1124
Bioconjugate Chem. 2002, 13, 1124−1133
Copolymers of Ethylene Imine and N-(2-Hydroxyethyl)-ethylene Imine as Tools To Study Effects of Polymer Structure on Physicochemical and Biological Properties of DNA Complexes Dagmar Fischer,†,‡ Anke von Harpe,‡,§ Klaus Kunath,† Holger Petersen,† Youxin Li,| and Thomas Kissel*,† Department of Pharmaceutics and Biopharmacy, University of Marburg, Ketzerbach 63, 35032 Marburg, Germany, Procter & Gamble Pharmaceuticals, Dr. Otto Ro¨hm-Strasse 2-4, 64331 Weiterstadt, Germany, and Schwarz Pharma AG, Alfred-Nobel-Strasse 10, 40789 Monheim, Germany. Received May 14, 2002; Revised Manuscript Received June 27, 2002
A series of five poly[(ethylene imine)-co-N-(2-hydroxyethyl-ethylene imine)] copolymers with similar molecular weights and different degrees of branching was established to study structure-function relationship with regard to physicochemical and biological properties as gene delivery systems. Copolymers were synthesized by acid-catalyzed ring-opening copolymerization of aziridine and N-(2hydroxyethyl)-aziridine in aqueous solution and characterized by GPC-MALLS, 1H- and 13C NMR, IR, potentiometric titration, and ion exchange chromatography. Complexation of DNA was determined by agarose gel electrophoresis, and complex sizes were quantitated by PCS. Cytotoxicity of the copolymers in fibroblasts was assessed by MTT-assay, LDH-assay, and hemolysis. The transfection efficiency was determined using the reporter plasmid pGL3 in 3T3 mouse fibroblasts. The copolymers obtained by solution polymerization had relatively low molecular weights of about 2000 Da, and the degree of branching increased with increasing ethylene imine ratio. The pKa as well as the buffer capacity increased proportional to the number of primary and secondary amines. Higher branched polymers showed stronger complexation and condensation of DNA, formed smaller polymer/DNA complexes, and induced the expression of plasmids to a higher extent than less branched polymers. In vitro cytotoxic effects and the hemolysis of erythrocytes decreased with decreased branching. Our results indicate that the basicity and degree of protonation of the polymers depending on their amount of primary and secondary amines seem to be important factors both for their transfection efficiency and for their cytotoxicity in gene transfer.
INTRODUCTION 1
The polycation polyethylene imine (PEI) is generally regarded as a useful reagent for nonviral delivery of DNA and RNA under both in vitro and in vivo conditions (Remy et al., 1998). PEI, compared to other nonviral vectors, is assumed to affect endosomal escape and the enzymatic protection of DNA and RNA (Behr, 1997). PEI is commercially available with different average molecular weights and degrees of branching. The transfection efficiencies, cytotoxicity, and DNA binding characteristics * Address correspondence to this author at the Department of Pharmaceutics and Biopharmacy, Philipps-University, Ketzerbach 63, D-35032 Marburg/Lahn, Germany. Tel: (0049)-6421282-5881; Fax: (0049)-6421-282-7016; E-mail:
[email protected]. † University of Marburg. ‡ These authors contributed equally to this work. § Procter & Gamble Pharmaceuticals. | Schwarz Pharma AG. 1 Abbreviations: DNA deoxyribonucleic acid, EI ethylene imine, HEEI N-(2-hydroxyethyl) ethylene imine, HMW high molecular weight, IR infrared, LDH lactate dehydrogenase, LMW low molecular weight, MALLS multiple angle laser light scattering, MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, MW molecular weight, NMR nuclear magnetic resonance, PCS photon correlation spectroscopy, PEI poly(ethylene imine), PEIHEEI poly[(ethylene imine)-co-N-(2hydroxyethyl-ethylene imine)], SEC size exclusion chromatography.
of PEI have been discussed somewhat controversially in the literature (Fischer et al., 2000; Gebhart and Kabanov, 2001). Systematic studies probing the relationship between the structure of PEI, especially the molecular weight, the degree of branching, the relative ratio of amino functions, and physicochemical and biological effects, are rare. DNA delivery using PEI was found to depend on the molecular weight of the polymer. Often highly branched PEIs with molecular weights in the range from 25 to 800 kDa were reported to be useful for gene delivery. These PEI/DNA complexes yield high levels of transgene expression in many cell lines and primary cells under in vitro conditions (Boussif et al., 1996; Erbacher et al., 1999), as well as in vivo, e.g., in kidney, lung, and brain (Abdallah et al., 1997; Boletta et al., 1997; Lecocq et al., 2000). Cytotoxic effects, however, limit their utility. Comparing the potential for transgene expression of highly branched PEI with molecular weights in the range from 0.6 to 70 kDa, Godbey et al. (1999) observed an increase in gene expression with increasing molecular weight of the polymers. The transfection efficiency for 1.8 kDa PEI was essentially zero. The inability of PEIs 1 µm with a broad size distribution. One exception was the 7/93 copolymer with only little branching (6%) and a high content of hydroxyethyl substituents, forming complexes of about 650 nm. Increasing the N/P ratio of the complexes, particle sizes were reduced according to the polymer composition. LMW-PEI formed complexes
