Stabilization of Liposomes through Enzymatic Polymerization of DNA Tristan Ruysschaert, † Laurent Paquereau, † Mathias Winterhalter, †,‡ and Didier Fournier* ,† Institut Pharmacologie et Biologie Structurale, UMR CNRS UniVersite ´ Paul Sabatier, 5089 Toulouse, France, and International UniVersity Bremen, D-28725 Bremen, Germany Received July 25, 2006; Revised Manuscript Received October 21, 2006 ABSTRACT Combining supramolecular self-assembly of lipids with enzymatic triggered DNA interfacial polymerization allows construction of composite nanocapsules. Covalent grafting of oligonucleotides functionalizes the surface of liposomes. Subsequent addition of an enzyme called terminal deoxynucleotidyl transferase elongates the single-stranded DNA. The elongated DNA hybridizes, creating a random network. The short segments of double-stranded DNA provides a substrate for the Klenow fragment of E. coli DNA polymerase, which synthesizes a double-strand DNA, reinforcing the network. Alternate action of both enzymes leads to a three-dimensional network anchored on the liposome surface. Since 1960, liposomes are used in life science as models for cells and have been found to have various applications as a delivery system in cosmetics, gene therapy, or medicine. 1 However, their poor chemical and mechanical stability often restricts possible applications. Many approaches for liposome stabilization were suggested. Among them, addition of cholesterol or ABA copolymer to the lipids enhances the mechanical stability. 2,3 Coating of various polymers such as poly(acrylic acid) derivatives, chitosan, carboxymethyl chi- tosan, poloxamer, or carboxymethyl chitin on the outer leaflet of the lipid membrane provided some mechanical stabiliza- tion and prolongation of the lifetime in the blood stream. 4-10 However, the above-mentioned methods were usually not sufficient. Here, we introduce crosslinked DNA as a polymer coat (Figure 1). DNA offers two interesting properties for building mate- rial. First, DNA interacts via the Watson-Crick pairing, a specific and strong interaction. Second, a large variety of highly specific biological tools exists to polymerize, to cut, or to ligate DNA. DNA polymerases allow polymerization with or without template. Cutting DNA may be sequence specific with restriction enzymes: single- or double-strand- specific depending on the DNAse, DNA extremities may be ligated, etc. Enzymes, described in detail by molecular biologists and commercially available, catalyze all of these reactions. Liposomes were prepared using the film hydration tech- nique. 11,12 Briefly, 45 μmol of phosphatidyl choline (egg-PC) and 5 μmol maleimide-functionalized lipids (1,2-dipalmitoyl- sn-glycero-3-phosphoethanolamine- N-[4-(p-maleimido- phenyl)butyramide]), product 870013 of Avanti Polar Lipids, Birmingham, AL) dissolved in CHCl 3 were placed in a glass tube, dried under a stream of N 2 , and then placed under vacuum for 3 h to form a lipid film. The film was solubilized by addition of 1 mL 145 mM NaCl, 2.5 mM HEPES, pH * Corresponding author. E-mail: fournier@ipbs.fr. † Institut de Pharmacologie et de Biologie Structurale. ‡ International University Bremen. Figure 1. (A) Combined action of two polymerases creates a DNA shell on a liposome. Single-strand oligonucleotides step were grafted on lipid heads (magenta). (B) Elongation of single-stranded DNA is catalyzed by terminal deoxynucleotidyl transferase (orange). Depending on the available nucleotides for elongation, the single strands hybridize. (C) Subsequent addition of Klenow DNA polymerase recognizing double-stranded fragments and comple- ments the strands (green). NANO LETTERS 2006 Vol. 6, No. 12 2755-2757 10.1021/nl061724x CCC: $33.50 © 2006 American Chemical Society Published on Web 11/01/2006