Liposomes for Inhalation.

Inhalation of liposomes formulated with phospholipids similar to endogenous lung surfactants and lipids offers biocompatibility and versatility within the pulmonary medicine field to treat a range of diseases such as lung cancer, cystic fibrosis and lung infections. Manipulation of the physicochemical properties of liposomes enables innovative design of the carrier to meet specific delivery, release and targeting requirements. This delivery system offers several benefits: improved pharmacokinetics with reduced toxicity, enhanced therapeutic efficacy, increased delivery of poorly soluble drugs, taste masking, biopharmaceutics degradation protection and targeted cellular therapy. This section provides an overview of liposomal formulation and delivery, together with their applications for different disease states in the lung.

Nasal delivery of encapsulated recombinant ACE2 as a prophylactic drug for SARS-CoV-2.

Angiotensin-converting enzyme 2 (ACE2) is responsible for cell fusion with SARS-CoV viruses. ACE2 is contained in different areas of the human body, including the nasal cavity, which is considered the main entrance for different types of airborne viruses. We took advantage of the roles of ACE2 and the nasal cavity in SARS-CoV-2 replication and transmission to develop a nasal dry powder. Recombinant ACE2 (rhACE2), after a proper encapsulation achieved via spray freeze drying, shows a binding efficiency with spike proteins of SARS-CoV-2 higher than 77 % at quantities lower than 5 µg/ml. Once delivered to the nose, encapsulated rhACE2 led to viability and permeability of RPMI 2650 cells of at least 90.20 ± 0.67 % and 47.96 ± 4.46 %, respectively, for concentrations lower than 1 mg/ml. These results were validated using nasal dry powder containing rhACE2 to prevent or treat infections derived from SARS-CoV-2.

Insulin Delivery to the Brain via the Nasal Route: Unraveling the Potential for Alzheimer's Disease Therapy.

This comprehensive review delves into the potential of intranasal insulin delivery for managing Alzheimer's Disease (AD) while exploring the connection between AD and diabetes mellitus (DM). Both conditions share features of insulin signalling dysregulation and oxidative stress that accelerate inflammatory response. Given the physiological barriers to brain drug delivery, including the blood-brain barrier, intranasal administration emerges as a non-invasive alternative. Notably, intranasal insulin has shown neuroprotective effects, impacting Aß clearance, tau phosphorylation, and synaptic plasticity. In preclinical studies and clinical trials, intranasally administered insulin achieved rapid and extensive distribution throughout the brain, with optimal formulations exhibiting minimal systemic circulation. The detailed mechanism of insulin transport through the nose-to-brain pathway is elucidated in the review, emphasizing the role of olfactory and trigeminal nerves. Despite promising prospects, challenges in delivering protein drugs from the nasal cavity to the brain remain, including enzymes, tight junctions, mucociliary clearance, and precise drug deposition, which hinder its translation to clinical settings. The review encompasses a discussion of the strategies to enhance the intranasal delivery of therapeutic proteins, such as tight junction modulators, cell-penetrating peptides, and nano-drug carrier systems. Moreover, successful translation of nose-to-brain drug delivery necessitates a holistic understanding of drug transport mechanisms, brain anatomy, and nasal formulation optimization. To date, no intranasal insulin formulation has received regulatory approval for AD treatment. Future research should address challenges related to drug absorption, nasal deposition, and the long-term effects of intranasal insulin. In this context, the evaluation of administration devices for nose-to-brain drug delivery becomes crucial in ensuring precise drug deposition patterns and enhancing bioavailability.

Oscillating high aspect ratio micro-channels can effectively atomize liquids into uniform aerosol droplets and dial their size on-demand.

Ultrasonic atomization of liquids into micrometer-diameter droplets is crucial across multiple fields, ranging from drug delivery, to spectrometry and printing. Controlling the size and uniformity of the generated droplets on-demand is crucial in all these applications. However, existing systems lack the required precision to tune the droplet properties, and the underlying droplet formation mechanism under high-frequency ultrasonic actuation remains poorly understood due to experimental constraints. Here, we present an atomization platform, which overcomes these current limitations. Our device utilizes oscillating high aspect ratio micro-channels to extract liquids from various inlets (ranging from sessile droplets to large beakers), bound them in a precisely defined narrow region, and, controllably atomize them on-demand. The droplet size can be precisely dialled from 3.6 µm to 6.8 µm by simply tuning the actuation parameters. Since the approach does not need nozzles, meshes or impacting jets, stresses exerted on the liquid samples are reduced, hence it is gentler on delicate samples. The precision offered by the technique allows us for the first time to experimentally visualise the oscillating fluid interface at the onset of atomization at MHz frequencies, and demonstrate its applications for targeted respiratory drug delivery.

An overview of in vitro and in vivo techniques for characterization of intranasal protein and peptide formulations for brain targeting.

The surge in neurological disorders necessitates innovative strategies for delivering active pharmaceutical ingredients to the brain. The non-invasive intranasal route has emerged as a promising approach to optimize drug delivery to the central nervous system by circumventing the blood-brain barrier. While the intranasal approach offers numerous advantages, the lack of a standardized protocol for drug testing poses challenges to both in vitro and in vivo studies, limiting the accurate interpretation of nasal drug delivery and pharmacokinetic data. This review explores the in vitro experimental assays employed by the pharmaceutical industry to test intranasal formulation. The focus lies on understanding the diverse techniques used to characterize the intranasal delivery of drugs targeting the brain. Parameters such as drug release, droplet size measurement, plume geometry, deposition in the nasal cavity, aerodynamic performance and mucoadhesiveness are scrutinized for their role in evaluating the performance of nasal drug products. The review further discusses the methodology for in vivo characterization in detail, which is essential in evaluating and refining drug efficacy through the nose-to-brain pathway. Animal models are indispensable for pre-clinical drug testing, offering valuable insights into absorption efficacy and potential variables affecting formulation safety. The insights presented aim to guide future research in intranasal drug delivery for neurological disorders, ensuring more accurate predictions of therapeutic efficacy in clinical contexts.

A counter-swirl design concept for dry powder inhalers.

A swirling airflow is incorporated in several dry powder inhalers (DPIs) for effective powder de-agglomeration. This commonly requires the use of a flow-straightening grid in the DPI to reduce drug deposition loss caused by large lateral spreading of the emerging aerosol. Here, we propose a novel grid-free DPI design concept that improves the aerosol flow characteristics and reduces the aforementioned drug loss. The basis of this design is the implementation of a secondary airflow that swirls in the opposite direction (counter-swirl) to that of a primary swirling airflow. In-vitro deposition, computational fluid dynamics simulations and particle image velocimetry measurements are used to evaluate the counter-swirl DPI aerosol performance and flow characteristics. In comparison with a baseline-DPI that has only a primary swirling airflow, the counter-swirl DPI has 20% less deposition of the emitted drug dose in the induction port and pre-separator of a next generation impactor (NGI). This occurs as a result of the lower flow-swirl generated from the counter-swirl DPI which eliminates the axial reverse flow outside of the mouthpiece and substantially reduces lateral spreading in the exiting aerosol. Modifications to the counter-swirl DPI design were made to prevent drug loss from the secondary airflow tangential inlets, which involved the addition of wall perforations in the tangential inlets and the separation of the primary and secondary swirling airflows by an annular channel. These modified DPI devices were successful in that aspect but had higher flow-swirl than that in the counter-swirl DPI and thus had higher drug mass retained in the device and deposited in the induction port and pre-separator of the NGI. The fine particle fraction in the aerosols generated from all the counter-swirl-based DPIs and the baseline-DPI are found to be statistically similar to each other.

Challenges and current advances in in vitro biofilm characterization.

Biofilms are structured communities of bacterial cells encased in a self-produced polymeric matrix, which develop over time and exhibit temporal responses to stimuli from internal biological processes or external environmental changes. They can be detrimental, threatening public health and causing economic loss, while they also play beneficial roles in ecosystem health, biotechnology processes, and industrial settings. Biofilms express extreme heterogeneity in their physical properties and structural composition, resulting in critical challenges in understanding them comprehensively. The lack of detailed knowledge of biofilms and their phenotypes has deterred significant progress in developing strategies to control their negative impacts and take advantage of their beneficial applications. A range of in vitro models and characterization tools have been developed and used to study biofilm growth and, specifically, to investigate the impact of environmental and growth factors on their development. This review article discusses the existing knowledge of biofilm properties and explains how external factors, such as flow condition, surface, interface, and host factor, may impact biofilm growth. The limitations of current tools, techniques, and in vitro models that are currently used for biofilms are also presented.

Engineered dry powders for the nose-to-brain delivery of transforming growth factor-beta.

Nose-to-brain delivery is increasing in popularity as an alternative to other invasive delivery routes. However, targeting the drugs and bypassing the central nervous system are challenging. We aim to develop dry powders composed of nanoparticles-in-microparticles for high efficiency of nose-to-brain delivery. The size of microparticles (between 250 and 350 µm), is desired for reaching the olfactory area, located below the nose-to-brain barrier. Moreover, nanoparticles with a diameter between 150 and 200 nm are desired for traveling through the nose-to-brain barrier. The materials of PLGA or lecithin were used in this study for nanoencapsulation. Both types of capsules showed no toxicology on nasal (RPMI 2650) cells and a similar permeability coefficient (Papp) of Flu-Na, which was about 3.69 ± 0.47 × 10-6 and 3.88 ± 0.43 × 10-6 cm/s for TGF-ß-Lecithin and PLGA, respectively. The main difference was related to the location of deposition; the TGF-ß-PLGA showed a higher drug deposition in the nasopharynx (49.89 ± 25.90 %), but the TGF-ß-Lecithin formulation mostly placed in the nostril (41.71 ± 13.35 %).

A Safe-by-Design Approach for the Synthesis of a Novel Cross-Linked Hyaluronic Acid with Improved Biological and Physical Properties.

Hyaluronic acid (HA) is a polymer with unique biological properties that has gained in interest over the years, with applications in pharmaceutical, cosmetic, and biomedical fields; however, its widespread use has been limited by its short half-life. Therefore, a new cross-linked hyaluronic acid was designed and characterized using a natural and safe cross-linking agent, such as arginine methyl ester, which provided improved resistance to enzymatic action, as compared to the corresponding linear polymer. The antibacterial profile of the new derivative was shown to be effective against S. aureus and P. acnes, making it a promising candidate for use in cosmetic formulations and skin applications. Its effect on S. pneumoniae, combined with its excellent tolerability profile on lung cells, also makes this new product suitable for applications involving the respiratory tract.

The development of a 3D-printed in vitro integrated oro-pharyngeal air-liquid interface cellular throat model for drug transport.

To simulate the deposition of drugs in the oro-pharynx region, several in vitro models are available such as the United States Pharmacopeia-Induction Port (USP-IP) throat and the Virginia Commonwealth University (VCU) models. However, currently, there is no such in vitro model that incorporates a biological barrier to elucidate drug transport across the pharyngeal cells. Cellular models such as in vitro air-liquid interface (ALI) models of human respiratory epithelial cell lines are extensively used to study drug transport. To date, no studies have yet been performed to optimise the ALI culture conditions of the human pharyngeal cell line Detroit 562 and determine whether it could be used for drug transport. Therefore, this study aimed to develop a novel 3D-printed throat model integrated with an ALI cellular model of Detroit 562 cells and optimise the culture conditions to investigate whether the combined model could be used to study drug transport, using Lidocaine as a model drug. Differentiating characteristics specific to airway epithelia were assessed using 3 seeding densities (30,000, 60,000, and 80,000 cells/well (c/w), respectively) over 21 days. The results showed that Detroit 562 cells completely differentiates on day 18 of ALI for both 60,000 and 80,000 c/w with significant mucus production, showing response to bacterial and viral stimuli and development of functional tight junctions and Lidocaine transport with no significant differences observed between the ALI models with the 2 cell seeding densities. Results showed the suitability of the Low density (60,000 c/w or 1.8 × 105 cells/cm2) ALI model to study drug transport. Importantly, the developed novel 3D-printed throat model integrated with our optimised in vitro Detroit 562 ALI model showed transport of Lidocaine throat spray. Overall, the study highlights the potential of the novel 3D-printed bio-throat integrated model as a promising in vitro system to investigate the transport of inhalable drug therapies targeted at the oro-pharyngeal region.

Microfluidics assembly of inhalable liposomal ciprofloxacin characterised by an innovative in vitro pulmonary model.

Respiratory tract infections (RTIs) are reported to be the leading cause of death worldwide. Delivery of liposomal antibiotic nano-systems via the inhalation route has drawn significant interest in RTIs treatment as it can directly target the site of infection and reduces the risk of systemic exposure and side effects. Moreover, this formulation system can improve pharmacokinetics and biodistribution and enhance the activity against intracellular pathogens. Microfluidics is an innovative manufacturing technology that can produce nanomedicines in a homogenous and scalable way. The objective of this study was to evaluate the antibiofilm efficacy of two liposomal ciprofloxacin formulations with different vesicle sizes manufactured by using a 3D-printed microfluidic chip. Each formulation was characterised in terms of size, polydispersity index, charge and encapsulation. Moreover, the aerosolisation characteristics of the liposomal formulations were investigated and compared with free ciprofloxacin solution using laser diffraction and cascade impaction methods. The in vitro drug release was tested using the dialysis bag method. Furthermore, the drug transport and drug release studies were conducted using the alveolar epithelial H441 cell line integrated next-generation impactor in vitro model. Finally, the biofilm eradication efficacy was evaluated using a dual-chamber microfluidic in vitro model. Results showed that both liposomal-loaded ciprofloxacin formulations and free ciprofloxacin solution had comparable aerosolisation characteristics and biofilm-killing efficacy. The liposomal ciprofloxacin formulation of smaller vesicle size showed significantly slower drug release in the dialysis bag technique compared to the free ciprofloxacin solution. Interestingly, liposomal ciprofloxacin formulations successfully controlled the release of the drug in the epithelial cell model and showed different drug transport profiles on H441 cell lines compared to the free ciprofloxacin solution, supporting the potential for inhaled liposomal ciprofloxacin to provide a promising treatment for respiratory infections.

In vitro and in vivo applications of a universal and synthetic thermo-responsive drug delivery hydrogel platform.

A synthetic and thermo-responsive polymer, poly(N-isopropylacrylamide)-co-(polylactide/2-hydroxy methacrylate)-co-(oligo (ethylene glycol)), is used to formulate a universal carrier platform for sustained drug release. The enabling carrier, denoted as TP, is prepared by dissolving the polymer in an aqueous solution at a relatively neutral pH. A wide range of therapeutic moieties can be incorporated without the need for the addition of surfactants, organic solvents, and other reagents to the carrier system. The resulting solution is flowable through fine gauge needle, allowing accurate administration of TP to the target site. After injection, TP carrier undergoes a coil to globe phase transition to form a hydrogel matrix at the site. The benign nature of the polymer carrier and its physical gelation process are essential to preserve the biological activity of the encapsulated compounds while the adhesive hydrogel nature of the matrix allows sustained elusion and controlled delivery of the incorporated therapeutics. The TP carrier system has been shown to be non-toxic and elicits a minimal inflammatory response in multiple in vitro studies. These findings suggest the suitability of TP as an enabling carrier of therapeutics for localized and sustained drug delivery. To confirm this hypothesis, the capabilities of TP to encapsulate and effectively deliver multiple therapeutics of different physicochemical characteristics was evaluated. Specifically, a broad range of compounds were tested, including ciprofloxacin HCl, tumor necrosis factor-alpha (TNF-α), transforming growth factor beta 1 (TGF-ß1), and recombinant human bone morphogenetic protein 2 (BMP2). In vitro studies confirmed that TP carrier is able to control the release of the encapsulated drugs over an extended period of time and mitigate their burst release regardless of the compounds' physiochemical properties for the majority of the loaded therapeutics. Importantly, in vitro and in vivo animal studies showed that the released drugs from the TP hydrogel matrix remained potent and bioactive, confirming the high potential of the TP polymer system as an enabling carrier.

Real-time in-situ electrochemical monitoring of Pseudomonas aeruginosa biofilms grown on air-liquid interface and its antibiotic susceptibility using a novel dual-chamber microfluidic device.

Biofilms are communities of bacterial cells encased in a self-produced polymeric matrix that exhibit high tolerance toward environmental stress. Despite the plethora of research on biofilms, most P. aeruginosa biofilm models are cultured on a solid-liquid interface, and the longitudinal growth characteristics of P. aeruginosa biofilm are unclear. This study demonstrates the real-time and noninvasive monitoring of biofilm growth using a novel dual-chamber microfluidic device integrated with electrochemical detection capabilities to monitor pyocyanin (PYO). The growth of P. aeruginosa biofilms on the air-liquid interface (ALI) was monitored over 48 h, and its antibiotic susceptibility to 6 h exposure of 50, 400, and 1600 µg/ml of ciprofloxacin solutions was analyzed. The biofilm was treated directly on its surface and indirectly from the substratum by delivering the CIP solution to the top or bottom chamber of the microfluidic device. Results showed that P. aeruginosa biofilm developed on ALI produces PYO continuously, with the PYO production rate varying longitudinally and peak production observed between 24 and 30 h. In addition, this current study shows that the amount of PYO produced by the ALI biofilm is proportional to its viable cell numbers, which has not been previously demonstrated. Biofilm treated with ciprofloxacin solution above 400 µg/ml showed significant PYO reduction, with biofilms being killed more effectively when treatment was applied to their surfaces. The electrochemical measurement results have been verified with colony-forming unit count results, and the strong correlation between the PYO electrical signal and the viable cell number highlights the usefulness of this approach for fast and low-cost ALI biofilm study and antimicrobial tests.

In vitro interactions of aerosol formulations with human nasal epithelium using real-time monitoring of drug transport in a nasal mucosa-on-a-chip.

The current organ-on-chip platforms used for studying respiratory drug delivery are limited to the administration of drug solutions and suspensions, lacking the in vivo aerosol drug administration and aerosol interaction with the respiratory tract barrier. Moreover, they mostly rely on conventional assays that require sample collection and 'off the chip' analyses, which can be labor-intensive and costly. In this study, a human nasal epithelial mucosa (NEM)-on-a-chip is developed that enables the deposition of aerosolized nasal formulations while emulating realistic shear stresses (0.23 and 0.78 Pa), exerted to the inferior and middle turbinate of the human nasal cavity. Under these different dynamic conditions in the donor channel of the NEM-on-a-chip, the deposited dose of aerosols and particle size distributions varied. In addition, the increase in the shear stress to 0.78 Pa adversely affected the cells' viability, reflected by a 36.9 ± 5.4% reduction in the transepithelial electrical resistance. The epithelial transport profiles of aerosolized ibuprofen formulations under 0.23 Pa shear stress were successfully monitored in real-time by an electrochemical sensor embedded in the acceptor channel, where the NEM-on-a-chip was able to monitor the effect of permeation enhancer in the test formulation on the rate of drug transport. The novel NEM-on-a-chip can potentially be a promising physiologically relevant tool for reliable nasal aerosol testing in vitro.

Generic dry powder inhalers bioequivalence: Batch-to-batch variability insights.

Active pharmaceutical ingredient(s) [API(s)] of dry powder inhalers (DPIs) deposition and their fate in the respiratory system are influenced by a complex matrix of formulation, device, manufacturing and physiological variations. DPIs on the market have shown bioinequivalence between batches of the same product. Despite being clinically insignificant, they affect bioequivalence studies when a generic product is compared with the originator. This review discusses implications of batch-to-batch variability on bioequivalence study outcomes and shortcomings of current regulatory requirements. Possible formulation and manufacturing factors resulting in batch-to-batch variability highlight the inherent nature of this issue. Despite scholarly investigations and official regulatory guidance, there remains a need for reliable and realistic in vitro tests that accurately guide a representative reference product batch selection.

Thermoresponsive and Injectable Hydrogel for Tissue Agnostic Regeneration.

Injectable hydrogels can support the body's innate healing capability by providing a temporary matrix for host cell ingrowth and neovascularization. The clinical adoption of current injectable systems remains low due to their cumbersome preparation requirements, device malfunction, product dislodgment during administration, and uncontrolled biological responses at the treatment site. To address these challenges, a fully synthetic and ready-to-use injectable biomaterial is engineered that forms an adhesive hydrogel that remains at the administration site regardless of defect anatomy. The product elicits a negligible local inflammatory response and fully resorbs into nontoxic components with minimal impact on internal organs. Preclinical animal studies confirm that the engineered hydrogel upregulates the regeneration of both soft and hard tissues by providing a temporary matrix to support host cell ingrowth and neovascularization. In a pilot clinical trial, the engineered hydrogel is successfully administered to a socket site post tooth extraction and forms adhesive hydrogel that stabilizes blood clot and supports soft and hard tissue regeneration. Accordingly, this injectable hydrogel exhibits high therapeutic potential and can be adopted to address multiple unmet needs in different clinical settings.

In vitro evaluation of nebulized eucalyptol nano-emulsion formulation as a potential COVID-19 treatment.

Coronavirus is a type of acute atypical respiratory disease representing the leading cause of death worldwide. Eucalyptol (EUC) known also as 1,8-cineole is a potential inhibitor candidate for COVID-19 (main protease-Mpro) with effective antiviral properties but undergoes physico-chemical instability and poor water solubility. Nano-emulsion (NE) is a promising drug delivery system to improve the stability and efficacy of drugs. This work focuses on studying the anti- COVID-19 activity of EUC by developing nebulized eucalyptol nano-emulsion (EUC-NE) as a potentially effective treatment for COVID-19. The EUC -NE formulation was prepared using Tween 80 as a surfactant. In vitro evaluation of the EUC-NE formulation displayed an entrapment efficiency of 77.49 %, a droplet size of 122.37 nm, and an EUC % release of 84.7 %. The aerodynamic characterization and cytotoxicity of EUC-NE formulation were assessed, and results showed high lung deposition and low inhibitory concentration. The antiviral mechanism of the EUC-NE formulation was performed, and it was found that it exerts its action by virucidal, viral replication, and viral adsorption. Our results confirmed the antiviral activity of the EUC-NE formulation against COVID-19 and the efficacy of nano-emulsion as a delivery system, which can improve the cytotoxicity and inhibitory activity of EUC.

Development of excipients free inhalable co-spray-dried tobramycin and diclofenac formulations for cystic fibrosis using two and three fluid nozzles.

This study aims to investigate the effect of physicochemical properties and aerosol performance of two (2FN) and three-fluid nozzles (3FN) on the inhalable co-formulation of tobramycin and diclofenac dry powders. Combination formulations of tobramycin and diclofenac at 2:1 and 4:1 w/w ratios were prepared at a laboratory scale using a spray dryer in conjunction with a 2FN or 3FN. Powder size, morphology, solid-state characteristics, and aerodynamic and dissolution properties were characterised. The nozzle types and the formulation composition influenced the yield, particle size, solid-state properties, aerosolization behaviour and dissolution of the co-spray dried formulations. In particular, using the 2FN the co-spray dried formulation of tobramycin and diclofenac at 2:1 w/w showed smaller particle size (D50, 3.01 ± 0.06 µm), high fine particle fractions (FPF) (61.1 ± 3.6% for tobramycin and 65.92 ± 3 for diclofenac) and faster dissolution with approx. 70% diclofenac released within 3 h and approx. 90% tobramycin was released within 45 min. However, the 3FN for the co-spray dried formulation of tobramycin and diclofenac at a 2:1 w/w ratio showed a larger particle size (D50, 3.42 ± 0.02 µm), lower FPF (40.6 ± 3.4% for tobramycin and 36.9 ± 0.84 for diclofenac) and comparative slower dissolution with approx. 60% diclofenac was released within 3 h and 80% tobramycin was released within 45 min. A similar trend was observed when the tobramycin to diclofenac ratio was increased to 4:1 w/w. Overall results suggest that spray drying with 2FN showed a superior and viable approach to producing excipients-free inhalable co-spray dried formulations of tobramycin and diclofenac. However, the formulation produced using the 3FN showed higher enrichment of hydrophobic diclofenac and an ability to control the tobramycin drug release in vitro.

Validation of a cell integrated next-generation impactor to assess in vitro drug transport of physiologically relevant aerosolised particles.

The development of novel inhaled formulations in the pre-clinical stage has been impeded by a lack of meaningful information related to drug dissolution and transport at the lung epithelia due to the absence of physiologically relevant in vitro respiratory models. The objective of the present study was to develop an in vitro experimental model, which combined the next generation impactor (NGI) and two respiratory epithelial cell lines, for examining the aerodynamic performance of dry powder inhalers and the fate of aerosolised drugs following lung deposition. The NGI impaction plates of stage 3 (i.e., a cut-off diameter of 2.82-4.46 µm) and stage 7 (i.e., a cut-off diameter of 0.34-0.55 µm) were modified to accommodate 3 cell cultures inserts. Specifically, Calu-3 cells and H441 cells, which are representative of the bronchial and alveolar epithelia in the lung, respectively, were cultivated at the air-liquid interface on SnapwellsTM with polycarbonate membranes. The aerodynamic particle size distribution of the modified NGI was investigated using resveratrol dry powder formulation (as a model drug). The suitability of such an in vitro model was confirmed by examining the in vitro aerodynamic performance of the model drug as compared to the conventional NGI setup (i.e., without the integrated Snapwell inserts), as well as the effect of experimental conditions (e.g., 60 L/min airflows) on the cells in the integrated Snapwell inserts. After deposition of the aerodynamically fractioned resveratrol, the permeation of the drug across the cell layer to the basolateral chamber of the Snapwell inserts was evaluated over 24 h. Results obtained from the drug transport study showed that the cell-integrated NGI provided realistic drug delivery conditions to the cells that can be used to assess the fate of fractionated aerosol particles. This system enables a better understanding of the in vitro drug deposition in the lungs and allows studies on both aerodynamic characterisation and drug transport (drug biological interactions with the cells) to be performed simultaneously.

Protective Abilities of an Inhaled DPI Formulation Based on Sodium Hyaluronate against Environmental Hazards Targeting the Upper Respiratory Tract.

The exposure of lung epithelium to environmental hazards is linked to several chronic respiratory diseases. We assessed the ability of an inhaled dry powder (DPI) medical device product (PolmonYDEFENCE/DYFESATM, SOFAR SpA, Trezzano Rosa, Italy), using a formulation of sodium hyaluronate (Na-Hya) as the key ingredient as a defensive barrier to protect the upper respiratory tract. Specifically, it was evaluated if the presence of the barrier formed by sodium hyaluronate present on the cells, reducing direct contact of the urban dust (UD) with the surface of cells can protect them in an indirect manner by the inflammatory and oxidative process started in the presence of the UD. Cytotoxicity and the protection capability against the oxidative stress of the product were tested in vitro using Calu-3 cells exposure to UD as a trigger for oxidative stress. Inflammation and wound healing were assessed using an air-liquid interface (ALI) culture model of the Calu-3 cells. Deposition studies of the formulation were conducted using a modified Anderson cascade impactor (ACI) and the monodose PillHaler® dry powder inhaler (DPI) device, Na-Hya was detected and quantified using high-performance-liquid-chromatography (HPLC). Solubilised PolmonYDEFENCE/DYFESATM gives protection against oxidative stress in Calu-3 cells in the short term (2 h) without any cytotoxic effects. ALI culture experiments, testing the barrier-forming (non-solubilised) capabilities of PolmonYDEFENCE/DYFESATM, showed that the barrier layer reduced inflammation triggered by UD and the time for wound closure compared to Na-Hya alone. Deposition experiments using the ACI and the PillHaler® DPI device showed that the majority of the product was deposited in the upper part of the respiratory tract. Finally, the protective effect of the product was efficacious for up to 24 h without affecting mucus production. We demonstrated the potential of PolmonYDEFENCE/DYFESATM as a preventative barrier against UD, which may aid in protecting the upper respiratory tract against environmental hazards and help with chronic respiratory diseases.