Avidity-Dependent Organ Selective Delivery of VCAM-Targeted Lipid Nanoparticles

Introduction: Conjugation of antibodies to the surface of nanoparticles is a widely studied approach to achieve organ- or cell-selective delivery. While most studies have focused on the relationship between an antibody and delivery to a single organ, we investigated the role of antibody conjugation density on targeting when multiple organs express high levels of the target molecule. Vascular Cell Adhesion Molecule-1 (VCAM) is primarily expressed in lymphoid tissue, notably in the spleen, but also has significant basal expression in vascular endothelial cells of the brain. Conjugation of monoclonal antibodies (mAb) targeted to VCAM introduces affinity for both VCAM itself as well as to cells expressing FcγR, namely those present in the reticuloendothelial system. We hypothesize that balancing VCAM- and Fc-dependent delivery mechanisms by tuning antibody conjugation density will allow precise control of delivery to the spleen and brain.

Materials and

Methods: Liposomes were formulated with an azide functionality to permit conjugation with DBCO-modified mAbs via strain-promoted alkyne-azide cycloaddition (click chemistry). Azide-liposomes were incubated with DBCO-mAb (VCAM or control IgG) at ratios of 0 – 200 mAb/liposome overnight at 37°C with rotation. Liposome size and polydispersity were assessed via dynamic light scattering. Conjugation efficiency was measured using gel filtration chromatography. Liposomes (radioisotope or fluorescent labeled) were intravenously injected into mice and pharmacokinetics (PK) and whole organ biodistribution were assessed by gamma counting. Cell specificity within organs was evaluated by flow cytometry and immunohistology. Herein, a specific coating density of liposome is designated as ‘liposome with # (VCAM or IgG)’.

Results, Conclusions, and Discussions: As we tuned the density of VCAM mAb on the liposome surface we observed: 1) increasing density led to decreased blood circulation and increased liver uptake; 2) increasing density led to increased brain targeting (0.05 to 1.7%ID/g); and, 3) splenic uptake peaked at 1 – 3 mAb/liposome and decreased to the level of untargeted control as density increased beyond that point (3 VCAM: 174%ID/g, 200 VCAM: 63%ID/g). To elucidate effects of Fc receptors, liposomes were formulated with 200 total antibody molecules/liposome in various ratios of VCAM/untargeted control. This study showed that high Fc density diverts VCAM-targeted liposomes away from the spleen, with 3 VCAM/197 IgG having uptake at the level of untargeted control (200 IgG). This did not hold true for brain targeting, as coating liposomes with 50 VCAM/150 IgG did not abrogate targeting compared to those coated with just 50 VCAM. Blocking of FcγRII/III by preinjection of an inhibitory antibody did not abrogate the superior targeting of liposomes with 3 VCAM mAb compared to higher densities completely. Further distribution studies revealed that at a density of 3 VCAM mAb/liposome, ~60% of liposomes in the spleen were localized to the interstitial space, and not internalized by cells. We hypothesize that the low coating density may eliminate CAM-mediated endocytosis by target cells.
Next, we performed a PK study of liposomes with 3, 25, and 200 VCAM mAb. For 3 VCAM liposomes, splenic uptake peaked at 1 hour and maintained above ~150% ID/g for at least 2 hours, allowing local release of dexamethasone into the splenic interstitial space. For 25 and 200 VCAM liposomes, similar brain uptake was observed at early time points. However, 25 VCAM liposomes had prolonged blood PK, allowing for sustained delivery to the brain, resulting in higher delivery of SOD to the brain endothelium.
In conclusion, we have identified 2 mechanisms for VCAM-targeted liposome delivery: (1) immediate brain targeting is correlated with VCAM mAb density alone; (2) splenic targeting is associated with both VCAM and Fc densities. By tuning VCAM and Fc on liposomes, we can achieve spleen or brain specific targeting for local drug delivery.

Acknowledgements (Optional): J.N. received funding from the American Heart Association (Grant 916172). P.M.G. received funding from the National Institutes of Health (R00HL153696).