Effect of Thiol Pendant Conjugates on Plasmid DNA Binding, Release, and Stability of Polymeric Delivery Vectors

Abstract

Polymers have attracted much attention as potential gene delivery vectors due to their chemical and structural versatility. However, several challenges associated with polymeric carriers, including low transfection efficiencies, insufficient cargo release, and high cytotoxicity levels have prevented clinical implementation. Strong electrostatic interactions between polymeric carriers and DNA cargo can prohibit complete cargo release within the cell. As a result, cargo DNA never reaches the cell's nucleus where gene expression takes place. In addition, highly charged cationic polymers have been correlated with high cytotoxicity levels, making them unsuitable carriers in vivo. Using poly(allylamine) (PAA) as a model, we investigated how pH-sensitive disulfide cross-linked polymer networks can improve the delivery potential of cationic polymer carriers. To accomplish this, we conjugated thiol-terminated pendant chains onto the primary amines of PAA using 2-iminothiolane, developing three new polymer vectors with 5, 13, or 20% thiol modification. Unmodified PAA and thiol-conjugated polymers were tested for their ability to bind and release plasmid DNA, their capacity to protect genetic cargo from enzymatic degradation, and their potential for endolysosomal escape. Our results demonstrate that polymer-plasmid complexes (polyplexes) formed by the 13% thiolated polymer demonstrate the greatest delivery potential. At high N/P ratios, all thiolated polymers (but not unmodified counterparts) were able to resist decomplexation in the presence of heparin, a negatively charged polysaccharide used to mimic in vivo polyplex-protein interactions. Further, all thiolated polymers exhibited higher buffering capacities than unmodified PAA and, therefore, have a greater potential for endolysosomal escape. However, 5 and 20% thiolated polymers exhibited poor DNA binding-release kinetics, making them unsuitable carriers for gene delivery. The 13% thiolated polymers, on the other hand, displayed high DNA binding efficiency and pH-sensitive release.

Overcoming Nonviral Gene Delivery Barriers: Perspective and Future

“…7, 70 In summary, efforts to improve complexation and subsequent gene delivery have resulted in the synthesis of CPs with amine groups intermittently interchanged with thiol moieties, 71 surface attachment of pendant hydroxyl groups, 51, 72 backbone alkylation, 73 and imidazole grafting (upon a chitosan backbone). 35 In addition to improved complexation, imidazole-functionalized CPs possessed pKa values of ~6, which is expected to facilitate enhanced gene delivery by polyplex escape from endosomal compartments (to be further explained below).…”

Synthesis and Characterization of Glycol Chitosan DNA Nanoparticles for Retinal Gene Delivery

“…We next assessed the zeta potential of the NPs, an important index for average surface charge of the NPs in solution. [13a, 21] The NPs have a positive surface potential of 24.17±0.74 mV (Fig. 1D), compared to the negative potential of naked pDNA (-25.90±0.32 mV).…”

Journey to the Center of the Cell: Current Nanocarrier Design Strategies Targeting Biopharmaceuticals to the Cytoplasm and Nucleus

“…This causes osmotic swelling and eventual bursting of the endosome, releasing the nanocarrier into the cytosol [75]. Although numerous publications support this buffering technique for inducing endosomal escape [22, 76-80], its validity has been questioned. Several studies demonstrate that amine containing polymers do not induce changes in pH as previously suggested, and argue that the osmotic pressure built during endosome acidification is theoretically insufficient to cause membrane bursting on its own [81].…”