Professor Georgia Institute of Technology Atlanta, Georgia, United States
Introduction:: The lymphatic vasculature provides essential physiologic support to a variety of tissues throughout the body through its roles in maintaining fluid balance, providing a route for protein and wast clearance from the interstitium, facilitating immune cell and antigen trafficking to lymph nodes, and absorbing dietary lipid from the intestine. The functions are achieved through specialized structural features of initial lymphatics, which allow for the entry of large molecules and cells, as well as specialized collecting lymphatics, which through intrinsic contractility provide the pumping working necessary to move fluid against adverse pressure gradients. While significant advances have been made in the last two decades in our understanding of many of the molecular mechanism underlying lymphatic formation and function, translating this knowledge into functional therapies has been limited. I will summarize two different technologies developed by our research lab and network of collaborators that deliver mRNA to lymphatic endothelial cells, and deliver small molecule calcium channel agonists to lymphatics to enhance lymphatic pumping.
Materials and Methods:: S-(−)-Bay K8644 (BayK), a small-molecule agonist of L-type calcium channels was loaded into plutonic-propylene sulfide based nanoparticles of 30 nm in diameter, that have previously been shown to have high lymphatic targeting when delivered intradermally in vivo. These BayK-NP were tested on isolated vessels, in healthy mice, and in a mouse model of lymphedema to quantify their efficacy on lymphatic contractility. In a second study, a large in vivo screen of over 100 different lipid nanoparticle formations were tested in the mouse to screen for LNPs that would deliver mRNA to lymphatic endothelial cells in vivo. Each formulation was loaded with a unique DNA barcode and mRNA that encoded for an exogenous protein that would be synthesized and expressed on the cell surface. Flow cytometry was utilized to sort on LEC in the lymph node that expressed the exogenous protein and cells underwent sequencing to identify DNA barcodes associated with these cell populations. LEC-targeting LNP formulations were then further tested to quantify their enhancement of mRNA delivery to LEC in collecting lymphatic vessels. Lastly, LNP were loaded with mRNA encoding for the lymphatic specific growth factor VEGF-C, and delivered intradermally in a mouse model of lymphatic injury.
Results, Conclusions, and Discussions:: When formulated within lymph-draining nanoparticles (NPs), BayK acutely improved lymphatic vessel function, affects not seen from treatment with BayK in its free form. By preventing rapid drug access to the circulation, NP formulation also reduced BayK’s dose-limiting side effects. When applied to a mouse model of lymphedema, treatment with BayK formulated in lymph-draining NPs, but not free BayK, improved pumping pressure generated by intact lymphatic vessels and tissue remodeling associated with the pathology.
Upon screening 100 different LNP formulations through intradermal injection in vivo, several unique formulations, including LNP7, were identified that were highly effective at enhancing mRNA delivery to LEC. When compared to a standard commercially available LNP formulation of similar size, made with the lipid MC3, LNP7 showed a 3-fold increase in mRNA delivery to LEC in the draining lymph node and in the collecting lymphatic vessel. Lastly, when loaded with VEGF-C mRNA, LNP7 resulted in targeted, enhanced VEGF-C production in vivo and improved lymphatic drainage in an injury model.
This work reveals the utility of two different lymph-targeting NP platforms to pharmacologically enhance lymphatic pumping in vivo with small molecules and to deliver mRNA to LEC in vivo and highlights promising new approaches to treating lymphatic dysfunction.
