Design of experiment (DoE) of mucoadhesive valproic acid-loaded nanostructured lipid carriers (NLC) for potential nose-to-brain application.

Epilepsy is a highly prevalent neurological disease and valproic acid (VPA) is used as a first-line chronic treatment. However, this drug has poor oral bioavailability, which requires the administration of high doses, resulting in adverse effects. Alternative routes of VPA administration have therefore been investigated, such as the nose-to-brain route, which allows the drug to be transported directly from the nasal cavity to the brain. Here, the use of nanostructured lipid carriers (NLC) to encapsulate drugs administered in the nasal cavity has proved advantageous. The aim of this work was to optimise a mucoadhesive formulation of VPA-loaded NLC for intranasal administration to improve the treatment of epilepsy. The Design of Experiment (DoE) was used to optimise the formulation, starting with component optimisation using Mixture Design (MD), followed by optimisation of the manufacturing process parameters using Central Composite Design (CCD). The optimised VPA-loaded NLC had a particle size of 76.1 ± 2.8 nm, a polydispersity index of 0.190 ± 0.027, a zeta potential of 28.1 ± 2.0 mV and an encapsulation efficiency of 85.4 ± 0.8%. The in vitro release study showed VPA release from the NLC of 50 % after 6 h and 100 % after 24 h. The in vitro biocompatibility experiments in various cell lines have shown that the optimised VPA-loaded NLC formulation is safe up to 75 µg/mL, in neuronal (SH-SY5Y), nasal (RPMI 2650) and hepatic (HepG2) cells. Finally, the interaction of the optimised VPA-loaded NLC formulation with nasal mucus was investigated and mucoadhesive properties were observed. The results of this study suggest that the use of intranasal VPA-loaded NLC may be a promising alternative to promote VPA targeting to the brain, thereby improving bioavailability and minimising adverse effects.

Tangential velocity constraint for orbital maneuvers with Theory of Functional Connections.

Maneuvering a spacecraft in the cislunar space is a complex problem, since it is highly perturbed by the gravitational influence of both the Earth and the Moon, and possibly also the Sun. Trajectories minimizing the needed fuel are generally preferred in order to decrease the mass of the payload. A classical method to constrain maneuvers is mathematically modeling them using the Two Point Boundary Value Problem (TPBVP), defining spacecraft positions at the start and end of the trajectory. Solutions to this problem can then be obtained with optimization techniques like the nonlinear least squares conjugated with the Theory of Functional Connections (TFC) to embed the constraints, which recently became an effective method for deducing orbit transfers. In this paper, we propose a tangential velocity (TV) type of constraints to design orbital maneuvers. We show that the technique presented in this paper can be used to transfer a spacecraft (e.g. from the Earth to the Moon) and perform gravity assist maneuvers (e.g. a swing-by with the Moon). In comparison with the TPBVP, solving the TV constraints via TFC offers several advantages, leading to a significant reduction in computational time. Hence, it proves to be an efficient technique to design these maneuvers.

Design of experiment (DoE) as a quality by design (QbD) tool to optimise formulations of lipid nanoparticles for nose-to-brain drug delivery.

INTRODUCTION: The nose-to-brain route has been widely investigated to improve drug targeting to the central nervous system (CNS), where lipid nanoparticles (solid lipid nanoparticles - SLN and nanostructured lipid carriers - NLC) seem promising, although they should meet specific criteria of particle size (PS) <200 nm, polydispersity index (PDI) <0.3, zeta potential (ZP) ~|20| mV and encapsulation efficiency (EE) >80%. To optimize SLN and NLC formulations, design of experiment (DoE) has been recommended as a quality by design (QbD) tool. AREAS COVERED: This review presents recently published work on the optimization of SLN and NLC formulations for nose-to-brain drug delivery. The impact of different factors (or independent variables) on responses (or dependent variables) is critically analyzed. EXPERT OPINION: Different DoEs have been used to optimize SLN and NLC formulations for nose-brain drug delivery, and the independent variables lipid and surfactant concentration and sonication time had the greatest impact on the dependent variables PS, EE, and PDI. Exploring different DoE approaches is important to gain a deeper understanding of the factors that affect successful optimization of SLN and NLC and to facilitate future work improving machine learning techniques.

Lipid nanoparticles strategies to modify pharmacokinetics of central nervous system targeting drugs: Crossing or circumventing the blood-brain barrier (BBB) to manage neurological disorders.

The main limitation to the success of central nervous system (CNS) therapies lies in the difficulty for drugs to cross the blood-brain barrier (BBB) and reach the brain. Regarding its structure and enzymatic complexity, crossing the BBB is a challenge, although several alternatives have been identified. For instance, the use of drugs encapsulated in lipid nanoparticles has been described as one of the most efficient approaches to bypass the BBB, as they allow the passage of drugs through this barrier, improving brain bioavailability. In particular, solid lipid nanoparticles (SLN) and nanostructured lipid carriers (NLC) have been a focus of research related to drug delivery to the brain. These systems provide protection of lipophilic drugs, improved delivery and bioavailability, having a major impact on treatments outcomes. In addition, the use of lipid nanoparticles administered via routes that transport drugs directly into the brain seems a promising solution to avoid the difficulties in crossing the BBB. For instance, the nose-to-brain route has gained considerable interest, as it has shown efficacy in 3D human nasal models and in animal models. This review addresses the state of the art on the use of lipid nanoparticles to modify the pharmacokinetics of drugs employed in the management of neurological disorders. A description of the structural components of the BBB, the role of the neurovascular unit and limitations for drugs to entry into the CNS is first addressed, along with the developments to increase drug delivery to the brain, with a special focus on lipid nanoparticles. In addition, the obstacle of BBB complexity in the creation of new effective drugs for the treatment of the most prevalent neurological disorders is also addressed. Finally, the proposed strategies for lipid nanoparticles to reach the CNS, crossing or circumventing the BBB, are described. Although promising results have been reported, especially with the nose-to-brain route, they are still ongoing to assess its real efficacy in vivo in the management of neurological disorders.

Aeromonas piscicola AH-3 expresses an extracellular collagenase with cytotoxic properties.

UNLABELLED: The aim of this study was to investigate the presence and the phenotypic expression of a gene coding for a putative collagenase. This gene (AHA_0517) was identified in Aeromonas hydrophila ATCC 7966 genome and named colAh. We constructed and characterized an Aeromonas piscicola AH-3::colAh knockout mutant. Collagenolytic activity of the wild-type and mutant strains was determined, demonstrating that colAh encodes for a collagenase. ColAh-collagen interaction was assayed by Far-Western blot, and cytopathic effects were investigated in Vero cells. We demonstrated that ColAh is a gluzincin metallopeptidase (approx. 100 kDa), able to cleave and physically interact with collagen, that contributes for Aeromonas collagenolytic activity and cytotoxicity. ColAh possess the consensus HEXXH sequence and a glutamic acid as the third zinc binding positioned downstream the HEXXH motif, but has low sequence similarity and distinct domain architecture to the well-known clostridial collagenases. In addition, these results highlight the importance of exploring new microbial collagenases that may have significant relevance for the health and biotechnological industries. SIGNIFICANCE AND IMPACT OF THE STUDY: Collagenases play a central role in processes where collagen digestion is needed, for example host invasion by pathogenic micro-organisms. We identified a new collagenase from Aeromonas using an integrated in silico/in vitro strategy. This enzyme is able to bind and cleave collagen, contributes for AH-3 cytotoxicity and shares low similarity with known bacterial collagenases. This is the first report of an enzyme belonging to the gluzincin subfamily of the M9 family of peptidases in Aeromonas. This study increases the current knowledge on collagenolytic enzymes bringing new perspectives for biotechnology/medical purposes.

Anguilla anguilla L. liver EROD induction and genotoxic responses after retene exposure.

Anguilla anguilla L. (European eel) were exposed for 8, 16, 24, and 72 h to 0 (control), 0.1, 0.3, 0.9, and 2.7 microM retene. A. anguilla L. liver ethoxyresorufin-O-deethylase EROD activity significantly increased during the whole exposure period to all retene concentrations, when compared to their controls. The liver cytochrome P450 content only increased after exposure to high retene concentrations (0.9 and 2.7 microM) from 8 to 24 and 72 h, respectively. Generally, liver DNA integrity decreased with increased retene concentrations. Thus, a low retene concentration (0.1 microM) was only effective at 16 h, 0.3 and 0.9 microM had an early and prolonged effect up to 24h, and 2.7 microM decreased liver DNA integrity during the whole exposure period. However, blood DNA integrity decrease was observed in eels after 24h exposure to 0.1 microM retene, and at 16 h to 0.3 and 0.9 microM retene, despite an early blood DNA integrity decrease at 8 and 16 h exposure to 2.7 microM retene. An early genotoxic response to retene was also observed as erythrocyte nuclear abnormalities plus Notched (ENA+Not) frequency increase at 8, 16, and 24h exposure to 0.1 and 0.3 microM retene as well as at 8, 16, 24, and 72 h to 0.9 microM retene. Though, the highest retene concentration (2.7 microM) only induced ENA+Not and erythrocyte nuclear abnormalities minus Notched (ENA-Not) at 16 h exposure. The eel ENA+Not increase was more sensitive than the ENA-Not increase as a measure of retene genotoxicity. Eel liver alanine amino transferase (ALT) increased activity reveals its enhanced transamination capacity after short-term exposure to retene. The A. anguilla L. ratio between hemoglobin concentration and red blood cells count (Hb/RBC) increased at 8h exposure to 0.1, 0.3, and 0.9 microM retene, suggesting an initial homeostatic process.

Anguilla anguilla L. genotoxic and liver biotransformation responses to abietic acid exposure.

Adult eels (Anguilla anguilla L.) were exposed for 8, 16, 24, and 72 h to 0, 0.1, 0.3, 0.9, and 2.7 microM abietic acid (AA). Genotoxicity was measured as erythrocytic nuclear abnormalities (ENA), as well as DNA strand breaks in blood and liver. Liver cytochrome P450 (P450) content, liver ethoxyresorufin O-deethylase (EROD), and glutathione S-transferase (GST) activities were determined as biotransformation biomarkers. Liver alanine transaminase (ALT) activity was also measured as an indication of tissue damage. Low AA concentrations, such as 0.1 and 0.3 microM, result in a delayed induction of A. anguilla L. liver EROD activity, whereas the higher AA concentration (2.7 microM AA) also has a delayed effect probably as a consequence of liver tissue high inhibitory concentration. The current eel liver GST activity results demonstrate that only low AA concentrations promote liver increases in GST, whereas high AA concentrations, such as 0.9 and 2.7 microM, do not alter it. The results concerning eel liver ALT activity indicate that significant liver damage is induced by high AA concentrations, such as 2.7 and 0.9 microM. The eel ENA result analysis reveals that AA is a weak ENA inducer in A. anguilla L. Blood DNA integrity results suggest that low AA concentrations promote late decreases in blood DNA integrity; nevertheless, high AA concentrations are early blood genotoxic inducers compared with low AA doses. According to the present research results with respect to eel liver DNA damage, all of the AA exposure concentrations decreased liver DNA integrity.

Genotoxic and biochemical responses in caged eel (Anguilla anguilla L.) after short-term exposure to harbour waters.

European eel (Anguilla anguilla L.) were caged and exposed in situ for 8 and 48 h to the Aveiro offward fishing harbour water (HW) and to clean seawater under laboratory conditions (Control). Eel liver biotransformation (Phase I) was measured as ethoxyresorufin-O-deethylase (EROD) activity, cytochrome P450 (P450) and glutathione S-transferase (GST) activity (Phase II). Genotoxic responses were determined as blood, liver and kidney DNA strand breaks as well as erythrocytic nuclear abnormalities (ENAs). HW failed to significant increase liver EROD, GST activities and ENA frequency. Nevertheless, P450 content was significantly increased after 8 and 48 h exposure. Genotoxicity measured as DNA integrity decrease was found in blood after 8 and 48 h exposure to HW, whereas in liver and kidney, it was observed after 48 h exposure to HW. Blood, kidney and liver genotoxicity may be due to the presence of polycyclic aromatic hydrocarbons (PAHs) which are genotoxic compounds and the main HW organic contaminants.

Genotoxic and hepatic biotransformation responses induced by the overflow of pulp mill and secondary-treated effluents on Anguilla anguilla L.

Pulp and paper mill effluent compounds pollute the aquatic environment and are responsible for increased biochemical alterations and genotoxicity in aquatic organisms such as fish. Adult eels (Anguilla anguilla L) were exposed during 8, 16, 24, and 72 h to the following conditions: (1) aerated, filtered, and dechlorinated tap water (C); (2) 2.5% (v/v) sewage water previously treated with activated sludge (T); (3) bleached kraft pulp and paper mill effluent collected at the river Vouga, close to an ancient sewage outlet (Portucel), diluted in tap water [25% (E25) and 50% (E50)]; and (4) bleached kraft pulp and paper mill effluent sediment [water-soluble fraction (S)]. Liver biotransformation induced by the above conditions was measured as ethoxyresorufin-O-deethylase (EROD), cytochrome P450 (P450) (Phase I), and glutathione-S-transferase (GST) (Phase II). Genotoxicity was also determined as blood/liver DNA strand breaks and erythrocytic nuclear abnormalities (ENA) induced on European eel (A. anguilla L). Liver EROD activity was significantly increased in eels at 8 and 16 h exposure to E25, as well as at 16, 24, and 72 h exposure to E50. S exposure induced liver EROD activity only at 24h. A significant decrease in liver P450 was observed at 72 h exposure to T, whereas a significant P450 increase at 16 h was followed by a significant decrease at 24h exposure to E25. Another P450 significant increase was noticed at 72 h exposure to S. Liver GST activity (Phase II) demonstrated a significant increase at 72 h exposure to E50 and to S. A significant decrease in blood DNA integrity was observed at 72 h exposure to T and at 24 and 72 h to S. Blood DNA integrity significantly decreased at 16 and 24 h exposure to E25, as well as at 8, 16, and 24 h exposure to E50. Liver DNA integrity significantly decreased at 72 h exposure to T and at 16 h exposure to S. Moreover, liver DNA integrity was significantly decreased at 24h exposure to E25 and E50, and 72 h to E50. A. anguilla L. increased ENA frequency was detected in T at 16, 24, and 72 h, whereas in E25 and S it was observed at 8, 16, and 24 h. Furthermore, E50 ENA frequency increased at 24 h exposure.

Benzo[a]pyrene and beta-naphthoflavone mutagenic activation by European eel (Anguilla anguilla L.) S9 liver fraction.

Is Anguilla anguilla L. (eel) liver ethoxyresorufin O-deethylase (EROD) induction absolutely necessary in order to convert promutagens as benzo[a]pyrene (BaP) into a mutagenic compound? Eels were exposed for 8 h to clean (control) and 0.3 microM beta-naphthoflavone (BNF)-contaminated water. The 8-h exposure to 0.3 microM BNF brought about a very high EROD induction (10 pmol/min/mg protein) compared to control animals (1 pmol/min/mg protein). The Ames test (Maron and Ames, 1983) was carried out with Salmonella typhimurium TA 98 strain (TA98 His-) and eel isolated S9 liver fraction was used as a metabolic BaP activator. The BaP and BNF dose range concentrations tested were 0 (blank), 0.015, 0.08, 0.15, 0.38, 0.75, 1.5, 3.8, and 7.5 microM/plate and 0 (blank), 0.412, 1.235, 3.704, 11.1, 33.0, and 100 nM BNF, respectively. A dose-response relationship between BaP concentration and mutagenic activity was observed in the presence of S9 fractions in control and 0.3 microM BNF-exposed eels. Significant positive results, as TA98 His+ revertants, were observed at 0.38, 0.75, 1.5, 3.8, and 7.5 microM BaP/plate induced by BNF S9 fractions. Significant BaP mutagenic activation by liver control S9 was detected only at 1.5, 3.8, and 7.5 microM/plate. The BaP 1.5, 3.8, and 7.5 microM/plate mutagenic activation by BNF S9 and control S9 were not significantly different. Relative to BNF activation, it was only possible to detect His+ reversion at 11.1 nM BNF concentration with 0.3 microM BNF-induced S9. The above results demonstrate that the eel S9 liver fraction has the capacity to biotransform high BaP concentrations and convert it into a mutagenic compound with or without previous liver BNF biotransformation induction. The same does not apply to low BaP concentrations, where liver S9 induction by BNF is necessary to promote mutagenesis.

Anguilla anguilla L. biochemical and genotoxic responses to benzo[a]pyrene.

Eels (Anguilla anguilla L.) were exposed for 2, 4, 6, 8, 16, 24, 48, 72, 144, and 216 h to 0 (control), 0.3, 0.9, and 2.7 microM benzo[a]pyrene (BaP). The biotransformation induced by BaP was measured as liver ethoxyresorufin O-deethylase (EROD) activity and cytochrome P450 content, and compared with the genotoxic effects, such as erythrocytic nuclear abnormalities (ENA), and blood and liver DNA strand breaks. The liver exhibited a highly significant EROD activity increase from 2 up to 216 h exposure to 0.9 and 2.7 microM BaP, whereas 0.3 microM BaP exposure induced a significant liver EROD increase from 2 up to 144 h. Liver cytochrome P-450 content was significantly increased at 8 h to 2.7 microM BaP exposure. Liver DNA integrity was decreased at 16 h, from 8 up to 144 h and 8 up to 72 h exposure to 0.3, 0.9, and 2.7 microM BaP, respectively. A significant decrease in blood DNA integrity was observed at 48, 72, 144 h, from 8 up to 72, and from 6 up to 72 h exposure to 0.3, 0.9, and 2.7 microM BaP, respectively. The A. anguilla L. genotoxic response to BaP, measured as ENA induction, was significantly increased at 144 h exposure to 0.3 microM BaP. The intermediate BaP concentration tested (0.9 microM) induced a significant three fold ENA increase at 48 and 72 h exposure compared to their controls. The highest BaP concentration (2.7 microM) induced a significant increase in ENA frequency at 72, 144 and 216 h exposure.

The four final rotation states of Venus.

Venus rotates very slowly on its axis in a retrograde direction, opposite to that of most other bodies in the Solar System. To explain this peculiar observation, it has been generally believed that in the past its rotational axis was itself rotated to 180 degrees as a result of core-mantle friction inside the planet, together with atmospheric tides. But such a change has to assume a high initial obliquity (the angle between the planet's equator and the plane of the orbital motion). Chaotic evolution, however, allows the spin axis to flip for a large set of initial conditions. Here we show that independent of uncertainties in the models, terrestrial planets with dense atmosphere like Venus can evolve into one of only four possible rotation states. Moreover, we find that most initial conditions will drive the planet towards the configuration at present seen at Venus, albeit through two very different evolutionary paths. The first is the generally accepted view whereby the spin axis flips direction. But we have also found that it is possible for Venus to begin with prograde rotation (the same direction as the other planets) yet then develop retrograde rotation while the obliquity goes towards zero: a rotation of the spin axis is not necessary in this case.

Genetic characterization of a new thermotolerant Bacillus licheniformis strain.

A potentially new thermotolerant B. licheniformis strain (code name I89), producer of an antibiotic active against Gram-positive bacteria, was genetically characterized and compared with the type strain B. licheniformis ATCC 10716, producer of bacitracin. Studies on DNA base composition (G + C content) and DNA reassociation revealed that the two strains show around 76% homology. Nevertheless, results obtained by rRNA hybridization, with a heterologous probe coding for most of the 16S region of the rRNA operon of Bacillus subtilis, revealed differences in the number of copies for that gene and in the hybridization pattern. Additionally, a different restriction digestion pattern was obtained when DNA was digested with the enzymes NotI, SmaI and analyzed by PFGE. The I89 strain holds a 7.6-kb plasmid not present in the reference strain. The existence of various unique restriction sites and also the stability of this plasmid make it ideal for the future development of a cloning and expression vector.