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A Novel Approach to Making the Gas-Filled Liposome Real: Based on the Interaction of Lipid with Free Nanobubble within the Solution Jilai Tian,† Fang Yang,*,†,‡ Huating Cui,† Ying Zhou,† Xiaobo Ruan,§ and Ning Gu*,†,‡ †

State Key Laboratory of Bioelectronics, Jiangsu Key Laboratory for Biomaterials and Devices, School of Biological Sciences & Medical Engineering, Southeast University, Nanjing 210096, China ‡ Collaborative Innovation Center of Suzhou Nano-Science and Technology, Suzhou Key Laboratory of Biomaterials and Technologies, Suzhou 215123, China § Xuzhou Central Hospital, Xuzhou 221009, China S Supporting Information *

ABSTRACT: Nanobubbles with a size less than 1 μm could make a promising application in ultrasound molecular imaging and drug delivery. However, the fabrication of stable gas encapsulation nanobubbles is still challenging. In this study, a novel method for preparation of lipid- encapsulated nanobubbles was reported. The dispersed phospholipid molecules in the prefabricated free nanobubbles solution can be assembled to form controllable stable lipid encapsulation gas-filled ultrasound-sensitive liposome (GU-Liposome). The optimized preparation parameters and formation mechanism of GU-Liposome were investigated in detail. Results showed that this type of GU-Liposome had mean diameter of 194.4 ± 6.6 nm and zeta potential of −25.2 ± 1.9 mV with layer by layer self-assembled lipid structure. The acoustic imaging analysis in vitro indicated that ultrasound imaging enhancement could be acquired by both perfusion imaging and accumulation imaging. The imaging enhancement level and duration time was related with the ratios of lipid to gas in the GU-Liposome structure. All in all, by this novel and controllable nanobubble construction technique, it will broaden the future theranostic applications of nanobubbles. KEYWORDS: nanobubbles, liposome, assembly, ultrasound imaging, theranostic shell of the bubbles, which is more flexible and highly sensitive to acoustic waves than the hard shells of the cross-linked or entangled polymers.4 Among them, lipid shell offers the excellent characters of readily expand, rupture, reseal, compress, buckle, or respread under ultrasound exposure. 3 The formulations of commercially available UCAs, such as Definity, Imagent, Sonovue, and Levovist are all mainly composited by lipids.5 However, almost all the commercial available UCAs have diameters on the micrometer scale and, thus, are restricted to just enhance the visibility of blood vessels and cannot enter surrounding tissues or cells.6 Besides, microsized bubbles often remain the short circulation half- life and are easily arrested by liver and spleen. Nanobubbles, with sizes less than 1 μm, may be expected to have some priorities in ultrasound molecular imaging. Through the enhanced permeability and retention (EPR) effects, nanobubbles could be transferred from vessels into surrounding tissues even cells to be potentially imaged by ultrasound after accumulation,7 which triggers researchers’ great interest to

1. INTRODUCTION Due to the advantages of freely utilizing, noninvasive, dynamic observing, real-time detection, and the high priority of biological safety without radio contamination, ultrasonic imaging has been considered as a promising diagnosis tool for a variety of diseases. Gas-filled bubbles could be conducive to acquiring enhanced ultrasound contrast image due to the strong backscattering and the unique nonlinear effects under exposure to ultrasound. Bubbles were first suggested to have the effects of contrast enhancement coming from the observation of echocardiography of the aortic root by Gramiak and Shah.1 Since then, gas-filled bubbles taken as ultrasonic contrast agents (UCAs) have been paid more and more attention and have progressively gained development in clinical use. Gas bubbles commonly comprise with gas core and stabilizing shells. The high molecular weight and low-solubility filling gas such as SF6 or C3F8 is selected as the gas component in majority of UCAs, which exhibits less prone to outward loss than air. Coating materials of bubbles are always composed of lipid, polymer and/or protein since all these materials are assured of the safety when intravenous administration.2,3 Phospholipids or proteins are often chosen as the thin soft © 2015 American Chemical Society

Received: August 25, 2015 Accepted: November 16, 2015 Published: November 16, 2015 26579

DOI: 10.1021/acsami.5b07778 ACS Appl. Mater. Interfaces 2015, 7, 26579−26584

Research Article

ACS Applied Materials & Interfaces

described the manufacturing method. Before being mixed with gas, the water was purified by ion-exchange resin and degassed by a vacuum pump system to obtained deionized and degassed water. First, free nanobubbles aqueous solutions were prepared as depicted in Figure 1a. The generation of free bubbles was realized by gas−liquid mixing pump (20WSC04D, Shanghai Daimler Machinery Equipment Co., Ltd., China) and SF6 was adopted as gas phase. Then, it was followed by the second step displayed in Figure 1b. ePC and DSPE-PEG2k (95:5, w/w) were dissolved in absolute ethyl alcohol, and then were subjected to drying under vacuum to form dry lipid films. After free nanobubbles solution was subsequently added into the dry lipid bottle, the lipid container was sealed and placed upside down immediately. It was left for hours to allow full assembly between the lipid molecules and free bubbles. Shaking was taken as a supplementary step to enhance the incubation performance. After that, the final uniform lipid nanobubble product was obtained by membrane filtration (0.8 μm) to obtain uniform nanobubble size. Degassed water rather than the free nanobubbles water was operated to prepare the conventional liposomes in parallel as controls. To optimize the ultrasound imaging enhancement of prepared GULiposomes, we also investigated the formulation screening. Based on the fixed total input amount of gas, the total lipids (ePC: DSPEPEG2k = 95:5, w/w) were set into four groups: 1.5, 3.0, 6.0, and 9.0 mg/mL. All of them were prepared in parallel. 2.3. Physicochemical Characterization of GU-Liposomes. Morphology of ultimately obtained GU-Liposomes was observed by transmission electron microscopy (TEM, JEM-2100, JEOL, Japan) and scanning electron microscopy (SEM, Hitachi S-4800, Japan). Samples were dispersed on amorphous carbon-coated copper grids followed by negative stained using 2% aqueous phosphotungstic acid (pH 7.3) for TEM analysis. For SEM analysis, a drop of sample solution was dispersed onto a 1 × 1 cm silicon wafer with sequential drying at room temperature. The size, size distribution, and zeta potential were tested by Zeta-sizer Nano ZS90 (Malvern Instrument, Ltd., Co., United Kingdom). To fully understand the lipid assembling process of the formation the GU-Liposomes, we determined phospholipids concentration during the preparation by using ammonium ferrothiocyanate method.26 Briefly, both ferric chloride hexahydrate (FeCl3·6H2O, 27.03 g) and ammonium thiocyanate (NH4SCN, 30.40 g) were dissolved in 1 L of deionized distilled water to be taken as color− substrate solution. Then through color reaction between lipid and substrate, the concentration of lipid was determined at 488 nm using UV/vis spectrophotometer (UV-3600, Shimadzu, Japan). 2.4. In Vitro Ultrasound Imaging Evaluation. For acoustic imaging evaluation of GU-Liposomes in vitro, a self-made agar phantom was used which was composited with 3% agar, 86% distilled degassed water, and 11% glycerol. A silicone tube was embedded in the gel phantom to mimic the vascular structure and to load samples. The test samples were imaged by using VisualSonics microimaging Vevo 2100 systems (FUJIFILM VisualSonics, Inc.) and a transducer of MS250 was employed. Frequency was set at 18 MHz and the acquisition contrast gain was 30 dB. All parameters were not changed throughout all imaging acquisition. Before sampling, degassed water was injected and scanned to confirm a clear background signal. Then GU-Liposomes were bolus injected into the silicone tube to be imaged. The mean power intensity in B-mode were analyzed in the Region of Interest (ROI).

develop nanoscaled bubbles for early diagnosis of extravascular lesions. An alternative consideration of the nanobubbles is their synchronously potentiality as drug vectors in regions of interests. In recent years, in order to overcome a challenge of low drug-loading amount in the bubbles, researchers have conducted significant work,8−11 such as lipospheres,12 liposome-loaded microbubbles,13 bubble liposome,14 acoustic liposome,15 nested liposome (SHERPAs),16 pluronic nanobubbles,17 lung surfactant microbubbles,18 and eliposome.19 However, either the carrier structure or manufacturing method is so complicated that it is needed further improvement. Traditionally, methods of sonication, high shear emulsification, membrane emulsification, and so on20 have been applied to prepare microscaled bubbles. Thus, to explore the operational novel preparation method of encapsulated nanobubbles has become a continuous improvement goal. On the other hand, conclusive evidence had demonstrated that free gas nanobubbles were stable in the bulk water21,22 or on the surface.23,24 Therefore, in this study, based on the preprepared free gas nanobubbles aqueous solutions,25 we developed a novel and controllable preparation technique to make lipid encapsulated nanobubbles. Results demonstrated that the structure of about 200 nm diameter nanobubbles was multilayer lipid encapsulation due to lipid assembly on the surface of free bubbles. Due to the gas core and the multilayer lipid loading capability, such gas-filled ultrasound-sensitive liposome (GU-Liposome) would be beneficial for extravascular ultrasound imaging and drug delivery in the future.

2. MATERIALS AND METHODS 2.1. Materials. Egg phosphatidylcholine (ePC; PC-98T, CPC > 98%, injection grade) was obtained from Shanghai Advanced Vehicle Technology Pharmaceutical Ltd., Co. (AVT, Shanghai, China). Poly(ethylene glycol-2000)- grafted distearoylphosphatidylethanolamine (DSPE-PEG2k, purity >99%) was from Southeast Pharmaceuticals Co., Ltd. (Suzhou, China). Sulfur hexafluoride (SF6) with the purity of 99.99% was purchased from Anhui Qiangyuan Gas Co., Ltd. (Wuhu, China). All other solvents and reagents were analytical purity. 2.2. Preparation and Formulation Screening of GU-Liposomes. GU-Liposomes were constructed by self-assembled lipid molecular on the surface the free SF6 gas nanobubbles. Figure 1

Figure 1. Schematic diagram of manufacturing method of GULiposomes based on free nanobubbles aqueous solutions. (a) First, free nanobubbles aqueous solutions were prepared: (A) water tank, (B) SF6 gas reservoir, (C) pump, (D) gas−liquid separator, (E) nanobubbles aqueous solutions tank, and (F) the obtained nanobubbles aqueous solutions. (b) Illustration of preparing method of GU-Liposomes from (F) the obtained nanobubbles aqueous solutions; (G) dry mixture of ePC and DSPE-PEG2k (95:5, w/w). After (F) filling, the container is immediately sealed and placed upside-down and allowed to sit for hours to incubate fully. Then, (H) the final GULiposomes were obtained.

3. RESULTS AND DISCUSSION 3.1. Morphological Characterization of GU-Liposomes. TEM and SEM images of GU-Liposomes structure were displayed in Figure 2a−c. Figure 2a showed the mean size of GU-Liposomes was 194.4 ± 6.6 nm after membrane filtration (0.8 μm). The size distribution became narrower after membrane filtration. The zeta potential of GU-Liposomes was −25.2 ± 1.9 mV. Figure 2b indicated the spherical shape of the GU-Liposomes. Conventionally, SF6 as a hydrophobic gas, is 26580


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be seen from the picture at 8 h. Remarkably, at the time point of 12 h, substantial fragments of the lipid banded assembly was prone to be adsorbed on the membrane surface of a bubble attributed to saturated gas/liquid interface energy.29 While in the control group (Figure S2), a mass of assembled particles could be seen at 2 h, which were about 20−30 nm in size and no significant morphological change at 5 h. Until incubation for 8 h, because a huge amount of phospholipids were swelled into solutions, lipid molecules could be assembled into a banded bilayer structure. For the degassed water group, because no gas bubbles or no gas/liquid interface existed as free nanobubbles solution, the lipid tubular micelles or small granular aggregates were spontaneously converted into spherical vesicles, as reported before.30,31 Further, to fully understand the formation mechanism of GU-Liposomes based on the layer by layer process which had been displayed in Figure 3a, we measured phospholipids Figure 2. Morphology of the obtained GU-Liposomes structure. (a) Size distribution of lipid nanobubbles before and after the membrane filtration (0.8 μm), taking lipid formulation group of 1.5 mg/mL for example. Narrower distribution was observed after the membrane filtration. (b) SEM and (c) TEM images of GU-Liposome and (d) the typical enlarged TEM image of GU-Liposomes, in which the multilayer of bubble can be seen clearly.

often encapsulated by the monolayer lipids; however, multilayer structures with vacant core were found for the GU-Liposomes, which can be seen clearly in the enlarged TEM image shown in Figure 2 b. 3.2. Mechanism of GU-Liposomes Formation. It was reported that free nanobubbles with a respective diameter of less than 200 nm had been revealed the interface of hydrogen bonds. The hydrogen atoms pointing toward the water phase and oxygen atoms toward the gas phase. Thus, the negatively charged surface can remain nanobubbles for months,27,28 Whereas the free gas microbubbles or macrobubbles were unstable, tending to shrink, disappear, or coalesce and burst. On this basis, the GU-Liposomes were prepared by assembling the lipid molecules on the surface of prefabricated free SF6 nanobubbles by using gas−liquid mixing pump method. The existence and stability of SF6 free bubbles in the deionized and degassed water were observed without incubation with dry lipids film. The results are displayed in Figure S1, and indicate that the SF6 free nanobubbles formed in the water and can maintain enough stability for the following incubation. To reveal the self-assembling process and mechanism between the preprepared free nanobubbles and lipid molecules, we monitored the morphological transformation during the assembly process using TEM. Samples were taken at time point of 2, 5, 8, and 12 h during the reaction. The results shown in Figure S2 display the morphology change when the free bubbles or degassed water incubation with dry lipid at different reaction time. For GU-Liposomes shown in Figure S2, it could be obviously found that there are several air gaps in an integrated phospholipid particle at 2 h. The reason may be that the existing free SF6 nanobubbles with few incomplete lipid molecule encapsulation can be deformed or fractured during the vacuum process of the TEM observation. At 5 h, some separated gaps disappeared, and in turn, a complete hollow core encapsulated in lipid layers could be seen. As time went on, a mass of phospholipids was swelling and dispersing into free bubble solution to form banded bilayer structure, which could

Figure 3. Illustration of the formation process of GU-Liposomes through (a) TEM images at different reaction time points (2, 5, 8, 12 h). The scale bars represent 100 nm. (b) Measurement the total phospholipids in the water solution at different incubation times; 200 μL of each samples were taken after separation of the large lipids that dispersed incompletely through separation (n = 3). (c) Schematic of the hypothetical formation; (blue) gas core and (red) phospholipids. PC molecule was dispersed into gas bubbles interface, and then, several bubbles would coalesce into a whole encapsulated bubble. As time went on, lots of dispersed phospholipids would be assembled into banded bilayer fracture, and then, the fracture would be absorbed into the shell surface. As the processes repeated, the multilayer lipid encapsulated bubbles would be fabricated.

concentration of each sample at different times (0.25, 0.6, 1, 2, 5, 8, 12 h), and the results are shown in Figure 3b. Generally, the dispersed lipid concentration in the free bubble solution was higher than in the degassed water. Owing to the existing gas core, once the dry lipid was transferred into solution, the dispersed free lipid molecules tended to be adsorbed on the gas/liquid interface, which ensured the concentration gradient to enhance dry lipid to enter into the solution much faster. However, for the degassed water, only the lipid concentration 26581


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Figure 4. Results of (a) mean diameter, (b) the polydispersity index, and (c) zeta potential of different formulations where the filling gas was fixed and total lipid concentrations were set at 1.5, 3.0, 6.0, and 9.0 mg/mL.

Figure 5. Ultrasonic evaluation in vitro of GU-Liposomes. Total lipids (ePC: DSPE-PEG2k = 95:5, w/w) were selected as 1.5, 3.0, 6.0, 9.0 mg/mL, while the inlet gas was fixed. (a) Schematic of experimental apparatus. (b) The beginning perfusion images of the four groups. (c) The terminative aggregation images, where the enclosed upper pipe section provided the ROI, whose mean power was recorded quantitatively in panels e and f. (d) The B-mode mean power change of selected upper whole pipe section, and (g) the bubbles gathering duration time of the ROI.

constant (6.02 × 1023), and Ao is the surface area of one PC molecule (0.65 nm2). Thus, if all bubbles are encapsulated by one-layer lipid molecules, then the calculated total lipid molar amount in the whole solution should be 10.79 μmol. The experimental results shown in Table S1 indicate that the lipid concentration after reacting for 5 h was 10.09 μmol. That means at this time point, most of the one-layer bubbles come into being. After that, excessive phospholipid into the solution is bound to be assembled to form multilayer structures. This process is indeed confirmed by TEM images in Figure S2. Thus, according to the aforementioned theoretical and experimental results, the GU-Liposome formation can be illuminated as Figure 3c. First, once PC molecules were dissolved into the solution, the free bubbles may immediately

was above the lowest micelle concentration,32 and he conventional liposomes could be formed, which needed longer time and a greater amount of lipid molecules. Therefore, it seemed that the pre-existing gas bubbles in water could trigger the lipid molecules more easily to transfer into bulk solution phase and form nanobubble structure. Theoretically, the total lipid molar amount (MPC) for one layer bubbles can be calculated as eq 1 (the detailed derivation process is shown in Supporting Information): MPC =

3Vgas r × A 0 × NA

(1)

where Vgas is the total amount of input gas in a vial (0.1413 mL), r is the mean radius (about 100 nm), NA is Avogadro’s 26582


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the drug loading capability of the bubbles. The about 200 nm diameter would endow bubbles with excellent nanoscaled properties, such as long circulation time and desirable extravasation. The formulation screening further optimized the ultrasonic imaging enhancement of GU-Liposomes. This preparation technique is feasible and controllable to construct lipid encapsulated nanobubbles, which makes a promising platform for future theranostics of ultrasound molecular imaging and therapy.

recruit them to the gas/liquid interface until taking shape of one layer bubble structure. As time went on, the second lipid molecules then can be adsorbed on the surface to form banded bilayer structure. Layer by layer, the stable multilayer lipid encapsulated bubbles would finally be constructed by controlling the incubation time and lipid concentration. 3.3. Formulation Screening and Echogenic Properties of GU-Liposomes in Vitro. The shell structure can be controlled by lipid concentration, which, in turn, will influence the acoustic characteristics due to different surface properties. Lipid amounts of 1.5, 3.0, 6.0, and 9.0 mg/mL are equal to 26, 52, 104, and 156 μmol, respectively. According to the calculation of eq 1, the total lipid molar amount for one-layer bubbles is 10.79 μmol. Theoretically, there should be appropriate 2, 4, 9, and 14 layers for these four formulations accordingly, which may result in the increased mean size shown in Figure 4. The group of 6.0 mg/mL possessed the highest value in size and zeta potential, which was 194.4 ± 6.6 nm and −25.2 ± 1.9 mV. For the fourth group (9.0 mg/mL), the mean size decreased a little bit. The reason maybe that the excess lipid amount was forced the formation of some conventional liposomes but not lipid bubbles. The echogenic properties of different lipid formulations were characterized by using the experimental apparatus shown in Figure 5a. Each group was injected into the sampling pipeline. It was found that, first, GU-Liposomes were fully distributed in the whole pipeline in the view of ultrasonic probe scattering (Figure 5b), then they were floated upward closing to the probe caused by the ultrasonic flow shear stress. Finally, ultrasonic shinning GU-Liposomes were accumulated in upside area. Figure 5b,c dictated the very beginning perfusion and the terminative accumulation images, respectively. The selected ROI (Figure 5c) explained the accumulation effect. Taken group of 6.0 mg/mL for instance (Figure 5d), it was conferred that the curve went through the perfusion (t = 0), the accumulation to peak value (t = 20−30 min), and subsequently debilitating imaging (t = 30−110 min). This dynamic changing may be entailed by the bubble burst or moving far away from probe caused by acoustic radiation force impulse.33 Figure 5e showed that the mean echogenic power level of perfusion images in B-mode at the very beginning injection of each group. The whole pipelines were designated as the ROI. Results indicated that power level increased significantly from the group of 1.5 mg/mL to group of 6.0 mg/mL but not for the group of 9.0 mg/mL. As mentioned before, for the group of 9.0 mg/mL, there were some conventional liposomes to coexist with GU-Liposome to decrease the imaging enhancement at the perfusion step. Although the coexistence of conventional liposomes and GU-Liposome was not helpful for perfusion enhancement, it was beneficial for resisting bubble shrinkage and disruption.34 Furthermore, limited by the ultrasound imaging resolution, single nanoscaled bubbles may be not ideal UCAs. However, the accumulation or aggregation of some amount of nanobubbles favored the targeting imaging enhancement. In this study, groups of 6.0 and 9.0 mg/mL held for the longer time than that in groups of 1.5 and 3.0 mg/mL.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.5b07778. Observation of SF6 free gas bubbles without dry lipids at the bottom of the vials in the daylight; TEM images of liposomes obtained by using free nanobubbles water (GU-Liposomes), degassed water (conventional liposomes) and using distilled water (another conventional liposomes), operated in parallel at different stand hours and subjected to the ultimate shaking; phospholipids concentration and lipid count in the solution at different reaction time; and derivation process of eq 1. (PDF)

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AUTHOR INFORMATION

Corresponding Authors

* Tel./Fax: (+86) 25-83272460. E-mail: yangfang2080@seu. edu.cn. *E-mail: [email protected]. Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This investigation was financially funded by the project of National Key Basic Research Program of China (2011CB933503 and 2013CB733804), the National Natural Science Foundation of China (31370019), National High Technology Research and Development Program (“863” Program) of China (2013AA032205), Research Innovation Program for College Graduates of Jiangsu Province (No. KYLX15_0219). Partial funding also came from the Author of National Excellent Doctoral Dissertation of China (201259), as well as from the Fundamental Research Funds for the Central Universities.

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REFERENCES

(1) Gramiak, R.; Shah, P. Echocardiography of the Aortic Root. Invest. Radiol. 1968, 3, 356−366. (2) Cavalli, R.; Bisazza, A.; Lembo, D. Micro- and Nanobubbles: A Versatile Non-Viral Platform for Gene Delivery. Int. J. Pharm. 2013, 456, 437−445. (3) Cavalieri, F.; Zhou, M. F.; Tortora, M.; Lucilla, B.; Ashokkumar, M. Methods of Preparation of Multifunctional Microbubbles and their in Vitro/in Vivo Assessment of Stability, Functional and Structural Properties. Curr. Pharm. Des. 2012, 18, 2135−2151. (4) Sirsi, S. R.; Borden, M. A. State-of-the-Art Materials for Ultrasound-Triggered Drug Delivery. Adv. Drug Delivery Rev. 2014, 72, 3−14.

4. CONCLUSIONS In summary, a novel preparing method of encapsulated nanobubbles using prefabricated free nanobubble aqueous solution was reported. Results demonstrated the layer-bylayer assembling process to form multilayer gas-containing ultrasound sensitive liposomes, which was expected to increase 26583


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ACS Applied Materials & Interfaces (5) Zhao, Y. Z.; Du, L. N.; Lu, C. T.; Jin, Y. G.; Ge, S. P. Potential and Problems in Ultrasound-Responsive Drug Delivery Systems. Int. J. Nanomed. 2013, 8, 1621−1633. (6) Christiansen, J. P.; French, B. A.; Klibanov, A. L.; Kaul, S.; Lindner, J. R. Targeted Tissue Transfection with Ultrasound Destruction of Plasmid-Bearing Cationic Microbubbles. Ultrasound Med. Biol. 2003, 29, 1759−1767. (7) Lanza, G. M.; Wickline, S. A. Targeted Ultrasonic Contrast Agents for Molecular Imaging and Therapy. Prog. Cardiovasc. Dis. 2001, 44, 13−31. (8) Ibsen, S.; Schutt, C. E.; Esener, S. Microbubble-mediated Ultrasound Therapy: A Review of Its Potential in Cancer Treatment. Drug Des., Dev. Ther. 2013, 7, 375−388. (9) Martin, K. H.; Dayton, P. A. Current Status and Prospects for Microbubbles in Ultrasound Theranostics. WIREs Nanomed. Nanobio. 2013, 5, 329−345. (10) Husseini, G. A.; Pitt, W. G.; Martins, A. M. Ultrasonically Triggered Drug Delivery: Breaking the Barrier. Colloids Surf., B 2014, 123, 364−386. (11) Rychak, J. J.; Klibanov, A. L. Nucleic Acid Delivery with Microbubbles and Ultrasound. Adv. Drug Delivery Rev. 2014, 72, 82− 93. (12) Saad, A. H.; Williams, A. R. Effects of Therapeutic Ultrasound on Clearance Rate of Blood Borne Colloidal Particles in Vivo. Br. J. Cancer Suppl. 1982, 5, 202−205. (13) Geers, B.; De Wever, O.; Demeester, J.; Bracke, M.; De Smedt, S. C.; Lentacker, I. Targeted Liposome-loaded Microbubbles for Cellspecific Ultrasound-Triggered Drug Delivery. Small 2013, 9, 4027− 4035. (14) Suzuki, R.; Takizawa, T.; Negishi, Y.; Hagisawa, K.; Tanaka, K.; Sawamura, K.; Utoguchi, N.; Nishioka, T.; Maruyama, K. Gene Delivery by Combination of Novel Liposomal Bubbles with Perfluoropropane and Ultrasound. J. Controlled Release 2007, 117, 130−136. (15) Sax, N.; Kodama, T. Optimization of Acoustic Liposomes for Improved In Vitro and In Vivo Stability. Pharm. Res. 2013, 30, 218− 224. (16) Ibsen, S.; Benchimol, M.; Simberg, D.; Schutt, C.; Steiner, J.; Esener, S. A Novel Nested Liposome Drug Delivery Vehicle Capable of Ultrasound Triggered Release of Its Payload. J. Controlled Release 2011, 155, 358−366. (17) Wu, H.; Rognin, N. G.; Krupka, T. M.; Solorio, L.; Yoshiara, H.; Guenette, G.; Sanders, C.; Kamiyama, N.; Exner, A. A. Acoustic Characterization and Pharmacokinetic Analyses of New Nanobubble Ultrasound Contrast Agents. Ultrasound Med. Biol. 2013, 39, 2137− 2146. (18) Sirsi, S. R.; Fung, C.; Garg, S.; Tianning, M. Y.; Mountford, P. A.; Borden, M. A. Lung Surfactant Microbubbles Increase Lipophilic Drug Payload for Ultrasound-Targeted Delivery. Theranostics 2013, 3, 409−419. (19) Javadi, M.; Pitt, W. G.; Tracy, C. M.; Barrow, J. R.; Willardson, B. M.; Hartley, J. M.; Tsosie, N. H. Ultrasonic Gene and Drug Delivery Using eLiposomes. J. Controlled Release 2013, 167, 92−100. (20) Stride, E.; Edirisinghe, M. Novel Microbubble Preparation Technologies. Soft Matter 2008, 4, 2350−2359. (21) Martinez, J.; Stroeve, P. Transient Behavior of the Hydrophobic Surface/Water Interface: From Nanobubbles to Organic Layer. J. Phys. Chem. B 2007, 111, 14069−14072. (22) Jin, F.; Li, J.; Ye, X.; Wu, C. Effects of pH and Ionic Strength on the Stability of Nanobubbles in Aqueous Solutions of α-Cyclodextrin. J. Phys. Chem. B 2007, 111, 11745−11749. (23) Lou, S. T.; Ouyang, Z. Q.; Zhang, Y.; Li, X.-J.; Hu, J.; Li, M.-Q.; Yang, F.-J. Nanobubbles on Solid Surface Imaged by Atomic Force Microscopy. J. Vac. Sci. Technol., B: Microelectron. Process. Phenom. 2000, 18, 2573−2575. (24) Ishida, N.; Inoue, T.; Miyahara, M.; Higashitani, K. Nano Bubbles on a Hydrophobic Surface in Water Observed by TappingMode Atomic Force Microscopy. Langmuir 2000, 16, 6377−6380.

(25) Ohgaki, K.; Khanh, N. Q.; Joden, Y.; Tsuji, A.; Nakagawa, T. Physicochemical Approach to Nanobubble Solutions. Chem. Eng. Sci. 2010, 65, 1296−1300. (26) Stewart, J. C. Colorimetric Determination of Phospholipids with Ammonium Ferrothiocyanate. Anal. Biochem. 1980, 104, 10−14. (27) Agarwal, A.; Ng, W. J.; Liu, Y. Principle and Applications of Microbubble and Nanobubble Technology for Water Treatment. Chemosphere 2011, 84, 1175−1180. (28) Takahashi, M. ζ Potential of Microbubbles in Aqueous Solutions: Electrical Properties of the Gas-Water Interface. J. Phys. Chem. B 2005, 109, 21858−21864. (29) Ho, C. C.; Chen, P. Y.; Lin, K. H.; Juan, W. T.; Lee, W. L. Fabrication of Monolayer of Polymer/Nanospheres Hybrid at a WaterAir Interface. ACS Appl. Mater. Interfaces 2011, 3, 204−208. (30) Brea, R. J.; Cole, C. M.; Devaraj, N. K. In Situ Vesicle Formation by Native Chemical Ligation. Angew. Chem., Int. Ed. 2014, 53, 14102− 14105. (31) Yang, G.; O'Duill, M.; Gouverneur, V.; Krafft, M. P. Recruitment and Immobilization of a Fluorinated Biomarker Across an Interfacial Phospholipid Film Using a Fluorocarbon Gas. Angew. Chem., Int. Ed. 2015, 54, 8402−8406. (32) Takakura, K.; Yamamoto, T.; Kurihara, K.; Toyota, T.; Ohnuma, K.; Sugawara, T. Spontaneous Transformation from Micelles to Vesicles Associated with Sequential Conversions of Comprising Amphiphiles Within Assemblies. Chem. Commun. 2014, 50, 2190− 2192. (33) Marston, P. L.; Thiessen, D. B. Manipulation of Fluid Objects with Acoustic Radiation Pressure. Ann. N. Y. Acad. Sci. 2004, 1027, 414−434. (34) Kodama, T.; Tomita, N.; Yagishita, Y.; Horie, S.; Funamoto, K.; Hayase, T.; Sakamoto, M.; Mori, S. Volumetric and Angiogenic Evaluation of Antitumor Effects with Acoustic Liposome and HighFrequency Ultrasound. Cancer Res. 2011, 71, 6957−6964.

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