Targeted Liposome Delivery to Plasmacytoid Dendritic Cells Using Noncompetitive Transferrin Receptor Ligands

Introduction:: Plasmacytoid dendritic cells (pDCs) are the most potent producers of interferon alpha in the body, and a crucial component of antiviral immune response and development of cellular and humoral responses to new antigens. Further, dysregulation of pDC function is associated with autoimmune diseases such as systemic lupus erythematosus (SLE). Despite their centrality to immune response, previous attempts to target delivery to pDCs have had limitations such as relying on nonspecific receptors, or on endocytosis through non-natural mechanisms. Recent work has demonstrated that pDCs uniquely and homogenously express high levels of CD71, an endocytosing iron transporter. While this provides a potential mechanism to target specific delivery to pDCs, transferrin, the natural ligand of CD71 is ubiquitous and will likely dominate binding at a competitive site. The T7 peptide (HAIYPRH) is a noncompetitive ligand for CD71. By modifying this peptide to incorporate an N-terminal cysteine, it can be ligated to the surface of maleimide functionalized liposomes. These liposomes effectively target plasmacytoid dendritic cells in lymphoid organs better than control liposomes functionalized with plain cysteine rather than cys-T7 in vitro and in vivo.


Materials and Methods:: Liposomes were synthesized with a ratio of 6:3:1 of DPPC:DC-cholesterol:DSPE-PEG-Maleimide with the addition of 100 ug of DiD per 10 mg of lipid using thin-film rehydration with DI water and extruded to a diameter of 200 nm, which was confirmed by dynamic light scattering. Liposomes were functionalized to either cys-T7 or cysteine using a maleimide-thiol ligation reaction in the presence of excess peptide, which was subsequently removed by dialysis. To test the ability of cys-T7 liposomes to target pDCs in vitro, single cell splenocyte suspensions were treated with either cys-T7 liposomes, cysteine liposomes or were left untreated for 1, 4, or 16 hours. After treatment the suspensions were analyzed using flow cytometry to determine the fraction of DiD⁺ cells among different populations. To determine targeting precision in vivo, mice were injected with either cys-T7 liposomes, cysteine liposomes or sterile PBS into both hind footpads and after 24 hours the spleens, and livers of those mice were collected and analyzed by flow cytometry using a similar method to that for in vitro treated cells. To confirm that T7 liposomes drain to the lymphatic vasculature, cys-T7 or cysteine labeled liposomes were synthesized labeled with cy7 rather than DiD and were injected into the right footpad of mice, while the left footpad was injected with PBS. The distribution of Cy7 was then measured at time points ranging up to 3 days post injection using whole-animal imaging techniques. N=3 biological replicates were used for experiments.


Results, Conclusions, and Discussions:: DLS data confirmed that the liposomes were synthesized with approximately 200 nm diameter. Based on expression of B220, CD317, Siglec-H, CD11c, CD11b and GR-1 five leukocyte populations were resolved: B-cells (B220⁺, CD11c^-), conventional DCs (B220^-, CD11c⁺), pDCs (B220⁺, CD11c^int, CD317⁺, Siglec-H⁺), Macrophages(B220^-,CD11c^-, CD11b⁺,GR-1^-) and Neutrophils(B220^-,CD11c^-, CD11b⁺,GR-1⁺). In vitro after 1 hour both pDCs and cDCs took up cys-T7 liposomes as a similar rate with only minimal uptake of cys liposomes. After 4 and 16 hours the uptake of T7 liposomes increased in pDCs but not cDCs leading to significantly greater uptake by pDCs than cDCs of T7 liposomes with no significant change in uptake of cysteine liposomes. In vivo, although cys liposomes were taken up at greater rates by all cell types they did not target pDCs very precisely but rather were taken up at high levels by several different leukocyte populations in the spleen. By contrast, T7 labeled liposomes were only taken up by pDCs at high levels among the observed splenocyte cell types, with all other cell types including cDCs and macrophages having low uptake. Together these indicate that T7 conjugated liposomes are more precisely targeted to uptake by pDCs than control liposomes and are overall less likely to be taken up by off-target cells. In the liver all analyzed cell types took up cysteine conjugated liposomes at higher rates than t7 liposomes. This effect was particularly pronounced in neutrophils and macrophages, both of which had far greater uptake of cysteine liposomes than T7 ones. This indicates that T7 liposomes are less likely to be cleared by the body than cysteine liposomes and particularly less likely to be taken up by off-target cell types during this clearance, reinforcing the interpretation that T7 can effectively target liposome delivery to pDCs in vivo. Whole-animal imaging demonstrated that cysteine and T7 liposomes had similar biodistribution patterns, indicating that the presence of CD71-high cells in adipose and endothelial tissue does not substantially impair the ability of T7 liposomes to drain into the lymphatic vasculature.