Near-infrared triggered multifunctional nano-platform, construction method and application of nano-platform in preparation of triple negative breast cancer resisting drugs

Abstract

The invention discloses a near-infrared triggered multifunctional nano platform, a construction method and application of the near-infrared triggered multifunctional nano platform in preparation of a medicine for resisting triple negative breast cancer. The nano platform DOX-PAAP is of a core-shell structure, an Au-Au-PEG nano assembly structure prepared from gold nanoparticles AuNPs and gold nanoclusters AuNCs is used as a core, a polydopamine coating is used as an intermediate response layer, and adriamycin molecules are loaded on the surface of the polydopamine coating to form a functional shell. According to the invention, photo-thermal and catalytic characteristics of gold nanoparticles and gold nanoclusters are fully utilized, and an intelligent response coating of polydopamine is combined, so that a nano platform with a quadruple synergistic function is constructed: photo-thermal conversion efficiency not less than 60% is realized through near-infrared triggering, precise drug controlled release is completed by utilizing a pH / GSH dual-response mechanism, a self-supply type ROS generation system is established, and the self-supply type ROS generation system is used for preparing a self-supply type ROS nano-platform. GSH depletion is synchronously activated, and a ferroptosis pathway is strengthened.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of nanomedicine, and provides a near-infrared triggered multifunctional nano platform, a construction method and application thereof in preparation of anti-triple-negative breast cancer drugs. BACKGROUND

[0002] The field of breast cancer treatment currently faces severe clinical challenges. Triple-negative breast cancer, due to its high invasiveness and lack of effective targets, mainly relies on anthracycline drugs such as doxorubicin (DOX) for chemotherapy. However, the inherent multiple drug resistance mechanisms of the tumor microenvironment seriously restrict the treatment effect: tumor cells form drug efflux pumps by overexpressing P-glycoprotein, resulting in insufficient intracellular drug concentration; at the same time, the up-regulated glutathione (GSH) antioxidant system can quickly eliminate reactive oxygen species (ROS), weakening the oxidative stress killing effect of chemotherapeutic drugs; more critically, the hypoxic microenvironment further limits the generation efficiency of ROS. This drug resistance dynamic leads to a high recurrence rate of up to 40% of triple-negative breast cancer patients within one year after initial treatment, and the tumor after recurrence is often more invasive, doubling the difficulty of treatment. Therefore, a breakthrough strategy must directly target the synergistic integration of these drug resistance mechanisms, such as achieving dynamic synergies through the combination of multifunctional modules, rather than relying solely on single therapy.

[0003] In the exploration of solutions, nanoscale synergistic therapy technologies have shown great potential in recent years, which is based on the integration of multiple treatment modalities such as phototherapy, chemotherapy and ferroptosis induction to overcome the limitations of single therapy. Photothermal-chemotherapy synergistic systems use the plasmonic resonance effect of gold or copper-based nanomaterials to convert near-infrared light into heat energy, enhancing drug penetration and tumor ablation, but the tissue penetration depth of phototherapy is usually only 3 to 5 millimeters, and it is difficult to achieve precise drug release in deep tumors; enzyme kinetics therapy generates ROS by catalyzing endogenous hydrogen peroxide (H2O2) in tumors through transition metal nanoscale enzymes such as iron-based catalysts, which can bypass hypoxic limitations, but the concentration of hydrogen peroxide at the tumor site is often less than 50 µM, and the catalytic efficiency is affected by pH and enzyme activity, resulting in unstable therapeutic effect; iron-dependent cell death triggered by ferroptosis induction strategies focuses on depleting GSH or inhibiting GPX4 enzyme, however, the low bioavailability and systemic toxicity of existing inducers such as erastin or sorafenib analogs, including liver damage, limit their clinical translation. Although multi-mechanism synergistic frameworks have been widely proposed, such as simultaneous integration of photothermal conversion, enzyme catalysis and GSH depletion, practical applications still face three technical 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; the spatiotemporal control problem leads to the inability to precisely synchronize drug release and ROS generation in the tumor site, for example, photothermal-triggered drug release may precede the active oxygen burst, reducing synergistic efficiency; and the biocompatibility limitation is reflected in the long-term in vivo retention of metal-based nanocarriers such as liver accumulation, which can cause inflammation or fibrosis, and several preclinical studies have shown that more than 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 new intelligent platforms that need to achieve simultaneous release of drugs and ROS through synergistic coupling of core modules for precise spatiotemporal control, thereby breaking through the existing technical bottlenecks.

[0004] In summary, although the prior art has its own advantages, the treatment efficiency is restricted by physical barriers such as tissue depth, biochemical interference such as glutathione level fluctuation, and cell defense mechanisms such as P-glycoprotein overexpression; toxic side effects are caused by non-targeted chemotherapy drugs such as DOX cardiotoxicity and potential immunogenicity of nanomaterials; drug resistance persists due to the dynamic adaptability of tumor cells such as activation of DNA repair or metabolic reprogramming. The development of the next generation of multifunctional synergistic platform design should have microenvironment response capabilities such as pH or active oxygen sensitive polymer shell to automatically adapt to hypoxia or high GSH area, real-time monitoring function embedded with fluorescence or MRI contrast agents to achieve treatment feedback, and integration of phototherapy, chemotherapy, and ferroptosis modules. Emerging intelligent nanocarriers can reduce complexity through modular design, while triggering on-demand release using tumor-specific stimuli such as enzyme overexpression or low pH to enhance spatial and temporal control. Therefore, there is an urgent need to develop a multifunctional synergistic platform that integrates multiple treatment modalities, has microenvironment response capabilities, and can monitor in real time. SUMMARY

[0005] Therefore, the present application aims to provide a near-infrared triggered multifunctional nanoplatform, a construction method thereof, and an application thereof in the preparation of an anti-triple-negative breast cancer drug, so as to solve the problem of drug resistance of breast cancer.

[0006] To achieve the above-mentioned purpose, the present application provides the following technical solutions. 1. A near-infrared triggered multifunctional nanoplatform, wherein the nanoplatform is a core-shell structure, the Au-Au@PEG nanometer assembly structure made of gold nanoparticles AuNPs and gold nanoclusters AuNCs is used as the core, a polydopamine coating layer is used as an intermediate response layer, and an adriamycin molecule is loaded on the surface of the polydopamine coating layer to form a functional shell.

[0007] 2. A construction method of the near-infrared triggered multifunctional nanoplatform, comprising the following steps: (1) Au-Au@PEG nanometer assembly structure is first prepared from gold nanoparticles AuNPs and gold nanoclusters AuNCs; (2) then a polydopamine coating layer (PDA coating layer) is uniformly coated on the surface of the Au-Au@PEG nanometer assembly structure to obtain PAAP (polydopamine-coated Au-Au@PEG nanometer assembly structure); (3) finally, an adriamycin molecule (DOX) is non-covalently loaded on the PDA coating layer through π-π stacking to obtain DOX-PAAP.

[0008] Preferably, in step (1), the average particle size of the Au-Au@PEG nanometer assembly structure is 90 nm.

[0009] Preferably, the specific method of step (1) is as follows: (1-1) First, mix and stir gold nanoparticles AuNPs with mercapto polyethylene glycol amino solution (SH-PEG-NH2 solution), and dialyze with deionized water to remove unreacted SH-PEG-NH2; (1-2) Then add MES (2-morpholinoethanesulfonic acid) buffer solution, first stir, continue to add EDC (1-ethyl-(3-dimethylaminopropyl) carbodiimide) aqueous solution, second stir, then add NHS (N-hydroxysuccinimide) aqueous solution, third stir, dialyze with deionized water to remove unreacted EDC and NHS, to obtain PEG@AuNPs solution; (1-3) Then adjust the pH of gold nanoclusters AuNCs to 7-8, and mix and stir with the PEG@AuNPs solution at room temperature (25°C), dialyze with deionized water, and store at low temperature.

[0010] Further preferably, the molar ratio of AuNPs to AuNCs is 1:1.5-1:2.5.

[0011] Further preferably, the volume ratio of AuNPs, mercapto polyethylene glycol amino solution, MES buffer solution, EDC aqueous solution, and NHS aqueous solution is 2:1:1:2:2; wherein the concentration of the mercapto polyethylene glycol amino solution is 0.25-0.5 mmol / L, the concentration of the MES buffer solution is 0.1-0.2 mol / L, pH=6.5, the concentration of the EDC aqueous solution is 0.05-0.1 mol / L, and the concentration of the NHS aqueous solution is 0.05-0.1 mol / L.

[0012] Further preferably, in step (1-1), the mixing and stirring time is 6-8 hours.

[0013] Further preferably, in step (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.

[0014] Further preferably, in step (1-3), the mixing and stirring time is 10-12 hours, and the dialysis time with deionized water is 10-12 hours.

[0015] Further preferably, the average particle size of the gold nanoparticles AuNPs is 25-35 nm.

[0016] Further preferably, the gold nanoparticles AuNPs are prepared by the following method: mixing 10 mg / mL HAuCl4·3H2O aqueous solution and 10 mg / mL PVP aqueous solution and injecting into a flask; then, adding deionized water, stirring for 2 minutes, heating to 85℃, and quickly adding 1.7 mg / mL NaBH4 aqueous solution; continuing to heat to 90℃, and incubating and stirring for 15-20 minutes; deionized water dialysis for 10-12 hours to remove excess NaBH4, and storing under low temperature conditions.

[0017] Further preferably, the volume ratio of the HAuCl4·3H2O aqueous solution, the PVP aqueous solution, and the deionized water is 3:3:92-95; and the NaBH4 aqueous solution is obtained by dissolving 11.7 mg NaBH4 in 10 mL water.

[0018] Further preferably, the dialysis bag used for dialysis has a molecular weight cut-off of 3500 Da.

[0019] Further preferably, the average particle size of the gold nanoclusters AuNCs is 1.8-2.5 nm.

[0020] Further preferably, the gold nanoclusters AuNCs are prepared by the following method: mixing HAuCl4·3H2O solution and GSH (glutathione) solution, heating at 80℃ for 5 hours, deionized water dialysis for 10-12 hours to completely remove excess GSH, and storing under low temperature conditions.

[0021] Further preferably, the volume ratio of the HAuCl4·3H2O solution and the GSH solution is 0.3:50; the concentration of the HAuCl4·3H2O solution is 0.1 g / mL, and the concentration of the GSH solution is 3 mM.

[0022] Further preferably, the dialysis bag used for dialysis has a molecular weight cut-off of 3500 Da.

[0023] Preferably, in step (2), the thickness of the polydopamine coating layer is 10-15 nm.

[0024] Preferably, the specific method of step (2) is: first, adding a dopamine hydrochloride solution (HCl-DA solution) into a Tris buffer solution and mixing thoroughly, then slowly injecting into the Au-Au@PEG nano-assembly structure, stirring for 5-7 hours under light-shielded aerobic conditions, then adding 1 mol / L dilute hydrochloric acid to adjust the pH to 7.0 to terminate the reaction, thereby obtaining PAAP.

[0025] Further preferably, the volume ratio of the dopamine hydrochloride solution, the Tris buffer solution and the Au-Au@PEG nano-assembly structure is 1:2:4; wherein the concentration of the dopamine hydrochloride solution is 0.6 mg / mL, the concentration of the Tris buffer solution is 10 mmol / L, and the pH is 8.0-9.0.

[0026] Further preferably, the obtained product after the reaction is finished is centrifuged for three times to remove the excessive dopamine hydrochloride, and is then dispersed in a PBS buffer solution with a concentration of 0.1 M and a pH of 7.4 for standby.

[0027] Preferably, in step (3), the average particle size of the DOX-PAAP is 120-150 nm.

[0028] Preferably, the specific method of step (3) is as follows: first, the doxorubicin is prepared into a 1 mg / mL doxorubicin aqueous solution by using water, and then the doxorubicin aqueous solution is mixed with the PAAP, and is stirred for 4-6 hours in the dark to realize the drug adsorption by using the π-π stacking effect; after the loading, the mixture is centrifuged at 12,000 rpm for 15-20 minutes to remove the free DOX, and the supernatant is determined for the loading efficiency by using ultraviolet spectrum (480 nm).

[0029] Further preferably, the mass ratio of the doxorubicin to the PAAP is 1:4-1:6, and further preferably is 1:5.

[0030] Further preferably, the centrifugation condition is 10,000-15,000 rpm for 10-20 minutes, and more further preferably is 12,000 rpm for 15 minutes.

[0031] Preferably, in step (3), the DOX-PAAP is dispersed in a phosphate buffer solution, and is stored in a dark environment at 4℃.

[0032] 3. The use of the aforementioned near-infrared triggered multifunctional nano-platform in the preparation of anti-triple-negative breast cancer drugs.

[0033] The present application has the following beneficial effects: The present application provides a near-infrared (NIR) triggered multifunctional nano-platform DOX-PAAP, a construction method and the use thereof in the preparation of anti-triple-negative breast cancer drugs. The nano-platform integrates photothermal therapy, photodynamic therapy and enzyme therapy, and the photo-thermal conversion efficiency under near-infrared light irradiation is 60-65%; under the conditions of a pH of 5.8 and a glutathione concentration of ≥8 mM, the drug release rate is increased to more than 3 times of that in an ordinary environment.

[0034] The present application makes full use of the photo-thermal and catalytic properties of gold nanoparticles (AuNPs) and gold nanoclusters (AuNCs), and combines the intelligent response coating of polydopamine (PDA) to construct a nano-platform with four synergistic functions: a photo-thermal conversion efficiency of not less than 60% is achieved through near-infrared triggering, precise drug release is achieved by using a pH / GSH dual-response mechanism, a self-supply type ROS generation system is established, and GSH depletion is simultaneously activated, thereby strengthening the ferroptosis pathway. This innovative architecture successfully overcomes the space-time control obstacles and biological toxicity defects of traditional carriers, and provides a new solution to break through the breast cancer drug resistance dilemma.

[0035] The present application has a breakthrough structural design. The AuNPs-AuNCs provide cascade catalytic active sites, the polydopamine intermediate layer realizes the pH / glutathione dual-response degradation mechanism, and the doxorubicin shell precisely targets tumor cells. This three-level structure first solves the space-time control problem that traditional carriers cannot simultaneously coordinate the photo-thermal effect, enzyme catalysis and drug release.

[0036] The present application has a multi-mechanism synergistic effect. Under the triggering of near-infrared light, the gold-based core simultaneously generates local high temperature (ΔT > 40°C) and hydroxyl radicals (·OH), and the polydopamine layer simultaneously releases doxorubicin and consumes glutathione. Experiments have proved that this synergistic effect makes the apoptosis rate of tumor cells increase to 4.2 times that of single chemotherapy, and significantly activates the ferroptosis pathway.

[0037] The present application has excellent biocompatibility. The dual modification of polyvinylpyrrolidone and polyethylene glycol and the coating of PDA greatly reduce the liver accumulation toxicity, and animal experiments show that there is no pathological damage to the main organs of the administration group, thereby breaking through the technical bottleneck of organ damage caused by long-term retention of metal nano-carriers.

[0038] Based on the innovative design of the nano-platform DOX-PAAP of the present application in the field of tumor treatment, the application field mainly covers the following aspects: through the multi-component synergistic effect of AuNPs, AuNCs, PDA and DOX, the platform efficiently integrates photothermal therapy, photodynamic therapy and enzyme therapy, is suitable for targeted treatment of triple-negative breast cancer, and solves the problem of drug resistance; at the same time, its microenvironment response characteristics (such as pH / GSH triggered release) enable it to achieve precise release in a drug delivery system, thereby reducing non-targeted toxicity; in addition, the platform can be extended to the field of medical imaging, realizes real-time treatment monitoring through near-infrared triggering, and provides multifunctional support for tumor diagnosis and personalized medicine. The synergistic effect of these applications (such as far exceeding the effect of single therapy) ensures a broad prospect in clinical transformation.

[0039] Additional advantages, objects, and features of the application will be apparent to those skilled in the art upon examination of the following detailed description, it being understood that each BRIEF DESCRIPTION OF DRAWINGS

[0040] In order to make the objectives, technical solutions and advantages of the present application clearer, the preferred embodiments of the present application will be described in detail below with reference to the accompanying drawings, in which: Figure 1 The preparation flow chart of the present application.

[0041] Figure 2 The transmission electron microscope (TEM) image of the product of Example Step S1 (A), the TEM image of the product of Example Step S2 (B), the TEM image of the product of Example Step S3 (C), the scanning electron microscope (SEM) image (D), the high-angle annular dark-field scanning transmission (HAADF-STEM) electron microscope image (E), and the corresponding element distribution mapping image (F) of the product of Example Step S3 are shown.

[0042] Figure 3 The TEM image of the product of Example Step S4 is shown.

[0043] Figure 4 The ultraviolet absorption spectrum of the product of Example Step S5 is shown.

[0044] Figure 5 The ultraviolet absorption spectrum (A), the photoluminescence spectrum (B), the infrared spectrum (C), the thermogravimetric curve (D), the Au 4f high-resolution X-ray photoelectron spectrum (E), and the Zeta potential (F) of the product of Example Step S3 are shown.

[0045] Figure 6 The DOX release curve of the product of Example Step S5 at pH 5.8 and 7.4 for 48 hours is shown.

[0046] Figure 7 The DOX release curve of the product of Example Step S5 at pH 5.8 (A) and 7.4 (B) for 48 hours after adding different GSH concentrations is shown.

[0047] Figure 8 The photothermal curve of the product of Example Step S4 under irradiation of near-infrared laser at different concentrations (A), the photothermal curve of the product of Example Step S4 under irradiation of near-infrared laser at different laser power densities (B), the photothermal cycling curve in three on / off irradiation cycles (C), the linear fitting curve of the cooling time and the driving temperature negative natural logarithm under photothermal cycling in a single on / off irradiation cycle (D), and the infrared thermal image of the product of Example Step S4 under irradiation of near-infrared laser at different concentrations (E) are shown.

[0048] Figure 9 UV absorption spectra (A), (B) and relative production amount (C), (D) of the product of Example Step S3, the product of Example Step S4 for the evaluation of the degradation efficiency of MB.

[0049] Figure 10 Different time-UV absorption spectra (A) and relative production amount of singlet oxygen (B) and electron spin resonance wave (ESR) spectra (C) of the product of Example Step S4 for the production of singlet oxygen.

[0050] Figure 11 Biocompatibility evaluation (A) and killing ability evaluation (B) of 4T1 cancer cells of the products of Example Steps S1, S2, S3, S4 and S5 and PAAP+NIR and DOX-PAAP+NIR for a total of 7 groups.

[0051] Figure 12 Fluorescence microscope images (A) and fluorescence semi-quantitative analysis chart (B) of the ROS production ability of 4T1 cancer cells of the products of Example Step S4 and Example Step S5 of different groups G1: Control; G2: DOX; G3: NIR; G4: PAAP; G5: DOX-PAAP; G6: PAAP+NIR; and G7: DOX-PAAP+NIR.

[0052] Figure 13 Fluorescence microscope images (A) and AM / PI ratio semi-quantitative analysis chart (B) of the live and dead cell staining (AM / PI) of 4T1 cancer cells of the products of Example Step S4 and Example Step S5 of different groups G1: Control; G2: DOX; G3: NIR; G4: PAAP; G5: DOX-PAAP; G6: PAAP+NIR; and G7: DOX-PAAP+NIR.

[0053] Figure 14 Analysis of the apoptosis of 4T1 cells in different treatment groups by flow cytometry of 4T1 cancer cells of the products of Example Step S4 and Example Step S5 of different groups G1: Control; G2: DOX; G3: NIR; G4: PAAP; G5: DOX-PAAP; G6: PAAP+NIR; and G7: DOX-PAAP+NIR.

[0054] Figure 15 Evaluation of the in vitro PA imaging performance of the product of Example Step S5.

[0055] Figure 16Different time evaluation for in vivo PA imaging of the product of Example Step S5.

[0056] Figure 17 For in vivo photothermal performance of the product of Example Step S4, the product of Example Step S5, the photothermal images (A) and the temperature curves (B) of the 4T1 tumor of BALB / c mice after different treatments were evaluated.

[0057] Figure 18 For the different groups of the product of Example Step S4, the product of Example Step S5, G1: Control; G2: DOX; G3: NIR; G4: PAAP; G5: DOX-PAAP; G6: PAAP+NIR; and G7: DOX-PAAP+NIR, the representative ROS fluorescence images (A), H&E staining images (B), TUNEL immunofluorescence staining images (C), Ki-67 immunofluorescence staining (D) and fluorescence semi-quantitative analysis chart (E), TUNEL (% nuclei) semi-quantitative analysis chart (F) and Ki-67 (% nuclei) semi-quantitative analysis chart (G) of the tumor sections of mice. DETAILED DESCRIPTION

[0058] The present application will be further described below in conjunction with the specific embodiments.

[0059] It should be understood that the following examples are merely illustrative and explanatory of the present application and should not be construed as limiting the scope of protection of the present application. Any technology realized based on the above description of the present application is encompassed within the scope of the present application intended to be protected.

[0060] In addition, unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present application can be purchased from the market or prepared by existing methods.

[0061] EXAMPLE In conjunction Figure 1 As shown in the following examples, a method for constructing a near-infrared (NIR) triggered multifunctional nano-platform DOX-PAAP is described in detail, and the specific steps are as follows: S1. AuNPs preparation: Mix 3 mL of aqueous HAuCl4·3H2O solution (10 mg / mL) with 3 mL of aqueous PVP solution (10 mg / mL) and inject into a flask. Then, add 92 mL of deionized water, stir for 2 minutes, and heat to 85 °C. Quickly add aqueous NaBH4 solution (11.7 mg dissolved in 10 mL of water), continue heating to 90 °C, and stir for 15 minutes. The synthesized AuNPs are dialyzed in deionized water overnight (molecular weight cut-off value: 3500 Da) to remove excess NaBH4 and stored under low temperature conditions.

[0062] See Figure 2 In FIG. A, TEM can see that AuNPs are successfully prepared.

[0063] S2. AuNCs preparation: Mix 0.3 mL of aqueous HAuCl4·3H2O solution (0.1 g / mL) with 50 mL of GSH solution (3 mM) and heat at 80 °C for 5 hours. The obtained AuNCs are dialyzed in deionized water for 12 hours (molecular weight cut-off value: 3500 Da) to completely remove excess GSH and stored under low temperature conditions. See Figure 2 In FIG. B, TEM can see that AuNCs are successfully prepared.

[0064] S3. Preparation of gold-based nano-assembly structure of AuNPs and AuNCs: Mix 5 mL of SH-PEG-NH2 solution (0.25 mM) with 10 mL of AuNPs and stir for 6 hours. Dialyze in deionized water for 12 hours to remove excess SH-PEG-NH2. To activate the -COOH groups on AuNCs, add 5 mL of MES buffer (0.2 M, pH 6.5) and stir for 2 minutes to stabilize the solution. Then, add 10 mL of aqueous EDC solution (0.1 M) and stir for 1 hour. Then, add 10 mL of aqueous NHS solution (0.1 M) and continue stirring at room temperature for 4 hours. Dialyze the mixture in deionized water overnight to remove unreacted EDC and NHS, obtaining a PEG@AuNPs solution. After activation, adjust the pH of the dialyzed AuNCs (molar ratio of AuNPs to AuNCs is 1:2) to 7-8, mix with the PEG@AuNPs solution at room temperature, and stir for 12 hours to obtain Au-Au@PEG. Dialyze the final product in deionized water overnight to remove residual impurities and store under low temperature conditions. See Figure 2 In FIGS. C, D, E, and F, TEM and SEM can see that gold-based nano-assembly structures are successfully prepared, and element mapping also proves that the assembly structure is successful.

[0065] S4. Preparation of PDA-coated gold-based nanoassemblies: The HCl-DA solution (0.6 mg / mL) was added into Tris buffer (10 mM, pH 8.5) and mixed thoroughly, and then the Au-Au@PEG solution was slowly injected. After stirring for 6 hours in the dark under aerobic conditions, the reaction was terminated by adding dilute hydrochloric acid to adjust the pH to ≈7.0, and PDA-coated gold-based nanoassemblies (PAAP) were obtained. The resulting product was centrifuged three times to remove excess HCl-DA and then redispersed in an aqueous solution at pH 7.4 or PBS buffer for later use. See Figure 3 , TEM showed that the PDA-coated gold-based nanoassemblies were successfully prepared, and the coating thickness was about 12 nm.

[0066] S5. Preparation of multifunctional nano-platform (DOX-PAAP): A 1 mg / mL DOX aqueous solution was mixed with PAAP at a mass ratio of 1:5, and the mixture was stirred in the dark for 4 hours to achieve drug adsorption by π-π stacking. After loading, the mixture was centrifuged at 12,000 rpm for 15 minutes to remove free DOX, and the supernatant was determined for loading efficiency by ultraviolet spectroscopy (480 nm). The final product DOX-PAAP was dispersed in PBS and stored in the dark at 4°C. See Figure 4 , UV-vis absorption spectroscopy showed that DOX-PAAP was successfully prepared.

[0067] Experimental Example 1: The AuNCs, PEG@AuNPs, and Au-Au@PEG in the steps S2 and S3 of the embodiment of the present application were subjected to UV-vis absorption spectroscopy, photoluminescence (PL) fluorescence spectroscopy, and Fourier (FT-IR) infrared spectroscopy, respectively, as shown in A, B, and C in Figure 5 . It can be seen that Au-Au@PEG has a plasmonic resonance peak and fluorescence activity, and the presence of an amide bond is also observed, which further proves that the Au-Au@PEG assembly structure is successful.

[0068] Experimental Example 2: The AuNCs, AuNPs, Au-Au@PEG, and PAAP in the steps S1, S2, S3, and S4 of the embodiment of the present application were subjected to thermogravimetric (TG) analysis, XPS valence state analysis, and Zeta potential distribution detection, respectively, as shown in D, E, and F in Figure 5 . It can be seen that the corresponding mass loss under different weight loss platforms, the structure electron binding energy conforms to the XPS fine spectrum of Au 4f, and the potential change, which further proves that the Au-Au@PEG assembly structure is successful.

[0069] Experimental Example 3: The DOX-PAAP in step S5 of the embodiment of the present application was dispersed in PBS buffer with pH values of 5.8 and 7.4, respectively. The samples were placed in a constant temperature shaking incubator at 37°C. At the predetermined time points (0.5, 1, 2, 3, 4, 8, 12, 24, 36, 48 hours), equal volumes of solution were taken and supplemented with equal volumes of fresh PBS buffer. The release kinetics of DOX was monitored by measuring the absorbance of DOX at 480 nm using UV spectroscopy. The experiment was repeated three times to ensure reproducibility. See Figure 6 It can be seen that DOX-PAAP has the ability of pH-responsive drug release due to the cleavage of PDA coating under acidic conditions.

[0070] Experimental Example 4: To evaluate the effect of GSH, the DOX-PAAP in step S5 of the embodiment of the present application was dispersed in PBS buffer with pH values of 5.8 and 7.4, respectively, and GSH was added to a final concentration of 1 mM, 5 mM and 10 mM, respectively. The samples were placed in a 37°C water bath shaker. At the time points (0.5, 1, 2, 3, 4, 8, 12, 24 hours), samples were taken and supplemented with fresh PBS buffer. The absorbance of DOX was measured at 412 nm using UV spectroscopy. The experiment was repeated three times. See Figure 7 It can be seen that DOX-PAAP has the ability of pH and GSH dual-responsive drug release due to the Schiff base reaction with GSH.

[0071] Experimental Example 5: The photothermal performance of PAAP in step S4 of the embodiment of the present application was evaluated using near-infrared laser irradiation (wavelength 808 nm, power density 1 W / cm²). Different concentrations of PAAP aqueous solution (final concentrations of 50, 100, 200, 400, 500 μg / mL, respectively) were irradiated at room temperature, and the temperature change and thermal image of the sample were recorded using a FLIR A6700 infrared thermal imager. Each heating process lasted for 10 minutes. In addition, the photothermal performance of 500 μg / mL PAAP solution under different laser powers (1.0, 1.2, 1.5 W / cm²) was evaluated. To investigate the photothermal stability of PAAP, a cyclic heating experiment was performed on 500 μg / mL PAAP aqueous solution under the same conditions, including three on / off cycles, each cycle lasting 10 minutes. See Figure 8 It can be seen that PAAP aqueous solution has excellent light stability, heat preservation ability and excellent 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 the present application.

[0072] Experimental Example 6: Au-Au@PEG and PAAP in Example step S3, Example step S4 were dispersed in water (final concentration of 50, 100, 200, 400, 500 μg / mL), H2O2 solution (10 mM) and MB solution (10 μg / mL) were added, and incubated in a 37°C air bath constant temperature oscillator for 1 hour. The absorbance was measured at 664 nm wavelength by UV spectroscopy to monitor the degradation of MB by ·OH. To make a comparison, blank MB solution and H2O2 / MB mixed solution were tested under the same conditions. The experiment was repeated three times in parallel, and the relative amount of ·OH was calculated by the absorbance difference at 664 nm. See Figure 9 It can be seen that Au-Au@PEG and PAAP have the ability to consume H2O2, and the PDA coating can synergize with ·OH to degrade MB, indicating that the present application has a class of enzyme activity of enzyme therapy. At the same time of having the class of enzyme activity, compared with traditional Fenton reaction reagents, the biological safety and dosage of the nano platform of the present application are more excellent.

[0073] Experimental Example 7: The PAAP aqueous solution (500 μg / mL) in Example step S4 was irradiated by near-infrared light (808 nm, 1.0 W / cm²) for different lengths of time (0, 2, 4, 6, 8, 10 minutes), and then mixed with a DPBF solution with a final concentration of 10 mM. The absorbance was measured at 416 nm wavelength by UV spectroscopy to monitor the generation of ¹O2. The experiment was repeated three times in parallel, and the relative amount of ¹O2 was calculated by the absorbance difference at 416 nm. See Figure 10 It can be seen that PAAP itself does not have the ability to generate ¹O2, but will react to generate ¹O2 under NIR irradiation, which shows that the PAAP nano platform of the present application can synergize to generate more ROS than the previous nano platform, and the generated ¹O2 has more killing performance and activity than the traditional ·OH. The present application can synergize to generate more ROS to promote the improvement of the tumor hypoxic microenvironment.

[0074] Experimental Example 8: 4T1 cells (from the Chinese Academy of Sciences Cell Bank) were inoculated in a 96-well plate, 100 μL of 1640 culture medium (containing 10% fetal bovine serum and 1% streptomycin / penicillin) was added, and incubated at 37°C, 5% CO2 overnight; then the fresh culture medium containing the steps S1, 2, 3, 4 and 5 of the present application was replaced, and incubated for 24 hours; then 0.25 mg / mL of 10% CCK-8 solution was added per well for incubation for 1 hour, and the absorbance at 490 nm wavelength was measured by using an enzyme label instrument to calculate the cell survival rate; after administration, near-infrared light irradiation (808 nm, 1 W / cm², 10 minutes) was applied, and the cell survival rate was detected after 24 hours of incubation according to the foregoing steps; a blank control was set for all experiments, and repeated three times to ensure repeatability. See Figure 11 It can be seen that, compared with PAAP alone, DOX-PAAP can synergistically promote cancer cell death by releasing DOX, and PAAP+NIR and DOX-PAAP+NIR have the ability to further kill cancer cells under photothermal conditions. The present application has the synergistic ability of chemotherapy-photothermal-photodynamic therapy and enzyme therapy, and compared with other nano platforms, the cascade and spatiotemporal sequence of the integrated synergistic ability have been greatly promoted.

[0075] Experimental Example 9: 4T1 cells were inoculated in a 6-well plate at a density, 1.0 mL of 1640 culture medium (containing 10% fetal bovine serum and 1% streptomycin / penicillin) was added, and incubated at 37°C, 5% CO2 overnight; after different treatment groups (grouping and concentration are the same as the foregoing scheme) of the steps S4 and S5 of the present application were treated for 4 hours, the cells were washed with PBS buffer three times; then co-incubated with 10 μM DCFH-DA for 30 minutes in the dark; finally, the green fluorescence intensity was observed by using a laser confocal microscope, and the fluorescence semi-quantitative analysis was performed by using LAS-X software. See Figure 12 It can be seen that PAAP and DOX-PAAP have the ability to produce ROS under photothermal conditions, and compared with the DOX group alone, the ROS of DOX-PAAP is enhanced, and compared with PAAP and DOX-PAAP, more ROS can be produced under photothermal conditions to enhance the microenvironment and improve hypoxia in combination with photodynamic therapy. The columnar chart data of fluorescence semi-quantitative analysis also supports this point.

[0076] Experimental Example 10: 4T1 cells were seeded in 6-well plates, 1.0 mL of 1640 medium (containing 10% fetal bovine serum and 1% streptomycin / penicillin) was added, and incubated at 37°C, 5% CO2 overnight; after removing the culture medium, washing with phosphate buffered saline (PBS) for three times; then applying different treatment groups of the present application: DOX group at a concentration of 10 μg / mL, PAAP group and DOX-PAAP group at a concentration of 200 μg / mL; for the near-infrared group, applying 808 nm laser irradiation (power density 1 W / cm², time length 10 minutes); after 4 hours of treatment, Calcein-AM and PI were used for staining, and then imaged by laser confocal microscope. See Figure 13 It can be seen that PAAP and DOX-PAAP have the ability to kill cancer cells under photothermal conditions, compared with the DOX alone group and the NIR alone group, DOX combined with photothermal therapy can effectively kill cancer cells. The column chart data of the ratio analysis of Calcein-AM and PI also supports this point.

[0077] Experimental Example 11: The cells of the different treatment groups of the present application were collected, washed with PBS to remove the culture medium and impurities, and then resuspended; Annexin V-FITC and PI dye were used for staining, and incubated at 4°C for 15-20 minutes in the dark; flow cytometry was used to collect data, and early apoptotic cells, late apoptotic cells and necrotic cells were distinguished according to the Annexin V / PI staining mode; finally, the proportion of each type of cell was analyzed and calculated by FlowJo software. See Figure 14 It can be seen that compared with the DOX alone group, the proportion of late apoptotic cells in the DOX-PAAP group increased, and under photothermal conditions, the proportion of late apoptotic cells in PAAP+NIR and DOX-PAAP+NIR reached the maximum, indicating that PAAP can release DOX under photothermal conditions to induce apoptosis in cancer cells.

[0078] Experimental Example 12: The Vevo LAZR photoacoustic imaging system (Visual Sonics) was used to evaluate the photoacoustic imaging of the present application. The wavelength range (5 nm interval) of 680-970 nm was selected for photoacoustic imaging scanning of DOX-PAAP, and the best excitation wavelength was determined. Under the excitation wavelength of 710 nm, the in vitro photoacoustic signals of different concentrations of DOX-PAAP suspension (1, 2, 3, 4, 5 mg / mL) in the present application were detected. The 4T1 tumor mice were injected intravenously with the suspension of the present application, and the photoacoustic images of the tumor site were collected before and 2, 8, 24, and 48 hours after injection. See Figure 15As can be seen from FIG. 16, DOX-PAAP has the ability of PA imaging in vitro and in vivo and has excellent imaging quality.

[0079] Experimental Example 13: The 4T1 tumor-bearing mice were intravenously injected with the suspension of step S5 of the embodiment of the application. The near-infrared laser (wavelength 808 nm, power density 1 W / cm²) was used for irradiation, and the photothermal performance of DOX-PAAP in vivo was evaluated. The temperature change and thermal image of the sample were recorded using a FLIR A6700 infrared thermal imager. Each heating process lasted for 10 minutes. See Figure 17 As can be seen, compared with the NIR group alone, PAAP and DOX-PAAP still have good photothermal performance in vivo and can produce photothermal effect under NIR laser irradiation.

[0080] Experimental Example 14: The tumor tissues of the 4T1 tumor-bearing mice were fixed with 40 g / L 4% paraformaldehyde PBS solution for 24 hours, dehydrated with gradient ethanol and then paraffin-embedded to prepare 5 μm continuous sections. For active oxygen (ROS) detection, after the sections were deparaffinized and hydrated, 10 μM DCFH-DA working solution (purchased from Biyun Tian Biotechnology Co., Ltd.) was added dropwise, and the sections were incubated at 37°C in the dark for 30 minutes. After being washed with PBS for 3 times, the sections were mounted and the green fluorescence images were collected using a laser confocal microscope (excitation / emission wavelength 488 / 525 nm); H&E staining was performed by staining with hematoxylin for 5 minutes, differentiating with 4.4 g / L 1% hydrochloric acid ethanol and staining with eosin for 3 minutes. After being mounted with neutral balsam, the morphological changes of the tissues were observed by an optical microscope (Nikon Eclipse E100, 200x); TUNEL apoptosis detection was performed according to the Roche kit instructions: proteinase K (20 μg / mL) was used for 37°C digestion for 15 minutes, and TUNEL reaction mixture was incubated in the dark for 60 minutes. After the cell nucleus was stained with DAPI, the fluorescence images were collected using a laser confocal microscope (Zeiss LSM 900); Ki-67 proliferation detection was performed by incubating rabbit anti-Ki-67 monoclonal antibody (1:200, Abeam ab16667) at 4°C overnight, reacting with Cy3-labeled secondary antibody (1:500) at 37°C for 1 hour, and imaging after staining with DAPI. All image analyses were performed by double-blind evaluation: the ROS average fluorescence intensity was calculated using Image Pro Plus 6.0 software (≥3 fields per group), the tissue damage was quantified according to the proportion of necrotic area; the apoptosis rate was calculated by counting the proportion of TUNEL⁺ / DAPI⁺ cells; the proliferation activity was evaluated by counting the proportion of Ki-67 positive cells; ≥5 sections were analyzed for each group, 3 fields were randomly selected for each section, and PBS negative control and positive tissue control were set to ensure the reliability of the results. See Figure 18It can be seen that, in terms of ROS generation in vivo, compared with the PAAP and DOX-PAAP groups alone, the ROS generation of the PAAP+NIR and DOX-PAAP+NIR treatment groups was enhanced under NIR irradiation; in terms of H&E staining, although compared with the DOX group alone, the DOX-PAAP group could promote apoptosis, under NIR irradiation, the H&E staining of the PAAP+NIR and DOX-PAAP+NIR treatment groups showed that a large number of tumor cells were apoptotic and the nuclei disappeared; finally, the synergistic effect between the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and Ki-67 labeling showed that the damage of the PAAP+NIR and DOX-PAAP+NIR groups to the cancer cells was more serious than that of the other groups (the DOX group, the PAAP group and the DOX-PAAP group), and the anti-proliferation effect was stronger. This shows that the DOX-PAAP has a strong ability to promote cancer cell apoptosis under near-infrared light irradiation.

[0081] Finally, it should be pointed out that the above examples are only used to illustrate the technical solutions of the present application and not to limit it, although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application can be modified or replaced by equivalents without departing from the purpose and scope of the technical solutions, and they should all be covered in the scope of the claims of the present application.

Claims

1. A near-infrared triggered multifunctional nano-platform, 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.

2. The method for constructing a near-infrared triggered multifunctional nano-platform according to claim 1, characterized in that: The specific steps are as follows: (1) First, gold nanoparticles AuNPs and gold nanoclusters AuNCs were made into Au-Au@PEG nanoassembly structures; (2) Then, the polydopamine coating is evenly coated on the surface of the Au-Au@PEG nanoassembly 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 construction method according to claim 2, characterized in that In step (1), the average particle size of the Au-Au@PEG nanoassembly structure is 90 nm.

4. The construction method according to claim 2, characterized in that The specific method of step (1) is: (1-1) First, gold nanoparticles (AuNPs) and thiol-polyethylene glycol amino solution were mixed and stirred, and then dialyzed against deionized water to remove unreacted SH-PEG-NH2; (1-2) Then, MES buffer was added, stirred for the first time, and EDC aqueous solution was added. Stirred for the second time, and then NHS aqueous solution was added. Stirred for the third time, and dialyzed against deionized water to remove unreacted EDC and NHS to obtain PEG@AuNPs solution; (1-3) The pH of the gold nanoclusters AuNCs was then adjusted to 7–8, mixed with the PEG@AuNPs solution at room temperature, dialyzed against deionized water, and stored at low temperature.

5. The construction method according to claim 4, characterized in that The gold nanoparticles AuNPs were prepared by the following method: 10 mg / mL HAuCl4·3H2O aqueous solution and 10 mg / mL PVP 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 a NaBH4 aqueous solution was quickly added; the mixture was further heated to 90°C, stirred at this temperature for 15 minutes, dialyzed against deionized water to remove excess NaBH4, and stored under low temperature conditions.

6. The construction method according to claim 4, characterized in that The gold nanoclusters AuNCs were prepared by the following method: 0.1 g / mL HAuCl4·3H2O solution was mixed with 3 mM GSH solution, heated at 80°C for 5 hours, dialyzed against deionized water to completely remove excess GSH, and stored at low temperature.

7. The construction method according to claim 2, characterized in that: The specific method of step (2) is as follows: first, add the dopamine hydrochloride solution into the Tris buffer solution and mix thoroughly, then slowly inject the Au-Au@PEG nanoassembly structure, stir for 5 to 7 hours under aerobic conditions in the dark, then add 1 mol / L dilute hydrochloric acid to adjust the pH to 7.0 to terminate the reaction, and thus obtain PAAP.

8. The construction method 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, and then the doxorubicin aqueous solution is mixed with PAAP, stirred for 4 hours under dark conditions, and drug adsorption is achieved by π-π stacking. After loading, the mixture is centrifuged at 12,000 rpm for 15 minutes to remove free DOX, and the supernatant is used to measure the loading efficiency by ultraviolet spectroscopy.

9. Use of the near-infrared triggered multifunctional nanoplatform according to claim 1 in the preparation of drugs against triple-negative breast cancer.

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