Su-Jin Cheong

Oleyl-Chitosan Nanoparticles Based on a Dual Probe for Optical/MR Imaging in Vivo

Oleic acid-conjugated chitosan (oleyl-chitosan) is a powerful platform for encapsulating oleic acid-decorated iron oxide nanoparticles (ION), resulting in a good magnetic resonance imaging (MRI) probe. Oleyl-chitosan could self-assemble into core-shell structures in aqueous solution and provide the effective core compartment for loading ION. ION-loaded oleyl-chitosan nanoparticles showed good enhanced MRI sensitivity in a MR scanner. Cy5.5 dye was accessed to the oleyl-chitosan conjugate for near-infrared (NIR) in vivo optical imaging. After intravenous injection of ION-loaded Cy5.5-conjugated oleyl-chitosan (ION-Cy5.5-oleyl-chitosan) nanoparticles in tumor-bearing mice, both NIRF and MR imaging showed the detectable signal intensity and enhancement in tumor tissues via enhanced permeability and retention (EPR) effect. Tumor accumulation of the nanoparticles was confirmed through ex vivo fluorescence images and Prussian blue staining images in tumor tissues. It is concluded that ION-Cy5.5-oleyl-chitosan nanoparticle is highly an effective imaging probe for detecting tumor in vivo.

Superparamagnetic iron oxide nanoparticles-loaded chitosan-linoleic acid nanoparticles as an effective hepatocyte-targeted gene delivery system.

The goal of this study was to develop a gene delivery imaging system that targets hepatocytes to help diagnose and treat various liver diseases. To this end, we prepared superparamagnetic iron oxide nanoparticles (SPIO)-loaded with water-soluble chitosan (WSC)-linoleic acid (LA) nanoparticles (SCLNs) that formed gene complexes capable of localizing specifically to hepatocytes. We confirmed that (99m)Tc-labeled SCLNs delivered into mice via intravenous injection accumulated mainly in the liver using nuclear and magnetic resonance imaging. SCLN/enhanced green fluorescence protein (pEGFP) complexes were also successfully formed and were characterized with a gel retardation assay. SCLN/pEGFP complexes were transfected into primary hepatocytes, where GFP expression was observed in the cytoplasm. In addition, the injection of the gene complexes into mice resulted in significantly increased expression of GFP in hepatocytes in vivo. Furthermore, gene silencing was effectively achieved by administration of gene complexes loaded with specific siRNAs. In conclusion, our results indicate that the SCLNs have the potential to be useful for hepatocyte-targeted imaging and effective gene delivery into hepatocytes.

Prostate Cancer-Targeted Imaging Using Magnetofluorescent Polymeric Nanoparticles Functionalized with Bombesin

PurposeIn this work, the aim was to prepare and characterize a magnetofluorescent polymeric nanoparticle for prostate cancer imaging in vivo.MethodsGlycol chitosan (GC) was chemically modified with N-acetyl histidine (NAHis) as a hydrophobic moiety, and bombesin (BBN) was conjugated to the hydrophobically modified GC for use in targeting gastric-releasing peptide receptors (GRPR) overexpressed in prostate cancer cells. NAHis-GC conjugates were labeled with the near-infrared (NIR) fluorophore Cy5.5 (C-NAHis-GC conjugate).ResultsBBN-conjugated C-NAHis-GC nanoparticles (BC-NAHis-GC nanoparticles) showed significantly higher binding to the PC3 cell surface than nanoparticles without BBN, and the cellular binding was clearly inhibited by BBN. The tumor-to-muscle ratios of C- and BC-NAHis-GC nanoparticles were 2.26 ± 0.66 and 5.37 ± 0.43, respectively. The tumor accumulation of BC-NAHis-GC nanoparticles was clearly reduced by co-injection of BBN. Further, iron oxide nanoparticles (IO) were loaded into BC-NAHis-GC nanoparticles to investigate the possibility of use as a probe for MRI. IO-BC-NAHis-GC nanoparticles were well observed in the PC3 cells, and the blocking with BBN significantly reduced the cellular binding of the nanoparticles.ConclusionThese results demonstrate that the BBN conjugation to NAHis-GC nanoparticles improves their tumor accumulation in PC3-bearing mice in comparison to nanoparticles without BBN, suggesting that BC-NAHis-GC nanoparticles may be useful for prostate cancer imaging.

Characterization, biodistribution and small-animal SPECT of I-125-labeled c-Met binding peptide in mice bearing c-Met receptor tyrosine kinase-positive tumor xenografts

c-Met is a receptor tyrosine kinase involved in tumor cell growth, invasion, metastases and angiogenesis. Overexpression of c-Met is frequently observed in several tumor types. Here, we report the in vitro cell-binding properties and biodistribution and SPECT/CT imaging in glioma (U87MG) xenograft-bearing mice of (125)I-labeled c-Met-binding peptides (cMBPs) including analogs conjugated to amino acid and aliphatic carbon linkers. In vitro assays showed that the peptide without any linker and those with GGG and 8-aminooctanoic acid linkers had low cellular internalization and that IC(50) values of peptides were 1.5 microM, 65 nM and 85.3 nM, respectively. Biodistribution studies showed the GGG-containing peptide had higher tumor uptake and a higher tumor-to-blood activity concentration ratio than other receptor-binding ligands. SPECT/CT studies with a dedicated small-animal imaging system were performed in U87MG-bearing athymic mice. Although U87MG tumor xenografts could be visualized by SPECT/micro-CT using the various (125)I labeled cMBPs, image contrast and overall quality were unremarkable.

In Vivo Imaging of Mesenchymal—Epithelial Transition Factor (c-Met) Expression using an Optical Imaging System

Mesenchymal-epithelial transition factor (c-Met) is a receptor tyrosine kinase that has been shown to be overexpressed and mutated in a variety of malignancies, such as glioma. We have recently found that an (125)I-radiolabeled Gly-Gly-Gly (GGG)- or 8-aminooctanoic acid (AOC)-containing c-Met binding peptide (cMBP) specifically targets c-Met receptor in vivo and in vitro. In this report, cyanine dye 5.5 (Cy5.5)-conjugated GGG- or AOC-containing cMBPs were evaluated in human cancer cell xenografts in order to investigate the possibility of c-Met receptor targeting using an optical imaging system. The receptor binding affinity of Cy5.5-conjugated peptides was tested in 96-well plates coated with a c-Met/Fc chimeric protein. Optical imaging studies were performed in U87MG and Ramos bearing athymic mice. The binding affinities of Cy5.5-conjugated GGG- or AOC-containing cMBPs were determined to be 0.318 and 0.342 microM, respectively. Confocal images show that Cy5.5-conjugated peptides bound mainly to the cell surface and that peptide binding was clearly inhibited by free cMBP. Subcutaneous U87MG tumors were clearly visualized with each of the two fluorescent probes. Of the two, cMBP-AOC-Cy5.5 displayed higher tumor uptake and tumor-to-normal tissue ratios at 10 min to 24 h postinjection in the U87MG tumor model. For the in vivo blocking study, cMBP-AOC-Cy5.5 (4 nmol) was co-injected with cold cMBP (0.13 micromol) into the U87MG xenograft mice. Image-based tumoral uptake decreased up to approximately 35%. These results suggest that Cy5.5-conjugated cMBP could potentially be used to detect c-Met-positive cancers in vivo. However, additional modifications to this optical imaging agent are needed to further improve its efficacy.

Surface engineering of quantum dots for in vivo imaging.

The aim of this study was to investigate the effect of gluconic acid (GA) conjugation on the biodistribution of cysteamine-capped quantum dots (amino-QDots) in vivo. Cadmium selenide/zinc sulfide (CdSe/ZnS) was capped with cysteamine through a thiol exchange method, and different amounts of GA were conjugated to the amine groups of cysteamine via the formation of an amide bond. The emission maxima of the synthesized QDots, the amino-QDots and the GA-conjugated amine-QDots (GA-QDots) were located at 720, 600 and 610 nm, respectively. In the cell viability studies, the GA-QDots showed very low toxicity against CHO cells as compared to the cytotoxicity of the amino-QDots. The QDots were next intravenously injected into normal mice and then we performed ex vivo optical imaging. The majority of the amino-QDots were accumulated in the lung. In contrast, the GA-QDots were cleared out of the body through the kidney. Therefore, we expect that the conjugation of GA onto the amino-QDots can create opportunities for using amino-QDots for in vivo imaging.

The effect of mannosylation of liposome-encapsulated indocyanine green on imaging of sentinel lymph node

Abstract The imaging of sentinel lymph nodes (SLN) has been researched for its role in assessing cancer progression and postsurgical lymphedema. Indocyanine green (ICG) is a near-infrared (NIR) optical dye that has been approved by the Food and Drug Administration. It is known that liposome-encapsulated ICG (LP-ICG) has improved stability and fluorescence signal compared with ICG. We designed mannosylated liposome-encapsulated ICG (M-LP-ICG) as an optical contrast agent for SLN. M-LP-ICG has a higher UV absorbance spectrum and fluorescence intensity than LP-ICG. The stability of M-LP-ICG measured in 50% fetal bovine serum solution by a dialysis method was better than that of LP-ICG. M-LP-ICG demonstrated a high uptake in RAW 264.7 macrophage cell because the density of mannose is high. There were differences between M-LP-ICG and glucosylated liposome-encapsulated ICG (G-LP-ICG), which are geometrical isomers. The result of an inhibition study of M-LP-ICG showed a statistically significant decrease in uptake in RAW 264.7 cells after either co-treatment or pre-treatment with d-(+)-mannose as an inhibitor. Results from an in vitro experiment demonstrated that M-LP-ICG was specifically taken up by macrophage cells through the mannose receptor on its surface. The time-series images acquired from a normal mouse model after subcutaneous injection showed that the signal from M-LP-ICG in SLN and other organs appeared early and disappeared quickly in comparison with signals from LP-ICG. Not only the sentinel but also the draining lymph nodes were observed partly in M-LP-ICG. M-LP-ICG appears to increase the specificity of uptake and retention in macrophages, making it a good candidate contrast agent for an optic imaging system for SLN and the lymphatic system.

Optical imaging of MMP expression and cancer progression in an inflammation‐induced colon cancer model

The purpose of this study was to use a near‐infrared (NIR) fluorescent cyclic His‐Try‐Gly‐Phe peptide to characterize and image the expressions of matrix metalloproteinases (MMPs), which are correlated with cancer promotion, in an inflammation‐induced colorectal cancer (ICRC) model. We explored the relationship between the development of colon cancer and the expression of MMPs at the same colonic sites in ICRC models. To develop ICRC models, mice were administered a single intraperitoneal dose (10 mg/kg) of azoxymethane (AOM) and exposed orally to 2% dextran sodium sulfate (DSS) for one week. MMP‐2 expression and β‐catenin activation in colonic lesions were characterized by immunohistochemical (IHC) staining. After being treated with inducers for some time, cancerous lesions were found to express high β‐catenin and MMP‐2. The profiles of MMP expression were correlated with β‐catenin activation in the colonic lesions. c(KAHWGFTLD)NH2 (C6) peptide was prepared by standard Fmoc peptide synthesis to target MMPs. Molecular weight of Cy5.5‐C6 was 1,954.78 g/mol (calculated MW = 1955.23 g/mol). The in vitro characterization of Cy5.5‐C6 showed MMP binding specificity in a cell experiment. In vivo NIRF imaging showed high accumulation of Cy5.5‐C6 in tumors with associated expression of MMP‐2 in colonic lesions after intravenous injection. The MMP‐2 specificity of Cy5.5‐C6 was confirmed by successful inhibition of probe uptake in the tumor due to the presence of excess C6 peptide. The use of Cy5.5‐C6 to target MMP‐2 has the potential to be developed into an effective molecular imaging agent to monitor ICRC progress.

Nonpolymeric Surface–Coated Iron Oxide Nanoparticles for In Vivo Molecular Imaging: Biodegradation, Biocompatibility, and Multiplatform

A new approach to the surface engineering of superparamagnetic iron oxide nanoparticles (SPIONs) may encourage their development for clinical use. In this study, we demonstrated that nonpolymeric surface modification of SPIONs has the potential to be an advanced biocompatible contrast agent for biomedical applications, including diagnostic imaging in vivo. Methods: Adenosine triphosphate (ATP), which is an innate biomaterial derived from the body, was coated onto the surface of SPIONs. An in vivo degradation study of ATP-coated SPIONs (ATP@SPIONs) was performed for 28 d. To diminish phagocytosis, ATP@SPIONs were surface-modified with gluconic acid. We next studied the ability of the SPIONs to serve as a specific targeted contrast agent after conjugation of cMet-binding peptide. The SPIONs were conjugated with Cy5.5 and labeled with 125I for multimodality imaging. In vivo and in vitro tumor-targeted binding studies were performed on U87MG cells or a U87MG tumor model using animal SPECT/CT, an optical imaging system, and a 1.5-T clinical MR scanner. Results: ATP@SPIONs showed rapid degradation in vivo and in vitro, compared with ferumoxides. ATP@SPIONs modified with gluconic acid reduced phagocytic uptake, showed improved biodistribution, and provided good targetability in vivo. The gluconic acid–conjugated ATP@SPIONs, when conjugated with cMet-binding peptide, were successfully visualized on the U87MG tumors implanted in mice via multimodality imaging. Conclusion: We suggest that ATP@SPIONs can be used as a multiplatform to target a region of interest in molecular imaging. When we consider the biocompatibility of contrast agents in vivo, ATP@SPIONs are superior to polymeric surface–modified SPIONs.

Evaluation of the therapeutic efficacy of a VEGFR2-blocking antibody using sodium-iodide symporter molecular imaging in a tumor xenograft model.

PURPOSE Vascular endothelial growth factor receptor 2-blocking antibody (DC101) has inhibitory effects on tumor growth and angiogenesis in vivo. The human sodium/iodide symporter (hNIS) gene has been shown to be a useful molecular imaging reporter gene. Here, we investigated the evaluation of therapeutic efficacy by molecular imaging in reporter gene transfected tumor xenografts using a gamma imaging system. METHODS The hNIS gene was transfected into MDA-MB-231 cells using Lipofectamine. The correlation between the number of MDA-MB-231-hNIS cells and the uptake of (99m)Tc-pertechnetate or (125)I was investigated in vitro by gamma imaging and counting. MDA-MB-231-hNIS cells were injected subcutaneously into mice. When the tumor volume reached 180-200 mm(3), we randomly assigned five animals to each of three groups representing different tumor therapies; no DC101 (control), 100 μg, or 150 μg DC101/mouse. One week and 2 weeks after the first injection of DC101, gamma imaging was performed. Mice were sacrificed 2 weeks after the first injection of DC101. The tumor tissues were used for reverse transcriptase-polymerase chain reaction (RT-PCR) and CD31 staining. RESULTS Uptake of (125)I and (99m)Tc-pertechnetate into MDA-MB-231-hNIS cells in vitro showed correlation with the number of cells. In DC101 treatment groups, the mean tumor volume was smaller than that of the control mice. Furthermore, tumor uptake of (125)I was lower than in the controls. The CD31 staining and RT-PCR assay results showed that vessel formation and expression of the hNIS gene were significantly reduced in the tumor tissues of treatment groups. CONCLUSION This study demonstrated the power of molecular imaging using a gamma imaging system for evaluating the therapeutic efficacy of an antitumor treatment. Molecular imaging systems may be useful in evaluation and development of effective diagnostic and/or therapeutic antibodies for specific target molecules.