Continuous sensing of tumor-targeted molecular probes with a vertical cavity surface emitting laser-based biosensor.

Molecular optical imaging is a widespread technique for interrogating molecular events in living subjects. However, current approaches preclude long-term, continuous measurements in awake, mobile subjects, a strategy crucial in several medical conditions. Consequently, we designed a novel, lightweight miniature biosensor for in vivo continuous optical sensing. The biosensor contains an enclosed vertical-cavity surface-emitting semiconductor laser and an adjacent pair of near-infrared optically filtered detectors. We employed two sensors (dual sensing) to simultaneously interrogate normal and diseased tumor sites. Having established the sensors are precise with phantom and in vivo studies, we performed dual, continuous sensing in tumor (human glioblastoma cells) bearing mice using the targeted molecular probe cRGD-Cy5.5, which targets αVß3 cell surface integrins in both tumor neovasculature and tumor. The sensors capture the dynamic time-activity curve of the targeted molecular probe. The average tumor to background ratio after signal calibration for cRGD-Cy5.5 injection is approximately 2.43±0.95 at 1 h and 3.64±1.38 at 2 h (N=5 mice), consistent with data obtained with a cooled charge coupled device camera. We conclude that our novel, portable, precise biosensor can be used to evaluate both kinetics and steady state levels of molecular probes in various disease applications.

Family of enhanced photoacoustic imaging agents for high-sensitivity and multiplexing studies in living mice.

Photoacoustic imaging is a unique modality that overcomes to a great extent the resolution and depth limitations of optical imaging while maintaining relatively high contrast. However, since many diseases will not manifest an endogenous photoacoustic contrast, it is essential to develop exogenous photoacoustic contrast agents that can target diseased tissue(s). Here we present a family of novel photoacoustic contrast agents that are based on the binding of small optical dyes to single-walled carbon nanotubes (SWNT-dye). We synthesized five different SWNT-dye contrast agents using different optical dyes, creating five "flavors" of SWNT-dye nanoparticles. In particular, SWNTs that were coated with either QSY(21) (SWNT-QSY) or indocyanine green (SWNT-ICG) exhibited over 100-times higher photoacoustic contrast in living animals compared to plain SWNTs, leading to subnanomolar sensitivities. We then conjugated the SWNT-dye conjugates with cyclic Arg-Gly-Asp peptides to molecularly target the α(v)ß(3) integrin, which is associated with tumor angiogenesis. Intravenous administration of these tumor-targeted imaging agents to tumor-bearing mice showed significantly higher photoacoustic signal in the tumor than in mice injected with the untargeted contrast agent. Finally, we were able to spectrally separate the photoacoustic signals of SWNT-QSY and SWNT-ICG in living animals injected subcutaneously with both particles in the same location, opening the possibility for multiplexing in vivo studies.

Pharmacokinetically stabilized cystine knot peptides that bind alpha-v-beta-6 integrin with single-digit nanomolar affinities for detection of pancreatic cancer.

PURPOSE: Detection of pancreatic cancer remains a high priority and effective diagnostic tools are needed for clinical applications. Many cancer cells overexpress integrin α(v)ß(6), a cell surface receptor being evaluated as a novel clinical biomarker. EXPERIMENTAL DESIGN: To validate this molecular target, several highly stable cystine knot peptides were engineered by directed evolution to bind specifically and with high affinity (3-6 nmol/L) to integrin α(v)ß(6). The binders do not cross-react with related integrin α(v)ß(5), integrin α(5)ß(1), or tumor-angiogenesis-associated integrin, α(v)ß(3). RESULTS: Positron emission tomography showed that these disulfide-stabilized peptides rapidly accumulate at tumors expressing integrin α(v)ß(6). Clinically relevant tumor-to-muscle ratios of 7.7 ± 2.4 to 11.3 ± 3.0 were achieved within 1 hour after radiotracer injection. Minimization of off-target dosing was achieved by reformatting α(v)ß(6)-binding activities across various natural and pharmacokinetically stabilized cystine knot scaffolds with different amino acid content. We show that the primary sequence of a peptide scaffold directs its pharmacokinetics. Scaffolds with high arginine or glutamic acid content suffered high renal retention of more than 75% injected dose per gram (%ID/g). Substitution of these amino acids with renally cleared amino acids, notably serine, led to significant decreases in renal accumulation of less than 20%ID/g 1 hour postinjection (P < 0.05, n = 3). CONCLUSIONS: We have engineered highly stable cystine knot peptides with potent and specific integrin α(v)ß(6)-binding activities for cancer detection. Pharmacokinetic engineering of scaffold primary sequence led to significant decreases in off-target radiotracer accumulation. Optimization of binding affinity, specificity, stability, and pharmacokinetics will facilitate translation of cystine knots for cancer molecular imaging.

Ultrahigh sensitivity carbon nanotube agents for photoacoustic molecular imaging in living mice.

Photoacoustic imaging is an emerging modality that overcomes to a great extent the resolution and depth limitations of optical imaging while maintaining relatively high-contrast. However, since many diseases will not manifest an endogenous photoacoustic contrast, it is essential to develop exogenous photoacoustic contrast agents that can target diseased tissue(s). Here we present a novel photoacoustic contrast agent, Indocyanine Green dye-enhanced single walled carbon nanotube (SWNT-ICG). We conjugated this contrast agent with cyclic Arg-Gly-Asp (RGD) peptides to molecularly target the alpha(v)beta(3) integrins, which are associated with tumor angiogenesis. Intravenous administration of this tumor-targeted contrast agent to tumor-bearing mice showed significantly higher photoacoustic signal in the tumor than in mice injected with the untargeted contrast agent. The new contrast agent gave a markedly 300 times higher photoacoustic contrast in living tissues than previously reported SWNTs, leading to subnanomolar sensitivities. Finally, we show that the new contrast agent can detect approximately 20 times fewer cancer cells than previously reported SWNTs.

Photoacoustic ocular imaging.

We developed a photoacoustic ocular imaging device and demonstrated its utility in imaging the deeper layers of the eye including the retina, choroid, and optic nerve. Using safe laser intensity, the photoacoustic system was able to visualize the blood distribution of an enucleated pig's eye and an eye of a living rabbit. Ultrasound images, which were simultaneously acquired, were overlaid on the photoacoustic images to visualize the eye's anatomy. Such a system may be used in the future for early detection and improved management of neovascular ocular diseases, including wet age-related macular degeneration and proliferative diabetic retinopathy.

A comparison between time domain and spectral imaging systems for imaging quantum dots in small living animals.

PURPOSE: We quantified the performance of time-domain imaging (TDI) and spectral imaging (SI) for fluorescence imaging of quantum dots (QDs) in three distinct imaging instruments: eXplore Optix (TDI, Advanced Research Technologies Inc.), Maestro (SI, CRi Inc.), and IVIS-Spectrum (SI, Caliper Life Sciences Inc.). PROCEDURE: The instruments were compared for their sensitivity in phantoms and living mice, multiplexing capabilities (ability to resolve the signal of one QD type in the presence of another), and the dependence of contrast and spatial resolution as a function of depth. RESULTS: In phantoms, eXplore Optix had an order of magnitude better sensitivity compared to the SI systems, detecting QD concentrations of ~40 pM in vitro. Maestro was the best instrument for multiplexing QDs. Reduction of contrast and resolution as a function of depth was smallest with eXplore Optix for depth of 2-6 mm, while other depths gave comparable results in all systems. Sensitivity experiments in living mice showed that the eXplore Optix and Maestro systems outperformed the IVIS-Spectrum. CONCLUSION: TDI was found to be an order of magnitude more sensitive than SI at the expense of speed and very limited multiplexing capabilities. For deep tissue QD imaging, TDI is most applicable for depths between 2 and 6 mm, as its contrast and resolution degrade the least at these depths.