Current Nanocarrier Strategies Improve Vitamin B12 Pharmacokinetics, Ameliorate Patients' Lives, and Reduce Costs.

Vitamin B12 (VitB12) is a naturally occurring compound produced by microorganisms and an essential nutrient for humans. Several papers highlight the role of VitB12 deficiency in bone and heart health, depression, memory performance, fertility, embryo development, and cancer, while VitB12 treatment is crucial for survival in inborn errors of VitB12 metabolism. VitB12 is administrated through intramuscular injection, thus impacting the patients' lifestyle, although it is known that oral administration may meet the specific requirement even in the case of malabsorption. Furthermore, the high-dose injection of VitB12 does not ensure a constant dosage, while the oral route allows only 1.2% of the vitamin to be absorbed in human beings. Nanocarriers are promising nanotechnology that can enable therapies to be improved, reducing side effects. Today, nanocarrier strategies applied at VitB12 delivery are at the initial phase and aim to simplify administration, reduce costs, improve pharmacokinetics, and ameliorate the quality of patients' lives. The safety of nanotechnologies is still under investigation and few treatments involving nanocarriers have been approved, so far. Here, we highlight the role of VitB12 in human metabolism and diseases, and the issues linked to its molecule properties, and discuss how nanocarriers can improve the therapy and supplementation of the vitamin and reduce possible side effects and limits.

Novel Therapeutic Delivery of Nanocurcumin in Central Nervous System Related Disorders.

Nutraceuticals represent complementary or alternative beneficial products to the expensive and high-tech therapeutic tools in modern medicine. Nowadays, their medical or health benefits in preventing or treating different types of diseases is widely accepted, due to fewer side effects than synthetic drugs, improved bioavailability and long half-life. Among herbal and natural compounds, curcumin is a very attractive herbal supplement considering its multipurpose properties. The potential effects of curcumin on glia cells and its therapeutic and protective properties in central nervous system (CNS)-related disorders is relevant. However, curcumin is unstable and easily degraded or metabolized into other forms posing limits to its clinical development. This is particularly important in brain pathologies determined blood brain barrier (BBB) obstacle. To enhance the stability and bioavailability of curcumin, many studies focused on the design and development of curcumin nanodelivery systems (nanoparticles, micelles, dendrimers, and diverse nanocarriers). These nanoconstructs can increase curcumin stability, solubility, in vivo uptake, bioactivity and safety. Recently, several studies have reported on a curcumin exosome-based delivery system, showing great therapeutical potential. The present work aims to review the current available data in improving bioactivity of curcumin in treatment or prevention of neurological disorders.

Lifestyle, Oxidative Stress, and Antioxidants: Back and Forth in the Pathophysiology of Chronic Diseases.

Oxidative stress plays an essential role in the pathogenesis of chronic diseases such as cardiovascular diseases, diabetes, neurodegenerative diseases, and cancer. Long term exposure to increased levels of pro-oxidant factors can cause structural defects at a mitochondrial DNA level, as well as functional alteration of several enzymes and cellular structures leading to aberrations in gene expression. The modern lifestyle associated with processed food, exposure to a wide range of chemicals and lack of exercise plays an important role in oxidative stress induction. However, the use of medicinal plants with antioxidant properties has been exploited for their ability to treat or prevent several human pathologies in which oxidative stress seems to be one of the causes. In this review we discuss the diseases in which oxidative stress is one of the triggers and the plant-derived antioxidant compounds with their mechanisms of antioxidant defenses that can help in the prevention of these diseases. Finally, both the beneficial and detrimental effects of antioxidant molecules that are used to reduce oxidative stress in several human conditions are discussed.

Cytotoxicity of temozolomide on human glioblastoma cells is enhanced by the concomitant exposure to an extremely low-frequency electromagnetic field (100Hz, 100G).

Glioblastoma multiforme (GBM) is the most malignant brain cancer that causes high mortality in humans. It responds poorly to the most common cancer treatments, such as surgery, chemo- and radiation therapy. Temozolomide (TMZ) is an alkylating agent that has been widely used to treat GBM; resistance to this drug is often found. One unexplored possibility for overcoming this resistance is a treatment based on concomitant exposure to electromagnetic fields (EMF) and TMZ. Indeed, many evidences show that EMF affects cancer cells and drug performance. In this study, we evaluated the potential synergistic effect of 100µM TMZ and EMF (100Hz, 100G) on two human glioma cells line, i.e., U87 and T98G above single treatments, TMZ or EMF. Co-treatment synergistically enhanced apoptosis in U87 and T98G cells, by increasing the expression of P53, Bax, and Caspase-3 and decreasing that of Bcl-2 and Cyclin-D1. We also observed an increase in reactive oxygen species (ROS) production and the overexpression of the heme oxygenase-1 (HO-1) gene in comparison to controls. In conclusion, since EMF enhanced the apoptotic effect of TMZ, possibly through a redox regulation mechanism, the TMZ/EMF combination may be effective for glioma cancer treating. Further studies are needed to reveal the action mechanism of this possible novel therapeutic approach.

Rose Bengal acetate photodynamic therapy (RBAc-PDT) induces exposure and release of Damage-Associated Molecular Patterns (DAMPs) in human HeLa cells.

The new concept of Immunogenic Cell Death (ICD), associated with Damage Associated Molecular Patterns (DAMPs) exposure and/or release, is recently becoming very appealing in cancer treatment. In this context, PhotoDynamic Therapy (PDT) can give rise to ICD and to immune response upon dead cells removal. The list of PhotoSensitizers (PSs) able to induce ICD is still short and includes Photofrin, Hypericin, Foscan and 5-ALA. The goal of the present work was to investigate if Rose Bengal Acetate (RBAc), a powerful PS able to trigger apoptosis and autophagy, enables photosensitized HeLa cells to expose and/or release pivotal DAMPs, i.e. ATP, HSP70, HSP90, HMGB1, and calreticulin (CRT), that characterize ICD. We found that apoptotic HeLa cells after RBAc-PDT exposed and released, early after the treatment, high amount of ATP, HSP70, HSP90 and CRT; the latter was distributed on the cell surface as uneven patches and co-exposed with ERp57. Conversely, autophagic HeLa cells after RBAc-PDT exposed and released HSP70, HSP90 but not CRT and ATP. Exposure and release of HSP70 and HSP90 were always higher on apoptotic than on autophagic cells. HMGB1 was released concomitantly to secondary necrosis (24 h after RBAc-PDT). Phagocytosis assay suggests that CRT is involved in removal of RBAc-PDT generated apoptotic HeLa cells. Altogether, our data suggest that RBAc has all the prerequisites (i.e. exposure and/or release of ATP, CRT, HSP70 and HSP90), that must be verified in future vaccination experiments, to be considered a good PS candidate to ignite ICD. We also showed tha CRT is involved in the clearance of RBAc photokilled HeLa cells. Interestingly, RBAc-PDT is the first cancer PDT protocol able to induce the translocation of HSP90 and plasma membrane co-exposure of CRT with ERp57.

Administration Dependent Antioxidant Effect of Carica papaya Seeds Water Extract.

Carica papaya is widely used in folk medicine as herbal remedy to prevent, protect against, and cure several diseases. These curative properties are based on the presence in different parts of the plant of phytochemical nutrients with antioxidant effect. Seeds are the less exploited part; thus this study is aimed at assessing the antioxidant activities of the C. papaya seeds water extract against hydrogen peroxide (H2O2) oxidative stress in human skin Detroit 550 fibroblasts. C. papaya seeds water extract is not toxic and acts as a potent free radical scavenger, providing protection to Detroit 550 fibroblasts that underwent H2O2 oxidative stress. Data show that (i) the maximum protective effect is achieved by the simultaneous administration of the extract with 1 mM H2O2; (ii) the extract in presence of an oxidative stress does not increase catalase activity and prevents the release of cytochrome C and the inner mitochondrial transmembrane potential (Δψ m ) loss; (iii) the extract is more efficient than vitamin C to hamper the oxidative damage; (iv) the purified subfractions of the seeds water extract exert the same antioxidant effect of whole extract. In conclusion, C. papaya seeds water extract is potentially useful for protection against oxidative stress.

In vitro and in vivo clearance of Rose Bengal Acetate-PhotoDynamic Therapy-induced autophagic and apoptotic cells.

This study focuses on the clearance of Rose Bengal Acetate (RBAc)-PhotoDynamic Therapy (PDT)-generated apoptotic and autophagic HeLa cells by murine and human macrophages. Indeed, phagocytosis of dead cells drives the therapeutic efficacy of PDT through both efficient removal of dead/dying cells and macrophages response evoked during engulfment and, up to now, clearance of dying photosensitized cells has been less investigated than PDT mechanisms of cell death induction. RBAc-PDT ensures a long onset of cytotoxicity and a time-related cell death of HeLa cells by signals originating from or converging on almost all intracellular organelles. On this basis, to clarify whether the efficacious cell death commitment is followed by an efficient clearance mechanism, we primarily focused on the analysis of 'eat me' signals exposure and 'find me' signals release, and then investigated the migration, recognition, engulfment and response of murine Raw 264.7 and human blood isolated macrophages. Dead cells secreted 'find me' signals, i.e. fractalkine and Heat Shock Protein 70 (HSP 70), to recruit macrophages and promote their fast phagocytosis. Macrophages phagocytosed apoptotic and autophagic PDT-treated cells more efficiently than the respective positive controls, i.e. puromycin-induced apoptotic and Earle's balanced salt solution-starved autophagic cells. Phagocytosis depends on the glycans exposed on dead cells. The macrophages internalization of photokilled cells elicits the production of Interleukin-10, Transforming Growth Factor-ß and Tumour Necrosis Factor-α by macrophages. TNFα production, along with HSP70 release and plasma membrane translocation on dead cells, suggest an immunogenic impact of RBAc-PDT. In fact, macrophages, activated fibroblasts and endothelial cells colonized the inoculum site of photosensitized cells in rat calf muscles, endorsing the hypothesis of immunogenic elicitation of RBAc-PDT.

Rose bengal acetate photodynamic therapy-induced autophagy.

Photodynamic therapy (PDT), an anticancer therapy requiring the exposure of cells or tissue to a photosensitizing drug followed by irradiation with visible light of the appropriate wavelength, induces cell death by the efficient induction of apoptotic as well as non-apoptotic mechanisms, such as necrosis and autophagy, or a combination of all three mechanisms. However, the exact role of autophagy in photodynamic therapy is still a matter of debate. To understand the role of autophagy in PDT, we investigated the induction of autophagy in HeLa cells photosensitized with Rose Bengal Acetate (RBAc). After incubation with Rose Bengal Acetate (10-5 M), HeLa cells were irradiated for 90 seconds (green LED DPL 305, emitting at 530 +15 nm to obtain 1.6 J/cm2 as the total light dose) and allowed to recover for 72 h. Induction of autophagy and apoptosis were observed with peaks at 8 h and 12 h after irradiation, respectively. Autophagy was detected by biochemical (Western Blotting for the LC3B protein) and morphological criteria (TEM, cytochemistry). In addition, the pan-caspase inhibitor, z-VAD, was unable to completely prevent cell death. The simultaneous onset of apoptosis and autophagy following Rose Bengal Acetate PDT is of remarkable interest in light of the findings that autophagy can result in the class II presentation of antigens and thus, explain why low dose PDT can yield anti-tumor immune responses.

Chronic treatment with ethanolic extract of the leavesof Azadirachta indica ameliorates lesions of pancreatic islets in streptozotocin diabetes / Tratamiento crónico con extracto etanólico de hojas de Azadirachta indica disminuye las lesiones de los islotes pancreáticos en diabetes por estreptozotocina

Botanical drugs are complementary therapies in the management of diabetes mellitus. In this work, we studied the effects of chronic treatment of diabetic rats with A. indica (neem) on blood glucose, pancreatic islet histopathology, and oxidative status of the pancreas. Fifty-four Wistar rats (5-8 weeks old) were randomly assigned to 5 treatment groups. Hyperglycemia was induced in 34 fasted rats with a single i.p. injection of STZ (70 mg/kg bw/d). Ethanolic extract of A. indica leaves (500 mg/kg bw/d) was given orally to diabetic rats (n=12) for 50d. Glibenclamide was given (p.o) at 600 µg/ kg bw/d. In each group, blood glucose, islet histopathology, and pancreatic oxidative status, were assessed. All hyperglycemic rats in the neem-treated group had become normoglycemic at the end of week 2. By 50d, the number of viable b cells was highest in the neem-treated diabetic rats (compared with the diabetic and glibenclamide groups). Similarly, islet histology showed marked improvement in this group, in addition to improved oxidative stress. Our findings confirmed the hypoglycemic effect of neem. Besides, the improved islet morphology and oxidative status in neem-treated diabetic rats suggest the potential of this herb at improving lesions of the pancreatic islet in diabetes mellitus.

Los medicamentos a base de plantas son terapias complementarias en el manejo de la diabetes mellitus. En este trabajo se estudiaron los efectos del tratamiento crónico de ratas diabéticas con A. indica (Neem) sobre la glucosa de la sangre, la histopatología de los islotes pancreáticos, y el estado oxidativo del páncreas. Cincuenta y cuatro ratas Wistar (5-8 semanas de edad) fueron asignadas aleatoriamente a 5 grupos de tratamiento. La hiperglucemia fue inducida en 34 ratas en ayunas con una única inyección IP de STZ (70 mg/kg peso corporal/d). El extracto etanólico de hojas de A. indica (500 mg/kg de peso corporal/día) fue administrado por vía oral a ratas diabéticas (n=12) por 50d. Glibenclamida fue dada (PO) a 600 mg/kg peso corporal/d. En cada grupo, la glucosa en la sangre, la histopatología de los islotes, y el estado oxidativo de páncreas, se evaluaron. Todas las ratas de hiperglucemia en el grupo tratado con el Neem se habían convertido en normoglucémicas al final de la semana 2. Por 50d, el número de células b viables fue mayor en el Neem ratas tratadas con diabetes (en comparación con los grupos de diabéticos y glibenclamida). Del mismo modo, la histología de los islotes mostró una notable mejoría en este grupo, además de mejorar el estrés oxidativo. Nuestros resultados confirman el efecto hipoglucemiante de Neem. Además, la mejora de la morfología de los islotes y el estado de oxidación en el neem tratados con ratas diabéticas sugieren el potencial de esta hierba en la mejora de las lesiones de los islotes pancreáticos en la diabetes mellitus.

Photodynamic therapy-induced apoptosis of HeLa cells.

Photodynamic therapy (PDT), which is a treatment for cancer and certain noncancerous conditions, requires exposure of cells or tissue to a photosensitizing drug followed by irradiation with visible light of the appropriate wavelength. By using Rose Bengal Acetate (RBAc) as the photosensitizer and an innovative green light-emitting diode, we investigated the efficiency with which apoptosis is induced in HeLa cells, focusing our study on mitochondria alteration and cytochrome c release. Indeed, RBAc is a very efficient fluorogenic substrate and easily enters the cells where the original photoactive molecule is restored by specific esterases. HeLa cells after PDT underwent a consistent rate of apoptosis (peaked at 12 h of recovery post-PDT). Necrosis was observed at the longest times of recovery as a result of secondary necrosis. PDT gave rise to a series of shape modifications, mainly referable to apoptotic-related changes (i.e., extensive blebs formation) involving both F-actin and tubulin networks. Soon after PDT, mitochondria lose their potential membranes and release large quantities of cytochrome c.