Targeted magnetic ultrasound microbubbles, and methods of making and using the same

Abstract

The application discloses a targeted magnetic ultrasonic microbubble for regulating telomerase activity and a preparation method and application thereof. The targeted magnetic ultrasonic microbubble is in a hollow bubble structure. The targeted magnetic ultrasonic microbubble has a shell layer and a targeting material modified on the outer surface of the shell layer. The shell layer comprises a magnetic material and a telomerase inhibitor. The targeting material recognizes M1 type macrophages. The targeted magnetic ultrasonic microbubble for regulating telomerase activity and the preparation method and application thereof can improve the targeting property of an atherosclerosis drug.

Description

Technical Field

[0001] The present application relates to the field of nano-biomedicine technology, and in particular to a targeted magnetic ultrasound microbubble and a preparation method and application thereof. Background Art

[0002] Atherosclerosis (AS) is a chronic, progressive pathological process characterized by the deposition of lipids (primarily cholesterol and triglycerides), inflammatory cell infiltration, smooth muscle cell proliferation, and fibrous tissue accumulation within the arterial wall, resulting in the formation of plaques. These plaques gradually enlarge, leading to arteriosclerosis and stenosis, impairing blood flow and, in severe cases, causing thrombosis and even arterial obstruction. Currently, lipid-lowering, blood pressure-lowering, and antiplatelet therapy are routine clinical treatments for atherosclerosis, but the incidence and mortality of cardiovascular and cerebrovascular events caused by atherosclerosis remain high.

[0003] Research suggests that the activation and proliferation of M1 macrophages are key to the development of atherosclerosis. M1 macrophages promote the progression of atherosclerosis by secreting a large number of pro-inflammatory cytokines. Furthermore, during this process, the activity of telomerase and telomerase reverse transcriptase in M1 macrophages increases. Previous studies have shown that modulating the activity of telomerase and telomerase reverse transcriptase in cells with telomerase inhibitors can treat atherosclerosis.

[0004] However, how to better deliver telomerase inhibitors to target cells and enhance the targeting effect is the key and difficulty in the implementation of this technology. Summary of the Invention

[0005] The purpose of this application is to provide a targeted magnetic ultrasound microbubble and its preparation method and application.

[0006] This application adopts the following technical solutions:

[0007] In the first aspect, the present application discloses a targeted magnetic ultrasound microbubble for regulating telomerase activity. The targeted magnetic ultrasound microbubble has a hollow bubble structure. The targeted magnetic ultrasound microbubble has a shell layer and a targeting material modified on the outer surface of the shell layer. The shell layer contains a magnetic material and a telomerase inhibitor. The targeting material recognizes M1 macrophages.

[0008] It should be noted that the targeted magnetic ultrasound microbubbles of the present application have a hollow bubble structure, which can facilitate real-time visualization of lesions in a specific target area under ultrasound; the outer surface is modified with a targeting material capable of recognizing M1 macrophages, which can improve the targeting of the microbubbles and promote precise drug delivery. In addition, it should be noted that when the targeted magnetic ultrasound microbubbles are applied in vivo, the presence of blood flow shear force in the blood vessel can easily cause "axial flow phenomenon", which can greatly affect the targeting efficiency of the microbubbles. The targeted magnetic ultrasound microbubbles of the present application contain magnetic materials, which can change and guide the distribution of the targeted magnetic ultrasound microbubbles under the action of an external magnetic field, increase the contact opportunity of the targeted magnetic ultrasound microbubbles with the lesions, for example, change the axial distribution characteristics of the microbubbles in the blood vessel under the action of the magnetic field, guide the microbubbles to adhere to the tube wall, increase the contact opportunity of the microbubbles with the target points on the blood vessel endothelium, thereby improving the targeting transmission efficiency of the microbubbles.

[0009] In an implementation manner of the present application, the particle size of the targeted magnetic ultrasound microbubbles is 2134-2448 nm. It should be noted that in this case, it is beneficial to ultrasound contrast, beneficial to guarantee the permeability of the microbubbles and carry enough drugs, and beneficial to improve the stability in blood circulation.

[0010] In an implementation manner of the present application, the shell layer further contains a shell layer base material, and the magnetic material and the telomerase inhibitor are embedded in the shell layer base material.

[0011] In an implementation manner of the present application, the mass ratio of the shell layer base material, the magnetic material and the telomerase inhibitor is (8-10):(1-2):(1-2). It should be noted that in this case, the overall performance of the targeted magnetic ultrasound microbubbles can be improved.

[0012] In an implementation manner of the present application, the shell layer base material includes at least one of polylactic acid-glycolic acid copolymer and polylactic acid.

[0013] In an implementation manner of the present application, the magnetic material includes at least one of ferroferric oxide, iron, cobalt and nickel.

[0014] In an implementation manner of the present application, the telomerase inhibitor includes at least one of BIBR1532, Imetelstat and TMP1363.

[0015] In an implementation manner of the present application, the targeting material includes at least one of anti-CD86 antibody and anti-CD80 antibody. It should be noted that M1 macrophages specifically express CD80 and CD86 on the surface, and by modifying the surface of the targeted magnetic ultrasound microbubbles with anti-CD86 antibody and / or anti-CD80 antibody, the targeted magnetic ultrasound microbubbles can target M1 macrophages.

[0016] In one implementation of the present application, the targeted magnetic ultrasound microbubbles can be used as ultrasound contrast agents. It should be noted that the targeted magnetic ultrasound microbubbles of the present application have a hollow bubble structure and can achieve real-time visualization of lesions in a specific target area under ultrasound.

[0017] In one implementation of the present application, the targeted magnetic ultrasound microbubbles contain at least one of octafluoropropane, perfluorobutane and sulfur hexafluoride.

[0018] The second aspect of the present application discloses a method for preparing targeted magnetic ultrasonic microbubbles for regulating telomerase activity, comprising: dissolving a shell matrix material, a magnetic material and a telomerase inhibitor in an organic solvent to obtain a shell solution; adding a foaming agent to the shell solution and performing ultrasonic vibration; collecting a precipitate by centrifugation, and drying the precipitate in an atmosphere containing a first gas to obtain magnetic ultrasonic microbubbles; and modifying the surface of the magnetic ultrasonic microbubbles with a targeting material to obtain the targeted magnetic ultrasonic microbubbles.

[0019] In one implementation of the present application, the shell matrix material includes polylactic acid-glycolic acid copolymer and polylactic acid.

[0020] In one implementation of the present application, the magnetic material includes at least one of ferroferric oxide, iron, cobalt and nickel.

[0021] In one implementation of the present application, the telomerase inhibitor includes at least one of BIBR1532, Imetelstat and TMP1363.

[0022] In one implementation of the present application, the targeted material includes at least one of an anti-CD86 antibody and an anti-CD80 antibody.

[0023] In one implementation of the present application, the first gas includes at least one of octafluoropropane, perfluorobutane, and sulfur hexafluoride. It should be noted that by filling the interior of the targeted magnetic ultrasound microbubbles with inert gases such as octafluoropropane, perfluorobutane, and sulfur hexafluoride, the stability of the targeted magnetic ultrasound microbubbles can be improved. Furthermore, these gases can act as ultrasound contrast agents, facilitating enhanced ultrasound signals and real-time monitoring of blood flow within blood vessels.

[0024] It should be noted that through the generation of bubbles by the foaming agent and the action of ultrasonic vibration, the shell matrix material (such as polylactic acid-glycolic acid copolymer), the magnetic material (such as ferroferric oxide) and the telomerase inhibitor (such as BIBR1532) form a hollow bubble structure. Under the hydrophobic interaction force, the magnetic material and the telomerase inhibitor are embedded in the shell matrix material.

[0025] In one implementation of the present application, the mass ratio of the shell matrix material, the magnetic material and the telomerase inhibitor is (8 to 10): (1 to 2): (1 to 2).

[0026] In one implementation of the present application, the mass ratio of the shell matrix material, the magnetic material and the telomerase inhibitor is 8:1:1.

[0027] In one implementation of the present application, the foaming agent is ammonium bicarbonate. It should be noted that the ammonia gas generated by ammonium bicarbonate can help form microbubbles with a hollow bubble structure. At the same time, ammonia gas is easily soluble in water and can be discharged from the interior of the microbubbles.

[0028] In one implementation of the present application, after the ultrasonic vibration, a dispersant is further added. It should be noted that the addition of a dispersant is beneficial to making the microbubbles more dispersed and reducing the aggregation of microbubbles.

[0029] In one implementation of the present application, the dispersant includes polyvinyl alcohol.

[0030] In one implementation of the present application, polyethyleneimine is added to the magnetic ultrasound microbubbles, and then the targeting material is added and incubated to obtain magnetic ultrasound microbubbles modified with the targeting material, that is, the targeted magnetic ultrasound microbubbles are obtained. It should be noted that the addition of polyethyleneimine can make the surface of the magnetic ultrasound microbubbles have a positive charge, which is conducive to modifying the targeting material (e.g., anti-CD86 antibody) to the surface of the magnetic ultrasound microbubbles.

[0031] The third aspect of the present application discloses a use of the targeted magnetic ultrasound microbubbles disclosed in the first aspect of the present application in the preparation of a product for treating atherosclerosis.

[0032] In one implementation of the present application, the targeted magnetic ultrasound microbubbles, under the influence of an external magnetic field and a targeting material, target M1 macrophages in the atherosclerotic plaque area and subsequently release telomerase inhibitors under the influence of ultrasonic cavitation. It should be noted that ultrasonic cavitation can cause the targeted magnetic ultrasound microbubbles to burst, triggering the release of the telomerase inhibitor. Cavitation also increases cell membrane permeability, promoting the entry of the telomerase inhibitor into the cells.

[0033] In one implementation of the present application, the targeted magnetic ultrasound microbubbles are injected into the subject via intravenous injection.

[0034] In one implementation of the present application, the targeted magnetic ultrasound microbubble dosage is 0.04 ng / kg to 0.06 ng / kg of a telomerase inhibitor, once daily. It should be noted that the dosage of the targeted magnetic ultrasound microbubble dosage is based on the telomerase inhibitor content, and 0.04 ng / kg means the dosage of the telomerase inhibitor (ng) is the subject's weight (kg) multiplied by 0.04.

[0035] The beneficial effects of this application are:

[0036] The targeted magnetic ultrasound microbubbles of the present application are modified on their outer surface with a targeting material that can recognize M1 macrophages, which can improve the targeting of the microbubbles and promote the precise delivery of drugs; the targeted magnetic ultrasound microbubbles contain magnetic materials, which can change and guide the distribution of the targeted magnetic ultrasound microbubbles under the action of an external magnetic field, increase the contact chance of the targeted magnetic ultrasound microbubbles with the lesions, and improve the targeted transmission efficiency of the microbubbles. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 It is a schematic flow chart of the method for preparing targeted magnetic ultrasound microbubbles involved in this application.

[0038] Figure 2 This is a schematic diagram of the structure and component determination results of the targeted magnetic ultrasound microbubbles involved in this application.

[0039] Figure 3 This is a graph showing the concentration and potential changes of the targeted magnetic ultrasound microbubble solution at different time points involved in this application.

[0040] Figure 4 This is a schematic diagram of the targeted magnetic ultrasound microbubble solution involved in this application under the action of a magnet.

[0041] Figure 5 It is a schematic diagram of the plate flipping experiment and parallel plate flow chamber experiment of the targeted magnetic ultrasonic microbubble involved in this application.

[0042] Figure 6 It is a schematic diagram of the results of the plate flipping experiment of targeted magnetic ultrasound microbubbles involved in this application.

[0043] Figure 7 It is a schematic diagram of the results of the parallel plate flow chamber experiment of targeted magnetic ultrasound microbubbles involved in this application.

[0044] Figure 8 This is a schematic diagram of the in vitro and in vivo performance test results of the targeted magnetic ultrasound microbubbles involved in this application. DETAILED DESCRIPTION

[0045] The present invention is further described in detail below by specific embodiments in conjunction with the accompanying drawings. In the following embodiments, many detailed descriptions are intended to enable the present application to be better understood. However, those skilled in the art can readily appreciate that some of the features may be omitted in different circumstances, or may be replaced by other materials or methods. In some cases, some operations related to the present application are not shown or described in the specification. This is to avoid overwhelm the core of the present application with excessive descriptions, and for those skilled in the art, it is not necessary to describe these related operations in detail. The related operations can be fully understood based on the description in the specification and the general technical knowledge in the art.

[0046] In addition, the features, operations, or characteristics described in the specification may be combined in any appropriate manner to form various embodiments. Furthermore, the steps or actions in the method description may be reordered or adjusted in a manner readily apparent to those skilled in the art. Therefore, the various sequences in the specification and drawings are provided solely for the purpose of clearly describing a particular embodiment and are not intended to be mandatory, unless otherwise specified.

[0047] Atherosclerosis is a disease that primarily affects large and medium-sized arteries, causing thickening and hardening of the vascular lining, and narrowing of the lumen. Currently, lipid-lowering, blood pressure-lowering, and antiplatelet therapy are routine clinical treatments, but the incidence and mortality of cardiovascular and cerebrovascular events associated with its progression remain high. Therefore, there is an urgent need for more effective and safe methods for the diagnosis and treatment of atherosclerosis. Studies have shown that the activation and proliferation of M1 macrophages are central to the development and progression of atherosclerosis. M1 macrophages specifically express CD80 and CD86 on their surface, promoting the progression of atherosclerosis by secreting large amounts of proinflammatory cytokines. Furthermore, during this process, the activities of telomerase and human telomerase reverse transcriptase (hTERT) are increased. Studies have shown that modulating the activity of telomerase and hTERT in cells with telomerase inhibitors can treat atherosclerosis. However, the delivery of telomerase inhibitors to target cells remains a key and challenging aspect of this approach.

[0048] The targeted ultrasound microbubble delivery system can not only visualize lesions through ultrasound angiography, but also achieve safe targeted drug delivery in vivo. However, when the targeted ultrasound microbubble delivery system is used in vivo, the "axial flow phenomenon" caused by the shear force of blood flow in the blood vessels will greatly affect its targeting efficiency. Under the influence of an external magnetic field, the axial distribution characteristics of microbubbles in the blood vessels are changed, guiding the microbubbles closer to the vessel wall, increasing their chances of contact with the target points on the vascular endothelium, and greatly improving the targeted transmission efficiency of the entire system. However, in the absence of a magnetic field, the use of magnetic ultrasound microbubbles alone is prone to miss the target again due to the shear force of blood flow.

[0049] In view of this, the present application creatively provides a targeted magnetic ultrasound microbubble with the ability to regulate telomerase activity, a preparation method thereof, and an application in the treatment of atherosclerosis. The targeted magnetic ultrasound microbubble delivery system constructed in the present application promotes better targeting and entry of drugs into target cells through dual targeting (magnetic and biological targeting) and ultrasonic cavitation, thereby achieving the purpose of efficiently controlling atherosclerosis. Specifically, the targeted magnetic ultrasound microbubble can be specifically targeted to the M1 macrophages in the atherosclerotic plaque area under dual targeting of an external magnetic field and anti-CD86 antibodies (or anti-CD80 antibodies), and the specific targeting process can be observed in real time under ultrasound. Thereafter, under ultrasonic cavitation, telomerase inhibitors such as BIBR1532 are released and promoted to enter cells, and atherosclerosis is treated by inhibiting telomerase reverse transcriptase activity, upregulating Caspace-3 protein expression, and promoting M1 macrophage apoptosis. The targeted magnetic ultrasound microbubbles constructed in this application have the advantages of good targeting effect, strong inhibition of telomerase activity, high efficiency in promoting apoptosis and strong anti-atherosclerosis therapeutic effect, and can provide new ideas for targeted and efficient anti-atherosclerosis.

[0050] The present invention is further described in detail below by means of specific examples. The following examples are only provided to further illustrate the present invention and should not be construed as limiting the present invention. In the following examples, unless otherwise specified, the reagents and instruments used are all commercially available, and the experimental procedures are all carried out in accordance with the product instructions and conventional experimental specifications.

[0051] Example:

[0052] (1) Construction of targeted magnetic ultrasound microbubbles

[0053] Figure 1 This is a schematic diagram of the process for preparing the targeted magnetic ultrasound microbubbles involved in this application, such as Figure 1 As shown, polylactic acid-glycolic acid copolymer (PLGA), Fe3O4 and BIBR1532 can be dissolved in chloroform, (NH4)HCO3 solution is added and ultrasonic vibration is performed, and then polyvinyl alcohol is added to obtain a hollow bubble structure with PLGA as the shell and Fe3O4 and BIBR1532 embedded in the PLGA. The hollow part of the hollow bubble structure is filled with chloroform gas, and then the shell surface is modified with anti-CD86 antibodies using polyethyleneimine to obtain targeted magnetic ultrasonic microbubbles.

[0054] Specifically, poly(lactic-co-glycolic acid) (PLGA): Fe₃O₄ powder: BIBR1532 (40 mg, 5 mg, and 5 mg, respectively) can be accurately weighed in a mass ratio of 8:1:1 (PLGA, Fe₃O₄, and BIBR1532, respectively). These can be dissolved in 1 mL of chloroform and placed in an ice bath. Once fully dissolved, 0.2 mL of (NH₄)HCO₃ (6% w / v) solution is added to the organic phase. In an ice bath, ultrasonic cell disruptor is used for sonication emulsification at 55W, operating for 3 seconds, and resting for 3 seconds, for a total of 2 minutes. Then, 5 mL of poly(vinyl alcohol) (PVA, 2% w / v) solution is added and thoroughly homogenized at 50,000 rpm / min for 10 minutes. Then, 10 mL of deionized water is added and magnetic stirring is applied for 4 hours to evaporate the organic solvent. The precipitate is collected by centrifugation at 5,000 rpm / min for 10 minutes at 4°C. Resuspend with appropriate amount of deionized water, freeze at -80℃ overnight, transfer to freeze dryer, freeze-dry in C3F8 gas environment in the dark for 48 hours to obtain BIBR1532-loaded magnetic ultrasonic microbubble powder.

[0055] Add polyethyleneimine (PEI, Mw=25000) to deionized water to obtain a PEI aqueous solution with a concentration of 0.1 mg / ml. Weigh 10 mg of the above-mentioned lyophilized powder (magnetic ultrasonic microbubble powder loaded with BIBR1532) and dissolve it in the polyethyleneimine (PEI, Mw=25000) aqueous solution, and stir magnetically in the dark for 4 hours. Centrifuge at 5000 rpm / min for 10 minutes at 4°C to collect the precipitate. Resuspend with an appropriate amount of deionized water, freeze at -80°C overnight, and transfer to a freeze dryer. Freeze-dry in the dark under a C3F8 gas environment for 48 hours to obtain magnetic cationic ultrasonic microbubble powder loaded with BIBR1532.

[0056] Dissolve 10 mg of the above powder (BIBR1532-loaded magnetic cationic ultrasound microbubble powder) in 0.1 mL of PBS. Add 0.01 mL of a 200 μg / mL anti-CD86 antibody solution. Incubate at 4°C in the dark for 10 minutes. Centrifuge at 5000 rpm for 5 minutes to collect the precipitate and remove unbound free anti-CD86 antibody. Resuspend the solution in an appropriate amount of PBS to prepare the BIBR1532-loaded targeted magnetic ultrasound microbubble solution.

[0057] (2) Characterization of the physical and chemical properties of targeted magnetic ultrasound microbubbles

[0058] Structure, composition, and potential measurements: Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were used to observe the size and morphology of the microbubbles. A fluorescent secondary antibody of the same species as the anti-CD86 antibody was co-incubated with the microbubbles, and confocal microscopy was used to observe whether the microbubbles were successfully loaded with the antibody. The BIBR1532 loading capacity in the microbubbles was calculated using a UV spectrophotometer and Lambert-Beer's law. The particle size and surface potential of the BIBR1532-loaded targeted magnetic ultrasound microbubbles were measured using a Malvern Zetasizer Nano particle size potentiometer. Changes in the microbubble size and potential after loading with Fe₃O₄ and anti-CD86 antibody were also observed. Figure 2 This is a schematic diagram of the structure and component determination results of the targeted magnetic ultrasound microbubbles involved in this application. Figure 2 Part A is a schematic diagram of scanning electron microscopy, Part B is a schematic diagram of transmission electron microscopy, Part C is a schematic diagram of particle size results, Part D is a schematic diagram of confocal immunofluorescence, and Part E is a schematic diagram of Zeta potential. Figure 2 It can be seen that the prepared BIBR1532-loaded targeted magnetic ultrasound microbubble solution is brown, and both SEM and TEM show that the microbubbles have a hollow bubble structure, a rough surface, and a particle size of 2291±157.09nm. Before PEI modification, the zeta potentials of untargeted microbubbles (containing no magnetic material or targeting material) and single-targeted microbubbles (only magnetic) were -18.33±0.86mV and -21.23±1.46mV, respectively. After positive charge modification, the potentials of the two were significantly increased to 14.43±1.34mV and 20.27±1.12mV, respectively. After adding anti-CD86 antibodies for co-incubation, the potentials of the two decreased significantly, which indirectly indicates that the antibodies can be successfully connected to the microbubbles. Confocal immunofluorescence showed that the red microbubbles co-localized with the green anti-CD86 antibodies, proving that the anti-CD86 antibodies were successfully loaded onto the microbubbles.

[0059] Stability analysis: At room temperature, the concentration and potential of the microbubble solution were measured at 0 h, 4 h, 8 h, 12 h, 24 h and 7 d to evaluate the stability of the microbubbles. Figure 3 This is a graph showing the concentration and potential changes of the targeted magnetic ultrasound microbubble solution at different time points involved in this application, such as Figure 3 As shown, there was no significant difference in the concentration and potential of the microbubbles measured at different time points (p < 0.05), which proved that the prepared BIBR1532-loaded targeted magnetic ultrasound microbubbles had good stability.

[0060] Magnetic responsiveness analysis: The microbubble solution was thoroughly mixed and the ability of the microbubbles to be captured by the magnetic field was observed under the action of a magnet (≈1.2 T) to evaluate the magnetic responsiveness of the microbubbles. Figure 4 This is a schematic diagram of the targeted magnetic ultrasound microbubble solution involved in this application under the action of a magnet, such as Figure 4As shown in the figure, the targeted magnetic ultrasound microbubbles uniformly distributed in PBS quickly gathered on one side of the magnet after 120s, proving that the prepared BIBR1532-loaded targeted magnetic ultrasound microbubbles have good magnetic responsiveness.

[0061] Targeting analysis: The targeting ability of microbubbles was evaluated by observing the number of microbubbles around cells under the action of magnetic field and antibody targeting through plate flipping experiment and parallel plate flow chamber experiment. Figure 5 Schematic diagram of the plate flipping experiment and parallel plate flow chamber experiment of the targeted magnetic ultrasound microbubble involved in this application, such as Figure 5 As shown in the figure: ① The steps of the plate flipping experiment are as follows: prepare a 24-well plate, inoculate M1 macrophages at 5×10 4 The cells were seeded in a well plate at a density of 100 cells / well. When the cell density reached 80%, the cells were fixed with formaldehyde and the nuclei were stained with DAPI. Then 3 mL of 2.5×10 7 / mL, and pre-labeled with DiI microbubble suspension (control group, magnetic single target group, antibody single target group and dual target group). Use a PCR-specific well plate membrane to seal the well plate, then place a magnet at the bottom of the well plate, slowly rotate 360° along the long axis of the well plate for 10 minutes, and remove the magnet. Aspirate the microbubble suspension and gently rinse twice with PBS to remove free, untargeted microbubbles. Finally, count the number of microbubbles (red fluorescence) attached to the cells in each group under a fluorescence microscope. ②The steps of the parallel plate flow chamber experiment are as follows: M1 macrophages were inoculated into the culture dish according to the above method. When the cell density reached 80%, the cells were fixed with formaldehyde and the cell nuclei were stained with DAPI. The culture dish was turned upside down on the negative pressure suction platform, one end of the liquid interface was connected to the microinjection pump, and the other end was connected to the negative pressure suction pump. The air in the flow chamber pipeline was removed, and a magnet (≈1.2T) was placed above the culture dish. Subsequently, the microbubble suspensions (control group, magnetic single target group, antibody single target group and dual target group) pre-labeled with DiI were injected into the microinjection pump at different shear stresses (6, 12, 18, 24, 48 dyn / cm 2 ) through a parallel plate flow chamber. As the microbubble suspension flowed through the culture dish, a timer was started for 2.5 minutes. The magnetic field was then removed, and the flow chamber system was flushed with PBS for 1 minute, maintaining the original flow rate. Finally, the number of microbubbles adhered to cells in each group (red fluorescence) was counted under a fluorescence microscope. Figure 6 Schematic diagram of the results of the plate flipping experiment of the targeted magnetic ultrasound microbubble involved in this application, such as Figure 6 As shown in the figure, compared with non-targeted microbubbles (Non-target) or single-targeted microbubbles (Single-target), BIBR1532-loaded targeted magnetic ultrasound microbubbles (i.e., double-target microbubbles) have the highest number of cell pericellular adhesions. Figure 7Schematic diagram of the results of the parallel plate flow chamber experiment of the targeted magnetic ultrasound microbubble involved in this application, such as Figure 7 As shown in the figure, at each shear force, BIBR1532-loaded targeted magnetic ultrasound microbubbles maintained the highest cell adhesion number compared to untargeted microbubbles or single-targeted microbubbles. The advantage of targeted magnetic ultrasound microbubbles was particularly evident at the highest shear force, demonstrating that BIBR1532-loaded targeted magnetic ultrasound microbubbles have excellent M1 macrophage targeting performance.

[0062] (3) Performance characterization of targeted magnetic ultrasound microbubbles

[0063] In vitro study of the effect of promoting M1 cell apoptosis: After the plate flipping experiment, the supernatant was removed and washed three times with PBS to remove free microbubbles. Fresh culture medium was then added, and ultrasound (sound intensity: 1.2 W / cm2, duty cycle: 30%, treatment time: 40 s) was used to blast the microbubbles targeted to M1 macrophages to release BIBR1532. The cell membrane permeability was increased under the action of cavitation, which promoted the entry of BIBR1532 into the cells. The expression of telomerase reverse transcriptase and apoptosis-related proteins was analyzed by Western Blot to evaluate the performance of BIBR1532-loaded targeted magnetic ultrasound microbubbles in promoting M1 cell apoptosis.

[0064] In vivo anti-atherosclerotic effect study: ApoE- / - mice were fed a high-fat diet for 8 weeks to establish an atherosclerosis model. 0.2 mL of targeted magnetic ultrasound microbubble solution was injected through the tail vein and ultrasound (sound intensity: 1.5 W / cm 2 Treatment was conducted three times per week for four weeks (with a duty cycle of 30% and a treatment duration of 10 minutes). After treatment, the mice were sacrificed, and whole blood was collected for routine blood tests and biochemical analysis. Frozen sections of major organs (heart, liver, spleen, lungs, and kidneys) were prepared. The heart was stained with immunofluorescence, immunohistochemistry, Oil Red O, and hematoxylin and eosin (HE), while the remaining organs were stained with HE. This was done to evaluate the anti-atherosclerotic properties and biosafety of the BIBR1532-loaded targeted magnetic ultrasound microbubbles.

[0065] Figure 8 This is a schematic diagram of the in vitro and in vivo performance test results of the targeted magnetic ultrasound microbubbles involved in this application. Figure 8 In the figure, part A is the in vitro experimental results of targeted magnetic ultrasound microbubbles, and part B is the in vivo experimental results of targeted magnetic ultrasound microbubbles. Figure 8 As shown in Part A, compared with the control group (no microbubbles added), free drugs, non-targeted microbubbles or single-targeted microbubbles, targeted magnetic ultrasound microbubbles can significantly downregulate the expression of telomerase reverse transcriptase (TERT) protein and promote the expression of apoptosis-related protein Cleaved-caspase3, thereby promoting the apoptosis of M1 macrophages; Figure 8As shown in part B of FIG. 6, the results of the heart frozen section showed that, compared with the control group (without adding any microbubbles) and the single-targeting microbubble group, the plaque area of the targeting magnetic microbubble group was the smallest, the lipid deposition was the least, and the number of M1 macrophage infiltration in the plaque was the least; in addition, after the treatment, the blood routine and HE staining of the main organs of the mice showed no obvious changes compared with the control group. It is proved that the BIBR1532-loaded targeting magnetic microbubble has good biological safety and excellent anti-atherosclerosis performance without affecting the blood routine and the function of the main organs.

[0066] The above is a further detailed description of the present application in combination with specific embodiments, and the specific implementation of the present application cannot be limited to these descriptions. For ordinary skilled persons in the technical field to which the present application belongs, some simple deductions or replacements can be made without departing from the concept of the present application.

Claims

1. A targeted magnetic ultrasound microbubble for regulating telomerase activity, characterized in that: The targeted magnetic ultrasound microbubbles have a hollow bubble structure and have a shell layer and a targeting material modified on the outer surface of the shell layer. The shell layer contains a magnetic material and a telomerase inhibitor, and the targeting material recognizes M1 macrophages. The shell layer also contains a shell matrix material, and the magnetic material and the telomerase inhibitor are embedded in the shell matrix material. The shell matrix material includes at least one of polylactic acid-glycolic acid copolymer and polylactic acid. The telomerase inhibitor is BIBR1532. The targeted material is an anti-CD86 antibody.

2. The targeted magnetic ultrasound microbubble according to claim 1, characterized in that: The particle size of the targeted magnetic ultrasound microbubbles is 2134-2448 nanometers.

3. The targeted magnetic ultrasound microbubble according to claim 1, characterized in that: The magnetic material includes at least one of ferroferric oxide, cobalt and nickel.

4. The targeted magnetic ultrasound microbubble according to any one of claims 1 to 3, characterized in that: The targeted magnetic ultrasound microbubbles serve as ultrasound contrast agents.

5. The targeted magnetic ultrasound microbubble according to claim 4, characterized in that: The targeted magnetic ultrasound microbubbles contain at least one of octafluoropropane, perfluorobutane and sulfur hexafluoride.

6. A method for preparing targeted magnetic ultrasound microbubbles for regulating telomerase activity as claimed in claim 1, characterized in that: include: dissolving a shell matrix material, a magnetic material and a telomerase inhibitor in an organic solvent to obtain a shell solution; adding a foaming agent to the shell solution and performing ultrasonic vibration; collecting the precipitate by centrifugation, and drying the precipitate in an atmosphere containing the first gas to obtain magnetic ultrasonic microbubbles; The targeted magnetic ultrasound microbubbles are obtained by modifying the surface of the magnetic ultrasound microbubbles with a targeting material.

7. The preparation method according to claim 6, characterized in that The magnetic material includes at least one of ferroferric oxide, cobalt and nickel.

8. The preparation method according to claim 6, characterized in that The first gas includes at least one of octafluoropropane, perfluorobutane and sulfur hexafluoride.

9. The preparation method according to claim 6, characterized in that The mass ratio of the shell matrix material, the magnetic material and the telomerase inhibitor is 8-10:1-2:1-2.

10. The preparation method according to claim 9, characterized in that The mass ratio of the shell matrix material, the magnetic material and the telomerase inhibitor is 8:1:

1.

11. The preparation method according to claim 6, characterized in that The foaming agent is ammonium bicarbonate.

12. The preparation method according to claim 6, characterized in that After the ultrasonic vibration, a dispersant is added, and the dispersant includes polyvinyl alcohol.

13. The preparation method according to claim 6, characterized in that The targeted magnetic ultrasonic microbubbles are obtained by adding polyethyleneimine to the magnetic ultrasonic microbubbles and then adding the targeting material for incubation to obtain magnetic ultrasonic microbubbles modified with the targeting material.

14. Use of the targeted magnetic ultrasound microbubble according to any one of claims 1 to 5 in preparing a product for treating atherosclerosis.

15. The use according to claim 14, characterized in that The targeted magnetic ultrasound microbubbles target the M1 macrophages in the atherosclerotic plaque area under the action of an external magnetic field and a targeting material, and then release the telomerase inhibitor under the action of ultrasonic cavitation.

16. The use according to claim 14, characterized in that The targeted magnetic ultrasound microbubbles are injected into the subject's body through intravenous injection.

17. The use according to claim 14, characterized in that The targeted magnetic ultrasound microbubbles are administered at a dosage of 0.04-0.06 ng / kg of telomerase inhibitor, once a day.

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