Molecular Imaging

There is a dire need for a reliable, non-invasive imaging tool to detect, diagnose and characterize cancer – one of the leading causes of death in the United States. The early detection of cancer is absolutely necessary for effective therapeutic outcome and is a primary indicator for long term survival. Moreover, demarcating tumor boundaries with high specificity is required to direct therapeutic interventions to tumor location and cause less or no damage to the surrounding healthy tissue. This project aims to utilize the photoacoustic imaging technique and molecularly targeted plasmonic gold nanoparticles to detect highly proliferative cancerous cells [1].

Figure 1: Extinction spectra of targeted and non-targeted tissue phantoms.

Epithelial cancer cells tend to overexpress epithelial growth factor receptor (EGFR), causing the specifically targeted nanoparticles to cluster on the cell surface. This clustering leads to plasmon resonance coupling between nanoparticles and a red shift in the plasmon resonance frequency of the gold nanoparticle assembly [2-4]. The absorbance spectra of the tissue phantoms specifically targeted (red solid line) with gold nanoparticles and non-targeted tissue phantoms (green solid line) are shown in Fig. 1. The non-targeted phantom has an absorbance peak at 520 nm, which is in excellent agreement with the absorbance spectrum of a suspension of isolated gold nanoparticles. The targeted phantom has the peak red-shifted and broadened due to EGFR-mediated aggregation of gold nanoparticles. The red-shift provides the opportunity to differentiate cancer cells from surrounding benign cells by using a combination of labeling with gold nanoparticles and multi-wavelength illumination.

The application of molecular targeted gold nanoparticles in photoacoustic imaging was demonstrated on tissue phantoms prepared with human keratinocyte cell line. The 532 nm and 680 nm pulsed laser illuminations were chosen for the photoacoustic experiments due to overlap with the absorbance spectra of the isolated and aggregated gold nanoparticles. Our study shows that photoacoustic imaging can differentiate between cancer cells labeled with the molecular targeted gold nanoparticles and the cells mixed with non-specific gold nanoparticles, even when the concentration of isolated non-targeted particles is much higher.

Figure 2: Darkfield and photoacoustic images of targeted and non-targeted tissue phantom.

The darkfield image of the targeted phantom (Fig. 2A) shows orange colored cells caused by the plasmon-resonance scattering of anti-EGFR conjugated gold nanoparticles which interact with EGFR molecules on the cytoplasmic membrane of A431 cells. The photoacoustic images of the targeted tissue phantom. The non-targeted tissue phantom (Fig. 2D) has gold particles in suspension surrounding the cells as shown in the darkfield image. These isolated gold particles are associated with the greenish haze in the background surrounding the unlabeled A431 cells which appear bluish in the image.  At 532 nm laser irradiation, the photoacoustic image of the non-targeted phantom (Fig. 2E) indicates higher optical absorbance than the targeted phantom (Fig. 2B). At 680 nm illumination, very little optoacoustic response was obtained in the non-targeted phantom (Fig. 2F), unlike the targeted tissue phantom that produced signal from the entire thickness of approximately 1 mm (Fig. 2C).  Hence in congruence with the hyperspectral analysis (Fig. 1), a relatively low overall bulk extinction coefficient was observed in the non‑targeted phantom as compared to targeted phantom at 680 nm.

Gold nanoparticles have excellent biocompatibility [5] and the conjugation protocols to attach proteins to gold nanoparticles are also well developed [6-8]. The optoacoustic imaging with gold nanoparticles demonstrated in this report can be potentially extended to a combined diagnostic imaging and therapy approach. Photothermal therapy with plasmonic nanoparticles was previously demonstrated for both pulsed [9] and continuous light sources [10]. Based on the information obtained with optoacoustic imaging, pulsed or continuous wave photothermal therapy could be performed to induce localized tumor necrosis, potentially even using the same light source as was used in optoacoustic imaging.  Moreover, ultrasound based strain imaging can be performed together with optoacoustic imaging and phototherapy at no additional cost to monitor tumor necrosis over time [11].

In conclusion, the optoacoustic imaging technique could detect tumors by selectively targeting cancerous cells that overexpress EGFR. In our studies, optoacoustic imaging was performed at 532 nm and 680 nm on three tissue phantoms prepared using A431 skin cancer cells targeted with anti-EGFR gold bioconjugates. The results of our study demonstrate that using molecular targeted gold nanoparticles and optoacoustic imaging, specific molecular differentiation and highly sensitive and selective detection of cancer could be achieved. Further studies are required to evaluate this molecular specific imaging technique in vivo and its potential in combination with phototherapy.

References:

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[7]        G. T. Hermanson, Bioconjugate techniques. San Diego, CA: Academic, 1996.

[8]        M. Horisberger, "Colloidal gold: a cytochemical marker for light and fluorescent microscopy and for transmission and scanning electron microscopy," Scan. Electron Microsc, vol. 2, pp. 9-31, 1981.

[9]        C. M. Pitsillides, E. K. Joe, X. Wei, R. R. Anderson, and C. P. Lin, "Selective cell targeting with light-absorbing microparticles and nanoparticles," Biophys. J., vol. 84, pp. 4023-4032, 2003.

[10]      L. R. Hirsch, R. J. Stafford, J. A. Bankson, S. R. Sershen, B. Rivera, R. E. Price, J. D. Hazle, N. J. Halas, and J. L. West, "Nanoshell-mediated near-infrared thermal therapy of tumors under magnetic resonance guidance," Proc. Natl. Acad. Sci. U S A, vol. 100, pp. 13549-13554, 2003.

[11]      J. Shah, S. R. Aglyamov, K. Sokolov, T. E. Milner, and S. Y. Emelianov, "Ultrasound-based thermal and elasticity imaging to assist photothermal cancer therapy - Preliminary study," presented at Proceedings of the IEEE International Ultrasonics Symposium, Vancouver, Canada, 2006. PDF