Disulfide Cross-Linked Polymeric Redox-Responsive Nanocarrier Based on Heparin, Chitosan and Lipoic Acid Improved Drug Accumulation, Increased Cytotoxicity and Selectivity to Leukemia Cells by Tumor Targeting via “Aikido” Principle

Abstract

We have developed a micellar formulation of anticancer drugs based on chitosan and heparin grafted with lipoic and oleic acids that can release the cytotoxic cargo (doxorubicin) in response to external stimuli, such as increased glutathione concentration—a hallmark of cancer. Natural polysaccharides (heparin and chitosan) provide the pH sensitivity of the nanocarrier: the release of doxorubicin (Dox) is enhanced in a slightly acidic environment (tumor microenvironment). Fatty acid residues are necessary for the formation of nanoparticles (micelles) and solubilization of cytostatics in a hydrophobic core. Lipoic acid residues provide the formation of a labile S-S cross-linking between polymer chains (the first variant) or covalently attached doxorubicin molecules through glutathione-sensitive S-S bridges (the second variant)—both determine Redox sensitivity of the anticancer drugs carriers stable in blood circulation and disintegrate after intracellular uptake in the tumor cells. The release of doxorubicin from micelles occurs slowly (20%/6 h) in an environment with a pH of 7.4 and the absence of glutathione, while in a slightly acidic environment and in the presence of 10 mM glutathione, the rate increases up to 6 times, with an increase in the effective concentration up to 5 times after 7 h. The permeability of doxorubicin in micellar formulations (covalent S-S cross-linked and not) into Raji, K562, and A875 cancer cells was studied using FTIR, fluorescence spectroscopy and confocal laser scanning microscopy (CLSM). We have shown dramatically improved accumulation, decreased efflux, and increased cytotoxicity compared to doxorubicin control with three tumor cell lines: Raji, K562, and A875. At the same time, cytotoxicity and permeability for non-tumor cells (HEK293T) are significantly lower, increasing the selectivity index against tumor cells by several times.

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

“…Reduced glutathione (GSH) is the most important antioxidant in cells [ 56 , 57 ], and was found in all cell compartments in millimolar concentrations (1–10 mM). Chitosan-based polymeric micelles use this feature of cancer cells: GSH as a trigger causes accelerated release of cytostatic [ 44 ]. Evidence of the formation of S-S bonds and the possibility of their destruction by a reducing agent (glutathione excess—tumor microenvironment model) are shown in Figure 5 .…”

“…Polymeric micelles are promising carriers of a wide range of drugs, since they have a number of properties [37][38][39][40][41][42][43]: (1) the external hydrophilic shell ensures the colloidal stability of the system; (2) the internal hydrophobic core is necessary for the solubilization of drugs, which are often poorly soluble (which limits their use in medicine); (3) thermodynamic stability; (4) the possibility of obtaining biocompatible micellar structures; (5) increased permeability of the drug to target cells due to fatty acids; (6) wide possibilities for creating stimulus-sensitive delivery systems, for example, in tumors. For the latter, chitosan demonstrated pH sensitivity to a slightly acidic environment (tumors), and lipoic acid residues with S-S bonds between various polymer chains provided glutathione sensitivity [44]. Here, we propose to study the mechanisms of formation of such micelles using the FRET probe technique based on changes in the FRET efficiency and the distance between fluorophores during aggregation and disaggregation of amphiphilic molecules.…”