International Journal of Pharmaceutics 384 (2010) 148–153 Contents lists available at ScienceDirect International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm Pharmaceutical Nanotechnology Preparation of nanobubbles for ultrasound imaging and intracelluar drug delivery Ye Wang a,∗ , Xiang Li a , Yan Zhou c , Pengyu Huang a , Yuhong Xu a,b a Zhejiang-California International Nanosystems Institute Molecular Imaging Platform, Zhejiang University, 268 Kaixuan Road, Hangzhou 310029, PR China b School of Pharmacy, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, PR China c Shandong Provincial Research Center for Bioinformatic Engineering and Technique, 12 Zhangzhou Road, Zibo 255049, PR China article info Article history: Received 26 May 2009 Received in revised form 13 September 2009 Accepted 15 September 2009 Available online 23 September 2009 Keywords: Nanobubbles Ultrasound imaging Cellular uptake Drug delivery abstract Echogenic bubble formulations have wide applications in both disease diagnosis and therapy. In the cur- rent study, nanobubbles were prepared and the contrast agent function was evaluated in order to study the nanosized bubble’s property for ultrasonic imaging. Coumarin-6 as a model drug was loaded into nanobubbles to investigate the drug delivery potential to cells. The results showed that the nanobub- bles composed of 1% of Tween 80, and 3 mg/ml of lipid worked well as an ultrasonic contrast agent by presenting a contrast effect in the liver region in vivo. The drug-loaded nanobubbles could enhance drug delivery to cells significantly, and the process was analyzed by sigmoidally fitting the pharmacokinetic curve. It can be concluded that the nanobubble formulation is a promising approach for both ultrasound imaging and drug delivery enhancing. © 2009 Elsevier B.V. All rights reserved. 1. Introduction The development of nanomedicine has emerged with the marriage of nanotechnology and medicine (Sanhai et al., 2008). Nanovectors show significant importance for healthcare in both diagnostic and therapeutic applications, due to the many special features in nanosized particles. In the human body, nanoparticles could accumulate in organs and tissues important for diagno- sis or therapy, such as the liver, spleen, and tumor tissues. A large number of polymeric nanoparticles were applied in basic and clinical medical studies. Many of them improved the drugs’ distribution profile, which usually influenced positively the drug delivery properties (Farokhzad and Langer, 2006). In the diagnos- tic field, nanotechnology has impacted nearly all aspects of the imaging methodology. Nanoagents were designed and used as probes for the early detection of malignant diseases. There have been many papers published that demonstrated their applications in magnetic resonance (MR) (Sun et al., 2008), positron emission tomography (PET) (Lee et al., 2008), ultrasound (US) (Rapoport et al., 2007) imaging and others. It is believed that nanotechnology Abbreviations: SF6, sulphur hexafluoride; UBM, ultrasonic biological microscopy; SPC, soybean lipid; CHO, cholesterol; DPPG, dipalmitoyl phos- phatidylglycerol; MPP, sodium poly phosphate; CNP, chitosan nanoparticle; ROI, region of interest; TOI, time of interest; NBS, number of bright spots; MNTI, mean NBS of TOI; LiposomeA, the liposomes without Tween 80 modification. ∗ Corresponding author. Tel.: +86 571 86971897; fax: +86 571 86971897. E-mail addresses: wangyeph@hotmail.com, wangyeph@yahoo.com.cn (Y. Wang). has led the diagnostic and therapeutic work into the molecular level. Among the many novel nano-carriers being developed, echogenic bubble formulations have been gaining lots of atten- tion in recent decades (Saad et al., 2008). Bubbles are filled with gas, spherically shaped and stable in aqueous (Ferrara et al., 2007). Compared to other particles, bubbles have the special properties of being “explosive” under ultrasound-energy illumination, prompt- ing the destruction of bubbles and cellular membrane permeability changes. Various bubble formulations were used for local drug delivery (Bull, 2007), for gene delivery (Chen et al., 2003) and tar- geting drug delivery (Lum et al., 2006). They were developed based on micron-scale (m) bubbles commercially available as ultra- sound contrast agent for imaging diagnosis. Besides drug delivery, bubbles have also attracted investiga- tors on ultrasonic imaging work. Ultrasonic imaging is one of the most important technologies in medical diagnosis. It presents such advantages as freely utilizing, dynamic observing, real-time detecting, and the high priority of biological safety without radio- contamination. Ultrasonic imaging has been demonstrated as a promising tool for diagnosis of many diseases, gas filled bubbles were always taken as ultrasonic contrast agents. In recent decade, nanobubbles were developed as the contrast agent or drug vector mainly for tumor- related molecular imaging by the size effect (Pitt et al., 2004; Liu et al., 2006). For example, octafluoropropane filled nanobubbles were generated by Span 60 and Tween 80, the bubble size ranged from 450 nm to 700 nm, this kind of nanobubbles displayed dose- response echo enhancement both in vitro (Oeffinger and Wheatley, 0378-5173/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.ijpharm.2009.09.027