Transfection Efficacy and Cellular Uptake of Lipid-Modified Polyethyleneimine Derivatives for Anionic Nanoparticles as Gene Delivery Vectors

Abstract

Cationic polyethylenimine (PEI)-based nonviral gene carriers have been desirable to overcome the limitations of viral vectors in gene therapy. A range of PEI derivatives were designed, synthesized, and evaluated for nonviral delivery applications of plasmid DNA (pDNA). Linolenic acid, lauric acid, and oleic acid were covalently conjugated with low-molecular-weight PEI (M w ∼ 1200 Da) via two different linkers, gallic acid (GA) and p-hydroxybenzoic acid (PHPA), that allows a differential loading of lipids per modified amine (3 vs 1, respectively). 1H NMR spectrum confirmed the expected structure of the conjugates as well as the level of lipid substitution. SYBR Green binding assay performed to investigate the 50% binding concentration (BC50) of lipophilic polymers to pDNA revealed increased BC50 with an increased level of lipid substitution. The particle analysis determined that GA- and PHPA-modified lipopolymers gave pDNA complexes with ∼300 and ∼100 nm in size, respectively. At the polymer/pDNA ratio of 5.0, the ζ-potentials of the complexes were negative (−6.55 to −10.6 mV) unlike the complexes with the native PEI (+11.2 mV). The transfection experiments indicated that the prepared lipopolymers showed higher transfection in attachment-dependent cells than in suspension cells based on the expression of the reporter green fluorescent protein (GFP) gene. When loaded with Cy3-labeled pDNA, the lipopolymers exhibited effective cellular uptake in attachment-dependent cells while the cellular uptake was limited in suspension cells. These results demonstrate the potential of lipid-conjugated PEI via GA and PHPA linkers, which are promising for the modification of anchorage-dependent cells.

Alkylated Sulfonium Modification of Low Molecular Weight Polyethylenimine to Form Lipopolymers as Gene Vectors

“…Hydrophobic modification can facilitate DNA compacting via additional hydrophobic interaction and increase the lipophilicity of polymer/DNA particles which could benefit the cell membrane interaction and cellular uptake. − Through modification, the charge density of PEI is reduced, and the cytotoxicity is changed. The essential features of the hydrophobic modification on PEI could be achieved by changing the length of the hydrophobic alkyl − or aryl fragments, by varying the modification degree, and by switching the linker between the charged backbone and the hydrophobic groups …”

“… 12 − 14 Through modification, the charge density of PEI is reduced, and the cytotoxicity is changed. The essential features of the hydrophobic modification on PEI could be achieved by changing the length of the hydrophobic alkyl 15 − 20 or aryl 21 fragments, by varying the modification degree, 22 and by switching the linker between the charged backbone and the hydrophobic groups. 23 …”

Biodistribution of Therapeutic Small Interfering RNAs Delivered with Lipid-Substituted Polyethylenimine-Based Delivery Systems

“…The PEI and PEI-derived polymers (>20 kDa) in a high-molecular-weight form display multivalent binding to nucleic acids that lead to stable nanoparticles that can withstand membrane penetration. , However, they also display significant toxicity on sensitive human cells, although PEI cytotoxicity has been successfully reduced by modifying it with peptides, vitamins, amino acids, etc. − The low-molecular-weight PEI (<2 kDa) is relatively nontoxic due to weaker cell membrane interactions but also unable to act as a carrier. Our studies have shown that lipid modifications on low-molecular-weight PEI can convert the noneffective small-molecular-weight PEI into an effective carrier for a range of nucleic acids due to the increased uptake of resultant nanoparticles, so that it can be safely used to deliver siRNA, pDNA, and mRNA in vitro . ,− However, the in vivo delivery efficiency of the modified PEIs for nucleic acids remained to be explored.…”

Tuning the Potency of Farnesol-Modified Polyethylenimine with Polyanionic Trans-Booster to Enhance DNA Delivery

“…30 In our previous work, we incorporated polyanions into lipid-modified PEI/pDNA complexes as an efficient delivery system owing to its high efficiency in transfection. 31 While several polyanions appear to be effective for enhancing the transgene expression, the influence of polyplex details affecting the final transfection efficiency remains under studied. Better understanding of the formulation parameters and their effect on transfection efficiency remains to be reported.…”

“…Several attempts have been made to develop an effective therapeutic delivery system by combination of both PEI/DNA complexes and an additive, for example, by using polyanions such as hyaluronic acid, , polyamino acids such as α-polyaspartic acid, α-polyglutamic acid and γ-polyglutamic acid, , poly­(acrylic acid), poly­(ethylene glycol) derivatives with carboxylic acid side chains, and poly­( l -lactic acid) . In our previous work, we incorporated polyanions into lipid-modified PEI/pDNA complexes as an efficient delivery system owing to its high efficiency in transfection . While several polyanions appear to be effective for enhancing the transgene expression, the influence of polyplex details affecting the final transfection efficiency remains under studied.…”