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. 2021 Dec 16;9:786354. doi: 10.3389/fchem.2021.786354

FIGURE 7.

FIGURE 7

NIR-II/CRISPR-Cas9 system photothermal genome-editing strategy for cancer immunotherapy. (A) The frame of ANP/HSP-Cas9 complex synthesis, photothermal activation for PD-L1 genome editing and how to change the immunosuppressive tumor environment. ANR is composed of Au nanorods, biocompatible polystyrene sulfonate (PSS), and a supramolecular polymer (termed as PCM). Au nanorods act as a photothermal converter to induce unactive HSF monomer to active HSF trimer. The efficient intracellular delivery of ANP/HSP-Cas9 plasmid, can induce the transcriptional activation of Cas9 and sgRNA targeting PD-L1 upon NIR-II light irradiation, thereby enabling the precise genome editing of PD-L1. The heat produced by irradiation provide a mild condition which promotes immune memory, activates T cells, and improves T cell infiltration, transforms immunogenic-cold tumors into immunogenic-hot ones. This reprogramming tumor environment is beneficial for killing other distant tumor. (B) The schedule of tumor therapy by NIRII-controlled ANP/HSP-Cas9 in vivo. (C) Immunofluorescence analysis of HMGB1, CRT and PD-L1 protein expression from tumor tissue at different tumor depth. The significant CRT exposure within tumors can be implemented at depths up to 6 mm under NIR-II laser irradiation. (D) T7E1 assay was carried out to indicate indel mutations of PD-L1 in the tumor tissue. A high degree of genome editing was detected in tumor tissues. Western blot analysis of HMGB1, CRT, and PD-L1 proteins expression after different treated. The PD-L1 expression in tumors is significantly decreased by ANP-mediated genome editing. (E) Sanger sequencing results at PD-L1 locus from tumor tissue. Significant deletions and insertions are detected at the targeted loci around the PAM. ANP(+), ANP with NIR-II laser irradiation. ANP(−), ANP without NIR-II laser irradiation. ANP/P(+), ANP/HSP-Cas9 with NIR-II laser irradiation. ANP/P(−), ANP/HSP-Cas9 without NIR-II laser irradiation. (F) Representative in vivo bioluminescence images of mice after different treatment at day 21. (G) Tumor growth curves after being treated by ANP/HSP-Cas9 complexes with or without irradiation. (H) Body weight and Kaplan–Meier survival curves of tumor-bearing mice. ANP(+) significantly inhibit tumor growth, while the mice treated with ANP/HSP-Cas9 are more potent in delaying tumor growth after NIR-II laser irradiation. ANP(−) and ANP/P(−) show similar trend of tumor growth in comparison with the control group (reproduced from (Tang et al., 2021) with permission from Advanced Materials (Deerfield Beach, Fla.)).