Triple branched RGD modification on liposomes: A prospective strategy to enhance the glioma targeting efficiency

Glioma, as one of the most common primary intracranial tumors, is in an urgent need for specific targeting agents. Multi-branched RGD ligand is a promising alternative for liposome functionalization which combines the benefits of high affinity with α_vβ₃ receptors and proper branching structure in response to the receptor clustering. Herein, we designed and synthesized single branched, double branched and triple branched RGD ligand (1RGD-Chol, 2RGD-Chol and 3RGD-Chol) respectively, which were then modified on the liposomes to prepare six different kinds of liposomes (including 1RGD-Lip, 2RGD-Lip, 3RGD-Lip, 2 × 1RGD-Lip, 3 × 1RGD-Lip and unmodified Lip). Subsequently, a series of assays were conducted. The results exhibited that the liposome decorated with 3RGD-Chol ligand possessed superior cellular internalization ability in C6 cells and bEnd.3 cells, suggesting the strongest ability of 3RGD-Lip to target the blood-brain barrier (BBB) and glioma cells. Besides, both the cytotoxicity and pro-apoptotic assays revealed that PTX-3RGD-Lip had the strongest ability to inhibit the survival of C6 cells. Moreover, the enrichment of liposomes at tumor site was 3RGD-Lip > 3 × 1RGD-Lip ≈ 2RGD-Lip ≈ 2 × 1RGD-Lip > 1RGD-Lip > Lip according to the in vivo imaging of C6-bearing mice, which was consistent with the result of in vitro targeting experiments. To sum up, the targeting efficiency of liposomes can be strongly promoted by improving the amount of targeting molecules, whereas the branching structure and spatial distance of RGD residues also accounted for the affinity between liposomes and α_vβ₃ receptors. Collectively, PTX-3RGD-Lip would be a prospective strategy in glioma treatment.