In vivo imaging with fluorescent smart probes to assess treatment strategies for acute pancreatitis.

BACKGROUND AND AIMS: Endoprotease activation is a key step in acute pancreatitis and early inhibition of these enzymes may protect from organ damage. In vivo models commonly used to evaluate protease inhibitors require animal sacrifice and therefore limit the assessment of dynamic processes. Here, we established a non-invasive fluorescence imaging-based biomarker assay to assess real-time protease inhibition and disease progression in a preclinical model of experimental pancreatitis. METHODS: Edema development and trypsin activation were imaged in a rat caerulein-injection pancreatitis model. A fluorescent "smart" probe, selectively activated by trypsin, was synthesized by labeling with Cy5.5 of a pegylated poly-L-lysine copolymer. Following injection of the probe, trypsin activation was monitored in the presence or absence of inhibitors by in vivo and ex vivo imaging. RESULTS: We established the trypsin-selectivity of the fluorescent probe in vitro using a panel of endopeptidases and specific inhibitor. In vivo, the probe accumulated in the liver and a region attributed to the pancreas by necropsy. A dose dependent decrease of total pancreatic fluorescence signal occurred upon administration of known trypsin inhibitors. The fluorescence-based method was a better predictor of trypsin inhibition than pancreatic to body weight ratio. CONCLUSIONS: We established a fluorescence imaging assay to access trypsin inhibition in real-time in vivo. This method is more sensitive and dynamic than classic tissue sample readouts and could be applied to preclinically optimize trypsin inhibitors towards intrapancreatic target inhibition.

Remote triggered release of doxorubicin in tumors by synergistic application of thermosensitive liposomes and gold nanorods.

Delivery of chemotherapeutic agents after encapsulation in nanocarriers such as liposomes diminishes side-effects, as PEGylated nanocarrier pharmacokinetics decrease dosing to healthy tissues and accumulate in tumors due to the enhanced permeability and retention effect. Once in the tumor, however, dosing of the chemotherapeutic to tumor cells is limited potentially by the rate of release from the carriers and the size-constrained, poor diffusivity of nanocarriers in tumor interstitium. Here, we report the design and fabrication of a thermosensitive liposomal nanocarrier that maintains its encapsulation stability with a high concentration of doxorubicin payload, thereby minimizing "leak" and attendant toxicity. When used synergistically with PEGylated gold nanorods and near-infrared stimulation, remote triggered release of doxorubicin from thermosensitive liposomes was achieved in a mouse tumor model of human glioblastoma (U87), resulting in a significant increase in efficacy when compared to nontriggered or nonthermosensitive PEGylated liposomes. This enhancement in efficacy is attributed to increase in tumor-site apoptosis, as was evident from noninvasive apoptosis imaging using Annexin-Vivo 750 probe. This strategy affords remotely triggered control of tumor dosing of nanocarrier-encapsulated doxorubicin without sacrificing the ability to differentially dose drugs to tumors via the enhanced permeation and retention effect.

Rational identification of a novel peptide for targeting nanocarriers to 9L glioma.

Traditional therapies for high grade gliomas are limited in part by collateral damage to normal tissues. Selective delivery of therapies to tumors is, therefore, needed. Here, we report that liposomal nanocarriers coated with a novel oligopeptide enhance uptake by 9L gliosarcoma. A targeting nine amino acid peptide sequence (RSI) was identified by differential panning of random peptide phage display libraries on 9L cells and rat blood cells and plasma. Peptides were coupled to the surface of liposomal nanocarriers which were subsequently loaded with doxorubicin. The ability of RSI coated liposomes to facilitate drug uptake and cytotoxicity was compared with conventional liposomal nanocarriers and controls. In addition, plasma clearance profiles of the RSI peptide coupled liposomal nanocarriers were evaluated in adult immuno-competent rats. RSI peptide-coupled liposomal nanocarriers enhanced drug uptake by 9L cells by 500% compared with conventional liposomal nanocarriers, and significantly increased cytotoxicity. The plasma half-lives confirmed that the presence of the RSI peptide on the liposomal nanocarriers did not compromise circulation time in the blood in comparison with Stealth liposomal nanocarriers. These data suggest that phage-identified oligopeptides could lead to the development of new tumor selective nanocarriers.

MRI mediated, non-invasive tracking of intratumoral distribution of nanocarriers in rat glioma.

Nanocarrier mediated therapy of gliomas has shown promise. The success of systemic nanocarrier-based chemotherapy is critically dependent on the so-called leaky vasculature to permit drug extravasation across the blood-brain barrier. Yet, the extent of vascular permeability in individual tumors varies widely, resulting in a correspondingly wide range of responses to the therapy. However, there exist no tools currently for rationally determining whether tumor blood vessels are amenable to nanocarrier mediated therapy in an individualized, patient specific manner today. To address this need for brain tumor therapy, we have developed a multifunctional 100 nm scale liposomal agent encapsulating a gadolinium-based contrast agent for contrast-enhanced magnetic resonance imaging with prolonged blood circulation. Using a 9.4 T MRI system, we were able to track the intratumoral distribution of the gadolinium-loaded nanocarrier in a rat glioma model for a period of three days due to improved magnetic properties of the contrast agent being packaged in a nanocarrier. Such a nanocarrier provides a tool for non-invasively assessing the suitability of tumors for nanocarrier mediated therapy and then optimizing the treatment protocol for each individual tumor. Additionally, the ability to image the tumor in high resolution can potentially constitute a surgical planning tool for tumor resection.

Two-wave-plate compensator method for full-field retardation measurements.

The two-wave-plate compensator (TWC) method is expanded for full-field retardation measurements by use of a polarization microscope. The sample image is projected onto a CCD camera connected to a computer, allowing the retardation to be measured at all pixels. The retardation accuracy of this implementation of the TWC is evaluated to be 0.06 nm. The method is applied to polarization-maintaining fibers and long-period fiber gratings. The measured retardation is in good agreement with the crossed-polarizer images of the fibers. The method achieves a spatial resolution of 0.45 microm and a retardation resolution of 0.07 nm. The full-field TWC method can thus be a useful tool for characterizing and monitoring the fabrication of optical devices.