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The delivery of peptides via non-invasive administration routes remains a challenge to be addressed. In this regard, chitosan nanoparticles (CS NPs) have shown promise. However, their current batch preparation methods (ionotropic gelation, polyelectrolyte complexing, emulsification solvent diffusion or micro emulsification) have proven difficult to scale up. Here, we established a microfluidic-assisted ionotropic gelation method for the manufacturing of CS NPs, ionically crosslinked with sodium tripolyphosphate (TPP), and loaded with a model peptide, Argireline. The microfluidic process was optimized through a design of experiments approach. CS concentration and pH have the greatest effect on particle size, while CS and TPP concentrations and pH on PDI. The optimum formulation was successfully loaded with the peptide (90 % EE) and characterized by a size of 186.0 ± 1.0 nm and a PDI of 0.440 ± 0.002. Subsequently, Argireline-loaded CS-TPP NPs suspension was converted into a gel for a potential topical application, considering the non-toxic, biocompatible, and biodegradable properties of the components used in the formulation. The NPs gel demonstrated appropriate mechanical properties for Argireline transdermal delivery, along with improved control over its release and enhanced skin permeation for up to 48 h, compared to NPs suspension and free drug solution. Hence, this study demonstrated that the microfluidic-assisted ionotropic gelation method could be an easy-scalable platform for the manufacturing of peptide-loaded CS-TPP NPs which could be potentially applied for the transdermal delivery of biologics.

Here, we present novel biocompatible poly(butylene trans-1,4-cyclohexanedicarboxylate) (PBCE)-based random copolymer nanostructured scaffolds with tailored stiffness and hydrophilicity. The introduction of a butylene diglycolate (BDG) co-unit, containing ether oxygen atoms, along the PBCE chain remarkably improved the hydrophilicity and chain flexibility. The copolymer containing 50 mol% BDG co-units (BDG50) and the parent homopolymer (PBCE) were synthesized and processed as electrospun scaffolds and compression-molded films, added for the sake of comparison. We performed thermal, wettability, and stress–strain measures on the PBCE-derived scaffolds and films. We also conducted biocompatibility studies by evaluating the adhesion and proliferation of multipotent mesenchymal/stromal cells (hBM-MSCs) on each polymeric film and scaffold. We demonstrated that solid-state properties can be tailored by altering sample morphology besides chemical structure. Thus, scaffolds were characterized by a higher hydrophobicity and a lower elastic modulus than the corresponding films. The three-dimensional nanostructure conferred a higher adsorption protein capability to the scaffolds compared to their film counterparts. Finally, the PBCE and BDG50 scaffolds were suitable for the long-term culture of hBM-MSCs. Collectively, the PBCE homopolymer and copolymer are good candidates for tissue engineering applications.

In membrane-based water purification technology, control of the membrane pore structure is fundamental to defining its performance. The present study investigates the effect of the preparation conditions on the final pore size distribution and on the dye removal efficiency of cellulose acetate membranes. The membranes were fabricated by means of phase inversion (using different speeds of film casting and different thicknesses of the casted solution) and introducing modifications in the preparation conditions, such as the use of a coagulation bath instead of pure water and the addition of a surfactant as a solution additive. Both isotropic and anisotropic membranes could be fabricated, and the membranes’ pore size, porosity, and water permeability were found to be greatly influenced by the fabrication conditions. The removal capacity towards different types of water contaminants was investigated, considering, as model dyes, Azure A and Methyl Orange. Azure A was removed with higher efficiency due to its better chemical affinity for cellulose acetate, and for both dyes the uptake could be fitted using a pseudo-second order model, evidencing that the rate-limiting step is chemisorption involving valency forces through the sharing or exchange of electrons between the dye and the membrane.

The delivery of therapeutics across biological membranes (e.g., mucosal barriers) by avoiding invasive routes (e.g., injection) remains a challenge in the pharmaceutical field. As such, there is the need to discover new compounds that act as drug permeability enhancers with a favorable toxicological profile. A valid alternative is represented by the class of sugar-based ester surfactants. In this study, sucrose and lactose alkyl aromatic and aromatic ester derivatives have been synthesized with the aim to characterize them in terms of their physicochemical properties, structure–property relationship, and cytotoxicity, and to test their ability as permeability enhancer agents across Calu-3 cells. All of the tested surfactants showed no remarkable cytotoxic effect on Calu-3 cells when applied both below and above their critical micelle concentration. Among the explored molecules, lactose p-biphenyl benzoate (URB1420) and sucrose p-phenyl benzoate (URB1481) cause a reversible ~30% decrease in transepithelial electrical resistance (TEER) with the respect to the basal value. The obtained result matches with the increased in vitro permeability coefficients (Papp) calculated for FTIC-dextran across Calu-3 cells in the presence of 4 mM solutions of these surfactants. Overall, this study proposes sucrose- and lactose-based alkyl aromatic and aromatic ester surfactants as novel potential and safe permeation enhancers for pharmaceutical applications.

Skin disorders are widespread around the world, affecting people of all ages, and oxidative stress represents one of the main causes of alteration in the normal physiological parameters of skin cells. In this work, we combined a natural protein, fibroin, with antioxidant compounds extracted in water from pomegranate waste. We demonstrate the effective and facile fabrication of bioactive and eco-sustainable films of potential interest for skin repair. The blended films are visually transparent (around 90%); flexible; stable in physiological conditions and in the presence of trypsin for 12 days; able to release the bioactive compounds in a controlled manner; based on Fickian diffusion; and biocompatible towards the main skin cells, keratinocytes and fibroblasts. Furthermore, reactive oxygen species (ROS) production tests demonstrated the high capacity of our films to reduce the oxidative stress induced in cells, which is responsible for various skin diseases.

In spite of an extensive body of academic initiatives and innovative products, the toolkit of wound dressing has always revolved around a few common concepts such as adhesive patches and stitches and their variants. Our work aims at an alternative solution for an immediate restitutio ad integrum of the mechanical functionality in cutaneous repairs. We describe the fabrication and the application of electrospun mats of bioactive nanofibers all made of biocompatible components such as a natural polysaccharide and a cyanine dye for use as laser-activatable plasters, resembling the ultrastructure of human dermis. In particular, we investigate their morphological features and mechanical moduli under conditions of physiological relevance, and we test their use to bind a frequent benchmark of connective tissue as rabbit tendon and a significant case of clinical relevance as human dermis. Altogether, our results point to the feasibility of a new material for wound dressing combining translational potential, strength close to human dermis, extensibility exceeding 15% and state-of-art adhesive properties.

In recent years, several studies have focused their attention on the preparation of biocompatible and biodegradable nanocarriers of potential interest in the biomedical field, ranging from drug delivery systems to imaging and diagnosis. In this regard, natural biomolecules—such as proteins—represent an attractive alternative to synthetic polymers or inorganic materials, thanks to their numerous advantages, such as biocompatibility, biodegradability, and low immunogenicity. Among the most interesting proteins, keratin extracted from wool and feathers, as well as fibroin extracted from Bombyx mori cocoons, possess all of the abovementioned features required for biomedical applications. In the present review, we therefore aim to give an overview of the most important and efficient methodologies for obtaining drug-loaded keratin and fibroin nanoparticles, and of their potential for biomedical applications.

High-resolution ultrasound spectroscopy (HR-US) is a spectroscopic technique using ultrasound waves at high frequencies to investigate the structural properties of dispersed materials. This technique is able to monitor the variation of ultrasound parameters (sound speed and attenuation) due to the interaction of ultrasound waves with samples as a function of temperature and concentration. Despite being employed for the characterization of several colloidal systems, there is a lack in the literature regarding the comparison between the potential of HR-US for the determination of phospholipid thermal transitions and that of other common techniques both for loaded or unloaded liposomes. Thermal transitions of liposomes composed of pure phospholipids (dimyristoylphosphatidylcholine, DMPC; dipalmitoylphosphatidylcholine, DPPC and distearoylphosphatidylcholine, DSPC), cholesterol and their mixtures were investigated by HR-US in comparison to the most commonly employed microcalorimetry (mDSC) and dynamic light scattering (DLS). Moreover, tramadol hydrochloride, caffeine or miconazole nitrate as model drugs were loaded in DPPC liposomes to study the effect of their incorporation on thermal properties of a phospholipid bilayer. HR-US provided the determination of phospholipid sol-gel transition temperatures from both attenuation and sound speed that are comparable to those calculated by mDSC and DLS techniques for all analysed liposomal dispersions, both loaded and unloaded. Therefore, HR-US is proposed here as an alternative technique to determine the transition temperature of phospholipid membrane in liposomes.

In this work we report the preparation and characterization of free-standing keratin-based films containing Au/Ag nanorods. The effect of nanorods surface chemistry on the optical and mechanical properties of keratin composite films is fully investigated. Colloid nanorods confer to the keratin films interesting color effects due to plasmonic absorptions of the metal nanostructures. The presence of metal NRs induces also substantial change in the protein fluorescence emission. In particular, the relative contribution of the ordered-protein aggregates emission is enhanced by the presence of cysteine and thus strictly related to the surface chemistry of nanorods. The presence of more packed supramolecular structures in the films containing metal nanorods (in particular cysteine modified ones) is confirmed by ATR measurements. In addition, the films containing nanorods show a higher Young's modulus compared to keratin alone and again the effect is more pronounced for cysteine modified nanorods. Collectively, the reported results indicate the optical and mechanical properties of keratin composites films are related to a common property and can be tuned simultaneously, paving the way to the optimization and improvement of their performances and enhancing the exploitation of keratin composites in highly technological optoelectronic applications.

In this work, keratin sponges were prepared by freeze-drying method and tested for adsorption of Azure A and Methyl Orange dyes. The obtained materials showed a porosity of 99.92% and a mean pore size dimension of about 91 μm. The use of oxidized sucrose with a heating treatment at 150°C was demonstrated to be a useful crosslinking procedure alternative to the conventional glutaraldehyde. Keratin sponges showed a maximum adsorption capacity of 0.063 and of 0.037 mmol/g for Azure A and Methyl Orange, respectively. The absorption of the cationic dye Azure A onto keratin sponges was better described by Freundlich model while the isotherm adsorption of the anionic Methyl Orange was found to correlate with both Langmuir and Freundlich models. The mean free energies evaluated by using the D-R model indicated a physisorption of Methyl Orange and a chemisorptions of Azure A onto keratin sponges. Finally, the functionalization of keratin sponges with Zn Al hydrotalcites nanoparticles did not affect the adsorption performances of the adsorbent toward the cationic dye Azure A, while it improved those toward the anionic Methyl Orange, increasing the related removal efficiencies from 43 to 96%. Collectively, the reported data indicates that the combination of keratin with hydrotalcites nanoparticles is a good strategy to obtain more functional adsorbent materials of potential interest for water treatment and purification.

Federica Foglietta,1 Giulio C Spagnoli,2,3 Manuele Giuseppe Muraro,2 Marco Ballestri,4 Andrea Guerrini,4 Claudia Ferroni,4 Annalisa Aluigi,4 Giovanna Sotgiu,4 Greta Varchi4 1Department of Drug Science & Technology, University of Torino, Torino, Italy; 2Department of Biomedicine, University of Basel and University Hospital of Basel, Basel, Switzerland; 3Institute of Translational Pharmacology, CNR, Rome, Italy; 4Institute for Organic Synthesis & Photoreactivity, National Research Council, Bologna, Italy Purpose: Taxanes are highly effective cytotoxic drugs for progressing breast cancer treatment. However, their poor solubility and high toxicity urge the development of innovative formulations of potential clinical relevance. Materials and methods: By using a simple and straightforward aggregation method, we have generated paclitaxel (PTX) loaded in keratin nanoparticles (KER-NPs-PTX). Their activities were tested against human breast cancer MCF-7 and MDA MB 231 cell lines in conventional two-dimensional (2D) cultures and in a dynamic three-dimensional (3D) model with perfused bioreactor (p3D). Moreover, KER-NPs-PTX activity was compared to free PTX and to PTX loaded in albumin nanoparticles (HSA-NPs-PTX). Cell viability, induction of apoptosis, and gene expression analysis were used as readouts. Results: In 2D cultures, KER-NPs-PTX was able to inhibit tumor cell viability and to induce apoptosis similarly to PTX and HSA-NPs-PTX. In the p3D model, a lower sensitivity of tumor cells to treatments was observed. Importantly, only KER-NPs-PTX was able to induce a statistically significant increase in apoptotic cell percentages following 24 h treatment for MCF-7 (16.7±4.0 early and 11.3±4.9 late apoptotic cells) and 48 h treatment for MDA MB 231 (21.3±11.2 early and 10.5±1.8 late apoptotic cells) cells. These effects were supported, at least for MCF-7 cells, by significant increases in the expression of proapoptotic BAX gene (5.8±0.5) 24 h after treatment and of cleaved caspase 3 (CC3) protein. Conclusion: KER-NPs-PTX, generated by a simple procedure, is characterized by high water solubility and enhanced PTX-loading ability, as compared to HSA-NPs-PTX. Most importantly, it appears to be able to exert effective anticancer activities on breast cancer cells cultured in 2D or in p3D models. Keywords: keratin, nanoparticles, paclitaxel, breast cancer, perfused three-dimensional models

In this study, a bio-based polymeric system loaded with fruit by-products was developed. It was based on silk fibroin produced by the silkworm Bombyx mori and pomegranate peel powder, selected as active agent. The weight ratio between fibroin and pomegranate powder was 30:70. Pads also contained 20% w/w of glycerol vs. fibroin to induce water insolubility. Control systems, consisting of only fibroin and glycerol, were produced as reference. Both control and active systems were characterized for structural and morphological characterization (Fourier-transform infrared spectroscopy and optical microscope), antioxidant properties and antimicrobial activity against two foodborne spoilage microorganisms. Results demonstrate that under investigated conditions, an active system was obtained. The pad showed a good water stability, with weight loss of about 28% due to the release of the active agent and not to the fibroin loss. In addition, this edible system has interesting antioxidant and antimicrobial properties. In particular, the pad based on fibroin with pomegranate peel recorded an antioxidant activity of the same order of magnitude of that of vitamin C, which is one of the most well-known antioxidant compounds. As regards the antimicrobial properties, results underlined that pomegranate peel in the pad allowed maintaining microbial concentration around the same initial level (104 CFU/mL) for more than 70 h of monitoring, compared to the control system where viable cell concentration increased very rapidly up to 108 CFU/mL.

In recent years there has been a growing interest in the use of proteins as biocompatible and environmentally friendly biomolecules for the design of wound healing and drug delivery systems. Keratin is a fascinating protein, obtainable from several keratinous biomasses such as wool, hair or nails, with intrinsic bioactive properties including stimulatory effects on wound repair and excellent carrier capability. In this work keratin/poly(butylene succinate) blend solutions with functional properties tunable by manipulating the polymer blending ratios were prepared by using 1,1,1,3,3,3-hexafluoroisopropanol as common solvent. Afterwards, these solutions doped with rhodamine B (RhB), were electrospun into blend mats and the drug release mechanism and kinetics as a function of blend composition was studied, in order to understand the potential of such membranes as drug delivery systems. The electrophoresis analysis carried out on keratin revealed that the solvent used does not degrade the protein. Moreover, all the blend solutions showed a non-Newtonian behavior, among which the Keratin/PBS 70/30 and 30/70 ones showed an amplified orientation ability of the polymer chains when subjected to a shear stress. Therefore, the resulting nanofibers showed thinner mean diameters and narrower diameter distributions compared to the Keratin/PBS 50/50 blend solution. The thermal stability and the mechanical properties of the blend electrospun mats improved by increasing the PBS content. Finally, the RhB release rate increased by increasing the keratin content of the mats and the drug diffused as drug-protein complex.

Our work concerns the actual problem of spread of SARS- CoV-2 outbreak which requires fast and correct as possible answer. In current scenario, the need of rapid answer put away the imperative of proper methodology. We focus on the serogical immunoassay for diagnosis of Covid-19 as an important weapon not only for diagnostic purpose, but also for epidemiologic one. The right equilibrium between high speed, low cost and accuracy is obtained with easy-to-use decentralized point-of-care test as the colloidal gold-based immunochromatographic strip assay which detects IgM and IgG antibodies directed against SARS-CoV-2. As our aim is to evaluate the efficacy of Covid-19 rapid tests and of serological assays in real-life settings, we designed a research protocol aimed to establish how to use correctly these diagnostics, taking into account the different possible clinical and epidemiological scenarios.