Nano-molecular system and preparation method and application thereof

Abstract

According to the invention, a nano molecular system (ACu-NPs) based on amifostine and copper ions is innovatively constructed. According to the system, Cu < 2 + > is specifically released through a tumor microenvironment, the problem of non-specific distribution of copper ions in vivo is effectively solved, and toxicity to normal cells is avoided. Through a Fenton-like reaction catalyzed by Cu (II) / Cu (I), the system can consume glutathione (GSH) in tumor cells, promote generation of reactive oxygen species (ROS), remarkably reverse a tumor immunosuppression microenvironment and overcome the problem of low response rate of an immune checkpoint blocking (ICB) therapy. Besides, ACu-NPs generates WR-1065 through metabolism of alkaline phosphatase (ALP) in normal cells, ROS is removed, the normal cells are protected against oxidative damage, and the problem of toxicity of a traditional copper ion carrier to normal tissues is effectively solved. The invention provides an innovative dual treatment strategy for treating primary and metastatic tumors.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and particularly relates to a preparation method of a supramolecular chemistry self-assembled nanoscale molecular system and its application in tumor treatment. Background Art

[0002] Immune Checkpoint Inhibitors (ICB), as a class of innovative anti-tumor drugs, significantly enhance the anti-tumor immune response by specifically inhibiting the interaction between inhibitory receptors (such as PD-1, CTLA-4) on the surface of T cells and their corresponding ligands (such as PD-L1, B7). However, clinical data show that approximately 80% of tumor patients do not achieve a continuously significant therapeutic effect, and this heterogeneity of treatment response is closely related to immune escape mediated by the Tumor Microenvironment (TME). Many tumors exhibit an immune "cold tumor" phenotype, presenting an immunosuppressive TME, resulting in drug resistance to existing immunotherapy drugs. Glutathione (GSH) is an important antioxidant within cells and plays a key role in regulating the redox balance. In tumor cells, excessive GSH exhibits strong reducing properties, which is associated with the inhibition of processes such as ferroptosis, chemotherapy, and photodynamic therapy. Although GSH depletion can increase oxidative stress, the effect of single GSH depletion is limited. Reactive Oxygen Species (ROS), as a key oxidative regulator in tumor cells, its accumulation can significantly enhance intracellular oxidative stress, induce endoplasmic reticulum stress response, and then promote the exposure of calreticulin (CRT), trigger Immunogenic Cell Death (ICD), and enhance the systemic anti-tumor immune response. Therefore, synergistically increasing ROS generation and GSH depletion can effectively reverse the immunosuppressive microenvironment of tumors, thereby improving the cancer treatment effect.

[0003] Copper (Cu) is an important intracellular nutritional metal and participates in maintaining the redox balance of cells. The copper (II) / copper (I)-catalyzed Fenton-like reaction exhibits high ROS generation ability under acidic conditions, which makes copper-based nanoparticles have good catalytic properties in the acidic tumor microenvironment. Therefore, developing copper-based nanocarriers with high catalytic activity and tumor targeting is of great significance for enhancing ROS generation. Recent studies have discovered a new type of copper-dependent cell death mode - cuproptosis, which is different from ferroptosis and apoptosis. Cuproptosis is caused by excessive Cu 2+Triggering, by targeting lipoylated components (such as DLAT) in the tricarboxylic acid cycle, leads to the aggregation of lipoylated mitochondrial proteins and the reduction of Fe-S cluster proteins, ultimately triggering mitochondrial toxic stress and cell death. The occurrence of cuproptosis is closely related to the concentration of intracellular copper ions, and the deposition of copper ions is regulated by copper ion carriers. However, existing copper ion carriers (such as disulfiram, 8-hydroxyquinoline, etc.) have the disadvantage of insufficient tumor targeting, resulting in indiscriminate killing of normal cells and tumor cells, which limits their clinical application. Therefore, developing a copper ion carrier that can specifically target tumor cells and induce immunogenic cuproptosis is the key to promoting the immunotherapy of copper-based nanomaterials. Summary of the Invention

[0004] The purpose of the present invention is to solve the disadvantages existing in the prior art, and provide a preparation method and application of a brand-new nanoscale copper ion carrier, and develop a nanoscale co-delivery system with "normal cell protection function" and "inducing immunogenic cuproptosis function".

[0005] The present invention is achieved through the following technical solutions: A nanomolecular system, which is formed by supramolecular self-assembly of amifostine and copper ions, and the structural formula is shown in Formula I: I.

[0006] A preparation method of a nanomolecular system as described above, which includes the following steps: Add a copper ion-containing solution to the amifostine solution, stir and mix at a speed of 800-1000 rpm for 12-48 h, and then centrifuge at a speed of 9000-11000 rpm to obtain a nanoscale crude product; Wash the nanoscale crude product with deionized water to remove free molecular monomers, and then centrifuge at a speed of 11000-13000 rpm to obtain a nanoscale pure solid product; Add the nanoscale pure solid product to deionized water containing mouse serum albumin, and ultrasonically dissolve and resuspend it at a power of 600-700 W and an intensity of 40-50% to obtain the nanoscale copper ion carrier.

[0007] Among them, if the stirring speed during the mixing reaction is too low, insufficient stirring will lead to non-uniform reaction system; if it is too high, it will cause liquid splashing and affect the stability of the sample. If the mixing reaction time is too short, the reaction will be insufficient, affecting the yield and purity of the product. If the centrifugation speed during centrifugation is lower than 9000 rpm, not all reaction products can be centrifuged and precipitated. If the ultrasonic power and intensity are too small, the material cannot be ultrasonically dispersed evenly; if they are too high, more heat will be generated at the probe, causing the mouse serum albumin in the material to deteriorate, affecting the purity and performance of the material. As a preferred embodiment, the molar ratio of amifostine to copper ions is 1:1.

[0008] As a preferred embodiment, the copper ion-containing solution is a copper chloride solution.

[0009] As a preferred embodiment, the molar concentration of the amifostine solution is 10 mM and pH = 7.4; the molar concentration of the copper ion-containing solution is 10 mM. As a preferred embodiment, in the deionized water containing mouse serum albumin, the mass concentration of mouse serum albumin is 2%.

[0010] Use of a nanomolecular system as described above in a drug for inhibiting the growth, metastasis and spread of tumor cells.

[0011] A tumor therapeutic drug, which contains αPD-L1 (anti-programmed death-1 antibody) and the nanomolecular system described in claim 1.

[0012] The implementation principle of the present invention is as follows: Amifostine (WR-2721) is the first broad-spectrum cytoprotective prodrug approved by the FDA. In normal cells, amifostine is catalytically hydrolyzed by alkaline phosphatase (ALP) to generate WR-1065, which effectively protects normal cells from oxidative stress damage as a ROS scavenger. Due to the acidic microenvironment in tumor tissues, the ALP activity is low, resulting in a much lower concentration of WR-1065 in tumor tissues than in normal tissues, thus achieving selective cytoprotection.

[0013] In tumor cells, ACu-NPs specifically release Cu 2+ , target lipoylated mitochondrial proteins and reduce Fe-S cluster proteins, triggering cuproptosis. At the same time, Cu 2+ -mediated glutathione (GSH) depletion maintains the Fenton-like reaction, continuously generates reactive oxygen species (ROS), reverses the tumor immunosuppressive microenvironment (TME), amplifies oxidative stress and induces endoplasmic reticulum stress (ER), promotes the exposure of calreticulin (CRT), enhances immunogenic cell death (ICD), and further activates the systemic anti-tumor immune response. In normal cells, ACu-NPs are metabolized by alkaline phosphatase (ALP) into the antioxidant WR-1065, scavenging ROS and protecting normal cells from oxidative damage. When combined with immune checkpoint blockade therapy (ICB), ACu-NPs significantly enhance the CD8 + T cell-mediated anti-tumor immune response and effectively inhibit the growth of primary and metastatic tumors.

[0014] The beneficial effects obtained by the present invention are as follows: 1. The raw materials of the present invention are cheap and easily available, and the preparation process is simple, easy to operate and has good controllability; 2. The present invention innovatively constructs a nano-molecular system (ACu-NPs) based on amifostine and Cu 2+ . By specifically releasing Cu in the tumor microenvironment, 2+ the problem of normal cell toxicity caused by the non-specific distribution of copper ions is solved; The Cu(II) / Cu(I)-catalyzed Fenton-like reaction successfully consumes GSH and promotes the generation of ROS, effectively reversing the tumor immunosuppressive microenvironment and solving the problem of low response rate of ICB therapy; In addition, using ALP-mediated metabolism of amifostine to generate WR-1065 to scavenge ROS generated in normal cells and protect normal cells from oxidative damage, solving the toxicity problem of traditional copper ion carriers to normal tissues; Combined with immune checkpoint blockade therapy, it induces ICD and enhances the activity of CD8 + T cells, solving the problem of insufficient immune activation of single therapy and providing a new strategy for the treatment of primary and metastatic tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] By reading the following detailed description of the non-limiting embodiments with reference to the accompanying drawings, other features, objects and advantages of the present invention will become more apparent: FIG. 1 is a high-resolution transmission electron microscope photograph of the nano-molecular system ACu-NPs; Figure 2a is the particle size distribution diagram of the nano-molecular system ACu-NPs; Figure 2b is the Zeta potential diagram of the nano-molecular system ACu-NPs; Figure 3a is the infrared spectrum diagram of the nano-molecular system ACu-NPs; Figure 3b is the powder X-ray diffraction spectrum diagram of the nano-molecular system ACu-NPs; Figure 3c is the X-ray electron energy spectrum diagram of the nano-molecular system ACu-NPs; Figure 3d is the high-resolution X-ray electron energy spectrum diagram of the nano-molecular system ACu-NPs; Figure 4a is the relationship curve of glutathione content and reaction time under the same conditions for different groups; Figure 4b is the difference diagram of free radicals generated after the nano-molecular system ACu-NPs react for the same time under different pH conditions; Figure 5a is the statistical data diagram of the treatment toxicity of mouse fibroblasts (3T3) after different treatment processes; Figure 5b Statistical data graph of the treatment toxicity of mouse colorectal cancer cells (CT26) after different treatment processes; Figure 6a Curve of the relationship between the relative tumor volume of mice and time after different treatment methods of the nanomolecular system ACu-NPs; Figure 7 is a histological section of H&E staining of the heart, liver, spleen, lung, kidney and tumor sites of mice after different treatment methods. Specific implementation mode

[0016] The present invention will be described in detail below with reference to specific embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but do not limit the present invention in any form. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made. These all belong to the protection scope of the present invention.

[0017] A preparation method of a nanomolecular system, the nanomolecular system is composed of the cytoprotectant amifostine (AT) and the key nutrient metal copper ion Cu 2+ Prepared by using the supramolecular self-assembly technology; its supramolecular self-assembly structural formula is as follows: The molar ratio of AT to Cu 2+ is 1:1.

[0018] The preparation method includes the following specific steps: AT:Cu 2+ The stirring and mixing time is 12 hours, and the nanoscale crude product is obtained by centrifugal separation at 10000 rpm×10 min; then it is washed 3 times with deionized water to remove free molecular monomers, and then the nanoscale pure solid product is obtained by centrifugal separation at 12000 rpm×10 min; finally, mouse serum albumin (MSA) is used as an emulsifier and aggregation inhibitor, and it is dissolved and resuspended with 10 mL of deionized water solution containing 2% MSA, and ultrasonicated for 15-20 minutes at a power of 650W and an intensity of 45% to obtain the MSA-modified nanoscale copper ion carrier ACu-NPs.

[0019] Example 1 This example provides a preparation method of a nanomolecular system ACu-NPs, which specifically includes the following steps: Prepare 100 mL of deionized aqueous solution of CuCl2•2H2O (10.0 mM, pH = 7.4) and 100 mL of AT (10.0 mM, pH = 7.4) HEPES buffer in advance for later use. Take 10 mL of CuCl2•2H2O (10.0 mM, pH = 7.4) in a 50 mL beaker, add 10 mL of AT (10 mM, pH = 7.4) HEPES buffer, and magnetically stir at a speed of 900 rpm for 12 hours to form a light blue solution. The nanoscale crude product is obtained by centrifugation (10000 rpm × 10 min), and then washed with deionized water (10 mL × 3 times) to remove free molecular monomers. The nanoscale pure solid product is obtained by centrifugation (12000 rpm × 10 min). Finally, it is dissolved and ultrasonically resuspended in 10 mL of deionized aqueous solution containing 2% MSA to finally obtain the nanoscale copper ion carrier ACu-NPs with a concentration of 10 mM ACu-NPs, with AT and Cu 2+ Quantify.

[0020] Characterization of nanoscale copper ion carrier ACu-NPs After diluting the prepared ACu-NPs nanoscale copper ion carrier by 100 times, it is dropped onto a carbon-coated support film (nickel mesh), and after natural drying, its morphology is observed with a high-resolution transmission electron microscope (HRTEM). The particle size of the solution is measured by a dynamic light scattering (DLS, Brookhaven 90 plus Zeta) particle size analyzer. The results are as Figure 1 shown. ACu-NPs are in the shape of irregular nanosheets with a length of about 200 nm. As Figure 2a shown in -b, the average particle size measured by the dynamic light scattering particle size analyzer is 183.01 nm, and the Zeta potential is -23.56 mV. The results are consistent with those obtained by high-resolution field emission scanning electron microscopy.

[0021] Optical study and elemental characterization of nanoscale copper ion carrier ACu-NPs The infrared spectrum of ACu-NPs is measured by an FTIR-650 Fourier transform infrared spectrometer using a pressed tablet of freeze-dried ACu-NPs powder. Powder X-ray diffraction is obtained by a D8 advanced XRD (Bruker, Germany) and copper Kα radiation (λ = 1.54 A). Elemental characterization is measured by a K-Alpha X-ray photoelectron spectrometer using freeze-dried ACu-NPs powder.

[0022] As Figure 3aAs shown, Fourier transform infrared spectroscopy (FT-IR) demonstrated the coordination relationship of AT in the nanoparticles, and ACu-NPs had characteristic absorption peaks of CuCl2 and AT. Characteristic absorption peaks belonging to the in-plane vibration of N-H and the stretching vibration of N-H of AT were observed at 1045 cm -1 , 1656 cm -1 and 3345 cm -1 . These absorption peaks still existed after the formation of ACu-NPs, indicating that Cu 2+ could not replace the hydrogen protons. Subsequently, the PXRD pattern of ACu-NPs was detected, and the results are as Figure 3b shown, indicating that ACu-NPs were amorphous. X-ray photoelectron spectroscopy (XPS) also confirmed the existence of the metal element copper, and its characteristic binding energy was consistent with the standard XPS of Cu 2+ ( Figure 3d ). In addition, other non-metal elements such as C, N, O, P, and S in ACu-NPs were further confirmed ( Figure 3c ).

[0023] Study on the Specific Consumption of Glutathione and the Ability to Generate Reactive Oxygen Species by the Nanoscale Copper Ion Carrier ACu-NPs To detect the consumption of glutathione and the generation of free radicals under different conditions, a classical colorimetric method based on the decay of methylene blue (MB) in a physiological environment was used for determination.

[0024] The sulfhydryl group of glutathione (GSH) can react with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) to produce 5'-thio-2'-nitrobenzoic acid, which is yellow, and its relative content change can be determined by ultraviolet absorption. The specific steps for determining GSH are as follows: GSH, as an important antioxidant in cells, plays a key role in maintaining the intracellular redox homeostasis with its excellent antioxidant and detoxification abilities. Equal amounts of the same concentration of materials (PBS, CuCl2, ACu-NPs) were added to the NaHCO3 buffer containing 1 mM·mL -1 GSH; after different reaction times (0, 30, 60, 90, 120, 150, 180 min), appropriate amounts of the above mixtures were taken to react with 4 mg·mL -1 DTNB, and the ultraviolet absorption intensity at 412 nm was immediately detected to evaluate the GSH scavenging ability. The experimental results showed that CuCl2 and ACu-NPs could effectively release Cu 2+ and degrade GSH, indicating that ACu-NPs could still consume GSH at the pH simulating the tumor microenvironment ( Figure 4a ).

[0025] Hydroxyl radical (•OH) is the free radical species that generates the main cytotoxicity in the Fenton-like reaction and can be detected by the methylene blue (MB) degradation method. The specific steps for measuring ROS are as follows: Place the ACu-NPs solution containing 15 μg·mL -1 MB in buffer solutions of different pH values (pH = 5.5, 6.5, 7.4). After 30 min, measure the ultraviolet absorption of MB at 664 nm to detect the degradation rate of MB. The more MB is degraded, the more free radicals are generated. The hydroxyl radical generation rates of ACu-NPs at various pH values were compared, as Figure 4b shown. The •OH generated by the Fenton-like reaction catalyzing H2O2 itself causes the slow decay of methylene blue; as the pH decreases, the rate of ACu-NPs generating ROS to degrade MB becomes faster, indicating that ACu-NPs can still promote the generation of •OH at the pH value simulating the tumor microenvironment.

[0026] Example 2 This example provides an in vitro pharmacodynamic test of the nano-scale copper ion carrier prepared in Example 1 on colorectal cancer cells (CT26) and fibroblasts (3T3). The cytotoxic effect of ACu-NPs on cells was determined using a CCK-8 kit. Inoculate 2×10 5 cells on a 96-well plate. After culturing at 37 °C for 24 h, dilute ACu-NPs with PBS to 9 gradients of 800, 400, 200, 100, 50, 25, 12.5, 6.25, 3.125 μM according to the Cu 2+ concentration, and set PBS as the control group. Take 100 μL and add it to the corresponding wells respectively. After incubating for 24 h, aspirate the culture medium and add CCK-8 reagent. After incubating in the incubator in the dark for two hours, detect the 96-well plate with an enzyme-labeled instrument. Set the wavelength to 450 nm to read the OD value and calculate the cell survival rate.

[0027] Figure 5a As shown, the cell survival rates of ACu-NPs at each concentration are greater than those of CuCl2, indicating that ACu-NPs have less toxicity to normal cells compared with the single-drug group, have high biosafety, and have an obvious protective effect. In Figure 5b , the anti-tumor effect of ACu-NPs appears; as the concentration increases, ACu-NPs show an obvious concentration-dependent cytotoxicity, indicating that ACu-NPs can effectively kill tumor cells.

[0028] Example 3 This example provides a test on the therapeutic effect of the nano-scale copper ion carrier prepared in Example 1 on CT26 tumor-bearing mice. CT26 tumor cells were taken, centrifuged, and resuspended in sterile normal saline to prepare a suspension containing 5×10 6 cells. 50 μL of the suspension was inoculated subaxillarily on the right side of Balb / c mice and they were fed normally. Wait for the tumors in the mice to grow to about 80 mm 3 -100 mm 3 or so, and then the tumor-bearing mice were randomly divided into 6 groups according to Saline, AT, CuCl2, ACu-NPs, ACu-NPs + αCD8a, and ACu-NPs + αPD-L1. The dosing dose was ([Cu 2+ = 34.3 mg kg -1 , [AT] = 12.5 mg kg -1 ), with 8 mice in each group. Drugs were injected into the caudal vein of the mice on days 0, 3, and 6 respectively. The tumor volume and body weight of the mice were measured every day for 14 days. After collecting the data according to the above grouping, the mice were euthanized. The hearts, livers, spleens, lungs, kidneys, and tumor tissues of the mice were taken, fixed in 4% formaldehyde, and embedded in paraffin for sectioning. After H&E staining, the structural changes of each organ and tumor tissue were observed under a microscope.

[0029] After drug treatment on the CT26 tumor-bearing mouse model, the changes in the tumor volume of the mice were recorded. As shown in Figure 6, in the blank control group, the tumor grew rapidly during the 14-day monitoring period. In the experimental group, ACu-NPs had a certain inhibitory effect on tumor growth. In the ACu-NPs + αPD-L1 combination treatment group, the growth of the tumor volume in the mice was significantly inhibited. Especially after the 14-day treatment cycle ended, the tumor volume basically did not grow, indicating that the ACu-NPs nanodrug could effectively combine with the immune checkpoint blockade therapy and significantly inhibit the growth of tumor cells.

[0030] Subsequently, H&E staining section analysis was performed on the hearts, livers, spleens, lungs, kidneys, and tumor tissues of the treated mice. From the H&E section results of the hearts, livers, spleens, lungs, and kidneys of the mice, it can be seen that, such as Figure 7 , AT, ACu-NPs, and ACu-NPs + αPDL1 had high biosafety and basically no toxicity. On the contrary, the CuCl2 group caused greater damage to the kidney and liver tissues. Comparing the H&E section results of the tumor tissues with other groups, the tissue space in the tumor tissue section of the ACu-NPs group became larger, the nucleus shrank, and there were obvious pathological changes. After combination with αPDL1, this pathological change was more obvious, indicating that the ACu-NPs nanodrug could effectively inhibit the growth of tumor cells while protecting normal tissue cells.

[0031] Comparative Example 1 The difference between this comparative example and Example 1 is only that mesna is used instead of amifostine as a cytoprotectant.

[0032] Using the preparation method of Example 1 for this comparative example, no effective product precipitation can be obtained; through various optimization methods (changing the feed ratio, changing the dissolution buffer, adjusting the pH, adjusting the reaction temperature, etc.), no reaction product with the Tyndall phenomenon can be obtained.

[0033] Comparative Example 2 The difference between this comparative example and Example 1 is only that copper chloride is replaced with copper nitrate.

[0034] Repeating the DLS and Zeta tests of Example 1 on the product obtained from this comparative example, DLS shows an average particle size of 369.72 nm, which is relatively large and not conducive to the implementation of systemic administration via the mouse tail vein in Example 3; and the Zeta potential result is -3.89 mV, with a relatively low absolute value, and the biological stability is poorer compared to Example 1.

[0035] Comparative Example 3 The difference between this comparative example and Example 1 is only that the reaction condition of amifostine and copper ions has a pH < 6.

[0036] Using the preparation method of Example 1 for this comparative example, less product precipitation is obtained and the yield is low; repeating the study on the ability to specifically consume glutathione and generate reactive oxygen species in Example 1, the results show that compared with Example 1, less GSH is consumed within the same reaction time, and the generation rate of hydroxyl radicals is low; the reason may be that low pH inhibits the deprotonation of amifostine, resulting in weakened coordination ability.

[0037] The specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art can make various deformations or modifications within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. A nano-molecular system, characterized in that, It is formed by supramolecular self-assembly of amifostine and copper ions, and the structural formula is as shown in Formula I: I。 2. A method for preparing a nanomolecular system as described in claim 1, characterized in that, It includes the following steps: Add a copper ion-containing solution to the amifostine solution, stir and mix at a rotation speed of 800-1000 rpm for 12-48 h, and then centrifuge at a rotation speed of 9000-11000 rpm to obtain a nanoscale crude product; Wash the nanoscale crude product with deionized water to remove free molecular monomers, and then centrifuge at a rotation speed of 11000-13000 rpm to obtain a nanoscale pure solid product; Add the nanoscale pure solid product to deionized water containing mouse serum albumin, and ultrasonically dissolve and resuspend it at a power of 600-700 W and an intensity of 40-50% to obtain the nanoscale copper ion carrier.

3. The preparation method of the nano-molecular system according to claim 2, wherein, The molar ratio of amifostine to copper ions is 1:(1-3).

4. The preparation method of the nano-molecular system according to claim 2, characterized in that, The copper ion-containing solution is a copper chloride solution.

5. The preparation method of the nano-molecular system according to claim 2, characterized in that, The molar concentration of the amifostine solution is 10 mM, pH = 7.4; the molar concentration of the copper ion-containing solution is 10 mM.

6. The preparation method of the nano-molecular system according to claim 2, characterized in that, In the deionized water containing mouse serum albumin, the mass concentration of mouse serum albumin is 2%.

7. Use of a nanomolecular system as described in claim 1 in a drug for inhibiting the growth, metastasis and spread of tumor cells.

8. A tumor treatment drug, characterized in that, Containing αPD-L1 and the nanomolecular system described in claim 1.

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