Copper-chelated nanodrugs and methods of making the same

Abstract

This invention belongs to the interdisciplinary fields of nanomedicine, tumor immunotherapy, and chemotherapy, and discloses a copper chelate nanomedicine and its preparation method. The preparation method of the copper chelate nanomedicine includes the following steps: S1, firstly, a mercaptopropionic acid intermediate is prepared by performing a thiol addition reaction on a mitochondrial targeting group using mercaptopropionic acid; S2, secondly, the mercaptopropionic acid intermediate is coupled to a copper ion chelating group via an amidation reaction to obtain the compound IR-TIE; S3, the obtained IR-TIE is self-assembled with albumin to form IR-TIE@Alb nanoparticles. This invention exhibits precise tumor mitochondrial targeting, effectively downregulating free copper ions in tumor cells at extremely low doses, thereby downregulating IDO1 and LOX, achieving innate immune activation, reducing tumor collagen cross-linking, loosening the tumor cell extracellular matrix, and promoting drug penetration.

Description

Technical Field

[0001] This invention belongs to the interdisciplinary fields of nanomedicine, tumor immunotherapy and chemotherapy, and specifically relates to a copper chelate nanomedicine and its preparation method. Background Technology

[0002] Traditional copper-lowering drugs (such as penicillamine and triethylamine) can reduce blood copper, but they have poor tumor selectivity, require high doses, have serious side effects, and their biological effects are unclear. There are no reports of drugs that can simultaneously regulate IDO1 / LOX.

[0003] Specifically, existing copper-chelating drugs have the following shortcomings:

[0004] 1. Insufficient targeting and efficiency: Conventional copper-chelating drugs have difficulty specifically enriching tumors and mitochondria, limiting their precise copper chelation ability.

[0005] 2. Dosage and safety conflict: Higher systemic exposure is required to achieve efficacy, increasing the risk of liver and kidney toxicity and systemic side effects.

[0006] 3. Limited biological function: Most strategies focus on PD-L1-related regulation. In biological function research, there is a lack of comprehensive immune remodeling strategies that can enhance T cell immunity (IDO1 downregulation) and improve T cell penetration into tumor tissue (LOX downregulation).

[0007] 4. Insufficient treatment compatibility and synergy: There is a lack of evidence to inhibit and synergize with chemotherapy-induced IDO1 / LOX upregulation, making it difficult to support the translational application of combined standard therapy.

[0008] Therefore, there is an urgent need for a novel copper-free nanomedicine system that is mitochondrial-targeted, low-dose and highly effective, with strong tumor selectivity, bidirectional downregulation of IDO1 / LOX, and compatible with sensitization by chemotherapy / immunotherapy. Summary of the Invention

[0009] The purpose of this invention is to provide a copper chelate nanomedicine and its preparation method, so as to solve the problems of insufficient targeting and efficiency, contradiction between dosage and safety, single biological function, and insufficient therapeutic compatibility and synergy of existing copper-chelating drugs mentioned in the background art.

[0010] To achieve the above objectives, the present invention provides a method for preparing copper chelate nanomedicines, comprising the following steps:

[0011] S1. First, mercaptopropionic acid (MPA) is used to perform a thiol addition reaction on the mitochondrial targeting group (IR) to prepare a mercaptopropionic acid intermediate.

[0012] S2. Next, the mercaptopropionic acid intermediate is coupled to a copper ion chelating group via an amidation reaction to obtain the compound IR-TIE.

[0013] S3. The obtained IR-TIE is self-assembled with albumin (Alb) to form IR-TIE@Alb nanoparticles.

[0014] In one specific embodiment, the mitochondrial targeting group (IR) is heptamethylene cyanide dye (IR780).

[0015] In one specific implementation, step S1 consists of the following steps:

[0016] In anhydrous dimethylformamide (DMF), mercaptopropionic acid (MPA) is added to heptamethine cyanide dye and reacted at 30-45°C for 6-8 hours under nitrogen protection. After precipitation with diethyl ether and solvent removal by rotary evaporation, IR780-MPA with free carboxyl groups is generated.

[0017] In one specific embodiment, the copper ion chelating group is tripyridinemethylamine (TIE).

[0018] In one specific implementation, step S2 consists of the following steps:

[0019] The mercaptopropionic acid intermediate was dissolved in DMF or dichloromethane, and its carboxyl group was activated using EDC / NHS. Then, tripyridine methylamine containing a primary amine group was added.

[0020] The primary amine undergoes amidation coupling with the activated carboxyl group after stirring at 40-50℃ for 12-24 hours.

[0021] After the reaction was completed, the compound IR-TIE was obtained by silica gel column chromatography.

[0022] In one specific implementation, step S3 consists of the following steps:

[0023] The obtained IR-TIE was dissolved in dimethyl sulfoxide and then added to albumin solution. The mixture was stirred at room temperature in the dark for 2-3 hours. The solution was then poured into an ultrafiltration vessel to remove dimethyl sulfoxide and concentrate the volume. The concentrated solution was then lyophilized and then redissolved in deionized water to obtain IR-TIE@Alb.

[0024] In one specific embodiment, the concentration of the albumin solution is 5 mg / ml.

[0025] The present invention also provides a copper chelate nanomedicine, which is prepared by the copper chelate nanomedicine preparation method described above.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] This invention provides a method for preparing copper chelate nanomedicines. The prepared copper chelate nanomedicines have precise tumor mitochondrial targeting and can effectively downregulate free copper ions in tumor cells at extremely low doses, thereby downregulating IDO1 and LOX to achieve innate immune activation. At the same time, by inhibiting the LOX-mediated collagen cross-linking process, the density of collagen fibers in tumor tissue is reduced, thereby remodeling and loosening the tumor cell extracellular matrix and promoting drug penetration.

[0028] This invention constructs a tumor-selectively enriched and biocompatible nano-formulation, forming a scalable, component-controllable, albumin self-assembly formulation pathway, which has the translational potential and industrialization feasibility of imaging tracing (near-infrared) + therapy.

[0029] This invention overcomes the secondary immunosuppression induced by chemotherapy-induced IDO1 / LOX upregulation and achieves synergistic sensitization through copper-depleted immune remodeling and chemotherapy.

[0030] This invention provides a small molecule IR-TIE conjugated with a near-infrared dye and a functional chelating group, which self-assembles with albumin (Alb) to form nanoparticles (IR-TIE@Alb). In vivo, IR-TIE@Alb leverages albumin's active / passive tumor homing and mitochondrial affinity fragments to achieve dual tumor-mitochondrial targeting. At extremely low doses, it selectively removes copper, increasing the ADP / ATP ratio → activating AMPK → inhibiting C-MYC → downregulating IDO1. Simultaneously, due to its precise targeting of the mitochondrial "copper pool," it efficiently chelates copper ions, leading to downregulation of the copper-dependent enzyme LOX → disrupting LOX-collagen cross-links, thereby enhancing T cell killing, improving T cell and drug penetration into tumor tissue, and blocking the tumor immunosuppressive microenvironment induced by chemotherapy.

[0031] This invention enhances T-cell tumor cell killing through a precise copper chelation strategy in two ways: first, by inhibiting IDO1 to enhance T-cell activity; and second, by inhibiting LOX to promote T-cell infiltration into tumor cells. Simultaneously, it reverses chemotherapy-induced IDO1 / LOX upregulation, improves the immune microenvironment induced by chemotherapy, and promotes tumor penetration of chemotherapeutic drugs.

[0032] In addition to the objectives, features, and advantages described above, the present invention has other objectives, features, and advantages. The present invention will now be described in further detail. Attached Figure Description

[0033] The accompanying drawings, which form part of this application, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings:

[0034] Figure 1This is a schematic diagram of the synthesis of IR-TIE according to an embodiment of the present invention.

[0035] Figure 2 This is a schematic diagram of the synthesis of IR-TIE@Alb according to an embodiment of the present invention.

[0036] Figure 3 This is a scanning electron microscope (SEM) image of IR-TIE@Alb nanoparticles according to an embodiment of the present invention. The SEM image shows that the IR-TIE@Alb nanoparticles have a uniformly dispersed spherical structure with uniform particle size and smooth surface.

[0037] Figure 4 This is a dynamic light scattering (DLS) image of IR-TIE@Alb nanoparticles according to an embodiment of the present invention. The DLS image results show that the average hydrated particle size of IR-TIE@Alb is 86.95±0.88nm, with a narrow distribution and good stability.

[0038] Figure 5 This is a UV-Vis absorption spectrum of IR-TIE@Alb nanoparticles according to an embodiment of the present invention. Analysis shows that IR-TIE@Alb exhibits a distinct characteristic absorption peak at 780 nm, corresponding to the near-infrared absorption signal of IR-TIE molecules, proving that IR-TIE was successfully loaded into the albumin carrier (Alb) to form a stable complex.

[0039] Figure 6 This is a comparison of Western blot results of MB49 cells treated with different concentrations of IR-TIE@Alb according to an embodiment of the present invention. The results show that after treating MB49 cells with different concentrations of IR-TIE@Alb (0~2μM), the expression of IDO1 and LOX proteins decreased in a dose-dependent manner.

[0040] Figure 7 This is a comparison of Western blot results of MB49 cells treated with different drugs (Vehicle, TIE, IR780@Alb, IR-TIE@Alb) according to one embodiment of the present invention. The results show that the expression levels of IDO1 and LOX proteins were most significantly downregulated in the IR-TIE@Alb group.

[0041] Figure 8 This is a confocal immunofluorescence staining image of the expression level of IDO1 in cells after treatment with different drugs (Vehicle, TIE, IR780@Alb, IR-TIE@Alb) according to one embodiment of the present invention. It further verifies that IR-TIE@Alb can improve the tumor microenvironment by inhibiting immunosuppression-related pathways. Scale bar = 20 μm.

[0042] Figure 9This is a confocal immunofluorescence staining image of the expression level of LOX in cells after treatment with different drugs (Vehicle, TIE, IR780@Alb, IR-TIE@Alb) according to one embodiment of the present invention. It further verifies that IR-TIE@Alb can improve the tumor microenvironment by inhibiting immunosuppression-related pathways. Scale bar = 20 μm.

[0043] Figure 10 This is a confocal laser microscope image showing the co-localization of IR-TIE@Alb and the mitochondrial fluorescent probe MitoTracker in tumor cells according to an embodiment of the present invention. The results show that the IR-TIE@Alb signal and the MitoTracker signal highly overlap, and the co-localization scatter plot shows a clear diagonal distribution, indicating that IR-TIE@Alb has good mitochondrial targeting.

[0044] Figure 11 This is a fluorescence imaging image of IR-TIE@Alb in mice according to an embodiment of the present invention. It shows that after intravenous injection, IR-TIE@Alb can gradually accumulate in the tumor site within 1 to 12 hours, and the signal intensity reaches its peak at 24 hours. It still maintains obvious tumor site retention after 48 hours.

[0045] Figure 12 This is an in vitro fluorescence imaging image of the main organs and tumors of IR-TIE@Alb in mice 24 hours after administration according to an embodiment of the present invention. The results show that IR-TIE@Alb is mainly distributed in tumor tissue, while the fluorescence signal in other organs is weak, further verifying its significant tumor targeting.

[0046] Figure 13 This is a visual image showing the results of an in vitro hemolysis experiment of IR-TIE@Alb according to an embodiment of the present invention.

[0047] Figure 14 This is a hemolysis rate diagram of an in vitro hemolysis experiment of IR-TIE@Alb according to an embodiment of the present invention. Different concentrations of IR-TIE@Alb (50, 100, 150, 300 mg / mL) were incubated with red blood cell suspensions, with distilled water and physiological saline as positive and negative controls, respectively. The results showed that the hemolysis rate of the distilled water group was close to 100%, while the hemolysis rate of each concentration of IR-TIE@Alb treatment group was less than 5%, which was comparable to that of the physiological saline group, indicating that IR-TIE@Alb nanoparticles have good blood compatibility and biosafety.

[0048] Figure 15This is an organ HE staining image according to an embodiment of the present invention; after mice were injected with IR-TIE@Alb nanoparticles via the tail vein, histological sections of major organs such as the heart, liver, spleen, lung, and kidney were taken after administration, and the morphological changes of the tissues were observed by hematoxylin-eosin (H&E) staining; the results showed that, compared with the control group, the tissue structure of each organ was intact, and no obvious inflammatory cell infiltration, necrosis or pathological damage was observed, suggesting that IR-TIE@Alb nanoparticles have good biocompatibility and in vivo safety.

[0049] Figure 16 This is an image showing the immunohistochemical staining results of IDO1 and LOX in tumor tissue of the same patient before and after cisplatin chemotherapy, according to an embodiment of the present invention with IR-TIE@Alb combined with chemotherapy; IDO1 and LOX were significantly upregulated after chemotherapy.

[0050] Figure 17 This is a diagram showing the results of a colony formation experiment of IR-TIE@Alb combined with chemotherapy according to an embodiment of the present invention. The results show that the combination of IR-TIE@Alb and chemotherapy drugs can significantly reduce the number of clones, suggesting that IR-TIE@Alb can enhance the cell-killing effect of chemotherapy.

[0051] Figure 18 This is a schematic diagram of the animal experiment process of IR-TIE@Alb combined with cisplatin according to one embodiment of the present invention; after subcutaneous inoculation of the primary tumor in mice, drug injection was performed on days 0, 2, 4 and 8, and cisplatin (Cis-Pt) was administered on days 1, 3, 6 and 9. The primary tumor was removed on day 14, and after a week of rest, homologous tumor cells were inoculated again on the contralateral side to evaluate the immune memory effect.

[0052] Figure 19 This is a comparison image of primary tumors after removal in different treatment groups (1, Vehicle; 2, Cis-Pt; 3, IR-TIE@Alb; 4, Cis-Pt+IR-TIE@Alb) according to an embodiment of the present invention.

[0053] Figure 20 This is a graph showing the change in primary tumor volume over time in different treatment groups according to an embodiment of the present invention; the results show that the Cis-Pt+IR-TIE@Alb combination group has the most significant tumor suppression effect.

[0054] Figure 21 This is a statistical result of tumor endpoint quality in different treatment groups according to an embodiment of the present invention; the tumor quality in the combination group was significantly lower than that in the single-drug group and the control group (n=5, mean ± standard deviation), indicating that IR-TIE@Alb can significantly enhance the in vivo antitumor efficacy of cisplatin and may induce long-term immune memory.

[0055] Figure 22This diagram illustrates the pathological changes and immune-related protein expression in tumor tissues from different treatment groups (control group, cisplatin group, IR-TIE@Alb group, and cisplatin + IR-TIE@Alb group) according to one embodiment of the present invention. HE and immunohistochemical (IHC) staining were used for evaluation. HE staining results showed that the combination therapy group exhibited significantly increased disordered tumor cell arrangement, nuclear pyknosis, and necrotic areas, indicating the most significant cell damage. The single-drug groups also showed some degree of tissue necrosis, but it was less pronounced than in the combination group. Immunohistochemical results showed that IDO1 and LOX expression was enhanced in the cisplatin group, but significantly decreased in the IR-TIE@Alb single-drug and combination therapy groups, especially the lowest expression in the combination group. This indicates that IR-TIE@Alb can effectively inhibit cisplatin-induced immunosuppression pathways, thereby enhancing its anti-tumor effect.

[0056] Figure 23 This is a comparison image of the tumors removed from mice subjected to secondary tumor-bearing processes in different treatment groups (1, Vehicle; 2, Cis-Pt; 3, IR-TIE@Alb; 4, Cis-Pt+IR-TIE@Alb) according to one embodiment of the present invention.

[0057] Figure 24 This is a secondary tumor volume change curve over time according to an embodiment of the present invention; it shows that tumor growth in the Cis-Pt+IR-TIE@Alb combination group was significantly inhibited, with significant differences compared to the single-drug group and the control group.

[0058] Figure 25 This is a statistical result of the secondary tumor endpoint quality in one embodiment of the present invention. It shows that the tumor quality in the combined group is the lowest, suggesting that IR-TIE@Alb and cisplatin can significantly inhibit tumor recurrence and regeneration, and exhibit a durable anti-tumor and immune memory effect.

[0059] Figure 26 This is a graph illustrating the effect of IR-TIE@Alb combined with cisplatin treatment on T-cell immune infiltration in tumor tissue according to an embodiment of the present invention; flow cytometry was used to detect the distribution of T-cell subsets in tumor tissues of different treatment groups (control group, cisplatin group, IR-TIE@Alb group, cisplatin + IR-TIE@Alb group); the top row shows CD3+. + The proportion of T cells in the total tumor cell population, shown in the bottom row, is derived from CD3. + CD4 after group + With CD8 + Cell distribution; results showed that CD3 in the combination therapy group (cisplatin + IR-TIE@Alb) was significantly higher than that in the control group. + The proportion of cell infiltration increased significantly, CD8 + The proportion of effector T cells increased from 14.5% to 46.5%, while CD4... +The relative decrease in the proportion of cells suggests that combination therapy can effectively promote the growth of cytotoxic T cells (CD8+) within the tumor. + It infiltrates and enhances anti-tumor immune activity.

[0060] Figure 27 This is a comparative graph of cisplatin content in tumor tissue detected by ICP-MS according to an embodiment of the present invention. The platinum content in tumor tissue of different treatment groups was determined by inductively coupled plasma mass spectrometry (ICP-MS) to assess the enrichment of the drug in the tumor. The results showed that compared with the cisplatin-only (Cis-Pt) treatment group, the platinum content in the tumor tissue of the combination drug treatment group (Cis-Pt+IR-TIE@Alb) was significantly increased. This indicates that IR-TIE@Alb promotes the accumulation of cisplatin in the tumor site by inhibiting LOX-collagen junctions, which leads to a loosening of the extracellular matrix of the tumor tissue and improves the drug delivery efficiency and local chemotherapy concentration. Detailed Implementation

[0061] The embodiments of the present invention will be described in detail below. The specific embodiments described herein are merely illustrative of the present invention and are not intended to limit the present invention.

[0062] Example 1

[0063] The present invention provides a method for preparing a copper chelate nanomedicine, comprising the following steps:

[0064] S1. First, mercaptopropionic acid (MPA) is used to perform a thiol addition reaction on the mitochondrial targeting group (IR) to introduce a carboxyl functional group and prepare a mercaptopropionic acid intermediate.

[0065] Preferably, the mitochondrial targeting group (IR) is heptamethylene cyanide dye (IR780). As a parent dye for near-infrared absorption and emission, IR780 has a molecular backbone with positively charged polymethyl chains and terminal carboxyl or sulfonic acid groups, enabling: mitochondrial membrane potential-driven targeted enrichment, near-infrared imaging through in vivo tissue penetration, and self-assembly ability with albumin binding.

[0066] The specific steps of step S1 are as follows:

[0067] In anhydrous dimethylformamide (DMF), mercaptopropionic acid (MPA) was added to heptamethine cyanide dye (IR780), and the reaction was carried out at 30°C for 6-8 hours under nitrogen protection. After precipitation with diethyl ether and solvent removal by rotary evaporation, IR780-MPA with free carboxyl groups was generated.

[0068] S2. Next, the mercaptopropionic acid intermediate is coupled with a copper ion chelating group through an amidation reaction to obtain the compound IR-TIE.

[0069] Preferably, the copper ion chelating group is tripyridinemethylamine (TIE). TIE is a typical polydentate ligand that can chelate divalent copper ions (Cu). 2+ It coordinates stably in a 1:1 molar ratio to form a five- or six-coordinate chelate structure. Its nitrogen coordinating atoms are symmetrically distributed, exhibiting high affinity and selectivity, and can effectively capture free copper ions and achieve precise copper removal or controlled copper delivery.

[0070] The specific steps of step S2 are as follows:

[0071] The mercaptopropionic acid intermediate IR780-MPA was dissolved in DMF or dichloromethane, and its carboxyl group was activated using EDC / NHS. Then, tripyridine methylamine containing a primary amine group was added.

[0072] When the reaction is stirred at room temperature to 50°C for 12 to 24 hours, the primary amine undergoes amidation coupling with the activated carboxyl group to form a stable IR-TIE conjugated small molecule.

[0073] After the reaction was completed, the compound IR-TIE was purified by silica gel column chromatography (mobile phase: CH2Cl2 / methanol = 10:1) to obtain the compound.

[0074] IR-TIE possesses both an IR780 backbone and a TIE coordination terminus. The IR780 backbone provides mitochondrial targeting and near-infrared fluorescence visualization capabilities; the TIE coordination terminus can efficiently chelate copper ions and induce a sharp reduction in the copper ion content of the mitochondrial "copper pool".

[0075] S3. The obtained IR-TIE is self-assembled with albumin (Alb) to form IR-TIE@Alb nanoparticles.

[0076] The specific steps of step S3 are as follows:

[0077] 3.58 mg of IR-TIE was dissolved in 0.5 ml of dimethyl sulfoxide (DMSO), and then added to 50 ml of albumin solution (5 mg / ml). The solution was stirred at room temperature in the dark for 2 h, and then poured into an ultrafiltration cup equipped with a 30 kDa ultrafiltration membrane to remove the dimethyl sulfoxide and concentrate the volume to 10 ml. To obtain nanoparticles with uniform particle size, the concentrated solution was lyophilized and then redissolved in deionized water to obtain IR-TIE@Alb.

[0078] The IR-TIE molar mass was 915.4779; the albumin molar mass was 64000. A 50ml albumin solution with a concentration of 5mg / ml was prepared by adding 250mg of bovine serum albumin to 50ml of DDW.

[0079] The present invention also provides a copper chelate nanomedicine, which is prepared by the copper chelate nanomedicine preparation method described above.

[0080] The nanomedicine of this invention possesses the following characteristics: precise chelation and regulation of copper ions, mitochondrial-specific localization and visual tracking, albumin-mediated long-term in vivo circulation and tumor-targeted enrichment, high stability and excellent biocompatibility.

[0081] The above description, in conjunction with specific preferred embodiments, provides a further detailed explanation of the present invention. It should not be construed that the specific implementation of the present invention is limited to these descriptions. For those skilled in the art, various simple deductions and substitutions can be made without departing from the concept of the present invention, and all such modifications and substitutions should be considered within the scope of protection of the present invention.

Claims

1. A copper-chelating nanodrug, characterized in that, The copper chelated nanomedicine was prepared by the following method: S1. First, mercaptopropionic acid is used to perform a thiol addition reaction on a mitochondrial targeting group to prepare a mercaptopropionic acid intermediate; the mitochondrial targeting group is heptamethine cyanide dye. The structural formula of the heptamethylene cyan dye is: ; The structural formula of the mercaptopropionic acid intermediate is: ; S2. Next, the mercaptopropionic acid intermediate is coupled to a copper ion chelating group via an amidation reaction to obtain compound IR-TIE; the copper ion chelating group is tripyridinemethylamine; the specific steps are as follows: The mercaptopropionic acid intermediate was dissolved in DMF or dichloromethane, and its carboxyl group was activated using EDC / NHS. Then, tripyridine methylamine containing a primary amine group was added. The primary amine undergoes amidation coupling with the activated carboxyl group after stirring at 40-50℃ for 12-24 hours. After the reaction was completed, the compound IR-TIE was obtained by silica gel column chromatography for purification. The structural formula of tripyridinemethylamine is: ; The structural formula of the compound IR-TIE is: ; S3. The obtained IR-TIE is self-assembled with albumin to form IR-TIE@Alb nanoparticles.

2. The copper chelate nanomedicine according to claim 1, characterized in that, The specific steps of step S1 are as follows: In anhydrous dimethylformamide, mercaptopropionic acid was added to heptamethine cyanide dye and reacted at 30-45°C for 6-8 hours under nitrogen protection. The product was precipitated with diethyl ether and the solvent was removed by rotary evaporation to generate IR780-MPA with free carboxyl groups.

3. The copper chelate nanomedicine according to claim 1, characterized in that, The specific steps of step S3 are as follows: The obtained IR-TIE was dissolved in dimethyl sulfoxide and then added to albumin solution. The mixture was stirred at room temperature in the dark for 2-3 hours. The solution was then poured into an ultrafiltration vessel to remove dimethyl sulfoxide and concentrate the volume. The concentrated solution was then lyophilized and then redissolved in deionized water to obtain IR-TIE@Alb.

4. The copper chelate nanomedicine according to claim 3, characterized in that, The concentration of the albumin solution is 5 mg / ml.

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