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Hierarchically tumor-activated nanoCRISPR-Cas13a facilitates efficient microRNA disruption for multi-pathway-mediated tumor suppression

ABSTRACT: Rationale: CRISPR-Cas13a is an efficient tool for robust RNA knockdown with lower off-target effect, which may be a potentially powerful and safe tool for cancer gene therapy. However, therapeutic effect of current cancer gene therapy that targeting monogene was compromised by the multi-mutational signal pathway alterations of tumorigenesis. Methods: Here, hierarchically tumor-activated nanoCRISPR-Cas13a (CHAIN) is fabricated for multi-pathway-mediated tumor suppression by efficient microRNA disruption in vivo. A fluorinated polyetherimide (PEI; Mw=1.8KD) with graft rate of 33% (PF33) was utilized to compact the CRISPR-Cas13a megaplasmid targeting microRNA-21 (miR-21) (pCas13a-crRNA) via self-assemble to constitute a nanoscale 'core' (PF33/pCas13a-crRNA), which was further wrapped by modified hyaluronan (HA) derivatives (galactopyranoside-PEG2000-HA, GPH) to form CHAIN. Results: The dual-tumor-targeting and tumor-activated CHAIN not only manifested long-term circulation, but augmented tumor cellular uptake and endo/lysosomal escape, thus achieving efficient transfection of CRISPR-Cas13a megaplasmid (~ 13 kb) in tumor cells with minimal toxity. Efficient knockdown of miR-21 by CHAIN restored programmed cell death protein 4 (PDCD4) and reversion‐inducing‐cysteine‐rich protein with Kazal motifs (RECK) and further crippled downstream matrix metalloproteinases-2 (MMP-2), which undermined cancer proliferation, migration and invasion. Meanwhile, the miR-21-PDCD4-AP-1 positive feedback loop further functioned as an enhanced force for anti-tumor activity. Conclusion: Treatment with CHAIN in hepatocellular carcinoma mouse model achieved significant inhibition of miR-21 expression and rescued multi-pathway, which triggered substantial tumor growth suppression. By efficient CRISPR-Cas13a induced interference of one oncogenic microRNA, the CHAIN platform exerted promising capabilities in cancer treatment.

SUBMITTER: Liu X

PROVIDER: S-EPMC10240819 | biostudies-literature | 2023 Jan

REPOSITORIES: biostudies-literature

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Project description:The delivery of therapeutics into tumors remains a challenge in nanoparticle-mediated drug delivery. However, effective therapies such as photothermal therapy (PTT) are limited by quick systemic clearance and non-specific biodistribution. Anti-tumor strategies tailored to accommodate both tumor accumulation/retention and cellular internalization under a single platform would be a promising strategy. This work demonstrates a hierarchical activating strategy that would exhibit enhanced circulation and rapid tumor-tropism as well as facilitate tumor penetration, followed by tumor-specific drug release to realize trackable photothermal/chemotherapy. Methods: We engineered a lithocholic acid-conjugated disulfide-linked polyethyleneimine micelle (LAPMi) loaded with paclitaxel (LAPMi-PTX, L), followed by the electrostatic adsorption of indocyanine green (ICG, I) on LAPMI-PTX and subsequently coated them with thermosensitive DPPC and DSPE-PEG-NH2 lipids (L), producing Lipid/ICG/LAPMi-PTX (LIL-PTX) nanoparticles (NPs). The characteristics of NPs, including physicochemical characterization, photothermal & pH responsiveness, cell uptake, tumor spheroid penetration, anti-tumor efficacy and hierarchical activation of LIL-PTX NPs were investigated in vitro and in vivo by using CT26 cell line. The anti-metastatic potential of LIL-PTX NPs were demonstrated using 4T1 orthotopic tumor model. Results: The NPs synthesized possessed charge switchability in the mildly acidic pH, and were laser- and pH-responsive. Dual stimuli-responsive nature of LIL-PTX NPs improved the disposition of therapeutics to the tumor, reflected by enhanced intracellular uptake, tumor spheroid penetration and in vitro cytotoxicity studies. LIL-PTX NPs readily switched its surface charge from neutral to positive upon reaching the tumor milieu, thus resulting in rapid tumor tropism and accumulation. Under near-infrared laser irradiation, the thermosensitive lipids on LIL-PTX NPs were deshielded, and the tumor-penetrating LAPMi-PTX was subsequently exposed to the tumor milieu, thus resulting in enhanced intracellular internalization. Next, LAPMi-PTX evaded the endo-lysosomes, thereby releasing the PTX through the degradation of LAPMi mediated by intracellular GSH in the tumor. LIL-PTX NPs significantly improved the therapy by eradicating primary tumors completely and suppressing their subsequent lung metastasis. Conclusion: The improved therapeutic index is due to enhanced passive targeting by rapid tumor-tropic accumulation and tumor penetration by laser-driven exposure of LAPMi, thereby improving the therapeutic delivery for image-guided photothermal/chemotherapy.

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Hierarchically tumor-activated nanoCRISPR-Cas13a facilitates efficient microRNA disruption for multi-pathway-mediated tumor suppression

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