Protein Attachment Mechanism for Improved Functionalization of Affinity Monolith Chromatography (AMC).

This work aims at understanding the attachment mechanisms and stability of proteins on a chromatography medium to develop more efficient functionalization methodologies, which can be exploited in affinity chromatography. In particular, the study was focused on the understanding of the attachment mechanisms of bovine serum albumin (BSA), used as a ligand model, and protein G on novel amine-modified alumina monoliths as a stationary phase. Protein G was used to develop a column for antibody purification. The results showed that, at lower protein concentrations (i.e., 0.5 to 1.0 mg·mL-1), protein attachment follows a 1st-order kinetics compatible with the presence of covalent binding between the monolith and the protein. At higher protein concentrations (i.e., up to 10 mg·mL-1), the data preferably fit a 2nd-order kinetics. Such a change reflects a different mechanism in the protein attachment which, at higher concentrations, seems to be governed by physical adsorption resulting in a multilayered protein formation, due to the presence of ligand aggregates. The threshold condition for the prevalence of physical adsorption of BSA was found at a concentration higher than 1.0 mg·mL-1. Based on this result, protein concentrations of 0.7 and 1.0 mg·mL-1 were used for the functionalization of monoliths with protein G, allowing a maximum attachment of 1.43 mg of protein G/g of monolith. This column was then used for IgG binding-elution experiments, which resulted in an antibody attachment of 73.5% and, subsequently, elution of 86%, in acidic conditions. This proved the potential of the amine-functionalized monoliths for application in affinity chromatography.

Production of α-Tocopherol-Chitosan Nanoparticles by Membrane Emulsification.

α-tocopherol (α-T) has the highest biological activity with respect to the other components of vitamin E; however, conventional formulations of tocopherol often fail to provide satisfactory bioavailability due to its hydrophobic characteristics. In this work, α-tocopherol-loaded nanoparticles based on chitosan were produced by membrane emulsification (ME). A new derivative was obtained by the cross-linking reaction between α-T and chitosan (CH) to preserve its biological activity. ME was selected as a method for nanoparticle production because it is recognized as an innovative and sustainable technology for its uniform-particle production with tuned sizes and high encapsulation efficiency (EE%), and its ability to preserve the functional properties of bioactive ingredients operating in mild conditions. The reaction intermediates and the final product were characterized by 1HNMR, Fourier-transform infrared spectroscopy (FTIR) and differential scanning calorimetry (DSC), while the morphological and dimensional properties of the nanoparticles were analyzed using electronic scanning microscopy (SEM) and dynamic light scattering (DLS). The results demonstrated that ME has high potential for the development of α-tocopherol-loaded nanoparticles with a high degree of uniformity (PDI lower than 0.2), an EE of almost 100% and good mechanical strength, resulting in good candidates for the production of functional nanostructured materials for drug delivery. In addition, the chemical bonding between chitosan and α-tocopherol allowed the preservation of the antioxidant properties of the bioactive molecule, as demonstrated by an enhanced antioxidant property and evaluated through in vitro tests, with respect to the starting materials.

Multifunctional Membranes Based on ß-Glucans and Chitosan Useful in Wound Treatment.

In this work, bio-based membranes prepared using a crosslinked ß-glucans-chitosan dispersed in the chitosan matrix useful in promoting wound healing were studied for the first-time. Wound healing is a process that includes sequential steps designed to restore the structure and function of damaged cells and tissue. To minimize damage and the risk of infection during the healing process and to promote restoration of the integrity of damaged tissue, the wound should be dressed. Generally, according to their function in the wound, dressings are classified on the basis of type of material and physical form. The substances used to make a dressing are generally natural polymers such as hydrocolloids, alginates, polyurethane, collagen, chitosan, pectin and hyaluronic acid. The combination of polymeric substances, with antibacterial and antioxidant properties, could be exploited in the biomedical field for the development of biocompatible materials able to act as a barrier between the wound and the external environment, protecting the site from bacterial contamination and promoting healing. To this aim, bio-based membranes were prepared by the phase inversion induced by solvent evaporation, using the crosslinked ß-glucans-chitosan obtained by esterification reactions as a functional additive in the chitosan membrane. The reaction intermediates and the final products were characterized by Fourier transform infrared spectroscopy (FTIR) and differential scanning calorimetry (DSC) while the morphological properties of membranes were analyzed using electronic scanning microscopy (SEM). The chemical bonding between chitosan and ß-glucans allowed for the obtainment of a better dispersion of the combined new material into the membrane's matrix and as a consequence, an enhanced antibacterial property evaluated through in vitro tests, with respect to the starting materials.

Chitosan Membranes Filled with Cyclosporine A as Possible Devices for Local Administration of Drugs in the Treatment of Breast Cancer.

The aim of this work is the design, preparation and characterization of membranes based on cyclosporine A (CsA) and chitosan carboxylate (CC) to be used as an implantable subcutaneous medical device for a prolonged therapeutic effect in the treatment of breast cancer. The choice to use CsA is due to literature data that have demonstrated its possible antitumor activity on different types of neoplastic cells. To this end, CsA was bound to CC through an amidation reaction to obtain a prodrug to be dispersed in a chitosan-based polymeric membrane. The reaction intermediates and the final product were characterized by Fourier transform infrared spectroscopy (FT-IR) and proton nuclear magnetic resonance (1H-NMR). Membranes were analyzed by differential scanning calorimetry (DSC) and scanning electron microscopy (SEM). The data obtained showed the effective formation of the amide bond between CsA and CC and the complete dispersion of CsA inside the polymeric membrane. Furthermore, preliminary tests, conducted on MDA-MB-231, a type of breast cancer cell line, have shown a high reduction in the proliferation of cancer cells. These results indicate the possibility of using the obtained membranes as an interesting strategy for the release of cyclosporin-A in breast cancer patients.

Production of Plant-Derived Oleuropein Aglycone by a Combined Membrane Process and Evaluation of Its Breast Anticancer Properties.

Natural products and herbal therapies represent a thriving field of research, but methods for the production of plant-derived compounds with a significative biological activity by synthetic methods are required. Conventional commercial production by chemical synthesis or solvent extraction is not yet sustainable and economical because toxic solvents are used, the process involves many steps, and there is generally a low amount of the product produced, which is often mixed with other or similar by-products. For this reason, alternative, sustainable, greener, and more efficient processes are required. Membrane processes are recognized worldwide as green technologies since they promote waste minimization, material diversity, efficient separation, energy saving, process intensification, and integration. This article describes the production, characterization, and utilization of bioactive compounds derived from renewable waste material (olive leaves) as drug candidates in breast cancer (BC) treatment. In particular, an integrated membrane process [composed by a membrane bioreactor (MBR) and a membrane emulsification (ME) system] was developed to produce a purified non-commercially available phytotherapic compound: the oleuropein aglycone (OLA). This method achieves a 93% conversion of the substrate (oleuropein) and enables the extraction of the compound of interest with 90% efficiency in sustainable conditions. The bioderived compound exercised pro-apoptotic and antiproliferative activities against MDA-MB-231 and Tamoxifen-resistant MCF-7 (MCF-7/TR) cells, suggesting it as a potential agent for the treatment of breast cancer including hormonal resistance therapies.

Application of NF Polymeric Membranes for Removal of Multicomponent Heat-Stable Salts (HSS) Ions from Methyl Diethanolamine (MDEA) Solutions.

This study presents an efficient and scalable process for removing the heat-stable salts (HSS) ions from amine solution while recovering methyl diethanolamine (MDEA) solution for its reuse in gas sweetening plants. The presence of HSS in the amine solution causes the loss of solvent capacity, foaming, fouling, and corrosion in gas sweetening units so their removal is crucial for a more well-performing process. Furthermore, the recovery of the amine solution can make the sweetening step a more sustainable process. In this study, for the first time, the removal of a multicomponent mixture of HSS from MDEA solution was investigated via a nanofiltration process using flat-sheet NF-3 membranes. The impact of operating parameters on salts and amine rejection, and flux, including the operating pressure, HSS ions concentration, and MDEA concentration in the feed solution was investigated. Results based on the nanofiltration of an amine stream with the same composition (45 wt.% MDEA solution) as that circulating in a local gas refinery (Ilam Gas refinery), demonstrated a removal efficiency of HSS ions in the range from 75 to 80% and a MDEA rejection of 0% indicating the possibility of reusing this stream in the new step of gas sweetening.

Membrane Processes for Microplastic Removal.

Plastic pollution of the aquatic environment is a major concern considering the disastrous impact on the environment and on human beings. The significant and continuous increase in the production of plastics causes an enormous amount of plastic waste on the land entering the aquatic environment. Furthermore, wastewater treatment plants (WWTPs) are reported as the main source of microplastic and nanoplastic in the effluents, since they are not properly designed for this purpose. The application of advanced wastewater treatment technologies is mandatory to avoid effluent contamination by plastics. A concrete solution can be represented by membrane technologies as tertiary treatment of effluents in integrated systems for wastewater treatment, in particular, for the plastic particles with a smaller size (< 100 nm). In this review, a survey of the membrane processes applied in the plastic removal is analyzed and critically discussed. From the literature analysis, it was found that the removal of microplastic by membrane technology is still insufficient, and without the use of specially designed approaches, with the exception of membrane bioreactors (MBRs).

Influence of Lipase Immobilization Mode on Ethyl Acetate Hydrolysis in a Continuous Solid-Gas Biocatalytic Membrane Reactor.

Solid-gas biocatalysis was performed in a specially designed continuous biocatalytic membrane reactor (BMR). In this work, lipase from Candida rugosa (LCR) and ethyl acetate in vapor phase were selected as model enzyme and substrate, respectively, to produce acetic acid and ethanol. LCR was immobilized on functionalized PVDF membranes by using two different kinds of chemical bond: electrostatic and covalent. Electrostatic immobilization of LCR was carried out using a membrane functionalized with amino groups, while covalent immobilization was carried out using membrane, with or without surface-immobilized polyacrylamide (PAAm) microgels, functionalized with aldehyde groups. These biocatalytic membranes were tested in a solid-gas BMR and compared in terms of enzyme specific activity, catalytic activity, and volumetric reaction rate. Results indicated that lipase covalently immobilized is more effective only when the immobilization is mediated by microgels, showing catalytic activity doubled with respect to the other system with covalently bound enzyme (4.4 vs 2.2 µmol h-1). Enzyme immobilized by ionic bond, despite a lower catalytic activity (3.5 vs 4.4 µmol h-1), showed the same specific activity (1.5 mmol·h-1·g-1ENZ) of the system using microgels, due to a higher enzyme degree of freedom coupled with an analogously improved enzyme hydration. Using the optimized operating conditions regarding immobilized enzyme amount, ethyl acetate, and molar water flow rate, all three BMRs showed continuous catalytic activity for about 5 months. On the contrary, the free enzyme (in water/ethyl acetate emulsion) at 50 °C was completely inactive and at 30 °C (temperature optimum) has a specific activity 2 orders of magnitude lower (8.4 × 10-2 mmol h-1 g-1) than the solid-gas biocatalytic membrane reactor. To the best of our knowledge, this is the first example of solid-gas biocatalysis, working in the gaseous phase in which a biocatalytic membrane reactor, with the enzyme/substrate system lipase/ethyl acetate, was used.

Chemical Vapor Deposition of Photocatalyst Nanoparticles on PVDF Membranes for Advanced Oxidation Processes.

The chemical binding of photocatalytic materials, such as TiO2 and ZnO nanoparticles, onto porous polymer membranes requires a series of chemical reactions and long purification processes, which often result in small amounts of trapped nanoparticles with reduced photocatalytic activity. In this work, a chemical vapor deposition technique was investigated in order to allow the nucleation and growth of ZnO and TiO2 nanoparticles onto polyvinylidene difluoride (PVDF) porous membranes for application in advanced oxidation processes. The thickness of obtained surface coatings by sputtered nanoparticles was found to depend on process conditions. The photocatalytic efficiency of sputtered membranes was tested against both a model drug and a model organic pollutant in a small continuous flow reactor.

Integration of organic electrochemical transistors and immuno-affinity membranes for label-free detection of interleukin-6 in the physiological concentration range through antibody-antigen recognition.

We demonstrate the label-free and selective detection of interleukin-6 (IL-6), a key cell-signaling molecule in biology and medicine, by integrating an OECT with an immuno-affinity regenerated cellulose membrane. The objective of the membrane is to increase the local concentration of IL-6 at the sensing electrode and, thereby, enhance the device response for concentrations falling within the physiological concentration range of cytokines. The OECT gate electrode is functionalized with an oligo(ethylene glycol)-terminated self-assembled alkanethiolate monolayer (SAM) for both the immobilization of anti IL-6 antibodies and the inhibition of non-specific biomolecule binding. The OECT gate/electrolyte interface is exploited for the selective detection of IL-6 through the monitoring of antigen-antibody binding events occurring at the gate electrode.

Development of biohybrid immuno-selective membranes for target antigen recognition.

Membranes are gaining increasing interest in solid-phase analytical assay and biosensors applications, in particular as functional surface for bioreceptors immobilization and stabilization as well as for the concentration of target molecules in microsystems. In this work, regenerated cellulose immuno-affinity membranes were developed and they were used for the selective capture of interleukin-6 (IL-6) as targeted antigen. Protein G was covalently linked on the membrane surface and it was successfully used for the oriented site-specific antibody immobilization. The antibody binding capacity of the protein G-coupled membrane was evaluated. The specific anti IL-6 antibody was immobilized and a quantitative analysis of the amount of IL-6 captured by the immuno-affinity membrane was performed. The immobilization procedure was optimized to eliminate the non-specific binding of antigen on the membrane surface. Additionally, the interaction between anti IL-6 antibody and protein G was stabilized by chemical cross-linking with glutaraldehyde and the capture ability of immuno-affinity membranes, with and without the cross-linker, was compared. The maximum binding capacity of the protein G-coupled membrane was 43.8µg/cm2 and the binding efficiency was 88%. The immuno-affinity membranes showed a high IL-6 capture efficiency at very low antigen concentration, up to a maximum of 91%, the amount of captured IL-6 increased linearly as increasing the initial concentration. The cross-linked surface retained the antigen binding capacity demonstrating its robustness in being reused, without antibody leakage or reduction in antibody binding capacity. The overall results demonstrated the possibility of a reliable application of the immuno-affinity membrane developed for biosensors and bioassays also in multiple use.

Microencapsulation by Membrane Emulsification of Biophenols Recovered from Olive Mill Wastewaters.

Biophenols are highly prized for their free radical scavenging and antioxidant activities. Olive mill wastewaters (OMWWs) are rich in biophenols. For this reason, there is a growing interest in the recovery and valorization of these compounds. Applications for the encapsulation have increased in the food industry as well as the pharmaceutical and cosmetic fields, among others. Advancements in micro-fabrication methods are needed to design new functional particles with target properties in terms of size, size distribution, and functional activity. This paper describes the use of the membrane emulsification method for the fine-tuning of microparticle production with biofunctional activity. In particular, in this pioneering work, membrane emulsification has been used as an advanced method for biophenols encapsulation. Catechol has been used as a biophenol model, while a biophenols mixture recovered from OMWWs were used as a real matrix. Water-in-oil emulsions with droplet sizes approximately 2.3 times the membrane pore diameter, a distribution span of 0.33, and high encapsulation efficiency (98% ± 1% and 92% ± 3%, for catechol and biophenols, respectively) were produced. The release of biophenols was also investigated.

Influence of protein bulk properties on membrane surface coverage during immobilization.

Biomolecules immobilization is a key factor for many biotechnological applications. For this purpose, the covalent immobilization of bovine serum albumin (BSA), lipase from Candida rugosa and protein G on differently functionalized regenerated cellulose membranes was investigated. Dynamic light scattering and electrophoresis measurements carried out on biomolecules in solution indicated the presence of monomers, dimers and trimers for both BSA and protein G, while large aggregates were observed for lipase. The immobilization rate and the surface coverage on functionalized regenerated cellulose membranes were studied as a function of biomolecule concentration. Results indicated that the saturation coverage of BSA and protein G was concentration independent (immobilized protein amount of 2.40±0.03mg/g and 2.65±0.07mg/g, respectively). Otherwise, a different immobilization kinetics trend was obtained for lipase, for which the immobilized amount increases as a function of time without reaching a saturation value. Atomic force microscopy (AFM) micrographs showed the formation of monolayers for both BSA and protein G on the membrane surface, while a multilayer structure is found for lipase, in agreement with the trends observed in the related immobilization kinetics. As a result, the morphology of the proteins layer on the membrane surface seems to be strictly dependent on the proteins behavior in solution. Besides, the surface coverage has been described for BSA and protein G by the pseudo second order models, the results indicating the surface reaction as the controlling step of immobilization kinetics. Finally, enzyme activity and binding capacity studies indicated the preservation of the biomolecule functional properties.

Preparation of Pd-Loaded Hierarchical FAU Membranes and Testing in Acetophenone Hydrogenation.

Pd-loaded hierarchical FAU (Pd-FAU) membranes, containing an intrinsic secondary non-zeolitic (meso)porosity, were prepared and tested in the catalytic transfer hydrogenation of acetophenone (AP) to produce phenylethanol (PE), an industrially relevant product. The best operating conditions were preliminarily identified by testing different solvents and organic hydrogen donors in a batch hydrogenation process where micron-sized FAU seeds were employed as catalyst support. Water as solvent and formic acid as hydrogen source resulted to be the best choice in terms of conversion for the catalytic hydrogenation of AP, providing the basis for the design of a green and sustainable process. The best experimental conditions were selected and applied to the Pd-loaded FAU membrane finding enhanced catalytic performance such as a five-fold higher productivity than with the unsupported Pd-FAU crystals (11.0 vs. 2.2 mgproduct gcat(-1)·h(-1)). The catalytic performance of the membrane on the alumina support was also tested in a tangential flow system obtaining a productivity higher than that of the batch system (22.0 vs. 11.0 mgproduct gcat(-1)·h(-1)).

Selective separation of copper(II) and nickel(II) from aqueous media using the complexation-ultrafiltration process.

The polyethylenimine (PEI) as complexing agent was used to study the complexation-ultrafiltration (CP-UF) process in the selective removal of Cu(II) from Ni(II) contained in aqueous media. Preliminary tests showed that optimal chemical conditions for Cu(II) and Ni(II) complexation by the PEI polymer were pH>6.0 and 8.0, respectively, and polymer/metal weight ratio of 3.0 and 6.0, respectively. The effect of some important operating parameters on process selectivity was studied by performing UF tests at different parameters: pH, polymer/metal weight ratio, transmembrane pressure (TMP), and membrane cut-off in a batch experimental set-up. It was observed that process selectivity was achieved by choosing the pH value for obtaining a preferential copper complexation (pH 6.0), and the polymer/metal ratio needed to bound only the copper ion (3.0). The selective separation by UF tests was performed by using both a laboratory aqueous solution and a real aqueous effluent (water from Emoli torrent, Rende (CS)). The Iris 30 membrane at TMP of 200 kPa (2 bar) for both aqueous media gave the best results. A complete nickel recovery was reached, and copper recovery was the highest for this membrane (94% and 92%). Besides at this pressure, a lower water amount was needed to obtain total nickel recovery by diafiltration. A little higher membrane fouling was obtained by using the river effluent due to the presence of dissolved organic and inorganic matter.