Preparation method of imaging and treatment integrated nanoprobe based on biomass transformation

Abstract

The invention aims to provide an imaging and treatment integrated nanoprobe based on biomass transformation and a preparation method, the nanoprobe is of a sphere-like core-shell structure, the core is nano vinegar, metal ions are coupled, and the exterior of the nanoprobe is coated with a 4T1 tumor cell membrane, and the core of the sphere-like structure of the nanoprobe is finely screened nano vinegar, so that the nano vinegar has the advantages that the nano vinegar has the advantages that the structure is simple, the cost is low, and the application range is wide. According to the nano-probe, the anti-tumor effect is enhanced by coupling metal ion manganese, tumor cells are killed through generated OH, singlet oxygen (1O2) and heat, in addition, the aggregation of tumor parts is increased by externally coating 4T1 tumor cell membranes, and finally, the nano-probe integrating efficient tumor killing, high targeting and fluorescence imaging is realized.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical nanomaterials, and in particular relates to an imaging and treatment integrated nanoprobe based on biomass transformation and a preparation method thereof. Background Art

[0002] Cancer is one of the main causes of death in humans. Although the means of treating tumors have increased with the continuous development of medicine, they still cannot significantly prolong the survival of tumor patients or significantly improve their quality of life. Moreover, the treatment process cannot be visualized clinically at present. The only way to evaluate the treatment effect is to observe the tumor size by imaging after treatment.

[0003] Independent of existing clinical surgery, radiotherapy, chemotherapy, targeted therapy and immunotherapy, chemodynamic therapy (CDT) is an emerging treatment strategy that relies on endogenous H2O2 in tumors to convert it into highly toxic hydroxyl radicals (•OH) through Fenton or Fenton-like reactions. It can effectively kill tumor cells by destroying DNA chains, proteins and other biological molecules in cells. Photothermal and photodynamic therapy are also emerging anti-tumor treatment methods. They often use laser energy and work together with optical imaging to cleverly exert anti-tumor effects while imaging. The above new methods are expected to overcome the high toxicity of traditional radiotherapy and chemotherapy, providing a new option for anti-tumor treatment.

[0004] The in vivo visualization of anti-tumor drugs is of great significance for clinical treatment. It can directly observe the drug enrichment in tumor lesions in the body, and monitor its distribution in other organs to evaluate the occurrence of adverse reactions. Drug visualization methods include radionuclide imaging, magnetic resonance imaging (MRI), computed tomography (CT) and fluorescence imaging. The most commonly used method is to label the imaging elements on the drug molecules, so as to understand the distribution of drugs in the body in real time. Near-infrared II (NIR-II) fluorescence imaging has the characteristics of high signal-to-noise ratio, low autofluorescence, and deep penetration, and has attracted widespread attention. In addition, there are some emerging technologies for in vivo visualization of drugs, such as photoacoustic imaging and multiphoton microscopy. The development of these new technologies provides more tools and approaches for drug research. Summary of the invention

[0005] The purpose of the present invention is to provide an integrated imaging and therapeutic nanoprobe based on biomass transformation and a preparation method. The nanoprobe can synergistically kill tumor cells with efficient POD enzyme activity, CDT, photothermal and strong photodynamic therapy, and at the same time release fluorescence to play a role in drug visualization and tumor imaging.

[0006] The present invention adopts the following technical solution: A nanoprobe for integrated imaging and treatment based on biomass transformation. The nanoprobe is a quasi-spherical core-shell structure, wherein the inner core is nanoacetic acid coupled with metal ions and the outer surface is coated with a 4T1 tumor cell membrane.

[0007] A method for preparing an integrated imaging and therapeutic nanoprobe based on biomass transformation comprises the following steps: In the first step, the aged vinegar of more than 5 years is centrifuged at 12000 rpm for 20 min, the supernatant is aspirated and the precipitate is discarded, and the supernatant is retained; In the second step, a dialysis bag with a molecular weight of 8000-14000Da was placed in boiling water at 100°C for 5 minutes, and the dialysis bag was fully rinsed with ultrapure water; The third step is to place the supernatant obtained in the first step into the dialysis bag described in the second step, clamp both ends of the dialysis bag, place it in ultrapure water, put magnetic beads in the ultrapure water, stir at a speed of 1000 rpm under room temperature, and regularly replace the ultrapure water until the color of the ultrapure water becomes clear; Step 4: After the dialysis is completed, the liquid in the dialysis bag is packaged, frozen and dried at -20°C to obtain SAV, which is sealed and stored at room temperature; Step 5: The SAV aqueous solution was adjusted to pH 2, placed at 60°C, incubated for 2 h, dialyzed to remove acidity, and frozen to obtain HSAV; Step 6, preparing HSAV aqueous solution and manganese chloride aqueous solution respectively, mixing the HSAV aqueous solution and the manganese chloride aqueous solution, adding magnetic beads, stirring overnight, and then dialyzing and freeze-drying to obtain HSAVMn; Step 7: The 4T1 cells were broken and centrifuged to obtain the supernatant protein; In the eighth step, the supernatant of the seventh step was mixed with the HSAVMn of the sixth step, separated by a 30 KD ultrafiltration separation tube at a speed of 3500 rpm for 10 min, and washed three times with deionized water to obtain the nanocomplex HSAVMn@CM coated with 4T1 cell membrane protein.

[0008] Furthermore, the time for the scheduled replacement in the third step is once every 1 hour, which is changed to once every 3 hours after 3 replacements, and then changed to once every 6 hours after 3 replacements.

[0009] Furthermore, in the HSAV aqueous solution in the sixth step, the mass of HSAV is 5-25 mg, and the volume of deionized water is 1 mL; in the manganese chloride aqueous solution, the mass of manganese chloride is 1.799 mg, and the volume of deionized water is 1 mL.

[0010] Furthermore, the number of 4T1 cells in the seventh step is 8-12 million.

[0011] The nanoprobe of the present invention has a spherical structure with a finely selected nanoester as the core, which can enhance the anti-tumor effect by coupling metal ions, thereby generating •OH, singlet oxygen ( 1 O2) and heat to kill tumor cells. In addition, the external coating of 4T1 tumor cell membrane increases the aggregation of tumor sites, ultimately realizing a nanoprobe that combines efficient tumor killing, high targeting and fluorescence imaging.

[0012] The nanoprobe prepared by the preparation method of the present invention can cooperate with efficient POD enzyme activity, CDT, photothermal and strong photodynamic therapy to kill tumor cells, and release fluorescence to play the role of drug visualization and tumor imaging.

[0013] The beneficial effects of the present invention are as follows: The present invention discloses a diagnosis and treatment integrated nanoprobe, which has the EPR effect in the solid tumor area and the targeting effect on the tumor cell membrane, can produce •OH in the tumor H2O2 environment, and can produce after 808 nm laser irradiation. 1 O2 and heat generation, thus killing tumor cells. In addition, the nanoprobe can also perform NIR-Ⅱ imaging while playing an anti-tumor role.

[0014] In addition, since the emitted light of this material is NIR-Ⅱ fluorescence, it has a deeper penetration depth, which can achieve better imaging and guided treatment effects. At the same time, this material has POD enzyme activity, chemical kinetics, photodynamic and photothermal properties, which can effectively kill tumor cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is a transmission electron micrograph of the biomass nano vinegar prepared in Example 1 of the present invention.

[0016] Figure 2 The POD enzyme activity of an integrated imaging and therapeutic nanoprobe based on biomass nanovinegar modification synthesized in Example 1 of the present invention.

[0017] Figure 3 The photodynamic capability of an integrated imaging and therapeutic nanoprobe based on biomass vinegar transformation synthesized in Example 1 of the present invention. DETAILED DESCRIPTION

[0018] The technical solution of the present invention will be clearly and completely described below in combination with the embodiments of the present invention and the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0019] Example 1 Preparation of an integrated imaging and therapeutic nanoprobe based on biomass transformation: Take 20 mL of Shanxi vinegar that has been aged for more than 5 years, carefully observe its properties and smell its odor, and keep it for later use if there is no abnormality; Place the prepared vinegar in a high-speed centrifuge at a speed of 12,000 rpm for 20 min. After centrifugation, keep the supernatant and discard the precipitate. Repeat high-speed centrifugation according to the above steps, discard the precipitate, and keep the final supernatant for later use; Place a dialysis bag with a molecular weight of 8000-14000 Da in boiling water at 100 °C for 5 min, and rinse the dialysis bag thoroughly with ultrapure water; Place the prepared supernatant in the prepared dialysis bag and clamp both ends to prevent leakage; Place the large rotor at the bottom of a 2000 mL beaker, add 1800 mL of deionized water, and place the dialysis bag clamped in the previous step in the deionized water; Place the large beaker obtained in the previous step on a stirrer and set the speed to 1000 rpm. Adjust the speed as appropriate based on the rotation of the rotor and dialysis bag. Replace the above dialysate every 1 hour. After 3 changes, change to every 3 hours. After 3 changes, change to every 6 hours until the dialysate becomes clear. Take the final dialyzed liquid and divide it into individual flash bottles, each containing 2 mL, and freeze it in a -20 ℃ refrigerator; The above-mentioned product is placed in a freeze dryer and freeze-dried to obtain SAV, i.e., nano vinegar, which is finally packaged into EP tubes, sealed with sealing film and stored at room temperature; SAV was placed in a solution at 60 °C and pH = 2 for 2 h, then dialyzed to remove the acidity and freeze-dried to obtain HSAV; Weigh 1.799 mg of manganese chloride tetrahydrate and 1 mL of deionized water, add them into the EP tube and mix thoroughly to obtain a manganese chloride aqueous solution for later use; Weigh 5 mg of the above HSAV powder and 1 mL of deionized water, add them into the EP tube and mix thoroughly to obtain a HSAV aqueous solution for later use; Take 1 mL of the above HSAV aqueous solution and 1 mL of manganese chloride aqueous solution, add magnetic beads, and stir vigorously (880 rpm) overnight; then dialyze and freeze-dry to obtain HSAVMn.

[0020] The number of 4T1 cells was cultured to 10 million, digested and centrifuged, and dissolved in ultrapure water as a carrier. The ultrasonic crushing instrument power was 10%, the crushing time was 5 s, the interval time was 5 s, and the total time was 20 min. The crushing was carried out on ice. After crushing, the centrifugation speed was 12000 rpm, the time was 20 min, and the supernatant was retained for high-speed centrifugation at 4 ℃.

[0021] The supernatant obtained in the above steps was mixed with 10 mg HSAVMn, separated by 30 KD ultrafiltration separation tube at 3500 rpm for 10 min, and washed three times with deionized water to obtain the 4T1 cell membrane protein-coated nanocomplex HSAVMn@CM.

[0022] Figure 1 This is a transmission electron microscope image of the biomass nano vinegar prepared in Example 1 of the present invention. It can be seen from the figure that the nano particles are evenly distributed and have a spherical structure.

[0023] Figure 2 The imaging and treatment integrated nanoprobe based on biomass nanovinegar modification synthesized in Example 1 of the present invention generates ·OH. Figure 2 It can be seen that in the presence of H2O2, the probe HSAVMn@CM can catalyze the decomposition of H2O2 to produce ·OH.

[0024] Figure 3 Production of an integrated imaging and therapeutic nanoprobe based on biomass nanovinegar modification synthesized in Example 1 of the present invention 1 As can be seen from the figure, the ultraviolet absorbance of the material changes at around 410nm after laser irradiation, indicating that the nanoprobe HSAVMn@CM of the present invention has 1 O2 release capacity.

[0025] Example 2 Preparation of an integrated imaging and therapeutic nanoprobe based on biomass transformation: Take 20 mL of Shanxi vinegar that has been aged for more than 5 years, carefully observe its properties and smell its odor, and keep it for later use if there is no abnormality; Place the prepared vinegar in a high-speed centrifuge at a speed of 12,000 rpm for 20 min. After centrifugation, keep the supernatant and discard the precipitate. Repeat high-speed centrifugation according to the above steps, discard the precipitate, and keep the final supernatant for later use; Place a dialysis bag with a molecular weight of 8000-14000 Da in boiling water at 100 °C for 5 min, and rinse the dialysis bag thoroughly with ultrapure water; Place the prepared supernatant in the prepared dialysis bag and clamp both ends to prevent leakage; Place the large rotor at the bottom of a 2000 mL beaker, add 1800 mL of deionized water, and place the dialysis bag clamped in the previous step in the deionized water; Place the large beaker obtained in the previous step on a stirrer and set the speed to 1000 rpm. Adjust the speed as appropriate based on the rotation of the rotor and dialysis bag. Replace the above dialysate every 1 hour. After 3 changes, change to every 3 hours. After 3 changes, change to every 6 hours until the dialysate becomes clear. Take the final dialyzed liquid and divide it into individual flash bottles, each containing 2 mL, and freeze it in a -20℃ refrigerator; The above-obtained product was placed in a freeze dryer and freeze-dried to obtain SAV, which was then dispensed into EP tubes, sealed with sealing film and stored at room temperature; SAV was placed in a solution at 60 °C and pH = 2 for 2 h, then dialyzed to remove the acidity and freeze-dried to obtain HSAV; Weigh 1.799 mg of manganese chloride tetrahydrate and 1 mL of deionized water, add them into the EP tube and mix thoroughly to obtain a manganese chloride aqueous solution for later use; Weigh 5 mg of the above HSAV powder and 1 mL of deionized water, add them into the EP tube and mix thoroughly to obtain a HSAV aqueous solution for later use; Take 1 mL of the above HSAV aqueous solution and 1 ml of manganese chloride aqueous solution, add magnetic beads, and stir vigorously (880 r / min) overnight; then dialyze and freeze-dry to obtain HSAVMn.

[0026] The number of 4T1 cells was 8 million, digested and centrifuged, and dissolved in ultrapure water as a carrier. The ultrasonic crushing instrument power was 10%, the crushing time was 5s, the interval time was 5s, and the total time was 20min. The crushing was carried out on ice. After crushing, the centrifugation speed was 12000rpm, the time was 20min, and the centrifugation was carried out at 4 degrees low temperature and high speed, and the supernatant was retained.

[0027] The supernatant obtained in the above steps was mixed with 10 mg HSAVMn, separated by 30 KD ultrafiltration separation tube at 3500 r / min for 10 min, and washed with deionized water three times to obtain the 4T1 cell membrane protein-coated nanocomplex HSAVMn@CM.

[0028] Example 3 Preparation of an integrated imaging and therapeutic nanoprobe based on biomass transformation: Take 20mL of Shanxi vinegar that has been aged for more than 5 years, carefully observe its properties and smell its odor, and keep it for later use if there is no abnormality; Place the prepared vinegar in a high-speed centrifuge at a speed of 12000 rpm for 20 min. After centrifugation, retain the supernatant and discard the precipitate. Repeat high-speed centrifugation according to the above steps, discard the precipitate, and keep the final supernatant for later use; Place a dialysis bag with a molecular weight of 8000-14000 Da in boiling water at 100°C for 5 minutes, and rinse the dialysis bag thoroughly with ultrapure water; Place the prepared supernatant in the prepared dialysis bag and clamp both ends to prevent leakage; Place the large rotor at the bottom of a 2000mL beaker, add 1800mL of deionized water, and place the dialysis bag clamped in the previous step in the deionized water; Place the large beaker obtained in the previous step on a stirrer and set the speed to 1000 rpm. Adjust the speed as appropriate according to the rotation of the rotor and dialysis bag. Replace the above dialysate every 1 hour. After 3 changes, change to every 3 hours. After 3 changes, change to every 6 hours until the dialysate becomes clear. Take the final dialyzed liquid and divide it into individual flash bottles, each containing 2 mL, and freeze it in a -20℃ refrigerator; The above-obtained product was placed in a freeze dryer and freeze-dried to obtain SAV, which was then dispensed into EP tubes, sealed with sealing film and stored at room temperature; SAV was placed in a solution at 60 °C and pH = 2 for 2 h, then dialyzed to remove the acidity and freeze-dried to obtain HSAV; Weigh 1.799 mg of manganese chloride tetrahydrate and 1 mL of deionized water, add them into the EP tube and mix thoroughly to obtain a manganese chloride aqueous solution for later use; Weigh 5 mg of the above HSAV powder and 1 mL of deionized water, add them into the EP tube and mix thoroughly to obtain a HSAV aqueous solution for later use; Take 1 mL of the above HSAV aqueous solution and 1 mL of manganese chloride aqueous solution, add magnetic beads, and stir vigorously (880 r / min) overnight; then dialyze and freeze-dry to obtain HSAVMn.

[0029] The number of 4T1 cells cultured was 12 million, digested and centrifuged, and dissolved in ultrapure water as a carrier. The ultrasonic crushing instrument power was 10%, the crushing time was 5s, the interval time was 5s, and the total time was 20min. The crushing was carried out on ice. After crushing, the centrifugation speed was 12000 rpm, the time was 20min, and the centrifugation was carried out at 4 degrees low temperature and high speed, and the supernatant was retained.

[0030] The supernatant obtained in the above steps was mixed with 10 mg HSAVMn, separated by 30 KD ultrafiltration separation tube at 3500 r / min for 10 min, washed three times with deionized water, and freeze-dried to obtain a quantitative 4T1 cell membrane protein-encapsulated nanocomplex HSAVMn@CM.

[0031] Example 4 Preparation of an integrated imaging and therapeutic nanoprobe based on biomass transformation: Take 20mL of Shanxi vinegar that has been aged for more than 5 years, carefully observe its properties and smell its odor, and keep it for later use if there is no abnormality; Place the prepared vinegar in a high-speed centrifuge at a speed of 12000 rpm for 20 min. After centrifugation, retain the supernatant and discard the precipitate. Repeat high-speed centrifugation according to the above steps, discard the precipitate, and keep the final supernatant for later use; Place a dialysis bag with a molecular weight of 8000-14000 Da in boiling water at 100°C for 5 minutes, and rinse the dialysis bag thoroughly with ultrapure water; Place the prepared supernatant in the prepared dialysis bag and clamp both ends to prevent leakage; Place the large rotor at the bottom of a 2000mL beaker, add 1800mL of deionized water, and place the dialysis bag clamped in the previous step in the deionized water; Place the large beaker obtained in the previous step on a stirrer and set the speed to 1000 rpm. Adjust the speed as appropriate according to the rotation of the rotor and dialysis bag. Replace the above dialysate every 1 hour. After 3 changes, change to every 3 hours. After 3 changes, change to every 6 hours until the dialysate becomes clear. Take the final dialyzed liquid and divide it into individual flash bottles, each containing 2 mL, and freeze it in a -20℃ refrigerator; The above-obtained product was placed in a freeze dryer and freeze-dried to obtain SAV, which was then dispensed into EP tubes, sealed with sealing film and stored at room temperature; SAV was placed in a solution at 60 °C and pH = 2 for 2 h, then dialyzed to remove the acidity and freeze-dried to obtain HSAV; Weigh 1.799 mg of manganese chloride tetrahydrate and 1 mL of deionized water, add them into the EP tube and mix thoroughly to obtain a manganese chloride aqueous solution for later use; Weigh 15 mg of the above HSAV powder and 1 mL of deionized water, add them into the EP tube and mix thoroughly to obtain a HSAV aqueous solution for later use; Take 1 mL of the above HSAV aqueous solution and 1 mL of manganese chloride aqueous solution, add magnetic beads, and stir vigorously (880 r / min) overnight; then dialyze and freeze-dry to obtain HSAVMn.

[0032] The number of 4T1 cells was 8 million, digested and centrifuged, and dissolved in ultrapure water as a carrier. The ultrasonic crushing instrument power was 10%, the crushing time was 5s, the interval time was 5s, and the total time was 20min. The crushing was carried out on ice. After crushing, the centrifugation speed was 12000rpm, the time was 20min, and the centrifugation was carried out at 4 degrees low temperature and high speed, and the supernatant was retained.

[0033] The supernatant obtained in the above steps was mixed with 10 mg HSAVMn, separated by 30 KD ultrafiltration separation tube at 3500 r / min for 10 min, and washed with deionized water three times to obtain the 4T1 cell membrane protein-coated nanocomplex HSAVMn@CM.

[0034] Example 5 Preparation of an integrated imaging and therapeutic nanoprobe based on biomass transformation: Take 20mL of Shanxi vinegar that has been aged for more than 5 years, carefully observe its properties and smell its odor, and keep it for later use if there is no abnormality; Place the prepared vinegar in a high-speed centrifuge at a speed of 12000 rpm for 20 min. After centrifugation, retain the supernatant and discard the precipitate. Repeat high-speed centrifugation according to the above steps, discard the precipitate, and keep the final supernatant for later use; Place a dialysis bag with a molecular weight of 8000-14000 Da in boiling water at 100°C for 5 minutes, and rinse the dialysis bag thoroughly with ultrapure water; Place the prepared supernatant in the prepared dialysis bag and clamp both ends to prevent leakage; Place the large rotor at the bottom of a 2000mL beaker, add 1800mL of deionized water, and place the dialysis bag clamped in the previous step in the deionized water; Place the large beaker obtained in the previous step on a stirrer and set the speed to 1000 rpm. Adjust the speed as appropriate according to the rotation of the rotor and dialysis bag. Replace the above dialysate every 1 hour. After 3 changes, change to every 3 hours. After 3 changes, change to every 6 hours until the dialysate becomes clear. Take the final dialyzed liquid and divide it into individual flash bottles, each containing 2 mL, and freeze it in a -20℃ refrigerator; The above-obtained product was placed in a freeze dryer and freeze-dried to obtain SAV, which was then dispensed into EP tubes, sealed with sealing film and stored at room temperature; SAV was placed in a solution at 60 °C and pH = 2 for 2 h, then dialyzed to remove the acidity and freeze-dried to obtain HSAV; Weigh 1.799 mg of manganese chloride tetrahydrate and 1 mL of deionized water, add them into the EP tube and mix thoroughly to obtain a manganese chloride aqueous solution for later use; Weigh 15 mg of the above HSAV powder and 1 mL of deionized water, add them into the EP tube and mix thoroughly to obtain a HSAV aqueous solution for later use; Take 1 mL of the above HSAV aqueous solution and 1 mL of manganese chloride aqueous solution, add magnetic beads, and stir vigorously (880 r / min) overnight; then dialyze and freeze-dry to obtain HSAVMn.

[0035] The number of 4T1 cells was cultured to 10 million, digested and centrifuged, and dissolved in ultrapure water as a carrier. The ultrasonic crushing instrument power was 10%, the crushing time was 5s, the interval time was 5s, and the total time was 20min. The crushing was carried out on ice. After crushing, the centrifugation speed was 12000 rpm, the time was 20min, and the centrifugation was carried out at 4 degrees low temperature and high speed, and the supernatant was retained.

[0036] The supernatant obtained in the above steps was mixed with 10 mg HSAVMn, separated by 30 KD ultrafiltration separation tube at 3500 r / min for 10 min, and washed with deionized water three times to obtain the 4T1 cell membrane protein-coated nanocomplex HSAVMn@CM.

[0037] Example 6 Preparation of an integrated imaging and therapeutic nanoprobe based on biomass transformation: Take 20mL of Shanxi vinegar that has been aged for more than 5 years, carefully observe its properties and smell its odor, and keep it for later use if there is no abnormality; Place the prepared vinegar in a high-speed centrifuge at a speed of 12000 rpm for 20 min. After centrifugation, retain the supernatant and discard the precipitate. Repeat high-speed centrifugation according to the above steps, discard the precipitate, and keep the final supernatant for later use; Place a dialysis bag with a molecular weight of 8000-14000 Da in boiling water at 100°C for 5 minutes, and rinse the dialysis bag thoroughly with ultrapure water; Place the prepared supernatant in the prepared dialysis bag and clamp both ends to prevent leakage; Place the large rotor at the bottom of a 2000mL beaker, add 1800mL of deionized water, and place the dialysis bag clamped in the previous step in the deionized water; Place the large beaker obtained in the previous step on a stirrer and set the speed to 1000 rpm. Adjust the speed as appropriate according to the rotation of the rotor and dialysis bag. Replace the above dialysate every 1 hour. After 3 changes, change to every 3 hours. After 3 changes, change to every 6 hours until the dialysate becomes clear. Take the final dialyzed liquid and divide it into individual flash bottles, each containing 2 mL, and freeze it in a -20℃ refrigerator; The above-obtained product was placed in a freeze dryer and freeze-dried to obtain SAV, which was then dispensed into EP tubes, sealed with sealing film and stored at room temperature; SAV was placed in a solution at 60 °C and pH = 2 for 2 h, then dialyzed to remove the acidity and freeze-dried to obtain HSAV; Weigh 1.799 mg of manganese chloride tetrahydrate and 1 mL of deionized water, add them into the EP tube and mix thoroughly to obtain a manganese chloride aqueous solution for later use; Weigh 15 mg of the above HSAV powder and 1 mL of deionized water, add them into the EP tube and mix thoroughly to obtain a HSAV aqueous solution for later use; Take 1 mL of the above HSAV aqueous solution and 1 ml of manganese chloride aqueous solution, add magnetic beads, and stir vigorously (880 r / min) overnight; then dialyze and freeze-dry to obtain HSAVMn.

[0038] The number of 4T1 cells cultured was 12 million, digested and centrifuged, and dissolved in ultrapure water as a carrier. The ultrasonic crushing instrument power was 10%, the crushing time was 5s, the interval time was 5s, and the total time was 20min. The crushing was carried out on ice. After crushing, the centrifugation speed was 12000 rpm, the time was 20min, and the centrifugation was carried out at 4 degrees low temperature and high speed, and the supernatant was retained.

[0039] The supernatant obtained in the above steps was mixed with 10 mg HSAVMn, separated by 30 KD ultrafiltration separation tube at 3500 r / min for 10 min, and washed with deionized water three times to obtain the 4T1 cell membrane protein-coated nanocomplex HSAVMn@CM.

[0040] Example 7 Preparation of an integrated imaging and therapeutic nanoprobe based on biomass transformation: Take 20mL of Shanxi vinegar that has been aged for more than 5 years, carefully observe its properties and smell its odor, and keep it for later use if there is no abnormality; Place the prepared vinegar in a high-speed centrifuge at a speed of 12000 rpm for 20 min. After centrifugation, retain the supernatant and discard the precipitate. Repeat high-speed centrifugation according to the above steps, discard the precipitate, and keep the final supernatant for later use; Place a dialysis bag with a molecular weight of 8000-14000 Da in boiling water at 100°C for 5 minutes, and rinse the dialysis bag thoroughly with ultrapure water; Place the prepared supernatant in the prepared dialysis bag and clamp both ends to prevent leakage; Place the large rotor at the bottom of a 2000mL beaker, add 1800mL of deionized water, and place the dialysis bag clamped in the previous step in the deionized water; Place the large beaker obtained in the previous step on a stirrer and set the speed to 1000 rpm. Adjust the speed as appropriate according to the rotation of the rotor and dialysis bag. Replace the above dialysate every 1 hour. After 3 changes, change to every 3 hours. After 3 changes, change to every 6 hours until the dialysate becomes clear. Take the final dialyzed liquid and divide it into individual flash bottles, each containing 2 mL, and freeze it in a -20℃ refrigerator; The above-obtained product was placed in a freeze dryer and freeze-dried to obtain SAV, which was then dispensed into EP tubes, sealed with sealing film and stored at room temperature; SAV was placed in a solution at 60 °C and pH = 2 for 2 h, then dialyzed to remove the acidity and freeze-dried to obtain HSAV; Weigh 1.799 mg of manganese chloride tetrahydrate and 1 mL of deionized water, add them into the EP tube and mix thoroughly to obtain a manganese chloride aqueous solution for later use; Weigh 25 mg of the above HSAV powder and 1 mL of deionized water, add them into the EP tube and mix thoroughly to obtain a HSAV aqueous solution for use; Take 1 mL of the above HSAV aqueous solution and 1 mL of manganese chloride aqueous solution, add magnetic beads, and stir vigorously (880 r / min) overnight; then dialyze and freeze-dry to obtain HSAVMn.

[0041] The number of 4T1 cells was 8 million, digested and centrifuged, and dissolved in ultrapure water as a carrier. The ultrasonic crushing instrument power was 10%, the crushing time was 5s, the interval time was 5s, and the total time was 20min. The crushing was carried out on ice. After crushing, the centrifugation speed was 12000rpm, the time was 20min, and the centrifugation was carried out at 4 degrees low temperature and high speed, and the supernatant was retained.

[0042] The supernatant obtained in the above steps was mixed with 10 mg HSAVMn, separated by 30 KD ultrafiltration separation tube at 3500 r / min for 10 min, and washed with deionized water three times to obtain the 4T1 cell membrane protein-coated nanocomplex HSAVMn@CM.

[0043] Example 8 Preparation of an integrated imaging and therapeutic nanoprobe based on biomass transformation: Take 20mL of Shanxi vinegar that has been aged for more than 5 years, carefully observe its properties and smell its odor, and keep it for later use if there is no abnormality; Place the prepared vinegar in a high-speed centrifuge at a speed of 12000 rpm for 20 min. After centrifugation, retain the supernatant and discard the precipitate. Repeat high-speed centrifugation according to the above steps, discard the precipitate, and keep the final supernatant for later use; Place a dialysis bag with a molecular weight of 8000-14000 Da in boiling water at 100°C for 5 minutes, and rinse the dialysis bag thoroughly with ultrapure water; Place the prepared supernatant in the prepared dialysis bag and clamp both ends to prevent leakage; Place the large rotor at the bottom of a 2000mL beaker, add 1800mL of deionized water, and place the dialysis bag clamped in the previous step in the deionized water; Place the large beaker obtained in the previous step on a stirrer and set the speed to 1000 rpm. Adjust the speed as appropriate according to the rotation of the rotor and dialysis bag. Replace the above dialysate every 1 hour. After 3 changes, change to every 3 hours. After 3 changes, change to every 6 hours until the dialysate becomes clear. Take the final dialyzed liquid and divide it into individual flash bottles, each containing 2 mL, and freeze it in a -20℃ refrigerator; The above-obtained product was placed in a freeze dryer and freeze-dried to obtain SAV, which was then dispensed into EP tubes, sealed with sealing film and stored at room temperature; SAV was placed in a solution at 60 °C and pH = 2 for 2 h, then dialyzed to remove the acidity and freeze-dried to obtain HSAV; Weigh 1.799 mg of manganese chloride tetrahydrate and 1 mL of deionized water, add them into the EP tube and mix thoroughly to obtain a manganese chloride aqueous solution for later use; Weigh 25 mg of the above HSAV powder and 1 mL of deionized water, add them into the EP tube and mix thoroughly to obtain a HSAV aqueous solution for use; Take 1 mL of the above HSAV aqueous solution and 1 ml of manganese chloride aqueous solution, add magnetic beads, and stir vigorously (880 r / min) overnight; then dialyze and freeze-dry to obtain HSAVMn.

[0044] The number of 4T1 cells was cultured to 10 million, digested and centrifuged, and dissolved in ultrapure water as a carrier. The ultrasonic crushing instrument power was 10%, the crushing time was 5s, the interval time was 5s, and the total time was 20min. The crushing was carried out on ice. After crushing, the centrifugation speed was 12000 rpm, the time was 20min, and the centrifugation was carried out at 4 degrees low temperature and high speed, and the supernatant was retained.

[0045] The supernatant obtained in the above steps was mixed with 10 mg HSAVMn, separated by 30 KD ultrafiltration separation tube at 3500 r / min for 10 min, and washed with deionized water three times to obtain the 4T1 cell membrane protein-coated nanocomplex HSAVMn@CM.

[0046] Example 9 Take 20mL of Shanxi vinegar that has been aged for more than 5 years, carefully observe its properties and smell its odor, and keep it for later use if there is no abnormality; Place the prepared vinegar in a high-speed centrifuge at a speed of 12000 rpm for 20 min. After centrifugation, retain the supernatant and discard the precipitate. Repeat high-speed centrifugation according to the above steps, discard the precipitate, and keep the final supernatant for later use; Place a dialysis bag with a molecular weight of 8000-14000 Da in boiling water at 100°C for 5 minutes, and rinse the dialysis bag thoroughly with ultrapure water; Place the prepared supernatant in the prepared dialysis bag and clamp both ends to prevent leakage; Place the large rotor at the bottom of a 2000mL beaker, add 1800mL of deionized water, and place the dialysis bag clamped in the previous step in the deionized water; Place the large beaker obtained in the previous step on a stirrer and set the speed to 1000 rpm. Adjust the speed as appropriate according to the rotation of the rotor and dialysis bag. Replace the above dialysate every 1 hour. After 3 changes, change to every 3 hours. After 3 changes, change to every 6 hours until the dialysate becomes clear. Take the final dialyzed liquid and divide it into individual flash bottles, each containing 2 mL, and freeze it in a -20℃ refrigerator; The above-obtained product was placed in a freeze dryer and freeze-dried to obtain SAV, which was then dispensed into EP tubes, sealed with sealing film and stored at room temperature; SAV was placed in a solution at 60 °C and pH = 2 for 2 h, then dialyzed to remove the acidity and freeze-dried to obtain HSAV; Weigh 1.799 mg of manganese chloride tetrahydrate and 1 mL of deionized water, add them into the EP tube and mix thoroughly to obtain a manganese chloride aqueous solution for later use; Weigh 25 mg of the above HSAV powder and 1 mL of deionized water, add them into the EP tube and mix thoroughly to obtain a HSAV aqueous solution for use; Take 1 mL of the above HSAV aqueous solution and 1 ml of manganese chloride aqueous solution, add magnetic beads, and stir vigorously (880 r / min) overnight; then dialyze and freeze-dry to obtain HSAVMn.

[0047] The number of 4T1 cells cultured was 12 million, digested and centrifuged, and dissolved in ultrapure water as a carrier. The ultrasonic crushing instrument power was 10%, the crushing time was 5s, the interval time was 5s, and the total time was 20min. The crushing was carried out on ice. After crushing, the centrifugation speed was 12000 rpm, the time was 20min, and the centrifugation was carried out at 4 degrees low temperature and high speed, and the supernatant was retained.

[0048] The supernatant obtained in the above steps was mixed with 10 mg HSAVMn, separated by 30 KD ultrafiltration separation tube at 3500 r / min for 10 min, and washed with deionized water three times to obtain the 4T1 cell membrane protein-coated nanocomplex HSAVMn@CM.

Claims

1. An integrated imaging and therapeutic nanoprobe based on biomass transformation, characterized by: The nanoprobe is a spherical core-shell structure, the inner core is nanoacetate coupled with metal ions, and the outer surface is coated with 4T1 tumor cell membrane.

2. A method for preparing an integrated imaging and therapeutic nanoprobe based on biomass transformation as claimed in claim 1, characterized in that: The steps include: In the first step, the aged vinegar of more than 5 years is centrifuged at 12000 rpm for 20 min, the supernatant is aspirated and the precipitate is discarded, and the supernatant is retained; In the second step, a dialysis bag with a molecular weight of 8000-14000Da was placed in boiling water at 100°C for 5 minutes, and the dialysis bag was fully rinsed with ultrapure water; The third step is to place the supernatant obtained in the first step into the dialysis bag described in the second step, clamp both ends of the dialysis bag, place it in ultrapure water, put magnetic beads in the ultrapure water, stir at a speed of 1000 rpm under room temperature, and regularly replace the ultrapure water until the color of the ultrapure water becomes clear; Step 4: After the dialysis is completed, the liquid in the dialysis bag is packaged, frozen and dried at -20°C to obtain SAV, which is sealed and stored at room temperature; Step 5: The SAV aqueous solution was adjusted to pH 2, placed at 60°C, incubated for 2 h, dialyzed to remove acidity, and freeze-dried to obtain HSAV; Step 6, preparing HSAV aqueous solution and manganese chloride aqueous solution respectively, mixing the HSAV aqueous solution and the manganese chloride aqueous solution, adding magnetic beads, stirring overnight, and then dialyzing and freeze-drying to obtain HSAVMn; Step 7: The 4T1 cells were broken and centrifuged to obtain the supernatant protein; In the eighth step, the supernatant of the seventh step was mixed with the HSAVMn of the sixth step, separated by a 30 KD ultrafiltration separation tube at a speed of 3500 rpm for 10 min, and washed three times with deionized water to obtain the nanocomplex HSAVMn@CM coated with 4T1 cell membrane protein.

3. The method for preparing a nanoprobe for integrated imaging and treatment based on biomass transformation according to claim 2, characterized in that: The time for scheduled replacement in the third step is once every 1 hour. After 3 replacements, it is changed to once every 3 hours. After 3 more replacements, it is changed to once every 6 hours.

4. The method for preparing a nanoprobe for integrated imaging and treatment based on biomass transformation according to claim 2, characterized in that: In the HSAV aqueous solution in the sixth step, the mass of HSAV is 5-25 mg, and the volume of deionized water is 1 mL; in the manganese chloride aqueous solution, the mass of manganese chloride is 1.799 mg, and the volume of deionized water is 1 mL.

5. The method for preparing a nanoprobe for integrated imaging and treatment based on biomass transformation according to claim 2, characterized in that: The number of 4T1 cells in step 7 is 8-12 million.

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