Nanopartículas magnéticas revestidas para depleção de albumina de soro sanguíneo humano

Abstract

In the present work, a process for the separation of human serum proteins by adsorption using magnetite magnetic nanoparticles (Fe3O4) with different coatings was developed. In the first step of the process, a less complex supernatant is obtained in which human albumin (HSA) can be depleted in different amounts. Unlike available commercial kits, which discard the proteins adsorbed in this depletion step, the proposed method allows the recovery of proteins codepleted in the nanoparticles (depletome) and of the HSA itself, so that the samples obtained can be used for studies of the depletome and even albuminoma, which is the fraction of proteins that bind to HSA. The magnetite magnetic nanoparticles (Fe3O4) were synthesized by the coprecipitation method and coated with tetraethylorthosilicate (Fe3O4@TEOS) and bovine serum albumin (Fe3O4@TEOS@BSA) and it were characterized by X-ray diffraction, thermogravimetry, spectroscopy in the infrared region by Fourier transform, zeta potential, scanning electron microscopy, which confirmed surface modifications, it revealed high thermal stability and a pH-dependent surface charge. The nanoparticles obtained were used for the development of a sample preparation method for human serum albumin (HSA) depletion. The optimization studies of the adsorption of HSA on nanoparticles were carried out using the central composite rotational design (RCCD), and the results showed that the pH, the nanoparticle mass and the HSA concentration influence in the adsorption process. Theoptimal adsorption pHs were 3.5 and 4.5 for Fe3O4@TEOS@BSA and Fe3O4@TEOS respectively. For both nanoparticles, the adsorption kinetics was described by the Avrami fractional order model, with the equilibrium time reached in 10 minutes. Regarding the adsorption equilibrium data, the models that best fit were the Freundlich and Sips isotherms for Fe3O4@TEOS@BSA and Freundlich and Harkins-Jura for Fe3O4@TEOS. The maximum adsorption capacities of HSA by nanoparticles were estimated by the Langmuir isotherm at approximately 11.93 and 14.89 mg g-1

respectively. The desorption conditions of the proteins adsorbed on the surface and the reuse of the nanoparticles were also optimized with RCCDs, for both nanoparticles the desorption was influenced by the pH (desorption optimum pH 9.2 and 8.3 respectively) and volume of the desorption eluent, but the percentages of HSA recovery were low, it makes the reuse of nanoparticles still unfeasible. Electrophoresis in polyacrylamide gel containing sodium dodecyl sulfate (SDS-PAGE) showed different patterns of serum

protein bands when consecutive depletions were performed. It was evident that Fe3O4@TEOS showed greater affinity for HSA and efficiency in depletion. An important characteristic presented by the process is its versatility, in which the amount of HSA to be depleted can be controlled by varying the mass of nanoparticle used. In summary, this study showed that depletion using nanoparticles, mainly Fe3O4@TEOS, proved to be a sample preparation technique for rapid (around 1 hour), reliable and specific depletion of HSA, even though they have demonstrated co-depletion of other proteins, according to studies on SDS-PAGE. However, in the electrophoresis of the depleted proteins desorbed from the nanoparticles, most codepleted protein could be recovered, which reinforces the efficiency and viability of the nanoparticles.