A near-infrared triggered multifunctional nanoplatform, its construction method, and its application in the preparation of drugs against triple-negative breast cancer.

Abstract

This invention discloses a near-infrared triggered multifunctional nanoplatform, its construction method, and its application in the preparation of drugs for triple-negative breast cancer. The nanoplatform, DOX-PAAP, has a core-shell structure, with an Au-Au@PEG nanoassembly structure made of gold nanoparticles (AuNPs) and gold nanoclusters (AuNCs) as the core, a polydopamine coating as the intermediate responsive layer, and doxorubicin molecules loaded on the surface of the polydopamine coating to form a functional shell. This invention fully utilizes the photothermal and catalytic properties of gold nanoparticles and gold nanoclusters, combined with the intelligent responsive coating of polydopamine, to construct a nanoplatform with four synergistic functions: achieving a photothermal conversion efficiency of no less than 60% through near-infrared triggering; achieving precise controlled drug release using a pH / GSH dual-response mechanism; establishing a self-supplied ROS generation system; and simultaneously activating GSH depletion and enhancing the ferroptosis pathway.

Description

Technical Field

[0001] This invention belongs to the field of nanomedicine technology, and provides a near-infrared triggered multifunctional nanoplatform, its construction method, and its application in the preparation of anti-triple-negative breast cancer drugs. Background Technology

[0002] The treatment of breast cancer currently faces severe clinical challenges. Triple-negative breast cancer, due to its high invasiveness and lack of effective targets, primarily relies on anthracycline drugs such as doxorubicin (DOX) for chemotherapy. However, the inherent multidrug resistance mechanisms of the tumor microenvironment severely limit treatment efficacy: tumor cells overexpress P-glycoprotein to form drug efflux pumps, leading to insufficient intracellular drug concentrations; simultaneously, the upregulated glutathione (GSH) antioxidant system can rapidly scavenge reactive oxygen species (ROS), weakening the oxidative stress killing effect of chemotherapy drugs; more critically, the hypoxic microenvironment further limits the efficiency of ROS generation. This dynamic of drug resistance leads to recurrence in up to 40% of triple-negative breast cancer patients within one year of initial chemotherapy, and recurrent tumors are often more aggressive, significantly increasing the difficulty of treatment. Therefore, breakthrough strategies must directly target the synergistic integration of these drug resistance mechanisms, such as achieving dynamic synergistic effects through the combination of multifunctional modules, rather than relying solely on single therapies.

[0003] In exploring solutions, nanosynthetic technologies have shown great potential in recent years. Their core lies in integrating multiple treatment modalities, such as phototherapy, chemotherapy, and ferroptosis induction, to overcome the limitations of single therapies. Photothermal-chemotherapy synergistic systems utilize the plasma resonance effect of gold or copper-based nanomaterials to convert near-infrared light into heat energy, enhancing drug penetration and tumor ablation. However, phototherapy typically only penetrates 3 to 5 millimeters into the tissue, making precise drug release into deep tumors difficult. Enzyme kinetic therapy uses transition metal nanozymes, such as iron-based catalysts, to catalyze the production of ROS from endogenous tumor-derived hydrogen peroxide (H2O2), bypassing hypoxia limitations. However, the concentration of hydrogen peroxide at the tumor site is often insufficient (below 50 µM), and catalytic efficiency is low, affected by pH and enzyme activity, leading to unstable efficacy. Ferrroptosis induction strategies focus on consuming GSH or inhibiting GPX4 enzymes to trigger iron-dependent cell death. However, existing inducers, such as erastin or sorafenib analogs, have low bioavailability and systemic toxicity, including liver injury, limiting their clinical translation. Despite the widespread proposal of multi-mechanism synergistic frameworks, such as simultaneously integrating photothermal conversion, enzymatic catalysis, and GSH consumption, practical applications still face three major technological barriers: the complexity of carrier construction makes it difficult for traditional nanoplatforms to efficiently integrate multiple functional modules; for example, the synthesis of gold-iron composite nanoparticles requires precise control of size and surface chemistry. Spatiotemporal control challenges prevent precise synchronization of drug release and ROS generation at the tumor site; for example, photothermally triggered drug release may precede reactive oxygen species bursts, reducing synergistic efficiency. Furthermore, biocompatibility limitations manifest in the long-term in vivo retention of metal-based nanocarriers, such as accumulation in the liver leading to inflammation or fibrosis. Multiple preclinical studies have shown that over 30% of nanocarriers remain in the liver and spleen after a single dose, increasing the risk of organ damage. These limitations highlight the importance of developing novel intelligent platforms—which need to achieve spatiotemporally precise drug-ROS synchronous release through the synergistic coupling of core modules, thereby overcoming existing technological bottlenecks.

[0004] In summary, while existing technologies each have their advantages, their therapeutic efficiency is limited by physical barriers such as tissue depth, biochemical interferences such as fluctuations in glutathione levels, and cellular defense mechanisms such as P-glycoprotein overexpression. Toxic side effects stem from the non-targeting nature of chemotherapy drugs, such as the cardiotoxicity of DOX, and the potential immunogenicity of nanomaterials. Drug resistance persists due to the dynamic adaptation of tumor cells, such as the activation of DNA repair or metabolic reprogramming. The development of next-generation multifunctional synergistic platforms should possess microenvironment-responsive capabilities, such as pH or reactive oxygen species-sensitive polymer shells that automatically adapt to hypoxic or high GSH regions, real-time monitoring functions embedded with fluorescence or MRI contrast agents for therapeutic feedback, and integration of phototherapy, chemotherapy, and ferroptosis modules. Emerging intelligent nanocarriers can reduce complexity through modular design while utilizing tumor-specific stimuli such as enzyme overexpression or low pH triggering for on-demand release, enhancing spatiotemporal control. Therefore, there is an urgent need to develop a multifunctional synergistic platform that integrates multiple treatment modalities, possesses microenvironment-responsive capabilities, and can be monitored in real time. Summary of the Invention

[0005] In view of this, the purpose of this invention is to provide a near-infrared triggered multifunctional nanoplatform, its construction method, and its application in the preparation of anti-triple-negative breast cancer drugs, aiming to solve the problem of breast cancer drug resistance.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] 1. A near-infrared triggered multifunctional nanoplatform, wherein the nanoplatform has a core-shell structure, with an Au-Au@PEG nano-assembly structure made of gold nanoparticles AuNPs and gold nanoclusters AuNCs as the core, a polydopamine coating as an intermediate response layer, and doxorubicin molecules loaded on the surface of the polydopamine coating to form a functional shell.

[0008] 2. The specific steps of constructing the aforementioned near-infrared triggered multifunctional nanoplatform are as follows:

[0009] (1) First, gold nanoparticles AuNPs and gold nanoclusters AuNCs are made into Au-Au@PEG nano-assemblies;

[0010] (2) Then, a polydopamine coating (PDA coating) is uniformly coated on the surface of the Au-Au@PEG nano-assembly structure to obtain PAAP (polydopamine-coated Au-Au@PEG nano-assembly structure).

[0011] (3) Finally, doxorubicin molecules (DOX) are non-covalently loaded onto the PDA coating through π-π stacking to obtain DOX-PAAP.

[0012] Preferably, in step (1), the average particle size of the Au-Au@PEG nano-assembly structure is 90 nm.

[0013] Preferably, the specific method of step (1) is as follows:

[0014] (1-1) First, gold nanoparticles AuNPs are mixed with mercapto polyethylene glycol amino solution (SH-PEG-NH2 solution) and stirred, and then dialyzed with deionized water to remove unreacted SH-PEG-NH2;

[0015] (1-2) Then add MES (2-morpholinoethanesulfonic acid) buffer, stir for the first time, continue to add EDC (1-ethyl-(3-dimethylaminopropyl)carbodiimide) aqueous solution, stir for the second time, then add NHS (N-hydroxysuccinimide) aqueous solution, stir for the third time, and dialyze with deionized water to remove unreacted EDC and NHS to obtain PEG@AuNPs solution;

[0016] (1-3) Next, adjust the pH of the gold nanoclusters AuNCs to 7-8, mix and stir with the PEG@AuNPs solution at room temperature (25℃), dialyze with deionized water, and store at low temperature.

[0017] More preferably, the molar ratio of AuNPs to AuNCs is 1:1.5 to 1:2.5.

[0018] More preferably, the volume ratio of AuNPs, mercaptopolyethylene glycol amino solution, MES buffer, EDC aqueous solution, and NHS aqueous solution is 2:1:1:2:2; wherein the concentration of mercaptopolyethylene glycol amino solution is 0.25–0.5 mmol / L, the concentration of MES buffer is 0.1–0.2 mol / L, pH=6.5, the concentration of EDC aqueous solution is 0.05–0.1 mol / L, and the concentration of NHS aqueous solution is 0.05–0.1 mol / L.

[0019] More preferably, in step (1-1), the mixing time is 6 to 8 hours.

[0020] More preferably, in steps (1-2), the first stirring time is 2-5 minutes, the second stirring time is 1-2 hours, and the third stirring time is 4-6 hours.

[0021] More preferably, in steps (1-3), the mixing and stirring time is 10-12 hours, and the deionized water dialysis time is 10-12 hours.

[0022] More preferably, the average particle size of the gold nanoparticles (AuNPs) is 25–35 nm.

[0023] More preferably, the gold nanoparticles (AuNPs) are prepared by the following method: a 10 mg / mL aqueous solution of HAuCl4·3H2O is mixed with a 10 mg / mL aqueous solution of PVP and injected into a flask; then, deionized water is added, stirred for 2 minutes, heated to 85°C, and a 1.7 mg / mL aqueous solution of NaBH4 is quickly added; the mixture is further heated to 90°C, stirred for 15–20 minutes, dialyzed against deionized water for 10–12 hours to remove excess NaBH4, and stored at low temperature.

[0024] More preferably, the volume ratio of HAuCl4·3H2O aqueous solution, PVP aqueous solution, and deionized water is 3:3:92-95; the NaBH4 aqueous solution is obtained by dissolving 11.7 mg of NaBH4 in 10 mL of water.

[0025] Even more preferably, the molecular weight cutoff of the dialysis bag used for dialysis is 3500 Da.

[0026] More preferably, the average particle size of the gold nanoclusters AuNCs is 1.8–2.5 nm.

[0027] More preferably, the gold nanoclusters AuNCs are prepared by the following method: mixing HAuCl4·3H2O solution with GSH (glutathione) solution, heating at 80°C for 5 hours, dialyzing with deionized water for 10-12 hours to completely remove excess GSH, and storing under low temperature conditions.

[0028] More preferably, the volume ratio of HAuCl4·3H2O solution to GSH solution is 0.3:50; the concentration of HAuCl4·3H2O solution is 0.1 g / mL; and the concentration of GSH solution is 3 mM.

[0029] Even more preferably, the molecular weight cutoff of the dialysis bag used for dialysis is 3500 Da.

[0030] Preferably, in step (2), the thickness of the polydopamine coating is 10-15 nm.

[0031] Preferably, the specific method of step (2) is as follows: first, add the hydrochloric acid dopamine solution (HCl-DA solution) to Tris buffer and mix thoroughly, then slowly inject Au-Au@PEG nano-assembly structure, stir for 5 to 7 hours under light-proof and oxygen-containing conditions, then add 1 mol / L dilute hydrochloric acid to adjust the pH to 7.0 to terminate the reaction, and PAAP is obtained.

[0032] Further preferably, the volume ratio of dopamine hydrochloride solution, Tris buffer, and Au-Au@PEG nano-assembly structure is 1:2:4; wherein the concentration of dopamine hydrochloride solution is 0.6 mg / mL, the concentration of Tris buffer is 10 mmol / L, and the pH is 8.0–9.0.

[0033] In a further preferred embodiment, after the reaction is complete, the product is centrifuged and washed three times to remove excess dopamine hydrochloride, and then redispersed in 0.1 M, pH 7.4 PBS buffer for later use.

[0034] Preferably, in step (3), the average particle size of DOX-PAAP is 120~150 nm.

[0035] Preferably, the specific method of step (3) is as follows: first, doxorubicin is prepared into a 1 mg / mL doxorubicin aqueous solution using water, then the doxorubicin aqueous solution is mixed with PAAP and stirred for 4 to 6 hours in the dark to achieve drug adsorption by π-π stacking effect; after loading, the mixture is centrifuged at 12,000 rpm for 15 to 20 minutes to remove free DOX, and the loading efficiency of the supernatant is determined by ultraviolet spectroscopy (480 nm).

[0036] More preferably, the mass ratio of doxorubicin to PAAP is 1:4 to 1:6, and more preferably 1:5.

[0037] More preferably, the centrifugation conditions are 10,000 to 15,000 rpm for 10 to 20 minutes; even more preferably, 12,000 rpm for 15 minutes.

[0038] Preferably, in step (3), DOX-PAAP is dispersed in phosphate buffer and stored at 4°C in the dark.

[0039] 3. Application of the aforementioned near-infrared triggered multifunctional nanoplatform in the preparation of anti-triple-negative breast cancer drugs.

[0040] The beneficial effects of this invention are:

[0041] This invention provides a near-infrared (NIR) triggered multifunctional nanoplatform, DOX-PAAP, its construction method, and its application in the preparation of drugs for triple-negative breast cancer. This nanoplatform integrates photothermal therapy, photodynamic therapy, and enzyme therapy, achieving a photothermal conversion efficiency of 60–65% under NIR irradiation; and under conditions of pH 5.8 and a glutathione concentration ≥8 mM, the drug release rate is increased to more than three times that under normal environmental conditions.

[0042] This invention fully utilizes the photothermal and catalytic properties of gold nanoparticles (AuNPs) and gold nanoclusters (AuNCs), combined with a smart responsive coating of polydopamine (PDA), to construct a nanoplatform with four synergistic functions: achieving a photothermal conversion efficiency of no less than 60% through near-infrared triggering; achieving precise controlled drug release using a pH / GSH dual-response mechanism; establishing a self-supplied ROS generation system; and simultaneously activating GSH depletion to enhance the ferroptosis pathway. This innovative architecture successfully overcomes the spatiotemporal control barriers and biotoxicity defects of traditional carriers, providing a novel solution for overcoming the challenges of breast cancer drug resistance.

[0043] This invention features a groundbreaking structural design. AuNPs-AuNCs provide cascaded catalytic active sites, a polydopamine intermediate layer enables a pH / glutathione dual-response degradation mechanism, and the doxorubicin shell precisely targets tumor cells. This tertiary structure solves for the first time the spatiotemporal control challenge of traditional carriers in simultaneously coordinating photothermal effects, enzymatic catalysis, and drug release.

[0044] This invention exhibits a multi-mechanism synergistic effect. Under near-infrared light triggering, the gold-based core simultaneously generates local high temperatures (ΔT > 40°C) and hydroxyl radicals (·OH), while the polydopamine layer simultaneously releases doxorubicin and consumes glutathione. Experiments have confirmed that this synergistic effect increases the tumor cell apoptosis rate to 4.2 times that of chemotherapy alone and significantly activates the ferroptosis pathway.

[0045] The present invention exhibits excellent biocompatibility. The dual modification with polyvinylpyrrolidone and polyethylene glycol, along with the PDA coating, significantly reduces cumulative liver toxicity. Animal experiments showed no pathological damage to major organs in the treatment group, overcoming the technical bottleneck of organ damage caused by long-term retention of metal nanocarriers.

[0046] Based on the innovative design of the DOX-PAAP nanoplatform of this invention in the field of tumor therapy, its applications mainly cover the following aspects: This platform, through the synergistic effect of multiple components including AuNPs, AuNCs, PDAs, and DOX, efficiently integrates photothermal therapy, photodynamic therapy, and enzyme therapy, making it suitable for targeted therapy of triple-negative breast cancer and addressing drug resistance issues. Simultaneously, its microenvironment-responsive characteristics (such as pH / GSH-triggered release) enable precise controlled release in drug delivery systems, reducing off-target toxicity. Furthermore, this platform can be extended to the field of medical imaging, achieving real-time treatment monitoring through near-infrared triggering, providing multifunctional support for tumor diagnosis and personalized medicine. The synergistic effects of these applications (such as effects far exceeding those of single therapies) ensure broad prospects for clinical translation.

[0047] Other advantages, objectives, and features of the invention will be set forth in part in the description which follows, and in part will be apparent to those skilled in the art from the following examination, or may be learned from practice of the invention. The objectives and other advantages of the invention can be realized and obtained through the following description. Attached Figure Description

[0048] To make the objectives, technical solutions, and advantages of the present invention clearer, the preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings, wherein:

[0049] Figure 1 This is a flowchart illustrating the preparation process of the present invention.

[0050] Figure 2 The images shown are: transmission electron microscope (TEM) image (A) of the product from step S1 of the example, TEM image (B) of the product from step S2 of the example, TEM image (C) of the product from step S3 of the example, scanning electron microscope (SEM) image (D), high-angle annular dark field scanning transmission electron microscope (HAADF-STEM) image (E), and corresponding elemental distribution mapping diagram (F).

[0051] Figure 3 This is a TEM image of the product from step S4 of the example.

[0052] Figure 4 The UV absorption spectrum of the product from step S5 of the example is shown.

[0053] Figure 5 The ultraviolet absorption spectrum (A), photoluminescence spectrum (B), infrared spectrum (C), thermogravimetric curve (D), Au 4f high-resolution X-ray photoelectron spectrum (E), and Zeta potential (F) of the product from step S3 of the example are shown.

[0054] Figure 6 The DOX release curves of the product from step S5 of the example at pH 5.8 and 7.4 for 48 hours are shown.

[0055] Figure 7 The DOX release curves of the product from step S5 of the example at pH 5.8 (A) and 7.4 (B) after adding different GSH concentrations for 48 hours are shown.

[0056] Figure 8 The following are examples of photothermal curves (A) of the product from step S4 of the embodiment irradiated with near-infrared laser at different concentrations, (B) of the product irradiated with near-infrared laser at different laser power densities, (C) of the photothermal cycle curve in three on / off irradiation cycles, (D) of the linear fitting curve of cooling time versus driving force temperature under the photothermal cycle in a single on / off irradiation cycle, and (E) of the infrared thermogram of near-infrared laser irradiated with different concentrations.

[0057] Figure 9 The UV absorption spectra (A) and (B) of the product of step S3 and step S4 of the example, and their relative production amounts (C) and (D), are used to evaluate the degradation efficiency of MB.

[0058] Figure 10 The time-UV absorption spectra (A), relative yield of singlet oxygen (B), and electron paramagnetic resonance (ESR) spectra (C) of the product generated in step S4 of the example are shown.

[0059] Figure 11 The biocompatibility assessment (A) of the products of steps S1, S2, S3, S4 and S5, as well as the assessment of the killing ability against 4T1 cancer cells (B) of the seven groups including PAAP+NIR and DOX-PAAP+NIR.

[0060] Figure 12 Fluorescence microscopy images (A) and semi-quantitative fluorescence analysis diagrams (B) of the different groups of the products from step S4 and step S5 of Example, G1: Control; G2: DOX; G3: NIR; G4: PAAP; G5: DOX-PAAP; G6: PAAP+NIR; and G7: DOX-PAAP+NIR, show the ability of these products to generate ROS in 4T1 cancer cells.

[0061] Figure 13 The fluorescence microscopy images (A) and semi-quantitative analysis diagram (B) of different groups of the products of step S4 and step S5 of Example G1: Control; G2: DOX; G3: NIR; G4: PAAP; G5: DOX-PAAP; G6: PAAP+NIR; and G7: DOX-PAAP+NIR on the live / dead cell staining (AM / PI) of 4T1 cancer cells are shown in step S4 of Example S5.

[0062] Figure 14 The apoptosis of 4T1 cancer cells in different treatment groups (G1: Control; G2: DOX; G3: NIR; G4: PAAP; G5: DOX-PAAP; G6: PAAP+NIR; and G7: DOX-PAAP+NIR) was analyzed by flow cytometry for the products of step S4 and step S5 in the examples.

[0063] Figure 15 This is for the in vitro PA imaging performance evaluation of the product from step S5 of the example.

[0064] Figure 16This is an evaluation of the in vivo PA imaging of the product from step S5 of the embodiment at different time points.

[0065] Figure 17 The in vivo photothermal properties of the products from step S4 and step S5 of the examples were evaluated by assessing the photothermal images (A) and temperature curves (B) of 4T1 tumors in BALB / c mice after different treatments.

[0066] Figure 18 The images show representative ROS fluorescence (A), H&E staining (B), TUNEL immunofluorescence staining (C), Ki-67 immunofluorescence staining (D), and semi-quantitative fluorescence analysis (E), TUNEL (% nuclei) semi-quantitative analysis (F), and Ki-67 (% nuclei) semi-quantitative analysis (G) plots of mouse tumor sections from different groups of the products of step S4 and step S5 of Example 1: Control; G2: DOX; G3: NIR; G4: PAAP; G5: DOX-PAAP; G6: PAAP+NIR; and G7: DOX-PAAP+NIR. Detailed Implementation

[0067] The present invention will be further described below with reference to specific embodiments.

[0068] It should be understood that the following embodiments are merely illustrative and explanatory of the present invention, and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above description of the present invention are covered within the scope of protection intended by the present invention.

[0069] In addition, unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be obtained by purchasing them from the market or prepared by existing methods.

[0070] Example

[0071] Combination Figure 1 As shown in the following embodiments, a method for constructing a near-infrared (NIR) triggered multifunctional nanoplatform, DOX-PAAP, is described in detail. The specific steps are as follows:

[0072] S1. Preparation of AuNPs:

[0073] 3 mL of HAuCl4·3H2O aqueous solution (10 mg / mL) and 3 mL of PVP aqueous solution (10 mg / mL) were mixed and injected into a flask. Then, 92 mL of deionized water was added, and the mixture was stirred for 2 minutes before heating to 85 °C. NaBH4 aqueous solution (11.7 mg dissolved in 10 mL of water) was quickly added, and the mixture was heated to 90 °C and stirred for 15 minutes. The synthesized AuNPs were dialyzed overnight in deionized water (molecular weight cutoff 3500 Da) to remove excess NaBH4 and stored at low temperature.

[0074] See Figure 2 The results from TEM show that AuNPs were successfully prepared.

[0075] S2. Preparation of AuNCs:

[0076] 0.3 mL of HAuCl4·3H2O solution (0.1 g / mL) was mixed with 50 mL of GSH solution (3 mM), and heated at 80 °C for 5 hours. The resulting AuNCs were dialyzed against deionized water for 12 hours (molecular weight cutoff: 3500 Da) to completely remove excess GSH and stored at low temperature. See also Figure 2 The B-mode TEM image shows that AuNCs were successfully prepared.

[0077] S3. Preparation of gold-based nano-assemblies of AuNPs and AuNCs:

[0078] 5 mL of SH-PEG-NH2 solution (0.25 mM) was mixed with 10 mL of AuNPs and stirred for 6 hours. The mixture was then dialyzed against deionized water for 12 hours to remove excess SH-PEG-NH2. To activate the -COOH groups on the AuNCs, 5 mL of MES buffer (0.2 M, pH 6.5) was added and stirred for 2 minutes to stabilize the solution. Then, 10 mL of EDC aqueous solution (0.1 M) was added and stirred for 1 hour. Next, 10 mL of NHS aqueous solution (0.1 M) was added, and stirring continued for 4 hours at room temperature. The mixture was dialyzed against deionized water overnight to remove unreacted EDC and NHS, yielding a PEG@AuNPs solution. After activation, the pH of the dialyzed AuNCs (AuNPs to AuNCs molar ratio of 1:2) was adjusted to 7-8, and the mixture was mixed with the PEG@AuNPs solution at room temperature and stirred for 12 hours to obtain Au-Au@PEG. The final product was dialyzed against deionized water overnight to remove residual impurities and stored at low temperature. See also Figure 2 The C, D, E, and F values, along with TEM and SEM images, demonstrate the successful fabrication of the gold-based nano-assembly structure. The elemental mapping diagram also confirms the successful assembly.

[0079] S4. Preparation of PDA coating on gold-based nano-assembled structures:

[0080] HCl-DA solution (0.6 mg / mL) was added to Tris buffer (10 mM, pH 8.5) and mixed thoroughly, then Au-Au@PEG solution was slowly injected. After stirring for 6 hours under light-protected and aerobic conditions, dilute hydrochloric acid was added to adjust the pH to approximately 7.0 to terminate the reaction, yielding a PDA-coated gold-based nanoassembly structure (PAAP). The obtained product was washed three times by centrifugation to remove excess HCl-DA, and then redispersed in an aqueous solution at pH 7.4 or PBS buffer for later use. See also... Figure 3 TEM showed that the PDA coating with gold-based nano-assembly structure was successfully prepared, with a coating thickness of about 12 nm.

[0081] S5. Preparation of the multifunctional nanoplatform (DOX-PAAP):

[0082] A 1 mg / mL aqueous solution of DOX was mixed with PAAP at a mass ratio of 1:5 and stirred in the dark for 4 hours to achieve drug adsorption via π-π stacking. After loading, the mixture was centrifuged at 12,000 rpm for 15 minutes to remove free DOX, and the loading efficiency of the supernatant was determined by UV spectroscopy (480 nm). The final product, DOX-PAAP, was dispersed in PBS and stored at 4°C protected from light. See also... Figure 4 The UV-Vis absorption spectrum shows that DOX-PAAP was successfully prepared.

[0083] Experimental Example 1:

[0084] The AuNCs, PEG@AuNPs, and Au-Au@PEG from steps S2 and S3 of this embodiment were subjected to UV-vis absorption spectroscopy, photoluminescence (PL) fluorescence spectroscopy, and Fourier transform (FT-IR) infrared spectroscopy, respectively. See [link to relevant documentation]. Figure 5 A, B, and C. It can be seen that Au-Au@PEG possesses plasmonic resonance peaks and fluorescence activity. The presence of amide bonds is also observed, further confirming the successful assembly of the Au-Au@PEG structure.

[0085] Experimental Example 2:

[0086] The AuNCs, AuNPs, Au-Au@PEG, and PAAP in steps S1, S2, S3, and S4 of this embodiment of the invention were subjected to thermogravimetric (TG) analysis, XPS valence state analysis, and Zeta potential distribution detection, respectively. (See [link to relevant documentation]). Figure 5Figures D, E, and F show that the mass loss corresponding to different weightlessness platforms, the structural electronic binding energy, and the XPS fine spectrum and potential changes of Au 4f all confirm the successful assembly of the Au-Au@PEG structure.

[0087] Experimental Example 3:

[0088] The DOX-PAAP from step S5 of this embodiment was dispersed in PBS buffer at pH values ​​of 5.8 and 7.4. The samples were placed in a 37°C constant-temperature shaking incubator. Equal volumes of solution were taken at predetermined time points (0.5, 1, 2, 3, 4, 8, 12, 24, 36, and 48 hours), and an equal volume of fresh PBS buffer was added. The absorbance of DOX was measured at 480 nm using UV spectroscopy to monitor its release kinetics. The experiment was repeated three times to ensure reproducibility. See also... Figure 6 It can be seen that DOX-PAAP has the ability to release drugs in a pH-responsive manner due to the pyrolysis and release of the PDA coating under acidic conditions.

[0089] Experiment Example 4:

[0090] To assess the effect of GSH, DOX-PAAP from step S5 of this embodiment was dispersed in PBS buffer. GSH was added to final concentrations of 1 mM, 5 mM, and 10 mM at pH 5.8 and 7.4, respectively. Samples were placed in a 37°C water bath with a shaker. Samples were taken at time points (0.5, 1, 2, 3, 4, 8, 12, and 24 hours), and fresh PBS buffer was added. DOX absorbance was measured at 412 nm using UV spectroscopy. The experiment was repeated three times. See [link to relevant documentation]. Figure 7 As can be seen, DOX-PAAP undergoes a Schiff base reaction with GSH, exhibiting the ability to release drugs in both pH and GSH-responsive states.

[0091] Experimental Example 5:

[0092] The photothermal performance of PAAP in step S4 of this embodiment was evaluated using near-infrared laser irradiation (wavelength 808 nm, power density 1 W / cm²). At room temperature, PAAP aqueous solutions of different concentrations (final concentrations of 50, 100, 200, 400, and 500 μg / mL) were irradiated, and temperature changes and thermal images of the samples were recorded using a FLIR A6700 infrared thermal imager. Each heating process lasted 10 minutes. Furthermore, the photothermal performance of the 500 μg / mL PAAP solution at different laser powers (1.0, 1.2, and 1.5 W / cm²) was evaluated. To investigate the photothermal stability of PAAP, a cyclic heating experiment was conducted on the 500 μg / mL PAAP aqueous solution under the same conditions, including three on / off cycles, each cycle lasting 10 minutes. See [link to relevant documentation]. Figure 8 As can be seen, the PAAP aqueous solution possesses excellent photostability, heat retention capacity, and superior photothermal conversion efficiency. Compared with traditional photothermal agents and pure PDA nanoparticles or metal nanoparticles, the excellent photothermal conversion efficiency of 64.8% is one of the advantages of this invention.

[0093] Experimental Example 6:

[0094] In steps S3 and S4 of this embodiment, Au-Au@PEG and PAAP were dispersed in water (final concentrations of 50, 100, 200, 400, and 500 μg / mL, respectively). H2O2 solution (10 mM) and MB solution (10 μg / mL) were added, and the mixture was incubated for 1 hour in a 37°C air bath shaker. The absorbance was measured at 664 nm using UV spectroscopy to monitor the degradation effect of ·OH on MB. For control, a blank MB solution and an H2O2 / MB mixed solution were tested under the same conditions. The experiment was performed in triplicate, and the relative amount of ·OH generated was calculated from the difference in absorbance at 664 nm. See also... Figure 9 As can be seen, Au-Au@PEG and PAAP possess the ability to consume H2O2, and the PDA coating can synergistically generate ·OH to degrade MB, indicating that the present invention possesses enzyme-like activity for enzyme therapy. While possessing enzyme-like activity, compared with traditional Fenton-like reaction reagents, the nanoplatform of the present invention exhibits superior biosafety and dosage.

[0095] Experiment Example 7:

[0096] The PAAP aqueous solution (500 μg / mL) from step S4 of this embodiment was irradiated with near-infrared light (808 nm, 1.0 W / cm²) for different durations (0, 2, 4, 6, 8, 10 minutes), and then mixed with a DPBF solution of 10 mM final concentration. The absorbance was measured at 416 nm using ultraviolet spectroscopy to monitor ¹O₂ formation. The experiment was performed in triplicate, and the relative amount of ¹O₂ formed was calculated from the difference in absorbance at 416 nm. See [link to relevant documentation]. Figure 10 It can be seen that PAAP itself does not have the ability to generate ¹O2, but it will only react and generate ¹O2 under NIR irradiation. This indicates that the PAAP nanoplatform of the present invention can synergistically generate more ROS compared with the previous nanoplatform, and the generated ¹O2 has more killing properties and activity than traditional ·OH. The present invention can synergistically generate more ROS to promote the improvement of the tumor hypoxic microenvironment.

[0097] Experimental Example 8:

[0098] 4T1 cells (derived from the Cell Bank of the Chinese Academy of Sciences) were seeded in 96-well plates, and 100 μL of 1640 medium (containing 10% fetal bovine serum and 1% streptomycin / penicillin) was added. The plates were cultured overnight at 37°C and 5% CO2. The medium was then replaced with fresh medium containing steps S1, 2, 3, 4, and 5 of this embodiment, and cultured for another 24 hours. Afterward, 0.25 mg / mL of 10% CCK-8 solution was added to each well and incubated for 1 hour. Cell viability was calculated by measuring the absorbance at 490 nm using a microplate reader. Following drug administration, near-infrared light irradiation (808 nm, 1 W / cm², 10 min) was applied, followed by 24 hours of further culture. Cell viability was then assessed again following the aforementioned steps. All experiments included blank controls and were repeated three times to ensure reproducibility. See also... Figure 11 As can be seen, compared with PAAP alone, DOX-PAAP can synergistically promote cancer cell death by releasing DOX, while PAAP+NIR and DOX-PAAP+NIR have the ability to further kill cancer cells under photothermal conditions. This invention possesses the synergistic capabilities of chemotherapy-photothermal-photodynamic therapy and enzyme therapy. Compared with other nanoplatforms, the cascading and spatiotemporal sequence of synergistic capabilities after integration are greatly improved.

[0099] Experiment Example 9:

[0100] 4T1 cells were seeded at a density in 6-well plates, and 1.0 mL of 1640 medium (containing 10% fetal bovine serum and 1% streptomycin / penicillin) was added. The cells were cultured overnight at 37°C and 5% CO2. After treatment for 4 hours with different treatment groups (grouping and concentration as described above) in steps S4 and S5 of this embodiment, the cells were washed three times with PBS buffer. Subsequently, they were co-incubated with 10 μM DCFH-DA under light-protected conditions for 30 minutes. Finally, the green fluorescence intensity was observed using a laser confocal microscope, and semi-quantitative fluorescence analysis was performed using LAS-X software. See also... Figure 12 As can be seen, both PAAP and DOX-PAAP possess the ability to generate ROS under photothermal conditions. Compared with the DOX-only group, DOX-PAAP shows enhanced ROS. Furthermore, compared with PAAP, DOX-PAAP, under photothermal conditions, combined with photodynamic therapy, can generate more ROS to enhance the microenvironment and improve hypoxia. The bar chart data from the semi-quantitative fluorescence analysis also support this.

[0101] Experimental Example 10:

[0102] 4T1 cells were seeded in 6-well plates, and 1.0 mL of 1640 medium (containing 10% fetal bovine serum and 1% streptomycin / penicillin) was added. The cells were cultured overnight at 37°C and 5% CO2. After removing the medium, the cells were washed three times with phosphate-buffered saline (PBS). Then, different treatment groups were applied according to steps S4 and S5 of this embodiment: the DOX group had a concentration of 10 μg / mL, and the PAAP and DOX-PAAP groups both had a concentration of 200 μg / mL. For the near-infrared group, 808 nm laser irradiation (power density 1 W / cm², duration 10 min) was applied. After 4 hours of treatment, staining was performed using Calcein-AM and PI, followed by imaging using a laser confocal microscope. See also... Figure 13 As can be seen, PAAP and DOX-PAAP possess the ability to kill cancer cells under photothermal conditions. Compared with the DOX-only group and the NIR-only group, DOX combined with photothermal therapy can effectively kill cancer cells. The bar chart data of the ratio analysis of Calcein-AM and PI also supports this point.

[0103] Experimental Example 11:

[0104] Cells from different treatment groups in steps S4 and S5 of this embodiment were collected, washed with PBS to remove culture medium and impurities, and resuspended. They were stained with Annexin V-FITC and PI dye and incubated at 4°C for 15-20 minutes in the dark. Data were collected using flow cytometry, and early apoptotic cells, late apoptotic cells, and necrotic cells were distinguished according to the Annexin V / PI staining pattern. Finally, the proportions of each cell type were analyzed and calculated using FlowJo software. See also... Figure 14 It can be seen that, compared with the DOX-only group, the proportion of late apoptotic cells increased in the DOX-PAAP group. Under photothermal conditions, the proportion of late apoptotic cells in PAAP+NIR and DOX-PAAP+NIR reached the maximum, indicating that PAAP can induce apoptosis in cancer cells under photothermal conditions in combination with DOX release.

[0105] Experimental Example 12:

[0106] The photoacoustic imaging evaluation of step S5 in this embodiment of the invention was performed using the Vevo LAZR photoacoustic imaging system (Visual Sonics). The optimal excitation wavelength for DOX-PAAP was determined by screening wavelengths within the range of 680–970 nm (5 nm intervals). In vitro photoacoustic signals of different concentrations of DOX-PAAP suspensions (1, 2, 3, 4, and 5 mg / mL) in step S5 of this embodiment of the invention were detected at an excitation wavelength of 710 nm. The suspension from step S5 of this embodiment of the invention was intravenously injected into 4T1 tumor mice, and photoacoustic images of the tumor site were acquired before injection and at 2, 8, 24, and 48 hours after injection. See also... Figure 15 As can be seen from 16, DOX-PAAP has the ability to perform in vivo and in vitro PA imaging and has excellent imaging quality.

[0107] Experimental Example 13:

[0108] The suspension from step S5 of this embodiment was intravenously injected into 4T1 tumor-bearing mice. The in vivo photothermal performance of DOX-PAAP was evaluated using near-infrared laser irradiation (wavelength 808 nm, power density 1 W / cm²). Temperature changes and thermograms of the samples were recorded using a FLIR A6700 infrared thermal imager. Each heating process lasted 10 minutes. See [link to documentation] Figure 17 As can be seen, compared with the NIR group alone, PAAP and DOX-PAAP still have good photothermal properties in vivo and can generate photothermal effects under NIR laser irradiation.

[0109] Experimental Example 14:

[0110] Tumor tissues from mice carrying 4T1 tumors were fixed in 40 g / L 4% paraformaldehyde PBS solution for 24 hours, dehydrated with graded ethanol, and then embedded in paraffin to prepare 5 μm serial sections. For reactive oxygen species (ROS) detection, after dewaxing and hydration of sections, 10 μM MDCFH-DA working solution (purchased from Beyotime Biotechnology Co., Ltd.) was added, and the sections were incubated at 37°C in the dark for 30 minutes. After rinsing three times with PBS and mounting, green fluorescence images were acquired using a laser confocal microscope (excitation / emission wavelengths 488 / 525 nm). H&E staining involved hematoxylin staining for 5 minutes, differentiation with 4.4 g / L 1% hydrochloric acid ethanol, and eosin staining for 3 minutes. After mounting with neutral resin, tissue morphology was observed using an optical microscope (Nikon Eclipse E100, 200×). TUNEL apoptosis detection was performed according to the Roche kit instructions: proteinase K (20 μg / mL) was digested at 37°C for 15 minutes, and the TUNEL reaction mixture was incubated in the dark for 60 minutes. After counterstaining the cell nuclei with DAPI, fluorescence images were acquired using a laser confocal microscope (Zeiss LSM 900). Ki-67 proliferation detection was performed using rabbit anti-Ki-67 monoclonal antibody (1:200, Abcam). (ab16667) Incubated overnight at 4℃, Cy3-labeled secondary antibody (1:500) reacted at 37℃ for 1 hour, and imaged after DAPI counterstaining. All image analyses were performed under double-blind evaluation: ROS mean fluorescence intensity (≥3 fields / group) was calculated using Image Pro Plus 6.0 software, and tissue damage was quantified based on the proportion of necrotic areas; the apoptosis rate was calculated by statistically analyzing the TUNEL⁺ / DAPI⁺ cell ratio; and the proliferation activity was assessed by counting the proportion of Ki-67 positive cells; ≥5 sections were analyzed per group, with 3 fields of view randomly selected from each section, and PBS negative and positive tissue controls were included to ensure the reliability of the results. See also Figure 18 As can be seen, regarding ROS generation in vivo, compared to the PAAP and DOX-PAAP groups alone, ROS generation was enhanced in the PAAP+NIR and DOX-PAAP+NIR treatment groups under NIR irradiation. Regarding H&E staining, although the DOX-PAAP group promoted apoptosis compared to the DOX group alone, H&E staining in the PAAP+NIR and DOX-PAAP+NIR treatment groups under NIR irradiation showed extensive apoptosis and nucleus loss in tumor cells. The synergistic effect between terminal deoxynucleotidyl transferase (dUTP) nick end labeling (TUNEL) and Ki-67 labeling indicated that the PAAP+NIR and DOX-PAAP+NIR groups caused more severe damage to cancer cells than the other groups (DOX group, PAAP group, and DOX-PAAP group), exhibiting a stronger anti-proliferative effect. This suggests that DOX-PAAP has a strong ability to promote cancer cell apoptosis under near-infrared light irradiation.

[0111] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. The application of a near-infrared triggered multifunctional nanoplatform in the preparation of drugs against triple-negative breast cancer, characterized in that, This nanoplatform has a core-shell structure, with an Au-Au@PEG nanoassembly structure made of gold nanoparticles (AuNPs) and gold nanoclusters (AuNCs) as the core, a polydopamine coating as the intermediate response layer, and doxorubicin molecules loaded on the surface of the polydopamine coating to form a functional shell. The average particle size of the Au-Au@PEG nanoassembly structure is 90 nm, and the thickness of the polydopamine coating is 12 nm. The triggering conditions for the drug are: near-infrared radiation, pH 5.8, and glutathione concentration ≥8 mM.

2. The application according to claim 1, characterized in that, The specific steps for constructing the nanoplatform are as follows: (1) First, gold nanoparticles AuNPs and gold nanoclusters AuNCs are made into Au-Au@PEG nano-assemblies; (2) Then, the polydopamine coating is uniformly coated on the surface of the Au-Au@PEG nano-assembly structure to obtain PAAP; (3) Finally, doxorubicin molecules were non-covalently loaded onto the PDA coating through π-π stacking to obtain DOX-PAAP.

3. The application according to claim 2, characterized in that, The specific method for step (1) is as follows: (1-1) First, gold nanoparticles AuNPs are mixed and stirred with mercapto polyethylene glycol amino solution, and then dialyzed with deionized water to remove unreacted SH-PEG-NH2. (1-2) Then add MES buffer, stir for the first time, continue to add EDC aqueous solution, stir for the second time, add NHS aqueous solution, stir for the third time, dialyze with deionized water to remove unreacted EDC and NHS, and obtain PEG@AuNPs solution; (1-3) Next, adjust the pH of the gold nanoclusters AuNCs to 7-8, mix and stir with the PEG@AuNPs solution at room temperature, dialyze with deionized water, and store at low temperature.

4. The application according to claim 3, characterized in that, The gold nanoparticles (AuNPs) were prepared by the following method: 10 mg / mL HAuCl4·3H2O aqueous solution and 10 mg / mL LVP aqueous solution were mixed and injected into a flask; then, 92 mL of deionized water was added, stirred for 2 minutes, heated to 85 °C, and NaBH4 aqueous solution was quickly added; the temperature was further increased to 90 °C, and stirred for 15 minutes; the solution was dialyzed with deionized water to remove excess NaBH4, and stored at low temperature.

5. The application according to claim 3, characterized in that, The gold nanoclusters AuNCs were prepared by mixing 0.1 g / mL HAuCl4·3H2O solution with 3 mM GSH solution, heating at 80°C for 5 hours, dialyzing with deionized water to completely remove excess GSH, and storing at low temperature.

6. The application according to claim 2, characterized in that, The specific method of step (2) is as follows: First, add the hydrochloric acid dopamine solution to Tris buffer and mix thoroughly. Then, slowly inject Au-Au@PEG nano-assembly structure. After stirring for 5 to 7 hours under light-protected and oxygen-enriched conditions, add 1 mol / L dilute hydrochloric acid to adjust the pH to 7.0 to terminate the reaction, and PAAP is obtained.

7. The application according to claim 2, characterized in that, The specific method of step (3) is as follows: first, doxorubicin is prepared into a 1 mg / mL doxorubicin aqueous solution using water, then the doxorubicin aqueous solution is mixed with PAAP and stirred for 4 hours in the dark to achieve drug adsorption by using π-π stacking effect; after loading, the mixture is centrifuged at 12,000 rpm for 15 minutes to remove free DOX, and the loading efficiency of the supernatant is determined by ultraviolet spectroscopy.

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