Identification of Molecular-Mimicry-Based Ligands for Cholera Diagnostics using Magnetic Relaxation

Abstract

When covalently bound to an appropriate ligand, iron oxide nanoparticles can bind to a specific target of interest. This interaction can be detected through changes in the solution's spin-spin relaxation times (T2) via magnetic relaxation measurements. In this report, a strategy of molecular mimicry was used in order to identify targeting ligands that bind to the cholera toxin B subunit (CTB). The cellular CTB-receptor, ganglioside GM1, contains a pentasaccharide moiety consisting in part of galactose and glucose units. We therefore predicted that CTB would recognize carbohydrate-conjugated iron oxide nanoparticles as GM1 mimics, thus producing a detectable change in the T2 relaxation times. Magnetic relaxation experiments demonstrated that CTB interacted with the galactose-conjugated nanoparticles. This interaction was confirmed via surface plasmon resonance studies using either the free or nanoparticle-conjugated galactose molecule. The galactose conjugated nanoparticles were then used as CTB sensors achieving a detection limit of 40 pM. Via magnetic relaxation studies, we found that CTB also interacted with dextran-coated nanoparticles and surface plasmon resonance studies also confirmed this interaction. Additional experiments demonstrated that the dextran-coated nanoparticle can also be used as CTB sensors and that dextran can prevent the internalization of CTB into GM1-expressing cells. Our work indicates that magnetic nanoparticle conjugates and magnetic relaxation detection can be used as a simple and fast method to identify targeting ligands via molecular mimicry. Furthermore, our results show that the dextran-coated nanoparticles represent a low-cost approach for CTB detection.

Assessment of Molecular Interactions through Magnetic Relaxation

“…Using our competition method, we found that the K D of the dextran–CTB interaction is 4.9 μ M . This value is substantially lower than the value we previously determined using SPR (14 m M ) 28. In that study, CTB was immobolized to the SPR gold plate using a ganglioside as a linker.…”

“…Next, we utilized the bMR‐nanosensor‐based competition assay to study the interaction between toxins and small molecules. Recent reports describe the interaction of doxorubicin with the tetanus toxin C fragment (TTC)27 and galactose or dextran with cholera toxin B subunit (CTB) 28. The study of these interactions is important for the development of small‐molecule‐based therapeutics.…”

Magnetic nanoparticles with diblock glycopolymer shells give lectin concentration-dependent MRI signals and selective cell uptake

“…Carbohydrates have previously been used on the surface of functional nanoparticles to enhance cellular uptake efficiency and specicity. 23,85,86 Overexpression of specic mannose receptors on cancer cells has been investigated as target for therapeutics delivery, in a number of reports on cell lines such as MCF-7/Adr-res (breast cancer), SKOV-3 (ovarian adenocarcinoma), B16F10, B16F1, (murine melanoma), A549 (lung cancer), and A498 (kidney cancer). 53 Firstly, the cytotoxicity of IONPs and polymer functionalized IONPs was investigated for two different cell lines (in human lung broblast (MRC 5) and lung cancer cell (A549 cells)) using a standard method: Alamar Blue assay.…”

Assessment of Molecular Interactions through Magnetic Relaxation

“…Recent reports describe the interaction of doxorubicin with the tetanus toxin C fragment (TTC) [27] and galactose or dextran with cholera toxin B subunit (CTB). [28] The study of these interactions is important for the development of small-molecule-based therapeutics. For the TTCdoxorubicin interaction, a doxorubicin-carrying iron oxide nanoparticle was designed.…”

“…This value is substantially lower than the value we previously determined using SPR (14 mm). [28] In that study, CTB was immobolized to the SPR gold plate using a ganglioside as a linker. Likely, this approach may block sites at which dextran binds, therefore affecting the interaction between CTB and dextran.…”