Functionalized drug-loaded nanoparticles as well as preparation method and application thereof

Abstract

The invention belongs to the field of biological medicine, and particularly relates to functionalized drug-loaded nanoparticles as well as a preparation method and application thereof. The macromolecule coupled folic acid modified quercetin-loaded mesoporous calcium carbonate nanoparticle T (at) QU (at) CaCO3 constructed by the invention not only can be used as a'calcium bomb 'to provide calcium ions for tumor calcium overload in a tumor slightly acidic environment, but also can be used for highly loading a traditional Chinese medicine anticancer active ingredient quercetin; besides, nanoparticles are wrapped based on polymer coupled folic acid modification, the stability of mesoporous calcium carbonate can be improved, irritation of quercetin can be reduced, in-vivo circulation can be enhanced, tumor cells can be further targeted based on folic acid modification, accumulation of drugs in TNBC cells can be promoted, and therefore the targeted delivery capacity is improved, and the purposes of effect enhancing and toxicity reducing are achieved. And a feasible scheme is provided for accurate targeted treatment of breast cancer, especially triple negative breast cancer.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a functionalized drug-loaded nanoparticle, its preparation method, and its uses. Background Technology

[0002] Breast cancer is one of the most common malignant tumors in the world, seriously affecting people's health. According to the latest global cancer data, the incidence of breast cancer ranks first among female malignant tumors ([1] Nolan E, Lindeman G, Visvader J. Deciphering breast cancer: from biology to the clinic. Cell 2023, 186(8): 1708-1728.). Among them, triple-negative breast cancer (TNBC) is a subtype of breast cancer that lacks the expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2). It accounts for about 15-20% of the incidence of breast cancer. It has the characteristics of high mortality, strong metastasis and easy recurrence, and extremely poor clinical prognosis ([2] Leon-Ferre R, Goetz M. Advances in systemic therapies for triple negative breast cancer. BMJ 2023, 381: e071674.). Currently, the commonly used treatments in clinical practice are surgery, chemotherapy, and radiotherapy. Although these traditional treatments have made some progress with the advancement of technology, many challenges still exist: (1) Surgical resection has limitations, being limited to early-stage breast cancer patients, and mostly involving total mastectomy, which causes great physiological and psychological trauma to women; (2) Traditional chemotherapy has significant toxic side effects, lacks targeting, and is prone to drug resistance, leading to a serious decrease in patient compliance; (3) Radiotherapy has low precision, significant side effects, and toxic side effects on normal cells. Therefore, developing a safe, effective, and biocompatible new targeted TNBC treatment to achieve tumor targeted therapy, inhibit tumor recurrence and metastasis, prolong patient survival, and improve patient quality of life has become an urgent need for TNBC treatment. However, because TNBC patients lack ER / PR / HER-2 expression, they cannot benefit from small molecule targeted drugs, and the FDA has not yet approved any targeted therapies for TNBC. Therefore, finding effective TNBC treatment drugs and strategies is urgent.Over the past few decades, the emergence of drug delivery systems has become an effective carrier for precise drug delivery ([3] Xu M, Li S. Nano-drug delivery system targeting tumor microenvironment: A prospective strategy for melanoma treatment. Cancer Lett 2023, 574: 216397.), and can prolong the half-life of drugs, increase drug accumulation, and deliver drugs to specific targets, thereby minimizing the impact on non-target tissues, thus providing a more precise and targeted treatment modality for TNBC.

[0003] The rise of metal immunology has brought new ideas and methods to tumor immunotherapy. Among them, calcium ions, as second messengers, participate in the differentiation of immune cells, gene transcription and effector function regulation, and play an important role in tumor immune regulation. Studies have found that the disruption of intracellular calcium ion homeostasis can induce immunogenic death of tumor cells, which can significantly improve the effect of local tumor treatment and can also cause anti-tumor immune response in the body ([4] Zhu Y, Pan Z, Hao Y, Wang C, Dong Z, Li Q, et al. Metallo-alginate hydrogel can potentiate microwave tumor ablation for synergistic cancer treatment. Science Advances 2022, 8(31): eabo5285.). In addition, calcium ions can amplify immune signals by changing the charge in the environment, thereby achieving TCR-mediated T cell activation and enhancing the effect of anti-tumor immunotherapy ([5] Shi X, Bi Y, Yang W, Guo X, Jiang Y, Wan C, et al. Ca2+ regulates T-cell receptor activation by modulating the charge property of lipids. Nature 2013, 493(7430):111-115.). The maturation of DC cells has a positive effect on tumor immunotherapy. Zhao et al. found that the significant increase in calcium ion concentration in triple-negative breast cancer 4T1 cells can further induce DC maturation, activate anti-tumor immunity, and significantly inhibit the growth of primary tumors and distant tumor metastasis ([6] Zhao P, Wang M, Chen M, Chen Z, Peng X, Zhou F, et al. Programming cell pyroptosis with biomimetic nanoparticles for solid tumor immunotherapy. Biomaterials 2020, 254:120142.). Therefore, by regulating the intracellular calcium ion level of tumor cells, the tumor immune response can be significantly enhanced. Introducing the metal immune strategy into the treatment of TNBC not only has the potential to improve the tumor immune response, but may also enhance the efficacy of drug treatment, which brings potential benefits to the treatment of TNBC.

[0004] Folic acid participates in the synthesis of nucleic acids and amino acids, and is essential for maintaining cellular function, especially for the uncontrolled proliferation of tumor cells. The folate receptor is overexpressed in many cancers but is little or absent in most normal tissues, providing a theoretical basis for targeting tumors through interaction with the folate receptor (FR). Folate-modified nanomedicine delivery systems achieve targeted delivery to tumor cells by triggering receptor-mediated endocytosis through the specific binding of folate to FR.

[0005] Quercetin (QU) is a naturally occurring polyhydroxy flavonoid compound that is widely found in traditional Chinese medicines such as Sophora tonkinensis, Scutellaria baicalensis, and Sophora japonica. It has a significant inhibitory effect on various tumors, including breast cancer. The mechanisms involved include inducing apoptosis, inhibiting tumor metastasis, reversing tumor drug resistance, regulating autophagy, and regulating tumor immunity ([7] Zang X, Cheng M, Zhang X, Chen X. Quercetin nanoformulations: a promising strategy for tumor therapy. Food Funct 2021, 12(15), 6664-6681.).

[0006] To address the current pain points of lacking targeted drugs and the difficulty of targeted therapy for TNBC, it is necessary to find a biocompatible and highly targeted nanomedicine delivery system to achieve the goal of enhancing efficacy and reducing toxicity, thus providing a new feasible solution for the treatment of TNBC. Summary of the Invention

[0007] To address the problems of existing technologies, this invention provides a functionalized drug-loaded nanoparticle, its preparation method, and its applications.

[0008] A functionalized drug-loaded nanoparticle, wherein the functionalized drug-loaded nanoparticle is a folic acid-modified mesoporous calcium carbonate nanoparticle loaded with quercetin, comprising the following raw materials in parts by weight:

[0009] 100-200 servings of calcium source

[0010] CO3 2- 10,000-15,000 parts of precursor

[0011] Quercetin 100-200 parts

[0012] 100-300 parts of polymer-folic acid conjugate.

[0013] Preferably, the above-mentioned functionalized drug-loaded nanoparticles comprise the following raw materials in parts by weight:

[0014] 150 servings of calcium source

[0015] CO3 2-10,000 precursors

[0016] Quercetin 150 parts

[0017] 100 parts of polymer-folic acid conjugate.

[0018] Preferably, the calcium source is selected from at least one of calcium chloride, calcium nitrate, and calcium acetate;

[0019] And / or, the CO3 2- The precursor is selected from ammonium bicarbonate;

[0020] And / or, the polymer-folic acid conjugate is selected from at least one of polyethylene glycol-folic acid conjugate, hyaluronic acid-folic acid conjugate, polyvinylpyrrolidone-folic acid conjugate, and dextran-folic acid conjugate.

[0021] Preferably, the polyethylene glycol-folic acid coupling compound is methoxy polyethylene glycol-folic acid.

[0022] Preferably, the mass ratio of the mesoporous calcium carbonate nanoparticles to quercetin is 1:1-2.

[0023] The present invention also provides a method for preparing the above-mentioned functionalized drug-loaded nanoparticles, comprising the following steps:

[0024] Step 1, mix the calcium source with CO3 2- Precursor reaction yields mesoporous calcium carbonate nanoparticles;

[0025] Step 2: React mesoporous calcium carbonate nanoparticles with quercetin to obtain quercetin-loaded mesoporous calcium carbonate nanoparticles.

[0026] Step 3: React quercetin-loaded mesoporous calcium carbonate nanoparticles with a polymer-folic acid conjugate to obtain the final product.

[0027] Preferably, in step 3, the mass ratio of the quercetin-loaded mesoporous calcium carbonate nanoparticles to the polymer-folic acid conjugate is 1:1-2.

[0028] Preferably, in step 1, the solvent for the reaction is anhydrous ethanol;

[0029] And / or, in step 1, the reaction is carried out under vacuum conditions, at a temperature of 40°C-50°C, for a time of 2-3 days;

[0030] And / or, in step 2, the solvent for the reaction is dimethyl sulfoxide;

[0031] And / or, in step 2, the reaction temperature is 37℃-42℃, and the incubation time is 24h-48h;

[0032] And / or, in step 3, the reaction temperature is 20-25°C and the stirring time is 12-16 h.

[0033] The present invention also provides the use of the above-described functionalized drug-loaded nanoparticles in the preparation of drugs for treating breast cancer.

[0034] Preferably, the breast cancer is triple-negative breast cancer.

[0035] The present invention also provides a pharmaceutical composition for treating breast cancer, which is prepared by adding pharmaceutically acceptable excipients to the above-mentioned functionalized drug-loaded nanoparticles as active ingredients.

[0036] In the early stages of this invention, through experiments on the ratio of three components—mesoporous calcium carbonate, quercetin, and polyethylene glycol-folic acid conjugate—and the screening of mesoporous materials, a functionalized drug-loaded nanoparticle, T@QU@CaCO3, with good biocompatibility and high drug loading capacity, was constructed. This nanoparticle not only acts as a "calcium bomb" to provide calcium ions for tumor calcium overload in the microacidic environment of tumors, but also has a high loading capacity for quercetin, a traditional Chinese medicine anticancer active ingredient. Furthermore, folic acid modification of the nanoparticles enhances the stability of the mesoporous calcium carbonate, reduces the irritant effect of quercetin, and improves in vivo circulation. It also further targets tumor cells, promoting drug accumulation in TNBC cells, thereby improving targeted delivery capability and achieving synergistic efficacy and reduced toxicity, providing a feasible solution for precise targeted therapy of breast cancer.

[0037] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0038] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0039] Figure 1 To observe the structure of the mesoporous calcium carbonate prepared in Comparative Example 3 using transmission electron microscopy.

[0040] Figure 2 To observe the structure of the mesoporous calcium carbonate prepared in Comparative Example 4 using transmission electron microscopy.

[0041] Figure 3 To observe the structure of the mesoporous calcium carbonate prepared in Comparative Example 1 using transmission electron microscopy.

[0042] Figure 4TEM images of folic acid-modified mesoporous calcium carbonate nanoparticles loaded with quercetin. A, TEM image of mesoporous calcium carbonate; B, TEM image of mesoporous calcium carbonate loaded with quercetin; C, TEM image of folic acid-modified mesoporous calcium carbonate loaded with quercetin.

[0043] Figure 5 To observe intracellular calcium ion staining in different treatment groups using laser confocal microscopy (CLSM).

[0044] Figure 6 To assess the toxicity of different treatment groups to 4T1 cells at 24h and 48h.

[0045] Figure 7 To observe cell migration in different treatment groups using a cell scratch assay.

[0046] Figure 8 This is a T@QU@CaCO3-induced ICD experiment in 4T1 cells. A. Image showing CRT expression in cells after drug treatment; B. Image showing HMGB1 expression in cells after drug treatment. Detailed Implementation

[0047] In the following examples and experimental cases, reagents and raw materials not specifically described are all commercially available products.

[0048] Example 1: Folic acid-modified mesoporous calcium carbonate nanoparticles loaded with quercetin and their preparation method

[0049] Step 1: Preparation of mesoporous calcium carbonate (MCaCO3)

[0050] Dissolve 150 mg CaCl2 in 100 mL of anhydrous ethanol solution, sonicate to mix, add 10 g of ammonium bicarbonate granules, place in a vacuum drying oven, react at 40 °C for 2 days, ultrafilter, centrifuge, wash 3 times with pure water, and finally redissolve in pure water for later use.

[0051] Step 2: Loading quercetin (QU@CaCO3) onto mesoporous calcium carbonate

[0052] After centrifuging the mesoporous calcium carbonate, it was redissolved in 10 mL of DMSO solution. 50 mg of mesoporous calcium carbonate and 50 mg of quercetin (mass ratio 1:1) were added. After incubation on a shaker at 37 °C for 24 h, the mixture was centrifuged at 11,000 rpm, washed three times with pure water, and redissolved in pure water for later use.

[0053] Step 3: Folic acid-modified quercetin-loaded mesoporous calcium carbonate nanoparticles (T@QU@CaCO3)

[0054] Load 60 mg of quercetin onto the above-mentioned mesoporous calcium carbonate, redissolve it in 10 mL of pure water, add 100 mg of polyethylene glycol-folic acid coupling compound (methoxy polyethylene glycol-folic acid (mPEG-FA), molecular weight 2000 Da), stir and react overnight in a three-necked flask, centrifuge at 11000 rpm, and wash three times with pure water to obtain the final product.

[0055] This comparative example provides the control sample used in the experiment:

[0056] Comparative Example 1: Mesoporous calcium carbonate (MCaCO3, which can be abbreviated as CaCO3)

[0057] Prepared according to step 1 in Example 1.

[0058] Comparative Example 2: Quercetin loaded onto mesoporous calcium carbonate

[0059] Prepared according to steps 1 and 2 in Example 1.

[0060] Comparative Example 3: Preparation of Mesoporous Calcium Carbonate

[0061] Dissolve 200 mg CaCl2 in 300 mL of anhydrous ethanol solution, sonicate to mix, add 15 g of ammonium bicarbonate granules, place in a vacuum drying oven, react at 40 °C for 48 h, ultrafilter, centrifuge, wash three times with pure water, and finally redissolve in pure water for later use.

[0062] Comparative Example 4: Preparation of Mesoporous Calcium Carbonate

[0063] Dissolve 100 mg CaCl2 in 200 mL of anhydrous ethanol solution, sonicate to mix, add 15 g of ammonium bicarbonate granules, place in a vacuum drying oven, react at 42 °C for 24 h, ultrafilter, centrifuge, wash 3 times with pure water, and finally redissolve in pure water for later use.

[0064] The technical solution of the present invention will be further explained through experiments below.

[0065] Experiment Example 1: Screening Experiment of Mesoporous Materials

[0066] I. Experimental Methods

[0067] Mesoporous calcium carbonate was prepared using the methods of Comparative Examples 1, 3, and 4, and its structure was observed by transmission electron microscopy.

[0068] II. Experimental Results

[0069] like Figure 1 As shown, the mesoporous calcium carbonate prepared in Comparative Example 3 is spherical, but its morphological uniformity is poor; Figure 2 As shown, cubic mesoporous calcium carbonate was obtained in Comparative Example 4; as Figure 3As shown, the mesoporous calcium carbonate prepared by Comparative Example 1 is spherical and uniform in morphology. Therefore, the mesoporous calcium carbonate prepared by the method of Comparative Example 1 has the best morphology, and this nanoparticle was used for subsequent experiments.

[0070] Experimental Example 2: Structural Characterization of Functionalized Drug-Loaded Nanoparticles

[0071] In this experimental example, mesoporous calcium carbonate, mesoporous calcium carbonate loaded with quercetin, and folic acid-modified mesoporous calcium carbonate nanoparticles loaded with quercetin were prepared according to the methods of Comparative Example 1, Comparative Example 2, and Example 1.

[0072] I. Experimental Methods

[0073] The microstructure of nanoparticles was observed using transmission electron microscopy (TEM).

[0074] II. Experimental Results

[0075] The results are as follows Figure 4 As shown, MCaCO3 exhibits a uniform spherical shape, indicating successful preparation of mesoporous calcium carbonate nanoparticles. Figure 4 A); In mesoporous calcium carbonate QU@CaCO3 loaded with quercetin, the pore size of the nanoparticles decreases or even disappears due to the adsorption of quercetin. Figure 4 B) indicates that quercetin has successfully entered the mesopores and been adsorbed; the folic acid-modified mesoporous calcium carbonate T@QU@CaCO3 loaded with quercetin is coated with a membrane. Figure 4 C) indicates that the folic acid modification was successful. These results demonstrate the successful construction of folic acid-modified mesoporous calcium carbonate nanoparticles loaded with quercetin.

[0076] Experimental Example 3: Folic acid-modified mesoporous calcium carbonate nanoparticles loaded with quercetin can be used to treat triple-negative breast cancer.

[0077] In this experimental example, mesoporous calcium carbonate, mesoporous calcium carbonate loaded with quercetin, and folic acid-modified mesoporous calcium carbonate nanoparticles loaded with quercetin were prepared according to the methods of Comparative Example 1, Comparative Example 2, and Example 1.

[0078] I. Experimental Methods

[0079] (1) Cytotoxicity assay: The cytotoxicity of blank mesoporous calcium carbonate (CaCO3), free drug (QU), QU@CaCO3, and T@QU@CaCO3 was investigated using the CCK-8 assay. Mouse breast cancer cells (4T1) were plated when their density was approximately 80% or higher and they were in good condition. All cell manipulations were performed in a clean bench. First, the culture medium was discarded, and the cell surface was washed with PBS. Then, trypsin was added to digest the cells for 3-4 min. When the cells slid off the wall in sheets, complete culture medium was added to stop the digestion. The cell digestion solution was collected using a pipette, centrifuged at 1200 rpm for 3 min, and the supernatant was discarded. The cells were resuspended in fresh complete culture medium and counted using a cell counting chamber. The cells were then diluted with fresh complete culture medium to a concentration of approximately 50,000-60,000 cells / mL and added to 96-well plates at 100 μL per well using a pipette. Place the cells in an incubator. When the cells are in the logarithmic growth phase, add solutions containing different concentrations (12.5, 25, 50, 100, 200, 400, 800 μmol / L) of quercetin (QU), blank calcium carbonate solution (CaCO3), drug-loaded calcium carbonate solution (QU@CaCO3), and folic acid-modified drug-loaded calcium carbonate solution (T@QU@CaCO3). Incubate for 24 and 48 hours, respectively. Remove the 96-well plate from the incubator, add 10 μl of CCK8 reagent to each well under light-protected conditions, and incubate at 37°C for 30 minutes. Measure the absorbance (OD value) at 450 nm using a microplate reader and record the values. Calculate cell viability: (Experimental well OD value - Blank well OD value) / (Control well OD value - Blank well OD value) × 100%.

[0080] (2) Cell scratch assay: 4T1 cells in the culture dish were washed, digested, centrifuged, resuspended, counted, and then seeded into 12-well plates at 1×10⁻⁶ cells / well. 6 Cells were cultured per well for 24 hours. The culture medium was discarded, and the cells were washed once with PBS. A 10 μL pipette tip was used to make a scratch, followed by washing away the scratched cells with PBS. Cells were then photographed for observation. The CaCO3 group was treated with 50 μmol / L mesoporous calcium carbonate solution. The QU, QU@CaCO3, and T@QU@CaCO3 groups were treated with different formulations of quercetin containing the same concentration (60 μmol / L). After 24 hours, the culture medium was discarded, and the cells were washed once with PBS. Cell migration was then observed under a microscope.

[0081] (3) Calcium ion staining experiment: The experiment was performed when the 4T1 cells were in good growth condition and the density was above 85%. First, the 4T1 cells were digested, resuspended, counted, and then seeded into 6-well plates, 2 × 10⁶ cells per well. 5Cells were cultured in an incubator until they reached the logarithmic growth phase. The culture medium was discarded, and the cells were washed once with PBS. Then, each well was treated with different formulations of quercetin containing the same concentration (60 μmol / L) for 24 hours. Intracellular calcium ion staining was performed using the Fluo-4 AM calcium ion kit, and the cells were observed using a laser confocal microscope.

[0082] (4) Immunofluorescence assay: Immerse cell smears in 75% alcohol for 30 min, wipe clean with alcohol swabs, and then place in a 24-well plate. Mix the cell suspension with the culture medium evenly and gently add it to the wells along the well wall. After the cells adhere, add different drug-containing culture media containing the same concentration (60 μmol / L) of quercetin to each well. After 24 h, remove the plate and add pre-cooled PBS to prevent excessive detachment. Fix with 4% paraformaldehyde for 20 min, and wash three times with PBS for 5 min each time. Perforate the wells with 0.2% Trixton-X100 for 15 min, and wash three times with PBS for 5 min each time. Block with 10% goat serum at 37°C for 1 h, and wash three times with PBST for 5 min each time. Add primary antibody and incubate at 4°C overnight. After recovering the antibody, wash three times with PBST for 5 min each time. Add secondary antibody and incubate for 1-2 h, and wash three times with PBST for 5 min each time. Counterstain with DAPI for 10 min, and wash three times with PBST for 5 min each time. The cell smears were placed on a glass slide with a small amount of anti-quenching agent and observed using a laser confocal microscope.

[0083] II. Experimental Results

[0084] like Figure 5 As shown, the green fluorescence intensity of the QU@CaCO3 group was stronger than that of the PBS group, CaCO3 group, and QU group, and the green fluorescence intensity of the T@QU@CaCO3 group was more significant than that of the QU@CaCO3 group. This indicates that mesoporous calcium carbonate, after targeted modification, can better target and enter tumor cells, and QU can promote the entry of exogenous calcium ions into cells, causing an imbalance in intracellular calcium ion homeostasis and inducing apoptosis.

[0085] like Figure 6As shown, CCK-8 assays were performed to explore the cytotoxicity of the material itself and the cytotoxicity of the interaction between QU and released calcium ions. The results showed that mesoporous CaCO3 alone had no significant cytotoxicity to 4T1 cells. The QU group exhibited concentration-time-dependent killing effects on 4T1 cells within the range of 12.5–800 μmol / L. Compared with the QU group, the QU@CaCO3 group showed significantly enhanced cytotoxicity, indicating that mesoporous CaCO3 improved the intracellular delivery efficiency of quercetin. Compared with the QU group and the QU@CaCO3 group, the T@QU@CaCO3 group showed the strongest cytotoxicity against 4T1 cells. These results indicate that folic acid-modified quercetin-loaded mesoporous calcium carbonate nanoparticles can significantly enhance the in vitro antitumor activity of quercetin against triple-negative breast cancer 4T1 cells.

[0086] like Figure 7 As shown, the inhibitory effect of the drug on the migration of 4T1 cells was evaluated by cell scratch assay. The results showed that the cell growth and migration status of the mesoporous CaCO3 group and the PBS group were similar, indicating that the inhibitory effect of simply adding mesoporous CaCO3 on cell migration was very low. Migration was inhibited in the QU group, and the QU@CaCO3 group and T@QU@CaCO3 group showed more significant inhibitory effects on cell migration than the QU group, with the T@QU@CaCO3 group showing the best inhibitory effect. These results indicate that QU promotes an increase in intracellular calcium ion concentration, thereby inhibiting cell movement and migration. 2+ The inhibitory effect is better when added and modified with folic acid.

[0087] like Figure 8 As shown, the ability of the drug to induce ICD in 4T1 cells was evaluated by immunofluorescence experiments. It was found that the mesoporous CaCO3 group had almost no effect on the expression of CRT and HMGB1 in 4T1 cells, but the QU group showed a trend of increasing HMGB1 expression compared to the PBS group. The QU@CaCO3 and T@QU@CaCO3 groups had a good promoting effect on the expression of CRT and HMGB1 in 4T1 cells, with the T@QU@CaCO3 group showing the strongest fluorescence expression. The results indicate that QU promotes an increase in intracellular calcium ion concentration, thereby causing intracellular calcium ion disorder in the presence of Ca... 2+ When added, the effect is better, and it can better induce the expression of CRT and HMGB1 in 4T1 cells. At the same time, folic acid can directly kill breast cancer cells by targeting the mesoporous calcium carbonate delivery system and promote the occurrence of immunogenic cell death (ICD).

[0088] In summary, this invention, through preliminary experiments on the ratio of mesoporous calcium carbonate, quercetin, and polyethylene glycol-folic acid conjugate, and screening of mesoporous materials, constructed a functionalized drug-loaded nanoparticle, T@QU@CaCO3, with good biocompatibility and high drug loading capacity. This nanoparticle not only acts as a "calcium bomb" to provide calcium ions for tumor calcium overload after degradation in the microacidic environment of tumors, but also has a high loading capacity for quercetin, a traditional Chinese medicine anticancer active ingredient. Furthermore, folic acid modification of the nanoparticles improves the stability of the mesoporous calcium carbonate, reduces the irritant effect of quercetin, enhances in vivo circulation, and further targets tumor cells to promote drug accumulation in TNBC cells, thereby improving targeted delivery capability and achieving synergistic efficacy and reduced toxicity. This provides a feasible solution for precise targeted therapy of breast cancer, especially triple-negative breast cancer.

Claims

1. A functionalized drug-loaded nanoparticle, characterized in that, The functionalized drug-loaded nanoparticles are folic acid-modified mesoporous calcium carbonate nanoparticles loaded with quercetin, comprising the following raw materials in parts by weight: 100-200 servings of calcium source CO3 2- 10,000-15,000 parts of precursor Quercetin 100-200 parts 100-300 parts of polymer-folic acid conjugate.

2. The functionalized drug-loaded nanoparticles according to claim 1, characterized in that: The ingredients include the following parts by weight: 150 servings of calcium source CO3 2- 10,000 precursors Quercetin 150 parts 100 parts of polymer-folic acid conjugate.

3. The functionalized drug-loaded nanoparticles according to claim 1 or 2, characterized in that: The calcium source is selected from at least one of calcium chloride, calcium nitrate, and calcium acetate; And / or, the CO3 2- The precursor is selected from ammonium bicarbonate; And / or, the polymer-folic acid conjugate is selected from at least one of polyethylene glycol-folic acid conjugate, hyaluronic acid-folic acid conjugate, polyvinylpyrrolidone-folic acid conjugate, and dextran-folic acid conjugate.

4. The functionalized drug-loaded nanoparticles according to claim 1, characterized in that: The mass ratio of the mesoporous calcium carbonate nanoparticles to quercetin is 1:1-2.

5. The method for preparing functionalized drug-loaded nanoparticles according to any one of claims 1-4, characterized in that: Includes the following steps: Step 1, mix the calcium source with CO3 2- Precursor reaction yields mesoporous calcium carbonate nanoparticles; Step 2: React mesoporous calcium carbonate nanoparticles with quercetin to obtain quercetin-loaded mesoporous calcium carbonate nanoparticles. Step 3: React quercetin-loaded mesoporous calcium carbonate nanoparticles with a polymer-folic acid conjugate to obtain the final product.

6. The preparation method according to claim 5, characterized in that: In step 3, the mass ratio of the quercetin-loaded mesoporous calcium carbonate nanoparticles to the polymer-folic acid conjugate is 1:1-2.

7. The preparation method according to claim 5, characterized in that: In step 1, the solvent for the reaction is anhydrous ethanol; And / or, in step 1, the reaction is carried out under vacuum conditions, at a temperature of 40°C-50°C, for a time of 2-3 days; And / or, in step 2, the solvent for the reaction is dimethyl sulfoxide; And / or, in step 2, the reaction temperature is 37℃-42℃, and the incubation time is 24h-48h; And / or, in step 3, the reaction temperature is 20-25°C and the stirring time is 12-16 h.

8. Use of the functionalized drug-loaded nanoparticles according to any one of claims 1-4 in the preparation of a medicament for treating breast cancer.

9. The use according to claim 8, characterized in that: The breast cancer mentioned is triple-negative breast cancer.

10. A pharmaceutical composition for treating breast cancer, characterized in that: It is made by adding pharmaceutically acceptable excipients to functionalized drug-loaded nanoparticles as described in any one of claims 1-4 as the active ingredient.

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