A DNA tetrahedron-amifostine complex and its preparation method and use in radiation protection

Abstract

The present invention provides a DNA tetrahedron-aminofostine complex and a preparation method thereof and use thereof in radiation protection, and belongs to the field of biomedical technology. The DNA tetrahedron-aminofostine complex of the present invention is a complex formed by mixing a DNA tetrahedron framework nucleic acid and amifostine. The complex of the present invention uses a DNA tetrahedron as a carrier to carry out the coordinated delivery of amifostine. The DNA tetrahedron-aminofostine complex of the present invention overcomes the problem of short metabolism time of amifostine, and enhances the radiation protection effect of amifostine, achieving a synergistic radiation protection effect. The complex of the present invention can protect DNA in a radiation environment, has a protective effect on multiple organs at the same time, and can improve the survival rate under radiation, has an excellent radiation protection effect, and has excellent application prospects in various fields.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and specifically relates to a DNA tetrahedron-amifostine complex and a preparation method thereof and use thereof in radiation protection. Background Art

[0002] Ionizing radiation (IR) is widely used in industry, agriculture, and medicine, resulting in an increased risk of radiation exposure. Excessive radiation can cause serious physiological consequences to the body, even life-threatening, such as acute radiation syndrome, chronic radiation syndrome, and cancer. Currently, more than half of cancer patients receive radiotherapy in routine clinical treatment. Radiotherapy, as the main treatment for malignant tumors, kills cancer cells by destroying their DNA and inducing apoptosis, while also causing adverse damage to nearby healthy tissues. Early toxicity includes skin and mucosal diseases; long-term toxicity, such as chronic inflammation and tissue fibrosis, may not appear until months or years later, and may even eventually cause secondary cancers. In addition, radiotherapy to the lower abdomen or pelvis is likely to affect the body's fertility.

[0003] In order to reduce the health hazards caused by radiation damage, the research and development of high-efficiency and low-toxic radiation protectors has great application prospects. At present, there are many types of drugs being studied, mainly aminothiol drugs, cytokines, hormone drugs, nitroxide free radicals, natural medicines, etc. However, these are still far from ideal radiation protectors, and some drugs have serious side effects after use, which limits their clinical use. Among them, amifostine (AMF) is a broad-spectrum cytoprotectant that can resist the damage of ionizing radiation and chemotherapy drugs to DNA. It is approved as the only radiation protector for narrow clinical indications related to radiotherapy. Normal tissues have higher alkaline phosphatase activity than tumor tissues, which can convert AMF into the active form of WR-1065 to exert its effect. Therefore, AMF exhibits selective radiation protection in normal tissues, but not tumors. However, AMF has a short elimination half-life and will soon be metabolized and excreted by the human body, limiting its bioavailability and clinical application. Increasing the dosage will cause systemic toxicity and complications such as hypotension, nausea and vomiting. Therefore, the development of highly efficient and low-toxic radiation protectors still has great application prospects and social benefits.

[0004] Tetrahedral framework nucleic acid (tFNA), also known as tetrahedral DNA (TDN) and tetrahedral DNA nanostructure, is formed by four single-stranded DNAs through complementary base pairing between chains, and its shape is similar to a tetrahedron. The DNA tetrahedron has a stable structure and good biocompatibility, and can be used as a drug carrier. However, the special structure of the DNA tetrahedron itself and its certain medicinal activity make it difficult to predict how the two will interact after loading small molecule drugs. It is unknown whether they will play a synergistic or antagonistic role, and the effects of small molecule drugs with different structures may be quite different. At present, DNA tetrahedrons have not been loaded with amifostine. Whether DNA tetrahedrons can be used to load amifostine to achieve better radiation protection requires further study. Summary of the invention

[0005] The purpose of the present invention is to provide a DNA tetrahedron-amifostine complex and a preparation method thereof and use thereof in radiation protection.

[0006] The invention provides a DNA tetrahedron-amifostine complex, which is a complex formed by mixing DNA tetrahedron framework nucleic acid and amifostine.

[0007] Furthermore, when the DNA tetrahedron and amifostine are mixed, the molar ratio of the DNA tetrahedron to amifostine is 1:160 to 1:1280.

[0008] Furthermore, the molar ratio of the DNA tetrahedron to amifostine is 1:640.

[0009] Furthermore, the DNA tetrahedron is synthesized by self-assembly of four DNA single strands; the sequences of the four DNA single strands are shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, and SEQ ID NO.4, respectively.

[0010] Furthermore, the method for synthesizing the DNA tetrahedron comprises the following steps: adding four DNA single strands into TM buffer, maintaining at 95° C. for 10 min, and maintaining at 4° C. for more than 20 min, to obtain the tetrahedron.

[0011] Furthermore, the four DNA single strands are four DNA single strands in equal molar ratio.

[0012] The present invention also provides a method for preparing the aforementioned DNA tetrahedron-amifostine complex, which comprises the following steps:

[0013] The tetrahedral framework nucleic acid and amifostine are added into a solvent and mixed for reaction to obtain the product.

[0014] Further,

[0015] The solvent is one or more of water, PBS, and DMSO;

[0016] and / or, the concentration of the tetrahedral framework nucleic acid is 200-250 nM;

[0017] And / or, the reaction temperature is 4 to 10°C;

[0018] And / or, the reaction time is 4 to 6 hours.

[0019] The present invention also provides the use of the aforementioned DNA tetrahedron-amifostine complex in preparing a drug for preventing radiation damage.

[0020] Furthermore, the drug is a drug for preventing damage to the blood system and / or organs during radiation;

[0021] And / or, the drug is a drug for preventing DNA damage.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] (1) The DNA tetrahedron-amifostine complex of the present invention can protect DNA from radiation damage;

[0024] (2) The DNA tetrahedron-amifostine complex of the present invention can improve the survival rate of an organism under lethal radiation;

[0025] (3) The DNA tetrahedron-amifostine complex of the present invention can achieve multi-organ protection under sublethal radiation.

[0026] In summary, the present invention provides a DNA tetrahedron-amifostine complex, which uses DNA tetrahedron as a carrier to carry out the coordinated delivery of amifostine. The DNA tetrahedron-amifostine complex of the present invention overcomes the problem of short metabolism time of amifostine, and enhances the radiation protection effect of amifostine, achieving a synergistic radiation protection effect. The complex of the present invention can protect DNA in a radiation environment, has a protective effect on multiple organs, and can improve the survival rate under radiation, has an excellent radiation protection effect, and has excellent application prospects in various fields.

[0027] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0028] The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Schematic diagram of the synthesis of the DNA tetrahedron-amifostine complex (nanosuit) of the present invention.

[0030] Figure 2 Capillary electrophoresis diagrams of ssDNAs, tFNAs and nanosuit.

[0031] Figure 3 The particle size results of tFNAs and composite nanosuit: A is the particle size distribution of tFNAs; B is the particle size distribution of composite nanosuit; C is the particle size statistical result of tFNAs and nanosuit.

[0032] Figure 4 Zeta potential results of tFNAs and composite nanosuit: A is the Zeta potential of tFNAs; B is the Zeta potential of composite nanosuit; C is the statistical result of Zeta potential of tFNAs and nanosuit.

[0033] Figure 5 Transmission electron microscopy images of tFNAs and composite nanosuit: A is a transmission electron microscopy image of tFNAs; B is a transmission electron microscopy image of composite nanosuit.

[0034] Figure 6 The effects of different concentrations of amifostine combined with tFNAs to form a complex nanosuit: A is the encapsulation rate of the complex obtained by combining different concentrations of amifostine with tFNAs; B is the fluorescence intensity of the complex obtained by combining different concentrations of amifostine with tFNAs.

[0035] Figure 7 The results of immunofluorescence and flow cytometry detection of the cell entry ability of ssDNAs, tFNAs and nanosuit: A is a representative image of immunofluorescence detection; B is the quantitative detection result of flow cytometry.

[0036] Figure 8The cell viability test of A31 cells pretreated with different materials under different irradiation doses and the representative graph of reactive oxygen fluorescence detection: A is the cell viability test of A31 cells pretreated with different materials under different irradiation doses (n=5); B is the representative graph of reactive oxygen fluorescence detection. Statistical analysis: ***P<0.001, ****P<0.0001.

[0037] Fig. 9 The expression of apoptotic proteins in A31 cells pretreated with different materials under irradiation: A is a representative image of Caspase-3 immunofluorescence detection; B is a representative image of Bcl-2 immunofluorescence detection; C is a representative image of Bax immunofluorescence detection.

[0038] Fig.10 Results of DNA damage in A31 cells pretreated with different materials under irradiation: A is a representative figure of comet assay; B is a statistical analysis of DNA (%) in comet assay (n=3); C is a representative figure of γ-H2A.X immunofluorescence assay; D is a representative figure of DNA PKcs immunofluorescence assay. Statistical analysis: *P<0.05, **P<0.01.

[0039] Fig.11 Schematic diagram of the design of the mouse radiation protection experiment.

[0040] Fig.12 The effects of different materials pretreatment on the survival rate and hematological parameters of BALB / C mice: A is the 30-day survival rate of BALB / C mice exposed to 6.5Gy after pretreatment with different materials using Kaplan-Meier survival analysis (n=10); BD are the hematological parameters of leukocytes (B), erythrocytes (C), and platelets (D) of BALB / C mice exposed to 5Gy after pretreatment with different materials at 1, 7, and 30 days (n=5), and the black and red dotted lines are the normal range of the parameters; E and F are the results of serum SOD activity (E) and MDA level (F) of BALB / C mice exposed to 5Gy after pretreatment with different materials at 7 days (n=5). Statistical analysis: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0041] Fig.13Effects of different materials pretreatment on the bone marrow of BALB / C mice: A is a representative image of H&E-stained femoral sections of BALB / C mice exposed to 5Gy after being pretreated with different materials for 7 days and 30 days; B is the detection of DNA content in bone marrow cells at 1, 7 and 30 days (n=5); C and D are the detection of SOD activity (C) and MDA level (D) of bone marrow cells at 7 days (n=5). Statistical analysis: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0042] Fig.14 Effects of different materials pretreatment on the thymus of BALB / C mice: A is a representative image of H&E-stained thymus sections of BALB / C mice exposed to 5Gy 7 days and 30 days after pretreatment with different materials; B and C are the results of thymus index (B) and spleen index (C) at 7 days (n=5). Statistical analysis: **P<0.01, ***P<0.001, ****P<0.0001.

[0043] Fig.15 The effects of different materials pretreatment on the spleen of BALB / C mice: A is a representative image of H&E-stained spleen sections of BALB / C mice exposed to 5Gy 7 days and 30 days after pretreatment with different materials; B and C are the results of SOD activity (B) and MDA level (C) in the spleen at 7 days (n=5). Statistical analysis: *P<0.05, **P<0.01, ****P<0.0001.

[0044] Fig.16 The effects of different materials pretreatment on the liver of BALB / C mice: A is a representative image of H&E-stained liver sections of BALB / C mice exposed to 5Gy 7 days and 30 days after pretreatment with different materials; B and C are the results of liver SOD activity (B) and MDA level (C) at 7 days (n=5). Statistical analysis: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0045] Fig.17 Results of liver function tests of BALB / C mice exposed to 5Gy after pretreatment with different materials 7 days later (n=5): A is the result of ALT; B is the result of AST. The black and red dotted lines are the normal range of the parameters. Statistical analysis: *P<0.05, **P<0.01, ****P<0.0001.

[0046] Fig.18The effects of different materials pretreatment on the testis of BALB / C mice: A is a representative picture of H&E-stained testis sections of BALB / C mice exposed to 5Gy after 7 days and 30 days of pretreatment with different materials; B and C are the results of testicular SOD activity (B) and MDA level (C) at 7 days (n=5). Statistical analysis: *P<0.05, **P<0.01, ****P<0.0001.

[0047] Fig.19 The effects of different materials pretreatment on the small intestine of BALB / C mice: A is a representative image of H&E-stained small intestinal sections of BALB / C mice exposed to 5Gy after 7 days and 30 days of pretreatment with different materials; B and C are the results of 7-day small intestinal SOD activity (B) and MDA level (C) detection (n=5). Statistical analysis: *P<0.05, ***P<0.001, ****P<0.0001.

[0048] Fig. 20 Effects of pretreatment with different materials on the lungs, kidneys, and ovaries of BALB / C mice: AC are representative images of H&E-stained lung sections (A), kidney sections (B), and ovary sections (C) of BALB / C mice pretreated with different materials and exposed to 5 Gy 7 days and 30 days after exposure. DETAILED DESCRIPTION

[0049] The raw materials and equipment used in the specific embodiments of the present invention are all known products and are obtained by purchasing commercially available products.

[0050] Example 1. Preparation and characterization of the DNA tetrahedron-amifostine complex of the present invention

[0051] The synthesis schematic diagram of the DNA tetrahedron-amifostine complex of the present invention is as follows Figure 1 shown.

[0052] 1. Preparation of DNA tetrahedrons (tFNAs)

[0053] Four DNA single strands (S1, S2, S3, S4) were dissolved in TM Buffer (10mM Tris-HCl, 50mM MgCl2, pH=8.0), the final concentration of the four DNA single strands was 1μM, and they were fully mixed, and then quickly heated to 95°C for 10 minutes, and then quickly cooled to 4°C and maintained for more than 20 minutes to obtain DNA tetrahedrons (tFNAs). The sequences of the four DNA single strands are shown in Table 1.

[0054] Table 1. Sequences of the four DNA strands

[0055]

[0056] 2. Preparation of DNA tetrahedron-amifostine complex (nanosuit)

[0057] Preparation of amifostine (AMF) solution: AMF was purchased from MCE and directly dissolved in ddH2O to obtain AMF solution.

[0058] Preparation of DNA tetrahedron-amifostine complex (nanosuit): The synthesized DNA tetrahedron (tFNAs) and the amifostine solution were evenly mixed to make the concentration of DNA tetrahedron in the solution 250nM and the concentration of amifostine 160μM (the molar ratio of DNA tetrahedron to amifostine in the solution was 1:640). The mixed solution was incubated at 4°C for 6 hours to obtain a DNA tetrahedron-amifostine complex (nanosuit).

[0059] 3. Characterization and results

[0060] tFNAs and nanosuit were characterized by capillary electrophoresis (HPCE), dynamic light scattering (DLS), transmission electron microscopy (TEM) and charge measurement.

[0061] Figure 2 Capillary electrophoresis (HPCE) of ssDNA, tFNAs and nanosuit. Figure 3 Particle size results for tFNAs and composite nanosuit. Figure 4 Zeta potential results for tFNAs and the composite nanosuit. Figure 5 Transmission electron microscopy images of tFNAs and the composite nanosuit.

[0062] HPCE showed that the size of tFNAs was about 180 bp, and dynamic light scattering analysis showed that the particle size of tFNAs was about 10 nm. Transmission electron microscopy results showed that tFNAs were evenly distributed and about 10 nm in size. Charge determination proved that tFNAs were negatively charged. From the above results, it can be seen that the synthesis of tFNAs was successful.

[0063] HPCE showed that the size of the composite nanosuit was about 180-200bp, the particle size was about 18nm, and the Zeta potential was -7.42mV. The nanosuit was observed using a transmission electron microscope, and a uniform characteristic structure was observed under the microscope. The results showed that the molecular size, particle size and Zeta potential of the composite nanosuit could be stably measured, and were slightly larger than those of the simple tFNAs, indicating the successful synthesis of the nanosuit.

[0064] Example 2: Preparation of DNA Tetrahedron-Amifostine Complex of the Present Invention

[0065] 1. Preparation of DNA tetrahedrons (tFNAs)

[0066] The preparation method of DNA tetrahedron (tFNAs) is the same as that in Example 1.

[0067] 2. Preparation of DNA tetrahedron-amifostine complex (nanosuit)

[0068] The preparation method of nanosuit was the same as that of Example 1, except that the concentration of DNA tetrahedron was 250 nM, and the concentration of amifostine was changed from 160 μM to 40 μM, and the molar ratio of DNA tetrahedron to amifostine in the solution was 1:160.

[0069] Example 3: Preparation of DNA Tetrahedron-Amifostine Complex of the Present Invention

[0070] 1. Preparation of DNA tetrahedrons (tFNAs)

[0071] The preparation method of DNA tetrahedron (tFNAs) is the same as that in Example 1.

[0072] 2. Preparation of DNA tetrahedron-amifostine complex (nanosuit)

[0073] The preparation method of nanosuit was the same as that of Example 1, except that the concentration of DNA tetrahedron was 250 nM, and the concentration of amifostine was changed from 160 μM to 80 μM, and the molar ratio of DNA tetrahedron to amifostine in the solution was 1:320.

[0074] Example 4: Preparation of the DNA Tetrahedron-Amifostine Complex of the Present Invention

[0075] 1. Preparation of DNA tetrahedrons (tFNAs)

[0076] The preparation method of DNA tetrahedron (tFNAs) is the same as that in Example 1.

[0077] 2. Preparation of DNA tetrahedron-amifostine complex (nanosuit)

[0078] The preparation method of nanosuit was the same as that of Example 1, except that the concentration of DNA tetrahedron was 250 nM, and the concentration of amifostine was changed from 160 μM to 120 μM, and the molar ratio of DNA tetrahedron to amifostine in the solution was 1:480.

[0079] Example 5: Preparation of the DNA Tetrahedron-Amifostine Complex of the Present Invention

[0080] 1. Preparation of DNA tetrahedrons (tFNAs)

[0081] The preparation method of DNA tetrahedron (tFNAs) is the same as that in Example 1.

[0082] 2. Preparation of DNA tetrahedron-amifostine complex (nanosuit)

[0083] The preparation method of nanosuit was the same as that of Example 1, except that the concentration of DNA tetrahedron was 250 nM, and the concentration of amifostine was changed from 160 μM to 320 μM, and the molar ratio of DNA tetrahedron to amifostine in the solution was 1:1280.

[0084] The encapsulation efficiency of the composite nanosuit prepared in Examples 1 to 5 was calculated based on the HPLC results. Figure 6 As shown in A: With the increase of AMF concentration, the encapsulation rate increases, reaches a peak and then decreases, and the encapsulation rate is the highest when the AMF concentration is 160 μM. In order to further verify the successful synthesis of the composite nanosuit of Examples 1 to 5, the fluorescence spectra of Gel-red and Hochest 33342 were measured by a fluorescence spectrophotometer. Figure 6 B shows that as the AMF concentration increases to 160 μM, the fluorescence intensity of Gel-red and Hochest 33342 both decreases, proving the groove binding and intercalation binding of AMF to tFNAs. In summary, the AMF concentration of 160 μM was selected for subsequent studies.

[0085] The beneficial effects of the present invention are demonstrated below through specific experimental examples.

[0086] Experimental Example 1: Cell Experiment

[0087] 1. Cell Type

[0088] Mouse embryonic fibroblasts (BALB / 3T3 clone A31 cells).

[0089] 2. Related testing methods and results

[0090] (1) Detection of nanosuit entering A31 cells

[0091] 250nM ssDNAs, 250nM tFNAs and 250nM nanosuits with Cy5 (red fluorescent group) were incubated with equal amount of A31 cells for 6h and 12h respectively, without irradiation, and then immunofluorescence and flow cytometry were used to detect the cellular entry of each sample. Among them, ssDNAs with Cy5 were grafted with Cy5 on the S1 single strand; tFNAs with Cy5 were prepared according to the method described in Example 1, except that the S1 single strand was replaced with the aforementioned S1 grafted with Cy5; nanosuits with Cy5 were prepared according to the method described in Example 1, except that tFNAs were replaced with the aforementioned tFNAs with Cy5.

[0092] Immunofluorescence: cells were fixed with 4% paraformaldehyde, phalloidin (green) and DAPI (blue) were used to stain the cytoskeleton and nucleus, respectively. Finally, confocal microscopy was used for observation and image collection.

[0093] Flow cytometry: Cells were collected and the uptake of materials by cells was detected using a flow cytometer. The cellular uptake data was analyzed using FlowJov10.0.7 software.

[0094] Immunofluorescence and flow cytometry results Figure 7 As shown, the results showed that the cell penetration rate of nanosuit reached the highest level at 12 hours, which was significantly stronger than that of ssDNAs and tFNAs.

[0095] (2) Detection of the effect of nanosuit on the viability of radiation-damaged cells

[0096] The cell viability test was used to evaluate the effect of nanosuit on promoting cell survival under different radiation doses (2Gy, 4Gy, 6Gy, 8Gy and 10Gy).

[0097] Experimental groups:

[0098] Model group (IR): Mouse embryonic fibroblasts were cultured for 36 hours and then irradiated with different doses. Corresponding tests were performed 12 hours after irradiation.

[0099] Experimental group (tFNAs, AMF and nanosuit): After culturing mouse embryonic fibroblasts for 24 hours, 250nM tFNAs, 160μM AMF and 250nM nanosuit were added for pretreatment for 12 hours, and then irradiated with different doses. Corresponding tests were performed 12 hours after irradiation. tFNAs and nanosuit were prepared by the method of Example 1.

[0100] CCK8 reagent was used for detection, and the absorbance was detected by ELISA instrument to calculate the cell activity results of each group.

[0101] Depend on Figure 8 A shows that: the activity of cells decreased significantly after irradiation, and the decrease in cell activity became more serious as the irradiation dose increased; tFNAs pretreatment alone could not protect the cells; AMF pretreatment alone could rescue radiation-induced cell death to a certain extent; but compared with AMF, nanosuit's effect on rescuing radiation-induced cell death was significantly improved, indicating that nanosuit has a strong radiation protection effect on cells.

[0102] (3) Detection of the anti-oxidative stress effect of nanosuit

[0103] Under the influence of radiation, ROS will increase sharply, causing cellular oxidative stress damage. The anti-oxidative stress effect of nanosuit was tested as follows:

[0104] Control group (Control): mouse embryonic fibroblasts were cultured for 48 hours.

[0105] Model group (IR): Mouse embryonic fibroblasts were cultured for 36 hours and then irradiated with 6 Gy. Corresponding tests were performed 12 hours after irradiation.

[0106] Experimental groups (tFNAs, AMF and nanosuit): After culturing mouse embryonic fibroblasts for 24 hours, 250nM tFNAs, 160μM AMF and 250nM nanosuit were added for pretreatment for 12 hours, and then irradiated with 6Gy. Corresponding tests were performed 12 hours after irradiation. They were tFNAs+IR, AMF+IR and nanosuit+IR. tFNAs and nanosuit were prepared by the method of Example 1.

[0107] The ROS detection kit was used to detect the intracellular ROS level: the cells were loaded with DCFH-DA probe and incubated at 37°C for 20 minutes. Finally, confocal laser scanning microscopy was used for observation and image collection.

[0108] Active oxygen test results ( Figure 8 B) shows that nanosuit pretreatment reduced the level of intracellular reactive oxygen species induced by IR to some extent, indicating that the radiation protection ability of nanosuit is related to its effective free radical scavenging activity.

[0109] (4) Detection of the anti-apoptotic effect of nanosuit

[0110] Apoptosis is the main mechanism of large-scale cell loss in radiosensitive tissues. The specific experimental groups and methods are as shown above (3). After reaching the time point, the samples were fixed with 4% paraformaldehyde solution for 15-20 minutes, perforated with 0.5% TritonX-100 for 10 minutes, blocked with 5% sheep serum for 1 hour, and Caspase-3 (1:200), Bcl-2 (1:200), and Bax (1:350) primary antibody dilutions were added dropwise, and incubated at 4°C overnight; the next day, the samples were rewarmed for 30 minutes, incubated with fluorescent secondary antibodies in the dark for 1 hour, stained with phalloidin for 30 minutes, stained with DAPI for 10 minutes, and sealed, and images were collected using a confocal laser microscope.

[0111] The fluorescence intensity analysis results of Caspase-3, Bcl-2 and Bax showed that ( Fig. 9A-9C), nanosuit pretreatment significantly reduced the fluorescence intensity of Caspase-3 and Bax, and enhanced the expression of Bcl-2, indicating that nanosuit inhibited radiation-induced cell apoptosis by regulating the expression level of apoptotic proteins.

[0112] (5) Detection of the radiation protection effect of nanosuit on intracellular DNA

[0113] The hallmark of radiation-induced cell damage is uncontrolled DNA breakage. Therefore, the present invention tests the radiation protection effect of nanosuit on intracellular DNA. The specific experimental grouping and methods are as shown in (3) above. After reaching the time point, cells were collected for comet assay detection: ① Preparation of gel plates: first layer of gel (180 μL of 1% normal melting point agarose NMA preheated to 45°C was dripped onto the frosted surface of the same preheated slide, quickly covered with a clean coverslip, and left at 4°C for 10 min to solidify); second layer of gel (100 μL of cell suspension and 150 μL of 0.8% low melting point agarose LMA were mixed at 37°C, then the coverslip was gently removed, the LMA containing cells was dripped onto the first layer of gel plate, and immediately covered with a clean coverslip, and solidified at 4°C for 20 min); third layer of gel (remove the slide, quickly drip 100 μL of 0.5% low melting point agarose at 37°C onto the second layer of agarose, cover with a coverslip, and solidify at 4°C for 20 min). ② Lysis: Remove the slide and immerse the slide in the newly prepared 4℃ lysis solution for 2h. ③ Unwinding: Pour the newly prepared electrophoresis buffer into the electrophoresis tank, cover the slide by 0.25cm, cover the lid, and place it in the electrophoresis buffer for 30min. ④ Electrophoresis: At room temperature, adjust the liquid level of the buffer, and electrophores at 300mA and 25V for 15min. ⑤ Washing and staining: After electrophoresis, place the slide in a flat dish and rinse with neutralization buffer for 30min. After neutralization, stain the slide with 50-100μL acridine orange solution (EB) for 10min and rinse with distilled water for more than 2 times. ⑥ Result observation: Use fluorescence microscopy. Under certain conditions, the distribution of DNA migration distance (comet tail length) and DNA content (fluorescence intensity) is linearly correlated with the degree of DNA damage.

[0114] Comet assay detection images Fig.10 As shown in A, the quantitative analysis results of DNA tail moment are as follows Fig.10As shown in B. The quantitative analysis results of DNA tail moment showed that the protective effect of pretreatment with AMF (33.27±7.14%) or tFNAs (50.16±4.73%) on DNA damage was not significantly different from that of the model group, indicating that pretreatment with AMF or tFNAs alone could hardly avoid DNA damage in cells. However, the DNA of cells pretreated with nanosuit (9.35±3.23%) recovered to a level comparable to that of non-irradiated DNA. This indicates that pretreatment with nanosuit has a significant protective effect on cellular DNA damage caused by radiation, and has a synergistic effect compared with the use of AMF or tFNAs alone.

[0115] γ-H2A.X, as a product of DNA damage formation, is another key biomarker of DNA damage. In this study, the γ-H2A.X level in the nanosuit pretreatment group was lower than that in other groups ( Fig.10 C). In addition, DNAPKcs was detected by immunofluorescence to observe the extent of DNA damage. The intensity of DNA PKcs in the nanosuit pretreatment group was weaker than that in other groups ( Fig.10 D), indicating that the nanosuit has a strong radiation protection effect on intracellular DNA.

[0116] The results of this experimental example show that the nanosuit of the present invention can protect DNA from radiation damage by exerting anti-oxidative stress and anti-apoptosis effects, thereby promoting cell survival. Compared with the use of AMF or tFNAs alone, the nanosuit exerts a synergistic radiation protection effect.

[0117] Experimental Example 2: In vivo experiment

[0118] 1. Experimental Animals

[0119] BALB / C mice (18-22 g, 75 males and 75 females).

[0120] 2. Experimental groups (5 groups*30 mice)

[0121] A: Control: no medication and no irradiation;

[0122] B: IR group: no medication, only irradiation

[0123] C: tFNAs+IR group: irradiation followed by administration of tFNAs;

[0124] D: AMF+IR group: irradiation followed by AMF administration;

[0125] E: nanosuit+IR group: irradiation followed by nanosuit administration;

[0126] The drug-treated groups were given 10μM tFNAs, 6.4mM AMF or 10μM nanosuit, each intravenously injected at 200μL / time / day for 3 consecutive days, followed by whole-body irradiation at 6.5Gy / 5Gy. Fig.11 tFNAs and nanosuit were prepared by the method of Example 1.

[0127] 3. Related testing methods and results

[0128] (1) Detecting the effect of nanosuit on the survival of mice exposed to lethal radiation doses

[0129] After the BALB / C mice were treated in the corresponding groups, they were given 6.5Gy whole-body irradiation and observed for 30 days. The 30-day survival of the mice was detected by Kaplan-Meier survival analysis. The results showed that the 30-day survival rate after tFNAs pretreatment was 0%, and the average survival time was 9.8 days; the 30-day survival rate after AMF pretreatment was 20%, and the average survival time was 15.4 days; the 30-day survival rate after nanosuit pretreatment was 70%, and the average survival time was 25.1 days ( Fig.12 A). Therefore, the nanosuit effectively increased the survival rate of mice under lethal radiation doses and reduced radiation toxicity.

[0130] (2) Detecting the effect of nanosuit on the blood system of mice under sublethal radiation doses

[0131] After the BALB / C mice were treated according to the corresponding groups, they were given 5Gy whole-body irradiation. After reaching the 1st, 7th and 30th day time points, the mice were anesthetized and ocular venous blood was collected for hematological testing. The hematological parameters of white blood cells, red blood cells and platelets on days 1, 7 and 30 showed that the blood system damage of mice in the nanosuit pretreatment group was effectively alleviated, and the effect was significantly better than the use of AMF or tFNAs alone, playing a synergistic role: the white blood cell count decreased most significantly on the 7th day after irradiation, while the white blood cell count of mice in the nanosuit pretreatment group was 2-3 times that of the IR group. On the 30th day after irradiation, the nanosuit pretreatment group almost recovered to a healthy level. At the same time, the change trend of red blood cells and platelets was consistent with that of white blood cells ( Fig.12 B-12D).

[0132] (3) Detecting the effect of nanosuit on the bone marrow of mice at sublethal radiation doses

[0133] BALB / C mice were treated in the corresponding groups and given 5Gy whole-body irradiation. The mice were killed at 1, 7, and 30 days. The femurs were decalcified, embedded in paraffin, and sliced ​​(4μm). The tissue sections were stained with hematoxylin-eosin (H&E). Representative images of H&E-stained femoral sections at 7 and 30 days showed that the bone marrow cells in the femoral medullary cavity almost disappeared on the 7th day of irradiation, and the medullary cavity was filled with red blood cells; while nanosuit pretreatment significantly reduced the bone marrow suppression and bleeding caused by radiation. By 30 days, the cell composition in the medullary cavity almost returned to the healthy level close to that of the control group ( Fig.13 A).

[0134] Bone marrow cells were flushed from the femur, separated using a BM-MNCs extraction kit, and the DNA content of bone marrow cells was detected. The results of bone marrow cell DNA content on days 1, 7, and 30 showed that the relative content of total bone marrow cell DNA decreased significantly to 0.461±0.103 and 0.446±0.071 on days 1 and 7 after radiation compared with healthy mice. In contrast, the relative content of total DNA in the nanosuit pretreatment group had a higher level of 0.742±0.066 on day 1, and rose to 0.905±0.103 on day 7, almost returning to the healthy value ( Fig.13 B), the effect was also significantly better than AMF or tFNAs alone.

[0135] (4) Detecting the effects of nanosuit on important organs of mice under sublethal radiation doses

[0136] BALB / C mice were treated according to the corresponding groups and given 5Gy whole-body irradiation. The mice were killed at 1, 7, and 30 days. Multiple organs (thymus, spleen, liver, testis, small intestine, lung, kidney, and ovary) were embedded in paraffin and sliced ​​(4 μm). The tissue sections were stained with hematoxylin-eosin (H&E). Representative images of multiple organs stained with H&E at 7 and 30 days are shown ( Figures 14 to 20): The thymus and spleen showed obvious atrophy and structural damage under irradiation, while nanosuit pretreatment could significantly reduce spleen atrophy and thymus atrophy in mice; the liver showed hepatocyte degeneration and necrosis, nuclear enrichment and fragmentation under irradiation, while nanosuit pretreatment improved hepatocyte morphology, and ALT and AST related liver function tests were consistent with pathological results; the testis is one of the most sensitive tissues to radiation, and the spermatogenic cells of irradiated mice showed obvious degeneration and reduction in number. After nanosuit pretreatment, the morphology of the testis basically returned to normal; the small intestine is also one of the most sensitive organs to radiation. The small intestine of irradiated mice showed obvious pathological histological damage, including extensive villous atrophy, loose arrangement, intestinal mucosal damage, and reduced number of intestinal crypt cells, but in the nanosuit pretreatment group, the small intestinal villi were long and intact, and the crypts were normal. Other organs including lungs, kidneys and ovaries also showed moderate lesions under radiation, and corresponding improvements were found in mice pretreated with nanosuit. These pathological results show that nanosuit promotes the survival of important organs under irradiation. Compared with the use of AMF or tFNAs alone, nanosuit exerts a synergistic radiation protection effect.

[0137] (5) Detection of changes in SOD activity and MDA levels in mice by nanosuit

[0138] In order to understand the protective mechanism of nanosuit in vivo, the present invention detected the SOD activity and MDA level in the serum, bone marrow cells, spleen, liver, testis and small intestine of mice on the 7th day after 5Gy irradiation. The results showed that ( Fig.12 E, 12F, 13C, 13D, 14B, 14C, 15B, 15C, 16B, 16C, 18B, 18C, 19B, and 19C) The SOD activity of the irradiation group alone was reduced, and the MDA level was increased; in contrast, the nanosuit pretreatment significantly slowed down these trends. Therefore, the nanosuit achieves free radical scavenging by restoring SOD activity and inhibiting MDA levels, thereby protecting tissues and organs.

[0139] The results of this experimental example show that the nanosuit of the present invention can achieve multi-organ protection under sublethal radiation and promote the survival of the body under lethal radiation by restoring SOD activity and inhibiting MDA levels. Consistent with the cell experiment, compared with the use of AMF or tFNAs alone, the nanosuit exerts a synergistic radiation protection effect.

[0140] In summary, the present invention provides a DNA tetrahedron-amifostine complex, which uses DNA tetrahedron as a carrier to carry out the coordinated delivery of amifostine. The DNA tetrahedron-amifostine complex of the present invention overcomes the problem of short metabolism time of amifostine, and enhances the radiation protection effect of amifostine, achieving a synergistic radiation protection effect. The complex of the present invention can protect DNA in a radiation environment, has a protective effect on multiple organs, and can improve the survival rate under radiation, has an excellent radiation protection effect, and has excellent application prospects in various fields.

Claims

1. Use of a DNA tetrahedron-amifostine complex in the preparation of a drug for preventing radiation damage; the drug is a drug for preventing DNA damage; The DNA tetrahedron-amifostine complex is a complex formed by mixing a DNA tetrahedron framework nucleic acid and amifostine; the DNA tetrahedron is synthesized by self-assembly of four DNA single strands; the sequences of the four DNA single strands are shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, and SEQ ID NO.4 respectively; The molar ratio of the DNA tetrahedron to amifostine is 1:

640.

2. The use according to claim 1, characterized in that: The method for synthesizing the DNA tetrahedron comprises the following steps: adding four DNA single strands into TM buffer, maintaining at 95° C. for 10 min, and maintaining at 4° C. for more than 20 min, to obtain the tetrahedron.

3. The use according to claim 2, characterized in that: The four DNA single strands are four DNA single strands in equal molar ratio.

4. The use according to any one of claims 1 to 3, characterized in that: The preparation method of the DNA tetrahedron-amifostine complex comprises the following steps: The tetrahedral framework nucleic acid and amifostine are added into a solvent and mixed for reaction to obtain the product.

5. The use according to claim 4, characterized in that: The solvent is one or more of water, PBS, and DMSO; And / or, the concentration of the tetrahedral framework nucleic acid is 200-250 nM; And / or, the reaction temperature is 4-10°C; And / or, the reaction time is 4 to 6 hours.

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