Hydrogel scaffolds based on k-Carrageenan/xyloglucan blends to host spheroids from human adipose stem cells

Hydrogels are water-swollen networks of hydrophilic polymer. They can be fabricated in various shapes and swell in water or aqueous solutions maintaining their original shape or undergo progressive erosion; can exibit large volume phase transitions with the change of one environmental parameter (stimuli-responsivness), shock absorption and low sliding friction properties (1). The morphology and mechanical properties of hydrogels are strongly affected by the network composition, the nature and degree of crosslinking and the degree of swelling. Indeed, when hydrogels are designed as scaffolds for human tissues remodeling, they must have sufficient mechanical integrity to provide support to the cells from the time of implantation to the completion of the process. The large amount of water present in the hydrogels and its microscopic pores interconnectivity allows transportation of nutrients, oxygen and metabolites, that ensures cells viability, and permits cells migration and scaffold colonization. The polymeric network can immobilize biomolecules that may affect cells growth or differentiation, control drug release profiles and enzymatic degradation (2,3). The combination of two hydrogelforming polymers with different chemistries and crosslinking densities can be used to tailor the morphology, mechanical strength and toughness of the scaffold to meet specific requirements (1). This work investigates the physico-chemical, morphological and mechanical properties of hydrogels formed by the blend of two polysaccharides, k-Carrageenan (k-C) and Degalactosylated Xyloglucan (Deg-XG) undergoing salt-induced and temperature-induced solgel transition, respectively. It also studies the compatibility of the two biopolymers with spheroids from adipose-derived stem cells (S-ASCs) in the prospect of developing instructive scaffolds for use in regenerative medicine

Moringa oleifera Leaf Powder as Functional Additive in Cookies to Protect SH-SY5Y Cells

The aim of this work is the evaluation of the addition of Moringa leaf powder (MLP) in cookies in terms of antioxidant properties, dough processability and sensorial properties of the cookies. The total content of biophenols and flavonoids in MLP was detected and the identification of the bioactive molecules was performed by HPLC-ESI-TOF-MS measurements, before and after oven treatment at 180 ◦C for 20 min. After a preliminary evaluation of the MLP water soluble fraction (MLPsf) cytotoxicity, its protective effect against an oxidative injury induced in the SH-SY5Y cells was assessed. Data evidence that the bioactive molecules present in MLPsf are effective in preventing ROS production and in protecting neuronal cells against oxidative stress. Prototypes of cookies containing MLP in different concentrations were then produced and evaluated by a consumer panel. Selected doughs containing MLP were analysed to determine the total content of biophenols in the cookies after baking and their enrichment in terms of valuable chemical elements. The influence of MLP on the viscoelastic behaviour and morphology of the doughs was also assessed. Finally, the potential role in counteracting the insurgence of not treatable neurodegenerative pathologies of two main MLP components, glucomoringin and kaempferol derivatives, present also after the thermal treatment, was discussed

From Small Peptides to Large Proteins against Alzheimer'sDisease

Alzheimer's disease (AD) is the most common neurodegenerative disorder in the elderly. The two cardinal neuropathological hallmarks of AD are the senile plaques, which are extracellular deposits mainly constituted by beta-amyloids, and neurofibrillary tangles formed by abnormally phosphorylated Tau (p-Tau) located in the cytoplasm of neurons. Although the research has made relevant progress in the management of the disease, the treatment is still lacking. Only symptomatic medications exist for the disease, and, in the meantime, laboratories worldwide are investigating disease-modifying treatments for AD. In the present review, results centered on the use of peptides of different sizes involved in AD are presented

Radiation-tailored xyloglucan-doxorubicin nanoparticles for cancer therapy

Polysaccharides are long chains of monosaccharides linked by glycosidic bonds. They are widely utilized in biomedical applications, for tissue engineering and wound management, and as excipients of pharmaceutical formulations. In cancer therapy, the development of nanoscale drug delivery systems aims at addressing issues related to the low efficacy of chemotherapeutics and protein drugs due to poor solubility and stability, and to off-target effects that severely affect patients’ body conditions. Biocompatibility, availability of functional groups, amenability of chemical derivatisation and multifunctional conjugation with drugs and targeting ligands make polysaccharide nanoparticles interesting drug delivery devices for cancer treatment. Targeting and controlling the drug release at the tumour site are promising approaches to maximise the anti-tumour effects and reduce side-effects. Xyloglucan is a highly branched, hydrophilic polysaccharide composed of a -D-glucan main chain and -D-xylose branches, partially substituted by -D-galactoxylose. Xyloglucan extracted from tamarind seeds (TS-XG) is commercially available, non-toxic, biodegradable and FDA-approved as food additive. Due to the high molecular weight and tendency to associate in ribbon-like aggregates, aqueous colloidal dispersions of xyloglucan are characterised by relatively large nanoparticles with a broad size distribution. Gamma irradiation of either the colloidal dispersion of xyloglucan (XG) or the solid powder is a very effective and clean methodology to resize the molecular weight of the polysaccharide and reduce the particle size of its colloidal dispersions. When xyloglucan is irradiated as aqueous dispersion, irradiation causes extensive chain scission, with a dramatic reduction of molecular weight already at low doses (50-500 Gy). In contrast, when it is irradiated in the solid state, higher doses are required to change the molecular weight (20-40 kGy), with moisture playing a protective role. Selected systems have been conjugated to doxorubicin (DOX), a powerful anticancer drug that is extensively applied in the clinical treatment of human malignancies, such as leukemia and cancer of the liver, ovary and breast. This drug causes severe peripheral toxicity that poses limits to the maximum dosage that can be administered, and the emergence of multidrug resistance. DOX has been bound to irradiated XG through a cleavable bond and then further loaded with DOX by self-assembly. Xyloglucan contains galactose, which can be recognised and internalised by asialoglycoprotein receptor (ASGP-R). In order to increase the targeting efficiency, XG has been also conjugated to folic acid. Drug release efficiency and tumour cells selectivity are under investigation

Poly(vinyl alcohol)/κ-Carrageenan-based hydrogels enriched with the adhesive mussel protein Pvfp5β as 3D cell culture scaffold for tissue engineering applications

Many marine organisms such as sandcastle worms, barnacles and mussels, produce natural adhesives to attach to wet surfaces in aqueous tidal environments. In mussels, the adhesion is possible through the secretion of a protein-based water-resistant glue, composed of a mixture of proteins called mussel adhesive proteins (MAPs) or mussel foot proteins (mfps), that allow anchoring to almost any kind of surface in wet conditions [1]. The proteins confined to adhesive plaques are mfp-2, -3, -4, -5, and -6. All these proteins contain an atypically high concentration of the catecholic amino acid 3,4- dihydroxy-l-phenylalanine (DOPA), obtained by the post-translational enzymatic hydroxylation of tyrosine (Tyr) [2]. DOPA is the key molecule allowing the underwater mussel adhesion to different surfaces through the formation of irreversible covalent and reversible noncovalent bonds [3]. However, growing evidences show that the presence of DOPA is not a sufficient condition to generate strong underwater adhesion. A synergistic effect of catechols and adjacent lysine in bioadhesion of mussels has been suggested. The pair structure of Tyr/DOPA with basic residues of lysine, rather than the post-translational modification of Tyr to DOPA, should be responsible for the strong binding ability of mussel adhesive proteins [4]. We have recently fine-tuned the expression of the recombinant Pvfp5β protein in Escherichia coli [5]. Furthermore, the structural characterization showed that the produced protein was correctly folded as a β-rich protein with precise pairing of the sulfur bridges. We have also demonstrated that surfaces coated with recombinant Pvfp5β are not toxic, and improve cell adhesion, proliferation and spreading of NIH-3T3 and HeLa cell lines [5]. Thus, adhesive protein/peptide coatings and efficient exposure of bioactive domains might improve the biological properties of scaffold materials. Hydrogels, a unique class of polymeric materials, are three-dimensional porous networks constituted by polymeric chains crosslinked by chemical and/or physical bonds. They exhibit remarkable structure-derived properties, including high surface area, stimuli-responsiveness, inherent flexibility, controllable mechanical strength, and excellent biocompatibility. They are capable of absorbing and retaining large amounts of water in their networks and can extensively swell without dissolution due to the presence of crosslinks, that are at the basis of the network structure, and maintain macroscopic integrity [6]. k-carrageenan (kCar) is a naturally occurring polysaccharide extracted from marine red algae (Rhodophyceae). Chemically it consists of an alternating linear chain of (1->3)-β-D-galactose-4SO3- - (1->4)-3,6, anhydro-α-D-galactose. Due to its half-ester sulphate moieties, it is a strong anionic polymer and resembles the natural glycosaminoglycans (GAGs), which are an important component of the connective tissue. kCar is biocompatible, biodegradable, non-toxic, and gel-forming. Thus, kCar has been investigated for a wide range of biomedical puropses, such as wound dressing [7] and tissue engineering [8] applications. The generally accepted model of the gelling process of carrageenan solutions involves a coil-to-helix transition, followed, in the presence of certain cations, by aggregation of double helices to form a stiff, extended network [9]. Blending kCar with polyvinyl alcohol (PVA) allows obtaining composite scaffolds with higher, interconnected porosity, high swelling degree, improved toughness and degradability. PVA can be chemically crosslinked, with glutaraldehyde, or physically crosslinked by inducing the formation of crystalline domains via freeze-thawing [10]. In the present study, we used the crosslinking-agent free, freeze–thaw approach for the fabrication of PVA/kCar composite hydrogels as 3D scaffolds for cell culture, whose adhesion was enhanced by simple adsorption of cationic recombinant Pvfp5β protein. The physico-chemical properties of the PVA/kCar hydrogel (morphology, swelling and degradation, water absorption, mechanical strength) were evaluated. Furthermore, biocompatibility, adhesion, proliferation and morphology of the cells grown in presence of PVA/kCar-Pvfp5β hydrogel were investigated. All gathered information demonstrates the great potential of PVA/kCar-Pvfp5β scaffolds for tissue engineering applications. Prospects for the future PVA/kCar formulations are also being evaluated as bioinks for 3D bioprinting, a key enabling technology for the manufacture of complex tissue structures to mimic native organs and tissues. The bioprinting involves layer by layer deposition of cells-laden biomaterials in a predetermined structural architecture to generate functional tissues or organs. They provide structure for the bioprinted tissue and support and nutrients for the cells, creating an environment in which the cells can survive, grow, and proliferate. The research will then explore the influence of Pvfp5β on 3D inkjet bioprinted PVA/kCar scaffolds integrated with human adipose stem-cell spheroids (SASCs) in terms of cell survival and differentiation and scaffold colonization. References (max. 10 references) [1] Y. He, C. Sun, F. Jiang, B. Yang, J. Li, C. Zhong, L. Zheng, H. Ding. Lipids as integral components in mussel adhesion. Soft Matter, 14:7145–7154, 2018 [2] W. Zhang, H. Yang, F. Liu, T. Chen, G. Hu, D. Guo, Q. Hou, X. Wu, Y. Su, J. Wang. Molecular interactions between DOPA and sur- faces with different functional groups: a chemical force microscopy study. RSC Adv., 7:32518 –32527, 2017 [3] S.M. Kelly, T.J. Jess, Price N.C. How to study proteins by circular dichroism. Biochim. Biophys. Acta, 1751: 119–139 [4] X Ou, B Xue, Y Lao, Y Wutthinitikornkit, R Tian, A Zou, L Yang, W Wang, Y Cao, Jingyuan Li. Structure and sequence features of mussel adhesive protein lead to its salt-tolerant adhesion ability. Sci. Adv. 6: eabb7620, 2020 [5] R. Santonocito, F. Venturella, F. Dal Piaz, M.A. Morando, A. Provenzano, E. Rao, M.A. Costa, D. Bulone, P.L. San Biagio, D. Giacomazza, A. Sicorello, C. Alfano, R. Passantino, A. Pastore. Recombinant mussel protein Pvfp-5β: A potential tissue biohadesive. J. Biol. Chem., 294:12826:12835, 2019 [6] G.M. Kavanagh, S.B. Ross-Murphy. Rheological characterization of polymer gels. Prog. Polym. Sci., 23:533-562, 1998 [7] L.A. Ditta, E. rao, F. Provenzano, J. Lozano Sanchez, R. Santonocito, R. Passantino, M.A. Costa, M.A. Sabatino, C. Dispenza, D. Giacomazza, P.L. San Biagio, R. Lapasin. Agarose/kCarrageenan-based hydrogel film enriched with natural plant extracts for the treatment of cutaneous wounds. Int. J. Biol. Macromol., 164:2818-2830 [8] S.M. Mihaila, A.K. Gaharwar, R.L. Reis, A.P. Marques, M.E. Gomes, A. Kademhosseini. Photocrosslinkable kappa-carrageenan hydrogels for tissue engineering applications. Advanced Healthcare Materials, 2:895-907, 2013 [9] L. Du, T. Brenner, J. Xie, S. Matsukawa. A study on phase separation behavior in kappa/iota carrageenan mixtures by micro DSC, rheological measurements and simulating water and cations migration between phases. Food Hydrocolloids, 55:8188, 2016 [10] S.R. Stauffer, N.A: Peppast. Poly(vinyl ancohol) hydrogels prepared by freezing-thawing cyclic processing. Polymer, 33:3932-3936, 199

Devices for the capture of rare cells from biological samples for diagnostic purposes

The chance of surviving to a disease often depends on early diagnosis and effective therapy. In the field of early prenatal diagnosis, micromanipulation is a reliable technique for manual selection and isolation of rare fetal cells in maternal biological fluids for molecular or cytogenetic analysis. This technique allows obtaining pure cell populations for analysis, but it is expensive and time consuming, as it requires qualified and experienced staff and specific equipment [1]. This research aims at making the prenatal diagnosis more economical and reproducible in the hospital environment, by creating a device that allows selecting rare cells from biological samples in a semi-automated way. The device consists in electrospun nanofiber mats with surface functional groups conjugated to antibodies capable of selectively binding to the antigens present on the surface of target cells. Nanofiber mats were produced from polymer mixtures of Nylon 6.6 and Polyacrylic Acid (PAA) in a suitable solvent, with or without the addition of a third polymer, synthetized in house, that should prevent non-specific cell binding. The first phase of the work was devoted to the determination of the operating parameters for electrospinning in order to optimize the morphology of the mats, their mechanical resistance and handling characteristics. Bioconjugation protocols, based on EDC/NHS (1-ethyl-3-(3- dimethylaminopropyl) carbodiimide hydrochloride)/(Sulfo-(N- hydroxysulfosuccinimide) chemistry were developed to provide the electrospun mats with specific cell capture abilities. In order to facilitate the recognition of the antibody by the receptors expressed by the target cells, the functional groups of the mat were previously reacted to a linker that acts as a spacer arm. Antibodies labelled with fluorescent probe were used to be able to assess the success of the conjugation reactions by fluorimetry and spectrofluorimetry analyses. Protocols for cell capture tests on antibody-decorated mats were devised using different cell suspensions: fetal cells (CF), mesenchymal stem cells (MSC) and lymphocytes (WBCs, White Blood Cells). The results of the capture tests were obtained by observing the mats under the optical and electron microscopes (SEM). References [1] G. Makrydimas, G. Damiani., C. Jakil, V. Cigna., M. Orlandi, F. Picciotto, K. H. Nicolaides, 2020, Ultrasound in Obstetrics & Gynecology, 56(5), 672-677

Rare cell capture platforms based on antibody-conjugated electrospun nanofiber mats for noninvasive prenatal diagnostics

The chance of surviving to a disease often depends on early diagnosis and effective therapy. In the field of early prenatal diagnosis, micromanipulation is a reliable technique for manual selection and isolation of rare fetal cells in maternal biological fluids for molecular or cytogenetic analysis. This technique allows obtaining pure cell populations for analysis, but it is expensive and time consuming, as it requires qualified and experienced staff and specific equipment [1]. The aim of this study is to make the prenatal diagnosis more economical and reproducible in the hospital environment, by creating a device that allows selecting rare cells from biological samples in a semi-automated way. The device consists in electrospun nanofiber mats with surface functional groups conjugated to antibodies capable of selectively binding to the antigens present on the surface of target cells. Nanofiber mats were produced from polymer mixtures of Nylon 6.6 and Polyacrylic Acid (PAA) in a suitable solvent, with or without the addition of a third polymer, synthetized in house, that should prevent non-specific cell binding. The first phase of the work was devoted to the determination of the operating parameters for electrospinning to optimize the morphology of the mats, their mechanical resistance and handling characteristics. Bioconjugation protocols, based on EDC/NHS (1-ethyl-3-(3- dimethylaminopropyl) carbodiimide hydrochloride)/(Sulfo-(N-hydroxysulfosuccinimide) chemistry were developed to provide the electrospun mats with specific cell capture abilities. In order to facilitate the recognition of the antibody by the receptors expressed by the target cells, the functional groups of the mat were previously reacted to a linker that acts as a spacer arm. Antibodies labelled with fluorescent probe were used to be able to assess the success of the conjugation reactions by fluorimetry and spectrofluorimetry analyses. Protocols for cell capture tests on antibody-decorated mats were devised using different cell suspensions: fetal cells (CF), mesenchymal stem cells (MSC) and lymphocytes (WBCs, White Blood Cells). The results of the capture tests were obtained by observing the mats under the optical and electron microscopes (SEM)