An osteocyte-specific targeted delivery system has the potential to improve the efficacy and safety profile of therapeutic candidates for osteoporosis and other skeletal diseases.
Poor cytosolic delivery is a major rate-limiting step in delivery of biological therapies. For effective intracellular drug-delivery, the delivery system should specifically bind to a cell membrane-bound protein and internalise after binding. Although our knowledge of osteocytes and the factors that they release has grown over the last decades, little is known about their ability to internalise materials.
To quantify the binding, internalisation, and recycling of materials delivered to osteocytes, we applied an innovative sensor technology developed by our group known as SHIP (Specific Hybridization Internalization Probe) to osteocyte-like cells. We measured the internalisation kinetics of antibodies against transferrin receptor (TfR), which mediates cellular iron uptake, and E11, which is an osteocyte-specific transmembrane glycoprotein, in undifferentiated (stromal cells) and differentiated (osteocyte-like) Kusa 4b10 cells.
Differentiated Kusa 4b10 cells not only expressed higher E11 levels, but they also had higher uptake (% of total associated antibody - TAA) of the anti-E11 antibody than undifferentiated cells at 0.5, 1, and 2 h after incubation. Differentiated cells also had significantly greater internalisation of anti-TfR antibody (76% of TAA) compared to undifferentiated cells (36% of TAA) as early as 10 minutes after incubation. Only a limited portion of anti-TfR (<20% of TAA) and anti-E11 (<10% of TAA) antibodies were recycled to the surface of differentiated cells during the 40 minutes after initial internalisation, suggesting both antibodies are trafficked into non-recycling endosomes.
Our innovative molecular sensor, SHIP, enables high-throughput and quantitative analysis of the uptake of therapeutics in osteocytes. Our data indicate that osteocytes have greater and more rapid internalisation of anti-E11 antibody than undifferentiated cells without recycling, suggesting that E11 is a promising approach to target drug delivery to osteocytes.