Multifunctional Core-Shell Tecto Dendrimers For CRISPR/Cas9-Mediated Immune Checkpoint Blocking Therapy

Immune checkpoint blocking(ICB)therapy could activate the immune system to inhibit the development and metastasis of tumors,and hence has become a promising therapeutic strategy.Programmed death ligand 1(PD-L1)is considered to be a key regulator in the development of tumors.Regulatory strategies,such as PD-L1 monoclonal antibody or small interfering RNA,are still unsatisfied due to the temporary and inefficient blocking and the poor immune responsiveness.Clustered regularly interspersed short palindromic repeats/associated nuclease 9(CRISPR/Cas9)gene editing techniques could knockout the PD-L1 gene permanently and specifically,leading to more durable and effective ICB therapy.It is an urgent need to efficiently and specifically deliver the CRISPR/Cas9 system to tumor cells.In this paper,multifunctional Au nanoparticles loaded core-shell tecto dendrimers(Au CSTDs)were synthesized to deliver plasmid-based CRISPR/Cas9 system(Cas9-PD-L1)for permanent disruption of PD-L1 gene and efficient immunotherapy.The Au CSTDs were constructed by using lactobionic acid(LA)modified gold nanoparticles entrapped generation 5 PAMAM dendrimers as core and phenylboronic acid(PBA)conjugated generation 3 PAMAM dendrimers as shell,via the formation of responsive phenylborate ester bonds.Then Cas9-PD-L1 could be efficiently compacted and specifically taken up by cancer cells overexpressing sialic acids due to the PBA-mediated targeting,and be responsively released in cancer cells due to the responsive dissociation of the Au CSTDs,leading to the successful endosomal escape and efficient knockout of PD-L1 gene(59.8%).Further in vivo delivery in a mouse melanoma model reveals that the developed Au CSTDs/plasmidCRISPR/Cas9 complexes can be specifically accumulated at tumor site for enhanced CT imaging of tumors owing to the X-ray attenuation effect of Au,and disrupt the PD-L1 expression in tumor cells,thus promoting the ICB-based antitumor immunity.In conclusion,we successfully prepared multifunctional Au CSTDs to deliver plasmidCRISPR/Cas9 system for effective immune checkpoint blocking therapy and CT imaging,providing a new idea for dendritic macromolecular nanoplatform delivery of CRISPR/Cas9 system for disease treatment.