Probing the Immunogenicity of and Functionalizing Erythrocyte-Derived Optical Particles

There has been a recent increase in the design of delivery vehicles fabricated from cell membranes in order to equip systems with immune evasion capabilities. The membranes of erythrocytes have been used to camouflage various cargos to reduce their immune system uptake. We have developed erythrocyte-derived optical particles loaded with the FDA-approved near-infrared (NIR) dye, indocyanine green (ICG). We refer to these constructs as NIR erythrocyte-derived transducers (NETs). These NETs can be excited by NIR light, resulting in the generation of heat, fluorescence, and reactive oxygen species (ROS). We review the current field of light-based erythrocyte-derived particles for imaging and therapeutic applications. Next, we examine the acute innate immune response to micro- and nano-sized NETs (µNETs and nNETs, respectively) by measuring the cytokine production in response to these particles at different doses, and at different times post-injection. We look at the effect of functionalizing the nNETs with clinically relevant targeting moieties and the effect of a second injection of µNETs or nNETs one week after the first injection. The results show that up to 6 hours post-injection NETs do not induce the production of inflammatory cytokines to the same extent as a positive control injection of lipopolysaccharides. Functionalizing the nNETs was associated with a decrease in the cytokine response, and in general there were no significant differences between a single injection and dual injections of the µNETs or nNETs. These results show the potential for NETs as non-immunogenic delivery vehicles for ICG. We also investigate the targeting capacity of nano-sized NETs functionalized with the ligand binding domain of erythropoietin-production human hepatocellular receptor (Eph) B1. We show that these functionalized NETs (F-NETs) are able to target cells that over-express ephrin-B2 ligands. F-NETs can therefore be used to target diseased cell states characterized by the over-expression of ephrin-B2 for phototherapy. Finally, we demonstrate the successful functionalization of nano-sized NETs with a peptide that binds to EphA2, and is co-loaded with the chemotherapeutic Taxol in addition to ICG. Our results show the particles are able to target breast cancer cells expressing the EphA2 receptor, and facilitate receptor degradation upon binding to the cells