Anal Bioanal Chem (2010) 397:2109–2116 DOI 10.1007/s00216-010-3775-8 ORIGINAL PAPER Synchrotron microangiography studies of angiogenesis in mice with microemulsions and gold nanoparticles Chia-Chi Chien & C. H. Wang & C. L. Wang & E. R. Li & K. H. Lee & Y. Hwu & Chien-Yi Lin & Shing-Jyh Chang & C. S. Yang & Cyril Petibois & G. Margaritondo Received: 7 February 2010 /Revised: 17 April 2010 /Accepted: 22 April 2010 /Published online: 6 June 2010 # Springer-Verlag 2010 Abstract We present an effective solution for the problem provides the best contrast and minimal distortion of the of contrast enhancement in phase-contrast microangiogra- circulation and vessel structure. Such emulsions are phy, with the specific objective of visualising small reasonably biocompatible and, with sizes of 0±0.8 µm, (<8 µm) vessels in tumor-related microangiogenesis. sufficient to diffuse to the smallest vessel and still provide Different hydrophilic and hydrophobic contrast agents were reasonable contrast. We also explored the use of Au explored in this context. We found that an emulsified nanoparticle colloids that could be used not only to enhance version of the hydrophobic contrast agents Lipiodol® contrast but also for interesting applications in nano- medicine. Both the Lipiodol microemulsions and Au nanoparticle colloids can be conjugated with medicines or C.-C. Chien : C. H. Wang : C. L. Wang : E. R. Li : K. H. Lee : Y. Hwu (*) cell specific labeling agents and their small size can allow Institute of Physics, Academia Sinica, the study of the diffusion of contrast agents through the Nankang, vessel leakage. This enables direct imaging of drug delivery Taipei 115, Taiwan, China which is important for cancer treatment. e-mail: [email protected] : C.-C. Chien Y. Hwu Keywords Microradiology . Microangiography. Department of Engineering and System Science, Angiogenesis . Micro-emulsion . Gold nanoparticles . National Tsing Hua University, Contrast agent Hsinchu 300, Taiwan, China Y. Hwu Institute of Optoelectronic Sciences, Introduction National Taiwan Ocean University, Keelung 202, Taiwan, China Microradiology based on coherent synchrotron X-rays can : C.-Y. Lin S.-J. Chang provide the required lateral and time resolution for angiog- Hsinchu Mackay Memorial Hospital, raphy studies of microangiogenesis [1–3]. With bright Hsinchu 300, Taiwan, China unmonochromatized synchrotron X-rays, the image taking C. S. Yang is fast enough to limit the motion blurring and to allow high Center for Nanomedicine, National Health Research Institutes, resolution observation of the smallest vessels in live Miaoli 350, Taiwan, China animals [4]. However, finding a suitable contrast agent is not a trivial problem since those used in conventional C. Petibois Université de Bordeaux 2, radiology do not work well for phase-contrast micro- CNRS UMR 5248, CBMN, B8, Avenue des faculties, angiography with high lateral resolution. Most certified 33405 Talence-Cedex, France contrast agents for clinical use are nonionic, such as Iomeprol®, Imeron®, and Hexabrix®, whose fast diffusion G. Margaritondo Ecole Polytechnique Fédérale de Lausanne (EPFL), rate limits their application to image small capillaries 1015 Lausanne, Switzerland (<20 µm) in small animals [5–9]. Therefore, they are not 2110 C.-C. Chien et al. satisfactory for the purpose of enhancing the weak contrast In our search for a suitable contrast agent for synchrotron of the microvessels with respect to the surrounding tissues microangiography, we found that viscosity and solubility are to study tumor angiogenesis with whole tumor profiling in the most relevant factors. Viscous hydrophobic agents are vivo without losing important information of the three- resistant to dilution and therefore easier to detect. However, dimensional structure of the smallest vessels. such agents do not flow smoothly and are likely to deform We solved this problem after testing different hydrophilic small vessels affecting size and position measurements. and hydrophobic contrast agents. One of the good candidate Aqueous hydrophilic agents are quickly diluted in the of such solution is the emulsification of a hydrophobic and blood stream, making it difficult to detect in real time their otherwise viscous contrast agent, Lipiodol®; this enabled us circulation. On the positive side, such agents have limited to reach not only a lateral resolution ∼2 µm and a time adverse effects on the vascular structure as well as the resolution ∼1 ms, but also to detect tumor microvessels as health of animals. It is also possible to use these agents to small as 7–8 µm in live mice. Nanoparticle systems with study the diffusion through the leaky vessels. good colloidal stability and high enough X-ray absorption Based on these observations, we successfully tested a new can perform a similar function. type of contrast agent by emulsifying viscous hydrophobic These results are quite important for microangiogenesis Lipiodol®. The result is better microvessel detectability than studies—and for many similar issues in biomedical re- with hydrophilic agents and a natural circulation suitable for search. Important syndromes, notably vascular diseases and real-time microradiology. With this agent, we achieved cancer, are related to the growth of small vessels and to performances similar to Lipiodol in the detection of small changes in their fine morphology [2, 3]. In tumor growth, vessels. And we were able to observe its diffusion through permeable small vessels are believed to provide nutrients to small vessels. tumor cells [10]. Angiogenesis control can thus be an Finally, we also explored the use of aqueous PEG-Au effective way to fight cancer [11, 12]. But the implemen- nanoparticle colloids [43]. They behave like hydrophilic tation of new therapies requires a detailed knowledge of contrast but provide additional functions. The small nano- angiogenesis on a scale <8 µm with the use of suitable particle size and the leaky nature of microvessels lead to imaging techniques. highly selective localization at tumor sites by the EPR Widely used approaches such as magnetic resonance (enhanced retention and permeation) effect. After injecting imaging (MRI) [13, 14], single photon-emission computed 35 µl of PEG-Au solution (55 mg/ml) into a mouse tail tomography (SPECT), positron emission tomography (PET) vein, we found the accumulation to start within seconds [15], ultrasonography [16, 17], optical coherent tomography (120 s) and the differential concentration to increase for at (OCT) [18, 19], and computed tomography (CT) [20–24] least 12 h [44]. This made it possible to highlight small suffer from insufficient lateral resolution. The permeability vessels of 10 µm size sufficient for microradiology. of fluorescent macromolecules can be observed with the Note that both types of agents, microemulsions and intravital imaging method [25–28]—but this approach is nanoparticle colloids can be used as drug carriers [45–47]. limited to near-surface regions due to the low penetration of If combined with suitable molecules for treatment, our visible light. Post-mortem examinations using other types of imaging strategy could thus increase the probability of microscopies, such as optical, electron, or infrared [29–31], coordinated application to effective cancer diagnosis and cannot study time-evolving phenomena except with a large nanomedicine delivery. Furthermore, the possibility to number of killed animals. The fast circulation rate in mice study microvascular structures on a very small scale could [32], with a velocity in the pulmonary artery as high 35 cm/s by itself open new door to the study of microangiogensis in in the systolic phase [33], complicates the problem. full three dimensions with high lateral and time resolution. We show here that synchrotron microradiology [1, 34] can solve this problem if a suitable answer is found for the question of contrast enhancement. Thanks to the adjustable Experimental image acquisition geometry and the high coherence of synchrotron sources, phase (or refractive index) contrast is All procedures involving animals were approved by very effective in enhancing features with low absorption Academia Sinica Institutional Animal Care and Utilization contrast [35] such as blood vessels [4, 36–40]. The high Committee (AS IACUC). BALB/cByJNarl mice were source brightness makes it possible to achieve fast imaging provided by National Laboratory Animal Center, Taiwan. at the microsecond level with approximately micrometer All mice were housed in individual ventilated cages (five level lateral resolution [41, 42]. The fast image taking is per cage) with wood chip bedding and kept at 24±2 °C essential since small movements even for deep anesthetized with a humidity of 40–70% and a 12-h light/dark cycle. The animals would cause motion blur and jeopardize the tests were performed on 5-weeks old Balb/c mice after detection of <10 µm vessels. injection of the highly metastatic cancer cell strain CT-26, Angiogenesis in mice with microemulsions and gold nanoparticles 2111 suitable for tumor angiogenesis studies. CT-26 cells (1×106) tion was increased to 36 mg/ml of Au with several rounds in 10 µl RPMI-1640 medium were subcutaneous-injected in of centrifuge separation and cleaning. the left thigh region. Tumor volume was estimated by PE-05 catheters (BB31695, Scientific Commodities, adopting a formula v ¼ 0:5  a  b2, where a and b are the Inc., I.D.: 0.2 mm, O.D.: 0.36 mm) were used to inject smallest and the largest diameters, respectively. Treatment the contrast agents. The catheter was placed under of tumors was started after about 7 days, for example as anesthesia induced by intramuscular injection of 10 µl of shown in Fig. 1, when they reached a size of 100 to Zoletil 50 (50 mg/kg; Virbac Laboratories, Carros, France) 120 mm3 and after 26 days when they reached a size of per mouse (weight ∼20–25 g). The anterior tight skin was 1,000 to 1,200 mm3. incised along a 1-cm2 circle and after a sharp dissection, the The explored contrast agents included commercial catheter was inserted into the femoral artery and secured by Hexabrix® (Guerbet, Aulnay-sous-Bios, France) and Lipiodol® a 6-0 nylon ligature.