Fine-tuning DNA/albumin polyelectrolyte interactions to produce the efficient transfection agent cBSA-147

Eisele, Klaus; Gropeanu, Radu A.; Zehendner, Christoph M.; Rouhanipour, Ali; Ramanathan, Arvind; Mihov, George; Koynov, Kaloian; Kuhlmann, Christoph; Vasudevan, Subhash G.; Luhmann, Heiko J.; Weil, Tanja

doi:10.1016/j.biomaterials.2010.07.088

Cited by 64 publications

(95 citation statements)

References 29 publications

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“…However, fine-tuning the number and density of positive charges for cell uptake is not always trivial. In contrast, polycationic proteins, such as supercharged albumins, were applied in comparatively high quantities before cell toxicity was observed which has been attributed to their low charge density dissipated within a larger volume [45,46]. Therefore, the cationic ELP tags and in particular GFP-K72 offers an increased molecular weight and hydrodynamic volume, lower charge densities and no increase in cytotoxicity compared with the conventional polycationic tags, such as the polyarginine tag, which display high density of positive charges.…”

Section: Cytotoxicity Studymentioning

confidence: 99%

Enhancing cellular uptake of GFP via unfolded supercharged protein tags

Pesce

Kolbe

et al. 2013

Biomaterials

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a b s t r a c tOne of the barriers to the development of protein therapeutics is effective delivery to mammalian cells. The proteins must maintain a careful balance of polar moieties to enable administration and distribution and hydrophobic character to minimize cell toxicity. Numerous strategies have been applied to this end, from appending additional cationic peptides to supercharging the protein itself, sometimes with limited success. Here we present a strategy that combines these methods, by equipping a protein with supercharged elastin-like polypeptide (ELP) tags. We monitored cellular uptake and cell viability for GFP reporter proteins outfitted with a range of ELP tags and demonstrated enhanced uptake that correlates with the number of positive charges, while maintaining remarkably low cytotoxicity and resistance to degradation in the cell. GFP uptake proceeded mainly through caveolae-mediated endocytosis and we observed GFP emission inside the cells over extended time (up to 48 h). Low toxicity combined with high molecular weights of the tag opens the way to simultaneously optimize cell uptake and pharmacokinetic parameters. Thus, cationic supercharged ELP tags show great potential to improve the therapeutic profile of protein drugs leading to more efficient and safer biotherapeutics.

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Section: Cytotoxicity Studymentioning

confidence: 99%

Enhancing cellular uptake of GFP via unfolded supercharged protein tags

Pesce

Kolbe

et al. 2013

Biomaterials

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“…9). Extended observation times for achieving the maximum biological activity have been reported for other delivery systems as well [33] and are most likely due to slower cell uptake and drug release inside the cell. The attractive in vitro cytotoxic activity of dcBSA-147-PEO(5000) 28 -(DOX) 14 micelles could be attributed to their high membrane permeability benefits from multiple positive charges, as well as efficient carrier degradation and drug release in lysosomes.…”

Section: Enhanced Cellular Uptakementioning

confidence: 97%

“…Rf 0.56 (EtOAc/heptane, 1:1 v/v), 1 2.3 Preparation of cBSA-147-PEO(5000) 28 (2) dcBSA-147-PEO(5000) 28 (2) was prepared using a similar procedure reported in our previous paper [30]. Briefly, cationic bovine serum albumin, cBSA-147 [33] (1, 10 mg, 0.15 lmol) was first denatured in degassed urea-phosphate buffer (10 mL, 10 mM phosphate buffer, 5 M urea and 2 mM EDTA, pH 7.4) for 10 min, and then reducing agent TCEP (4.3 mg, 15 lmol) was added under argon atmosphere for 30 min. Subsequently, PEG-5000-MI (77 mg, 15 lmol) was added to the reaction and stir at RT for 3 h. Finally, the capping reagent N-propargyl maleimide (30 lmol) was given to the reaction and stir for another 3 h. The reaction mixture was first purified by ultrafiltration with Tris-HCl buffer (20 mM Tris, 150 mM NaCl, 2 mM EDTA, pH 7.4), and followed by further size exclusion purification using HiPrep TM Sephacyl TM S-100 HR gel filtration column on AKTÄ Purifier flash protein liquid chromatography with Tris-HCl buffer (20 mM Tris, 150 mM NaCl, pH 7.4).…”

Section: Synthesis Of N-propargyl Maleimidementioning

confidence: 99%

“…cBSA-147 stands for cationic BSA, which was obtained after converting about 87 out of 100 carboxylic acid groups of aspartic and glutamic acid residues of native BSA into primary amino groups according to published procedures [33]. cBSA-147 serves as efficient gene delivery carrier of low cytotoxicity that reveals fast and efficient cell uptake and even allows stable gene transfections [33] and it has been applied successfully as biocompatible surface coating [34]. However, due to the potential immunogenicity of cationic albumin derivatives [35], further modifications are required to efficiently shield epitopes to allow multiple dose applications in vivo.…”

Section: Dox Micellesmentioning

confidence: 99%

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Nano-Sized Albumin-Copolymer Micelles for Efficient Doxorubicin Delivery

Wu¹,

Shih²,

Ramanathan³

et al. 2012

Biointerphases

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We present the discovery of a nano-sized protein-derived micellar drug delivery system based on the polycationic albumin precursor protein cBSA-147. The anticancer drug doxorubicin (DOX) was efficiently encapsulated into nanosized micelles based on hydrophobic interactions with the polypeptide scaffold. These micelles revealed attractive stabilities in various physiological buffers and a wide pH range as well as very efficient uptake into A549 cells after 1 h incubation time only. In vitro cytotoxicity was five-times increased compared to free DOX also indicating efficient intracellular drug release. In addition, multiple functional groups are available for further chemical modifications. Based on the hydrophobic loading mechanism, various classical anti-cancer drugs, in principle, could be delivered even synergistically in a single micelle. Considering these aspects, this denatured albumin-based drug delivery system represents a highly attractive platform for nanomedicine approaches towards cancer therapy.

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“…The cationized BSA carries an average number of 113 or 147 amino groups, which results in a calculated positive net charge of +90 mV or +158 mV in aqueous solution at pH 7.4. 36 Since the number of charges within the protein scaffold strongly depends on the buffer conditions ͑pH, ionic strength͒, the surface zeta potential of adsorbed BSA in ultrapure water ͑see Ref. 37 for Fig.…”

Section: Glass Surface Coatingmentioning

confidence: 99%

Cationized albumin-biocoatings for the immobilization of lipid vesicles

et al. 2010

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Tethered lipid membranes or immobilized lipid vesicles are frequently used as biomimetic systems. In this article, the authors presented a suitable method for efficient immobilization of lipid vesicles onto a broad range of surfaces, enabling analysis by quantitative methods even under rigid, mechanical conditions-bare surfaces such as hydrophilic glass surfaces as well as hydrophobic polymer slides or metal surfaces such as gold. The immobilization of vesicles was based on the electrostatic interaction of zwitterionic or negatively charged lipid vesicles with two types of cationic chemically modified bovine serum albumin ͑cBSA͒ blood plasma proteins ͑cBSA-113 and cBSA-147͒. Quantitative analysis of protein adsorption was performed as the cBSA coatings were characterized by atomic force microscopy, surface zeta potential measurement, fluorescence microscopy, and surface plasmon spectroscopy, revealing a maximal surface coverage 270-280 ng/ cm 2 for 0.02 mg/ml cBSA on gold. Small unilamellar vesicles as well as giant unilamellar vesicles ͑GUVs͒ were readily immobilized ͑ϳ15 min͒ on cBSA coated surfaces. GUVs with 5-10 mol% negatively charged 1,2,-dipalmitoyl-sn-glycero-3-phosphoglycerol remained stable in liquid for at least 5 weeks.

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Fine-tuning DNA/albumin polyelectrolyte interactions to produce the efficient transfection agent cBSA-147

Cited by 64 publications

References 29 publications

Enhancing cellular uptake of GFP via unfolded supercharged protein tags

Enhancing cellular uptake of GFP via unfolded supercharged protein tags

Nano-Sized Albumin-Copolymer Micelles for Efficient Doxorubicin Delivery

Cationized albumin-biocoatings for the immobilization of lipid vesicles

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