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

Measuring the size of the lipid vesicles made with extrusion

Študije veziklov so pomembne za boljše razumevanje delovanja organizmov, saj imajo vezikli številne pomembne naloge. Med drugim skrbijo za medcelično komunikacijo in transport snovi. Vezikli so sestavljeni iz lipidnega dvosloja, v katerem je navadno ujeta tekočina skupaj z makromolekulami kot so proteini in RNA. V zadnjih letih se znanstveniki veliko ukvarjajo s pripravo umetnih veziklov ali liposomov, ki jih lahko izkoriščamo za različne namene. Ena od tehnik priprave velikih umetnih veziklov, zgrajenih iz enega lipidnega dvosloja, je ekstruzija. V diplomskem delu smo se posvetili veziklom iz treh različnih lipidov: iz jajčnega lecitina, lipida 1,2-dioleoil-sn-glicero-3-fosfatidiletanolamin ter mešanice lipidov 1,2-dioleoil-sn-glicero-3-fosfatidiletanolamin in 1,2-di-palmitoil-sn-glicero-3-fosfatidilholin. S pomočjo ekstruzije smo pripravili vezikle različnih velikosti lipida 1,2-dioleoil-sn-glicero-3-fosfatidiletanolamin. Velikost veziklov smo določali z metodo dinamičnega sipanja svetlobe, kjer po Stokes-Einsteinovi enačbi preko izmerjenega translacijskega difuzijskega koeficienta izračunamo hidrodinamski radij delcev. Po prvi ekstruziji so bile vse izmerjene velikosti radijev delcev občutno prevelike, kar smo pripisali nečistočam v ekstrudorju. Ostanek jajčnega lecitina smo zato ponovno ekstrudirali z drugim ekstrudorjem in dobili vezikle željene velikosti, kljub temu pa se pri ponovnih ekstruzijah lipida 1,2-dioleoil-sn-glicero-3-fosfatidiletanolamin velikosti delcev niso skladale z velikostjo por uporabljenega filtra za ekstruzijo. Po ponovni ekstruziji že ekstrudiranih vzorcev se je namreč vzpostavila približno enaka velikost hidrodinamskega radija ne glede na uporabljen filter.Studies of vesicles are important to better understand the functioning of living organisms, as they have a wide range of important roles like cell-cell communication and transport of substances. They are composed of a phospholipid bilayer consisted of water and macromolecules like proteins and RNA. Lately scientists have been working on preparation of artificial vesicles or liposomes which can be used for many different purposes. One of the techniques to prepare large vesicles formed from one lipid bilayer is the extrusion technique. We used extrusion for liposomes preparation with the aim of synthesizing vesicles of different sizes, composed of three different phospholipid bilayers. For the structure of phospholipid bilayers, we used natural or synthetic surfactants which reduce the surface tension. We made vesicles from egg lecithin, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine lipid, and a mixture of the 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine lipids. We measured the size of vesicles via dynamic light scattering and thus we were able to calculate the hydrodynamic radius of the particles with the help of measured translation diffusion coefficient (using the Stokes-Einstein equation). According to the first extrusion, all the measured sizes of the particles were too large, presumably due to the impurities in the extruder. We have repeated extrusion with the egg lecithin residues using another cleaner extruder. The vesicles were of the right size. However, with repeated extrusions of 2-dioleoyl-sn-glycero-3-phosphoethanolamine lipid, sizes of vesicles did not agree with the pore size of filters that were used for the extrusion. After another extrusion of samples that had already been extruded, the hydrodynamic radius of vesicles wase almost the same, irrespective of the filter used

Stability of artificial and natural vesicles

V organizmih ves čas poteka medcelična komunikacija pri kateri sodelujejo zunajcelični vezikli. Preko veziklov se prenašajo nukleinske kisline, proteini in različni metaboliti. Interakcije veziklov s tarčnimi celicami imajo različne posledice, kot sta na primer zaviranje tumorske aktivnosti in stimulacija signalnih poti. Umetne vezikle lahko enostavno pripravimo, hkrati pa njihove lastnosti omogočajo raznoliko uporabo. Med drugim jih uporabljamo kot sistem za dostavo zdravil in cepiv ter za prognostiko in diagnostiko številnih bolezni. V magistrskem delu smo s pomočjo ekstruzije pripravili umetne vezikle iz 1-palmitoil-2-oleoil-sn-glicero-3-fosfoholin lipida (POPC), ki smo jim dodajali po naboju različne surfaktante in polielektrolite za proučitev vpliva teh spojin na njihovo stabilnost. Spremljali smo vpliv surfaktantov in polielektrolitov na velikost in obliko veziklov, ter njihovo stabilnost pri različnih temperaturah. Velikost veziklov, intenziteto sipanja svetlobe in njihovo temperaturno stabilnost smo zasledovali z metodo dinamičnega sipanja svetlobe. Ko smo raztopini POPC veziklov dodali kationski surfaktant N-cetilpiridinijev klorid (CPC), anionski surfaktant natrijevega dodecil sulfata (SDS), ali neionski surfaktant Triton X-100 vse do kritične micelne koncentracije (cmc) surfaktanta, ni prišlo do velikih sprememb v velikosti hidrodinamskega radija (Rh) in intenziteti sipane svetlobe. Po preseženi cmc pa je v primeru dodatkov vseh surfaktantov intenziteta močno padla, porazdelitev delcev v suspenzijah pa je postala vedno bolj polidisperzna. Ob dodatkih polielektrolitov ni prišlo do večjih sprememb v velikosti Rh in intenziteti sipanja. Vezikli POPC so bili kljub dodatkom temperaturno izjemno stabilni. Z naraščanjem temperature je prišlo le do razširitve v razporeditvi Rh. S kalorimetrijo smo spremljali toplotne efekte vezave surfaktantov in polielektrolitov na vezikle POPC. Pri vezavah vseh treh surfaktantov na POPC so toplotni efekti eksotermni, pri vezavah polielektrolitov pa so toplotni efekti zanemarljivo majhni. Vezikle smo naredili še na enostavnejši način, to je z enostavnim mešanjem komercialnih lipidov (sojinega in jajčnega lecitina), ter preučevali, kako različne snovi vplivajo na spontano tvorbo hibridnih veziklov ali hibridosomov in njihovo stabilnost. Pri enostavnejšem načinu priprave veziklov ekstruzije nismo uporabili. Hibridosomi so nastali tako v odsotnosti kot tudi v prisotnosti glicerola. Glede na rezultate sklepamo, da glicerol prispeva k izoblikovanju veziklov in bolj homogenim lipidnim strukturam. Zanimalo nas je, kako vplivamo na tvorbo hibridosomov, če mešanici namesto vode dodamo ekstrakt iz smrekovih iglic, ki vsebujejo številne zdravilne učinkovine. Hibridosomi so nastali tudi v tem primeru, do razlik je prišlo le v intenziteti sipanja in velikosti hidrodinamskega radija.In organisms, intercellular communication takes place all the time via extracellular vesicles. Nucleic acids, proteins and various metabolites are transported via vesicles. Interactions of vesicles with target cells have various consequences, such as inhibition of tumour activity and stimulation of signalling pathways. Artificial vesicles can be easily prepared and their properties allow for a wide range of applications. They are used, among other things, as a delivery system for medicines and vaccines, and for the prognosis and diagnosis of many diseases. In the master thesis, artificial vesicles were prepared from 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine lipid (POPC) by extrusion, to which different surfactants and polyelectrolytes were added according to their charge in order to study the effect of these compounds on their stability. The influence of surfactants and polyelectrolytes on the size and shape of vesicles and their stability at different temperatures was monitored. The size of the vesicles, the light scattering (LS) intensity of vesicle suspensions and their temperature stability were determined using the dynamic light scattering method. When the cationic surfactant N-cetylpyridinium chloride (CPC), the anionic surfactant sodium dodecyl sulfate (SDS), or the nonionic surfactant Triton X-100 were added to the solution of POPC vesicles up to the critical micelle concentration (cmc) of the surfactant, there were no large changes in the size of the hydrodynamic radius (Rh) and intensities. After the cmc was exceeded, however, in the case of additions of all surfactants, the intensity dropped sharply, the samples became more and more polydisperse. With the addition of polyelectrolytes, there were no major changes in Rh size and intensity. Despite the additions, the POPC vesicles were extremely stable with respect to temperature increase. The thermal effects of surfactants and polyelectrolytes binding to POPC vesicles were monitored by calorimetry. In case of binding all three surfactants to POPC, the thermal effects were exothermic, while when binding polyelectrolytes, the thermal effects were negligible. We prepared the vesicles in an even simpler way, by simply mixing commercial lipids (soy and egg lecithin), and studied how different substances affect the spontaneous formation of hybrid vesicles or hybridosomes and their stability. In this simpler method of preparing vesicles, we did not use extrusion. Hybridosomes were formed both in the absence and presence of glycerol. Based on the results, we conclude that glycerol contributes to the formation of vesicles and more homogeneous lipid structures. We were interested in how we influence the formation of hybridosomes if, instead of water, an extract from spruce needles, which contain many active ingredients, is added to the mixture. Hybridosomes were formed also in this case, the differences were only in the scattering intensity and the size of the hydrodynamic radius