A drug-loaded nanoparticle with reversible charge response to low-intensity ultrasound and its preparation method

Abstract

The present invention relates to the field of biomedical technologies, and provides a drug-loaded nanoparticle with low-intensity ultrasound response and reversible charge, and a preparation method thereof. The components include nanoscale titanium dioxide, a chemotherapeutic drug, and a shell material, and the shell material is prepared by treating a chitosan-polypyrrole-thioketal nanogel with a sodium hydroxide solution. The drug-loaded nanoparticle with low-intensity ultrasound response and reversible charge provided by the present invention undergoes charge inversion after entering the tumor microenvironment from the plasma and is absorbed by tumor cells, and a cascade reaction occurs to release the drug after being induced by ultrasound. It has good biocompatibility, tumor targeting, stimulus responsiveness, stability, and low toxicity, which is conducive to ultrasound-controlled release and precise drug delivery during the chemotherapy process, and improves the curative effect.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and more particularly, to a drug-loaded nanoparticle with reversible charge responsive to low-intensity ultrasound and a preparation method thereof. Background Art

[0002] In order to solve the long-term drug resistance problem of first-line chemotherapy drugs for cancer such as oxaliplatin (Oxa), drug-loaded nanoparticles targeting cancer cells, tumor microenvironment and immune system have become a research and development hotspot in the medical field. The ability of such nanocarriers to overcome biological barriers is related to particle size, charge and hydrophilic-hydrophobic properties, and mainly depends on the surface charge. Positively charged nanocarriers, such as traditional lipid nanoparticles (LNPs), may directly damage negatively charged cell membranes through electrostatic interaction, resulting in cytotoxicity to normal tissues; while neutral or negatively charged nanocarriers can avoid protein adsorption and reticuloendothelial system clearance, thus effectively accumulating at the tumor site. However, negatively charged nanocarriers will repel negatively charged cell membranes, which is not conducive to tumor penetration and cellular internalization. Therefore, an ideal drug-loaded nanoparticle should have the property of reversible charge, being negatively charged in blood and normal tissue interstitium, and positively charged in the tumor microenvironment.

[0003] The charge-reversible drug-loaded nanoparticles provided by the prior art are prepared by hydrophobic self-assembly of some stimulus-responsive charge-reversible components, and then the stimulus triggers protonation / deprotonation or the covalent bond cleavage of the main functional groups of the carrier to result in charge reversibility. Although the surface charge conversion can be achieved by protonation under acidic conditions, the carrier structure will be damaged due to the increase in hydrophilicity. And the chemical bond cleavage process triggered by the stimulus usually takes several hours to complete, and the drug-loaded nanoparticles may be cleared before the required stimulus triggering process, resulting in limited drug delivery and therapeutic effects.

[0004] After the drug-loaded nanoparticles overcome biological barriers and enter the tumor site, the means and efficiency of drug release become the key factors restricting the curative effect. Existing studies have developed a variety of stimulus-responsive drug release nanocarriers, and their release behaviors can be activated or accelerated by endogenous or exogenous stimuli at the tumor site. Compared with endogenous stimuli (such as pH, enzymes, etc.), exogenous stimuli can more precisely manipulate the structure and other physical properties of the nanocarrier. Ultrasound can penetrate deep tissues, provide non-invasive exogenous stimuli, and has the advantages of spatiotemporal controllability, promoting the release of drugs from drug-loaded nanoparticles through thermal effects or non-thermal effects. Although high-intensity focused ultrasound has significant effects in triggering drug release, there is a risk of damaging normal tissues, which will cause a series of complications. In contrast, although the thermal effect and mechanical effect of low-intensity ultrasound are poor, it reduces the risk of injury and is sufficient to activate the photosensitizer to continuously produce reactive oxygen species, achieving the effect of promoting drug release. Summary of the Invention

[0005] The present invention aims to overcome at least one of the above-mentioned deficiencies of the prior art, and provides a drug-loaded nanoparticle with reversible charge response to low-intensity ultrasound and a preparation method thereof, achieving the effects of stable circulation, tumor targeting and controllable drug release, and solving the problems of ultrasound-controlled release and precise drug delivery in cancer chemotherapy.

[0006] The technical solution adopted by the present invention is to provide a tumor-absorbable nanoparticle shell material with reversible charge. It is characterized in that the backbone of the charge-reversible nanoparticle shell material (R-NGs) is chitosan-polypyrrole polymer (CS-PPy), and the glucosamine residues of the chitosan-polypyrrole polymer are connected by ketothiol bonds (TK bonds). The charge-reversible nanoparticle shell material contains a pyrrole ring (Py) structure that releases protons. Due to the selective adsorption of OH - on the pyrrole ring, in the plasma microenvironment at pH 7.4, R-NGs carry negative charges; while in the tumor microenvironment at pH 6.5, the pyrrole ring is protonated and R-NGs carry positive charges, so that R-NGs realize a reversible and rapid conversion of surface charges induced by pH, providing biocompatibility and tumor targeting for the drug-loaded nanoparticles. The glucosamine residues of the R-NGs are connected by ketothiol bonds. The purpose is that when there is a high concentration of reactive oxygen species in the environment, the ketothiol bonds will be broken by the reactive oxygen species and the glucosamine residues will be separated from each other, so that the shell is lysed and the drugs carried by the R-NGs are released. Therefore, the TK bond provides stimulus responsiveness for the drug-loaded nanoparticles.

[0007] Further, the chemical formula of the R-NGs is , where n ∈ [500, 1000], x ∈ [30, 200].

[0008] Another object of the present invention is to provide a preparation method of the tumor-absorbable nanoparticle shell material with reversible charge, including the following steps:

[0009] S1: Prepare solutions of chitosan and ammonium persulfate respectively, mix them, add pyrrole solution, stir, precipitate and dry to obtain chitosan-polypyrrole polymer (CS-PPy);

[0010] S2: Dissolve the chitosan-polypyrrole polymer obtained in step S1, mix it with ketothiol, active esterification reagent, condensing agent and emulsifier, dissolve and stir to obtain chitosan-polypyrrole-ketothiol nanogel (CS-PPy-TK NGs);

[0011] S3: Centrifuge the chitosan-polypyrrole-ketothiol nanogel obtained in step S2 to collect the precipitate, dialyze to retain macromolecules with a molecular weight cut-off above 12,000 - 14,000, mix with sodium hydroxide solution and stir to obtain the charge-reversible nanoparticle shell material (R-NGs).

[0012] Further, in step S1, the molar ratio of pyrrole, ammonium persulfate, and chitosan monomer is 1: (0.4 - 0.6): (0.1 - 0.4).

[0013] Further, in step S2, the mass ratio of chitosan-polypyrrole polymer to ketothiol is 1: (0.4 - 0.6).

[0014] Further, in step S2, the active esterification reagent is N-hydroxysuccinimide, the condensing agent is 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and the emulsifier is sorbitan monooleate; the molar ratio of ketothiol, N-hydroxysuccinimide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and sorbitan monooleate is (0.2 - 0.3): (0.12 - 0.16): 1: (40 - 60).

[0015] Further, in step S3, the mass ratio of chitosan-polypyrrole-ketothiol nanogel to the sodium hydroxide solution is 1: (2 - 4). An appropriate concentration of sodium hydroxide solution can provide an appropriate number of OH- adsorbed on the pyrrole ring to ensure the charge conversion ability of R-NGs from pH 7.4 to pH 6.5; after treatment with excessive sodium hydroxide, the charge reversal ability of R-NGs will disappear.

[0016] Another object of the present invention is to provide a low-intensity ultrasound-responsive charge-reversible drug-loaded nanoparticle, the components of which include nanoscale titanium dioxide, a chemotherapeutic drug, and the tumor-absorbable charge-reversible nanoparticle shell material as described above. The tumor-absorbable charge-reversible nanoparticle shell material encapsulates the nanoscale titanium dioxide and the chemotherapeutic drug. Under plasma physiological pH conditions, the surface of R-NGs is negatively charged, thus showing good biocompatibility and reducing circulatory loss; while in the acidic tumor microenvironment, the surface charge of R-NGs converts to positive, which is conducive to tumor cell uptake and cellular internalization, thus achieving tumor targeting. The nanoscale titanium dioxide encapsulated in the drug-loaded nanoparticle generates reactive oxygen species under low-intensity ultrasound irradiation, causing the ketothiol bond in the shell structure of the drug-loaded nanoparticle to undergo oxidative cleavage, and the shell to fragment and release the drug, thus realizing an ultrasound-induced cascade reaction and achieving more precise and effective local drug release.

[0017] Further, the chemotherapeutic drug is one or more of doxorubicin, paclitaxel, oxaliplatin, or regorafenib.

[0018] Furthermore, the intensity of the low-intensity ultrasound applicable to the R-NGs does not exceed 1 W / cm 2 , 50% duty cycle, and 1 MHz, and the duration does not exceed 5 min. Currently developed ultrasound-responsive drug delivery systems mainly utilize the thermal effect and cavitation effect of ultrasound to achieve drug release. However, the utilization of the thermal effect requires high-intensity focused ultrasound energy, and the realization of the cavitation effect depends on the precise adjustment of parameters such as frequency and intensity. Compared with directly utilizing the thermal effect and cavitation effect of ultrasound, the drug release induced by reactive oxygen species generated by ultrasound is more accurate, easier to control, requires lower intensity, and causes less damage to normal tissues surrounding tumor tissues.

[0019] Compared with the prior art, the technical effects of the present invention are as follows:

[0020] The low-intensity ultrasound-responsive charge-reversible drug-loaded nanoparticles provided by the present invention undergo charge inversion after entering the tumor microenvironment from plasma and are absorbed by tumor cells, and a cascade reaction occurs after ultrasound induction to release drugs. They have good biocompatibility, tumor targeting, stimulus responsiveness, stability, and low toxicity, which are beneficial to ultrasound-controlled release and precise drug delivery during chemotherapy, and improve the curative effect. Description of the Drawings

[0021] Figure 1 A is a schematic diagram of the synthesis process of CS-PPy-TK NGs.

[0022] Figure 1 B is the infrared spectrogram of CS, CS-PPy, and CS-PPy-TK NGs.

[0023] Figure 1 C-E are electron micrographs and particle size analyses before and after trypsin degradation.

[0024] Figure 1 F is the Zeta potential of CS-PPy-TK NGs and R-NGs in pH 7.4 solution or pH 6.5 solution, respectively.

[0025] Figure 1 G is the protein resistance detection of CS-PPy-TK NGs and R-NGs.

[0026] Figure 2 A is a schematic diagram of the drug release principle of Oxa-R-NGs.

[0027] Figure 2 B is the quantitative determination of the level of reactive oxygen species generated under ultrasound irradiation for different times by fluorescence spectrophotometry.

[0028] Figure 2 C-F are electron micrographs and particle size analyses before and after ultrasound treatment.

[0029] Figure 2 G is the determination of the release ratio of Oxa by ultraviolet spectrophotometry. Detailed implementation manners

[0030] It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present application belongs.

[0031] It should be noted that the terms used herein are only for describing the specific implementation manners and are not intended to limit the exemplary embodiments according to the present application. As used herein, unless the context clearly indicates otherwise, the singular forms are also intended to include the plural forms. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0032] The present invention will be further described below in conjunction with specific examples. The following examples are only for explaining the present invention and do not constitute a limitation to the present invention. If the specific experimental conditions are not indicated in the examples, they are usually in accordance with conventional conditions or the conditions recommended by the reagent company; the reagents, consumables, etc. used in the following examples can be obtained from commercial sources without special instructions.

[0033] Examples

[0034] I. Synthesis of low-intensity ultrasound-responsive charge-reversible drug-loaded nanoparticles

[0035] 1. Synthesis of chitosan-polypyrrole polymer (CS-PPy)

[0036] Weigh 0.832 g of pyrrole (Py) and dissolve it in 40 mL of 1 mol / L hydrochloric acid to obtain a pyrrole solution. Weigh 1.416 g of ammonium persulfate and dissolve it in 20 mL of 1 mol / L hydrochloric acid. Weigh 0.4 g of chitosan and dissolve it in 40 mL of 0.1 mol / L acetic acid. Mix the chitosan solution with the ammonium persulfate solution. Under dark conditions, dropwise add the pyrrole solution to the above mixed solution at a dropping rate of 50 mL / h. After the dropping is completed, stir for 10 hours, add 1600 mL of ethanol to precipitate and purify the polymer, filter to obtain the precipitate, and dry it at 60 °C for 2 days to obtain chitosan-polypyrrole polymer (CS-PPy).

[0037] 2. Synthesis of chitosan-polypyrrole-thioketal nanogels (CS-PPy-TK NGs)

[0038] Weigh 200 mg of CS-PPy and dissolve it in 20 mL of 1 mol / L hydrochloric acid. Weigh 89.4 mg of thioxoketone carboxyl (TK), 28.6 mg of N-hydroxysuccinimide (NHS), and 96 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) respectively, and mix them with the CS-PPy solution. Weigh 5.16 g of sorbitan monooleate (Span-80), dissolve it in 200 mL of cyclohexane, and mix it with the above solution. Ultrasonically treat the mixture at low temperature for 10 minutes, and then stir it at room temperature for 10 hours in the dark to obtain chitosan-polypyrrole-thioxoketone nanogels (CS-PPy-TK NGs). The synthesis process is as Figure 1 shown in A.

[0039] 3. Synthesis of R-NGs with charge reversal and ultrasonic response properties

[0040] After centrifuging CS-PPy-TK NGs at 10000 rpm for 10 minutes, take the precipitate, disperse it in 200 mL of water, and dialyze it with a dialysis bag with a molecular weight cut-off of 12000 - 14000 for 3 days using 6 L of deionized water, changing the water at least 3 times a day. Mix 1 mL of CS-PPy-TK NGs (1 mg / mL) with a 4-fold concentration of sodium hydroxide (4 mg / mL) solution, and stir it in the dark for 24 h to obtain R-NGs.

[0041] 4. Synthesis of drug-loaded nanoparticles

[0042] Add nano-titanium dioxide (1 mg / mL) to R-NGs, ultrasonically dissolve it with an ultrasonic dissolver for 10 minutes, and then stir it for 10 minutes to obtain a gel mixture. Dissolve 1 mg of the gel mixture and 1 mg of the chemotherapeutic drug oxaliplatin (Oxa) in 1 mL of deionized water, stir it in the dark for 24 hours, and centrifuge it at 12000 rpm for 10 minutes to obtain drug-loaded nanoparticles Oxa-R-NGs.

[0043] II. Detection of physicochemical properties

[0044] 1. Product structure characterization: Fourier transform infrared spectroscopy was used to collect the infrared spectra of CS, CS-PPy, and CS-PPy-TK NGs, as Figure 1 shown in B: The peaks at approximately 1535 cm -1 , 1170 cm -1 and 940 cm -1 correspond to the C=C, C=N, and C-H bonds on the pyrrole (Py) ring, proving the successful connection of chitosan (CS). CS-PPy-TK NGs show a peak at 1690 cm -1A new peak at [specific location] is attributed to the amide bond (-CO-NH-), indicating successful crosslinking between the amino group of CS and the carboxyl group of TK.

[0045] 2. Evaluation of ultrasonic responsiveness: Scanning electron microscopy was used to characterize the morphology of R-NGs before and after ultrasonic treatment, and dynamic light scattering was used to measure the particle size of R-NGs before and after ultrasonic treatment; ② Analysis of electron microscopy images and particle sizes before and after ultrasonic treatment ( Figure 2 C-F): Before ultrasound, the morphology of NGs was uniform ( Figure 2 C), and the particle size distribution was relatively uniform, with an average diameter of 138.39 nm ( Figure 2 E). After ultrasonic irradiation, the morphology of R-NGs was uneven ( Figure 2 D), and the particle size distribution was uneven, ranging from 7.5 nm to 190.1 nm ( Figure 2 F). These changes indicate the ultrasonic responsiveness of R-NGs.

[0046] 3. Evaluation of biodegradability: Scanning electron microscopy was used to characterize the morphology of R-NGs before and after trypsin treatment, and dynamic light scattering was used to measure the particle size of R-NGs before and after trypsin treatment; ③ Analysis of electron microscopy images and particle sizes before and after trypsin degradation ( Figure 1 C-E): After 24 hours of trypsin treatment, the morphology of R-NGs was uneven ( Figure 1 D), and the particle size was uneven ( Figure 1 E), indicating that R-NGs can be gradually degraded in the physiological environment.

[0047] 4. Protein resistance detection: CS-PPy-TK NGs or R-NGs (1 mg / mL) and bovine serum albumin (BSA, 2 mg / mL) were respectively mixed with 1 mL of PBS solution at pH 7.4 and 6.5, and the protein resistance of NGs was measured at pH 7.4 and 6.5. The mixture was incubated at 37 °C for 2 hours, and then centrifuged at 3000 rpm for 5 minutes to obtain the supernatant. The concentration of adsorbed protein was measured using a BCA protein assay kit, and the absorbance was measured at 570 nm using a microplate reader. The results are as Figure 1 shown in G. At pH 7.4 and pH 6.5, the protein adsorption of CS-PPy-TK NGs was significantly higher than that of R-NGs (p < 0.001). It shows that R-NGs reduce protein adsorption in the physiological environment and have cyclic stability.

[0048] 5. Potential detection: CS-PPy-TK NGs and R-NGs (1 mg / mL) were respectively incubated in PBS solution at pH 7.4 or 6.5. After incubating for 10 s, the Zeta potential was measured. The results are as Figure 1As shown in Fig. F, when the pH value of the solution changes from 7.4 to 6.5 (entering the tumor microenvironment), the CS-PPy-TK NGs remain positively charged, while the charge of the R-NGs changes from -9.5 mV to +10.4 mV. This indicates that the R-NGs have the ability of rapid charge inversion.

[0049] 6. Reactive oxygen species release experiment: Using 2’, 7’-DCFH-DA as a probe, the level of reactive oxygen species was measured by fluorescence spectrophotometry. First, 0.01 mL of DCFH-DA was mixed with 0.04 mL of sodium hydroxide solution (0.01 mol / L) and incubated for 30 minutes in the dark to obtain DCFH. Then, 5 mL of PBS (25 mmol / L) was added to terminate the reaction. To compare the levels of reactive oxygen species produced by R-NGs under different ultrasonic irradiation times, the R-NGs solution was mixed with DCFH (10 μmol) at a ratio of 1:1. Then, the mixture was ultrasonically irradiated for 0, 1, 3, and 5 minutes respectively under the ultrasonic parameters of 1 W / cm2, 50% duty cycle, and 1 MHz. The mixed solution was centrifuged at 12,000 rpm for 10 minutes, and the supernatant was obtained for the quantification of reactive oxygen species using a microplate multi-functional enzyme-labeling instrument. The fluorescence spectrophotometry was used to quantitatively determine the levels of reactive oxygen species produced under different ultrasonic irradiation times. The results are as Figure 2 shown in Fig. B. With the extension of the ultrasonic irradiation time, the reactive oxygen species increased, and the fluorescence intensity of reactive oxygen species in the group irradiated ultrasonically for 5 minutes was significantly higher than that of other groups (p<0.001).

[0050] 7. Encapsulation efficiency detection: Prepare 10 mL of an Oxa solution with a concentration of 1 mg / mL, and dilute it into a series of standard solutions with concentrations of 0.016, 0.032, 0.063, 0.125, 0.25, and 0.5 mg / mL respectively to establish a standard absorbance standard curve of Oxa. According to the standard curve, the concentration of free Oxa in the collected supernatant was determined by ultraviolet spectrophotometry at 250 nm. The drug loading efficiency can be calculated using the following formula:

[0051] Drug loading efficiency (%) = [(total mass of added drug - mass of drug in supernatant) / total mass of NGs] * 100%.

[0052] Measured as above, the drug loading rate of the R-NGs was 14.8%.

[0053] 8. Drug release detection:

[0054] The drug release principle is as Figure 2 shown in Fig. A. Under ultrasonic irradiation, titanium dioxide adsorbed in the nanogel induces the production of reactive oxygen species in water, causing the disulfide bond contained in TK to be oxidized and broken, and the R-NG structure to be damaged, releasing Oxa.

[0055] Determine the in vitro release kinetics of Oxa-R-NGs under the conditions of pH 7.4, pH 6.5, and pH 6.5 (ultrasound +). 1 mg of R-NGs was dissolved in 1 mL of PBS at pH 7.4 or pH 6.5. After centrifugation (12,000 rpm), the supernatant was collected at different time points (0, 0.5, 1, 2, 4, 6, 10, 14, 18, 24 h), and the concentration was determined by ultraviolet spectrophotometry at 250 nm according to the standard curve. In the pH 6.5 (ultrasound +) group, the sample was sonicated (1 W / cm2, 50% duty cycle, 1 MHz) for 5 minutes at the 4-hour time point, centrifuged (12,000 rpm, 5 minutes), and the supernatant was collected and measured as described above.

[0056] The results are as Figure 2 shown in Figure G. Under the condition of pH 7.4, Oxa was slowly released, and the cumulative release rate at 24 h was only 21.5%. While at pH 6.5, the release rate of Oxa was higher, and the cumulative release rate at 24 h was 43.4% (p < 0.001). Under the condition of pH 6.5 combined with ultrasonic irradiation, the 24-hour cumulative release rate of Oxa increased to 77.2% (p < 0.001). These results indicate that Oxa-R-NGs can effectively release Oxa in the acidic tumor microenvironment.

[0057] Obviously, the above embodiments of the present invention are only examples for clearly illustrating the technical solutions of the present invention, and are not limitations on the specific implementation manners of the present invention. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the claims of the present invention shall be included within the protection scope of the claims of the present invention.

Claims

1. A low-intensity ultrasound-responsive, charge-reversible drug-loaded nanoparticle, characterized in that: The components include nano-sized titanium dioxide, chemotherapeutic drugs and charge-reversible nanoparticle shell materials, wherein the charge-reversible nanoparticle shell materials encapsulate the nano-sized titanium dioxide and the chemotherapeutic drugs; The method for preparing the charge reversible nanoparticle shell material comprises the following steps: S1: chitosan and ammonium persulfate are prepared into solutions respectively and then mixed, pyrrole solution is added, stirred, precipitated and dried to obtain chitosan-polypyrrole polymer; S2: dissolving the chitosan-polypyrrole polymer obtained in step S1, mixing with thioketal, active esterification agent, condensation agent and emulsifier, dissolving and stirring to obtain chitosan-polypyrrole-thioketal nanogel; S3: centrifuging the chitosan-polypyrrole-thioketal nanogel obtained in step S2 to obtain a precipitate, dialyzing macromolecules with a molecular weight cutoff of more than 12,000 to 14,000, mixing with a sodium hydroxide solution and stirring to obtain the charge-reversible nanoparticle shell material; In step S1, the molar ratio of pyrrole, ammonium persulfate and chitosan monomer is 1:(0.4-0.6):(0.1-0.4); In the step S2, the mass ratio of chitosan-polypyrrole polymer to thioketal is 1: (0.4-0.6).

2. The low-intensity ultrasound-responsive, charge-reversible drug-loaded nanoparticles according to claim 1, characterized in that: In the step S2, the active esterification agent is N-hydroxysuccinimide, the condensation agent is 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and the emulsifier is sorbitan monooleate; the molar ratio of thioketal, N-hydroxysuccinimide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and sorbitan monooleate is (0.2-0.3):(0.12-0.16): 1: (40-60).

3. The low-intensity ultrasound-responsive, charge-reversible drug-loaded nanoparticles according to claim 1, characterized in that: In the step S3, the mass ratio of the chitosan-polypyrrole-thioketal nanogel to the sodium hydroxide solution is 1:(2-4).

4. The low-intensity ultrasound-responsive and charge-reversible drug-loaded nanoparticles according to claim 1, characterized in that: The chemotherapy drugs are one or more of doxorubicin, paclitaxel, oxaliplatin or regorafenib.

5. The low-intensity ultrasound-responsive charge-reversible drug-loaded nanoparticles according to claim 1, characterized in that: The intensity of the applicable low-intensity ultrasound does not exceed 1W / cm 2 , 50% duty cycle and 1 MHz, with a duration not exceeding 5 min.

Images