Preclinical evaluation of an 18F-labeled Tenascin-C aptamer for PET imaging of atherosclerotic plaque in mouse models of atherosclerosis.

Tenascin-C is an extracellular matrix glycoprotein strongly expressed in coronary atherosclerotic plaque. Aptamers are single-stranded oligonucleotides that bind to specific target molecules with high affinity. This study hypothesized that tenascin-C expression at atherosclerotic plaque in vivo could be detected by tenascin-C specific aptamers using positron emission tomography (PET). This paper reports the radiosynthesis of a fluorine-18 (18F)-labeled tenascin-C aptamer for the biodistribution and PET imaging of the tenascin-C expression in apolipoprotein E-deficient (ApoE-/-) mice. The aortas ApoE-/- mice showed significantly increased positive areas of Oil red O staining than control C57BL/6 mice, and tenascin-C expression was detected in foam cells accumulated in the subendothelial lesions of ApoE-/- mice. The ex vivo biodistribution of the 18F-labeled tenascin-C aptamer showed significantly increased uptake at the aorta of ApoE-/- mice, and ex vivo autoradiography of aorta revealed the high accumulation of the 18F-labeled tenascin-C aptamer in the atherosclerotic lesions of ApoE-/- mice, which was consistent with the location of the atherosclerotic plaques detected by Oil red O staining. PET imaging of the 18F-labeled tenascin-C aptamer revealed a significantly higher mean standardized uptake in the aorta of the ApoE-/- mice than the control C57BL/6 mice. These data highlight the potential use of tenascin-C aptamer to diagnose atherosclerotic lesions in vivo.

Enhancement of in vivo targeting properties of ErbB2 aptamer by chemical modification.

Aptamers have great potential for diagnostics and therapeutics due to high specificity to target molecules. However, studies have shown that aptamers are rapidly distributed and excreted from blood circulation due to nuclease degradation. To overcome this issue and to improve in vivo pharmacokinetic properties, inverted deoxythymidine (idT) incorporation at the end of aptamer has been developed. The goal of this study was to evaluate the biological characterization of 3'-idT modified ErbB2 aptamer and compare with that of unmodified aptamer via nuclear imaging. ErbB2-idT aptamer was labeled with radioisotope F-18 by base-pair hybridization using complementary oligonucleotide platform. The hyErbB2-idT aptamer demonstrated specific binding to targets in a ErbB2 expressing SK-BR-3 and KPL4 cells in vitro. Ex vivo biodistribution and in vivo imaging was studied in KPL4 xenograft bearing Balb/c nu/nu mice. 18F-hyErbB2-idT aptamer had significantly higher retention in the tumor (1.36 ± 0.17%ID/g) than unmodified 18F-hyErbB2 (0.98 ± 0.19%ID/g) or scrambled aptamer (0.79 ± 0.26% ID/g) at 1 h post-injection. 18F-hyErbB2-idT aptamer exhibited relatively slow blood clearance and delayed excretion by the renal and hepatobiliary system than 18F-hyErbB2 aptamer. In vivo PET imaging study showed that 18F-hyErbB2-idT aptamer had more stronger PET signals on KPL4 tumor than 18F-hyErbB2 aptamer. The results of this study demonstrate that attachment of idT at 3'-end of aptamer have a substantial influence on biological stability and extended blood circulation led to enhanced tumor uptake of aptamer.

In vivo positron emission tomography imaging for PD-L1 expression in cancer using aptamer.

The programmed death-1 (PD-1) receptor is an immunosuppressive receptor expressed on activated T-cells that elicits an inhibitory signal upon the engagement of its ligand, which is the programmed death ligand 1 (PD-L1). Recent studies have shown that PD-1/PD-L1 blockade can enhance endogenous antitumor immunity. Thus, the PD-1/PD-L1 axis may be a potential therapeutic target for cancer immunotherapy. Aptamers are oligonucleotides with high specificity and affinity for target molecules and promising candidates for molecular imaging and targeted therapy. 68Ga is an attractive radionuclide that serves as a low-cost alternative to cyclotron-produced positron emission tomography (PET) radionuclides. In this study, we developed a 68Ga-labeled PD-L1 aptamer and investigated its target specificity and utility for in vivo PET scanning. In the first part of our study, we evaluated the binding affinity of three PD-L1 aptamers in PD-L1-positive (H1975 and B16F10) and negative (A549 and HT-29) tumor cells by flow cytometry and confocal microscopy. Optical imaging studies of PD-L1 aptamers were performed in H1975 tumor-bearing mice, and the aptamer with the highest binding affinity to PD-L1 positive tumors was selected. PD-L1 aptamers were radiolabeled with 68Ga. PET was performed for in vivo imaging of the 68Ga-NOTA-PD-L1 aptamer in H1975 tumor-bearing mice (PD-L1-positive cells) and A549 tumor-bearing mice (PD-L1-negative cells). Flow cytometry and confocal microscopy showed that PD-L1 aptamers had strong binding to PD-L1-positive H1975 and B16F10 cells. In contrast, PD-L1-negative A549 and HT-29 cells showed low binding to PD-L1 aptamers. Optical imaging studies of H1975 tumor-bearing mice showed the highest uptake of the 2198-06-07 PD-L1 aptamer. PET of 68Ga-NOTA-PD-L1 aptamers demonstrated increased uptake into PD-L1-positive H1975 tumors compared with PD-L1-negative A549 tumors. We confirmed that 68Ga-NOTA-PD-L1 aptamers facilitated the visualization of PD-L1 expression by in vivo PET scanning. These data suggest that 68Ga-NOTA-PD-L1 aptamers could potentially act as tracers for imaging for PD-L1-positive cancers.

Targeted Therapy of Hepatocellular Carcinoma Using Gemcitabine-Incorporated GPC3 Aptamer.

Hepatocellular carcinoma (HCC) is the most common malignancy of the liver, which can progress rapidly and has a poor prognosis. Glypican-3 (GPC3) has been proposed to be an important diagnostic biomarker and therapeutic target for HCC. Aptamers have emerged as promising drug delivery vehicles because of their high binding affinity for target molecules. Herein, we developed G12msi, a gemcitabine-incorporated DNA aptamer, targeting GPC3, and evaluated its binding specificity and anti-tumor efficacy in GPC3-overexpressing HCC cell lines and murine xenograft models. GPC3-targeted aptamers were selected by using the SELEX process and the chemotherapy drug gemcitabine was internally incorporated into the aptamer. To determine the binding affinity and internalization of the G12msi, flow cytometry and confocal microscopy were performed on GPC3-positive HepG2, Hep3B, and Huh7 cells, as well as a GPC3-negative A431 cell. The anti-tumor activities of G12msi were evaluated with in vitro and in vivo models. We found that G12msi binds to GPC3-overexpressing HCC tumor cells with high specificity and is effectively internalized. Moreover, G12msi treatment inhibited the cell proliferation of GPC3-positive HCC cell lines with minimal cytotoxicity in control A431 cells. In vivo systemic administration of G12msi significantly inhibited tumor growth of HCC HepG2 cells in xenograft models without causing toxicity. These results suggest that gemcitabine-incorporated GPC3 aptamer-based drug delivery may be a promising strategy for the treatment of HCC.

PET imaging of HER2 expression with an 18F-fluoride labeled aptamer.

BACKGROUND/PURPOSE: Aptamers are oligonucleotide or peptide molecules that bind to a target molecule with high affinity and specificity. The present study aimed to evaluate the target specificity and applicability for in vivo molecular imaging of an aptamer labeled with a radioisotope. METHODS: The human epidermal growth factor receptor 2 (HER2/ErbB2) aptamer was radiolabeled with 18F-fluoride. HER2-positive tumor cell uptake of the aptamer was evaluated in comparison to negative controls by flow cytometry and confocal microscopy. Using 18F-labeled HER2-specific aptamer positron emission tomography (PET), in vivo molecular images of BT474 tumor-bearing mice were taken at 60, 90 and 120 minutes after injection. RESULTS: In flow cytometric analysis, HER2 aptamer showed strong binding to HER2-positive BT474 cells, while binding to HER2-negative MDA-MB231 cells was quite low. Likewise, in confocal microscopic images, the aptamer was bound to HER2-positive breast cancer cells, with minimal binding to HER2-negative cells. In vivo PET molecular imaging of BT474 tumor-bearing mice revealed significant higher uptake of the 18F-labeled HER2 specific aptamer into the tumor compared to the that of HER2-negative cell tumor(p = 0.033). HER2 aptamer was able to preferentially bind to HER2-positive breast cancer cells both in vitro and in vivo, by recognizing HER2 structure on the surface of these cells. CONCLUSION: The 18F-labeled aptamer enabled appropriate visualization of HER2 expression by human breast cancer cells. The results suggest that a radiolabeled HER2 aptamer could potentially be applied in the development of treatment strategies or in targeted therapy against HER2-positive breast cancer cells.

Gemcitabine-Incorporated G-Quadruplex Aptamer for Targeted Drug Delivery into Pancreas Cancer.

Gemcitabine has been considered a first-line chemotherapy agent for the treatment of pancreatic cancer. However, the initial response rate of gemcitabine is low and chemoresistance occurs frequently. Aptamers can be effectively internalized into cancer cells via binding to target molecules with high affinity and specificity. In the current study, we constructed an aptamer-based gemcitabine delivery system, APTA-12, and assessed its therapeutic effects on pancreatic cancer cells in vitro and in vivo. APTA-12 was effective in vitro and in vivo in pancreatic cancer cells with high expression of nucleolin. The results of in vitro cytotoxicity assays indicated that APTA-12 inhibited the growth of pancreatic cancer cell lines. In vivo evaluation showed that APTA-12 effectively inhibited the growth of pancreatic cancer in Capan-1 tumor-bearing mice compared to mice that received gemcitabine alone or vehicle. These results suggest that the gemcitabine-incorporated APTA-12 aptamer may be a promising targeted therapeutic strategy for pancreatic cancer.

Hybridization-based aptamer labeling using complementary oligonucleotide platform for PET and optical imaging.

Aptamers are promising next-generation ligands used in molecular imaging and theragnosis. Aptamers are synthetic nucleic acids that can be held together with complementary sequences by base-pair hybridization. In this study, the complementary oligonucleotide (cODN) hybridization-based aptamer conjugation platform was developed to use aptamers as the molecular imaging agent. The cODN was pre-labeled with fluorescent dye or radioisotope and hybridized with a matched sequence containing aptamers in aqueous conditions. The cODN platform-hybridized aptamers exhibited good serum stability and specific binding affinity towards target cancer cells both in vitro and in vivo. These results suggest that the newly designed aptamer conjugation platform offers great potential for the versatile application of aptamers as molecular imaging agents.

64Cu-ATSM Hypoxia Positron Emission Tomography for Detection of Conduit Ischemia in an Experimental Rat Esophagectomy Model.

BACKGROUND: We designed a hypoxia-imaging modality to detect ischemia of the gastric conduit after esophagectomy. MATERIALS AND METHODS: A rat esophagectomy model was created using 12-16-week-old, 300-350 g male Sprague-Dawley rats. In the operation group (n=6), partial gastric devascularization was performed by ligating the left gastric artery and the short gastric arteries and an esophagogastric anastomosis was performed. In the control group (n=6), the esophageal-gastric junction was incised and suturing was performed without gastric devascularization. Positron emission tomography (PET) images were taken using a microPET rodent model scanner, 24 h after the initial operation, after injection of 200 µCi 64Cu-diacetyl-bis (N4-methylsemicarbazone) (64Cu-ATSM) and pimonidazole 120 mg/kg. After microPET imaging, autoradiography and immunohistochemistry were performed. RESULTS: The PET image revealed 64Cu-ATSM uptake at the fundus in the operation group 3 h after 64Cu-ATSM injection. The maximum percentage of the injected dose per gram of tissue was higher in the operation group (0.047±0.015 vs. 0.026±0.006, p=0.021). The fundus/liver ratio was also higher in the operation group (0.541±0.126 vs. 0.278±0.049, p=0.002). Upon autoradiography, 64Cu-ATSM uptake was observed in the fundus in the operation group, and was well-correlated to that observed on the PET image. Upon immunohistochemistry, expression of hypoxia-inducible factor 1a and pimonidazole were significantly increased at the fundus and lesser curvature compared to the greater curvature in the operation group. CONCLUSION: Hypoxia PET imaging with 64Cu-ATSM can detect ischemia in a rat esophagectomy model. Further clinical studies are needed to verify whether hypoxia imaging may be useful in humans.

Dual optical biosensors for imaging microRNA-1 during myogenesis.

Dual optical microRNA (miRNA) imaging systems, bioluminescent reporter gene (a signal-off mechanism)- or fluorescent molecular beacon (MB) (a signal-on mechanism)-based miRNA imaging system, have individually allowed us to sense miRNA biogenesis in a noninvasive and iterative manner. Both of these imaging systems have shortcomings with respect to image quality. Therefore, we designed a dual optical imaging systems by simultaneous imaging of a miRNA-1 reporter gene (CMV/Gluc/3xPT_miR-1) and miRNA-1 MB in a single cell to overcome these limitations and used it to visualize miRNA-1, a highly expressed miRNA in cardiac and skeletal muscle. During myogenic differentiation of C2C12 cells, the bioluminescence intensity from CMV/Gluc/3xPT_miR-1 revealed a miRNA-1-dependent gradual decrease and the fluorescence intensity from miR-1 MB demonstrated a miRNA-1-dependent gradual increase both in vitro and in vivo. The dual miRNA-1 imaging systems, which provides the complementary imaging information about miRNA biogenesis, could be useful to sense miRNA expression during various biologic processes.

Molecular beacon-based bioimaging of multiple microRNAs during myogenesis.

MicroRNAs (miRNAs, miR) are associated with multiple cellular processes and diseases. Here, we designed fluorescent DNA probes composed of stem loop-structured DNA complementary to miRNAs and fluorophore-quencher pairs [molecular beacon (MB)] to simultaneously monitor the biogenesis of miR-206 and miR-26a, which are highly expressed during myogenic differentiation. C2C12 cells were transfected with an MB targeting miR-26a and containing a 6-FAM-BHQ1 pair (miRNA-26a MB) or an MB targeting miR-206 with a Texas Red-BHQ2 pair (miRNA-206 MB). In vitro and in vivo fluorescence analysis revealed that, only in differentiated single C2C12 cell, significantly increased fluorescence signals of miRNA-26a MB, miRNA-206 MB or simultaneous incubation of both beacons were detected due to the hybridization of miR-206 or miR-26a with their respective beacons, resulting in activation of fluorescence. Our MB-based miRNA imaging system may serve as a new imaging probe for monitoring multiple miRNAs during various cellular or disease processes associated with miRNAs.

The native metastability and misfolding of serine protease inhibitors.

The native metastability of serine protease inhibitors (serpins) is believed to facilitate the conformational change required for biological function. However, energetically unfavorable structural features that contribute to metastability of the native serpin conformation, such as buried polar groups, cavities, and over-packing of side-chains, also appear to hinder proper folding. Hence, folding of serpin polypeptides appears prone to error; in particular, the folding polypeptides are readily diverted toward a non-productive folding pathway culminating in a more stable but inactive conformation. In a survey of deficient serpin mutants, various folding defects, such as retarded protein folding, destabilized native conformation, and spontaneous conversion into more stable, inactive conformations such as the latent form and loop-sheet polymers, have been discovered.