Search Results (3)

Choline-acetyltransferase (ChAT) is the key cholinergic enzyme responsible for the biosynthesis of acetylcholine (ACh), a crucial signaling molecule with both canonical neurotransmitter function and auto- and paracrine signaling activity in non-neuronal cells, such as lymphocytes and astroglia. Cholinergic dysfunction is linked to both neurodegenerative and inflammatory diseases. In this study, we investigated a serendipitous observation, namely that the catalytic rate of human recombinant ChAT (rhChAT) protein greatly differed in buffered solution in the presence and absence of Triton X-100 (TX100). At a single concentration of 0.05% (v/v), TX100 boosted the specific activity of rhChAT by 4-fold. Dose–response analysis within a TX100 concentration range of 0.8% to 0.008% (accounting for 13.7 mM to 0.013 mM) resulted in an S-shaped response curve, indicative of an over 10-fold boost in the catalytic rate of rhChAT. This dramatic boost was unlikely due to a mere structural stabilization since it remained even after the addition of 1.0 mg/mL gelatin to the ChAT solution as a protein stabilizer. Furthermore, we found that the catalytic function of the ACh-degrading enzyme, AChE, was unaffected by TX100, underscoring the specificity of the effect for ChAT. Examination of the dose–response curve in relation to the critical micelle concentration (CMC) of TX100 revealed that a boost in ChAT activity occurred when the TX100 concentration passed its CMC, indicating that formation of micelle–ChAT complexes was crucial. We challenged this hypothesis by repeating the experiment on Tween 20 (TW20), another non-ionic surfactant with ~3-fold lower CMC compared to TX100 (0.06 vs. 0.2 mM). The analysis confirmed that micelle formation is crucial for ultra-boosting the activity of ChAT. In silico molecular dynamic simulation supported the notion of ChAT–micelle complex formation. We hypothesize that TX100 or TW20 micelles, by mimicking cell–membrane microenvironments, facilitate ChAT in accessing its full catalytic potential by fine-tuning its structural stabilization and/or enhancing its substrate accessibility. These insights are expected to facilitate research toward the development of new cholinergic-enhancing therapeutics through the formulation of micelle-embedded ChAT nanoparticles. Full article

Polyethyleneimine (PEI) is a synthetic cationic polymer recognized as a non-viral gene carrier with high transfection efficiency. However, cytotoxicity issues limit its use. Pluronic block-copolymers conjugated with PEI have demonstrated promising results for multiparametric target gene/drug co-delivery in cancer with reduced side effects. The goal of this work was to synthesize and characterize a novel nanosystem Pluronic L121-PEI for gene/drug co-delivery. For this purpose, hydroxyl groups from Pluronic were activated using acryloyl chloride leading to the synthesis of a diacrylate intermediate, which was further conjugated with PEI. FTIR and ¹H-NMR spectroscopy were used for structural characterization. Particle size, polydispersity index (PDI) and zeta potential were assessed by dynamic and electrophoretic light scattering, respectively. A fluorescent pyrene probe was used to evaluate the critical micellar concentration (CMC). A hemolysis experiment was performed to estimate the in vitro biocompatibility of the nanosystem. FTIR analysis showed that Pluronic diacrylate was successfully synthetized as a new band around 1730 cm⁻¹ (C=O bond) appears. Its conjugation with PEI was confirmed by the presence of a band between 3380 and 3390 cm⁻¹ (N–H bond). ¹H-NMR results showed characteristic proton peaks from Pluronic (-CH3 at δ1.1 ppm) and from PEI (-CH2-CH2NH- between δ2.7–3.4 ppm) and the molar ratio Pluronic–PEI was 1:2. The nanoparticles’ hydrodynamic diameter was ca. 125 nm with a PDI below 0.250, and a charge nearby +30 mV. The CMC was around 50 μg/mL. The hemolysis ratio of a 5 mg/mL nanomicellar solution was less than 5%. A novel Pluronic L121-PEI was successfully synthesized, which was able to self-assemble in aqueous solution leading to the formation of biocompatible cationic polymeric micelles. Their small size is suitable for tumor-targeting and as they are positively charged they can be also valuable for gene delivery. Overall, this new nanosystem could be a promising multiparametric nanoapproach for gene/drug co-delivery in cancer therapy. Full article

The cationic Pt complex (Pt(NC₆H₄-C₆H₄N-(CH₂)₁₀-O(C₆H₃-3,5-(OMe)₂)(MeN-(CH₂CH₂NMe₂)₂))⁺ was prepared by the reaction of alkylbipyridinium ligand with a nitrateplatinum(II) complex. Mixing the complex and α- and β-cyclodextrins in aqueous media produced the corresponding [2]rotaxanes with 1:1 stoichiometry. γ-Cyclodextrin and the Pt complex formed a rotaxane having components in a 1:1 or 2:1 molar ratio. The results of mass and nuclear magnetic resonance (NMR) measurements confirmed the rotaxane structures of the Pt complexes. Transmission electron microscopy (TEM) and atomic force microscope (AFM) analyses revealed the formation of micelles or vesicles. The addition of NaBH₄ to the rotaxanes in aqueous media formed Pt nanoparticles with diameters of 1.3–2.8 nm, as characterized by TEM. The aggregated size of the nanoparticles formed from the rotaxane did not change even at 70 °C, and they showed higher thermal stability than those obtained from the reduction of the cyclodextrin-free Pt complex. Full article