The biological membrane molecular landscape manages almost all interactions of the cell with its environment. Thus, manipulating biological membranes has the potential to improve our understanding of how cells interact with one another and provide new avenues for diagnosis and treatment.
Kode technology is a passive modification technology which uses function, spacer, lipid constructs (FSL) to modify biological membranes. Despite various FSL constructs being known for many years for effective cell modification many aspects of their dynamics of cell modification, cytotoxicity profile, and subsequent fate are not known. Additionally, FSL construct retention behaviour on kodecytes has not been explored. If the dynamics of FSL cellular labelling are better understood, improved methods of Kode transformation may be possible, or at least the impact of variables in koding experiments will be better understood and controlled.
Three FSL constructs, FSL-FLRO4, FSL-BODIPY (different fluorophore as functional group), and FSL-biotin (biotin as functional group) were chosen for this study as they could be identified by flow cytometry, fluorescence spectroscopy, fluorescence microscopy methodologies. This research initially explored the dynamics of FSL uptake by red blood cells and Jurkat cells, and considered factors like concentration, temperature, time, protein/serum presence, and the glycocalyx. The impact of each of these variables around the micellar concentration of FSL constructs during FSL uptake was also analysed.
Retention studies showed that at 37°C, kodecytes lose FSLs when stored in protein or BSA-supplemented media. Extended experiments found that the FSLs lost to the media were not able to efficiently re-label fresh RBC and appeared to have been inactivated. It was found that the storage medium in which koding reaction takes place actually acquires proteins and lipids shed from 37°C incubated cells and this capture released FSLs and prevent them from relabelling cells. Additionally, media supplemented with protein/plasma-lipid although not necessarily causing the loss of FSL from kodecytes, do instead interfere with the ability to relabel.
Thus, one of the important observations was that the act of incubation at 37°C leads to the formation of RBC-derived membranous entities released to the media (e.g. proteins, lipids, and microvesicles), which then interfere with the uptake of FSL. Hence, it could be said that the act of koding inhibits the uptake of the FSL construct, and thus changes the dynamics of FSL uptake over time.
Scanning Electron Microscopy (SEM) studies of visible characteristics of kodecytes did not identify any specific consequence due to the FSL, but rather the observed changes are due to the methodology - primarily 37°C incubation.
The conclusions from this research are that although many stages of the koding process are predictable, some of the underlying mechanisms are complex, and influenced by multiple external factors. Overall, this research has led to a greater depth in understanding of the koding process, and new observation will inform future experiments.
