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.

Hydrothermal Processing of Laminaria ochroleuca for the Production of Crude Extracts Used to Formulate Polymeric Nanoparticles.

A green extraction process using only water was proposed for the simultaneous extraction of alginate and bioactive compounds from Laminaria ochroleuca. Operation was carried out during non-isothermal heating up to maximal temperatures over the range of 70 °C to 100 °C. Once separated, the alginate and the crude extract were characterised and the biological activities and cytotoxicity of the extracts was studied, the latter in intestinal epithelial cells. Those alginates obtained at 90 °C exhibited the highest extraction yields and viscoelastic features of the corresponding hydrogels. The obtained results show that the extracts obtained by non-isothermal extraction were suitable to formulate nanoparticles, which showed the smallest size (≈ 250-350 nm) when the higher content of fucoidan extract was present. Given the evidenced properties, the extracts may find an application in the formulation of nanoparticulate carriers for drug delivery.

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.

Microencapsulated Solid Lipid Nanoparticles as a Hybrid Platform for Pulmonary Antibiotic Delivery.

Solid lipid nanoparticles (SLN) containing rifabutin (RFB), with pulmonary administration purposes, were developed through a technique that avoids the use of organic solvents or sonication. To facilitate their pulmonary delivery, the RFB-loaded SLN were included in microspheres of appropriate size using suitable excipients (mannitol and trehalose) through a spray-drying technique. Confocal analysis microscopy showed that microspheres are spherical and that SLN are efficiently microencapsulated and homogeneously distributed throughout the microsphere matrices. The aerodynamic diameters observed an optimal distribution for reaching the alveolar region. The dry powder's performance during aerosolization and the in vitro drug deposition were tested using a twin-impinger approach, which confirmed that the microspheres can reach the deep lung. Isothermal titration calorimetry revealed that SLN have higher affinity for mannitol than for trehalose. Upon microsphere dissolution in aqueous media, SLN were readily recovered, maintaining their physicochemical properties. When these dry powders reach the deep lung, microspheres are expected to readily dissolve, delivering the SLN which, in turn, will release RFB. The in vivo biodistribution of microencapsulated RFB-SLN demonstrated that the antibiotic achieved the tested organs 15 and 30 min post pulmonary administration. Their antimycobacterial activity was also evaluated in a murine model of infection with a Mycobacterium tuberculosis strain H37Rv resulting in an enhancement of activity against M. tuberculosis infection compared to nontreated animals. These results suggest that RFB-SLN microencapsulation is a promising approach 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.

Inhalable Antitubercular Therapy Mediated by Locust Bean Gum Microparticles.

Tuberculosis remains a major global health problem and alternative therapeutic approaches are needed. Considering the high prevalence of lung tuberculosis (80% of cases), the pulmonary delivery of antitubercular drugs in a carrier system capable of reaching the alveoli, being recognised and phagocytosed by alveolar macrophages (mycobacterium hosts), would be a significant improvement to current oral drug regimens. Locust bean gum (LBG) is a polysaccharide composed of galactose and mannose residues, which may favour specific recognition by macrophages and potentiate phagocytosis. LBG microparticles produced by spray-drying are reported herein for the first time, incorporating either isoniazid or rifabutin, first-line antitubercular drugs (association efficiencies >82%). Microparticles have adequate theoretical properties for deep lung delivery (aerodynamic diameters between 1.15 and 1.67 µm). The cytotoxic evaluation in lung epithelial cells (A549 cells) and macrophages (THP-1 cells) revealed a toxic effect from rifabutin-loaded microparticles at the highest concentrations, but we may consider that these were very high comparing with in vivo conditions. LBG microparticles further evidenced strong ability to be captured by macrophages (percentage of phagocytosis >94%). Overall, the obtained data indicated the potential of the proposed system for tuberculosis therapy.

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.

Natural carriers for application in tuberculosis treatment.

Tuberculosis remains the leading cause of preventable deaths worldwide and unsuccessful therapy is mainly due to non-compliance with very prolonged treatments, often associated with severe side-effects. Overcoming this problem demands the introduction of drug carriers releasing the antimicrobial agents in a targeted and sustained manner, allowing reduction in frequency and dosing numbers. Nano- and microparticles have taken the forefront of this approach, providing the means for the desired improvement of therapeutic schedules. Natural polymers are strong candidates as matrix-forming materials, usually exhibiting biocompatibility, biodegradability, low cost and some technological advantages as compared with synthetic counterparts. In this review, natural particulate carriers developed for tuberculosis therapy are presented, mainly focusing on the use of polysaccharides and lipids. Their effectiveness is discussed taking into account their composition. Finally, considerations on the general potential of natural materials for this application, as well as key factors still to be addressed, are discussed.

Microencapsulation of selenium by spray-drying as a tool to improve bioaccessibility in food matrix.

Se in the form of sodium selenite was microencapsulated by spray - drying and added to a food matrix (yogurt) to study the potential improvement of its bioaccessibility. Yogurt samples were also supplemented with Se in free salt form. Se-loaded microparticles were successfully prepared by spray-drying with production yields above 70%. The supplementation of yogurt with Se in the form of free sodium selenite had a low effect on improving the bioaccessibility of this micronutrient (1%). In turn, Se microencapsulation with mannitol or mannitol/gastro-resistant polymer (Eudragit®) had a strong impact on bioaccessibility results. After the gastric phase, Se bioaccessibility reached values of 21 and 40% for the microencapsulated formulations, respectively. This percentage rose to 55% at the end of intestinal phase, showing no differences between both formulations. Our results show the relevance of microencapsulation as an effective tool to improve the bioaccessibility of micronutrients when they are used in food supplementation.

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.

Engineering of konjac glucomannan into respirable microparticles for delivery of antitubercular drugs.

Few medically-approved excipients are available for formulation strategies to endow microcarriers with improved performance in lung drug targeting. Konjac glucomannan (KGM) is a novel, biocompatible material, comprising mannose units potentially inducing macrophage uptake for the treatment of macrophage-mediated diseases. This work investigated spray-dried KGM microparticles as inhalable carriers of model antitubercular drugs, isoniazid (INH) and rifabutin (RFB). The polymer was characterised and different polymer/drug ratios tested in the production of microparticles for which respirability was assessed in vitro. The swelling of KGM microparticles and release of drugs in simulated lung fluid were characterised and the biodegradability in presence of ß-mannosidase, a lung hydrolase, determined. KGM microparticles were drug loaded with 66-91% association efficiency and had aerodynamic diameter around 3 µm, which enables deep lung penetration. The microparticles swelled upon liquid contact by 40-50% but underwent size reduction (>62% in 90 min) in presence of ß-mannosidase, indicating biodegradability. Finally, drug release was tested showing slower release of RFB compared with INH but complete release of both within 24 h. This work identifies KGM as a biodegradable polymer of natural origin that can be engineered to encapsulate and release drugs in respirable microparticles with physical and chemical macrophage-targeting properties.

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.

Nanocomposite sponges for enhancing intestinal residence time following oral administration.

In this work, nanocomposites that combine mucopenetrating and mucoadhesive properties in a single system are proposed as innovative strategy to increase drug residence time in the intestine following oral administration. To this aim, novel mucoadhesive chitosan (CH) sponges loaded with mucopenetrating nanoemulsions (NE) were developed via freeze-casting technique. The NE mucopenetration ability was determined studying the surface affinity and thermodynamic binding of the nanosystem with mucins. The ability of nanoparticles to penetrate across a preformed mucins layer was validated by 3D-time laps Confocal Laser Scanning Microscopy imaging. Microscopy observations (Scanning Electron Microscopy and Optical Microscopy) showed that NE participated in the structure of the sponge affecting its stability and in vitro release kinetics. When incubated with HCT 116 and Caco-2 cell lines, the NE proved to be cytocompatible over a wide concentration range. Finally, the in vivo biodistribution of the nanocomposite was evaluated after oral gavage in healthy mice. The intestinal retention of NE was highly enhanced when loaded in the sponge compared to the NE suspension. Overall, our results demonstrated that the developed nanocomposite sponge is a promising system for sustained drug intestinal delivery.

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.

Inhalable Spray-Dried Chondroitin Sulphate Microparticles: Effect of Different Solvents on Particle Properties and Drug Activity.

Spray-drying stands as one of the most used techniques to produce inhalable microparticles, but several parameters from both the process and the used materials affect the properties of the resulting microparticles. In this work, we describe the production of drug-loaded chondroitin sulphate microparticles by spray-drying, testing the effect of using different solvents during the process. Full characterisation of the polymer and of the aerodynamic properties of the obtained microparticles are provided envisaging an application in inhalable tuberculosis therapy. The spray-dried microparticles successfully associated two first-line antitubercular drugs (isoniazid and rifabutin) with satisfactory production yield (up to 85%) and drug association efficiency (60%-95%). Ethanol and HCl were tested as co-solvents to aid the solubilisation of rifabutin and microparticles produced with the former generally revealed the best features, presenting a better ability to sustainably release rifabutin. Moreover, these presented aerodynamic properties compatible with deep lung deposition, with an aerodynamic diameter around 4 µm and fine particle fraction of approximately 44%. Finally, it was further demonstrated that the antitubercular activity of the drugs remained unchanged after encapsulation independently of the used solvent.

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 locust bean gum microparticles co-associating isoniazid and rifabutin: Therapeutic assessment in a murine model of tuberculosis infection.

Tuberculosis is a leading cause of death worldwide. Although the development of new antimycobacterial drugs is an obvious and necessary strategy to address the disease, improving the therapeutic performance of drugs already approved constitutes a valuable alternative approach. As the lung is the most affected organ, where M. tuberculosis is able to survive and proliferate, the direct pulmonary delivery of antitubercular drugs comprises a highly promising therapeutic strategy. In this work, spray-dried locust bean gum (LBG) microparticles were used to deliver a combination of two first line antitubercular drugs, isoniazid (INH) and rifabutin (RFB), to the alveolar zone, where macrophages hosting the bacteria reside. LBG is expected to mediate favoured macrophage uptake of microparticles, leading to enhanced therapeutic effect. The therapeutic effect of LBG/INH/RFB microparticles was evaluated in a murine model infected with M. tuberculosis, strain H37Rv and compared with oral co-therapy of INH and RFB in the free form. The pulmonary administration of LBG/INH/RFB microparticles 5 times per week was the only treatment schedule that provided negative growth index values in lung (-0.22), spleen (-0.14) and liver (-0.26) even using a lower therapeutic dose for both antibiotics. For the control group, the respective values were +1.95, +0.75 and +0.96.

Microspheres containing lipid/chitosan nanoparticles complexes for pulmonary delivery of therapeutic proteins.

Chitosan/tripolyphosphate nanoparticles have already been demonstrated to promote peptide absorption through several mucosal surfaces. We have recently developed a new drug delivery system consisting of complexes formed between preformed chitosan/tripolyphosphate nanoparticles and phospholipids, named as lipid/chitosan nanoparticles (L/CS-NP) complexes. The aim of this work was to microencapsulate these protein-loaded L/CS-NP complexes by spray-drying, using mannitol as excipient to produce microspheres with adequate properties for pulmonary delivery. Results show that the obtained microspheres are spherical and present appropriate aerodynamic characteristics for lung delivery (aerodynamic diameters around 2-3 microm and low apparent tap density of 0.4-0.5 g/cm3). The physicochemical properties of the L/CS-NP complexes are affected by the phospholipids composition. Phospholipids provide a controlled release of the encapsulated protein (insulin), which was successfully associated to the system (68%). The complexes can be easily recovered from the mannitol microspheres upon incubation in aqueous medium, maintaining their morphology and physicochemical characteristics. Therefore, this work demonstrates that protein-loaded L/CS-NP complexes can be efficiently microencapsulated, resulting in microspheres with adequate properties to provide a deep inhalation pattern. Furthermore, they are expected to release their payload (the complexes and, consequently, the encapsulated macromolecule) after contacting with the lung aqueous environment.