CN116983279A - Spherical nucleic acid preparation method and application based on circular template strategy - Google Patents

Spherical nucleic acid preparation method and application based on circular template strategy Download PDF

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CN116983279A
CN116983279A CN202310795034.3A CN202310795034A CN116983279A CN 116983279 A CN116983279 A CN 116983279A CN 202310795034 A CN202310795034 A CN 202310795034A CN 116983279 A CN116983279 A CN 116983279A
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hema
nucleic acid
parts
rna
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魏华
刘洪兵
喻翠云
陶正好
黎爽
王敦
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University of South China
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Abstract

The invention discloses a spherical nucleic acid preparation method and application based on an annular template strategy, comprising the following steps: a) By means of cocoaMethod for controlling radical polymerization (ATRP) and copper-catalyzed intramolecular click chemistry of alkynyl and azido groups, cyclic polymersc‑P(HEMA) 30 . B) The hydroxyl at the tail end of the side chain of the cyclic polymer is replaced by azido by utilizing continuous esterification and substitution reaction to obtain the cyclic templatec‑P(HEMA‑N 3 ) 30 . C) Covalent coupling of nucleic acids to a cyclic template using copper-free click chemistry of dibenzocyclooctynyl groups (DBCO) and azido groups to yield a circular brush-like macromoleculec‑P(HEMA‑RNA) 30 . Dialyzing the obtained circular brush-shaped macromolecules against water, and preparing spherical nucleic acid through self-assembly. The spherical nucleic acid prepared by the preparation method has good repeatability, good in vitro stability, can better protect nucleic acid from degradation of RNA degrading enzyme, and can greatly improve the uptake capacity of cells on nucleic acid.

Description

Spherical nucleic acid preparation method and application based on circular template strategy
Technical Field
The invention relates to the field of medical biological materials, in particular to a construction method and a preparation method of novel spherical nucleic acid based on an annular template strategy.
Background
Spherical Nucleic Acids (SNAs) are three-dimensional spherical geometries consisting of a central core layer and highly oriented, dense oligonucleotide layers (Mokhtarzadeh A, vahidnezhad H, youssefan L, et al Trends in molecular medicine,25 (12): 1066-1079, 2019). In the past 20 years, the SNAs structure has thoroughly changed the application of gene drugs in gene regulation, drug delivery, gene therapy, molecular diagnosis and the like, and has shown very good prospects in gene therapy. Spherical nucleic acid nanoparticles are a novel class of intracellular delivery systems for bioactive molecules that can be used as a more promising carrier for delivery of antisense oligonucleotide strands and immunomodulators without significant off-target effects, immunogenicity or cytotoxicity (Cutler J I, zhang K, zheng D, et al Journal of the American Chemical Society, 133 (24): 9254-9257, 2011). A variety of single-stranded (ss) and double-stranded (ds) oligonucleotides, typically nucleic acids with a base number of 25-40 nt 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, domiiguez D, et al Small,14 (49): 1803284, 2018). And SNA structures can elicit minimal immune stress response (25-fold reduction in immune response) compared to cationic nanocarriers. As the SNAs have high-density oligonucleotide chains on the surfaces, compared with linear nucleic acid, the SNAs are not easy to degrade by nuclease, so that the SNAs have 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 the high density of oligonucleotides (zeta potential of 30 mV), the 3D globular structure they form can be recognized by class a scavenger receptors to enter cells in endocytic form and can be rapidly internalized in almost all cell types by the process of cellular endocytosis mediated by the small cell proteins.
However, the developed SNAs form protein crowns due to the fact that the surface-dense nucleic acid shells easily adsorb positively charged proteins during blood circulation, and are finally cleared by the immune system, while spherical nucleic acid analogues formed by self-assembly of amphiphilic linear molecules are not easily formed into protein crowns due to the fact that the surface nucleic acid chains are not as dense as conventional SNAs, but nanoparticles formed by self-assembly of linear molecules are not stable under high salt concentration and a large amount of dilution conditions of body fluids.
With the progress of polymer chemical synthesis technology and the intensive research in this field, it has become possible to prepare nonlinear polymers which can have various topological structures such as star-shaped, branch-shaped, crown-shaped and ring-shaped structures. Topology polymer chemistry is currently being transformed from a synthetic approach to powerful tools for the design of advanced materials, particularly in biomedical applications, the topology effects of polymers due to complex structures have been increasingly used to fabricate a variety of materials and coatings that can provide biomedical functions such as diagnostics, bioimaging, drug and gene delivery, tissue engineering, and antimicrobial. Notably, although many literature reports comparing different performances of linear, star, graft and branched polymers in biomedical applications, etc., cyclic polymers have been of great interest in research because of the lack of chain ends, which are characteristic of exhibiting very different physicochemical properties and properties than linear polymers. The nanoparticles formed by self-assembly of the cyclic polymer in an aqueous solution can have smaller size and better stability than the linear analogues.
Disclosure of Invention
In order to solve the problems, the invention discloses a preparation method and application of spherical nucleic acid based on a circular template strategy. The spherical nucleic acid prepared by the preparation method has good repeatability, good in vitro stability, can better protect nucleic acid from degradation of RNA degrading enzyme, and can greatly improve the uptake capacity of cells on nucleic acid.
In order to achieve the above purpose, the technical scheme of the invention is as follows:
a spherical nucleic acid preparation method based on a circular template strategy comprises the following steps:
step one, preparing a cyclic polymerc-P(HEMA) 30 ；
Step two, enabling azido substituted cyclic polymerc-P(HEMA) 30 Terminal hydroxyl group to obtain a cyclic templatec-P(HEMA-N 3 ) 30 ；
Step three, chemically coupling the nucleic acid to the circular templatec-P(HEMA-N 3 ) 30 Obtaining the cyclic macromoleculec-P(HEMA-RNA) 30 The method comprises the steps of carrying out a first treatment on the surface of the The cyclic macromolecules are reacted withc-P(HEMA-RNA) 30 The Spherical Nucleic Acids (SNAs) were prepared by self-assembly by dialysis against water.
Further improvements, in step one, the cyclic polymerc-P(HEMA) 30 The preparation method of (2) is as follows:
1.1, obtained by ATRP polymerizationl-P(HEMA) 30 -Br；
1.2 by nucleophilic substitution reactionl-P(HEMA) 30 Conversion of the Br-terminal bromine atom to an azide group to givel-P(HEMA) 30 -N 3 ；
1.3 byl-P(HEMA) 30 -N 3 Intramolecular clicking chemical reaction to obtain cyclic polymerc-P(HEMA) 30 。
Further, the methodImprovements in or relating tol-P(HEMA) 30 The preparation method of Br is as follows:
410 parts by weight of small molecular initiator 2-bromoisobutyric acid propyne ester, 6.64 parts by weight of hydroxyethyl methacrylate and 425 parts by volume of N, N, N ', N, ' ' N ' ' -pentamethyldiethylenetriamine are dissolved in the mixed solution and stirred uniformly, and N is introduced 2 Bubbling for 30min, adding 290 parts by weight of cuprous bromide at 65 ℃ and stirring for reaction for 20 min, adding excessive glacial ethyl ether for precipitation to obtain a crude product, dissolving the crude product with DMF solution, transferring to a 500Da dialysis bag, dialyzing with water for 48h, and lyophilizing to obtain white solidl-P(HEMA) 30 -Br; wherein the mixed solution is prepared from DMF and IPA according to the volume ratio of 1:9; the ratio of parts by weight to parts by volume is in g/ml.
Further improved, the nucleophilic substitution reaction comprises the following steps:
1 part by weight ofl-P(HEMA) 30 -Br and 0.158 parts by weight NaN 3 Respectively dissolving in 2 parts by volume of DMF and 0.5 part by volume of water, reacting at 45 ℃ for 48h to obtain a reaction solution, transferring the reaction solution into a 500Da dialysis bag, dialyzing with water for 48 hours, and freeze-drying to obtain pale yellow solid, namelyl-P(HEMA) 30 -N 3 。
Further improved, the intramolecular click chemistry takes place as follows:
into a flask, 850 parts by volume of DMF was added, heated to 100deg.C, N 2 Bubbling 1.1 h, then adding 0.506 parts by volume of PMDETA and 0.418 parts by weight of CuBr in sequence, dissolving 0.5 parts by weight with 10 parts by volume of DMFl-P(HEMA) 30 -N 3 ，N 2 Bubbling for 1h to obtainl-P(HEMA) 30 -N 3 A solution; then will bel-P(HEMA) 30 -N 3 The solution is injected into a flask for reaction to continue to react 48h, excessive glacial ethyl ether is used for precipitation after the reaction is finished, DMF is used for dissolving the precipitation and is transferred into a 500Da dialysis bag for dialysis on water, after 48h dialysis, the white product is obtained by freeze-dryingc-P(HEMA) 30 。
Further improvement, the specific steps of the second step are as follows:
will be 0.1 part by weightc-P(HEMA) 30 Dissolving in 4 volume parts of DMF, stirring in ice water bath for 10 min, dropwise adding 0.324 volume parts of 2-bromoisobutyryl bromide, reacting at room temperature for 24 hr, adding excessive diethyl ether to precipitate after reaction, dissolving the precipitate in DMF, transferring to 500Da dialysis bag, dialyzing with water for 48h, and lyophilizing to obtain white solid powderc-P(HEMA-Br) 30 ；
Will be 0.135 weight partc-P(HEMA-Br) 30 Dissolving 0.311 weight part of sodium azide in 3 volume parts of DMF solution, reacting at 45 ℃ for 48h, transferring the reaction solution to a 500Da dialysis bag after the reaction is finished, dialyzing 48h against water, and freeze-drying to obtain light yellow solid powderc-P(HEMA-N 3 ) 30 。
Further improvement, in the third step, the annular templatec-P(HEMA-N 3 ) 30 Synthesis of cyclic macromolecules by copper-free click chemistry of dibenzocyclooctynyl and azidoc-P(HEMA-RNA) 30 。
Further improvement, the specific steps of the third step are as follows: 4 OD DBCO-RNA was dissolved in 3. Mu.L of DEPC water, 7.49. Mu.g c-P(HEMA-N 3 ) 30 Dissolving in 30 μL DMSO, mixing, reacting at 50deg.C for 48 hr, dialyzing to remove DMSO, ultrafiltering unreacted DBCO-RNA with 50 kDa ultrafiltering centrifuge tube to remove to obtain cyclic macromoleculec-P(HEMA-RNA) 30 。
Further improvements, the nucleic acid is RNA.
Further improvements, the RNA is miRNA, and the miRNA comprises miR-122.
Use of a spherical nucleic acid as set forth above; the spherical nucleic acid is used as a carrier for preventing degradation of the nucleic acid carried by the spherical nucleic acid by a nucleic acid degrading enzyme.
The invention has the advantages that:
the technical proposal of the invention has the beneficial effects that
The SNAs prepared by the invention has good reproducibility, greatly improves the in vitro stability of nucleic acid, can protect the nucleic acid from degradation of nucleic acid degrading enzyme to a certain extent, and can greatly improve the uptake capacity of cells on the nucleic acid.
Drawings
FIG. 1 is a cyclic macromoleculec-synthetic roadmap of P (HEMA-RNA) 30.
FIG. 2 is a schematic view ofl-P(HEMA) 30 -Br 1 H NMR spectra (DMSO)d 6 )。
FIG. 3l-P(HEMA) 30 -Br andl-P(HEMA) 30 -N 3 FT-IR diagram of (c).
FIG. 4c-P(HEMA) 30 -Br 1 H NMR spectra (DMSO)d 6 )。
FIG. 5 is a schematic view of a display l-P (HEMA) 30 -Br, l-P(HEMA) 30 -N 3 Andc-P(HEMA) 30 SEC elution profile of (c).
FIG. 6 is a diagram ofc-P(HEMA-Br) 30 A kind of electronic device 1 H NMR spectra (DMSO)d 6 )。
FIG. 7 is a diagram ofc-P(HEMA-N 3 ) 30 A kind of electronic device 1 H NMR spectra (DMSO)d 6 )。
FIG. 8 is a diagram ofc-P(HEMA-Br) 30 Andc-P(HEMA-N 3 ) 30 FT-IR diagram of (c).
FIG. 9 is free miR-122 (left), circular templatec-P(HEMA-N 3 ) 30 (neutralization) andc-P(HEMA-miR-122) 30 non-denaturing polyacrylamide gel electrophoresis pattern of (right).
FIG. 10 is a diagram ofc-P(HEMA-RNA) 30 DLS particle size plot of SNAs.
FIG. 11c-P(HEMA-RNA) 30 Transmission electron microscopy of SNAs.
FIG. 12 is a diagram ofc-P(HEMA-RNA) 30 SNAs were placed in particle size change patterns for different times. Data are expressed as mean ± standard deviation, n=3.
FIG. 13 is a diagram of c-P(HEMA-RNA) 30 The particle size of SNAs was varied by dilution with ultrapure water at various concentrations. Data are expressed as mean ± standard deviation, n=3.
FIG. 14 is a diagram ofc-P(HEMA-RNA) 30 Particle size variation of SNAs after dilution in different media. Data sheetShown as mean ± standard deviation, n=3.
FIG. 15 is free miR-122 andc-P(HEMA-RNA) 30 gel electrophoresis patterns of the nanocomplex after incubation with RNase a for different times, respectively.
Figure 16 shows the haemolysis rate of different formulations after incubation with erythrocytes at 37 ℃ for 3 h. Data are expressed as mean ± standard deviation, n=3. *P＜0.05，**P＜0.01，***P＜0.001。
FIG. 17 is a diagram ofc-P(HEMA-RNA) 30 Fluorescence imaging of SNAs after incubation with Bel-7402 cells for 8h (blue for Hoechst33342 labeled nuclei and green for FAM labeled miR-122; scale 200 μm; miR-122 dosing concentration 100 nM).
Description of the embodiments
The invention is further described below with reference to the drawings and examples.
Examples
The invention provides a preparation method of novel SNAs based on an annular template strategy, which comprises the following steps:
a) Method for obtaining cyclic polymer by utilizing atomic controlled radical polymerization (ATRP) and subsequent copper-catalyzed intramolecular click chemistry of alkynyl and azidoc-P(HEMA) 30 ;
B) The terminal hydroxyl of the cyclic polymer is replaced by azido by utilizing esterification reaction and substitution reaction to obtain the cyclic templatec-P(HEMA-N 3 ) 30 ；
C) The method comprises the steps of chemically coupling nucleic acid onto a cyclic template by using copper-free click chemistry reaction of Dibenzocyclooctyne (DBCO) and azido to obtain a cyclic macromoleculec-P(HEMA-RNA) 30 . And (3) performing water-proof dialysis on the obtained annular macromolecules, and performing self-assembly under hydrophilic and hydrophobic acting force to finally form SNAs.
The invention firstly utilizes atomic controlled radical polymerization (ATRP) and a subsequent copper-catalyzed intramolecular click chemistry method of alkynyl and azido to obtain a cyclic polymerc-P(HEMA) 30 . Specifically, the linear polymer is synthesized by ATRP polymerizationl-P(HEMA) 30 Br followed by terminating the linear polymer chain with sodium azideBromine atom substituted by azido group, synthesisl-P(HEMA) 30 -N 3 . Then, the copper-catalyzed click chemistry reaction of alkynyl and azido is utilized to lead the head and tail clicks of the linear polymer to be coupled to synthesize the cyclic polymerc-P(HEMA) 30 。
In embodiments of the present invention, the ATRP polymerization solvent is DMF/IPA (V/v=1:9), and in certain embodiments, the solvent is acetone.
In embodiments of the invention, the reaction temperature for ATRP polymerization is 65 ℃ and the reaction time is 20 minutes, in some embodiments 60 ℃ and 50 ℃ and the reaction time is 1 hour and 30 minutes.
In the examples of the present invention, the intramolecular click chemistry reaction temperature was 100deg.C for 48 hours and the solvent was DMF.
Subsequent in cyclic polymersc-P(HEMA) 30 Based on (1), the terminal hydroxyl of the cyclic polymer is replaced by azido by utilizing esterification reaction and substitution reaction to obtain the cyclic templatec-P(HEMA-N 3 ) 30 . Specifically, hydroxyl at the tail end of a branched chain of a cyclic polymer and bromoisobutyryl bromide are subjected to esterification reaction to synthesize a cyclic moleculec-P(HEMA-Br) 30 . The cyclic molecules are reacted with sodium azidec-P(HEMA-Br) 30 The bromine atom at the tail end of the branched chain is substituted by an azide group to synthesize the final cyclic templatec-P(HEMA-N 3 ) 30 。
In an embodiment of the present invention, the temperature of the esterification reaction was 25℃and the time of the reaction was 24h.
In the examples of the present invention, the substitution reaction solvent was anhydrous DMF at 45℃for 48h.
Finally, the copper-free click chemical reaction of Dibenzocyclooctyne (DBCO) and azido is utilized to couple the nucleic acid to the annular template through clicking, thus obtaining the annular macromoleculec-P(HEMA-RNA) 30 . And (3) performing water-proof dialysis on the obtained annular macromolecules, and performing self-assembly under hydrophilic and hydrophobic acting force to finally form SNAs. Specifically, DBCO modified RNA (DBCO-RNA) and circular templatesc-P(HEMA-N 3 ) 30 Alkynyl and stackingCopper-free click chemistry reaction of nitrogen group, coupling RNA click to the annular template to synthesize annular macromoleculec-P(HEMA-RNA) 30 Ultrafiltration removes unreacted DBCO-RNA. The obtained cyclic macromoleculec-P(HEMA-RNA) 30 Dissolved in DMSO and dialyzed against water for 48h, and allowed to self-assemble by hydrophilic and hydrophobic to form SNAs.
In embodiments of the present invention, the click chemistry reaction solvent is Dimethylsulfoxide (DMSO) and water (V: v=5:1), and in certain embodiments, the reaction solvent is DMSO: DMF (V/v=1:1).
In embodiments of the invention, the click chemistry reaction temperature is 50 ℃, the reaction time is 48h, and in certain embodiments, the reaction temperature is 40 ℃, 60 ℃.
In embodiments of the invention, the ratio of the annular template to the nucleic acid is 1:30, and in certain embodiments, the ratio is 1:1, 1:1.2.
In embodiments of the invention, the method of purification of the spherical nucleic acid is ultrafiltration, and in certain embodiments, the method of purification is high performance liquid phase separation.
The source of the raw materials used in the present invention is not particularly limited, and may be generally commercially available.
The invention also provides novel SNAs prepared by the preparation method. The spherical nucleic acid prepared by the preparation method has good repeatability and in-vitro stability, can better protect nucleic acid from degradation of RNA degrading enzyme, and can greatly improve the uptake of nucleic acid by cells.
The preparation method of the SNAs provided by the invention comprises the following steps: a) Method for obtaining cyclic polymer by utilizing atomic controlled radical polymerization (ATRP) and subsequent copper-catalyzed intramolecular click chemistry of alkynyl and azidoc-P(HEMA) 30 . B) The terminal hydroxyl of the cyclic polymer is replaced by azido by utilizing esterification reaction and substitution reaction to obtain the cyclic templatec-P(HEMA-N 3 ) 30 . C) The method comprises the steps of chemically coupling nucleic acid onto a cyclic template by using copper-free click chemistry reaction of Dibenzocyclooctyne (DBCO) and azido to obtain a cyclic macromoleculec-P(HEMA-RNA) 30 . Dialyzing the obtained cyclic macromolecule with water, and performing hydrophilic and hydrophobic treatmentSelf-assembly under force eventually forms spherical nucleic acids. The SNAs prepared by the preparation method provided by the invention has good reproducibility and in-vitro stability, can better protect nucleic acid from degradation of RNA degrading enzyme, and can greatly improve the uptake capacity of cells on nucleic acid.
Experimental results show that the SNAs prepared by the invention have good reproducibility, greatly improve the in vitro stability of nucleic acid, protect the nucleic acid from degradation of nucleic acid degrading enzyme to a certain extent, and compared with free nucleic acid groups, the constructed novel SNA structure can greatly improve the uptake capacity of cells on nucleic acid.
In order to further illustrate the present invention, the following examples are provided to describe in detail the construction of a novel spherical nucleic acid based on a circular template strategy and a method for preparing the same, but should not be construed as limiting the scope of the present invention.
The raw materials used in the following examples are all generally commercially available.
Examples
l-P(HEMA) 30 The synthesis of Br is carried out by taking hydroxyethyl methacrylate (HEMA) as a monomer, taking 2-bromoisobutyric acid propyne (alkyne-Br) as an initiator, taking N, N, N ', N, ' ' N ' ' -pentamethyldiethylenetriamine/CuBr as a catalyst, and carrying out ATRP polymerization. The specific operation is as follows: the small molecular initiator 2-bromoisobutyric acid propynyl ester 410 mg, hydroxyethyl methacrylate 6.64 g and 425 μl of N, N, N ', N, ' ' N ' ' -pentamethyldiethylenetriamine are dissolved in a mixed solution of 25 ml DMF and IPA (V/V=1/9), stirred at room temperature for 10 min to ensure complete dissolution and uniform mixing of all raw materials, and then the mixed solution is transferred to a 50 mL round bottom flask. N (N) 2 Bubbling for 30min. Then 290 mg cuprous bromide is added, the reaction is stirred for 20 min at 65 ℃, and the mixture is precipitated in excessive glacial diethyl ether. The crude product was dissolved in a small amount of DMF solution and transferred to a dialysis bag (500 Da), dialyzed against water 48, h, and lyophilized to give a white solidl-P(HEMA) 30 -Br. As shown in FIGS. 2 and 5, successful synthesis of the product was verified by nuclear magnetism and GPC (yield, 63.3%)
Examples
The linear polymeric terminal bromine atom is converted to an azide group by nucleophilic substitution. 1g is takenl-P(HEMA) 30 -Br and 158 mg NaN 3 Respectively, in 2mL DMF and 0.5. 0.5 mL water, 48h was reacted at 45 ℃. Transferring the reaction solution into a dialysis bag (500 Da), dialyzing with water for 48 hr, and lyophilizing to obtain pale yellow solidl-P(HEMA) 30 -N 3 . As shown in FIG. 3, successful substitution of the bromine atom with the azide group was confirmed by infrared (yield, 87.6%)
Examples
Under extremely dilute conditions byl-P(HEMA) 30 -N 3 Intramolecular clicking chemical reaction to obtain cyclic polymerc-P(HEMA) 30 . The specific process is as follows: 850 mL of DMF was added to the flask, heated to 100deg.C, N2 bubbled with 1h, then 506. Mu.L of PMDETA followed by 418 mg of CuBr. At the same time, 500 mg%l-P(HEMA) 30 -N 3 ) Polymers, N 2 Bubbling for 1h. Then using a syringe pump to pumpl-P(HEMA) 30 -N 3 The solution was slowly injected into the flask to continue the reaction 48 and h. After the reaction was completed, excess glacial diethyl ether was precipitated, the precipitate was dissolved in DMF and transferred to a dialysis bag (500 Da) for dialysis against water. Dialysis 48h, freeze drying to obtain white productc-P(HEMA) 30 . As shown in FIGS. 4 and 5, the cyclic polymer was verified by nuclear magnetism and GPCc-P(HEMA) 30 Is a successful synthesis of (a).
Examples
Synthesis by esterificationc-P(HEMA-Br) 30 . The method comprises the following specific steps: will be 100 mgc-P(HEMA) 30 Dissolved in 4 mL DMF, stirred in ice water bath for 10 min, followed by dropwise addition of 324. Mu.L 2-bromoisobutyryl bromide and reaction at room temperature for 24h. After the reaction was completed, excess diethyl ether was precipitated, and the precipitate was dissolved in DMF and transferred to a dialysis bag (500 Da) for dialysis against water 48h. Lyophilizing to obtain white solid powderc-P(HEMA-Br) 30 . As shown in FIG. 6, the nuclear magnetism is used for verificationc-P(HEMA-Br) 30 Is a successful synthesis of (a). (yield, 62.6%)
Examples
Will be 135 mgc-P(HEMA-Br) 30 With 311 mg sodium azide in 3 mL DMF solution, 48h was reacted at 45 ℃. After the completion of the reaction, the reaction solution was transferred to a dialysis bag (500D)a) Dialyzing 48, h with water, and lyophilizing to obtain yellowish solid powderc-P(HEMA-N 3 ) 30 . As shown in FIG. 7, the nuclear magnetism is used for verificationc-P(HEMA-Br) 30 Is a successful synthesis of (a). As shown in fig. 7 and 8, the nuclear magnetism and infrared are used for verificationc-P(HEMA-N 3 ) 30 Is a successful synthesis of (a). (yield, 88.7%)
Examples
Cyclic macromoleculesc-P(HEMA-RNA) 30 Synthesized by copper-free click chemistry of dibenzocyclooctynyl and azido. The specific procedure was as follows, 4 OD DBCO-RNA was dissolved in 3. Mu.L of DEPC water, 7.49. Mu.g c-P(HEMA-N 3 ) 30 Dissolved in 30. Mu.L DMSO, mixed well and reacted for 48h at 50. After the reaction was completed, DMSO was removed by dialysis. Unreacted DBCO-RNA was removed by ultrafiltration with an ultrafiltration centrifuge tube (50 kDa). As shown in FIG. 9, the cyclic macromolecules were verified by non-denaturing polyacrylamide gel electrophoresisc-P(HEMA-RNA) 30 Is a successful synthesis of (a).
Examples
Verification by Dynamic Light Scattering (DLS) and Transmission Electron Microscopy (TEM)c-P(HEMA-miR-122) 30 Particle size and morphology of the SNAs.
Taking a proper amount ofc-P(HEMA-miR-122) 30 The SNAs mother liquor was diluted to 10. Mu.M with ultrapure water, passed through a 450 nm filter membrane with a 1ml syringe, and 60. Mu.L was placed in a sample cell of a nanoparticle analyzer, and the average hydrated particle size of the sample solution was measured by DLS, and the results are shown in FIG. 10.
Dyeing by phosphotungstic acid negative dyeing method, and observing by TEMc-P(HEMA-miR-122) 30 Morphology of SNAs. Taking miR-122 with final concentration of 2 mu Mc-P(HEMA-miR-122) 30 SNAs 10 microliters was added dropwise to the copper mesh, and left to stand overnight to allow it to dry naturally (the copper mesh was covered with a cardboard box during this period to prevent contamination with foreign matter). Before photographing, 1% phosphotungstic acid solution was added dropwise over the sample solution for negative staining, and then photographing was performed by TEM observation, and the result is shown in FIG. 11.
Examples
The stability of the spherical nucleic acid was verified by dynamic light scattering, enzyme-labeled instrument and non-denaturing polyacrylamide gel electrophoresis.
The ability of nano-drugs to remain relatively stable for a period of time is particularly important for drug storage and placement, so we have determined by DLSc-P(HEMA-RNA) 30 The SNAs were allowed to stand for 1, 2, 3, 4, 5, 6, and 7 days to obtain the results shown in FIG. 12.
When the nano-drug is administered in vitro or in vivo, the nano-drug is usually diluted to a certain concentration, particularly when the nano-drug is administered in vivo, a great dilution process is usually carried out, the structure of the nano-composite is likely to be changed, the particle size is increased or the nano-micelle is disintegrated, so that the nano-drug has a certain anti-dilution capability and is particularly important.c-P(HEMA-RNA) 30 The particle size change after dilution of SNAs to different concentrations is shown in FIG. 13.
The nanocomposite is usually administered after dilution with medium before in vitro or in vivo administration, and the stability of the nanocomposite is affected by different liquid environments, so that the stability of the nanocomposite may be different in different dilution mediums, and thus we use several common dilution mediums including water, 1640 medium, PBS, and RNA final concentration of 2. Mu.Mc-P(HEMA-RNA) 30 The SNAs were diluted to 200 nM and the particle sizes were measured by DLS, respectively, and the results are shown in fig. 14.
c-P(HEMA-miR-122) 30 The nanocomposite after self-assembly by hydrophilic-hydrophobic can possess a structure similar to a spherical nucleic acid, a dense nucleic acid shell of the spherical nucleic acid can help protect grafted miR-122 from degradation by endonucleases, and protecting miRNA from degradation by nucleases is a key step in successful delivery of miRNA to a target site. Detection by examining the stability of miR-122 in the presence of a nucleasec-P(HEMA-miR-122) 30 The effect of SNAs on protecting miR-122, preventing miR-122 from being degraded by RNase A, and the result is shown in FIG. 15.
The effect of nanomedicine on red blood cells is a very important method to examine the toxicity of drugs on normal cells. After incubating the preparations of each fraction with red blood cells at 37℃for 3 h, the supernatant was centrifuged to measure the absorbance at 540 and nm, and the results are shown in FIG. 16.
Examples
Uptake of c-P (HEMA-RNA) 30 SNAs by hepatoma cells was examined qualitatively by fluorescence imaging. Human liver cancer Bel-7402 cells are inoculated into 48 pore plates at the density of 10 ten thousand per pore respectively, 37 ℃ and 5 percent CO 2 After the growth density of the cells reached 60% of the bottom of the flask, the complete medium was discarded, 500 μl of PBS was gently added along the walls, the cell plates were gently shaken to remove the dead cells and cell metabolites that may be present, the remaining PBS was aspirated, the procedure was repeated 2 times, 0.2mL of each set of preparations diluted with Opti-MEM was added in the dark, ensuring a final FAM-RNA concentration of 100 nM, and incubation was continued for 8h. After the time, the drug-containing medium was discarded, 0.5 mL of PBS was gently added along the wall, and the cell plate was gently shaken side to wash the drug solution that may remain. After washing, the used PBS was sucked off by a 1mL pipette, the procedure was repeated 5 times, 100. Mu.L of 4% paraformaldehyde was added to each well for cell immobilization, the paraformaldehyde was discarded after shaking for 15min on a shaker in the absence of light, washing with PBS 5 times, 100. Mu.L of hoechst33342 dye solution was added for staining the nuclei, the hoechst33342 dye solution was discarded after 20 min, 0.5 mL of PBS was added for 2 times, and finally an appropriate amount of PBS was added to each well for observation on a cell imager, and the whole procedure was protected from light, and the results are shown in FIG. 17.
Although embodiments of the present invention have been disclosed above, it is not limited to the details and embodiments shown and described, it is well suited to various fields of use for which the invention would be readily apparent to those skilled in the art, and accordingly, the invention is not limited to the specific details and illustrations shown and described herein, without departing from the general concepts defined in the claims and their equivalents.

Claims (10)

1. A preparation method of spherical nucleic acid based on a circular template strategy is characterized by comprising the following steps:
step one, preparing a cyclic polymerc-P(HEMA) 30 ；
Step two, enabling azido substituted cyclic polymerc-P(HEMA) 30 Terminal hydroxyl group to obtain a cyclic templatec-P(HEMA-N 3 ) 30 ；
Step three, chemically coupling the nucleic acid to the circular templatec-P(HEMA-N 3 ) 30 Obtaining the cyclic macromoleculec-P(HEMA-RNA) 30 The method comprises the steps of carrying out a first treatment on the surface of the The cyclic macromolecules are reacted withc-P(HEMA-RNA) 30 The Spherical Nucleic Acids (SNAs) were prepared by self-assembly by dialysis against water.
2. The method for preparing spherical nucleic acid based on circular template strategy according to claim 1, wherein in the first step, a circular polymerc-P(HEMA) 30 The preparation method of (2) is as follows:
1.1, obtained by ATRP polymerizationl-P(HEMA) 30 -Br；
1.2 by nucleophilic substitution reactionl-P(HEMA) 30 Conversion of the Br-terminal bromine atom to an azide group to givel-P(HEMA) 30 -N 3 ；
1.3 byl-P(HEMA) 30 -N 3 Intramolecular clicking chemical reaction to obtain cyclic polymerc-P(HEMA) 30 。
3. The method for preparing spherical nucleic acid based on circular template strategy according to claim 2, wherein the method comprises the steps ofl-P(HEMA) 30 The preparation method of Br is as follows:
410 parts by weight of small molecular initiator 2-bromoisobutyric acid propyne ester, 6.64 parts by weight of hydroxyethyl methacrylate and 425 parts by volume of N, N, N ', N, ' ' N ' ' -pentamethyldiethylenetriamine are dissolved in the mixed solution and stirred uniformly, and N is introduced 2 Bubbling for 30min, adding 290 parts by weight of cuprous bromide at 65 ℃ and stirring for reaction for 20 min, adding excessive glacial ethyl ether for precipitation to obtain a crude product, dissolving the crude product with DMF solution, transferring to a 500Da dialysis bag, dialyzing with water for 48h, and lyophilizing to obtain white solidl-P(HEMA) 30 -Br; wherein the mixed solution is prepared from DMF and IPA according to the volume ratio of 1:9; the ratio of parts by weight to parts by volume is in g/ml.
4. The method for preparing a spherical nucleic acid based on a circular template strategy according to claim 2, wherein the nucleophilic substitution reaction comprises the steps of:
1 part by weight ofl-P(HEMA) 30 -Br and 0.158 parts by weight NaN 3 Respectively dissolving in 2 parts by volume of DMF and 0.5 part by volume of water, reacting at 45 ℃ for 48h to obtain a reaction solution, transferring the reaction solution into a 500Da dialysis bag, dialyzing with water for 48 hours, and freeze-drying to obtain pale yellow solid, namelyl-P(HEMA) 30 -N 3 。
5. The method for preparing spherical nucleic acid based on circular template strategy according to claim 2, wherein the intramolecular click chemistry reaction is performed as follows:
into a flask, 850 parts by volume of DMF was added, heated to 100deg.C, N 2 Bubbling 1.1 h, then adding 0.506 parts by volume of PMDETA and 0.418 parts by weight of CuBr in sequence, dissolving 0.5 parts by weight with 10 parts by volume of DMFl-P(HEMA) 30 -N 3 ，N 2 Bubbling for 1h to obtainl-P(HEMA) 30 -N 3 A solution; then will bel-P(HEMA) 30 -N 3 The solution is injected into a flask for reaction to continue to react 48h, excessive glacial ethyl ether is used for precipitation after the reaction is finished, DMF is used for dissolving the precipitation and is transferred into a 500Da dialysis bag for dialysis on water, after 48h dialysis, the white product is obtained by freeze-dryingc-P(HEMA) 30 。
6. The method for preparing spherical nucleic acid based on circular template strategy according to claim 1, wherein the specific steps of the second step are as follows:
will be 0.1 part by weightc-P(HEMA) 30 Dissolving in 4 volume parts of DMF, stirring in ice water bath for 10 min, dropwise adding 0.324 volume parts of 2-bromoisobutyryl bromide, reacting at room temperature for 24 hr, adding excessive diethyl ether to precipitate after reaction, dissolving the precipitate in DMF, transferring to 500Da dialysis bag, dialyzing with water for 48h, and lyophilizing to obtain white solid powderc-P(HEMA-Br) 30 ；
Will be 0.135 weight partc-P(HEMA-Br) 30 Dissolving 0.311 weight part of sodium azide in 3 volume parts of DMF solution, reacting at 45 ℃ for 48h, transferring the reaction solution to a 500Da dialysis bag after the reaction is finished, dialyzing 48h against water, and freeze-drying to obtain light yellow solid powderc-P(HEMA-N 3 ) 30 。
7. The method for preparing spherical nucleic acid based on circular template strategy according to claim 1, wherein in the third step, circular templatec-P(HEMA-N 3 ) 30 Synthesis of cyclic macromolecules by copper-free click chemistry of dibenzocyclooctynyl and azidoc-P(HEMA-RNA) 30 。
8. The method for preparing spherical nucleic acid based on circular template strategy according to claim 7, wherein the specific steps of the third step are as follows: 4 OD DBCO-RNA was dissolved in 3. Mu.L of DEPC water, 7.49. Mu.g c-P(HEMA-N 3 ) 30 Dissolving in 30 μL DMSO, mixing, reacting at 50deg.C for 48 hr, dialyzing to remove DMSO, ultrafiltering unreacted DBCO-RNA with 50 kDa ultrafiltering centrifuge tube to remove to obtain cyclic macromoleculec-P(HEMA-RNA) 30 。
9. The method for preparing spherical nucleic acid based on circular template strategy according to claim 8, wherein the RNA is miRNA, the miRNA comprises miR-122, and the nucleic acid is RNA.
10. Use of a spherical nucleic acid as set forth in any one of claims 1-9; the spherical nucleic acid is used as a carrier for preventing degradation of the nucleic acid carried by the spherical nucleic acid by a nucleic acid degrading enzyme.
CN202310795034.3A 2023-06-30 2023-06-30 Spherical nucleic acid preparation method and application based on circular template strategy Pending CN116983279A (en)

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