This study presents molecularly self-assembled nucleic acid nanoparticles for targeted in vivo delivery of siRNA. The nanoparticles, composed of DNA tetrahedrons with precisely controlled size and surface ligands, efficiently deliver siRNA into cells and silence target genes in tumors. The DNA strands are self-assembled into tetrahedral structures, allowing precise control over the spatial orientation and density of targeting ligands such as peptides and folate. The study shows that at least three folate molecules per nanoparticle are required for optimal siRNA delivery and gene silencing, with the ligand orientation playing a critical role in the efficiency of gene silencing. The nanoparticles demonstrated a longer blood circulation time (t₁/₂ ~24.2 min) compared to the parent siRNA (t₁/₂ ~6 min). In vivo testing showed that the nanoparticles accumulated primarily in the tumor and kidney, with minimal accumulation in other organs. The study also demonstrated that the nanoparticles could effectively silence the firefly luciferase gene in KB xenograft tumors, with a 60% decrease in bioluminescent intensity. The nanoparticles showed no significant immune response, indicating their potential as safe and effective gene delivery systems. The study highlights the advantages of the self-assembled DNA/siRNA nanoparticles, including their ability to deliver siRNA with high specificity and efficiency, their ability to control the spatial orientation and density of targeting ligands, and their ability to avoid renal clearance while penetrating tumor vasculature. The nanoparticles were shown to be effective in both systemic and local delivery, with the ability to target various cancers through modification of size and ligand type. The study also demonstrates the potential of these nanoparticles for other tissue applications through modification of size and ligand type. The results suggest that these nanoparticles could be a promising platform for targeted gene therapy.This study presents molecularly self-assembled nucleic acid nanoparticles for targeted in vivo delivery of siRNA. The nanoparticles, composed of DNA tetrahedrons with precisely controlled size and surface ligands, efficiently deliver siRNA into cells and silence target genes in tumors. The DNA strands are self-assembled into tetrahedral structures, allowing precise control over the spatial orientation and density of targeting ligands such as peptides and folate. The study shows that at least three folate molecules per nanoparticle are required for optimal siRNA delivery and gene silencing, with the ligand orientation playing a critical role in the efficiency of gene silencing. The nanoparticles demonstrated a longer blood circulation time (t₁/₂ ~24.2 min) compared to the parent siRNA (t₁/₂ ~6 min). In vivo testing showed that the nanoparticles accumulated primarily in the tumor and kidney, with minimal accumulation in other organs. The study also demonstrated that the nanoparticles could effectively silence the firefly luciferase gene in KB xenograft tumors, with a 60% decrease in bioluminescent intensity. The nanoparticles showed no significant immune response, indicating their potential as safe and effective gene delivery systems. The study highlights the advantages of the self-assembled DNA/siRNA nanoparticles, including their ability to deliver siRNA with high specificity and efficiency, their ability to control the spatial orientation and density of targeting ligands, and their ability to avoid renal clearance while penetrating tumor vasculature. The nanoparticles were shown to be effective in both systemic and local delivery, with the ability to target various cancers through modification of size and ligand type. The study also demonstrates the potential of these nanoparticles for other tissue applications through modification of size and ligand type. The results suggest that these nanoparticles could be a promising platform for targeted gene therapy.