A boronene-based nanotherapeutic platform, its construction method, and its applications.

Abstract

This invention discloses a borene-based nanotherapeutic platform, its construction method, and its applications, belonging to the field of cancer diagnosis and treatment technology. The construction method of the borene-based nanotherapeutic platform includes the following steps: S1, preparing BNSs; S2, preparing BNSs-Au; S3, preparing BNSs-Au-Ag2S-HA. The borene-based multifunctional nanotherapeutic platform provided by this invention exhibits excellent sonodynamic effects; it can effectively improve the hypoxic and highly reducing tumor microenvironment, while possessing CAT-like and GOX-like activities, as well as the effect of laser irradiation heating. This promises to achieve combined SDT-CDT-PTT antitumor therapy, obtaining good therapeutic effects on deep tumors; it also has fluorescence emission, enabling visualization of tumor detection, treatment, and monitoring, thus laying the foundation for optimizing cancer treatment conditions, reducing drug toxicity and side effects, and improving treatment and evaluation outcomes.

Description

Technical Field

[0001] This invention relates to the field of cancer diagnosis and treatment technology, and in particular to a boronene-based nanotherapeutic platform, its construction method, and its applications. Background Technology

[0002] Cancer is a serious disease that threatens human life and health. Currently, chemotherapy and radiotherapy remain the most commonly used methods in clinical cancer treatment. These traditional cancer treatments have many limitations, such as a lack of tumor specificity, low treatment efficiency, and severe toxic side effects. Therefore, there is an urgent need to develop a treatment method with better efficacy and fewer side effects to assist or replace traditional treatments.

[0003] Currently, a series of new technologies based on nanomaterials have emerged for cancer treatment, including chemodynamic therapy, gene therapy, immunotherapy, and photodynamic therapy. Among them, photodynamic therapy (PDT) utilizes the energy of light, aided by photosensitizers, to transfer this energy to surrounding oxygen molecules, generating cytotoxic reactive oxygen species (ROS), thereby killing tumor cells and achieving anti-tumor effects. Compared to traditional cancer therapies such as radiotherapy and chemotherapy, PDT has advantages such as being less invasive, highly controllable, and having fewer side effects. However, the poor penetration of light into biological tissues limits the actual effectiveness of PDT in treating deep-seated tumors.

[0004] Sonodynamic therapy (SDT), developed from photodynamic therapy (PDT), is a promising non-invasive treatment method. Ultrasound has stronger tissue penetration than lasers, capable of penetrating soft tissues up to tens of centimeters deep. Therefore, SDT exhibits unparalleled advantages in treating deep tumors. Ultrasound-driven SDT not only has strong penetrating power, enabling it to reach lesions and sites non-invasively in a short time, but also uses low-intensity ultrasound waves, ensuring that prolonged treatment does not damage surrounding normal tissues and providing good safety for non-lesion sites. Thus, it demonstrates enormous potential in the treatment of deep tumors.

[0005] In recent years, two-dimensional nanomaterials have attracted significant attention from researchers in related fields as photosensitizers (PTAs) and sonosensitizers. Two-dimensional nanomaterials possess ultrathin planar structures and, due to their unique physicochemical properties, including significant photothermal conversion performance, large specific surface area, ease of functionalization, and high biocompatibility, show great potential in biomedical applications. In 2004, Novoselov discovered two-dimensional graphene from graphite, opening a new chapter in the application of two-dimensional nanomaterials. Recently, two-dimensional nanomaterials used in biomedicine mainly include graphene oxide (GO) and its derivatives, black phosphorus (BP), two-dimensional borenes (BNSs), and transition metal-based chalcogenides (TMDs). Among these, BNSs are emerging two-dimensional nanomaterials with enormous potential for biomedical applications. Due to their high chemical stability, thermoelectric properties, superconductivity, and excellent biocompatibility, some scholars have already explored the applications of BNSs in nanomedicine.

[0006] However, single inorganic somatosensitizers often have poor therapeutic effects. It has been proven that by conjugating SDT with other therapeutic modalities, nanomedicines with combined therapeutic functions can be constructed, which can effectively improve their tumor-killing effect. Therefore, developing a composite nanomaterial with high SDT efficacy that can be combined with multiple therapeutic modalities has important practical significance.

[0007] While achieving good therapeutic effects, another worthy research question is how to visualize the treatment and obtain a high-performance integrated diagnostic and therapeutic platform. Currently, CT imaging, magnetic resonance imaging, and fluorescence imaging are commonly used imaging techniques. Fluorescence imaging, as a non-invasive visualization detection technique capable of in vivo, cellular, and molecular-level imaging research, can acquire images with high signal-to-noise ratio and spatiotemporal resolution, which is of great significance for disease diagnosis and intraoperative navigation. However, fluorescence imaging faces key challenges such as tissue autofluorescence, photobleaching, fluorescence quenching, and low tissue penetration depth.

[0008] Penetration depth primarily depends on tissue absorption of light, but the tissue's own autofluorescence and scattered photons generate interference noise and background radiation, limiting the practical application of fluorescence imaging. For example, active components in the visible light band (400-700 nm) have higher light absorption and scattering, reducing visible light penetration. Biomolecules that produce non-specific fluorescence signals under visible light excitation can also interfere with the fluorescence signal. In contrast, near-infrared (NIR, 700-1700 nm) fluorescence imaging offers significant advantages in biological tissues, particularly with a marked reduction in photon scattering and autofluorescence. This allows for more effective penetration into biological tissues such as skin, resulting in deeper tissue penetration and better avoiding background interference from tissue autofluorescence and photon scattering. Therefore, near-infrared II (NIR-II) fluorescence imaging holds great potential for tumor monitoring and visualized treatment. Summary of the Invention

[0009] The purpose of this invention is to provide a boronene-based nanodiagnostic platform, its construction method, and its application, so as to provide a nanodiagnostic platform that combines multiple treatment methods, is low-invasive, highly controllable, has few side effects, provides visualization of detection and treatment, and has good therapeutic effects on tumors, especially deep tumors.

[0010] To achieve the above objectives, this invention provides a borene-based nanotherapeutic platform, its construction method, and its applications. A method for establishing a borene-based multifunctional nanotherapeutic platform, wherein the multifunctional nanotherapeutic platform is BNSs-Au-Ag2S-HA, is described below:

[0011] S1, Preparation of B NSs

[0012] Boron powder was dispersed in N-methylpyrrolidone and mixed thoroughly. After treatment with a cell disruptor, the mixture was centrifuged. The supernatant was collected and centrifuged again. The mixture was then washed twice with ultrapure water. The resulting product was placed in a refrigerator for pre-freezing for 3 hours and then freeze-dried.

[0013] S2, Preparation of B NSs-Au

[0014] The B NSs prepared in step S1 were dispersed in ultrapure water, then chloroauric acid solution was added, and the mixture was stirred at room temperature for 2 hours. Sodium borohydride solution was then added, and the mixture was stirred for another 18 hours. The supernatant was removed by centrifugation, and the resulting product was placed in a refrigerator for pre-freezing for 3 hours and then freeze-dried.

[0015] S3, Preparation of BNSs-Au-Ag2S-HA

[0016] The B NSs-Au obtained in step S2 was dispersed in ethylene glycol and stirred evenly. Then, under an inert gas atmosphere, the temperature was raised to 90°C and vacuumed. The temperature was then raised to 110°C, and mercaptopropionic acid and silver nitrate were added in sequence. The temperature was raised to 145°C and held for 1 hour before heating was stopped. The temperature was allowed to cool naturally to room temperature. The supernatant was removed by centrifugation. The precipitate was washed twice with ultrapure water by centrifugation and then pre-frozen in a refrigerator for 3 hours. Finally, it was freeze-dried to obtain B NSs-Au-Ag2S.

[0017] Disperse B NSs-Au-Ag2S in ultrapure water. Separately disperse HA in ultrapure water. Add the B NSs-Au-Ag2S dispersion dropwise to the HA solution. Stir for 12 hours in the dark. Centrifuge the obtained product to remove the supernatant. Then wash twice with ultrapure water. Place the product in a refrigerator for pre-freezing for 3 hours and freeze-dry.

[0018] Preferably, in step S1, the mass-to-volume ratio of boron powder to N-methylpyrrolidone is 1g:200mL; the parameters of the cell disruptor are set as follows: power of 1200W, ultrasonic duration of 2s, interval of 2s, and continuous ultrasonic treatment for 6h; after treatment with the cell disruptor, the centrifugation conditions are 5000r / min for 10min, and the centrifugation conditions for the supernatant and after washing with ultrapure water are 11000r / min for 30min.

[0019] Preferably, the pre-freezing temperature in steps S1-S3 is -20℃.

[0020] Preferably, in step S2, the mass-to-volume ratio of B NSs to ultrapure water is 1 mg:10 mL, the mass fraction of solute in the chloroauric acid solution is 1%, the mass fraction of solute in the sodium borohydride solution is 0.38%, and the mass-to-volume ratio of B NSs, chloroauric acid solution, and sodium borohydride solution is 0.5 mg:0.5 mL:0.3 mL; the centrifugation conditions are 11000 r / min for 25 min.

[0021] Preferably, in step S3, the mass-to-volume ratio of BNSs-Au:ethylene glycol:mercaptopropionic acid is 1 mg:2 mL:2 μL; the mass ratio of BNSs-Au:silver nitrate is 1:1.688; and the vacuuming time is 20 min.

[0022] Preferably, in step S3, the centrifugation conditions for centrifugation after cooling to room temperature, centrifugation and washing with ultrapure water, and centrifugation after stirring in the dark are all 11000 r / min and 30 min.

[0023] Preferably, in step S3, the mass-to-volume ratio of BNSs-Au-Ag2S to ultrapure water is 1 mg: 5 mL; and the mass-to-volume ratio of HA to ultrapure water is 1 mg: 1 mL.

[0024] A multifunctional nanotherapeutic platform based on borophene, established by the method described above.

[0025] Application of a borene-based multifunctional nanodiagnostic and therapeutic platform established by the method described above in tumor detection, treatment, and monitoring.

[0026] The tumor microenvironment is characterized by relative hypoxia and high levels of hydrogen peroxide (H2O2), which to some extent hinders the therapeutic effects of conventional methods. Nanozymes are nanomaterials with intrinsic enzyme-like activity, possessing inherent enzyme-like catalytic activity, lower cost, simpler preparation, higher handling capability, and greater versatility.

[0027] With the rapid development of nanotechnology, most nanoparticles, such as magnetic nanoparticles, cerium oxide nanoparticles, and gold nanoparticles, have shown their inherent nanozyme catalytic activity. Among them, gold nanoparticles have glucose oxidase-like (GOx-like) and catalase-like (CAT-like) activities, which can consume glucose in tumor cells to produce H2O2, thus achieving the effect of starvation therapy. They can also combine with CAT-like to decompose H2O2 to produce O2, thereby relieving hypoxia in the tumor site and improving the anti-tumor effect.

[0028] This invention provides a multifunctional nanotherapeutic platform based on borone (BNSs). Firstly, it utilizes the excellent photothermal properties of BNSs to provide photothermal therapeutic effects. Secondly, it anchors [the material] on BNSs...

[0029] AuNPs (AuNPs) are used to construct Schottky junctions, suppressing electron-hole recombination and enhancing [the structure / function]. 1 O2 generation efficiency is improved, enhancing the effectiveness of SDT (Surgical Treatment Therapy). Simultaneously, utilizing the GOx-like activity of AuNPs, glucose within tumor cells is consumed, acting as a "starvation therapy" and generating endogenous H2O2. The AuNPs then utilize their CAT-like activity to convert H2O2 into O2, providing raw materials for ROS (Reactive Oxygen Spectroscopy) generation, thereby enhancing the tumor-killing effect. Furthermore, Ag2S quantum dots bound to BNSs-Au emit NIR-II fluorescence for visualized treatment. Hyaluronic acid (HA) is used to modify the nanomedicine, actively targeting the highly expressed CD44 receptor in tumor cells, increasing the accumulation rate of the nanomedicine at the tumor site, and laying the foundation for improving biomedical diagnostic capabilities and evaluating treatment efficacy.

[0030] Therefore, the present invention provides a boronene-based nanotherapeutic platform, its construction method, and its application, the specific technical effects of which are as follows:

[0031] (1) The preparation method provided by the present invention can successfully anchor Au NPs onto B NSs, thereby significantly improving the acoustic dynamic effect of B NSs and enhancing tissue penetration, thus showing great potential for the treatment of deep tumors.

[0032] (2) The multifunctional nanotherapeutic platform provided by this invention can inhibit electron-hole recombination and enhance [the following text is incomplete and likely refers to a different topic] by constructing Schottky junctions. 1 O2 production efficiency, and under its own CAT-like and GOx-like activity, it can also generate a large amount of reactive oxygen species, thereby effectively alleviating the hypoxia and strong reducing properties in the tumor microenvironment, thus significantly improving the efficiency of SDT treatment.

[0033] (3) The multifunctional nano-therapeutic platform provided by the present invention has GOx-like activity that consumes glucose in tumor cells, thus having the effect of "starvation therapy";

[0034] (4) The present invention utilizes the excellent photothermal properties of B NSs to prepare a multifunctional nano-therapeutic platform with excellent heating capacity. It can be heated to 50°C in 600s by 1064nm laser irradiation, thereby producing excellent photothermal therapeutic effect on tumors.

[0035] (5) The multifunctional nanomedicine platform provided by the present invention can actively target the CD44 receptor highly expressed in tumor cells through modified hyaluronic acid, thereby increasing the enrichment rate of nanomedicines at the tumor site and significantly improving the killing ability of tumors.

[0036] (6) The multifunctional nano-diagnostic platform provided by the present invention has the characteristic of emitting NIR-II fluorescence through the combined Ag2S quantum, which can realize the visualization of tumor detection, treatment and monitoring, thereby laying the foundation for improving the biomedical diagnostic and treatment capabilities and evaluating the treatment effect;

[0037] (7) The method for constructing the multifunctional nano-diagnosis and treatment platform provided by the present invention is simple, low-cost, safe and effective.

[0038] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0039] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments of the present invention will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0040] Figure 1This is a schematic diagram illustrating the working principle of BAA-HA prepared in Example 1;

[0041] Figure 2 This is a schematic diagram of the process for preparing BAA-HA in Example 1;

[0042] Figure 3 These are SEM images of B NSs prepared in Example 1;

[0043] Figure 4 These are AFM images of B NSs prepared in Example 1;

[0044] Figure 5 The XRD pattern of B NSs prepared in Example 1;

[0045] Figure 6 This is the DLS spectrum of B NSs prepared in Example 1;

[0046] Figure 7 These are SEM images of BA prepared in Example 1;

[0047] Figure 8 These are EDS images of BA prepared in Example 1;

[0048] Figure 9 This is a SEM image of BAA-HA prepared in Example 1;

[0049] Figure 10 This is the DLS spectrum of BAA-HA prepared in Example 1;

[0050] Figure 11 The XRD pattern of BAA-HA prepared in Example 1;

[0051] Figure 12 The FTIR spectra of HA and BAA-HA prepared in Example 1 are as follows:

[0052] Figure 13 The image is an EDS image of the BAA-HA prepared in Example 1;

[0053] Figure 14 These are ESR detection results, where part a represents the results of B NSs-loaded Au nanoparticles before and after ultrasonic stimulation. 1 A comparison chart of O2 production capacity, with part b showing BAA-HA under different stimulus conditions. 1 A comparison chart of O2 generation capabilities;

[0054] Figure 15 The materials under ultrasonic stimulation within 0-5 minutes 1 A comparison of O2 generation capabilities, where part a represents B NSs. 1O2 production capacity results, part b is BA 1 O2 production capacity results, part c is BAA-HA 1 The results of O2 generation capacity show that part d represents the decreasing trend of absorbance of the three materials at 415 nm.

[0055] Figure 16 The CAT-like activity of BAA-HA was determined using the titanium sulfate colorimetric method. Part a shows the CAT-like activity of BAA-HA under different stimuli at pH 5.4, part c shows the CAT-like activity of BAA-HA under different stimuli at pH 7.4, part d shows the activity at pH 8.5, part e shows the color change of the reaction system at different time points within 0-180s when the reaction was terminated (pH 5.4), and part f shows the comparison of gas production after 60s of reaction in different reaction systems.

[0056] Figure 17 The GOx-like activity of BAA-HA was determined using the titanium sulfate colorimetric method. Part a compares the enzyme activities of B NSs, BA, BAA-HA and glucose substrate after co-incubation for 3 hours, while part b compares the enzyme activities of different concentrations of BAA-HA after co-incubation for 3 hours.

[0057] Figure 18 This is a graph showing the temperature rise of different concentrations of BAA-HA over 600 seconds under 1064nm laser irradiation.

[0058] Figure 19 The results are NIR-II fluorescence performance tests of BAA-HA. Part a shows the fluorescence spectra of different concentrations of BAA-HA under 808nm laser excitation, and part b shows the actual imaging effects of different concentrations of BAA-HA tested by the NIR-II small animal in vivo imaging system.

[0059] Figure 20 These are images of material uptake by GL261 cells, where part a shows the uptake effect of GL261 cells on BAA-FITC, and part b shows the uptake effect of GL261 cells on BAA-HA-FITC.

[0060] Figure 21 The killing ability of B NSs, BA, and BAA-HA against GL261 cells was determined by the MTT assay.

[0061] Figure 22This is a graph showing the results of various indicators verifying the ICD effect in tumor cells. Part a is the immunofluorescence image of CRT, part b is the immunofluorescence intensity statistics of CRT, part c is the immunofluorescence image of HSP90, part d is the immunofluorescence intensity statistics of HSP90, part e is the immunofluorescence image of HMGB1, part f is the immunofluorescence intensity statistics of HMGB1, part g is the trend of intracellular ATP content, and part h is the trend of cellular ATP release. Detailed Implementation

[0062] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0063] To make the objectives, technical solutions, and advantages of this application clearer, more thorough, and more complete, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings and embodiments. The following detailed descriptions are all illustrations of embodiments, intended to provide further detailed explanation of the present invention. Unless otherwise specified, all technical terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0064] The instruments, equipment, materials, and reagents used in the examples were all obtained commercially.

[0065] Example 1

[0066] A multifunctional nano-therapeutic platform based on borophene was established, as follows:

[0067] S1, Preparation of B NSs

[0068] 1g of boron powder was dispersed in 200mL of N-methylpyrrolidone and thoroughly mixed. The mixture was then subjected to a cell disruptor with the following parameters: power 1200W, sonication duration 2s, interval 2s, and continuous sonication for 6h. The resulting product was centrifuged at 5000r / min for 10min, and the supernatant was collected. The supernatant was then centrifuged at 11000r / min for 30min, and the supernatant was discarded. Ultrapure water was added to the product, and the mixture was centrifuged at 11000r / min for 30min. This process of removing the supernatant, adding ultrapure water, and centrifuging was repeated twice. The resulting product was pre-frozen at -20℃ for 3h, and then freeze-dried for 12h to obtain B NSs.

[0069] The obtained B NSs were observed using a scanning electron microscope (SEM), and the results are shown in the figure. Figure 3 .Depend on Figure 3 It can be seen that the obtained B NSs are in the form of nanosheets. The results were observed using atomic force microscopy (AFM). Figure 4 ,Depend on Figure 4It can be seen that the prepared B NSs have a thickness of about 5-7 nm, exhibiting the characteristics of ultrathin two-dimensional nanosheets.

[0070] The crystal structure of B NSs was analyzed using X-ray diffraction (XRD), and the results are shown in [Figure 1]. Figure 5 ,Depend on Figure 5 As can be seen, the XRD pattern of the prepared BNSs corresponds to that of the standard card (JCPDF:031-0207), indicating that the BNSs were successfully prepared. Analysis was performed using a nanoparticle size potentiometer (DLS), and the results are shown below. Figure 6 ,Depend on Figure 6 It can be seen that the particle size of B NSs dispersed in PBS is approximately 106 nm.

[0071] S2, Preparation of B NSs-Au

[0072] 5 mg of B NSs was dispersed in 50 mL of water. 500 μL of chloroauric acid solution (1% by mass) was added to the solution, and the mixture was stirred at room temperature for 2 h. Then, 300 μL of sodium borohydride solution (0.38% by mass) was added to the system, and the mixture was stirred at room temperature for another 18 h. The dispersion was then centrifuged at 11000 r / min for 25 min, the supernatant was removed, and the resulting product was pre-frozen at -20℃ for 3 h and then freeze-dried for 12 h to obtain B NSs-Au(BA).

[0073] The obtained BA was observed using SEM, and the results are shown below. Figure 7 ,Depend on Figure 7 It can be seen that the gold nanoparticles (Au NPs) are distributed in a dotted pattern on the surface of the nanosheets. The prepared BA was subjected to elemental analysis using energy-dispersive X-ray spectroscopy (EDS), and the results are shown below. Figure 8 ,Depend on Figure 8 It can be seen that Au NPs are successfully anchored on B NSs.

[0074] S3, Preparation of BNSs-Au-Ag2S-HA

[0075] 10 mg of B NSs-Au was dispersed in 20 mL of ethylene glycol and stirred for 20 min to ensure uniform dispersion. Under an Ar atmosphere, the mixture was gradually heated to 90 °C and evacuated for 20 min to remove water and gas. The temperature was then increased to 110 °C, and 20 μL of mercaptopropionic acid was added, followed by 16.88 mg of silver nitrate. The temperature was raised to 145 °C and maintained for 1 h. Heating was then stopped, and the mixture was allowed to cool naturally to room temperature. The reactants were centrifuged at 11000 rpm for 30 min, and the supernatant was removed. The product was then added to ultrapure water and centrifuged at 11000 rpm for 30 min. This process of removing the supernatant, adding ultrapure water, and centrifuging was repeated twice. The product was then pre-frozen at -20 °C for 3 h and then freeze-dried for 12 h to obtain B NSs-Au-Ag2S (BAA).

[0076] 10 mg of BAA was weighed and dispersed in 50 mL of ultrapure water to a concentration of 200 μg / mL. 50 mg of HA was also weighed and dispersed in 50 mL of ultrapure water. The BAA dispersion was added dropwise to the HA solution, and the mixture was stirred in the dark for 12 h. The resulting product was centrifuged at 11000 rpm for 30 min, and the supernatant was removed. 20 mL of ultrapure water was added to the product, and the mixture was centrifuged at 11000 rpm for 30 min. This process of removing the supernatant, adding ultrapure water, and centrifuging was repeated twice to remove excess HA. The product was pre-frozen at -20°C for 3 h, and then freeze-dried for 12 h to obtain BNSs-Au-Ag2S-HA (BAA-HA). The preparation process is described in [link to preparation procedure]. Figure 2 .

[0077] The prepared BAA-HA was observed using SEM, and the results are shown in the figure. Figure 9 ,Depend on Figure 9 It can be seen that Ag₂S is distributed on the surface of the nanosheets. The prepared BAA-HA was tested using nanoparticle size analysis and a zeta potential analyzer (DLS), and the results are shown in the figure. Figure 10 ,Depend on Figure 10 It can be seen that the particle size of BAA-HA dispersed in PBS is approximately 135 nm. XRD analysis of the prepared BAA-HA is shown in the figure below. Figure 11 ,Depend on Figure 11 It can be seen that its characteristic peaks match the characteristic peaks shown on the standard cards of each component, proving that BAA-HA was successfully synthesized.

[0078] The prepared BAA-HA was analyzed using Fourier transform infrared spectroscopy (FTIR), and the results are shown in the figure. Figure 12 ,Depend on Figure 12 The characteristic peaks of HA are clearly visible, indicating that HA modification on the material surface was successful. Elemental analysis of the prepared BAA-HA was performed using EDS, and the results are shown below. Figure 13 , Figure 13 This indicates that Ag2S and B NSs-Au have successfully recombinated.

[0079] Example of effect 1

[0080] Electron spin resonance (ESR) was used to test whether anchoring AuNPs with B NSs in Example 1 would improve the acoustic-dynamic effect of B NSs. The test results are shown in […]. Figure 14 .

[0081] Depend on Figure 14 As can be seen from part a, B NSs exhibited a peak intensity ratio of 1:1:1 after ultrasonic stimulation. 1 The characteristic peak of O2 showed a significant increase in intensity after ultrasonic stimulation was applied to BA, indicating that anchoring AuNPs has an enhancing effect on acoustic dynamics.

[0082] like Figure 14 As shown in part b, BAA-HA appears after the application of ultrasound stimulation. 1 The characteristic peaks of O2 indicate that BAA-HA possesses acoustic and dynamic properties, and this is further demonstrated by the addition of exogenous H2O2. 1 The enhanced characteristic peak of O2 is mainly due to the CAT-like activity of BAA-HA itself catalyzing the decomposition of H2O2 into O2, providing raw materials for the acoustic dynamic process and thus enhancing the characteristic peak. 1 The production of O2 enhances its ability to kill cancer cells.

[0083] Example of effect 2

[0084] 1,3-Diphenylisobenzofuran (DPBF) was used as... 1 O2 indicator, investigating the effects of BNSs, BA, and BAA-HA on ultrasound (1MHz, 1W / cm²). 2 (5 min, 50% duty cycle) excitation under these conditions 1 The effect of O2 yield, results are shown in Figure 15 DPBF has an absorption peak at 415 nm, which produces 1 O2 can degrade DPBF, resulting in a decrease in its absorbance at 415 nm. Figure 15 As can be seen from the middle d part, the absorption peak of DPBF at 415 nm gradually decreases with the increase of US application time, and the BA group ( Figure 15 The rate of decline in part b) was stronger than that in the BNSs group ( Figure 15 Part a), BAA-HA group ( Figure 15 The c-group showed no significant change compared to the BA group, further demonstrating that anchoring Au NPs can significantly improve the reactive oxygen species production efficiency, and that the modification of Ag2S and HA did not show a significant effect on the reactive oxygen species yield.

[0085] Example of effect 3

[0086] The CAT-like activity of BAA-HA was determined using a titanium sulfate colorimetric method. The method was as follows: A 200 μg / mL BAA-HA dispersion was prepared using 0.1 M acetate-sodium acetate buffer solutions at different pH values. H₂O₂ solution was added to maintain a final concentration of 5 mM. Under different stimuli, the reaction was allowed to proceed for different time periods, after which a 5% titanium sulfate solution was added to terminate the reaction and develop the color. The absorbance at 410 nm was measured. The results are shown below. Figure 16 As shown in the ad section, it can be seen from the figure that the CAT-like activity of the material decreases as the pH increases.

[0087] At different reaction times within 0-180 s, titanium sulfate solution (5% by mass) was added to terminate the reaction, with a final H2O2 concentration of 5 mM. The results are as follows: Figure 16 As shown in part e, when pH = 5.4, the color of the color reaction becomes noticeably lighter over time, indicating that the amount of H₂O₂ decomposed decreases significantly with increasing time. Figure 16 f shows that in different reaction systems (water, 50 μg / ml B NSs aqueous solution, 50 μg / ml BAA-HA aqueous solution), the addition of a 50 mM H2O2 solution resulted in a large number of bubbles being generated and adhering to the centrifuge tube wall after 60 seconds. The BAA-HA group showed the most significant bubble generation, indicating its strongest CAT-like effect. Therefore, in the acidic microenvironment of tumors, BAA-HA has a stronger ability to decompose H2O2, providing better raw materials for sonodynamic therapy and enhancing its therapeutic effect.

[0088] Example of effect 4

[0089] GOx-like activity can catalyze the conversion of glucose into H2O2 within tumor cells, thereby cutting off the energy supply to cancer cells. The experiment investigated whether the BAA-HA prepared in Example 1 possessed GOx-like catalytic activity. The method was as follows:

[0090] 50 mg of glucose was added to a 200 μg / ml BAA-HA aqueous solution, and oxygen was introduced. After incubation at 37°C for 3 hours, the supernatant was collected by centrifugation to remove the material. Then, a prepared titanium sulfate solution (5% by mass) was added to the supernatant, and the absorbance of the supernatant with added titanium sulfate solution was measured at 410 nm. The results are as follows. Figure 17As shown in sections a and b, the BA and BAA-HA groups showed an absorption peak at 410 nm, indicating that the anchored Au NPs have GOx-like activity, which can competitively consume glucose in the body by cancer cells, playing the role of "starvation therapy". At the same time, the generated H2O2 provides raw materials for sonodynamic therapy indirectly.

[0091] Example of effect 5

[0092] The photothermal properties of BAA-HA prepared in Example 1 were investigated using the following method:

[0093] The temperature rise curves of the material at different concentrations were obtained by irradiating an aqueous solution of BAA-HA with a 1064nm laser, as shown below. Figure 18 As shown in the figure, when the concentration of BAA-HA is 200 μg / mL, after irradiation with a 1064 nm laser for 600 s, its temperature can rise to above 50℃, which can kill tumor cells.

[0094] Example of effect 6

[0095] The NIR-II fluorescence properties of BAA-HA prepared in Example 1 were investigated using the following method:

[0096] First, the fluorescence spectra of BAA-HA at different concentrations (0-200 μg / mL) were measured using a fluorescence spectrometer. The test results are as follows: Figure 19 As shown in section a, BAA-HA at a concentration of 200 μg / mL exhibits a strong fluorescence peak at 1106 nm under 808 nm laser excitation. Therefore, BAA-HA prepared in Example 1 has the potential to achieve deeper imaging through NIR-II fluorescence.

[0097] The NIR-II fluorescence imaging effect of BAA-HA prepared in Example 1 was verified by observation using a small animal in vivo imaging system: Real-time imaging of different concentrations (0-200 μg / mL) of BAA-HA was performed using a small animal in vivo imaging system, and observations were conducted using a 1000 nm cutoff filter. The results are as follows: Figure 19 As shown in part b, it can be seen from the figure that under 808nm laser excitation, the NIR-II fluorescence intensity of BAA-HA gradually increases with the increase of BAA-HA concentration, indicating that the BAA-HA prepared in Example 1 exhibits excellent NIR-II fluorescence imaging effect.

[0098] Example of effect 7

[0099] The targeting effect of BAA-HA prepared in Example 1 on tumor cells was investigated using the following method:

[0100] The targeting effect of BAA-HA prepared in Example 1 on mouse glioblastoma GL261 cells was verified by a cell uptake experiment. By binding FITC to BAA-HA as a fluorescent molecule, the uptake of BAA-FITC (non-targeted group) and BAA-HA-FITC (HA-targeted group) by mouse glioblastoma GL261 cells at 2h, 4h, and 6h was observed. The results are as follows: Figure 20 Parts a and b are shown.

[0101] As can be seen from the figure, there was almost no drug accumulation in the untargeted group, while a large amount of drug accumulated around the tumor cells in the HA-targeted group at 4 hours, indicating that HA coating can significantly improve the targeting ability of nanoparticles to cancer cells.

[0102] Example of effect 8

[0103] The killing ability of BAA-HA prepared in Example 1 against cancer cells was investigated. The method was as follows: the killing ability of BNSs, BNSs-Au, and BAA-HA against mouse glioblastoma GL261 cells was evaluated by MTT assay. After co-incubating GL261 cells with 200 μg / mL of the above materials for 4 h, different stimulation conditions were applied. The results are as follows. Figure 21 As shown.

[0104] Figure 21 The results showed that without the added material, the cell survival rate in all groups was above 90%. After the addition of BAA-HA, the cell survival rate decreased significantly, with the US+H2O2+Laser group showing a survival rate below 15%. The survival rate of the BNSs group was higher than that of the BNSs-Au group, indicating that AuNPs anchoring significantly enhanced the killing ability of tumor cells by improving SDT efficiency and coupling with starvation therapy. The mechanism of action is described in [link to relevant documentation]. Figure 1 .

[0105] Example of effect 9

[0106] Experiments verified the performance of BAA-HA in inducing ICD effects in mouse glioblastoma GL261 cells.

[0107] CRT protein is one of the most abundant proteins in the endoplasmic reticulum, and its eversion is an early indicator of ICD effects. Additionally, HMGB1 is an abundant nuclear non-histone chromatin-binding protein released from the nucleus upon cell death. The interaction between HSP90 and CD91 can enhance immune stimulation by promoting dendritic cell (DC) maturation through antigen cross-presentation. Tumor cells release ATP in all three stages of ICD: early, middle, and late. CRT exposure and the release of HMGB1 and ATP are considered surrogate markers of ICD-related immunogenicity. Therefore, measuring the levels of CRT, HMGB1, HSP90, and ATP can demonstrate the occurrence of ICD.

[0108] The results are as follows Figure 22 As shown, green fluorescence increases with the application of stimulation, and higher intensity indicates a greater degree of CRT translocation. Both ultrasound and heat stimulation induce HSP90 expression; the intensity of red fluorescence increases with the degree of ICD effect. HMGB1 is gradually released extracellularly with increasing ICD effect; the more it is released, the lower its intracellular content, and the lower its green fluorescence intensity. ATP, on the other hand, shows a trend of decreasing intracellular content and increasing extracellular content with the occurrence of the ICD effect.

[0109] The results showed that various ICD effect markers changed significantly with the application of stimulation conditions, therefore BAA-HA can induce tumor ICD effects to kill tumor cells.

[0110] Therefore, the borene-based multifunctional nano-therapeutic platform provided by this invention has excellent sonodynamic effects; it can effectively improve the hypoxic and highly reducing state of the tumor microenvironment, while also possessing CAT-like and GOx-like activities, as well as the effect of laser irradiation heating, thus potentially achieving combined SDT-CDT-PTT anti-tumor therapy and obtaining good therapeutic effects on deep tumors; it also has fluorescence emission effects, enabling visualization of tumor detection, treatment, and monitoring, thereby laying the foundation for optimizing cancer treatment conditions, reducing drug toxicity and side effects, and improving treatment and evaluation effects.

[0111] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method for establishing a multifunctional nanotherapeutic platform based on borophene, characterized in that, The multifunctional nanotherapeutic platform is B NSs-Au-Ag2S-HA, and its establishment steps are as follows: S1, Preparation of B NSs Boron powder was dispersed in N-methylpyrrolidone and mixed thoroughly. After treatment with a cell disruptor, the mixture was centrifuged. The supernatant was collected and centrifuged again. The mixture was then washed twice with ultrapure water. The resulting product was placed in a refrigerator for pre-freezing for 3 hours and then freeze-dried. S2, Preparation of B NSs-Au The B NSs prepared in step S1 were dispersed in ultrapure water, then chloroauric acid solution was added, and the mixture was stirred at room temperature for 2 hours. Sodium borohydride solution was then added, and the mixture was stirred for another 18 hours. The supernatant was removed by centrifugation, and the resulting product was placed in a refrigerator for pre-freezing for 3 hours and then freeze-dried. S3, Preparation of BNSs-Au-Ag2S-HA The B NSs-Au obtained in step S2 was dispersed in ethylene glycol and stirred evenly. Then, under an inert gas atmosphere, the temperature was raised to 90°C and vacuumed. The temperature was then raised to 110°C, and mercaptopropionic acid and silver nitrate were added in sequence. The temperature was raised to 145°C and held for 1 hour before heating was stopped. The temperature was allowed to cool naturally to room temperature. The supernatant was removed by centrifugation. The precipitate was washed twice with ultrapure water by centrifugation and then pre-frozen in a refrigerator for 3 hours. Finally, it was freeze-dried to obtain B NSs-Au-Ag2S. Disperse B NSs-Au-Ag2S in ultrapure water. Separately disperse HA in ultrapure water. Add the B NSs-Au-Ag2S dispersion dropwise to the HA solution. Stir for 12 hours in the dark. Centrifuge the obtained product to remove the supernatant. Then wash twice with ultrapure water. Place the product in a refrigerator for pre-freezing for 3 hours and freeze-dry.

2. The method for establishing a multifunctional nano-therapeutic platform based on borophene according to claim 1, characterized in that, In step S1, the mass-to-volume ratio of boron powder to N-methylpyrrolidone is 1g:200mL; the parameters of the cell disruptor are set as follows: power of 1200W, ultrasonic duration of 2s, interval of 2s, and continuous ultrasonic treatment for 6h; after treatment with the cell disruptor, the centrifugation conditions are 5000r / min for 10min, and the centrifugation conditions of the supernatant and the centrifugation conditions after washing with ultrapure water are 11000r / min for 30min.

3. The method for establishing a multifunctional nano-therapeutic platform based on borophene according to claim 1, characterized in that: The pre-freezing temperature in steps S1-S3 is -20℃.

4. The method for establishing a multifunctional nano-therapeutic platform based on borophene according to claim 1, characterized in that: In step S2, the mass-to-volume ratio of B NSs to ultrapure water is 1 mg:10 mL, the mass fraction of solute in the chloroauric acid solution is 1%, the mass fraction of solute in the sodium borohydride solution is 0.38%, and the mass-to-volume ratio of B NSs, chloroauric acid solution, and sodium borohydride solution is 0.5 mg:0.5 mL:0.3 mL; the centrifugation conditions are 11000 r / min for 25 min.

5. The method for establishing a multifunctional nano-therapeutic platform based on borophene according to claim 1, characterized in that: In step S3, the mass-to-volume ratio of BNSs-Au:ethylene glycol:mercaptopropionic acid is 1 mg:2 mL:2 μL; the mass ratio of BNSs-Au:silver nitrate is 1:1.688; and the vacuuming time is 20 min.

6. The method for establishing a multifunctional nanotherapeutic platform based on borophene according to claim 1, characterized in that: In step S3, the centrifugation conditions for centrifugation after cooling to room temperature, centrifugation with ultrapure water for washing, and centrifugation after stirring in the dark are all 11000 r / min for 30 min.

7. The method for establishing a multifunctional nanotherapeutic platform based on borophene according to claim 1, characterized in that: In step S3, the mass-to-volume ratio of BNSs-Au-Ag2S to ultrapure water is 1 mg: 5 mL; the mass-to-volume ratio of HA to ultrapure water is 1 mg: 1 mL.

8. A multifunctional nanotherapeutic platform based on borophene, established by the method of any one of claims 1-7.

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