Preparation method and application of nanoprobes for multimodal imaging and synergistic therapy

Abstract

This invention relates to a method for preparing and applying a nanoprobe for multimodal imaging and synergistic therapy, belonging to the field of bioprobe technology. The method includes: loading the acoustic sensitizer MnTTP and the hypoxia-activating drug TPZ onto PLGA via an emulsion-solvent evaporation method, modifying it to an aqueous phase using PVA, using dopamine hydrochloride to increase biocompatibility, obtaining the nanoprobe TMP@D, and then... (The sentence is incomplete in the original text). 131 I-labeling on TMP@D yields radionuclide-loaded nanoprobes. 131 I-TMP@D. 131 I-TMP@D passively targets and enters tumor cells, enabling synergistic treatment of malignant hypoxic tumors by combining MR and SPECT multimodal imaging with radionuclide therapy, sonodynamic therapy, and hypoxia-activated chemotherapy.

Description

Technical Field

[0001] This invention relates to the field of nanobioprobe technology, and in particular to a method for preparing and applying a nanoprobe for multimodal imaging and synergistic therapy. Background Technology

[0002] Recent research indicates that cancer-related deaths have increased by 20% in recent years. Pancreatic cancer, often considered the "king of cancers," has a survival rate of only 12%, with most cases diagnosed at a time when it is inoperable or metastatic. The low oxygen content and dense structure of malignant tumor tissue are major reasons for resistance to conventional radiotherapy and chemotherapy. Therefore, improving the hypoxia level of tumor tissue can significantly improve the prognosis of cancer treatment. Reactive oxygen species (ROS), as products of aerobic respiration, have been shown to have significant killing effects on malignant tumors through multiple pathways. However, the production of ROS is accompanied by a further reduction in the oxygen content of tumor tissue, making drug resistance even more pronounced. Therefore, developing a probe capable of generating large amounts of ROS and alleviating hypoxia is essential.

[0003] Internal radiation therapy, which concentrates radioactive isotopes at the tumor site, is a highly effective method for treating malignant tumors. This treatment utilizes the energy accumulation of high-energy rays and the close-range action of the rays to efficiently kill tumor cells. 131 I-12, a "star radionuclide" in internal radiotherapy, boasts an excellent half-life (7.6 days), and its released beta rays exhibit superior linear energy transfer compared to X-rays. Beta rays not only damage tumor cell DNA double-strands but also radiate surrounding water molecules to generate reactive oxygen species (ROS), thereby damaging tumor cells. Simultaneously, 131 I-rays emitted by radionuclide radiation have been widely used in functional imaging SPECT (single-photon emission computed tomography). However, radionuclide therapy still needs to consider issues such as uneven biological distribution and radiation blind spots. Furthermore, the radiation resistance of hypoxic tumors and the self-repair of tumor cells' DNA further limit the efficacy of radionuclide therapy. Utilizing nanomaterials to deliver drugs and target radionuclides to tumor sites can significantly improve the shortcomings of radionuclide therapy.

[0004] Polylactic acid-glycolic acid copolymer (PLGA) is a biodegradable high-molecular-weight organic compound with good biocompatibility as a drug carrier, and is widely used in the biomedical field. Sonodynamic therapy (SDT) damages tumor cells by activating a sonosensitive agent with ultrasound to generate reactive oxygen species (ROS). Common sonosensitive agents include porphyrins, phthalocyanines, and porphyrins. Manganese porphyrin (MnTTP) can not only be used as a sonosensitive agent, but the Mn group in the porphyrin ring... 2+ Sensitive to T1-weighted magnetic resonance imaging (MRI), using Mn 2+The accumulation effect of nanoprobes at the tumor site can be observed at different times using MRI (magnetic resonance imaging). The therapeutic effect of SDT is largely affected by the location, concentration, and duration of the sonosensitive agent. Loading drugs into PLGA allows them to enter the tumor tissue under passive targeting and release them at the tumor site. 131 I and MnTTP, upon ultrasound activation, generate a large amount of ROS. Simultaneously, the bioreducing drug telatazamine (TPZ) loaded on the PLGA is extensively activated under the aggravated hypoxia state induced by radionuclide therapy and SDT to promote bioreducing chemotherapy.

[0005] Therefore, develop a 131 The use of I-labeled nanoprobes in conjunction with sonodynamics and chemotherapy to provide a new treatment approach for malignant tumors is of great clinical value. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides a method for preparing and applying a nanoprobe for multimodal imaging and synergistic therapy. The technical solution of this invention is as follows:

[0007] In a first aspect, a method for preparing a nanoprobe for multimodal imaging and synergistic therapy is provided, comprising: loading a sonosensitive agent MnTTP and a hypoxia-activating drug TPZ onto a PLGA via an emulsion-solvent evaporation method, modifying it to an aqueous phase under the action of PVA, using dopamine hydrochloride to increase biocompatibility, obtaining the nanoprobe TMP@D, and then... 131 I-labeling on TMP@D yields radionuclide-loaded nanoprobes. 131 I-TMP@D.

[0008] Optionally, the synthesis steps of the TMP@D are as follows:

[0009] S1, PLGA, MnTTP and TPZ are co-dissolved in dichloromethane, ultrasonically vibrated and then PVA is added dropwise while stirring in the dark to form an oil-in-water structure;

[0010] S2, the solution obtained from S1 is adjusted with Tris base;

[0011] S3, add dopamine hydrochloride to the solution obtained in S2, and continue stirring after ultrasonic vibration;

[0012] S4. The solution obtained in S3 was filtered through a liposome extruder, and then repeatedly ultrafiltered to retain 10kd molecular weight nanoprobes. After freeze-drying, TMP@D was obtained.

[0013] Optionally,

[0014] In S1: 20 mg PLGA, 2 mg TPZ, and 1 mg MnTTP were weighed and added to 1 mL of dichloromethane. After ultrasonic oscillation for 10 min, 50 mg PVA with a molecular weight of 20,000-40,000 was added dropwise to a solution formed by dissolving 5 mL of pure water. The mixture was stirred in the dark for 12 h, and the reaction system changed from a water-oil separation structure to a yellow, clear, oil-in-water structure.

[0015] In S2: the pH of the solution obtained in S1 is adjusted to 8.5 using Tris base;

[0016] In S3: 8 mg of dopamine hydrochloride powder was weighed and added to the solution obtained in S2. After the reaction system was ultrasonically vibrated for 5 min, it was transferred to a magnetic stirrer and stirred continuously at room temperature in the dark for 12 h. The solution became a black aqueous solution without precipitate.

[0017] In step S4, the solution obtained in step S3 is filtered through a liposome extruder and a 0.4 μm filter membrane, and then subjected to ultrafiltration centrifugation at 5000 rpm for 10 min using a 10 kd ultrafiltration tube. The solution is repeatedly filtered and washed with pure water until the filtrate is clear. The final solution is then freeze-dried in a freeze dryer for 48 h to obtain TMP@D, which is stored at 4 °C.

[0018] Optionally, the preparation method of the nanoprobe for multimodal imaging and synergistic therapy further includes: testing the effect of TMP@D in releasing singlet oxygen, specifically by using different concentrations of TMP@D at the same ultrasonic power and the same concentration of TMP@D at different ultrasonic frequencies as experimental groups; using 1.5 W·cm -2 Ultrasound was used as the probe excitation source, and pure water was used as the control group. The TMP@D concentrations were 50, 100, 200, and 400 μg·mL, respectively. -1 As one of its experimental groups; 1.5 W·cm -2 Ultrasound was used as the probe excitation source, with 0 ultrasounds serving as the control group. The number of ultrasounds was 1, 2, 3, 4, 5, and 6, respectively, forming two experimental groups with an interval of 1 minute. SOSG was used as the singlet oxygen detection reagent.

[0019] Optionally, the radionuclide 131 The I tag on TMP@D includes:

[0020] radioactive nuclides 131 I was labeled on TMP@D using the chloramine-T method.

[0021] Optionally, the radionuclide 131 I was labeled onto TMP@D via the chloramine-T method, including:

[0022] 3mg TMP@D, 2mCi Na131 I and 400 μL of chloramine T aqueous solution were added sequentially to a 25 mL reaction flask and mixed and stirred at room temperature in the dark. The resulting solution was then subjected to repeated ultrafiltration and centrifugation using a 10 kDa ultrafiltration tube at a speed of 5000 rpm for 10 min, followed by washing, until the filtrate was inactive.

[0023] Optionally, the concentration of TMP@D is 3 mg·mL. -1 The concentration of chloramine T aqueous solution is 10 mg·mL -1 The stirring time is 15 minutes.

[0024] Optionally, the preparation method of the nanoprobe for multimodal imaging and synergistic therapy further includes: detection 131 The nuclide stability efficiency of I-TMP@D is specifically detected by the following method: [The text abruptly ends here, likely due to an incomplete sentence or a missing section.] 131 I-TMP@D was placed in 2 mL of PBS and 2 mL of 1640 medium containing 10% fetal bovine serum, respectively; measurements were taken at different time points. 131 The radioactivity before and after I-TMP@D ultrafiltration is recorded as CPM0 before centrifugation and CPM1 after centrifugation with the probe retained. The formula for calculating the nuclide stabilization efficiency is: Nuclide stabilization efficiency = CPM1 / CPM0*100%.

[0025] Optionally, the preparation method of the nanoprobe for multimodal imaging and synergistic therapy further includes: [further details needed - likely related to the preparation of nanoprobes]. 131 I-TMP@D was used for toxicity testing and synergistic therapy testing against pancreatic cancer cells; 131 The specific detection method for I-TMP@D toxicity is as follows: using different concentrations... 131 I-TMP@D was used as the experimental group, and after incubation with BxPC3 cells for 4 hours, cell viability was detected using CCK-8 assay. The specific detection method for synergistic treatment of pancreatic cancer cells was as follows: 131 I, TMP@D, 131 I-TMP@D, TMP@D+US and 131 I-TMP@D+US was used as the experimental group, and the control group was added with an equal amount of culture medium. After incubation for 4 hours, the power was increased to 1.5 W·cm⁻¹. -2 Cells were irradiated with US radiation at 15-second intervals for 3 minutes, and then cell viability was detected using CCK-8 assay.

[0026] Secondly, an application of a nanoprobe for multimodal imaging and synergistic therapy is provided, specifically as described above. 131 Application of I-TMP@D in MRI and SPECT imaging of pancreatic cancer.

[0027] All of the above-mentioned optional technical solutions can be combined arbitrarily, and the present invention will not provide a detailed description of the structure after each combination.

[0028] By means of the above solution, the beneficial effects of the present invention are as follows:

[0029] The acoustic sensitizer MnTTP and the hypoxia-activating drug TPZ were loaded onto PLGA via an emulsion-solvent evaporation method, and then modified into an aqueous phase using PVA. Dopamine hydrochloride was used to enhance biocompatibility, resulting in the nanoprobe TMP@D. Then, a radionuclide was... 131 I-labeling on TMP@D yields radionuclide-loaded nanoprobes. 131 I-TMP@D. Because the tumor microenvironment is weakly acidic, dopamine will break down under acidic conditions, releasing MnTTP and TPZ from the core. Under ultrasound stimulation, MnTTP releases ROS, consuming more oxygen and making the tumor tissue even more hypoxic. 131 I-radiation splits water molecules to produce ROS, which, in turn, further reduces the oxygen content of tumor tissue, activates more TPZ to convert into toxic TPZ, and generates more ROS that act on the tumor tissue; in addition... 131 The gamma rays generated during the decay of I-TMP@D can be used for SPECT imaging; simultaneously, the Mn in the MnTTP porphyrin ring... 2+ It can be used for MR-T1 imaging. In summary, 131 I-TMP@D passively targets and enters tumor cells, enabling synergistic treatment with MR and SPECT multimodal imaging, radionuclide therapy, sonodynamic therapy, and hypoxia-activated chemotherapy, especially in the integrated diagnosis and treatment of malignant hypoxic tumors.

[0030] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it in accordance with the contents of the specification, the preferred embodiments of the present invention are described in detail below with reference to the accompanying drawings. Attached Figure Description

[0031] Figure 1 This is a particle size distribution diagram of TMP and TMP@D;

[0032] Figure 2 These are the UV-Vis absorption spectra of TPZ, MnTTP, and TMP@D;

[0033] Figure 3 These are the fluorescence emission spectra of TPZ, MnTTP, and TMP@D;

[0034] Figure 4 This is a potential diagram of TPZ, MnTTP, and TMP@D;

[0035] Figure 5TMP@D at concentrations of 0, 50, 100, 200, and 400 μg / mL at 1.5 W·cm⁻¹ -2 Schematic diagram of singlet oxygen release intensity under ultrasonic power;

[0036] Figure 6 It is TMP@D at a concentration of 100 μg / mL at 1.5 W·cm -2 Schematic diagram of singlet oxygen release intensity at ultrasonic power levels 0, 1, 2, 3, 4, 5, and 6 times;

[0037] Figure 7 yes 131 A schematic diagram showing the radiostability of I-TMP@D over time in PBS and 1640 medium containing 10% FBS;

[0038] Figure 8 yes 131 I and 131 A schematic diagram of the radiochemical purity of I-TMP@D using acetone as the developing solvent;

[0039] Figure 9 This is a schematic diagram showing the cell survival rate of BxPC-3 cells after treatment with different concentrations of probes.

[0040] Figure 10 This is a schematic diagram showing the cell survival rate of BxPC-3 cells after treatment in different treatment groups;

[0041] Figure 11 Intratumoral injection in mice 131 I-TMP@D acquired MR-T1 images of mice at different time points;

[0042] Figure 12 Injected into the tumor of mice separately 131 I and 131 I-TMP@D acquired whole-body SPECT images of mice at different time points;

[0043] Figure 13 This is a flowchart of the preparation method of nanoprobes for multimodal imaging and synergistic therapy provided in the embodiments of the present invention. Detailed Implementation

[0044] The specific embodiments of the present invention will be described in further detail below with reference to the accompanying drawings and examples. The following examples are for illustrative purposes only and are not intended to limit the scope of the invention.

[0045] The present invention provides a method for preparing nanoprobes for multimodal imaging and synergistic therapy. This method involves loading the acoustic sensitizer MnTTP and the hypoxia-activating drug TPZ onto a PLGA via an emulsion-solvent evaporation method, modifying it to an aqueous phase using PVA, and using dopamine hydrochloride to increase biocompatibility, resulting in the nanoprobe TMP@D. Then, a radionuclide is... 131 I-labeling on TMP@D yields radionuclide-loaded nanoprobes. 131 I-TMP@D.

[0046] In one specific embodiment, the synthesis steps of TMP@D are as follows:

[0047] S1, PLGA, MnTTP and TPZ are co-dissolved in dichloromethane, ultrasonically vibrated and then PVA is added dropwise while stirring in the dark to form an oil-in-water structure;

[0048] S2, the solution obtained from S1 is adjusted with Tris base;

[0049] S3, add dopamine hydrochloride to the solution obtained in S2, and continue stirring after ultrasonic vibration;

[0050] S4. The solution obtained in S3 was filtered through a liposome extruder, and then repeatedly ultrafiltered to retain 10kd molecular weight nanoprobes. After freeze-drying, TMP@D was obtained.

[0051] More specifically, in S1: 20 mg PLGA, 2 mg TPZ, and 1 mg MnTTP are weighed and added to 1 mL of dichloromethane. After ultrasonic oscillation for 10 min, 50 mg PVA with a molecular weight of 20,000-40,000 is added dropwise to a solution formed by dissolving 5 mL of pure water. The mixture is stirred in the dark for 12 h, and the reaction system changes from a water-oil separation structure to a clear yellow water-in-oil structure.

[0052] In S2: the pH of the solution obtained in S1 is adjusted to 8.5 using Tris base;

[0053] In S3: 8 mg of dopamine hydrochloride powder was weighed and added to the solution obtained in S2. After the reaction system was ultrasonically vibrated for 5 min, it was transferred to a magnetic stirrer and stirred continuously at room temperature in the dark for 12 h. The solution became a black aqueous solution without precipitate.

[0054] In step S4, the solution obtained in step S3 is filtered through an Avanti Polar Lipids liposome extruder and a 0.4 μm filter membrane. Then, it is ultrafiltered using a 10 kDa Millipore ultrafiltration tube at 5000 rpm for 10 min. The solution is washed three times with pure water until the filtrate is clear. The final solution is then freeze-dried for 48 h to obtain TMP@D, which is stored at 4°C.

[0055] In another embodiment, when TMP@D is prepared, a radionuclide is further prepared. 131 The I-marked TMP@D is... 131 The I-TMP@D method involves: [the radionuclide is then...] 131 I was labeled onto TMP@D using the chloramine-T method. Specifically: 3 mg TMP@D and 2 mCi Na were added. 131 I and 400 μL of chloramine T aqueous solution were sequentially added to a 25 mL reaction flask and mixed and stirred at room temperature in the dark. The resulting solution was then subjected to repeated ultrafiltration and centrifugation using a 10 kDa Millipore ultrafiltration tube at 5000 rpm for 10 min, followed by washing, until the filtrate was inactive. Preferably, the concentration of TMP@D is 3 mg·mL⁻¹. -1 The concentration of chloramine T aqueous solution is 10 mg·mL -1 The stirring time is 15 minutes.

[0056] To verify the performance of TMP@D prepared by the preparation method provided in this embodiment of the invention, the components of TMP@D were also characterized, referring to... Figure 1-4 .

[0057] The specific method is as follows: TMP (formed by stirring for 12 hours before adding dopamine) and TMP@D are dispersed in deionized water, and the particle size is measured using a Malvern particle size analyzer. The results are as follows: Figure 1 As shown. By Figure 1 The average particle size of TMP@D is 101.6 ± 9.851 nm, making it suitable for tumor treatment via the EPR effect.

[0058] The TMP@D, MnTTP, and TPZ prepared above were used to form suspensions. Baseline correction was performed with pure water, and the UV-Vis spectrum from 230 nm to 600 nm was scanned. The results are shown in the appendix. Figure 2 As shown. By Figure 2It was found that TMP@D exhibited characteristic absorption peaks at 274 nm and 474 nm. The characteristic absorption peak at 274 nm was consistent with that of TPZ, while the characteristic absorption peak at 474 nm was consistent with that of both TPZ and MnTTP. This indicates that TPZ was ultimately loaded into TMP@D, but the UV-Vis spectroscopy results did not confirm successful loading of MnTTP.

[0059] Furthermore, the TMP@D, MnTTP, and TPZ solutions were excited at 360 nm, and the emission spectra of each component in the 420-700 nm range were measured. The results are shown in the appendix. Figure 3 As shown. By Figure 3 It can be seen that the emission spectrum of TMP@D in the 470-550nm range is consistent with that of MnTTP, but inconsistent with that of TPZ, indicating that TMP@D was successfully loaded with MnTTP.

[0060] Ultimately, the combined ultraviolet-visible spectroscopy and fluorescence emission spectroscopy demonstrated that TMP@D was successfully loaded with TPZ and MnTTP.

[0061] In addition, the potentials of TMP@D, MnTTP, and TPZ were measured respectively, and the results are attached. Figure 4 As shown. By Figure 4 It can be seen that TMP@D exhibits a negative potential and is stable at -26.46±1.93mV, indicating that TMP@D carries a negative charge on its surface and can be dispersed stably in solution.

[0062] Furthermore, the preparation method of the nanoprobe for multimodal imaging and synergistic therapy provided in this embodiment of the invention also includes testing the effect of TMP@D in releasing singlet oxygen. Specifically, the testing method involves testing the generation of singlet oxygen by different concentrations of TMP@D at the same ultrasonic power and by the same concentration of TMP@D at different ultrasonic cycles. (Using 1.5 W·cm⁻¹) -2 Ultrasound was used as the probe excitation source, pure water as the control group, and TMP@D concentrations of 50, 100, 200, and 400 μg / mL were used as experimental groups; 1.5 W·cm -2 Ultrasound was used as the probe excitation source. Zero ultrasound cycles served as the control group, and ultrasound cycles of 1, 2, 3, 4, 5, and 6 cycles were used as the two experimental groups, with a 1-minute interval between each. SOSG was used as the singlet oxygen detection reagent. The test results are as follows: Figure 5 and Figure 6 As shown.

[0063] Depend on Figure 5 It can be seen that, under the same ultrasonic power, the higher the TMP@D concentration, the more singlet oxygen is released. From... Figure 6 It can be seen that for the same concentration of TMP@D, under different sonication times, the more sonication times, the more singlet oxygen is produced.

[0064] In another embodiment, the method for preparing the nanoprobe for multimodal imaging and synergistic therapy further includes: detecting... 131 The nuclide stability efficiency of I-TMP@D is specifically detected by the following method: [The text abruptly ends here, likely due to an incomplete sentence or a missing section.] 131 I-TMP@D was placed in 2 mL of PBS and 2 mL of 1640 medium containing 10% fetal bovine serum (FBS), respectively; measurements were taken at different time points. 131 The radioactivity before and after I-TMP@D ultrafiltration is recorded as CPM0 before centrifugation and CPM1 after centrifugation with the probe retained. The formula for calculating the nuclide stabilization efficiency is: Nuclide stabilization efficiency = CPM1 / CPM0*100%.

[0065] Radiostability over time, such as Figure 7 As shown, by Figure 7 We can obtain, 131 The high labeling efficiency and radiostability of I-TMP@D within 7 days give it a high labeling efficiency and stability. 131 I-TMP@D offers better biocompatibility, reducing the release of radionuclides into other normal tissues during in vivo treatment and providing a safety guarantee for radionuclide therapy.

[0066] Furthermore, 131 I and 131 I-TMP@D was spotted onto a silica gel thin-layer chromatography plate, and acetone was used as the developing solvent. The radiochemical purity was determined using an FC-3600 Flow-Count system (Bioscan, America). The results are as follows: Figure 8 As shown. By Figure 8 It can be obtained that, with 131 I. Compare with (Figure a) to illustrate 131 The radionuclides on I-TMP@D (Figure b) exist stably in a form chelated onto nanoparticles.

[0067] Furthermore, the method for preparing nanoprobes for multimodal imaging and synergistic therapy provided in this embodiment of the invention further includes: [further details needed - likely related to the preparation of nanoprobes for multimodal imaging and synergistic therapy]. 131 I-TMP@D was used for toxicity testing and synergistic therapy testing against pancreatic cancer cells. 131 The specific detection method for I-TMP@D toxicity is as follows: using different concentrations... 131 I-TMP@D was used as the experimental group. After incubating BxPC3 (human orthotopic pancreatic cancer cells) cells for 4 hours, cell viability was detected using CCK-8 assay. The specific detection method for synergistic treatment of pancreatic cancer cells was as follows: 131 I, TMP@D, 131 I-TMP@D, TMP@D+US and 131I-TMP@D+US was used as the experimental group, and the control group was added with an equal amount of culture medium. After incubation for 4 hours, the power was increased to 1.5 W·cm⁻¹. -2 Cells were irradiated with US radiation at 15-second intervals for 3 minutes, and then cell viability was detected using CCK-8 assay.

[0068] Specifically, 131 During I-TMP@D toxicity assay: BxPC3 cells were cultured at 5 × 10⁻⁶ 3 Cells were seeded at high density in 96-well plates and incubated overnight at 37°C. After washing away the culture medium with PBS, 100 μL of different concentrations of TMP@D (100, 200, 400, 800, 1000 μg / mL) were added to the experimental groups, 100 μL of 1640 medium was added to the control group, and 100 μL of 1640 medium was added to the blank group (which contained no cells). Five parallel sample wells were set up for each group. Different concentrations... 131 After incubating I-TMP@D and BxPC3 cells in an incubator for 4 hours, the cells were washed three times with PBS, incubated with fresh 1640 medium for 20 hours, and then 90 μL of 1640 and 10 μL of CCK-8 were added to each well. After 4 hours, the OD value was measured at 450 nm using a microplate reader, and the cell viability was calculated. The experimental results are as follows. Figure 9 As shown.

[0069] Depend on Figure 9 It can be concluded that the cytotoxicity of TMP@D itself can be judged by cell survival rate. Under normoxic conditions, the cell survival rate of high concentration of TMP@D exceeds 80%, indicating that it has almost no cytotoxicity. Under hypoxic conditions, the cell survival rate is low, indicating that TMP@D is beneficial for the treatment of hypoxic tumors.

[0070] Specifically, when performing synergistic therapy testing on pancreatic cancer cells: BxPC3 cells were administered at a concentration of 5 × 10⁻⁶ cells / year. 3 The culture medium was seeded at a density in 96-well plates and incubated overnight at 37°C. After washing away the culture medium with PBS, 100 μL of the culture medium was added to the experimental group. 131 I, TMP@D, 131 I-TMP@D, TMP@D+US and 131 I-TMP@D+US (50 μCi·mL) -1 2 mg·mL -1 The control group received 100 μL of 1640 medium, while the blank group (containing no cells) received 100 μL of 1640 medium. Five parallel sample wells were set up for each group. After cell treatment, each experimental group was washed three times with PBS, incubated with fresh 1640 medium for 20 h, and then 90 μL of 1640 and 10 μL of CCK-8 were added to each well. After 4 h, the OD value was measured at 450 nm using a microplate reader, and cell viability was calculated. Experimental results are as follows: Figure 10 As shown.

[0071] Depend on Figure 10 It can be seen that the cell survival rate after synergistic treatment under hypoxic conditions was only 22.36±6.626%, indicating that the treatment method of radionuclide therapy + sonodynamic therapy + hypoxic activating chemotherapy can greatly kill tumor cells.

[0072] In addition, this embodiment of the invention also performed in vivo imaging of the nanoprobe. Specifically, the tumor-bearing mouse model used 4-week-old male mice, and 1×10⁻⁶ nanoprobes were used to image the nanoprobes in vivo. 7 Cells were subcutaneously inoculated into the backs of mice. The tumors were allowed to grow to 800 mm. 3 At that time, TMP@D (10 mg / mL) was injected intratumorally. -1 MRI imaging was performed at different time points (0, 1, 3, 6, 12, and 24 hours) using 100 μL of ammonium chloride (m³). The imaging results are as follows: Figure 11 As shown. By Figure 11 It can be seen that TMP@D has a T1 relaxation response in vivo, and after intratumoral injection, the signal at the tumor site gradually diffuses and maintains a high signal over time.

[0073] The tumor volume reached 800 mm. 3 Tumor-bearing mice were randomly divided into two groups, and intratumoral injections were administered to each group. 131 I and 131 SPECT imaging was performed on I-TMP@D (200 μCi, 100 μL) at different time points: 0, 0.5, 1, 3, 6, 12, and 24 h. The imaging results are as follows: Figure 12 As shown. By Figure 12 It can be obtained that, with 131 Compared to I, 131 I-TMP@D can remain stable at the tumor site for at least 12 hours, while 131 I was present at the tumor site for less than 3 hours, proving 131 I-TMP@D can remain stably present at the tumor site for synergistic therapy.

[0074] It should be noted that in the above-mentioned embodiments of the present invention, statistical analysis was performed using Graphpad 10.1.1 software. The independent samples t-test was used for comparisons between two groups, and one-way ANOVA was used for comparisons among multiple groups. P < 0.05 was considered statistically significant.

[0075] In summary, the multimodal imaging and diagnostic probe provided in this embodiment of the invention... 131 I-TMP@D, with a core consisting of a nanocluster composite structure formed by PLGA-loaded MnTTP and TPZ, was modified with polyvinyl alcohol (PVA) to an aqueous phase and then coated with dopamine hydrochloride (PDA). Radionuclides were then labeled using the chloramine-T method. 131 I. Aqueous nanoclusters facilitate endocytosis into tumor cells, while the externally encapsulated dopamine hydrochloride provides... 131I labeling sites also increases the biocompatibility of nanoprobes, improves their size, and enhances their permeability and retention effect (EPR effect) at tumor sites.

[0076] Due to the hypoxia-activated properties of the probe, 131 I-TMP@D demonstrates excellent multimodal imaging and synergistic therapeutic capabilities in pancreatic cancer, a highly hypoxic malignant tumor, and is particularly suitable for MRI and SPECT imaging of pancreatic cancer.

[0077] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the technical principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for the preparation of a nanoprobes for multimodal imaging and synergistic therapy, characterized by, Comprise: The radiosensitizer MnTTP and the hypoxia-activated drug tirapazamine TPZ are loaded on PLGA by emulsion-solvent evaporation method, and modified into water phase under the action of PVA, and the biocompatibility is increased by using dopamine hydrochloride, to obtain the nanoprobe TMP@D, and then the radionuclide 131 I is labeled on TMP@D to obtain the radionuclide-loaded nanoprobe 131 I-TMP@D; The synthesis steps of the TMP@D are as follows: S1, PLGA, MnTTP and TPZ are dissolved in dichloromethane, and PVA is added dropwise after ultrasonic oscillation to form an oil-in-water structure under dark stirring; S2, adjust the solution obtained in S1 with Tris base; S3, add dopamine hydrochloride to the solution obtained in S2, and continue stirring after ultrasonic oscillation; S4, filter the solution obtained in S3 through a liposome extruder, then repeatedly ultrafiltrate the 10 kd molecular weight nanoprobes, and obtain TMP@D after freeze-drying.

2. The preparation method of the nanoprobes for multi-modal imaging and synergistic treatment according to claim 1, characterized in that, In S1, 20 mg of PLGA, 2 mg of TPZ and 1 mg of MnTTP are weighed and added into 1 mL of dichloromethane, ultrasonic oscillation is performed for 10 min, then 50 mg of PVA with a molecular weight of 20,000-40,000 is dissolved in 5 mL of pure water to form a solution, which is added dropwise into the reaction system, and dark stirring is performed for 12 h, and the reaction system changes from a water-oil separation structure to a yellow clear oil-in-water structure; In S2, the pH of the solution obtained in S1 is adjusted to 8.5 using Tris base; In S3, 8 mg of dopamine hydrochloride powder is added to the solution obtained in S2, the reaction system is ultrasonic oscillated for 5 min, then transferred to a magnetic stirrer, and continuously stirred at room temperature in the dark for 12 h, and the solution becomes a black water solution without precipitation; In S4, the solution obtained in S3 is filtered through a liposome extruder and a 0.4 μm filter membrane, then ultrafiltration centrifugation is performed at a speed of 5,000 rpm for 10 min using a 10 kd ultrafiltration tube, and the solution is repeatedly filtered and washed with pure water until the filtrate is clear, then the final solution is subjected to freeze-drying in a freeze-drying machine for 48 h to obtain TMP@D, which is stored in a 4 °C refrigerator.

3. The method for preparation of nanoprobe for multimodal imaging and synergistic therapy as claimed in claim 1, wherein, Also include: The effect of TMP@D on the release of singlet oxygen was tested. The specific test method was as follows: different concentrations of TMP@D, same ultrasonic power and same concentration of TMP@D with different ultrasonic times as experimental groups; 1.5 W·cm -2 Pure water as a control group, TMP@D concentrations were 50, 100, 200, and 400 μg·mL -1 as one of the experimental groups; 1.5 W·cm -2 Ultrasonic as probe excitation source, 0 times of ultrasonic as control group, 1, 2, 3, 4, 5, and 6 times of ultrasonic as the second experimental group with an interval of 1 min; SOSG as a singlet oxygen detection reagent.

4. The method for preparation of nanoprobe for multimodality imaging and synergistic therapy as claimed in claim 1 wherein, The radionuclide 131 I is labeled on TMP@D, comprising: The radionuclide 131 I was labeled on TMP@D by the chloramine-T method.

5. The method for preparation of nanoprobe for multimodality imaging and synergistic therapy as claimed in claim 4 wherein, The radionuclide 131 I was labeled on TMP@D by the chloramine-T method, including: 3 mg of TMP@D, 2 mCi of Na 131 I and 400 μL of an aqueous solution of chloramine T were sequentially put into a 25 mL reaction flask, mixed and stirred at room temperature in the dark, and the resulting solution was repeatedly ultrafiltration centrifuged using a 10 kd ultrafiltration tube at a rotation speed of 5000 rpm for 10 min, washed until the filtrate was not active.

6. The method for preparation of nanoprobe for multimodality imaging and synergistic therapy as claimed in claim 5 wherein, The concentration of the TMP@D is 3 mg·mL -1 The concentration of the chloramine T aqueous solution is 10 mg·mL -1 The stirring time is 15 min.

7. The method of claim 1 to 6, wherein, Also include: Detection 131 The nuclide stability efficiency of I-TMP@D was detected. The specific detection method was as follows: the same activity of I-TMP@D was respectively placed in 2 mL PBS and 2 mL 1640 culture medium containing 10% fetal bovine serum; the radioactivity of I-TMP@D before and after ultrafiltration was measured at different time points, and the radioactivity before centrifugation was recorded as CPM0, and the radioactivity of the intercepted probe after centrifugation was recorded as CPM1. 131 The nuclide stability efficiency of I-TMP@D was detected. The specific detection method was as follows: the same activity of I-TMP@D was respectively placed in 2 mL PBS and 2 mL 1640 culture medium containing 10% fetal bovine serum; the radioactivity of I-TMP@D before and after ultrafiltration was measured at different time points, and the radioactivity before centrifugation was recorded as CPM0, and the radioactivity of the intercepted probe after centrifugation was recorded as CPM1. 131 The nuclide The calculation formula of the nuclide stabilization efficiency is: nuclide stabilization efficiency = CPM1 / CPM0 x 100%. Also include: Also include: The calculation formula of the nuclide stabilization efficiency is: nuclide stabilization efficiency = CPM1 / CPM0 x 100%.

8. The method of claim 1 to 6, wherein, Also including: toxic detection of I-TMP@D and synergistic treatment detection of pancreatic cancer cells; 131 I-TMP@D, and synergistic treatment detection of pancreatic cancer cells; 131 The specific detection method of I-TMP@D toxicity is: using different concentrations of I-TMP@D to incubate BxPC3 cells for 4 hours, and then using CCK-8 to detect cell survival rate. 131 I-TMP@D as the experimental group, and BxPC3 cells were incubated for 4 hours, and then CCK-8 was used to detect cell survival rate; the specific detection method of synergistic treatment of pancreatic cancer cells is: using different concentrations of I-TMP@D to incubate BxPC3 cells for 4 hours, and then using CCK-8 to detect cell survival rate. 131 I, TMP@D, 131 I-TMP@D, TMP@D+US and 131 I-TMP@D+US as the experimental group, and the control group was added with the same amount of culture medium, and then incubated for 4 hours, and then irradiated with US with power of 1.5 W·cm -2 , interval 15s, time 3min, and then using CCK-8 to detect cell survival rate.

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