Influence of fluorophore and linker length on the localization and trafficking of fluorescent sterol probes

Fluorescent sterol probes, comprising a fluorophore connected to a sterol backbone by means of a linker, are promising tools for enabling high-resolution imaging of intracellular cholesterol. In this study, we evaluated how the size of the linker, site of its attachment and nature of the fluorophore, affect the localization and trafficking properties of fluorescent sterol probes. Varying lengths of linker using the same fluorophore affected cell penetration and retention in specific cell compartments. A C-4 linker was confirmed as optimal. Derivatives of heterocyclic sterol precursors attached with identical C-4 linker to different fluorophores at diverse positions also showed significant differences in their binding properties to various intracellular compartments and kinetics of trafficking. Two novel red-emitting probes with good cell permeability, fast intracellular labelling and slightly different distribution displayed very promising characteristics for sterol probes. These probes also strongly labelled endo/lysosomal compartment in cells with pharmacologically disrupted cholesterol transport, or with a genetic mutation of cholesterol transporting protein NPC1, that overlapped with filipin staining of cholesterol. Overall, the present study demonstrates that the physicochemical properties of the fluorophore/linker pairing determine the kinetics of uptake and distribution and subsequently influence the applicability of final probes.

Tuning cell behavior with nanoparticle shape

We investigated how the shape of polymeric vesicles, made by the exact same material, impacts the replication activity and metabolic state of both cancer and non-cancer cell types. First, we isolated discrete geometrical structures (spheres and tubes) from a heterogeneous sample using density-gradient centrifugation. Then, we characterized the cellular internalization and the kinetics of uptake of both types of polymersomes in different cell types (either cancer or non-cancer cells). We also investigated the cellular metabolic response as a function of the shape of the structures internalized and discovered that tubular vesicles induce a significant decrease in the replication activity of cancer cells compared to spherical vesicles. We related this effect to the significant up-regulation of the tumor suppressor genes p21 and p53 with a concomitant activation of caspase 3/7. Finally, we demonstrated that combining the intrinsic shape-dependent effects of tubes with the delivery of doxorubicin significantly increases the cytotoxicity of the system. Our results illustrate how the geometrical conformation of nanoparticles could impact cell behavior and how this could be tuned to create novel drug delivery systems tailored to specific biomedical application.

The different ways to chitosan/hyaluronic acid nanoparticles: templated vs direct complexation. Influence of particle preparation on morphology, cell uptake and silencing efficiency

This study is about linking preparative processes of nanoparticles with the morphology of the nanoparticles and with their efficiency in delivering payloads intracellularly. The nanoparticles are composed of hyaluronic acid (HA) and chitosan; the former can address a nanoparticle to cell surface receptors such as CD44, the second allows both for entrapment of nucleic acids and for an endosomolytic activity that facilitates their liberation in the cytoplasm. Here, we have systematically compared nanoparticles prepared either A) through a two-step process based on intermediate (template) particles produced via ionotropic gelation of chitosan with triphosphate (TPP), which are then incubated with HA, or B) through direct polyelectrolyte complexation of chitosan and HA. Here we demonstrate that HA is capable to quantitatively replace TPP in the template process and significant aggregation takes place during the TPP–HA exchange. The templated chitosan/HA nanoparticles therefore have a mildly larger size (measured by dynamic light scattering alone or by field flow fractionation coupled to static or dynamic light scattering), and above all a higher aspect ratio (R g/R H) and a lower fractal dimension. We then compared the kinetics of uptake and the (antiluciferase) siRNA delivery performance in murine RAW 264.7 macrophages and in human HCT-116 colorectal tumor cells. The preparative method (and therefore the internal particle morphology) had little effect on the uptake kinetics and no statistically relevant influence on silencing (templated particles often showing a lower silencing). Cell-specific factors, on the contrary, overwhelmingly determined the efficacy of the carriers, with, e.g., those containing low-MW chitosan performing better in macrophages and those with high-MW chitosan in HCT-116.

Uptake and metabolism of β-apo-8′-carotenal, β-apo-10′-carotenal, and β-apo-13-carotenone in Caco-2 cells

β-Apocarotenoids are eccentric cleavage products of carotenoids formed by chemical and enzymatic oxidations. They occur in foods containing carotenoids and thus might be directly absorbed from the diet. However, there is limited information about their intestinal absorption. The present research examined the kinetics of uptake and metabolism of β-apocarotenoids. Caco-2 cells were grown on 6-well plastic plates until a differentiated cell monolayer was achieved. β-Apocarotenoids were prepared in Tween 40 micelles, delivered to differentiated cells in serum-free medium, and incubated at 37°C for up to 8 h. There was rapid uptake of β-apo-8′-carotenal into cells, and β-apo-8′-carotenal was largely converted to β-apo-8′-carotenoic acid and a minor metabolite that we identified as 5,6-epoxy-β-apo-8′-carotenol. There was also rapid uptake of β-apo-10′-carotenal into cells, and β-apo-10′-carotenal was converted into a major metabolite identified as 5,6-epoxy-β-apo-10′-carotenol and a minor metabolite that is likely a dihydro-β-apo-10′-carotenol. Finally, there was rapid cellular uptake of β-apo-13-carotenone, and this compound was extensively degraded. These results suggest that dietary β-apocarotenals are extensively metabolized in intestinal cells via pathways similar to the metabolism of retinal. Thus, they are likely not absorbed directly from the diet.

Long-Term Ecological Research in the Arctic: Where Science Never Sleeps

When the Arctic (ARC) Long-Term Ecological Research (LTER) project began, I was an aquatic ecologist with experience in managing large projects in freshwaters and estuaries and a specialization in microbes. This project, which studies lakes, streams, and tundras, has greatly increased my breadth as an ecologist and allowed me to take part in terrestrial modeling, microbial studies in streams, and the role of soil mycorrhizal fungi in providing nutrients to many species of plants. As a mentor to several postdoctoral fellows, my LTER research has enabled me to learn about other fields such as the application of molecular biology to microbial ecology. The Arctic LTER project data, the long-term field experiments, and the facilities available at the University of Alaska field station brought me in contact with ecologists from many countries. One result of this association with experts was my coauthorship of a book on Arctic natural history aimed at communicating scientific knowledge to scientists and the general public unfamiliar with the Arctic (Huryn and Hobbie 2012). I have always collaborated extensively with many scientists and encouraged collaboration as the best way to carry out ecosystem research. The Arctic LTER project brought many opportunities to broaden the scope of my collaboration to include terrestrial ecologists and microbiologists. My PhD research was about year-round primary productivity of an Arctic lake but while on a postdoctoral fellowship at Uppsala University, Sweden, I switched to an emphasis on bacterial uptake kinetics in lakes. The techniques I helped develop in freshwater worked in the ocean and estuaries too (Hobbie and Williams 1984). In addition we developed the epifluorescence method for quantifying the abundance of planktonic bacteria. Our paper (Hobbie, Daley, and Jasper 1977) finally convinced oceanographers that bacteria are abundant (at 10⁹ per liter) and important. Recently, I have used my understanding of kinetics of uptake to analyze microbial activity in the soil. My Arctic expertise led to leadership of the aquatic part of the International Biological Program (IBP) at Barrow, Alaska, beginning in 1970. We (28 scientists, graduate students, and postdoctoral fellows) studied shallow ponds to quantify the carbon, nitrogen, and phosphorus cycles.

Molecular diffusion in the human nail measured by stimulated Raman scattering microscopy

The effective treatment of diseases of the nail remains an important unmet medical need, primarily because of poor drug delivery. To address this challenge, the diffusion, in real time, of topically applied chemicals into the human nail has been visualized and characterized using stimulated Raman scattering (SRS) microscopy. Deuterated water (D2O), propylene glycol (PG-d8), and dimethyl sulphoxide (DMSO-d6) were separately applied to the dorsal surface of human nail samples. SRS microscopy was used to image D2O, PG-d8/DMSO-d6, and the nail through the O-D, -CD2, and -CH2 bond stretching Raman signals, respectively. Signal intensities obtained were measured as functions of time and of depth into the nail. It was observed that the diffusion of D2O was more than an order of magnitude faster than that of PG-d8 and DMSO-d6. Normalization of the Raman signals, to correct in part for scattering and absorption, permitted semiquantitative analysis of the permeation profiles and strongly suggested that solvent diffusion diverged from classical behavior and that derived diffusivities may be concentration dependent. It appeared that the uptake of solvent progressively undermined the integrity of the nail. This previously unreported application of SRS has permitted, therefore, direct visualization and semiquantitation of solvent penetration into the human nail. The kinetics of uptake of the three chemicals studied demonstrated that each altered its own diffusion in the nail in an apparently concentration-dependent fashion. The scale of the unexpected behavior observed may prove beneficial in the design and optimization of drug formulations to treat recalcitrant nail disease.