M.V. Efremova

Changing the Enzyme Reaction Rate in Magnetic Nanosuspensions by a Non‐Heating Magnetic Field

Weak magnetic fields (< 1 T) are known to change the paths and kinetics of some radical chemical reactions at room temperature through spin conversion of short-lived radical pairs. [1] Magnetic fields were also used to tune enzyme activity (oxidation of glucose). Specifically, exposure of magnetic nanoparticles functionalized with enzymes to magnetic field allowed the movement of particles in proximity to electrode, thus activating the enzyme. [2] Moreover, radio-frequency (RF) alternating current (AC) magnetic fields can induce the heating of single-domain magnetic nanoparticles (MNPs), and this can be used in magnetic hyperthermia to kill tumors. [3] Herein we describe a distinct effect of non-heating superlow-frequency magnetic fields as well as more conventional RF magnetic fields on the kinetics of chemical reactions catalyzed by the enzymes a-chymotrypsin (ChT) or bgalactosidase (b-GaL) immobilized on nanoscale MNP aggregates. Magnetite MNPs (7 to 12 nm diameter) were synthesized by reducing tris(acetylacetonato)iron(III), then they were coated with anionic poly(ethylene glycol)-bpolyacrylate (PEG-PAA), or poly(ethylene glycol)-b-polymethacrylate (PEG-PMA) copolymers. The polymers were bound to the magnetite surfaces by the carboxylate groups. [4] According to thermogravimetric analysis, the content of Fe3O4 in both coated MNP materials was about 35 wt %, and this was in excellent agreement with that measured by inductively coupled plasma-mass spectrometry. Upon dispersion in water (pH 6.5), they formed nanoscale, negatively charged aggregates (hydrodynamic intensity average diameters and zeta potentials of 194 nm, 70 mV and 39 nm, 49 mV for PEG-PAA/MNP and PEG-PMA/MNP, respectively, as determined by dynamic light scattering). Based on TEM, they contained clusters of 7 to 20 MNP grains. Enzymes were conjugated to these aggregates using 1-ethyl-3-(3dimethylaminopropyl)-carbodiimide and N-hydroxysulfosuccinimide to couple amines on the protein to carboxylic acids on the copolymer (Scheme 1).

Enzyme-functionalized gold-coated magnetite nanoparticles as novel hybrid nanomaterials: Synthesis, purification and control of enzyme function by low-frequency magnetic field

The possibility of remotely inducing a defined effect on NPs by means of electromagnetic radiation appears attractive. From a practical point of view, this effect opens horizons for remote control of drug release systems, as well as modulation of biochemical functions in cells. Gold-coated magnetite nanoparticles are perfect candidates for such application. Herein, we have successfully synthesized core-shell NPs having magnetite cores and gold shells modified with various sulphur containing ligands and developed a new, simple and robust procedure for the purification of the resulting nanoparticles. The carboxylic groups displayed at the surface of the NPs were utilized for NP conjugation with a model enzyme (ChT). In the present study, we report the effect of the low-frequency AC magnetic field on the catalytic activity of the immobilized ChT. We show that the enzyme activity decreases upon exposure of the NPs to the field.

A new approach to the control of biochemical reactions in a magnetic nanosuspension using a low-frequency magnetic field

A new approach to the control of biochemical reactions in magnetic nanosuspensions exposed to a low-frequency (nonheating) magnetic field, which has a nanomechanical effect on macro-molecules chemically linked to magnetic nanoparticles (MNPs), is described. Experimental verification of this approach showed that a magnetic field with an intensity of from 15 to 220 kA/m and a frequency of 50 Hz affected the kinetics of a chemical reaction in an aqueous solution containing suspended MNPs of magnetite (FeO · Fe2O3) and chymotrypsin molecules linked to them through polymer bridges. The field dependence of the effect is shown. The effect is interpreted within the framework of a nanomechanical model taking into account the deformations, conformational change, and destruction of weak bonds in the enzyme macromolecule under the action of the forces applied to it during the orientation of MNPs in the field.

In Situ Observation of Chymotrypsin Catalytic Activity Change Actuated by Nonheating Low-Frequency Magnetic Field

Magnetomechanical modulation of biochemical processes is a promising instrument for bioengineering and nanomedicine. This work demonstrates two approaches to control activity of an enzyme, α-chymotrypsin immobilized on the surface of gold-coated magnetite magnetic nanoparticles (GM-MNPs) using a nonheating low-frequency magnetic field (LF MF). The measurement of the enzyme reaction rate was carried out in situ during exposure to the magnetic field. The first approach involves α-chymotrypsin-GM-MNPs conjugates, in which the enzyme undergoes mechanical deformations with the reorientation of the MNPs under LF MF (16-410 Hz frequency, 88 mT flux density). Such mechanical deformations result in conformational changes in α-chymotrypsin structure, as confirmed by infrared spectroscopy and molecular modeling, and lead to a 63% decrease of enzyme initial activity. The second approach involves an α-chymotrypsin-GM-MNPs/trypsin inhibitor-GM-MNPs complex, in which the activity of the enzyme is partially inhibited. In this case the reorientation of MNPs in the field leads to disruption of the enzyme-inhibitor complex and an almost 2-fold increase of enzyme activity. The results further demonstrate the utility of magnetomechanical actuation at the nanoscale for the remote modulation of biochemical reactions.

Сore–shell magnetite–gold nanoparticles: Preparing and functionalization by chymotrypsin

In this work we present the results of the synthesis of magnetite–gold nanoparticles with a core–shell structure. The preparation is carried out in several stages: synthesis of the core, coating with a gold shell, purification of magnetite–gold particles from an uncoated magnetite, and functionalization of the surface with sulfur-containing ligands. The conditions needed for the functionalization of nanoparticles with lipoic acid and mercapto-methoxy polyethylene glycol are indicated in detail, making it possible to determine the optimal conditions needed to achieve an efficient purification and a maximum concentration of the particles in a solution required for biological tests. The possibility of remotely controlling the chymotrypsin properties using an alternating magnetic field has been demonstrated by the example of magnetite–gold nanoparticles. The magnetite–gold nanoparticles which we have obtained are promising for future biomedical applications.

Novel method for rapid toxicity screening of magnetic nanoparticles

Iron oxide nanoparticles have attracted a great deal of research interest and have been widely used in bioscience and clinical research including as contrast agents for magnetic resonance imaging, hyperthermia and magnetic field assisted radionuclide therapy. It is therefore important to develop methods, which can provide high-throughput screening of biological responses that can predict toxicity. The use of nanoelectrodes for single cell analysis can play a vital role in this process by providing relatively fast, comprehensive, and cost-effective assessment of cellular responses. We have developed a new method for in vitro study of the toxicity of magnetic nanoparticles (NP) based on the measurement of intracellular reactive oxygen species (ROS) by a novel nanoelectrode. Previous studies have suggested that ROS generation is frequently observed with NP toxicity. We have developed a stable probe for measuring intracellular ROS using platinized carbon nanoelectrodes with a cavity on the tip integrated into a micromanipulator on an upright microscope. Our results show a significant difference for intracellular levels of ROS measured in HEK293 and LNCaP cancer cells before and after exposure to 10 nm size iron oxide NP. These results are markedly different from ROS measured after cell incubation with the same concentration of NP using standard methods where no differences have been detected. In summary we have developed a label-free method for assessing nanoparticle toxicity using the rapid (less than 30 minutes) measurement of ROS with a novel nanoelectrode.

Magnetite-Gold nanohybrids as ideal all-in-one platforms for theranostics

High-quality, 25 nm octahedral-shaped Fe3O4 magnetite nanocrystals are epitaxially grown on 9 nm Au seed nanoparticles using a modified wet-chemical synthesis. These Fe3O4-Au Janus nanoparticles exhibit bulk-like magnetic properties. Due to their high magnetization and octahedral shape, the hybrids show superior in vitro and in vivo T2 relaxivity for magnetic resonance imaging as compared to other types of Fe3O4-Au hybrids and commercial contrast agents. The nanoparticles provide two functional surfaces for theranostic applications. For the first time, Fe3O4-Au hybrids are conjugated with two fluorescent dyes or the combination of drug and dye allowing the simultaneous tracking of the nanoparticle vehicle and the drug cargo in vitro and in vivo. The delivery to tumors and payload release are demonstrated in real time by intravital microscopy. Replacing the dyes by cell-specific molecules and drugs makes the Fe3O4-Au hybrids a unique all-in-one platform for theranostics.

Size-selected Fe3O4–Au hybrid nanoparticles for improved magnetism-based theranostics

Size-selected Fe3O4–Au hybrid nanoparticles with diameters of 6–44 nm (Fe3O4) and 3–11 nm (Au) were prepared by high temperature, wet chemical synthesis. High-quality Fe3O4 nanocrystals with bulk-like magnetic behavior were obtained as confirmed by the presence of the Verwey transition. The 25 nm diameter Fe3O4–Au hybrid nanomaterial sample (in aqueous and agarose phantom systems) showed the best characteristics for application as contrast agents in magnetic resonance imaging and for local heating using magnetic particle hyperthermia. Due to the octahedral shape and the large saturation magnetization of the magnetite particles, we obtained an extraordinarily high r 2-relaxivity of 495 mM−1·s−1 along with a specific loss power of 617 W·gFe −1 and 327 W·gFe −1 for hyperthermia in aqueous and agarose systems, respectively. The functional in vitro hyperthermia test for the 4T1 mouse breast cancer cell line demonstrated 80% and 100% cell death for immediate exposure and after precultivation of the cells for 6 h with 25 nm Fe3O4–Au hybrid nanomaterials, respectively. This confirms that the improved magnetic properties of the bifunctional particles present a next step in magnetic-particle-based theranostics.