Fluorescence labelling of Bacterial Ghosts to analyse cellular uptake and distribution in cancer cell lines

The employment of microorganisms for cancer treatment has been of great interest in the scientific community. Bacterial Ghosts (BGs), for example, can be a feasible alternative to nanocarriers. BGs are envelopes of bacteria produced by the expression of the lysis gene E. In this way, a lysis tunnel structure is formed within the envelope of the living bacteria. They are mostly known as vaccine delivery systems, but they could potentially be used for cancer therapy due to their bacterial-like tumour-targeting properties. In this project, BG cytotoxicity and association were investigated using three cell lines (CT26luc, HeLa, and KP). BGs derived from Escherichia coli were labelled with a fluorescent dye, Alexa Fluor 488, and added to cells. Cells were co-incubated for 24 or 48 hours, and a cell viability assay was performed. Cellular uptake and distribution were also analysed. The experiment was repeated to enlighten if BGs were inside the cells or attached to the external surface of their membrane, washing the cells after 4 hours of incubation. In this way, only the BGs already associated with cells were incubated for 20 or 44 more hours. Cells were analysed using a widefield-fluorescence microscope (FLM) and a flow cytometer (FCM). BGs showed no cytotoxicity for any of the tested cell lines. The percentage of cells associated to BG was already significant when cells were incubated for 4 hours, then washed and incubated for another 20 hours, but improved by prolonging the incubation time to a total of 48 hours. Among the three cell lines, CT26luc was the one which exhibited the highest association, with a percentage of cells associated to BGs above 70% after 24 hours of co-incubation, whereas it was around 60% for both HeLa and KP. Z-stacks were acquired to study the position of BGs in relation to cells. Additionally to the above-mentioned cell lines, HEK293T was included. After a co-incubation period of 24 hours, nuclei and membranes were stained and cells were imaged on a FLM. Results showed that the majority of BGs seemed to be attached to the external surface of the cell’s membrane. It appeared that the BG uptake in HEK293T cells was quantitatively comparable to CT26luc, but a flow cytometric analysis should be performed to confirm this. BGs were also coated with three different concentrations a cationic polymer constituted by linear polyethylenimine, polyethylene glycol and Cysteine (LPC). The potential aggregation of LPC-coated-BGs was assessed on a FLM, either immediately after the coating or after a 24-hour storage at 4°C. No significant aggregation was visualised when BGs were coated with all the tested concentrations of LPC. After a co-incubation of 4 and 24 hours with coated BGs, cell association was analysed in CT26luc and KP cell lines. Compared to uncoated BG-treated cells, the percentage of cell association using BGs coated with the highest concentration of LPC was remarkably higher. Both cell lines reached a percentage of association above 85% after 24 hours of co-incubation. Further research could focus on the cytotoxicity of coated BGs and on investigating whether their cell internalisation is higher than the one of uncoated BGs.