Degradable Polymer-Coated Gold Nanoparticles for Co-Delivery of DNA and siRNA

Purpose: There is a need for improved nanobiotechnologies that enable intracellular delivery of difficult-to-deliver biologics such as nucleic acids. Ideally, a delivery material should deliver both large molecules such as DNA and small molecules such as siRNA—enabling both positive and negative regulation of genes—while being non-cytotoxic and capable of imaging/theranostic multifunctionality. Gold nanoparticles (AuNPs) are attractive cores due to their synthesis, monodispersity, biocompatibility, optical properties, and facile thiol functionalization. Layer-by-layer (LbL) approaches can coat AuNPs with charge-alternating polyelectrolytes to complex nucleic acids into enhanced permeability and retention (EPR)-relevant sizes.

Hypothesis: Starting with gold nanoparticles as a core, layer-by-layer degradable polymer coatings enable co-delivery of both DNA and siRNA simultaneously. Polymers that degrade through two different mechanisms—hydrolytic cleavage of ester groups and environmentally triggered degradation of disulfide linkages—can be used to engineer release kinetics. The resulting hybrid inorganic/polymeric nanoparticles will be internalizable by human primary brain cancer cells, reach the cytoplasm and nucleus, mediate exogenous DNA expression and siRNA-mediated knockdown, with knockdown efficacy superior to Lipofectamine® 2000.

Aims: Synthesize and characterize citrate-stabilized AuNPs and 11-mercaptoundecanoic acid (11-MUA)-conjugated AuNPs. - Construct LbL degradable polymer-coated AuNPs for simultaneous DNA and siRNA co-delivery. - Characterize nanoparticle diameter, zeta potential, nucleic acid loading, and layering efficiency. - Evaluate cellular uptake by transmission electron microscopy (TEM). - Assess siRNA-mediated knockdown and exogenous DNA expression in human primary glioblastoma multiforme (GBM) cells. - Compare knockdown/expression efficacy and cytotoxicity against Lipofectamine® 2000.

Approach: In vitro only. No in vivo animal studies. - Cell model: Human primary glioblastoma multiforme cell line GBM319. eGFP-negative cells used for DNA expression; stably eGFP-positive GBM319 cells used for siRNA knockdown. - Transfection: 96-well plates; 10,000 cells/well seeded 1 day prior; formulations delivered as 20 µL; media changed 2 h after delivery. - Controls: Lipofectamine® 2000 as positive control; untreated cells; scrambled siRNA as negative control for knockdown. - Doses: Low dose (LD) and high dose (HD) nucleic acid; outer 447 polymer concentrations 1.25, 2.5, 5 mg/mL; DNA and siRNA co-delivery formulations assessed. - Disease context: Brain cancer/glioblastoma; proof-of-concept theranostic platform.

Key methods: Physicochemical characterization: Dynamic light scattering (DLS), nanoparticle tracking analysis (NanoSight NS500), zeta potential (Malvern Zetasizer), TEM, UV-Vis spectroscopy. - Nucleic acid loading/layering efficiency: Fluorescence intercalating dyes (YO-PRO®-1 for DNA alone; Picogreen® for DNA in presence of siRNA; Ribogreen® for siRNA in presence of DNA), heparin/salt displacement, NanoDrop. - Polymer quantification: Fluoraldehyde™ OPA assay for primary amines; GPC for polymer molecular weight. - Transfection/knockdown: Fluorescence microscopy, plate reader, flow cytometry (BD Accuri C6) for eGFP and dsRed expression; knockdown calculated by comparing anti-eGFP siRNA vs scrambled siRNA. - Cytotoxicity: MTS assay (CellTiter 96®) for relative metabolic activity (RMA). - Cellular uptake: TEM of GBM319 cells after 2 h incubation with HD5 formulation.

Key results: Particle size: CAu = 23 ± 1 nm; MAu = 27 ± 2 nm; layered particles reached ~200 nm. Largest increase at DNA layer (~230 nm). - Zeta potential: Reversed charge with each polyelectrolyte layer, confirming successful LbL assembly. - Nucleic acid loading/layering efficiency: - One DNA layer: 24.1 ± 0.4% - Two DNA layers with degradable SS37 or 447: 5.8 ± 0.5% - Two DNA layers with PEI: 29 ± 1% - Co-delivery DNA/siRNA: DNA = 12 ± 2%; siRNA = 80 ± 3% - siRNA-mediated knockdown: Maximum 44 ± 5% by plate reader and 34 ± 3% by flow cytometry. HD5 and LD5 formulations were superior to Lipofectamine® 2000 at comparable dosages: - vs. Lipo 160 ng (2.5:1): 1.4–1.8-fold higher - vs. Lipo 240 ng (0.5:1): HD2.5, LD5, HD5 were 5.8-, 4.7-, and 2.3-fold higher - DNA-mediated expression: Co-delivery formulations reached up to 10.8 ± 0.5% expression. HD5 was not statistically different from Lipofectamine® 2000 at 100 ng (2.5:1). DNA-only formulations reached up to 37 ± 2%; MAu-P-D-SS37-D-447 achieved 28 ± 1% with RMA 91 ± 2%. - Cytotoxicity: RMA ranged from 73 ± 4% to 91 ± 6% among layered NP formulations. Degraded HD5 after 18 h was statistically similar to non-degraded HD5 (p = 0.34). - Cellular uptake: TEM showed ~200 nm particles in cells, with aggregates in putative endosomes and particles reaching cytoplasm/nucleus.

Interpretation: The authors claim that LbL degradable polymer-coated gold nanoparticles are an enabling theranostic platform technology capable of delivering combinations of genetic therapies to human cells. The system achieved simultaneous DNA expression and siRNA knockdown, with knockdown efficacy superior to Lipofectamine® 2000. The use of two uniquely degrading polymers—hydrolytically degradable 447 and reducible SS37—allows release kinetics to be engineered. The gold core enables imaging and potential photothermal therapy, supporting applications in cancer therapeutics, overcoming drug resistance, promoting apoptosis, inhibiting migration, and rectifying diseases caused by aberrant proteins.

Limitations: In vitro only: No in vivo validation, biodistribution, or therapeutic efficacy in animal models. - Single cell line: Human GBM319 glioblastoma cells only; generalizability to other cell types is unclear. - No targeting ligand: Delivery relies on nonspecific electrostatic interactions; no active tumor targeting. - Modest DNA expression: Co-delivery DNA expression reached ~10.8%; DNA-only formulations reached higher (~37%), indicating co-delivery trade-offs. - Layering efficiency: Two DNA layers with degradable polymers had low layering efficiency (~5.8%) compared to PEI (~29%). - No direct correlation: Relationship between number of particles internalized and expression/knockdown was not established. - No long-term storage/stability data in this manuscript, though prior work by group suggests lyophilization possible. - No in vivo safety or toxicity data. - No targeting or PEGylation optimization, though authors suggest PEG-conjugated 447 or hyaluronic acid as future directions.