Cationic copolymers nanoparticles for nonviral gene vectors: Synthesis, characterization, and application in gene delivery Giovanna Gomez d’Ayala, 1 * Anna Calarco, 2 * Mario Malinconico, 1 Paola Laurienzo, 1 Orsolina Petillo, 2 Angela Torpedine, 2 Gianfranco Peluso 2 1 Institute of Polymers Chemistry and Technology, CNR, Via Campi Flegrei 34, 80078 Pozzuoli, Naples, Italy 2 Institute of Protein Biochemistry, CNR, Via Pietro Castellino 111, 80131 Naples, Italy Received 19 May 2009; revised 9 September 2009; accepted 4 December 2009 Published online 2 March 2010 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jbm.a.32752 Abstract: The major aim of nonviral delivery systems for gene therapy is to mediate high levels of gene expression with low toxicity. Nowadays, one of the most successful syn- thetic polycations used in gene delivery research is poly(ethy- lenimine) (PEI) in its high-molecular weight (HMW) branched form. However, PEI is not the ideal transfection agent in vivo because of its overwhelming cytotoxicity. To overcome its toxic effects with a minimal impact on transfection efficiency, PEI has been conjugated with several nonionic biocompatible polymers. Here, we describe the synthesis of nanosized par- ticles consisting of HMW PEI (25 kDa) crosslinked with poly- (e-caprolactone) (PCL, 50–60 kDa), a biodegradable aliphatic polyester. PCL was modified by the insertion of glycidyl groups able to condense with the amines of PEI to chemically bind PEI onto PCL. The nanoparticles obtained have been characterized in relation to their physicochemical and biologi- cal properties, and the results are extremely promising in terms of low cell toxicity and high transfection efficiency. These biological effects might be related to the peculiar DNA binding to covalently connected polymeric nanoparticles, without the formation of entangled DNA/polymer-soluble aggregates. V C 2010 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 94A: 619–630, 2010 Key Words: polycaprolactone, poly(ethylenimine), gene deliv- ery, nanoparticles, nonviral DNA vectors INTRODUCTION Poly(ethylenimine) (PEI) is a synthetic cationic polymer that is largely known in literature for its applications in nonviral vector systems for gene delivery. 1,2 PEI spontaneously asso- ciates with phosphates of DNA through electrostatic interac- tions because of the protonated amine groups forming sta- ble DNA/PEI polyplexes. 3,4 Highly branched, high-molecular weight (HMW) PEI shows a good transfection efficiency because of its buffering capacity, but it has also been reported to be toxic for many cellular lines. To avoid this adverse effect, low-molecular weight (LMW) PEI has been copolymerized with nonionic biocompatible polymers. Diblock copolymers of PEI with hydrophilic macromolecules forming DNA polyplexes with neutral surface charge as well as ternary block copolymers obtained by grafting diblock copolymers of poly(ethylene glycol) (PEG) and poly(e-capro- lactone) (PCL) onto branched PEI have been extensively investigated. 5,6 Recently, it has been reported that copoly- mers obtained by the reaction between PCL diacrylate and LMW PEI showed effective and stable DNA condensation with particle sizes below 200 nm. 7 A different approach resides in the combination of HMW PEI with biopolymers, either natural or synthetic, that allows for an easier manufacturing of the materials to obtain devices such as nanocarriers. Examples of the real- ization of vectors based on solid nanoparticles of blends of PEI with biodegradable polymers are reported in litera- ture. 8,9 In nanoparticle-based gene delivery systems, DNA can be encapsulated inside the nanoparticle 10,11 or adsorbed onto nanoparticle cationic surface. 12,13 This last system has the advantage of ease binding with DNA, DNA protection, avoidance of the direct contact of plasmid with organic sol- vents during particles preparation; furthermore, a rapid release of DNA inside the target cells is facilitated. More- over, DNA/nanoparticle complexes have a much lower tend- ency to aggregate when compared with soluble DNA/polyca- tion polyplexes. The objective of this work is to prepare and characterize graft copolymers of PCL and PEI suitable for their applica- tions in gene delivery. PCL was previously modified by the insertion of glycidyl groups able to condense with the amines of PEI. Chemical bonds with PCL avoid the release of toxic free PEI in the living cell after delivery of the DNA. Nanoparticles of controlled size and persistent shape have been obtained by an emulsion method and have been char- acterized by scanning electron microscopy (SEM). The nano- particles have been characterized in relation to their physi- cochemical and biological properties. Some experimental results are extremely promising in terms of low cell toxicity and high transfection efficiency. *These authors contributed equally to this work. Correspondence to: P. Laurienzo; e-mail: paola.laurienzo@ictp.cnr.it or G. Peluso; e-mail: g.peluso@ibp.cnr.it Contract grant sponsor: Italian Ministry of University and Research; contract grant number: 2006-prot. RBIP06ZJ78_002 V C 2010 WILEY PERIODICALS, INC. 619