Linear Poly(ethylenimine-propylenimine) Random Copolymers for Gene Delivery: From Polymer Synthesis to Efficient Transfection with High Serum Tolerance

Abstract

Naturally occurring oligoamines, such as spermine, spermidine, and putrescine, are well-known regulators of gene expression. These oligoamines frequently have short alkyl spacers with varying lengths between the amines. Linear polyethylenimine (PEI) is a polyamine that has been widely applied as a gene vector, with various formulations currently in clinical trials. In order to emulate natural oligoamine gene regulators, linear random copolymers containing both PEI and polypropylenimine (PPI) repeat units were designed as novel gene delivery agents. In general, statistical copolymerization of 2-oxazolines and 2-oxazines leads to the formation of gradient copolymers. In this study, however, we describe for the first time the synthesis of near-ideal random 2-oxazoline/2-oxazine copolymers through careful tuning of the monomer structures and reactivity as well as polymerization conditions. These copolymers were then transformed into near-random PEI–PPI copolymers by controlled side-chain hydrolysis. The prepared PEI–PPI copolymers formed stable polyplexes with GFP-encoding plasmid DNA, as validated by dynamic light scattering. Furthermore, the cytotoxicity and transfection efficiency of polyplexes were evaluated in C2C12 mouse myoblasts. While the polymer chain length did not significantly increase the toxicity, a higher PPI content was associated with increased toxicity and also lowered the amount of polymers needed to achieve efficient transfection. The transfection efficiency was significantly influenced by the degree of polymerization of PEI–PPI, whereby longer polymers resulted in more transfected cells. Copolymers with 60% or lower PPI content exhibited a good balance between high plasmid–DNA transfection efficiency and low toxicity. Interestingly, these novel PEI–PPI copolymers revealed exceptional serum tolerance, whereby transfection efficiencies of up to 53% of transfected cells were achieved even under 50% serum conditions. These copolymers, especially PEI–PPI with DP500 and a 1:1 PEI/PPI ratio, were identified as promising transfection agents for plasmid DNA.

Balancing gene transfection and cytotoxicity of nucleic acid carriers with focus on ocular and hepatic disorders: evaluation of hydrophobic and hydrophilic polyethyleneimine derivatives

“…Depending on the degree of substitution, succinylation of PEI chains may optimize the polymer/DNA interaction strength to balance stability-lability, and they provide stability to the vector at the serum medium, 33 thus allowing for enhanced transfection levels. 45 This output can also be linked, at least to some extent, to lower polymer toxicity. 37 Truly, these assumptions seem not to be clear enough at this moment, and we are conducting investigations in this regard to better identify the main reason linked to the outstanding performance of the BPEI-Succ chains.…”

Designing Synthetic Polymers for Nucleic Acid Complexation and Delivery: From Polyplexes to Micelleplexes to Triggered Degradation

“…These copolymers exhibited serum tolerance and were successful in transfection of the majority of C2C12 cells in the presence of serum. In this work, higher molecular weight polymers yielded greater transfection efficiency: copolymers with a degree of polymerization of 500, with ∼40–60% PPI, were most promising, resulting in 69% transfection efficiency without substantial cytotoxicity . In another study, Guégan and co-workers found that poly­(vinylamine) (PVA), an isomer of PEI, achieved efficient transfection on a variety of cell lines, including H2K2B4, C2C12, 16HBE, and DC2.4 cells …”

“…In this work, higher molecular weight polymers yielded greater transfection efficiency: copolymers with a degree of polymerization of 500, with ∼40−60% PPI, were most promising, resulting in 69% transfection efficiency without substantial cytotoxicity. 21 In another study, Gueǵan and co-workers found that poly(vinylamine) (PVA), an isomer of PEI, achieved efficient transfection on a variety of cell lines, including H2K2B4, C2C12, 16HBE, and DC2.4 cells. 22 The potential of polymeric vectors in gene delivery is increasingly recognized, while shortcomings are continually uncovered (Figure 1).…”

Polytrimethylenimines: Highly Potent Antibacterial Agents with Activity and Toxicity Modulated by the Polymer Molecular Weight

“… 24 − 28 Different N -methylated PTMI derivatives were synthesized; 24 , 29 , 30 however, they have not been the subject of antimicrobial studies. Both PEI and PTMI may be obtained in the branched, 20 linear, 27 , 31 and dendrimeric forms. 23 Linear PEI (L-PEI) and linear PTMI (L-PTMI) are usually obtained by hydrolysis of poly(alkyl-2-oxazoline)s 32 , 33 and poly(alkyl-2-oxazine)s, 28 , 34 respectively.…”

“…Its dendrimeric form has gained popularity as a nonviral transfection vector and in drug delivery systems, , although there are very limited reports on a linear form of PTMI. − Different N -methylated PTMI derivatives were synthesized; ,, however, they have not been the subject of antimicrobial studies. Both PEI and PTMI may be obtained in the branched, linear, , and dendrimeric forms . Linear PEI (L-PEI) and linear PTMI (L-PTMI) are usually obtained by hydrolysis of poly­(alkyl-2-oxazoline)­s , and poly­(alkyl-2-oxazine)­s, , respectively.…”