Engineering Fusogenic Lipid-Patched Cell Carriers for Lymphatic Drug Delivery 

• Aaron Maniyatte, Biomedical Engineering, University of Delaware

• Jason Gleghorn, Biomedical Engineering, University of Delaware

Lymph nodes are frequent sites of cancer metastasis, but drug delivery into the LN lobule is restricted by high endothelial venules (HEVs) that tightly regulate what crosses into the tissue1. Nanoparticle carriers delivered intravenously or subcutaneously have improved targeting to some degree, but they suffer from low drug-carrying capacity, poor accumulation at the node, and limited reach beyond a handful of superficial nodes2-3. T cells, a common immune cell, cross into lymph nodes routinely by virtue of their membrane properties. Building on prior work with a T cell-mimetic microparticle platform that improved LN drug delivery but faced durability and immunocompatibility limitations, we developed a carrier that patches T-cell membrane material onto red blood cells (RBCs). Fusogenic liposomes were made by combining the cationic lipid DOTAP with membranes isolated from Jurkat T cells, using freeze-thaw cycling to drive fusion and extrusion to control particle size. Fusion was confirmed by fluorescence resonance energy transfer (FRET), which showed roughly 37% dequenching consistent with lipid mixing, and dynamic light scattering showed a shift in particle size between that of pure DOTAP and pure Jurkat membrane (JM) liposomes consistent with fusion. These liposomes were then patched onto RBCs through electrostatic interaction between the cationic liposome surface and the anionic RBC membrane. Fluorescence and confocal microscopy confirmed patching of both synthetic and JM liposomes, and CD3 staining verified that T-cell membrane components were retained on the RBC surface. A liposome to RBC ratio of 1000:1 produced the most consistent surface coverage. Additionally, the loading of the chemotherapeutic doxorubicin (DOX) into the RBC was done through diffusion. DOX loading was confirmed through fluorescent microscopy and plate reader measurements. These results establish that RBCs can be feasibly engineered to carry T-cell membrane patches. 

References:

1. Lan, HR., Zhang, YN., Han, YJ. et al. Multifunctional nanocarriers for targeted drug delivery and diagnostic applications of lymph nodes metastasis: a review of recent trends and future perspectives. J Nanobiotechnol 21, 247 (2023). https://doi.org/10.1186/s12951-023-01990-4
2. Trac N, Chung EJ. Overcoming physiological barriers by nanoparticles for intravenous drug delivery to the lymph nodes. Exp Biol Med (Maywood). 2021;246(22):2358-2371. doi:10.1177/15353702211010762
3. Schudel, A., Francis, D.M. & Thomas, S.N. Material design for lymph node drug delivery. Nat Rev Mater 4, 415–428 (2019). https://doi.org/10.1038/s41578-019-0110-7