Research Article Cite This: ACS Appl. Mater. Interfaces 2019, 11, 23436−23444
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Photodegradable CuS SERS Probes for Intraoperative Residual Tumor Detection, Ablation, and Self-Clearance Yuanyuan Qiu,†,⊥ Miao Lin,†,⊥ Gaoxian Chen,†,⊥ Chenchen Fan,† Mingwang Li,† Xiajing Gu,† Shan Cong,∥ Zhigang Zhao,∥ Lei Fu,‡ Xiaohong Fang,*,§ and Zeyu Xiao*,†
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Department of Nuclear Medicine, Clinical and Fundamental Research Center, Ren Ji Hospital, School of Medicine & Department of Pharmacology and Chemical Biology, School of Medicine, ‡Shanghai Key Laboratory for Molecular Engineering of Chiral Drugs, School of Pharmacy, Shanghai Jiao Tong University, Shanghai 200025, P. R. China § Beijing National Laboratory for Molecular Sciences, Key Laboratory of Molecular Nanostructure and Nanotechnology, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, P. R. China ∥ Key Lab of Nanodevices and Applications, Suzhou Institute of Nano-Tech and Nano-Bionics, Chinese Academy of Sciences, Suzhou 215123, P. R. China S Supporting Information *
ABSTRACT: Surface-enhanced Raman scattering (SERS) probes have exhibited great potential in biomedical applications. However, currently reported SERS probes are mainly fabricated by nondegradable Au or Ag nanostructures, which are not favorably cleared from the imaged tissues. This bottleneck hinders their in vivo applications. We herein explore a degradable SERS probe consisting of hollow CuS nanoparticles (NPs) to circumvent the current limitation. We identify, for the first time, the Raman enhancement effects of hollow CuS NPs as a SERS probe for Raman imaging of residual tumor lesions. Uniquely, CuS SERS probes are degradable, which stems from laser-induced photothermal effects of CuS NPs, leading to their disintegration from shell structures into individual crystals, thus facilitating their self-clearance from imaged tissues. This novel CuS SERS probe with photodegradation characteristics opens avenues for applying Raman imaging toward a myriad of biomedical applications. KEYWORDS: CuS nanoparticles, surface-enhanced Raman scattering (SERS), intraoperative imaging, theranostics, Raman probes, nanomedicine, phototherapy
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INTRODUCTION Raman imaging utilizing surface-enhanced Raman scattering (SERS) probes has received great attention in recent years.1,2 Principled by the inelastic scattering of photons interacting with molecules, Raman imaging generates fingerprint-like spectra of suitable molecules (Raman reporters) with distinct composition.3−5 Despite the intrinsically weak signals of Raman effects (merely ∼10−7 scattered photons is Raman shift), SERS probes bring Raman reporters in proximity to their surfaces, leading to dramatically enhanced sensitivity of Raman signals with detected limits of 10−9−10−12 M.5 This SERS phenomenon allows for highly sensitive and stable signal for Raman imaging,6−8 while maintaining detailed information on chemical composition due to their molecular Raman “fingerprints”.9,10 Numerous efforts have been focused on designing highly sensitive SERS probes, including most dominant metal-based gold or silver nanostructures,11,12 and a few semiconducting materials.13,14 These SERS probes have demonstrated great promise in biomedical imaging, such as in delineating tumor margins for precise resection.2,15 © 2019 American Chemical Society
Nevertheless, currently reported SERS probes are nondegradable16,17 and thus they would not be completely eliminated from the imaged tissue. Such long-term persistence would raise concerns of chronic toxicity owing to the possibility for SERS probes to aggregate, produce deleterious metabolites, and redistribute to major organs.5,18,19 To date, a few studies have demonstrated SERS probes to satisfy efficient clearance. This material bottleneck has long existed and is impeding the clinical translation of SERS probes for a wide range of biomedical applications. Herein, we explore a photodegradable SERS probe to circumvent the current limitation. This SERS probe consists of hollow CuS nanoparticles (NPs). We, for the first time, discover the Raman enhancement effect of hollow CuS NPs for a serial of Raman reporters, thus serving as a novel SERS probe for Raman imaging (Scheme 1). Uniquely, upon continuous Received: January 9, 2019 Accepted: June 5, 2019 Published: June 5, 2019 23436
DOI: 10.1021/acsami.9b00469 ACS Appl. Mater. Interfaces 2019, 11, 23436−23444
Research Article
ACS Applied Materials & Interfaces
redispersed in water, yielding PEGylated and Raman reporter-coated hollow CuS NPs, termed as CuS SERS probes. The morphology was observed using transmission electron microscopy (TEM) imaging (Tecnai G2 Spirit Biotwin, FEI). The region of interest in hollow CuS NP sample was selected for energy-dispersive spectroscopy (EDS) analysis (Falion 60S, EDAX). UV−vis spectra were acquired by a UV/ VIS/NIR spectrophotometer (Lamda 950, PerkinElmer). The hydrodynamic diameter was determined by a laser particle size analyzer (Zetasizer Nano ZSP, Malvern, U.K.). The concentration of Cu ions was measured using an inductively coupled plasma optical analyzer (ICAP7600, Thermo). The concentration of CuS SERS probes was determined using a nanoparticle tracking analyzer (NanoSight NS300, Malvern, U.K.). Raman Enhancement of CuS SERS Probes. Detection of the Raman signals of CuS SERS probes was performed using a Renishaw inVia Raman system, which is coupled to a Leica DM-2700M microscope (Leica) and controlled by WiRE 4.2 software. To evaluate the photostability of Raman signals, CuS SERS probe suspension (560 pM) was continuously irradiated for 35 min with a 10 mW laser power. The Raman spectra were acquired (10 mW, 3 s acquisition time, 20× objective) at an interval of 5 min. To determine the serum stability of their Raman signals, CuS probes (560 pM) were incubated in 10% fetal bovine serum (100 μL) and SERS signals were determined at 0, 2, 4, 6, 8, 10, 24, 30, 36, 48, and 72 h, respectively. To test the limit of detection (LOD), CuS SERS probes were redispersed in 10 μL of water on a phantom at increasing concentrations of 0, 1, 4, 8, 20, 35, 140, and 280 pM (n = 3 samples for each concentration) and the Raman detection parameters were a 10 mW laser power, a 3 s acquisition time, and a 20× objective. The calibration curve was fitted with the Raman intensity at 506 cm−1 as a function of the logarithmic concentration of the CuS SERS probes. A serial of concentration of CuS SERS probes were made from 8 to 280 pM. In the linear range, the regression coefficient (R2) was 91% and the calibration curve was Y = −49.06 + 75.57X, where Y is the Raman intensity at the 506 cm−1 band and X is the logarithmic concentration of the CuS SERS probes. With reference to the IUPAC (i.e., International Union of Pure and Applied Chemistry) standard,23,24 the limit of detection (LOD) was calculated as the 3 s/m standard, where s represents the standard deviation of the blank and m indicates the slope of the linear region. The Raman spectra were analyzed by a signal to baseline algorithm with WiRE 4.2 software (Renishaw). Photodegradation Characteristics of CuS SERS Probes. CuS SERS probes were dispersed in water solutions (200 μg/mL, ∼200 μL) in the 1.5 mL tubes and then irradiated with a 980 nm laser (GCSLS-06-7W00, Daheng Science & Technology, Beijing, China) at a power intensity of 800 mW for indicated time periods of 0−5 min (n = 3 samples for each timepoint). The adjustment of laser distance allowed the spot to cover the entire surface of samples. The morphology of CuS SERS probes after irradiation was then observed using TEM imaging. The size distributions were measured using a laser particle size analyzer. Temperature change was monitored using a FLIR Ax5 camera and quantified by IRMeter software at an interval of 30 s. The temperature and morphological changes of CuS SERS probes (200 μg/mL, ∼20 μL) under the Raman detection parameter were also recorded for periods of 0−5 min.
Scheme 1. Schematic Design of CuS SERS Probes for Intraoperative Residual Tumor Detection, Ablation, and Self-Clearancea
a
CuS SERS probes consist of hollow CuS NPs incorporated with Raman reporters. In different laser irradiation conditions, CuS SERS probes sequentially exhibit enhanced Raman signals, hyperthermia effects, and dissociation behaviors. Taken the advantage of these triple features, CuS SERS probes manifest their potential applications in oncology surgery. CuS SERS probes are preoperatively injected into tumor tissues. After removing the visible tumor tissues, operators utilized the 785 nm laser (10 mW) to elicit the Raman signals of CuS SERS probes for residual tumor detection and then applied the 980 nm laser (800 mW, 3 min) to trigger hyperthermia of CuS SERS probes for residual tumor ablation, followed by continuous irradiation till 5 min for the self-clearance of CuS SERS probes from the tumors.
near-infrared (NIR) laser irradiation at a higher power, the CuS SERS probes dissociate from CuS shells into individual CuS crystals, resulting from their notable photothermal absorption within the NIR region.5,18,20,21 Simultaneously, the photothermal transition of CuS NPs contributes to hyperthermia-based therapeutic effects. Collectively, we use orthotopic prostate tumor as a model disease and delineate the CuS SERS probes capable of intraoperative detection of residual tumor lesions by Raman imaging, eradication of residual tumor lesions by hyperthermia, and degradation into small particles, facilitating their self-clearance from the tumor tissue and attenuating long-term tissue toxicity.
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MATERIALS AND METHODS
Materials. Poly(vinylpyrrolidone) (PVP-K40), 3,3′-diethylthiatricarbocyanine iodide (DTTC), 3,3′-diethylthiacarbocyanine iodide (DTC), and 3,3′-diethylthiadicarbocyanine iodide (DTDC) were purchased from Sigma-Aldrich Chemicals. Copper chloride (CuCl2), sodium sulfide (Na2S), and hydrazine hydrate were purchased from J&K Scientific (China). All reagents and consumables related to cell culture were ordered from Thermo Fisher Scientific. Preparation and Characterization of CuS SERS Probes. CuS SERS Probes were synthesized following a previously described method with minor modifications.22 Briefly, poly(vinylpyrrolidone) (PVP-K40, 0.48 g) was added to distilled water (50 mL) in a round bottom flask. CuCl2 solution (200 μL, 0.5 M) was then added to PVP solution under stirring at 660 rpm at 25 °C. Subsequently, NaOH solution (50 mL, pH 9.18) was added, followed by the addition of hydrazine hydrate (12.5 μL) to form a suspension of aggregate Cu2O spheres. After 6 min, Na2S stock solution (400 μL, 180 mg/mL) was injected into the suspension and the mixture was heated at 60 °C with gentle stirring at 660 rpm for 3.5 h. Subsequently, the mixture was centrifuged (13 500 rpm, 30 min) and washed twice with distilled water. The pellet (100 μL, 22 nM) was redispersed and mixed with 2 mL of deionized water and 20 μL of Raman reporters (16 mM) with 24 h of magnetic stirring. Subsequently, methoxy−PEG5000−thiol (400 μL, 10 mg/mL) was added and followed by another 24 h of rotation. Finally, the products were centrifuged, washed, and
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RESULTS AND DISCUSSION Preparation and Characterization CuS SERS Probes. The SERS probes were synthesized by initially preparing Cu2O spheres and sequentially reacting with Na2S solution.22,25 The yielding hollow CuS NPs are adsorbed with the Raman reporters and protected by an encapsulating poly(ethylene glycol) (PEG) layer (Figure 1a). The synthetic NPs had a hydrodynamic diameter of ∼126 nm with high monodispersity (Figure S1). As illustrated by transmission electron microscopic (TEM) imaging, the NPs contain a hollow interior encompassing a porous shell of 10−12 nm diameter (Figure 1b), formed by small CuS crystals through sulfidation due to 23437
DOI: 10.1021/acsami.9b00469 ACS Appl. Mater. Interfaces 2019, 11, 23436−23444
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Figure 1. Hollow CuS NPs as SERS substrates. (a) Schematic presentation illustrating the three-dimensional structure of CuS SERS probes. (b) TEM images of CuS SERS probes. Screening of a serial of Raman-active reporters including (c) DTTC, (d) DTDC, and (e) DTC with hollow CuS NPs as SERS substrates. Energy-level diagram of (f) DTTC, (g) DTDC, and (h) DTC adsorbed on hollow CuS NPs.
displays 3−30-fold higher Raman signal intensity at 506 cm−1 than that of DTDC and DTC (Figure S3a,b). Therefore, we used DTTC as the representative Raman reporter for further investigation. These DTTC-adsorbed, PEGylated hollow CuS NPs are refereed as CuS SERS probes hereinafter, and their enhancement factor was calculated to be within the scope of 3.9 × 102−4.6 × 104 for different characteristic peaks (Supporting Information, Table 1). We next explored the enhancement mechanism of reporter molecule−hollow CuS NP system. Two primary theories are widely accepted for SERS effects, the electromagnetic mechanism and the charge-transfer (CT) mechanism.30−32 Considering that the plasmon resonances of CuS lie in the second near-infrared region,33 which is far from the 785 nm exciting laser; thus, the CT mechanism, by forming chargetransfer complexes between DTTC and CuS NPs, may be dominantly responsible for the enhancement activity of CuS SERS probes.34−36 To gain insight into the CT mechanism in the hollow CuS NP−molecule system, the energy-level
the Kirkendall effect.26,27 Energy-dispersive spectroscopy (EDS) analysis verified that these hollow NPs were CuS crystallites (Figure S2). To evaluate their Raman enhancement effects, hollow CuS NPs were respectively adsorbed with a series of widely used Raman reporters, including 3,3′-diethylthiatricarbocyanine iodide (DTTC), 3,3′-diethylthiadicarbocyanine iodide (DTDC), and 3,3′-diethylthiacarbocyanine iodide (DTC). The Raman reporter-adsorbed hollow CuS NPs were subsequently exposed to a 785 nm laser with the imaging conditions of 3 s acquisition time, 10 mW laser power, and a 20× objective. A smooth single crystal is considered to be lack of SERS effect,28,29 and thus, we coated Raman reporters on the single-crystal CuS for comparison. Substantial Raman enhancement was observed for all of the three reporter molecules adsorbed on hollow CuS NPs, while negligible Raman signal was obtained from a reporter molecule-adsorbed CuS single crystal (Figures 1c−e and S3a). Thus, hollow CuS NPs could serve as a SERS-active substrate. Notably, DTTC 23438
DOI: 10.1021/acsami.9b00469 ACS Appl. Mater. Interfaces 2019, 11, 23436−23444
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ACS Applied Materials & Interfaces
Figure 2. Characterization of DTTC-adsorbed CuS SERS probes. (a) SERS spectra of CuS probes in the region of 480−560 cm−1 with the concentrations ranging from 280 to 0 pM. (b) SERS intensity at 506 cm−1 was linearly fitted as a function of logarithmic concentration of CuS SERS probes, and the limit of detection (LOD) was 1 pM. (c) Raman signal intensity of CuS SERS probes were kept stable during 72 h of incubation in the serum. Data are means ± SD (n = 3). (d) Raman signal intensity of CuS SERS probes remained constant under continuous laser irradiation (785 nm, 10 mW laser power) for 35 min.
Figure 3. In vitro photodegradation characteristics of CuS SERS probes. (a) Schematic presentation illustrating the photodegradation of CuS SERS probes upon laser irradiation. (b) Morphological changes of CuS SERS probes under continuous laser irradiation (980 nm, 800 mW) for 1−5 min. (c) Thermal imaging of CuS SERS probes and water. (d) Temperature elevations (ΔT) of CuS SERS probes and water under continuous laser irradiation (980 nm, 800 mW) for 5 min. (e) Laser irradiation dramatically decreased viabilities of PC3 cells incubated with CuS SERS probes at different concentrations (**p < 0.01 and ***p < 0.001). (f) Calcein AM/PI staining of PC3 cells indicate the therapeutic effects of CuS SERS probes upon laser irradiation.
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DOI: 10.1021/acsami.9b00469 ACS Appl. Mater. Interfaces 2019, 11, 23436−23444
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Figure 4. CuS SERS probes for in vivo Raman imaging, therapy, and self-clearance. (a) Schematic illustration of CuS SERS probes for intraoperative residual tumor detection, eradication, and self-clearance. (b) Raman imaging of residual tumor cells. The white dotted line represents resected bed after surgery. The yellow dashed box displays the Raman imaging regions. The magnified overlay of Raman and white light image shows multiple submillimeter positive foci of CuS probes based on the “fingerprints-like” Raman spectra. H&E staining confirms that these Raman signal-positive foci are tumor tissues (area 1), rather than normal bladder tissues (area 2), and that these Raman signals are originated from the existing CuS probes (brown arrows in TEM image) in the tumor focis (area 1). (c) Thermal images of PC3 tumor-bearing mice with laser irradiation of CuS SERS while using phosphate-buffered saline (PBS) as the control. (d) Bioluminescence images of monitoring PC3 tumors’ regrowth in different treatment groups (PBS, surgery resection, and surgery resection with laser irradiation; n = 5 mice per group). (e) Tumor growth of mice monitored by bioluminescence signals in three treatment groups. (f) Curves of mice body weight in three treatment groups. (g) Survival curves of mice in three treatment groups. (h) TEM images demonstrating the morphological changes of CuS SERS probes (brown arrows) distributed in tumor tissues before and after laser irradiation. (i) Laser irradiation accelerated the clearance of CuS SERS from tumors over day 15 and day 28 (***p < 0.001). 23440
DOI: 10.1021/acsami.9b00469 ACS Appl. Mater. Interfaces 2019, 11, 23436−23444
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ACS Applied Materials & Interfaces
3c,d). Additionally, the temperature increase of the CuS SERS probe is in a laser power-dependent manner, ranging from 500 to 900 mW (Figure S4b). The photothermal transition efficiency of CuS SERS probes was then calculated to be 52.3% according to Roper’s method (Figures S4c,d),43 which is higher than that of most existing NPs with the d−d energy band transition (typically less than 30%).5,44 CuS SERS probes maintained little variation of temperature elevation of 60 °C during the five repeated laser on/off cycles (Figure S4e), exhibiting robust photothermal transition stability. Notably, the temperature of CuS SERS probes uninterruptedly increased to 85 °C, regardless of its structure disintegration in 4−5 min (Figure S4f). This is mainly because the photothermal transition effects are derived from the d−d energy band transition of Cu2+ ions rather than structuredependent plasmonic resonance.45,46 Although the probes were dissembled into small CuS crystals, the overall amount of Cu2+ transition is kept the same and thus the photothermal effects. Simultaneously, the photothermal effects contribute to the cell-based therapeutic effects of CuS SERS probes (Figure 3e,f). Prostate cancer PC3 cells (PC-3M-luc-C6) were incubated with the probes at a serial of concentration for 4 h, allowing for efficient cellular uptake. Cells were then exposed to a 5 min, 980 nm laser irradiation, followed by monitoring the cell viability. CuS SERS probes caused grievous photothermal destruction to tumor cells in a dose-dependent fashion (Figure 3e). The remarkable therapeutic effects were further confirmed by fluorescence imaging, where calcein AM with green color labels living cells and PI with red color labels dead cells. CuS SERS under the laser irradiation group manifested a complete cell death, whereas cells with SERS probes or laser exposure alone remained alive (Figure 3f). Notably, photothermally induced dissemination of CuS SERS probes would not happen under the Raman imaging condition and thus would not affect their Raman detection. Within the 3−5 s exposure time at the laser condition for Raman imaging, CuS SERS probes would merely elicit weak photothermal effect with a slight temperature increase (ΔT) of less than 1 °C and the morphology would remain unchanged even upon continuous 5 min irradiation (Figure S5). Therefore, we can obtain a stable signal for Raman detection (Figure 2d). CuS SERS Probes for in Vivo Raman Imaging, Therapy, and Self-Clearance. We next explored the in vivo application of CuS SERS Probes. Given that CuS SERS probes could combine SERS imaging with photothermal therapy, and could be photodegradable in a laser-manipulated manner, it would have tremendous potential in the localized complete cancer surgery. As a proof of concept, we established an orthotopic mouse model of luciferase-transfected prostate tumor (PC-3M-luc-C6) to simulate tumor surgery in the clinic (Figure 4a).47 CuS SERS probes were injected locally into the mice tumor prior to the surgery. Subsequently, surgical resection of observable bulk tumors under white light illumination was performed (Figure 4b). Until the maximal surgical resection was achieved, we corroborated the capability of CuS SERS probes for Raman imaging-based detection of residual tumor lesions. In Figure 4b, although light imaging did not reveal any residual tumor tissue, Raman imaging evidently identified multiple submillimeter tumor lesions harboring CuS SERS probes around the resection bed. Hematoxylin and eosin (H&E) staining substantiated the correlation of Raman signalpositive areas with the presence of residual microscopic tumor
diagram of these three molecules on hollow CuS NPs were obtained (Figure 1f−h). The conduction band (CB) and valence band (VB) of semiconductor CuS are documented as −4.29 and −6.29 eV, respectively.37 Meanwhile, the energy gap between lowest unoccupied molecular orbital (LUMO) and the highest occupied molecular orbital (HOMO) levels of DTTC, DTDC, and DTC are estimated as 1.59, 1.9, and 2.1 eV, respectively.38−40 As shown in Figure 1f−h, the overall Raman enhancement in the CuS NPs may result from the following category of charge-transfer resonance, including molecule resonance (μmol) of reporter molecules, the photoninduced charge-transfer resonance (μPICT), exciton resonance (μex) of surface state energy levels (Ess), and the ground-state charge-transfer resonance (μGSCT) from matched energy level between hollow CuS NPs and Raman reporters. In detail, the DTTC molecule exhibits the CT process as follows: (I) electrons from the HOMO of the adsorbed DTTC molecule are excited to the LUMO by the 785 nm laser (1.58 eV) and then injected into CuS CB (conduction band). (II) Electrons of CuS VB (valence band) are excited to surface state energy levels (Ess) by the 785 nm laser (1.58 eV). (III) Electrons from the HOMO of the adsorbed DTTC molecule are excited to CuS CB by the incident light (785 nm, 1.58 eV). (IV) Electrons from the HOMO of the adsorbed DTTC molecule are excited to surface state energy levels (Ess) of CuS by the incident light (785 nm, 1.58 eV). Additionally, DTDC and DTC molecules on hollow CuS NPs demonstrated similar CT modes to DTTC except for CT process I; this is because that the energy gap between HOMO and LUMO of DTDC (1.9 eV) or DTC (2.1 eV) is higher than the incident light (785 nm, 1.58 eV), and thus, the electrons transitions from molecules HOMO to LUMO were hardly excited. We subsequently determined the detection threshold of CuS SERS probes by analyzing their Raman spectra in the region of 480−560 cm−1 with the concentrations of 0−280 pM (Figure 2a). Figure 2b depicts the intensity of the SERS peak at 506 cm−1 as a function of logarithmic concentrations of CuS SERS probes, illustrating a linear range from 8 to 280 pM. The limit of detection (LOD) was estimated to be 1 pM. Moreover, the CuS SERS probes demonstrate minimal variation in terms of Raman signal intensity in serum at 37 °C for 72 h (Figure 2c) and upon continuous laser irradiation for 35 min (Figure 2d), and thus, the CuS SERS probes have the potential to serve as a photostable Raman imaging nanoplatform for in vivo applications. Photodegradation Characteristics of CuS SERS Probes. The photodegradation characteristics of CuS SERS probes are depicted in Figure 3a. TEM imaging illustrates morphological changes of CuS SERS probes upon continuous NIR laser irradiation (980 nm, 800 mW) for 5 min (Figure 3b). In the first 1−3 min, the hollow spherelike CuS SERS probes emerged a slight accumulation. In the 4th min, the dense shell of CuS SERS probes became loose and the hollow area was filled with flocculelike small CuS clusters. In the 5th min, the loose CuS SERS probes completely broke down into small NPs of less than 20 nm (magnified image). The photodegradation of CuS SERS probe might result from the elevated surface temperature of CuS by its strong absorbance in the range of 700−1100 nm5,41,42 (Figure S4a), thus promoting disintegration of the CuS SERS probe from polycrystalline structures into individual crystals. Indeed, the photothermal effects of CuS SERS probes contributed to a rapid temperature increase (ΔT) of 58.6 °C in 5 min (Figure 23441
DOI: 10.1021/acsami.9b00469 ACS Appl. Mater. Interfaces 2019, 11, 23436−23444
Research Article
ACS Applied Materials & Interfaces
contrast, there was no obvious damage to the harvested organs in the surgery-with-laser intervention group. To further assess the in vivo safety of CuS SERS probes, we evaluated their biodistribution and elimination via systematic delivery in PC3 tumor models. At 24 h postinjection, CuS SERS probes were distributed into the targeted tumors and other organs including heart, liver, spleen, lungs, and kidneys (Figure S8a). Particularly, CuS SERS probes showed a good targeting ability with 3.6% ID/g (i.e., percentage of the injected dose per gram of the tissue) of Cu level in tumor due to the widely accepted enhanced permeability and retention effects. We subsequently examined the elimination of CuS SERS probes from main organs within 30 days (Figure S8b). At day 1, CuS SERS probes demonstrated obvious retention in most of the organs; however, at day 30 postinjection, only 0.6, 0.4, 0.9, 0.5, and 0.3% ID/g of Cu retained in heart, liver, spleen, lungs, and kidneys, respectively (Figure S8). This result illustrated that CuS SERS probes were efficiently eliminated from body, further confirming the biosafety of CuS SERS probes. A similar observation has been reported in a previous comparison toxicity study on CuS NPs with Au NPs.18 In this study, CuS NPs are effectively eliminated through hepatobiliary and renal excretion at day 30 postinjection and do not demonstrate any toxicity in the histological or blood chemistry analysis; in contrast, Au NPs are long-term accumulated in the liver, thus leading to elevated serum lactate dehydrogenase and irreversible changes, indicating potential long-term toxicity.18
lesions. Meanwhile, the TEM image revealed the existence of CuS SERS probes inside these tumor lesions (Figure 4b). To illustrate the photothermal effects of CuS SERS probes in residual tumors, we utilized a 980 nm laser (800 mW, 5 min) to irradiate the signal-positive areas. In the CuS group, the temperature rapidly reached ∼43.4 °C in 1 min and continuously increased to ∼49.1 °C in 3 min (Figure 4c), revealing that a trace amount of CuS SERS probes (42 °C); irradiation was then stopped intermittently for 5 s in 4−5 min, maintaining the temperature of 42−50 °C to avoid overheating (>50 °C).48 In contrast, the PBS group exhibited a negligible temperature increase of 27.6−36.8 °C in 1−3 min and remained almost unchanged in the following 4−5 min, suggesting that the irradiation itself would not elicit hyperthermia. We subsequently evaluated the hyperthermia of CuS SERS probes for eradicating the residual tumors. At surgery day (0 day), mice with distinct treatments were divided into three groups (PBS, resection, and resection with laser irradiation). After the surgery, the wounds were closed and tumor recurrence recorded by the bioluminescent signals was continuously monitored for 16 days (Figures 4d−f and S6). The PBS group without surgical resection resulted in sustainable growth of tumor volumes. Due to surgical resection of bulk tumors, the CuS SERS probe group without laser irradiation exhibited declined tumor signals at day 3; however, the regrowth of residual tumor lesions led to a rapid signal relapse after day 6. Comparably, the CuS SERS probe group with laser irradiation displayed entire tumor ablation with no recurrence during the observation, mainly due to the remarkable hyperthermia destruction to residual tumors by CuS SERS probes. The monitoring of mice survival in three groups further confirmed therapeutic effectiveness. All of the mice in the CuS SERS with laser treatment group obtained exhaustive tumor-free survival, whereas the mice in the other two groups died of disease (Figure 4g). In addition, CuS SERS probes had no effects on the body weight of the mice in the three groups. We then verified the degradation of CuS SERS probes by laser irradiation (980 nm laser, 800 mW, 5 min) in tumor tissues, thus facilitating their self-clearance from tumors. In Figure 4h, CuS SERS probes without laser treatment remained intact in tumors; in comparison, laser irradiation triggered disintegration of CuS probes into small particles, sporadically dispersing in the tumor tissues. We subsequently tested the clearance of CuS SERS probes from tumors by quantitatively analyzing the leftover amount of Cu inside tumors at indicated timepoints.20 For the CuS probe group without laser intervention, about 43.5% injected dose (ID) and 21.7% ID of Cu were remained inside tumors at day 15 day and day 28 postinjection, respectively; in contrast, upon laser irradiation, majority of CuS SERS probes were eliminated from tumors, with merely one-third (∼16.5% ID) of Cu at day 15 and onefifth (∼4.2% ID) of Cu at day 28 left inside tumor tissues (Figure 4i), denoting that hyperthermia-induced dissemination of CuS SERS probes accelerated their clearance from the tumors. To further evaluate whether the clearance ameliorate the potential toxicity, H&E staining was performed on the main organs (heart, liver, spleen, lung, and kidney) of the mice (Figure S7a). Notably, metastasis of residual tumors to the livers and dramatic inflammation of spleens were observed in the mice without the laser intervention group (Figure S7b). In
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CONCLUSIONS In summary, we reported the employment of hollow CuS NPs to serve as SERS substrate materials, resulting in significant amplification in Raman signature signals. We, for the first time, demonstrate that hollow CuS NPs could serve as SERS probes and achieve biomedical applications in vivo. Importantly, these hollow CuS-based SERS probes have been confirmed to render two unique features over current SERS nanoplatforms, photodegradability and robust therapeutic functionality.18,20 In detail, the hollow CuS NPs can be disseminated into small clusters upon NIR light irradiation, thus facilitating their postimaging clearance from the tissues and avoiding potential long-term toxicity. CuS NPs acquire absorption in the NIR region from the d−d energy band transition of Cu2+ ions, and thus, their photothermal therapeutic efficacy remain consistent regardless of shape transition, dissociation, and encompassing environment.5 This allows for coincidently achieving satisfactory SERS enhancement and photothermal therapeutic efficacy. With the triple Raman imaging−therapy−photodegradation features of this novel CuS SERS probe, we have demonstrated its in vivo application of intraoperative theranostics of residual tumors, further facilitating its postimaging clearance from tumors. Despite using local injection in this study, we anticipate that the application of CuS SERS probes may be broadened to other biomedical applications with systematic administration. Importantly, due to the hollow structures of CuS SERS probes capable of incorporating multiple drugs and imaging agents, a variety of new theranostic-related drug delivery applications for clinical translation can be envisioned.
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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.9b00469. 23442
DOI: 10.1021/acsami.9b00469 ACS Appl. Mater. Interfaces 2019, 11, 23436−23444
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Adiabatic Compression of Surface Plasmon Polaritons. Nat. Nanotechnol. 2010, 5, 67−72. (5) Yang, T.; Wang, Y.; Ke, H.; Wang, Q.; Lv, X.; Wu, H.; Tang, Y.; Yang, X.; Chen, C.; Zhao, Y.; Chen, H. Protein-Nanoreactor-Assisted Synthesis of Semiconductor Nanocrystals for Efficient Cancer Theranostics. Adv. Mater. 2016, 28, 5923−5930. (6) Andreou, C.; Neuschmelting, V.; Tschaharganeh, D. F.; Huang, C. H.; Oseledchyk, A.; Iacono, P.; Karabeber, H.; Colen, R. R.; Mannelli, L.; Lowe, S. W.; Kircher, M. F. Imaging of Liver Tumors Using Surface-Enhanced Raman Scattering Nanoparticles. ACS Nano 2016, 10, 5015−5026. (7) Zhang, Y. Q.; Qiu, Y. Y.; Lin, L.; Gu, H. C.; Xiao, Z. Y.; Ye, J. Ultraphotostable Mesoporous Silica-Coated Gap-Enhanced Raman Tags (GERTs) for High-Speed Bioimaging. ACS Appl. Mater. Interfaces 2017, 9, 3995−4005. (8) Qiu, Y.; Zhang, Y.; Li, M.; Chen, G.; Fan, C.; Cui, K.; Wan, J.-B.; Han, A.; Ye, J.; Xiao, Z. Intraoperative Detection and Eradication of Residual Microtumors with Gap-Enhanced Raman Tags. ACS Nano 2018, 12, 7974−7985. (9) Pazos, E.; Garcia-Algar, M.; Penas, C.; Nazarenus, M.; Torruella, A.; Pazos-Perez, N.; Guerrini, L.; Vazquez, M. E.; Garcia-Rico, E.; Mascarenas, J. L.; Alvarez-Puebla, R. A. Surface-Enhanced Raman Scattering Surface Selection Rules for the Proteomic Liquid Biopsy in Real Samples: Efficient Detection of the Oncoprotein C-MYC. J. Am. Chem. Soc. 2016, 138, 14206−14209. (10) Song, J.; Duan, B.; Wang, C.; Zhou, J.; Pu, L.; Fang, Z.; Wang, P.; Lim, T. T.; Duan, H. SERS-Encoded Nanogapped Plasmonic Nanoparticles: Growth of Metallic Nanoshell by Templating RedoxActive Polymer Brushes. J. Am. Chem. Soc. 2014, 136, 6838−6841. (11) Xu, W. G.; Xiao, J. Q.; Chen, Y. F.; Chen, Y. B.; Ling, X.; Zhang, J. Graphene-Veiled Gold Substrate for Surface-Enhanced Raman Spectroscopy. Adv. Mater. 2013, 25, 928−933. (12) Masetti, M.; Xie, H. N.; Krpetic, Z.; Recanatini, M.; AlvarezPuebla, R. A.; Guerrini, L. Revealing DNA Interactions with Exogenous Agents by Surface-Enhanced Raman Scattering. J. Am. Chem. Soc. 2015, 137, 469−476. (13) Shan, C.; Yuan, Y.; Chen, Z.; Hou, J.; Mei, Y.; Su, Y.; Zhang, Y.; Liang, L.; Li, Q.; Geng, F. Noble Metal-Comparable SERS Enhancement from Semiconducting Metal Oxides by Making Oxygen Vacancies. Nat. Commun. 2015, 6, No. 7800. (14) Li, W.; Zamani, R.; Gil, P. R.; Pelaz, B.; Ibanez, M.; Cadavid, D.; Shavel, A.; Alvarez-Puebla, R. A.; Parak, W. J.; Arbiol, J.; Cabot, A. CuTe Nanocrystals: Shape and Size Control, Plasmonic Properties, and Use as SERS Probes and Photothermal Agents. J. Am. Chem. Soc. 2013, 135, 7098−7101. (15) Gao, X.; Yue, Q.; Liu, Z.; Ke, M.; Zhou, X.; Li, S.; Zhang, J.; Zhang, R.; Chen, L.; Mao, Y.; Li, C. Guiding Brain-Tumor Surgery via Blood-Brain-Barrier-Permeable Gold Nanoprobes with Acid-Triggered MRI/SERRS Signals. Adv. Mater. 2017, 29, No. 1603917. (16) Qian, J.; Jiang, L.; Cai, F. H.; Wang, D.; He, S. L. FluorescenceSurface Enhanced Raman Scattering co-Functionalized Gold Nanorods as Near-infrared Probes for Purely Optical In Vivo Imaging. Biomaterials 2011, 32, 1601−1610. (17) Jung, K.; Hahn, J.; In, S.; Bae, Y.; Lee, H.; Pikhitsa, P. V.; Ahn, K.; Ha, K.; Lee, J. K.; Park, N.; Choi, M. Hotspot-Engineered 3D Multipetal Flower Assemblies for Surface-Enhanced Raman Spectroscopy. Adv. Mater. 2014, 26, 5924−5929. (18) Guo, L. R.; Panderi, I.; Yan, D. D.; Szulak, K.; Li, Y. J.; Chen, Y. T.; Ma, H.; Niesen, D. B.; Seeram, N.; Ahmed, A.; Yan, B. F.; Pantazatos, D.; Lu, W. A Comparative Study of Hollow Copper Sulfide Nanoparticles and Hollow Gold Nanospheres on Degradability and Toxicity. ACS Nano 2013, 7, 8780−8793. (19) Sharifi, S.; Behzadi, S.; Laurent, S.; Forrest, M. L.; Stroeve, P.; Mahmoudi, M. Toxicity of Nanomaterials. Chem. Soc. Rev. 2012, 41, 2323−2343. (20) Guo, L. R.; Yan, D. D.; Yang, D. F.; Li, Y. J.; Wang, X. D.; Zalewski, O.; Yan, B. F.; Lu, W. Combinatorial Photothermal and Immuno Cancer Therapy Using Chitosan-Coated Hollow Copper Sulfide Nanoparticles. ACS Nano 2014, 8, 5670−5681.
Experiment details and materials: Raman measurements of Raman reporter-adsorbed hollow CuS NPs and single-crystal CuS; calculation of the enhancement factor; photodegradation characteristics of CuS SERS probes, photothermal effect, photostability, and photothermal conversion efficiency; calcein AM/PI staining; in vitro photothermal cytotoxicity of CuS SERS probes; in vivo Raman imaging and photothermal ablation of residual tumor tissues; degradation characteristics of CuS SERS probes and their clearance from tumors; and biodistribution of CuS SERS probes in mice by systematic delivery and statistical analysis (PDF)
AUTHOR INFORMATION
Corresponding Authors
*E-mail:
[email protected] (X.F.). *E-mail:
[email protected] (Z.X.). ORCID
Zhigang Zhao: 0000-0002-9327-9893 Lei Fu: 0000-0002-0802-6446 Xiaohong Fang: 0000-0002-2018-0542 Zeyu Xiao: 0000-0002-3457-5772 Author Contributions ⊥
Y.Q., M. L., and G.C. contributed equally to this work.
Author Contributions
Z.X. and Y.Q. designed experiments. Y.Q., M.L., and G.C. carried out experiments. M.L. assisted with cell imaging and schematic figure design. X.G. assisted with nanoparticle synthesis. S.C. and Z.Z. assisted with the analysis of enhancement mechanism. All authors analyzed and discussed experimental results. Z.X., Y.Q., and X.F. wrote the manuscript. All authors edited the manuscript and approved the final version. Z.X. obtained the funding support and supervised all studies. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We gratefully acknowledge the National Natural Science Foundation of China (Nos. 31671003, 21874092, and 81471779), the Thousand Young Talents Program, the Program for Professor of Special Appointment (Eastern Scholar) at Shanghai Institutions of Higher Learning (No. TP2014028) for their financial support. We gratefully acknowledge Prof. Bin Zhao from Jilin University and Prof. Haifeng Yang from Shanghai Normal University.
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REFERENCES
(1) Gandra, N.; Singamaneni, S. Bilayered Raman-Intense Gold Nanostructures with Hidden Tags (BRIGHTs) for High-Resolution Bioimaging. Adv. Mater. 2013, 25, 1022−1027. (2) Harmsen, S.; Huang, R.; Wall, M. A.; Karabeber, H.; Samii, J. M.; Spaliviero, M.; White, J. R.; Monette, S.; O’Connor, R.; Pitter, K. L.; et al. Surface-Enhanced Resonance Raman Scattering Nanostars for High-Precision Cancer Imaging. Sci. Transl. Med. 2015, 7, No. 271ra7. (3) Lin, L.; Tian, X.; Hong, S.; Dai, P.; You, Q.; Wang, R.; Feng, L.; Xie, C.; Tian, Z. Q.; Chen, X. A Bioorthogonal Raman Reporter Strategy for SERS Detection of Glycans on Live Cells. Angew. Chem., Int. Ed. 2013, 52, 7266−7271. (4) De Angelis, F.; Das, G.; Candeloro, P.; Patrini, M.; Galli, M.; Bek, A.; Lazzarino, M.; Maksymov, I.; Liberale, C.; Andreani, L. C.; Di Fabrizio, E. Nanoscale Chemical Mapping Using Three-Dimensional 23443
DOI: 10.1021/acsami.9b00469 ACS Appl. Mater. Interfaces 2019, 11, 23436−23444
Research Article
ACS Applied Materials & Interfaces (21) Li, Y.; Cupo, M.; Guo, L.; Scott, J.; Chen, Y.-T.; Yan, B.; Lu, W. Enhanced Reactive Oxygen Species through Direct Copper Sulfide Nanoparticle-Doxorubicin Complexation. Nanotechnology 2017, 28, No. 505101. (22) Ramadan, S.; Guo, L.; Li, Y.; Yan, B.; Lu, W. Hollow Copper Sulfide Nanoparticle-Mediated Transdermal Drug Delivery. Small 2012, 8, 3143−3150. (23) Li, M.; Cushing, S. K.; Liang, H.; Suri, S.; Ma, D.; Wu, N. Plasmonic Nanorice Antenna on Triangle Nanoarray for SurfaceEnhanced Raman Scattering Detection of Hepatitis B Virus DNA. Anal. Chem. 2013, 85, 2072−2078. (24) Gilfrich, J. V.; Birks, L. S. Estimation of Detection Limits in Xray Fluorescence Spectrometry. Anal. Chem. 1984, 56, 77−79. (25) Bai, L.; Ye, X.; Song, C.; Chen, H.; Cai, B.; Bai, Y.; Xu, C.; Li, Z. The Surfactant-Free Synthesis of Hollow CuS Nanospheres Via Clean Cu2O Templates and Their Catalytic Oxidation of Dye Molecules with H2O2. RSC Adv. 2016, 6, 83885−83889. (26) Lee, Y. I. Selective Transformation of Cu Nanowires to Cu2S or CuS Nanostructures and the Roles of the Kirkendall Effect and Anion Exchange Reaction. Mater. Chem. Phys. 2016, 180, 104−113. (27) Zhu, H.; Wang, J.; Wu, D. Fast Synthesis, Formation Mechanism, and Control of Shell Thickness of CuS Hollow Spheres. Inorg. Chem. 2009, 48, 7099−7104. (28) García-Vidal, F. J.; Pendry, J. B. Collective Theory for Surface Enhanced Raman Scattering. Phys. Rev. Lett. 1996, 77, 1163−1166. (29) Ren, B.; Picardi, G.; Pettinger, B.; Schuster, R.; Ertl, G. TipEnhanced Raman Spectroscopy of Benzenethiol Adsorbed on Au and Pt Single-Crystal Surfaces. Angew. Chem., Int. Ed. 2005, 44, 139−142. (30) Xu, H.; Xie, L.; Zhang, H.; Zhang, J. Effect of Graphene Fermi Level on the Raman Scattering Intensity of Molecules on Graphene. ACS Nano 2011, 5, 5338−5344. (31) Lin, J.; Shang, Y.; Li, X.; Yu, J.; Wang, X.; Guo, L. Ultrasensitive SERS Detection by Defect Engineering on Single Cu2O Superstructure Particle. Adv. Mater. 2016, 29, No. 1604797. (32) Wang, X.; Shi, W.; She, G.; Mu, L. Using Si and Ge Nanostructures as Substrates for Surface-Enhanced Raman Scattering Based on Photoinduced Charge Transfer Mechanism. J. Am. Chem. Soc. 2011, 133, 16518−16523. (33) Wei, T.; Liu, Y.; Dong, W.; Zhang, Y.; Huang, C.; Sun, Y.; Chen, X.; Dai, N. Surface-Dependent Localized Surface Plasmon Resonances in CuS Nanodisks. ACS Appl. Mater. Interfaces 2013, 5, 10473−10477. (34) Wang, X.; Shi, W.; She, G.; Mu, L. Surface-Enhanced Raman Scattering (SERS) on Transition Metal and Semiconductor Nanostructures. Phys. Chem. Chem. Phys. 2012, 14, 5891−5901. (35) Lin, J.; Hao, W.; Shang, Y.; Wang, X.; Qiu, D.; Ma, G.; Chen, C.; Li, S.; Guo, L. Direct Experimental Observation of FacetDependent SERS of Cu2O Polyhedra. Small 2017, 14, No. 1703274. (36) Wang, X.; She, G.; Xu, H.; Mu, L.; Shi, W. The SurfaceEnhanced Raman Scattering from ZnO Nanorod Arrays and Its Application for Chemosensors. Sens. Actuators, B 2014, 193, 745− 751. (37) He, H. Y. Facile Synthesis of Ultrafine CuS Nanocrystalline/ TiO2: Fe Nanotubes Hybrids and Their Photocatalytic and Fentonlike Photocatalytic Activities in the Dye Degradation. Microporous Mesoporous Mater. 2016, 227, 31−38. (38) Ho, C. C.; Chen, H. F.; Ho, Y. C.; Liao, C. T.; Su, H. C.; Wong, K. T. Phosphorescent Sensitized Fluorescent Solid-State NearInfrared Light-Emitting Electrochemical Cells. Phys. Chem. Chem. Phys. 2011, 13, 17729−17736. (39) Kolic, P. E.; Siraj, N.; Cong, M.; Regmi, B. P.; Luan, X.; Ying, W.; Warner, M. I. Improving Energy Relay Dyes for Dye-Sensitized Solar Cells by Use of a Group of Uniform Materials Based on Organic Salts (GUMBOS). RSC Adv. 2016, 6, 95273−95282. (40) Pepe, G.; Cole, J. M.; Waddell, P. G.; McKechnie, S. Molecular Engineering of Cyanine Dyes to Design a Panchromatic Response in Co-Sensitized Dye-Sensitized Solar Cells. Mol. Syst. Des. Eng. 2016, 1, 86−98.
(41) Tian, Q.; Tang, M.; Sun, Y.; Zou, R.; Chen, Z.; Zhu, M.; Yang, S.; Wang, J.; Wang, J.; Hu, J. Hydrophilic Flower-Like CuS Superstructures as an Efficient 980 nm Laser-Driven Photothermal Agent for Ablation of Cancer Cells. Adv. Mater. 2011, 23, 3542−3547. (42) Riedinger, A.; Avellini, T.; Curcio, A.; Asti, M.; Xie, Y.; Tu, R.; Marras, S.; Lorenzoni, A.; Rubagotti, S.; Iori, M.; Capponi, P. C.; Versari, A.; Manna, L.; Seregni, E.; Pellegrino, T. Post-Synthesis Incorporation of 64Cu in CuS Nanocrystals to Radiolabel Photothermal Probes: A Feasible Approach for Clinics. J. Am. Chem. Soc. 2015, 137, 15145−15151. (43) Roper, D. K.; Ahn, W.; Hoepfner, M. Microscale Heat Transfer Transduced by Surface Plasmon Resonant Gold Nanoparticles. J. Phys. Chem. C 2007, 111, 3636−3641. (44) Li, Y.; Lu, W.; Huang, Q.; Li, C.; Chen, W. Copper Sulfide Nanoparticles for Photothermal Ablation of Tumor Cells. Nanomedicine 2010, 5, 1161−1171. (45) Wang, Z. T.; Huang, P.; Jacobson, O.; Wang, Z.; Liu, Y. J.; Lin, L. S.; Lin, J.; Lu, N.; Zhang, H. M.; Tian, R.; Niu, G.; Liu, G.; Chen, X. Y. Biomineralization-Inspired Synthesis of Copper Sulfide-Ferritin Nanocages as Cancer Theranostics. ACS Nano 2016, 10, 3453−3460. (46) Zhou, M.; Zhang, R.; Huang, M.; Lu, W.; Song, S.; Melancon, M. P.; Tian, M.; Liang, D.; Li, C. A Chelator-Free Multifunctional [64Cu]CuS Nanoparticle Platform for Simultaneous Micro-PET/CT Imaging and Photothermal Ablation Therapy. J. Am. Chem. Soc. 2010, 132, 15351−15358. (47) Lin, Q. Y.; Jin, C. S.; Huang, H.; Ding, L. L.; Zhang, Z. H.; Chen, J.; Zheng, G. Nanoparticle-Enabled, Image-Guided Treatment Planning of Target Specific RNAi Therapeutics in an Orthotopic Prostate Cancer Model. Small 2014, 10, 3072−3082. (48) Chen, F.; Hong, H.; Goel, S.; Graves, S. A.; Orbay, H.; Ehlerding, E. B.; Shi, S. X.; Theuer, C. P.; Nickles, R. J.; Cai, W. B. In Vivo Tumor Vasculature Targeting of CuS@MSN Based Theranostic Nanomedicine. ACS Nano 2015, 9, 3926−3934.
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