Comparison of PET tracing and biodistribution between 64Cu-labeled micro-and nano-polystyrene in a murine inhalation model.

INTRODUCTION: Recent studies showed the presence of microplastic in human lungs. There remains an unmet need to identify the biodistribution of microplastic after inhalation. In this study, we traced the biodistribution of inhaled micro-sized polystyrene (mPS) and/or nano-sized PS (nPS) using 64Cu with PET in mice. METHODS: We used 0.2-0.3-µm sized mPS and 20-nm sized nPS throughout. 64Cu-DOTA-mPS, 64Cu-DOTA-nPS and/or 64CuCl2 were used to trace the distribution in the murine inhalation model. PET images were acquired using an INVEON PET scanner at 1, 12, 24, 48, and 72 h after intratracheal instillation, and the SUVmax for interesting organs were determined, biodistribution was then determined in terms of percentage injected dose/gram of tissue (%ID/g). Ex vivo tissue-radio thin-layer chromatography (Ex vivo-radioTLC) was used to demonstrate the existence of 64Cu-DOTA-PS in tissue. RESULTS: PET image demonstrated that the amount of 64Cu-DOTA-mPS retained within the lung was significantly higher than 64Cu-DOTA-nPS until 72 h; SUVmax values of 64Cu-DOTA-mPS in lungs was 11.7 ± 5.0, 48.3 ± 6.2, 65.5 ± 2.3, 42.2 ± 13.1, and 13.2 ± 2.3 at 1, 12, 24, 48, and 72 h respectively whereas it was 31.2 ± 3.1, 17.3 ± 5.9, 10.0 ± 3.4, 8.1 ± 2.4 and 8.9 ± 3.6 for 64Cu-DOTA-nPS at the corresponding timepoints. The biodistribution data supported the PET data with a similar pattern of clearance of the radioactivity from the lung. nPS cleared rapidly post instillation in comparison to mPS within the lungs. Higher accumulation of %ID/g for nPS (roughly 2 times) were observed compared to mPS in spleen, liver, intestine, thymus, kidney, brain, salivary gland, ovary, and urinary bladder. Ex vivo-radioTLC was used to demonstrate that the detected gamma rays originated from 64Cu-DOTA-mPS or nPS. CONCLUSION: PET image demonstrated the differences in accumulations of mPS and/or nPS between lungs and other interesting organs. The information provided may be used as the basis for future studies on the toxicity of mPS and/or nPS.

Porphyrin-Based Brain Tumor-Targeting Agents: [64Cu]Cu-porphyrin and [64Cu]Cu-TDAP.

The aim of this study is to evaluate a radioactive metal complex platform for brain tumor targeting. Herein, we introduce a new porphyrin derivative, 5,10,15,20-(tetra-N,N-dimethyl-4-aminophenyl)porphyrin (TDAP), in which four N,N-dimethyl-4-p-phenylenediamine (DMPD) moieties are conjugated to the porphyrin labeled with the radiometal 64Cu. DMPD affected the pharmacokinetics of porphyrin in terms of retention time in vivo and tumor-targeting ability relative to those of unmodified porphyrin. [64Cu]Cu-TDAP showed stronger enhancement than [64Cu]Cu-porphyrin in U87MG glioblastoma cells, especially in the cytoplasm and nucleus, indicating its tumor-targeting properties and potential use as a therapeutic agent. In the subcutaneous and orthotopic models of brain-tumor-bearing mice, [64Cu]Cu-TDAP was clearly visualized in the tumor site via positron emission tomography imaging and showed a tumor-to-brain ratio as high as 13. [64Cu]Cu-TDAP deserves attention as a new diagnostic agent that is suitable for the early diagnosis and treatment of brain tumors.

d-Amino Acid Peptide Residualizing Agents for Protein Radioiodination: Effect of Aspartate for Glutamate Substitution.

The residualizing prosthetic agent Nε-(3-[*I]iodobenzoyl)-Lys5-Nα-maleimido-Gly¹-d-GEEEK ([*I]IB-Mal-d-GEEEK) showed promise for the radioiodination of monoclonal antibodies (mAbs) that bind to internalizing molecular targets. Although enhanced tumor uptake was achieved in these studies, elevated kidney accumulation also was observed, particularly with low-molecular-weight, single-domain antibody fragments (sdAbs). Here, we developed an analogous agent (IB-Mal-d-GDDDK), in which glutamate residues (E) were replaced with aspartates (D) to determine whether this modification could decrease renal uptake. [125I]IB-Mal-d-GDDDK and [131I]IB-Mal-d-GEEEK were synthesized with similar radiochemical yields (60⁻80%) and coupled to the anti-HER2 sdAb 5F7 at 50⁻60% efficiency. Paired-label internalization assays in vitro indicated similar levels of intracellular activity residualization in HER2-expressing BT474M1 cells for [125I]IB-Mal-d-GDDDK-5F7 and [131I]IB-Mal-d-GEEEK-5F7. A paired-label biodistribution comparison of the two labeled conjugates was performed in mice with HER2-expressing SKOV-3 xenografts, and the results of this study indicated that renal uptake at 1 h was 127.5 ± 18.7% ID/g and 271.4 ± 66.6% ID/g for [125I]IB-Mal-d-GDDDK-5F7 and [131I]IB-Mal-d-GEEEK-5F7, respectively. The tumor uptake of the two radioconjugates was not significantly different. These results demonstrate that substitution of E with D in the IB-Mal-d-GEEEK construct reduced kidney accumulation of the sdAb. However, renal activity levels need to be reduced further if d-amino acid derived prosthetic agents are to be of practical value for labeling low molecular weight biomolecules such as sdAbs.

Catabolism of (64)Cu and Cy5.5-labeled human serum albumin in a tumor xenograft model.

Human serum albumin (HSA), the most abundant protein in blood plasma, has been used as a drug carrier for the last few decades. Residualizingly radiolabeled serum albumin has been reported to be avidly taken up by tumors of sarcoma-bearing mice and to most likely undergo lysosomal degradation. In this study, we prepared (64)Cu-1,4,7,10-tetraazacyclododecane-N,N',N″,N'″-tetraacetic acid (DOTA) and Cy5.5-conjugated HSA (dual probe), and evaluated its tumor uptake and catabolism. Two dual probes were prepared using different DOTA conjugation sites of HSA (one via Lys residues and the other via the Cys residue). (64)Cu-DOTA-Lys-HSA-Cy5.5 (dual probe-Lys) exhibited higher uptake by RR1022 sarcoma cells in vitro than (64)Cu-DOTA-Cys-HSA-Cy5.5 (dual probe-Cys). In RR1022 tumor-bearing mice, the two dual probes showed a similar level of tumor uptake, but uptake of dual probe-Lys was reduced in the liver and spleen compared to dual probe-Cys, probably because of the presence of a higher number of DOTA molecules in the former. At 24 and 48 h after injection, dual probe-Lys was intact or partially degraded in blood, liver, kidney, and tumor samples, but (64)Cu-DOTA-Lys was observed in the urine using radioactivity detection. Similarly, Cy5.5-Lys was observed in the urine using fluorescence detection. These results indicate that dual probe-Lys may be useful for predicting the catabolic fate of drug-HSA conjugates.

Tumor-homing glycol chitosan-based optical/PET dual imaging nanoprobe for cancer diagnosis.

Imaging techniques including computed tomography, magnetic resonance imaging, and positron emission tomography (PET) offer many potential benefits to diagnosis and treatment of cancers. Each method has its own strong and weak points. Therefore, multimodal imaging techniques have been highlighted as an alternative method for overcoming the limitations of each respective imaging method. In this study, we fabricated PET/optical activatable imaging probe based on glycol chitosan nanoparticles (CNPs) for multimodal imaging. To prepare the dual PET/optical probes based on CNPs, both (64)Cu radiolabeled DOTA complex and activatable matrix metalloproteinase (MMP)-sensitive peptide were chemically conjugated onto azide-functionalized CNPs via bio-orthogonal click chemistry, which was a reaction between azide group and dibenzyl cyclooctyne. The PET/optical activatable imaging probes were visualized by PET and optical imaging system. Biodistribution of probes and activity of MMP were successfully measured in tumor-bearing mice.

In vivo characterization of 68Ga-NOTA-VEGF 121 for the imaging of VEGF receptor expression in U87MG tumor xenograft models.

PURPOSE: Vascular endothelial growth factor receptors (VEGFRs) are associated with tumor growth and induction of tumor angiogenesis and are known to be overexpressed in various human tumors. In the present study, we prepared and evaluated (68)Ga-1,4,7-triazacyclononane-1,4,7-triacetic acid-benzyl (NOTA)-VEGF(121) as a positron emission tomography (PET) radioligand for the in vivo imaging of VEGFR expression. METHODS: (68)Ga-NOTA-VEGF(121) was prepared by conjugation of VEGF(121) and p-SCN-NOTA, followed by radiolabeling with (68)GaCl(3) and then purification using a PD-10 column. Human aortic endothelial cell (HAEC) binding of (68)Ga-NOTA-VEGF(121) was measured as a function of time. MicroPET and biodistribution studies of U87MG tumor xenografted mice were performed at 1, 2, and 4 h after injection of (68)Ga-NOTA-VEGF(121). The tumor tissues were then sectioned and subjected to immunostaining. RESULTS: The decay-corrected radiochemical yield of (68)Ga-NOTA-VEGF(121) was 40 ± 4.5 % and specific activity was 243.1 ± 104.6 GBq/µmol (8.6 ± 3.7 GBq/mg). (68)Ga-NOTA-VEGF(121) was avidly taken up by HAECs in a time-dependent manner, and the uptake was blocked either by 32 % with VEGF(121) or by 49 % with VEGFR2 antibody at 4 h post-incubation. In microPET images of U87MG tumor xenografted mice, radioactivity was accumulated in tumors (2.73±0.32 %ID/g at 2 h), and the uptake was blocked by 40 % in the presence of VEGF(121). In biodistribution studies, tumor uptake (1.84±0.14 %ID/g at 2 h) was blocked with VEGF(121) at a similar level (52 %) to that of microPET images. Immunostaining analysis of U87MG tumor tissues obtained after the microPET imaging showed high levels of VEGFR2 expression. CONCLUSION: These results demonstrate that (68)Ga-NOTA-VEGF(121) has potential for the in vivo imaging of VEGFR expression. In addition, our results also suggest that the in vivo characteristics of radiolabeled VEGF depend on the properties of the radioisotope and the chelator used.

Facile method to radiolabel glycol chitosan nanoparticles with (64)Cu via copper-free click chemistry for MicroPET imaging.

An efficient and straightforward method for radiolabeling nanoparticles is urgently needed to understand the in vivo biodistribution of nanoparticles. Herein, we investigated a facile and highly efficient strategy to prepare radiolabeled glycol chitosan nanoparticles with (64)Cu via a strain-promoted azide-alkyne cycloaddition strategy, which is often referred to as click chemistry. First, the azide (N3) group, which allows for the preparation of radiolabeled nanoparticles by copper-free click chemistry, was incorporated to glycol chitosan nanoparticles (CNPs). Second, the strained cyclooctyne derivative, dibenzyl cyclooctyne (DBCO) conjugated with a 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) chelator, was synthesized for preparing the preradiolabeled alkyne complex with (64)Cu radionuclide. Following incubation with the (64)Cu-radiolabeled DBCO complex (DBCO-PEG4-Lys-DOTA-(64)Cu with high specific activity, 18.5 GBq/µmol), the azide-functionalized CNPs were radiolabeled successfully with (64)Cu, with a high radiolabeling efficiency and a high radiolabeling yield (>98%). Importantly, the radiolabeling of CNPs by copper-free click chemistry was accomplished within 30 min, with great efficiency in aqueous conditions. In addition, we found that the (64)Cu-radiolabeled CNPs ((64)Cu-CNPs) did not show any significant effect on the physicochemical properties, such as size, zeta potential, or spherical morphology. After (64)Cu-CNPs were intravenously administered to tumor-bearing mice, the real-time, in vivo biodistribution and tumor-targeting ability of (64)Cu-CNPs were quantitatively evaluated by microPET images of tumor-bearing mice. These results demonstrate the benefit of copper-free click chemistry as a facile, preradiolabeling approach to conveniently radiolabel nanoparticles for evaluating the real-time in vivo biodistribution of nanoparticles.

PET Tracing of Biodistribution for Orally Administered 64Cu-Labeled Polystyrene in Mice.

Plastics are used commonly in the world because of their convenience and cost effectiveness. Microplastics, an environmental threat and human health risk, are widely detected in food and consequently ingested. However, degraded plastics are found everywhere, creating an environmental threat and human health risk. Therefore, real-time monitoring of orally administered microplastics to trace them in the body is tremendously important. Methods: In this study, to visualize their absorption path, we labeled polystyrene with [64Cu]Cu-DOTA. We prepared radiolabeled polystyrene with 64Cu. Afterward, [64Cu]Cu-DOTA-polystyrene was orally administered to mice, and we evaluated its transit and absorption using PET imaging. The absorption path and distribution of [64Cu]Cu-DOTA-polystyrene were determined using PET over 48 h. Ex vivo tissue radio-thin-layer chromatography (TLC) was used to demonstrate the existence of [64Cu]Cu-DOTA-polystyrene in tissue. Results: PET images demonstrated that [64Cu]Cu-DOTA-polystyrene began to transit to the intestine within 1 h. Accumulation of [64Cu]Cu-DOTA-polystyrene in the liver was also observed. The biodistribution of [64Cu]Cu-DOTA-polystyrene confirmed the distribution of [64Cu]Cu-DOTA-polystyrene observed on the PET images. Ex vivo radio-TLC demonstrated that the detected γ-rays originated from [64Cu]Cu-DOTA-polystyrene. Conclusion: This study provided PET evidence of the existence and accumulation of microplastics in tissue and cross-confirmed the PET findings by ex vivo radio-TLC. This information may be used as the basis for future studies on the toxicity of microplastics.

The Prediction of HER2-Targeted Treatment Response Using 64Cu-Tetra-Azacyclododecanetetra-Acetic Acid (DOTA)-Trastuzumab PET/CT in Metastatic Breast Cancer: A Case Report.

A 45-year-old woman diagnosed with breast cancer reported disease progression in the form of metastatic lung and recurrent breast lesions following chemotherapy and human epidermal growth factor receptor 2 (HER2)-targeted therapy. The patient underwent 64Cu-tetra-azacyclododecanetetra-acetic acid (DOTA)-trastuzumab positron emission tomography/computed tomography (PET/CT) to evaluate the HER2 expression status. 64Cu-DOTA-trastuzumab accumulated in the left breast and lymph nodes but not in the lung lesions. Following trastuzumab emtansine treatment, there was a significant improvement in the lesions with 64Cu-DOTA-trastuzumab accumulation. However, the lesions that did not accumulate 64Cu-DOTA-trastuzumab aggravated. Therefore, it was concluded that 64Cu-DOTA-trastuzumab PET/CT can be used to predict the outcome of HER2-targeted treatment by evaluating HER2 expression in breast cancer patients.

An Improved 211At-Labeled Agent for PSMA-Targeted α-Therapy.

α-Particle emitters targeting the prostate-specific membrane antigen (PSMA) proved effective in treating patients with prostate cancer who were unresponsive to the corresponding ß-particle therapy. 211At is an α-emitter that may engender less toxicity than other α-emitting agents. We synthesized a new 211At-labeled radiotracer targeting PSMA that resulted from the search for a pharmacokinetically optimized agent. Methods: A small series of 125I-labeled compounds was synthesized from tin precursors to evaluate the effect of the location of the radiohalogen within the molecule and the presence of lutetium in the chelate on biodistribution. On that basis, 211At-3-Lu was selected and evaluated in cell uptake and internalization studies, and biodistribution and PSMA-expressing (PSMA+) PC3 PIP tumor growth control were evaluated in experimental flank and metastatic (PC3-ML-Luc) models. A long-term (13-mo) toxicity study was performed for 211At-3-Lu, including tissue chemistries and histopathology. Results: The radiochemical yield of 211At-3-Lu was 17.8% ± 8.2%. Lead compound 211At-3-Lu demonstrated total uptake within PSMA+ PC3 PIP cells of 13.4 ± 0.5% of the input dose after 4 h of incubation, with little uptake in control cells. In SCID mice, 211At-3-Lu provided uptake that was 30.6 ± 4.8 percentage injected dose per gram (%ID/g) in PSMA+ PC3 PIP tumor at 1 h after injection, and this uptake decreased to 9.46 ± 0.96 %ID/g by 24 h. Tumor-to-salivary gland and tumor-to-kidney ratios were 129 ± 99 at 4 h and 130 ± 113 at 24 h, respectively. Deastatination was not significant (stomach, 0.34 ± 0.20 %ID/g at 4 h). Dose-dependent survival was demonstrated at higher doses (>1.48 MBq) in both flank and metastatic models. There was little off-target toxicity, as demonstrated by hematopoietic stability, unchanged tissue chemistries, weight gain rather than loss throughout treatment, and favorable histopathologic findings. Conclusion: Compound 211At-3-Lu or close analogs may provide limited and acceptable toxicity while retaining efficacy in management of prostate cancer.

Synthesis and preliminary evaluation of 211At-labeled inhibitors of prostate-specific membrane antigen for targeted alpha particle therapy of prostate cancer.

INTRODUCTION: The high potency and short tissue range of α-particles are attractive features for targeted radionuclide therapy, particularly for cancers with micro-metastases. In the current study, we describe the synthesis of a series of 211At-labeled prostate-specific membrane antigen (PSMA) inhibitors and their preliminary evaluation as potential agents for metastatic prostate cancer treatment. METHODS: Four novel Glu-urea based PSMA ligands containing a trialkyl stannyl group were synthesized and labeled with 211At, and for comparative purposes, 131I, via halodestannylation reactions with N-chlorosuccinimide as the oxidant. A PSMA inhibitory assay was performed to evaluate PSMA binding of the unlabeled, iodinated compounds. A series of paired-label biodistribution experiments were performed to compare each 211At-labeled PSMA ligand to its 131I-labeled counterpart in mice bearing subcutaneous PC3 PSMA+ PIP xenografts. RESULTS: Radiochemical yields ranged from 32% to 65% for the 211At-labeled PSMA inhibitors and were consistently lower than those obtained with the corresponding 131I-labeled analogue. Good localization in PC3 PSMA+ PIP but not control xenografts was observed for all labeled molecules studied, which exhibited a variable degree of in vivo dehalogenation as reflected by thyroid and stomach activity levels. Normal tissue uptake and in vivo stability for several of the compounds was markedly improved compared with the previously evaluated compounds, [211At]DCABzL and [*I]DCIBzL. CONCLUSIONS AND IMPLICATIONS FOR PATIENT CARE: Compared with the first generation compound [211At]DCABzL, several of the novel 211At-labeled PSMA ligands exhibited markedly improved stability in vivo and higher tumor-to-normal tissue ratios. [211At]GV-620 has the most promising characteristics and warrants further evaluation as a targeted radiotherapeutic for prostate cancer.

Astatine-211 labeled anti-HER2 5F7 single domain antibody fragment conjugates: radiolabeling and preliminary evaluation.

INTRODUCTION: Derived from heavy chain only camelid antibodies, ~15-kDa single-domain antibody fragments (sdAbs) are an attractive platform for developing molecularly specific imaging probes and targeted radiotherapeutics. The rapid tumor accumulation and normal tissue clearance of sdAbs might be ideal for use with 211At, a 7.2-h half-life α-emitter, if appropriate labeling chemistry can be devised to trap 211At in cancer cells after sdAb binding. This study evaluated two reagents, [211At]SAGMB and iso-[211At]SAGMB, for this purpose. METHODS: [211At]SAGMB and iso-[211At]SAGMB, and their radioiodinated analogues [131I]SGMIB and iso-[131I]SGMIB, were synthesized by halodestannylation and reacted with the anti-HER2 sdAb 5F7. Radiochemical purity, immunoreactivity and binding affinity were determined. Paired-label internalization assays on HER2-expressing BT474M1 breast carcinoma cells directly compared [131I]SGMIB-5F7/[211At]SAGMB-5F7 and iso-[131I]SGMIB-5F7/iso-[211At]SAGMB-5F7 tandems. The biodistribution of the two tandems was evaluated in SCID mice with subcutaneous BT474M1 xenografts. RESULTS: Radiochemical yields for Boc2-iso-[211At]SAGMB and Boc2-[211At]SAGMB synthesis, and efficiencies for coupling of iso-[211At]SAGMB and [211At]SAGMB to 5F7 were similar, with radiochemical purities of [211At]SAGMB-5F7 and iso-[211At]SAGMB-5F7 >98%. iso-[211At]SAGMB-5F7 and [211At]SAGMB-5F7 had immunoreactive fractions >80% and HER2 binding affinities of less than 5 nM. Internalization assays demonstrated high intracellular trapping of radioactivity, with little difference observed between corresponding 211At- and 131I-labeled 5F7 conjugates. Higher BT474M1 intracellular retention was observed from 1-6 h for the iso-conjugates (iso-[211At]SAGMB-5F7, 74.3 ± 2.8%, vs. [211At]SAGMB-5F7, 63.7 ± 0.4% at 2 h) with the opposite behavior observed at 24 h. Peak tumor uptake for iso-[211At]SAGMB-5F7 was 23.4 ± 2.2% ID/g at 4 h, slightly lower than its radioiodinated counterpart, but significantly higher than observed with [211At]SAGMB-5F7. Except in kidneys and lungs, tumor-to-normal organ ratios for iso-[211At]SAGMB-5F7 were greater than 10:1 by 2 h, and significantly higher than those for [211At]SAGMB-5F7. CONCLUSION: These 211At-labeled sdAb conjugates, particularly iso-[211At]SAGMB-5F7, warrant further evaluation for targeted α-particle radiotherapy of HER2-expressing cancers.

Brush border enzyme-cleavable linkers: Evaluation for reducing renal uptake of radiolabeled prostate-specific membrane antigen inhibitors.

INTRODUCTION: Radiolabeled, low-molecular-weight prostate-specific membrane antigen (PSMA) inhibitors based on the Glu-ureido pharmacophore show promise for the detection and treatment of castration-resistant prostate cancer; however, high renal retention of activity, related in part to overexpression of PSMA in kidneys can be problematic. The goal of the current study was to investigate the use of brush border enzyme-cleavable linkers as a strategy for reducing kidney activity levels from radiolabeled PSMA inhibitors. METHODS: PSMA-769 (6), a derivative of the prototypical PSMA inhibitor (((S)­1­carboxy­5­(4­iodobenzamido)pentyl)carbamoyl)glutamate (12) modified to contain a Gly-Tyr linker, and its protected tin precursor (11) were synthesized starting from the basic pharmacophore molecule Lys-urea-Glu. An analogue of 6 containing d­tyrosine in lieu of l­tyrosine (PSMA-769-d-tyrosine) and the corresponding tin precursor (d-11) also were synthesized. Both radioiodinated and 211At-labeled 6 were synthesized by radiohalogenation of 11 and deprotection in situ. Similarly, radioiodinated d-6 was synthesized from d-11. Paired label biodistribution of [125I]12 and [131I]6 was performed in normal mice and in SCID mice bearing both PC3 PIP (PSMA+) and PC3 flu (PSMA-) subcutaneous prostate carcinoma xenografts. The biodistribution of [131I]6 and [211At]6 was also evaluated in this tumor model. Biodistribution of the two radioiodinated diastereomers of 6 was evaluated in normal mice and urine samples were analyzed for the presence of 4­iodohippuric acid. RESULTS: Compounds [131I]6 and [211At]6 were synthesized from 11 in overall radiochemical yields of 32.5 ±â€¯0.1% (n = 4) and 22% (n = 1), respectively; radiochemical purity was >95%. In normal mice, renal uptake of [131I]6 was 1.4-, 2.8- and 161-fold lower than that seen for co-injected [125I]12 at 1 h, 4 h and 21 h, respectively. In tumor-bearing mice, kidney uptake of [131I]6 was similar to that for [125I]12 (P > 0.05) at 1 h and 4 h but was 6- to 7-fold lower at 21 h; however, [131I]6 uptake in PC3 PIP tumors was also lower than that seen for [125I]12 at 21 h (12.6 ±â€¯3.4%ID/g vs. 36.8 ±â€¯12.4%ID/g). Uptake of [211At]PSMA-769 in PC3 PIP tumors was slightly higher than that seen for [131I]PSMA-769 at 4 h (9.6 ±â€¯1.6%ID/g versus 7.8 ±â€¯1.6%ID/g; P = 0.002); its uptake in a number of normal tissues also was higher. In normal mice, kidney uptake of [125I]PSMA-769 at 4 h was about 73% of that seen for [131I]PSMA-769-d-tyrosine. Activity in the urine of mice receiving [125I]PSMA-769 contained mainly 4­[125I]iodohippuric acid while unmetabolized intact molecule was present in the case of [125I]PSMA-769-d-tyrosine. CONCLUSION: Use of this brush border enzyme-cleavable linker reduced kidney uptake and resulted in improved tumor:kidney uptake ratios. Although further structural improvements are needed, this linker approach might be useful as a component in strategies for reducing renal uptake of radiolabeled PSMA inhibitors.

Fluorine-18 Labeling of the HER2-Targeting Single-Domain Antibody 2Rs15d Using a Residualizing Label and Preclinical Evaluation.

PURPOSE: Our previous studies with F-18-labeled anti-HER2 single-domain antibodies (sdAbs) utilized 5F7, which binds to the same epitope on HER2 as trastuzumab, complicating its use for positron emission tomography (PET) imaging of patients undergoing trastuzumab therapy. On the other hand, sdAb 2Rs15d binds to a different epitope on HER2 and thus might be a preferable vector for imaging in these patients. The aim of this study was to evaluate the tumor targeting of F-18 -labeled 2Rs15d in HER2-expressing breast carcinoma cells and xenografts. PROCEDURES: sdAb 2Rs15d was labeled with the residualizing labels N-succinimidyl 3-((4-(4-[18F]fluorobutyl)-1H-1,2,3-triazol-1-yl)methyl)-5-(guanidinomethyl)benzoate ([18F]RL-I) and N-succinimidyl 4-guanidinomethyl-3-[125I]iodobenzoate ([125I]SGMIB), and the purity and HER2-specific binding affinity and immunoreactivity were assessed after labeling. The biodistribution of I-125- and F-18-labeled 2Rs15d was determined in SCID mice bearing subcutaneous BT474M1 xenografts. MicroPET/x-ray computed tomograph (CT) imaging of [18F]RL-I-2Rs15d was performed in this model and compared to that of nonspecific sdAb [18F]RL-I-R3B23. MicroPET/CT imaging was also done in an intracranial HER2-positive breast cancer brain metastasis model after administration of 2Rs15d-, 5F7-, and R3B23-[18F]RL-I conjugates. RESULTS: [18F]RL-I was conjugated to 2Rs15d in 40.8 ± 9.1 % yield and with a radiochemical purity of 97-100 %. Its immunoreactive fraction (IRF) and affinity for HER2-specific binding were 79.2 ± 5.4 % and 7.1 ± 0.4 nM, respectively. [125I]SGMIB was conjugated to 2Rs15d in 58.4 ± 8.2 % yield and with a radiochemical purity of 95-99 %; its IRF and affinity for HER2-specific binding were 79.0 ± 12.9 % and 4.5 ± 0.8 nM, respectively. Internalized radioactivity in BT474M1 cells in vitro for [18F]RL-I-2Rs15d was 43.7 ± 3.6, 36.5 ± 2.6, and 21.7 ± 1.2 % of initially bound radioactivity at 1, 2, and 4 h, respectively, and was similar to that seen for [125I]SGMIB-2Rs15d. Uptake of [18F]RL-I-2Rs15d in subcutaneous xenografts was 16-20 %ID/g over 1-3 h. Subcutaneous tumor could be clearly delineated by microPET/CT imaging with [18F]RL-I-2Rs15d but not with [18F]RL-I-R3B23. Intracranial breast cancer brain metastases could be visualized after intravenous administration of both [18F]RL-I-2Rs15d and [18F]RL-I-5F7. CONCLUSIONS: Although radiolabeled 2Rs15d conjugates exhibited lower tumor cell retention both in vitro and in vivo than that observed previously for 5F7, given that it binds to a different epitope on HER2 from those targeted by the clinically utilized HER2-targeted therapeutic antibodies trastuzumab and pertuzumab, F-18-labeled 2Rs15d has potential for assessing HER2 status by PET imaging after trastuzumab and/or pertuzumab therapy.

Hybrid lymph node imaging using 64Cu-labeled mannose-conjugated human serum albumin with and without indocyanine green.

OBJECTIVE: Human serum albumin (HSA), which has 58 Lys residues, one Cys residue, and indocyanine green (ICG) adsorption sites, can be used as a multifunctional platform for the development of hybrid imaging probes. In this study, we prepared 64Cu-labeled mannose-conjugated HSA with and without ICG ([64Cu]1-ICG and [64Cu]1, respectively) and compared hybrid PET/near-infrared fluorescence (NIRF) imaging with positron emission tomography (PET)/Cerenkov luminescence (CL) imaging of lymph nodes (LNs). MATERIALS AND METHODS: 1,4,7,10-Tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA)/mannose-conjugated HSA (1) was synthesized by conjugating mannose molecules to Lys residues and a DOTA molecule to a Cys residue of HSA. Compound 1 was then labeled with Cu ([64Cu]1), and the resulting [64Cu]1 was adsorbed with ICG ([64Cu]1-ICG). PET/NIRF or PET/CL imaging and subsequent biodistribution studies were performed in ICR mice after injection of the probes into the foot pads. RESULTS: The numbers of mannose and DOTA molecules conjugated to HSA were 7.17 ± 0.49 and 0.95 ± 0.18, respectively. The site-specific conjugation of one DOTA molecule to HSA was sufficient for 64Cu-labeling with high efficiency (96.0 ± 1.1%). PET/NIRF and PET/CL imaging and subsequent biodistribution studies demonstrated that the probes were avidly taken up by the popliteal LNs (PO), with a slightly higher uptake ratio of the PO to the lumbar LNs by [64Cu]1. CONCLUSION: In-vivo studies suggest that [64Cu]1 has more specific and selective binding to mannose receptors in the PO than [64Cu]1-ICG.

Hybrid PET/optical imaging of integrin αVß3 receptor expression using a (64)Cu-labeled streptavidin/biotin-based dimeric RGD peptide.

BACKGROUND: Hybrid PET/optical imaging provides quantitative and complementary information for diagnosis of tumors. Herein, we developed a (64)Cu-labeled AlexaFluor 680-streptavidin ((AF)SAv)/biotin-based dimeric cyclic RGD peptide (RGD2) for hybrid PET/optical imaging of integrin αVß3 expression. METHODS: (64)Cu-1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA)-(AF)SAv/biotin-PEG-RGD2 was prepared by formation of a complex comprising DOTA-(AF)SAv and biotin-PEG-RGD2, followed by radiolabeling with (64)Cu. Receptor binding studies of DOTA-(AF)SAv/biotin-PEG-RGD2 were performed using U87MG cells and (125)I-RGDyK as the radioligand, and cellular uptake studies of (64)Cu-DOTA-(AF)SAv/biotin-PEG-RGD2 were also performed. MicroPET imaging followed by optical imaging of U87MG tumor-bearing mice was acquired after injection of the hybrid probe, and region of interest (ROI) analysis of tumors was performed. Ex vivo PET/optical imaging and biodistribution studies of the major tissues were performed after the in vivo imaging, and immunofluorescence staining of the tumor tissue sections was carried out. RESULTS: (64)Cu-DOTA-(AF)SAv/biotin-PEG-RGD2 was prepared in 52.1 ± 5.4 % radiochemical yield and with specific activity of 1.0 ± 0.1 GBq/mg. Receptor binding studies showed that DOTA-(AF)SAv/biotin-PEG-RGD2 had higher binding affinity for integrin αVß3 than RGD2, reflecting a possible polyvalency effect. Moreover, the hybrid probe revealed time-dependent uptake by U87MG cells. In a microPET/optical imaging study, the hybrid probe demonstrated high accumulation in tumors; ROI analysis revealed 2.7 ± 0.2 % ID/g at 1 h and 4.7 ± 0.2 % ID/g at 21 h after injection, and subsequently acquired optical images showed tumors with strong fluorescence intensity. Ex vivo PET/optical images of the major tissues confirmed the in vivo imaging data, and biodistribution studies demonstrated high and specific uptake in tumors (4.8 ± 0.1 % ID/g). Immunofluorescence staining showed the formation of new blood vessels in tumor tissues, suggesting that the tumor uptake was due to specific binding of the hybrid probe to integrin αVß3 expressed on tumor cells. CONCLUSIONS: These results indicate that a (64)Cu-DOTA-(AF)SAv/biotin-PEG-RGD2 is able to provide quantitative information on hybrid PET/optical imaging of integrin αVß3 expression.

68Ga-NODAGA-VEGF121 for in vivo imaging of VEGF receptor expression.

PURPOSE: Vascular endothelial growth factor (VEGF) is a crucial regulator of angiogenesis. In this study, we labeled VEGF121 with (68)Ga using a hydrophilic chelating agent, NODAGA and evaluated the resulting (68)Ga-NODAGA-VEGF121 for in vivo imaging of VEGF receptor (VEGFR) expression. METHODS: NODAGA-VEGF121 was prepared and its binding affinity for VEGFR2 was measured using (125)I-VEGF121. (68)Ga-NODAGA-VEGF121 was prepared by labeling NODAGA-VEGF121 with (68)GaCl3 followed by purification using a PD-10 column. Human aortic endothelial cell (HAEC) binding studies of (68)Ga-NODAGA-VEGF121 were performed at 37°C for 4 h. MicroPET imaging followed by biodistribution studies were performed in U87MG tumor-bearing mice injected with (68)Ga-NODAGA-VEGF121. Immunofluorescence staining of the tumor tissues was performed to verify VEGFR2 expression. RESULTS: Binding affinity of NODAGA-VEGF121 for VEGFR2 was found to be comparable to that of VEGF121. (68)Ga-NODAGA-VEGF121 was prepared in 47.8% yield with specific activity of 3.4 GBq/mg. (68)Ga-NODAGA-VEGF121 was avidly taken up by HAECs with a time-dependent increase from 9.88 %ID at 1 h to 20.86 %ID at 4h. MicroPET imaging of mice demonstrated high liver and spleen uptake with clear visualization of tumor at 1h after injection. ROI analysis of tumors revealed 2.53 ± 0.11 %ID/g at 4 h after injection. In the blocking study, tumor uptake was inhibited by 29% at 4 h. Subsequent biodistribution studies demonstrated tumor uptake of 2.38 ± 0.15 %ID/g. Immunofluorescence staining of the tumor tissues displayed high level of VEGFR2 expression. CONCLUSIONS: These results demonstrate that (68)Ga-NODAGA-VEGF121 led to VEGFR-specific distribution in U87MG tumor-bearing mice. This study also suggests that altered physicochemical properties of VEGF121 after radiolabeling may affect biodistribution of the radiolabeled VEGF121.

Characterization of oleanolic acid derivative for colon cancer targeting with positron emission tomography.

Oleanolic acid (OA) is a pentacyclic triterpenoid found in various plant species. Triterpenoid compounds have been shown to inhibit tumor proliferation and to induce apoptosis in cancer cells. We synthesized an OA derivative and evaluated its inhibitory effects on cell proliferation in human colon cancer. Radioisotope-labeled OA was prepared for noninvasive monitoring of tumor progression in vitro and in vivo. The OA derivative decreased cell survival in a concentration-dependent manner and increased apoptosis in HT-29 cells. Furthermore, it induced downregulation of cyclin D1, Cox-2, Bcl-2 and Bcl-xL mRNA expression and upregulation of the mRNA expression of the anti-apoptotic Bax protein in HT29 cells. NF-κB p65 and IκB expression also decreased, whereas expression of the apoptosis marker, the cleaved form of PARP-1, significantly increased in OA derivative-treated HT-29 cells. Radioisotope-labeled OA (68Ga-NOTA-OA) showed significantly high tumor uptake, as assessed by biodistribution and positron emission tomography imaging analyses, at 1 h post-injection in the human colon cancer xenograft model. Our results demonstrate that the OA derivative has promising properties as an anticancer drug and as an imaging tool for tumor targeting.

64Cu-Labeled tetraiodothyroacetic acid-conjugated liposomes for PET imaging of tumor angiogenesis.

INTRODUCTION: We synthesized and evaluated (64)Cu-labeled tetraiodothyroacetic acid (tetrac)-conjugated liposomes for PET imaging of tumor angiogenesis, because tetrac inhibits angiogenesis via integrin αVß3. METHODS: Tetrac-PEG-DSPE and DOTA-PEG-DSPE were synthesized and formulated with other lipids into liposomes. The resulting tetrac/DOTA-liposomes were labeled with (64)Cu at 40 °C for 1 h and purified using a PD-10 column. (64)Cu-DOTA-liposomes were also prepared for comparison. Human aortic endothelial cell (HAEC) binding studies were performed by incubating the liposomes with the cells at 37 °C. MicroPET imaging followed by tissue distribution study was carried out using U87MG tumor-bearing mice injected with tetrac/(64)Cu-DOTA-liposomes or (64)Cu-DOTA-liposomes. RESULTS: HAEC binding studies exhibited that tetrac/(64)Cu-DOTA-liposomes were avidly taken up by the cells from 1.02 %ID at 1 h to 11.89 %ID at 24 h, while (64)Cu-DOTA-liposomes had low uptake from 0.47 %ID at 1 h to 1.57 %ID at 24 h. MicroPET imaging of mice injected with tetrac/(64)Cu-DOTA-liposomes showed high radioactivity accumulation in the liver and spleen. ROI analysis of the tumor images revealed 1.93 ± 0.12 %ID/g at 1 h and 2.70 ± 0.36 %ID/g at 22 h. In contrast, tumor ROI analysis of (64)Cu-DOTA-liposomes revealed 0.54 ± 0.08 %ID/g at 1 h and 0.52 ± 0.09 %ID/g at 22 h. Tissue distribution studies confirmed that the tumor uptakes of tetrac/(64)Cu-DOTA-liposomes and (64)Cu-DOTA-liposomes were 1.75 ± 0.03 %ID/g and 0.36 ± 0.01 %ID/g at 22 h, respectively. CONCLUSION: These results demonstrate that tetrac/(64)Cu-DOTA-liposomes have significantly enhanced tumor uptake compared to (64)Cu-DOTA-liposomes due to tetrac conjugation. Further studies are warranted to reduce the liver and spleen uptake of tetrac/(64)Cu-DOTA-liposomes.

Cholesteryl hyaluronic acid-coated, reduced graphene oxide nanosheets for anti-cancer drug delivery.

Here, we report hyaluronyl reduced graphene oxide (rGO) nanosheets as a tumor-targeting delivery system for anticancer agents. Hyaluronyl-modified rGO nanosheets were prepared by synthesizing cholesteryl hyaluronic acid (CHA) and using it to coat rGO nanosheets, yielding CHA-rGO. Compared with rGO, CHA-rGO nanosheets showed increased colloidal stability under physiological conditions and improved in vivo safety, with a survival rate of 100% after intravenous administration of 40 mg/kg in mice. The doxorubicin (Dox) loading capacity of CHA-rGO was 4-fold greater than that of rGO. Uptake of Dox by CD44-overexpressing KB cells was higher for CHA-rGO than for rGO, and was decreased in the presence of hyaluronic acid through competition for CD44 receptor binding. After intravenous administration in tumor-bearing mice, CHA-rGO/Dox showed higher tumor accumulation than rGO/Dox. The in vivo antitumor efficacy of Dox delivered by CHA-rGO was significantly increased compared with free Dox or rGO/Dox. In CHA-rGO/Dox-treated mice, tumor weights were reduced to 14.1% ± 0.1% of those in untreated mice. Our findings indicate that CHA-rGO nanosheets possess greater stability, safety, drug-loading capacity, and CD44-mediated delivery of Dox than rGO nanosheets. These beneficial properties of CHA-rGO improved the distribution of Dox to tumors and facilitated the cellular uptake of Dox by CD44-overexpressing tumor cells, resulting in enhanced anticancer effects.