A novel PolyAr87-based cell transfection protocol for nanobody expression optimized via a targeted design of transfection approach.

Introduction: Transfection is a fundamental technique for introducing foreign nucleic acids into eukaryotic cells, widely used in biotechnology for recombinant protein expression. Nanobodies fused to enzymes are key reagents in the development of diagnostic tests such as ELISA. Here, we optimized a novel, low-cost, ready-to-use linear polyethyleneimine (PEI)-based transfection reagent, PolyAr87, for the efficient delivery of a plasmid encoding a nanobody-HRP fusion protein into HEK293T cells. Results: When compared with other commonly used transfection reagents-including branched and linear PEI and FuGene® 6-PolyAr87 showed superior performance over PEI powders and comparable efficacy to FuGene® 6 at a substantially lower cost. Then, using a Design of Experiments (DoE) approach, specifically the Design of Transfection (DoT) model, we applied a two-phase optimization strategy comprising a Full Factorial Design (FFD) and a Box-Behnken Design (BBD) to identify and fine-tune key factors affecting transfection efficiency. PolyAr87 concentration and DNA concentration were found to significantly influence outcomes, with optimal efficiency achieved at 1.75 μg/mL of DNA and 5.0 μg/ mL of PolyAr87. Discussion: Model validation demonstrated strong predictive power and reproducibility. These findings confirm both the effectiveness of PolyAr87 as a cost-efficient transfection reagent and the utility of DoT-based optimization for enhancing gene delivery protocols in mammalian cell systems.