Purpose: Systemic delivery of RNA therapeutics with polymer-based nanocarriers is limited by poor colloidal stability in blood and inefficient intracellular delivery of siRNA to the cytosol. There is a need for nanoparticles that remain stable extracellularly but rapidly release RNA intracellularly.
Hypothesis: If bioreducible poly(β-amino ester) (PBAE) nanoparticles are photocrosslinked, then they will have improved colloidal stability, reduced serum protein adsorption, enhanced cellular uptake, and efficient siRNA-mediated knockdown under high-serum conditions and after systemic administration in vivo.
Aims: Synthesize acrylate-terminated crosslinking PBAE and amine-terminated cationic PBAEs containing disulfide bonds. - Form siRNA-loaded nanoparticles and photocrosslink them via UV/photoinitiator. - Characterize size, surface charge, protein adsorption, serum stability, and bioreducible release. - Evaluate in vitro siRNA delivery in glioma and melanoma cells under high-serum conditions. - Assess cellular uptake, endosomal escape, biodistribution, in vivo knockdown, and safety.
Delivery system:
Component: Polymer class; Details: Bioreducible poly(β-amino ester)s (PBAEs) with ester bonds for hydrolysis and disulfide bonds for cytosolic release
Component: Crosslinking polymer; Details: Acrylate-terminated R64-Ac
Component: Cationic end-capped polymers; Details: R646 (E6 endcap) or R647 (E7 endcap)
Component: Payload; Details: siRNA (eGFP, luciferase, Bcl-2, scrambled control)
Component: Crosslinking; Details: Photoinitiator Irgacure 2959 + UV light (350 nm, 0.5–2 min)
Component: Nanoparticle size; Details: ~204–207 nm
Component: Surface charge; Details: Shielded relative to non-crosslinked formulations
Component: Targeting ligand; Details: None
Component: Key feature; Details: Photocrosslinked bioreducible nanoparticles (XbNPs) with reduced serum protein adsorption and tunable organ targeting by polymer/siRNA ratio
Approach: In vitro: Patient-derived glioblastoma cells (GBM319, GBM1A), murine glioma (GL261), murine melanoma (B16F10); high serum conditions (50–100%). - In vivo: C57BL/6 mice; B16F10 metastatic lung melanoma model; repeated i.v. injections of XbNPs carrying siLuc, siBcl-2, or scRNA (n = 7). - Biodistribution: IR-labeled siRNA; organ imaging 18 h after i.v. injection (n = 3–5). - Safety: Serum AST and ALT after four repeated i.v. injections (n = 4). - No large-animal validation.
Key methods: Polymer/nanoparticle characterization: GPC, ¹H NMR, DLS, NTA, TEM, zeta potential. - Crosslinking: Acrylate peak integration by NMR. - Stability/release: Gel electrophoresis in 50% serum and 10 mM GSH. - Protein adsorption: BCA assay, SDS-PAGE. - In vitro knockdown/viability: Flow cytometry for GFP, MTS assay. - Uptake/endosomal escape: Flow cytometry, confocal microscopy, Gal8-GFP recruitment assay. - In vivo: IVIS biodistribution and bioluminescence; flow cytometry of lung cell populations; AST/ALT assays.
Key results: Crosslinking: Molecular weight increased by 42.1% (Mn) and 27.7% (Mw); acrylate peak intensity decreased by 79.1% ± 0.3%. - Serum stability: XbNPs retained siRNA encapsulation in 50% serum; non-crosslinked formulations largely dissociated by 2 h. - In vitro knockdown: XbNPs achieved up to 96% ± 2% GFP knockdown in GBM319 cells under high serum; >50% in GBM1A and GL261; near-complete knockdown in B16F10 at 400 w/w. - Long-term serum stability: After 6 h pre-incubation in 100% serum, XbNPs still caused 82% ± 2% GFP knockdown. - Uptake: XbNPs showed higher cellular uptake than non-crosslinked nanoparticles at 6 and 24 h in 50% and 100% serum; endosomal escape was not significantly different. - In vivo biodistribution: XbNPs at 900 w/w targeted lungs; at 400 w/w targeted spleen. - In vivo knockdown: Systemic siLuc and siBcl-2 XbNPs reduced B16F10 lung tumor bioluminescence (p < 0.005; n = 7). - Safety: No significant increase in AST or ALT after four repeated i.v. injections (p = 0.13 and p = 0.65).
Interpretation: The authors claim that photocrosslinked bioreducible PBAE nanoparticles address the key limitations of cationic polymeric siRNA carriers by improving colloidal stability, shielding surface charge, reducing serum protein adsorption, enabling efficient endosomal escape, and facilitating environmentally triggered cytosolic release. They conclude that XbNPs are a promising platform for systemic RNA therapeutics and that the photocrosslinking strategy may be generalizable to other cationic nanocarriers.
Limitations: In vivo model: Only a murine metastatic melanoma lung model; no survival or tumor volume endpoints. - No large-animal validation. - No targeting ligand: Organ targeting achieved by tuning polymer/siRNA ratio, not active targeting. - Limited safety data: Only AST/ALT after repeated dosing; no comprehensive toxicity or long-term safety. - Mechanism partly unresolved: Endosomal escape was unchanged; enhanced efficacy mainly attributed to improved uptake and stability. - Payload scope: Only siRNA tested; no mRNA, plasmid DNA, or CRISPR cargo. - No direct comparison with clinically approved lipid nanoparticles (e.g., Onpattro). - Potential UV/photoinitiator concerns not fully addressed for translation. - No human data.
