Semi‐Automated Synthesis and Screening of a Large Library of Degradable Cationic Polymers for Gene Delivery

Abstract

Higher delivery efficiency than conventional nonviral systems (such as poly(ethyleneimine)) have been identified in 46 polymers through cell-based screening of a large, 2350-member library by a high-throughput, semi-automated process. The transfection potential of the polymers was assessed by testing their ability to deliver luciferase expressing plasmid and green fluorescent protein plasmid (see picture) to a monkey kidney fibroblast cell line. The combinatorial, automated high-throughput synthesis and evaluation of small molecules has revolutionized modern drug discovery. Here we describe the first high-throughput, semi-automated methodology for the synthesis and screening of a large library of polymers for gene delivery. A key feature of these methods is that synthesis, storage, and cell-based testing are all performed without removing solvent, thereby allowing high- throughput manipulations using simple, easily automated fluid-handling systems. By using these methods, we synthesized a library of 2350 structurally unique, degradable, cationic polymers. High-throughput, cell-based screening has identified 46 new polymers that transfect with a higher efficiency than conventional nonviral delivery systems such as poly(ethyleneimine). The major barrier to the success of gene therapy in the clinic remains the lack of safe and efficient DNA-delivery methods. Currently, the majority of clinical trials use modified viruses as delivery vehicles, which, while effective at transferring DNA to cells, suffer from potentially serious toxicity and production problems.1, 2 In contrast, nonviral systems offer a number of potential advantages, including ease of production, stability, low immunogenicity and toxicity, and reduced vector size limitations.3 Despite these advantages, however, existing nonviral delivery systems are far less efficient than viral vectors.4 One promising group of nonviral delivery compounds are cationic polymers, which spontaneously bind and condense DNA.5–12 We have been particularly interested in poly(β-amino ester)s as delivery agents which have been shown to generally possess low cytotoxicity and are easily synthesized by the conjugate addition of a primary amine or bis(secondary amine) to a diacrylate (Scheme 1).11, 12 Synthesis of poly(β-amino ester)s. Poly(β-amino ester)s were synthesized by the conjugate addition of primary or bis(secondary amines) to diacrylates. The traditional development of new biomedical polymers has been an iterative process: polymers typically are designed one at a time and then individually tested for their properties. More recently, attention has focused on the development of parallel, combinatorial approaches that facilitate the generation of structurally diverse libraries of polymeric biomaterials.12–15 However, the methods used in all previous studies are still relatively low-throughput, and therefore were not able to generate large amounts of structure–function information. Among the primary factors limiting the throughput and automation of poly(β-amino ester) synthesis and testing was the viscosity of the monomer and polymer solutions, and the difficulty with manipulating the solid polymer products. While automation of liquid handling is straightforward using conventional robotics, the manipulation of solids and viscous liquids on a small scale is not. Therefore, we sought to develop a general procedure with which polymers could be synthesized and screened in cell-based assays without leaving the solution phase. Since this would require the presence of residual solvent in the cell assays, we chose to use a relatively nontoxic solvent: dimethyl sulfoxide (DMSO). DMSO is a widely used solvent in the culturing of cells, and is commonly used when storing frozen stocks of cells. It is miscible with water and is generally well-tolerated. The first step in preparing for high-throughput synthesis was to identify conditions that would allow for the production of polymer and yet possess a