ANALYTICAL BIOCHEMISTRY
Analytical Biochemistry 334 (2004) 196–198 www.elsevier.com/locate/yabio

Notes & Tips

A spectrophotometric assay for the quantiWcation of polyethylenimine in DNA nanoparticles
Martin Bertschinger, Sophie Chaboche, Martin Jordan¤, Florian M. Wurm
Swiss Federal Institute of Technology Lausanne, Laboratory of Cellular Biotechnology, Lausanne VD1015, Switzerland Received 29 April 2004 Available online 28 August 2004

Since the Wrst description of polyethylenimine (PEI)1 as a gene delivery vehicle, there has been a strong interest in its use for in vivo and in vitro transfections of mammalian cells [1]. The mechanism of gene transfer by PEI is still unclear, and little is known about the formation of PEI– DNA nanoparticles. Such questions are diYcult to address because a rapid and sensitive assay for the quantiWcation of PEI in diVerent transfection media is not available. The only spectrophotometric assay for the quantiWcation of PEI is based on the dark blue cuprammonium complex that is formed when copper(II) ions are added to PEI [2,3]. This complex has a strong absorbance peak at 285 nm and a weaker one at 630 nm. Unfortunately, the absorption maximum at 285 nm is close to that of DNA (260 nm). In addition, most cell culture media contain components that absorb at this wavelength. At 630 nm, there is much less interference from biological molecules, but the signal from the cuprammonium complex is also low. We successfully adapted a commercially available protein detection assay for the quantiWcation of PEI. All experiments described below were performed with linear 25-kDa PEI (Polysciences, Warrington, PA, USA) that we routinely use for the transfection of mammalian cells [4], but it was possible to quantitate other PEIs (diVerent molecular weights; branched) with this method (data not shown). The mixing of a PEI solution with the Advanced Protein Assay (APA) reagent (Cytoskeleton, Denver, CO, USA) resulted in the formation of a colored product that absorbed in the range of 570– 615 nm, with the absorbance being proportional to the
Corresponding author. E-mail address: martin.jordan@epX.ch (M. Jordan). 1 Abbreviations used: PEI, polyethylenimine; QOD, limit of quantiWcation; LOD, limit of detection. 0003-2697/$ - see front matter  2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2004.07.020
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PEI concentration. BrieXy, 200 l of the APA reagent was mixed with 800 l of the PEI sample by vortexing for 5 s. The solution was then incubated for 30 min at room temperature before the absorbance was measured at 590 nm. For incubation periods of less than 30 min, the absorbance signal was more variable than was observed for a 30-min incubation. Incubation periods of more than 30 min frequently resulted in visible precipitates. We initially quantiWed PEI in 150 mM NaCl because DNA and PEI are frequently mixed in this salt solution prior to transfection. The limit of quantiWcation (QOD, estimated to be 10 times the standard deviation of blanked values) was approximately 50 ng/ml or 100 times lower than had been determined previously [3]. The limit of detection (LOD, estimated to be three times the standard deviation of blanked values) was 16 ng/ml. The absorption increased linearly up to a PEI concentration of 1 g/ml (Table 1). Using a nonlinear four-parameter curve Wt (Softmax Pro 2.4, Molecular Devices, Sunnyvale, CA, USA), PEI concentrations of up to 4 g/ ml were measured without dilution (Fig. 1). PEI standard curves were also established in diVerent cell culture media. The absorbance maximum of the APA reagent is suitable for these measurements. Because the only component in media that absorbs at 590 nm is phenol red, the low pH of the APA solution resulted in a complete protonation of phenol red, eliminating its interference at this wavelength. In RPMI 1640 (Applichem, Darmstadt, Germany), the PEI concentration was measured with nearly the same sensitivity as in 150 mM NaCl (QOD D 0.2 g/ml). However, chemically deWned media such as EX CELL 293 (JRH Bioscience, Lenexa, KS, USA) and ProCHO5 (Cambrex, East Rutherford, NJ, USA) reacted with the APA reagent. To reduce the background due to unknown medium components, the

Notes & Tips / Analytical Biochemistry 334 (2004) 196–198 Table 1 Optical characteristics and limitations of the PEI assay at D 590 nm Medium NaCl (150 mM) Dilution LOD ( g/ml) QOD ( g/ml) Linear range ( g/ml) — 0.016 0.053 0.05–1 EXCELL 293 1:25 1.13 3.8 3.8–50 RPMI 1640 — 0.059 0.2 0.2–2.0 ProCHO5 1:25 0.57 1.9 2.0–50

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Fig. 1. Standard curve of PEI concentration in 150 mM NaCl. The absorbances of solutions with known concentrations of PEI were determined at 590 nm following incubation with the APA reagent. The absorbance values were blanked against air and Wtted using a nonlinear four-parameter curve as described in the given equation. The linear range of the standard curve is shown in magniWcation.

samples were Wrst diluted 25-fold with 150 mM NaCl. Unfortunately, this decreased the QOD for PEI to 4 g/ ml in EX CELL 293 and to 2 g/ml in ProCHO5 (Table 1). The linear ranges for the three diVerent media are summarized in Table 1. We also analyzed the inXuence of DNA on PEI quantiWcation using the APA reagent and found that the reagent has a more than 150-fold higher aYnity for PEI than for DNA ( OD/ g of DNA D 0.002 vs. OD/ g of PEI D 0.36). Therefore the eVect of DNA on the measurement was neglected for the experiment described below. When plasmid DNA and PEI are mixed, they immediately form a complex [5]. At higher PEI concentrations, these complexes are further compacted into PEI–DNA nanoparticles that can be sedimented in a microcentrifuge at 12,800g for 2 min. Nanoparticle formation was observed when the PEI:DNA ratio (w/w) in the mixture was greater than 0.25 (Fig. 2). The complete incorporation of DNA into particles was demonstrated by spectrophotometric analysis of the supernatant after centrifugation. All of the DNA was present in the pellet fraction at PEI:DNA ratios of 0.25 or more (Fig. 2). Using the APA reagent, it was also possible to quantify the PEI in solution. Without sedimentation, both soluble

Fig. 2. QuantiWcation of PEI and DNA in diVerent mixtures after removal of PEI–DNA nanoparticles by centrifugation. Two equal volumes of 150 mM NaCl with either 50 g/ml DNA or varying amounts of PEI were mixed, incubated for 10 min at room temperature, and then centrifuged for 2 min at 12,800g in a microcentrifuge. Undiluted supernatants were used to quantify the DNA concentration at 260 nm. For the PEI–APA assay, supernatants were diluted 50-fold in 150 mM NaCl to be in the range of the calibration curve. The dashed line represents the added amount of PEI.

PEI and PEI complexed to DNA were detected. After removal of the nanoparticles by centrifugation, only the PEI remaining in solution was detected (Fig. 2). At the critical PEI:DNA ratio of 0.25, no PEI remained in solution because all of it was present in nanoparticles. Increasing the PEI did not result in a further incorporation of PEI into nanoparticles. At PEI:DNA ratios greater than 0.25, the PEI concentration in the supernatant increased linearly with the amount of PEI added, demonstrating that the PEI:DNA complexes formed at ratios of 0.25 to 2 contained the same amount of PEI. This result was surprising given that the PEI–DNA complexes formed at ratios between 0.25 and 0.50 were not competent for transfection, but high transfection eYciencies were obtained with PEI:DNA ratios of 2 or more [4]. We showed that the APA reagent is an excellent tool for quantiWcation of PEI in a concentration range from 0.05 to 4 g/ml in 150 mM NaCl. Moreover, the interaction between this reagent and PEI was not disturbed by most cell culture media or by the presence of DNA. The

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Notes & Tips / Analytical Biochemistry 334 (2004) 196–198

method is fast, involves little manipulation, and exceeds the sensitivity of previously published spectrophotometric methods. With this assay, it was demonstrated that PEI:DNA particles form at a critical PEI:DNA ratio of 0.25 while incorporating all of the available PEI and DNA molecules. At this ratio, the DNA was fully saturated with PEI, and further addition of soluble PEI did not result in incorporation into the nanoparticles. With this assay, a systematic analysis of PEI:DNA complex formation in diVerent standard cell culture media is possible and may help to optimize transfection.

References
[1] O. Boussif, F. Lezoualc’h, M. Zanta, M. Mergny, D. Scherman, B. Demeneix, J.P. Behr, A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine, Proc. Natl. Acad. Sci. USA 92 (1995) 7297–7301. [2] T. Perrine, W. Landis, Analysis of polyethylenimine by spectrophotometry of its copper chelate, J. Polymer Sci. 5 (1967) 1993–2003. [3] F. Ungaro, G. De Rosa, A. Miro, F. Quaglia, Spectrophotometric determination of polyethylenimine in the presence of an oligonucleotide for the characterization of controlled release formulations, J. Pharmacol. Biomed. Anal. 31 (2003) 143–149. [4] M. Derouazi, P. Girard, F. Van Tilborgh, K. Iglesias, N. Muller, M. Bertschinger, F. Wurm, Serum-free large scale transient transfection of CHO cells, Biotechnol. Bioeng. 87 (2004) 537–545. [5] J. Jeong, S. Song, D. Lim, H. Lee, T. Park, DNA transfection using linear poly(ethylenimine) prepared by controlled acid hydrolysis of poly(2-ethyl-2-oxazoline), J. Control Release 73 (2001) 391–399.

Acknowledgment The authors thank David Hacker for critically reading the manuscript.