Imitated IgM multivalent nucleic acid aptamer-albumin nano-carrier as well as preparation method and application thereof

Abstract

The invention discloses an IgM (Immunoglobulin M)-imitated multivalent nucleic acid aptamer-albumin nano-carrier, and belongs to the technical field of anti-tumor drug delivery. The nano-carrier is formed by self-assembly of an IgM-like nucleic acid aptamer (TYA) and albumin (HSA) through hydrophobic interaction; the TYA is prepared by carrying out amidation reaction on poly (maleic anhydride-alt-1-octadecene) (C18PMH), Boc-PEG-NH2 and a COOH <-> modified nucleic acid aptamer through covalent coupling on the poly (maleic anhydride-alt-1-octadecene) (C18PMH) and the Boc-PEG-NH2. According to the present invention, the IgM-imitated series Y-shaped structure design is adopted, such that the problems of poor stability and insufficient targeting binding force of the existing aptamer modified albumin carrier are solved, the occupation of the albumin hydrophobic cavity is reduced, the drug loading rate is increased to 1.686%, and the in-vivo retention time is prolonged to 8 h. The nano-carrier has excellent tumor targeting property and anti-tumor effect, and a novel efficient carrier is provided for targeted delivery of anti-tumor drugs.

Description

Technical Field

[0001] This invention relates to the field of antitumor drug delivery technology, specifically to an IgM-like multivalent nucleic acid aptamer-albumin nanocarrier, its preparation method, and its application. Background Technology

[0002] The information disclosed in the background section of this invention is intended only to enhance the understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Albumin (HSA) is a commonly used drug carrier material. To enhance its accumulation effect at tumor sites, it is often necessary to modify its surface with targeting ligands. Nucleic acid aptamers, as short-chain single-chain oligonucleotides, can specifically recognize tumor-related proteins, cells, and other targets, and have high affinity, making them ideal targeting ligands.

[0004] In existing technologies, nucleic acid aptamer modification of albumin is mainly divided into two categories: covalent modification and non-covalent modification. Covalent modification is complex, requiring multiple purification steps, which not only increases preparation costs but may also damage the native structure of albumin. Non-covalent modification (such as hydrophobic interactions and electrostatic adsorption), although simple to operate and under mild conditions, conventional non-covalent modification adopts a "one hydrophobic long chain binds to one targeting ligand" model, which makes the nucleic acid aptamer prone to detachment from the albumin surface, resulting in insufficient binding force to the target. At the same time, conventionally modified nucleic acid aptamers compete with hydrophobic drugs for the limited hydrophobic cavity of albumin, significantly reducing the drug loading rate and limiting its application in antitumor drug delivery.

[0005] Therefore, developing an albumin nanocarrier that is easy to prepare, highly stable, has good targeting properties, and high drug loading capacity has become a technical problem that urgently needs to be solved in this field. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide an IgM-like multivalent nucleic acid aptamer-albumin nanocarrier, which solves the technical problems of poor stability, insufficient targeting binding force, and low drug loading rate of existing nucleic acid aptamer-modified albumin carriers, while improving the targeted delivery efficiency of antitumor drugs by albumin carriers.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: An IgM-mimicking multivalent nucleic acid aptamer-albumin nanocarrier comprises an IgM-mimicking nucleic acid aptamer (TYA) and albumin (HSA), wherein the IgM-mimicking nucleic acid aptamer is composed of poly(maleic anhydride-alt-1-octadecene) (C 18PMH), Boc-PEG-NH2 and COOH-modified nucleic acid aptamers (COOH-Aptamer) are covalently coupled via an amide reaction; the IgM-like nucleic acid aptamer and albumin are self-assembled through hydrophobic interactions to form a tandem Y-type multivalent nucleic acid aptamer-albumin nanocarrier (TYA-HSA), which is used for targeted delivery of antitumor drugs.

[0008] Furthermore, the COOH-modified nucleic acid aptamers include COOH-Sgc8 and COOH-AS1411.

[0009] Furthermore, the aforementioned anti-tumor drugs include paclitaxel (PTX).

[0010] This invention also provides a method for preparing the IgM-mimicking multivalent nucleic acid aptamer-albumin nanocarrier as described above, comprising the following steps: S1, Poly(maleic anhydride-alt-1-octadecene) (C 18 PMH), Boc-PEG-NH2 and 1-ethyl-3-dimethylaminopropylcarbodiimide were dissolved in an organic solvent, stirred at room temperature for 24 hours, and then the solvent was dried. S2, add trifluoroacetic acid and stir at room temperature for 4 hours to remove the Boc protecting group, synthesizing C. 18 PMH-PEG-NH2 was evaporated to dryness, then dialyzed for 2 days using a dialysis bag, and then freeze-dried. S3. The dried product was mixed with the COOH-modified nucleic acid aptamer in DMSO and reacted at room temperature for 24 hours. After dialysis with water, the mixture was freeze-dried to obtain the IgM-like multivalent nucleic acid aptamer. S4. Prepare an aqueous solution of albumin and disperse it by ultrasonication. Add the IgM-like multivalent nucleic acid aptamer and incubate for 4 hours to obtain the IgM-like multivalent nucleic acid aptamer-albumin nanocarrier.

[0011] Furthermore, Boc-PEG-NH2 and the COOH-modified nucleic acid aptamer are added in a molar ratio of 10:1.

[0012] Preferably, in step S2, the molecular weight cutoff of the dialysis bag is 14 kDa; in step S3, a dialysis membrane with a molecular weight cutoff of 20 kDa is used in the water dialysis process.

[0013] Preferably, in step S4, the incubation conditions are: 37°C and 300 rpm / min.

[0014] The present invention also provides the application of the above-described IgM-like multivalent nucleic acid aptamer-albumin nanocarrier in the preparation of targeted antitumor drugs.

[0015] The beneficial effects of this invention are as follows: 1) Significantly improved stability: Achieving C through amide reaction 18 The covalent coupling of PMH-PEG-NH2 and COOH-Aptamer forms an IgM-like tandem Y-shaped structure, which prevents the nucleic acid aptamer from detaching from the albumin surface and prolongs the in vivo retention time to 8 hours (current HSA vectors only last 2 hours). 2) Enhanced targeting: The IgM-like nucleic acid aptamer can specifically recognize tumor surface targets, and its targeting binding force and tissue penetration ability are superior to conventional modified vectors; 3) Improved drug loading: The tandem Y-shaped structure design reduces the occupation of the hydrophobic cavity of albumin, avoiding competition with hydrophobic drugs, and the drug loading rate increases from 1.392% with conventional modification to 1.686%; 4) Simple preparation process: It uses a non-covalent self-assembly method to bind albumin, which is simple to operate, mild under mild conditions, and does not require complicated purification steps, thus reducing preparation costs and not destroying the natural structure of albumin.

[0016] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it in accordance with the contents of the specification, the preferred embodiments of the present invention are described in detail below with reference to the accompanying drawings. Attached Figure Description

[0017] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, some of the drawings in the following description are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0018] Figure 1 This is a schematic diagram of the structure of PTX@TYS-HSA as shown in Embodiment 1 of the present invention; Figure 2 The image shown in Example 2 of this invention is a gel electrophoresis diagram of TYS. Channel 1 is the Marker, channel 2 is COOH-Sgc8, and channel 3 is TYS. Figure 3 The ultraviolet spectrum of TYS-HSA shown in Example 2 of this invention; Figure 4 This is a confocal image of a tumor sphere as shown in Embodiment 3 of the present invention; Figure 5 This is a confocal image of tumor sphere targeted killing as shown in Embodiment 3 of the present invention. Detailed Implementation

[0019] To better describe the present invention, specific embodiments are provided below for further explanation. Unless otherwise specified, the methods in the following embodiments are conventional methods.

[0020] Unless otherwise specified, the technical solutions described in this invention are all conventional solutions in the field; unless otherwise specified, the reagents or materials described are all from commercial sources.

[0021] The following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains. The following examples are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental conditions not specifically stated in the examples are generally performed under conventional conditions or as recommended by the reagent company; reagents, consumables, etc., used in the following examples are commercially available unless otherwise specified.

[0022] Example 1: Preparation of IgM-like nucleic acid aptamer-albumin (TYS-HSA) vector 1.1 Preparation of IgM-like nucleic acid aptamers (TYS): 10mgC 18 PMH was dissolved in 4 mL of dichloromethane and stirred until fully dissolved. Then, 193 mg of Boc-PEG(5k)-NH2 and 11 mg of EDC, which had been pre-dissolved in 1 mL of dichloromethane, were added. After stirring at room temperature for 24 hours, the dichloromethane solvent was dried under nitrogen.

[0023] Next, 2 mL of trifluoroacetic acid was added under magnetic stirring, and the mixture was stirred at room temperature for 4 hours to remove the Boc protecting group. After evaporating the trifluoroacetic acid solvent, the remaining solid was dissolved in water and dialyzed for 2 days in a dialysis bag with a molecular weight cutoff of 14 kDa to remove unreacted PEG polymers and other reagents.

[0024] After freeze-drying, the final product (C 18 PMH-PEG-NH2) is stored as a white solid at -20°C for later use. It is used to connect the COOH-modified nucleic acid aptamer with C... 18 PMH-PEG-NH2 coupling, drying product C 18 PMH-PEG-NH2 and COOH-Sgc8 were mixed in DMSO, wherein the amount of COOH-Sgc8 added was 1 / 10 (molar amount) of Boc-PEG(5k)-NH2. After reacting at room temperature for 24 hours, the product was dialyzed through a 20kDa membrane with water and then freeze-dried to obtain C. 18 PMH-PEG-Sgc8(TYS).

[0025] 1.2 Preparation of TYS-HSA nanocarriers: An albumin solution (40 mg / mL, dissolved in distilled water) was prepared and ultrasonically dispersed for 5 minutes. Subsequently, 1 μM TYS was added to the albumin solution and incubated for 4 hours at 37°C and 300 rpm / min on an Eppendorf thermostat to obtain TYS-HSA.

[0026] The TYS-HSA nanocarriers prepared above can be used to load and deliver antitumor drugs such as paclitaxel. Figure 1 The assembly relationship between the IgM-like nucleic acid aptamer and albumin and the drug loading mode after paclitaxel was loaded onto the TYS-HSA nanocarrier were demonstrated.

[0027] Example 2: Synthesis and Verification of TYS-HSA Nanocarriers 2.1 Synthesis Verification of TYS: C was verified using 10% polyacrylamide gel. 18 To check the successful preparation of PMH-PEG-Sgc8: Mix ddH2O, 5×TBE buffer, 30% Acr-Bis (29:1), ammonium sulfate (10% APS), and tetramethylethylenediamine (TETMD) in a specific ratio, quickly pour into a gel preparation rack, and let stand at room temperature for 10 min until it solidifies. Mix 10 μL of COOH-Sgc8 and TYS thoroughly with 2 μL of 6×GelRed loading buffer, then add them sequentially to the sample wells of the gel. Run at 120V for 30 min. After completion, select the GelRed channel for imaging. See [link to documentation]. Figure 2 The TYS band lagged significantly behind COOH-Sgc8, indicating that COOH-Sgc8 and C... 18 PMH-PEG-NH2 successfully covalently bonded, resulting in a significant increase in molecular weight.

[0028] 2.2 Synthesis Verification of TYS-HSA: See Figure 3 Using a UV-Vis spectrophotometer, TYS-HSA was scanned, and characteristic absorption peaks of HSA-FITC and TYS-Cy5 were detected at 488 nm and 646 nm, respectively, proving that TYS and HSA were successfully coupled.

[0029] Example 3: Drug delivery and antitumor effects of TYS-HSA 3.1 In vitro targeting experiment: MB49 cells were seeded in ultra-low adsorption 96-well plates and cultured into tumor spheroids by centrifugation. Fluorescent isothiocyanate (FITC)-labeled HSA was used for fluorescence imaging experiments.

[0030] The experiment was divided into two groups: HSA-C 18S (MS-HSA) and HSA-C 18 PMH-PEG-Sgc8 (TYS-HSA). Tumor spheres were incubated with 0.4 mg / mL MS-HSA and TYS-HSA at 4°C in the dark for 1 hour. Cells were then gently washed three times with washing buffer to remove unbound dye. Imaging was performed using a Leica confocal scanning microscope (Leica, TCS SP8, Germany) to verify the targeting and penetration capabilities of TYS-HSA.

[0031] Please see Figure 4 The fluorescence intensity inside the tumor spheres in the TYS-HSA group was significantly higher than that in the MS-HSA group, indicating that TYS-HSA has better targeting and deeper penetration.

[0032] 3.2 In vivo targeting experiments: A mouse orthotopic bladder cancer model was constructed. A 0.7×19 mm indwelling needle was gently inserted into the mouse bladder through the urethra. 200 μL of TYS-HSA (concentration of 20 mg / mL, calculated as HSA-Cy5) was injected into the mice with bladder cancer. Fluorescence accumulation at the tumor site was observed at 1, 2, 4, and 8 hours using an IVIS Lumina device. After 24 hours, liver, spleen, kidney, heart, lung, and bladder tissues were collected for in vitro imaging, and fluorescence intensity was measured using ImageJ software. The IVIS Lumina device results showed that the fluorescence intensity at the tumor site remained high even after 8 hours, and after 24 hours, the fluorescence intensity of the bladder tissue was significantly higher than that of other organs. This indicates that the nanocarrier can significantly enhance the binding force to the target and prolong its retention time in vivo.

[0033] 3.3 Antitumor effect experiment: Female mice with MB49-luc tumors were randomly divided into four groups (n=4 per group): PBS, PTX@HSA, PTX@TYR-HSA, and PTX@TYS-HSA groups. Each treatment involved urethral instillation of 200 μL of the drug (at a concentration of 13 mg / kg PTX@HSA) for 1 hour. Mice were treated on days 0, 3, 7, 10, 14, 16, 21, and 24. Tumors were visualized using the IVIS Lumina system on days 0, 5, 12, 19, and 25 via intraperitoneal injection of D-fluorescein potassium (10 mg / kg). On day 26, the mice were euthanized, and their bladders were collected.

[0034] Bladder tissue was collected post-treatment for histopathological analysis. It was first immersed in 4% paraformaldehyde, followed by paraffin embedding. The paraffin-embedded tumor sections were stained with hematoxylin and eosin (H&E). TUNEL assay, Ki67 and γ-H2AX immunohistochemical staining, and CD8 immunofluorescence staining were performed according to standard procedures. Please refer to [link to relevant documentation]. Figure 5 The results showed that the PTX@TYS-HSA group had a larger area of ​​tumor tissue necrosis, more apoptotic cells, and CD8+. + Increased T-cell infiltration significantly enhances the therapeutic effect on tumors compared to other agents.

[0035] In summary, this invention provides an IgM-inspired multivalent nucleic acid aptamer-albumin nanocarrier and its preparation method, which achieves C0.05 through an amide reaction. 18 The covalent coupling of PMH-PEG-NH2 and COOH-Aptamer forms an IgM-like tandem Y-shaped structure, preventing the nucleic acid aptamer from detaching from the albumin surface and extending the in vivo retention time to 8 hours (compared to only 2 hours for existing HSA vectors). The IgM-like nucleic acid aptamer can specifically recognize tumor surface targets, and its targeting binding force and tissue penetration are superior to conventional modified vectors. The tandem Y-shaped structure design reduces the occupation of the hydrophobic cavity of albumin, avoiding competition with hydrophobic drugs, and increasing the drug loading rate from 1.392% in conventional modifications to 1.686%. The albumin is bound by a non-covalent self-assembly method, which is simple to operate, mild under mild conditions, requires no complex purification steps, reduces preparation costs, and does not damage the native structure of albumin.

[0036] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0037] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A nanocarrier mimicking an IgM-based multivalent nucleic acid aptamer-albumin, characterized in that, Includes an IgM-like nucleic acid aptamer and albumin, wherein the IgM-like nucleic acid aptamer is composed of poly(maleic anhydride-alt-1-octadecene) (C 18 The nucleic acid aptamers modified with PMH, Boc-PEG-NH2 and COOH- are covalently coupled via an amide reaction; the IgM-like nucleic acid aptamer and albumin self-assemble through hydrophobic interactions to form a tandem Y-type multivalent nucleic acid aptamer-albumin nanocarrier.

2. The IgM-mimicking multivalent nucleic acid aptamer-albumin nanocarrier as described in claim 1, characterized in that, The COOH-modified nucleic acid aptamers include COOH-Sgc8 and COOH-AS1411.

3. A method for preparing an IgM-mimicking multivalent nucleic acid aptamer-albumin nanocarrier as described in any one of claims 1-2, characterized in that, Includes the following steps: S1, Poly(maleic anhydride-alt-1-octadecene) (C 18 PMH), Boc-PEG-NH2 and 1-ethyl-3-dimethylaminopropylcarbodiimide were dissolved in an organic solvent, stirred at room temperature for 24 hours, and then the solvent was dried. S2, add trifluoroacetic acid and stir at room temperature for 4 hours to remove the Boc protecting group, synthesizing C. 18 PMH-PEG-NH2 was evaporated to dryness, then dialyzed for 2 days using a dialysis bag, and then freeze-dried. S3. The dried product was mixed with the COOH-modified nucleic acid aptamer in DMSO and reacted at room temperature for 24 hours. After dialysis with water, the mixture was freeze-dried to obtain the IgM-like multivalent nucleic acid aptamer. S4. Prepare an aqueous solution of albumin and disperse it by ultrasonication. Add the IgM-like multivalent nucleic acid aptamer and incubate for 4 hours to obtain the IgM-like multivalent nucleic acid aptamer-albumin nanocarrier.

4. The preparation method according to claim 3, characterized in that, Boc-PEG-NH2 and the COOH-modified nucleic acid aptamer were added at a molar ratio of 10:

1.

5. The preparation method according to claim 3, characterized in that, In step S2, the molecular weight cutoff of the dialysis bag is 14 kDa; in step S3, a dialysis membrane with a molecular weight cutoff of 20 kDa is used in the water dialysis process.

6. The preparation method according to claim 3, characterized in that, In step S4, the incubation conditions are: 37℃ and 300 rpm / min.

7. The application of an IgM-like multivalent nucleic acid aptamer-albumin nanocarrier prepared by any one of claims 1-2 or any one of claims 3-6 in the preparation of targeted antitumor drugs.

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