Effects of Conjugation Strategy on the Anticancer Activity and Selectivity of Melittin Bioconjugates

Melittin is a membrane-active anticancer peptide with broad cytotoxicity, but its therapeutic application is limited by low tumor selectivity and toxicity toward non-tumor cells. This study evaluated whether conjugation with the CD44-targeting aptamer peptide P7 could modulate melittin activity according to peptide orientation and spacer incorporation.

BP7Mel-C, BP7Mel-N, and BP7(AEEA)Mel-N were synthesized by Fmoc-based solid-phase peptide synthesis, purified by reverse-phase HPLC, and characterized by mass spectrometry. In silico ADMET and half-life predictions, circular dichroism, fluorescence spectroscopy, serum degradation, flow cytometry, MTT cytotoxicity, clonogenic assays, and membrane permeabilization assays were performed.

The bioconjugates retained the α-helical conformation associated with melittin membrane interaction, whereas peptide orientation affected predicted and experimental stability. P7-FAM showed greater fluorescence in tumor cell lines than in MRC-5 cells under the tested conditions. Cytotoxicity varied with peptide architecture and cell line. BP7(AEEA)Mel-N showed an IC₅₀ of 16.02 μg/mL in MRC-5 cells, compared with 5.82 μg/mL for melittin, whereas BP7Mel-N showed an IC₅₀ of 6.50 μg/mL. In MDA-MB-231 cells, BP7Mel-N showed reduced potency compared with melittin (32.38 vs. 7.74 μg/mL). All peptides induced substantial CF release in POPC vesicles at concentrations corresponding to the MRC-5 IC₅₀.

P7-melittin conjugation modulated the physicochemical and biological properties of melittin in an architecture- and cell-line-dependent manner. BP7(AEEA)Mel-N showed reduced cytotoxicity toward MRC-5 cells, whereas BP7Mel-N displayed reduced potency in MDA-MB-231 cells. These findings indicate that membrane permeabilization alone does not explain the cellular responses. A key limitation is that targeting, pharmacokinetic predictions, and therapeutic efficacy were not validated in vivo.