Nanotechnology offers innovative solutions for addressing the challenges posed by biofilm-forming bacteria, which are highly resistant to conventional antimicrobial therapies. This review explores the integration of pharmaceutical nanotechnology with antimicrobial peptides (AMPs) to enhance the treatment of biofilm-related infections. The use of various nanoparticle systems—including inorganic/metallic, polymeric, lipid-based, and dendrimer nanostructures—provides promising avenues for improving drug delivery, targeting, and biofilm disruption. These nanocarriers facilitate the penetration of biofilms, down-regulate biofilm-associated genes, such as ALS1, ALS3, EFG1, and HWP1, and inhibit bacterial defense mechanisms through membrane disruption, reactive oxygen species generation, and intracellular targeting. Furthermore, nanoparticle formulations such as NZ2114-NPs demonstrate enhanced efficacy by reducing biofilm bacterial counts by several orders of magnitude. This review highlights the potential of combining nanotechnology with AMPs to create novel, targeted therapeutic approaches for combatting biofilm-related infections and overcoming the limitations of traditional antimicrobial treatments.

Background/Objectives: Tuberculosis remains a major public health challenge due to the persistence of Mycobacterium tuberculosis (Mtb) and the emergence of multidrug-resistant strains. In this study, Pep-H, an HNP-1-derived antimicrobial peptide with the sequence RRYGTCIYQGRLWAF-NH₂, was used as a compact scaffold to examine how single-residue substitutions at the Cys position affected its biological and functional profile. Methods: A focused single-position substitution panel was generated by replacing Cys with Trp, Ala, Arg, or Met while preserving peptide length and sequence context, and the analogs were computationally prioritized according to their predicted antitubercular potential and contrasting side-chain properties. The peptides were synthesized, purified, characterized by HPLC and mass spectrometry, and evaluated for activity against Mtb H37Rv, cytotoxicity, hemolysis, ethidium bromide accumulation, and DPPH radical scavenging. Results: Pep-H retained the most favorable profile, showing the highest antimycobacterial potency, low hemolysis, favorable selectivity indices, enhanced ethidium bromide accumulation, and the strongest antioxidant response. All Cys substitutions reduced antimycobacterial activity, indicating that none of the tested residues reproduced the integrated biological profile of Pep-H. Conclusions: The contrasting outcomes of the Arg- and Met-containing analogs suggest that increased cationicity or sulfur retention alone was insufficient, while supporting a multifactorial contribution of Cys side-chain chemistry and the local GTCIY environment.