Synchrotron microangiography studies of angiogenesis in mice with microemulsions and gold nanoparticles
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- Chien, C., Wang, C.H., Wang, C.L. et al. Anal Bioanal Chem (2010) 397: 2109. doi:10.1007/s00216-010-3775-8
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We present an effective solution for the problem of contrast enhancement in phase-contrast microangiography, with the specific objective of visualising small (<8 µm) vessels in tumor-related microangiogenesis. Different hydrophilic and hydrophobic contrast agents were explored in this context. We found that an emulsified version of the hydrophobic contrast agents Lipiodol® provides the best contrast and minimal distortion of the circulation and vessel structure. Such emulsions are reasonably biocompatible and, with sizes of 0 ± 0.8 µm, sufficient to diffuse to the smallest vessel and still provide reasonable contrast. We also explored the use of Au nanoparticle colloids that could be used not only to enhance contrast but also for interesting applications in nanomedicine. Both the Lipiodol microemulsions and Au nanoparticle colloids can be conjugated with medicines or cell specific labeling agents and their small size can allow the study of the diffusion of contrast agents through the vessel leakage. This enables direct imaging of drug delivery which is important for cancer treatment.
KeywordsMicroradiologyMicroangiographyAngiogenesisMicro-emulsionGold nanoparticlesContrast agent
Microradiology based on coherent synchrotron X-rays can provide the required lateral and time resolution for angiography studies of microangiogenesis [1–3]. With bright unmonochromatized synchrotron X-rays, the image taking is fast enough to limit the motion blurring and to allow high resolution observation of the smallest vessels in live animals . However, finding a suitable contrast agent is not a trivial problem since those used in conventional radiology do not work well for phase-contrast microangiography with high lateral resolution. Most certified contrast agents for clinical use are nonionic, such as Iomeprol®, Imeron®, and Hexabrix®, whose fast diffusion rate limits their application to image small capillaries (<20 µm) in small animals [5–9]. Therefore, they are not satisfactory for the purpose of enhancing the weak contrast of the microvessels with respect to the surrounding tissues to study tumor angiogenesis with whole tumor profiling in vivo without losing important information of the three-dimensional structure of the smallest vessels.
We solved this problem after testing different hydrophilic and hydrophobic contrast agents. One of the good candidate of such solution is the emulsification of a hydrophobic and otherwise viscous contrast agent, Lipiodol®; this enabled us to reach not only a lateral resolution ∼2 µm and a time resolution ∼1 ms, but also to detect tumor microvessels as small as 7–8 µm in live mice. Nanoparticle systems with good colloidal stability and high enough X-ray absorption can perform a similar function.
These results are quite important for microangiogenesis studies—and for many similar issues in biomedical research. Important syndromes, notably vascular diseases and cancer, are related to the growth of small vessels and to changes in their fine morphology [2, 3]. In tumor growth, permeable small vessels are believed to provide nutrients to tumor cells . Angiogenesis control can thus be an effective way to fight cancer [11, 12]. But the implementation of new therapies requires a detailed knowledge of angiogenesis on a scale <8 µm with the use of suitable imaging techniques.
Widely used approaches such as magnetic resonance imaging (MRI) [13, 14], single photon-emission computed tomography (SPECT), positron emission tomography (PET) , ultrasonography [16, 17], optical coherent tomography (OCT) [18, 19], and computed tomography (CT) [20–24] suffer from insufficient lateral resolution. The permeability of fluorescent macromolecules can be observed with the intravital imaging method [25–28]—but this approach is limited to near-surface regions due to the low penetration of visible light. Post-mortem examinations using other types of microscopies, such as optical, electron, or infrared [29–31], cannot study time-evolving phenomena except with a large number of killed animals. The fast circulation rate in mice , with a velocity in the pulmonary artery as high 35 cm/s in the systolic phase , complicates the problem.
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 image acquisition geometry and the high coherence of synchrotron sources, phase (or refractive index) contrast is very effective in enhancing features with low absorption contrast  such as blood vessels [4, 36–40]. The high source brightness makes it possible to achieve fast imaging at the microsecond level with approximately micrometer level lateral resolution [41, 42]. The fast image taking is essential since small movements even for deep anesthetized animals would cause motion blur and jeopardize the detection of <10 µm vessels.
In our search for a suitable contrast agent for synchrotron microangiography, we found that viscosity and solubility are the most relevant factors. Viscous hydrophobic agents are resistant to dilution and therefore easier to detect. However, such agents do not flow smoothly and are likely to deform small vessels affecting size and position measurements.
Aqueous hydrophilic agents are quickly diluted in the blood stream, making it difficult to detect in real time their circulation. On the positive side, such agents have limited adverse effects on the vascular structure as well as the health of animals. It is also possible to use these agents to study the diffusion through the leaky vessels.
Based on these observations, we successfully tested a new type of contrast agent by emulsifying viscous hydrophobic Lipiodol®. The result is better microvessel detectability than with hydrophilic agents and a natural circulation suitable for real-time microradiology. With this agent, we achieved performances similar to Lipiodol in the detection of small vessels. And we were able to observe its diffusion through small vessels.
Finally, we also explored the use of aqueous PEG-Au nanoparticle colloids . They behave like hydrophilic contrast but provide additional functions. The small nanoparticle size and the leaky nature of microvessels lead to highly selective localization at tumor sites by the EPR (enhanced retention and permeation) effect. After injecting 35 µl of PEG-Au solution (55 mg/ml) into a mouse tail vein, we found the accumulation to start within seconds (120 s) and the differential concentration to increase for at least 12 h . This made it possible to highlight small vessels of 10 µm size sufficient for microradiology.
Note that both types of agents, microemulsions and nanoparticle colloids can be used as drug carriers [45–47]. If combined with suitable molecules for treatment, our imaging strategy could thus increase the probability of coordinated application to effective cancer diagnosis and nanomedicine delivery. Furthermore, the possibility to study microvascular structures on a very small scale could by itself open new door to the study of microangiogensis in full three dimensions with high lateral and time resolution.
The explored contrast agents included commercial Hexabrix® (Guerbet, Aulnay-sous-Bios, France) and Lipiodol® (Guerbet, Aulnay-sous-Bios, France). Typically, 70 µl of Hexabrix® (320 mg/ml of iodine) or 40 µl (480 mg/ml of iodine) Lipiodol® were injected via thigh artery.
In the case of emulsified Lipiodol®, the iodine concentration was ∼240 mg/ml and a volume of 50 µl was injected. The emulsified Lipiodol® was prepared by mixing two solutions: solution A, which is 0.1 ml of span-80 mixed with 1 ml of Lipiodol®, and solution B, which is 0.6 ml Tween-20 mixed with 3 ml of phosphate buffer solution. By adding the hydrophobic solution A with the aqueous solution B in a 2:1 ratio, and stirring for 8 h, the emulsified solution is formed with small droplets of size in the µm range. The two surfactants, Span-80 and Tween-20, are polysorbate emulsifiers widely used in food additive and cosmetics. These emulsifiers interface Lipiodol and water and stabilize and suspended the encapsulated Lipiodol droplets. Before injection, this emulsified Lipiodol® solution was sonicated for 15 min to ensure a homogenized suspension.
In a separate series of tests, we injected ∼35 µl of a colloidal solution of PEG-Au nanoparticles synthesized by a special X-ray irradiation method , whose concentration was increased to 36 mg/ml of Au with several rounds of centrifuge separation and cleaning.
PE-05 catheters (BB31695, Scientific Commodities, Inc., I.D.: 0.2 mm, O.D.: 0.36 mm) were used to inject the contrast agents. The catheter was placed under anesthesia induced by intramuscular injection of 10 µl of Zoletil 50 (50 mg/kg; Virbac Laboratories, Carros, France) per mouse (weight ∼20–25 g). The anterior tight skin was incised along a 1-cm2 circle and after a sharp dissection, the catheter was inserted into the femoral artery and secured by a 6-0 nylon ligature. With the mouse in the imaging position, one of the aforementioned contrast agents was injected at a 0.5 µl/s rate. During imaging, the mice were kept under anesthesia using 1% isoflurane in oxygen.
Microradiology was implemented with unmonochromatized (white) synchrotron X-rays emitted by the 01-A beamline wavelength shifter of the National Synchrotron Radiation Research Center (Taiwan) . The photon energy ranged from 4 keV to 30 keV with a peak intensity at energy ∼12 keV and the beam current was kept constant at 300 mA with the top-up operation mode. To obtain 3 × 3 mm images, the X-rays were first converted to visible light by a CdWO4 single crystal scintillator and then captured by an optical microscope with a CCD camera (Andor, 1,000 × 1,000 pixels). The radiation dose was reduced by attenuating the emitted X-ray beam with two pieces of 550 µm single crystalline silicon wafers placed before the animal.
The exposure time was ∼100 ms and the distance between the sample and the scintillator was ∼5 cm; a ×2, ×5, or ×10 lens in the optical microscope was used to obtain the desired field of view. The size of each pixel in the final image taken with the ×5 lens was ∼2.4 × 2.4 µm2.
Results and discussion
The strong Hexabrix® diffusion is observed not only in the normal vessels but also in the tumor area. In the tumor area, marked by letter T at the left part of Fig. 2a, we can only detect the whole area become darker with respect to surrounding tissue, where the boundary is marked by an arrow head and to that before injection. The parallel vessel structure in muscle site (marked by letter N) also showed strongly diffused Hexabrix® (marked by arrows). Driven by the concentration gradient, Hexabrix® diffused to the interstitial tissues and the abnormal vessels with intercellular openings and transvascular holes . This causes it to be rapidly diluted by blood before producing sufficient contrast for small vessels. The diffusion smears even the sharp phase-contrast edges observed with no contrast agent .
Figure 5 shows images with emulsified Lipiodol in mouse tumor area taken 26 days after tumor planting and reveal the abnormal vessel structure. This is particularly clear in the whole leg picture of Fig. 5c. Figure 5a and b show a vessel microstructure, marked by arrow heads in the close vicinity of the tumor site, clearly resulted from the tumor microangiogenesis. We also identified areas, such as that marked in Fig. 5d, as blood pools which show no clear vessel structure. Such image implies that the irregular vessel structure allows the emulsified hydrophobic Lipiodol droplets diffusing out of the vessels to the interstitial tissue.
Finally, we would like to present results for PEG-Au nanoparticles selectively bound because of the EPR effect. The aforementioned new colloidal synthesis method by X-ray irradiation [49, 50] yielded excellent stability and biocompatibility plus high concentration. An aqueous colloid with ∼36 mg/ml of PEG-Au with an average Au core size ∼7 ± 2 nm  was administrated to mice without any adversary effects.
Although emulsified hydrophobic agent are inexpensive and easy to make, it is well known that one of the most important application of microemulsions and nanoparticle colloid is not only to enhance the contrast but also to obtain other functions such as specific cancer targeting, diagnosis, and radiotherapy [41, 53–56]. The PEG-Au nanoparticles can be easily conjugated with cancer drugs such as TNF-α (tumor necrosis factor-alpha)  or doxorubicin  and it is not difficult to encapsulate drugs with microemulsions , either. Similar imaging approaches can also be applied to other type of nanoparticles [57–59] and delivery system like liposome, micelles, or microspheres as long as high X-ray contrast materials can also be incorporated at the same time.
In this work, we demonstrated a practical solution for the problem of contrast enhancement in phase-contrast microangiography by emulsifying a hydrophobic and otherwise viscous contrast agent. We were thus able to detect vessels as small as ∼7–8 µm, thanks in particular to the short image taking time and limited motion blurring. We also explored the use of colloidal PEG-Au for contrast with mildly positive results compensated by the potential use of nanoparticles for diagnosis and therapy.
This work is supported by National Science and Technology Program for Nanoscience and Nanotechnology, the Thematic Research Project of Academia Sinica, the Biomedical Nano-Imaging Core Facility at National Synchrotron Radiation Research Center (Taiwan), the Blonc Project of ANR-NSC (French National Research Agency and Taiwan National Science Council, the Center for Biomedical Imaging (CIBM) in Lausanne, partially funded by the Leenaards and Jeantet foundations and by the Swiss Fonds National de la Recherche Scientifique and by the EPFL.