Extraction method of blueberry source exosome-like nanoparticles

Abstract

The invention relates to the technical field of exosome separation, and particularly discloses an extraction method of blueberry source exosome-like nanoparticles. The extraction method comprises the following steps: performing differential centrifugation on blueberry juice to obtain blueberry centrifugate; adding magnetic polymer microspheres into the blueberry centrifugate, and incubating to obtain an incubation solution; and carrying out solid-liquid separation on the incubation liquid by adopting a magnetic field to obtain the blueberry source exosome-like nanoparticles. According to the method, differential centrifugation and magnetic separation are combined, so that the complex exosome extraction process is effectively simplified, the operation difficulty and the time cost are reduced, the extraction concentration and the purity of the blueberry exosome-like nanoparticles are remarkably improved, and the structure of the exosome cannot be damaged. Meanwhile, the process has good repeatability and stability, large-scale production and extraction are facilitated, the extraction efficiency is further improved, the extraction of the blueberry source exosome-like nanoparticles can be rapidly and efficiently carried out, and the process has a relatively high industrial prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of exosome separation, and in particular to a method for extracting blueberry-derived exosome-like nanoparticles. Background Art

[0002] Exosomes (exosomes) are nanoscale extracellular vesicles secreted by cells. They contain a variety of proteins, lipids, and nucleic acids and play important physiological roles by mediating cell-to-cell communication. They are secreted by virtually all eukaryotic and prokaryotic cell types. Plant exosomes are morphologically similar to mammalian exosomes, but their composition and function are less well-studied. Studies have found that compared with animal exosomes, plant exosomes offer advantages such as lower cost, reduced immunogenicity, greater stability, and higher natural medicinal value.

[0003] Blueberry, a flowering plant belonging to the Ericaceae family, contains polyphenols, anthocyanins, and flavonoids. The exosome-like vesicles it secretes, called blueberry exosome-like nanoparticles (BELNs), possess antioxidant, anti-inflammatory, and anti-tumor activities. Furthermore, the unique structure of vitamin C, abundant in blueberries, provides liver protection and improves liver dysfunction.

[0004] At present, exosome extraction methods still face some technical bottlenecks. Ultracentrifugation is one of the mainstream methods for isolating exosomes. Although it is widely used, it has obvious disadvantages: on the one hand, its extraction concentration is low; on the other hand, the high-speed centrifugation process easily destroys the membrane structure of the exosomes, affecting the integrity and activity of the exosomes. The polymer precipitation method is often used in commercial exosome extraction kits. This method uses polyethylene glycol (PEG) as a medium to reduce the solubility of exosomes and achieve sedimentation separation under low-speed centrifugation conditions. However, this method has problems such as poor extraction purity and long operation time. Given the limitations of existing extraction methods, it is urgent to develop a new exosome extraction technology to overcome the above technical difficulties. Summary of the Invention

[0005] In response to the problems of existing exosome extraction methods in the art, such as low extraction concentration or purity, easy destruction of exosome structure, and long operation time, the present invention provides a method for extracting blueberry-derived exosome-like nanoparticles.

[0006] In order to solve the above technical problems, the technical solution provided by the present invention is:

[0007] A method for extracting blueberry-derived exosome-like nanoparticles comprises the following steps:

[0008] Step a, crushing fresh blueberries, squeezing juice, and filtering to obtain blueberry juice; performing differential centrifugation on the blueberry juice to obtain blueberry centrifuge liquid;

[0009] Step b, adding magnetic polymer microspheres to the blueberry centrifuge, mixing evenly, and incubating to obtain an incubation solution; wherein the magnetic polymer microspheres include Fe3O4 nanoparticle cores and poly (N-isopropylacrylamide-co-PEG methacrylate) cross-linked polymers coated on the outside;

[0010] Step c: using a magnetic field to perform solid-liquid separation on the incubation solution to obtain blueberry-derived exosome-like nanoparticles.

[0011] Compared with the prior art, the extraction method of blueberry-derived exosome-like nanoparticles provided by the present invention first uses differential centrifugation to perform preliminary separation of blueberry juice, effectively removing impurities such as cell debris and larger organelles, reducing interference with subsequent separation; then, magnetic polymer microspheres containing Fe3O4 nanoparticles coated with poly(N-isopropylacrylamide-co-PEG methacrylate) cross-linked polymer are used as a separation agent. Among them, the poly(N-isopropylacrylamide-co-PEG methacrylate) cross-linked polymer has unique molecular recognition ability and affinity, and can specifically bind to characteristic molecules on the surface of blueberry-derived exosome-like nanoparticles, reducing nonspecific adsorption with miscellaneous proteins in the sample, thereby accurately enriching exosome-like nanoparticles from complex blueberry centrifuge fluid, effectively avoiding co-precipitation of other impurity particles, and greatly improving the purity and extraction concentration of the extraction product. At the same time, the poly(N-isopropylacrylamide-co-PEG methacrylate) cross-linked polymer has excellent biocompatibility. During the binding process with exosomes, it does not chemically react with the lipids and proteins on the exosome membrane, thus avoiding damage to the exosome structure caused by chemical reactions and ensuring the integrity and functional activity of the exosomes. Finally, under the action of an external magnetic field, the magnetic microspheres combined with exosome-like nanoparticles can quickly move and aggregate in a directional manner within a short period of time, achieving efficient separation from the liquid. Compared with traditional mechanical separation methods such as filtration and high-speed centrifugation, this avoids physical damage to the exosome structure caused by severe mechanical forces, maximally protecting the structural integrity of the exosomes, greatly shortening the separation time, and significantly improving the overall extraction efficiency.

[0012] Specifically, in step a, fresh blueberries are washed, added into PBS buffer, juiced using a wall breaking machine, and filtered through gauze to obtain blueberry juice.

[0013] Furthermore, the mass ratio of the PBS buffer solution to blueberries is (1-4):1.

[0014] Specifically, the pH of the PBS buffer is 7.2-7.6.

[0015] Furthermore, in step a, the differential centrifugation specifically comprises the following steps:

[0016] The blueberry juice is centrifuged at 800g-1200g for 5min-10min, the supernatant is centrifuged at 2800g-3200g for 10min-20min, and finally the supernatant is centrifuged at 9000g-11000g for 20min-30min, filtered, and blueberry centrifuge liquid is obtained.

[0017] The optimized differential centrifugation process allows impurities such as cell fragments and larger organelles in blueberries to be gradually separated in a relatively mild environment. While ensuring that the impurity content is minimized, it effectively reduces the risk of damage to the exosome structure, creates favorable conditions for the subsequent addition of magnetic polymer microspheres for targeted enrichment, and is conducive to improving the subsequent magnetic separation efficiency.

[0018] It should be noted that the entire centrifugation process was carried out at 3°C ​​to 6°C.

[0019] Furthermore, in step a, after the differential centrifugation is completed, the supernatant is filtered through a 0.22 μm filter membrane to obtain a blueberry centrifuge liquid.

[0020] Furthermore, in step b, the mass ratio of the blueberry centrifuge liquid to the magnetic polymer microspheres is (10-15):1.

[0021] Furthermore, in step b, the specific steps of incubation include: first incubating at 3°C ​​to 6°C for 30 minutes to 40 minutes; and then incubating at 35°C to 40°C for 5 minutes to 10 minutes.

[0022] At the same time, the incubation temperature was initially controlled between 3°C and 6°C. The poly(N-isopropylacrylamide-co-PEG methacrylate) cross-linked polymer expands at low temperatures, allowing its active groups to more fully recognize and bind to characteristic molecules on the exosome surface. Furthermore, the relatively long incubation time at low temperatures provides ample time for the exosomes to bind to the magnetic polymer microspheres, ensuring the capture of as many exosomes as possible, significantly improving the extraction efficiency and subsequent enrichment concentration of exosomes. Furthermore, the low temperature inhibits nonspecific adsorption of impurities to the magnetic polymer microspheres, further enhancing the purity of the extracted product. The temperature was then raised to 35°C to 40°C for 5 to 10 minutes. Within this temperature range, the cross-linked polymer transitions from an expanded state to a contracted state, further shortening the distance to the exosome binding site and strengthening the binding force between the two. This results in a more secure bond between the exosomes and the magnetic polymer microspheres, reducing exosome shedding due to external forces during the subsequent separation process. This ensures efficient enrichment of exosomes during the separation process, further improving extraction efficiency and purity.

[0023] In addition, since the step-by-step incubation effectively improves the specificity of the binding of exosomes to magnetic polymer microspheres and reduces the nonspecific adsorption of impurities, it is beneficial to simplify the subsequent washing steps. It also reduces the loss of exosomes during the washing process, further improving the extraction efficiency and final yield of exosomes.

[0024] It should be noted that in step c, the incubation solution is subjected to solid-liquid separation using a magnetic field to obtain magnetic microspheres bound to exosome-like nanoparticles, which are then washed and redispersed in pre-cooled PBS buffer. The mixture is then allowed to stand at 3°C ​​to 6°C for 15 to 30 minutes, and then placed next to a strong magnet again. The mixture is allowed to stand at 3°C ​​to 6°C for 5 to 10 minutes and filtered to obtain a blueberry exosome-like nanoparticle suspension.

[0025] Specifically, step c includes the following steps: placing the incubation solution next to a strong magnet, letting it stand for 5 minutes to 10 minutes, discarding the supernatant to obtain a precipitate; gently washing the obtained precipitate 2 to 3 times with PBS buffer precooled to 3°C to 6°C, and then redispersing it in PBS buffer precooled to 3°C to 6°C, letting it stand for 15 minutes to 30 minutes, placing it next to a strong magnet, letting it stand for 5 minutes to 10 minutes, taking the supernatant and filtering it with a 0.22μm filter membrane to obtain a blueberry exosome-like nanoparticle suspension.

[0026] It should be noted that the ambient temperature is maintained at 3°C ​​to 6°C during the above-mentioned standing process.

[0027] Under low temperature conditions, the molecular chains of poly (N-isopropylacrylamide-co-PEG methacrylate) cross-linked polymers will stretch again, weakening the binding force with exosomes and releasing exosomes.

[0028] As a specific embodiment of the present invention, the preparation method of the magnetic polymer microspheres comprises the following steps:

[0029] S1, synthesis of Fe3O4 nanoparticles;

[0030] S2, dissolving N-isopropyl propionamide, polyethylene glycol methacrylate, and N,N'-methylenebisacrylamide in water, adding an initiator and an organic amine reducing agent, and performing a polymerization reaction to obtain a poly(N-isopropyl acrylamide-co-PEG methacrylate) cross-linked polymer;

[0031] S3, dispersing the Fe3O4 nanoparticles and poly (N-isopropylacrylamide-co-PEG methacrylate) cross-linked polymer in water, adding sodium lauryl sulfate to obtain a mixed dispersion; adding the mixed dispersion to cyclohexane containing Span 80, mixing evenly, drying, and magnetically separating the microspheres to obtain magnetic polymer microspheres.

[0032] Furthermore, S1 specifically includes the following steps:

[0033] Dissolve ferric salt and ferrous salt in water, adjust the pH of the system to 10-11, add sodium citrate, stir and react at 70-80°C for 1-1.5 hours, separate the solid and liquid, wash and dry to obtain Fe3O4 nanoparticles.

[0034] Specifically, the ferric salt is ferric chloride, and the ferrous salt is ferrous chloride.

[0035] Furthermore, in combination with the above, the molar ratio of the iron salt to the ferrous salt is (1.8-2.2):1.

[0036] Furthermore, in combination with the above, the mass ratio of the sodium citrate to the ferrous salt is 1:(1-2).

[0037] Furthermore, in combination with the above, the mass ratio of the iron salt to water is (40-60):1.

[0038] Furthermore, in S2, the mass ratio of the N-isopropylpropionamide to polyethylene glycol methacrylate is (4-6):1.

[0039] Furthermore, in S2, the mass ratio of N-isopropylpropionamide to N,N'-methylenebisacrylamide is (40-50):1.

[0040] Furthermore, in S2, the mass ratio of the N-isopropylpropionamide to the initiator is (40-50):1.

[0041] Furthermore, in S2, the mass ratio of the N-isopropylpropionamide to the organic amine reducing agent is (40-50):1.

[0042] Furthermore, in S2, the mass ratio of the N-isopropylpropionamide to water is 1:(20-25).

[0043] Furthermore, in S2, the polymerization reaction temperature is 10°C to 30°C, and the reaction time is 6h to 8h.

[0044] Furthermore, in S2, the initiator is ammonium persulfate.

[0045] Furthermore, in S2, the organic amine reducing agent is tetramethylethylenediamine.

[0046] Furthermore, in S3, the mass ratio of the poly(N-isopropylacrylamide-co-PEG methacrylate) cross-linked polymer to sodium lauryl sulfate is (10-15):1.

[0047] Furthermore, in S3, the mass ratio of the Fe3O4 nanoparticles to the poly(N-isopropylacrylamide-co-PEG methacrylate) cross-linked polymer is 1:(4-6).

[0048] Furthermore, in S3, the mass ratio of the mixed dispersion to the cyclohexane containing Span 80 is 1:(1-2).

[0049] Furthermore, in S3, the mass ratio of cyclohexane to Span 80 is (50-60):1.

[0050] By combining differential centrifugation and magnetic separation, this method effectively simplifies the complex exosome extraction process, reducing operational difficulty and time costs, significantly improving the concentration and purity of extracted blueberry exosome-like nanoparticles without damaging the exosome structure. Furthermore, this process exhibits excellent repeatability and stability, facilitating large-scale production and extraction, further improving extraction efficiency. This allows for rapid and efficient extraction of blueberry-derived exosome-like nanoparticles, and holds great promise for industrialization. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 This is the particle size distribution diagram of the blueberry-derived exosome nanoparticles extracted in Example 1;

[0052] Figure 2 This is the particle size distribution diagram of blueberry-derived exosome nanoparticles extracted in Comparative Example 1;

[0053] Figure 3 This is the particle size distribution diagram of blueberry-derived exosome nanoparticles extracted in Comparative Example 2;

[0054] Figure 4 This is the particle size distribution diagram of blueberry-derived exosome nanoparticles extracted in Comparative Example 3;

[0055] Figure 5 This is the particle size distribution diagram of blueberry-derived exosome nanoparticles extracted in Comparative Example 4;

[0056] Figure 6 TEM images of blueberry-derived exosome nanoparticles extracted from Example 1 and Comparative Examples 1 to 4, wherein (a) Comparative Example 1, (b) Example 1, (c) Comparative Example 2, (d) Comparative Example 3, and (e) Comparative Example 4;

[0057] Figure 7 This is a comparison chart of the protein concentrations of blueberry-derived exosome nanoparticles extracted in Example 1 and Comparative Examples 1 to 4. DETAILED DESCRIPTION

[0058] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0059] In order to better illustrate the present invention, further examples are given below.

[0060] The pH of the PBS buffer used in the following examples and comparative examples is 7.2-7.6.

[0061] Example 1

[0062] This embodiment provides a method for preparing blueberry-derived exosome nanoparticles, comprising the following steps:

[0063] Step a: wash fresh blueberries, add them to PBS buffer in an amount twice their weight, add them to a juicer to extract the juice, and filter with gauze to obtain blueberry juice;

[0064] Step b, centrifuging the blueberry juice at 1000 g for 8 minutes at 4° C., centrifuging the supernatant at 3000 g for 15 minutes, and finally centrifuging the supernatant at 10000 g for 25 minutes. The supernatant was filtered through a 0.22 μm filter membrane to obtain a blueberry centrifuge;

[0065] Step c, adding the blueberry centrifuge liquid to a sample tube, and then adding 1 / 10 of the mass of the blueberry centrifuge liquid to the sample tube, inverting and mixing thoroughly, incubating at 4°C for 35 minutes, and then incubating at 38°C for 8 minutes to obtain an incubation solution;

[0066] Step d, place the sample tube next to a strong magnet, let it stand at 4°C for 10 minutes, discard the supernatant to obtain a precipitate; the obtained precipitate is gently washed twice with PBS buffer precooled to 4°C to remove unbound impurities, and then redispersed in PBS buffer precooled to 4°C, let it stand at 4°C for 20 minutes, place it next to a strong magnet, and let it stand at 4°C for another 10 minutes. Take the supernatant and filter it with a 0.22μm filter membrane to obtain a blueberry exosome-like nanoparticle suspension.

[0067] The preparation method of the magnetic polymer microspheres comprises the following steps:

[0068] S1, 1.09 g of FeCl3·6H2O and 0.4 g of FeCl2·4H2O were dissolved in 50 mL of deionized water, magnetically heated to 75°C, ammonia was added to adjust the pH to 10.6, 0.5 g of sodium citrate was added, and the mixture was kept warm and stirred for 1 h. The Fe3O4 nanoparticles were separated using a magnet, then washed with deionized water and dispersed in deionized water to obtain a 10 mg / mL Fe3O4 nanoparticle dispersion.

[0069] S2, dissolving 2 g of N-isopropylacrylamide, 0.5 g of PEG methacrylate, and 0.05 g of N,N'-methylenebisacrylamide in 40 mL of deionized water, adding 0.05 g of ammonium persulfate and 0.05 g of tetramethylethylenediamine, and magnetically stirring at room temperature for 6 h. The reaction solution was dialyzed (molecular weight cut-off 10 kDa) for 48 h to remove unreacted monomers, and lyophilized to obtain a poly(N-isopropylacrylamide-co-PEG methacrylate) cross-linked polymer;

[0070] S3, disperse 1 g of the poly (N-isopropylacrylamide-co-PEG methacrylate) cross-linked polymer prepared above in 50 mL of deionized water, add 20 mL of the Fe3O4 nanoparticle dispersion prepared above and 0.1 g of sodium dodecyl sulfate, ultrasonically disperse for 30 min, add it to cyclohexane (110 g) containing Span 80 (2 g), ultrasonically treat for 15 min in an ice bath, react in a vacuum drying oven at 60°C for 12 h, collect the microspheres by magnetic separation, wash with anhydrous ethanol and acetone in sequence, and freeze-dry to obtain magnetic polymer microspheres.

[0071] Example 2

[0072] This embodiment provides a method for preparing blueberry-derived exosome nanoparticles, comprising the following steps:

[0073] Step a: wash fresh blueberries, add them to 1 times the weight of PBS buffer, add them to a juicer to extract the juice, and filter with gauze to obtain blueberry juice;

[0074] Step b, centrifuging the blueberry juice at 800 g for 10 min at 6° C., centrifuging the supernatant at 2800 g for 20 min, and finally centrifuging the supernatant at 9000 g for 30 min. The supernatant was filtered through a 0.22 μm filter membrane to obtain a blueberry centrifuge;

[0075] Step c, adding the blueberry centrifuge liquid to a sample tube, and then adding 1 / 15 of the blueberry centrifuge liquid by weight of magnetic polymer microspheres thereto, inverting and mixing thoroughly, incubating at 6° C. for 30 minutes, and then incubating at 35° C. for 10 minutes to obtain an incubation solution;

[0076] Step d, place the sample tube next to a strong magnet, let it stand at 6°C for 5 minutes, discard the supernatant to obtain a precipitate; the obtained precipitate is gently washed three times with PBS buffer precooled to 6°C to remove unbound impurities, and then redispersed in PBS buffer precooled to 6°C, let it stand at 6°C for 15 minutes, place it next to a strong magnet, and let it stand at 6°C for another 5 minutes. Take the supernatant and filter it with a 0.22μm filter membrane to obtain a blueberry exosome-like nanoparticle suspension.

[0077] The preparation method of the magnetic polymer microspheres comprises the following steps:

[0078] S1, 0.98 g of FeCl3·6H2O and 0.4 g of FeCl2·4H2O were dissolved in 58 mL of deionized water, magnetically heated to 70°C, and ammonia was added to adjust the pH to 10.8. 0.4 g of sodium citrate was added, and the mixture was kept warm and stirred for 1.5 h. The Fe3O4 nanoparticles were separated using a magnet, washed with deionized water, and dispersed in deionized water to obtain an 8 mg / mL Fe3O4 nanoparticle dispersion.

[0079] S2, dissolving 2.5 g of N-isopropylacrylamide, 0.5 g of PEG methacrylate, and 0.05 g of N,N'-methylenebisacrylamide in 60 mL of deionized water, adding 0.05 g of ammonium persulfate and 0.05 g of tetramethylethylenediamine, and magnetically stirring at room temperature for 7 h. The reaction solution was dialyzed (molecular weight cut-off 10 kDa) for 48 h to remove unreacted monomers, and lyophilized to obtain a poly(N-isopropylacrylamide-co-PEG methacrylate) cross-linked polymer;

[0080] S3, disperse 1 g of the poly (N-isopropylacrylamide-co-PEG methacrylate) cross-linked polymer prepared above in 50 mL of deionized water, add 25 mL of the Fe3O4 nanoparticle dispersion prepared above and 0.08 g of sodium dodecyl sulfate, ultrasonically disperse for 30 minutes, add it to cyclohexane (120 g) containing Span 80 (2 g), ultrasonically treat for 15 minutes in an ice bath, react in a vacuum drying oven at 60°C for 13 hours, collect the microspheres by magnetic separation, wash with anhydrous ethanol and acetone in sequence, and freeze-dry to obtain magnetic polymer microspheres.

[0081] Example 3

[0082] This embodiment provides a method for preparing blueberry-derived exosome nanoparticles, comprising the following steps:

[0083] Step a: wash fresh blueberries, add them to PBS buffer in an amount 4 times their weight, add them to a juicer to extract the juice, and filter with gauze to obtain blueberry juice;

[0084] Step b, centrifuging the blueberry juice at 1200 g for 5 min at 3° C., centrifuging the supernatant at 3200 g for 10 min, and finally centrifuging the supernatant at 11000 g for 20 min. The supernatant was filtered through a 0.22 μm filter membrane to obtain a blueberry centrifuge;

[0085] Step c, adding the blueberry centrifuge liquid to a sample tube, and then adding 1 / 12 of the blueberry centrifuge liquid by weight of magnetic polymer microspheres thereto, inverting and mixing thoroughly, incubating at 3°C ​​for 40 minutes, and then incubating at 40°C for 5 minutes to obtain an incubation solution;

[0086] Step d, place the sample tube next to a strong magnet, let it stand at 3°C ​​for 10 minutes, discard the supernatant to obtain a precipitate; the obtained precipitate is gently washed three times with PBS buffer precooled to 3°C to remove unbound impurities, and then redispersed in PBS buffer precooled to 3°C, let it stand at 3°C ​​for 30 minutes, place it next to a strong magnet, and let it stand at 3°C ​​for another 10 minutes. Take the supernatant and filter it with a 0.22μm filter membrane to obtain a blueberry exosome-like nanoparticle suspension.

[0087] The preparation method of the magnetic polymer microspheres comprises the following steps:

[0088] S1, 1.20 g of FeCl3·6H2O and 0.4 g of FeCl2·4H2O were dissolved in 48 mL of deionized water, magnetically heated to 80°C, and ammonia was added to adjust the pH to 10.2. 0.8 g of sodium citrate was added, and the mixture was kept warm and stirred for 1 h. The Fe3O4 nanoparticles were separated using a magnet, washed with deionized water, and dispersed in deionized water to obtain a 12 mg / mL Fe3O4 nanoparticle dispersion.

[0089] S2, 3 g of N-isopropylacrylamide, 0.5 g of PEG methacrylate, and 0.08 g of N,N'-methylenebisacrylamide were dissolved in 75 mL of deionized water, 0.06 g of ammonium persulfate and 0.06 g of tetramethylethylenediamine were added, and the mixture was magnetically stirred at room temperature for 8 h. The reaction solution was dialyzed (molecular weight cut-off 10 kDa) for 48 h to remove unreacted monomers, and then lyophilized to obtain a poly(N-isopropylacrylamide-co-PEG methacrylate) cross-linked polymer;

[0090] S3, disperse 1 g of the poly (N-isopropylacrylamide-co-PEG methacrylate) cross-linked polymer prepared above in 50 mL of deionized water, add 20 mL of the Fe3O4 nanoparticle dispersion prepared above and 0.07 g of sodium dodecyl sulfate, ultrasonically disperse for 30 min, add it to cyclohexane (100 g) containing Span 80 (2 g), ultrasonically treat for 15 min in an ice bath, react in a vacuum drying oven at 60°C for 15 h, collect the microspheres by magnetic separation, wash with anhydrous ethanol and acetone in sequence, and freeze-dry to obtain magnetic polymer microspheres.

[0091] Comparative Example 1

[0092] This comparative example provides a method for preparing blueberry-derived exosome nanoparticles, comprising the following steps:

[0093] Step a: wash fresh blueberries, add them to PBS buffer in an amount twice their weight, add them to a juicer to extract the juice, and filter with gauze to obtain blueberry juice;

[0094] Step b, centrifuging the blueberry juice at 1000 g for 8 minutes at 4° C., centrifuging the supernatant at 3000 g for 15 minutes, and finally centrifuging the supernatant at 10000 g for 25 minutes. The supernatant was filtered through a 0.22 μm filter membrane to obtain a blueberry centrifuge;

[0095] Step c: centrifuging the blueberry centrifuge at 100,000 g for 60 min, and redispersing the resulting precipitate in PBS buffer precooled to 4° C. to obtain a blueberry-derived exosome nanoparticle suspension.

[0096] Comparative Example 2

[0097] This comparative example provides a method for preparing blueberry-derived exosome nanoparticles, comprising the following steps:

[0098] Step a: wash fresh blueberries, add them to PBS buffer in an amount twice their weight, add them to a juicer to extract the juice, and filter with gauze to obtain blueberry juice;

[0099] Step b, centrifuging the blueberry juice at 1000 g for 8 minutes at 4° C., centrifuging the supernatant at 3000 g for 15 minutes, and finally centrifuging the supernatant at 10000 g for 25 minutes. The supernatant was filtered through a 0.22 μm filter membrane to obtain a blueberry centrifuge;

[0100] Step c: add an equal volume of 16% PEG 8000 to the above blueberry centrifuge, invert and mix thoroughly, incubate at 4°C overnight, centrifuge at 10,000g for 30 minutes, and redisperse the resulting precipitate in PBS buffer precooled to 4°C to obtain a blueberry-derived exosome nanoparticle suspension.

[0101] Comparative Example 3

[0102] This comparative example provides a method for preparing blueberry-derived exosome nanoparticles, comprising the following steps:

[0103] Step a: wash fresh blueberries, add them to PBS buffer in an amount twice their weight, add them to a juicer to extract the juice, and filter with gauze to obtain blueberry juice;

[0104] Step b, centrifuging the blueberry juice at 1000 g for 8 minutes at 4° C., centrifuging the supernatant at 3000 g for 15 minutes, and finally centrifuging the supernatant at 10000 g for 25 minutes. The supernatant was filtered through a 0.22 μm filter membrane to obtain a blueberry centrifuge;

[0105] Step c, adding the blueberry centrifuge liquid to a sample tube, and then adding 1 / 10 of the weight of Fe3O4 nanoparticles thereto, inverting and mixing thoroughly, incubating at 4°C for 35 minutes, and then incubating at 38°C for 8 minutes to obtain an incubation solution;

[0106] Step d, place the sample tube next to a strong magnet, let it stand at 4°C for 10 minutes, discard the supernatant to obtain a precipitate; the obtained precipitate is gently washed twice with PBS buffer precooled to 4°C to remove unbound impurities, and then redispersed in PBS buffer precooled to 4°C, let it stand at 4°C for 20 minutes, place it next to a strong magnet, and let it stand at 4°C for another 10 minutes. Take the supernatant and filter it with a 0.22μm filter membrane to obtain a blueberry exosome-like nanoparticle suspension.

[0107] The preparation method of the above-mentioned Fe3O4 nanoparticles comprises the following steps:

[0108] 1.09 g of FeCl3·6H2O and 0.4 g of FeCl2·4H2O were dissolved in 50 mL of deionized water, magnetically heated to 75°C, ammonia was added to adjust the pH to 10.6, 0.5 g of sodium citrate was added, and the mixture was kept warm and stirred for 1 h. Fe3O4 nanoparticles were separated using a magnet, then washed with deionized water and dried to obtain Fe3O4 nanoparticles.

[0109] Comparative Example 4

[0110] This comparative example provides a method for preparing blueberry-derived exosome nanoparticles, which differs from Example 1 only in that the magnetic polymer microspheres are replaced with methoxypolyethylene glycol-coated Fe3O4 nanoparticles, comprising the following steps:

[0111] Step a: wash fresh blueberries, add them to PBS buffer in an amount twice their weight, add them to a juicer to extract the juice, and filter with gauze to obtain blueberry juice;

[0112] Step b, centrifuging the blueberry juice at 1000 g for 8 minutes at 4° C., centrifuging the supernatant at 3000 g for 15 minutes, and finally centrifuging the supernatant at 10000 g for 25 minutes. The supernatant was filtered through a 0.22 μm filter membrane to obtain a blueberry centrifuge;

[0113] Step c, adding the above blueberry centrifuge liquid to a sample tube, and then adding 1 / 10 of the weight of the methoxy polyethylene glycol-coated Fe3O4 nanoparticles thereto, inverting and mixing thoroughly, incubating at 4°C for 35 minutes, and then incubating at 38°C for 8 minutes to obtain an incubation solution;

[0114] Step d, place the sample tube next to a strong magnet, let it stand at 4°C for 10 minutes, discard the supernatant to obtain a precipitate; the obtained precipitate is gently washed twice with PBS buffer precooled to 4°C to remove unbound impurities, and then redispersed in PBS buffer precooled to 4°C, let it stand at 4°C for 20 minutes, place it next to a strong magnet, and let it stand at 4°C for another 10 minutes. Take the supernatant and filter it with a 0.22μm filter membrane to obtain a blueberry exosome-like nanoparticle suspension.

[0115] The preparation method of the methoxypolyethylene glycol-coated Fe3O4 nanoparticles comprises the following steps:

[0116] S1, 1.09 g of FeCl3·6H2O and 0.4 g of FeCl2·4H2O were dissolved in 50 mL of deionized water, heated magnetically to 75 °C, and ammonia was added to adjust the pH to 10.6. 0.5 g of sodium citrate was added, and the mixture was kept warm and stirred for 1 h. Fe3O4 nanoparticles were separated using a magnet, washed with deionized water, and dried to obtain Fe3O4 nanoparticles;

[0117] S2, Fe3O4 nanoparticles and methoxy polyethylene glycol were mixed in a mass ratio of 1:5, and PBS buffer 50 times the mass of Fe3O4 nanoparticles was added. The mixture was stirred at 25°C for 12 h, and the particles were collected using a magnet and washed three times with PBS buffer to obtain methoxy polyethylene glycol-coated Fe3O4 nanoparticles.

[0118] The blueberry exosome-like nanoparticle suspensions prepared in Example 1 and Comparative Examples 1 to 4 were tested for particle size distribution using a Malvern laser particle size analyzer. The results are as follows: Figures 1 to 5 As shown in the figure, it can be seen that the particle size of the blueberry exosome-like nanoparticles prepared in Example 1 and Comparative Examples 1 to 4 is about 100 nm, which is consistent with the exosome particle size range. However, the particle size distribution of the blueberry exosome-like nanoparticles prepared in Example 1 is more concentrated and uniform.

[0119] The TEM images of the blueberry exosome-like nanoparticle suspensions prepared in Example 1 and Comparative Examples 1 to 4 are as follows: Figure 6 As shown. Figure 6 As can be seen, the blueberry exosome-like nanoparticles prepared in Example 1 and Comparative Examples 1-4 all exhibited a saucer-like appearance and a double-layer membrane structure, consistent with exosome morphology. However, the blueberry exosome-like nanoparticles extracted using the methods of Comparative Examples 2-4 showed significant protein aggregate impurities, indicating adsorption of contaminants during the extraction process. In comparison, the blueberry exosomes extracted using the methods of Example 1 and Comparative Example 1 exhibited higher purity.

[0120] The protein concentration of the blueberry exosome-like nanoparticle suspension prepared in Example 1 and Comparative Examples 1 to 4 was detected using a BCA protein concentration assay kit. The assay method is as follows:

[0121] Calculate the required volume of working solution and thoroughly mix reagent A and reagent B (A:B=50:1) in the BCA kit in a 15mL centrifuge tube. Dilute the protein standard (2mg / mL) with 1×PBS according to the instructions. Take 25μL of the standard and the test solution and add them to the enzyme-labeled plate. Add 200μL of BCA working solution to each well, shake and mix, and incubate at 37°C for 30min. Measure the absorbance at a wavelength of 562nm on the enzyme reader. Draw a standard curve based on the absorbance of the protein standard and calculate the protein concentration of blueberry exosome-like nanoparticles. The results are as follows: Figure 7 shown.

[0122] A comparison revealed that the blueberry exosome-like nanoparticles extracted in Example 1 had the highest concentration. In contrast, the extraction method in Example 1 reduced specific adsorption, significantly improving the protein concentration and purity of the extracted blueberry exosome-like nanoparticles, making the particle size more uniform, and significantly reducing extraction time, suggesting broad application prospects.

[0123] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions or improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for extracting blueberry-derived exosome-like nanoparticles, characterized in that: The steps include: Step a, crushing fresh blueberries, squeezing juice, and filtering to obtain blueberry juice; performing differential centrifugation on the blueberry juice to obtain blueberry centrifuge liquid; Step b, adding magnetic polymer microspheres to the blueberry centrifuge, mixing evenly, and incubating to obtain an incubation solution; wherein the magnetic polymer microspheres include Fe3O4 nanoparticle cores and poly (N-isopropylacrylamide-co-PEG methacrylate) cross-linked polymers coated on the outside; Step c: using a magnetic field to perform solid-liquid separation on the incubation solution to obtain blueberry-derived exosome-like nanoparticles.

2. The method for extracting blueberry-derived exosome-like nanoparticles according to claim 1, wherein: In step a, the differential centrifugation specifically comprises the following steps: The blueberry juice is centrifuged at 800g-1200g for 5min-10min, the supernatant is centrifuged at 2800g-3200g for 10min-20min, and finally the supernatant is centrifuged at 9000g-11000g for 20min-30min, filtered, and blueberry centrifuge liquid is obtained.

3. The method for extracting blueberry-derived exosome-like nanoparticles according to claim 1, wherein: In step b, the mass ratio of the blueberry centrifuge to the magnetic polymer microspheres is (10-15):1; and / or In step b, the specific steps of incubation include: first incubating at 3°C ​​to 6°C for 30 minutes to 40 minutes; and then incubating at 35°C to 40°C for 5 minutes to 10 minutes.

4. The method for extracting blueberry-derived exosome-like nanoparticles according to claim 1 or 3, wherein: The preparation method of the magnetic polymer microspheres comprises the following steps: S1, synthesis of Fe3O4 nanoparticles; S2, dissolving N-isopropyl propionamide, polyethylene glycol methacrylate, and N,N'-methylenebisacrylamide in water, adding an initiator and an organic amine reducing agent, and performing a polymerization reaction to obtain a poly(N-isopropyl acrylamide-co-PEG methacrylate) cross-linked polymer; S3, dispersing the Fe3O4 nanoparticles and poly (N-isopropylacrylamide-co-PEG methacrylate) cross-linked polymer in water, adding sodium lauryl sulfate to obtain a mixed dispersion; adding the mixed dispersion to cyclohexane containing Span 80, mixing evenly, drying, and magnetically separating the microspheres to obtain magnetic polymer microspheres.

5. The method for extracting blueberry-derived exosome-like nanoparticles according to claim 4, wherein: S1 specifically includes the following steps: Dissolve ferric salt and ferrous salt in water, adjust the pH of the system to 10-11, add sodium citrate, stir and react at 70-80°C for 1-1.5 hours, separate the solid and liquid, wash and dry to obtain Fe3O4 nanoparticles.

6. The method for extracting blueberry-derived exosome-like nanoparticles according to claim 5, wherein: The molar ratio of the iron salt to the ferrous salt is (1.8-2.2):1; and / or The mass ratio of the sodium citrate to the ferrous salt is 1:(1-2).

7. The method for extracting blueberry-derived exosome-like nanoparticles according to claim 4, wherein: In S2, the mass ratio of N-isopropylpropionamide to polyethylene glycol methacrylate is (4-6):1; and / or In S2, the mass ratio of N-isopropylpropionamide to N,N'-methylenebisacrylamide is (40-50):1; and / or In S2, the mass ratio of the N-isopropylpropionamide to the initiator is (40-50):

1.

8. The method for extracting blueberry-derived exosome-like nanoparticles according to claim 4, wherein: In S2, the mass ratio of the N-isopropylpropionamide to the organic amine reducing agent is (40-50):1; and / or In S2, the mass ratio of N-isopropylpropionamide to water is 1:(20-25); and / or In S2, the polymerization reaction temperature is 10°C to 30°C, and the reaction time is 6h to 8h.

9. The method for extracting blueberry-derived exosome-like nanoparticles according to claim 4, characterized in that: In S2, the initiator is ammonium persulfate; and / or In S2, the organic amine reducing agent is tetramethylethylenediamine.

10. The method for extracting blueberry-derived exosome-like nanoparticles according to claim 4, characterized in that: In S3, the mass ratio of the poly (N-isopropylacrylamide-co-PEG methacrylate) cross-linked polymer to sodium lauryl sulfate is (10-15):1; and / or In S3, the mass ratio of the Fe3O4 nanoparticles to the poly(N-isopropylacrylamide-co-PEG methacrylate) cross-linked polymer is 1:(4-6); and / or In S3, the mass ratio of the mixed dispersion to the cyclohexane containing Span 80 is 1:(1-2).

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