Аннтотация

© 2018 Tomsk State University. All Rights Reserved.  Sanguinarine is a benzophenanthridine alkaloid with antimicrobial, antiviral, antiparasitic, anti-inflammatory, antiplatelet, antiangiogenic, and antitumor activity. One of the important properties of sanguinarine is a pronounced ability to suppress thrombogenesis, tumor growth and metastasis. However, the low solubility of sanguinarine in biological fluids limits its medical use. The present research was devoted to the development of the liposomal form of sanguinarine and the study of its biological activity. We obtained liposomes with sanguinarine on the basis of lecithin and cholesterol by the method of hydration of a thin film with buffer, followed by sonication and extrusion through a polycarbonate membrane with a pore size of 100 nm. Purification of liposomal dispersion from a drug that was not included in the vesicles was performed by gel filtration chromatography. We studied the morphology of the obtained liposomal particles by scanning electron microscopy; particle size and zeta potential were determined by dynamic light scattering. The study of the dynamics of sanguinarine release was conducted using the method of dialysis; quantitative analysis of the released sanguinarine from liposomes was performed using reverse-phase HPLC. The cytotoxic activity (CTA) of liposomal preparation against tumor cells of human breast carcinoma MCF-7 line was determined by the MTT assay. The toxicity and biological effects of liposomal sanguinarine on the cultures of Paramecium caudatum Ehrenberg and Tetrahymena pyriformis WH1, as well as the study of the effect of the drug on the complement system, were evaluated using the automated video registration system “BioLaT” (Russia). According to electron microscopy data, the obtained liposomes were spherical nanosized particles (See Fig. 1). The mean size of the obtained liposomal particles with sanguinarine included in their composition, determined using the method of the dynamic light scattering, was 108.5±2.2 nm, and the zeta potential was –34.7±1.4 mV. The effectiveness of sanguinarine inclusion in liposomes was quite high and amounted to 72.8±4.8%. The study of the dynamics of sanguinarine release from liposomes in conditions close to physiological (pH 7.4; 37°C) showed that this process occurs at the highest rate in the first 2 h of incubation. Then, the process is prolonged (release of about 50% sanguinarine after 6 h of incubation, and about 93% after 70 h) (See Fig. 2). Liposomal sanguinarine showed dose-dependent cytotoxic activity against tumor cells of human carcinoma MCF-7 in the micromolar concentration range (Seе Fig. 3). The CTA of liposomal sanguinarine (IC 50 14.5 mM) was slightly lower than the activity of free sanguinarine (IC 50 9.4 mM), which can be explained by the prolonged release of sanguinarine from liposomes into the cell medium, as well as by the specificity of compartmentalization and intracellular release of the drug when it is absorbed by tumor cells by endocytosis. The prolonged release and the property of preferential accumulation of liposomes in tumor tissue can have a positive effect on therapeutic efficacy in the application of liposomal sanguinarine in vivo. The effect of liposomal sanguinarine on the survival of P. caudatum ciliates was dose-dependent (See Fig. 4). The minimum inhibitory concentration of liposomal sanguinarine was 0.49 mM. At concentrations from 0.245 mM and below, the drug did not cause cell death for 2 h; over the next 24 h, the death of the ciliates was neither observed. Thus, liposomal sanguinarine has a pronounced cytotoxic effect on P. caudatum, a representative of the protozoa, which can serve as the basis for the development of antiprotozoal drugs. To identify pathogenic Protozoa species spectrum vulnerable to the action of liposomal sanguinarine, additional research is required. We also assessed the influence of liposomal sanguinarine on the protective blood systems - coagulation and the complement system. The effect of liposomal sanguinarine on thrombus formation in vitro was evaluated in citrate plasma after its recalcification according to the time of the onset of thrombus formation and the resulting clot density (See Fig. 5). The clot size in plasma solutions with the addition of the drug was significantly smaller compared with the control. At the same time, liposomal sanguinarine induces the formation of a clot after 7 min of incubation, whereas in the control the formation of a clot begins only after 14 min of incubation. Thus, under the conditions of this experiment, liposomal sanguinarine had a pronounced stimulating effect on thrombus formation. Stimulation of thrombosis by liposomal sanguinarine can be caused both by direct activation of coagulation enzymes and by the induction of enzymatic reactions of the coagulation system, which can efficiently proceed on the surface of liposomal nanoparticles. The study of the effect of liposomal sanguinarine in a non-toxic concentration of 60 ng/ml on the functional activity of the complement system against T. pyriformis ciliates showed that the half-life of the ciliates as a target of the complement system in the medium containing serum and liposomal sanguinarine (T 50 21.7 min) reduced approximately twice compared with the control (T 50 41.6 min) (See Fig. 6). In the absence of serum in the samples, liposomal sanguinarine at a concentration of 60 ng/ml, on the contrary, had a stimulating effect on T. pyriformis growth - the value of the proliferation coefficient for native cells was 2.1±0.2, and for the treated cells it was 6.4±0.8. The obtained data may indicate the activating effect of liposomal sanguinarine with respect to the assembly of the membrane attack complex of the complement system on the surface of T. pyriformis cells, causing their death. This effect allows to envisage the prospect of using liposomal sanguinarine as an immunostimulating agent. Thus, the pronounced cytotoxic antitumor and antiprotozoal activity, demonstrated in experiments in vitro, makes it possible to consider liposomal sanguinarine as a promising antitumor and antiprotozoal agent. The detected effect of thrombosis stimulation by liposomal sanguinarine seems to be important when selecting the dose of the drug introduced into the bloodstream.