The researchers built two small libraries of cell-penetrating peptides composed of alternating arginine and tryptophan residues. Arginine carries a positive charge and binds the negatively charged RNA backbone, while tryptophan is hydrophobic and helps the carrier interact with the lipid bilayer of the cell membrane. Each peptide contained a constant five alternating tryptophan-arginine units, either in linear form or as a cyclic ring, attached through a lysine linker to an increasing number of additional linear arginine-tryptophan units. This produced five linear peptides, designated L1 through L5, and five hybrid cyclic/linear peptides, designated C1 through C5, allowing the team to compare the effect of cyclization and of increasing molecular weight within a consistent chemical framework.

The first surprise came from binding measurements. The team mixed each peptide with scrambled siRNA in saline across fourteen nitrogen-to-phosphate (N/P) ratios and quantified binding with a SYBR Green II dye-exclusion assay, calculating the ratio needed for fifty percent binding, known as BR50. The values ranged from 1.21 to 2.08, considerably tighter than the BR50 values above 2.7 typically seen with the group’s earlier peptide carriers. As expected, adding more arginine residues lowered BR50, and the hybrid cyclic peptides generally bound more strongly than their linear counterparts, with statistically significant differences for the L2/C2 and L3/C3 pairs. On paper, this looked like excellent carrier performance: strong binding means good encapsulation and protection from degrading enzymes.

But strong binding has a dark side. When the researchers measured the size and surface charge of the peptide/siRNA complexes, they found hydrodynamic diameters of roughly 98 to 115 nanometers at an N/P ratio of 10, squarely within the 50 to 200 nanometer range considered useful for cancer applications and the enhanced permeation and retention effect. Zeta potentials turned positive at N/P ratios of 10 and above, and the hybrid peptides C2, C4, and C5 showed significantly higher positive charges than their linear counterparts, likely because cyclization makes the peptide more rigid and redistributes its charge. Everything about the particles suggested they should enter cells efficiently.

And enter cells they did. In three breast cancer cell lines, including the triple-negative lines MDA-MB-231 and MDA-MB-468 and the HER2-positive AU 565, flow cytometry with fluorescently labeled siRNA showed that several linear peptides, particularly L1 and L3, achieved internalization comparable to or better than Lipofectamine, a commercial transfection reagent. Confocal microscopy confirmed that the fluorescent signal sat inside the cells, not merely stuck to their surfaces. Yet when the team tested whether this uptake translated into actual gene silencing, targeting the STAT3 protein in MDA-MB-231 cells, the answer was a resounding no. Lipofectamine reduced STAT3 expression to about 61 percent of baseline, but the lead peptide L1 left STAT3 essentially untouched, at 92.5 percent expression.

The explanation lay in the serum stability experiments. When peptide/siRNA complexes were exposed to fetal bovine serum for 24 hours and then treated with heparin, a polyanion normally capable of displacing siRNA from carriers, most complexes refused to release their cargo. The siRNA bands barely moved through the gel, staying trapped in the loading wells, and even raising the heparin concentration from 1.4 to 4 milligrams per liter did not help. Only L1 and, to a lesser extent, L2 showed partial release. This was direct evidence that the peptides gripped siRNA so tightly that neither heparin in the lab nor the cytoplasm inside the cell could pry the cargo loose. The siRNA was being delivered into cells and then effectively imprisoned there, vulnerable to degradation before it could reach the RNA-induced silencing complex.

The solution was to add a third component: Trans-Booster, a commercially available negatively charged transfection aid developed in Hasan Uludag’s laboratory and supplied by RJH Biosciences. When mixed into the peptide/siRNA complexes at a 1:2 siRNA-to-Trans-Booster weight ratio, the polyanion reduced binding affinity, lowered zeta potentials at lower N/P ratios, and, crucially, restored the free siRNA band in the release assays for complexes that had previously shown no release at all. The functional consequences were striking. With Trans-Booster added at 2, 4, and 8 micrograms per milliliter, STAT3 expression in MDA-MB-231 cells dropped to approximately 82, 45, and 41 percent of untreated levels, respectively. In a quantitative GFP-silencing assay in engineered MDA-231 cells, L1 combined with Trans-Booster left only 23.7 percent of the GFP signal remaining, essentially matching Lipofectamine’s 23.6 percent, while the peptides alone left 90 to 96 percent of the signal intact.

The team then asked whether the same principle applied to CRISPR/Cas9 delivery, a far more demanding cargo because the ribonucleoprotein must reach the nucleus. Using fluorescently labeled tracrRNA in HEK-293 cells, they found that the linear peptide L3 at 25 micromolar achieved the highest uptake among the peptides, though the commercial reagent CRISPRMAX still outperformed all peptide formulations. When the peptides were used to deliver RNPs targeting the housekeeping gene HPRT, none of the tested peptides alone produced the two diagnostic cleavage bands detected by the T7 endonuclease assay. But in the groups that included Trans-Booster, faint cleavage bands appeared, most noticeably for L3 and C3, providing the first hint that charge modulation could extend beyond siRNA to genome editing cargo.

The toxicity profile offered additional reassurance. Across all three breast cancer cell lines, the peptide/siRNA complexes were significantly milder than Lipofectamine, which reduced viability in AU 565 cells to 42 percent under the manufacturer’s recommended conditions. The worst peptide complex, C3 in MDA-MB-231 cells, still left 80 percent viability even at the highest N/P ratio of 80. The authors also noted interesting structure-activity nuances: increasing arginine content did not monotonically improve uptake, and in some cell lines the lower N/P ratio of 20 outperformed 40, possibly because excess free peptide disrupts complex organization or because higher tryptophan content promotes compact peptide conformations that bury the very arginine residues needed for cargo binding.

The broader message is that intermediate, tunable binding affinity, not maximal affinity, is the design target for nucleic acid carriers. The finding aligns with reports from other groups, including work showing that excessively tight siRNA binding impairs the in vivo antitumor efficacy of polyplexes, and with the observation that some cell-penetrating peptides require heparin concentrations as high as 4 milligrams per milliliter for complete cargo release. At the same time, the literature contains opposing examples in which ultra-high-affinity RNA-binding proteins enhanced cytosolic delivery, underscoring that the optimum is system-dependent. The authors acknowledge that their study was conducted entirely in vitro, that direct mechanistic evidence of the intracellular release event is still lacking, and that techniques such as isothermal titration calorimetry, circular dichroism spectroscopy, and cryo-electron microscopy will be needed to fully characterize the carrier/cargo interaction. Still, the design principle they establish, that a polyanionic additive can convert a binding-locked carrier into a functional one, offers a practical and generalizable lever for the next generation of peptide-based gene delivery systems.

Subject of Research: Charge modulation of peptide/nucleic acid complexes: an anionic additive enhances gene silencing and CRISPR/Cas9 editing by promoting intracellular nucleic acid release

Article Title: Charge modulation of peptide/nucleic acid complexes: an anionic additive enhances gene silencing and CRISPR/Cas9 editing by promoting intracellular nucleic acid release

Article References: Alhazza, A., Shoushtari, S. K., Uludag, H., Parang, K., & Aliabadi, H. M. (2026). Charge modulation of peptide/nucleic acid complexes: an anionic additive enhances gene silencing and CRISPR/Cas9 editing by promoting intracellular nucleic acid release. Frontiers in Nanotechnology, 8, Article 1925723. https://doi.org/10.3389/fnano.2026.1925723

Image Credits: AI Generated

DOI: 10.3389/fnano.2026.1925723

Keywords: siRNA delivery, CRISPR/Cas9, cell-penetrating peptides, gene silencing, nanomedicine, charge modulation, Trans-Booster, STAT3, breast cancer, nucleic acid release, polyanionic additive, drug delivery