ELSWIFLUOPRO

ABOUT THE PROJECT

The ability to translocate the cellular membranes and gain access to the cell interior, including the different cellular compartments, remains a major obstacle in current drug development. The hydrophobic nature of cellular membranes protects cells from the surrounding environment and does not enable internalization of bigger hydrophilic species including bioactive molecules. Several strategies have been developed to deliver therapeutic agents across cellular membranes such as microinjection, electroporation, or receptor-mediated endocytosis; however, each method is associated with various drawbacks (e.g. low efficiency, high toxicity). In 1988, cell-penetrating peptides (CPPs) emerged as a valuable novel class of short peptide sequences capable of crossing the plasma membrane. Since their discovery, they have become one of the most popular and efficient tool to access the intracellular medium. CPPs, which vary greatly in size, amino acid sequence, and charge, can deliver a wide range of bioactive molecules such as proteins, peptides, oligonucleotides, nanoparticles, or liposomes to a variety cell types and different cellular compartments, both in vitro and in vivo. Reported high delivery yields, low toxicity and the possibility to add diverse modification to the peptide backbone make CPPs an excellent candidate for drug delivery platforms.

The visualization of the cell penetrating peptide translocation is of high importance. It can be achieved with fluorescence microscopy, which has become an essential tool for biological research. The bonding of a CPP to a small organic dye allows determination of the CPP localization and uptake. A wide range of fluorescent markers can be used for this purpose including rhodamine and fluorescein derivatives, BODIPY, or cyanine dyes. Nevertheless, the fluorescence labelling of CPPs with current fluorophores is connected with several drawbacks. The CPP uptake does not always correlate with bioavailability; therefore, the interpretation of the translocation process can be misleading. Moreover, CPPs are known to bind to the external face of the cell membrane and remain associated with cells even after repeated washings. The analysis can thereby give false positive results, since fluorescence cannot easily discriminate between internalized and surface-bound peptides.

To reduce signals from externally bound peptides, a protocol based on CPPs functionalized with nitrobenzoxadiazole (NBD) was developed. The outer NBD can be reduced by dithionite which results in its fluorescence quenching. The residual fluorescence of the sample, after the dithionite treatment, should therefore correspond to the intracellular species. However, dithionite must be used in excess, because it is air sensitive and undergoes rapid disproportionation in aqueous solution. Moreover, dithionite is not absolutely impermeant and may cross membranes, which leads to the extinction of fluorescence emitted by internalized peptides. In addition, once “switched off”, the NBD probe cannot be “switched on” due to the irreversibility of the reduction process. Therefore, search for a more reliable CPP-fluorophore system which would exhibite reversible on/off switching properties is very desirable.

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The aim of this project is the development of novel versatile electrochemically switchable fluorescent probes for the detection of CPP translocation which is crucial within the context of the monitoring of drug delivery through cell membranes and, more generally, in biological research. The general idea is to couple a bright fluorophore with an electro-controlled fluorescent quencher and subsequently with a cell penetrating peptide. So prepared CPP is expected to penetrate to the cell interior as well as bind to the external face of the cell membrane. The application of an adequate value of electric potential should result in the activation of the redox sensor and fluorescence quenching of photoactivated fluorophore through the photoinduced electron transfer (PET). As the electron transfer cannot occur through the membrane, the external probes would be switched off and fluorescence only of internalized CPPs could be study with high accuracy. The use of the redox sensor which could be reduced/oxidized reversibly would allow reversible on/off switching of the fluorescent probe.