Novel Branched Poly(Ethylenimine)−Cholesterol Water-Soluble Lipopolymers for Gene Delivery

Abstract

A novel water-soluble lipopolymer was synthesized by linking cholesteryl chloroformate to the secondary amino groups of branched poly(ethylenimine) (PEI) of 1,800 and 10,000 Da. Conjugation through PEI secondary amines gives this newly synthesized lipopolymer (abbreviated as PEI-Chol) special advantage over our previously synthesized lipopolymers, which utilized the primary amino groups for conjugation, as the primary amino groups have a significant role in DNA condensation. Also, significantly, only one cholesterol molecule was grafted onto each PEI molecule (confirmed by (1)H NMR and MALDI-TOF mass spectrometry), leaving enough space for the steric interactions of the PEI's primary amines with the DNA. The PEI-Chol lipopolymer was characterized for the critical micellar concentration (cmc), buffer capacity, DNA condensation (by band retardation and circular dichroism), in vitro transfection efficiency, and cell viability. The cmcs of PEI-Chol 1,800 and PEI-Chol 10,000 were 496.6 and 1,330.5 microg/mL, respectively. The acid-base titration indicated high buffering capacity of the polymers around the pH range of 5-7, which indicated their potential for buffering in the acidic pH environment of the endosomes. The band retardation studies indicated that efficient condensation of the plasmid DNA could be achieved using these lipopolymers. The circular dichroism spectra indicated a change in DNA conformation and adoption of lower energy state upon condensation with these lipopolymers when an N/P ratio of 2.5/1 or above was formulated. The mean particle size of these complexes was in the range 110-205 nm, except for the complexes prepared using PEI of 1,800 Da, which had a mean particle size of 384 +/- 300 nm. The zeta potential of DNA complexes prepared using PEI-Chol 1,800, PEI-Chol 10,000 and PEI of 1,800, 10,000, and 25,000 Da at an N/P ratio of 15/1 was in the range 23-30 mV and was dependent on the N/P ratios. The in vitro transfection of PEI-Chol/pCMS-EGFP complexes in Jurkat cells showed high levels of expressed Green Fluorescent Protein (GFP) with little toxicity as determined by flow cytometry. These novel water-soluble lipopolymers provided good transfection efficiency with other desirable characteristics such as water solubility, free primary amino groups for efficient DNA condensation and high buffer capacity that indicated the possibility of efficient endosomal release.

Non-Viral Gene Delivery Systems Based on Cholesterol Cationic Lipids: Structure-Activity Relationships

“…The same authors endeavored to design a more effective transfection reagent based on branched PEI (bPEI) using chemically protected primary amine and conjugation of cholesterol at the secondary amino groups of PEI. However, contrary to expectations, the presence of the non-modified primary amino groups only resulted in a slight increase (1.5-fold) of the TE (Wang et al, 2002). Kim and co-workers obtained partially contradictory data .…”

Preparation, characterization, and transfection efficiency of low molecular weight polyethylenimine-based nanoparticles for delivery of the plasmid encoding CD200 gene

“…It is supposed that the critical pH range of the endosomal compartment is around 5.5-7 which acts as a driving force for osmotic burst mechanism by which swelling and disruption of the endosomal/lysosomal membranes lead to the early escape of polyplexes. 40 It is obvious that PEI-SUC-PEI derivative exhibited the highest buffering capacity in the critical pH range.…”

Stability and toxicity of empty or gene-loaded lipopolysaccharide-amine nanopolymersomes

“…5,9,10 Alternatively, cholesterol modification has been proved to be an effective strategy to increase both the stability and the gene transfection efficacy of polyplexes, which may be realized by alleviating serum inhibition, enhancing the cellular/nuclear uptake, and decreasing cytotoxicity. 4,5,7,[11][12][13][14][15][16][17] Additionally, introducing anionic polymer such as anionic polysaccharide is able to improve the polyplex stability and the transfection efficiency by conferring the charge shielding and biodegradability, aiding the rupture of endo-/lysosome and gene release and decreasing cytotoxicity. 5,[18][19][20][21][22] Based on these studies, we engineered a novel brush copolymer of lipopolysaccharide-amine (LPSA) as a cytosolic delivery vector by introducing anionic polysaccharide of oxidized alginate (OA) and Cho to polyethyleneimine (PEI) with molecular weight of 1.8k dalton (PEI 1.8k), where OA and Cho-graft-PEI act as the backbone and side chains, respectively.…”