ABSTRACT
In this work, we report the use of bioorthogonal chemistry, specifically the strain-promoted click azide-alkyne cycloaddition (SPAAC) for the covalent attachment of magnetic nanoparticles (MNPs) on living cell membranes. Four types of MNPs were prepared, functionalized with two different stabilizing/passivation agents (a polyethylene glycol derivative and a glucopyranoside derivative, respectively) and two types of strained alkynes with different reactivities: a cyclooctyne (CO) derivative and a dibenzocyclooctyne (DBCO) derivative. The MNPs were extensively characterized in terms of physicochemical characteristics, colloidal stability, and click reactivity in suspension. Then, the reactivity of the MNPs toward azide-modified surfaces was evaluated as a closer approach to their final application in a living cell scenario. Finally, the DBCO-modified MNPs, showing superior reactivity in suspension and on surfaces, were selected for cell membrane immobilization via the SPAAC reaction on the membranes of cells engineered to express azide artificial reporters. Overall, our work provides useful insights into the appropriate surface engineering of nanoparticles to ensure a high performance in terms of bioorthogonal reactivity for biological applications.
Subject(s)
Azides , Magnetite Nanoparticles , Alkynes/chemistry , Azides/chemistry , Cell Membrane , Click Chemistry , Cycloaddition Reaction , Polyethylene Glycols/chemistryABSTRACT
The hypothesis of rescuing FKBP12/RyR1 interaction and intracellular calcium homeostasis through molecular "reshaping" of FKBP12 was investigated. To this end, novel 4-arylthioalkyl-1-carboxyalkyl-1,2,3-triazoles were designed and synthesized, and their efficacy was tested in human myotubes. A library of 17 compounds (10a-n) designed to dock the FKBP12/RyR1 hot-spot interface contact residues, was readily prepared from free α-amino acids and arylthioalkynes using CuAAC "click" protocols amenable to one-pot transformations in high overall yields and total configurational integrity. To model nitro-oxidative stress, human myotubes were treated with the peroxynitrite donor SIN1, and evidence was found that some triazoles 10 were able to normalize calcium levels, as well as FKBP12/RyR1 interaction. For example, compound 10 b at 150 nM rescued 46% of FKBP12/RyR1 interaction and up to 70% of resting cytosolic calcium levels in human myotubes under nitro-oxidative stress. All compounds 10 analyzed showed target engagement to FKBP12 and low levels of cytotoxicity in vitro. Compounds 10b, 10c, 10h, and 10iR were identified as potential therapeutic candidates to protect myotubes in muscle disorders with underlying nitro-oxidative stress, FKBP12/RyR1 dysfunction and calcium dysregulation.
Subject(s)
Calcium/metabolism , Drug Discovery , Muscle, Skeletal/drug effects , Tacrolimus Binding Protein 1A/metabolism , Triazoles/pharmacology , Dose-Response Relationship, Drug , HEK293 Cells , Humans , Molecular Structure , Muscle, Skeletal/metabolism , Oxidative Stress/drug effects , Ryanodine Receptor Calcium Release Channel/chemistry , Ryanodine Receptor Calcium Release Channel/metabolism , Structure-Activity Relationship , Tacrolimus Binding Protein 1A/chemistry , Triazoles/chemical synthesis , Triazoles/chemistryABSTRACT
To improve the selectivity of magnetic nanoparticles for tumor treatment by hyperthermia, Fe3O4 nanoparticles have been functionalized with a peptide of the type arginine-glycine-aspartate (RGD) following a "click" chemistry approach. The RGD peptide was linked onto the previously coated nanoparticles in order to target αvß3 integrin receptors over-expressed in angiogenic cancer cells. Different coatings have been analyzed to enhance the biocompatibility of magnetic nanoparticles. Monodispersed and homogeneous magnetite nanoparticles have been synthesized by the seed growth method and have been characterized using X-ray diffraction, thermogravimetric analysis, infrared spectroscopy, transmission electron microscopy and magnetic measurements. The magnetic hyperthermia efficiency of the nanoparticles has also been investigated and cytotoxicity assays have been perfomed for functionalized nanoparticles.