<i>In Situ</i> 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 <i>in situ</i> 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

Accumulation of exosomes secreted from macrophages in Cath.A neurons and genetic material transfer.

<p><b>A</b>: Cath.A neurons grown on slides were fixed and stained with Anti-NeuN Antibodies (blue, left picture); exosomes were isolated from Raw 264.7 macrophages media, stained with lipophilic fluorescent dye, DIO (green), and added to Cath.A neurons for 24 hours (right picture). <b>B</b>: Raw 264.7 macrophages were transfected with fluorescently-labeled with YOYO-1 tomato protein <i>p</i>DNA (green), and then cultured in complete media. Confocal images of transfected macrophages on day 3 show incorporation of <i>p</i>DNA (green) in the nucleus and expression of tomato protein (red) in the cytoplasm. <b>C</b>: Media from macrophages transfected as described above with tomato protein <i>p</i>DNA (labeled with YOYO-1) was collected over 24 hours, and isolated exosomes were added to Cath.A neurons for various times. Then, the neurons were fixed and stained with Anti-NeuN Antibodies (blue). Confocal images of neurons incubated with exosomal fraction demonstrated relatively constant amount of YOYO-1-labeled <i>p</i>DNA (green), and increasing in time expression levels of tomato protein (red) confirmed by the quantification of green and red fluorescence on confocal images (graph). Co-localization of YOYO-1-labeled genetic material and expressed tomato protein in neurons is manifested by yellow staining. Statistical significance of tomato protein expression levels (shown by asterisk: <i>p</i><0.05) was assessed by a standard t-test compared to day one after transfection. The bar: 20 µm.</p

Therapeutic effect of catalase-transfected macrophages on motor functions in a PD mouse model.

<p>BALB/c mice were <i>i.c.</i> injected with 6-OHDA. Forty eight hours later, the animals were <i>i.v.</i> injected with catalase-transfected macrophages (bars with diagonal pattern) or PBS (black bars), or empty-transfected macrophages (white bars). Control group was <i>i.c.</i> injected with PBS, and then 48 hours later <i>i.v.</i> injected with PBS (grey bars). Apomorphine (<b>A</b>) and rotaroid (<b>B</b>) tests demonstrated statistically significant improvements in motor functions upon treatment with catalase-transfected macrophages. Number of rotations (<b>A</b>) was significantly decreased in 6-OHDA-intoxicated mice treated with catalase-transfected macrophages compared to non-treated PD mice. No rotations were detected in control PBS-injected mice in apomorphine test. Time spent on the rotarod (<b>B</b>) in 6-OHDA intoxicated mice treated with catalase-transfected macrophages was the same as in healthy non-intoxicated control mice on the seventh week after the intoxication. In contrast, significant decreases were observed in 6-OHDA-intoxicated mice injected with PBS. No effect on motor functions was recorded in 6-OHDA-intoxicated mice treated with empty-transfected macrophages. Statistical significance was calculated using one-way ANOVA test. Values are means ± SEM (<i>N</i> = 10), and <i>p</i><0.05 compared with ^aPBS, and ^b6-OHDA.</p

Exosomes secreted from GFP-transfected macrophages contain GFP DNA, RNA, the transcription factor, and expressed protein.

<p>Exosomes from GFP-transfected cells were collected over two days and evaluated for (<b>A</b>): GFP DNA (<b>1</b>) and RNA (<b>2</b>) by PCR analysis. Exosomes secreted from macrophages transfected with empty vector were used as a control (<b>3</b>). (<b>B</b>): Levels of GFP DNA and RNA in exosomes from GFP-transfected macrophages were compared to those from empty vector-transfected macrophages (<b>1</b>), or non-transfected cells (<b>2</b>) by Real-Time PCR analysis. <b>C</b>: expression levels of GFP (30K) in exosomes from GFP-transfected cells (<b>1</b>) or empty vector-transfected macrophages (<b>2</b>) were examined by western blot and compared to the levels of CD63 (53K). Exosomes released from GFP-transfected macrophages contained four orders of magnitude more of GFP DNA and RNA compared to non-transfected macrophages or those transfected with empty vector (<b>A, B</b>); and 6.1 times greater levels of the expressed protein, GFP (<b>C</b>). Exosomes contain substantially higher levels of NF-kb, a transcription factor that involved in GFP <i>p</i>DNA expression, compared to macrophages as demonstrated by western blot (<b>D</b>). AFM images of exosomes revealed differences between: (<b>E</b>) small donut-shaped (empty) exosomes released from non-transfected macrophages, and (<b>F</b>) large spherical (likely filled with the expressed proteins and genetic material) exosomes from catalase-transfected macrophages. The bar: 200 nm.</p