An electroporation buffer and its application in loading peptides onto exosomes

Abstract

The present invention discloses an electrotransfer buffer and its application in exosome loading polypeptide, and relates to the field of biotechnology. The electrotransfer buffer is made of the following components: 80-120 mM trehalose, 6-10 mM potassium dihydrogen phosphate, 12-16% iodixanol and buffer. The present invention also provides a method for exosome drug loading using the above-mentioned electrotransfer buffer, the present invention realizes more efficient loading of palmitoyl pentapeptide-4 by exosomes, increases the loading amount of molecules without losing exosomes, and can obtain a higher collagen expression level, and the method of the present invention can be scaled up to meet subsequent production requirements.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to an electrotransfer buffer and an application thereof in exosome loading with polypeptides. Background Art

[0002] Exosomes are membrane vesicles that are released from the cell after the fusion of intracellular multivesicular bodies with the cell membrane. They are 30-150nm in diameter and have a phospholipid bilayer. Exosomes contain a variety of components, including proteins, lipids, mRNA, mitochondrial DNA, miRNA, and many other non-coding RNAs. Due to their low immunogenicity, low toxicity, tissue targeting, and engineering transformation, they have become a very potential drug delivery carrier.

[0003] Exosomes have the advantages of good biocompatibility, small size, and low immunogenicity. They can be used as natural carriers for small molecule drugs, transmembrane proteins, and nucleic acid drugs. There are currently a large number of related 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 separation) and endogenous drug delivery (during exosome biogenesis). Exogenous loading is to first separate the exosomes, and then load the drug molecules directly onto the surface or cavity of the exosome membrane by passive or active means. Exogenous exosome loading can be divided into active loading and passive loading. Passive loading includes co-incubation method, which is fast and simple, 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, etc. Endogenous loading is to load drug molecules into parent cells before isolating exosomes, so that the exosomes secreted by them carry drug molecules. These methods also have disadvantages such as affecting the quality of exosomes, low loading efficiency, and easy aggregation of exosomes.

[0004] Chinese patent CN111378685A discloses a method for preparing drug-loaded exosomes and drug-loaded exosomes, the method comprising the following steps: step (1): culture and transform HEK293T cells using a culture medium without exosomes and collect the cells; step (2): culture the HEK293T cells in a substrate having multiple channels with a diameter of 200-600 nm, then place the substrate in a buffer containing the target plasmid DNA, first perform electroporation, and then stimulate with a directional current for 8-12 hours; step (3): ultracentrifuge the obtained buffer, collect the precipitate and resuspend it in a buffer solution to obtain exosomes loaded with gene drugs. This method combines the exosome purification and drug loading steps into one, so that the exosomes secreted by the cells are loaded with target gene drugs, while greatly improving their yield, targeting and applicability, but its operation is relatively complicated.

[0005] Palmitoyl Pentapeptide-4 is a messenger peptide that can stimulate the synthesis of collagen (type I, type II, type IV), elastic fibers and hyaluronic acid. It can also increase the skin's moisture content and water retention, increase skin thickness and reduce fine lines. It reverses the skin aging process by rebuilding from the inside out. Therefore, it has the effect of anti-wrinkle, anti-aging and improving skin quality.

[0006] 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. Summary of the invention

[0007] The object of the present invention is an electrotransfer buffer and its application in exosome loading polypeptide.

[0008] To achieve the above-mentioned object of the invention, the technical solution of the present invention is as follows:

[0009] In one aspect, the present invention provides an electrotransfer buffer, which is made of the following components: 80-120 mM trehalose, 6-10 mM dipotassium hydrogen phosphate, 12-16% iodixanol and a buffer.

[0010] Furthermore, the electrotransfer buffer is made of the following components: 90-110 mM trehalose, 6-8 mM dipotassium hydrogen phosphate, 14-16% iodixanol and buffer.

[0011] Furthermore, the electrotransfer buffer is made of the following components: 100 mM trehalose, 7 mM dipotassium hydrogen phosphate, 15% iodixanol and buffer.

[0012] Specifically, the buffer is selected from one or more of PBS buffer, Tris buffer, MES buffer, HEPES buffer, citric acid-sodium citrate buffer, acetic acid-sodium acetate buffer, glycine-hydrochloric acid buffer, and MOPS buffer.

[0013] Furthermore, the buffer solution is PBS buffer solution.

[0014] In another aspect, the present invention provides a method for drug loading in exosomes, comprising the following steps:

[0015] (1) Add exosomes and drugs to the electroporation buffer and perform electroporation;

[0016] (2) After the electroporation is completed, incubation buffer and drugs are added and incubated to obtain loaded exosomes;

[0017] (3) Purifying the loaded exosomes.

[0018] Specifically, the electrotransfer buffer described in step (1) is made of the following components: 80-120 mM trehalose, 6-10 mM potassium dihydrogen phosphate, 12-16% iodixanol and buffer.

[0019] Furthermore, the electrotransfer buffer is made of the following components: 90-110 mM trehalose, 6-8 mM dipotassium hydrogen phosphate, 14-16% iodixanol and buffer.

[0020] Furthermore, the electrotransfer buffer is made of the following components: 100 mM trehalose, 7 mM dipotassium hydrogen phosphate, 15% iodixanol and buffer.

[0021] Specifically, the final concentration of the exosomes described in step (1) is 1E+9-1E+11 particles / mL.

[0022] Furthermore, the final concentration of the exosomes described in step (1) is 1E+11 particles / mL.

[0023] Specifically, the drugs in step (1) and step (2) are polypeptides.

[0024] Furthermore, the drug in step (1) and step (2) is palmitoyl pentapeptide-4.

[0025] Furthermore, the final concentration of the palmitoyl pentapeptide-4 is 0.5-3 mg / ml;

[0026] Furthermore, the final concentration of the palmitoyl pentapeptide-4 is 0.5-1.5 mg / ml;

[0027] Furthermore, the final concentration of the palmitoyl pentapeptide-4 is 1 mg / ml.

[0028] Specifically, the electroporation in step (1) is performed using an electroporator.

[0029] Specifically, the conditions for electroporation in step (1) are: voltage 300-500 V, pulse width 100-500 us, and number of electric shocks 5-8 times.

[0030] Furthermore, the conditions for electroporation in step (1) are as follows: voltage 350-450V, pulse width 100-200us, and number of electric shocks 6-8 times.

[0031] Furthermore, the conditions for electroporation in step (1) are as follows: voltage 400 V, pulse width 150 us, and number of electric shocks 6 times.

[0032] Specifically, the incubation buffer in step (2) is made of buffer and magnesium chloride.

[0033] Furthermore, the concentration of magnesium chloride in step (2) is 1-3 mM;

[0034] Furthermore, the concentration of magnesium chloride in step (2) is 1 mM.

[0035] Specifically, the buffer is selected from one or more of PBS buffer, Tris buffer, MES buffer, HEPES buffer, citric acid-sodium citrate buffer, acetic acid-sodium acetate buffer, glycine-hydrochloric acid buffer, and MOPS buffer.

[0036] Furthermore, the buffer solution is PBS buffer solution.

[0037] Specifically, the incubation time in step (2) is 30-100 min;

[0038] Furthermore, the incubation time in step (2) is 60 minutes.

[0039] Specifically, the purification step described in step (3) is: centrifuging the loaded exosomes, taking the supernatant, washing, and concentrating.

[0040] Furthermore, the centrifugal speed is 8000-12000g, and the centrifugal time is 5-15min;

[0041] Furthermore, the centrifugal speed is 10000g and the centrifugal time is 10min.

[0042] Specifically, the washing is performed using a 100 kD ultrafiltration tube and PBS at a ratio of 1:5 for 5 times.

[0043] In another aspect, the present invention provides an application of the above-mentioned electroporation buffer or the above-mentioned exosome drug loading method in improving drug loading efficiency.

[0044] In another aspect, the present invention provides the use of the above-mentioned electroporation buffer or the above-mentioned exosome drug loading method in improving the expression level of collagen.

[0045] The beneficial effects of the present invention are:

[0046] The electrotransfer buffer and electrotransfer method used in the present invention can efficiently load polypeptide molecules into exosomes, achieve more efficient loading of palmitoyl pentapeptide-4 by exosomes, increase the loading amount of molecules without losing exosomes, and obtain higher collagen expression levels in in vitro experiments. The method of the present invention can be scaled up to meet subsequent production requirements. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 This is the analysis result of palmitoyl pentapeptide-4 content.

[0048] Figure 2 This is a detection chart of the content of palmitoyl pentapeptide-4 loaded in exosomes.

[0049] Figure 3 The particle size distribution of exosomes after ultrasonic loading.

[0050] Figure 4 This is the result graph of collagen content. DETAILED DESCRIPTION

[0051] In order to make the technical means, creative features, purpose and efficacy of the present invention easy to understand, the present invention is further explained in conjunction with specific embodiments below, but the following embodiments are only preferred embodiments of the present invention, not all. Based on the embodiments in the implementation mode, other embodiments obtained by those skilled in the art without creative work all belong to the protection scope of the present invention. In the following embodiments, unless otherwise specified, the operating methods used are conventional operating methods, the equipment used are conventional equipment, and the equipment materials used in each embodiment are the same.

[0052] The manufacturer and item number of the experimental materials:

[0053] Iodixanol was purchased from Aladdin Reagent Company, catalog number I342725;

[0054] Magnesium chloride was purchased from Aladdin Reagent Company, catalog number M140788;

[0055] Palmitoyl pentapeptide-4 was purchased from Aladdin Reagent Company with the catalog number P292764.

[0056] Example 1

[0057] 1. Buffer Preparation

[0058] Electroporation buffer: PBS solution with 100 mM trehalose, 7 mM potassium dihydrogen phosphate (adjusted to pH 7.4 with phosphoric acid) and 15 (W / V)% iodixanol.

[0059] The above formula is prepared at a 2X concentration for mixing with exosomes and other molecules.

[0060] Incubation buffer: PBS solution with 1 mM MgCl2 added.

[0061] 2. Electroporation method:

[0062] First, use the above 2X electroporation buffer to prepare 400uL of electroporation system, add exosomes (purchased from Yinguan Biotechnology Co., Ltd., catalog number: ST08002) to a final concentration of 1E+11 particles / mL, and palmitoyl pentapeptide-4 to a final concentration of 1mg / mL, and make up the rest of the volume with PBS.

[0063] Electroporation was performed using an X-Porator H1 electroporator (Yida Biotechnology Co., Ltd.); the above electroporation system was added to the electroporation cup, and electroporation was performed using a voltage of 400V, a pulse width of 150us, and 6 electric shocks.

[0064] After the electroporation, add 4 times the volume of incubation buffer to the sample, and the final concentration of palmitoyl pentapeptide-4 is 1 mg / mL. Incubate on ice for 1 hour to promote the recovery of exosome membrane.

[0065] Complete the purification step.

[0066] 3. Purification:

[0067] The loaded exosomes were first centrifuged at 10000g for 10 minutes, and the supernatant was collected. Then, the supernatant was washed five times with PBS at a ratio of 1:5 using a 100kD ultrafiltration tube and concentrated to 400uL or the required volume.

[0068] Comparative Example 1

[0069] The only difference from Example 1 is that the electrotransfer buffer is different. The formula of the electrotransfer buffer in Comparative Example 1 is shown in Table 1 below.

[0070] Table 1 Electroporation buffer recipe

[0071]

[0072] Comparative Example 2

[0073] The only difference from the embodiment is that the electroporation parameters are different. The electroporation parameters in Comparative Example 2 are as follows:

[0074] Table 2 Electroporation parameter settings

[0075]

[0076] Experimental Example 1 Loading Efficiency Analysis

[0077] Palmitoyl pentapeptide-4 in the sample was quantitatively analyzed using a high performance liquid chromatography system HPLC (brand: Agilent Technologies, Inc., model: 1260 Infinity II) and a chromatographic column (brand: GL Sciences, model: Inertsustain AQ-C18 5um 250x4.6mm).

[0078] Mobile phase: A: 0.3% acetic acid in 50 mM ammonium acetate aqueous solution; mobile phase B: acetonitrile, gradient elution was performed, and the gradient settings were as shown in Table 3 below.

[0079] Table 3 Gradient settings

[0080]

[0081] A UV detector was used to detect the absorption peak at a wavelength of 214 nm.

[0082] The palmitoyl pentapeptide-4 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 content of palmitoyl pentapeptide-4 in the samples was calculated.

[0083] Palmitoyl pentapeptide-4 was analyzed by HPLC. Figure 1 As shown, the characteristic absorption peak of palmitoyl pentapeptide-4 can be obtained at around 9 minutes.

[0084] The results of purification after exosome loading are as follows Figure 2 As shown in the figure, it can be seen that the sample contains the characteristic absorption of palmitoyl pentapeptide-4, indicating that the molecule is effectively loaded.

[0085] The particle size distribution of exosomes after electroporation is as follows Figure 3 As shown in the figure, it can be seen that the particle size distribution of exosomes did not change significantly after electroporation.

[0086] The amount of exosomes retained and the loading content of different ratios of buffer were measured by nanoflow cytometry (manufacturer: Xiamen Fuliu Biotechnology Co., Ltd., model: vNTA-S20) and HPLC, respectively, as shown in Table 4 below. Among them, Example 1 (100 mM trehalose, 7 mM potassium dihydrogen phosphate, 15% iodixanol) can obtain the maximum loading amount with less loss of exosomes. The control group using the comparative buffer had a certain loss of exosomes, and the loading efficiency was significantly lower.

[0087] Table 4 Exosome loading efficiency

[0088]

[0089] Experimental Example 2 In vitro experiment

[0090] Collagen secretion test: Fibroblasts were plated in 24-well plates, and exosomes (2.36E+10 particles / mL exosomes 20ug / mL palmitoyl pentapeptide-4) and control drugs (unloaded exosomes 2.36E+10 particles / mL, palmitoyl pentapeptide-4 raw materials 10ug / mL and 100ug / mL) were added after 24 hours. After 72 days of drug incubation, the cell supernatant was collected and the content was detected using a collagen ELISA kit (purchased from Beijing Solebow Technology Co., Ltd., catalog number SEKH-0401).

[0091] After stimulating cells with exosomes, palmitoyl pentapeptide-4 (10ug / mL and 100ug / mL) and loaded palmitoyl pentapeptide-4 (20ug / mL), the collagen content in the cell supernatant was as follows Figure 4 , it can be seen that the loading group significantly stimulated the secretion of collagen.

[0092] Comparative Example 3

[0093] The electrotransfer method in Example 1 is different in that the electrotransfer buffer is different. The electrotransfer buffer in Comparative Example 1 is prepared as follows: 21% OptiPrep is added to PBS, the final concentration of exosomes added is 1E+11 particles / mL, and the final concentration of palmitoyl pentapeptide-4 is 1 mg / mL.

[0094] Electroporation parameters: voltage 400V, pulse width 150us, electric shock 6 times.

[0095] After electroporation, incubate on ice for 1 hour.

[0096] The purification steps and collagen secretion test steps were the same as in Example 1, and the results were as follows: Figure 4 .

[0097] 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, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for drug delivery of exosomes, characterized in that: The following steps are involved: (1) Add exosomes and drugs to the electroporation buffer and perform electroporation; (2) After the electroporation is completed, incubation buffer and drugs are added and incubated to obtain loaded exosomes; (3) Purifying the loaded exosomes; The electrotransfer buffer described in step (1) is made of the following components: 90-110 mM trehalose, 6-8 mM potassium hydrogen phosphate, 14-16% iodixanol and PBS buffer; The conditions for electroporation in step (1) are as follows: voltage 300-500 V, pulse width 100-500 us, and number of electric shocks 5-8 times; The drug in step (1) and step (2) is palmitoyl pentapeptide-4, and the final concentration of palmitoyl pentapeptide-4 is 0.5-3 mg / ml; The final concentration of exosomes described in step (1) was 1×10 9 -1×10 11 particles / mL.

2. The exosome drug delivery method according to claim 1, characterized in that: The electrotransfer buffer is made of the following components: 100 mM trehalose, 7 mM dipotassium hydrogen phosphate, 15% iodixanol and buffer.

3. The exosome drug delivery method according to claim 1, characterized in that: The final concentration of the palmitoyl pentapeptide-4 is 1 mg / ml.

4. The exosome drug delivery method according to claim 1, characterized in that: The conditions for electroporation in step (1) are as follows: voltage 400 V, pulse width 150 us, and number of electric shocks 6 times.

5. The exosome drug delivery method according to claim 1, characterized in that: The incubation buffer described in step (2) is made of buffer and magnesium chloride.

6. The exosome drug delivery method according to claim 5, characterized in that: The concentration of magnesium chloride in step (2) is 1-3 mM.

7. Use of the exosome drug loading method according to any one of claims 1 to 6 in the preparation of a drug for improving drug loading efficiency.

8. Use of the exosome drug loading method according to any one of claims 1 to 6 in the preparation of a drug for improving the expression level of collagen.

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