Nanosystems for gene therapy targeting brain damage caused by viral infections.

Several human pathogens can cause long-lasting neurological damage. Despite the increasing clinical knowledge about these conditions, most still lack efficient therapeutic interventions. Gene therapy (GT) approaches comprise strategies to modify or adjust the expression or function of a gene, thus providing therapy for human diseases. Since recombinant nucleic acids used in GT have physicochemical limitations and can fail to reach the desired tissue, viral and non-viral vectors are applied to mediate gene delivery. Although viral vectors are associated to high levels of transfection, non-viral vectors are safer and have been further explored. Different types of nanosystems consisting of lipids, polymeric and inorganic materials are applied as non-viral vectors. In this review, we discuss potential targets for GT intervention in order to prevent neurological damage associated to infectious diseases as well as the role of nanosized non-viral vectors as agents to help the selective delivery of these gene-modifying molecules. Application of non-viral vectors for delivery of GT effectors comprise a promising alternative to treat brain inflammation induced by viral infections.

Pluronic® F127 Thermoresponsive Viscum album Hydrogel: Physicochemical Features and Cellular In Vitro Evaluation.

Viscum album L., popularly known as mistletoe, is well known for its anti-cancer properties, and the pharmaceutical application of hydroalcoholic dry extracts is still limited due to its low solubility in aqueous media, and physicochemical instability. The Pluronic® F127 is an amphiphilic polymer, which permits the solubilization of lipophilic and hydrophilic compounds. In this investigation, physicochemical features of hydrogel containing V. album dry extract (VADE-loaded-hydrogel) were performed by: dynamic light scattering (DLS), thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), and transmission electron microscopy (TEM). VADE-loaded-hydrogel presented nanometer-size micelles with volume distribution ranging from 10.58 nm to 246.7 nm, and a polydispersity index of 0.441. The sample thermal analyses (TG and DSC) showed similar decomposition curves; however, the thermal events indicated an increase in thermal stability in relation to the presence of the extract. In addition to these interesting pharmaceutical features, IC50 values of 333.40 µg/mL and >1000 µg/mL were obtained when tumor (SCC-25) and non-tumor (L929) cells were incubated with VADE-loaded-hydrogel, respectively. The optical and ultrastructural cellular analysis confirmed the tumor selectivity since the following alterations were detected only in SCC-25 cells: disorganization of plasmatic membrane; an increase of cytoplasmatic vacuole size; alteration in the cristae mitochondrial shape; and generation of amorphous cellular material. These results emphasize the promising antitumoral potential of VADE-loaded-hydrogel as an herbal drug delivery system via in vitro assays.

Physicochemical Properties of Zinc and Lactose in Solid Mixtures: Influence of Trituration Process.

BACKGROUND: Recent experimental results supporting the dynamization process show modification in the characteristics of solid mixtures. OBJECTIVE: The present work aims to evaluate the physicochemical properties of metallic zinc and lactose, evidencing the interactions between all chemical components presented in dynamized solid mixtures by analytical techniques. METHODS: Mixtures of zinc and lactose (1:9 w/w) were successively triturated at the same proportion according to the Brazilian Homeopathic Pharmacopoeia, receiving the designation of 10-1 - 10-6 (1dH - 6dH). All samples were submitted to the following characterization techniques: Atomic Absorption Spectrometry (AAS), Scanning Electron Microscopy (SEM), X-ray Diffraction (XRD), Differential Scanning Calorimetry (DSC), Thermogravimetry (TG), and Raman Spectroscopy (RS). RESULTS: AAS results detected 97.0% of zinc in the raw material, and the triturated zinc lactose system (ZnMet) presented mean values similar to those expected for the physical mixtures: i.e., 9.94%, 1.23%, and 0.11% in the three first proportions (10-1, 10-2, 10-3), respectively. SEM images showed particle size reduction due to the trituration process. The XRD assays of ZnMet 10-3 and 10-6 indicated peak changes at 12.3° and 43.26°, probably associated with modifications of inter-atomic crystalline spacing. The thermal analysis results of dynamized samples suggest modifications in the chemical interaction between zinc and lactose induced by the physical forces applied. RS experiments showed variation in vibration frequencies due to the dynamization procedure, in which marked ZnMet 10-6 spectral modifications were detected at 357, 477, 1086 and 1142 cm-1, and in the wavelength range 860-920 cm-1. CONCLUSION: These results highlight the importance of applying suitable characterization methods to improve our understanding of the properties of homeopathic solid mixtures, whereas the uses of sensitive tools evidence the influence of trituration on the crystalline properties and in the enthalpy variation of dynamized samples.

Distinguishing Activities in the Photodynamic Arsenals of the Pigmented Ciliates Blepharisma sinuosum Sawaya, 1940 and Blepharisma japonicum Suzuki, 1954 (Ciliophora: Heterotrichea).

Blepharismins are photodynamic hypericin-like dianthrones produced as a variable pigment blend in Blepharisma ciliates and mostly studied in the Afro-Asiatic Blepharisma japonicum. The present work describes the bioactivity of pigments from the Brazilian Blepharisma sinuosum. Comparative analyses showed that the pigments from both species can trigger photo-induced modifications in phospholipids, but different redox properties and biological activities were assigned for each pigment blend. Stronger activities were detected for B. sinuosum pigments, with the lethal concentration LC50 10 × lower than B. japonicum pigments in light-irradiated tests against Bacillus cereus and less than half for treatments on the human HeLa tumor cells. HPLC showed B. sinuosum producing a simpler pigment blend, mostly with the blepharismin-C (~ 70%) and blepharismin-E (~ 30%) types. Each blepharismin engaged a specific dose-response profile on sensitive cells. The blepharismin-B and blepharismin-C were the most toxic pigments, showing LC50  ~ 2.5-3.0 µm and ~ 100 µm on B. cereus and HeLa cells, respectively, after illumination. Similarity clustering analysis compiling the bioactivity data revealed two groups of blepharismins: the most active, B and C, and the less active, A, D and E. The B. sinuosum pigment blend includes one representative of each clade. Functional and medical implications are discussed.

Influence of levofloxacin and clarithromycin on the structure of DPPC monolayers.

Research on lipid/drug interactions at the nanoscale underpins the emergence of synergistic mechanisms for topical drug administration. The structural understanding of bio-mimetic systems employing 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) as a lung surfactant model mixed with antibiotics, as well as their biophysical properties, is of critical importance to modulate the effectiveness of therapeutic agents released directly to the airways. In this paper, we investigate the structural details of the interaction between Levofloxacin, 'a respiratory quinolone', and the macrolide Clarithromycin, with DPPC monolayers at the air-water interface, using a combination of Brewster angle microscopy, polarization modulation-infrared reflection-adsorption spectroscopy (PM-IRRAS), surface pressure isotherms and neutron reflectometry (NR) to describe the structural details of this interaction. The results allowed association of changes in the π-A isotherm profile with changes in the molecular organization and the co-localization of the antibiotics within the lipid monolayer by NR measurements. Overall, both antibiotics are able to increase the thickness of the acyl tails in DPPC monolayers with a corresponding reduction in tail tilt as well as to interact with the phospholipid headgroups as shown by PM-IRRAS experiments. The effects on the DPPC monolayers are correlated with the physical-chemical properties of each antibiotic and dependent on its concentration.

Interaction of levofloxacin with lung surfactant at the air-water interface.

The molecular-level interaction of levofloxacin with lung surfactant was investigated using Langmuir monolayers and atomistic molecular dynamics (MD) simulations. In the simulation, the DPPC/POPC mixed monolayer was used as a lung surfactant model and the molecules of levofloxacin were placed at the air-lipid interface to mimic the adsorption process on the lung surfactant model. The simulation results indicate that amphoteric levofloxacin expands the lung surfactant, also stabilizing the film for levofloxacin fractions until 10% w/w at least. The Langmuir monolayers made with the lung surfactant Curosurf had expanded isotherms upon incorporation of levofloxacin, without changes in monolayer elasticity. In fact, levofloxacin induced film stability with increased collapse pressures in the Curosurf isotherms and delayed the phase transition, according to Brewster angle microscopy (BAM) imaging. Using polarization-modulated infrared reflection-absorption spectroscopy (PM-IRRAS), we found that levofloxacin is preferentially located in the head group region, inducing an increased organization of the Curosurf film. This location of levofloxacin was confirmed with MD simulations. The stability inferred demonstrates that the lung surfactant can be used as a drug delivery system for the administration via inhalation or intratracheal instillation of levofloxacin to treat lung diseases such as pneumonia and respiratory distress syndrome.

Three dimensional electrospun PCL/PLA blend nanofibrous scaffolds with significantly improved stem cells osteogenic differentiation and cranial bone formation.

Nanofibrous scaffolds that are morphologically/structurally similar to natural ECM are highly interested for tissue engineering; however, the electrospinning technique has the difficulty in directly producing clinically relevant 3D nanofibrous scaffolds with desired structural properties. To address this challenge, we have developed an innovative technique of thermally induced nanofiber self-agglomeration (TISA) recently. The aim of this work was to prepare (via the TISA technique) and evaluate 3D electrospun PCL/PLA blend (mass ratio: 4/1) nanofibrous scaffolds having high porosity of ∼95.8% as well as interconnected and hierarchically structured pores with sizes from sub-micrometers to ∼300 µm for bone tissue engineering. The hypothesis was that the incorporation of PLA (with higher mechanical stiffness/modulus and bioactivity) into PCL nanofibers would significantly improve human mesenchymal stem cells (hMSCs) osteogenic differentiation in vitro and bone formation in vivo. Compared to neat PCL-3D scaffolds, PCL/PLA-3D blend scaffolds had higher mechanical properties and in vitro bioactivity; as a result, they not only enhanced the cell viability of hMSCs but also promoted the osteogenic differentiation. Furthermore, our in vivo studies revealed that PCL/PLA-3D scaffolds considerably facilitated new bone formation in a critical-sized cranial bone defect mouse model. In summary, both in vitro and in vivo results indicated that novel 3D electrospun PCL/PLA blend nanofibrous scaffolds would be strongly favorable/desired for hMSCs osteogenic differentiation and cranial bone formation.