Polymeric materials provide particularly attractive scaffolds for the creation of supramolecular bioconjugates for the delivery of nucleic acids but typically lack the efficiency and biocompatibility to be clinically relevant. To address both issues, we produced zwitterion-like derivatives of polyethylenimine via succinylation of primary and secondary amines (zPEI). Polymers were generated with 9−55% of the amines modified (zPEI X, where X indicates the percentage of amines succinylated). Characterization of polymer/DNA interactions revealed that the presence of succinyl groups decreased the protonation constant of zPEI, resulting in both a decreased buffering capacity and polyplexes that dissociated in the presence of lower amounts of a competing counteranion compared to unmodified PEI. zPEI polyplexes also exhibited decreased aggregation in the presence of serum proteins. In the absence of serum, transfections with zPEI/DNA polyplexes exhibited similar or slightly improved transgene expression compared to unmodified PEI/DNA polyplexes. More importantly, zPEI 9−25 increased transgene expression up to 51-fold upon transfection in the presence of serum compared to PEI/DNA, while higher succinylation decreased gene delivery activity. Gene delivery mediated by zPEI 9/DNA polyplexes in the presence of serum was equal to or greater than unmodified PEI/DNA polyplexes in the absence of serum. The data suggest that succinylation increased gene transfection by decreasing polymer/DNA interaction strength, which may allow for more facile polyplex unpackaging, and/or increased stability of polyplex size and inhibition of aggregation in the presence of serum. However, it appears there exists a balance between the positive effects of succinylation and the need for sufficient polymer/DNA binding to condense and protect the cargo.
Gene therapy aims to treat patients
by altering or controlling
gene expression. The field of gene therapy has had increasing success
in recent years primarily using viral-based approaches; however, there
is still significant interest toward the use of polymeric materials
due to their potential as flexible, low-cost scaffolds for gene delivery
that do not suffer the mutagenesis and immunogenicity concerns of
viral vectors. To address the challenges of efficiency and biocompatibility,
a series of zwitterion-like polyethylenimine derivatives (zPEIs) were
produced via the succinylation of 2–11.5% of polyethylenimine
(PEI) amines. With increasing modification, zPEI polyplexes exhibited
decreased serum-protein aggregation and dissociated more easily in
the presence of a competitor polyanion when compared to unmodified
PEI.
Surprisingly, the gene delivery mediated in the presence of serum
showed that succinylation of as few as 2% of PEI amines resulted in
transgene expression 260- to 480-fold higher than that of unmodified
PEI and 50- to 65-fold higher than that of commercial PEI-PEG2k in HEK293 and HeLa cells, respectively. Remarkably, the
same zPEIs also produced 16-fold greater efficiency of CRISPR/Cas9
gene knock-in compared to unmodified PEI in the presence of serum.
In addition, we show that 2% succinylation does not significantly
decrease polymer/DNA binding ability or serum protein interaction
to a significant extent, yet this small modification is still sufficient
to provide a remarkable increase in transgene expression and gene
knock-in in the presence of serum.
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