Abstract
In the biomedical applications of nanoparticles (NPs), the proper choice of surface chemistry is a crucial aspect in their design. The nature of the coating can heavily impact the interaction of NPs with biomolecules, affect the state of aggregation, and ultimately determine their biological fate. As such, protein corona formation and the aggregation behaviour of gold NPs (Au NPs) are studied here. Au NPs are prepared with four distinct surface functionalisations, namely mercaptosuccinic acid (MSA), N-4-thiobutyroil glucosamine, HS-PEG5000 and HS-alkyl-PEG600. Corona formation, aggregation, and the intracellular behaviour of the Au NPs are then investigated by means of Fluorescence Correlation Spectroscopy (FCS) in cell culture media and in live cells. To evaluate the state of aggregation and the formation of a protein corona, the Au NPs are incubated in cell media and the diffusion coefficient is determined via FCS. The in vitro behaviour is compared with the level of aggregation of the NPs in cells. Diffusion times of the NPs are estimated at different positions in the cell after a one hour incubation period. It is found that the majority of MSA and glucose-Au NPs are present inside the cell as slowly diffusing species with diffusion times (τD) greater than 6000 μs (hydrodynamic diameter >250 nm). PEGylated Au NPs adsorb a small amount of protein and manifest low agglomeration both in media and in living cells. In particular, the HS-alkyl-PEG600 coating shows an excellent correlation between lower protein adsorption, 4-fold lower compared to the MSA coated NPs, and limited intracellular aggregation. In the case of single HS-alkyl-PEG600 coated NPs, it is found that typical intracellular τD values range from 500 to 1500 μs, indicating that these particles display reduced aggregation in the intracellular environment.
Highlights
The reaction was performed under dry conditions and Argon atmosphere
HAuCl4·3H2O, AgNO3, hydroquinone, 4,4′-dithiodibutyric acid, glucosamine hydrochloride, butylamine, 1Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), N,N'-Dicyclohexylcarbodiimide (DCC), NHydroxysuccinimide (NHS), 1,4-Dithiothreitol (DTT) and NaOH were purchased from Sigma-Aldrich, and used without further purification
HAuCl4·3H2O was stored at 4 °C, shielded from light, as 10 mM solution and NaOH was stored as 1 M water solution
Summary
HAuCl4·3H2O, AgNO3, hydroquinone, 4,4′-dithiodibutyric acid, glucosamine hydrochloride, butylamine, 1Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), N,N'-Dicyclohexylcarbodiimide (DCC), NHydroxysuccinimide (NHS), 1,4-Dithiothreitol (DTT) and NaOH were purchased from Sigma-Aldrich, and used without further purification. Dynamic Light Scattering (DLS) measurements were performed employing a Malvern Zetasizer Nano ZS90. At least three independent measurements of 10 runs (10 s each one) were performed for each sample. A reduced volume plastic cuvette was employed for DLS experiments loaded with 450 μl of sample. A disposable cuvette with 1 cm optical path length was used for the measurements. 4 ml of a DCC (756 mg, 3.67 mmol, 2.2, eq) solution in dioxane were slowly dropped in the reaction mixture and left, under vigorous stirring, at room temperature for 8 hours. The solvent was evaporated at reduced pressure and the obtained product dissolved in Et2O-Acetone (1:1) solution. The mixture was filtrated to remove the white insoluble solid and the 4,4′-Dithiodibutyroil NHS ester was recovered evaporating the solvent at reduced pressure.
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