Preparation method and application of programmable DNA hydrogel for inhibiting tumor activity of breast cancer

Abstract

The invention discloses a preparation method and application of programmable DNA hydrogel for inhibiting breast cancer tumor activity. The DNA hydrogel is composed of an RCA long chain AL containing a multivalent AS411 aptamer sequence and an RCA long chain CL hybridized with a cholesterol single chain ss-chol. Through reasonable DNA programming sequence design, a three-dimensional DNA structure can be formed on the surface of the cell. The DNA hydrogel specifically targets cancer cells with high expression of nucleolin receptors through the synergistic effect of multivalent AS1411 and chol, and is weak in targeting to normal cells with low expression of nucleolin, so that toxic and side effects on the normal cells are effectively avoided. The DNA hydrogel has excellent programmability, hydrogel with high, medium and low pore densities can be simply prepared through flexible DNA sequence design, and the breast cancer resistance activity of the DNA hydrogel is in positive correlation with the pore density of the DNA hydrogel. In animal in-vivo experiments, the DNA hydrogel under three-dimensional and high pore density significantly inhibits breast cancer activity, and a new strategy is provided for tumor treatment.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of hydrogel materials, and particularly relates to a preparation method and application of a programmable DNA hydrogel for inhibiting breast cancer tumor activity. BACKGROUND

[0002] The incidence and mortality of malignant tumors are continuously increasing, which has become one of the key public health problems that seriously threaten people's health. Nucleolin (NCL) is a protein that exists universally in the nucleus, cytoplasm and cell membrane surface, and is also one of the most abundant phosphoproteins in the nucleolus. It can shuttle from the nucleolus to the nucleus, cytoplasm and cell membrane surface, and has strong shuttle properties. NCL is selected as a commonly used drug targeting delivery receptor protein. NCL plays an important role in the life activities of cancer cells, and inhibiting the effect of NCL on the surface of cancer cells has a profound impact on inducing programmed death of cancer cells and developing corresponding cancer treatment methods.

[0003] At present, most of the applications of receptor aggregation rely on external stimulation, such as light control and magnetic control, and the process is complex and easy to produce toxic side effects. In recent years, DNA programming technology has been applied to receptor aggregation research. However, most of the existing methods rely on one-dimensional linear or two-dimensional DNA structures, and are limited to using aptamers for monovalent recognition, which is easily affected by receptor internalization and enzyme degradation. Especially for high-mobility membrane proteins like NCL that shuttle rapidly between the nucleolus and the cytoplasmic membrane, it is difficult to stably bind to the cell surface. In view of the three-dimensional characteristics of cells, programmable and controllable three-dimensional DNA nanomaterials can be constructed on the cell surface, and the physical properties (such as elastic modulus and network density) can be precisely and programmably controlled. Through multivalent enhancement of the binding effect, higher density of receptor aggregation can be promoted, which can ultimately inhibit cancer cell proliferation, migration and induce cancer cell apoptosis, thereby treating tumors. SUMMARY

[0004] In view of the above shortcomings of the prior art, the present application provides a preparation method and application of a programmable DNA hydrogel for inhibiting breast cancer tumor activity.

[0005] To achieve the above-mentioned purposes, the technical scheme adopted by the present application is as follows: A programmable DNA hydrogel for inhibiting breast cancer tumor activity is provided, which is composed of RCA long chain AL containing multivalent AS411 aptamer sequence and RCA long chain CL hybridized with cholesterol single-stranded ss-chol. The RCA long chain AL can hybridize with the RCA long chain CL, and the number of hybridized bases of the RCA long chain AL and the RCA long chain CL is positively correlated with the density of the DNA hydrogel and the number of controlled cancer cells. The sequence of the cholesterol single-stranded ss-chol is shown in SEM ID NO. 1.

[0006] Further, the RCA long chain CL is obtained by hybridizing the RCA long chain L with the cholesterol single strand ss-chol based on the rolling circle amplification technology, and the RCA long chain AL and the circular template of the RCA long chain are synthesized by using the template strand and the primer strand; In the synthesis of the circular template CT-AL, the primer strand is Primer 1 as shown in SEM ID NO. 2, and the template strand is Template 1 as shown in SEM ID NO. 3. In the synthesis of the circular template CT-L, the primer strand is Primer 2 as shown in SEM ID NO. 4, and the template strand is Template 2 as shown in SEM ID NO. 5.

[0007] Further, when the hybridization base number of the RCA long chain AL and the RCA long chain CL is 26, the template strand of the circular template CT-AL for synthesizing the RCA long chain AL is Template 2-26 as shown in SEM ID NO. 6.

[0008] Further, when the hybridization base number of the RCA long chain AL and the RCA long chain CL is 37, the template strand of the circular template CT-AL for synthesizing the RCA long chain AL is Template 2-37 as shown in SEM ID NO. 7.

[0009] Further, when the hybridization base number of the RCA long chain AL and the RCA long chain CL is 52, the template strand of the circular template CT-AL for synthesizing the RCA long chain AL is Template 2-52 as shown in SEM ID NO. 8.

[0010] The application also provides a preparation method of the DNA hydrogel for inducing the nucleolin receptor on the cell surface. S1: respectively synthesizing and preparing the circular template CT-AL and the circular template CT-L; S2: synthesizing the RCA long chain AL according to the CT-AL, and synthesizing the RCA long chain L according to the CT-L; S3: cross-linking the RCA long chain L with the cholesterol single strand ss-chol to obtain the DNA chain CL; S4: mixing the RCA long chain AL and the DNA chain CL in a mass ratio of 1:1, and then stirring at 37 DEG C for 1h at 600 rpm to prepare the DNA hydrogel.

[0011] Further, the synthesis method of the circular template CT-AL or the circular template CT-L is specifically as follows: A1: respectively dissolving the primer strand and the template strand in the 80mM NaCl aqueous solution to obtain the 10μM primer strand solution and the 10μM template strand solution; A2: take 80 muL of primer chain solution, 40 muL of template chain solution and 20 muL of 10xT4 Buffer, and supplement with ultrapure water to 200 muL and mix evenly; A3: the reaction solution is heated at 95 DEG C for 10 min, then naturally cooled to 25 DEG C, then 3 muL of T4 DNA ligase is added, mixed, then circularized at 16 DEG C for 16 h, and finally inactivated by heating at 65 DEG C for 10 min to obtain the circular template CT-AL or the circular template CT-L.

[0012] Further, the synthesis method of the RCA long chain AL and the RCA long chain L is specifically as follows: Add 30 muL of the circular template CT-AL or the circular template CT-L, 10 muL of dNTP with a concentration of 10 mM, 10 muL of 10xphi29 DNA polymerase Buffer and 2.5 muL of phi29 DNA polymerase to an EP tube, supplement with ultrapure water to 100 muL, mix evenly, then incubate at 30 DEG C for 4 h, and finally inactivated by heating at 65 DEG C for 10 min to obtain the RCA long chain AL or the RCA long chain L.

[0013] Further, the step S3 is specifically as follows: the RCA long chain L and the cholesterolsingle strand ss-chol are mixed at a molar ratio of 1:2, then crosslinked by water bath at 55 DEG C for 30 min.

[0014] The application further provides application of the above-mentioned DNA hydrogel in preparation of a medicine for inhibiting tumors.

[0015] The application has the following beneficial effects: The application does not add any external stimulation and drug molecules, and only needs to form a three-dimensional DNA structure on the cell surface through reasonable DNA coding sequence design. The DNA hydrogel specifically targets the cancer cells with high expression of nucleolin receptors through the synergistic effect of the multivalent AS1411 and chol, and has weak targeting effect on normal cells with low expression of nucleolin, thereby effectively avoiding the toxic side effects on normal cells.

[0016] The DNA hydrogel prepared by the application has good stability and multivalent effect, and the three-dimensional structure of the hydrogel can effectively match the three-dimensional characteristics of cancer cells to effectively capture the cancer cells and induce the aggregation of the nucleolin receptors on the cell surface, and the multivalent effect enhances the recognition and action ability on the cancer cells.

[0017] The DNA hydrogel of the present application has excellent programmability, and through flexible DNA sequence design, it can be easily prepared into hydrogels with high, medium and low three different porosities, thereby realizing the best capture effect on breast cancer cells, and precisely regulating the behavior of cancer cells, and effectively inhibiting their survival, proliferation and migration. The anti-breast cancer activity of the DNA hydrogel is positively correlated with its porosity, that is, under the three-dimensional DNA hydrogel, higher porosity corresponds to stronger breast cancer cell inhibition capacity. In animal in vivo experiments, the DNA hydrogel under three-dimensional and high porosity significantly inhibits breast cancer activity, providing a new strategy for tumor treatment. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 The ultraviolet-visible light morphology diagram, confocal microscope imaging diagram and SEM characterization diagram of the DNA hydrogel AC-DNet prepared in Example 1 after SYBR Green II staining; Figure 2 The apparent dissociation constant diagram for MCF-7 cells in Example 2; Figure 3 The gel property characterization diagram of three hydrogels in Example 3; Figure 4 The laser confocal microscope imaging diagram of three hydrogels in Example 4; Figure 5 The cell survival rate result diagram of three hydrogels on MCF-7 cells in Example 5; Figure 6 The tumor inhibition result diagram of different groups on breast cancer tumors in mice in Example 5; Figure 7 The cell proliferation determination result diagram of three hydrogels on MCF-7 cells in Example 5; Figure 8 The cell migration determination result diagram of three hydrogels on MCF-7 cells in Example 5; Figure 9 The cell survival rate result diagram of HAC-DNet on THLE-2 cells and MCF-7 cells in Example 6; Figure 10 The surface nucleolin receptor laser confocal microscope imaging of MCF-7 cells treated by different groups in Example 7; Figure 11 The reaction related principle diagram of the DNA hydrogel of the present application for regulating the behavior of cancer cells; DETAILED DESCRIPTION The specific embodiments of the present application are described below to facilitate the understanding of the present application for those skilled in the art, but it should be clear that the present application is not limited to the scope of the specific embodiments, and for those skilled in the art, it is obvious that various changes are within the spirit and scope of the present application defined and determined by the appended claims, and all the inventions utilizing the concept of the present application are within the scope of protection.

[0019] Example 1 A method for preparing the above-mentioned DNA hydrogel for inducing nucleolin receptors on the surface of cells, the specific steps are as follows: S1: Synthetically prepare circular templates CT-AL and CT-L respectively; specifically: A1: Dissolve the primer strand and the template strand in 80mM NaCl aqueous solution to obtain a 10μM primer strand solution and a 10μM template strand solution; A2: Take 80μL of the primer strand solution, 40μL of the template strand solution and 20μL of 10×T4 Buffer, and add ultrapure water to 200μL and mix well; A3: Heat the reaction solution at 95℃ for 10min, then naturally cool to 25℃, then add 3μL of T4 DNA ligase, mix well, then circle at 16℃ for 16h, and finally heat at 65℃ for 10min to inactivate, to obtain the circular templates CT-AL and CT-L; In the synthesis of the circular template CT-AL, the primer strand is Primer1 as shown in SEM ID NO. 2, and the template strand is Template1 as shown in SEM ID NO. 3; In the synthesis of the circular template CT-L, the primer strand is Primer2 as shown in SEM ID NO. 4, and the template strand is Template2 as shown in SEM ID NO. 5; S2: Synthetically obtain RCA long chain AL according to CT-AL, and synthetically obtain RCA long chain L according to CT-L; specifically: add 30μL, 0.2μM of the circular template CT-AL or the circular template CT-L, 10μL of dNTP with a concentration of 10mM, 10μL of 10×phi29 DNA polymerase Buffer and 2.5μL of phi29 DNA polymerase into an EP tube, add ultrapure water to 100μL, mix well, then incubate at 30℃ for 4h, and after the reaction is completed, heat at 65℃ for 10min to inactivate, to obtain the RCA long chain AL and the RCA long chain L; S3: RCA long chain L and cholesterol single chain ss-chol were mixed at a molar ratio of 1:2, and then crosslinked at 55℃ for 30 min in a water bath to obtain DNA chain CL; the sequence of the cholesterol single chain ss-chol is shown in SEM ID NO. 1; in specific implementation, the RCA long chain L obtained in S2 was diluted to 25 nM, and then mixed with the same volume of 25 nM RCA long chain L and 50 nM cholesterol single chain ss-chol; S4: RCA long chain AL and DNA chain CL were mixed at a mass ratio of 1:1, and then reacted at 37℃ for 1 h with continuous stirring at 600 rpm to obtain DNA hydrogel AC-DNet.

[0020] The obtained AC-DNet was subjected to SYBR Green II staining, and the ultraviolet-visible light morphology after staining was detected as shown in Figure 1 a, the confocal microscope imaging as shown in Figure 1 b, and the SEM characterization as shown in Figure 1 c; it can be seen from Figure 1 a that the RCA long chain AL and the DNA chain CL mixed and hybridized to form a gel precipitate; it can be seen from Figure 1 b that the morphology of the gel precipitate was observed under confocal imaging, and it can be seen that a clear fibrous network structure was formed; and it can be seen from Figure 1 c that the SEM characterization structure also proved that the DNA hydrogel mainly presented an irregular network structure. In summary, the prepared DNA hydrogel mainly presented a fibrous network structure.

[0021] Example 2 In order to compare the binding ability of the DNA hydrogel AC-DNet to the cell surface NCL, the fluorescence intensity of the monovalent structure AS411, the multivalent linear structure RCA long chain AL, and the multivalent network structure DNA hydrogel AC-DNet after binding to the MCF-7 cell surface nucleolin was measured by flow cytometry at different concentrations, and the apparent dissociation constant of the three structures to the cell surface nucleolin was fitted. The results are shown in Figure 2 It can be seen from Figure 2 that the dissociation constant Kd of AS1411 to the MCF-7 cell surface nucleolin was 413.1 nM, the Kd value of RCA long chain AL was 130.2 nM, and the Kd value of DNA hydrogel AC-DNet was 58.21 nM. Therefore, the affinity of DNA hydrogel AC-DNet is about 7.1 times higher than that of aptamer AS411, and about 2.4 times higher than that of RCA long chain AL. The above results show that the multivalent network structure AC-DNet has a higher ability to target and bind to the cell surface nucleolin than the monovalent aptamer, which is conducive to the long-term regulation of nucleolin aggregation on the cell surface by AC-DNet.

[0022] Example 3 The number of hybridization between RCA long chain AL and DNA chain CL can be adjusted by the design of RCA long chain AL, and three different hybridization numbers of corresponding circular templates are given in this embodiment, specifically: when the hybridization base number of RCA long chain AL and RCA long chain CL is 26, the template chain of the circular template CT-AL for synthesizing RCA long chain AL is Template2-26 as shown in SEM ID NO. 6.

[0023] When the hybridization base number of RCA long chain AL and RCA long chain CL is 37, the template chain of the circular template CT-AL for synthesizing RCA long chain AL is Template2-37 as shown in SEM ID NO. 7.

[0024] When the hybridization base number of RCA long chain AL and RCA long chain CL is 52, the template chain of the circular template CT-AL for synthesizing RCA long chain AL is Template2-52 as shown in SEM ID NO. 8.

[0025] The method of Example 1 was used to synthesize, and the template chain Template2-26 was used to synthesize low-density hydrogel LAC-DNet, the template chain Template2-37 was used to synthesize medium-density hydrogel MAC-DNet, and the template chain Template2-52 was used to synthesize high-density hydrogel HAC-DNet.

[0026] The rheology, mercury injection experiment, and scanning electron microscope determination of the three kinds of DNA hydrogels were performed, and the determination results are shown in Figure 3 , wherein Figure 3 a is the rheology characterization of the three kinds of hydrogels; Figure 3 b is the time scanning rheology determination result of the three kinds of hydrogels; Figure 3 c is the G’ and G” value results of the three density hydrogels under strain amplitude scanning (strain range from 0.1 to 1000%). Figure 3 d is the SEM characterization result of the three density hydrogels.

[0027] Specifically, the rheology determination method is: the rheometer (MCR 302) is used to measure the rheological properties of the DNA network, and the sample loading amount is 200 μL. Parameters: time scanning mode, strain 1%, frequency 1 Hz, and environmental temperature 25°C.

[0028] The mercury porosimetry assay was performed as follows: the DNA hydrogel was rapidly frozen in liquid nitrogen and then dried in a vacuum freeze dryer (SCIENTZ, SCIENTZ-10N). The porosity and pore size of the DNA hydrogel were measured using a mercury porosimeter (Micromeritics, MicroActive AutoPore V 9600).

[0029] The SEM measurement method was as follows: The silicon wafer was thoroughly cleaned with ultrapure water and then dried with nitrogen. The sample was washed three times with ultrapure water to remove excess salt ions. 10 μL of sample was dropped onto the silicon wafer, rapidly frozen in liquid nitrogen for 20 seconds, and then freeze-dried overnight in a vacuum freeze dryer. Morphological images were taken using a scanning electron microscope (Hitachi, S-4800).

[0030] Depend on Figure 3 It can be seen that the hydrogels at all three densities exhibit a storage modulus greater than the loss modulus, proving that the gels in all three states are in a gel state. With the increase of the number of hybridizations, the G' value increases accordingly, indicating that increasing the number of hybridizations enhances the gel's hardness. However, the highest value is only 5 Pa, below 10 Pa, proving that the hydrogel prepared in this application is an ultrasoft structure, causing almost no mechanical damage to cells and maintaining stability within 100%. Mercury intrusion porosimetry was used to determine the differences in pore size among the three hydrogels. It was found that the low-density hydrogel, with the fewest hybridizations, had an average pore size of 50 μm, the medium-density hydrogel had an average pore size of 5 μm, while the high- and low-density hydrogels had an average pore size of approximately 50 nm. Furthermore, SEM characterization gradually showed a denser network. In summary, adjusting the number of hybridizations between the two chains can control the gel's pore size and properties.

[0031] Example 4 The number of cells captured by the three hydrogels prepared in Example 3 was determined. Specifically, long-chain AL and long-chain CL synthesized with primer chains corresponding to the three template chains were stained with 1×SYBR Green II dye for 30 min in the dark, and then washed twice with water. MCF-7 cells were incubated with 1×DPC-Cy5 dye at 37°C for 15 min. First, the stained long-chain AL was incubated with stained MCF-7 cells at 25°C for 30 min, and then the stained long-chain CL was added and incubated at 37°C and 450 rpm for 1 hour, and washed once with water. Images were captured using an inverted fluorescence microscope (Nikon, Ti-E), and the images were processed using ImageJ. The number of cells captured in the DNA hydrogel was counted to obtain the results shown below. Figure 4 The laser confocal microscope imaging described above; by Figure 4It can be seen that among the three hydrogels, HAC-DNet can capture more cells, and the number of captured cells increases with increasing density, proving that the pore size difference of the gel network can affect the number of captured cells.

[0032] Example 5 Using the three hydrogels prepared in Example 3 for cell survival rate determination of breast cancer MCF-7 cells, first, a tumor-bearing mouse model was established: 5-week-old female NOD / scid IL2rgnullmice (NSG) were selected, and MCF-7 cells (1×10 7 cells per mouse) were injected into the mammary pad, and 3 mg / kg of estrogen aqueous solution was injected intraperitoneally every 3 days until the end of the experiment. When the tumor volume reached ≈100 mm 3 , the mice were randomly divided into 6 groups (5 mice per group), and a breast cancer tumor model was established.

[0033] The three hydrogels prepared in Example 3 were injected intratumorally into the mouse tumor, and the drug was administered once every 3 days, repeated 4 times. The tumor volume in nude mice was measured in two dimensions every 3 days using a vernier caliper, and the mouse body weight was measured, with a maximum measurement of 18 days. The tumor volume was calculated using the formula 0.5×(a×b 2 ), where a is the maximum diameter and b is the minimum diameter. All mice were euthanized after treatment. When MCF-7 cells were treated with three density hydrogels for 24 h, the absorbance (OD) at 450 nm was measured in a microplate reader using a CCK-8 kit, and the MCF-7 cell survival rate was calculated according to the formula , where Control is PBS treatment, and the rest of the treatment conditions are the same. sample is the experimental group with sample treatment, and blank is the blank group without cells and with culture medium. The results are shown in Figure 5 , compared with the control group treated with PBS, the survival rate of breast cancer MCF-7 cells was negatively correlated with the density of the hydrogel, and the survival rate of MCF-7 cells with high-density hydrogel HAC-DNet was only 25%, which fully proved that by adjusting the number of hybrids, the survival of breast cancer MCF-7 cells could be regulated.

[0034] At the same time, the anti-tumor effect of A-DNet containing only AS1411 (using the method of Example 1, the difference is that AL is directly hybridized with L, and ss-chol is not hybridized), C-DNet containing only chol (using the method of Example 1, the difference is that T sequence is used instead of AS1411 sequence in the Template sequence of AL), high-density HAC-DNet, medium-density MAC-DNet and low-density LAC-DNet was also determined. The results are shown in Figure 6 , where, Figure 6a is a tumor growth trend comparison chart; Figure 6 b is a mouse weight comparison chart; Figure 6 c is a mouse tumor characterization; Figure 6 d is a mouse tumor weight comparison chart. From Figure 6 It can be seen that the weight curve of all mice has no obvious fluctuation within 18 days, proving that the experimental material has biological safety. The tumor volume curve shows that the HAC-DNet group has the most obvious inhibitory effect on tumor growth due to the simultaneous presence of AS1411 and chol, while the MAC-DNet group and the LAC-DNet group have similar tumor growth trends. The analysis results of the tumor weight of the 5 groups of mice show that compared with the PBS group, the tumor inhibition rates of the C-DNet, A-DNet, and HAC-DNet groups are 34.57%, 38.48%, and 65.72%, respectively, while the tumor inhibition rates of the MAC-DNet and LAC-DNet groups are 46.48% and 38.09%, respectively, proving that the HAC-DNet with the simultaneous presence of AS1411 and chol has the most optimal inhibitory effect on tumors.

[0035] The CCK-8 kit was also used to determine the absorbance values of MCF-7 cells after 48h and 72h of action, and the cell viability at 24h, 48h, and 72h was statistically analyzed. The results are shown in Figure 7 Compared with the control group treated with PBS, the cell proliferation rate under HAC-DNet gradually decreased with time, and the cell proliferation rate under low, medium, and high density gradually decreased, which also indicates that by adjusting the number of hybrids, the cell proliferation behavior of breast cancer MCF-7 cells can be further inhibited.

[0036] The scratch test was also used to observe the cell migration of breast cancer MCF-7 cells after 24h of action of the three density hydrogels. The specific experimental method is as follows: MCF-7 cells were inoculated in a 12-well plate at a concentration of 4x10 5 / mL, allowed to adhere for 24h, and a scratch was made using a gun head. AL and CL were added in turn, and after 4h of action at 37°C, the culture medium was removed, and the remaining cells were washed with PBS. The culture medium was replaced with a low serum concentration culture medium for continuous culture, and the scratch area after 24h was observed, and the image was processed using Image J. The results are shown in Figure 8 Figure 8 a is a scratch test characterization chart of MCF-7 cells, Figure 8 b is a comparison chart of the migration inhibition rate of MCF-7 cells under the action of three density hydrogels. From Figure 8 It can be seen that the increase in gel density caused by the increase in the number of hybrids leads to a decrease in the migration rate of MCF-7 cells, and the migration rate of HAC-DNet is only 15%.

[0037] ​In summary, in the present application, by adjusting the hybridization number, the migration behavior of breast cancer MCF-7 cells can be effectively regulated.

[0038] Example 6 Toxicity of the high-density hydrogel HAC-DNet prepared in Example 3 on human normal liver cells THLE-2 was investigated, specifically: CCK-8 kit (Tongren Chemical Institute Dojindo Molecular Technologies) was used for determination. THLE-2 / MCF-7 cells were inoculated in a 96-well plate at a density of 1x10 4 cells per well, and incubated in a 37°C, 5% carbon dioxide environment for 24 hours. First, 15 μL of AL was added to 100 μL of culture medium and reacted at 25°C for 20 minutes. Then, 15 μL of CL was added for hybridization reaction at 37°C, and the PBS treatment group was used as a control, with the rest being consistent. After incubation for 24 hours, the culture medium was discarded, and the cells were washed twice with PBS. Then, 10% CCK-8 reagent was added and reacted at 37°C for 1 hour, and the absorbance of CCK-8 at 450 nm was determined using an enzyme label instrument (Bio-Tek, USA). The results are shown in Figure 9 Figure 9 a is the cell survival rate of the high-density hydrogel HAC-DNet acting on normal cells THLE-2, Figure 9 b is the cell survival rate of the high-density hydrogel HAC-DNet acting on cancer cells MCF-7. It can be known from Figure 9 the above that the cell survival rate of the high-density hydrogel HAC-DNet on THLE-2 cells is about 90%, indicating that it has almost no toxicity to normal cells, and the survival rate of MCF-7 cells with high expression of nucleolin is about 20%. This result fully demonstrates that the DNA hydrogel of the present application has good targeting cancer cell effect ability, and almost no toxic side effects on normal cells.

[0039] Example 7 In order to verify that the present application can induce the aggregation of nucleolin receptors on the cell surface, we used nucleolin antibodies (red) to observe and analyze the localization of nucleolin receptors. The specific method is as follows: MCF-7 cells were inoculated in a 96-well plate at a density of 0.5x10 5 ​The cells were seeded in imaging dishes at 1 x 104 / mL, treated with PBS (control), AL, CL, C-DNet without AS1411, A-DNet without ss-chol, AC-DNet with both AS1411 and chol for 4 h, then the medium was removed and the cells were washed with PBS for 3 times, and the cells were fixed with 4% paraformaldehyde. After washing with PBS for 3 times, the cells were blocked with 2% BSA for 1 h. After treatment, the NCL primary antibody was added for reaction and combination at 4°C, then PBS was added for washing for 3 times, and the cells were stained with lgG 647 modified secondary antibody. The nucleus was stained with DAPI. Confocal fluorescence imaging was performed on an Olympus laser scanning confocal imaging system (Olympus, Melville, NY), and the images were processed using Image J software, and the results are shown in Figure 10 As shown in Figure 10 It can be seen that in the control group Control treated with PBS, nucleolin (red) exists in the cytoplasm, nucleus and cell surface. Compared with the untreated control group, the use of RCA long chain AL can cause a moderate change in the fluorescence fluctuation around the cells, but the traction of a single multivalent polymer is not enough to cause significant aggregation of receptors. It can be observed that the AC-DNet treatment with the simultaneous presence of AS1411 and Chol shows significant aggregation of cell surface nucleolin receptors. These results prove that AC-DNet significantly enhances the clustering of cell surface nucleolin receptors, disrupts their spatial distribution on the cell surface, and fully demonstrates that the DNA hydrogel of the application significantly induces the aggregation of cell surface nucleolin receptors. It can be inferred that the reaction principle of the DNA hydrogel of the application for regulating the behavior of cancer cells is as shown in Figure 11 Wherein Figure 11 a is a schematic diagram showing the principle of forming a long chain AL containing a multivalent AS1411 and a L containing a plurality of sequences capable of being complementary to ss-chol by rolling circle amplification technology, and then the L is hybridized with ss-chol to form CL; Figure 11 b is a schematic diagram showing the principle that AL targets the surface of NCL highly expressed cancer cells through the targeting of multivalent AS1411, and further hybridizes with CL to form a DNA hydrogel AC-DNet; Figure 11 c is a schematic diagram showing the principle of further regulating the behavior of cancer cells by increasing the number of hybridization between AL and CL to obtain low-density hydrogel (LAC-DNet), intermediate-density hydrogel (MAC-DNet), and high-density hydrogel (HAC-DNet), respectively.

Claims

1. A programmable DNA hydrogel for inhibiting breast cancer tumor activity, characterized in that, The DNA hydrogel is composed of RCA long chain AL containing a multivalent AS411 aptamer sequence and RCA long chain CL hybridized with cholesterol single-strand ss-chol; the RCA long chain AL can be hybridized with the RCA long chain CL, and the number of hybridized bases of the RCA long chain AL and the RCA long chain CL is positively correlated with the density of the DNA hydrogel and the number of regulated cancer cells; the sequence of the cholesterol single-strand ss-chol is shown as SEM ID NO.

1.

2. The DNA hydrogel of claim 1, wherein, The RCA long chain CL is obtained by hybridizing the RCA long chain L with the cholesterol single-strand ss-chol based on the rolling circle amplification technology, and the circular template of the RCA long chain AL and the RCA long chain is synthesized by using a template strand and a primer strand; In the synthesis of the circular template CT-AL, the primer strand is Primer 1 shown as SEM ID NO. 2, and the template strand is Template 1 shown as SEM ID NO. 3; In the synthesis of the circular template CT-L, the primer strand is Primer 2 shown as SEM ID NO. 4, and the template strand is Template 2 shown as SEM ID NO.

5.

3. The DNA hydrogel of claim 1, wherein, When the number of hybridized bases of the RCA long chain AL and the RCA long chain CL is 26, the template strand of the circular template CT-AL for synthesizing the RCA long chain AL is Template 2-26 shown as SEM ID NO.

6.

4. The DNA hydrogel of claim 1, wherein, When the number of hybridized bases of the RCA long chain AL and the RCA long chain CL is 37, the template strand of the circular template CT-AL for synthesizing the RCA long chain AL is Template 2-37 shown as SEM ID NO.

7.

5. The DNA hydrogel of claim 1, wherein, When the number of hybridized bases of the RCA long chain AL and the RCA long chain CL is 52, the template strand of the circular template CT-AL for synthesizing the RCA long chain AL is Template 2-52 shown as SEM ID NO.

8.

6. A method for preparing a DNA hydrogel for inducing nucleolin receptors on cell surfaces as described in claim 2, characterized in that, The specific steps are as follows: S1: respectively synthesize and prepare the circular template CT-AL and the circular template CT-L; S2: synthesize the RCA long chain AL according to the CT-AL, and synthesize the RCA long chain L according to the CT-L; S3: crosslink the RCA long chain L with the cholesterol single-strand ss-chol to obtain the DNA chain CL; S4: mix the RCA long chain AL and the DNA chain CL in a mass ratio of 1:1, and then continuously stir at 600 rpm at 37℃ for 1h to prepare the DNA hydrogel.

7. The preparation method according to claim 6, characterized in that, The synthesis method of the circular template CT-AL or the circular template CT-L is as follows: A1: dissolve the primer strand and the template strand in 80mM NaCl aqueous solution to obtain 10μM primer strand solution and 10μM template strand solution; A2: take 80μL of the primer strand solution, 40μL of the template strand solution and 20μL of 10×T4 Buffer, and supplement with ultrapure water to 200μL and mix well; A3: The reaction solution was heated at 95℃ for 10 min, then naturally cooled to 25℃, 3 μL of T4 DNA ligase was added, mixed, circularized at 16℃ for 16 h, and finally inactivated by heating at 65℃ for 10 min to obtain the circular template CT-AL or the circular template CT-L.

8. The preparation method according to claim 6, characterized in that, The synthesis method of the RCA long chain AL and the RCA long chain L is specifically as follows: 30 μL of the circular template CT-AL or the circular template CT-L, 10 μL of dNTP with a concentration of 10 mM, 10 μL of 10× phi29 DNA polymerase Buffer and 2.5 μL of phi29 DNA polymerase were added into an EP tube, ultrapure water was added to make up to 100 μL, mixed, incubated at 30℃ for 4 h, and then inactivated by heating at 65℃ for 10 min after the reaction was completed, so as to obtain the RCA long chain AL or the RCA long chain L.

9. The preparation method according to claim 6, characterized in that, The step S3 is specifically as follows: the RCA long chain L and the cholesterol single strand ss-chol were mixed at a molar ratio of 1:2, and then crosslinked by water bath at 55℃ for 30 min.

10. Use of the DNA hydrogel of claim 1 in the preparation of a drug for treating and inhibiting tumors.

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