Role of Cell Membrane−Vector Interactions in Successful Gene Delivery Sriram Vaidyanathan, † Bradford G. Orr, §,∥ and Mark M. Banaszak Holl* ,†,‡,∥,⊥ † Departments of Biomedical Engineering, ‡ Chemistry, and § Physics, ∥ Program in Applied Physics and ⊥ Macromolecular Science and Engineering, University of Michigan, Ann Arbor, Michigan 48109, United States CONSPECTUS: Cationic polymers have been investigated as nonviral vectors for gene delivery due to their favorable safety profile when compared to viral vectors. However, nonviral vectors are limited by poor efficacy in inducing gene expression. The physicochemical properties of cationic polymers enabling successful gene expression have been investigated in order to improve expression efficiency and safety. Studies over the past several years have focused on five possible rate-limiting processes to explain the differences in gene expression: (1) endosomal release, (2) transport within specific intracellular pathways, (3) protection of DNA from nucleases, (4) transport into the nucleus, and (5) DNA release from vectors. However, determining the relative importance of these processes and the vector properties necessary for optimization remain a challenge to the field. In this Account, we describe over a decade of studies focused on understanding the interaction of cationic polymer and cationic polymer/oligonucleotide (polyplex) interactions with model lipid membranes, cell membranes, and cells in culture. In particular, we have been interested in how the interaction between cationic polymers and the membrane influences the intracellular transport of intact DNA to the nucleus. Recent advances in microfluidic patch clamp techniques enabled us to quantify polyplex cell membrane interactions at the cellular level with precise control over material concentrations and exposure times. In attempting to relate these findings to subsequent intracellular transport of DNA and expression of protein, we needed to develop an approach that could distinguish DNA that was intact and potentially functional for gene expression from the much larger pool of degraded, nonfunctional DNA within the cell. We addressed this need by developing a FRET oligonucleotide molecular beacon (OMB) to monitor intact DNA transport. The research highlighted in this Account builds to the conclusion that polyplex transported DNA is released from endosomes by free cationic polymer intercalated into the endosomal membrane. This cationic polymer initially interacts with the cell plasma membrane and appears to reach the endosome by lipid cycling mechanisms. The fraction of cells displaying release of intact DNA from endosomes quantitatively predicts the fraction of cells displaying gene expression for both linear poly(ethylenimine) (L- PEI; an effective vector) and generation five poly(amidoamine) dendrimer (G5 PAMAM; an ineffective vector). Moreover, intact OMB delivered with G5 PAMAM, which normally is confined to endosomes, was released by the subsequent addition of L-PEI with a corresponding 10-fold increase in transgene expression. These observations are consistent with experiments demonstrating that cationic polymer/membrane partition coefficients, not polyplex/membrane partition coefficients, predict successful gene expression. Interestingly, a similar partitioning of cationic polymers into the mitochondrial membranes has been proposed to explain the cytotoxicity of these materials. Thus, the proposed model indicates the same physicochemical property (partitioning into lipid bilayers) is linked to release from endosomes, giving protein expression, and to cytotoxicity. ■ INTRODUCTION Recent advances in genome editing such as the CRISPR/Cas system has sparked a renewed interest in gene therapy. 1,2 The clinical success of gene therapy depends on the successful delivery of intact plasmid DNA (pDNA) to the nucleus of target cells. 3 Viral and nonviral delivery agents (vectors) are used to protect DNA from nucleases present in blood and tissue and also to promote cellular uptake. 3,4 Although viral vectors enable tissue specific delivery and induce greater gene expression than nonviral vectors, concerns about adverse immune reactions remain. 3 Thus, strategies to improve the serum stability, targeted cellular uptake, and nuclear transport of nonviral vectors are being pursued. 4,5 In vivo delivery is also complicated by potential formation of protein coronas onto polyplexes. 6,7 The challenges are substantially fewer for gene delivery to cultured cells since serum stability and targeted delivery are not a concern. This Account focuses on the key challenges common to both gene therapy and biotechnology applications of a particular class of nonviral vectors polycationic polymers. Although not discussed in detail herein, the general observations of transport and release are also relevant for the transport of siRNA and miRNA for purposes of gene silencing. 3,4 Received: April 28, 2016 Published: July 26, 2016 Article pubs.acs.org/accounts © 2016 American Chemical Society 1486 DOI: 10.1021/acs.accounts.6b00200 Acc. Chem. Res. 2016, 49, 1486−1493