Maximization of Loading and Stability of ssDNA:Iron Oxide Nanoparticle Complexes Formed through Electrostatic Interaction

摘要

The use of inorganic nanoparticles (NPs) as vectors for the delivery of oligonucleotides for in vitro and in vivo applications is rapidly gaining momentum. Some of the reasons making them especially good candidates for this purpose are their ease of synthesis in a range of sizes and surface coatings, their propensity to penetrate cell membranes, their stability and biocompatibility and their unique size-dependent physical properties that impart additional diagnostic and therapeutic tools. Notwithstanding these notable attributes, a major obstacle to their practical use is given by the typically low oligonucleotide loading levels attainable through conventional bioconjugation procedures. This shortcoming is especially worrisome as toxicity concerns have been associated with co-delivery of NPs. In this paper we are analytically analyzing the formation of electrostatic complexes between negatively charged ssDNA and positively charged iron oxide nanoparticles (SPIO-NP) with the purpose of identifying the optimal conditions leading to stable formulations at high oligo loading levels. The formation and loading levels of ssDNA:SPIO-NP complexes have been investigated at different oligo:NP ratios and under different ionic strengths through Dynamic Light Scattering, fluorescence quenching experiments and pull-down assays. Through these studies we have identified optimal conditions for attaining maximal oligo loading levels and we are proposing a simple model to explain an unusual behavior observed in the formation of the complexes. Finally we introduce an alternative loading method relying on the electrostatic co-loading of an oligo sequence in the presence of a negatively charged PEGylated block copolymer, yielding very stable and high loading PEGylated ssDNA:SPIO-NPs. The findings that we are reporting are of general validity and similar conditions could be easily translated to the electrostatic formation of ssDNA:NP complexes consisting of different NP materials and sizes.