Nanoprobe based on hollow mesoporous carbon nitride as well as preparation method and application of nanoprobe

Abstract

The invention discloses a nanoprobe based on hollow mesoporous carbon nitride as well as a preparation method and application of the nanoprobe, and belongs to the technical field of medicines. The preparation method comprises the following steps: S1, synthesizing hollow mesoporous carbon nitride (HCNS) by using a hard template method; s2, carrying out a hydrothermal reaction on the HCNS and KSCN to synthesize modified hollow mesoporous carbon nitride HCNxS; and S3, dispersing the HCNxS in a DOX / PBS solution, fully loading the DOX on the HCNxS to obtain DOX-coated HCNxS, and closing the pore channels of the HCNxS by using the activated HA to obtain the DOX-coated HCNxS-HA. The hollow structure of the DOX-coated HCNxS-HA endows the DOX with 19.05% of loading rate, the surface amphoteric characteristic and HA modification realize CD44 receptor mediated tumor targeting delivery, and trigger pH response drug release in an acidic microenvironment; the DOX-coated HCNxS-HA can destroy a lysosome membrane and a proton sponge effect through ROS mediation, the co-localization rate of the lysosome is remarkably reduced (the Pearson's correlation coefficient is reduced from 0.73 to 0.18), enrichment of DOX in tumor cells is promoted, the survival rate of MDA-MB-231 cells is reduced to 13.8%, and the DOX-coated HCNxS-HA has a killing effect on breast cancer cells.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and particularly to a nanosensor based on hollow mesoporous carbon nitride, and a preparation method and application thereof. Background Art

[0002] As a very common malignant tumor among women globally, breast cancer has a wide range of impacts. Common treatment methods for breast cancer include surgical treatment, chemotherapy, radiotherapy, and immunotherapy. These traditional diagnosis and treatment methods have problems such as large trauma, high toxic and side effects, and insufficient targeting. In response to this situation, personalized medicine has received great attention because it can customize treatment according to the needs and characteristics of patients, minimizing side effects. Therapeutics combines diagnostic drugs and therapeutic drugs through real-time monitoring of drugs to provide patient-centered care for the treatment of breast cancer and other diseases, thereby establishing cancer treatment plans and obtaining better treatment effects. Currently, nanotherapy is being widely explored as an effective method for treating breast cancer.

[0003] Currently, cancer diagnosis uses a variety of cancer biomarkers as detection molecules. In the case of breast cancer, many detection methods have been developed to identify these biomarkers in physiological amounts from serum or other body fluids. Currently, techniques for evaluating HER-2 status include gene methods such as fluorescence in situ hybridization (FISH), chromogenic in situ hybridization (CISH), and PCR-based gene copy analysis, biosensors, evaluating protein expression by immunohistochemistry (IHC) or Western blotting, and evaluating messenger RNA by PCR or Northern blotting. In addition, it has been reported that lipase can support the proliferation of breast cancer cells, promote the invasiveness of triple-negative breast cancer, and its level is significantly increased in tumor patients such as triple-negative breast cancer, colon cancer, and gastric cancer. Free fatty acids are produced by the decomposition of lipase, which can activate the PI3K / AKT signaling pathway, promoting the proliferation, survival, and anti-apoptotic ability of breast cancer cells. In addition, the TAZ protein (a key molecule in the Hippo signaling pathway) in adipocytes promotes the proliferation of breast cancer cells by upregulating the expression of lipase-related proteins (such as Resistin). Lipase can also break down stored lipids to provide adenosine triphosphate (ATP) for rapidly proliferating cancer cells. Therefore, lipase plays an important role in the signaling pathway of breast cancer.

[0004] Graphitic carbon nitride (g-CN) is a polymeric semiconductor material with a two-dimensional layered structure, composed of C and N elements. Its unique electronic structure stems from the triazine ring units formed by sp2 hybridized carbon and nitrogen atoms, which are connected by covalent bonds within the layer and interact through van der Waals forces between layers. This material has a π-π conjugated electron system with a bandgap energy of approximately 2.7 eV (conduction band -1.1 eV, valence band +1.6 eV), can respond to visible light, and exhibits excellent optoelectronic properties. Thanks to its low toxicity, tunable bandgap, high biocompatibility, and photoluminescence characteristics, g-CN shows great potential in the biomedical field. Especially in the fields of biomedical imaging, drug delivery, PDT, and combination therapy, g-CN materials have attracted extensive attention.

[0005] Bulk g-CN usually selects compounds containing triazine structures (such as melamine) as precursors and is synthesized by polymerization reactions, solvothermal methods, or pyrolysis at high temperatures. However, the extensive application of bulk g-CN is limited due to the interlayer stacking during the polycondensation process.

[0006] Bulk g-CN has a low specific surface area and poor crystallization quality, which have some limitations in practical applications. Therefore, researchers have turned to preparing nanostructured nCN materials (g-CN nanomaterials, nCN), which have diverse morphologies, commonly including nanosheets, nanotubes, nanorods, quantum dots, porous microspheres, etc.

[0007] However, the biomedical applications of nCN are still limited by inherent defects: low drug loading efficiency of nanosheets and quantum dots, difficulty for ultraviolet-visible light-excited PDT to penetrate deep tissues, and easy capture and degradation of nanosheets by lysosomes due to size effects, etc. Summary of the Invention

[0008] The object of the present invention is to provide a nanosensor based on hollow mesoporous carbon nitride and its preparation method and application. By combining the special morphology of hollow mesoporous carbon nitride (HCNS) with hydrothermal reactions to introduce nitrogen defects, its water dispersibility and material properties are improved. Further, a nanodrug is constructed to achieve chemotherapy-PDT, and a nanosensor is used for highly sensitive detection, providing a new solution for breast cancer detection and treatment.

[0009] To achieve the above object, the present invention provides a preparation method of a nanosensor based on hollow mesoporous carbon nitride, including the following steps:

[0010] S1. Synthesize hollow mesoporous carbon nitride HCNS using the hard template method;

[0011] S2. Perform a hydrothermal reaction on HCNS and KSCN to synthesize modified hollow mesoporous carbon nitride HCN x S;

[0012] S3. Disperse HCN x S in the DOX / PBS solution so that DOX is fully loaded onto HCN x S to obtain DOX@HCN x S, and use activated HA to block the pores of HCN x S to obtain DOX@HCN x S-HA.

[0013] Preferably, the steps for synthesizing HCNS by the hard template method in S1 include:

[0014] S11. Using tetraethyl orthosilicate (TEOS) as a precursor, synthesize SiO2 microspheres by a method;

[0015] S12. Add TEOS and a pore-forming agent to the SiO2 microspheres synthesized in S11, and calcine at 550 °C to generate mesoporous SiO2;

[0016] S13. Load monocyanamide (CY) into the pores of the mesoporous SiO2 shell to obtain a CY / SiO2 hybrid, and the CY / SiO2 hybrid is transformed into a g-CN / SiO2 nanocomposite through the thermal-induced self-condensation of CY at 550 °C under a N2 atmosphere;

[0017] S14. Add 4M NH4HF2 to the g-CN / SiO2 nanocomposite obtained in S13, mix and stir for 12 h to remove the SiO2 microspheres to obtain HCNS.

[0018] Preferably, in S2, the steps for synthesizing HCN x S include:

[0019] Stir 1 mg / mL HCNS and 0.2 mM KSCN at room temperature for 2 h to obtain a mixed solution; place the Teflon liner containing the mixed solution in a reaction kettle, and put the reaction kettle into an oven at 160 °C for a hydrothermal reaction for 2 - 5 h; after the reaction, cool naturally and wash with water 3 - 5 times.

[0020] Preferably, put the reaction kettle into an oven at 160 °C for a hydrothermal reaction for 3 h.

[0021] Preferably, in S3, the preparation of DOX@HCN x S includes:

[0022] Disperse HCN x S in a 1 mg / mL DOX / PBS solution, stir for 36 h in the dark to load DOX, and after reaching adsorption equilibrium, collect DOX@HCN x S by centrifugation.

[0023] Preferably, HCN x S: The mass-volume ratio of the DOX / PBS solution is 2 mg: 1 mL.

[0024] Preferably, the activation of HA includes: adding EDC and NHS to the 3 mg / mL HA / PBS solution respectively, and stirring for 1 h under dark conditions to activate the carboxyl group of HA.

[0025] Preferably, in S3, DOX@HCN x S reacts with the activated HA for 12 h to block HCN x the pore channels of S.

[0026] Preferably, the volume-mole ratio of the 3 mg / mL HA / PBS solution: EDC: NHS is 10 mL: 0.75 mol: 0.75 mol.

[0027] On the other hand, the present invention provides an application of the above-mentioned nano-probe based on hollow mesoporous carbon nitride in the preparation of anti-breast cancer drugs.

[0028] Therefore, the nano-probe based on hollow mesoporous carbon nitride, its preparation method and application of the present invention have the following beneficial effects:

[0029] (1) By using the hard template method and regulating the precursor and reaction conditions, HCNS with a hollow mesoporous structure is prepared, and further uniformly dispersed hollow mesoporous carbon nitride (HCN x S) is obtained through the hydrothermal reaction of KSCN. The hollow structure of HCN x S significantly improves the drug loading capacity. The stability, water dispersibility and fluorescence of HCN x S are optimized, and it has significant pH responsiveness and the ability to generate ROS under NIR.

[0030] (2) Based on HCN x S, the anti-cancer drug doxorubicin (DOX) is loaded and HA is connected to construct the nano-drug platform DOX@HCN x S-HA; the hollow spherical structure of HCN x S significantly improves the drug loading rate and near-infrared responsiveness; HA is modified on the surface of HCN x S, endowing the nano-drug with the ability to specifically recognize CD44; through the detection of FTIR, UV-vis and visualization characterization, the successful preparation of the nano-drug based on hollow mesoporous carbon nitride (DOX@HCN x S-HA) is proved. According to the standard curve of DOX, the loading rate of DOX loaded on HCN x S is calculated to be 19.05%, realizing the efficient loading of DOX.

[0031] (3) In vitro cell experiments were used to show that after modification with HA, the prepared nano-drugs could promote the active targeted uptake of the drug delivery system and enhance the intracellular drug accumulation; under the irradiation of an 808 nm near-infrared laser, HCN x S generated highly efficient ROS through defect intermediate energy levels and achieved pH responsiveness in the simulated tumor microenvironment, releasing 75.48% of DOX, realizing dual responsiveness of NIR / pH.

[0032] (4) According to CLSM imaging and lysosome tracing experiments, it was confirmed that after the drug entered the lysosome, HCN x S material would be phagocytosed by the lysosome and co-localization occurred, but HCN x ROS generated by S under the excitation of near-infrared light damaged the integrity of the lysosome membrane, and the co-localization situation weakened; after modification with HA, the CD44 receptor-mediated endocytosis of nanoparticles in MDA-MB-231 breast cancer cells was significantly enhanced, and HCN x The Pearson correlation coefficient of lysosome co-localization of S-HA further decreased to 0.18 after irradiation. This process effectively avoided the risk of drug degradation or efflux by lysosomes, significantly increased the enrichment concentration of DOX in the cytoplasm, and thus enhanced the killing effect on breast cancer cells.

[0033] (5) Based on the CCK-8 cytotoxicity experiment, it was confirmed that DOX@HCN x S-HA nano-drugs had a significant killing effect on MCF-7 / MDA-MB-231 breast cancer cells in vitro, and its efficacy was concentration-dependent; HA modification significantly enhanced drug intracellular enrichment by targeting the CD44 receptor, while HCN x The nitrogen defect engineering of S endows it with near-infrared light response ability, realizes efficient ROS generation and chemotherapy-PDT synergistic therapy, and the ROS-mediated lysosome membrane is damaged, synergistically promoting the escape of DOX from the lysosome.

[0034] The technical solutions of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Description of the Drawings

[0035] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for the description of the embodiments of the present invention will be briefly introduced below. Obviously, the accompanying drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0036] Figure 1 Is the preparation flow chart of nitrogen-deficient hollow mesoporous carbon nitride HCN x S;

[0037] Figure 2 Mesoporous SiO2, g-CN / SiO2, HCNS and HCN x S-1 to HCN x TEM images of S-6, where a) is mesoporous SiO2, b) is g-CN / SiO2, c) is HCNS, and d) is HCN x S-1, and e) is HCN x S-2, and f) is HCN x S-3, and g) is HCN x S-4, and h) is HCN x S-5, and i) is HCN x S-6;

[0038] Figure 3 is HCN x SEM image of S-5;

[0039] Figure 4 is HCNS and HCN x S-1 to HCN x Zeta potential diagram of S-6;

[0040] Figure 5 is HCNS and HCN x S-1 to HCN x XRD pattern of S-6;

[0041] Figure 6 is HCNS and HCN x S-1 to HCN x FTIR spectrum of S-6;

[0042] Figure 7 is HCNS and HCN x XPS spectrum of S-5, where a) is the XPS spectrum at a resolution of C 1s and b) is the XPS spectrum at a resolution of N 1s at that time;

[0043] Figure 8 is HCNS and HCN x Fluorescence property analysis of S-5, where a) is the down-conversion fluorescence spectrum of HCNS and b) is HCN x Down-conversion fluorescence spectrum of S-5, c) is the up-conversion fluorescence spectrum of HCNS, and d) is HCN x Up-conversion fluorescence spectrum of S-5;

[0044] Figure 9 is HCNS, HCN x S-5 and HCN x Zeta potential of S-5 and HCN at different pH values;

[0045] Figure 10 HCN x Characterization of the photocatalytic ROS production performance of S-5, where a) is DCFH+0.15 mg / mL HCN x Fluorescence spectra of S-5 after mixing at different irradiation times; b) DCFH and DCFH+0.15 mg / mL HCN under 808 nm near-infrared laser irradiation. x Changes of S-5 fluorescence intensity over time;

[0046] Figure 11 HCN x Analysis of the photothermal conversion capacity of S, where a) is HCN x Temperature variation curves of S-5 heating and natural cooling, b) HCN x S-5 cooling time and - relationship curve and linear fitting;

[0047] Figure 12 HCN x S and HCN x FTIR image of S-HA;

[0048] Figure 13 DOX@HCN x S-HA, Free DOX and HCN x UV-visible absorption spectrum of S;

[0049] Figure 14 is the standard curve of DOX;

[0050] Figure 15 DOX@HCN x Release efficiency of S-HA at different pH;

[0051] Figure 16 CLSM images of MDA-MB-231 cells after incubation with DOX and DOX@HCNxS-HA for 6 h;

[0052] Figure 17 DOX, HCN x S, DOX@HCN x CLSM images of ROS generated in MDA-MB-231 cells after 6 h of S-HA incubation;

[0053] Figure 18 HCN under dark conditions x Analysis of the lysosomal escape mechanism of S, where a) is the reaction between MDA-MB-231 and HCN under dark conditions. xCLSM images of the co-localization of S and Lyso-Tracker Red (40×). b) Analysis results of the co-localization using Image-J. c) Pearson correlation coefficients of the fluorescence signals of the two colors under dark conditions;

[0054] Figure 19 For HCN under the irradiation of an 808 nm near-infrared laser x Analysis of the lysosomal escape mechanism of S. Among them, a) shows MDA-MB-231 and HCN under the irradiation of an 808 nm laser x CLSM images of the co-localization of S and Lyso-Tracker Red (40×). b) Analysis results of the co-localization using Image-J. c) Pearson correlation coefficients of the fluorescence signals of the two colors under the irradiation of an 808 nm laser;

[0055] Figure 20 For HCN under dark conditions x Analysis of the lysosomal escape mechanism of S-HA. Among them, a) shows MDA-MB-231 and HCN under dark conditions x CLSM images of the co-localization of S-HA and Lyso-Tracker Red (40×). b) Analysis results of the co-localization using Image-J. c) Pearson correlation coefficients of the fluorescence signals of the two colors under dark conditions;

[0056] Figure 21 For HCN under the irradiation of an 808 nm laser x Analysis of the lysosomal escape mechanism of S-HA. Among them, a) shows MDA-MB-231 and HCN under the irradiation of an 808 nm laser x CLSM images of the co-localization of S-HA and Lyso-Tracker Red (40×). b) Analysis results of the co-localization using Image-J. c) Pearson correlation coefficients of the fluorescence signals of the two colors under the irradiation of an 808 nm laser;

[0057] Figure 22 For DOX@HCN x Analysis of the cell killing ability of S-HA. Among them, a) shows the viability of MCF-7 cells, and b) shows the viability of MDA-MB-231 cells. Detailed implementation manners

[0058] The technical solutions of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0059] In order to make the objectives, technical solutions, and advantages of the present application clearer, more thorough, and more complete, the technical solutions of the present invention will be clearly and completely described below through the accompanying drawings and embodiments. The following detailed descriptions are all descriptions of embodiments, aiming to provide further detailed explanations of the present invention. Unless otherwise specified, all technical terms used in the present invention have the same meanings as those commonly understood by those of ordinary skill in the art to which this application belongs.

[0060] The instruments, equipment, reagents, and materials used in the examples were all obtained through commercial channels.

[0061] Example 1

[0062] A preparation method of nitrogen-deficient hollow mesoporous carbon nitride (HCN x S), the flow chart is as Figure 1 shown, and specifically includes the following steps:

[0063] S1. Synthesize hollow mesoporous carbon nitride (HCNS) by the hard template method.

[0064] Using tetraethyl orthosilicate (TEOS) as the precursor and the silicon spheres synthesized by the method as the sacrificial core material. After the SiO2 core is formed, another batch of TEOS is added to C 18 TMOS as the pore-forming agent, and calcined at 550 °C to generate a thin mesoporous silicon shell. Loading melamine (CY) into the pores of the mesoporous SiO2 shell to obtain a CY / SiO2 hybrid, and then converting it into a g-CN / SiO2 nanocomposite by the thermal-induced self-condensation polymerization of CY at 550 °C under N2, and then removing the SiO2 template by stirring with 4M NH4HF2 for 12 h to obtain HCNS, and then centrifugally washing with pure water 5 times. Finally, the obtained pale yellow HCNS powder is dried overnight in a vacuum oven at 80 °C.

[0065] S2. Synthesize HCN x S by the hydrothermal method of KSCN.

[0066] Stir 1 mg / mL HCNS and 0.2 mM KSCN at room temperature on a magnetic stirrer for 2 h; place the Teflon liner containing the mixed solution in a reaction kettle, and then put the reaction kettle into an oven at 160 °C for a hydrothermal reaction for 2 - 5 h; after natural cooling, wash with water 3 - 5 times.

[0067] Example 2

[0068] A preparation method of nitrogen-deficient hollow mesoporous carbon nitride (HCN x S), which is different from Example 1 in that: synthesize HCN xS: Adjust the 1 mg / mL HCNS aqueous solution to pH = 8 with 1 mM NaOH, and place it in the inner lining of a hydrothermal autoclave made of Teflon. Put the hydrothermal autoclave into an oven at 160 °C for a hydrothermal reaction for 2 - 5 h.

[0069] The numbers of nitrogen-deficient hollow mesoporous carbon nitride obtained by different hydrothermal treatment methods in Examples 1 and 2 are shown in Table 1 below:

[0070] Table 1 Numbers of nitrogen-deficient hollow mesoporous carbon nitride corresponding to different hydrothermal treatment methods

[0071] <![CDATA[HCN x Serial number of S]]> Hydrothermal treatment method <![CDATA[HCN x S-1]]> Adjust 10 mg / mL HCNS to pH = 8 with NaOH solution and hydrothermal treat at 160 °C for 2 h <![CDATA[HCN x S-2]]> Adjust 10 mg / mL HCNS to pH = 8 with NaOH solution and hydrothermal treat at 160 °C for 3 h <![CDATA[HCN x S-3]]> Adjust 10 mg / mL HCNS to pH = 8 with NaOH solution and hydrothermal treat at 160 °C for 4 h <![CDATA[HCN x S-4]]> 10 mg / mL HCNS + 0.2 mM KSCN solution, hydrothermal treat at 160 °C for 2 h <![CDATA[HCN x S-5]]> 10 mg / mL HCNS + 0.2 mM KSCN solution, hydrothermal treat at 160 °C for 3 h <![CDATA[HCN x S-6]]> 10 mg / mL HCNS + 0.2 mM KSCN solution, hydrothermal treat at 160 °C for 4 h

[0072] Test Example 1

[0073] Characterize the micro-morphologies of mesoporous SiO2, g-CN / SiO2, HCNS and HCN x S-1 to HCN x S-6, as Figure 2 shown.

[0074] HCNS has a hollow mesoporous microsphere structure, with a particle size of about 300 - 400 nm. The hollow mesoporous spheres are relatively sticky to each other, and the dispersibility is poor.

[0075] Comparing the effects of different hydrothermal modification conditions, it is found that: for the NaOH treatment group (HCN x S-1 to HCN x S-3): As the hydrothermal time prolongs, the integrity of the hollow structure is gradually damaged. For HCN x S-3 and HCN x S-6 (hydrothermal treatment for 4 h), shell collapse occurs, and the improvement of particle dispersibility is limited, indicating that too long hydrothermal time will damage the cavity structure of HCN x S, and excessive corrosion in an alkaline environment will lead to a decrease in structural stability. For the KSCN treatment group (HCN x S-4 to HCN x S-6): After introducing K + , the hollow mesoporous structure is retained, and the dispersibility is significantly improved. Among them, for HCN x S-5, less particle agglomeration appears in the micro-morphology, the hollow spheres are more evenly distributed, the dispersibility is improved, and the size of the spheres is also reduced.

[0076] Further perform SEM characterization on HCN x S-5, as Figure 3 shown, and the spherical structure is well maintained.

[0077] Test Example 2

[0078] The dispersibility and stability of HCNS and HCN x S-1 to HCN x S-6 were compared by using Zeta potential measurement and static experiment. The Zeta potential diagrams of each sample solution are shown as Figure 4 follows.

[0079] Among them, the average Zeta potential of HCNS is -24.33 mV, while the average Zeta potential of the optimized HCN x S-5 drops to -34.5 mV, which is attributed to the enhanced surface negative charge induced by nitrogen defects and the electrostatic repulsion effect. The experimental results show that the hydrothermal treatment of KSCN can improve the water dispersibility of HCNS while maintaining the structural integrity of HCNS.

[0080] Test Example III

[0081] XRD analysis was performed on HCNS and HCN x S-1 to HCN x S-6, as shown in Figure 5 the figure. The results show that all materials exhibit peaks at 12.9° and 27.4°. The peak at 27.4° corresponds to the strong diffraction peak of the interlayer stacking of the C-N conjugated aromatic heterocyclic layer, and the peak at 12.9° corresponds to the diffraction peak of the 3-s-triazine ring unit. Compared with HCNS, the diffraction peaks of HCN x S-5 on the (100) and (002) crystal planes show a significant weakening phenomenon, which indicates that the conjugated length of the repeating structure in the plane represented by the (100) crystal plane increases, changing from long-range order to short-range order, and the degree of layered stacking reflected by the (002) crystal plane decreases significantly, meaning that the size of the conjugated region in the plane is reduced.

[0082] FTIR analysis was performed on HCNS and HCN x S-1 to HCN x S-6, as shown in Figure 6 the figure. The results show that all materials exhibit peaks at 3000 - 3600 cm -1 , corresponding to the stretching of N-H and O-H, at 1650 - 1250 cm -1 corresponding to carbon-nitrogen heterocycles (C-N, C=N), and at 811 cm -1 corresponding to the characteristic absorption peak of the triazine ring, indicating that the basic structure of the material has not changed during the hydrothermal treatment. It is worth noting that HCN x S-4 and HCN x S-5 exhibit a characteristic absorption peak corresponding to -C≡N at 2180 cm -1 .

[0083] Test Example IV

[0084] XPS spectra of HCNS and HCN x S-5 were analyzed, as Figure 7 shown, where a) is the XPS spectrum at a resolution of C 1s and b) is the XPS spectrum at a resolution of N 1s .

[0085] In the high-resolution XPS spectrum of C 1s , the sp 2 carbon (284.8 eV, C-C) increases, while in the XPS survey spectrum of HCN x S-5, the N / C ratio decreases, indicating the introduction of nitrogen defects. Additionally, new K2 x and K2 p3 / 2 peaks are observed in the XPS spectrum of HCN p1 / 2 S-5, which appear at 292.8 and 295.5 eV respectively, and are characteristic peaks of N-K bonds, indicating that K + binds to the C-N ring formed by the triple N-bridges connecting the triazine units. In summary, the introduction of -C≡N groups, -OH groups, nitrogen defects, and K + makes the structure of HCN x S-5 more porous and improves the water dispersibility of the material.

[0086] Test Example Five

[0087] Fluorescence properties of HCNS and HCN x S-5 were analyzed, as Figure 8 shown, where a) is the down-conversion fluorescence spectrum of HCNS, b) is the down-conversion fluorescence spectrum of HCN x S-5, c) is the up-conversion fluorescence spectrum of HCNS, and d) is the up-conversion fluorescence spectrum of HCN x S-5.

[0088] HCNS exhibits different fluorescence peaks at different excitation wavelengths (280 - 360 nm): the weak peak at 365 nm represents the n-π transition of the free melem groups (residues of triazine ring precursors) on the surface of HCNS, and the main peak at 446 nm originates from the π-π conjugate structure of the g-CN bulk. However, HCN x S-5 modified by hydrothermal treatment with KSCN only retains a single fluorescence peak at 446 nm under the same excitation conditions, and the intensity is significantly enhanced (about 1.8 times that of HCNS), indicating that the hydrothermal reaction can remove the melem groups on the material surface, and the introduction of nitrogen defects enhances the electron transition efficiency of the π-conjugated system. In addition, the improved crystallinity of HCN x S- (the XRD full-width at half-maximum decreases) and the optical confinement effect of the hollow structure further synergistically amplify the fluorescence signal.

[0089] Under 780nm near-infrared light excitation, HCNS only produces weak blue-green light emission (400-500nm), while HCN x The upconversion fluorescence intensity of S-5 is enhanced by about 1.5 times. This phenomenon is attributed to the intermediate energy level formed by nitrogen defects, which converts low-energy near-infrared photons into high-energy visible light through a multiphoton absorption mechanism. The introduction of nitrogen defects not only broadens the light absorption range, but also acts as an electron trap to suppress non-radiative recombination, thereby improving the upconversion quantum yield. This characteristic makes HCN x S-5 has significant advantages in deep tissue photodynamic therapy and multimodal imaging.

[0090] Test Example 6

[0091] HCNS and HCN x The amphoteric properties of S were analyzed. The pH of 1 mg / mL material solution was gradually adjusted using 0.2M Na2HPO4 and NaH2PO4 buffer solutions to avoid changes in sample properties caused by rapid changes in pH. The pH range of the sample solution was adjusted from 4 to 10. The Zeta potential and hydrated particle size of the two at different pH levels were tested to analyze their surface charge characteristics and their stability in different environments. x S-5, HCN x Zeta potential of S-6, such as Figure 9 shown.

[0092] HCN x The isoelectric point of S is between 6.3 and 6.4. x The amphoteric nature of S is due to the dual acid-base properties of the surface functional groups. In an acidic environment (pH < 6.3), the amino groups are protonated (-NH3 + ), giving HCN x S positive charge (Zeta potential>0); in alkaline environment (pH>6.3), the carboxyl group is deprotonated (-COO - ) or oxygen defects adsorb hydroxyl groups (-OH - ), resulting in a negative surface charge (Zeta potential < 0). HCN x The amphoteric property of S is the coexistence of surface amino and carboxyl groups and the coordinated regulation of nitrogen defects. Its isoelectric point (pH 6.3-6.4) just matches the tumor microenvironment (pH = 6.5), enabling it to stably load drugs at physiological pH and trigger targeted drug release through charge reversal in the acidic microenvironment of the tumor, providing a key physical and chemical basis for the design of intelligent nano-drug delivery systems.

[0093] Test Example 7

[0094] 0.5 mL of 1 mM DCFH-DA was mixed with 2 mL of 0.01 M NaOH in methanol and stirred vigorously at room temperature in the dark for 30 min to produce DCFH (2',7'-dichlorodihydrofluorescein). PBS buffer solution with a pH of 7.4 was then added to adjust the pH of the solution to neutral. 1 mL of the prepared DCFH solution was then mixed with 1 mL of 0.15 mg / mL HCN. x S-5 mixed and transferred to a quartz cell. DCFH and HCN x The mixed solution of S-5 was exposed to 808 nm near-infrared laser for different time (1.0 W / cm 2 ) was irradiated (in a light-proof state). The photoluminescence spectrum of the dispersion (emission at 488 nm) was measured every 5 min, and the HCN-free x The DCFH solution of S-5 was used as a control.

[0095] like Figure 10 As shown, wherein a) is DCFH+0.15mg / mL HCN x Fluorescence spectra of S-5 after mixing at different irradiation times; b) DCFH and DCFH+0.15 mg / mL HCN under 808 nm near-infrared laser irradiation. x Changes in the fluorescence intensity of S-5 over time.

[0096] The results showed that under the irradiation of 808nm near-infrared laser, HCN x The fluorescence intensity of the mixed solution of S-5 and DCFH increased significantly with the irradiation time, while the fluorescence intensity of the pure DCFH solution (without HCN) x The fluorescence signal of S-5) was almost unchanged, indicating that HCN x S-5 can efficiently generate ROS under 808nm near-infrared excitation, and its generation amount is positively correlated with the illumination time.

[0097] HCN x The ROS generation ability of S-5 is due to its unique energy band structure and defect engineering regulation. First, the near-infrared light absorption and upconversion effect: nitrogen defects introduce intermediate energy levels in the energy band, making HCN x S-5 can convert 808nm near-infrared light into high-energy visible light (400-500nm) through a multiphoton absorption mechanism. The electrons in the valence band are excited to jump to the conduction band, thereby generating electron-hole pairs. Secondly, nitrogen vacancies act as active sites, promoting the transfer of electrons to dissolved oxygen (O2), generating superoxide anions (·O2 - ); at the same time, water molecules are oxidized by holes to generate hydroxyl radicals (·OH), and the two work together to increase ROS production.

[0098] Test Example 8

[0099] Disperse 1 mg / mL of HCN x S-5 nanoparticles into PBS solution. Then, irradiate a cuvette containing 1 mL of the test solution with an 808 nm laser with a power density of 2 W / cm 2 . Measure the instantaneous temperature of the sample every 30 s, and plot the temperature change curve based on the illumination time to analyze the photothermal performance of the sample. To detect the photothermal conversion effect of HCN x S-5, continue to test the natural cooling temperature curve of HCN x S-5 after 10 min of illumination, and calculate the photothermal efficiency of HCN x S-5 according to the following formula:

[0100]

[0101] where is the time constant of the sample cooling cycle system;

[0102] T is the real-time temperature of the HCN x S-5 dispersion, T sur is the ambient temperature, T max is the highest temperature of the HCN x S-5 dispersion, T max,water is the highest temperature of water;

[0103] m D is for T sur the mass of water, C D is for T sur the heat capacity of water;

[0104] h is the heat transfer coefficient, S is the surface area of the cuvette;

[0105] I is the power density of the 808 nm laser, A 808 is the absorbance of HCN x S-5 at 808 nm wavelength corresponding to the corresponding concentration.

[0106] The results are as Figure 11 shown, where a) is the temperature change curve of HCN x S-5 during heating and natural cooling, and b) is the relationship curve and linear fitting between the cooling time of HCN x S-5 and -.

[0107] The results show that as the irradiation time of the 808 nm laser increases, the temperature of HCN x S-5 increases, verifying that the material itself has photothermal activity. Use the temperature change curve of its natural cooling to perform a linear fitting with time (t), and calculate according to the formula for photothermal performance testing that HCN xThe photothermal conversion efficiency of S-5 is 10.3%. Although the efficiency of photothermal conversion is not particularly prominent, HCN x As a kind of graphitic carbon nitride, S-5 also has good biocompatibility, which can also endow it with important application potential in the field of photothermal therapy and other fields.

[0108] Example 3

[0109] A hollow mesoporous carbon nitride HCN based on nitrogen defects x A nanosensor DOX@HCN of S x A preparation method of S-HA, applying the above-screened HCN x Prepared with S-5, specifically including the following steps:

[0110] S1. Add 0.75 mol of EDC and NHS to 10 mL of 3 mg / mL HA / PBS solution respectively, and stir for 1 h under dark conditions to activate the carboxyl group of hyaluronic acid (HA).

[0111] S2. Disperse 30 mg of HCN x S samples in 15 mL of 1 mg / mL DOX / PBS solution, and stir for 36 h in the dark to load DOX. After reaching the adsorption equilibrium, collect the DOX-loaded carrier (DOX@HCN x S) by centrifugation.

[0112] The PBS buffer used in the experiment is 0.01 M, pH 7.2 - 7.4.

[0113] S3. React DOX@HCN x S with the activated HA for 12 h to block the pores of HCN x S, and collect DOX@HCN x S-HA by centrifugation, and wash it multiple times.

[0114] Example 4

[0115] HCN x A preparation method of S-HA, including the following steps:

[0116] S1. Add 0.75 mol of EDC and NHS to 10 mL of 3 mg / mL HA / PBS solution respectively, and stir for 1 h under dark conditions to activate the carboxyl group of hyaluronic acid (HA).

[0117] S2. React HCN x S with the activated HA for 12 h, and collect HCN x S-HA by centrifugation, and wash it multiple times.

[0118] Test Example 9

[0119] Analysis of HCN by FTIR x S, HCN x Analyze the surface chemical properties of S-HA, such as Figure 12 shown. The results show that HCN x Characteristic absorption peaks of S-HA: An obvious absorption peak appears at 1145 cm -1 , corresponding to the symmetric stretching vibration of the glycosidic bond (C-O-C) in the HA polysaccharide chain, while the original HCN x S has no characteristic signal in this region; HCN x S-HA does not show a significant amide I band (C=O) or amide II band (N-H) in the range of 1550 - 1650 cm -1 , which may be related to the weak signal of the N-acetylglucosamine unit of HA or the overlap with the carbon-nitrogen heterocyclic peak (near 1600 cm x ) of HCN -1 S. The appearance of the 1145 cm -1 characteristic peak confirms that HA is covalently modified on the surface of HCN x S.

[0120] Test Example Ten

[0121] DOX@HCN x The UV-visible absorption spectra of S-HA, Free DOX and HCN x S are as Figure 13 shown. It can be seen that free DOX presents a typical broad absorption band at 480 nm, which is due to the π-π electronic transition of its anthraquinone structure. Pure HCN x S does not peak at 480 nm, while the loaded DOX@HCN x S-HA still retains this characteristic peak at the same wavelength, and there is no peak position shift or broadening, indicating that DOX exists in the carrier in the form of drug molecules intactly, without structural changes such as protonation or chemical degradation. DOX is successfully loaded on HCN x S because the planar anthraquinone structure of DOX forms a π-π conjugate with the aromatic carbon-nitrogen heterocycle of HCN x S, enhancing the adsorption stability and the role of hydrogen bonds.

[0122] Test Example Eleven

[0123] Using a UV-visible absorption spectrometer, measure the absorbance of the supernatant obtained by washing DOX@HCN x S-HA multiple times at a wavelength of 480 nm. Using the pre-drawn DOX standard curve, determine the amount of DOX loaded on HCN x S, and then calculate the drug loading rate of HCN x S using the formula.

[0124] The drug loading calculation formula is as follows:

[0125]

[0126] wherein, it represents HCN x m1 is the total mass of DOX initially added when S is loaded with DOX, m2 refers to the mass of DOX in the supernatant obtained by centrifugation after loading, and m0 is the total mass of HCN x S.

[0127] Drawing of the DOX standard curve: Using deionized water as the solvent, a series of doxorubicin (DOX) standard solutions with gradually increasing concentrations (5, 10, 20, 25, 30, 40, 50 μg / mL) were prepared. The absorbance of DOX solutions with different concentrations was scanned at a wavelength of 480 nm using UV-vis, and its standard curve was drawn.

[0128] The standard curve of DOX is as Figure 14 shown.

[0129] HCN x The drug loading rate and encapsulation rate of HCN

[0130] S are shown in Table 2 below: x Drug loading rate and encapsulation rate of HCN

[0131] Drug carrier name Drug loading rate (%) Entrapment efficiency (%) <![CDATA[HCN x S]]> 19.05 22.13

[0132] Test Example XII

[0133] Under the conditions of PBS at 37 °C and different pH values, the pH responsiveness of the drug carrier was studied.

[0134] 2 mL of 1 mg / mL DOX@HCN x S-HA solution was separately transferred into two dialysis bags (MW = 10 KDa), and then immersed in 40 mL of release media (10 mM PBS at pH = 7.2 and 10 mM PBS at pH = 6), respectively. At selected time intervals (t = 0.5, 1, 1.5, 2, 3, 4, 6, 8, 10, 12, 24, 36, 48, 60, 72 h), the release media was analyzed using UV-vis and replaced with fresh release media. The DOX concentration was calculated based on the absorbance at 480 nm. In the release curve of the drug release behavior, the cumulative release percentage of the drug from HCN x S was plotted against time. The release efficiency was calculated according to the formula:

[0135]

[0136] wherein, m t is DOX@HCN xThe mass of DOX cumulatively released from S-HA at different time points, m 总 is the complex DOX@HCN x The total mass of DOX in S-HA.

[0137] DOX@HCN x The release efficiency of S-HA at different pH values is as Figure 15 shown.

[0138] DOX@HCN x S-HA releases 75.48% and 37.05% of DOX in the simulated tumor microenvironment (pH 6.0) and physiological environment (pH 7.2), respectively, showing significant pH responsiveness. This difference stems from the dynamic regulation of the surface charge of the material: in an acidic environment (pH 6.0), the -NH2 of HCN x S is protonated to -NH3 + (the isoelectric point of HCN x S is 6.3 - 6.4), resulting in an increase in surface positive charge, weakening the electrostatic attraction between DOX and the carrier, and at the same time accelerating the drug diffusion due to the swelling of the hollow mesoporous structure. Secondly, the tumor is in a slightly acidic environment, and the pH responsiveness of the DOX@HCN x S-HA complex enables it to specifically release DOX in the tumor microenvironment, thereby increasing the accumulation concentration of DOX in tumor tissues and effectively enhancing the therapeutic effect. The pH of the tumor microenvironment is about 4.0 - 6.5, while the pH of normal tissues is about 7.4, providing a natural targeting mechanism for the intelligent delivery of DOX@HCN x S-HA. Moreover, the pH-responsive nanodrug platform can also integrate imaging functions (such as fluorescence imaging, magnetic resonance imaging, etc.) to achieve the pH responsiveness of nanodrugs.

[0139] Test Example XIII

[0140] In a 35 mm confocal culture dish, inoculate well-growing MDA-MB-231 cells, inoculate 1.0×10 5 cells per well. When the cells are cultured to about 70%, remove the cell culture medium, wash with PBS solution, and then use 1.0 mL of 100 μg / mL HCN x S or DOX@HCN xIncubate with fresh serum-free culture medium of S-HA for 6 h (using pure DOX as a control). After drug incubation, remove the culture medium, wash 3 times with PBS solution, and sequentially add 500 μL of paraformaldehyde to fix for 10 min; aspirate the paraformaldehyde solution, wash 3 times with PBS, stain the cells with DAPI according to the manufacturer's instructions (stain for 30 min), wash 3 times with PBS, and add 1 mL of PBS. Under the DOX channel with an excitation wavelength fixed at 488 nm and the DAPI channel at 405 nm, directly observe the targeting efficiency of the nanodrug by CLSM.

[0141] CLSM images of MDA-MB-231 cells after incubation with DOX and DOX@HCNxS-HA for 6 h are as Figure 16 shown. The red fluorescence of DOX (excitation wavelength 488 nm) is highly co-localized with the DAPI-labeled cell nuclei (blue fluorescence, excitation wavelength 405 nm), indicating that DOX@HCN x S-HA can effectively penetrate the cell membrane and accumulate in the cell nuclei. The specific binding of HA and the CD44 receptor triggers clathrin-dependent endocytosis, enabling the nanodrug to efficiently enter lysosomes and escape to the cytoplasm through the pH / ROS response mechanism. Finally, the drug successfully diffuses into the cell nuclei, significantly improving the delivery efficiency in MDA-MB-231 cells and reducing the toxic effect on normal cells.

[0142] DOX, HCN x S, DOX@HCN x CLSM images of the generation of ROS in MDA-MB-231 cells after incubation with DOX, HCN Figure 17 S, DOX@HCN x S-HA for 6 h are as x shown. Compared with the DOX control group, green fluorescence is generated in the HCN x S, DOX@HCN x S-HA treatment groups, indicating that they can all effectively stimulate the generation of ROS, which reveals from the side that HCN x S accelerates the process of apoptosis by inducing the generation of ROS. Further, the treatment group of DOX@HCN x S-HA combined with NIR shows the brightest green fluorescence signal. In contrast, the fluorescence signal of HCN x S alone under NIR irradiation is weak.

[0143] Through cell uptake experiments, it is observed that the DOX@HCN x S-HA nanodrug exhibits higher cell uptake efficiency and longer retention time in cells. Based on DOX@HCN xThe nanoformulation of S-HA can increase the production of ROS, thus optimizing the PDT performance and significantly enhancing its efficacy in anti-tumor phototherapy. In the study of the lysosomal escape mechanism, the photochemical internalization strategy generates ROS by photosensitizers under specific light irradiation conditions to damage the lysosomal membrane and promote drug release into the cytoplasm. Therefore, it is further demonstrated that DOX@HCN x S-HA is expected to achieve lysosomal escape.

[0144] Test Example XIV

[0145] Using HCN x The characteristic that S emits blue-green fluorescence at Ex = 360 nm was used. Taking MDA-MB-231 cells as a model, HCN x S or HCN x S-HA was co-incubated with the lysosome-specific fluorescent probe Lyso-Tracker Red. Through the CLSM technique, the co-localization between lysosomes (red fluorescence) and nanomaterials (blue fluorescence) was observed, and the Pearson correlation coefficient between the red lysosome probe and the blue nanomaterial fluorescence signal was calculated to quantitatively evaluate the distribution of nanomaterials in lysosomes and their escape ability. The lower the Pearson correlation coefficient, the lower the co-localization degree between nanomaterials and lysosomes, that is, the stronger the lysosomal escape ability of nanomaterials.

[0146] Lysosomes were observed through the red channel, and HCN x S and HCN x S-HA were observed through the blue channel. As Figure 18 shown, among them, a) is the CLSM image (40×) of the co-localization of MDA-MB-231 with HCN x S and Lyso-Tracker Red under dark conditions, b) is the analysis result of the co-localization using Image-J, and c) is the Pearson correlation coefficient of the two-color fluorescence signals under dark conditions.

[0147] The results showed that under dark conditions, the blue and red fluorescence signals overlapped well, indicating that the nanoparticles of HCN x S were captured by lysosomes when co-incubated with breast cancer cells. The Pearson correlation coefficient of the two-color fluorescence signals under dark conditions was as high as 0.73. Thus, it can be seen that due to the endocytosis of lysosomes, HCN x S was difficult to rapidly escape from lysosomes.

[0148] As Figure 19 shown, among them, a) is MDA-MB-231 with HCN under 808 nm laser irradiation xCLSM images of the co-localization of S and Lyso-Tracker Red (40×), b) is the analysis result of the co-localization using Image-J, and c) is the Pearson correlation coefficient of the fluorescence signals of the two colors under the irradiation of an 808 nm laser.

[0149] The results showed that under the irradiation of an 808 nm near-infrared laser, HCN x S-treated MDA-MB-231 cells, in which there was still a large amount of overlap between the blue fluorescence signal of HCN x S and the red lysosome fluorescence signal. However, compared with the CLSM image of the co-localization of HCN x S and MDA-MB-231 cells in the dark, it could still be seen that a small amount of blue fluorescence signal was separated from the lysosome, and the overlap was also weaker than that of HCN x S in the dark.

[0150] Further co-localization analysis of lysosomes and HCN x S was performed. By calculation, the Pearson correlation coefficient was 0.58, which was smaller than that in the dark condition, indicating that the overlapping signal of HCN x S with lysosomes was weakened under the action of NIR. Combining with the fact that the material itself could generate ROS under NIR, and in the study of lysosomal escape mechanism, the photochemical internalization strategy was to generate ROS by photosensitizers under specific light conditions to damage the lysosomal membrane and promote the release of drugs into the cytoplasm. It was further verified that the ROS generated by HCN x S under the irradiation of an 808 nm near-infrared laser could induce the rupture of the lysosomal membrane, enabling HCN x S to escape from the lysosome.

[0151] As Figure 20 shown, among them, a) is the CLSM image (40×) of the co-localization of MDA-MB-231 and HCN x S-HA and Lyso-Tracker Red in the dark, b) is the analysis result of the co-localization using Image-J, and c) is the Pearson correlation coefficient of the fluorescence signals of the two colors in the dark condition.

[0152] The results showed that in the dark condition, the overlapping part of the blue fluorescence signal of HCN x S-HA and the red lysosome probe fluorescence signal was less. After calculation, the Pearson correlation coefficient of the fluorescence signals of the two colors in the dark condition was 0.36, indicating the specific targeting ability of HA, which could make the nanodrug more precisely target the cell nucleus. Combining with the pH responsiveness of HCN x S itself, DOX@HCN xThe S-HA nanodrug platform can target nanodrugs to the cell nucleus in tumor cells, has a certain killing effect on tumor cells, and is expected to achieve the treatment of breast cancer.

[0153] As Figure 21 shown, among them, a) is the CLSM image (40×) of the co-localization of MDA-MB-231 and HCN x S-HA and Lyso-Tracker Red under 808nm laser irradiation, b) is the analysis result of the co-localization using Image-J, and c) is the Pearson correlation coefficient of the fluorescence signals of the two colors under 808nm laser irradiation.

[0154] The results show that under the irradiation of an 808nm near-infrared laser, the overlapping part of the blue fluorescence signal of HCN x S-HA and the fluorescence signal of red lysosomes has significantly decreased, and the nanoparticles can be more targeted to the cell nucleus. The Pearson correlation coefficient of their co-localization is 0.18, further verifying that HCN x S connected to HA can more effectively promote the escape of drugs from lysosomes. Combining with the co-localization of HCN x S without connecting HA, its Pearson correlation coefficient gradually decreases, indicating that the ability of NIR to generate ROS can cause lysosome rupture, which is in line with the strategy of photochemical internalization of the lysosome mechanism. And HCN x After connecting HA, S combines the ability of HA to specifically bind to the cell receptor CD44 of MDA-MB-231 cells, enabling the nanodrug platform to more accurately target the cell nucleus. Coupled with the pH-responsive property of the nanoparticles themselves, it can cause the proton sponge effect.

[0155] Regarding the un-verified DOX@HCN x S-HA nanodrug platform, while only verifying the lysosome escape ability of HCN x S-HA and HCNxS: First, the lysosome probe selected is Lyso-Tracker Red, which shows a red fluorescence signal, the same as the fluorescence signal shown by DOX under CLSM, and the co-localization analysis of the two cannot clearly show. If the Lyso-Tracker Green probe is selected, the channel is 488nm, and the optimal excitation wavelength of the HCN x S material itself is around 360nm, and the fluorescence signal of HCN x S can be clearly observed in the 405 and 488 channels of CLSM, which will be confused with the color of Lyso-Tracker Green.

[0156] In summary, the DOX@HCN constructed in the present invention xThe S-HA nanomedicine platform can utilize the ability of NIR to generate ROS and its amphoteric properties to cause the proton sponge effect, achieve lysosomal escape, and the nanomedicine can target the cell nucleus of tumor cells and has a killing effect on tumor cells.

[0157] Test Example XV

[0158] Inoculate well-grown MDA-MB-231 cells or MCF-7 cells in a 96-well plate, inoculate 5000 - 1.0×10 4 cells per well, and culture for 24 h. Add about 100 μL of PBS around the wells to be tested to reduce evaporation. Prepare a series of drugs with gradually increasing concentrations: 100, 125, 150, 200, 250, 300 μg / mL (diluted with basal medium), add the drug-containing medium with different concentrations to the 96-well plate. The control group only has basal medium, 100 μL per well, and incubate for 24 h. For the NIR group, use an 808 nm near-infrared laser with a power density of 1.5 W / cm 2 to irradiate the samples and then continue to incubate.

[0159] Determination: Prepare CCK-8 pre-formulated in proportion (prepared with basal medium). Then carefully take out the drug medium in the 96-well plate, wash it 2 - 3 times with PBS to exclude the influence caused by the drug and CCK-8. Discard the PBS in 5 wells (blank group). Add basal medium + CCK-8 to each well, 100 μL per well, place the 96-well plate in an incubator for 1 - 4 hours. When measuring, place the 96-well plate in the dark, and measure the absorbance at 450 nm with an enzyme-labeled instrument and calculate the cell viability value.

[0160] As Figure 22 shown, where a) is the viability of MCF-7 cells and b) is the viability of MDA-MB-231 cells. The results show that the therapeutic effects of each experimental group are concentration-dependent. Even at a high concentration of 300 μg / mL HCN x S, the relative survival rate of MCF-7 cells can still be maintained above 85% respectively, indicating that pure HCN x S has good biocompatibility and can be used as a delivery carrier for tumor drugs in biomedicine. And HCN x S combined with DOX does not change the pharmacological effect of DOX. After MCF-7 and MDA-MB-231 cells are incubated with DOX@HCN x S-HA for 24 h, as the concentration increases, the cell survival rate decreases, showing concentration-dependence, and the cell viability decreases significantly. However, when HCN x S and DOX@HCN xWhen incubated under 808 nm laser irradiation, the cell viability of S-HA decreased significantly. Especially for DOX@HCN x The survival rate of MCF-7 cells in the S-HA + 808 nm laser group (13.75%) was much lower than that of HCN x The S + 808 nm laser group (67.52%), indicating DOX@HCN x The cytotoxic effect of S-HA + 808 nm laser on MCF-7 cells was much higher, attributed to the active targeting effect mediated by HA modification through the CD44 receptor, which significantly improved the uptake efficiency of nanodrugs in breast cancer cells and reduced the non-specific toxicity to normal tissues; NIR-driven efficient ROS generation, breaking through the tissue penetration depth limitation of traditional photodynamic therapy and achieving deep tumor killing. HCN x The "proton sponge effect" of S and the ROS-mediated lysosomal membrane damage synergistically reduced the retention of DOX in lysosomes, promoted the rapid release of DOX into the cytoplasm, and then targeted to the nucleus, thereby enhancing the chemo-photodynamic combined treatment effect.

[0161] 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 them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that they can still modify or equivalently replace the technical solutions of the present invention, and these modifications or equivalent replacements cannot make the modified technical solutions deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A preparation method of a nano-probe based on hollow mesoporous carbon nitride, characterized in that, It includes the following steps: S1. Synthesize hollow mesoporous carbon nitride (HCNS) by the hard template method; S2, Hydrothermal reaction of HCNS and KSCN to synthesize modified hollow mesoporous carbon nitride HCN x S; S3. Disperse HCN x S in the DOX / PBS solution, and allow DOX to be fully loaded onto HCN x S to obtain DOX@HCN x S. Use activated HA to block the pores of HCN x S to obtain DOX@HCN x S-HA.

2. The preparation method of a nano-probe based on hollow mesoporous carbon nitride according to claim 1, wherein, The steps of synthesizing HCNS by the hard template method in S1 include: S11. Using tetraethyl orthosilicate (TEOS) as a precursor, synthesize SiO2 microspheres by using the method; S12. Add TEOS and a pore-forming agent to the SiO2 microspheres synthesized in S11, and calcine at 550 °C to generate mesoporous SiO2; S13. Load cyanamide (CY) into the pores of the mesoporous SiO2 shell to obtain a CY / SiO2 hybrid, and the CY / SiO2 hybrid is transformed into a g-CN / SiO2 nanocomposite through the thermal-induced self-condensation of CY at 550 °C under a N2 atmosphere; S14. Add 4M NH4HF2 to the g-CN / SiO2 nanocomposite obtained in S13, mix and stir for 12 h to remove the SiO2 microspheres to obtain HCNS.

3. The preparation method of a nano-probe based on hollow mesoporous carbon nitride according to claim 1, wherein, In S2, hydrothermal reaction is used to synthesize HCN x The steps of S include: Stir 1 mg / mL HCNS and 0.2 mM KSCN at room temperature for 2 h to obtain a mixed solution; place the Teflon liner containing the mixed solution in a reaction kettle, and put the reaction kettle into an oven at 160 °C for a hydrothermal reaction for 2 - 5 h; after the reaction is completed, cool naturally and wash 3 - 5 times with water.

4. The preparation method of a nano-probe based on hollow mesoporous carbon nitride according to claim 3, characterized in that: Put the reaction kettle into an oven at 160 °C for a hydrothermal reaction for 3 h.

5. The preparation method of a nano-probe based on hollow mesoporous carbon nitride according to claim 1, characterized in that, In S3, DOX@HCN x The preparation of S includes: Disperse HCN x S in 1 mg / mL DOX / PBS solution and stir for 36 h under dark conditions to load DOX. After reaching adsorption equilibrium, collect DOX@HCN x S by centrifugation.

6. The preparation method of a nano-probe based on hollow mesoporous carbon nitride according to claim 5, characterized in that: HCN x S: The mass-volume ratio of the DOX / PBS solution is 2 mg: 1 mL.

7. The preparation method of a nano-probe based on hollow mesoporous carbon nitride according to claim 1, characterized in that, The activation of HA includes: adding EDC and NHS to a 3 mg / mL HA / PBS solution respectively, and stirring for 1 h under dark conditions to activate the carboxyl group of HA.

8. The preparation method of a nano-probe based on hollow mesoporous carbon nitride according to claim 1, characterized in that, In S3, DOX@HCN x S reacts with activated HA for 12 h to block HCN x channels of S.

9. The preparation method of a nano-probe based on hollow mesoporous carbon nitride according to claim 7, characterized in that, The volume molar ratio of the 3 mg / mL HA / PBS solution: EDC: NHS is 10 mL: 0.75 mol: 0.75 mol.

10. Use of a nanosensor based on hollow mesoporous carbon nitride as described in any one of claims 1 - 9 in the preparation of an anti-breast cancer drug.

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