Integrated Nanoparticle Platform of Gold Adjuvant Combined with XPO1 Inhibitor and ATR Inhibitor, and Its Preparation and Application

Abstract

An integrated nanoplatform of gold adjuvant combined with XPO1 inhibitor and ATR inhibitor, with the general formula [AA@G], and its preparation method: First, add TCEP to human serum albumin solution, and then add ATR inhibitor to synthesize HSA-ATR inhibitor. Then add chloroauric acid solution to HEPES buffer solution, stir under heating conditions until the solution turns purplish red to synthesize nano-gold adjuvant. After fully mixing AP, NH2-PEG2000-SH and HEPES, add chloroauric acid solution and continue to stir until the solution turns from light yellow to colorless to synthesize AP@G. Finally, mix the HSA-ATR inhibitor solution, AP@G solution and nano-gold adjuvant solution to form AA@G. The nanoplatform of the present invention has excellent drug-loading capacity, can unify the pharmacokinetics of different drugs, and ultimately plays a role in enhancing the sensitivity of tumor treatment.

Description

Technical Field

[0001] The present invention belongs to the technical field of bioengineering, and particularly relates to an integrated nano-platform of gold adjuvant combined with XPO1 inhibitor and ATR inhibitor, and its preparation and application. Background Art

[0002] Cancer poses a major threat to human health. In the past few years, many treatment methods have been approved for the treatment of cancer. However, many methods can only provide limited survival benefits. The treatment of cancer remains challenging because there are few drugs targeting key pathogenic factors. Scientists have proposed that combination therapy can help make up for treatment deficiencies and improve the response to drugs. Therefore, there is an urgent need for effective drug combinations to improve the treatment effect of liver cancer. However, traditional drug combination therapies are often affected by the respective pharmacokinetic characteristics of each drug, and their biodistributions are different, resulting in low treatment efficiency.

[0003] Nuclear localization signal (NLS) and nuclear export signal (NES) are the core features of proteins that control nucleocytoplasmic transport. Exportin 1 (XPO1) is a eukaryotic nucleocytoplasmic transporter that directly binds to proteins containing NES and translocates them to the cytoplasm. XPO1 is overexpressed in many cancers and is an effective drug target. However, due to tumor heterogeneity and rapid adaptation to targeted therapy, tumor recurrence and metastasis often occur in patients. Akira Inoue et al. demonstrated that the response of tumor cells to DNA damage induced by XPO1 inhibitors can be restored because cell cycle arrest helps DNA damage repair. Therefore, the combined application of XPO1 inhibitors and other DNA damage response drugs targeting the cell cycle can improve the treatment effect of liver cancer. Apoptin encoded by the chicken anemia virus (CAV) VP3 gene can selectively induce apoptosis. Apoptin96-110 contains a nuclear export signal (NES), enabling it to compete with XPO1, bind to proteins containing NES and inhibit their transport, and is an XPO1 inhibitor. The DNA damage response (DDR) signaling pathway depends on the activation of ataxia-telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3 related (ATR), which is crucial for DNA damage repair. Therefore, inhibiting ATM or ATR is a promising cancer treatment strategy. Recently, combination therapy with cell cycle checkpoint inhibitors, especially ATR inhibitors, is being clinically studied for many cancers. Ideally, inhibiting ATR will abrogate homologous recombination (HR) repair. Therefore, combining Apoptin96-110 with ATR inhibitors is likely to achieve good tumor treatment effects.

[0004] To break the limitations of combination therapy, a nanotechnology strategy has emerged, providing new opportunities for drug combination cocktails by using nanoparticles as adjuvants to achieve unified pharmacokinetics of drug carriers. Gold has been used as an adjuvant and carrier in nanomedicine due to its therapeutic effects on many diseases. According to previous research results, gold nanoparticle-based nanodrugs can reduce toxicity, enhance immunogenicity, and are widely used in cancer treatment because it shows deep tumor penetration ability by loosening the extracellular matrix of tumor cells and provides storage stability. Summary of the Invention

[0005] To overcome the above-mentioned disadvantages of the prior art, the purpose of the present invention is to provide an integrated nanoplatform of a gold adjuvant combined with an XPO1 inhibitor and an ATR inhibitor, which can block the cell cycle, promote DNA damage of tumor cells, inhibit DNA damage repair of tumor cells, and promote apoptosis of tumor cells, thereby improving the sensitivity of tumor treatment.

[0006] To achieve the above purpose, the technical solution adopted by the present invention is:

[0007] An integrated nanoplatform of a gold adjuvant combined with an XPO1 inhibitor and an ATR inhibitor, with the general formula [AA@G], where the first A is an XPO1 inhibitor, which is the 96-110 functional fragment of the polypeptide Apoptin, and Ac and NH2 are modified at the head and tail. To modify NH2, amino acid C is also added to the tail, and the final amino acid sequence is Ac-RVSELKESLITTTPSC-NH2. The second A is an ATR inhibitor, and @G is a gold adjuvant. The gold adjuvant serves as a nanocarrier to carry the XPO1 inhibitor and the ATR inhibitor.

[0008] In one embodiment, the 96-110 functional fragment of Apoptin has an NES sequence and can inhibit the transport of XPO1 to NES-related proteins, thereby inhibiting the action of XPO1.

[0009] In one embodiment, the ATR inhibitor can inhibit the DNA damage repair function of ATR.

[0010] In one embodiment, the gold adjuvant has tissue and cell penetration ability and can carry the XPO1 inhibitor and the ATR inhibitor into the cell interior to play a role together.

[0011] The present invention also provides a preparation method of the integrated nanoplatform of the gold adjuvant combined with the XPO1 inhibitor and the ATR inhibitor, including the following steps:

[0012] Step 1, add TCEP to the human serum albumin solution, and then add the ATR inhibitor to synthesize HSA-ATR inhibitor;

[0013] Step 2: Add chloroauric acid solution to HEPES buffer solution, stir under heating conditions until the solution turns purplish red to synthesize nano-gold adjuvant.

[0014] Step 3: After thoroughly mixing AP, NH2-PEG2000-SH and HEPES, add chloroauric acid solution and continuously stir until the solution turns from light yellow to colorless to synthesize AP@G.

[0015] Step 4: Mix HSA-ATR inhibitor, AP@G and nano-gold adjuvant to form AA@G.

[0016] In one embodiment, in Step 1, the mass-volume ratio of TCEP to HSA is 1:1 - 1:5, and the molar ratio of HSA to ATR inhibitor is 1:1 - 1:5. In Step 2, the molar ratio of chloroauric acid to HEPES is 1:1 - 1:5. In Step 3, the molar ratio of AP, NH2-PEG2000-SH, HEPES and chloroauric acid is 2:2:1:5 - 3:3:1:5. In Step 4, the molar ratio of HSA-ATR inhibitor, AP@G and gold adjuvant is 1:1:1 - 1:3:3.

[0017] The integrated nano-platform of the gold adjuvant combined with XPO1 inhibitor and ATR inhibitor in the present invention can be used to prepare drugs for blocking cell cycle, promoting DNA damage of tumor cells, inhibiting DNA damage repair of tumor cells, promoting apoptosis of tumor cells, and improving the sensitivity of tumor treatment.

[0018] Compared with the prior art, the nano-platform obtained in the present invention blocks cell cycle, promotes DNA damage of tumor cells, inhibits DNA damage repair of tumor cells, promotes apoptosis of tumor cells, and improves the sensitivity of tumor treatment. The nano-platform prepared by the preparation method of the present invention has the advantages of excellent drug loading capacity and unified pharmacokinetics. Description of the Drawings

[0019] Figure 1 It is a schematic diagram of the synthesis of AA@G.

[0020] Figure 2 It is the preparation and characterization of AA@G. Among them:

[0021] A is the transmission electron microscopy image (TEM) of AA@G;

[0022] B is the elemental analysis result of AA@G;

[0023] C is the photo of AA@G;

[0024] D is the particle size of AA@G

[0025] E and F are the changes in Zeta potential during the synthesis of AA@G;

[0026] G is the UV-Vis absorption spectrum;

[0027] H is the FT-IR absorption spectrum;

[0028] I is the hydrodynamic diameter distribution of AA@G;

[0029] J is the drug release ability of AA@G.

[0030] Figure 3 are the performance detections of AA@G. Among them:

[0031] A is the targeted fluorescence microscopy detection of AA@G;

[0032] B is the targeted flow cytometry detection of AA@G;

[0033] C is the detection of the tissue penetration ability of AA@G;

[0034] D and E are the in vivo metabolism detections of AA@G.

[0035] Figure 4 are the in vitro treatment effects of AA@G. Among them: A is the CCK8 result;

[0036] B and C are the analysis of the drug synergy effect;

[0037] D is the detection result of cell apoptosis;

[0038] E is the analysis of the cell cycle result;

[0039] F and G are the statistical analysis results of D and E;

[0040] H is the detection of the treatment effect of AA@G in the 3D tumor tissue model. Figure 5 is the analysis of the action mechanism of AA@G. Among them: A is the schematic diagram of the action mechanism of AA@G;

[0041] B is the WB result;

[0042] C is the immunofluorescence result.

[0043] Figure 6 is the in vivo action effect of AA@G. Among them: A is the flow chart of the in vivo experiment of AA@G

[0044] B is the statistical analysis of the tumor volume of mice after treatment with different treatment groups;

[0045] C is the statistical analysis of the body weight of mice after treatment with different treatment groups;

[0046] D is the statistical analysis of the tumor weight of mice after treatment with different treatment groups;

[0047] E is the tumor volume of mice after treatment in different treatment groups;

[0048] F is the HE staining and Tunel staining of tumor tissues after treatment in different treatment groups;

[0049] G is the change in protein expression level verified by immunohistochemistry after treatment in different treatment groups. Specific implementation manners

[0050] The implementation manners of the present invention will be described in detail below with reference to the accompanying drawings and embodiments.

[0051] Nanotechnology provides new opportunities for drug combination cocktails by using nanoparticles as adjuvants to achieve unified pharmacokinetics of drug carriers. Gold has been used as an adjuvant and carrier in nanomedicine due to its therapeutic effects on many diseases. According to previous research results, gold nanoparticle-based nanodrugs can reduce toxicity, enhance immunogenicity, and are widely used in cancer treatment because it shows deep tumor penetration ability by loosening the extracellular matrix of tumor cells and provides storage stability.

[0052] Nuclear localization signals (NLSs) and nuclear export signals (NESs) are core features of proteins that control nucleocytoplasmic transport. Exportin 1 (XPO1) is a eukaryotic nucleocytoplasmic transporter that directly binds to NES-containing proteins and translocates them to the cytoplasm. XPO1 is overexpressed in a variety of cancers and is a valid drug target. However, due to tumor heterogeneity and rapid adaptation to targeted therapies, tumor recurrence and metastasis often occur in patients. Akira Inoue et al. demonstrated that the response of tumor cells to XPO1 inhibitor-induced DNA damage can be restored because cell cycle arrest contributes to DNA damage repair. Therefore, the combination of XPO1 inhibitors with other DNA damage response drugs targeting the cell cycle can improve the therapeutic efficacy of liver cancer. Apoptin, encoded by the chicken anemia virus (CAV) VP3 gene, can selectively induce apoptosis. Apoptin96-110 contains a nuclear export signal (NES), enabling it to compete with XPO1, bind to NES-containing proteins, and inhibit their transport, making it an XPO1 inhibitor. The DNA damage response (DDR) signaling pathway depends on the activation of ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3-related (ATR), which is crucial for DNA damage repair. Therefore, inhibiting ATM or ATR is a promising cancer treatment strategy. Recently, combination therapies with cell cycle checkpoint inhibitors, especially ATR inhibitors, are being clinically investigated for many cancers. Ideally, inhibiting ATR would abrogate homologous recombination (HR) repair. Therefore, combining Apoptin96-110 with an ATR inhibitor is likely to achieve good tumor treatment effects.

[0053] This invention attempts to synthesize stable size-controlled nano-gold adjuvant particles and, leveraging their powerful drug-loading capacity, load XPO1 inhibitors and ATR inhibitors, ultimately constructing a composite nanoparticle to provide experimental materials for the next step of tumor treatment sensitization research.

[0054] Specifically, this invention provides an integrated nano-platform of a gold adjuvant combined with an XPO1 inhibitor and an ATR inhibitor, with the general formula [AA@G], where the sequence of A is RVSELKESLITTTPSC. Among them, the first A is an XPO1 inhibitor, which is the 96-110 functional fragment of the polypeptide Apoptin, and Ac and NH2 are modified at the beginning and end. To modify NH2, the amino acid C is also added at the end, so its final amino acid sequence is Ac-RVSELKESLITTTPSC-NH2. The second A is an ATR inhibitor, and @G is a gold adjuvant. The gold adjuvant serves as a nanocarrier to carry the XPO1 inhibitor and the ATR inhibitor.

[0055] In the present invention, "integrated" means that the gold adjuvant carrying the XPO1 inhibitor and the ATR inhibitor forms a unified whole. Here, "carrying" means that the gold adjuvant, as a nanocarrier, can carry the XPO1 inhibitor, the ATR inhibitor and other drugs.

[0056] The present invention also discloses a specific preparation process thereof, and the steps are as follows: First, add 1 mg of TCEP to 0.5 mL of human serum albumin (HSA, 10 mg / mL) solution. Then, add the ATR inhibitor (1 mM) to the above HSA solution. Finally, use 5 mL of ultrapure water as a dilution solution for dilution to synthesize HSA-ATR inhibitor. Then, add 0.5 mL of chloroauric acid solution (10 mM) to 4.5 mL of 50 mM HEPES buffer solution, stir under heating conditions until the solution turns purple-red to synthesize nano gold adjuvant. Then, after fully mixing 2.5 mg of AP, 2 mg of NH2-PEG2000-SH and 4.5 mL of HEPES (50 mM), add 0.5 mL of chloroauric acid solution (10 mM) and continuously stir until the solution turns from light yellow to colorless to synthesize AP@G. Finally, mix 2 mL of HSA-ATR inhibitor solution, 2 mL of AP@G solution and 4 mL of nano gold adjuvant solution for 10 minutes to form AA@G, as Figure 1 shown.

[0057] The application of the integrated nano-platform of the gold adjuvant combined with the XPO1 inhibitor and the ATR inhibitor blocks the cell cycle, promotes DNA damage of tumor cells, inhibits DNA damage repair of tumor cells, promotes apoptosis of tumor cells, and improves the sensitivity of tumor treatment. The following verification tests are carried out on AA@G:

[0058] 1. Synthesis of AA@G

[0059] 1) HSA-ART inhibitor

[0060] (1) Prepare 0.5 mL of 10 mg / mL HSA solution and add 1 mg of TECP, and ultrasonicate for 5 min.

[0061] (2) Add the ATR inhibitor to the above HSA solution to make its final concentration 1 mM.

[0062] (3) Dilute the above solution to 5 mL (dilute 10 times) and ultrasonicate for 10 min.

[0063] 2) Preparation of nano gold adjuvant solution

[0064] (1) Add 0.5 mL of 10 mM chloroauric acid solution to 4.5 mL of 50 mM HEPES solution, heat and stir until the solution turns purple-red, indicating that the gold core is successfully prepared

[0065] 3) Preparation of AP@G

[0066] (1) Add 2.5 mg of AP and 2 mg of NH2-PEG2000-SH to 4.5 mL of 50 mM HEPES. After mixing, add 0.5 mL of 10 mM chloroauric acid solution and stir until the solution changes from light yellow to colorless, indicating the formation of AP@G

[0067] 4) Preparation of AA@G

[0068] (1) Add 2 mL of HSA-ART inhibitor and 2 mL of gold-peptide polymer to 4 mL of AP@G solution, and stir for 10 min to complete the preparation of AA@G. (Final concentration of ATR inhibitor: 200 μM, polypeptide concentration: 0.2 mg / mL)

[0069] 2. Characterization of AA@G

[0070] 1) Preparation of electron microscope samples

[0071] (1) Take a 1.5 mL Eppendorf tube, take 1 mL of the prepared AA@G solution and place it in the centrifuge tube, and use a vortex mixer or ultrasonic device to disperse AA@G evenly;

[0072] (2) Take another 1.5 mL Eppendorf tube, add 100 μL of ethanol solution, then inject 1 μL of the original solution, and ultrasonically disperse for 3 minutes to disperse AA@G evenly;

[0073] (3) Use forceps to take out the carbon film copper grid and place it on the experimental table with the front side facing up;

[0074] (4) Use a 10 μL pipette to take the dispersed nanoparticle solution and drop it on the carbon film copper grid, dry it in the air, repeat 4 - 5 times, and observe using a transmission electron microscope with an accelerating voltage of 200 KV and a test temperature of 23 + 2 °C;

[0075] (5) After observation, EDS analysis can be performed on the sample to detect the chemical elements contained in the nanoparticles.

[0076] 2) Determination of particle size, dispersibility and Zeta potential

[0077] (1) Determination of particle size and dispersibility:

[0078] a) Take a 1.5 mL Eppendorf tube, add 1 mL of ethanol solution, then inject 1 μL of the prepared AA@G solution and place it in the centrifuge tube, and ultrasonically disperse AA@G evenly;

[0079] b) Prepare a cuvette, turn on the instrument for preheating, use a 1 mL pipette to transfer the prepared sample to the cuvette, and perform the test on the machine, noting that the triangular part of the cuvette should face forward;

[0080] (2) Potential measurement:

[0081] a) Take a 1.5 mL EPP tube, add 1 mL of ethanol solution, and then inject 5 μL of the prepared AA@G solution and place it in a centrifuge tube. Ultrasonic to disperse the nanoparticles evenly;

[0082] b) Prepare a disposable folding capillary cell. Use a 2 mL syringe to add the sample into the cell, pay attention not to generate bubbles during the process, and at the same time make the liquid level exceed the conductive part of the cell;

[0083] c) Turn on the instrument for preheating, place the part with the Malvern logo of the cell forward, and start the test.

[0084] 3) Ultraviolet absorption spectrum detection

[0085] (1) Turn on the ultraviolet-visible spectrometer and preheat it for half an hour;

[0086] (2) Prepare PBS for baseline scanning and clean the cuvette in time;

[0087] (3) Take a 5 mL EPP tube, add 3 mL of PBS solution, and then inject 10 μL of the prepared AA@G solution and place it in a centrifuge tube;

[0088] (4) Use a 1 mL pipette to transfer the prepared sample to a cuvette, place it in an ultraviolet-visible spectrophotometer, use the buffer solution as the blank solution, and scan the absorption spectrum in the wavelength range of 250 - 800 nm.

[0089] 4) Infrared spectrum analysis

[0090] (1) Turn on the ultraviolet-visible spectrometer and preheat it for half an hour;

[0091] (2) Vacuum freeze-dry the prepared AA@G particle solution. After the sample becomes dry powder, wrap it with weighing paper and gently press it with a glass slide to make the sample more uniform;

[0092] (3) Scan after loading the sample.

[0093] 5) High performance liquid chromatography analysis

[0094] (1) Instrument preparation:

[0095] a) Select the chromatographic column and mobile phase according to the analysis object. Use a C18 column (Nova-Pak 3.9×250 mm, Waters, Milford, MA); the mobile phase includes water and acetonitrile, and the volume ratio is 1:9.

[0096] b) Filter the mobile phase, select different filter membranes according to needs, and perform ultrasonic degassing on the filtered mobile phase for 30 min;

[0097] c) Connect the mobile phase pipeline, turn on the HPLC workstation (including computer software and chromatograph), and connect the detection system;

[0098] d) Flush the pump and injection valve. To flush the pump, directly draw with a syringe at the outlet of the pump. To flush the injection valve, under the manual menu, first click purge, then click start, and the flushing speed should not exceed 10 mL / min;

[0099] e) Design the sample run method. Click file, select select users and methods, click new method, and select the required accessories (injection valve, pump, detector, etc.). A complete sample run method should include:

[0100] (a) Stabilize the flow before injection, generally for 2 - 5 minutes;

[0101] (b) Zero the baseline;

[0102] (c) Convert the injection valve from loading to inject;

[0103] (d) The sample run time varies with different samples.

[0104] (2) Sample preparation:

[0105] a) Prepare a 2 mL syringe, a 0.45 μm filter, and a sample vial;

[0106] b) Remove the syringe needle, draw in the sample, inject it into the filter, and filter it into the sample vial. The sample volume should be about 200 μL;

[0107] c) Observe the sample vial. There must be no bubbles. If there are bubbles, shake the sample vial until the bubbles are discharged

[0108] (3) Injection

[0109] a) Select the sample run method, click start, and start the injection;

[0110] b) First inject pure solvent as the baseline, then start injecting the test sample. After all samples are detected, run a baseline for a while in the same way to clean the system.

[0111] (4) Shutdown

[0112] First shut down the computer, then shut down the liquid chromatography.

[0113] 3. In vivo application of AA@G

[0114] Five-week-old healthy C57 mice weighing approximately 18 g were selected. After culturing in a laminar flow chamber for 1 week, Hep1-6 cells diluted with PBS (phosphate buffer) (2×10 6 / mouse) were subcutaneously injected into the root of the right hind limb of nude mice under sterile conditions to establish a mouse liver cancer xenograft model. The mice were raised until the tumor volume reached approximately 100 mm 3 . Then, they were used as the modeled nude mice for subsequent experiments.

[0115] 1) Grouping: Control, @G, AP@G, AT@G, AA@G;

[0116] 2) Intraperitoneal injection of nano-drugs for each group

[0117] (1) Fix the mice and expose their abdomens; prepare a 1-ml syringe;

[0118] (2) Dissolve the nano-drugs for each group with PBS and filter them through a 0.22-μm filter membrane;

[0119] (3) Hold the syringe with the right hand, draw an appropriate amount of the drug solution, insert the needle into the lower right abdomen, and push the solution into the abdomen;

[0120] (4) Pull out the syringe, press the puncture site with a gauze, and fold the tail to stop bleeding;

[0121] Results and Discussion:

[0122] As shown in A of Figure 2 , the synthesized AA@G is spherical, with uniform size and good dispersibility. The EDS analysis of AA@G with an energy dispersive spectrometer shows that AA@G mainly contains Au, S, O, and N elements, and they are evenly distributed, exactly matching the shape of AA@G, indicating the formation of Au-S bonds ( Figure 2 B of Figure 2 ). C of Figure 2 shows the color change during the synthesis of AA@G. Particle size analysis shows that the size of AA@G is distributed around 22.3 nm ( Figure 2 D of

[0123] . Detection of the change in surface potential during the preparation of the nano-complex shows that @G is negatively charged, approximately -8.95 ± 6.80 mV, and AA@G is negatively charged, approximately 21.60 ± 4.45 mV ( Figure 2 E and F of

[0123] . The ultraviolet-visible absorption spectrum shows that the ultraviolet-visible absorption peak λmax of AP is located near 220 nm; the ultraviolet-visible absorption peaks λmax of ATR inhibitors are located near 330 nm and 520 nm; while @G does not have an ultraviolet-visible absorption peak; when @G encapsulates AP and ATR inhibitors, the ultraviolet-visible absorption peak λmax shows a red shift, and the maximum absorption peaks are located near 230 nm and 530 nm respectively ( Figure 2In G). It is illustrated that @G has good drug-loading performance and can successfully load AP and ATR inhibitors. The results of infrared spectroscopy analysis show that there are changes in the absorption peaks of AA@G after loading AP and VP, indicating the generation of new functional groups. This further proves that AA@G can successfully load AP and ATR inhibitors. Figure 2 In H). Figure 2 In I, it is indicated that AA@G can stably exist in serum, while it decomposes in tumor tissues with high glutathione concentration. Figure 2 In J, it is indicated that AA@G can successfully release drugs in a glutathione environment.

[0124] To verify the cell-penetrating ability of AA@G, the present invention incubated AA@G with cancer cells. The results show that AA@G can smoothly enter the cells. Figure 3 In A and B). The present invention also constructed a 3D tumor tissue model to observe the penetrating ability of AA@G in the tissue. The results show that AA@G can reach the interior of the tissue and has strong tissue-penetrating ability. Figure 3 In C). To further observe the in vivo metabolism of AA@G, the present invention injected AA@G into tumor-bearing mice and detected the distribution of AA@G in various organs by ICP-MS. It can be found that AA@G accumulates in tumor tissues, while in normal organs, although there is a transient increase, it finally returns to the normal level, indicating that AA@G can be smoothly metabolized from the organism. The results of CCK8, flow cytometry apoptosis, and live-dead cell staining show that AA@G can effectively promote cell apoptosis and improve sensitivity. Figure 4 In A to H).

[0125] Figure 5 In A shows the mechanism of action of AA@G. Figure 5 The results of the WB experiment in B show that compared with the control group and the @G group, the expression levels of γ-H2AX and... increase, while the expression levels of Rad51, p-chk1, and CyclinD1 decrease in the AA@G treatment group. These results indicate that AA@G promotes DNA damage and inhibits HR repair after DSB. The results of immunofluorescence (IF) are consistent with the WB results, as shown in Figure 5 In C.

[0126] To determine the in vivo effect of AA@G, the present invention randomly divided Hep1-6 tumor-bearing mice into six groups: control group, @G, AP@G, AT@G, and AA@G groups. As Figure 6 In A shows, the mice were treated. The body weight and tumor growth of each group were recorded every 2 days to evaluate the treatment effect. The body weight measurement shows that the mice in each group have similar growth curves. Figure 6 In C); the tumor growth measurement results show that the tumor growth in the AA@G treatment group is significantly slower than that in other treatment groups.Figure 6 in B, E); the tumor weight results showed that the tumor weight in the AA@G treatment group was the lowest ( Figure 6 in D). As Figure 6 in F, G, the HE tissue staining results of tumor sections showed that compared with other groups, there was more necrosis in the tumor tissue in the AA@G treatment group. The TUNEL fluorescence staining results showed that there were more green dots in the tumor sections of the AA@G treatment group, indicating that its apoptosis rate was significantly increased and the tumor killing effect was more obvious. Immunohistochemistry (IHC) staining was used to evaluate the expression levels of γ-H2AX, Ki67, p-ATM / ATR, and CyclinD1 to further study the role of AA@G in improving treatment sensitivity in vivo. The immunohistochemical staining results showed that the expression level of γ-H2AX was the highest and the expression levels of Ki67, p-ATM / ATR, and CyclinD1 were the lowest in the AA@G group.

[0127] In summary, the application of the integrated nanoplatform of the gold adjuvant combined with the XPO1 inhibitor and the ATR inhibitor proposed by the present invention blocks the cell cycle, promotes tumor cell DNA damage, inhibits tumor cell DNA damage repair, promotes tumor cell apoptosis, and improves tumor treatment sensitivity. The nanoplatform of the present invention can also carry other drugs to achieve multiple therapeutic effects. The nanoplatform prepared by the preparation method of the present invention has the advantages of excellent drug loading capacity and unified pharmacokinetics.

Claims

1. An integrated nanoplatform of gold adjuvant combined with XPO1 inhibitor and ATR inhibitor, characterized in that, The general formula is [AA@G], wherein the first A is an XPO1 inhibitor, which is a 96-110 functional fragment of the polypeptide Apoptin, and is modified with Ac and NH2 at the head and tail. In order to modify NH2, an amino acid C is also added to the tail, so that the final amino acid sequence is Ac-RVSELKESLITTTPSC-NH2. The second A is an ATR inhibitor, and @G is a gold adjuvant. The gold adjuvant serves as a nanocarrier to carry the XPO1 inhibitor and the ATR inhibitor, and the ATR inhibitor is AZD6738.

2. The preparation method of the integrated nano-platform of the gold adjuvant combined with the XPO1 inhibitor and the ATR inhibitor according to claim 1, characterized in that, The steps include: Step 1, adding TCEP to a human serum albumin solution, and then adding an ATR inhibitor to synthesize an HSA-ATR inhibitor; Step 2, adding chloroauric acid solution to HEPES buffer and stirring under heating conditions until the solution turns purple-red to synthesize nanogold adjuvant; Step 3, after XPO1 inhibitor, NH2-PEG2000-SH and HEPES are fully mixed, chloroauric acid solution is added and stirred continuously until the solution turns light yellow to colorless to synthesize AP@G; Step 4: HSA-ATR inhibitor, AP@G and nanogold adjuvant are mixed to form AA@G.

3. The preparation method according to claim 2, characterized in that, In the step 1, the mass volume ratio of TCEP to HSA is 1:1-1:5, and the molar ratio of HSA to ATR inhibitor is 1:1-1:

5.

4. The preparation method according to claim 2, characterized in that, In the step 2, the molar ratio of chloroauric acid to HEPES is 1:1-1:

5.

5. The preparation method according to claim 2, characterized in that, In the step 3, the molar ratio of the XPO1 inhibitor, NH2-PEG2000-SH, HEPES and chloroauric acid is 2:2:1:5-3:3:1:

5.

6. The preparation method according to claim 2, characterized in that, In step 4, the molar ratio of the HSA-ATR inhibitor, AP@G and gold adjuvant is 1:1:1-1:3:

3.

7. Use of the integrated nanoplatform of the gold adjuvant combined with XPO1 inhibitor and ATR inhibitor as claimed in claim 1 for preparing drugs for treating liver cancer.

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