The development and application of novel nanospheres based on cationic and anionic random amphiphilic polypeptides with prolonged stability were proposed. The random copolymers, e.g., poly(l-lysine-co-d-phenylalanine) (P(Lys-co-dPhe)) and poly(l-glutamic acid-co-d-phenylalanine) (P(Glu-co-dPhe)), with different amount of hydrophilic and hydrophobic monomers were synthesized. The polypeptides obtained were able to self-assemble into nanospheres. Such characteristics as size, PDI and ζ-potential of the nanospheres were determined, as well as their dependence on pH was also studied. Additionally, the investigation of their biodegradability and cytotoxicity was performed. The prolonged stability of nanospheres was achieved via introduction of d-amino acids into the polypeptide structure. The cytotoxicity of nanospheres obtained was tested using HEK-293 cells. It was proved that no cytotoxicity up to the concentration of 500 µg/mL was observed. C-peptide delivery systems were realized in two ways: (1) peptide immobilization on the surface of P(Glu-co-dPhe) nanospheres; and (2) peptide encapsulation into P(Lys-co-dPhe) systems. The immobilization capacity and the dependence of C-peptide encapsulation efficiency, as well as maximal loading capacity, on initial drug concentration was studied. The kinetic of drug release was studied at model physiological conditions. Novel formulations of a long-acting C-peptide exhibited their effect ex vivo by increasing activity of erythrocyte Na+/K+-adenosine triphosphatase.
The early diagnostics of hepatitis C virus (HCV) infections is currently one of the most highly demanded medical tasks. This study is devoted to the development of biochips (microarrays) that can be applied for the detection of HCV. The analytical platforms of suggested devices were based on macroporous poly(glycidyl methacrylate-co-di(ethylene glycol) dimethacrylate) monolithic material. The biochips were obtained by the covalent immobilization of specific probes spotted onto the surface of macroporous monolithic platforms. Using the developed biochips, different variants of bioassay were investigated. This study was carried out using hepatitis C virus-mimetic particles (VMPs) representing polymer nanoparticles with a size close to HCV and bearing surface virus antigen (E2 protein). At the first step, the main parameters of bioassay were optimized. Additionally, the dissociation constants were calculated for the pairs “ligand–receptor” and “antigen–antibody” formed at the surface of biochips. As a result of this study, the analysis of VMPs in model buffer solution and human blood plasma was carried out in a format of direct and “sandwich” approaches. It was found that bioassay efficacy appeared to be similar for both the model medium and real biological fluid. Finally, limit of detection (LOD), limit of quantification (LOQ), spot-to-spot and biochip-to-biochip reproducibility for the developed systems were evaluated.
In present work, the development of macroporous monolithic layers bearing the artificial recognition sites toward L‐phenylalanine has been carried out. The set of macroporous poly(2‐aminoethyl methacrylate‐co‐2‐hydroxyethyl methacrylate‐co‐ethylene glycol dimethacrylate) materials with average pore size ranged in 340–1200 nm was synthesized. The applicability of Hildebrand's and Hansen's theories for the prediction of polymer compatibility with porogenic solvents was evaluated. The dependences of average pore size on theoretically calculated parameters were plotted. The linear trend detected for Hansen's theory has indicated the high suitability of this approach to select appropriate porogens. The synthesized monolithic MIP layers were tested toward the ability to rebind phenylalanine‐derivative in microarray format. The influence of such factors as average pore size of the material, the concentration of template molecule in polymerization mixture, interaction time of analyte with its imprinted sites on binding efficiency were studied. The developed materials demonstrated good analyte rebinding from buffer solution with recognition factors 2.5–3.4 depending on the MIP sample. The comparable rebinding efficiency was also detected when the analysis was carried using complex biological media. The selectivity of phenylalanine binding from the equimolar mixture of structural analogues was 81.9% for free amino acid and 91.2% for labeled one.
Nowadays, macroporous polymer monoliths represent widely used stationary phases for a number of dynamic interphase mass exchange processes such as high-performance liquid chromatography, gas chromatography, electrochromatography, solid-phase extraction, and flow-through solid-state biocatalysis. This review represents the first summary in the field of current achievements on the preparation of macroporous polymer monolithic layers, as well as their application as solid phases for thin-layer chromatography and different kinds of microarray.
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