A Quick Reverse Transfection and Dual-Imaging Protocol for Simultaneous Viability and Oligonucleotide Delivery Assessment in 4T1 Cells

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Abstract
The 4T1 cell line is a widely used model for triple-negative breast cancer (TNBC) research due to its high metastatic potential and resemblance to clinical progression. However, its biological characteristics often limit the efficiency and reproducibility of conventional nucleic acid delivery protocols. Here, we present an optimized non-viral delivery framework for efficient cell-associated uptake in 4T1 cells. Five carrier systems—Xfect RNA polymer (Xfect), polyethyleneimine (PEI), chitosan (CHI), and gold nanoparticle conjugates (AuPEI and AuCHI)—were systematically evaluated using both standard (adherent) and reverse (suspension) delivery approaches. A fluorescently labeled oligonucleotide (5′-TYE563) served as a reporter cargo at final concentrations of 5 nM and 10 nM, comparing complexation times of 15 s and 15 min. The proposed protocol introduces a rapid complexation strategy combined with reverse delivery, significantly reducing preparation time while enhancing cell-associated uptake. Under optimized conditions (10 nM, 15 s, reverse), Xfect achieved near-complete cellular uptake with minimal cytotoxicity. Crucially, the colocalization of dual-fluorescence signals enabled the simultaneous validation of metabolic viability and internalized cargo within a single field of view, bypassing the need for independent bulk cytotoxicity assays. This methodology provides a rapid, reproducible, and cost-effective framework to standardize early-stage delivery conditions for difficult-to-transfect cell lines such as 4T1.

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