Targeted theranostic nano-system integrating functions of both diagnosis and therapy shows great potential for improving diagnosis and therapeutic efficacy. Herein, multifunctional nanoparticle based on activatable hyaluronic acid (HA) conjugating two near-infrared (NIR) dyes of Cy5.5 and IR825 was successfully designed and fabricated, and simultaneously used as a carrier for encapsulating perfluorooctylbromide (PFOB). In this system, PFOB showed good capability to absorb the X-rays, Cy5.5 on the outer surface acted as a fluorescent dye activatable by hyaluronidases (Hyals) in the tumor, and IR825 in the core as a photothermal agent. The obtained nanoparticles (NPs) of PFOB@IR825-HA-Cy5.5 can be utilized for triple X-ray computed tomography (CT), fluorescence and photoacoustic imaging. When PFOB@IR825-HA-Cy5.5 NPs were intravenously injected into the mice bearing HT-29 tumor, efficient tumor accumulation was clearly observed, as revealed by the triple modal imaging. An in vivo tumor treatment experiment was conducted by combination of PFOB@IR825-HA-Cy5.5 and near-infrared laser irradiation, achieving effective tumor ablation in mice. Therefore, PFOB@IR825-HA-Cy5.5 NPs is a safe, efficient, imageable photothermal nanoprobe, showing great potential for cancer theranostics.Theranostic agent of PFOB@IR825-HA-Cy5.5 NPs was successfully fabricated. PFOB showed good capability to absorb the X-rays, Cy5.5 on the outer surface acted as fluorescent probe, while IR825 in the core as photothermal agent. By utilizing the fluorescence quench effect of the two dyes and degradability of HA by hyaluronidase (Hyal-1) in the tumor, strong fluorescence enhancement could be achieved to delineate the tumor more clearly. Such theranostic agent demonstrated excellent diagnostics capability without compromising the photothermal therapy efficacy, and can be utilized as an active targeting agent for triple X-ray computed tomography (CT), photoacoustic and activatable fluorescence imaging guided photothermal ablation of tumors.Download high-res image (217KB)Download full-size image

An efficient chemical reduction protocol has been developed for the synthesis of hyaluronic acid-coated silver nanoparticles (HA-Ag NPs) that are spherical, ultrasmall and monodisperse. The as-synthesized HA-Ag NPs not only exhibited excellent long-term stability and low cytotoxicity but also could be used as a nanoplatform for X-ray computed tomography (CT) and single-photon emission computed tomography (SPECT) imaging after being radiolabeled with 99mTc.Keywords: HA-Ag NPs; long-term stability; low cytotoxicity; nanoplatform; SPECT imaging; X-ray computed tomography (CT)

Integrins, a large family of cell adhesion receptors, have been shown to play an important role for glioma proliferation and invasion. Several integrin receptors, including αvβ3, αvβ5, and α5β1, have generated clinical interest for glioma diagnosis and antitumor therapy. Integrin α5β1 has been highlighted as a prognostic and diagnostic marker in glioma, and its expression is correlated with a worse prognosis in high-grade glioma. However, unlike extensively studied integrins αvβ3 and αvβ5, very few integrin α5β1-specific radiotracers have been reported. Developing α5β1-specific radiotracers may provide alternative diagnosis and evaluation options in addition to well-studied αvβ3/αvβ5-specific tracers, and they may add new documents for profiling tumor progression. Here, a novel integrin α5β1-specific probe 99mTc-HisoDGR was fabricated for SPECT (single-photon emission computed tomography) imaging of glioma. To confirm its selective targeting of integrin α5β1 in vivo, the mouse models of α5β1-positive U87MG human glioma were subjected to SPECT/CT scans, and biodistribution experiments and blocking studies were performed. Small-animal SPECT/CT imaging experiments demonstrated that the tumors were clearly visualized in both subcutaneous and orthotopic glioma tumor models with clear background at 0.5, 1, and 2 h p.i. The tumor accumulation of 99mTc-HisoDGR showed significant reduction when excess cold isoDGR peptide was coinjected, suggesting that the tumor uptake was specifically mediated. Our work revealed that 99mTc-HisoDGR represented a powerful molecular probe for integrin α5β1-positive cancer imaging; moreover, it might be a promising tool for evaluating malignancy, predicting prognosis, selecting subpopulations of patients who might be sensitive to integrin α5β1-targeted drugs, and assessing and monitoring the response to integrin α5β1-targeted drugs in clinical trials.

This Article reported the fabrication of a robust theranostic cerasome encapsulating indocyanine green (ICG) by incorporating 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)2000]-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid monoamide (DSPE-PEG2000-DOTA), followed by chelating radioisotope of 177Lu. Its applications in optical and nuclear imaging of tumor uptake and biodistribution, as well as photothermal killing of cancer cells, were investigated. It was found that the obtained cerasome could act efficiently as fluorescence contrast agent as well as nuclear imaging tracer. Encapsulating ICG into cerasome could protect ICG from degradation, aggregation, and fast elimination from body, resulting in remarkable improvement in near-infrared fluorescence imaging, photothermal stability, and in vivo pharmacokinetic profile. Both fluorescence and nuclear imaging showed that such agent could selectively accumulate in tumor site after intravenous injection of the cerasome agent into Lewis lung carcinoma tumor bearing mice, resulting in efficient photothermal ablation of tumor through a one-time NIR laser irradiation at the best time window. The ability to track the uptake of cerasomes on a whole body basis could provide researchers with an excellent tool for developing cerasome-based drug delivery agents, especially the strategy of labeling cerasomes with theranostic radionuclide 177Lu, enabling the ability of the 177Lu-labeled cerasomes for radionuclide cancer therapy and even the combined therapy.Keywords: cerasome; indocyanine green; multimodal imaging; nanotheranostics; photothermal therapy

β-Emitters can produce Cerenkov radiation that is detectable by Cerenkov luminescence imaging (CLI), allowing the combination of PET and CLI with one radiotracer for both tumor diagnosis and visual guidance during surgery. Recently, the clinical feasibility of CLI with the established therapeutic reagent Na131I and the PET tracer 18F-FDG was demonstrated. 68Ga possesses a higher Cerenkov light output than 18F and 131I, which would result in higher sensitivity for CLI and improve the outcome of CLI in clinical applications. However, the research on 68Ga-based tumor-specific tracers for CLI is limited. In this study, we examined the use of 68Ga-radiolabeled DOTA-3PRGD2 (68Ga-3PRGD2) for dual PET and CLI of orthotopic U87MG human glioblastoma. For this purpose, the Cerenkov efficiencies of 68Ga and 18F were measured with the IVIS Spectrum system (PerkinElmer, USA). The CLI signal intensity of 68Ga was 15 times stronger than that of 18F. PET and CLI of 68Ga-3PRGD2 were performed in U87MG human glioblastoma xenografts. Both PET and CLI revealed a remarkable accumulation of 68Ga-3PRGD2 in the U87MG human glioblastoma xenografts at 1 h p.i. with an extremely low background in the brain when compared with 18F-FDG. Furthermore, 68Ga-3PRGD2 was used for dual PET and CLI of orthotopic human glioblastoma. The orthotopic human glioblastoma was clearly visualized by both imaging modalities. In addition, the biodistribution of 68Ga-3PRGD2 was assessed in normal mice to estimate the radiation dosimetry. The whole-body effective dose is 20.1 ± 3.3 μSv/MBq, which is equal to 3.7 mSv per whole-body PET scan with a 5 mCi injection dose. Thus, 68Ga-3PRGD2 involves less radiation exposure in patients when compared with 18F-FDG (7.0 mSv). The use of 68Ga-3PRGD2 in dual PET and CLI shows great promise for tumor diagnosis and image-guided surgery.

Cetuximab is an antiepidermal growth factor receptor (EGFR) monoclonal antibody that has received the approval of the Food and Drug Administration (FDA) for cancer treatment. However, most clinical studies indicate that cetuximab can only elicit positive effects on a subset of cancer patients. In this study, we investigated whether near-infrared fluorescence (NIRF) imaging of tumor vascular endothelial growth factor (VEGF) expression could be a biomarker for tumor early response to cetuximab therapy in preclinical wild-type and mutant tumor models of the KRAS gene. The treatment efficacy of cetuximab was determined in both HT-29 (wild-type KRAS) and HTC-116 (mutant KRAS) human colon cancer models. A VEGF-specific optical imaging probe (Dye755-Ran) was synthesized by conjugating ranibizumab (an anti-VEGF antibody Fab fragment) with a NIRF dye. Serial optical scans with Dye755-Ran were performed in HT-29 and HTC-116 xenograft models. By using longitudinal NIRF imaging, we were able to detect early tumor response on day 3 and day 5 after initiation of cetuximab treatment in the cetuximab-responsive HT-29 tumor model. Enzyme-linked immunosorbent assay (ELISA) confirmed that cetuximab treatment inhibited human VEGF expression in the KRAS wild-type HT-29 tumor but not in the KRAS mutant HCT-116 tumor. We have demonstrated that the antitumor effect of cetuximab can be noninvasively monitored by serial fluorescence imaging using Dye755-Ran. VEGF expression detected by optical imaging could serve as a sensitive biomarker for tumor early response to drugs that directly or indirectly act on VEGF.

The reversible combination of a ligand with specific sites on the surface of a receptor is one of the most important processes in biochemistry. A classic equation with a useful simple graphical method was introduced to obtain the equilibrium constant, Kd, and the maximum density of receptors, Bmax. The entire 125I-labeled ligand binding experiment includes three parts: the radiolabeling, cell saturation binding assays and the data analysis. The assay format described here is quick, simple, inexpensive, and effective, and provides a gold standard for the quantification of ligand-receptor interactions. Although the binding assays and quantitative analysis have not changed dramatically compared to the original methods, we integrate all the parts to calculate the parameters in one concise protocol and adjust many details according to our experience. In every step, several optional methods are provided to accommodate different experimental conditions. All these refinements make the whole protocol more understandable and user-friendly. In general, the experiment takes one person less than 8 h to complete, and the data analysis could be accomplished within 2 h.

Antiangiogenic therapy is an effective strategy to inhibit tumor growth. Endostar, as an approved antiangiogenesis agent, inhibits the newborn vascular endothelial cells, causing the decrease of integrin αvβ3 expression. Radiolabeled 3PRGD2, a novel PEGlayted RGD dimer probe (PEG4-E[PEG4-c(RGDfK)]2) showed highly specific targeting capability to integrin αvβ3, which could be used for monitoring the efficacy of Endostar antiangiogenic therapy. In this study, 68Ga-3PRGD2 PET imaging was performed in Endostar treated/untreated Lewis Lung Carcinoma (LLC) mice on days 3, 7, 14, and 21 post-treatment for monitoring the tumor response to Endostar treatment, with the 18F-FDG imaging as control. As a result, 68Ga-3PRGD2 PET reflected the tumor response to Endostar antiangiogenic therapy much earlier (day 3 post-treatment vs day 14 post-treatment) and more accurately than that of 18F-FDG metabolic imaging, which provides new opportunities to develop individualized therapeutic approaches, establish optimized dosages and dose intervals for effective treatment that improve the survival rate of patients.Keywords: antiangiogenic therapy; endostar; in vivo monitoring; molecular imaging; radiotracer;

Epidermal growth factor receptor (EGFR) has been well characterized as an important target for cancer therapy. Immunotherapy based on EGFR-specific antibodies (e.g., panitumumab and cetuximab) has shown great clinical promise. However, increasing evidence has indicated that only a subgroup of patients receiving these antibodies will benefit from them, and even patients who do initially experience a major response may eventually develop therapeutic resistance. In this study, we investigated whether panitumumab and cetuximab can serve as delivery vehicles for tumor-targeted radionuclide therapy in a preclinical tumor model that did not respond to immunotherapy. The in vitro toxicity and cell binding properties of panitumumab and cetuximab were characterized. Both antibodies were conjugated with 1,4,7,10-tetraazadodecane-N,N′,N″,N‴-tetraacetic acid (DOTA) and radiolabeled with 177Lu. Small-animal SPECT/CT, biodistribution, and radioimmunotherapy (RIT) studies of 177Lu-DOTA–panitumumab (177Lu-Pan) and 177Lu-DOTA–cetuximab (177Lu-Cet) were performed in the UM-SCC-22B tumor model. Both 177Lu-Pan and 177Lu-Cet exhibited favorable tumor targeting efficacy. The tumor uptake was 20.92 ± 4.45, 26.10 ± 5.18, and 13.27 ± 1.94% ID/g for 177Lu-Pan, and 15.67 ± 3.84, 15.72 ± 3.49, and 7.82 ± 2.36% ID/g for 177Lu-Cet at 24, 72, and 120 h p.i., respectively. RIT with a single dose of 14.8 MBq of 177Lu-Pan or 177Lu-Cet significantly delayed tumor growth. 177Lu-Pan induced more effective tumor growth inhibition due to a higher tumor uptake. Our results suggest that panitumumab and cetuximab can function as effective carriers for tumor-targeted delivery of radiation, and that RIT is promising for targeted therapy of EGFR-positive tumors, especially for those tumors that are resistant to antibody-based immunotherapy.Keywords: antibody; epidermal growth factor receptor; small-animal imaging; targeted therapy;

Optical imaging is emerging as a powerful tool for the noninvasive imaging of the biological processes in living subjects. This study aimed to investigate whether optical imaging of integrin αvβ3 and vascular endothelial growth factor (VEGF) expression can serve as sensitive biomarkers for tumor early response to antiangiogenic therapy.We synthesized two near-infrared fluorescence (NIRF) imaging agents, CF680R-3PRGD2 and CF750-BevF(ab')2, which were designed to specifically bind to integrin αvβ3 and VEGF, respectively. The ability of optical imaging using the two imaging agents for early monitoring the antiangiogenic effect of sunitinib was evaluated.CF680R-3PRGD2 and CF750-BevF(ab')2 specifically bound to their respective targets in vitro and in HT-29 tumor-bearing nude mice. Sunitinib treatment led to significantly decreased tumor uptake of CF680R-3PRGD2 (e.g., 7.47 ± 1.62 % vs. 4.24 ± 0.16 % on day 4; P < 0.05) and CF750-BevF(ab')2 (e.g., 7.43 ± 2.43 % vs. 4.04 ± 1.39 % on day 2; P < 0.05) in vivo. Immunofluorescence staining and an enzyme-linked immunosorbent assay confirmed that sunitinib-induced changes in tumor uptake of CF680R-3PRGD2 and CF750-BevF(ab')2 were correlated with changes in the levels of integrin αvβ3 and VEGF. Radiobiodistribution of 99mTc-3PRGD2 and 125I-BevF(ab')2, the radiocounterparts of CF680R-3PRGD2 and CF750-BevF(ab')2, respectively, also validated optical imaging results.Longitudinal monitoring of tumor integrin αvβ3 and VEGF expression could be used as early biomarkers for tumor response to antiangiogenic therapy. This strategy may facilitate the development of new antiangiogenic drugs, and be used for elucidation of the underlying mechanisms of therapies involving the integrin and the VEGF signaling pathway.

There is growing interest in the development of radiolabeled multivalent ligands because of their higher tumor uptake versus that of the corresponding monomer. This report presents the synthesis of a [Tyr3]octreotide dimer conjugate, HYNIC-E([Tyr3]octreotide)2 {HYNIC-TOC2, HYNIC = 6-[2-(2-sulfonatobenzaldehyde)hydrazono]nicotinyl}, and its biological evaluation in the AR42J tumor model. The binding affinity of HYNIC-TOC2 for somatostatin receptor subtype 2 (SSTR2) was determined in AR42J rat pancreatic cancer cells, using 125I-[Tyr3]octreotide as the radiotracer. 99mTc-HYNIC-TOC2 was prepared by using tricine and EDDA as coligands (EDDA = ethylenediamine-N,N′-diacetic acid). Biodistribution and γ imaging were performed in nude mice bearing AR42J tumors. 99mTc-HYNIC-TOC2 was obtained in >95% labeling yield with favorable stability. Compared with those of HYNIC-TOC (IC50 = 3.74 ± 0.82 nM), HYNIC-TOC2 showed significantly increased SSTR2 binding affinity (IC50 = 0.74 ± 0.19 nM), and 99mTc-HYNIC-TOC2 showed significantly increased tumor uptake [13.31 ± 3.14%ID/g vs 5.32 ± 0.94%ID/g 1 h postinjection (p.i.) and 12.05 ± 2.92%ID/g vs 5.87 ± 1.96%ID/g 4 h p.i.]. Although the level of accumulation of 99mTc-HYNIC-TOC2 in kidneys was significantly increased (94.40 ± 6.51%ID/g vs 32.27 ± 4.51%ID/g 1 h p.i.), this high uptake was inhibited by the injection of l-lysine before the administration of 99mTc-HYNIC-TOC2 (30.99 ± 5.05%ID/g 1 h p.i.) while tumor uptake decreased only slightly. Consistent with biodistribution data, in vivo planar γ imaging showed that the tumors were clearly visualized, while the background signal was much weaker except for that of the kidneys and bladder. The new radiotracer 99mTc-HYNIC-TOC2 with a higher binding affinity and good stability was designed and evaluated. The higher tumor uptake of 99mTc-HYNIC-TOC2 suggests that 90Y/177Lu-labeled TOC2 might have an advantage for the radiotherapy of SSTR2-positive tumors. These data merit the translation of 99mTc-HYNIC-TOC2 to a clinical setting.Keywords: 99mTc; dimer; SPECT; SSTR2-positive tumor; [Tyr3]octreotide;

Viral nanoparticles (VNPs) have shown great potential as platforms for biomedical applications. Before using VNPs for further biomedical applications, it is important to clarify their biological behavior in vivo, which is rare for rod-like VNPs. In this paper, a study of tobacco mosaic virus (TMV), a typical rod-like VNP, is performed on blood clearance kinetics, biodistributions in both normal and tumor-bearing mice, histopathology and cytotoxicity. TMV was radiolabeled with 125I using Iodogen method for in vivo quantitative analysis and imaging purpose. In the normal mice, the accumulation of TMV in the immune system led to a rapid blood clearance. The uptake of TMVs in the liver was less than that in the spleen, which is opposite to the results observed in the case of spherical VNPs. No signs of overt toxicity were observed in examined tissues according to the results of histological analysis. In addition, similar biodistribution patterns were observed in U87MG tumor-bearing mice.

Since their discovery in 2006, induced pluripotent stem cells (iPSCs) have gained increasing interest for tissue regeneration and transplantation therapies. However, teratoma formation after iPSC transplantation is one of the most serious drawbacks that may limit their further clinical application. We investigated here whether human iPSC-derived teratomas could be detected by an integrin-targeting agent 99mTc-PEG4-E[PEG4-c(RGDfK)]2 (99mTc-3PRGD2).Human-induced pluripotent stem cells (hiPSCs) were generated and characterized. In vitro integrin αvβ3 expression levels of hiPSC- and hiPSC-derived teratoma cells were determined by flow cytometry. 99mTc-3PRGD2 was prepared, and planar gamma imaging and biodistribution studies were carried out in teratoma-bearing severe combined immunodeficient (SCID) mice. Positron emission tomography (PET) imaging of teratomas with 2-deoxy-2-[18F]fluoro-d-glucose (18F-FDG) was also performed for comparison. Integrin αvβ3 expression in teratoma tissues was determined by immunofluorescence staining.99mTc-3PRGD2 showed high (2.82 ± 0.21 and 2.69 ± 0.73%ID/g at 0.5 and 1 h pi, respectively) and specific (teratoma uptake decreased from 2.69 ± 0.73 to 0.53 ± 0.26%ID/g after blocking with cold 3PRGD2) uptake in teratoma tissues, and planar gamma imaging demonstrated the feasibility of noninvasively detecting the teratoma formation with 99mTc-3PRGD2. 18F-FDG showed low teratoma uptake and thus failed to detect the teratomas. Ex vivo immunofluorescence staining validated the integrin αvβ3 expression in the vasculature during teratoma formation.Gamma imaging with 99mTc-3PRGD2 is a promising approach for the noninvasive monitoring of tumorigenicity after hiPSCs transplantation.

We recently designed and synthesized a Glu-c(RGDyK)-bombesin (RGD-BBN) heterodimeric peptide exhibiting a dual integrin αvβ3 and gastrin-releasing peptide receptor (GRPR) targeting property. In this study, we investigated whether 99mTc-labeled RGD-BBN peptide could be used for the noninvasive detection of lung carcinoma by using small-animal single-photon emission computed tomography (SPECT)/CT. RGD-BBN peptide was conjugated with 6-hydrazinonicotinyl (HYNIC) and then radiolabeled with 99mTc using tricine and TPPTS as the coligands (TPPTS = trisodium triphenylphosphine-3,3′,3″-trisulfonate). The biodistribution, planar gamma imaging, and small-animal SPECT/CT studies of 99mTc-HYNIC(tricine)(TPPTS)-RGD-BBN (99mTc-RGD-BBN) were performed in C57/BL6 mice bearing Lewis lung carcinoma (LLC) or bearing both inflammation and LLC. HYNIC-RGD-BBN possessed a dual integrin αvβ3 and GRPR binding capacity. 99mTc-RGD-BBN was prepared with a high radiochemical purity (>98%), and it exhibited specific tumor imaging with high contrast to the contralateral background. 99mTc-RGD-BBN was superior to 18F-FDG for distinguishing lung carcinoma from inflammation. The uptake of 99mTc-RGD-BBN in LLC xenografts was 2.69 ± 0.66% ID/g at 1 h postinjection (p.i.) and was decreased to 1.99 ± 0.61% ID/g at 2 h p.i. The inflammation uptake of 99mTc-RGD-BBN was 1.20 ± 0.32% ID/g at 1 h and 0.56 ± 0.17% ID/g at 2 h p.i., respectively. High pancreas uptake (25.76 ± 5.49%ID/g and 19.56 ± 6.78% ID/g at 1 and 2 h p.i., respectively) was also found due to the high GRPR expression of this organ. Small-animal SPECT/CT using 99mTc-RGD-BBN can specifically detect the LLC pulmonary metastases. Our results suggested that SPECT/CT with 99mTc-RGD-BBN would provide an effective approach for the noninvasive detection of lung cancer.Keywords: GRPR; integrin αvβ3; lung carcinoma; RGD-bombesin; single-photon emission computed tomography (SPECT);

Targeting radiopeptides are promising agents for radio-theranostics. However, in vivo evaluation of their targeting specificity is often obscured by their short biologic half-lives and low binding affinities. Here, we report an approach to efficiently examine targeting radiopeptides with a new class of octavalent peptide fluorescent nanoprobe (Octa-FNP) platform, which is composed of candidate targeting peptides and a tetrameric far-red fluorescent protein (tfRFP) scaffold. To shed light on this process, 125I-Octa-FNP, 125I-tfRFP and 125I-peptide were synthesized, and their targeting functionalities were compared. Both fluorescence imaging and radioactive quantification results confirmed that 125I-Octa-FNP had a significantly higher cellular binding capability than 125I-tfRFP. In vivo biodistribution studies show that at 6 h post-injection, 125I-Octa-FNP had 2-fold and 30-fold higher tumor uptake than that of 125I-tfRFP and 125I-peptide, respectively. Moreover, γ-imaging at 24 h post-injection revealed a remarkable accumulation of 125I-Octa-FNP in the tumor while maintaining an extremely low background contrast, which was further confirmed by immunofluorescence analysis. These data suggested that, as an engineered and multivalent platform, Octa-FNP could enhance the tumor targeting of a designed peptide and provide excellent contrast radioimaging, making it a valuable tool for the evaluation of the targeting ability of specifically designed radiopeptides for cancer theranostics.

We have recently developed a series of new Arg-Gly-Asp (RGD) dimeric peptides for specific targeting of integrin αvβ3 with enhanced tumor uptake and improved pharmacokinetics. In this study, we investigated 90Y-labeled RGD tetramer (RGD4) and the new type of RGD dimer (3PRGD2), for the radionuclide therapy of integrin αvβ3-positive tumors. Biodistribution and gamma imaging studies of 111In labeled RGD4 and 3PRGD2 were performed. Groups of nude mice were used to determine maximum tolerated dose (MTD) of 90Y-DOTA−RGD4 and 90Y-DOTA−3PRGD2. The radionuclide therapeutic efficacy of 90Y-DOTA−RGD4 and 90Y-DOTA−3PRGD2 was evaluated in U87MG tumor-bearing nude mice. The U87MG tumor uptake of 111In-DOTA−3PRGD2 was slightly lower than that of the 111In-DOTA−RGD4 (e.g., 6.13 ± 0.82%ID/g vs 6.43 ± 1.6%ID/g at 4 h postinjection), but the uptake of 111In-DOTA−3PRGD2 in normal organs, such as liver and kidneys, was much lower than that of 111In-DOTA−RGD4, which resulted in much higher tumor-to-nontumor ratios and lower toxicity. The MTD of 90Y-DOTA−RGD4 in nude mice is less than 44.4 MBq, while the MTD of 90Y-DOTA−3PRGD2 in mice is more than 55.5 MBq. 90Y-DOTA−3PRGD2 administration exhibited a similar tumor inhibition effect as compared with 90Y-DOTA−RGD4 at the same dose. The tumor vasculature in the 90Y-DOTA−3PRGD2 treatment group was much less than the control groups. Radionuclide therapy studies exhibited that both 90Y-DOTA−RGD4 and 90Y-DOTA−3PRGD2 caused significant tumor growth delay in the U87MG tumor model. Compared to 90Y-DOTA−RGD4, the low accumulation of 90Y-DOTA−3PRGD2 in normal organs led to lower toxicity and higher MTD in nude mice, which would make it more suitable for high dose or multiple-dose regimens, in order to achieve maximum therapeutic efficacy.Keywords (keywords): 90Y; Arg-Gly-Asp (RGD); integrin αvβ3; radionuclide therapy; tumor;

99mTc-3PRGD2 is a 99mTc-labeled dimeric cyclic RGD peptide with increased receptor binding affinity and improved kinetics for in vivo imaging of integrin αvβ3 expression in nude mouse model. To accelerate its clinical translation, we reported here the evaluation of the kit-formulated 99mTc-3PRGD2 in healthy cynomolgus primates for its blood clearance kinetics, biodistribution, and radiation dosimetry.Healthy cynomolgus primates (4.1 ± 0.7 kg, n = 5) were anesthetized, and the venous blood samples were collected via a femoral vein catheter at various time points after injection of ~555 MBq of 99mTc-3PRGD2. Serial whole-body scans were performed with a dual-head single photon emission computed tomography system after administering ~555 MBq of 99mTc-3PRGD2 in the non-human primates, and the radiation dosimetry estimate was calculated.99mTc-3PRGD2 could be easily obtained from freeze-dried kits with high radiochemical purity (>95%) and high specific activity (~5 Ci/μmol). 99mTc-3PRGD2 had a rapid blood clearance with less than 1% of the initial radioactivity remaining in the blood circulation at 60 min postinjection. No adverse reactions were observed up to 4 weeks after the repeated dosing. The whole-body images exhibited high kidney uptake of 99mTc-3PRGD2 and high radioactivity accumulation in the bladder, demonstrating the rapid renal clearance of this tracer. The highest radiation doses of 99mTc-3PRGD2 were found in the kidneys (13.2 ± 1.08 μGy/MBq) and the bladder wall (33.1 ± 1.91 μGy/MBq).99mTc-3PRGD2 can be readily available using the kit formulation. This tracer is safe and well tolerated, and no adverse events occurred in non-human primates. Further clinical testing and translation of 99mTc-3PRGD2 for noninvasive imaging of integrin αvβ3 in humans are warranted.

This study describes a quantitative method to estimate the migratory capacity of neural stem cells (NSCs) using magnetic resonance imaging.NSCs were labeled with ferumoxides and injected stereotaxically into the corpus callosum of the normal rat brain. Control subjects received either free ferumoxides or nonviable labeled cells. Subjects were scanned after initial injection and at 1 week. Image sets were coregistered, compared morphologically, and analyzed parametrically to determine migration speed.Subjects receiving injections of viable cells had a significantly greater spread of the tracer after 1 week than either control group (p < 0.05). The speed of migration for viable NSCs was significantly higher than that of controls along the corpus callosum (p < 0.05). Migratory speeds estimated from histology and imaging were significantly correlated.This study provides a quantitative assessment of posttransplantation neural stem cell migration that is clearly distinguishable from tracer clearance.

The cell adhesion molecule integrin αvβ3 is an important player in the process of tumor angiogenesis and metastasis. Abegrin™, a fully humanized anti-integrin αvβ3 monoclonal antibody, was currently in clinical trials for cancer therapy. Herein, we labeled Abegrin™ with 111In, evaluated the in vitro and in vivo characteristics, and investigated whether the expression of integrin αvβ3 in tumors could be imaged with 111In-labeled Abegrin™.The binding affinity and specificity of Abegrin™ was analyzed using U87MG glioblastoma cells. Abegrin™ was coupled with 1,4,7,10-tetraazadodecane-N,N′,N″,N′″-tetraacetic acid (DOTA) for 111In radiolabeling. γ Imaging of 111In-DOTA–Abegrin™ was carried out in nude mice bearing both integrin αvβ3-positive U87MG and integrin αvβ3-negative HT-29 tumors. Biodistribution and blocking studies of 111In-DOTA–Abegrin™ were investigated in U87MG tumor-bearing nude mice.Abegrin™ exhibited high-binding affinity to human integrin αvβ3 expressed on U87MG cells (Kd of 0.35 ± 0.06 nM). The antibody retained antigen-binding affinity/specificity after DOTA conjugation. γ Imaging showed that the tumor uptake of 111In-DOTA–Abegrin™ in integrin αvβ3-positive U87MG tumors was much higher than that in integrin αvβ3-negative HT-29 tumors. In the HT-29 tumors, Abegrin™ was mainly nonspecifically accumulated around the blood vessels, while in the U87MG tumors, besides the nonspecific tumor retention, Abegrin™ also specifically bound the human integrin αvβ3 expressed on the tumor cells. Biodistribution and blocking studies exhibited that the U87MG tumor uptake of 111In-DOTA–Abegrin™ decreased from 14.12 ± 0.44 to 6.93 ± 0.94 percentage of injected dose per gram of tissue after coinjection of excess dose of cold Abegrin™, which confirmed the in vivo integrin αvβ3 binding specificity of 111In-DOTA–Abegrin™.Abegrin™ showed specific binding to human integrin αvβ3 expressed on the tumor cells. 111In-DOTA–Abegrin™ can specifically target the human integrin αvβ3 expression in the nude mouse model. 111In-DOTA–Abegrin™ has a potential for clinical translation as an agent for integrin αvβ3-positive tumor imaging, evaluating tumor angiogenic status and monitoring the therapeutic efficacy of Abegrin™-based cancer therapy.

Integrin αvβ3 has been well-documented as one of the key players in the process of tumor angiogenesis. Radiolabeled RGD (Arg-Gly-Asp) peptides that specifically target integrin αvβ3 have great potential for tumor early detection and noninvasively monitoring the status of tumor angiogenesis. We developed a cyclic RGD dimeric probe 99mTc-HYNIC-Gly3-E[PEG4-c(RGDfK)]2 (99mTc-G3-2P4-RGD2) (using tricine and TPPTS as the coligands, TPPTS = trisodium triphenylphosphine-3,3′,3′′-trisulfonate), and investigated whether it could be used to noninvasively visualize and quantify integrin αvβ3 expression in vivo. HYNIC-Gly3-E[PEG4-c(RGDfK)]2 was synthesized and labeled with 99mTc. The biodistribution and planar γ-imaging studies of 99mTc-G3-2P4-RGD2 were performed in both U87MG (human integrin αvβ3 positive/murine integrin αvβ3 positive) and HT-29 (human integrin αvβ3 negligible /murine integrin αvβ3 positive) tumor-bearing nude mouse models. The correlation of 99mTc-G3-2P4-RGD2 tumor uptake values (measured by ex vivo biodistribution) with expression levels of human integrin αvβ3 or murine integrin αvβ3 (measured by Western blot) were determined in U87MG and HT-29 tumor models, respectively. 99mTc-G3-2P4-RGD2 exhibited increased receptor binding affinity and in vivo tumor uptake as compared with previously reported RGD dimeric tracer 99mTc-RGD2 (without Gly3 and PEG4 spacers). The tumor uptake of 99mTc-G3-2P4-RGD2 was related to the expression levels of both human integrin αvβ3 (expressed on tumor cells) and murine integrin αvβ3 (expressed on newborn tumor vasculature). Our results demonstrate that 99mTc-G3-2P4-RGD2 is a useful agent for integrin αvβ3 imaging. The relationship between 99mTc-G3-2P4-RGD2 uptake and integrin αvβ3 expression level as determined by this study would provide useful information for clinical translation of RGD probes.

Radioimmunotherapy (RIT) is a promising approach for the treatment of a wide variety of malignancies. The aim of this study was to investigate the therapeutic efficacy of 131I-labeled anticarcinoembryonic antigen (CEA) monoclonal antibody CL58 in a human colon cancer mouse model. In vitro and in vivo characteristics of 125I-CL58 were evaluated in LS180 human colon cancer cells and the nude mouse model. 131I-CL58 was prepared and its in vivo therapeutic efficacy was tested. 125I-CL58 showed high affinity to LS180 cells, as well as high tumor uptake and long tumor retention in LS180 tumor xenografts. 131I-CL58 exhibited dose-dependent inhibition of LS180 tumor growth. With the excellent in vitro and in vivo characteristics, and the effective therapy for colon cancer in animal model, 131I-CL58 is a promising agent for RIT of CEA-positive tumors including colon cancer.

In this study, we reported the preparation and evaluation of 177Lu-DOTA-RGD2, 177Lu-DOTA-Bz-RGD2 and 177Lu-DTPA-Bz-RGD2 (RGD2 = E[c(RGDfK)]2) as a potential therapeutic radiotracers for the treatment of integrin αvβ3-positive tumors. The BALB/c nude mice bearing the U87MG human glioma xenografts were used to evaluate the biodistribution characteristics and excretion kinetics of 177Lu-DOTA-RGD2, 177Lu-DOTA-Bz-RGD2 and 177Lu-DTPA-Bz-RGD2. It was found that there were no major differences in their lipophilicity and biodistribution characteristics, particularly at latter time points. A major advantage of using DTPA-Bz as the bifunctional chelator (BFC) was its high radiolabeling efficiency (fast and high yield radiolabeling) at room temperature. Using DOTA and DOTA-Bz as BFCs, the radiolabeling kinetics was slow, and heating at 100°C and higher DOTA-conjugate concentration were needed for successful 177Lu-labeling. Therefore, DTPA-Bz is an optimal BFC for routine preparation of 177Lu-labeled cyclic RGDfK peptides, and 177Lu-DTPA-Bz-RGD2 is worthy of further investigation for targeted radiotherapy of integrin αvβ3-positive tumors.

In this report, we present in vitro and in vivo evaluation of three 111In-labeled DTPA conjugates of a cyclic RGDfK dimer: DTPA-Bn-SU016 (SU016 = E[c(RGDfK)]2; DTPA-Bn = 2-(p-isothioureidobenzyl)diethylenetriaminepentaacetic acid), DTPA-Bn-E-SU016 (E = glutamic acid) and DTPA-Bn-Cys-SU016 (Cys = cysteic acid). The integrin αvβ3 binding affinities of SU016, DTPA-Bn-SU016, DTPA-Bn-E-SU016, and DTPA-Bn-Cys-SU016 were determined to be 5.0 ± 0.7 nM, 7.9 ± 0.6 nM, 5.8 ± 0.6 nM, and 6.9 ± 0.9 nM, respectively, against 125I-c(RGDyK) in binding to integrin αvβ3, suggesting that E or Cys residue has little effect on the integrin αvβ3 affinity of E[c(RGDfK)]2. It was also found that the 111In-labeling efficiency of DTPA-Bn-SU016 and DTPA-Bn-E-SU016 is 3–5 times better than that of DOTA analogues due to fast chelation kinetics and high-yield 111In-labeling under mild conditions (e.g., room temperature). Biodistribution studies were performed using BALB/c nude mice bearing U87MG human glioma xenografts. 111In-DTPA-Bn-SU016, 111In-DTPA-Bn-E-SU016, and 111In-DTPA-Bn-Cys-SU016 all displayed rapid blood clearance. Their tumor uptake was comparable between 0.5 and 4 h postinjection (p.i.) within experimental error. 111In-DTPA-Bn-E-SU016 had a significantly lower (p < 0.01) kidney uptake than 111In-DTPA-Bn-SU016 and 111In-DTPA-Bn-Cys-SU016. The liver uptake of 111In-DTPA-Bn-SU016 was 1.69 ± 0.18% ID/g at 24 h p.i., while the liver uptake values of 111In-DTPA-Bn-E-SU016 and 111In-DTPA-Bn-Cys-SU016 were 0.55 ± 0.11% ID/g and 0.79 ± 0.15% ID/g at 24 h p.i., respectively. Among the three 111In radiotracers evaluated in this study, 111In-DTPA-Bn-E-SU016 has the lowest liver and kidney uptake and the best tumor/liver and tumor/kidney ratios. Results from metabolism studies indicated that there is little metabolism (<10%) for three 111In radiotracers at 1 h p.i. Imaging data showed that tumors can be clearly visualized at 4 h p.i. with good contrast in the tumor-bearing mice administered with 111In-DTPA-Bn-E-SU016. It is concluded that using a glutamic acid linker can significantly improve excretion kinetics of the 111In-labeled E[c(RGDfK)]2 from liver and kidneys.

In this report, we present the synthesis and evaluation of the 99mTc-labeled β-Ala-BN(7−14)NH2 (ABN = β-Ala-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH2) as a new radiotracer for tumor imaging in the BALB/c nude mice bearing HT-29 human colon cancer xenografts. The gastrin releasing peptide receptor binding affinity of ABN and HYNIC-ABN (6-hydrazinonicotinamide) was assessed via a competitive displacement of 125I-[Tyr4]BBN bound to the PC-3 human prostate carcinoma cells. The IC50 values were calculated to be 24 ± 2 nM and 38 ± 1 nM for ABN and HYNIC-ABN, respectively. HYNIC is the bifunctional coupling agent for 99mTc-labeling, while tricine and TPPTS (trisodium triphenylphosphine-3,3′,3′′-trisulfonate) are used as coligands to prepare the ternary ligand complex [99mTc(HYNIC-ABN)(tricine)(TPPTS)] in very high yield and high specific activity. Because of its high hydrophilicity (log P = −2.39 ± 0.06), [99mTc(HYNIC-ABN)(tricine)(TPPS)] was excreted mainly through the renal route with little radioactivity accumulation in the liver, lungs, stomach, and gastrointestinal tract. The tumor uptake at 30 min postinjection (p.i.) was 1.59 ± 0.23%ID/g with a steady tumor washout over the 4 h study period. As a result, it had the best T/B ratios in the blood (2.37 ± 0.68), liver (1.69 ± 0.41), and muscle (11.17 ± 3.32) at 1 h p.i. Most of the injected radioactivity was found in the urine sample at 1 h p.i., and there was no intact [99mTc(HYNIC-ABN)(tricine)(TPPTS)] detectable in the urine, kidney, and liver samples. Its metabolic instability may contribute to its rapid clearance from the liver, lungs, and stomach. Despite the steady radioactivity washout, the tumors could be clearly visualized in planar images of the BALB/c nude mice bearing the HT-29 human colon xenografts at 1 and 4 h p.i. The favorable excretion kinetics from the liver, lungs, stomach, and gastrointestinal tract makes [99mTc(HYNIC-ABN)(tricine)(TPPTS)] a promising SPECT radiotracer for imaging colon cancer.

A series of new O6-BG derivatives (14–21,23–30) were synthesized as inactivators of O6-Alkylguanine-DNA alkyltransferase (AGT), and their ability to inhibit AGT was preliminary evaluated by MTT with O6-BG as the control. The result suggested compound 30 displayed a higher activity than O6-BG.