PolyMag Transfection Reagent

Clusters of iron oxide particles are excellent contrast agents for molecular and cellular MRI because of their large effect on water proton transverse relaxation. Their efficiency depends on the magnetization of the particles, the size of the cluster and the intra-aggregate volume fraction occupied by the iron oxide particles inside the cluster. After optimization of these different parameters, a relaxivity of ∼ 750 s−1mM−1 can theoretically be achieved. Polymag?™ clusters are initially intended for magnetofection but are here shown to present excellent relaxation properties. Magnetometry shows that the particles constituting the clusters have a high saturation magnetization (Mv = 371000 A/m) and present a broad log-normal size distribution (d0,c = 4.9 and σ = 0.53). The clusters have a hydrodynamic diameter of 180 nm and also present a large polydispersity index (PDI = 0.15). The transverse relaxivity of the clusters is remarkably high: r2 = 470 s−1mM−1 at 1.41 T and 37°C. As their relaxation properties are independent of temperature and interecho time, the clusters should be mainly in the Static Dephasing Regime (SDR), even if some large clusters are probably closer to the Partial Refocusing Model (PRM), as suggested by the large estimated ΔωtτD ∼ 55. Since they present a large magnetic moment, the Polymag™ clusters are sensitive to the magnetic field of the NMR device, in which they probably form linear chains: a reversible increase of the relaxation times is observed after the insertion in the NMR system. They could be good candidates for cellular imaging since they should be easily internalized in cells using the magnetofection protocol and then easily detected by MRI thanks to their high relaxivity.

Embryonic stem (ES) cells are recognized as an excellent cell culture model for studying developmental mechanisms and their therapeutic modulations. The aim of this work was to define whether using magnetofection was an efficient way to manipulate stem cells genetically without adversely affecting their proliferation or self-renewal capacity. We compared our magnetofection results to those of a conservative method using FuGENE 6. Using enhanced green fluorescent protein (eGFP) as a reporter gene in D3 mouse ES (mES) cells, we found that magnetofection gave a significantly higher efficiency (45%) of gene delivery in stem cells than did the FuGENE 6 method (15%), whereas both demonstrated efficient transfection in NIH-3T3 cells (60%). Although the transfected D3 (D3-eGFP) mES cells had undergone a large number of passages (>50), a high percentage of cells retained ES markers such as Oct-4 and stage-specific embryonic antigen-1 (SSEA-1). They also retained the ability to form embryoid bodies and differentiated in vitro into cells of the three germ layers. eGFP expression was sustained during stem cell proliferation and differentiation. This is the first transfection report using magnetofection in ES cells. On the basis of our results, we conclude that magnetofection is an efficient and reliable method for the introduction of foreign DNA into mouse ES cells and may become the method of choice.

Polyhexamethylene guanidine phosphate (PHMG-p), a humidifier disinfectant, is known to cause lung toxicity, including inflammation and pulmonary fibrosis. In this study, we aimed to investigate the effect of PHMG-p on human lung tissue models (2D epithelial cells and 3D organoids) under conditions of oxidative stress and viral infection. The effect of PHMG-p was studied by evaluating the formation of stress granules (SGs), which play a pivotal role in cellular adaptation to various stress conditions. Under oxidative stress and respiratory syncytial virus (RSV) infection, exposure to PHMG-p remarkably increased eIF2α phosphorylation, which is essential for SG-related signalling, and significantly increased SG formation. Furthermore, PHMG-p induced fibrotic gene expression and caused cell death due to severe DNA damage, which was further increased under oxidative stress and RSV infection, indicating that PHMG-p induces severe lung toxicity under stress conditions. Taken together, toxicity evaluation under various stressful conditions is necessary to accurately predict potential lung toxicity of chemicals affecting the respiratory tract.

Alzheimer’s disease (AD) is a complex disease with an unknown etiology. Available treatments, limited to cholinesterase inhibitors and N-methyl-d-aspartate receptor (NMDAR) antagonists, provide symptomatic relief only. As single-target therapies have not proven effective, rational specific-targeted combination into a single molecule represents a more promising approach for treating AD, and is expected to yield greater benefits in alleviating symptoms and slowing disease progression. In the present study, we designed, synthesized, and biologically evaluated 24 novel N-methylpropargylamino-quinazoline derivatives. Initially, compounds were thoroughly inspected by in silico techniques determining their oral and CNS availabilities. We tested, in vitro, the compounds’ effects on cholinesterases and monoamine oxidase A/B (MAO-A/B), as well as their impacts on NMDAR antagonism, dehydrogenase activity, and glutathione levels. In addition, we inspected selected compounds for their cytotoxicity on undifferentiated and differentiated neuroblastoma SH-SY5Y cells. We collectively highlighted II-6h as the best candidate endowed with a selective MAO-B inhibition profile, NMDAR antagonism, an acceptable cytotoxicity profile, and the potential to permeate through BBB. The structure-guided drug design strategy applied in this study imposed a novel concept for rational drug discovery and enhances our understanding on the development of novel therapeutic agents for treating AD.

NMDA receptors (NMDARs) are ionotropic glutamate receptors that play a key role in excitatory neurotransmission. The number and subtype of surface NMDARs are regulated at several levels, including their externalization, internalization, and lateral diffusion between the synaptic and extrasynaptic regions. Here, we used novel anti-GFP (green fluorescent protein) nanobodies conjugated to either the smallest commercially available quantum dot 525 (QD525) or the several nanometer larger (and thus brighter) QD605 (referred to as nanoGFP-QD525 and nanoGFP-QD605, respectively). Targeting the yellow fluorescent protein-tagged GluN1 subunit in rat hippocampal neurons, we compared these two probes to a previously established larger probe, a rabbit anti-GFP IgG together with a secondary IgG conjugated to QD605 (referred to as antiGFP-QD605). The nanoGFP-based probes allowed faster lateral diffusion of the NMDARs, with several-fold increased median values of the diffusion coefficient (D). Using thresholded tdTomato-Homer1c signals to mark synaptic regions, we found that the nanoprobe-based D values sharply increased at distances over 100 nm from the synaptic edge, while D values for antiGFP-QD605 probe remained unchanged up to a 400 nm distance. Using the nanoGFP-QD605 probe in hippocampal neurons expressing the GFP-GluN2A, GFP-GluN2B, or GFP-GluN3A subunits, we detected subunit-dependent differences in the synaptic localization of NMDARs, D value, synaptic residence time, and synaptic-extrasynaptic exchange rate. Finally, we confirmed the applicability of the nanoGFP-QD605 probe to study differences in the distribution of synaptic NMDARs by comparing to data obtained with nanoGFPs conjugated to organic fluorophores, using universal point accumulation imaging in nanoscale topography and direct stochastic optical reconstruction microscopy.SIGNIFICANCE STATEMENT Our study systematically compared the localization and mobility of surface NMDARs containing GFP-GluN2A, GFP-GluN2B, or GFP-GluN3A subunits expressed in rodent hippocampal neurons, using anti-green fluorescent protein (GFP) nanobodies conjugated to the quantum dot 605 (nanoGFP-QD605), as well as nanoGFP probes conjugated with small organic fluorophores. Our comprehensive analysis showed that the method used to delineate the synaptic region plays an important role in the study of synaptic and extrasynaptic pools of NMDARs. In addition, we showed that the nanoGFP-QD605 probe has optimal parameters for studying the mobility of NMDARs because of its high localization accuracy comparable to direct stochastic optical reconstruction microscopy and longer scan time compared with universal point accumulation imaging in nanoscale topography. The developed approaches are readily applicable to the study of any GFP-labeled membrane receptors expressed in mammalian neurons.