Targeted Delivery Inside the Cells Directly Visualized with Förster Resonance Energy Transfer (FRET)

We established a real-time Förster resonance energy transfer (FRET) based assay to evaluate targeted drug delivery using polymeric micelles. Red fluorescent protein (RFP)-expressing <i>E. coli</i> cells were used as a test system to monitor the delivery of drug-fluorophore such as curcumin and umbelliferones (MUmb and AMC) encapsulated in the polymeric micellar formulations. The efficiency of the drug delivery was quantified using the FRET efficiency, measured as the degree of energy transfer from the drug to the RFP. FRET efficiency directly provides the determination of the delivery efficacy, offering a versatile platform adaptable to various drugs and cell types. We used polymer micelles as a carrier for targeted delivery of fluorescent drugs to bacterial cells expressing RFP. The physicochemical characterization of the interaction between the drugs and the micelles including spectral properties, and the solubility and binding constants, were determined. We revealed a stronger affinity of MUmb for heparin-based micelles (<i>K</i>_d~10^-5 M) compared to chitosan-based micelles (<i>K</i>_d~10^-4 M), underscoring the influence of polymer composition on drug loading efficiency. For micelles containing MUmb, a FRET efficiency significantly exceeds (by three times) the efficiency for non-micellar MUmb, which have minimal penetration into bacterial cells. The most noticeable effect was observed with the use of the micellar curcumin providing pronounced activation of the RPF fluorescence signal, due to the interaction with curcumins (fluorophore-donor). Curcumin delivery using Chit5-OA micelle resulted in a 115% increase in RFP fluorescence intensity, and Hep-LA showed a significant seven-fold increase. These results highlight the significant effect of micellar composition on the effectiveness of drug delivery. In addition, we have developed a visual platform designed to evaluate the effectiveness of a pharmaceutical product through the visualization of the fluorescence of a bacterial culture on a Petri dish. This method allows us to quickly and accurately assess the penetration of a drug into bacteria, or those located inside other cells, such as macrophages, where the intercellular latent forms of the infection are located. Micellar formulations show enhanced antibacterial activity compared to free drugs, and formulations with Hep-OA micelles demonstrate the most significant reduction in <i>E. coli</i> viability. Synergistic effects were observed when combining curcumin and MUmb with moxifloxacin, resulting in a remarkable 40-50% increase in efficacy. The presented approach, based on the FRET test system with RFP expressed in the bacterial cells, establishes a powerful platform for development and optimizing targeted drug delivery systems.