Method for opening blood-brain barrier and accurately delivering medicine by combining microbubbles with nano liquid drops

Abstract

The invention relates to the technical field of ultrasonic combined ultrasonic response drug carriers, in particular to a method for opening a blood-brain barrier through microbubbles combined with nano liquid drops to achieve precise drug delivery. By using the drug-loaded microbubble, the nano-droplet and the method provided by the invention, the explosion of the microbubble in the brain can be realized through the stimulation of ultrasonic energy at an imaging level, the explosion of the drug-loaded nano-droplet is induced while a blood brain barrier is opened, and the liquid core of the nano-droplet enables the nano-droplet to have higher drug loading and longer blood stability, so that the drug-loaded nano-droplet has higher drug loading capacity and higher blood stability. Therefore, the blasting of the nano liquid drops can enable the loaded medicine to be delivered into brain parenchyma more accurately and efficiently, and meanwhile, the gradient ultrasonic activation method provided by the invention can ensure that the blood brain barrier is safely opened without influencing normal tissues.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and specifically relates to a method for opening the blood-brain barrier and delivering drugs by combining microbubbles with nanodroplets, and its use in the treatment of central nervous system degenerative diseases and brain tumors. Background Art

[0002] Central nervous system diseases are the diseases that cause the highest loss of healthy life, and the number of cases is on the rise. The main difficulty in its treatment is that it is difficult for drugs to pass through the blood-brain barrier and cannot act quickly and efficiently on the diseased brain parenchyma. If hydrophobic central nervous system drugs are directly injected into the blood, they will face the risk of not being evenly dispersed in the hydrophilic blood system. When the diameter of the drug droplets is greater than 5 μm, it is very easy to cause vascular embolism. Therefore, hydrophilic preparations need to be made, but hydrophilic modification will cause the challenge of low BBB permeability. Some hydrophilic drugs can achieve efficacy by increasing the dosage, but their adverse reactions are serious.

[0003] In order to reduce its toxic side effects, drug-loaded micro-nanoparticles came into being. However, micro-nanoparticles rely on endocytosis to cross the blood-brain barrier, which is inefficient and takes a long time to take effect, and still cannot solve the problem of drug delivery in the brain. Among many emerging drug delivery systems, ultrasound combined with microbubble technology can reversibly and non-invasively open the blood-brain barrier, opening a new door for drug delivery in the brain. The degree of BBB opening is related to the parameters of ultrasound and microbubbles.

[0004] However, the commonly used microbubbles are mostly inert fluorocarbon or fluorine-sulfur gas microbubbles wrapped by phospholipids and proteins. Solid drugs can only be loaded in the shell layer of microbubbles, resulting in low loading capacity and poor stability. Nanodroplets, which have similar properties to microbubbles, have better stability, and drugs can be dissolved into the core of nanodroplets after fluorination, thereby achieving efficient drug loading. However, the activation threshold of nanodroplets is high, making it difficult to achieve the explosion of nanodroplets and release of drugs in the presence of skull bones.

[0005] Therefore, current research urgently needs to develop a set of efficient drug delivery carriers that can efficiently deliver drugs to the brain while applying ultrasound to open the blood-brain barrier. Summary of the invention

[0006] In view of the above technical problems, the present invention provides a method for safely opening the blood-brain barrier and delivering drugs by combining microbubbles with nanodroplets.

[0007] Specifically, the present invention combines drug-loaded microbubbles and drug-loaded nanodroplets. When ultrasonic stimulation is applied, microbubbles with a low explosion threshold explode first, and while reversibly opening the blood-brain barrier, they can also serve as initiators for the explosion of drug-loaded nanodroplets, thereby achieving the release of drugs in the nanodroplets and enabling their efficient enrichment in the brain parenchyma.

[0008] The present invention also provides a safe and reversible ultrasound stimulation mode to ensure the safety of cerebral blood vessels during ultrasound stimulation, wherein the ultrasound frequency range is 3.0-11 MHz. The ultrasound stimulation mechanical index is variable, ranging from 0.4-1.6, and the trend is a step-by-step increase from small to large, with a step of 0.1-0.4 / 10 s, and remains unchanged after the ultrasound mechanical index reaches a peak value of 1.6. Since both microbubbles and nanodroplets have a particle size distribution, the step-by-step increase in the mechanical index can ensure that the microbubbles and liquids are exploded in batches, thereby avoiding the irreversible destruction of the blood-brain barrier and tissue bleeding caused by the instantaneous high energy-induced explosion of a large number of microbubbles and nanodroplets.

[0009] Technical solution explanation The microbubble of the present invention is characterized in that the microbubble is composed of a lipid shell and a gas encapsulated by the shell, wherein the lipid is a phospholipid, the polar head group of the phospholipid is PC (acylcholine), PE (acylethanolamine), PG (sodium acylglycerate), PS (acylserine) and PA (glycerophosphatidic acid), and the non-polar polar group is a long carbon chain with a length of 16-28, selected from 1,2-distearoyl-sn-glyceryl-3-phosphocholine (DSPC), distearoylphosphatidylethanolamine (DSPE), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylcholine (DP ... One or more of diacylphosphatidylcholine (DAPC), 1,2-bis(diphenylphosphino)ethane (DPPE), dioleoylphosphatidylethanolamine (DOPE), distearoylphosphatidylglycerol (DSPG), dipalmitoylphosphatidylglycerol (DPPG), dimyristoylphosphatidylglycerol (DMPG), distearoylphosphatidic acid (DSPA), dipalmitoylphosphatidic acid (DPPA), dipalmitoylphosphatidylserine (DPPS), distearoylphosphatidylserine (DSPS) and distearoylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG).

[0010] In addition, to ensure the stability of the microbubbles, the lipid shell also contains an emulsifier and a stabilizer. Preferably, relative to 100 parts by weight of the lipid, the content of the emulsifier is 25-45 parts by weight, and the content of the stabilizer is 15-30 parts by weight; more preferably, relative to 100 parts by weight of the lipid, the content of the emulsifier is 30-40 parts by weight, and the content of the stabilizer is 22-28 parts by weight.

[0011] In the present invention, the emulsifier may be an emulsifier conventionally used in the art. Preferably, the emulsifier is selected from one or more of polyethylene glycol 4000 (PEG4000), polyethylene glycol 40s (PEG40s), polyoxypropylene polyoxyethylene block polyether (Pluronic), polyethylene glycol 1400 (PEG1400) and polysorbate 80.

[0012] The stabilizers are fatty acids and polyols.

[0013] The gas encapsulated by the shell layer is an inert gas, specifically one or more of perfluoropropane, perfluorobutane, sulfur hexafluoride and nitrogen.

[0014] The gaseous drug loaded in the microbubbles is a gas with active ingredients, specifically selected from one or more of oxygen, hydrogen, nitric oxide, carbon monoxide, and xenon.

[0015] The volume ratio of inert gas to gaseous drug is 1:(0.05-0.5).

[0016] The solid drug loaded in the microbubble is a commonly used therapeutic agent for brain diseases, specifically selected from vincristine, vinblastine, vinfoline, vindesine, vinorelbine, temozolomide, carmustine, lomustine, cabazitaxel, docetaxel, larotaxel, ortataxel, paclitaxel, tesetaxel, ixabepilone, lomustine, procarbazine, rituximab, tocilizumab, temozolomide, carboplatin, erlotinib, irinotecan, enzastaurin, vorinostat, doxorubicin, cisplatin, Gleevec, 5-fluorouracil, tamoxifen, The invention includes, but is not limited to, oxifene, topotecan, belotecan, imatinib, floxuridine, gemcitabine, leuprolide, flutamide, zoledronic acid, methotrexate, camptothecin, hydroxyurea, streptozocin, valrubicin, retinoic acid, mechlorethamine, chlorambucil, busulfan, doxefoxazole, mitomycin, prednisone, everolimus, mitoxantrone, levodopa, carbidopa, entacapone, tolcapone, dopamine agonists, donepezil, galantamine, rivastigmine, memantine, anticholinergics and amantadine.

[0017] The mass ratio of microbubble membrane material to solid drug is 1:(0.01-0.4).

[0018] The injection dose of microbubbles in vivo is 1-5 x 10 7 / kg.

[0019] The nano droplet of the present invention is composed of a shell layer and a liquid encapsulated by the shell layer, wherein the shell layer is mainly composed of a skeleton material, an emulsifier and a surface charge modifier, and the skeleton material is selected from high molecular polymers and lipids.

[0020] The liquid encapsulated by the shell is a fluorine phase liquid, which is specifically selected from substances that can be liquid under normal temperature and pressure conditions or under certain environmental conditions at normal temperature, but can be converted into gaseous state at normal temperature under specific environmental or excitation conditions, such as perfluorocarbons; and perfluorocarbons also have the characteristics of high molecular weight, stable properties, and good biosafety. The contrast agent obtained with liquid perfluorocarbon as the content has the unique performance of liquid-gas phase transition, and can be excited to form gaseous state under conditions such as ultrasound and light, thereby forming a microbubble contrast agent; the microbubbles can undergo inertial cavitation when ruptured, so that they can load drugs and burst at a fixed point in the target area and release drugs locally, exerting the therapeutic effect of targeted drug delivery; microbubbles generate microflows and shear forces locally when cavitating, stimulating the opening of pores in the endothelial cell membrane and channels between cells, and promoting the delivery of therapeutic drugs into cells. Therefore, the contrast agent obtained with liquid perfluorocarbon as the content can not only achieve similar effects as traditional microbubble ultrasound contrast agents, but also its liquid core allows the nanodroplets to have a smaller particle size and higher stability, thereby providing huge application prospects for tissue imaging and targeted precision treatment. Therefore, it is specially studied in the present invention. Therefore, according to a preferred specific embodiment of the present invention, the content is liquid perfluorocarbon.

[0021] Preferably, the perfluorocarbon is selected from one or more of perfluoropropane, perfluorobutane, perfluoropentane and perfluorohexane. When the perfluorocarbon is perfluoropentane and / or perfluorohexane, since these two substances are in liquid state at room temperature and pressure, no special operation is required in the process of preparing the contrast agent film-forming agent and the contrast agent; and when the perfluorocarbon is perfluoropropane and / or perfluorobutane, since these two substances are in gaseous state at room temperature and pressure, the temperature and / or pressure need to be controlled in the process of preparing the contrast agent film-forming agent and the contrast agent to keep them in liquid state.

[0022] The solid drug loaded in the nanodroplet is a commonly used therapeutic agent for brain diseases, specifically selected from vincristine, vinblastine, vinfoline, vindesine, vinorelbine, temozolomide, carmustine, lomustine, cabazitaxel, docetaxel, larotaxel, ortataxel, paclitaxel, tesetaxel, ixabepilone, lomustine, procarbazine, rituximab, tocilizumab, temozolomide, carboplatin, erlotinib, irinotecan, enzastaurin, vorinostat, doxorubicin, cisplatin, Gleevec, 5-fluorouracil, tadalafil, tadalafil, cisplatin, tadalafil ... The invention includes, but is not limited to, oxime, topotecan, belotecan, imatinib, floxuridine, gemcitabine, leuprolide, flutamide, zoledronic acid, methotrexate, camptothecin, hydroxyurea, streptozocin, valrubicin, retinoic acid, mechlorethamine, chlorambucil, busulfan, doxefoxime, mitomycin, prednisone, everolimus, mitoxantrone, levodopa, carbidopa, entacapone, tolcapone, dopamine agonists, donepezil, galantamine, rivastigmine, memantine, anticholinergics and amantadine.

[0023] The mass ratio of the nano-droplet membrane material to the solid drug is 1:(0.1-1).

[0024] The injection dose of nanodroplets in vivo is 1-20 x 10 9 / kg.

[0025] The neurodegenerative diseases and brain tumors described in the present invention can be at least one selected from the group consisting of: brain tumors, brain infections caused by bacteria or viruses, Parkinson's disease, encephalitis, stroke, paralysis, Alzheimer's disease, Lougerie's disease, Huntington's disease, Pick's disease, Creutzfeldt-Jakob disease, epilepsy, thrombosis, embolism, cerebral infarction, arteriolar occlusion and brain metabolic disorders, but are not limited thereto.

[0026] Compared with the prior art, the beneficial effects of the present invention are: 1) By combining microbubble technology, microbubble explosion at low activation energy can be achieved, thereby inducing the explosion of nanodroplets and releasing drugs; 2) Low activation energy ensures that the blood-brain barrier can be opened using an ultrasound imaging probe, and the strategy of increasing the mechanical index gradient provided by the present invention can make the blood-brain barrier open more safely and effectively.

[0027] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Schematic diagram of ultrasound intervention treatment using the present method in Example 1 and a top view and cross-sectional view of Evans blue entering the mouse brain DETAILED DESCRIPTION

[0029] The technical scheme in the embodiment of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiment of the present invention. The embodiments described in the present invention are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the art without making creative work are within the scope of protection of the present invention. Unless otherwise specified, the materials used in the following examples are all commercially available analytical pure.

[0030] The core gas prepared was perfluoropropane lipid microbubbles with an average particle size of 3 μm; the core liquid obtained was perfluoropentane, and the core contained doxorubicin hydrochloride nanodroplets, the average particle size of the nanodroplets was 432 nm, and the drug loading was 15%. In the following examples, we conducted experimental tests on the above microbubbles and nanodroplets.

[0031] Example 1 1) Balb / c-nude mice were used as intervention subjects, and the hippocampus region of the mouse brain was selected as the ultrasound irradiation window. The ultrasound imaging probe (L12-3) was placed close to the skin at the acoustic window, and an ultrasound coupling agent was applied to the skin contact surface to reduce acoustic attenuation. The imaging mode parameters were adjusted to focused wave and contrast mode through the ultrasound system (Verasonics vantage 256), the ultrasound frequency was 7MHz, and the mechanical index was 0.08, and the ultrasound image of the brain was obtained in the imaging interface; 2) Intravenous injection of a suspension of perfluoropropane lipid microbubbles and doxorubicin hydrochloride nanodroplets, with a microbubble injection dose of 3 x 10 7 / kg, the injection dose of nanodroplets is 1×10 10 / kg, the concentration of microbubbles and nanodroplets in the suspension was adjusted so that the injection volume of the suspension was 100 μL / mouse, and the changes in brain ultrasound signals were observed in the imaging interface; 3) When the ultrasound signal changes in the image and contrast agent is seen flowing into the section of the mouse hippocampus, the imaging parameters are adjusted to the stimulation mode, where the ultrasound waveform is a focused wave, the ultrasound frequency is 4 MHz, the mechanical index is variable, the range is from 0.4 to 1.6, and the trend is a step-by-step increase from small to large, the step is 0.2 / 10s, and the ultrasound mechanical index remains unchanged after reaching the peak value of 1.6. The ultrasound irradiation time is 5 min. Ultrasound irradiation is performed to open the blood-brain barrier by using the cavitation effect of microbubble explosion, and stimulate the explosion of nanodroplets and release drugs to cross the blood-brain barrier and enter the brain tissue.

[0032] Example 2 1) C57 mice were used as intervention subjects, and the hippocampus region of the mouse brain was selected as the ultrasound irradiation window. The ultrasound imaging probe (L12-3) was placed close to the skin at the acoustic window, and an ultrasound coupling agent was applied to the skin contact surface to reduce acoustic attenuation. The imaging mode parameters were adjusted to focused wave and contrast mode through the ultrasound system (Verasonics vantage 256), the ultrasound frequency was 7 MHz, and the mechanical index was 0.08, and the ultrasound image of the brain was obtained in the imaging interface; 2) Intravenous injection of a suspension of perfluoropropane lipid microbubbles and doxorubicin hydrochloride nanodroplets, with a microbubble injection dose of 3 x 10 7 / kg, the injection dose of nanodroplets is 1×10 10 / kg, the concentration of microbubbles and nanodroplets in the suspension was adjusted so that the injection volume of the suspension was 100 μL / mouse, and the changes in brain ultrasound signals were observed in the imaging interface; 3) When the ultrasound signal changes in the image and contrast agent is seen flowing into the section of the mouse hippocampus, the imaging parameters are adjusted to the stimulation mode, where the ultrasound waveform is a focused wave, the ultrasound frequency is 4 MHz, the mechanical index is variable, the range is from 0.4 to 1.6, and the trend is a step-by-step increase from small to large, the step is 0.2 / 10s, and the ultrasound mechanical index remains unchanged after reaching the peak value of 1.6. The ultrasound irradiation time is 5 min. Ultrasound irradiation is performed to open the blood-brain barrier by using the cavitation effect of microbubble explosion, and stimulate the explosion of nanodroplets and release drugs to cross the blood-brain barrier and enter the brain tissue.

[0033] Example 3 Refer to the method of Example 1, except that the step size of the ultrasonic stimulation mode is 0.4 / 10s.

[0034] Comparative Example 1 The method of Example 1 was used with the exception that only the microbubble suspension was injected into the mice.

[0035] Comparative Example 2 Refer to the method of Example 1, except that only the nanodroplet suspension is injected into the mice.

[0036] Comparative Example 3 The method of Example 1 was used with reference to the above, except that only microbubble suspension was injected into the mice, and the ultrasound stimulation mode was fixed mechanical index stimulation with a mechanical index of 1.6.

[0037] Test Case

[0038] 1) Comparative analysis of the penetration effect of Evans blue after blood-brain barrier opening This experiment uses Evans blue to evaluate the degree of opening of the blood-brain barrier using this method. Evans blue is a dye with a molecular weight of 961 Da. In scientific research, it is widely used to evaluate the degree of opening of the blood-brain barrier. This experiment uses Blab / c-nude mice. After intravenous injection of a suspension of microbubbles and nanodroplets, ultrasound irradiation is immediately performed to open the blood-brain barrier and release drugs. Evans blue solution (100 mg / kg) is immediately injected intravenously, and brain tissue is extracted 8 hours after injection. Cardiac perfusion is performed before extracting brain tissue to determine the effect of EB in the blood on the experimental results. The mouse brain is then placed in a paraformaldehyde solution for fixation, and the section obtained by ultrasound intervention is cut by a brain locator for analysis, and then the quantitative results of EB leakage are obtained by tissue homogenization.

[0039] 2) HE staining to determine bleeding The ultrasound section in test case 1 was stained with HE, and the red blood cell leakage in the section was observed under a fluorescence microscope and graded, where grade 0 indicated no red blood cell extravasation, grade 1 indicated a small amount of red blood cell extravasation but no neuronal loss, and grade 2 indicated severe red blood cell extravasation. Finally, the bleeding grade relationship corresponding to different ultrasound and microbubble parameters was obtained.

[0040] 3) IVIS characterizes drug delivery efficiency in the brain In order to characterize the drug release efficiency of this method in the brain, we used near-infrared dye DiD fluorescent dye as a model drug and then loaded it into the core of the nanodroplet. This experiment was conducted using Blab / c-nude mice. After intravenous injection of a suspension of microbubbles and nanodroplets containing DiD dye, they were immediately irradiated with ultrasound to open the blood-brain barrier and release the drug. 24 hours after ultrasound intervention, the heart, liver, spleen, lung, kidney, and brain organs were extracted and subjected to fluorescence photography using IVIS. By analyzing the fluorescence images and quantitatively analyzing the fluorescence intensity of each organ, the fluorescence intensity obtained by all organs was set as 1, and the brain drug delivery efficiency was calculated.

[0041] Table 1 Evans blue leakage quantitative results (μg / g) Bleeding safety grade assessment Analysis of drug delivery efficiency in the brain Example 1 278 0 15.5% Example 2 200 0 11.2% Example 3 326 1 18.6% Comparative Example 1 258 1 0 Comparative Example 2 86 0 2.3% Comparative Example 3 350 3 0

[0042] As can be seen from Table 1, compared with the prior art, the method of opening the blood-brain barrier and achieving drug delivery proposed in the present invention has better safety and higher drug delivery efficiency. Specifically, the use of imaging-level ultrasonic energy stimulation can induce microbubble explosion, open the blood-brain barrier, and induce the explosion of high-drug-loaded nanodroplets, thereby releasing the drug into the brain parenchyma. In addition, the gradually increasing step ultrasonic stimulation can cause the ultrasonic microbubbles to explode in batches, avoiding the intracerebral hemorrhage caused by the explosion of a large number of microbubbles, and has higher safety.

[0043] The preferred embodiments of the present invention are described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be regarded as the contents disclosed by the present invention and belong to the protection scope of the present invention.

Claims

1. A method for opening the blood-brain barrier and delivering drugs using microbubbles and nanodroplets, characterized in that: The following steps are involved: a) Select a part that is easy to transmit ultrasound as the ultrasound irradiation window, place the ultrasound imaging probe close to the skin at the acoustic window, apply ultrasound coupling agent on the skin contact surface to reduce acoustic attenuation, adjust the imaging parameters to the imaging mode through the ultrasound system, and obtain an ultrasound image of the brain in the imaging interface; b) injecting a suspension of microbubbles and nanodroplets intravenously and observing changes in brain ultrasound signals in the imaging interface; c) When the ultrasonic signal changes in the image, the imaging parameters are adjusted to the stimulation mode, and ultrasonic irradiation is performed to open the blood-brain barrier by using the cavitation effect of the microbubble explosion, and stimulate the explosion of nanodroplets and release drugs to pass through the blood-brain barrier and enter the brain tissue.

2. The ultrasonic irradiation acoustic window according to claim 1 comprises the temporal side of a normal skull, a skull drilling hole or the location of a craniotomy.

3. The imaging parameter according to claim 1, characterized in that: Its frequency range is 3.0-11 MHz, the range of mechanical index is 0.05-1.6, the imaging waveform is focused wave or plane wave, and the imaging mode is ordinary grayscale imaging or contrast imaging.

4. The imaging modality according to claim 1, characterized in that The mechanical index is 0.05-0.

2.

5. The microbubble according to claim 1, characterized in that The microbubbles are composed of a lipid shell and a gas encapsulated by the shell. The microbubbles are loaded with drugs that are gaseous or solid at room temperature and pressure and have therapeutic functions. The particle size of the microbubbles is 0.5-10 μm, preferably 0.8-5 μm.

6. The nanodroplet according to claim 1, characterized in that The nano droplets consist of a shell layer and a liquid encapsulated by the shell layer. The nano droplets are loaded with drugs that are solid at room temperature and pressure and have therapeutic functions. The particle size of the nano droplets is 0.1-0.9 μm, preferably 0.2-0.5 μm.

7. The stimulation modality according to claim 1, characterized in that The mechanical index is variable, ranging from 0.4 to 1.6, with a trend of increasing from small to large in steps, with a step size of 0.1 to 0.4 / 10 s. After the ultrasonic mechanical index reaches a peak value of 1.6, it remains unchanged.

8. The ultrasonic irradiation according to claim 1, characterized in that The ultrasonic irradiation time is 3-10 min.

9. The method of using microbubbles and nanodroplets for opening the blood-brain barrier and delivering drugs according to claim 1, characterized in that: This method can achieve drug delivery of multiple drugs to brain tissue and can be used to treat neurodegenerative diseases and brain tumors.

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