Loaded vesicles, methods of making, kits, pharmaceuticals and uses thereof

Abstract

The present application relates to a kind of loaded vesicle and its preparation method, kit, medicine and purposes.Loaded vesicle includes DNA modified nanoparticle, vesicle layer, the vesicle layer is coated in the outside of the DNA modified nanoparticle, antisense DNA, the antisense DNA is loaded on the nanoparticle and / or the vesicle layer;Preparation method includes: DNA modified nanoparticle and vesicle layer are assembled, obtain frame, antisense DNA is mixed with the frame and is reacted, so that the antisense DNA is loaded on the DNA modified nanoparticle or the vesicle layer, and loaded vesicle is prepared.After the loaded vesicle enters cell, it can release antisense DNA, it can target after mRNA complementary combination, inhibit mRNA translation, so as to promote cancer cell apoptosis, with the advantages such as strong stability and controllable release antisense DNA;And, the preparation method is simple to operate.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to a loaded vesicle and a preparation method, a kit, a medicine and uses thereof. Background Art

[0002] In recent years, a variety of anticancer drugs, including small molecule chemotherapeutics, have been developed. However, traditional chemotherapeutics carry serious side effects, including systemic toxicity and multidrug resistance (MDR). The emergence of nucleic acid-based therapeutics offers an alternative anticancer treatment that helps improve the side effects of small molecule therapeutics.

[0003] Therefore, there is an urgent need to provide a stable and effective nucleic acid drug. Summary of the Invention

[0004] The present invention aims to, at least to some extent, address at least one of the technical problems existing in the prior art. To this end, the present invention provides a loaded vesicle, a preparation method, a kit, a pharmaceutical, and uses thereof. The loaded vesicle is loaded with antisense DNA, which, upon entry into cells, can promote apoptosis of cancer cells. The loaded vesicle exhibits advantages such as high stability and controlled release of antisense DNA. Furthermore, the preparation method has advantages such as ease of operation.

[0005] It should be noted that the present invention is completed based on the following work of the inventors:

[0006] Antisense oligonucleotides are mainly composed of 15-25 nucleotides, including antisense DNA and antisense RNA. The chemotherapeutic mechanism of antisense DNA provides a genetic level approach. Through the rational design of highly sequence-specific nucleic acid drugs, it can target and bind to messenger RNA (mRNA), thereby blocking mRNA translation through space. However, the inventors found that antisense DNA has a large molecular weight and carries a negative charge. It is difficult to penetrate the cell membrane without an artificial carrier. In addition, traditional antisense DNA is easily degraded by enzymes in the intracellular environment, which greatly reduces its effect of inhibiting mRNA translation.

[0007] In view of this, the inventors discovered through a large number of experiments that by using poly(propylene) glycol (PPO) chemical conjugation and reducible disulfide bonds to modify antisense DNA, and then loading it onto a vesicle layer or nanoparticles, the antisense DNA can smoothly pass through the cell membrane and enter the cell, inhibiting mRNA translation and achieving the purpose of promoting cancer cell apoptosis. Moreover, the loaded vesicles contain antisense DNA and have the advantages of strong stability and controllable release of antisense DNA.

[0008] Thus, in one aspect of the present application, the present application provides a loaded vesicle. According to an embodiment of the present application, the loaded vesicle comprises: a DNA-modified nanoparticle; a vesicle layer, the vesicle layer being coated outside the DNA-modified nanoparticle; and antisense DNA, the antisense DNA being loaded on the nanoparticle and / or the vesicle layer.

[0009] The inventors have found through a large number of experiments that the loaded vesicle contains antisense DNA to improve the targeting effect thereof. Specifically, after the loaded vesicle enters a cell, it can be disassembled in an intracellular reducing environment to release antisense DNA, which, after targeted and complementary combined with mRNA, inhibits the translation of mRNA under the action of RNAse H enzyme, thereby promoting the apoptosis of cancer cells; and the loaded vesicle containing antisense DNA has the advantages of strong stability and controllable release of antisense DNA.

[0010] According to an embodiment of the present application, the above-mentioned vesicle further comprises at least one of the following additional technical features:

[0011] According to an embodiment of the present application, the vesicle layer comprises assembly DNA, at least a part of the assembly DNA being complementary to at least a part of the modified DNA on the nanoparticle. Thus, by making at least a part of the assembly DNA complementary to at least a part of the modified DNA on the nanoparticle, the assembly DNA and the modified DNA are complementary combined, thereby realizing the assembly of the vesicle layer and the DNA-modified nanoparticle into the framework of the loaded vesicle.

[0012] According to an embodiment of the present application, the assembly DNA is PPO-S-S-b-DNA or PPO-S-S-b-DNA. Thus, the redox-responsive disulfide bond is introduced by using PPO-S-S-b-DNA, and the responsive loaded vesicle is obtained by using the strategy of framework-induced self-assembly in the later stage.

[0013] According to an embodiment of the present application, the nanoparticle is a gold nanoparticle, and the modified DNA is thiol DNA. Thus, the thiol DNA is linked to the nanoparticle by Au-S bond to form the DNA-modified nanoparticle.

[0014] According to an embodiment of the present application, the diameter of the gold nanoparticle is 10-15 nm.

[0015] According to an embodiment of the present invention, the nanoparticles are gold nanoparticles protected by dihydrated bis(p-sulfonylphenyl)phenylphosphine dipotassium salt. Thus, the nanoparticles are protected by dihydrated bis(p-sulfonylphenyl)phenylphosphine dipotassium salt (BSPP), so that the gold nanoparticles can be stably present and not easy to precipitate. According to an embodiment of the present invention, the sequence of the antisense DNA is the same as that of the assembly DNA. The inventors have found through a large number of experiments that when the sequence of the antisense DNA is the same as that of the assembly DNA, when the loaded vesicle enters the cell, the DNA sequence on the assembly DNA is released, which can target and complementarily bind to mRNA, inhibit mRNA translation, and further promote cancer cell apoptosis.

[0016] According to an embodiment of the present invention, the antisense DNA and the assembled DNA have the nucleotide sequence shown in SEQ ID NO:1, or a nucleotide sequence with at least 80% homology thereto. The inventors have discovered through extensive experiments that this antisense DNA sequence can inhibit the translation of Bcl-2 mRNA, thereby promoting apoptosis in breast cancer cells. SEQ ID NO:1 is TCTCCCAGCGTGCGCCAT.

[0017] According to an embodiment of the present invention, the modified DNA has a nucleotide sequence as shown in SEQ ID NO: 2 or a nucleotide sequence having at least 80% homology thereto. Thus, a portion of the modified DNA sequence is complementary to a portion of the assembled DNA sequence, thereby complementary binding of the DNA-modified gold nanoparticles to the assembled DNA to form a framework. SEQ ID NO: 2 is TTTATGGCGCACGCTGGG.

[0018] According to an embodiment of the present invention, the antisense DNA is PPO-SSb-DNA or PPO-SS-DNA. The inventors have found through experiments that chemical conjugation with PPO and modification of the antisense DNA with a reducible disulfide bond can improve the stability of the antisense DNA while also enabling controlled release of the antisense DNA.

[0019] According to an embodiment of the present invention, the diameter of the loaded vesicle is 30-50 nm. Thus, the loaded vesicle contains antisense DNA, has advantages such as strong stability and controllable release of antisense DNA, and can further promote cancer cell apoptosis.

[0020] In another aspect, the present invention provides a method for preparing the aforementioned loaded vesicles. According to an embodiment of the present invention, the method comprises: (1) assembling DNA-modified nanoparticles and a vesicle layer to obtain a framework; and (2) mixing antisense DNA with the framework to react, so that the antisense DNA is loaded onto the DNA-modified nanoparticles or the vesicle layer to obtain the loaded vesicles.

[0021] According to an embodiment of the present invention, DNA-modified nanoparticles and a vesicle layer are first assembled to form a framework, and then antisense DNA is added, so that the antisense DNA forms loaded vesicles around the framework through hydrophobic forces. This preparation method can effectively load antisense DNA onto the framework, is simple to operate, and the resulting loaded vesicles are highly stable.

[0022] According to an embodiment of the present invention, in step (2), the antisense DNA and the framework are reacted at 25-40°C for 30-60 minutes. The inventors have found through extensive experiments that the above temperature can convert the hydrophilic framework into a hydrophobic framework. Under the induction of the hydrophobic groups, the antisense DNA can form loaded vesicles around the framework through hydrophobic forces.

[0023] According to an embodiment of the present invention, the molar ratio of the antisense DNA to the framework is 600-700: 1, preferably 660-670: 1. The inventors have found through experiments that the above ratio can fully load the antisense DNA onto the framework, thereby increasing the yield of loaded vesicles.

[0024] According to an embodiment of the present invention, the preparation steps of the DNA-modified nanoparticles include: adding thiol DNA and gold nanoparticles to a buffer solution and letting it stand for 12-24 hours, then adding NaCl to a final concentration of 400-600mM, and continuing the reaction to obtain a crude solution containing DNA-modified nanoparticles. The inventors found through experiments that standing for 6-12 hours can fully combine the thiol DNA and gold nanoparticles to form Au-S bonds; by adding NaCl to a final concentration of 400-600mM, the charges can be shielded, the structure of the chains can be stabilized, the repulsive forces between each other can be reduced, and the DNA can be more easily attached to the gold particles. The inventors found through experiments that if the final concentration of NaCl is too low, the number of DNA links attached to the gold particles will be reduced due to inadequate shielding; if the final concentration of NaCl is too high, it will cause coagulation due to excessive ionic strength.

[0025] According to an embodiment of the present invention, the molar amount of the thiol DNA is 150-300 times the molar amount of the gold nanoparticles, preferably 200 times. The above ratio can fully combine the thiol DNA and the gold nanoparticles, thereby improving the utilization rate of the raw materials.

[0026] According to an embodiment of the present invention, the buffer solution comprises 0.4-0.5×TBE and 30-60 mM NaCl, and the pH value of the buffer solution is 8-9. Thus, the use of the above buffer solution can improve the efficiency of preparing loaded vesicles.

[0027] According to an embodiment of the present application, the concentration of NaCl is gradually increased to 400-600 mM within 4-6 hours. The inventors have found through experiments that if NaCl is added quickly, aggregation is likely to occur; however, if NaCl is gradually added, aggregation is prevented because the ion strength is not too high at any moment.

[0028] According to an embodiment of the present application, the crude solution containing the DNA-modified nanoparticles is subjected to first centrifugation and first redissolution in sequence to obtain the DNA-modified nanoparticles. In this way, the crude solution containing the DNA-modified nanoparticles is subjected to first centrifugation and first redissolution to obtain DNA-modified nanoparticles with higher purity.

[0029] According to an embodiment of the present application, the first centrifugation is performed at 8000-12000 rpm for 25-35 min. In this way, the DNA-modified nanoparticles are precipitated, and the supernatant containing impurities is removed.

[0030] According to an embodiment of the present application, the first centrifugation is repeated more than twice, preferably twice. In this way, impurities are further removed.

[0031] According to an embodiment of the present application, the precipitate obtained after the first centrifugation is subjected to the first redissolution in a first redissolution solution. In this way, a DNA-modified nanoparticle solution with higher purity is obtained.

[0032] According to an embodiment of the present application, the first redissolution solution comprises 0.4-0.5×TBE and 30-60 mM NaCl, and the pH value of the first redissolution solution is 8-9. In this way, the DNA-modified nanoparticles can be stably stored in the first redissolution solution.

[0033] According to an embodiment of the present application, in step (1), the DNA-modified nanoparticles are mixed with the assembled DNA, and are allowed to stand at 3-5 °C for 12-24 hours to obtain a crude solution containing a frame. The inventors have found through a large number of experiments that under the above conditions, the modified DNA and the assembled DNA are sufficiently complementary to form a frame.

[0034] According to an embodiment of the present application, the crude solution containing the frame is subjected to second centrifugation and second redissolution in sequence to obtain the frame. In this way, the crude solution containing the frame is subjected to second centrifugation and second redissolution to obtain a frame with higher purity.

[0035] According to an embodiment of the present application, the second centrifugation is performed at 8000-12000 rpm for 25-35 min. In this way, the frame is sufficiently precipitated, and impurities are removed.

[0036] According to an embodiment of the present application, the second centrifugation is repeated more than twice, preferably three times. In this way, the assembled DNA that is not combined in the frame is removed as much as possible.

[0037] According to the embodiment of the present application, the second re-dissolution is performed on the precipitate obtained after the second centrifugation in a second re-dissolution solution. In this way, the framework solution with higher purity can be obtained.

[0038] According to the embodiment of the present application, the second re-dissolution solution comprises 0.4-0.5×TBE and 30-60mM NaCl, and the pH value of the second re-dissolution solution is 8-9. In this way, the framework can be stored stably in the second re-dissolution solution.

[0039] According to the embodiment of the present application, the molar amount of the assembled DNA is 800-900 times, preferably 850-870 times, of the molar amount of the DNA-modified nanoparticles. The inventor has found through experiments that the above-mentioned ratio can improve the yield of the framework.

[0040] In another aspect of the present application, a kit or a medicine is provided. According to the embodiment of the present application, the kit or the medicine comprises the above-mentioned loaded vesicle or the loaded vesicle prepared by the above-mentioned method.

[0041] According to the embodiment of the present application, after the kit or the medicine comprising the above-mentioned loaded vesicle is co-incubated with cells, the loaded vesicle is reduced by the reduced glutathione (GSH) in the cells, the loaded vesicle starts to disassemble, the antisense DNA is released, and the mRNA antisense is inhibited after the antisense DNA is complementary to the mRNA, so as to achieve the purpose of promoting the apoptosis of the cancer cells. In the experiment, the glutathione used is at a concentration of 10mM in the cells, which also provides the basis for the effect of the loaded vesicle in the cancer cells.

[0042] According to the embodiment of the present application, the final concentration of the loaded vesicle is 0.5-4nM (the final concentration added in the cell solution), preferably 2-3nM. The inventor has found through a large number of experiments that when the concentration of the loaded vesicle is 0.5-3nM, the survival rate of the cancer cells is reduced to below 80%; and when the concentration of the loaded vesicle is 2-3nM, the survival rate of the cancer cells is reduced to below 60%.

[0043] In another aspect of the present application, the use of the above-mentioned loaded vesicle or the loaded vesicle prepared by the above-mentioned method or the above-mentioned kit or medicine in the preparation of a product for promoting the apoptosis of cancer cells is provided. According to the embodiment of the present application, after the loaded vesicle is co-incubated with cells, the loaded vesicle is reduced by the reduced glutathione (GSH) in the cells, the loaded vesicle starts to disassemble, the antisense DNA is released, and the mRNA antisense is inhibited after the antisense DNA is complementary to the mRNA, so as to achieve the purpose of promoting the apoptosis of the cancer cells.

[0044] According to an embodiment of the present invention, the cancer cells include breast cancer cells, lung adenocarcinoma cells, human glioma cells, human prostate cells, and human cervical cancer cells. Therefore, the antisense DNA of the present invention is applicable to the above cancer cells.

[0045] Additional aspects and advantages of the present invention will be set forth in part in the description which follows and, in part, will be obvious from the description which follows, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the following description of the embodiments with reference to the accompanying drawings, in which:

[0047] Figure 1 The DLS and TEM images of the assembly process of the antisense DNA-loaded vesicles in Example 1 show the formation of the antisense DNA-loaded vesicles and the successful loading of the antisense DNA onto the vesicles.

[0048] Figure 2 This is the DLS graph of the antisense DNA-loaded vesicles after treatment with 10 mM GSH in Example 1;

[0049] Figure 3 This is a graph showing the cell viability of the blank group, experimental group, and control group in Example 3 detected using MTS reagent at different times;

[0050] Figure 4 This is a diagram showing the cell survival status of the blank group and the experimental group in Example 4, respectively, using Calcein-AM / PI staining to mark the cell survival status;

[0051] Figure 5 For example 5, the blank group, experimental group and control group were respectively Flow cytometric analysis of apoptosis rates of cells labeled with Fluor 488Annexin V-FITC and PI. DETAILED DESCRIPTION

[0052] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be understood as limiting the present invention.

[0053] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood to indicate or imply relative importance or implicitly specify the number of the technical features indicated. Therefore, features defined as "first" or "second" may explicitly or implicitly include one or more of such features. Furthermore, in the description of the present invention, unless otherwise specified, "plurality" means two or more.

[0054] If no specific techniques or conditions are specified in the examples, the experiments were carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. All reagents or instruments used without specifying the manufacturer are commercially available conventional products.

[0055] Experimental Materials

[0056] polypropylene oxide (PPO) with a number-average molecular weight of 2000 (Sigma-Aldrich, Merck, USA);

[0057] thiol monomer (GenePharma, Suzhou GenePharma Co., Ltd., China);

[0058] The cell culture medium was high-glucose Dulbecco's modified eagle medium (DMEM) (Gibco, Thermo Fisher Scientific, USA);

[0059] fetal bovine serum (Gibco, Thermo Fisher Scientific, USA);

[0060] MTS Reagent CellTiter AQueous One Solution Cell Proliferation Assay (MTS) (Promega, USA);

[0061] The cell live-death dye kit was Calcein-AM / PI (Solarbio, Solarbio, Beijing);

[0062] The cell apoptosis kit was Dead Cell Apoptosis Kit with Annexin V FITC and PI, for flow cytometry (ThermoFisher, Thermo Fisher Scientific, USA);

[0063] Laser scanning confocal microscope (LSM 710Met, Zeiss, Germany);

[0064] Dynamic light scattering was performed using a Zetasizer Nano-ZS (Malvern, UK);

[0065] flow cytometer (ACEA Biosciences, Aeson Biosciences, USA);

[0066] The DNA sequence was synthesized using the standard phosphoramidite DNA solid phase synthesis method (Mermade-12 DNA synthesizer, BioAutomation, USA). The specific sequence is shown in Table 1. The DNA and PPO block copolymer was synthesized by solid phase synthesis and purified by 20% Denature PAGE.

[0067] Table 1: DNA sequences

[0068] sequence name Absorption coefficient (L / mol·cm) Mw(g / mol) Sequence information (5'-3') D6 (6nt) 49200 1763 TTTTTT D18 (18nt) 164800 5547 TTTATGGCGCACGCTGGG Z18 (18nt) 176300 5489 TTTTAATCCAGTAGTGAC S18 (18nt) 156000 5412 TCTCCCAGCGTGCGCCAT C18 (18nt) 179100 5514 TAGGTCACTACTGGATTA

[0069] A disulfide bond monomer was added to the 5' end of the D6, D18, Z18, S18, and C18 sequences used, among which S18 was antisense DNA.

[0070] D6, D18, and S18 sequences were used to form antisense DNA-loaded vesicles (also known as antisense FGA vesicles), and D6, Z18, and C18 sequences were used to form random DNA-loaded vesicles (also known as random FGA vesicles).

[0071] Example 1

[0072] 1. Preparation of antisense DNA-loaded vesicles

[0073] 13nm gold nanoparticles were mixed with D6 and D18 in a 0.5×TBE and 50mM NaCl mixed solution (pH 8.3) to a final volume of 20 μL. The final concentrations of the gold nanoparticles, D6, and D18 in the mixed solution were 60nM, 12μM, and 12μM, respectively. After overnight reaction (the overnight time in this application is 12-24 hours, the same below), 4M NaCl was added, and the concentration of NaCl was gradually increased to 500mM within 5 hours. The mixture was then centrifuged at 10,000 rpm for 30 minutes to remove the supernatant. The centrifugal removal supernatant step was repeated twice, and the precipitate was collected. The red precipitate was redissolved in 20 μL of a 0.5×TBE, 50mM NaCl mixed solution (pH 8.3) to obtain DNA-modified gold nanoparticles.

[0074] The DNA-modified gold nanoparticles (DNA-AuNPs) obtained above (60 nM, 10 μL) were mixed with the redox-responsive amphiphilic molecule S18-SSb-PPO. The mixture was then incubated at 4°C for 12-24 hours to allow for complete base pairing. The mixture was then centrifuged at 10,000 rpm for 30 minutes, the supernatant removed, and the precipitate dissolved in a mixture of 0.5× TBE and 50 mM NaCl. This process was repeated three times to remove as much excess S18-SSb-PPO as possible. Finally, the framework was dissolved in 20 μL of a mixture of 0.5× TBE and 50 mM NaCl and stored at 4°C until ready for use.

[0075] Then, S18-SSb-PPO (10 μM, 40 μL, buffer solution: 0.5×TBE and 50 mM NaCl) was added to the framework and heated to 37°C for 30 min to make PPO hydrophobic. After 30 min, it was naturally cooled to room temperature and placed for 12-24 h. The formation of vesicles can be observed by transmission electron microscopy. For details, see Figure 1 .

[0076] 2. Controlled release test of antisense DNA-loaded vesicles

[0077] Take 60nM of the antisense DNA-loaded vesicles formed above and add them to 10mM reduced glutathione (GSH). Add buffer (1×PBS) to a final volume of 10μL. After standing for 12-24h, use dynamic light scattering to detect the change in particle size. For details, see Figure 2 .

[0078] Figure 2 The experimental results showed that the size of the antisense DNA-loaded vesicle system changed from 43nm to 28nm. This size distribution is consistent with the previously reported system, proving that GSH destroys the antisense DNA-loaded vesicles. It should be noted that the size of the disassembly system (referring to the substance generated after the antisense nucleic acid-loaded vesicles are added with GSH) is different from that of DNA-AuNPs, which can be explained by the anchoring of AuNPs by double-stranded DNA. These results indicate that the prepared antisense DNA-loaded vesicles have good controllable disassembly properties, which can promote the release of antisense DNA and provide an efficient delivery system.

[0079] Example 2: Random DNA-loaded vesicles

[0080] The preparation method is the same as that of Example 1, except that the modified DNA is D6 and Z18, and the assembly DNA and antisense DNA are both C18 sequences, namely C18-SSb-PPO.

[0081] Example 3: Cell viability test after incubation of cells with antisense DNA-loaded vesicles

[0082] MCF-7 cells were selected. After recovery, they were cultured in DMEM medium for two passages. When they reached 75-80% growth, the DMEM medium was removed, and the cells were washed once with PBS (pH 7.4). After digestion with 0.25% trypsin, the cells were counted using a cell counter, and 1500 cells were plated per well of a 96-well plate and incubated overnight. MCF-7 cells were divided into five groups: 1. Blank group: MCF-7 cells alone; 2. Experimental group 1: 0.8 nM antisense DNA-loaded vesicles; 3. Experimental group 2: 2.5 nM antisense DNA-loaded vesicles; 4. Control group 1: 0.8 nM random DNA-loaded vesicles; 5. Control group 2: 2.5 nM random DNA-loaded vesicles. Each group had 5 parallel samples, the total volume of the culture medium was 100 μL, the day of addition was marked as time 0, 20 μL of MTS reagent was added after incubation for 24 h, 48 h, and 72 h, and the absorbance at 490 nm was recorded using a microplate reader after incubation for 1 h. Figure 3 .

[0083] The experimental results showed that after three days of incubation, the cell viability of control group 1 was 92.8%, comparable to that of the blank group. In contrast, the cell viability of experimental group 1 was 79.2%, a significant decrease compared to both control group 1 and the blank group, indicating that the antisense DNA-loaded vesicles indeed delivered the antisense DNA and promoted cancer cell apoptosis.

[0084] To verify whether the therapeutic effect was concentration-dependent, the concentration of random DNA-loaded vesicles was increased. The results showed that the survival rate of control group 2 was similar to that of the blank group, consistent with the above experimental results, indicating that even at higher concentrations, the vesicle carriers maintained good biocompatibility.

[0085] Experimental Group 2 showed that cell viability decreased to 59.8% after 3 days, demonstrating a superior apoptosis-promoting effect compared to Experimental Group 1, Control Groups 1-2, and the blank control group. These experimental results can be interpreted as the fact that delivery of antisense DNA-loaded vesicles inhibited Bcl-2 mRNA translation, ultimately promoting cancer cell apoptosis. These results demonstrate that antisense DNA delivery by antisense DNA-loaded vesicles is concentration-dependent, with increasing concentrations effectively promoting apoptosis. Furthermore, the delivery vector itself has low cytotoxicity, making it a promising antisense DNA delivery vehicle.

[0086] Example 4: Live-dead staining experiment after incubation of cells with antisense DNA-loaded vesicles

[0087] When the MCF-7 cells grow to 75-80%, the DMEM medium of the MCF-7 cells is removed, and the cells are washed once with PBS (pH 7.4), then digested with 0.25% trypsin, counted by a cell counter, and seeded into 96-well plates at 1500 cells per well, and incubated overnight. The DMEM medium is removed, and the antisense DNA-loaded vesicles are added to each group, and the groups are as follows: 1, blank group: MCF-7 cells; 2, experimental group: antisense DNA-loaded vesicles at a concentration of 2.5 nM. Each group is in DMEM medium, and the total volume is 100 μL. After incubation overnight, the cells are treated according to the procedure in the Calcein AM / PI kit (Solebo, China), and then imaged by laser scanning confocal microscopy. See Figure 4 .

[0088] The experimental results show that in the blank group, most of the cells are stained green, representing that most of the cells are living cells. In the experimental group, some of the cells are labeled by red fluorescence, indicating that after treatment with the antisense DNA-loaded vesicles, some cells die, which is consistent with the MTS analysis results, further supporting the successful delivery of the antisense DNA-loaded vesicles prepared in Example 1.

[0089] Example 5: Flow cytometry experiment after incubation of antisense DNA-loaded vesicles with cells

[0090] When the MCF-7 cells grow to 75-80%, the DMEM medium of the MCF-7 cells is removed, and the cells are washed once with PBS (pH 7.4), then digested with 0.25% trypsin, counted by a cell counter, and seeded into 96-well plates at 1500 cells per well, and incubated overnight. The DMEM medium is removed, and the antisense DNA-loaded vesicles are added to each group, and the groups are as follows: 1, blank group: MCF-7 cells; 2, experimental group: antisense DNA-loaded vesicles at a concentration of 2.5 nM. Each group is in DMEM medium, and the total volume is 100 μL. After incubation overnight, the cells are treated according to the procedure in the Calcein AM / PI kit (Solebo, China), and then imaged by laser scanning confocal microscopy. See 4 Figure 5 .

[0091] ​Under flow cytometry, AV fluorescence intensity was high and PI fluorescence intensity was low, defining the Q2-2 region as late apoptotic cells. Q2-1 represented necrotic cells, Q2-3 represented live cells, and Q2-4 represented early apoptotic cells. The blank group had 10.54% apoptotic cells in the Q2-2 region, similar to the 10.42% apoptotic cells in the control group. In the experimental group, 20.31% of cells were apoptotic. These results demonstrate that antisense DNA-loaded vesicles can deliver antisense DNA, thereby downregulating the expression of the anti-apoptotic Bcl-2 mRNA. These results also support cell proliferation and live / dead staining assays.

[0092] In the description of this specification, the reference terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

[0093] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. A loaded vesicle, characterized in that Include: DNA-modified nanoparticles; A vesicle layer, wherein the vesicle layer is coated on the outside of the DNA-modified nanoparticles; Antisense DNA, wherein the antisense DNA is loaded on the nanoparticles and / or the vesicle layer; The vesicle layer includes assembly DNA, at least a portion of which is complementary to at least a portion of the modified DNA on the nanoparticle; The assembled DNA is PPO-SSb-DNA or PPO-SS-DNA; The nanoparticles are gold nanoparticles, and the modified DNA is thiol DNA; The antisense DNA and the assembly DNA have the nucleotide sequence shown in SEQ ID NO: 1; The modified DNA has a nucleotide sequence as shown in SEQ ID NO: 2; The antisense DNA is PPO-SSb-DNA or PPO-SS-DNA.

2. The loaded vesicle according to claim 1, characterized in that The nanoparticles are gold nanoparticles protected by dipotassium dihydrate bis(p-sulfonylphenyl)phenylphosphine.

3. The loaded vesicle according to claim 2, characterized in that The diameter of the loaded vesicles is 30-50 nm.

4. A method for preparing the loaded vesicle according to any one of claims 1 to 3, characterized in that: Include: (1) Assembling DNA-modified nanoparticles and vesicle layers to obtain a framework; (2) mixing the antisense DNA with the framework to react so that the antisense DNA is loaded on the DNA-modified nanoparticles or the vesicle layer to prepare the loaded vesicle; The steps for preparing the DNA-modified nanoparticles include: Add the thiol DNA and gold nanoparticles to a buffer solution and let it stand for 12-24 hours, then add NaCl to a final concentration of 400-600 mM and continue the reaction to obtain a crude solution containing DNA-modified nanoparticles; The crude solution containing the DNA-modified nanoparticles is subjected to a first centrifugation and a first redissolution in sequence to obtain the DNA-modified nanoparticles; In step (1), the DNA-modified nanoparticles are mixed with the assembled DNA and allowed to stand at 3-5°C for 12-24 hours to obtain a crude solution containing the framework; The crude solution containing the framework is subjected to a second centrifugation and a second redissolution in sequence to obtain the framework.

5. The method according to claim 4, characterized in that In step (2), the antisense DNA reacts with the framework at 25-40° C. for 15-60 min.

6. The method according to claim 4, characterized in that The molar ratio of the antisense DNA to the framework is 600-1000:

1.

7. The method according to claim 4, characterized in that The molar ratio of the antisense DNA to the framework was 660-670:

1.

8. The method according to claim 4, characterized in that The molar amount of the thiol DNA is 100-400 times the molar amount of the gold nanoparticles.

9. The method according to claim 4, characterized in that The molar amount of the thiol DNA is 200 times the molar amount of the gold nanoparticles.

10. The method according to claim 4, characterized in that The buffer comprises 0.4-0.5×TBE and 30-60 mM NaCl, and the pH value of the buffer is 8-9.

11. The method according to claim 4, characterized in that The NaCl concentration was gradually increased to 400-600 mM within 4-6 h.

12. The method according to any one of claims 4 to 11, characterized in that: The first centrifugation is performed at 8000-12000 rpm for 25-35 min.

13. The method according to any one of claims 4 to 11, characterized in that: The first centrifugation is repeated two or more times.

14. The method according to any one of claims 4 to 11, characterized in that: The first centrifugation was repeated twice.

15. The method according to any one of claims 4 to 11, characterized in that: The precipitate obtained after the first centrifugation is placed in the first reconstitution solution for the first reconstitution.

16. The method according to claim 15, characterized in that The first reconstitution solution contains 0.4-0.5×TBE and 30-60 mM NaCl, and the pH value of the first reconstitution solution is 8-9.

17. The method according to any one of claims 4 to 11, characterized in that: The second centrifugation is performed at 8000-12000 rpm for 25-35 min.

18. The method according to any one of claims 4 to 11, characterized in that: The second centrifugation was repeated two or more times.

19. The method according to any one of claims 4 to 11, characterized in that: The second centrifugation was repeated 3 times.

20. The method according to any one of claims 4 to 11, characterized in that The precipitate obtained after the second centrifugation is placed in the second reconstitution solution for the second reconstitution.

21. The method according to claim 20, characterized in that The second reconstitution solution contains 0.4-0.5×TBE and 30-60 mM NaCl, and the pH value of the second reconstitution solution is 8-9.

22. The method according to any one of claims 4 to 11, characterized in that The molar amount of the assembled DNA is 800-900 times the molar amount of the DNA-modified nanoparticles.

23. The method according to any one of claims 4 to 11, characterized in that The molar amount of the assembled DNA is 850-870 times the molar amount of the DNA-modified nanoparticles.

24. A kit or medicine, characterized in that: The invention comprises the loaded vesicles according to any one of claims 1 to 3 or the loaded vesicles prepared by the method according to any one of claims 4 to 23.

25. The kit or medicine according to claim 24, characterized in that The final concentration of the loaded vesicles was 0.5-4 nM.

26. The kit or medicine according to claim 24, characterized in that The final concentration of the loaded vesicles was 2-3 nM.

27. Use of the loaded vesicle according to any one of claims 1 to 3, or the loaded vesicle prepared by the method according to any one of claims 4 to 23, or the kit or drug according to any one of claims 24 to 26 in preparing a product for promoting apoptosis of cancer cells.

28. The use according to claim 27, characterized in that The cancer cells include breast cancer cells, lung adenocarcinoma cells, human brain glioma cells, human prostate cells, and human cervical cancer cells.

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