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.

EGFRvIII is expressed only in tumor cells and strongly in glioblastoma and is considered a promising target in cancer diagnosis and therapy. Aptamers are synthetic single-stranded oligonucleotides that bind to biochemical target molecules with high binding affinity and specificity. This study examined the potential of the ⁶⁸Ga-NOTA-EGFRvIII aptamer as a nuclear imaging probe for visualizing EGFRvIII-expressing glioblastoma by positron emission tomography (PET). EGFRvIII aptamer was selected using the SELEX technology, and flow cytometry and fluorescence microscopy verified the high binding affinity to EGFRvIII positive U87MG vIII 4.12 glioma cells but not to EGFRvIII negative U87MG cells. The EGFRvIII aptamer was conjugated with a chelator (1,4,7-triazanonane-1,4,7-triyl)triacetic acid (NOTA) for ⁶⁸Ga-labeling. The ⁶⁸Ga-NOTA-EGFRvIII aptamer was prepared using the preconcentration-based labeling method with a high radiolabeling yield at room temperature. Ex vivo biodistribution analyses confirmed the significantly higher tumor uptake of the ⁶⁸Ga-NOTA-EGFRvIII aptamer in EGFRvIII-expressing xenograft tumors than that in EGFRvIII negative tumors, confirming the specific tumor uptake of the ⁶⁸Ga-NOTA-EGFRvIII aptamer in vivo. PET imaging studies revealed a high retention rate of the ⁶⁸Ga-NOTA-EGFRvIII aptamer in U87MG vIII 4.12 tumors but only low uptake levels in U87-MG tumors, suggesting that the ⁶⁸Ga-NOTA-EGFRvIII aptamer may be used as a PET imaging agent for EGFRvIII-expressing glioblastoma.

Background/Objectives: Pulmonary fibrosis is a progressive and fatal lung disease with limited diagnostic and therapeutic options. Fibroblast activation protein (FAP) has emerged as a promising molecular imaging target for the non-invasive assessment of fibrotic activity. This study aimed to evaluate the diagnostic feasibility of [⁶⁸Ga]Ga-FAP inhibitor (FAPI) and [¹⁸F]fluorodeoxyglucose ([¹⁸F]FDG) positron emission tomography (PET) for imaging pulmonary fibrosis in a mouse model. Methods: A pulmonary fibrosis model was established by intratracheal administration of polyhexamethylene guanidine-phosphate (PHMG-p) to C57BL/6 mice. Fibrosis severity was quantified by the Ashcroft scoring system using hematoxylin and eosin and Masson’s trichrome staining and evaluated by computed tomography (CT) imaging at 7, 14, and 21 days after PHMG-p exposure. PET imaging was performed, and ex vivo biodistribution was assessed after injection of [⁶⁸Ga]Ga-FAPI-04 and [¹⁸F]FDG. Results: Histological analysis and Ashcroft scoring revealed greater fibrosis severity in the PHMG-p-treated group. Western blot analysis demonstrated upregulation of FAP expression after PHMG-p exposure. CT showed increased mean lung density, while [⁶⁸Ga]Ga-FAPI-04 PET revealed significantly elevated pulmonary uptake of [⁶⁸Ga]Ga-FAPI-04 in the PHMG-p-treated group compared with the controls. [¹⁸F]FDG PET imaging also showed higher uptake of [¹⁸F]FDG in the PHMG-p-treated group than in the controls. Ex vivo biodistribution confirmed greater [⁶⁸Ga]Ga-FAPI-04 accumulation in the lungs of PHMG-p-treated mice. Conclusions: [⁶⁸Ga]Ga-FAPI-04 PET serves as a sensitive imaging biomarker for evaluation of fibrotic activity in PHMG-p-induced pulmonary fibrosis and complements [¹⁸F]FDG PET for assessing disease progression and therapeutic response.