Study of the impact of dry powder insufflation over respiratory epithelial cells

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Dry powders: developing tools to study the impact of inhalation over respiratory epithelium

Publication . Pontes, Jorge Filipe Rodrigues; Grenha, Ana; Santos, Rui

The lung is responsible for the gaseous exchange of carbon dioxide (CO2) with oxygen (O2). Furthermore, within the lung there is an equilibrium that is maintained by a great number of mechanisms that filter the air, making it free of materials that can induce damage. The knowledge of the different lung physiological processes helps understand diseases and promote adequate and efficacious therapies. Additionally, the study of the physics and mechanics of the lungs can work favourably when lung is used as a route of administration for drugs. Nowadays, there is a myriad of marketed inhalers that are mainly used in the therapy of asthma and chronic obstructive pulmonary disease. These essentially include metered dose inhalers and dry powder inhalers, the latter currently observing increased popularity. Research on the topic of inhalable dry powders is intense, with many formulations being proposed in the literature. After the intrinsic characterisation of formulations, their study continues focusing on behaviour aspects, particularly addressing the interactions with biological structures, such as cells, tissues or animals. These studies require the existence of methodologies and tests that generate reproducible data for a potential approval by the competent authorities. These methodologies can be divided in three categories: in vivo, ex vivo and in vitro tests. When the study of dry powders is focused, it is easier to replicate data in in vivo platforms, not only because animals are used after a careful selection of the best model and subsequent documentation for validation of an experimental setting, but also because dry powders are used and delivered as such. However, in in vitro assays, it is not easy to mimic the lung environment and inhalation itself, as the delivery of powders over cells cultured on an air-liquid interface is required for the drawing of robust conclusions. Often, cell-based in vitro assays entail testing of dry powders in suspension, meaning that the powder is added to the liquid media where cells grow, and the exposure is provided with the mediation of the media. Regrettably, this does not mimic the occurrences in the lung, as the organ is devoid of abundant liquid. In vitro platforms allowing to comply with realistic conditions are relatively scarce and those existing are expensive and technically complex, which is not feasible in early stages of research. The design and development of an affordable and simple in vitro platform capable of testing dry powders, ensuring reproducible experimentation and data, would be highly helpful. This PhD project proposed the development of tools to improve lung delivery research using dry powders. The development of a device that allows the delivery of dry powders onto cell surfaces, thus simulating more appropriately the lung environment, was envisaged. Moreover, a quartz crystal microbalance was used to establish a technique enabling the determination of dry powder deposition profiles. In parallel, the determination of the viability of respiratory cells after the insufflation of a dry powder using the developed device was performed. Locust bean gum (LBG) is a polysaccharide that has been proposed as excipient in several drug formulation strategies. It has been included in drug delivery systems such as microparticles and nanocarriers, but regrettably LBG does not have the best features for this end, with a low solubility in cold water, the high viscosity of solutions at concentrations above 1% (w/v), and the neutral character, hinder its use. Chemically modified LBG could be a strategy to overcome these limitations. A sulphated derivative of LBG was thus prepared and used in the production of lipid nanocapsules (LNC) by a technique of solvent displacement. Despite adequate properties of the carriers (size around 200 nm, PdI < 0.2, highly negative ζ-potential), encapsulation of the model drug rifabutin failed to achieve satisfactory loadings (< 2%). Moreover, the conversion of LNC into inhalable microparticles by spray-drying using mannitol as carrier matrix, was also not successful, resulting in a negligible amount of powder being recovered. The focus on the formulation used in this PhD project shifted, and the establishment of a spray-drying protocol permitting the production of LBG-based microparticles suitable for lung delivery purposes was investigated. In parallel, an exhaustive study of LBG characteristics, relevant for its role as matrix material, was performed. LBG from three different suppliers was tested, along with a range of spray-drying inlet temperatures, varying between 103 ºC and 160 ºC. The various dry powders were prepared and observed to have similar aspect from a macroscopical visualization. Considering the yields obtained for different temperatures, the chosen formulation was that prepared with an inlet temperature of 130 ± 1 ºC. This temperature ensured a compromise with an inlet temperature that is intermediate and an acceptable production yield around 55-60%. The geometric diameters of the microparticles were determined to vary within 3.8 and 4.5 μm. Further chemical characterisation of LBG was performed (Fourier-transform infrared spectroscopy (FT-IR), molecular weight distribution by High Performance Size Exclusion Chromatography (HPSEC), thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC)), evidencing that the heating cycles (for solubilisation and processing in the spray-dryer) might induce LBG depolymerization to a certain extent. However, additional tests are required to confirm these observations, and aerodynamic characterisation of the different LBG-based microparticles is also considered necessary. Notwithstanding, an LBG-based microparticle formulation was chosen to advance to cell-based and in vivo assays. The former relied on cell viability determination, where a concentration-dependent effect was observed. In fact, as concentrations increment, so does the viscosity of LBG, impacting negatively on cell viability. However, testing of LBG did not induce the disruption of the cell membrane and, thus, the release of the cytoplasmatic enzyme lactate dehydrogenase (LDH). As for the in vivo assays, no allergic reaction was initiated in the sequence of the inhalation of LBG microparticles, thus constituting a strong indication of LBG safety for lung drug delivery applications, although more studies, addressing different indicators, are needed. This work demonstrated that a thorough characterisation of polymers works favourably in the process of preparing dry powders, its optimisation and subsequent biological risk assessment. The design and development of a device enabling the insufflation of dry powders onto cell supports followed the successful preparation of LBG-based microparticles. After several optimisations concerning the model, the method by which the dry powder is weighed and loaded into the device and the air insufflation mechanism, a final model comprised of a funnel and a weighing accessory was conceived. Different 3D printing techniques were also tested to obtain the device (fused deposition modelling and stereolithography), as well as several air pumps. An air compressor was selected as air pump, being tested at different outlet air pressures and using several polysaccharide-based dry powders, but the insufflation yields generally did not go beyond 20-30%, signalling this as a feature requiring future optimisation. Alveolar epithelial cells were cultured on a petri dish and exposed for 24 h to LBG microparticles, insufflated with the developed device. No significant effect was found on cell viability (always > 70%) and the same was observed after pumping air, indicating potential safety of LBG microparticles and of the proper insufflation method. Future assays assessing other parameters are anyway required to further support these findings. An application of the device was further explored by means of inclusion of a Quartz Crystal Microbalance (QCM) into the experimental setting, which permitted evaluating the deposition profile of different dry powders in real time, allowing for a more thorough analysis of the device performance. The use of the QCM in cell-based tests, where cells are cultured over the crystal, using the developed device can provide more insight into the real interaction between cells and dry powders. Overall, the inclusion of this technology in experimentation, especially concerning the in vitro analysis of dry powders, can provide more precise data when compared with conventional techniques. In dry powder formulation development, it is advantageous for choosing the most adequate excipients that translates into the best deposition profile. At more advanced stages, the quantification of interactions between cells and particles will be certainly crucial, as the determination of the best excipients for formulations will help on the improvement of the therapeutic approaches. Further developments concerning this PhD project are envisaged, but all that was achieved and the discussion that followed, will certainly guide future endeavours in aerosol research.

2023-06-28Doctoral thesis

Open access

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

Publication . Pontes, Jorge Filipe; Diogo, Hermínio P.; Conceição, Eusébio; Almeida, Maria P.; Borges dos Santos, Rui M.; Grenha, Ana

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.

2024-06Journal article

Open access