title: 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.
authors.affiliation: Fischell Department of Bioengineering, University of Maryland, College Park, Maryland 20742, USA.
authors.forename: Irene
authors.initials: I
authors.lastname: Bacalocostantis
authors.forename: Viraj P
authors.initials: VP
authors.lastname: Mane
authors.forename: Michael S
authors.initials: MS
authors.lastname: Kang
authors.forename: Addison S
authors.initials: AS
authors.lastname: Goodley
authors.forename: Silvia
authors.initials: S
authors.lastname: Muro
authors.forename: Peter
authors.initials: P
authors.lastname: Kofinas
mesh_terms: D001665:Binding Sites; D004247:DNA / Q000737:chemistry*; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000737:chemistry*; D010957:Plasmids / Q000737:chemistry; D011073:Polyamines / Q000737:chemistry*; D013438:Sulfhydryl Compounds / Q000737:chemistry*
chemical_list: D011073:Polyamines; D013438:Sulfhydryl Compounds; C063994:polyallylamine; D004247:DNA