Nano preparation as well as preparation method and application thereof

Abstract

The invention discloses a nano preparation as well as a preparation method and application thereof, relates to the field of pharmaceutical preparations, and solves the problems of high toxic and side effects when copper death and gas treatment are independently used for treating tumors and insufficient synergism when the gas treatment is combined with other treatment methods in the prior art. The preparation method comprises the following steps: connecting 2, 4-dinitrobenzenesulfonyl (DNs) with L-arginine (Arg) through sulfonation reaction to form a double-gas donor molecule DNs-Arg capable of releasing NO and SO2 at the same time; the preparation method comprises the following steps: constructing a copper complex DNs-Arg-Cu with GSH responsiveness by utilizing the coordination effect of negative charges of carboxyl in Arg molecules and Cu < 2 + >, and preparing a nano preparation DP-DA-Cu NPs by utilizing DSPE-mPEG2k as a carrier. The nano preparation DP (at) DA-Cu NPs provided by the invention realizes an efficient anti-tumor effect, and can be applied to preparation of anti-tumor nano drugs.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical preparations, and particularly relates to a nano - preparation, a preparation method thereof and an application thereof. Background Art

[0002] Malignant tumors seriously endanger human health. Currently, the main clinical treatment methods include surgical treatment, chemotherapy, radiotherapy, etc. Although these treatment methods can delay the progression of the disease, they will produce serious toxic and side effects. Surgical treatment is not applicable to middle and late - stage tumors. Due to the invasion and metastasis of tumor cells, surgery often fails to completely remove tumor tissues and is likely to cause complications. Tumor cells are prone to develop drug resistance to chemotherapy drugs, greatly reducing the treatment effect. Radiotherapy may cause radiation damage to normal tissues around the tumor, thus limiting the treatment effect.

[0003] In recent years, the rise of gas therapy has brought new opportunities for tumor treatment. Currently, there have been research reports that L - arginine (L - Arg) is a nitric oxide (NO) donor with good biocompatibility. The guanidine group contained in its structure can produce NO through enzymatic reactions in tumor cells. High - concentration NO can effectively inhibit the growth of tumor cells, induce apoptosis, and can enhance the killing effect of immune cells on tumor cells, improving the body's anti - tumor immune response. However, NO has certain toxicity. Excessive concentration will damage normal tissues, and too low concentration will not achieve the ideal anti - tumor effect. In addition, some researchers have combined NO treatment with traditional tumor treatment methods (such as radiotherapy, chemotherapy, etc.), but their synergistic effect is not good. For example, chemotherapy drugs will change the metabolic state of tumor cells, affecting the action target of NO on tumor cells; radiotherapy will cause an inflammatory reaction in tumor tissues, changing the distribution and activity of NO.

[0004] Copper (Cu) is a trace metal inside cells and plays an important role in different cell metabolic processes. In 2022, the team of Golub proposed the concept of "cuproptosis", which refers to a form of cell death. The main process is the accumulation of intracellular copper ions and their entry into mitochondria, followed by binding to the relevant lipoylated components involved in the tricarboxylic acid cycle (TCA), thereby triggering a protein toxicity stress response and ultimately leading to cell death (Copper induces cell death by targeting lipoylated TCA cycle proteins, Science 2022, 375, 1254 - 1261). Compared with traditional treatment methods, cuproptosis therapy has significant advantages such as avoiding drug resistance. However, during the application of cuproptosis, the intracellular endogenous copper content is low, and exogenous copper ions are easily offset by the high level of glutathione (GSH) in tumor cells, thus limiting the effect of cuproptosis therapy.

[0005] To address the defects existing in the above gas therapy and cuproptosis therapy, further improve the treatment effect and biological safety, develop a nanomedicine based on the combination of cuproptosis and gas therapy, promote the development of a multifunctional cuproptosis nanoplatform, and provide a more effective strategy for the preparation of primary tumor inhibitory drugs. Summary of the Invention

[0006] To solve the problems of high toxic and side effects when cuproptosis and gas therapy treat tumors alone and insufficient synergy when gas therapy is combined with other treatment methods in the prior art, the present invention proposes a nanoplatform and its preparation method and application.

[0007] A preparation method of a nanoplatform, comprising the following preparation steps:

[0008] S1: Add 2,4 - dinitrobenzenesulfonyl chloride (DNs) and sodium hydroxide to a tetrahydrofuran (THF)-H2O mixed solution, stir until clear, then continue to add a THF-H2O mixed solution containing an amino acid for reaction. After the reaction, rotate and evaporate to remove THF, adjust the pH, extract, retain the organic phase, rotate and evaporate to remove the organic solvent to obtain a mixture, then perform deprotection and sedimentation. After removing the supernatant, dry with a cold trap to remove the excess solvent, and prepare DNs-Arg;

[0009] S2: Mix the DNs-Arg aqueous solution and the CuSO4 aqueous solution, adjust the pH and stir, centrifuge, remove the supernatant, and dry the lower solid to prepare the DNs-Arg-Cu complex;

[0010] S3: Self-assemble methoxypolyethylene glycol-phosphatidylethanolamine and DNs-Arg-Cu complex in an N,N-dimethylformamide (DMF)-H2O solution, then dialyze the mixed solution overnight, and make up the volume after dialysis is completed to prepare the nanoformulation DP@DA-Cu NPs.

[0011] Further, the molar ratio of the DNs, NaOH, and amino acid in S1 is 1.1-3.5:2-3:1; the amino acid is N α -tert-butoxycarbonyl-L-arginine (Boc-Arg-OH);

[0012] Further, the temperature of the rotary evaporation in S1 is 30-40 °C; the pH adjustment is to adjust the pH to 1; the extraction is to perform extractions using dichloromethane (DCM), saturated NaCl solution, and H2O respectively;

[0013] Further, the deprotection process in S1 is to add trifluoroacetic acid (TFA) and DCM to the mixture and react for 4 h; the sedimentation is to add the mixture to a mixed solution of ether and n-hexane for sedimentation;

[0014] Further, the molar ratio of the DNs-Arg and CuSO4 in S2 is 2-3:1;

[0015] Further, the pH adjustment in S2 is to adjust the pH to 7.5; the stirring time is 30 min; the centrifugation speed is 4500 rpm, and the centrifugation time is 5 min; the drying temperature is 45 °C;

[0016] Further, the mass ratio of the methoxypolyethylene glycol-phosphatidylethanolamine and DNs-Arg-Cu complex in S3 is 1-5:1;

[0017] Further, the self-assembly time in S3 is 2.5 h.

[0018] A nanoformulation is prepared by the above preparation method.

[0019] An application of the above nanoformulation in the preparation of anti-tumor nano-drugs.

[0020] Compared with the prior art, the present invention solves the problems of high toxicity and side effects when cuproptosis and gas therapy are used to treat tumors alone, and the lack of synergy when gas therapy is combined with other treatment methods. The specific beneficial effects are as follows:

[0021] 1. High anti-tumor effect: In the present invention, a drug supply agent DNs-Arg containing NO and sulfur dioxide (SO2) is prepared through a sulfonation reaction, and copper ion loading is further achieved by the coordination between the negatively charged carboxyl group on the amino acid and free divalent copper ions to obtain a copper complex DNs-Arg-Cu. Further, DSPE-mPEG 2k is used as a drug carrier to prepare a nanoformulation DP@DA-Cu NPs. DP@DA-Cu NPs release SO2 through the GSH response of their DNs in cancer cells. SO2 can inhibit the activity of superoxide dismutase (SOD), significantly increasing the intracellular O2 - concentration. In addition, the naked L-Arg is oxidized by the high concentration of H2O2 in tumor cells to release NO, and finally NO consumes superoxide anion (O2 - ), generating peroxynitrite anion (ONOO - ) with strong oxidizing properties, aggravating DNA damage, and thus triggering apoptosis of tumor cells. Moreover, the DP@DA-Cu NPs nanoparticles can effectively promote the delivery of copper ions, resulting in the enrichment of copper ions in tumor cells, thereby affecting the tricarboxylic acid cycle (TCA) in mitochondria and then triggering cuproptosis. The combination of gas therapy and cuproptosis achieves a high anti-tumor effect.

[0022] 2. Low drug toxicity: The nanoformulation DP@DA-Cu NPs prepared in the present invention has almost no toxic effect on normal cells (such as fibroblast L929), while showing strong toxicity to tumor cells (such as colorectal cancer cell CT26). The non-toxic DSPE-mPEG 2k is selected as the drug carrier, which can not only stabilize the nano-micelles and increase blood circulation in vivo, but also significantly reduce drug toxicity, providing a new idea for the development of anti-tumor nano-drugs.

[0023] 3. Simple preparation method: The synthesis method of the nanoformulation DP@DA-Cu NPs provided in the present invention is simple, low-cost, and has good biocompatibility, reducing the technical threshold and economic cost of large-scale production, and providing a solid foundation for its further research and clinical transformation in the biomedical fields such as tumor treatment. Description of the Drawings

[0024] Figure 1 is the 1H NMR spectrum of DNs-Arg;

[0025] Figure 2 is the preparation flow chart of the DNs-Arg-Cu complex;

[0026] Figure 3 are the infrared spectra of DNs-Arg and DNs-Arg-Cu;

[0027] Figure 4 Transmission electron microscopy and dynamic light scattering diagrams of DP@DA-Cu NPs;

[0028] Figure 5 Intracellular gas release and ROS laser confocal diagrams;

[0029] Figure 6 Toxicity test results of nanoparticles against CT26 cells;

[0030] Figure 7 Toxicity test results of nanoparticles against L929 cells;

[0031] Figure 8 Results of in vivo tumor suppression experiments of nanoparticles;

[0032] Figure 9 In vivo tumor suppression curve diagram of nanoparticles;

[0033] Figure 10 Tumor mass diagram after nanoparticle treatment;

[0034] Figure 11 Mouse body weight curve diagram after nanoparticle treatment. Detailed implementation manners

[0035] To make the technical solutions of the present invention clearer, the following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings of the present invention. It should be noted that the following embodiments are only used to better understand the technical solutions of the present invention and should not be construed as a limitation to the present invention.

[0036] Example 1.

[0037] S1: Add 20.4 g of DNs and 1.5 g of NaOH to the reaction flask in sequence, add a mixed solution of 40 mL of THF and 10 mL of H2O, stir in an ice-water bath until clear, and continue to dropwise add a mixed solution of THF (40 mL) / H2O (10 mL) containing 10 g of Boc-Arg-OH to the reaction flask under stirring conditions. After restoring to room temperature, react overnight; after the reaction is completed, rotate and evaporate to remove THF at 40 °C, then add dilute hydrochloric acid dropwise to the mixture to adjust the pH value to 1; extract with DCM, saturated NaCl solution and H2O respectively, retain the organic phase and rotate and evaporate to remove DCM at 35 °C; then continue to add 30 mL of TFA and 30 mL of DCM to the mixture and continue to react for 4 h; precipitate the reaction mixture in a mixed solution of 300 mL of ether and 300 mL of n-hexane, remove the supernatant, connect to a cold trap and pump dry the excess solvent to prepare the supply drug DNs-Arg containing NO and SO2. The yield is 42.8%. As Figure 1It is the 1H NMR spectrum of DNs-Arg.

[0038] S2: Add 0.4 g of DNs-Arg and 8 mL of H2O into a reaction flask, and stir to completely dissolve it; then add 2.5 mL of an aqueous CuSO4 solution (0.16 mol / L) into the reaction flask, and adjust the pH value of the mixed solution to 7.5 with 30 wt% NaOH solution, and continuously stir at room temperature for 30 min; after the reaction is completed, centrifuge the mixture at a speed of 4500 rpm for 5 min to remove the supernatant. Place the solid in the lower layer in an oven at 45 °C to dry, and prepare the DNs-Arg-Cu complex. As Figure 2 It is the preparation flow chart of the DNs-Arg-Cu complex.

[0039] As Figure 3 It is the infrared spectra of DNs-Arg and DNs-Arg-Cu. It can be seen from the figure that the characteristic peak of DNs-Arg-Cu at 1586 cm -1 is the bending vibration of -NH2, and the characteristic peak at 1132 cm -1 is the stretching vibration of C-N. This is because the lone pair electrons of the nitrogen atom in the guanidine group on the side chain of arginine (Arg) contribute to the copper atom, resulting in an increase in the bipolarity of the C-N bond; while the band at 592 cm -1 of DNs-Arg-Cu is the characteristic peak of the symmetric vibration of Cu-N, indicating that the α-N atom participates in coordination. By comparing the infrared spectra of DNs-Arg and DNs-Arg-Cu, the formation of the DNs-Arg-Cu complex is confirmed.

[0040] S3: Add 4.3 g of DSPE-mPEG 2k and 1.2 g of the DNs-Arg-Cu complex into a reaction flask, dissolve them ultrasonically with 2.2 mL of DMF, continue to drop 20 mL of water into the reaction flask, and self-assemble for 2.5 h; transfer the mixed solution after the reaction to a dialysis bag with a molecular weight cut-off of 3500 Da, change the water every 1 h, and dialyze overnight; after dialysis is completed, make up the volume to 26 mL (the concentration of DSPE-mPEG 2k is 0.15 mg / mL), and prepare the nanoformulation DP@DA-Cu NPs.

[0041] As Figure 4 It is the transmission electron microscopy and dynamic light scattering aqueous phase particle size of DP@DA-Cu NPs. It can be seen from the figure that the DNs-Arg-Cu NPs are spherical, and the aqueous phase diameter is: 139.1 ± 0.2 nm; this is because the hydrophobic DNs-Arg-Cu and the hydrophilic DSPE-mPEG 2kThe structure self-assembles into nanoparticles through hydrophilic-hydrophobic interaction, and the resulting morphological changes intuitively reflect the self-assembly mechanism of DP@DA-Cu NPs.

[0042] Comparative Example 1:

[0043] S1: 20.4 g of DNs and 1.5 g of NaOH were successively added to a reaction flask, and 40 mL of THF and 10 mL of H2O mixed solution were added. The mixture was stirred in an ice-water bath until clear, and then a THF (40 mL) / H2O (10 mL) mixed solution containing 9 g of N α -tert-Butoxycarbonyl-L-lysine (Boc-Lys-OH) was added dropwise to the reaction flask under stirring. After returning to room temperature, the reaction was carried out overnight. After the reaction was completed, THF was removed by rotary evaporation at 40 °C. Subsequently, dilute hydrochloric acid was added dropwise to the mixture to adjust the pH value to 1.0; the mixture was extracted with DCM, saturated NaCl solution and H2O respectively, and the organic phase was retained and DCM was removed by rotary evaporation at 35 °C; then 30 mL of TFA and 30 mL of DCM were added to the mixture, and the reaction was continued for 4 h; the reaction mixture was precipitated in a mixed solution of 300 mL of ether and 300 mL of n-hexane, the supernatant was removed, and the cold trap was connected to dry the excess solvent to obtain the solid product DNs-Lys. The yield was 30.2%.

[0044] S2: 0.4 g of DNs-Lys and 8 mL of H2O were added to a reaction flask and completely dissolved under stirring; then 2.5 mL of CuSO4 aqueous solution (0.16 mol / L) was added to the reaction flask, and the pH value of the reaction mixture was adjusted to 7.5 with 30 wt% NaOH solution, and the mixture was continuously stirred at room temperature for 30 min. After the reaction was completed, the mixture was centrifuged at 4500 rpm for 5 min to remove the supernatant. The lower layer solid was dried in an oven at 45 °C to prepare the DNs-Lys-Cu complex.

[0045] S3: 4.3 g of DSPE-mPEG 2k and 1.1 g of DNs-Lys-Cu complex were successively added to a reaction flask and ultrasonically dissolved with 2.2 mL of DMF. Then 20 mL of water was added dropwise to the reaction flask, and self-assembly was carried out for 2.5 h; the reaction mixture after completion was transferred to a dialysis bag with a cut-off molecular weight of 3500 Da, and the water was changed every 1 h, and dialysis was carried out overnight; after dialysis was completed, the volume was fixed to 26 mL (DSPE-mPEG 2k concentration was 0.15 mg / mL) to prepare the nanopreparation DP@DL-Cu NPs.

[0046] Comparative Example 2:

[0047] S1: Add 0.2 g of L-Arg and 8 mL of H2O into a reaction flask, and completely dissolve it under stirring; then add 2.5 mL of aqueous CuSO4 solution (0.16 mol / L) into the reaction flask, and adjust the pH value of the mixed solution to 7.5 with 30 wt% NaOH solution, and continuously stir at room temperature for 30 min; after the reaction is completed, centrifuge the mixture at a speed of 4500 rpm for 5 min to remove the supernatant. Place the lower-layer solid in an oven at 45 °C for drying to prepare the L-Arg-Cu complex.

[0048] S2: Sequentially add 4.3 g of DSPE-mPEG 2k and 0.6 g of L-Arg-Cu complex into a reaction flask, ultrasonically dissolve it with 2.2 mL of DMF, continue to drop 20 mL of water into the reaction flask, and self-assemble for 2.5 h; transfer the mixed solution after the reaction is completed into a dialysis bag with a cut-off molecular weight of 3500 Da, change the water every 1 h, and dialyze overnight; after dialysis is completed, make up the volume to 26 mL (the concentration of DSPE-mPEG 2k is 0.15 mg / mL) to prepare the nanoformulation DP@LA-Cu NPs.

[0049] Intracellular ROS detection:

[0050] Inoculate CT26 cells in a confocal culture dish at a density of 1.0×10 5 cells / dish. After incubating overnight, replace the medium with fresh RPMI-1640 containing 10 μM DP@DA-Cu NPs for 6 h. Stain the cells with DEACA for 8 min to reach a final concentration of 1 μM. Subsequently, rinse the cells 3 times with PBS (stain the cells with DHE for 10 min to reach a final concentration of 5 μM; stain the cells with 3-amino-4-aminomethyl-2',7'-difluorofluorescein diacetate (DAF-FM DA) for 20 min to reach a final concentration of 5 μM; stain the cells with HKGreen-4I for 15 min to reach a final concentration of 3 μM; stain the cells with DCFH-DA for 20 min to reach a final concentration of 1 μM). Finally, use a laser confocal microscope to photograph the labeled cells.

[0051] As Figure 5 shown, using diethylaminocoumarin acrylate (DEACA), dihydroethidium (DHE), DAF-FM DA, HKGreen-4I, and 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) fluorescent probes, with the aid of laser confocal technology, for SO2, O2 - 、NO、OONO -The generation mechanism of SO2 was further investigated. Nanoparticles NPs containing DNs groups, such as DNs-Arg, DP@DL-Cu NPs, and DP@DA-Cu NPs, can generate SO2 in CT26 tumor cells under the action of glutathione (GSH) at high concentrations (2-10 mM). SO2 can inhibit the activity of SOD, thereby significantly increasing the concentration of O2 - in the cells. As GSH is consumed, the intracellular ROS level becomes unbalanced. The excess ROS will further oxidize the exposed guanidine groups to produce NO. A part of NO is consumed by O2 - to generate OONO with strong oxidizing ability - . Finally, the total intracellular ROS was detected using the DCFH-DA fluorescent probe. The results showed that after DP@DA-Cu NPs were internalized by cells, they could rapidly release SO2 and NO, and through a series of biochemical reactions in the cells, generate more toxic OONO - , damage nuclear DNA, and then induce apoptosis of cancer cells.

[0052] Cytotoxicity test:

[0053] Mouse colorectal cancer cells CT26 and mouse fibroblast cells L929 (5000 cells / well) were placed in a 96-well plate, with 180 μL of culture medium in each well, and incubated overnight in a 37 °C incubator. After incubation, DNs-Arg, DP@LA-Cu NPs, DP@DL-Cu NPs, and DP@DA-Cu NPs were added to the cells respectively and incubated for another 24 h. Then, 20 μL of MTT solution was added to each well, and after incubation for 4 h, the waste liquid was aspirated. 150 μL of dimethyl sulfoxide (DMSO) solvent was added to each well, and it was gently shaken for 5 min to completely dissolve it. The absorbance at 492 nm was measured using an enzyme-linked immunosorbent assay (ELISA) reader. The cells treated without drugs were used as the control group (cell viability was recorded as 100%), and the cell survival rate at each concentration was calculated. Each experiment was repeated three times, and the average value was calculated.

[0054] As Figure 6 shown in the cytotoxicity test results of DNs-Arg, DP@LA-Cu NPs, DP@DL-Cu NPs, and DP@DA-Cu NPs on CT26 cells; Figure 7Toxicity test results of DNs-Arg, DP@LA-Cu NPs, DP@DL-Cu NPs, and DP@DA-Cu NPs on L929 cells. As can be seen from the figure, DNs-Arg, DP@LA-Cu NPs, DP@DL-Cu NPs, and DP@DA-Cu NPs do not show toxicity to normal cells L929, while DNs-Arg, DP@LA-Cu NPs, DP@DL-Cu NPs, and DP@DA-Cu NPs all show toxicity to CT26 cells. Among them, DP@DA-Cu NPs have the strongest toxicity to CT26 cells, and with the increase of the added dose of the nanoparticle DP@DA-Cu NPs, stronger toxicity to CT26 cells is shown. This is because DP@DA-Cu NPs release SO2 through the GSH response of their DNs in cancer cells, and SO2 can inhibit the activity of superoxide dismutase (SOD), significantly increasing the concentration of superoxide anion (O2 - ) in cells. In addition, naked L-Arg is oxidized by high concentrations of H2O2 in tumor cells to release NO, and finally NO will consume O2 - , generating peroxynitrite anion (ONOO - ) with strong oxidizing properties, aggravating DNA damage, and then triggering apoptosis of tumor cells. And this nanoparticle can effectively promote the transport of copper ions, resulting in the enrichment of copper ions in tumor cells, thus affecting the tricarboxylic acid cycle (TCA) in mitochondria and then triggering cuproptosis.

[0055] In vivo tumor suppression experiment:

[0056] Female Balb / c mice (6 - 8 weeks old, 17 - 18 g) were subcutaneously implanted with 1.0×10 6 CT26 cells in their abdominal area, suspended in 0.1 mL PBS. Once the tumor volume reached 100 mm 3 , the mice were randomly divided into five groups: PBS, DNs-Arg, DP@LA-Cu NPs (0.5 mg / kg Cu), DP@DL-Cu NPs (0.5 mg / kg Cu), and DP@DA-Cu NPs (0.5 mg / kg Cu), with 5 mice in each group. PBS and NPs were injected via the tail vein on days 0, 4, 8, and 12. The body weight and tumor volume of the mice were systematically recorded every two days. On day 16, the mice were euthanized, and their tumor tissues were collected for photographing and weighing. The formula for calculating the tumor volume (V) is as follows:

[0057]

[0058] In the above formula, a and b (mm) are the length and width of the tumor, respectively.

[0059] AsFigure 8 The results of the anti-tumor experiment of nanoparticles in vivo Figure 9 The anti-tumor curve graph of nanoparticles in vivo. It can be seen from the graph that DP@LA-Cu NPs, DP@DL-Cu NPs and DP@DA-Cu NPs can effectively inhibit tumor growth, and the average tumor inhibition rates are 62.5%, 70.4% and 96.2% respectively. Compared with the PBS group, the inhibitory effect of DNs-Arg on tumor growth is minimal, and the tumor inhibition rate is 24.3%. As Figure 10 The tumor mass graph after nanoparticle treatment. The average tumor weight of the DP@DA-Cu NPs group is 0.21 g, which is much lower than that of the PBS group (1.78 g), the DNs-Arg group (1.27 g), the DP@LA-Cu NPs group (0.58 g) and the DP@DL-Cu NPs group (0.57 g). This result is consistent with Figure 8 the results. As Figure 11 The body weight curve graph of mice after nanoparticle treatment. It can be seen from the graph that the body weights of the five groups of mice have little difference, further verifying the biosafety of the nanoformulation prepared by the present invention.

[0060] In summary, the nanoformulation DP@DA-Cu NPs provided by the present invention achieves an efficient anti-tumor effect through gas therapy synergistic cuproptosis, and also significantly reduces drug toxicity; the synthesis method is simple, the cost is low, and the biocompatibility is good, reducing the technical threshold and economic cost of large-scale production, providing a solid foundation for its further research and clinical transformation in the biomedical fields such as tumor treatment.

[0061] The description of the above embodiments is only used to help understand the method and its core idea of the present invention. It should be noted that for those of ordinary skill in the art of this technology, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention.

[0062] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be obvious to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but will be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A preparation method of a nano preparation, characterized in that, It includes the following preparation steps: S1: Add 2,4-dinitrobenzenesulfonyl chloride and sodium hydroxide into a tetrahydrofuran-water mixed solution, stir until clear, then continue to add a tetrahydrofuran-water mixed solution containing amino acid for reaction. After the reaction, rotary evaporate to remove tetrahydrofuran, adjust the pH, extract, retain the organic phase and rotary evaporate to remove the organic solvent to obtain a mixture. Subsequently, perform deprotection and sedimentation, remove the supernatant, and dry with a cold trap to remove the excess solvent to prepare DNs-Arg; S2: Mix the DNs-Arg aqueous solution and the CuSO4 aqueous solution, adjust the pH and stir, centrifuge, remove the supernatant, and dry the solid in the lower layer to prepare the DNs-Arg-Cu complex; S3: Self-assemble methoxypolyethylene glycol-phosphatidylethanolamine and the DNs-Arg-Cu complex in an N,N-dimethylformamide-aqueous solution. Subsequently, dialyze the mixed solution overnight, and make up the volume after dialysis to prepare the nano-formulation DP@DA-CuNPs.

2. The preparation method of the nano - preparation according to claim 1, characterized in that, The molar ratio of the total amount of 2,4-dinitrobenzenesulfonyl chloride and sodium hydroxide to the amino acid described in S1 is 1.1 to 3.5: 2 to 3: 1, and the amino acid is N α -tert-butoxycarbonyl-L-arginine.

3. The preparation method of the nano - preparation according to claim 1, characterized in that, In S1, the temperature of the rotary evaporation is 30-40 °C; the pH adjustment is to adjust the pH to 1; the extraction is to perform extraction using dichloromethane, saturated NaCl solution and H2O respectively.

4. The preparation method of the nano - preparation according to claim 1, characterized in that, In S1, the deprotection process is to add trifluoroacetic acid and dichloromethane to the mixture for reaction for 4 h; the sedimentation is to add the mixture to a mixed solution of ether and n-hexane for sedimentation.

5. The preparation method of the nano - preparation according to claim 1, characterized in that, In S2, the molar ratio of DNs-Arg to CuSO4 is 2-3:

1.

6. The preparation method of the nano - preparation according to claim 1, characterized in that, In S2, the pH adjustment is to adjust the pH to 7.5; the stirring time is 30 min; the centrifugation speed is 4500 rpm, and the centrifugation time is 5 min; the drying temperature is 45 °C.

7. The preparation method of the nano preparation according to claim 1, wherein, In S3, the mass ratio of methoxypolyethylene glycol-phosphatidylethanolamine to the DNs-Arg-Cu complex is 1-5:

1.

8. The preparation method of the nano - preparation according to claim 1, characterized in that, In S3, the self-assembly time is 2.5 h.

9. A nano - preparation, characterized in that, Prepared by the preparation method described in any one of claims 1-8.

10. Use of a nano - preparation as described in claim 9, characterized in that, Applied to the preparation of anti-tumor nano-drugs.

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