Syntheses and preliminary evaluation of [18F]AlF‐NOTA‐G‐TMTP1 for PET imaging of high aggressive hepatocellular carcinoma

The goal of this study is to evaluate a new ¹⁸F‐labeled imaging agent for diagnosing high metastatic (aggressive) hepatocellular carcinoma using positron emission tomography (PET). The new ¹⁸F‐labeled imaging agent [¹⁸F]AlF‐NOTA‐G‐TMTP1 was synthesized and radiolabeled with ¹⁸F using NOTA‐AlF chelation method. The tumor‐targeting characteristics of [¹⁸F]AlF‐NOTA‐G‐TMTP1 was assessed in HepG2, SMCC‐7721, HCC97L and HCCLM3 xenografts. The total synthesis time was about 20 min with radiochemical yield of 25 ± 6%. The specific activity was about 11.1–14.8 GBq/µmol at the end of synthesis based on the amount of peptide used and the amount of radioactivity trapped on the C₁₈ column. The log P value of [¹⁸F]AlF‐NOTA‐G‐TMTP1 was ‐3.166 ± 0.022. [¹⁸F]AlF‐NOTA‐G‐TMTP1 accumulated in SMCC‐7721 and HCCLM3 tumors (high metastatic potential) in vivo and result in tumor/muscle (T/M) ratios of 4.5 ± 0.3 and 4.7 ± 0.2 (n = 4) as measured by PET at 40 min post‐injection (p.i.). Meanwhile, the tumor/muscle (T/M) ratios of HepG2 and HCC97L tumors (low metastatic potential) were1.6 ± 0.3 and 1.8 ± 0.4. The tumor uptake of [¹⁸F]AlF‐NOTA‐G‐TMTP1 could be inhibited 61.9% and 57.6% by unlabeled G‐TMTP1 in SMCC‐7721 and HCCLM3 xenografts at 40 min p.i., respectively. Furthermore, [¹⁸F]AlF‐NOTA‐G‐TMTP1 showed pretty low activity in the liver and intestines in all tumor bearing mice, such in vivo distribution pattern would be advantageous for the detection of hepatic carcinoma. Overall, [¹⁸F]AlF‐NOTA‐G‐TMTP1 may specifically target high metastatic or/and aggressive hepatocellular carcinoma with low background activity and, therefore, holds the potential to be used as an imaging agent for detecting tumor lesions within the liver area. Copyright © 2016 John Wiley & Sons, Ltd.     

PET imaging of prostate tumors with 18F‐Al‐NOTA‐MATBBN

Overexpression of the gastrin‐releasing peptide receptor (GRPR) in prostate cancer provides a promising target for detection the disease. MATBBN is a new bombesin analog originating from the GRPR antagonists with a hydrophilic linker. In this study NOTA‐conjugated MATBBN was labeled by the Al¹⁸F method and the potential of ¹⁸F‐Al‐NOTA‐MATBBN for prostate tumor PET imaging was also evaluated. NOTA‐MATBBN was radiolabeled with ¹⁸F using Al¹⁸F complexes. Partition coefficient, in vitro stability and GRPR binding affinity were also determined. PET studies were performed with ¹⁸F‐Al‐NOTA‐MATBBN in PC‐3 tumor‐bearing mice. ¹⁸F‐Al‐NOTA‐MATBBN can be produced within 30 min with a decay‐corrected yield of 62.5 ± 2.1% and a radiochemical purity of >98%. The logP octanol–water value for the Al¹⁸F‐labeled BBN analog was ?2.40 ± 0.07 and the radiotracer was stable in phosphate‐buffered saline and human serum for 2 h. The IC₅₀ values of displacement for the ¹⁸F‐Al‐NOTA‐MATBBN with MATBBN was 126.9 ± 2.75 nm . The PC‐3 tumors were clearly visible with high contrast after injection of the labeled peptide. At 60 min post‐injection, the tumor uptakes for ¹⁸F‐Al‐NOTA‐MATBBN and ¹⁸F‐FDG were 4.59 ± 0.43 and 1.98 ± 0.35% injected dose/g, and tumor to muscle uptake radios for two tracers were 6.77 ± 1.10 and 1.78 ± 0.32, respectively. Dynamic PET revealed that ¹⁸F‐Al‐NOTA‐MATBBN was excreted mainly through the kidneys. GRPR‐binding specificity was also demonstrated by reduced tumor uptake of ¹⁸F‐Al‐NOTA‐MATBBN after coinjection with excess unlabeled MATBBN peptide at 1 h post‐injection. NOTA‐ MATBBN could be labeled rapidly with ¹⁸F using one step method. ¹⁸F‐Al‐NOTA‐MATBBN may be a promising PET imaging agent for prostate cancer. Copyright © 2014 John Wiley & Sons, Ltd.     

Dual‐isotope 111In/177Lu SPECT imaging as a tool in molecular imaging tracer design

The synthesis, design and subsequent pre‐clinical testing of new molecular imaging tracers are topic of extensive research in healthcare. Quantitative dual‐isotope SPECT imaging is proposed here as a tool in the design and validation of such tracers, as it can be used to quantify and compare the biodistribution of a specific ligand and its nonspecific control ligand, labeled with two different radionuclides, in the same animal. Since the biodistribution results are not blurred by experimental or physiological inter‐animal variations, this approach allows determination of the ligand's net targeting effect. However, dual‐isotope quantification is complicated by crosstalk between the two radionuclides used and the radionuclides should not influence the biodistribution of the tracer. Here, we developed a quantitative dual‐isotope SPECT protocol using combined ¹¹¹Indium and ¹⁷⁷Lutetium and tested this tool for a well‐known angiogenesis‐specific ligand (cRGD peptide) in comparison to a potential nonspecific control (cRAD peptide). Dual‐isotope SPECT imaging of the peptides showed a similar organ and tumor uptake to single‐isotope studies (cRGDfK–DOTA, 1.5 ± 0.8%ID cm^?3; cRADfK–DOTA, 0.2 ± 0.1%ID cm^?3), but with higher statistical relevance (p‐value 0.007, n = 8). This demonstrated that, for the same relevance, seven animals were required in case of a single‐isotope test design as compared with only three animals when a dual‐isotope test was used. Interchanging radionuclides did not influence the biodistribution of the peptides. Dual‐isotope SPECT after simultaneous injection of ¹¹¹In and ¹⁷⁷Lu‐labeled cRGD and cRAD was shown to be a valuable method for paired testing of the in vivo target specificity of ligands in molecular imaging tracer design. Copyright © 2012 John Wiley & Sons, Ltd.     

In vivo evaluation of the dopaminergic neurotransmission system using [123I]FP‐CIT SPECT in 6‐OHDA lesioned rats

The 6‐hydroxydopamine (6‐OHDA) rodent model of Parkinson's disease (PD) has been used to evaluate the nigrostriatal pathway. The aim of this work was to explore the relationship between the degree of 6‐OHDA‐induced dopaminergic degeneration and [¹²³I]FP‐CIT binding using single photon emission computed tomography (SPECT). Fourteen rats received a 6‐OHDA injection (4 or 8 µg) into the left medial forebrain bundle. After 3 weeks, magnetic resonance imaging and scans with a small‐animal SPECT system were performed. Finally, the nigrostriatal lesion was assessed by immunohistochemical analysis. Immunohistochemical analysis confirmed two levels of dopaminergic degeneration. Lesions induced by 6‐OHDA diminished the ipsilateral [¹²³I]FP‐CIT binding by 61 and 76%, respectively. The decrease in tracer uptake between control and lesioned animals was statistically significant, as was the difference between the two 6‐OHDA lesioned groups. Results concluded that [¹²³I]FP‐CIT SPECT is a useful technique to discriminate the degree of dopaminergic degeneration in a rat model of PD. Copyright © 2014 John Wiley & Sons, Ltd.     

SPECT imaging of fibrin using fibrin‐binding peptides

Noninvasive detection of fibrin in vivo using diagnostic imaging modalities may improve clinical decision‐making on possible therapeutic options in atherosclerosis, cancer and thrombus‐related pathologies such as pulmonary embolism and deep venous thrombosis. The aim of this study was to assess the potential of a novel ¹¹¹In‐labeled fibrin‐binding peptide (FibPep) to visualize thrombi in mice noninvasively using single‐photon emission computed tomography (SPECT). FibPep and a negative control peptide (NCFibPep) were synthesized and their fibrin‐binding properties were assessed in vitro. FibPep showed enhanced binding compared with NCFibPep to both fibrin and blood clots. FibPep bound to fibrin with a dissociation constant (K_d) of 0.8 μ m , whereas NCFibPep displayed at least a 100‐fold lower affinity towards fibrin. A FeCl₃‐injury carotid artery thrombosis mouse model was used to evaluate the peptides in vivo. FibPep and NCFibPep displayed rapid blood clearance and were eliminated via the renal pathway. In vivo SPECT imaging using FibPep allowed clear visualization of thrombi. Ex vivo biodistribution showed significantly increased uptake of FibPep in the thrombus‐containing carotid in comparison to the noninjured carotid (5.7 ± 0.7 and 0.6 ± 0.4% injected dose per gram (%ID g^?1), respectively; p < 0.01; n = 4), whereas nonspecific NCFibPep did not (0.4 ± 0.2 and 0.3 ± 0.0%ID g^?1, respectively; n = 4). In conclusion, FibPep displayed high affinity towards fibrin in vitro and rapid blood clearance in vivo, and allowed sensitive detection of thrombi using SPECT imaging. Therefore, this particular imaging approach may provide a new tool to diagnose and monitor diseases such as atherosclerosis and cancer. Copyright © 2013 John Wiley & Sons, Ltd.     

131I SPECT/CT用于131I治疗后分化型甲状腺癌研究进展

对于分化型甲状腺癌(DTC)目前主要采用外科手术切除及术后¹³¹I治疗等,¹³¹I治疗后DTC复发及转移可影响临床后续治疗及其效果。¹³¹I治疗后全身显像(Rx-WBS)为常规影像学监测疗效手段,而¹³¹I SPECT/CT将功能代谢与解剖结构信息相结合,可提供更准确、更全面的影像学信息。本文就¹³¹I SPECT/CT用于¹³¹I治疗DTC后研究进展进行综述。     

64CuS‐labeled nanoparticles: a new sentinel‐lymph‐node‐mapping agent for PET–CT and photoacoustic tomography

Determining sentinel lymph node (SLN) status is critical to cancer staging and treatment decisions. Currently, in clinical practice, ^99mTc‐radiocolloid‐mediated planar scintigraphy and single‐photon emission computed tomography (SPECT) are used to guide the biopsy and resection of SLNs. Recently, an emerging technique that combines positron emission tomography (PET) and photoacoustic tomography (PAT; PET–PAT) may offer accurate information in detecting SLNs. Herein, we report a kind of ⁶⁴CuS‐labeled nanoparticle (⁶⁴CuS‐NP) for the detection of SLNs with PET–PAT. We subcutaneously injected ⁶⁴CuS‐NPs into the rats’ forepaw pads. After 24 h, the rats’ first draining axillary lymph nodes (i.e. the SLNs) could be clearly visualized with micro‐PET (μPET)–CT. Rats were sacrificed after μPET–CT imaging, their axillary lymph nodes were surgically identified, and then PAT was employed to discover ⁶⁴CuS‐NP‐avid SLNs, which were embedded inside tissues. Biodistribution, autoradiography, and copper staining analyses confirmed the SLNs’ high uptake of ⁶⁴CuS‐NPs. Our study indicates that ⁶⁴CuS‐NPs are a promising dual‐function agent for both PET–CT and PAT and could be used with multi‐modal imaging strategies such as PET–PAT to identify SLNs in a clinical setting. Copyright © 2016 John Wiley & Sons, Ltd.     

The laminin‐211‐derived PPFEGCIWN motif accelerates wound reepithelialization and increases phospho‐FAK‐Tyr397 and Rac1‐GTP levels in a rat excisional wound splinting model

We previously reported that the PPFEGCIWN motif (Ln2‐LG3‐P2‐DN3), residues 2678–2686 of the human laminin α2 chain, promotes cell attachment of normal human epidermal keratinocytes (NHEKs) and dermal fibroblasts (NHDFs); however, its in vivo effects on cutaneous wound healing have not yet been examined. In this study, we sought to determine whether Ln2‐LG3‐P2‐DN3 could promote full‐thickness cutaneous wound healing by accelerating wound reepithelialization and wound closure in vivo. Ln2‐LG3‐P2‐DN3 had significantly higher cell attachment and spreading activities than vehicle or scrambled peptide control in both NHEKs and NHDFs in vitro. The wound area was significantly smaller in rats treated with Ln2‐LG3‐P2‐DN3 than in those treated with vehicle or scrambled peptide in the early phase of wound healing. Furthermore, Ln2‐LG3‐P2‐DN3 significantly accelerated wound reepithelialization relative to vehicle or scrambled peptide and promoted FAK‐Tyr397 phosphorylation and Rac1 activation. Collectively, our findings suggest that the PPFEGCIWN motif has potential as a therapeutic agent for cutaneous regeneration via the acceleration of wound reepithelization and wound closure.     

Antibody‐functionalized nanoparticles for imaging cancer: influence of conjugation to gold nanoparticles on the biodistribution of 89Zr‐labeled cetuximab in mice

Antibody‐labeled gold nanoparticles represent a promising novel tool regarding cancer imaging and therapy. Nevertheless, the characterization of biodistribution of such immunonanocarriers has been poorly documented. In this study, the biodistribution of ⁸⁹Zr‐labeled cetuximab before and after the coupling reaction to gold nanoparticles (AuNPs) was compared and the quantitative imaging performance of ⁸⁹Zr immuno‐PET was evaluated. Cetuximab was functionalized with the desferal moiety and labeled with ⁸⁹Zr (⁸⁹Zr–Df–Bz–NCS–cetuximab). AuNPs with a mean diameter of 5 nm were synthesized according a new method developed in the laboratory, and conjugated to ⁸⁹Zr–Df–Bz–NCS–cetuximab using carbodiimide chemistry (AuNPs–PPAA–cetuximab–⁸⁹Zr). The two tracers were injected in A431 xenograft‐bearing mice. Tumor and liver uptakes were assessed at different times after injection using quantitative PET imaging. The in vivo specificity of the binding was investigated using a saturating dose of unlabeled cetuximab. Radiolabeled cetuximab was conjugated to AuNPs with a coupling reaction yield >75%. All conjugates were stable in vitro and to a lesser extent in plasma. In vivo distribution studies revealed no significant difference in tumor uptake for cetuximab conjugated to nanoparticles up to 72 h after injection, compared with unconjugated cetuximab. Immuno‐PET studies showed that AuNPs–PPAA–cetuximab–⁸⁹Zr provided high tumor‐to‐background ratio. The liver uptake of AuNPs–PPAA–cetuximab–⁸⁹Zr was higher, compared with ⁸⁹Zr–Df–Bz–NCS–cetuximab. In vivo blocking experiments demonstrated selective tumor targeting after coupling reaction. This study showed that the conjugation of AuNPs to cetuximab did not affect its tumor accumulation and that the efficacy of EGFR‐targeted nanoparticles was unaltered. The ⁸⁹Zr‐labeled cetuximab‐targeted gold nanoparticles could be a valuable tool for theranostic purposes. Copyright © 2013 John Wiley & Sons, Ltd.     

Affinity Maturation of an ERBB2-Targeted SPECT Imaging Peptide by In Vivo Phage Display

Purpose

The goal of this study was to improve the pharmacokinetic properties and specificity of an ERBB2-targeted peptide for SPECT imaging.

Procedures

Bacteriophages (phages) displaying the ERBB2 targeting sequence, KCCYSL, flanked by additional random amino acids were used for in vivo selections in mice-bearing ERBB2-expressing MDA-MB-435 human breast xenografts. Phage-displayed peptides were evaluated for ERBB2 and cancer cell binding affinity and specificity in vitro, and one peptide was radiolabeled with ¹¹¹In-DOTA and biodistribution and SPECT imaging properties were compared to the first generation peptide, ¹¹¹In-DOTA-KCCYSL.

Results

In vivo phage display selected two peptides, 1-D03 (MEGPSKCCYSLALSH) and 3-G03 (SGTKSKCCYSLRRSS), with higher breast carcinoma cell specificity and similar ErbB2 affinity (236 and 289 nM, respectively) to the first generation peptide. The corresponding radiolabeled probes bound with higher affinity to target cancer cells than ¹¹¹In-DOTA-KCCYSL; however, only ¹¹¹In-DOTA-1-D03 demonstrated higher specificity for MDA-MB-435 cells. Biodistribution analysis demonstrated that although ¹¹¹In-DOTA-1-D03 had slightly reduced tumor uptake (0.661 % ID/g) in comparison to ¹¹¹In-DOTA-KCCYSL (0.78 %/ID/g), its dramatic improvement in blood clearance led to a significantly higher tumor/blood ratio (6.02:1). Non-specific uptake was also reduced in most organs including heart, lung, muscle, bone, and kidneys. SPECT imaging revealed tumor-specific uptake of ¹¹¹In-DOTA-1-D03, which was confirmed by blocking with unlabeled 1-D03 peptide.

Conclusions

This is the first evidence that SPECT imaging peptides with improved tumor specificity and pharmacokinetics can be obtained by in vivo phage display affinity maturation. The combination of ERBB2-specific binding, rapid clearance, and tumor specificity may make 1-D03 a viable candidate for clinical imaging studies.     

Targeted radiotherapy with tumor vascular homing trimeric GEBP11 peptide evaluated by multimodality imaging for gastric cancer

Targeted radiopharmaceutical is an effective treatment for solid tumors. By labeling with radionuclides, targeting peptide could achieve both noninvasive diagnosis and targeted radionuclide therapy. In order to evaluate the potential applicability of GEBP11 peptides in diagnosis and radiotherapy of gastric cancer, in this study, iodine 131 labeled GEBP11 peptides, including a novel bifid PEGlylated GEBP11 trimer and its corresponding monomer, were developed. The clinical potential of GEBP11 peptides, such as tumor binding affinity and antitumor efficacy was demonstrated and assessed with multimodality imaging methods. Cerenkov and SPECT imaging showed higher tumor uptake for ¹³¹I-2PEG-(GEBP11)₃ (P < 0.05, day 1; P < 0.01, day 2; vs. monomer). Biodistribution studies indicated higher tumor accumulation and better T/NT of ¹³¹I-2PEG-(GEBP11)₃. Bioluminescence imaging exhibited a significant tumor growth suppression in ¹³¹I-2PEG-(GEBP11)₃ treated group (P < 0.001 vs. control; P < 0.01 vs. monomer). After treatment with ¹³¹I-2PEG-(GEBP11)₃, the tumor volume and vasculature decreased significantly, and the survival time was prolonged to 75.5 days. Meanwhile, no significant hepatic or renal toxicity was observed with ¹³¹I-2PEG-(GEBP11)₃ administered. In conclusion, ¹³¹I-2PEG-(GEBP11)₃ could be a promising candidate for peptide-based targeting therapy of gastric cancer. 2PEG-(GEBP11)₃ might be a potential drug delivery vehicle for the antiangiogenic therapy of gastric cancer.     

SPECT/CT诊断低危甲状腺乳头状癌术后131I治疗后淋巴结转移发生率及其影响因素

目的 评估低危甲状腺乳头状癌（PTC）患者术后¹³¹I治疗后SPECT/CT诊断转移淋巴结的发生率,并探讨其影响因素。方法 回顾性分析术后¹³¹I治疗前根据2015版美国甲状腺协会指南分为低危的409例PTC患者资料,¹³¹I治疗后5天行全身显像（WBS）和断层融合显像（SPECT/CT）,根据显像结果及随访资料判断SPECT/CT上有无转移淋巴结的存在。比较有转移淋巴结及无转移淋巴结患者的临床资料,分析SPECT/CT上存在转移淋巴结的影响因素,利用ROC曲线预测存在淋巴结转移的最佳界值。结果 ¹³¹I治疗后SPECT/CT发现91例患者存在转移淋巴结,发生率为22.25%（91/409）。单因素分析显示肿瘤大小、刺激性甲状腺球蛋白（sTg）及T分期是存在转移淋巴结的影响因素（P均<0.05）;Logistic分析显示sTg水平是影响转移淋巴结存在的独立危险因素。ROC曲线分析显示,sTg预测转移淋巴结存在的最佳界值为4.01 ng/ml,敏感度87.3%,特异度60.1%。结论 低危PTC患者术后及¹³¹I治疗后SPECT/CT显示转移淋巴结的发生率较高,肿瘤较大及sTg水平高是其影响因素,sTg水平高是预测淋巴结转移的重要指标。     

[68Ga]‐HP‐DO3A‐nitroimidazole: a promising agent for PET detection of tumor hypoxia

The goal of this study is to evaluate a new ⁶⁸Ga‐based imaging agent for detecting tumor hypoxia using positron emission tomography (PET). The new hypoxia targeting agent reported here, [⁶⁸Ga]‐HP‐DO3A‐nitroimidazole ([⁶⁸Ga]‐HP‐DO3A‐NI), was constructed by linking a nitroimidazole moiety with the macrocyclic ligand component of ProHance®, HP‐DO3A. The hypoxia targeting capability of this agent was evaluated in A549 lung cancer cells in vitro and in SCID mice bearing subcutaneous A549 tumor xenografts. The cellular uptake assays showed that significantly more [⁶⁸Ga]‐HP‐DO3A‐NI accumulates in hypoxic tumor cells at 30, 60 and 120 min than in the same cells exposed to 21% O₂. The agent also accumulated in hypoxic tumors in vivo to give a tumor/muscle ratio (T/M) of 5.0 ± 1.2 (n = 3) as measured by PET at 2 h post‐injection (p.i.). This was further confirmed by ex vivo biodistribution data. In addition, [⁶⁸Ga]‐HP‐DO3A‐NI displayed very favorable pharmacokinetic properties, as it was cleared largely through the kidneys with little to no accumulation in liver, heart or lung (%ID/g < 0.5%) at 2 h p.i. The specificity of the agent for hypoxic tissues was further validated in a comparative study with a control compound, [⁶⁸Ga]‐HP‐DO3A, which lacks the nitroimidazole moiety, and by PET imaging of tumor‐bearing mice breathing air versus 100% O₂. Given the commercial availability of cGMP ⁶⁸Ge/⁶⁸Ga generators and the ease of ⁶⁸Ga labeling, the new agent could potentially be widely applied for imaging tumor hypoxia prior to radiation therapy. Copyright © 2015 John Wiley & Sons, Ltd.     

Preliminary evaluation of novel 68Ga‐DOTAVAP‐PEG‐P2 peptide targeting vascular adhesion protein‐1

Introduction: Expression of vascular adhesion protein‐1 (VAP‐1) is induced at the sites of inflammation where extravasation of leukocytes from blood to the peripheral tissue occurs. VAP‐1 is a potential target for anti‐inflammatory therapy and for in vivo imaging of inflammation. Purpose of this study was to preliminarily evaluate a novel VAP‐1‐targeting peptide as a potential PET imaging agent. Methods: Cyclic 17‐amino‐acid peptide selected from phage display libraries was 1,4,7,10‐tetraazacyclododecane‐N,N′,N′′,N′′′‐tetraacetic acid (DOTA) conjugated via 8‐amino‐3,6‐diooxaoctanoyl linker (polyethylene glycol, PEG derivative) and labelled with ⁶⁸Ga (⁶⁸Ga‐DOTAVAP‐PEG‐P2). In vitro stability of ⁶⁸Ga‐DOTAVAP‐PEG‐P2 was determined in saline, rat plasma and human plasma by radio‐HLPC. Lipophilicity was measured by calculating octanol‐water partition coefficient (logP). Whole‐body distribution kinetics and stability after intravenous injection in healthy rats was studied in vivo by PET imaging, ex vivo by measuring radioactivity of excised tissues, and by radio‐HPLC. Results: In vitro the ⁶⁸Ga‐DOTAVAP‐PEG‐P2 remained stable >4 h in saline and rat plasma, but degraded slowly in human plasma after 2 h of incubation. The logP value of ⁶⁸Ga‐DOTAVAP‐PEG‐P2 was ?1·3. In rats, ⁶⁸Ga‐radioactivity cleared rapidly from blood circulation and excreted quickly in urine. At 120 min after injection the fraction of intact ⁶⁸Ga‐DOTAVAP‐PEG‐P2 were 77 ± 6·0% and 99 ± 1·0% in rat plasma and urine, respectively. Conclusions: These basic and essential in vitro and in vivo studies of the new VAP‐1 targeting peptide revealed promising properties for an imaging agent. Further investigations to clarify in vivo VAP‐1 targeting are warranted.     

Radiolabelled GLP‐1 analogues for in vivo targeting of insulinomas

Internalizing agonists are usually selected for peptide receptor targeting. There is increasing evidence that non‐internalizing receptor antagonists can be used for this purpose. We investigated whether the glucagon‐like peptide‐1 receptor (GLP‐1R) antagonist exendin(9–39) can be used for in vivo targeting of GLP‐1R expressing tumours and compared the in vitro and in vivo characteristics with the GLP‐1R agonists exendin‐3 and exendin‐4. The binding and internalization kinetics of labelled [Lys⁴⁰(DTPA)]exendin‐3, [Lys⁴⁰(DTPA)]exendin‐4 and [Lys⁴⁰(DTPA)]exendin(9–39) were determined in vitro using INS‐1 cells. The in vivo targeting properties of [Lys⁴⁰(¹¹¹In‐DTPA)]exendin‐3, [Lys⁴⁰(¹¹¹In‐DTPA)]exendin‐4 and [Lys⁴⁰(¹¹¹In‐DTPA)]exendin(9–39) were examined in BALB/c nude mice with subcutaneous INS‐1 tumours. ^natIn‐labelled [Lys⁴⁰(DTPA)]exendin‐3, [Lys⁴⁰(DTPA)]exendin‐4 and [Lys⁴⁰(DTPA)]exendin(9–39) exhibited similar IC₅₀ values (13.5, 14.4 and 13.4 n m , respectively) and bound to 26 × 10³, 41 × 10³ and 37 × 10³ receptors per cell, respectively. [Lys⁴⁰(¹¹¹In‐DTPA)]exendin‐3 and [Lys⁴⁰(¹¹¹In‐DTPA)]exendin‐4 showed rapid in vitro binding and internalization kinetics, whereas [Lys⁴⁰(¹¹¹In‐DTPA)]exendin(9–39) showed lower binding and minimal internalization in vitro. In mice, high specific uptake of [Lys⁴⁰(¹¹¹In‐DTPA)]exendin‐3 [25.0 ± 6.0% injected dose (ID) g^?1] in the tumour was observed at 0.5 h post‐injection (p.i.) with similar uptake up to 4 h p.i. [Lys⁴⁰(¹¹¹In‐DTPA)]exendin‐4 showed higher tumour uptake at 1 and 4 h p.i. (40.8 ± 7.0 and 41.9 ± 7.2% ID g^?1, respectively). Remarkably, [Lys⁴⁰(¹¹¹In‐DTPA)]exendin(9–39) showed only low specific uptake in the tumour at 0.5 h p.i. (3.2 ± 0.7% ID g^?1), rapidly decreasing over time. In conclusion, the GLP‐1R agonists [Lys⁴⁰(DTPA)]exendin‐3 and [Lys⁴⁰(DTPA)]exendin‐4 labelled with ¹¹¹In could be useful for in vivo GLP‐1R targeting, whereas [Lys⁴⁰(DTPA)]exendin(9–39) is not suited for in vivo targeting of the GLP‐1R. Copyright © 2012 John Wiley & Sons, Ltd.     

A radiolabeled nonapeptide probe targeting PC‐3 cells and bone metastases of prostate cancer in mice

Previous investigations showed that interleukin‐11 (IL‐11) and the IL‐11 receptor (IL‐11R) are correlated with regulation of tumor progression and may play significant roles in bone metastases. The nonapeptide structure c(CGRRAGGSC) is a phage‐display‐selected IL‐11 mimic that binds to IL‐11R. The aim of this study was to synthesize radiolabeled c(CGRRAGGSC) and to investigate the possible interaction between this radioactive probe and an IL‐11R‐positive bone metastasis model of PC‐3 prostate cancer. The molecular probe ^99mTc–DTPA–c(CGRRAGGSC) was radiolabeled with ^99mTc using the diethylenetriaminepentaacetic acid (DTPA) chelate. Counterstaining was performed with LSS670, a near‐infrared dye. The binding sites of the molecular probe in PC‐3 cells were observed under a fluorescence microscope. The binding characteristics of the labeled probe were analyzed using radioreceptor analysis. Single photon emission tomography imaging and biodistribution of the probe were investigated using xenografts of PC‐3 cells into tibias of nude mice. The labeled product, ^99mTc–DTPA–c(CGRRAGGSC), was obtained with high labeling efficiency, high radiochemical purity and good stability. The molecular probe was combined with the PC‐3 cell membrane and cytoplasm through fluorescence tracing. In the saturation and competitive inhibition experiments performed in vitro, the K_d value was 0.32 ± 0.02 n m and the B_max value was 754 ± 34 fmol mg^?1 pro. The probe exhibited a high tumor uptake in vivo. The radioactive molecular probe ^99mTc–DTPA–c(CGRRAGGSC) may be used as a specific molecular imaging agent for detecting IL‐11R overexpression in tumors and bone metastasis, such as prostate cancers. Copyright © 2012 John Wiley & Sons, Ltd.     

Insulin/IGF‐1 hybrid receptor expression on human platelets: consequences for the effect of insulin on platelet function

Summary. Objectives: As platelets express both insulin and insulin‐like growth factor‐1 (IGF‐1) receptors, their subunits may randomly heterodimerize to form insulin/IGF‐1 receptor hybrids, which avidly bind IGF‐1, but not insulin. This study investigated the possibility that platelets express hybrid receptors, which may affect insulin action on platelet function. Methods: Platelets were incubated with insulin and IGF‐1. Expression and phosphorylation of insulin/IGF‐1 receptors was determined by western blotting of immunoprecipitates, and compared with platelet functional responses. Relative expression of insulin and IGF‐1 receptors was estimated by competitive ligand binding and quantitative polymerase chain reaction. Results: We demonstrated the presence of insulin/IGF‐1 hybrid receptors on human platelets by detecting both insulin and IGF‐1 receptor β subunits in coimmunoprecipitation studies. Stimulation of platelets with insulin (1–100 nm ) resulted in tyrosine phosphorylation of insulin receptors, but not of hybrid receptors. High insulin concentrations (50–100 nm ) stimulated weak phosphorylation of IGF‐1 receptors and protein kinase B (Akt), and correlated with moderately increased aggregation and fibrinogen binding, whereas low insulin concentrations (1–10 nm ) had no effect. In contrast, IGF‐1 (1–100 nm ) induced strong phosphorylation of both hybrid and IGF‐1 receptors, and potentiated platelet aggregation and fibrinogen binding. Specific binding of [¹²⁵I]IGF‐1 (1.08% ± 0.16%) was significantly higher than that of [¹²⁵I]insulin (0.15% ± 0.03%). Accordingly, IGF‐1 receptor mRNA was more abundant than insulin receptor mRNA (IGF‐1 receptor/insulin receptor ratio 69 ± 3.8). Conclusions: Insulin has minimal effects on platelet function, which can be explained by the relatively low insulin receptor expression levels resulting in the majority of insulin receptor subunits being expressed as insulin/IGF‐1 hybrids.     

Nature‐inspired nanoformulations for contrast‐enhanced in vivo MR imaging of macrophages

Magnetic resonance imaging (MRI) of macrophages in atherosclerosis requires the use of contrast‐enhancing agents. Reconstituted lipoprotein particles that mimic native high‐density lipoproteins (HDL) are a versatile delivery platform for Gd‐based contrast agents (GBCA) but require targeting moieties to direct the particles to macrophages. In this study, a naturally occurring methionine oxidation in the major HDL protein, apolipoprotein (apo) A‐I, was exploited as a novel way to target HDL to macrophages. We also tested if fully functional GBCA–HDL can be generated using synthetic apo A‐I peptides. The fluorescence and MRI studies reveal that specific oxidation of apo A‐I or its peptides increases the in vitro macrophage uptake of GBCA–HDL by 2–3 times. The in vivo imaging studies using an apo E‐deficient mouse model of atherosclerosis and a 3.0 T MRI system demonstrate that this modification significantly improves atherosclerotic plaque detection using GBCA–HDL. At 24 h post‐injection of 0.05 mmol Gd kg^?1 GBCA–HDL containing oxidized apo A‐I or its peptides, the atherosclerotic wall/muscle normalized enhancement ratios were 90 and 120%, respectively, while those of GBCA–HDL containing their unmodified counterparts were 35 and 45%, respectively. Confocal fluorescence microscopy confirms the accumulation of GBCA–HDL containing oxidized apo A‐I or its peptides in intraplaque macrophages. Together, the results of this study confirm the hypothesis that specific oxidation of apo A‐I targets GBCA–HDL to macrophages in vitro and in vivo. Furthermore, our observation that synthetic peptides can functionally replace the native apo A‐I protein in HDL further encourages the development of these contrast agents for macrophage imaging. Copyright © 2014 John Wiley & Sons, Ltd.     

Modification of MR molecular imaging probes with cysteine‐terminated peptides and their potential for in vivo tumour detection

One of the challenges facing superparamagnetic iron oxide (SPIO) nanoparticles is to improve their biological compatibility. While highly uniform SPIOs can be manufactured, the surfaces are hydrophobic as a result of the surfactants used in their fabrication. In this study, we developed a general strategy to fabricate an MR molecular imaging probe in one step by replacing hydrophobic surfactants with small peptides terminated with cysteine. The hydrophobic SPIO surface was transformed into a hydrophilic one by exchanging surface oleic acids with the peptides RGD–Cys or RGD–PEG–Cys. After the RGD–Cys and RGD–PEG–Cys peptide exchange, both RGD–Cys–SPIO and RGD–PEG–Cys–SPIO specifically targeted α_vβ₃‐expressing cells (A549) in vitro, with RGD–Cys–SPIO achieving this more efficiently. Furthermore, MR imaging of A549 tumors receiving RGD–Cys–SPIO or RGD–PEG–Cys–SPIO demonstrated that both the targeted particles could reach and label the α_vβ₃‐expressing tumor, much more efficiently than the non‐targeted particles (Cys–SPIO). Histology showed that the probes not only target the tumor neovasculature but also extravasate from vessels and address the tumor cells. Our study shows that directly replacing oleic acid with cysteine or cysteine‐terminated small peptides is a general strategy to transforming the hydrophobic surface of SPIO into a hydrophilic one, as well as providing targeting ligands. Such SPIOs are of interest as MR molecular imaging probes to detect for cancer in vivo. Copyright © 2010 John Wiley & Sons, Ltd.     

Incorporation of an apoE‐derived lipopeptide in high‐density lipoprotein MRI contrast agents for enhanced imaging of macrophages in atherosclerosis

Magnetic resonance (MR) imaging is becoming a pivotal diagnostic method to identify and characterize vulnerable atherosclerotic plaques. We previously reported a reconstituted high‐density lipoprotein (rHDL) nanoparticle platform enriched with Gd‐based amphiphiles as a plaque‐specific MR imaging contrast agent. Further modification can be accomplished by inserting targeting moieties into this platform to potentially allow for improved intraplaque macrophage uptake. Since studies have indicated that intraplaque macrophage density is directly correlated to plaque vulnerability, modification of the rHDL platform may allow for better detection of vulnerable plaques. In the current study we incorporated a carboxyfluoresceine‐labeled apolipoprotein E‐derived lipopeptide, P2fA2, into rHDL. The in vitro macrophage uptake and in vivo MR efficacy were demonstrated using murine J774A.1 macrophages and the apolipoprotein E knock‐out (apoE^?/?) mouse model of atherosclerosis. The in vitro studies indicated enhanced association of murine macrophages to P2fA2 enriched rHDL (rHDL–P2A2) nanoparticles, relative to rHDL, using optical techniques and MR imaging. The in vivo studies showed a more pronounced and significantly higher signal enhancement of the atherosclerotic wall 24 h after the 50 µmol Gd/kg injection of rHDL–P2A2 relative to administration of rHDL. The normalized enhancement ratio for atherosclerotic wall of rHDL–P2A2 contrast agent injection was 90%, while that of rHDL was 53% 24 h post‐injection. Confocal laser scanning microscopy revealed that rHDL–P2A2 nanoparticles co‐localized primarily with intraplaque macrophages. The results of the current study confirm the hypothesis that intraplaque macrophage uptake of rHDL may be enhanced by the incorporation of the P2fA2 peptide into the modified HDL particle. Copyright © 2008 John Wiley & Sons, Ltd.