Investigation of Silver Nanoparticle Induced Lipids Changes on a

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Investigation of silver nanoparticle induced lipids changes on single cell surface by ToF-SIMS Xin Hua, Hao-Wen Li, and Yi-Tao Long Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b04591 • Publication Date (Web): 20 Dec 2017 Downloaded from http://pubs.acs.org on December 20, 2017

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Analytical Chemistry

Investigation of Silver Nanoparticle Induced Lipids Changes on Single Cell Surface by ToF-SIMS Xin Hua, Hao-Wen Li, Yi-Tao Long* Key Laboratory of Advanced Materials & School of Chemistry and Molecular Engineering, School of Chemistry and Molecular Engineering, East China University of Science and Technology, Shanghai, 200237 P. R. China. Email: [email protected]; Phone: +86-021-6425-2339. ABSTRACT: Lipids are the main component of cell membrane. They not only provide structural support of cells, but also directly participate in complex cellular metabolic processes. Lipid signaling is an important part of cell signaling. Evidence showed that abnormal cellular metabolism may induce lipids changes. Besides, owing to single cell heterogeneity, it is necessary to distinguish different behaviors of individual cells. Time-of-flight secondary ion mass spectrometry (ToF-SIMS) is a sensitive surface analysis technique with high spatial resolution, which is useful in single cell surface analysis. Herein, we used ToF-SIMS to investigate silver nanoparticles induced lipids changes on the surface of single macrophage cells. Delayed extraction mode of ToF-SIMS was used to simultaneously obtain high mass resolution of mass spectra and high spatial resolution of single cell chemical imaging. Principle component analysis (PCA) results showed good agreement with the cytotoxicity assay results. Clear distinctions were observed between the cell groups treated with high or low dose of silver nanoparticles. The loadings plots revealed that the separation was mainly due to changes of cholesterol, diacylglycerol (DAG) as well as monoacylglycerol (MAG). Meanwhile, the chemical mapping of single cell components showed that cholesterol and DAG tend to migrate to the surrounding of cells after high dose Ag NPs treatment. Our results demonstrated the feasibility of ToF-SIMS for characterizing the changes of the lipids on single cell surface, providing a better understanding of the mechanism of cell-nanoparticle interactions at molecular level. Single cell analysis is increasingly attracting attentions, since ensemble analysis may neglect the heterogeneities among individual cells1. Even though the ensemble analysis results for two cell samples are absolutely the same, the biological behavior of these cells could be totally different. Owing to the complexity of single cell structure and chemical composition, as well as the extremely small amount of target analytes, single cell analysis remains a challenge2. Lipids are a class of biomolecules that play important roles in biomembrane formation, energy storing and cell signaling3. Previous work demonstrated that the interactions between cells and exogenous stimuli might result in composition changes of cell lipids. For example, it was shown that hepatitis C virus (HCV) affects desmosterol homeostasis by increasing its intracellular abundance and affecting its localization4. The change of human breast cancer microenvironment could lead to lipids heterogeneity resulting from fatty acid processing5. Certain food was also proved to induced changes of lipids in rat neuronal tissue6. Silver nanoparticles (Ag NPs) were widely used in various practical applications such as antimicrobial agents and therapeutics. It was reported that Ag NPs smaller than 50 nm showed broad spectrum inhibition of bacteria and fungi7. Cytotoxicity of Ag NPs is of great interest in the concern of its adverse effect on human health. Previous studies showed that oxidative stress play a prominent role in Ag NPs-induced cytotoxicity, which may cause the changes of intracellular components such as DNAs, proteins and lipids8. The level of lipid peroxidation is an indicator of oxidative stress in cells8. Thus, the study of lipids changes

in single cells is very important for evaluating the effect of Ag NPs exposure for human beings. Various analysis techniques have been applied in single cell analysis, including imaging techniques9,10, optical spectroscopy10-12, electrochemical biosensors13,14 and mass spectrometry15,16. These techniques provided rich morphological or chemical information of single cells. Recent years have witnessed great advances for single cell mass spectrometry with the advantages of label-free, high sensitivity and enormous information on chemical identity in a single measurement17,18. Time-of-flight secondary ion mass spectrometry (ToF-SIMS) is a high spatial resolution chemical imaging technique for single cell analysis without the need for cell labeling or lysis. This technique is becoming increasingly popular for biological applications such as the study of cell differentiation19,20, lipids metabolism6,21 and drug targeting16. Scheme 1. Schematic illustration of ToF-SIMS characterization of silver nanoparticles induced lipids changes.

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In this work, the cytotoxicity of Ag NPs with different doses were studied. The corresponding lipids changes were investigated by ToF-SIMS (Scheme 1). Principal component analysis (PCA) was used to unravel the differences among cell samples treated with different Ag NPs doses. ToF-SIMS chemical imaging further showed the spatial distribution of lipids on the surface of Ag NPs treated cells. These results are complementary to previous findings regarding cell-Ag NPs interaction for a better understanding of Ag NPs induced cytotoxicity.

EXPERIMENTAL SECTION RAW264.7 macrophage cells were seeded on goldcoated silicon wafers. These cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum, 100 μg mL−1 streptomycin and 100 μg mL−1 penicillin in a humid atmosphere with 5% CO2 at 37 °C. The cell samples were cultured for 24 h before treated with 10 nm Ag NPs with a final concentration of 2000, 500, 50, 5 or 0 ng mL-1 for another 24 h, respectively. Next, the cell samples were removed from the culture media and were gently rinsed for 30 s in 150 mM ammonium acetate solution at pH 7.4. Prior to cryofixation, the edges of the silicon wafers were touched with a Kimwipe to remove excess liquid. A drop (10 μL) of 0.1 mg mL-1 2,5-dihydroxybenzoic acid (DHB) solution was casted on top of the cells. The obtained cell samples were immediately transferred into liquid nitrogen for fast frozen at -196 °C to maintain the cell shape. The frozen samples were then placed in the vacuum chamber of a manifold type freeze drier. The samples were lyophilized for 6 h before ToF-SIMS analysis. More experimental details are presented in the Supporting Information (Experimental section). RESULTS AND DISCUSSION ToF-SIMS was initially applied in the elemental mapping of inorganic materials, owing to poor ionization yield of intact molecules. It was not until recent years that cluster primary ion sources were introduced to ToF-SIMS and significantly improved the ionization efficiency for organic molecules22,23. Since then, ToF-SIMS has become an emerging technique in the analysis of biological samples such as tissues and single cells5,16,24. The capability of high resolution chemical imaging of ToF-SIMS was proved to be especially useful for the visualization of spatial distribution of intracellular species. However, a major problem lies in the relatively low secondary ion yield of intracellular species25. To increase secondary ion yield, gold film coated silicon substrates were used for cell culturing in this work. Be-

sides, a drop of DHB solution was cast onto the sample surface before subsequent frozen-dehydration process. This method is called “matrix-enhanced SIMS (MESIMS)”.26,27 By doing so, ionization yield of single cells significantly increased (see Figure S-1). It was known that DHB application may cause delocalization of chemical compounds in tissue samples28. This could potentially happen in dispersed single cell samples as well. To reduce this effect, we transferred the samples to liquid nitrogen for fast frozen immediately after DHB deposition. Figure S-2 showed the scanning electron microscopy (SEM) images of single cells without (Figure S-2A) and with (Figure S-2B) DHB coating. A uniform DHB crystal layer was observed covering cell surface. Some water-soluble components (such as K+, Na+) may easily delocalize in the process of ammonium acetate washing and DHB application. However, it was not likely lipids were easily delocalized. Chemical imaging of DHBcoated macrophage cells by ToF-SIMS were shown in Figure S-1(B) and Figure S-2(C). Phosphatidylcholine was found uniformly distributed on cell surface in micrometer scale. Thus, we believe in our analysis, delocalization of lipids was negligible. Another technical issue for ToF-SIMS imaging was the trade-off between high lateral resolution and high mass resolution. In conventional instrumental setup, it was challenging to obtain high mass resolution and high lateral resolution simultaneously. In this work, delayed extraction mode was applied to maintain both high lateral resolution and high mass resolution 29. Moreover, it was known that delayed extraction mode was useful in reducing the so called “topographic effect” in the imaging of topographic samples30, which was important for high quality single cell mapping.

Figure 1. Positive ToF-SIMS spectra of macrophage cells treated with Ag NPs with a concentration of 0, 5 and 500 ng mL-1, respectively.

UV-vis spectrum and TEM characterization of asprepared Ag NPs were presented in Figure S-3. The maximum absorption peak was 395 nm. A mean particle diameter of ca. 10 nm as well as good dispersion were obtained. After treated with Ag NPs of different


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Analytical Chemistry concentrations, macrophage cells were analysed with ToF-SIMS. As shown in Figure 1, with the increase of Ag NPs concentration, the signal intensity of m/z 313.31, 369.38, 551.52, 577.51 and 603.52 showed a clear increase. Peak assignments were listed in Table S-1. These results indicated an increase of MAG, cholesterol and DAG on the macrophage cell surface after interacted with Ag NPs. The increase of DAG could be a result of phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis31. The increase of cholesterol was in good agreement with previous ensemble analysis results32. MAG could be a fragment of DAG. These results revealed the lipids changes of Ag NPs-treated macrophage cells at single cell level. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2Htetrazolium bromide (MTT) assay was used to illustrate the cytotoxicity of Ag NPs. As displayed in Figure 2(A), the MTT assay indicated the cell viability of each cell sample group after Ag NPs treatment. With the increase of Ag NPs concentration, a significant decrease of cell viability was observed in the 500 ng mL-1 and 2000 ng mL-1 group. The cell survival rate of these two sample groups were 44% and 46% of the control group, respectively, indicating severe cell damage. Lots of previous studies demonstrated that Ag NPs-induced cytotoxicity was mainly caused by oxidative stress, which was in-

duced when the generation of ROS exceeds the cell’s antioxidant capacity33. To investigate the cytotoxicity of Ag NPs at molecular level, changes of surface components of macrophage cells before and after Ag NPs treatment were measured by ToF-SIMS. PCA was frequently used in the simplification of multivariate data sets such as ToF-SIMS data34. In this work, PCA was performed for the comparison of different cell sample groups. To avoid the interference of non-cell components from substrate, region of interest (ROI) with a diameter of 20 μm was chosen at the central region of each cell. ToF-SIMS data was reconstructed within each ROI before PCA. Figure 2(B) showed the PCA score plot of PC1 vs. PC2. The total variance captured by PC1 and PC2 was 90.23%. The result revealed a clear separation between cell samples treated with high dose (500 ng mL-1 and 2000 ng mL-1 group) or low dose (0 ng mL-1, 5 ng mL-1 and 50 ng mL-1 group) of Ag NPs. This classification was consistent with the MTT result. Corresponding loading plot of PC1 in Figure 2(C) demonstrated a decrease of MAG, DAG and cholesterol in the central region of cells in high dose groups. However, the mass spectra of whole cell showed an increase of MAG, DAG and cholesterol after Ag NPs treatment (Figure 1). Considering the ROI chosen in PCA, it was necessary to investigate the spatial distribution of these lipids.

Figure 2. (A) MTT assay of macrophage cell viability treated with Ag NPs with different concentrations; (B) PCA score plot of PC1 vs. PC2 from ToF-SIMS spectra of Ag NPs treated macrophage cells. A 95% confidence limit for each sample group was defined by an ellipse with the same color to the corresponding sample group clusters; (C) Loading plot of PC1 corresponding to (B).

Figure 3 ToF-SIMS imaging of macrophage cells untreated (A) and treated (B) with 500 ng mL-1 Ag NPs. C27H45+ (m/z 369.38) is a characteristic fragment of cholesterol. C39H71O4+ (m/z 603.52) is a representative fragment of DAG. The overlay images are a merge of C27H45+ (red), C39H71O4+ (green) and total ions (blue).



Displayed in Figure 3 are the distributions of cholesterol (C27H45+) and DAG (C39H71O4+) on the surface of macrophage cells incubated with or without Ag NPs exposure. The shape of entire cell was clearly visible in total ion images with a lateral resolution of ~200 nm. It was shown that in the cell sample without Ag NPs treatment, both cholesterol and DAG distributed uniformly on the cell surface. While after treated with 500 ng mL-1 Ag NPs, much cholesterol and DAG migrated to the surrounding of cell, which was the reason for the decrease of lipids in the PCA result. Besides, due to the migration of cell surface components, DHB-related species, i.e. m/z 246.09 (C13H10O5+) and m/z 290.09 (C14H10O7+), became the dominant component on cell surface (Figure S-4). This could explain the negative loading of these two species in PC1. The decrease of Au3+ signal in Figure 1 after Ag NPs treatment was also caused by the migration. In addition, the Ag NPs treated cell looked shrunken. These are clear indications of unhealthy cells, as a result of the increase of cell membrane fluidity as well as permeability. These results gave direct visualization of cell morphology and component distribution changes in the process of cell death in a label-free and high-resolution manner. CONCLUSIONS In this work we demonstrated the feasibility of ToFSIMS in characterizing the cytotoxicity of Ag NPs. In combination with PCA, ToF-SIMS was able to analyse the cytotoxicity of Ag NPs from molecular level. It was found that cell surface lipids such as DAG and cholesterol increased after Ag NPs treatment. However, due to the increase of cell membrane fluidity and permeability, most lipids migrated to the surrounding of cells, indicating cell death. ToF-SIMS provided a direct visualization of chemical changes on cell membrane in the process of cell-nanoparticles interaction, showing great potential for further studies of nanoparticle metabolism and the health impact.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Experimental details, ToF-SIMS imaging of a single cell under optimized instrumental condition, and UV-vis spectrum, TEM characterization of as-prepared Ag NPs.

AUTHOR INFORMATION Corresponding Author * E-mail: [email protected]

ORCID Yi-Tao Long: 0000-0003-2571-7457 Xin Hua: 0000-0003-1064-083X

Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENT This research was supported by the National Natural Science Foundation of China (21421004, 21327807, 21705046), the Program of Introducing Talents of Discipline to Universities (B16017), Innovation Program of Shanghai Municipal Education Commission (2017-01-07-00-02-E00023), the Fundamental Research Funds for the Central Universities (222201718001, 222201717003), Shanghai Sailing Program (17YF1403000), Shanghai Natural Science Foundation (17ZR1407700) and China Postdoctoral Science Foundation (2017M611470).

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Figure 1. Positive ToF-SIMS spectra of macrophage cells treated with Ag NPs with a concentration of 0, 5 and 500 ng mL-1, respectively. 203x288mm (300 x 300 DPI)



Figure 2. (A) MTT assay of macrophage cell viability treated with Ag NPs with different concentrations; (B) PCA score plot of PC1 vs. PC2 from ToF-SIMS spectra of Ag NPs treated macrophage cells. A 95% confidence limit for each sample group was defined by an ellipse with the same color to the corresponding sample group clusters; (C) Loading plot of PC1 corresponding to (B). 465x131mm (72 x 72 DPI)


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Figure 3 ToF-SIMS imaging of macrophage cells untreated (A) and treated (B) with 500 ng mL-1 Ag NPs. C27H45+ (m/z 369.38) is a characteristic fragment of cholesterol. C39H71O4+ (m/z 603.52) is a representative fragment of DAG. The overlay images are a merge of C27H45+ (red), C39H71O4+ (green) and total ions (blue). 503x216mm (72 x 72 DPI)


Investigation of Silver Nanoparticle Induced Lipids Changes on a

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