Cascade‐Responsive “Oxidative Stress Amplifiers” Simultaneously Destroy Lysosomes and Co‐Deliver CRISPR/Cas9 to Enhance Oxidative Damage in Tumor

Abstract

Amplifying intracellular oxidative stress by organelle‐targeted reactive oxygen species (ROS) production combined with tumor cell‐specific gene disruption is a promising strategy for tumor treatment. However, due to the vulnerability of CRISPR/Cas9 ribonucleoproteins (RNPs) to ROS, co‐delivery of CRISPR/Cas9 RNPs and ROS generators to enhance the sensitivity of tumor cells to oxidative stress remains challenging. Herein, a cascade‐responsive “oxidative stress amplifier” (named DR‐TAF‐pHT/FA) is proposed, which can successively respond to cathepsin B, localized laser irradiation and ATP to generate ROS on the lysosomal membrane of tumor cells and release Cas9/sgNrf2 RNPs for efficient gene disruption. It is demonstrated that, under near infrared (NIR) irradiation, DR‐TAF‐pHT/FA achieves targeted rupture of lysosomal membranes, inducing significant intracellular oxidative stress. Meanwhile, due to the protective function of TAF coating (TA‐Fe3+ coordination self‐assembled networks), Cas9/sgNrf2 RNPs can safely escape into the cytoplasm and be released in response to ATP, further amplifying oxidative stress and promoting tumor cell apoptosis through efficient Nrf2 gene disruption. Treatment with DR‐TAF‐pHT/FA + NIR significantly improves tumor ablation efficiency and extends median survival time (over 70 days) in Hela xenograft models. This “oxidative stress amplifier” provides a new paradigm for multimodal and synergistic tumor therapy through precise lysosomal membrane bursting together with efficient Nrf2 gene disruption.

Amplification of Oxygen-Independent Free Radicals Based on a Glutathione Depletion and Biosynthesis Inhibition Strategy for Photothermal and Thermodynamic Therapy of Hypoxic Tumors

“…As displayed in Figure f, cells treated with GZABEF + L had the weakest red fluorescence compared with other treatments, confirming the lowest MMP and the most severe mitochondrial damage. High levels of ROS in cells may also disrupt the lysosomal membrane, which is essential for maintaining cell proliferation . As shown in Figure S7, the strongest green fluorescence was found in the GZABEF + L group, indicating the most serious lysosomal damage.…”

“…High levels of ROS in cells may also disrupt the lysosomal membrane, which is essential for maintaining cell proliferation. 23 As shown in Figure S7, the strongest green fluorescence was found in the GZABEF + L group, indicating the most serious lysosomal damage. These results demonstrated that GZABEF could disrupt intracellular redox homeostasis under laser irradiation through the production of a large number of alkyl radicals, the consumption of endogenous and exogenous GSH, and the inhibition of GPX4 activity, ultimately inducing mitochondriamediated cell apoptosis.…”

A quaternary ammonium-based nanosystem enables delivery of CRISPR/Cas9 for cancer therapy

“…9 In contrast, CRISPR/Cas9 RNP complexes could be directly delivered into cells for rapid and highly efficient genome editing without transcription and translation. 10 The preformed RNP also reduces the possibility of off-target events and minimizes immunogenicity. 11 Nevertheless, the delivery of the RNP also faces many difficulties: RNP is easily denatured and has negligible intracellular delivery activity due to the high molecular weight of Cas9 (160 kDa) and sgRNA (∼31 kDa, ∼130 bases).…”