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Photoacoustics

Photoacoustic Molecular Imaging for the Diagnosis and Treatment of Prostate Cancer: Photoacoustic (PA) imaging can sample optical phenomena within tissue to a depth of several cm [Oraevsky 1994, Kruger 1994].Absorbed pulsed laser light in the near infrared (NIR) creates acoustic sources whose strength is proportional to the local absorption at the incident optical wavelength. An image is formed using ultrasound (US) technology, with PA contrast directly related to optical absorption. Overall, PA holds great promise as a molecular imaging tool, marrying the molecular sensitivity of optics with the penetration, spatial resolution, and real-time capability of US. Moreover, PA imaging can be integrated with molecular therapy to produce low-cost tools for molecular medicine. Our immediate focus is the diagnosis and treatment of prostate cancer. Worldwide in males, only lung cancer has a higher incidence and prostate cancer has the highest revalence [Parkin 2005]. Nearly one in six American men will be diagnosed with prostate cancer during his lifetime. Current screening tests based on prostate specific antigen (PSA) are highly sensitive, but not very specific [Eastham 2003]. A follow up measurement to augment PSA tests and monitor treatment is greatly needed. The prostate is also an ideal target because simultaneous US/PA imaging is possible using a potentially low-cost, integrated US/PA transrectal ultrasound (TRUS) probe. Finally, these cancers may be easily treated using molecularly targeted photothermal therapies guided by real-time US/PA imaging.

In previous work, we have investigated PA imaging for prostate cancer diagnosis using nanoprobes (NP) such as gold nanorods (GNRs) targeted to membrane-bound proteins identified as cancer biomarkers [Agarwal 2007]. Typical PA images are presented in the left panel of figure 5 for cell culture experiments testing the NP’s sensitivity and specificity. The signal in the top compartment is 20 dB higher than that in the control, indicating an order of magnitude higher GNR concentration due to specific binding of the targeting antibody. We have also demonstrated that GNRs can be detected at depth, as illustrated in the center panel of figure 5 in which a color-coded PA image is superimposed on an ultrasound scan of an excised prostate injected with a GNR solution The PA image clearly shows high NP concentration near the injection site.

Figure 5. Left panel: PA micrographs showing enhanced uptake of molecularly targeted GNRs; center panel: PA color image of GNR injected into prostate interior superimposed on a ultrasound scan of an excised prostate; right panel: PA imaging during photothermal therapy monitors temperature changes.

An important feature of this technology is that the same nanoparticle can also be used for cancer treatment, enabling low-cost, image-guided therapy. Since diffuse tumors in the prostate are not well suited to surgical resection, molecularly-targeted photothermal ablation therapy provides a minimally invasive alternative. PA imaging can monitor GNR uptake and also map temperature changes due to therapeutic laser light. The PA pressure depends on temperature. Figure 5 (right panel) illustrates how PA imaging can assess temperature change during molecularly-targeted photothermal therapy [Emelianov 2009]. A continuous wave laser tuned to the GNR absorption peak provided the therapeutic source. A tumor bearing mouse was injected with GNRs while simultaneous US/PA images were acquired. Thermal images derived from PA signals overlaid on US images show the gradual temperature increase as therapy progresses.

For many clinical applications, background tissue signals limit both the sensitivity and specificity of molecular imaging studies. To suppress these signals, we have explored a new class of PA agents, called coupled agents. Here we focus on a coupled agent combining magnetomotive forces with tuned NIR absorption. Magnetomotive manipulation suppresses background signals, while high NIR absorption provides a large PA signal per particle. Leveraging recent advances in coupled agents, our initial studies on molecular US/PA imaging and photothermal therapy to manage prostate cancer have two specific aims.

Aim 1: Design and construct an integrated magnetomotive PA/US imaging system producing a time varying (DC to kHz) magnetic field of at least one Tesla at a depth of 1 cm from the electromagnet. This field can induce controlled motion with a penetration depth of several centimeters. This should be sufficient for clinical translation to human prostate imaging using an integrated PA/US transrectal ultrasound (TRUS) probe.

Aim 2: We will also develop and characterize a new magnetic core – gold shell nanoprobe with combined magnetic and optical properties. It enables the magnetomotive PA imaging methods developed in aim 1 and also provides a vehicle for molecularly targeted photothermal therapy.

We have tested magnetomotive PA imaging using 30 nm iron oxide magnetic NPs (MNPs). A polyvinyl alcohol (PVA) phantom with two inclusions, one containing MNPs and the other black ink (background), was imaged at 532 nm optical wavelength for 50 seconds at a frame rate of 20 Hz. Between 15–30 seconds, an electromagnet provided magnetomotive force inducing motion within the MNP inclusion. The top panel in figure 6 shows an averaged PA image, in which the ink region dominates. To increase MNP contrast, a motion-enhanced PA image was created by inputting pixel-wise displacements into a slow-time matched filter and variance estimator to generate a weighting image. The bottom panel shows the product of PA and weighting images. Clearly, the background was suppressed by more than 40 dB. Studies for specific aim 1 will build on these results to move toward a translatable imaging protocol.

Figure 6. Averaged PA image in which the ink region is dominant (top); product of the original PA image and the weighting image based on magnetomotive-induced motion (bottom). The background is suppressed by over 40 dB.

Conventional MNPs do not have sufficient NIR absorption for clinical applications. To overcome this limitation, we have collaborated with Professor Xiaohu Gao on a new contrast agent addressing the long sought-after goal of an Au-coated MNP with precisely controlled shell thickness and smooth surface. Professor Gao’s lab recently invented a method enabling ultrathin Au coatings on isolated nanoparticles. Absorption per particle will be quantified using a spectrophotometer. We will also measure the magnetomotive force possible at depth. PVA phantoms with controlled elastic, acoustic, optical, and magnetic properties will be fabricated using established techniques. Inclusions placed at different depths and with different particle concentrations will be imaged. Imaging depth and magnetomotive contrast enhancement will then be quantified. We will also develop an integrated PA/US imaging protocol using time varying magnetic fields to maximize NP contrast. Magnetomotive force generation will be synchronized with PA/US image acquisition. PA and US images before, during, and after energizing the electromagnet will be interleaved and acquired continuously. They will be input to a motion estimation algorithm to enhance regions with MNP-Au core-shell NPs. We will design motion patterns combined with optimal slow-time filtering to maximize NP contrast.

In parallel, we will further develop our NP platform and functionalize it for the prostate. Monodisperse Fe₂O₃ and Fe₃O₄ NPs will be synthesized via thermolysis of iron precursors in organic solvents. We will produce 5-40 nm diameter hydrophobic MNPs to probe the optimal size for imaging and targeting. For Au nanoshell growth, MNPs will be coated with a layer of positively charged peptide, poly-L-histidine (PLH). The imidazole groups in PLH immobilize Au³⁺ ions at high packing density. Optical absorption is tuned by the ratio of Au shell thickness to overall NP size. The smallest NP with high NIR absorption (800-850 nm) and ample stability under laser irradiation and magnetization will be used for all subsequent studies.

For tumor imaging and treatment, NPs must be targeted. We will test a non-antibody approach using RNA aptamers targeting prostate specific membrane antigen (PSMA). Chemically modified aptamers will be used to enhance stability against nucleases [Lin 1994]. They will be custom-made with a thiol group at the 3’ end, away from the aptamer binding site, and stably linked to the MNP-Au NPs through the strong Au-thiolate interaction. Targeting specificity will be tested on PSMA positive prostate cancer cell lines such as LNCaP and controls using primarily optical and PA methods. These same NPs will be tested for photothermal therapy, providing a minimally invasive alternative to prostate surgery. This procedure is relatively straightforward if monitored and controlled in real time. Initial studies will be performed in tissue phantoms and cell cultures to quantify the accuracy, sensitivity, and spatial resolution of PA-derived temperature maps.