Diagnosis and treatment integrated nano platform, preparation method and application thereof

Abstract

The invention provides a diagnosis and treatment integrated nano platform as well as a preparation method and application thereof. According to the diagnosis and treatment integrated nano platform, an organic-inorganic nano hybrid is constructed through structure optimization and function integration, four modes of PTT, PDT, SDT and CDT are organically combined innovatively, and efficient synergistic treatment of GBM is achieved through real-time monitoring of photoacoustic imaging. First, a dOMV encapsulation strategy significantly improves the ability to penetrate through the blood brain barrier. Meanwhile, the IEICO-4F has a strong near-infrared absorption characteristic and has a synergistic effect with the high catalytic activity of the mesoporous platinum nanoparticles. Under the laser / ultrasonic excitation condition, the diagnosis and treatment integrated nano platform shows excellent photo-thermal conversion performance, the Fenton reaction rate can be increased, endogenous hydrogen peroxide can be continuously catalyzed to be decomposed to generate oxygen, and the tumor hypoxia state is effectively relieved. The series of reactions further enhance the generation of ROS, activate a Caspase-1 / GSDMD mediated pyroptosis pathway, finally achieve an efficient tumor inhibition effect, and successfully break through the limitation of a single treatment mode.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to a diagnosis and treatment integrated nano platform and application thereof. BACKGROUND

[0002] Glioblastoma (GBM) is one of the most aggressive and most difficult to treat malignant tumors, with a very poor prognosis, a median survival of only 14 months, and a 5-year survival rate of less than 5%. Due to the inherent characteristics of GBM: high invasiveness, immunosuppression, genetic heterogeneity, and strong drug resistance leading to rapid recurrence, the current standard therapy (surgery, radiotherapy and chemotherapy) cannot significantly prolong the survival of patients. In the latest research progress of GBM treatment, the potential of new therapies such as photodynamic therapy (PDT), sonodynamic therapy (SDT), photothermal therapy (PTT) and chemical dynamic therapy (CDT) is highlighted. However, these existing technologies face significant challenges. The application of photodynamic therapy is restricted by factors such as tumor hypoxic environment and shallow light penetration; although sonodynamic therapy overcomes the limitation of penetration depth, it has the problem of low reactive oxygen species (ROS) production; the heat treatment effect of photothermal therapy is hindered by problems such as poor tissue penetration and off-target damage; and the Fenton reaction of chemical dynamics therapy is not only slow, but also limited by space conditions. Recently, related research has focused on the exploration of simple multi-mode combined treatment strategies, such as the combined application of PDT and SDT. However, the presence of the blood-brain barrier significantly affects the delivery efficiency of nanomedicines; at the same time, the severe hypoxic microenvironment inside the tumor constitutes a serious limitation to the efficacy of PDT and SDT combined therapy.

[0003] In view of the many defects exposed by the prior art in treating GBM, it is urgent to develop a new method or new technology that can effectively overcome the barrier of blood-brain barrier, improve the hypoxic microenvironment inside the tumor, and integrate the advantages of multiple treatment modes to achieve efficient and safe treatment of GBM, so as to improve the treatment effect of GBM, prolong the survival of patients and improve their quality of life. SUMMARY

[0004] In view of this, the purpose of the present application is to provide a diagnosis and treatment integrated nano platform, a preparation method, and the application of the diagnosis and treatment integrated nano platform in preparing a drug for treating glioblastoma. The diagnosis and treatment integrated nano platform provided by the present application is an organic-inorganic nano hybrid based on structure optimization and function integration, which innovatively combines four modes of PTT, PDT, SDT and CDT, realizes efficient synergistic treatment of GBM through real-time monitoring by photoacoustic imaging (PAI).

[0005] Specifically, the application provides a diagnosis and treatment integrated nano platform, which comprises a nanoparticle composition containing IEICO-4F (CAS: 2089044-02-8) and mesoporous platinum nanoparticles, and an E. coli DH5α lipopolysaccharide-free bacterial outer membrane vesicle coated on the surface of the mesoporous platinum nanoparticles.

[0006] In some specific embodiments of the application, the diagnosis and treatment integrated nano platform comprises the following preparation steps: loading IEICO-4F into mesoporous platinum nanoparticles by physical or chemical methods to obtain Pt-IEICO-4F nanoparticles; combining E. coli DH5α lipopolysaccharide-free bacterial outer membrane vesicles with the Pt-IEICO-4F nanoparticles to form composite nanoparticles Pt-IEICO-4F@dOMV; and separating the composite nanoparticles Pt-IEICO-4F@dOMV, i.e. the diagnosis and treatment integrated nano platform.

[0007] In some specific embodiments of the application, the preparation step of the mesoporous platinum nanoparticles comprises: mixing a non-ionic block copolymer template agent, an electrolyte additive and a reducing organic acid in an aqueous phase, and forming a homogeneous precursor solution by ultrasonic treatment; adding a platinum precursor to the precursor solution, mixing uniformly, and then reacting at 50-90°C for 12-48 hours to cause in-situ reduction of platinum ions in the template self-assembly structure; and after the reaction is completed, performing solid-liquid separation and washing to obtain the mesoporous platinum nanoparticles.

[0008] In some specific embodiments of the application, the non-ionic block copolymer template agent is block polyether F-127 (CAS: 9003-11-6); the electrolyte additive is KBr; the reducing organic acid is ascorbic acid; and the platinum precursor is chloroplatinic acid.

[0009] In some specific embodiments of the application, the mesoporous platinum nanoparticles are prepared as follows: block polyether F-127 and KBr are dissolved in water to form a first solution; the first solution is mixed with an ascorbic acid solution under ultrasonic conditions to obtain a second solution; chloroplatinic acid is added to the second solution and mixed uniformly to obtain a third solution; the third solution is reacted at 50-90°C for 12-48 hours; and after the reaction is completed, solid-liquid separation and washing are performed to obtain the mesoporous platinum nanoparticles.

[0010] In some specific embodiments of the application, the mass ratio of the block polyether F-127, KBr, ascorbic acid and chloroplatinic acid is (13-16):(29-36):(7-10):1.

[0011] In some specific embodiments of the application, the solid-liquid separation and washing are centrifugal separation and water washing, respectively.

[0012] In some embodiments of the present application, the physical or chemical method is nanoscale co-precipitation.

[0013] In some embodiments of the present application, the preparation process of the Pt-IEICO-4F nanoparticles comprises: dissolving DSPE-PEG2000-COOH in water, mixing it with mesoporous platinum nanoparticles under ultrasonic; then, dissolving IEICO-4F in a tetrahydrofuran solution, stirring the above-mentioned mixed solution at room temperature for 8-24 h, after the reaction is completed, performing solid-liquid separation and washing to obtain Pt-IEICO-4F nanoparticles.

[0014] In some embodiments of the present application, the mass ratio of the DSPE-PEG2000-COOH, mesoporous platinum nanoparticles and IEICO-4F is (3-7): 1: 1.

[0015] In some embodiments of the present application, the extraction method of the E. coli DH5α lipopolysaccharide-free bacterial outer membrane vesicles comprises the following steps:

[0016] Performing first centrifugal treatment on the E. coli DH5α bacterial liquid to collect bacterial precipitate; resuspending the precipitate with a buffer solution to perform cell disruption; then, performing second centrifugal treatment after ultrasonic to remove large fragments and collect wet precipitate; resuspending the wet precipitate with a Tris buffer solution to generate E. coli DH5α lipopolysaccharide-free bacterial outer membrane vesicles (dOMV) on the spot; performing third centrifugal treatment to remove insoluble impurities and collect supernatant; performing ultracentrifugal treatment on the supernatant to collect precipitate, thereby obtaining precipitated dOMV.

[0017] In some embodiments of the present application, the extraction method of the E. coli DH5α lipopolysaccharide-free bacterial outer membrane vesicles comprises the following steps:

[0018] Performing first centrifugal treatment on the E. coli DH5α bacterial liquid to collect bacterial precipitate; resuspending the precipitate with 3-6 times the volume of a PBS buffer solution to perform cell disruption; then, performing second centrifugal treatment after ultrasonic in an ice water bath to remove large fragments and collect wet precipitate; resuspending the wet precipitate with a Tris buffer solution (0.1 M Tris, 10 mM EDTA and 5 mg / mL sodium deoxycholate) to generate E. coli DH5α lipopolysaccharide-free bacterial outer membrane vesicles (dOMV) on the spot; performing third centrifugal treatment to remove insoluble impurities and collect supernatant; performing ultracentrifugal treatment on the supernatant to collect precipitate, thereby obtaining precipitated dOMV.

[0019] In some embodiments of the present application, the specific operation of the S2 step comprises: mixing E. coli DH5a lipopolysaccharide-free bacterial outer membrane vesicles and Pt-IEICO-4F nanoparticles, and ultrasonicating in an ice water bath, and then incubating in a low temperature environment for 8-24 h to form composite nanoparticles Pt-IEICO-4F@dOMV; and then centrifuging the mixture to collect the precipitate to obtain the composite nanoparticles Pt-IEICO-4F@dOMV, i.e. the diagnosis and treatment integrated nano platform.

[0020] In some embodiments of the present application, the mass ratio of E. coli DH5a lipopolysaccharide-free bacterial outer membrane vesicles to Pt-IEICO-4F nanoparticles in the S2 step is (1-2): 1.

[0021] The present application also provides a preparation method of a diagnosis and treatment integrated nano platform, comprising the following preparation steps:

[0022] S1. loading IEICO-4F into mesoporous platinum nanoparticles by physical or chemical methods to obtain Pt-IEICO-4F nanoparticles;

[0023] S2. combining E. coli DH5a lipopolysaccharide-free bacterial outer membrane vesicles and Pt-IEICO-4F nanoparticles to form composite nanoparticles Pt-IEICO-4F@dOMV; and separating the composite nanoparticles Pt-IEICO-4F@dOMV, i.e. the diagnosis and treatment integrated nano platform.

[0024] In some embodiments of the present application, the preparation step of the mesoporous platinum nanoparticles comprises: mixing a non-ionic block copolymer template agent, an electrolyte additive and a reducing organic acid in an aqueous phase, and ultrasonicating to form a homogeneous precursor solution; adding a platinum precursor to the precursor solution, mixing uniformly, and then reacting at 50-90°C for 12-48 hours to cause in-situ reduction of platinum ions in the template self-assembly structure; and after the reaction is completed, performing solid-liquid separation and washing to obtain mesoporous platinum nanoparticles.

[0025] In some embodiments of the present application, the non-ionic block copolymer template agent is block polyether F-127 (CAS: 9003-11-6); the electrolyte additive is KBr; the reducing organic acid is ascorbic acid; and the platinum precursor is chloroplatinic acid.

[0026] In some specific embodiments of the present invention, the preparation process of the mesoporous platinum nanoparticles is as follows: block polyether F-127 and KBr are dissolved in water to form a first solution; the first solution is mixed with ascorbic acid solution under ultrasonic conditions to obtain a second solution; chloroplatinic acid is added to the second solution and mixed well to obtain a third solution; the third solution is reacted at 50℃-90℃ for 12h-48h; after the reaction is completed, mesoporous platinum nanoparticles are obtained by solid-liquid separation and washing.

[0027] In some specific embodiments of the present invention, the mass ratio of the block polyether F-127, KBr, ascorbic acid and chloroplatinic acid is (13-16):(29-36):(7-10):1.

[0028] In some specific embodiments of the present invention, the solid-liquid separation and washing are centrifugal separation and water washing, respectively.

[0029] In some specific embodiments of the present invention, the physical or chemical method is a nano-coprecipitation method.

[0030] In some specific embodiments of the present invention, the preparation process of the Pt-IEICO-4F nanoparticles includes: dissolving DSPE-PEG2000-COOH in water and mixing it with mesoporous platinum nanoparticles under ultrasound; subsequently, dissolving IEICO-4F (CAS: 2089044-02-8) in tetrahydrofuran solution and stirring the above mixed solution at room temperature for 8-24 hours; after the reaction is completed, Pt-IEICO-4F nanoparticles are obtained by solid-liquid separation and washing.

[0031] In some specific embodiments of the present invention, the mass ratio of DSPE-PEG2000-COOH, mesoporous platinum nanoparticles and IEICO-4F is (3-7):1:1.

[0032] In some specific embodiments of the present invention, the method for extracting the outer membrane vesicles of Escherichia coli DH5α lipopolysaccharide bacteria includes the following steps:

[0033] The *E. coli* DH5α bacterial culture was centrifuged for the first time, and the bacterial precipitate was collected. The precipitate was resuspended in buffer and the cells were disrupted. Then, after sonication, a second centrifugation was performed to remove large fragments, and the wet precipitate was collected. The wet precipitate was resuspended in Tris buffer to generate the *E. coli* DH5α lipopolysaccharide-free bacterial outer membrane vesicles (dOMV). A third centrifugation was performed to remove insoluble impurities, and the supernatant was collected. The supernatant was ultracentrifuged, and the precipitate was collected to obtain the precipitate dOMV.

[0034] In some specific embodiments of the present invention, the method for extracting the outer membrane vesicles of Escherichia coli DH5α lipopolysaccharide bacteria includes the following steps:

[0035] The *E. coli* DH5α bacterial culture was centrifuged for the first time, and the bacterial precipitate was collected. The precipitate was resuspended in 3-6 volumes of PBS buffer to disrupt the cells. The cells were then sonicated in an ice-water bath and centrifuged a second time to remove large fragments, and the wet precipitate was collected. The wet precipitate was resuspended in Tris buffer (0.1 M Tris, 10 mM EDTA, and 5 mg / mL sodium deoxycholate) to generate the *E. coli* DH5α lipopolysaccharide-free bacterial outer membrane vesicles (dOMV). A third centrifugation was performed to remove insoluble impurities, and the supernatant was collected. The supernatant was then ultracentrifuged, and the precipitate was collected to obtain the dOMV precipitate.

[0036] In some specific embodiments of the present invention, the specific operation of step S2 includes: mixing the outer membrane vesicles of Escherichia coli DH5α lipopolysaccharide bacteria with Pt-IEICO-4F nanoparticles, sonicating in an ice-water bath, and then incubating at low temperature for 8-24 hours to form composite nanoparticles Pt-IEICO-4F@dOMV; subsequently, centrifuging the mixture, collecting the precipitate, and obtaining composite nanoparticles Pt-IEICO-4F@dOMV, i.e., a diagnostic and therapeutic nanoplatform.

[0037] The present invention also provides a medicament or pharmaceutical composition for treating glioblastoma, comprising the therapeutic nanoplatform described in any one of the claims or the therapeutic nanoplatform prepared by any one of the preparation methods described in the claims.

[0038] In some specific embodiments of the present invention, a pharmaceutically acceptable carrier is also included.

[0039] The present invention also provides the application of any of the therapeutic nanoplatforms described in any one of the claims or the therapeutic nanoplatforms prepared by any one of the preparation methods described in any one of the claims in the preparation of drugs or pharmaceutical compositions for glioblastoma.

[0040] The present invention has the following significant advantages and effects compared with the prior art:

[0041] In traditional treatments for GBM, conventional drugs struggle to overcome the physiological limitations of the body's biological barrier (BBB) ​​to achieve effective therapeutic concentrations at the tumor site, and surgical treatment also fails to completely eradicate the tumor. Furthermore, the complex biological characteristics of GBM limit the efficacy of single treatment modalities, resulting in traditional therapies failing to significantly prolong patient survival. To address these shortcomings, this invention proposes a biomimetic nanoplatform (Pt-IEICO-4F@dOMV) that coordinates four synergistic treatment modalities (PDT, PTT, SDT, and CDT) for GBM treatment. Mesoporous platinum nanoparticles, acting as multifunctional centers, not only efficiently perform photothermal conversion for tumor ablation but also enhance the Fenton reaction to promote chemotherapy drug delivery and alleviate tumor hypoxia by decomposing H2O2. The dual-mode sensitizer (IEICO-4F) further enhances the therapeutic performance of platinum-based drugs, exhibiting excellent near-infrared absorption and intramolecular charge transfer (ICT) capabilities, enabling deep tissue penetration, and serving as a photosensitizer / sound sensitizer for PDT and SDT. The dOMV coating ensures drug penetration of the blood-brain barrier and precise targeting of the tumor. The therapeutic advantage of this system lies in the fact that platinum-enhanced hyperthermia improves tumor oxygenation levels, thereby significantly increasing ROS production in both PDT and SDT. Furthermore, the combined oxidative stress of ·OH (CDT) and 1O2 (PDT and SDT) leads to intense pyroptosis and apoptosis. Real-time photoacoustic imaging technology allows for precise spatiotemporal control of these effects. Notably, the mesoporous platinum structure forms a self-sustaining therapeutic cycle by continuously converting tumor-generated H2O2 into oxygen and cytotoxic ROS. This comprehensive approach not only demonstrates superior tumor accumulation capacity, multimodal therapeutic efficacy, and biocompatibility but also opens new avenues for glioblastoma treatment, effectively addressing delivery challenges and treatment resistance issues. Attached Figure Description

[0042] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art 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.

[0043] Figure 1 In Figure (a), the structural formula of IEICO-4F is shown; (b) shows the ground-state energy geometry; and (c) and (d) show the pre-molecular orbital electron density distribution.

[0044] Figure 2 This is a schematic diagram of the EICO-4F energy level transition.

[0045] Figure 3The UV-Vis spectra of mPt, IEICO-4F, and Pt-IEICO-4F are shown.

[0046] Figure 4 This is a transmission electron microscope image of IEICO-4F.

[0047] Figure 5 The image shows the X-ray powder diffraction pattern of mPt nanoparticles.

[0048] Figure 6 The X-ray photoelectron spectroscopy of mPt nanoparticles is in the C1s region.

[0049] Figure 7 The X-ray photoelectron spectroscopy of mPt nanoparticles is in the Pt4f region.

[0050] Figure 8 Transmission electron microscopy image of Pt-IEICO-4F.

[0051] Figure 9 Transmission electron microscopy image of Pt-IEICO-4F@dOMV.

[0052] Figure 10 Particle size distribution diagrams for mPt, Pt-IEICO-4F, and Pt-IEICO-4F@dOMV

[0053] Figure 11 The zeta potentials are mPt, Pt-IEICO-4F, and Pt-IEICO-4F@dOMV.

[0054] Figure 12 This is a schematic diagram of an in vitro blood-brain barrier model.

[0055] Figure 13 The results of flow cytometry analysis of GL261 cells cultured on the basal side after co-incubating with Pt-IEICO-4F@Ce6 and Pt-IEICO-4F@Ce6@dOMV (50 μg / mL) for 6 hours in Transwell chambers are as follows.

[0056] Figure 14 Quantitative flow cytometry analysis of basal GL261 cells.

[0057] Figure 15 Confocal laser scanning microscopy images of Pt-IEICO-4F@Ce6 and Pt-IEICO-4F@Ce6@dOMV (50 μg / mL) co-incubated with GL261 cells for 0, 1, 2, 4, 6, 8, and 10 hours.

[0058] Figure 16In the figure, a and b are the flow cytometry results after Pt-IEICO-4F@Ce6 and Pt-IEICO-4F@Ce6@dOMV (50 μg / mL) were co-incubated with GL261 cells for 0, 1, 2, 4, 6, 8, and 10 hours, respectively.

[0059] Figure 17 Quantitative flow cytometry analysis of GL261 cells co-incubated with Pt-IEICO-4F@Ce6 and Pt-IEICO-4F@Ce6@dOMV at different time points. (Values ​​are expressed as mean ± standard deviation, n = 3. **p < 0.01; ***p < 0.001; ****p < 0.0001).

[0060] Figure 18 CLSM plot of ROS generation in GL261 cells under different conditions. (Scale bar = 20 μm).

[0061] Figure 19 To perform quantitative analysis of CLSM plots used to detect intracellular ROS generation.

[0062] Figure 20 To detect the generation of ROS in GL261 cells under different conditions using flow cytometry.

[0063] Figure 21 The results are used for quantitative analysis of intracellular ROS generation by flow cytometry.

[0064] Figure 22 Viability of GL261 cells under PBS, different concentrations of Pt-IEICO-4F@dOMV, and different treatment conditions. (Numbers are expressed as mean ± SD, n = 3. **p < 0.01; ***p < 0.001; ****p < 0.0001)

[0065] Figure 23 CLSM images of GL261 cells after co-staining with Calcein-AM / PI under different conditions.

[0066] Figure 24 To quantitatively analyze the number of live and dead cells in GL261 cells after co-staining with Calcein-AM / PI under different conditions.

[0067] Figure 25 To analyze the apoptosis of GL261 cells under different treatment conditions after Annexin V-FITC / PI staining, flow cytometry was used.

[0068] Figure 26To monitor the changes in mitochondrial membrane potential in GL261 cells under different conditions using the JC-1 method (scale bar = 20 μm).

[0069] Figure 27 To quantitatively analyze the fluorescence intensity of GL261 cells under different conditions using the JC-1 method.

[0070] Figure 28 Western blot was used to detect the expression levels of Caspase-1, GSDMD-FL, and GSDMD-N in GL261 cells.

[0071] Figure 29 Quantitative analysis of Caspase-1, GSDMD-FL, and GSDMD-N expression levels. (Numbers are expressed as mean ± standard deviation, n = 3. **p < 0.01; ***p < 0.001; ****p < 0.0001) (I: PBS, II: PBS + L + US, III: Pt-IEICO-4F@dOMV, IV: Pt-IEICO-4F@dOMV + US, V: Pt-IEICO-4F@dOMV + L, VI: Pt-IEICO-4F@dOMV + L + US. Laser: 808 nm, 1 W / cm²) 2 5 min; Ultrasound: 1.0 MHz, 0.5 W / cm 2 20% duty cycle, 5 min).

[0072] Figure 30 Brain fluorescence images at different time points after mice were injected with Pt-IEICO-4F@Ce6 and Pt-IEICO-4F@Ce6@dOMV.

[0073] Figure 31 In vitro fluorescence images of major organs in mice 24 hours after injection of Pt-IEICO-4F@Ce6 and Pt-IEICO-4F@Ce6@dOMV.

[0074] Figure 32 Photoacoustic imaging of the brain at different time points after injection of Pt-IEICO-4F and Pt-IEICO-4F@dOMV in mice. (Scale bar = 25 mm).

[0075] Figure 33Table 9a shows the quantitative analysis of fluorescence intensity at different time points after injection of t-IEICO-4F@Ce6 and Pt-IEICO-4F@dOMV; table 9b shows the quantitative analysis of fluorescence intensity in major organs of mice 24 hours after injection of Pt-IEICO-4F@Ce6 and Pt-IEICO-4F@dOMV; and table 9c shows the quantitative analysis of photoacoustic intensity at different time points after injection of Pt-IEICO-4F and Pt-IEICO-4F@dOMV.

[0076] Figure 34 In Figure a, laser irradiation of the tumor site at different time points after tail vein injection of PBS and Pt-IEICO-4F@dOMV (808nm, 1W / cm²) is shown. 2 b) is an infrared thermal image; b) is a temperature distribution map of the tumor site.

[0077] Figure 35 This is a schematic diagram of the treatment process in mice with an orthotopic glioblastoma model.

[0078] Figure 36 These are in vivo bioluminescent images of brain tumors on days 1, 3, 5, 7, and 10 during treatment.

[0079] Figure 37 The results show the quantitative analysis of bioluminescence intensity in tumor-bearing mice from different groups. (Values ​​are expressed as mean ± standard deviation, n = 3. **p < 0.01; ***p < 0.001; ****p < 0.0001).

[0080] Figure 38 To monitor changes in body weight in the treatment group mice during the treatment period. Detailed Implementation

[0081] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are merely some embodiments of the present invention and should not be used to limit the scope of protection of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0082] Protection range.

[0083] Example 1: Preparation of a therapeutic nanoplatform (Pt-IEICO-4F@dOMV nanoparticles)

[0084] The specific steps are as follows:

[0085] 1) Synthesis of mesoporous platinum nanoparticles

[0086] First, F-127 (180 mg) and KBr (400 mg) were weighed and dissolved in 5 mL of water. This solution was then mixed with 6 mL of ascorbic acid solution (0.1 M) under sonication for 10 min. Next, 0.15 mL of chloroplatinic acid (0.2 M) was added and the mixture was shaken thoroughly. The mixture was then reacted at 70 °C for 24 h. After the reaction was complete, the reaction solution was collected, centrifuged (8000 rpm, 10 min), and washed three times with water to obtain mesoporous platinum nanoparticles.

[0087] 2) Preparation of Pt-IEICO-4F nanoparticles

[0088] To modify the surface of mesoporous platinum nanoparticles, 5 mg of DSPE-PEG2000-COOH was dissolved in 1 mL of water and mixed with 1 mg of mesoporous platinum nanoparticles under sonication for 5 min. Subsequently, 1 mg of IEICO-4F was dissolved in 1 mL of tetrahydrofuran solution and stirred with the above mixture overnight at room temperature. The Pt-IEICO-4F nanoparticles were collected by centrifugation at 8000 rpm for 10 min and washed three times with water.

[0089] 3) Preparation of Pt-IEICO-4F@dOMV nanoparticles

[0090] The *E. coli* DH5α bacterial culture was centrifuged for 20 min (4℃, 5000g), and the precipitate was collected and resuspended in four volumes of PBS. The precipitate was then sonicated in an ice-water bath for 20 min and centrifuged for 1 h (4℃, 2900g). The wet precipitate was resuspended in 7.5 volumes of Tris buffer (0.1 M Tris, 10 mM EDTA, and 5 mg / mL sodium deoxycholate) to generate dOMV. Centrifugation was performed at 4℃ (20000g) for 1 h to remove bacterial debris, followed by ultracentrifugation of the supernatant for 2 h (4℃, 125000g) to obtain the precipitate dOMV. Finally, 100 μL of the dOMV solution (300 μg / mL) was mixed with Pt-IEICO-4F, sonicated in an ice-water bath for 15 min, and incubated overnight at 4℃. The mixture was centrifuged for 10 minutes (4℃, 12000rpm) the next day, and the precipitate was collected to obtain Pt-IEICO-4F@dOMV nanoparticles, namely the integrated diagnostic and therapeutic nanoplatform.

[0091] Example 2: Characterization of the Therapeutic Nanoplatform (Pt-IEICO-4F@dOMV Nanoparticles)

[0092] 1) IEICO-4F is a high-performance non-fullerene small molecule acceptor material. The molecular structure of IEICO-4F is as follows: Figure 1 As shown in (a), the ground state energy geometry is as follows: Figure 1As shown in (b), a large conjugated ADA structure is formed by a terminal electron acceptor group (fluorodicyanoethylene ketone), an electron-rich alkoxythiophene bridge, and a core backbone (IDT units containing benzene alkyl side chains). Based on density functional theory, the high occupied molecular orbital (HOMO) of IEICO-4F can be calculated to be -5.07 eV, the lowest unoccupied molecular orbital (LUMO) to be -3.40 eV, and the band gap to be 1.67 eV. Figure 1 c and d). The energy difference (ΔES1-T1) between S1 and T1 is a key factor influencing intersystem crossing (ISC) processes. The ΔES1-T1 of IEICO-4F is 0.5969 eV. Figure 2 The smaller the energy difference, the more favorable the transition of molecules from the singlet to the triplet state. Due to the large ADA conjugated structure and narrow band gap of IEICO-4F, it exhibits a redshift in the absorption spectrum, displaying strong near-infrared absorption at 899 nm. Figure 3 This indicates that the molecule has good light absorption and photothermal conversion capabilities in the near-infrared region.

[0093] 2) Transmission electron microscopy (TEM) revealed that the mesoporous platinum nanoparticles (mPt nanoparticles, prepared in the examples) exhibited a typical sea urchin-like shape, with an average diameter of 43.82 ± 1 nm. Figure 4 The crystal structure of mPt was detected by X-ray powder diffraction (XRD). The results showed that the diffraction peaks at 39.5°, 45.8°, 67.2°, 81.4°, and 85.7° correspond to the (111), (200), (220), (311), and (222) crystal planes of Pt (PDF#04-0802). Figure 5 A C1s peak was observed in the X-ray photoelectron spectroscopy (XPS) of mPt. Figure 6 Furthermore, in the Pt4f spectrum, the two peaks of Pt are composed of Pt04f5 / 2 and Pt04f7 / 2 (72.8 and 69.4 eV). Figure 7 Due to their large specific surface area, mesoporous platinum nanoparticles can effectively load IEICO-4F. Meanwhile, IEICO-4F, as a narrow-bandgap non-fullerene acceptor material, exhibits strong electron-withdrawing properties that can interact electronically with the mesoporous structure of platinum. Therefore, IEICO-4F can be loaded into mesoporous platinum nanoparticles via nanoprecipitation, resulting in Pt-IEICO-4F nanoparticles. TEM images show that Pt-IEICO-4F nanoparticles are urchin-like spherical particles with a diameter of 50.75 ± 3 nm. Figure 8 The encapsulation efficiency of IEICO-4F was calculated to be 81.9%, and the drug loading rate was 41.3% according to the formula. Furthermore, in the UV-Vis absorption spectrum (… Figure 3mPt showed a broad absorption band, with absorption peaks of IEICO-4F and Pt-IEICO-4F located at approximately 899 nm and 877 nm, respectively, indicating the successful preparation of Pt-IEICO-4F. Finally, the transmission electron microscope image of the multifunctional radioactive nanoparticles Pt-IEICO-4F@dOMV prepared in the examples is shown below. Figure 9 As shown. Dynamic light scattering (DLS) curves show that the average sizes of mPt, Pt-IEICO-4F, and Pt-IEICO-4F@dOMV are 47, 60, and 71 nm, respectively. Figure 10 Due to hydration and Brownian motion, the particle size is larger than that shown in the TEM image. The Zeta potentials of mPt, Pt-IEICO-4F, and Pt-IEICO-4F@dOMV were -27.7, -29.5, and -28.6 mV, respectively. Figure 11 This indicates that the nanoparticles maintained good dispersibility in different solutions.

[0094] Example 3: In vitro killing effect test of the therapeutic nanoplatform (Pt-IEICO-4F@dOMV nanoparticles)

[0095] 1) This embodiment constructed an in vitro BBB model to investigate the ability of Pt-IEICO-4F@dOMV nanoparticles to penetrate the BBB. For example... Figure 12 As shown, an in vitro BBB model was constructed using Transwell cell culture plates. First, bEnd.3 cells were seeded into the upper chambers of the Transwell plates. During cell culture, the transendothelial resistance (TEER) of the chambers was monitored periodically to determine the success of the model construction. TEER is the most typical indicator for assessing BBB integrity and tight junctions. When the TEER value stabilized between 150-300 Ωcm... 2 Within this range, the tight junctions of the constructed in vitro model can be considered similar to those of the in vivo BBB, thus proving the successful construction of the model. Pt-IEICO-4F@dOMV nanoparticles were prepared by labeling Pt-IEICO-4F@Ce6@dOMV with dihydroporphyrin e6 (Ce6). Subsequently, GL261 cells were seeded in the basal side chamber and cultured for 24 hours to ensure sufficient cell adhesion and growth. Then, the original culture medium in the Transwell chamber was replaced with fresh medium containing Pt-IEICO-4F / Ce6 and Pt-IEICO-4F@dOMV / Ce6. After 6 hours of incubation, the uptake of nanoparticles by GL261 cells in the basal side chamber was analyzed in detail using flow cytometry. Figure 13 Based on the quantitative analysis results ( Figure 14As can be seen, the average fluorescence intensity of the Pt-IEICO-4F@Ce6@dOMV group was significantly higher than that of the control group (6.11 times) and the uncoated group (3.10 times). This result reveals that Pt-IEICO-4F modified with dOMV can effectively improve the delivery efficiency of nanoparticles. Next, to further investigate the uptake characteristics of Pt-IEICO-4F@dOMV in GL261 cells, this example uses CLSM and flow cytometry for evaluation. Under the CLSM field of view, the morphology and structure of the GL261 cell nuclei are clearly visible as intact and normal. Furthermore, with the gradual extension of incubation time, the red fluorescence intensity of both groups of cells continuously increases. Figure 15 Furthermore, this embodiment also used flow cytometry to further detect the uptake of Pt-IEICO-4F@Ce6 and Pt-IEICO-4F@Ce6@dOMV in cells. Figure 16 a, b). The quantitative analysis results were highly consistent with the CLSM observation results, both indicating that the nanoparticles could be rapidly and effectively taken up by cells. With increasing incubation time, the amount of nanoparticles taken up by cells gradually increased. At each time point, the fluorescence intensity of the dOMV-coated group was consistently higher than that of the uncoated group ( Figure 17 The above series of experimental results, from different perspectives, confirm that dOMV coating can significantly promote the internalization of Pt-IEICO-4F nanoparticles into cells.

[0096] 2) GL261 cells were differentially treated according to experimental groups (groups I: PBS group, II: PBS+L+US group, III: Pt-IEICO-4F@dOMV group, IV: Pt-IEICO-4F@dOMV+US group, V: Pt-IEICO-4F@dOMV+L group, VI: Pt-IEICO-4F@dOMV+L+US group; L refers to 808nm laser (1W / cm²). 2 US refers to ultrasound (1.0MHz, 0.5W / cm). 2 (50% duty cycle), both L and US were irradiated for 5 minutes, followed by co-incubation with DCFH-DA to trigger fluorescence. ROS generation in each treatment group was assessed using CLSM. CLSM images showed strong green fluorescence in the Pt-IEICO-4F@dOMV+L+US group, directly demonstrating that, under the synergistic effect of laser and US, Pt-IEICO-4F@dOMV can induce more abundant ROS generation in GL261 cells. Figure 18 Further quantitative analysis of the green fluorescence intensity in the CLSM images was performed. Figure 19The results more intuitively support the above argument. Furthermore, to ensure the accuracy and reliability of the experimental results, this embodiment also used flow cytometry to further verify the ROS generation in cells of different treatment groups. Figure 20 Quantitative analysis results show ( Figure 21 The results are highly consistent with those observed by CLSM. These experimental results reveal, at the cellular level, the superior ROS generation efficiency of Pt-IEICO-4F@dOMV under laser-ultrasound dual-modal excitation.

[0097] 3) In this embodiment, the Cell Count Kit-8 (CCK8) assay was used to systematically evaluate the cytotoxic effects of different concentrations of Pt-IEICO-4F@dOMV solution on GL261 cells. Figure 22 Experimental data showed that even at concentrations as high as 100 μg / ml, cell viability remained above 80%, demonstrating that it had no significant cytotoxicity to GL261 cells and good biocompatibility. In the control group without Pt-IEICO-4F@dOMV, no significant decrease in cell viability was observed regardless of whether laser treatment, ultrasound treatment, or combined laser-ultrasound treatment was used alone. This result confirms that laser or ultrasound irradiation alone cannot effectively kill tumor cells, thus highlighting Pt-IEICO-4F@dOMV as a good photosensitizer. Under the same concentration conditions, the cell viability of the combined laser-ultrasound treatment group showed a decreasing trend compared to the laser treatment group or the ultrasound treatment group alone. In particular, when the Pt-IEICO-4F@dOMV solution concentration reached 100 μg / ml, cell viability decreased to 24.17%, which directly reflects that the inhibitory effect of Pt-IEICO-4F@dOMV on tumor cells was significantly enhanced under the synergistic effect of laser and ultrasound.

[0098] 4) In addition, this embodiment used Calcein-AM and PI double staining methods, and further observed the inhibitory effect of Pt-IEICO-4F@dOMV on GL261 cells using CLSM. Figure 23 In the ultrasound and laser treatment groups, CLSM images showed a dispersed distribution of green and red fluorescence, indicating that these two treatments only had a moderate inhibitory effect on GL261 cells. In contrast, the laser-ultrasound combined treatment group generally exhibited strong red fluorescence, which fully demonstrates the outstanding tumor cell killing ability of this combined treatment. Meanwhile, quantitative analysis results of live / dead cell assays (…) Figure 24The results were consistent with those obtained from the CCK-8 assay, further validating the above observations. To further evaluate the apoptosis-inducing effect of Pt-IEICO-4F@dOMV, Annexin V-FITC / PI staining was performed, followed by quantitative analysis using flow cytometry. Figure 25 As shown, the apoptosis rate in the laser-ultrasound combined treatment group reached 40.09%, significantly higher than other treatment groups. This result is corroborated by CCK-8 assay and live / dead cell staining results. JC-1 staining was used to investigate Pt-IEICO-4F@dOMV-mediated mitochondrial damage; JC-1 staining can reveal changes in mitochondrial transmembrane potential by the transition from red to green fluorescence. Figure 26 As shown, the laser-ultrasound combined treatment group emitted strong green fluorescence and weak red fluorescence, indicating that ROS generated under laser-ultrasound treatment can cause severe mitochondrial damage. Quantitative analysis of the green fluorescence of JC-1 monomers and the red fluorescence of JC-1 aggregates also visually revealed this result. Figure 27 Subsequently, the underlying mechanism of Pt-IEICO-4F@dOMV-induced pyroptosis was investigated. Western blot analysis was used to detect the intracellular expression levels of GSDMD-N in various treatment groups. Figure 28 ).like Figure 29 It is evident that the GSDMD-N content in the Pt-IEICO-4F@dOMV+L+US group was significantly higher than that in other groups, indicating that GSDMD-N was effectively activated.

[0099] Example 4: In vivo killing effect test of the theotherapy nanoplatform (Pt-IEICO-4F@dOMV nanoparticles)

[0100] 1) To further investigate the targeted delivery efficiency of Pt-IEICO-4F@dOMV, an in situ GBM model was constructed in this embodiment. After drug administration, the biodistribution of the nanoparticles in vivo was observed using animal fluorescence imaging and photoacoustic imaging. Pt-IEICO-4F@Ce6 and Pt-IEICO-4F@Ce6@dOMV were injected into mice via the tail vein, and in vivo fluorescence imaging was performed at different time points after injection. Figure 30As time progressed, the fluorescence signal intensity continuously increased, reaching its peak at 8 hours in both groups, before gradually weakening. At each time point, the fluorescence signal intensity of the dOMV-coated group was significantly higher than that of the uncoated group. This phenomenon indicates that Pt-IEICO-4F@dOMV exhibits good biocompatibility under dOMV modification, successfully overcoming the BBB and significantly improving targeted delivery efficiency. Twenty-four hours after injection, a weak fluorescence signal could still be detected at the tumor site in the dOMV-coated mice, which is attributed to dOMV effectively enhancing the long-term stability of the nanoparticles in blood circulation. Subsequently, the mice were sacrificed, and their major organs (heart, liver, spleen, lung, kidney, and brain) were collected for fluorescence imaging analysis. Figure 31 The imaging results showed a high degree of agreement with in vivo imaging, with nanoparticles mainly concentrated in detoxification organs. This finding clearly suggests that these organs play a crucial role in the metabolism and detoxification of nanoparticles. Pt-IEICO-4F possesses unique optical properties, enabling photoacoustic imaging under 808nm laser excitation. Based on this property, this embodiment used a photoacoustic imager to repeatedly verify the targeting performance of Pt-IEICO-4F@dOMV. Figure 32 The experimental results were consistent with expectations. Eight hours after injection of Pt-IEICO-4F and Pt-IEICO-4F@dOMV nanoparticles, respectively, the photoacoustic signals reached their peak values, and the photoacoustic signal intensity of the dOMV-coated group was higher than that of the uncoated group. Mouse brain at different time points ( Figure 33 a) and major organs ( Figure 33 b) Quantitative results of fluorescence imaging, and quantitative results of photoacoustic signal intensity at the site of mouse brain tumor (b) Figure 33 c) further validates the above arguments. To evaluate the in vivo photothermal conversion capability of Pt-IEICO-4F@dOMV, thermal imaging and temperature changes of mouse tumor sites under 808nm laser irradiation were recorded using a thermal imager. Figure 34 (a) and (b) The experimental results clearly show that the temperature at the tumor site in mice injected with Pt-IEICO-4F@dOMV rose rapidly, eventually reaching 48.0±1.4℃. This data strongly indicates that Pt-IEICO-4F@dOMV also possesses excellent photothermal conversion capabilities in mice, generating sufficient heat to achieve effective thermal ablation of tumor tissue.

[0101] 2) To further investigate the anti-tumor effect of Pt-IEICO-4F@dOMV in vivo, this embodiment also constructed a mouse model carrying GL261-Luc tumors and randomly divided them into 6 groups as follows: (1) PBS control group; (2) PBS+US+L group; (3) Pt-IEICO-4F@dOMV group; (4) Pt-IEICO-4F@dOMV+US group; (5) Pt-IEICO-4F@dOMV+L group; (6) Pt-IEICO-4F@dOMV+US+L group ( Figure 35 According to the group assignments, mice received either PBS buffer or Pt-IEICO-4F@dOMV via tail vein injection. Eight hours after injection, the nanoparticles successfully penetrated the BBB and effectively accumulated at the tumor site. Subsequently, according to the group settings, an 808nm laser (1W / cm²) was used... 2 ) and ultrasound (1.0MHz, 0.5W / cm) 2 The tumor site was irradiated for 5 minutes (50% duty cycle). During laser treatment, the temperature of the tumor site was monitored in real time using a thermal imager to ensure that the temperature threshold required for effective photothermal therapy was reached. Throughout the treatment cycle, tumor growth dynamics were indirectly monitored by observing the bioluminescence of GL261-luc cells in the mouse brain every other day, and changes in the mouse's tumor weight were recorded. The results were obtained from mouse bioluminescence imaging (…). Figure 36 ) and quantitative analysis results of luminescence intensity ( Figure 37 In the study, it was observed that, compared to the control group, the Pt-IEICO-4F@dOMV+US and Pt-IEICO-4F@dOMV+L groups only showed weak inhibitory effects on tumor growth. This result suggests that single PTT, PDT, or SDT treatments have limited efficacy in anti-tumor therapy. Under the synergistic effect of laser and ultrasound, Pt-IEICO-4F@dOMV effectively inhibited tumor growth. This is because the thermal effect of PTT enhanced the efficiency of ROS generation during PDT and SDT, and also increased the Fenton reaction rate of CDT, catalyzing the generation of oxygen from H2O2, forming a cyclic amplification effect of ROS, thereby continuously inhibiting tumor cell proliferation. Furthermore, during the entire treatment period, the body weight of mice in each group did not change significantly, indicating that Pt-IEICO-4F@dOMV has good biocompatibility. Figure 38 ).

[0102] The above description is merely a preferred embodiment of this application and an explanation of the technical principles employed. Those skilled in the art should understand that the scope of the invention involved in this application is not limited to the technical solutions formed by specific combinations of the above-described technical features, but should also cover other technical solutions formed by arbitrary combinations of the above-described technical features or their equivalents without departing from the inventive concept. For example, the above-described features have similar functions to (but are not limited to) those disclosed in this application.

Claims

1. A diagnostic and therapeutic nanoplatform, characterized in that, The integrated diagnostic and therapeutic nanoplatform includes a nanoparticle composition comprising IEICO-4F and mesoporous platinum nanoparticles, as well as Escherichia coli DH5α lipopolysaccharide bacterial outer membrane vesicles coated on the surface of the mesoporous platinum nanoparticles.

2. The integrated diagnostic and therapeutic nanoplatform as described in claim 1, characterized in that, The therapeutic nanoplatform comprises the following preparation steps: loading IEICO-4F into mesoporous platinum nanoparticles using physical or chemical methods to obtain Pt-IEICO-4F nanoparticles; combining the outer membrane vesicles of Escherichia coli DH5α lipopolysaccharide bacteria with the Pt-IEICO-4F nanoparticles to form composite nanoparticles Pt-IEICO-4F@dOMV; and separating the composite nanoparticles Pt-IEICO-4F@dOMV, which constitutes the therapeutic nanoplatform.

3. The integrated diagnostic and therapeutic nanoplatform as described in any one of claims 1-2, characterized in that, The preparation steps of the mesoporous platinum nanoparticles include: mixing a nonionic block copolymer template agent, an electrolyte additive, and a reducing organic acid in an aqueous phase, and then ultrasonically treating the mixture to form a homogeneous precursor solution; adding a platinum precursor to the precursor solution, mixing it evenly, and then reacting it at 60–90°C for 12–48 hours to allow platinum ions to be reduced in situ in the self-assembled structure of the template agent; after the reaction is completed, solid-liquid separation and washing are performed to obtain mesoporous platinum nanoparticles.

4. A method for preparing a therapeutic nanoplatform, characterized in that, The preparation steps include the following: S1. IEICO-4F is loaded into mesoporous platinum nanoparticles by physical or chemical methods to obtain Pt-IEICO-4F nanoparticles. S2. Combine the outer membrane vesicles of Escherichia coli DH5α lipopolysaccharide bacteria with Pt-IEICO-4F nanoparticles to form composite nanoparticles Pt-IEICO-4F@dOMV; separate the composite nanoparticles Pt-IEICO-4F@dOMV, which is the diagnostic and therapeutic nanoplatform.

5. The method for preparing the integrated diagnostic and therapeutic nanoplatform as described in claim 4, characterized in that, The preparation steps of the mesoporous platinum nanoparticles include: mixing a nonionic block copolymer template agent, an electrolyte additive, and a reducing organic acid in an aqueous phase, and then ultrasonically treating the mixture to form a homogeneous precursor solution; adding a platinum precursor to the precursor solution, mixing it evenly, and then reacting it at 50–90°C for 12–48 hours to allow platinum ions to be reduced in situ within the template agent's self-assembled structure; after the reaction is complete, solid-liquid separation and washing are performed to obtain mesoporous platinum nanoparticles.

6. The method for preparing the integrated diagnostic and therapeutic nanoplatform as described in claim 5, characterized in that, The nonionic block copolymer template agent is block polyether F-127; the electrolyte additive is KBr; the reducing organic acid is ascorbic acid; and the platinum precursor is chloroplatinic acid.

7. The method for preparing the integrated diagnostic and therapeutic nanoplatform as described in claim 4, characterized in that, The physical or chemical method is a nano-coprecipitation method.

8. The method for preparing the integrated diagnostic and therapeutic nanoplatform as described in any one of claims 4-7, characterized in that, The method for extracting the outer membrane vesicles of Escherichia coli DH5α lipopolysaccharide bacteria includes the following steps: The Escherichia coli DH5α bacterial culture was centrifuged for the first time to collect the bacterial precipitate; the precipitate was resuspended in buffer to disrupt the cells; then sonicated and centrifuged a second time to remove large fragments and collect the wet precipitate; the wet precipitate was resuspended in Tris buffer to generate the dOMV of Escherichia coli DH5α lipopolysaccharide bacteria. A third centrifugation is performed to remove insoluble impurities, and the supernatant is collected. The supernatant is then subjected to ultracentrifugation, and the precipitate is collected to obtain the precipitate dOMV.

9. A medicament or pharmaceutical composition for treating glioblastoma, characterized in that, Therapeutic nanoplatforms include the therapeutic nanoplatforms described in any one of claims 1-3 or prepared by the preparation method described in any one of claims 4-8.

10. The use of the therapeutic nanoplatform according to any one of claims 1-3 or the therapeutic nanoplatform prepared by the preparation method according to any one of claims 4-8 in the preparation of a drug or pharmaceutical composition for glioblastoma.

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