Ag nps-dox-dna hydrogel triggered by atp and h2s synergistically and preparation method and application thereof

Abstract

The application discloses Ag NPs-DOX-DNA hydrogel triggered by ATP and H2S in cooperation, a preparation method and application thereof, and the hydrogel is a temperature-sensitive type and comprises DOX, Ag NPs and DNA hydrogel; wherein the DNA hydrogel comprises ATP aptamer, cDNA and N-isopropyl acrylamide; the sequence of the ATP aptamer is SEQ ID NO.1: CACCTGGGGGAGTATTGCGGAGGAAGG-Acrydite, and the sequence of the cDNA is SEQ ID NO.2: CTCCCCCAGGTGTTT-Acrydite. Compared with the prior art, the application has the following advantages: (1) the Ag NPs-DOX-DNA hydrogel firstly proposes to apply ATP and H2S as a biomarker group for targeted release of DOX and Ag NPs imaging and treatment of CRC, is successfully applied to specific imaging of human colon cancer HCT 116 cells, realizes double treatment of CRC cells, and has good specificity and practicability; (2) the Ag NPs-DOX-DNA hydrogel has good biocompatibility and can provide a safe and effective strategy for diagnosis and treatment of CRC.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of cancer treatment, and relates to a CRC diagnosis and treatment system, in particular to Ag NPs-DOX-DNA hydrogel triggered by ATP and H2S in cooperation and a preparation method and application thereof. BACKGROUND

[0002] Traditional methods for diagnosis and treatment of colorectal cancer (CRC) include endoscopic observation, surgical resection, drug chemotherapy and radiotherapy. However, invasive examination and surgery often bring painful experience to patients, affect the quality of life of patients, and are prone to cause recurrence and metastasis of tumors. The side effects of chemotherapy and radiotherapy are also obvious, for example, blind drug treatment can make cancer cells develop drug resistance; in addition, indiscriminate drug release can cause damage to normal cells.

[0003] In recent years, DNA-based hydrogels have attracted the attention of many researchers as drug delivery tools. Oligonucleotides are cross-linked through base complementary pairing to form a highly stable three-dimensional network structure, and such a gel can bear the role of drug transport and allow drug release by collapsing the scaffold in a specific environment. For example, in a study by Chen et al., a hydrogel coated metal organic framework (MOF) material was developed using nucleic acid hairpins, wherein the hairpin chain contains an aptamer sequence of adenosine triphosphate (ATP). The role of ATP in the tumor microenvironment makes the hydrogel coating be degraded, and the drug wrapped in the MOF material is released in a targeted manner. In addition, a chemical inert polymer such as polyacrylamide can be used as the skeleton of the DNA network to achieve the purpose of adjusting the transition of the hydrogel between solid and liquid states.

[0004] In order to enhance the targeted release of drugs, it is necessary to make full use of the biomarkers of CRC. ATP is the main source of energy in cells and is one of the main components of the tumor microenvironment, which has an important influence on the growth, movement, diffusion and anti-tumor immune response of cancer cells. Studies have shown that the content of ATP in cells is about 106 times that of extracellular content, and the content in the tumor microenvironment is 100-500 μM, which is much higher than 10-100 nM in non-tumor tissues, so ATP can be used as one of the biomarkers indicating the occurrence of CRC. However, a single biomarker is not sufficient for the direction of CRC, and lacks specificity. Hydrogen sulfide (H2S) is considered to be the third endogenous gas signal molecule after nitric oxide and carbon monoxide, and is involved in various physiological and pathological processes in the human body, such as antioxidant, anti-inflammatory, diabetes and Alzheimer's disease. Endogenous H2S is usually generated through enzymatic reaction, especially under the action of cysteine-β-synthase (CBS) in CRC, a large amount of H2S is produced, so endogenous H2S is often regarded as a biomarker of CRC.

[0005] In addition, studies have found that high expression of H2S in CRC tissue can promote the proliferation of cancer cells and induce the occurrence of tumors, so it is necessary to remove H2S from CRC tissue. Many nanomaterials have been shown to have H2S scavenging effects in the prior art, such as ZnO, Cu2O, Fe3O4, etc. Compared with these metal oxides, silver nanoparticles (Ag NPs) have lower cytotoxicity and higher biocompatibility, and are a functional nanomaterial commonly used in vivo. Bi et al. reported a H2S-responsive SiO2@Ag probe for near-infrared / photoacoustic dual-mode specific imaging of CRC, in which Ag can undergo sulfuration with endogenous H2S and be converted to SiO2@Ag2S after in situ biosynthesis, so Ag NPs can be used as H2S scavenging materials.

[0006] The prior art generally only detects or treats a single biomarker of CRC, which results in a high misdiagnosis rate of CRC diagnosis, and the existing drug delivery platform non-targeted releases drugs in the cell environment, which will cause damage to normal cells. The present application uses the good biocompatibility and temperature-sensitive properties of DNA hydrogel as a carrier for drugs and nanomaterials, and through the specific expression of double biomarkers in CRC cells, the gel is sequentially digested to release the contents, the fluorescence is turned on, and the double treatment effect is achieved. The present application will solve the problems of high cytotoxicity, single action, poor specificity, insufficient treatment effect, etc. in the prior art.

[0007] The previous work of the research group, "miRNA-211 and H2S synergistically triggered 3D DNA network (3D DNA network) structure", is an accurate early screening and detection method for CRC, and is also used for in situ imaging in CRC cancer cells, achieving accurate positioning and efficient diagnosis of CRC. The present application realizes the therapeutic effect on the basis of cancer cell imaging, and the "Ag NPs-DOX-DNA hydrogel triggered by ATP and H2S synergistically" is a diagnosis and treatment strategy with strong specificity and effective treatment of CRC. Through targeted release of Ag NPs and DOX, on the one hand, H2S is largely removed, causing a dramatic increase in the content of reactive oxygen species (ROS) in the cell, producing cytotoxicity, and on the other hand, the anticancer drug DOX synergistically treats, achieving double treatment of CRC cells, with good specificity and practicality. SUMMARY

[0008] Technical problems solved: In order to overcome the shortcomings of the prior art, the present application uses DNA hydrogel as a drug release platform, loads nanomaterial Ag NPs and cancer treatment drug DOX therein, and selectively releases Ag NPs and DOX through the characteristics of CRC tissue and cell-specific release of high concentrations of ATP and H2S, and activates the generation of ROS while clearing H2S and reducing its content, so as to achieve the purpose of synergistic treatment of CRC with DOX. In view of this, the present application provides Ag NPs-DOX-DNA hydrogel triggered by ATP and H2S synergistically, and a preparation method and application thereof.

[0009] Technical scheme: The Ag NPs-DOX-DNA hydrogel triggered by ATP and H2S synergistically is a temperature-sensitive type, and comprises: DOX, Ag NPs and DNA hydrogel; wherein the DNA hydrogel comprises ATP aptamer, cDNA and N-isopropyl acrylamide; the sequence of the ATP aptamer is SEQ ID NO. 1: CACCTGGGGGAGTATTGCGGAGGAAGG-Acrydite, and the sequence of the cDNA is SEQ ID NO. 2: CTCCCCCAGGTGTTT-Acrydite.

[0010] The preparation method of the Ag NPs-DOX-DNA hydrogel triggered by ATP and H2S synergistically comprises the following steps:

[0011] S1, preparing a temperature-sensitive DNA hydrogel

[0012] N-isopropyl acrylamide and ammonium persulfate are dissolved in a buffer solution, and after ultrasonic complete dissolution, mixed with 100 mu M ATP aptamer, 100 mu M cDNA and N, N, N', N'-tetramethyl ethylenediamine, and the obtained mixture is subjected to polymerization reaction at room temperature; wherein the molar ratio of N-isopropyl acrylamide and ammonium persulfate is 200-300:1, and the molar ratio of ATP aptamer and cDNA is 1:1-3;

[0013] S2, preparing Ag NPs

[0014] 3-6 mM AgNO3 solution is slowly added to an equal volume of freshly prepared NaBH4 solution under the condition of magnetic stirring, the reaction time is 10-15 min, centrifugation is carried out at a speed of 6000-8000 rpm for 5-10 min, water washing is carried out to remove excess unreacted reducing agent, and the internal reaction is terminated; wherein the molar ratio of NaBH4 and AgNO3 is 1:1-2;

[0015] S3, preparing Ag NPs-DOX-DNA hydrogel

[0016] The DOX is dissolved in the hydrogel prepared by S1, and then the Ag NPs prepared by S2 is added and mixed uniformly by ultrasonic to prepare the Ag NPs-DOX-DNA hydrogel; wherein the volume ratio of the hydrogel prepared by S1 to the Ag NPs prepared by S2 is 1-2:1, and the concentration of the DOX in the Ag NPs-DOX-DNA hydrogel is 0.01-0.1 mg / mL.

[0017] Preferably, the buffer in S1 is Tris-HCl 10 mM, EDTA 10 mM, and pH=8.0.

[0018] Preferably, the molar ratio of the ATP aptamer and the cDNA in S1 is 1:1.

[0019] Preferably, the concentration of the NaBH4 aqueous solution in S2 is 4 mM, and the molar ratio of NaBH4 to AgNO3 is 1:1.

[0020] The application of the Ag NPs-DOX-DNA hydrogel triggered by ATP and H2S in the early screening of CRC.

[0021] The application of the Ag NPs-DOX-DNA hydrogel triggered by ATP and H2S in the treatment of CRC.

[0022] The principle of the Ag NPs-DOX-DNA hydrogel triggered by ATP and H2S for the early screening and treatment of CRC is that: first, the ATP aptamer and the complementary DNA single strand (cDNA) are modified by acrylic acid and form copolymers with acrylamide, respectively. At low temperature, the DNA hydrogel is in a liquid state and can be delivered to the tumor site by in-situ injection. At 37℃, the DNA hydrogel rapidly changes into a solid state and adheres to the surface of the tumor. The local high concentration of ATP can degrade the DNA hydrogel through the interaction with its aptamer, thereby releasing the Ag NPs and DOX into the CRC cells. After that, the high level of H2S reacts with the Ag NPs to generate silver sulfide quantum dots (Ag2S QDs), on the one hand, a large amount of H2S is removed, so that the content of reactive oxygen species (ROS) in the cells increases dramatically, producing cytotoxicity, on the other hand, the characteristics of Ag2S QDs are used for fluorescence imaging of CRC cells.

[0023] Beneficial effects: (1) The Ag NPs-DOX-DNA hydrogel first proposes to apply ATP and H2S as a biomarker group for targeted release of DOX and Ag NPs imaging and treatment of CRC, is successfully applied to specific imaging of human colon cancer HCT 116 cells, realizes the dual treatment of CRC cells, and has good specificity and practicability; (2) The Ag NPs-DOX-DNA hydrogel has good biocompatibility, and can provide a safe and effective strategy for the diagnosis and treatment of CRC. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 It is a schematic diagram for diagnosing and treating CRC based on Ag NPs-DOX-DNA gel.

[0025] Figure 2 (a), (b) SEM images of DNA hydrogel; (c) TEM image of Ag NPs; (d) HRTEM image of Ag NPs-DNA gel; Element mapping of Ag NPs-DNA gel: (e) Ag, (f) P, (g) C, (h) N, (i) O.

[0026] Figure 3 It is a real object diagram of ordinary gel and DNA hydrogel under different conditions. (a) Hydrogel at 25 DEG C, (b) hydrogel at 37 DEG C, (c) DNA hydrogel at 37 DEG C, (d) (e) (f) are DNA hydrogels added with 0.5 μM, 1 μM, 10 μM ATP respectively.

[0027] Figure 4 (a) Fluorescence micrograph of Ag NPs-DNA gel in HCT 116 cells and Hela cells under different concentrations of ATP conditions; (b) Standard curve of ATP content and average gray value, the scale is 100 μm.

[0028] Figure 5 (a) Fluorescence micrograph of HCT 116 cells after incubation with PBS, Ag NPs-DNA gel, DOX-DNA gel and Ag NPs-DOX-DNA gel for 24 h and staining with DCFH-DA (10 μM); (b) Flow cytometry analysis of ROS level.

[0029] Figure 6 It is a diagram of (a) treatment effect, (b) body weight change and (c) change of tumor diameter of mice within 21 days after injection of normal saline, Ag NPs-DNA gel, DOX-DNA gel and Ag NPs-DOX-DNA gel.

[0030] Figure 7The MTT method was used to evaluate the HCT 116 cell viability values (%) after incubation with PBS, Ag NPs, DOX, DNA hydrogel, Ag NPs-DNA gel, DOX-DNA gel and Ag NPs-DOX-DNA gel for 24 h. DETAILED DESCRIPTION

[0031] The following examples further illustrate the content of the present application, but should not be understood as limiting the application. Modifications and substitutions of the methods, steps or conditions of the present application, without departing from the spirit and scope of the present application, all belong to the scope of the present application. If not specifically indicated, the technical means used in the examples are conventional means known to those skilled in the art.

[0032] Example 1 Preparation of Ag NPs-DOX-DNA hydrogel

[0033] The preparation method of Ag NPs-DOX-DNA hydrogel triggered by ATP and H2S in cooperation includes the following steps:

[0034] S1, Preparation of temperature-sensitive DNA hydrogel

[0035] 0.15 g of N-isopropyl acrylamide (NIPAM) and 0.01 g of ammonium persulfate (APS) were dissolved in 1 mL of buffer, and ultrasonic was used to completely dissolve them. 167 μL of the above solution was mixed with 10 μL of 100 μM ATP aptamer, 10 μL of 100 μM cDNA and 10 μL of N,N,N',N'-tetramethyl ethylenediamine (TEMED), and buffer was added to make the volume reach 1 mL. The resulting mixture was polymerized at room temperature for 20 min.

[0036] S2, Preparation of Ag NPs

[0037] Ag NPs were synthesized using NaBH4 as a reducing agent. 5 mL of 4 mM AgNO3 solution was slowly added to an equal volume of freshly prepared NaBH4 solution under magnetic stirring, and the reaction time was 10 min. Centrifugation was performed at 8000 rpm for 5 min, and water washing was performed twice to remove excess unreacted reducing agent, and the internal reaction was terminated.

[0038] S3, Preparation of Ag NPs-DOX-DNA gel

[0039] 0.05 g of DOX was dissolved in 1 mL of DNA hydrogel solution and mixed well, then 500 μL of Ag NPs after centrifugation and removal of supernatant was added to the above solution, ultrasonic was performed for 10 min, and after mixing well, it was used for experiment.

[0040] As Figure 2(a) and (b) show that the prepared DNA hydrogel has a loose porous reticular crosslinked structure after freeze-drying, which is conducive to the efficient loading of Ag NPs and DOX. Figure 2 (c) is a TEM image of Ag NPs, with a particle size of about 25 nm and good dispersion in solution. Figure 2 (d) is an HRTEM image of Ag NPs-DNA gel. Figure 2 (e) (f) (g) (h) (i) correspond to Ag, P, C, N, and O element mapping images, respectively, confirming the successful preparation of Ag NPs-DNA gel.

[0041] Figure 3 Actual images of gels and DNA hydrogels synthesized using NIPAM as raw material under different conditions are shown. Figure 3 (a) is the state of the ordinary gel at 25°C, at which time the gel is in a transparent liquid state. When the temperature is raised to 37°C, as shown in Figure 3 (b), the transparent liquid gradually changes to a solid gel, indicating that the gel synthesized using NIPAM as raw material has the property of temperature-controlled physical form, but at this time only part of it is converted into a white solid gel. The DNA hydrogel at 37°C can be completely converted into a solid gel in a milky white opaque state, as shown in Figure 3 (c), which is because the ATP aptamer in the DNA gel binds to the cDNA, enhancing the polymerization force between NIPAM. As shown in Figure 3 (d) (e) (f), at 37°C, 0.5 μM, 1 μM, and 10 μM of ATP were added to equal amounts of DNA hydrogel, respectively, and it was observed that the DNA hydrogel was digested to different degrees, and the greater the concentration of ATP, the stronger the degree of conversion of the DNA hydrogel to a liquid state.

[0042] Example 2 In vitro imaging of HCT 116 cells

[0043] Human colorectal cancer HCT 116 cells were cultured using RPMI-1640 medium containing 10% fetal bovine serum (containing double antibodies). The cells were seeded in a 60 mm 2 culture dish and placed in a 37°C, 5% CO2 incubator. When the cells grew to about 80% confluence, Ag NPs, DOX, DNA hydrogel, and Ag NPs-DOX-DNA gel were added to the culture dish, and after 12 h of incubation, the medium was aspirated and discarded. After washing the cells with PBS, an inverted fluorescence microscope was used for observation, with an excitation wavelength of 460-550 nm.

[0044] As shown in Figure 4As shown, red fluorescence was observed in all four groups of HCT 116 cells, and the fluorescence intensity increased with the increasing concentration of ATP, which indicated that ATP played a role in inducing the release of Ag NPs in the Ag NPs-DNA gel system. High concentration of ATP caused more Ag NPs to be released from the DNA hydrogel into HCT 116 cells, and to be etched into Ag2S QDs with red fluorescence properties under the action of H2S. In addition, since the content of ATP in the CRC tissue environment is much higher than that in non-cancerous tissue, obvious red fluorescence was also observed in the control group. On the other hand, no obvious fluorescence signal was observed in Hela cells, so it can be determined that the imaging of Ag NPs in CRC cells is specific.

[0045] In addition, by using ImageJ software to convert the fluorescence intensity in the fluorescence micrograph of each group into the average gray value, the concentration of ATP in HCT 116 cells was quantified, and the average gray value of the picture was proportional to the logarithm of the ATP concentration. Through calculation, it was found that the content of ATP in HCT 116 cells was 0.735 μM.

[0046] Example 3 In vivo treatment of HCT 116 cells

[0047] The experimental mice used in this method were Bal / c-Nu type 4-5 week old female mice. The culture dish containing 5.2 x 10 6 HCT 116 cells was discarded of the medium, and after digestion, it was uniformly dispersed in 100 μL PBS buffer solution, and then the cell suspension was injected into the subcutaneous position of the right hind limb of the nude mouse to establish a CRC tumor model.

[0048] In order to verify the process of Ag NPs removing H2S and generating ROS, 2,7-dichlorodi-hydrofluorescein diacetate (DCFH-DA) probe was used to explore the ability of different nanomaterials to generate ROS. As shown in FIG. 8, the fluorescence intensity of the control group was the highest, followed by the Ag NPs group, and the fluorescence intensity of the Ag NPs+H2S group was the lowest. This result indicated that Ag NPs could remove H2S and generate ROS. Figure 5(a) shows that PBS, Ag NPs-DNA gel, DOX-DNA gel and Ag NPs-DOX-DNA gel were incubated with HCT 116 cells for 24 h, and then the ROS content in the cells was detected using a ROS detection kit. PBS was used as a control group, and it was observed that the Ag NPs-DNA gel group had more obvious green fluorescence, which indicated that the decrease of H2S by Ag NPs led to a significant increase in intracellular ROS. In addition, the fluorescence intensity of DCFH-DA in the Ag NPs-DOX-DNA gel treated HCT 116 cells was much higher than that in the other three groups, thus confirming the synergistic therapeutic effect of Ag NPs and DOX on CRC cells. The fluorescence signal of ROS in the four groups of HCT 116 cells was monitored using a flow cytometer, as shown in Figure 5 (b) shows similar results.

[0049] Figure 6 The therapeutic effects of different nanomaterials on CRC model mice within 21 days are shown in Figure 6 (a) and (c), it can be seen that the tumor volume of the mice treated with PBS increased significantly after 21 days. The tumor growth rate of the mice treated with Ag NPs-DNA gel slowed down after 21 days, the tumor volume of the mice treated with DOX-DNA gel decreased slightly, and the tumor volume of the mice treated with Ag NPs-DOX-DNA gel decreased significantly after 21 days, which indicated that Ag NPs-DOX-DNA gel had a good synergistic therapeutic effect on CRC. The body weight of the mice injected with different materials was detected, and the results are shown in Figure 6 (b), the body weight of the four groups of mice increased steadily within 21 days, which indicated that Ag NPs-DOX-DNA gel had good biocompatibility and had no special effect on the growth of mice.

[0050] Figure 7 The MTT method was used to evaluate the HCT 116 cell viability value after incubation with PBS, Ag NPs, DOX, DNA hydrogel, Ag NPs-DNA gel, DOX-DNA gel and Ag NPs-DOX-DNA gel for 24 h. As shown in Figure 7 PBS buffer was used as a control, and after incubation with HCT 116 cells for 24 h, the cytotoxicity of Ag NPs, DNA hydrogel and Ag NPs-DNA gel was low, the cytotoxicity of DOX and DOX-DNA gel was slightly higher than that of the former, and the cytotoxicity of Ag NPs-DOX-DNA gel was higher, which confirmed that the ROS produced by the H2S scavenging effect of Ag NPs and DOX had a synergistic therapeutic effect on HCT 116.

Claims

1. Ag NPs-DOX-DNA hydrogel triggered by ATP and H2S synergistically, characterized in that, The hydrogel is temperature-sensitive, and comprises: DOX, Ag NPs and a DNA hydrogel; wherein the DNA hydrogel comprises an ATP aptamer, cDNA and N-isopropyl acrylamide; the sequence of the ATP aptamer is SEQ ID NO. 1: CACCTGGGGGAGTATTGCGGAGGAAGG-Acrydite, and the sequence of the cDNA is SEQ ID NO. 2: CTCCCCCAGGTGTTT-Acrydite. The hydrogel is prepared by the following method: S1, preparing a temperature-sensitive DNA hydrogel N-isopropyl acrylamide and ammonium persulfate are dissolved in a buffer, and after ultrasonic complete dissolution, 100 μM ATP aptamer, 100 μM cDNA and N,N,N',N'-tetramethyl ethylenediamine are mixed, and the obtained mixture is subjected to a polymerization reaction at room temperature; wherein the molar ratio of N-isopropyl acrylamide and ammonium persulfate is 200-300:1, and the molar ratio of ATP aptamer and cDNA is 1:1-3; S2, preparing Ag NPs 3-6 mM AgNO3 solution is slowly added to an equal volume of freshly prepared NaBH4 solution under the condition of magnetic stirring, the reaction time is 10-15 min, centrifugation is carried out at a speed of 6000-8000 rpm for 5-10 min, water washing is carried out to remove excess unreacted reducing agent, and the internal reaction is terminated; wherein the molar ratio of NaBH4 and AgNO3 is 1:1-2; S3, preparing Ag NPs-DOX-DNA hydrogel DOX is dissolved in the hydrogel prepared in S1 and mixed uniformly, then Ag NPs prepared in S2 are added, ultrasonic mixing is carried out to prepare Ag NPs-DOX-DNA hydrogel; wherein the volume ratio of the hydrogel prepared in S1 and the Ag NPs prepared in S2 is 1-2:1, and the concentration of DOX in the Ag NPs-DOX-DNA hydrogel is 0.01-0.1 mg / mL.

2. The Ag NPs-DOX-DNA hydrogel triggered by ATP and H2S synergistically according to claim 1, characterized in that, The buffer in S1 is Tris-HCl 10 mM, EDTA 10 mM, pH=8.

0.

3. The Ag NPs-DOX-DNA hydrogel triggered by ATP and H2S synergistically according to claim 1, characterized in that, The molar ratio of ATP aptamer and cDNA in the system in S1 is 1:

1.

4. The Ag NPs-DOX-DNA hydrogel triggered by ATP and H2S synergistically according to claim 1, characterized in that, The concentration of NaBH4 aqueous solution in S2 is 4 mM, and the molar ratio of NaBH4 and AgNO3 is 1:

1.

5. The application of the Ag NPs-DOX-DNA hydrogel triggered by ATP and H2S in the preparation of a CRC early screening reagent.

6. The application of the Ag NPs-DOX-DNA hydrogel triggered by ATP and H2S in the preparation of a CRC treatment drug.

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