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Background pH-low Insertion Peptides (pHLIPs) can serve as a targeting moiety that enables pH-sensitive probes to detect solid tumors. Using these probes in conjunction with multispectral optoacoustic tomography (MSOT) is a promising approach to improve imaging for pancreatic cancer. Methods A pH-sensitive pHLIP (V7) was conjugated to 750 NIR fluorescent dye and evaluated as a targeted probe for pancreatic adenocarcinoma. The pH-insensitive K7 pHLIP served as an untargeted control. Probe binding was assessed in vitro at pH 7.4, 6.8, and 6.6 using human pancreatic cell lines S2VP10 and S2013. Using MSOT, semi-quantitative probe accumulation was then assessed in vivo with a murine orthotopic pancreatic adenocarcinoma model. Results In vitro, the V7–750 probe demonstrated significantly higher fluorescence at pH 6.6 compared to pH 7.4 (S2VP10, p=0.0119; S2013, p=0.0160), while no difference was observed with the K7–750 control (S2VP10, p=0.8783; S2013, p=0.921). In the in vivo S2VP10 model, V7–750 probe resulted in 782.5 MSOT a.u. signal compared to 5.3 MSOT a.u. in K7–750 control in tumor (p= 0.0001). Similarly, V7–750 probe signal was 578.3 MSOT a.u. in the S2013 model compared to K7–750 signal at 5.1 MSOT a.u. (p=0.0005). There was minimal off-target accumulation of the V7–750 probe within the liver or kidney, and probe distribution was confirmed with ex vivo imaging. Conclusion Compared to pH-insensitive controls, V7–750 pH-sensitive probe specifically targets pancreatic adenocarcinoma, and has minimal off-target accumulation. The non-invasive detection of pH-targeted probes by means of MSOT represents a promising modality to improve the detection and monitoring of pancreatic cancer.

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Background Advances in small animal imaging have improved the detection and monitoring of cancer in vivo, although with orthotopic models precise localization of tumors remains a challenge. In this study, we evaluated multispectral optoacoustic tomography (MSOT) as an imaging modality to detect pancreatic adenocarcinoma in an orthotopic murine model. Methods In vitro binding of syndecan-1 probe to the human pancreatic cancer cell line S2VP10 was evaluated on flow cytometry. For in vivo testing, S2VP10 cells were orthotopically implanted into the pancreas of SCID mice. At 7 days post-implantation, the mice were intravenously injected with syndecan-1 probe and tumor uptake was evaluated with multispectral optoacoustic tomography (MSOT) at multiple time points. Comparison was made to a free-dye control, indocyanine green (ICG). Probe uptake was verified ex vivo with fluorescent imaging. Results Syndecan-1 probe demonstrated partial binding to S2VP10 cells in vitro. In vivo, syndecan-1 probe preferentially accumulated in the pancreas tumor (480 MSOT a.u.) compared to off-target organs, including the liver (67 MSOT a.u.) and kidney (96 MSOT a.u.). Syndecan-1 probe accumulation peaked at 6 hours (480 MSOT a.u.), while the ICG control dye failed to demonstrate similar retention within the tumor bed (0.0003 MSOT a.u.). At peak accumulation, signal intensity was 480 MSOT a.u., resulting in several times greater signal in the tumor bed than in the kidney or liver. Ex vivo fluorescent imaging comparing tumor signal to that within off-target organs confirmed the in vivo results. Conclusion MSOT demonstrates successful accumulation of syndecan-1 probe within pancreatic tumors, and provides high resolution images which allow non-invasive, real time comparison of signal within individual organs. Syndecan-1 probe preferentially accumulates within a pancreatic adenocarcinoma model, with minimal off-target effects.

Detection of orthotopic xenograft tumors is difficult due to poor spatial resolution and reduced image fidelity with traditional optical imaging modalities. In particular, light scattering and attenuation in tissue at depths beyond subcutaneous implantation hinder adequate visualization. We evaluate the use of multispectral optoacoustic tomography (MSOT) to detect upregulated epidermal growth factor (EGF) receptor in orthotopic pancreatic xenografts using a near-infrared (NIR) EGF-conjugated CF-750 fluorescent probe. MSOT is based on the photoacoustic effect and thus not limited by photon scattering, resulting in high-resolution tomographic images. Pancreatic tumor-bearing mice with luciferase-transduced S2VP10L tumors were intravenously injected with EGF-750 probe prior to MSOT imaging. We characterized probe specificity and bioactivity via immunoblotting, immunocytochemistry, and flow cytometric analysis. In vitro data along with optical bioluminescence/fluorescence imaging were used to validate acquired MSOT in vivo images of probe biodistribution. Indocyanine green dye was used as a non-specific control to define specificity of EGF-probe accumulation. Maximum accumulation occurred at six hours post-injection, demonstrating specific intra-tumoral probe uptake and minimal liver and kidney off-target accumulation. Optical bioluminescence and fluorescence imaging confirmed tumor-specific probe accumulation consistent with MSOT images. These studies demonstrate the utility of MSOT to obtain volumetric images of ligand probe biodistribution in vivo to detect orthotopic pancreatic tumor lesions through active targeting of EGF receptor.

Morphological change of a micelle of poly(styrene)-b-poly(2-vinylpyridine)-b-poly(ethylene oxide) (PS-PVP-PEO) polymer was induced by binding sodium dodecyl sulfate (SDS) to the PVP block in acidic aqueous solutions. The change in the size of SDS/PS-PVP-PEO complexes was detected by dynamic light scattering measurements and atomic force microscopy, and the binding of SDS was confirmed by zeta-potential measurements. When the micelle was free from SDS in acidic aqueous solutions, the hydrodynamic diameter of the micelle was 216 nm, reflecting the extended conformation of the PVP block due to the repulsion between protonated pyridine units. As the cationic PVP block was electrically neutralized with anionic SDS, the diameter was gradually reduced concomitant with the decrease in zeta-potential and finally reached 175 nm when the PVP block was completely neutralized. The decrease in the diameter shows the morphological change of the PVP block from extended to shrunken forms. Further addition of SDS did not cause the changes of the diameter nor zeta-potential. This indicates that SDS was not bound to the PS-PVP-PEO polymer after the PVP block was fully neutralized and that the hydrophobic binding of SDS to the polymer was negligible due to the low concentration of SDS.

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Kinetic folding experiments by pulsed hydrogen/deuterium exchange (HDX) mass spectrometry (MS) are a well-established tool for water-soluble proteins. To the best of our knowledge, the current study is the first that applies this approach to an integral membrane protein. The native state of bacteriorhodopsin (BR) comprises seven transmembrane helices and a covalently bound retinal cofactor. BR exposure to sodium dodecyl sulfate (SDS) induces partial unfolding and retinal loss. We employ a custom-built three-stage mixing device for pulsed-HDX/MS investigations of BR refolding. The reaction is triggered by mixing SDS-denatured protein with bicelles. After a variable folding time (10 ms to 24 h), the protein is exposed to excess D(2) O buffer under rapid exchange conditions. The HDX pulse is terminated by acid quenching after 24 ms. Subsequent off-line analysis is performed by size exclusion chromatography and electrospray MS. These measurements yield the number of protected backbone N-H sites as a function of folding time, reflecting the recovery of secondary structure. Our results indicate that much of the BR secondary structure is formed quite late during the reaction, on a time scale of 10 s and beyond. It is hoped that in the future it will be possible to extend the pulsed-HDX/MS approach employed here to membrane proteins other than BR.