Neural stem cell exosome and application thereof in preparation of liver drugs

Abstract

The invention provides a neural stem cell exosome, the surface of the exosome is modified with liver targeting molecules and immunomodulatory proteins, the average particle size of the exosome is 40-200 nanometers, and the Zeta potential is-30 to-10 millivolts. Liver targeting molecules and immunomodulatory protein CD47 are co-modified on the surface of the exosome, and a mild membrane insertion method is adopted, so that the two technical problems of insufficient liver targeting and short in-vivo circulation time of the exosome are solved at the same time, and the treatment effect of the neural stem cell exosome as a liver drug delivery carrier is improved.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to a neural stem cell exosome and a use thereof in preparation of liver drugs. BACKGROUND

[0002] Exosomes are nanoscale extracellular vesicles actively secreted by cells, usually with a diameter of 30-200 nanometers, and have a typical double-membrane structure. They are widely involved in intercellular information transmission, immune regulation, cell differentiation and other physiological processes. Due to the characteristics of natural biocompatibility, low immunogenicity, good tissue permeability and the ability to carry a variety of bioactive substances, in recent years, as a very potential drug delivery system and therapeutic agent, exosomes have attracted widespread attention in various disease treatment fields.

[0003] In the field of liver disease treatment, exosomes show certain application prospects. Some studies have explored exosomes from different cell sources, such as mesenchymal stem cell (MSC) exosomes, liver cell-derived exosomes, etc., in the treatment of acute liver injury, liver fibrosis, non-alcoholic steatohepatitis (NASH), and even hepatocellular carcinoma models. These exosomes may exert their effects by delivering specific miRNAs, mRNAs or functional proteins, regulating immune responses, promoting hepatocyte proliferation, inhibiting fibrosis or inducing cancer cell apoptosis. For example, there are studies using bone marrow mesenchymal stem cell exosomes to encapsulate norcantharidin for anti-liver cancer treatment, showing enhanced targeting and anti-tumor effects; there are also patents involving membrane vesicles (exosomes) derived from umbilical cord mesenchymal stem cells for improving liver, kidney injury and skin condition.

[0004] However, there are still many limitations and challenges in the existing technology of liver treatment strategies based on exosomes. First, the commonly used exosome sources may not have sufficient targeting ability to specific cell types in the liver, resulting in low enrichment efficiency of drugs in the lesion site, affecting the therapeutic effect and possibly increasing off-target effects. Second, exosomes are easily cleared by the mononuclear phagocyte system (MPS) in the body, especially in the liver, Kupffer cells will uptake a large amount of exosomes, shortening their half-life and reducing their bioavailability. In addition, the drug loading method of exosomes may cause damage to their membrane structure, affecting their integrity and function, and the drug loading capacity and encapsulation efficiency often need to be improved. Although there have been studies trying to improve the targeting and phagocytosis avoidance of exosomes through surface modification, these methods are usually complex in process, and the repeatability and stability need to be further verified, and the long-term safety and potential immunogenicity still need to be evaluated in depth.

[0005] Furthermore, most current research focuses on exosomes derived from MSCs or somatic cells, with relatively little exploration into the application of neural stem cell (NSC)-derived exosomes in the treatment of liver diseases. Neural stem cells possess a unique secretome, and their exosomes may carry active cargo different from those from other sources. This could potentially offer unique mechanisms and advantages in treating liver diseases, such as more effectively modulating the neuroendocrine-immune network to influence the liver's inflammatory microenvironment. However, research and applications in this area are currently lacking. Therefore, there is a need to design neural stem cell exosomes and their application in preparing liver drugs. Summary of the Invention

[0006] To overcome the shortcomings of the prior art, a neural stem cell exosome and its use in the preparation of liver drugs are provided.

[0007] To achieve the above objectives, the present invention provides the following technical solution: A neural stem cell exosome, the surface of which is modified with liver-targeting molecules and immunomodulatory proteins, the exosome having an average particle size of 40-200 nanometers and a zeta potential of -30 to -10 millivolts.

[0008] The liver-targeting molecule is attached to the surface of exosomes via membrane insertion, with a binding density of 50-500 pmol of liver-targeting molecule per milligram of exosome protein.

[0009] The specific steps for attaching the liver-targeting molecule to the surface of exosomes via membrane insertion are as follows: reacting the amphiphilic molecule and the liver-targeting molecule at a molar ratio of 1:1-2 in phosphate buffer at pH 6.5-7.5 for 2-4 hours to obtain the targeting molecule complex; incubating the targeting molecule complex with exosomes at a mass ratio of 1:5-20, allowing the targeting molecule complex to insert into the exosome membrane through hydrophobic interactions, thus completing the modification of the liver-targeting molecule on the surface of the exosomes.

[0010] The amphiphilic molecule is one of DMPE-PEG2000-MAL, DSPE-PEG2000-NHS, and cholesterol-PEG2000-MAL.

[0011] The incubation temperature is 35-38℃, the incubation time is 2-4 hours, and the stirring speed is 50-100 rpm.

[0012] The immunomodulatory protein was modified onto the surface of exosomes via an in vitro linking method, with a modification density of 20-200 pmol of immunomodulatory protein per milligram of exosome protein.

[0013] The specific steps for modifying the surface of exosomes with the immunomodulatory protein via in vitro linking are as follows: the immunomodulatory protein and a bifunctional cross-linking agent are reacted at 4°C for 1-2 hours at a molar ratio of 1:1-3, and then reacted with exosomes at room temperature for 2-4 hours. After purification, exosomes modified with the immunomodulatory protein are obtained.

[0014] The purification was performed by sucrose density gradient centrifugation at 100,000 × g and 4 °C for 16-18 hours, collecting exosomes with a sucrose density range of 30%-45%.

[0015] The immunomodulatory protein is CD47 protein, and the bifunctional cross-linking agent is sulfonyl-SMCC.

[0016] Application of a neural stem cell exosome, wherein the neural stem cell exosome is used to prepare liver drugs.

[0017] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows: 1. This invention significantly enhances the targeting specificity of exosomes to liver lesions by co-modifying the surface of exosomes with liver-targeting molecules and immunomodulatory proteins. The liver-targeting molecules introduced in this invention can actively recognize and bind to receptors overexpressed on the surface of hepatocytes, hepatic stellate cells, etc., making it easier for exosomes to accumulate in the liver, thereby improving therapeutic efficacy and reducing systemic side effects.

[0018] 2. The membrane insertion method used in this invention for surface modification can better maintain the integrity and stability of the exosome membrane structure. Unlike some chemical cross-linking methods that may damage the integrity of the exosome lipid bilayer, this method utilizes the hydrophobic ends of amphiphilic molecules to spontaneously embed into the membrane structure. The entire process is under mild conditions, which is beneficial for maintaining the natural biological functions and drug loading capacity of exosomes.

[0019] 3. This invention effectively reduces the risk of exosomes being rapidly cleared by the mononuclear phagocytic system by introducing the immunomodulatory protein CD47. The CD47 protein can interact with the signal regulatory protein α on the surface of macrophages, transmitting a "don't eat me" signal, thereby inhibiting the phagocytic activity of exosomes by the immune system, prolonging their circulating half-life in the blood, and providing a more sufficient time window for the drug to exert its long-term therapeutic effect. Detailed Implementation

[0020] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0021] In the specific embodiments of this application, the sources of various main raw materials are briefly described as follows: Phosphate buffer: purchased from Shanghai Kanglang Biotechnology Co., Ltd.

[0022] DMPE-PEG2000-MAL: Purchased from Xi'an Weiyi Biotechnology Co., Ltd.

[0023] DSPE-PEG2000-NHS: Purchased from Xi'an Qiyue Biotechnology Co., Ltd.

[0024] Cholesterol-PEG2000-MAL: Purchased from Chongqing Yusi Pharmaceutical Technology Co., Ltd.

[0025] CD47 protein: purchased from Beijing Yiqiao Shenzhou Technology Co., Ltd.

[0026] Sulfonated SMCC: Purchased from Wuhan AmyJet Technology Co., Ltd.

[0027] The technical solution of this application is as follows: A neural stem cell exosome, the surface of which is modified with liver-targeting molecules and immunomodulatory proteins, the exosome having an average particle size of 40-200 nanometers and a zeta potential of -30 to -10 millivolts.

[0028] The liver-targeting molecule is attached to the surface of exosomes via membrane insertion, with a binding density of 50-500 pmol of liver-targeting molecule per milligram of exosome protein.

[0029] The specific steps for attaching the liver-targeting molecule to the surface of exosomes via membrane insertion are as follows: reacting the amphiphilic molecule and the liver-targeting molecule at a molar ratio of 1:1-2 in phosphate buffer at pH 6.5-7.5 for 2-4 hours to obtain the targeting molecule complex; incubating the targeting molecule complex with exosomes at a mass ratio of 1:5-20, allowing the targeting molecule complex to insert into the exosome membrane through hydrophobic interactions, thus completing the modification of the liver-targeting molecule on the surface of the exosomes.

[0030] The amphiphilic molecule is one of DMPE-PEG2000-MAL, DSPE-PEG2000-NHS, and cholesterol-PEG2000-MAL.

[0031] The incubation temperature is 35-38℃, the incubation time is 2-4 hours, and the stirring speed is 50-100 rpm.

[0032] The immunomodulatory protein was modified onto the surface of exosomes via an in vitro linking method, with a modification density of 20-200 pmol of immunomodulatory protein per milligram of exosome protein.

[0033] The specific steps for modifying the surface of exosomes with the immunomodulatory protein via in vitro linking are as follows: the immunomodulatory protein and a bifunctional cross-linking agent are reacted at 4°C for 1-2 hours at a molar ratio of 1:1-3, and then reacted with exosomes at room temperature for 2-4 hours. After purification, exosomes modified with the immunomodulatory protein are obtained.

[0034] The purification was performed by sucrose density gradient centrifugation at 100,000 × g and 4 °C for 16-18 hours, collecting exosomes with a sucrose density range of 30%-45%.

[0035] The immunomodulatory protein is CD47 protein, and the bifunctional cross-linking agent is sulfonyl-SMCC.

[0036] Application of a neural stem cell exosome, wherein the neural stem cell exosome is used to prepare liver drugs.

[0037] This application addresses two major technical challenges—insufficient liver targeting of exosomes and short in vivo circulation time—by co-modifying the surface of exosomes with liver-targeting molecules and the immunomodulatory protein CD47, and employing a mild membrane insertion method. This improves the therapeutic efficacy of neural stem cell exosomes as liver drug delivery carriers.

[0038] The technical solutions of the present invention are further illustrated below through examples and comparative examples, but the scope of protection of the present invention is not limited thereto.

[0039] Example 1 This embodiment provides a method for preparing neural stem cell exosomes. First, neural stem cells are cultured using serum-free medium containing 50 ng / mL epidermal growth factor, 50 ng / mL basic fibroblast growth factor, and 2% B27 additive (v / v). When cell confluence reaches 90%, the medium is replaced with basal medium and cultured for another 48 hours. The conditioned medium is then collected. Exosomes are purified by differential centrifugation: first, centrifuging at 10,000 × g for 35 minutes at 4°C, then centrifuging at 120,000 × g for 70 minutes at 4°C. In practical applications, other methods can be used to prepare exosomes, or commercially available exosomes can be used; specific details are not elaborated here.

[0040] Next, surface modification was performed. The amphiphilic molecule DMPE-PEG2000-MAL was reacted with a liver-targeting molecule at a molar ratio of 1.5:1 in phosphate buffer at pH 6.5 for 2 hours to obtain a targeting molecule complex. In this example, the liver-targeting molecule used was a self-made peptide with the sequence CRLTRKRGLK. This complex was then incubated with exosomes at a mass ratio of 1:20 at 38°C with a stirring speed of 100 rpm for 2 hours, allowing the complex to insert into the exosome membrane through hydrophobic interactions. Subsequently, immunomodulatory protein modification was performed. CD47 protein was reacted with the bifunctional cross-linking agent sulfonyl-SMCC at a molar ratio of 3:1 at 4°C for 1 hour, and then reacted with exosomes at room temperature for 2 hours. Finally, purification was performed by sucrose density gradient centrifugation. Exosomes in the 30% sucrose density range were collected by centrifugation at 120,000 × g at 4°C for 16 hours.

[0041] Example 2 In this embodiment, the similarities to those in Embodiment 1 will not be repeated, and the differences are as follows: During surface modification, cholesterol-PEG2000-MAL and liver-targeting molecules were reacted at a molar ratio of 1:2 in phosphate-buffered saline (pH 7.5) for 4 hours. The targeting molecule complex and exosomes were incubated at a mass ratio of 1:12 at 35°C with stirring at 50 rpm for 4 hours. Immunomodulatory protein modification involved reacting CD47 protein and sulfonyl-SMCC at a molar ratio of 2:1 at 4°C for 2 hours, followed by reaction with exosomes at room temperature for 3 hours. For purification, exosomes were collected by centrifugation at 80,000 × g and 4°C for 17 hours, yielding a 45% sucrose density range.

[0042] Example 3 In this embodiment, the similarities to those in Embodiment 1 will not be repeated, and the differences are as follows: For surface modification, DSPE-PEG2000-NHS and liver-targeting molecules were reacted at a molar ratio of 2:1 in phosphate-buffered saline (pH 7.0) for 3 hours. The targeting molecule complex and exosomes were incubated at a mass ratio of 1:5 at 36.5°C with stirring at 75 rpm for 3 hours. For immunomodulatory protein modification, CD47 protein and sulfonyl-SMCC were reacted at a molar ratio of 1:1 at 4°C for 1.5 hours, followed by reaction with exosomes at room temperature for 4 hours. Purification was performed by centrifugation at 100,000 × g at 4°C for 18 hours, collecting exosomes with a sucrose density of 37.5%.

[0043] Comparative Example 1 In this comparative example, the similarities with Example 1 will not be repeated, and the differences are as follows: No liver-targeting molecules are modified; only the CD47 protein is modified.

[0044] Comparative Example 2 In this comparative example, the similarities with Example 2 will not be repeated, and the differences are as follows: No modification of CD47 protein was performed; only liver-targeting molecules were modified.

[0045] Comparative Example 3 In this comparative example, the similarities with Example 3 will not be repeated, and the differences are as follows: Mesenchymal stem cells were used instead of neural stem cells as the source of exosomes.

[0046] Comparative Example 4 In this comparative example, the similarities with Example 1 will not be repeated, and the differences are as follows: A chemical cross-linking method was used instead of membrane insertion for the modification of liver-targeting molecules, with glutaraldehyde as the cross-linking agent.

[0047] Comparative Example 5 In this comparative example, the similarities with Example 1 will not be repeated, and the differences are as follows: No surface modifications were used; only unmodified neural stem cell exosomes were used.

[0048] Performance Test Results and Analysis

[0049] Exosomes were prepared according to the parameters of the examples and comparative examples, respectively. To evaluate the performance of the exosomes prepared in each example and comparative example, the following test methods were used: exosome particle size and zeta potential were measured using dynamic light scattering; protein concentration was measured and surface molecular modification density was calculated using the BCA method; drug loading and encapsulation efficiency were determined using high-performance liquid chromatography; the distribution of exosomes in liver tissue was observed using confocal microscopy; the circulating half-life of exosomes in vivo was monitored using a small animal in vivo imaging system; and the therapeutic effect was evaluated by liver function index detection and pathological section analysis. The specific test results are shown in Table 1.

[0050] Table 1 Analysis of Test Results

[0051] The results in Table 1 show that the neural stem cell exosomes provided by this invention exhibit excellent performance characteristics. Data from Examples 1 to 3 show that by adjusting the preparation parameters, exosomes with different properties can be obtained, but all maintain good liver targeting and in vivo circulation characteristics. Example 1 achieved high levels of both targeting molecule modification density and CD47 modification density, and correspondingly showed the best liver enrichment rate and circulating half-life.

[0052] Comparative Example 1, lacking liver-targeting molecular modifications, exhibited a longer circulation time but a significantly reduced liver accumulation rate, resulting in a poorer overall efficacy improvement. Comparative Example 2, lacking CD47 modification, while achieving better liver targeting, was rapidly cleared, leading to insufficient actual duration of action and limited efficacy improvement. This indicates that a single modification strategy cannot simultaneously address both the key issues of targeting and circulation time.

[0053] Comparative Example 3 illustrates the unique advantages of neural stem cell-derived exosomes. Even with the same modification methods, exosomes derived from mesenchymal stem cells showed lower liver enrichment and efficacy compared to those derived from neural stem cells, which may be related to the unique active ingredients carried by neural stem cell exosomes. Comparative Example 4 demonstrates the advantages of membrane insertion over chemical cross-linking. Although chemical cross-linking can also achieve surface modification, the cross-linking process may affect the integrity of the exosome membrane, leading to a slight increase in particle size and a decrease in the absolute value of the zeta potential, ultimately resulting in inferior circulation time and efficacy compared to membrane insertion. Comparative Example 5, serving as a blank control, fully demonstrates the importance of surface modification. While unmodified exosomes exhibit good basic properties, their lack of active targeting ability and immune evasion limits their effectiveness in practical applications.

[0054] Test results show that the present invention has successfully prepared neural stem cell exosomes with good liver targeting and long circulation characteristics, providing a new solution for the treatment of liver diseases.

[0055] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A neural stem cell exosome, characterized in that, The exosomes are modified with liver-targeting molecules and immunomodulatory proteins on their surface. The average particle size of the exosomes is 40-200 nanometers, and the zeta potential is -30 to -10 millivolts.

2. The neural stem cell exosome according to claim 1, characterized in that, The liver-targeting molecule is attached to the surface of exosomes via membrane insertion, with a binding density of 50-500 pmol of liver-targeting molecule per milligram of exosome protein.

3. The neural stem cell exosome according to claim 2, characterized in that, The specific steps for attaching the liver-targeting molecule to the surface of exosomes via membrane insertion are as follows: reacting the amphiphilic molecule and the liver-targeting molecule at a molar ratio of 1:1-2 in phosphate buffer at pH 6.5-7.5 for 2-4 hours to obtain the targeting molecule complex; incubating the targeting molecule complex with exosomes at a mass ratio of 1:5-20, allowing the targeting molecule complex to insert into the exosome membrane through hydrophobic interactions, thus completing the modification of the liver-targeting molecule on the surface of the exosomes.

4. The neural stem cell exosome according to claim 3, characterized in that, The amphiphilic molecule is one of DMPE-PEG2000-MAL, DSPE-PEG2000-NHS, and cholesterol-PEG2000-MAL.

5. The neural stem cell exosome according to claim 3, characterized in that, The incubation temperature is 35-38℃, the incubation time is 2-4 hours, and the stirring speed is 50-100 rpm.

6. The neural stem cell exosome according to claim 1, characterized in that, The immunomodulatory protein was modified onto the surface of exosomes via an in vitro linking method, with a modification density of 20-200 pmol of immunomodulatory protein per milligram of exosome protein.

7. The neural stem cell exosome according to claim 6, characterized in that, The specific steps for modifying the surface of exosomes with the immunomodulatory protein via in vitro linking are as follows: the immunomodulatory protein and a bifunctional cross-linking agent are reacted at 4°C for 1-2 hours at a molar ratio of 1:1-3, and then reacted with exosomes at room temperature for 2-4 hours. After purification, exosomes modified with the immunomodulatory protein are obtained.

8. The neural stem cell exosome according to claim 7, characterized in that, The purification was performed by sucrose density gradient centrifugation at 100,000 × g and 4 °C for 16-18 hours, collecting exosomes with a sucrose density range of 30%-45%.

9. A neural stem cell exosome according to claim 7, characterized in that, The immunomodulatory protein is CD47 protein, and the bifunctional cross-linking agent is sulfonyl-SMCC.

10. The application of a neural stem cell exosome as described in any one of claims 1-9, characterized in that, The neural stem cell exosomes are used to prepare liver drugs.

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