Manganese diethyldithiocarbamate loaded nanoparticles and pharmaceutical composition

Abstract

The invention belongs to the technical field of biological medicine and tumor diagnosis and treatment, and particularly relates to manganese diethyldithiocarbamate loaded nanoparticles and a pharmaceutical composition. The nanoparticles loaded with the manganese diethyldithiocarbamate are prepared from an amphiphilic polymer and diethyldithiocarbamate according to the mass ratio of (0.25 to 4) to 1; the manganese diethyldithiocarbamate accounts for more than 50% of the molar mass of the diethyldithiocarbamate; the amphiphilic polymer is one or more of DSPE (Distearoyl Polyethylene)-PEG (Polyethylene Glycol), DSPE-PEG-folic acid, DSPE-PEG-phenylboronic acid and DSPE-PEG-arginyl glycyl aspartic peptide. According to the application, the amphiphilic polymer of the DSPE-PEG and the derivative of the DSPE-PEG and the ethyl dithiocarbamate are adopted to form the nano-particles, and hydrophobic manganese ethyl dithiocarbamate can be wrapped in a hydrophobic micro-area.

Description

Technical Field

[0001] This application belongs to the field of biomedicine and tumor diagnosis and treatment technology, specifically relating to nanoparticles loaded with manganese diethyldithiocarbamate and a pharmaceutical composition. Background Technology

[0002] Disulfiram (DSF), a traditional alcohol remedy, has recently been found to possess significant antitumor activity. The Cu(DDTC)2 complex (CuET), formed by the binding of diethyldithiocarbamate (DDTC) to copper ions during its metabolism in vivo, is considered a key active substance in its anticancer effects. Studies have shown that CuET can inhibit tumor growth through multiple mechanisms, including inducing copper death (cuproptosis), mitochondrial dysfunction, and immunogenic cell death (ICD).

[0003] To improve tumor targeting and reduce systemic toxicity, various nanosystems have been developed for the in-situ generation of CuET at tumor sites. However, most nanosystems cannot monitor the CuET generation process at tumor sites, severely limiting the clinical translation and personalized treatment evaluation of this strategy. A few existing technologies can monitor the CuET generation process (e.g., the literature "A fibroblast activation protein α-activatable nanoagent co-delivering diethyldithiocarbamate and copper for tumor therapy and imaging"), but due to their complex synthesis steps, complicated preparation, high cost, and limited fluorescence imaging depth, they cannot achieve non-invasive detection of deep tumor tissue, thus limiting their clinical application.

[0004] Existing technologies include DSPE-PEG nanoparticles that can achieve MRI monitoring (e.g., the literature AMagnetofluorescent Carbon Dot Assembly as an Acidic H2O2-Driven Oxygenerator to Regulate Tumor Hypoxia for Simultaneous Bimodal Imaging and Enhanced Photodynamic Therapy). However, the function of these nanoparticles is limited to in vivo tracking and cannot be combined with CuET in situ synthesis technology, thus preventing their application in the CuET system.

[0005] Patent document CN116392455A discloses CuET nanoparticles co-stabilized with hydroxyalkyl starch and polydopamine. These nanoparticles are formed by co-stabilizing CuET nanocrystals with hydroxyalkyl starch and polydopamine, wherein hydroxyalkyl starch and polydopamine coat the surface of the CuET nanocrystals to form the co-stabilized CuET nanoparticles. However, the preparation process is complex, and the serum stability of CuET prepared from hydroxyalkyl starch is poor, requiring polydopamine co-stabilization, thus limiting its clinical translation potential. Summary of the Invention

[0006] Therefore, there is an urgent need to develop a novel therapeutic strategy that can both achieve in situ generation of CuET and report its formation process in real time, so as to ensure that the drug development process can be monitored and ultimately enhance the anti-tumor immune response.

[0007] This application provides a reportable sequential drug delivery system that uses Mn² + The release coupled with CuET generation enabled, for the first time, magnetic resonance imaging (MRI) visualization and monitoring of CuET formation, while simultaneously enhancing the anti-tumor immune response.

[0008] To achieve the above objectives, in a first aspect, this application provides nanoparticles loaded with manganese diethyldithiocarbamate, comprising an amphiphilic polymer in a mass ratio of (0.25~4):1 and a manganese diethyldithiocarbamate; the manganese diethyldithiocarbamate accounts for more than 50% of the molar mass of the manganese diethyldithiocarbamate. The amphiphilic polymer is one or more of DSPE-PEG (1,2-distearate-sn-glycerol-3-phosphoethanolamine-polyethylene glycol), DSPE-PEG-folic acid, DSPE-PEG-phenylboronic acid, and DSPE-PEG-arginylglycyl aspartic peptide.

[0009] Preferably, the amphiphilic polymer is DSPE-PEG with a molecular weight of 1000-5000.

[0010] Preferably, the mass ratio of the amphiphilic polymer to diethyldithiocarbamate is (0.5~1):1.

[0011] Preferably, the particle size of the nanoparticles is 70 nm to 1000 nm.

[0012] As a further preferred embodiment, the nanoparticles have a particle size of 100 nm to 120 nm.

[0013] Secondly, this application provides a method for preparing the above-mentioned nanoparticles, comprising the following steps: A 0.2%–0.3% (w / w) amphiphilic polymer solution was uniformly mixed with a 0.12%–0.15% (w / w) DDTC solution, and then Mn²⁺ solution with a concentration of 0.009 mol / L–0.015 mol / L was added. + The solution is subjected to ultrasound to obtain the nanoparticles; the solute in the DDTC solution is one or more of sodium diethyldithiocarbamate, potassium diethyldithiocarbamate, or ammonium diethyldithiocarbamate. In the amphiphilic polymer solution, the mass ratio of the amphiphilic polymer to the solute in the DDTC solution is (2~20):1, and the Mn... 2+ The molar mass ratio of ions to the solute in the DDTC solution is (0.25~0.85):1.

[0014] Preferably, the power of the ultrasound is 100 W to 300 W, and the duration is 30 seconds to 90 seconds.

[0015] Thirdly, this application also provides a pharmaceutical composition comprising the nanoparticles loaded with diethyldithiocarbamate, and further comprising Cu 2+ Solution, the Cu 2+ Cu in solution 2+ The molar mass ratio of the nanoparticles to manganese diethyldithiocarbamate is greater than or equal to 1.

[0016] Preferably, the Cu 2+ Cu in solution 2+ The molar concentration is 0.1 mM ~ 10 mM.

[0017] As a further preferred embodiment, the Cu 2+ Cu in solution 2+ The molar concentration is 0.25 mM ~ 1 mM.

[0018] Preferably, the Cu 2+ The solute in the solution is one or more of CuCl2, CuSO4, Cu(NO3)2, or copper gluconate.

[0019] Preferably, the pharmaceutical composition is used to treat breast cancer, lung cancer, liver cancer, or colon cancer.

[0020] In summary, compared with the prior art, the technical solutions conceived in this application have the following main technical advantages: 1. Nanoparticles are formed by combining amphiphilic polymers of DSPE-PEG and its derivatives with ethyl dithiocarbamate. By controlling the mass ratio of the amphiphilic polymer to diethyl dithiocarbamate at (0.25~4):1, a highly efficient loading effect of diethyl dithiocarbamate is achieved. The amphiphilic polymer is used to target and deliver the nanoparticles to the tumor site, allowing the nanoparticles to accumulate in the tumor tissue through enhanced permeation and retention (EPR) effects. The amphiphilic polymer can encapsulate hydrophobic manganese ethyl dithiocarbamate in hydrophobic microdomains, making it less prone to aggregation and fully utilizing its drug function. 2. The amphiphilic polymer is preferably DSPE-PEG with a molecular weight between 1000 and 5000. The polymer steric hindrance hinders the adsorption of manganese ethyl dithiocarbamate to serum proteins, thereby reducing the risk of clearance by the mononuclear phagocytic system in vivo. 3. During the preparation process, the amphiphilic polymer solution, DDTC solution, and Mn²⁺ are thoroughly mixed using ultrasound. + The solution allows the formed manganese ethyl dithiocarbamate to be fully dispersed; Mn 2+ The molar mass of the ions is 25% to 85% of that of sodium / potassium / ammonium diethyldithiocarbamate, to prevent the generated manganese diethyldithiocarbamate from breaking through the hydrophobic microregion and exceeding the stability limit of the amphiphilic polymer to aggregate. 4. Cu in the pharmaceutical composition 2+ This is used to react with the DDTC ligand in manganese diethyldithiocarbamate in the tumor microenvironment to generate CuET and release Mn²⁺. + The Mn² + Enhanced T1-weighted magnetic resonance imaging (MRI) signals can be used for non-invasive monitoring of CuET formation; CuET and Mn 2+ It induces copper death and / or apoptosis in tumor cells and disrupts mitochondrial function. Mitochondrial dysfunction leads to the release of mitochondrial DNA (mtDNA), activating the cGAS-STING signaling pathway, which further induces immunogenic cell death (ICD), dendritic cell maturation, macrophage repolarization to M1 type, and CD8+. + T cell activation. Attached Figure Description

[0021] Figure 1 This serves as a verification of the observations in Example 1 of this application; Figure 2 This demonstrates the linear relationship between the amount of manganese ions released and the amount of copper ions added in Example 2 of this application. Figure 3 This is to verify the results of the ultraviolet-visible spectroscopic spectroscopy in Example 2 of this application; Figure 4 This is to verify the results of T1-weighted imaging performed by the MRI scanner in Example 3 of this application; Figure 5 This is the result of T1-weighted imaging and pseudocolor images of the tumor site at different time points in Example 3 of this application; Figure 6 This application verifies the effect of MDDP or MDDP combined with CuCl2 on the viability of 4T1 cells in Example 4. Figure 7 This is to verify the Western blot quantification results of copper death-related proteins in Example 4 of this application; Figure 8 Example 4 serves as a verification example for this application, showing the expression and aggregation of dihydrolipoic acid acetyltransferase (DLAT) through co-focusing imaging. Figure 9 This is the result of mitochondrial membrane potential detection in Example 4 of this application; Figure 10 This application verifies the apoptosis of 4T1 cells under different treatments in Example 4. Figure 11 The image and magnified view of the laser confocal image in Example 5 of this application are shown. Figure 12 This is to verify the detection results of the relative content of cytoplasmic mitochondrial DNA after different treatments in Example 5 of this application; Figure 13 The results of the Western blot analysis of proteins related to the cyclic guanosine monophosphate-adenosine monophosphate synthase-interferon gene stimulatory factor pathway (cGAS-STING pathway) in Example 5 of this application are as follows: Figure 14 This is a bar chart showing the relative expression levels of interferon-β (IFN-β) in Example 5 of this application. Figure 15 This is the result of the immunogenicity and cell death detection of 4T1 cells in Example 5 of this application. Figure 16 The test results were used to verify the antitumor therapeutic efficacy and safety of different treatment groups in Example 6 of this application; Figure 17 The results of flow cytometry analysis of the antitumor immune responses induced by different treatment groups in Example 7 of this application are used to verify the present application. Detailed Implementation

[0022] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application.

[0023] In the description of this application, it should be understood that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of this application, "multiple" means two or more, unless otherwise explicitly specified.

[0024] Furthermore, throughout this specification, references to "an embodiment"; "an embodiment," "an example," or similar language indicate that a particular feature, structure, or characteristic described in connection with that embodiment is included in at least one embodiment of this application. Therefore, the appearance of the phrase "in one embodiment;" throughout this specification, and similar language, may, but not necessarily, refer to the same embodiment.

[0025] This application specifically relates to a sequential drug delivery system based on the in-situ generation of the disulfiram metabolite CuET and the linkage of magnetic resonance imaging (MRI) signals. More particularly, it relates to a strategy that combines MnDDTC3-loaded nanoparticles with exogenous copper ions to achieve non-invasive monitoring of CuET generation and synergistic enhancement of anti-tumor immune responses. This sequential drug delivery system is implemented using nanoparticles loaded with manganese diethyldithiocarbamate and a pharmaceutical composition thereof.

[0026] The nanoparticles loaded with manganese diethyldithiocarbamate have a particle size of 70 nm to 1000 nm (preferably 100 nm to 120 nm) and comprise a uniformly mixed amphiphilic polymer and diethyldithiocarbamate in a mass ratio of (0.25 to 4): 1; wherein more than 50% of the diethyldithiocarbamate is manganese diethyldithiocarbamate; in one embodiment, the mass ratio of the amphiphilic polymer to the diethyldithiocarbamate is preferably (0.5 to 1): 1.

[0027] The amphiphilic polymer is one or more of DSPE-PEG, DSPE-PEG-folic acid, DSPE-PEG-phenylboronic acid, and DSPE-PEG-arginylglycyl aspartic peptide; in one embodiment, the amphiphilic polymer is DSPE-PEG with a molecular weight of 1000-5000.

[0028] The preparation method of the above-mentioned nanoparticles includes the following steps: A 0.2%–0.3% (w / w) amphiphilic polymer solution was uniformly mixed with a 0.12%–0.15% (w / w) DDTC solution, and then Mn²⁺ solution with a concentration of 0.009 mol / L–0.015 mol / L was added. +The solution is subjected to continuous ultrasonication at a power of 100 W to 300 W for 30 to 90 seconds, and nanoparticles are obtained after ultrafiltration purification; the solute of the DDTC solution is one or more of sodium diethyldithiocarbamate, potassium diethyldithiocarbamate, or ammonium diethyldithiocarbamate. Because of reactant losses during the preparation process, the amphiphilic polymer is lost in the largest amount, followed by the solute in the DDTC solution, and then Mn. 2+ The highest retention rate was observed; to control the mass ratio of amphiphilic polymer to diethyldithiocarbamate in the formed nanoparticles to be (0.25~4):1, and with more than 50% of diethyldithiocarbamate being manganese diethyldithiocarbamate; during preparation, the mass ratio of amphiphilic polymer to solute in DDTC solution was (2~20):1, Mn 2+ The molar mass ratio of ions to solute in the DDTC solution is (0.25~0.4):1.

[0029] These nanoparticles can be used to prepare drug compositions for integrated cancer diagnosis and treatment. In addition to nanoparticles loaded with manganese diethyldithiocarbamate, the drug composition also includes Cu at a molar concentration of 0.1 mM to 10 mM. 2+ Solution, Cu 2+ Cu in solution 2+ It can react with the DDTC ligand in manganese diethyldithiocarbamate in nanoparticles to generate CuET, therefore when Cu 2+ When the molar mass ratio of Cu to manganese diethyldithiocarbamate is greater than or equal to 1, the nanoparticles can fully exert their function; 2+ The solution can be prepared using one or more of CuCl2, CuSO4, Cu(NO3)2, or copper gluconate as a solute; in one embodiment, Cu 2+ Cu in solution 2+ The molar concentration is 0.25 mM ~ 1 mM.

[0030] The above-mentioned pharmaceutical composition can be used to treat breast cancer, lung cancer, liver cancer, or colon cancer; when this pharmaceutical composition is used for diagnosis and treatment, the process is as follows: (1) Nanoparticles are administered to the patient and are enriched in the patient's tumor tissue through the enhanced penetration and retention (EPR) effect; (2) Cu was administered to the patient via intravenous injection. 2+ Solution, Cu 2+ Ions react with DDTC ligands released from nanoparticles in the tumor microenvironment to generate the anticancer active substance CuET, while simultaneously causing Mn² to... + Displacement release from MnDDTC3; following the chemical process: MnDDTC3+ Cu² + → Cu(DDTC)2(CuET) + Mn² + + Byproducts (3) Released Mn² + As a T1-weighted MRI contrast agent, CuET enhances the MRI signal intensity in the tumor region, thus providing non-invasive, real-time reporting of CuET generation. CuET induces copper death and apoptosis in tumor cells, disrupts mitochondrial function, releases mitochondrial DNA (mtDNA), thereby activating the cGAS-STING signaling pathway, promoting type I interferon secretion, inducing dendritic cell maturation, macrophage repolarization to M1 type, and activating CD8. + T cells trigger a strong anti-tumor immune response; (4) Systemic induction of immunogenic cell death (ICD), manifested as calreticulin (CRT) eversion, HMGB1 release and ATP secretion.

[0031] The present application will be described in detail below with reference to specific embodiments. It should be understood that these embodiments are only used to explain the present application and are not intended to limit its scope of protection. All technical solutions obtained by equivalent substitution or equivalent transformation fall within the scope of protection of the present application.

[0032] Example 1 Step 1: Preparation of MDDP nanoparticles DSPE-PEG nanoparticles (MDDP) loaded with MnDDTC3 (manganese diethyldithiocarbamate, molecular weight 499.7) were prepared using a one-pot method. Weigh 22 mg of DSPE-PEG (produced by Shanghai Pengshuo Biotechnology, model PS1-E1-2K, PEG molecular weight 2000) and dissolve it in 10 mL of ultrapure water or physiological saline. Weigh 3.83 mg of NaDDTC3 (i.e., sodium diethyldithiocarbamate, molecular weight 171.259) and dissolve it in 3 mL of ultrapure water or physiological saline. Mix them together. Under room temperature sonication, add 1 mL of MnCl2·4H2O (1.97 mg / mL) ultrapure water or physiological saline solution. The sonication power is 200 W and the sonication time is 60 seconds. After ultrafiltration purification (100 kDa filter membrane), MDDP nanoparticles with uniform particle size of about 100 nm to 120 nm are obtained. These nanoparticles can be uniformly dispersed in physiological saline and remain clear after being placed at 4 °C for 3 days.

[0033] Step Two: Use of the Pharmaceutical Composition Mix 2 mL of MDDP aqueous solution (1.3 mM Mn) with an equal volume of CuCl2·2H2O solution with a concentration of 0.25 mM.

[0034] Example 2 The same steps as described in Example 1 were repeated, except that in step two, the concentration of the CuCl2·2H2O solution was 0.5 mM.

[0035] Example 3 The same steps as described in Example 1 were repeated, except that in step two, the concentration of the CuCl2·2H2O solution was 1.0 mM.

[0036] Example 4 Weigh out mPEG 2000 -PLGA 5000 (50:50) 30 mg was dissolved in 10 mL of ultrapure water, and 3.83 mg of NaDDTC3 was weighed and dissolved in 3 mL of ultrapure water. The mixture was then added to a 1 mL solution of MnCl2·4H2O (concentration 1.97 mg / mL) under ultrasonic conditions of 180 W at room temperature, and the mixture was sonicated for 90 seconds. The reaction solution was purified by a 100 kDa ultrafiltration tube to obtain MnDDTC3-loaded polymer nanoparticles. These nanoparticles had a wide particle size distribution, ranging from 150 nm to 350 nm.

[0037] Comparative Example 1 Repeat Example 1 using the same steps, except that in step two, the concentration of the CuCl2·2H2O solution is 0 mM.

[0038] Comparative Example 2 30 mg of hydroxyethyl starch (HES130 / 0.4) was dissolved in 10 mL of ultrapure water, and 3.83 mg of NaDDTC3 was dissolved in 3 mL of ultrapure water. The mixture was then added to a 1 mL solution of MnCl2·4H2O (concentration 1.97 mg / mL) under ultrasonic conditions at 180 W at room temperature, and the mixture was sonicated for 90 seconds. During the reaction, precipitation rapidly occurred, failing to form a stable and clear nanodispersion. Centrifugation yielded only a small amount of precipitate, confirming that hydroxyethyl starch could not effectively stabilize the MnDDTC3 complex under these preparation conditions and could not form uniform nanoparticles suitable for drug delivery.

[0039] Comparative Example 3 15 mg of Tween-80 and 5 mg of cholesterol were dissolved in 10 mL of ultrapure water, and 3.83 mg of NaDDTC3 was dissolved in 3 mL of ultrapure water. The mixture was then added to a 1 mL solution of MnCl2·4H2O (1.97 mg / mL) under ultrasonic conditions at 250 W at room temperature, and the mixture was sonicated for 60 seconds. The reaction solution was purified by centrifugation using a 100 kDa ultrafiltration tube to obtain MnDDTC3 nanomicelles. These micelles exhibited non-uniform particle size; while they could be uniformly dispersed in physiological saline, visible precipitation occurred after 3 days at 4 °C, indicating poor long-term stability and a tendency to aggregate.

[0040] Comparative Example 4 50 mg of bovine serum albumin (BSA) and 5.0 mg of NaDDTC3 were dissolved separately in phosphate buffer (pH 7.4) and stirred for 30 minutes. Then, MnCl2 solution was slowly added dropwise, and the reaction was continued with stirring for 2 hours. During the reaction, precipitation rapidly occurred, failing to form a stable, clear nanodispersion. Centrifugation yielded only a small amount of precipitate, confirming that BSA could not effectively stabilize the MnDDTC3 complex under these preparation conditions and could not form uniform nanoparticles suitable for drug delivery.

[0041] Experimental results verification Verification Example 1 MDDP aqueous solution (2 mL, 1.3 mM Mn) was mixed with equal volumes of CuCl2·2H2O solutions of different concentrations (0, 0.25, 0.5, 1.0 mM) (i.e., Examples 1-3, Comparative Example 1); after incubation, the solution was diluted to a suitable concentration, and the hydrated particle size was determined by dynamic light scattering (DLS). The results are as follows: Figure 1 As shown, the reaction between MDDP and CuCl2 did not significantly change the particle size of the nanoparticles. The particle size of the MDDP nanoparticles was around 110 nm. Even with the addition of CuCl2, the nanoparticle size remained around 110 nm, indicating that the reaction between MDDP and CuCl2 did not significantly alter the nanoparticle size. 2000 Under stable conditions, the entire system still retains the properties of nanoparticles.

[0042] Verification Example 2: In vitro CuET generation and Mn² + Release verification MDDP nanoparticles (2 mL, 1.3 mM Mn) were co-incubated with different concentrations of CuCl2 (2 mL, 0, 0.25, 0.5, 1.0 mM) (equivalent to Examples 1-3, Comparative Example 1). The filtrate was collected by ultrafiltration, and the amount of Mn²⁺ released in the filtrate was detected by ICP-MS. + Concentration was determined by detecting CuET formation using UV-Vis.

[0043] After ultrafiltration and centrifugation, the Mn²⁺ content in the supernatant was determined by inductively coupled plasma mass spectrometry (ICP-MS). + Concentration. CuET formation was also detected using UV-Vis. For example... Figure 2 As shown, with the increase of copper ion input, manganese ions are gradually released, and the amount of manganese ion released is linearly related to the amount of copper ion input. Figure 3 As shown, when co-incubated with 1.0 mM CuCl2, sufficient CuET can be generated, causing the characteristic absorption peak of CuET at 450 nm to gradually rise. This indicates that the system can efficiently trigger in-situ generation of CuET and simultaneously release Mn²⁺. + .

[0044] Verification Example 3: T1-weighted MRI in vitro and in vivo imaging In vitro: MDDP (2 mL, 1.3 mM Mn) was mixed with 2 mL of physiological saline and Cu²⁺ at different concentrations. + The reacted solutions (2 mL, 0.25, 0.5, 1.0 mM) were placed in NMR tubes (i.e., equivalent to Examples 1-3, Comparative Example 1), and each sample was then diluted to Mn concentrations of 0.65 mM, 0.325 mM, 0.1625 mM, 0.08 mM, and 0.04 mM, respectively. T1-weighted imaging (TR / TE = 500 ms / 6 ms) was performed using a 4.7 TMRI scanner. The longitudinal relaxation rate r1 was calculated. Figure 4 The results showed that the r1 value of the system after the reaction increased from 2.842 mM. - ¹s - ¹Increased to 7.811 mM - ¹s - ¹, significantly higher than without Cu² + Group (i.e., Comparative Example 1) shows that Mn² + It effectively enhances T1 signal and has good MRI imaging potential.

[0045] In vivo: A 4T1 tumor-bearing mouse model was established, and 4T1 tumor-bearing BALB / c mouse models were randomly divided into two groups: the experimental group ( Figure 5 Below) Mice were injected via tail vein with MDDP (Mn dose: 2 mg / kg) obtained in step one of Example 1, followed by injection of CuCl2 (Cu dose: 3 mg / kg) 2 h later; control group ( Figure 5 (Top): MDDP + saline. MRI scans were performed at 0, 2, and 4 hours. Figure 5ROI analysis showed that the signal intensity in the tumor region of the control group peaked at 2 hours and then decreased; while the magnetic resonance imaging signal of the experimental group was further enhanced 2 hours after injection of copper chloride. The results demonstrate that this system can achieve non-invasive, dynamic, and visual monitoring of the CuET generation process.

[0046] Validation Example 4: Study on the antitumor mechanism of MDDP combined with copper chloride The cytotoxicity of MDDP alone or in combination with CuCl2 was evaluated using the CCK-8 assay. 4T1 cells were seeded at 8000 cells / well in 96-well plates. After overnight adhesion, 100 μL of physiological saline and 100 μL of different concentrations of MDDP solution prepared in step one of Example 1 were added, with Mn concentrations equivalent to 15.6 nM, 31.3 nM, 62.5 nM, 125 nM, 250 nM, and 500 nM, respectively, and incubated for 2 h. Subsequently, 0.05 µmol CuCl2 was added to the MDDP+CuCl2 group, and the cells were cultured for another 24 h. 10 µL of CCK-8 was added to each well, and the cells were incubated at 37 ℃ for 1 h. The absorbance at 450 nm was measured using a microplate reader, and the relative viability was calculated. Figure 6 The results of CCK-8 assays showed that MDDP significantly inhibited the activity of tumor cells after reacting with CuCl2, while MDDP itself had low toxicity.

[0047] Western blot detection of copper death-related proteins: 4T1 cells were treated with the drug for 12 h, then lysed with RIPA (containing protease / phosphatase inhibitor), quantified by BCA, separated by SDS-PAGE, transferred to a membrane, blocked with 5% skim milk, and incubated overnight at 4 ℃. FDX1, LIAS primary antibody, HRP-labeled secondary antibody were added, and the cells were visualized by ECL (GelView 6000Pro II). β-actin was used as an internal control. Figure 7 Western blot results showed that the expression of copper death marker proteins FDX1 and LIAS was significantly downregulated in the MDDP + CuCl2 group. In the figure, Control represents 1640 complete medium containing 10% fetal bovine serum, CuCl2 represents a 500 nM copper chloride complete medium solution, MDDP represents a 500 nM MDDP complete medium dispersion, and MDDP + CuCl2 represents treatment with 500 nM MDDP complete medium dispersion for 2 hours followed by treatment with 500 nM copper chloride complete medium solution, with a final volume of 2 mL.

[0048] Immunofluorescence observation of DLAT aggregation: 4% paraformaldehyde fixation, 0.1% Triton X-100 permeabilization, 5% BSA blocking, anti-DLAT antibody incubation at 4 ℃ overnight, CoraLite® Plus 647 labeled secondary antibody incubation in the dark, Hoechst 33342 counterstaining, and Olympus FV3000 confocal microscopy imaging. Figure 8 Laser confocal microscopy revealed significant DLAT aggregation in the MDDP + CuCl2 group. In the figure, Control represents 1640 complete medium containing 10% fetal bovine serum, CuCl2 represents a 500 nM copper chloride complete medium solution, MDDP represents a 500 nM MDDP complete medium dispersion, and MDDP + CuCl2 represents treatment with a 500 nM MDDP complete medium dispersion for 2 hours followed by treatment with a 500 nM copper chloride complete medium solution, with a final volume of 2 mL.

[0049] Mitochondrial membrane potential: JC-1 probe was incubated at 37 ℃ for 20 min, and red / green fluorescence conversion was monitored. Simultaneously, Figure 9 To obtain the fluorescence intensity of the mitochondrial membrane potential probe JC-1 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazole carbonyl cyanine iodide monomer and aggregate under different treatments using confocal imaging, this study demonstrated that combined treatment with MDDP and CuCl2 could reduce the mitochondrial membrane potential of 4T1 cells; mitochondrial membrane potential detection (JC-1 staining) showed severely impaired mitochondrial function.

[0050] Annexin V-FITC / PI double staining method: After 24 h of drug treatment, cells were collected, Annexin V-FITC and PI were added according to the instructions, and the cells were detected by flow cytometry. Figure 10 Apoptosis staining showed elevated levels of early and late apoptosis, indicating that this system kills tumor cells through a dual mechanism of inducing copper death and apoptosis. Figure 10 Content A represents a flow cytometry analysis plot. Figure 10Content B represents a statistical chart of the proportion of early and late apoptotic cells. PI-PE represents the fluorescence intensity of propidium iodide in the PE channel, Annexin V-FITC represents the fluorescence intensity of Annexin V in the FITC channel, and numbers such as 2.35 and 2.85 indicate the proportion of different cell populations in the parent population. In the figure, Control represents 1640 complete culture medium containing 10% fetal bovine serum, CuCl2 represents a 500 nM copper chloride complete culture medium solution, MDDP represents a 500 nM MDDP complete culture medium dispersion, and MDDP +CuCl2 represents treatment with a 500 nM MDDP complete culture medium dispersion for 2 hours followed by treatment with a 500 nM copper chloride complete culture medium solution, with a final volume of 2 mL.

[0051] Verification Example 5: Investigation into the effects of MDDP combined with copper chloride on activating the cGAS-STING pathway and inducing ICD 4T1 mouse breast cancer cells were seeded in culture dishes and, after adhesion, were divided into three groups: a control group (treated with 1640 complete medium), a group treated with 2 ml of 0.5 µM copper chloride 1640 complete medium solution, 2 ml of 250 nM MDDP complete medium dispersion, and a group treated with MDDP + CuCl2 (2 ml of 250 nM MDDP complete medium dispersion followed by 2 ml of 0.5 µM copper chloride 1640 complete medium solution two hours after administration of MDDP). To detect the co-localization of mitochondrial and cytoplasmic DNA, mitochondria were labeled using MitoTrackerRed incubation at 37°C in the dark. After washing with PBS, double-stranded DNA (dsDNA) was labeled using PicoGreen dye incubation at room temperature in the dark. Confocal laser scanning microscopy was used to observe whether dsDNA was released from the mitochondria into the cytoplasm. In this embodiment, the combined treatment of MDDP and CuCl2 effectively improved the cGAS-STING pathway in 4T1 tumor cells. Figure 11 Confocal laser scanning microscopy revealed that in the MDDP combined with CuCl2 treatment group (G4), double-stranded DNA (dsDNA) was significantly released from the mitochondria into the cytoplasm, while in the control group, dsDNA was mainly located in the mitochondria and nucleus, indicating that this treatment can induce the cytoplasmic release of mitochondrial DNA (mtDNA).

[0052] To further quantify the level of mitochondrial DNA (mtDNA) in the cytoplasm, cells were lysed using a mild lysis buffer containing digitonin, and the mtDNA copy number was detected by qPCR after DNA extraction. Figure 12 qPCR quantitative analysis further confirmed that the mtDNA content in the cytoplasm of the G4 group was significantly increased, approximately 1.46 times that of the control group. The released mtDNA reacted with Mn²⁺ produced during the reaction. +Co-activate the cGAS-STING signaling pathway.

[0053] To assess the activation of the cGAS-STING pathway, cells were collected 6 hours after drug treatment for Western blot analysis to detect the expression levels of STING, TBK1, IRF3 and their phosphorylated proteins, with GAPDH as an internal reference protein. Figure 13 Western blot results showed significantly enhanced phosphorylation levels of STING, TBK1, and IRF-3.

[0054] Simultaneously, cell supernatant was collected, and the absorbance was measured at 450 nm to quantify IFN-β secretion levels, following the instructions for the mouse IFN-β ELISA kit. Furthermore, IFN-β secretion was significantly increased. Figure 14 The results showed that the experimental group was approximately 3.16 times that of the control group, indicating that the combined treatment of MDDP and CuCl2 could promote the release of IFN-β from 4T1 cells. For the assessment of immunogenic cell death (ICD), CoraLite® Plus 555-labeled anti-calreticulin (CRT) antibody was incubated at 4°C in the dark for 30 minutes. After washing, the exposure of CRT on the cell membrane surface was detected by flow cytometry. For the HMGB1 release assay, cells were fixed and perforated using BD Cytofix / Cytoperm™ reagent, then stained with PE-labeled anti-HMGB1 antibody, and the changes in intracellular HMGB1 protein levels were analyzed by flow cytometry. For the ATP release assay, the cell supernatant was collected, the assay reagents in the Enhanced ATP Assay Kit were added, and the relative luminescent units (RLU) were measured using a chemiluminescence analyzer to assess the amount of extracellular ATP released.

[0055] also, Figure 15 Content A shows that the combined treatment also induced typical ICD features: extracellular ATP levels increased to 8 times that of the control group; Figure 15 Content B shows that flow cytometry analysis revealed that the proportion of calreticulin (CRT) exposed on the cell membrane surface increased to approximately 20%. Figure 15 Content C shows that, simultaneously, the level of high-mobility group box 1 (HMGB1) in the cells decreased to 50% of that in the control group, indicating that it was released into the extracellular space in large quantities. In summary, co-treatment with MDDP and CuCl2 significantly induced mtDNA release in 4T1 cells, activated the cGAS-STING pathway, and was accompanied by the release and exposure of key ICD molecules such as ATP, CRT, and HMGB1. This fully demonstrates that the combined treatment of MDDP and CuCl2 can induce immunogenic cell death in 4T1 cells and has good potential for anti-tumor immune activation.

[0056] Verification Example 6: Antitumor Therapy Experiment 4T1 tumor-bearing mice were randomly divided into four groups: G1: control group (saline); G2: CuCl2 group; G3: MDDP group; G4: MDDP + CuCl2 group (sequential administration, with CuCl2 treatment given two hours after MDDP administration). The concentrations of MDDP and CuCl2 were the same as in Case 5. The dosage of copper was 3 mg / kg, and the dosage of manganese was 2 mg / kg. MDDP saline dispersion and copper chloride solution were administered every 3 days for a total of 4 rounds. Tumor volume and body weight were measured regularly. After treatment, the mice were sacrificed, and the tumor weight was measured. Figure 16 The results showed that the MDDP + CuCl2 group had a significantly better tumor inhibition effect than the other groups, and the tumor volume and weight were significantly lower than the other three groups. Figure 16 Contents A and B), and no significant weight loss was observed. Figure 16 Content C) indicates that the system has excellent antitumor activity and good safety.

[0057] Validation Example 7: Assessment of the immune response induced by MDDP combined with copper chloride treatment 4T1 tumor-bearing mice were randomly divided into four groups: G1: control group (saline); G2: CuCl2 group; G3: MDDP group; G4: MDDP + CuCl2 group (sequential administration, with CuCl2 treatment given two hours after MDDP administration). The concentrations of MDDP and CuCl2 were the same as in Case 5. The dosage of copper was 3 mg / kg, and the dosage of manganese was 2 mg / kg. One cycle of administration was performed every 3 days, for a total of 4 cycles. To systematically evaluate the effect of the sequential administration strategy of this application on the tumor immune microenvironment, tumor tissue and tumor draining lymph nodes (tdLNs) of 4T1 tumor-bearing mice after treatment were collected, and single-cell suspensions were prepared. Flow cytometry was used to detect the maturation status of dendritic cells (DCs), macrophage polarization phenotype, and CD8+. + T cell activation level. Specific antibody combinations are as follows: DC maturity marker: CD11c + CD80 + / CD86 + ; Macrophage polarization: F4 / 80 + CD86 + (M1 type); CD8 + T cell activation: IFN-γ + CD3 + CD8 + .

[0058] After completing the treatment according to the aforementioned protocol, the mice were euthanized, and the tumor draining lymph nodes (tdLNs) and tumor tissue were separated. Single-cell suspensions were prepared for flow cytometry analysis.

[0059] The results showed that CD80 levels were higher in the sequential dosing group (G4). + / CD86 + The proportion of mature dendritic cells reached 30.2%, significantly higher than the other three groups (G1: 12.8%; G2: 17.5%; G3: 18%), indicating that this strategy effectively promoted DC maturation and enhanced antigen presentation capacity. Figure 17 ContentA); Figure 17 Content B shows: M1 macrophages (CD86) in the tumor of the sequential drug administration group + The proportion of G1 was 26.3%, significantly higher than the other three groups (G1: 14.3%; G2: 19.3%; G3: 18.3%), suggesting that macrophages are repolarizing towards an anti-tumor phenotype; Figure 17 Content C shows: IFN-γ in the sequential dosing group + CD8 + The proportion of T cells reached 3.8%, significantly higher than other groups, indicating that CD8+... + T cells are effectively activated and secrete the key effector IFN-γ.

[0060] Based on step one of Example 1, while ensuring that the mass ratio of DSPE-PEG to sodium diethyldithiocarbamate is (2~20):1, and that the Mn 2+ Under the premise that the molar mass ratio of ions to sodium diethyldithiocarbamate is (0.25~0.85):1; the PEG in DSPE-PEG... 2000 Replace with PEG 1000 or PEG 5000 Alternatively, adjusting the mass fraction of DSPE-PEG to between 0.2% and 0.3%, or adjusting the mass fraction of sodium diethyldithiocarbamate solution to between 0.12% and 0.15%, or adjusting the molar concentration of MnCl2 solution to between 0.009 mol / L and 0.015 mol / L, or adjusting the ultrasonic power to between 100 W and 300 W and the ultrasonic time to between 30 and 90 seconds, can all produce MDDP nanoparticles identical to those in Example 1. Chromatographic analysis showed that the mass ratio of DSPE-PEG to sodium diethyldithiocarbamate in these MDDP nanoparticles was (0.25–4):1; and 50%–90% of the sodium diethyldithiocarbamate was converted to manganese diethyldithiocarbamate; the particle size of the above nanoparticles was found to be distributed between 70 nm and 1000 nm.

[0061] Based on step one of Example 1, DSPE-PEG can be replaced with DSPE-PEG-folic acid, DSPE-PEG-phenylboronic acid, or DSPE-PEG-arginylglycyl aspartic acid peptide of the same molecular weight; or sodium ethyl dithiocarbamate can be replaced with potassium or ammonium salt of ethyl dithiocarbamate of the same molar mass. It has been verified that MDDP nanoparticles with the same physical properties and therapeutic functions can be prepared. Based on step two of Example 1, the CuCl2·2H2O solution was replaced with CuSO4, Cu(NO3)2 or copper gluconate solution of the same molar concentration. It was found that when used in combination with MDDP nanoparticles, they all had the same therapeutic effect as in Examples 1-3.

[0062] Based on step two of Example 1, the molar concentration of CuCl2·2H2O solution was adjusted between 0.1 mM and 10 mM. It was found that when used in combination with MDDP nanoparticles, it exhibited the same therapeutic effect as in Examples 1-3, and in Cu... 2+ When the molar mass of the nanoparticle is greater than the molar mass of manganese diethyldithiocarbamate in the nanoparticle, its therapeutic effect is comparable to that in Example 3.

[0063] In summary, the embodiments of this application describe the preparation of nanoparticles, and the reaction of nanoparticles with Cu. 2+ The solution-formed drug composition can not only directly kill tumor cells, but also reshape the immunosuppressive microenvironment and synergistically activate innate and adaptive anti-tumor immune responses.

[0064] The embodiments of this application implement the following functions: Magnetic resonance imaging: For the first time, magnetic resonance imaging was used for non-invasive monitoring of CuET generation: Compared with monitoring methods such as fluorescence imaging, magnetic resonance imaging has advantages such as deep imaging depth and multi-planar monitoring; Integrated diagnosis and treatment design: Synchronizes the drug activation process with imaging reports to support individualized treatment assessment; The mechanism of switching from "low toxicity to high toxicity" is as follows: the prodrug form is safe, and the highly toxic CuET is activated only locally in the tumor, thus improving the safety of treatment; Powerful immune activation: It induces mtDNA release through mitochondrial damage, thereby activating the cGAS-STING pathway and inducing ICD, generating a T cell response cascade to achieve sustained anti-tumor immunity; High convertibility: The materials used (DSPE-PEG, Mn²) + Cu² + All of these are clinically usable or approved substances, facilitating clinical translation; Sequential dosing is flexible and controllable: the therapeutic window can be optimized by adjusting the order and dosage of administration.

[0065] Those skilled in the art should understand that the above embodiments are for illustrative purposes only and are not intended to limit the scope of this application. Any equivalent substitutions, modifications, or improvements made to this application within the spirit and principles of this application should be included within the scope of protection of this application.

Claims

1. A nanoparticle loaded with manganese diethyldithiocarbamate, characterized in that, It includes an amphiphilic polymer in a mass ratio of (0.25~4):1 and a diethyldithiocarbamate; wherein more than 50% of the diethyldithiocarbamate is manganese diethyldithiocarbamate. The amphiphilic polymer is one or more of DSPE-PEG, DSPE-PEG-folic acid, DSPE-PEG-phenylboronic acid, and DSPE-PEG-arginylglycyl aspartic peptide.

2. The nanoparticles as described in claim 1, characterized in that, The amphiphilic polymer is DSPE-PEG with a molecular weight of 1000~5000.

3. The nanoparticles as described in claim 1, characterized in that, The mass ratio of the amphiphilic polymer to diethyldithiocarbamate is (0.5~1):

1.

4. The nanoparticles as described in claim 1, characterized in that, The nanoparticles have a particle size of 70 nm to 1000 nm.

5. A method for preparing the nanoparticles according to any one of claims 1-4, characterized in that, Includes the following steps: A 0.2%–0.3% (w / w) amphiphilic polymer solution was uniformly mixed with a 0.12%–0.15% (w / w) DDTC solution, and then Mn²⁺ solution with a concentration of 0.009 mol / L–0.015 mol / L was added. + The solution is sonicated to obtain the nanoparticles; the solute in the DDTC solution is one or more of sodium diethyldithiocarbamate, potassium diethyldithiocarbamate, or ammonium diethyldithiocarbamate.

6. The preparation method according to claim 5, characterized in that, The ultrasound power is 100 W to 300 W, and the duration is 30 to 90 seconds.

7. A pharmaceutical composition, characterized in that, The pharmaceutical composition includes the nanoparticles according to any one of claims 1-4, and further includes Cu. 2+ Solution, the Cu 2+ Cu in solution 2+ The molar mass ratio of the nanoparticles to manganese diethyldithiocarbamate is greater than or equal to 1.

8. The pharmaceutical composition of claim 7, characterized in that, The Cu 2+ Cu in solution 2+ The molar concentration is 0.1 mM ~ 10 mM.

9. The pharmaceutical composition according to claim 7, characterized in that, The Cu 2+ The solute in the solution is one or more of CuCl2, CuSO4, Cu(NO3)2, or copper gluconate.

10. The pharmaceutical composition according to claim 7, characterized in that, The pharmaceutical composition is used to treat breast cancer, lung cancer, liver cancer, or colon cancer.

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