Enhancement of siRNA transfection by the optimization of fatty acid length and histidine content in the CPP

Abstract

Extracellular synthetic nucleic acids, such as siRNAs, are unable to reach their intended targets efficiently.Therefore, delivery methods such as cell-penetrating peptides (CPP), which increase their transport, could enhance the potency of siRNA as therapeutic agents. The CPP NickFect55 (NF55) is an efficient peptide-based delivery vector, which has been previously used to deliver plasmid DNA into cells in vivo.To achieve higher intracellular delivery and bioactivity from the delivered cargo, we designed a series of histidine-containing peptides by optimizing pH-sensitivity, net charge, hydrophobicity, and charge distribution in the sequence of the CPP NF55. In the current work, we have applied a strategy where we have replaced amino acids in the C-terminus of the peptide in order to distribute hydrophobic and hydrophilic amino acids into distinct regions along the alpha-helical projection, including histidine amino acids into the sequence at the N-terminus, and optimizing the N-terminal fatty acid modification to suit the specific peptide sequence. We tested the CPPs based on the transfection efficacy, CPP/siRNA complex stability, and the properties of the CPPs, such as hemolytic activity, buffering capability and cell toxicity. As a result, we have introduced a new peptide with a completely redesigned N-terminus that displays adaptive response to its physical environment. This peptide -NickFect70 (NF70)efficiently condenses siRNA, protects the cargo against degradation and effectively mediates target gene knockdown both in mammalian cell culture and in vivo, in a mouse model. Fig. 4 Membrane activity of nickfect peptides. Hemolytic activity of peptides was measured after 1 h-incubation of murine red blood cells with peptide solutions at 37°C. After incubation released hemoglobin was quantified in the supernatant by measuring absorbance at 540 nm. (a) Cells in PBS pH 7.5 supplemented with 5% glucose at 37°C. as controls RBC in 2× PBS treated with 0.1% Triton X-100 with 5% glucose (100% hemolysis) and mQ with 5% glucose (0% hemolysis) were used. (b) Modeling of endosomolytic activity of CPPs using buffer solutions with different pH. Peptides were used at 0.75 µm concentrations. Hemolytic activity was assessed in PBS buffer at pH 5.5-7.5.

The Multifaceted Histidine-Based Carriers for Nucleic Acid Delivery: Advances and Challenges

“…Porosk et al, prepared a series of histidine-containing lipopolymers to optimize siRNA delivery [ 81 ]. Previously, the group modified KALA containing lipopolymers with a pH-buffering chloroquine analog to enhance siRNA delivery [ 82 ].…”

“…They determined that some histidine patterns that were N-terminal to the KALA group were effective carriers in vitro and in vivo. After a single intravenous injection, one histidine-containing analog, H6-20 (also named NickFect70), silenced factor VII in the liver of mice by about 60% compared to untreated controls [ 81 ]. Chen et al demonstrated that a stearylated histidine-containing cationic peptide in complex with siGADPH was as effective as silencing its target in vitro as lipofectamine [ 83 ].…”

Design of acid‐activated cell‐penetrating peptides with nuclear localization capacity for anticancer drug delivery

“…Therefore, many efforts have been undertaken to promote the endosomal escape of CPPs 46–48 . Incorporation of histidine residues is a simple and effective approach for this purpose 49–52 . To evaluate the endosomal escape of HNLS‐3 containing histidine residues, we used confocal microscopy to observe the colocalization between FITC‐labeled HNLS‐3 and LysoTracker Red‐labeled endosomes/lysosomes.…”

Internalisation and Biological Activity of Nucleic Acids Delivering Cell-Penetrating Peptide Nanoparticles Is Controlled by the Biomolecular Corona

“…Recently, the preformed protein corona was harnessed to convert nanoparticles targetable to tumour cells. For that, particles were coated with tumour-specific antibodies and human serum albumin that helps to prevent phagocytosis of the particles [ 27 , 28 , 29 ].…”