In vivo Fluorescence Imaging of Solid Tumor-Bisphosphonate Interaction.
Rao V. L. Papineni*1, Thirupandiyur Udayakumar2, Mansoor Ahmed2, John Pizzonia1, and Alan Pollack2
1. Carestream Molecular Imaging, 4 Research Dr., Woodbridge, CT, 06525, USA. 2. Department of Radiation Oncology, University of Miami, Miami, FL, USA
* rao.papineni1@carestreamhealth.com

Bisphosphonates are used for the osteoporosis and in palliative cancer related bone pain management. A role in the cancer cell proliferation and metastases prevention has been proposed. Here, we determined the possibility of utilizing NIR dye conjugated bisphosphonate drug as a potential theragnostic agent. Alendronate was conjugated to water soluble, near-infrared tricarbocyanine, cyclic enamine-functionalized dye, and the imaging probe was used for the purpose. Near-infrared (NIR) fluorescence imaging of both LNCaP cells in cell culture and grown orthotopically in nude mice indicate NIR-bisphosphonate binding. Both in vivo and ex vivo NIRF image analysis of 4 nmol of NIR conjugate delivery show substantial tumor residence time and routine biodistribution to areas of high metabolic bone activity. We present here early evidence of NIR-bisphosphonate analog as a potential diagnostic tool. 15 min 18 min 24 min 48 min 82 min





Ex Vivo Tumor Imaging. The excised tumors of control and NIR-alendronate injected mice were imaged (NIRF:exp: 60 sec; 80 FOV). The X-ray (upper left) and NIRF-X-ray overlay (upper right) are shown. The fluorescence data is plotted to the right.



NIRF Imaging of Orthotopic Tumor

Non-invasive in vivo imaging of NIR-Alendronate binding at orthotopically grown prostate tumor. Prolonged residence (18 hrs) of 4 nmoles NIR-alendronate injected into the tumor was determined by Time-Lapse Fluorescence and X-ray imaging using Carestream In-Vivo Multispectral FX PRO.
[EX: 650 EM: 790; exp: 15 sec (above panels only); FOV: 180. X-ray : exp: 60 sec 0.4 mm filter; Energy (Kev): 12.79]



N H 640 LSS Xsight dye

640 LSS Xsight dye

in vivo NIRF Imaging: LNCaP cells were grown orthotopically in nude mice. NIR tricarbocyanine, cyclic enamine dye conjugated alendronate (4 nmoles) was injected into the tumor after providing anesthesia. Control mice were injected with free dye. X-Ray and NIRF imaging using Carestream in-Vivo Multispectral FX Pro with parameters as described below.

Overlay images of the NIR fluorescence signals (time shown) on X-ray are shown in the upper and the lower two panels. Negative control- the NIR dye alone was injected into tumor of anesthetized mice (not shown here). The in-vivo and ex-vivo imaging were performed simultaneously with the NIR-alendronate analysis. The ex-vivo sample analysis (3-D plot) are shown in the panel to the right.

NIR- Bisphosphonate Cancer Cell binding

Preparation of the bisphosphonate-Near-IR dye Conjugate. A solution containing 5.57 mg (5.O mol) of LSS dye (nearinfrared tricarbocyanine, cyclic enamine functionalized dye) as its NHS ester in 1.0 mL of PBS buffer at pH 8.7 and 0.5 mL of THF was treated with 35.2 g (100 mol) of alendronate and 25.9 mg (200 umol) of diisopropylethylamine, then stirred at ambient temperature overnight. The resultant product was purified directly by preparative HPLC using a 20mm x 20 cm Phenomenex Luna, 10 m C-18 with a gradient over 15 minutes of 10-50 % methanol versus 0.1 M ammonium acetate buffer at pH 6.9. The result product was lyophilized three times to yield 2.4 mg of the product as a light, fluffy dark blue-green powder, 99 % purity by HPLCMS with a consistent mass spectra. The excitation spectra  max observed = 642 nm, emission spectra  max = 740 nm, thus displaying the large Stokes shift.

Representative Fluorescence Imaging Parameters. Exposure Type: 15.000 sec. Exposure: X-binning: Y-binning: Illumination Source: f-Stop: FOV: Focal Plane: Vertical Resolution: Horizontal Resolution: Magnification Stage: Excitation Filter: Emission Filter: Standard Exposure Time: 1 of 1 No No Multi-wavelength 2.58 180 mm 14.1 mm 290 ppi 290 ppi No 650 790

30 sec

60 sec

4 hrs

18 hrs
LNCaP prostate cancer cell lines grown were incubated with 0.4 M (upper panels) and 0.2 M (lower panels) of NIR - tricarbocyanine, cyclic enamine dye conjugated alendronate. Binding of NIR-alendronate was observed using NIR microscopy (Phase Contrast in Green; NIRF in Red).

Theragnostics - Seek, See, Subdue and Salve

 NIR - tricarbocyanine, cyclic enamine dye conjugated alendronate is a potential tumor theragnostic agent - bisphosphonates has been shown to prevent cancer cell proliferation and prevent metastasis. Analogs of NIR alendronate consequently are capable to detect bone lesions and secondary metastasis sites outside of the solid malignant site.  Screening of high affinity analogs of bisphosphonates for tumor binding activity is now feasible with the noninvasive approach demonstrated here. 

Bisphophonate analogs the new class of “theragnostic” genre of drugs?
Courtesy Dr. Michael Bolognese

Ex Vivo Imaging: X-ray (left) and NIRF-X-ray overlay (right panel). to show binding of NIR –alendronate at orthotopic tumor after 18 hrs.

Carestream is a trademark of Carestream Health, Inc. Carestream Molecular Imaging is a division of Carestream Health, Inc. Although the Carestream In-Vivo Multispectral Imaging System FX can be used for in vivo and in vitro molecular imaging of materials, researchers should be aware that the methods of preparing and viewing the materials for molecular imaging may be subject to various patent rights.

"Molecular Imaging - Wisdom To See For Maladies To Flee" Dr. Rao V. L. Papineni

© Carestream Health, Inc.

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