Megh Bhatt
New York University
Dual-Modality Labeling of Collagen Type I Binding Thermo-Responsive Assembled Protein for Imaging Fibrosis in Metabolic Dysfunction-Associated Steatohepatitis (MASH)
Molecular imaging tools capable of in vivo visualization enable real-time, high-resolution observation of cellular and molecular interactions. Here, we advance a self-assembling protein nanomicelle, collagen type I binding thermo-responsive assembled protein (Col1-TRAP), for noninvasive imaging of metabolic dysfunction-associated steatohepatitis (MASH). Col1-TRAP exhibits high specificity for type I collagen with nanomolar affinity. Building on prior near-infrared Col1-TRAP studies, we develop dual-modality radiometal labeling strategies for magnetic resonance imaging (MRI) and single photon emission computed tomography (SPECT). For gadolinium labeling, the N-terminal methionine was replaced with azidohomoalanine (Aha) to enable click conjugation of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-PEG5-dibenzocyclooctyne (DOTA-PEG5-DBCO), followed by gadolinium incorporation. DOTA conjugation and gadolinium labeling are confirmed by ultraviolet-visible spectroscopy (UV-Vis), matrix-assisted laser desorption ionization (MALDI), and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). For technetium-99m labeling, Col1-TRAP is labeled using technetium tricarbonyl and confirmed by SDS-PAGE, instant thin-layer chromatography (iTLC), and TLC imaging. These results support the development of Col1-TRAP as a dual-modality molecular imaging probe with strong potential for the early detection and management of fibrosis in MASH.
Molecular imaging tools capable of in vivo visualization enable real-time, high-resolution observation of cellular and molecular interactions. Here, we advance a self-assembling protein nanomicelle, collagen type I binding thermo-responsive assembled protein (Col1-TRAP), for noninvasive imaging of metabolic dysfunction-associated steatohepatitis (MASH). Col1-TRAP exhibits high specificity for type I collagen with nanomolar affinity. Building on prior near-infrared Col1-TRAP studies, we develop dual-modality radiometal labeling strategies for magnetic resonance imaging (MRI) and single photon emission computed tomography (SPECT). For gadolinium labeling, the N-terminal methionine was replaced with azidohomoalanine (Aha) to enable click conjugation of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-PEG5-dibenzocyclooctyne (DOTA-PEG5-DBCO), followed by gadolinium incorporation. DOTA conjugation and gadolinium labeling are confirmed by ultraviolet-visible spectroscopy (UV-Vis), matrix-assisted laser desorption ionization (MALDI), and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). For technetium-99m labeling, Col1-TRAP is labeled using technetium tricarbonyl and confirmed by SDS-PAGE, instant thin-layer chromatography (iTLC), and TLC imaging. These results support the development of Col1-TRAP as a dual-modality molecular imaging probe with strong potential for the early detection and management of fibrosis in MASH.
