A method for preparing spherical nucleic acids based on a cyclic template strategy and uses thereof

Abstract

The application discloses a spherical nucleic acid preparation method based on a cyclic template strategy and application, and comprises the following steps: A) using controllable free radical polymerization (ATRP) and intramolecular click chemistry of copper-catalyzed alkyne and azido, a cyclic polymer is prepared c -P(HEMA) 30 B) using continuous esterification and substitution reaction, the cyclic polymer side chain terminal hydroxyl group is substituted by azido, and a cyclic template c -P(HEMA-N3) 30 C) using copper-free click chemistry reaction of a dibenzo cyclooctyne group (DBCO) and azido, nucleic acid is covalently coupled to the cyclic template, and a cyclic brush macromolecule c -P(HEMA-RNA) 30 The obtained cyclic brush macromolecule is subjected to water dialysis, and spherical nucleic acid is prepared through self-assembly. The spherical nucleic acid prepared by the preparation method has good reproducibility, good in-vitro stability, can better protect the nucleic acid from degradation of RNA degradation enzyme, and can greatly improve the nucleic acid uptake capacity of cells.

Description

Technical Field

[0001] This invention relates to the field of medical biomaterials, and in particular to a novel method for constructing and preparing spherical nucleic acids based on a circular template strategy. Background Technology

[0002] Spherical nucleic acids (SNAs) are three-dimensional spherical geometries composed of a central core layer and a highly oriented, dense oligonucleotide layer (Mokhtarzadeh A, Vahidnezhad H, Youssefian L, et al. Trends inmolecular medicine, 25(12): 1066-1079, 2019). Over the past 20 years, the structure of SNAs has revolutionized the application of gene drugs in gene regulation, drug delivery, gene therapy, and molecular diagnostics, showing great promise in gene therapy. Spherical nucleic acid nanoparticles are a novel intracellular delivery system for bioactive molecules. Without significant off-target effects, immunogenicity, or cytotoxicity, they can serve as a promising carrier for delivering antisense oligonucleotide chains and immunomodulators (Cutler JI, Zhang K, Zheng D, et al. Journal of the American Chemical Society, 133(24): 9254-9257, 2011). Various single-stranded (ss) and double-stranded (ds) oligonucleotides, typically nucleic acids with 25-40 nt bases and an actual length of 7-12 nm, such as DNA, RNA, peptide nucleic acids (PNA), miRNA, small interfering RNA (siRNA), and long chain nucleic acids (LNA), have been coupled to inorganic nanoparticle cores to generate spherical nucleic acid structures (Guan C, Chernyak N, Dominguez D, et al. Small, 14(49): 1803284, 2018). Furthermore, compared to cationic nanocarriers, SNA structures elicit minimal immune stress (a 25-fold reduction in immune response). Due to the high density of oligonucleotide chains on the surface of SNAs, they are less susceptible to degradation by nucleases compared to linear nucleic acids, thus exhibiting better stability. Unlike linear DNA, SNAs can be taken up by cells without the aid of transfection reagents. Although SNAs are negatively charged due to their high density of oligonucleotides (zeta potential of 30 mV), the 3D spherical structures they form can be recognized by class A scavenger receptors, allowing them to enter cells via endocytosis. They can be rapidly internalized in almost all cell types through caveolin-mediated endocytosis.

[0003] However, developed SNAs are prone to forming protein crowns by adsorbing positively charged proteins during blood circulation due to their dense nucleic acid shells, which are eventually cleared by the immune system. In contrast, spherical nucleic acid analogs formed by the self-assembly of amphiphilic linear molecules are less likely to form protein crowns because their surface nucleic acid chains are not as dense as those of traditional SNAs. However, nanoparticles formed by the self-assembly of linear molecules are not stable under conditions of high salt concentration and large dilution in body fluids.

[0004] With advancements in polymer synthesis techniques and deeper research in this field, the preparation of nonlinear polymers has become possible. These polymers can possess various topological structures, including star-shaped, branched, canopy-like, and cyclic structures. Currently, topological polymer chemistry is transforming from a synthetic method into a powerful tool for designing advanced materials, particularly in biomedical applications. The topological effects resulting from the complex structures of polymers are increasingly being used to manufacture materials and coatings that can provide functions such as diagnostics, bioimaging, drug and gene delivery, tissue engineering, and antibacterial properties. Notably, although numerous studies have reported comparing the different performances of linear, star-shaped, grafted, and branched polymers in biomedical applications, cyclic polymers, due to the lack of chain ends, exhibit significantly different physicochemical properties and performance compared to linear polymers, attracting widespread research interest. Cyclic polymer nanoparticles, formed through self-assembly in aqueous solutions, exhibit smaller size and better stability compared to linear analogs. Summary of the Invention

[0005] To address the aforementioned problems, this invention discloses a method for preparing spherical nucleic acids based on a circular template strategy and its applications. The spherical nucleic acids prepared using this method exhibit good reproducibility, good in vitro stability, and effectively protect nucleic acids from degradation by RNA-degrading enzymes, while also significantly enhancing the cellular uptake of nucleic acids.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows:

[0007] A method for preparing spherical nucleic acids based on a circular template strategy includes the following steps:

[0008] Step 1: Preparation of cyclic polymers c -P(HEMA) 30 ;

[0009] Step 2: Substitute the cyclic polymer with azide groups. c -P(HEMA) 30 Terminal hydroxyl groups yield cyclic templates c -P(HEMA-N3) 30 ;

[0010] Step 3: Chemically couple nucleic acids to a circular templatec -P(HEMA-N3) 30 Above, cyclic macromolecules were obtained. c -P(HEMA-RNA) 30 ; cyclic macromolecules c -P(HEMA-RNA) 30 Spherical nucleic acids (SNAs) were prepared by self-assembly through water dialysis.

[0011] A further improvement is made in step one, where the cyclic polymer... c -P(HEMA) 30 The preparation method is as follows:

[0012] 1.1 Obtained using ATRP polymerization reaction l -P(HEMA) 30 -Br;

[0013] 1.2. Through nucleophilic substitution reactions... l -P(HEMA) 30 The -Br terminal bromine atom is converted into an azide group, resulting in l -P(HEMA) 30 -N3;

[0014] 1.3, by l -P(HEMA) 30 -N3 undergoes an intramolecular click chemistry reaction to yield a cyclic polymer. c -P(HEMA) 30 .

[0015] Further improvements, the l -P(HEMA) 30 The preparation method of -Br is as follows:

[0016] 410 parts by weight of the small molecule initiator 2-bromoisobutyrate propynyl ester, 6.64 parts by weight of hydroxyethyl methacrylate, and 425 parts by volume of N,N,N',N,'N''-pentamethyldiethylenetriamine were dissolved in a mixed solution and stirred until homogeneous. N2 was bubbled through the solution for 30 min, then 290 parts by weight of cuprous bromide were added and the mixture was stirred at 65°C for 20 min. Excess ice-cold diethyl ether was then added to precipitate the crude product. The crude product was dissolved in DMF solution and transferred to a 500 Da dialysis bag. Dialysis was performed with water for 48 h, followed by lyophilization to obtain a white solid, which is the product. l -P(HEMA) 30 -Br; where the mixed solution is prepared by mixing DMF and IPA in a volume ratio of 1:9; the ratio of parts by weight to parts by volume is expressed in g / ml.

[0017] A further improvement is made to the nucleophilic substitution reaction as follows:

[0018] Take 1 part by weight l- P(HEMA) 30 -Br and 0.158 parts by weight of NaN3 were dissolved in 2 parts by volume of DMF and 0.5 parts by volume of water, respectively. The mixture was reacted at 45°C for 48 h to obtain a reaction solution. The reaction solution was transferred to a 500 Da dialysis bag and dialyzed against water for 48 h. After lyophilization, a light yellow solid was obtained, which is the product. l -P(HEMA) 30 -N3.

[0019] Further improvements, the steps for intramolecular click chemistry are as follows:

[0020] Add 850 parts by volume of DMF to a flask, heat to 100 °C, bubble with N2 for 1 h, then add 0.506 parts by volume of PMDETA and 0.418 parts by weight of CuBr sequentially, and dissolve 0.5 parts by weight of [unspecified substance] in 10 parts by volume of DMF. l -P(HEMA) 30 -N3, N2 bubbled for 1 hour, to obtain l- P(HEMA) 30 -N3 solution; then... l- P(HEMA) 30 -N3 solution was injected into the flask and the reaction continued for 48 h. After the reaction was completed, excess ice-cold diethyl ether precipitated the product. The precipitate was dissolved in DMF and transferred to a 500 Da dialysis bag for dialyzing with water. After dialysis for 48 h, the product was lyophilized to obtain a white product. c -P(HEMA) 30 .

[0021] Further improvements are made, and the specific steps of step two are as follows:

[0022] Take 0.1 parts by weight c- P(HEMA) 30 Dissolve in 4 parts by volume of DMF, stir in an ice-water bath for 10 min, then add 0.324 parts by volume of 2-bromoisobutyryl bromide dropwise, and continue the reaction at room temperature for 24 h. After the reaction is complete, add excess diethyl ether to precipitate, dissolve the precipitate in DMF and transfer to a 500 Da dialysis bag for dialyzing against water for 48 h, then freeze-dry to obtain a white solid powder. c -P(HEMA-Br) 30 ;

[0023] 0.135 parts by weight c -P(HEMA-Br) 300.311 parts by weight of sodium azide was dissolved in 3 parts by volume of DMF solution and reacted at 45°C for 48 h. After the reaction was completed, the reaction solution was transferred to a 500 Da dialysis bag and dialyzed against water for 48 h. The solution was then lyophilized to obtain a pale yellow solid powder. c -P(HEMA-N3) 30 .

[0024] A further improvement is made in step three, using an annular template. c -P(HEMA-N3) 30 Synthesis of cyclic macromolecules via copper-free click chemistry of dibenzocyclooctynyl and azide groups. c -P(HEMA-RNA) 30 .

[0025] Further improvements are made, and the specific steps of step three are as follows: Dissolve 4 OD DBCO-RNA in 3 μL of DEPC water, 7.49 μg c -P(HEMA-N3) 30 Dissolve in 30 μL DMSO, mix well, and react at 50 ℃ for 48 h. After the reaction is complete, remove DMSO by dialyzing. Remove unreacted DBCO-RNA by ultrafiltration using a 50 kDa ultrafiltration centrifuge tube to obtain the cyclic macromolecule. c -P(HEMA-RNA) 30 .

[0026] A further improvement is that the nucleic acid is RNA.

[0027] A further improvement is that the RNA is a miRNA, and the miRNA includes miR-122.

[0028] One use of a spherical nucleic acid, as shown above; the spherical nucleic acid is used as a carrier to prevent the nucleic acid loaded on the spherical nucleic acid from being degraded by nucleic acid-degrading enzymes.

[0029] Advantages of this invention:

[0030] Beneficial effects of the technical solution of this invention

[0031] The SNAs prepared by this invention have good reproducibility, greatly improve the in vitro stability of nucleic acids, protect nucleic acids from degradation by nucleic acid degrading enzymes to a certain extent, and greatly improve the cells' ability to take up nucleic acids. Attached Figure Description

[0032] Figure 1 It is a cyclic macromolecule c Synthetic route of -P(HEMA-RNA)30.

[0033] Figure 2 forl -P(HEMA) 30 -Br 1 H NMR spectrum (DMSO- d 6).

[0034] Figure 3 l- P(HEMA) 30 -Br and l- P(HEMA) 30 FT-IR plot of -N3.

[0035] Figure 4 c -P(HEMA) 30 -Br 1 H NMR spectrum (DMSO- d 6).

[0036] Figure 5 for l -P (HEMA) 30 -Br, l -P(HEMA) 30 -N3 and c -P(HEMA) 30 SEC rinsing curve.

[0037] Figure 6 for c -P(HEMA-Br) 30 of 1 H NMR spectrum (DMSO-) d 6).

[0038] Figure 7 for c -P(HEMA-N3) 30 of 1 H NMR spectrum (DMSO- d 6).

[0039] Figure 8 for c -P(HEMA-Br) 30 and c -P(HEMA-N3) 30 FT-IR plot.

[0040] Figure 9 Free miR-122 (left), annular template c -P(HEMA-N3) 30 (Medium) and c -P(HEMA-miR-122) 30(Right) Non-denaturing polyacrylamide gel electrophoresis image.

[0041] Figure 10 for c -P(HEMA-RNA) 30 DLS particle size distribution of SNAs.

[0042] Figure 11 c -P(HEMA-RNA) 30 Transmission electron microscopy image of SNAs.

[0043] Figure 12 for c -P(HEMA-RNA) 30 Particle size variation of SNAs placed for different time periods. Data are expressed as mean ± standard deviation, n=3.

[0044] Figure 13 for c -P(HEMA-RNA) 30 The particle size variation of SNAs after dilution with ultrapure water at different concentrations. Data are expressed as mean ± standard deviation, n=3.

[0045] Figure 14 for c -P(HEMA-RNA) 30 Particle size variation of SNAs after dilution in different media. Data are expressed as mean ± standard deviation, n=3.

[0046] Figure 15 For free miR-122 and c -P(HEMA-RNA) 30 Gel electrophoresis images of the nanocomposite after incubation with RNase A for different times.

[0047] Figure 16 Hemolysis rates of different formulations after incubation with red blood cells at 37°C for 3 hours. Data are expressed as mean ± standard deviation, n=3. * P <0.05,** P <0.01, *** P <0.001.

[0048] Figure 17 for c -P(HEMA-RNA) 30 Fluorescence imaging of SNAs after incubation with Bel-7402 cells for 8 h (blue represents Hoechst 33342-labeled cell nuclei, green represents FAM-labeled miR-122; scale bar is 200 μm, miR-122 concentration is 100 nM). Implementation

[0049] The present invention will be further described below with reference to the accompanying drawings and embodiments. Example

[0050] This invention provides a novel method for preparing SNAs based on a ring template strategy, comprising the following steps:

[0051] A) Cyclic polymers were obtained using atom-controlled radical polymerization (ATRP) followed by copper-catalyzed intramolecular click chemistry of alkynyl and azido groups. c -P(HEMA) 30 ;

[0052] B) Using esterification and substitution reactions, the terminal hydroxyl groups of the cyclic polymer are replaced with azide groups to obtain a cyclic template. c -P(HEMA-N3) 30 ;

[0053] C) Using a copper-free click chemistry reaction between dibenzocyclooctynyl (DBCO) and an azide group, nucleic acids are chemically coupled to a cyclic template to obtain cyclic macromolecules. c -P(HEMA-RNA) 30 The obtained cyclic macromolecules were dialyzed in a hydrophobic environment and self-assembled under hydrophilic-hydrophobic interactions to eventually form SNAs.

[0054] This invention first utilizes atom-controlled radical polymerization (ATRP) followed by copper-catalyzed intramolecular click chemistry of alkynyl and azide groups to obtain cyclic polymers. c -P(HEMA) 30 Specifically, linear polymers were synthesized using ATRP polymerization. l -P(HEMA) 30 -Br, then using sodium azide to replace the terminal bromine atom of the linear polymer chain with an azide group, synthesizing l -P(HEMA) 30 -N3. Then, using copper-catalyzed click chemistry between alkynyl and azide groups, the linear polymer is coupled head-to-tail to form a cyclic polymer. c -P(HEMA) 30 .

[0055] In embodiments of the present invention, the solvent for the ATRP polymerization reaction is DMF / IPA (V / V=1:9), and in some embodiments, the solvent is acetone.

[0056] In some embodiments of the present invention, the reaction temperature of the ATRP polymerization reaction is 65°C and the reaction time is 20 min. In some embodiments, the reaction temperature is 60°C or 50°C and the reaction time is 1 h or 30 min.

[0057] In an embodiment of the present invention, the intramolecular click chemistry reaction temperature is 100°C, the time is 48 h, and the solvent is DMF.

[0058] Subsequent in cyclic polymers c -P(HEMA) 30 Based on this, esterification and substitution reactions are used to replace the terminal hydroxyl groups of the cyclic polymer with azide groups to obtain a cyclic template. c -P(HEMA-N3) 30 Specifically, this involves esterifying the hydroxyl groups at the ends of the side chains of a cyclic polymer with bromoisobutyryl bromide to synthesize a cyclic molecule. c -P(HEMA-Br) 30 Using sodium azide to cyclic molecules c -P(HEMA-Br) 30 The bromine atom at the end of the branch chain is replaced with an azide group to synthesize the final cyclic template. c -P(HEMA-N3) 30 .

[0059] In an embodiment of the present invention, the esterification reaction temperature is 25°C and the reaction time is 24 h.

[0060] In an embodiment of the present invention, the substitution reaction solvent is anhydrous DMF, the reaction temperature is 45°C, and the reaction time is 48h.

[0061] Finally, using a copper-free click chemistry reaction between dibenzocyclooctynyl (DBCO) and an azide group, nucleic acids are click-coupled to a cyclic template to obtain a cyclic macromolecule. c -P(HEMA-RNA) 30 The obtained circular macromolecules were dialyzed in a hydrophobic environment and self-assembled under hydrophilic-hydrophobic interactions to form SNAs. Specifically, DBCO-modified RNA (DBCO-RNA) and a circular template were used. c -P(HEMA-N3) 30 A copper-free click chemistry reaction occurs between the alkyne and azide groups, which click-couples RNA to a circular template, synthesizing a cyclic macromolecule. c -P(HEMA-RNA) 30 Unreacted DBCO-RNA was removed by ultrafiltration. The resulting cyclic macromolecules were then... c -P(HEMA-RNA) 30 Dissolve in DMSO and dialyze in water for 48 hours to allow it to form SNAs through hydrophilic-hydrophobic self-assembly.

[0062] In embodiments of the present invention, the reaction solvents for the click chemical reaction are dimethyl sulfoxide (DMSO) and water (V:V=5:1), and in some embodiments, the reaction solvents are DMSO:DMF (V / V=1:1).

[0063] In embodiments of the present invention, the click chemical reaction temperature is 50°C and the reaction time is 48 h. In some embodiments, the reaction temperature is 40°C or 60°C.

[0064] In embodiments of the present invention, the ratio of the circular template to nucleic acid is 1:30, and in some embodiments, the ratio is 1:1 or 1:1.2.

[0065] In embodiments of the present invention, the purification method for spherical nucleic acids is ultrafiltration, and in some embodiments, the purification method is high-performance liquid chromatography (HPLC).

[0066] The present invention does not impose any special restrictions on the source of the raw materials used above, and they can be commercially available.

[0067] This invention also provides a novel SNAs prepared by the method described above. The spherical nucleic acids prepared by the method of this invention exhibit good reproducibility, good in vitro stability, and can effectively protect nucleic acids from degradation by RNA-degrading enzymes, while significantly improving cellular uptake of nucleic acids.

[0068] The method for preparing SNAs provided by this invention includes: A) obtaining cyclic polymers by using atom-controlled radical polymerization (ATRP) and subsequent copper-catalyzed intramolecular click chemistry of alkynyl and azide groups. c -P(HEMA) 30 B) By using esterification and substitution reactions, the terminal hydroxyl groups of the cyclic polymer are replaced with azide groups to obtain a cyclic template. c -P(HEMA-N3) 30 C) Using a copper-free click chemistry reaction between dibenzocyclooctynyl (DBCO) and an azide group, nucleic acids are chemically coupled to a cyclic template to obtain cyclic macromolecules. c -P(HEMA-RNA) 30 The obtained cyclic macromolecules were dialyzed in a hydrothermal environment, and under the influence of hydrophilic and hydrophobic interactions, they self-assembled to form spherical nucleic acids. The SNAs prepared using the method of this invention exhibit good reproducibility, good in vitro stability, and can effectively protect nucleic acids from degradation by RNA-degrading enzymes, while also significantly improving the cellular uptake of nucleic acids.

[0069] Experimental results show that the SNAs prepared by this invention have good reproducibility, greatly improve the in vitro stability of nucleic acids, protect nucleic acids from degradation by nucleic acid degrading enzymes to a certain extent, and compared with free nucleic acid groups, the constructed novel SNA structure can greatly improve the cells' ability to take up nucleic acids.

[0070] To further illustrate the present invention, the following detailed description of the construction and preparation method of a novel spherical nucleic acid based on a circular template strategy provided by the present invention is provided in conjunction with embodiments, but it should not be construed as limiting the scope of protection of the present invention.

[0071] The raw materials used in the following examples are all commercially available. Example

[0072] l -P(HEMA) 30 The synthesis of Br-2-bromoisobutyrate (HEMA) was carried out using hydroxyethyl methacrylate (HEMA) as the monomer, propyne 2-bromoisobutyrate (alkyne-Br) as the initiator, and N,N,N',N,'N''-pentamethyldiethylenetriamine / CuBr as the catalyst via ATRP polymerization. The specific procedure was as follows: 410 mg of the small molecule initiator propyne 2-bromoisobutyrate (HEMA), 6.64 g of hydroxyethyl methacrylate (HEMA), and 425 μL of N,N,N',N,'N''-pentamethyldiethylenetriamine (PDETA) were dissolved in a 25 mL mixture of DMF and IPA (V / V = 1 / 9). The mixture was stirred at room temperature for 10 min to ensure complete dissolution and mixing of all raw materials. The solution was then transferred to a 50 mL round-bottom flask. N2 was bubbled for 30 min. Then, 290 mg of cuprous bromide was added, and the mixture was stirred at 65 °C for 20 min, precipitating the bromide in excess ice-cold diethyl ether. The crude product was dissolved in a small amount of DMF solution, transferred to a dialysis bag (500 Da), dialyzed in water for 48 h, and then lyophilized to obtain a white solid. l -P(HEMA) 30 -Br. For example... Figure 2 and Figure 5 As shown, the successful synthesis of the product was verified by NMR and GPC (yield, 63.3%). Example

[0073] The terminal bromine atom of linear polymerization is converted to an azide group via a nucleophilic substitution reaction. Take 1g l- P(HEMA) 30 Br and 158 mg NaN3 were dissolved in 2 mL DMF and 0.5 mL water, respectively, and reacted at 45 °C for 48 h. The reaction solution was transferred to a dialysis bag (500 Da), dialyzed against water for 48 h, and then lyophilized to obtain a pale yellow solid. l -P(HEMA) 30 -N3. For example... Figure 3 As shown, infrared spectroscopy confirmed that the bromine atom was successfully substituted by an azide group (yield, 87.6%). Example

[0074] Under extremely rare conditions, l -P(HEMA)30 -N3 undergoes an intramolecular click chemistry reaction to yield a cyclic polymer. c -P(HEMA) 30 The specific procedure is as follows: 850 mL of DMF was added to a flask, heated to 100 °C, and bubbled with N2 for 1 h. Then, 506 μL of PMDETA and 418 mg of CuBr were added sequentially. Simultaneously, 500 mg of (…) was dissolved in 10 mL of DMF. l -P(HEMA) 30 -N3) polymer, N2 bubbled for 1 hour. Then use a syringe pump to... l- P(HEMA) 30 -N3 solution was slowly injected into the flask, and the reaction continued for 48 h. After the reaction was complete, excess ice-cold diethyl ether precipitated the product. The precipitate was dissolved in DMF and transferred to a dialysis bag (500 Da) for dialyzing with water. After dialysis for 48 h, the product was lyophilized to give a white product. c -P(HEMA) 30 .like Figure 4 and Figure 5 As shown, the cyclic polymer was verified using NMR and GPC. c -P(HEMA) 30 Successful synthesis. Example

[0075] Synthesized via esterification reaction c -P(HEMA-Br) 30 The specific steps are as follows: Take 100 mg c- P(HEMA) 30 Dissolve in 4 mL DMF, stir in an ice-water bath for 10 min, then add 324 μL of 2-bromoisobutyryl bromide dropwise, and continue the reaction at room temperature for 24 h. After the reaction is complete, excess diethyl ether precipitates the precipitate, which is dissolved in DMF and transferred to a dialysis bag (500 Da) for dialyzing with water for 48 h. Lyophilize to obtain a white solid powder. c -P(HEMA-Br) 30 .like Figure 6 As shown, NMR confirmed this. c -P(HEMA-Br) 30 Successful synthesis. (Yield, 62.6%) Example

[0076] 135 mg c -P(HEMA-Br) 30 311 mg of sodium azide was dissolved in 3 mL of DMF solution and reacted at 45 °C for 48 h. After the reaction was complete, the reaction solution was transferred to a dialysis bag (500 Da), dialyzed against water for 48 h, and lyophilized to obtain a pale yellow solid powder. c-P(HEMA-N3) 30 .like Figure 7 As shown, NMR confirmed this. c -P(HEMA-Br) 30 Successful synthesis. For example... Figure 7 and Figure 8 As shown, NMR and IR were used to verify... c -P(HEMA-N3) 30 Successful synthesis. (Yield, 88.7%) Example

[0077] Cyclic macromolecules c -P(HEMA-RNA) 30 Synthesized via a copper-free click chemistry reaction of dibenzocyclooctynyl and azide groups. The specific steps are as follows: 4 OD DBCO-RNA was dissolved in 3 μL of DEPC water, and 7.49 μg... c -P(HEMA-N3) 30 Dissolve in 30 μL DMSO, mix well, and react at 50°C for 48 h. After the reaction, remove DMSO by dialyzing. Remove unreacted DBCO-RNA using an ultrafiltration centrifuge tube (50 kDa). Figure 9 As shown, the cyclic macromolecules were verified by non-denaturing polyacrylamide gel electrophoresis. c -P(HEMA-RNA) 30 Successful synthesis. Example

[0078] Verification was performed using dynamic light scattering (DLS) and transmission electron microscopy (TEM). c -P(HEMA-miR-122) 30 Particle size and morphology of SNAs.

[0079] Take an appropriate amount c -P(HEMA-miR-122) 30 The SNA stock solution was diluted to 10 μM with ultrapure water, filtered through a 450 nm filter using a 1 ml syringe, and 60 μL was placed in the sample cell of a nanoparticle size analyzer. The average hydrated particle size of the sample solution was measured by DLS. The results are as follows: Figure 10 As shown.

[0080] Staining was performed using phosphotungstic acid negative staining, and observation was conducted using TEM. c -P(HEMA-miR-122) 30 The morphology of SNAs. The final concentration of miR-122 was 2 μM. c -P(HEMA-miR-122) 3010 μL of SNAs was added dropwise to a copper grid and left to dry overnight (the copper grid was covered with a cardboard box during this time to prevent contamination). Before photographing, 1% phosphotungstic acid solution was added to the sample solution for negative staining, followed by TEM observation and photography. The results are as follows. Figure 11 As shown. Example

[0081] The stability of the spherical nucleic acids was verified by dynamic light scattering, microplate reader, and non-denaturing polyacrylamide gel electrophoresis.

[0082] The ability of nanomedicines to maintain relative stability over a certain period is crucial for drug storage and placement; therefore, we used DLS to determine... c -P(HEMA-RNA) 30 The particle size of SNAs after 1, 2, 3, 4, 5, 6, and 7 days of storage is as follows: Figure 12 As shown.

[0083] When nanomedicines are administered in vitro or in vivo, they are usually diluted to a certain concentration. In particular, when administered in vivo, they usually undergo a significant dilution process, which may alter the structure of the nanocomposite, increase the particle size, or cause the nanomicelles to disintegrate. Therefore, it is especially important for nanomedicines to have a certain degree of resistance to dilution. c -P(HEMA-RNA) 30 The particle size changes of SNAs after dilution to different concentrations are as follows: Figure 13 As shown.

[0084] Nanocomposites typically require dilution with a medium before in vitro or in vivo administration. However, the stability of nanocomposites is affected by different liquid environments, and therefore their stability may vary in different dilution media. We used several commonly used dilution media, including water, 1640 medium, and PBS, to dilute the RNA to a final concentration of 2 μM. c -P(HEMA-RNA) 30 SNAs were diluted to 200 nM, and then the particle size was determined by DLS. The results are as follows: Figure 14 As shown.

[0085] c -P(HEMA-miR-122) 30 The nanocomposite assembled via hydrophilic-hydrophobicity possesses a structure similar to spherical nucleic acids. The dense nucleic acid shell of these spherical nucleic acids helps protect the grafted miR-122 from degradation by nucleases, and protecting miRNA from nuclease degradation is a crucial step for successful miRNA delivery to the target site. The stability of miR-122 in the presence of nucleases was investigated to determine its effectiveness. c-P(HEMA-miR-122) 30 SNAs protect miR-122 from being degraded by RNase A, as shown in the following results. Figure 15 As shown.

[0086] The effect of nanomedicines on erythrocytes is a crucial method for investigating the toxicity of drugs to normal cells. After incubating each component of the formulation with erythrocytes at 37°C for 3 hours, the supernatant was centrifuged, and its absorbance at 540 nm was measured. The results are as follows: Figure 16 As shown. Example

[0087] The uptake of cP(HEMA-RNA)30 SNAs by hepatocellular carcinoma cells was qualitatively investigated using fluorescence imaging. Human hepatocellular carcinoma Bel-7402 cells were seeded at a density of 100,000 cells / well in 48-well plates and cultured at 37°C with 5% CO2 for 24 hours. Once the cell density reached 60% of the bottom of the flask, the complete culture medium was discarded, and 500 μL of PBS was gently added along the plate wall. The plate was gently shaken to remove any dead cells and cell metabolites. The remaining PBS was removed, and this process was repeated twice. Then, 0.2 mL of each formulation diluted with Opti-MEM was added in the dark to ensure a final FAM-RNA concentration of 100 nM, and incubation continued for 8 hours. After this time, the drug-containing culture medium was discarded, and 0.5 mL of PBS was gently added along the plate wall. The plate was gently shaken to wash away any remaining drug solution. After washing, tilt the cell plate and aspirate the used PBS with a 1ml pipette. Repeat this process 5 times. Add 100μL of 4% paraformaldehyde to each well to fix the cells. Shake on a shaker for 15 min in the dark, then discard the paraformaldehyde. Wash 5 times with PBS, then add 100μL of Hoechst 33342 staining solution to stain the cell nuclei. After 20 min, discard the Hoechst 33342 staining solution, and wash twice with 0.5mL of PBS. Finally, add an appropriate amount of PBS to each well and observe on a cell imaging system. The entire procedure was performed in the dark. The results are as follows: Figure 17 As shown.

[0088] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. Other modifications can be easily made by those skilled in the art. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details and the illustrations shown and described herein.

Claims

1. A method for preparing spherical nucleic acids based on cyclic template strategy, comprising the following steps: Step 1, preparing a cyclic polymer c -P(HEMA) 30 ; Step 2, substituting azido group for the cyclic polymer c -P(HEMA) 30 terminal hydroxyl group to obtain a cyclic template c -P(HEMA-N3) 30 ; Step 3, chemically coupling nucleic acids to the cyclic template c -P(HEMA-N3) 30 to obtain a cyclic macromolecule c -P(HEMA-RNA) 30 ; and c -P(HEMA-RNA) 30 dialyzing the cyclic macromolecule against water to prepare spherical nucleic acids (SNAs) by self-assembly; In the step 1, the cyclic polymer c -P(HEMA) 30 is prepared by the following method: 1.

1. Obtained by ATRP polymerization reaction l - P(HEMA) 30 - Br; 1.

2. Through nucleophilic substitution reactions... l -P(HEMA) 30 The -Br terminal bromine atom is converted into an azide group, resulting in l -P(HEMA) 30 -N3; 1.

3. by l -P(HEMA) 30 N3 intramolecular click chemistry reaction to give a cyclic polymer c -P(HEMA) 30 ; The specific steps of the step two are as follows: 0.1 parts by weight of c- P(HEMA) 30 in 4 parts by volume of DMF, stirred in an ice-water bath for 10 min, then 0.324 parts by volume of 2-bromoisobutyryl bromide was added dropwise, and the reaction was continued at room temperature for 24 h. After the reaction was completed, an excess of diethyl ether was added to precipitate, the precipitate was dissolved in DMF and transferred to a 500 Da dialysis bag for dialysis against water for 48 h, and then lyophilized to obtain a white solid powder c P(HEMA-Br) 30 ; 0.135 parts by weight of c -P(HEMA-Br) 30 with 0.311 parts by weight of sodium azide in 3 parts by volume of DMF solution at 45°C for 48 h, after the reaction, the reaction solution was transferred to a 500 Da dialysis bag, dialyzed against water for 48 h, lyophilized to obtain a light yellow solid powder c -P(HEMA-N3) 30 .

2. The method for preparing spherical nucleic acids based on a circular template strategy as described in claim 1, characterized in that, The l - P(HEMA) 30 -Br was prepared as follows: The small molecule initiator 2-bromoisobutyric acid propargyl ester 410 90 parts by weight, hydroxyethyl methacrylate 6.64 parts by weight and N,N,N',N',N''-pentamethyldiethylenetriamine 425 parts by volume were dissolved in a mixed solution and stirred uniformly, N2 was bubbled for 30 min, then 290 parts by weight of cuprous bromide was added and stirred at 65°C for 20 min, then an excess of ice ethyl ether was added to precipitate the crude product, the crude product was dissolved in a DMF solution and then transferred to a 500 Da dialysis bag, dialyzed against water for 48 h, freeze-dried to obtain a white solid, which is l - P(HEMA) 30 -Br; wherein the mixed solution is prepared by mixing DMF and IPA in a volume ratio of 1:9; the ratio units of parts by weight and parts by volume are g / ml.

3. The method for preparing spherical nucleic acids based on a circular template strategy as described in claim 1, characterized in that, The specific steps of the nucleophilic substitution reaction are as follows: Take 1 part by weight l- P(HEMA) 30 -Br and 0.158 parts by weight NaN3were dissolved in 2 volumes of DMF and 0.5 volumes of water, respectively, and reacted at 45°C for 48 h to obtain a reaction solution. The reaction solution was transferred to a 500 Da dialysis bag and dialyzed against water for 48 h, and then lyophilized to obtain a light yellow solid, which was l -P(HEMA) 30 -N3.

4. The method for preparing spherical nucleic acids based on a circular template strategy as described in claim 1, characterized in that, The specific steps of the intramolecular click chemistry reaction are as follows: In a flask was added 850 parts by volume of DMF, heated to 100 °C, N2 sparging for 1 h, then added 0.506 parts by volume of PMDETA and 0.418 parts by weight of CuBr in turn, dissolved 0.5 parts by weight of l P(HEMA) 30 N3, N2 sparging for 1 h, to obtain l- P(HEMA) 30 N3 solution; then the l- P(HEMA) 30 N3 solution was injected into the flask reaction continued to react for 48 h, after the reaction was completed, the excess ethyl ether precipitated, dissolved in DMF and transferred to the 500 Da dialysis bag dialysis, dialysis for 48 h, lyophilized to obtain a white product c P(HEMA) 30 .

5. The method for preparing spherical nucleic acids based on a circular template strategy as described in claim 1, characterized in that, In the third step, the cyclic template c -P(HEMA-N3) 30 Synthesis of cyclic macromolecules by copper-free click chemistry reaction of dibenzocyclooctyne and azido groups c -P(HEMA-RNA) 30 .

6. The method for preparing spherical nucleic acids based on a circular template strategy as described in claim 5, characterized in that, The specific steps of step three are as follows: 4 OD DBCO-RNA is dissolved in 3 μL of DEPC water, 7.49 μg c -P(HEMA-N3) 30 is dissolved in 30 μL of DMSO, mixed uniformly, and reacted at 50 °C for 48 h. After the reaction is completed, DMSO is removed by dialysis, and unreacted DBCO-RNA is removed by ultrafiltration with a 50 kDa ultrafiltration centrifuge tube, to obtain a cyclic macromolecule c -P(HEMA-RNA) 30 .

7. The method of claim 6, wherein the circular template strategy-based spherical nucleic acid preparation method is characterized by, The RNA is miRNA, and the miRNA includes miR-122, and the nucleic acid is RNA.

8. Use of the spherical nucleic acid as shown in any one of claims 1-7; the spherical nucleic acid is used as a carrier for preventing the nucleic acid loaded by the spherical nucleic acid from being degraded by a nucleic acid degradation enzyme.

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