Influence of Alkyl Chains of Modified Polysuccinimide-Based Polycationic Polymers on Polyplex Formation and Transfection.

Marcelo H Kravicz,Mrityunjoy Kar,Stefanie Wedepohl,Marcelo Calderón,Debora T Balogh,Maria Vitoria L B Bentley

doi:10.1002/mabi.201900117

Marcelo H Kravicz, Mrityunjoy Kar + Show 4 more

Open Access

https://doi.org/10.1002/mabi.201900117

Copy DOI

Abstract

The development of polymers with low toxicity and efficient gene delivery remains a significant barrier of nonviral gene therapy. Modification and tuning of chemical structures of carriers is an attractive strategy for efficient nucleic acid delivery. Here, polyplexes consisting of plasmid DNA (pDNA) and dodecylated or non-dodecylated polysuccinimide (PSI)-based polycations are designed, and their transfection ability into HeLa cells is investigated by green fluorescent protein (GFP) expressing cells quantification. All cationic polymers show lower cytotoxicity than those of branched polyethyleneimine (bPEI). PSI and bPEI-based polyplexes have comparable physicochemical properties such as size and charge. Interestingly, a strong interaction between dodecylated polycations and pDNA caused by the hydrophobic moiety is observed in dodecylated PSI derivatives. Moreover, the decrease of GFP expression is associated with lower dissociation of pDNA from polyplexes according to the heparin displacement assay. Besides, a hydrophobization of PSI cationic derivatives with dodecyl side chains can modulate the integrity of polyplexes by hydrophobic interactions, increasing the binding between the polymer and the DNA. These results provide useful information for designing polyplexes with lower toxicity and greater stability and transfection performance.

Highlights

The development of polymers with low toxicity and efficient gene delivery and the ability to transfer and express exogremains a significant barrier of nonviral gene therapy
The strategy has expanded from the plasmid DNA transference to messenger RNA, microRNA, and small interfering RNA,[4,7] different therapeutic payloads dodecylated PSI derivatives
It is known that increasing the hydrophobicity of a polymer chain to a certain degree would facilitate micellization of amphiphilic polymers and a minimal hydrophobicity is indicated for branched polyethyleneimine (bPEI) 25.[44]. Here, the Critical micellar concentration (CMC) of all polycations 1An and 2An was determined according to previous studies,[45,46] and for convenience, we describe the 1:3 ratio as I3/I1 ratio versus log of polymer concentration

Summary

Polymers Synthesis and Characterization

To elucidate the ability of polyplex formation of dodecylated and non-dodecylated PSI derivatives, we synthesized a series of polycations with different amine moieties. The actual dodecylamine ratio found was ≈12 mol% and both PSI and its dodecylated copolymers grafted with amines An resulted in a total aminolysis and ring opening of PSI backbone, observed with the chemical shift of methine protons signal at 5.25 ppm to a new signal at 4.5 ppm. To confirm the non-toxicity of the alkyl chain in the polymers, cytotoxicity assays of bare dodecylated PSI derivatives obtained after amylolysis with amines 1-(3-aminopropyl) imidazole (A5) and 1-(2-aminoethyl) piperazine (A6) were performed (Figure S6, Supporting Information) Both imidazole and piperazine have lower in vitro cytotoxicity and are used in polymer and drug design.[48,49,50] For the imidazole side chain (A5), higher cell viability was obtained until 250 μg mL−1, and a decreased cell viability was observed for the piperazine side group (A6), presumably due to the secondary amine in the piperazine ring. The concentration range was between 2 and 20 μg mL−1

Physicochemical Properties and DNA Binding Ability

Cytotoxicity of Polyplexes

Conclusion

Experimental Section

Conflict of Interest