RESUMEN
Biophysical coupling between the inner and outer leaflets, known as inter-leaflet or transbilayer coupling, is a fundamental organizational principle in the plasma membranes of live mammalian cells. Lipid-based interactions between the two leaflets are proposed to be a primary mechanism underlying transbilayer coupling. However, there are only a few experimental evidence supporting the existence of such interactions in live cells. This is seemingly due to the lack of experimental strategies to perturb the lipid composition in one leaflet and quantitative techniques to evaluate the biophysical properties of the opposite leaflet. The existing strategies often dependent on immobilization and clustering a component in one of the leaflets and technically demanding biophysical tools to evaluate the effects on the opposing leaflet. In the recent years, the London group developed a simple but elegant method, namely methyl-alpha-cyclodextrin catalyzed lipid exchange (LEX), to efficiently exchange outer leaflet lipids with an exogenous lipid of choice. Here, we adopted this method to perturb outer leaflet lipid composition. The corresponding changes in the inner leaflet is evaluated by comparing the diffusion of lipid probes localized in this leaflet in unperturbed and perturbed conditions. We employed highly multiplexed imaging fluorescence correlation spectroscopy (ImFCS), realized in a commercially available or home-built total internal reflection fluorescence microsocope equipped with a fast and sensitive camera, to determine diffusion coefficient of the lipid probes. Using the combination of LEX and ImFCS, we directly demonstrate lipid-based transbilayer coupling that does not require immobilization of membrane components in live mast cells in resting conditions. Overall, we present a relatively straightforward experimental strategy to evaluate transbilayer coupling quantitively in live cells.
Asunto(s)
Espectrometría de Fluorescencia , Espectrometría de Fluorescencia/métodos , Animales , Membrana Dobles de Lípidos/metabolismo , Membrana Dobles de Lípidos/química , Membrana Celular/metabolismo , Membrana Celular/química , Mastocitos/metabolismo , HumanosRESUMEN
Oral delivery, while a highly desirable form of nanoparticle-drug administration, is limited by challenges associated with overcoming several biological barriers. Here, the authors study how fluorescent and poly(ethylene glycol)-coated (PEGylated) core-shell silica nanoparticles sized 5 to 50 nm interact with major barriers including intestinal mucus, intestinal epithelium, and stomach acid. From imaging fluorescence correlation spectroscopy studies using quasi-total internal reflection fluorescence microscopy, diffusion of nanoparticles through highly scattering mucus is progressively hindered above a critical hydrodynamic size around 20 nm. By studying Caco-2 cell monolayers mimicking the intestinal epithelia, it is observed that ultrasmall nanoparticles below 10 nm diameter (Cornell prime dots, [C' dots]) show permeabilities correlated with high absorption in humans from primarily enhanced passive passage through tight junctions. Particles above 20 nm diameter exclusively show active transport through cells. After establishing C' dot stability in artificial gastric juice, in vivo oral gavage experiments in mice demonstrate successful passage through the body followed by renal clearance without protein corona formation. Results suggest C' dots as viable candidates for oral administration to patients with a proven pathway towards clinical translation and may generate renewed interest in examining silica as a food additive and its effects on nutrition and health.
Asunto(s)
Portadores de Fármacos , Nanopartículas , Humanos , Ratas , Ratones , Animales , Portadores de Fármacos/química , Células CACO-2 , Ratas Sprague-Dawley , Dióxido de Silicio/química , Nanopartículas/químicaRESUMEN
Stochastic optical reconstruction microscopy (STORM) is an optical super-resolution microscopy (SRM) technique that traditionally requires toxic and non-physiological imaging buffers and setups that are not conducive to live-cell studies. It is observed that ultrasmall (<10 nm) fluorescent core-shell aluminosilicate nanoparticles (aC' dots) covalently encapsulating organic fluorophores enable STORM with a single excitation source and in a regular (non-toxic) imaging buffer. It is shown that fourfold coordinated aluminum is responsible for dye blinking, likely via photoinduced redox processes. It is demonstrated that this phenomenon is observed across different dye families leading to probes brighter and more photostable than the parent free dyes. Functionalization of aC' dots with antibodies allows targeted fixed cell STORM imaging. Finally, aC' dots enable live-cell STORM imaging providing quantitative measures of the size of intracellular vesicles and the number of particles per vesicle. The results suggest the emergence of a powerful ultrasmall, bright, and photostable optical SRM particle platform with characteristics relevant to clinical translation for the quantitative assessment of cellular structures and processes from live-cell imaging.