Synthesis and Physicochemical Characterization of Gelatine-Based Biodegradable Aerogel-like Composites as Possible Scaffolds for Regenerative Medicine.

Regenerative medicine is an interdisciplinary field aiming at restoring pathologically damaged tissues and whole organs by cell transplantation in combination with proper supporting scaffolds. Gelatine-based ones are very attractive due to their biocompatibility, rapid biodegradability, and lack of immunogenicity. Gelatine-based composite hydrogels, containing strengthening agents to improve their modest mechanical properties, have been demonstrated to act as extracellular matrices (ECMs), thus playing a critical role in "organ manufacturing". Inspired by the lysyl oxidase (LO)-mediated process of crosslinking, which occurs in nature to reinforce collagen, we have recently developed a versatile protocol to crosslink gelatine B (Gel B) in the presence or absence of LO, using properly synthesized polystyrene- and polyacrylic-based copolymers containing the amine or aldehyde groups needed for crosslinking reactions. Here, following the developed protocol with slight modifications, we have successfully crosslinked Gel B in different conditions, obtaining eight out of nine compounds in high yield (57-99%). The determined crosslinking degree percentage (CP%) evidenced a high CP% for compounds obtained in presence of LO and using the styrenic amine-containing (CP5/DMAA) and acrylic aldehyde-containing (CPMA/DMAA) copolymers as crosslinking agents. ATR-FTIR analyses confirmed the chemical structure of all compounds, while optical microscopy demonstrated cavernous, crater-like, and labyrinth-like morphologies and cavities with a size in the range 15-261 µm. An apparent density in the range 0.10-0.45 g/cm3 confirmed the aerogel-like structure of most samples. Although the best biodegradation profile was observed for the sample obtained using 10% CP5/DMAA (M3), high swelling and absorption properties, high porosity, and good biodegradation profiles were also observed for samples obtained using the 5-10% CP5/DMAA (M4, 5, 6) and 20% CPMA/DMAA (M9) copolymers. Collectively, in this work of synthesis and physicochemical characterization, new aerogel-like composites have been developed and, based on their characteristics, which fit well within the requirements for TE, five candidates (M3, M4, M5, M6, and M9) suitable for future biological experiments on cell adhesion, infiltration and proliferation, to confirm their effective functioning, have been identified.

Synthesis and Characterization of Amine and Aldehyde-Containing Copolymers for Enzymatic Crosslinking of Gelatine.

In tissue engineering (TE), the support structure (scaffold) plays a key role necessary for cell adhesion and proliferation. The protein constituents of the extracellular matrix (ECM), such as collagen, its derivative gelatine, and elastin, are the most attractive materials as possible scaffolds. To improve the modest mechanical properties of gelatine, a strategy consists of crosslinking it, as naturally occurs for collagen, which is stiffened by the oxidative action of lysyl oxidase (LO). Here, a novel protocol to crosslink gelatine has been developed, not using the commonly employed crosslinkers, but based on the formation of imine bonds or on aldolic condensation reactions occurring between gelatine and properly synthesized copolymers containing amine residues via LO-mediated oxidation. Particularly, we first synthesized and characterized an amino butyl styrene monomer (5), its copolymers with dimethylacrylamide (DMAA), and its terpolymer with DMAA and acrylic acid (AA). Three acryloyl amidoamine monomers (11a-c) and their copolymers with DMAA were then prepared. A methacrolein (MA)/DMAA copolymer already possessing the needed aldehyde groups was finally developed to investigate the relevance of LO in the crosslinking process. Oxidation tests of amine copolymers with LO were performed to identify the best substrates to be used in experiments of gelatine reticulation. Copolymers obtained with 5, 11b, and 11c were excellent substrates for LO and were employed with MA/DMAA copolymers in gelatine crosslinking tests in different conditions. Among the amine-containing copolymers, that obtained with 5 (CP5/DMMA-43.1) afforded a material (M21) with the highest crosslinking percentage (71%). Cytotoxicity experiments carried out on two cell lines (IMR-32 and SH SY5Y) with the analogous (P5) of the synthetic constituent of M21 (CP5/DMAA) had evidenced no significant reduction in cell viability, but proliferation promotion, thus establishing the biocompatibility of M21 and the possibility to develop it as a new scaffold for TE, upon further investigations.

Reactive Oxygen Species (ROS)-Mediated Antibacterial Oxidative Therapies: Available Methods to Generate ROS and a Novel Option Proposal.

The increasing emergence of multidrug-resistant (MDR) pathogens causes difficult-to-treat infections with long-term hospitalizations and a high incidence of death, thus representing a global public health problem. To manage MDR bacteria bugs, new antimicrobial strategies are necessary, and their introduction in practice is a daily challenge for scientists in the field. An extensively studied approach to treating MDR infections consists of inducing high levels of reactive oxygen species (ROS) by several methods. Although further clinical investigations are mandatory on the possible toxic effects of ROS on mammalian cells, clinical evaluations are extremely promising, and their topical use to treat infected wounds and ulcers, also in presence of biofilm, is already clinically approved. Biochar (BC) is a carbonaceous material obtained by pyrolysis of different vegetable and animal biomass feedstocks at 200-1000 °C in the limited presence of O2. Recently, it has been demonstrated that BC's capability of removing organic and inorganic xenobiotics is mainly due to the presence of persistent free radicals (PFRs), which can activate oxygen, H2O2, or persulfate in the presence or absence of transition metals by electron transfer, thus generating ROS, which in turn degrade pollutants by advanced oxidation processes (AOPs). In this context, the antibacterial effects of BC-containing PFRs have been demonstrated by some authors against Escherichia coli and Staphylococcus aureus, thus giving birth to our idea of the possible use of BC-derived PFRs as a novel method capable of inducing ROS generation for antimicrobial oxidative therapy. Here, the general aspects concerning ROS physiological and pathological production and regulation and the mechanism by which they could exert antimicrobial effects have been reviewed. The methods currently adopted to induce ROS production for antimicrobial oxidative therapy have been discussed. Finally, for the first time, BC-related PFRs have been proposed as a new source of ROS for antimicrobial therapy via AOPs.

Development of Phytochemical Delivery Systems by Nano-Suspension and Nano-Emulsion Techniques.

The awareness of the existence of plant bioactive compounds, namely, phytochemicals (PHYs), with health properties is progressively expanding. Therefore, their massive introduction in the normal diet and in food supplements and their use as natural therapeutics to treat several diseases are increasingly emphasized by several sectors. In particular, most PHYs possessing antifungal, antiviral, anti-inflammatory, antibacterial, antiulcer, anti-cholesterol, hypoglycemic, immunomodulatory, and antioxidant properties have been isolated from plants. Additionally, their secondary modification with new functionalities to further improve their intrinsic beneficial effects has been extensively investigated. Unfortunately, although the idea of exploiting PHYs as therapeutics is amazing, its realization is far from simple, and the possibility of employing them as efficient clinically administrable drugs is almost utopic. Most PHYs are insoluble in water, and, especially when introduced orally, they hardly manage to pass through physiological barriers and scarcely reach the site of action in therapeutic concentrations. Their degradation by enzymatic and microbial digestion, as well as their rapid metabolism and excretion, strongly limits their in vivo activity. To overcome these drawbacks, several nanotechnological approaches have been used, and many nanosized PHY-loaded delivery systems have been developed. This paper, by reporting various case studies, reviews the foremost nanosuspension- and nanoemulsion-based techniques developed for formulating the most relevant PHYs into more bioavailable nanoparticles (NPs) that are suitable or promising for clinical application, mainly by oral administration. In addition, the acute and chronic toxic effects due to exposure to NPs reported so far, the possible nanotoxicity that could result from their massive employment, and ongoing actions to improve knowledge in this field are discussed. The state of the art concerning the actual clinical application of both PHYs and the nanotechnologically engineered PHYs is also reviewed.

Hydrogel Formulations of Antibacterial Pyrazoles Using a Synthesized Polystyrene-Based Cationic Resin as a Gelling Agent.

Here, to develop new topical antibacterial formulations to treat staphylococcal infections, two pyrazoles (3c and 4b) previously reported as antibacterial agents, especially against staphylococci, were formulated as hydrogels (R1-HG-3c and R1HG-4b) using a cationic polystyrene-based resin (R1) and here synthetized and characterized as gelling agents. Thanks to the high hydrophilicity, high-level porosity, and excellent swelling capabilities of R1, R1HG-3c and R1HG-4b were achieved with an equilibrium degree of swelling (EDS) of 765% (R1HG-3c) and 675% (R1HG-4b) and equilibrium water content (EWC) of 88% and 87%, respectively. The chemical structure of soaked and dried gels was investigated by PCA-assisted attenuated total reflectance (ATR) Fourier transform infrared (FTIR) spectroscopy, while their morphology was investigated by optical microscopy. Weight loss studies were carried out with R1HG-3c and R1HG-4b to investigate their water release profiles and the related kinetics, while their stability was evaluated over time both by monitoring their inversion properties to detect possible impairments of the 3D network and by PCA-assisted ATR-FTIR spectroscopy to detect possible structural changes. The flow and dynamic rheological characterization of the gels was assessed by determining their viscosity vs. shear rate, applying the Cross rheological equation to achieve the curves of shear stress vs. shear rate, and carrying out amplitude and frequency sweep experiments. Finally, their content in NH3+ groups was determined by potentiometric titrations. Due to their favorable physicochemical characteristic and the antibacterial effects of 3c and 4b possibly improved by the cationic R1, the pyrazole-enriched gels reported here could represent new weapons to treat severe skin and wound infections sustained by MDR bacteria of staphylococcal species.

Cationic Polystyrene-Based Hydrogels as Efficient Adsorbents to Remove Methyl Orange and Fluorescein Dye Pollutants from Industrial Wastewater.

Water pollution from dyes is harmful to the environment, plants, animals, and humans and is one of the most widespread problems afflicting people throughout the world. Adsorption is a widely used method to remove contaminants derived from the textile industry, food colorants, printing, and cosmetic manufacturing from water. Here, aiming to develop new low-cost and up-scalable adsorbent materials for anionic dye remediation and water decontamination by electrostatic interactions, two cationic resins (R1 and R2) were prepared. In particular, they were obtained by copolymerizing 4-ammonium methyl and ethyl styrene monomers (M1 and M2) with dimethylacrylamide (DMAA), using N-(2-acryloylamino-ethyl)-acrylamide (AAEA) as cross-linker. Once characterized by several analytical techniques, upon their dispersion in an excess of water, R1 and R2 provided the R1- and R2-based hydrogels (namely R1HG and R2HG) with equilibrium degrees of swelling (EDS) of 900% and 1000% and equilibrium water contents (EWC) of 90 and 91%, respectively. By applying Cross' rheology equation to the data of R1HG and R2HG's viscosity vs. shear rate, it was established that both hydrogels are shear thinning fluids with pseudoplastic/Bingham plastic behavior depending on share rate. The equivalents of -NH3+ groups, essential for the electrostatic-based absorbent activity, were estimated by the method of Gaur and Gupta on R1 and R2 and by potentiometric titrations on R1HG and R2HG. In absorption experiments in bulk, R1HG and R2HG showed high removal efficiency (97-100%) towards methyl orange (MO) azo dye, fluorescein (F), and their mixture (MOF). Using F or MO solutions (pH = 7.5, room temperature), the maximum absorption was 47.8 mg/g in 90' (F) and 47.7 mg/g in 120' (MO) for R1, while that of R2 was 49.0 mg/g in 20' (F) and 48.5 mg/g in 30' (MO). Additionally, R1HG and R2HG-based columns, mimicking decontamination systems by filtration, were capable of removing MO, F, and MOF from water with a 100% removal efficiency, in different conditions of use. R1HG and R2HG represent low-cost and up-scalable column packing materials that are promising for application in industrial wastewater treatment.

Imidazo-Pyrazole-Loaded Palmitic Acid and Polystyrene-Based Nanoparticles: Synthesis, Characterization and Antiproliferative Activity on Chemo-Resistant Human Neuroblastoma Cells.

Neuroblastoma (NB) is a childhood cancer, commonly treated with drugs, such as etoposide (ETO), whose efficacy is limited by the onset of resistance. Here, aiming at identifying new treatments for chemo-resistant NB, the effects of two synthesized imidazo-pyrazoles (IMPs) (4G and 4I) were investigated on ETO-sensitive (HTLA-230) and ETO-resistant (HTLA-ER) NB cells, detecting 4I as the more promising compound, that demonstrated IC50 values lower than those of ETO on HTLA ER. Therefore, to further improve the activity of 4I, we developed 4I-loaded palmitic acid (PA) and polystyrene-based (P5) cationic nanoparticles (P5PA-4I NPs) with high drug loading (21%) and encapsulation efficiency (97%), by a single oil-in-water emulsification technique. Biocompatible PA was adopted as an emulsion stabilizer, while synthesized P5 acted as an encapsulating agent, solubilizer and hydrophilic-lipophilic balance (HLB) improver. Optic microscopy and cytofluorimetric analyses were performed to investigate the micromorphology, size and complexity distributions of P5PA-4I NPs, which were also structurally characterized by chemometric-assisted Fourier transform infrared spectroscopy (FTIR). Potentiometric titrations allowed us to estimate the milliequivalents of PA and basic nitrogen atoms present in NPs. P5PA-4I NPs afforded dispersions in water with excellent buffer capacity, essential to escape lysosomal degradation and promote long residence time inside cells. They were chemically stable in an aqueous medium for at least 40 days, while in dynamic light scattering (DLS) analyses, P5PA-4I showed a mean hydrodynamic diameter of 541 nm, small polydispersity (0.194), and low positive zeta potentials (+8.39 mV), assuring low haemolytic toxicity. Biological experiments on NB cells, demonstrated that P5PA-4I NPs induced ROS-dependent cytotoxic effects significantly higher than those of pristine 4I, showing a major efficacy compared to ETO in reducing cell viability in HTLA-ER cells. Collectively, this 4I-based nano-formulation could represent a new promising macromolecular platform to develop a new delivery system able to increase the cytotoxicity of the anticancer drugs.

A Self-Forming Hydrogel from a Bactericidal Copolymer: Synthesis, Characterization, Biological Evaluations and Perspective Applications.

Objects touched by patients and healthcare workers in hospitals may harbor pathogens, including multi-drug resistant (MDR) staphylococci, enterococci (VRE), Escherichia coli, Acinetobacter, and Pseudomonas species. Medical devices contaminated by these pathogens may also act as a source of severe and difficult-to-treat human infections, thus becoming a critical public health concern requiring urgent resolutions. To this end, we recently reported the bactericidal effects of a cationic copolymer (CP1). Here, aiming at developing a bactericidal formulation possibly to be used either for surfaces disinfection or to treat skin infections, CP1 was formulated as a hydrogel (CP1_1.1-Hgel). Importantly, even if not cross-linked, CP1 formed the gel upon simple dispersion in water, without requiring gelling agents or other additives which could be skin-incompatible or interfere with CP1 bactericidal effects in possible future topical applications. CP1_1.1-Hgel was characterized by attenuated-total-reflectance Fourier transform infrared (ATR-FTIR) and UV-Vis spectroscopy, as well as optic and scanning electron microscopy (OM and SEM) to investigate its chemical structure and morphology. Its stability was assessed by monitoring its inversion properties over time at room temperature, while its mechanical characteristics were assessed by rheological experiments. Dose-dependent cytotoxicity studies performed on human fibroblasts for 24 h with gel samples obtained by diluting CP_1.1-Hgel at properly selected concentrations established that the 3D network formation did not significantly affect the cytotoxic profile of CP1. Also, microbiologic investigations carried out on two-fold serial dilutions of CP1-gel confirmed the minimum inhibitory concentrations (MICs) previously reported for the not formulated CP1.Selectivity indices values up to 12 were estimated by the values of LD50 and MICs determined here on gel samples.

One-Step, Low-Cost, Operator-Friendly, and Scalable Procedure to Synthetize Highly Pure N-(4-ethoxyphenyl)-retinamide in Quantitative Yield without Purification Work-Up.

It is widely reported that N-(4-hydroxyphenyl)-retinamide or fenretinide (4-HPR), which is a synthetic amide of all-trans-retinoic acid (ATRA), inhibits in vitro several types of tumors, including cancer cell lines resistant to ATRA, at 1-10 µM concentrations. Additionally, studies in rats and mice have confirmed the potent anticancer effects of 4-HPR, without evidencing hemolytic toxicity, thus demonstrating its suitability for the development of a new chemo-preventive agent. To this end, the accurate determination of 4-HPR levels in tissues is essential for its pre-clinical training, and for the correct determination of 4-HPR and its metabolites by chromatography, N-(4-ethoxyphenyl)-retinamide (4-EPR) has been suggested as an indispensable internal standard. Unfortunately, only a consultable old patent reports the synthesis of 4-EPR, starting from dangerous and high-cost reagents and using long and tedious purification procedures. To the best of our knowledge, no article existed so far describing the specific synthesis of 4-EPR. Only two vendors worldwide supply 4-ERP, and its characterization was incomplete. Here, a scalable, operator-friendly, and one-step procedure to synthetize highly pure 4-EPR without purification work-up and in quantitative yield is reported. Additionally, a complete characterization of 4-EPR using all possible analytical techniques has been provided.

Broad-Spectrum Bactericidal Activity of a Synthetic Random Copolymer Based on 2-Methoxy-6-(4-Vinylbenzyloxy)-Benzylammonium Hydrochloride.

Low-molecular-weight organic ammonium salts exert excellent antimicrobial effects by interacting lethally with bacterial membranes. Unfortunately, short-term functionality and high toxicity limit their clinical application. On the contrary, the equivalent macromolecular ammonium salts, derived from the polymerization of monomeric ammonium salts, have demonstrated improved antibacterial potency, a lower tendency to develop resistance, higher stability, long-term activity, and reduced toxicity. A water-soluble non-quaternary copolymeric ammonium salt (P7) was herein synthetized by copolymerizing 2-methoxy-6-(4-vinylbenzyloxy)-benzylammonium hydrochloride monomer with N, N-di-methyl-acrylamide. The antibacterial activity of P7 was assessed against several multidrug-resistant (MDR) clinical isolates of both Gram-positive and Gram-negative species. Except for colistin-resistant Pseudomonas aeruginosa, most isolates were susceptible to P7, also including some Gram-negative bacteria with a modified charge in the external membrane. P7 showed remarkable antibacterial activity against isolates of Enterococcus, Staphylococcus, Acinetobacter, and Pseudomonas, and on different strains of Escherichia coli and Stenotrophomonas maltophylia, regardless of their antibiotic resistance. The lowest minimal inhibitory concentrations (MICs) observed were 0.6-1.2 µM and the minimal bactericidal concentrations (MBC) were frequently overlapping with the MICs. In 24-h time-kill and turbidimetric studies, P7 displayed a rapid non-lytic bactericidal activity. P7 could therefore represent a novel and potent tool capable of counteracting infections sustained by several bacteria that are resistant to the presently available antibiotics.

Bactericidal Activity of a Self-Biodegradable Lysine-Containing Dendrimer against Clinical Isolates of Acinetobacter Genus.

The genus Acinetobacter consists of Gram-negative obligate aerobic pathogens, including clinically relevant species, such as A. baumannii, which frequently cause hospital infections, affecting debilitated patients. The growing resistance to antimicrobial therapies shown by A. baumannii is reaching unacceptable levels in clinical practice, and there is growing concern that the serious conditions it causes may soon become incurable. New therapeutic possibilities are, therefore, urgently needed to circumvent this important problem. Synthetic cationic macromolecules, such as cationic antimicrobial peptides (AMPs), which act as membrane disrupters, could find application in these conditions. A lysine-modified cationic polyester-based dendrimer (G5-PDK), capable of electrostatically interacting with bacterial surfaces as AMPs do, has been synthesized and characterized here. Given its chemical structure, similar to that of a fifth-generation lysine containing dendrimer (G5K) with a different core, and previously found inactive against Gram-positive bacterial species and Enterobacteriaceae, the new G5-PDK was also ineffective on the species mentioned above. In contrast, it showed minimum inhibitory concentration values (MICs) lower than reported for several AMPs and other synthetic cationic compounds on Acinetobacter genus (3.2-12.7 µM). Time-kill experiments on A. baumannii, A. pittii, and A. ursingii ascertained the rapid bactericidal effects of G5-PDK, while subsequent bacterial regrowth supported its self-biodegradability.

Development and characterization of a mucoadhesive sublingual formulation for pain control: extemporaneous oxycodone films in personalized therapy.

OBJECTIVE: The aim of this work was the development of mucoadhesive sublingual films, prepared using a casting method, for the administration of oxycodone. MATERIALS AND METHODS: A solvent casting method was employed to prepare the mucoadhesive films. A calibrated pipette was used to deposit single aliquots of different polymeric solutions on a polystyrene plate lid. Among the various tested polymers, hydroxypropylcellulose at low and medium molecular weight (HPC) and pectin at two different degrees of esterification (PC) were chosen for preparing solutions with good casting properties, capable of producing films suitable for mucosal application. RESULTS AND DISCUSSION: The obtained films showed excellent drug content uniformity and stability and rapid drug release, which, at 8 min, ranged from 60% to 80%. All films presented satisfactory mucoadhesive and mechanical properties, also confirmed by a test on healthy volunteers, who did not experience irritation or mucosa damages. Pectin films based on pectin at lower degrees of esterification have been further evaluated to study the influence of two different amounts of drug on the physicochemical properties of the formulation. A slight reduction in elasticity has been observed in films containing a higher drug dose; nevertheless, the formulation maintained satisfactory flexibility and resistance to elongation. CONCLUSIONS: HPC and PC sublingual films, obtained by a simple casting method, could be proposed to realize personalized hospital pharmacy preparations on a small scale.

Attempts to Improve Lipophilic Drugs' Solubility and Bioavailability: A Focus on Fenretinide.

The development of numerous drugs is often arrested at clinical testing stages, due to their unfavorable biopharmaceutical characteristics. It is the case of fenretinide (4-HPR), a second-generation retinoid, that demonstrated promising in vitro cytotoxic activity against several cancer cell lines. Unfortunately, response rates in early clinical trials with 4-HPR did not confirm the in vitro findings, mainly due to the low bioavailability of the oral capsular formulation that was initially developed. Capsular 4-HPR provided variable and insufficient drug plasma levels attributable to the high hepatic first-pass effect and poor drug water solubility. To improve 4-HPR bioavailability, several approaches have been put forward and tested in preclinical and early-phase clinical trials, demonstrating generally improved plasma levels and minimal systemic toxicities, but also modest antitumor efficacy. The challenge is thus currently still far from being met. To redirect the diminished interest of pharmaceutical companies toward 4-HPR and promote its further clinical development, this manuscript reviewed the attempts made so far by researchers to enhance 4-HPR bioavailability. A comparison of the available data was performed, and future directions were proposed.

AmBisome® Formulations for Pediatrics: Stability, Cytotoxicity, and Cost-Effectiveness Studies.

Liposomal amphotericin B (Ambisome®) is the gold standard for the treatment and prevention of fungal infections both in the adult and pediatric populations. The lyophilized dosage form has to be reconstituted and diluted by hospital staff, but its management can be challenging due to the spontaneous tendency of amphotericin B to form aggregates with different biological activity. In this study, the colloidal stability of the liposomes and the chemical stability of amphotericin B were investigated over time at storage conditions. Three liposomal formulations of amphotericin B at 4.0 mg/mL, 2.0 mg/mL, and 0.2 mg/mL were prepared and assayed for changes regarding the dimensional distribution, zeta potential, drug aggregation state, and onset of by-products. Our analyses highlighted that the most diluted formulation, kept at room temperature, showed the greatest changes in the aggregation state of the drug and accordingly the highest cytotoxicity. These findings are clinically relevant since the lower dosages are addressed to the more vulnerable patients. Therefore, the centralization of the dilution of AmBisome® at the pharmacy is of fundamental importance for assuring patient safety, and at the same time for reducing medication waste, as we demonstrated using the cost-saving analysis of drug expense per therapy carried out at the G. Gaslini children hospital.

NaDES Application in Cosmetic and Pharmaceutical Fields: An Overview.

Natural deep eutectic solvents (NaDES) represent a new generation of green, non-flammable solvents, useful as an efficient alternative to the well-known ionic liquids. They can be easily prepared and exhibit unexpected solubilizing power for lipophilic molecules, although those of a hydrophilic nature are mostly used. For their unique properties, they can be recommend for different cosmetic and pharmaceutical applications, ranging from sustainable extraction, obtaining ready-to-use ingredients, to the development of biocompatible drug delivery responsive systems. In the biomedical field, NaDES can be used as biopolymer modifiers, acting as delivery compounds also known as "therapeutic deep eutectic systems", being able to solubilize and stabilize different chemical and galenical formulations. The aim of this review is to give an overview of the current knowledge regarding natural deep eutectic solvents specifically applied in the cosmetic and pharmaceutical fields. The work could help to disclose new opportunities and challenges for their implementation not only as green alternative solvents but also as potential useful pathways to deliver bioactive ingredients in innovative formulations.

Development of Biotinylated Liposomes Encapsulating Metformin for Therapeutic Targeting of Inflammation-Based Diseases.

Inflammation is a physiological response to a damaging stimulus but sometimes can be the cause of the onset of neurodegenerative diseases, atherosclerosis, and cancer. These pathologies are characterized by the overexpression of inflammatory markers like endothelial adhesion molecules, such as Vascular Cell Adhesion Molecule-1 (VCAM-1). In the present work, the development of liposomes for therapeutic targeted delivery to inflamed endothelia is described. The idea is to exploit a three-step pretargeting system based on the biotin-avidin high-affinity interaction: the first step involves a previously described biotin derivative bearing a VCAM-1 binding peptide; in the second step, the avidin derivative NeutrAvidinTM, which strongly binds to the biotin moiety, is injected; the final step is the administration of biotinylated liposomes that would bind to NeutravidinTM immobilized onto VCAM-1 overexpressing endothelium. Stealth biotinylated liposomes, prepared via the thin film hydration method followed by extrusion and purification via size exclusion chromatography, have been thoroughly characterized for their chemico-physical and morphological features and loaded with metformin hydrochloride, a potential anti-inflammatory agent. The three-step system, tested in vitro on different cell lines via confocal microscopy, FACS analysis and metformin uptake, has proved its suitability for therapeutic applications.

Development of Medicines for Rare Pediatric Diseases.

To date, approximately 7000 rare diseases exist, affecting between 6% and 8% of the global population and >30 million people in the European Union [...].

Correction: Alfei et al. 4-Hydroxybenzoic Acid as an Antiviral Product from Alkaline Autoxidation of Catechinic Acid: A Fact to Be Reviewed. Plants 2022, 11, 1822.

In the original publication [...].

Cationic Polystyrene-Based Hydrogels: Low-Cost and Regenerable Adsorbents to Electrostatically Remove Nitrites from Water.

Nitrites are metastable anions that are derived from the oxidation of ammonia by agricultural pollution, sewage, decaying protein, and other nitrogen sources. They are a recognized environmental issue due to their role in eutrophication, as well as in surface and groundwater contamination, being toxic to almost all living creatures. Recently, we reported on the high efficiency of two cationic resins (R1 and R2) forming hydrogels (R1HG and R2HG) by dispersion in water in removing anionic dyes from water by electrostatic binding. Here, aiming at developing adsorbent materials for nitrite remediation, R1, R2, R1HG, and R2HG were first tested in adsorption experiments in batches monitored by UV-Vis methods, using the Griess reagent system (GRS) in order to assess their removal efficiency by contact over time. Particularly, samples of water appositely contaminated with nitrites were analyzed by UV-Vis before and during treatment with the hydrogels. The initial concentration of nitrites was quantified (118 mg/L). Then, the removal of nitrites over time, the removal efficiency of R1HG (89.2%) and of R2HG (89.6%), their maximum adsorption (21.0 mg/g and 23.5 mg/g), as well as the adsorption kinetics and mechanisms were evaluated. Additionally, R1HG- and R2HG-based columns (h = 8-10 cm, ØE = 2 cm) mimicking mini-scale decontamination systems by filtration were used to rapidly filter samples of water polluted with nitrite that were under pressure. R1HG and R2GH were capable of totally removing nitrites (99.5% and 100%) from volumes of nitrite solutions that were 118 mg/L that is 10 times the volumes of resins used. Additionally, when extending filtration to increasing volumes of the same nitrite solution up to 60 times the volume of resins used, the removal efficiently of R1HG decreased, and that of R2HG remained stable at over 89%. Interestingly, both the worn-out hydrogels were regenerable by 1% HCl washing, without a significant reduction in their original efficiency. There is a lack of studies in the literature reporting on novel methods to remove nitrite from water. R1HG and especially R2HG represent low-cost, up-scalable, and regenerable column-packing materials with promise for applications in the treatment of drinking water contaminated by nitrites.

Pyrazole-Enriched Cationic Nanoparticles Induced Early- and Late-Stage Apoptosis in Neuroblastoma Cells at Sub-Micromolar Concentrations.

Neuroblastoma (NB) is a severe form of tumor occurring mainly in young children and originating from nerve cells found in the abdomen or next to the spine. NB needs more effective and safer treatments, as the chance of survival against the aggressive form of this disease are very small. Moreover, when current treatments are successful, they are often responsible for unpleasant health problems which compromise the future and life of surviving children. As reported, cationic macromolecules have previously been found to be active against bacteria as membrane disruptors by interacting with the negative constituents of the surface of cancer cells, analogously inducing depolarization and permeabilization, provoking lethal damage to the cytoplasmic membrane, and cause loss of cytoplasmic content and consequently, cell death. Here, aiming to develop new curative options for counteracting NB cells, pyrazole-loaded cationic nanoparticles (NPs) (BBB4-G4K and CB1H-P7 NPs), recently reported as antibacterial agents, were assayed against IMR 32 and SHSY 5Y NB cell lines. Particularly, while BBB4-G4K NPs demonstrated low cytotoxicity against both NB cell lines, CB1H-P7 NPs were remarkably cytotoxic against both IMR 32 and SHSY 5Y cells (IC50 = 0.43-0.54 µM), causing both early-stage (66-85%) and late-stage apoptosis (52-65%). Interestingly, in the nano-formulation of CB1H using P7 NPs, the anticancer effects of CB1H and P7 were increased by 54-57 and 2.5-4-times, respectively against IMR 32 cells, and by 53-61 and 1.3-2 times against SHSY 5Y cells. Additionally, based on the IC50 values, CB1H-P7 was also 1-12-fold more potent than fenretinide, an experimental retinoid derivative in a phase III clinical trial, with remarkable antineoplastic and chemopreventive properties. Collectively, due to these results and their good selectivity for cancer cells (selectivity indices = 2.8-3.3), CB1H-P7 NPs represent an excellent template material for developing new treatment options against NB.