ORIGINAL RESEARCH article
Front. Bioeng. Biotechnol.
Sec. Bioprocess Engineering
A novel PolyAr87-based cell transfection protocol for nanobody expression optimized via a targeted Design of Transfection approach
Provisionally accepted
Lautaro Fidel Bracco1*
Giovanna L. Liguori2*
ANTONELLA LANATI3
Juan Manuel Lázaro-Martínez4,5
Mariela Bollini6
Leonardo Poggio7
Marina Bok7Lorena Itatí Ibañez8
Viviana Parreño7- 1Centro de Investigación en Ciencias Veterinarias y Agronómicas (CICVYA), INTA, Hurlingham, Argentina
- 2Istituto di Genetica e Biofisica Adriano Buzzati Traverso Consiglio Nazionale delle Ricerche, Naples, Italy
- 3Valore Qualità, . Pavia, Italy
- 4Universidad de Buenos Aires Facultad de Farmacia y Bioquimica, Buenos Aires, Argentina
- 5CONICET-Universidad de Buenos Aires, Instituto de Química y Metabolismo del Fármaco (IQUIMEFA), Ciudad Autónoma de Buenos Aires, Argentina
- 6Centro de Investigaciones en Bionanociencias (CIBION – CONICET), Polo Científico Tecnológico, Ciudad Autónoma de Buenos Aires, Argentina
- 7Instituto de Virología e Innovación Tecnológica, IVIT, CONICET-INTA, Hurlingham, Argentina
- 8Instituto de Quimica Fisica de los Materiales Medio Ambiente y Energia, Buenos Aires, Argentina
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Transfection is a fundamental technique for introducing foreign nucleic acids into eukaryotic cells, widely used in biotechnology for recombinant protein expression. 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. Nanobodies fused to enzymes are key reagents in the development of diagnostic tests such as ELISA. 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, DNA concentration, and complexation time were found to significantly influence outcomes, with optimal efficiency achieved at 1.75 µg/mL of DNA and 5.0 µg/mL of PolyAr87. 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.
Keywords: Design of Experiment - DoE, HEK293, Method Optimization, PolyAR reagent, Transfection
Received: 07 Oct 2025; Accepted: 19 Dec 2025.
Copyright: © 2025 Bracco, Liguori, LANATI, Lázaro-Martínez, Bollini, Poggio, Bok, Ibañez and Parreño. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
* Correspondence:
Lautaro Fidel Bracco
Giovanna L. Liguori
Disclaimer: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher.