A polydopamine modified nifedipine nano-platform, and a preparation method and application thereof

Abstract

The application discloses a kind of polydopamine modified nifedipine nano platform and its preparation method and application, its particle size is 100-300 nm, present hexagon, by as carrier hollow polydopamine nanoparticle and the nifedipine loaded on it constitute, wherein, hollow polydopamine nanoparticle is made by ZIF-8 and dopamine response.The application has excellent drug loading capacity and the targeting ability to tumor, can target tumor cells by EPR effect, effectively kill breast cancer cells;Firstly, it is clear that nifedipine inhibits breast cancer through GnRH signal pathway, provides new drug selection and combined drug strategy for breast cancer treatment;Give consideration to antitumor and cardiovascular protective effect, possibly reduce the risk of cardiovascular complications in breast cancer patient treatment.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a polydopamine-modified nifedipine nanoplatform, its preparation method, and its application. Background Technology

[0002] Breast cancer is one of the most common malignant tumors among women worldwide, and despite continuous advancements in diagnosis and treatment, its mortality rate remains high. Current anti-tumor therapies often trigger cardiovascular complications, becoming a leading cause of non-tumor death in patients. Therefore, developing drugs with both anti-tumor and cardiovascular protective effects is of significant clinical importance. Nifedipine, a commonly used dihydropyridine calcium channel blocker, is primarily used to treat hypertension and angina. Recent studies have found potential applications of nifedipine in various solid tumors, but its use in breast cancer remains highly controversial.

[0003] Multiple studies have shown that nifedipine can promote the proliferation and migration of breast cancer cells, for example, by stimulating cancer cell growth and metastasis, leading to metastatic lesions, as observed in in vitro and in vivo experiments. Specifically, nifedipine activates cancer cell proliferation and migration mechanisms through the miRNA-524-5p-BRI3-Erk signaling pathway, resulting in accelerated tumor progression. Other reports indicate that calcium channel blockers such as nifedipine may increase the risk of breast cancer or worsen prognosis; for example, epidemiological studies have observed an increased risk of lung cancer, colorectal cancer, kidney cancer, and breast cancer associated with the use of such drugs. A few studies have explored its role in reversing chemotherapy resistance, such as in combination with doxorubicin or vincristine, but no significant response was observed, and it may exacerbate myelosuppression or other toxic reactions.

[0004] Although high concentrations of nifedipine have shown potential therapeutic effects in some studies, such as reducing proliferation in certain breast cancer cell lines, overall evidence suggests that its promoting effect is more dominant, and direct use faces biosafety issues, including systemic toxicity and increased cardiovascular risk.

[0005] Furthermore, its molecular mechanism of action in breast cancer cells remains unclear, and dose-dependency leads to conflicting results; for example, low concentrations may promote tumor growth, while high concentrations occasionally show inhibitory effects, but these are inconsistent. These controversies stem from the complexity of calcium signaling pathways, with nifedipine's effects on proliferation and migration varying significantly across different cancer cell lines. Compared to other calcium channel blockers such as verapamil or diltiazem, nifedipine exhibits a more pronounced pro-cancer effect, while the latter sometimes shows inhibition of tumor metastasis.

[0006] Overall, existing research on nifedipine mainly focuses on its negative effects, limiting its potential for reuse as an anticancer drug. Summary of the Invention

[0007] The purpose of this invention is to overcome the shortcomings of the prior art and provide a polydopamine-modified nifedipine nanoplatform.

[0008] Another object of the present invention is to provide a method for preparing the above-mentioned polydopamine-modified nifedipine nanoplatform.

[0009] Another object of the present invention is to provide the application of the above-mentioned polydopamine-modified nifedipine nanoplatform.

[0010] The technical solution of the present invention is as follows:

[0011] A polydopamine-modified nifedipine nanoplatform with a particle size of 100-300 nm and a hexagonal shape is provided. It consists of hollow polydopamine nanoparticles as a carrier and nifedipine loaded thereon. The hollow polydopamine nanoparticles are prepared by reacting ZIF-8 and dopamine.

[0012] In a preferred embodiment of the present invention, the molar ratio of nifedipine, dopamine and ZIF-8 is 2-3:4-5:1.

[0013] The preparation method of the above-mentioned polydopamine-modified nifedipine nanoplatform includes the following steps:

[0014] (1) Synthesize ZIF-8 solution;

[0015] (2) Add dopamine and formaldehyde to the ZIF-8 solution obtained in step (1), and stir at a constant speed for 3-5 h at 55-64 °C to obtain a hollow polydopamine nanoparticle solution.

[0016] (3) Dissolve nifedipine in formaldehyde, add the hollow polydopamine nanoparticle solution obtained in step (2), stir and centrifuge, take the precipitate, and repeatedly disperse and centrifuge with PBS to obtain the final product.

[0017] In a preferred embodiment of the present invention, in step (2), the temperature of the uniform stirring reaction is 60°C and the time is 4 h.

[0018] The use of the above-mentioned polydopamine-modified nifedipine nanoplatform in the preparation of cancer therapeutic compositions.

[0019] In a preferred embodiment of the present invention, the cancer is breast cancer, and the polydopamine-modified nifedipine nanoplatform induces apoptosis of breast cancer cells by inhibiting GnRHR protein expression.

[0020] A cancer treatment composition comprising the above-mentioned polydopamine-modified nifedipine nanoplatform as its active ingredient.

[0021] In a preferred embodiment of the present invention, the cancer is breast cancer, and the polydopamine-modified nifedipine nanoplatform induces apoptosis of breast cancer cells by inhibiting GnRHR protein expression.

[0022] The beneficial effects of this invention are:

[0023] 1. This invention possesses excellent drug loading capacity and tumor targeting ability, effectively killing breast cancer cells through the EPR effect. Specifically, the nanoplatform is hexagonal with uniform size and a particle size distribution of approximately 200 nm. It exhibits good dispersibility, and the particle size and dispersibility do not change over time, ensuring stable drug delivery and high-concentration accumulation in the tumor area. This inhibits the growth, proliferation, and migration of breast cancer cells in in vitro experiments and significantly slows tumor growth and reduces tumor volume and weight in in vivo experiments.

[0024] 2. This invention is the first to clearly demonstrate that nifedipine inhibits breast cancer through the GnRH signaling pathway, providing a new drug option and combination therapy strategy for breast cancer treatment. The specific mechanism includes inhibiting the expression of GnRH1 and GnRHHR proteins, inducing apoptosis in breast cancer cells. Bioinformatics analysis identified the direct protein targets (DPTs) of nifedipine and its core KEGG pathway (GnRH signaling pathway), and the inhibitory effect was confirmed in various breast cancer cell lines (such as MCF-7, T47D, MDA-MB-468, and MDA-MB-231) through experiments including Western blot, flow cytometry, and TUNEL staining.

[0025] 3. This invention combines anti-tumor and cardiovascular protective effects, potentially reducing the risk of cardiovascular complications in breast cancer patients during treatment. As a commonly used calcium channel blocker in clinical practice, nifedipine retains its cardiovascular protective function while exerting its anti-tumor effect. In in vivo animal experiments, it did not cause significant changes in mouse body weight, indicating its safety and providing a potential strategy for addressing non-tumor deaths (such as cardiovascular complications) caused by anti-tumor therapy. Attached Figure Description

[0026] Figure 1 This invention presents the DPTs and KEGG pathway analysis of nifedipine in Example 1. Specifically: A shows a list of 10 direct protein targets, and B shows the core KEGG pathway (GnRH signaling pathway).

[0027] Figure 2 This invention demonstrates the preparation and characterization of NP@PDA obtained in Example 1. A is a transmission electron microscope (TEM) image of NP@PDA, B shows the particle size distribution of NP@PDA, and C shows the temporal dispersion and hydrodynamic diameter distribution of NP@PDA.

[0028] Figure 3 This invention demonstrates the in vitro antitumor effect of nifedipine in Example 1. Specifically: A shows the cell proliferation inhibition rate as detected by CCK8 assay; B shows the results of the colony formation assay; and C shows the cell migration ability as detected by the scratch assay.

[0029] Figure 4 This illustrates the flow cytometry detection of cell apoptosis rate in Example 1 of the present invention.

[0030] Figure 5 This shows the results of Western blot detection of GnRH1 and GnRHR protein expression in Example 1 of the present invention.

[0031] Figure 6 This invention demonstrates the in vivo antitumor effect of nifedipine in Example 1 of this invention. Wherein: A is a schematic diagram of the animal experimental design; B and C show tumor volume comparisons; D shows changes in mouse body weight; E shows TUNEL staining for apoptosis detection; and F shows IHC staining for GnRH1 / GnRHR expression detection. Detailed Implementation

[0032] The technical solution of the present invention will be further explained and described below with reference to specific embodiments and accompanying drawings.

[0033] Example 1

[0034] I. Experimental Materials and Methods

[0035] 1. Synthesis and Characterization of NP@PDA

[0036] 1) Synthesis of ZIF-8: ZIF-8 was synthesized according to the technical solution disclosed in CN114522151A, and a ZIF-8 solution was obtained;

[0037] 2) Synthesis of PDA: Add 1 g of dopamine to 4 mL of ZIF-8 solution, add 30 mL of formaldehyde, transfer to a three-necked flask and place in a 60 ℃ oil bath for uniform stirring and reaction for 4 h to obtain a hollow polydopamine nanoparticle solution.

[0038] 3) Synthesis of NP@PDA: Dissolve 1 mg of nifedipine in 10 mL of formaldehyde, add the hollow polydopamine nanoparticle solution obtained in step 2), stir for 24 h, centrifuge, and collect the precipitate. Disperse repeatedly with PBS, centrifuge and wash 3 times to obtain NP@PDA dispersion.

[0039] 4) Electron microscope sample preparation:

[0040] a) Take a 1 mL EP tube, put 100 μL of the NP@PDA dispersion prepared in step 3) into a centrifuge tube, and use a vortex mixer or sonication to disperse the NP@PDA evenly;

[0041] b) Remove the carbon film copper mesh with sample tweezers and place it on the experimental table with the front side facing up;

[0042] d) Use a 10 µL pipette to drop the NP@PDA dispersion prepared in step a) onto a carbon film copper grid, dry it in air, repeat 3 times, and observe it using a transmission electron microscope. The accelerating voltage is 200 kV and the test temperature is 23±2 ℃.

[0043] 5) Particle size and dispersibility determination:

[0044] a) Take a 1.5 mL EP tube, add 1 mL of 95% ethanol solution, and then inject 1 µL of the NP@PDA dispersion prepared in step 3) into a centrifuge tube and sonicate to disperse the NP@PDA evenly.

[0045] b) Prepare cuvettes, turn on the instrument to preheat, use a 1 mL pipette to transfer the prepared NP@PDA sample into the cuvette, and test it on the instrument. Note that the triangular part of the cuvette should be facing forward.

[0046] 2. Cell lines and reagents

[0047] 1) Breast cancer cell lines (MCF-7, T47D, MDA-MB-468, MDA-MB-231) were purchased from ATCC.

[0048] 2) Nifedipine and CCK8 kits were purchased from MedChemExpress; antibodies were purchased from CST.

[0049] 3. Bioinformatics Analysis

[0050] 1) Obtain nifedipine DPTs (ID: DB01115) through DrugBank.

[0051] 2) Use STRING to construct a protein interaction network, and Cytoscape to enrich and visualize pathways.

[0052] 4. In vitro functional experiments

[0053] 1) CCK8 experiment: to detect the effect of different concentrations of nifedipine (0-200 μmol / L) on cell viability.

[0054] 2) Colony formation experiment: After inoculating cells, they were treated with nifedipine for 24 h and cultured for 14 days before counting the number of clones.

[0055] 3) Flow cytometry: Annexin V / 7-AAD staining was used to detect apoptosis rate.

[0056] 4) Western blot: Extract total protein and detect the expression of proteins such as GnRH1 and GnRHR.

[0057] 5. In vivo animal experiments

[0058] 1) Nude mouse tumor model: MCF-7 cells were inoculated into the axilla of nude mice. When the tumor volume reached 100 mm³, the mice were randomly divided into a control group (PBS) and a nifedipine group (20 mg / kg, intraperitoneal injection, 5 times a week).

[0059] 2) Tumor measurement: Tumor volume was measured every day, and samples were taken for TUNEL and IHC staining after the experiment.

[0060] II. Results and Discussion:

[0061] In this embodiment, drug bank detection of nitroglycerin plains-origin DPT revealed 10 nitroglycerin plains-origin DPTs (CACNA1C, CACNA1D, CACNA1G, CACNA1H, CACNA1I, CACNA1S, CACNB2, NR1I2, CALM1, KCND3). Figure 1 A). This embodiment identified the KEGG pathway of nifedipine DPTs using STRING and Cytoscape, and found that the GnRH signaling pathway is one of the most important signaling pathways of nifedipine (A). Figure 1 B). For example... Figure 2 As shown in Figure A, the NP@PDA synthesized in this embodiment is hexagonal, uniform in size, and has good dispersibility. Figure 2 Figure B shows that the size distribution of NP@PDA is around 200 nm. The time stability and dispersibility of NP@PDA were tested, and the results showed that the particle size and dispersibility of NP@PDA did not change over time. Figure 2 C).

[0062] Further experimental results validated the accuracy of the bioinformatics analysis predictions. CCK8 and colony formation experiments confirmed that certain concentrations of nifedipine can inhibit the growth and proliferation of breast cancer cells. Figure 3 A, 3B), and inhibit breast cancer cell migration ( Figure 3 C). Apoptosis experiments confirmed that nifedipine induced apoptosis in breast cancer cells. Figure 4 Western blotting analysis showed that nifedipine promotes apoptosis in breast cancer cells through the GnRH signaling pathway. Figure 5 Therefore, in vitro experiments have demonstrated that NP@PDA can affect the GnRH signaling pathway in breast cell lines, inhibiting the growth and proliferation of breast cancer cells.

[0063] To determine the in vivo effects of NP@PDA, MCF-7 tumor-bearing mice were randomly divided into two groups: a control group and an NP@PDA group. Figure 6 As shown in Figure A, mice were treated. Body weight and tumor growth were recorded every two days for each group to assess treatment efficacy. Tumor growth measurements showed that the NP@PDA treatment group exhibited significantly slower tumor growth compared to other treatment groups. Figure 6 (British BC); Body weight measurements showed that mice in each group had similar growth curves ( Figure 6 (D); such as Figure 6 TUNEL fluorescence staining results showed that the tumor slices treated with NP@PDA had more green spots, indicating a significantly increased apoptosis rate and a more pronounced tumor-killing effect. Immunohistochemical staining (IHC) was used to assess the expression levels of GnRH1 / GnRH to further investigate the mechanism of action of NP@PDA in vivo, demonstrating that NP@PDA can inhibit the GnRH signaling pathway.

[0064] This embodiment employs a combination of bioinformatics analysis and experimental methods for prediction. The results demonstrate that NP@PDA inhibits breast cancer through the GnRH signaling pathway, opening new avenues for exploring new applications of existing drugs and elucidating the molecular mechanisms of drug action in breast cancer cells. This embodiment is the first to clearly demonstrate that NP@PDA inhibits breast cancer through the GnRH signaling pathway, providing new drug options and combination therapy strategies for breast cancer treatment. This embodiment also considers both anti-tumor and cardiovascular protective effects, potentially reducing the risk of cardiovascular complications in breast cancer patients during treatment.

[0065] The above description is merely a preferred embodiment of the present invention, and therefore should not be construed as limiting the scope of the present invention. All equivalent changes and modifications made in accordance with the scope of the patent and the contents of the specification should still fall within the scope of the present invention.

Claims

1. A polydopamine-modified nifedipine nanoplatform, characterized in that: Its particle size is 100-300 nm, and it is hexagonal. It consists of hollow polydopamine nanoparticles as a carrier and nifedipine loaded on them. The hollow polydopamine nanoparticles are made by reacting ZIF-8 and dopamine. The preparation method of this polydopamine-modified nifedipine nanoplatform includes the following steps: (1) Synthesize ZIF-8 solution; (2) Add dopamine and formaldehyde to the ZIF-8 solution obtained in step (1), and stir at a constant speed at 55-64 °C for 3-5 h to obtain a hollow polydopamine nanoparticle solution. (3) Dissolve nifedipine in formaldehyde, add the hollow polydopamine nanoparticle solution obtained in step (2), stir and centrifuge, take the precipitate, and repeatedly disperse and centrifuge with PBS to obtain the final product.

2. The polydopamine-modified nifedipine nanoplatform according to claim 1, characterized in that: The molar ratio of nifedipine, dopamine, and ZIF-8 is 2-3: 4-5:

1.

3. The method for preparing a polydopamine-modified nifedipine nanoplatform according to claim 1 or 2, characterized in that: Includes the following steps: (1) Synthesize ZIF-8 solution; (2) Add dopamine and formaldehyde to the ZIF-8 solution obtained in step (1), and stir at a constant speed at 55-64 °C for 3-5 h to obtain a hollow polydopamine nanoparticle solution. (3) Dissolve nifedipine in formaldehyde, add the hollow polydopamine nanoparticle solution obtained in step (2), stir and centrifuge, take the precipitate, and repeatedly disperse and centrifuge with PBS to obtain the final product.

4. The preparation method according to claim 3, characterized in that: In step (2), the temperature of the uniform stirring reaction is 60 °C and the time is 4 h.

5. The use of the polydopamine-modified nifedipine nanoplatform according to claim 1 or 2 in the preparation of cancer therapeutic compositions, characterized in that: The cancer is breast cancer, and the polydopamine-modified nifedipine nanoplatform induces apoptosis in breast cancer cells by inhibiting GnRHR protein expression.

6. A cancer treatment composition, characterized in that: Its active ingredient includes the polydopamine-modified nifedipine nanoplatform as described in claim 1 or 2.

7. The cancer treatment composition as described in claim 6, characterized in that: The cancer is breast cancer, and the polydopamine-modified nifedipine nanoplatform induces apoptosis in breast cancer cells by inhibiting GnRHR protein expression.

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