Cholesterol Domains Enhance Transfection

Betker, Jamie L.; Kullberg, Max; Gomez, Joe; Anchordoquy, Thomas J.

doi:10.4155/tde.13.16

Cited by 21 publications

(37 citation statements)

References 37 publications

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“…Indeed it was reported recently by Betker et al that the cholesterol domain formation significantly improves the transfection by serum protein binding in certain formulations [45]. Further, the difference in transfection efficiency in presence of serum among all the geminis could be explained on the basis of their biophysical characteristics.…”

Section: Resultsmentioning

confidence: 94%

Gene Transfection in High Serum Levels: Case Studies with New Cholesterol Based Cationic Gemini Lipids

et al. 2013

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BackgroundSix new cationic gemini lipids based on cholesterol possessing different positional combinations of hydroxyethyl (-CH2CH2OH) and oligo-oxyethylene -(CH2CH2O)n- moieties were synthesized. For comparison the corresponding monomeric lipid was also prepared. Each new cationic lipid was found to form stable, clear suspensions in aqueous media.Methodology/Principal FindingsTo understand the nature of the individual lipid aggregates, we have studied the aggregation properties using transmission electron microscopy (TEM), dynamic light scattering (DLS), zeta potential measurements and X-ray diffraction (XRD). We studied the lipid/DNA complex (lipoplex) formation and the release of the DNA from such lipoplexes using ethidium bromide. These gemini lipids in presence of a helper lipid, 1, 2-dioleoyl phophatidyl ethanol amine (DOPE) showed significant enhancements in the gene transfection compared to several commercially available transfection agents. Cholesterol based gemini having -CH2-CH2-OH groups at the head and one oxyethylene spacer was found to be the most effective lipid, which showed transfection activity even in presence of high serum levels (50%) greater than Effectene, one of the potent commercially available transfecting agents. Most of these geminis protected plasmid DNA remarkably against DNase I in serum, although the degree of stability was found to vary with their structural features.Conclusions/Significance-OH groups present on the cationic headgroups in combination with oxyethylene linkers on cholesterol based geminis, gave an optimized combination of new genera of gemini lipids possessing high transfection efficiency even in presence of very high percentage of serum. This property makes them preferential transfection reagents for possible in vivo studies.

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Section: Resultsmentioning

confidence: 94%

Gene Transfection in High Serum Levels: Case Studies with New Cholesterol Based Cationic Gemini Lipids

et al. 2013

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“…Luciferase signal was measured as previously described using Cell Lysis Buffer (Promega, Madison, WI, USA), Luciferase Assay Reagent (Promega) and a Monolight 2010 luminometer (Analytical Luminescence Laboratory, Ann Arbor, MI, USA) to detect output luminescence. 16 To determine the co-localization of liposomes and PL/DNA particles within cellular endosomes, cells were imaged using an Operetta high content imaging system with Harmony cellular analysis software (Perkin-Elmer, Santa Clara, CA, USA). The software requires a nuclear stain and membrane stain to locate cells for analysis.…”

Section: Methodsmentioning

confidence: 99%

Gene delivery to Her-2+ breast cancer cells using a two-component delivery system to achieve specificity

Kullberg

McCarthy

Anchordoquy

2014

Nanomedicine: Nanotechnology, Biology and Medicine

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Current liposomal gene delivery systems predominately utilize cationic lipids, which efficiently bind and deliver DNA plasmid, but also result in nonspecific gene expression in lung and liver tissue. To improve specificity, a two-component delivery strategy employing neutral liposomes was used to target breast cancers positive for the human epidermal growth factor receptor 2 (Her-2). The first component consisted of plasmid DNA condensed with cationic polyethylene glycol (PEG) modified polylysine (PL/DNA). The second component was a neutral Her-2 targeting liposome conjugated to the pore-forming protein, Listeriolysin O (LLO). Independently, PL/DNA delivery resulted in low expression of plasmid DNA. However, when PL/DNA and LLO/liposomes co-localized within an endosome, LLO disrupted endosome integrity, leading to cytoplasmic delivery and expression of the plasmid. When used to deliver a plasmid encoding the luciferase gene, this two-component system resulted in gene expression that was 268-fold greater in Her-2 positive cells than in Her-2 negative cells.

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“…Dialyzed proteins from the pull-down experiments were prepared for mass spectrometry using Millipore C4 ZipTips (Billerica, MA) and trifluoroacetic acid (TFA) as previously described [26]. Each sample was then mixed 1:1 v/v with saturated matrix solution (α-cyano-4-hydroxycinnamic [CHCA] for protein detection at ~10 mg/ml or saturated in 0.1%TFA/50% ACN/water).…”

Section: Methodsmentioning

confidence: 99%

“…The 1:1 matrix/protein solutions (1 μl) were then spotted on a clean plate. The plate was loaded in the Bruker Omniflex MALDI-TOF (Bruker Corp., Fremont, CA) and spectra analyzed by Flex Analysis software as previously described [26]. Due to the mass accuracy and possible modification/degradation of proteins extracted from the protein corona, it is difficult to definitively identify proteins associated with each approximate mass with this procedure.…”

Section: Methodsmentioning

confidence: 99%

“…We have previously documented the effect of lipoplex cholesterol content on serum protein interactions, and shown that high cholesterol contents greatly reduce the amount of protein adsorbed to lipoplexes [25,26]. In the current study, we utilize mass spectrometry to identify the individual proteins from fetal calf serum that are adsorbed to lipoplex formulations possessing different cholesterol contents.…”

Section: Introductionmentioning

confidence: 99%

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The effects of lipoplex formulation variables on the protein corona and comparisons with in vitro transfection efficiency

Betker

Gomez

Anchordoquy

2013

Journal of Controlled Release

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The use of lipoplexes for the intracellular delivery of nucleic acids typically involves the optimization of several parameters that are known to affect delivery. Researchers commonly vary charge ratio, and often incorporate different amounts of helper lipids (e.g., cholesterol) to optimize formulations for transfection in cell culture and in vivo. The results of such experiments are often interpreted in the context of nuclease resistance and cell association, but effects on the protein corona are usually not considered. While many studies have demonstrated that lipoplex structure and function can be dramatically compromised in the presence of serum, little attention has been paid to the adsorption of specific proteins and how this might be affected by formulation parameters. In this study, we characterize changes in the protein corona that occur as DOTAP-based lipoplexes are formulated with different amounts of cholesterol and prepared at different charge ratios. Our results demonstrate a significant effect of lipid composition on both total protein adsorption as well as the individual proteins from fetal calf serum that are associated with lipoplexes. In addition, we show that PEGylation increases protein adsorption with our formulations; effects that depend on the type of PEG conjugate employed in the lipoplex. Attempts to identify a specific protein responsible for enhancing transfection were unsuccessful.

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Cholesterol Domains Enhance Transfection

Cited by 21 publications

References 37 publications

Gene Transfection in High Serum Levels: Case Studies with New Cholesterol Based Cationic Gemini Lipids

Gene Transfection in High Serum Levels: Case Studies with New Cholesterol Based Cationic Gemini Lipids

Gene delivery to Her-2+ breast cancer cells using a two-component delivery system to achieve specificity

The effects of lipoplex formulation variables on the protein corona and comparisons with in vitro transfection efficiency

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