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Back to Journal »International Journal of Nanomedicine» Volume 16

The blood-brain barrier is induced to open the brain delivery of curcumin through low-intensity ultrasound through lipid-PLGA nanobubbles

Authors: Yan Ying, Chen Ying, Liu Zhong, Cai Fei, Niu Wei, Song Li, Liang Hong, Su Zhong, Yu B, Yan Fei

Published on November 4, 2021, Volume 2021: 16 pages, 7433-7447 pages

DOI https://doi.org/10.2147/IJN.S327737

Single anonymous peer review

Editor approved for publication: Dr. Yan Shen

Yiran Yan,1,* Yan Chen,2,* Zhongxun Liu,1 Feiyan Cai,3 Wanting Niu,4,5 Liming Song,1 Haifeng Liang,1 Su Zhiwen,1 Boyu,1 Fei Yan6 1Department of Orthopedics, Zhujiang Hospital, South China Medical University, Guangzhou 510282, Guangdong; 2Department of Ultrasound Diagnostics, Zhujiang Hospital, Southern Medical University, Guangzhou 510282, Guangdong; 3Paul C. Lauterbur Biomedical Imaging Research Center, Institute of Biomedicine and Health Engineering, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, Guangdong; 4VA Boston Healthcare System, Boston, Massachusetts, 02130, USA; 5 Department of Orthopedics, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA; 6 Quantitative Engineering, Shenzhen Institute of Synthetic Biology, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences Key Laboratory of Biology, Shenzhen, Guangdong 518055, People’s Republic of China *These authors contributed equally to this work. Corresponding author: Fei Yan, Shenzhen Research Institute, Advanced Technology of the Chinese Academy of Sciences, Shenzhen, Guangdong Province, No. 1068 Xueyuan Avenue, Shenzhen University Town, Zip Code: 518055 Tel 86 755 86392284 Fax 86 755 96382299 Email [email protected] Boyu Orthopedics, Zhujiang Hospital, Southern Medical University, No.253 Industrial Avenue, Haizhu District, Guangzhou City, Guangdong Province, Post Code: 510280 Tel 86 20 67282573 Fax 86 20 61643010 Email [email protected ] Background: Parkinson's disease (PD) is a progressive neurodegenerative disease. Due to the existence of the blood-brain barrier (BBB), it is difficult for conventional drugs to act on the nucleus of diseased cells and exert its effect on inhibiting or delaying the progression of PD. Recent literature indicates that curcumin shows great potential for the treatment of PD. However, due to its poor druggability and low bioavailability through BBB, its application in the body is still difficult. Method: The melt crystallization method was used to improve the solubility of curcumin, and curcumin was wrapped in lipid-PLGA nanobubbles to prepare curcumin-loaded lipid-PLGA nanobubbles (Cur-NBs). A series of analysis methods were used to characterize the bubble size, zeta potential, ultrasound imaging capability and drug encapsulation efficiency of Cur-NBs. Low-intensity focused ultrasound (LIFU) combined with Cur-NB was used to open the blood-brain barrier and promote the delivery of curcumin to the deep brains of PD mice, and then conduct behavioral evaluation of the treatment effect. Results: The melt crystallization method increased the solubility of curcumin, which was 2627 times higher than pure curcumin. The resulting Cur-NB has a nanometer size of about 400 nm and shows excellent contrast imaging performance. After LIFU irradiates Cur-NBs under the optimized sound pressure, the curcumin drug wrapped in Cur-NBs can be effectively released, and the cumulative release rate reaches 30% within 6 hours. Importantly, the combination of Cur-NBs and LIFU can open the BBB and deliver curcumin locally to the deep brain nucleus. Compared with only Cur-NBs and LIFU group, it significantly improves curcumin in the Parkinson’s C57BL/6J mouse model. In the efficacy. Conclusion: In this work, we greatly improved the solubility of curcumin and developed Cur-NBs for brain delivery of curcumin against PD by combining with LIFU-mediated BBB. Cur-NBs provide a platform for the treatment of PD diseases or other central nervous system (CNS) diseases for these potential drugs that are difficult to cross the BBB. Keywords: nanobubbles, blood-brain barrier, low-intensity focused ultrasound, curcumin, Parkinson's disease

Parkinson’s disease (PD) is a chronic progressive neurodegenerative disease. Its pathological features are the death of dopaminergic neurons in the substantia nigra compact area (SNC) and the formation of inclusion bodies in neurons called Lewy bodies. It is mainly composed of α-synuclein (a-syn).1-3 Although endogenous dopamine is the current drug therapy for the treatment of PD, it has no neuroprotective effect, and continuous neuronal degeneration eventually leads to recurrence of symptoms. 4 Nowadays, multiple pathogenic mechanisms have been shown to be related to PD,5,6 and many emerging new therapies are developed for these pathogenic mechanisms. 7-9 However, many potential PD drugs have no or limited therapeutic effects due to their low druggability and poor permeability across the blood-brain barrier (BBB). 10-12 Therefore, there is an urgent need to find new strategies to improve their druggability And facilitate their brain delivery in the body, with minimal adverse effects on the central nervous system.

In recent years, the combination of microbubbles (MB) and transcranial low-intensity focused ultrasound (LIFU) has been developed as a promising strategy to overcome BBB and locally deliver drugs for central nervous system diseases. 13,14 In this technology, the low-frequency transducer can be transmitted to the skull and focus on specific brain regions or cell nuclei, inducing MBs circulating in these brain regions to produce cavitation, such as stable cavitation and inertial cavitation. 15 The former includes microfluidics, radiation forces and the latter involves microjets, shear forces and shock waves. 16 The combination of stable cavitation and inertial cavitation exerts a powerful mechanical effect on the cerebrovascular, resulting in a short and modifiable opening of the BBB. 17 Cell permeability of mannitol compared to other brain drug delivery strategies (such as endothelium), 18 receptor-mediated endocytosis/transcytosis 19 and nose-to-brain transmission, 20 LIFU-induced bl blood-brain barrier opening It has many obvious advantages, including non-invasive, local and efficient drug delivery, and is suitable for small and large molecule drugs. 21 In addition, some drugs can be coated on the surface of MBs through electrostatic interactions, or encapsulated in bubbles and shells through hydrophobic interactions. 22 In this way, the drug can be prevented from being degraded by exogenous enzymes and its druggability can be expanded, thereby greatly improving its usability. Traditionally, the particle size of MB ranges from 1 μm to 8 μm, and it cannot travel freely in the liver and spleen. 23 In addition, lipid-based MB has poor drug encapsulation efficiency due to its unilamellar lipid shell structure. 24 Recently, nano-sized (<1000 nm) nanobubbles have attracted the attention of researchers due to their unique advantages such as higher drug encapsulation efficiency, more stable and longer blood circulation. Therefore, it is necessary to develop a nanobubble with high drug encapsulation efficiency for LIFU-mediated BBB open treatment of neurodegenerative diseases.

Curcumin is a polyphenol compound isolated from the rhizomes of the Zingiberaceae plant. It has a variety of biological effects in anti-tumor, antibacterial, anti-oxidation and anti-inflammatory aspects. 25 Curcumin has been shown to have great potential in preventing many different types of cancer (including multiple myeloma) and colorectal cancer, pancreatic cancer, breast cancer, prostate cancer, lung cancer, head and neck cancer. 26 In addition, it is reported that curcumin can also be used as a neuroprotective agent for the treatment of neurological diseases. 27-29 The literature of different experimental PD models in vitro also shows that curcumin can effectively inhibit and remove a-syn, leading to the reduction of the toxicity of a-syn oligomers in cells. 30,31 In addition, it has been proven that curcumin is non-toxic even at high doses. However, curcumin has low bioavailability due to its poor solubility in aqueous solutions (20.0 μg/mL in water)32 and poor permeability to BBB. 33 Solid dispersion is a state in which fine particles are dispersed in a highly soluble carrier. It is used to improve solubility. 34 PEG-6K is a toxic and highly soluble reagent, which can be widely used for solid dispersion by melt crystallization. In order to solve the above problems, we used a melt crystallization method to improve the solubility of curcumin, and prepared curcumin-loaded lipid-PLGA hybrid nanobubbles (Cur-NBs) in this study. Due to the relatively hard shell and uniform size distribution of Cur-NBs compared with lipid bubbles, the stable cavitation of BBB opening and the drug release from the brain can be better controlled by adjusting the input of sound energy. The combination of Cur-NBs and LIFU is used to induce the opening of BBB and evaluate its efficacy on PD. Our research provides a new strategy that can be used to induce BBB opening by ultrasound to deliver curcumin to the brain of mice non-invasively, safely and locally to treat PD (Figure 1). Figure 1 Schematic diagram of Cur-NBs combined with LIFU non-invasive local delivery of curcumin for PD treatment.

Figure 1 Schematic diagram of Cur-NBs combined with LIFU non-invasive local delivery of curcumin for PD treatment.

50:50 Poly(lactide-co-glycolide) (PLGA), dichloromethane and ammonium bicarbonate were purchased from Jinan Daigang Biomaterials Co., Ltd. (Jinan, Shandong, China). 1,2-Distearoyl-sn-glycerol-3-phosphatidylcholine (DSPC) and 1,2-distearoyl-sn-glycerol-3-phosphoethanolamine-N-[methoxy (polyethylene Diol)-2000] (DSPE-PEG2000) was obtained from Avanti Polar Lipids Inc. (Alabaster, AL, USA). Polyvinyl alcohol (PVA, Mw, 30,000-70,000), ammonium bicarbonate ((NH4)HCO3), sodium pentobarbital, curcumin (Cur), Evans blue (EB) and 1-Methyl-4-phenyl- 1,2,3,6-Tetrahydropyridine (MPTP) was purchased from Sigma Aldrich (St Louis, MO, USA). Polyethylene glycol 6000 (PEG-6000) was purchased from Solarbio Co., Ltd. (Beijing, China). All other chemicals are of analytical grade and can be used without any further purification. Female C57BL6 mice aged 6-8 weeks (weighing about 20 g) were purchased from Guangdong Medical Laboratory Animal Center (Guangzhou, Guangdong Province, China).

The solid dispersion is prepared from curcumin and PEG-6000 at a weight ratio of 1:10, and is used for future research through the melt crystallization method. 35,36 In short, a mixture of curcumin and PEG-6000 is heated in a 65°C water bath until it initially reaches complete melting, while stirring for 0.5 hours to obtain a clear and homogeneous solution. The obtained homogeneous liquid is rapidly frozen in a centrifuge tube, soaked in liquid nitrogen for 5 minutes to form a PEG-modified curcumin solid dispersion (SD-Cur), and then the SD-Cur is collected and ground into a powder. The curcumin-PEG6k physical mixture (PM-Cur) is obtained by mixing equal amounts of curcumin and PEG-6000 in a mortar for 10 minutes. The samples are stored at 25°C before use.

In order to evaluate the release behavior of various mixed modes, 5 mL of pure curcumin, PM-Cur or SD-Cur samples were added to PBS with 5 mg curcumin and added to the dialysis membrane (MW 100000 Da, Yuanye, Shanghai, China) to face PBS and rotating at 37 °C at a speed of 50 rpm. At different time points (5, 15, 30, 60, 90, and 120 minutes), 1 mL of each sample solution was collected to pass through a microplate reader (Infinite M1000 PRO, Tecan, Männedorf, Switzerland) and 1 was then supplemented with mL of PBS. The released drug was separated by centrifugation at 4500g for 10 minutes, 100 μL of clear liquid was transferred to a 96-well plate, and the drug concentration was measured by a microplate reader. The cumulative release rate is calculated as follows:

Cur-NBs are prepared by slightly improving the double emulsion evaporation process. In short, first use the melt crystallization method to synthesize SD-Cur with a weight ratio of 1:10. Then, dissolve 55 mg of SD-Cur in 1 mL of double distilled water. After that, 60 mg of ammonium bicarbonate was added as an aqueous phase to the SD-Cur solution. At room temperature, 50 mg PLGA (50:50), 2 mg DSPC and 0.5 mg DSPE-PEG2000 were dissolved in 1 mL dichloromethane (DCM, Shanghai Lingfeng Chemical Reagent Co., Ltd., China). Add 0.2 mL of the freshly prepared water phase to the previous oil phase. The mixed solution was emulsified by ultrasonic treatment (130 W, Sonics and Materials, Newtown, CT, USA) for 2 minutes (40% amplitude, 30 ms on, 30 ms off), then iced water bath, and 5 mL 3.5% w/v The aqueous PVA solution was added to the initial emulsion and homogenized by a homogenizer (T25 digital, IKA, Staufen, Germany) at 7000 rpm for 6 minutes. Immediately after adding 8 mL of double distilled water, the resulting emulsion was homogenized at 7000 rpm for 6 minutes. The final suspension was then stirred at room temperature for 4 hours. The Cur-NB was then separated by centrifugation at 4000g for 10 minutes. The obtained nanobubbles were resuspended in double distilled water, rinsed 3 times, and freeze-dried for 48 hours. Finally, the sample was refilled with air and stored at 4°C.

A scanning electron microscope (SEM) (HITACHI SU8010, Hitachi, Tokyo, Japan) was used to confirm the morphology of Cur-NBs. The internal structure of Cur-NBs was characterized by a transmission electron microscope (TEM) (FEI Spirit T12, FEI Company, Hillsboro, OR, USA). In short, the prepared Cur-NBs were diluted with distilled water to an appropriate concentration for easy observation, and then dropped onto the TEM copper net. Then, 1% phosphotungstic acid solution was dropped on the sample for 2 minutes, and then washed with PBS on the filter paper. Evaporate the solvent of the sample, and dry the remaining solid nanobubbles overnight before TEM observation. Dynamic light scattering (DLS) (MS2000, Malvern Instruments Ltd., Malvern, Worcestershire, UK) was used to characterize the average diameter and size distribution of Cur-NB. In order to determine the fluorescence excitation spectrum change between curcumin itself and the Cur-NBs rehydration solution, an FS5 spectrofluorometer (Edinburgh Instruments, Edinburgh, UK) was used, and a confocal laser scanning microscope (A1R, Nikon, Tokyo) was used to observe Fluorescence imaging, Japan). In order to observe the floating behavior of nanobubbles, the resuspension solution of Cur-NBs was allowed to stand at room temperature for 5 hours, and a camera was used to record the floating behavior.

The serum is removed from the mouse blood by centrifugation to obtain a working solution of mouse red blood cells (RBC). After washing 3 times with PBS, the cells were diluted 10 times with PBS solution for further testing. Then, (a) prepare a negative control mixed with 0.2 mL of diluted RBC suspension in 0.8 mL of PBS, (b) also prepare a positive control mixed with 0.2 mL of diluted RBC suspension in 0.8 mL of deionized water, and (c) Mix 0.8 mL of Cur-NBs solution with various solution concentrations of 25, 50, 100, 200, 400, 800, 1600 μg/mL with 0.2 mL of diluted RBC suspension. The supernatant was obtained by centrifugation, and then the above mixture was incubated for 3 hours. According to the absorbance value detected by an ultraviolet-visible spectrophotometer (UV-2600, Shimadzu Corporation, Tokyo, Japan) at a wavelength of 450 nm, the percentage of hemolysis is calculated according to the following formula:

In order to evaluate the drug release behavior of LIFU nanobubbles, 1 mg/mL Cur-NBs were stimulated with various sound intensities of 0, 0.31, 0.38 and 0.45 MPa at 37 °C for 60 seconds. The LIFU system consists of a spherical transducer (1.0 MHz; focal length: 38.0 mm), a waveform generator (DG4000, Rigol, Beijing, China) and a 50 dB power amplifier (LZY-22, Mini-circuits, New York, USA). We separated the released drug from Cur-NB by centrifugation at 4500g for 10 minutes, and transferred 100 μL of the clear liquid to a 96-well plate, and measured the drug concentration with a microplate reader. Use Vevo2100 imaging system (Visual Sonics, Toronto, Ontario, Canada) to acquire real-time B-mode and contrast-mode images, and then observe the destruction of Cur-NBs under a scanning electron microscope.

In order to find the optimal sound pressure to induce stable cavitation and avoid inertial cavitation, we used the equipment setup shown in Figure 4D to study the sub-harmonic power spectrum generated by nanobubble cavitation. Passive cavitation detection (PCD) 37 type ultrasonic cavitation open BBB dedicated monitoring platform consists of waveform generator, power amplifier, preamplifier (type 600, Millar Instruments, Houston, Texas, USA), ultrasonic transducer Device (V310-SU, Olympus, New York, USA) and digital oscilloscope (DPO4034, Tektronix, Beaverton, Oregon, USA). The ultrasonic transmitting transducer is fixedly connected to the waveform generator and the power amplifier, and the ultrasonic receiving transducer is connected to the preamplifier and the digital oscilloscope. Before each test, 100 ul Cur-NBs solution was added to the membrane chamber containing 200 mL PBS. A 1 MHz transducer is used to emit ultrasonic waves to excite the Cur-NB, and another identical transducer placed at a relatively right angle is used to receive the acoustic spectrum signal.

The ultrasound imaging capabilities of Cur-NBs were tested by placing different concentrations of Cur-NBs (0, 0.0625, 0.125, 0.25, and 0.5 mg/ml) into 1.5% (w/v) agarose phantom wells. Then put the 18 MHz transducer connected to the Vevo 2100 imaging system against the side of the hole to obtain an image including B mode and contrast mode. The actual strength of the signal is calculated by the Vevo 2100 instrument software.

Before the in vivo ultrasound imaging experiment, the mouse body temperature was maintained at 37.0°C with a heating blanket. After the anesthesia was stabilized, the fur on the head was removed, and the skin was cut to expose the skull. Use a suitable micro drill (RWD Life Science, Shenzhen, Guangdong, China) to perform partial craniectomy in the area between bregma and lambda at a constant speed. The ultrasound probe is perpendicular to the plane of the resection area, and is fixed on the fixture to obtain better imaging after fine adjustments. Since the hair, skin and skull are partially removed, the surface imaging mode uses a 7 MHz ultrasound probe (Resona 7, Mindray, Shenzhen, Guangdong, China), and the contrast mode image and B mode image are immediately taken by the Resona 7 ultrasound system under the following settings Collection: The depth is 2.5 cm, the contrast mode gain is 65%, the B mode gain is 55%, the contrast harmonic (FCH) frequency is 5.6 MHz, and the dynamic range is 115 dB. With the help of a stereotaxic instrument (68030, RWD Life Science, Shenzhen, Guangdong, China), the deep parts of the brain irradiated by ultrasound are located below the projection surface of the dorsal and lateral cortex, including the striatum and substantia nigra. The settings of ultrasonic detection parameters, including position, plane and depth, remain unchanged throughout the imaging process.

In order to evaluate the ability of Cur-NBs combined with LIFU to open the brain blood barrier, the mouse head was depilated under 1% pentobarbital anesthesia, and then fixed on a commercial stereotaxic instrument to mark the location of the striatum in the mouse brain. Install the focused ultrasound transducer (1.0 MHz; focal length: 38 mm) connected to the waveform generator and power amplifier on the external fixation device and align it with the original target positioning mark. The LIFU pulse duration is about 200 ms and the pulse frequency is 1 Hz. After administering the mixed solution of Cur-NBs and Evans Blue through the tail vein, ultrasound irradiation with different sound pressures (0.24-0.45 MPa) was performed on the deep side of the brain through the above-mentioned fixed transducer. After 4 hours of ultrasound irradiation, the BBB opening was confirmed by observation of the tissue section with a fluorescence microscope.

The subacute mouse PD model was established by intravenous injection of MPTP every day for 7 days. Curcumin is delivered to Cur-NB via LIFU-mediated BBB opening once every two days. Conduct behavioral tests on day 7 and day 21.

Randomly divide C57BL/6 mice into 5 groups, each with 6 mice (healthy control, MPTP, LIFU/MPTP, Cur-NBs/MPTP and LIFU/Cur-NBs/MPTP), and perform subacute PD modeling plan. MPTP (40 mg/kg) was injected intravenously every day for 7 consecutive days. At the same time, for the Cur-NBs group, Cur-NBs solution was administered once every other day for a total of 6 times. For the LIFU group, ultrasound-induced BBB opening was performed every other day after Cur-NBs administration, a total of 6 times. Behavioural tests were performed on the 7th and 21st days after the first MPTP injection.

Behavioral testing is used to evaluate the relief of PD symptoms in C57BL/6 mice after various treatments on the 7th and 21st days. Apparatus, Huaibei, Anhui, China) After one training session, it was recorded at least 3 times at a speed of 25 rpm to evaluate the exercise ability and coordination of mice in all groups. Record the rod climbing test duration of each mouse from the top of the 50cm rod (1cm in diameter) to the bottom of the rod at least 3 times, and observe the neurogenic dyskinesia of each group of mice.

Using OriginLab 2018, the experimental data are expressed as mean ± standard error of the mean (SEM). Use SPSS Statistics software (* *P <0.01, ***P <0.001; ##P <0.01, ###P <0.001; ΔP <0.05; ns = not significant P> 0.05).

Curcumin has low solubility in physiological solutions, so it is difficult to use in most pharmaceutical preparations. 38 In order to improve the solubility of curcumin, we first obtained PEG-modified curcumin solid dispersion (SD-Cur) by manual grinding and melt crystallization (Figure 2A). After melting and crystallization, the appearance of an orange solid can be observed (Figure 2B). After melting and crystallization, the solubility of curcumin in PBS was significantly improved, showing a darker color compared with curcumin and curcumin-PEG6k physical mixture (PM-Cur) (Figure 2C). The solubility of curcumin in SD-Cur aqueous solution is 52.54%, 1.82 times higher than that of PM-Cur (28.9%), and 2627 times higher than that of pure curcumin, which greatly improves the solubility of pure curcumin. 0.02%) after 2 hours (Figure 2D). Compared with pure curcumin solution (emission wavelength: 495 nm), a slight red shift of the fluorescence emission band of SD-Cur product (emission wavelength: 550 nm) was observed (Figure 2E). Afterwards, the dissolved curcumin was used to make curcumin-loaded lipid-PLGA nanobubbles (Cur-NBs) by slightly modifying the double emulsion evaporation process. The resulting Cur-NBs are shown in Figure 2F. Obviously, the yellow Cur-NBs can float in the PBS solution, indicating that the Cur-NBs were successfully obtained (Figure 2G). Figure 2 Preparation of PEG-modified curcumin solid dispersion and Cur-NBs. (A) Schematic diagram of the preparation of Cur-PEG solid dispersion using the melt maximization method. (B) The obtained PEG-modified curcumin solid dispersion (SD-Cur). (C) Curcumin solution sample containing pure curcumin, curcumin-PEG6k physical mixture (PM-Cur) and PEG-modified curcumin solid dispersion (SD-Cur). (D) The percentage of curcumin dissolved in different formulations over time. (E) Shift of fluorescence emission wavelength between curcumin and SD-Cur. (F) Cur-NBs produced in freeze-dried powder. (G) The resulting Cur-NBs are resuspended in PBS.

Figure 2 Preparation of PEG-modified curcumin solid dispersion and Cur-NBs. (A) Schematic diagram of the preparation of Cur-PEG solid dispersion using the melt maximization method. (B) The obtained PEG-modified curcumin solid dispersion (SD-Cur). (C) Curcumin solution sample containing pure curcumin, curcumin-PEG6k physical mixture (PM-Cur) and PEG-modified curcumin solid dispersion (SD-Cur). (D) The percentage of curcumin dissolved in different formulations over time. (E) Shift of fluorescence emission wavelength between curcumin and SD-Cur. (F) Cur-NBs produced in freeze-dried powder. (G) The resulting Cur-NBs are resuspended in PBS.

A series of characterization methods were used to study the physical and chemical properties of Cur-NBs. SEM observed a spherical morphology with a porous structure, and TEM showed a hollow structure of Cur-NBs (Figure 3A and B). The encapsulation rate of Cur-NBs was 88.23%, while the drug loading of Cur-NBs was 1.76%. The size of Cur-NBs is 436 ± 58.3 nm (PDI: 0.31 ± 0.16) (Figure 3C), and the zeta potential of dynamic light scattering (DLS) analysis is -27.5 ± 0.6 mV. In addition, Cur-NBs can remain stable for at least 3 weeks because their size and zeta potential remain almost unchanged (Figure 3D). In addition, during the synthesis process, with the different ratios of PLGA and SD-Cur, the particle size and zeta potential data changed slightly. As shown in the fluorescence microscope image, green fluorescence can be observed in the inner cavity of Cur-NBs, which proves that curcumin is trapped in bubbles (Figure 3E). Hemolysis experiments confirmed the biocompatibility, indicating that Cur-NBs have excellent biocompatibility, even at a concentration of 1600 μg/mL (Figure 3F and G). Figure 3 Characterization of Cur-NB. (A) SEM of Cur-NBs after lyophilization. 500 nm (left scale bar), 250 nm (right scale bar). (B) TEM image of Cur-NBs. 100 nm (left scale bar), 200 nm (right scale bar). (C) DLS particle size distribution of Cur-NBs. (D) The zeta potential and size change of Cur-NBs measured within 3 weeks. (E) Observation of Cur-NBs under a confocal fluorescence microscope (from left to right: bright field image, shell and cavity containing curcumin fluorescence, merged). The scale bar is 200 nm. (F and G) The hemolysis rate of Cur-NBs at different concentrations (25, 50, 100, 200, 400, 800, 1600 μg/mL).

Figure 3 Characterization of Cur-NB. (A) SEM of Cur-NBs after lyophilization. 500 nm (left scale bar), 250 nm (right scale bar). (B) TEM image of Cur-NBs. 100 nm (left scale bar), 200 nm (right scale bar). (C) DLS particle size distribution of Cur-NBs. (D) The zeta potential and size change of Cur-NBs measured within 3 weeks. (E) Observation of Cur-NBs under a confocal fluorescence microscope (from left to right: bright field image, shell and cavity containing curcumin fluorescence, merged). The scale bar is 200 nm. (F and G) The hemolysis rate of Cur-NBs at different concentrations (25, 50, 100, 200, 400, 800, 1600 μg/mL).

To test the ultrasound-mediated bubble destruction ability, Cur-NBs were exposed to acoustic negative pressure of 0, 0.31, 0.38, or 0.45 MPa for 1 minute. The contrast signal was found to be significantly reduced in these ultrasound-irradiated Cur-NBs (Figure 4A, middle). SEM images showed obvious bubble destruction in Cur-NBs, which were accepted under 0.45 MPa acoustic exposure compared to 0.31 MPa (Figure 4A, bottom). The quantitative analysis of the acoustic signal intensity of Cur-NBs shows that the higher the acoustic negative pressure is used, the lower the acoustic signal will be (Figure 4B). In order to test the ability of ultrasound-mediated drug release, Cur-NBs were exposed to ultrasound irradiation under an acoustic negative pressure of 0.38 MPa. Compared with Cur-NBs that were not irradiated by ultrasound, irradiated Cur-NBs significantly improved the drug release efficiency (Figure 4C). Figure 4 Ultrasound-mediated destruction and drug release of Cur-NB. (A) The destruction of Cur-NBs under different sound intensities (0, 0.31, 0.38, 0.45 MPa) and the images evaluated in B mode, contrast mode and SEM. The scale bar is 250 μm. (B) Quantitative analysis of b-mode and contrast mode signal intensity under various sound intensities. *** P <0.001 vs 0 MPa group. (C) Time curve of curcumin release from Cur-NBs under natural conditions or under ultrasonic damage. Data are expressed as mean ± SD. (D) Schematic diagram of sub-harmonic detection settings. (E) The characteristics of sub-harmonics are detected under various sound intensities (0.31, 0.38 and 0.45 MPa).

Figure 4 Ultrasound-mediated destruction and drug release of Cur-NB. (A) The destruction of Cur-NBs under different sound intensities (0, 0.31, 0.38, 0.45 MPa) and the images evaluated in B mode, contrast mode and SEM. The scale bar is 250 μm. (B) Quantitative analysis of b-mode and contrast mode signal intensity under various sound intensities. *** P <0.001 vs 0 MPa group. (C) Time curve of curcumin release from Cur-NBs under natural conditions or under ultrasonic damage. Data are expressed as mean ± SD. (D) Schematic diagram of sub-harmonic detection settings. (E) The characteristics of sub-harmonics are detected under various sound intensities (0.31, 0.38 and 0.45 MPa).

The appearance of subharmonics and broadband noise is considered to be able to distinguish the characteristics of cavitation events (steady cavitation or inertial cavitation). In view of the fact that inertial cavitation is related to the emission of broadband noise, stable cavitation is evidenced by the radiation of fundamental frequency, higher harmonics and sometimes harmonic frequencies. 39 To further examine the ultrasonic energy of the acoustic spectrum of Cur-NB excited by different excitations, the design and construction of the passive cavitation detection (PCD) system is shown in Figure 4D. Here, a 1 MHz transducer is used to emit ultrasonic waves to excite the Cur-NB, and another identical transducer placed at a relatively right angle is used to receive the sound spectrum signal. Obviously, when the sound intensity increases from 0.31 MPa to 0.45 MPa, the peak amplitude of the sound spectrum of Cur-NBs increases with the increase of sound intensity. Interestingly, the broadband noise spectrum of Cur-NB at sub-harmonic frequencies is more obvious at 0.45 MPa, indicating that there are more bubbles with inertial cavitation (Figure 4E).

In order to determine the ultrasound imaging capabilities of Cur-NBs, we performed in vivo and in vitro imaging experiments. Various concentrations of Cur-NB from 0 to 0.5 mg/ml were added to the agar phantom wells and imaged by ultrasound. The results showed that the B-mode imaging signal and the ultrasound contrast signal gradually increased with the increase of bubble concentration (Figure 5A-C). In vivo imaging was also detected in the brains of mice whose skulls were partially removed to facilitate the transmission of ultrasound beams into the brain. The contrast-enhanced ultrasound echo signals of these Cur-NBs can be observed immediately after injection through the tail vein vein and can last for more than 3 minutes (Figure 5D and E). In contrast, injecting PBS in the same way cannot produce any contrast-enhanced signal. These results indicate that Cur-NBs have good in vitro and in vivo ultrasound contrast imaging capabilities. Figure 5 In vitro and in vivo acoustic imaging of Cur-NBs. (A) Ultrasound images of Cur-NB at different concentrations (0.0625 mg/ml, 0.125 mg/ml, 0.25 mg/ml, 0.5 mg/ml) in in vitro B mode and non-linear contrast mode. (B) Quantization of B-mode sound signal (n = 3). (C) Quantization of acoustic signals in nonlinear contrast mode (n = 3). (D) Acoustic signal of intracranial contrast imaging of Cur-NBs injected with PBS (white dotted line is the skull removed). (E) The time curve of the acoustic signal injected into Cur-NBs or PBS.

Figure 5 In vitro and in vivo acoustic imaging of Cur-NBs. (A) Ultrasound images of Cur-NB at different concentrations (0.0625 mg/ml, 0.125 mg/ml, 0.25 mg/ml, 0.5 mg/ml) in in vitro B mode and non-linear contrast mode. (B) Quantization of B-mode sound signal (n = 3). (C) Quantization of acoustic signals in nonlinear contrast mode (n = 3). (D) Acoustic signal of intracranial contrast imaging of Cur-NBs injected with PBS (white dotted line is the skull removed). (E) The time curve of the acoustic signal injected into Cur-NBs or PBS.

In order to determine whether Cur-NBs combined with LIFU can open the blood-brain barrier, adult male wild-type mice (C57/BL6J) were used and exposed to different acoustic pressure tail veins after intravenous injection of Cur-NBs and Evans blue (EB) ( Figure 6A). Figure 6B shows that Cur-NBs combined with ultrasound can effectively open the BBB under various appropriate sound pressures. When the sound pressure exceeds 0.31 MPa, significantly enhanced penetration is observed after the ultrasound-mediated BBB opening treatment (Figure 6C). In terms of quantity, when the BBB is opened at 0.45 MPa, the EB content is 2.04 and 11.24 times higher than that at 0.38 MPa and 0.31 MPa, respectively. Without ultrasound, Evans Blue cannot enter the brain. Similarly, the amount of curcumin delivered to the brain under 0.45 MPa sound pressure was also significantly higher than that of the 0.38 MPa and 0.31 MPa groups (Figure 6D). Interestingly, it was found that more curcumin was transported to deep brain regions, such as the cerebral cortex or striatum, at 0.45 MPa, which was 1.37 and 1.45 times higher than the 0.38 MPa and 0.31 MPa groups. Therefore, LIFU-induced BBB opening can deliver curcumin to the brain, including deep brain regions, and will be used to enhance curcumin's efficacy. Figure 6 BBB opening and drug permeability of Cur-NBs combined with LIFU. (A) Schematic diagram of the BBB opening of Cur-NBs combined with LIFU. (B) Cur-NBs combined with LIFU to evaluate BBB openings at various sound intensities (0, 0.24, 0.31, 0.38, and 0.45 MPa). (Cortex: cerebral cortex; striatum: striatum). The scale bar is 250 μm. (C and D) Quantitative analysis of the fluorescence intensity of each group of Evans blue and curcumin. **P <0.01 and ***P <0.001 vs 0 MPa group, ##P <0.01 and ###P <0.001 between the two groups.

Figure 6 Cur-NBs combined with LIFU's BBB opening and drug permeability. (A) Schematic diagram of the BBB opening of Cur-NBs combined with LIFU. (B) Cur-NBs combined with LIFU to evaluate BBB openings at various sound intensities (0, 0.24, 0.31, 0.38, and 0.45 MPa). (Cortex: cerebral cortex; striatum: striatum). The scale bar is 250 μm. (C and D) Quantitative analysis of the fluorescence intensity of each group of Evans blue and curcumin. **P <0.01 and ***P <0.001 vs 0 MPa group, ##P <0.01 and ###P <0.001 between the two groups.

Next, we further examined the therapeutic effect of PD. The subacute mouse PD model was established by intravenous injection of MPTP every day for 7 days. Curcumin is delivered to Cur-NB via LIFU-mediated BBB opening once every two days. The behavioral test was performed on the 7th and 21st days (Figure 7A). Our results showed that all the MPTP-treated mouse groups showed poor neuromuscular coordination, indicating that the retention time on the rotating rod was significantly reduced on day 7 and day 21 compared to the wide control group (Figure 7B). However, mice that received Cur-NBs LIFU stayed on the rotating rod much longer than mice that received only Cur-NBs, LIFU and PD model groups. In addition, compared with the retention time of 7 days, the mice treated with Cur-NBs LIFU stayed on the rotating rod significantly longer at 21 days. However, there was no significant difference in the rotating rod test between 7 days and 21 days in only the Cur-NBs, only LIFU and PD model groups. In addition, we used the pole climbing test to further evaluate the movement ability of the PD mouse. Compared with the wide control group, MPTP management resulted in a significant loss of agile exercise and prolonged their total trial time (Figure 7C). However, the Cur-NBs LIFU group mice showed a significant advantage in exercise ability, and the test time was shorter than that of the Cur-NBs, LIFU and PD model groups only. Therefore, our results indicate that curcumin can alleviate PD symptoms through LIFU-mediated BBB opening and the brain delivery of Cur-NBs. Figure 7 Behavior analysis. (A) Schematic diagram of the treatment plan of MPTP-induced Parkinson's disease mouse model (n = 6 per group). (B) Rotating rod test was performed on PD and healthy control mice on the 7th and 21st day after the first MPTP injection (n = 6 per group). (C) Pole-climbing test of PD and healthy control mice on the 7th and 21st day after the first MPTP injection (n=6 per group). **P <0.01 vs control group, ##P <0.01 vs MPTP group, ΔP <0.05 between the two groups, ns = not significant between the two groups (P> 0.05).

Figure 7 Behavior analysis. (A) Schematic diagram of the treatment plan of MPTP-induced Parkinson's disease mouse model (n = 6 per group). (B) Rotating rod test was performed on PD and healthy control mice on the 7th and 21st day after the first MPTP injection (n = 6 per group). (C) Pole-climbing test of PD and healthy control mice on the 7th and 21st day after the first MPTP injection (n=6 per group). **P <0.01 vs control group, ##P <0.01 vs MPTP group, ΔP <0.05 between the two groups, ns = not significant between the two groups (P> 0.05).

In recent years, with the development of polymer materials and nanoscience, nano-scale ultrasound contrast agents have become popular due to their longer circulation time and larger surface area for carrying drugs. 40,41 In this study, we prepared an ultrasound contrast agent loaded with curcumin. Lipid-PLGA nanobubbles (Cur-NBs) deliver curcumin to the brain for PD treatment through an improved double emulsion solvent evaporation method. Compared with traditional lipid microbubbles, our nanobubbles have some obvious advantages. First of all, Cur-NBs have nano-scale particle size and relatively uniform particle size distribution. This feature not only enables these bubbles to have a more stable drug formulation, but also enables them to have a more uniform response to US excitation. Secondly, the bubble shell is made of lipid-PLGA polymer, which has a harder shell structure than lipid bubbles, but softer than the shell of pure PLGA bubbles. 42 This will make it possible to integrate the advantages of lipid bubbles and PLGA bubbles. As shown in Figure 4 and Figure 5, our Cur-NB not only has excellent ultrasound contrast imaging capabilities, but also has the ability to release drugs triggered by US Good performance. At the same time, Cur-NBs showed better pressure resistance at medium and high acoustic energy, indicating that more stable cavitation rather than inertial cavitation occurs when they are exposed to US. Because stable cavitation is safer than inertial cavitation, it is valuable to them in the BBB opening. 43 It will not produce extreme physical reactions such as micro jets and shock waves, greatly reducing the damage to the brain caused by bubble cavitation. 44,45 In addition, we have prepared a PEG-modified curcumin solid dispersion that is encapsulated in the cavity of NBs by melt crystallization, which can prevent drug leakage. Compared with pure curcumin and PM-SD, with polyethylene glycol 6000 as a hydrophilic carrier, the water solubility of curcumin dispersed on SD-Cur is greatly improved. This will increase the availability of the drug, because soluble curcumin is more easily released from the cavity of NB than its lipid form.

Since the recognized mechanism of LIFU in the opening of the blood-brain barrier is mainly the cavitation effect, physical events such as microjets and blasting shock waves related to inertial cavitation may cause capillary damage or edema in fragile brain tissues, triggering a series of safety concerns Controversy about the impact of LIFU on brain tissue. 46 Steady-state cavitation means that cavitation bubbles expand or contract only at low sound pressure. This instantaneously expanded but unruptured bubble mechanically stretches the blood vessel wall of the brain tissue, thereby expanding the tight junctions of various microcirculation endothelial cells, increasing the membrane pores of endothelial cells, and inducing transcellular vomiting and vomiting. . 47 To detect the most suitable ultrasonic parameters, this study optimizes the acoustic energy parameters. Our research results found that some cracks are formed in the bubble shell of Cur-NBs, and the bubble structure does exist after LIFU irradiation. It is very different from the cavitation of lipid bubbles, in which it collapses into a large number of fragments. Therefore, when the lipid bubbles produce inertial cavitation, Cur-NB can avoid a large number of micro jets and shock waves under high sound pressure. 48 In addition, the better solubility of curcumin can also increase its bioavailability and reduce the risk of hazardous chemical reagents.

In our research, our results show that the combination of Cur-NBs and LIFU can selectively open the blood-brain barrier and deliver curcumin to deep brain regions with optimized acoustic parameters. In our mouse model of Parkinson's, curcumin may be significantly beneficial for delaying or reversing disease progression. 49 In the animal experiment shown above, the mice in the Cur-NBs LIFU group showed significantly better curative effects in the rotating rod and climbing rod tests. PD mice are better than Cur-NBs only, LIFU only and PD model groups, indicating that curcumin can alleviate PD symptoms through LIFU-mediated BBB open brain delivery.

Our research successfully synthesized Cur-NBs containing curcumin in the lipid-PLGA nanobubble cavity. The resulting Cur-NBs have excellent contrast imaging and can be used for LIFU-mediated BBB opening to deliver curcumin locally to diseased brain regions. Significantly improved efficacy was achieved in the Parkinson’s C57BL/6J mouse model. Our research provides a platform for these potential drugs that are difficult to cross the blood-brain barrier to treat PD or other central nervous system diseases.

PD, Parkinson's disease; BBB, blood-brain barrier; Cur-NBs, curcumin-loaded lipid-PLGA nanobubbles; LIFU, low-intensity focused ultrasound; NB, nanobubbles; CNS, central nervous system; SNC, substantia nigra compacts ; A-syn, α-synuclein; MB, microbubbles; PLGA, poly(lactide-co-glycolide); DSPC, 1,2-distearoyl-sn-glycero-3-phospholipid Acylcholine; DSPE-PEG2000, 1,2-Distearoyl-sn-glycerol-3-phosphoethanolamine-N-[Methoxy(polyethylene glycol)-2000]; (NH4)HCO3, ammonium bicarbonate ; PEG-6000, polyethylene glycol 6000; SD, solid dispersion; PM, physical mixture; SD-Cur, PEG-modified curcumin solid dispersion; PM-Cur, curcumin-PEG6k physical mixture; DCM, dichloro Methane; PVA, polyvinyl alcohol; SEM, scanning electron microscope; TEM, transmission electron microscope; DLS, dynamic light scattering; RBC, red blood cell; MPTP, 1-methyl-4-phenyl-1,2,3,6- Tetrahydropyridine; PCD, passive cavitation detection; EB, Evans blue.

All animal work was carried out in accordance with the "Experimental Animals-Guidelines for the Ethical Review of Animal Welfare" (National Standards of the People’s Republic of China, GB/T 35892-2018), and was approved by the Animal Welfare Committee of Shenzhen Research Institute (approved by the Ministry of Advanced Technology, Chinese Academy of Sciences) No.: SIAT-IRB-180208-YGS-YF-A0442). The female C57BL6 mice are housed in isolation pens that meet the national standards (experimental animals: environmental and pen facility requirements [GB 14925-2010]).

The author thanks the National Key Research and Development Program (2018YFC0115900, 2020YFA0908800), the National Natural Science Foundation of China (81871376, 81660381, 81371931, 819741323, 819741323) and the Guangdong Natural Science Foundation of China 81974132324 for their support of this research 201804010082), the Guangdong Province Cerebrovascular Disease Translational Research Innovation Platform , Shenzhen Science and Technology Innovation Committee (JCYJ20190812171820731 and ZDSYS201802061806314), Shenzhen Strategic Emerging Industry Project (CXZZ20198505) (CXZZ20198505), Shenzhen Science and Technology Planning Project (CXZZ20198505), 1804018051.

Yiran Yan and Yan Chen contributed equally to this work and shared the co-first author. Yiran Yan conducted experiments and wrote the manuscript. Liming Song, Haifeng Liang, and Zhiwen Su contributed to data interpretation and modified the experimental procedure. Liu Zhongxun and Niu Wanting conducted the synthesis and supporting technology of Cur-NBs. Cai Feiyan measured the sound pressure distribution. Yan Chen, Bo Yu, and Fei Yan proposed hypotheses, approved the data analysis, and revised the manuscript. All authors participated in data analysis, drafting or revising the article, finally approved the version to be published, agreed to the journal to be submitted, and agreed to be responsible for all aspects of the work.

The authors report no conflicts of interest in this work.

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