Biomaterials 28 (2007) 1267–1279 Nanostructured calcium phosphates (NanoCaPs) for non-viral gene delivery: Influence of the synthesis parameters on transfection efficiency Dana Olton a , Jinhua Li c , Mary E. Wilson a,b , Todd Rogers a,b , John Close d , Leaf Huang e , Prashant N. Kumta a,b,1 , Charles Sfeir c,Ã,1 a Department of Biomedical Engineering, Carnegie Mellon University, Pittsburgh, PA 15213, USA b Materials Science and Engineering, Carnegie Mellon University, Pittsburgh, PA 15213, USA c Department of Oral Biology, Center for Craniofacial Regeneration, University of Pittsburgh, Pittsburgh, PA 15261, USA d Dental Public Health and Information Management, University of Pittsburgh, Pittsburgh, PA 15261, USA e Division of Molecular Pharmaceutics, School of Pharmacy, University of North Carolina, Chapel Hill, NC 27599, USA Received 11 August 2006; accepted 25 October 2006 Abstract Calcium phosphate (CaP) based approaches remain an attractive option for delivering plasmid DNA (pDNA) into cultured cells. However, despite their appeal, current synthesis methodologies typically yield lower, less consistent transfection efficiencies when compared to viral approaches. Therefore, we report here a novel method to consistently synthesize efficient, nano-sized, mono-dispersed CaP–pDNA particles; accomplished by optimizing both the stoichiometry (Ca/P ratio) of the CaP particles as well as the mode in which the calcium and phosphate precursor solutions are mixed. Our results indicate that calcium and phosphate precursors when mixed in a controlled and regulated manner reproducibly result in nano-sized particles that consistently yield higher transfection efficiencies when compared to particles synthesized via manual mixing (a two-fold increase was observed). Also, maximum transfection efficiencies in both HeLa and MC3T3-E1 cells lines were obtained when a Ca/P ratio between 100 and 300 was used. Particles synthesized within this optimum Ca/P ratio range were between 25 and 50 nm. Our data suggests that these maximized transfection efficiencies were obtained because these particles not only effectively condensed (70% efficient) but also efficiently bound (90% efficient) the pDNA. In addition, X-ray diffraction and Fourier transform infrared spectroscopy analyses confirmed that all of the synthesized CaP structures exhibited the hydroxyapatite phase. r 2006 Published by Elsevier Ltd. Keywords: Nanoparticles; Calcium phosphate; Non-viral vectors; Gene therapy 1. Introduction There is an acknowledged need for the development of novel, safe and effective non-viral gene delivery therapies to be used for tissue regeneration, cancer treatment, DNA vaccines, etc. Current non-viral gene therapies possess several potential advantages, such as tissue-specific target- ing [1], ease of large-scale production [1–3], the capacity to carry large DNA inserts [4] as well as the potential to exhibit relatively lower immunogenicity [1,2,5] in compar- ison to viral approaches. However, despite these advan- tages, non-viral vectors typically exhibit relatively lower transfection efficiencies when compared to viral ap- proaches [2,5]. There is therefore a need to identify new, non-viral material forms to package and deliver plasmid DNA (pDNA) in an effective, safe, efficient and relevant manner for in vivo applications. A variety of non-viral gene delivery vectors, including naked DNA injection [6,7], electroporation [8], gene gun administration [9,10], cationic [11,12] and anionic [13,14] lipids, cationic polymers [1,15,16], peptides [17,18] as well as ceramic particles of calcium phosphate (CaP) [19–23] are ARTICLE IN PRESS www.elsevier.com/locate/biomaterials 0142-9612/$ - see front matter r 2006 Published by Elsevier Ltd. doi:10.1016/j.biomaterials.2006.10.026 Ã Corresponding author. Department of Oral Biology, Center for Craniofacial Regeneration, University of Pittsburgh, Pittsburgh, PA 15261, USA. Tel.: +1 412 648 1949; fax: +1 412 624 6685. E-mail address: csfeir@pitt.edu (C. Sfeir). 1 Prashant N. Kumta and Charles Sfeir contributed equally.