Lipid- and polymer-based nanoparticle systems for the delivery of CRISPRCas9

Purpose: CRISPR/Cas9 genome editing holds major therapeutic promise, but in vivo delivery is arguably the most difficult barrier to clinical translation. Efficient, selective transport of editing components to target cells/tissues while minimising off-target effects remains unresolved. This review critiques lipid- and polymer-based nanoparticle delivery strategies for CRISPR/Cas9.

Hypothesis: As a review, no formal hypothesis is tested. The central working premise is that engineered lipid- and polymeric nanoparticles—with optimised size, shape, surface charge, endosomal escape, and targeting—can safely and efficiently deliver CRISPR/Cas9 cargo (plasmid DNA, mRNA, Cas9 protein/sgRNA, or RNP) and achieve genome editing in vitro and in vivo.

Aims: Introduce CRISPR/Cas9 mechanisms, repair pathways, and delivery barriers. - Review limitations of viral and physical delivery approaches. - Critically discuss lipid-based and polymeric nanoparticle systems for CRISPR/Cas9 delivery. - Highlight key carrier properties, targeting strategies, stimuli-responsive systems, and future translation challenges.

Delivery system: Lipid-based: liposomes, solid lipid nanoparticles (SLNs), nanostructured lipid carriers (NLCs), cationic/ionizable lipid nanoparticles, Lipofectamine, PLNP, zwitterionic amino lipids (ZALs), bioreducible lipid BAMEA-O16B, lipid/gold nanoclusters (LGCP). Polymer-based: PEI, PEI-β-cyclodextrin, PAMAM, PBAE hyperbranched copolymers, chitosan, PLGA, cationic lipid-assisted nanoparticles (CLAN), liposome-templated hydrogel nanoparticles (LHNPs), polymeric capsules, DNA-responsive hydrogels. Payloads: Cas9 protein, Cas9 mRNA, sgRNA, plasmid DNA, minicircle DNA. Targeting/functionalization: antibodies (e.g., EGFR), aptamers (e.g., A10), peptides (TAT, iRGD, mHph3), albumin, biomimetic cell membranes, exosome–liposome hybrids.

Approach: Review article; no original experimental groups, n, doses, or controls. It synthesises published in vitro studies (HEK293, A375, CT26, U87, GS5, HepG2, HeLa, etc.) and in vivo rodent models (mouse TTR knockdown, melanoma, intracranial U87 tumours, type 2 diabetes, chronic myeloid leukaemia, hepatocellular carcinoma).

Key methods: Summarises techniques from cited studies: transfection/gene-editing efficiency, GFP knockout, mRNA/protein knockdown (TTR, PCSK9, PLK1, VEGFR2, NLRP3, Ntn1, BCR-ABL), tumour growth inhibition, biodistribution/imaging, and cytotoxicity/viability assays.

Key results: LPO1 lipid nanoparticle: >97% knockdown of mouse TTR protein. - BAMEA-O16B bioreducible LNP: up to 90% GFP knockout in HEK cells; 80% PCSK9 knockdown in mouse serum after IV injection. - Cationic lipid NP with protamine/Cas9-sgPLK-1: 47.4% transfection in A375 cells; >67% tumour growth suppression after intratumoral injection. - LHNPs: 79.3% and 80.2% growth inhibition in U87 and GS5 cells; 60.4% PLK1 inhibition in intracranial U87 tumours. - EGFR-targeted lipid nanoparticles: up to 80% PLK1 gene editing in vivo. - Polymeric/hybrid SiO₂ capsules: >70% eGFP-mRNA transfection vs <50% for a commercial liposome reagent.

Interpretation: No single optimal CRISPR/Cas9 delivery platform exists; lipid- and polymer-based nanoparticles each offer distinct advantages and disadvantages. These systems can accommodate diverse cargo, be functionalised for cell-specific targeting, and improve endosomal escape, but clinical translation requires better understanding of circulation, pharmacokinetics, systemic editing, safety, and scalable manufacturing.

Limitations: Review only; no new primary data. Cited studies vary widely in cell types, animal models, doses, and endpoints. Long-term safety, off-target effects, immunogenicity, large-animal validation, and clinical data remain limited. Some carriers (PEI, PAMAM) have cytotoxicity; exosome/hybrid systems face low yield, heterogeneity, and scale-up challenges.