Biology subjects

Benedetti, C.

Publications and source records attributed to Benedetti, C..

2 recordsLinked to original sources

bioRxiv2024

Investigation of Lipid-PEG Anchorage of Proteins onto Mesenchymal Stem Cell Derived Extracellular Vesicles for Intracellular Delivery: A Quantitative Assessment

Despite biomolecule delivery is a natural function of Extracellular Vesicles (EVs), low loading of exogenous macromolecules such as proteins into EVs limits their interest as convincing protein delivery systems for health applications. In this context, lipid-anchorage of exogenous cargo into EV membrane recently emerged as a promising option to enable their vectorization into cells. Nevertheless, this option was not explored for protein intracellular delivery, and further characterization of critical parameters governing the association of a lipid-anchored cargo protein to EVs stills needed to confirm the relevance of this anchorage strategy. Therefore, we sought to identify these parameters in a precise and quantitative manner, using bulk and single nanoparticle analysis methods to identify protein loading capacity and subsequent intracellular delivery. We identified incubation temperature, cargo concentration, Lipid Anchor (LA) structure (lipid nature, linker) and EV origin as critical factors influencing maximal EV loading capacity. Precise control of these parameters enabled to load cargo protein close to EV saturation without hindering cellular delivery. Structural properties of LA influenced not only cargo protein/EV association, but also intracellular delivery into different carcinoma cell lines. By thoroughly characterizing Lipid-PEG-protein anchorage, this study evidences the interest of this tunable and controllable approach for efficient EV protein delivery. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=58 SRC="FIGDIR/small/610030v3_ufig1.gif" ALT="Figure 1"> View larger version (15K): org.highwire.dtl.DTLVardef@1fed681org.highwire.dtl.DTLVardef@1b4d728org.highwire.dtl.DTLVardef@1998e3aorg.highwire.dtl.DTLVardef@488be3_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2022

Alternative culture systems for bovine oocyte in vitro maturation: liquid marbles and differentially shaped 96-well plates

In vivo matured oocytes exhibit higher developmental competence than those matured in vitro, but mimicking the in vivo environment by in vitro conditions has been challenging. Till now, conventional two-dimensional (2D) systems have been used for in vitro maturation of bovine cumulus-oocytes-complexes (COCs). However, using such systems may cause cell flattening and does not allow cumulus expansion in all dimensions, which is less physiological. Therefore, implementing a low-cost and highly effective in vivo-like microenvironment methodology may help to optimize oocyte in vitro maturation. Here, we used two different systems to culture COCs and evaluate their potential influence on embryo development and quality. In the first system, we used treated fumed silica particles to create a 3D microenvironment (liquid marbles; LM) to mature COCs. In the second system, we cultured COCs in 96-well plates with different dimensions (flat, ultra-low attachment round-bottom, and V-shaped 96-well plates). In both systems, the nuclear maturation rate remained similar to the control in 2D, showing that most oocytes reached metaphase II. However, the subsequent blastocyst rate remained lower in the liquid marble system compared to 96-well plates and control 2D systems. Interestingly, a lower total cell number was found in the resulting embryos from both systems (LM and 96-well plates) compared to the control. In conclusion, oocytes matured in liquid marbles or 96-well plates showed no remarkable change in terms of meiotic resumption, embryo development, and quality in both systems. None of the surface geometries influenced embryo development. These findings provide important inferences in many aspects of oocyte and embryo development. Further investigation is needed to determine other aspects like toxicity testing and ultrastructural changes in oocytes.

developmental biology↗