Bioreducible polymers with cell penetrating and endosome buffering functionality for gene delivery systems

Area of Science:

• Biomaterials Science
• Polymer Chemistry
• Gene Delivery Systems

Background:

• Cationic polymers are crucial for gene delivery, but their efficiency is often limited by cellular uptake and endosomal escape.
• Bioreducible polymers offer advantages in controlled release and reduced toxicity.
• Arginine and imidazole moieties are known to play roles in cell penetration and endosomal buffering, respectively.

Purpose of the Study:

• To synthesize and characterize bioreducible cationic polymers (p(DAH-R/API)s) with varying ratios of arginine-grafted diaminohexane (DAH-R) and 1-(3-aminopropyl) imidazole (API) monomers.
• To investigate the impact of monomer composition on polymer properties, including buffering capacity, cytotoxicity, and gene delivery efficiency.
• To elucidate the mechanism of endosomal escape mediated by these polymers.

Main Methods:

• Synthesis of p(DAH-R/API)s via Michael reaction of N,N'-cystaminebisacrylamide (CBA) with DAH-R and API monomers at different ratios (2:1, 1:1, 1:2).
• Evaluation of polymer characteristics: pDNA condensation, Zeta-potential, buffering capacity, cellular uptake, and cytotoxicity assays.
• In vitro transfection efficiency studies, including experiments with endosomal escape inhibitors (chloroquine, nigericin).

Main Results:

• Polymer properties, including buffering capacity, cytotoxicity, pDNA condensation, Zeta-potential, cellular uptake, and transfection efficiency, were strongly correlated with the monomer composition ratios.
• Increased API content enhanced endosome buffering but decreased arginine residues, leading to reduced cytotoxicity, pDNA condensation, cellular uptake, and transfection efficiency.
• Polymers with a predominance of arginine residues demonstrated superior gene delivery capabilities.

Conclusions:

• Arginine moieties are critical for efficient gene delivery, primarily through direct endosome membrane penetration and enhanced cellular uptake.
• While imidazole moieties contribute to endosome buffering, their increased presence can negatively impact gene delivery outcomes due to reduced arginine content.
• The findings emphasize the importance of optimizing the balance between arginine and buffering moieties for effective polymeric gene delivery systems.