Polymeric Micelles Formulation of Combretastatin Derivatives with Enhanced Solubility, Cytostatic Activity and Selectivity against Cancer Cells

Abstract

Combretastatin derivatives is a promising class of antitumor agents, tubulin assembly inhibitors. However, due to poor solubility and insufficient selectivity to tumor cells, we believe, their therapeutic potential has not been fully realized yet. This paper describes polymeric micelles based on chitosan (a polycation that causes pH and thermosensitivity of micelles) and fatty acids (stearic, lipoic, oleic and mercaptoundecanoic), which were used as a carrier for a range of combretastatin derivatives and reference organic compounds, demonstrating otherwise impossible delivery to tumor cells, at the same time substantially reduced penetration into normal cells. Polymers containing sulfur atoms in hydrophobic tails form micelles with a zeta potential of about 30 mV, which increases to 40–45 mV when cytostatics are loaded. Polymers with tails of oleic and stearic acids form poorly charged micelles. The use of polymeric 400 nm micelles provides the dissolution of hydrophobic potential drug molecules. Micelles could significantly increase the selectivity of cytostatics against tumors, which has been shown using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, Fourier transform infrared (FTIR) spectroscopy, flow cytometry and fluorescence microscopy. Atomic force microscopy presented the difference between the unloaded micelles and those loaded with the drug: the size of the former was 30 nm on average, while the latter had a “disc-like” shape and a size of about 450 nm. The loading of drugs into the core of micelles was confirmed by UV and fluorescence spectroscopy methods; shifts of absorption and emission maxima into the long-wavelength region by tens of nm was observed. With FTIR spectroscopy, a high interaction efficiency of micelles with the drug on cells was demonstrated, but at the same time, selective absorption was observed: micellar cytostatics penetrate into A549 cancer cells 1.5–2 times better than the simple form of the drugs. Moreover, in normal HEK293T, the penetration of the drug is reduced. The proposed mechanism for reducing the accumulation of drugs in normal cells is the adsorption of micelles on the cell surface and the preservation of cytostatics to penetrate inside the cells. At the same time, in cancer cells, due to the structural features of the micelles, they penetrate inside, merging with the membrane and releasing the drug by pH- and glutathione-sensitive mechanisms. From a methodological point of view, we have proposed a powerful approach to the observation of micelles using a flow cytometer, which, in addition, allows us to quantify the cells that have absorbed/adsorbed cytostatic fluorophore and distinguish between specific and non-specific binding. Thus, we present polymeric micelles as drug delivery systems in tumors using the example of combretastatin derivatives and model fluorophore-cytostatic rhodamine 6G.

Specific FRET Probes Sensitive to Chitosan-Based Polymeric Micelles Formation, Drug-Loading, and Fine Structural Features

“…The synthesis of chitosan grafted with fatty acids was carried out using the carbodiimide approach described earlier [ 45 , 47 ]. The Chit5 and lipoic acid (LA) or oleic acid (OA) ( Figure 3 ) chemical conjugation was confirmed by a significant decrease in intensity of the absorption band of carboxylic acid group (1730–1700 cm −1 ) of lipoic acid, and the appearance of characteristic peaks of ν(C=O) at 1630 cm −1 and δ(N–H) at 1560 cm −1 oscillation in amide bond between chitosan and acid residues.…”

“…Of particular interest is the aspect of the formation of the polymeric micelles (as a smart drug delivery system), where the kinetics data of formation/destruction of S-S bonds are of great importance. This can be considered as the basis for creating stimulus-sensitive drug delivery systems to tumor cells, where drug molecules will be selectively released due to the higher glutathione level in cancer cells [ 45 , 47 ]. The visualization of the effective penetration of R6G into A549 cancer cells in the micellar form compared with a free cytostatic is shown in Figure S4 .…”

“…The synthesis of modified chitosan was carried out as described by us earlier with some modifications [ 45 , 46 , 47 ]. Chitosan was dissolved in 1 mM HCl solution (10 mg/mL) and then the pH was adjusted to 7.4 using 0.1 M phosphate buffer.…”

Mucosal Adhesive Chitosan Nanogel Formulations of Antibiotics and Adjuvants (Terpenoids, Flavonoids, etc.) and Their Potential for the Treatment of Infectious Diseases of the Gastrointestinal Tract

“…The formation of a gel nanoparticle is confirmed by flow cytometry data for chitosan particles cross-linked with genipin and loaded with FITC (Figure 2c,d). We have previously proposed the flow cytometry method as a way to characterize nanoparticles and their effects on cells [72]. Side sca ering is approximately equal to front sca ering (Figure 2c,d Table 1 presents data on the bacterial growth-inhibiting ability for various forms of drugs, and it also shows the selectivity coefficients of the action against E. coli vs. Lactobacillus as a model (or surrogate) of the therapeutic window.…”

Application of Pluronics for Enhancing Aqueous Solubility of Lipophilic Microtubule Destabilizing Compounds on the Sea Urchin Embryo Model

“…For these experiments, four potent antimitotic microtubule destabilizing agents with different chemical structures and water solubility were selected, namely albendazole 1, diarylisoxazole 2 [3], and chalcones 3 and 4 [34,35] (Figure 1). The reported aqueous solubility of 1 was <1 µg/mL at pH 7.0 [45] and that of 2 was <0.2 µg/mL in PBS at 37 • C [46]. The solubility of 3 and 4 was determined as 344.36 mg/L (1 mM) and 88.66 mg/L (0.27 mM), respectively, using the procedure described in Section 2.2.…”