Module 1: Anchoring to cancer cells
Anchoring our liposomes to cancer cells is one of the main challenges to ensure local production of the anticancer drug. One of our targeting strategies consisted in decorating liposomes with anti-HER2 nanobodies (anti-HER2 nb), which specifically bind to HER2 receptors that are overexpressed in cancer cells, (read our Design page). We experimentally test anchoring of liposomes functionalized with anti-HER2 nb on cancer cells cultured in the laboratory.
To produce anti-HER2 nanobodies (anti-HER2 nb), we used the E. coli strain BL21(DE3). The expression of anti-HER2 nb was induced using IPTG and purification was carried out using Cobalt resin (see the Nanobody expression and purification protocol).
Two separate batches of protein expression and purification were prepared in duplicate, and both were successful.
Figure 1: SDS-PAGE analysis (15% acrylamide) of protein samples after purification of periplasmic extract and Coomassie Blue staining. Periplasmic extract (With IPTG or No IPTG), flowthrough (FT), wash (W), elution with 20 mM imidazole, 200 mM imidazole, 250 mM imidazole and 500 mM imidazole (respectively E1/20, E1/200, E1/250 and E1/500).
The expected size of anti-HER2 nb is approximately 17 kDa. In Figure 1 one can observe a clear band around this size in the extract and flowthrough, demonstrating expression of the anti-HER2 nb. The presence of a similar band in the negative control (No IPTG, lane 2, gel 2 in Figure 1) probably reflects an uncontrolled expression of the nanobody due to promoter leakage. Approximately 2 mL of the nanobody at 17.65 µM was produced for each sample.
400-nm fluorescent liposomes were prepared and coated with anti-HER2 nb, see Protocol page. Colorectal adenocarcinoma cells Caco-2, which are HER2-positive, were used to test the anchoring of liposomes by anti-HER2 nb see Protocol page. Figure 1 shows a microscopy image of adherent and non-adherent Caco-2 cells observed in brightfield. For education purposes we appended on the image some features of living eukaryotic cells that can be distinguished with a standard optical microscope.
Figure 2: Optical imaging of adherent and non-adherent Caco-2 cells. Cells were cultured in a petri dish and imaged with an inverted fluorescence microscope in the brightfield mode with a 40X magnification.
Fluorescent liposomes were incubated on top of Caco-2 cells as described in our Protocol page. Figure 3 shows the trajectory of liposomes over time. It is possible to differentiate between diffusing liposomes and anchored liposomes on cancer cells.
Figure 3: Optical imaging of Caco-2 cells (brightfield) and 400-nm fluorescent liposomes (red fluorescence) functionalized with anti-HER2 nb after 1 hour incubation. This gif animation taken from a movie allows for categorizing liposomes as diffusing or immobile (anchored) during the lifespan of the movie.
Qualitatively, this experiment comforted us in the liposomes capabilities to anchor on cancer cells. However, it does not allow us to ascertain the specificity of the interaction between the liposome and the cancer cell. Control liposome samples without anti-HER2 nanobodies or competitive assays with soluble extracellular domain of HER2 added in solution will have to be performed.
These experiments provide evidence that the anti-HER2 nanobodies production was successful and that it can be used to promote liposome anchoring on cancer cells. However, we would recommend performing more experiments to better characterize the anti-HER2 nanobodies and its interaction with HER2 on cancer cells.
Through this experiment, we have also demonstrated the capacity of our liposome to be immobile close to cancer cells, which is decisive for local production of the anticancer drug.
