Preparation method and application of a microalgae exosome-fucoxanthin composite nanoparticle

Abstract

The present invention discloses a preparation method and application of microalgae exosome-fucoxanthin composite nanoparticles, which are characterized by including the following steps: adding a fucoxanthin ethanol solution and a microalgae exosome solution into a cuvette according to a mass ratio of 0.5-1.5:1, co-incubating for 30 min, adding an electroporation buffer solution with a volume 1-3 times that of the mixed solution, then using an electroporator to perform electroporation on the mixed solution in the cuvette, controlling the voltage at 100-500 V and the capacitance at 300-700 μF for electric shock, then incubating at 35-40 °C for 0.5-2 h, and then centrifuging the mixed solution at 80,000-120,000 g at 4 °C for 20-40 min to collect the precipitate particles, thus obtaining the microalgae exosome-fucoxanthin composite nanoparticles. The application of the composite nanoparticles in the preparation of antioxidants, ABTS radical scavengers and / or DPPH radical scavengers has the advantages of improving the stability and bioavailability of fucoxanthin and producing a synergistic antioxidant effect.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a preparation method and application of a microalgae exosome-fucoxanthin composite nanoparticle. Background Art

[0002] Fucoxanthin (FX) is a fat-soluble natural xanthophyll carotenoid, which can be widely isolated from marine brown algae and other large or microalgae. It has a wide range of bioavailability and functions, such as significant antioxidant, anti-obesity, anti-diabetic and anti-inflammatory properties, and has important application potential in the treatment and prevention of various cancers and tumors.

[0003] The highly unsaturated conjugated double bonds, alkene bonds and epoxy structures in the chemical structure of fucoxanthin result in its poor stability. It is prone to structural changes when exposed to heat, light and gastric acid, affecting its bioavailability. During daily processing, storage and use, fucoxanthin is very sensitive to temperature and light. High temperature will destroy the molecular structure of fucoxanthin, resulting in the cleavage or isomerization of double bonds, and its degradation will accelerate with the increase of temperature. Ultraviolet and visible light irradiation will cause the rich conjugated double bonds in its molecular structure to undergo photochemical oxidation, isomerization and degradation reactions, significantly reducing the biological activity and stability of fucoxanthin. The gastric acid environment will also affect its structure, resulting in structural changes, which limits its application in drug research and development and the food industry. At the same time, the dual problems of poor stability and low water solubility directly lead to its low bioavailability, which is not conducive to absorption, distribution and metabolism in the body, cannot fully exert its potential bioavailability, resulting in a low effective concentration in the body and unable to achieve the expected therapeutic or health care effects. In addition, there are also many challenges in the development of oral consumer dietary products, such as the difficulty in achieving ideal effects in dosage form design and drug release control. In summary, the market prospect of fucoxanthin is very broad, but due to its highly unsaturated structure, it has defects such as poor water solubility and strong environmental sensitivity, which greatly limit the actual production and application of fucoxanthin functional drugs. Therefore, developing an effective delivery system can reduce the instability of fucoxanthin under adverse conditions, thereby improving its oral bioavailability.

[0004] Extracellular vesicles (EVs) are natural membranous nanoparticles with a diameter of 30-150 nm that are naturally released by cells and have a typical structure with a round shape and some depressions, resembling a "tea saucer". EVs are secreted by cells and can carry various substances such as nucleic acids, proteins, amino acids, and lipids. According to size, composition, subcellular origin, and secretion mode, extracellular vesicles can be divided into the following three types: exosomes, microvesicles, and apoptotic bodies. After being released by cells, exosomes can participate in the regulation of various physiological activities, such as development, immunity, tissue homeostasis, cancer, and neurodegenerative diseases, and play an important role in cell-to-cell communication under physiological and pathological conditions. Due to their unique phospholipid bilayer structure, exosomes can encapsulate or adsorb various drugs and enclose different types of drugs in their lumen. These exosomes have become an ideal drug delivery tool due to their natural biocompatibility, low immunogenicity, potential cancer cell targeting properties, and enhanced cell uptake efficiency through direct fusion with the plasma membrane.

[0005] Chlorella Chlorella is a single-celled freshwater microalga of the genus Chlorella in the phylum Chlorophyta. It has the characteristics of fast growth rate, short reproduction cycle, and easy artificial cultivation. As an important aquatic source plant, microalgae are easy to cultivate on a large scale, have low cultivation costs, and are rich in active substances. They show great advantages in drug delivery, tumor treatment, wound healing, etc., and can produce exosomes more quickly and easily in large quantities. There is currently no relevant research report on using microalgae exosomes to encapsulate fucoxanthin drug delivery systems. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a preparation method and application of microalgae exosome-fucoxanthin composite nanoparticles that can improve the stability and bioavailability of fucoxanthin and produce a synergistic antioxidant effect.

[0007] The technical solution adopted by the present invention to solve the above technical problems is as follows: A preparation method of microalgae exosome-fucoxanthin composite nanoparticles, comprising the following steps: adding a fucoxanthin ethanol solution and a microalgae exosome solution in a mass ratio of 0.5-1.5:1 to a cuvette and co-incubating for 30 min, then adding an electroporation buffer with a volume 1-3 times that of the mixed solution, and then using an electroporator to perform electroporation on the mixed solution in the cuvette, controlling the voltage at 100-500 V and the capacitance at 300-700 μF for electric shock, then incubating at 35-40 °C for 0.5-2 h, and then centrifuging the mixed solution at 80,000 - 120,000 g at 4 °C for 20-40 min to collect the precipitated particles, thus obtaining the microalgae exosome-fucoxanthin composite nanoparticles.

[0008] Further, the preparation method of the fucoxanthin ethanol solution is as follows: Dissolve fucoxanthin in 95% ethanol, and stir it with a magnetic stirrer until it is completely dissolved to obtain a fucoxanthin ethanol solution with a concentration of 5 mg / mL.

[0009] Further, the preparation method of the microalgae exosome solution is as follows: For the Chlorella vulgaris algal solution obtained through cultivation, successively centrifuge it at 200 - 400 g for 8 - 12 min, 1500 - 2500 g for 15 - 25 min, and 8,000 - 12,000 g for 25 - 35 min at 4°C using differential centrifugation to remove cells and cell debris. Take the supernatant, filter it through a 0.22 μm aqueous filter membrane, and then ultracentrifuge it at 80,000 - 1,200,000 g for 80 - 100 min. Take the precipitate and resuspend it with sterile PBS buffer to obtain a microalgae exosome solution with a final protein concentration of 1 mg / mL.

[0010] Further, the Chlorella vulgaris is cultured in an autotrophic mode, specifically as follows: Inoculate a single colony of Chlorella sp FACHB - 9 into 200 mL of BG11 medium and culture it in a shaker at 25°C and 120 rpm for 6 - 8 days. Then, continue to inoculate the seed liquid into 600 mL of BG11 medium and culture it in a shaker at 25°C and 120 rpm for 5 - 6 days. Collect the Chlorella vulgaris FACHB - 9 algal solution. The formula of the BG11 medium is as follows: 1.5 g of NaNO3, 0.04 g of K2HPO4, 0.075 g of MgSO4·7H2O, 0.036 g of CaCl2·2H2O, 0.02 g of Na2CO3, 0.006 g of citric acid, 0.006 g of ferric citrate, 1 mL of trace element solution A5, and 1000 mL of distilled water. The formula of the trace element solution A5 is: 2.86 g / L of H3BO3, 1.81 g / L of MnCl2·4H2O, 0.222 g / L of ZnSO4, 0.39 g / L of Na2MoO4, 0.079 g / L of CuSO4·5H2O, and 0.049 g / L of Co(NO3)2·6H2O.

[0011] Further, the Chlorella vulgaris is cultured in a mixotrophic mode, specifically as follows: Chlorella sp FACHB-9 Inoculate it into 200 mL of BG11 medium containing 2 g / L of glucose and culture it in a shaker at 25°C and 120 rpm for 6 - 8 days. Then, continue to inoculate the seed liquid into 600 mL of BG11 medium containing 2 g / L of glucose and culture it in a shaker at 25°C and 120 rpm for 5 - 6 days. Collect the Chlorella vulgaris FACHB - 9 algal solution.

[0012] Further, the Chlorella is cultured in a heterotrophic mode as follows: A single colony of Chlorella sp FACHB-9 is inoculated into 200 mL of modified Endo medium and cultured in a shaker at 25°C and 120 rpm for 6 - 8 days. Then, the seed liquid is continuously inoculated into 600 mL of modified Endo medium and cultured in a shaker at 25°C and 120 rpm for 5 - 6 days to collect Chlorella FACHB - 9 algal liquid. The formula of the modified Endo medium is as follows: 28 g / L glucose, 1.2 g / L potassium dihydrogen phosphate, 4.8 g / L urea, 1.2 g / L magnesium sulfate, 105 mg / L calcium chloride dihydrate, 16 g / L ferrous sulfate heptahydrate, 2.1 mg / L disodium ethylenediaminetetraacetate, 0.2 g / L trisodium citrate, 2.86 mg / L boric acid, 0.222 mg / L zinc sulfate heptahydrate, 1.81 mg / L manganese chloride tetrahydrate, 0.025 mg / L sodium molybdate, 0.07 mg / L copper sulfate pentahydrate, and 1.2 mg / L cobalt nitrate hexahydrate.

[0013] Further, the formula of the electroporation buffer is as follows: 1.15 mM potassium dihydrogen phosphate, 25 mM potassium chloride, 1.46 M sucrose, pH 7.2.

[0014] Further, the mass ratio of the fucoxanthin ethanol solution to the microalgal exosome solution is 1:1.

[0015] The present invention also provides the application of the microalgal exosome - fucoxanthin composite nanoparticles prepared by the above method in the preparation of antioxidants.

[0016] The present invention also provides the application of the microalgal exosome - fucoxanthin composite nanoparticles prepared by the above method in the preparation of 2,2'-azino - bis(3 - ethylbenzothiazoline - 6 - sulfonic acid) (abbreviated as ABTS) radical scavengers and / or 1,1 - diphenyl - 2 - picrylhydrazyl (abbreviated as DPPH) radical scavengers.

[0017] Compared with the prior art, the advantages of the present invention are as follows: The present invention discloses for the first time a preparation method and application of a microalgal exosome-fucoxanthin composite nanoparticle. In this method, Chlorella vulgaris in heterotrophic mode is screened as the algal strain with high exosome production, and then exosomes are extracted by a combination of differential centrifugation and ultracentrifugation. Using the microalgal exosomes as the wall material and fucoxanthin as the core material, fucoxanthin is loaded into the heterotrophic Chlorella vulgaris exosomes by electroporation. The conjugated double bond system in the chemical structure of fucoxanthin itself makes it vulnerable to oxidation and loss of activity. The encapsulation of exosomes can reduce the damage of these adverse factors to fucoxanthin, thereby maintaining its structural integrity and keeping its antioxidant activity before reaching the action site, and further enhancing its ability to exert antioxidant effects in vivo. In addition, when microalgal exosomes act together with fucoxanthin, a synergistic antioxidant effect may be produced to improve the stability and bioavailability of fucoxanthin. The invention is simple and convenient to operate, has low cost and short production cycle, and is suitable for batch processing and industrialization. Description of the Drawings

[0018] Figure 1 Growth curves of Chlorella vulgaris FACHB-9 in autotrophic, mixotrophic, and heterotrophic modes in Example 1;

[0019] Figure 2 Protein concentrations of exosomes extracted from Chlorella vulgaris FACHB-9 in autotrophic, mixotrophic, and heterotrophic modes in Example 1;

[0020] Figure 3 TEM micrograph of exosomes extracted from Chlorella vulgaris FACHB-9 in heterotrophic mode in Example 2;

[0021] Figure 4 Fucoxanthin loading rates in microalgal exosome-fucoxanthin composite nanoparticles under the electroshock action of different voltages and capacitances in Example 3;

[0022] Figure 5 TEM micrograph of microalgal exosome-fucoxanthin composite nanoparticles in Example 4;

[0023] Figure 6 Particle sizes and Zeta potential diagrams of microalgal exosomes and microalgal exosome-fucoxanthin composite nanoparticles in three culture modes in Example 4;

[0024] Figure 7 Results of drug loading rate, entrapment rate, protein recovery rate, and FX loss rate of microalgal exosome-fucoxanthin composite nanoparticles in Example 5;

[0025] Figure 8Results of the stability determination of microalgal exosome-fucoxanthin composite nanoparticles in Example 6, where a shows the effect of ultraviolet lamp irradiation time on the stability of fucoxanthin in different samples, b shows the effect of heating time at 60 °C on the retention rate of fucoxanthin in different samples, and c shows the effect of storage time at room temperature on the retention rate of fucoxanthin in different samples;

[0026] Figure 9 Results of the antioxidant activity determination of different samples in Example 7, where a shows the ABTS scavenging rate of different samples and b shows the DPPH scavenging rate of different samples. Detailed implementation manners

[0027] The present invention will be further described in detail below in conjunction with the accompanying drawings and examples.

[0028] Example 1: Screening the culture mode for Chlorella vulgaris to produce more exosomes.

[0029] Autotrophic mode of Chlorella vulgaris: Inoculate a single colony of Chlorella sp FACHB-9 into 200 mL of BG11 medium and culture it in a shaker at 25 °C and 120 rpm for 6 - 8 days. Then, inoculate the seed solution into 600 mL of BG11 medium and culture it in a shaker at 25 °C and 120 rpm for 5 - 6 days. Collect the Chlorella vulgaris FACHB-9 algal solution. The formula of the BG11 medium is as follows: 1.5 g of NaNO3, 0.04 g of K2HPO4, 0.075 g of MgSO4·7H2O, 0.036 g of CaCl2·2H2O, 0.02 g of Na2CO3, 0.006 g of citric acid, 0.006 g of ferric citrate, 1 mL of trace element solution A5, and 1000 mL of distilled water. The formula of the trace element solution A5 is: 2.86 g / L of H3BO3, 1.81 g / L of MnCl2·4H2O, 0.222 g / L of ZnSO4, 0.39 g / L of Na2MoO4, 0.079 g / L of CuSO4·5H2O, and 0.049 g / L of Co(NO3)2·6H2O.

[0030] Mixotrophic mode of Chlorella vulgaris: Inoculate a single colony of Chlorella sp FACHB-9 into 200 mL of BG11 medium containing 2 g / L of glucose and culture it in a shaker at 25 °C and 120 rpm for 6 - 8 days. Then, inoculate the seed solution into 600 mL of BG11 medium containing 2 g / L of glucose and culture it in a shaker at 25 °C and 120 rpm for 5 - 6 days. Collect the Chlorella vulgaris FACHB-9 algal solution.

[0031] Heterotrophic mode of Chlorella vulgaris: Inoculate a single colony of Chlorella sp FACHB-9Inoculate into 200 mL of modified Endo medium and culture in a shaker at 25°C and 120 rpm for 6 - 8 days. Then, continue to inoculate the seed liquid into 600 mL of modified Endo medium and culture in a shaker at 25°C and 120 rpm for 5 - 6 days to collect the Chlorella vulgaris FACHB - 9 algal solution. The formula of the modified Endo medium is as follows: 28 g / L glucose, 1.2 g / L potassium dihydrogen phosphate, 4.8 g / L urea, 1.2 g / L magnesium sulfate, 105 mg / L calcium chloride dihydrate, 16 g / L ferrous sulfate heptahydrate, 2.1 mg / L disodium ethylenediaminetetraacetate, 0.2 g / L trisodium citrate, 2.86 mg / L boric acid, 0.222 mg / L zinc sulfate heptahydrate, 1.81 mg / L manganese chloride tetrahydrate, 0.025 mg / L sodium molybdate, 0.07 mg / L copper sulfate pentahydrate, and 1.2 mg / L cobalt nitrate hexahydrate.

[0032] Biomass determination method: Take 10 mL of the Chlorella vulgaris FACHB - 9 algal solution obtained under different culture modes, respectively filter through a 0.45 μm aqueous filter membrane by membrane filtration under suction. Take the retained matter, place it in an oven at 85°C for drying, and weigh it after 24 hours. Subtract the weight of the 0.45 μm aqueous filter membrane from the dried weight to obtain the biomass of the algal solution obtained under different culture modes.

[0033] Algal solution exosome extraction method: Collect 800 mL of the Chlorella vulgaris FACHB - 9 algal solution obtained under different culture modes, and use differential centrifugation method to centrifuge at 300 g for 10 min, 2,000 g for 20 min, and 10,000 g for 30 min in sequence at 4°C to remove cells and cell debris. Take the supernatant, filter through a 0.22 μm aqueous filter membrane, and then ultra - centrifuge at 100,000 g for 90 min. Take the precipitate and resuspend it with 2 mL of sterile PBS buffer to obtain an exosome suspension, and store it at - 80°C for subsequent experiments.

[0034] Protein concentration determination method: Take 100 μL of the exosome suspension extracted from the algal solution under different culture modes respectively, add 100 μL of lysis buffer according to a volume ratio of 1:1, ultrasonicate 3 times for 10 s, and then let it stand on ice for 30 min. The formula of the lysis buffer is as follows: 50 mM Tris - HCl (pH 7.4 - 8.0), 1% Triton X - 100, 10% sodium deoxycholate, 1 mM phenylmethylsulfonyl fluoride, 1 mM ethylenediaminetetraacetic acid.

[0035] Take 20 μL of different sample lysates into a 96-well microplate respectively, and then add 200 μL of BCA working solution (MA0082-2, Meilun Biotech) to each well. Incubate at 37 °C for 30 min, and measure the absorbance of the samples at 562 nm using a microplate reader. Calculate the exosome protein concentration according to the linear relationship between the absorbance value after subtracting the blank control and the exosome protein concentration.

[0036] Figure 1 are the growth curves of Chlorella FACHB-9 under autotrophic, mixotrophic, and heterotrophic modes. As Figure 1 can be seen, under the heterotrophic mode, Chlorella FACHB-9 reached the maximum biomass on the tenth day, which was 1.64 ± 0.06 g / L, 2.51 times and 1.33 times that of autotrophic and mixotrophic modes respectively, indicating that the biomass of Chlorella FACHB-9 under the heterotrophic mode was significantly higher than that of the other two culture modes.

[0037] Figure 2 are the exosome protein concentrations extracted from the algal solutions of Chlorella FACHB-9 under autotrophic, mixotrophic, and heterotrophic modes. The exosome concentration of Chlorella was measured by the BCA method. As Figure 2 can be seen, the exosome concentration of Chlorella extracted under the heterotrophic mode was 1.87 ± 0.08 mg / g algal dry weight, 3.01 and 1.76 times that of autotrophic and mixotrophic modes respectively, indicating that the protein concentration extracted from Chlorella FACHB-9 under the heterotrophic mode was significantly higher than that of the other two modes.

[0038] The above results show that Chlorella FACHB-9 cultured under the heterotrophic mode has relatively higher biomass and exosomes with higher protein concentration. Therefore, Chlorella FACHB-9 cultured under the heterotrophic mode was selected for subsequent experiments.

[0039] Example 2: Structural characterization of exosomes extracted from Chlorella cultured under the heterotrophic mode.

[0040] Transmission electron microscopy (TEM) measurement: Use a JEM-2100 transmission electron microscope to observe the morphological structure of exosomes of Chlorella FACHB-9 under the heterotrophic mode. Drop the exosome suspension extracted from the algal solution cultured under the heterotrophic mode in Example 1 onto a carbon-coated copper grid, let it stand at room temperature for 10 min, then remove the excess sample with filter paper. Then stain the copper grid with phosphotungstic acid for 10 s, remove the excess liquid with filter paper, wash it with PBS buffer for 1 min, remove the excess liquid with filter paper, dry it under an incandescent lamp, and observe the morphology of the newly prepared sample using a transmission electron microscope. Figure 3 is the transmission electron micrograph of exosomes of Chlorella FACHB-9 under the heterotrophic mode, as Figure 3As shown, the exosomes of Chlorella FACHB-9 have a typical structure with a round shape and partial depressions, resembling a "cathode ray tube" shape, and have a lipid bilayer structure, which is consistent with the structure of conventional exosomes.

[0041] Example 3. A method for preparing fucoxanthin nanoparticles using microalgal exosomes, the steps are as follows:

[0042] Step 1. Dissolve 5 mg of fucoxanthin in 1 mL of 95% ethanol, and stir with a magnetic stirrer at 800 rpm for 2 h to completely dissolve it, obtaining a fucoxanthin ethanol solution with a concentration of 5 mg / mL, denoted as FX.

[0043] Step 2. Add the exosome suspension extracted from the algal solution cultured in the heterotrophic mode in Example 1 to PBS buffer to obtain a microalgal exosome solution with a final protein concentration of 1 mg / mL, denoted as EV.

[0044] Step 3. Add the fucoxanthin ethanol solution and the microalgal exosome solution to a cuvette in a mass ratio of 1:1, co-incubate for 30 min, then add 2 volumes of electroporation buffer (1.15 mM potassium dihydrogen phosphate, 25 mM potassium chloride, 1.46 M sucrose, pH 7.2, filtered through a 0.22 μm filter) to increase the capacitance. Then use a BioRad electroporator to perform electroporation on the mixed solution in the cuvette, control the voltage at 100 - 500 V and the capacitance at 300 - 700 μF for electroshock, and then incubate at 37 °C for 1 h to restore the EV membrane structure. Centrifuge the mixed solution at 100,000 g at 4 °C for 30 min to remove the electroporation buffer and free FX, and collect the precipitate particles, which are the microalgal exosome-fucoxanthin composite nanoparticles, denoted as FX-EV. Figure 4 For the statistical results of the loading efficiency under different conditions, it can be seen that as the voltage increases, the drug loading rate first increases and then gradually decreases; as the capacitance increases, a similar result is also presented. From Figure 4 It can be known that under the conditions of a voltage of 200 V and a capacitance of 500 μF, the optimal fucoxanthin loading rate is 15.64 ± 3.12%. Therefore, this electroporation condition is selected for subsequent experiments. The mass ratio of the above fucoxanthin ethanol solution to the microalgal exosome solution can also be 0.5:1, 1.5:1, and any value within 0.5 - 1.5:1.

[0045] Example 4. Structural characterization of the microalgal exosome-fucoxanthin composite nanoparticles prepared in Example 3.

[0046] 1. Transmission electron microscopy (TEM) measurement: The JEM-2100 transmission electron microscope (TEM) was used to observe the morphological structure of the composite nanoparticles. The composite nanoparticles prepared in Example 3 were dissolved in PBS buffer and then dropped onto a carbon-coated copper grid. After standing at room temperature for 10 min, the excess sample was removed with filter paper. Then, the copper grid was stained with phosphotungstic acid for 10 s, and the excess liquid was removed with filter paper. After washing with PBS buffer for 1 min, the excess liquid was removed with filter paper, and the sample was dried under an incandescent lamp. The morphology of the newly prepared sample was observed using a transmission electron microscope. Figure 5 The transmission electron microscopy image of the FX-EV composite nanoparticles is shown in Figure 5 As shown, the microalgal exosome-fucoxanthin composite nanoparticles are spherical core-shell nanoparticles, and fucoxanthin is adsorbed in the lipid bilayer structure of the exosomes, which can prove the successful construction of this system.

[0047] 2. Particle size and ζ-potential measurement: The exosome suspension extracted from the algal solution cultured under different modes in Example 1, and the microalgal exosome-fucoxanthin composite nanoparticles prepared in Example 3 were dissolved in PBS buffer, and then the particle size and zeta potential of the samples to be measured were measured using a Malvern particle size analyzer. The purpose of dilution is to avoid the multiple scattering effect caused by the instrument. Each sample was measured three times, and the results were averaged. The results are shown in Figure 6 as follows.

[0048] As can be seen from Figure 6 it, the particle sizes of Chlorella vulgaris FACHB-9 and the FX-EV composite nanoparticles and their Zeta potentials under different culture modes are all within the range of 30 - 200 nm for exosome particle size and -10 - -30 mV for potential. It can be speculated that there may be a positive proportional relationship between the particle size of Chlorella vulgaris FACHB-9 and the protein concentration. The particle sizes of exosomes extracted under heterotrophic, mixotrophic, and autotrophic modes increase with the increase of protein concentration. Since fucoxanthin is encapsulated, the particle size of exosome protein further increases. Therefore, the FX-EV composite nanoparticles have the largest particle size.

[0049] Determination of the drug loading rate of the microalgal exosome-fucoxanthin composite nanoparticles prepared in Example 5 and Example 3.

[0050] Mix the FX-EV composite nanoparticles with methanol at a volume ratio of 1:1, and then use a probe-type ultrasonic crusher to ultrasonically treat the FX-EV mixture in an ice bath, set at 30% amplitude, on / off 5 / 10 s, for 50 cycles. Then centrifuge at 100,000 g for 30 min at 4 °C. After centrifugation, take the supernatant and measure the concentration and mass of FX in FX-EV using a high-performance liquid chromatograph (HPLC). The entire experimental process is carried out in the dark. The chromatographic column used is YMCC-30 (250×4.6 mm, 3 μm, Waters, Ireland). The mobile phase: A is methanol, B is water. The detection wavelength is 450 nm, the flow rate is 0.7 μm / min, the detection wavelength is 450 nm, the column temperature is 35 °C, the injection volume is 10 μL, and the total elution time is 25 min. At the same time, collect the precipitate and measure the protein content of the precipitate according to the BCA method in Example 1, which is the mass of EV in FX-EV.

[0051] Calculate the drug loading rate, encapsulation rate, protein recovery rate, and fucoxanthin loss rate of the FX-EV sample according to the following formulas.

[0052] Drug loading rate (%) = Mass of FX in FX-EV / Total mass of FX-EV * 100%;

[0053] Encapsulation rate (%) = Concentration of FX in FX-EV / Total concentration of FX * 100%, where the total concentration of FX is the concentration of FX in the mixed solution before electroporation in Step 3 of Example 3;

[0054] Protein recovery rate (%) = Mass of EV in FX-EV / Total mass of EV * 100%, where the total mass of FX is the concentration of the EV solution in the mixed solution before electroporation in Step 3 of Example 3 multiplied by the volume of the EV solution;

[0055] Fucoxanthin loss rate (%) = 100% - Encapsulation rate of FX.

[0056] Figure 7 For the statistical results of the drug loading rate, encapsulation rate, protein recovery rate, and fucoxanthin loss rate of the FX-EV composite nanoparticles, as Figure 7 shown, the drug loading rate of FX in the FX-EV composite nanoparticles is 15.64 ± 3.12%, the encapsulation rate is 87.03 ± 2.82%, the protein recovery rate is 97.82 ± 2.04%, and the fucoxanthin loss rate is 12.98 ± 2.82%.

[0057] Determination of the stability of the microalgae exosome-fucoxanthin composite nanoparticles prepared in Example 6 and Example 3.

[0058] 1. Photostability determination: Dissolve FX in methanol and dissolve the FX-EV composite nanoparticle sample in PBS buffer, with a final concentration of 2 mg / mL for both. Expose the samples to 5 W, 352 nm ultraviolet light at 25 °C for 1, 2, 3, 4, and 5 h to determine the photostability. Take 1 mL of the sample every 1 h, set up three parallels, and then use the method in Example 5 to determine the fucoxanthin content in the sample. The calculation formula for the fucoxanthin retention rate is as follows: Retention rate (%) = content of fucoxanthin in the sample at different treatment time points / original content of fucoxanthin in the untreated sample.

[0059] Figure 8 In a, it shows the effect of different times on the photostability of fucoxanthin in different states. After 5 h of ultraviolet lamp irradiation, the retention rate of free FX is 14.01 ± 1.14%, while the retention rate of FX in the FX-EV sample is 36.52 ± 1.75%, which is significantly higher than that of free fucoxanthin, indicating that the construction of this system can effectively improve the photostability of fucoxanthin and overcome the instability of fucoxanthin under light conditions.

[0060] 2. Thermal stability determination: Dissolve FX in methanol and dissolve the FX-EV composite nanoparticle sample in PBS buffer, with a final concentration of 2 mg / mL for both. Heat the samples in a water bath at 60 °C for 30, 60, 90, 120, and 150 minutes. Take 1 mL of the sample every 30 min, set up three parallels, and then determine the fucoxanthin content in the sample, and further calculate the fucoxanthin retention rate using the above formula.

[0061] Figure 8 In b, it shows the effect of different times on the thermal stability of fucoxanthin in different states. After heating at 60 °C for 150 min, the retention rate of free FX is 47.62 ± 2.26%, while the retention rate of FX in the FX-EV sample is 63.27 ± 1.84%, which is significantly higher than that of free fucoxanthin, indicating that the construction of this system can effectively improve the thermal stability of fucoxanthin and overcome the instability of fucoxanthin under heating conditions.

[0062] 3. Room temperature stability determination: Dissolve FX in methanol and dissolve the FX-EV composite nanoparticle sample in PBS buffer, with a final concentration of 2 mg / mL for both. Store the samples in the dark at room temperature for 3, 6, 9, 12, and 15 days to determine the storage stability. Take 1 mL of the sample every 3 days, set up three parallels, and then determine the fucoxanthin content in the sample, and further calculate the fucoxanthin retention rate using the above formula.

[0063] Figure 8In c, the effect of different storage times at room temperature on the stability of fucoxanthin in different states was studied. After 12 days of storage in the dark at room temperature, the retention rate of free FX was 13.28 ± 2.20%, while the retention rate of FX in the FX-EV sample could be maintained above 70%, which was significantly higher than that of free fucoxanthin. This indicates that the construction of this system can effectively improve the storage stability of fucoxanthin and overcome the instability of fucoxanthin during long-term storage at room temperature. The above results show that the construction of the Chlorella vulgaris exosome-fucoxanthin composite nanoparticle system can overcome the instability of fucoxanthin under light, heat, and room temperature storage, and the construction of this system can effectively improve the stability of fucoxanthin.

[0064] Example 7: Determination of the antioxidant activity of the microalgal exosome-fucoxanthin composite nanoparticles prepared in Example 3.

[0065] ABTS radical scavenging experiment:

[0066] Step 1: After ABTS (2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid) is oxidized, a relatively stable blue-green cationic radical ABTS·⁺ is generated. A 7 mM ABTS solution is mixed with an equal volume of a 140 mM potassium persulfate solution and allowed to react statically for 12 hours at room temperature in the dark. The above mixture is diluted with distilled water to an absorbance of 0.7 at 734 nm to obtain the ABTS working solution.

[0067] Step 2: The FX-EV composite nanoparticles prepared in Example 3 are dissolved in PBS buffer to obtain an FX-EV solution with a loaded FX concentration of 200 μg / mL and a wall material EV concentration of 1 mg / mL. 50 μL of the FX-EV solution, 50 μL of an FX solution with a concentration of 200 μg / mL, 50 μL of an EV solution with a concentration of 1 mg / mL, and 150 μL of the ABTS working solution are mixed as experimental group Ai, distilled water is used instead of the ABTS working solution as control group Aj, and distilled water is used instead of the sample as blank group A0. At the same time, 50 μL of a Vc solution with a concentration of 200 μg / mL is weighed as a positive control. All group samples are added to a 96-well microplate and reacted in the dark for 30 min. Three parallels are set. After the reaction, the absorbance value (A) of the solution in each well is measured using a microplate reader at a wavelength of 734 nm.

[0068] According to the measured absorbance value, the scavenging rate (%) of the antioxidant substance for ABTS·⁺ is calculated using the following formula: ABTS scavenging rate (%) = [1 - (Ai - Aj) / A0] * 100%. Figure 9As can be seen from a, the ABTS radical scavenging rates of FX and EV were 38.21±1.11% and 26.62±1.02% respectively, while that of FX-EV was 50.58±1.05%. The synergistic effect between FX and EV in the FX-EV composite nanoparticles significantly improved the ABTS radical scavenging rate of the composite nanoparticles.

[0069] 2. DPPH radical scavenging experiment:

[0070] DPPH (1,1-diphenyl-2-picrylhydrazyl) is a stable free radical, which shows a deep purple color in ethanol solution and has a strong absorption peak at 517 nm. When an antioxidant is present, its lone pair of electrons is paired, causing the color of the DPPH radical solution to fade and the absorbance to decrease. The ability of the antioxidant to scavenge DPPH radicals can be evaluated by measuring the change in absorbance.

[0071] The FX-EV composite nanoparticles prepared in Example 3 were dissolved in PBS buffer to obtain an FX-EV solution with a loaded FX concentration of 200 μg / mL and a wall material EV concentration of 1 mg / mL. 50 μL of the FX-EV solution, 50 μL of the FX solution with a concentration of 200 μg / mL, 50 μL of the EV solution with a concentration of 1 mg / mL were respectively mixed with 0.5 mL of 0.2 mM DPPH ethanol solution as experimental group Ai, anhydrous ethanol was used instead of DPPH ethanol solution as control group Aj, and distilled water was used instead of the sample as blank group A0. 50 μL of a Vc solution with a concentration of 200 μg / mL was weighed as a positive control. All group samples were added to a 96-well microplate and reacted in the dark for 30 min. Three parallels were set. After the reaction, the absorbance value (A) of the solution in each well was measured at a wavelength of 517 nm using a microplate reader.

[0072] According to the measured absorbance value, the scavenging rate (%) of the antioxidant against DPPH was calculated using the following formula: DPPH scavenging rate (%) = [1-(Ai-Aj) / A0]*100%. As Figure 9 can be seen from b, the DPPH radical scavenging rates of FX and EV were 31.31±1.08% and 26.21±1.12% respectively, while that of FX-EV was 44.48±1.04%. The synergistic effect between FX and EV in the FX-EV composite nanoparticles significantly improved the DPPH radical scavenging rate of the composite nanoparticles.

[0073] The above results indicate that the construction of the Chlorella vulgaris exosome-fucoxanthin composite nanoparticle system can effectively improve the bioavailability of fucoxanthin. The conjugated double bond system in the chemical structure of fucoxanthin itself makes it vulnerable to oxidation and lose its activity. The encapsulation of exosomes can reduce the damage of these adverse factors to fucoxanthin, thus maintaining its structural integrity, keeping its antioxidant activity before reaching the action site, and then potentially enhancing its ability to exert antioxidant effects in vivo. In addition, exosomes themselves contain some components with antioxidant activity, such as certain proteins, nucleic acids, and lipids, which may produce a synergistic antioxidant effect when acting together with fucoxanthin.

[0074] The above description is not a limitation of the present invention, nor is the present invention limited to the above examples. Changes, modifications, additions, or substitutions made by those of ordinary skill in the art within the scope of the essence of the present invention shall also fall within the protection scope of the present invention.

Claims

1. A preparation method of a microalgae exosome-fucoxanthin composite nanoparticle, characterized in that Including the following steps: After adding the fucoxanthin ethanol solution and the microalgal exosome solution into a cuvette at a mass ratio of 0.5 - 1.5:1 and co-incubating for 30 min, add an electroporation buffer with a volume 1 - 3 times that of the mixed solution. Then use an electroporator to perform electroporation on the mixed solution in the cuvette, control the voltage at 100 - 500 V and the capacitance at 300 - 700 μF for electric shock. Then incubate at 35 - 40 °C for 0.5 - 2 h, and then centrifuge the mixed solution at 80,000 - 120,000 g at 4 °C for 20 - 40 min to collect the precipitate particles, thus obtaining microalgal exosome-fucoxanthin composite nanoparticles. The preparation method of the fucoxanthin ethanol solution is as follows: Dissolve fucoxanthin in 95% ethanol and stir with a magnetic stirrer until it is completely dissolved to obtain a fucoxanthin ethanol solution with a concentration of 5 mg / mL. The preparation method of the microalgal exosome solution is as follows: For the Chlorella vulgaris algal solution obtained by cultivation, successively centrifuge at 200 - 400 g for 8 - 12 min, 1500 - 2500 g for 15 - 25 min, and 8,000 - 12,000 g for 25 - 35 min at 4 °C by differential centrifugation to remove cells and cell debris. Take the supernatant, filter it through a 0.22 μm aqueous filter membrane, and then ultracentrifuge at 80,000 - 1,200,000 g for 80 - 100 min. Take the precipitate and resuspend it with sterile PBS buffer to obtain a microalgal exosome solution with a final protein concentration of 1 mg / mL. The Chlorella vulgaris is cultured in a heterotrophic mode as follows: Inoculate the Chlorella spFACHB-9 single colony into 200 mL of modified Endo medium and culture it in a shaker at 25 °C and 120 rpm for 6 - 8 days. Then continue to inoculate the seed solution into 600 mL of modified Endo medium and culture it in a shaker at 25 °C and 120 rpm for 5 - 6 days to collect the Chlorella vulgaris FACHB-9 algal solution. The formula of the modified Endo medium is as follows: 28 g / L glucose, 1.2 g / L potassium dihydrogen phosphate, 4.8 g / L urea, 1.2 g / L magnesium sulfate, 105 mg / L calcium chloride dihydrate, 16 g / L ferrous sulfate heptahydrate, 2.1 mg / L disodium ethylenediaminetetraacetate, 0.2 g / L sodium citrate, 2.86 mg / L boric acid, 0.222 mg / L zinc sulfate heptahydrate, 1.81 mg / L manganese chloride tetrahydrate, 0.025 mg / L sodium molybdate, 0.07 mg / L copper sulfate pentahydrate, and 1.2 mg / L cobalt nitrate hexahydrate. The formula of the electroporation buffer is as follows: 1.15 mM potassium dihydrogen phosphate, 25 mM potassium chloride, 1.46 M sucrose, pH 7.

2.

2. The preparation method of a microalgae exosome-fucoxanthin composite nanoparticle according to claim 1, wherein: The mass ratio of the fucoxanthin ethanol solution to the microalgal exosome solution is 1:

1.

3. Use of the microalgal exosome-fucoxanthin composite nanoparticles prepared by the method according to claim 1 in the preparation of an antioxidant.

4. Use of the microalgal exosome-fucoxanthin composite nanoparticles prepared by the method according to claim 1 in the preparation of an ABTS radical scavenger and / or a DPPH radical scavenger.

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