The NHS esters of pHrodo and TAMRA were purchased from Invitrogen Corporation (Carlsbad, CA). Avidin was purchased from Pierce Biochemical, Inc. (Milwaukee, WI). Trastuzumab, an FDA-approved humanized anti-HER-2 antibody, was purchased from Genentech Inc. (South San Francisco, CA). All other chemicals used were of reagent grade.
Determination of pHrodo chemical structure
The structure of pHrodo has been determined with mass spectroscopic analysis and NMR. The NHS ester of pHrodo was used for this purpose. Briefly, mass spectroscopic analysis was performed with LCMS-IT-TOF systems (Shimadzu America Co., Columbia, MD). 1
H- and 13
C-NMR analyses were performed with a Gemini or Mercury 300 MHz spectrometer (Varian, Palo Alto, CA). By summarizing both results with reference to the patent document,11
the structure of pHrodo was suggested as shown in .
NMR 1H (300 MHz, DMSO) δ: 7.35 (1H, d, J 1Hz, Bz –CH–). 7.32 (1H, d, J 1Hz, Bz –CH–). 7.18 (1H, d, J 1Hz, Ar –CH–). 7.14 (1H, d, J 1Hz, Ar –CH–). 6.93 (2H, d, J 1Hz, 2Ar –CH–). 6.82 (2H, d, J 1Hz, 2Ar –CH–). 3.65 (1H, s –CH3), 3.41 (2H, q, –N–CH2–CH3), 3.30 (6H, s, Bz–N–(CH3)2), 3.28 (12H, s, 2Ar–N–(CH3)2), 2.82 (2H, s, –CH3), 2.75 (2H, t, –N–CH2CH2–), 2.65 (4H, s, suc –CH2CH2–), 2.27 (2H, t, sucO2C–CH2CH2–), 1.89 (2H, m, –CH2CH2CH2–), 1.09 (3H, t, –N–CH2–CH3). LCMS-IT-TOF (m/z) 642.33.
Synthesis of fluorophore conjugated avidin and trastuzumab
Avidin (14 nmol) was incubated with the NHS ester of pHrodo or TAMRA (70 nmol) in 0.1 M Na2HPO4 (pH 8.5) at room temperature for 30 min. Each mixture was purified with a Sephadex G50 column (PD-10; GE Healthcare, Piscataway, NJ). Trastuzumab (6.5 nmol) was treated with NHS esters (65 nmol) and purified in the same manner as above. The number of fluorophore molecules per avidin or trastuzumab was ~2–3.
Determination of fluorescence activation capacities in vitro
The quenching–dequenching abilities of each conjugate were investigated by treating the conjugates with 5% SDS to disassociate molecular interactions between fluorophores. For pHrodo probes, H3PO4 was added to the solution (430 mM) to make it acidic (pH ~ 4). For antibody conjugates, 2-mercaptoethanol (2-ME) was also added to uncouple S–S bonds between the light and heavy chains and the mixture was heated at 95 °C for 2 min. The fluorescence signal intensity of each sample was measured with a fluorescence spectrometer (Perkin-Elmer LS55, Perkin-Elmer, Shelton, CT, USA). The absorption spectrum was measured with a UV-Vis system (8453 Value UV-Visible Value System; Agilent Technologies, Santa Clara, CA).
A d-galactose receptor positive ovarian cancer cell line, SHIN3, and a HER2 gene transfected cell line, NIH3T3/HER2+ were used. The cells were grown in RPMI 1640 (Invitrogen Corporation, Carlsbad, CA) containing 10% fetal bovine serum (Invitrogen Corporation, Carlsbad, CA), 0.03% l-glutamine, 100 units mL−1 penicillin, and 100 µg mL−1 streptomycin in 5% CO2 at 37 °C.
Fluorescence microscopy studies
3T3/HER2+ cells (1 × 104) were plated on a cover glass-bottomed culture well and incubated for 16 h. Then, trastuzumab-TAMRA or trastuzumab-pHrodo were added to the medium (30 µg mL−1), and the cells were incubated for either 1, 8 or 24 h. Cells were washed once with PBS, and fluorescence microscopy was performed using an Olympus BX51 microscope (Olympus America, Inc., Melville, NY) equipped with the following filters: excitation wavelength 530 to 570 nm, emission wavelength 590 nm long pass. Transmitted light differential interference contrast images were also acquired.
In vivo fluorescence imaging in peritoneal tumor bearing mice
All procedures were carried out in compliance with the Guide for the Care and Use of Laboratory Animal Resources (1996), National Research Council, and approved by the National Cancer Institute Animal Care and Use Committee. Intraperitoneal (i.p.) tumor implants were introduced by an i.p. injection of 2 × 106 SHIN3 (d-galactose receptor positive ovarian cancer cells) in female nude mice (National Cancer Institute Animal Production Facility, Frederick, MD). The imaging studies were performed 3 weeks after the injection of tumor cells.
-Galactose targeting probe, pHrodo conjugated avidin (Av-pHrodo) or TAMRA conjugated avidin (Av-TM) was injected into tumor bearing nude mice (50 µg, i.p., n
= 4 for each group). The mice were anesthetized with pentobarbital (30 mg kg−1
i.v., with 0.1% scopolamine butyl bromide) 1 or 2 h after the probe injection, and fluorescence endoscopy was performed using the Olympus EVIS ExERA-II CLV-180 system (Olympus Corp., Tokyo, Japan). The excitation wavelength used was 543/25 nm and the emission filter was 597.5/55 nm. The fluorescence intensities of tumor and background were measured on the images (n
= 20) using Image-J software (National Institutes of Health, Bethesda, MD, http://rsb.info.nih.gov/ij/
) and the tumor to background ratio was calculated. After the endoscopy, the mice were sacrificed with carbon dioxide and ex vivo
imaging of the peritoneal implants was performed on a Maestro In-Vivo Imaging System (CRi, Inc., Woburn, MA). A band-pass excitation filter from 503 to 555 nm and a long-pass emission filter over 580 nm were used. The tunable emission filter was automatically stepped in 10 nm increments from 500 to 800 nm while the camera captured images at each wavelength interval with constant exposure. The spectral fluorescence images consisting of auto-fluorescence spectra and the spectra from pHrodo or TAMRA were obtained and then unmixed based on their spectral patterns.