The large size of many near infrared (NIR) fluorescent nanoparticles prevents rapid extravasation from blood vessels and subsequent diffusion to tumors. This confines in vivo uptake to the peritumoral space and results in high liver retention. We developed a viscosity modulated approach to synthesize ultrasmall silver sulfide quantum dots (QDs) with distinct tunable light emission from visible to near-infrared in spectrum and a QD core diameter between less than 5 nm. Further functionalization of these Ag2S QDs with different type of molecules such as targeting peptides, retains monodisperse, relatively small water soluble QDs without loss of the functionality of the peptide’s high binding affinity to cancerous tumor. Fluorescence and electron microscopy showed that selective integrin-mediated internalization was observed only in cancer cells treated with the peptide-labeled QDs, demonstrating that the unlabeled hydrophilic nanoparticles exhibit characteristics of negatively charged fluorescent dye molecules, which typically do not internalize in cells. The biodistribution profiles of intravenously administered QDs in different mouse models of cancer reveal an exceptionally high tumor-to-liver uptake ratio, suggesting that the small sized QDs evaded conventional opsonization and subsequent high uptake in the liver and spleen. The seamless tunability of the QDs over a wide spectral range with only a small increase in size, as well as the ease of labeling the bright and non-cytotoxic QDs with biomolecules, provides a platform for multiplexing information, tracking the trafficking of single molecules in cells, and selectively targeting disease biomarkers in living organisms without premature QD opsonization in circulating blood.
The clinical diagnosis of most cancers is based on evaluation of histology microscopic slide to view the size and shape of
cellular nuclei, and morphological structure of tissue. To achieve this goal in vivo and in deep tissue, near infrared (NIR)
dyes-bovine serum albumin (BSA) and immunoglobulin G (IgG) conjugates were synthesized. The spectral study show
that the absorption and fluorescence of the dye-conjugates are in the “tissue optical window” between 650 nm and 1100
nm. The internalization and pinocytosis of the synthesized compound were investigated in cell level using fluorescence
microscopy to obtain the optimal concentration and staining time scale.
We have developed a novel real-time intraoperative fluorescence imaging device that can detect near-infrared (NIR)
fluorescence and map sentinel lymph nodes (SLNs). In contrast to conventional imaging systems, this device is compact,
portable, and battery-operated. It is also wearable and thus allows hands-free operation of clinicians. The system directly
displays the fluorescence in its goggle eyepiece, eliminating the need for a remote monitor. Using this device in murine
lymphatic mapping, the SLNs stained with indocyanine green (ICG) can be readily detected. Fluorescence-guided SLN
resection under the new device was performed with ease. Ex vivo examination of resected tissues also revealed high
fluorescence level in the SLNs. Histology further confirmed the lymphatic nature of the resected SLNs.
Biochemical processes frequently involve protonation and deprotonation, resulting in pH changes that can be monitored with pH indicators. In heterogenous media such as tissue where indicator concentration or visual observation is limited, highly sensitive pH indicators with reduced tissue autofluorescence are needed. Because of the reduced tissue autofluorescence in the near infrared (NIR) region, NIR fluorescence dyes such as indocyanine green (ICG) and its
derivatives have been used to image molecular processes. Removing one of the N-subsitituent of ICG led to pHsensitive dyes operating in the NIR region and at physiologically relevant range. Further modification of the compound and synthetic procedure allowed their conjugation to peptides for specific delivery to target cells and tissues. Synthetic strategy and preliminary results on the spectral and biological properties of such dyes and their bioconjugates are described.
Numerous studies have shown that basic Tat peptide (48-57) internalized non-specifically in cells and localized in the nucleus. However, localization of imaging agents in cellular nucleus is not desirable because of the potential mutagenesis. When conjugated to the peptides that undergo receptor-mediated endocytosis, Tat peptide could target specific cells or pathologic tissue. We tested this hypothesis by incorporating a somatostatin receptor-avid peptide (octreotate, Oct) and two different fluorescent dyes, Cypate 2 (Cy2) and fluorescein 5'-carboxlic acid (5-FAM), into the Tat-peptide sequence. In addition to the Cy2 or 5-FAM-labeled Oct conjugated to Tat peptide (Tat) to produce Tat-Oct-Cypate2 or Tat-Oct-5-FAM, we also labeled the Tat the Tat peptide with these dyes (Tat-Cy2 and Tat-5-FAM) to serve as positive control. A somatostatin receptor-positive pancreatic tumor cell line, AR42J, was used to assess cell internalization. The results show that Tat-5-FAM and Tat-Cypate2 localized in both nucleus and cytoplasm of the cells. In contrast to Tat-Oct-Cypate2, which localized in both the cytoplasm and nucleus, Tat-Oct-5-FAM internalized in the cytoplasm but not in the nucleus of AR42J cells. The internalizations were inhibited by adding non-labeled corresponding peptides, suggesting that the endocytoses of each group of labeled and the corresponding unlabeled compounds occurred through a common pathway. Thus, fluorescent probes and endocytosis complex between octreotate and somatostatin receptors in cytoplasm could control nuclear internalization of Tat peptides.
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