Targeted delivery inside the cells directly visualized with FRET
Authors: Zlotnikov Igor D. , Kudryashova Elena V. , Kudryashova Elena Vadimovna 
Published: February 2025

In many areas of biomedical research, a targeted delivery to certain types of cells may be required. Polymeric micelles have been described as one of the possibilities for such delivery. Here we describe a model system, demonstrating the possibility to visualize this process in real time. E.coli cells were transfected to produce the red fluorescent protein (RFP) inside the cell. Three different substances, selected for their ability to form the FRET pair with RFP, were encapsulated in polymeric micelles and delivered inside the E.coli, yielding FRET fluorescent signal immediately upon delivery. Obviously, for practical application, depending on the substance to be delivered and target cells, RFP can be replaced with a different fluorescent protein able to yield FRET effect with the substance in question; and E.coli to be replaced with different target cells able to transiently express the necessary fluorescent protein. Interestingly, we have observed FRET effect inside the E.coli cells at concentrations, which are normally not sufficient to yield such effect in-situ, indicating that cell membrane may play an important role to facilitate the spatial proximity of the donor organic molecules with the RFP fluorescence acceptor epitope. So, we employ FRET to directly measure drug delivery efficacy, by monitoring the interactions of RFP-expressing bacteria with bioactive compounds curcumin, 4-methylumbelliferone (MUmb), and 7-amino-4-methylcoumarin (AMC) – as stand-alone drugs or as adjuvants for antibiotics. Four micellar systems (Chit-OA, Chit-LA, Hep-OA, and Hep-LA) based on chitosan and heparin, modified with oleic (OA) or lipoic (LA) acid, were synthesized and characterized using FTIR and NMR spectroscopy. Physicochemical characterization, of the interaction between the drugs (curcumin, MUmb, AMC) and the micelles including binding constants and stoichiometry, were determined using fluorimetry. We revealed a stronger affinity of MUmb for heparin-based micelles (Kd ~ 10⁻⁵ M) compared to chitosan-based micelles (Kd ~ 10⁻⁴ M), underscoring the influence of polymer composition on drug interaction. Drug-loaded micelles achieved a FRET efficiency of 0.3, substantially surpassing the 0.1 efficiency of non-micellar MUmb, which barely penetrated bacterial cells. Notably, Chit5-OA elicited 115% increase in RFP fluorescence, while Hep-LA showed a remarkable 7-fold enhancement, further emphasizing the impact of micelle composition on drug delivery efficiency. Micellar formulations show enhanced antibacterial activity compared to free drugs: formulations with Hep-OA micelles demonstrate the most significant reduction in E. coli viability. Synergistic effects were observed when combining curcumin and MUmb with moxifloxacin, resulting in a remarkable 40-50% increase in efficacy, particularly at low (≤5 µM) moxifloxacin concentrations. The presented approach, combining FRET analysis with structural and binding studies, establishes a powerful platform for development and optimizing targeted drug delivery systems.