An amphiphilic nanoassembly based on hydrophobic molecules and oligodenucleotide chains, preparation method and application

Abstract

The present invention proposes an amphiphilic nanoassembly based on a hydrophobic molecule and an oligodenucleotide chain, a preparation method, and an application thereof. The invention also provides a preparation method for an amphiphilic nanoassembly in which a hydrophilic oligodenucleotide single chain is covalently linked to a hydrophobic organic small molecule. A hydrophobic tetraphenylethylene derivative (TPE) having aggregation-induced emission properties is covalently linked to the 5' end of a hydrophilic oligodenucleotide single chain (DNA) to prepare an amphiphilic TPE-DNA molecule. The reaction system is purified and characterized by gel electrophoresis, HPLC, time-of-flight mass spectrometry, DLS, TEM, and other characterization methods, as well as the morphology and size of the nanoassembly. At room temperature, the molecule can self-assemble in a specific solvent to form micelles. The self-assembled micelles can be used for loading hydrophobic drugs and releasing drugs after enzymatic hydrolysis, as well as having strong fluorescence emission.

Description

Technical Field

[0001] The present invention relates to the field of organic molecule synthesis, in particular to the field of self-assembly of DNA nanomaterials. Background Art

[0002] Micelles, which are spherical, lamellar, or rod-shaped nanoparticles composed of amphiphilic surfactants (non-polymer micelles) or amphiphilic copolymers (polymer micelles), have attracted the interest of researchers as a new type of drug carrier. Micelles have stable, long-lasting effects, and high biodistribution. They can form core-shell structures ranging in size from 10 to 100 nm in specific environments. They are mainly composed of an outer hydrophilic shell and an inner hydrophobic core, so they can be loaded with hydrophobic drugs. In addition, the outer hydrophilic shell can provide stability to the micelles in aqueous environments. Since the 1980s, DNA has been widely used in the construction of nanomaterials based on the predictable and programmable sequence-specific hybridization between DNA chains of Watson-Crick base pairs. With the development of DNA nanotechnology, different DNA nanostructures have emerged and have been applied in many fields, such as biomedicine, nanomaterials, and biomimetic materials.

[0003] In the early 1980s, Seeman creatively used DNA as a building block for nanomaterials, bringing significant changes to the biological field. Since then, numerous oligonucleotide-based nanomaterials have been used in the field of drug delivery. DNA self-assembly is a widely used method. DNA amphiphiles, formed by self-assembly in specific solvents, can form a variety of shapes and can encapsulate hydrophilic or hydrophobic drug molecules within their structures. Although DNA's low immunogenicity and negligible cytotoxicity generally make it superior to metal- and polymer-based drug delivery platforms, nucleases in body fluids and cells rapidly degrade DNA, resulting in poor therapeutic or delivery effects. Current research has demonstrated the use of DNA coupled to organic small molecules to achieve the assembly of nanoparticles with different morphologies in aqueous solution. Different organic small molecules can be designed to impart different functions to nanoassemblies, such as imaging and targeting. Summary of the Invention

[0004] In order to overcome the shortcomings of existing nano drug carrier preparation technology, such as poor water solubility of drug monomers, poor circulation characteristics of drug carriers, high in vivo biological toxicity, and single functionality, the present invention provides a simple and universal method, which uses amphiphilic molecules after coupling aggregation-induced luminescence molecules with imaging capabilities with endogenous biological macromolecules DNA as building units, and self-assembles in a specific solvent to form TPE-nC-DNA nano micelles with autofluorescence and good biocompatibility. These TPE-nC-DNA micelles are constructed from endogenous biological macromolecules, so the assembly has excellent biocompatibility. TPE-nC-DNA molecules can achieve drug loading and delivery through hydrophobic interactions. After the TPE-nC-DNA nano micelles are enzymatically hydrolyzed by DNA enzymes, the designed hydrophobic molecules can produce strong fluorescence at the drug release site through aggregation-induced emission.

[0005] One of the purposes of the present invention is to construct nanomicelles through hydrophobic interactions.

[0006] The second purpose of the present invention is to construct a nano-micelle with good biocompatibility and no toxicity.

[0007] The second purpose of the present invention is to construct nanoassemblies for efficient drug loading.

[0008] The third purpose of the present invention is to enable the constructed nanoassembly to realize imaging by utilizing the fluorescent molecules it carries.

[0009] In order to achieve the above objectives, the technical solution adopted by the present invention is:

[0010] The preparation of TPE-nC-DNA nanomicelles is mainly divided into two parts. The first part is the synthesis of tetraphenylethylene derivatives and oligonucleotide single chains modified with tetraphenylethylene derivative molecules, and the second part is the formation of nanomicelles.

[0011] Tetraphenylethylene derivative molecules are obtained through substitution reactions between monohydroxytetraphenylethylene and 5-bromo-1-pentanol, 10-bromo-1-decanol, or 16-bromo-hexadecanol, or through substitution reactions between monobromosubstituted tetraphenylethylene and 1,5-pentanediol or 1,10-decanediol. Single-stranded oligonucleotides modified with tetraphenylethylene derivatives are obtained via solid-phase synthesis. The sequence of the single-stranded oligodenucleotide is OH-D18-CPG, where D18 is shown in SEQ ID No. 1. ① First, a certain amount of TPE-nC-OH is reacted with a phosphochlorine reagent in anhydrous and oxygen-free dichloromethane. ② The single-stranded oligonucleotide with a hydroxyl group at the 5' end, supported on a CPG bead, is mixed with the solution obtained in ① under anhydrous and oxygen-free conditions and reacted at room temperature for 8 hours. ③ The mixture is washed with acetonitrile and dichloromethane, respectively, and then reacted with an oxidizing reagent (I2|THF|H2O|Py) for 1 minute. ④ The oxidized crude product was reacted with a certain amount of ammonia water for 3 hours to remove CPG and obtain a crude DNA product modified with a tetraphenylethylene derivative.

[0012] ⑤ The crude product was purified by HPLC and the concentration was determined by UV test.

[0013] TPE-nC-DNA nanomicelles were prepared by the following method: a certain amount of TPE-10C-DNA stored in aqueous solution was added to a mixed solution of CH3CN / H2O, maintained at 90°C for 30 min, then cooled to 25°C at a rate of 1°C / min and stored at 25°C.

[0014] Compared with the prior art, the present invention has the following outstanding effects:

[0015] 1. The present invention provides an amphiphilic nanoassembly based on hydrophobic molecules and oligodenucleotide chains, and its micelles. The amphiphilic TPE-nC-DNA molecules, obtained by coupling endogenous macromolecular DNA with tetraphenylethylene derivatives, serve as building blocks and self-assemble in a mixed solvent to form nanomicelles. Compared to conventional polymer micelles, the amphiphilic nanoassembly TPE-nC-DNA prepared in the present invention utilizes a specific DNA sequence containing 18 bases, resulting in a micellar shape. The micelles of this amphiphilic nanoassembly exhibit excellent biocompatibility and are non-toxic and harmless, making them widely applicable for drug loading and delivery.

[0016] 2. The main body of the nanomicelle is a tetraphenylethylene derivative in the hydrophobic part and an oligodeoxyribonucleic acid single chain in the hydrophilic part. The amphiphilic core-shell structure enables it to load hydrophobic drugs in the hydrophobic region. After hydrolysis by DNA hydrolase, it can release the drug while promoting the aggregation-induced emission of tetraphenylethylene molecules, thereby bringing about strong fluorescence emission.

[0017] 3. The present invention also provides a method for preparing the nanomicelles, which features a simple and easy-to-use preparation process. By utilizing hydrophobic interactions as the driving force, functionalized nanomicelles are constructed in a specific solvent. The simplicity and universality of this preparation method opens the possibility of covalently linking more functional molecules to DNA single strands, greatly expanding the application range of nanomicelles. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is the H NMR spectrum of TPE-10C-OH;

[0019] Figure 2 This is the NMR carbon spectrum of TPE-10C-OH;

[0020] Figure 3 The electrophoresis results of the target product TPE-10C-DNA are shown in Figure 1. Lane 1 is the DNA raw material, and lane 2 is the TPE-10C-DNA product.

[0021] Figure 4 High performance liquid chromatography (HPLC) characterization of TPE-10C-DNA. The retention time of TPE-10C-DNA is 25 min.

[0022] Figure 5 This is the time-of-flight mass spectrum of TPE-10C-DNA.

[0023] Figure 6 This is the dynamic light scattering diagram of TPE-10C-DNA nanomicelles.

[0024] Figure 7 This is the transmission electron microscopy (TEM) image of TPE-10C-DNA nanomicelles.

[0025] Figure 8 The fluorescence results of drug loading.

[0026] Figure 9 The fluorescence results of drug release. DETAILED DESCRIPTION

[0027] The invention is further described below through examples in combination with the accompanying drawings. The examples are only used to further explain the technical solutions of the present invention and cannot be considered as limiting the scope of protection of the present invention. Non-essential improvements or adjustments made by those skilled in the art based on the contents of the above invention are all within the scope of protection of the present invention.

[0028] Example 1

[0029] In this example, the target product TPE-10C-DNA was prepared by the following method, the specific steps are:

[0030] Step 1. Synthesis of TPE-10C-OH and solid-phase synthesis of TPE-10C-DNA:

[0031]

[0032] Catalyst: potassium carbonate, potassium iodide. Solvent: anhydrous acetonitrile.

[0033] Specific experimental procedures: TPE-OH (200 mg, 0.6 mmol), K2CO3 (260 mg, 1.8 mmol), 10-bromo-1-decanol (170 mg, 0.72 mmol), and a catalytic trace of KI (2% mol) were weighed into a 100 mL two-necked flask. Under a nitrogen atmosphere, 25 mL of anhydrous acetonitrile was added and stirred at 78°C for 10 h. After cooling to room temperature, 25 mL of saturated sodium bicarbonate solution was added and stirred vigorously for 0.5 h. After stirring, the mixture was extracted with dichloromethane (20 mL x 3). The organic layer was collected, the solvent evaporated under reduced pressure, and then column chromatography was performed using a 1:1.5 ratio of petroleum ether to dichloromethane. The product obtained after column chromatography was a white powder with a yield of 46%.

[0034] Step 2. Solid-phase synthesis of TPE-10C-DNA:

[0035]

[0036] Catalyst: N,N-diisopropylethylamine, 5-ethylmercaptotetrazolyl.

[0037] The following DNA sequences are from Huzhou Hippo Biotechnology Co., Ltd.

[0038] Specific experimental steps: Weigh TPE-10C-OH (0.106 g, 0.2 mmol) obtained in step 1 into a 10 mL round-bottom flask, add 2 mL of anhydrous and oxygen-free dichloromethane and N, N-diisopropylethylamine (204.48 μL, 1.2 mmol) under an argon atmosphere, stir for five minutes, then add 2-cyanoethyl-N, N-diisopropylethylamine chlorophosphoramidite (140.88 μL, 0.6 mmol), react at room temperature for 40 minutes, and monitor the reaction degree by TLC during this period. After the reaction is completed, add 2 mL of anhydrous dichloromethane to dilute, wash with 5 mL of saturated sodium bicarbonate, 5 mL of deionized water, and 5 mL of saturated sodium chloride in sequence, dry the organic layer with anhydrous sodium sulfate, concentrate under reduced pressure, and then add 2 mL of anhydrous and oxygen-free dichloromethane to dissolve the product under an argon atmosphere. Under an argon atmosphere, the resulting product was added to a 5 mL shaped vial containing DNA-CPG (1 μmol) and 5-ethylmercaptotetrazole (13.017 mg, 100 μmol) and allowed to react at room temperature for 8 h. After the reaction was complete, the product was washed twice with 5 mL of acetonitrile and 5 mL of dichloromethane, followed by oxidation with 1 mL of an oxidizing agent (I2|THF|H2O|Py) for 1 min. After oxidation, the product was washed twice with 5 mL of acetonitrile. After removing all liquids, 1.25 mL of ammonia was added and the reaction was continued at 55°C for 3 h. After the reaction was complete, the product was purified by HPLC.

[0039] The products obtained in steps 1 and 2 above were characterized and analyzed, and the results were as follows:

[0040] (1) Using a nuclear magnetic resonance spectrometer to characterize the product obtained in step 1, such as Figure 1 、 Figure 2 shown.

[0041] (2) Prepare 20% denaturing polyacrylamide gel to characterize the product obtained in step 2. The electrophoresis condition is 150V for 1h. The results are as follows: Figure 3 As shown in the figure, the band of TPE-10C-DNA is obviously different from the band of D18 raw material, and the band of TPE-10C-DNA has a significantly lower migration rate than that of D18, which indicates the successful coupling of TPE-10C-OH and D18.

[0042] (3) The product obtained in step 2 was characterized by HPLC using 0.1 M TEAA and anhydrous acetonitrile as eluents. Figure 4 After collecting the product at a retention time of 25 min, the product was concentrated using a DNA concentrator and the product concentration was adjusted to 100 μM.

[0043] (4) The purified product of step 2 is characterized by time-of-flight mass spectrometry, and the proton peak of the target product can be obtained. Figure 5The vertical axis of the figure is the percentage of ion signal intensity, and the highest peak of the time-of-flight mass spectrum represents the content of the target product TPE-10C-DNA.

[0044] When 10-bromo-1-decanol in this example is replaced by 5-bromo-1-pentanol, 1,5-pentanediol, 1,10-decanediol, and 16-bromo-hexadecanol, the target products are TPE-5C-OH, TPE-10C-OH, and TPE-16C-OH, respectively.

[0045] Example 2

[0046] In this example, the target product TPE-10C-DNA nanomicelles was prepared by the following method. The specific preparation steps are as follows:

[0047] The purified TPE-10C-DNA molecules from step 2 of Example 1 were dissolved in deionized water at a concentration of 100 μM and stored at 4°C. Next, 42 μL of the preservative solution was added to a mixture containing 5 μL of anhydrous acetonitrile and 58 μL of water. The three were mixed thoroughly to obtain a solution with a TPE-10C-DNA concentration of 40 μM. The resulting solution was then maintained at 90°C for 30 minutes, then cooled to 25°C at a rate of 1°C / min and stored.

[0048] like Figure 6 and Figure 7 Figure 2 shows the characterization of the nanomicelles obtained in the above steps using dynamic light scattering (DLS) and transmission electron microscopy (TEM). DLS shows a hydrated particle size of 34 ± 5 nm, while TEM shows a diameter of 25 ± 2 nm. Generally, the particle size indicated by DLS is slightly larger than that indicated by TEM because DLS measures the hydrated particle size of the substance.

[0049] In this embodiment, TPE-10C-DNA can also be replaced by TPE-5C-OH or TPE-16C-OH.

[0050] Example 3

[0051] In this example, the loading and release of drugs by the target products TPE-5C-OH, TPE-10C-OH, and TPE-16C-OH nanomicelles obtained in Example 2 were detected by the following method:

[0052] (1) Drug loading: 40 μM, 105 μL TPE-10C-DNA solution was added to a 200 μL centrifuge tube containing 31.5 ng camptothecin (anticancer drug CPT), maintained at 90°C for 30 min, then cooled to 25°C at a rate of 1°C / min and stored. The blank group was an assembly without camptothecin, and its treatment steps were the same as the drug-loaded group. The pure drug group did not contain TPE-10C-DNA, and its treatment steps were the same as the drug-loaded group. The DNA concentration of all groups was 40 μM, the volume was 105 μL, and the CPT concentration was 0.3 μg / mL.

[0053] The target products TPE-10C-DNA@CPT, TPE-10C-DNA, and CPT obtained in the above steps were detected by fluorescence spectrophotometry in the presence of CH3CN / H2O=1 / 20 and a final volume of 105 μL. The results are as follows: Figure 8 The fluorescence of the CPT-encapsulated nanoparticles was stronger than that of the blank carrier (TPE-10C-DNA group) because the fluorescent properties of CPT itself enhanced the overall fluorescence. On the other hand, since the CPT molecules were encapsulated in the hydrophobic domains of the amphiphilic nanoparticles, the intermolecular charge transfer and π-π stacking between the TPE and CPT molecules made the fluorescence of the drug-loaded nanoparticles weaker than that of the pure drug group.

[0054] (2) Drug release: 3 μL of 2000 U / mL DNase I and 3 μL of reaction buffer were added to the solution obtained in step (1). After incubation at 37°C for 1.5 h, EDTA was added to quench the reaction and the resulting sample was named TPE-10C-DNA@CPT+DNase. The control group, the CPT group, and the TPE-10C-DNA@CPT group were left untreated.

[0055] The target products CPT, TPE-10C-DNA@CPT, and TPE-10C-DNA@CPT+DNase obtained in the above steps were detected by fluorescence spectrophotometry in the presence of CH3CN / H2O=1 / 20 and a final volume of 105 μL. The results are as follows: Figure 9 As shown in the figure, the TPE-10C-DNA amphiphilic nanocarriers in the enzymatic hydrolysis group were digested by DNase, resulting in the release of TPE molecules originally in the hydrophobic domain of the amphiphilic carrier into the mixed solution of acetonitrile and water. The aggregation of the hydrophobic TPE molecules caused them to produce a strong fluorescence of 2.5 times that of the drug-loaded group through aggregation-induced luminescence.

Claims

1. Amphiphilic nanoassemblies based on hydrophobic molecules and oligodenucleotide chains, characterized in that The hydrophobic tetraphenylethylene TPE derivative TPE-10C-OH was coupled with the hydrophilic oligodenucleotide chain by chemical bonding to obtain the amphiphilic TPE-10C-DNA molecule, whose structure is as follows: ，n=8； The amphiphilic TPE-10C-DNA molecules self-assembled into spherical nanoassemblies in a mixed solvent, wherein the mixed solvent was a CH3CN / H2O solution, and the volume ratio of the organic solvent to water in the CH3CN / H2O solution was 1:20; The number of bases of the oligonucleotide single chain is 18, and the sequence of the oligonucleotide single chain is OH-D18-CPG:5'-OH-TTCACAGGTCAACGCGCG-CPG; The molecular structure of tetraphenylethylene TPE derivative TPE-10C-OH is 。 2. The method for preparing an amphiphilic nanoassembly based on hydrophobic molecules and oligodenucleotide chains according to claim 1, wherein: The method comprises the following steps: taking an appropriate amount of tetraphenylethylene TPE derivative and the oligodeoxynucleotide chain for solid phase synthesis reaction, coupling the tetraphenylethylene derivative to the 5' end of the oligodeoxynucleic acid single chain, removing the CPG ball, evaporating the solvent under reduced pressure, and purifying to obtain the target product TPE-10C-DNA.

3. The preparation method according to claim 2, characterized in that The tetraphenylethylene (TPE) derivative TPE-10C-OH is obtained by reacting monohydroxytetraphenylethylene with 10-bromo-1-decanol, or by reacting monobromo-substituted tetraphenylethylene with 1,10-decanediol; the molar ratio of monohydroxytetraphenylethylene to alcohol is 5:6, and the molar ratio of monobromo-substituted tetraphenylethylene to diol is 5:

7.

4. The preparation method according to claim 2, characterized in that The oligonucleotide chain is CPG-D18-OH, and the tetraphenylethylene TPE derivative and CPG-D18-OH are fully reacted at room temperature in the presence of a phosphochlorine reagent at a molar ratio of 200:

1. The mixed system after the reaction is washed and then reacted with an oxidizing reagent. The crude product obtained after oxidation is added with ammonia water and incubated at 55° C. for 3-5 hours to remove the CPG beads. The solvent is then evaporated under reduced pressure, and the concentrated product obtained after the reduced pressure evaporation is dissolved in deionized water to obtain a solution of the crude product. The solution of the crude product is purified by HPLC, and the eluent includes 0.1M TEA and anhydrous acetonitrile. After purification, the solvent is evaporated under reduced pressure and desalted to obtain the target product TPE-10C-DNA.

5. The preparation method according to claim 4, characterized in that The preparation steps of the crude product before oxidation include: adding the tetraphenylethylene TPE derivative to anhydrous and oxygen-free dichloromethane and N,N-diisopropylethylamine, mixing them, adding 2-cyanoethyl-N,N-diisopropylethylamine chlorophosphoramidite to react, adding anhydrous dichloromethane to dilute the mixture after the reaction, washing with saturated sodium bicarbonate, deionized water, and saturated sodium chloride in sequence, then removing water and dissolving in anhydrous and oxygen-free dichloromethane, mixing with CPG-D18-OH at a molar ratio of 200:1 under an oxygen-free environment, adding 5-ethylmercaptotetrazole to react, and washing the mixed system after the reaction and reacting with an oxidizing reagent I2|THF|H2O|Py to obtain an oxidized crude product.

6. A method for preparing micelles based on the amphiphilic nanoassembly of hydrophobic molecules and oligodenucleotide chains according to claim 1, characterized in that: The method comprises the following steps: taking a certain amount of an aqueous solution of TPE-10C-DNA amphiphilic molecules and dissolving it in the mixed solvent; mixing the mixture evenly; retaining the mixture at 90°C for 30 minutes; and cooling the mixture to 25°C at a rate of 1°C / min and storing the mixture to obtain nanomicelles of the TPE-10C-DNA amphiphilic nanoassembly.

7. A micelle comprising the amphiphilic nanoassembly based on self-assembly of hydrophobic molecules and oligodenucleotide chains according to claim 1, characterized in that: The morphology of the assembly in a CH3CN / H2O solution is micellar, the storage temperature of the micelles of the amphiphilic nanoassembly is 25°C, and the micelles of the amphiphilic nanoassembly disassemble in the presence of DNA enzyme.

8. Use of the micelle of the amphiphilic nanoassembly based on self-assembly of hydrophobic molecules and oligodenucleotide chains as claimed in claim 7 in the preparation of drugs for loading, delivering and releasing camptothecin.

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