Development of a dry powder insufflation device with application in in vitro cell-based assays in the context of respiratory delivery.

Research on pharmaceutical dry powders has been increasing worldwide, along with increased therapeutic strategies for an application through the pulmonary or the nasal routes. In vitro methodologies and tests that mimic the respiratory environment and the process of inhalation itself are, thus, essential. The literature frequently reports cell-based in vitro assays that involve testing the dry powders in suspension. This experimental setting is not adequate, as both the lung and the nasal cavity are devoid of abundant liquid. However, devices that permit powder insufflation over cells in culture are either scarce or technically complex and expensive, which is not feasible in early stages of research. In this context, this work proposes the development of a device that allows the delivery of dry powders onto cell surfaces, thus simulating inhalation more appropriately. Subsequently, a quartz crystal microbalance (QCM) was used to establish a technique enabling the determination of dry powder deposition profiles. Additionally, the determination of the viability of respiratory cells (A549) after the insufflation of a dry powder using the developed device was performed. In all, a prototype for dry powder insufflation was designed and developed, using 3D printing methods for its production. It allowed the homogenous dispersion of the insufflated powders over a petri dish and a QCM crystal, and a more detailed study on how dry powders disperse over the supports. The device, already protected by a patent, still requires further improvement, especially regarding the method for powder weighing and the efficiency of the insufflation process, which is being addressed. The impact of insufflation of air and of locust bean gum (LBG)-based microparticles revealed absence of cytotoxic effect, as cell viability roughly above 70 % was always determined.

Respirable konjac glucomannan microparticles as antitubercular drug carriers: Effects of in vitro and in vivo interactions.

Pulmonary delivery of drugs is potentially beneficial in the context of lung disease, maximising drug concentrations in the site of action. A recent work proposed spray-dried konjac glucomannan (KGM) microparticles as antitubercular drug (isoniazid and rifabutin) carriers to treat pulmonary tuberculosis. The present work explores in vitro and in vivo effects of these microparticles, focusing on the ability for macrophage uptake, the exhibited antibacterial activity and safety issues. Efficient uptake of KGM microparticles by macrophages was demonstrated in vitro, while the antitubercular activity of the model drugs against Mycobacterium bovis was not affected by microencapsulation in KGM microparticles. Despite the good indications provided by the developed system, KGM is not yet approved for pulmonary applications, which is a limiting characteristic. To reinforce the available data on the performance of the material, safety parameters were evaluated both in vitro and in vivo, showing promising results. No significant cell toxicity was observed at concentrations considered realistic for lung delivery approaches (up to 125 µg/mL) when lung epithelial cells and macrophages were exposed to KGM microparticles (both drug-loaded and unloaded). Finally, no signs of systemic or lung inflammatory response were detected in mice after receiving 10 administrations of unloaded KGM microparticles.

Fucoidan from Fucus vesiculosus: Evaluation of the Impact of the Sulphate Content on Nanoparticle Production and Cell Toxicity.

The composition of seaweeds is complex, with vitamins, phenolic compounds, minerals, and polysaccharides being some of the factions comprising their structure. The main polysaccharide in brown seaweeds is fucoidan, and several biological activities have been associated with its structure. Chitosan is another marine biopolymer that is very popular in the biomedical field, owing to its suitable features for formulating drug delivery systems and, particularly, particulate systems. In this work, the ability of fucoidan to produce nanoparticles was evaluated, testing different amounts of a polymer and using chitosan as a counterion. Nanoparticles of 200-300 nm were obtained when fucoidan prevailed in the formulation, which also resulted in negatively charged nanoparticles. Adjusting the pH of the reaction media to 4 did not affect the physicochemical characteristics of the nanoparticles. The IC50 of fucoidan was determined, in both HCT-116 and A549 cells, to be around 160 µg/mL, whereas it raised to 675-100 µg/mL when nanoparticles (fucoidan/chitosan = 2/1, w/w) were tested. These marine materials (fucoidan and chitosan) provided features suitable to formulate polymeric nanoparticles to use in biomedical applications.

Nanoencapsulation of Gla-Rich Protein (GRP) as a Novel Approach to Target Inflammation.

Chronic inflammation is a major driver of chronic inflammatory diseases (CIDs), with a tremendous impact worldwide. Besides its function as a pathological calcification inhibitor, vitamin K-dependent protein Gla-rich protein (GRP) was shown to act as an anti-inflammatory agent independently of its gamma-carboxylation status. Although GRP's therapeutic potential has been highlighted, its low solubility at physiological pH still constitutes a major challenge for its biomedical application. In this work, we produced fluorescein-labeled chitosan-tripolyphosphate nanoparticles containing non-carboxylated GRP (ucGRP) (FCNG) via ionotropic gelation, increasing its bioavailability, stability, and anti-inflammatory potential. The results indicate the nanosized nature of FCNG with PDI and a zeta potential suitable for biomedical applications. FCNG's anti-inflammatory activity was studied in macrophage-differentiated THP1 cells, and in primary vascular smooth muscle cells and chondrocytes, inflamed with LPS, TNFα and IL-1ß, respectively. In all these in vitro human cell systems, FCNG treatments resulted in increased intra and extracellular GRP levels, and decreased pro-inflammatory responses of target cells, by decreasing pro-inflammatory cytokines and inflammation mediators. These results suggest the retained anti-inflammatory bioactivity of ucGRP in FCNG, strengthening the potential use of ucGRP as an anti-inflammatory agent with a wide spectrum of application, and opening up perspectives for its therapeutic application in CIDs.

Supersaturable self-microemulsifying delivery systems: an approach to enhance oral bioavailability of benzimidazole anticancer drugs.

This study explored the design of supersaturable self-microemulsifying drug delivery systems (S-SMEDDS) to address poor solubility and oral bioavailability of a novel benzimidazole derivative anticancer drug (BI). Firstly, self-microemulsifying drug delivery systems SMEDDS made of Miglyol® 812, Kolliphor® RH40, Transcutol® HP, and ethanol were prepared and loaded with the BI drug. Upon dispersion, the systems formed neutrally charged droplets of around 20 nm. However, drug precipitation was observed following incubation with simulated gastric fluid (pH 1.2). Aiming at reducing this precipitation and enhancing drug payload, supersaturable systems were then prepared by adding 1% hydroxypropyl cellulose as precipitation inhibitor. Supersaturable systems maintained a higher amount of drug in a supersaturated state in gastric medium compared with conventional formulations and were stable in simulated intestinal medium (pH 6.8). In vitro cell studies using Caco-2 cell line showed that these formulations reduced in a transient manner the transepithelial electrical resistance of the monolayers without toxicity. Accordingly, confocal images revealed that the systems accumulated at tight junctions after a 2 h exposure. In vivo pharmacokinetic studies carried out following oral administration of BI-loaded S-SMEDDS, SMEDDS, and free drug to healthy mice showed that supersaturable systems promoted drug absorption compared with the other formulations. Overall, these data highlight the potential of using the supersaturable approach as an alternative to conventional SMEDDS for improving oral systemic absorption of lipophilic drugs.

Multifunctional Nanocarriers for Lung Drug Delivery.

Nanocarriers have been increasingly proposed for lung drug delivery applications. The strategy of combining the intrinsic and more general advantages of the nanostructures with specificities that improve the therapeutic outcomes of particular clinical situations is frequent. These include the surface engineering of the carriers by means of altering the material structure (i.e., chemical modifications), the addition of specific ligands so that predefined targets are reached, or even the tuning of the carrier properties to respond to specific stimuli. The devised strategies are mainly directed at three distinct areas of lung drug delivery, encompassing the delivery of proteins and protein-based materials, either for local or systemic application, the delivery of antibiotics, and the delivery of anticancer drugs-the latter two comprising local delivery approaches. This review addresses the applications of nanocarriers aimed at lung drug delivery of active biological and pharmaceutical ingredients, focusing with particular interest on nanocarriers that exhibit multifunctional properties. A final section addresses the expectations regarding the future use of nanocarriers in the area.

Dual antibiotherapy of tuberculosis mediated by inhalable locust bean gum microparticles.

Despite the existence of effective oral therapy, tuberculosis remains a deadly pathology, namely because of bacterial resistance and incompliance with treatments. Establishing alternative therapeutic approaches is urgently needed and inhalable therapy has a great potential in this regard. As pathogenic bacteria are hosted by alveolar macrophages, the co-localisation of antitubercular drugs and pathogens is thus potentiated by this strategy. This work proposes inhalable therapy of pulmonary tuberculosis mediated by a single locust bean gum (LBG) formulation of microparticles associating both isoniazid and rifabutin, complying with requisites of the World Health Organisation of combined therapy. Microparticles were produced by spray-drying, at LBG/INH/RFB mass ratio of 10/1/0.5. The aerodynamic characterisation of microparticles revealed emitted doses of more than 90% and fine particle fraction of 38%, thus indicating the adequacy of the system to reach the respiratory lung area, thus partially the alveolar region. Cytotoxicity results indicate moderate toxicity (cell viability around 60%), with a concentration-dependent effect. Additionally, rat alveolar macrophages evidenced preferential capture of LBG microparticles, possibly due to chemical composition comprising mannose and galactose units that are specifically recognised by macrophage surface receptors.

Species composition and density estimates of the anurofauna of a site within the northernmost large Atlantic Forest remnant (Parque Estadual do Desengano) in the state of Rio de Janeiro, Brazil / Composição de espécies e estimativa de densidade da anurofauna em uma área dentro do mais setentrional dos grandes remanescentes de Mata Atlântica (Parque Estadual do Desengano) do Estado do Rio de Janeiro, Brasil

We present data on species composition, relative abundance and estimated densities for leaf-litter anuran amphibians from an Atlantic Forest area within the Parque Estadual do Desengano, in the state of Rio de Janeiro, Brazil, based on results of a short-term survey carried out during the winter at altitudes of 1000-1500 m. Three sampling methods were used: plot sampling, visual encounter surveys (VES; performed during the day, at the twilight, and at night), and pitfall traps. We recorded 13 species, with the direct-developer Ischnocnema parva (Girard, 1853) being the most abundant. Most frogs (ca. 90% of all individuals) sampled by VES were captured during the crepuscular and nocturnal periods. The estimated density of the local leaf-litter frog assemblage based on plot sampling was 13.2 ind.100 m-2, which is one of the highest values currently reported for Atlantic Rainforest areas. This is the first study analyzing the anuran fauna of the Parque Estadual do Desengano and adds to the knowledge of the fauna of the northern region of the state of Rio de Janeiro, which is still poorly studied.

Apresentamos dados sobre a composição de espécies, abundância relativa e densidade estimada para anfíbios anuros de serrapilheira de uma área de Mata Atlântica no Parque Estadual do Desengano, no Estado do Rio de Janeiro, Brasil, com base em resultados de um estudo de curto prazo realizado durante o inverno a altitudes de 1000-1500 m. Três métodos de amostragem foram utilizados: amostragem em parcelas, encontros visuais (realizados durante o dia, no crepúsculo e à noite) e armadilhas de queda. Foram registradas 13 espécies, com Ischnocnema parva (Girard, 1853), uma forma com desenvolvimento direto, sendo a mais abundante. A maioria dos anuros (ca. 90% de todos os indivíduos) amostrados pelo método de encontros visuais foi coletada durante os períodos crepuscular e noturno. A densidade estimada para anuros de serrapilheira foi de 13,2 ind.100 m-2, que é um dos valores mais altos atualmente registrados para áreas de Mata Atlântica. Este é o primeiro estudo que analisa a fauna de anuros do Parque Estadual do Desengano e contribui para o conhecimento da fauna da região norte do estado do Rio de Janeiro, que ainda é pouco estudada.

Parameters from the community of leaf-litter frogs from Estação Ecológica Estadual Paraíso, Guapimirim, Rio de Janeiro State, southeastern Brazil

We studied the leaf-litter frog community of Estação Ecológica Estadual Paraíso, in Guapimirim, Rio de Janeiro State, southeastern Brazil. Herein we combined three sampling methods (large plots, visual encounter surveys and pit-fall traps) to present data on species composition, richness, relative abundance and densities. The local assemblage of frogs associated to the leaf-litter was composed by 14 species, belonging to nine families. Haddadus binotatus, a direct-developing frog, was the most abundant species in the community. The estimated density of the local leaf-litter frog assemblage based on plot sampling was 4.3 frogs/100 m². Haddadus binotatus had the highest density (1.1 ind/100 m²). Frogs were predominantly found at night. Thoropa miliaris had the largest values of SVL (39.0 ± 10.3 mm), whereas the smallest species were Euparkerella brasiliensis (16.7 ± 2.2 mm) and E. cochranae (16.0 ± 2.7 mm). Rhinella ornata had the highest mean body mass (12.1 ± 7.5 g), and E. cochranae the lowest (0.4 ± 0.2 g). The overall frog mass was 938.6 g/ha. Our data support that higher densities of leaf-litter frogs tend to occur in the Neotropical region compared to the OldWorld tropics, tending to be higher in Central America than in South America.

Estudamos a comunidade de anuros de folhiço da EstaçãoEcológica Estadual Paraíso, em Guapimirim, estado do Rio de Janeiro, no sudeste do Brasil. Combinamos três métodosde amostragem (plots, transectos e armadilhas de queda) para apresentar dados sobre a composição de espécies, riqueza,abundância relativa e densidade. A assembleia local foi composta por 14 espécies de anuros, pertencentes a nove famílias. Haddadus binotatus, espécie de desenvolvimento direto, foi a mais abundante durante o estudo. A densidade de anuros de folhiço estimada com base na amostragem por plots foi de4,3 ind/100m². Haddadus binotatus apresentou a maior densidade (1,1 ind/100m²). Os anuros foram registrados predominantemente durante a noite. Thoropa miliaris apresentou os maiores valores de CRC (39,0 ± 10,3 mm). As menores espécies foram Euparkerella brasiliensis (16,7 ± 2,2 mm) e E. cochranae (16,0 ± 2,7mm). Rhinella ornata apresentou a maior massa corporal média (12,1 ± 7,5 g) e E. cochranae (0,4 ± 0,2 g) a menor. A massa média total foi de 938,6 g/ha. Nossos resultados corroboram com a tendência de maiores densidades de anuros de folhiço na região Neotropical quando comparado com áreas Tropicais do Velho Mundo, tendendo a serem maiores na América Central do que na América do Sul.

Parameters from the community of leaf-litter frogs from Estação Ecológica Estadual Paraíso, Guapimirim, Rio de Janeiro State, southeastern Brazil.

We studied the leaf-litter frog community of Estação Ecológica Estadual Paraíso, in Guapimirim, Rio de Janeiro State, southeastern Brazil. Herein we combined three sampling methods (large plots, visual encounter surveys and pit-fall traps) to present data on species composition, richness, relative abundance and densities. The local assemblage of frogs associated to the leaf-litter was composed by 14 species, belonging to nine families. Haddadus binotatus, a direct-developing frog, was the most abundant species in the community. The estimated density of the local leaf-litter frog assemblage based on plot sampling was 4.3 frogs/100 m(2). Haddadus binotatus had the highest density (1.1 ind/100 m(2)). Frogs were predominantly found at night. Thoropa miliaris had the largest values of SVL (39.0 ± 10.3 mm), whereas the smallest species were Euparkerella brasiliensis (16.7 ± 2.2 mm) and E. cochranae (16.0 ± 2.7 mm). Rhinella ornata had the highest mean body mass (12.1 ± 7.5 g), and E. cochranae the lowest (0.4 ± 0.2 g). The overall frog mass was 938.6 g/ha. Our data support that higher densities of leaf-litter frogs tend to occur in the Neotropical region compared to the OldWorld tropics, tending to be higher in Central America than in South America.