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.

Core polymer optimization of ternary siRNA nanoparticles enhances in vivo safety, pharmacokinetics, and tumor gene silencing.

Gene silencing with siRNA nanoparticles (si-NPs) is promising but still clinically unrealized for inhibition of tumor driver genes. Ternary si-NPs containing siRNA, a single block NP core-forming polymer poly[(2-(dimethylamino)ethyl methacrylate)-co-(butyl methacrylate)] (DMAEMA-co-BMA, 50B), and an NP surface-forming diblock polymer 20 kDa poly(ethylene glycol)-block-50B (20kPEG-50B) have the potential to improve silencing activity in tumors due to the participation of both 50B and 20kPEG-50B in siRNA electrostatic loading and endosome disruptive activity. Functionally, single block 50B provides more potent endosomolytic activity, while 20kPEG-50B colloidally stabilizes the si-NPs. Here, we systematically explored the role of the molecular weight (MW) of the core polymer and of the core:surface polymer ratio on ternary si-NP performance. A library of ternary si-NPs was formulated with variation in the MW of the 50B polymer and in the ratio of the core and surface forming polymeric components. Increasing 50B core polymer MW and ratio improved si-NP in vitro gene silencing potency, endosome disruptive activity, and stability, but these features also correlated with cytotoxicity. Concomitant optimization of 50B size and ratio resulted in the identification of lead ternary si-NPs 50B4-DP100, 50B8-DP100, and 50B12-DP25, with potent activity and minimal toxicity. Following intravenous treatment in vivo, all lead si-NPs displayed negligible toxicological effects and enhanced pharmacokinetics and tumor gene silencing relative to more canonical binary si-NPs. Critically, a single 1 mg/kg intravenous injection of 50B8-DP100 si-NPs silenced the tumor driver gene Rictor at the protein level by 80% in an orthotopic breast tumor model. 50B8-DP100 si-NPs delivering siRictor were assessed for therapeutic efficacy in an orthotopic HCC70 mammary tumor model. This formulation significantly inhibited tumor growth compared to siControl-NP treatment. 50B8-DP100 si-NPs were also evaluated for safety and were well-tolerated following a multi-dose treatment scheme. This work provides new insight on ternary si-NP structure-function relationships and identifies core polymer optimization strategies that can yield safe si-NP formulations with potent oncogene silencing.

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.

Structural Optimization of Polymeric Carriers to Enhance the Immunostimulatory Activity of Molecularly Defined RIG-I Agonists.

RNA ligands of retinoic acid-inducible gene I (RIG-I) hold significant promise as antiviral agents, vaccine adjuvants, and cancer immunotherapeutics, but their efficacy is hindered by inefficient intracellular delivery to the cytosol where RIG-I is localized. Here, we address this challenge through the synthesis and evaluation of a library of polymeric carriers rationally designed to promote the endosomal escape of 5'-triphosphate RNA (3pRNA) RIG-I agonists. We synthesized a series of PEG-block-(DMAEMA-co-A n MA) polymers, where A n MA is an alkyl methacrylate monomer ranging from n = 2-12 carbons, of variable composition, and examined effects of polymer structure on the intracellular delivery of 3pRNA. Through in vitro screening of 30 polymers, we identified four lead carriers (4-50, 6-40, 8-40, and 10-40, where the first number refers to the alkyl chain length and the second number refers to the percentage of hydrophobic monomer) that packaged 3pRNA into ∼100-nm-diameter particles and significantly enhanced its immunostimulatory activity in multiple cell types. In doing so, these studies also revealed an interplay between alkyl chain length and monomer composition in balancing RNA loading, pH-responsive properties, and endosomal escape, studies that establish new structure-activity relationships for polymeric delivery of 3pRNA and other nucleic acid therapeutics. Importantly, lead carriers enabled intravenous administration of 3pRNA in mice, resulting in increased RIG-I activation as measured by increased levels of IFN-α in serum and elevated expression of Ifnb1 and Cxcl10 in major clearance organs, effects that were dependent on polymer composition. Collectively, these studies have yielded novel polymeric carriers designed and optimized specifically to enhance the delivery and activity of 3pRNA with potential to advance the clinical development of RIG-I agonists.

Modifying Cell Membranes with Anionic Polymer Amphiphiles Potentiates Intracellular Delivery of Cationic Peptides.

Rapid, facile, and noncovalent cell membrane modification with alkyl-grafted anionic polymers was sought as an approach to enhance intracellular delivery and bioactivity of cationic peptides. We synthesized a library of acrylic acid-based copolymers containing varying amounts of an amine-reactive pentafluorophenyl acrylate monomer followed by postpolymerization modification with a series of alkyl amines to afford precise control over the length and density of aliphatic alkyl side chains. This synthetic strategy enabled systematic investigation of the effect of the polymer structure on membrane binding, potentiation of peptide cell uptake, pH-dependent disruption of lipid bilayers for endosome escape, and intracellular bioavailability. A subset of these polymers exhibited pKa of ∼6.8, which facilitated stable membrane association at physiological pH and rapid, pH-dependent endosomal disruption upon endocytosis as quantified in Galectin-8-YFP reporter cells. Cationic cell penetrating peptide (CPP) uptake was enhanced up to 15-fold in vascular smooth muscle cells in vitro when peptide treatment was preceded by a 30-min pretreatment with lead candidate polymers. We also designed and implemented a new and highly sensitive assay for measuring the intracellular bioavailability of CPPs based on the NanoLuciferase (NanoLuc) technology previously developed for measuring intracellular protein-protein interactions. Using this split luciferase class of assay, polymer pretreatment enhanced intracellular delivery of the CPP-modified HiBiT peptide up to 30-fold relative to CPP-HiBiT without polymer pretreatment (p < 0.05). The overall structural analyses show that polymers containing 50:50 or 70:30 molar ratios of carboxyl groups to alkyl side chains of 6-8 carbons maximized peptide uptake, pH-dependent membrane disruption, and intracellular bioavailability and that this potentiation effect was maximized by pairing with CPPs with high cationic charge density. These results demonstrate a rapid, mild method for polymer modification of cell surfaces to potentiate intracellular delivery, endosome escape, and bioactivity of cationic peptides.

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.

An anionic, endosome-escaping polymer to potentiate intracellular delivery of cationic peptides, biomacromolecules, and nanoparticles.

Peptides and biologics provide unique opportunities to modulate intracellular targets not druggable by conventional small molecules. Most peptides and biologics are fused with cationic uptake moieties or formulated into nanoparticles to facilitate delivery, but these systems typically lack potency due to low uptake and/or entrapment and degradation in endolysosomal compartments. Because most delivery reagents comprise cationic lipids or polymers, there is a lack of reagents specifically optimized to deliver cationic cargo. Herein, we demonstrate the utility of the cytocompatible polymer poly(propylacrylic acid) (PPAA) to potentiate intracellular delivery of cationic biomacromolecules and nano-formulations. This approach demonstrates superior efficacy over all marketed peptide delivery reagents and enhances delivery of nucleic acids and gene editing ribonucleoproteins (RNPs) formulated with both commercially-available and our own custom-synthesized cationic polymer delivery reagents. These results demonstrate the broad potential of PPAA to serve as a platform reagent for the intracellular delivery of cationic cargo.