Elsevier

Biomaterials

Volume 31, Issue 9, March 2010, Pages 2617-2626
Biomaterials

Photoacoustic imaging of living mouse brain vasculature using hollow gold nanospheres

https://doi.org/10.1016/j.biomaterials.2009.12.007Get rights and content

Abstract

Photoacoustic tomography (PAT) also referred to as optoacoustic tomography (OAT) is a hybrid imaging modality that employs nonionizing optical radiation and ultrasonic detection. Here, we describe the application of a new class of optical contrast agents based on mesoscopic hollow gold nanospheres (HAuNS) to PAT. HAuNS are ∼40 nm in diameter with a hollow interior and consist of a thin gold wall. They display strong resonance absorption tuned to the near-infrared (NIR) range, with an absorption peak at 800 nm, whose photoacoustic efficiency is significantly greater than that of blood. Following surface conjugation with thiolated poly(ethylene glycol), the pegylated HAuNS (PEG-HAuNS) had distribution and elimination half-lives of 1.38 ± 0.38 and 71.82 ± 30.46 h, respectively. Compared with PAT images based on the intrinsic optical contrast in nude mice, the PAT images acquired within 2 h after intravenous administration of PEG-HAuNS showed the brain vasculature with greater clarity and detail. The image depicted brain blood vessels as small as ∼100 μm in diameter using PEG-HAuNS as contrast agents. Preliminary results showed no acute toxicity to the liver, spleen, or kidneys in mice following a single imaging dose of PEG-HAuNS. Our results indicate that PEG-HAuNS are promising contrast agents for PAT, with high spatial resolution and enhanced sensitivity.

Introduction

Noninvasive molecular and functional imaging techniques show promise for detecting and monitoring various physiological and pathological conditions in animals and, ultimately, humans. Among these imaging modalities, photoacoustic imaging offers higher spatial resolution and allows deeper tissues to be imaged than most optical imaging techniques [1], [2], [3], [4], [5], [6], [7], [8]. Photoacoustic tomography (PAT) referred to as optoacoustic tomography (OAT) is a hybrid, nonionizing imaging modality that combines the merits of both optical and ultrasonic imaging methods [6]. PAT detects absorbed photons ultrasonically through the photoacoustic effect: a short-pulsed laser irradiates biological tissues and induces wideband ultrasonic waves (photoacoustic waves) as a result of transient thermoelastic expansion [8]. Owing to the optical absorption of hemoglobin, PAT has been successfully applied to the visualization of different structures in biological tissues, especially to imaging of the cerebral cortex in small animals [9], [10], human blood vessels [10], [11], microvasculature of tumors [4], [12] and quantifying oxygen hemoglobin saturation in tumors [2], [5], [9], [13].

PAT is capable of monitoring exogenous optical contrast agents with high sensitivity and specificity [5], [11], [14]. Optical contrast allowed spectroscopic separation of signal contributions from multiple optical absorbers such as oxyhemoglobin, deoxyhemoglobin, and molecular contrast agents, thus enabling simultaneous molecular and functional imaging [5]. Recently, gold nanoparticles such as gold nanoshells [7], nanocages [15], [16], nanorods [17], [18], [19], [20], [21], [22], [23], and nanobeacons [24], as well as carbon nanotubes [25], [26], [27], have been employed as molecular contrast enhancement agents in the near-infrared (NIR) range, where intrinsic optical absorption in tissue is minimal and penetration is optimal [28]. These gold nanoparticles can generate greater photoacoustic signals due to light excitation in the NIR spectral region, where the signal ratio of gold nanoparticles to hemoglobin is higher, thus showing greater contrast to endogenous chromophores [14], [26]. Moreover, the gold nanoparticles are not susceptible to photobleaching, a problem commonly associated with the use of organic dyes. These nanoparticles showed enhanced photoacoustic mapping of cerebral vasculature [7], [15], sentinel lymph node [16], [18], [27] and tumor in vivo [25], [26], joint tissue ex vivo [29], and macrophages in atherosclerotic plaques [30]. PAT also can noninvasively image the progressive extravasation of gold nanoshells through solid tumor vasculature in vivo [31]. In addition, gadolinium (Gd)-doped, gold-speckled silica nanoparticles were synthesized as multimodal nanoparticulate contrast agents for noninvasive imaging using both magnetic resonance imaging (MRI) and PAT [32].

Hollow gold nanospheres (HAuNS) have the unique combination of small size (outer diameter, 40–50 nm), spherical shape, and a hollow interior that results from their highly uniform structure [33], [34], [35]. The HAuNS have a strong and precisely tunable absorption band peaked at ∼800 nm and are coated with polyethylene glycol (PEG, MW 5000) to increase their blood circulation half-life. The purpose of this study was to evaluate the use of these HAuNS as a new molecular contrast agent for PAT. The accuracy of PAT with pegylated HAuNS (PEG-HAuNS) of mouse brain vasculature was confirmed through histological analysis. The pharmacokinetics, biodistribution, and acute toxicity of PEG-HAuNS were assessed to provide preliminary data for future translation in clinical applications.

Section snippets

Materials

Biotinylated secondary antibody, straptavidin-horseradish peroxidase (straptavidin-HRP), 3,3′-diaminobensidine (DAB), hematoxylin, luciferin, and 4% paraformaldehyde were purchased from Fisher Scientific (Waltham, MA). Rat anti-mouse CD31 monoclonal antibody was purchased from Millipore (Billerica, MA). Alexa Fluor 594-tagged goat anti-rat IgG and RPMI-1640 phenol red free cell culture medium were purchased from Invitrogen (Carlsbad, CA). Rat anti-mouse CD 68 antibody was obtained from AbD

PEG-HAuNS characterizations

A transmission electron microscopy (TEM) image of PEG-HAuNS shows that the nanoparticles had a mean outer diameter of 43–46 nm and a mean shell thickness of 2–4 nm (Fig. 1A). The resonance absorbance of these nanoparticles was tuned to peak at 800 nm (Fig. 1B). The optical absorption and scattering coefficients of gold nanospheres (i.e., absorption and scattering cross-sections per unit volume) were calculated (Fig. 1C). Our calculations showed that HAuNS with an outer diameter of 45 nm and

Conclusion

In this study, we evaluated a new nanoparticulate optical contrast agent based on HAuNS for photoacoustic imaging. The particles can be fabricated with surface plasmon resonance tunable at the NIR wavelength (∼800 nm), which allows deeper penetration of laser light and lowers the intrinsic background noise. The photoacoustic efficiency of HAuNS is significantly greater than that of blood. The pegylated HAuNS showed the mouse brain vasculature with greater clarity and more detailed structures,

Acknowledgements

We thank Dawn Chalaire for editing the manuscript. This work was supported in part by a grant from the National Institutes of Health (R01 CA119387 and R44 CA110137), a Seed Grant through the Alliance for NanoHealth by the Department of Army Telemedicine and Advanced Technology Research Center (W81XWH-07-2-0101), and by the John S. Dunn Foundation.

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    Present address: Grodno State University, Grodno, Belarus.

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