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    Drug Delivery

    Invasion of Jurkat Cells Conjugated with E-Selectin Modified Liposomes

    (L-442) Invasion of Jurkat Cells Conjugated with E-Selectin Modified Liposomes

    Saturday, October 14, 2023
    9:30 AM – 10:30 AM PDT
    Location: Exhibit Hall - Row L - Poster # 442
    Introduction::

    Introduction: Many groups have studied liposomes loaded with chemotherapeutics for drug delivery purposes. However, a major limitation is delivering and targeting those liposomes from the bloodstream into the tumor. Since T cells are major mediators of the adaptive immune response and have been of interest for immunoengineers as a mechanism to target tumors, several groups have proposed the use of T cells as transporters of drugs. Recent work has conjugated liposomes to white blood cells to transport the liposomes into the tumor. Building from this work, we engineered liposomes conjugated with E-selectin to attach them to T cells. Using shear stress, we attached the liposomes to Jurkat cells, an immortalized T cell line, to evaluate their ability to carry and remain bound to the liposomes through a collagen coated transwell, as a model of migration into the tumor.




    Materials and Methods::

    Liposome Fabrication and e-selectin conjugation: Liposomes were prepared via an extrusion method using a Mini-Extruder (Avanti Polar Lipids). A phosphorous lipid 18:1 DGS-NTA(Ni2+) (Avanti Polar Lipids) was included in the formulation for the conjugation with E-selectin that bears a his-tag tail. Preformed liposomes were incubated with E-selectin at 37°C for 1 h to complete the conjugation. The size distribution of liposomes was measured by dynamic light scattering (DLS) using a Zetasizer (Malvern Panalytical).




     




    Jurkat Cell Culture: Jurkat cells were cultured in complete media (RPMI with 10% FBS). Media was changed every 3 days. Cells were activated using anti-CD3/CD28 conjugated magnetic beads (DynabeadsTM), mixed, and cultured until E-selectin liposome attachment. Using a magnet, Dynabeads were pulled to the bottom of the tube so the cells could be separated from the beads.




     




    Liposome Conjugation to the Jurkat Cells: E-selectin coated liposomes were conjugated to Jurkat cells using fluid shear in a Brookfield cone-and-plate viscometer for 30 minutes at a rate of 188 s-1, and compared to those incubated in static conditions.




     




    Transwell: 6-well transwell inserts were coated with 150 L of 1 and 3 mg/mL rat tail type I collagen. 106 cells were plated in each transwell, and 0.5% serum media was placed in the transwell and 10% serum was used in the well to create a serum gradient. Cells migrated through the collagen and transwell for 24-48 hours. Invasive fractions calculated as (Migrated Cells)/(Migrated Cells + Non-migrated Cells).




    Results, Conclusions, and Discussions:: The hydrodynamic size of liposomes was measured to be ~110 nm in average (data not shown). The size increased to ~120 nm after the conjugation with E-selectin (data not shown). When attaching the liposomes to Jurkat cells, the static condition resulted in 12.7% attachment, which is significantly lower than the 37.6% attachment seen under shear conditions. (Figure 1A,C). Jurkats were then placed in transwells containing 1 and 3 mg/mL collagen.  Our data indicates that invasion is decreased in 3 mg/mL compared to 1 mg/mL collagen density (Figure 1B). To measure the ability of Jurkat cells to hold onto the liposome after invasion, we compared the liposome attachment per cell after 1mg/mL transwell migration and we see a statistically significant decrease in liposome attachment post collagen invasion (Figure 1C) and liposome fluorescent intensity (Figure 1D).



    Conclusions: E-selectin mediated conjugation of liposomes to T cells is enhanced by shear stress exposure.  When then induced invasion through collagen using a serum gradient, migration is reduced in higher density collagen compared to lower density, and we observed lower liposome attachment and fluorescent intensity of the liposomes post collagen trans-migration.




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