WRAP5 peptide-based nanoparticles achieve robust siRNA silencing and pDNA expression in cells with scalable microfluidic formulation.

Efficient nucleic acid delivery remains a critical challenge for gene therapies, particularly for conditions like Familial Adenomatous Polyposis (FAP) or Inherited Retinal Diseases (IRDs), which require precise genetic modulation. While lipid nanoparticles (LNPs) are clinically advanced, they often struggle with specific tissue targeting, such as penetrating the blood-brain barrier (BBB), and have limitations in packaging capacity. Peptide-based nanoparticles (PBNs) offer a promising alternative, potentially overcoming these hurdles due to their tunable properties and biocompatibility. This study explores the formulation and efficacy of WRAP5, a W- and R-rich amphipathic peptide, as a novel PBN platform.

Researchers evaluated the formulation of WRAP5-based peptide nanoparticles (PBN) using a microfluidic device. They systematically assessed the impact of key process parameters: flow rate ratio (FRR), total flow rate (TFR), and mixing channel design. The study involved 72 formulations encapsulating either small interfering RNA (siRNA) or plasmid DNA (pDNA). Nanoparticle characteristics were analyzed using dynamic light scattering (DLS) for size and polydispersity. Biological activity was confirmed in GIST-T1 cells for siRNA delivery (measuring CDK4 silencing) and in HeLa cells for pDNA delivery (measuring mCHERRY expression). Stability was monitored during storage at 4°C for up to 70 days.

WRAP5-based PBNs consistently formed with mean sizes ranging from 50 to 70 nm, exhibiting a low polydispersity index (PdI < 0.22), irrespective of the varied FRR, TFR, or mixer type. This indicates a highly robust and scalable formulation process. Stability studies revealed that siRNA-loaded PBNs showed moderate size increases during storage at 4°C, while pDNA-loaded PBNs maintained high stability for up to 70 days. Biological assays confirmed the functional efficacy of the formulated nanoparticles. > WRAP5:siRNA PBNs achieved approximately 50% CDK4 silencing in GIST-T1 cells, and WRAP5:pDNA PBNs mediated efficient mCHERRY expression in HeLa cells. Crucially, these biological activities were consistent regardless of the specific formulation method or storage duration, highlighting the inherent resilience of the WRAP5 platform. This robustness contrasts sharply with LNP systems, which often demand stringent control over FRR and TFR for optimal performance.