Elsevier

Journal of Controlled Release

Volume 69, Issue 2, 3 November 2000, Pages 309-322
Journal of Controlled Release

Characterization of commercially available and synthesized polyethylenimines for gene delivery

https://doi.org/10.1016/S0168-3659(00)00317-5Get rights and content

Abstract

Five new polyethylenimines (PEI) were synthesized by polymerization of aziridine in aqueous solution and compared to several commercially available PEI used for gene transfer. Polymers were characterized by 13C NMR spectroscopy, capillary viscosimetry, potentiometric titration and Cu(II) complex formation to gain insight into structural and functional properties. 13C NMR analysis revealed differences in the extent of branching based on the ratio of primary, secondary and tertiary amino groups. An amino group ratio 1°:2°:3°=1:2:1 was obtained for the synthesized PEI, whereas commercially available PEI generally showed a higher degree of branching (1:1:1). Capillary viscosimetry of aqueous PEI solutions with a sufficient amount of salt gave Mark–Houwink parameters of α=0.26 and KV=1.00 cm3/g for the commercially available polymers. In case of the synthesized polymers, variation of reaction conditions yielded viscosity average molar masses (Mv) in the range of 8000–24 000 g/mol. PEI solutions were investigated by potentiometric titration analysis showing that their buffer capacity was not significantly influenced by molar mass or polymer structure. The pKa values (8.18–9.94) and the buffer capacity β (0.08–0.014 mol/l) were of comparable magnitude. This study highlights the necessity of more detailed characterization methods for PEI used in gene transfer protocols since physico-chemical properties do not reflect the vast differences found in transfection efficiencies.

Introduction

Polyethylenimine (PEI) is a cationic polymer exhibiting the highest positive charge density when fully protonated in aqueous solution [1]. These properties are useful for a variety of industrial applications, e.g. in the paper industry for flocculation of negatively charged fibers [2] or as an additive for production of ink-jet paper [3]. Its chelating properties are exploited in waste water treatment to remove metal ions [4].

Branched polyethylenimines are obtained by cationic polymerization of aziridine either at elevated temperatures in aqueous or alcoholic solution or in bulk at low temperatures following the reaction scheme shown in Fig. 1 [5]. The weight average molecular weights (Mw) achieved with this synthesis are typically in the range 20 000–50 000 g/mol [5]. To generate higher molecular weights of branched polyethylenimine in technical synthesis, bifunctional linkers such as dichloroethane or epichlorhydrine derivatives are used [6].

Meanwhile, new applications for PEI have emerged in biology and medicine. Branched and linear polyethylenimines are considered to be promising candidates as non-viral vectors for plasmid [7], [8], [9] and oligonucleotide delivery [10], [11], both in vitro and in vivo. They provide an attractive alternative to cationic lipid formulations because they combine remarkable transfection efficiencies with high complex stability [12] and allow transfection in the presence of serum [13]. Recently, linear PEI has become commercially available as transfection reagent under the trademark ExGen 500®, with an apparently high transfection efficiency [14].

Different commercially available polymers have been described as transfection reagents which are offered with various molar masses by different suppliers. These PEI have not been characterized in detail with respect to their physico-chemical properties and in the literature conflicting information is provided regarding structural requirements and molecular weights necessary for efficient gene delivery. While some authors argue that lower molecular weight PEI are more effective transfection reagents [9], [15], others report failing transfection for very low molecular weights in the range 600–1800 g/mol and increasing efficiency with increasing molecular weights of PEI [16]. The optimal molecular weight specifications for gene transfer is still not known with certainty, as it is difficult to compare different studies historically using various sources of PEI, transfection protocols, cell lines etc. Additionally, the purity, toxicity and biocompatibility of PEI is a matter of concern, e.g. high molecular weight polyethylenimine (800 000 g/mol) as non-biodegradable polymer can not be renally excreted and presents, therefore, problems for in vivo applications.

Moreover, the issue of the optimal PEI architecture for gene delivery is not known. The concept explaining the transfection efficiency of PEI is based on the so-called proton sponge hypothesis [17]. After uptake of PEI/DNA complexes into the endo-/lysosomal compartment PEI should buffer the acidic pH of the lysosome, protecting the DNA from degradation and causing an osmotic swelling/rupture of the vesicles by which DNA is released into the cytoplasm. Protonation of the polycation leads to an expansion of the polymeric network due to intramolecular charge repulsion. A branched polymer structure is thought to be a prerequisite for this behavior. The proton sponge hypothesis does not explain why unbranched, linear PEI was demonstrating high transfection efficiencies in several studies [9], [14], [18].

Moreover, little is known about the relationship between molar masses or branching structure of PEI and their effect on the buffer capacity. Different molar masses of PEI as well as the choice of linear or branched PEI affect transfection efficiency as well as cytotoxicity of PEI/DNA complexes [19], [20]. The buffer capacity of PEI solutions can be determined by the potentiometric titration with hydrochloric acid and the influence of branching and molecular weight on the buffer capacity of PEI can be investigated.

The complexation and condensation of oligonucleotides and DNA is thought to occur mainly through electrostatic interactions of the positively charged PEI and negatively charged DNA [21]. These processes are influenced by the level of protonation on one hand and the flexibility of the polymer chains on the other. As a simple model for studying polymer chain flexibility, we investigated the ability of PEI to form metal complexes with Cu(II) ions in solution. The formation of these complexes requires a steric coordination of the nitrogen atoms of PEI and was used to probe the accessibility of the nitrogen atoms of these polymers as a function of the degree of branching and molecular weight.

In this study, a more detailed characterization of commercially available branched PEI was carried out with respect to the use of these polymers for gene transfer. Additionally, we synthesized PEI using different reaction conditions in aqueous solution to modify molar masses and the degree of branching.

Section snippets

Materials

Eight commercially available polyethylenimines were studied. These are referred to as PEI 1 (Fluka, Deisenhofen, Germany Cat. No. 03880, Lot No. 345312/1 995, Mr=600 000–1 000 000 g/mol (no method disclosed)), PEI 2 (Sigma, Deisenhofen, Germany Cat. No. P-3143, Lot No. 126H0142, Mw=750 000, Mn=60 000 (no method disclosed)), PEI 3 (Aldrich, Deisenhofen, Germany Cat. No. 40, 872-7, Lot No. AR 05601DQ, Mw=25 000 (light scattering, LS), Mn=10 000 (SEC)), PEI 4 (Aldrich Cat. No. 40,870-0, Lot No. DR

Synthesis

Acid-catalyzed polymerization of aziridine has been described previously [5]. Protonation of the aziridine monomer leads to an immonium cation which reacts with additional monomer for propagation or with an already formed polymer chain to form branches (Fig. 1). Bulk polymerization of anhydrous aziridine upon addition of initiator proceeds with violence and leads to higher molecular weight products. Polymerization in aqueous solution occurs more slowly and can be influenced by variation of

Discussion

The aim of this study was to validate methods for the characterization of polycations such as PEI. Such methods should allow determination of the degree of branching and molar mass of PEI used for gene or oligonucleotide transfer both in vitro and in vivo. Transfection efficiencies, polymer cytotoxicity and DNA binding characteristics seem to be influenced by molar mass and structural components of the polycations, such as PEI [16], [19]. Therefore, a more detailed characterization of the

Conclusions

The physico-chemical characterization of commercially available PEI is insufficient to predict their performance as transfection reagents. A more detailed description would be helpful based on analytical methods, such as 13C NMR spectroscopy to describe not only molecular weight and molecular weight distribution but also information about the structure of the PEI used as gene delivery system. A detailed study of the branched structure of PEI using 2D- and inverse gated-decoupled spectra

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    Present address: Schwarz Pharma AG; Alfred-Nobel-Str.10, 40789 Monheim, Germany.

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