Encapsulation of echinomycin in cyclodextrin inclusion complexes into liposomes: in vitro anti-proliferative and anti-invasive activity in glioblastoma

Abstract

Echinomycin, a DNA bis-intercalator peptide, belongs to the family of quinoxaline antibiotics. Echinomycin exhibits potent antitumor and antimicrobial activity. However, it is highly water insoluble and suffers from low bioavailability and unwanted side effects. Therefore, developing new formulations and delivery systems that can enhance echinomycin solubility and therapeutic potency is needed for further clinical application. In this study, echinomycin has been complexed into the hydrophobic cavity of γ-cyclodextrin (γCD) then encapsulated into PEGylated liposomes. The anti-proliferative and anti-invasive effect has been evaluated against U-87 MG glioblastoma cells. Echinomycin-in-γCD inclusion complexes have been characterized by phase solubility assay, TLC, and ¹H-NMR. The echinomycin-in-γCD inclusion complexes have been loaded into liposomes using a thin film hydration method to end up with echinomycin-in-γCD-in-liposomes. Drug-loaded liposomes were able to inhibit cell proliferation with IC₅₀ of 1.0 nM. Moreover, echinomycin-in-γCD-in-liposomes were found to inhibit the invasion of U-87 MG cells using the spheroid gel invasion assay. In conclusion, the current work describes for the first time γCD-echinomycin complexes and their encapsulation into PEGylated liposomes.

Fig. 1. The chemical structure of echinomycin (A) and γCD (B).

Fig. 2. Formation of echinomycin-in-γCD inclusion complexes. (A) Phase solubility diagram of echinomycin 0.055 mM concentration vs. different γCD concentration from 0.025–0.22 mM (n = 3). (B) Scanning for absorbance of Echi-γCD from 290–350 nm is showing an increase in UV spectra by increasing the concentration of γCD. (C) Echinomycin 0.035 mM concentration 1/(A − A₀) vs. 1/[γCD] of different γCD concentration from 0.025–0.22 mM (n = 3). (D) Echinomycin 0.023 mM concentration 1/(A − A₀) vs. 1/[γCD]² of different γCD concentration from 0.012–0.1 mM (n = 3).

Fig. 3. Inclusion mechanism of echinomycin in γCD. ¹H-NMR spectrums for γCD, echinomycin and echinomycin-in-γCD complex in DMSO.

Fig. 4. The proposed model for echinomycin-in-γCD inclusion. Showing the inner and outer cavity hydrogen atoms of γCD and the chemical structure of echinomycin representing the major hydrophobic parts, (1) quinoxaline group and (2) isopropyl group.

Fig. 5. A representative DLS and TEM graphs of (A) blank liposomes and (B) echinomycin-in-γCD-in-liposomes.

Fig. 6. Colloidal stability of blank liposomes and echinomycin-in-γCD-in-liposomes at 4 °C and 37 °C. (A) and (B) mean diameter (nm) and the polydispersity index (PDI) of blank liposomes. (C) and (D) mean diameter (nm) and the polydispersity index (PDI) of echinomycin-in-γCD-in-liposomes. (E) In vitro release of echinomycin from echinomycin-in-γCD-in-liposomes monitored over 72 h at 37 °C. All values represent the average ± SD of three independent experiments.

Fig. 7. IC₅₀ values after treatment with different preparations of echinomycin. (A) The dose–response curve for U-87 MG cells treated with free echinomycin. (B) The dose–response curve for U-87 MG cells treated with echinomycin-in-γCD inclusion complexes. (C) The dose–response curve for U-87 MG cells treated with echinomycin-in-γCD-in-liposomes. (D) Confocal imaging microscopy treated with rhodamine labeled showing the localization of liposomes in the cytoplasm. All cytotoxicity values represent the average ± SD of three independent experiments.

Fig. 8. The spheroid gel invasion assay. The invasion area of U-87 MG spheroids monitored until day 4 after treatment, (A) untreated U-87 MG spheroids, (B) U-87 MG spheroids treated with blank liposomes, (C) U-87 MG spheroids treated with echinomycin-in-γCD inclusion complexes, (D) U-87 MG spheroids treated echinomycin-in-γCD-in-liposomes. (E) The average invasion area from day 0 to day 4. All values represent the average ± SD of three independent experiments.