present in both polymers were exploited for efficient crosslinking via Diels-Alder ligation with PEG-Star-maleimide under cell-compatible conditions. The obtained chitosan-gelatin hybrid was employed to formulate hydrogels and 3D printable biopolymers and its processability and biocompatibility were preliminarily investigated.

3D Extracellular Matrix Mimics: Fundamental Concepts and Role of Materials Chemistry to Influence Stem Cell Fate.

Synthetic 3D extracellular matrices (ECMs) find application in cell studies, regenerative medicine, and drug discovery. While cells cultured in a monolayer may exhibit unnatural behavior and develop very different phenotypes and genotypes than in vivo, great efforts in materials chemistry have been devoted to reproducing in vitro behavior in in vivo cell microenvironments. This requires fine-tuning the biochemical and structural actors in synthetic ECMs. This review will present the fundamentals of the ECM, cover the chemical and structural features of the scaffolds used to generate ECM mimics, discuss the nature of the signaling biomolecules required and exploited to generate bioresponsive cell microenvironments able to induce a specific cell fate, and highlight the synthetic strategies involved in creating functional 3D ECM mimics.

Integration of nano- and biotechnology for beta-cell and islet transplantation in type-1 diabetes treatment.

Regenerative medicine using human or porcine ß-cells or islets has an excellent potential to become a clinically relevant method for the treatment of type-1 diabetes. High-resolution imaging of the function and faith of transplanted porcine pancreatic islets and human stem cell-derived beta cells in large animals and patients for testing advanced therapy medicinal products (ATMPs) is a currently unmet need for pre-clinical/clinical trials. The iNanoBIT EU H2020 project is developing novel highly sensitive nanotechnology-based imaging approaches allowing for monitoring of survival, engraftment, proliferation, function and whole-body distribution of the cellular transplants in a porcine diabetes model with excellent translational potential to humans. We develop and validate the application of single-photon emission computed tomography (SPECT) and optoacoustic imaging technologies in a transgenic insulin-deficient pig model to observe transplanted porcine xeno-islets and in vitro differentiated human beta cells. We are progressing in generating new transgenic reporter pigs and human-induced pluripotent cell (iPSC) lines for optoacoustic imaging and testing them in transplantable bioartificial islet devices. Novel multifunctional nanoparticles have been generated and are being tested for nuclear imaging of islets and beta cells using a new, high-resolution SPECT imaging device. Overall, the combined multidisciplinary expertise of the project partners allows progress towards creating much needed technological toolboxes for the xenotransplantation and ATMP field, and thus reinforces the European healthcare supply chain for regenerative medicinal products.

Coupling quaternary ammonium surfactants to the surface of liposomes improves both antibacterial efficacy and host cell biocompatibility.

By functionalizing the surface of PEG-liposomes with linkers bearing quaternary ammonium compounds (QACs), we generated novel bacteria disruptors with anti-adhesive properties and reduced cytotoxicity compared to free QACs. Furthermore, QAC-functionalized liposomes are a promising platform for future drug encapsulation. The QAC (11-mercaptoundecyl)-N,N,N-trimethylammonium bromide (MTAB) was attached to maleimide-functionalized liposomes (DSPE-PEG) via thiol linker. The MTAB-functionalized liposomes were physicochemically characterized and their biological activity, in terms of anti-adherence activity and biofilm prevention in Escherichia coli were assessed. The results showed that MTAB-functionalized liposomes inhibit bacterial adherence and biofilm formation while reducing MTAB toxicity.

A New Approach for Glyco-Functionalization of Collagen-Based Biomaterials.

The cell microenvironment plays a pivotal role in mediating cell adhesion, survival, and proliferation in physiological and pathological states. The relevance of extracellular matrix (ECM) proteins in cell fate control is an important issue to take into consideration for both tissue engineering and cell biology studies. The glycosylation of ECM proteins remains, however, largely unexplored. In order to investigate the physio-pathological effects of differential ECM glycosylation, the design of affordable chemoselective methods for ECM components glycosylation is desirable. We will describe a new chemoselective glycosylation approach exploitable in aqueous media and on non-protected substrates, allowing rapid access to glyco-functionalized biomaterials.

Phage-displayed peptides targeting specific tissues and organs.

Phage display is a powerful and widely used technique to find novel peptide ligands. A massive amount of peptide sequences have been identified for all kinds of materials, and peptides that may have targeting capabilities towards specific cells and tissues have received special attention in biomedical sciences. As a result, it is increasingly harder to follow all the work that has been done, which sometimes leads to many promising ligands receiving little attention, together with the publication of false positives that have already been found. The aim of this review is to provide an updated and comprehensive list of phage-displayed peptides targeting different tissues and organs. The limitations of the technique are carefully analysed and the future perspectives envisaged.

Glycans in nanomedicine, impact and perspectives.

Glycans have been selected by nature for both structural and 'recognition' purposes. Taking inspiration from nature, nanomedicine exploits glycans not only as structural constituents of nanoparticles and nanostructured biomaterials but also as selective interactors of such glyco-nanotools. Surface glycosylation of nanoparticles finds application in targeting specific cells, whereas recent findings give evidence that the glycan content of cell microenvironment is able to induce the cell fate. This review will highlight the role of glycans in nanomedicine, schematizing the different uses and roles in drug-delivery systems and in biomaterials for regenerative medicine.

Flavonoids and Their Glycosides as Anti-amyloidogenic Compounds: Aß1-42 Interaction Studies to Gain New Insights into Their Potential for Alzheimer's Disease Prevention and Therapy.

Combining NMR spectroscopy, transmission electron microscopy, biochemical and in vitro toxicity assays, we characterized the effect of flavonoid glycosylation, a chemical modification found very frequently in nature, on their ability to recognize and bind Aß1-42 oligomers, preventing their aggregation and their neurotoxicity. Our data allow the elucidation of their structure-activity relationships, showing that glycosylation has a modest impact on flavonoid affinity for Aß oligomers but, at the same time, increases both solubility and chemical stability, thus promoting their beneficial properties against Alzheimer's disease (AD). As flavonoids and their glycosides are widely available in natural foods, our results provide important information for the evaluation of the role of a flavonoid-rich diet for the prevention of AD. In addition, the structural data collected can be exploited for the rational design of more potent Aß oligomer inhibitors, useful for the development of new therapies against AD.

(18)F-labeling syntheses and preclinical evaluation of functionalized nanoliposomes for Alzheimer's disease.

The aim of the present study was to synthesize functionalized (18)F-labeled NLs ((18)F-NLs) and evaluate their biological behavior in mouse models of Alzheimer's disease (AD) using positron emission tomography (PET) and ex vivo brain autoradiography. (18)F-fluorine was introduced to (18)F-NLs either by using a core forming (18)F-lipid or by encapsulating a (18)F-tracer, (18)F-treg-curcumin inside the NLs. Phosphatidic acid (PA) and curcumin derivative (Curc) functionalized (18)F-NLs with or without additional mApoE functionalization were produced using thin film hydration. The biodistribution and ß-amyloid plaque-binding ability of (18)F-NLs were studied in wild type mice and AD mouse models using in vivo PET imaging and ex vivo brain autoradiography at 60min after (18)F-NL injection. Functionalized (18)F-NLs were successfully synthesized. The preclinical evaluation in mice showed that the functional group affected the biodistribution of (18)F-NLs. Further functionalization with mApoE increased the brain-to-blood ratio of (18)F-NLs but the overall brain uptake remained low with all functionalized (18)F-NLs. The liposomal encapsulation of (18)F-treg-curcumin was not successful and preclinical results of encapsulated (18)F-treg-curcumin and plain (18)F-treg-curcumin were identical. Although the studied functionalized (18)F-NLs were not suitable for PET imaging as such, the synthesis techniques introduced in this study can be utilized to modify the biological behavior of (18)F-labeled NLs.

Protein Kinase A Activation Promotes Cancer Cell Resistance to Glucose Starvation and Anoikis.

Cancer cells often rely on glycolysis to obtain energy and support anabolic growth. Several studies showed that glycolytic cells are susceptible to cell death when subjected to low glucose availability or to lack of glucose. However, some cancer cells, including glycolytic ones, can efficiently acquire higher tolerance to glucose depletion, leading to their survival and aggressiveness. Although increased resistance to glucose starvation has been shown to be a consequence of signaling pathways and compensatory metabolic routes activation, the full repertoire of the underlying molecular alterations remain elusive. Using omics and computational analyses, we found that cyclic adenosine monophosphate-Protein Kinase A (cAMP-PKA) axis activation is fundamental for cancer cell resistance to glucose starvation and anoikis. Notably, here we show that such a PKA-dependent survival is mediated by parallel activation of autophagy and glutamine utilization that in concert concur to attenuate the endoplasmic reticulum (ER) stress and to sustain cell anabolism. Indeed, the inhibition of PKA-mediated autophagy or glutamine metabolism increased the level of cell death, suggesting that the induction of autophagy and metabolic rewiring by PKA is important for cancer cellular survival under glucose starvation. Importantly, both processes actively participate to cancer cell survival mediated by suspension-activated PKA as well. In addition we identify also a PKA/Src mechanism capable to protect cancer cells from anoikis. Our results reveal for the first time the role of the versatile PKA in cancer cells survival under chronic glucose starvation and anoikis and may be a novel potential target for cancer treatment.