Rapidly Transducing and Spatially Localized Magnetofection Using Peptide-Mediated Non-Viral Gene Delivery Based on Iron Oxide Nanoparticles.

Non-viral delivery systems are generally of low efficiency, which limits their use in gene therapy and editing applications. We previously developed a technology termed glycosaminoglycan (GAG)-binding enhanced transduction (GET) to efficiently deliver a variety of cargos intracellularly; our system employs GAG-binding peptides, which promote cell targeting, and cell penetrating peptides (CPPs), which enhance endocytotic cell internalization. Herein, we describe a further modification by combining gene delivery and magnetic targeting with the GET technology. We associated GET peptides, plasmid (p)DNA, and iron oxide superparamagnetic nanoparticles (MNPs), allowing rapid and targeted GET-mediated uptake by application of static magnetic fields in NIH3T3 cells. This produced effective transfection levels (significantly higher than the control) with seconds to minutes of exposure and localized gene delivery two orders of magnitude higher in targeted over non-targeted cell monolayers using magnetic fields (in 15 min exposure delivering GFP reporter pDNA). More importantly, high cell membrane targeting by GET-DNA and MNP co-complexes and magnetic fields allowed further enhancement to endocytotic uptake, meaning that the nucleic acid cargo was rapidly internalized beyond that of GET complexes alone (GET-DNA). Magnetofection by MNPs combined with GET-mediated delivery allows magnetic field-guided local transfection in vitro and could facilitate focused gene delivery for future regenerative and disease-targeted therapies in vivo.

Enhanced Cellular Transduction of Nanoparticles Resistant to Rapidly Forming Plasma Protein Coronas.

Nanoparticles (NPs) are increasingly being developed as biomedical platforms for drug/nucleic acid delivery and imaging. However, in biological fluids, NPs interact with a wide range of proteins that form a coating known as protein corona. Coronae can critically influence self-interaction and binding of other molecules, which can affect toxicity, promote cell activation, and inhibit general or specific cellular uptake. Glycosaminoglycan (GAG)-binding enhanced transduction (GET) is developed to efficiently deliver a variety of cargoes intracellularly; employing GAG-binding peptides, which promote cell targeting, and cell penetrating peptides (CPPs) which enhance endocytotic cell internalization. Herein, it is demonstrated that GET peptide coatings can mediate sustained intracellular transduction of magnetic NPs (MNPs), even in the presence of serum or plasma. NP colloidal stability, physicochemical properties, toxicity and cellular uptake are investigated. Using label-free snapshot proteomics, time-resolved profiles of human plasma coronas formed on functionalized GET-MNPs demonstrate that coronae quickly form (<1 min), with their composition relatively stable but evolving. Importantly GET-MNPs present a subtly different corona composition to MNPs alone, consistent with GAG-binding activities. Understanding how NPs interact with biological systems and can retain enhanced intracellular transduction will facilitate novel drug delivery approaches for cell-type specific targeting of new nanomaterials.

Characterization of flavins in roots of Fe-deficient strategy I plants, with a focus on Medicago truncatula.

The root accumulation and excretion of riboflavin (Rbfl) and Rbfl derivatives have been studied in the model legume species Medicago truncatula, grown in hydroponics in two different Fe deficiency conditions, with and without CaCO(3). Using high resolution mass spectrometry techniques coupled to liquid chromatography, three different flavin derivatives not previously reported in plants, putatively identified as 7-hydroxy-Rbfl, 7α-hydroxy-Rbfl and 7-carboxy-Rbfl, were found along with Rbfl in Fe-deficient M. truncatula roots. In the presence of CaCO(3) most of the flavins were accumulated in the roots, whereas in the absence of CaCO(3) there was partial export to the nutrient solution. The major flavins in roots and nutrient solution were Rbfl and 7-hydroxy-Rbfl, respectively. Flavins were located in the root cortex and epidermal cells, preferentially in a root region near the apex that also exhibited increased ferric chelate reductase (FCR) activity. Six out of 15 different species of horticultural interest showed root increases in both Rbfl (four of them also having Rbfl derivatives) and FCR. No significant correlation was found between Rbfl and either phosphoenolpyruvate carboxylase or FCR activities, whereas the latter two showed a good correlation between them. The possible roles of Rbfl and Rbfl derivatives in roots and nutrient solutions are discussed. Medicago truncatula is proposed as a model system for flavin studies.