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Research Article Cite This: ACS Appl. Mater. Interfaces 2018, 10, 7910−7918

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Organelle-Targeting Gold Nanorods for Macromolecular Profiling of Subcellular Organelles and Enhanced Cancer Cell Killing Yanting Shen,† Lijia Liang,† Shuqin Zhang,‡ Dianshuai Huang,‡ Rong Deng,† Jing Zhang,† Huixin Qu,‡ Shuping Xu,*,† Chongyang Liang,*,† and Weiqing Xu† †

State Key Laboratory of Supramolecular Structure and Materials, Institute of Theoretical Chemistry, Jilin University, Changchun 130012, China ‡ Institute of Frontier Medical Science, Jilin University, Changchun 130021, People’s Republic of China S Supporting Information *

ABSTRACT: Subcellular organelles, for example, nucleus, mitochondria, and lysosome, are the vital organelles with responsibilities that maintain cell operation and metabolism. Owing to their roles in energy production and programmed cell death, these organelles have become prime therapeutic targets in different diseases and states. In this study, biocompatible, organelle-targeting nanoprobes were developed by modifying gold nanorods (AuNRs) with specific targeting peptides. These nanoprobes were employed to directly profile subcellular biomolecules and vital organelles by surfaceenhanced Raman scattering (SERS) spectroscopy. Macromolecular spectral profiles of subcellular organelles were achieved and compared. Further, these organelle-targeting AuNRs were used for the photothermal treatment of cancer cells (HepG2, HeLa, and MCF-7 cell lines). The cell viability assays show that the nucleus- and mitochondria-targeting AuNRs provide higher photothermal efficiencies under an 808 nm laser relative to the lysosome-targeting ones. This study makes critical insights into the spectral profiles of subcellular organelles and also inspires people in the development of high-efficacy cancer therapeutic strategies by subcellular organelle-targeting drugs. KEYWORDS: in situ analysis, organelle-targeting, SERS, PTT, nucleus, mitochondria, lysosome

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INTRODUCTION As the vital organelles of cells, the nucleus, mitochondria, and lysosome can participate in many important physiological and pathological processes including cell proliferation, organism metabolism, and intracellular transportation and play essential roles in regulating cellular biological functions.1,2 The dysfunction of these organelles would lead to a variety of aberrant regulations and multiple diseases. For example, any turbulence to the nucleus may lead to the abnormal regulation of cell activity and even cause the programmed death of the cell.3 Inflammation, tumor, silicosis, and various lysosomal storage diseases are related to lysosomal dysfunction strictly. Besides, deficiency in mitochondrial function would accompany with multifarious pathological symptoms, such as the cardiovascular, neurodegenerative diseases and cancers as well.1,4 Therefore, the concept of organelle and subcellular targeting has gained more and more attention in the near future. To explore the molecular mechanism and the specific functions of the organelles, a variety of organelle-targeting molecules and ligands were developed.5−7 For example, importing exotic materials to lysosome seems more accessible because of the superior accumulation effect of lysosomes.8 Small molecules (such as N,N-dimethyl ethylenediamine) can © 2018 American Chemical Society

also help nanoparticles to achieve a precisely targeting feature based on the permeation and accumulation of the monobasic amine.9 For nucleus targeting, small molecules can enter and escape from the nuclear pore complex (NPC) freely in a passive way because the NPCs are equipped with 9 nm channels. However, macromolecules with a diameter of over 9 nm or with a weight of above 45 kDa require the assistance of the nuclear location sequence (NLS) to the target nucleus, which is an active transport way.10 Similar to nucleus targeting, transporting nanoparticles to the mitochondria is not easy. The most common-used ligands for targeting the mitochondria are the lipophilic cations and the mitochondrial targeting sequence peptides.11−13 On the basis of the targeting strategies, more and more organelle-targeting nanoplatforms were developed to detect subcellular analytes and to monitor intracellular environments. As a powerful analytical tool, surface-enhanced Raman scattering (SERS) spectroscopy can not only provide detailed fingerprint spectral information but also combine the advantages of high sensitivity, showing notable superiority in Received: January 23, 2018 Accepted: February 13, 2018 Published: February 13, 2018 7910

DOI: 10.1021/acsami.8b01320 ACS Appl. Mater. Interfaces 2018, 10, 7910−7918

Research Article

ACS Applied Materials & Interfaces

Scheme 1. Schematic Illustration of the SERS Detections and the NIR Photothermal Therapy of Cancer Cells with the AuNRBased Subcellular Organelle-Targeting Nanoprobes; ①−④; Preparation of the Organelle-Targeting Nanoprobes, Cell Culture with the Organelle-Targeting Nanoprobes, the Organelle-Selective SERS Detection, and Organelle-Targeting PTT

designed nanoprobes toward their organelles were achieved by superresolution fluorescence microscopy and biotransmission electron microscopy (Bio-TEM), which is of great importance in accurately obtaining the molecular information of organelles. (2) The inherent macromolecular Raman profiles of the nucleus, mitochondria, and lysosome were obtained simultaneously and compared for the first time, which are helpful in obtaining infromation on the cells at their molecular level and creating comprehensive reference maps of cancer cells. (3) The PTT efficacies of different organelle-targeting platforms were investigated under the same condition. Nuclear-targeting and mitochondria-targeting probes possess a relatively higher killing ability, which plays a vital role in the design of targeting antitumor drugs.

the analysis of biosamples, such as cells and tissues, as well as living bodies.14,15 Recently, efforts have been made for obtaining information on the subcellular organelles at the molecular level.9,16 For example, Lim and co-workers designed three SERS-tag nanoprobes with nucleus-targeting, mitochondria-targeting, and cytoplasm-targeting functions for subcellular imaging.17 Hu et al. also presented single SERS probes (membrane- or nucleus-targeting nanoprobes) with either labeling or label-free strategies to achieve the targeted SERS images (nucleus and cell membranes) by a confocal Raman system.18 However, owing to an indirect exploration of their method, very little information on cell organelles can be achieved. Also, these studies care less about the precise locations of these targeting nanoprobes. Thus, the obtained label-free SERS spectra may lack accuracy in the interested detection positions. Therefore, the direct and precise detections and analysis of the biomolecular information from specific organelles are still challenging, which is of great significance in expounding molecular mechanisms during critical physiological and pathological processes. Photothermal therapy (PTT) is a widely used therapeutic approach that converts light energy into heat to generate hyperthermia and further cause cell death. As one of the critical treatments, metal nanoparticles were widely adopted as a PTTsensitive agent to improve the light collection efficiency due to the plasmonic property of metal nanomaterials.18−21 Recently, PTT nanomaterials combined with organelle-targeting agents, for example, nucleus, have been extensively exploited to obtain the maximum therapeutic efficacy.22−26 However, no one compares the PTT nanotherapy effect among different organelle-targeting strategies. In this paper, with the aim of obtaining precise bimolecular information on subcellular organelles and exploring the different efficacy among different organelle-targeting platforms in the PTT treatment toward cancer cells, we designed biocompatible and organelle-targeting nanoprobes based on the gold nanorods (AuNRs) modified with specific targeting peptides to target the nucleus, mitochondria, and lysosome. The profound plasmonic effects of these targeting nanoprobes are found in two aspects: profiling the molecular information of specific organelles and performing PTT to induce cell death. Valuable insights of our work can be outlined as (1) coincident experimental lines of evidence on the successful targeting of our

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EXPERIMENTAL SECTION

Preparation of the AuNR-Based Nanoprobes. AuNRs were synthesized using a seed growth method,27 and the achieved AuNR has a length of 66 nm and a width of 30 nm. The detailed procedures are described in the Supporting Information. Transmission electron microscopy (TEM), ultraviolet−visible (UV−vis) spectroscopy, and dynamic light scattering (DLS) spectroscopy were used to measure the size, morphology, plasmonic property, and zeta potential of the obtained AuNRs. Then, the AuNRs were modified with methoxy poly(ethylene glycol)-thiol (mPEG-SH) with MW = 5000 to inhibit aggregation and improve biocompatibility.28 The number of the mPEG-SH on each particle is about 1000. Then, the lysosome-, mitochondria-, and nucleus-targeting (ALT, AMT, and ANT) nanoprobes were obtained by modifying RGD, RGD/MLS, and RGD/NLS, respectively, on the surface of PEGylated AuNRs (1 in Scheme 1) via the covalent linking between gold and the thiol group of cysteine (bold in the peptide sequence of mitochondrial localization sequence MLS), NLS, and RGD). The mixture was incubated overnight at room temperature. In addition, the molar ratio of MLS (or NLS) with AuNRs is 104:1, whereas the molar ratio of RGD is 103:1 according to the literature studies.29,30 Following a step of centrifugation (4000 rpm, 8 min), the cleaned AuNRs were redispersed in water for use. The AuNR concentration was eventually evaluated by the absorbance value by using UV−vis spectroscopy. Observation of Intracellular Distribution of Targeting Nanoprobes by Super-resolution Confocal Fluorescence Microscopy. To obtain the confocal fluorescent images of nanoprobes, the fluorescein isothiocyanate (FITC)-labeled targeting nanoprobes were prepared first, using our previously published method.28 The FITC-labeled nanoprobes (0.1 nM) were cultured with cancer cells for 24 h, and then the mitochondria, nucleus, and lysosome of the 7911


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Figure 1. (a) TEM image of the synthesized AuNRs. (b,c) UV−vis spectra and zeta potentials of the AuNRs, AMT, ANT, and ALT nanoprobes, respectively. (d) Cell viabilities of HepG2 cells after they were incubated with different nanoprobes with a concentration of 0.1 nM for 24 h. (e) Temperature elevation profiles of water and AMT-, ANT-, and ALT-contained solutions (2.0 nM) after they were irradiated with an 808 nm laser under a power density of 4.0 W/cm2 for 10 min. (f) UV−vis spectra of the AMT, ANT, and ALT nanoprobes before (solid) and after 6-month storage (dash). For the WST-1 assay, 104 cells were seeded in 96-well plates and cultured with AMT, ANT, and ALT with a concentration of 0.2 nM for 24 h. Finally, the cell viabilities without and with irradiation were measured by a standard WST-1 assay. Statistical Analysis. According to standard deviation, a two-paired t-test was conducted for statistical analysis. *** means significantly different with the p-value < 0.001. ** means significantly different with the p-value < 0.01. * means significantly different with the p-value < 0.05.

cells were stained with commercial organelle dyes for 15 min. Then, we used super-resolution fluorescence microscopy and analysis software to record and deal with the fluorescent images of an individual cell, using a method we developed in a previously published paper.28 The nucleus (blue, Hoechst 33342, excited by a 405 nm laser), mitochondria (red, MitoTracker red CMXRos, excited by a 543 nm laser), and lysosome (red, LysoTracker red DND-99, excited by a 543 nm laser) will give colors on the fluorescent images. Bio-TEM Imaging of Cells. After HepG2 cells were cultured with 0.1 nM of AMT, ANT, and ALT nanoprobes for 24 h incubation, the cells were harvested and fixed with 0.25% trypsin and glutaraldehyde at 4 °C. Afterward, the cells were washed two times with phosphatebuffered saline (PBS) and then dehydrated with increasing ethanol gradients. Then, they were treated with propylene oxide and embedded in Epon. Finally, the cells were cut into thick sections of about 80 nm and placed on a carbon film supported by the copper grids, followed by a staining procedure with uranyl acetate and lead citrate for Bio-TEM observation. SERS Detections. The confocal Raman system (LabRAM Aramis, Horiba Jobin Yvon) was used for SERS detections of the mitochondria, nucleus, and lysosome. The laser is a He−Ne laser (λex = 632.8 nm), and the power is about 7.1 mW. After the cells were cultured with nanoprobes for 24 h, they were fixed on coverslips for SERS detections. Experimental conditions, t = 10 s and accumulations = 2 times, were set. Photothermal Performance of the AMT, ANT, and ALT. PTT efficacies of the AuNRs, AMT, ANT, and ALT were investigated by irradiating a 2 mL quartz plate containing 1.0 mL of AuNRs (or AMT, ANT, or ALT nanoprobes) with a concentration of 2.0 nM under a laser (808 nm, 4.0 W/cm2) for 10 min irradiation. The temperature of the irradiated aqueous dispersion was recorded on a thermocouple combined with a digital temperature controller. Then, the cells cultured in glass-bottom dishes (φ 30 mm, NEST) were incubated with the AMT, ANT, and ALT nanoprobes for 24 h, and the concentration is 0.2 nM. After irradiation for 5 min, the culture medium was removed and 1.0 mL of PBS mixed with 2.0 μM of propidium iodide (PI) and 3.0 μM of calcein-AM were added. After incubation for 30 min, the cells were washed two times with PBS and further imaged with the confocal fluorescence microscope (Olympus). Calcein-AM (green) and PI (red) were used to stain living and dead cells, respectively.

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RESULTS AND DISCUSSION Synthesis and Characteristics of Organelle-Targeting Nanoprobes. To in situ obtain the SERS spectra of organelles, the plasmon-based nanoprobes should be firstly transferred to the targeting locations, where the Raman signal of adjacent molecules around nanoprobes can be significantly enhanced million times under the laser.30 Here, AuNRs work for a Raman-signal enhancement substrate. The detailed preparation procedures of AuNRs can be found in the experimental sections. The achieved AuNRs are observed to be relatively monodispersed (Figure 1a) with the localized surface plasmon resonance (LSPR) peak located at 629 nm (Figure 1b), which are utilized for the preparation of organelle-targeting nanoprobes. To improve the biocompatibility, reduce the toxicity, and prevent the absorption of the proteins in the culture medium to AuNRs, the AuNRs were coated with mPEG-SH first. To achieve organelle-targeting nanoprobes and further improve their biocompatibility, cell membrane penetration peptides (RGD, CGPDGRDGRDGRDGR)31−38 that have the cancer cell-targeting activity were fixed on the PEG-coated AuNRs, which increases the cellular uptake by targeting αvβ6 as well as other αv integrins on the cell surface. We also compared the cell uptake of the naked AuNRs and the RGD-modified AuNRs by dark-field imaging (Figure S1), and the results indicate that the coating of RGD is helpful for the internalization of nanoparticles. The obtained AuNRs−PEG−RGD complex (without further surface modification) was regarded as the 7912


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Figure 2. Dark-field/fluorescent images of HepG2 cells treated with our prepared organelle-targeting probes for 4, 8, and 24 h. (a−c) Cells were cultured with ANT, AMT, and ALT, while the nucleus, mitochondria, and lysosome were stained with blue, red, and red with the commercial dyes, respectively. The scale bar is 10 μm.

prolonged to 24 h, most of the nanoprobes have been delivered to the near regions of the nuclei, as shown in Figure 2a3 (an enlarged image showing nanoprobes in a single cell is presented in Figure S2). For AMT, we can observe that when cultured with cells for 4 h, the probes can be selectively accumulated in the mitochondria, as shown in Figure 2b1. When the culture time prolonged from 4 to 24 h, these nanoprobes still remained in the mitochondria without fleeing. Figure 2b2,b3 shows no apparent difference in the AMT number, indicating that the saturation of the targeting nanoprobes can be achieved within 4 h. The uptake of the ALT is similar to that of the AMT, as shown in Figure 2c1−c3. Therefore, we consider that the appropriate culture time for the ANT is 24 h, whereas 4 h is enough for mitochondria- and lysosome-targeting probes. To ensure the consistency of the culture time, we used cells incubated with nanoprobes for 24 h in the next experiments. Next, the super-resolution fluorescence imaging was performed to investigate the precise locations of the targeting nanoprobes. To trace the nanoprobes, the FITC-labeled organelle-targeting nanoprobes (AMT-FITC and ANT-FITC) were first prepared by immobilizing the FITC-labeled MLS and NLS on the surface of AuNRs. The ALT-FITC was achieved through the chemical reaction of FITC with PEG-NH2 as described in the experimental section. After the nanoprobes were cultured with cells for 24 h, the nucleus, mitochondria, and lysosome were stained with dyes in blue, red, and red, respectively, to colocalize with the FITC-labeled nanoprobes (green). Figure 3A1−A3 illustrates the mitochondria, nucleus, and lysosome treated with the FITC-labeled targeting nanoprobes for 24 h. B1−B3 are the simulation images of a single HepG2 cell A1−A3, handled and analyzed via Imaris X 64 software from Bitplane (Zurich, Switzerland). The magnified images of B1−B3 are shown in C1−C3. From B2 and C2, we can find that the nanoprobes have arrived in the nucleus, whereas some of them were stopped by the nuclear membrane, which is also consistent with our previous work.41 For mitochondria (B1,C1) and lysosome (B3,C3), owing to their small sizes, the locations of nanoprobes and organelles are overlapped (as highlighted by red circles). We can find some free organelles that are not occupied by nanoprobes. However, it is hard to find free nanoprobes. These imaging data indicate that our

ALT nanoprobe because the lysosome, as one of the vesicles, is the site where most exterior NPs (such as AuNPs and AuNRs) had superior accumulation after internalization.39,40 To prepare the ANL and AML nanoprobes, we further enriched the AuNRs−PEG−RGD particles with the nuclear localization signal peptides (NLS, GGVKRKKKPGGC)3 and mitochondria localization peptides (MLS, MLALLGWWWFFSRKKC) which can transport the molecular cargo to the near nuclear region and mitochondria specifically and precisely according to the published paper.17 These successful conjunctions were preliminarily investigated by UV−vis spectroscopy (Figure 1b) and DLS (Figure 1c). Besides, the cell viability of HepG2 cells treated with nanoprobes was illustrated by the WST-1 assay. From Figure 1d, the cell viabilities of cells after incubation with the AMT, ANT, and ALT are 92.5 ± 3.5, 89.1 ± 4.2, and 95.5 ± 0.7%, respectively. The results indicate that these nanoprobes have minor damage on cells after co-incubation with cells for 24 h at our experimental concentration (0.1 nM). Furthermore, the stabilities of the nanoprobes were testified by UV−vis spectroscopy (Figure 1f). Generally, the broadening or the red shift of the LSPR band would be observed with the storage time, which resulted from the aggregation of the metal nanoparticles. However, in the present study, these two phenomena were not observed except for a little decrease of the LSPR band due to the sedimentation of AuNRs, indicating that our nanoprobes have relatively excellent stability for at least 6 months. Precise Locations of the AMT, ANT, and ALT in Living Cells. As we used in previous work,28 several imaging methods were used to confirm the targeting ability of the nanoprobes and to precisely identify the locations of the AMT, ANT, and ALT in cells, such as dark-field/fluorescence microscopy, the super-resolution imaging system, and Bio-TEM. First, the culture time of cells incubated with the targeting nanoprobes was optimized. With the assistance of the self-developed darkfield/fluorescence co-imaging apparatus, the AuNRs provide brighter scattering light under the dark-field imaging, while the cell organelles were stained with their specific tracers, displaying bright fluorescence (Figure 2). With the increase of the incubation time, more and more nucleus-targeting probes were observed in or around the nuclei. When the incubation time 7913


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molecular information on the organelles in HepG2 cells. To exclude the interference of the modified peptides on the AuNRs’ surface, we first measured and compared the SERS spectra of the three targeting nanoprobes using a confocal Raman microspectrometer with a 632.8 nm excitation wavelength as shown in Figure S5. When the concentration is 0.1 nM (experimental condition), there are no apparent peaks in their SERS spectrum. Considering that the AuNRs in the specific organelles might be in an aggregation state, we further recorded the SERS spectra of the AuNRs under a higher concentration, 4.0 nM. Thus, we obtained many identifiable peaks of these targeting probes, which reveal the vibration information of the targeting peptides that had been modified on AuNRs. Unexpectedly, we did not find these peaks when we measured the intracellular SERS spectra (also enhanced with these targeting AuNRs). It might be because these targeting peptides were digested to some extent after they were exposed to the enzymes within the 24 h culture time, or the intracellular components have stronger adsorption effect, leading to the desorption of these targeting peptides from the surface of the AuNRs ultimately. Therefore, we think that there is almost no distinct interference in the SERS detections of the intracellular components. To obtain accurate spectral signals, we collected the SERS spectra from different cells and different points of one individual cell (several of them are shown in Figure S6a−c). We can find that the SERS spectra of the nucleus and mitochondria have better reproducibility compared with that of the lysosome. The relatively weak reproducibility of the lysosome may result from the fusion of the lysosome and endosome. It is well-known that once internalized, nanoparticles will be transported to early endosomes, then later to endosomes, and finally, the endosomes will fuse. It is reported that the SERS spectra of these endosomes at different stages showed slight differences.39 Therefore, the fusion at different degrees among lysosomes may lead to a difference in the SERS spectral behaviors. To achieve a better identification and analysis of molecular compositions of specific organelles, the mean SERS spectra from ten cells of three organelles were obtained and analyzed as shown in Figure 4, and the complete band assignments are listed in Table 1. Contributions from amino acids, protein backbone, nucleotides, lipids, and carbohydrates can be identified by the SERS spectra. The nucleus contains some characteristic peaks of DNA, adenine bases at 733 and 1333 cm−1, 1575 cm−1 (guanine and adenine),42 especially the information of bases. The DNA skeleton vibration information was detected, such as O−P−O stretching at 912 cm−1 and deoxyribose C−O stretching at 1027 and 1462 cm−1.43 Similar bands were found in other spectra. On the other hand, as another main component of the nucleus, the conformational information of proteins was given. The C−C twisting and ring breathing of phenylalanine is located at 626 and 999 cm−1.44 Peaks at 655, 694, 818, and 852 cm−1 are assigned to C−S stretching of cysteine, leucine, and tryptophan; and ring breathing of tyrosine,45 the C−N and C− C stretching; and CH2 wagging at 1073, 1549, and 1370 cm−1.46 In addition, some vibrations relevant to carbohydrates at low wavenumbers were explored. The spectra of the mitochondria are mainly attributed to the contribution of protein, lipid, and a few bases. For example, 999, 1073, 1137, and 1176 cm−1 vibrations previously were assigned to the ring breathing of

Figure 3. (A1−A3) Confocal fluorescent images of the HepG2 cell treated with FITC-labeled nanoprobes (green) and the mitochondria, nucleus, and lysosome were stained, which give colors of dark yellow, blue, and dark yellow, respectively. (B1−B3) Panorama simulation images of a single cell (A1−A3). (C1−C3) Local amplification images of the cell in A2,B2, and C2. The red circles represent the nanoprobes that arrive in their target organelles. (D−F) Bio-TEM images of the HepG2 cells after incubation with 0.1 nM AMT, ANT, and ALT for 24 h. The red, white, and yellow arrows represent M, N, and L, respectively. C = cytoplasm, N = nucleoplasm, L = lysosome, and M = mitochondria.

designed nanoprobes can be successfully delivered to their specific organelles. Moreover, to have a clear insight of the targeting performance of the nanoprobes in HepG2 cells, Bio-TEM was employed and the results are shown in Figure 3D−F. We can obviously find that most of the AMT, ANT, and ATL nanoprobes have arrived in their targeted organelles and formed nanoaggregates because of the successful modification of the targeting peptides. The results also demonstrate that even after being cultured with cells for 24 h, these nanoprobes still remain in the cells rather than undergoing degradation. More than that, we also testified the distributions of the nanoprobes in cells by confocal fluorescence spectroscopy (Figure S3 in Supporting Information), and very similar consequences have also been acquired, confirming the targeting functions of our designed nanoprobes. As a control, we also obtain Bio-TEM images of HepG2 cells without any nanoprobes to see the structure of subcellular organelles. We can find that the nucleoplasm has a clear and high contrast boundary, whereas the lysosomes (yellow arrows point out) can also be identified as dark circles with a smaller size of about 300 nm. As to mitochondria, they display brighter contrast domains (red arrows point out). With the internalization of the AuNRs that had been decorated with the targeting peptides, we can clearly observe that these AuNRs have been involved in their target organelles (B−D in Figure S4), confirming the targeting feature of our designed nanoprobes. In Situ SERS Spectra of the Organelles. On the basis of the targeting organelle nanoprobes, we obtained macro7914


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phenylalanine,29 C−N stretching vibration, C−N backbone, and C−H bending, respectively. The characteristic peaks of amide III are present at 1278 (α-helix) and 1218 cm−1 (βsheet).44 Peaks located in 1547 and 1583 cm−1 are assigned to the N−H deformation and tryptophan, respectively. A few assignments come from lipids and bases, which are located at 1137 and 1027 cm−1. The SERS information of lysosomes is relatively less than those on the nucleus and mitochondria, containing 999, 1027, 1073, 1162, 1279, 1317, and 1563 cm−1, from the main contents of proteins or carbohydrates. The assignment of 999, 1027, and 1073 cm−1 was same with the ascriptions of the other two organelles. The peaks at 1162, 1279, and 1317 cm−1 are assigned to the C−C stretching, CH2 skeletal stretching twisting, and twisting of proteins,44 whereas 1563 cm−1 is assigned to tryptophan. Finally, we directly compared and analyzed the distinct differences of the SERS spectra of specific organelles (Figure 4) based on their roles in cell physical and physiological processes. These spectra have been normalized with the band intensity at 999 cm−1. By comparison, we can conclude that the nucleus, mitochondria, and lysosome share same bands at 999, 1027, and 1073 cm−1 (green bar in Figure 4). These data agree that protein, lipid, and carbohydrates are the main components of cells and organelles. Figure 4 also indicates many differences, mainly in the regions of 400−500 cm−1, 733, 1160−1180, 1333−1371, and 1570−1600 cm−1. Differences among the three organelles are marked in orange. Among them, 733, 1333, 1576, and 1583 cm−1 correspond to the various vibrational modes of the DNA. The first two are assigned to adenine (A), whereas the latter two belong guanine and adenine (G, A). As the genetic control center, almost all DNA sequences exist in the nucleus and no or few in lysosomes and mitochondria. The bands in 1160−1180 cm−1 belong to protein or lipid. Their differences may come from the different kinds of contents in these specific organelles. Interestingly, the SERS spectra of C− O stretching of DNA at 1027 cm−1 were observed in the lysosome. However, as we all know, the lysosome contains no genetic material such as DNA or base. Therefore, we suppose that the abnormal phenomenon may result from the cell autophagy. In addition, in this pathway, the DNA and RNA could be transported to lysosomes and were degraded, which have been investigated by Fujiwara and co-workers.47,48 Photothermal Therapy with the Organelle-Targeting Nanoprobes. AuNRs have been widely used in PTT applications because of their advantages of prominent biocompatibility and favorable LSPR properties of AuNPs (Figure S7).49 Recently, subcellular targeting PTT platforms or that combined with other therapeutic strategies on the basis of the AuNRs have been developed for improving the treatment efficiency.30,50,51 In these studies, the AuNRs was usually modified with the targeting peptide for targeting a specific organelle to enhance cell death. However, the comparative study of the photothermal damage of cancer cells with AuNRs located in different organelles is not reported yet. Here, we developed AMT, ANT, and ALT nanoprobes to target the mitochondria, nucleus, and lysosome and compared the photothermal damage with different subcellular locations. We first tested the NIR laser illumination-induced temperature elevation profile of the probe nanoparticles in solution. Figure 1e shows the temperature evolution of water, AMT, ANT, and ALT, respectively, with the same concentration (2.0 nM) under the laser irradiation for 10 min (4.0 W/cm2). The results demonstrate that water shows almost no photothermal

Figure 4. Top panel: Right-field images of the organelles, and the red circles represent some of the detection spots. The scale bars indicate 20 μm. Bottom panel: The mean SERS spectra of the nucleus, mitochondria, and lysosome with the assistance of 0.1 nM of AMT, ANT, and ALT nanoprobes, respectively. These spectra were normalized according to the 999 cm−1 band. The green and pink band indicate the common and different ranges. λex = 632.8 nm, t = 10 s, accumulations = 2 times, and laser power = 7.1 mW.

Table 1. Assignments of the SERS Peaks Raman shift (cm−1) nucleus

mitochondria

lysosome

417 478 626 645 655 694 733 759 818 852 912 961 999 1027 1073

999 1027 1073 1137

999 1027 1073 1162

1176 1218 1245 1278

1279 1317

1333 1354 1370

assignment GluA GlcNac C−C twisting C−S stretching C−S twisting Leu A Trp, T Trp Tyr O−P−O stretching random coil Phe ring breathing GlcNac, Glc,GluA, C−O stretching C−C twisting C−N stretching C−N backbone C−C stretching Tyr, C−H bend amide III (β-sheet) amide III amide III (α-helix) −CH2 twisting A Trp Trp CH2 wagging

1413 1465

1549

1482 1507 1549 1563

1575 1583 1641

R amide II, C−N protein C−C stretching Trp G, A G, A, Trp coil conformation of polypeptides 7915


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Figure 5. The cell viability of HepG2 (A), HeLa (B), and MCF-7 (C) cells cultured with AMT, ANT, and ALT nanoprobes before and after laser irradiation. (D) HepG2 cells stained with calcein-AM and PI after being cultured with or without targeting nanoprobes and with or without the irradiation for 5 min (4.0 W/cm2). The scale bar is 100 μm.

may produce higher local heat effect in the process of PTT.52 The latter is on the basis of the structure and the roles of different organelles. As we all know, the nucleus is the control center of a cell’s genetic material, which has been regarded as the useful target of cancer therapy, and the PTT mechanism is that the structure and amount of proteins such as replication protein A 70 are related to DNA replication, repair, and recombination. If these functions were influenced and inhibited by PTT, irreversible cell apoptosis under mild hyperthermia would happen.26,30 Therefore, the cancer cell death treated with ANT is higher than the cells treated with ALT without nucleus targeting. For cells treated with AMT, the higher PTT effect might be attributed to the destruction of mitochondria.53,54 Mitochondria is the gateway of cell apoptosis. Many apoptosis models have been built based on the destruction of mitochondria. In present study, the AMT nanoprobes can enter into mitochondria; the local hyperthermia produced from AMT nanoprobes can directly destroy the mitochondria and release proteins related to apoptosis. In summary, the reason why AMT and ANT nanoprobes can induce more efficient cell death may be derived from the accumulation of probes in the organelle, which directly influence the function and structure and further lead to the cell death. However, more details about molecular information during PTT are still unclear and need further exploration in the future.

response. When these nanoprobes have been irradiated for 10 min, the temperatures of the solutions containing different targeting nanoprobes arrive in about 55 °C, indicating a high enough temperature that fulfills the requirement of photothermal ablation of cancer cells. Then, HepG2 cells were cultured with AMT, ANT, and ALT nanoprobes for 24 h with 0.2 nM concentration, and then they were irradiated by an 808 laser for 5 min. To distinguish live and dead cells visually, the cells after were incubated with 3.0 μM calcein-AM (green, live cell dye) and 2.0 μM PI (red, dead cell dye), respectively. Figure 5D shows the fluorescent images of these treated cells. For a control sample that contains no nanoprobes, the laser irradiation has almost no damage on the HepG2 cells. However, for the cells treated with AMT, ANT, and ALT nanoprobes, after laser irradiation, cell death percent significantly increases (P < 0.001, Figure 5A), especially the cells treated with AMT and ANT nanoprobes, suggesting that all these three targeting probes are efficient photothermal agents that can lead to the death of the cancer cell. To further compare the PTT efficiency of AMT, ANT, and ALT nanoprobes, the WST-1 assay was performed as illustrated in Figure 5C. The cell death proportions are 48.4, 64.2 and 28.7% for AMT, ANT, and ALT nanoprobes, respectively. Next, to confirm the general applicability of the nanoprobes, the PTT efficacies of another two types of cell lines, HeLa and MCF-7, were also tested, and the results are shown in Figure 5B,C. The PTT-induced cell death with AMT, ANT, and ALT nanoprobes are 46.4, 48.8, and 32.3% for HeLa cells and 22.9, 23.3, and 7.0% for MCF-7, respectively. The above data reveal that the AMT and ANT are more efficient in cancer cell killing compared with the ALT nanoprobes. We speculate that the different cancer therapeutic effects may include two aspects: the different aggregation states and the different functions of the organelles play a vital role in the progress of cell physiological and pathological processes. The former can be concluded by the imaging results in Figures 4A, 2, and S4. From these images, we can find that the AMT and ANT nanoprobes can form more aggregates in cells compared with ALT nanoprobes. Moreover, the aggregates

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CONCLUSIONS In summary, ANT, AMT, and ALT nanoprobes were designed by assembling the polymer (PEG) and biocompatible, targeting peptides (NLS, MLS, and RGD) on the surface of the AuNRs, which possess excellent biological compatibility, specific targeting ability, as well as remarkable SERS enhancement, and PTT effect. Super-resolution imaging and Bio-TEM characterizations prove congruously the targeting ability of these nanoprobes. Under the assistance of these AuNR-based nanoprobes, the macromolecular profiles of the three subcellular organelles were partly profiled, compared for the first time, and assigned according to their structure and function in cell activities and physical processes. Moreover, the 7916


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Li from Key Laboratory of Pathobiology, Ministry of Education, Jilin Univ. for TEM analysis.

PTT treatment for the three cancer cells with different subcellular targeting nanoprobes was investigated, demonstrating that mitochondria- and nucleus-targeting probes have stronger cancer-killing effects. We also speculate that the differences among the three nanoprobes are associated with the aggregate station and the direct damage for specific organelle during the PTT process, which further enhance the cell death compared with the ALT nanoprobe. Our study achieves biomolecular information and compositions of three specific organelles and provides a useful tool for the early diagnosis, targeted drug action mechanisms, and treatment progresses of diseases at subcellular molecular levels. Moreover, the PTT study makes a primary assessment on the difference of subcellular organelle-targeting nanoplatforms, which will be a reference for exploring novel targeting therapy strategies.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.8b01320. Supporting Information includes (1) materials, instruments, the synthesis of AuNRs, and the extraction of proteins and the DNA genome, (2) the dark-field images of uptake of HepG2 cells treated with AuNRs with and without RGD modification, (3) the enlarged dark-field, fluorescent, and merged images of cells treated with AMT, ANT, and ALT for 24 h, (4) the colocalization of nanoprobes with organelle by confocal fluorescence microscopy, (5) Bio-TEM images of single cells treated with AMT, ANT, and ALT for 24 h, (6) the SERS spectra of AMT, ANT, and ALT nanoprobes, (7) the SERS spectra of organelles in HepG2 cells, (8) the SERS spectra of proteins and DNA genome extracted from HepG2 cells, and (9) the photothermal response AuNPs and AuNRs (PDF)

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REFERENCES

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (S.X.). *E-mail: [email protected] (C.L.). ORCID

Shuping Xu: 0000-0002-6216-6175 Weiqing Xu: 0000-0002-1947-317X Author Contributions

Y. Shen, L. Liang, S. Xu, C. Liang, and W. Xu designed research; Y. Shen, S. Zhang, H. Qu, and D. Huang performed research; Y. Shen, L. Liang, D. Huang, J. Zhang, and R. Deng analyzed data; Y. Shen, L. Liang, and S. Xu wrote the paper. All authors have approved the final version of the manuscript. Funding

This work was supported by the National Natural Science Foundation of China NSFC grant nos. 21373096, 91441105, 21573087 and 21573092. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Prof. Wei Shi, Key Lab for Molecular Enzymology & Engineering, Ministry of Education of Jilin Univ., and his team for helping on cell cultures. We also thank Yanru Li and Shuang 7917


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