68Ga-labelled NOTA-RGD-GE11 peptide for dual integrin and EGFR-targeted tumour imaging.

INTRODUCTION: Multiple peptide receptors are co-expressed in many types of cancers. Arg-Gly-Asp (RGD) and GE11 peptides specifically target integrin αVß3 and EGFR, respectively. Recently, we designed and synthesized a heterodimer peptide NOTA-c(RGDyK)-GE11 (NOTA-RGD-GE11). The aim of this study was to investigate the characteristics of NOTA-RGD-GE11 for dual receptor imaging. METHODS: NOTA-RGD-GE11 heterodimer was labelled with 68Ga. The dual receptor binding affinity was investigated by antibody competition binding assay. The in vitro and in vivo characteristics of [68Ga]Ga-NOTA-RGD-GE11 were investigated and compared with that of monomeric peptides [68Ga]Ga-NOTA-RGD and [68Ga]Ga-NOTA-GE11. RESULTS: NOTA-RGD-GE11 had binding affinities with both integrin αVß3 and EGFR. The dual receptor targeting property of [68Ga]Ga-NOTA-RGD-GE11 was validated by blocking studies in a NCI-H292 tumour model. [68Ga]Ga-NOTA-RGD-GE11 showed higher tumour uptake than [68Ga]Ga-NOTA-RGD and [68Ga]Ga-NOTA-GE11 in biodistribution and PET/CT imaging studies. CONCLUSION: The dual receptor targeting and enhanced tumour uptake of [68Ga]Ga-NOTA-RGD-GE11 warrant its further investigation for dual integrin αVß3 and EGFR-targeted tumour imaging.

Development of single vial kits for preparation of 68 Ga-labelled hexavalent lactoside for PET imaging of asialoglycoprotein receptor.

The aim of this study was to formulate and evaluate the freeze-dried kit of NOTA-hexavalent lactoside (NOTA-HL) for the preparation of 68 Ga-labeled glycoligand for PET imaging of the asialoglycoprotein receptor (ASGPR). 68 GaCl3 was obtained from a commercial 68 Ge/68 Ga generator. Single-vial kits of HL were formulated. Optimization of radiolabeling with 68 Ga, various evaluations of NOTA-HL kits, and in vitro stability study of 68 Ga-HL were carried out. PET/CT imaging of normal mice injected with 68 Ga-NOTA-HL was performed. NOTA-HL kit was successfully formulated. High radiochemical yields (>95%) were obtained by 68 Ga radiolabeling. The NOTA-HL kits were stable for at least 12 months, and 68 Ga-NOTA-HL exhibited good in vitro stability. PET studies in normal mice revealed high specific accumulation of activity in the liver. The NOTA-HL kit was developed for fast 68 Ga labeling. 68 Ga-NOTA-HL showed high specific uptake in liver. The availability of ready-to-use NOTA-HL kits combined with 68 Ge/68 Ga generators would provide an efficient approach for PET imaging of ASGPR.

Use of 111In-Hexavalent Lactoside for Liver Reserve Estimation in Rodents with Thioacetamide-Induced Hepatic Fibrosis.

Many biochemical tests detecting the presence of liver disease are not liver-specific and may be abnormal in nonhepatic conditions. The asialoglycoprotein receptor (ASGPR) is a hepatocyte-specific receptor for Gal/GalNAc-terminated glycopeptide or glycoprotein. The number of these receptors decreases in patients with chronic liver diseases. Here, we aimed to evaluate the use of 111In-hexavalent lactoside, a known ASGPR imaging biomarker, as a more sensitive probe to detect small changes in liver reserve in animal models of chronic liver injury. Thioacetamide (TAA) treatment via intraperitoneal injection every 2 days in BALB/c mice continued for 1, 2, 3, or 4 months. The liver fibrosis stages were determined by Sirius Red staining and were based on the METAVIR classification method. Serum transaminase enzymes (alanine transaminase (ALT) and aspartate transaminase (AST)), alkaline phosphatase, albumin, and bilirubin were measured using a FUJI FDC3500 i/s analyzer. The ASGPR staining was performed by immunohistocytochemical stain. The percentages of fibrosis and ASGPR were calculated using ImageJ software after collagen staining and anti-ASGPR staining, respectively. A nanoSPECT/CT was used for molecular imaging and liver uptake measurement. We observed fibrosis grades of F0-F1 in mice treated with TAA for 1 month, F2 in mice treated for 2 months, F3-F4 in mice treated for 3 months, and F4 in mice treated for 4 months. The levels of ALT and albumin were not significantly different in the TAA groups from those in the controls. Although the average levels of AST, alkaline phosphatase, and bilirubin in the TAA groups were different from those in the control group, there was little difference between TAA groups. More sensitive distinctions among TAA groups were detected in 111In-hexavalent lactoside uptake of ASGPR, ASGPR staining, and fibrosis % than when using the conventional AST, ALT, albumin, alkaline phosphatase, and bilirubin tests. The absorption and distribution of 111In-hexavalent lactoside were lower in the chronic hepatitis models than the normal controls. The liver reserves measured by 111In-hexavalent lactoside uptake were 71.7 ± 7.5% and 50.9 ± 5.6% after 1 and 2 months, respectively, of TAA treatment. As an ASGPR biomarker, 111In-hexavalent lactoside has higher sensitivity than traditional liver function tests and collagen stain to provide more objective data for evaluating compensated cirrhosis or changes in liver damage. ASGPR staining can reflect the regenerated hepatocytes, but the need for a biopsy limits its use. 111In-hexavalent lactoside measurement is comparable with ASGPR staining, which suggests that 111In-hexavalent lactoside measurement will be more useful as a practical, noninvasive test of chronic liver injury.

The specificity and accuracy of (111)In-hexavalent lactoside in estimating liver reserve and its threshold value for mortality in mice.

BACKGROUND & AIMS: The asialoglycoprotein receptor on hepatocyte membranes recognizes the galactose residues of glycoproteins. We investigated the specificity, accuracy and threshold value of asialoglycoprotein receptor imaging for estimating liver reserve via scintigraphy using (111)In-hexavalent lactoside in mouse models. METHODS: (111)In-hexavalent lactoside scintigraphy for asialoglycoprotein receptor imaging was performed on groups of normal mice, orthotopic SK-HEP-1-bearing mice, subcutaneous HepG2-bearing mice, mice with 20-80% partial hepatectomy and mice with acute hepatitis induced by acetaminophen. Liver reserve was measured by relative liver uptake and compared with normal mice. Asialoglycoprotein receptor blockade was performed via an in vivo asialofetuin competitive binding assay. RESULTS: A total of 73.64±7.11% of the injection dose accumulated in the normal liver tissue region, and radioactivity was barely detected in the hepatoma region. When asialoglycoprotein receptor was blocked using asialofetuin, less than 0.41±0.04% of the injection dose was detected as background in the liver. Asialoglycoprotein receptor imaging data revealed a linear correlation between (111)In-hexavalent lactoside binding and residual liver mass (R(2)=0.8548) in 20-80% of partially hepatectomized mice, demonstrating the accuracy of (111)In-hexavalent lactoside imaging for measuring the functional liver mass. Asialoglycoprotein receptor imaging data in mice with liver failure induced using 600mg/kg acetaminophen revealed 19-45% liver reserve relative to normal mice and a fatal threshold value of 25% liver reserve. CONCLUSION: The (111)In-hexavalent lactoside imaging method appears to be a good, specific, visual and quantitative predictor of functional liver reserve. The diagnostic threshold for survival was at 25% liver reserve in mice.

Polyethylene glycol-coated graphene oxide attenuates antigen-specific IgE production and enhanced antigen-induced T-cell reactivity in ovalbumin-sensitized BALB/c mice.

BACKGROUND: Graphene oxide (GO) is a promising nanomaterial for potential application in the versatile field of biomedicine. Graphene-based nanomaterials have been reported to modulate the functionality of immune cells in culture and to induce pulmonary inflammation in mice. Evidence pertaining to the interaction between graphene-based nanomaterials and the immune system in vivo remains scarce. The present study investigated the effect of polyethylene glycol-coated GO (PEG-GO) on antigen-specific immunity in vivo. METHODS: BALB/c mice were intravenously administered with a single dose of PEG-GO (0.5 or 1 mg/kg) 1 hour before ovalbumin (OVA) sensitization, and antigen-specific antibody production and splenocyte reactivity were measured 7 days later. RESULTS: Exposure to PEG-GO significantly attenuated the serum level of OVA-specific immunoglobulin E. The production of interferon-γ and interleukin-4 by splenocytes restimulated with OVA in culture was enhanced by treatment with PEG-GO. In addition, PEG-GO augmented the metabolic activity of splenocytes restimulated with OVA but not with the T-cell mitogen concanavalin A. CONCLUSION: Collectively, these results demonstrate that systemic exposure to PEG-GO modulates several aspects of antigen-specific immune responses, including the serum production of immunoglobulin E and T-cell functionality.

Mouse intragastric infusion (iG) model.

Direct intragastric delivery of a diet, nutrient or test substance can be achieved in rodents (mice and rats) on a long-term (2-3 months) basis using a chronically implanted gastrostomy catheter and a flow-through swivel system. This rodent intragastric infusion (iG) model has broad applications in research on food intake, gastrointestinal (GI) physiology, GI neuroendocrinology, drug metabolism and toxicity, obesity and liver disease. It achieves maximal control over the rate and pattern of delivery and it can be combined with normal ad libitum feeding of solid diet if so desired. It may be adopted to achieve infusion at other sites of the GI system to test the role of a bypassed GI segment in neuroendocrine physiology, and its use in genetic mouse models facilitates the genetic analysis of a central question under investigation.