Preparation method of natural exosome system carrying fluorescent polydopamine nanoparticles

Abstract

The invention provides a preparation method of a natural exosome system carrying fluorescent polydopamine nanoparticles, and relates to the technical field of biomedicine, and the preparation method comprises the following steps: S1, preparing fluorescent PDA: dissolving dopamine hydrochloride in a Tris-HCl solution with the pH value of 8.5, carrying out a stirring reaction by a magnetic stirrer, then adding glutathione for a reaction, finally adding DNTB for a reaction, and after the reaction is completed, carrying out suction filtration to obtain fluorescent PDA; dialyzing and freeze-drying the obtained reaction solution to obtain the fluorescent PDA; s2, preparing the exosome, namely culturing macrophages, inducing the macrophages into M2, adding the fluorescent PDA prepared in the S1, culturing by using a complete culture medium prepared from exosome-free serum, taking supernatant, and obtaining an exosome system by using an ultracentrifugation method. The exosome disclosed by the invention can effectively resist inflammation, has an excellent deoxidation effect, is better in biocompatibility, has high bioavailability, biological stability, targeting specificity, low toxicity and low immunogenicity, and can be widely applied to treatment of inflammatory diseases.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a method for preparing an exosome system naturally carrying fluorescent polydopamine nanoparticles. Background Art

[0002] Systemic lupus erythematosus (SLE) is a systemic multisystem autoimmune disease characterized by the presence of autoantibodies against nuclear antigens, immune complex deposition, and chronic inflammation of classic target organs such as the skin, joints, and kidneys. Despite great advances in the diagnosis and treatment of SLE, the disease burden remains high. Understanding the typical manifestations and diagnostic process is important to facilitate early referral and diagnosis of patients. For most patients, the earliest complaints are systemic symptoms, mucocutaneous symptoms, and musculoskeletal symptoms. These symptoms may include fatigue, lupus-specific rash, oral ulcers, alopecia, joint pain, and myalgia. Currently, glucocorticoids, antimalarials, cyclophosphamide, and methotrexate have been widely used in the treatment of SLE patients. However, these drugs cannot completely control the disease, and long-term use may cause severe toxic reactions and complications. In addition, biologics such as belimumab and rituximab have been used to treat SLE. However, biologics currently have limited efficacy in SLE, may cause serious hematologic side effects, and their high prices make them unaffordable for patients. Summary of the invention

[0003] The purpose of the present invention is to solve the technical problems in the prior art that biological agents currently have limited efficacy in SLE, may cause serious blood system side effects, and are expensive and unaffordable for patients.

[0004] In order to achieve the above object, the present invention adopts the following technical solutions:

[0005] A method for preparing an exosome system naturally carrying fluorescent polydopamine nanoparticles comprises the following steps:

[0006] S1: Preparation of fluorescent PDA:

[0007] Dopamine hydrochloride was dissolved in a Tris-HCl solution with a pH of 8.5, and a magnetic stirrer was used to stir the reaction, and then glutathione was added to react, and finally DNTB was added to react. After the reaction was completed, the obtained reaction solution was dialyzed and freeze-dried to obtain fluorescent PDA;

[0008] S2: Preparation of exosomes:

[0009] Macrophages were cultured and induced into M2, and the fluorescent PDA prepared in S1 was added. The cells were cultured using complete medium prepared with exosome-free serum, and the supernatant was collected and the exosome system was obtained by ultracentrifugation.

[0010] Preferably, the reaction temperature in S1 is 22°C-28°C.

[0011] Preferably, the stirring reaction time of the stirrer in S1 is 2 minutes, the reaction time after adding glutathione is 2 hours, and the reaction time after adding DNTB is 1 hour.

[0012] Preferably, the dialysis time in S1 is 24 hours, and the dialysis is performed using a 1000Da dialysis bag.

[0013] Preferably, the ultracentrifugation method in S2 is density gradient ultracentrifugation.

[0014] Preferably, the density gradient ultracentrifugation method comprises the following specific steps:

[0015] Centrifuge at 300 × g for 15 min and collect the supernatant;

[0016] Centrifuge at 2000 × g for 15 min and collect the supernatant;

[0017] Centrifuge at 10,000 × g for 30 minutes and collect the supernatant;

[0018] The mixture was centrifuged at 150,000 × g for 90 minutes, the supernatant was removed, the remaining precipitate was resuspended in PBS, and filtered with a 0.22 μm filter to obtain exosomes naturally carrying fluorescent polydopamine nanoparticles.

[0019] Preferably, after adding fluorescent PDA to S2, when the cell confluence density reaches more than 70%, the cells are washed 2-3 times with PBS to remove residual bovine serum, and then cultured with complete culture medium without exosome serum for 24 hours.

[0020] The present application also provides an exosome system naturally carrying fluorescent polydopamine nanoparticles, and the exosome system is prepared using the preparation method described above.

[0021] Preferably, it comprises fluorescent polydopamine, exosomes and anti-inflammatory components.

[0022] The present application also provides an application of the exosome system naturally carrying fluorescent polydopamine nanoparticles as described above in the preparation of a product for reducing the verified level of systemic lupus erythematosus.

[0023] Compared with the prior art, this application has the following beneficial effects:

[0024] The present invention provides a method for preparing an exosome system that naturally carries fluorescent polydopamine nanoparticles. The exosome has unique advantages in the treatment of SLE. It has a long half-life and exists in the body for a long time. It can be stored at 4°C and -20°C for a short period of time, or stored at -80°C for a long period of time. The exosome can transport proteins and nucleic acids between cells and protect them from being degraded when entering cells. It is a membrane vesicle secreted by various cells in the body, which is decomposed by enzymes after inactivation and will not accumulate in large quantities for a long time. It has little toxic side effects and is small enough to pass through biological membranes and even has the ability to pass through the blood-brain barrier (BBB) ​​and blood-cerebrospinal fluid barrier (BCSFB). BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a particle size diagram of an exosome system naturally carrying fluorescent polydopamine nanoparticles in one embodiment of the present invention;

[0026] Figure 2 This is a potential diagram of an exosome system naturally carrying fluorescent polydopamine nanoparticles in one embodiment of the present invention;

[0027] Figure 3 This is a transmission electron microscopy image of an exosome system naturally carrying fluorescent polydopamine nanoparticles in one embodiment of the present invention;

[0028] Figure 4 In one embodiment of the present invention, an exosome system naturally carrying fluorescent polydopamine nanoparticles is used to treat the serum TNF-α level of SLE mice

[0029] Figure 5 FIG. 1 is a transmission electron microscope image of a fluorescent PDA in one embodiment of the present invention. DETAILED DESCRIPTION

[0030] The present invention is further described in detail below in conjunction with specific embodiments.

[0031] A method for preparing an exosome system naturally carrying fluorescent polydopamine nanoparticles comprises the following steps:

[0032] S1: Preparation of fluorescent PDA:

[0033] Dopamine hydrochloride was dissolved in a Tris-HCl solution with a pH of 8.5, and the mixture was stirred with a magnetic stirrer for 2 minutes to react at a temperature of 22°C-28°C;

[0034] Then glutathione is added, and the reaction temperature of glutathione is 22°C-28°C, and the reaction time is 2h;

[0035] Finally, DNTB was added, and the reaction temperature of the added DNTB was 22° C.-28° C., and the reaction time was 1 h.

[0036] The product was dialyzed for 24 hours using a 1000Da dialysis bag and freeze-dried to obtain fluorescent PDA.

[0037] S2: Preparation of exosome system:

[0038] Macrophages were cultured and induced into M2, and the fluorescent PDA prepared in S1 was added at the same time. The cells were cultured using complete medium prepared with exosome-free serum, and the supernatant was collected and the exosome system was obtained by ultracentrifugation.

[0039] Based on the above preparation method, the present application provides an exosome system naturally carrying fluorescent polydopamine nanoparticles, the exosome system comprising fluorescent polydopamine, exosomes and anti-inflammatory components. The exosome system naturally carrying fluorescent polydopamine nanoparticles prepared by the above preparation method has high bioavailability, biological stability, targeting specificity, low toxicity and low immunogenicity.

[0040] In addition, the present application also provides the use of the above-mentioned exosome system naturally carrying fluorescent polydopamine nanoparticles in the preparation of a product for reducing the verified level of systemic lupus erythematosus.

[0041] The above contents are described below in conjunction with specific embodiments:

[0042] Example 1: Preparation of exosome system naturally carrying fluorescent polydopamine nanoparticles:

[0043] First prepare the fluorescent PDA:

[0044] 20 mg of dopamine hydrochloride was dissolved in 10 mL of Tris-HCl solution with a pH of 8.5, and stirred for 2 min with a magnetic stirrer. The stirring reaction was carried out at room temperature, and then 24 mg of glutathione was added and stirred for 2 h at room temperature. Finally, 31 mg of DNTB was added and stirred for 1 h at room temperature. The reaction was dialyzed with a 1000 Da dialysis bag for 24 h and freeze-dried to obtain fluorescent PDA.

[0045] See also Figure 5 The working concentration of the fluorescent PDA prepared by the above method is 50 μg / mL, and the prepared fluorescent PDA has spontaneous fluorescence, which is blue-green. It also has the effect of promoting the polarization of macrophages to M2 macrophages.

[0046] Then prepare the exosome system:

[0047] Macrophages were cultured and induced to become M2, and the fluorescent PDA was added at the same time. When the cell confluence density reached more than 70%, the cells were washed 2-3 times with PBS to remove the residual bovine serum, serum-free medium was added, and the culture was continued for 24 hours. The supernatant was taken and centrifuged using density gradient ultracentrifugation to prepare exosomes. The density gradient ultracentrifugation was performed in an environment of 4°C, and the specific steps were as follows:

[0048] Centrifuge at 300 × g for 15 min and collect the supernatant;

[0049] Centrifuge at 2000 × g for 15 min and collect the supernatant;

[0050] Centrifuge at 10,000 × g for 30 minutes and collect the supernatant;

[0051] The mixture was centrifuged at 150,000 × g for 90 min at 4°C, the supernatant was removed, the remaining precipitate was resuspended in PBS, and filtered with a 0.22 μm filter to obtain exosomes naturally carrying fluorescent polydopamine nanoparticles.

[0052] The average diameter of the nanoparticles of the exosome system naturally carrying fluorescent polydopamine nanoparticles prepared by the above preparation method is 100-200nm. It has inherent advantages such as long stability, convenient storage, good content protection, avoidance of immune monitoring, and crossing biological barriers. Please refer to Figure 1 and Figure 2 The dynamic light scattering method was used to detect and study the particle size distribution of exosomes. The collected exosomes were diluted to a particle concentration of 1010L-1. After filtering with a 0.22μm microporous filter membrane, they were injected into the Nanosight nanoparticle tracking analyzer for analysis to obtain the potential and particle size diagram of the exosomes.

[0053] Example 2: Electron microscopy detection:

[0054] The M2@PDA exosomes prepared in Example 1 were stored at -80°C for 1 month and then dispersed by ultrasound to form a suspension. A few drops were dropped on the copper mesh of the electron microscope using a pipette and dried (or blotted with filter paper) to form a sample for electron microscope observation. The test results are shown in Figure 2. Figure 3 As shown, it also shows that exosomes can be stored at -80℃ for a long time.

[0055] Embodiment 3:

[0056] After treating SLE mice with exosomes, serum samples of mice were obtained. Whole blood was collected into a test tube without anticoagulant and placed at room temperature for 1 hour. After the whole blood naturally coagulated and the serum was separated, it was centrifuged at about 1500g for 10 minutes at 4°C, and the yellow supernatant was obtained as serum. Then the expression level of TNF-α of each component was obtained by the following operation method:

[0057] 1. Prepare reagents, samples and standards and dilute them in proportion.

[0058] 2. Sample addition: Set up blank wells (blank control wells do not add samples and enzyme-labeled reagents, and the rest of the steps are the same), standard wells, and test sample wells. Accurately add 50μL of the standard sample to the enzyme-labeled plate, add 40μL of sample diluent to the test sample wells, and then add 10μL of the test sample (the final dilution of the sample is 5 times). Add the sample to the bottom of the well of the enzyme-labeled plate, try not to touch the well wall, and gently shake to mix.

[0059] 3. Incubation: Seal the plate with a sealing film and incubate at 37°C for 30 minutes.

[0060] 4. Liquid preparation: Dilute the 30 times concentrated washing solution with 30 times distilled water and set aside.

[0061] 5. Washing: Carefully peel off the sealing film, discard the liquid, spin dry, fill each well with washing solution, let it stand for 30 seconds and then discard, repeat this 5 times and pat dry.

[0062] 6. Add enzyme: Add 50 μL of enzyme-labeled reagent to each well, except for the blank well.

[0063] 7. Incubation: Same operation as 3.

[0064] 8. Washing: Same operation as 5.

[0065] 9. Color development: First add 50μL of color developer A to each well, then add 50μL of color developer B, gently shake to mix, and develop the color at 37℃ in the dark for 10 minutes.

[0066] 10. Termination: Add 50 μL of stop solution to each well to terminate the reaction (the blue color immediately turns yellow).

[0067] 11. Determination: Use the blank well as zero, and measure the absorbance (OD value) of each well in sequence at a wavelength of 450nm. The determination should be performed within 15 minutes after adding the stop solution. The expression level of TNF-α in each component is obtained.

[0068] The results are as follows Figure 4 As shown in the figure, TNF-α, as a proinflammatory cytokine, plays an important role in the occurrence and development of inflammation in vivo. Exosomes were used to treat SLE mice, and serum samples of SLE mice were obtained to detect the expression level of TNF-α. It can be seen that the level of TNF-α was reduced in the treatment group compared with the pathological group, which was statistically significant.

[0069] The experimental materials and their sources in the above examples are as follows:

[0070] GSH was purchased from Aladdin, DTNB and DA were purchased from Sigma, and Tris-HCl was purchased from Bio-Tech.

[0071] The present application provides an exosome system that naturally carries fluorescent polydopamine nanoparticles, wherein polydopamine is a substance similar to melanin and has multiple properties such as antioxidant, photothermal conversion, adhesion, biocompatibility and biodegradability.

[0072] The exosome system prepared in this application has unique advantages in the treatment of SLE. It has a long half-life and exists in the body for a long time. It can be stored at 4°C and -20°C for a short time, or stored at -80°C for a long time. Exosomes can transport proteins and nucleic acids between cells and protect them from degradation when entering cells. They are membrane vesicles secreted by various cells in the body. After inactivation, they are decomposed by enzymes and will not accumulate in large quantities for a long time. They have small toxic side effects and are small enough to pass through biological membranes, and even have the ability to pass through the blood-brain barrier (BBB) ​​and blood-cerebrospinal fluid barrier (BCSFB). Exosomes can carry different drugs to meet the needs of treatment, prolong the half-life of drugs, and increase the stability of their release. In addition, exosomes have a variety of administration routes, and appropriate administration methods can be selected according to the site of the lesion, such as subcutaneous injection, intravenous injection, intraperitoneal injection, intranasal injection, oral administration, etc. As a drug carrier, exosomes have inherent advantages such as long stability, convenient storage, good content protection, avoidance of immune monitoring, and crossing biological barriers. The exosomes are effective in anti-inflammatory and have excellent deoxidation effects. At the same time, the exosomes have better biocompatibility, high bioavailability, biostability, targeting specificity, low toxicity and low immunogenicity, and can be widely used in the treatment of inflammatory diseases. The exosomes show great prospects as a drug or gene delivery carrier for the treatment of SLE, making them an excellent candidate for the treatment of SLE and other autoimmune diseases.

Claims

1. A method for preparing an exosome system naturally carrying fluorescent polydopamine nanoparticles, characterized in that: The following steps are involved: S1: Preparation of fluorescent PDA: Dopamine hydrochloride was dissolved in a Tris-HCl solution with a pH of 8.5, and a magnetic stirrer was used to stir the reaction, and then glutathione was added to react, and finally DNTB was added to react. After the reaction was completed, the obtained reaction solution was dialyzed and freeze-dried to obtain fluorescent PDA; S2: Preparation of exosomes: Macrophages were cultured and induced into M2, and the fluorescent PDA prepared in S1 was added. The cells were cultured using complete medium prepared with exosome-free serum, and the supernatant was collected and the exosome system was obtained by ultracentrifugation.

2. The method for preparing an exosome system naturally carrying fluorescent polydopamine nanoparticles according to claim 1, characterized in that: The reaction temperature in S1 is 22°C-28°C.

3. The method for preparing an exosome system naturally carrying fluorescent polydopamine nanoparticles according to claim 2, characterized in that: The stirring reaction time of the stirrer in S1 is 2 minutes, the reaction time after adding glutathione is 2 hours, and the reaction time after adding DNTB is 1 hour.

4. The method for preparing an exosome system naturally carrying fluorescent polydopamine nanoparticles according to claim 3, characterized in that: The dialysis time in S1 is 24 h, and the dialysis is performed using a 1000 Da dialysis bag.

5. The method for preparing an exosome system naturally carrying fluorescent polydopamine nanoparticles according to claim 4, characterized in that: The ultracentrifugation method in S2 is density gradient ultracentrifugation.

6. The method for preparing an exosome system naturally carrying fluorescent polydopamine nanoparticles according to claim 5, characterized in that: The specific steps of the density gradient ultracentrifugation method are as follows Centrifuge at 300 × g for 15 min and collect the supernatant; Centrifuge at 2000 × g for 15 min and collect the supernatant; Centrifuge at 10,000 × g for 30 minutes and collect the supernatant; The mixture was centrifuged at 150,000 × g for 90 minutes, the supernatant was removed, the remaining precipitate was resuspended in PBS, and filtered with a 0.22 μm filter to obtain exosomes naturally carrying fluorescent polydopamine nanoparticles.

7. The method for preparing an exosome system naturally carrying fluorescent polydopamine nanoparticles according to claim 6, characterized in that: After adding fluorescent PDA to the S2, when the cell confluence density reaches more than 70%, the cells are washed 2-3 times with PBS to remove residual bovine serum, and then cultured with complete culture medium without exosome serum for 24 hours.

8. An exosome system naturally carrying fluorescent polydopamine nanoparticles, characterized in that: The exosome system is prepared using the preparation method described in any one of claims 1 to 7.

9. The exosome system naturally carrying fluorescent polydopamine nanoparticles according to claim 8, characterized in that: Includes fluorescent polydopamine, exosomes and anti-inflammatory ingredients.

10. Use of the exosome system naturally carrying fluorescent polydopamine nanoparticles as claimed in claim 8 in the preparation of a product for reducing the verified level of systemic lupus erythematosus.

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