WO2022160987A1 - 离子通道靶向微泡及其制备方法和应用 - Google Patents

离子通道靶向微泡及其制备方法和应用 Download PDF

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WO2022160987A1
WO2022160987A1 PCT/CN2021/138062 CN2021138062W WO2022160987A1 WO 2022160987 A1 WO2022160987 A1 WO 2022160987A1 CN 2021138062 W CN2021138062 W CN 2021138062W WO 2022160987 A1 WO2022160987 A1 WO 2022160987A1
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ion channel
ultrasonic
microbubbles
targeted
microbubble
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严飞
谢丽婷
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Shenzhen Institute of Advanced Technology of CAS
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Shenzhen Institute of Advanced Technology of CAS
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/22—Echographic preparations; Ultrasonic imaging preparations
    • A61K49/222—Echographic preparations; Ultrasonic imaging preparations characterised by a special physical form, e.g. emulsions, liposomes
    • A61K49/223—Microbubbles, hollow microspheres, free gas bubbles, gas microspheres
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/555—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound pre-targeting systems involving an organic compound, other than a peptide, protein or antibody, for targeting specific cells
    • A61K47/557—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound pre-targeting systems involving an organic compound, other than a peptide, protein or antibody, for targeting specific cells the modifying agent being biotin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/22—Echographic preparations; Ultrasonic imaging preparations
    • A61K49/221—Echographic preparations; Ultrasonic imaging preparations characterised by the targeting agent or modifying agent linked to the acoustically-active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/08—Antiepileptics; Anticonvulsants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/20—Hypnotics; Sedatives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/24—Antidepressants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/30—Drugs for disorders of the nervous system for treating abuse or dependence

Definitions

  • the application belongs to the technical field of ultrasonic nerve stimulation, and in particular relates to an ion channel targeting microbubble and a preparation method and application thereof.
  • Ultrasound-based non-invasive neuromodulation technology is considered to be one of the next-generation neuromodulation technologies with the most promising clinical translation. It has the advantages of non-invasiveness, large penetration depth, and high spatiotemporal distribution rate. Ultrasound neuromodulation can stimulate or inhibit the central nervous system at the stimulation site through different intensities, frequencies, pulse repetition pulse widths and durations, resulting in reversible changes in two-way regulation of nerve function. Its regulatory effect has been verified on various scale targets such as neurons, nematodes, mice, and non-human primates.
  • Mechanosensitive ion channel is a new type of ion channel discovered in recent years. It is different from traditional voltage-sensitive and ligand-gated ion channels. It senses changes in membrane tension caused by cell deformation and other ways to open, causing The transmembrane transport of ions inside and outside the cell is involved in mediating many life activities, and its function has been paid more and more attention. Studies have shown that ultrasound can generate a tensile force on the cell membrane, activate mechanosensitive ion channels on the membrane, and change the membrane potential of the cell through the transmembrane current generated by the channel. Recent studies have shown that ultrasound, as a mechanical stimulus, can open mechanosensitive ion channels at low frequency and low energy, causing ion influx.
  • Ultrasonic microbubbles are microbubbles with a particle size in the micrometer scale (2 ⁇ m to 8 ⁇ m), or even nanoscale, composed of a core gas surrounded by a shell membrane. When the targeted microbubbles are excited by ultrasound, the microbubbles will shrink and expand regularly with the periodic pressure changes of the acoustic waves. Ultrasound targeting microbubble is to connect target molecule-specific antibodies or ligands to the surface of ultrasound microbubbles to construct targeted ultrasound microbubbles, so that the ultrasound microbubbles can actively bind to the target tissue. There is a close connection between ion channels and neural stimulation. By regulating ion channels, neural stimulation can be regulated to a certain extent. However, targeted microbubbles for ion channels are currently lacking.
  • the purpose of the present application is to provide an ion channel-targeted microbubble and a preparation method thereof, and its application, aiming to solve the problem that there is currently no ion-channel-targeted microbubble.
  • a first aspect of the present application provides an ion channel targeting ultrasonic microbubble, comprising a shell membrane and a biologically inert gas, the biologically inert gas is enclosed in a capsule formed by the shell membrane, wherein the outer surface of the shell membrane is Ion channel antibodies are bound to the surface.
  • a second aspect of the present application provides a method for preparing an ion channel-targeted ultrasonic microbubble, comprising the following steps:
  • Avidin protein is added to the biotinylated microbubbles for incubation, biotin-labeled ion channel antibodies are added, and incubated to obtain the ion channel-targeted ultrasonic microbubbles.
  • a third aspect of the present application provides the use of ion channel-targeted ultrasound microbubbles in the preparation of therapeutic formulations for neurological diseases.
  • the present application provides a novel ion channel targeting ultrasound microbubble.
  • ultrasound stimulation before ultrasound stimulation, the extracellular ion concentration is higher than the intracellular ion concentration, and the ion channel targeting microbubble binds to receptor ligands on the cell membrane
  • the microbubble is targeted to bind to the ion channel through the ion channel antibody, which can reduce the ultrasonic radiation force to open the ultrasonic energy of the ion channel, and promote the flow of extracellular ions into the cell, thereby enhancing the sensitivity of the ultrasonic nerve stimulation response.
  • the present application provides a novel method for preparing ion channel-targeted ultrasonic microbubbles.
  • biotinylated microbubbles are prepared, and then avidin is introduced on the surface of the biotinylated microbubbles to construct a biotin-avidin system.
  • biotin-avidin conjugated ion channel antibody ion channel targeted ultrasonic microbubbles were prepared.
  • the method has a simple operation process, and the ion channel-targeted ultrasonic microbubbles are easy to obtain. More importantly, the obtained ion channel-targeted ultrasonic microbubbles can target the opening of ion channels under ultrasonic conditions and enhance the response to ultrasonic nerve stimulation. sensitivity.
  • the ultrasonic microbubbles are targeted by means of ion channels, and the ion channels are targeted to be opened under ultrasonic conditions, so as to achieve a sensitization effect on nerve stimulation, thereby improving the feeling effect of nerve stimulation.
  • Fig. 1 is the action principle diagram of the ion channel-targeted ultrasonic microbubble provided in the embodiment of the present application under ultrasonic conditions;
  • Fig. 2 is the preparation flow chart of the biotinylated phospholipid microbubbles provided in the embodiment of the present application;
  • 3A is a particle size distribution diagram of the ion channel targeting microbubbles provided in Example 1 of the present application;
  • 3B is a bright field image of the ion channel targeting microbubble provided in Example 1 of the present application;
  • Fig. 3C is the Fluo 4-AM staining diagram of ion channel-targeted microvesicles provided in Example 1 of the present application;
  • Figure 4A is a bright field image, a Fluo 4 staining image, a PI staining image, a Fluo 4 staining image, and an ultrasound-stimulated N 2 A cell under the condition that the Piezol ion channel-targeted microvesicles provided in Example 2 of the present application adhere to N 2 A cells.
  • PI staining fusion map
  • 4B is a graph showing the change of time fluorescence intensity of N 2 A cells bound to the Piezol ion channel-targeted microbubble before, immediately or 5 minutes after ultrasonic irradiation of the Piezol ion channel-targeted microbubble provided in Example 2 of the present application;
  • Figure 4C is a graph of the average maximum relative fluorescence intensity (df/F0) after ultrasound irradiation of N 2 A cells incubated with non-targeted microbubbles provided in Comparative Example 2;
  • 5A is a bright field image after ultrasound stimulation of neurons adhered to Piezol ion channel targeting microbubbles provided in Example 3 of the present application;
  • Figure 5B is a diagram of Fluo 4 staining before ultrasound stimulation of neurons adhering to Piezol ion channel targeting microbubbles provided in Example 3 of the present application;
  • Figure 5C is a diagram of Fluo 4 staining after ultrasound stimulation of neurons adhered to Piezol ion channel targeting microbubbles provided in Example 3 of the present application;
  • 5D is a graph of the average relative fluorescence intensity of ultrasound-stimulated N 2 A cells as a function of time when Piezol ion channel-targeted microvesicles adhere to N 2 A cells provided in Example 3 of the present application;
  • 5E is a graph of the average maximum relative fluorescence intensity of N 2 A cells in the Piezol ion channel targeting microvesicle group provided in Example 1 and the non-target microvesicle group provided in Comparative Example 1 in Example 3 of the present application;
  • Figure 5F is a graph showing the correlation between the number of targeted microvesicle adherent cells and the relative fluorescence intensity provided in Example 3 of the application;
  • Figure 6A is a graph of the relative fluorescence intensity of a single N 2 A cell in the control group provided in Example 4 of the present application as a function of time;
  • Figure 6B is a graph of the relative fluorescence intensity of a single N 2 A cell in the GsMTx-4 group provided in Example 4 of the present application as a function of time;
  • FIG. 6C is a graph of the average maximum relative fluorescence intensity of N 2 A cells in the GsMTx-4 group and N 2 A cells in the control group provided in Example 4 of the present application.
  • At least one means one or more, and “plurality” means two or more.
  • At least one item(s) below” or similar expressions thereof refer to any combination of these items, including any combination of single item(s) or plural items(s).
  • at least one (a) of a, b, or c or, “at least one (a) of a, b, and c” can be expressed as: a, b, c, a-b ( That is, a and b), a-c, b-c, or a-b-c, where a, b, and c can be single or multiple respectively.
  • first and second are only used for descriptive purposes to distinguish objects such as substances from each other, and cannot be understood as indicating or implying relative importance or implying the number of indicated technical features.
  • first XX may also be referred to as the second XX
  • second XX may also be referred to as the first XX.
  • a feature defined as “first” or “second” may expressly or implicitly include one or more of that feature.
  • Ultrasonic microbubbles are microbubbles with a particle size in the micrometer scale (2 ⁇ m to 8 ⁇ m), or even nanoscale, composed of a core gas surrounded by a shell membrane.
  • the targeted microbubbles When the targeted microbubbles are excited by ultrasound, the microbubbles will shrink and expand regularly with the periodic pressure changes of the acoustic waves.
  • microbubbles After calculating the magnitude of the ultrasonic radiation force received by microbubbles and cells, some scientists found that compared with cells, microbubbles have a higher ability to respond to the mechanical stimulation of ultrasonic radiation force.
  • the sound radiation power is 20 times higher. That is, compared to cells, ultrasound microbubbles require less ultrasound energy to bind cells and open mechanosensitive channels.
  • Ultrasound targeting microbubbles can connect target molecule-specific antibodies or ligands to the surface of ultrasound microbubbles to construct targeted ultrasound microbubbles, so that the ultrasound microbubbles can actively bind to the target tissue.
  • current targeted microbubbles cannot yet target mechanosensitive ion channels and cannot enhance the response of nerve cells to ultrasound stimulation.
  • the embodiment of the present application provides a novel ion channel targeted ultrasonic microbubble
  • the targeted ultrasonic microbubble includes a shell membrane and a biologically inert gas, and the biologically inert gas is enclosed in a capsule formed by the shell membrane.
  • the ion channel antibody is bound to the outer surface of the shell membrane, and the ion channel antibody can be combined with the ion channel.
  • the extracellular ion concentration is higher than the intracellular ion concentration, and the ion channel targeting microbubble and the receptor ligand on the cell membrane are closed; under ultrasound stimulation, the microbubble is targeted to the ion channel through the ion channel antibody Combined, it can reduce the ultrasonic energy of the ultrasonic radiation force to open the ion channel, and under the action of the ultrasonic radiation force, the "gate" of the ion channel can be opened, and the ion channel can be opened to promote the flow of extracellular ions into the cell, thereby enhancing the response of ultrasonic nerve stimulation. sensitivity.
  • the ion channel-targeted ultrasonic microbubble includes the basic structure of the ultrasonic microbubble, that is, a shell membrane and a biologically inert gas, and the biologically inert gas is enclosed in a capsule enclosed by the shell membrane.
  • the shell membrane can be a shell membrane material that can be used by ultrasonic microbubbles.
  • the shell membrane is a phosphoester-based shell membrane or a phosphoester-derivative-based shell membrane.
  • the shell film is a phospholipid shell film, and the phospholipid shell film includes the following components: distearate phosphatidylcholine (DSPC), distearate phosphatidylethanolamine poly-ethyl acetate Diol 2000 (DSPE-PEG2000), biotinylated polyethylene glycol 2000 and distearate phosphatidylethanolamine.
  • DSPC distearate phosphatidylcholine
  • DSPE-PEG2000 distearate phosphatidylethanolamine poly-ethyl acetate Diol 2000
  • biotinylated polyethylene glycol 2000 and distearate phosphatidylethanolamine.
  • the biologically inert gas is sealed in the shell membrane, and various biologically inert gases used in the ultrasonic microbubble can be selected.
  • the biologically inert gas is a fluoroalkane, preferably a perfluoroalkane.
  • the biologically inert gas is perfluoropropane, of course, the biologically inert gas is not limited thereto.
  • the outer surface of the shell membrane contains binding sites for ion channel antibodies, so that under ultrasonic conditions, the ion channel antibodies can be targeted and bound to the ion channels of the cell membrane, so that the targeted ultrasonic microbubbles can be targeted by ultrasonic waves.
  • Ion channels at the binding site are opened by the radiation force generated by ultrasound.
  • ion channel antibodies are bound to the outer surface of the shell membrane.
  • the shell membrane of the ion channel targeting ultrasonic microbubble provided in the embodiments of the present application includes two sides, one side relative to the gas inner core is the inner side, and the corresponding shell membrane surface is the inner surface; One side is the outer side, and the corresponding shell membrane surface is the outer surface.
  • the ion channel antibody is bound to the outer surface of the shell membrane via the biotin-avidin system. That is, the ion channel is targeted to the outer surface of the shell membrane of the ultrasonic microbubble, and at least part of the area is bound with a biotin-avidin structure. By binding the ion channel antibody in this way, the ion channel antibody has better binding stability.
  • ion channel antibodies can also be bound to the outer surface of the shell membrane by other linking systems.
  • the ion channel antibody is an antibody capable of acting on the ion channel, and in some embodiments, the ion channel is a mechanosensitive ion channel, but not limited thereto. Therefore, after the ion channel targeting ultrasonic microbubble binds to the cell membrane ion channel under ultrasonic conditions, the ultrasonic energy for opening the ion channel is reduced by the ultrasonic radiation force, and the opening of the ion channel is promoted by the radiation force generated by the ultrasonic wave.
  • the ion channel targeted by the ultrasonic microbubble can be an exogenous mechanosensitive ion channel or an endogenous mechanosensitive ion channel.
  • the ion channel is selected from the group consisting of Piezol ion channel, MscL ion channel, DEG/E NaC/ASIC channel, TRP channel and K2P channel.
  • the ion channel-targeted ultrasonic microbubbles provided in the embodiments of the present application can target the above-mentioned channels under ultrasonic conditions, and open the channels under the action of ultrasonic radiation force.
  • the ion channel is not limited to the channel types listed above, and can also be other ion channels that are beneficial to activate nerve cells.
  • the ion channel targeting ultrasonic microbubble can be introduced into one or more ions including calcium ions, sodium ions, and potassium ions. flow (into the cell), thereby sensitizing the response to ultrasound nerve stimulation.
  • the embodiments of the present application can provide ion channel-targeted ultrasound microbubbles for application in neurological or other systemic diseases such as movement disorders, pain, epilepsy, Parkinson's disease, and psychiatric diseases.
  • the microbubbles are not limited to micron-scale targeted biological bubbles, and may also include other acoustically corresponding materials including nanobubbles.
  • the size of the microbubbles is microscale or nanoscale. Among them, the size of the micro-scale microbubbles is 2 ⁇ m to 8 ⁇ m.
  • the ion channel-targeted ultrasound microbubbles provided in the examples of the present application can act on nerve cells but are not limited to nerve cells.
  • the nerve cells include but are not limited to nerve cells in the deep brain region and nerve cells in the superficial brain region.
  • the effect of the ion channel-targeted ultrasonic microbubbles provided in the embodiments of the present application can be evaluated by live cell fluorescence imaging, and whether the ion-channel-targeted ultrasonic microbubbles are targeted to open the ion channel can be verified by observing the changes in intracellular and extracellular fluorescence intensity. .
  • the shell film is a phosphoester shell film
  • the phosphoester shell film includes the following components: distearate phosphatidyl choline (DSPC), distearate phosphatidyl ethanolamine poly-ethylene glycol 2000 (DSPE -PEG2000), biotinylated polyethylene glycol 2000 and distearate phosphatidylethanolamine (DSPE-PEG2000-Biotin), the biological inert gas is fluoroalkane as an example, the obtained ion channel targeting ultrasonic microbubbles are in The principle of action under ultrasonic conditions is shown in Figure 1 (Avidin is avidin, and Biotinylated Antibody is biotinylated antibody or biotinylated antibody).
  • DSPC distearate phosphatidyl choline
  • DSPE -PEG2000 distearate phosphatidyl ethanolamine poly-ethylene glycol 2000
  • the preparation method of the ion channel-targeted ultrasonic microbubble provided in the embodiment of the present application can be prepared by the following method.
  • a second aspect of the embodiments of the present application provides a method for preparing an ion channel-targeted ultrasonic microbubble, comprising the following steps:
  • the novel method for preparing ion channel-targeted ultrasonic microbubbles includes first preparing biotinylated microbubbles, and then introducing avidin on the surface of the biotinylated microbubbles to construct a biotin-avidin system , and with the help of biotin-avidin conjugated ion channel antibody, ion channel targeted ultrasonic microbubbles were prepared.
  • the method has a simple operation process, and the ion channel-targeted ultrasonic microbubbles are easy to obtain. More importantly, the obtained ion-channel-targeted ultrasonic microbubbles can target the opening of ion channels under ultrasonic conditions, and sensitize ultrasonic nerve stimulation. the response to.
  • ultrasonic microbubbles can be prepared with reference to the preparation methods of non-targeted ultrasonic microbubbles or other targeted ultrasonic microbubbles; Biotin.
  • biotinylated microvesicles are prepared by:
  • the shell membrane raw material and the biotin raw material are mixed, and the mixing can be performed by direct mixing, and after dissolving in an organic solvent, further mixing is performed to obtain a first mixed solution.
  • the first mixed solution is obtained by dissolving in an organic solvent and then mixing on a vortex mixer.
  • the container avoids forming a uniform film, and further drying treatment is performed to remove the remaining solvent.
  • the drying process can be accomplished in a vacuum oven for 2-6 hours.
  • the buffer solution is a Tris buffer solution, thereby obtaining the second solution.
  • the second solution is heated to more than the phase transition temperature, and the water bath ultrasonic wave is made into the third solution, which is subpackaged and processed, and adopts biological inert gas to replace the gas in the subpackage container, and mechanical vibration obtains biotinylated microbubbles.
  • the biologically inert gas can be a fluoroalkane, including but not limited to perfluoropropane.
  • biotinylated microbubbles are biotinylated phospholipid microbubbles
  • the shell membrane of the phospholipid microbubble is a phospholipid shell membrane
  • the biologically inert gas therein is a fluoroalkane
  • the preparation method of biotinylated phospholipid microbubbles is:
  • DSPC distearate phosphatidylcholine
  • DSPE-PEG2000 20mg/ml distearate phosphatidylethanolamine poly-ethylene glycol 2000
  • DSPE-PEG2000-Biotin 20mg/ml biological Mixed with plain acylated polyethylene glycol 2000 modified distearate phosphatidylethanolamine (DSPE-PEG2000-Biotin), dissolved in chloroform and mixed on a vortex mixer.
  • the antibody carrying biotin is further coupled to the surface of the biotinylated microbubble by means of biotin-avidin bridging.
  • avidin protein was added to the biotinylated microbubbles for incubation, and before the addition of avidin, PBS (abbreviation for phosphate buffer saline, representing phosphate buffered saline solution) was used for washing 3 to 4 times. After adding biotin-labeled ion channel antibody, incubate for 15-30 minutes and wash with PBS to remove unbound avidin protein.
  • PBS abbreviation for phosphate buffer saline, representing phosphate buffered saline solution
  • biotin-labeled ion channel antibody exemplarily, an antibody to Piezol channel
  • room temperature 5-40° C.
  • step S02 includes: washing the biotinylated phospholipid microbubbles 3-4 times with PBS by means of centrifugal flotation (400 g), adding avidin protein for incubation for 15- 30min, wash 3-4 times with PBS to remove unbound avidin protein; add Biotin-labeled antibody (antibody of corresponding mechanosensitive ion channel, such as Piezo1 ion channel antibody), incubate again at room temperature for 15-30min to obtain ions The channel targets microbubbles.
  • Biotin-labeled antibody antibody of corresponding mechanosensitive ion channel, such as Piezo1 ion channel antibody
  • the particle size and distribution of the ion channel-targeted ultrasonic microbubbles prepared in this example can be measured by a particle counting analyzer, and the shape of the microbubbles can be observed with a light microscope.
  • the third aspect of the embodiments of the present application provides the application of ion channel-targeted ultrasonic microbubbles in the preparation of therapeutic preparations for neurological diseases.
  • ion channels target ultrasound microbubbles as ultrasound neurostimulation potentiators.
  • neurostimulation field disorders include movement disorders, pain, epilepsy, Parkinson's disease, depression, drug addiction, sleep dysfunction. That is, the ion channel-targeted ultrasonic microbubbles provided in the embodiments of the present application, after entering the biological environment, can open the ion channels under ultrasonic conditions, regulate and enhance the nerve stimulation effect, and realize the treatment of the above diseases.
  • the ion channel-targeted ultrasonic microbubbles target the opening of ion channels under ultrasonic conditions.
  • the ultrasonic sound pressure of the ultrasonic treatment is 0.01-0.50 MPa, preferably 0.03-0.17 MPa, so that the ion channel targeted ultrasonic microbubbles are bound to the surface of the ion channel, and the ion channel is opened by the ultrasonic radiation force.
  • ion channels are used to target ultrasonic organisms, and ion channels are targeted to be opened under ultrasonic conditions, so as to achieve a sensitization effect on nerve stimulation, thereby improving the feeling effect of nerve stimulation.
  • N 2 A nerve cells are stimulated by ultrasound to generate calcium ion influx, and calcium ion fluorescent probes are used for imaging to illustrate the present application.
  • a preparation method of an ion channel targeting ultrasonic microbubble comprises:
  • DSPC distearate phosphatidylcholine
  • DSPE-PEG2000 20mg/ml distearate phosphatidylethanolamine poly-ethylene glycol 2000
  • DSPE-PEG2000-Biotin 20mg/ml biological Mixed with plain acylated polyethylene glycol 2000 modified distearate phosphatidylethanolamine (DSPE-PEG2000-Biotin), dissolved in chloroform and mixed on a vortex mixer.
  • the ion channel thus obtained is targeted to ultrasonic microbubbles, and the particle size and distribution are determined by a particle counting analyzer, and the microbubble morphology is observed with a light microscope.
  • the particle size distribution diagram of the ion channel-targeted microbubbles prepared in Example 1 is shown in Figure 3A (the ordinate "Number” is the number, and the abscissa "Diameter” is the diameter), and the bright field image under the microscope is shown in Figure 3B, Fluo 4-AM staining is shown in Figure 3C.
  • the average particle size of Piezo1 ion channel-targeted ultrasound microbubbles is 1.82um, and the distribution is concentrated; from Figure 3B, it can be seen from Figure 3B that the N 2 A cells are spindle-shaped in the bright field, and elongated protrusions can be seen around the cells, and N 2 A cells can be seen in the bright field. Piezo1 ion channel-targeted ultrasound microbubbles adhered around the 2 A cell membrane; Figure 3C uses the calcium ion fluorescent indicator Fluo 4 to load N 2 A cells, and observed under a fluorescence microscope, the N 2 A cells show green fluorescence.
  • a method for preparing ultrasonic microbubbles differs from Example 1 in that the surface of the microbubbles does not bind to the antibody of the Piezol ion channel labeled with Biotin.
  • N 2 A cells were cultured and seeded in a 6-well plate. When the cell density reached 75-85%, the cells were labeled with Ca 2+ fluorescent probe Fluo 4-AM.
  • step (3) Incubating the Piezol ion channel targeting ultrasonic microbubbles of step (1) with the cells of step (2).
  • the cells were cultured and seeded in a 6-well plate. When the cell density reached 75-85%, the cells were labeled with the Ca 2+ fluorescent probe Fluo 4-AM; the ultrasonic microbubbles prepared in Comparative Example 1 were incubated with the cells.
  • Example 2 and Comparative Example 2 Under the voltage condition of 0.03-0.17MPa, the target cells obtained in Example 2 and Comparative Example 2 were subjected to ultrasonic stimulation, and the method was as follows: the cells were cultured, digested with trypsin in the log phase, and inoculated in a 6-well plate, Incubate in a constant temperature incubator at 37°C for 24-48 hours; label cells with ion channel fluorescent probes, and stain at room temperature for 30 minutes in the dark.
  • Figure 4 The Ca 2+ imaging after ultrasound stimulation was observed with a fluorescence microscope, and the results are shown in Figure 4 (the effect of Piezol ion channel targeting microbubbles to sensitize N 2 A cells by ultrasound stimulation).
  • Figure 4A is the brightfield image, Fluo 4 staining, PI staining, Fluo 4 staining and PI staining fusion image of ultrasound-stimulated N 2 A cells under the condition of Piezo1 ion channel targeting microvesicles to adhere to N 2 A cells
  • Figure 4B (the abscissa "Time” is time) is the time fluorescence intensity change diagram of Piezo1-targeted microbubble-bound N 2 A cells before, immediately or 5 min after ultrasound irradiation of Piezo1 ion channel-targeted microbubbles
  • Figure 4C is N 2 Average maximum relative fluorescence intensity map (df/F0) after ultrasound irradiation of A cells incubated with non-targeted microbubbles provided in Compar
  • the stimulation effect of Piezo1 ion channel-targeted microbubble sensitization ultrasound on neurons is the same as that in Example 2.
  • FIG. 5A is a bright field image of neurons adhered to Piezo1 ion channel-targeted microbubbles after ultrasonic stimulation
  • Figure 5B is a Fluo 4 staining image of neurons adhered to Piezo1 ion channel-targeted microbubbles before ultrasonic stimulation
  • Figure 5C is the ultrasound-stimulated Fluo 4 staining of the neuron adhered to Piezo1-targeted microbubbles
  • the cells were cultured and seeded in a 6-well plate. When the cell density reached 75-85%, the cells were labeled with the Ca 2+ fluorescent probe Fluo 4-AM.
  • step (3) Incubating the Piezol ion channel targeting ultrasonic microbubbles of step (1) with the cells of step (2).
  • FIG. 6 is a graph of the relative fluorescence intensity of a single N 2 A cell in the control group (control group, N 2 A cells were treated without the blocking agent GsMTx-4) with time;
  • Figure 6B is a graph of a single N 2 A cell in the GsMTx-4 group Changes of relative fluorescence intensity of A cells with time;
  • Fig. 6C is a graph of the average maximum relative fluorescence intensity of GsMTx-4 group N 2 A cells and control (control) N 2 A cells.

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Abstract

一种离子通道靶向微泡及其制备方法和应用，其包括壳膜和生物惰性气体，生物惰性气体封于壳膜围合形成的囊体内，壳膜外表面结合有离子通道抗体。该离子通道靶向微泡通过离子通道抗体与离子通道靶向结合，可增强超声神经刺激响应的灵敏度。

Description

离子通道靶向微泡及其制备方法和应用 技术领域
本申请属于超声神经刺激技术领域，尤其涉及一种离子通道靶向微泡及其制备方法和应用。
背景技术
近年来，基于物理场的神经调控技术(如电、磁和光遗传技术等)成为动神经科学快速发展的重要动力，并为治疗脑疾病提供了新的方向。基于超声的无创神经调控技术，被认为是最具有临床转化前景的下一代神经调控技术之一，它具有无创、大穿透深度、高时空分布率等优点。超声神经调控通过不同的强度、频率、脉冲重复脉冲宽度、持续时间使刺激部位的中枢神经产生刺激或抑制效应，对神经功能产生双向调节的可逆性变化。其调控效果在神经元、线虫、小鼠、非人灵长类动物等多种尺度目标上得到了验证。
机械敏感性离子通道是近年来发现的一种新型离子通道，有别于传统的电压敏感，以及配体门控类型的离子通道，它感受细胞形变等方式导致的膜张力的变化而开放，引起细胞内外离子的跨膜运输，参与介导众多的生命活动，其功能愈来愈受到重视。研究表明，超声可以对细胞膜产生一个牵张力，激活膜上的机械敏感离子通道，通过通道产生的跨膜电流来改变细胞的膜电位。最新研究表明，超声作为一种机械刺激，可在低频低能量条件下打开机械敏感离子通道，引起离子内流。
超声微泡是由壳膜包裹核心气体构成的粒径在微米级 (2μm～8μm)，甚至纳米级的微气泡。当靶向微泡受到超声激励时，微泡会随着声波周期性压力变化发生有规律的收缩与膨胀。超声靶向微泡是将目的分子特异性抗体或配体连接到超声微泡表面构筑靶向超声微泡，使超声微泡主动结合到靶标组织。离子通道与神经刺激之间有着密切的联系，通过调控离子通道，可以一定程度调控神经刺激。然而，目前尚缺乏针对离子通道的靶向微泡。
技术问题
本申请的目的在于提供一种离子通道靶向微泡及其制备方法其应用，旨在解决目前没有针对离子通道的靶向微泡的问题。
技术解决方案
为实现上述申请目的，本申请采用的技术方案如下：
本申请第一方面提供一种离子通道靶向超声微泡，包括壳膜和生物惰性气体，所述生物惰性气体封于所述壳膜围合形成的囊体内，其中，所述壳膜的外表面结合有离子通道抗体。
本申请第二方面提供一种离子通道靶向超声微泡的制备方法，包括如下步骤：
制备生物素化的微泡；
将所述生物素化的微泡中加入亲和素蛋白进行孵育，加入生物素标记的离子通道抗体，孵育处理，得到所述离子通道靶向超声微泡。
本申请第三方面提供离子通道靶向超声微泡在制备用于神经疾病的治疗制剂中的应用。
有益效果
本申请提供了一种新型的离子通道靶向超声微泡，在超声刺激下(超声刺激前，细胞外离子浓度高于细胞内离子浓度，离子通道靶向微泡与细胞膜上受体配体结合的通道关闭)，该微泡通过离子通道抗体与离子通道靶向结合，可降低超声辐射力开启离子通道的超声能量，促使胞外离子向胞内流动，从而增强超声神经刺激响应的灵敏度。
本申请提供了一种新型的离子通道靶向超声微泡的制备方法，先制备生物素化的微泡，然后在生物素化的微泡表面引入亲和素，构建生物素-亲和素系统，并借助生物素-亲和素结合离子通道抗体，制备离子通道靶向超声微泡。该方法操作流程简单，离子通道靶向超声微泡易于获得，更重要的是，由此得到的离子通道靶向超声微泡，能够在超声条件下靶向开启离子通道，并增强超声神经刺激响应的灵敏度。
本申请提供的应用，借助离子通道靶向超声微泡，在超声条件下靶向开启离子通道，以实现对神经刺激的增敏作用，从而提高神经刺激的感受效果。
附图说明
为了更清楚地说明本发明实施例中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动性的前提下，还可以根据这些附图获得其他的附图。
图1是本申请实施例提供的离子通道靶向超声微泡在超声条件下的作用原理图；
图2是本申请实施例提供的生物素化的磷脂微泡的制备流程图；
图3A是本申请实施例1提供的离子通道靶向微泡的粒径分布图；
图3B是本申请实施例1提供的离子通道靶向微泡的镜下明场图；
图3C是本申请实施例1提供的离子通道靶向微泡的Fluo 4-AM染色图；
图4A是本申请实施例2提供的Piezo1离子通道靶向微泡黏附N 2A细胞的情况下，超声刺激N 2A细胞的明场图、Fluo 4染色图、PI染色图、Fluo 4染色和PI染色融合图；
图4B是本申请实施例2提供的Piezo1离子通道靶向微泡超声照射前、即刻或5min后，Piezo1离子通道靶向微泡结合的N 2A细胞的时间荧光强度变化图；
图4C是N 2A细胞与对比例2提供的非靶向微泡孵育，超声照射后的平均最大相对荧光强度图(df/F0)；
图5A是本申请实施例3提供的粘附了Piezo1离子通道靶向微泡的神经元超声刺激后明场图；
图5B是本申请实施例3提供的粘附了Piezo1离子通道靶向微泡的神经元超声刺激前Fluo 4染色图；
图5C是本申请实施例3提供的粘附了Piezo1离子通道靶向微泡的神经元超声刺激后Fluo 4染色图；
图5D是本申请实施例3提供的在Piezo1离子通道靶向微泡黏附N 2A细胞的情况下，超声刺激的N 2A细胞平均相对荧光强度随时间变化图；
图5E为本申请实施例3中，实施例1提供的Piezo1离子通道靶向微泡组和对比例1提供的非靶微泡组的N 2A细胞的平均最大相对荧光强度图；
图5F为本申请实施例3提供的靶向微泡黏附细胞的数量与相对荧光强度的相关关系图；
图6A为本申请实施例4提供的对照组单个N 2A细胞的相对荧光强度随时间变化图；
图6B为本申请实施例4提供的GsMTx-4组单个N 2A细胞的相对荧光强度随时间变化图；
图6C为本申请实施例4提供的GsMTx-4组N 2A细胞和对照组N 2A细胞的平均最大相对荧光强度图。
本发明的最佳实施方式
为了使本申请要解决的技术问题、技术方案及有益效果更加清楚明白，以下结合实施例，对本申请进行进一步详细说明。应当理解，此处所描述的具体实施例仅仅用以解释本申请，并不用于限定本申请。
本申请中，术语“和/或”，描述关联对象的关联关系，表示可以存在三种关系，例如，A和/或B，可以表示：单独存在A，同时存在A和B，单独存在B的情况。其中A，B可以是单数或者复数。字符“/”一般表示前后关联对象是一种“或”的关系。
本申请中，“至少一个”是指一个或者多个，“多个”是指两个或两个以上。“以下至少一项(个)”或其类似表达，是指的这些项中的任意组合，包括单项(个)或复数项(个)的任意组合。例如，“a,b, 或c中的至少一项(个)”，或，“a,b,和c中的至少一项(个)”，均可以表示：a,b,c,a-b(即a和b),a-c,b-c,或a-b-c，其中a,b,c分别可以是单个，也可以是多个。
应理解，在本申请的各种实施例中，上述各过程的序号的大小并不意味着执行顺序的先后，部分或全部步骤可以并行执行或先后执行，各过程的执行顺序应以其功能和内在逻辑确定，而不应对本申请实施例的实施过程构成任何限定。
在本申请实施例中使用的术语是仅仅出于描述特定实施例的目的，而非旨在限制本申请。在本申请实施例和所附权利要求书中所使用的单数形式的“一种”、“所述”和“该”也旨在包括多数形式，除非上下文清楚地表示其他含义。
术语“第一”、“第二”仅用于描述目的，用来将目的如物质彼此区分开，而不能理解为指示或暗示相对重要性或者隐含指明所指示的技术特征的数量。例如，在不脱离本申请实施例范围的情况下，第一XX也可以被称为第二XX，类似地，第二XX也可以被称为第一XX。由此，限定有“第一”、“第二”的特征可以明示或者隐含地包括一个或者更多个该特征。
超声微泡是由壳膜包裹核心气体构成的粒径在微米级(2μm～8μm)，甚至纳米级的微气泡。当靶向微泡受到超声激励时，微泡会随着声波周期性压力变化发生有规律的收缩与膨胀。有科学家计算微泡和细胞受到的超声辐射力的大小后发现，相比细胞，微泡对超声辐射力的机械刺激有更高的反应能力，具体的，微泡受到的声辐射 力比细胞受到的声辐射力高20倍。即：相比细胞，超声微泡结合细胞并开启机械敏感通道所需要的超声能量更低。
超声靶向微泡可以将目的分子特异性抗体或配体连接到超声微泡表面构筑靶向超声微泡，使超声微泡主动结合到靶标组织。然而，目前的靶向微泡尚且不能靶向至机械敏感型离子通道，无法增强神经细胞对超声刺激的响应。
鉴于此，本申请实施例提供了一种新型的离子通道靶向超声微泡，该靶向超声微泡包括壳膜和生物惰性气体，生物惰性气体封于壳膜围合形成的囊体内。其中，壳膜的外表面结合有离子通道抗体，离子通道抗体可以与离子通道结合。超声刺激前，细胞外离子浓度高于细胞内离子浓度，离子通道靶向微泡与细胞膜上受体配体结合的通道关闭；在超声刺激下，该微泡通过离子通道抗体与离子通道靶向结合，可降低超声辐射力开启离子通道的超声能量，并在超声辐射力的作用下拉动离子通道的“闸门”，开启离子通道，促使胞外离子向胞内流动，从而增强超声神经刺激响应的灵敏度。
具体的，本申请实施例中，离子通道靶向超声微泡包括超声微泡基本结构，即壳膜和生物惰性气体，生物惰性气体封于壳膜围合形成的囊体内。
其中，壳膜可以采用超声微泡可采用的壳膜材料，示例性的，壳膜为磷酯类壳膜或磷酯衍生物类壳膜。在一些实施例中，壳膜为磷酯类壳膜，且磷酯类壳膜包括如下组分：二硬脂酸磷酯酰胆碱(DSPC)、二硬脂酸磷酯酰乙醇胺聚-乙二醇2000(DSPE-PEG2000)、生物素酰化 聚乙二醇2000和二硬脂酸磷酯酰乙醇胺。由此形成的离子通道靶向超声微泡，在超声作用下，更容易推动与其结合的离子通道的开启。
本申请实施例中，生物惰性气体封锁在壳膜中，可以选择超声微泡使用的各种生物惰性气体。在一些实施例中，生物惰性气体为氟代烷烃，优选为全氟代烷烃。示例性的，生物惰性气体为全氟丙烷，当然，生物惰性气体并不限于此。
在此基础上，壳膜的外表面含有离子通道抗体的结合位点，使得在超声条件下，离子通道抗体能够靶向结合在细胞膜离子通道处，从而使得靶向超声微泡在超声作用下，借助超声产生的辐射力开启结合位点处的离子通道。
本申请实施例中，壳膜的外表面结合有离子通道抗体。应当注意的是，本申请实施例提供的离子通道靶向超声微泡的壳膜，包括两侧，相对于气体内核的一侧为内侧，对应的壳膜表面为内表面；与气体内核相反的一侧为外侧，对应的壳膜表面为外表面。
在一些实施例中，离子通道抗体通过生物素-亲和素系统结合在壳膜的外表面。即：离子通道靶向超声微泡的壳膜外表面，至少部分区域结合有生物素-亲和素结构。通过这种方式结合离子通道抗体，离子通道抗体具有较好的结合稳定性。当然，离子通道抗体也可以通过其它连接系统结合在壳膜的外表面。
应当注意的是，本申请实施例中，离子通道抗体为能够作用于离子通道的抗体，在一些实施例中，离子通道为机械敏感型离子通道，但不限于此。由此，离子通道靶向超声微泡在超声条件下结合细胞膜 离子通道后，降低超声辐射力开启离子通道的超声能量，并借助超声产生的辐射力来推动离子通道的开启。
本申请实施例中，离子通道靶向超声微泡靶向作用的离子通道，可以为外源性机械敏感性离子通道，也可以为内源性机械敏感性离子通道。在一些实施例中，离子通道选自Piezo1离子通道、MscL离子通道、DEG/E Na C/ASIC通道、TRP通道和K2P通道。本申请实施例提供的离子通道靶向超声微泡，在超声条件下，能够靶向作用于上述通道，并在超声辐射力作用下开启通道。当然，离子通道不限于上述列举的通道类型，还可以为有利于激活神经细胞的其他离子通道。示例性的，在超声条件下，离子通道靶向超声微泡靶向开启Piezo1离子通道后，可以引入包括钙离子、钠离子、钾离子在内的多种离子中的一种或多种离子内流(往细胞内流动)，从而增敏超声神经刺激的响应。在这种情况下，可以将本申请实施例提供离子通道靶向超声微泡，用于运动性障碍、疼痛、癫痫、帕金森病、精神疾病等神经或其他系统疾病的应用中。
本申请实施例中，微泡并不局限与微米级靶向生物气泡，还可以包括纳泡在内的其它声学相应材料。在一些实施例中，微泡的尺寸为微米级或纳米级。其中，微米级的微泡的尺寸为2μm～8μm。
本申请实施例提供的离子通道靶向超声微泡，可以作用于神经细胞但不限于神经细胞。其中，神经细胞包括但不限于深脑区域的神经细胞和浅脑区域的神经细胞。
本申请实施例提供的离子通道靶向超声微泡的作用效果，可以通 过活细胞荧光成像方法进行评估，通过观察细胞内外荧光强度的变化来验证离子通道靶向超声微泡是否靶向开启离子通道。
以壳膜为磷酯类壳膜，且磷酯类壳膜包括如下组分：二硬脂酸磷酯酰胆碱(DSPC)、二硬脂酸磷酯酰乙醇胺聚-乙二醇2000(DSPE-PEG2000)、生物素酰化聚乙二醇2000和二硬脂酸磷酯酰乙醇胺(DSPE-PEG2000-Biotin)，生物惰性气体为氟代烷烃为例，得到的离子通道靶向超声微泡在超声条件下的作用原理图1所示(Avidin为亲和素，Biotinylated Antibody为生物素标记抗体或生物素化抗体)。由图可见：在离子通道靶向超声微泡形成后(A)，在超声条件下，离子通道靶向超声微泡表面的特异性抗体结合在细胞表面(B)，最终进入开启离子通道，使细胞内外离子浓度发生变化(C)。
本申请实施例提供的离子通道靶向超声微泡的制备方法，可以通过下述方法制备获得。
本申请实施例第二方面提供一种离子通道靶向超声微泡的制备方法，包括如下步骤：
S01.制备生物素化的微泡；
S02.将生物素化的微泡中加入亲和素蛋白进行孵育，加入生物素标记的离子通道抗体，孵育处理，得到离子通道靶向超声微泡。
本申请实施例提供的新型的离子通道靶向超声微泡的制备方法，先制备生物素化的微泡，然后在生物素化的微泡表面引入亲和素，构建生物素-亲和素系统，并借助生物素-亲和素结合离子通道抗体，制备离子通道靶向超声微泡。该方法操作流程简单，离子通道靶向超声 微泡易于获得，更重要的是，由此得到的离子通道靶向超声微泡，能够在超声条件下靶向开启离子通道，并增敏超声神经刺激的响应。
具体的，上述步骤S01中，制备生物素化的微泡，可以参照非靶向的超声微泡或其他靶向作用的超声微泡的制备方法，制备超声微泡；并在超声微泡表面结合生物素。
在一些实施例中，生物素化的微泡的制备方法为：
S011.取壳膜原料，混合处理后溶解于有机溶剂中，得到第一混合溶液。
该步骤中，将壳膜原料和生物素原料进行混合，混合可以采用直接混合处理，溶于有机溶剂中后，进一步混合，得到第一混合溶液。在一些实施例中，溶于有机溶剂后在涡旋混合器上混匀，得到第一混合溶液。
S012.采用干燥的氮气流去除第一混合溶液中的有机溶剂后，干燥处理；加入缓冲溶液，得到第二溶液。
该步骤中，采用干燥的氮气流去除第一混合溶液中的有机溶剂后，容器避免形成一层均匀的薄膜，进一步进行干燥处理，去除剩余的溶剂。在一些实施例中，干燥处理可以在真空烘箱中实现，时间为2～6小时。
干燥结束后，加入脱气的缓冲溶液，在一些实施例中，缓冲溶液为Tris缓冲溶液，由此得到第二溶液。
S013.加热第二溶液至相转变温度以上，水浴超声制成第三溶液，分装处理，并采用生物惰性气体置换分装容器中的气体，机械震荡得 到生物素化的微泡。
该步骤，生物惰性气体可以为氟代烷烃，包括但不限于全氟丙烷。
示例性的，生物素化的微泡为生物素化的磷脂微泡，磷脂微泡的壳膜为磷酯壳膜，其中的生物惰性气体为氟代烷烃。
在一些实施例中，如图2所示，生物素化的磷脂微泡的制备方法为：
(1)将20mg/ml的二硬脂酸磷酯酰胆碱(DSPC)、20mg/ml的二硬脂酸磷酯酰乙醇胺聚-乙二醇2000(DSPE-PEG2000)和20mg/ml的生物素酰化聚乙二醇2000修饰二硬脂酸磷酯酰乙醇胺(DSPE-PEG2000-Biotin)混合，溶解于三氯甲烷中并在涡旋混合器上混匀。
(2)使用干燥的氮气流除去有机溶剂使其在试管壁上形成一层均匀的薄膜，真空烘箱中干燥2-6小时；然后加入一定体积脱气的Tris缓冲溶液，得到磷脂溶液。
(3)加热磷脂溶液到其相转变温度(55-60℃)以上，并用水浴超声制成磷脂溶液，分装入小西林瓶中，将瓶中的空气置换成全氟丙烷，用机械震荡器震荡45s获得生物素化的超声微泡。
上述步骤S02中，将携带生物素的抗体利用生物素-亲和素桥接的方式进一步偶连到生物素化的微泡表面。
具体的，将生物素化的微泡中加入亲和素蛋白进行孵育，加入亲和素之前，采用PBS(phosphate buffer saline的缩写，表示磷酸缓冲盐溶液)进行洗涤3～4次。加入生物素标记的离子通道抗体后孵育 15～30分钟，用PBS洗涤以去除未结合的亲和素蛋白。
进一步的，加入生物素标记的离子通道抗体，示例性的，Piezo1通道的抗体，室温(5～40℃)条件下继续孵育15～30分钟，得到离子通道靶向超声微泡。
以上文制备的生物素化的磷脂微泡为例，步骤S02包括：采用PBS借助离心漂浮法(400g)将生物素化的磷脂微泡洗涤3-4次，加入亲和素蛋白进行孵育15-30min，用PBS洗涤3-4次去除未结合的亲和素蛋白；加入Biotin标记的抗体(相应机械敏感型离子通道的抗体，比如Piezo1离子通道的抗体)，室温再次孵育15-30min，获得离子通道靶向微泡。
本实施例制备得到的离子通道靶向超声微泡，可以通过颗粒计数分析仪测定其粒径大小和分布，光镜观测其微泡形态。
本申请实施例第三方面提供离子通道靶向超声微泡制备用于神经疾病的治疗制剂中的应用。
在一些实施例中，离子通道靶向超声微泡作为超声神经刺激增效剂。
在一些实施例中，神经刺激领域疾病包括运动性障碍疾病、疼痛、癫痫、帕金森病、抑郁症、药物成瘾症、睡眠功能障碍。即本申请实施例提供的离子通道靶向超声微泡，进入生物环境后，可以在超声条件下开启离子通道，调控并增效神经刺激作用，实现对上述疾病的治疗。
在一些实施例中，离子通道靶向超声微泡在超声条件下靶向开启 离子通道。该实施例中，超声处理的的超声波声压为0.01-0.50MPa，优选0.03-0.17MPa，从而有利于离子通道靶向超声微泡结合在离子通道表面，并借助超声辐射力开启离子通道。
本申请实施例提供的应用，借助离子通道靶向超声生物，在超声条件下靶向开启离子通道，以实现对神经刺激的增敏作用，从而提高神经刺激的感受效果。
下面以Piezo1离子通道为例，超声刺激N 2A神经细胞后产生钙离子内流，使用钙离子荧光探针进行成像，对本申请进行说明。
实施例1
一种离子通道靶向超声微泡的制备方法包括：
(1)将20mg/ml的二硬脂酸磷酯酰胆碱(DSPC)、20mg/ml的二硬脂酸磷酯酰乙醇胺聚-乙二醇2000(DSPE-PEG2000)和20mg/ml的生物素酰化聚乙二醇2000修饰二硬脂酸磷酯酰乙醇胺(DSPE-PEG2000-Biotin)混合，溶解于三氯甲烷中并在涡旋混合器上混匀。
(2)使用干燥的氮气流除去有机溶剂使其在试管壁上形成一层均匀的薄膜，真空烘箱中干燥2-6小时；然后加入一定体积脱气的Tris缓冲溶液，得到磷脂溶液。
(3)加热磷脂溶液到其相转变温度(55-60℃)以上，并用水浴超声制成磷脂溶液，分装入小西林瓶中，将瓶中的空气置换成全氟丙烷，用机械震荡器震荡45s获得生物素化的超声微泡。
(4)采用PBS借助离心漂浮法(400g)将生物素化的磷脂微泡洗涤3-4次，加入亲和素蛋白进行孵育15-30min，用PBS洗涤3-4次去除未结合的亲和素蛋白；加入Biotin标记的Piezo1离子通道的抗体，室温再次孵育15-30min，获得离子通道靶向微泡。
由此得到的离子通道靶向超声微泡，通过颗粒计数分析仪测定其粒径大小和分布，光镜观测其微泡形态。实施例1制备的离子通道靶向微泡的粒径分布图如图3A所示(纵坐标“Number”为数量，横坐标“Diameter”表示直径)，镜下明场图如图3B所示，Fluo 4-AM染色图如图3C所示。由图3A可见，Piezo1离子通道靶向超声微泡的平均粒径为1.82um，分布集中；由图3B可见，明场可见N 2A细胞成梭形，细胞周边可见细长的突起，并且N 2A细胞膜周边粘附了Piezo1离子通道靶向超声微泡；图3C使用钙离子荧光指示剂Fluo 4负载N 2A细胞，荧光显微镜下观察，N 2A细胞呈绿色荧光。
对比例1
一种超声微泡的制备方法，与实施例1的不同之处在于：微泡表面不结合Biotin标记的Piezo1离子通道的抗体。
实施例2
Piezo1离子通道靶向微泡增敏超声对N 2A细胞的刺激效应：
(1)提供上述实施例1制备的Piezo1离子通道靶向超声微泡；
(2)培养N 2A细胞并接种于6孔板，待长到75-85％的细胞密度 时，使用Ca 2+荧光探针Fluo 4-AM标记细胞。
(3)将步骤(1)的Piezo1离子通道靶向超声微泡与步骤(2)的细胞孵育。
对比例2
培养细胞并接种于6孔板，待长到75-85％的细胞密度时，使用Ca 2+荧光探针Fluo 4-AM标记细胞；将对比例1制备的超声微泡与细胞孵育。
在0.03-0.17MPa的电压条件下，对实施例2和对比例2得到的靶向细胞进行超声刺激，方法如下：培养细胞，在对数期用胰酶消化，并接种于6孔板内，在37℃恒温培养箱中培养24-48h；使用离子通道荧光探针标记细胞，室温避光染色30min，染色结束后，吸出染液，用PBS洗三遍；将一定浓度的离子通道靶向微泡加入细胞内进行孵育，室温孵育一段时间，然后用PBS洗去未黏附的微泡；超声刺激：启动超声刺激系统，用于刺激孔板上的细胞。
用荧光显微镜观察超声刺激后的Ca 2+成像，结果如图4所示(Piezo1离子通道靶向微泡增敏超声刺激N 2A细胞的效果图)。其中，图4A是Piezo1离子通道靶向微泡黏附N 2A细胞的情况下，超声刺激N 2A细胞的明场图、Fluo 4染色图、PI染色图、Fluo 4染色和PI染色融合图；图4B(横坐标“Time”为时间)是Piezo1离子通道靶向微泡超声照射前、即刻或5min后Piezo1靶向微泡结合的N 2A细胞的时间荧光强度变化图；图4C是N 2A细胞与对比例2提供的非靶向微泡 孵育，超声照射后的平均最大相对荧光强度图(df/F0)。
由图可见：Piezo1离子通道靶向微泡黏附N 2A细胞的情况下，当脉冲超声声压为0.03MPa时，超声可快速刺激N 2A细胞内Fluo 4荧光强度显著增强，超声刺激结束后N 2A细胞荧光强度下降，PI染色发现死亡N 2A细胞很少。实验结果提示：Piezo1离子通道靶向微泡可以增敏超声对N 2A细胞的刺激效应。
实施例3
Piezo1离子通道靶向微泡增敏超声对神经元的刺激效应：操作方法同实施例2。
在0.03-0.17MPa的电压条件下，对实施例3得到的靶向细胞进行超声刺激，用荧光显微镜观察Ca 2+成像，结果如图5所示(Piezo1离子通道靶向微泡增敏超声对神经元的刺激效应图)。其中，图5A为粘附了Piezo1离子通道靶向微泡的神经元超声刺激后明场图；图5B为粘附了Piezo1离子通道靶向微泡的神经元超声刺激前Fluo 4染色图；图5C为粘附了Piezo1靶向微泡的神经元超声刺激Fluo 4染色；图5D(横坐标“Time”表示时间)为在Piezo1离子通道靶向微泡黏附N 2A细胞的情况下，超声刺激的N 2A细胞平均相对荧光强度随时间变化图，红色箭头表示超声刺激时刻；图5E为实施例1提供的Piezo1离子通道靶向微泡组和对比例1提供的非靶微泡组的N 2A细胞的平均最大相对荧光强度图；图5F(横坐标“Bubble nunber of binding to cells”表示：与细胞结合的靶向微泡)为靶向微泡黏附细胞的数量与相对荧 光强度的相关关系图。
由图可见，无论是Piezo1离子通道靶向微泡黏附N 2A细胞，还是黏附神经元，超声刺激下，细胞荧光强度明显增加，超声刺激结束后，细胞荧光强度下降。Piezo1离子通道靶向微泡组N 2A细胞的平均最大相对荧光强度远高于非靶微泡组N 2A细胞的平均最大相对荧光强度。结果提示：Piezo1离子通道靶向微泡可以增敏超声对神经元的刺激效应。
实施例4
细胞膜上的Piezo1离子通道在超声刺激的已粘附Piezo1靶向微泡的N 2A细胞钙水平升高中的作用
(1)提供上述实施例1制备的Piezo1离子通道靶向超声微泡；
(2)培养细胞并接种于6孔板，待长到75-85％的细胞密度时，使用Ca 2+荧光探针Fluo 4-AM标记细胞。
(3)将步骤(1)的Piezo1离子通道靶向超声微泡与步骤(2)的细胞孵育。
(4)Piezo1离子通道抑制剂处理细胞：用3umol/L的Piezo1通道特异性的阻断剂GsMTx-4处理N 2A细胞30min。
(5)启动超声刺激系统，设置超声频率为2MHz，声压为0.17MPa的脉冲超声刺激N 2A细胞10s。
用活细胞钙荧光成像观测钙离子浓度改变，结果如图6所示(在Piezo1离子通道靶向微泡黏附N 2A细胞的情况下，细胞膜上的Piezo1 离子通道在超声刺激的N 2A细胞钙水平升高中的作用)。图6A为对照组(control组，不加阻断剂GsMTx-4对N 2A细胞进行处理)单个N 2A细胞的相对荧光强度随时间的改变图；图6B为GsMTx-4组单个N 2A细胞的相对荧光强度随时间的改变图；图6C为GsMTx-4组N 2A细胞和对照组(control)N 2A细胞的平均最大相对荧光强度图。
结果显示：在Piezo1离子通道靶向微泡黏附N 2A细胞的情况下，3umol/L的Piezo1通道特异性的阻断剂GsMTx-4处理N 2A细胞30min降低了超声刺激的N 2A细胞钙水平升高(p<0.05)。可见，Piezo1离子通道靶向微泡能够在超声条件下靶向作用于Piezo1离子通道，并开启Piezo1离子通道。
以上仅为本申请的较佳实施例而已，并不用以限制本申请，凡在本申请的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本申请的保护范围之内。

Claims (10)

  1. 一种离子通道靶向超声微泡，其特征在于，包括壳膜和生物惰性气体，所述生物惰性气体封于所述壳膜围合形成的囊体内，其中，所述壳膜的外表面结合有离子通道抗体。
  2. 如权利要求1所述的离子通道靶向超声微泡，其特征在于，所述离子通道抗体为能够作用于离子通道的抗体，且所述离子通道为机械敏感型离子通道。
  3. 如权利要求2所述的离子通道靶向超声微泡，其特征在于，所述离子通道选自Piezo1离子通道、MscL离子通道、DEG/E Na C/ASIC通道、TRP通道和K2P通道。
  4. 如权利要求1至3任一项所述的离子通道靶向超声微泡，其特征在于，所述微泡的尺寸为微米级或纳米级。
  5. 如权利要求1至3任一项所述的离子通道靶向超声微泡，其特征在于，所述离子通道抗体通过生物素-亲和素系统结合在所述壳膜的外表面。
  6. 一种离子通道靶向超声微泡的制备方法，其特征在于，包括如下步骤：
    制备生物素化的微泡；
    将所述生物素化的微泡中加入亲和素蛋白进行孵育，加入生物素标记的离子通道抗体，孵育处理，得到所述离子通道靶向超声微泡。
  7. 如权利要求6所述的离子通道靶向超声微泡的制备方法，其特征在于，所述生物素化的微泡的制备方法为：
    取壳膜原料，混合处理后溶解于有机溶剂中，得到第一混合溶液；
    采用干燥的氮气流去除所述第一混合溶液中的有机溶剂后，干燥处理；加入缓冲溶液，得到第二溶液；
    加热所述第二溶液至相转变温度以上，水浴超声制成第三溶液，分装处理，并采用生物惰性气体置换分装容器中的气体，机械震荡得到所述生物素化的微泡。
  8. 一种离子通道靶向超声微泡在制备用于神经疾病的治疗制剂中的应用。
  9. 如权利要求8所述的应用，其特征在于，所述离子通道靶向超声微泡作为超声神经刺激增效剂。
  10. 如权利要求9所述的应用，其特征在于，所述神经领域疾病包括运动性障碍疾病、疼痛、癫痫、帕金森病、抑郁症、药物成瘾症、睡眠功能障碍；和/或
    所述离子通道靶向超声微泡在超声条件下靶向开启离子通道。
PCT/CN2021/138062 2021-01-28 2021-12-14 离子通道靶向微泡及其制备方法和应用 Ceased WO2022160987A1 (zh)

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