Hyperthemia-Promoted Cytosolic and Nuclear Delivery of Copper

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Hyperthemia-Promoted Cytosolic and Nuclear Delivery of Copper/Carbon Quantum Dot-Crosslinked Nanosheets: Multimodal Imaging-Guided Photothermal Cancer Therapy Yan-Wen Bao, Xian-Wu Hua, Yan-Hong Li, Hao-Ran Jia, and Fu-Gen Wu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b15332 • Publication Date (Web): 20 Dec 2017 Downloaded from http://pubs.acs.org on December 21, 2017

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ACS Applied Materials & Interfaces

Hyperthemia-Promoted Cytosolic and Nuclear Delivery of Copper/Carbon Quantum

Dot-Crosslinked

Nanosheets:

Multimodal

Imaging-Guided

Photothermal Cancer Therapy

Yan-Wen Bao,† Xian-Wu Hua,† Yan-Hong Li, Hao-Ran Jia, and Fu-Gen Wu*

State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University, 2 Sipailou Road, Nanjing 210096, P. R. China

KEYWORDS: carbon dots (CDs), copper-containing photothermal agents, high photothermal

conversion

efficiency,

hyperthemia-induced

photothermal therapy

1 ACS Paragon Plus Environment

lysosomal

escape,

ACS Applied Materials & Interfaces 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT Copper-containing nanomaterials have been applied in various fields due to their appealing physical, chemical, and biomedical properties/functions. Herein, for the first time, a facile, room-temperature and one-pot method of simply mixing copper ions and sulfur-doped CDs is developed for the synthesis of copper/carbon quantum dot (or carbon dot, CD)-crosslinked nanosheets (CuCD NSs). The thus-obtained CuCD NSs with the size of 20–30 nm had a high photothermal conversion efficiency of 41.3% and good photothermal stability. Especially, after coating with thiol-polyethylene glycol and fluorescent molecules, the resultant CuCD NSs could selectively target tumor tissues and realize multimodal (photoacoustic, photothermal, and fluorescence) imaging-guided cancer therapy. More importantly, our CuCD NSs exhibited laser-triggered cytosolic delivery, lysosomal escape, and nuclear-targeting properties, which greatly enhanced their therapeutic efficacy. The significantly enhanced tumor accumulation of CuCD NSs after in situ tumor-site laser irradiation was also observed in in vivo experiments. These in vitro and in vivo events occurring during the continuous laser irradiation have not been observed. Overall, this work develops a CDs-assisted synthetic method of photothermal nanoagents for triple-modal imaging-guided phototherapy and deepens our understanding of the action mechanism of photothermal therapy, which will promote the development of nanomedicine and beyond.


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1. INTRODUCTION

Owing to their fascinating physical, chemical, and biomedical properties/functions, copper-containing nanomaterials are fabricated and applied in numerous fields such as electronics,1 catalysis,2,3 multiple modality cancer imaging,4–7 photo-/radiotherapy,8–11 and bacterial treatment.12 In photothermal therapy (PTT), a minimally invasive or noninvasive treatment with high efficiency,13 copper-containing nanomaterials possess some appealing advantages over several other metal nanomaterials14–16 (e.g. Au, Pd, and Mo) and organic dyes17–20 (e.g. cypate, IR825, and indocyanine green). First, as an earth-abundant element, copper is less expensive as compared with other noble metals. Second, copper-based nanoagents are biodegradable and can release vital trace copper element for maintaining good health.21 Third, when compared to organic compounds, copper-containing nanomaterials, especially copper sulfide nanoparticles (CuS NPs),10,22,23 have attracted increasing attention due to their superior photostability, easily modifiable surface, and good PT properties. However, the biomedical applications of CuS NPs may be limited because of their dose-dependent cytotoxicity.24 Besides, although various synthetic approaches of copper-containing nanoparticles have been reported,12,25–27 their wide applications still suffer from harsh preparation conditions including relative high temperature, high pressure, oxygen-free environment, and long reaction time. Therefore, it is highly desirable to develop a facile preparation method of copper-containing nanoagents with excellent photothermal properties and low cytotoxicity for cancer therapy. Carbon quantum dots (or carbon dots, CDs) represent a type of highly appealing



carbon nanomaterials due to their inherent advantages including excellent water dispersibility, unique optical features, superior photostability, and low toxicity.28–31 These properties make CDs promising alternatives in numerous applications such as bioimaging,32–35 detection,36–39 drug delivery,40–43 catalysis,44,45 light-emitting devices,46 and phototheranostics.47,48 Moreover, there are a few reports on using metal-containing CD composites for catalysis,2,49 quantitative detection,50 magnetic resonance imaging (MRI),51 antibacterial agents,52 and photothermal agents.53 However, there have been no reports on CDs-assisted preparation of metal-containing nanoagents with excellent photothermal property for multimodal imaging-guided photothermal cancer therapy. Here, we report a novel and facile preparation method of copper/CD-crosslinked nanosheets (CuCD NSs) using copper ions and sulfur-doped CDs for multimodal imaging-guided photothermal cancer therapy (Scheme 1). The CDs acted as both the reductant and the template, which were synthesized using o-phenylenediamine and L-cysteine

by hydrothermal treatment. The CuCD NSs were then obtained through

simply mixing of Cu2+ solution and CDs solution without stirring at 25 °C for 2 h. The as-synthesized CuCD NSs show several advantages over conventional photothermal nanomaterials. First, the fabrication of CuCD NSs is facile and can be easily scaled up with good repeatability considering its low requirements of reaction conditions, facilities, and raw materials. Second, owning to their high near-infrared (NIR) optical absorption capability, excellent photothermal property (photothermal conversion efficiency: 41.3%; extinction coefficient at 808 nm: 19.5 L g–1 cm–1), good


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photothermal stability, proper size (20–30 nm), as well as good biocompatibility, CuCD NSs can successfully realize photoacoustic (PA) imaging and photothermal (PT)

imaging/therapy.

Besides,

after

thiol-polyethylene

glycol

(HS-PEG)

modification and fluorescence (FL) labeling, CuCD NSs efficiently target tumor sites through the enhanced permeability and retention (EPR) effect and realize FL imaging-guided PTT. Last but not least, CuCDs NSs can achieve light-triggered cytosolic and nuclear drug delivery. It is known that the limited cellular uptake and endo/lysosomal capture are two key barriers in drug delivery because nanodrugs are usually internalized by cells via endocytosis, entrapped in endosomes, and eventually accumulated within lysosomes, resulting in unexpected therapeutic efficacy.54–56 To overcome

the

endo/lysosomal

entrapment

problem,

organelle

(especially

mitochondria and nucleus)-targeted cancer treatment has become a hot topic because of the susceptibility of mitochondria/nucleus to reactive oxygen species (ROS) or hyperthermia.57–59 Surprisingly, our CuCD NSs, without extra modifications of organelle-targeting ligands, could escape from lysosomes with increased endocytosis and realize nucleus-targeted PTT under NIR laser irradiation, which significantly enhanced the therapeutic efficacy of the PT nanoagents with minimal side effects. The present work provides the first example of fabricating CuCD NSs as novel nanotheranostic agents for multimodal (PA, PT, and FL) imaging-guided photothermal cancer therapy.

Scheme 1. Schematic Illustrating the Fabrication of CuCD NSs and Their Application in



Multimodal Imaging-Guided Photothermal Cancer Therapy

2. EXPERIMENTAL DETAILS 2.1. Synthesis of Sulfur-Doped CDs and PEG-Modified CuCD NSs. First, o-phenylenediamine (300 mg) and L-cysteine (675 mg) were dissolved in 30 mL of sodium hydroxide (NaOH) solution (0.2 M, to increase the solubility of L-cysteine). Then the solution was transferred into a Teflon reactor. After experiencing the hydrothermal process at 160 °C for 10 h, the orange-yellow CDs solution was obtained. To synthesize CuCD NSs, Cu2+ solution (60 mmol) was simply mixed with the as-synthesized CDs solution (containing 50 mg CDs), and the formed mixture was kept at 25 °C without stirring for 2 h. After dialysis (MWCO: 1 kDa), the purified 6 ACS Paragon Plus Environment

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CuCD NSs solution was obtained. It is worth noting that the time-dependent reaction process was monitored by measuring the ultraviolet–visible (UV–vis) absorption spectra of the reacting solution at different time points (0, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 130, 140, 150, 160, 170, and 180 min). To improve the stability of CuCD NSs, 3 mL of CuCD NSs solution and 9 mg of thiol-PEG2k (HS-PEG2k) were mixed and the reaction was kept stirring at 25 °C overnight. The PEG-modified CuCD NSs solution was dialyzed (MWCO: 7 kDa) against deionized (DI) water for 48 h before final use. In addition, fluorescein isothiocyanate (FITC)/rhodamine B isothiocyanate (RITC)-labeled PEG-modified CuCD NSs were also synthesized for monitoring the cellular uptake of PEG-modified CuCD NSs in cells and in vivo, respectively. Briefly, the mixture of 1.0 mg of thiol-PEG2k-amine (HS-PEG2k-NH2) and 0.125 mg/0.268 mg of FITC/RITC was allowed to react in the sodium carbonate buffer (100 mM, pH 9.5) at 25 °C overnight, and then dialyzed for the next use. For 3 mL of CuCD NSs solution, 8.55 mg of HS-PEG2k and 0.51 mg of HS-PEG2k-FITC/0.57 mg of HS-PEG2k-RITC were added, the obtained mixture was kept stirring at 25 °C for 24 h and dialyzed (MWCO: 7 kDa) against dimethyl sulfoxide (DMSO) and then DI water. 2.2. Evaluation of the Photothermal Performance. Aqueous solutions (200 µL) containing the PEG-modified CuCD NSs with varied Cu concentrations (0 to 30 µg mL–1) were added into eppendorf (EP) tubes (500 µL). The temperature changes of the solutions under 808 nm laser irradiation (2 W cm–2) were monitored (with a time



interval of 15 s and a duration of 10 min) using an infrared thermal imaging camera. 2.3. Cellular Uptake of the PEG-Modified CuCD NSs. For the cellular uptake experiment, MCF-7 breast cancer cells were first incubated in a 96-well plate for 24 h, and then treated with the medium containing FITC-labeled PEG-modified CuCD NSs (Cu concentration: 10 µg mL–1). After further incubation for a certain time period (0, 2, 4, 6, 9, 12, or 24 h), the cells in each well were rinsed for three times with phosphate-buffered saline (PBS), and then digested, collected, and analyzed using a flow cytometer with FITC channel. Furthermore, to confirm the subcellular localization of PEG-modified CuCD NSs, MCF-7 cells, which had been incubated in confocal dishes overnight, were treated with FITC-labeled PEG-modified CuCD NSs (10 µg mL–1 of Cu) for 4 h, and then costained with LysoTracker (to label lysosome) for 30 min and observed under a confocal laser scanning microscope (CLSM). The cellular uptake induced by laser irradiation was also studied. MCF-7 cells were pretreated with FITC-labeled PEG-modified CuCD NSs (10 µg mL–1 of Cu) for 4 h followed by laser irradiation for different time periods (0, 1, 3, 5, 7, and 10 min), and then co-stained with Hoechst 33342 and observed by CLSM. Afterward, the cells were detached from wells using trypsin. The green FL signal within cells from FITC-labeled PEG-modified CuCD NSs and cellular side scatter (SSC) signal were quantified via flow cytometry. 2.4. In Vitro PTT. The photothermal cytotoxicity of PEG-modified CuCD NSs was evaluated with MCF-7 cells. The cells were seeded in a 96-well culture plate (5 × 103 cells well–1) and cultured overnight. After pre-treatment with PEG-modified CuCD


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NSs solutions with varied Cu concentrations (0, 5, 10, 20, and 30 µg mL–1) for 4 h, the cells were subsequently exposed to an 808 nm laser irradiation (2 W cm–2) for 10 min and cultured for another 12 h. The viability of MCF-7 cells was evaluated by 3-(4,5-dimethylthiazol-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium

(MTT)

assay.

Moreover, to quantify the percentage of dead cells by live/dead cell staining, MCF-7 cells incubated without or with PEG-modified CuCD NSs (Cu concentration: 5, 10, and 20 µg mL–1) were treated in the same way as mentioned above. Afterwards, the treated cells were co-stained with commercial live/dead staining kits (in which calcein acetoxymethyl ester (calcein-AM) stains live cells green and propidium iodide (PI) stains dead ones red) for 30 min and then imaged by CLSM.

3. RESULTS AND DISCUSSION 3.1. Synthesis and Characterization of CDs. The sulfur-doped CDs were synthesized using o-phenylenediamine and L-cysteine as the carbon sources by a hydrothermal route. The transmission electron microscopy (TEM) image of CDs (Figure 1A) illustrates their uniformity and good dispersion, with an average diameter of 3.4 ± 0.5 nm. In ultraviolet–visible (UV–vis) absorption spectra, both CDs (Figure 1B) and their precursors (Figure S1) have an obvious absorption band in the wavelength range of 230−280 nm and a peak at 340 nm, which originate from the π−π* transition of the aromatic ring structure and the n−π* transition of the C=O group, respectively. Besides, the excitation-dependent FL behavior of CDs was observed in Figure S2, agreeing well with the CDs reported in other



literature.34,35,60–62 The surface structure and composition of CDs were further characterized by Fourier transform infrared (FTIR) and X-ray photoelectron spectroscopy (XPS) analyses. In Figure 1C, the band from 3500 to 3200 cm–1 originates from the stretching vibrations (ν) of O–H and N–H, while the bands at 3100–2800 cm–1 come from ν(C−H). The peaks observed at 1583, 1500, and 1450 cm−1 are assigned to the stretching vibrations of benzene skeleton, suggesting the presence of benzene ring. The peaks at 2551, 1668, 1420, and 1270 cm−1 are attributable to ν(S−H), ν(C=O), ν(C–N), and ν(C–S), respectively, which may come from the amide bond (–CONH–) and/or L-cysteine. For the XPS spectrum in Figure 1D, the four peaks at 284.6, 531.6, 397.9, and 164.1 eV come from C1s, O1s, N1s, and S2p, respectively. In the high-resolution XPS spectra (Figure 1E), the C1s peaks of sp2 C, sp3 C, and oxidized C reside at 284.5, 285.8, and 288.1 eV. The O1s peaks (Figure 1F) of C–OH/C–O–C, C=O, and C–O center at 530.0, 531.8, and 534.0 eV, respectively, while the N1s peaks (Figure 1G) of pyridinic N, amino N, and pyrrolic N locate at 398.2, 399.4, and 400.4 eV, respectively. In addition, the S2p peaks (Figure 1H) at 164.0 and 167.6 eV confirm the presence of C–S and C–SOx, respectively. The two peaks at 163.5 and 164.6 eV resolved from the former peak correspond to the 2p3/2 and 2p1/2 of the C–S covalent bond, respectively. These results strongly suggest that the CDs are functionalized with various functional groups as –COOH, –OH, –NH2, –SH, and –SOx. Moreover, a negative zeta potential (−40.9 ± 2.3 mV) of the CDs was recorded in DI water, proving the existence of massive carboxyl groups on CDs surface.


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Figure 1. Characterization of CDs. (A) TEM image. Inset: The corresponding size histogram. (B) UV–vis spectrum of the CDs solution. Inset: Photographs of the CDs aqueous solution under white light (left) and a UV lamp (right, 365 nm). (C) FTIR spectra of CDs and their precursors. XPS total (D) and high-resolution (E–H) spectra of the dried CDs.

3.2. Synthesis and Characterization of CuCD NSs. To obtain CuCD NSs, Cu2+ solution and CDs solution were first mixed, and then the obtained mixture was placed without stirring at 25 °C, during which the initial brown solution turned black green. The time-dependent absorption spectra of the CuCD NSs solution during the reaction period of 180 min were measured (Figure 2A). It can be seen that the absorbance in the NIR region (see the inset of Figure 2A) increased significantly in the initial reaction stage of 60 min, and reached around 1.0 after 100 min. The high NIR



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absorption might suggest that CuCD NSs hold great potential for converting NIR light into heat. It was inferred that copper ions crosslinked negatively charged CDs (testified by the change of zeta potential from −40.9 ± 2.3 mV to –33.9 ± 2.4 mV) and bound to sulfur element on CDs via strong Cu–S bonding interaction during the reaction, which resulted in the formation of CuCD NSs. The TEM image (Figure 2B) displayed sheet-like morphology of as-obtained CuCD NSs with an average size of 23.4 nm (see more clearer TEM images in Figure S3), which exhibited negligible change after PEGylation (Figure S4). The selected area electron diffraction (SAED) pattern

demonstrated

the

formation

of

randomly

oriented

crystallites

of

polycrystalline CuCD NSs and the well-defined peaks in X-ray diffraction (XRD) pattern (Figure 2C) indicated the formation of CuCD NSs with high crystallinity. Interestingly, the obtained powder XRD pattern roughly matched the standard diffraction pattern of Cu9S8 [79-2321], indicating the composition of the nanosheets. The chemical structure and surface composition of CuCD NSs were characterized by XPS. Figure 2D showed the characteristic peaks of C, O, N, S, and Cu. Specially, the content of S atom in CuCD NSs was significantly higher than that in CDs, suggesting that the successful synthesis of CuCD NSs is attributed to the Cu–S bonding between copper ions and sulfur element on CDs. The high-resolution C1s, O1s, N1s, and S2p XPS spectra (Figure 2E–H) of CuCD NSs were similar to those of CDs. Besides, the Cu2p spectra (Figure 2I) revealed the presence of Cu2p3/2 (932.3 eV) and Cu2p1/2 (952.2 eV), confirming the incorporation of Cu (I) and Cu (II) in CuCD NSs.


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Figure 2. Characterization of CuCD NSs. (A) Time-dependent absorption spectra of CuCD NSs solution during a reaction period of 180 min. (B) TEM image (a), the corresponding size histogram (b), and SAED pattern (c) of the as-synthesized CuCD NSs. The white dotted circles show the CuCD NSs perpendicular to the TEM grid. (C) The powder XRD patterns of CuCD NSs and Cu9S8 [79-2321]. (D) XPS spectrum of CuCD NSs. (E–I) High-resolution XPS C1s, O1s, N1s, S2p, and Cu2p spectra of CuCD NSs.

3.3. Fabrication of PEG-Modified CuCD NSs and Their PT/PA Properties. For biomedical applications, CuCD NSs were modified with HS-PEG2k though the strong Cu−S bonding to improve their stability in physiological media. The successful PEGylation of CuCD NSs was confirmed by the changes in zeta potential (from –33.9 ± 2.4 mV in CuCD NSs to –14.9 ± 1.5 mV in PEG-Modified CuCD NSs) and



hydrodynamic diameter (from 33.7 ± 9.4 nm in CuCD NSs to 45.7 ± 12.8 nm in PEG-Modified CuCD NSs). As expected, the as-obtained PEG-modified CuCD NSs showed excellent stability in DI water, PBS, and cell culture medium (Figure 3A). We then examined the temperature elevation of the PEG-modified CuCD NSs solutions with varied concentrations under the irradiation of an 808 nm laser (2 W cm–2). After laser irradiation for 10 min, the temperature of the PEG-modified CuCD NSs solutions with the Cu concentration of 0, 5, 10, 20, and 30 µg mL–1 increased by 3.0, 27.0, 35.7, 47.1, and 52.1 °C, respectively (Figure 3B). Furthermore, we have demonstrated that PEG-modified CuCD NSs exhibited good reusability after five cycles of laser on/off treatments (Figure 3C). To further explore the advantages of PEG-modified CuCD NSs, we chose PEG-modified CuS NPs as a comparison. The PEG-modified CuS NPs (9.1 ± 1.5 nm, see the TEM result in Figure S5A) were synthesized based on a previously reported method.12 To reduce liver capture and renal filtration, the optimal size of a nano-sized agent is reported to be 10–100 nm in diameter, which can realize the most efficient therapeutic effect.63 In the present work, the PEG-modified CuS NPs are relatively small, so that they may be quickly cleared by renal filtration; in contrast, the PEG-modified CuCD NSs possess the proper size (20–30 nm) for in vivo cancer therapy. As shown in Figure 3D, the UV–vis spectra of PEG-modified CuCD NSs exhibited the strong absorption in the NIR region due to the localized surface plasmon resonance (LSPR) of valence-band free carriers (positive holes),64 and the extinction coefficient was 19.5 L g–1 cm–1 (at 808 nm), higher than that of various photothermal


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nanoagents, such as CuS NPs (15.9 L g–1 cm–1, Figure S5B), graphene oxide (3.6 L g–1 cm–1),65 gold nanorods (13.9 L g–1 cm–1),66 and ultrasmall black phosphorus quantum dots (14.8 L g–1 cm–1).66 Besides, according to the previous method,67–69 the photothermal conversion efficiency (η) of PEG-modified CuCD NSs was calculated to be 41.3% (Figure 3E), higher than that of PEG-modified CuS NPs (39.2%, Figure S6). The high light-to-heat conversion efficiency makes CuCD NSs be able to rapidly and efficiently convert the NIR light energy into hyperthemia to kill cancerous cells. A direct comparison of photothermal performance of PEG-modified CuCD NSs and PEG-modified CuS NPs was shown in Figure S7 and S8. It was found that PEG-modified CuCD NSs exhibited stronger absorption in the NIR region, higher photothermal conversion efficiency, and better biocompatibility as compared with PEG-modified CuS NPs. We hypothesized that these results could be attributed to the large amount of carbon element originated from CDs in CuCD NSs. We next investigated the PA property of PEG-modified CuCD NSs since they have high NIR optical absorption. Figure 3F clearly shows a linear correlation between PA signal intensity and Cu concentration, which is beneficial for their quantitative PA imaging.



Figure 3. Aqueous stability and photothermal properties of CuCD NSs. (A) Photographs of the CuCD NSs and the PEG-modified CuCD NSs dispersed in DI water (i), PBS (ii), and cell culture medium (iii) for 8 h. (B) Temperature changes of DI water and the PEG-modified CuCD NSs aqueous solutions with varied Cu concentrations during the 808 nm laser irradiation (2 W cm–2, 10 min). (C) Temperature change of the PEG-modified CuCD NSs solution during five laser irradiation on/off cycles. (D) UV–vis spectra of the aqueous solutions containing the PEG-modified CuCD NSs with varied Cu concentrations. Inset: Relationship of A/L value (at λ = 808 nm) and Cu concentration. (E) Temperature profile of the PEG-modified CuCD NSs solution irradiated by an 808 nm laser for 10 min, followed by natural cooling. Inset: The linear fitting of time from the cooling period versus negative natural logarithm of driving force temperature. (F) PA signal intensity of the PEG-modified CuCD NSs solutions with varied Cu concentrations upon excitation at 808 nm. Inset: The corresponding PA images.

3.4. Cytotoxicity Evaluation of PEG-Modified CuCD NSs. For biomedical applications, it is essential to evaluate the cytotoxicity of PEG-modified CuCD NSs.


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Consequently, the MTT assays were conducted to study the relative viabilities of HepG2, A549, MCF-7, L02, and AT II cells after treatment with the PEG-modified CuCD NSs (Cu concentration: 1, 5, 10, 20, 30, 40 or 50 µg mL–1) for 24 h (Figure 4A). Even when the Cu concentration reached 30 µg mL–1, the viabilities of treated cells were still over 80%, indicating the good cytocompatibility of PEG-modified CuCD NSs. Furthermore, no obvious hemolysis was observed when red blood cells (RBCs) were incubated with PEG-modified CuCD NSs at varied Cu concentrations of 0–100 µg mL–1 (Figure S9). All these results showed the suitability of PEG-modified CuCD NSs for both in vitro and in vivo PTT.

Figure 4. (A) Viabilities of various cells incubated with the PEG-modified CuCD NSs solutions at varied Cu concentrations for 24 h. (B) Cellular uptake of FITC-labeled PEG-modified CuCD NSs measured by flow cytometry. (C) CLSM images of FITC-labeled PEG-modified CuCD NSs/LysoTracker-costained MCF-7 cells.



3.5. Cellular Uptake and Subcellular Fate of CuCD NSs. From the flow cytometric analyses (Figure 4B), it is found that the cellular uptake of FITC-labeled PEG-modified CuCD NSs increased with time. The merged CLSM image in Figure 4C reveals that PEG-modified CuCD NSs were internalized by MCF-7 cells via lysosomal capture before laser irradiation. 3.6. In Vitro PTT. To quantitatively assess the photothermal cytotoxicity of PEG-modified CuCD NSs in vitro, cells cultured with varied concentrations of PEG-modified CuCD NSs were irradiated with laser for 10 min (808 nm, 2 W cm–2). The MTT results shown in Figure 5A indicate that the laser irradiation did not elicit cytotoxicity toward MCF-7 cells, and PEG-modified CuCD NSs without laser treatment exerted a negligible degree of toxicity toward MCF-7 cells. However, upon irradiation, the MCF-7 cell viability decreased with increasing concentration of PEG-modified CuCD NSs, and < 10% of cells were alive at 10–30 µg mL–1 (based on Cu), which was consistent with the live/dead staining result (Figure 5B). In addition, the photohtermal effect of PEG-modified CuCD NSs was quantitatively studied via the flow cytometry-based apoptosis/necrosis assay. From Figure 5C, we found that PEG-modified CuCD NSs (10 µg mL−1) plus laser remarkably increased the cell apoptosis rate from 5.28% to 80.77%, indicating that the cell death was mainly caused by hyperthemia-induced apoptosis.


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Figure 5. In vitro PTT. (A) Viability of MCF-7 cells treated with varied Cu concentrations of PEG-modified CuCD NSs without or with 808 nm laser irradiation (2 W cm–2) for 10 min. (B) Live/dead staining of MCF-7 cells treated with varied Cu concentrations of PEG-modified CuCD NSs and laser irradition. (C) Flow cytometry-based apoptosis/necrosis assay of MCF-7 cells treated by laser, PEG-modified CuCD NSs, and PEG-modified CuCD NSs + laser.

3.7. Hyperthemia-Promoted Cytosolic and Nuclear Delivery of CuCD NSs. To further investigate the effects of hyperthemia on the delivery and release of CuCD NSs, we monitored the cellular uptake and intracellular localization of CuCD NSs with continuous laser irradiation using CLSM and flow cytometry. From Figure 4C and 6A, FITC-labeled PEG-modified CuCD NSs were captured by lysosomes via



endocytosis without laser treatment. However, with the continuous 808 nm laser irradiation, more PEG-modified CuCD NSs were internalized into cells (resulting in an 18-fold to 32-fold increase in celllular uptake amount), detached from lysosomes, and released into the cytosol and even the nucleus (Figure 6A–D and S10). Besides, the MTT result (Figure S11) demonstrated that a short laser irradiation time of 5 min elicited low cytotoxicity toward cells pre-treated with PEG-modified CuCD NSs. It was hypothesized that the hyperthemia resulted from the photothermal agent and laser could destabilize the cell membrane structures (especially plasma, endo/lysosomal, and nuclear membranes), which promoted PEG-modified CuCD NSs to release into to subcellular organelles susceptible to hyperthermia (such as mitochondria and nucleus). Moreover, the in vivo experiments also proved the enhanced tumor accumulation of the nanomaterials after 10 min of in situ tumor site laser irradiation (Figure 6E): The FL intensity analysis in tumor sites indicated that the amount of the PEG-modified CuCD NSs increased to 2-fold of the original value after laser irradiation, and then sightly decreased 12 h later possibly because of the destruction of tumor tissues. Collectively, we observe for the first time the laser-triggered lysosomal escape, nuclear-targeting, cellular uptake, and tumor accumulation of PT nanoagents in real time during the continuous laser irradiation, highlighting the importance of laser-controlled drug delivery and release in photothermal cancer therapy.


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Figure 6. Hyperthemia-promoted cytosolic and nuclear delivery of CuCD NSs. (A) CLSM images of MCF-7 cells. Cells were pretreated with FITC-labeled PEG-modified CuCD NSs (10 µg mL–1 of Cu) for 4 h, irradiated (2 W cm–2) for varied time periods (0–10 min), and then stained with Hoechst 33342 before imaging. The temperature values of the media recorded during the treatments were also indicated in the images. (B) The line-scan FL intensity profiles of the marked positions (red dotted arrows) at different time points. The green FL is from FITC-labeled PEG-modified CuCD NSs, and the blue FL is from Hoechst 33342. The gray shadings reveal the



nucleus areas. (C) Effect of laser irradiation time on the cellular FL. (D) The corresponding quantitative analysis of the cellular FL intensity in C). The “Pre” indicates the sample before drug and laser treatments. (E) FL intensity in tumor sites before and after laser irradiation. Inset: The corresponding FL images of tumors from Figure S12. The “Pre” indicates the sample before laser treatments, while “0 h” and “12 h” indicate the samples after laser treatment for 0 h and 12 h, respectively.

3.8. In Vivo Multimodal Imaging-Guided PTT. To study the biodistribution of PEG-modified CuCD NSs (RITC-labeled), the nude mice bearing subcutaneous C6 cancer xenograft were sacrificed at 0, 1, 2, 3, 6, 12, and 24 h postinjection, and the collected major organs and tumors were imaged using the in vivo imaging system. Before tracing the RITC-labeled PEG-modified CuCD NSs, we first demonstrated that free RITC had no evident tumor-targeting ability (Figure S13). As shown in Figure 7A, PEG-modified CuCD NSs could rapidly and effectively target and accmulate in the tumor site with increasing time caused by the blood circulation and the EPR effect. After 12 h, PEG-modified CuCD NSs were gradually cleared out by liver and kidneys. In addition, the excretion study indicated that the additional PEG-modified CuCD NSs could be effectively cleared from the mouse body via both the biliary (into the feces) and the renal (into urine) pathways. (Figure S14). Regarding the PA imaging, a series of PA images captured preinjection and at 1, 3, and 24 h postinjection are shown in Figure 7B. The tumor contrast rapidly enhanced within 3 h postinjection, due to the greatly increased accumulation of PEG-modified


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CuCD NSs in the tumor, and the contrast was decreased at 24 h postinjection, which was in accordance with the FL imaging results. Encouraged by the efficient passive tumor targeting of PEG-modified CuCD NSs, in vivo PTT experiments were conducted to further evaluate their PT therapeutic efficacy. The nude mice bearing subcutaneous C6 cancer xenograft were i.v. injected with 150 µL of PBS or PEG-modified CuCD NSs in PBS (Cu dose: 2 mg kg–1), followed by 10-min laser irradiation (1 W cm–2) on tumor sites at 3 h postinjection. Meanwhile, the time-dependent temperature changes of the tumor sites during PTT process were monitored using an infrared thermal imager. Both the temperature curves and the thermographs (Figure 7C and D) suggested that the rapid temperature rise from 37.0 °C (0 min) to 49.0 °C (10 min) in the PEG-modified CuCD NSs-treated group, resulting in severe damage to the tumor. In contrast, the tumor site temperature in the PBS-treated group only increased from 37.0 °C to 42.2 °C within 10 min. Subsequently, the tumors in PBS (control)-, laser-, and PEG-modified CuCD NSs-treated groups showed remarkable growths; whereas in the PEG-modified CuCD NSs + laser group, the tumor growth was completely suppressed (Figure 7E). The further therapeutic effect was validated by H&E staining of tumor slices 3 days after various treatments and the distinct necrosis was found only in the “PEG-modified CuCD NSs + laser”-treated group (Figure 7F). Finally, we examined the potential toxic effects of the PEG-modified CuCD NSs to mice. No obvious body weight loss (Figure S15) or abnormal behavior was seen for the PEG-modified CuCD NSs-treated mice during our experiments, suggesting the



low side effects of the nanoagents. In addition, organ damages and inflammatory lesions were absent in the H&E-stained slices taken from the major organs (heart (H), liver (Li), spleen (S), lung (Lu), kidneys (K)) of mice 20 days after PTT treatment, which also confirmed the low systemic toxicity of the CuCD NSs-based photothermal cancer therapy (Figure 7G). To sum up, the above results demonstrate that PEG-modified CuCD NSs have superior PTT efficacy in vitro and hold great potential as a PTT agent with rapid tumor-targeting ability, high tumor-ablation efficiency, and excellent biocompatibility.

Figure 7. Biodistribution of PEG-modified CuCD NSs and in vivo PTT. (A) Ex vivo FL images of major organs and tumors dissected from the mice at the indicated time points after the injection of


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RITC-labeled PEG-modified CuCD NSs (150 µL; Cu dose: 2 mg kg–1). (B) In vivo PA images of tumor (irradiated under an 808 nm laser) taken at different time points (0, 1, 3, or 24 h) after the tumor-bearing mouse was i.v. injected with PEG-modified CuCD NSs. (C) Photothermal heating curves in the tumor sites during the irradiation (808 nm, 1 W cm–2, 10 min). (D) Thermal images of tumor-bearing mice after 10-min irradiation. (E) The growth curves of C6 tumors of PBS (control)-, laser-, PEG-modified CuCD NSs-, and “PEG-modified CuCD NSs + laser”-treated mice. ***P < 0.001, one-way ANOVA. (F) Images of H&E-stained tumor slices taken 3 days after four kinds of treatments. (G) H&E-stained slices of major organ slices 20 days after treatment without (control) or with PEG-modified CuCD NSs.

4. CONCLUSION In summary, we first prepared a novel type of sulfur-doped CDs using o-phenylenediamine and L-cysteine as precursors by one-step hydrothermal treatment. Then the CDs were used as both the reductant and the reaction template to fabricate CuCD NSs by adding copper ions to the CDs solution at 25 °C without stirring. Compared with other photothermal agents, our CuCD NSs show the following advantages: (1) the easy-operating, time-saving, and cost-effective synthesis using common substances of copper ions, o-phenylenediamine, and L-cysteine is suitable for mass production; (2) the high NIR optical absorption, excellent photothermal property (excellent photothermal conversion efficiency and stability), proper size (20–30 nm), as well as good biocompatibility ensure their satisfying PA imaging and PT imaging/therapy; (4) the laser-triggered cytosolic/nuclear delivery property of CuCD



NSs makes it possible to greatly increase the photothermal therapeutic efficiency, which will help to develop novel nanoplatforms for realizing light-controlled drug delivery systems. Besides, this work also investigated the detailed events occurring during the continuous laser irradiation at both in vitro and in vivo levels, which has not been clarified before. This work opens a new window for the applications of CDs, develops a new synthetic approach of functional metal-based nanotheranostic agents, and deepens our understanding of the action mechanism of PTT, which may hold great promise in the field of nanomedicine and beyond.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publication website at DOI: Materials and instruments, other experimental details, UV–vis spectra, FL spectra, TEM images, photothermal heating curves, cell viabilities, hemolysis analysis, quantitative analysis of SSC intensity, ex vivo FL images of major organs and tumors, Cu mass in feces and urine, and body weights of mice (PDF)

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


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†

Y.W.B. and X.W.H. contributed equally to this work.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was supported by grants from the Innovative and Entrepreneurial Talent Recruitment Program of Jiangsu Province, Natural Science Foundation of Jiangsu Province (BK20170078), and National Natural Science Foundation of China (21673037). The authors would also like to thank Dr. Yan Wu for operating the photoacoustic instrument.

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Hyperthemia-Promoted Cytosolic and Nuclear Delivery of Copper

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