Intracellular vesicles obtained by cracking mesenchymal stem cells as well as preparation method and application of intracellular vesicles

Abstract

The invention provides intracellular vesicles obtained by cracking mesenchymal stem cells as well as a preparation method and application of the intracellular vesicles, and belongs to the technical field of medicines. Comprising the following steps: S1, taking out a culture bottle with menstrual blood stem cells, resuscitating, and removing a culture medium supernatant; s2, adding a proliferation culture medium, incubating, and removing supernate of the culture medium; s3, adding a digestive enzyme solution, digesting, stopping digesting, shaking the culture bottle to enable the cells to fall off from the culture bottle, pouring liquid, transferring the liquid into a liquid storage bottle, and blowing and beating the cell suspension into single cells by using a transfer pipette; s4, dispersing the single cells into a suspension solvent, splitting, removing cell membrane and organelle fragments, and taking supernate; and S5, ultracentrifugal treatment, and taking the precipitate as the intracellular vesicles. The intracellular vesicles prepared by the invention can be used as a drug carrier, the administration route is relatively wide, and the drug loading stability, the drug solubilization amount and the drug bioavailability are relatively high.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to an intracellular vesicle obtained by lysing mesenchymal stem cells, and a preparation method and application thereof. Background Art

[0002] Menstrual stem cells are a type of mesenchymal stem cells that exist in menstrual blood and have strong proliferation potential and extensive differentiation ability. They have a unique source and can be collected from women's menstrual blood in a non-invasive manner, avoiding the ethical controversy in the traditional stem cell acquisition process. The collection process is simple, safe, and has low immunogenicity. Compared with other types of stem cells, menstrual stem cells have significant advantages and are gradually gaining attention in regenerative medicine and tissue repair. Compared with placental, umbilical cord or bone marrow stem cells, the collection of menstrual stem cells not only does not cause ethical issues, but also avoids the health risks brought about by invasive surgery, and has broad clinical application potential.

[0003] At present, studies have shown that menstrual stem cells show great potential in tissue repair and regeneration, especially in the fields of bones, cartilage, nerves and muscles, and have achieved certain application results. For example, menstrual stem cells have been used in experimental studies in many fields such as fracture repair, cartilage regeneration, and nerve damage repair, and have shown good biological properties. Its high proliferation rate enables it to expand rapidly and provide a sufficient number of cells for clinical treatment. Compared with other types of stem cells, menstrual stem cells have higher immune compatibility, can effectively avoid immune rejection reactions, and reduce complications after transplantation. In addition, menstrual stem cells can also secrete a variety of growth factors and cytokines to promote the healing and regeneration of damaged tissues, thereby accelerating the tissue repair process.

[0004] Menstrual stem cells have shown good effects in wound repair and regeneration of aged tissues. Especially in the treatment of aging-related diseases such as osteoporosis and joint degeneration, menstrual stem cells have shown considerable prospects. Studies have shown that menstrual stem cells can not only directly participate in the regeneration of damaged tissues, but also play a regulatory role in the local microenvironment through the secretion of cytokines, promoting the repair and reconstruction of local tissues. Its unique immunomodulatory function makes it a potential new tool for the treatment of immune system-related diseases, especially in the fields of autoimmune diseases and immune aging, and has high research value.

[0005] In addition, the application of menstrual stem cells in immune regulation has gradually attracted the attention of the scientific community. Menstrual stem cells can secrete a variety of immunomodulatory factors to regulate the balance of the immune system. By regulating the immune system, the body's immune tolerance is enhanced, the symptoms of autoimmune diseases are alleviated, and even the occurrence of immune rejection reactions after organ transplantation is reduced. Current research shows that the inhibitory effect of menstrual stem cells on immune cells is expected to provide new ideas for the treatment of immune-related diseases, especially in the clinical application prospects of autoimmune diseases and allergic diseases.

[0006] In recent years, vesicles released by cells into the extracellular environment or blood circulation, the so-called "extracellular vesicles" (EVs), have received widespread attention. Extracellular vesicles mainly include exosomes, microvesicles or microparticles and apoptotic bodies, among which exosomes are the focus of research. Exosomes are formed by cell membrane invagination, carry specific biological molecules of the source cells, and participate in intercellular information transmission and material transport. However, the collection of exosomes faces many challenges, such as difficulty in purification, susceptibility to contamination and poor storage stability, which to some extent limits its clinical transformation. According to the "Minimum Information on Extracellular Vesicle Research" (MISEV) guidelines, it is difficult to completely separate purified exosomes by traditional ultracentrifugation methods, and it is recommended to use more precise terms such as "small extracellular vesicles" (sEVs).

[0007] In addition, there are abundant nanoscale vesicles inside cells, namely intracellular vesicles (IVs). These vesicles originate from a variety of organelles, including the endoplasmic reticulum, Golgi apparatus, endosomes and plasma membrane, and play a key role in the transport and secretion of substances in cells. IVs can be composed of multiple subtypes such as constitutive secretory vesicles, synaptic vesicles, and coated vesicles. They are involved in the transport of biological molecules such as proteins, RNA and lipids, and play an important role in regulating cell functions. However, there are currently few studies on how to effectively isolate and utilize menstrual stem cells and their related IVs, and the exploration of their specific mechanisms needs to be further deepened. Summary of the invention

[0008] The present invention aims to provide an intracellular vesicle obtained by lysing mesenchymal stem cells and a preparation method and application thereof, so as to effectively solve the deficiencies of drugs in terms of water solubility, biocompatibility, immunogenicity, toxic side effects and duration of action. The obtained intracellular vesicle has a significant solubilization ability, and its bilayer lipid membrane exhibits excellent firmness and stability. As a drug carrier, the vesicle has a variety of administration routes, and at the same time shows significant advantages in terms of drug loading stability, drug solubilization efficiency and bioavailability.

[0009] The technical solution of the present invention is achieved in this way:

[0010] The present invention provides a method for preparing intracellular vesicles obtained by lysing mesenchymal stem cells, comprising the following steps:

[0011] S1. providing mesenchymal stem cells and pre-treating them to remove the culture medium;

[0012] S2. Proliferate and culture the mesenchymal stem cells to obtain the required number of cells;

[0013] S3. digesting the cells by enzymatic digestion to form a single cell suspension;

[0014] S4. Lyse the single cells to release the cell contents and remove non-target components by centrifugation;

[0015] S5. The supernatant after centrifugation is subjected to ultracentrifugation to separate and collect the precipitated intracellular vesicles.

[0016] More specifically, the method for preparing intracellular vesicles obtained by lysing mesenchymal stem cells comprises the following steps:

[0017] S1. Take out the culture bottle containing menstrual blood stem cells, revive at room temperature, and discard the culture medium supernatant;

[0018] S2. Add proliferation medium to the culture flask, incubate, and discard the culture medium supernatant;

[0019] S3. Add digestion enzyme solution to the culture flask, digest, pour in culture medium to stop digestion, shake the culture flask to make the cells fall off the culture flask, transfer the liquid to the storage bottle, and use a pipette to blow the cell suspension to single cells;

[0020] S4. Disperse the single cells into a suspension solvent, add a lysis solution to lyse the cells, centrifuge to remove cell membranes and organelle fragments, and collect the supernatant;

[0021] S5. The supernatant is subjected to ultracentrifugation and the precipitate is the intracellular vesicles.

[0022] As a further improvement of the present invention, the proliferation culture medium is a DMEM culture medium with a vesicle-inducing factor added thereto, and the vesicle-inducing factor is a combination of hemolytic streptolysin and fibronectin.

[0023] As a further improvement of the present invention, the concentration of fibronectin is about 1-20 μg / mL, and the concentration of hemolytic streptolysin O can be from 50 ng / mL to 200 ng / mL. Exemplarily, the concentration (μg / mL) of fibronectin is 1, 2, 3, 5, 7.5, 10, 12.5, 15, 17.5, 20 μg / mL; the concentration of hemolytic streptolysin O is 50, 60, 75, 90, 100, 120, 150, 175, 190, 200 ng / mL.

[0024] As a combination, a low-dose combination, a medium-dose combination and a high-dose combination can be considered. For example, a low-dose combination can be: 2 μg / mL of fibronectin + 50 ng / mL of hemolytic streptolysin; a medium-dose combination: 10 μg / mL of fibronectin + 100 ng / mL of hemolytic streptolysin; a high-dose combination: 20 μg / mL of fibronectin + 200 ng / mL of hemolytic streptolysin.

[0025] As a further improvement of the present invention, the incubation conditions are 35-38°C, saturated humidity, 3-6v / v% CO 2 Incubate at the concentration for 12-24h.

[0026] As a further improvement of the present invention, the digestive enzyme solution is a mixed solution of 1.5-2.5wt% trypsin and 0.5-1wt% neutral protease.

[0027] As a further improvement of the present invention, the digestion temperature is 35-38°C and the time is 3-5h.

[0028] As a further improvement of the present invention, the lysate is a mixed solution prepared by adding 100-200 mmol / L NaCl, 0.5-1.5 wt% Tween-85, 0.5-1.5 wt% sodium deoxycholate, and 0.05-0.15 wt% sodium dodecylbenzene sulfonate to a Tris-HCl solution with a pH of 7.4-7.9, and the lysate temperature is 35-40° C. and the time is 20-40 min.

[0029] As a further improvement of the present invention, the ultracentrifugation treatment is centrifugation at 2000-3000g for 10-20min, or centrifugation at 40000-50000g for 5-10min.

[0030] The present invention further relates to a combination of hemolytic streptolysin and fibronectin for obtaining intracellular vesicles from mesenchymal stem cells.

[0031] The present invention further relates to an intracellular vesicle obtained by lysing mesenchymal stem cells prepared by the above preparation method.

[0032] Intracellular vesicles (IVs) obtained by lysing mesenchymal stem cells using the preparation method described in this article are nano-sized vesicles with a double-layer lipid membrane structure. They are lysed and purified from mesenchymal stem cells, retaining many important biological functional characteristics of the parent cells, including carrying a variety of active proteins, RNA molecules and lipid components. These vesicles have a wide range of potential applications. Due to the use of optimized lysis and ultracentrifugation processes, the obtained vesicles are of high purity and high yield, and have higher biological stability and functional consistency compared with extracellular vesicles extracted by traditional methods. The main features of these vesicles include: (1) High-efficiency carriers: vesicles can be absorbed by target cells in vivo through endocytosis and deliver the active molecules inside them directly to the target site; (2) Broad-spectrum applicability: Because they are derived from mesenchymal stem cells, vesicles show low immunogenicity and are suitable for different patient groups, including those with impaired immunity; (3) Easy to store and transport: vesicles can be stored for a long time under low temperature conditions (such as -80°C), and their functional and structural integrity are maintained. This vesicle preparation method provides a new tool for research in the fields of regenerative medicine and drug delivery, and offers potential innovative solutions for the treatment of complex diseases.

[0033] The present invention further relates to a use of the intracellular vesicle as a drug carrier.

[0034] The bilayer lipid membrane structure of intracellular vesicles and the bioactive molecules encapsulated in them make them ideal drug carriers. The present invention proposes to use the prepared intracellular vesicles as carriers to solve the bottleneck problems of traditional drug carriers in terms of water solubility, biocompatibility, stability, etc. Specifically, these vesicles can be used to encapsulate small molecule drugs, nucleic acid drugs (such as siRNA, mRNA) or protein drugs, and deliver them to target tissues by intravenous injection or local administration.

[0035] Compared with other drug delivery systems (such as polymer nanoparticles, liposomes, etc.), intracellular vesicles have unique biocompatibility and low immunogenicity, can circulate in the body for a long time, and reduce the side effects of drugs. In addition, the surface of vesicles can be loaded with targeting ligands such as antibodies, peptide chains or small molecules through genetic engineering or chemical modification, thereby achieving efficient targeted delivery to specific tissues or cells.

[0036] This vesicle carrier can be widely used in the treatment of various diseases, such as improving the delivery efficiency of immunomodulatory molecules in immunotherapy and enhancing the bioavailability of antibiotics in anti-infection treatment. In addition, vesicles can also be used to deliver drugs across the blood-brain barrier, providing innovative solutions for neurological diseases.

[0037] The present invention further relates to a cell culture medium comprising a combination of hemolytic streptolysin and fibronectin.

[0038] The present invention proposes a novel cell culture medium containing hemolytic streptolysin and fibronectin, which is designed to optimize the proliferation and differentiation environment of mesenchymal stem cells. This culture medium significantly improves the proliferation activity and survival rate of cells by combining the cell membrane repair properties of hemolytic streptolysin and the cell adhesion function of fibronectin.

[0039] Hemolytic streptolysin can act gently on the cell membrane at low concentrations, enhancing the fluidity of the cell membrane, thereby promoting the absorption of nutrients while avoiding damage to the cell structure. Fibronectin, as an extracellular matrix protein, can enhance the cell's ability to adhere to the wall by binding to integrin receptors, providing a more stable growth environment for cells. The synergistic effect of these two components can significantly improve the culture medium's ability to support mesenchymal stem cells.

[0040] The present invention further relates to a drug comprising the intracellular vesicles prepared according to the method described herein as a drug carrier.

[0041] The intracellular vesicles can be used as drug carriers for delivering a variety of active ingredients, including but not limited to small molecule drugs, nucleic acids (such as DNA, RNA or siRNA), proteins, peptides and other biologically active molecules with therapeutic or diagnostic functions. By adopting the preparation method described herein, these intracellular vesicles have efficient loading capacity, stability and biocompatibility.

[0042] In addition, the drug can further include targeting molecules adapted to the surface of the vesicle, such as antibodies, ligands or peptides, to enhance the targeting ability to specific cells or tissues. This targeted delivery capability can significantly increase the concentration of the drug at the target site, thereby reducing systemic side effects.

[0043] The invented intracellular vesicles can be used to treat a wide range of diseases, including but not limited to inflammatory diseases, autoimmune diseases, infectious diseases, tumors, and neurodegenerative diseases. As a natural intercellular information transmission system, intracellular vesicles can accurately transmit specific signals or therapeutic molecules to target cells by carrying a variety of biological molecules (such as proteins, RNA, lipids, etc.), and play a role in regulating immunity, promoting repair, and inhibiting inflammation. Therefore, intracellular vesicles have shown significant therapeutic effects in the treatment of various diseases.

[0044] In the treatment of inflammatory diseases, intracellular vesicles can carry molecules with anti-inflammatory effects, such as anti-inflammatory factors, immunosuppressive factors or specific small molecule drugs, and deliver them to the site of inflammation, thereby alleviating local or systemic inflammatory responses. Through this targeted delivery, intracellular vesicles can not only effectively reduce adverse side effects, but also improve the specificity and effectiveness of treatment. Such applications have great therapeutic potential in chronic inflammatory diseases such as rheumatoid arthritis and inflammatory bowel disease.

[0045] In terms of autoimmune diseases, the immunomodulatory effects of intracellular vesicles have also been widely studied. Intracellular vesicles can regulate the activity of the immune system, restore immune tolerance, and reduce the attack of immune cells on autologous tissues. For example, in autoimmune diseases such as systemic lupus erythematosus and type 1 diabetes, intracellular vesicles can adjust the function of immune cells and reduce pathological immune responses, thereby providing new treatments for these patients.

[0046] For infectious diseases, intracellular vesicles can carry antibacterial, antiviral or immunostimulatory molecules to enhance the body's defense against infection. Especially in bacterial, viral and fungal infections, intracellular vesicles can not only increase the local concentration of drugs, but also help drugs penetrate the lesion area and promote the clearance of pathogens. In addition, the natural properties of intracellular vesicles enable them to circumvent the drug resistance mechanisms of certain pathogens, providing a promising treatment strategy.

[0047] In tumor treatment, intracellular vesicles have the potential to be used as delivery vehicles, which can accurately deliver anticancer drugs, gene therapy or immunotherapy molecules to tumor cells. This targeted delivery can significantly reduce the toxicity of drugs to normal tissues and improve the efficacy of treatment. Studies have shown that intracellular vesicles can promote the effective release of anti-tumor drugs by fusing with tumor cells, thereby enhancing the death response of tumor cells.

[0048] In addition, intracellular vesicles have also shown potential in the treatment of neurodegenerative diseases. By delivering neurotrophic factors, neuroprotective molecules or repair factors, intracellular vesicles are expected to help restore damaged neural function. For example, for neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease, intracellular vesicles can effectively deliver specific therapeutic molecules, promote the repair and regeneration of nerve cells, and slow down the progression of the disease.

[0049] In addition to therapeutic functions, intracellular vesicles also have broad application prospects in the field of diagnosis. As a natural carrier, intracellular vesicles can be used to deliver contrast agents, fluorescent markers or specific diagnostic molecules for in vivo imaging or for the detection of disease markers. In this way, intracellular vesicles can help doctors accurately diagnose the type, location and stage of development of the disease. Its application in in vivo imaging, such as magnetic resonance imaging (MRI), ultrasound imaging or fluorescence imaging, can provide real-time, non-invasive diagnostic information. In addition, intracellular vesicles can also be used as a delivery carrier for disease biomarkers to help identify early disease characteristics and improve the early diagnosis rate of diseases, especially in the fields of cancer, cardiovascular disease and neurological diseases.

[0050] As a multifunctional and controllable biological carrier, intracellular vesicles have both therapeutic and diagnostic functions, providing new possibilities for future precision medicine and personalized treatment. With further in-depth research, intracellular vesicles are expected to become a widely used therapeutic tool, especially in clinical treatment, disease monitoring and precision diagnosis, playing an important role.

[0051] The proliferation medium described herein can be selected from the following culture media: DMEM (Dulbecco's Modified Eagle Medium), α-MEM (Minimum Essential Medium α), RPMI 1640, commercial MSC-specific serum-free culture media (such as StemPro, MSCGro, MesenCult, etc.). The embodiments herein only use DMEM as an example.

[0052] The present invention has the following beneficial effects:

[0053] The present invention adopts a proliferation culture medium to carry out proliferation culture on menstrual blood stem cells, and increases the generation amount of vesicles by adding vesicle inducing factors, and can also increase the drug loading capacity.

[0054] Fibronectin is a macromolecular glycoprotein that can bind to receptors, collagen and other matrix components on the cell surface to regulate cell adhesion, migration and signal transduction. Fibronectin plays an important role in biological processes such as cell morphological changes, cell-cell adhesion and vesicle generation. Fibronectin can be extracted from animal sources (such as cattle, pigs or chickens), or expressed from Escherichia coli or mammalian cells through recombinant DNA technology. There is high-purity fibronectin extracted from mammalian cells on the market, which is often used in cell culture and research. For example, fibronectin can be purchased from many biological reagent companies such as Sigma-Aldrich, Thermo Fisher, Abcam, etc., usually sold as liquid or lyophilized powder. It can be used in cell culture medium to support cell attachment and growth.

[0055] Streptolysin O (SLO) is a toxin produced by Streptococcus pyogenes, which can destroy the membrane of cells and induce the formation of holes in the cell membrane. It destroys the structure of the membrane by binding to the cell membrane, causing the cell contents to leak and then destroy the cells. Streptolysin is extracted or synthesized from Streptococcus pyogenes, and can be produced by fermentation and cultivation of these bacteria. Exemplarily, streptolysin can be purchased through commercial suppliers, usually provided in purified form, and is widely used in cell research, immunology and molecular biology experiments. For example, it can be purchased from Sigma-Aldrich, Thermo Fisher, etc.

[0056] Hemolytic bacterial toxins can induce the formation of pores in the cell membrane and promote the release of vesicles. The extracellular matrix component fibronectin can bind to receptors on the cell membrane, regulate the interaction between cells and the surrounding matrix, and promote the formation of vesicles. By combining hemolytic bacterial toxins and extracellular matrix components, the production of vesicles can be effectively increased and their function can be enhanced.

[0057] On the other hand, the present invention digests menstrual stem cells at room temperature and pressure, which is simple to operate and improves the proliferation activity of cells, thereby improving the production efficiency of menstrual stem cells. After digestion, lysis solution is added for lysis, and ultracentrifugation is performed to obtain the precipitate to obtain the intracellular vesicles.

[0058] The intracellular vesicles prepared by the present invention can be used as drug carriers to solve the problems of water solubility, biocompatibility, immunogenicity, toxic side effects, duration of action, etc. of drugs. They have strong solubilization ability, and their double-layer lipid membranes have good firmness and stability. As drug carriers, they have a wide range of administration routes, and have high drug loading stability, drug solubilization amount, and drug bioavailability. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 is a comparative graph of intracellular vesicle production;

[0060] Figure 2 It is a comparison chart of drug loading. DETAILED DESCRIPTION

[0061] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0062] Example 1

[0063] This embodiment provides a method for preparing intracellular vesicles obtained by lysing mesenchymal stem cells, comprising the following steps:

[0064] S1. Take out the number of (1-2)×10 7 The culture bottles of menstrual blood stem cells were revived at room temperature and the culture medium supernatant was discarded;

[0065] S2. Add proliferation medium to the culture flask, 35°C, saturated humidity, 3v / v% CO 2 Incubate at 1:1 concentration for 12 h, and discard the culture supernatant;

[0066] The proliferation medium is a DMEM medium in which vesicle-inducing factors are added, wherein the vesicle-inducing factors are hemolytic streptolysin (50 ng / mL) and fibronectin (20 μg / mL);

[0067] S3. Add digestive enzyme solution to the culture flask and digest at 35°C for 3 h. Pour in culture medium to terminate digestion. Shake the culture flask to remove cells from the flask. Pour the solution into a storage bottle and pipette the cell suspension to single cells.

[0068] The digestive enzyme solution is a mixed solution of 1.5wt% trypsin and 0.5wt% neutral protease;

[0069] S4. Disperse the single cells into the suspension solvent, add the lysis solution, lyse at 35°C for 20 min, centrifuge to remove the cell membrane and organelle fragments, and collect the supernatant;

[0070] The lysate is a mixed solution prepared by adding 100 mmol / L NaCl, 0.5 wt% Tween-85, 0.5 wt% sodium deoxycholate, and 0.05 wt% sodium dodecylbenzene sulfonate to a Tris-HCl solution at pH=7.4;

[0071] S5. Centrifuge the supernatant at 2000g for 10 min and 40000g for 5 min, and take the precipitate as the intracellular vesicles.

[0072] Example 2

[0073] This embodiment provides a method for preparing intracellular vesicles obtained by lysing mesenchymal stem cells, comprising the following steps:

[0074] S1. Take out the number of (1-2)×10 7 The culture bottles of menstrual blood stem cells were revived at room temperature and the culture medium supernatant was discarded;

[0075] S2. Add proliferation medium to the culture flask, 38°C, saturated humidity, 6v / v% CO 2 Incubate at 100 μg / mL for 24 h, and discard the culture supernatant;

[0076] The proliferation medium is a DMEM medium with vesicle inducing factors added thereto, wherein the vesicle inducing factors are hemolytic streptolysin (100 ng / mL) and fibronectin (10 μg / mL);

[0077] S3. Add digestive enzyme solution to the culture flask and digest at 38°C for 5 h. Pour in culture medium to terminate digestion. Shake the culture flask to remove cells from the flask. Pour the solution into a storage bottle and pipette the cell suspension to single cells.

[0078] The digestive enzyme solution is a mixed solution of 2.5wt% trypsin and 1wt% neutral protease;

[0079] S4. Disperse the single cells into the suspension solvent, add the lysis solution, lyse at 40°C for 40 min, centrifuge to remove the cell membrane and organelle fragments, and collect the supernatant;

[0080] The lysate is a mixed solution prepared by adding 200 mmol / L NaCl, 1.5 wt% Tween-85, 1.5 wt% sodium deoxycholate, and 0.15 wt% sodium dodecylbenzene sulfonate to a Tris-HCl solution at pH=7.9;

[0081] S5. Centrifuge the supernatant at 3000g for 20 min and 50000g for 10 min, and take the precipitate as the intracellular vesicles.

[0082] Example 3

[0083] This embodiment provides a method for preparing intracellular vesicles obtained by lysing mesenchymal stem cells, comprising the following steps:

[0084] S1. Take out the number of (1-2)×10 7The culture bottles of menstrual blood stem cells were revived at room temperature and the culture medium supernatant was discarded;

[0085] S2. Add proliferation medium to the culture flask, 37°C, saturated humidity, 5v / v% CO 2 Incubate at 1:1 concentration for 18 h, and discard the culture supernatant;

[0086] The proliferation medium is a DMEM medium with vesicle inducing factors added thereto, wherein the vesicle inducing factors are hemolytic streptolysin (200 ng / mL) and fibronectin (10 μg / mL);

[0087] S3. Add digestion enzyme solution to the culture flask and digest at 37°C for 4 hours. Pour in culture medium to terminate digestion. Shake the culture flask to remove cells from the flask. Pour the solution into a storage bottle and pipette the cell suspension to single cells.

[0088] The digestive enzyme solution is a mixed solution of 2wt% trypsin and 0.7wt% neutral protease;

[0089] S4. Disperse the single cells into the suspension solvent, add the lysis solution, lyse at 37°C for 30 min, centrifuge to remove the cell membrane and organelle fragments, and collect the supernatant;

[0090] The lysate is a mixed solution prepared by adding 150 mmol / L NaCl, 1 wt% Tween-85, 1 wt% sodium deoxycholate, and 0.1 wt% sodium dodecylbenzene sulfonate to a Tris-HCl solution at pH=7.6;

[0091] S5. Centrifuge the supernatant at 2500 g for 15 min and 45000 g for 10 min, and take the precipitate as the intracellular vesicles.

[0092] Example 4

[0093] Compared with Example 3, the difference is that the vesicle inducing factor is a single hemolytic streptolysin.

[0094] Example 5

[0095] Compared with Example 3, the difference is that the vesicle inducing factor is a single fibronectin.

[0096] Comparative Example 1

[0097] Compared with Example 3, the difference is that no vesicle inducing factor is added.

[0098] Specifically include the following steps:

[0099] S1. Take out the number of (1-2)×10 7The culture bottles of menstrual blood stem cells were revived at room temperature and the culture medium supernatant was discarded;

[0100] S2. Add DMEM medium to the culture flask, 37°C, saturated humidity, 5v / v% CO 2 Incubate at 1:1 concentration for 18 h, and discard the culture supernatant;

[0101] S3. Add digestion enzyme solution to the culture flask and digest at 37°C for 4 hours. Pour in culture medium to terminate digestion. Shake the culture flask to remove cells from the flask. Pour the solution into a storage bottle and pipette the cell suspension to single cells.

[0102] The digestive enzyme solution is a mixed solution of 2wt% trypsin and 0.7wt% neutral protease;

[0103] S4. Disperse the single cells into the suspension solvent, add the lysis solution, lyse at 37°C for 30 min, centrifuge to remove the cell membrane and organelle fragments, and collect the supernatant;

[0104] The lysate is a mixed solution prepared by adding 150 mmol / L NaCl, 1 wt% Tween-85, 1 wt% sodium deoxycholate, and 0.1 wt% sodium dodecylbenzene sulfonate to a Tris-HCl solution at pH=7.6;

[0105] S5. Centrifuge the supernatant at 2500 g for 15 min and 45000 g for 10 min, and take the precipitate as the intracellular vesicles.

[0106] The extracellular vesicles (EVs) obtained in Examples 1-5 and Comparative Example 1 were observed under a transmission electron microscope (TEM) and all showed round or oval structures. The diameter range of the vesicles in Examples 1-5 was smaller than that in Comparative Example 1, with a diameter of about 20-120 nm. In the TEM image, the lipid membrane structure of the vesicles treated with negative staining was clearly visible, showing edges with obvious light and dark contrast.

[0107] Test Example 1

[0108] The intracellular vesicles prepared in Examples 1-5 or Comparative Example 1 were collected and weighed. The results are shown in Table 1.

[0109] Table 1

[0110] Group Intracellular vesicle weight (g) Example 1 0.28±0.05 Example 2 0.20±0.02 Example 3 0.24±0.03 Example 4 0.13±0.01 Example 5 0.15±0.05 Comparative Example 1 0.09±0.02

[0111] The results are shown in Table 1 and Figure 1 .Depend on Figure 1 It can be seen that the intracellular vesicles obtained by the method in Examples 1-3 of the present invention have a high yield and a high yield.

[0112] Test Example 2

[0113] 0.1 g of the intracellular vesicles prepared in Examples 1-5 or Comparative Example 1 was added to 50 mL of water, and 0.5 g of doxorubicin was added. The mixture was stirred and mixed evenly. The mixture was centrifuged at 2500 g for 15 min and 45000 g for 10 min. The supernatant was collected and the absorbance was tested. The content of doxorubicin in the supernatant was calculated to obtain the drug loading capacity of the intracellular vesicles for doxorubicin. The results are shown in Tables 2 and Figure 2 .

[0114] Table 2

[0115] Group Drug loading of doxorubicin (mg / g) Example 1 335 Example 2 359 Example 3 342 Example 4 324 Example 5 316 Comparative Example 1 232

[0116] It can be seen from the above table that the intracellular vesicles obtained by the methods in Examples 1-3 of the present invention have a higher drug loading capacity, which may be closely related to the smaller particle size of the vesicles obtained by the present invention.

[0117] 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 preparing intracellular vesicles obtained by lysing mesenchymal stem cells, characterized in that: The following steps are involved: S1. providing mesenchymal stem cells and pre-treating them to remove the culture medium; S2. Proliferate and culture the mesenchymal stem cells to obtain the required number of cells; S3. digesting the cells by enzymatic digestion to form a single cell suspension; S4. Lyse the single cells to release the cell contents and remove non-target components by centrifugation; S5. The supernatant after centrifugation is subjected to ultracentrifugation to separate and collect the precipitated intracellular vesicles.

2. The preparation method according to claim 1, characterized in that: Vesicle inducing factors are added into the proliferation culture medium, and the vesicle inducing factors are hemolytic streptolysin and fibronectin.

3. The preparation method according to claim 2, characterized in that: The concentration of fibronectin is about 1-20 μg / mL, and the concentration of hemolytic streptolysin is 50-200 ng / mL.

4. The preparation method according to claim 1, characterized in that: The incubation conditions are 35-38° C., saturated humidity, and 3-6 v / v% CO 2 concentration for 12-24 hours.

5. The preparation method according to claim 1, characterized in that: The digestive enzyme solution is a mixed solution of 1.5-2.5wt% trypsin and 0.5-1wt% neutral protease.

6. The preparation method according to claim 1, characterized in that: The digestion temperature is 35-38°C and the digestion time is 3-5h.

7. The preparation method according to claim 1, characterized in that: The lysate is a mixed solution prepared by adding 100-200 mmol / L NaCl, 0.5-1.5 wt% Tween-85, 0.5-1.5 wt% sodium deoxycholate and 0.05-0.15 wt% sodium dodecylbenzene sulfonate to a Tris-HCl solution with a pH of 7.4-7.

9. The lysate temperature is 35-40° C. and the lysate time is 20-40 min.

8. The preparation method according to claim 1, characterized in that: The ultracentrifugation treatment is performed by centrifugation at 2000-3000 g for 10-20 min, or at 40000-50000 g for 5-10 min.

9. Use of a combination of hemolytic streptolysin and fibronectin in the preparation of intracellular vesicles from mesenchymal stem cells.

10. Use of the intracellular vesicles prepared according to the method of claims 1-8 as a drug carrier.

11. A cell culture medium comprising a combination of hemolytic streptolysin and fibronectin.

12. A drug comprising the intracellular vesicles prepared according to the method of claims 1-8 as a drug carrier.

Images