pH-triggered endosomal escape of pore-forming Listeriolysin O toxin-coated gold nanoparticles

The research leading to these results has received funding from the European Research Council under the European Union's Seventh Framework Programme (ERC grant agreement n° 338133)Background: A major bottleneck in drug delivery is the breakdown and degradation of the delivery system through the endosomal/lysosomal network of the host cell, hampering the correct delivery of the drug of interest. In nature, the bacterial pathogen Listeria monocytogenes has developed a strategy to secrete Listeriolysin O (LLO) toxin as a tool to escape the eukaryotic lysosomal system upon infection, allowing it to grow and proliferate unharmed inside the host cell. Results: As a “proof of concept”, we present here the use of purifed His-LLO H311A mutant protein and its conjuga tion on the surface of gold nanoparticles to promote the lysosomal escape of 40 nm-sized nanoparticles in mouse embryonic fbroblasts. Surface immobilization of LLO was achieved after specifc functionalization of the nanoparti cles with nitrile acetic acid, enabling the specifc binding of histidine-tagged proteins. Conclusions: Endosomal acidifcation leads to release of the LLO protein from the nanoparticle surface and its self-assembly into a 300 Å pore that perforates the endosomal/lysosomal membrane, enabling the escape of nanoparticles.Depto. de Química FísicaFac. de Ciencias QuímicasTRUEUnión Europea. FP7Ministerio de Ciencia e Innovación (MICINN)Comunidad de MadridUniversidad Complutense de Madridpu

Hybridosomes from spruce needle homogenate

Introduction: Being of compatible structure with biomembranes, lipid–based nanoparticles are considered as convenient platforms for drug delivery systems. In the proposed work we considered formation of lipid nanovesicles associated with bioactive phytochemicals from spruce needle homogenate (here called hybridosomes). We formed hybridosomes by mixing appropriate amounts of lecithin, supernatant of isolation of extracellular particles from spruce needle homogenate and glycerol. Methods: We visualized hybridosomes by light microscopy and cryogenic transmission electron microscopy and assessed them by flow cytometry, dynamic light scattering, ultraviolet–visual spectroscopy and interferometric microscopy. Results: We found that the particles consisted of a bilayer membrane and a fluid-like interior. Flow cytometry and interferometric light microscopy measurements showed that the majority of the particles were nano-sized. Dynamic light scattering and interferometric light microscopy measurements agreed well with the determined average hydrodynamic radius of the particles Rh (between 140 and 180 nm) while their number densities were in the range between 10^13 and 10^14/mL indicating that hybridosomes present about 2/3 of the mixture, excluding solvent and other small molecules. Discussion: Simple and low-cost preparation method, non-demanding saving process and efficient formation procedure suggest that large scale production of hybridosomes from lipids and spruce needle homogenate is feasible.Small New World 2.0 4-5 September 2023., Graz, Austri

Hybridosomes from spruce needle homogenate

Introduction: Being of compatible structure with biomembranes, lipid–based nanoparticles are considered as convenient platforms for drug delivery systems. In the proposed work we considered formation of lipid nanovesicles associated with bioactive phytochemicals from spruce needle homogenate (here called hybridosomes). We formed hybridosomes by mixing appropriate amounts of lecithin, supernatant of isolation of extracellular particles from spruce needle homogenate and glycerol. Methods: We visualized hybridosomes by light microscopy and cryogenic transmission electron microscopy and assessed them by flow cytometry, dynamic light scattering, ultraviolet–visual spectroscopy and interferometric microscopy. Results: We found that the particles consisted of a bilayer membrane and a fluid-like interior. Flow cytometry and interferometric light microscopy measurements showed that the majority of the particles were nano-sized. Dynamic light scattering and interferometric light microscopy measurements agreed well with the determined average hydrodynamic radius of the particles Rh (between 140 and 180 nm) while their number densities were in the range between 10^13 and 10^14/mL indicating that hybridosomes present about 2/3 of the mixture, excluding solvent and other small molecules. Discussion: Simple and low-cost preparation method, non-demanding saving process and efficient formation procedure suggest that large scale production of hybridosomes from lipids and spruce needle homogenate is feasible.Small New World 2.0 4-5 September 2023., Graz, Austri

Characterization of Nanohybridosomes from Lipids and Spruce Homogenate Containing Extracellular Vesicles

Introduction: Lipid nanovesicles associated with bioactive phytochemicals from spruce needle homogenate (here called nano-sized hybridosomes or nanohybridosomes, NSHs) were considered. Methods: We formed NSHs by mixing appropriate amounts of lecithin, glycerol and supernatant of isolation of extracellular vesicles from spruce needle homogenate. We visualized NSHs by light microscopy and cryogenic transmission electron microscopy and assessed them by flow cytometry, dynamic light scattering, ultraviolet–visual spectroscopy, interferometric light microscopy and liquid chromatography–mass spectrometry. Results: We found that the particles consisted of a bilayer membrane and a fluid-like interior. Flow cytometry and interferometric light microscopy measurements showed that the majority of the particles were nano-sized. Dynamic light scattering and interferometric light microscopy measurements agreed well on the average hydrodynamic radius of the particles Rh (between 140 and 180 nm), while the concentrations of the particles were in the range between 1013 and 1014/mL indicating that NSHs present a considerable (more than 25%) of the sample which is much more than the yield of natural extracellular vesicles (EVs) from spruce needle homogenate (estimated less than 1%). Spruce specific lipids and proteins were found in hybridosomes. Discussion: Simple and low-cost preparation method, non-demanding saving process and efficient formation procedure suggest that large-scale production of NSHs from lipids and spruce needle homogenate is feasible. Plain Language Summary: Cells shed into their exterior nanoparticles (here referred to as extracellular vesicles – EVs) that are free to move, reach distant cells and are taken up by them. As they carry bioactive constituents, EVs may have important impact on the recipient cells. The mechanisms of EV formation and mediation can be employed in designing therapeutic, prophylactic and diagnostic methods for various medical issues. EVs can be harvested from biological samples; however, their yield is small,12 and there are potential side effects. Artificial vesicles – liposomes – have high yield; however, in vivo, they can be degraded before reaching the target and their reproducibility is yet insufficient. In order to combine advantages of both types of nanoparticles, we have composed nanohybridosomes (NSHs) from soya lecithin, water and supernatant of isolation of EVs from spruce needle homogenate, visualized them by cryogenic electron microscopy and characterized them with respect to their size, concentration and protein/nucleic acid content. We have applied a recently developed interferometric light microscopy to determine the hydrodynamic radius and the concentration of EVs. We found that the majority of composed particles are nano-sized and that they enclose more than 25% of the incoming volume of liquid, which is considerably more than about 1% that can be harvested by isolation of EVs from spruce needle homogenate by (ultra) centrifugatio

Design of protein logic gate system operating on lipid membranes

Lipid membranes are becoming increasingly popular in synthetic biology due to their biophysical properties and crucial role in communication between different compartments. Several alluring protein–membrane sensors have already been developed, whereas protein logic gates designs on membrane-embedded proteins are very limited. Here we demonstrate the construction of a two-level protein–membrane logic gate with an OR-AND logic. The system consists of an engineered pH-dependent pore-forming protein listeriolysin O and its DARPin-based inhibitor, conjugated to a lipid vesicle membrane. The gate responds to low pH and removal of the inhibitor from the membrane either by switching to a reducing environment, protease cleavage, or any other signal depending on the conjugation chemistry used for inhibitor attachment to the membrane. This unique protein logic gate vesicle system advances generic sensing and actuator platforms used in synthetic biology and could be utilized in drug delivery

Cytotoxic activity of LLO Y406A is targeted to the plasma membrane of cancer urothelial cells

Identification of novel agents for bladder cancer treatment is highly desirable due to the high incidence of tumor recurrence and the risk of progression to muscle-invasive disease. The key feature of the cholesterol-dependent toxin listeriolysin O mutant (LLO Y406A) is its preferential activity at pH 5.7, which could be exploited either directly for selective targeting of cancer cells or the release of accumulated therapeutics from acidic endosomes. Therefore, our goal was to compare the cytotoxic effect of LLO Y406A on cancer cells (RT4) and normal urothelial cells (NPU), and to identify which cell membranes are the primary target of LLO Y406A by viability assays, life-cell imaging, fluorescence, and electron microscopy. LLO Y406A decreased viability, altered cell morphology, provoked membrane blebbing, and induced apoptosis in RT4 cells, while it did not affect NPU cells. LLO Y406A did not cause endosomal escape in RT4 cells, while the plasma membrane of RT4 cells was revealed as the primary target of LLO Y406A. It has been concluded that LLO Y406A has the ability to selectively eliminate cancer urothelial cells through pore-forming activity at the plasma membrane, without cytotoxic effects on normal urothelial cells. This promising selective activity merits further testing as an anti-cancer agent