Hyaluronic Acid‐Coated DNA Nanoflowers as Smart Nanocarriers for Deep Tumor Penetration and Enhanced Gene Therapy in Pancreatic Cancer

ABSTRACT The dense stroma barrier of pancreatic tumors significantly impedes the delivery of therapeutic agents, presenting major challenges to effective gene therapy in pancreatic cancer. Herein, we introduce a strategy to overcome this limitation by using the pancreatic tumor extracellular matrix component hyaluronic acid (HA) to coat dual‐enzyme responsive DNA nanoflowers as a smart nanocarrier capable of responding to multiple tumor‐associated endogenous nucleases, which is designed for the co‐delivery of Cas9/sgRNA ribonucleoproteins (RNPs) and antisense oligonucleotides (ASOs) into deep pancreatic tumors, enabling efficient synergistic gene therapy for pancreatic cancer. The DNA nanoflowers, assembled from ultralong single‐stranded DNA with multiple sgRNA recognition sequences, efficiently load and deliver Cas9/sgRNA RNPs and ASOs. HA coating significantly enhances the penetration of DNA nanoflowers into pancreatic tumors by approximately 5‐fold, and improves their accumulation in cancer cells by approximately 15.8‐fold. In pancreatic cancer cells, overexpressed ribonuclease H and flap structure‐specific endonuclease 1, respectively, trigger the release of Cas9/sgRNA RNPs and ASOs, facilitating precise and effective gene editing and silencing. In vivo studies in a pancreatic cancer mouse model demonstrate the remarkable therapeutic efficacy with a tumor inhibition rate of approximately 91.9%, highlighting the potential of HA‐coated DNA nanoflowers as a promising treatment strategy for pancreatic cancer.