The effect of addition of two targeted vectors, cRGD peptide and folic acid, with the same linker length on the properties of the doxorubicin phospholipid composition: a study of properties in vitro.

Abstract

Serious side effects of the chemotherapeutic drug doxorubicin prompt researchers to develop systems for its targeted delivery to cells. In this work, we continued the study on the effect of using two vectors in a phospholipid delivery system of doxorubicin (Dox) for targeted therapy of breast cancer. We have obtained a composition NPh-Dox-cRGD-Fol(2.0) with the same linker length for both targeting ligands, cRGD and folic acid (PEG 2000). The resulting composition NPh-Dox-cRGD-Fol(2.0) with a particle size less than 50 nm and with 99% Dox incorporated into nanoparticles in an experiment on drug release at different pH values (5.0 and 7.4) showed a faster release and a high level of Dox compared to the phospholipid nanoform and a composition containing only the cRGD peptide. In vitro experiments on MDA-MB-231 breast cancer cells expressing the folate receptor and integrin αvβ3 demonstrated an increase in the total accumulation and internalization of Dox upon incubation with the dual-vector composition compared to the control samples. On the MCF-7 breast cancer cell line (expressing only the folate receptor), a similar effect was observed upon incubation with the single-vector composition containing folic acid (NPh-Dox-Fol(2.0)). In experiments with normal Wi-38 cell line, the internalization and total accumulation of the drug were comparable for both the free substance and the vector compositions. After 24 h-incubation of MDA-MB-231 cells with Dox-containing (10 μg/ml DOX) samples, the lowest percentage of living cells was observed for the studied dual-vector composition NPh-Dox-cRGD-Fol(2.0). On MCF-7 cells, the cytotoxic effect was manifested equally for the studied samples. The study of the cell death pathway on MDA-MB-231 cells showed the predominance of the apoptotic pathway (late apoptosis), while in the case of MCF-7 the necrosis pathway predominated. The cell cycle study performed using MDA-MB-231 cells (folate receptor (+) and integrin αvβ3 (+)) revealed an increase in the percentage of cells in the G0/G1 phase was noted thus indicating apoptotic cell death during incubation with NPh-Dox-cRGD-Fol(2.0). No differences were found between the samples in experiments performed on MCF-7 cells (folate receptor (+) and integrin αvβ3 (-)).