2.1. Advantages of the pulmonary route

Despite the natural barriers to prevent invasion of unwanted airborne particles or living entities, such as airway geometry, humidity, mucociliary clearance and resident populations of macrophages, the lungs are constantly challenged with infectious agents, including Mtb, which make these air-filled organs an attractive target for drug delivery strategies as it provides direct access to the infection site. Delivery systems for TB treatment through the pulmonary route present advantages when compared to the conventional oral and injectable routes. These include drug delivery directly to the infected area, increasing local drug concentration which will impact in bacterial burden and reduce the systemic dosage of the drug [[4], [5], [6], [7], [8], [9], [10], [11], [12], [13]]. Additionally, inhalable systems avoid unwanted side effects often caused by drug metabolism in the gastrointestinal tract before the drug reaches the systemic circulation [[4], [5], [6], [7], [8], [9], [10], [11], [12], [13]]. Unlike the injectable route, inhalation is a pain free and self-administrable delivery means favoring patient convenience and compliance to the treatment.

2.2. Particulate carriers as inhalable delivery systems

Pure drug formulations are typically burst released in the lungs and rapidly undergo unspecific distribution [5,[14], [15], [16], [17], [18]]. In order to achieve a more efficient solution, therapeutic agents can be formulated into particulate carrier systems such as microparticles, nanoparticles, liposomes, micelles or dendrimers. Such systems allow protection of drugs from direct contact with the lung tissue, avoiding early degradation, preclude rapid clearance from the body and assist the control and sustain release of drugs over long periods of time. Particulate carriers also reduce drug toxicity, circumvent undesirable physicochemical properties of the drugs (e.g. low water solubility) and improve drug up-take by macrophages [19].

In recent years the advantages of inhalable particulate carrier systems have been allied to the benefits of dry powder formulations to be delivered by dry powder inhalers (DPIs) for an improved drug delivery system. Dry powder formulations have improved stability as a result of its dry form, do not require refrigeration and allow longer storage periods. Formulations are often combined with pharmaceutically suitable excipients such as lactose, leucine, mannitol or trehalose to improve processing and aerodynamic properties [20,21]. DPIs are propellant free, portable, easy to use and cost-effective devices. Moreover, DPIs are activated by the inspiration effort of patients, allowing a rapid and higher dose administration and a more efficient pulmonary drug deposition [22,23]. Thus, DPIs are currently considered the most convenient and suitable alternative for inhaling anti-TB drugs [24].

2.3. Design and features of inhalable particulate systems

Medicinal particles are designed and developed so their deposition in the respiratory tract can be predicted rather precisely (Fig. 1). The behavior of inhaled particles and their deposition depend of several parameters including particle dimensions, density, shape, composition, concentration, surface properties such as particle charge and the breathing pattern (air flow) of the individual [13,25].

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Fig. 1. Schematic representation of the particle deposition within the upper or lower airways considering their dimensions and the type of carrier systems used for their delivery.

The aerodynamic diameter is an important parameter affecting the pulmonary trajectory and depends on both particle dimension and density. Particles with an aerodynamic diameter in the range 1–5 μm are deposited in the lower airways, which is a desirable location for anti-TB drugs [13,[26], [27], [28]]. On the other hand, particles larger than 5 μm undergo deposition in the upper regions, such as the nasopharynx and they are consequently swallowed, while particles smaller than 1 μm are likely to be exhaled or retained in the alveoli if transported by a carrier system [13,26,29]. After reaching the lower airways, particulate systems are likely to be either phagocytized by alveolar macrophages, adhere to the lung tissue or enter the blood circulation via lung vascular tree. Another potential destination is the entrapment of these particles within the granulomas which are compact and organized aggregates of immune cells where Mtb persists [30,31].

2.4. Types of microparticulate dry powder systems

Liposomes, micelles and polymeric particles have been developed as inhalable dry powder particulate systems aiming at anti-TB drug delivery strategies. Liposomes and micelles studies were reviewed elsewhere [32,33]. Polymeric particles offer great opportunities in target drug delivery and controlled and sustained release of drugs. The possibility of surface modification of polymeric particles can give rise to systems with a great diversity of physicochemical properties and, if desired, favor a more efficient targeting approach [34]. Additionally, the production of polymeric particles is considerably more cost-effective than lipidic systems.

Polymeric particles include microparticles and nano-in-microparticles and these can further be divided into nanocomposites (also referred as nano-embedded microparticles) and porous nanoaggregate particles (PNAPs) (Fig. 2). Nano-in-microparticle systems have been developed to utilize nanoparticulate systems, often exhaled from the lungs, into successful inhalable drug carrier systems (Fig. 1). In nanocomposites, drug loaded nanoparticles are incorporated in a micronized sugar matrix (e.g. lactose, mannitol, leucine, maltodextrin) to form micro-scaled particles of 1–5 μm. These nanocomposites can then be decomposed into nanoparticles in the lower airways, since the sugar moiety is soluble in the lung lining fluid. On the other hand, PNAPs consist of spherical nanoaggregates at the micron-size level that also re-disperse into the elementary nanoparticles in the lung lining fluid.

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Fig. 2. Developed inhalable dry powder systems for anti-TB drug delivery based on polymeric particles.

A considerable focus has been given to synthetic polymers poly(lactide-co-glycolide) (PLGA) and polylactide (PLA) due to their biocompatible and biodegradable properties, non-toxicity [[35], [36], [37]] and to the ability to encapsulate hydrophobic anti-TB drugs. Synthetic polymers also benefit from high purity products and batch-to-batch uniformity. However, more recently, the trend for dry powder production has been shifted towards the use of natural polymers such as chitosan, gelatin or guar gum due to their abundance and availability in nature, cost effectiveness and the fact that these raw materials require a minimal use of organic solvents to be processed. Also, combinations of polymers have been tested to merge properties of the polymers involved in order to obtain more efficient delivery systems.

2.5. Techniques used to develop polymeric microparticulate dry powders

Emulsion followed by solvent evaporation is commonly used to produce microparticles, in particular PLGA microparticles, however these are produced with a relatively wide size distribution [38]. Makino et al. have used this technique with a Shirasu porous glass (SPG) membrane to formulate microparticles with lower size dispersion [39,40]. In order to overcome the low yield of this process, a premix membrane emulsification method was used to prepare monodispersed PLGA microparticles [41,42]. Alternatively to SPG membrane emulsification, an adjuvant strategy based on glass beads facilitated stirring, improved the homogeneity of PLGA microparticles produced by the emulsion/solvent evaporation method [43]. Although emulsion/solvent evaporation is often used to produce anti-TB drug loaded microparticles, it is labor intensive and not practicable for large production, which can limit translational strategies into therapies.

Inhalable microparticles and nano-in-microparticles were successfully obtained using the spray-drying (SD) technique [38,[44], [45], [46]], the most used technique for production of inhalable dry powders for anti-TB delivery. SD is a simple, fast and versatile technique that can be used with different spray nozzles. For instance, rifampicin dihydrate microcrystals were coated with PLGA and/or PLA by SD with a three-fluid spray nozzle producing core-shell microcapsules with the appropriate aerodynamic properties to reach the alveoli and with controlled release properties not achieved by using only rifampicin dihydrate microcrystals [14,15]. Due to the existence of three independent channels, one for the core (drug), one for the shell (polymer) and the other for the atomizing gas, the process can be done in a single step.

Nanocomposites containing rifampicin-PLGA nanoparticles and mannitol have also been prepared in a one-step approach using a four-fluid nozzle spray drier [53]. In this technique two liquid and two gas passages allowed the drug and the carrier to be prepared in different solvents avoiding the limitations associated to using a common solvent as in the traditional two fluid spray-drier.

Spray-drying can also be combined with techniques such as anti-solvent precipitation and ionotropic gelation that are oriented to nanoparticle production, to fabricate nano-in-microparticulate systems. Inhalable guar gum nanoparticles were prepared using the precipitation technique with ethanol as anti-solvent [50]. Chitosan and alginate nanoparticles have been prepared by the ionotropic gelation, a method based on the complexation between oppositely charged species namely chitosan and tripolyphosphate anion or calcium cation and alginate [51,52,54]. The physical cross-linking by electrostatic interactions avoids toxic reagents used in the chemical cross-linking such as glutaraldeyde.

The supercritical technique has also been proposed for the production of inhalable polymeric microparticles. Patomchaiviwat et al. prepared rifampicin loaded PLA microparticles, in a size range suitable for lower airways deposition, using a supercritical anti-solvent process [49]. Supercritical anti-solvent is a quite recent and barely explored technique for anti-TB drug delivery. Although it is a single step process this technology is expensive and labor intensive and requires deeper investigation to assess its full potential for anti-TB drug delivery.

2.6. Carrier systems oriented for macrophage targeting

Phagocytosis of drug carrier systems by macrophages especially by infected macrophages is a very interesting approach leading to the internalization of these systems, and to an intracellular influx of the drug in the phagosome, where the bacilli reside, which could result in a more efficient anti-TB approach.

Among the physical properties, the size of the particulate carrier system is one of the most important characteristics affecting up-take via phagocytosis (Fig. 3) [55].

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Fig. 3. Physico-chemical properties of microparticulate systems undergoing phagocytosis by alveolar macrophages.

Phagocytosis ranged particles (1–6 μm) enter the macrophage and can potentially deliver larger amounts of anti-TB drugs directly to the infection than oral or injected drug doses [56]. Particle shape also plays an important role in the up-take process as the local shape determines the initial contact with macrophages and their phagocytic fate [57]. Using shape-switching particles it was shown that elliptical disks have potential to mitigate phagocytosis, however when they switched their shape into spheres they were internalized by macrophages [58]. In relation to surface charge an increase up-take was observed when PLGA microparticles were coated with cationic polymer polyethylenimine relative to uncoated ones [43]. Furthermore, harder and non-porous particles are more efficiently taken up than soft and porous particles and hydrophobic and insoluble particles are more easily opsonized and this fact increases the probability of recognition by alveolar macrophages [55]. The chemical composition of the particle is also a significant feature for macrophage response. PLGA microparticles prepared with different proportions of lactide and glycolide and with different PLGA molecular weights result in different interactions with alveolar macrophages [59]. The presence of mannose moiety in the structure of polymers such as guar gum and mannan results in higher up-take by macrophages [51,60,61].

The carrier surface can be functionalized to incorporate ligands that target macrophage surface receptors in a process that is known as active targeting. Mannosylated gelatin microparticles have been developed to target macrophage mannose membrane receptor [62]. Other ligands including carbohydrate binding receptors such as galactose, β-glucan, N-acetylglycosamine and folic acid have been explored for macrophage targeting aiming at a wide range of diseases [63]. However, their potential towards therapeutic TB strategies requires further investigation.