EXTRACELLULAR VESICLES FROM MICROALGAE

Abstract

Extracellular vesicles derived from native, photosynthetic, non-fermenting microalgae are provided. A method for isolating extracellular vesicles from native, photosynthetic, non-fermenting microalgae involving growth, centrifugation and ultracentrifugation steps is also provided. Use of the isolated extracellular vesicles as carriers for deliverling diagnostic, therapeutic, nutraceutic and/or cosmetic agents is further provided.

Claims

1. An extracellular vesicle (EV) derived from a microalga, wherein the extracellular vesicle (EV) is a biogenic lipidic membranous nanovesicle having a particle size ranging from 50 to 300 nm, being a small extracellular vesicle (sEV) or from 300 nm to 2 μm, being a large extracellular vesicle (IEV) and is comprised in a lipidic bilayer membrane, and wherein the extracellular vesicle (EV) is derived from a native, photosynthetic, non-fermenting microalga and contains at least the extracellular vesicle protein marker Alix and optionally one or more protein markers selected from the group consisting of enolase, actin and any combination thereof.

2. The extracellular vesicle (EV) of claim 1, wherein the microalga is selected from photosynthetic microalgal phyla Chlorophytes and Bacillariophytes.

3. The extracellular vesicle (EV) of claim 1, wherein the microalga is selected from the photosynthetic microalgal divisions Euglenophyta, Cryptophyta, Rhodophyta, Glaucophyta, Chromophyta and Chlorophyta.

4. The extracellular vesicle (EV) of claim 1, wherein said extracellular vesicle (EV) is used as a carrier for delivering a diagnostic, therapeutic, nutraceutic and/or cosmetic agent.

5. A method of isolating extracellular vesicles (EVs) containing at least the extracellular vesicle protein marker Alix and optionally one or more protein markers selected from the group consisting of enolase, actin and any combination thereof, from native, photosynthetic, non-fermenting microalgae, the method comprising the steps of: a) inoculating a microalgal culture medium with a native, photosynthetic, non-fermenting microalgal biomass inoculum at a final concentration of from 0.5 to 2 mg per ml of wet biomass; b) cultivating the inoculated microalgal biomass with illumination provided by a LED lighting system at about 30-200 microEinstein per second per square meter (μE m.sup.−2 s.sup.−1), whereby microalgae-derived extracellular vesicles (EVs) are produced; c) isolating a first fraction of the microalgae-derived extracellular vesicles (EVs) from the microalgae culture of step b) by a first separation step including low-velocity centrifugation, whereby a pellet containing the microalgae-derived extracellular vesicles (EVs) and a low-velocity supernatant are obtained; and d) isolating a second fraction of the microalgae-derived extracellular vesicles (EVs) from the low-velocity supernatant obtained in step c), by a second separation step including ultracentrifugation, whereby a pellet containing further microalgae-derived extracellular vesicles (EVs) is obtained.

6. The method of claim 5, wherein in step b) the inoculated microalgal biomass is cultured under controlled temperature and photoperiod, wherein the temperature is within a range of from 13 to 22° C. and the photoperiod is about 14:10 (light:dark), and for a predetermined period of time ranging from 10 to 60 days.

7. The method of claim 5, wherein in step c) the first separation step comprises at least a first low-velocity centrifugation step, a second low-velocity centrifugation step and a third low-velocity centrifugation step, each at a different centrifugation velocity comprised between 300×g and 10,000×g.

8. The method of claim 7, wherein the first low-velocity centrifugation step is performed at about 300×g, the second low-velocity centrifugation step is performed at about 2,000×g, and the third low-velocity centrifugation step is performed at about 10,000×g.

9. The method of claim 8, wherein the first low-velocity centrifugation step is performed at a temperature of about 4° C. for about 10 minutes, the second low-velocity centrifugation step is performed at a temperature of about 4° C. for about 10 minutes and the third low-velocity centrifugation step is performed at a temperature of about 4° C. for about 30 minutes.

10. The method of claim 5, wherein in step d) the second separation step is performed by ultracentrifugation at about 118,000×g.

11. The method of claim 10, wherein the ultracentrifugation is performed at a temperature of about 4° C. for about 2 hours.

12. The method of claim 5, wherein the microalgae-derived extracellular vesicles (EVs) isolated in step c) are larger in size than the further microalgae-derived extracellular vesicles (EVs) isolated in step d).

13. The method of claim 5, wherein the microalgae-derived extracellular vesicles (EVs) isolated in step c) have a particle size comprised between 50 and 300 nm, being small extracellular vesicles (sEVs) and the further microalgae-derived extracellular vesicles isolated in step d) have a particle size comprised between 300 nm and 2 μm, being large extracellular vesicles (IEVs).

Description

BRIEF DESCRIPTION OF THE FIGURES

[0031] FIG. 1 is a graph reporting the results obtained with the Diatom Odontella sp. (LA-CW-28) derived sEVs by Dynamic Light Scattering (DLS) experiments on CW-28 Nanovesicles;

[0032] FIG. 2 shows Atomic Force Microscopy (AFM) images on Chlorophyte Tetraselmis sp. (LA-CW-02) derived sEVs;

[0033] FIG. 3 shows western blot images obtained by using established antibodies; and

[0034] FIG. 4 is a graph reporting the results of experiments using Iodixanol gradient to determine the nanoalgosome density; also shown are representative immunoblot images from nanoalgosomes isolated by dUC, and loaded on iodixanol density gradient.

DETAILED DESCRIPTION

[0035] The microalgae-derived EVs of the present invention have a particle size comprised between 50 and 300 nm (small extracellular vesicles (sEV), obtainable by ultracentrifugation) or comprised between 300 nm and 2 μm (large extracellular vesicles (lEV), obtainable by low-velocity centrifugation). They contain the established extracellular vesicle protein marker Alix, and optionally the protein markers enolase and/or actin. As experimentally demonstrated by their sensitivity to the detergent sodium dodecyl sulfate (SDS), the microalgae-derived EVs of the invention are biogenic lipidic membranous nanovesicles with a lipidic bilayer membrane.

[0036] As it will be further illustrated herein below, the microalgae-derived EVs of the present invention are isolated from the conditioned media of microalgal cultures, such as, but not limited to, the marine, photosynthetic Diatom Odontella sp. or the Chlorophyte Tetraselmis sp.

[0037] The novel product is obtainable by the method of the present invention, which comprises the following steps: [0038] a) inoculating a photosynthetic microalgal culture medium with a microalgal biomass inoculum at a final concentration of from 0.5 to 2 mg per ml of wet biomass; [0039] b) cultivating the inoculated microalgal biomass with illumination provided by a LED lighting system at about 30-200 microEinstein per second and square meter (μE m.sup.−2 s.sup.−1), whereby microalgae-derived extracellular vesicles are produced; [0040] c) isolating a first fraction of microalgae-derived extracellular vesicles from the microalgae culture of step b) by a first separation step including low-velocity centrifugation, whereby a pellet containing microalgae-derived extracellular vesicles and a low-velocity supernatant are obtained; and [0041] d) isolating a second fraction of microalgae-derived extracellular vesicles from the low-velocity supernatant obtained in step c), by a second separation step including ultracentrifugation, whereby a pellet containing further microalgae-derived extracellular vesicles is obtained.

[0042] A detailed description of the isolation method of the present invention is provided herein below. The following detailed description is provided by way of illustration only and is not intended to limit the scope of the present invention, which is determined by the appended claims.

[0043] 1. Microalgal Cultivation

[0044] Step a): Inoculation

[0045] The microalgal culture media (filter-sterilized f/2 or BG11 media for marine and freshwater species) are inoculated with a microalgal biomass inoculum at a final concentration of 0.5 to 1.5 mg per ml of wet biomass at beginning of experiments (concentration in cultivation vessel on day-0) for a final volume of 0.1-15 liters in a glass flasks or cylindrical reactor. The final total volume may be scaled up and down according to the desired yield.

[0046] Step b): Growth

[0047] The inoculated microalgal biomass are maintained under controlled temperature (13-22° C., preferably 17° C.) and photoperiod conditions (14:10 light:dark) and for a predetermined period of time (10-50 days, preferably 30 days), with illumination provided by a LED lighting system at about 30-200 microEinstein per second and square meter (μE m.sup.−2 s.sup.−1) (preferably 100 μE m.sup.−2 s.sup.−1).

[0048] In a typical batch system the growth dynamics are monitored typically over a duration of 10-50 days, preferably 30 days, in the cultures using a well plate spectrophotometer, an electronic particle counter, gravimetry or light microscopy as appropriate. The temperature and the photoperiod may be varied to optimize growth in different species of microalgae, and different measurements may be used to monitor cell growth. Modulation of cultivation parameters will affect quality and quantity of EVs produced.

[0049] Steps c) and d): Isolation of Microalgae-Derived Extracellular Vesicles

[0050] An essential step of the method of the present invention is to separate the extracellular vesicles from the microalgal cells and culture supernatant containing soluble secreted materials comprising but not limited to secreted proteins, metabolites and the molecular components of microalgae culture medium. Different separation procedures based on different physical principles, i.e. filtration, centrifugation, precipitation may in principle be applied. Preferentially, differential centrifugation for the isolation of extracellular vesicles (EVs) from a microalgae culture. Differential centrifugation is composed of a series of centrifugation steps with increasing velocity to remove cells, cellular debris and to obtain the fraction containing micro vesicles at the end of low-velocity centrifugation and smaller EVs containing fraction at the end of ultracentrifugation step. This step is essential to the isolation procedure; indeed, the velocity and time of centrifugation are critical. For instance, if the initial centrifugation velocity is too high, cell can lyse and contaminate the sample.

[0051] Step c): Low Velocity Centrifugation

[0052] Low-velocity centrifugation is preferably performed by at least three separate centrifugation steps at increasing velocity. The velocity (range 200-15000×g), number of the centrifugation steps (range 1-10 steps, preferably 6 steps), temperature (range 2-20° C., preferably 4° C.), type of rotor (fixed angle or swingout rotors, preferably swinging bucket rotors), tube and centrifuge employed in the low velocity centrifugation step may vary. Preferably, the first low-velocity centrifugation step is performed at the velocity of 300×g, at 4° C. for 10 min in swinging bucket rotors. The second low-velocity centrifugation step is preferably performed at 2,000×g at 4° C. for 10 min. The third low-velocity centrifugation step is preferably performed at 10,000×g at 4° C. for 30 min. Preferably, each low velocity centrifugation steps is performed two times. Pellets are obtained by careful decantation. Alternatively, in some instances, the supernatant can be removed by aspiration with the help of pipette. Pellets resulting from the last low velocity centrifugation step at 10,000×g contain the larger extracellular vesicles (e.g., microvesicles). Preferably, the 10,000×g pellet is washed two times with an appropriate buffer. Generally, PBS buffer filtered through a 20 nm sterile filter is employed. Washing steps are performed by careful resuspension of the pellet followed by re-centrifugation using the conditions used for obtaining the pellet itself.

[0053] Step d): Ultracentrifugation

[0054] Ultracentrifugation of the 10,000×g supernatant of step c) is performed in order to obtain a fraction containing the smaller EVs. Ultracentrifugation is preferably performed at 118,000×g (range 100,000-120,000×g) using swinging bucket rotors at 4° C. for 2 h, but the duration of the centrifugation may vary (range 1-24 hours). Preferably, a washing step is performed using the preferred buffer in which the pellet is carefully resuspended. The supernatant is usually removed by careful decantation but, in some instances, it may be removed by aspiration. After ultracentrifugation, the pellet may be resuspended in generally low volume of buffer.

[0055] Although the disclosed product has been obtained by using differential ultracentrifugation, other fractional isolation methods could in principle be used to separate small EVs (e.g. tangential flow fractionation, gradient ultracentrifugation).

[0056] Within the present description, the terms “extracellular vesicles” or “EVs” is used with reference to both large (e.g., microvesicles) and small extracellular vesicles E.G., exosomes, ectosomes).

[0057] The term “microalgae” is usually used to indicate any eukaryotic microscopic algae, typically found in freshwater or marine systems, living in both the water column and sediment. They are unicellular species which exist individually, or in chains or groups. Microalgae include, for ex-ample but are not limited to, photosynthetic Diatoms, Dinoflagellates, Haptophytes or Chlorophytes.

[0058] Furthermore, within the present description the term “microalgae” is used to indicate photosynthetic microalgae including, for example, species with silica exoskeleton and/or ornamented cell walls and with promising antioxidant or antimicrobial activities or signatures of pigments and fatty acids for example but not limited to the Chlorophyte Haematococcus pluvialis, diatom Stauroneis sp., Haptophyte Pavlova sp. or the model species Chlamydomonas reinhardtii. Further examples of microalgae include the Chlorophyte Tetraselmis sp. (such as the strain Tetraselmis sp. LA-CW-02) and the Diatom Odontella sp. (such as the strain Odontella sp. LA-CW-28).

[0059] The production method of the present invention differs from other EV-related methods of the prior art in that its focus is targeted to nanovesicles refined for the first time from unicellular eukaryotic, photosynthetic microalgae. In addition, irrespective of their content, the goal of the invention is also to produce microalgae-derived EVs in a large amount for industry application/s. This aim could not be achieved with mammalian cell-derived EVs, which are conversely indicated for different purposes which require low amount of EVs, including personalized cell-free therapy.

[0060] The method of the present invention advantageously allows to generate a broad range of new products based on membranous biogenic nanomaterials (i.e., EVs) from a sustainable and renewable bioresource (i.e., microalgae), which can be used as new natural delivery system for high-value microalgal substances (such as antioxidants, pigments, lipids and complex carbohydrates), bioactive bio-logical molecules (e.g., proteins, miRNA, siRNA, mRNA, lncRNA, peptides) and/or synthetic drugs.

[0061] The following experimental part is provided by way of illustration only and is not intended to limit the scope of the invention as defined by the appended claims.

EXPERIMENTAL PART

[0062] Samples of marine photosynthetic Chlorophyte (strain Tetraselmis sp. LA-CW-02) and Diatom (strain Odontella sp. LA-CW-28) including cellular pellets and conditioned media were used. Cell pellets were solubilized in RIPA buffer and small and large extracellular vesicles (exosomes and microvesicles) were isolated by ultracentrifugation from the conditioned media.

[0063] Microalgal strains are grown for 30 days in triplicate glass tubes containing 60 ml of filter-sterilized medium. All the cultures are subjected to a photoperiod of 14:10 (light:dark) under an irradiance of 60-70 μE s.sup.−1. The corresponding conditioned media are processed at the end of the microalgal cultivation as follows:

[0064] Series of low speed centrifugation rounds (300×g and 2,000×g) to remove cells and large organic agglomerates. The biogenic extracellular nanoparticles are recovered by 10,000×g (large EVs) and 118,000×g (small EVs) centrifugation using swinging bucket rotors at 4° C. for 2 h.

[0065] EV protein content measured by microbicinchoninic (BCA) colorimetric assay. To examine the size distribution of microalgal EVs, Dynamic Light Scattering (DLS) analyses of sEVs was applied. Scattered light intensity and its time autocorrelation function g2(t) were measured simultaneously on different EV samples at T=20° C. using a Brookhaven BI-9000 correlator (Brookhaven Instruments, Holtsville, N.Y., USA) equipped with a solid-state laser tuned to λ0=532 nm. The samples were diluted to a final total protein content of 50 μg/ml in order to avoid vesicle interaction and multiple scattering artefacts. Lipid bilayer presence was assessed by treating sEV preparation with SDS and analyzing the sEV size distribution following detergent treatment. EV morphology was assessed by Atomic force microscopy (AFM).

[0066] Results: Starting from a conditioned media volume of 60 ml and a final microalgal biomass of 1.5 mg/ml the following product content is obtained, in terms of total small extracellular vesicle protein amount: CW-02 small extracellular vesicle total proteins=8+2 μg, CW-28 small extracellular vesicle total proteins=3+0.7 μg.

[0067] DLS analyses (FIG. 1) demonstrated that membranous nanovesicles (sensitive to SDS) of 50-350 nm in size (moda of 160 nm) are present in the sEV preparation.

[0068] FIG. 3 shows a representative image of immunoblot analyses of lysates (Lys) of the microalgal strains (LA CW2, LA CW23, LA CW28), large EV fraction (lEV), small EV fractions (sEV). The loaded protein amount is indicated in each lane. EVs positive for an EV protein marker (e.g., Alix) were isolated from the microalgal conditioned-media.

[0069] The results obtained are illustrated in the appended drawings, in which:

[0070] FIG. 1 shows the results obtained with the Diatom Odontella sp. (LA-CW-28) derived sEVs by Dynamic Light Scattering (DLS) experiments on CW-28 Nanovesicles. The upper panel shows the intensity autocorrelation functions for sEVs, sEVs after incubation with 0.5% SDS and the reference 0.5% SDS solution; the functions are analyzed to derive the size distributions; the lower panel shows the size distributions normalized to the initial amount of sEVs. sEV samples have been measured in the buffer used for their resuspension (Phosphate Buffer Saline, PBS, with 0.5M NaCl) or upon addition of SDS (to a final concentration of 0.5%). SDS affects the concentration of sEVs upon incubation, confirming the lipid composition of microalgae-derived nanovesicles membrane.

[0071] FIG. 2 shows Atomic Force Microscopy (AFM) images on Chlorophyte Tetraselmis sp. (LA-CW-02) derived sEVs. AFM images (in tapping mode) of sEVs from microalgae (100 times dilution and 10 minutes incubation on mica sample substrate). The upper panel displays a large field 2 μm image, where many round-shaped EVs can be observed (marked by arrows) with a size between 40 and 100 nm. The lower panel displays a close-up 500 nm image displaying in more detail a sEV of 75 nm.

[0072] FIG. 3 shows the results obtained from western blot analysis using established antibodies. Immunoblots of total protein extracts (Lys), large vesicles (1EV) and small vesicles (sEV) from LA CW2, LA CW23, LA CW28 microalgae strains using specific antibody recognizing an EV marker (anti-Alix, clone 3A9), and anti-Enolase and anti-Hsp70. The lower panel shows the Ponceau red stained PVDF membrane, as loading control.

[0073] FIG. 4 shows the results of experiments using Iodixanol gradient to determine the nanoalgosome density. (A) The density of the ten fractions measured in the gradient ultracentrifugation (gUC) of dUC-isolated samples. (B) The quantity of protein measured in each gUC fraction. (C) Representative immunoblot analyses of nanoalgosomes isolated by dUC, and loaded on iodixanol density gradient. 20 μg of microalgae lysate and equal fraction volumes were loaded on gel. Fraction 5 of dUC-separated nanoalgosomes and at a less extent the fraction 6 are positive for EV specific biomarkers (Alix). Two independent technical replicas (n=2) were performed.