7 research outputs found
Extracellular vesicles from blood plasma as mediators of anti-inflammatory effects, oxidative stress and angiogenesis in HUVEC
Introduction: Blood plasma is used in regenerative medicine for more than 30 years as it was found to have
impact on angiogenesis and proliferation of endothelial cells. This effect was initially ascribed to growth factors
and cytokines, however, particularly in the last decade researchers are focusing on platelet-derived
extracellular vesicles (PEVs) that are also present in the platelet rich plasma.
Methods: We prepared platelet and extracellular vesicles-rich plasma (PVRP) by centrifugation of the
human blood, and differential (ultra) centrifugation of PVRP to isolate PEVs. We exposed Human Umbilical Vein
Endothelial Cells (HUVEC) to 5% PEVs for 24 hours and assessed inflammation markers (Interleukin(IL)-1beta,
IL-6 and Tumor necrosis factor (TNF)-alpha using ELISA tests), oxidative stress markers (Cholinesterase
(ChE) and glutathione S-transferase (GST) activity by spectrophotometry, as well reactive oxygen species
(ROS) and lipid droplets (LD) by flow cytometry). We observed morphological changes in HUVEC indicating
angiogenesis by using optical microscopy.
Results: We found that after 24 hours caused a decrease of the concentration of IL-6, IL-1beta and TNF-alpha.
Also, ChE and GST activity and ROS count were decreased. LD production, which is triggered in need to protect
the cells from free radicals and oxidative stress damage, was however higher. Treatment of the cells had
impact on cell morphology with progressed formation of the tubes and cell connecting, which is regarded as the
beginning of HUVEC angiogenesis process.
Discussion: Beneficial effect of PVRP in healing and regeneration may include suppression of inflammation
and oxidative stress by PEVs.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
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
Autologous Platelet and Extracellular Vesicle-Rich Plasma as Therapeutic Fluid: A Review
The preparation of autologous platelet and extracellular vesicle-rich plasma (PVRP) has been explored in many medical fields with the aim to benefit from its healing potential. In parallel, efforts are being invested to understand the function and dynamics of PVRP that is complex in its composition and interactions. Some clinical evidence reveals beneficial effects of PVRP, while some report that there were no effects. To optimize the preparation methods, functions and mechanisms of PVRP, its constituents should be better understood. With the intention to promote further studies of autologous therapeutic PVRP, we performed a review on some topics regarding PVRP composition, harvesting, assessment and preservation, and also on clinical experience following PVRP application in humans and animals. Besides the acknowledged actions of platelets, leukocytes and different molecules, we focus on extracellular vesicles that were found abundant in PVRP
Autologous platelet and extracellular vesicle-rich plasma as therapeutic fluid
The preparation of autologous platelet and extracellular vesicle-rich plasma (PVRP) has been explored in many medical fields with the aim to benefit from its healing potential. In parallel, efforts are being invested to understand the function and dynamics of PVRP that is complex in its composition and interactions. Some clinical evidence reveals beneficial effects of PVRP, while some report that there were no effects. To optimize the preparation methods, functions and mechanisms of PVRP, its constituents should be better understood. With the intention to promote further studies of autologous therapeutic PVRP, we performed a review on some topics regarding PVRP composition, harvesting, assessment and preservation, and also on clinical experience following PVRP application in humans and animals. Besides the acknowledged actions of platelets, leukocytes and different molecules, we focus on extracellular vesicles that were found abundant in PVRP