Extending the Luminescence Lifetime in Breast Cancer Diagnostics

Kansas State University, Manhattan KS

Investigators

Abstract

CBET-1159966 Bossmann Intellectual Merit. Developing cancer diagnostics for recognizing breast cancer at the localized stage, in combination with the exact identification of the tumor boundaries during surgery, would be very advantageous, because virtually all breast cancer mortality occurs after the cancer has metastasized. In 2012, the National Cancer Institute anticipates approx. 230,000 new breast cancer cases and 40,000 deaths. The 5-year relative survival of breast cancer decreases to 23 percent at the distant stage, from 83 percent at the regional stage and 98 percent at the localized stage. Numerous proteases are overexpressed in breast cancer and surrounding tissue. Luminescence assays have the potential to be three orders of magnitude more sensitive, when compared to immunoassays. Furthermore, luminescence assays are able to detect only chemically active proteases, whereas immunoassays detect a mixture of active proteases and zymogens. The major impediment that has prevented the breakthrough of luminescence assays for in-vitro and in-vivo protease detection is the autofluorescence of blood and especially tissue. Autofluorescence of biospecimens limits the sensitivity, specificity and statistical reproducibility of optical cancer detection methods. This is especially valid for the detection of early cancers (in blood tests) and early metastases (in-vivo). Using the current state-of-the-art cancer treatment, early detection of cancer remains the only valid option for decreasing cancer mortality. Whereas spin-allowed energy transfer proceeds within nanoseconds, spin-forbidden energy transfer requires significantly more time (50 to 250 ns). The autofluorescence of blood and tissue decays within less than 25ns. Therefore, only the signal from the luminescent protease sensor will be visible after that time. The absence of autofluorescence will greatly enhance the signal-to-noise ratio. It will also significantly shorten the time required for in-vitro and in-vivo protease measurements. Spin-forbidden energy transfer occurs between a ruthenium-poly-pyridyl sensitizer and a chemically attached cyanine-dye upon excitation of the sensitizer. The sensitizer-dye diad will be attached to iron/iron oxide core/shell nanoparticles. Intense plasmon quenching of all luminescence occurring from the diad will occur as long as the diad is attached to the nanoparticle. The linker between diad and nanoparticle will consist of a consensus sequence (oligopeptide) that can be cleaved quickly only by its respective protease. Up to four different cyanine dyes featuring different absorption and emission maxima will be used as components in a diad. Upon release by a protease from the nanoparticle, each diad will exhibit a characteristic, long-lived luminescence signal (Light Switch for Protease Detection). The nanoplatforms capable of spin-forbidden energy transfer mediated luminescence will be tested in BALB C mice bearing 4T1 tumors. The 4T1 metastatic breast cancer model is a syngeneic model. We will image the tumor location, surgically excise the tumors, characterize the excised tumor tissue and conduct a mouse survival study after tumor excision. The core/shell iron/iron oxide NPs can also potentially be used for MRI imaging. Broader Impacts. Significance and Broader Impact: The results from the project could potentially lead to a new and relatively inexpensive method for early cancer diagnostics and a new standard of care in breast cancer surgery. In addition, the project will provide quantitative data with respect to how the activity of cancer-related proteases varies depending on in-vitro and in-vivo conditions. These findings will be of importance for the development of enzyme-activatable anticancer drugs. The Departments of Anatomy&Physiology and Chemistry at Kansas State University (KSU) will create one block course for the duration of one week in order to enhance their students? perspective on biophotonics, biosensing and nanomedicine: Dr. Bossmann will develop and teach this block course entitled ?Theranostics? during the spring semester as a part of the ?Materials Chemistry Course? (CHM 852) of Chemistry at KSU. This course is open to all graduate and undergraduate students in veterinary medicine, chemistry, biochemistry, biology and physics. The proposed study will help extend this research to ongoing collaborative efforts on cancer therapy and diagnostics at Kansas State University. The research will also create opportunities to incoming REU (Research Experience of Undergraduates), SUROP (Summer Undergraduate Research Opportunity Program) and DSP (Developing Scholars? Program) students at KSU. The proposed instrumental development will be open to collaborations with other Universities and Research Institutions.