Cytocompatibility and cellular interactions of chondroitin sulfate microparticles designed for inhaled tuberculosis treatment.

Tuberculosis remains a leading cause of death, therapeutic failure being mainly due to non-compliance with prolonged treatments, often associated with severe side-effects. New therapeutic strategies are demanded and, considering that the lung is the primary site of infection, direct lung delivery of antibiotics is possibly an effective approach. Therapeutic success in this context depends on suitable carriers that reach the alveoli where Mycobacterium hosts (macrophages) reside, as well as on their ability to promote macrophage capture and intracellular accumulation of drugs. In this work, we propose inhalable polymeric microparticles produced from chondroitin sulfate, a polymer composed by moieties recognized by macrophage receptors. Spray-drying of chondroitin sulfate in combination with two first-line antitubercular drugs (isoniazid and rifabutin) yielded respirable microparticles that evidenced no cytotoxic effects on lung epithelial cells (A549) and macrophages (dTHP1 and J744A.1). The microparticles exhibited tendency for macrophage capture in a dose-dependent manner, which was validated through imaging. High content image analysis revealed that rifabutin induced a dose-dependent increase in phospholipid content of macrophages, which could be prevented by formulation in chondroitin sulfate microparticles. This work provides indications on the potential of chondroitin sulfate carriers to interact with macrophages, thus providing a platform for drug delivery in the context of macrophage intracellular diseases, namely tuberculosis.

Carrageenan from red algae: an application in the development of inhalable tuberculosis therapy targeting the macrophages.

Macrophages have unique surface receptors that might recognize preferentially several moieties present on the surface of infecting organisms, including in the bacterial cell wall. Benefiting from a similar composition regarding the referred moieties, polysaccharides might be good candidates to compose the matrix of drug carriers aimed at macrophage targeting, as they can use the same recognition pathways of the infecting organisms. Carrageenan (CRG), a polysaccharide extracted from red edible seaweed, is an interesting possibility for the approach of directly targeting alveolar macrophages, as its composition is reported to be recognized by several macrophage lectin receptors. Inhalable starch/CRG microparticles were successfully produced, effectively associating isoniazid (96%) and rifabutin (74%) simultaneously. Furthermore, the produced microparticles presented adequate aerodynamic properties for pulmonary delivery with potential to reach the respiratory zone, with a mass median aerodynamic diameter (MMAD) between 3.3 and 3.9 µm. It was further demonstrated that the antitubercular activity of the drugs remained unchanged after encapsulation. The formulation evidenced no cytotoxic effects on lung epithelial cells (A549), although mild toxicity was observed on macrophage-differentiated THP-1 cells for the drug-loaded formulation. Starch/CRG microparticles also exhibited a propensity to be captured by macrophages in a dose-dependent manner, as well as an ability to activate the target cells. This work provides indications on the potential of the starch/CRG carriers to interact with macrophages, thus providing a platform for drug delivery in the context of macrophage intracellular diseases. Additionally, if tuberculosis is focused, these microparticles can be used as inhalable drug carriers. Graphical abstractz.

Inhalable chitosan microparticles for simultaneous delivery of isoniazid and rifabutin in lung tuberculosis treatment.

The direct delivery of antibiotics to the lung has been considered an effective approach to treat pulmonary tuberculosis, which represents approximately 80% of total cases. In this sense, this work aimed at producing inhalable chitosan microparticles simultaneously associating isoniazid and rifabutin, for an application in pulmonary tuberculosis therapy. Spray-dried chitosan microparticles were obtained with adequate flow properties for deep lung delivery (aerodynamic diameter of 4 µm) and high drug association efficiencies (93% for isoniazid and 99% for rifabutin). The highest concentration of microparticles that was tested (1 mg/mL) decreased the viability of macrophage-differentiated THP-1 cells to around 60% after 24 h exposure, although no deleterious effect was observed in human alveolar epithelial (A549) cells. The release of LDH was, however, increased in both cells. Chitosan microparticles further evidenced capacity to activate macrophage-like cells, inducing cytokine secretion well above basal levels. Moreover, the propensity of macrophages to internalize microparticles was demonstrated, with uptake levels over 90%. Chitosan microparticles also inhibited bacterial growth by 96%, demonstrating that the microencapsulation preserved drug antibacterial activity in vitro. Overall, the obtained data suggest the potential of chitosan microparticles for inhalable lung tuberculosis therapy.

Spray-dried fucoidan microparticles for pulmonary delivery of antitubercular drugs.

Pulmonary tuberculosis accounts for 80% of cases and the delivery of antitubercular drugs into the lungs allows targeting the infected organ and, possibly, reducing systemic drug toxicity. This work aimed at using fucoidan as matrix of inhalable microparticles that associate two first-line antitubercular drugs, for an application in pulmonary tuberculosis therapy. Fucoidan is composed of fucose and sulphated sugar residues, moieties described as being recognised by surface receptors of alveolar macrophages, which host mycobacteria. Inhalable fucoidan microparticles loaded with antitubercular drugs were successfully produced with high association efficiencies of either isoniazid (95%) or rifabutin (81%). The microparticles evidenced no cytotoxicity on lung epithelial cells (A549). However, rifabutin-loaded microparticles showed a certain degree of toxicity on macrophage-like cells (THP-1) at the highest tested concentration (1 mg/mL). Furthermore, microparticles showed favourable aerodynamic properties for deep lung delivery (MMAD 2.0-3.8 µm) and, thus, show potential for an application as inhalable tuberculosis therapy.

Inhalable Fucoidan Microparticles Combining Two Antitubercular Drugs with Potential Application in Pulmonary Tuberculosis Therapy.

The pulmonary delivery of antitubercular drugs is a promising approach to treat lung tuberculosis. This strategy not only allows targeting the infected organ instantly, it can also reduce the systemic adverse effects of the antibiotics. In light of that, this work aimed at producing fucoidan-based inhalable microparticles that are able to associate a combination of two first-line antitubercular drugs in a single formulation. Fucoidan is a polysaccharide composed of chemical units that have been reported to be specifically recognised by alveolar macrophages (the hosts of Mycobacterium). Inhalable fucoidan microparticles were successfully produced, effectively associating isoniazid (97%) and rifabutin (95%) simultaneously. Furthermore, the produced microparticles presented adequate aerodynamic properties for pulmonary delivery with potential to reach the respiratory zone, with a mass median aerodynamic diameter (MMAD) between 3.6⁻3.9 µm. The formulation evidenced no cytotoxic effects on lung epithelial cells (A549), although mild toxicity was observed on macrophage-differentiated THP-1 cells at the highest tested concentration (1 mg/mL). Fucoidan microparticles also exhibited a propensity to be captured by macrophages in a dose-dependent manner, as well as an ability to activate the target cells. Furthermore, drug-loaded microparticles effectively inhibited mycobacterial growth in vitro. Thus, the produced fucoidan microparticles are considered to hold potential as pulmonary delivery systems for the treatment of tuberculosis.

Sulfated Seaweed Polysaccharides as Multifunctional Materials in Drug Delivery Applications.

In the last decades, the discovery of metabolites from marine resources showing biological activity has increased significantly. Among marine resources, seaweed is a valuable source of structurally diverse bioactive compounds. The cell walls of marine algae are rich in sulfated polysaccharides, including carrageenan in red algae, ulvan in green algae and fucoidan in brown algae. Sulfated polysaccharides have been increasingly studied over the years in the pharmaceutical field, given their potential usefulness in applications such as the design of drug delivery systems. The purpose of this review is to discuss potential applications of these polymers in drug delivery systems, with a focus on carrageenan, ulvan and fucoidan. General information regarding structure, extraction process and physicochemical properties is presented, along with a brief reference to reported biological activities. For each material, specific applications under the scope of drug delivery are described, addressing in privileged manner particulate carriers, as well as hydrogels and beads. A final section approaches the application of sulfated polysaccharides in targeted drug delivery, focusing with particular interest the capacity for macrophage targeting.

RNA interference with EAG1 enhances interferon gamma injury to glioma cells in vitro.

AIM: The aim of this study was to silence Ether à go-go 1 (EAG1) in glioma cells by RNAi in order to further analyze whether silencing this channel would improve injury caused by interferon gamma (IFN-γ). MATERIALS AND METHODS: EAG1 silencing by the siRNAs EAG1hum_287 and EAG1hum_1727 (sequence targets 5'-GGCCTATTGTGTACAGCAATT-3' and 5'-GGGACTTCCTGAAGCTCTATT-3', respectively) was determined by reverse transcription real-time quantitative polymerase chain reaction (RT-qPCR). Cell viability was measured by the 3-(4,5)-dimethylthiazol-2-y1)-2,5-diphenyltetrazolium bromide (MTT) assay. U-138MG glioma cells were injured by IFN-γ (25 ng/ml, 24 h) with or without the RNAi for EAG1 by a non-viral vector (pKV10.1-3, 0.2 µg). RESULTS: EAG1hum_287 and EAG1hum_1727 caused 0.46- and 0.52-fold decrease in EAG1 mRNA content, respectively. RNAi for EAG1 by pKv10.1-3 strengthened the reduction in cell viability caused by IFN-γ (11.4% versus 40.4%, p<0.05). CONCLUSION: The present study reinforces the notion that EAG1 has a role in glioma biology, suggesting that this channel is a relevant player preserving the cell viability during IFN-γ injury.