Liposome nano‐formulation with cationic polar lipid DOTAP and cholesterol as a suitable pH‐responsive carrier for molecular therapeutic drug (all‐trans retinoic acid) delivery to lung cancer cells

The molecular targeted drug ATRA demands a suitable carrier that delivers to the cancer site due to its poor bioavailability and drug resistance. ATRA, being a lipid with carboxylic acid, has been nano‐formulated as a cationic lipo‐ATRA with DOTAP:cholesterol:ATRA (5:4:1) and its pH‐responsive release, intracellular drug accumulation, and anticancer effect on human lung cancer (A549) cell line analysed. The analysis of the physicochemical characteristics of the developed lipo‐ATRA (0.8 µmol) revealed that the size of 231 ± 2.35 d.nm had a zeta potential of 6.4 ± 1.19 and an encapsulation efficiency of 93.7 ± 3.6%. The ATRA release from lipo‐ATRA in vitro was significantly (p ≤ 0.05) higher at acidic pH 6 compared to pH 7.5. The intracellular uptake of ATRA into lipo‐ATRA‐treated A549 cells was seven‐fold higher (0.007 ± 0.001 mg/ml) while only three‐fold uptake was observed in free ATRA treatment (0.003 ± 0.002 mg/ml). The lipo‐ATRA treatment caused a highly significant (p ≤ 0.001) decrease in percent cell viability at 48 h when compared with the free ATRA treatment. Overall, the results proved that the developed lipo‐ATRA has suitable physicochemical properties with enhanced ATRA release at acidic pH, while maintaining stability at physiologic pH and temperature. This resulted in an increased ATRA uptake by lung cancer cells with enhanced treatment efficiency. Hence, it is concluded that DOTAP lipo‐ATRA is a suitable carrier for ATRA delivery to solid cancer cells.     

Small Extracellular Vesicles Derived from Human Chorionic MSCs as Modern Perspective towards Cell-Free Therapy

Mesenchymal stem cells (MSCs) are of great interest to scientists due to their application in cell therapy of many diseases, as well as regenerative medicine and tissue engineering. Recently, there has been growing evidence surrounding the research based on extracellular vesicles (EVs), especially small EVs (sEVs)/exosomes derived from MSCs. EVs/exosomes can be secreted by almost all cell types and various types of EVs show multiple functions. In addition, MSCs-derived exosomes have similar characteristics and biological activities to MSCs and their therapeutic applications are considered as a safe strategy in cell-free therapy. The aim of this study was the characterization of MSCs isolated from the chorion (CHo-MSCs) of human full-term placenta, as well as the isolation and analysis of small EVs obtained from these cells. Accordingly, in this study, the ability of small EVs’ uptake is indicated by synovial fibroblasts, osteoblasts and periosteum-derived MSCs. Improvement in the understanding of the structure, characteristics, mechanism of action and potential application of MSCs-derived small EVs can provide new insight into improved therapeutic strategies.     

Some physico-chemical properties of nine commercial or semi-commercial whey protein concentrates, isolates and fractions

Summary The physico-chemical properties are reported for a group of whey protein powders prepared on a commercial or semi-commercial scale by three companies and chemically characterized as described elsewhere (Holt et al ., 1999). The dependence of the apparent β-lactoglobulin % on the recovered % showed that the nine samples could be placed in three distinct groups with β-lactoglobulin weight % of 70.9 ± 1.1 (Group 1), 62.0 ± 3.4 (Group 2) and 39.5 ± 4.9 (Group 3). Measurements by ¹H-NMR spectroscopy, on 3 of the samples confirmed that the native fold still predominated in the β-lactoglobulin. β-lactoglobulin could be crystallized from all the powders and the normal space group and cell dimensions were determined for the 8 samples that gave crystals of good enough quality for X-ray studies. Differential scanning microcalorimetry of samples dispersed in a phosphate buffer showed a clear difference between Goups 1 and 2 with a more prominent peak due to α-lactalbumin in the Group 2 samples. Light scattering and size exclusion chromatography showed that two types of aggregates were present to a variable extent in all the samples and after a heat treatment, the larger aggregates tended to predominate in Group 2. The rheology measurements, also made in the phosphate buffer, showed a difference of gel stiffness during heat treatment between the Group 1 and Group 2 samples with the exception of the BORCwpc+ sample. Within each group, gel stiffness increased with the degree of lactoslylation of the β-lactoglobulin. Interfacial measurements on samples dispersed in water presented a more complex pattern of behaviour although surface tension measurements at the air water interface of the Group 2 samples showed a two-step pattern of surface tension decrease with time, compared to a single step pattern in the Group 1 samples.     

16S rRNA of Mucosal Colon Microbiome and CCL2 Circulating Levels Are Potential Biomarkers in Colorectal Cancer

Colorectal cancer (CRC) is one of the most common malignancies in the Western world and intestinal dysbiosis might contribute to its pathogenesis. The mucosal colon microbiome and C-C motif chemokine 2 (CCL2) were investigated in 20 healthy controls (HC) and 20 CRC patients using 16S rRNA sequencing and immunoluminescent assay, respectively. A total of 10 HC subjects were classified as overweight/obese (OW/OB_HC) and 10 subjects were normal weight (NW_HC); 15 CRC patients were classified as OW/OB_CRC and 5 patients were NW_CRC. Results: Fusobacterium nucleatum and Escherichia coli were more abundant in OW/OB_HC than in NW_HC microbiomes. Globally, Streptococcus intermedius, Gemella haemolysans, Fusobacterium nucleatum, Bacteroides fragilis and Escherichia coli were significantly increased in CRC patient tumor/lesioned tissue (CRC_LT) and CRC patient unlesioned tissue (CRC_ULT) microbiomes compared to HC microbiomes. CCL2 circulating levels were associated with tumor presence and with the abundance of Fusobacterium nucleatum, Bacteroides fragilis and Gemella haemolysans. Our data suggest that mucosal colon dysbiosis might contribute to CRC pathogenesis by inducing inflammation. Notably, Fusobacterium nucleatum, which was more abundant in the OW/OB_HC than in the NW_HC microbiomes, might represent a putative link between obesity and increased CRC risk.     

CHIR99021, trough GSK-3β Targeting,Reduces Epithelioid Sarcoma Cell Proliferation by Activating Mitotic Catastrophe and Autophagy

Epithelioid sarcoma (ES) is a rare disease representing <1% of soft tissue sarcomas. Current therapies are based on anthracycline alone or in combination with ifosfamide or other cytotoxic drugs. ES is still characterized by a poor prognosis with high rates of recurrence. Indeed, for years, ES survival rates have remained stagnant, suggesting that conventional treatments should be revised and improved. New therapeutic approaches are focused to target the key regulators of signaling pathways, the causative markers of tumor pathophysiology. To this end, we selected, among the drugs to which an ES cell line is highly sensitive, those that target signaling pathways known to be dysregulated in ES. In particular, we found a key role for GSK-3β, which results in up-regulation in tumor versus normal tissue samples and associated to poor prognosis in sarcoma patients. Following this evidence, we evaluated CHIR99021, a GSK-3 inhibitor, as a potential drug for use in ES therapy. Our data highlight that, in ES cells, CHIR99021 induces cell cycle arrest, mitotic catastrophe (MC) and autophagic response, resulting in reduced cell proliferation. Our results support the potential efficacy of CHIR99021 in ES treatment and encourage further preclinical and clinical studies.     

Subcritical water extraction of thyreostats from bovine muscle followed by liquid chromatography-tandem mass spectrometry

Thyreostats can be used fraudulently to promote a rapid increase in weight of breeding animals at low cost. Their severe toxicological effects impose the development of reliable analytical methods to be used in monitoring plans. This work describes an alternative approach to isolate residues of thiouracil, methyl-thiouracil, propyl-thiouracil, phenyl-thiouracil, tapazole and mercaptobenzimidazole from bovine muscle tissue. The developed procedure is based on the following three steps: i) matrix solid-phase dispersion with C₁₈ for the preliminary sample preparation; ii) subcritical water extraction (SWE) at 160°C and 100 bar; iii) clean-up on an Oasis HLB cartridge. The quantitative determination was performed by LC-MS/MS in dual polarity ionization by using internal standardization. The SWE-LC-MS/MS method was validated according to the identification criteria of the Commission decision 2002/657/EC. The relative recoveries ranged from 72 to 97%; within-lab reproducibility was less than 18%. The decision limit and the detection capability of all analytes were below the recommended concentration, set at 10 µg kg^?1, but the validation results demonstrated that this method could only be applied for screening of thiouracil and methyl-thiouracil.

Besides the analytical advantages related to the use of water as solvent extraction, the procedure allowed significant removal of lipids, whose detrimental effects on instrumentation and MS sensitivity are well-known.     

A step forward towards the development of stable freeze-dried liposomes: a quality by design approach (QbD)

This study highlights the advantages of using a Quality by Design (QbD) approach in order to gain a more comprehensive understanding of the freeze-drying process of pravastatin-loaded long-circulating liposomes (LCL-PRAV). Within the QbD paradigm, the present study aimed to establish the design space for the optimization of freeze-dried LCL-PRAV by means of Design of Experiment (DOE). The encapsulated solute retention (ESR), the average particle size, and zeta potential after freeze-drying, the residual moisture content, the macroscopic cake appearance, the glass transition temperature (T_g) of the freeze-dried cake, and the primary drying time were defined as critical quality attributes (CQAs) for the freeze-dried final product. Further on, the influence of lyoprotectant type, freezing rate, shelf temperature during primary drying, and the presence of an annealing step on the CQAs was investigated through a 21-run D-optimal experimental design. Three-dimensional response surfaces were generated to complete the statistical analysis and for a better understanding of the influence of variables and their interactions on the responses. The developed model was then used to build the design space for the freeze-dried liposomes, within which the product quality was assured and the process variability was minimized.     

From Brain to Heart: Possible Role of Amyloid-β in Ischemic Heart Disease and Ischemia-Reperfusion Injury

Ischemic heart disease (IHD) is among the leading causes of death in developed countries. Its pathological origin is traced back to coronary atherosclerosis, a lipid-driven immuno-inflammatory disease of the arteries that leads to multifocal plaque development. The primary clinical manifestation of IHD is acute myocardial infarction (AMI),) whose prognosis is ameliorated with optimal timing of revascularization. Paradoxically, myocardium re-perfusion can be detrimental because of ischemia-reperfusion injury (IRI), an oxidative-driven process that damages other organs. Amyloid-β (Aβ) plays a physiological role in the central nervous system (CNS). Alterations in its synthesis, concentration and clearance have been connected to several pathologies, such as Alzheimer’s disease (AD) and cerebral amyloid angiopathy (CAA). Aβ has been suggested to play a role in the pathogenesis of IHD and cerebral IRI. The purpose of this review is to summarize what is known about the pathological role of Aβ in the CNS; starting from this evidence, we will illustrate the role played by Aβ in the development of coronary atherosclerosis and its possible implications in the pathophysiology of IHD and myocardial IRI. Better elucidation of Aβ’s contribution to the molecular pathways underlying IHD and IRI could be of great help in developing new therapeutic strategies.     

Deficit in Adipose Differentiation in Mesenchymal Stem Cells Derived from Chronic Rhinosinusitis Nasal Polyps Compared to Nasal Mucosal Tissue

Chronic rhinosinusitis of the nasal mucosa is an inflammatory disease of paranasal sinuses, which causes rhinorrhea, nasal congestion, and hyposmia, and in some cases, it can result in the development of nasal polyposis. Nasal polyps are benign lobular-shaped growths that project in the nasal cavities; they originate from inflammation in the paranasal mucous membrane and are associated with a high expression of interleukins (IL)-4, IL-5, IL-13, and IgE. Polyps derive from the epithelial–mesenchymal transition of the nasal epithelium resulting in a nasal tissue remodeling. Nasal polyps from three patients with chronic rhinosinusitis as well as control non-polyp nasal mucosa were used to isolate and cultivate mesenchymal stem cells characterized as CD73⁺, CD90⁺, CD105⁺/CD14⁻, CD34⁻, and CD45⁻. Mesenchymal stem cells (MSCs) cultures were induced to differentiate toward adipocytes, where lipid droplets and adipocyte genes PPARγ2, ADIPO-Q, and FABP4 were observed in control non-polyp nasal mucosa-derived mesenchymal cells but were scarcely present in the cultures derived from the nasal polyps, where apoptosis was evident. The modulation of the response to adipogenic stimulus in polyps represents a change in the molecular response that controls the cascade required for differentiation as well as possible means to specifically target these cells, sparing the normal mucosa of the nasal sinuses.