Nonviral In Vivo Delivery of CRISPR-Cas9 Using Protein-Agnostic, High-Loading Porous Silicon and Polymer Nanoparticles.

The complexity of CRISPR machinery is a challenge to its application for nonviral in vivo therapeutic gene editing. Here, we demonstrate that proteins, regardless of size or charge, efficiently load into porous silicon nanoparticles (PSiNPs). Optimizing the loading strategy yields formulations that are ultrahigh loading─>40% cargo by volume─and highly active. Further tuning of a polymeric coating on the loaded PSiNPs yields nanocomposites that achieve colloidal stability under cryopreservation, endosome escape, and gene editing efficiencies twice that of the commercial standard Lipofectamine CRISPRMAX. In a mouse model of arthritis, PSiNPs edit cells in both the cartilage and synovium of knee joints, and achieve 60% reduction in expression of the therapeutically relevant MMP13 gene. Administered intramuscularly, they are active over a broad dose range, with the highest tested dose yielding nearly 100% muscle fiber editing at the injection site. The nanocomposite PSiNPs are also amenable to systemic delivery. Administered intravenously in a model that mimics muscular dystrophy, they edit sites of inflamed muscle. Collectively, the results demonstrate that the PSiNP nanocomposites are a versatile system that can achieve high loading of diverse cargoes and can be applied for gene editing in both local and systemic delivery applications.

Kupffer cell release of platelet activating factor drives dose limiting toxicities of nucleic acid nanocarriers.

This work establishes that Kupffer cell release of platelet activating factor (PAF), a lipidic molecule with pro-inflammatory and vasoactive signaling properties, dictates dose-limiting siRNA nanocarrier-associated toxicities. High-dose intravenous injection of siRNA-polymer nano-polyplexes (si-NPs) elicited acute, shock-like symptoms in mice, associated with increased plasma PAF and consequently reduced PAF acetylhydrolase (PAF-AH) activity. These symptoms were completely prevented by prophylactic PAF receptor inhibition or Kupffer cell depletion. Assessment of varied si-NP chemistries confirmed that toxicity level correlated to relative uptake of the carrier by liver Kupffer cells and that this toxicity mechanism is dependent on carrier endosome disruptive function. 4T1 tumor-bearing mice, which exhibit increased circulating leukocytes, displayed greater sensitivity to these toxicities. PAF-mediated toxicities were generalizable to commercial delivery reagent in vivo-jetPEI® and an MC3 lipid formulation matched to an FDA-approved nanomedicine. These collective results establish Kupffer cell release of PAF as a key mediator of siRNA nanocarrier toxicity and identify PAFR inhibition as an effective strategy to increase siRNA nanocarrier tolerated dose.

Tuning Composition of Polymer and Porous Silicon Composite Nanoparticles for Early Endosome Escape of Anti-microRNA Peptide Nucleic Acids.

Porous silicon nanoparticles (PSNPs) offer tunable pore structure and easily modified surface chemistry, enabling high loading capacity for drugs with diverse chemicophysical properties. While PSNPs are also cytocompatible and degradable, PSNP integration into composite structures can be a useful approach to enhance carrier colloidal stability, drug-cargo loading stability, and endosome escape. Here, we explored PSNP polymer composites formed by coating of oxidized PSNPs with a series of poly[ethylene glycol-block-(dimethylaminoethyl methacrylate-co-butyl methacrylate)] (PEG-DB) diblock copolymers with varied molar ratios of dimethylaminoethyl methacrylate (D) and butyl methacrylate (B) in the random copolymer block. We screened and developed PSNP composites specifically toward intracellular delivery of microRNA inhibitory peptide nucleic acids (PNA). While a copolymer with 50 mol % B (50B) is optimal for early endosome escape in free polymer form, its pH switch was suppressed when it was formed into 50B polymer-coated PSNP composites (50BCs). We demonstrate that a lower mol % B (30BC) is the ideal PEG-DB composition for PSNP/PEG-DB nanocomposites based on having both the highest endosome disruption potential and miR-122 inhibitory activity. At a 1 mM PNA dose, 30BCs facilitated more potent inhibition of miR-122 in comparison to 40BC (p = 0.0095), 50BC (p < 0.0001), or an anti-miR-122 oligonucleotide delivered with the commercial transfection reagent Fugene 6. Using a live cell galectin 8-based endosome disruption reporter, 30BCs had greater endosomal escape than 40BCs and 50BCs within 2 h after treatment, suggesting that rapid endosome escape correlates with higher intracellular bioactivity. This study provides new insight on the polymer structure-dependent effects on stability, endosome escape, and cargo intracellular bioavailability for endosomolytic polymer-coated PSNPs.

Gal8 Visualization of Endosome Disruption Predicts Carrier-Mediated Biologic Drug Intracellular Bioavailability.

Endolysosome entrapment is one of the key barriers to the therapeutic use of biologic drugs that act intracellularly. The screening of prospective nanoscale endosome-disrupting delivery technologies is currently limited by methods that are indirect and cumbersome. Here, we statistically validate Galectin 8 (Gal8) intracellular tracking as a superior approach that is direct, quantitative, and predictive of therapeutic cargo intracellular bioactivity through in vitro high-throughput screening and in vivo validation. Gal8 is a cytosolically dispersed protein that, when endosomes are disrupted, redistributes by binding to glycosylation moieties selectively located on the inner face of endosomal membranes. The quantitative redistribution of a Gal8 fluorescent fusion protein from the cytosol into endosomes is demonstrated as a real-time, live-cell assessment of endosomal integrity that does not require labeling or modification of either the carrier or the biologic drug and that allows quantitative distinction between closely related, endosome-disruptive drug carriers. Through screening two families of siRNA polymeric carrier compositions at varying dosages, we show that Gal8 endosomal recruitment correlates strongly ( r = 0.95 and p < 10-4) with intracellular siRNA bioactivity. Through this screen, we gathered insights into how composition and molecular weight affect endosome disruption activity of poly[(ethylene glycol)- b-[(2-(dimethylamino)ethyl methacrylate)- co-(butyl methacrylate)]] [PEG-(DMAEMA- co-BMA)] siRNA delivery systems. Additional studies showed that Gal8 recruitment predicts intracellular bioactivity better than current standard methods such as Lysotracker colocalization ( r = 0.35, not significant), pH-dependent hemolysis (not significant), or cellular uptake ( r = 0.73 and p < 10-3). Importantly, the Gal8 recruitment method is also amenable to fully objective high-throughput screening using automated image acquisition and quantitative image analysis, with a robust estimated Z' of 0.6 (whereas assays with Z' > 0 have high-throughput screening utility). Finally, we also provide measurements of in vivo endosomal disruption based on Gal8 visualization ( p < 0.03) of a nanocarrier formulation confirmed to produce significant cytosolic delivery and bioactivity of siRNA within tumors ( p < 0.02). In sum, this report establishes the utility of Gal8 subcellular tracking for the rapid optimization and high-throughput screening of the endosome disruption potency of intracellular delivery technologies.