A Custom-Engineered Water-Soluble Peptide-Based Macrocyclic Polynuclear Gd ³⁺ -Chelated Paramagnetic Multimodal Contrast Agent for Mitochondria-Targeted Fluorescence Imaging Integrated with MRI

The strategic development of cellular organelle-targeted multimodal imaging probes, which integrate fluorescent biomarkers with magnetic resonance (MR) contrast agents to harvest synergistic imaging information, holds transformative potential in biomedical diagnostics. In this pursuit, a custom-engineered peptide-based multimodal contrast agent, featuring the integration of an orange-red emissive synthetic hemicyanine (HCy) fluorescent dye with a preorganized macrocyclic polynuclear tri-Lys(DOTA-Gd³⁺) chelated MRI contrast agent, HCy-Lys(DOTA-Gd³⁺)-Lys(DOTA-Gd³⁺)-Lys(DOTA-Gd³⁺)-Gly, has been meticulously synthesized via the Fmoc-solid phase peptide synthesis methodology on Wang resin. This rational design paradigm harnesses synergistic advantages to transcend the inherent boundaries of traditional single-modality imaging methods. This paramagnetic peptide-based multimodal contrast agent, HCy-Lys(DOTA-Gd³⁺)-Lys(DOTA-Gd³⁺)-Lys(DOTA-Gd³⁺)-Gly, exhibits a substantial magnetic moment (7.54 μ_B per Gd³⁺) alongside pronounced magnetic susceptibility. The peptide-multimodal contrast agent HCy-Lys(DOTA-Gd³⁺)-Lys(DOTA-Gd³⁺)-Lys(DOTA-Gd³⁺)-Gly has been successfully employed for living cell mitochondrial-targeted confocal laser scanning microscopy, three-dimensional imaging, real-time dynamic monitoring, and multicolor imaging, in conjunction with <i>T</i>₁-weighted MRI, demonstrating a notably high longitudinal relaxivity (<i>r</i>₁) of 7.65 mM^-1 s^-1. The Lys amino acid-based preorganized macrocyclic scaffold confers exceptional kinetic stability to Gd³⁺ chelation, while the polynuclear architecture enhances MRI contrast. The water-soluble, biocompatible, noncytotoxic, bright orange-red emissive, paramagnetic, preorganized macrocyclic polynuclear tri-Lys(DOTA-Gd³⁺) chelates embodied in HCy-Lys(DOTA-Gd³⁺)-Lys(DOTA-Gd³⁺)-Lys(DOTA-Gd³⁺)-Gly represent a promising multifunctional contrast agent for live-cell mitochondria-targeted multimodal imaging techniques.