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Materials Letters 62 (2008) 121 – 124 www.elsevier.com/locate/matlet
Multiple emulsion microbubbles for ultrasound imaging Fang Yang a , Aiyuan Gu b , Zhongping Chen a , Ning Gu a,⁎, Min Ji c,⁎ a
State Key Laboratory of Bioelectronics, Jiangsu Laboratory for Biomaterials and Devices, School of Biological Science and Medical Engineering, Southeast University, Nanjing 210096, PR China b Belson Imaging Technology Co., Ltd, Wuxi 214091, PR China c Institute of Pharmaceutical Engineering, Southeast University, Nanjing 210096, PR China Received 30 March 2007; accepted 24 April 2007 Available online 25 May 2007
Abstract In order to improve the sensitivity of ultrasound imaging, the contrast agents, a powerful non-invasive and real-time medical imaging technique, are used. However, air or N2 or perfluorocarbon only encapsulated microbubbles which are currently used have lower efficiency and short imaging time. So the novel contrast agents with a higher efficiency are required. To achieve this objective, the strategy that we have explored involves the use of superparamagnetic iron oxide (SPIO) Fe3O4 nanoparticles multilayer emulsion microbubbles. This multilayer structure consists of three layers. The core is poly-D, L-lactide (PLA) encapsulated N2 nanobubble with the SPIO nanoparticles forming oil-in-water (W/O) layer. The outermost is water-in-oil-in-water ((W/O)/W) emulsion layer with PVA solution. Herein we describe the synthesis and characterization of ultrasound imaging microstructure with an overall diameter of around 2μm–8μm. On the one hand, the stable gas encapsulated microstructure can provide a high scattering intensity resulting in high echogenicity, On the other hand, SPIO nanoparticles have shown the potential of highresolution sonography. So the multiple emulsion microbubbles with SPIO can have double action to enhance the ultrasound imaging. Besides, because SPIO can also serve as magnetic resonance imaging (MRI) contrast agents, such microstructure may be useful for multimodality imaging studies in ultrasound imaging and MRI. © 2007 Elsevier B.V. All rights reserved. Keywords: Ultrasound imaging; Composite materials; Multilayer structure; Microbubbles
1. Introduction Diagnostic medical ultrasound is now a well-established technique for clinical diagnostics. Ultrasound images, however, do not have a very sharp contrast and sometimes the area being imaged is buried and shadowed by tissue. Recently, the researches have shown that ultrasound contrast agent (UCA) can resolve in part when imaging. UCA alters ultrasound image in a meaningful way that helps the diagnostician to distinguish normal and abnormal conditions more obviously . It is a versatile, non-invasive, low risk, low cost, and portable realtime imaging technique. The basic rationale is using the gas as the enhanced reflective medium [2,3]. The first generation UCA contains just the dissociated air or gas suspended in liquid, which is a little unstable in the bloodstream and can not tolerate ⁎ Corresponding authors. Tel.: +86 25 83792576; fax: +86 25 83794960. E-mail addresses: [email protected]
(N. Gu), [email protected]
(M. Ji). 0167-577X/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.matlet.2007.04.111
enough imaging time. The second generation UCA contains encapsulated ultrasound microbubbles. Such coated microbubbles have the advantage of being stable in the body for a significant period of time, as the shells serve to protect the gases of the microbubbles from diffusion into the bloodstream [4,5]. The third generation UCA contains perfluorocarbon gas  rather than air or N2 which results in a longer life span of the contrast agents within the circulatory system . Nowadays, the development of UCA involves the application of ultrasound energy for targeting or controlling drug release. This new concept of using ultrasound energy can enhance effects of various drugs, such as thromboly agents, transdermal drug delivery, anticancer drugs, and the most exciting applications of gene therapy [8,9]. Although microbubbles have limited use because of too large size and short blood half-life, microbubbles can produce a strong backscattered sound signal because of a large acoustic impedance mismatch compared with the surrounding tissue.
F. Yang et al. / Materials Letters 62 (2008) 121–124
With the development of other research, Nolte, et al.  demonstrated that the detection of Fe3O4 nanoparticles in brain tumours using high-resolution intraoperative ultrasound can improve tumour definition in recorded images. Junghwan Oh et al.  studied the ultrasound reflective signal increased slightly with higher SPIO doses. The rationale for this study derives from the need to differentiate the SPIO nanoparticles multilayer emulsion microbubbles from that of the commonly used gaseous only microbubbles. PLA, one of the polymer shell layers, can slowly degrade in an aqueous solution with no toxicity. Therefore, the goal of this study was to attain the stable and longer span multiple UCA using the gas and SPIO, which may enhance the ultrasound image more distinctly. Fig. 1 is the schematic diagram of the design idea. 2. Experimental 2.1. Materials
first emulsion microbubbles solution under an external magnetic field. The first W/O microbubble emulsion was then poured into a 1% PVA (w/v) solution and mixed mechanically for 2h to form (W/O)/ W multiple emulsion microbubbles and to eliminate the organic solution. After reaction, the final emulsion became milk-white. 2.3. Separation of multiple emulsion microbubbles The multiple emulsion microbubbles solution was transferred to a custom made centrifuge tube. Suspended microbubbles were centrifuged for 3min at 500rpm. In most cases, the solution was separated into two distinct layers: the upper layer containing mostly bubbles, and the lower layer containing suspended bubbles in solution. The lower layer of the solution was collected for size and acoustic analysis, and the top layer was discarded. The collected multiple emulsion microbubbles were stored at 4°C in tightly capped vials sealed with parafilm. The collected microbubble agents were diluted 1:1 with deionized water prior to use.
Poly(D, L-lactide) (PLA) (7000MW) was purchased from Shandong Key Laboratory of Medical polymer Materials. Poly (vinyl alcohol) (PVA), 88mol% hydrolyzed with a MW of 25,000 was from Alfa Aesar® a Johnson Matthey company. Superparamagnetic iron oxide (SPIO) Fe3O4 was from Jiangsu Laboratory for Biomaterials and Devices. Span 80, Tween 80 and N2 were reagent grade.
2.2. Preparation of multiple emulsion microbubbles
Experiments were performed initially with an in vitro model in order to provide insight for interpreting later measurements of in vivo ultrasound imaging. Acoustic analysis was performed by exposing a sample by the self-made acoustic setup using a system in vitro. A 2.5MHz focused ultrasound transducer sending a pulse of ultrasound into a suspension of microbubbles was used. The solution was contained in a silica gel tube of 1.5cm in diameter. A pulser–receiver and computation detection system were provided by Belson Imaging Technology Co., Ltd, Wu Xi. The backscattered signal was received by the same transducer, and the returned signal was then amplified by the pulser–receiver unit. The acoustic data was collected by the computer soft detection system. In order to simulate the in vivo process as far as possible, the circulating dynamic mode was designed. The sample solution was circulated by wriggling pump in the silica gel tube. Each tube was filled with either deioned water, or deioned water with SPIO multiple emulsion microbubbles or deioned water without SPIO multiple emulsion microbubbles. There should be no air in the circulating tube. When the three samples were pumped into the silica gel tube respectively, we can see the flow signal imaging in the tube from the computer soft system.
The methylene chloride organic solution (10.00ml) was prepared containing PLA (0.50g) and hydrophobic SPIO Fe3O4 nanoparticles (0.5g) at 25°C. To generate the first W/O microbubble emulsion, 1.00mL deionized water and a few Tween 80 (about 1.00ml) were added to the organic solution and sonicated continuously by ultrasound probe at 100W with constant purging using a steady (4ml/min) stream of N2 gas for 5min. The W/O microbubble emulsion is brown and visibly homogeneous. The dissociated Fe3O4 can be separated from the
The morphology and structure of this multiple emulsion were determined by transmission electron microscopy (TEM) and scanning electron microscopy (SEM). 2.5. Acoustic test in vitro
3. Results and discussion 3.1. Characterization of Fe3O4/PLA/N2 multiple emulsion microbubbles
Fig. 1. Schematic diagram of multiple emulsion microbubble design.
3.1.1. TEM analysis The morphology of Fe3O4 multiple emulsion microbubbles is shown in Fig. 2. The results show that the Fe3O4 nanoparticles were
F. Yang et al. / Materials Letters 62 (2008) 121–124
Fig. 2. TEM image of the SPIO encapsulated multiple emulsion microbubbles.
dispersed uniformly around the polymer layer. The microbubbles are spherical and the particle size ranges from 2 to 8μm. 3.1.2. SEM analysis A representative SEM image of the emulsion microbubbles solution is shown in Fig. 3. The samples were gold coated under reduced pressure with a sputter coater prior to examination. The imaged picture was demonstrated essentially spherical in shape. 3.2. In vitro acoustic testing The ultrasound imagings of the deioned water, the multiple emulsion microbubbles without SPIO and the multiple emulsion microbubbles with SPIO are shown in Fig. 4. Because the deioned water has the uniform ultrasonic propagation performance, there is no
Fig. 4. The ultrasound imaging in the different samples in vitro (A) deioned water; (B) the multiple emulsion microbubbles without SPIO; (C) the multiple emulsion microbubbles with SPIO.
fluid flow signal in the tube. The brighter area can be seen distinctly in the multiple emulsion microbubbles without SPIO and the multiple emulsion microbubbles with SPIO. However, the result shows a brighter area in the multiple emulsion microbubbles with SPIO than in the multiple emulsion microbubbles without SPIO. It seems that the microbubbles with SPIO have more contribution to ultrasound echo effect.
4. Conclusions In this study, we synthesized multilayer emulsion microbubbles structure containing SPIO Fe3O4 nanoparticles in the oil layer and a N2 gas core. The diameter of microbubble can be controlled by the reaction conditions, resulting in particles with diameters of approximately 2μm–8μm. The microstructure may be useful to enhance the sensitivity of ultrasound imaging. The utilization of Fe3O4/PLA/N2 multiple emulsion stable microbubbles will be applicable to intravascular ultrasound imaging. The gas and SPIO may have double ultrasound cavitation effects. Combining SPIO Fe3O4 nanoparticles with ultrasound imaging technique may be more attractive in ultrasound molecular imaging and also may provide a dramatic increase in resolution over conventional clinical diagnostic ultrasound scanners. Acknowledgements
Fig. 3. SEM image showing the surface morphology.
The authors acknowledge Belson Imaging Technology Co., Ltd, Wuxi, for providing the ultrasound detection system. The
F. Yang et al. / Materials Letters 62 (2008) 121–124
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