Transferrin modified targeted lipid nano bubble as well as preparation method and application thereof

Abstract

The invention provides a transferrin modified targeted lipid nano bubble as well as a preparation method and application thereof, and relates to the technical field of synthesis of ultrasonic medical imaging contrast agents. The transferrin modified targeted lipid nano bubble provided by the invention has a bubble-like structure; the shell of the transferrin modified targeted lipid nano bubble is made of single-layer phospholipid; the surface of a shell of the transferrin modified targeted lipid nano bubble is coupled with biotinylated TF through streptavidin; and perfluoropropane gas is wrapped in the transferrin modified targeted lipid nano bubble. The transferrin modified targeting lipid nano bubble provided by the invention has good stability and strong targeting property, and can target ferroptosis cells.

Description

Technical Field

[0001] The present invention relates to the technical field of ultrasonic medical imaging contrast agent synthesis, and in particular to a transferrin-modified targeted lipid nanobubble and a preparation method and application thereof. Background Art

[0002] In recent years, problems such as drug resistance and poor efficacy of cancer cells due to dysfunctional cell apoptosis or defective mechanisms remain prominent. Some new treatment models, such as inducing ferroptosis in tumor cells, have begun to attract attention. Ferroptosis is highly sensitive to some cancer cell subtypes and apoptosis-resistant chemotherapy. Therefore, early prediction of the anti-cancer efficacy of ferroptosis is the key and prerequisite for treatment. Studies have found that the number of transferrin receptors (Transferrin receptor protein 1, TfR1) on cancer cell membranes and the iron content in cancer cells are generally higher than those in normal cells. The absorption of cellular iron is mainly controlled by the transmembrane glycoprotein TfR1, which carries transferrin-bound iron into the cell through receptor-mediated endocytosis. Some scholars have found that blocking this process by knocking out TfR1 can prevent cell ferroptosis. Therefore, TfR1 is considered to be a marker protein for the occurrence of ferroptosis, has good selectivity for the recognition of cell ferroptosis, and is a specific marker of ferroptosis.

[0003] Existing ferroptosis monitoring methods are mostly limited to in vitro invasive levels, and there is still a lack of molecular probes for in vivo specific non-invasive exploration of ferroptosis mechanisms, which hinders the early prediction and real-time evaluation of therapeutic efficacy. Ultrasound molecular imaging tools are one of the preferred diagnostic methods in clinical practice, with advantages such as safety, real-time, visualization, and repeatable diagnosis. Ultrasound molecular imaging is based on the preparation of ultrasound microbubbles that target specific molecules in the body. In addition to evaluating tumor blood perfusion, targeted ultrasound microbubbles can also closely bind to specific cell surface receptors to achieve imaging of specific molecules in the target area. At present, the invention of ultrasound molecular imaging diagnostic models based on the characteristics of cell ferroptosis is still blank. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a transferrin-modified targeted lipid nanobubble and its preparation method and application. The transferrin-modified targeted lipid nanobubble provided by the present invention has good stability and strong targeting, and can be used for ultrasonic targeted imaging of ferroptotic cells, thereby evaluating the ferroptosis of breast cancer cells.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a transferrin-modified targeted lipid nanobubble, which has a bubble-like structure; the shell of the transferrin-modified targeted lipid nanobubble is a single layer of phospholipid; the shell surface of the transferrin-modified targeted lipid nanobubble is coupled to biotinylated TF via streptavidin; and the interior of the transferrin-modified targeted lipid nanobubble is wrapped with perfluoropropane gas.

[0007] The present invention provides a method for preparing transferrin-modified targeted lipid nanobubbles described in the above technical solution, comprising the following steps:

[0008] Filling perfluoropropane gas into the lipid film suspension and shaking it to obtain a lipid microbubble mixture;

[0009] centrifuging the lipid microbubble mixture to obtain lipid nanobubbles;

[0010] The lipid nanobubbles are co-incubated with a biotinylated TF solution and a streptavidin solution to obtain transferrin-modified targeted lipid nanobubbles.

[0011] Preferably, the method for preparing the lipid film suspension comprises: mixing a dry lipid film and a hydrating solution containing Poloxamer 188 to obtain a lipid film suspension; the hydrating solution containing Poloxamer 188 comprises a Poloxamer 188 solution, PBS buffer and glycerol.

[0012] Preferably, the preparation method of the dry lipid film comprises: mixing 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotinylated (polyethylene glycol) 2000] and chloroform, adding a fluorescent dye DiR solution under light-proof conditions, and removing the chloroform to obtain a dry lipid film.

[0013] Preferably, the perfluoropropane gas is injected into the lipid film suspension, comprising: placing the lipid film suspension in a tube and sealing it, removing the air in the tube, and automatically injecting the perfluoropropane gas into the sealed tube by utilizing the pressure difference between the inside and outside of the tube.

[0014] Preferably, the centrifugation includes a first centrifugation and a second centrifugation performed sequentially; the rotation speed of the first centrifugation is 1000-1500 rpm, and the time of the first centrifugation is 3-5 minutes; the rotation speed of the second centrifugation is 800-1000 rpm, and the time of the second centrifugation is 3-5 minutes.

[0015] Preferably, the centrifugation temperature is 4-8°C.

[0016] Preferably, the co-incubation further comprises standing, and the suspension obtained after standing is transferrin-modified targeted lipid nanovesicles.

[0017] The present invention provides the use of the transferrin-modified targeted lipid nanobubbles described in the above technical solution or the transferrin-modified targeted lipid nanobubbles prepared by the preparation method described in the above technical solution as ultrasound contrast agents in ultrasound imaging.

[0018] The present invention provides the use of the transferrin-modified targeted lipid nanovesicles described in the above technical solution or the transferrin-modified targeted lipid nanovesicles prepared by the preparation method described in the above technical solution in the preparation of drugs targeting ferroptotic cells.

[0019] The present invention provides a transferrin-modified targeted lipid nanobubble. The transferrin-modified targeted lipid nanobubble provided by the present invention has a single-layer membrane structure with a perfluoropropane gas-filled core inside. The surface of the transferrin-modified targeted lipid nanobubble is coupled to biotinylated TF via a "biotin-avidin" structure. The transferrin-modified targeted lipid nanobubble provided by the present invention has good stability and strong targeting, and can target ferroptotic cells.

[0020] The present invention provides a method for preparing transferrin-modified targeted lipid nanobubbles. The present invention uses perfluoropropane gas to prepare microbubbles, which can improve the stability of the lipid microbubble mixture. The lipid microbubble mixture is centrifuged to obtain lipid nanobubbles. The present invention adopts a "biotin-avidin" coupling method, utilizing the interaction between biotin and avidin, and co-incubates the lipid nanobubbles with a biotinylated TF solution and a streptavidin solution to obtain the transferrin-modified targeted lipid nanobubbles. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Transferrin modified targeted lipid nanobubbles NB TF Preparation flow chart of

[0022] Figure 2 Figure 1 is a diagram of the cross-linking pattern of streptavidin-biotin TF;

[0023] Figure 3 For NB TF Particle size distribution (above) and NB TF Surface potential (bottom);

[0024] Figure 4 Observe NB under ordinary bright field microscope TF The morphology (left) and scanning electron microscopy observation of NB TF The shape of (right);

[0025] Figure 5 For NB TF In vitro ultrasound echo characteristics;

[0026] Figure 6Expression of TfR1 molecules in control cells and ferroptotic cells, as well as transferrin-modified targeted lipid nanobubbles NB TF The experimental results were compared with those of control cells and breast cancer ferroptosis cells by targeted binding;

[0027] Figure 7 Transferrin modified targeted lipid nanobubbles NB TF and control lipid nanobubbles NB lgG The experimental results were compared with those of breast cancer ferroptosis cell targeting;

[0028] Figure 8 For NB TF Ultrasound imaging results in in vivo animal experiments;

[0029] Figure 9 right Figure 8 Comparative analysis results of C-related parameters in;

[0030] Figure 10 It is a homemade simple three-way pipe gas exchange device; Figure 10 1 represents the first syringe (10 mL) connected to the tee, 2 represents the second syringe (50 mL) connected to the tee, 3 represents the needle connected to the tee (needle specification 0.45×16RWLB), and 4 represents the tee. DETAILED DESCRIPTION

[0031] The present invention provides a transferrin-modified targeted lipid nanobubble, which has a bubble-like structure; the shell of the transferrin-modified targeted lipid nanobubble is a single layer of phospholipid; the shell surface of the transferrin-modified targeted lipid nanobubble is coupled to biotinylated TF via streptavidin; and the interior of the transferrin-modified targeted lipid nanobubble is wrapped with perfluoropropane gas.

[0032] In the present invention, the particle size of the transferrin-modified targeted lipid nanobubbles is preferably 300-700 nm, more preferably 500 nm.

[0033] The present invention provides a method for preparing transferrin-modified targeted lipid nanobubbles, comprising the following steps:

[0034] Filling perfluoropropane gas into the lipid film suspension and shaking it to obtain a lipid microbubble mixture;

[0035] centrifuging the lipid microbubble mixture to obtain lipid nanobubbles;

[0036] The lipid nanobubbles are co-incubated with a biotinylated TF solution and a streptavidin solution to obtain transferrin-modified targeted lipid nanobubbles.

[0037] Unless otherwise specified, the materials and equipment used in the present invention are all commercially available products in the art.

[0038] The present invention injects perfluoropropane gas into a lipid film suspension and vibrates the mixture to obtain a lipid microbubble mixture. In the present invention, the method for preparing the lipid film suspension preferably includes mixing a dry lipid film with a hydrating solution containing Poloxamer 188 to obtain the lipid film suspension. The present invention hydrates the dry lipid film by microvibration to obtain the lipid film suspension.

[0039] In the present invention, the method for preparing the dry lipid film preferably comprises: mixing 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotinylated (polyethylene glycol) 2000] (DSPE-PEG2000-Biotin), and chloroform, adding a fluorescent dye DiR solution under light-proof conditions, and removing the chloroform to obtain a dry lipid film. In the present invention, the mixing is preferably performed under light-proof conditions. In the present invention, the lipid film-forming material is mixed with chloroform and the dry lipid film is prepared using an optimized "thin film hydration method."

[0040] In the present invention, the DPPC and DSPE-PEG2000-Biotin are preferably solid powders; the mass ratio of the DPPC and DSPE-PEG2000-Biotin is preferably 5:2. In the present invention, the dosage ratio of the DPPC and chloroform is preferably 10 mg:1-2 mL, more preferably 10 mg:2 mL. In the present invention, the dosage ratio of the DPPC and the fluorescent dye DiR solution is preferably 10 mg:5-10 μL, more preferably 10 mg:10 μL; the concentration of the fluorescent dye DiR solution is preferably 10 μM. In the present invention, the method for removing chloroform preferably includes rotary evaporation (rotary evaporation); the temperature of the rotary evaporation is preferably 45-50°C, more preferably 50°C; the time of the rotary evaporation is preferably 40-50 min, more preferably 40 min; the speed of the rotary evaporation is preferably 120-130 rpm, more preferably 120 rpm.

[0041] The present invention mixes a dry lipid film and a hydrating solution containing Poloxamer 188 to obtain a lipid film suspension. In the present invention, the hydrating solution containing Poloxamer 188 preferably includes a Poloxamer 188 solution, a PBS buffer solution, and glycerol. In the present invention, the solvent of the Poloxamer 188 solution is preferably ultrapure water; the concentration of the Poloxamer 188 solution is preferably 10wt%; and the Poloxamer 188 solution is preferably sterile filtered before use. In the present invention, in the hydrating solution containing Poloxamer 188, the volume proportion of the Poloxamer 188 solution is preferably 2-4%, more preferably 2%. In the present invention, the concentration of the PBS buffer solution is preferably 0.01M (mo / L). In the present invention, in the hydrating solution containing Poloxamer 188, the volume proportion of the PBS buffer solution is preferably 80-90%, more preferably 88%. In the present invention, the volume proportion of glycerol in the hydrating solution containing Poloxamer 188 is preferably 5-10%, more preferably 10%. In the present invention, the hydrating solution containing Poloxamer 188 is preferably preheated before use, and the preheating temperature is preferably 37°C. In the present invention, the mixing is preferably performed on a thermostatic shaker; the mixing temperature is preferably 37°C; and the mixing time is preferably 1 hour.

[0042] In the present invention, before the perfluoropropane gas is injected into the lipid film suspension, the air in the lipid film suspension is preferably removed. In the present invention, the perfluoropropane gas is injected into the lipid film suspension, which preferably includes: placing the lipid film suspension in a tube and sealing it, then removing the air in the tube, and automatically injecting the perfluoropropane gas into the sealed tube by utilizing the pressure difference between the inside and outside of the tube. In a specific embodiment of the present invention, Figure 10 The homemade, simple three-way pipe gas exchange device shown here fills perfluoropropane gas into a lipid film suspension. One end of a needle 3 is connected to a three-way pipe 4, and the other end is inserted into a sealed tube containing the lipid film suspension. The three-way pipe switch is adjusted, and manual suction is performed to allow air in the sealed tube to enter through the needle 3, pass through the three-way pipe 4, and enter the first syringe 1 to be evacuated. The air in the sealed tube is repeatedly evacuated. When the pressure in the tube is minimum (close to vacuum), the three-way pipe switch is adjusted again, allowing the perfluoropropane gas in the second syringe 2 to enter the sealed tube containing the lipid film suspension under the action of the pressure difference. In the present invention, the number of times the air in the sealed tube is evacuated is preferably twice to maximize the removal of residual air in the sealed tube.

[0043] The present invention injects perfluoropropane gas into a lipid film suspension and oscillates the suspension to obtain a lipid microbubble mixture. In the present invention, the oscillation is preferably mechanical. In the present invention, the oscillation is preferably performed on a high-speed blender; the frequency of the high-speed blender is 50 Hz; the frequency of the oscillation is preferably 45 to 50 seconds per time; the number of oscillations is preferably 2; and the oscillation time is preferably 90 to 100 seconds.

[0044] In a specific embodiment of the present invention, the lipid film suspension is divided into three sealed tubes, and perfluoropropane gas is injected into each sealed tube, followed by shaking to obtain three tubes of milky white lipid microbubble mixture; the three tubes of milky white lipid microbubble mixture are then combined into one tube, and a hydration solution containing Poloxamer 188 is added for dilution to obtain a lipid microbubble mixture. In the present invention, the injection method of the perfluoropropane gas and the composition of the hydration solution containing Poloxamer 188 are consistent with those described above and will not be repeated here. In the present invention, the dilution multiple is preferably 10 times.

[0045] After obtaining the lipid microbubble mixture, the present invention centrifuges the lipid microbubble mixture to obtain lipid nanobubbles. In the present invention, the centrifugation preferably includes a first centrifugation and a second centrifugation performed sequentially; the speed of the first centrifugation is preferably 1000-1500 rpm, more preferably 1000 rpm, and the time of the first centrifugation is preferably 3-5 minutes, more preferably 3 minutes; the speed of the second centrifugation is preferably 800-1000 rpm, more preferably 800 rpm, and the time of the second centrifugation is preferably 3-5 minutes, more preferably 3 minutes. In the present invention, the temperature of the centrifugation is preferably 4-8°C, more preferably 4°C. The present invention uses two low-speed step centrifugations to remove large bubbles and impurities in the lipid microbubble mixture to obtain lipid nanobubbles.

[0046] After obtaining the lipid nanobubbles, the present invention co-incubates the lipid nanobubbles with a biotinylated TF solution and a streptavidin solution to obtain transferrin-modified targeted lipid nanobubbles. In the present invention, the biotinylated TF solution refers to a biotin-coupled transferrin (TF) solution. In the present invention, the concentration of the lipid nanobubbles is preferably 6×10 8 / mL; the concentration of the biotinylated TF solution is preferably 0.5 mg / mL; the concentration of the streptavidin solution is preferably 0.5 mg / mL. In the present invention, the ratio of the lipid nanobubbles to the biotinylated TF solution and the streptavidin solution is preferably 500 μL:5 μL:10 μL. In the present invention, the co-incubation preferably comprises: a first incubation of the biotinylated TF solution and the streptavidin solution, followed by a second incubation with the lipid nanobubbles. In the present invention, the temperature of the first incubation is preferably 4°C; the temperature of the second incubation is preferably room temperature; the time of the first incubation is preferably 10 to 20 minutes, more preferably 20 minutes; and the time of the second incubation is preferably 20 to 30 minutes, more preferably 30 minutes. In the present invention, the co-incubation preferably includes a resting step after the resting step. The resulting suspension is the transferrin-modified targeted lipid nanobubbles. In the present invention, the resting step is preferably performed on ice; the resting step is preferably 5 to 10 minutes, more preferably 5 minutes. In the present invention, the middle and lower layers of the suspension are preferably taken after the standing to obtain transferrin-modified targeted lipid nanobubbles.

[0047] The present invention provides the use of the transferrin-modified targeted lipid nanobubbles described in the above technical solution as an ultrasound contrast agent in ultrasound imaging, preferably used in ultrasound molecular imaging, specifically used in targeted ultrasound molecular imaging of TfR1 molecules in ferroptotic cells.

[0048] The present invention provides the application of the transferrin-modified targeted lipid nanobubbles described in the above technical solution or the transferrin-modified targeted lipid nanobubbles prepared by the preparation method described in the above technical solution in the preparation of targeted ferroptosis cell drugs, specifically used in the preparation of targeted triple-negative breast cancer ferroptosis cell drugs. In the present invention, the transferrin-modified targeted lipid nanobubbles are evaluated by targeting triple-negative breast cancer ferroptosis cells at the cell level in vitro and by ultrasonic molecular imaging of ferroptosis tumor tissues of breast cancer mice at the animal level in vivo. In the present invention, the transferrin-modified targeted lipid nanobubbles are preferably treated with Co before use. 60 Irradiate for 15 minutes and sterilize. In the present invention, the transferrin-modified targeted lipid nanobubbles are preferably targeted ultrasound contrast agents.

[0049] The transferrin-modified targeted lipid nanobubbles prepared by the present invention have good stability and strong targeting, and can be used for non-invasive evaluation of the expression of chemotherapy-induced ferroptosis cells in triple-negative breast cancer.

[0050] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions of the present invention. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0051] Example 1

[0052] 10 mg of commercially purchased 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (abbreviated as DPPC, molecular weight: 734.04, CAS No.: 63-89-8, Sigma) solid powder and 4 mg of commercially purchased 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotinylated (polyethylene glycol) 2000] (abbreviated as DSPE-PEG2000-Biotin, molecular weight: 3016.81, CAS No.: 474964-24-4, Avanti Polar Lipids (Shanghai) solid powder was placed in a 25 mL rotary evaporation flask, 2 mL of chloroform was added in the dark, and the mixture was shaken. Then, 10 μL of fluorescent dye DiR solution (chloroform, concentration 10 μM) was added in the dark, and the mixture was thoroughly mixed and dispersed. After thorough mixing and dispersion, the chloroform was removed by rotary evaporation for 40 min (rotary evaporation water bath temperature 50°C, speed 120 rpm) to obtain a dry lipid film attached to the wall and bottom of the rotary evaporation flask;

[0053] 1 mL of a hydration solution containing Poloxamer 188 preheated to 37° C. (the hydration solution containing Poloxamer 188 consists of a Poloxamer 188 solution, a PBS buffer solution, and glycerol; the concentration of the Poloxamer 188 solution is 10 wt %, the concentration of the PBS buffer solution is 0.01 M, and the volume ratio of the PBS buffer solution, glycerol, and the Poloxamer 188 solution is 88:10:2) was added to the rotary evaporator with the dry lipid film attached thereto. The flask was placed in a 37° C. constant temperature shaker and slowly shaken (at a speed of 120 rpm) for 1 h until the lipid film fell off, thereby obtaining a lipid film suspension.

[0054] The lipid film suspension was evenly divided into 3 sealed tubes with airtight rubber stoppers; Figure 10The homemade simple three-way pipe gas exchange device shown in the figure repeatedly evacuates the air from each sealed tube. When the pressure inside the tube is at its lowest (close to vacuum), the valve of the three-way pipe is switched, and perfluoropropane gas (C3F8, purity 99.9%) is automatically injected into each sealed tube by utilizing the pressure difference between the inside and outside of the tube, so that the pressure inside and outside the tube reaches equilibrium. The sealed tubes with airtight rubber stoppers are placed on a high-speed blender and mechanically oscillated twice at a frequency of 50 Hz at a speed of 45 seconds per time for a total of 90 seconds to obtain a milky white lipid microbubble mixture.

[0055] The three tubes of milky white lipid microbubble mixture were combined into one tube, and 9 mL of a hydration solution containing Poloxamer 188 pre-cooled to 4°C was added (the hydration solution containing Poloxamer 188 consists of a Poloxamer 188 solution, a PBS buffer solution, and glycerol; the concentration of the Poloxamer 188 solution is 10 wt %, the concentration of the PBS buffer solution is 0.01 M, and the volume ratio of the PBS buffer solution, the glycerol, and the Poloxamer 188 solution is 88:10:2). The mixture was then centrifuged at 1000 rpm for 3 min at 4°C, then at 800 rpm and centrifuged for 3 min to obtain lipid nanobubbles (NB).

[0056] 2 mL of pure water was added to 1 mg of biotinylated TF (Cat. No. 010-0634, Rockland), and the mixture was dissolved to obtain a 0.5 mg / mL biotinylated TF solution. 5 μL of the biotinylated TF solution was incubated with 10 μL of streptavidin solution (0.5 mg / mL, Invitrogen) at 4°C for 20 min, and then gently shaken and mixed with 500 μL of the lipid nanobubbles (6×10 8 The suspension was incubated at room temperature for 30 min, and the obtained suspension was placed on ice for 5 min. The middle and lower layers of the suspension were taken and the obtained solution was the transferrin-modified targeted lipid nanobubble, which was recorded as NB. TF .

[0057] Comparative Example 1

[0058] The preparation method is basically the same as that of Example 1, except that biotinylated IgG solution (0.5 mg / mL) is used instead of biotinylated TF solution to connect with the streptavidin solution and lipid nanobubbles. The control lipid nanobubbles NB lgG Used for subsequent comparative experiments.

[0059] Test Example 1

[0060] Figure 1 Transferrin modified targeted lipid nanobubbles NB prepared in Example 1 TFThe preparation flow chart shows that the transferrin-modified targeted lipid nanobubbles prepared by the present invention are ultrasound contrast agents with a single-layer phospholipid shell, a shell surface modified with streptavidin and coupled with specific biotinylated TF, and an interior encapsulated with stable perfluoropropane gas.

[0061] Figure 2 Figure 3 is a diagram of the cross-linking pattern of streptavidin-biotinylated TF. Figure 1 In the biotin-streptavidin cross-linking method, streptavidin has a tetrameric conformation and has a very high binding affinity to biotin. The multivalent nature of streptavidin can bind to four biotinylated TF molecules with high affinity. The dissociation constant (Kd) between streptavidin and biotin is about 10 -14 mol / L, of which Figure 2 The B represents the biotin molecule.

[0062] Figure 3 NB prepared in Example 1 TF Particle size distribution (above) and NB TF The surface potential (below) shows that NB TF The particle size is about 512.9±10.2nm, NB TF The surface potential is -26.33 mV.

[0063] Figure 4 Observe NB under ordinary bright field microscope TF The morphology (left) and scanning electron microscopy observation of NB TF The morphology (right) shows that NB TF Appears as single spherical particles, evenly dispersed.

[0064] Figure 5 NB prepared in Example 1 TF The ultrasound echo characteristics in vitro show that NB TF It has a good imaging effect and lays the foundation for further ultrasound imaging research in in vivo animal experiments.

[0065] Test Example 2

[0066] Expression of TfR1 molecules and NB prepared in Example 1 TF Application of targeting ferroptosis cells in breast cancer Figures 6-7 As shown in the figure, the level of ferroptosis in tumor tissue cells of breast cancer mice was evaluated by ultrasound molecular imaging signals. Figures 8-9 shown.

[0067] Figure 6 Expression of TfR1 molecules in control cells and ferroptotic cells, as well as transferrin-modified targeted lipid nanobubbles NB TFThe experimental results were compared with those of control cells and breast cancer ferroptosis cells. Figure 7 Transferrin modified targeted lipid nanobubbles NB TF and control lipid nanobubbles NB lgG The experimental results were compared with those of breast cancer ferroptosis cell targeting.

[0068] The experimental process of TfR1 molecule expression is as follows: (1) First, 4T1 mouse breast cancer cells (Cell Bank of the Chinese Academy of Sciences, catalog number: SCSP-5056) incubated with 10 μM Erastin solution for 24 hours were used as iron-depleted cells, and 4T1 mouse breast cancer cells incubated with an equal amount of normal saline for 24 hours were used as control cells; after the incubation, the cell culture medium was discarded, and 200 μL of 4°C pre-cooled PBS buffer (concentration 0.01 M) was added to wash the remaining culture medium, followed by adding 100 μL of pre-cooled 4% paraformaldehyde solution, fixing the cells at room temperature for 20 minutes and then discarding them, and then adding 200 μL of PBS buffer (concentration 0.01 M) each time, washing 3 times, each time for 5 minutes, and removing the PBS solution. (2) Then, the cells were permeabilized by adding 100 μL of 0.1% Triton X-100 solution (0.1% Triton X-100 solution was obtained by adding 10 mL of PBS to 10 μL of Triton X-100 stock solution) to the culture dish, incubating at room temperature for 20 minutes and then discarding it. Goat serum was then added and the cells were incubated at 37°C for 30 minutes and then discarding the blocking solution. TfR1 primary antibody working solution (antibody diluted with PBS at a dilution ratio of 1:200) was added dropwise and incubated on a 4°C flatbed shaker for 24 hours. The primary antibody was then discarded and the cells were washed three times with PBS buffer for 5 minutes each. (3) Finally, FITC-labeled secondary antibody (antibody diluted with PBS buffer at a dilution ratio of 1:50) was added to the culture dish and incubated in the dark at room temperature for 1 hour. The secondary antibody was then discarded and the cells were washed three times with PBS for 5 minutes each and then discarded. The cells were then incubated with the nuclear dye DAPI (1 μg / mL) in the dark for 3 minutes, washed with distilled water for 1 minute, and dried. The cells were then sealed with anti-fluorescence attenuation mounting media and the expression of TfR1 was observed under a laser confocal microscope.

[0069] The targeted binding experiment process is as follows: (1) First, 4T1 mouse breast cancer cells were incubated with 10 μM Erastin solution for 24 hours as ferroptotic cells, and 4T1 mouse breast cancer cells were incubated with an equal amount of saline for 24 hours as control cells; after the incubation, the cell culture medium was discarded and the cells were washed three times with 0.01M PBS buffer, 200 μL each time, and 5 minutes each time. (2) Nuclear dye DAPI staining solution (concentration of 1 μg / mL) was added to the cells, incubated in the dark for 10 minutes, and then washed three times with 0.01M PBS buffer, 5 minutes each time. (3) 200 μL of 6×10 8 NB / mL TF The suspension was added to control cells and ferroptotic cells respectively; equal amounts (200 μL volume, concentration 6×10 8 NB lgG The suspension was added to the ferroptotic cells and incubated at 4°C in the dark for 30 minutes. The excess nanobubble solution was then gently poured off and the cells were gently washed twice with 0.01M PBS buffer, each for 3 minutes. (4) Finally, the NB was observed under a laser confocal microscope. TF Targeted binding to control cells and breast cancer ferroptosis cells, as well as NB TF and NB lgG Targeted binding with breast cancer ferroptosis cells.

[0070] Figure 6 and Figure 7 Transferrin-modified targeted lipid nanobubbles are abbreviated as NB TF , control lipid nanobubbles are abbreviated as NB lgG ; DAPI is a nuclear dye used to locate the position of cells; DiR means NB TF and NB lgG Stained with red DiR dye, DiR-NB TF Indicates NB stained with red DiR dye TF , used to locate the position of lipid nanobubbles; Merge represents the overlap of the two images.

[0071] Figure 6 The results of A showed that compared with the control cells, a large amount of green-labeled TfR1 was expressed on the surface of ferroptotic cells.

[0072] Figure 6 The results of B showed that compared with control cells, a large number of DiR-labeled NBs were tightly bound to the periphery of ferroptotic cells. TF .

[0073] Figure 7 NB lgG and NB TFComparison between the two revealed that lgG In contrast, a large number of DiR-labeled NBs can be seen TF Tightly bound to the periphery of ferroptotic cells, no NB was observed lgG Significantly binds to ferroptotic cells.

[0074] Figure 8 For NB TF Ultrasound imaging results in in vivo animal experiments. The experimental process is as follows: 4T1 mouse breast cancer cells were orthotopically inoculated under the left second mammary pad of female BALB / c mice (6-8 weeks old, weighing approximately 16-18g) to form tumors. 3 According to the established experimental plan, the tumors of transplanted mice were divided into ferroptosis tumor group and control tumor group. The treatment method of the ferroptosis tumor group was as follows: each mouse was intraperitoneally injected with a 6.8mM Erastin solution at a dose of 0.1mL / 10g in mice to induce ferroptosis in the tumor tissue of the mouse breast cancer orthotopic transplanted tumor. The control tumor group was obtained by treating with an equal amount of saline instead of Erastin solution. Before ultrasound imaging, the mice were anesthetized with 2% isoflurane inhalation, the tumor site was depilated, and then the limbs were fixed on the operating table and kept in a prone position. The imaging area was exposed and 1.5% isoflurane was used to maintain respiration during the imaging process (heart rate 380-450 beats / min, respiratory curve was stable). First, ultrasound coupling gel was applied between the mouse tumor site and the transducer to reduce the attenuation of ultrasound in the air. Subsequently, a Vevo2100 high-resolution ultrasound imager (MS250 ultrasound probe, frequency 13-24 MHz) was used to perform two-dimensional imaging (conventional grayscale imaging) of the target tumor area of ​​the mouse, and then switched to ultrasound contrast imaging mode. The ultrasound contrast imaging mode parameter optimization settings are as follows: mechanical index 0.065, contrast gain 38 dB, dynamic range 35 dB, depth 20 mm. 150 μL of NB was injected into the tail vein of the ferroptosis tumor mice. TF Suspension (concentration of 6×10 8 The images were taken immediately after observation for 3 minutes. The images were saved. The mice with control tumors were also given the same amount of NB. TF Observe the imaging contrast within the target tumor area, using the same parameters as above. Perform offline quantitative analysis of the contrast images to compare the imaging differences between ferroptotic tumors and control tumors.

[0075] Figure 8 A is the ultrasound targeted contrast imaging mode diagram; Figure 8 B stands for NB TF Comparison of ultrasound contrast imaging between control tumors and ferroptotic tumors. Figure 8 C is NB TFTime-intensity change curves corresponding to ultrasound imaging in control tumors (top) and ferroptotic tumors (bottom). Figure 8 In C, the horizontal axis is time in seconds, and the vertical axis is the ultrasonic echo intensity in dB. Figure 9 A, B, and C are extracted Figure 8 The area under the curve (AUC), peak intensity (PI), and peak intensity half-time obtained from the C-related parameters between the groups were statistically different. The analysis results showed that NB TF The ultrasound signal intensity of ferroptotic tumors was significantly higher than that of control tumors. Quantitative analysis of the time-intensity curves of contrast perfusion also revealed that the AUC, PI, and peak half-time in ferroptotic tumors were significantly higher than those in control tumors. **** indicates P < 0.0001, indicating statistically significant differences.

[0076] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A transferrin-modified targeted lipid nanovesicle, characterized in that: The transferrin-modified targeted lipid nanobubble has a bubble-like structure; the outer shell of the transferrin-modified targeted lipid nanobubble is a single layer of phospholipids; the outer shell surface of the transferrin-modified targeted lipid nanobubble is coupled to biotinylated TF via streptavidin; and the interior of the transferrin-modified targeted lipid nanobubble is wrapped with perfluoropropane gas.

2. The method for preparing the transferrin-modified targeted lipid nanovesicles according to claim 1, comprising the following steps: Filling perfluoropropane gas into the lipid film suspension and shaking it to obtain a lipid microbubble mixed solution; Centrifuging the lipid microvesicle mixture to obtain lipid nanovesicles; The lipid nanobubble is co-incubated with a biotinylated TF solution and a streptavidin solution to obtain a transferrin-modified targeted lipid nanobubble.

3. The preparation method according to claim 2, characterized in that: The preparation method of the lipid film suspension comprises: mixing a dry lipid film and a hydrating solution containing Poloxamer 188 to obtain the lipid film suspension; the hydrating solution containing Poloxamer 188 comprises a Poloxamer 188 solution, a PBS buffer solution and glycerol.

4. The preparation method according to claim 3, characterized in that: The preparation method of the dry lipid film comprises: mixing 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotinylated (polyethylene glycol) 2000] and chloroform, adding a fluorescent dye DiR solution under light-proof conditions, and removing the chloroform to obtain a dry lipid film.

5. The preparation method according to claim 2, characterized in that: The method of injecting the perfluoropropane gas into the lipid film suspension comprises: placing the lipid film suspension in a tube and sealing it, removing the air in the tube, and automatically injecting the perfluoropropane gas into the sealed tube by utilizing the pressure difference between the inside and outside of the tube.

6. The preparation method according to claim 2, characterized in that: The centrifugation includes a first centrifugation and a second centrifugation performed sequentially; the rotation speed of the first centrifugation is 1000-1500 rpm, and the time of the first centrifugation is 3-5 minutes; the rotation speed of the second centrifugation is 800-1000 rpm, and the time of the second centrifugation is 3-5 minutes.

7. The preparation method according to claim 2 or 6, characterized in that: The centrifugal temperature is 4-8°C.

8. The preparation method according to claim 2, characterized in that: The co-incubation further includes standing, and the suspension obtained after standing is the transferrin-modified targeted lipid nanovesicles.

9. Use of the transferrin-modified targeted lipid nanobubble according to claim 1 or the transferrin-modified targeted lipid nanobubble prepared by the preparation method according to any one of claims 2 to 8 as an ultrasound contrast agent in ultrasound imaging.

10. Use of the transferrin-modified targeted lipid nanovesicles according to claim 1 or the transferrin-modified targeted lipid nanovesicles prepared by the preparation method according to any one of claims 2 to 8 in the preparation of drugs targeting ferroptotic cells.

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