Microfluidic Assembly of pDNA/Cationic Liposome Lipoplexes with High pDNA Loading for Gene Delivery Tiago A. Balbino, † Juliana M. Serafin, † Antonio A. Malfatti-Gasperini, ‡ Cristiano L. P. de Oliveira, § Leide P. Cavalcanti, † Marcelo B. de Jesus, ∥ and Lucimara G. de La Torre* ,† † School of Chemical Engineering, University of Campinas, UNICAMP, Campinas, SP 13083-970, Brazil ‡ Brazilian Synchrotron Light Laboratory, CNPEM, Campinas, Sã o Paulo 13083-100, Brazil § Institute of Physics, University of Sã o Paulo, USP, Sã o Paulo, SP 05508, Brazil ∥ Department of Biochemistry and Tissue Biology, Institute of Biology, University of Campinas, Campinas, SP 13083-970, Brazil ABSTRACT: Microfluidics offers unique characteristics to control the mixing of liquids under laminar flow. Its use for the assembly of lipoplexes represents an attractive alternative for the translation of gene delivery studies into clinical trials on a sufficient throughput scale. Here, it was shown that the microfluidic assembly of pDNA/cationic liposome (CL) lipoplexes allows the formation of nanocarriers with enhanced transfection efficiencies compared with the conventional bulk- mixing (BM) process under high pDNA loading conditions. Lipoplexes generated by microfluidic devices exhibit smaller and more homogeneous structures at a molar charge ratio (R ±) of 1.5, representing the ratio of lipid to pDNA content. Using an optimized model to fit small-angle X-ray scattering (SAXS) curves, it was observed that large amounts of pDNA induces the formation of aggregates with a higher number of stacked bilayers (N ∼ 5) when the BM process was used, whereas microfluidic lipoplexes presented smaller structures with a lower number of stacked bilayers (N ∼ 2.5). In vitro studies further confirmed that microfluidic lipoplexes achieved higher in vitro transfection efficiencies in prostate cancer cells at R ± 1.5, employing a reduced amount of cationic lipid. The correlation of mesoscopic characteristics with in vitro performance provides insights for the elucidation of the colloidal arrangement and biological behavior of pDNA/CL lipoplexes obtained by different processes, highlighting the feasibility of applying microfluidics to gene delivery. 1. INTRODUCTION Since it was first reported in 1972, gene therapy has been pursued as a promising strategy for the treatment of several diseases, with ongoing clinical trials. As an example, this technology has been shown to be a safe and effective treatment for 1.5 years after vector administration in Leber congenital amaurosis, the most severe inherited retinal dystrophy, which causes blindness or visual impairment. 1,2 DNA-based gene therapy relies upon the insertion of a functional plasmid DNA (pDNA) into the nucleus of cells, which in turn enables the expression of therapeutic proteins; this phenomenon is called transfection. 3 For systematic gene delivery to be effective, the pDNA requires appropriate protection due to its poor cellular internalization and fast enzymatic degradation. Many materials, such as chitosan, 4 cationic lipids, 5 and proteins, 6 have been employed as nonviral gene carriers. Such carriers are capable of electrostatically binding and condensing pDNA into cationic nanosized particles with optimal characteristics to be internalized and processed by cells. 7 Among nonviral cationic carriers, liposomes have long been explored for the delivery of nucleic acids. 8 Liposomes are self-assembled polar lipid vesicles of colloidal dimensions whose bilayer structure is similar to that of human cell membranes. 9 The use of cationic lipids in the lipid blend allows the formation of cationic liposomes (CLs) that can electrostatically interact with the negatively charged pDNA, forming lipoplexes. The spontaneous interactions between CLs and pDNA can lead to the formation of lipoplexes with different characteristics. Several factors, such as the individual concentrations of the species, the ionic strength of the media, the characteristics and composition of the lipid mixture, and the lipid/DNA charge ratio (R±), can affect the physicochemical, structural and biological properties of lipoplexes. 10,11 In a given R± condition, when positive charges from the cationic lipid balance negative charges from the DNA’s phosphate groups, lipoplexes are considered to be at the isoneutrality ratio. 12 At this point, the surface net charge of the lipoplexes shifts from positive to negative values as R± decreases. Additionally, close to the isoneutrality ratio, lipoplexes tend to form agglomerates with larger particle sizes and high polydispersity indexes (PdIs) and can also exhibit structures with a higher number of stacked bilayers. 13−15 Received: November 13, 2015 Revised: January 7, 2016 Published: January 27, 2016 Article pubs.acs.org/Langmuir © 2016 American Chemical Society 1799 DOI: 10.1021/acs.langmuir.5b04177 Langmuir 2016, 32, 1799−1807