Multifunctional DNA nanosphere, preparation method therefor, and use thereof

Abstract

A multifunctional DNA nanosphere, a preparation method therefor, and use thereof. An acid-responsive i-motif sequence, an EGFRvIII aptamer A32 sequence, and an MRI contrast agent Gd-DOTA-N3 are combined to design the DNA nanosphere to achieve MRI enhancement / fluorescence integrated dual development of glioma, so as to obtain a glioma tumor development signal that can meet clinical requirements. Meanwhile, the acid-responsive i-motif sequence, the EGFRvIII aptamer A32 sequence, the MRI contrast agent Gd-DOTA-N3, and a chemotherapeutic drug doxorubicin (DOX) are combined, Gd-DOTA-N3 and Cy5-BHQ fluorescent pairs are modified on specific bases, and using the principle of DNA complementary base pairing, novel multifunctional DNA nanospheres for glioma fluorescence / MRI imaging and treatment are prepared.

Description

Multifunctional DNA nanoball and preparation method and application thereof TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, in particular to a multifunctional DNA nanoball and preparation method and application thereof. BACKGROUND

[0002] At present, magnetic resonance (MRI) is the most effective examination method for imaging the range of glioma and judging the tumor adjacent relationship. Through different sequence scanning and gadolinium contrast agent enhanced scanning, MRI can assist glioma imaging and guide glioma surgery. However, the current MRI examination including enhanced scanning still has the following disadvantages in glioma imaging:

[0003] (1) MRI shows the preoperative tumor image range, which cannot represent the actual tumor boundary observed during surgery. At present, the MRI intraoperative navigation technology guided by MRI for glioma range also has the problem of "brain tissue deformation and displacement". Due to the high running cost and the objective prolongation of anesthesia time, the navigation combined with intraoperative MRI (iMRI) technology greatly limits its generalization in clinical practice.

[0004] (2) The current principle of MRI enhanced imaging is mainly to use the incomplete wall of tumor neovasculature in glioma. The gadolinium contrast agent can penetrate the tumor blood brain barrier (tBBB) and gather in the extracellular space of the tumor to realize the enhanced imaging of glioma. However, it is not a real sense of targeted into glioma tumor cells. Therefore, in the case of low-grade glioma with relatively complete tBBB, the enhanced MRI is not ideal for imaging the range of glioma. In addition, due to the short retention time of gadolinium in glioma tumor tissue, the actual gadolinium dose used to ensure the enhancement effect will remain in the kidneys, liver and other organs, thereby increasing the risk of secondary damage to the kidneys, liver and other organs.

[0005] (3) It is also difficult to effectively distinguish between tumor pseudoprogression, local radiation necrosis and glioma recurrence which occur after glioma resection and adjuvant radiotherapy by MRI enhanced scanning.

[0006] Therefore, it is necessary to solve the above problems to fully exert the advantages of MRI in glioma imaging. For tumor fluorescence imaging of glioma, although fluorescence guided surgery (FGS) based on 5-aminolevulinic acid (5-ALA), fluorescein sodium (FLS-Na) and other fluorescent agents has been used to define the range of glioma during surgery, there are still many disadvantages:

[0007] (1) Clinical application found that the range of 5-ALA intraoperative derived fluorescence cannot match the range of enhanced MRI visualization: usually more than the enhanced area of glioma tumor shown by MRI; at the same time, the duration of 5-ALA fluorescence is still not sufficient, and the range and intensity of visualization are constantly decaying, plus the interference of the surrounding environment light, which makes the accuracy of 5-ALA tumor fluorescence visualization in surgery be affected to varying degrees;

[0008] (2) FLS-Na visualization of glioma is achieved by entering the tumor through the damaged tBBB, so the fluorescence intensity of FLS-Na in low-grade glioma with relatively intact tBBB is not high, and the visualization of tumor is not ideal; in addition, the duration of FLS-Na fluorescence is also limited, and FLS-Na will diffuse and attenuate intraoperative fluorescence, making the fluorescence staining of meningeal structures in the surgical area more obvious, which brings difficulties to long-time intraoperative visualization and identification of glioma range;

[0009] (3) More importantly, both 5-ALA and FLS-Na belong to non-specific glioma visualization, and lack the ability of targeted recognition of glioma tumor cells, which will inevitably cause non-specific fluorescence staining of glioma tumor cells to a certain extent, thereby affecting the accurate judgment of glioma range.

[0010] From the above, whether it is glioma magnetic resonance visualization or intraoperative glioma fluorescence visualization is a single tumor visualization mode independently, and how to realize integrated multi-modal visualization of glioma, realize effective fusion of intraoperative tumor fluorescence visualization and preoperative MRI visualization, especially MRI enhanced visualization, mutual confirmation and mutual assistance, overcome the respective shortcomings of current MRI and fluorescence visualization for glioma tumor visualization, and realize precise resection of glioma under multi-modal visualization, is the urgent problem to be solved in current clinical glioma visualization assisted surgery.

[0011] Chemotherapy is an important supplement for glioma treatment, but even if the maximum safe range of surgical resection and chemotherapy drug combination therapy for glioma, the postoperative survival of most patients is still less than 2 years, the reasons include: (1) conventional chemotherapy prevents the growth and division of cancer cells by interfering with DNA replication and cell mitosis, but most chemotherapy drugs are non-specific, which inevitably brings side effects, and the serious adverse reactions caused by the patients are closely related to the high mortality rate; (2) the existence of blood-brain barrier and blood-tumor barrier prevents the delivery of macromolecular drugs and small molecular non-fat-soluble drugs, at the same time, the existence of various unfavorable factors such as damage of tumor site blood vessels, acidic microenvironment of tumor tissue and highly invasive growth of glioma makes the delivery of chemotherapy drugs for glioma a difficult problem; (3) in order to achieve effective drug concentration, high-dose chemotherapy drugs are often used, which is easy to induce tumor resistance and lead to poor efficacy and prognosis. Therefore, in recent years, a large number of nanoparticle-based drug delivery systems have emerged, which can carry anticancer drugs across the blood-brain barrier to reach the lesion site. Nanotechnology provides more diverse options and strategies for drug development, and it is urgent to develop drug delivery systems with specific glioma cells, light side effects and good efficacy.

[0012] Glioma is the most common primary intracranial tumor and most of them are malignant, although there are various treatment methods including surgical resection, radiotherapy and chemotherapy, unfortunately, the comprehensive treatment effect of glioma has stagnated in recent years, and gene therapy and immunotherapy have not been successful, the reasons include the following three points: (1) glioma cells have high invasiveness, which makes it difficult to completely resect glioma by surgery, which is an important reason for postoperative recurrence of glioma; (2) due to the existence of blood-brain barrier and blood-tumor barrier, chemotherapy drugs are often difficult to reach intracranial tumor area, and have greater toxic side effects on normal tissues; (3) chemoresistance is one of the main reasons for low survival rate of patients, and 40-50% of patients have drug resistance to temozolomide.

[0013] Therefore, the present application focuses on the above problems, realizes MRI enhancement / fluorescence integrated double imaging of glioma by designing a multifunctional DNA nanosphere, so as to obtain a glioma tumor imaging signal that can meet the clinical needs, and at the same time realizes precise killing of glioma cells by loading chemotherapy drugs, and then obtains a significant tumor synergistic chemotherapy effect. SUMMARY

[0014] The purpose of the present application is to provide a multifunctional DNA nanosphere and a preparation method and application thereof, so as to solve the problems existing in the prior art. The preparation method provided by the present application can obtain a multifunctional DNA nanosphere with double imaging and capable of treating glioma.

[0015] To achieve the above object, the present application provides the following scheme:

[0016] The present application provides a preparation method of DNA nanoballs for glioma dual imaging, comprising the following steps:

[0017] Gd-DOTA-N3 and Y1-1 are covalently connected through click chemistry reaction to obtain Y1-1 modified by Gd-DOTA-N3; the nucleotide sequence of Y1-1 is shown as SEQ ID NO. 7;

[0018] Y-DNA, L-DNA and A32 are mixed to obtain a mixed system; the Y-DNA comprises Y1-1 modified by Gd-DOTA-N3 with a nucleotide sequence as shown in SEQ ID NO. 7, Y2 with a nucleotide sequence as shown in SEQ ID NO. 2, and Y3 with a nucleotide sequence as shown in SEQ ID NO. 3; the L-DNA comprises Linker 1 with a nucleotide sequence as shown in SEQ ID NO. 4 and Linker 2 with a nucleotide sequence as shown in SEQ ID NO. 5; the nucleotide sequence of A32 is shown as SEQ ID NO. 6;

[0019] After the mixed system is heated and treated, it is cooled to obtain a solution containing the DNA nanoballs.

[0020] Preferably, the molar ratio of Y1-1 modified by Gd-DOTA-N3, Y2 and Y3 in the Y-DNA is 1:1:1;

[0021] The molar ratio of Linker 1 and Linker 2 in the L-DNA is 1:1;

[0022] The molar ratio of Y-DNA and L-DNA in the mixed system is 3:5, the molar ratio of A32 and L-DNA is 1:5, and the final concentration of Gd-DOTA-N3 is 9 μM.

[0023] Preferably, the heating treatment is performed at 95℃ for 5 min.

[0024] The present application provides an application of the DNA nanoballs prepared by the above preparation method in preparing a diagnostic glioma product.

[0025] The present application provides a preparation method of multifunctional DNA nanoballs for glioma dual imaging and treatment, comprising the following steps:

[0026] Gd-DOTA-N3 and Y1-1 are covalently connected through click chemistry reaction to obtain Y1-1 modified by Gd-DOTA-N3; the nucleotide sequence of Y1-1 is shown as SEQ ID NO. 7;

[0027] mixing Y-DNA, L-DNA and A32 to obtain a mixed system; the Y-DNA comprises Y1-1 modified by Gd-DOTA-N3, Y2 and Y3, wherein the Y1-1 is nucleotides as shown in SEQ ID NO. 7, the Y2 is nucleotides as shown in SEQ ID NO. 2, and the Y3 is nucleotides as shown in SEQ ID NO. 3; the L-DNA comprises Linker 1 and Linker 2, wherein the Linker 1 is nucleotide sequence as shown in SEQ ID NO. 4, and the Linker 2 is nucleotide sequence as shown in SEQ ID NO. 5; the A32 is nucleotide sequence as shown in SEQ ID NO. 6;

[0028] cooling the mixed system after the heating treatment to obtain a solution of the multifunctional DNA nanosphere without loading DOX;

[0029] mixing and incubating the multifunctional DNA nanosphere solution without loading DOX and doxorubicin to obtain the multifunctional DNA nanosphere.

[0030] Preferably, the molar ratio of Y1-1, Y2 and Y3 modified by Gd-DOTA-N3 in the Y-DNA is 1:1:1.

[0031] Preferably, the molar ratio of Linker 1 and Linker 2 in the L-DNA is 1:1.

[0032] Preferably, the molar ratio of Y-DNA and L-DNA in the mixed system is 3:5, the molar ratio of A32 and L-DNA is 1:5, and the final concentration of Gd-DOTA-N3 is 9 μM.

[0033] Preferably, the heating treatment is performed at 95℃ for 5 min.

[0034] Preferably, the concentration of the DNA nanosphere in the multifunctional DNA nanosphere solution without loading DOX is 3 μM, and the concentration of the doxorubicin is 50 μM.

[0035] The application provides a multifunctional DNA nanosphere prepared by the preparation method.

[0036] The application provides application of the multifunctional DNA nanosphere in preparation of a product for diagnosing and / or treating glioma.

[0037] The application discloses the following technical effects:

[0038] The present application focuses on the above several problems, combines acid-responsive i-motif sequence, EGFRvIII aptamer A32 sequence and MRI contrast agent Gd-DOTA-N3, designs DNA nanospheres, realizes MRI enhancement / fluorescence integrated double imaging of glioma, so as to obtain glioma tumor imaging signals that can meet clinical needs.

[0039] Meanwhile, the acid-responsive i-motif sequence, EGFRvIII aptamer A32 sequence, MRI contrast agent Gd-DOTA-N3 and chemotherapeutic drug doxorubicin (DOX) are combined, Gd-DOTA-N3 and Cy5-BHQ fluorescent pairs are modified on specific bases, the principle of DNA base complementary pairing is used, and a novel multifunctional DNA nanosphere for glioma fluorescence / MRI imaging and treatment is prepared. The present application designs three modules based on the principle of DNA base complementary pairing, which are Y-shaped DNA (Y-DNA), connecting DNA (L-DNA) and aptamer DNA for preparing nanospheres. Y-DNA is composed of three single-stranded DNAs (Y1, Y2 and Y3), and each single-stranded DNA contains a sticky end. In order to realize MRI enhanced imaging, a sequence containing an alkyne group is designed at the end of Y1, that is, Y1-1, which can be covalently connected with Gd-DOTA-N3 through click chemistry reaction, so as to introduce the MRI enhancer into the DNA nanosphere. The aptamer DNA is composed of an aptamer A32 which can specifically bind to EGFRvIII and a sticky end. Therefore, the aptamer DNA (A32) can inhibit the extension of the nanosphere and target EGFRvIII positive glioma cells. L-DNA is a linear double-stranded assembled by Linker 1 and Linker 2, and contains two sticky ends which are complementary to the sticky ends of Y-DNA and A32. In order to realize glioma fluorescence imaging, Cy5 is used to modify Linker 1, and considering that the continuous fluorescence signal of Cy5 will cause strong background interference, a BHQ-3 quenching group is modified on the Cy5 pairing base to inhibit its fluorescence emission. When in an acidic environment, the Linker 1 containing the i-motif sequence folds itself to form an i-motif structure, thereby separating from the Linker 2, so that the DNA nanosphere disintegrates, and the Cy5 fluorescence is restored. It can be seen that the present application realizes precise killing of glioma cells by loading chemotherapeutic drugs, and thus obtains a significant synergistic chemotherapy effect on tumors. The nanosphere has a rapid pH response ability, can control the recovery of Cy5 fluorescence and the release of DOX, and the fluorescence signal-to-noise ratio thereof can reach 1:6 at pH=5. The targeting ability of the aptamer A32 can increase the enrichment of the nanosphere in the glioma area, thereby improving the dual imaging effect of the DNA nanosphere. The slow release of DOX by the DNA nanosphere and the targeting ability of A32 can improve the killing effect of DOX on tumors. Through experiments, it is verified that the DNA nanosphere has good three-in-one functions of glioma dual imaging and targeted treatment, and has the characteristics of strong specificity, high sensitivity, long duration, good stability, better chemotherapy effect and lower side effects, and is expected to realize precise guidance for surgical resection of glioma and synchronous postoperative chemotherapy.

[0040] It can be seen that the multifunctional DNA nanosphere is prepared, can target glioma cells, realizes fluorescence / magnetic resonance dual imaging of glioma tumor, improves the efficacy of doxorubicin (DOX) against glioma, realizes precise guidance of surgical resection of glioma and simultaneously assists postoperative chemotherapy. BRIEF DESCRIPTION OF DRAWINGS

[0041] In order to more clearly illustrate the technical solutions of the embodiments of the present application or the prior art, the drawings needed in the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor.

[0042] Fig. 1 is a schematic diagram of construction of the multifunctional DNA nanosphere and its dual imaging and treatment function for glioma; wherein Cy5 is a Cy5 fluorescent group, BHQ3 is a BHQ-3 fluorescent group, GD is Gd-DOTA-N3, DOX is doxorubicin, and EGFRvIII is EGFRvIII protein on the membrane of glioma cells;

[0043] Fig. 2 is a characterization result of the multifunctional DNA nanosphere; wherein A is PAGE verification of assembly of DNA, bands 1-7 are Y1, Y1+Y2, Y1+Y2+Y3, Linker 1, Linker 2, Linker 1+Linker 2 and multifunctional DNA nanoparticles respectively; B is particle size analysis of Y-DNA and A32 different molar ratio DNA nanosphere, a is the molar ratio of Y-DNA and A32 is 4:1, b is the molar ratio of Y-DNA and A32 is 3:1, c is the molar ratio of Y-DNA and A32 is 2:1; C is a TEM image of Y-DNA; D is multifunctional DNA nanosphere of Y-DNA and A32 molar ratio of 3:1; E is a scanning electron microscope observation result; F is an atomic force microscope observation result; the scale of C-F is 100nm;

[0044] Fig. 3 is a TEM image of multifunctional DNA nanosphere of Y-DNA and A32 different molar ratio; wherein A is the molar ratio of Y-DNA and A32 is 4:1; B is the molar ratio of Y-DNA and A32 is 2:1; the scale of A and B is 100nm;

[0045] Figure 4 is the result of performance study of DOX-loaded DNA nanospheres; wherein, A is the fluorescence spectrum of multifunctional DNA nanospheres with pH change; B is the comparison of pH response ability of DNA nanospheres prepared by L-DNA containing i-motif sequence and L'-DNA with random sequence; C is the fluorescence spectrum of multifunctional DNA nanospheres with different concentrations after incubation with 50 μM DOX; D is the fluorescence spectrum of 3 μM multifunctional DNA nanospheres after incubation with 50 μM DOX at pH 7.25 and 5.5; L-DNA includes nucleotide sequence of Linker 1 as shown in SEQ ID NO. 4 and nucleotide sequence of Linker 2 as shown in SEQ ID NO. 5, L'-DNA includes nucleotide sequence of Linker 1-1 as shown in SEQ ID NO. 8 and nucleotide sequence of Linker 2-1 as shown in SEQ ID NO. 9; i-motif is multifunctional nanosphere of L-DNA containing i-motif sequence, and control is DNA nanosphere prepared by L'-DNA with random sequence;

[0046] Figure 5 is the reversibility study of multifunctional DNA nanospheres between pH neutral (7.25) and acidic (5.00);

[0047] Figure 6 is the T1 weighted imaging diagram of DNA nanospheres modified with different concentrations of Gd-DOTA-N3; wherein, DNA-Gd is DNA nanosphere modified with Gd-DOTA-N3, and DNA (control) is DNA nanosphere without modification of Gd-DOTA-N3;

[0048] Figure 7 is the results of cytological behavior study of DOX-loaded DNA nanospheres; A is the fluorescence intensity of Cy5 after U87 cells are incubated with 3 μM multifunctional DNA nanospheres for 1 h, 3 h and 5 h to quantify the absorption of multifunctional DNA nanospheres; B is the quantification of the amount of multifunctional DNA nanospheres taken up by U87 or U87-E cells after 1 μM multifunctional DNA nanospheres are incubated for 1 h; C is the amount of DNA nanospheres taken up by U87 and U87-E cells detected by flow cytometry after 1 μM multifunctional DNA nanospheres are incubated for 1 h; D is the pH-responsive multifunctional DNA nanospheres imaging the lysosomes in living cells within 1-6 hours, the yellow fluorescence is the colocalization of DNA nanospheres (red) and lysosomes (green) (bar = 30 μm); D is, (bar = 30 μm), Hoechst is a nuclear stain, Cy5 is a Cy5 fluorescent group, Lysotrack is a lysosome tracer, Merge is a fusion image; E is a representative fluorescence image of U87-E cells treated with DOX and A32-DNA-DOX (2.5 μg / mL) for 1 h, 2 h, 4 h and 6 h, DNA-DOX is multifunctional DOX-loaded DNA nanospheres, Free DOX is free DOX;

[0049] Figure 8 is the results of cytotoxicity study of DNA nanospheres; wherein, A is the CCK8 detection of the toxicity of DNA nanospheres to U87-E, U87, Hela, HA and 293T cells, PBS is PBS, DNA nanospheres are multifunctional DNA nanospheres; B is the IC 50 values of free DOX and A32-DNA-DOX to U87-E cell viability;

[0050] Figure 9 is the results of anti-tumor efficacy evaluation in mice; wherein, A is the T2-weighted MRI image after A32-DNA-DOX, DNA-DOX, free DOX, DNA nanospheres and PBS treatment; B is the fluorescence imaging of glioma mouse brain sections after 2 hours of tail vein injection of DNA nanospheres; C is the fluorescence image of glioma tissue and various organs collected after 2 hours of tail vein injection of nanospheres;

[0051] Figure 10 is the results of anti-tumor efficacy evaluation in mice; wherein, A is the T2-weighted MRI image after A32-DNA-DOX, DNA-DOX, free DOX, DNA nanospheres and PBS treatment; B is the survival cycle of intracranial glioma-bearing mice in A32-DNA-DOX, DNA-DOX, free DOX, DNA nanospheres and PBS groups; C is the weight change curve of glioma-bearing mice in A32-DNA-DOX, DNA-DOX, free DOX, DNA nanospheres and PBS groups;

[0052] Figure 11 is in vivo magnetic resonance imaging: T1 enhanced magnetic resonance imaging of glioma mice at different time points after intravenous injection of DNA nanoballs, wherein A is T1 weighted imaging; B is T2 weighted imaging; C is the T1 scan result of glioma mice 0.5 h after tail vein injection of Gd-DOTA-N3 modified DNA nanoballs; D is the T1 scan result of glioma mice 1 h after tail vein injection of Gd-DOTA-N3 modified DNA nanoballs; E is the T1 scan result of glioma mice 2 h after tail vein injection of Gd-DOTA-N3 modified DNA nanoballs; F is the T1 scan result of glioma mice 4 h after tail vein injection of Gd-DOTA-N3 modified DNA nanoballs; G is the T1 scan result of glioma mice 6 h after tail vein injection of Gd-DOTA-N3 modified DNA nanoballs; H is the T1 scan result of glioma mice 12 h after tail vein injection of Gd-DOTA-N3 modified DNA nanoballs. DETAILED DESCRIPTION

[0053] The following detailed description of various example embodiments of the application will not be considered limiting of the application, but rather a description of certain aspects, features, and embodiments of the application.

[0054] It should be understood that the terms used herein are for the purpose of describing particular embodiments and are not intended to limit the application. Additionally, for numerical ranges that are expressed in a range format, it is intended that any and every unit of the lower limit of that range to the upper limit of that range is also specifically contemplated. For example, if a range of amounts is stated to be 1% to 90%, it is intended that amounts such as 2% to 89%, 3% to 88%, 4% to 87%, and so on, are expressly enumerated in this specification. These are only examples of what is specifically enumerated herein and are not intended to limit the application in any way.

[0055] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, the preferred methods and materials are described. All patents, patent applications, publications, and descriptions mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the patents, patent applications, publications, and descriptions are cited.

[0056] Many modifications and variations of this application can be made in the light of the above teachings without departing from the spirit and scope thereof. Other implementations of this application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. The examples and embodiments described herein are exemplary only and are not intended to be limiting.

[0057] As used herein, the terms "comprise", "include", "have", "contain", and the like are open-ended terms, i.e., meaning "including but not limited to".

[0058] Example 1

[0059] A preparation method of a glioma dual imaging and treatment DNA nanosphere, the construction schematic diagram is shown in Figure 1, and the steps are as follows:

[0060] Step 1: Gd-DOTA-N3 (manufacturer: Xi'an Qiyue Biological Technology Co., Ltd., product connection: https: / / www.afzhan.com / chanpin / 12679228.html) is covalently connected with Y1-1 through click chemistry reaction (the nucleotide sequence of Y1-1 is shown in Table 1, and the reaction system is shown in Table 2), and then the reaction product is purified with a 3kDa centrifugal filter to remove the reaction substrate and catalyst, and the relative molecular mass of the product is detected by a mass spectrometer. The relative molecular mass of the product is 15180.4, and it is denoted as Gd-DOTA-N3 modified Y1-1.

[0061] Step 2: Y-DNA (Gd-DOTA-N3 modified Y1-1, Y2 and Y3), L-DNA (Linker 1 and Linker 2) and A32 are added into 1×PBS buffer (10mM Mg 2+ ) to obtain a mixed system, wherein the nucleotide sequences of Gd-DOTA-N3 modified Y1-1, Y2, Y3, Linker 1, Linker 2 and A32 are shown in Table 1; the molar ratio of Gd-DOTA-N3 modified Y1-1, Y2 and Y3 in Y-DNA is 1:1:1, and the molar ratio of Linker 1 and Linker 2 in L-DNA is 1:1; the molar ratio of Y-DNA to L-DNA is 3:5, and the molar ratio of A32 to L-DNA is 1:5; in the mixed system, the final concentration of L-DNA is 5μM, the final concentration of Gd-DOTA-N3 is 9μM, and the final concentration of DNA nanosphere is 3μM. In this step, the size of the DNA nanosphere can be controlled by adjusting the molar ratio of Y-DNA to A32, and the average size of the DNA nanosphere in this step is about 150nm.

[0062] Step 3: The mixed system in step 2 is heated to 95℃, and after 5min, it is slowly cooled to room temperature (to perform DNA single-strand hybridization reaction) to obtain a DNA nanosphere mixed solution, and the concentration of the DNA nanosphere in the mixed solution is 3μM, i.e., multifunctional DNA nanosphere without loading DOX. In this step, heating to 95℃ denatures the DNA, and then it is annealed, which is beneficial to DNA hybridization.

[0063] Step 4: 50 μΜ of DOX was mixed with the DNA nanoballs mixture from Step 3 and incubated at room temperature for 1 h. The product was purified and concentrated by 10 kDa centrifugal filter, resuspended in PBS buffer and stored at 4 °C. The multifunctional DNA nanoballs loaded with DOX were obtained. The concentration of DNA nanoballs in the mixture used in this step was 3 μΜ, and the concentration of DOX used was 50 μΜ, which could saturate the DNA nanoballs with DOX.

[0064] Table 1 Single-stranded DNA sequences used in this example Note: If no modification with contrast agent is needed, the sequence of Y1 can be used.

[0065] Table 2 Click chemistry reaction system

[0066] Example 2 Characterization of multifunctional DNA nanoballs

[0067] Polyacrylamide gel electrophoresis (PAGE) was used to verify the successful self-assembly of multifunctional DNA nanoballs without DOX prepared in Example 1, and the results are shown in Figure 2A. Band 7 represents the successful assembly of DNA nanoballs, which has the largest molecular weight and the slowest migration compared to other components, indicating that DNA has successfully self-assembled into nanoballs.

[0068] Next, the effect of the addition ratio of Y-DNA and A32 on the size of DNA nanoballs was studied by dynamic light scattering (DLS) and transmission electron microscopy (TEM). Y-DNA and A32 were mixed at different molar ratios of 4:1, 3:1 and 2:1, while the concentration of L-DNA was 5 μΜ. Multifunctional DNA nanoballs without DOX were prepared according to the preparation method of Example 1. The ratio of sticky ends of Y-DNA and A32 to sticky ends of L-DNA was 1:1, which ensured the complete reaction of ssDNA. DLS results (Figure 2B) showed that different ratios of Y-DNA and A32 could lead to different nanoball sizes; a higher proportion of A32 led to smaller nanoballs. TEM showed that the DNA nanoballs had a 3D spherical shape, and the diameter was consistent with the DLS results (Figure 2C-D and Figure 3). When the ratio of Y-DNA to A32 was 3:1, the average particle size of the DNA nanoballs was about 134 nm.

[0069] The size was further confirmed by scanning electron microscopy (SEM) and atomic force microscopy (AFM) (Figure 2E and F). The size of the nanoparticles is of great clinical significance, as a size of <200 nm can prolong the blood circulation time. Therefore, multifunctional nanoballs of 134 nm were used in subsequent experiments.

[0070] Linker 1 tends to form i-motif structure in acidic environment, thus dissociates from its complementary strand, increasing the distance between the fluorescent pair of Cy5 and BHQ-3, resulting in the recovery of Cy5 fluorescent signal, thus generating fluorescent signal. Therefore, the pH-responsive ability of the multifunctional DNA nanospheres prepared in Example 1 without loading DOX was detected by fluorescence emission spectrum, while the DNA nanospheres prepared by L'-DNA without i-motif structure were used as control. The multifunctional DNA nanospheres containing i-motif structure and the DNA nanospheres without i-motif structure were placed in buffers with different pH, and the fluorescence intensity was measured. The results showed that the fluorescence intensity of Cy5 gradually increased with the decrease of pH, and the signal-to-noise ratio reached 1:6 (A in FIG. 4). On the contrary, Linker 1-1 and Linker 2-1 without i-motif structure did not induce the dissociation of DNA nanospheres (B in FIG. 4). In addition, the fluorescence intensity of the multifunctional DNA nanospheres prepared in Example 1 without loading DOX showed good reproducibility between neutral solution (pH = 7.25) and acidic solution (pH = 5.0) (FIG. 5). This study showed that fluorescence was only generated after the multifunctional DNA nanospheres entered the lysosomes of glioma cells.

[0071] DOX can be excited to emit green or red fluorescence, and this fluorescence can be quenched when DOX is embedded in DNA double strands, so the ability of DNA nanospheres to carry DOX was evaluated by fluorescence spectrum analysis. Different concentrations of DOX-loaded multifunctional DNA nanosphere solutions were co-incubated with 50 μM DOX for 1 h, and fluorescence analysis was performed. The spectrum showed that when the concentration of the multifunctional DNA nanospheres reached 2 μM, the DOX loading was almost saturated (C in FIG. 4). Therefore, 3 μM DNA nanospheres were used to oversaturate 50 μM DOX in the subsequent experiments. Since Linker 1 can dissociate from its complementary strand in an acidic environment, part of the DOX embedded in the DNA double strand can be rapidly released in an acidic environment (D in FIG. 4).

[0072] For enhanced MRI imaging, Gd-DOTA-N3 was covalently linked to Y1 modified with alkyne through click chemistry reaction, thereby introducing MRI enhancer into multifunctional DNA nanospheres. T1 weighted scanning showed that Gd-DOTA-N3 modified multifunctional DNA nanosphere solution had good T1 enhanced imaging effect, and was positively correlated with the concentration of Gd-DOTA-N3 in the solution (FIG. 6).

[0073] Example 3 Cell uptake and targeting ability of multifunctional DNA nanospheres

[0074] To verify whether the cells can uptake the multifunctional DNA nanoballs actively, the DNA nanoballs were assembled using the Linker 2 sequence without modification of BHQ-3, and the preparation method was the same as that in Example 1, so that the uptake of the DNA nanoballs can be quantified by measuring the fluorescence intensity of Cy5. After U87 cells were incubated with the multifunctional DNA nanoballs (3 μM) for 1 h, 3 h and 5 h respectively, it was found that the Cy5 fluorescence was obviously located outside the cell nucleus, and the red fluorescence intensity increased with the prolongation of the incubation time, indicating that the cells can uptake the nanoballs actively, and the uptake amount is positively correlated with the time (A in FIG. 7). U87-E cells (U87-EGFRv III cells, purchased from the ATCC cell library) can increase the uptake of the DNA nanoballs by the cells due to the high expression of EGFRv III. After the multifunctional DNA nanoballs were incubated with U87 and U87-E cells for 1 h, the column chart and flow chart show that the U87-E cells have a higher uptake of the multifunctional DNA nanoballs than the U87 cells (B and C in FIG. 7).

[0075] Example 4 Co-localization analysis of multifunctional nanoballs and lysosomes

[0076] Lysosomes are acidic organelles in cells, and the internal pH thereof is about 5.0. When the multifunctional DNA nanoballs without loading of DOX are phagocytosed into cells, they will dissociate and release Cy5 fluorescence. To verify this assumption, the multifunctional DNA nanoballs without loading of DOX (3 μM) were incubated with U87-E cells for 1 h, 3 h and 6 h, and the lysosomes were labeled with a green fluorescent probe. The fluorescence images show that the red fluorescence of Cy5 and the green fluorescence of lysosomes are co-located (yellow part) (D in FIG. 7). A considerable part of the red fluorescence and the green fluorescence are separated, which may be due to the escape of part of the DNA structure from the lysosomes.

[0077] Example 5 Release and toxicity of multifunctional nanoballs in U87-E cells

[0078] To study the release of DOX in cells by the multifunctional DNA nanoballs (A32-DNA-DOX) loaded with DOX prepared in Example 1, U87-E cells were co-cultured with A32-DNA-DOX or free DOX for 1 h, 2 h, 4 h and 6 h. The fluorescence images show that after incubation for 1 h, the free DOX can enter the inside of the cell nucleus, while most of the A32-DNA-DOX remains in the cytoplasm. After further incubation, the A32-DNA-DOX also gradually shows nuclear localization (E in FIG. 7).

[0079] The cytotoxicity of the DOX-loaded multifunctional DNA nanoballs to different cells (U87-E cells, U87 cells, Hela cells, HA cells and 293T cells) was studied by CCK8 experiment, with PBS as a control. The results showed that the multifunctional DNA nanoballs with a concentration lower than 10 μM had no cytotoxicity to various cells, showing good biocompatibility (A in Fig. 8). In contrast, the results of CCK8 experiment showed that the cytotoxicity of free DOX and A32-DNA-DOX was concentration-dependent, and A32-DNA-DOX showed higher cytotoxicity than free DOX (B in Fig. 8). This can be attributed to the controlled release of DOX in the cytoplasm. In an acidic environment, the acid-responsive part in the DOX-loaded multifunctional DNA nanoballs releases the loaded DOX rapidly, equivalent to the first dose of clinical administration, while the remaining DOX can be gradually released from the double-stranded under acidic conditions, providing a maintenance dose, which makes the retention rate of DOX in the cells higher, thus increasing the cytotoxicity.

[0080] Example 6 Targeted fluorescence imaging of multifunctional DNA nanoballs in vivo in mice

[0081] To confirm whether the multifunctional DNA nanoballs without loading DOX prepared in Example 1 can enter the tumor area through blood circulation, 0.1 mL of the multifunctional nanoball solution (3 μM) was injected into the tail vein of a glioma mouse, and detected by an in vivo imaging system (schematic diagram of the dual imaging and treatment function of the multifunctional DNA nanoballs on glioma). The results showed that obvious fluorescence signals were observed in the tumor area (A in Fig. 9). In addition, the brain tissue of the mouse was collected 2 h after injection for frozen sectioning and Cy5 fluorescence detection. Compared with normal brain tissue, significant fluorescence was observed in the tumor area, indicating that the DNA nanoballs can effectively accumulate in the tumor area (B in Fig. 9). Similarly, the imaging ability of the DNA nanoballs was explored in a mouse with subcutaneous glioma, and ex vivo organ imaging was performed 2 h after tail vein injection, and Cy5 fluorescence signals were observed in the subcutaneous glioma tissue (C in Fig. 9). In summary, the multifunctional DNA nanoballs have good glioma-targeted imaging ability.

[0082] Example 7 Anti-glioma effect of DOX-loaded multifunctional DNA nanoballs in vivo

[0083] To investigate the anti-glioma efficacy of the multifunctional DNA nanospheres without DOX and the multifunctional DNA nanospheres with DOX prepared in Example 1 in vivo, the mice in each experimental group (A32-DNA-DOX group: the multifunctional DNA nanospheres with DOX prepared in Example 1 were injected; DNA-DOX group: the DNA nanospheres without A32 were injected, the preparation method was the same as that in Example 1, except that the aptamer A32 was not used; Free DOX group: DOX was injected; DNA nanospheres group: the multifunctional DNA nanospheres without DOX were injected; PBS group: PBS was injected; the injection amount of DOX in each treatment was 4 mg / kg, and the injection was performed every two days, for a total of 14 days) were subjected to MRI scanning at 2, 3 and 4 weeks after the U87-E cells were implanted into the mice intracranially, and the results showed that the glioma tumor in the A32-DNA-DOX and DNA-DOX groups grew more slowly than that in the multifunctional DNA nanospheres, free DOX and PBS groups (A in Fig. 10).

[0084] In addition, the survival time and body weight change of 7 tumor-bearing mice in each group were recorded, and the results showed that the survival time of the A32-DNA-DOX or DNA-DOX group was significantly longer than that of the other groups (B in Fig. 10), indicating that the multifunctional DNA nanospheres with DOX had better efficacy in inhibiting tumor growth. The intracranial glioma-bearing mice in the A32-DNA-DOX and DNA-DOX groups had a slower body weight loss rate than those in the other groups, which also indicated the effectiveness of the treatment (C in Fig. 10).

[0085] Example 8 Magnetic resonance enhancement ability of the multifunctional DNA nanospheres in vivo

[0086] The multifunctional DNA nanospheres modified with Gd-DOTA-N3 with a concentration of 30 μM were prepared by the method prepared in Example 1. After 0.1 mL of the multifunctional DNA nanospheres were injected into the tail vein for different time (0.5, 1, 2, 4, 6 and 12 h), the glioma-bearing mice were subjected to T1 scanning, and the results showed that the glioma area appeared uneven enhancement imaging, indicating that the multifunctional DNA nanospheres had certain enhancement imaging ability (Fig. 11).

[0087] The above-described examples only describe the preferred modes of the present application, and do not limit the scope of the present application. Without departing from the design spirit of the present application, various modifications and improvements to the technical solutions of the present application made by those skilled in the art shall fall within the protection scope determined by the claims of the present application.

Claims

1. A method for preparing DNA nanoballs for glioma dual imaging, characterized in that, The method comprises the following steps: covalently connecting Gd-DOTA-N3 and Y1-1 through a click chemistry reaction to obtain Y1-1 modified by Gd-DOTA-N3; the nucleotide sequence of Y1-1 is shown as SEQ ID NO. 7; mixing Y-DNA, L-DNA and A32 to obtain a mixed system; the Y-DNA comprises Y1-1 modified by Gd-DOTA-N3 with a nucleotide sequence shown as SEQ ID NO. 7, Y2 with a nucleotide sequence shown as SEQ ID NO. 2, and Y3 with a nucleotide sequence shown as SEQ ID NO. 3; the L-DNA comprises Linker 1 with a nucleotide sequence shown as SEQ ID NO. 4 and Linker 2 with a nucleotide sequence shown as SEQ ID NO. 5; the nucleotide sequence of A32 is shown as SEQ ID NO. 6; after heating treatment of the mixed system, cooling is performed to obtain a solution containing the DNA nanoball.

2. The production method according to claim 1, characterized by, The molar ratio of Y1-1 modified by Gd-DOTA-N3, Y2 and Y3 in the Y-DNA is 1:1:1; The molar ratio of Linker 1 and Linker 2 in the L-DNA is 1:1; In the mixed system, the molar ratio of Y-DNA and L-DNA is 3:5, the molar ratio of A32 and L-DNA is 1:5, and the final concentration of Gd-DOTA-N3 is 9 μM.

3. The preparation method according to claim 1, characterized in that, The heating treatment is performed at 95 ℃ for 5 min.

4. Application of the DNA nanoball prepared by the preparation method in any one of claims 1-3 in preparation of a product for diagnosing glioma.

5. A method for preparing multifunctional DNA nanoballs for glioma dual imaging and therapy, characterized in that, The method comprises the following steps: covalently connecting Gd-DOTA-N3 and Y1-1 through a click chemistry reaction to obtain Y1-1 modified by Gd-DOTA-N3; the nucleotide sequence of Y1-1 is shown as SEQ ID NO. 7; mixing Y-DNA, L-DNA and A32 to obtain a mixed system; the Y-DNA comprises Y1-1 modified by Gd-DOTA-N3 with a nucleotide sequence shown as SEQ ID NO. 7, Y2 with a nucleotide sequence shown as SEQ ID NO. 2, and Y3 with a nucleotide sequence shown as SEQ ID NO. 3; the L-DNA comprises Linker 1 with a nucleotide sequence shown as SEQ ID NO. 4 and Linker 2 with a nucleotide sequence shown as SEQ ID NO. 5; the nucleotide sequence of A32 is shown as SEQ ID NO. 6; after heating treatment of the mixed system, cooling is performed to obtain a solution containing the DNA nanoball. mixing and incubating the solution of the multifunctional DNA nanoball not loaded with DOX and doxorubicin to obtain the multifunctional DNA nanoball.

6. The production method according to claim 5, wherein The molar ratio of Y1-1 modified by Gd-DOTA-N3, Y2 and Y3 in the Y-DNA is 1:1:1; The molar ratio of Linker 1 and Linker 2 in the L-DNA is 1:1; The molar ratio of Y-DNA and L-DNA in the mixed system is 3:5, the molar ratio of A32 and L-DNA is 1:5, and the final concentration of Gd-DOTA-N3 is 9 μM.

7. The preparation method according to claim 5, characterized in that, The temperature of the heating treatment is 95℃, and the time is 5 min.

8. The preparation method according to claim 5, characterized in that, The concentration of DNA nanospheres in the solution of the multifunctional DNA nanospheres without loading DOX is 3 μM, and the concentration of adriamycin is 50 μM.

9. Multifunctional DNA nanospheres prepared by the preparation method in any one of claims 5-8.

10. Use of the multifunctional DNA nanospheres in claim 9 in the preparation of a product for diagnosing and / or treating glioma.

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