Intracellular delivery of more than one protein with spatio-temporal control

Transient, non-integrative modulation of cell function by intracellular delivery of proteins has high potential in cellular reprogramming, gene editing and therapeutic medicine applications. Unfortunately, the capacity to deliver multiple proteins intracellularly with temporal and spatial control has not been demonstrated. Here, we report a near infrared (NIR) laser-activatable nanomaterial that allows for precise control over the release of two proteins from a single nanomaterial. The nanomaterial is formed by gold nanorods (AuNRs) modified with single stranded DNA (ssDNA) to which complementary DNA-conjugated proteins are hybridized. Using DNA strands with distinct melting temperatures we are able to control independently the release of each protein with a laser using the same wavelength but with different powers. Studies in mammalian cells show that AuNRs conjugated with proteins are internalized by endocytosis and NIR laser irradiation promotes endosomal escape and the release of the proteins from the AuNRs simultaneously. Our results further demonstrate the feasibility of protein release from a carrier that has been accumulated within the cell up to 1 day while maintaining its activity.