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Our research group has recently developed microparticles of a hyaluronic acid derivative used for bottom-up growth of microparticles/human mesenchymal stem cells (hMSCs). In this work, we investigated a strategy to increase the stiffening of aggregated constructs between microparticles and hMSCs. In particular, we applied a Michael-type crosslinking procedure between microparticles to allow a chemically driven and cell-compatible stiffening of constructs. Two batches of microparticles were functionalized with thiol and maleimide groups, respectively, and were then mixed to allow chemical crosslinking. The adhesion of hMSCs was controlled through addition of the adhesive peptide cyclo(-Arg-Gly-Asp-D-Phe-Cys) (cyRGDC). Rheological measurements performed in this study showed that the chemical stiffening strategy allows the G ' modulus of bottom-up growing constructs to be increased, while viability tests suggest that the chemical procedure did not negatively affect cell viability compared with constructs obtained without chemical crosslinking. (c) 2018 Society of Chemical Industry

The aim of this work was to evaluate the potential use of hydrogels based on hyaluronic acid (HA) chemically cross-linked with ±,²-poly(N-2-hydroxyethyl) (2-aminoethylcarbamate)-D,L-aspartamide (PHEA-EDA) as substitutes for the amniotic membrane able to release limbal cells for corneal regeneration. Hydrogels, shaped as films, with three different molar ratios (X) between PHEA-EDA and HA (X = 0.5, 1.0 and 1.5) have been investigated. First, it has been evaluated their swelling ability, hydrolytic resistance in simulated physiological fluid and cell compatibility by using human dermal fibroblasts chosen as a model cell line. Then adhesion studies in comparison with collagen gel, have been performed by using immortalized cells, such as human corneal epithelial cells (HCEC) or primary cells, such as rabbit limbal epithelial cells (RLEC) and/or rabbit limbal fibroblasts (RLF). HA/PHEA-EDA hydrogels allow a moderate/poor adhesion of all investigated cells thus suggesting their potential ability to act as cell delivery systems. Finally, commercial contact lenses have been coated, in their inner surface, with each HA/PHEA-EDA film and it has been found that in these conditions, a greater cell adhesion occurs, particularly when RLEC are in co-culture with RLF. However, this adhesion is only transitory, in fact after three days, viable cells are released in the culture medium thus suggesting a potential application of HA/PHEA-EDA hydrogels, for delivering limbal cells in the treatment of corneal damage

Neurodegenerative diseases affect millions of people worldwide and the presence of various physiological barriers limits the accessibility to the brain and reduces the efficacy of various therapies. Moreover, new carriers having targeting properties to specific brain regions and cells are needed in order to improve therapies for the brain disorder treatment. In this study, for the first time, Myelin nanoVesicles (hereafter defined MyVes) from brain-extracted myelin were produced. The MyVes have an average diameter of 100–150 ​nm, negative zeta potential, spheroidal morphology, and contain lipids and the key proteins of the myelin sheath. Furthermore, they exhibit good cytocompatibility. The MyVes were able to target the white matter and interact mainly with the microglia cells. The preliminary results here presented allow us to suppose the employment of MyVes as potential carrier to target the white matter and microglia in order to counteract white matter microglia-related diseases., Graphical abstract Image 1, Highlights • Bio-fabrication of brain tissue derived nanovesicles: myelin nanovesicles. • Myelin nanovesicles contain the main proteins of the myelin sheath (myelin basic protein and myelin proteolipid protein). • Myelin nanovesicles can lade a drug/molecule and cross a blood–brain barrier model. • Myelin nanovesicles target white matter and microglia cells.

Background There is increasing interest in the development of vessel substitutes, and many studies are currently focusing on the development of biodegradable scaffolds capable of fostering vascular regeneration. We tested a new biocompatible and biodegradable material with mechanical properties similar to those of blood vessels. Methods The material used comprises a mixture of α,β-poly(N-2-hydroxyethyl)- d,l -aspartamide (PHEA) and polylactic acid (PLA), combined with polycaprolactone (PCL) by means of electrospinning technique. Low-molecular-weight heparin was also linked to the copolymer. A tubular PHEA-PLA/PCL sample was used to create an arteriovenous fistula in a pig model with the use of the external iliac vessels. The flow was assessed by means of Doppler ultrasound examination weekly, and 1 month after the implantation we removed the scaffold for histopathologic evaluation. Results The implants showed a perfect leak-proof seal and adequate elastic tension to blood pressure. About ∼3 weeks after the implantation, Doppler examination revealed thrombosis of the graft, so we proceeded to its removal. Histologic examination showed chronic inflammation, with the presence of foreign body cells and marked neovascularization. The material had been largely absorbed, leaving some isolated spot residues. Conclusions The biocompatibility of PHEA-PLA/PCL and its physical properties make it suitable for the replacement of vessels. In the future, the possibility of functionalizing the material with a variety of molecules, to modulate the inflammatory and coagulative responses, will allow obtaining devices suitable for the replacement of native vessels.

Bacterial colonization of implanted biomaterials remains one of the most challenging complications in orthopedics and trauma surgery, with extremely high social and economic costs. Antibacterial coating of implants has been advocated by many experts as a possible solution to reduce the burden of implant-related infection and several different solutions have been proposed in the last decades. However, while most of the investigated technologies have shown their efficacy in vitro and/or in vivo, only few were able to reach the market, due to clinical, industrial, economic and regulatory issues. Hyaluronic acid composites have been previously shown to possess antifouling capabilities and have been used in various clinical settings to reduce bacterial adhesion and mitigate biofilm-related infections. Recently, a fast-resorbable, hyaluronic-based hydrogel coating was developed to protect implanted biomaterials in orthopedics, trauma and maxillofacial surgery. Preclinical and clinical testing did show the safety and efficacy of the device that can be intraoperatively loaded with one or more antibiotics and directly applied by the surgeon to the implant surface, at the time of surgery. Here, we review the current evidence concerning this very first antibacterial coating of implants and outline the economic impact of the possible large-scale application of this technology.

α,β-Poly( N -2-hydroxyethyl)- dl -aspartamide grafted with polylactic acid (PHEA- g -PLA) is a biocompatible and biodegradable amphiphilic copolymer that has been already employed to prepare a drug delivery system. In this study we have prepared for the first time a fibrillar scaffold from PHEA- g -PLA by the electrospinning of a solution of this copolymer in a mixture of N , N -dimethyl formamide (DMF) and acetone (80:20 vol/vol). The average diameter and the morphology of electrospun fibers were detected by scanning electron microscopy. Chemical degradation studies in phosphate buffer solution pH 7.4 have been performed until 15 days in order to obtain a preliminary information about the hydrolytic resistance of the prepared scaffold.

In situ gel forming systems have been prepared by linking polylactic acid (PLA) to a water soluble and polyfunctional polymer, such as α,β-poly( N -2-hydroxyethyl)- d , l -aspartamide (PHEA). Three graft copolymers PHEA–PLA with a different derivatization degree in PLA, have been synthesized and characterized. PHEA–PLA graft copolymer with the highest amount in PLA has been used to prepare solutions in organic solvents able to give rise to gel-like matrices when injected into phosphate buffered saline solution. The chemical degradation of these gels has been evaluated and in vitro tests have been performed to evaluate the cell compatibility of the hydrolysis products. The possibility to use these gels for drug release has been investigated by incorporating leuprolide as a peptide model drug and by evaluating its in vitro release. To improve the drug release profile, PHEA–PLA graft copolymer has been derivatized with pendant carboxylic groups that are able to form an ion pair with the leuprolide thus reducing the burst effect and prolonging its release.

Conclusion: HA-g-PDLLA hydrogel is suitable to coat a prosthesis remaining adherent to it and distributing itself in the bone cavity. The hydrogel has been proven effective in a rabbit model of highly contaminated implant. In vivo studies have demonstrated how the intraoperative coating of implants with DAC is safe and feasible. HA-EDA-MA hydrogel is able to protect PEP from alteration during storage and it seems a good candidate for a potential once-daily oral formulation in the treatment of celiac disease.

Introduction. The reaction between !,"-poly(aspartylhydrazide) (PAHy), a water soluble synthetic polymer and 3-(carboxypropyl)trimethyl-ammonium chloride (CPTACl) produced copolymers bearing permanent positive charges (PAHy–CPTA) with molecular weight of 10 kDa and PAHy–CPTA copolymers differing in positive charge amount (18–58%) were chosen for biological investigations. Materials and methods. Biophysical properties of DNA/PAHy–CPTA polyplexes were evaluated in terms of DNA condensation, zeta potential and size distribution. Cytotoxicity studies on Neuro2A murine neuroblastoma cells evidenced absence of toxicity of these copolymers up to 300 2g/ml unlike linear polyethylenimine (LPEI) that was highly toxic already at 20 2g/ml. Results and Discussion. PAHy–CPTA copolymers did not induce any erythrocyte aggregation up to 1 mg/ml. Cellular interaction studies of PAHy–CPTA polyplexes evidenced a faster binding of these polyplexes with cells compared to DNA/LPEI polyplexes. The in vitro transfection ability of PAHy– CPTA polyplexes was strongly affected by experimental conditions reaching about 10% of the transfection efficiency of optimized LPEI polyplexes. Conclusions. Finally, in vivo application studies confirmed the biocompatibility of PAHy–CPTA copolymers. With LPEI, clear signs of microvesicular fatty liver were observed and with LPEI polyplexes significant weight loss. In strong contrast, PAHy–CPTA did not induce histopathological changes or weight loss.

The preparation and physicochemical and biological characterization of a novel polyaminoacid hydrogel have been reported. The ,-poly(N-2- hydroxyethyl)-dl-aspartamide (PHEA) has been used as a starting polymer for a derivatization reaction with methacrylic anhydride (MA) to give rise to the methacrylate derivative named PHM. Photocrosslinking of PHM has been performed in aqueous solution at 313 nm and in the absence of toxic initiators. PHM-based hydrogel has been characterized by scanning electron microscopy, X-ray diffractometry, swelling measurements in aqueous media; the degradation of PHM-based hydrogel has been evaluated as a function of time in the absence or in the presence of esterase. Besides, the biocompatibility of this hydrogel and of its degradation products has been evaluated by performing in vitro assays on human chronic myelogenous leukaemia cells (K-562), chosen as a model cell line. Finally, ATR-FTIR measurements have showed that interaction between PHMbased hydrogel and each of four plasma proteins (albumin, -globulin, transferrin and fibrinogen) does not cause change in protein conformation thus supporting its potential use as a material to prepare parenteral drug delivery systems. © 2006 Elsevier B.V. All rights reserved.

The aim of this work has been the preparation and characterization of novel hydrogels with polysaccharide-poly(amino acid) structure having suitable physicochemical properties for pharmaceutical applications. In the first step, hyaluronic acid (HA) and alpha,beta-poly(N-2-hydroxyethyl)-DL-aspartamide (PHEA) have been derivatized with methacrylic anhydride (AMA), thus obtaining HA-AMA and PHM derivatives, respectively. In the second step, aqueous solutions of both these derivatives have been irradiated at 313 nm to obtain chemical hydrogels. The hydrogel obtained by irradiating for 15 min an aqueous solution containing 4% w/v of HA-AMA and 4% w/v of PHM resulted in the highest yield. Its swelling ability was dependent on the pH and nature of the external medium. Besides, this hydrogel undergoes a partial hydrolysis, especially in the presence of enzymes, such as esterase or hyaluronidase, but the entity of this degradation is lower than that observed for a hydrogel based on HA-AMA alone. The ability of this hydrogel to entrap drug molecules has been evaluated by using thrombin as a model drug. In vitro release studies and a platelet aggregation test demonstrated that the HA-AMA/PHM hydrogel is able to release thrombin in the active form, thus suggesting its suitability for the treatment of hemorrhages.

Hyaluronic acid, celiac disease

Here, the synthesis of a galactosylated amphiphilic copolymer bearing rhodamine (RhB) moieties and its use for the preparation of polymeric fluorescent nanoparticles for potential applications in therapy and diagnosis are described. To do this, firstly, a fluorescent derivative of α,β-poly( N -2-hydroxyethyl)- d , l -aspartamide (PHEA) was synthesized by chemical reaction with RhB, and with polylactic acid (PLA), to obtain PHEA-RhB-PLA. Then, the derivatization of PHEA-RhB-PLA with GAL-PEG-NH 2 allows obtaining PHEA-RhB-PLA-PEG-GAL copolymer, with derivatization degrees in -PLA and -PEG-GAL equal to 1.9 mol% and 4.5 mol%, respectively. Starting from this copolymer, liver-targeted fluorescent nanoparticles were prepared by high pressure homogenization–solvent evaporation method, and showed nanoscaled size, slightly negative zeta potential and spherical shape. Chemical and enzymatic stability of fluorescent dye covalently linked to the copolymer backbone by ester linkage was demonstrated until 4 days of incubation. Finally, thanks to the covalently-linked fluorescent RhB, it was demonstrated that these galactosylated nanoparticles interact with HepG2 cells that are positive for the asialoglycoprotein receptor (ASGPR), while these do not interact with HeLa cells that are negative for the same receptor, demonstrating the contributor of ASGPR to the internalization process.

With the high occurrence of cardiovascular disease and increasing numbers of patients requiring vascular access, there is a significant need for small-diameter (

In recent years there has been a growing interest for the development of tubular scaffolds employed to assist the replacement of small blood vessels. Materials designed for this purpose need to be biodegradable, have good mechanical properties and improve cell adhesion, proliferation and differentiation. To obtain biomaterials with these properties, electrospinning seems to be one of the most useful technique. Several biodegradable synthetic polymers or constituents of the extracellular matrix (ECM) have been electrospun showing optimal mechanical properties and biodegradability. However, such polymers are lacking in versatile chemical structure affordable to immobilize growth factors or chemokines. The glycosaminoglycan heparin is able to bind several growth factors like vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) and, when grafted onto the scaffold surface it is able to attract cells thus improving their proliferation and differentiation. Aim of this research was the production and the preliminary in vitro biological characterization of a new biodegradable material, obtained by electrospun a polyaminoacid-graft-polyester copolymer. The electrospun biomaterial has been successfully grafted with heparin exploiting the better chemical reactivity of the polyaminoacid portions of the graft copolymer. Then its morphology has been investigated by scanning electron microscopy (SEM) and the potential biodegradability of the material has been studied until 60 days. Preliminary biological data in vitro, on human endothelial cells, show a good compatibility of the scaffold obtained by electrospinning, with regard to cell adhesion and proliferation. Experiments are in progress to evaluate the effects of heparin on cell differentiation.

A new chemical strategy is described to link ethylenediamino (EDA) groups to primary hydroxyl groups of hyaluronic acid (HA) and the obtained derivatives have been characterized by 1H-NMR and 13C-NMR analyses. Such HA–EDA derivatives have been exploited to control the functionalization degree in benzoyl-cysteine (BC) groups, chosen as moieties able to allow both self-assembling in aqueous media and an oxidative crosslinking. In particular, the kinetics of oxidation of thiol groups in HA–EDA–BC derivatives has been studied in Dulbecco's Phosphate Buffer Solution (DPBS) pH 7.4 by colorimetric assays and rheological measurements. Mechanical properties of chemical hydrogels obtained after oxidative crosslinking have been evaluated by using two different concentration values (1 and 1.5% w/v) of HA–EDA–BC. These hydrogels show a dense interconnected fibrillar structure similar to the extracellular matrix of soft tissue and a resistance to chemical and enzymatic hydrolysis. Therefore, the potential suitability of HA–EDA–BC hydrogels as scaffolds for cartilage regeneration has been preliminarily assessed using primary human chondrocytes and evaluating their viability and ability to produce extracellular collagen.

The mechanical properties of hydrogel scaffolds based on hyaluronic acid (HA) that were chemically crosslinked with α,β-poly(N-2-hydroxyethyl)(2-aminoethylcarbamate)-D,L-aspartamide (PHEA-EDA) were investigated. Variation of these properties as a function of three different PHEA-EDA amounts used to crosslink HA has been related to the reaction efficiency evaluated using a colorimetric assay. Moreover, the amount of unreacted amino groups that was still present in the hydrogels was related to the attachment behavior of human dermal fibroblasts to the hydrogel surface. The mechanical data and biological results suggest the suitability of the investigated hydrogels as scaffolds for the regeneration of soft tissues such as skin. Open image in new window

Polymeric micelles obtained by self-assembling of amphiphilic hyaluronic acid (HA) graft copolymers have been prepared and characterized. In particular, hyaluronic acid (HA) has been grafted to polylactic acid (PLA) and polyethylenglycol chains (PEG), then the copolymers able to form micelles in aqueous medium have been chosen to entrap the antitumoral drug Doxorubicin. The critical aggregation concentration of HA-g-PLA or HA-g-PLA-g-PEG micelles has been determined by using pyrene as a fluorescent probe, whereas their shape and size have been evaluated by light scattering measurements, scanning and transmission electron microscopies. The selective cytotoxicity of drug loaded micelles toward the CD-44 over-expressing HCT-116 cells compared to receptor deficient human derm fibroblasts has been demonstrated. Pegylated micelles showed better stability and drug loading capacity and they were able to escape from macrophage phagocytosis.

A hyaluronic acid derivative bearing pendant l -benzoyl-cysteine portions (with a derivatization degree equal to 10 mol.%) was synthesized by linking N,N′-dibenzoyl- l -cystine to the polysaccharide and then reducing its disulfide bridge to thiol groups. The formation of π–π stacking interactions between the benzoyl moieties was studied by fluorescence spectroscopy as a function of polymer concentration and oxidation time. The efficiency of oxidation of thiol groups to disulfide bridges occurring in phosphate buffer pH 7.4, was determined by colorimetric assays. The hydrogel formed by means of oxidative crosslinking has shown the presence of fibrillar aggregates as detected by light and scanning electron microscopy. Human derm fibroblasts were encapsulated into hydrogel-forming solution, and their ability to proliferate was tested during 3 days of culture.