A method for separating and preparing an exosome of chlorella

Abstract

This invention discloses a method for isolating and preparing Chlorella exovesicles. Specifically, Chlorella, conventionally cultured to the early logarithmic growth phase, is induced to develop in a nitrogen-limited medium. The culture medium is then concentrated using a tangential flow filtration system. Finally, the concentrate is subjected to density gradient centrifugation with iodixanol. After centrifugation, components within the density range of 1.10–1.12 g / mL are collected to obtain the Chlorella exovesicle product. This invention achieves concentrated and precise collection of Chlorella exovesicles by regulating membrane lipids during the early induction culture of Chlorella, combined with TFF concentration and density gradient centrifugation. This method effectively eliminates impurities, achieving a recovery rate of over 60% and a purity of 2.56 × 10⁻⁶. 10 The particle / μg method provides an efficient, controllable, and scalable solution for the industrial-scale separation and preparation of microalgal exovesicles.

Description

Technical Field

[0001] This invention belongs to the field of algal exosome extraction technology, specifically relating to a method for separating and preparing exovesicles from Chlorella. Background Technology

[0002] Chlorella vulgaris is a single-celled eukaryotic microalga characterized by its short growth cycle, high photosynthetic efficiency, and rich nutritional content, making it valuable for applications in the food, cosmetics, and biopharmaceutical industries. Research indicates that Chlorella can secrete nanoscale membrane vesicles (EVs) during cultivation. These EVs act as "messengers" for intercellular communication, carrying abundant lipids, proteins, and nucleic acids. They exhibit low immunogenicity, strong targeting, and the ability to cross biological barriers, leading to their widespread exploration for applications in drug delivery, healthcare, and functional products. Compared to EVs derived from mammalian cells, the research and utilization of Chlorella EVs is still in its early stages, particularly with the underdeveloped large-scale preparation technology system, which severely restricts subsequent industrial applications.

[0003] Currently, the main method for separating and obtaining microalgal exovesicles is still centrifugation, including ultracentrifugation, differential centrifugation, and centrifugal ultrafiltration concentration. These centrifugation methods have the following main problems:

[0004] 1) Low recovery rate: Excessive centrifugation during the separation process can cause vesicles to deform and rupture, resulting in a recovery rate that is usually less than 30%.

[0005] 2) Insufficient purity: Differential centrifugation cannot distinguish chloroplast fragments and polysaccharide aggregates with similar densities, resulting in a low particle / protein ratio and easy co-precipitation of impurities.

[0006] 3) Polarization clogging: During the centrifugation and ultrafiltration process, the microalgae culture medium is highly viscous and the polysaccharide system will develop a severe polarization layer in a short period of time (5-15 min), resulting in a significant decrease in flux (usually more than 90%), making it impossible to scale up.

[0007] Recent studies have also attempted to replace the traditional centrifugal separation methods with membrane filtration or density gradient centrifugation, but none of them have fundamentally solved the problems of low recovery rate, low purity, and significant flux reduction caused by polarization blockage of the high-viscosity microalgae system during ultrafiltration. Summary of the Invention

[0008] To address the aforementioned problems, the present invention aims to provide a highly efficient method for the separation and preparation of Chlorella exovesicles, which overcomes the limitations of existing separation methods, such as low recovery rate and purity, as well as the technical obstacles of membrane separation caused by polarization blockage in high-viscosity systems.

[0009] The specific technical solution of this invention is as follows:

[0010] A method for isolating and preparing microalgal exovesicles includes the following steps:

[0011] 1) Transfer Chlorella vulgaris cultured to the early logarithmic growth phase to a nitrogen-limited medium, and expose it to 450 nm blue light (50-70% concentration) with a total light intensity of 100-150 μmol photons / m². -2 s -1 Induced culture under light for 36–60 h;

[0012] 2) Concentrate the culture medium through a tangential flow filtration system using a 50–150 kDa hollow fiber membrane, with an inlet pressure of 8–12 psi, a transmembrane pressure difference of 4–6 psi, a tangential flow rate of 80–120 mL / min, and dialysis washing with 2–4 times the volume of the filter.

[0013] 3) Centrifuge the concentrate using an iodixanol density gradient. First, centrifuge the concentrate at 2–8℃ and 100,000–150,000×g for 2–3 hours. Then, resuspend the precipitate and spread it on an iodixanol density gradient. Centrifuge at 2–8℃ and 130,000–170,000×g for 16–20 hours. Collect the components in the density range of 1.10–1.12 g / mL to obtain the Chlorella exovesicle product.

[0014] Furthermore, in step 1 above, the nitrogen-limiting culture medium uses NaNO3 as the sole nitrogen source, with a concentration of 6.5–7.5 mg / L; during induction culture, 450 nm blue light accounts for 60% of the light and the total light intensity is 120 μmol photons m. -2 s -1 The light exposure was 12h:12h, the temperature was 25℃, and the incubation time was 48h.

[0015] Furthermore, in step 2 above, the concentration conditions are as follows: the molecular weight cutoff of the fiber membrane is 100 kDa, the inlet pressure is 10 psi, the transmembrane pressure difference is 5 psi, the tangential flow rate is 100 mL / min, and the dialysis wash is 3 times the volume.

[0016] Further, in step 3 above, the iodixanol density gradient is set as follows: 4 discontinuous density gradients: 1.08 g / mL, 1.10 g / mL, 1.12 g / mL, and 1.14 g / mL. The centrifugation operation of the concentrate is as follows: first, the concentrate is centrifuged at 4℃ and 120,000 × g for 2.5 h, then the precipitate is resuspended and spread on the iodixanol density gradient, and centrifuged at 4℃ and 150,000 × g for 18 h, and the components in the density range of 1.10 to 1.12 g / mL are collected.

[0017] Furthermore, the purity (particle / protein ratio) of the Chlorella exovesicle product obtained in step 3 above is 1.2–1.6 × 10⁻⁶. 10 particles / μg.

[0018] Furthermore, step 3 above also includes SEC (size exclusion chromatography) purification of the Chlorella exovesicle product. The separation range of size exclusion chromatography purification is 10 kDa to 20 MDa, and the purity of the purified product is 2.0 to 2.5 × 10⁻⁶. 10 particles / μg.

[0019] Furthermore, the SEC purification uses propylene dextran gel S-400 HR packing material with a column volume of 20–50 mL. The equilibration buffer and eluent are phosphate buffer at pH 7.4. The flow rate is 0.3–0.7 mL / min, and 0.8–1.2 column volumes of eluent are collected.

[0020] This invention achieves concentrated and precise collection of Chlorella exovesicles by regulating membrane lipids during the early induction culture of Chlorella, combined with TFF concentration and density gradient centrifugation. This process largely eliminates interference from chloroplast fragments, achieving a recovery rate of over 60% and a purity of 2.5 × 10⁻⁶. 10 The particle / μg ratio is significantly higher than that of existing extraction technologies, providing an efficient, controllable, and scalable solution for the industrial-scale separation and preparation of microalgal exovesicles. Attached Figure Description

[0021] Figure 1 Example 1: Particle size distribution analysis results in the supernatant of the induction culture medium.

[0022] Figure 2 Transmission electron microscopy (TEM) image of Chlorella exovesicles in the supernatant of the induction culture medium in Example 1.

[0023] Figure 3 Western blot results of Chlorella exovesicle products from Examples 1-3 and Examples 5-6. Detailed Implementation Plan

[0024] The invention's technical solution will be further explained below with reference to specific embodiments.

[0025] Example 1

[0026] 1) Sample Source and Initial Culture: Seawater samples from the Tianjin coastal area were filtered through a 0.22 μm filter membrane for sterilization. Under an inverted microscope, single cells of Chlorella with typical morphology were picked using a micromanipulator and transferred to 48-well plates (each well containing 1 mL of sterile filtered seawater) for culture, with at least 10 cells per well. After 3–5 days of culture, microscopic examination was performed. Algal cultures with uniform cell morphology and good growth were transferred to 25 mL of F / 2 medium for amplification and identification, while gene sequencing was performed to ensure pure culture.

[0027] 2) Induction culture: Pure cultured Chlorella was inoculated into 2L of standard F / 2 medium and incubated at 25℃ with 120μmol photons m -2 s -1 Light intensity and 12-hour light-dark cycle: Cultured for 12 hours until the early logarithmic growth phase, then transferred to nitrogen-limited medium (NaNO3 as the sole nitrogen source, concentration 7.5 mg / L, other components the same as standard F / 2 medium). Simultaneously, the light quality was adjusted to 60% blue light at 450 nm (LED light source, total light intensity maintained at 120 μmol photons / m²). -2 s -1 The culture was induced and cultured for 48 hours. After induction, the culture medium was centrifuged at 5000×g and 4℃ for 20 min, and the supernatant (1.8 L) was collected. The supernatant of the induced culture medium was diluted appropriately and NTA (nanoparticle tracer analysis) was performed. The particle size analysis results are as follows: Figure 1 As shown, the particle size exhibits a unimodal distribution, with the main peak concentrated in the 100–200 nm range, which is within the typical particle size range of Chlorella exovesicles. The particle size is relatively uniform, with no obvious polydispersity. Transmission electron microscopy (Hitachi TEM system) reveals typical spherical vesicle structures. Figure 2 It has a relatively clear outer lipid bilayer membrane and homogeneous contents. The total number of particles in the collected induction culture supernatant was 7.0 × 10⁻⁶. 13 particles.

[0028] 3) TFF Concentration: An ÄKTA flux s tangential flow filtration (TFF) system was used, equipped with a 100 kDa hollow fiber membrane (UMP-1533, effective area 330 cm²). The supernatant was pretreated by sequential 0.45 μm and 0.22 μm microfiltration in series, followed by concentration at an inlet pressure of 10 psi, a transmembrane pressure differential of 5 psi, and a tangential flow rate of 100 mL / min. The permeate flow rate was monitored in real time during concentration. When the concentration reached 1 / 10 of the original volume (approximately 180 mL), sterile PBS (pH 7.4) was added for dialysis and washing. The washing volume was three times the original concentrated volume (approximately 540 mL). After continuous filtration to remove impurities, approximately 200 mL of TFF concentrate was obtained.

[0029] 4) Density gradient centrifugation: Dilute 60% OptiPrep stock solution to 50% (w / v) with 10mM Tris-HCl (pH 8.6) to obtain the iodixanol working solution for density gradient preparation. Prepare four discontinuous gradient layers: from bottom to top, 1.08 g / mL (2 mL), 1.10 g / mL (2 mL), 1.12 g / mL (2 mL), and 1.14 g / mL (2 mL). Centrifuge the TFF concentrate at 4℃ and 120,000 × g for 2.5 h, then resuspend the precipitate in Tris-HCl and place it on the top layer of the iodixanol density gradient, centrifuging at 4℃ and 150,000 × g for 18 h. After centrifugation, collect samples layer by layer from the top using capillary pipette, collecting 12 tubes of samples, 500 μL each. Measure the refractive index (20℃) of each sample and convert it to the actual density. The remaining tubes are used for auxiliary positioning. Combine tube samples with a density of 1.10–1.12 g / mL, and dilute with PBS to obtain Chlorella exovesicle products.

[0030] NTA was used to determine the total number of particles in the product, and the recovery rate was calculated using the following formula:

[0031] Recovery rate (%) = Total number of product particles / Total number of particles in the induction culture supernatant × 100

[0032] The supernatant of the induction culture medium was obtained by centrifuging the culture medium at 5000×g and 4℃ for 20 min after the aforementioned induction culture. The total number of particles in the product was determined to be 5.1 × 10⁻⁶. 13 Particles; recovery rate 73%.

[0033] The protein concentration in the product was determined using the BCA method, and the purity was calculated using the following formula:

[0034] Purity (particles / μg) = Product particle concentration / Product protein concentration

[0035] The particle concentration of the product was obtained through the aforementioned NTA determination. The calculation results show that the purity of the exovesicle product obtained in this density gradient centrifugation step is 1.62 × 10⁻⁶. 10 particles / μg.

[0036] 5) SEC Purification: An S-400 HR propylene dextran gel chromatography column (20 mL column volume, 1.6 × 10 cm) was mounted onto the ÄKTA pure system, ensuring a tight column connection and no air bubbles. 1×PBS buffer was passed through the column at a flow rate of 0.5–1.0 mL / min to equilibrate the three column volumes. The Chlorella exovesicle product obtained by the aforementioned density gradient centrifugation was filtered through a 0.22 μm filter and loaded onto the column at a flow rate of 0.5 mL / min, with a UV 280 nm detector for monitoring. 0.8–1.2 column volumes of eluent were collected and concentrated to 2 mL using an ultrafiltration tube with a molecular weight cutoff of 100 kDa. The concentrated sample was then filtered again through a 0.22 μm sterile filter to obtain the final purified exovesicle product. The total number of particles in the final purified product was determined using NTA; the protein concentration in the final purified product was determined using the BCA method. The recovery rate and purity after final purification were calculated based on the results, using the same calculation method as the aforementioned density gradient centrifugation steps.

[0037] The final purified product contained 4.4 × 10⁻⁶ particles. 13 The particles were calculated to have a final purity recovery rate of 62% and a purity of 2.56 × 10⁻⁶. 10 particles / μg.

[0038] In this embodiment and the following embodiments and experiments, the particle concentration, total number of particles, recovery rate and purity index were all calculated based on a particle population with a particle size range of 100-200 nm, and the detection method was nanoparticle tracking analysis (NTA).

[0039] It is known in the prior art that nitrogen limitation can induce lipid accumulation in microalgae. However, this application has found through experiments that the degree of nitrogen limitation has a non-linear relationship with the specific enrichment of Chlorella membrane lipid SQDG (sulfoquinoylglycerol). Furthermore, the inventors have discovered that under nitrogen limitation, specific light quality can become a necessary condition for inducing "high-density unsaturated membrane lipid synthesis," exhibiting a synergistic effect with nitrogen limitation. Specific experimental results are shown in Tables 1 and 2.

[0040] Table 1 shows the effects of different nitrogen (NaNO3) concentrations on the accumulation of SQDG membrane lipids in *Chlorella vulgaris* and the buoyancy density, recovery rate, and purity of external vesicles under white light irradiation (other culture conditions, TFF concentration, density gradient centrifugation, and index determination were the same as in Example 1). The SQDG membrane lipids were determined by UPLC-QTOF-MS (extraction using the Bligh & Dyer method, quantification using the LipidSearch-assisted internal standard method); the buoyancy density was determined according to the actual density calculation method after density gradient centrifugation in Example 1. As shown in Table 1, when the NaNO3 concentration was 75 mg / L (standard F / 2 medium), the total lipid content of SQDG, the recovery rate of external vesicles, and the purity were the lowest. With increasing nitrogen restriction, cellular stress responses were induced, triggering external vesicles as a signal transduction or clearance mechanism. Simultaneously, membrane lipid remodeling (SQDG accumulation) may enhance vesicle stability and release efficiency, manifested as an increase in particle concentration and a decrease in buoyancy density in the culture supernatant, leading to an increase in vesicle recovery rate and purity, which was most pronounced when the NaNO3 concentration decreased to 7.5 mg / L. However, when the NaNO3 concentration further decreased to 6.3 mg / L, algal cells may experience metabolic inhibition due to excessive nitrogen restriction, resulting in decreased secretory capacity, and the vesicle recovery rate and purity subsequently reversed from an increase to a decrease.

[0041] Table 1. Effects of different nitrogen concentration limits on the accumulation of SQDG membrane lipids in Chlorella vulgaris and the recovery rate and purity of exovesicles.

[0042]

[0043] a. "Recovery rate" refers to the recovery rate of external vesicles after density gradient centrifugation;

[0044] b. “Purity” refers to the purity of the sample after density gradient centrifugation. “P / Pr” means “particles / μg protein”, which is the number of vesicle particles per microgram of protein.

[0045] Table 2 shows the effects of changing the light quality composition (all other culture conditions, TFF concentration, density gradient centrifugation, and index measurements were the same as in Example 1) on the lipid unsaturation, chemical composition, and fluidity of Chlorella membranes while maintaining a nitrogen (NaNO3) concentration of 7.5 mg / L. Table 2 shows that when blue light accounted for 60%, the degree of lipid unsaturation was highest (DBI 2.7), the C18:3 content increased to the highest level, and the density shift was most significant. Comparison with Table 1 also shows that under nitrogen-limited conditions, a 60% blue light concentration further reduced the buoyancy density to 1.120 g / mL. Compared to other light quality conditions, 60% blue light maximally activated FAD desaturase, increasing lipid unsaturation and decreasing membrane density. Conversely, 100% red light may inhibit fatty acid desaturase activity through the phytochrome signaling pathway, significantly reducing lipid unsaturation, making the outer vesicle membrane structure more compact, thereby increasing buoyancy density. 90% of blue light may cause photoinhibition. Excessive blue light stress can lead to oxidative damage, causing lipid peroxidation of polyunsaturated fatty acids, making membrane lipids more tightly packed, and thus reversing the membrane fluidity advantage.

[0046] Table 2. Effects of different light qualities on lipid unsaturation, chemical composition, and fluidity of Chlorella membranes.

[0047]

[0048] a. "DBI", double bond index, measures the degree of unsaturation of membrane lipids;

[0049] b. “C18:3 / C16:0”, the relative abundance of linolenic acid (C18:3) and palmitic acid (C16:0).

[0050] Example 2

[0051] 1) Sample source and initial culture: Same as in Example 1.

[0052] 2) Standard culture: Pure cultured Chlorella was inoculated into 2L of standard F / 2 medium (NaNO3 concentration of 75mg / L) and incubated at 25℃ with 120μmol photons m -2 s -1 Cultured under white light and light-dark cycles of 12 hours until the logarithmic growth phase (10–14 days), the culture medium was centrifuged at 5000 × g at 4℃ for 20 min, and the supernatant was collected. The total number of particles in the supernatant was 2.2 × 10⁻⁶. 13 particles.

[0053] 3) TFF concentration, density gradient centrifugation, and SEC purification: Same as in Example 1; however, after the density gradient centrifugation step, tube samples with a density of 1.10–1.12 g / mL are still collected together; the total number of particles in the SEC-purified product is 2.8 × 10⁻⁶.12 The particles had a recovery rate of 13% and a purity of 0.40 × 10⁻⁶. 10 particles / μg.

[0054] Example 3

[0055] 1) Sample source, initial culture and induction culture: Same as in Example 1.

[0056] 2) Concentration using ultrafiltration tubes: The supernatant was subjected to tandem microfiltration at 0.45 μm and 0.22 μm, then aliquoted into 100 kDa ultrafiltration tubes (Millipore Amicon Ultra-15). The tubes were centrifuged at 3200 × g for 15 min at 4 °C, repeatedly centrifuged until the volume was reduced to 1 / 10 of the original volume. Then, sterile PBS (pH 7.4) was added for dialysis. Centrifugation was performed after each addition of PBS, repeated three times, discarding the filtrate each time. Severe polarization and clogging of the ultrafiltration tube membrane were observed during this concentration step. Approximately 200 mL of concentrated solution was obtained in this step.

[0057] 3) Density gradient centrifugation and SEC purification: Same as in Example 1. The total number of particles in the product after SEC purification was 2.0 × 10⁻⁶. 13 The particles had a recovery rate of 29% and a purity of 2.15 × 10⁻⁶. 10 particles / μg.

[0058] Example 4

[0059] 1) Sample source, initial culture, induction culture and TFF concentration: Same as in Example 1.

[0060] 2) Density gradient centrifugation: Dilute 60% OptiPrep stock solution to 50% (w / v) with 10mM Tris-HCl (pH 8.6) to prepare a discontinuous three-layer gradient: from bottom to top, 1.10 g / mL (2 mL), 1.13 g / mL (2 mL), and 1.16 g / mL (2 mL). Centrifuge the TFF concentrate at 4℃ and 120,000×g for 2.5 h, then resuspend the precipitate in Tris-HCl and place it on top of the iodixanol density gradient, centrifuging at 4℃ and 150,000×g for 18 h. After centrifugation, collect samples from the 1.10–1.13 g / mL density range, dilute with PBS, and obtain the Chlorella exovesicle product.

[0061] 3) SEC purification: Same as in Example 1. The total number of particles in the product after SEC purification was 3.1 × 10⁻⁶. 13 The particles were recovered at a rate of 43%, with a purity of 1.92 × 10⁻⁶. 10 particles / μg.

[0062] Example 5

[0063] The sample source, culture, TFF concentration, density gradient centrifugation, and SEC purification were basically the same as in Example 1, except that samples with a density range of 1.08–1.10 g / mL were collected as the product after density gradient centrifugation. The total number of particles in the SEC-purified product was 4.3 × 10⁻⁶. 12 The particles were recovered at a rate of 6%, with a purity of 0.29 × 10⁻⁶. 10 particles / μg.

[0064] Example 6

[0065] The sample source, culture, TFF concentration, density gradient centrifugation, and SEC purification were basically the same as in Example 1, except that samples with a density range of 1.13–1.16 g / mL were collected as the product after density gradient centrifugation. The total number of particles in the SEC-purified product was 7.1 × 10⁻⁶. 12 The particles had a recovery rate of 10% and a purity of 0.48 × 10⁻⁶. 10 particles / μg.

[0066] Western blot analysis was performed on the products from Examples 1-3 and Examples 5-6 above, using Chlorella cell lysate as a positive control and blank culture medium as a negative control to verify the proteins Alix and H. + The antibody efficacy against ATPase, LHCB, HSP70, and β-actin was assessed. The specific method was as follows: RIPA lysis buffer (containing protease inhibitors) was added to the products of each example and the Chlorella cell lysis buffer, incubated on ice for 30 min, centrifuged at 12000×g for 15 min at 4°C, and the supernatant was collected. Protein concentration was determined using the BCA method and adjusted to a uniform 2 μg / μL; 30 μg of total protein was subjected to 10% SDS-PAGE electrophoresis and transferred to a PVDF membrane, blocked with 5% skim milk powder for 1 h, and then primary antibodies (rabbit anti-Alix, rabbit anti-H) were added. + After adding ATPase, rabbit anti-LHCB, rabbit anti-HSP70, and rabbit anti-β-actin, the mixture was incubated overnight at 4°C. HRP-labeled goat anti-rabbit secondary antibody was added, and the mixture was incubated at room temperature for 1 hour. After washing with TBST, ECL chemiluminescence reagent was used for color development, and images were acquired using a ChemiDoc imaging system. Each protein was hybridized independently, and the band gray values ​​were measured using ImageJ software. Normalization was performed using β-actin as an internal control, with Example 1 as a 100% reference.

[0067] Western blot results are as follows Figure 3 As shown: Alix and H in Example 1 +-ATPase showed a strong positive result, with normalized relative values ​​of 100% and 90%, respectively. LHCB and HSP70 were both negative, indicating that the product obtained in Example 1 was a high-purity functional microalgal exovesicle. In contrast, the product in Example 2 showed negative results for Alix and H... + -ATPase is extremely weak (normalized value only about 10%), and functional vesicles are almost undetectable. In Example 3, Alix and H... + Both ATPase and HSP70 showed some positivity, with normalized values ​​of approximately 50%, 20%, and 40%, respectively, indicating that the product contained a mixture of numerous ruptured extravesicles and free proteins. In Example 5, LHCB showed strong positivity with a normalized value of approximately 90%, while Alix showed very weak positivity (normalized value of approximately 10%), indicating that the main component of its product was chloroplast fragments. In Example 6, HSP70 showed relatively strong positivity with a normalized value of approximately 60%, while Alix showed very weak positivity, indicating the presence of a large number of dead cells and protein aggregates in the product.

[0068] Example 7

[0069] 1) Sample source and initial culture: Same as in Example 1.

[0070] 2) Culture (standard conditions): Pure cultured Chlorella was inoculated into 2L F / 2 medium and incubated at 20±2℃ with 100μmol photons m -2 s -1 The cells were cultured for 30 days under white light and a light-dark cycle of 14:10. During culture, aeration was provided through a 0.22 μm filter, and the cells were manually shaken every 3–4 days. After culture, the culture medium was centrifuged to remove cells, yielding a supernatant (approximately 1.8 L). The total number of particles in the supernatant was 4.3 × 10⁻⁶. 13 particles.

[0071] 3) TFF Concentration: An ÄKTA flux s tangential flow filtration (TFF) system equipped with a 50 kDa hollow fiber membrane was used. The supernatant was pretreated by sequential microfiltration at 450 nm and 200 nm. Concentration was then carried out at an inlet pressure of 10 psi, a transmembrane pressure differential of 5 psi, and a tangential flow rate of 100 mL / min. The permeate flow rate was monitored in real time during the concentration process. When the concentration reached 1 / 10 of the original volume (approximately 180 mL), sterile PBS (pH 7.4) was added for dialysis and washing. The washing volume was three times the original concentrated volume (approximately 540 mL). After continuous filtration to remove impurities, approximately 200 mL of TFF concentrate was obtained.

[0072] 4) Density gradient centrifugation: Dilute 60% OptiPrep stock solution with 10mM Tris-HCl (pH 8.6) to prepare a discontinuous three-layer gradient: 1.10 g / mL (2 mL), 1.13 g / mL (2 mL), and 1.16 g / mL (2 mL). Centrifuge the TFF concentrate at 4℃ and 110,000×g for 2 h. Resuspend the precipitate in Tris-HCl and place it on the top layer of the gradient, then centrifuge at 4℃ and 110,000×g for 24 h. After centrifugation, collect samples from the density range of 1.10–1.16 g / mL, dilute with PBS to obtain the Chlorella exovesicle product.

[0073] 5) SEC purification: Same as in Example 1. The total number of particles in the product after SEC purification was 1.1 × 10⁻⁶. 13 The particles had a recovery rate of 26% and a purity of 1.4 × 10⁻⁶. 10 particles / μg.

[0074] Example 8

[0075] 1) Sample source and initial culture: Same as in Example 1.

[0076] 2) Cultivation (standard conditions): Same as Example 7.

[0077] 3) After culturing, the supernatant (about 1.8L) was centrifuged at 4℃ and 10000×g for 30 min. The supernatant was then collected and ultracentrifuged at 4℃ and 118000×g (Beckman SW28 rotor) for 70 min. After centrifugation, the supernatant was discarded, and the precipitate was resuspended in PBS to obtain the sample.

[0078] 4) SEC purification: Same as in Example 1. The total number of particles in the product after SEC purification was 8.0 × 10⁻⁶. 12 The particles had a recovery rate of 19% and a purity of 0.9 × 10⁻⁶. 10 particles / μg.

Claims

1. A method for isolating and preparing Chlorella exovesicles, characterized in that... Includes the following steps: 1) Transfer Chlorella vulgaris cultured to the early logarithmic growth phase to a nitrogen-limited medium, and expose it to 450 nm blue light (50-70% concentration) with a total light intensity of 100-150 μmol photons / m². -2 s -1 Induced culture under light for 36–60 h; 2) Concentrate the culture medium through a tangential flow filtration system using a 50–150 kDa hollow fiber membrane, with an inlet pressure of 8–12 psi, a transmembrane pressure difference of 4–6 psi, a tangential flow rate of 80–120 mL / min, and dialysis washing with 2–4 times the volume of the filter. 3) Centrifuge the concentrate using an iodixanol density gradient. First, centrifuge the concentrate at 2–8℃ and 100,000–150,000×g for 2–3 hours. Then, resuspend the precipitate and spread it on an iodixanol density gradient. Centrifuge at 2–8℃ and 130,000–170,000×g for 16–20 hours. Collect the components in the density range of 1.10–1.12 g / mL to obtain the Chlorella exovesicle product.

2. The method for isolating and preparing Chlorella exovesicles according to claim 1, characterized in that: In step 1, the nitrogen-limiting medium uses NaNO3 as the sole nitrogen source, with a concentration of 6.5–7.5 mg / L; during induction culture, 450 nm blue light accounts for 60% of the light and the total light intensity is 120 μmol photons m. -2 s -1 The light exposure was 12h:12h, the temperature was 25℃, and the incubation time was 48h.

3. The method for isolating and preparing Chlorella exovesicles according to claim 1, characterized in that: In step 2, the concentration conditions are as follows: the molecular weight cutoff of the fiber membrane is 100 kDa, the inlet pressure is 10 psi, the transmembrane pressure difference is 5 psi, the tangential flow rate is 100 mL / min, and the dialysis wash is 3 times the volume.

4. The method for isolating and preparing Chlorella exovesicles according to claim 1, characterized in that: In step 3, the iodixanol density gradient is set as follows: 4 discontinuous density gradients: 1.08 g / mL, 1.10 g / mL, 1.12 g / mL, and 1.14 g / mL. The centrifugation operation of the concentrate is as follows: first, the concentrate is centrifuged at 4℃ and 120,000 × g for 2.5 h, then the precipitate is resuspended and spread on the iodixanol density gradient, and centrifuged at 4℃ and 150,000 × g for 18 h, and the components in the density range of 1.10 to 1.12 g / mL are collected.

5. The method for isolating and preparing Chlorella exovesicles according to claim 4, characterized in that: The purity of the Chlorella exovesicle product obtained in step 3 is 1.2–1.6 × 10⁻⁶. 10 particles / μg.

6. The method for isolating and preparing Chlorella exovesicles according to claims 1 to 5, characterized in that: Step 3 further includes size exclusion chromatography purification of the Chlorella exovesicle product. The separation range of size exclusion chromatography purification is 10 kDa to 20 MDa, and the purity of the purified product is 2.0 to 2.5 × 10⁻⁶. 10 particles / μg.

7. The method for isolating and preparing Chlorella exovesicles according to claim 6, characterized in that: Size exclusion chromatography purification was performed using propylene dextran gel S-400 HR packing material with a column volume of 20–50 mL. The equilibration buffer and eluent were phosphate buffer at pH 7.

4. The flow rate was 0.3–0.7 mL / min, and 0.8–1.2 column volumes of eluent were collected.

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