Biomedical Chemistry: Research and Methods 2023, 6(4), e00204

Influence of Adding cRGD Peptide to Phospholipide Nanoparticles with Doxorubicin Included on Apoptosis in Glioblastoma Cells in Vitro

L.V.Kostryukova*, Yu.A.Tereshkina, A.M.Gisina, F.N.Bedretdinov, A.M.Pyatigorsky

Institute of Biomedical Chemistry, Pogodinskaya str., 10, Moscow 119121, Russia; *e-mail: kostryukova87@gmail.com

Keywords: glioblastoma; phospholipid nanoparticles; cRGD; integrin αvβ3; doxorubicin; apoptosis

DOI:10.18097/BMCRM00204

The whole version of this paper is available in Russian.

One of the methods of treating glioblastoma after surgery is chemotherapy; the drugs used in this case, due to their nonspecific distribution, lead to a number of complications. One way to overcome this drawback is to supply drugs with delivery systems with targeted molecules. This approach allows increasing the accumulation of therapeutic agents directly at the lesion site, minimizing side effects. This work is a continuation of the study of the mechanism of action of the previously obtained phospholipid composition of doxorubicin with a targeted cRGD peptide (NPh-Dox-cRGD). This peptide is capable of selectively interacting with integrin αvβ3, a receptor expressed on the surface of a number of tumor cells, including glioblastoma. The work assessed the cytotoxic effect of the NPh-Dox-cRGD composition in comparison with the free substance (Dox) and embedded in phospholipid nanoparticles without a targeted ligand (NPh-Dox). It was shown that after 24 h of incubation of U-87 MG cells with substances at the maximum concentration of Dox (30 μg/ml), the percentage of viability cells was 6% for Dox, 21% for NPh-Dox-cRGD, and 21% for NPh-Dox – 17%, i.e. When Dox was incorporated into phospholipid NPs, its cytotoxic effect was observed to a lesser extent. No statistically significant differences were noted in the control line HeLa. Assessment of tumor cell death using flow cytometry indicated that most of the cells died via apoptosis. When incubated with a composition containing a targeting peptide, NPh-Dox-cRGD, at a concentration (Dox) of 0.5 μg/ml, the percentage of cells susceptible to late apoptosis was 29.7%, for the free form - 24.4%. An assessment of cells susceptible to early apoptosis (Dox concentration 0.5 µg/ml) showed that the percentage of these cells for the sample with the peptide was higher and amounted to 11.4%.

Figure 1. Viability of U-87 MG (A, C) and HeLa (B, G) cells incubated with free Dox, embedded in phospholipid NPs (NPh-Dox), Dox embedded in phospholipid NPs with a targeted peptide
(NPh-Dox-cRGD ) for 24 h (A, B) and 48 h (V, G) in different concentrations. Data are presented as mean ± standard deviation (n = 4).
Figure 2. Apoptosis assay in U-87 MG cells culture incubated with free Dox; Dox embedded in phospholipid NPs (NPh-Dox); and Dox embedded in a phospholipid composition with a targeted peptide (NPh-Dox-cRGD). Dox concentrations were 0.5 and 5 µg/ml. Quadrant design: upper left (Q1) – necrosis, cells stained with propidium iodide; top right (Q2) – late apoptosis; bottom right (Q3) – early apoptosis, cells stained with Annexin V; bottom left (Q4) – fluorescence signal at the level of autofluorescence of unstained cells.
Figure 3. The percentage of cells undergoing early apoptosis when incubated with U-87 MG cells at a Dox concentration of 0.5 μg/ml, determined after 24 h of incubation with samples of Dox free (Dox) embedded in phospholipid NPs (NPh- Dox), and integrated into phospholipid NPs with a targeted peptide (NPh-Dox-cRGD). Control - cells not treated with substances.

CLOSE
Table 1. Calculated value IC50 µg/ml obtained on the U-87 MG cell line after 24 h of incubation.

FUNDING

The work was performed within the framework of the Program for Basic Research in the Russian Federation for a long-term period (2021-2030) ((№ 122030100170-5).

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