A Biocompatible Synthetic Lung Fluid Based on Human Respiratory Tract Lining Fluid Composition.

PURPOSE: To characterise a biorelevant simulated lung fluid (SLF) based on the composition of human respiratory tract lining fluid. SLF was compared to other media which have been utilized as lung fluid simulants in terms of fluid structure, biocompatibility and performance in inhalation biopharmaceutical assays. METHODS: The structure of SLF was investigated using cryo-transmission electron microscopy, photon correlation spectroscopy and Langmuir isotherms. Biocompatibility with A549 alveolar epithelial cells was determined by MTT assay, morphometric observations and transcriptomic analysis. Biopharmaceutical applicability was evaluated by measuring the solubility and dissolution of beclomethasone dipropionate (BDP) and fluticasone propionate (FP), in SLF. RESULTS: SLF exhibited a colloidal structure, possessing vesicles similar in nature to those found in lung fluid extracts. No adverse effect on A549 cells was apparent after exposure to the SLF for 24 h, although some metabolic changes were identified consistent with the change of culture medium to a more lung-like composition. The solubility and dissolution of BDP and FP in SLF were enhanced compared to Gamble's solution. CONCLUSION: The SLF reported herein constitutes a biorelevant synthetic simulant which is suitable to study biopharmaceutical properties of inhalation medicines such as those being proposed for an inhaled biopharmaceutics classification system.

Differences in the coronal proteome acquired by particles depositing in the lungs of asthmatic versus healthy humans.

Most inhaled nanomedicines in development are for the treatment of lung disease, yet little is known about their interaction with the respiratory tract lining fluids (RTLFs). Here we combined the use of nano-silica, as a protein concentrator, with label-free snapshot proteomics (LC-MS/MS; key findings confirmed by ELISA) to generate a quantitative profile of the RTLF proteome and provided insight into the evolved corona; information that may be used in future to improve drug targeting to the lungs by inhaled medicines. The asthmatic coronal proteome displayed a reduced contribution of surfactant proteins (SP-A and B) and a higher contribution of α1-antitrypsin. Pathway analysis suggested that asthmatic RTLFs may also be deficient in proteins related to metal handling (e.g. lactoferrin). This study demonstrates how the composition of the corona acquired by inhaled nanoparticles is modified in asthma and suggests depressed mucosal immunity even in mild airway disease.

Enrichment of immunoregulatory proteins in the biomolecular corona of nanoparticles within human respiratory tract lining fluid.

When inhaled nanoparticles deposit in the lungs, they transit through respiratory tract lining fluid (RTLF) acquiring a biomolecular corona reflecting the interaction of the RTLF with the nanomaterial surface. Label-free snapshot proteomics was used to generate semi-quantitative profiles of corona proteins formed around silica (SiO2) and poly(vinyl) acetate (PVAc) nanoparticles in RTLF, the latter employed as an archetype drug delivery vehicle. The evolved PVAc corona was significantly enriched compared to that observed on SiO2 nanoparticles (698 vs. 429 proteins identified); however both coronas contained a substantial contribution from innate immunity proteins, including surfactant protein A, napsin A and complement (C1q and C3) proteins. Functional protein classification supports the hypothesis that corona formation in RTLF constitutes opsonisation, preparing particles for phagocytosis and clearance from the lungs. These data highlight how an understanding of the evolved corona is necessary for the design of inhaled nanomedicines with acceptable safety and tailored clearance profiles. FROM THE CLINICAL EDITOR: Inhaled nanoparticles often acquire a layer of protein corona while they go through the respiratory tract. Here, the authors investigated the identity of these proteins. The proper identification would improve the understanding of the use of inhaled nanoparticles in future therapeutics.

Development of new in vitro models of lung protease activity for investigating stability of inhaled biological therapies and drug delivery systems.

Proteases play a vital role in lung health and are critically important to the metabolic clearance of inhaled protein-based therapeutics after inhalation. Surprisingly little is known about lung fluid protease composition and there is a consequent lack of biorelevant experimental models, which limits research and development in the burgeoning field of inhaled biologics. The aim of this study was to quantify proteases in human lung fluid and to use this data to design novel in vitro experimental models of lung lining fluid possessing biorelevant lung protease activity for use in biopharmaceutical stability studies. As a proof of concept, these novel models were used to investigate the effect of proteolytic activity on the stability of albumin nanoparticles, a biologic nanoparticle formulation widely investigated as a pulmonary drug delivery system. Bronchoalveolar lavage fluid was collected from healthy human volunteers and proteomic analysis was used to quantify the predominant proteases. Based on these data, four new lung protease models were constructed based on: (i) trypsin as a sole protease, (ii) dipeptidyl peptidase IV, cathepsin D, cathepsin H, and angiotensin converting enzyme in ratio and concentration to mimic the protease concentration in healthy lungs. Neutrophil elastase was used to model protease activity in inflammation. Albumin nanoparticles of 100 nm diameter remained intact over 48 h in phosphate buffered saline, but were degraded more rapidly in trypsin (50% reduction in 10 min) compared to the healthy lung protease model (50% reduction in 150 min). The addition of neutrophil elastase to the healthy lung protease model resulted in a similar, but more variable degradation profile. Nanoparticle degradation was associated with concomitant appearance of small fragments and aggregates. In conclusion, we have characterised the protease concentration in the lungs of healthy humans, designed models of lung protease activity and demonstrated their utility in studying albumin nanoparticle degradation. These methods and models have wide application to study the influence of proteases in lung disease, expression of proteases in respiratory cell culture models, stability of peptide and protein-based drugs and inhaled drug delivery systems.