Spherical nucleic acid nanoparticles based on aptamer modification as well as preparation method and application of spherical nucleic acid nanoparticles

Abstract

The invention belongs to the technical field of targeted protein degradation, and particularly relates to spherical nucleic acid nanoparticles based on aptamer modification and a preparation method and application thereof.The preparation method comprises the steps that gold nanoparticles serve as carriers, aptamers for specifically recognizing target membrane protein are covalently connected to the surfaces of the gold nanoparticles through gold-sulfur bonds, and multivalent Apt-SNAs are formed; the prepared Apt-SNAs can be selectively taken in by cells overexpressing target membrane protein through a gridding protein mediated endocytosis pathway, the target membrane protein is transported to lysosome for degradation, and the Apt-SNAs can be used for preparing drugs for targeted degradation of programmed cell death-ligand 1 on a cell membrane and drugs for treating cancers, and has a wide application prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of targeted protein degradation, and particularly relates to a spherical nucleic acid nanoparticle based on aptamer modification, and a preparation method and application thereof. Background Art

[0002] Overexpression of cell surface receptors or proteins plays a crucial role in the occurrence and progression of cancer. These proteins are often closely related to cell signaling pathways, regulating cell proliferation, differentiation and survival. When these receptors or proteins are abnormally overexpressed, it leads to the continuous activation of signaling pathways, which triggers the excessive proliferation and anti-apoptotic properties of tumor cells. Traditional drugs usually block the function of proteins by inhibiting their active sites, but in some cases, the drug effect is limited or it is difficult to target due to the structural characteristics of the protein. Targeted protein degradation technology can not only effectively reduce the levels of cancer-related proteins, but also overcome the limitations of traditional inhibitors, especially in dealing with "undruggable" targets. In addition, these degraders can promote the degradation of target proteins through specific mechanisms, thereby fundamentally eliminating the function of harmful proteins. In this way, targeted degradation technology provides a new approach for cancer treatment, which can significantly improve the specificity and effectiveness of treatment and reduce the impact on normal cells, thus hopefully improving the prognosis of patients. Therefore, the development of drugs for targeted protein degradation to degrade these overexpressed proteins is of great significance for cancer treatment.

[0003] Lysosomal targeting chimeras (LYTACs) are an emerging protein degradation technology that aims to regulate biological processes by selectively degrading target proteins outside the cell and on the cell surface. In recent years, advances in LYTACs and related technologies have provided powerful tools for degrading cell membrane proteins. These methods use bifunctional molecules to connect the extracellular domain of the target protein to the lysosomal targeting receptor, thereby triggering the endocytosis of the target protein and degradation in the lysosome. In cancer research, LYTACs can be used to degrade overexpressed receptors or signaling molecules, thereby inhibiting the growth and metastasis of tumor cells. Due to its high selectivity and effectiveness, LYTACs are expected to become an innovative strategy for the treatment of cancer and immune diseases, and promote the development of personalized precision medicine.

[0004] Currently, most LYTACs rely on specific lysosomal targeting receptors to achieve lysosomal targeted delivery, which may cause complexity and challenges in clinical therapeutic applications, especially due to the variability of receptor expression between cells and between different patients, which may lead to inconsistent therapeutic effects. In addition, the design and production process of most LYTACs is complex, involving multi-step chemical synthesis or protein recombination, which further limits their feasibility in large-scale clinical applications. Therefore, it is urgent to develop more universal degradation platforms to reduce dependence on specific lysosomal targeting receptors, and to optimize the applicability and safety of these platforms through further research to overcome potential therapeutic limitations. These improvements will help improve the effectiveness and wide application potential of LYTACs in complex biological environments, and provide more flexible and efficient means for the treatment of cancer and other diseases. Summary of the invention

[0005] The present invention solves the above-mentioned technical problems existing in the prior art, and provides a spherical nucleic acid nanoparticle (AptSNA) based on aptamer modification and its preparation method and application. Based on the natural receptor-mediated endocytosis of cells, the present invention has developed a spherical nucleic acid based on aptamer-modified gold nanoparticles (AuNPs), which can pull the cell programmed death-ligand 1 (PD-L1) on the cell membrane into the lysosome for degradation. Therefore, without the need to rely on the lysosomal targeting function of the lysosomal targeting receptor, efficient degradation of cell membrane target proteins can be achieved, reducing dependence on specific lysosomal targeting receptors. In addition, the design and production process of this aptamer spherical nucleic acid is simple, does not involve multi-step chemical synthesis or protein recombination, has broad application prospects, and provides a new research direction for the efficient degradation of different cell membrane target proteins.

[0006] The technical solution of the present invention is as follows:

[0007] An aptamer-modified spherical nucleic acid nanoparticle (AptSNA), comprising a thiol-modified aptamer probe (SH-Aptamer) and gold nanoparticles (AuNPs),

[0008] The thiol-modified aptamer probe is covalently linked to the surface of the gold nanoparticles via a gold-sulfur bond;

[0009] The sequence of the thiol-modified aptamer probe is:

[0010] SH-C6-TTTTTTTTTTTACAGGTTCTGGGGGGTGGGTGGGGAACCTGTT, as shown in SEQ No. 1.

[0011] Preferably, the gold nanoparticles are prepared by sodium citrate reduction method.

[0012] Preferably, the particle size of the gold nanoparticles is 13.0 to 15.0 nm.

[0013] The preparation method of the spherical nucleic acid nanoparticles based on aptamer modification comprises the following steps:

[0014] Step 1, adding tri(2-carboxyethyl)phosphine to the thiol-modified aptamer probe solution, incubating at room temperature in the dark for 1 hour, and then annealing to form an activated SH-Aptamer solution;

[0015] Step 2, adding the activated SH-Aptamer solution to the gold nanoparticle solution, incubating with shaking, adding sodium chloride to the buffer system, and continuing to incubate to obtain the aptamer-modified spherical nucleic acid nanoparticles.

[0016] Preferably, in step 1, the molar ratio of the thiol-modified aptamer probe to tris(2-carboxyethyl)phosphine is 1:5 to 1:10.

[0017] More preferably, the thiol-modified aptamer probe solution is a sodium acetate buffer, which contains 5 mM sodium acetate and has a pH of 5.2.

[0018] Preferably, in step 1, the annealing conditions are heating to 95° C. for 5 minutes, decreasing to 25° C. at a rate of 0.1° C. / s, and continuing to stabilize for 1 hour or more.

[0019] Preferably, in step 2, the concentration of the activated SH-Aptamer solution is 100 μM, and the volume is 100 μL; the concentration of the gold nanoparticle solution is 10 nM, and the volume is 3 mL.

[0020] More preferably, the specific steps of step 2 are: adding the activated SH-Aptamer solution to the gold nanoparticle solution, shaking and incubating overnight, adding Tris-acetate buffer A, and slowly adding sodium chloride solution to the reaction system, continuing to incubate overnight, and resuspending with Tris-acetate buffer B after centrifugation;

[0021] The concentration of the Tris-acetate buffer A is 500 mM Tris, pH=8.2, and the final concentration is 5 mM Tris; the concentration of the sodium chloride solution is 1 M sodium chloride, and the final concentration is 300 mM sodium chloride; the concentration of the Tris-acetate buffer B is 300 mM sodium chloride, 25 mM Tris, pH=8.2.

[0022] The spherical nucleic acid nanoparticles modified with aptamers can be used to prepare drugs for targeted degradation of programmed cell death-ligand 1 (PD-L1) on cell membranes.

[0023] The spherical nucleic acid nanoparticles modified with aptamers can be used to prepare drugs for treating cancer.

[0024] Compared with the prior art, the advantages of the present invention are as follows:

[0025] Based on the natural receptor-mediated endocytosis of cells, the present invention has developed a spherical nucleic acid nanoparticle modified with an aptamer, which can pull the PD-L1 protein on the cell membrane into the lysosome for degradation. Therefore, without the need for the lysosome targeting function of the lysosome targeting receptor, efficient degradation of the cell membrane target protein can be achieved, reducing the dependence on specific lysosome targeting receptors.

[0026] The design and production process of the aptamer-modified spherical nucleic acid nanoparticles developed by the present invention are simple, do not involve multi-step chemical synthesis or protein recombination, have broad application prospects, and provide a new research direction for the efficient degradation of different cell membrane target proteins. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 (a) The preparation process flow chart of AptSNA and the transmission electron microscopy images of AuNPs (b) and AptSNA (c).

[0028] Scale bar: 100 nm;

[0029] Figure 2 Schematic diagram of AptSNA used for targeted degradation of cell membrane PD-L1 protein;

[0030] Figure 3 The results of immunoblotting analysis of the expression level of cell membrane PD-L1 protein after incubation of MDA-MB-231 cells with AptSNA (3 nM) for different time periods;

[0031] Figure 4 Flow cytometry was used to analyze the effects of clathrin-mediated endocytosis inhibitor (chlorpromazine) and lysosomal inhibitor (chloroquine) on the degradation of PD-L1 on the MDA-MB-231 cell membrane by AptSNA. DETAILED DESCRIPTION

[0032] Embodiment 1:

[0033] Preparation of aptamer-modified spherical nucleic acid nanoparticles (AptSNA). Programmed cell death ligand 1 (PD-L1) is a transmembrane protein that is mainly expressed on the surface of tumor cells and some immune cells. In cancer treatment, blocking the interaction between PD-1 / PD-L1 can effectively activate T cells and improve the ability of the immune system to recognize and attack tumor cells. Therefore, PD-L1 was selected as a model target protein. Experimental principle Figure 1As shown in Figure a, first, AuNPs were prepared by sodium citrate reduction method; then, SH-Aptamer was modified on the surface of AuNPs by gold-sulfur covalent bonding to obtain AptSNA; the design of SH-Aptamer mainly includes three parts: (i) the 5-terminal modified thiol group, which can covalently bind to AuNPs; (ii) the 3-terminal aptamer part, which can effectively recognize and target PD-L1 protein; (iii) the linker connecting the 3-terminal thiol group and the 5-terminal aptamer, which is composed of 10 thymines (T) and extends the distance between the aptamer and AuNPs, providing sufficient space for the multivalent binding of a large number of aptamers on AptSNA to PD-L1.

[0034] The sequence of SH-Aptamer is:

[0035] SH-C6-TTTTTTTTTTTACAGGTTCTGGGGGGTGGGTGGGGAACCTGTT, as shown in SEQ No. 1.

[0036] The specific preparation method is as follows:

[0037] 1. Preparation of AuNPs

[0038] First, AuNPs were prepared by sodium citrate reduction method. 98 mL of secondary water and 2 mL of 50 mM HAuCl4 solution were added to a 200 mL three-necked flask in sequence. The above solution was heated to reflux under vigorous stirring, and 10 mL of 38.8 mM trisodium citrate solution was quickly added. The color of the reaction solution changed from light yellow to wine red within 1 minute. After continuing to heat and reflux for 20 minutes, the solution was naturally cooled to room temperature under vigorous stirring. Figure 1 b is the transmission electron microscopy image of AuNPs. The results show that AuNPs have good dispersibility and their average particle size is about 13nm.

[0039] 2. Preparation of activated SH-Aptamer

[0040] First, 100 μL of 100 μM SH-Aptamer was mixed with 5 μL of 10 mM tris(2-carboxyethyl)phosphine, and then 5 μL of sodium acetate buffer (500 mM sodium acetate, pH = 5.2) was added and incubated at 37°C for 1 hour. Next, the solution was heated to 95°C for 5 minutes, then cooled to 25°C at a rate of 0.1°C / s and stabilized for 1 hour to obtain activated SH-Aptamer.

[0041] 3. Preparation of AptSNA

[0042] First, add the above 3mL of 10nM AuNPs solution and activated SH-Aptamer in a clean glass bottle, mix well and shake in the dark for 16 hours. Then, add 300μL of Tris-acetate buffer A (500mMTris, pH=8.2), and slowly add 300μL of 1M sodium chloride solution to the reaction system, mix well and shake in the dark for 24 hours. Next, centrifuge the above reaction solution at 16100xg for 10 minutes, discard the supernatant, resuspend with Tris-acetate buffer B (300mM sodium chloride, 25mM Tris, pH=8.2), repeat centrifugation and washing three times to obtain AptSNA. Figure 1 c is the transmission electron microscopy image of AptSNA. The results show that AptSNA has good dispersibility, and the successful modification of SH-Aptamer can be seen in the enlarged image.

[0043] Embodiment 2:

[0044] like Figure 2 As shown, the present invention successfully prepared AptSNA containing targeted programmed cell death ligand 1 (PD-L1), and verified that it can directly degrade PD-L1 protein on the membrane through the clathrin-mediated endocytosis process. The present invention uses AptSNA constructed by gold nanoparticles modified with PD-L1-specific aptamers, and this mechanism is significantly different from traditional lysosome targeting chimeras (LYTAC) in degrading cell membrane proteins. Traditional LYTAC relies on lysosomal targeting receptors for lysosomal targeting to achieve degradation of target proteins, while AptSNA of the present invention directly utilizes endogenous clathrin-mediated endocytic activity to degrade cell membrane proteins. Since AptSNA does not contain lysosomal targeting receptors, this process does not involve the occupancy of lysosomal targeting receptors on the cell membrane. The present invention provides new insights into the lysosome-based membrane protein targeted degradation strategy and demonstrates the application potential of AptSNA in targeted protein degradation. This discovery lays the foundation for the future development of more efficient and specific protein degradation technologies.

[0045] AptSNA for immunoblotting analysis of cell membrane PD-L1 degradation: MDA-MB-231 cells were inoculated in 6-well cell culture plates and grown overnight to a confluence of 60% to 70%. The original culture medium was removed, and culture medium containing AptSNA (final concentration of 3nM) was added thereto respectively, and incubated at 37°C for different times. The culture medium was removed, and the membrane proteins of the obtained cells were prepared using a cell membrane / cytoplasm / nuclear membrane protein step-by-step extraction kit (Shanghai Beibo Biotechnology Co., Ltd.) according to the manufacturer's instructions. Then, immunoblotting analysis was performed to calculate the cell membrane PD-L1 degradation rate. The specific steps of immunoblotting analysis were as follows: 20 μg of total protein was added to the channel of 12% stain-free SDS-PAGE (Bio-Rad Life Sciences Products Co., Ltd.) for vertical electrophoresis, the voltage was set to 90V, and the electrophoresis time was 90 minutes. Then, total protein imaging was performed using an XRS+ chemiluminescence imager (Bio-Rad Life Sciences Products Co., Ltd.). Next, the proteins on the gel were transferred to a polyvinylidene fluoride (PVDF) membrane by transfer, the transfer current was set to 150mA, and the transfer time was 75 minutes. After the transfer was completed, the PVDF membrane was incubated in a wash buffer (137mM sodium chloride, 2.7mM potassium chloride, 25mM Tris, and 0.05% Tween-20) containing 5% bovine serum albumin at room temperature for 1 hour. After blocking, the PVDF membrane was washed three times with wash buffer (5 minutes each time) and incubated with the PD-L1 primary antibody at 4°C overnight. After the incubation, the PVDF membrane was washed three times with wash buffer (5 minutes each time) and then incubated with a horseradish peroxidase-labeled secondary antibody at room temperature for 1 hour. Finally, the protein blot image was obtained by chemiluminescence using an XRS+ chemiluminescence imager. The WB bands were quantitatively analyzed using Image J software. The target protein level in each group was first normalized with the total protein in the same lane, and finally normalized with the control group cells to obtain the relative quantitative analysis results of cell membrane PD-L1 protein expression. Figure 3 As shown in the figure, the expression level of cell membrane PD-L1 was analyzed by immunoblotting after incubating MDA-MB-231 cells with 3nM AptSNA for different time periods and extracting membrane proteins. The statistical analysis results showed that the expression level of PD-L1 on MDA-MB-231 cells decreased with the increase of incubation time with AptSNA, showing a significant time dependence, indicating that AptSNA can effectively degrade PD-L1 protein on the cell membrane.

[0046] Analysis of the mechanism by which AptSNA achieves cell membrane PD-L1 degradation through cellular clathrin-mediated endocytosis: MDA-MB-231 cells were seeded in 6-well cell culture plates and grown overnight to a confluence of 60% to 70%. Next, the cells were washed three times with PBS buffer, to which medium containing AptSNA (final concentration of 3nM) was added, and incubated at 37°C for 24 hours. Afterwards, the cells were washed three times with PBS buffer again and digested with 0.25% trypsin (1000μL) at 37°C for 10 minutes. The treated cells were collected and incubated in complete DMEM medium with 500μL of Cy5 fluorescently modified Aptamer (200nM) at 4°C for 30 minutes, and then washed with PBS buffer. Finally, 10,000 cells of each sample were analyzed by flow cytometry at an excitation wavelength of 638 nm using a CytoFlex flow cytometer (Beckman Coulter, USA), and the data were processed by FlowJo_V10 software (positive group). In order to study the effect of clathrin-mediated endocytosis inhibitor (chlorpromazine group) on the degradation of AptSNA in the cell membrane PD-L1, 5 μg / mL of chlorpromazine was added and incubated with the cells for 0.5 hours before adding AptSNA (3 nM) for treatment, and then the analysis was continued according to the same process; in order to study the effect of lysosomal inhibitor (chloroquine group) on the degradation of AptSNA in the cell membrane PD-L1, when AptSNA (3 nM) was added for treatment, 10 μM chloroquine was added and incubated with the cells at the same time, and then the analysis was continued according to the same process; in addition, untreated cells (blank group, no inhibitors and AptSNA were added) were used as controls and analyzed according to the same process. The results are as follows Figure 4 As shown in the figure, compared with the blank group with the strongest Cy5 mean fluorescence intensity, the Cy5 fluorescence intensity of the positive group cells was significantly reduced, indicating that AptSNA effectively degraded the PD-L1 protein on the MDA-MB-231 cell membrane. When the clathrin-mediated endocytosis inhibitor (chlorpromazine group) was used, the degradation effect of AptSNA on the PD-L1 protein on the MDA-MB-231 cell membrane was inhibited. In addition, after MDA-MB-231 cells were co-incubated with AptSNA and lysosomal inhibitors (chloroquine group), the degradation of PD-L1 on the cell membrane was the lowest, and the Cy5 mean fluorescence intensity was close to that of the blank group cells. The above results show that the degradation of PD-L1 protein on the MDA-MB-231 cell membrane by AptSNA is based on clathrin-mediated endocytosis and endosomal-lysosomal pathways.

[0047] The sequence of Cy5 fluorescently modified Aptamer is:

[0048] TACAGGTTCTGGGGGGTGGGTGGGGAACCTGTT-Cy5, as shown in SEQ No.2.

[0049] Sequence Listing

[0050]

[0051] It should be noted that the above embodiments are merely preferred embodiments of the present invention and are not intended to limit the protection scope of the present invention. Equivalent replacements or substitutions made on the basis of the above embodiments all fall within the protection scope of the present invention.

Claims

1. A spherical nucleic acid nanoparticle modified with an aptamer, characterized in that: Including thiol-modified aptamer probe gold nanoparticles, The thiol-modified aptamer probe is connected to the surface of the gold nanoparticle; The sequence of the thiol-modified aptamer probe is: SH-C6-TTTTTTTTTTTACAGGTTCTGGGGGGTGGGTGGGGAACCTGTT, as shown in SEQ No.

1.

2. The spherical nucleic acid nanoparticle based on aptamer modification according to claim 1, characterized in that: The particle size of the gold nanoparticles is 13.0-15.0 nm.

3. The method for preparing spherical nucleic acid nanoparticles based on aptamer modification according to claim 1, characterized in that: The following steps are involved: Step 1, adding tri(2-carboxyethyl)phosphine to the thiol-modified aptamer probe solution, incubating at room temperature in the dark for 1 hour, and then annealing to form an activated SH-Aptamer solution; Step 2, adding the activated SH-Aptamer solution to the gold nanoparticle solution, incubating with shaking, adding sodium chloride to the buffer system, and continuing to incubate to obtain the aptamer-modified spherical nucleic acid nanoparticles.

4. The preparation method according to claim 3, characterized in that: In the step 1, the molar ratio of the thiol-modified aptamer probe to tris(2-carboxyethyl)phosphine is 1:5 to 1:

10.

5. The preparation method according to claim 4, characterized in that: The thiol-modified aptamer probe solution is a sodium acetate buffer containing 5 mM sodium acetate, with a pH of 5.

2.

6. The preparation method according to claim 3, characterized in that: In the step 1, the annealing conditions are heating to 95° C. for 5 minutes, decreasing to 25° C. at a rate of 0.1° C. / s, and continuing to stabilize for 1 hour or more.

7. The preparation method according to claim 3, characterized in that: In the step 2, the concentration of the activated SH-Aptamer solution is 100 μM, and the volume is 100 μL; the concentration of the gold nanoparticle solution is 10 nM, and the volume is 3 mL.

8. The preparation method according to claim 3, characterized in that: The specific steps of step 2 are: adding the activated SH-Aptamer solution to the gold nanoparticle solution, shaking and incubating overnight, adding Tris-acetate buffer A, and slowly adding sodium chloride solution to the reaction system, continuing to incubate overnight, and resuspending with Tris-acetate buffer B after centrifugation; The concentration of the Tris-acetate buffer A is 500 mM Tris, pH=8.2, and the final concentration is 5 mM Tris; the concentration of the sodium chloride solution is 1 M sodium chloride, and the final concentration is 300 mM sodium chloride; the concentration of the Tris-acetate buffer B is 300 mM sodium chloride, 25 mM Tris, pH=8.

2.

9. Use of the spherical nucleic acid nanoparticles modified with aptamers according to any one of claims 1 to 8 in the preparation of drugs for targeted degradation of programmed cell death-ligand 1 on cell membranes.

10. Use of the aptamer-modified spherical nucleic acid nanoparticles according to any one of claims 1 to 8 in preparing drugs for treating cancer.

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