DNA aptamer-mediated cell targeting

Abstract

One important issue using cells as therapeutics is targeted delivery. Engineering cell surfaces to improve delivery efficiency is thus of great interest. Here we report a simple, efficient and effective way to modify the cell surface with target-specific ligands, i.e., DNA aptamers, while minimizing the effects on the modified cells. We demonstrated that after incubating with lipo-aptamer probes (shown in expansion), immune cells (red) recognize cancer cells (blue) in the cell mixture, and kill cancer cells.

Figure 1

Modification of cell membranes with aptamers. (a) Schematic representation of targeting cancer cells (blue) with aptamer-modified immune cells (red). After incubating with lipo-aptamer probes (shown in expansion), immune cells recognize cancer cells in the cell mixture, and kill cancer cells. (b) Confocal microscope image of lipo-Lib-TMR-treated CEM cells. Red fluorescent probes were found only on the cell surface. Scale bar: 10 µm. (c) CEM cells were treated with lipo-Lib-FITC for different time intervals in cell culture medium. The maximum insertion was reached in 2 hours. (d) Ramos cells spontaneously aggregate after treatment with lipo-TD05-TMR. Scale bar: 100 µm. (e) Control experiments showed no assembly when Ramos cells were treated with lipo-lib-TMR. Scale bar: 100 µm.

Figure 2

Aptamer-directed assembly and disassembly of cell aggregates and quantitative analysis of aggregation. (a) 1:10 mixture of lipo-sgc8-TMR-modified Ramos and CEM cells. Scale bar: 100 µm. (b) Discrete cell clusters at higher magnification. Scale bar: 20 µm. (c) Confocal micrograph of selective cell assembly. Lipo-TD05-TMR-modified K562 cells (red fluorescence) were incubated with CellTracker Green-labeled Ramos cells (green fluorescence) and unlabeled CEM cells. No mismatched cell assembly (K562 and CEM cells or Ramos and CEM cells) was observed. Scale bar: 50 µm. (d) Confocal micrograph of selective cell assembly of lipo-Sgc8-TMR-modified K562 cells (red fluorescence) and unlabeled CEM cells. (e) Aggregation percentage of CEM cells in different sample sets. CEM-to-Ramos cell ratios are represented by blue (1:1), red (1:5) and green (1:10) bars. (f) 1:5 mixture of lipo-TD05-TMR-modified CEM (red) cells and Ramos (green) cells after 20 min incubation. CEM and Ramos cells formed aggregates. Scale bar: 100 µm. (g) Mixture of Lipo-TD05-TMR-modified CEM (red) cells and Ramos (green) cells from (f) treated with DNase I. Scale bar: 100 µm. (h) 1:5 mixture of lipo-Lib-TMR-modified CEM (red) cells and Ramos (green) cells after 25 min incubation. CEM and Ramos cells remained apart. Scale bar: 100 µm. (i) Mixture of Lipo-Lib-TMR-modified CEM (red) cells and Ramos (green) cells from (h) treated with DNase I. Scale bar: 100 µm.

Figure 3

Aptamer-assisted immune effector cell targeting and killing of leukemia cells (a) NK-K562 cell binding assay. NK cells recognize K562 cells spontaneously; however, aptamer modification improved the targeting efficiency by 50%. (b) NK-K562 cell killing assay. Aptamer-modified NK cells killed K562 cells more efficiently, especially in the presence of background cells (Ramos). (c) CTL-Ramos cell binding assay. Without presenting the specific MHC1:peptide on Ramos cell surfaces, CTL cannot recognize Ramos cells. On the other hand, aptamer-modified CTL recognized 80% Ramos cells. (d) CTL-Ramos cell killing assay. Assisted by membrane-anchored aptamers, CTL targeted and killed Ramos cells via cell-mediated immunity. CTL-mediated cytotoxicity was confirmed by blocking the perforin/granzyme pathway with antibody. Values are means with SD (n=3). The single asterisk indicates a significant difference between aptamer-modified and unmodified or Lib-modified groups determined by the one-tailed t-test at P<0.01. The double asterisks indicate a significant difference between aptamer-modified and anti-Perforin treated groups determined by the one-tailed t-test at P<0.01.