Preparation method of exosome loaded active substance

Abstract

The invention discloses a preparation method of an exosome loaded active substance, and belongs to the technical field of biological medicines. According to the method, a brand new method is provided for loading the NAD < + > to the exosome, the exosome is used as a delivery carrier, efficient delivery of the NAD < + > in vivo and in cells is achieved, and the method is a better delivery mode compared with direct intravenous infusion of the NAD < + >. According to the method, under the assistance of the fat-soluble coenzyme Q10, the exosome can be promoted to realize high-efficiency active substance loading. NAD < + > and coenzyme Q10 are mixed, so that lossless loading can be realized, an exosome membrane is not damaged, and the technical problem that the combined synergistic effect of NAD < + > and exosome cannot be realized in the prior art is solved; meanwhile, by mixing the NAD < + > and the coenzyme Q10, the combined synergistic effect of the NAD < + > and the exosome can be realized, so that a better anti-aging biological effect of the NAD < + > is exerted.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a method for preparing exosomes loaded with active substances. Background Art

[0002] In the field of biomedicine, nicotinamide adenine dinucleotide (NAD+) is a key molecule that plays an indispensable role in the production of cellular energy, DNA repair, and the maintenance of cellular health. NAD+ can activate sirtuins, poly (ADP-ribose) polymerase (PARP), and differentiation antigen 38 (CD38), and is involved in DNA damage repair, immune regulation, and cell proliferation. It is also closely related to age-related diseases such as cardiovascular disease, neurodegenerative diseases, and metabolic diseases. When DNA damage occurs, PARP-1 consumes a large amount of NAD+, resulting in reduced energy production. In addition, high levels of NAD+ can activate sirtuins, enabling them to carry out metabolic and stress protection responses, which has the effect of delaying aging and preventing and treating aging-related diseases.

[0003] In the field of nanotechnology, exosomes, as disc-shaped vesicles with a size of 40 to 130 nm, are widely studied for drug delivery. Exosomes have a variety of biological functions, including intercellular communication, promotion of tissue regeneration, anti-inflammatory effects, anti-tumor effects, and as biomarkers. Due to their unique advantages such as strong penetration ability, long blood circulation time, good biocompatibility, and wide biodistribution, exosomes are considered to be natural drug carriers with more advantages than liposomes. In the field of drug delivery, the delivery of NAD+ is an important and valuable research direction.

[0004] Since NAD+ is a water-soluble molecule with a short half-life in the body, it enters cells mainly relying on concentration differences, passively diffuses into cells, and is then transported to mitochondria through the solute transporter MCART1 / SLC25A51 to exert its effect, but its efficiency is not high.

[0005] Secondly, the existing exosome drug loading methods, such as the co-incubation method, are inefficient and difficult to achieve high-efficiency loading. In addition, electroporation or ultrasound methods damage the integrity of the exosome membrane, and there are some problems such as leakage of contents. Therefore, how to utilize the advantages of exosomes such as strong biocompatibility, high stability, and easy passage through the blood-brain barrier to efficiently and non-destructively deliver NAD+ to cells is the main problem facing current technology. Summary of the invention

[0006] The first purpose of the present invention is to provide a method for preparing exosomes loaded with active substances. The method of loading NAD+ into exosomes uses exosomes as delivery carriers to achieve efficient delivery of NAD+, thereby solving the technical problem of low efficiency of NAD+ delivery into cells and difficulty in achieving efficient intracellular delivery.

[0007] The second purpose of the present invention is to provide a method for preparing exosomes loaded with active substances. This scheme can achieve lossless loading of exosomes by mixing NAD+ with coenzyme Q10 and using coenzyme Q10 to assist NAD+, thereby improving safety and solving the technical problem that the prior art cannot achieve the combined synergistic effect of NAD+ and exosomes; at the same time, by mixing NAD+ with coenzyme Q10, the combined synergistic effect of NAD+ and exosomes can be achieved, thereby exerting a better anti-aging biological effect of NAD+.

[0008] The present invention is achieved through the following technical solutions:

[0009] A method for preparing exosomes loaded with active substances comprises the following steps:

[0010] S1. Active substances and auxiliary agents are selected and physically mixed to obtain a mixed solution A;

[0011] S2, using a co-incubation method to load the active substance in the mixed solution A into exosomes, to obtain;

[0012] The exosomes are derived from the secretion of mesenchymal stem cells or other stem cells and are obtained by separation and extraction;

[0013] The active substance is loaded into exosomes.

[0014] Preferably, the active substance is nicotinamide adenine dinucleotide;

[0015] The auxiliary agent is coenzyme Q10 injection.

[0016] Coenzyme Q10 (COQ10) is a fat-soluble molecule produced endogenously in the human body and is considered to be an important component of the mitochondrial electron transport chain and an electron acceptor for complexes I and II. Coenzyme Q10 plays a key role in mitochondrial oxidative phosphorylation and ATP production, so it is essential for all energy-dependent processes in the body, boosting the immune system and acting as a free radical scavenger. Exosomes have a variety of biological functions, such as intercellular communication, promoting tissue regeneration, anti-inflammatory effects, anti-tumor effects and serving as biomarkers.

[0017] Therefore, the method of the present invention utilizes the fat solubility of coenzyme Q10 to encapsulate the active substance, and then loads it into exosomes, which can achieve the combined synergistic effect of the active substance and exosomes and improve the therapeutic effect.

[0018] Preferably, the mixing volume ratio of the active substance to the auxiliary agent is 1:0-10.

[0019] Preferably, the mixing volume ratio of the active substance to the auxiliary agent is 1:2.

[0020] Preferably, the physical mixing process in S1 is: select active substances and auxiliary agents according to a volume ratio, mix them, stir them with a magnetic stirrer at a speed of 300 r / min for 25 to 30 minutes, and let them stand for 30 to 35 minutes to obtain a mixed solution A;

[0021] The mixed solution A is a clear liquid.

[0022] Preferably, the method for loading the active substance in the mixed solution A into exosomes in S2 is:

[0023] After the mixed solution A and the exosomes are mixed, the mixture is stirred magnetically at a speed of 300 r / min for 25 to 30 min at room temperature to obtain;

[0024] The mixing volume ratio of the mixed solution A to the exosomes is 1:1-10.

[0025] Preferably, the mixing volume ratio of the mixed solution A to the exosomes is 1:10.

[0026] Compared with the prior art, the present invention has at least the following technical effects:

[0027] (I) The present invention provides a novel method for preparing exosomes secreted by mesenchymal stem cells for efficiently loading active substances. This method provides a new method for loading NAD+ into exosomes, which uses exosomes as delivery carriers to achieve in vivo and intracellular delivery of NAD+, and is a better delivery method than direct intravenous infusion of NAD+.

[0028] The preparation method of the exosome loaded with active substances is a method that can promote the exosomes to load active substances efficiently with the help of fat-soluble coenzyme Q10 through the exosomes secreted by mesenchymal stem cells or other stem cells. Mixing NAD+ with fat-soluble coenzyme Q10 can achieve lossless loading to exosomes, thereby improving safety and solving the technical problem that the existing technology cannot achieve the combined synergistic effect of NAD+ and exosomes; at the same time, by mixing NAD+ with coenzyme Q10, the combined synergistic effect of NAD+ and exosomes can be achieved, thereby exerting a better anti-aging biological effect of NAD+.

[0029] The method of the present invention uses exosomes as carriers. Exosomes are a natural drug carrier with good biocompatibility. At the same time, coenzyme Q10 injection is a commercially available product. During the loading and preparation process, it can be directly injected into the body without going through a purification process, and has good safety. Therefore, the method of the present invention is relatively safe. This advantage makes the method of the present invention more feasible in clinical applications.

[0030] (ii) The method of the present invention adopts a co-incubation method to load drugs, which is simple to operate and easy to industrialize. This advantage makes the method of the present invention more feasible in industrial production.

[0031] (III) Due to the advanced nature of this technical solution, it can be widely used in the fields of biomedicine, nanotechnology and molecular biology. Specifically:

[0032] (1) In the field of biomedicine, this technical solution provides a method for efficiently and safely delivering NAD+ into cells. By mixing NAD+ with fat-soluble coenzyme Q10, the efficiency of exosome loading NAD+ can be significantly improved, thereby increasing the level of NAD+ in cells and exerting a better anti-aging biological effect.

[0033] (2) Due to the unique advantages of exosomes such as strong penetration ability, long blood circulation time, good biocompatibility, and wide biodistribution, this technical solution can also reduce the dose of intravenous infusion of NAD+ and reduce the burden on patients. Therefore, this technical solution has broad application prospects in anti-aging and prevention and treatment of aging-related diseases.

[0034] (3) In the field of nanotechnology, this technical solution provides a new method for loading drugs into exosomes. Existing methods for loading drugs into exosomes, such as direct co-incubation, electroporation, ultrasound, transfection, etc., are inefficient and difficult to achieve high-efficiency loading without leakage of exosome contents. However, this technical solution can significantly improve the efficiency of loading NAD+ into exosomes by mixing NAD+ with fat-soluble coenzyme Q10 and assisting with fat-soluble coenzyme Q10, thus providing a new idea and method for loading drugs into exosomes. Therefore, this technical solution has broad application prospects in the field of nanotechnology.

[0035] (4) In the field of molecular biology, this technical solution provides a new method for NAD+ delivery. Since NAD+ is a water-soluble molecule with a short half-life in the body, it requires frequent intravenous infusion, which is a heavy burden on patients. However, this technical solution can significantly improve the efficiency of exosome loading NAD+ by mixing NAD+ with fat-soluble coenzyme Q10, thereby increasing the level of NAD+ in cells and providing new ideas and methods for NAD+ delivery. Therefore, this technical solution has broad application prospects in the field of molecular biology.

[0036] In general, due to its advantages of high efficiency, safety and simplicity, this technical solution can not only improve the delivery efficiency of NAD+ and reduce the burden on patients, but also provide new ideas and methods for exosome loading of drugs and NAD+ delivery. Therefore, it has broad application prospects in application fields such as biomedicine, nanotechnology and molecular biology. DETAILED DESCRIPTION

[0037] The embodiments of the present invention will be described in detail below in conjunction with examples. However, those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention. The specific conditions not specified in the examples are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used without indicating the manufacturer are all conventional products that can be purchased commercially.

[0038] A technical solution of a specific implementation of the present invention is:

[0039] A method for preparing exosomes loaded with active substances comprises the following steps:

[0040] S1. Active substances and auxiliary agents are selected and physically mixed to obtain a mixed solution A;

[0041] S2, using a co-incubation method to load the active substance in the mixed solution A into exosomes, to obtain;

[0042] The exosomes are derived from the secretion of mesenchymal stem cells or other stem cells and are obtained by separation and extraction;

[0043] The active substance is loaded into exosomes.

[0044] The active substance is nicotinamide adenine dinucleotide;

[0045] The auxiliary agent is coenzyme Q10 injection.

[0046] The mixing volume ratio of the active substance to the auxiliary agent is 1:0-10.

[0047] The mixing volume ratio of the active substance to the auxiliary agent is 1:2.

[0048] The physical mixing process in S1 is: select active substances and auxiliary agents according to volume ratio, mix them, stir them with magnetic force at a speed of 300 r / min for 25 to 30 minutes, and let them stand for 30 to 35 minutes to obtain mixed solution A;

[0049] The mixed solution A is a clear liquid.

[0050] The above-mentioned mixing method can be physical mixing, and the physical mixing method can be mixing NAD+ and coenzyme Q10 in a solvent.

[0051] The method for loading the active substance in the mixed solution A into exosomes in S2 is:

[0052] After the mixed solution A and the exosomes are mixed, the mixture is stirred magnetically at a speed of 300 r / min for 25 to 30 min at room temperature to obtain;

[0053] The mixing volume ratio of the mixed solution A to the exosomes is 1:1-10.

[0054] The mixing volume ratio of the mixed solution A to the exosomes is 1:10.

[0055] The above-mentioned loading method can be a combination of physical adsorption and membrane permeation, or the mixed NAD+ and coenzyme Q10 can be mixed with exosomes.

[0056] How to use the product: Infuse NAD+-loaded exosomes into the body. The infusion method can be intravenous injection or intramuscular injection. The intravenous injection method is to dissolve the NAD+-loaded exosomes in a solvent.

[0057] Product Monitoring: Monitor the level of NAD+ in the body or cells.

[0058] The monitoring method is composed of NAD+ detection protein probe and supporting reagents.

[0059] The dosage and infusion frequency of NAD+ are adjusted according to the level of NAD+ in the body and cells, providing a basis for clinical application plans.

[0060] Example 1: Mixing Coenzyme Q10 and NAD+

[0061] Coenzyme Q10 injection was produced by Guangzhou Baiyunshan Mingxing Pharmaceutical Co., Ltd., with a specification of 5 mg / 2 mL), and coenzyme I (NAD+) for injection, with a specification of 5 mg / vial, was purchased from Kangnuo Biopharmaceutical Co., Ltd.

[0062] NAD+ and fat-soluble coenzyme Q10 were mixed in different volume ratios (1:0, 1:1, 1:2, 1:5, 1:10).

[0063] Coenzyme Q10 is used as a solvent for the coenzyme Q10 injection preparation.

[0064] The mixing method is physical mixing, mixing NAD+ and coenzyme Q10, mixing with magnetic stirring (speed 300r / min, time 30min), and then standing for 30min after stirring to allow them to fully contact and become a clear liquid.

[0065] It was observed that the volume ratio of NAD+ and coenzyme Q10 was 1:2. After standing, the solution was relatively stable, and there was no obvious stratification of the liquid. Therefore, this ratio was selected as the preparation parameter.

[0066] Example 2: NAD+ and Coenzyme Q10 mixture loaded exosomes

[0067] A physical method of co-incubation was used.

[0068] Exosomes secreted by mesenchymal stem cells were provided by Beijing Enze Kangtai Biotechnology Co., Ltd., with a specification of 1×10 11 Granules / 5ml / tube, batch EK1003-20240601. NAD+ detection was commissioned by China Science Coenzyme Medical Technology (Shenzhen) Co., Ltd.

[0069] As in Example 1, a mixture of NAD+ and coenzyme Q10 solution (1:2 volume ratio) was mixed with exosomes, wherein the ratios of the mixture to the exosomes were 1:1, 1:2, 1:5, and 1:10 (volume ratio), respectively. The mixed solution was magnetically stirred at room temperature (speed 300 r / min, time 30 min) to allow NAD+ to fully contact the exosomes, and part of the NAD+ entered the exosomes.

[0070] At the same time, a batch of NAD+ directly loaded exosome samples were prepared as a comparative example for comparison.

[0071] Then the exosome particle identification, encapsulation efficiency and loading rate were tested respectively.

[0072] The loaded exosomes were extracted using a kit (Invitrogen, Catalog No. 4478359), and RIPA lysis and extraction buffer (Thermo Scientific, Catalog No. 89900) was used to detect the mass of NAD+ loaded in the exosomes.

[0073] The loading rate and encapsulation efficiency were determined as follows:

[0074] The luminescence value of the biological probe method was used to calculate the mass concentration of NAD+ after exosome lysis according to the standard curve equation.

[0075] The loading rate and encapsulation rate of NAD+ in exosomes are respectively as follows:

[0076] Loading rate (%) = NAD+ amount in exosomes / total mass of the reaction system after drying;

[0077] Encapsulation efficiency (%) = (total amount of NAD+ added to the system - amount of free NAD+) / total amount of NAD+ added to the system.

[0078] It can be seen from Table 1 that after Coenzyme Q10 is mixed with NAD+, and then NAD+ is loaded into exosomes, its loading rate and encapsulation rate are higher than when NAD+ is directly loaded into exosomes.

[0079] Comprehensive analysis shows that the appropriate volume ratio of exosomes to coenzyme Q10 and NAD+ mixture is 1:10.

[0080] Table 1 Loading rate and encapsulation rate of exosomes loaded with NAD+

[0081]

[0082] Note: Compared with the corresponding ratio of NAD+:exosome groups, *p<0.05, **p<0.01.

[0083] Example 3: Verification of the NAD+ molar concentration of coenzyme Q10-assisted NAD+-loaded exosomes in mice

[0084] 3.1 C57BL / 6J mice, half male and half female, 12 weeks old, were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd.

[0085] 3.2 The sources and specifications of NAD+ injection and exosomes are the same as those in Example 1 and Example 2.

[0086] Coenzyme Q10 assists NAD+ in loading exosomes. A mixed solution of NAD+ and coenzyme Q10 is prepared according to the method of Example 1, and then prepared according to the method of Example 2, that is, the ratio (volume) of the mixed solution to the exosomes is 1:10.

[0087] The subjects were randomly divided into three groups: direct NAD+ injection group, NAD+-loaded exosomes group, and coenzyme Q10-assisted NAD+-loaded exosomes group.

[0088] Each group had 12 mice, both male and female, who were injected with NAD+ via tail vein (according to the uniform NAD+ amount of 10 mg / kg). Blood was collected immediately (0 min), 10 min, 60 min, and 180 min after injection, and the levels of NAD+ in peripheral blood and peripheral blood nucleated cells were dynamically monitored.

[0089] 3.3 The bioluminescent probe method (commissioned by China Science Coenzyme Medical Technology (Shenzhen) Co., Ltd.) was used to detect the total level of NAD+ in the peripheral blood of animals and the level of NAD+ in isolated peripheral blood mononuclear cells.

[0090] Combining Table 2 and Table 3, the results show that in the group assisted by coenzyme Q10 in exosome loading with NAD+, the peak concentration of intracellular NAD+ was higher and the amount of distribution was larger. When the same amount of NAD+ entered the animal body, the intracellular NAD+ lasted longer.

[0091] Table 2 NAD+ levels in peripheral blood (μmol / L)

[0092]

[0093] Note: Compared with the direct injection of NAD+ group, *p<0.05; compared with the exosome-loaded NAD+ group, #p<0.05.

[0094] Table 3 NAD+ levels in peripheral blood cells (pmol / mg)

[0095]

[0096] Note: Compared with the direct injection of NAD+ group, *p<0.05; compared with the exosome-loaded NAD+ group, #p<0.05.

[0097] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the protection scope of the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for preparing exosomes loaded with active substances, characterized in that: The steps include: S1. Active substances and auxiliary agents are selected and physically mixed to obtain a mixed solution A; S2, using a co-incubation method to load the active substance in the mixed solution A into exosomes, to obtain; The exosomes are derived from the secretion of mesenchymal stem cells or other stem cells and are obtained by separation and extraction; The active substance is loaded into exosomes.

2. The method for preparing exosomes loaded with active substances according to claim 1, characterized in that: The active substance is nicotinamide adenine dinucleotide; The auxiliary agent is coenzyme Q10 injection.

3. The method for preparing exosomes loaded with active substances according to claim 1, characterized in that: The mixing volume ratio of the active substance to the auxiliary agent is 1:0-10.

4. The method for preparing exosomes loaded with active substances according to claim 3, characterized in that: The mixing volume ratio of the active substance to the auxiliary agent is 1:

2.

5. The method for preparing exosomes loaded with active substances according to claim 1, characterized in that: The physical mixing process in S1 is: select active substances and auxiliary agents according to volume ratio, mix them, stir them with magnetic force at a speed of 300 r / min for 25 to 30 minutes, and let them stand for 30 to 35 minutes to obtain mixed solution A; The mixed solution A is a clear liquid.

6. The method for preparing exosomes loaded with active substances according to claim 1, characterized in that: The method for loading the active substance in the mixed solution A into exosomes in S2 is: After the mixed solution A and the exosomes are mixed, the mixture is stirred magnetically at a speed of 300 r / min for 25 to 30 min at room temperature to obtain; The mixing volume ratio of the mixed solution A to the exosomes is 1:1-10.

7. The method for preparing exosomes loaded with active substances according to claim 6, characterized in that: The mixing volume ratio of the mixed solution A to the exosomes is 1:10.

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