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Estimation of SPIO Nanoparticles Uptakes by Macrophages Using Transmission Electron Microscopy

https://doi.org/10.3390/ijms232213801

“Due to their interesting size-dependent magnetic characteristics and relative biocompatibility, magnetic superparamagnetic iron oxide (SPIO) nanoparticles have been widely exploited as probes for cell and subcellular structure identification, as well as medication and gene delivery. A thorough understanding of the mechanics of the interaction between nanoparticles and macrophages is vital in managing dynamic processes in nanomedicine. In this study, the interaction behavior and uptake of SPIO nanoparticles by M1- and M2-type macrophages were investigated. Mice monocytes were differentiated into M1 and M2 macrophages, and the uptake of SPIO nanoparticles was studied using a TEM microscope. A high resolution image of 1 nm resolution, an image processing technique, was developed to extract the SPIO-NPs from tomographic TEM microscopic images. Lysosomes appear to be the zones of high concentrations of SPIO inside macrophages. Lysosomes were first selected in each image, and then segmentation by the Otsu thresholding method was used to extract the SPIO-NPs. The Otsu threshold method is a global thresholding technique used to automatically differentiate SPIOs from the background. The SPIO-NPs appear in red colors, and the other pixels in the image are considered background. Then, an estimation of the SPIO-NP uptakes by lysosomes is produced. Higher uptake of all-sized nanoparticles was observed in M1- and M2-type macrophages. An accurate estimation of the number of SPIO-NPs was obtained. This result will help in controlling targeted drug delivery and assessing the safety impact of the use of SPIO-NPs in nanomedicine for humans.

Figure 1a shows a sample of the control tomographic tilt series, and Figure 1b shows the macrophage with SPIO1-M1 obtained from the TEM Hitachi H7500.
Figure 1. Transmission electron microscopy micrographs; (a) a control image without SPIO-NP. (b) AMNP-labeled M2 macrophages revealing the intralysosomal localization of nanoparticles or microvesicles (100 nm–1 μm), which directly bud from the plasma membrane. The red box englobes a lysosome or a microvesicle. The black scale bar on the left corresponds to 200 nm, and the blue scale bar represents 100 nm.
The background image in Figure 2 shows the individual SPIO in the background from which the size of the SPIO is estimated. The Otsu method was utilized to determine the threshold. The SPIO-NPs are represented by the red pixels in (b), where (c) includes the red particles representing the SPIOs and (d) the threshold used to separate the NPs is shown. The quantity of red pixels in each lysosome estimates how many SPIO-NPs the macrophage has ingested if we can count the total number of lysosomes and/or microvesicles in the macrophage.
Figure 2. The white rectangle region from the background extracted from Figure 1. (a) The original image with a scale bar of 100 nm. (b) Image after selection of the SPIO nanoparticles in red color, (c) SPIOs were extracted from (b) using threshold method, and (d) is the threshold selected at the first minimum of the histogram.
Figure 3 shows the lysosomes/microvesicles in the first column; they seem slightly of different sizes, and the diameters vary between 160 and 250 nm. The SPIO NPs are represented by the red dots in column 2 of Figure 3. In the picture, varying amounts of NPs are shown in red inside each lysosome. SPIOs are seen inside the lysosomes in these images. Therefore, it is simple to estimate the amount of SPIOs engulfed by each lysosome/microvesicles by counting the red dots or pixels inside it and then estimating the number of NPs inside macrophages by counting the total number of NPs for all lysosomes/microvesicles inside the macrophages. The image resolution and the diameter of each SPIO (10 to 13 nm) were considered. The results are shown in the last column of Table 1.
Figure 3. The first column represents 4 single microphages after uptake of the SPIO nanoparticles. The blue scale bar on the first column of the images represents 100 nm. The second column represents the segmented regions in red color where the SPIOs NPs inside the lysosomes or microvesicles. The third column is the histogram of the image. The red line represents the threshold chosen at the first minimum. The vertical axis represents the number of pixels for each gray level. The horizontal axis represents gray levels (0 to 255).

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