Dopamine- and Grape-Seed-Extract-Loaded Solid Lipid Nanoparticles: Interaction Studies between Particles and Differentiated SH-SY5Y Neuronal Cell Model of Parkinson's Disease.

Parkinson's disease (PD) is a prevalent neurodegenerative disorder, primarily associated with dopaminergic neuron depletion in the Substantia Nigra. Current treatment focuses on compensating for dopamine (DA) deficiency, but the blood-brain barrier (BBB) poses challenges for effective drug delivery. Using differentiated SH-SY5Y cells, we investigated the co-administration of DA and the antioxidant Grape Seed Extract (GSE) to study the cytobiocompability, the cytoprotection against the neurotoxin Rotenone, and their antioxidant effects. For this purpose, two solid lipid nanoparticle (SLN) formulations, DA-co-GSE-SLNs and GSE-ads-DA-SLNs, were synthesized. Such SLNs showed mean particle sizes in the range of 187-297 nm, zeta potential values in the range of -4.1--9.7 mV, and DA association efficiencies ranging from 35 to 82%, according to the formulation examined. The results showed that DA/GSE-SLNs did not alter cell viability and had a cytoprotective effect against Rotenone-induced toxicity and oxidative stress. In addition, this study also focused on the evaluation of Alpha-synuclein (aS) levels; SLNs showed the potential to modulate the Rotenone-mediated increase in aS levels. In conclusion, our study investigated the potential of SLNs as a delivery system for addressing PD, also representing a promising approach for enhanced delivery of pharmaceutical and antioxidant molecules across the BBB.

Deconstructing SARS-CoV-2 neutralization: A modular molecular framework for computational design and comparison of antibodies and nanobodies targeting the spike RBD.

Since 2020 the COVID-19 pandemic has led scientists to search for strategies to predict the transmissibility and virulence of new severe acute respiratory syndrome coronavirus 2 variants based on the estimation of the affinity of the spike receptor binding domain (RBD) for the human angiotensin-converting enzyme 2 (ACE2) receptor and/or neutralizing antibodies. In this context, our lab developed a computational pipeline to quickly quantify the free energy of interaction at the spike RBD/ACE2 protein-protein interface, reflecting the incidence trend observed in the transmissibility/virulence of the investigated variants. In this new study, we used our pipeline to estimate the free energy of interaction between the RBD from 10 variants, and 14 antibodies (ab), or 5 nanobodies (nb), highlighting the RBD regions preferentially targeted by the investigated ab/nb. Our structural comparative analysis and interaction energy calculations allowed us to propose the most promising RBD regions to be targeted by future ab/nb to be designed by site-directed mutagenesis of existing high-affinity ab/nb, to increase their affinity for the target RBD region, for preventing spike-RBD/ACE2 interactions and virus entry in host cells. Furthermore, we evaluated the ability of the investigated ab/nb to simultaneously interact with the three RBD located on the surface of the trimeric spike protein, which can alternatively be in up- or down- (all-3-up-, all-3-down-, 1-up-/2-down-, 2-up-/1-down-) conformations.

Solid Lipid Nanoparticles Containing Dopamine and Grape Seed Extract: Freeze-Drying with Cryoprotection as a Formulation Strategy to Achieve Nasal Powders.

(1) Background: DA-Gelucire® 50/13-based solid lipid nanoparticles (SLNs) administering the neurotransmitter dopamine (DA) and the antioxidant grape-seed-derived proanthocyanidins (grape seed extract, GSE) have been prepared by us in view of a possible application for Parkinson's disease (PD) treatment. To develop powders constituted by such SLNs for nasal administration, herein, two different agents, namely sucrose and methyl-ß-cyclodextrin (Me-ß-CD), were evaluated as cryoprotectants. (2) Methods: SLNs were prepared following the melt homogenization method, and their physicochemical features were investigated by Raman spectroscopy, Scanning Electron Microscopy (SEM), atomic force microscopy (AFM) and X-ray Photoelectron Spectroscopy (XPS). (3) Results: SLN size and zeta potential values changed according to the type of cryoprotectant and the morphological features investigated by SEM showed that the SLN samples after lyophilization appear as folded sheets with rough surfaces. On the other hand, the AFM visualization of the SLNs showed that their morphology consists of round-shaped particles before and after freeze-drying. XPS showed that when sucrose or Me-ß-CD were not detected on the surface (because they were not allocated on the surface or completely absent in the formulation), then a DA surfacing was observed. In vitro release studies in Simulated Nasal Fluid evidenced that DA release, but not the GSE one, occurred from all the cryoprotected formulations. Finally, sucrose increased the physical stability of SLNs better than Me-ß-CD, whereas RPMI 2650 cell viability was unaffected by SLN-sucrose and slightly reduced by SLN-Me-ß-CD. (4) Conclusions: Sucrose can be considered a promising excipient, eliciting cryoprotection of the investigated SLNs, leading to a powder nasal pharmaceutical dosage form suitable to be handled by PD patients.

Small-molecule drugs for cystic fibrosis: Where are we now?

The cystic fibrosis (CF) lung disease is due to the lack/dysfunction of the CF Transmembrane Conductance Regulator (CFTR), a chloride channel expressed by epithelial cells as the main regulator of ion and fluid homeostasis. More than 2000 genetic variation in the CFTR gene are known, among which those with identified pathomechanism have been divided into six mutation classes. A major advancement in the pharmacotherapy of CF has been the development of small-molecule drugs hitting the root of the disease, i.e. the altered ion and fluid transport through the airway epithelium. These drugs, called CFTR modulators, have been advanced to the clinics to treat nearly 90% of CF patients, including the CFTR potentiator ivacaftor, approved for residual function mutations (Classes III and IV), and combinations of correctors (lumacaftor, tezacaftor, elexacaftor) and ivacaftor for patients bearing at least one the F508del mutation, the most frequent mutation belonging to class II. To cover the 10% of CF patients without etiological therapies, other novel small-molecule CFTR modulators are in evaluation of their effectiveness in all the CFTR mutation classes: read-through agents for Class I, correctors, potentiators and amplifiers from different companies for Class II-V, stabilizers for Class VI. In alternative, other solute carriers, such as SLC26A9 and SLC6A14, are the focus of intensive investigation. Finally, other molecular targets are being evaluated for patients with no approved CFTR modulator therapy or as means of enhancing CFTR modulatory therapy, including small molecules forming ion channels, inhibitors of the ENaC sodium channel and potentiators of the calcium-activated chloride channel TMEM16A. This paper aims to give an up-to-date overview of old and novel CFTR modulators as well as of novel strategies based on small-molecule drugs. Further investigations in in-vivo and cell-based models as well as carrying out large prospective studies will be required to determine if novel CFTR modulators, stabilizers, amplifiers, and the ENaC inhibitors or TMEM16A potentiators will further improve the clinical outcomes in CF management.

The Non-Gastric H+/K+ ATPase (ATP12A) Is Expressed in Mammalian Spermatozoa.

H+/K+ ATPase Type 2 is an heteromeric membrane protein involved in cation transmembrane transport and consists of two subunits: a specific α subunit (ATP12A) and a non-specific ß subunit. The aim of this study was to demonstrate the presence and establish the localization of ATP12A in spermatozoa from Bubalus bubalis, Bos taurus and Ovis aries. Immunoblotting revealed, in all three species, a major band (100 kDa) corresponding to the expected molecular mass. The ATP12A immunolocalization pattern showed, consistently in the three species, a strong signal at the acrosome. These results, described here for the first time in spermatozoa, are consistent with those observed for the ß1 subunit of Na+/K+ ATPase, suggesting that the latter may assemble with the α subunit to produce a functional ATP12A dimer in sperm cells. The above scenario appeared to be nicely supported by 3D comparative modeling and interaction energy calculations. The expression of ATP12A during different stages of bovine sperm maturation progressively increased, moving from epididymis to deferent ducts. Based on overall results, we hypothesize that ATP12A may play a role in acrosome reactions. Further studies will be required in order to address the functional role of this target protein in sperm physiology.

Camel (Camelus spp.) Urine Bioactivity and Metabolome: A Systematic Review of Knowledge Gaps, Advances, and Directions for Future Research.

Up to the present day, studies on the therapeutic properties of camel (Camelus spp.) urine and the detailed characterization of its metabolomic profile are scarce and often unrelated. Information on inter individual variability is noticeably limited, and there is a wide divergence across studies regarding the methods for sample storage, pre-processing, and extract derivatization for metabolomic analysis. Additionally, medium osmolarity is not experimentally adjusted prior to bioactivity assays. In this scenario, the methodological standardization and interdisciplinary approach of such processes will strengthen the interpretation, repeatability, and replicability of the empirical results on the compounds with bioactive properties present in camel urine. Furthermore, sample enlargement would also permit the evaluation of camel urine's intra- and interindividual variability in terms of chemical composition, bioactive effects, and efficacy, while it may also permit researchers to discriminate potential animal-intrinsic and extrinsic conditioning factors. Altogether, the results would help to evaluate the role of camel urine as a natural source for the identification and extraction of specific novel bioactive substances that may deserve isolated chemical and pharmacognostic investigations through preclinical tests to determine their biological activity and the suitability of their safety profile for their potential inclusion in therapeutic formulas for improving human and animal health.

Personalized Medicine in Mitochondrial Health and Disease: Molecular Basis of Therapeutic Approaches Based on Nutritional Supplements and Their Analogs.

Mitochondrial diseases (MDs) may result from mutations affecting nuclear or mitochondrial genes, encoding mitochondrial proteins, or non-protein-coding mitochondrial RNA. Despite the great variability of affected genes, in the most severe cases, a neuromuscular and neurodegenerative phenotype is observed, and no specific therapy exists for a complete recovery from the disease. The most used treatments are symptomatic and based on the administration of antioxidant cocktails combined with antiepileptic/antipsychotic drugs and supportive therapy for multiorgan involvement. Nevertheless, the real utility of antioxidant cocktail treatments for patients affected by MDs still needs to be scientifically demonstrated. Unfortunately, clinical trials for antioxidant therapies using α-tocopherol, ascorbate, glutathione, riboflavin, niacin, acetyl-carnitine and coenzyme Q have met a limited success. Indeed, it would be expected that the employed antioxidants can only be effective if they are able to target the specific mechanism, i.e., involving the central and peripheral nervous system, responsible for the clinical manifestations of the disease. Noteworthily, very often the phenotypes characterizing MD patients are associated with mutations in proteins whose function does not depend on specific cofactors. Conversely, the administration of the antioxidant cocktails might determine the suppression of endogenous oxidants resulting in deleterious effects on cell viability and/or toxicity for patients. In order to avoid toxicity effects and before administering the antioxidant therapy, it might be useful to ascertain the blood serum levels of antioxidants and cofactors to be administered in MD patients. It would be also worthwhile to check the localization of mutations affecting proteins whose function should depend (less or more directly) on the cofactors to be administered, for estimating the real need and predicting the success of the proposed cofactor/antioxidant-based therapy.

Crestal bone changes around early vs. conventionally loaded implants with a multi-phosphonate coated surface: A randomized pilot clinical trial.

OBJECTIVES: To compare the marginal bone level around implants with a thin multi-phosphonate coated surface after either an early or conventional loading protocol. MATERIAL AND METHODS: A randomized pilot clinical trial was conducted. Dental impressions were obtained after either 4 (test) or 8 weeks (control) and single crowns screwed-in 2 weeks later. Several variables were evaluated including radiographical marginal bone level (MBL), patient's level variables, and those related to the restoration and surrounding tissues. These data were obtained at several time points up to a 1-year follow-up. RESULTS: Thirty-four patients were included in the study, 18 assigned to the test group. No differences at implant placement were detected for tissue thickness, keratinized mucosa, nor any other clinical or radiological variable. At the time of impressions, tissue was thinner in the test group (2.30 (0.46) versus 2.78 (0.66) mm, test versus control, respectively; p = .012) so shorter abutments were used in this group. Regardless, no significant changes in marginal bone level were detected neither within group along time nor between groups. The average MBL at the 1-year follow-up was -0.15 (0.32) versus -0.22 (0.37) (p = .443) (test versus control, respectively). None of the clinical or radiological variables evaluated had a determinant influence on the MBL at any visit nor group. CONCLUSION: The use of implants with a multi-phosphonate coated surface for early loading offers successful radiographical outcomes 1 year after loading. MBL over time was not affected by taking the impressions 4 or 8 weeks after implant placement and loading them 2 weeks later.

Environmental Exposure during Pregnancy: Influence on Prenatal Development and Early Life: A Comprehensive Review.

Preconception and prenatal exposure to environmental contaminants may affect future health. Pregnancy and early life are critical sensitive windows of susceptibility. The aim of this review was to summarize current evidence on the toxic effects of environment exposure during pregnancy, the neonatal period, and childhood. Alcohol use is related to foetal alcohol spectrum disorders, foetal alcohol syndrome being its most extreme form. Smoking is associated with placental abnormalities, preterm birth, stillbirth, or impaired growth and development, as well as with intellectual impairment, obesity, and cardiovascular diseases later in life. Negative birth outcomes have been linked to the use of drugs of abuse. Pregnant and lactating women are exposed to endocrine-disrupting chemicals and heavy metals present in foodstuffs, which may alter hormones in the body. Prenatal exposure to these compounds has been associated with pre-eclampsia and intrauterine growth restriction, preterm birth, and thyroid function. Metals can accumulate in the placenta, causing foetal growth restriction. Evidence on the effects of air pollutants on pregnancy is constantly growing, for example, preterm birth, foetal growth restriction, increased uterine vascular resistance, impaired placental vascularization, increased gestational diabetes, and reduced telomere length. The advantages of breastfeeding outweigh any risks from contaminants. However, it is important to assess health outcomes of toxic exposures via breastfeeding. Initial studies suggest an association between pre-eclampsia and environmental noise, particularly with early-onset pre-eclampsia. There is rising evidence of the negative effects of environmental contaminants following exposure during pregnancy and breastfeeding, which should be considered a major public health issue.

Cyto/Biocompatibility of Dopamine Combined with the Antioxidant Grape Seed-Derived Polyphenol Compounds in Solid Lipid Nanoparticles.

Background: The loss of nigrostriatal neurons containing dopamine (DA) together with the "mitochondrial dysfunction" in midbrain represent the two main causes related to the symptoms of Parkinson's disease (PD). Hence, the aim of this investigation is to co-administer the missing DA and the antioxidant grape seed-derived proanthocyanidins (grape seed extract, GSE) in order to increase the levels of the neurotransmitter (which is unable to cross the Blood Brain Barrier) and reducing the oxidative stress (OS) related to PD, respectively. Methods: For this purpose, we chose Solid Lipid Nanoparticles (SLN), because they have been already proven to increase DA uptake in the brain. DA-SLN adsorbing GSE (GSE/DA-SLN) were formulated and subjected to physico-chemical characterization, and their cytocompatibility and protection against OS were examined. Results: GSE was found on SLN surface and release studies evidenced the efficiency of GSE in preventing DA autoxidation. Furthermore, SLN showed high mucoadhesive strength and were found not cytotoxic to both primary Olfactory Ensheathing and neuroblastoma SH-SY5Y cells by MTT test. Co-administration of GSE/DA-SLN and the OS-inducing neurotoxin 6-hydroxydopamine (100 µM) resulted in an increase of SH-SY5Y cell viability. Conclusions: Hence, SLN formulations containing DA and GSE may constitute interesting candidates for non-invasive nose-to-brain delivery.

Treatment of Cystic Fibrosis Patients Homozygous for F508del with Lumacaftor-Ivacaftor (Orkambi®) Restores Defective CFTR Channel Function in Circulating Mononuclear Cells.

The treatment of cystic fibrosis (CF) patients homozygous for the F508del mutation with Orkambi®, a combination of a corrector (lumacaftor) and a potentiator (ivacaftor) of the mutated CFTR protein, resulted in some amelioration of the respiratory function. However, a great variability in the clinical response was also observed. The aim of this study was to evaluate the response to Orkambi® in a small cohort of F508del/F508del patients (n = 14) in terms of clinical and laboratory parameters, including ex vivo CFTR activity in mononuclear cells (MNCs), during a 12-month treatment. Patients responded with an increase in percent predicted forced expiratory volume in 1 s (FEV1%) and body mass index (BMI) as well as with a decrease in white blood cell (WBC) total counts and serum C-reactive protein (CRP) levels, although not significantly. Sweat chloride and CFTR-dependent chloride efflux were found to decrease and increase, respectively, as compared with pre-therapy values. CFTR and BMI showed a statistically significant correlation during Orkambi® treatment. Clustering analysis showed that CFTR, BMI, sweat chloride, FEV1%, and WBC were strongly associated. These data support the notion that CFTR-dependent chloride efflux in MNCs should be investigated as a sensitive outcome measure of Orkambi® treatment in CF patients.

Correctors of mutant CFTR enhance subcortical cAMP-PKA signaling through modulating ezrin phosphorylation and cytoskeleton organization.

The most common mutation of the cystic fibrosis transmembrane regulator (CFTR) gene, F508del, produces a misfolded protein resulting in its defective trafficking to the cell surface and an impaired chloride secretion. Pharmacological treatments partially rescue F508del CFTR activity either directly by interacting with the mutant protein and/or indirectly by altering the cellular protein homeostasis. Here, we show that the phosphorylation of ezrin together with its binding to phosphatidylinositol-4,5-bisphosphate (PIP2) tethers the F508del CFTR to the actin cytoskeleton, stabilizing it on the apical membrane and rescuing the sub-membrane compartmentalization of cAMP and activated PKA. Both the small molecules trimethylangelicin (TMA) and VX-809, which act as 'correctors' for F508del CFTR by rescuing F508del-CFTR-dependent chloride secretion, also restore the apical expression of phosphorylated ezrin and actin organization and increase cAMP and activated PKA submembrane compartmentalization in both primary and secondary cystic fibrosis airway cells. Latrunculin B treatment or expression of the inactive ezrin mutant T567A reverse the TMA and VX-809-induced effects highlighting the role of corrector-dependent ezrin activation and actin re-organization in creating the conditions to generate a sub-cortical cAMP pool of adequate amplitude to activate the F508del-CFTR-dependent chloride secretion.

Effect of cariporide on ram sperm pH regulation and motility: possible role of NHE1.

Sperm motility, a feature essential for in vivo fertilization, is influenced by intracellular pH (pHi) homeostasis. Several mechanisms are involved in pHi regulation, among which sodium-hydrogen exchangers (NHEs), a family of integral transmembrane proteins that catalyze the exchange of Na+ for H+ across lipid bilayers. A preliminary characterization of NHE activity and kinetic parameters, followed by analysis of the expression and localization of the protein in ram spermatozoa was performed. NHE activity showed an apparent Km for external Na+ of 17.61 mM. Immunoblotting revealed a molecular mass of 85 kDa. Immunolocalization pattern showed some species-specific aspects, such as positive labeling at the equatorial region of the sperm head. Cariporide, a selective NHE1 inhibitor, significantly reduced pHi recovery (85%). Similarly, exposure to cariporide significantly inhibited different motility parameters, including those related to sperm capacitation. In vitro fertilization (IVF) was not affected by cariporide, possibly due to the non-dramatic, although significant, drop in motility and velocity parameters or due to prolonged exposure during IVF, which may have caused progressive loss of its inhibitory effect. In conclusion, this is the first study documenting, in a large animal model (sheep) of well-known translational relevance, a direct functional role of NHE on sperm pHi and motility. The postulated specificity of cariporide toward isoform 1 of the Na+/H+ exchanger seems to suggest that NHE1 may contribute to the observed effects on sperm cell functionality.

Characterization of mitochondrial function in cells with impaired cystic fibrosis transmembrane conductance regulator (CFTR) function.

Evidence supporting the occurrence of oxidative stress in Cystic Fibrosis (CF) is well established and the literature suggests that oxidative stress is inseparably linked to mitochondrial dysfunction. Here, we have characterized mitochondrial function, in particular as it regards the steps of oxidative phosphorylation and ROS production, in airway cells either homozygous for the F508del-CFTR allele or stably expressing wt-CFTR. We find that oxygen consumption, ΔΨ generation, adenine nucleotide translocator-dependent ADP/ATP exchange and both mitochondrial Complex I and IV activities are impaired in CF cells, while both mitochondrial ROS production and membrane lipid peroxidation increase. Importantly, treatment of CF cells with the small molecules VX-809 and 4,6,4'-trimethylangelicin, which act as "correctors" for F508del CFTR by rescuing the F508del CFTR-dependent chloride secretion, while having no effect per sè on mitochondrial function in wt-CFTR cells, significantly improved all the above mitochondrial parameters towards values found in the airway cells expressing wt-CFTR. This novel study on mitochondrial bioenergetics provides a springboard for future research to further understand the molecular mechanisms responsible for the involvement of mitochondria in CF and identify the proteins primarily responsible for the F508del-CFTR-dependent mitochondrial impairment and thus reveal potential novel targets for CF therapy.

Trimethylangelicin promotes the functional rescue of mutant F508del CFTR protein in cystic fibrosis airway cells.

Cystic fibrosis transmembrane conductance regulator (CFTR) carrying the F508del mutation is retained in endoplasmic reticulum and fails to traffic to the cell surface where it functions as a protein kinase A (PKA)-activated chloride channel. Pharmacological correctors that rescue the trafficking of F508del CFTR may overcome this defect; however, the rescued F508del CFTR still displays reduced chloride permeability. Therefore, a combined administration of correctors and potentiators of the gating defect is ideal. We recently found that 4,6,4'-trimethylangelicin (TMA), besides inhibiting the expression of the IL-8 gene in airway cells in which the inflammatory response was challenged with Pseudomonas aeruginosa, also potentiates the cAMP/PKA-dependent activation of wild-type CFTR or F508del CFTR that has been restored to the plasma membrane. Here, we demonstrate that long preincubation with nanomolar concentrations of TMA is able to effectively rescue both F508del CFTR-dependent chloride secretion and F508del CFTR cell surface expression in both primary or secondary airway cell monolayers homozygous for F508del mutation. The correction effect of TMA seems to be selective for CFTR and persisted for 24 h after washout. Altogether, the results suggest that TMA, besides its anti-inflammatory and potentiator activities, also displays corrector properties.

CFTR regulation in human airway epithelial cells requires integrity of the actin cytoskeleton and compartmentalized cAMP and PKA activity.

The cystic fibrosis transmembrane conductance regulator (CFTR) mutation ΔF508CFTR still causes regulatory defects when rescued to the apical membrane, suggesting that the intracellular milieu might affect its ability to respond to cAMP regulation. We recently reported that overexpression of the Na(+)/H(+) exchanger regulatory factor NHERF1 in the cystic fibrosis (CF) airway cell line CFBE41o-rescues the functional expression of ΔF508CFTR by promoting F-actin organization and formation of the NHERF1-ezrin-actin complex. Here, using real-time FRET reporters of both PKA activity and cAMP levels, we find that lack of an organized subcortical cytoskeleton in CFBE41o-cells causes both defective accumulation of cAMP in the subcortical compartment and excessive cytosolic accumulation of cAMP. This results in reduced subcortical levels and increased cytosolic levels of PKA activity. NHERF1 overexpression in CFBE41o-cells restores chloride secretion, subcortical cAMP compartmentalization and local PKA activity, indicating that regulation of ΔF508CFTR function requires not only stable expression of the mutant CFTR at the cell surface but also depends on both generation of local cAMP signals of adequate amplitude and activation of PKA in proximity of its target. Moreover, we found that the knockdown of wild-type CFTR in the non-CF 16HBE14o-cells results in both altered cytoskeletal organization and loss of cAMP compartmentalization, whereas stable overexpression of wt CFTR in CF cells restores cytoskeleton organization and re-establishes the compartmentalization of cAMP at the plasma membrane. This suggests that the presence of CFTR on the plasma membrane influences the cytoskeletal organizational state and, consequently, cAMP distribution. Our data show that a sufficiently high concentration of cAMP in the subcortical compartment is required to achieve PKA-mediated regulation of CFTR activity.

MED1101: a new dialdehydic compound regulating P2×7 receptor cell surface expression in U937 cells.

BACKGROUND INFORMATION: P2×7R is a member of the ionotropic family of purinergic receptors activated by millimolar concentrations of extracellular ATP such as induced by inflammatory stimuli. The receptor is widely expressed in cells of haematopoietic origin such as monocytes, macrophages and microglia. There is growing interest in anta-gonist compounds of the P2×7R since it has been demonstrated to be a viable therapeutic target for inflammatory diseases. Here, we tested the possible P2×7 antagonist effect of MED1101, a newly synthesised dialdehydic compound on U937 monocyte cells. RESULTS: Human U937 cells express the full-length P2×7A receptor isoform. Treatment with lipopolysaccharide (LPS), a potent inducer of inflammation, significantly increased the expression of the receptor in the plasma membrane. Importantly, MED1101 induced internalisation of the P2×7R already after 30 min incubation in both physiological conditions and in presence of the inflammatory stimulus (LPS) and this effect was observable for up to 12 h after its removal. Moreover, MED1101 induced an impairment of monocyte migration/transmigration through direct P2×7R antagonism and subsequent inhibition of the intracellular signal transduction processes of Ca2+ influx and MAPK phosphorylation. CONCLUSIONS: Our results clearly demonstrate that in U937 monocyte cells MED1101 acts as a P2×7R antagonist through the induction of receptor internalisation and subsequent inhibition of down-stream signal transduction pathways that regulate monocyte migration/transmigration, thus playing a potential therapeutic role in inflammatory diseases.

Slightly viscous dispersions of mucoadhesive polymers as vehicles for nasal administration of dopamine and grape seed extract-loaded solid lipid nanoparticles.

With the aim to find an alternative vehicle to the most used thermosensitive hydrogels for efficient nanotechnology-based nose-to-brain delivery approach for Parkinson's disease (PD) treatment, in this work we evaluated the Dopamine (DA) and the antioxidant grape seed-derived pro-anthocyanidins (Grape Seed Extract, GSE) co-loaded solid lipid nanoparticles (SLNs) put in slight viscous dispersions (SVDs). These SVDs were prepared by dispersion in water at low concentrations of mucoadhesive polymers to which SLN pellets were added. For the purpose, we investigated two polymeric blends, namely Poloxamer/Carbopol (PF-127/Carb) and oxidized alginate/Hydroxypropylmethyl cellulose (AlgOX/HPMC). Rheological studies showed that the two fluids possess Newtonian behaviour with a viscosity slightly higher that water. The pH values of the SVDs were mainly within the normal range of nasal fluid as well as almost no osmotic effect was associated to both SVDs. All the SVDs were capable to provide DA permeation through nasal porcine mucosa. Moreover, it was found that PF-127/Carb blend possesses penetration enhancer capability better than the Alg OX/HPMC combination. Flow cytometry studies demonstrated the uptake of viscous liquids incorporating fluorescent SLNs by human nasal RPMI 2650 cell in time-dependent manner. In conclusion, the SVD formulations may be considered promising alternatives to thermosensitive hydrogels strategy. Moreover, in a broader perspective, such SVD formulations may be also hopeful for treating various neurological diseases beyond PD treatment.

Combined Dopamine and Grape Seed Extract-Loaded Solid Lipid Nanoparticles: Nasal Mucosa Permeation, and Uptake by Olfactory Ensheathing Cells and Neuronal SH-SY5Y Cells.

We have already formulated solid lipid nanoparticles (SLNs) in which the combination of the neurotransmitter dopamine (DA) and the antioxidant grape-seed-derived proanthocyanidins (grape seed extract, GSE) was supposed to be favorable for Parkinson's disease (PD) treatment. In fact, GSE supply would reduce the PD-related oxidative stress in a synergic effect with DA. Herein, two different methods of DA/GSE loading were studied, namely, coadministration in the aqueous phase of DA and GSE, and the other approach consisting of a physical adsorption of GSE onto preformed DA containing SLNs. Mean diameter of DA coencapsulating GSE SLNs was 187 ± 4 nm vs. 287 ± 15 nm of GSE adsorbing DA-SLNs. TEM microphotographs evidenced low-contrast spheroidal particles, irrespective of the SLN type. Moreover, Franz diffusion cell experiments confirmed the permeation of DA from both SLNs through the porcine nasal mucosa. Furthermore, fluorescent SLNs also underwent cell-uptake studies by using flow cytometry in olfactory ensheathing cells and neuronal SH-SY5Y cells, evidencing higher uptake when GSE was coencapsulated rather than adsorbed onto the particles.

Targeting mitochondrial impairment for the treatment of cardiovascular diseases: From hypertension to ischemia-reperfusion injury, searching for new pharmacological targets.

Mitochondria and mitochondrial proteins represent a group of promising pharmacological target candidates in the search of new molecular targets and drugs to counteract the onset of hypertension and more in general cardiovascular diseases (CVDs). Indeed, several mitochondrial pathways result impaired in CVDs, showing ATP depletion and ROS production as common traits of cardiac tissue degeneration. Thus, targeting mitochondrial dysfunction in cardiomyocytes can represent a successful strategy to prevent heart failure. In this context, the identification of new pharmacological targets among mitochondrial proteins paves the way for the design of new selective drugs. Thanks to the advances in omics approaches, to a greater availability of mitochondrial crystallized protein structures and to the development of new computational approaches for protein 3D-modelling and drug design, it is now possible to investigate in detail impaired mitochondrial pathways in CVDs. Furthermore, it is possible to design new powerful drugs able to hit the selected pharmacological targets in a highly selective way to rescue mitochondrial dysfunction and prevent cardiac tissue degeneration. The role of mitochondrial dysfunction in the onset of CVDs appears increasingly evident, as reflected by the impairment of proteins involved in lipid peroxidation, mitochondrial dynamics, respiratory chain complexes, and membrane polarization maintenance in CVD patients. Conversely, little is known about proteins responsible for the cross-talk between mitochondria and cytoplasm in cardiomyocytes. Mitochondrial transporters of the SLC25A family, in particular, are responsible for the translocation of nucleotides (e.g., ATP), amino acids (e.g., aspartate, glutamate, ornithine), organic acids (e.g. malate and 2-oxoglutarate), and other cofactors (e.g., inorganic phosphate, NAD+, FAD, carnitine, CoA derivatives) between the mitochondrial and cytosolic compartments. Thus, mitochondrial transporters play a key role in the mitochondria-cytosol cross-talk by leading metabolic pathways such as the malate/aspartate shuttle, the carnitine shuttle, the ATP export from mitochondria, and the regulation of permeability transition pore opening. Since all these pathways are crucial for maintaining healthy cardiomyocytes, mitochondrial carriers emerge as an interesting class of new possible pharmacological targets for CVD treatments.