1. Engineering of functional nanosnowflakes from gold nanocarriers capped with amino-modified DNA oligonucleotidesAlexandre Loukanov, Velichka Arahangelova, Saim Emin, Chavdar Filipov, 2023, izvirni znanstveni članek Opis: Abstract
The design, engineering and electron microscopic characterization of anisotropic nanosized snowflake‐like structural assemblies (nanosnowflakes) is reported. They were fabricated through immobilization of double stranded amine‐modified and thiol‐terminated DNA oligonucleotides on the surface of ultra‐small isotropic gold nanoparticles used as nanocarriers. The transmission electron microscopy images combined with spectrophotometric data revealed the formation of self‐assembled structural aggregation between individual ligands‐coated nanoparticles. They act as seeds for the further spontaneous dendritic growth in different directions. Their anisotropic morphology is formed due to the occurrence of facilitated electrostatic interactions between positive charged amino‐groups and the negative sugar‐phosphate backbone of oligonucleotides. Thus, nanosnowflakes with size distribution between 40 and 80 nm were obtained. The microscopic analysis demonstrated also that the stable nanosnowflakes structure was highly dependent on the solution ionic strength, which effect the charge fluctuation within the assembly. The reported DNA functionalized nanostructures have potential to be applied as a platform for development of therapeutic materials, as well as drug delivery nanosystems.
Research Highlights
The engineering, fabrication, and microscopic characterization of DNA nanosnowflakes is reported.
The electron microscopy analysis revealed formation of self‐assemblies with anisotropic morphology.
The nanosnowflakes size distribution was between 40 and 80 nm.
Ključne besede: DNA, nanocarrier, functionalization, drug delivery
Objavljeno v RUNG: 18.03.2024; Ogledov: 291; Prenosov: 0
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3. Pharmacological application of thermal Lens technique - A thermal diffusivity studyMohanachandran Nair Sindhu Swapna, 2018, izvirni znanstveni članek Opis: The photothermal phenomenon has emerged as a potential tool for the nondestructive evaluation of thermal and optical properties of materials. Thermal analysis of drugs is an unavoidable part of preformulation study, as temperature variations can induce structural changes of the constituents of drugs. Techniques based on photothermal phenomena are highly sensitive, as only the absorbed radiation contributes to the signal. Periodic illumination and subsequent nonradiative de-excitation generate thermal lens signals of various types within and around the sample. Variation of thermal diffusivity with a concentration of the commonly used drug terbutaline is studied through the single-beam thermal lens technique. The ultraviolet–visible spectrum of the drug shows strong absorption around 500 nm, which suggests the possible wavelengths that can be used for the study. It is found that concentration of the drug in liquid form decides its thermal stability, as its thermal diffusivity varies with concentration. The study gives information about the optimum value for the concentration of the drug noted above for which the chance of thermal stability is high.
Ključne besede: thermal lens, thermal diffusivity, pharmacology, drug
Objavljeno v RUNG: 30.06.2022; Ogledov: 1075; Prenosov: 0
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10. RAPID UPLC-ESI-MS/MS BASED ASSAY FOR DISCOVERY OF UDP-N-ACETYLMURAMOYL-L-ALANINE:D-GLUTAMATE (MurD) LIGASE INHIBITORSVjekoslava Car, 2016, magistrsko delo Opis: A rapid, selective, robust and sensitive analytical assay method, operating in a short time frame with acceptable levels of precision, linear range and the accuracy necessary for successful Mur ligases inhibitors discovery, was developed.
An LC-MS/MS analytical procedure was designed for the determination of a MurD ligase reaction product (UMAG). The special focus of this work was on UDP-N-acetylmuramyl-L-alanine:D-glutamate ligase (MurD) activity. The assay method is especially valuable as an orthogonal (secondary) assay for the primary high throughput fluorescent-based assay screening of potential Mur ligase inhibitors. The LC-MS/MS assay is fully compatible with the components from the primary fluorescent-based assay and enables the analysis of the same samples by both methodologies. The presented LC-MS/MS assay procedure is used for the evaluation of the false positive hits (molecules) from the primary, fluorescence based, high throughput screening assay experiments. This is important for the elimination of false positive hits from the prohibitively expensive and time-consuming investigation process.
Method development describes the evaluation and optimization of the various stages of sample preparation, chromatographic separation, MS/MS determination and quantification. An enzyme reaction is performed in a 96-well plate. The quenched reaction mixture samples were spiked with an internal standard (phenacetin). The permeate was injected onto the U(H)PLC-MS/MS triple quadrupole system after sample ultrafiltration. Chromatographic separation was achieved on the ACQUITY UPLCTM HSS T3 column (100 x 2.1 mm i.d., 1.8 µm particle size) using an ammonium format buffer at pH 2.8 and acetonitrile as eluent. Elution initiated with an isocratic-hold for 1.1 min, followed by a two-step linear gradient of up to 3 min, giving a total run time of 5 min including equilibration. The flow rate was kept at a constant 0.4 mL/min.
UMAG quantitative analysis was performed by positive electrospray ionization, followed by tandem mass spectrometry (ESI-MS/MS). The analytical assay quantifies UMAG in a linear range from 0.25 to 20 µM using 70 µL of samples. Validation results demonstrated that UMAG concentrations can be accurately and precisely determined in samples from the primary assay.
Evaluation of inhibitory activities of compounds measured by both the fluorescence and the LC-MS/MS method demonstrated that the values were in a very good agreement. This analytical method can be used to screen a compound library at a defined concentration of each compound to obtain the percentage of inhibition, or with a series of compound concentrations to obtain inhibition potency of a compound (IC50). The selected Lek compounds no. 1 and 2 from the virtual screening campaign were presented, tested and further investigated due to the expression of significant MurD ligase inhibitory action acquired by primary high throughput tests.
This assay has been developed for MurD, but during development, chromatographic and MS/MS conditions for UM and UMA were studied and defined as well. Therefore, this analytical assay method can easily be applied to other Mur ligases (i.e. MurC, MurE) enzyme activity monitoring in the process of bacteria cell wall peptidoglycan formation. This method enables the identification of many different Mur ligase inhibitors in a continued search for new Gram positive and Gram negative bacteria antibiotics.
Ključne besede: Mur ligases, UDP-N-acetylmuramyl-L-alanine:D-glutamate (MurD) inhibitors, UNAM-Ala-Glu, LC-MS/MS, liquid chromatography, tandem mass spectrometry, antibiotics, drug discovery
Objavljeno v RUNG: 23.09.2016; Ogledov: 6894; Prenosov: 273
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