Dual-responsive chondroitin sulfate self-assembling nanoparticles for combination therapy in metastatic cancer cells.

In this study, we developed self-assembling nanoparticles (LCPs) able to trigger the release of Chlorambucil (Chl) and Doxorubicin (DOX) to MDA-MB-231 cells by exploiting the enzyme and redox signals. The DOX loaded LCPs was prepared by the self-assembly of two chondroitin sulphate (CS) derivatives, obtained by the covalent conjugation of Lipoic Acid (LA) and Chlorambucil (Chl) to the CS backbone. After the physic-chemical characterization of the conjugates by FT-IR, 1H NMR, and determination of the critical aggregation concentration, spherical nanoparticles with mean hydrodynamic diameter of 45 nm (P.D.I. 0.24) and Z-potential of - 44 mV were obtained by water addition/solvent evaporation method. In vitro experiments for the release of Chl and DOX were performed in healthy and cancer cells, using a cell culture media to maintain the physiological intracellular conditions (pH 7.4) (and concentration of esterase and GSH. The results allowed the selective release of the payloads to be detected: Chl release of 0 and 41% were obtained after 2 h incubation in normal and in cancer cells respectively, while values of 35 (in healthy cells) and 60% (in cancer cells) were recorded for DOX release after 96 h. Finally, viability studies proved the ability of the newly proposed nanosystem to enhance the cytotoxic activity of the two drugs against cancer cells.

Curcumin-Sodium Alginate and Curcumin-Chitosan Conjugates as Drug Delivery Systems: An Interesting Rheological Behaviour.

The conjugation of polyphenols is a valuable strategy with which to confer tailored properties to polymeric materials of biomedical interest. Within this investigation, we aim to explore the possibility to use this synthetic approach to increase the viscosity of conjugates, thus allowing the release of a loaded therapeutic to be better controlled over time than in neat polyphenols. Curcumin (CUR) was conjugated to sodium alginate (CA) and chitosan (CS) with functionalisation degrees of 9.2 (SA-CUR) and 15.4 (CS-CUR) mg g-1. Calorimetric analyses showed higher degrees of chain rigidity upon conjugation, with a shift of the degradation peaks to higher temperatures (from 239 to 245 °C and from 296 to 303 °C for SA-CUR and CS-CUR, respectively). Rheological analyses were used to prove the enhanced interconnection between the polymer chains in the conjugates, confirmed by the weak gel parameters, A and z. Moreover, the typical non-Newtonian behaviour of the high-molecular-weight polysaccharides was recorded, together with an enhancement of the activation energy, Ea, in CS-CUR vs. CS (opposite behaviour recorded for SA-CUR vs. SA). The evaluation of the delivery performance (of Doxorubicin as a model drug) showed sustained release profiles, opening opportunities for the development of controlled delivery systems.

Multivariate Curve Resolution Methodology Applied to the ATR-FTIR Data for Adulteration Assessment of Virgin Coconut Oil.

Virgin coconut oil (VCO) is a functional food with important health benefits. Its economic interest encourages fraudsters to deliberately adulterate VCO with cheap and low-quality vegetable oils for financial gain, causing health and safety problems for consumers. In this context, there is an urgent need for rapid, accurate, and precise analytical techniques to detect VCO adulteration. In this study, the use of Fourier transform infrared (FTIR) spectroscopy combined with multivariate curve resolution-alternating least squares (MCR-ALS) methodology was evaluated to verify the purity or adulteration of VCO with reference to low-cost commercial oils such as sunflower (SO), maize (MO) and peanut (PO) oils. A two-step analytical procedure was developed, where an initial control chart approach was designed to assess the purity of oil samples using the MCR-ALS score values calculated on a data set of pure and adulterated oils. The pre-treatment of the spectral data by derivatization with the Savitzky-Golay algorithm allowed to obtain the classification limits able to distinguish the pure samples with 100% of correct classifications in the external validation. In the next step, three calibration models were developed using MCR-ALS with correlation constraints for analysis of adulterated coconut oil samples in order to assess the blend composition. Different data pre-treatment strategies were tested to best extract the information contained in the sample fingerprints. The best results were achieved by derivative and standard normal variate procedures obtaining RMSEP and RE% values in the ranges of 1.79-2.66 and 6.48-8.35%, respectively. The models were optimized using a genetic algorithm (GA) to select the most important variables and the final models in the external validations gave satisfactory results in quantifying adulterants, with absolute errors and RMSEP of less than 4.6% and 1.470, respectively.

Wine Lees as Source of Antioxidant Molecules: Green Extraction Procedure and Biological Activity.

An ultrasound-assisted extraction method, employing ethanol and water as solvents at low temperature (30 °C) and reduced time (15 min), was proposed to extract bioactive molecules from different cultivars (Magliocco Canino, Magliocco Rosato, Gaglioppo, and Nocera Rosso) of wine lees. All the extract yields were evaluated and their contents of phenolic acids, flavonoids, and total polyphenols were determined by means of colorimetric assays and high-performance liquid chromatography coupled with diode-array detection (HPLC-DAD) and Fourier transform infrared (FTIR) techniques. Radical scavenging assays were performed and the Magliocco Canino extracted with a hydroalcoholic mixture returned the best results both against ABTS (0.451 mg mL-1) and DPPH (0.395 mg mL-1) radicals. The chemometric algorithms principal component analysis (PCA) and partial least square regression (PLS) were used to process the data obtained from all qualitative-quantitative sample determinations with the aim of highlighting data patterns and finding possible correlations between composition and antioxidant features of the different wine lees cultivars and the extraction procedures. Wine lees from Magliocco Canino and Magliocco Rosato were found to be the best vegetable matrices in terms of metabolite content and antioxidant properties. The components extracted with alcoholic or hydroalcoholic solvents, specifically (-)-epigallocatechin gallate, chlorogenic acid, and trans-caftaric acid, were found to be correlated with the antioxidant capacity of the extracts. Multivariate data processing was able to identify the compounds related to the antioxidant features. Two PLS models were optimized by using their concentration levels to predict the IC50 values of the extracts in terms of DPPH and ABTS with high values of correlation coefficient R2, 0.932 and 0.824, respectively, and a prediction error lower than 0.07. Finally, cellular (SH-SY5Y cells) antioxidant assays were performed on the best extract (the hydroalcoholic extract of Magliocco Canino cv) to confirm its biological performance against radical species. All these recorded data strongly outline the aptness of valorizing wine lees as a valuable source of antioxidants.

Recent advances in PI3K/PKB/mTOR inhibitors as new anticancer agents.

The biochemical role of the PI3K/PKB/mTOR signalling pathway in cell-cycle regulation is now well known. During the onset and development of different forms of cancer it becomes overactive reducing apoptosis and allowing cell proliferation. Therefore, this pathway has become an important target for the treatment of various forms of malignant tumors, including breast cancer and follicular lymphoma. Recently, several more or less selective inhibitors targeting these proteins have been identified. In general, drugs that act on multiple targets within the entire pathway are more efficient than single targeting inhibitors. Multiple inhibitors exhibit high potency and limited drug resistance, resulting in promising anticancer agents. In this context, the present survey focuses on small molecule drugs capable of modulating the PI3K/PKB/mTOR signalling pathway, thus representing drugs or drug candidates to be used in the pharmacological treatment of different forms of cancer.

Anticancer Drugs: Recent Strategies to Improve Stability Profile, Pharmacokinetic and Pharmacodynamic Properties.

In past decades, anticancer research has led to remarkable results despite many of the approved drugs still being characterized by high systemic toxicity mainly due to the lack of tumor selectivity and present pharmacokinetic drawbacks, including low water solubility, that negatively affect the drug circulation time and bioavailability. The stability studies, performed in mild conditions during their development or under stressing exposure to high temperature, hydrolytic medium or light source, have demonstrated the sensitivity of anticancer drugs to many parameters. For this reason, the formation of degradation products is assessed both in pharmaceutical formulations and in the environment as hospital waste. To date, numerous formulations have been developed for achieving tissue-specific drug targeting and reducing toxic side effects, as well as for improving drug stability. The development of prodrugs represents a promising strategy in targeted cancer therapy for improving the selectivity, efficacy and stability of active compounds. Recent studies show that the incorporation of anticancer drugs into vesicular systems, such as polymeric micelles or cyclodextrins, or the use of nanocarriers containing chemotherapeutics that conjugate to monoclonal antibodies can improve solubility, pharmacokinetics, cellular absorption and stability. In this study, we summarize the latest advances in knowledge regarding the development of effective highly stable anticancer drugs formulated as stable prodrugs or entrapped in nanosystems.

Exploring the anticancer and antioxidant properties of Vicia faba L. pods extracts, a promising source of nutraceuticals.

Background: Pulse crops are considered the major sources of proteins, dietary fiber, micronutrients, and bioactive phytochemicals. Among the numerous pulse crops, broad beans (Vicia faba L.) have received particular attention due to their nutraceutical, functional and economic importance. Our attention was mainly focused on the broad bean pods (VFs), which are the primary by-product of the domestic and industrial processing of broad beans and an attractive source of valuable ingredients. Methods: In order to investigate the VFs properties, the flours from broad beans of three different harvest periods were extracted with acetone, methanol and 70% aqueous ethanol and the dried extracts were analyzed, qualitatively and quantitatively, and tested for their antioxidant through DPPH and ABTS assay and anticancer activities using the MTT assay and immunofluorescence analysis. Results: The VF extracts demonstrated a good in vitro radical scavenging activity from the first stage of collection of all the V. faba L. extracts. Additionally, the extracts were tested for their cytotoxicity against a panel of cancer and normal cells and the outcomes indicated the ethanol extract as the most active against the melanoma cell line Sk-Mel-28, without affecting the viability of the normal cells. Finally, we found out that the ethanol extract interfered with the microtubules organization, leading to the cancer cells death by apoptosis.

Clonal analysis of human clonogenic keratinocytes.

Regenerative medicine has its roots in harnessing stem cells for permanent restoration of damaged or diseased tissues. The first procedure for the transplantation of epidermal cultures in massive full-thickness burns was established in the 1980s. Since then, epithelial stem cell-based therapies have been further developed in cell and gene therapy protocols aimed at restoring visual acuity in severe ocular burns and treating patients affected by genetic skin diseases, as Epidermolysis Bullosa. The clinical success of these Advanced Therapy Medicinal Products (ATMPs) requires the presence of a defined number of epithelial stem cells in the grafts, detected as holoclone-forming cells. To date, the most trustworthy method to identify and measure holoclones in a culture is the clonal analysis of clonogenic keratinocytes. Here we describe in detail how to perform such a clonal analysis and identify each epidermal clonal type.

Interaction of letrozole and its degradation products with aromatase: chemometric assessment of kinetics and structure-based binding validation.

Letrozole is one of the most prescribed drugs for the treatment of breast cancer in post-menopausal women, and it is endowed with selective peripheral aromatase inhibitory activity. The efficacy of this drug is also a consequence of its long-lasting activity, likely due to its metabolic stability. The reactivity of cyano groups in the letrozole structure could, however, lead to chemical derivatives still endowed with residual biological activity. Herein, the chemical degradation process of the drug was studied by coupling multivariate curve resolution and spectrophotometric methodologies in order to assess a detailed kinetic profile. Three main derivatives were identified after drug exposure to different degradation conditions, consisting of acid-base and oxidative environments and stressing light. Molecular docking confirmed the capability of these compounds to accommodate into the active site of the enzyme, suggesting that the sustained inhibitory activity of letrozole may be at least in part attributed to the degradation compounds.

Ultrasound-Assisted Extraction, Chemical Characterization, and Impact on Cell Viability of Food Wastes Derived from Southern Italy Autochthonous Citrus Fruits.

Citrus fruits are one of the principal fruits used to produce juices. Over the years, these fruits have been recognized as new health-promoting agents. In this work, food wastes derived from autochthonous citrus fruits of Southern Italy, named Limone di Rocca Imperiale, Arancia Rossa Moro, and Arancia Bionda Tardivo from Trebisacce, were analyzed. After fresh-squeezing juice, peel and pomace were employed to obtain six different extracts using an ultrasound-assisted method in a hydroalcoholic solvent. The extracts were analyzed in terms of qualitative composition, antioxidant properties, and antiproliferative activity on MCF-7, MDA-MB-231, and BJ-hTERT cell lines. GC-MS and LC-ESI-MS analyses showed different compounds: of note, limonin-hexoside, neodiosmin, obacunone glucoside, and diacetyl nomilinic acid glucoside have been identified as limonoid structures present in all the samples, in addition to different polyphenols including naringenin-glucoside, hesperetin-O-hexoside-O-rhamnoside-O-glucoside, diferuloyl-glucaric acid ester, chlorogenic acid, and the presence of fatty acids such as palmitic, myristic, and linoleic acids. These extracts were able to exert antioxidant activity as demonstrated by DPPH and ABTS assays and, although at higher doses, to reduce the cell viability of different solid tumor cell lines, as shown in MTT assays.

Transgenic Epidermal Cultures for Junctional Epidermolysis Bullosa - 5-Year Outcomes.

Inherited junctional epidermolysis bullosa is a severe genetic skin disease that leads to epidermal loss caused by structural and mechanical fragility of the integuments. There is no established cure for junctional epidermolysis bullosa. We previously reported that genetically corrected autologous epidermal cultures regenerated almost an entire, fully functional epidermis on a child who had a devastating form of junctional epidermolysis bullosa. We now report long-term clinical outcomes in this patient. (Funded by POR FESR 2014-2020 - Regione Emilia-Romagna and others.).

Hologene 5: A Phase II/III Clinical Trial of Combined Cell and Gene Therapy of Junctional Epidermolysis Bullosa.

Epidermolysis bullosa (EB) is a group of devastating genetic diseases characterized by skin and mucosal fragility and formation of blisters, which develop either spontaneously or in response to minor mechanical trauma. There is no definitive therapy for any form of EB. Intermediate junctional EB (JEB) caused by mutations in the gene LAMB3 has been the first genetic skin disease successfully tackled by ex vivo gene therapy. Here, we present a multicenter, open-label, uncontrolled phase II/III study that aims at confirming the efficacy of Hologene 5, a graft consisting of cultured transgenic keratinocytes and epidermal stem cells and meant to combine cell and gene therapy for the treatment of LAMB3-related JEB. Autologous clonogenic keratinocytes will be isolated from patients' skin biopsies, genetically corrected with a gamma-retroviral vector (γRV) carrying the full-length human LAMB3 cDNA and plated onto a fibrin support (144cm2). The transgenic epidermis will be transplanted onto surgically prepared selected skin areas of at least six JEB patients (four pediatric and two adults). Evaluation of clinical efficacy will include, as primary endpoint, a combination of clinical parameters, such as percentage of re-epithelialization, cellular, molecular, and functional parameters, mechanical stress tests, and patient-reported outcome (PRO), up to 12months after transplantation. Safety and further efficacy endpoints will also be assessed during the clinical trial and for additional 15years in an interventional non-pharmacological follow-up study. If successful, this clinical trial would provide a therapeutic option for skin lesions of JEB patients with LAMB3 mutations and pave the way to a combined cell and gene therapy platform tackling other forms of EB and different genodermatoses. Clinical Trial Registration: EudraCT Number: 2018-000261-36.

Toward Multitasking Pharmacological COX-Targeting Agents: Non-Steroidal Anti-Inflammatory Prodrugs with Antiproliferative Effects.

The antitumor activity of certain anti-inflammatory drugs is often attributed to an indirect effect based on the inhibition of COX enzymes. In the case of anti-inflammatory prodrugs, this property could be attributed to the parent molecules with mechanism other than COX inhibition, particularly through formulations capable of slowing down their metabolic conversion. In this work, a pilot docking study aimed at comparing the interaction of two prodrugs, nabumetone (NB) and its tricyclic analog 7-methoxy-2,3-dihydro-1H-cyclopenta[b]naphthalen-1-one (MC), and their common active metabolite 6-methoxy-2-naphthylacetic acid (MNA) with the COX binding site, was carried out. Cytotoxicity, cytofluorimetry, and protein expression assays on prodrugs were also performed to assess their potential as antiproliferative agents that could help hypothesize an effective use as anticancer therapeutics. Encouraging results suggest that the studied compounds could act not only as precursors of the anti-inflammatory metabolite, but also as direct antiproliferative agents.

Essential Oils of Foeniculum vulgare subsp. piperitum and Their in Vitro Anti-Arthritic Potential.

Wild Foeniculum vulgare subsp. piperitum (C.Presl) Bég. flowers, fruits and leaves were extracted with steam distillation and obtained essential oils (EOs) were characterized using GC/MS. The study was designed to verify the potential effectiveness of fennel EOs in the treatment of inflammation and arthritis. Since tissue proteins denaturation is a major cause of arthritic diseases, fennel EOs and their main constituents were evaluated for their ability to inhibit the heat-induced proteins degradation using bovine serum albumin as a protein model. Moreover, the in vitro inhibitory effects of the three EOs on the pro-inflammatory mediator nitric oxide (NO) production were verified in LPS-stimulated RAW 264.7 cells. Estragole (28.81-33.40 %), anethole (24.16-27.40 %), fenchone (9.76-18.48 %), α-phellandrene (1.63-8.37 %) and limonene (5.54-6.05 %) were the major constituents. All the EOs showed a concentration-dependent biological activity, being the flower EO the most effective in inhibiting NO production (IC50 =232.2±11.3 µg/mL). The leaf EO showed a very good bovine serum albumin (BSA) anti-denaturation activity (IC50 =95.9±2.4 µg/mL). Moreover, four components were proved to be effective in protecting protein from heat-induced degradation, being α-phellandrene the most active compound (IC50 =73.2±1.9 µg/mL).

Photostability study of multicomponent drug formulations via MCR-ALS: The case of the hydrochlorothiazide-amiloride mixture.

The kinetics and photodegradation mechanism of the pharmaceutical mixture of hydrochlorothiazide (HCT) and amiloride (AML) has been studied in depth using a chemometric approach. Water solutions of HCT and AML, separately or in binary mixtures, were irradiated with forced light at different pH values (3, 7, 9 and 12). Multivariate Curve Resolution - Alternating Least Squares (MCR-ALS) modelling has been applied to the experimental data recorded by UV spectrophotometry and HPLC-UV/MS. 78 data sets were collected and their chemometric processing has allowed the simultaneous determination of the behaviour of the two drugs in the mixture when exposed to light and the dependence of their photodegradation kinetics on pH. MCR-ALS has been applied using three different implementations. Soft-MCR-ALS and hybrid Hard/Soft-MCR-ALS have been used to resolve the experimental data and to get the equilibrium and kinetic parameters of the investigated chemical processes. A third implementation of the MCR-ALS method has been used in the analysis of the incomplete data sets obtained when UV spectrophotometric and HPLC-UV/MS data were simultaneously analysed, using a row- and column-wise incomplete augmented data matrix arrangement. In these matrices, information from HPLC-UV detector was used as a bridge between the data recorded by UV spectrophotometry (acid-base and kinetic reactions monitoring) and the data obtained by HPLC-MS.

Toward Combined Cell and Gene Therapy for Genodermatoses.

To date, more than 200 monogenic, often devastating, skin diseases have been described. Because of unmet medical needs, development of long-lasting and curative therapies has been consistently attempted, with the aim of correcting the underlying molecular defect. In this review, we will specifically address the few combined cell and gene therapy strategies that made it to the clinics. Based on these studies, what can be envisioned for the future is a patient-oriented strategy, built on the specific features of the individual in need. Most likely, a combination of different strategies, approaches, and advanced therapies will be required to reach the finish line at the end of the long and winding road hampering the achievement of definitive treatments for genodermatoses.

Advances in stem cell research and therapeutic development.

Despite many reports of putative stem-cell-based treatments in genetic and degenerative disorders or severe injuries, the number of proven stem cell therapies has remained small. In this Review, we survey advances in stem cell research and describe the cell types that are currently being used in the clinic or are close to clinical trials. Finally, we analyse the scientific rationale, experimental approaches, caveats and results underpinning the clinical use of such stem cells.

Photo and thermal stress of linseed oil and stabilization strategies.

The thermal and light stability of linseed oil has been studied by monitoring the concentrations of fatty acids and lignans, as main nutraceutical components. Linseed oil was subjected to stressing light and temperature conditions, in accordance with the ICH international rules, and monitored by UV-vis spectroscopy and HPLC-DAD. The change of UV spectra along the photodegradation tests, setting the irradiation power at 350 W/m2, confirmed a significant overall sensitivity of linseed oil to light. At the same time, the HPLC determination of the major fatty acids showed a marked variation in their concentration up to a residual concentration of 62.3 and 67.2% for α-linolenic and linoleic acid, respectively, after 18 h. In contrast, thermal tests at 60 °C showed some stability, with a concentration of residual fatty acids in the range 82-95% after 48 h. The examined lignans showed significant stability when exposed to both light and heat. Several photoprotection approaches have been also studied to increase the photostability of linseed oil. A significant increase in the stability of fatty acids has been observed using amber glass containers or ascorbic acid or by combining the two protection factors.

Living with Keratinocytes.

A feature distinguishing human hematopoietic and epithelial stem cells from other equally fascinating stem cells is perhaps their easier translation into a clinical setting. We have devoted nearly our entire scientific career in trying to turn our understanding of epithelial stem cell biology into something that could help people suffering from virtually untreatable diseases of squamous epithelia. We have done that as a team, together with our numerous students, postdocs, technicians and valuable collaborators, clinicians, regulators, and, lately, industrial partners. We had rewarding successes and burning failures, but we always did our best. This award, given by friends and colleagues deserving it more than us, has been the most important recognition of our work. Below, we summarize our story.