Development of an in vitro system to study the interactions of aerosolized drugs with pulmonary mucus

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. Mucus is the first biological component inhaled drugs encounter on their journey towards their pharmacological target in the upper airways. Yet, how mucus may influence drug disposition and efficacy in the lungs has been essentially overlooked. In this study, a simple in vitro system was developed to investigate the factors promoting drug interactions with airway mucus in physiologically relevant conditions. Thin layers of porcine tracheal mucus were prepared in Transwell® inserts and initially, the diffusion of various fluorescent dyes across those layers was monitored over time. A deposition system featuring a MicroSprayer® aerosolizer was optimized to reproducibly deliver liquid aerosols to multiple air-facing layers and then exploited to compare the impact of airway mucus on the transport of inhaled bronchodilators. Both the dyes and drugs tested were distinctly hindered by mucus with high logP compounds being the most affected. The diffusion rate of the bronchodilators across the layers was in the order: ipratropium ≈ glycopyronnium > formoterol > salbutamol > indacaterol, suggesting hydrophobicity plays an important role in their binding to mucus but is not the unique parameter involved. Testing of larger series of compounds would nevertheless be necessary to better understand the interactions of inhaled drugs with airway mucus

Rapid formulation of redox-responsive oligo-β-aminoester polyplexes with siRNA via jet printing

Here we describe a rapid inkjet formulation method for screening newly-synthesised cationic materials for siRNA delivery into cancer cells. Reduction responsive oligo-β-aminoesters were prepared and evaluated for their ability to condense siRNA into polyplexes through a fast inkjet printing method. A direct relationship between the oligomer structures and charge densities, and the final cell response in terms of uptake rate and transfection efficacy, was found. The oligo-β-aminoesters were well-tolerated by the cancer cells, compared to conventional cationic polymers so far employed in gene delivery, and were as active in silencing of a representative luciferase gene

Live imaging of cellular internalization of single colloidal particle by combined label-free and fluorescence total internal reflection microscopy

In this work we utilise the combination of label-free total internal reflection microscopy and total internal reflectance fluorescence (TIRM/TIRF) microscopy to achieve a simultaneous, live imaging of single, label-free colloidal particle endocytosis by individual cells. The TIRM arm of the microscope enables label free imaging of the colloid and cell membrane features, while the TIRF arm images the dynamics of fluorescent-labelled clathrin (protein involved in endocytosis via clathrin pathway), expressed in transfected 3T3 fibroblasts cells. Using a model polymeric colloid and cells with a fluorescently-tagged clathrin endocytosis pathway, we demonstrate that wide field TIRM/TIRF co-imaging enables live visualization of the process of colloidal particle interaction with the labelled cell structure, which is valuable for discerning the membrane events and route of colloid internalization by the cell. We further show that 500 nm model polystyrene colloid associates with clathrin, prior to and during its cellular internalisation. This association is not apparent with larger, 1 μm colloid, indicating an upper particle size limit for clathrin-mediated endocytosis

Investigating histidinylated highly branched poly(lysine) for siRNA delivery

The temporary silencing of disease-associated genes utilising short interfering RNA (siRNA) is a potent and selective route for addressing a wide range of life limiting disorders. However, the few clinically approved siRNA therapies rely on lipid based formulations, which although potent, provide limited chemical space to tune the stability, efficacy and tissue selectivity. In this study, we investigated the role of molar mass and histidinylation for poly(lysine) based non-viral vectors, synthesised through a fully aqueous thermal condensation polymerisation. Formulation and in vitro studies revealed that higher molar mass derivatives yielded smaller polyplexes attributed to a greater affinity for siRNA at lower N/P ratios yielding greater transfection efficiency, albeit with some cytotoxicity. Histidinylation had a negligible effect on formulation size, yet imparted a moderate improvement in biocompatibility, but did not provide any meaningful improvement over silencing efficiency compared to non-histidinylated derivatives. This was attributed to a greater degree of cellular internalisation for non-histidinylated analogues, which was enhanced with the higher molar mass material. This journal i

Enhanced uptake in 2D- and 3D- lung cancer cell models of redox responsive PEGylated nanoparticles with sensitivity to reducing extra-and intracellular environments

In the treatment of lung cancer, there is an urgent need of innovative medicines to optimize pharmacological responses of conventional chemotherapeutics while attenuating side effects. Here, we have exploited some relatively unexplored subtle differences in reduction potential, associated with cancer cell microenvironments in addition to the well-known changes in intracellular redox environment. We report the synthesis and application of novel redox-responsive PLGA (poly(lactic-co-glycolic acid)) -PEG(polyethylene glycol) nanoparticles (RR-NPs) programmed to change surface properties when entering tumor microenvironments, thus enhance cell internalization of the particles and their drug cargo. The new co-polymers, in which PEG and PLGA were linked by ‘anchiomeric effector’ dithiylethanoate esters were synthesized by a combination of ring-opening polymerization and Michael addition reactions and employed to prepare NPs. Non redox-responsive nanoparticles (nRR-NPs) based on related PLGA-PEG copolymers were also prepared as comparators. Spherical NPs of around 120 nm diameter with a low polydispersity index and negative zeta potentials as well as good drug loading of docetaxel were obtained. The NPs showed prolonged stability in relevant simulated biological fluids and a high ability to penetrate an artificial mucus layer due to the presence of the external PEG coating. Stability, FRET and drug release studies in conditions simulating intracellular reductive environments demonstrated a fast disassembly of the external shell of the NPs, thus triggering on-demand drug release. FACS measurements and confocal microscopy showed increased and faster uptake of RR-NPs in both 2D- and 3D- cell culture models of lung cancer compared to nRR-NPs. In particular, the ‘designed-in’ reductive instability of RR-NPs in conditioned cell media, the fast PEG release in the extracellular compartment, as well as a diminution of uptake rate in control experiments where extracellular thiols were neutralized, suggested a partial extracellular release of the PEG fringe that promoted rapid internalization of the residual NPs into cells. Taken together, these results provide further evidence of the effectiveness of PEGylated reducible nanocarriers to permeate mucus layer barriers, and establish a new means to enhance cancer cell uptake of drug carriers by extra-and intra-cellular cleavage of protein-and cell-shielding hydrophilic blocks

Rapid formulation of redox-responsive oligo-β-aminoester polyplexes with siRNA via jet printing

Here we describe a rapid inkjet formulation method for screening newly-synthesised cationic materials for siRNA delivery into cancer cells. Reduction responsive oligo-β-aminoesters were prepared and evaluated for their ability to condense siRNA into polyplexes through a fast inkjet printing method. A direct relationship between the oligomer structures and charge densities, and the final cell response in terms of uptake rate and transfection efficacy, was found. The oligo-β-aminoesters were well-tolerated by the cancer cells, compared to conventional cationic polymers so far employed in gene delivery, and were as active in silencing of a representative luciferase gene

Investigating the intracellular effects of hyperbranched polycation-DNA complexes on lung cancer cells using LC-MS-based metabolite profiling

Cationic polymers have emerged as a promising alternative to viral vectors in gene therapy. They are cheap to scale up, easy to functionalise and also presume to be safer than the viral vectors, however many of them are cytotoxic. The large number of polycations, designed to address the toxicity problem, raises a practical need to develop a fast and reliable method for assessing the safety of these materials. In this regard, metabolomics provides a detailed and comprehensive method that can assess the potential toxicity at the cellular and molecular level. Here, we applied metabolomics to investigate the impact of hyperbranched polylysine, hyperbranched polylysine-co-histidine and branched polyethyleneimine polyplexes at sub-toxic concentrations on the metabolic pathways of A459 and H1299 lung carcinoma cell lines. The study revealed that the polyplexes downregulated metabolites associated with glycolysis and the TCA cycle, and induced oxidative stress in both cell lines. The fold changes of the metabolites indicated that the polyplexes of polyethyleneimine and hyperbranched polylysine affected the metabolism much more than the polyplexes of hyperbranched polylysine-co-histidine. This was in line with transfection results, suggesting a correlation between the toxicity and transfection efficiency of these polyplexes. Our work highlights the importance of metabolomics approach not just to assess the potential toxicity of polyplexes but also to understand the molecular mechanism of them which could help to design more efficient vectors

Mechanism of mucosal permeability enhancement of CriticalSorb® (Solutol® HS15) investigated In Vitro in cell cultures.

Purpose CriticalSorb™, with the principal component Solutol® HS15, is a novel mucosal drug delivery system demonstrated to improve the bioavailability of selected biotherapeutics. The intention of this study is to elucidate mechanism(s) responsible for the enhancement of trans-mucosal absorption of biological drugs by Solutol® HS15. Methods Micelle size and CMC of Solutol® HS15 were determined in biologically relevant media. Polarised airway Calu-3 cell layers were used to measure the permeability of a panel of biological drugs, and to assess changes in TEER, tight junction and F-actin morphology. The rate of cell endocytosis was measured in vitro in the presence of Solutol® HS15 using a membrane probe, FM 2–10. Results This work initially confirms surfactant-like behaviour of Solutol® HS15 in aqueous media, while subsequent experiments demonstrate that the effect of Solutol® HS15 on epithelial tight junctions is different from a ‘classical’ tight junction opening agent and illustrate the effect of Solutol® HS15 on the cell membrane (endocytosis rate) and F-actin cytoskeleton. Conclusion Solutol® HS15 is the principle component of CriticalSorb™ that has shown an enhancement in permeability of medium sized biological drugs across epithelia. This study suggests that its mechanism of action arises primarily from effects on the cell membrane and consequent impacts on the cell cytoskeleton in terms of actin organisation and tight junction opening