Chain-Length-Optimized 1,8-Naphthalimide–Ibuprofen Fluorescent Probe for HSA Binding and Cellular Imaging

Fluorescent probes based on 1,8-naphthalimide (NI) are attractive molecular tools for sensing and imaging biomacromolecules in biological systems. In this study, a matched series of NI derivatives bearing H, n-hexyl, or n-octyl N-imide substituents and terminal acrylamide or propiolamide groups was designed and synthesized to empirically evaluate how these structural variables affect cell viability and imaging performance. Within this compound series, MTT assays across six cell lines generally yielded higher cell-viability values for the acrylamide-containing probes than for their matched propiolamide analogues, and the octyl-substituted Probe D3 provided a favorable balance between MTT-based cell viability and intracellular fluorescence. Probe D3 exhibited green fluorescence with an emission maximum at approximately 550–560 nm and was compatible with confocal cellular imaging. To introduce HSA-binding capability into the optimized NI scaffold, an ibuprofen-functionalized analogue, Probe D3–ibuprofen, was prepared based on the known binding preference of ibuprofen for Sudlow’s site II of human serum albumin (HSA). HSA caused modest changes in the absorption and fluorescence spectra of Probe D3–ibuprofen, while microscale thermophoresis confirmed a micromolar interaction with an apparent Kd value of 14.22 ± 4.43 μM. Quantitative image analysis showed a higher probe-to-DAPI fluorescence intensity ratio in HeLa cells treated with Probe D3–ibuprofen/HSA than in cells treated with the probe alone, supporting its potential for HSA-associated cellular imaging. This work provides a chain-length-guided strategy for engineering NI-based fluorescent probes for protein-oriented cellular imaging.