A complex of umbilical cord mesenchymal stem cell exosome and cisplatin and a preparation method and application thereof

Abstract

The application provides a compound of umbilical cord mesenchymal stem cell exosome and cisplatin, a preparation method and application thereof, the umbilical cord mesenchymal stem cell exosome is wrapped cisplatin to obtain the compound of umbilical cord mesenchymal stem cell exosome and cisplatin, and the preparation method of the compound comprises the following steps: 1) extracting umbilical cord mesenchymal stem cell exosome; 2) mixing the umbilical cord mesenchymal stem cell exosome and cisplatin to obtain a mixture according to a mass ratio of 1:1; 3) ultrasonic treatment is carried out on the mixture by using an ultrasonic method, and after ultrasonic treatment, centrifugal treatment is carried out to discard supernatant, to obtain the compound, and the compound can also be used as a drug for treating cervical cancer. The compound of umbilical cord mesenchymal stem cell exosome and cisplatin provided by the application has good biocompatibility, high stability and good targeting, can transport cisplatin to a lesion site in a directional manner, reduces the toxic side reaction of cisplatin, and has a good inhibitory effect on gynecological tumors.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, and particularly relates to a complex of umbilical cord mesenchymal stem cell exosomes and cisplatin, its preparation method and application. Background Technology

[0002] Cisplatin (cis-diamminedichloroplatinum(II), CDDP) was first approved by the U.S. Food and Drug Administration in 1978 for the treatment of testicular and bladder cancer, and has since been widely used in clinical practice. As a broad-spectrum antitumor drug, cisplatin exerts its inhibitory effect on tumor growth primarily by binding to tumor cell DNA to form a complex, causing DNA damage and subsequently leading to mitochondrial apoptosis, thus producing cytotoxicity.

[0003] Cisplatin has a relatively weak targeting effect, leading to significant toxic side effects in clinical practice and the development of drug resistance in tumors. Typically, in the early stages of cisplatin-based cancer treatment, tumor growth is significantly inhibited, and even solid tumors show a marked reduction in size, with the patient's condition remaining relatively stable during initial treatment. However, as treatment continues, a large proportion of tumors initially sensitive to cisplatin eventually develop drug resistance, a phenomenon particularly pronounced in ovarian cancer patients.

[0004] Exosomes are vesicles secreted by various cells, possessing a phospholipid bilayer structure and nanoparticle size. They exhibit good biocompatibility, high stability, and good targeting properties, playing a crucial role in targeted drug delivery and enhancing drug bioavailability. For example, cisplatin can be transported to lesion sites using nanomedicine delivery systems. However, there are no clear technical specifications for the use of mesenchymal stem cell exosomes for platinum-based drug delivery, and their application in gynecological tumors such as cervical cancer has not been studied. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a complex of umbilical cord mesenchymal stem cell exosomes and cisplatin, its preparation method and application, with the aim of using umbilical cord mesenchymal stem cell exosomes to encapsulate cisplatin and transport it to the lesion site, thereby greatly reducing the drug resistance of gynecological tumors to cisplatin.

[0006] To solve the above-mentioned technical problems, the present invention is implemented as follows: a complex of umbilical cord mesenchymal stem cell exosomes and cisplatin, wherein the umbilical cord mesenchymal stem cell exosomes encapsulate cisplatin.

[0007] This invention also provides a method for preparing a complex of umbilical cord mesenchymal stem cell exosomes and cisplatin, comprising the following steps:

[0008] 1) Extraction of exosomes from umbilical cord mesenchymal stem cells;

[0009] 2) The umbilical cord mesenchymal stem cell exosomes and cisplatin were mixed at a mass ratio of 1:1 to obtain a mixture;

[0010] 3) The mixture is subjected to ultrasonic treatment, and after ultrasonic treatment, the supernatant is discarded by centrifugation to obtain the complex.

[0011] Further, umbilical cord mesenchymal stem cell exosomes were extracted according to the following steps:

[0012] 1.1) Culture umbilical cord tissue to obtain umbilical cord mesenchymal stem cells;

[0013] 1.2) Passage culture of umbilical cord mesenchymal stem cells using complete human umbilical cord mesenchymal stem cell culture medium;

[0014] 1.3) The culture medium of umbilical cord mesenchymal stem cells obtained from passage culture was separated and extracted using an exosome extraction kit to obtain umbilical cord mesenchymal stem cell exosomes.

[0015] Furthermore, before performing ultrasonic treatment step 3), ethyl acetate, lysine, and fructose in a mass-volume ratio of 0.1%-15% are added to the mixture obtained in step 2).

[0016] Furthermore, in step 3), degassing is performed simultaneously with ultrasonic treatment.

[0017] Furthermore, in step 3), ultrasonic processing is performed at 20 kHz.

[0018] Furthermore, the ultrasound treatment is performed multiple times, 6 seconds per session, and after each ultrasound, the device is placed on ice for 3-15 minutes to equilibrate.

[0019] Furthermore, in step 3), after the ultrasound is completed, the sample is centrifuged at 13500g for 40 minutes.

[0020] The present invention also provides the use of a complex of umbilical cord mesenchymal stem cell exosomes and cisplatin in the preparation of a medicament for the treatment of cervical cancer.

[0021] Compared with existing technologies, the advantages of the complex of umbilical cord mesenchymal stem cell exosomes and cisplatin, its preparation method, and its application in this invention are as follows:

[0022] This invention provides a complex of umbilical cord mesenchymal stem cell exosomes and cisplatin, its preparation method, and its application. The umbilical cord mesenchymal stem cell exosomes encapsulate cisplatin to obtain the complex. The preparation method of the complex includes the following steps: 1) extracting umbilical cord mesenchymal stem cell exosomes; 2) mixing the umbilical cord mesenchymal stem cell exosomes and cisplatin at a 1:1 mass ratio to obtain a mixture; 3) sonicating the mixture using ultrasound, followed by centrifugation to discard the supernatant, obtaining the complex. The complex can also be used as a drug for treating cervical cancer. The umbilical cord mesenchymal stem cell exosome and cisplatin complex provided by this invention has good biocompatibility, high stability, and good targeting, enabling targeted delivery of cisplatin to the lesion site, reducing the toxic side effects of cisplatin, and exhibiting a good inhibitory effect on gynecological tumors. Attached Figure Description

[0023] Figure 1 This is a particle size distribution detection diagram of umbilical cord mesenchymal stem cell exosomes in Example 1 of the present invention;

[0024] Figure 2 This is a surface morphology diagram of umbilical cord mesenchymal stem cell exosomes in Embodiment 1 of the present invention;

[0025] Figure 3 This is a marker protein detection diagram of umbilical cord mesenchymal stem cell exosomes in Example 1 of the present invention;

[0026] Figure 4 A is the HPLC detection chromatogram of cisplatin standard in Example 2 of this invention;

[0027] Figure 4 B is an HPLC chromatogram of cisplatin detection in the supernatant of the complex of umbilical cord mesenchymal stem cell exosomes and cisplatin in Example 2 of the present invention.

[0028] Figure 5 A is a graph showing the encapsulation efficiency of cisplatin by umbilical cord mesenchymal stem cell exosomes in Example 3 of this invention;

[0029] Figure 5 B is a comparison chart of the cisplatin content in the complex of umbilical cord mesenchymal stem cell exosomes immediately after being treated with cisplatin and after being stably stored for seven days in Example 3 of the present invention.

[0030] Figure 6 This is a graph showing the inhibitory effect of the complex of umbilical cord mesenchymal stem cell exosomes and cisplatin on the proliferation of cervical cancer cells HeLa cells at 24h and 48h in Example 4 of this invention. Detailed Implementation

[0031] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0032] Example 1: Extraction and Detection of Exosomes from Umbilical Cord Mesenchymal Stem Cells

[0033] First, the specific steps for extracting exosomes from umbilical cord mesenchymal stem cells are as follows: A fresh, healthy umbilical cord is taken, rinsed thoroughly with PBS, and then the blood vessels in the cord are removed with scissors and forceps. The Fahrenheit jelly tissue inside the cord is then extracted to obtain umbilical cord tissue. The umbilical cord tissue is then finely minced to a size of 1mm*1mm and placed in α-MEM culture medium, which is incubated at 37°C in a 5% CO2 incubator. The α-MEM culture medium contains 15% FBS, 100U / ml penicillin, and 100U / ml streptomycin. After 5-7 days of umbilical cord tissue culture, some cells can be seen crawling out from around the umbilical cord tissue, appearing as small spindle-shaped cells. After 7 days, the cells begin to proliferate rapidly, forming cell colonies of varying sizes. Once the cells have reached confluence, they are passaged using 0.25% trypsin. Subsequent passages are performed using complete human umbilical cord mesenchymal stem cell culture medium (serum-free type II), purchased from Cyagen Biosciences Co., Ltd., catalog number HUXUC-90062. Umbilical cord mesenchymal stem cell culture medium is collected from the complete human umbilical cord mesenchymal stem cell culture medium, placed on ice, and centrifuged at 2000g for 10 min to remove residual cells. The supernatant is collected, and then centrifuged again at 10000g for 10 min to remove cell debris. The supernatant is collected again. Umbilical cord mesenchymal stem cell exosomes are extracted from the supernatant using a cell culture medium exosome extraction kit. The specific steps are as follows:

[0034] a. Transfer the supernatant to a new centrifuge tube, and add water at a volume ratio of 2:1 (V / V). 样 V A IsolationRegent A solution (ratio = 2:1);

[0035] b. After inverting and mixing 3-5 times, let stand at 4℃ for 5 minutes, then centrifuge at 10000g for 3 minutes; transfer the supernatant to a new centrifuge tube and add Isolation Regent B solution at a volume ratio of 1:1;

[0036] c. After inverting and mixing 3-5 times, let stand at 4℃ for 1 hour, then centrifuge at 13500 g for 0.5 hours, discard the supernatant, and collect the precipitate, which is the exosome of umbilical cord mesenchymal stem cells.

[0037] d. Add 200-500 μL of sterile PBS to resuspend the precipitate for subsequent experiments, or aliquot and store at -80°C.

[0038] After successful extraction of umbilical cord mesenchymal stem cells, the umbilical cord mesenchymal stem cells were identified:

[0039] Umbilical cord mesenchymal stem cell exosomes were resuspended and identified using nanoparticle size tracking (NTA), transmission electron microscopy (TEM), and Western blotting (WB). The particle size distribution, surface morphology, and marker proteins of the obtained umbilical cord mesenchymal stem cell exosomes were tested. Figure 1 , 2 And 3.

[0040] Example 2: Preparation of a complex of umbilical cord mesenchymal stem cell exosomes and cisplatin

[0041] Experimental group: Umbilical cord mesenchymal stem cell exosomes and cisplatin (Sangon Biotech) were mixed at a mass ratio of 1:1. For every 100 μL of mixture, 0.1-15 g of ethyl acetate, 0.2-5 g of lysine, and 0.2-2 g of fructose were added. The mixture was then subjected to sonication with the following parameters: 20 kHz, degassing, 6 seconds / time, 4 times (repeated 3 times). After each sonication, the mixture was equilibrated on ice for 5 minutes. After sonication, the mixture was equilibrated at room temperature for 30 minutes and then centrifuged at 13500 g for 40 minutes. The supernatant was separated from the precipitate, which was the complex of umbilical cord mesenchymal stem cell exosomes and cisplatin from the experimental group.

[0042] Control group: Umbilical cord mesenchymal stem cell exosomes and cisplatin (Sangon Biotech) were mixed at a mass ratio of 1:1, and the mixture was treated with sonication. The specific parameters for sonication were: 20 kHz, degassing, 6 seconds / time, 4 times (repeated 3 times), with the mixture placed on ice for 5 minutes after each sonication. After sonication, the mixture was equilibrated at room temperature for 30 minutes and then centrifuged at 13500g for 40 minutes. The supernatant was separated from the precipitate, and the precipitate obtained was the complex of umbilical cord mesenchymal stem cell exosomes and cisplatin from the control group.

[0043] The cisplatin-loaded complex of umbilical cord mesenchymal stem cell exosomes from the experimental group was centrifuged at 13500g for 30 min. The cisplatin content in the supernatant was collected, and the cisplatin loading content of the mesenchymal stem cell exosomes was analyzed using high-performance liquid chromatography (HPLC). This analysis further determined the efficiency of cisplatin loading in the mesenchymal stem cells. The HPLC detection parameters were as follows:

[0044] The chromatographic column was C18 (4.6 mm × 250 mm, 5 μm), the column temperature was 30 ℃, the mobile phase was acetonitrile:water = 30:70, the flow rate was 1.5 ml / min, the detection wavelength was 270 nm, and the injection volume was 20 μl. The detection results are attached. Figure 4 As shown, Figure 4 A is the HPLC detection chromatogram of cisplatin standard in this embodiment. Figure 4 B is an HPLC chromatogram of cisplatin detection in the supernatant of the complex of umbilical cord mesenchymal stem cell exosomes and cisplatin in this embodiment.

[0045] Example 3: Detection of the encapsulation efficiency and stability of cisplatin by umbilical cord mesenchymal stem cell exosomes.

[0046] The complexes of umbilical cord mesenchymal stem cell exosomes and cisplatin in the control and experimental groups of Example 2 were centrifuged at 13500g for 30 min. The cisplatin content in the supernatant was then determined by high-performance liquid chromatography (HPLC). The encapsulation efficiency of umbilical cord mesenchymal stem cell exosomes for cisplatin, i.e., the drug loading rate, was calculated as (1 - cisplatin content in supernatant / total cisplatin content) * 100%. The results of the cisplatin encapsulation efficiency of umbilical cord mesenchymal stem cell exosomes are attached. Figure 5 A. The encapsulation efficiency of the control group was 46%, while that of the experimental group was 63%. This shows that after the umbilical cord mesenchymal stem cell exosomes and cisplatin were mixed in a conventional manner, the addition of ethyl acetate (0.1%-15% by weight), lysine (0.2%-5% by weight), and fructose (0.2%-2% by weight) improved the cisplatin encapsulation efficiency of the umbilical cord mesenchymal stem cell exosomes.

[0047] The cisplatin-encapsulated complexes of umbilical cord mesenchymal stem cell exosomes from the control and experimental groups in Example 2 were stabilized at 4°C for seven days. Then, 200 μL of pure water was added, and the mixture was centrifuged at 13500 g for 30 min. The cisplatin content in the supernatant was collected, and the concentration in the exosomes after seven days of storage was measured and compared with the cisplatin concentrations in the control and experimental groups in Example 2. The stability of cisplatin encapsulation in umbilical cord mesenchymal stem cell exosomes was calculated. The stability test results of cisplatin encapsulation in umbilical cord mesenchymal stem cell exosomes are attached. Figure 5 B, where the cisplatin loading stability of the control group was approximately 78%, and that of the experimental group was approximately 83%. The experimental data indicate that the addition of ethyl acetate (0.1%-15%), lysine (0.2%-5%), and fructose (0.2%-2%) to the conventional mixture of umbilical cord mesenchymal stem cell exosomes and cisplatin effectively improves the cisplatin loading stability of the umbilical cord mesenchymal stem cell exosomes.

[0048] Example 4: Experiment on the inhibitory effect of umbilical cord mesenchymal stem cell exosomes-cisplatin complex on the proliferation of cervical cancer cells HeLa cells.

[0049] HeLa cells were cultured and digested with trypsin containing 0.25% EDTA. The cells were then incubated at 37°C in a 5% CO2 incubator for 5 min. When the cells were observed to become rounded and shrunken under an inverted microscope, DMEM medium containing 10% FBS was added to stop the trypsin digestion. The cells were then pipetted until completely detached. The culture medium was collected and transferred to a 5 ml sterile centrifuge tube. The tube was centrifuged at 1000 rpm for 5 minutes, and the supernatant was discarded.

[0050] HeLa cells were resuspended in medium containing 3% FBS, brought to a fixed volume, and counted using a hemocytometer. Cells were then seeded into 96-well plates (1×10⁶ cells / wells). 4 (Cells / well). The culture plates were transferred to an incubator and cultured at 37 ℃, 5% CO2, and saturated humidity for 12 h. The supernatant was discarded, and cisplatin solution with a final concentration of 10 μM, MSC-Exo-cisplatin complex culture medium, and an equal volume of 100 μg / ml MSC-Exo culture medium were added to each well. DMEM complete culture medium was used as a blank control group. Three replicates of different concentrations of culture medium were set up for each group. The culture plates were incubated for another 24 h. After 24 h, 20 μL of 5 g / L MTT was added to each well, and the plates were incubated for another 4 h. The supernatant was then aspirated, and 150 μL of DMSO solution was added. The plates were shaken for 10 min, and the absorbance was measured using a microplate reader at a wavelength of 490 nm. The absorbance (OD) value of each well was measured. The proliferation rate of HeLa cells = absorbance value per well of the experimental group / absorbance value per well of the control group * 100%. Correspondingly, the inhibition rate of HeLa cells = 100% - proliferation rate of HeLa cells. The inhibition rate of HeLa cells after 24 hours of culture was calculated.

[0051] The absorbance of each well in each group after 48 hours of culture was measured using the same method, and the inhibition rate of HeLa cells after 48 hours was calculated. Figure 6 This example shows the inhibitory effect of the complex of umbilical cord mesenchymal stem cell exosomes and cisplatin on the proliferation of cervical cancer HeLa cells at 24h and 48h. Figure 6The vertical axis represents the proliferation rate of HeLa cells. After 24 hours of culture, the proliferation rate of HeLa cells in MSC-Exo medium was approximately 105%, in cisplatin solution approximately 96%, and in MSC-Exo-cisplatin complex medium only 60%. This shows that while cisplatin solution can also inhibit HeLa cell proliferation, its inhibitory effect is far less than that of the MSC-Exo-cisplatin complex medium. After 48 hours of culture, the proliferation rate of HeLa cells in MSC-Exo medium was approximately 81%, and in cisplatin solution approximately 86%. This indicates that cisplatin can inhibit HeLa cells over time, but the efficiency of inhibition gradually decreases. At this point, the proliferation rate of HeLa cells in MSC-Exo-cisplatin complex medium was 47%, demonstrating that the inhibitory effect of the MSC-Exo-cisplatin complex medium on HeLa cells is far superior to that of cisplatin solution. These experimental results demonstrate that cisplatin-encapsulated umbilical cord mesenchymal stem cells have a significant inhibitory effect on gynecological tumor cells, and the inhibitory effect and long-term inhibitory ability are far superior to cisplatin.

[0052] 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, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing a complex of umbilical cord mesenchymal stem cell exosomes and cisplatin, characterized in that, Includes the following steps: 1) Extraction of exosomes from umbilical cord mesenchymal stem cells; 2) Mix the umbilical cord mesenchymal stem cell exosomes and cisplatin at a mass ratio of 1:1, and add ethyl acetate (0.1%-15% by mass), lysine (0.2%-5% by mass), and fructose (0.2%-2% by mass) to obtain a mixture; 3) The mixture is subjected to ultrasonic treatment, and after ultrasonic treatment, the supernatant is discarded by centrifugation to obtain the complex.

2. The method according to claim 1, characterized in that, Extract umbilical cord mesenchymal stem cell exosomes according to the following steps: 1.1) Culture umbilical cord tissue to obtain umbilical cord mesenchymal stem cells; 1.2) Passage culture of umbilical cord mesenchymal stem cells using complete human umbilical cord mesenchymal stem cell culture medium; 1.3) The culture medium of umbilical cord mesenchymal stem cells obtained from passage culture was separated and extracted using an exosome extraction kit to obtain umbilical cord mesenchymal stem cell exosomes.

3. The method according to claim 1, characterized in that, In step 3), degassing is performed simultaneously with ultrasonic treatment.

4. The method according to claim 1, characterized in that, In step 3), ultrasonic processing is performed at 20 kHz.

5. The method according to claim 4, characterized in that, The ultrasound treatment was performed multiple times, 6 seconds per session, and after each ultrasound, the device was placed on ice for 3-15 minutes to equilibrate.

6. The method according to claim 1, characterized in that, After the ultrasound is completed in step 3), the sample is centrifuged at 13500g for 40 minutes.

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