Ping Yu

In Vitro and In Vivo Evaluation of Alexa Fluor 680- Bombesin(7-14)NH 2 Peptide Conjugate, a High-Affinity Fluorescent Probe with High Selectivity for the Gastrin- Releasing Peptide Receptor

Gastrin-releasing peptide (GRP) receptors are overexpressed on several types of human cancer cells, including breast, prostate, small cell lung, and pancreatic cancers. Bombesin (BBN), a 14–amino acid peptide that is an analogue of human GRP, binds to GRP receptors with very high affinity and specificity. The aim of this study was to develop a new fluorescent probe based on BBN having high tumor uptake and optimal pharmacokinetics for specific targeting and optical imaging of human breast cancer tissue. In this study, solid-phase peptide synthesis was used to produce H2N-glycylglycylglycine-BBN[7–14]NH2 peptide with the following general sequence: H2N-G-G-G-Q-W-A-V-G-H-L-M-(NH2). This conjugate was purified by reversed-phase high-performance liquid chromatography and characterized by electrospray-ionization mass spectra. The fluorescent probe Alexa Fluor 680-G-G-G-BBN[7–14]NH2 conjugate was prepared by reaction of Alexa Fluor 680 succinimidyl ester to H2N-G-G-G-BBN[7–14]NH2 in dimethylformamide (DMF). In vitro competitive binding assays, using 125I-Tyr4-BBN as the radiolabeling gold standard, demonstrated an inhibitory concentration 50% value of 7.7 ± 1.4 nM in human T-47D breast cancer cells. Confocal fluorescence microscopy images of Alexa Fluor 680-G-G-G-BBN[7–14]NH2 in human T-47D breast cancer cells indicated specific uptake, internalization, and receptor blocking of the fluorescent bioprobe in vitro. In vivo investigations in SCID mice bearing xenografted T-47D breast cancer lesions demonstrated the ability of this new conjugate to specifically target tumor tissue with high selectivity and affinity.

Near-infrared fluorescence imaging of gastrin releasing peptide receptor targeting in prostate cancer lymph node metastases

Development of high affinity and specificity molecular imaging probes that increase accuracy for early detection of lymph node (LN) metastases is important for improving survivorship in prostate cancer. We evaluated the specificity, sensitivity, and accuracy of fluorescence‐labeled bombesin (BBN) peptides to detect LN and systematic metastases in orthotopic mouse models bearing gastrin releasing peptide receptor (GRPR)‐positive human prostate cancer.

Dual-band Fourier domain optical coherence tomography with depth-related compensations.

Dual-band Fourier domain optical coherence tomography (FD-OCT) provides depth-resolved spectroscopic imaging that enhances tissue contrast and reduces image speckle. However, previous dual-band FD-OCT systems could not correctly give the tissue spectroscopic contrast due to depth-related discrepancy in the imaging method and attenuation in biological tissue samples. We designed a new dual-band full-range FD-OCT imaging system and developed an algorithm to compensate depth-related fall-off and light attenuation. In our imaging system, the images from two wavelength bands were intrinsically overlapped and their intensities were balanced. The processing time of dual-band OCT image reconstruction and depth-related compensations were minimized by using multiple threads that execute in parallel. Using the newly developed system, we studied tissue phantoms and human cancer xenografts and muscle tissues dissected from severely compromised immune deficient mice. Improved spectroscopic contrast and sensitivity were achieved, benefiting from the depth-related compensations.

Spatial convolution for mirror image suppression in Fourier domain optical coherence tomography

We developed a spatial convolution approach for mirror image suppression in phase-modulated Fourier domain optical coherence tomography, and demonstrated it in vivo for small animal imaging. Utilizing the correlation among neighboring A-scans, the mirror image suppression process was simplified to a three-parameter convolution. By adjusting the three parameters, we can implement different Fourier domain sideband windows, which is important but complicated in existing approaches. By properly selecting the window size, we validated the spatial convolution approach on both simulated and experimental data, and showed that it is versatile, fast, and effective. The new approach reduced the computational cost by 32% and improved the mirror image suppression by 10%. We adapted the spatial convolution approach to a GPU accelerated system for ultrahigh-speed processing in 0.1 ms. The advantage of the ultrahigh speed was demonstrated in vivo for small animal imaging in a mouse model. The fast scanning and processing speed removed respiratory motion artifacts in the in vivo imaging.

Imaging site-specific peptide-targeting in tumor tissues using spectral-domain optical coherence tomography

We report imaging studies on site-specific peptide-targeting in tumor tissues using newly developed optical peptide probes and spectral-domain optical coherence tomography (SD-OCT). The system used two broadband superluminescent light emission diodes with different central wavelengths. An electro-optic modulation in the reference beam was used to get full-range deep imaging inside tumor tissues. The optical probes were based on Bombesin (BBN) that is a fourteen amino acid peptide. BBN has high binding affinity to gastrin-releasing peptide (GRP) receptors overexpressed on several human cancer cell lines. Fluorescence BBN probes were developed by conjugating the last eight residues of BBN, -Q-W-A-V-G-H-L-M-(NH2), with Alexa Flour 680 or Alexa Fluor 750 dye molecules via amino acid linker -G-G-G. The SD-OCT imaging can identify normal tissue and tumor tissue through the difference in scattering coefficient, and trace the BBN conjugate probes through the absorption of the dye molecules using the twowavelength algorithm. We performed the specific uptake and receptor-blocking experiments of the optical BBN probes in severely compromised immunodeficient mouse model bearing human PC-3 prostate tumor xenografts. Tumor and muscle tissues were collected and used for SD-OCT imaging. The SD-OCT images showed fluorescence traces of the BBN probes in the peptide-targeted tumor tissues. Our results demonstrated that SD-OCT is a potential tool for preclinical and clinical early cancer detection.

Design, Synthesis, and in Vitro and in Vivo Evaluation of High Affinity and Specificity Near-Infrared Fluorescent Bombesin Antagonists for Tumor Imaging

The bombesin (BBN) antagonist binds with high affinity to the gastrin releasing peptide receptor (GRPr), a receptor overexpressed on many human cancers. We present an investigation employing BBN antagonist for highly specific near-infrared fluorescence (NIRF) imaging of GRPr-positive tumors. Nine NIRF-dye labeled BBN antagonists with differing linkers and dyes were synthesized and characterized to screen for the optimal agent. Three novel agents, AF750-G-pip-Sta-BBN (1), AF750-GSG-Sta-BBN (2), and AF750-6Ahx-Sta-BBN (3), exhibited an excellent binding-specificity and affinity to human PC-3 prostate cancer cells in vitro, and a remarkable in vivo tumor-selectivity and NIRF imaging sensitivity in PC-3 tumor-bearing mice. Compound 1 showed the fastest, and 3, the slowest, pharmacokinetics on the tumor sites. Despite of high tumor uptake, 2 had a low pancreas uptake distinct from 1 and 3 at 0.44 nmol dose. This difference was attributed to the inherent linker properties such as the hydrophilicity, polarity, and charge.

Effects of wavelength and side lobes on Airy beam for optical coherence tomography

Optical coherence tomography system using a broad wavelength band Airy beam generated by a transmissive cubic phase mask. We experimentally show that the detrimental effects from the side lobes and wavelength dependent curvature are negligible.

A new near-infrared absorption and fluorescent probe based on bombesin for molecular imaging

We have developed a series of new dye bombesin conjugates for site-specific absorption and fluorescence imaging of human prostate and breast cancers. Bombesin (BBN), an amphibian analog to the endogenous ligand, binds to the gastrin releasing peptide (GRP) receptors with high specificity and affinity. Previously, we developed an Alexa Fluor 680-GGG-BBN peptide conjugate which demonstrated high binding affinity and specificity for breast cancer cells in the in vitro and in vivo tests (Ref: Ma et al., Molecular Imaging, vol. 6, no. 3, 2007: 171-180). This probe can not be used as an absorption probe in near-infrared imaging because its absorption peak is in the visible wavelength range. In addition, site specific longer wavelength fluorescent probe is desired for in vivo molecular imaging because long wavelength photons penetrate deeper into tissue. The new absorption and fluorescent probe we developed is based on the last eight-residues of BBN, -Q-W-A-V-G-H-L-M-(NH2), and labeled with AlexaFluor750 through a chemical linker, beta-alanine. The new probe, Alexa Fluor 750-BetaAla-BBN(7-14)NH2, exhibits optimal pharmacokinetics for specific targeting and optical imaging of the GRP receptor over-expressing cancer cells. Absorption spectrum has been measured and showed absorption peaks at 690nm, 720nm and 735nm. Fluorescent band is located at 755nm. In vitro and in vivo investigations have demonstrated the effectiveness of the new conjugates to specifically target human cancer cells overexpressing GRP receptors and tumor xenografts in severely compromised immunodeficient mouse model.

Using fluorescence molecular tomography for multimodality fusion imaging

Multimodality molecular imaging that combines anatomical and functional information has shown promise in development of tumor-targeted pharmaceuticals for cancer detection or therapy. Most multimodality imaging techniques are based on nuclear imaging modalities and MRI or CT. Fluorescence molecular tomography (FMT) is an emerging optical modality for non-invasive functional imaging and early diagnosis of carcinoma. Three-dimensional FMT can differentiate tissue physiological changes in vivo to provide functional information when used in conjunction with cancer cell selectively targeted probes. In this study, we present the design of such a system for multimodality molecular imaging. A frequency domain radio frequency technique based on commercial amateur radio equipment has been developed. A heterodyne method is used to transfer a low frequency oscillation into a single-side-band at radio frequency. The difference in phase, caused by fluorescence photon density wave, is detected between a transmitting fiber and a receiving fiber bundle, and then measured at lower frequency after demodulation. To achieve multimodality molecular imaging, a new fluorescent labeled tumor-targeting probe, fluorescent bombesin conjugates, has been developed with high affinity and specificity for targeting breast cancer cells. The developed multimodality fusion strategy will provide increased sensitivity/specificity for cancer cells, with respect to any single imaging modality.