Novel exosome composition as well as preparation method and application thereof

Abstract

The invention discloses a novel exosome composition as well as a preparation method and application thereof, and belongs to the technical field of biological medicines. The composition is obtained by mixing exosome, nicotinamide adenine dinucleotide or a precursor thereof and ginsenoside, can play a synergistic effect of different action mechanisms, and can significantly enhance the cognitive function to treat diseases related to brain hypofunction compared with single use of one component. Moreover, the components are all natural products existing in the nature, and clinical intravenous injection products have better safety in actual use. The composition can be applied to preparation of products for treating brain memory hypofunction, and is used for treating brain hypofunction caused by memory hypofunction, Alzheimer's disease, ischemic cerebral apoplexy sequelae and the like. The invention provides a detailed product preparation method and use guidance, and the effectiveness and safety of the traditional Chinese medicine composition are verified through a product pharmacodynamic embodiment.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a novel exosome composition and a preparation method and application thereof. Background Art

[0002] Brain function impairment caused by Alzheimer's disease has become a global health problem. Parkinson's disease and chronic ischemic stroke can also lead to varying degrees of brain function impairment. Traditional treatment methods have many limitations, such as unsatisfactory treatment effects, no effective drugs to reverse the development of pathology, difficulty in crossing the blood-brain barrier (BBB), large side effects, and inconvenience in use, especially for patients with significant memory impairment. In recent years, exosomes, as a new type of biological carrier, have shown great potential in the treatment of neurodegenerative diseases. Clinical studies have been conducted on exosome nasal drops for the treatment of AD. Exosomes can carry various biomolecules (such as miRNA, proteins, etc.), have anti-inflammatory, immunomodulatory and angiogenesis functions, and also have the ability to cross the blood-brain barrier. However, the mechanism of neurodegenerative diseases such as AD is complex and involves multiple links. Multi-target drugs are a development direction that play a role in multiple links and are expected to achieve better results (Liu Ruiting, Lv Qiujun. Acta Pharmaceutica Sinica, 2009, 44, 258-263). As an important coenzyme, NAD+ plays a key role in cellular energy metabolism and DNA repair, and has been shown to improve neuronal function. NAD+ precursors, such as nicotinamide mononucleotide (NMN), nicotinamide riboside (NR) (including nicotinamide riboside salts, such as nicotinamide riboside chloride), etc., can increase the level of NAD+ in the body, but most NAD+ precursors are taken orally, undergo intestinal metabolism and pass through the intestinal wall, and have low bioavailability. If these precursors are used to prevent and treat neurological diseases, they need to cross the blood-brain barrier, which affects the effectiveness of their use.

[0003] Exosomes, as cell-derived nanovesicles, can participate in the intercellular transport of substances, mediate intercellular communication and regulate the body's biological functions. They have the advantages of low immunogenicity, low toxicity and the ability to naturally cross the BBB, and play an important role in brain targeted delivery.

[0004] Public research has shown that ginsenosides Rg1 and Rb1 can improve the entire memory process, enhance synaptic transmission, and enhance synaptic transmission for a long time. They can also increase the acetylcholine content in the brain and increase the density of central M-choline receptors. Holistic, cellular and molecular biology experiments have proven that its mechanism is to enhance neural plasticity, increase the function of the central cholinergic system, promote protein synthesis in the brain, mobilize the body's protective mechanism, and improve age-related functional impairment.

[0005] Currently, it is common to use a single raw material, but the effect of using the raw material alone is always unsatisfactory. For example, the bioavailability of oral ginsenosides is low, it is difficult to cross the blood-brain barrier, and the clinical effect is not ideal. Therefore, although there is a tradition of using ginseng in the existing technology, its active ingredient saponin rarely becomes a real therapeutic drug. Summary of the invention

[0006] The first object of the present invention is to provide a novel exosome composition, which is a composition obtained by mixing exosomes, nicotinamide adenine dinucleotide or its precursor and ginsenosides, which can exert synergistic effects of different mechanisms of action, and can significantly enhance cognitive function to treat diseases related to brain dysfunction compared to using one component alone. In addition, these components are all natural products existing in nature, and there are also intravenous injection products in actual use in clinical practice, which have good safety.

[0007] The second object of the present invention is to provide an application of a novel exosome composition, which can be used in the preparation of a product for treating brain memory loss. The composition is used to treat brain dysfunction caused by memory loss, Alzheimer's disease (AD), sequelae of ischemic stroke, etc.

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

[0009] A novel exosome composition, wherein the exosome composition is prepared from the following raw materials in parts by weight:

[0010] Exosomes 33-44wt%, nicotinamide adenine dinucleotide or its precursor 55-65wt% and ginsenosides 1-2wt%.

[0011] Preferably, the exosome composition is prepared from the following raw materials in parts by weight:

[0012] Exosomes 40wt%, nicotinamide adenine dinucleotide or its precursor 58wt% and ginsenoside 2wt%.

[0013] Preferably, the exosomes are obtained by separation and purification from the culture supernatant of mesenchymal stem cells or other stem cells;

[0014] The mesenchymal stem cells include any one of umbilical cord mesenchymal stem cells, autologous adipose-derived mesenchymal stem cells and bone marrow mesenchymal stem cells;

[0015] The other stem cells include any one of placental or embryonic stem cells, induced pluripotent stem cells and neural stem cells;

[0016] The nicotinamide adenine dinucleotide is a nicotinamide adenine dinucleotide injection;

[0017] The nicotinamide adenine dinucleotide precursor includes any one of nicotinic acid, tryptophan, nicotinamide, nicotinamide mononucleotide, nicotinamide ribose chloride and other nicotinamide ribose salts;

[0018] The ginsenoside includes Rg1 or Rb1.

[0019] Preferably, the exosomes are separated and purified from the culture medium of umbilical cord-derived mesenchymal stem cells by tangential flow ultrafiltration;

[0020] The nicotinamide adenine dinucleotide precursor is nicotinamide ribose chloride;

[0021] The ginsenoside is Rg1.

[0022] Among them, exosomes can come from umbilical cord mesenchymal stem cells, autologous adipose mesenchymal stem cells, bone marrow mesenchymal stem cells, placenta or embryonic stem cells, or induced pluripotent stem cells, neural stem cells, etc. NAD+ precursors include nicotinic acid (NA), tryptophan (Trp), nicotinamide (NAM), nicotinamide mononucleotide (NMN) and nicotinamide riboside (NR). NA becomes NAD+ through the Preiss-Handler pathway, Trp enters the Preiss-Handler pathway through the de novo synthesis pathway and becomes NAD+, while NAM, NMN and NR become NAD+ through the salvage synthesis pathway.

[0023] A method for preparing a novel exosome composition comprises the following steps:

[0024] S1: Exosome solution was obtained by separation and purification from umbilical cord-derived mesenchymal stem cell culture medium using tangential flow ultrafiltration;

[0025] S2: Weigh nicotinamide adenine dinucleotide or its precursor and ginsenoside in proportion, add 1-1.5 mL of purified water to dissolve, mix well, and filter to sterilize;

[0026] S3: Mix S1 and S2 in proportion, make into freeze-dried powder, and store in the refrigerator as bottle A;

[0027] S4: Sterile saline, as bottle B.

[0028] Preferably, the mixing ratio of the lyophilized powder in S3 and the sterile saline in S4 is 1-1.5 mL:1 mL.

[0029] A novel exosome composition is used in the preparation of a product for treating brain memory impairment, wherein the brain memory impairment includes any one of Alzheimer's disease, chronic ischemic stroke and Parkinson's disease.

[0030] Preferably, the product is nasal drops;

[0031] The preparation method of the nasal drops is:

[0032] The physiological saline in bottle B obtained by the preparation method is squeezed into bottle A, mixed to form an exosome composition, and allowed to stand to allow the exosomes to encapsulate nicotinamide adenine dinucleotide or its precursor, and ginsenoside;

[0033] Then insert the drip head into bottle A.

[0034] Preferably, the method of using the nasal drops for non-therapeutic purposes is: select the product, and drip 2-3 drops into each nostril each time, 1-2 times a day.

[0035] Compared with the prior art, the present invention has at least the following technical effects:

[0036] The present invention provides a novel exosome composition, which is a composition obtained by mixing exosomes, nicotinamide adenine dinucleotide or its precursor and ginsenosides, which can exert synergistic effects of different mechanisms of action, and can significantly enhance cognitive function to treat diseases related to brain dysfunction compared with using one component alone. Moreover, these components are all natural products existing in nature, and there are also intravenous injection products in actual use in clinical practice, which have good safety.

[0037] The application of the novel exosome composition can be used in the preparation of a product for treating brain memory impairment. The composition is used for the treatment of brain dysfunction caused by memory impairment, Alzheimer's disease (AD), sequelae of ischemic stroke, etc.

[0038] The composition can reduce brain tissue damage to improve cognitive function and achieve the purpose of treatment. The invention provides a detailed product preparation method and use guide, and verifies its effectiveness and safety through product pharmacodynamic examples.

[0039] After in-depth exploration, research and verification by the inventors, it was found that the composition containing exosomes, NAD+ or its precursor and ginsenosides produced an unexpected synergistic effect in enhancing memory, that is, the effect of using the combination of the three produced a stronger and more significant effect than using exosomes, NAD+ or its precursor or ginsenosides separately.

[0040] At the same time, the three compositions can be administered through nasal drops, are convenient to use, and can produce good therapeutic effects. DETAILED DESCRIPTION

[0041] 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.

[0042] Example 1: Preparation of exosome composition nasal drops

[0043] Exosomes are derived from umbilical cord-derived mesenchymal stem cells and are prepared by Beijing Enze Kangtai Biotechnology Co., Ltd. 20 billion particles / mg of exosome lyophilized powder. The particle number is detected by nanoparticle tracking analysis (NTA) and the expression of characteristic marker proteins (such as CD63 and CD81) is confirmed by Western Blot.

[0044] NAD+ precursor nicotinamide riboside chloride (NR-Cl), CAS number: 23111-00-4, was purchased from Infant Pharmaceuticals (Shanghai) Co., Ltd.

[0045] Ginsenoside Rg1, CAS No.: 22427-39-0, was purchased from Chengdu Purifa Technology Development Co., Ltd. Physiological saline was used for preparation of the solution.

[0046] The composition includes the following component proportions in terms of mass percentage, as summarized in the following table:

[0047] Raw material category Composition content(%) Exosomes Exosomes secreted by umbilical cord-derived mesenchymal stem cells 40 NAD+ Precursors NR-Cl 58 Ginsenosides Rg1 2

[0048] Preparation method: Exosomes are obtained by separating and purifying stem cell culture fluid by tangential flow ultrafiltration; appropriate amounts of nicotinamide adenine dinucleotide precursor and ginsenoside are weighed and mixed, dissolved in purified water, mixed, and sterilized by filtration. The above two liquids are mixed in proportion to prepare freeze-dried powder (bottle A), and stored in brown vials in a refrigerator;

[0049] Sterile saline, 1-5mL (Bottle B).

[0050] When using, squeeze 1mL of physiological saline (bottle B) into bottle A to dissolve the exosome composition, mix well and let stand for 30 minutes, then insert the drip head and use it. According to the stability of exosomes in the solution state, it can be used for 3 days if stored at 2-8℃ after use. Discard the remaining and take new bottles A and B to re-prepare.

[0051] How to use: The user should drop 2-3 drops into each nostril 1-2 times a day for several weeks to several months. The specific course of treatment depends on the user's condition.

[0052] Example 2: Pharmacodynamics experiment of exosome composition nasal drops in the treatment of brain memory impairment

[0053] 2.1 This experiment aims to evaluate the effect of exosome composition nasal drops on improving memory impairment. The Alzheimer's Disease (AD) model was selected to verify the enhancing effect of exosome composition nasal drops on the memory function of AD mice through behavioral tests and biochemical index detection.

[0054] 2.2 Animals: 3-month-old male APP / PS1 double transgenic mice, strain name C57BL / 6Smoc-Tgtn(APP-PSEN1(P))1Smoc, purchased from Shanghai Model Organisms Technology Co., Ltd., catalog number: NM-KI-233918, a total of 70 mice. At the same time, 10 wild-type C57BL / 6J mice were purchased.

[0055] 2.3 In addition to the normal control group, the model animals were randomly divided into 7 groups: model control group, exosome group, NR-Cl group, ginsenoside group, exosome + NR-Cl group, exosome + ginsenoside group, exosome + NR-Cl + ginsenoside group, with 10 animals in each group.

[0056] The test substances used in the above exosomes + NR-Cl group, exosomes + ginsenoside group, and exosomes + NR-Cl + ginsenoside group were all prepared according to the preferred ratio, that is, the mass concentrations of exosomes, NR-Cl, and ginsenoside Rg1 in the test solution were 100 μg / mL, 145 μg / mL, and 5 μg / mL, respectively.

[0057] Dosage: Use 5 μL of nasal drops twice a day, once in the morning and once in the afternoon, for 4 consecutive weeks.

[0058] Drug administration method: The animal was placed on its back and fixed, and the drug was administered by nasal drops. The animal was fixed for 30 seconds after the drops to ensure that the exosomes entered the brain. The model control group was given a drop of normal saline.

[0059] 2.4 Behavioral testing:

[0060] Morris water maze test: used to assess spatial learning and memory abilities.

[0061] Y-maze test: used to assess short-term memory.

[0062] 2.4.1 Morris water maze test The instrument model is MazeEngineers Morris Water Maze (MazeEngineers Company). Test parameters: platform diameter 10 cm, pool diameter 120 cm, water temperature 22±1°C, recording system EthoVision XT video tracking system (Noldus Information Technology).

[0063] The test method is described as follows: (1) Training period: 5 days, 4 trials per day. Each trial lasts 60 seconds. If the mouse fails to find the platform, the experimenter guides the mouse to the platform and stays there for 15 seconds. (2) Spatial exploration test: On the 6th day after the training period, the platform is removed and the mouse is allowed to swim freely for 60 seconds. The time the mouse spends in the quadrant where the original platform is located (target quadrant time) is recorded. (3) Test indicators: The time (latency) for the mouse to find the platform, swimming speed, path length, and target quadrant time are recorded.

[0064] 2.4.2 Y-maze test, instrument model MazeEngineers Y-Maze (MazeEngineers). Test parameters: arm length × arm width × height 30 cm × 10 cm × 30 cm. Ambient temperature maintained at 22 ± 1 °C. Recording system SmartVideo Tracking System (Panlab / Harvard Apparatus).

[0065] The method is described as follows: (1) Habituation phase: The mice were placed in the central area of ​​the Y-maze and allowed to freely explore all three arms for 5 minutes to adapt to the environment. (2) Short-term memory test: Immediately after the habituation phase, the mice's stay time and number of entries in the new arm (arm that has not been visited) were recorded. The test time was 5 minutes. (3) Test indicators: The percentage of stay time in the new arm and the percentage of entries in the new arm were calculated to evaluate the short-term memory ability of the mice.

[0066] Percentage of time spent in the new arm = (time spent in the new arm / total time spent) × 100%;

[0067] Percentage of new arm entries = (new arm entries / total entries) × 100%.

[0068] 2.4.3 Biochemical index detection, β-amyloid protein (Aβ) level: ELISA method was used to detect the Aβ level in mouse brain tissue.

[0069] Western Blot was used to detect the phosphorylation status of tau protein.

[0070] The level of β-amyloid protein (Aβ) was detected using Mouse β-Amyloid (1-42) ELISA Kit (Invitrogen, catalog number: KHB3441).

[0071] The method is briefly described as follows: Mouse brain tissue samples were collected, homogenized with PBS buffer, centrifuged and the supernatant was obtained. The Aβ1-42 concentration was determined according to the instructions of the ELISA kit.

[0072] The results are expressed as pg / mg protein.

[0073] The tau antibody used to detect the phosphorylation level of tau protein was a rabbit polyclonal anti-tau antibody (Cat#AB1048, Millipore Sigma). The phosphorylated tau antibody (p-tau) was a rabbit monoclonal anti-p-tau (Ser202 / Thr205) antibody (Cat#44-748G, Invitrogen).

[0074] 2.5 The experimental method is described as follows: (1) Sample preparation: Collect mouse brain tissue, homogenize with RIPA lysis buffer and centrifuge to obtain the supernatant. (2) Protein quantification: Use BCA protein quantification kit (Pierce BCA Protein Assay Kit, Thermo Scientific) to determine the protein concentration. (3) Electrophoresis: Load equal amounts of protein onto 4-12% Bis-Tris precast gel (Invitrogen) for SDS-PAGE. (4) Transfer: Wet transfer to PVDF membrane (Millipore Sigma). (5) Blocking and incubation: Block with 5% skim milk powder for 1 hour, then add total tau and p-tau antibodies respectively, and incubate at 4°C overnight. (6) Secondary antibody incubation: Add HRP-labeled goat anti-rabbit IgG secondary antibody (Jackson ImmunoResearch) and incubate at room temperature for 1 hour. (7) Development: Use ECL luminescent solution (Thermo Scientific) for development and take pictures using chemiluminescence imaging system (Chemi Doc MP, Bio-Rad). (8) Data analysis: ImageJ software was used to quantify the grayscale value of the bands and calculate the ratio of p-tau to total tau.

[0075] 2.6 Combining Tables 1, 2 and 3, the results showed that the exosome group, NR-Cl, ginsenosides and multiple combinations of the above three ingredients all shortened the latency of AD model mice in the Morris water maze test to varying degrees, reduced the path length, and increased their stay time and number of entries in the new arm in the Y maze test, indicating that exosomes, NR-Cl and ginsenosides can improve the spatial learning and short-term memory ability of AD model mice to a certain extent.

[0076] However, the combined effect of exosomes + NR-Cl + Rg1 is more significant.

[0077] After treatment with exosomes, NR-Cl and ginsenosides respectively, the Aβ level and tau protein phosphorylation level in the brain tissue of AD model mice were significantly reduced, indicating that these three components may improve memory function by reducing pathological protein deposition, but the combined effect of the three components in the exosomes+NR-Cl+Rg1 group was the most obvious.

[0078] Table 1: Morris water maze test results

[0079] Group Average latency (s) Swimming speed (cm / s) Path length (cm) Normal control 15±2 20±1 300±50 AD model comparison <![CDATA[45±5 * ]]> <![CDATA[13+2 * ]]> <![CDATA[690±74 * ]]> Exosome group <![CDATA[33±4 # ]]> 15±2 <![CDATA[540±40 # ]]> NR-Cl group 38±5 14±3 580±47 Ginsenoside group <![CDATA[32±3 # ]]> 14±2 <![CDATA[380±40 #％ ]]> Exosomes+NR-Cl <![CDATA[30±3 # ]]> <![CDATA[16±3 # ]]> <![CDATA[430±34 #％ <!-- 5 -->]]> Exosomes + Ginsenosides <![CDATA[31±6 # ]]> <![CDATA[16±4 # ]]> <![CDATA[410±39 #％ ]]> Exosomes+NR-Cl+Rg1 <![CDATA[25±3 ##￥ ]]> <![CDATA[18±3 #￥ ]]> <![CDATA[350±60 ##￥& ]]>

[0080] Note: Compared with the normal control group, *p<0.05; compared with the model control group, #p<0.05, ##p<0.01; compared with the exosome group, %p<0.05; compared with the exosome+NR-Cl group, ￥p<0.05; compared with the exosome+ginsenoside group, &p<0.05

[0081] Table 2: Y-maze test results

[0082]

[0083]

[0084] Note: Compared with the normal control group, *p<0.05; compared with the model control group, #p<0.05, ##p<0.01; compared with the exosomes+NR-Cl group, ￥p<0.05; compared with the exosomes+ginsenoside group, &p<0.05.

[0085] Table 3: Aβ and tau protein phosphorylation levels

[0086] Group Aβ level (pg / mg) Tau phosphorylation level (OD value) Normal control 0.5±0.1 0.8±0.2 AD model comparison <![CDATA[2.6±0.4 * ]]> <![CDATA[2.8+0.3 * ]]> Exosome group <![CDATA[2.0±0.3 # ]]> <![CDATA[1.9±0.1 # ]]> NR-Cl group <![CDATA[2.1±0.3 # ]]> <![CDATA[1.8±0.2 # ]]> Ginsenoside group <![CDATA[1.9±0.2 # ]]> <![CDATA[1.7±0.3 # ]]> Exosomes+NR-Cl <![CDATA[1.8±0.4 # ]]> <![CDATA[1.6±0.1 # ]]> Exosomes + Ginsenosides <![CDATA[1.5±0.6 # ]]> 1.9±0.5 Exosomes+NR-Cl+Rg1 <![CDATA[1.0±0.2 #￥& ]]> <![CDATA[1.1±3 #￥& ]]>

[0087] Note: Compared with the normal control group, *p<0.05; compared with the model control group, #p<0.05; compared with the exosomes+NR-Cl group, ￥p<0.05; compared with the exosomes+ginsenoside group, &p<0.05.

[0088] 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 novel exosome composition, characterized in that: The exosome composition is prepared from the following raw materials in parts by weight: Exosomes 33-44wt%, nicotinamide adenine dinucleotide or its precursor 55-65wt% and ginsenosides 1-2wt%.

2. A novel exosome composition according to claim 1, characterized in that: The exosome composition is prepared from the following raw materials in parts by weight: Exosomes 40wt%, nicotinamide adenine dinucleotide or its precursor 58wt% and ginsenoside 2wt%.

3. A novel exosome composition according to any one of claims 1 or 2, characterized in that: The exosomes are obtained by separation and purification from the culture supernatant of mesenchymal stem cells or other stem cells; the mesenchymal stem cells include any one of umbilical cord mesenchymal stem cells, autologous adipose mesenchymal stem cells and bone marrow mesenchymal stem cells; The other stem cells include any one of placental or embryonic stem cells, induced pluripotent stem cells and neural stem cells; The nicotinamide adenine dinucleotide is a nicotinamide adenine dinucleotide injection; The nicotinamide adenine dinucleotide precursor includes any one of nicotinic acid, tryptophan, nicotinamide, nicotinamide mononucleotide, nicotinamide ribose chloride and other nicotinamide ribose salts; The ginsenoside includes Rg1 or Rb1.

4. A novel exosome composition according to claim 3, characterized in that: The exosomes are separated and purified from the culture medium of umbilical cord-derived mesenchymal stem cells by tangential flow ultrafiltration; The nicotinamide adenine dinucleotide precursor is nicotinamide ribose chloride; The ginsenoside is Rg1.

5. A method for preparing a novel exosome composition according to any one of claims 1 or 2, characterized in that: The steps include: S1: Exosome solution was obtained by separation and purification from umbilical cord-derived mesenchymal stem cell culture medium using tangential flow ultrafiltration; S2: Weigh nicotinamide adenine dinucleotide or its precursor and ginsenoside in proportion, add 1-1.5 mL of purified water to dissolve, mix well, and filter to sterilize; S3: Mix S1 and S2 in proportion, make into freeze-dried powder, and store in the refrigerator as bottle A; S4: Sterile saline, as bottle B.

6. The method for preparing a novel exosome composition according to claim 5, characterized in that: The mixing ratio of the lyophilized powder in S3 and the sterile saline in S4 is 1-1.5 mL:1 mL.

7. Use of the novel exosome composition according to any one of claims 1 or 2 in the preparation of a product for treating brain memory impairment, characterized in that: The brain memory impairment includes any one of Alzheimer's disease, chronic ischemic stroke and Parkinson's disease.

8. Use of a novel exosome composition according to claim 7 in the preparation of a product for treating brain memory impairment, characterized in that: The product is a nasal drop; The preparation method of the nasal drops is: The physiological saline in bottle B obtained by the preparation method according to any one of claims 5 to 6 is squeezed into bottle A, mixed to form an exosome composition, and allowed to stand to allow the exosomes to encapsulate nicotinamide adenine dinucleotide or its precursor, and ginsenoside; Then insert the drip head into bottle A.

9. Use of a novel exosome composition according to claim 8 in the preparation of a product for treating brain memory impairment, characterized in that: The method of using the nasal drops for non-therapeutic purposes is: select the product, and drip 2-3 drops into each nostril each time, 1-2 times a day.

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