Liver cancer targeting extracellular small vesicles as well as preparation method and application thereof
By using GPC3 aptamer modification technology on small extracellular vesicles, combining cholesterol residues and fluorescent groups, the targeting of liver cancer cells is significantly improved, and the problem of insufficient targeting in the prior art is solved. It has achieved accurate delivery of drugs and the effect of liver cancer treatment.
Patent Information
- Application Number
- CN202411124295.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-08-16
- Publication Date
- 2024-12-17
AI Technical Summary
The existing small extracellular vesicles lack targeting, resulting in uneven distribution of drugs, reducing therapeutic effects, and high preparation and modification costs, limiting their large-scale application.
By loading anti-tumor drugs in targeted extracellular vesicles for liver cancer, GPC3 aptamers are used to modify extracellular vesicles, so that they can bind GPC3 receptors on the surface of liver cancer cells with high affinity, improve targeting, and Stability and targeting are enhanced by cholesterol residues and fluorophore modification.
It significantly improves the targeting of small extracellular vesicles on liver cancer tissues, achieves accurate and efficient delivery of drugs, directly inhibits the proliferation and progress of liver cancer cells, reduces side effects, and provides a simple and effective treatment plan.
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Figure CN119139484A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the field of medical technology and relates to a liver cancer-targeted extracellular vesicle and a preparation method and application thereof. Background Art
[0002] Almost all prokaryotic and eukaryotic cells release extracellular vesicles (EVs). These nanoscale extracellular vesicles can carry many cellular components, including DNA, RNA, lipids, metabolites, cytoplasm and cell surface proteins, and produce corresponding physiological functions through interactions with surrounding cells. At present, extracellular vesicles have developed into a new type of nano drug delivery carrier that can improve the tissue penetration, retention and uptake efficiency of drugs. However, unmodified ordinary vesicles lack tissue targeting, which hinders the biomedical application of extracellular vesicles.
[0003] Targeted molecular modification of the surface of extracellular vesicles can improve tissue targeting. The modification materials currently used mainly include proteins, peptides, nanomaterials, etc. Through various extracellular vesicle membrane surface modification methods, the membrane modification materials are attached to the surface of extracellular vesicles, so that the modified extracellular vesicles are highly selectively enriched in specific tissues and organs, improving the efficacy and reducing the toxic side effects caused by ectopic drug enrichment.
[0004] Although surface modification can improve the targeting of extracellular vesicles, the vesicles still have the problem of insufficient targeting, which may lead to uneven drug distribution and greatly reduce the therapeutic effect. In addition, the current preparation and modification costs of extracellular vesicles are high, which also limits their large-scale application. Summary of the invention
[0005] The purpose of the present invention is to address the above-mentioned problems existing in the prior art. The present invention provides a liver cancer-targeted extracellular vesicle, which can exert precise and efficient anti-tumor effects by loading anti-tumor drugs in the liver cancer-targeted extracellular vesicles.
[0006] The purpose of the present invention can be achieved by the following technical solutions:
[0007] A liver cancer-targeted extracellular vesicle, wherein the extracellular vesicle is obtained from bone marrow mesenchymal stem cells and then modified with a GPC3 aptamer.
[0008] In the above-mentioned liver cancer-targeted extracellular vesicles, the bone marrow mesenchymal stem cells are bone marrow mesenchymal stem cells derived from BALB / c mice.
[0009] Preferably, obtaining extracellular vesicles from bone marrow mesenchymal stem cells specifically comprises the following steps:
[0010] S1. Bone marrow was obtained from the femur and tibia of BALB / c mice and cultured in DMEM / F12 culture medium, in which serum without extracellular vesicles was added;
[0011] S2. Observe the morphology of primary BALB / c mouse bone marrow mesenchymal stem cells under an optical microscope on the 14th day after they were extracted;
[0012] S3, flow cytometry was used to detect the third-generation cell surface markers Sca-1, CD44, CD45, CD11b and CD31 of the stable proliferation of bone marrow mesenchymal stem cells of primary BALB / c mouse cells;
[0013] S4. Collect the supernatant of the primary BALB / c mouse bone marrow mesenchymal stem cells of stable passage, use the "classical differential ultracentrifugation method", obtain the extracellular vesicles and resuspend them in PBS solution, and store them at -80°C for future use;
[0014] Among them, each 800-1000 mL of supernatant obtained after "classical differential ultracentrifugation" treatment is resuspended with 800 μL-1000 μL of PBS.
[0015] In the above-mentioned liver cancer-targeted extracellular vesicles, the GPC3 aptamer can bind with high affinity and strong specificity to the GPC3 receptor highly expressed on the surface of liver cancer cells.
[0016] In the above-mentioned liver cancer-targeted extracellular vesicles, the nucleotide sequence of the GPC3 aptamer is shown as SEQ ID NO.1.
[0017] In the above-mentioned liver cancer-targeted extracellular vesicles, the 3' end and the 5' end of the GPC3 aptamer each have three consecutive phosphodiester bonds modified by thiophosphate. The present invention replaces a non-bridging oxygen atom in the oligonucleotide backbone with a sulfur atom, thereby making it more resistant to degradation by nucleases (especially exonucleases), thereby greatly improving its stability in vivo.
[0018] Preferably, the 3' end of the GPC3 aptamer has three consecutive phosphodiester bonds starting from the first phosphodiester bond modified by phosphorothioate;
[0019] The 5' end of the GPC3 aptamer has three consecutive phosphodiester bonds starting from the first phosphodiester bond modified by phosphorothioate.
[0020] In the above liver cancer-targeted extracellular vesicles, the 3' end of the GPC3 aptamer is connected to a cholesterol residue, and the 5' end is connected to a fluorescent group. The present invention promotes its insertion and anchoring in the vesicle membrane by connecting a hydrophobic cholesterol residue to the 3' end of the oligonucleotide molecule.
[0021] Preferably, the fluorescent group is a Cy5 fluorescent probe.
[0022] The present invention also provides a method for preparing the liver cancer-targeted extracellular vesicles, which comprises the following steps: adding the extracellular vesicles and GPC3 aptamers to a PBS solution, incubating at room temperature, and then removing impurities to obtain the targeted extracellular vesicles.
[0023] In the above-mentioned method for preparing liver cancer-targeted extracellular vesicles, the amount of the aptamer in each 1 mg of the extracellular vesicle solution is 2-5 nmol, based on the protein content of the extracellular vesicles.
[0024] In the above-mentioned method for preparing liver cancer-targeted extracellular vesicles, dialysis or ultrafiltration is used to remove impurities.
[0025] The present invention also provides an application of the liver cancer-targeted extracellular vesicles in a drug for treating liver cancer.
[0026] Compared with the prior art, the present invention has the following beneficial effects:
[0027] 1. The present invention can significantly improve the targeting of extracellular vesicles to liver cancer tissues by modifying extracellular vesicles with small molecule GPC3 nucleic acid aptamers. This improved targeting enables extracellular vesicles to more effectively load anti-tumor drugs and deliver them to liver cancer cells, thereby exerting precise and efficient anti-tumor effects. The selection of GPC3 nucleic acid aptamers is based on their high affinity to the GPC3 receptors on the surface of liver cancer cells, ensuring that the drugs can specifically bind to liver cancer cells.
[0028] 2. The present invention utilizes GPC3 not only as a marker molecule for hepatocellular carcinoma, but also as a potential therapeutic target. By targeting the GPC3 receptor with a nucleic acid aptamer, the proliferation and progression of liver cancer cells can be directly inhibited. This direct mechanism of action helps to enhance the overall therapeutic effect and reduce the side effects caused by nonspecific drug delivery.
[0029] 3. The present invention uses extracellular vesicles modified with small molecule GPC3 nucleic acid aptamers as drug delivery carriers, which not only improves the targeting of liver cancer tissues, but also directly inhibits the progression of liver cancer. This method provides a new perspective for the treatment of hepatocellular carcinoma, with a clear theoretical basis, mature experimental methods and simple operation procedures. With further research and clinical verification, this innovative therapy is expected to bring more accurate and effective treatment options for liver cancer patients. BRIEF DESCRIPTION OF THE DRAWINGS
[0030] Figure 1 This is a schematic diagram of primary BALB / c mouse bone marrow mesenchymal stem cells under an optical microscope on the 14th day of the present application embodiment;
[0031] Figure 2 This is a diagram showing the expression of the third generation cell surface markers for the proliferation of primary BALB / c mouse bone marrow mesenchymal stem cells detected by flow cytometry in an embodiment of the present application;
[0032] Figure 3 This is a morphological image of liver cancer-targeting extracellular vesicles modified with aptamers characterized by transmission electron microscopy in an embodiment of the present application;
[0033] Figure 4 For the in vitro targeting experiment of the present application example, immunofluorescence co-localization characterization was performed in the Huh7 cell line;
[0034] Figure 5 For the in vivo targeting experiment of the present application example, Huh7 cell line-constructed subcutaneous tumor-bearing nude mice were injected with PBS-diluted aptamer-modified liver cancer-targeted extracellular vesicles solution through the tail vein, and in vivo imaging was performed 6 hours later. DETAILED DESCRIPTION
[0035] The following are specific embodiments of the present invention, which further describe the technical solutions of the present invention, but the present invention is not limited to these embodiments.
[0036] First, primary BALB / c mouse bone marrow mesenchymal stem cells and their derived extracellular vesicles were obtained:
[0037] (1) Take female BALB / c mice aged 4-5 weeks, kill them by cervical dislocation, and immediately soak them in 75% alcohol for 5-10 minutes. Then transfer the BALB / c mice to a clean bench. Fix the limbs of the BALB / c mice on sterile plastic foam boards so that their hind limbs can be fully exposed to the operator's field of vision. Use autoclaved surgical scissors and forceps to cut the skin of the hind legs of the BALB / c mice, carefully peel off the complete femur and tibia of the BALB / c mice, and separate the surrounding skin, muscle and connective tissue. The separated femur and tibia are placed in a culture dish containing DMEM / F12 complete culture medium for later use. Take the femur or tibia, cut both ends with scissors, and use a 1mL disposable sterile syringe to absorb DMEM / F12 complete culture medium, and flush the bone marrow out of the bone marrow cavity. Repeat the bone marrow cavity flushing until the femur and tibia become translucent and white. Collect the DMEM / F12 culture medium with bone marrow flushed out, filter twice with a 70μm sterile filter to remove large tissue blocks, broken bones and other impurities, then collect the filtered liquid and centrifuge at 300g for 5 minutes. After centrifugation, remove the supernatant, add 3-5 times the volume of the cell sediment red blood cell lysis solution, repeatedly blow and stand for 3 minutes. After the lysis is completed, add 5mL of DMEM / F12 complete culture solution, and centrifuge at 300g for 5 minutes after repeated blowing. After centrifugation, remove the supernatant and observe whether the precipitate is still red. If the red precipitate can still be seen, it means that the red blood cells are not completely lysed. Repeat the above red blood cell lysis steps until the precipitate completely turns white (Note: the red blood cell lysis solution is harmful to normal cells, and it is important to ensure that the lysis time is not too long). Finally, resuspend the cells with 5mL of DMEM / F12 complete culture solution, inoculate them into a T25 culture bottle, and culture them in a 37°C incubator. Change the solution the next day, wash away the floating cells, and the remaining attached cells are bone marrow mesenchymal stem cells. The primary BALB / c mouse bone marrow mesenchymal stem cells used in the method of the present invention can also be obtained by other extraction methods. The primary BALB / c mouse bone marrow mesenchymal stem cells are used as source cells of nanocarriers in the method of the present invention, and their extraction method and process do not constitute a limitation on the protection scope of the present invention.
[0038] (2) Extraction and purification of extracellular vesicles from bone marrow mesenchymal stem cells of primary BALB / c mice: 800-1000 mL of supernatant of bone marrow mesenchymal stem cells of primary BALB / c mice were collected and subjected to differential ultracentrifugation as follows: centrifuge at 300 g for 10 minutes, remove the precipitate, and take the supernatant; continue to centrifuge at 2000 g for 10 minutes, remove the precipitate, and take the supernatant; continue to centrifuge at 10000 g for 30 minutes, remove the precipitate, and take the supernatant; ultracentrifuge at 110000 g for 70 minutes twice, and remove the supernatant; the final precipitate was resuspended with 800 μL-1000 μL of PBS solution, and 50 μL of sample was taken to determine the protein content using the BCA method. The extracellular vesicles used in the method of the present invention can also be obtained by other extraction methods, and the extraction method and process of the extracellular vesicles do not constitute a limitation on the scope of protection of the present invention.
[0039] (3) Co-incubation of GPC3 aptamer with extracellular vesicles: GPC3 aptamer was obtained by chemical synthesis as shown in Table 1. After modification with cholesterol residues, phosphorothioate and Cy5 fluorescent probe, sterile deionized water was added to dilute the aptamer aqueous solution to 100 pmol / μL. Take 20 μL of aptamer aqueous solution and add it to 1 mL of extracellular vesicle solution. After blowing evenly, incubate at room temperature for 1 hour. Then centrifuge at 3000 rpm for 10 minutes in a 30Kda ultrafiltration tube to remove free aptamer solution. Then rinse twice with 500 μL PBS solution. Then carry out subsequent experiments:
[0040] Table 1: GPC3 aptamer sequences
[0041] sequence 5` to 3` SEQ ID NO.1 Cy5-TAACGCTGACCTTAGCTGCATGGCTTTACATGTTCCA-Chol
[0042] The 3' and 5' ends of the GPC3 aptamer each have three consecutive phosphodiester bonds modified by phosphorothioate starting from the first phosphodiester bond. The 3' end of the GPC3 aptamer is connected to a cholesterol residue, and the 5' end is connected to a fluorescent group.
[0043] (4) In vitro targeting verification: The targeting ability of targeted extracellular vesicles relative to ordinary extracellular vesicles was evaluated by fluorescence co-localization method. First, the Huh7 cell line was cultured in a confocal culture dish. When the confluence reached 30-40%, the culture medium was aspirated and treated with 4% paraformaldehyde at room temperature for 15 minutes, and blocked with 5% bovine serum albumin (BSA) solution at room temperature for 1 hour. After blocking, GPC3 immunofluorescence rabbit primary antibody (Affinity, Cat No: DF6710) was used for overnight incubation in a refrigerator at 4°C. Then, a fluorescent secondary antibody (Proteintech, Cat No: SA00013-2) excited at 488nm was used for incubation at room temperature in the dark for 1 hour. After incubation with the fluorescent secondary antibody, 0.5 mL of 5% BSA solution was mixed with 0.5 mL of aptamer-modified targeted extracellular vesicle solution (v / v=1:1), and then added to the confocal dish and incubated at room temperature in the dark for 1 hour. Finally, 2.5ug / mL Hochest33342 was used for incubation at room temperature for 10 minutes. After the fluorescence staining, confocal microscopy was used to compare the staining intensity of Huh7 cells by random sequence-modified extracellular vesicles (sEV-Random-Cy5) and GPC3 aptamer-modified extracellular vesicles (sEV-GPC3-apt-Cy5).
[0044] (5) In vivo targeting verification: The enrichment of targeted extracellular vesicles relative to ordinary extracellular vesicles in tumor tissues was evaluated by in vivo imaging tracing. First, a nude mouse subcutaneous tumor model was constructed using the Huh7 cell line. The nude mice were then randomly divided into two groups and injected with sterile sEV-Random-Cy5 and sEV-GPC3-apt-Cy5 solutions through the tail vein. Fluorescence imaging was performed using the IVIS small animal in vivo imaging system 6 hours after administration, and the fluorescence signal intensity in the tumors of the two groups of animals was compared.
[0045] The liver cancer-targeted extracellular vesicles prepared by the method of the present invention can be used to load various drugs and implement in vivo and in vitro drug delivery, and the related methods and processes of drug loading and delivery do not constitute a limitation on the protection scope of the present invention.
[0046] The present invention will be further described below in conjunction with specific examples. It should be understood that the following specific implementation methods are only used to illustrate the present invention and are not used to limit the scope of the present invention.
[0047] Embodiment 1:
[0048] S1 and GPC3 aptamers were obtained by chemical synthesis, and were modified with cholesterol residues, phosphorothioate and Cy5 fluorescent probes to prepare the Cy5 fluorescent probe-modified aptamer GPC3-apt-Cy5.
[0049] S2. Then add sterile deionized water to dilute it to 100 pmol / μL aqueous solution. Take 20 μL of aptamer aqueous solution, add it to 1 mL of extracellular vesicle solution with a concentration of 1 mg / mL, blow evenly, and incubate at room temperature for 1 hour. Then centrifuge at 3000 rpm for 10 minutes in a 30Kda ultrafiltration tube to remove free aptamer solution.
[0050] S3, and then eluted twice with 500 μL PBS solution to prepare aptamer-modified liver cancer-targeted extracellular vesicles GPC3-apt-Cy5, which were stored at -80°C for subsequent detection and experiments.
[0051] Embodiment 2:
[0052] The only difference from Example 1 is that the GPC3 aptamer is not linked to the Cy5 fluorescent probe.
[0053] Embodiment 3:
[0054] S1 and GPC3 aptamers were obtained by chemical synthesis, and were modified with cholesterol residues, phosphorothioate and Cy5 fluorescent probes to prepare the Cy5 fluorescent probe-modified aptamer GPC3-apt-Cy5.
[0055] S2. Then add sterile deionized water to dilute it to 100pmol / μL aqueous solution. Take 20μL of aptamer aqueous solution, add it to 1mL of extracellular vesicle solution with a concentration of 1mg / mL, blow evenly, and incubate at room temperature for 1h. After incubation, dialyze it with a 30kDa dialysis bag for 24h. Thus, aptamer-modified liver cancer-targeted extracellular vesicles are prepared. Store at -80℃ for subsequent detection and experiments;
[0056] Embodiment 4:
[0057] The only difference from Example 3 is that the GPC3 aptamer is not linked to the Cy5 fluorescent probe.
[0058] Comparative Example 1:
[0059] The only difference from Example 1 is that the GPC3 aptamer is replaced by a random nucleotide sequence, but the two sequences contain the same number of four bases, and the nucleotide sequence is shown in SEQ ID NO.2.
[0060] Table 2: Random nucleotide sequences of Comparative Example 1
[0061] sequence 5` to 3` SEQ ID NO.2 Cy5-ACGAGCCCATCTGGAAGATGACCTCTCGTTTTTTCAT-Chol
[0062] The 3' and 5' ends of the random sequence each have three consecutive phosphodiester bonds modified by phosphorothioate starting from the first phosphodiester bond, the 3' end is connected to a cholesterol residue, and the 5' end is connected to a fluorescent group.
[0063] Figure 1 This is the observation of primary BALB / c mouse bone marrow mesenchymal stem cells under an optical microscope on the 14th day. All cells are long spindle-shaped and arranged in regular parallel and spiral shapes, which is the typical morphology of mouse BMSCs under an optical microscope, proving that the present invention successfully extracted primary BALB / c mouse bone marrow mesenchymal stem cells.
[0064] Figure 2 Flow cytometry detection showed that: progenitor cell surface specific antigen positive expression: Sca-1 (79.3%); stem cell surface specific antigen high expression: CD44 (99.55%), hematopoietic cell specific surface antigen low expression: CD45 (8.04%), CD11b (10.97%), endothelial cell specific surface antigen low expression: CD31 (4.06%). The above results are consistent with the cell phenotype of BMSCs, proving that the present invention extracts primary BALB / c mouse bone marrow mesenchymal stem cells.
[0065] Figure 3 Electron microscopic images show multiple double-membrane vesicles, which present a typical saucer-like structure or a hemispherical shape with a depression on one side, and a particle size of about 100 nm. This result is also consistent with the morphology and particle size of extracellular vesicles.
[0066] Figure 4 The immunofluorescence co-localization experiment showed that the red fluorescence signal of the extracellular vesicles modified with GPC3-apt-Cy5 prepared in Example 1 of the present invention was stronger than that of the extracellular vesicles modified with Random-Cy5 in Comparative Example 1, indicating that the former significantly improved the targeting to liver cancer cells.
[0067] Figure 5 The results of in vivo imaging of nude mice show that the GPC3-apt-Cy5 modified extracellular vesicles prepared in Example 1 of the present invention are significantly enriched in liver cancer tissues, which shows that this type of extracellular vesicles still has a high liver cancer targeting ability in vivo.
[0068] In summary, it can be proved that the present invention successfully obtains primary BALB / c mouse bone marrow mesenchymal stem cells and extracts extracellular vesicles derived therefrom. After modification with GPC3 aptamers, the extracellular vesicles can improve their targeting to liver cancer cells in both in vivo and in vitro environments.
[0069] The parts of the embodiments herein that are not exhaustive of the midpoint values of the technical scope claimed for protection by the present invention and the new technical solutions formed by equivalent replacement of single or multiple technical features in the technical solutions of the embodiments are also within the scope claimed for protection by the present invention; at the same time, in all the listed or unlisted embodiments of the scheme of the present invention, each parameter in the same embodiment merely represents an example of its technical solution (i.e., a feasible solution), and there is no strict coordination and limitation relationship between the parameters, wherein the parameters can be replaced with each other without violating the axioms and the claims of the present invention, unless otherwise stated.
[0070] The technical means disclosed in the scheme of the present invention are not limited to the technical means disclosed in the above technical means, but also include technical schemes composed of any combination of the above technical features. The above is a specific implementation of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications are also regarded as the protection scope of the present invention.
[0071] The specific embodiments described herein are merely examples of the spirit of the present invention. Those skilled in the art may make various modifications or additions to the specific embodiments described or replace them in similar ways, but they will not deviate from the spirit of the present invention or exceed the scope defined by the appended claims.
Claims
1. A liver cancer-targeted extracellular vesicle, characterized in that: The extracellular vesicles are obtained from bone marrow mesenchymal stem cells and then modified with GPC3 aptamers.
2. The liver cancer-targeting extracellular vesicle according to claim 1, characterized in that: The bone marrow mesenchymal stem cells are bone marrow mesenchymal stem cells derived from BALB / c mice.
3. The liver cancer-targeting extracellular vesicle according to claim 1, characterized in that: The GPC3 aptamer can bind to the GPC3 receptor highly expressed on the surface of liver cancer cells with high affinity and strong specificity.
4. The liver cancer-targeting extracellular vesicle according to claim 1 or 3, characterized in that: The nucleotide sequence of the GPC3 aptamer is shown in SEQ ID NO.
1.
5. The liver cancer-targeting extracellular vesicle according to claim 4, characterized in that: The 3' end and the 5' end of the GPC3 aptamer each have three consecutive phosphodiester bonds modified by phosphorothioate.
6. The liver cancer-targeting extracellular vesicle according to claim 4, characterized in that: The 3' end of the GPC3 aptamer is connected to a cholesterol residue, and the 5' end is connected to a fluorescent group.
7. A method for preparing liver cancer-targeted extracellular vesicles as claimed in claim 1, characterized in that: The method comprises the following steps: adding extracellular vesicles and GPC3 aptamers into a PBS solution, incubating at room temperature, and then removing impurities to obtain targeted extracellular vesicles.
8. The method for preparing liver cancer-targeted extracellular vesicles according to claim 7, characterized in that: The amount of the aptamer in each 1 mg of the extracellular vesicle solution is 2-5 nmol based on the protein content of the extracellular vesicles.
9. The method for preparing liver cancer-targeted extracellular vesicles according to claim 7, characterized in that: Impurities are removed by dialysis or ultrafiltration.
10. Use of the liver cancer-targeted extracellular vesicles according to claim 1 in the preparation of nucleic acid drugs, small molecule targeted drugs or chemotherapy drugs.