Extrahepatic siRNA delivery to inflammatory macrophages remains limited by poor tissue selectivity and inefficient endosomal escape. A structure-guided, histidine-rich peptide platform was engineered to create pH-switchable endosomolytic carriers. Peptides were optimized for low membrane activity at physiological pH and high endosomal disruption at acidic pH. This study establishes histidine-rich, pH-responsive peptides as a promising platform for spleen-selective, extrahepatic RNA delivery.
ABSTRACT
Extrahepatic delivery of small interfering RNA (siRNA) remains a major translational challenge because most nanocarriers preferentially accumulate in the liver, while endosomal sequestration limits productive cytosolic release. Inflammatory macrophages in the spleen are attractive therapeutic targets in systemic inflammation, yet spleen-selective delivery systems with efficient endosomal escape remain underdeveloped. Here, a structure-guided peptide engineering workflow was used to generate histidine-rich, pH-switchable endosomolytic peptides for spleen-selective siRNA delivery. Sequence design integrated pH-dependent charge transition modeling, amphipathic helix prediction, membrane interaction scoring, and safety filtering. Six candidate peptides were synthesized and evaluated for pH-responsive structure, membrane disruption, hemocompatibility, siRNA complexation, serum stability, macrophage uptake, endosomal escape, biodistribution, and anti-inflammatory efficacy. The lead peptide, HSEP-6, showed a predicted net charge increase from +3.1 at pH 7.4 to +7.4 at pH 5.5, helix content increasing from 17% to 56%, and acidic calcein release increasing from 9% to 62%. In inflammatory macrophages and LPS-challenged mice, HSEP-6 enabled efficient siRNA delivery, spleen-selective accumulation, marked Irf5 silencing, reduced TNF-α and IL-6, and no measurable systemic toxicity, supporting histidine-rich pH-switchable peptides as a rational platform for extrahepatic RNA delivery.
