Nanodiamond-chymotrypsin and nanodiamond-papain conjugates, their synthesis and activity and visualization of their interaction with cells using optical and electron microscopyстатья
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Дата последнего поиска статьи во внешних источниках: 17 сентября 2018 г.
Аннотация:Two novel conjugates of detonation nanodiamonds (dNDs) with the proteolytic enzymes
chymotrypsin and papain were synthesized. The synthesis was performed via functionalization of
the dNDs’ surface with acidic/alkali treatment followed by carbodiimide-mediated protein binding.
Covalent binding of the enzymes was confirmed by Fourier transform infrared spectrographic analysis
and high-performance liquid chromatography (HPLC) amino acid analysis. HPLC also proved
the preservation of the enzymes’ composition during synthesis. The same assay was used to determine
the binding ratios. The ratios were 12% (mass to mass) for chymotrypsin and 7.4% for papain.
The enzymatic activity of the conjugates was measured using chromogenic substrates and appeared
to be approximately 40% of that of the native enzymes. The optimum pH values and stability under
various conditions were determined. The sizes of resulting particles were measured using dynamic
light scattering and direct electron microscopic observation. The enzyme conjugates were shown to
be prone to aggregation, resulting in micrometer-sized particles. The f-potentials were measured
and found to be positive for the conjugates. The conjugated enzymes were tested for biological
activity using an in vitro model of cultured transformed human epithelial cells (HeLa cell line). It
was shown that dND-conjugated enzymes effectively bind to the surface of the cells and that
enzymes attack exposed proteins on the plasma membrane, including cell adhesion molecules.
Incubation with conjugated enzymes results in morphological changes of the cells but does not
affect cell viability, as judged by monitoring the cell division index and conducting ultrastructural
studies. dNDs are internalized by the cells via endocytosis, being enclosed in forming coated
vesicles by chance, and they accumulate in single membrane-bound vacuoles, presumably late
endosomes/phagosomes, along with multimembranous onionlike structures. The authors propose a
model of a stepwise conjugate binding to the cell membrane and gradual release of the enzymes.