Assessing Transduction Efficiency And Cell Targeting By Cell-penetrating Peptides In The Mouse Lung

Cell-penetrating peptides (CPPs) are short peptides (5-30 amino acids) capable of crossing cell membranes and delivering macromolecular cargoes without the need for transfection reagents. However, defining in vivo. cellular selectivity requires validation at the cell-type level. An integrated workflow is presented to assess the transduction efficiency of a Cy5.5-labeled cyclized CPP, cR11A, within alveolar epithelial type II (AT2) cells following retro-orbital intravenous injection in mice. Lungs were harvested at 15 or 60 min post-injection and analyzed using complementary readouts, including paired-fraction fluorescence-activated cell sorting (FACS) to isolate Cy5.5⁺ and Cy5.5⁻ lung single-cell suspensions followed by single-cell RNA sequencing, flow cytometry of lung single cells from Sftpc-CreERT2^(+/-);LSL-GFP^(+/-) AT2 reporter mice to quantify GFP⁺/Cy5.5⁺ events, and confocal z-stack imaging of lung cryosections to confirm colocalization of cR11A-Cy5.5 with AT2-associated signal. This protocol yields concordant evidence of cell-type engagement using multiple complementary readouts and provides a reproducible framework for evaluating cell-specific targeting of CPPs, with modifications for different target organs.