(598f) Succinylated Polyethylenimine Derivatives Enhance Gene Expression and Serum Stability in vitro

Polyethylenimine (PEI) is considered the gold standard of polymeric, non-viral gene delivery vectors. The highly cationic polymer readily condenses DNA through electrostatic interactions and can subsequently be internalized and processed by cells to incorporate the genetic payload into the existing genome to produce a therapeutic effect. However, the standard properties of PEI may not be optimal when attempting to overcome common inter- and intracellular barriers. For example, the repeating amine structure of PEI results in a large positive charge density, which is conducive to condensing DNA and leads to favorable electrostatic interactions with the negatively charged cell membrane. Alternatively, the excess positive charge leads to nonspecific binding of negatively charged serum proteins and can produce a significant cytotoxicity. Intracellularly, the large number of amines in PEI results in a large buffering capacity, allowing for endosomal and lysosomal escape via the ‘proton-sponge’ mechanism, but the strong electrostatic interactions between PEI/DNA can prevent facile unpackaging. Thus, the degree of protonation can greatly affect the behavior of PEI polyplexes.

In an attempt to address these issues, we have previously shown that acetylating fractions of primary and secondary amines of PEI can lead to increases in gene delivery efficiency. It was shown that modifying up to 43% of the primary amines with acetic anhydride mediated a higher transgene expression, while further reaction led to lower levels of expression. Furthermore, with increasing degrees of acetylation, polymer/DNA interactions were weakened and resulted in a higher efficiency of polyplex unpackaging within the cell. However, the modified polymers all had significantly lower buffering capacities, suggesting that there may be an optimal balance between the strength of polymer/DNA interactions and buffering capacity. Despite the significant increases in gene delivery efficiency realized by acetylated PEI, transfections in the presence of serum resulted in gene delivery activity lower than that of unmodified PEI in the absence of serum, implying that the acetylated polymers still suffered from serum instability.

In this study we modified PEI by reacting varying fractions of primary and secondary amines with succinic anhydride, resulting in zwitterionic PEI derivatives. Derivatives with 5.8% (zPEI₅), 8.0% (zPEI₈), 15.4% (zPEI₁₅), 26.7% (zPEI₂₆), and 35.0% (zPEI₃₄) of the primary amines succinylated were successfully synthesized. In vitro gene delivery activity increased over that of unmodified PEI in zPEI₅, zPEI₈, and zPEI₁₅ in the absence of serum, with zPEI₅ resulting in an approximately 10-fold increase. More importantly, the succinylated polymers exhibited a higher stability in the presence of serum, with zPEI₅ gene delivery in serum surpassing gene delivery by unmodified PEI in the absence of serum. Cytotoxicity of the succinylated polymers decreased with increasing degrees of succinylation. Additionally, succinylation effectively reduced the ζ-potential from 15.1 mV to 15.0-9.6 mV while increasing particle diameter from 61 nm to 174-669 nm. The succinylated PEIs also exhibited decreased polymer/DNA interaction strengths, most likely attributed to the presence of negatively charged carboxyl groups which electrostatically repel the DNA. Similarly, the enhanced transfection activities suggest the presence of negatively charged functional groups shielded the succinylated polyplexes from the effects of the serum proteins. Much like the balance of polymer/DNA interactions to buffering capacity, our results suggest a similar balance between shielding particles from negatively charged serum proteins while maintaining favorable interactions with the negatively charged cell membrane. Future studies will explore and compare the differences between the acetylated and succinylated polymers to illuminate mechanisms and cellular pathways to identify design criteria for polymeric gene delivery vectors.