Combining brain-penetrating and anticancer peptides into bivalent-branched peptides conserves brain targeting and improves activity in vitro

Triple-negative breast cancer (TNBC) is a particularly challenging subtype of breast cancer (BC) due to the absence of common therapeutically targetable receptors, its tendency toward chemoresistance, and its high rates of recurrence and metastasis, particularly to the brain. The development of brain metastases (BM) poses an additional hurdle, largely due to the blood-brain barrier (BBB). Recently, we reported the therapeutic potential of a dual-acting peptide, PepH3-vCPP2319, that combines a BBB peptide shuttle (BBBpS – PepH3) and an anticancer peptide (ACP – vCPP2319) to eliminate TNBC-BM. In vitro , PepH3-vCPP2319 showed high translocation capacity in our transwell system (BBB cross > 40%), high anticancer activity (IC 50 ∼ 5.0 µM), and low toxicity (HC 50 > 25.0 µM). To further enhance its potency, we designed two bivalent-branched peptides that conjugate two PepH3 sequences and one vCPP2319 sequence, or vice versa. Our results indicated that, compared with PepH3-vCPP2319, both peptides translocated (∼ 40%) across a cellular BBB model and exhibited stronger anticancer activity (IC 50 ∼ 2.0 µM) while maintaining moderate toxicity (HC 50 > 15.0 µM). Compared with vCPP2319 and PepH3-vCPP2319, both bivalent-branched peptides are internalized quicker in cancer cells without membrane disruption, suggesting an intracellular mechanism of action. In conclusion, our study demonstrates the merit of branched peptide design in keeping the brain delivery and improving the efficacy of ACPs, although with slightly high toxicity, for the treatment of TNBC-BM.