¹Limanskaya L.A., ¹Pakhomova E.V., ¹Trusova V.M., ¹Gorbenko G.P.,

²Deligeorgiev T., ²Vasilev A., ²Kaloianova S., ²Lesev N.

¹V.N. Karazin Kharkiv National University, 4 Svobody Sq., Kharkiv, 61077, Ukraine

²Department of Applied Organic Chemistry, Faculty of Chemistry, University of Sofia, Sofia, 1164, Bulgaria

DELIVERY OF NEW POTENTIAL ANTITUMOR DRUG BY LIPOSOMAL NANOSYSTEMS

Europium chelate (EC) (here referred to as V10) belongs to a new class of potential antitumor drugs with high cytotoxic activity. Lanthanide complexes are of particular interest for biomedical investigations and diagnostics, since their spectral characteristics are optimal for decrease of light scattering in biological patterns and fluorescence background contribution. However, the application of such drugs in a free form is limited by their high toxicity and metabolic instability. One efficient way to increase drug efficiency is based on using different drug delivery systems such as dendrimers, nanotubes, nanoshells, quantum dots, liposomes, etc. Highly adaptable liposome-based nanocarriers currently attract increasing attention, because of their indisputable advantages, viz. complete biodegradability, ability to carry both hydrophilic and lipophilic payloads and protect them from chemical degradation and transformation, increased therapeutic index of drug, flexibility in coupling with targeting and imaging ligands, improved pharmacodynamic profiles compared to free drugs, etc.

The aim of current research was: 1) identifying the probes whose fluorescence is quenched by EC; 2) evaluating the most probable EC localization in liposomal membranes consisting of phosphatidylcholine (PC) by comparing the quenching efficiencies for probes differing in their location across the lipid bilayer. To achieve this goal, we evaluate V10 as a quencher for the fluorescent probes Prodan, 4-dimethylaminochalcone (DMC), Laurdan, 3-methoxybenzanthrone (MBA) and SQ-1, residing at different depths in the liposomal membranes.

At the first step of the study fluorescence spectra of DMC, Laurdan, MBÀ, Prodan and SQ-1 were recorded in the suspension of PC liposomes in the presence of increasing concentrations of V10 (Fig.1).

(A) (B)

(C) (D)

(E) (F)

Fig 1. Structural formula of europium chelate (A) and fluorescence spectra of DMC (B), Laurdan (C), MBÀ (D), Prodan (E) and SQ-1 (F) in suspension of PC liposomes in the presence of europium chelate V10.

As seen from Fig. 1, addition of europium chelate was followed by the decrease in fluorescence intensity of DMC, MBA, Laurdan and Prodan, only for squaraine probe SQ-1 an opposite effect takes place. These data were interpreted in terms of EC ability to serve as a quencher of DMC, Laurdan, MBÀ and Prodan fluorescence.

Fluorescence quenching has been widely studied both as a fundamental phenomenon, and as a source of information about biochemical systems. Both static and dynamic quenching requires molecular contact between the fluorophore and quencher. Collisional quenching of fluorescence is described by the Stern-Volmer equation:

,

where F₀ and F are the fluorescence intensities in the absence and presence of a quencher, respectively; k_m stands for the bimolecular quenching constant; τ₀ is the lifetime of the fluorophore in the absence of a quencher, and [Q] is the concentration of a quencher. By analyzing the obtained spectra according to the Stern-Volmer equation, we received Stern-Volmer plots (Fig. 2). Likewise, bimolecular quenching constants which reflect the efficiency of the quenching or the accessibility of the fluorophores to the quencher have been evaluated (Table 1).

Table 1. Quenching parameters of fluorescent probes

Fluorescent probe	Bimolecular quenching constant, M^-1×sec^-1
DMC	(6.1±1.7)×10^9
Prodan	(2.7±0.8)×10^9
Laurdan	(6.8±1.9)×10^10
MBA	(3.8±1.1)×10^10

The quenching efficiency of the probes was found to decrease in the order Laurdan>MBA>DMC>Prodan. Since Laurdan adopts the deepest location in liposomal membranes, embracing the glycerol backbone and initial acyl chain carbons, it can be assumed that europium chelate under study, being nonpolar in nature, partially penetrates in the hydrophobic region of the lipid bilayer.

(A) (B)

(Ñ) (D)

Fig 2. Structural formulas of the probes employed: DMC (A), MBA (B), Prodan (C), SQ-1 (D), and typical Stern-Volmer plots for fluorescence quenching by europium chelate V10 in suspension of PC liposomes.

This work was supported in part by the grant #4534 from the Science and Technology Center in Ukraine and Fundamental Research State Fund (project number F.28.4/007).

References

1. Lakowicz J.R. Principles of Fluorescent Spectroscopy, third ed. Plenum Press, New York. 2006.

2. Zhang X., Lei X., Dai H. Synthesis and characterization of light lanthanide complexes with 5-aminosalicylic acid // Synth. React. Inorg. Met.-Org. Chem. 2004. V. 34(6). P. 1123-1134.