CN110548140B - Use of microbubbles with oxygen for preparing set for inducing normalization of diseased tissue blood vessels - Google Patents

Use of microbubbles with oxygen for preparing set for inducing normalization of diseased tissue blood vessels Download PDF

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CN110548140B
CN110548140B CN201810555428.0A CN201810555428A CN110548140B CN 110548140 B CN110548140 B CN 110548140B CN 201810555428 A CN201810555428 A CN 201810555428A CN 110548140 B CN110548140 B CN 110548140B
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CN110548140A (en
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叶秩光
何奕儒
朱书苇
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Abstract

Use of oxygen-bearing microbubbles for preparing a set for inducing normalization of diseased tissue blood vessels, wherein the oxygen-bearing microbubbles comprise oxygen and a gas which is insoluble in water, and the particle size of the oxygen-bearing microbubbles is 0.5-20 μm; wherein, the set comprises an oxygen-carrying microbubble mixed liquid and an ultrasonic wave emitting device; an effective dose of the microbubbles with oxygen enters the organism through intravenous injection, and then the ultrasonic wave emitting device is used for irradiating the lesion tissue, so that the microbubbles with oxygen are broken at the lesion tissue to release oxygen.

Description

Use of microbubbles with oxygen for preparing set for inducing normalization of diseased tissue blood vessels
Technical Field
The invention relates to an application of microbubbles with oxygen in preparing a set for inducing normalization of blood vessels of a diseased tissue, in particular to a set for inducing normalization of blood vessels of the diseased tissue by injecting the microbubbles with oxygen intravenously and irradiating ultrasonic waves at the diseased tissue to stimulate the microbubbles with oxygen to release oxygen locally at the diseased tissue.
Background
After the tumor tissue grows to a certain extent, the tumor cell itself or the surrounding connective tissue secretes many growth factors for promoting angiogenesis so as to induce the formation of new blood vessels at the tumor site to supply nutrients for the tumor cell. However, the abnormal proliferation of new vessels inside the tumor leads to the bending and uneven thickness of the vessels, more pores in the vessel wall and reduced blood transport function of the vessels. Therefore, even when the drug is administered to a tumor tissue, the drug to be administered does not smoothly reach the inside of the tumor tissue, and the therapeutic effect is limited.
In order to increase the efficiency of tumor treatment, drugs for normalizing tumor blood vessels are usually administered before the administration of the treatment, however, conventional drugs usually have short effective period, so that the time for the therapeutic drugs to actually reach the inside of tumor tissues is limited, and the therapeutic effect of tumors is often limited.
Since the normalization of tumor blood vessels is helpful for the delivery of therapeutic drugs, there is a need for a kit for inducing the normalization of blood vessels, which can not only induce the normalization of blood vessels in tumor tissues, improve the blood vessel morphology and function of tumor tissues, but also prolong the time window for the normalization of blood vessels.
Disclosure of Invention
In order to achieve the above object, the present invention provides a use of microbubbles containing oxygen for preparing a set for inducing normalization of blood vessels in a diseased tissue, wherein the microbubbles containing oxygen comprise a gas hardly soluble in water and an oxygen gas, the microbubbles containing oxygen have a particle size of 0.5 to 20 μm, the set comprises a microbubble mixed solution containing oxygen and an ultrasonic wave emitting device, an effective dose of microbubbles containing oxygen is injected into a living body by veins, and the diseased tissue is irradiated by the ultrasonic wave emitting device, so that the microbubbles containing oxygen are ruptured at the diseased tissue to release the oxygen gas.
In a preferred embodiment, the particle size of the oxygen-carrying microbubbles is preferably 0.7 to 3.0 μm, wherein more than 3.0 μm of the oxygen-carrying microbubbles accounts for 0.5% of the total number.
In a preferred embodiment, the volume ratio of the water-insoluble gas and the oxygen contained in the oxygen-carrying microbubbles is 1:1 to 3: 1, wherein 1:1 to 1.4:1 is preferred.
In a preferred embodiment, the water-insoluble gas contained in the oxygen-bearing microbubbles is selected from perfluoropropane (C)3F8) Perfluorobutane (C)4F10) Nitrogen (N2), carbon dioxide (CO)2) And mixtures thereof. Among them, perfluoropropane is preferable.
In a preferred embodiment, the oxygen-containing microbubbles further comprise a shell of phospholipid encapsulating the water-insoluble gas and the oxygen gas. Among them, the phospholipid shell layer is preferably composed of 1, 2-Distearoyl-sn-glycerol-3-phosphocholine (1, 2-Distearoyl-sn-glycerol-3-phosphocholine; DSPC) and 1, 2-Distearoyl-sn-glycero-3-phosphoethanolamine-N- [10- (trimethoxysilyl) undecanamide (polyethylene glycol-2000) (1, 2-Distearoyl-sn-glycerol-3-phosphoethanolamine-N- [10- (trimethoxysilyl) undecanamide (polyethylene glycol-2000), DSPE-PEG-2000).
In a preferred embodiment, the dosage of the microbubbles with oxygen is 2.5 to 3.5 μ L/kg per day.
In a preferred embodiment, the ultrasound device is a high intensity focused ultrasound device. In a preferred embodiment, the parameters of the ultrasonic transmitter are: the sound pressure is 1.5-2.5 MPa; the period is 500-1500; the Pulse Repetition Frequency (PRF) is 1-5 Hz.
In a preferred embodiment, the diseased tissue is tumor tissue, normal tissue with vascular embolization resulting in hypoxia, or damaged blood vessels.
Drawings
Fig. 1 is a graph with different perfluoropropanes: the particle size distribution diagram of the oxygen-carrying microbubbles of the volume ratio of the oxygen gas;
fig. 2 is a graph with different perfluoropropanes: a volume distribution map of oxygen-carrying microbubbles of a volume ratio of oxygen;
FIG. 3 is a graph showing the change in dissolved oxygen of different groups of microbubbles;
FIG. 4 is a schematic diagram showing the change of image brightness map of the images taken by the perfluoropropane microbubble and the ultrasound imaging system with oxygen microbubbles;
FIG. 5 is a schematic diagram showing the perfusion ratio of perfluoropropane microbubbles and oxygen-carrying microbubbles to release gas to the local tumor part through ultrasonic stimulation;
FIG. 6 is a schematic graph showing the density of perfluoropropane microbubbles and tumor vessels with oxygen microbubbles releasing gas to tumor sites via ultrasonic stimulation;
FIG. 7 is a schematic graph of the variation of blood perfusion ratio for tumors for different doses of oxygen bearing microbubbles;
FIG. 8 is a graph showing the expression levels of PHD2, HIF-1. alpha., VEGF, and TGF-. beta.on day 4 after administration of perfluoropropane microbubbles and oxygen-bearing microbubbles.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to the accompanying drawings in combination with specific embodiments.
Preparation method of microbubbles with oxygen-film hydration method
In this embodiment, the oxygen-carrying microbubbles are prepared by a film hydration method, which includes the following steps: (1) preparing a phospholipid film: 2.5mg of 1, 2-Distearoyl-sn-glycerol-3-phosphocholine (1, 2-Distearoyl-sn-glycerol-3-phosphothiocholine; DSPC) and 1mg of 1, 2-Distearoyl-sn-glycerol-3-phosphoethanolamine-N- [10- (trimethoxysilyl) undecanamide (polyethylene glycol-2000)](ii) a DSPE-PEG-2000) was added to a 2mL sample bottle, DSPC and DSPE-PEG-2000 were uniformly dissolved and mixed with 0.25mL Chloroform (Chloroform) as a solvent, and the Chloroform was drained after heating at 60 ℃ for 1 hour by a water bath method, and then the sample bottle was placed in a vacuum concentrator and continuously drained under vacuum for 24 hours, after the organic solvent was completely removed, a lipid film was formed at the bottom of the sample bottle, which was then stored at-20 ℃. (2) Preparing oxygen-carrying microbubbles: phosphate buffer solution (Phosphate buffered)saline, PBS) and glycerin (Glycerol) at a volume ratio of 20: 0.1, 0.8mL of a PBS glycerin solution was added to the sample bottle, heated at 60 ℃ for 10 minutes by a water bath method, and after the phospholipid membrane was dissolved and mixed uniformly using a water bath ultrasonic homogenizer (Model 2510, Branson, NY, USA), the inside of the sample bottle was evacuated by an air pump, and perfluoropropane (C) was filled3F8) Gas is introduced into the sample bottle, then perfluoropropane gas is pumped out, oxygen is refilled into the sample bottle, and then the sample bottle is vibrated for 45 seconds at normal temperature by using a high-speed vibrator (Vialmix, Bristol-Myers liquid Imaging, NY, USA), and during the vibration, phospholipid molecules self-assemble and coat perfluoropropane and oxygen to form oxygen-carrying microbubbles. In this embodiment, the gas volume ratio of perfluoropropane to oxygen in the sample bottle is adjusted to 1:1, 1.4:1, 2: 1, 3: 1, 1: 0, and the optimal gas volume ratio of perfluoropropane to oxygen in the sample bottle is determined by the particle size distribution and concentration of the microbubbles with oxygen.
Particle size distribution and concentration of microbubbles with oxygen
The particle size distribution and concentration, average particle size and volume distribution of the above-mentioned oxygen-carrying microbubbles prepared with perfluoropropane/oxygen volume ratios of 1:1, 1.4:1, 2: 1, 3: 1 and 1: 0 and the perfluoropropane microbubbles as a control were measured using a particle size analyzer (Model Multisizer 3, Beckman Coulter inc., CA, USA). The average particle size and concentration are shown in table 1 below, and the particle size and volume distribution are shown in fig. 1 and fig. 2, respectively:
table 1, average particle size and concentration of oxygen-bearing microbubbles at different gas ratios.
Figure BDA0001681925670000041
As can be seen from FIGS. 1 and 2, the oxygen-carrying microbubbles having perfluoropropane/oxygen volume ratios of 1:1 and 3: 1 had a large number of microbubbles larger than 2 μm and had a low total concentration, whereas the oxygen-carrying microbubbles prepared using a 1.4:1 ratio had particle sizes and volume distributions similar to those of the 1: 0 perfluoropropane microbubbles (see Table 1), so that the following measurements and in vivo experiments were performed using the oxygen-carrying microbubbles prepared using a perfluoropropane/oxygen volume ratio of 1.4: 1.
Dissolved oxygen with microbubbles of oxygen
Measuring the dissolved oxygen amount of the microbubbles with oxygen by using a dissolved oxygen meter, and respectively measuring the degassed PBS, the PBS + perfluoropropane + oxygen, the perfluoropropane microbubbles and the microbubbles with oxygen (C)3F8∶O21.4: 1), cleaned microbubbles with oxygen (C)3F8∶O21.4: 1) in total. The PBS + perfluoropropane + oxygen refers to an aerated water solution without microbubble coated gas, the cleaned microbubbles with oxygen refer to that the prepared microbubbles with oxygen are centrifuged for one minute at 2000rcf, and then the supernatant is replaced by degassed PBS, so that the group is to eliminate the dissolved oxygen in the PBS solution and further calculate the oxygen concentration on the microbubbles with oxygen. The dissolved oxygen amount is measured by placing 800 μ L of full-concentration microbubbles with oxygen into a 20mL sample bottle, inserting a probe of a dissolved oxygen meter into the bottle, submerging a monitor at the top end of the probe into the microbubbles with oxygen, fixing the probe of the dissolved oxygen meter by a three-axis platform during measurement, and recording the change of the dissolved oxygen amount of each group after the value of the dissolved oxygen amount is stable. The oxygen content of the oxygen-carrying microbubbles prepared with a perfluoropropane/oxygen volume ratio of 1.4:1 is 8.9 ± 0.02mg/L, which is increased by 2.07 ± 0.14mg/L compared to the perfluoropropane microbubbles, and the comparison of the oxygen content of the two groups of oxygen-carrying microbubbles and cleaned oxygen-carrying microbubbles shows that the dissolved oxygen does not change significantly (p > 0.05) due to the replacement of PBS, and thus the measurement results show that most of the oxygen is encapsulated in the microbubbles and not dissolved in PBS.
Stability of microbubbles with oxygen
This example simulates the acoustic stability of microbubbles with oxygen and perfluoropropane at 37 ℃ in a living body using a commercial ultrasound imaging system (Model 3000, Terason, Burlington, Mass.) in conjunction with a self-made phantom.
In this embodiment, a replica is prepared from agar powder (ultrapurestargase, Invitrogen, CA, USA), agar powder with a weight percentage of 2% is uniformly mixed with degassed Distilled Deionized Water (DDW), the agar powder is completely dissolved in the heating and stirring process, when the mixed solution is clear, transparent and colorless, the mixed solution is poured into a self-made replica container for sizing, a solid cylindrical glass tube model with a diameter of 0.5mm is inserted before non-solidification to serve as a cavity of the replica, and after the replica is completely solidified, the glass tube model is removed, so that the preparation of the replica can be completed.
Then, the phantom was first immersed in degassed water, and an imaging probe of an ultrasonic imaging system was clamped by a jig, and microbubbles (C) containing oxygen diluted 4000 times with physiological saline were injected into the phantom chamber3F8∶O21.4: 1) and perfluoropropane microbubbles to perform imaging, in order to simulate the in vivo environment of a living body, the temperature of the water tank was controlled at 37 ℃ using a heating rod, one ultrasound image was taken every 10 minutes, continuously for 60 minutes, and after three experiments were repeated, MATLAB (2010 a; MathWorks, Natick, MA, USA), to quantify the contrast effect of the images generated by the microbubbles with oxygen and the microbubbles with perfluoropropane, selecting regions of interest (ROI) of the same size at each time point, and dividing the background intensity of the water at equal height positions to obtain the change of the acoustic intensity Signal (SNR) at different time points. As shown in fig. 4, it can be seen from the measurement results that no matter the microbubbles with oxygen or the perfluoropropane microbubbles have, no significant decrease in image brightness occurs within 60 minutes of the ultrasound imaging system, and it can be confirmed that the microbubbles do not become unstable due to the addition of oxygen.
In vivo experiment verifies that microbubbles with oxygen promote normalization of blood vessels
The present example used C57BL/6JNarl strain black mice, male sex, age between 6-8 weeks, body weight approximately 30g, provided by the animal center. Subcutaneous tumor model Mouse Prostate cancer cells (TRAMP) were used, and 1X 10 cells were taken6The TRAMP cells were implanted subcutaneously in the right leg of mice, and after waiting 7 days, tumors grewThe experiment was repeated until the diameter was about 7 mm. During the experiment, mice were anesthetized by intraperitoneal injection, and the anesthetic drug was 50 μ L of a mixture of sultam (Zoletil 50, Virbac, TW) and entacapone (Rompun 20, Bayer, TW) in a volume ratio of 1: 1. After the mice are anesthetized, the hairs at the tumor positions are shaved off by a shaver, and the hair removal ointment is uniformly smeared to completely clean the tumor epidermis areas. In the experiment, in order to avoid mouse temperature loss, the body temperature of the mouse is maintained at 37 ℃ by a heating pad.
Then, an ultrasonic image guidance treatment system is erected for in vivo verification, the right hind leg of a mouse planted with a tumor is placed below a self-made water tank containing a preservative film window, a 2MHz High intensity focused ultrasonic probe (High intensity focused ultrasound, HIFU) and an image probe of a commercial ultrasonic image system are placed into the water tank and are focused on the same section of the subcutaneous tumor together, the 2MHz High intensity focused ultrasonic probe stimulates oxygen-carrying micro bubbles to release oxygen, and meanwhile, an ultrasonic image provided by the image probe of the commercial ultrasonic image system is used for monitoring the treatment process and positioning the tumor position so as to adjust the treatment area.
The detailed experimental procedure is as follows:
(1) blood perfusion image of whole tumor before oxygen administration: first, perfluoropropane microbubbles are injected into the tumor to obtain information on blood perfusion, and in order to obtain the whole tumor containing perfluoropropane microbubbles with a fixed concentration when ultrasound images of the tumor are collected, the injection concentration of the perfluoropropane microbubbles is continuously 2 × 10 from the mouse eye socket by using a syringe pump in the embodiment9The injection flow rate of the/mL perfluoropropane microbubbles is 0.3mL/h, after the perfluoropropane microbubbles circulate for 1 minute, a commercial ultrasonic imaging system is matched with a three-axis platform to move the mouse, and a section image is collected at intervals of 0.5mm to obtain the blood perfusion image before oxygen supply of the whole tumor.
(2) Oxygen-carrying microbubbles release oxygen: after waiting 30 minutes for complete metabolism of perfluoropropane microbubbles, the first dose of 1X 10 is injected from the eye socket7With oxygen microbubbles (C)3F8∶O21.4: 1; n-9), after 1 minute of circulation, the band is broken with a high intensity focused ultrasound probeThe oxygen microbubbles release oxygen, and the parameters of the ultrasonic wave include a sound pressure of 2MPa, a period of 1000, and a Pulse Repetition Frequency (PRF) of 2 Hz. In order to avoid the invalidation of the high-intensity focused ultrasonic irradiation caused by the fact that the oxygen-carrying microbubbles in the blood vessel are not timely supplemented after being destroyed, the mouse is moved by a three-axis platform in a way of stopping irradiation for 6 seconds after irradiation for 6 seconds, and when half of the tumor is treated (about 10 minutes after injection), a second agent 1 multiplied by 10 is injected7And then scanning half of the tumor with high intensity focused ultrasound for a total scan time of about 20 minutes, two doses of 1 × 107The oxygen-carrying microbubbles can ensure the uniformity of the oxygen-carrying microbubbles at the front and rear sections of the tumor scanned by the high-intensity focused ultrasonic waves, and the total treatment dose is 2 multiplied by 10 for each mouse7Oxygen-carrying microbubbles in a safe dose range of 3.9 to 6.9 x 107In the range of one microbubble/mouse. In addition, the present embodiment further includes a control group (N-6) that does not perform any injection and irradiation of ultrasonic waves, and a comparison group (N-8) that injects perfluoropropane microbubbles and irradiates ultrasonic waves, and the experimental manner of the comparison group is to inject perfluoropropane microbubbles instead of the oxygen-carrying microbubbles of the experimental group.
(3) Whole tumor blood flow perfusion image after oxygen administration: the time points for collecting the whole tumor blood flow perfusion image are 0(1 minute later), 2, 4, 6 and 8 days after injecting oxygen-carrying microbubbles (experimental group) or perfluoropropane microbubbles (comparative group), the image capturing method is the same as the step (1) above, and the injection pump is used for continuously injecting the oxygen-carrying microbubbles (experimental group) or perfluoropropane microbubbles (comparative group) into the eye sockets of the mice with the concentration of 2 multiplied by 109And the injection flow rate of the/mL perfluoropropane microbubble is 0.3mL/h, after the perfluoropropane microbubble circulates for 1 minute, a commercial ultrasonic imaging system is matched with the three-axis platform to move the mouse, and a section image is collected at intervals of 0.5mm, so that the blood flow perfusion image of the whole tumor after oxygen supply is collected. The ultrasound images were then further analyzed to calculate the proportion of blood perfusion as shown in FIG. 5, and the density of tumor vessels as shown in FIG. 6, and thereby assess whether the change in tumor blood perfusion was due to functional repair accompanied by normalization of the vessels, or due to simple vascular proliferation.
As can be seen from the experimental results shown in fig. 5, the blood perfusion ratio was significantly increased in the experimental group injected with microbubbles of oxygen alone and irradiated with ultrasonic waves, and maintained at 1.95 ± 0.78 up to 8 days after the administration of oxygen, while the blood perfusion ratio was lower than 1 in the control group and the comparative group injected with microbubbles of perfluoropropane and irradiated with ultrasonic waves on days 2 to 4. Therefore, it is believed that the present embodiment uses microbubbles with oxygen and uses ultrasound to irradiate the tumor to release oxygen to induce tumor blood vessels to normalize, thereby resulting in increased tumor blood perfusion. Furthermore, as can be seen from the experimental results shown in fig. 6, the densities of tumor vessels in the experimental group (N-5), the comparative group (N-4) and the control group (N-6) did not significantly increase or decrease, and it can be inferred that the increase in the perfusion ratio of tumor blood flow was due to the normalization of the function of blood vessels, but not due to the proliferation of blood vessels.
In vivo experiment verifies that the time window length of the microbubbles with oxygen for promoting the normalization of blood vessels
Next, the present example was tested with different doses of microbubbles with oxygen, which was tested in a manner substantially the same as the experimental groups described above, except that in the different groups, the dose of microbubbles with oxygen was 0.5X 10 per mouse7(N＝2)、1×107(N＝4)、2×107(N＝8)、4×107(N-3) oxygen-carrying microbubbles, and in addition, the present embodiment includes a control group (N-6) in which microbubbles are not injected and ultrasound is not irradiated. FIG. 7 shows the change of tumor blood perfusion ratio at different doses, and from the results shown in FIG. 7, it can be seen that adjusting the dose of different microbubbles with oxygen can affect the normalization of blood vessels in a short time window, and the dose is 2X 10 per mouse7The group with microbubbles with oxygen showed a significant increase in the blood perfusion ratio from day 2 compared with the control group, and the time window for normalization of blood vessels was from day 2 to day 10 after oxygen administration, based on the perfusion ratio of 1.
Analysis of factors associated with normalization of blood vessels
In this example, day 4 after oxygen administration was taken as a time point for normalization of blood vessels, mice were sacrificed on day 4 after oxygen treatment, the whole tumor was taken off and tissue extraction was performed, and the expression levels of oxygen-detecting enzyme (PHD2), Hypoxia-inducible factor-1 α (HIF-1 α), Vascular Endothelial Growth Factor (VEGF), and Transforming growth factor- β (TGF- β) on Vascular endothelial cells were measured by Western blot analysis (Western blot). The measurement results are shown in fig. 8.
The data in the literature show that the normalization of tumor blood vessels can improve the delivery efficiency of oxygen, and under the environment of high oxygen concentration, oxygen detection enzyme (PHD2) on vascular endothelial cells can decompose hypoxia inducible factor (HIF-1 alpha), thereby reducing the expression of HIF-1 alpha, leading to the reduction of the expression level of Vascular Endothelial Growth Factor (VEGF) of downstream genes, slowing the growth rate of the tumor blood vessels, and repairing abnormal blood vessels in the tumor with time, so that the tumor blood vessels are normalized. In addition, the expression level of transforming growth factor (TGF-. beta.) can be used to assess whether the rate of tumor cell proliferation is affected after tumor blood vessel normalization.
Thus, the results of the measurements shown in FIG. 8 show that PHD2, HIF-1 α, and VEGF expression decreased after injection of oxygen-bearing microbubbles and vascular normalization by ultrasound to release oxygen at the tumor site, whereas TGF-. beta.was not significantly different. Among them, the decrease in the expression levels of PHD2, HIF-1 α and VEGF is consistent with the above theory, and it was further confirmed that the oxygen-carrying microbubbles released oxygen at the tumor site can induce the normalization of tumor blood vessels.
Statistical analysis
The data of this example were statistically analyzed using Student's t-test two-tailed assay.
By combining the test results, the injection of the microbubbles with oxygen can induce the normalization of blood vessels at the tumor part by releasing oxygen at the tumor part by using ultrasonic waves, so as to increase the blood perfusion at the tumor part, and the time window for the normalization of the blood vessels can reach 2 to 10 days after oxygen supply.
The above-mentioned embodiments are intended to illustrate the objects, technical solutions and advantages of the present invention in further detail, and it should be understood that the above-mentioned embodiments are only exemplary embodiments of the present invention and are not intended to limit the present invention, and any modifications, equivalents, improvements and the like made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (5)

1. An application of oxygen-carrying microbubbles for preparing a set for inducing normalization of lesion tissue blood vessels, wherein the oxygen-carrying microbubbles comprise oxygen and a gas which is difficult to dissolve in water, the volume ratio of the gas which is difficult to dissolve in water and the oxygen contained in the oxygen-carrying microbubbles is 1.4:1, and the particle size of the oxygen-carrying microbubbles is 0.5-3.0 μm; the oxygen-carrying microbubble further comprises a phospholipid shell layer which coats the oxygen and the gas which is difficult to dissolve in water; at least one of the hardly water-soluble gases contained in the oxygen-bearing microbubbles is selected from perfluoropropane (C)3F8) Perfluorobutane (C)4F10) And mixtures thereof;
wherein, the set comprises an oxygen-carrying microbubble mixed liquid and an ultrasonic wave emitting device; an effective dose of the microbubbles with oxygen enters the organism through intravenous injection, and then the ultrasonic wave emitting device is used for irradiating the lesion tissue, so that the microbubbles with oxygen are broken at the lesion tissue to release oxygen.
2. The use according to claim 1, wherein the oxygen-bearing microbubbles have a particle size of 0.7 to 3.0 μm.
3. The use according to claim 1, wherein the phospholipid shell layer consists of 1, 2-distearoyl-sn-glycero-3-phosphocholine and 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [10- (trimethoxysilyl) undecanamide (polyethylene glycol-2000) ].
4. Use according to claim 1, wherein the parameters of the ultrasound emitting device are: the sound pressure is 1.5-2.5 MPa; the period is 500-1500; the pulse repetition frequency is 1-5 Hz.
5. The use of claim 1, wherein the diseased tissue is tumor tissue, tissue that is hypoxic due to vascular embolization, or damaged blood vessels.
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