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 in vitro, with chemical transfection by cationic lipids being the most widely used for RNA interference (RNAi). However, translating these in vitro results into in vivo remains a significant challenge. In this study, we established an ex vivo 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 CIITA and the microRNA mir-3972 targeting HLA-DRA showed dose-dependent suppression in HLA-DR expression. This study provides the framework for preliminary testing of RNAi in a physiologically relevant ex vivo model, enabling assessment of key endpoints such as toxicity, transfection efficiency, and immune activation potential of gene delivery systems.