Fluorescent labels, especially FRET probes, are promising tools for studying a number of bio-chemical processes. In this paper, we promoted 2 important applications of FRET probes (MUTMAC-R6G and FITC-R6G): i) the formation of micelles from surfactants of various natures and polymers (chitosan – fatty acid), as well as ii) monitoring of enzymatic activity with improved parameters (increased analytical signal and improved selectivity due to shift to the long-wavelength region). The formation of micelles is accompanied by the convergence of fluorophores in the hydrophobic micelle core by a distance closer than in the buffer solution, thus r/R0 (where R0 – Foerster radius) is chosen as an analytical signal of micelle formation, including critical micelle concentration (CMC) and critical pre-micelle concentration (CPMC). The CMC values calculated using FRET probes are in a good agreement with literature data. At the same time, the r/R0 function provides valuable information about the nature and mechanism of micelle formation. With the second analytical application of FRET probes, we considered the optimization of techniques for studying enzymatic activity. The enzyme catalyses the reaction with the release of a fluorescent product, the signal from which may not be enough for detection, or it may be quenched, for example, in the reverse micelles (AOT-octane). Here we have proposed a solution – a FRET probe containing a rhodamine 6G (R6G) acceptor, which allows us to monitor the enzymatic reaction selectively (in the red region, 550-600 nm), and obtain a significantly higher fluorescence yield (potentially from 10 to 250 times). Thus, we have demonstrated a high potential for the FRET probes application as indicators of micelle formation as well as for the study of the enzyme catalytic activity. In the future, the method developed has prospects for application in the visualization of the enzyme functioning in cells due to the shift of the fluorescence signal to the long-wavelength region with an increase in the signal selectivity to suppress autofluorescence.

Chitosan and its derivatives as a biocompatible, biodegradable polymer that has been well-established in biotechnology and medicine as the nanogels and nanofilms forming material, has a number of practical applications in the field of the supply of medicines, food colloids, agrotechnology, as well. In this paper, the focus of attention is directed to the study of the physico-chemical principles of the formation of gels and nano-particles using fluorescence spectroscopy methods. We have obtained chitosan (Chit5) and pegylated chitosan (Chit5-PEG) particles with an average size of 170 or 400 nm, respectively by counterflow extrusion method. The paper presents three based fluorescent probes sensitive to the formation of chitosan particles: congo red, malachite green as well as covalently attached pyrene-label. Dyes fluoresce about an order of magnitude brighter in the composition of chitosan nanogel particles or in mscrogels. With these fluorescence probes, the pH- and temperature-dependent behavior of polymers was studied, and the influence of the genipin -based crosslinking agent on the gel formation parameters, such as kinetics and concentration dependence of the nanoparticles formation accompanied by sharply increases in the fluorescence intensity of dyes was evaluated. A sensitive approach for tracking gelation and including of small drug molecules and protein by FRET using a pyrene-tryptophan donor-acceptor pair is proposed using tryptophan or ovalbumin as fluorophores-donors. Upon nanogel formation FRET effect is observed manifested in sharp increase if the fluorescence intensity of the pyrene (acceptor). Thus, the application of the fluorescent probes with FRET function for monitoring the gelation and nanoparticles formation as well as drug encapsulation in chitosan-based parts, which have broad prospects in biomedical practice and food industry.

Human serum albumin (HSA) is multifunctional protein, known to be a natural carrier for a number of endogenous and exogenous compounds, including drugs. Formulating anticancer drugs with HSA is a clinically validated approach to improve the pharmacological properties and provide targeted delivery (passive and active) of cytotoxic payload to the tumor (such as in Abraxane). A number of receptors facilitating HSA uptake from systemic circulation have been characterized. What is interesting, such uptake may differ depending on the nature and quantity of ligands bound to HSA. Based on this, one might like to modify the HSA in a way that its distribution is more favorable for certain therapeutic purposes, thus ensuring more efficient delivery of the therapeutic cargo to disease site. E.g. Levofloxacin (LV), broad spectrum antibiotic drug, could benefit from extended systemic exposure, and stronger interaction with plasma proteins could be useful for this purpose. We have engrafted monomeric or polymeric CDs on the surface of HSA molecules to strengthen the LV adsorption (CD-LV dissociation constant is three orders of magnitude lower than that for HSA-LF). We have found that (HSA-HPolS)conj+LV exhibited the highest activity against E. coli, whereas (HSA-HPCD)conj+LV was the most effective against B. subtilis; and both HSA conjugates were more potent than LV alone or LV with HSA. Further fine-tuning of HSA could yield an improvement in biodistribution, and thus a more favorable risk/benefit ratio.

Potentially, such an approach would significantly increase the effectiveness of therapy for a number of infectious and other diseases, reduce the dosage of antibiotics, shorten the duration of treatment, and reduce the risk of developing resistance. Moreover, the use of a polymer carrier with covalently bound organic molecules of different structures avoids problems with different (suboptimal) solubility and bio-distribution, which would be almost inevitable when using the same compounds separately. It would be very difficult to find antibiotic/adjuvant pairs that simultaneously achieve optimal concentrations in the same target cells. In our case, terpenoids and alkylbenzenes used as adjuvants are practically insoluble as individual compounds and their unacceptable pharmacological properties would not allow them to be used as efflux pump inhibitors.