Characterization of Nanohybridosomes from Lipids and Spruce Homogenate Containing Extracellular Vesicles

Abstract

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 R_h (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)centrifugation.

Keywords:

• hybridosomes
• liposomes
• nanovesicles
• extracellular particles
• small cellular particles
• drug delivery

Disclosure

The authors report no conflicts of interest in this work.

Additional information

Funding

This research was funded by Slovenian Research Agency, grant numbers P2-0232, P3-0388, P1-0201, P1-0391, J3-3066, L3-2621, J2-4447, J2-4427 and IO-0003. Project no. SNN 138407 has been implemented with the support provided by the Ministry of Innovation and Technology of Hungary from the National Research, Development and Innovation Fund, financed under the SNN_21 funding scheme. The first and the second authors (Vesna Spasovski and Anna Romolo) contributed equally to the work. The last two authors (Ksenija Kogej and Veronika Kralj-Iglič contributed equally to the work.