Preparation method and application of polynucleotide nanocomposite

Abstract

The invention relates to the technical field of biology, in particular to a preparation method and application of a polynucleotide nanocomposite, and the preparation method of the polynucleotide nanocomposite comprises the following steps: assembling a DNA tetrahedron containing i-motif and MMP-9 restriction enzyme cutting sites through an annealing method; the DNA tetrahedron is anchored to the surface of the gold nanorod through ultraviolet light-controlled self-assembly, and a nano-composite is formed; a chemotherapeutic drug is embedded into a double-chain region of the nano-composite by a gradient pH loading method, and a photosensitizer is coupled to prepare the polynucleotide nano-composite. The polynucleotide nano-composite is prepared through light-operated self-assembly and the like, and the accumulation efficiency of the medicine in tumor tissues is improved by combining a dual targeting mechanism of an AS1411 aptamer and an MMP-9 restriction enzyme cutting site; meanwhile, according to the nanocomposite, the drug release time sequence is accurately controlled through input of three kinds of signals of pH, enzyme and near-infrared light, and the drug treatment effect is improved.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a preparation method and application of a polynucleotide nanocomplex. Background Art

[0002] In recent years, nano-drug carriers have shown great potential in the field of cancer treatment, especially delivery systems based on gold nanoparticles, liposomes, polymer nanoparticles and other materials, which have attracted widespread attention due to their adjustable size, surface functionalization capabilities and passive targeting effect (EPR effect); among them, polynucleotides have unique physicochemical properties and biocompatibility and are often used as nano-carriers, such as DNA tetrahedrons.

[0003] However, traditional nanocarriers mainly rely on passive targeting effects (such as the EPR effect), but due to the heterogeneity of tumor blood vessels, the accumulation efficiency of drugs in the tumor site is low. Although active targeting strategies (such as antibody modification) can improve targeting, there are problems such as off-target toxicity and low endocytosis efficiency. In addition, existing stimuli-responsive carriers mostly rely on a single signal trigger (such as pH or enzyme), but the dynamic complexity of the tumor microenvironment leads to imprecise release control. Summary of the invention

[0004] In view of the shortcomings of the prior art, the present invention aims to provide a method for preparing and applying a polynucleotide nanocomplex.

[0005] A method for preparing a polynucleotide nanocomplex comprises the following steps: (1) Synthesis of monodisperse gold nanorods using microfluidic dynamic template method; (2) Assembling a DNA tetrahedron containing the i-motif and the MMP-9 restriction site by annealing; (3) Using UV-controlled self-assembly to anchor DNA tetrahedrons to the surface of gold nanorods to form a nanocomposite; (4) The gradient pH loading method is used to embed the chemotherapy drug into the double-stranded region of the nanocomplex and couple it with a photosensitizer to prepare a polynucleotide nanocomplex; The polynucleotide nanocomplex has a gold nanorod as the core and is wrapped with a DNA tetrahedron framework; the surface of the gold nanorod is modified with a thiol-linked DNA anchoring chain; the vertices of the DNA tetrahedron contain a pH-sensitive i-motif sequence and an MMP-9 enzyme-cleaved peptide sequence, the double-stranded region of the DNA tetrahedron is embedded with a chemotherapeutic drug, the vertices are coupled with a photosensitizer, and the tetrahedron surface is spatially orthogonally modified with an AS1411 aptamer and a targeting peptide activated by MMP-9 enzyme cleavage.

[0006] Preferably, the aspect ratio of the gold nanorods is 4.0-4.5, wherein the length is 25±3 nm and the diameter is 6±1 nm; the i-motif sequence comprises at least three consecutive CCCTAA repeating units and melts at pH ≤ 6.5; and the MMP-9 cleavage peptide sequence is GPLGVRGK.

[0007] Preferably, the chemotherapy drug is doxorubicin (DOX) and / or cisplatin, with a loading amount of 25-35wt%; and the photosensitizer is porphyrin zinc@metal organic framework (ZnPc@MOF) with a particle size of 5-10nm.

[0008] Preferably, each side of the DNA tetrahedron is 15-20 nm long and the surface is modified with Gd 3+ Chelating sites (sequence is T3-C6-T3); 4-mercaptobenzoic acid (4-MBA) is embedded on the surface of the gold nanorods as a SERS reporter molecule.

[0009] Preferably, in step (1), the microfluidic core layer solution is 0.25 mM chloroauric acid and 0.1 M hexadecyltrimethylammonium bromide, with a flow rate of 0.8 mL / min; the shell layer solution is 0.01 M silver nitrate and 0.1 M ascorbic acid, with a flow rate of 0.2 mL / min.

[0010] Preferably, in step (2), among the four DNA chains of the DNA tetrahedron, the sequence of chain 1 is 5'-AS1411 aptamer-(CCCTAA)3-MMP-9 complementary region-3'; the sequence of chain 2 is 5'-T3-C6-T3-ZnPc@MOF coupling end-3'; and the sequences of chains 3 and 4 are 5'-GC repeat region (chemotherapeutic drug embedding site)-3'.

[0011] Preferably, in step (3), the UV irradiation conditions are 365 nm, 10 mW / cm 2 , lasting 30±5 minutes.

[0012] A use of a polynucleotide nanocomplex prepared by any of the above-mentioned methods for preparing a polynucleotide nanocomplex in the preparation of drugs for synergistic tumor therapy.

[0013] The polynucleotide nanocomplex is used in the preparation of a drug for synergistic tumor therapy. The polynucleotide nanocomplex drug is intravenously injected, and AS1411 aptamer and MMP-9 enzyme cleavage are used to activate targeting; chemotherapy drugs are released in a tumor microenvironment (pH 6.5-7.0, MMP-9 ≥ 50 ng / mL); 808 nm near-infrared light is applied at 1-1.5 W / cm 2 Synchronously trigger photothermal and photodynamic therapy.

[0014] Preferably, the synergistic parameters of photothermal and photodynamic therapy are: first, at 1 W / cm 2 Irradiation for 5 minutes induced an increase in vascular permeability, and then 0.5 W / cm 2 The drug penetration is enhanced for 10 minutes; the complete tumor remission rate is ≥90% and the metastasis inhibition rate is ≥95% during the treatment cycle.

[0015] Preferably, imaging guidance is also included, using MRI and SERS imaging technology to locate the tumor before surgery (detection limit ≤ 0.2mm 3 ), 808nm near-infrared light was used to trigger photothermal therapy (heating to 48±2℃) during the operation, and SERS was used to dynamically monitor residual lesions after the operation.

[0016] The beneficial effects achieved by the present invention are: 1. The present invention prepares a polynucleotide nanocomplex through light-controlled self-assembly, etc., and combines the dual targeting mechanism of AS1411 aptamer and MMP-9 cleavage site, so that the nanocomplex can remain stable in the blood circulation, avoiding nonspecific adsorption and off-target toxicity. Once it reaches the tumor site, the MMP-9 cleavage site is activated, and the AS1411 aptamer can fully exert its targeting function, thereby improving the accumulation efficiency of the drug in the tumor tissue; at the same time, the nanocomplex uses three signal inputs of pH, enzyme and near-infrared light to accurately control the timing of drug release, thereby improving the therapeutic effect of the drug.

[0017] 2. The present invention activates the AS1411 aptamer through MMP-9 enzymatic cleavage to achieve dynamic targeting of "passive enrichment (EPR effect) + active locking (aptamer)", thereby increasing the tumor accumulation rate and improving the targeting specificity. The enzymatic peptide (GPLGVRGK) is only cleaved in the tumor microenvironment (MMP-9 ≥ 50 ng / mL), reducing the nonspecific uptake of normal tissues, integrating pH (i-motif), enzyme (MMP-9) and photothermal (NIR) signals to achieve precise spatiotemporal release of drugs.

[0018] 3. The present invention combines multiple synergistic effects. The nanocomposite uses gold nanorods as the core component, which generates heat under 808nm near-infrared light irradiation to achieve photothermal therapy. At the same time, the embedded chemotherapy drugs and photosensitizers (ZnPc@MOF) play the roles of chemotherapy and photodynamic therapy respectively. In addition, photothermal therapy can also induce immune response and enhance anti-tumor immune effect, thereby achieving the spatiotemporal coupling effect of chemotherapy, photothermal and photodynamic therapy, and maximizing the synergistic effect of each therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The present invention is a flowchart of the method for preparing the polynucleotide nanocomplex used in the present invention. DETAILED DESCRIPTION

[0020] The present invention is further described in detail below in conjunction with specific embodiments.

[0021] Example: A method for preparing a polynucleotide nanocomplex, such as Figure 1 As shown, the following steps are included: (1) Synthesis of gold nanorods (AuNR): core solution: 0.25 mM chloroauric acid + 0.1 M hexadecyltrimethylammonium bromide, flow rate 0.8 mL / min; shell solution: 0.01 M silver nitrate + 0.1 M ascorbic acid, flow rate 0.2 mL / min; microfluidic dynamic template method, reaction temperature 75 °C, reaction time 30 min, to generate gold nanorods; 4-mercaptobenzoic acid was embedded on the surface of the gold nanorods as a surface enhanced Raman scattering (SERS) reporter molecule; (2) DNA tetrahedral assembly: Among the four DNA chains, chain 1: 5'-AS1411 aptamer-(CCCTAA)3-MMP-9 complementary region-3'; chain 2: 5'-T3-C6-T3-ZnPc@MOF coupling end-3'; chain 3 / chain 4: 5'-GC repeat region (DOX embedding site)-3'; Annealing procedure: Mix the four DNA strands (molar ratio 1:1:1:1) in Tris-Mg 2+ Buffer (10mM Tris, 5mM MgCl2, pH7.4); heated at 95℃ for 5min, then cooled to 4℃ at 4℃ / min, finally forming a DNA tetrahedron structure, which contains pH-sensitive i-motif sequence and MMP-9 cleavage peptide sequence (GPLGVRGK) at the vertex, double-stranded region for embedding chemical drugs, and vertex coupling photosensitizer (ZnPc@MOF); at the same time, Gd was modified on the surface of DNA tetrahedron 3+ The chelating site (sequence is T3-C6-T3) enables it to have the function of magnetic resonance imaging (MRI), and the AS1411 aptamer and the targeting peptide activated by MMP-9 cleavage are spatially orthogonally modified on the tetrahedron surface to improve the targeting specificity; (3) UV-controlled self-assembly: Gold nanorods (1 mg / mL) and DNA tetrahedrons (0.1 mM) were mixed and exposed to UV light at 365 nm and 10 mW / cm 2 The samples were irradiated with ultraviolet light for 30 min to induce a self-assembly reaction between the thiol-modified DNA anchor chains and the surface of the gold nanorods, thus firmly fixing the DNA tetrahedrons on the surface of the gold nanorods. (4) Drug and photosensitizer loading: Doxorubicin (DOX) was embedded into the double-stranded region of the DNA tetrahedron using the gradient pH loading method (pH was set between 6.0 and 8.0) and purified by ultrafiltration. The drug loading capacity was 32.7 wt%. ZnPc@MOF was coupled to the vertices of the DNA tetrahedron using EDC / NHS activation. The coupling efficiency was 89.5%, and a polynucleotide nanocomplex was prepared.

[0022] Performance testing: Experimental verification of the functions of polynucleotide nanocomplexes, including drug release behavior, photothermal-photodynamic synergistic effects, in vivo anti-tumor effects, imaging guidance capabilities, and safety evaluation; 1. Multiple responsive drug release: prepare polynucleotide nanocomplexes according to the method in the examples; divide the prepared nanocomplexes into four portions, each portion is used for the following four experimental groups; Group 1: pH 7.4 + no stimulation; in PBS buffer at pH 7.4, no additional stimulation was applied to simulate the physiological environment.

[0023] Group 2: pH 6.5 + 37 ° C; in an acidic environment of pH 6.5, simulating the pH value of the tumor microenvironment.

[0024] Group 3: pH 6.5 + MMP-9 (100 ng / mL); MMP-9 enzyme (100 ng / mL) was added to an acidic environment of pH 6.5 to simulate the high expression of MMP-9 in the tumor microenvironment.

[0025] Group 4: pH 6.5 + MMP-9 + NIR (808 nm, 1 W / cm 2 , 5min); MMP-9 enzyme (100ng / mL) was added in an acidic environment of pH 6.5, and 808nm near-infrared light irradiation (1W / cm 2 , 5 minutes), simulating the photothermal triggering conditions in the tumor microenvironment.

[0026] Each experimental group was sampled after 24 h, and the release of DOX and ZnPc@MOF was measured by high performance liquid chromatography (HPLC) or UV-visible spectrophotometer. The results are shown in Table 1.

[0027] Table 1 Response release performance test results (24h cumulative release rate)

[0028] 2. Photothermal-photodynamic synergistic effect: 4T1 breast cancer cells were used and divided into: control group (no treatment); simple DOX group (5 μg / mL); AuNR+NIR group (1 W / cm 2 , 5min); polynucleotide nanocomplex (referred to as complex) + NIR group (1W / cm2 The survival rate and ROS level of cells in each group were detected. The results are shown in Table 2.

[0029] Table 2 Photothermal-photodynamic synergistic effect test results (48h cell survival rate)

[0030] 3. Anti-tumor effect in vivo Animal model establishment: BALB / c mice were subcutaneously inoculated with 4T1 breast cancer cells. When the tumor volume reached about 100 mm 3 The groups were divided into groups, with n=8 in each group; The control group (normal saline); free DOX group (5 mg / kg); polynucleotide nanocomplex (referred to as complex) group (no light); polynucleotide nanocomplex (referred to as complex) + NIR group (808 nm, 1 W / cm 2 , 5min); The treatment regimen was tail vein injection (once every 3 days, 3 times in total). Light treatment was performed 24 hours after the first administration. The tumor volume was measured, the survival time of each group of mice was recorded, and the median survival time was calculated. The results are shown in Table 3.

[0031] Table 3 In vivo antitumor test results

[0032] 4. Multimodal imaging to guide treatment Preoperative localization: 6 h after intravenous injection of polynucleotide nanocomplexes, tumors were localized using MRI and SERS imaging techniques; MRI detection: T1-weighted images show that the signal in the tumor area is enhanced by 2.3 times (vs surrounding tissue); SERS imaging: Raman intensity at the tumor site (1130 cm -1 ) up to 1.2×10 6 counts / mm 2 ; Intraoperative navigation: Real-time PAI monitoring after NIR irradiation, recording the increase of tumor temperature from 37°C to 48°C; Post-treatment monitoring: 7 days after treatment, the SERS signal decreased to 12% of the initial value (indicating the completion of drug release).

[0033] Table 4 Comparison of imaging performance

[0034] 5. Safety evaluation Blood biochemical index detection: On the 7th day after treatment, blood samples of mice were collected to detect indicators such as ALT, AST and BUN, as shown in Table 5.

[0035] Table 5 Blood biochemical indexes (7th day after treatment)

[0036] Histopathological analysis: H&E staining of the heart, liver, spleen, lung, kidney and other major organs showed no obvious pathological damage; large areas of tumor tissue were necrotic (≥90%), and inflammatory cell infiltration was visible at the edge.

[0037] The above embodiments are merely illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Anyone familiar with the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by a person of ordinary skill in the art without departing from the spirit and technical concept disclosed by the present invention shall still be covered by the claims of the present invention.

Claims

1. A method for preparing a polynucleotide nanocomplex, characterized in that: The following steps are involved: (1) Synthesis of monodisperse gold nanorods using microfluidic dynamic template method; (2) Assembling a DNA tetrahedron containing the i-motif and the MMP-9 restriction site by annealing; (3) Using UV-controlled self-assembly to anchor DNA tetrahedrons to the surface of gold nanorods; (4) The gradient pH loading method is used to embed the chemotherapy drug into the double-stranded region and couple it with a photosensitizer to prepare a polynucleotide nanocomplex; The polynucleotide nanocomplex has a gold nanorod as the core and is wrapped with a DNA tetrahedron framework; the surface of the gold nanorod is modified with a thiol-linked DNA anchoring chain; the vertices of the DNA tetrahedron contain a pH-sensitive i-motif sequence and an MMP-9 enzyme-cleaved peptide sequence, the double-stranded region of the DNA tetrahedron is embedded with a chemotherapeutic drug, the vertices are coupled with a photosensitizer, and the surface of the tetrahedron is spatially orthogonally modified with an AS1411 aptamer and a targeting peptide activated by MMP-9 enzyme cleavage.

2. The method for preparing the polynucleotide nanocomplex according to claim 1, characterized in that: The aspect ratio of the gold nanorods is 4.0-4.5, wherein the length is 25±3nm and the diameter is 6±1nm; the i-motif sequence comprises at least three consecutive CCCTAA repeating units and is unzipped at pH≤6.5; and the MMP-9 enzyme cleavage peptide sequence is GPLGVRGK.

3. The method for preparing the polynucleotide nanocomplex according to claim 1, characterized in that: The chemotherapy drugs are doxorubicin and / or cisplatin, with a loading amount of 25-35wt%; the photosensitizer is porphyrin zinc@metal organic framework with a particle size of 5-10nm.

4. The method for preparing a polynucleotide nanocomplex according to claim 1, characterized in that: The length of each side of the DNA tetrahedron is 15-20 nm, and the surface is modified with Gd 3+ The chelating site has a sequence of T3-C6-T3; 4-mercaptobenzoic acid is embedded on the surface of the gold nanorod as a SERS reporter molecule.

5. The method for preparing a polynucleotide nanocomplex according to claim 1, characterized in that: In step (1), the microfluidic core layer solution is 0.25 mM chloroauric acid and 0.1 M hexadecyltrimethylammonium bromide, with a flow rate of 0.8 mL / min; the shell layer solution is 0.01 M silver nitrate and 0.1 M ascorbic acid, with a flow rate of 0.2 mL / min.

6. The method for preparing the polynucleotide nanocomplex according to claim 1, characterized in that: In step (2), among the four DNA chains of the DNA tetrahedron, the sequence of chain 1 is 5'-AS1411 aptamer-(CCCTAA)3-MMP-9 complementary region-3'; the sequence of chain 2 is 5'-T3-C6-T3-ZnPc@MOF coupling end-3'; and the sequences of chain 3 and chain 4 are 5'-GC repeat region (chemotherapeutic drug embedding site)-3'.

7. The method for preparing a polynucleotide nanocomplex according to claim 1, characterized in that: In step (3), the UV light conditions are 365nm, 10mW / cm 2 , lasting 30±5 minutes.

8. Use of a polynucleotide nanocomplex prepared by the method for preparing a polynucleotide nanocomplex according to any one of claims 1 to 7 in preparing drugs for synergistic tumor therapy.

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