RESUMEN
The use of targeted drug delivery systems, including those based on selective absorption by certain receptors on the surface of the target cell, can lead to a decrease in the minimum effective dose and the accompanying toxicity of the drug, as well as an increase in therapeutic efficacy. A fullerene C60 conjugate (FA-PVP-C60) with polyvinylpyrrolidone (PVP) as a biocompatible spacer and folic acid (FA) as a targeting ligand for tumor cells with increased expression of folate receptors (FR) was obtained. Using 13C NMR spectroscopy, FT-IR, UV-Vis spectrometry, fluorometry and thermal analysis, the formation of the conjugate was confirmed and the nature of the binding of its components was established. The average particle sizes of the conjugate in aqueous solutions and cell culture medium were determined using dynamic light scattering (DLS) and nanoparticle tracking analysis (NTA). The FA-PVP-C60 showed antiradical activity against â¢DPPH, â¢OH and O2â¢-, but at the same time, it was shown to generate 1O2. It was found that the conjugate in the studied concentration range (up to 200 µg/mL) is non-toxic in vitro and does not affect the cell cycle. To confirm the ability of the conjugate to selectively accumulate through folate-mediated endocytosis, its uptake into cells was analyzed by flow cytometry and confocal microscopy. It was shown that the conjugate is less absorbed by A549 cells with low FR expression than by HeLa, which has a high level of expression of this receptor.
Asunto(s)
Sistemas de Liberación de Medicamentos , Ácido Fólico , Fulerenos , Povidona , Ácido Fólico/química , Ácido Fólico/farmacología , Humanos , Povidona/química , Fulerenos/química , Fulerenos/farmacología , Sistemas de Liberación de Medicamentos/métodos , Línea Celular Tumoral , Células A549 , Células HeLa , Tamaño de la PartículaRESUMEN
BACKGROUND: To date, EVs characterization techniques are extremely diverse. The contribution of AFM, in particular, is often confined to size distribution. While AFM provides a unique possibility to carry out measurements in situ, nanomechanical characterization of EVs is still missing. METHODS: Blood plasma EVs were isolated by ultracentrifugation, analyzed by flow cytometry and NTA. Followed by cryo-EM, we applied PeakForce AFM to assess morphological and nanomechanical properties of EVs in liquid. RESULTS: Nanoparticles were subdivided by their size estimated for their suspended state into sub-sets of small S1-EVs (< 30 nm), S2-EVs (30-50 nm), and sub-set of large ones L-EVs (50-170 nm). Non-membranous S1-EVs were distinguished by higher Young's modulus (10.33(7.36;15.25) MPa) and were less deformed by AFM tip (3.6(2.8;4.4) nm) compared to membrane exosomes S2-EVs (6.25(4.52;8.24) MPa and 4.8(4.3;5.9) nm). L-EVs were identified as large membrane exosomes, heterogeneous by their nanomechanical properties (22.43(8.26;53.11) MPa and 3.57(2.07;7.89) nm). Nanomechanical mapping revealed a few non-deformed L-EVs, of which Young's modulus rose up to 300 MPa. Taken together with cryo-EM, these results lead us to the suggestion that two or more vesicles could be contained inside a large one being a multilayer vesicle. CONCLUSIONS: We identified particles similar in morphology and showed differences in nanomechanical properties that could be attributed to the features of their inner structure. GENERAL SIGNIFICANCE: Our results further elucidate the identification of EVs and concomitant nanoparticles based on their nanomechanical properties.
Asunto(s)
Exosomas , Nanopartículas , Módulo de Elasticidad , Microscopía de Fuerza Atómica , PlasmaRESUMEN
The crystal and supramolecular structure of the bacterial cellulose (BC) has been studied at different stages of cellobiohydrolase hydrolysis using various physical and microscopic methods. Enzymatic hydrolysis significantly affected the crystal and supramolecular structure of native BC, in which the 3D polymer network consisted of nanoribbons with a thickness T ≈ 8 nm and a width W ≈ 50 nm, and with a developed specific surface SBET ≈ 260 m2·g-1. Biodegradation for 24 h led to a ten percent decrease in the mean crystal size Dhkl of BC, to two-fold increase in the sizes of nanoribbons, and in the specific surface area SBET up to ≈ 100 m2·g-1. Atomic force and scanning electron microscopy images showed BC microstructure "loosening"after enzymatic treatment, as well as the formation and accumulation of submicron particles in the cells of the 3D polymer network. Experiments in vitro and in vivo did not reveal cytotoxic effect by the enzyme addition to BC dressings and showed a generally positive influence on the treatment of extensive III-degree burns, significantly accelerating wound healing in rats. Thus, in our opinion, the results obtained can serve as a basis for further development of effective biodegradable dressings for wound healing.