Investigation of Self-Emulsifying Drug-Delivery System Interaction with a Biomimetic Membrane under Conditions Relevant to the Small Intestine

Self-emulsifying drug-delivery systems (SEDDS) have been extensively shown to increase oral absorption of solvation-limited compounds. However, there has been little clinical and commercial use of these formulations, in large part because the demonstrated advantages of SEDDS have been outweighed by our inability to precisely predict drug absorption from SEDDS using current in vitro assays. To overcome this limitation and increase the biological relevancy of in vitro assays, an absorption function can be incorporated using biomimetic membranes. However, the effects that SEDDS have on the integrity of a biomimetic membrane are not known. In this study, a quartz crystal microbalance with dissipation monitoring and total internal reflection fluorescence microscopy were employed as complementary methods to in vitro lipolysis-permeation assays to characterize the interaction of various actively digested SEDDS with a liquescent artificial membrane comprising lecithin in dodecane (LiDo). Observations from surface analysis showed that interactions between the digesting SEDDS and LiDo membrane coincided with inflection points in the digestion profiles. Importantly, no indications of membrane damage could be observed, which was supported by flux profiles of the lipophilic model drug felodipine (FEL) and impermeable marker Lucifer yellow on the basal side of the membrane. There was a correlation between the digestion kinetics of the SEDDS and the flux of FEL, but no clear correlation between solubilization and absorption profiles. Membrane interactions were dependent on the composition of lipids within each SEDDS, with the more digestible lipids leading to more pronounced interactions, but in all cases, the integrity of the membrane was maintained. These insights demonstrate that LiDo membranes are compatible with in vitro lipolysis assays for improving predictions of drug absorption from lipid-based formulations

Fast and Accurate Nanoparticle Characterization Using Deep-Learning-Enhanced Off-Axis Holography

Characterization of suspended nanoparticles in their native environment plays a central role in a wide range of fields, from medical diagnostics and nanoparticleenhanced drug delivery to nanosafety and environmental nanopollution assessment. Standard optical approaches for nanoparticle sizing assess the size via the diffusion constant and, as a consequence, require long trajectories and that the medium has a known and uniform viscosity. However, in most biological applications, only short trajectories are available, while simultaneously, the medium viscosity is unknown and tends to display spatiotemporal variations. In this work, we demonstrate a label-free method to quantify not only size but also refractive index of individual subwavelength particles using 2 orders of magnitude shorter trajectories than required by standard methods and without prior knowledge about the physicochemical properties of the medium. We achieved this by developing a weighted average convolutional neural network to analyze holographic images of single particles, which was successfully applied to distinguish and quantify both size and refractive index of subwavelength silica andpolystyrene particles without prior knowledge of solute viscosity or refractive index. We further demonstrate how these features make it possible to temporally resolve aggregation dynamics of 31 nm polystyrene nanoparticles, revealing previously unobserved time-resolved dynamics of the monomer number and fractal dimension of individual subwavelength aggregates

Low-temperature fabrication and characterization of a symmetric hybrid organic–inorganic slab waveguide for evanescent light microscopy

Organic and inorganic solid materials form the building blocks for most of today\u27s high-technological instruments and devices. However, challenges related to dissimilar material properties have hampered the synthesis of thin-film devices comprised of both organic and inorganic films. We here give a detailed description of a carefully optimized processing protocol used for the construction of a three-layered hybrid organic–inorganic waveguide-chip intended for combined scattering and fluorescence evanescent-wave microscopy in aqueous environments using conventional upright microscopes. An inorganic core layer (SiO2 or Si3N4), embedded symmetrically in an organic cladding layer (CYTOP), aids simple, yet efficient in-coupling of light, and since the organic cladding layer is refractive index matched to water, low stray-light (background) scattering of the propagating light is ensured. Another major advantage is that the inorganic core layer makes the chip compatible with multiple well-established surface functionalization schemes that allows for a broad range of applications, including detection of single lipid vesicles, metallic nanoparticles or cells in complex environments, either label-free—by direct detection of scattered light—or by use of fluorescence excitation and emission. Herein, focus is put on a detailed description of the fabrication of the waveguide-chip, together with a fundamental characterization of its optical properties and performance, particularly in comparison with conventional epi illumination. Quantitative analysis of images obtained from both fluorescence and scattering intensities from surface-immobilized polystyrene nanoparticles in suspensions of different concentrations, revealed enhanced signal-to-noise and signal-to-background ratios for the waveguide illumination compared to the epi-illumination

Label-free spatio-temporal monitoring of cytosolic mass, osmolarity, and volume in living cells

Microorganisms adapt their biophysical properties in response to changes in their local environment. However, quantifying these changes at the single-cell level has only recently become possible, largely relying on fluorescent labeling strategies. In this work, we utilize yeast (Saccharomyces cerevisiae) to demonstrate label-free quantification of changes in both intracellular osmolarity and macromolecular concentration in response to changes in the local environment. By combining a digital holographic microscope with a millifluidic chip, the temporal response of cellular water flux was successfully isolated from the rate of production of higher molecular weight compounds, in addition to identifying the produced compounds in terms of the product of their refractive index increment (dndc) and molar mass. The ability to identify, quantify and temporally resolve multiple biophysical processes in living cells at the single cell level offers a crucial complement to label-based strategies, suggesting broad applicability in studies of a wide-range of cellular processes

Fluorescence excitation enhancement by waveguiding nanowires

The optical properties of vertical semiconductor nanowires can allow an enhancement of fluorescence from surface-bound fluorophores, a feature proven useful in biosensing. One of the contributing factors to the fluorescence enhancement is thought to be the local increase of the incident excitation light intensity in the vicinity of the nanowire surface, where fluorophores are located. However, this effect has not been experimentally studied in detail to date. Here, we quantify the excitation enhancement of fluorophores bound to a semiconductor nanowire surface by combining modelling with measurements of fluorescence photobleaching rate, indicative of the excitation light intensity, using epitaxially grown GaP nanowires. We study the excitation enhancement for nanowires with a diameter of 50-250 nm and show that excitation enhancement reaches a maximum for certain diameters, depending on the excitation wavelength. Furthermore, we find that the excitation enhancement decreases rapidly within tens of nanometers from the nanowire sidewall. The results can be used to design nanowire-based optical systems with exceptional sensitivities for bioanalytical applications

Single-vesicle imaging reveals lipid-selective and stepwise membrane disruption by monomeric α-synuclein

The interaction of the neuronal protein α-synuclein with lipid membranes appears crucial in the context of Parkinson\u27s disease, but the underlying mechanistic details, including the roles of different lipids in pathogenic protein aggregation and membrane disruption, remain elusive. Here, we used single-vesicle resolution fluorescence and label-free scattering microscopy to investigate the interaction kinetics of monomeric α-synuclein with surface-tethered vesicles composed of different negatively charged lipids. Supported by a theoretical model to account for structural changes in scattering properties of surface-tethered lipid vesicles, the data demonstrate stepwise vesicle disruption and asymmetric membrane deformation upon α-synuclein binding to phosphatidylglycerol vesicles at protein concentrations down to 10 nM (∼100 proteins per vesicle). In contrast, phosphatidylserine vesicles were only marginally affected. These insights into structural consequences of α-synuclein interaction with lipid vesicles highlight the contrasting roles of different anionic lipids, which may be of mechanistic relevance for both normal protein function (e.g., synaptic vesicle binding) and dysfunction (e.g., mitochondrial membrane interaction)

Time-Resolved and Label-Free Evanescent Light-Scattering Microscopy for Mass Quantification of Protein Binding to Single Lipid Vesicles

In-depth understanding of the intricate interactions between biomolecules and nanoparticles is hampered by a lack of analytical methods providing quantitative information about binding kinetics. Herein, we demonstrate how label-free evanescent light-scattering microscopy can be used to temporally resolve specific protein binding to individual surface-bound (∼100 nm) lipid vesicles. A theoretical model is proposed that translates protein-induced changes in light-scattering intensity into bound mass. Since the analysis is centered on individual lipid vesicles, the signal from nonspecific protein binding to the surrounding surface is completely avoided, offering a key advantage over conventional surface-based techniques. Further, by averaging the intensities from less than 2000 lipid vesicles, the sensitivity is shown to increase by orders of magnitude. Taken together, these features provide a new avenue in studies of protein-nanoparticle interaction, in general, and specifically in the context of nanoparticles in medical diagnostics and drug delivery

Control of Polymer Brush Morphology, Rheology, and Protein Repulsion by Hydrogen Bond Complexation

Polymer brushes are widely used to alter the properties of interfaces. In particular, poly(ethylene glycol) (PEG) and similar polymers can make surfaces inert toward biomolecular adsorption. Neutral hydrophilic brushes are normally considered to have static properties at a given temperature. As an example, PEG is not responsive to pH or ionic strength. Here we show that, by simply introducing a polymeric acid such as poly(methacrylic acid) (PMAA), the highly hydrated brush barrier can change its properties entirely. This is caused by multivalent hydrogen bonds in an extremely pH-sensitive process. Remarkably, it is sufficient to reduce the pH to 5 for complexation to occur at the interface, which is two units higher than in the corresponding bulk systems. Below this critical pH, PMAA starts to bind to PEG in large amounts (comparable to the PEG amount), causing the brush to gradually compact and dehydrate. The brush also undergoes major rheology changes, from viscoelastic to rigid. Furthermore, the protein repelling ability of PEG is lost after reaching a threshold in the amount of PMAA bound. The changes in brush properties are tunable and become more pronounced when more PMAA is bound. The initial brush state is fully recovered when releasing PMAA by returning to physiological pH. Our findings are relevant for many applications involving functional interfaces, such as capture-release of biomolecules

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