Targeted Liposomes Encapsulating miR-603 Complexes Enhance Radiation Sensitivity of Patient-Derived Glioblastoma Stem-Like Cells

Abstract

Despite potential for clinical efficacy, therapeutic delivery of microRNAs (miRNA) remains a major translational barrier. Here, we explore a strategy for miRNA delivery in the treatment of glioblastoma, the most common form of adult brain cancer, that involves complexation of miRNA with polyethylenimine (PEI) and encapsulation in targeted liposomes. miRNA 603 (miR-603) is a master regulatory miRNA that suppresses glioblastoma radiation resistance through down-regulation of insulin-like growth factor 1 (IGF1) signaling. miR-603 was complexed with PEI, a cationic polymer, and encapsulated into liposomes decorated with polyethylene glycol (PEG) and PR_b, a fibronectin-mimetic peptide that specifically targets the α₅β₁ integrin that is overexpressed in glioblastomas. Cultured patient-derived glioblastoma cells internalized PR_b-functionalized liposomes but not the non-targeted liposomes. The integrin targeting and complexation of the miRNA with PEI were associated with a 22-fold increase in intracellular miR-603 levels, and corresponding decreases in IGF1 and IGF1 receptor (IGF1R) mRNA expression. Moreover, treatment of glioblastoma cells with the PR_b liposomes encapsulating miR-603/PEI sensitized the cells to ionizing radiation (IR), a standard of care treatment for glioblastomas. These results suggest that PR_b-functionalized PEGylated liposomes encapsulating miR-603/PEI complexes hold promise as a therapeutic platform for glioblastomas.

Keywords: glioblastoma stem-cell state; miR-603; microRNA delivery; radiation therapy; stealth liposomes; targeting integrin α5β1.

Figure 1

(a) Components and schematic of the PR_b-functionalized liposome encapsulating a miR-603/PEI complex. Not drawn to scale. Cryo-TEM images of (b) miR-603/PEI complexes at N:P = 6; (c) empty PR_b liposomes; (d) PR_b liposomes encapsulating miR-603/PEI complexes at N:P = 6.

Figure 2

(a) Representative integrin α₅β₁ expression in GBM-CCC-001 cells. (b) Cell association of non-targeted liposomes and PR_b-functionalized liposomes was evaluated via flow cytometry in GBM-CCC-001 cells after 48 h incubation at 37 °C. Data are presented as mean ± SD (n = 2). Student’s t-test analysis was used to determine significance, * p < 0.05. Confocal microscopy images of (c) non-targeted liposomes and (d) PR_b-functionalized liposomes incubated for 48 h at 37 °C with GBM-CCC-001 cells. Liposomes with encapsulated calcein are shown in green, cell membranes in red and nuclei in blue.

Figure 3

mRNA levels of (a) miR-603 and (b) IGF1 and IGF1R relative to PBS control determined by qRT-PCR in GBM-CCC-001 cells following exposure to indicated treatments for 48 h at 37 °C. Data are presented as mean ± SD (n = 3). p-values from one-way ANOVA with Tukey’s HSD post-hoc analysis can be found in Tables S2–S4.

Figure 4

(a) Schematic representation of the limiting dilution assay. Limiting dilution analysis measured the clonogenic potential of GBM-CCC-001 cells, which were exposed to indicated liposomal formulations for either (b) 24 h or (c) 48 h with or without additional IR treatment. Limiting dilution assays were performed with 12 replicate wells (b) or 8 replicate wells (c) per dilution. The entire experiment was independently repeated twice. Statistical analysis was performed using the ELDA software and p-values can be found in Tables S5 and S6.