Cytosolic delivery of peptides is of great interest owing to their biological functions, which could be utilized for therapeutic applications. However, their susceptibility to enzymatic degradation and multiple cellular barriers generally hinders their clinical application. Integration into nanoparticles, which can enhance the stability and membrane permeability of bioactive peptides, is a promising strategy to overcome extracellular and intracellular obstacles. Herein, we present a versatile platform for the cellular delivery of various cargo peptides by integration into metallo-peptidic coordination nanoparticles. Both termini of cargo peptides were conjugated with gallic acid (GA) to assemble GA-modified peptides into nanostructures upon coordination of Fe(III). Initial pre-complexation of Fe(III) by poly-(vinylpolypyrrolidon) (PVP) as a template favored the formation of nanoparticles, which are able to deliver the peptides into cells efficiently. Iron–gallic acid peptide nanoparticles (IGPNs) are stable in water and are supposed to generate reactive oxygen species (ROS) from endogenous H₂O₂ in cells via the Fenton reaction. The strategy was successfully applied to an exemplary set of peptide sequences varying in length (1–7 amino acids) and charge (negative, neutral, positive). To confirm the capability of transporting bioactive cargos into cells, pro-apoptotic peptides were integrated into IGPNs, which demonstrated potent killing of human cervix carcinoma HeLa and murine neuroblastoma N2a cells at a 10 µM peptide concentration via the complementary mechanisms of peptide-triggered apoptosis and Fe(III)-mediated ROS generation. This study demonstrates the establishment of IGPNs as a novel and versatile platform for the assembly of peptides into nanoparticles, which can be used for cellular delivery of bioactive peptides combined with intrinsic ROS generation.

CRISPR-Cas9 genome editing is a versatile platform for studying and treating various diseases. Homology-directed repair (HDR) with DNA donor templates serves as the primary pathway for gene correction in therapeutic applications, but its efficiency remains a significant challenge. This study investigates strategies to enhance gene correction efficiency using a T-shaped lipo-xenopeptide (XP)-based Cas9 RNP/ssDNA delivery system combined with various HDR enhancers. Nu7441, a known DNA-PKcs inhibitor, was found to be most effective in enhancing HDR-mediated gene correction. An over 10-fold increase in HDR efficiency was achieved by Nu7441 in HeLa-eGFPd2 cells, with a peak HDR efficiency of 53% at a 5 nM RNP concentration and up to 61% efficiency confirmed by Sanger sequencing. Surprisingly, the total gene editing efficiency including non-homologous end joining (NHEJ) was also improved. For example, Nu7441 boosted exon skipping via NHEJ-mediated splice site destruction by 30-fold in a DMD reporter cell model. Nu7441 modulated the cell cycle by reducing cells in the G1 phase and extending the S and G2/M phases without compromising cellular uptake or endosomal escape. The enhancement in genome editing by Nu7441 was widely applicable across several cell lines, several Cas9 RNP/ssDNA carriers (LAF-XPs), and also Cas9 mRNA/sgRNA/ssDNA polyplexes. These findings highlight a novel and counterintuitive role for Nu7441 as an enhancer of both HDR and total gene editing efficiency, presenting a promising strategy for Cas9 RNP-based gene therapy.