Synthesis of (68)Ga-labeled NOTA-RGD-GE11 heterodimeric peptide for dual integrin and epidermal growth factor receptor-targeted tumor imaging.

Radiolabeled Arg-Gly-Asp (RGD) peptide analogs have been extensively studied for αvß3 integrin-targeted angiogenesis imaging. According to recently presented evidence, the dodecapeptide GE11 has high affinity to the epidermal growth factor receptor (EGFR), which is overexpressed in many types of cancer. Dual-receptor molecular imaging probes with two different heterodimeric peptides exhibit improved cancer targeting efficacy. In the present study, the design and synthesis of a new RGD-GE11 peptide heterodimer for dual αvß3 integrin/EGFR-targeted cancer imaging are described. The RGD-GE11 heterodimer was linked with 6-aminohexanoic acid (6-Ahx) and cysteine and conjugated with 1,4,7-triazacyclononane-N,N',N″-triacetic acid (NOTA) to form NOTA-RGD-cys-6-Ahx-GE11. The monomeric peptides, NOTA-cys-6-Ahx-GE11 and c(RGDyK), were formed by a peptide synthesizer. The peptide heterodimer NOTA-RGD-GE11 was obtained by NOTA-cys-6-Ahx-GE11 and maleimidopropyl-c(RGDyK) conjugation with a thioether linkage. The NOTA peptide conjugate was labeled with freshly eluted (68)Ga and purified using reversed-phase high-performance liquid chromatography. The (68)Ga-NOTA-RGD-cys-6-Ahx-GE11 was successfully prepared, in this study, with a radiochemical yield of 85% and a radiochemical purity of >98%. These results warrant further investigation of this heterodimeric peptide's binding affinity to the receptors.

In vitro selection and characterization of a novel Zn(II)-dependent phosphorothiolate thiolesterase ribozyme.

Here we present the in vitro selection of a novel ribozyme specific for Zn2+-dependent catalysis on hydrolysis of a phosphorothiolate thiolester bond. The ribozyme, called the TW17 ribozyme, was evolved and selected from an artificial RNA pool covalently linked to a biotin-containing substrate through the phosphorothiolate thiolester bond. The secondary structure for the evolved ribozyme consisted of three major helices and three loops. Biochemical and chemical studies of ribozyme-catalyzed reaction products provided evidence that the ribozyme specifically catalyzes hydrolysis of the phosphorothiolate thiolester linkage. A successful ribozyme construct with active catalysis in trans further supported the determined ribozyme structure and indicated the potential of the ribozyme for multiple-substrate turnover. The ribozyme also requires Zn2+ and Mg2+ for maximal catalysis. The TW17 ribozyme, in the presence of Zn2+ and Mg2+, conferred a rate enhancement of at least 5 orders of magnitude when compared to the estimated rate of the uncatalyzed reaction. The ribozyme completely lost catalytic activity in the absence of Zn2+, like Zn2+-dependent protein hydrolases. The discovery and characterization of the TW17 ribozyme suggest additional roles for Zn2+ in ribozyme catalysts.

Synthesis, Radiolabeling, and Preliminary in vivo Evaluation of [68Ga] IPCAT-NOTA as an Imaging Agent for Dopamine Transporter.

INTRODUCTION: Novel radiotracer development for imaging dopamine transporters is a subject of interest because although [99mTc]TRODAT-1, [123I]ß-CIT, and [123I]FP-CIT are commercially available; 99Mo/99mTc generator is in short supply and 123I production is highly dependent on compact cyclotron. Therefore, we designed a novel positron emission tomography (PET) tracer based on a tropane derivative through C-2 modification to conjugate NOTA for chelating 68Ga, a radioisotope derived from a 68Ge/68Ga generator. METHODS: IPCAT-NOTA 22 was synthesized and labeled with [68Ga]GaCl4 - at room temperature. Biological studies on serum stability, LogP, and in vitro autoradiography (binding assay and competitive assay) were performed. Furthermore, ex vivo autoradiography, biodistribution, and dynamic PET imaging studies were performed in Sprague Dawley rats. RESULTS: [68Ga]IPCAT-NOTA 24 obtained had a radiochemical yield of ≥90% and a specific activity of 4.25 MBq/nmol. [68Ga]IPCAT-NOTA 24 of 85% radiochemical purity (RCP%) was stable at 37°C for up to 60 minutes in serum with a lipophilicity of 0.88. The specific binding ratio (SBR%) reached 15.8 ± 6.7 at 60 minutes, and the 85% specific uptake could be blocked through co-injection at 100- and 1000-fold of the cold precursor in in vitro binding studies. Tissue regional distribution studies in rats with [68Ga]IPCAT-NOTA 24 showed striatal uptake (0.02% at 5 minutes and 0.007% at 60 minutes) with SBR% of 6%, 25%, and 62% at 5-15, 30-40, and 60-70 minutes, respectively, in NanoPET studies. The RCP% of [68Ga]IPCAT-NOTA 24 at 30 minutes in vivo remained 67.65%. CONCLUSION: Data described here provide new information on the design of PET probe of conjugate/pendent approach for DAT imaging. Another chelator or another direct method of intracranial injection must be used to prove the relation between [68Ga]IPCAT-NOTA 24 uptake and transporter localization.

A novel 111indium-labeled dual carbonic anhydrase 9-targeted probe as a potential SPECT imaging radiotracer for detection of hypoxic colorectal cancer cells.

Tumor hypoxia is a common feature in colorectal cancer (CRC), and is associated with resistance to radiotherapy and chemotherapy. Thus, a specifically targeted probe for the detection of hypoxic CRC cells is urgently needed. Carbonic anhydrase 9 (CA9) is considered to be a specific marker for hypoxic CRC diagnosis. Here, a nuclear imaging Indium-111 (111In)-labeled dual CA9-targeted probe was synthesized and evaluated for CA9 detection in in vitro, in vivo, and in human samples. The CA9-targeted peptide (CA9tp) and CA9 inhibitor acetazolamide (AAZ) were combined to form a dual CA9-targeted probe (AAZ-CA9tp) using an automatic microwave peptide synthesizer, which then was conjugated with 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) for radioisotope (111In) labeling (111In-DOTA-AAZ-CA9tp). The assays for cell binding, stability, and toxicity were conducted in hypoxic CRC HCT15 cells. The analyses for imaging and biodistribution were performed in an HCT15 xenograft mouse model. The binding and distribution of 111In-DOTA-AAZ-CA9tp were detected in human CRC samples using microautoradiography. AAZ-CA9tp possessed good CA9-targeting ability in hypoxic HCT15 cells. The dual CA9-targeted radiotracer showed high serum stability, high surface binding, and high affinity in vitro. After exposure of 111In-DOTA-AAZ-CA9tp to the HCT15-bearing xenograft mice, the levels of 111In-DOTA-AAZ-CA9tp were markedly and specifically increased in the hypoxic tumor tissues compared to control mice. 111In-DOTA-AAZ-CA9tp also targeted the areas of CA9 overexpression in human colorectal tumor tissue sections. The results of this study suggest that the novel 111In-DOTA-AAZ-CA9tp nuclear imaging agent may be a useful tool for the detection of hypoxic CRC cells in clinical practice.

Indium-111-labeled CD166-targeted peptide as a potential nuclear imaging agent for detecting colorectal cancer stem-like cells in a xenograft mouse model.

BACKGROUND: Cancer stem cells (CSCs) are involved in drug resistance, metastasis, and relapse of cancers, which can significantly affect tumor therapy. Hence, to develop specifically therapeutic target probe at CSCs for improvement of survival and quality of life of cancer patients is urgently needed. The CD166 protein has been suggested to be involved in colorectal cancer (CRC) tumorigenesis and to be considered a marker for colorectal CSCs (CRCSCs) detection. In this study, therefore, we attend to apply a nuclear imaging agent probe, Glycine18-Cystine-linked CD166-targeted peptides (CD166tp-G18C), to detect the changes of CD166 level in a CRC xenograft mouse model. RESULTS: We isolated the CD166-positive cells from the HCT15 CRC cell line (CD166+HCT15) and evaluated their morphology and ability of clone formation, migration, protein expression, and drug resistance. The CD166-positive HCT15 cells display the CSCs characteristics. We discovered and designed a CD166-targeted peptide (CD166tp-G18C) as a targeted probe of CRC stem-like cell for cell binding assay. The CD166tp-G18C confirmed the CD166 protein targeting ability in CD166+HCT15 cells. The diethylenetriaminopentaacetic acid (DTPA)-conjugated CD166tp-G18C further was labeled with indium-111 (111In-DTPA-CD166tp-G18C) as nuclear imaging agent for imaging and bio-distribution analysis in vivo. Finally, we observed that the 111In-DTPA-CD166tp-G18C was significantly enhanced in tumor tissues of CD166+HCT15 xenograft mice as compared to the non-CD166tp-G18C control. CONCLUSIONS: Our results indicated that the indium-111-labeled CD166tp-G18C may be served as a powerful tool for colorectal CSCs nuclear imaging in the CRC patients.

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

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

Corroboration of Zn(ii)-Mg(ii)-tertiary structure interplays essential for the optimal catalysis of a phosphorothiolate thiolesterase ribozyme.

The TW17 ribozyme, a catalytic RNA selected from a pool of artificial RNA, is specific for the Zn2+-dependent hydrolysis of a phosphorothiolate thiolester bond. Here, we describe the organic synthesis of both guanosine α-thio-monophosphate and the substrates required for selecting and characterizing the TW17 ribozyme, and for deciphering the catalytic mechanism of the ribozyme. By successively substituting the substrate originally conjugated to the RNA pool with structurally modified substrates, we demonstrated that the TW17 ribozyme specifically catalyzes phosphorothiolate thiolester hydrolysis. Metal titration studies of TW17 ribozyme catalysis in the presence of Zn2+ alone, Zn2+ and Mg2+, and Zn2+ and [Co(NH3)6]3+ supported our findings that Zn2+ is absolutely required for ribozyme catalysis, and indicated that optimal ribozyme catalysis involves the presence of outer-sphere and one inner-sphere Mg2+. A survey of the TW17 ribozyme activity at various pHs revealed that the activity of the ribozyme critically depends on the alkaline conditions. Moreover, a GNRA tetraloop-containing ribozyme constructed with active catalysis in trans provided catalysis and multiple substrate turnover efficiencies significantly higher than ribozymes lacking a GNRA tetraloop. This research supports the essential roles of Zn2+, Mg2+, and a GNRA tetraloop in modulating the TW17 ribozyme structure for optimal ribozyme catalysis, leading also to the formulation of a proposed reaction mechanism for TW17 ribozyme catalysis.

Imaging specificity of MR-optical imaging agents following the masking of surface charge by poly(ethylene glycol).

The coupling of specific antibodies to imaging agents often improves imaging specificity. However, free amine groups designed for the coupling can cause nonspecific binding of the imaging agents. We report here development of a nanocarrier, MnMEIO-silane-NH2-mPEG nanoparticles (NPs), consisting of a manganese-doped iron oxide nanoparticle core (MnMEIO), a copolymer shell of silane and amine-functionalized poly(ethylene glycol) (silane-EA-mPEG). The key feature in MnMEIO-silane-NH2-mPEG is the flexible PEG, which masks the non-conjugated reactive amine groups (-NH2 â†” -NH3(+)) and reduces nonspecific binding of MnMEIO-silane-NH2-mPEG to cells. The amine groups on MnMEIO-silane-NH2-mPEG were conjugated with the fluorescent dye, Cy777 or antibodies [Erbitux (Erb)] to form a MR-optical imaging contrast agent (MnMEIO-silane-NH2-(Erb)-mPEG) for EGFR-expressing tumors. Confocal microscopic and flow cytometric analyses showed that MnMEIO-silane-NH2-(Erb)-mPEG displayed low nonspecific binding. Moreover, TEM images showed that MnMEIO-silane-NH2-(Erb)-mPEG were endocytosed by EGFR-expressing cells. In line with their EGFR expression levels, A431, PC-3, and Colo-205 tumors treated with MnMEIO-silane-NH2-(Erb)-mPEG NPs showed -97.1%, -49.7%, and -2.8% contrast enhancement, respectively, in in vitro T2-weighted MR imaging. In vivo T2-weighted MR imaging and optical images showed that MnMEIO-silane-NH2-(Erb)-mPEG could specifically and effectively target to EGFR-expressing tumors in nude mice; the relative contrast enhancements were 7.94 (at 2 h) and 7.59 (at 24 h) fold higher in A431 tumors as compared to the EGFR-negative Colo-205 tumors. On the contrary, MnMEIO-silane-NH2-(Erb) NPs showed only 1.44 (at 2 h) and 1.52 (at 24 h) fold higher in EGFR-positive tumors as compared to the EGFR-negative tumors. Finally, antibodies can be readily changed to allow imaging of other tumors bearing different antigens. These data indicate that masking surface charges on contrast agents is a useful strategy to improve imaging efficacy.

Targeted RGD nanoparticles for highly sensitive in vivo integrin receptor imaging.

A new magnetic resonance imaging (MRI) contrast bearing RGD peptide is reported. In this study, ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles with various sizes were prepared. Particles sizes between 6 and 13 nm were tuned by varying the stirring rate. Remarkable negative contrast was observed because USPIO nanoparticles (13.1 ± 2.1 nm) generated high transversal relaxivity r2 (188 ± 3 m m(-1) s(-1) ) and saturation magnetization (94 emu g(-1) Fe). The USPIO nanoparticles were coated with PDA [2-(pyridyldithio)-ethylamine; PDA nanoparticles] containing functional polymer, which can be readily synthesized by Michael addition. The PDA nanoparticles were conjugated with RGD peptide (RGD nanoparticles) for targeting the specific site. The target specificity and high relaxivity allowed RGD nanoparticles to differentiate the expression level of integrin receptor on several cell lines and tumors (MCF-7, A-549, HT-29 and HT-1080) by in vitro and in vivo MR imaging. Importantly, a remarkable negative contrast (-51.3 ± 6.7%) was observed for in vivo MR imaging of MCF-7 tumor. This result implies that the RGD nanoparticles that greatly enhance the MR imaging are highly sensitive for early stage tumor detection.