Purpose: Endosomal escape remains a major barrier for polymeric gene delivery. Arginine-grafted bioreducible polymers (ABPs) show high transfection via cell-penetrating arginine, but the effect of adding endosome-buffering moieties on their biophysical and transfection properties was not well defined.
Hypothesis: If endosome-buffering 1-(3-aminopropyl)imidazole (API) is copolymerized with arginine-grafted diaminohexane (DAH-R) in bioreducible poly(amido amine)s, then increasing API content will increase endosome buffering capacity and reduce cytotoxicity, but may also reduce pDNA condensation, zeta potential, cellular uptake, and transfection. Arginine content will dominate transfection efficiency.
Aims: Synthesize bioreducible p(DAH-R/API)s with varying DAH-R:API ratios (2:1, 1:1, 1:2). - Characterize buffering capacity, pDNA condensation, particle size, zeta potential, cytotoxicity, cellular uptake, and transfection efficiency. - Investigate the endosomal escape mechanism using chloroquine and nigericin. - Identify the optimal monomer composition for gene delivery.
Delivery system:
Component: Polymer class; Details: Bioreducible cationic poly(amido amine)s (p(DAH-R/API)s)
Component: Backbone; Details: Michael addition of N,N′-cystaminebisacrylamide (CBA, disulfide-containing) with N-Boc-DAH and API
Component: Cell-penetrating moiety; Details: Arginine grafted onto DAH (DAH-R)
Component: Endosome-buffering moiety; Details: 1-(3-aminopropyl)imidazole (API); imidazole pKa ≈ 6
Component: Payload; Details: pCMV-Luc plasmid DNA (firefly luciferase reporter)
Component: Polyplex formation; Details: Electrostatic self-assembly
Component: Targeting ligand; Details: None
Component: Bioreducible feature; Details: Disulfide bonds cleaved by intracellular glutathione/DTT
Approach: In vitro only. C2C12 mouse myoblast cells. - Cytotoxicity: MTT assay; PEI25k as control; polymer concentrations 5–500 µg/mL. - Transfection: Luciferase reporter; serum-free and 10% serum conditions; weight ratios 20–80; PEI25k control at weight ratio 1. - Uptake: Flow cytometry with YOYO-1-labeled pDNA; PEI25k control. - Endosomal escape mechanism: Pretreatment with chloroquine (100 µM) or nigericin (10 µM); ABP as control. - No in vivo studies. No targeting ligand.
Key methods: Synthesis and characterization: ¹H NMR; SEC-MALS for molecular weight. - Buffering capacity: Acid-base titration (pH 7.4–5.1). - pDNA condensation: Agarose gel electrophoresis with and without DTT. - Particle size and zeta potential: Dynamic light scattering / Zetasizer. - Cytotoxicity: MTT assay. - Cellular uptake: Flow cytometry. - Transfection: Luciferase assay normalized to total protein. - Mechanism: Chloroquine and nigericin treatments.
Key results: Buffering capacity increased with API content: 35.4% for p(DAH₂-R/API₁), 39.1% for p(DAH₁-R/API₁), 42.9% for p(DAH₁-R/API₂). - Zeta potential maximum decreased with API content: 22–26 mV, 18–23 mV, and 13–16 mV, respectively. - Cytotoxicity: All p(DAH-R/API)s showed ~100% relative cell viability at 100 µg/mL; PEI25k dropped to 23% at 10 µg/mL and <6% above 100 µg/mL. - Cellular uptake: p(DAH₂-R/API₁) = 69.6%, p(DAH₁-R/API₁) = 52.0%, p(DAH₁-R/API₂) = 43.9%, PEI25k = 60.6%. - Transfection at weight ratio 80: Serum-free: 160-, 53-, and 50-fold higher than PEI25k for p(DAH₂-R/API₁), p(DAH₁-R/API₁), and p(DAH₁-R/API₂), respectively. In serum: 162-, 129-, and 79-fold higher. - Endosomal escape mechanism: Chloroquine reduced transfection to 46–71% of control; nigericin increased transfection to 140–220% for p(DAH-R/API)s and ~10-fold for ABP, suggesting direct endosome membrane penetration by arginine in addition to endosome buffering.
Interpretation: The authors claim that arginine moieties, rather than endosome-buffering API, are the dominant factor for efficient gene delivery in these bioreducible polymers. The p(DAH-R/API) polyplexes appear to escape endosomes by both direct membrane penetration (arginine) and endosome buffering (imidazole), emphasizing the importance of cell-penetrating arginine for polymeric gene delivery systems.
Limitations: In vitro only: No in vivo validation, biodistribution, or therapeutic efficacy. - Single cell line: C2C12 mouse myoblasts only. - No targeting ligand: Delivery relies on nonspecific electrostatic interactions. - pAPI excluded: The API-only polymer was insoluble at pH 7.4 and not tested beyond NMR. - Mechanism inferred pharmacologically: Chloroquine/nigericin data suggest a mechanism but no direct imaging or trafficking studies. - Short-term assays: Transfection measured after 2 days; long-term expression and toxicity not assessed. - No direct comparison to ABP across all ratios: ABP used mainly as a control in mechanism studies. - Citation DOI missing in supplied file.
