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Nucleus and Mitochondria Targeting Theranostic Plasmonic SERS Nanoprobes Reveal Molecular Stress Response Difference in Hyperthermia Cell Death between Cancerous and Normal Cells Guohua Qi, Ying Zhang, Shuping Xu, Chuanping Li, Dandan Wang, Haijuan Li, and Yongdong Jin Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b03034 • Publication Date (Web): 20 Sep 2018 Downloaded from http://pubs.acs.org on September 21, 2018
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Analytical Chemistry
Nucleus and Mitochondria Targeting Theranostic Plasmonic SERS Nanoprobes Reveal Molecular Stress Response Difference in Hyperthermia Cell Death between Cancerous and Normal Cells Guohua Qia,b，Ying Zhanga,c，Shuping Xud，Chuanping Lia,b, Dandan Wanga,c, Haijuan Lia, Yongdong Jina,b,c* a State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, Jilin, P. R. China. b University of Chinese Academy of Sciences, Beijing 100049, P. R. China c University of Science and Technology of China, Hefei 230026, P. R. China d State Key Laboratory of Supramolecular Structure and Materials, Jilin University, 2699 Qianjin Avenue, Changchun 130012, P. R. China ABSTRACT: Metallic plasmonic nanoparticles have been intensively exploited as theranostic nanoprobes for plasmonic photothermal therapy (PPT) and surface enhanced Raman spectroscopy (SERS) applications. But the underlying molecular mechanisms associated with PPT-induced apoptosis between cancerous and normal cells have remained largely unknown or disputed. In this study, we designed an organelle-targeting theranostic plasmonic SERS nanoprobe (CDs-Ag/Au NS) composed of porous Ag/Au nanoshell (p-Ag/Au NSs) and carbon dots (CDs) for nucleus- and mitochondria-targeted PPT of cells. The differences in molecular stress response in the PPT-induced hyperthermia cell death between cancerous HeLa and normal L929 and H8 cells have been revealed by site-specific single-cell SERS detection. The contents of tryptophan (Trp), phenylalanine (Phe), and tyrosine (Tyr) in HeLa cells were found more evidently increased than L929 and H8 cells during the PPT-induced cell-death process. And from the mitochondria point of view, we found that the PPT-induced cell apoptosis for HeLa cells may mainly stem from (or be regulated through) cellular thermal stress-responsive proteins, while for L929 and H8 cells it seems more related to DNA. Understanding molecular stress response difference of the PPT-induced cell apoptosis between cancerous and normal cells is helpful for diagnosis and treatment of cancer and the method will open an avenue for single cell studies.
INTRODUCTION The Au-based plasmonic nanoparticles (NPs) have attracted tremendous attention for plasmonic photothermal therapy (PPT) of cancer due to high therapeutic efficiency and minimal invasiveness for cancer treatment.1,2 Such NPs whose localized surface plasmon resonance (LSPR) display intense optical properties can rapidly convert the light to heat to destroy cancer cells, through the electron-electron, electronphonon, and phonon-phonon collisions.3,4 On the other hand, the strong electromagnetic field near the surface of NPs make them useful and powerful as surface enhanced Raman spectroscopy (SERS) probes for single cell studies to investigate in-situ the PPT process and may reveal underlying hyperthermia cell death mechanisms at molecule level,5-7 which remains a challenge to date. Although the PPT method has been successfully exploited for cancer therapy, the underlying molecular mechanism associated with PPT-induced apoptosis between the cancerous and normal cells have remained largely unknown or disputed. It has been found that cancerous cells are more sensitive to the heat compared to normal cells 8, 9 which implies that molecular apoptosis mechanisms between normal and cancerous cells in the PPT process may be different. Recently, El-Sayed’s group had tried to reveal cancer cell
death mechanisms associated with Au nanorod PPT from nucleus level by simultaneous time-dependent SERS, metabolomics, and proteomics, 10 but the role of mitochondria, another important thermal sensitive organelle in the PPT process, has not been studied. Indeed, the majority of the cell’s energy supply is produced from mitochondria which also play a key role in program cell death such as apoptosis.11,12 These organelles are sensitive to the change of the heat shock and cardiolipin, and meanwhile the reactive oxygen species (ROS) formation can readily damage the mitochondria which can provoke mitochondrial dysfunction.13 However, there is no report on using mitochondrial-targeting plasmonic SERS nanoprobes for PPT-induced cell apoptosis mechanisms study yet, and therefore it is still a challenge in the field. With these considerations in mind, as shown in Scheme 1, we designed herein a smart hybrid plasmonic NP based on porous Ag/Au bimetal nanoshells (NSs) with surface decoration of C-dots (denoted as CDs-Ag/Au NSs). The CDs-Ag/Au hybrid NSs display superior photothermal conversion ability and biocompatibility than the parent p-Ag/Au NSs since carbon nanomaterials have good light absorption capacity. The CDs-Ag/Au NSs were then assembled with mitochondria- and cell nucleus-targeting peptide to prepare organelle-targeting SERS nanoprobes. The as-prepared nanoprobes can absorb
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NIR laser light to effectively kill cells (cancerous or normal cells) during the PPT process. At the same time, the timedependent and selected-area SERS spectra were collected in real time from a single cell, and the difference in heat resistance between the randomly selected normal (H8 and L929) and cancerous HeLa cells were tested and explained from the organelle level. We found that the nucleus-targeting is a more effective way than mitochondria-targeting in the PPT process to induce cell apoptosis. Chemical information of structural or conformational changes of functional proteins and lipids in two organelles of cancerous cells during the PPT-induced apoptosis was revealed by time-dependent in-situ SERS detection on single cell level. The metabolism-related tryptophane (Trp) and phenylalanine (Phe) of cancerous HeLa cells were more evidently increased than normal L929 and H8 cells; whereas the metabolism-related carbohydrates for the L929 cells were more obviously increased in the PPT process than HeLa cells to resist this stimulus. Moreover, DNA molecules in both cancerous and normal cells were found deformed to a certain degree because of the high temperature generated in the PPT process. Scheme1: Schematic illustration of the preparation of nucleus- and mitochondria-targeting CDs-Ag/Au NSs and their use for plasmonic photothermal therapy of cancer cells and self-sensing SERS detection of the process.
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EXPERIMENTAL SECTION Synthesis of CDs-Ag/Au nanoshells The p-Ag/Au NSs were synthesized using our method developed previously.14-16 The positively charged CDs were obtained via the reported microwave method.17 The CDs were assembled on the surface of the p-Ag/Au NSs electrostatically to from the CDs-Ag/Au NSs. Firstly, the Ag/Au NSs (10 ml, 0.12 nM) were mixed with the CDs solution (8 μL, 40 mg/mL), and then stirred overnight. Lastly, excess ligands were removed by centrifugation (6000 r, 10 min) for twice. The particles were re-dispersed in DI water for use.
Preparation of nucleus and mitochondria targeting plasmonic nanoprobes The CDs-Ag/Au NSs were modified by targeting peptide of cell nucleus and mitochondria using the method reported pre-
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viously.10 Firstly, the (30 μL, 1 mM) mPEG-SH were added into the CDs-Ag/Au NSs solution with stirring overnight. The PEGylated NPs (10 mL) were simultaneously treated with the RGD (5 mM, 2 μL) and NLS (5 mM, 20 μL) stirring overnight at room temperature to achieve 104 and 105 molar excess, which was denoted as NLS@RGD-PEG-CDs-Ag/Au NSs (NT-nanoprobes) for targeting to the nucleus. The method of mitochondria targeting nanoprobes were prepared as the same as NT-nanoprobes. The targeting peptides were modified successfully through the covalent linking between gold and the thiol group of cysteine (bold in the peptide sequence of nucleus and mitochondrial localization sequence NLS, MLS and RGD). The prepared nucleus and mitochondria targeting plasmonic nanoprobes were denoted as NT-nanoprobes and MT-nanoprobes.
Cell culture HeLa, L929 and H8 cells were purchased from the American Type Culture Collection (ATCC, USA). The cells were grown in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) supplemented with 10% (v/v) fetal bovine serum (Gibco) and 1% (v/v) antibiotic solution which was contained 200 units mL−1 penicillin and 200 units mL−1 streptomycin at 37 °C, 5% CO2 in a humidified atmosphere.
Photothermal therapy of living cells The CDs-Ag/Au NSs with different concentrations were exposed at the laser (808 nm) for 10 min to record the changes of temperature using a digital thermometer (Traceable 14-64844 type). The photothermal efficiency of the hybrid CDsAg/Au NSs was compared to the parent p-Ag/Au NSs via the photothermal ablation under the same conditions (5 min, 2 W /cm2, 0.36 nM). At the same time, the thermal imaging of CDs-Ag/Au NSs (0.36 nM) under the different time were taken using the Fluke infrared thermometer (TiS40) for 5 min at 808 nm laser exposure (power density 2 W/cm2). All the cells were cultured in the 35 mm dishes. After 24 h incubation, the complete culture medium was removed, and washed three times using the phosphate buffer (PBS) (10 mM, pH=7.4) and then added the organelle-targeting nanoprobes to each dish incubated with a mount of complete culture medium overnight. After incubation, the free nanoprobes were removed using the PBS washing three times. Sample dishes were then exposed at the laser (808 nm, 2 W/cm2) for 5 min. The trypan blue can be accumulated on the dead cell stained the blue, but the living cell were not stained because of the integrity of cell membrane. The cells stained were imaged in the bright field under the 10× magnification.
SERS measurement in vitro Time-dependent single-cell SERS spectra were recorded with the organelle-targeted nanoprobes to monitor in-situ the cellular molecular stress response changes in the PPT process. The cells were seeded on the adhered glass slides containing lysine with cell coverage of about 70～80%, and washed three times using the cold PBS and then incubated with NT- or MTnanoprobes for 24 h. Before SERS detection, the cell media (with nanoprobes) were removed and washed. Timedependent SERS spectra recorded from cell nucleus or mitochondria were collected during the NIR laser exposure period by a confocal Raman system (LabRAM ARAMIS, HORIBA JobinYvon, USA) with a 7.1 mW/633 nm laser as an excita-
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tion source and actualized by Synapse Thermoelectric cooled charge coupled device (CCD) camera (HORIBA Jobin Yvon, USA). Laser excitation and Raman scattering light collection were through a × 50 microscope objective lens (numerical aperture = 0.5, LMLFLN, Olympus, Japan), leading to a 1.5～ 3 μm spot size and resulting in single cell irradiation/resolution. Outstretched scan spectra with a spectral from 400 to 1800 cm-1 were collected with an integration time of 20 s and one accumulation. The 520.7 cm-1 of vibrational band of silicon pellet was applied to set as a reference for the wavenumber calibration. For the PPT experiments, each spectrum was averaged from ten individual cells and three different places of each cell detected before and after heating.
RESULT AND DISCUSSION CDs-Ag/Au NS Synthesis and Characterization
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The carbon dots (CDs) with size of 1～5 nm used to enhance PPT efficiency of the hybrid NPs were synthesized and characterized thoroughly (Figure S1). The p-Ag/Au NSs with averaged diameter of ~ 40 nm (Figure S2) were synthesized. As seen from TEM images in Figure 1a and Figure S2c, there is no morphology change of the p-Ag/Au NSs after the modification with CDs. High resolution TEM image (Fig. 1b) also clearly exhibits the typical lattice fringe with spacing of ~ 0.2 nm, which can be attributed to the (102) diffraction planes of graphitic (sp2) carbon.18, 19 Figure 1c recorded the element analysis, of which the nitrogen element was due to the amidogen from CDs. The successful CDs decoration of the NSs was further characterized by zeta potential measurement, UVvis, Raman and IR spectra (Figure S3), and XPS spectra of the sample (Figure S4). Counts (a.u.)
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Figure 1. a) TEM images, b)-c) HRTEM image, and elemental analysis of the as-synthesized hybrid plasmonic CDs-Ag/Au NSs.
Photothermal Conversion Efficiency of the NSs 50 nm
The temperature elevation profiles of the CDs-Ag/Au NSs after the mixture of Ag/Au NSs with varied CD concentrations was optimized under the NIR laser exposure (808 nm, 2 W/cm2) for 10 min (Figure S5), to reach highest photothermal conversion efficiency. Therefore, for obtaining better photothermal efficiency, the concentration of admixture CDs for resulting CDs-Ag/Au NSs was set as 40 mg/mL in the whole experiments. The loading content of CDs (~ 0.13 mg/mL) on the surface of Ag/Au NSs was roughly estimated by SERS spectra in Figure S6, obtained from the linear relationship between the SERS intensity at 1350 cm-1 and concentration of CDs. Moreover, the good photostability of the as-synthesized CDs (2 mg/mL) and CDs-Ag/Au NSs solutions (0.36 nm) was also manifested by only slight intensity and position changes in the UV-vis spectra under the long-time irradiation with 808 nm laser (2 W/cm2) for 1 h (Figure S7 (a)-(d)). Meanwhile, photothermal effect of CDs-Ag/Au NSs treated with 808 nm illuminating at five on/off cycles at 2 W/ cm2 was not reduced,
as shown in Figure S 7e. Figure 2a records the temperature elevation profile of the solutions containing same amount of control CDs, p-Ag/Au NSs (0.24 nM) and CDs-Au NSs (0.24 nM), respectively, under the 808 nm CW laser (2 W/ cm2） irradiation for 10 min. The CDs-Ag/Au NSs showed significantly better photothermal efficiency, with the solution temperature increasing to ～ 50℃ after the successive 10 min laser irradiation. Figure 2b shows the temperature elevation profile of the CDs-Ag/Au NSs with different NP concentrations. Figure 2c displays the IR thermal images of the CDsAg/Au NSs (0.36 nM) solution in 96-well plate under successive light illumination. The optical-thermal conversion efficiency (η %) was calculated to be around 74% and 49% for CDs-Ag/Au NSs and p-Ag/Au NSs, respectively, with the maximum temperature reaches to ~ 49.9 ℃ at 2 W cm-2 for 5 min, based on a previously reported method.1 We then randomly selected HeLa and L929 cells in this study to check PPT efficiency of the NSs for cell application. As shown in Figure 2d, under the same NP dose (~ 0.24 nM) and irradiation conditions, more cells (both HeLa and L929 cells) were dead (stained with trypan blue) when treated with CDs-Ag/Au NSs and p-Ag/Au NSs. This suggests that the hybrid CDs-Ag/Au NS is a better cell PPT agent than single p-Ag/Au NS. Moreover, due to good biocompatibility of CDs, the resulting CDsAg/Au NSs showed reduced cytotoxicity as compared to Ag/Au NSs alone (Figure S8).
Preparation and Cell PPT Study of the Plasmonic Organelle-Targeting Nanoprobes The nucleus and mitochondria targeting nanoprobes were then prepared by surface modification of the as-prepared CDsAg/Au NSs with the organelle-targeting peptides, NLS@RGD-PEG (NT) and MLS@RGD-PEG (MT), 20 and will be denoted thereafter as NT-nanoprobes and MTnanoprobes, respectively. As shown in Figure S9 (a&c), the successful surface modifications of the NT and MT were confirmed by both slight LSPR band red-shift from UV-vis spectra and pronounced Zeta potential variation as a consequence of surface modification. Although a slight spectral broadening occurred, no obvious aggregation of NPs in solutions was observed (see inset photo in Figure S9a). Dynamic light scattering (DLS) was also performed to measure size distribution of CDs-Ag/Au NSs before and after the modifications. As shown in Figure S9b, the average hydrodynamic diameter of the Ag/Au NSs, CDs-Ag/Au NSs, and NT-nanoprobes and MTnanoprobes were measured to be 42.9 ± 4.4, 46.5 ± 1.3, 50.82 ± 4.38, and 50.6 ± 3.0 nm, respectively. The hydrodynamic diameters of the NPs were (reasonably) slightly larger than that obtained from TEM (~ 40 nm for the Ag/Au NSs), due to the existence of certain hydrophilic functional groups on the surface of NPs.21 The low cytotoxicity of the as-prepared nanoprobes was also manifested by the MTT assay, as shown in Figures S9d&10. At the same time, cell viability of the cells treated with different concentrations of nanoprobes were tested under a NIR laser with power of 2 W/cm2 for 5 min, as shown in Figure S11 & S12. The NT- and MT-nanoprobes with concentration of ~ 0.36 nM were then applied for cell PPT study. We selected cancerous HeLa cells and normal L929 cells in this study. The cells incubated with standard culture media were used as a control. After the treatment
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Figure 3. a)-c) The cell viability test of HeLa and L929 cells after the incubation with ~ 0.36 nM of no-targeting nanoprobes, NTnanoprobes, and MT-nanoprobes, respectively, for 24 h and followed by PPT treatment with a 808 nm laser (2 W/cm2) for 5 min at different time interval.
to heat than normal cells. As seen from Figure 3b&c, the NTnanoprobe showed better cell PPT efficiency because of the high heat resistance for mitochondria which are the places to generate energy with heat. This is not surprising since the maximum temperature for the mitochondria to survive is ~ 50 ℃ as previously reported.22, 23
To prove the site-specific targeting of the NT-nanoprobe to the nucleus, fluorescence (FL) and dark-field scattering imaging of living cells were conducted by microscopy. Figure 4b& c show the FL co-localization imaging of HeLa and L929 cells, by using Fluorescein isothiocyanate (FITC) as whole cell FL staining agent and 4,6-diamino-2-phenyl indole (DAPI) as nucleus-specific cell FL staining agent, respectively. As seen from the bright field images of living cells, after incubation with the NT-nanoprobes some small black particles were accumulated to the nucleus position (Figure 4e) as compared with those without incubation with the nanoprobes (Figure 4a). As distinctly shown in Figure 4f&g and compared to Figure 4b&c, the FL images of cell nucleus parts (both for HeLa b)
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with the NT- and MT-nanoprobes for 24 h, the cells were exposed under a CW 808 nm NIR laser with power of 2 W/cm2 for 5 min at different time intervals. The cell viability was performed using the MTT assay. As seen from Figure 3 a-c, the percentage of cell viability was obviously decreased with the time increasing. And the survival ratio of HeLa and L929 cells treated with no-targeted nanoprobes was much higher than that incubated with the NT- and MT-nanoprobes in the PPT process. We also have counted the percentage of cell viability between the normal L929 cells and cancerous HeLa cells incubated with the NT- or MT-nanoprobes and the results indicated that the normal L929 cells have higher survival rates (~ 70% and 75% for the NT- and MT-nanoprobes, respectively) than the cancerous HeLa cells (~ 42% and 63%, respectively) under the same PPT conditions. The influence of enhanced permeability and retention (EPR) effect of cancer cells can be ignored, as the contents of SERS nanoprobes devoured for 24 by two cell lines were investigated using ICP-MS (Figure S13 a), from which we found that the phagocytosis of different cell organelles of HeLa and L929 cells showed a small difference. Moreover, we tested the temperature elevation profiles of nanoprobes devoured within nucleus and mitochondria of HeLa and L929 cells. As shown in Figure S13 b, after the NIR laser exposure for 5 min all the temperature increased above 45 ℃, which is harmful for the cancerous cells. These results indicate that the cancer cells are more sensitive
Targeting Characterization of the Nanoprobes by Microscopy Co-Localization of the NT-nanoprobe for nucleus
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Figure 2. a) The temperature elevation profiles of the solution containing CDs, p-Ag/Au NSs and CD-Ag/Au NSs, respectively. b) concentration-dependent temperature elevation profiles of the CD-Ag/Au NSs solutions. c) The photothermal images of the CDs-Ag/Au NSs (0.36 nM) under the NIR laser exposure (808 nm, 2 W/cm2) for different time duration. d) Microscopy images of the HeLa cells (top row) and L929 cells (bottom row), stained with trypan blue, before and after the incubation with p-Ag/Au NSs (0.24 nM), CDs (0.4 mg/mL) and CDs-Ag/Au NSs (0.24 nM), respectively, for 24 h, followed by irradiation with 808 nm laser (power density 2 W/cm2) for 5 min. The scale bar is 20 μm.
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Figure 4. The bright field, fluorescence, and their merged images for living cells (HeLa and L929 cells) using the FITC (2.5 μM, 20 μL in 1 mL free fresh DMEM medium, Ex=488 nm) and DAPI (0.1 μM, 10 μL in 1 mL free fresh DMEM medium, Ex=360 nm) as cell FL staining agent, respectively, before (a-d) and after (e-h) the incubation with the NT-nanoprobes for 24 h. Scale bar: 20 μm.
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and L929 cells) with the NT-nanoprobes were weakened or even disappeared both for FITC staining (Figure 4f) and especially the nucleus-specific DAPI staining (Figure 4g) owing to the fluorescence quenching caused by the proximity of plasmonic NT-nanoprobes that located around the nucleus. Figure S14 shows the dark field scattering images of living cells, from which we can see that the scattering images arising from the NT-nanoprobes also centered on the nucleus parts. These results (FL co-localization and dark-field scattering image) therefore strongly proved the success of NT-nanoprobe targeting to the nucleus.
Co-Localization of the MT-nanoprobe for mitochondria To identify the intracellular localization of the MTnanoprobes, we performed dark-field and FL co-localizat imaging experiments employing the MitoTracker™ Green FM as mitochondria-specific cell FL staining agent. The FL of Mito Bright Field
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Figure 6. Bio-TEM images of HeLa cells before (Ⅰ) and after incubation with 0.36 nM MT-nanoprobes ( Ⅱ ) and NTnanoprobes (Ⅲ) for 24 h. The nanoprobes targeting to the organelles was labeled using the purple arrow and blue frame for nucleus and red arrow and yellow frame for mitochondria, respectively. N= nucleus, M=mitochondria, C= cytoplasm.
uptaken by HeLa cells and mainly distributed in lysosomes (Figure S16), which would result in poorer photothermal effect than targeted-nanoprobes. All the results proved that the as-prepared SERS nanoprobes are capable of targeting to the nucleus and mitochondria and beneficial for the PPT of cancer cells.
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Figure 5. The bright field, fluorescence and their merged images for living cells (HeLa and L929 cells) using the MitoTracker™ Green FM (1 μM, 10 μL, in fresh DMEM medium) as cell FL staining agent, before (a-c) and after (d-f) the incubation with the MT-nanoprobes for 24 h. Scale bar: 20 μm
Tracker™ Green FM was quenched after the cell incubated with the MT-nanoprobes for 24 h, as shown in Figure 5e&f (cf. Figure 5b). We further confirmed the mitochondrial localization of the MT-nanoprobes by dark-field scattering imaging (Figure S15), which revealed that the scattered light arising from MT-nanoprobes were accumulated mainly on the outer area of the nucleus. The dark-field image coincides well with the FL image of cells. These results confirm the success of MT-nanoprobe targeting to the mitochondria. The distribution of SERS nanoprobes was more clearly observed by bio-TEM. As shown in Figure 6 ( Ⅱ & Ⅲ ), the majority of MTnanoprobes was found singly dispersed in the mitochondria, while the NT-nanoprobes was accumulated in cell nucleus, respectively, so that the NT-nanoprobes showed better cell PPT efficiency than MT-nanoprobes. Cells have been found maintained their structure, and the SERS nanoprobes in the organelles have not been degraded after 24 h incubation. Meanwhile, we also performed the incubation of no-targeted nanoprobes with HeLa cells for 24 h and found by Bio-TEM that the no-targeted CDs-Ag/Au NSs was sparsely
It is known that normal cells usually have higher heat resistance than cancerous cells, but the underlying molecular stress response and mechanisms are still unclear and need to be explored. To this end, nucleus selected-area SERS detection for cancerous and normal cells during PPT process was performed, with NT-nanoprobes as nucleus-targeted SERS substrates. Firstly, to examine the possible cytoplasmic proteins adsorption effect on SERS detections, proteins in the endochylema were separated from HeLa and L929 cells and mixed with the CDs-Ag/Au NSs (0.36 nM) for different times and then tested through SERS. The results (Figure S17 b) manifested a negligible effect of cytoplasmic proteins on SERS detection since there was no observable background signal which may obstruct the SERS signal from two organelles collected. We also found that the CDs-Ag/Au NSs dispersed well in solution (0.36 nM) after mixing with cytoplasmic protein extracts (Figure S17a). We selected the cancerous HeLa cell and normal L929 cell as the models. We tested first the initial SERS spectra of PEGCDs-Ag/Au NSs and NT-nanoprobes, respectively, and also the cells lines incubated with NT-nanoprobes, before the PPT treatment. As shown in Figure S18, the NT-nanoprobes used have no interference to the nucleus SERS detection. Moreover, the SERS spectra of HeLa and L929 cells treated with notargeted nanoprobes for 24 h were also investigated. As shown in Figure S19, the SERS intensity of cell lines (L929 and HeLa) incubated with no-targeting nanoprobes was found much weaker than that obtained with the organelle-targeting nanoprobes, as the no-targeting nanoprobes were mainly endocytosed by lysosomes and were monodisperse within lysosomes, as seen in Figure S16. Therefore, non-targeted nanoprobes have negligible effect on our experiments to obtain organelle-targeting SERS spectra. For better clarification, the main vibrations in the mean SERS spectra of nucleus, which are mainly attributed to the specific vibrations corresponding to lipid, DNA and protein, have been assigned in Table S1. Figure 7b shows the time-dependent SERS spectra of the nucleus collected from a single HeLa cell in the
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Figure 7. (a) and (d) the Bright field images of Hela and L929 cells incubated with NT-nanoprobes for 24 h. The scale bar: 5 μm. Nucleus site-specific time-dependent SERS spectra collected from a single HeLa (b) and L929 cells (e) under the NIR 808nm laser exposure at 2 W/cm2 for 0, 1, 3, 5 min, respectively. (c) and (f) SERS intensity variations of related Raman bands for HeLa (583, 766,1007, 1105, 1240 cm-1) and L929 cells (560, 798, and 1525 cm-1) with the different irradiation time under the PPT process. All the SERS spectrum were averaged from ten individual cells and three locations of each cell recorded before and after heating.
PPT (from living to dead). The Raman bands at 491cm-1 And 627 cm-1 were assigned to the disulfide (S-S) and the C-S bonds of the sulfur-containing amino acids in cellular proteins surroundings on the NT-nanoprobes.24, 25As seen from the time-dependent SERS spectra the vibrations corresponding for C-S bond appeared a red-shift of ~ 4 nm due to the translating of torsional vibration of C-S bond to the tans vibration. Such small changes could be a reflection of the interactions between proteins and the nanoprobes as well as their conformational modifications in the dihedral angle of the C−S−S−C bonds. 26 The band at 583 cm-1 could be attributed to the tryptophan (Trp) residues which plays an important role in protein folding (due to its largest nonpolar surface area) and is sensitive to the strength of the van der Waals, hydrogen bonding, and π−π interactions with surrounding amino acid residues.27 In the PPT process, the intensity of SERS band at 583 cm-1 was gradually increased (Figure 7c), which indicated that the hyperthermia induced conformational change of cellular proteins in the PPT process leads to cell death gradually. The Raman band observed at 766 cm-1 in the initial spectra is mainly attributed to the Thymine (T) base of DNA.28 The SERS intensity at 766 cm-1 was decreased with the increase in illumination time, which means that the content of T base was reduced owing to the DNA fragmentation in the PPT process. The phenylalanine (Phe) vibration at around 1000 cm−1 which are attributed to the in-plane CH stretching vibration and side chain vibration was increased in the microenvironment around the nanoparticles during PPT process. 10 The feature band around the 1105 cm-1 in the initial spectrum mainly composed of the C−N vibration of proteins and the C−C stretching vibrations of the long-chain hydrocarbon backbone of the lipids,29 was gradually reduced due to the disrupture of long-chain hydrocarbon backbone of cellular lipids and their conformational change to the all-trans form.17 The amide III vibration of the β-sheet conformation of pro-
teins was grown around 1240 cm-1.30 While fairly strong in the initial SERS spectra, these bands disappeared gradually in intensity, because the β-sheet conformation of cellular proteins was destroyed in the PPT process (in Figure 7c).31 The Raman band at 1340 cm-1 in the initial spectra was attributed to the adenine (A) of DNA. The band appeared a pronounced blue-shift, primarily because the double helix structure of DNA in the nucleus was twisted during laser exposure.32 The above time-dependent SERS spectra reveal that the structures of associated proteins, DNAs and lipids of HeLa cells were destroyed or deformed during the nucleus-targeted PPT process, which ultimately results in hyperthermia-induced cell death. Since normal cells have better heat resistance than cancer cells, we then checked the time evolution of SERS spectra recorded from a single normal L929 cell during the nucleustargeted PPT process, in order to see the difference from the molecular perspective between the normal cell and cancer cell. As shown in Figure 7e, the time-dependent spectra recorded from the nucleus of a L929 cell showed a quite different SERS response during the nucleus-targeted PPT process. The Raman band around 560 cm-1 which is assigned to the carbohydrate, was slightly weaken in intensity because of the energy consumed in this process. Unlike that of HeLa cell, the Trp vibration located at 583 cm-1 has no obvious intensity changes. Meanwhile, the Raman band centered at 1617 cm-1 which is also attributed to the Phe, remained unchanged in intensity. The result strongly implies that the Trp and Phe for cancerous HeLa cells were overexpressed as compared with the normal L929 cells. As shown in Figure 7f, the SERS intensity of the two bands at 798 cm-1 and 1525 cm-1 that belonged to the O-PO and adenine (A) bases of DNA, were slightly increased with time in the PPT process, implying that the double helix structure of DNA was damaged or deformed to a certain extent. To further confirm the conclusion, a normal epithelial cell line, H8 cells, was also selected for the PPT experiments. Firstly,
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Mitochondria SERS Detection for HeLa and L929 Cells with MT-Nanoprobes During PPT Process In addition to the nucleus, mitochondria also play an important role in the process of apoptosis.33, 34 Mitochondria are important energy-producing cellular organelles and are highly sensitive to the heat shock.11, 35 Therefore, we designed the mitochondria targeting probes (MT-nanoprobes) for SERS study of the PPT process and tried to reveal the differences between normal L929 and H8 cells and cancerous HeLa cells. The chemical components of mitochondria mainly include, proteins, lipids, nucleic acids. We tested firstly the initial SERS spectra of PEG-CDs-Ag/Au NSs, MT-nanoprobes, respectively, and also the cells (both HeLa and L929 and cells) treated with MT-nanoprobes, before the PPT treatment. As shown in Figure S22, the MT-nanoprobes used have no interference to the mitochondria SERS detection and therefore the obtained SERS spectra are mainly a reflection of structural evolution of cellular proteins and DNAs. Details on the assignments of the main evolution of the mitochondria sitespecific SERS spectra mitochondria are given in Table S2. Figure 8b&e show the mean SERS spectra from a single HeLa and L929 cells incubated with MT-nanoprobes under the 808 nm laser exposure. Figure 8c shows the intensity changes of three specific Raman bands during the mitochondria-targeted. The intensity of bands centered at 632 and 996 cm -1 which are assigned to the phenylalanine (Phe) of cellular proteins was increased, implying that the Phe metabolism pathway might be significantly perturbed during the 5 min PPT process. In the SERS spectra, we also observed that the intensity of tyrosine （Tyr） at 1183 cm-1 increased with the PPT time, which was a reflection of Tyr production from the phenylalanine hydroxylation in vivo and was sensitive to the side-chain conformational change of proteins.36, 37 The SERS spectra revealed that the conformation of cellular proteins was changed or fractured owing to the temperature increase in the PPT process, which leads to the mitochondrial dysfunction to induce cell apoptosis. Meanwhile, the metabolizing of Phe, and Tyr were markedly increased as deduced from the SERS spectra analyses. Figure 8f shows the bar graphs of intensity variation of the Raman bands at 670 and 1340 cm-1, which were assigned to T and A bases of DNAs, respectively, of the L929 cell, the result of which indicates that the bases may be damaged and the
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we checked the endocytosis effect of H8 cells for SERS nanoprobes and found that the H8 cells own also good endocytosis effect as HeLa cells, as shown in Figure S20a. Similarly, the H8 cells also have higher heat resistance than cancerous cells (Figure S20b). As seen from the mean SERS spectra of nucleus collected from H8 cells (Figure S21, b&c), the contents of Trp (588 cm-1) and Phe (1000 cm-1) were slightly changed. However, the SERS band at 1525 cm-1 was evidently changed with the increasing of PPT time, similar to the case of L929 cells. Comparing SERS Spectra of normal L929 and H8 cells with that of cancerous HeLa cells during the nucleus-targeted PPT process, we found that the structure and conformation of proteins and lipids in or near the nucleus of cancerous cells were more vulnerable to be destructed than normal cells. Moreover, the contents of Trp and Phe of HeLa cells were more evidently increased than L929 and H8 cells during the PPT process. It also worth mentioned that carbohydrate metabolism for normal L929 cells increased in the PPT process.
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Figure 8. (a) and (d) the Bright field images of Hela and L929 cells incubated with MT-nanoprobes for 24h. The scale bar: 5 μm. Mitochondria site-specific time-dependent SERS spectra recorded from a single HeLa (b) and L929 cells (e) under the NIR 808nm laser exposure at 2 W/cm2 for 0, 1, 3, 5 min, respectively. (c) and (f) SERS intensity variations of related Raman bands for HeLa (632, 996 and 1183 cm-1) and L929 cells (670 and 1340 cm-1) with the different irradiation time under the PPT process. All the spectra were averaged from ten individual cells and three locations of each cell before and after PPT process. double helix structure of DNAs was changed into unordered single chain.38 The SERS spectra of mitochondria within H8 cells were also recorded during the PPT process (Figure S23b). As seen from Figure S23c, the SERS intensity at 595 and 1000 cm-1 was slightly increased during the PPT process, the SERS bands of which were assigned to trp and phe. Moreover, the intensity of Raman band at 667 cm-1 and 1340 cm-1 also has emerged the same variation trends as L929 cells. After comparative in-situ SERS analyses of cancerous HeLa cells and normal L929 and H8 cells during the mitochondriatargeted PPT process, we found that the PPT-induced hyperthermia cell death of the cancerous HeLa cells was regulated mainly by cellular proteins, while the cell death of normal L929 and H8 cells were mainly affected by the cellular DNAs. Moreover, the metabolizing of Tyr and Phe for HeLa cells was more evidently increased than normal L929 and H8 cells in the PPT process. All these results indicate the molecular stress response difference in the PPT-induced hyperthermia cell death for the normal L929 and H8 cells and cancerous HeLa cells from the organelles level. As shown in Figure S24, a good repeatability of the organelle-specific SERS detections during the PPT process was also manifested by the repeated tests.
CONCLUSION We designed an organelle-targeting theranostic plasmonic SERS nanoprobe that composed of p-Ag/Au nanoshell and carbon dots (CDs-Ag/Au NS) and used for photothermal treatment of single cell and self-sensing the PPT process in the organelle level (nucleus and mitochondria). The hybrid CDsAg/Au NSs have higher biocompatibility and photothermal conversion efficiency than single p-Ag/Au NSs. We analyzed the mechanism of heat tolerance of normal L929 cells from 7 the organelle level using the in-situ SERS technique.
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The difference in molecular stress response in the PPTinduced hyperthermia cell death process between cancerous HeLa cells and normal L929 and H8 cells has been revealed in-situ by nucleus and mitochondria site-specific and timedependent SERS detection of single cells. Compared the nucleus site-specific SERS analyses of normal L929 and H8 cells to cancerous HeLa cells, the contents of Trp and Phe in HeLa cells were more evidently increased than L929 cells during the PPT-induced cell apoptosis process. From the mitochondria site-specific SERS analyses, we found that the PPT-induced cell apoptosis for HeLa cells may stem from and regulated through cellular proteins, while for normal L929 and H8 cells it seems more related to cellular DNAs. Moreover, the metabolism-related Tyr and Phe of cancerous HeLa cells were more evidently increased than normal L929 and H8 cells. Our results also indicated that the nucleus-targeting is a more effective way than mitochondria-targeting in the PPT process to induce cell apoptosis. SERS monitoring the different cellular molecular stress responses of normal and cancer cells in single cell level and real-time during the PPT process will help understanding the underlying molecular mechanisms between normal and cancer cells involved in photothermal cancer cell death. ASSOCIATED CONTENT Supporting Information This supporting information is available free of charge via the Internet at http://pubs.acs.org.” Characterizations of materials and SERS nanoprobes, Photothermal effect of nanoprobes, Dark-field imaging of organelles probes in cells, Assignment of SERS spectra, The SERS spectra of organelles in H8 cells, The repeatability of SERS spectra. AUTHOR INFORMATION Corresponding Author *
[email protected] Notes Authors declare no competing financial interest ACKNOWLEDGMENT This work was supported by the National Key Research and Development Program of China (Grant No. 2016YFA0201300), the National Natural Science Foundation of China (grant Nos.21475125 and 21675146), and the Instrument Developing Project of the Chinese Academy of Sciences (Grant No. YZ201666).
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