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Tracing Therapeutic Process of Targeted Aptamer/Drug Con- jugate on Cancer Cell by SERS Spectroscopy. Rong Deng,a Huixin Qu,b Lijia Liang,a Jing Zhang...
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Tracing the Therapeutic Process of Targeted Aptamer/Drug Conjugate on Cancer Cells by Surface-Enhanced Raman Scattering Spectroscopy Rong Deng,† Huixin Qu,‡ Lijia Liang,† Jing Zhang,† Biying Zhang,† Dianshuai Huang,‡ Shuping Xu,*,† Chongyang Liang,*,‡ and Weiqing Xu† †

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

ABSTRACT: Targeted delivery of chemotherapeutic agents to pathology areas can improve drug efficiency and reduce serious side effects on normal regions. However, their treatment mechanism on cells or cell nuclei is still mysterious due to the lack of in situ characterization methods. In this paper, the specific diagnosis and treatment processes of a targeted antitumor agent (doxorubicin, Dox) functionalized aptamer complex (TLS11a-GC−Dox) toward HepG2 cells, a human hepatocellular carcinoma cell line, were tracked in real time by the surface-enhanced Raman scattering (SERS) spectroscopic technique and dark-field imaging with the assistance of gold nanorodbased nuclear targeted probes, which possess remarkable SERS enhancement ability, specific targeting, and excellent biological compatibility. This is the first time to explore the acting mechanism of an aptamer-based targeted drug on cell nucleus based on the spectral information on components inside the cell nucleus. The results demonstrate that this aptamer/drug conjugate has targeting and sustained-release actions and its therapeutic effect is achieved by the gradual damage of relevant proteins and DNA in nuclei. Better understanding of the mechanism of aptamer− drug conjugates acting on cancer cells is conductive to increasing cancer therapy efficiency and is also helpful for the design of highly effective drug delivery methods.

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sequences at the tail of the aptamer can effectively improve the drug loading of Dox.17 Therefore, in the present study, we selected an aptamer (TLS11a-GC) representative of the targeted drug carrier. Just like an antitumor drug, the action mechanism of this aptamer-based targeted drug deserves further investigation, not only because the optimization of drug dose is vital but also because its delivery is extremely important for us to build a highly effective therapy plan and find new methods for anticancer therapy. Assessment of drug efficacy on cells is generally based on cell viability assays, such as WST-1 and MTT tests.18,19 However, these methods cannot provide information on the process during cell apoptosis. As the nucleus is the most precious and dominating part of a cell, explorations of cell nuclear behavior are in high demand. However, it is not easy to probe a nucleus directly and in situ. Classical cytobiological approaches are mostly based on extraction of the nucleus and in vitro

he benefit of a drug depends on how it is administered or delivered. Though a growing number of novel drugs have been developed for disease treatment with the advancement of medicine, the targeted delivery of chemotherapy agents in cells, tissues, or diseases has not been adopted extensively yet, let alone to further explore drug effects. Carrier-based drug delivery is an excellent alternative with high expectations.1−3 The carriers commonly used in literature are liposomes,4 polymeric micelles,5 nanoparticles (NPs),6 and capsules.7 However, as exogenous substances, most of them are difficult to degrade in the body, and their special recognition of cancer cells mainly relies on other targeted molecules. As a single-stranded structured nucleic acid ligand, an aptamer is biodegradable and possesses high affinity and specific recognition ability for many biological compounds, such as small molecules,8,9 peptides,10 and proteins.11 In cellrelated studies, aptamers have been widely used to specifically target overexpressed proteins on the cell membrane to achieve targeting purpose.12−14 Moreover, it has been reported that an aptamer with GC sequences can be used as a carrier for doxorubicin (Dox, an anticancer drug) through noncovalent intercalation in its two-dimensional structure.15,16 More GC © 2017 American Chemical Society

Received: October 9, 2016 Accepted: January 31, 2017 Published: January 31, 2017 2844

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cell, and its fluorescence would recover. The released Dox will enter the nucleus (③) and highlight it. The transfer and therapeutic process of the TLS11a-GC−Dox conjugates can be monitored by dark-field (DF)/fluorescence colocalization imaging.42 Also, the features of intranuclear components are recorded by in situ SERS spectroscopy with the help of nucleartargeted nanoprobes. By means of SERS spectroscopic analysis, we can explore the effect of the aptamer-based anticancer drug on HepG2 cells during the treatment process.

measurements.20,21 Microenvironment changes and excess extraction procedures may bring many impacts on the nucleus. To obtain intranuclear information in situ, the surfaceenhanced Raman scattering (SERS) technique is considered in the present study. SERS spectroscopy is a sensitive vibrational spectral technique that can offer clear spectroscopic information on components inside the cells or tissues in situ.22−25 It shows high sensitivity of 106−1012 times beyond normal Raman signal. Such high sensitivity mainly originates from the enhanced local electromagnetic field where noble metal NPs or nanostructures lie.26 Noble metal NPs are one kind of typical plasmonic materials that possess unique localized surface plasmon resonance (LSPR) properties. The fingerprint information on components located near metal NPs will be greatly improved.27−29 Moreover, noble metal NPs display excellent biocompatibility, which allows them to be widely used for cellular studies, such as intracellular sensors, thermal therapeutic agents, drug delivery carriers, and contrast agents for medical imaging.30−32 Owing to these merits, many multiple-modified Au or Ag NPs with targeted molecules (e.g., aptamer, peptide, and antibody) have been developed as SERS nanoprobes, to specifically link to the cancer cell membrane33−35 and mediate the endocytosis process,36 or as nuclear-targeted probes, to investigate crucial physiological processes and drug action mechanisms.37−41 Our goal is to analyze the action mechanism of the targeted drug system (an aptamer/drug conjugate) with the assistance of these nuclear targeted probes. As shown in Scheme 1, nuclear-

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EXPERIMENTAL SECTION Synthesis of Nuclear-Targeted Nanoprobes. AuNRs with an aspect ratio of about 2.2 were synthesized according to the modified seed-mediated growth method reported in literature.43,44 Detailed procedures are shown in Supporting Information. Briefly, a seed solution was first synthesized by reduction reaction of HAuCl4, and then the seeds were added into a growth solution to produce AuNRs. AuNRs were further modified by PEG, NLS, and RGD step-by-step. The prepared AuNR-based nanoprobes [0.05 nM, in fresh Iscove’s modified Dulbecco’s medium (IMDM)] were cultured with HepG2 (a human hepatocellular carcinoma cell line) cells on coverslips for 24 h. After that, the coverslips were cleaned three times with phosphate-buffered saline (PBS), fixed with 4% formaldehyde for 20 min, and then sealed for further use. Observation of Intranuclear Distribution of NuclearTargeted Nanoprobes by High-Resolution 3D Confocal Fluorescence Microscopy. To take fluorescent images of the nuclear-targeted nanoprobes inside cells, fluorescein isothiocyanate (FITC)-labeled NLS-RGD-PEG-AuNRs were prepared by linking FITC-tagged NLS on the surface of AuNRs instead of pure NLS. After cells were cultured with 0.05 nM FITClabeled NLS-RGD-PEG-AuNRs for 24 h, individual cell fluorescent images were taken by a DeltaVision OMX imaging system with 3D SIM model from GE Healthcare (OM06051, Pittsburgh, PA). Analysis software, Imaris X 64 from Bitplane (Zurich, Switzerland), was further used to deal with image data and build 3D views. Conjugation of Aptamer/Dox. A cancer-cell-targeted aptamer, TLS11a-GC (the sequence is provided in Supporting Information), and a FITC-labeled TLS11a-GC were purchased from Sangon. The conjugation of aptamer and drug (TLS11aGC−Dox) was finished within several minutes through the physical bonding of Dox (5.0 μM) and TLS11a-GC in a buffer solution containing sodium acetate (0.10 M) and sodium chloride (0.05 M). The unbound Dox was removed by dialysis (6.0 kDa cutoff, Pierce). The fluorescence spectrum of Dox after reaction with aptamer was monitored via a Shimadzu 5301PC fluorescence spectrophotometer with excitation wavelength of 480 nm. By evaluating the fluorescence quenching of Dox when bound to TLS11a-GC (see Supporting Information), the binding ratio is determined to be 4:1 (the molar equivalent of Dox:TLS11a-GC). Assessment of Uptake of TLS11a-GC−Dox and Free Dox by Confocal Fluorescence Microscopy. HepG2 cells, which had been planted on coverslips, were cultured with the TLS11a-GC−Dox conjugates (containing 15.0 μM Dox) in IMDM medium for 2 h (or 12 h). Then the cells were cleaned three times with PBS. After that, they were fixed with 4% formaldehyde for 10 min and stained with Hoechst 33342 for 15 min to highlight cell nuclei. They were washed again and sealed before images were taken. A control experiment was carried out simultaneously with the same concentration of free

Scheme 1. Exploring the Therapeutic Effect of Aptamer/ Drug Conjugates Acting on HepG2 Cells by SERS Spectroscopy and Dark-Field Imaging with the Assistance of Nuclear-Targeted Nanoprobes

targeted SERS nanoprobes (NLS-RGD-PEG-AuNRs) were first synthesized by using gold nanorods (AuNRs) as the signal enhancement agent, followed by surface modifications of a thiol-modified poly(ethylene glycol) polymer (SH-PEG) ligand, a cell-membrane-targeted peptide (RGD), and a nuclear localizing signal (NLS) peptide that can increase targeting and internalization efficiency for HepG2 cells (①). Then the antitumor agent (Dox) was loaded onto an aptamer carrier (TLS11a-GC) that can specially bind to and act on HepG2 cells. Extra GC sequences were designed to improve the drugloading efficiency. It should be noted that the fluorescence of Dox loaded on TLS11a-GC would be quenched once they formed a physical bond (②). However, after the internalization of TLS11a-GC−Dox in the cell, Dox would be gradually released due to enzymatic degradation of TLS11a-GC in the 2845

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Analytical Chemistry Dox for HepG2 cells. Fluorescent images of cells with the Hoechst 33342-stained nuclei (in blue) and Dox distribution (in red) were obtained on a fluorescence microscope (IX71, Olympus) with 50×/0.75 numerical aperture (NA) objective and different filter cubes. SERS Detection of TLS11a-GC−Dox Acting on Cell Nuclei. HepG2 cells that had been planted on coverslips were incubated with 0.05 nM NLS-RGD-PEG-AuNRs for 24 h, and then they were treated with 4.0 μM TLS11a-GC−Dox. After TLS11a-GC−Dox was added, the SERS spectra of cell nuclei were measured by a confocal Raman system (LabRAM Aramis, Horiba JobinYvon) with a 633 nm laser as the excitation source. The laser was directed into the microscope and focused on the sample by a 50×/0.75 NA objective with an integration time of 20 s and detection occurring twice. The power of the laser on the sample was around 7.1 mW. The cells were fixed on coverslips. The size of the laser spot is around 0.7 μm under such objective, while the planar size of HepG2 cell nucleus is approximately 7 × 11 μm. So to obtain accurate spectral signals of the intracellular compounds, SERS spectra at similar sites of nuclei were collected. Each time-dependent spectrum is a statistical average of multiple sets of measurements and the spectral profiles are treated with baseline correction by NGSLabSpec1 software and further normalization for easy comparison.

Figure 1. (A) TEM image of AuNRs. (B) Dark-field image of HepG2 cells incubated with NLS-RGD-PEG-AuNRs for 24 h. (C) SERS spectra of HepG2 cell nuclei with enhancement of the NLS-RGDPEG-AuNRs (0.05 nM, top curve) and of functional peptides on the surface of NLS-RGD-PEG-AuNRs in aqueous solution (1.3 nM, bottom curve). (D) Comparison of SERS and Raman spectra of HepG2 cell nuclei that had been cultured (a) with or (b) without 0.05 nM NLS-RGD-PEG-AuNRs.

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As the nanoprobe could enter into the cell nucleus with the help of RGD and NLS, Figure 1B is the DF image of RGDNLS-PEG-AuNR distribution in HepG2 cells. It can be seen that some NLS-RGD-PEG-AuNRs have arrived in nuclei. To further prove this conclusion, we labeled the NLS with FITC and then used confocal fluorescence imaging to display the locations of NLS-RGD-PEG-AuNRs inside cells. Figure 2A is a

RESULTS AND DISCUSSION Synthesis of Nuclear-Targeted SERS-Active Nanoprobe. The nuclear-targeted nanoprobes (NLS-RGD-PEGAuNRs) need to satisfy the following requirements. (1) Strong SERS enhancement ability: The NLS-RGD-PEG-AuNRs we prepared have an absorption peak at 635 nm (Figure S1A), which is well-matched with the LSPR of the excitation wavelength (λex = 633 nm). This is critical for gaining highquality SERS spectra. (2) Good biocompatibility: The surface of AuNRs is further coated with PEG to increase the stability in a biological environment and minimize cytotoxicity according to literature and our previous studies.39 (3) Specific targeting to cell nuclei: The PEG-AuNRs were continuously modified with cancer-cell-specific targeted peptide (RGD, CGGGPKKKRKGC) and nuclear localization signal peptide (NLS, GGVKRKKKPGGC) to increase the internalization of AuNRs in cell nuclei. RGD can bind with the αvβ6 or αv integrins on the cell surface. So the RGD-decorated AuNRs can be delivered into cell by receptor-mediated endocytosis. NLS has a crucial sequence (KRKKK), with a similar function to the TAT sequence (YGRKKRRQRRRGGGC) that helps AuNRs enter into the nucleus through the nuclear pores.45,46 The doses of NLS and RGD are both controlled at 30% coverage of the whole surface area of AuNRs. The functional AuNRs were characterized by transmission electron microscopy (TEM; Figure 1A), ultraviolet−visible (UV−vis) spectroscopy, and dynamic light scattering (DLS) measurements. The maximum of the plasmonic band of AuNRs red-shifts from 626 to 631 nm for PEG coating and to 635 nm for NLS-RGD-PEG coating (Figure S1A). The surface potential of AuNRs also changes from 19.8 mV to 10.1 and 8.4 mV, respectively, after combination with PEG and NLS-RGD-PEG (Figure S1B). Beyond that, with regard to the effect of nanoprobes on cellular metabolism and the physiological activity of cells, the concentration of nanoprobes we used in this study is as low as 0.05 nM, which has almost no toxicity to our cells, as confirmed by WST-1 assay (Figure S1C).

Figure 2. (A) Confocal image of cancer cell treated with 0.05 nm NLS-RGD-PEG-AuNRs for 24 h. The blue color represents the nucleus of cancer cell, while the green is the NLS-RGD-PEG-AuNRs labeled by FITC. This image was detected by a DeltaVision OMX imaging system with 3D SIM model from GE Healthcare (No.OM06051, Pittsburgh, PA). (B−D) 3D simulation images of the cell in panel A, rendered and analyzed via Imaris X 64 software from Bitplane (Zurich, Switzerland). (B) Panorama of the cancer cell; (C) magnification of the encircled area in panel B; (D) perspectives of NLS-RGD-PEG-AuNRs within the nuclear area.

confocal image of a HepG2 cell cultured with 0.05 nM FITClabeled NLS-RGD-PEG-AuNRs for 24 h. The blue color represents the nucleus of cancer cell, while the green shows nanoprobe distribution. We also scanned the cell nuclei layerby-layer along the Z direction using an Olympus FV1000 confocal microscope; it also displayed that these two colors were overlaid to a great extent (Figure S2). Figure 2B−D 2846

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fluorescence with an excitation wavelength of 480 nm and emission wavelengths of 557 and 584 nm. Dox can be fixed on an aptamer (TLS11a) by intercalating its flat aromatic ring into the CG sequence of TLS11a. To increase the loading capacity of Dox, additional GC sequences are on demand, under the precondition of no impact on the recognition ability of TLS11a sequence. In this study, a repeated GC tail with a number of eight was added to the 5′-end of TLS11a. It should be noted that the fluorescence of Dox can be quenched by GC sequences, based on the strong interaction between them, and the molar equivalence of Dox to aptamer is 1.0:1.2. Here we adopted an aptamer with an 8-GC tail and 4:1 binding molar equivalence of Dox to TLS11a was achieved, which can be proved by maximal quenching of Dox’s fluorescence (Figure S4). Owing to this data, we can confirm that the drug-loading efficiency has been improved due to more GC sequences of the aptamer. When TLS11a-GC−Dox conjugates were internalized in the HepG2 cells, Dox was gradually released owing to the enzymatic degradation of TLS11a- GC in the cell. As a typical anticancer drug, Dox can act on deoxyribonucleic acid (DNA) in the nucleus, which would gradually light up the nuclei under fluorescence imaging (② in Scheme 1). Figure 3 shows the

displays high-resolution three-dimensional (3D) images of one cell, where panel B is the panorama of the cancer cell, panel C shows the magnification of the encircled area in panel B, and panel D is the perspectives of nanoprobes within the nuclear area. From these images, we can clearly see that many of the nanoprobes really entered into the nucleus. Moreover, we can find many AuNR nanoaggregates in Figure 2B−D, as well as in Figure 1B. It can be explained that when more and more nanoprobes were internalized in the cell, the possibility for AuNRs meeting increases, which makes AuNRs tend to aggregate. Interestingly, the aggregation of AuNRs is favorable for SERS measurements due to strong local electromagnetic coupling. After the nuclear-targeted probes were inside the cell, we measured the SERS spectrum of the HepG2 cell nucleus (top curve in Figure 1C). The band assignments are listed in Table S1. To make sure whether the targeted peptides on the nanoprobes would interfere the SERS signals of intranuclear biomolecules, we tried to measure the SERS spectrum of the nuclear-targeted probes. Since the concentration of NLS-RGDPEG-AuNRs we used in this study is too low to detect SERS signals, we increased the probe concentration to 1.3 nM and added salts to induce aggregation. Fortunately, the SERS spectrum of the nuclear-targeted probes at such a high concentration was achieved, shown as the bottom curve in Figure 1C. When these two spectra are compared, despite several overlaps, most peaks are different, indicating these nanoprobes would have less interference in the spectral analysis of intranuclear biomolecules. Figure 1D shows Raman and SERS spectra of the biological components in HepG2 cell nuclei. By comparison, we can find that with NLS-RGD-PEG-AuNRs as the Raman signal enhancement substrate, the band intensities have greatly improved. Several new bands that are invisible in the Raman spectrum appear: for example, 509, 655, 1172, and 1215 cm−1. The high detection sensitivity of SERS indicates that it is much easier to identify changes in nuclear components under drug treatments. However, additional changes also exist between these two spectra. For example, the 1689 cm−1 peak almost disappears and the 997 cm−1 band shows a blue shift in wavenumber compared with that in the Raman spectrum. The reason for these changes is still unknown. They may be caused by the interaction between NLS-RGD-PEG-AuNRs and cells or by interference from the culture environment. Since these changes were identified at the beginning, they will have almost no interference in the analysis of SERS spectra of nuclei under further drug-caused damage. Cell-Surface Binding and Drug-Loading Capability of TLS11a-GC. The specific recognition by aptamer (TLS11aGC) of HepG2 cells is crucial for drug delivery efficiency and targeting. To evaluate the binding ability of TLS11a-GC to HepG2 cells, we adopted another kind of cells (BNL.CL2 cells, a non-cancer liver cell line) for comparison. HepG2 and BNL.CL2 cells were both treated with FITC-labeled TLS11aGC (details are found in Supporting Information) and they were measured by confocal fluorescence microscopic images (Figure S3). The fluorescence (green) intensity of the FITC on the periphery of HepG2 cells is remarkably stronger than on the BNL.CL2 cells, indicating that TLS11a-GC possesses targeting recognition for HepG2 cells. On the basis of this result, an aptamer/Dox conjugate, TLS11a-GC−Dox, was prepared to deliver specifically an antitumor agent (Dox) to cancer cells. Dox exhibits

Figure 3. Confocal fluorescent images of HepG2 cells that were incubated with free Dox and TLS11a-GC−Dox for (a, b) 2 h and (c, d) 12 h. Blue and red colors represent the distributions of Hoechst 33342 and Dox in nuclei. Right panels show the overlaid images.

fluorescent images of HepG2 cells treated with TLS11a-GC− Dox and free Dox for 2 and 12 h, respectively. By comparison of Figure 3 rows a and b, it can be observed that the percent cumulative release of Dox from TLS11a-GC−Dox is relatively lower in nuclei than that of free Dox. However, as the culture time is prolonged to 12 h, the accumulations of Dox in Figure 3c,d become closer. This means that our designed drug conjugate (TLS11a-GC−Dox) has a property of sustained drug release. 2847

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and 1172 cm−1. From this result, we preliminarily infer that TLS11a-GC−Dox not only achieves targeted delivery of Dox but also has the same pharmacological effect as free Dox. Ro further investigate the drug action of TLS11a-GC−Dox on HepG2 cells, dynamic changes of biological molecules in HepG2 cell nuclei were monitored by SERS spectroscopy (Figure 5) and DF imaging (Figure 6). After 0.05 nM NLS-

Real-Time SERS Spectroscopic Detection. As it has been demonstrated that nanoprobes have been positioned in cancer nuclei, the SERS signal of the intranuclear components around the nanoprobes can be recorded and amplified. To investigate the anticancer effect of TLS11a-GC−Dox on cancer cells, we first compared the pharmacological effects of free Dox and TLS11a-GC−Dox on HepG2 cells. HepG2 cells with nucleartargeted nanoprobes were respectively treated with 15 μM free Dox and 4.0 μM TLS11a-GC−Dox (the Dox concentration is equivalent). Figure 4A−C shows DF images of HepG2 cells

Figure 4. (A−C) DF images and (D) SERS spectra of HepG2 cells with 0.05 nM nuclear-targeted nanoprobes, (A, a) before and (B, b) after they were treated with 4 μM TLS11a-GC−Dox conjugates or (C, c) 15 μM free Dox for 24 h, respectively.

before and after treatment with TLS11a-GC−Dox or free Dox. In Figure 4A, the cells with nanoprobes possess weak and disperse scattering before drug therapy. After Dox or TLS11aGC−Dox treatment for 24 h, the dying cells are obviously shrunken and give strong white scattering light. The increased scattering is caused by scattering effects of the uneven surface of the swelled cells, as well as cells lifting from the slide and out of the focal plane.38 Meanwhile, the fluorescence of targeted Dox in the nuclei can be excited simultaneously, which also influences the scattering of NLS-RGD-PEG-AuNRs and illuminates the whole cell. The visual variation in cell morphology indicates that both drugs can drive the cells to die. To assess the roles of nanoprobes and Dox on cell apoptosis, we compared the cytotoxicity of NLS-RGD-PEGAuNRs before and after Dox and TLS11a-GC−Dox addition (Figure S5). It was found that cell viabilities decreased once Dox and TLS11a-GC−Dox were added, proving that the pharmacological effect of Dox is the key reason for cell death. In addition, SERS spectra of nuclear components that were treated by TLS11a-GC−Dox and free Dox also show many obvious changes (Figure 4D). To check whether the SERS signals of Dox will interfere with the signal of cell nuclei, the SERS spectra of Dox were obtained by inducing the nanoprobes to aggregate in solution (Figure S6). The comparison indicates the SERS spectra of Dox and cell nucleus are absolutely different. In Figure 4D, by comparisons of a to b and a to c, we can find that many similar spectral variations between two drugs can be observed, such as 509, 1465, 1075,

Figure 5. Time-dependent intranuclear SERS spectra of HepG2 cells first cultured with 0.05 nM NLS-RGD-PEG-AuNRs for 24 h and then treated with 4 μM TLS11a-GC−Dox conjugates for 0, 6, 12, 18, and 24 h.

RGD-PEG-AuNRs were dispersed in the nuclei, HepG2 cells were treated with 4.0 μM TLS11a-GC−Dox. Since the Dox on TLS11a-GC−Dox conjugates can be released in the cell by enzymatic degradation, the therapeutic effect of Dox will be performed gradually after internalization, which can be observed from the increase of fluorescence brightness in cell nuclei with incubation times of 0−24 h (Figure 6C). Figure 5 shows time-dependent SERS spectra of the nuclear components obtained within this therapeutic process. These spectra were mainly collected from the specific spots where the nanoprobes were congregated. Each spectrum is a mean spectrum averaged from dozens of spots on several cells. The mean spectra would reduce these differences and support the acceptable repeatability for spectral analysis (Figure S7). The band at 509 cm−1 represents the −S−S− vibration from sulfurcontaining amino acids in proteins. Its intensity decreases at 6 h. This might mean breakage of disulfide bonds and dissolution of the tertiary protein structure owing to the therapeutic effect. However, during the treatment process, dissolution of the tertiary protein structure could lead to the dissociation and reconstitution of folding proteins. So more disulfide bonds may be exposed and accessible to the surface of the AuNRs. This 2848

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Figure 6. (A) Bright-field, (B) DF, and (C) fluorescent images of HepG2 cells that were first cultured with 0.05 nM NLS-RGD-PEG-AuNRs for 24 h and then treated with 4 μM TLS11a-GC−Dox conjugates for 0, 6, 12, 18, and 24 h.

might be the reason why the peak intensity at 509 cm−1 reaches a maximum at the treatment time of 12 h. When cells are continually treated by TLS11a-GC−Dox, the disulfide bonds start to break and the band intensity decreases again. The complexity of the environment may be another impacting factor. Besides, the Raman band at 1075 cm−1, assigned to C− N stretching vibrations of polypeptide chain, decrease obviously, while the 1171 cm−1 band belonging to tyrosine has almost no distinct changes, indicating the hydroxyphenyl ring of tyrosine is not seriously damage. At 0, 6, 12, 18, and 24 h, the intensity ratios of 1075 to 1171 cm−1 bands (I1075/I1171) are 1.16, 1.16, 1.14, 1.12, and 0.99, respectively. The weakening tendency of the intensity ratio (I1075/I1171) might be caused by the influence of microenvironment on the C−N stretching vibration of polypeptide chain. It also can be found from the variation tendency of these ratios that the TLS11a-GC−Dox starts to take pharmacological effect when the acting time is

about 12 h, causing the intensity ratio to decrease, which proves again the sustained release function of TLS11a-GC−Dox. In addition, the vibration at 1466 cm−1, corresponding to the ribose of DNA, shows a several-wavenumber shift and disappears entirely. It reflects that the covalent bonds between ribose and bases have been affected by TLS11a-GC−Dox. In addition to the ordered changes of peak intensity, the Raman shifts of several bands also vary regularly (Table S1). The bands of 510, 627, 1075, 1266, and 1354 cm−1 belong to components in protein, while the bands of 1212, 1331, and 1466 cm−1 represent DNA. The peaks at 627 and 1331 cm−1 shift to higher wavenumbers, while the bands at 1212, 1266, and 1354 cm−1 all shift to lower wavenumber. The 1212 and 1331 cm−1 vibrations belong to adenine (A) in DNA. Changes in these two peaks means the DNA structure is altered in this therapy process. The bands at 627, 1266, and 1354 cm−1 are assigned to the sensitive vibrational modes of C−C twisting of 2849

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Analytical Chemistry phenylalanine and to amide III vibrations of α-helix and tryptophan in proteins. All these changes in Raman shifts indicate that the conformations of these groups have been disturbed by TLS11a-GC−Dox. Therefore, variations in the Raman intensity and shift indicate that proteins and DNA in cancer cell nuclei had both been damaged during the process that HepG2 cells were treated with TLS11a-GC−Dox. Real-Time Dark-Field Imaging. The process of TLS11aGC−Dox acted on HepG2 cells can also be researched by persistent cell deformation, a visual process that is displayed on bright-field and dark-field images (Figure 6A,B). Since HepG2 cells are adhere-wall cultured, we can find many long microscopic filaments around individual cells and the shape is fusiform before drug treatment. After incubation with TLS11aGC−Dox conjugates, the cellular morphology gradually shrank and eventually turned into a small spherical shape. Also, the adherence of cells gradually reduced, which causes them to float in the culture medium when they are close to apoptosis. The scattering of the NLS-RGD-PEG-AuNRs inside HepG2 cells exhibits brighter and brighter light with the cells’ shrinkage and Dox accumulation. This visualization process demonstrates that TLS11a-GC−Dox conjugates have dosage-related and timedependent manners. Along with the prolongation of drug action time and the accumulation of drug concentration in body, the Dox loaded on the TLS11a-GC−Dox conjugates gradually performs its damaging action.

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AUTHOR INFORMATION

Corresponding Authors

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

Shuping Xu: 0000-0002-6216-6175 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by National Natural Science Foundation of China (21373096, 21573092, 21573087, 91441105, and 31271478). We appreciate Professors Kun Liu and Ying-Wei Yang (both of Jilin University) for teaching us the strategy of AuNR preparation.

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CONCLUSIONS In summary, we traced the therapeutic process of targeted aptamer/drug conjugate (TLS11a-GC−Dox) on cancer cells by SERS spectroscopy and dark-field images to especially explore the drug effect on cancer cell nuclei. A nuclear-targeted nanoprobe (NLS-RGD-PEG-AuNRs), which possesses remarkable SERS enhancement ability, specific targeting, and excellent biological compatibility, were employed for SERS measurements and DF imaging. A targeted drug, TLS11a-GC−Dox, was obtained by loading anticancer drug Dox on the TLS11aGC that can specifically identify HepG2 cells. Its targeted delivery and sustained release properties were demonstrated by fluorescent imaging and SERS analysis. The treatment process was clearly recorded by real-time SERS spectra of the components inside cancer cell nuclei and the visual morphological shrinkage of cells under DF imaging. By analyzing the time-dependent SERS spectra during the therapeutic process, we observed that many proteins and DNA molecules in cancer cell nuclei had been damaged, from which we can preliminarily deduce that TLS11a-GC−Dox conjugates may disturb cell division during the treatment process. This is the first time to reveal the treatment mechanism of a targeted drug by SERS spectroscopy. Better understanding of the mechanism of drug action is useful to optimize the administration of anticancer drugs and to improve the efficiency of cancer therapy.

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nanoprobes, assessment of intranuclear distribution of NLS-RGD-PEG-AuNRs, specific surface binding of aptamer for HepG2 cell, conjugation of aptamer/Dox, cytotoxicity of NLS-RGD-PEG-AuNRs and Dox to cell, SERS spectra of HepG2 cell nuclei and free Dox, and SERS analysis of cancer cell nuclei at different drug treatment times (PDF)

ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.6b03971. Additional text, seven figures, and one table describing synthesis and characterization of nuclear targeted 2850

DOI: 10.1021/acs.analchem.6b03971 Anal. Chem. 2017, 89, 2844−2851

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