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

Abstract

physical properties suitable for directly targeting therapeutic sites using magnetic fields. [2] Furthermore, the ability of MNPs (a subtype being superparamagnetic iron oxide NPs; SPIONs) to efficiently convert magnetic energy into thermal energy, makes them a focus of interest for use in hyperthermia-based cell ablation anticancer therapies (such as Magforce Nanotechnologies AG). [3-6] Other relevant applications include imaging (including a focus on regenerative medicine), tissue engineering, for mechanical stimulation for cell differentiation in vivo [7-17] and, most recently, their use as synthetic enzymes in biocatalytic processes. [18] Targeted delivery strategies for MNPs have been developed toward specific-or overexpressed receptors on diseased cells by functionalizing the NP surface with proteins, antibodies, or other biomolecules as targeting ligands. These strategies efficiently enhance NP delivery to the target cells in vitro; however, there is mounting evidence that targeting ability of functionalized NPs disappears when placed in an in vivo biological environment. [19-21] It is now well established that when any material surface encounters a biological system, interactions occur between the material and the system components (i.e. proteins, lipids, DNA) forming a layer termed the protein corona. The protein corona defines the biological identity of the particle or surface, and has proven to be an obstacle in the past for effective targeted delivery of ligands, since it affects the physicochemical 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.

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

“…Dextran-coated MNPs are approved by the Food and Drug Administration (FDA) for their use in vivo and are currently being optimized for multiple applications in biomedicine. ,− We have previously shown that positive GET peptides complex to the negatively charged dextran coating of Nanomag-D MNPs (MicroMod) and enhance the cell uptake of the MNPs. ,, In our previous studies, we focused on the characterization of the complexation of GET peptides and MNPs, establishing the minimum amount of GET peptides required in order to enhance cellular uptake of MNPs, as well as the concentration of GET at which MNPs would be saturated. We found that the optimal concentration of GET for MNP delivery (4 nmol GET per mg MNPs) was significantly inferior to the saturation maximum (40 nmol for GET per mg of MNPs).…”

“…Dextran-coated MNPs are approved by the Food and Drug Administration (FDA) for their use in vivo and are currently being optimised for multiple applications in biomedicine 16,[36][37][38] . We have previously shown that positive GET peptides complex to the negatively charged dextran coating of Nanomag-D MNPs (MicroMod) and enhance the cell uptake of the MNPs 26,37,39 . In our previous studies 39 , we focused on the characterization of the complexation of GET peptide and MNPs, establishing the minimum amount of GET peptide required in order to enhance cellular uptake of MNPs, as well as the concentration of GET at which MNPs would be saturated.…”

Effects of Microenvironment and Dosing on Efficiency of Enhanced Cell Penetrating Peptide Nonviral Gene Delivery

“…Cell viability from monolayers were assayed for cell metabolism using PrestoBlue (ThermoFisher, cat no: A13262) as described previously. , We employed 50 μL and 500 μL volumes for assays of 96-well plates and 12-well plates, respectively. Time of incubation was varied with appropriate controls to allow significant color changes before fluorometry in black 96-well plates (50 μL/sample).…”

Journey of micronanoplastics with blood components

“…187,188 There are well-accepted in vitro methods to measure protein binding. 189,190 These methods include mass spectrometry (to pinpoint the precise plasma proteins and associated amino acid residues that interact with ligand); 191 spectroscopic methods including UV-vis, uorescence, and circular dichroism spectroscopy (to investigate the modications in protein structure 192 brought on by the interaction with the ligand); electron microscopy (to visualize how MNPs interact with plasma proteins); 193 size-exclusion chromatography and highperformance liquid chromatography to separate proteins from ligands and analyze their binding. 194 However, in the last two decades, uorescence spectroscopy and its calculations to measure protein binding have revolutionized these research areas.…”