Preparation method and application of engineered exosome derived from ultrasonic response type brain glioma

Abstract

The invention discloses a preparation method and application of an engineered exosome derived from ultrasonic response type brain glioma, and belongs to the field of biomedical engineering. The engineered exosome has a nano-scale'shell '-'core' structure, the'core 'structure is formed by coating ZIF-8 nanoparticles internally loaded with a sound-sensitive agent and oxygen-carrying perfluorocarbon with the'shell' of the brain glioma exosome, and the engineered exosome is mainly characterized in that (1) by utilizing the biological activity of the brain glioma exosome, the tumor homologous targeting property can be exerted, the blood brain barrier can be penetrated, and the brain glioma can be effectively inhibited; reaching the glioma part; (2) under external ultrasonic stimulation, oxygen-carrying perfluorocarbon can release oxygen, slow hypoxia of tumor tissues, disturb brain glioma exosome membranes and release contents; and (3) in addition, the hypoxia degree of tumor tissues is relieved, and the sonodynamic performance of the sonosensitizer under ultrasonic stimulation is greatly improved, so that the growth of brain glioma is effectively inhibited.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical engineering, and specifically relates to a method for preparing engineered exosomes derived from ultrasound-responsive brain gliomas and applications thereof. Background Art

[0002] Intracranial tumors have the characteristics of high recurrence rate, high mortality rate and low cure rate, which seriously endanger the life and health of Chinese people. Among them, brain glioma, as the most common intracranial tumor, has the characteristics of high malignancy, fast growth and short course of disease. It can cause high intracranial pressure, epilepsy, mental disorder and other phenomena in patients in a short time, and its five-year survival rate is less than 5%. At present, clinical treatment methods are mainly surgery, radiotherapy and chemotherapy. However, since the brain is the most important central nervous system of the human body, surgery often causes irreversible damage to the patient's brain function and structure, resulting in poor prognosis and postoperative quality of life of patients. Traditional chemotherapy methods have problems such as poor drug accumulation effect in the lesion site and large off-target side effects, which makes the chemotherapy of brain glioma patients also suffer great challenges. Therefore, the development of an efficient brain glioma targeted treatment system is of great significance for effectively killing tumor cells while protecting normal tissue function.

[0003] Among them, as an important barrier to protect brain tissue from toxins and pathogens, the blood-brain barrier (BBB) ​​poses a major challenge to the targeted treatment of craniocerebral tumors. The blood-brain barrier refers to the barrier between plasma and brain cells formed by the walls of brain capillaries and glial cells, and the barrier between plasma and cerebrospinal fluid formed by the choroid plexus, which is of great significance for maintaining the normal function of brain tissue. However, this also greatly limits the accessibility of drugs in craniocerebral tumors. A large number of studies have shown that the level of drug molecules in brain gliomas is much lower than the level of drugs in plasma (less than 0.1%). In recent years, as an efficient cell signal transmission system, exosomes play an important role in signal transmission across biological barriers (such as the blood-brain barrier). They are nanovesicles with a lipid bilayer structure and a particle size of 30 to 150nm. Exosomes are widely used in the field of tumor treatment due to their good biocompatibility, excellent drug loading capacity, and tumor homing targeting ability. In addition, studies have shown that exosomes secreted by tumor cells have excellent homologous targeting properties and can effectively achieve tumor homing targeting. Therefore, the development of targeted drug delivery systems based on glioma exosomes through biomedical engineering is of great significance for the precise targeted treatment of glioma.

[0004] In addition, in recent years, sonodynamic therapy has been approved by the U.S. Food and Drug Administration (FDA) for the clinical treatment of malignant tumors as a new non-invasive tumor treatment method. It uses ultrasound to stimulate the sonosensitizer accumulated in the target area to produce reactive oxygen species (ROS) to kill the tumor to achieve the treatment purpose. However, the hypoxic characteristics of tumor tissue and the low accumulated dose of sonosensitizer greatly weaken the effect of tumor sonodynamic therapy. Therefore, based on the brain glioma exosome platform, loading sonosensitizers and oxygen supply substances through bioengineering means is of great value for achieving a high level of brain glioma sonodynamic therapy. Summary of the invention

[0005] Against the above background, in order to achieve a high level of targeted sonodynamic therapy for brain glioma, the purpose of the present invention is to provide a method for preparing ultrasound-responsive engineered exosomes derived from brain glioma.

[0006] The object of the present invention is achieved as follows: an ultrasound-responsive engineered exosome derived from brain glioma, characterized in that: the engineered exosome has a nanoscale "shell"-"core" structure, wherein (1) the "core" structure is a ZIF-8 nanoparticle loaded with a sonosensitizer and oxygen-carrying perfluorocarbon, and (2) the "shell" structure is a coating layer of the brain glioma exosome.

[0007] A method for preparing engineered exosomes derived from ultrasound-responsive brain glioma, characterized in that the method comprises the following steps:

[0008] Step 1): Preparation of ZIF-8 nanoparticles loaded with sonosensitizer and perfluorocarbon

[0009] Through zinc ions (Zn 2+ ) is reacted with 2-methylimidazole (2-MIM) to prepare ZIF-8 nanoparticles, which are then loaded with sonosensitizers and perfluorocarbons to obtain ZIF-8 nanoparticles loaded with sonosensitizers and perfluorocarbons.

[0010] Step 2): Isolation and extraction of exosomes from glioma cells

[0011] The glioma cell line was expanded by in vitro culture, and then the glioma cell exosomes were extracted.

[0012] Step 3): Preparation of ultrasound-responsive engineered exosomes from glioma

[0013] ZIF-8 nanoparticles loaded with sonosensitizer and perfluorocarbon were co-mixed with glioma cell exosomes, extruded, and filled with oxygen to prepare ultrasound-responsive glioma-derived engineered exosomes.

[0014] Preferably, the solvent used in the coordination reaction and loading of the sonosensitizer and perfluorocarbon involved in step 1) is one of water, methanol, ethanol, a mixed solvent of water and methanol, and a mixed solvent of water and ethanol.

[0015] Preferably, the glioma cells described in step 2) are one of U87MG cell line, U373MG cell line, A172 cell line, T98G cell line, C6 cell line, and GL261 cell line. The method for separating and extracting exosomes from glioma cells described in step 2) is one of ultracentrifugation and density gradient centrifugation.

[0016] Preferably, the sonosensitizer described in step 3) is one of verteporfin, photoporphyrin, tetramethylthienoporphyrin, zinc phthalocyanine, and indocyanine green. The perfluorocarbon described in step 3) is one of perfluorobutane, perfluoropentane, perfluorohexane, perfluoroheptane, perfluorooctane, perfluorododecane, and perfluoromethylcyclohexane.

[0017] Another object of the present invention is to provide the use of the above-mentioned engineered exosomes in tumor treatment.

[0018] Preferably, the application of the ultrasound-responsive engineered exosomes derived from glioma is characterized in that the ultrasound-responsive engineered exosomes can be applied to the treatment of, including but not limited to, gliomas, choroid plexus tumors, meningiomas, and pituitary tumors.

[0019] Preferably, the application of the ultrasound-responsive engineered exosomes derived from brain glioma is characterized in that the ultrasound frequency used is in the range of 0.1-20 MHz and the power is in the range of 0.1-5 W / cm 2 , ultrasonic time is 0.1-30min.

[0020] Working principle of the present invention

[0021] The ultrasound-responsive engineered exosomes derived from glioma described in the present invention have the following working principle: the engineered exosomes have a nanoscale "shell"-"core" structure, wherein the "core" structure is a ZIF-8 nanoparticle loaded with sonosensitizer and oxygen-carrying perfluorocarbon, which is then coated by the "shell" of glioma exosomes. Under the homologous targeting of glioma exosomes, it can effectively break through the blood-brain barrier and achieve tumor tissue homing targeting. Through external ultrasonic stimulation, the content ZIF-8 nanoparticles can be effectively released, and oxygen is released to improve local hypoxia of glioma, thereby achieving a high level of sonodynamic therapy for glioma.

[0022] Beneficial effects of the present invention:

[0023] 1. The present invention provides an ultrasound-responsive engineered exosome derived from brain glioma, which has a simple preparation process and good biocompatibility.

[0024] 2. The ultrasound-responsive engineered exosomes from glioma provided by the present invention have excellent tumor homing ability, can cross the blood-brain barrier, and target glioma tissue

[0025] 3. The ultrasound-responsive engineered exosomes derived from glioma provided by the present invention can effectively release oxygen under external ultrasonic stimulation to improve glioma tissue hypoxia, thereby greatly improving the sonodynamic therapy effect of glioma, which is an innovation in the field of biomedical engineering. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The particle size distribution and Zeta potential distribution test of the ultrasound-responsive brain glioma-derived engineered exosomes (SonoGliomaExo) prepared in Example 1;

[0027] Figure 2 The storage stability test of the ultrasound-responsive brain glioma-derived engineered exosomes (SonoGliomaExo) prepared in Example 1;

[0028] Figure 3 The ultrasound-responsive oxygen release curve of the ultrasound-responsive brain glioma-derived engineered exosomes (SonoGliomaExo) prepared in Example 1;

[0029] Figure 4 The in vitro sonodynamic effect test of the ultrasound-responsive engineered exosomes (SonoGliomaExo) derived from brain glioma prepared in Example 1;

[0030] Figure 5 Evaluation of the in vitro anti-tumor effect of the ultrasound-responsive engineered exosomes (SonoGliomaExo) derived from brain glioma prepared in Example 1; DETAILED DESCRIPTION

[0031] The present invention is further described below by way of examples, but the following examples do not limit the scope of rights of this patent.

[0032] Example 1

[0033] The preparation method of ultrasound-responsive brain glioma-derived engineered exosomes (SonoGliomaExo) is as follows:

[0034] (1) Preparation of ZIF-8 nanoparticles loaded with sonosensitizer and perfluorocarbon: 1.2 g of Zn(NO3)2·6H2O was dissolved in 10 mL of water, 2-methylimidazole was dissolved in 80 mL of water, the aqueous solution was filtered using a 0.8 μm microporous filter membrane, the two were mixed, stirred at room temperature for 5 min, the precipitate was collected by centrifugation (6500 rpm, 30 min), and washed 3 times with deionized water to obtain purified ZIF-8 nanoparticles, which were dried in an oven and then redispersed in methanol (10 mg / mL). 1 mg of sonosensitizer verteporfin and 10 μL of perfluorohexane were taken, dispersed in ZIF-8 nanoparticle methanol solution (10 mg / mL), stirred at room temperature overnight, and ZIF-8 nanoparticles loaded with sonosensitizer verteporfin and perfluorohexane were prepared.

[0035] (2) Isolation and extraction of exosomes from glioma cells: Human glioma cells (U87MG cells, purchased from the Cell Resource Center of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences) were cultured at a rate of 1×10 6 The cells were inoculated in 6-well plates at a density of 100 cells / well and grown at 37°C until 90% confluence. The culture supernatant was collected and centrifuged (4°C, 3000 g, 10 min). The lower exosome precipitate was further collected by high-speed centrifugation (4°C, 120000 g, 20 min), and purified by washing three times with PBS to obtain U87MG brain glioma cell exosomes.

[0036] (3) Preparation of ultrasound-responsive engineered exosomes from glioma: The ZIF-8 nanoparticles loaded with sonosensitizer verteporfin and perfluorohexane prepared in step (1) are mixed with the U87MG glioma cell exosomes separated and extracted in step (2), and extruded through 800 nm and 400 nm polycarbonate membranes respectively using an extruder, and further filled with oxygen to prepare ultrasound-responsive engineered exosomes SonoGliomaExo from glioma.

[0037] Example 2

[0038] Particle size distribution and Zeta potential distribution test of ultrasound-responsive brain glioma-derived engineered exosomes (SonoGliomaExo):

[0039] The ultrasound-responsive engineered exosomes SonoGliomaExo prepared in Example 1 were measured for particle size and potential by dynamic light scattering. The results are as follows: Figure 1 As shown, the particle size of SonoGliomaExo is 208±4.15nm and the potential is -29±2.59mV.

[0040] Example 3

[0041] Storage stability test of ultrasound-responsive brain glioma-derived engineered exosomes (SonoGliomaExo):

[0042] The ultrasound-responsive engineered exosomes SonoGliomaExo from glioma prepared in Example 1 were placed in a 4°C environment, and their particle sizes were measured by dynamic light scattering on days 0, 1, 2, 4, 7, 10, and 14, respectively, and a particle size change curve was plotted. Figure 2 As shown in the figure, the particle size of SonoGliomaExo did not change significantly within 14 days, indicating good storage stability.

[0043] Example 4

[0044] Ultrasound-responsive oxygen release curve of ultrasound-responsive brain glioma-derived engineered exosomes (SonoGliomaExo):

[0045] The ultrasound-responsive engineered exosomes SonoGliomaExo from glioma prepared in Example 1 were dispersed in deoxygenated water and stimulated with ultrasound (1 W / cm 2 ,1MHz,5min), the oxygen content in the solution is measured by an oxygen electrode, and the oxygen content-time curve is shown in Figure 3 The results showed that SonoGliomaExo could effectively release oxygen and increase the oxygen content in the medium under ultrasound stimulation.

[0046] Example 5

[0047] In vitro sonodynamic effect test of ultrasound-responsive brain glioma-derived engineered exosomes (SonoGliomaExo):

[0048] The ultrasound-responsive engineered exosomes SonoGliomaExo derived from glioma prepared in Example 1 were dispersed in deoxygenated water in normoxic and hypoxic states, respectively, and singlet oxygen indicator SOSG was added for co-incubation. The solution was stimulated with ultrasound (1 W / cm 2 ,1MHz,5min), and then detect the change of SOSG fluorescence intensity (Ex=504nm,Em=525nm). The results are as follows Figure 4 As shown, under hypoxic conditions, the sonodynamic effect of verteporfin was significantly inhibited, while SonoGliomaExo significantly improved the sonodynamic effect in hypoxic environments because it could responsively release oxygen to enhance the oxygen concentration in the medium.

[0049] Example 6

[0050] In vitro evaluation of the anti-tumor effect of ultrasound-responsive brain glioma-derived engineered exosomes (SonoGliomaExo):

[0051] Human glioma cells (U87MG cells, purchased from the Cell Resource Center of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences) were cultured at a rate of 5 × 10 3 The cells were seeded at a density of 100 cells / mL in a 96-well plate and placed in a hypoxic and normoxic environment induced by AnaeroPack (Mitsubishi Corporation, Japan) and cultured overnight at 37°C (5% CO2). Different concentrations of SonoGliomaExo were added, which were equivalent to verteporfin (0, 0.1, 0.2, 0.5, 1, and 2 μg / mL), and the solution was stimulated with ultrasound (1 W / cm 2 ,1MHz,5min), and after further incubation for 24h, 20μL of MTT solution (5mg / mL) was added to each well and incubated for another 4h. The upper culture medium was discarded, 150μL of DMSO was added, and after shaking to dissolve, the absorbance of each well was read at 570nm using an ELISA reader to calculate the cell survival rate of each well. The results are shown in Figure 5 As shown in the figure, the sonodynamic killing effect of free verteporfin in a hypoxic environment is severely hindered. However, due to the ultrasound-responsive oxygen supply capacity of SonoGliomaExo, the killing effect of brain glioma in a hypoxic environment is greatly improved.

Claims

1. An ultrasound-responsive engineered exosome derived from brain glioma, characterized in that: The engineered exosomes are nanoscale "shell"-"core" structures, wherein (1) the "core" structure is a ZIF-8 nanoparticle loaded with sonosensitizer and oxygen-carrying perfluorocarbon, and (2) the "shell" structure is a coating layer of the brain glioma exosomes.

2. The method for preparing ultrasound-responsive engineered exosomes derived from brain glioma according to claim 1, characterized in that: The following steps are involved: 1) Through zinc ions (Zn 2+ ) and 2-methylimidazole (2-MIM) to prepare ZIF-8 nanoparticles, and then load the sonosensitizer and perfluorocarbon to obtain ZIF-8 nanoparticles loaded with sonosensitizer and perfluorocarbon; 2) Expanding glioma cell lines by in vitro culture and then extracting glioma cell exosomes; 3) The ZIF-8 nanoparticles loaded with sonosensitizer and perfluorocarbon were co-mixed and extruded with brain glioma cell exosomes, and filled with oxygen to prepare ultrasound-responsive brain glioma-derived engineered exosomes.

3. The method for preparing ultrasound-responsive engineered exosomes derived from brain glioma according to claim 2, characterized in that: The coordination reaction and loading of the sonosensitizer and perfluorocarbon involved in step 1) use a solvent selected from the group consisting of water, methanol, ethanol, a mixed solvent of water and methanol, and a mixed solvent of water and ethanol.

4. The method for preparing ultrasound-responsive engineered exosomes derived from brain glioma according to claim 2, characterized in that: The glioma cells described in step 2) include but are not limited to U87MG cell line, U373MG cell line, A172 cell line, T98G cell line, C6 cell line, and GL261 cell line; the method for isolating and extracting exosomes from glioma cells described in step 2) includes but is not limited to ultracentrifugation and density gradient centrifugation.

5. The method for preparing ultrasound-responsive engineered exosomes derived from brain glioma according to claim 2, characterized in that: The sonosensitizer described in step 3) includes but is not limited to verteporfin, photoporphyrin, tetramethylthienoporphyrin, zinc phthalocyanine, and indocyanine green; the perfluorocarbon described in step 3) includes but is not limited to perfluorobutane, perfluoropentane, perfluorohexane, perfluoroheptane, perfluorooctane, perfluorododecane, and perfluoromethylcyclohexane.

6. The use of an ultrasound-responsive engineered exosome derived from brain glioma according to claim 1, characterized in that: The ultrasound-responsive engineered exosomes use an ultrasound frequency range of 0.1-20 MHz and a power range of 0.1-5 W / cm 2 , ultrasonic time is 0.1-30min.

7. The use of an ultrasound-responsive engineered exosome derived from brain glioma according to claim 1, characterized in that: The ultrasound-responsive engineered exosomes can be applied to the treatment of, including but not limited to, brain gliomas, choroid plexus tumors, meningiomas, and pituitary tumors.

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