d-limonene-loaded liposomes target malignant glioma cells via the downregulation of angiogenic growth factors

Glioblastoma multiforme is the most lethal form of a brain tumor in clinical oncology. The current treatment modalities possess their own pros and cons and hence, there is an urge in exploring alternative strategies. When nanocarriers loaded with drugs are directed towards the targeted site, they tend to show promising results. In our study, we aimed to evaluate the potency of d-limonene-loaded liposomes on U87MG cells and the signaling pathways involved in cell death. The results obtained from our study state the limonene-loaded liposomes had the potency to induce cell cycle arrest in the G2/M phase, whereas the quantitative real-time polymerase chain reaction (q-RTPCR) analysis confirmed that the nanoformulations could efficiently downregulate the gene expression of angiogenic growth factors such as vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), angiogenin, matrix metalloproteinase enzyme 2 (MMP 2), matrix metalloproteinase enzyme 3 (MMP 3), matrix metalloproteinase enzyme 9 (MMP 9), matrix metalloproteinase enzyme 13 (MMP13) and secreted protein acid rich in cysteine (SPARC). The Western blot assay confirms that the drug-loaded liposomes could downregulate the expression of angiogenic proteins VEGF, FGF, and angiogenin, which was later validated by immunofluorescence assay, further confirming the results. Cell internalization study using limonene-loaded liposomes was also carried out on glioma (U87MG) cells and human embryonic kidney (HEK293) cells. The results confirm that the drug-loaded liposomes could only internalize in U87MG cells and not in HEK293 cells. A molecular docking study reveals that the drug could efficiently bind to angiogenic proteins such as VEGF, FGF, and angiogenin and also onto claudin, junction adhesion molecule-1, and Occludin proteins which are overexpressed in the blood-brain barrier, thus confirming its ability to target and cross the barrier.