Antibiotics | Fluorescently Labeled Peptide Compounds Can Penetrate HeLa Cells and Eliminate Multidrug-Resistant Bacteria

This study investigates the application of fluorescently labeled antimicrobial peptides in combating multidrug-resistant (MDR) bacteria and cancer cells, revealing their potential for intracellular delivery and apoptosis induction, which provides critical insights for developing novel diagnostic and therapeutic tools.

 

Literature Overview
This article titled 'Fluorescent Peptides Internalize HeLa Cells and Kill Multidrug-Resistant Clinical Bacterial Isolates', published in the journal Antibiotics (2025, 14, 793), reviews and summarizes research on antimicrobial peptides' interactions with cancer cells and drug-resistant bacteria. The study systematically analyzes how different fluorescent labels affect antimicrobial and anticancer activity, as well as their cellular localization capabilities. Through synthesis and functional verification, the research provides new molecular tools for addressing multidrug resistance and cancer treatment.

Background Knowledge
Antimicrobial peptides (AMPs) and antimicrobial-anticancer peptides (ACPs) have emerged as critical tools for targeting pathogens and cancer cells due to their affinity for negatively charged cell membranes. The therapeutic challenges posed by multidrug-resistant bacteria and cancer cells have driven investigations into molecules with dual activity. Building on bovine lactoferricin-derived antimicrobial peptides (LfcinB), this study designed the peptide sequence RWQWRWQWR and its dimeric derivative (RRWQWR-hF-KKLG)2K-Ahx, which exhibit broad-spectrum antimicrobial and anticancer activity through membrane interaction, cytoplasmic/nuclear internalization, and apoptosis induction. The research further evaluates how fluorescent labeling affects peptide functionality and explores its application in cellular uptake and mechanism studies. Due to the structural characteristics of fluorophores, their conjugation position and linkage type significantly impact antimicrobial and cytotoxic activity, making this study a source of key data for developing novel targeted diagnostic and therapeutic tools.

 

 

Research Methods and Experiments
The study employed Fmoc/tBu solid-phase peptide synthesis (SPPS) to incorporate fluorescent probes including 2-aminobenzoic acid (Abz), 5(6)-carboxyfluorescein (FAM), and rhodamine B (RhB) into the RWQWRWQWR sequence and its dimeric derivative. Synthesis efficiency, purity, and bioactivity were systematically assessed. Purified peptides were validated through RP-HPLC and LC-MS analyses. Antimicrobial activity was determined by minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) assays, while cytotoxicity was evaluated via MTT assays. Confocal microscopy further characterized the intracellular localization of fluorescently labeled peptides.

Key Conclusions and Perspectives

• Peptides RhB-1 and RhB-2 exhibit two isomeric forms (open-ring and spirocyclic lactone) in RP-HPLC analysis, with equilibrium states influenced by peptide sequences. The dimeric peptide RhB-2 shows exclusively the open-ring conformation, indicating peptide structure regulates probe conformation
• Antimicrobial activity of fluorescent probes depends on fluorophore type and bacterial species: Abz-1 demonstrates effective activity against E. coli, S. aureus, and E. faecalis clinical isolates, whereas RhB-1 shows activity only against Gram-positive bacteria
• In anticancer applications, RhB-1 exhibits concentration-dependent cytotoxicity against HeLa and MCF-7 cells with IC50 values of 61 µM and 18 µM respectively, suggesting apoptosis-inducing mechanisms
• Fluorescence microscopy and confocal imaging confirm RhB-labeled peptides effectively penetrate HeLa cell membranes and localize within cytoplasm and nucleus, demonstrating their internalization capacity and potential for targeting intracellular mechanisms
• While some fluorescent labels reduce antimicrobial and cytotoxic activity, C-terminal labeling (e.g., 1-Abz) improves hemocompatibility, highlighting the importance of modification positions for functional retention

Research Significance and Prospects
This study establishes a synthetic and functional evaluation framework for developing novel fluorescent antimicrobial-anticancer peptides, revealing labeling strategies' potential in mechanism exploration and drug delivery. Future research should focus on optimizing fluorescent probe conjugation methods to preserve peptide activity while enhancing imaging sensitivity, thereby creating tools for personalized therapies and monitoring co-occurring infections in cancer patients.

 

 

Conclusion
This systematic evaluation of fluorescent probes in antimicrobial-anticancer peptides demonstrates that fluorophore type and conjugation position significantly affect functional properties. Certain labeled peptides maintain antimicrobial and cytotoxic activity while achieving cellular penetration and localization capabilities, offering new approaches for mechanism studies and targeted therapies. With rational design and functionalization, these fluorescent probes could serve as multifunctional tools for disease diagnostics, therapeutic monitoring, and target exploration.

 

Daniel Castellar-Almonacid, Kelin Johana Cuero-Amu, Jose David Mendoza-Mendoza, Javier García-Castañeda, and Ricardo Fierro-Medina. Fluorescent Peptides Internalize HeLa Cells and Kill Multidrug-Resistant Clinical Bacterial Isolates. Antibiotics.