(J-383) Lymphatic endothelial cell (LEC)-targeting nanocarriers for modulating vaccine-induced immunity

Introduction:: Vaccine-induced immunity wanes over time due to declining antibody levels and memory cell numbers. Vaccines targeting lymphatic endothelial cells (LECs) may help address this limitation. LECs line lymphatic vessels and lymph nodes (LNs), and are uniquely poised to interact with antigens and immune cells. They can capture and present antigen to T cells, preferentially generating memory-like T cells¹. LECs also act as antigen reservoirs, storing antigen and releasing it over several weeks². These processes may improve vaccine-induced immunity by biasing memory T cell generation, and enhancing germinal centre reactions and antibody production by B cells. To leverage these processes, we developed LEC-targeting nanocarriers using a set of highly expressed LEC-specific surface receptors with well-characterized binding peptides, including VEGFR3, NRP2, STAB2, and CAV1^3-8. Here we show that these binding peptides may be formulated as targeting ligands that allow their incorporation into self-assembling lipid-based nanocarriers. We show that targeting of VEGFR3, the most LEC-specific receptor, allows preferential nanocarriers uptake by LECs in vitro and in vivo, and that in vivo uptake occurs in a VEGFR3-dependent manner.

Materials and Methods::

Targeting ligands (TLs) were synthsized using standard Fmoc solid phase peptide synthesis, and consisted of the binding peptide, an ethylene glycol spacer, and palmitic acid tail (Fig. 1A). TLs were purified by RP-HPLC and purity assessed by analytical HPLC and LCMS. Liposomes were prepared by thin film hydration and extrusion. DSPC, DSPE-PEG2000, TLs, cholesterol, and fluorescent dye were dissolved in 1:1 CHCl₃:MeOH, dried under vacuum, rehydrated in PBS, sonicated, and extruded through a 100 nm filter. Liposome size was measured by DLS. To test LEC uptake in vitro, fluorescently labelled liposomes were added to murine LECs for 24 hours, then analyzed by flow cytometry and microscopy. To test LEC uptake in vivo, mice were injected intradermally with fluorescent nanocarriers, and after 24 hours the draining LNs were analyzed by flow cytometry. To test uptake dependency on VEGFR3 expression, we also injected nanocarriers in VEGFR3^lox/loxProx1^Cre/ERT2 mice.

Results, Conclusions, and Discussions::

Binding peptides and control (non-binding) peptides for LEC-specific receptors have previously been identified^3-8. We synthesized targeting ligands (TLs) consisting of binding or control peptide, an ethylene glycol (EG) spacer, and palmitic acid tails for incorporation into PEGylated liposomes (Fig 1A). EG spacers have been shown to promote cell targeting by improving peptide display above the PEG corona of liposomes⁹. Liposomes were 40-50 nm in diameter, meeting the 10-100 nm range known to allow optimal lymphatic drainage¹⁰. In in vitro studies, we observed preferential uptake of VEGFR3-TL liposomes by LECs compared to Ctrl-TL liposomes. We also determined the optimal targeting ligand incorporation to be 5-10 mol% of total liposome lipid content in vitro. In vivo, we observed a twofold increase in both the proportion of LECs containing VEGFR3-TL liposomes (Fig. 1B), and the amount of VEGFR3-TL liposomes taken up, compared to Ctrl-TL liposomes. In addition, we did not observe any differences between VEGFR3-TL and Ctrl-TL liposome uptake in any other LN cell compartment. We also confirmed that 5-10 mol% of VEGFR3-TL was the optimal targeting ligand concentration in vivo. Finally, to determine if this preferential uptake occurs through VEGFR3 targeting, we used a conditional VEGFR3 knockout model and showed a reduction in LEC uptake of VEGFR3-TL liposomes.

Here we show the development of several LEC-specific receptor targeting ligands (TLs) to deliver nanocarriers to LECs. One highly LEC-specific targeted receptor, VEGFR3, allows preferential liposomal delivery to LECs and we show that this uptake is VEGFR3-mediated. We are evaluating if combinations of targeting ligands can be used to further improve LEC-specific nanocarrier delivery. While TLs were used in liposomes, these targeting strategies are applicable to other types of self-assembling nanocarriers such as polymersomes, which have also been used to encapsulate various antigens and adjuvant for vaccination. Future studies will use LEC-targeting nanocarriers for vaccine delivery, to understand the potential role LECs can play in improving the robustness of vaccine-induced immunity.

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