Fe3O4SPIONs in cancer theranostics-structure versus interactions with proteins and methods of their investigation.

As the second leading cause of death worldwide, neoplastic diseases are one of the biggest challenges for public health care. Contemporary medicine seeks potential tools for fighting cancer within nanomedicine, as various nanomaterials can be used for both diagnostics and therapies. Among those of particular interest are superparamagnetic iron oxide nanoparticles (SPIONs), due to their unique magnetic properties,. However, while the number of new SPIONs, suitably modified and functionalized, designed for medical purposes, has been gradually increasing, it has not yet been translated into the number of approved clinical solutions. The presented review covers various issues related to SPIONs of potential theranostic applications. It refers to structural considerations (the nanoparticle core, most often used modifications and functionalizations) and the ways of characterizing newly designed nanoparticles. The discussion about the phenomenon of protein corona formation leads to the conclusion that the scarcity of proper tools to investigate the interactions between SPIONs and human serum proteins is the reason for difficulties in introducing them into clinical applications. The review emphasizes the importance of understanding the mechanism behind the protein corona formation, as it has a crucial impact on the effectiveness of designed SPIONs in the physiological environment.

Enhanced edible plant production using nano-manganese and nano-iron fertilizers: Current status, detection methods and risk assessment.

BACKGROUND: Nanotechnology offers many benefits in the globally important field of food production and human nutrition, particularly by implementing agricultural nanoproducts. Of these, edible plant fertilizers enriched with nanosized forms of essential metals, Mn and Fe, are growing in importance with the advantages of enhanced action on plant roots. SCOPE AND APPROACH: This review focuses on the importance of tracking the bioaccumulation and biodistribution of these pertinent nanofertilizers. An emphasis is given to the critical analysis of the state-of-the-art analytical strategies to examine the Mn and Fe nanoparticles in edible plant systems as well as to shedding light on the vast gap in the methodologies dedicated to the speciation, in vitro simulation, and safety testing of these promising nanomaterials. Also provided are guidances for the food chemists and technologists on the lights and shadows of particular analytical approaches as a matter of authors' expertise as analytical chemists. KEY FINDINGS AND CONCLUSIONS: While the use of nanotechnology in agriculture seems to be growing increasingly, there is still a lack of analytical methodologies capable of investigating novel Mn- and Fe-based nanomaterials as potential fertilizers. Only the advent of reliable analytical tools in the field could bridge the gaps in our knowledge about processes in which those materials participate in the plant systems and their effects on crop production and quality of the produced food.

Protein-Mediated Transformations of Superparamagnetic Nanoparticles Evidenced by Single-Particle Inductively Coupled Plasma Tandem Mass Spectrometry: A Disaggregation Phenomenon.

Progress toward translating superparamagnetic iron oxide nanoparticles (SPIONs) with specific diagnostic and therapeutic properties for clinical applications depends on developing and implementing appropriate methodologies that would allow in-depth characterizations of their behavior in a real biological environment. Herein, we report a versatile approach for studying interactions between SPIONs and proteins using single-particle inductively coupled plasma tandem mass spectrometry. By monitoring the changes in the size distribution upon exposure to human serum, the formation of stable protein corona is revealed, accompanied by particle disaggregation.

Current and emerging mass spectrometry methods for the preclinical development of metal-based drugs: a critical appraisal.

This Trends article highlights the multiple ways in which the state-of-the-art molecular mass spectrometry can support the preclinical development of novel metal-based anticancer drugs. Examples from the recent literature-beyond routine characterization applications-are presented to illustrate what analytical and experimental design challenges are to be addressed to facilitate the translation of promising drug candidates to clinical practice.

Metal-Specific Response of High-Resolution ICP-MS for Proteins Binding to Gold Nanoparticles in Human Serum.

Metalloproteins have many different functions such as storage and transport of proteins, enzymes, signal transduction proteins, etc. Herein, for a selection of gold nanoparticles differing in shape, size, charge, and surface modification, the binding behavior in human serum was assessed with respect to metal-containing proteins. Our results based on sector-field ICP-MS measurements and a simple calculation algorithm indicate the possible involvement of proteins, incorporating Cu and Fe, in the formation of the biomolecular layer around the particle surface. Given that such binding encompasses a substantial amount of copper and iron within the serum proteome (>50%) at a calculated nanoparticle dose, it may result in depleting their biological functions and should be taken into account when selecting lead candidates with an improved biocompatibility.

Toward a deeper and simpler understanding of serum protein-mediated transformations of magnetic nanoparticles by ICP-MS.

A great variety of magnetic nanomaterials are entering preclinical investigations with the objective to select the most promising candidates for diagnostic and therapeutic applications. For an analytical approach to be used as a high-throughput screening tool, simple and cost-efficient sample preparation protocol is a basiс prerequisite. Here, we demonstrate how the application of continuous magnetic field allows for quantitatively separating iron oxide magnetic nanoparticles from a mixture with human serum to facilitate monitoring of their biomolecular interactions with high-resolution inductively coupled plasma mass spectrometry. By measuring the signals of sulfur and metal isotopes, it is possible to monitor the formation of the protein corona and alterations in the concentrations of relevant metals due to binding of specific metalloproteins, respectively.

To-Do and Not-To-Do in Model Studies of the Uptake, Fate and Metabolism of Metal-Containing Nanoparticles in Plants.

Due to the increasing release of metal-containing nanoparticles into the environment, the investigation of their interactions with plants has become a hot topic for many research fields. However, the obtention of reliable data requires a careful design of experimental model studies. The behavior of nanoparticles has to be comprehensively investigated; their stability in growth media, bioaccumulation and characterization of their physicochemical forms taken-up by plants, identification of the species created following their dissolution/oxidation, and finally, their localization within plant tissues. On the basis of their strong expertise, the authors present guidelines for studies of interactions between metal-containing nanoparticles and plants.

High-resolution ICP-MS approach for characterization of magnetic nanoparticles for biomedical applications.

The potential of iron oxide-based nanoparticles (IONs) as theranostic agents is believed to be in a great part due to non-invasive diagnosis and therapeutic applications. However, there is still a lack of well-recognized methodology to assess bioresistance, hypotoxicity, reactivity toward pertinent biomolecules, as well as an eventual dose of IONs as prerequisites for their clinical use. In this study, we demonstrate how application of high-resolution ICP-MS in combination with conventional ultrafiltration can address these important issues in a simple and high-throughput way. Based upon interference-free and sensitive measurements of iron and sulfur isotopes ensured by sector-field ICP-MS mode, the comparative testing of a series of novel IONs modified by PEG or PEG and an ionic liquid, was performed. Satisfactory stability (less than 1 % of soluble Fe), minor toxicity (by virtue of releasing a free iron) and transit into bioconjugates in human serum, different in speed, proved the prospective of the tested IONs for in-depth preclinical development.

Characterization of quantum dots in cancer cytosol using ICP-MS-based combined techniques.

Knowledge of the intracellular behavior of quantum dots (QDs), which encompasses the antiproliferative effect on living cells, is still limited. For this reason, the transformations of CdSeS/ZnS-based QDs in cancer cytosol were examined using capillary electrophoresis (CE) and high-performance liquid chromatography (HPLC) hyphenated with inductively coupled plasma MS (ICP-MS). CE-ICP-MS method revealed the dose- and time-dependent speciation changes of QDs in the cytosol, while HPLC-ICP-MS (in the size-exclusion chromatography mode) allowed further characterization of the resulting Cd species. In such an appraisal, the decent CE advantage of high resolution is well complemented by higher sensitivity of HPLC (LOD 4.0 × 10-10 and 5.4 × 10-12 mol/L Cd, respectively). Additionally, the influence of serum protein corona on the surface of QDs on their uptake by Hep G2 cancer cells was investigated by direct ICP-MS analysis that revealed that the conjugated proteins greatly reduce the particle internalization.

An improved protocol for ICP-MS-based assessment of the cellular uptake of metal-based nanoparticles.

Along with a growing interest in biomedical applications of metal-based nanoparticles, there is a compelling need in systematic information on their behavior in human body systems, preferably at the cellular level. However, in most of the in-vitro uptake experiments, the nanomaterial was applied in its native form that in reality can hardly reach the cell. In this work, we developed an improved procedure in which prior to addition to the cells the particles are converted into the protein conjugates by incubation in human serum. The procedure was tested for gold nanoparticles of different size, chosen as a representative nanomaterial on multifunctional medicinal use, and MCF-7 cell line. Using ICP-MS to measure intracellular metal concentration, it was shown that an original state has significant effect on particle internalization. The protein corona significantly inhibits the uptake amount by MCF-7 cells, with the greatest influence (a 15-fold decrease compared to uncoated particles) being exerted over the smallest, 5-nm particles (3 pg Au/cell). Conjugates of larger particles (20 and 50 nm) are taken up more effectively (45 and 34 pg Au/cell, respectively). The advanced protocol makes the uptake results more reliable and its implementation may accelerate the preclinical development of metal-based nanoparticles as a viable theranostic implement.

Uptake, translocation, size characterization and localization of cerium oxide nanoparticles in radish (Raphanus sativus L.).

Due to their unique physical and chemical properties, the production and use of cerium oxide nanoparticles (CeO2 NPs) in different areas, especially in automotive industry, is rapidly increasing, causing their presence in the environment. Released CeO2 NPs can undergo different transformations and interact with the soil and hence with plants, providing a potential pathway for human exposure and leading to serious concerns about their impact on the ecosystem and human organism. This study investigates the uptake, bioaccumulation, possible translocation and localization of CeO2 NPs in a model plant (Raphanus sativus L.), whose edible part is in direct contact with the soil where contamination is more likely to happen. The stability of CeO2 NPs in plant growth medium as well as after applying a standard enzymatic digestion procedure was tested by single particle ICP-MS (SP-ICP-MS) showing that CeO2 NPs can remain intact after enzymatic digestion; however, an agglomeration process was observed in the growth medium already after one day of cultivation. An enzymatic digestion method was next used in order to extract intact nanoparticles from the tissues of plants cultivated from the stage of seeds, followed by size characterization by SP-ICP-MS. The results obtained by SP-ICP-MS showed a narrower size distribution in the case of roots suggesting preferential uptake of smaller nanoparticles which led to the conclusion that plants do not take up the CeO2 NPs agglomerates present in the medium. However, nanoparticles at higher diameters were observed after analysis of leaves plus stems. Additionally, a small degree of dissolution was observed in the case of roots. Finally, after CeO2 NPs treatment of adult plants, the spatial distribution of intact CeO2 NPs in the radish roots was studied by laser ablation ICP-MS (LA-ICP-MS) and the ability of NPs to enter and be accumulated in root tissues was confirmed.

Analytical methodology for studying cellular uptake, processing and localization of gold nanoparticles.

Interactions of gold nanoparticles (AuNPs) with live cells are known to exert a great impact on their functions, including cell signalling, genomic, proteomic, and metabolomic processes. Modern analytical techniques applied to studying nanoparticle-cell interactions are to improve our understanding of the mode of action of AuNPs, which is essential for their approval in disease therapeutics. Such methods may vary depending on what step of particle internalization is in question, i.e., cellular uptake, intracellular transport (accompanying by changes in the chemical state), translocation to different cell compartments, interaction with relevant subcellular structures and localization. This review focuses on the implementation and critical assessment of advanced analytical methodologies to investigate the cellular processing of AuNPs. Also addressed is a sought-after issue of accounting in in-vitro studies for a chemical form in which the AuNPs enter the cell in vivo.

Capillary-HPLC with tandem mass spectrometry in analysis of alkaloid dyestuffs - a new approach.

Development of the identification method of alkaloid compounds in Amur cork tree as well as not examined so far Oregon grape and European Barberry shrubs are presented. The novel approach to separation of alkaloids was applied and the capillary-high-performance liquid chromatography (capillary-HPLC) system was used, which has never previously been reported for alkaloid-based dyestuffs analysis. Its optimization was conducted with three different stationary phases (unmodified octadecylsilane-bonded silica, octadecylsilane modified with polar groups and silica-bonded pentaflourophenyls) as well as with different solvent buffers. Detection of the isolated compounds was carried out using diode-array detector (DAD) and tandem mass spectrometer with electrospray ionization (ESI MS/MS). The working parameters of ESI were optimized, whereas the multiple reactions monitoring (MRM) parameters of MS/MS detection were chosen based on the product ion spectra of the quasi-molecular ions. Calibration curve of berberine has been estimated (y = 1712091x + 4785.03 with the correlation coefficient 0.9999). Limit of detection and limit of quantification were calculated to be 3.2 and 9.7 ng/mL, respectively. Numerous alkaloids (i.e., berberine, jatrorrhizine and magnoflorine, as well as phellodendrine, menisperine and berbamine) were identified in the extracts from alkaloid plants and silk and wool fibers dyed with these dyestuffs, among them their markers.

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.

Combination of ICP-MS, capillary electrophoresis, and their hyphenation for probing Ru(III) metallodrug-DNA interactions.

Determination of the DNA-binding reactivity and affinity is an important part of a successful program for the selection of metallodrug candidates. For such assaying, a range of complementary analytical techniques was proposed and tested here using one of few anticancer metal-based drugs that are currently in clinical trials, indazolium trans-[tetrachloridobis(1H-indazole)ruthenate(III), and a DNA oligonucleotide. A high reactivity of the Ru drug was confirmed in affinity capillary electrophoresis (CE) mode, where adduct formation takes place in situ (i.e., in the capillary filled with an oligonucleotide-containing electrolyte). To further characterize the binding kinetics, a drug-oligonucleotide mixture was incubated for a different period of time, followed by ultrafiltration separation into two different in molecular weight fractions (>3 and <3 kDa). The time-dependent distribution profiles of the Ru drug were then assessed by CE-inductively coupled plasma mass spectrometry (ICP-MS), revealing that at least two DNA adducts exist at equilibrium conditions. Using standalone ICP-MS, dominant equilibrium amount of the bound ruthenium was found to occur in a fraction of 5-10 kDa, which includes the oligonucleotide (ca. 6 kDa). Importantly, in all three assays, the drug was used for the first time in in-vitro studies, not in the intact form but as its active species released from the transferrin adduct at simulated cancer cytosolic conditions. This circumstance makes the established analytical platform promising to provide a detailed view on metallodrug targeting, including other possible biomolecules and ex vivo samples.

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.