Patent Grant
11660342
This application claims priority to Korean Patent Application No. 10-2020-0033940 filed on Mar. 19, 2020, the disclosure of which is incorporated by reference herein in its entirety.
The present invention relates to a method for increasing the blood-brain barrier (BBB) permeability, and more particularly, to a method for increasing the blood-brain barrier permeability by opening the blood-brain barrier using a nitric oxide donor-carrying nanogenerator and a triggering stimulus to be applied thereto, and allowing a desired substance to be accumulated in the deep part of the brain.
The blood-brain barrier is a defense system existing in a living body and plays the role of preventing foreign materials such as bacteria and viruses, which have flowed in along the blood vessels, from penetrating into the deep part of the brain. Such a defense system is crucial for protecting the brain; however, there is also a problem that the defense system prevents an artificially administered functional drug from flowing into the brain. There are a method of directly administering a drug into the brain through a surgery and a method of injecting a drug into the cerebrospinal fluid; however, those methods have limitations in terms of high risk, high cost, and low level of convenience for the patient.
Many drugs have been developed for the treatment of various brain diseases; however, treatments using existing drug systems have significant limitations due to the blood-brain barrier. Therefore, there is a demand for a new composition or drug delivery system, which can deliver a drug in a non-invasive manner but does not interfere with the form or efficacy of the drug.
On the other hand, neurodegenerative diseases cause cognitive impairment and/or physical disability such as memory loss, dementia, and tremors, and these diseases are widely spreading among millions of people over the world. Deep brain stimulation (DBS) is one of clinical therapeutic methods effective for alleviating various neurological symptoms in Parkinson's disease, dystonia, or epilepsy by electrically stimulating the nerve system with a chronically transplanted electrode.
However, since such a method needs excising the deep part of the brain and transplanting an electrode, there is a problem that numerous side effects such as neuronal death, glial network disruption, and stroke may be induced during the course of operation.
Various alternative measures including optogenetic, magnetothermal, and magnetoelectric stimulators have been suggested; however, non-specific gene insertion and thermal damage to non-specific tissues are important problems that need to be solved.
Therefore, there has long been a demand for an on-demand, non-invasive, and wireless neural stimulation strategy for clinical treatment of neurodegenerative diseases.
An object of the present invention is to provide a method for increasing the blood-brain barrier permeability by opening the blood-brain barrier using a nitric oxide donor-carrying nanogenerator and a triggering stimulus to be applied thereto, and allowing a desired substance to be accumulated in the deep part of the brain.
Furthermore, it is an object of the invention to provide a method for increasing the blood-brain barrier permeability, which can alleviate degenerative brain disease symptoms, by delivering a triggering stimulus to a nanogenerator that has been delivered to the deep part of the brain and opening the Ca2+ influx channels of neurons (nerve cell) present around the nanogenerator through a piezoelectric effect of the nanogenerator.
In order to solve the problems described above, according to an aspect of the present invention, there is provided a method for increasing the blood-brain barrier permeability, the method including: (S1) a step of delivering a nanogenerator carrying nitric oxide (NO) donor to a site adjacent to the blood-brain barrier; (S2) a step of delivering a first triggering stimulus to the area where the nanogenerator has been delivered so as to release nitric oxide from the nanogenerator; and (S3) a step of allowing the released nitric oxide to activate matrix metallopeptidase-9 (MMP-9) and inducing the activated MMP-9 to weaken the tight junction between a cerebrovascular endothelial cell and another cerebrovascular endothelial cell.
At this time, the first triggering stimulus may be any one or more selected from the group consisting of ultrasound, light, heat, and glutathione (GSH).
Here, the ultrasound may be an ultrasound with a center frequency of 1.0 to 1.5 MHz and a duty cycle of 10% to 20%.
The nanogenerator may be a nanogenerator including barium titanate (BaTiO3) particles and a polydopamine layer covering the surface of the barium titanate particles.
The nitric oxide donor may be an ultrasound-responsive nitric oxide donor.
At this time, the ultrasound-responsive nitric oxide donor may be any one selected from the group consisting of N,N′-di-sec-butyl-N,N′-dinitroso-1,4-phenylenediamine (BNN6, C14H22N4O2), 1,3-bis-(2,4,6-trimethylphenyl)imidazolylidene nitric oxide (IMesNO), and mixtures of these.
In the step (S1), an active agent may be delivered to a site adjacent to the blood-brain barrier together with the nanogenerator.
At this time, the active agent may be a small molecule, a protein, a polysaccharide, a nucleic acid, a lipid, or a mixture of any two or more of these.
The active agent may be a pharmaceutically active agent, a diagnostically active agent, or a combination of these.
Here, the pharmaceutically active agent may be a drug, a neurotrophic factor or a growth factor for treating any one or more diseases selected from the group consisting of a neurodegenerative disease, a neuropsychiatric disease, a brain tumor, a traumatic brain injury and a stroke.
Meanwhile, the method for increasing the blood-brain barrier permeability according to the present invention may further include: (S4) a step of allowing the nanogenerator to be delivered to the deep part of the brain by passing through between a cerebrovascular endothelial cell and a cerebrovascular endothelial cell, the step being performed after the step (S3); and (S5) a step of delivering a second triggering stimulus to the nanogenerator that has been delivered to the deep part of the brain, so as to open the Ca2+ influx channels of neurons present around the nanogenerator through a piezoelectric effect of the nanogenerator.
At this time, the second triggering stimulus may be any one or more selected from the group consisting of ultrasound, sound, mechanical pressure, and resonance.
Furthermore, the method for increasing the blood-brain barrier permeability may be used for alleviating a degenerative brain disease.
According to the method for increasing the blood-brain barrier permeability of the present invention, the blood-brain barrier can be opened by using a nitric oxide donor-carrying nanogenerator and a triggering stimulus to be applied thereto, and thus a desired substance can be accumulated in the deep part of the brain.
As a result, the accumulated substance can be used as a drug, a neurotrophic factor or a growth factor for treating a disease such as a neurodegenerative disease, a neuropsychiatric disease, a brain tumor, a traumatic brain injury, a stroke or etc.
Furthermore, when the triggering stimulus is delivered to the nanogenerator that has been delivered to the deep part of the brain, and the Ca2+ influx channels of neurons present around the nanogenerator are opened through a piezoelectric effect of the nanogenerator, degenerative brain disease symptoms can be alleviated.
Specifically,
Specifically,
Hereinafter, embodiments of the present invention will be described in detail with reference to the attached drawings so that those ordinarily skilled in the art to which the present invention is pertained can easily carry out the invention. However, the present invention can be embodied in various different forms and is not intended to be limited to the embodiments and drawings described herein.
According to
According to embodiments of the present invention, by using a nanogenerator carrying a nitric oxide donor that includes nitric oxide (NO), which is a vasodilating gas, and a triggering stimulus to be applied thereto in order to activate MMP-9 (matrix metallopeptidase-9) with released nitric oxide, and weakening the tight function between a cerebrovascular endothelial cell and another cerebrovascular endothelial cell by the activated MMP-9, the blood-brain barrier can be opened, and a desired substance can be accumulated in the deep part of the brain. As a result, the accumulated substance can be used as a drug, a neurotrophic factor or a growth factor for treating a disease such as a neurodegenerative disease, a neuropsychiatric disease, a brain tumor, a traumatic brain injury, a stroke or etc.
At this time, the first triggering stimulus may be not only ultrasound but also any one or more selected from the group consisting of light, heat, and glutathione (GSH), and the first triggering stimulus is preferably ultrasound, and more preferably high-intensity focused ultrasound (HIFU).
The ultrasound may be an ultrasound with a center frequency of 1.0 to 1.5 MHz and a duty cycle of 10% to 20%. When the ranges of the center frequency and the duty cycle are less than the above-described value ranges, the nitric oxide release amount is not sufficient, and opening of the blood-brain barrier (BBB) may be limited. When the above-described ranges exceed the value ranges, there is a problem that hyperthermal damage caused by tissue burning and permanent BBB disruption may occur.
More specifically, the center frequency may be 1.5 MHz, the power may be 462.4 W/cm2, the duty cycle may be 10%, the exposure time may be 60 seconds, and the pulse repetition frequency may be 10 Hz.
The nanogenerator may be a nanogenerator including barium titanate (BaTiO3) particles and a polydopamine layer covering the surface of the barium titanate particles.
On the surface of the nanogenerator, a nitric oxide donor is supported, and later, detachment of nitric oxide from the nitric oxide donor can be achieved by the ultrasound delivered to the nanogenerator.
The nitric oxide donor is not particularly limited so long as it responds to the first triggering stimulus, and preferably, any ultrasound-responsive nitric oxide donor can all be used. The nitric oxide donor may be used as a permeation enhancer for enhancing the permeability of the inner wall of the brain blood vessels. Furthermore, the nitric oxide donor may be included in an amount effective for enhancing the permeability of the inner wall of the brain blood vessels.
The ultrasound-responsive nitric oxide donor may be more specifically any one selected from the group consisting of N,N′-di-sec-butyl-N,N′-dinitroso-1,4-phenylenediamine (BNN6, C14H22N4O2), 1,3-bis-(2,4,6-trimethylphenyl)imidazolylidene nitric oxide (IMesNO), and a mixture of these; however, the nitric oxide donor is not limited to these.
The following chemical reaction scheme discloses a method for synthesizing BNN6, which is an example of the ultrasound-responsive nitric oxide donor.
Meanwhile, in the step (S1), an active agent may be delivered together with the nanogenerator to a site adjacent to the blood-brain barrier.
In a case in which the nitric oxide donor is used in combination with the active agent, the nanogenerator may be administered according to the regimen of administration, for example, the duration of administration, of the active agent.
The brain blood vessels may be blood vessels that have strong tight junctions between vascular endothelial cells and vascular endothelial cells and do not allow or only partially allow substances to pass through the blood vessels. The above-mentioned blood vessels may form the blood-brain barrier (BBB).
The active agent may be a substance that is known to be unable to permeate, or is actually unable to permeate, through the inner membrane of the brain blood vessels. Furthermore, the active agent may be a substance that permeates through the inner membrane of the brain blood vessels only in a small amount and cannot be delivered in an effective amount in the absence of other permeation auxiliary agents.
The active agent may be a small molecule, a protein, a polysaccharide, a nucleic acid, a lipid, or a combination of these. The small molecule may be a non-polymer molecule. The molecule may be an organic compound molecule. The protein may be or may not be glycosylated. A glycoprotein may be, for example, erythropoietin (EPO). The protein may be a protein having a molecular weight of 500 Da to 1000 kDa, 500 Da to 500 kDa, 500 Da to 100 kDa, 500 Da to 500 kDa, 1 kDa to 500 kDa, 5 kDa to 500 kDa, 10 kDa to 500 kDa, or 20 kDa to 500 kDa. The polysaccharide may be dextran or starch. The nucleic acid may be a single-stranded or double-stranded polynucleotide. The nucleic acid may be siRNA, shRNA, miRNA, or an antisense oligonucleotide.
The nitric oxide donor-carrying nanogenerator and the active agent may be administered simultaneously or separately. The nanogenerator and the active agent may be administered as a single composition. This administration may be conducted such that the nanogenerator is administered first, followed by administration of the active agent. In this case, the active agent may be administered within 30 minutes, within 20 minutes, within 15 minutes, within 10 minutes, or within 5 minutes, after the administration of the nanogenerator. The active agent may be administered, for example, within 1 minute to 30 minutes, within 1 minute to 20 minutes, within 1 minute to 5 minutes, within 5 minutes to 15 minutes, within 5 minutes to 10 minutes, or within 3 minutes to 15 minutes, after the administration of the nanogenerator. The administration may be conducted such that the active agent is administered first, followed by administration of the nanogenerator. In this case, the nanogenerator may be administered within 30 minutes, within 20 minutes, within 15 minutes, within 10 minutes, or within 5 minutes, after the administration of the active agent. The nanogenerator may be administered within 1 minute to 30 minutes, within 1 minute to 20 minutes, within 1 minute to 5 minutes, within 5 minutes to 15 minutes, within 5 minutes to 10 minutes, or within 3 minutes to 15 minutes, after the administration of the active agent.
The active agent may be a pharmaceutically active agent, a diagnostically active agent, or a combination of these.
At this time, the pharmaceutically active agent may be a drug, a neurotrophic factor or a growth factor for treating any one disease selected from the group consisting of a neurodegenerative disease, a neuropsychiatric disease, a brain tumor, a traumatic brain injury and a stroke.
Meanwhile, the method for increasing the blood-brain barrier permeability according to the present invention may further include: (S4) a step of allowing the nanogenerator to be delivered to the deep part of the brain by passing through between a cerebrovascular endothelial cell and a cerebrovascular endothelial cell, the step being performed after the step (S3); and (S5) a step of delivering a second triggering stimulus to the nanogenerator that has been delivered to the deep part of the brain, so as to open the Ca2+ influx channels of neurons present around the nanogenerator through a piezoelectric effect of the nanogenerator.
At this time, the second triggering stimulus may be any one or more selected from the group consisting of ultrasound, sound, mechanical pressure, and resonance such as resonant frequency, and the second triggering stimulus may be preferably ultrasound, and more preferably high-intensity focused ultrasound (HIFU).
Here, the nitric oxide donor carried by the piezoelectric nanogenerator may be explained as a non-invasive wireless neural stimulator.
A piezoelectric material may be mechanically deformed and polarized in order to produce a direct current output under an external stimulation. At this time, the external stimulation may be preferably high-intensity focused ultrasound (HIFU) having deep penetrability and site-specificity.
As described above, nitric oxide that has been released due to a triggering stimulus (ultrasound or the like) temporarily induces destruction of the blood-brain barrier, and the systemically administered nanogenerator reaches to the deep part of the brain, electrically stimulates dopaminergic nerve in the living body, and induces fear memory against the triggering stimulus (ultrasound or the like). Ultimately, when the nanogenerator according to the present invention and a triggering stimulus (ultrasound or the like) to be applied thereto are used, non-invasive deep brain stimulation (DBS) can be achieved.
According to another aspect of the present invention, there is provided a method for delivering an active agent to an individual, the method including a step of administering a nitric oxide-carrying nanogenerator and the active agent to the individual.
With regard to the above-described method, the nanogenerator and the active agent are as described above.
The administration may be performed by methods that are known in the pertinent art. Regarding the administration, the nitric oxide-carrying nanogenerator and the active agent can be directly administered to an individual by any means through a route such as, for example, intravenous, intramuscular, oral, transdermal, transmucosal, intranasal, intratracheal, or subcutaneous administration. This administration may be conducted systemically or topically. The administration may involve applying the agents in a localized manner to a BBB site, for example, the brain, spinal cord, or retina.
The individual may be a mammal, for example, a human being, a cow, a horse, a pig, a dog, a sheep, a goat, or a cat. The individual may be an individual having a disease in the brain, spinal cord, or retina.
The administration may be conducted by administering a nanogenerator and an active agent respectively in an amount of 0.01 mg to 1,000 mg, for example, 0.1 mg to 500 mg, 0.1 mg to 100 mg, 0.1 mg to 50 mg, 0.1 mg to 25 mg, 1 mg to 1,000 mg, 1 mg to 500 mg, 1 mg to 100 mg, 1 mg to 50 mg, 1 mg to 25 mg, 5 mg to 1,000 mg, 5 mg to 500 mg, 5 mg to 100 mg, 5 mg to 50 mg, 5 mg to 25 mg, 10 mg to 1,000 mg, 10 mg to 500 mg, 10 mg to 100 mg, 10 mg to 50 mg, or 10 mg to 25 mg, per day for each individual.
The nanogenerator and the active agent may be administered simultaneously or separately. The nanogenerator and the active agent may be administered as a single composition or as separate compositions. The administration may be performed such that the nanogenerator is administered first, followed by administration of the active agent. In this case, the active agent may be administered within 30 minutes, within 20 minutes, within 15 minutes, within 10 minutes, or within 5 minutes, after the administration of the nanogenerator. The active agent may be administered, for example, within 1 minute to 30 minutes, within 1 minute to 20 minutes, within 1 minute to 5 minutes, within 5 minutes to 15 minutes, within 5 minutes to 10 minutes, or within 3 minutes to 15 minutes, after the administration of the nanogenerator. The administration may be conducted such that the active agent is administered first, followed by administration of the nanogenerator. In this case, the nanogenerator may be administered within 30 minutes, within 20 minutes, within 15 minutes, within 10 minutes, or within 5 minutes, after the administration of the active agent. The nanogenerator may be administered, for example, within 1 minute to 30 minutes, within 1 minute to 20 minutes, within 1 minute to 5 minutes, within 5 minutes to 15 minutes, within 5 minutes to 10 minutes, or within 3 minutes to 15 minutes, after the administration of the active agent.
The above-described method may be intended to diagnose the presence of a substance or a symptom, or to treat a symptom. The substance or symptom may be present in the brain, the retina, or the spinal cord.
The active agent may be a pharmaceutically active agent or a diagnostically active agent. The method may include administering a pharmaceutically or diagnostically acceptable carrier together.
The nitric oxide donor-carrying nanogenerator may be administered in an amount effective for enhancing the permeability of the blood-brain barrier.
According to another aspect of the present invention, there is provided a use of a nitric oxide donor-carrying nanogenerator for being used in a method for delivering an active agent to an individual. This use may be intended to deliver an active agent to an individual through the brain blood vessels.
Hereinafter, the present invention will be described in more detail by way of specific Examples. The following Examples are only for the purpose of illustrating the present invention and describe only that ultrasound is used as the first and second triggering stimuli. However, as described above, the first triggering stimulus can be light, heat, glutathione (GSH) or the like in addition to ultrasound, while the second triggering stimulus can be sound, mechanical pressure, resonance, or the like in addition to ultrasound, and the present invention is not intended to be limited by the following Examples.
1. Production of Ultrasound-Responsive Nanogenerator
The precondition for an on-demand, non-invasive wireless neuronal stimulator is that the stimulator should respond to ultrasound, preferably to HIFU (high-intensity focused ultrasound), and generate both nitric oxide and direct current output. BTNP-pDA-BNN6 that fully satisfies the above-described condition was produced by an oxidation polymerization step of gradually coating the surface of piezoelectric BTNP (BaTiO3 nanoparticles) with polydopamine; and a step of loading ultrasound-responsive BNN6 on the polydopamine coating layer (see
More particularly, piezoelectric BTNP having a size of 200 nm was selected as a starting substance. This BTNP exhibits high potential for producing output current inside a biological system; however, due to its hydrophobic properties, the clinical usage is limited, and the substance instantaneously aggregates under aqueous conditions (see
Subsequently, BNN6 as an ultrasound-responsive nitric oxide donor was prepared by synthesizing from N,N′-di-sec-butylamino-p-phenylenediamine (BPA), and synthesis of BNN6 was confirmed by 1H-NMR and an LC-MS (ESI+) analysis. In the 1H-NMR spectra, the secondary amine peaks disappearing at d=2.99 and 3.29 ppm show that two NO moieties replaced two hydrogen atoms in the p-phenylenediamine of BPA to form BNN6 (see
Furthermore, the LC-MS (ESI+) data showed that the molecular weight of the BNN6 thus synthesized was 279 g/mol, which suggested that the molecular weight was perfectly consistent with the chemical structure (see
In order to complete the production of a nanogenerator, BTNP-pDA and BNN6 were incubated together in a 10 wt % acetonitrile (ACN) solution so as to load water-insoluble BNN6 on the surface of polydopamine through π-π attraction as shown in
Subsequently, ultrasound-responsive NO release of the nanogenerator was monitored under physiological conditions (DPBS, pH 7.4, 37° C.). When ultrasound was absent, only a small overall amount (1.53 nmol/mg) of NO was released. In contrast, when ultrasound was irradiated, a larger overall amount of NO (74.5 nmol/mg) was released, and NO was released at a rate of 2.97 pmol/mg as the maximum instantaneous concentration per second (see
It was demonstrated that the nanogenerator produced according to the present invention maintained piezoelectric characteristics and has a potential of producing direct current output in response to ultrasound. From an X-ray diffraction (XRD) analysis, the crystallographic structure showed two different distinct peaks at 2θ≈45°, and this implies the presence of tetragonality after surface modification (see
2. Measurement of Electrochemical Behavior of Nanogenerator
The electrochemical behavior of the nanogenerator produced as described above was investigated, and the method developed by Ramya, et al was extended. A patch-clamp provided under physiological conditions was used for recording the electric potential generated by suspended ultrasound-responsive nanogenerator (see
3. Nerve Stimulation Using Nanogenerator
Extracorporeal ultrasound stimulation in the presence of the nanogenerator produced as described above was performed. A cytotoxicity test was performed in order to find the optimum ultrasound condition, and as a result, it was found that an ultrasound intensity higher than 0.8 W/cm2 affected the viability of differentiated SH-SY5Y neurons. Therefore, the cytotoxicity test was performed while the ultrasound intensity was maintained at 0.4 W/cm2 with a duty cycle of 50%. In order to investigate the extracorporeal nerve stimulation, the intracellular calcium dynamics irrespective of the presence of a nanogenerator was monitored. It was found from Fluo-4 AM fluorescence images that the nanogenerator (NG) can stimulate SH-SY5Y-derived neurons when ultrasound is applied (see
The intracellular calcium concentration plays an important role in the release of neurotransmitters and promotes gathering of ready-to-release vesicles. At the nerve endings, calcium induces release of neurotransmitters during the action potential period. An investigation was conducted to see whether the release of ultrasound-responsive neurotransmitters induced by calcium influx occurred, and after short irradiation of ultrasound, the concentration of dopamine, a neurotransmitter, in the medium was measured using ELISA. The concentration of dopamine increased significantly when the system was treated with both the nanogenerator and ultrasound, and this implies that the release of neurotransmitters from SH-SY5Y-derived dopaminergic neurons was accelerated (see
4. NO-Associated BBB Permeation of Nanogenerator
In order to evaluate the influence of NO on the BBB permeation in brain tissues, the nanogenerator in a volume of 80 mg/kg was intravenously administered to 8-week old female Balb/c mice, and then the mice were exposed to ultrasound (see
For an effective ultrasound treatment, the treated site was monitored in real time under ultrasonic guidance, using an ultrasonic imaging system-synchronized ultrasonication apparatus (see
Two hours after the treatment with injection and ultrasound treatment, the mice were victimized, and then the amount of the nanogenerator accumulated inside the brain was quantified. The brain was digested with aqua regia using an ICP-OES analysis, and then the barium ion concentration inside the brain tissue was measured.
The unwanted barium ion concentration detected from the mice in a saline-treated negative control group (Ctrl (−)) was subtracted from the respective values. It was discovered that the barium ion concentration dramatically increased only in Group D, and this implies that NO had permeated through the BBB, and the nanogenerator (NG) was accumulated (see
Meanwhile, the long-term in vivo removal and toxicity of the administered nanogenerator were additionally investigated. An ICP-OES analysis was conducted to measure the in vivo distribution and the in vivo residual amount of the nanogenerator. The barium ion concentrations in the brain tissue and other various tissues were noticeably reduced 60 days after the injection (see
5. Non-Invasive Ultrasound Deep Brain Stimulation
It was checked whether the electric potential generated by the nanogenerator upon irradiation with ultrasound could stimulate neurons in the body so as to change the behavior of awake mice.
1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced acute Parkinson's disease model mice were prepared, and their behavior was monitored according to the overall experiment timeline (see
Group II (MPTP-treated mice) exhibited a noticeably decreased freezing level while fear was induced, as compared to Group I. No significant different was observed while the mice acquired fear memory; however, among the MPTP-treated groups, Group IV and Group V exhibited significantly high freezing levels while fear was induced (see
In addition, the change in the tyrosine hydrolase (TH) level during treatment of brain fragments based on immunohistochemistry (IHC) was investigated (see
The above-given description is intended only for illustrating the present invention, and any person having ordinary skill in the art to which the present invention is pertained, will be able to understand that the present invention can be realized in any form that has been modified to the extent that the fundamental characteristics of the present invention are maintained. Therefore, the disclosed Examples and Experimental Examples should be considered not from a restrictive viewpoint but from an illustrative viewpoint. The scope of the present invention is disclosed not in the above-described embodiments but in the following claims, and it should be understood that any alterations made to an extent equivalent to the claims are included in the present invention.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10-2020-0033940 | Mar 2020 | KR | national |
| Number | Name | Date | Kind |
|---|---|---|---|
| 20050074506 | Natan et al. | Apr 2005 | A1 |
| 20140050718 | Thorne et al. | Feb 2014 | A1 |
| Number | Date | Country |
|---|---|---|
| 2003-523965 | Aug 2003 | JP |
| 2019-191518 | Oct 2019 | JP |
| 20190011615 | Feb 2019 | KR |
| 10 2020 0015251 | Feb 2020 | KR |
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| Number | Date | Country | |
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| 20210290762 A1 | Sep 2021 | US |
