Evaluation of Lipid-Based Transfection in Primary Monocytes Within an Ex Vivo Whole-Blood Model

Transfection is a fundamental method in biomedical research to study intracellular molecular mechanisms by manipulating target protein expression. Various methods have been developed to deliver nucleic acids into the cells of interest <i>in vitro</i>, with chemical transfection by cationic lipids being the most widely used for RNA interference (RNAi). However, translating these <i>in vitro</i> results into <i>in vivo</i> remains a significant challenge. In this study, we established an <i>ex vivo</i> transfection model using cationic lipids in human whole blood. Three different lipid-based reagents were evaluated regarding toxicity, transfection efficiency, and immunogenicity across leukocyte populations using spectral flow cytometry. CD14⁺ monocytes were identified as the primary population to be transfected by cationic lipids in whole blood. To assess immunogenicity, the monocyte-specific activation markers CD80 and human leukocyte antigen DR isotype (HLA-DR) were analyzed upon transfection. Our results demonstrated that Lipofectamine RNAiMAX outperforms the other two reagents, showing low toxicity and high transfection efficiency in combination with a minimal potential for monocyte activation. Functional knockdown experiments using siRNA targeting <i>CIITA</i> and the microRNA mir-3972 targeting <i>HLA-DRA</i> showed dose-dependent suppression in HLA-DR expression. This study provides the framework for preliminary testing of RNAi in a physiologically relevant <i>ex vivo</i> model, enabling assessment of key endpoints such as toxicity, transfection efficiency, and immune activation potential of gene delivery systems.