A GD2-targeted spherical nucleic acid nanoparticle and its preparation method and application

Abstract

The present invention provides a spherical nucleic acid nanoparticle targeting GD2 and its preparation method and application. The preparation method comprises: using siRNA nucleic acid chain, nucleic acid aptamer nucleic acid chain and small molecule inhibitor NVP-CGM097 to self-assemble to obtain spherical nucleic acid nanoparticle S-AGsM7; wherein the sequences of the sense chain and antisense chain of the siRNA nucleic acid chain are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively, and the sequence of the nucleic acid aptamer nucleic acid chain is shown in SEQ ID NO.3. The spherical nucleic acid nanoparticles using the technical solution of the present invention can accurately target retinoblastoma cell lines Y79 and WERI-RB1, deliver siRNA-MDM2 and small molecule inhibitor NVP-CGM097 into cells, block the interaction between MDM2 and p53, inhibit the degradation of p53, and thus inhibit RB proliferation.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a GD2-targeted spherical nucleic acid nanoparticle and a preparation method and application thereof. Background Art

[0002] Retinoblastoma (RB), a rare disease, is the most common intraocular malignancy in children. The diagnosis of RB primarily relies on imaging and specialized ophthalmological examinations. In recent years, with the widespread use of genetic testing, it has become a new diagnostic tool for clinicians. Traditional treatments for RB include surgery (enucleation), chemotherapy, radiotherapy, and local therapies. In recent years, gene therapy and vitrectomy have also been introduced.

[0003] p53 is a tumor suppressor gene located on chromosome 17p13-17, encoding a 53kD nuclear phosphoprotein. This protein participates in DNA replication and repair, acts as a transcriptional regulator, and can induce apoptosis upon demand. MDM2 is an oncogene, amplified in many tumors, particularly malignancies such as sarcomas. The MDM2 oncogene is located at 12q13-14, and multiple experiments have demonstrated its ability to transform cells in vitro and to be tumorigenic in animals. Studies have shown that MDM2 functions in tumors by binding to p53. Therefore, reducing MDM2 activity and blocking the p53-MDM2 interaction can inhibit the proliferation, invasion, and migration of RB.

[0004] Research has found that apoptosis caused by loss of Rb gene function can be mediated by p53. p53 transcriptionally activates WAF1 (a p53-related cell cycle gene), thereby inhibiting cell cycle progression, suggesting that p53 plays a regulatory role in retinoblastoma. MDM2 (mouse double microsome 2) is amplified and overexpressed in human tumors, suggesting that it promotes tumor formation and progression.

[0005] MDM2 has multiple functions, a primary one being its interaction with wild-type or mutant p53 proteins. Under the induction of the wild-type p53 gene, MDM2 can enhance transcription, leading to elevated MDM2 protein levels. Conversely, MDM2 binds to p53 to form a complex, promoting p53 degradation and inhibiting its function. Thus, p53 and MDM2 form a self-feedback regulatory loop. This regulation maintains a balance between p53 and MDM2 within the cell, facilitating DNA repair after damage while preventing cell growth stagnation after repair. Maintaining and regulating this balance depends on the presence (free or bound) and amounts of p53 and MDM2 proteins. Overexpression of MDM2 blocks p53 transcriptional activation, thereby inhibiting its function, similar to the effects of p53 mutations. Furthermore, MDM2 can bind to mutant p53 proteins, stabilizing them. Certain mutant p53 proteins may also activate or enhance MDM2 gene expression or MDM2 protein function. Data indicate that cells expressing mutant p53 protein are more tumorigenic, and MDM2 can also play a role in tumor development and progression in a p53-independent manner. Recent research indicates that a novel enzymatic pathway is involved in the regulatory pathway between MDM2 and p53, suggesting that the interaction between the two is not a single pathway. Studies have shown that the development and progression of retinoblastoma is the result of abnormal changes in multiple oncogenes and tumor suppressor genes, particularly MDM2 gene amplification or overexpression and p53 gene mutation, which play a significant role.

[0006] Ganglioside GD2 is a tumor-associated surface antigen widely present in human cancers and stem cells. It is abnormally expressed in childhood embryonic tumors (neuroblastoma, retinoblastoma, etc.). Therefore, GD2 has been proven to be a safe antibody target.

[0007] To date, there have been few studies and reports on biological targeted therapies for RB. Faced with the current status of RB treatment in my country and in response to the global call for "individualized treatment" and "precision treatment" strategies, the development of highly effective and low-toxic specific targeted drugs has become a key issue that needs to be urgently addressed in the clinical treatment of RB and saving patients' eyes and even their lives. Summary of the Invention

[0008] In response to the above technical problems, the present invention discloses a GD2-targeted spherical nucleic acid nanoparticle and its preparation method and application, which can effectively inhibit the growth of RB cells and can be used to prepare high-efficiency and low-toxic specific targeted drugs.

[0009] To this end, the technical solution adopted in the present invention is:

[0010] A method for preparing GD2-targeted spherical nucleic acid nanoparticles, comprising:

[0011] Taking advantage of the hydrophilicity of nucleic acids and the lipophilicity of cholesterol, spherical nucleic acid nanoparticles S-AGsM7 were self-assembled using siRNA nucleic acid chains, nucleic acid aptamer nucleic acid chains, and the small molecule inhibitor NVP-CGM097; wherein the sequences of the sense and antisense strands of the siRNA nucleic acid chains are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively, and the sequence of the nucleic acid aptamer nucleic acid chain is shown in SEQ ID NO.3, specifically as follows:

[0012] Chain of Justice:

[0013] 5-CCACCUCACAGAUUCCAGC(dT)(dT)(dT)(dT)(dA)(dG)(dC)(dT)(dT)(dG)(dC)(dA)(dG)(dT)(dC)(dC)(dT)-3(SEQ ID NO. 1);

[0014] Antisense strand: 5-GCUGGAAUCUGUGAGGUGGTT-3 (SEQ ID NO. 2);

[0015] Aptamer nucleic acid chain:

[0016] 5-AGGACTGCAAGCTAACCGCCCAAATCCCTAAGAGCACAAACACCAAACACAACCACCCCAACCAGACACACTACACACGCA-3 (SEQ ID NO. 3);

[0017] The 3' end of the sense strand is connected to cholesterol via a thioether bond, and the 5' end of the nucleic acid aptamer nucleic acid strand is connected to cholesterol via a thioether bond.

[0018] The GD2-targeted spherical nucleic acid nanoparticles obtained by this technical solution can use SNA technology to accurately block the MDM2-p53 pathway, and accurately inhibit RB proliferation at both the gene and protein levels. That is, by utilizing the hydrophilicity of nucleic acids and the lipophilicity of cholesterol, the 3' end of the nucleic acid chain containing the interfering RNA (siRNA) that interferes with the expression of the MDM2 protein is modified with cholesterol, and the 5' end of the nucleic acid chain containing the nucleic acid aptamer targeting GD2 is modified with cholesterol. The two nucleic acid chains are mixed with the small molecule inhibitor NVP-CGM097 that blocks MDM2-p53, and self-assemble into a spherical nucleic acid SNA with NVP-CGM097 loaded at the core through the hydrophilicity and lipophilicity and the complementary pairing characteristics of nucleic acid bases. This spherical nucleic acid SNA can specifically bind to cell surface GD2 through nucleic acid aptamers, accurately deliver and take up siRNA-MDM2 and NVP-CGM097 into target cells, and degrade the disulfide bonds of each nucleic acid chain under the action of reduced glutathione (GSH), releasing siRNA-MDM2 and NVP-CGM097. On the one hand, siRNA-MDM2 interferes with the expression of MDM2, and on the other hand, NVP-CGM097 competes with p53 for binding to MDM2, blocking the MDM2-p53 interaction and inhibiting the degradation of p53, thereby inhibiting RB proliferation.

[0019] As a further improvement of the present invention, the molar ratio of the siRNA nucleic acid chain and the nucleic acid aptamer nucleic acid chain is 1:1, and the molar ratio of the small molecule inhibitor NVP-CGM097 to the siRNA nucleic acid chain / nucleic acid aptamer nucleic acid chain is 5-15:1. Further preferably, the molar ratio of the small molecule inhibitor NVP-CGM097 to the siRNA nucleic acid chain / nucleic acid aptamer nucleic acid chain is 10:1. Using this technical solution, when the molar ratio of the small molecule inhibitor NVP-CGM097 to the siRNA nucleic acid chain / nucleic acid aptamer nucleic acid chain is 5:1 and 15:1, the inhibition rate on Y79 cells and WERI-RB1 cells is slightly weaker than 10:1, but higher than the inhibition rate on Y79 cells and WERI-RB1 cells of the spherical nucleic acid nanoparticles assembled from the siRNA nucleic acid chain and the nucleic acid aptamer nucleic acid chain without the addition of small molecule inhibitors.

[0020] As a further improvement of the present invention, the preparation method of the targeted GD2 spherical nucleic acid nanoparticles comprises:

[0021] Step S1, preparing siRNA nucleic acid chain freeze-dried powder and aptamer nucleic acid chain freeze-dried powder;

[0022] Step S2, dissolving the siRNA nucleic acid chain freeze-dried powder and the aptamer nucleic acid chain freeze-dried powder in RNase-free water to obtain a siRNA nucleic acid chain solution and a nucleic acid aptamer nucleic acid chain solution, respectively, adding NVP-CGM097 to an organic solvent and dissolving it to obtain an NVP-CGM097 solution, adding the siRNA nucleic acid chain solution, the nucleic acid aptamer nucleic acid chain solution, and the NVP-CGM097 solution to a PBS solution, and mixing them uniformly to obtain a mixed solution;

[0023] Step S3, placing the mixed solution obtained in step S2 in a PCR instrument to perform base pairing of the nucleic acid chains and self-assemble into spherical nucleic acids;

[0024] Step S4, purifying the obtained spherical nucleic acid to obtain spherical nucleic acid nanoparticles S-AGsM7.

[0025] As a further improvement of the present invention, the organic solvent is dimethyl sulfoxide (DMSO).

[0026] As a further improvement of the present invention, step S4 includes freeze-drying the spherical nucleic acid product obtained in step S3 into a powder, dissolving it in RNase-free water, adding chloroform and mixing it thoroughly, washing the remaining NVP-CGM097, centrifuging, aspirating the lower organic solution, and repeating the washing at least twice.

[0027] As a further improvement of the present invention, in step S2, the concentrations of the siRNA nucleic acid chain and the aptamer nucleic acid chain in the mixed solution are 20 μM, and the concentration of NVP-CGM097 is 200 μM.

[0028] As a further improvement of the present invention, in step S3, the PCR conditions are: 95° C. for 10 min, and 4° C. for 20 min.

[0029] The present invention also discloses a GD2-targeted spherical nucleic acid nanoparticle, which is prepared by using any one of the above methods for preparing the GD2-targeted spherical nucleic acid nanoparticle.

[0030] The present invention also discloses the application of the above-mentioned targeted GD2 spherical nucleic acid nanoparticles, which are used in the preparation of eye-protection therapeutic drugs for retinoblastoma, can perform specific targeted therapy on RB, and are highly effective and low in toxicity.

[0031] The present invention also discloses an eye-protecting therapeutic drug for retinoblastoma, which comprises the GD2-targeting spherical nucleic acid nanoparticles as described above.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] The spherical nucleic acid nanoparticles using the technical solution of the present invention have the effect of inhibiting the proliferation of retinoblastoma through the coordination between the components. Experimental results show that the spherical nucleic acid nanoparticles provided by the present invention can accurately target the retinoblastoma cell lines Y79 and WERI-RB1, delivering siRNA-MDM2 and the small molecule inhibitor NVP-CGM097 into the cells. On the one hand, siRNA-MDM2 interferes with the expression of MDM2, and on the other hand, NVP-CGM097 competitively binds to MDM2 with p53, blocking the MDM2-p53 interaction and inhibiting the degradation of p53, thereby inhibiting RB proliferation. It can be used as an eye-preserving treatment for retinoblastoma and can effectively inhibit the proliferation and metastasis of retinoblastoma. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 It is an agarose gel electrophoresis diagram of the self-assembly of SNA nanoparticles according to an embodiment of the present invention.

[0035] Figure 2 This is a DSL analysis chart of the particle size of S-AGsM7 nanoparticles according to an embodiment of the present invention.

[0036] Figure 3 It is a DSL analysis chart of the particle size of S-AGsM nanoparticles of the comparative example of the present invention.

[0037] Figure 4 These are TEM analysis diagrams of the particle size of S-AGsM7 of the embodiment of the present invention and the S-AGsM nanoparticles of the comparative example; wherein (a) is S-AGsM and (b) is S-AGsM7.

[0038] Figure 5 These are the results of flow cytometry detection of effective SNA uptake into RB cell lines by S-AGsM7 of the present invention and S-AGsM of the comparative example; wherein (a) is Y79 and (b) is WERI-RB1.

[0039] Figure 6 This is a comparative analysis chart of the GSSG content of S-AGsM7 of the embodiment of the present invention and the S-AGsM of the comparative example; wherein (a) is Y79 and (b) is WERI-RB1.

[0040] Figure 7 These are the results of the growth inhibition of RB cells by S-AGsM7 of the example of the present invention and S-AGsM of the comparative example; wherein (a) is Y79 and (b) is WERI-RB1. DETAILED DESCRIPTION

[0041] The preferred embodiments of the present invention are described in further detail below.

[0042] Example 1

[0043] A GD2-targeted spherical nucleic acid nanoparticle, the preparation method of which specifically includes:

[0044] Step S1, preparing siRNA nucleic acid chain freeze-dried powder and aptamer nucleic acid chain freeze-dried powder;

[0045] The sequences of the sense strand and antisense strand of the siRNA nucleic acid chain are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively, and the sequence of the nucleic acid aptamer nucleic acid chain is shown in SEQ ID NO.3, specifically as follows:

[0046] Chain of Justice:

[0047] 5-CCACCUCACAGAUUCCAGC(dT)(dT)(dT)(dT)(dA)(dG)(dC)(dT)(dT)(dG)(dC)(dA)(dG)(dT)(dC)(dC)(dT)-3(SEQ ID NO.1); 3'-end modification-SS-cholesterol;

[0048] Antisense strand: 5-GCUGGAAUCUGUGAGGUGGTT-3 (SEQ ID NO. 2);

[0049] Aptamer nucleic acid chain:

[0050] 5-AGGACTGCAAGCTAACCGCCCAAATCCCTAAGAGCACAAACACCAAACACAACCACCCCAACCAGACACACTACACACGCA-3 (SEQ ID NO. 3); 5'-end modification-SS-cholesterol.

[0051] Step S2, preparation of spherical nucleic acid nanoparticles S-AGsM7 self-assembled by nucleic acid and small molecule inhibitors.

[0052] Lyophilized siRNA and aptamer nucleic acid strand powders were dissolved in RNase-free water to obtain siRNA and aptamer nucleic acid strand solutions, respectively, and the final concentrations were adjusted to 100 μM. NVP-CGM097 powder was dissolved in the organic solvent dimethyl sulfoxide (DMSO) to obtain an NVP-CGM097 solution, and the final concentration was adjusted to 10 mg / mL.

[0053] Separately, add the siRNA nucleic acid chain solution, the aptamer nucleic acid chain solution, and the NVP-CGM097 solution to a 10 mM PBS solution, so that the final concentrations of the siRNA nucleic acid chain and the aptamer nucleic acid chain are both 20 μM, and the final concentration of NVP-CGM097 is 200 μM. The final volume is 100 μL. Mix thoroughly and centrifuge to concentrate the solution at the bottom of the tube.

[0054] The mixture was placed in a PCR instrument and set at 95°C for 10 minutes and 4°C for 20 minutes to allow the bases of the nucleic acid chains to be complementary and to self-assemble into spherical nucleic acids.

[0055] The product was freeze-dried to a powder in a vacuum freeze dryer. Dissolve the product in 50 μL of RNase-free water, wash the remaining NVP-CGM097 with chloroform, add 500 μL of chloroform, mix thoroughly, and centrifuge at 7500 rpm at 4°C for 10 minutes. Remove the lower organic layer and repeat the wash twice. The remaining solution was freeze-dried to a powder in a vacuum freeze dryer, dissolved in RNase-free water, named S-AGsM7, and stored at 4°C.

[0056] Example 2

[0057] Preparation of spherical nucleic acid nanoparticles FITC-S-AGsM7 that self-assemble with fluorescently labeled nucleic acids and small molecule inhibitors.

[0058] In this example, in order to facilitate subsequent detection, a fluorescently labeled aptamer nucleic acid chain freeze-dried powder was prepared. The sequence of the fluorescently labeled aptamer nucleic acid chain is shown in SEQ ID NO. 3.

[0059] 5-AGGACTGCAAGCTAACCGCCCAAATCCCTAAGAGCACAAACACCAAACACAACCACCCCAACCAGACACACTACACACGCA-3, 5'-end modified with SS-cholesterol, 3'-end modified with FITC.

[0060] Lyophilized powders of siRNA and fluorescently labeled aptamer nucleic acid chains were dissolved in RNase-free water to a final concentration of 100 μM. NVP-CGM097 powder was dissolved in dimethyl sulfoxide (DMSO) to a final concentration of 10 mg / mL. SNAs were prepared according to the steps of Example 1 to obtain spherical nucleic acid nanoparticles FITC-S-AGsM7, which self-assembled with fluorescently labeled nucleic acids and the small molecule inhibitor NVP-CGM097.

[0061] Comparative Example 1

[0062] Preparation of spherical nucleic acid nanoparticles S-AGsM self-assembled by nucleic acid.

[0063] The siRNA nucleic acid chain lyophilized powder and the nucleic acid aptamer nucleic acid chain lyophilized powder in Example 1 were dissolved in RNase-free water to a final concentration of 100 μM. The two solutions were added to a 10 mM PBS solution to a final concentration of 20 μM for the siRNA nucleic acid chain and the nucleic acid aptamer nucleic acid chain, respectively. The final volume was 100 μL, and the mixture was thoroughly mixed and centrifuged to concentrate the solution at the bottom of the tube. The mixture was placed in a PCR instrument and set to 95°C for 10 min and 4°C for 20 min to perform base complementary pairing of the nucleic acid chains, self-assemble into a spherical nucleic acid, named S-AGsM, and stored at 4°C.

[0064] Comparative Example 2

[0065] Preparation of spherical nucleic acid nanoparticles FITC-S-AGsM with fluorescently labeled nucleic acid self-assembly.

[0066] The siRNA nucleic acid chain freeze-dried powder in Example 1 and the fluorescently labeled nucleic acid aptamer nucleic acid chain freeze-dried powder in Example 2 were respectively dissolved in RNase-free water to adjust the final concentration to 100 μM, and SNA was prepared according to the steps of Comparative Example 1 to obtain FITC-S-AGsM.

[0067] Example 3

[0068] The S-AGsM7 obtained in Example 1 and the S-AGsM obtained in Comparative Example 1 were subjected to agarose electrophoresis to identify the self-assembled structure of the SNA nanoparticles, comprising the following steps:

[0069] Preparation of gel: Add 0.2g agarose and 20mL TAE (1×) electrophoresis buffer to a conical flask, heat and boil until clear, add 1μL of dye GoldView, shake well, pour the gel into the gel plate, insert the comb vertically, and let it stand until gelling. After the gel is completely solidified, transfer it to the electrophoresis tank, add a small amount of TAE (1×) electrophoresis buffer, and remove the comb;

[0070] Sample preparation: add 9 μL of S-AGsM solution and 9 μL of S-AGsM7 solution to 1 μL of 10× Loading Buffer respectively and mix well;

[0071] Sample loading: Add the mixed sample to the spotting wells and add 5 μL of nucleic acid marker to one of the spotting wells;

[0072] Turn on the power supply, 30-40mA constant current, voltage about 70V, electrophoresis for 30min;

[0073] The gel was removed and observed under UV light.

[0074] Since the assembly materials of SNA nanoparticles are nucleic acid molecules, agarose gel electrophoresis was used to identify the self-assembly of SNA nanoparticles. Figure 1 As shown in the figure, compared with the bands of nucleic acid aptamer (lane 2) and siRNA (lane 3), the bands of S-AGsM (lane 4) and S-AGsM7 (lane 5) appear at a larger molecular weight, indicating that the two SNA nanoparticles are highly aggregated and have high purity. This experiment shows that the SNA self-assembly is successful.

[0075] Example 4

[0076] The S-AGsM7 obtained in Example 1 and the S-AgsM obtained in Comparative Example 1 were characterized by SNA nanoparticles: dynamic light scattering (DSL) and transmission electron microscopy (TEM) were used to analyze the SNA particle size.

[0077] Sample preparation: prepare 100 μL of the product S-AGsM from step 4 and 100 μL of the product S-AGsM7 from step 5.

[0078] test:

[0079] (1) DSL: Open the computer and DSL instrument, run the software program, create a new experiment window, and set the corresponding parameters and file name. Parameters include the sample type, solvent type, temperature, concentration, etc. Add 100 μL of the prepared product S-AGsM from Example 4 to the sample cell. Place the sample cell in the sample chamber, close the sample chamber lid, and click "record" to begin data acquisition. Clean the sample cell and use the same procedure to acquire data for the product S-AGsM7 from Example 5 and analyze the data.

[0080] Since SNA is a nano-sized particle, DSL was used to analyze the particle size distribution of SNA nanoparticles. Figure 2 and Figure 3 As shown, the particle size of S-AGsM is about 20 nm, and the particle size of S-AGsM7 is about 20 nm, and the results are in line with expectations.

[0081] (2) TEM: Turn on the computer and TEM, evacuate the machine, perform a self-check, load the sample slice, adjust the magnification, position and brightness, take pictures, and analyze the data.

[0082] The results are as follows Figure 4 As shown in the figure, both S-AGsM and S-AGsM7 exhibit spherical structures with a particle size of about 20 nm, which is consistent with the DSL results and in line with expectations.

[0083] Example 5

[0084] Flow cytometry was used to detect the uptake of SNA into RB cells.

[0085] First, retinoblastoma cell lines Y79 and WERI-RB1 were cultured, including the following steps:

[0086] Retinoblastoma cell lines Y79 and WERI-RB1 were cultured in T25 culture flasks at 37°C in a 5% CO2 environment. The Y79 culture system was 1640 medium with 20% serum (FBS), and the WERI-RB1 culture system was 1640 medium with 10% serum. Cell viability experiments were performed when the cell viability was above 90%.

[0087] Then, flow cytometry was used to detect the uptake of SNA into RB cells.

[0088] Cell collection: The Y79 and WERI-RB1 cells of Example 9 were cultured in T-25 cell culture flasks until the logarithmic growth phase, the culture medium was discarded, the cells were washed twice with PBS, and then the cells were collected and resuspended.

[0089] Cell counting: After counting with trypan blue staining, cells were transferred to 1.5 mL EP tubes and 10 6 Centrifuge the cells at 1,000 rpm at 4°C for 5 min and discard the supernatant.

[0090] Blocking: Add 5% skim milk, 500 μL / tube, and block at 4°C for 30 min.

[0091] Washing: Add 2% FBS-PBS solution, 500 μL / tube, centrifuge at 4°C, 1,000 rpm for 5 min, discard the supernatant, and repeat twice.

[0092] Incubate with the primary antibody: add 20 μM of the test sample (S-AGsM of Comparative Example 1, S-AGsM7 of Example 1), 250 μL / tube, set up test groups at different time points for each sample, and 250 μL of 5% skim milk as a negative control. Since the nanoparticles can be taken into the cell at 37 ° C, and this process is terminated at 4 ° C. Therefore, all test groups are first incubated at 37 ° C, and the corresponding test groups are removed at the test time points (0, 2, 5, 15, 30, 40, 60, 90 min) and transferred to 4 ° C to terminate the internalization process.

[0093] Washing: Add 2% FBS-PBS solution, 500 μL / tube, centrifuge at 4°C, 1,000 rpm for 5 min, discard the supernatant, and repeat twice.

[0094] Flow cytometry was started and the uptake rate was calculated according to the formula: Uptake rate (%) = [1-(average fluorescence intensity at the time point to be measured-average fluorescence intensity of the negative control group) / (average fluorescence intensity at 0 minutes-average fluorescence intensity of the negative control group)] x 100%.

[0095] The results are as follows Figure 5 As shown in the figure, S-AGsM and S-AGsM7 were rapidly taken up into Y79 cells within 25 minutes, reaching a plateau after 30 minutes. Furthermore, S-AGsM and S-AGsM7 were rapidly taken up into WERI-RB1 cells within 30 minutes, reaching a plateau after 40 minutes. The uptake rates of both SNAs exceeded 50%, demonstrating that SNAs can be effectively taken up into RB cells.

[0096] Example 6

[0097] The intracellular GSSG content was determined after SNA treatment in RB cells.

[0098] Since SNA needs the action of intracellular reduced glutathione (GSH) to reduce the disulfide bonds in SNA, convert GSH into oxidized glutathione (GSSG), depolymerize the SNA structure, and release the globular center NVP-CGM097 to exert its effect, measuring the intracellular GSSG content can reflect whether GSH reacts with the disulfide bonds of SNA. The specific steps include the following:

[0099] Sample preparation: After the cells of Example 9 have grown to the logarithmic growth phase, the cells are collected and counted and then plated in a 6-well plate. 6 Cells were cultured overnight. S-AGsM, the product of Example 4, and S-AGsM7, the product of Example 5, were added to the culture medium to a final concentration of 20 μM. After continued culture in an incubator for 72 hours, the cells were harvested, washed once with PBS, and centrifuged. The supernatant was aspirated. Protein Removal Reagent M solution was added in an amount three times the volume of the cell pellet. The sample was then rapidly frozen and thawed twice using liquid nitrogen and a 37°C water bath. Incubate on ice for 5 minutes. Centrifuge at 10,000 g for 10 minutes at 4°C. The supernatant was used for total glutathione determination. The sample was temporarily stored at 4°C. 30 μL of the prepared sample was added to 6 μL of GSH scavenging auxiliary solution and immediately vortex mixed. 1.2 μL of GSH scavenging working solution was then added, immediately vortex mixed, and incubated at 25°C for 60 minutes. This can be used for subsequent GSSG content detection.

[0100] Prepare standards: Prepare 15, 10, 5, 2, 1, and 0.5 μM GSSG solutions and use these six points to create a standard curve. Take 30 μL of the prepared sample, add 6 μL of GSH scavenging auxiliary solution, and immediately vortex mix. Then add 1.2 μL of GSH scavenging working solution, immediately vortex mix, and incubate at 25°C for 60 minutes. This can be used for subsequent GSSG content testing.

[0101] Prepare each group of test solutions according to the system in Table 1 below:

[0102] Table 1

[0103] Blank group Standards / Samples Standards / Samples 0 10 Protein Removal Reagent M Solution 10 10

[0104] Add 150 μL of total glutathione detection working solution, mix well, and incubate at room temperature for 5 minutes. Add 50 μL of 0.5 mg / ml NADPH solution, mix well, and incubate at room temperature for 30 minutes. Measure A with a microplate reader. 412 . Calculate the GSSG content in the sample to be tested based on the standard curve.

[0105] The results are as follows Figure 6 As shown in the figure, compared with the negative control group, the concentration of GSSG increased to varying degrees after treatment with S-AGsM and S-AGsM7 in both RB cell lines, indicating that intracellular GSH was consumed by the disulfide bonds of SNA and oxidized to GSSG. This experiment shows that under the action of GSH, the disulfide bonds of SNA are reduced to sulfhydryl groups, disconnecting the nucleic acid chain from cholesterol and depolymerizing the SNA structure.

[0106] Example 7

[0107] To verify whether SNA can effectively inhibit the growth of RB cells, the CCK8 assay was used to determine the inhibitory effect of SNA on the growth of RB cells. The specific steps included the following:

[0108] Cell preparation: After the cells of Example 4 have grown to the logarithmic growth phase, the cells were collected and the culture medium was discarded. The cells were washed twice with PBS and then collected and resuspended. After counting the cells, the cells were transferred to a 96-well plate and 5×10 3 Cells were added to each well, and the serum concentration in the culture medium was reduced by half. The cells were cultured at 37°C overnight with 5% CO2. The culture medium was discarded after centrifugation, and serum-free medium was added to starve the cells for 4 hours. The cells were then centrifuged and the culture medium was discarded. The cells were washed once with PBS and then dosed.

[0109] Drug administration: Two drug administration groups (S-AGsM and S-AGsM7) and a blank control group were set up for each cell type. Five drug concentrations (1 μM, 5 μM, 10 μM, 20 μM, 30 μM) were prepared in 2% FBS medium, and 100 μL / well was used.

[0110] Detection: 72 hours after administration, add CCK8 solution, 10 μL / well, 5% CO2, and incubate at 37°C for 4 hours. Use a multifunctional microplate reader to detect the absorbance difference at wavelengths of 570 nm and 630 nm.

[0111] The results are as follows Figure 7As shown in the results, the inhibition rate of 30 μM S-AGsM on Y79 cells and WERI-RB1 cells did not reach 40%, while the inhibition rate of 10 μM S-AGsM7 on Y79 cells and WERI-RB1 cells reached 47% and 53%, respectively, indicating that S-AGsM7 has a stronger inhibitory effect on cell growth than S-AGsM.

[0112] The above experimental data show that S-AGsM7 can precisely target retinoblastoma cell lines Y79 and WERI-RB1, delivering siRNA-MDM2 and the small molecule inhibitor NVP-CGM097 into the cells. On the one hand, siRNA-MDM2 interferes with the expression of MDM2, and on the other hand, NVP-CGM097 competitively binds to MDM2 with p53, blocking the MDM2-p53 interaction and inhibiting the degradation of p53, thereby inhibiting RB proliferation.

[0113] The above is a further detailed description of the present invention in conjunction with specific preferred embodiments, and the specific implementation of the present invention should not be considered to be limited to these descriptions. For those skilled in the art of the present invention, without departing from the concept of the present invention, several simple deductions or substitutions can be made, which should be considered to fall within the scope of protection of the present invention.

Claims

1. A method for preparing GD2-targeted spherical nucleic acid nanoparticles, characterized in that: include: Spherical nucleic acid nanoparticles S-AGsM7 were self-assembled using siRNA nucleic acid chains, nucleic acid aptamer nucleic acid chains and small molecule inhibitor NVP-CGM097; The sequences of the sense strand and antisense strand of the siRNA nucleic acid chain are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively, and the sequence of the nucleic acid aptamer nucleic acid chain is shown in SEQ ID NO.3; The 3' end of the sense strand is linked to cholesterol via a thioether bond, and the 5' end of the aptamer nucleic acid strand is linked to cholesterol via a thioether bond.

2. The method for preparing the targeted GD2 spherical nucleic acid nanoparticles according to claim 1, wherein: The molar ratio of the siRNA nucleic acid chain and the nucleic acid aptamer nucleic acid chain is 1:1, and the molar ratio of the small molecule inhibitor NVP-CGM097 to the siRNA nucleic acid chain / the nucleic acid aptamer nucleic acid chain is 10:

1.

3. The method for preparing the targeted GD2 spherical nucleic acid nanoparticles according to claim 2, wherein: include: Step S1, preparing siRNA nucleic acid chain freeze-dried powder and aptamer nucleic acid chain freeze-dried powder; Step S2, dissolving the siRNA nucleic acid chain freeze-dried powder and the aptamer nucleic acid chain freeze-dried powder in RNase-free water to obtain a siRNA nucleic acid chain solution and a nucleic acid aptamer nucleic acid chain solution, respectively, adding NVP-CGM097 to an organic solvent and dissolving it to obtain an NVP-CGM097 solution, adding the siRNA nucleic acid chain solution, the nucleic acid aptamer nucleic acid chain solution, and the NVP-CGM097 solution to a PBS solution, and mixing them uniformly to obtain a mixed solution; Step S3, placing the mixed solution obtained in step S2 in a PCR instrument to perform base pairing of the nucleic acid chains and self-assemble into spherical nucleic acids; Step S4, purifying the obtained spherical nucleic acid to obtain spherical nucleic acid nanoparticles S-AGsM7.

4. The method for preparing the targeted GD2 spherical nucleic acid nanoparticles according to claim 3, wherein: The organic solvent is dimethyl sulfoxide.

5. The method for preparing the targeted GD2 spherical nucleic acid nanoparticles according to claim 3, wherein: Step S4 includes freeze-drying the spherical nucleic acid product obtained in step S3 into powder, adding RNase-free water to dissolve it, adding chloroform to mix thoroughly, washing the remaining NVP-CGM097, centrifuging, aspirating the lower organic solution, and repeating the washing at least twice.

6. The method for preparing the targeted GD2 spherical nucleic acid nanoparticles according to claim 3, wherein: In step S2, the concentrations of the siRNA nucleic acid chain and the aptamer nucleic acid chain in the mixed solution are 20 μM, and the concentration of NVP-CGM097 is 200 μM; In step S3, the PCR conditions were: 95°C for 10 min, and 4°C for 20 min.

7. A GD2-targeted spherical nucleic acid nanoparticle, characterized by: The targeted GD2 spherical nucleic acid nanoparticles are prepared by the preparation method according to any one of claims 1 to 6.

8. The use of the GD2-targeting spherical nucleic acid nanoparticles according to claim 7, characterized in that: The invention is used for preparing eye-protecting therapeutic drugs for retinoblastoma.

9. An eye-protecting therapeutic drug for retinoblastoma, characterized by: It comprises the GD2-targeting spherical nucleic acid nanoparticles as claimed in claim 7.

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