Low-Cost Potentiometric Sensor for Chloride Measurement in Continuous Industrial Process Control.

Recently, the new updates in legislation about drinking water control and human health have increased the demand for novel electrochemical low-cost sensors, such as potentiometric ones. Nowadays, the determination of chloride ion in aqueous solutions has attracted great attention in several fields, from industrial processes to drinking water control. Indeed, chloride plays a crucial role in corrosion, also influencing the final taste of beverages, especially coffee. The main goal is to obtain devices suitable for continuous and real-time analysis. For these reasons, we investigated the possibility to develop an easy, low-cost potentiometric chloride sensor, able to perform analysis in aqueous mediums for long immersion time and reducing the need of periodic calibration. We realized a chloride ion selective electrode made of Ag/AgCl sintered pellet and we tested its response in model solutions compatible with drinking water. The sensor was able to produce a stable, reproducible, and accurate quantification of chloride in 900 s, without the need for a preliminary calibration test. This opens the route to potential applications of this sensor in continuous, in situ, and real time measurement of chloride ions in industrial processes, with a reduced need for periodic maintenance.

Biodegradable paclitaxel-loaded microparticles prepared from novel block copolymers: influence of polymer composition on drug encapsulation and release.

This study covers the preparation of microspheres for the controlled and targeted release of paclitaxel, using novel degradable polymers as carrier materials. Paclitaxel-loaded microspheres were prepared by oil-in-water single-emulsion solvent extraction/evaporation technique by using a series of polyurethanes and a block copolymer; the physicochemical properties of these polymers were modulated by changing nature and composition of their structural units. The obtained microparticles showed a regular morphology and properties (diameter: 1-100 µm; resuspension index: 18.8-100%; encapsulation efficiency: 26.6-97.2%) depending on polymer hydrophilicity and emulsifier used. In vitro release curves showed in all cases almost zero-order kinetics after an initial low burst effect (from 1 to 8.4%), which is required to minimize the drug side effects. This work also proposes a novel strategy to combine a controlled and a targeted release through the functionalization of the polymer matrix with peptide sequences. An RGD-functionalized polyurethane was used to successfully prepare paclitaxel-loaded microparticles. Studies on the preparation of polymer microspheres are reported.

SOFT-MI: a novel microfabrication technique integrating soft-lithography and molecular imprinting for tissue engineering applications.

An innovative approach has been employed for the realization of bioactive scaffolds able to mimic the in vivo cellular microenvironment for tissue engineering applications. This method is based on the combination of molecular imprinting and soft-lithography technology to enhance cellular adhesion and to guide cell growth and proliferation due to presence of highly specific recognition sites of selected biomolecules on a well-defined polymeric microstructure. In this article polymethylmethacrylate (PMMA) scaffolds have been realized by using poly(dimethylsiloxane) (PDMS) microstructured molds imprinted with FITC-albumin and TRITC-lectin. In addition gelatin, an adhesion protein, was employed for the molecular imprinting of polymeric scaffolds for cellular tests. The most innovative aspect of this research was the molecular imprinting of whole cells for the development of substrates able to enhance the cell adhesion processes.

Analysis of mass transport in ionic liquids: a rotating disk electrode approach.

Ionic Liquids are a promising alternative to water electrolytes for the electrodeposition of metals. These solvents have a much larger electrochemical window than water that expands the potential of electrodeposition. However, mass transport in Ionic Liquids is slow. The slow mass transport dramatically affects the rate of reactions at the solid-liquid interface, hampering the exploitation of Ionic Liquids in high-throughput electrodeposition processes. In this paper, we clarify the origin of such poor mass transport in the diffusion-advection (convection) regime. To determine the extent and the dynamics of the convection boundary layers, we performed Rotating Disk Electrode (RDE) experiments on model reactions along with the finite element simulation. Both the experiments and the finite element modelling showed the occurrence of peaks in the RDE curves even at relatively high rotation rates (up to 2000 rpm). The peak in the RDE is the fingerprint of partial diffusion control that happens for the relative extent of the diffusion and convection boundary layers. In looking for a close match between the experiments and the simulations, we found that the ohmic drop plays a critical role and must be considered in the calculation to find the best match with the experimental data. In the end, we have shown that the combined approach consisting of RDE experiments and finite elements modelling providing a tool to unravel of the structure of the diffusion and convection boundary layers both in dynamic and stationary conditions.

Melt-extruded guides for peripheral nerve regeneration. Part I: poly(epsilon-caprolactone).

Melt-extruded guides for peripheral nerve repair based on poly(epsilon-caprolactone) (PCL) were realised and their physico-chemical properties were evaluated. Preliminarily, PCL cast films were found to support the attachment and proliferation of Neonatal Olfactory Bulb Ensheating Cells (NOBEC). S5Y5 neuroblastoma cells were cultured inside PCL guides in their uncoated form or coated with a non-specific adhesion protein (gelatin) and a specific peptide for nerve regeneration (poly(L-lysine)). Coating increased cell density (gelatin) and/or the cell density rate on substrates (poly(L-lysine); gelatin) as compared to uncoated guides. Various in vivo tests were carried out for the repair of small (0.5 cm), medium (1.5 cm) and long (4.5 cm) size defects in the peripheral nerves of Wistar rats. For the small nerve defects, uncoated and coated PCL guides were tested. Results from in vivo tests were subjected to histological examination after 45 days, 6 and 8 months postoperative for small, medium and large defects, respectively. Regeneration was found for small and medium size defects. For 0.5 cm defects, the coating did not affect regeneration significantly. Grip-tests also evidenced functional recovery for the 1.5 cm-long defects treated with PCL guides, after 6 months from implantation. On the other hand, mechanical stiffness of PCL conduits impaired the repair of 4.5 cm-long defects in 8-month period: the lack of flexibility of the guide to rat movements caused its detachment from the implant site. The research showed that PCL guides can be used for the successful repair of small and medium size nerve defects, with possible improvements by suitable bio-mimetic coatings.

PAM-microfabricated polyurethane scaffolds: in vivo and in vitro preliminary studies.

Polymeric scaffolds were realised with linear degradable PU in the form of square, hexagonal and octagonal grids. They were characterised in terms of their mechanical properties. Analysis shows that the mechanical properties of the scaffolds depend on their geometries which are easily modulated using PAM. In vitro biological assays showed that PU promotes the adhesion and proliferation of fibroblast cells and that cell activities are better on PU scaffolds than on PU films. In vivo implantation of PU and PLGA scaffolds and PU films demonstrated that the scaffolds are completely resorbed after three months with a slight inflammatory response, while the PU film was still present after six months with an intense granulomatous reaction.

Towards the design of highly selective recognition sites into molecular imprinting polymers: a computational approach.

A computational approach to simulate the formation of possible imprinted polymers in acetonitrile solution for theophylline (THO) is proposed, using combined molecular dynamics (MD), molecular mechanics (MM), docking and site mapping computational techniques. Methacrylic acid (MAA) and methylmethacrylate (MMA) monomers are used to simulate possible homo and copolymer structures. The model is able predict binding affinity and selectivity when considering THO analogues, such as caffeine, theobromine, xanthine and 3-methylxanthine. Comparison with available experimental data is proposed.

Supported imprinted nanospheres for the selective recognition of cholesterol.

The preparation of innovative polymeric systems using molecular imprinting technology for application in extracorporeal blood purification is described. Membranes based on a methylmethacrylate-co-acrylic acid copolymer, produced through the phase inversion method, were modified introducing into their structure specific binding sites for cholesterol molecule by adding molecularly imprinted nanoparticles in the membrane matrix. Membranes prepared are intended to selectively remove cholesterol from the blood by using interactions at a molecular level, between the membrane/nanoparticles devices and the template, created during the preparation of polymers. Three polymeric systems in form of nanoparticles were prepared differing in the polymerisation solvent (a mixture of acetonitrile and ethanol (1:1) or pure ethanol), and the molar ratio between the functional monomer and the cross-linker (2.3:1 and 1:1). Two out of three of the prepared polymers showed a very good template rebinding capacity both in phosphate buffer solution (pH 6.9) and in ethanol. In particular the nanoparticles rebound 115.4 mg cholesterol/g polymer in buffer solution, and 57 mg cholesterol/g polymer in ethanol. The deposition of the nanoparticles on the surface of the phase inversion membranes produced devices with interesting rebinding performances towards cholesterol in buffer solution: a specific recognition of 14.09 mg cholesterol/g system (membrane and nanoparticles) was detected, indicating maintained binding capacity of supported particles as well.

Enzymatic erosion of bioartificial membranes to control drug delivery.

The preparation of an enzymatic controlled drug release system from blends of PVA/starch/alphaA, in the form of films, is described. It was shown that alphaA hydrolyses the starch within these films, resulting in a time-dependent change of the porosity in the matrix. Films were characterized by calorimetric analysis to study the interactions between the enzyme and the polymeric constituents at the molecular level. The presence of alphaA, in fact, influenced the PVA crystallization in the blends. Release tests and permeability experiments were carried out to evaluate the transport properties of the films. An increase in porosity and permeability was observed by increasing alphaA content (16-28 wt.-%). Films loaded with theophylline and caffeine were also prepared to analyze drug release properties of the matrix. Drug release kinetics were coherent with the measured changes in porosity: at higher alphaA concentrations the amount of released drug increased under the influence of diffusion and erosion processes. The results obtained are promising for the realization of drug delivery devices for a rapid release or for the release of poorly soluble drugs which usually remain entrapped in the matrix.SEM images of a PVA/starch/alphaA film before (A) and after (B) the erosion.

Acrylonitrile-acrylic acid copolymer membrane imprinted with uric acid for clinical uses.

The preparation of new polymeric membranes using molecular imprinting technology for application in blood filtration devices is described. Membranes, based on an acrylic acid-acrylonitrile copolymer, produced through phase inversion, were modified by introducing specific binding sites for uric acid into their structure. The materials prepared are intended for use to selectively remove uric acid from the blood in the case of increased serum uric acid values associated with different pathologies. The interactions at a molecular level between the membrane forming copolymer and the template were investigated by means of calorimetry, infrared spectroscopy and morphological analysis. The presence of interactions between the template and the copolymer, and a good thermal stability of the imprinted membranes were observed. In addition, the results of rebinding tests on the imprinted membranes indicated a good capacity of molecular recognition for the template and satisfactory selectivity properties towards compounds of similar structure such as theophylline. Membrane permeability values suggest their application as (ultra) haemofiltration devices. Poly(acrylonitrile-co-acrylic acid) membrane.

Poly(ester urethane) guides for peripheral nerve regeneration.

A biocompatible and elastomeric PU was synthesized from low-molecular-weight PCL as macrodiol, CMD as chain extender and HDI as chain linker for applications in the field of peripheral nerve repair. PU cast films supported in vitro attachment and proliferation of NOBEC. The in vitro adhesion and proliferation of S5Y5 neuroblastoma cells on the inner surface of uncoated, gelatin- and PL-coated PU guides were compared. Due to their superior in vitro performance, PL-coated PU guides were tested in vivo for the repair of 1.8 cm-long defects in rat sciatic nerves. The progressive regeneration was confirmed by EMG and histological analysis showing the presence of regenerating fibers in the distal stumps.

Synthesis and characterization of a novel PNIPAAm-based copolymer with hydrolysis-dependent thermosensitivity.

The aim of this work was the synthesis and characterization of a novel poly(N-isopropylacrylamide)-based copolymer, with hydrolysis-dependent thermosensitivity, for bioengineering applications. For this purpose, N-isopropylacrylamide (NIPAAm) and 2-hydroxyethylmethacrylate-6-hydroxyhexanoate (HEMAHex) monomers were chosen. The poly(NIPAAm-co-HEMAHex) copolymer was synthesized by radical polymerization. The physicochemical, mechanical, functional and biological properties of the copolymer were investigated. The physicochemical characterization confirmed that the copolymerization was successfully carried out. In addition, the newly synthesized poly(NIPAAm-co-HEMAHex) copolymer showed temperature sensitivity, with a phase separation temperature under body temperature (at 23 °C). Fourier transform infrared spectroscopy and differential scanning calorimetry results after hydrolysis tests indicated that the incorporation of the HEMAHex ester groups provides the cleavage of the lateral chain, which leads to an increase in the hydrophilicity of the copolymer and, consequently, to an increase in the lower critical solution temperature (LCST) with time. Since the LCST increases above body temperature (up to 40.4 °C), the copolymer becomes soluble again and diffuses away. It was also demonstrated that the hydrolysis occurred on the peripheral ester bond of the lateral chain, with the release of 6-hydroxyhexanoic acid, whose bioresorbibility has been reported in the literature. Therefore, the properties of this copolymer are very interesting and make it particularly attractive for biomedical applications.

Three-dimensional microfabricated scaffolds with cardiac extracellular matrix-like architecture.

In recent years, research in the field of myocardial tissue engineering has advanced thanks to the development of new biomaterials and a more clear understanding of processes that are at the basis of cardiac tissue growth. However, classical porous scaffolds developed during these years to try to reconstruct and mimic heart function have proven to be inadequate because they are not able to reproduce the typical myocardial environment. One approach to increase functionality of tissue-engineered constructs relies on attempts to mimic the microarchitecture of natural tissues, since it is well known that topology is one of the principal stimuli that cells need to activate their functions. The aim of this work was the realization of three-dimensional microfabricated scaffolds, with cardiac extracellular matrix (ECM)-like architecture. For this purpose, samples of pig myocardium were decellularized, embedded in paraffin wax and analyzed under an optical microscope, in order to evaluate the geometrical features of the cardiac ECM. On the basis of these data, a simplified model of the cardiac ECM microarchitecture was designed. Microfabricated scaffolds were realized with Soft Lithography technique, using a bioartificial blend, based on alginate, gelatin and a novel poly(N-isopropylacrylamide)-based copolymer, which we synthesized. The scaffolds were characterized in terms of topological and mechanical properties. Moreover, cell adhesion, proliferation, and differentiation tests were performed. The microfabricated scaffolds showed they matched the anisotropic mechanical properties of adult human left ventricular myocardium, while at the same time being able to promote myoblast alignment in the absence of external stimuli.

Molecularly imprinted nanoparticles with recognition properties towards a laminin H-Tyr-Ile-Gly-Ser-Arg-OH sequence for tissue engineering applications.

Nanotechnology is an emerging field that promises to revolutionize medicine and is increasingly used in tissue engineering applications. Our research group proposed for the first time molecular imprinting as a new nanotechnology for the creation of advanced synthetic support structures for cell adhesion and proliferation. The aim of this work was the synthesis and characterization of molecularly imprinted polymers with recognition properties towards a laminin peptide sequence and their application as functionalization structures in the development of bioactive materials. Nanoparticles with an average diameter of 200 nm were synthesized by precipitation polymerization of methacrylic acid in the presence of the template molecule and trimethylpropane trimethacrylate as the cross-linking agent. The imprinted nanoparticles showed good performance in terms of recognition capacity and selectivity. The cytotoxicity tests showed normal vitality of C2C12 myoblasts cultured in the medium that was put in contact with the imprinted polymers. After the deposition on the polymeric film surface, the imprinted particles maintained their specific recognition and rebinding behaviour, showing an even higher quantitative binding than free nanoparticles. Preliminary in vitro cell culture tests demonstrated the ability of functionalized materials to promote cell adhesion, proliferation and differentiation, suggesting that molecular imprinting can be used as an innovative functionalization technique.

Enzymatically crosslinked porous composite matrices for bone tissue regeneration.

Three-dimensional porous hydroxyapatite/collagen (HA/Coll) composites with a random pore structure were obtained by freeze-drying and crosslinked by an enzymatic treatment using microbial transglutaminase (mTGase). The procedure resulted in improved mechanical strength and thermal stability of the scaffolds. The scaffolds were characterized in terms of their stability (Coll release, swelling, collagenase-mediated degradation), thermal properties (thermogravimetric analysis, differential scanning calorimetry), mechanical behavior under compression and cell compatibility. Enzymatic treatment stabilized the sponges to water vapors, with measurable swelling ratio between 100% for HA/Coll/mTGase 0/100 to 5% for HA/Coll/mTGase 80/20. Weight loss in water due to Coll release was between 2 and 10% in mTGase-crosslinked samples and decreased with increasing HA content. Cultures of MG63 osteoblast-like cells and human umbilical vein endothelial cells (HUVEC) showed good adhesion and proliferation on the scaffolds, good viability (through MTT test, 100-150% of control), and good differentiation (alkaline phosphatase, up to 40 UI/L with respect to 35 UI/L for control).

Synthesis and characterization of copolymers of methylmethacrylate and 2-hydroxyethyl methacrylate for the aqueous solubilization of Paclitaxel.

The aim of the present work is the modification of a hydrophobic polymeric macromolecule, polymethylmethacrylate, by introducing hydrophilic moieties of 2-hydroxyethyl methacrylate within the polymer chain. Synthesis, characterization, and drug delivery control capabilities exerted on a highly hydrophobic drug (Paclitaxel) are illustrated. In particular, the dependency of the drug delivery kinetic on the fraction of hydrophilic units inserted in the copolymer chain was studied. Results showed that it is possible to have an increase of the kinetic delivery introducing hydrophilic units. In addition, a double control, diffusive and due to the relaxation of the molecules, on drug delivery was obtained.

Preparation and characterization of alginate/gelatin blend films for cardiac tissue engineering.

The aim of this work was the preparation of blends based on alginate and gelatin, with different weight ratio, to combine the advantages of these two natural polymers for application in cardiac tissue engineering. The physicochemical characterization, performed by Fourier transform infrared spectroscopy, differential scanning calorimetry and thermogravimetric analysis, revealed a good miscibility and the presence of interactions among the functional groups of pure biopolymers. Concerning the swelling and degradation tests, performed in different solutions simulating body fluids, both swelling degree and weight losses were higher in phosphate buffer saline (PBS) and for the blends with a higher content of gelatin. These results indicated a better stability of the blends in cell culture medium than in PBS and suggested a mainly hydrolytic degradation process. Cell culture tests, carried out using C2C12 myoblasts, showed a good cell proliferation for all the blends containing more than 60% of gelatin, with the alginate/gelatin 20:80 showing the best response. The same blend was the only one on which cell differentiation was observed. The results obtained in the biological characterization allow to select the alginate/gelatin 20:80 blend as a suitable material to prepare scaffolds for myocardial tissue engineering.

Chitosan/gelatin blends for biomedical applications.

Blends between chitosan (CS) and gelatin (G) with various compositions (CS/G 0/100 20/80, 40/60, 60/40, 100/0 w/w) were produced as candidate materials for biomedical applications. Dehydro-thermal crosslinking was adopted to promote the formation of amide and ester bonds between the macromolecules ((CS/G)-t). The effect of composition and crosslinking on the physico-chemical properties of the samples was evaluated by scanning electron microscopy, thermogravimetry, contact angle measurements, dissolution and swelling tests. Mechanical properties of (CS/G)-t samples were also determined through stress-strain and creep-recovery tests. The elastic moduli of dry blend samples showed a positive deviation from the additive law of the in-series model, because of interactions and/or chemical bonds between components. The comparison between the elastic moduli of wet samples and those of different human tissues showed that (CS/G)-t substrates can be suitable for soft-tissue reconstruction. (CS/G)-t two-dimensional scaffolds were fabricated by micro-molding, based on the use of a polydimethylsiloxane mould to create patterns with micro-scale resolution on cast films. Biocompatibility of (CS/G)-t samples was studied by means of cell tests using NIH-3T3 fibroblasts. Finally, the evaluation of the affinity of (CS/G)-t samples towards neuroblastoma cells adhesion and proliferation was performed, showing promising results for the blend containing 80 wt % gelatin.

Genipin-crosslinked chitosan/gelatin blends for biomedical applications.

Blends between chitosan (CS) and gelatin (G) with various compositions (CS/G 0/100 20/80, 40/60, 60/40 100/0 w/w) were produced, as candidate materials for biomedical applications. Different amounts of genipin (0.5 wt.% and 2.5 wt.%) were used to crosslink CS/G blends, promoting the formation of amide and tertiary amine bonds between the macromolecules and the crosslinker. The effects of composition and crosslinking on the physico-chemical properties of samples were evaluated by infrared analysis, thermogravimetry, contact angle measurements, dissolution and swelling tests. Mechanical properties of crosslinked samples were also determined through stress-strain and creep tests: samples stiffness increased with increasing the crosslinker amount and the CS content. Blend composition affected mouse fibroblasts adhesion and proliferation on substrates, depending on the crosslinker amount. Finally, crosslinked CS/G blends containing 80 wt.% G were found to support neuroblastoma cells adhesion and proliferation which made them promising candidates for uses in the field of nerve regeneration.

Poly(3-hydroxybutyrate-co-3-hydroxyvalerate)/poly(epsilon-caprolactone) blends for tissue engineering applications in the form of hollow fibers.

In this work, hollow fibers to be used as guides for tissue engineering applications were produced by dry-jet-wet spinning of poly(3-hydroxybutyrate-co-3-hydroxyvalerate)/poly(epsilon-caprolactone) (PHBHV/PCL) solutions in chloroform with various weight ratios between the components (PHBHV/PCL 100/0; 80/20; 60/40; 50/50; 40/60; 20/80; 0/100 w/w). Fibers obtained from PHBHV/PCL blends had a low degree of surface and bulk porosity, depending on composition. Physicochemical characterization involving scanning electron microscopy and differential scanning calorimetry (DSC) showed that PHBHV/PCL blends are compatible. Interactions between blend components were studied by Fourier transform infrared total reflectance spectroscopy, DSC analysis, and polarized optical microscopy analysis. Homogeneity of blend composition was assessed by IR-chemical imaging analysis. PHBHV/PCL samples were found to be weakly hydrophilic and their biocompatibility was proved by in vitro tests using mouse fibroblasts. Mechanical properties of PHBHV/PCL blends were investigated by stress-strain tests, showing an increasing ductility of blend samples with increasing PCL amount. Hollow fibers supported fibroblasts attachment and proliferation depending on composition and porosity degree.