Cellular processing of gold nanoparticles: CE-ICP-MS evidence for the speciation changes in human cytosol.

The cellular uptake of gold nanoparticles (AuNPs) may (or may not) affect their speciation, but information on the chemical forms in which the particles exist in the cell remains obscure. An analytical method based on the use of capillary electrophoresis hyphenated with inductively coupled plasma mass spectrometry (ICP-MS) has been proposed to shed light on the intracellular processing of AuNPs. It was observed that when being introduced into normal cytosol, the conjugates of 10-50 nm AuNPs with albumin evolved in human serum stayed intact. On the contrary, under simulated cancer cytosol conditions, the nanoconjugates underwent decomposition, the rate of which and the resulting metal speciation patterns were strongly influenced by particle size. The new peaks that appeared in ICP-MS electropherograms could be ascribed to nanosized species, as upon ultracentrifugation, they quantitatively precipitated whereas the supernatant showed only trace Au signals. Our present study is the first step to unravel a mystery of the cellular chemistry for metal-based nanomedicines.

CE Separation and ICP-MS Detection of Gold Nanoparticles and Their Protein Conjugates.

A full understanding and mediation of nanoparticle-serum protein interactions is key to design nanoparticles with vivid functions within the body, and to solve this problem one needs to differentiate and characterize individual nano-protein conjugates. In this paper, the authors applied capillary electrophoresis combined with inductively coupled plasma mass spectrometry detection to study the behavior of gold nanoparticles of different geometry, size and surface functionalization upon interacting with serum proteins and their mixtures. Due to high-resolution and -sensitivity benefits of this combined technique baseline separations were attained for free nanoparticles (at real-life doses) and different protein conjugates, and the conversion into the protein-bound form was scrutinized in terms of reaction time.

The fate of differently functionalized gold nanorods in human serum: A response from capillary electrophoresis-inductively coupled plasma mass spectrometry.

A highly selective and sensitive method was developed to characterize intrinsic intramolecular interactions between potential theranostic agents, gold nanorods (AuNRs), and plasma proteins. The method is based on a hyphenation of capillary electrophoresis (CE) with inductively coupled plasma mass spectrometry (ICP-MS), which enables monitoring the speciation changes of AuNRs under physiologically compatible conditions. To improve the separation resolution between the intact nanorods and different gold-protein conjugates, the CE system was optimized by varying the type and concentration of background electrolyte, applied voltage, and sample loading. Optimization allowed also for acquiring the acceptable figures of merit such as migration time and peak area precision of 4.7-8.2% and 5.1-6.3%, respectively, detection limits in the range of 5.5-5.7µgL-1 Au, and recoveries on the order of 91-99%. With the developed method the metal-specific profiles were recorded for differently functionalized AuNPs in combinations with individual serum proteins and in human serum. In case of carboxy-modified AuNPs, proteinization in real-serum environment occurs without albumin participation, apo-transferrin dominating the protein corona under equilibrium conditions. On the contrary, the AuNRs with surface amino-groups first form the albumin conjugate but albumin in this "soft" corona becomes slowly replaced by other, less abundant proteins, exhibiting a higher affinity toward the aminated surface.

Characterization of the protein corona of gold nanoparticles by an advanced treatment of CE-ICP-MS data.

CE is well known not only as an efficient separation method, but also as a viable tool for studying chemical reactions, including kinetic assaying and analysis of chemical equilibria. In this communication, the latter feature of CE interfaced with ICP-MS was exploited to determine the stoichiometric composition of the protein corona of gold nanoparticles (AuNPs) at equilibrium conditions. For both individual albumin and human serum involved in binding, the number of protein molecules bound per AuNP (n) was calculated. Since the time scale of the corona formation was previously found to be dependent on the particle size, two calculation algorithms were adopted here. In the case of 5-nm AuNPs, rather slowly associating with the protein, the peak areas measured for the conjugated and free particles were taken in computation (the (34) S signal due to bound protein was also monitored simultaneously to confirm that equilibrium is reached). In binding labile systems (10-50 nm AuNPs), the particles are converted into the protein-bound form relatively fast due mostly to the favor of a much greater excess of the protein so that no peak of the free particles interacting with serum being recorded. Therefore, the n value was estimated by relating the sulfur peak area of each of these conjugates to that of 5-nm AuNPs to calculate the number of bound albumin molecules that was then divided by the number of AuNPs. The AuNPs were found to react with from 13 to 292 albumin molecules that is in good agreement with the literature data.

A sensitive and versatile method for characterization of protein-mediated transformations of quantum dots.

We report the development and application of an analytical system consisting of capillary electrophoresis (CE) interfaced with inductively coupled plasma mass spectrometry (ICP-MS) for sensitive and high-resolution characterization of quantum dots (QDs) interacting with serum proteins. Separation resolution between the intact CdSeS/ZnS QDs and their protein conjugates was optimized by varying the type and concentration of background electrolyte, applied voltage, and sample loading. Special attention was paid to the CE system compatibility with physiological conditions, avoiding aggregation effects, and analyte recovery. Optimization trials allowed for acquiring satisfactory stability of migration times (within 6.0% between different days), peak area precision of 5.2-8.0%, capillary recoveries in the range of 90-96%, and a lower limit of detection of 7.5 × 10(-9) mol L(-1) Cd. With the developed method distinct metal-specific profiles were obtained for the QDs in combination with individual serum proteins, their mixtures, and in human serum. Particularly, it was found that albumin binding to the particle surface is completed after 1 h, without noticeable disruption of the core-shell integrity. The transferrin adsorption is accompanied by the removal of the ZnS shell, resulting in evolving two different metal-protein conjugated forms. On the other hand, proteinization in real-serum environment occurs without binding to major transport proteins, the QDs also lose their the shell (the higher the dose the longer is the time they stay unbroken). The concomitant changes in migration behavior can be attributed to interactions with serum proteins other than albumin and transferrin. Speciation information provided by CE-ICP-MS may shed light on the mechanism of QD delivery to the target regions of the body.

A shotgun metalloproteomic approach enables identification of proteins involved in the speciation of a ruthenium anticancer drug in the cytosol of cancer cells.

The study reported herein focused on the development and optimization of a versatile analytical methodology for characterization of intracellular distribution of protein-bound species of ruthenium originating from an anticancer Ru-based drug, indazolium trans-[tetrachloridobis(1H-indazole)ruthenate(III)]. A direct analysis of the drug-treated cytosol of cancer cells using size-exclusion chromatography (SEC) interfaced with inductively coupled plasma mass spectrometry (ICP-MS) revealed that over 85% of ruthenium is converted into a high molecular-mass fraction. To further determine the ruthenium binding pattern, a shotgun approach was used, with the entire proteome being digested and the resulting peptides being analyzed by capillary high-performance liquid chromatography (µHPLC) combined with electrospray ionization triple quadrupole MS. This allowed for identification of the ruthenated proteins on the basis of characteristic MS/MS spectra of the respective peptides. It was found that both Ru(III)- and Ru(II)-ligated functionalities participate in adduct formation, the hydrolyzed forms of the drug being attached to the majority of the binding proteins. Of an array of proteins responding to drug treatment, the most important - from the viewpoint of unveiling the exact mode of action - are inhibitor, pro-apoptotic, and DNA reparation proteins.

Use of high-performance liquid chromatography-tandem electrospray ionization mass spectrometry to assess the speciation of a ruthenium(III) anticancer drug in the cytosol of cancer cells.

The discovery of intracellular active forms is a crucial issue for the approval of further anticancer metal-based drugs. This challenge calls for an apt analytical methodology to scrutinize the speciation changes of a metallodrug in cancer cytosol. In the current study, we have developed an approach for portraying low-molecular-mass cytosolic species of a Ru(III) drug, indazolium trans-[tetrachloridobis(1H-indazole)ruthenate(III)], based on using capillary high-performance liquid chromatography combined with tandem electrospray ionization mass spectrometry. The approach, which featured the use of the transferrin adduct as an eventual drug entity entering the cell, facilitated identification of components of the cytosol of cancer cells and their ruthenated forms in which the metal proved to be in +3 or +2 charge states. The Ru species released from the protein-bound form were also characterized with respect to the ligand environment.