Preparation method of an exosome system naturally carrying fluorescent polydopamine nanoparticles

Abstract

The present invention provides a method for preparing an exosome system naturally carrying fluorescent polydopamine nanoparticles, which relates to the field of biomedical technology and includes the following steps: S1: preparing fluorescent PDA: dissolving dopamine hydrochloride in a Tris-HCl solution with a pH of 8.5, stirring the reaction with a magnetic stirrer, then adding glutathione to react, and finally adding DNTB to react. After the reaction is completed, the obtained reaction solution is dialyzed and freeze-dried to obtain fluorescent PDA; S2: preparing exosomes: culturing macrophages and inducing them into M2, while adding the fluorescent PDA prepared in S1, culturing them using a complete culture medium without exosome serum, taking the supernatant, and obtaining the exosome system using ultracentrifugation. The exosomes of the present application can effectively fight inflammation and have excellent deoxidation effects. At the same time, the exosomes have better biocompatibility, high bioavailability, biostability, target specificity, low toxicity and low immunogenicity, and can be widely used in the 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 in classic target organs such as the skin, joints, and kidneys. Despite significant advances in the diagnosis and treatment of SLE, the disease burden remains high. Understanding the typical presentation and diagnostic process is crucial to facilitate early referral and diagnosis. For most patients, the initial complaints are systemic, mucocutaneous, and musculoskeletal symptoms. These symptoms may include fatigue, lupus-specific rash, oral ulcers, alopecia, joint pain, and myalgia. Currently, glucocorticoids, antimalarials, cyclophosphamide, and methotrexate are widely used to treat SLE. However, these medications do not fully control the disease, and long-term use can lead to severe toxicities and complications. Additionally, biologics such as belimumab and rituximab have been used to treat SLE. However, biologics currently have limited efficacy in SLE, can cause severe hematologic side effects, and are unaffordable due to their high cost. 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 stirred with a magnetic stirrer to react. 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. The fluorescent PDA prepared in S1 was added and cultured using complete medium containing exosome-free serum. 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 steps:

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

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

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

[0018] The cells were centrifuged at 150,000 × g for 90 minutes, the supernatant was discarded, 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 in a complete 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 a use of the above-described exosome system naturally carrying fluorescent polydopamine nanoparticles in the preparation of a product for reducing the inflammation level of systemic lupus erythematosus.

[0023] Compared with the existing technology, 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 exosomes have unique advantages in the treatment of systemic lupus erythematosus (SLE). They have a long half-life and exist in the body for a long time. They can be stored at 4°C and -20°C for a short period of time, or at -80°C for a long period of time. Exosomes can transport proteins and nucleic acids between cells, protecting 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 over a long period of time. They have low toxic side effects and are small enough to pass through biological membranes, and are even capable of crossing 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 micrograph 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 TEM image of fluorescent PDA in one embodiment of the present invention. DETAILED DESCRIPTION

[0030] The present invention is further described in detail below with reference to 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 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 using a 1000Da dialysis bag for 24 hours and freeze-dried to obtain fluorescent PDA.

[0037] S2: Preparation of exosome system:

[0038] Macrophages were cultured and induced into M2. The fluorescent PDA prepared in S1 was added and cultured using complete medium prepared with exosome-free serum. 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, comprising fluorescent polydopamine, exosomes, and an anti-inflammatory component. The exosome system naturally carrying fluorescent polydopamine nanoparticles prepared by the above preparation method exhibits high bioavailability, biostability, target 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 inflammation level of systemic lupus erythematosus.

[0041] The above contents are described below with reference to 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 with a magnetic stirrer for 2 min. The reaction was carried out at room temperature, and then 24 mg of glutathione was added and stirred at room temperature for 2 h. Finally, 31 mg of DNTB was added and stirred at room temperature for 1 h. The solution 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 autofluorescence, which is blue-green. It also has the effect of promoting the polarization of macrophages into M2 macrophages.

[0046] Then prepare the exosome system:

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

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

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

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

[0051] The cells were centrifuged at 150,000 × g for 90 minutes at 4°C, the supernatant was discarded, and 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, filtered through a 0.22μm microporous filter membrane, and then 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 ultrasonically dispersed to form a suspension. A few drops were pipetted onto the copper mesh of the electron microscope. After drying (or blotting with filter paper), the sample was used for electron microscopy observation. The test results were as follows: Figure 3 As shown, it also shows that exosomes can be stored at -80℃ for a long time.

[0055] Example 3:

[0056] After treating SLE mice with exosomes, serum samples were obtained from the mice. Whole blood was collected into a tube without anticoagulant and allowed to stand at room temperature for 1 hour. After the whole blood naturally coagulated and the serum was separated, it was centrifuged at approximately 1500g for 10 minutes at 4°C. The yellow supernatant was collected to obtain the serum. The expression levels of TNF-α in each component were then determined using the following procedure:

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

[0058] 2. Sample Addition: Set up blank wells (blank control wells without sample or enzyme-labeled reagent; all other steps remain the same), standard wells, and test sample wells. Accurately add 50 μL of the standard to the enzyme-labeled plate. Add 40 μL of sample diluent to the test sample wells, followed by 10 μL of the test sample (a final dilution of 5x). Add the sample to the bottom of the plate well, avoiding contact with the well walls. Gently shake to mix.

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

[0060] 4. Liquid preparation: Dilute the 30-fold concentrated washing solution with 30-fold 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 it. 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 50 μL of color developer A to each well, then add 50 μL of color developer B, gently shake to mix, and incubate at 37°C 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 will immediately turn yellow).

[0067] 11. Measurement: Use the blank well as the zero setting and measure the absorbance (OD) of each well at 450 nm. Measurement should be performed within 15 minutes after adding the stop solution. The expression level of TNF-α in each component will be determined.

[0068] The results are as follows Figure 4 As shown, TNF-α, a pro-inflammatory cytokine, plays a crucial role in the development and progression of inflammation in vivo. SLE mice were treated with exosomes, and serum samples were obtained to measure TNF-α expression. As can be seen, compared with the pathology group, the treatment group showed a statistically significant decrease in TNF-α levels.

[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 Beyotime.

[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 biodegradation.

[0072] The exosome system prepared in this application has unique advantages in the treatment of SLE. It has a long half-life and persists in the body for a long time. It can be stored at 4°C and -20°C for short periods of time, or at -80°C for long periods of time. Exosomes can transport proteins and nucleic acids between cells, protecting them from degradation upon entry. They are membrane vesicles secreted by various cells in the body. After inactivation, they are broken down by enzymes and do not accumulate in large quantities over long periods of time. They have minimal toxic side effects and are small enough to cross biological membranes, even the blood-brain barrier (BBB) ​​and the blood-cerebrospinal fluid barrier (BCSFB). Exosomes can carry different drugs to meet therapeutic needs, extending their half-life and increasing the stability of their release. In addition, exosomes have a variety of administration routes, and the appropriate route of administration can be selected according to the lesion site, such as subcutaneous injection, intravenous injection, intraperitoneal injection, intranasal injection, and oral administration. As drug carriers, exosomes have inherent advantages such as long-term stability, convenient storage, good content protection, avoidance of immune surveillance, and crossing biological barriers. These exosomes are effective anti-inflammatory and have excellent deoxidation properties. They also possess improved biocompatibility, high bioavailability, biostability, target specificity, low toxicity, and low immunogenicity, making them widely applicable in the treatment of inflammatory diseases. These exosomes show great promise as a drug or gene delivery vector for the treatment of SLE, making them excellent candidates for the treatment of SLE and other autoimmune diseases.

Claims

1. A method for preparing an exosome system naturally carrying fluorescent polydopamine nanoparticles, characterized by: 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 stirred with a magnetic stirrer to react. 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. The fluorescent PDA prepared in S1 was added and cultured using complete medium containing exosome-free serum. 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 was 24 h, and a 1000 Da dialysis bag was used for dialysis.

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 minutes and collect the supernatant; Centrifuge at 2000 × g for 15 minutes and collect the supernatant; Centrifuge at 10,000 × g for 30 minutes and collect the supernatant; The cells were centrifuged at 150,000 × g for 90 minutes, the supernatant was discarded, 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 by: The exosome system is prepared using the preparation method according to 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 components.

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

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