Supported Lipid Membranes and Their Use for the Characterization of Biological Nanoparticles

Biological nanoparticles (BNPs) are nano-sized lipid vesicles of biological origin, which are involved in multiple biological processes. BNP characterization techniques are critical for improving the understanding of how these particles contribute to cellular communication, viral infections and drug-delivery applications. However, due to their small size (between 50 and 200 nm in diameter) and molecular heterogeneity, quantitative characterization of their physical, chemical and biological properties is demanding, especially since their large structural and compositional heterogeneity calls for methods with single nanoparticle resolution. To address this challenge, work in this thesis has been focused on investigating and using supported lipid bilayers (SLBs) and their two-dimensional fluidity as a platform for nanoparticle characterization. To investigate SLBs, we combined confocal microscopy with microfluidics to identify the mechanisms by which lipid vesicles are spontaneously converted into various types of planar membranes on a multitude of surfaces (Paper\ua0I) and found that most of the studied materials can support lipid film formation. In the context of SLB formation, specific focus was put on using total internal reflection fluorescence (TIRF) microscopy to monitor the kinetics of vesicle adsorption, rupture and spreading of individual SLB patches on glass (Paper\ua0II), revealing that the SLB formation process was driven by the autocatalytic growth and merger of multiple small SLB patches at appreciably high vesicle coverage. TIRF was also successfully employed to monitor lipid-enveloped drug permeation through an SLB formed on a mesoporous silica thin film (Paper III). The insights gained from investigating SLBs was also used for in depth characterization of BNPs using the surface-based flow-nanometry method, allowing for independent determination of size and fluorescence emission of individual BNPs tethered to a laterally fluid SLB formed on the floor of a microfluidic channel. This way we could demonstrate that the fluorescence emission from lipophilic dyes depends in a non-trivial way on nanoparticle size, and varies significantly between the different types of BNPs (Paper IV). The flow-nanometry concept was also used to elucidate the effect of vesicle size on their diffusivity on the SLB in the limit of few tethers (Paper V). The insights gained in this thesis work on lipid self-assembly at different surfaces and the possibility to use SLBs on silica for in-depth characterization of BNPs demonstrate this as a promising approach in the field of single nanoparticle analytics, which in future work will be possible to extend into a novel means to probe interactions between BNPs and cell-membrane mimics representing a near-native situation

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

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

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. Graphical abstract : [Figure not available: see fulltext.

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

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

High throughput size-determination and multiplexed fluorescence analysis of single biological particles in a nanofluidic device

Biological nanoparticles, such as exosomes and viruses, are responsible for a multitude of important functions, but methods to characterize them on the single particle level are rare. We here present a nanofluidic platform for multi-parametric characterization of biological nanoparticles with high throughput. The device consists of feeding microchannels and an array of ~100 nanochannels where the nanoparticles can be characterized. We determine the size by analyzing the Brownian motion of the particles and quantify their content based on fluorescence imaging of up to three different colors. We successfully benchmark our method against existing techniques, such as Nanoparticle Tracking Analysis (NTA)

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