Exosome loaded resveratrol as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to exosome loaded resveratrol as well as a preparation method and application thereof. The preparation method comprises the following steps: carrying out pretreatment by using sodium carboxymethyl cellulose, and loading resveratrol into the exosome in combination with ultrasonic and co-incubation. The preparation method provided by the invention is simple to operate, high in loading efficiency and suitable for large-scale production. The exosome provided by the invention is loaded with the resveratrol, so that the bioavailability of the resveratrol is enhanced, and the exosome has remarkable effects of removing ROS (Reactive Oxygen Species) and inhibiting melanin secretion of cells.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to exosomes loaded with resveratrol, and a preparation method and application thereof. Background Art

[0002] Exosomes are extracellular vesicles secreted by cells with a diameter of 30-150nm. They have become very potential drug delivery carriers due to their low immunogenicity, low toxicity, tissue targeting, and ability to be engineered. There are a lot of relevant studies on exosome drug delivery. Looking at all the current drug delivery studies, the drug delivery methods of exosomes can be divided into exogenous drug delivery (after exosome isolation) and endogenous drug delivery (during exosome biogenesis). Exogenous exosome loading can be divided into active loading and passive loading. Passive loading includes co-incubation, which is fast and simple and has no effect on the integrity of exosomes, but has low efficiency and is more effective for hydrophobic molecules; active loading includes electroporation, extrusion, ultrasound, cyclic freeze-thaw, membrane penetration and dialysis. These methods also have disadvantages such as affecting the quality of exosomes, low loading efficiency, and easy aggregation of exosomes.

[0003] In summary, all current exosome drug delivery methods have certain limitations, so the efficient and stable loading of drugs into exosomes is also one of the bottlenecks for the widespread clinical application of exosomes as carriers. Therefore, finding an efficient exosome drug delivery method will greatly increase the scope of clinical application of exosomes and solve the current difficulties in clinical application.

[0004] Resveratrol is a polyphenolic compound. Resveratrol (RES) is a natural polyphenolic antitoxin secreted by at least 100 different plants after fungal infection or pathogen attack. Because it can effectively scavenge free radicals and regulate the expression and activity of antioxidant enzymes, RES has been used to treat a variety of diseases. Among these various biological effects of resveratrol, the antioxidant property is the most prominent. Resveratrol has been shown to protect cells from hydrogen peroxide-induced oxidative stress and ultraviolet irradiation-mediated cell death. However, resveratrol is not easily absorbed and utilized due to its poor water solubility. In addition, resveratrol is easily oxidized and degraded. Therefore, a suitable encapsulation and delivery technology can greatly promote the bioavailability of resveratrol. Summary of the invention

[0005] In order to solve the above problems, the present invention provides an exosome, which uses a specific pretreatment method combined with ultrasound and co-incubation process to load resveratrol into the exosome, thereby improving its loading efficiency and also increasing the stability and bioavailability of resveratrol.

[0006] In one aspect, the present invention provides a method for loading resveratrol with exosomes, comprising the following steps: S1. Pretreatment: dissolving resveratrol in an organic solvent to obtain a resveratrol solution; adding sodium carboxymethyl cellulose to the exosomes to obtain an exosome solution of sodium carboxymethyl cellulose; S2, mixing: mixing the above-mentioned sodium carboxymethyl cellulose exosome solution with the resveratrol solution to obtain A; S3. Loading: Sonicate and incubate the above A to obtain B.

[0007] S4, purification: purifying the above B to obtain purified exosomes loaded with resveratrol; The concentration of sodium carboxymethyl cellulose in step S1 is 5%.

[0008] Specifically, the organic solvent in step S1 can be one or more of ether, chloroform, methanol, ethanol, acetone or ethyl acetate.

[0009] Preferably, the organic solvent in step S1 may be ethanol.

[0010] Specifically, the concentration of the resveratrol solution in step S1 can be 5-15 mg / mL.

[0011] Preferably, the concentration of the resveratrol solution in step S1 may be 10-15 mg / mL.

[0012] Further preferably, the concentration of the resveratrol solution in step S1 may be 10-12 mg / mL.

[0013] More preferably, the concentration of the resveratrol solution in step S1 may be 10 mg / mL.

[0014] Specifically, the mixing ratio of the exosome solution of sodium carboxymethyl cellulose and the resveratrol solution in step S2 is 10-20:1-3.

[0015] Preferably, the mixing ratio of the exosome solution of sodium carboxymethyl cellulose and the resveratrol solution in step S2 is 10-15:1-2.

[0016] Further preferably, the mixing ratio of the exosome solution of sodium carboxymethyl cellulose and the resveratrol solution in step S2 is 10:1.

[0017] Specifically, the ultrasound in step S2 is cyclic ultrasound; the cyclic ultrasound is set to be on for 25-35s and off for 25-35s, and one cycle of the cyclic ultrasound is 2-5min.

[0018] Preferably, specifically, the ultrasound in step S2 is cyclic ultrasound; the cyclic ultrasound is set to be on for 30-35s, off for 30-35s, and one cycle of the cyclic ultrasound is 2-3min.

[0019] Further preferably, specifically, the ultrasound in step S2 is cyclic ultrasound; the cyclic ultrasound is set to be on for 30 seconds, off for 30 seconds, and one cycle of cyclic ultrasound is 3 minutes.

[0020] More specifically, the number of cyclic ultrasound is 4.

[0021] Specifically, the incubation in step S2 is performed on ice for 2-3 minutes per ultrasonic cycle.

[0022] Preferably, the incubation in step S2 is incubation on ice for 2 min per ultrasonic cycle.

[0023] More specifically, the incubation further includes incubating on ice for 40-80 minutes after transfer.

[0024] Preferably, the incubation further comprises incubating on ice for 50-60 min after transfer.

[0025] Further preferably, the incubation further comprises incubating on ice for 60 minutes after transfer.

[0026] In another aspect, the present invention provides exosomes loaded with resveratrol, obtained by the aforementioned method.

[0027] In another aspect, the present invention provides the use of the aforementioned exosomes loaded with resveratrol in medicines, health products and cosmetics.

[0028] In yet another aspect, the present invention provides a drug, characterized in that it comprises the aforementioned exosomes loaded with resveratrol.

[0029] Specifically, the drug also includes pharmaceutically acceptable excipients.

[0030] More specifically, the pharmaceutically acceptable excipient is selected from one or a combination of two or more of wetting agents, emulsifiers, preservatives, antioxidants, buffers, excipients, diluents, lubricants, antibacterial agents, suspending agents, suspending aids, solubilizers, thickeners, stabilizers, sweeteners and flavors.

[0031] Preferably, the pharmaceutically acceptable excipient is at least one selected from lactose, mannose, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, polyvinyl pyrrolidone, cellulose, water, syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, magnesium stearate and mineral oil.

[0032] Specifically, the medicine includes, but is not limited to, drops, ointments, emulsions, films, smears, gels, pastes, sprays, aerosols or patches.

[0033] In another aspect, the present invention provides a health product, characterized in that it comprises the aforementioned exosomes loaded with resveratrol.

[0034] Specifically, the health care product also includes excipients acceptable to health care products.

[0035] More specifically, the acceptable excipients for health care products are selected from one or a combination of two or more of wetting agents, emulsifiers, preservatives, antioxidants, buffers, excipients, diluents, lubricants, antibacterial agents, suspending agents, suspending aids, solubilizers, thickeners, stabilizers, sweeteners and flavors.

[0036] Preferably, the acceptable excipients for the health care product are at least one selected from lactose, mannose, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, polyvinyl pyrrolidone, cellulose, water, syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, magnesium stearate and mineral oil.

[0037] In yet another aspect, the present invention provides a cosmetic, characterized in that it comprises the aforementioned exosomes loaded with resveratrol.

[0038] Specifically, the cosmetic also includes cosmetically acceptable excipients.

[0039] More specifically, the cosmetically acceptable auxiliary material is selected from one or a combination of two or more of emulsifiers, moisturizers, sunscreens, preservatives, antioxidants, flavors, pigments, thickeners and film formers.

[0040] Specifically, the cosmetics include but are not limited to: facial masks, facial creams, lotions, essences or eye creams.

[0041] The technical effects achieved by the present invention are: (1) The technical solution provided by the present invention realizes the effective loading of resveratrol into exosomes. The loaded resveratrol promotes cell absorption and has higher cell activity than that of exosomes without resveratrol in in vitro experiments.

[0042] (2) The technical solution provided by the present invention is easy to operate and only requires simple equipment through ultrasound to complete the loading of resveratrol and subsequent purification.

[0043] (3) The technical solution provided by the present invention is easy to scale up, and the method can be easily scaled up to meet subsequent production requirements.

[0044] (4) The exosomes loaded with resveratrol provided by the present invention can significantly inhibit the ROS effect.

[0045] (5) The exosomes loaded with resveratrol provided by the present invention can effectively inhibit the secretion of cellular melanin. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 The number of exosomes loaded in the examples and comparative examples.

[0047] Figure 2 The loading efficiency and exosome quantity under different loading methods.

[0048] Figure 3 It is the characteristic absorption peak of resveratrol standard, and the retention time is 10.208min.

[0049] Figure 4 This is the concentration curve of resveratrol.

[0050] Figure 5 This is a detection diagram of the resveratrol content loaded in exosomes in Example 1, and the retention time is 10.132 min.

[0051] Figure 6 This is the particle size distribution of exosomes after ultrasonic loading in Example 1.

[0052] Figure 7 Comparison of ROS scavenging effects between exosomes loaded with resveratrol and the control group.

[0053] Figure 8 The relative expression of melanin in exosomes loaded with resveratrol and the control group. DETAILED DESCRIPTION

[0054] The present invention will be further described in detail below in conjunction with specific examples. The following examples are not intended to limit the present invention, but are only intended to illustrate the present invention. The experimental methods used in the following examples are generally conventional, unless otherwise specified, and the materials, reagents, etc. used in the following examples are commercially available, unless otherwise specified.

[0055] Example 1 1.1 Preprocessing Resveratrol has very low solubility in water and is soluble in ethanol, but exosomes cannot tolerate high concentrations of ethanol. To improve loading efficiency, the following treatments are required: Resveratrol is first dissolved in ethanol to prepare a 10 mg / mL solution. Purified HEK293 cell-derived exosomes (from InnoBio, catalog number: ST08002, concentration: 1E+11 particles / mL) are reconstituted at room temperature and sodium carboxymethyl cellulose is added to prepare an exosome solution containing 5% (w / v) sodium carboxymethyl cellulose.

[0056] Then the exosome solution was mixed with 10 mg / mL resveratrol ethanol solution at a ratio of 10:1. The loading operation should be performed immediately after mixing.

[0057] 1.2 Loading method After mixing, place on ice and sonicate the mixture using an ultrasonic cell disruptor (manufacturer: Ningbo Xinzhi; model: JY92-IIN). The preferred program is 20% amplitude, 30s on, 30s off for 3 minutes per cycle, and 4 cycles of sonication with 2 minutes of incubation on ice between each cycle. After loading, the exosomes were incubated on ice for 1 hour to promote loading and recovery of the exosome membrane.

[0058] 1.3 Purification The loaded exosomes were purified using an exosome purification kit (manufacturer: Yinguan Bio, catalog number: KY08001) to remove the remaining resveratrol and sodium carboxymethyl cellulose in the sample. According to the operating requirements of the kit, the ultrasonic sample was added to the purification column, then eluted with PBS, and specific fractions were collected to obtain the purified exosomes. Other components, resveratrol and sodium carboxymethyl cellulose, were separated due to different molecular weights.

[0059] 1.4 Loading efficiency analysis Resveratrol in the samples was quantitatively analyzed using a high performance liquid chromatography system HPLC (brand: Agilent, model: 1260 Infinity II) and a chromatographic column (brand: Techno, model: Inertsustain AQ-C18 5um250x4.6mm).

[0060] Mobile phase: A: 0.1% trifluoroacetic acid (TFA) water (v / v), B: 0.1% TFA acetonitrile (v / v), gradient elution, the gradient setting is shown in Table 1.

[0061] Table 1

[0062] A UV detector was used to detect the absorption peak at a wavelength of 306 nm.

[0063] Resveratrol standard was used to prepare solutions of 100ug / mL, 10ug / mL, 1ug / mL, 0.1ug / mL, and 0.01ug / mL. The peak area was measured using the above detection method and a standard curve was drawn. The loaded samples were detected under the same conditions and the resveratrol content in the samples was calculated.

[0064] 1.5 In vitro experiments The experimental groups are shown in Table 2: Table 2

[0065] 1.5.1 In vitro reactive oxygen species elimination test HaCAT cells were plated in 96-well plates, and exosomes and control drugs were added after 24 hours. After 24 hours of drug incubation, 250 μmol / L hydrogen peroxide was added to the 96-well plates and incubated at 37 degrees for 30 minutes. After the incubation, the ROS kit was used to detect reactive oxygen species. After collecting the cells, each group was detected using a fluorescence spectrophotometer with an excitation wavelength of 488 nm and an emission wavelength of 525 nm.

[0066] 1.5.2 Melanin inhibition test A375 cells were cultured with opti-MEM and loaded exosomes and control test articles for 4 days. The cells were then digested and collected, dissolved in 1 mL of 1M NaOH solution at 80°C for 30 minutes, and centrifuged at 16,000g for 20 minutes. The absorbance (OD) of the supernatant was measured at 410nm using an ELISA reader. Three standard synthetic melanin curves (0 to 300mg / mL) were prepared for each experiment.

[0067] Comparative Example A comparative example was set up according to the method of Example 1. The differences between the comparative example and the example are shown in Table 3: Table 3

[0068] Effect and analysis 1. Number of loaded exosomes The results are as follows Figure 1 As shown, during the pretreatment process, the addition of 5% sodium carboxymethyl cellulose (Example 1) helps to improve the loading efficiency, while the increase in the added concentration increases the loss of exosomes and reduces the loading amount. 5% PEG 2000 (Comparative Example 3) also has a high loading efficiency.

[0069] 2. Effect of loading on exosome content The results are shown in Table 4 and Figure 2 As shown, the loss of exosomes in Example 1 increased significantly after 4 cycles, while the loading efficiency showed a trend of first increasing and then decreasing with the increase in the number of cycles. Example 1 was significantly better than Comparative Examples 4-8 as a method for loading resveratrol exosomes.

[0070] Table 4

[0071] 3. Resveratrol content analysis results When resveratrol is analyzed by HPLC, the characteristic absorption peak of resveratrol can be obtained in about 10 minutes using the above analysis method. Figure 3 shown.

[0072] This method was used to generate a resveratrol concentration curve ( Figure 4 ), maintains high linearity in the range of 0.1-100ug, thus achieving accurate quantification of resveratrol in the sample.

[0073] 4. Results of purification after exosome loading like Figure 5-Figure 6 As shown, after 4 ultrasonic treatments and purification, the content of resveratrol in the exosomes in Example 1 was 7.9ug / mL, and the exosome particle size distribution did not change significantly, indicating that the loading method can effectively load resveratrol into the exosomes within the tolerance range of the exosomes.

[0074] 5. In vitro experimental results The results of ROS scavenging experiments showed that ( Figure 7 ) Exosomes loaded with resveratrol can achieve a significant ROS inhibition effect at a lower concentration (5ug / mL), which is higher than the resveratrol group (5ug / mL). And resveratrol loaded with sodium carboxymethylcellulose is more effective than resveratrol loaded with PEG2000 (5% w / v) (5ug / mL).

[0075] The results of melanin inhibition experiments showed that ( Figure 8 ), resveratrol has the activity of inhibiting cell melanin secretion at high concentrations, and exosome-loaded resveratrol increases the activity. 5ug / mL resveratrol loaded by exosomes is better than resveratrol directly used at the same concentration, and resveratrol loaded with sodium carboxymethylcellulose is more effective than resveratrol loaded with PEG2000.

[0076] The above detailed description is a specific description of one of the feasible embodiments of the present invention, and the embodiment is not intended to limit the scope of the present invention. It should be pointed out that any equivalent implementation or change that does not deviate from the present invention should be included in the scope of the technical solution of the present invention. Therefore, the protection scope of the patent of the present invention shall be based on the attached requirements.

Claims

1. A method for loading resveratrol with exosomes, characterized in that: The following steps are involved: S1. Pretreatment: dissolving resveratrol in an organic solvent to obtain a resveratrol solution; adding sodium carboxymethyl cellulose to the exosomes to obtain an exosome solution of sodium carboxymethyl cellulose; S2, mixing: mixing the above-mentioned sodium carboxymethyl cellulose exosome solution with the resveratrol solution to obtain A; S3, loading: ultrasonicating and incubating the above A to obtain B; S4, purification: purifying the above B to obtain purified exosomes loaded with resveratrol; The concentration of sodium carboxymethyl cellulose in step S1 is 5% w / v.

2. The method according to claim 1, characterized in that The organic solvent in step S1 is one or more of ether, chloroform, methanol, ethanol, acetone or ethyl acetate.

3. The method according to claim 1, characterized in that The concentration of the resveratrol solution in step S1 is 5-15 mg / mL.

4. The method according to claim 1, characterized in that In step S2, the mixing ratio of the exosome solution of sodium carboxymethyl cellulose and the resveratrol solution is 10-20:1-3.

5. The method according to claim 1, characterized in that The ultrasound in step S2 is cyclic ultrasound; the cyclic ultrasound is set to be on for 25-35s and off for 25-35s, and one cycle of cyclic ultrasound is 2-5min.

6. The method according to claim 5, characterized in that The number of the cyclic ultrasound is 4.

7. The method according to claim 1, characterized in that The incubation in step S2 is performed on ice for 2-3 min per ultrasonic cycle.

8. The method according to claim 7, characterized in that The incubation also includes incubating on ice for 40-80 minutes after transfer.

9. An exosome loaded with resveratrol, characterized in that: The exosomes loaded with resveratrol are obtained by the method according to any one of claims 1-8.

10. Use of the exosomes loaded with resveratrol according to claim 9 in the preparation of medicines, health products and cosmetics.

11. A drug, characterized in that The method comprises the exosomes loaded with resveratrol as claimed in claim 9.

12. The drug according to claim 11, characterized in that The drug also includes pharmaceutically acceptable excipients.

13. A health product, characterized in that: The method comprises the exosomes loaded with resveratrol as claimed in claim 9.

14. A cosmetic, characterized in that: The method comprises the exosomes loaded with resveratrol as claimed in claim 9.

15. The cosmetic according to claim 14, characterized in that The cosmetics include facial masks, facial creams, lotions, essences or eye creams.

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