Visualizing the cytosolic delivery of bioconjugated QDs into T cell lymphocytes

The aggregation state and endosomal trapping of engineered nanocarriers once internalized into cells remain poorly characterized. Here, we visualized the membrane penetrating dynamics of semiconductor quantum dots (QDs) into the cytosol of T cells on a single-cell and single-nanoparticle basis. We water solubilized CdSe/CdZnS QDs with polymer encapsulants functionalized with a cell-penetrating peptide composed of an Asp-Ser-Ser (DSS) repeat sequence. T cells tolerated the 24-h incubation with QDs at concentrations of 5 nM or lower. Single-particle imaging demonstrated that the number of internalized nanoparticles was dependent upon the concentration of the probes for both control (peptide-free) and DSS-QDs. DSS-QDs were mostly distributed as monomers, whereas the control QDs were aggregated into clusters. Single-particle tracking using total internal reflection and highly inclined illumination showed that DSS-QDs were stationary near the activating surface and mobile within the cytosol of the T cell. A correlation exhibited between the mobility and aggregation state of individual QD clusters, with monomeric DSS-QDs showing the highest mobility. In addition, monomeric DSS-QDs displayed much faster diffusion than the endosomes. A small-molecule endosome marker confirmed the absence of colocalization between endosomes and DSS-QDs, indicating their endosomal escape. The ability to deliver and track individual QDs in the cytosol of live T cells creates inroads for the optimization of drug delivery and gene therapy through the use of nanoparticles.