A membrane-free microfluidic approach to mucus permeation for efficient differentiation of mucoadhesive and mucopermeating nanoparticulate systems.

The gastrointestinal mucus barrier is a widely overlooked yet essential component of the intestinal epithelium, responsible for the body's protection against harmful pathogens and particulates. This, coupled with the increasing utilisation of biological molecules as therapeutics (e.g. monoclonal antibodies, RNA vaccines and synthetic proteins) and nanoparticle formulations for drug delivery, necessitates that we consider the additional absorption barrier that the mucus layer may pose. It is imperative that in vitro permeability methods can accurately model this barrier in addition to standardised cellular testing. In this study, a mucus-on-a-chip (MOAC) microfluidic device was engineered and developed to quantify the permeation kinetics of nanoparticles through a biorelevant synthetic mucus layer. Three equivalently sized nanoparticle systems, formulated from chitosan (CSNP), mesoporous silica (MSNP) and poly (lactic-co-glycolic) acid (PLGA-NP) were prepared to encompass various surface chemistries and nanostructures and were assessed for their mucopermeation within the MOAC. Utilising this device, the mucoadhesive behaviour of chitosan nanoparticles was clearly visualised, a phenomenon not often observed via standard permeation models. In contrast, MSNP and PLGA-NP displayed mucopermeation, with significant differences in permeation pattern due to specific mucus-nanoparticle binding. Further optimisation of the MOAC to include a more biorelevant mucus mimic resulted in 5.5-fold hindered PLGA-NP permeation compared to a mucin solution. Furthermore, tracking of PLGA-NP at a single nanoparticle resolution revealed rank-order correlations between particle diffusivity and MOAC permeation. This device, including utilisation of biosimilar mucus, provides a unique ability to quantify both mucoadhesion and mucopenetration of nano-formulations and elucidate mucus binding interactions on a microscopic scale.

Diffusion of Lipid Nanovesicles Bound to a Lipid Membrane Is Associated with the Partial-Slip Boundary Condition.

During diffusion of nanoparticles bound to a cellular membrane by ligand-receptor pairs, the distance to the laterally mobile interface is sufficiently short for their motion to depend not only on the membrane-mediated diffusivity of the tethers but also in a not yet fully understood manner on nanoparticle size and interfacial hydrodynamics. By quantifying diffusivity, velocity, and size of individual membrane-bound liposomes subjected to a hydrodynamic shear flow, we have successfully separated the diffusivity contributions from particle size and number of tethers. The obtained diffusion-size relations for synthetic and extracellular lipid vesicles are not well-described by the conventional no-slip boundary condition, suggesting partial slip as well as a significant diffusivity dependence on the distance to the lipid bilayer. These insights, extending the understanding of diffusion of biological nanoparticles at lipid bilayers, are of relevance for processes such as cellular uptake of viruses and lipid nanoparticles or labeling of cell-membrane-residing molecules.

TIRF Microscopy-Based Monitoring of Drug Permeation Across a Lipid Membrane Supported on Mesoporous Silica.

There is an urgent demand for analytic approaches that enable precise and representative quantification of the transport of biologically active compounds across cellular membranes. In this study, we established a new means to monitor membrane permeation kinetics, using total internal reflection fluorescence microscopy confined to a ≈500 nm thick mesoporous silica substrate, positioned underneath a planar supported cell membrane mimic. This way, we demonstrate spatiotemporally resolved membrane permeation kinetics of a small-molecule model drug, felodipine, while simultaneously controlling the integrity of, and monitoring the drug binding to, the cell membrane mimic. By contrasting the permeation behaviour of pure felodipine with felodipine coupled to the permeability enhancer caprylate (C8), we provide evidence for C8-facilitated transport across lipid membranes, thus validating the potential for this approach to successfully quantify carrier system-induced changes to cellular membrane permeation.

Influence of Bile Composition on Membrane Incorporation of Transient Permeability Enhancers.

Transient permeability enhancers (PEs), such as caprylate, caprate, and salcaprozate sodium (SNAC), improve the bioavailability of poorly permeable macromolecular drugs. However, the effects are variable across individuals and classes of macromolecular drugs and biologics. Here, we examined the influence of bile compositions on the ability of membrane incorporation of three transient PEs-caprylate, caprate, and SNAC-using coarse-grained molecular dynamics (CG-MD). The availability of free PE monomers, which are important near the absorption site, to become incorporated into the membrane was higher in fasted-state fluids than that in fed-state fluids. The simulations also showed that transmembrane perturbation, i.e., insertion of PEs into the membrane, is a key mechanism by which caprylate and caprate increase permeability. In contrast, SNAC was mainly adsorbed onto the membrane surface, indicating a different mode of action. Membrane incorporation of caprylate and caprate was also influenced by bile composition, with more incorporation into fasted- than fed-state fluids. The simulations of transient PE interaction with membranes were further evaluated using two experimental techniques: the quartz crystal microbalance with dissipation technique and total internal reflection fluorescence microscopy. The experimental results were in good agreement with the computational simulations. Finally, the kinetics of membrane insertion was studied with CG-MD. Variation in micelle composition affected the insertion rates of caprate monomer insertion and expulsion from the micelle surface. In conclusion, this study suggests that the bile composition and the luminal composition of the intestinal fluid are important factors contributing to the interindividual variability in the absorption of macromolecular drugs administered with transient PEs.

Independent Size and Fluorescence Emission Determination of Individual Biological Nanoparticles Reveals that Lipophilic Dye Incorporation Does Not Scale with Particle Size.

Advancements in nanoparticle characterization techniques are critical for improving the understanding of how biological nanoparticles (BNPs) contribute to different cellular processes, such as cellular communication, viral infection, as well as various drug-delivery applications. Since BNPs are intrinsically heterogeneous, there is a need for characterization methods that are capable of providing information about multiple parameters simultaneously, preferably at the single-nanoparticle level. In this work, fluorescence microscopy was combined with surface-based two-dimensional flow nanometry, allowing for simultaneous and independent determination of size and fluorescence emission of individual BNPs. In this way, the dependence of the fluorescence emission of the commonly used self-inserting lipophilic dye 3,3'-dioctadecyl-5,5'-di(4-sulfophenyl)oxacarbocyanine (SP-DiO) could successfully be correlated with nanoparticle size for different types of BNPs, including synthetic lipid vesicles, lipid vesicles derived from cellular membrane extracts, and extracellular vesicles derived from human SH-SY5Y cell cultures; all vesicles had a radius, r, of ∼50 nm and similar size distributions. The results demonstrate that the dependence of fluorescence emission of SP-DiO on nanoparticle size varies significantly between the different types of BNPs, with the expected dependence on membrane area, r2, being observed for synthetic lipid vesicles, while a significant weaker dependence on size was observed for BNPs with more complex composition. The latter observation is attributed to a size-dependent difference in membrane composition, which may influence either the optical properties of the dye and/or the insertion efficiency, indicating that the fluorescence emission of this type of self-inserting dye may not be reliable for determining size or size distribution of BNPs with complex lipid compositions.

Enhancing the Cellular Uptake and Antibacterial Activity of Rifampicin through Encapsulation in Mesoporous Silica Nanoparticles.

An urgent demand exists for the development of novel delivery systems that efficiently transport antibacterial agents across cellular membranes for the eradication of intracellular pathogens. In this study, the clinically relevant poorly water-soluble antibiotic, rifampicin, was confined within mesoporous silica nanoparticles (MSN) to investigate their ability to serve as an efficacious nanocarrier system against small colony variants of Staphylococcus aureus (SCV S. aureus) hosted within Caco-2 cells. The surface chemistry and particle size of MSN were varied through modifications during synthesis, where 40 nm particles with high silanol group densities promoted enhanced cellular uptake. Extensive biophysical analysis was performed, using quartz crystal microbalance with dissipation (QCM-D) and total internal reflection fluorescence (TIRF) microscopy, to elucidate the mechanism of MSN adsorption onto semi-native supported lipid bilayers (snSLB) and, thus, uncover potential cellular uptake mechanisms of MSN into Caco-2 cells. Such studies revealed that MSN with reduced silanol group densities were prone to greater particle aggregation on snSLB, which was expected to restrict endocytosis. MSN adsorption and uptake into Caco-2 cells correlated well with antibacterial efficacy against SCV S. aureus, with 40 nm hydrophilic particles triggering a ~2.5-log greater reduction in colony forming units, compared to the pure rifampicin. Thus, this study provides evidence for the potential to design silica nanocarrier systems with controlled surface chemistries that can be used to re-sensitise intracellular bacteria to antibiotics by delivering them to the site of infection.

Molecular Lipid Films on Microengineering Materials.

In this study, we have systematically investigated the formation of molecular phospholipid films on a variety of solid substrates fabricated from typical surface engineering materials and the fluidic properties of the lipid membranes formed on these substrates. The surface materials comprise of borosilicate glass, mica, SiO2, Al (native oxide), Al2O3, TiO2, ITO, SiC, Au, Teflon AF, SU-8, and graphene. We deposited the lipid films from small unilamellar vesicles (SUVs) by means of an open-space microfluidic device, observed the formation and development of the films by laser scanning confocal microscopy, and evaluated the mode and degree of coverage, fluidity, and integrity. In addition to previously established mechanisms of lipid membrane-surface interaction upon bulk addition of SUVs on solid supports, we observed nontrivial lipid adhesion phenomena, including reverse rolling of spreading bilayers, spontaneous nucleation and growth of multilamellar vesicles, and the formation of intact circular patches of double lipid bilayer membranes. Our findings allow for accurate prediction of membrane-surface interactions in microfabricated devices and experimental environments where model membranes are used as functional biomimetic coatings.

Spatiotemporal Kinetics of Supported Lipid Bilayer Formation on Glass via Vesicle Adsorption and Rupture.

Supported lipid bilayers (SLBs) represent one of the most popular mimics of the cell membrane. Herein, we have used total internal reflection fluorescence microscopy for in-depth characterization of the vesicle-mediated SLB formation mechanism on a common silica-rich substrate, borosilicate glass. Fluorescently labeling a subset of vesicles allowed us to monitor the adsorption of individual labeled vesicles, resolve the onset of SLB formation from small seeds of SLB patches, and track their growth via SLB-edge-induced autocatalytic rupture of adsorbed vesicles. This made it possible to perform the first quantitative measurement of the SLB front velocity, which is shown to increase up to 1 order of magnitude with time. This effect can be classified as dramatic because in many other physical, chemical, or biological kinetic processes the front velocity is either constant or decreasing with time. The observation was successfully described with a theoretical model and Monte Carlo simulations implying rapid local diffusion of lipids upon vesicle rupture.