A DNA/Upconversion Nanoparticle Complex Enables Controlled Co-Delivery of CRISPR-Cas9 and Photodynamic Agents for...

Photodynamic therapy (PDT) depends on the light-irradiated exciting of photosensitizer (PS) to generate reactive oxygen species (ROS), which faces challenges and limitations in hypoxia and antioxidant response of cancer cells, and limited tissue-penetration of light. Herein, a multifunctional DNA/upconversion nanoparticles (UCNPs) complex is developed which enables controlled co-delivery of CRISPR-Cas9, hemin, and protoporphyrin (PP) for synergistic PDT. An ultralong single-stranded DNA (ssDNA) is prepared via rolling circle amplification (RCA), which contains recognition sequences of single guide RNA (sgRNA) for loading Cas9 ribonucleoprotein (RNP), G-quadruplex sequences for loading hemin and PP, and linker sequences for combining UCNP. Cas9 RNP cleaves the antioxidant regulator nuclear factor E2-related factor 2 (Nrf2), improving the sensitivity of cancer cells to ROS, and enhancing the synergistic PDT effect. The G-quadruplex/hemin DNAzyme mimicks horseradish peroxidase (HRP) to catalyze the endogenous H₂O₂ to O₂, overcoming hypoxia condition in tumors. The introduced UCNP converts NIR irradiation with deep tissue penetration to light with shorter wavelength, exciting PP to transform the abundant O₂ to ¹O₂. The integration of gene editing and PDT allows substantial accumulation of ¹O₂ in cancer cells for enhanced cell apoptosis, and this synergistic PDT has shown remarkable therapeutic efficacy in a breast cancer mouse model.