Fluorescent Carbon Quantum Dots with Intrinsic ... - ACS Publications

Subscriber access provided by UNIV OF NEW ENGLAND ARMIDALE

Biological and Medical Applications of Materials and Interfaces

Fluorescent Carbon Quantum Dots with Intrinsic Nucleolus-Targeting Capability for Nucleolus Imaging and Enhanced Cytosolic and Nuclear Drug Delivery Xian-Wu Hua, Yan-Wen Bao, and Fu-Gen Wu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b19549 • Publication Date (Web): 06 Mar 2018 Downloaded from http://pubs.acs.org on March 6, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 39 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

ACS Applied Materials & Interfaces

Fluorescent Carbon Quantum Dots with Intrinsic Nucleolus-Targeting Capability for Nucleolus Imaging and Enhanced Cytosolic and Nuclear Drug Delivery

Xian-Wu Hua,†,§ Yan-Wen Bao,†,§ 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 ‡

Key Laboratory of Developmental Genes and Human Disease of the Ministry of Education

of China, Institute of Life Sciences, Southeast University, 2 Sipailou Road, Nanjing 210096, P. R. China

KEYWORDS: carbon dots, protoporphyrin IX, nucleolus tracker, photodynamic therapy (PDT), nuclear targeting, drug delivery

1

ACS Paragon Plus Environment

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

Page 2 of 39

ABSTRACT Nucleolus

tracking

and

nucleus-targeted

photodynamic

therapy

are

attracting

increasing attention due to the importance of nucleolus and the sensitivity of nucleus to various therapeutic stimuli. Herein, a new class of multifunctional fluorescent carbon quantum dots (or carbon dots, CDs) synthesized via the one-pot hydrothermal reaction of m-phenylenediamine and

L-cysteine,

was reported to effectively target nucleolus. The

as-prepared CDs possess superior properties such as low-cost and facile synthesis, good water dispersibility, various surface groups for further modifications, prominent photostability, excellent compatibility, and rapid/convenient/wash-free staining procedures. Besides, as compared with SYTO RNASelect (a commonly used commercial dye for nucleolus imaging) that can only image nucleolus in fixed cells, the CDs can realize high-quality nucleolus imaging in not only fixed cells but also living cells, allowing the real-time tracking of nucleolus-related biological behaviors. Furthermore, after conjugating with protoporphyrin IX (PpIX), a commonly used photosensitizer, the resultant CDs-PpIX nanomissiles showed remarkably increased cellular uptake and nucleus-targeting properties, and achieved greatly enhanced phototherapeutic efficiency since the nuclei show poor tolerance to reactive oxygen species (ROS) produced during photodynamic therapy. The in vivo experiments revealed that the negatively-charged CDs-PpIX nanomissiles could rapidly and specifically target tumor site after intravenous injection and cause efficient tumor ablation with no toxic side effects after laser irradiation. It is believed that the present CD-based nanosystem will hold great potential in nucleolus imaging and nucleus-targeted drug delivery and cancer therapy.

2


Page 3 of 39 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


1. INTRODUCTION As a key component in nucleus, the nucleolus is the ribosome factory of the cell and the site for ribosomal RNA (rRNA) production, processing and assembly with ribosomal proteins.1,2 Besides, for pathological detection of malignant lesions, the nucleolus is usually regarded as a diagnostic biomarker because the changes in the morphology (such as size and shape) and number of nucleoli often serve as the premonitors of some human disorders (including cancer).3,4 Furthermore, nucleolus has been recently regarded as an ideal target for cancer therapy.5,6 Despite the significant role of nucleolus for disease theranostics, SYTO RNASelect, the only commercial probe for nucleolus imaging, suffers from high cost, strict storage conditions, unknown functional groups (at least to consumers), and complicated staining procedures for cell imaging (cells must be fixed by methanol before imaging), which severely limit its applications for real-time live cell tracking and drug delivery. To address these problems, considerable effort has been devoted to the development of new nucleolar probes. In recent years, a variety of molecular probes, such as crescent-shaped fluorescent dyes

synthesized

by

hybridizing

coumarin

and

pyronin

moieties,7

Styryl-TO,8

ɑ-cyanostilbene derivative (ACSP),2 low molecular weight organic molecules (PY and IN),9 PNA FIT-probes,10 etc., have been developed to realize live-cell nucleolus imaging. Besides, metal-based nanomaterials, such as the phosphorescent silver(I)gold(I) cluster complex,11 chitosan–gold hybrid nanospheres12, and gold nanoclusters (AuNCs),4 were also explored for nucleolus imaging. However, the complicated synthesis, high cost, unsatisfying nucleolus-targeting performance, lack of modifiable functional groups, and/or potential cytotoxicity impede their applications in the biomedical filed.

3



On the other hand, the emergence of multidrug resistance (MDR) severely hampers the cellular uptake and nuclear invasion of many chemotherapeutic drugs, such as doxorubicin, cisplatin, and camptothecin, resulting in largely reduced therapeutic efficacy. Besides, cell nucleus is recognized as an ideal target in gene therapy, chemotherapy, radiotherapy, and phototherapy13–16 due to its central role in the control of cellular activities by regulating gene expression, and high susceptibility towards chemotherapeutics, hyperthermia, and reactive oxygen species. However, it is still highly challenging for drugs especially photosensitizers (PSs) to be delivered into nuclei with satisfactory therapeutic outcome due to lysosomal/endosomal entrapment and their drawbacks of poor water solubility, low cellular uptake, and/or inability of nucleus-targeting. To overcome these shortcomings, various nanotechnology-based delivery systems have been developed for nuclear drug delivery.17–19 The modification with nuclear location signal (NLS) is a common method to guide various nanosystems to enter the nucleus through nuclear pore complexes (NPCs).19–26 Among them, trans-activator of transcription (TAT) peptide is one of the most frequently used ligands to endow drugs with nucleus-targeting ability to overcome MDR and/or realize nucleus-targeted gene/phototherapy.15,27–37 However, the TAT functionalization of delivery systems may compromise the pharmacokinetic behavior since the existence of positively charged arginines and lysines may increase mononuclear phagocytic system recognition.38 Besides, since most NLSs are not fluorescent, the addition of a fluorescent tag is required for fluorescence tracking purpose; however, the conjugation of a fluorescent molecule may hinder the nucleus-targeting capability of the NLSs. Furthermore, the high cost and poor stability (necessity of harsh storage conditions) are also the barriers for their further applications in

4


Page 4 of 39

Page 5 of 39 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


biomedical research and clinical therapy. Considering the limitations of NLS modification, cationic amino acids, polyethylenimine, Hoechst dyes, and chitosan have been developed to realize nucleus-targeted therapy, since it is difficult for neutrally and negatively charged molecules to enter nucleus.39–44 However, a highly positively charged surface of nanomaterials may cause the undesirable interactions with negative charged intracellular proteins, thereby limiting the in vivo applications of these nanomaterials.45 The potential cytotoxicities of Hoechst dyes and polyethylenimine are also serious concerns for their uses in clinic therapy. Fluorescent carbon quantum dots (or carbon dots, CDs), the nanomaterials with fascinating properties, have generated much enthusiasm and have been explored for many potential applications such as bioimaging,46–52 catalysis,53–55 biosensing,56–58 drug carriers/therapeutics for cancer therapy,59–63 and antibacterial treatments.58,65 Zhang and Huang et al. reported a fluorescent carbogenic small molecular complex that can selectively stain the RNA-riched nucleolus.3 Lin et al. also prepared a novel type of CDs by microwave-assisted heating of citric acid formamide solution to realize nucleolus imaging and drug delivery (using fluorescein isothiocyanate, FITC, as a drug representative).66 However, there have been no reports on the development of negatively-charged CDs that have inherent nucleolus-targeting property and can overcome the barrier of nuclear membrane and ultimately realize nuclear-targeted drug delivery and photodynamic therapy (PDT). Herein, a novel type of muiltfunctional CDs, which can avoid lysosomal/endosomal entrapment, selectively target nucleolus, and successfully deliver protoporphyrin IX (PpIX, a widely used photosensitizer) into nucleus, was prepared through one-pot hydrothermal

5



Page 6 of 39

method using m-phenylenediamine and L-cysteine as the carbon sources. Compared with the commercial nucleolus dye, SYTO RNASelect, the as-prepared CDs exhibit many superior properties including low-cost and facile synthesis, good water-dispersibility and photostability,

easily

modifiable

surface

groups,

excellent

biocompatibility,

rapid/convenient/wash-free staining procedures, and real-time live cell tracking capability. Besides, the working concentration of our CDs (5 µg/mL) is much lower than that of the CDs reported previously, indicating the enhanced cytosolic and nuclear delivery of our CDs. Furthermore, after conjugating with PpIX, the resultant negatively-charged CDs-PpIX exhibit greatly increased cellular uptake as compared with free PpIX, and realize nucleus-targeted PDT. Besides, the in vivo experimental results indicate that CDs-PpIX possess excellent tumor targeting ability and satisfying fluorescence imaging-guided therapeutic performance. To the best of our knowledge, this work represents the first example of using CD-based nanoplatform for realizing nucleus-targeted drug delivery and PDT without the additional conjugation of a specific nucleus targeting ligand. Scheme 1. Schematics of the Synthetic Procedure of CDs, the Selective Nucleolus Imaging of CDs, and the Corresponding Enhanced Nucleus-Targeted PDT Application

6


Page 7 of 39 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


2. EXPERIMENTAL DETAILS 2.1. Synthesis of CDs. The CDs were prepared by the hydrothermal treatment of m-phenylenediamine and L-cysteine. In brief, L-cysteine (675 mg) dissolved in 20 mL 0.3 M NaOH aqueous solution was mixed with 10 mL m-phenylenediamine (300 mg) aqueous solution. Then, the mixture was transferred into a 50 mL hydrothermal reactor. After treatment at 160 oC for 10 h, the CD solution was further purified via centrifugation (15,000 rpm, 15 min) to remove the subsidence and dialysis (molecular weight cut-off, MWCO: 1000 Da) against deionized (DI) water for two days to eliminate the residual reagents. The obtained CD solution was stored in 4oC for further use. 2.2. Photostability Assessment of CDs. First, the pH values of CD solutions were adjusted by NaOH (1 M) and/or HCl (1 M) and the corresponding fluorescence intensities (Ex: 488 nm, Em: 520 nm) of the CD aqueous solutions (10 µg/mL) with different pH values (1–14) were measured and recorded by a spectrofluorophotometer. Besides, the

7



photobleaching assessment of CDs was conducted and a commercial dye, SYTO RNASelect, was used as the control group. Briefly, the fluorescence changes of CDs (10 µg/mL; Ex: 488 nm; Em: 520 nm) and SYTO RNASelect (1 µM; Ex: 488 nm; Em: 530 nm) induced by the continuous irradiation of UV light (365 nm, 6 W, 0–60 min) were monitored by measuring the fluorescence intensities at 520 and 530 nm at different time points, respectively. 2.3. Optimization of Live-Cell Staining by CDs. HeLa cells cultured overnight were stained by CDs (5 µg/mL) for different time periods (5 , 10 , 30 min, 1, 2, 4, 6, 9, 12, or 24 h), and then the cell imaging results and fluorescence intensities of stained cells were obtained by a confocal microscope (TCS SP8, Leica, Germany) and a flow cytometer (NovoCyte 2060, ACEA Bioscience, USA), respectively. Next, the influences of CD concentration on live-cell nucleolus imaging were investigated by staining live cells with CD solutions at concentrations of 1, 3, 5, 10, or 20 µg/mL for 30 min. The fluorescence images and intensities of stained cells at each condition were obtained by confocal imaging and flow cytometric analysis, respectively. 2.4. Confocal Imaging of Live Cells (Unfixed Cells) Using CDs/Hoechst 33342/SYTO RNASelect/TRITC-Phalloidin. Human epithelial carcinoma cells (HeLa), human lung cancer cells (A549), human liver cancer cells (HepG2), human liver cells (L02), human lung cells (AT II), and murine macrophages (RAW264.7) were employed for further cellular experiments. RAW264.7 cells were cultured in complete Roswell Park Memorial Institute (RPMI) 1640 medium and the others were incubated in complete Dulbecco’s modified Eagle’s medium (DMEM). For live-cell staining using CDs, cells were first cultured for 24 h followed by a 30-min staining with the corresponding media (containing 5 µg/mL

8


Page 8 of 39

Page 9 of 39 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


CDs) and then imaged under a confocal microscope without wash treatment. On the other hand, the live-cell imaging procedures using Hoechst 33342 (5 µg/mL, 10 min, washing before imaging, Ex: 405 nm) and SYTO RNASelect (1 µM, 30 min, washing before imaging, Ex: 488 nm) were carried out following their respective staining protocols. To realize the co-staining of CDs, Hoechst 33342, and TRITC-phalloidin, the fixed and permeabilized HeLa cells were incubated with CD solutions (5 µg/mL, 30 min), Hoechst 33342 (5 µg/mL, 10 min), and TRITC-phalloidin (100 nM, 30 min). After washing 3 times with phosphate-buffered saline (PBS), the co-stained cells were imaged by the confocal microscope. 2.5. Confocal Imaging of Fixed Cells Using CDs/Hoechst 33342/SYTO RNASelect. The fixed HeLa cells were harvested by the following methods from the staining protocol of SYTO RNASelect. Cells were first fixed by prechilled methanol (–20 oC) for 15 min and then permeabilized by 0.5% Triton X-100 for 2 min at ambient temperature. Afterwards, the fixed-cell staining procedures using CDs/Hoechst 33342/SYTO RNASelect were conducted following the methods of live-cell staining of these three dyes. 2.6. Interactions Between CDs and RNA/DNA. HeLa cells were first treated with the medium that containing 30 µg/mL DNase solution (Sigma) or 25 µg/mL RNase solution (GE) at 37ºC for 2 h, respectively, and then fixed and permeabilized by methanol and 0.5% Triton X-100. The pretreated cells were then rinsed with PBS for 3 times before staining by CDs/Hoechst 33342/SYTO RNASelect according to the staining methods mentioned above. Besides, we also assessed the changes of the fluorescence properties of CDs and SYTO RNASelect dye induced by RNA or DNA. CDs (or SYTO RNASelect dye) were dissolved in

9



PBS (as control experiment) or PBS containing RNA or DNA (150 µg/mL) at 37 oC for 30 min. Afterwards, the fluorescence emission spectra of the three groups of CDs (Ex: 488 nm) and the three groups of SYTO RNASelect dye (Ex: 488 nm) were recorded by a spectrofluorophotometer. 2.7. Subcellular Localization and PDT Efficacy of CDs-PpIX. HeLa cells were first incubated with PpIX or CDs-PpIX (PpIX concentration: 5 µg/mL) at 37 oC for 6, 12, 18, or 24 h. After washing with PBS for 3 times, the pretreated cells at each time point were further incubated with Hoechst 33342 for 10 min and then rinsed with complete DMEM medium for subsequent confocal imaging (PpIX/CDs-PpIX: Ex: 552 nm; Hoechst 33342: Ex: 405 nm). For cytotoxicity evaluations of PpIX and CDs-PpIX, we first assessed their dark toxicity to HeLa cells. The cells were treated with PpIX or CDs-PpIX (PpIX concentration: 1, 2, 5, 10, 15, or 20 µg/mL) for 24 h and then analyzed via MTT assay to measure the corresponding cell viabilities. To assess and compare the PDT efficacy of free PpIX and CDs-PpIX, HeLa cells were cultured for 24 h with DMEM and then incubated with free PpIX or CDs-PpIX (PpIX concentration: 0, 0.5, 1, 2, 3, or 4 µg/mL) for varied time periods (1, 4, 12, or 24 h). After rinsing twice, the preincubated cells in each group were irradiated with 635 nm (20 mW/cm2) laser for 10 min, followed by incubation at 37 oC for 4 h. The cell viability of each group was further quantified by MTT assay. Finally, the live/dead staining with two dyes of calcein acetoxymethyl ester (calcein-AM, which stains the live cells green) and propidium iodide (PI, which stains the dead cells red) was also applied to test the viability of HeLa cells, which were first incubated with CDs-PpIX (PpIX concetration: 2 µg/mL) for 24 h and then irradiated by a 635 nm laser (20 mW/cm2, 10 min).

10


Page 10 of 39

Page 11 of 39 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


2.8. In Vivo Imaging. For in vivo imaging, the U14 tumor-bearing mice were first intravenously (i.v.) injected with PBS, free PpIX, or CDs-PpIX (PpIX dose: 5 mg/kg). Then the in vivo fluorescence imaging experiments were conducted with a Cri Maestroin in vivo imaging system (Ex: 520 nm, Em: 640 nm) at different time points (0, 1, 3, 6, 9, 12, 18, or 24 h). Besides, to investigate the biodistribution of CDs-PpIX, the mice i.v. injected with CDs-PpIX (dose: 5 mg/kg of PpIX) were sacrificed at 1, 3, or 7 d postinjection and their major organs (hearts, livers, spleens, lungs, and kidneys) and tumors were excised for ex vivo fluorescence imaging. The corresponding fluorescence signals in the in vivo experiments were analyzed by the CRi Maestro Image software.

3. RESULTS AND DISCUSSION 3.1. Preparation and Characterization of CDs. The water-dispersible fluorescent CDs were prepared using m-phenylenediamine and L-cysteine (dissloved in 0.2 M NaOH solution) by one-step hydrothermal method. After purification by centrifugation and dialysis, the obtained CDs were further characterized by a variety of analytical techniques and then applied as a new nucleolus-targeting probe and nanocarrier for cancer imaging and nuclear-targeted PDT (Scheme 1). The transmission electron microscopy (TEM) image (Figure 1a) shows that the CDs are nearly spherical and well dispersed with an average diameter of 3.8 ± 0.5 nm. Besides, the ultraviolet–visible (UV−vis) spectrum (Figure 1b) exhibits two absorption peaks at 269 and 405 nm, indicating the presence of the π−π* transition (aromatic C=C and C=N) and the surface states of CDs, respectively.67 Similar with other previously reported CDs,54 the CDs in this work exhibit the excitation-dependent fluorescence emission property (Figure 1c). Besides, the fluorescence quantum yield (QY) of 11



CDs in water was calculated to be ~8.4% (using quinine sulfate as a reference). The fluorescence emission properties of CDs in different solvents at room temperature were also investigated (Figure S1). The results show that the CDs in different solvents (water, methanol, ethanol, dimethyl sulfoxide (DMSO), dichloromethane, and chloroform) exhibit wide disparities in the positions and/or intensities of the fluorescence peaks, revealing the polarity-sensitive fluorescence property of the CDs. The highest fluorescence emission of CDs in DMSO may be due to the best dispersity of the CDs in this solvent. The composition and surface functional groups of the CDs were investigated by Fourier transform infrared (FTIR) spectroscopy and X-ray photoelectron spectroscopy (XPS). In FTIR spectrum (Figure 1d), the broad band at 3600–3100 cm–1 is assigned to O–H and N–H stretching vibrations (denoted as n(O–H) and n(N–H), respectively). Besides, the peaks at ~2890 cm–1 (ranging from 3000 to 2800 cm–1) and 1480 cm–1 indicate the C–H stretching vibrations and C=C stretching vibrations/C–H bending vibration (denoted as n(C–H) and n(C=C)/δ(C–H), respectively), respectively. The peaks at 1620, 1350, 1100, and 980 cm–1 originated from the stretching vibrations of C=O, C–N, C–O, and C–S (denoted as n(C=O), n(C–N), n(C–O), and n(C–S), respectively), respectively. Furthermore, XPS spectra (Figure 1e–i) were recorded to explore the chemical compositions of CDs. From Figure 1e, it can be seen that the CDs contain the C, O, N, and S elements (the H element cannot be detected by XPS), and their atomic percentages are 61.6%, 34.1%, 1.7%, and 2.6%, respectively. The high resolution spectrum of C1s (Figure 1f) exhibits three peaks at 284.5, 286.1, and 287.6 eV, which can be attributed to C−C/C=C, C−O/C−N, and C=O, respectively. In the high resolution spectra of N1s and O1s (Figure 1g and h), the peaks at 398.9, 400.1, 530.6, 532.3

12


Page 12 of 39

Page 13 of 39 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


and 535.3 eV can be attributed to N−H, C−N−C, C=O, C−O, and S−O/S=O, respectively. The peaks at 162.9, 164.1, 167.9, 168.3, and 169.1 eV in high resolution spectrum of S2p (Figure 1i) confirm the presence of the sulfur-containing groups of C−S (S2p3/2), C−S (S2p1/2), and C−SOx (x = 2, 3, and 4), respectively. Moreover, the zeta potential of CDs was measured to be −23.2 mV, suggesting that the CDs are negatively charged, possibly due to the presence of −COOH and −SOx groups. To sum up, the CDs contain various elements (C, H, N, O, and S) and functional groups (e.g., −OH, −NH2, −COOH, and C–S).

Figure 1. Characterizations of CDs. (a) TEM image of CDs. The inset shows the corresponding size distribution histogram. (b) UV–vis absorption spectra of CDs. Inset: photographs of the CDs aqueous solutions under white light (left) and UV light (right), respectively. (c) Excitation-dependent photoluminescence behavior of CDs. (d) FTIR spectrum, (e) XPS survey scan, and (f) C1s, (g) N1s, (h) O1s, and (i) S2p XPS curves of CDs.

13



3.2. Nucleolus Imaging in Living Cells. In consideration of the possible application of the CDs in cell imaging, we first explored the intracellular localization of CDs in HeLa cells and then optimized their staining conditions (including staining time and CD concentration). As shown in Figure 2a–c and S2, after treating HeLa cells with CDs (5 µg/mL, which is much lower than the working concentrations of other CDs reported in recent years51,54,57) for 30 min, the CDs were located in some round areas, which were the nucleoli and the number of nucleoli in different cells was usually in the range of 2–4 per cell. Notably, CDs selectively stained the nucleolus within 5 min of incubation with no signs of lysosomal/endosomal entrapment, indicating that the CDs might have a direct and fast movement toward the nucleus after cellular internalization. Meanwhile, even after staining for 24 h, the CDs were still localized at nucleoli, without the translocation to other areas. These results suggest that the CDs are competent as a new nucleolus-targeting dye for wash-free, real-time, and long-term fluorescence imaging. Next, we evaluated the staining performance of CDs for other cell lines. Considering the structural and functional diversities among different cells (normal and cancerous cells), the two cancerous cells (HepG2 and A549) and the three normal cells (L02, AT II, and RAW264.7) were employed to assess the nucleolus-targeting performances of the CDs. After treatment with CDs (5 µg/mL) for 30 min, all nucleoli in the five kinds of cells were specifically stained by CDs with green fluorescence (Figure S3), indicating the highly universal and specific nucleolus imaging ability of the CDs. From Figure S4, it can be seen that the fluorescence intensity of CDs remained stable in solutions with the pH value ranging from 5 to 11, or under the continuous irradiation of a UV (365 nm) lamp for a duration of 60

14


Page 14 of 39

Page 15 of 39 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


min, while that of the SYTO RNASelect dye was quenched by ~15% after 60 min of irradiation, indicating the excellent pH stability and photostability of the CDs. In addition, the CDs exhibited negligigble cytotoxicity toward AT II cells, showing their suitability for biomedical applications (Figure S5).

Figure 2. (a) Confocal images of HeLa cells treated with the CDs (5 µg/mL) for different time periods. Scale bar: 20 µm. Effects of (b) staining time and (c) CD concentration on the fluorescence intensity of HeLa cells stained by CDs.

3.3. Comparison of CDs and Commercial Nucleus Dyes on Living/Fixed Cells Imaging. In some cellular experiments, such as immunostaining, cells usually need to be fixed by methanol or 4% paraformaldehyde to maintain their structure and the shape. However, the fixation step usually results in unsatisfying nucleus imaging by using common nucleus-targeting dyes such as Hoechst 33342 (a DNA dye) and SYTO RNASelect (the only 15



commercial dye with RNA targeting ability for nucleolus imaging). Herein, we systematically compared the nucleus imaging performances of CDs, Hoechst 33342, and SYTO RNASelect on unfixed (living) cells and fixed (dead) cells. As shown in Figure 3, in unfixed cells (living cells), only CDs could image nucleoli by emitting bright green fluorescence. In contrast, Hoechst 33342 stained the whole nucleus except the nucleoli; SYTO RNASelect even could not enter nucleus and failed in nucleus imaging. Nevertheless, all the three dyes could light on the nucleoli when incubated with fixed cells. On the other hand, similar results were also shown in Figure S6, in which the CDs, Hoechst 33342, and TRITC-phalloidin selectively stained the nucleolus, nucleus, and cytoskeleton with green, blue, and red fluorescence, respectively. These results show the significant influence of fixation on the staining of these two commercial nucleus dyes and point out the limitations for SYTO RNASelect to image the nucleoli in living cells, indicating the great promise of our CDs in applications of live-cell nucleolus tracking.

16


Page 16 of 39

Page 17 of 39 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


Figure 3. Confocal images of unfixed and fixed (by methanol) HeLa cells stained by CDs, Hoechst 33342 (denoted as Hoechst in the figure), or SYTO RNASelect (denoted as RNASelect in the figure). Scale bar: 20 µm.

3.4. Nucleolus-Targeting Mechanism of CDs. It is well known that most RNAs (especially the rRNA) in nucleus accumulate in the nucleolus. Besides, some DNAs are also present in the nucleolus. To investigate the nucleolus-targeting mechanism of CDs, the digest experiments using ribonuclease RNase (which hydrolyzes RNA) and DNase (which hydrolyzes DNA) were conducted on HeLa cells to investigate the influences of DNA and RNA on the cell imaging performances of CDs, SYTO RNASelect, and Hoechst 33342. Figure 4a shows that the three dyes still locate at the nucleoli of DNA-digested and fixed cells, which agrees with the results in Figure 3. After the cells were digested by RNAse and then fixed by methanol, all the three dyes were uniformly distributed in the whole nucleus. Based on the above mentioned results, it can be concluded that the nucleolus staining mechanism of CDs is the same as that of SYTO RNASelect dye, which selectively binds to RNA instead of DNA after entering the nucleus. For further exploration of the nucleolus-targeting mechanism of CDs, the fluorescence response of the two probes (i.e., CDs and SYTO RNASelect) to DNA and RNA were performed and the corresponding results are presented in Figure 4b and c. From the changes of fluorescence intensities of the two probes, it can be seen that in solution fluorescence measurements, both CDs and SYTO RNASelect have the “light-switch” effect of nucleic acids with higher response to RNA than DNA: the fluorescence intensities of CDs (at 520 nm) and SYTO RNASelect (at 530 nm) in the presence of RNA are approximately 1.2 and 1.5

17



times as high as that in the presence of DNA, respectively. Besides, the fluorescence intensity of CDs bound with RNA or DNA is markedly stronger than that of free CDs. A similar phenomenon was also reported by Song et al.,9 evidencing that this result may be resulted from the structural diversity of nucleic acids determining the specific interaction between CDs and nucleic acids. In summary, as compared with SYTO RNASelect, the CDs seem to have the similar RNA selectivity and “turn-on” fluorescence response after RNA binding. Since CDs and RNA are both negatively charged, such a specific RNA interaction of the CDs may not be due to the electrostatic attraction interaction, but may be attributed to the specific surface chemistry (especially the contribution from the carbon source of m-phenylenediamine) of the CDs.

18


Page 18 of 39

Page 19 of 39 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


Figure 4. (a) DNase or RNase digest experiments. Confocal images of fixed HeLa cells stained by CDs (5 µg/mL, 30 min, Ex = 488 nm), SYTO RNASelect (1 µM, 15 min, Ex = 488 nm), and Hoechst 33342 (10 min, Ex = 405 nm) before and after treatment with DNase (25 mg/mL) or RNase (25 mg/mL) at 37 oC for 2 h. Scale bar: 20 µm. (b) and (c): Fluorescence spectra of CDs (5 µg/mL, Ex = 488 nm) and SYTO RNASelect (1 µM, Ex = 488 nm) in water, RNA solution, and DNA solution. RNA and DNA concentration: 150 µg/mL.

3.5. Cellular Uptake Mechanism of CDs. Considering the high cellular uptake efficiency of CDs, various investigations were conducted to explore their cellular uptake pathways. Generally, the main factors affecting the cellular uptake of drugs/nanomaterials are temperature and energy supply, which can mostly inhibit active transport (such as cell 19



Page 20 of 39

endocytosis). Besides, cell endocytosis can be usually classified into the following four types: caveolae-mediated

endocytosis

(CvME),

lipid

raft-mediated

endocytosis

(LrME),

clathrin-mediated endocytosis (CME), and macropinocytosis. We therefore evaluated the influences of 4 oC, NaN3 (inhibitor of energy supply), and four endocytosis inhibitors (genistein,

inhibitor

of

CvME;

chlorpromazine,

CPZ,

inhibitor

of

CME;

methyl-β-cyclodextrin, MβCD, inhibitor of LrME; 5-(N,N-dimethyl)-amiloride hydrochloride, amiloride, inhibitor of macropinocytosis) on the cellular uptake of CDs. The cellular localization and fluorescence intensity of CDs in HeLa cells of each group (4 oC-, NaN3-, CPZ-, MβCD-, amiloride-, or genistein-group) were visualized and quantified by a confocal microscope and a flow cytometer, respectively. Compared with the fluorescence in the CDs-group (cells were stained CDs without any pretreatments), the fluorescence of 4oC- and NaN3-groups significantly declined by 70% and 52%, respectively, indicating the cellular uptake of CDs is temperature- and energy-dependent (Figure 5A and B). For endocytosis inhibition assays using the four inhibitors, compared with CDs-group, the CDs fluorescence intensities of CPZ-, amiloride-, and genistein-groups slightly declined by 13%, 26%, and 11%, respectively, but that of MβCD-group increased by 7%, suggesting that the CME, macropinocytosis, and CvME were the main routes for the cellular uptake of CDs and played an important role in the nucleolus targeting performance of CDs. In addition, the localization of CDs (nucleolus-targeting) in all groups was not affected by these 6 factors. These results demonstrate that the endocytosis of CDs involves pathways mainly including energy-/temperature-dependent transport and macropinocytosis-, CME-, CvME-, and/or LrME-involving endocytosis.

20


Page 21 of 39 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


Furthermore, we utilized two inhibitors of importazole and ivermectin to explore whether the CDs translocate into nucleus through the NPCs via a distinct mechanism mediated by the importin ɑ and/or β transport receptor. From Figure S7, after cells were pretreated separately with importazole and ivermectin, which inhibit importin β transport receptor and both importin ɑ and β transport receptors, respectively, the fluorescence intensities of the treated cells only slightly declined as compared with that of control group (without pretreatment). It seems that the two inhibitors have no influences on the nuclear translocation and nucleolus-targeting performance of CDs, indicating that a possible way of free diffusion may be the mechanism of CDs for realizing nuclear translocation.

Figure 5. Intracellular localization of CDs after blocking one of the different cell endocytosis pathways. (a) Confocal images of HeLa cells before (control) and after various treatments as indicated. Scale bar: 20 µm. (b) The corresponding flow cytometric results showing the fluorescence intensities of HeLa cells before (control) and after various treatments as indicated.

3.6. CDs for Nucleus-Targeted PDT. As an emerging medical technique for cancer therapy, PDT shows a highly promising non-invasive therapeutic strategy using PSs and light irradiation to induce the destruction of cancerous cells and tissues.68–71 Upon appropriate light irradiation, the PSs can transfer the absorbed energy to surrounding oxygen molecules,

21



resulting in the generation of massive cytotoxic reactive oxygen species (ROS), such as 1O2, hydrogen peroxide, superoxide, and hydroxyl radicals, which can ultimately and effectively kill cells by crucial and irreversible damages to biomolecules including lipids, proteins, and DNA. However, in the practical applications of classic PSs-based PDT, the undesirable hydrophpbicity, poor cellular uptake, and lysosomal/endosomal capture are serious problems which severely restrict the PDT efficiency and in vivo therapeutic outcomes. Considering the presence of amine groups on the surface of the CDs that can bind to carboxyl groups via the dicyclohexylcarbodiimide (DCC)/1-hydroxybenzotriazole (HOBt) coupling reaction, we select PpIX (a carboxyl-containing PS) as a representative anticancer drug to test the feasibility of constructing a CDs-based and nucleus-targeted drug delivery system that can overcome the drawbacks of PpIX and realize desirable therapeutic efficacy both in vitro and in vivo. The as-synthesized CDs-PpIX were first characterized by TEM, UV–vis spectroscopy, and FL spectroscopy. As displayed in the TEM image (Figure 6a), CDs-PpIX showed a larger size of 25.2 ± 5.7 nm than that (3.8 ± 0.5 nm) of CDs, which might be due to the crosslinking between the amine-containing CDs and the carboxyl-containing PpIX during the dicyclohexylcarbodiimide (DCC)/1-hydroxybenzotriazole (HOBt) treatment and/or the partial aggregation of the conjugates because of the presence of the relatively hydrophobic PpIX molecules on the surface of CDs-PpIX.. Besides, the drug (PpIX) loading capacity of CDs was calculated to be 23.3%, and such a high drug loading capacity of CDs might be attributed to the relatively low molecular weight of CDs (since the CDs were obtained after dialysis using a dialysis membrane with a MWCO of 1000 Da) and high molecular weight of

22


Page 22 of 39

Page 23 of 39 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


PpIX, high specific surface area of CDs, and the high content of amine groups on the surface of CDs (which enables the successful PpIX conjugation). Meanwhile, the singlet oxygen (1O2) quantum yield (Φ△) induced by CDs-PpIX under 635 nm light irradiation was measured to be 62.1% with 1,3-diphenylbenzofuran (DPBF) as the 1O2 indicator (Figure S9). The zeta potential measurement revealed that CDs-PpIX were a bit more negatively charged (–25.5 mV) as compared to CDs (–23.2 mV). After storage for over 3 months, no evident changes in the size (32.6 ± 7.0 nm, see the TEM result in Figure S10) and zeta potential (–27.3 mV) of CDs-PpIX were observed, indicating their excellent aqueous stability. Besides, the hydrodynamic diameters of CDs-PpIX dispersed in water or in 10% fetal bovine serum (FBS) were also measured to be 41.3 ± 16.1 and 43.7 ± 13.2 nm, respectively (Figure S11), suggesting that there were negligible interaction between CDs-PpIX and serum protein. On the other hand, CDs-PpIX exhibited three absorption peaks at 400, 500, and 570 nm (Figure 6b) and two fluorescence emission peaks at 500 nm (Ex: 379 or 405 nm) and 630 nm (Ex: 379, 405, or 570 nm) (Figure S8), which coincided with the UV–vis and fluorescence spectra results of free CDs and PpIX, respectively, indicating the successful synthesis of CDs-PpIX. Besides, the 1O2 generation of CDs-PpIX and PpIX (laser: 635 nm, 20 mW/cm2; PpIX concentration: 5 µg/mL) was evaluated with the singlet oxygen sensor green (SOSG) kit, which can emit green fluorescence (excitation/emission maxima: ~504/525 nm) in the presence of 1O2. As displayed in Figure 6c, under the same irradiation condition, the 1O2 generation of CDs-PpIX was much more than that of free PpIX, which can be ascribed to the improved aqueous dispersityof CDs-PpIX as compared to that of free PpIX (note that PpIX has a poor water solubility). In summary, the results above demonstrate the successful

23



preparation, desirable water dispersibility, and enhanced 1O2 generation of CDs-PpIX, suggesting the great potential of CDs-PpIX for efficient cancer therapy. Considering the nucleolus-targeting ability of CDs, it is possible for CDs to realize the nuclear delivery of PpIX, which can maximize the therapeutic efficacy of ROS (produced by PpIX under light irradiation) and result in more efficient tumor ablation. First, the time-dependent cellular uptake and subcellular localization of CDs-PpIX and free PpIX were investigated by co-staining HeLa cells with Hoechst 33342 and CDs-PpIX (or free PpIX) and the results were shown in Figure 6d and e. After incubation with HeLa cells for different time periods, CDs-PpIX were rapidly and massively endocytosed into the cells, while free PpIX molecules were scarcely internalized by the cells even after incubation for 24 h. Meanwhile, strong red fluorescence appeared in the cytosol with some red fluorescent dots occurring in the nuclei when cells were incubated with CDs-PpIX for 12 and 18 h. At 24 h, a significantly larger amount of red fluorescent dots could be seen in the nuclei, indicating the successful CDs-assisted nuclear delivery of the PpIX moieties. These results demonstrated the highly increased cellular uptake and successful nuclear delivery of PpIX in CDs-PpIX group as compared with the free PpIX group. To further quantify the time-dependent cellular uptake of free PpIX and CDs-PpIX, the fluorescence intensitie of HeLa cells, which were cultured for 24 h and then treated with free PpIX or CDs-PpIX for varied time periods (0, 0.5, 1, 2, 4, 6, 9, 12, 18, or 24 h), were measured with flow cytometry. From Figure 6f, it can be seen that the cellular uptake of CDs-PpIX is much higher than that of free PpIX at each incubation time point, and the cellular uptake amounts of the two samples reached the maxima at above 2 h. Based on the above results, we then further explored the dark toxicity and the PDT efficacy

24


Page 24 of 39

Page 25 of 39 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


of CDs-PpIX and free PpIX on HeLa cells. As shown in Figure 6g, free PpIX and CDs-PpIX (PpIX concentrations: 0–20 µg/mL) elicited no obvious cytotoxicity toward the cells after incubation for 24 h in the dark, indicating their excellent cytocompatibility. We then systematically evaluated the influences of cellular uptake and subcellular localization of CDs-PpIX or PpIX on their PDT efficiency. First, as shown in Figure 6h, under the same PpIX concentration, CDs-PpIX showed more efficient cell killing ability than free PpIX as the cell viability of CDs-PpIX group was much lower than that of free PpIX group. Besides, when cells were pretreated with CDs-PpIX (PpIX concentration: 2 µg/mL) for 1, 4, 12, or 24 h (corresponding to 1 h-, 4 h-, 12 h-, or 24 h-group, respectively) and then exposed under a 5-min irradiation with 635 nm laser (20 mW/cm2), the cell viability of 4 h-group was very close to that of 12 h-group but lower than that of 1 h-group and higher than that of 24 h-group. The results can be ascribed to the reasons that (1) the cellular uptake of CDs-PpIX reached the maximum and did not increase after 4 h incubation and (2) CDs-PpIX could not enter nuclei until incubation for 24 h. Besides, similar results were also obtained when the PpIX concentration in CDs-PpIX was 0.5, 1, or 3 µg/mL. However, when the PpIX concentration in CDs-PpIX reached 4 µg/mL, the cell viabilities of 4 h-, 12 h-, and 24 h-group were the same at 8%, whereas the cell viability of 1 h-group was still as high as 26%, indicating that the cellular uptake of CDs-PpIX (PpIX: 4 µg/mL) after 4 h incubation was sufficient to efficiently kill cells via PDT treatment. All these phenomena prove that the increased cellular uptake and the nucleus-targeting ability of PpIX endowed by the CDs can realize greatly enhanced PDT efficiency on cancerous cells, which will tremendously improve the treatment outcome with higher therapeutic efficiency and fewer side effects due to the decreased dose

25



of drug (PpIX). Finally, live/dead staining (using alcein-AM and PI) was utilized for the further exploration of the therapeutic efficacy of free PpIX and CDs-PpIX combined without and with laser treatment and the results are presented in Figure 6i. HeLa cells treated with CDs-PpIX and 635 nm laser irradiation showed substantial cellular destruction (with almost all the cells displaying intense red fluorescence), whereas the other groups (free PpIX, CDs-PpIX, and “free PpIX + laser”) showed almost no or few red fluorescent cells, further proving the high PDT efficacy of CDs-PpIX.

26


Page 26 of 39

Page 27 of 39 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


Figure 6. (a) Typical TEM image and the corresponding size distribution histogram (inset) of CDs. (b) UV–vis absorption spectra of CDs, PpIX, and CDs-PpIX solutions. (c) Changes of SOSG fluorescence intensities at 526 nm of PpIX, CDs, and CDs-PpIX solutions (Dose: 10 µg/mL of CDs and 5 µg/mL of PpIX) during the continuous 635 nm laser irradiation (20 mW/cm2). (d) Confocal images of HeLa cells treated with PpIX or CDs-PpIX (PpIX concentration: 5 µg/mL) for different time periods. The cells were co-stained with Hoechst 33342 before imaging. Scale bar: 20 µm. (e) The corresponding fluorescence-intensity-profile analysis of the marked arrows in (d). (e1): free PpIX, (e2): CDs-PpIX. Red line: PpIX, blue line: Hoechst 33342. (f) Fluorescence intensities of HeLa cells treated with CDs-PpIX or free PpIX for different time periods. (g) Viabilities of HeLa cells incubated with CDs-PpIX or free PpIX (PpIX concentration: 1, 2, 5, 10, 15, or 20 µg/mL) for 24 h. (h) Viabilities of HeLa cells incubated CDs-PpIX or free PpIX (PpIX concentration: 0, 0.5, 1, 2, 3, or 4 µg/mL) for different time periods (1, 4, 12, or 24 h) followed by laser irradiation (635 nm, 20 mW/cm2, 10 min). (i) Live/dead staining results of HeLa cells treated with CDs-PpIX or free PpIX (PpIX concentration: 2 µg/mL) for 24 h before (laser off) and after (laser on) laser irradiation (635 nm, 20 mW/cm2, 10 min). Scale bar: 20 µm.

3.7. In Vivo/Ex Vivo Fluorescence Tracking. Considering the remarkably enhanced in vitro PDT efficiency induced by the nucleus-targeting performance of CDs-PpIX, we further evaluated the in vivo PDT efficacy of CDs-PpIX. From Figure 7a and b, the tumor region emitted bright red fluorescence originated from CDs-PpIX during the first 1 h and then the corresponding fluorescence intensity continuously increased and reached maximum at 6 h. After that, the fluorescence intensity experienced a slightly decrease during 6–24 h. These results verified the efficient tumor accumulation and retention properties of CDs-PpIX, which may be attributed to their negative charge (which can prolong blood circulation), appropriate size, enhanced cellular uptake and nucleus-targeting ability. By contrast, free PpIX exhibited

27



much weaker fluorescence than CDs-PpIX at all the time points. The above results indicate the satisfying tumor-targeting ability of CDs-PpIX with high-contrast fluorescence imaging and great potential for efficient photodynamic ablation of the tumor region. Besides, to further investigate the in vivo biodistribution of CDs-PpIX, the major organs and tumor tissues of the treated mice were excised at 1, 3, and 7 d postinjection, and then imaged by the Cri Maestroin in vivo imaging system. As shown in Figure 7c, CDs-PpIX were mainly accumulated at the tumor site and liver at 1 d postinjection and then gradually cleared from these sites in the following days, as evidenced by the ex vivo fluorescence results at day 3 and 7. Interestingly, the fluorescence intensities of kidneys at day 3 were higher than those at 1 and 7 d postinjection. The quantitative biodistribution analysis suggest that CDs-PpIX were cleared mainly by liver and partially by kidneys within 7 days. On the other hand, the fluorescence intensity of tumor tissue was much higher than that of the other five organs at day 1, 3, and 7, demonstrating the efficient tumor-targeting ability and prolonged tumor retention properties of CDs-PpIX, which were very advantageous to realize imaging-guided PDT with high therapeutic efficiency and few toxic side effects. 3.8. In Vivo PDT. First, the nucleus-targeting performance of CDs-PpIX at the tumor site was evaluated by collecting the tumor cells from mice 24 h post intravenous injection of CDs-PpIX and co-staining the obtained tumor cells with Hoechst 33342. As shown in Figure S12, the red fluorescence emitted from CDs-PpIX partially coincides with the blue fluorescence of Hoechst 33342, proving the successful nuclear delivery of the nanoagents in the animal model. Afterwards, the in vivo PDT was conducted as follows: The tumor-bearing nude mice were first i.v. injected with PBS, free PpIX, or CDs-PpIX at the PpIX dose of 5

28


Page 28 of 39

Page 29 of 39 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


mg/kg, and then treated without/with 30-min laser irradiation (635 nm, 30 mW/cm2) at 24 h postinjection. The therapeutic outcomes were evaluated by monitoring the tumor volumes and body weights of the mice in 6 treated groups every other day for 20 days. From Figure 8a and c, the tumor on the mouse treated by “CDs-PpIX + laser” was completely eliminated with no regrowth observed within 20 days. By contrast, the tumor volumes of the mice in the other groups showed rapid and continuous growth. To further investigate the therapeutic mechanism, the hematoxylin and eosin (H&E) staining of tumor slices was performed. From the results shown in Figure 8c, the tumor tissue of mice after “CDs-PpIX + laser” treatment was completely destructed but no evident tumor tissue destruction was observed in the groups treated with “PBS + laser” or “free PpIX + laser”. Further, no evident body weight loss and negligible changes of blood indexes (obtained from the blood routine analysis) were observed in all the groups (Figure 8b and S13), suggesting the good biosafety of these treatments to the mice. For further evaluation on the in vivo biocompatibility of CDs-PpIX, the major organs were excised and collected from the sacrificed mice, which had been i.v. injected without (control) or with CDs-PpIX for 20 days, for H&E staining. The results displayed in Figure 8d show that there were no detectable pathological damage including cell apoptosis, cell necrosis, and inflammation in the major organs in all these groups, suggesting the excellent biocompatibility of the nanoagent. The above results demonstrate that CDs-PpIX are a promising nanoplatform for effective and safe PDT.

29



Figure 7. In vivo and ex vivo fluorescence imaging for U14 tumor-bearing nude mice. (a) In vivo fluorescence images and (b) quantitative fluorescence analysis of the tumors after intravenous injection of free PpIX and CDs-PpIX (dose: 5 mg/kg of PpIX), respectively. The blue dotted circles in (a) indicate the tumor areas. (c) Biodistribution of CDs-PpIX in tumor-bearing mice: ex vivo fluorescence images (inset) and the quantitative fluorescence analysis of CDs-PpIX in heart (H), liver (L), spleen (S), lung (Lu), kidneys (K), and tumor (T) of the mice i.v. injected with CDs-PpIX (dose: 5 mg/kg of PpIX) for different time periods (1, 3, or 7 days).

30


Page 30 of 39

Page 31 of 39 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


Figure 8. In vivo PDT and biosafety evaluation. (a) Tumor growth and (b) body weight changes of the mice in different groups after various treatments. ***P < 0.001, one-way analysis of variance (ANOVA). (c) Histopathologic tumor slices (after H&E-staining) and the representative photographs (inset) of U14 tumor-bearing mice (at the 20th day) treated with different methods as indicated. The mice were first i.v. injected with PBS, free PpIX, or CDs-PpIX (dose: 5 mg/kg of PpIX) and then irradiated by a laser (635 nm, 30 mW/cm2) for 30 min at 6 h postinjection. (d) Histopathologic slices (after H&E-staining) from the heart, liver, spleen, lung, and kidneys of the mice injected with PBS (set as control) and CDs-PpIX (dose: 5 mg/kg of PpIX) after 20 days of PDT treatment, respectively. Scale bar: 100 µm.

4. CONCLUSION

31



In summary, a new type of fluorescent carbon dots that can simultaneously realize nucleolus imaging and nucleus-targeted drug delivery has been prepared for the first time using a simple one-step hydrothermal treatment with the carbon sources of m-phenylenediamine and L-cysteine.

As a nucleolus imaging dye, the as-prepared CDs possess superior properties as

compared with the only commercially available dye, SYTO RNASelect. First, the CDs are suitable for large-scale production because their synthetic method is cost-effective, eco-friendly, and facile. Second, the CDs with excellent photostability can stain the nucleolus of both live and fixed cells, achieving rapid, real-time, and wash-free imaging, which are the main shortcomings of SYTO RNASelect. More importantly, various functional surface groups (such as –NH2, –COOH, and –SH) of CDs endow CDs with the great promise to be a new nanocarrier for realizing nucleus-targeted drug delivery and PDT after covalently modifying the CDs with PpIX. Because it is crucial in drug delivery field to fabricate nanoplatforms with intelligent targeting properties for cancer diagnosis and therapy, the nucleus-targeted drug delivery achieved by the CDs will foster the development of novel nuclear delivery strategies. Furthermore, in vivo experiments exhibit the excellent tumor-homing performance, long blood circulation and tumor retention, efficient tumor ablation, and few toxic side effects of CDs-PpIX, suggesting the great advantages of the nanoagents for cancer therapy. The present study not only provides a useful approach for realizing live-cell nucleolus imaging and nuclear drug delivery with significantly improved cancer therapeutic performance, but also demonstrates the great potential of CDs in organelle-targeted imaging and drug delivery for cancer therapy.

32


Page 32 of 39

Page 33 of 39 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


ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: Materials, characterization, and other experimental details; FL spectra, confocal images, stability assay, cell viabilities, 1O2 quantum yield, flow cytometry analysis, TEM results, dynamic light scattering data, and blood routine analysis (PDF)

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

X.W.H. and Y.W.B. contributed equally.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was supported by grants from the National Natural Science Foundation of China (21673037), Natural Science Foundation of Jiangsu Province (BK20170078), Innovative and Entrepreneurial Talent Recruitment Program of Jiangsu Province, and Fundamental Research Funds for the Central Universities.

REFERENCES (1) Németh, A.; Längst, G. Genome Organization in and around the Nucleolus. Cell 2011, 27, 149–156. (2) Yu, C. Y. Y.; Zhang, W. J.; Kwok, R. T. K.; Leung, C. W. T.; Lam, J. W. Y.; Tang, B. Z. A 33



Photostable AIEgen for Nucleolus and Mitochondria Imaging with Organelle-Specific Emission. J. Mater. Chem. B 2016, 4, 2614–2619. (3) He, H.; Wang, Z. C.; Cheng, T. T.; Liu, X.; Wang, X. J.; Wang, J. Y.; Ren, H.; Sun, Y. W.; Song, Y. Z.; Yang, J.; Xia, Y. Q.; Wang, S. J.; Zhang, X. D.; Huang, F. Visible and Near-Infrared Dual-Emission Carbogenic Small Molecular Complex with High RNA Selectivity and Renal Clearance for Nucleolus and Tumor Imaging. ACS Appl. Mater. Interfaces 2016, 8, 28529−28537. (4) Wang, X. J.; Wang, Y. N.; He, H.; Ma, X. Q.; Chen, Q.; Zhang, S.; Ge, B. S.; Wang, S. J.; Nau, W. M.; Huang, F. Deep-Red Fluorescent Gold Nanoclusters for Nucleoli Staining: Real-Time Monitoring of the Nucleolar Dynamics in Reverse Transformation of Malignant Cells. ACS Appl. Mater. Interfaces 2017, 9, 17799–17806. (5) Quin, J. E.; Devlin, J. R.; Cameron, D.; Hannan, K. M.; Pearson, R. B.; Hannan, R. D. Targeting the Nucleolus for Cancer Intervention. Biochim. Biophys. Acta-Mol. Cell Res. 2014, 1842, 802–816. (6) Woods, S. J.; Hannan, K. M.; Pearson R. B.; Hannan, R. D. The Nucleolus as a Fundamental Regulator of the p53 Response and a New Target for Cancer Therapy. Biochim. Biophys. Acta-Gene Regul. Mech. 2015, 1849, 821–829. (7) Liu, W. M.; Zhou, B. J.; Niu, G. L.; Ge, J. C.; Wu, J. S.; Zhang, H. Y.; Xu, H. T.; Wang, P. F. Deep-Red Emissive Crescent-Shaped Fluorescent Dyes: Substituent Effect on Live Cell Imaging. ACS Appl. Mater. Interfaces 2015, 7, 7421−7427. (8) Lu, Y. J.; Deng, Q.; Hu, D. P.; Wang, Z. Y.; Huang, B. H.; Du, Z. Y.; Fang, Y. X.; Wong, W. L.; Zhang, K.; Chow, C. F. A Molecular Fluorescent Dye for Specific Staining and Imaging of RNA in Live Cells: A Novel Ligand Integration from Classical Thiazole Orange and Styryl Compounds. Chem. Commun. 2015, 51, 15241−15244. (9) Song, G. F.; Sun, Y. M.; Liu, Y.; Wang, X. K.; Chen, M. L.; Miao, F.; Zhang, W. J.; Yu, X. Q.; Jin, J. L. Low Molecular Weight Fluorescent Probes with Good Photostability for Imaging RNA-Rich Nucleolus and RNA in Cytoplasm in Living Cells. Biomaterials 2014, 35, 2103−2112. (10) Kummer, S.; Knoll, A.; Socher, E.; Bethge, L.; Herrmann, A.; Seitz, O. PNA FIT-Probes for the Dual Color Imaging of Two Viral mRNA Targets in Influenza H1N1 Infected Live Cells. Bioconjugate Chem. 2012, 23, 2051−2060. (11) Chen, M.; Lei, Z.; Feng, W.; Li, C. Y.; Wang, Q. M.; Li, F. Y. A Phosphorescent Silver(I)–Gold (I) Cluster Complex that Specifically Lights Up the Nucleolus of Living Cells With FLIM Imaging. Biomaterials 2013, 34, 4284−4295. (12) Hu,Y.; Chen, Q.; Ding, Y.; Li, R. T.; Jiang, X. Q.; Liu, B. R. Entering and Lighting Up Nuclei Using Hollow Chitosan–Gold Hybrid Nanospheres. Adv. Mater. 2009, 21, 3639–3643. (13) Yang, X. Z.; Du, X. J.; Liu, Y.; Zhu, Y. H.; Liu, Y. Z.; Li, Y. P.; Wang, J. Rational Design of Polyion Complex Nanoparticles to Overcome Cisplatin Resistance in Cancer Therapy. Adv. Mater. 2014, 26, 931–936. (14) Han, K.; Zhang, W. Y.; Zhang, J.; Lei, Q.; Wang, S. B.; Liu, J. W.; Zhang, X. Z.; Han, H. Y. Acidity-Triggered Tumor-Targeted Chimeric Peptide for Enhanced Intra-Nuclear Photodynamic Therapy. Adv. Funct. Mater. 2016, 26, 4351–4361. (15) Peng, H. B.; Tang, J.; Zheng, R.; Guo, G. N.; Dong, A. G.; Wang, Y. J.; Yang, W. L. Nuclear-Targeted Multifunctional Magnetic Nanoparticles for Photothermal Therapy. Adv. 34


Page 34 of 39

Page 35 of 39 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


Healthcare Mater. 2017, 6, 1601289. (16) Park, K.; Yang, J. A.; Lee, M. Y.; Lee, H.; Hahn, S. K. Reducible Hyaluronic Acid-siRNA Conjugate for Target Specific Gene Silencing. Bioconjugate Chem. 2013, 24, 1201−1209. (17) Shen, S. Y.; Liu, M.; Li, T.; Lin, S. Q.; Mo, R. Recent Progress in Nanomedicine-Based Combination Cancer Therapy Using a Site-Specific Co-Delivery Strategy. Biomater. Sci. 2017, 5, 1367–1381. (18) Fan, W. P.; Yung, B.; Huang, P.; Chen, X. Y. Nanotechnology for Multimodal Synergistic Cancer Therapy. Chem. Rev. 2017, 117 , 13566−13638. (19) Hu, J. J.; Xiao, D.; Zhang, X. Z. Advances in Peptide Functionalization on Mesoporous Silica Nanoparticles for Controlled Drug Release. Small 2016, 12, 3344–3359. (20) Kang, B.; Mackey, M. A.; El-Sayed, M. A. Nuclear Targeting of Gold Nanoparticles in Cancer Cells Induces DNA Damage, Causing Cytokinesis Arrest and Apoptosis. J. Am. Chem. Soc. 2010, 132, 1517–1519. (21) Dozzo, P.; Koo, M. S.; Berger, S.; Forte, T. M.; Kahl, S. B. Synthesis, Characterization, and Plasma Lipoprotein Association of a Nucleus-Targeted Boronated Porphyrin. J. Med. Chem. 2005, 48, 357–359. (22) Anajafi, T.; Yu, J. R.; Sedigh, A.; Haldar, M. K.; Muhonen, W. W.; Oberlander, S.; Wasness, H.; Froberg, J.; Molla, M. D. S.; Katti, K. S.; Choi, Y.; Shabb, J. B.; Srivastava, D. K.; Mallik, S. Nuclear Localizing Peptide-Conjugated, Redox-Sensitive Polymersomes for Delivering Curcumin and Doxorubicin to Pancreatic Cancer Microtumors. Mol. Pharmaceutics 2017, 14, 1916–1928. (23) Fan, Y. B.; Li, C. Y.; Li, F. Y.; Chen, D. Y. pH-Activated Size Reduction of Large Compound Nanoparticles for in Vivo Nucleus-Targeted Drug Delivery. Biomaterials 2016, 85, 30–39. (24) Li, L.; Sun, W.; Zhong, J. J.; Yang, Q. Q.; Zhu, X.; Zhou, Z.; Zhang, Z. R.; Huang, Y. Multistage Nanovehicle Delivery System Based on Stepwise Size Reduction and Charge Reversal for Programmed Nuclear Targeting of Systemically Administered Anticancer Drugs. Adv. Funct. Mater. 2015, 25, 4101–4113. (25) Austin, L. A. Kang, B.; Yen, C. W.; El-Sayed, M. A. Plasmonic Imaging of Human Oral Cancer Cell Communities during Programmed Cell Death by Nuclear-Targeting Silver Nanoparticles. J. Am. Chem. Soc. 2011, 133, 17594–17597. (26) Ali, M. R.; Wu, K.Y.; Ghosh, D.; Do, B. H.; Chen, K. C.; Dawson, M. R.; Fang, N.; Sulchek, T. A.; El-Sayed, M. A. Nuclear Membrane-Targeted Gold Nanoparticles Inhibit Cancer Cell Migration and Invasion. ACS Nano 2017, 11, 3716–3726. (27) Jin, E.; Zhang, B.; Sun, X. R.; Zhou, Z. X.; Ma, X. P.; Sun, Q H.; Tang, J. B.; Shen, Y. Q.; Kirk, E. V.; Murdoch, W. J.; Radosz, M. Acid-Active Cell-Penetrating Peptides for in Vivo Tumor-Targeted Drug Delivery. J. Am. Chem. Soc. 2013, 135, 933–940. (28) Yuan, H.; Fales, Andrew, M.; Tuan, V. D. TAT Peptide-Functionalized Gold Nanostars: Enhanced Intracellular Delivery and Efficient NIR Photothermal Therapy Using Ultralow Irradiance. J. Am. Chem. Soc. 2012, 134, 11358–11361. (29) Xiong, L.; Du, X.; Kleitz, F.; Qiao, S. Z. Cancer-Cell-Specific Nuclear-Targeted Drug Delivery by Dual-Ligand-Modified Mesoporous Silica Nanoparticles. Small 2015, 11, 44, 5919–5926. (30) Li, J. M.; Liu, F.; Shao, Q.; Min, Y. Z.; Costa, M.; Yeow, E. K. L.; Xing, B. G. Enzyme-Responsive Cell-Penetrating Peptide Conjugated Mesoporous Silica Quantum Dot Nanocarriers for Controlled Release of Nucleus-Targeted Drug Molecules and Real-Time 35



Intracellular Fluorescence Imaging of Tumor Cells. Adv. Healthcare Mater. 2014, 3, 1230–1239. (31) Ruan, G.; Agrawal, A.; Marcus, A. I.; Nie, S. M. Imaging and Tracking of Tat Peptide-Conjugated Quantum Dots in Living Cells: New Insights into Nanoparticle Uptake, Intracellular Transport, and Vesicle Shedding. J. Am. Chem. Soc. 2007, 129, 14759–14766. (32) Pan, L. M.; Liu, J. N.; He, Q. J.; Shi, J. L. MSN-Mediated Sequential Vascular-to-Cell Nuclear-Targeted Drug Delivery for Efficient Tumor Regression. Adv. Mater. 2014, 26, 6742–6748. (33) Pan, L. M.; J. N.; He, Liu, Q. J.; Liu, J. N.; Chen, Y.; Ma, M.; Zhang, L. L.; Shi, J. L. Nuclear-Targeted Drug Delivery of TAT Peptide-Conjugated Monodisperse Mesoporous Silica Nanoparticles. J. Am. Chem. Soc. 2012, 134, 5722–5725. (34) Vankayala, R.; Kuo, C. L.; Nuthalapati, K.; Chiang, C. S.; Hwang. K. C. Nucleus-Targeting Gold Nanoclusters for Simultaneous in Vivo Fluorescence Imaging, Gene Delivery, and NIR-Light Activated Photodynamic Therapy. Adv. Funct. Mater. 2015, 25, 5934–5945. (35) Li, Z. Y.; Liu, Y.; Hu, J. J.; Xu, Q.; Liu, L. H.; Jia, H. Z.; Chen, W. H.; Lei, Q.; Rong, L.; Zhang, X. Z. Stepwise-Acid-Active Multifunctional Mesoporous Silica Nanoparticles for Tumor-Specific Nucleus-Targeted Drug Delivery. ACS Appl. Mater. Interfaces 2014, 6, 14568–14575. (36) Tkachenko, A. G.; Xie, H.; Coleman, D.; Glomm, W.; Ryan, J.; Anderson, M. F.; Franzen, S.; Feldheim, D. L. Multifunctional Gold Nanoparticle-Peptide Complexes for Nuclear Targeting. J. Am. Chem. Soc. 2003, 125, 4700–4701. (37) Zhao, J.; Li, Q.; Hao, X. F.; Ren, X. K.; Guo, J. T.; Feng, Y. K.; Shi, C. C. Multi-Targeting Peptides for Gene Carriers with High Transfection Efficiency. J. Mater. Chem. B 2017, 5, 8035–8051. (38) Sui, M. H.; Liu, W. E.; Shen, Y. Q. Nuclear Drug Delivery for Cancer Chemotherapy. J. Control. Release 2011, 155, 227–236. (39) Zhou, Z. X.; Murdoch, W. J.; Shen, Y. Q. A Linear Polyethylenimine (LPEI) Drug Conjugate with Reversible Charge to Overcome Multidrug Resistance in Cancer Cells. Polymer 2015, 76, 150–158. (40) Xu, P. S.; Kirk, E. A. V.; Zhan,Y. H.; Murdoch, W. J. Radosz, M.; Shen, Y. Q. Targeted Charge-Reversal Nanoparticles for Nuclear Drug Delivery. Angew. Chem., Int. Ed. 2007, 46, 4999–5002. (41) Chen, Z. Z.; Zhang, L. F.; He, Y. L.; Li, Y. F. Sandwich-Type Au-PEI/DNA/PEI-Dexa Nanocomplex for NucleusTargeted Gene Delivery in Vitro and in Vivo. ACS Appl. Mater. Interfaces 2014, 6, 14196–14206. (42) Yu, X. W.; Hou, J. H. Shi, Y. J.; Su, C.; Zhao, L. Preparation and Characterization of Novel Chitosan–Protamine Nanoparticles for Nucleus-Targeted Anticancer Drug Delivery. Int. J. Nanomed. 2016, 11, 6035–6046. (43) Zhao, D.; He, Z. K.; Chan, P. S.; Wong, R. N. S.; Mak, N. K.; Lee, A. W. M.; Chan, W. H. NAC-Capped Quantum Dot as Nuclear Staining Agent for Living Cells via an in Vivo Steering Strategy. J. Phys. Chem. C 2010, 114, 6216–6221. (44) Cai, Y. B.; Shen, H. S.; Zhan, J.; Lin, M. L.; Dai, L. H.; Ren, C. H.; Shi, Y.; Liu, J. F.; Gao, J.; Yang Z. M. Supramolecular “Trojan Horse” for Nuclear Delivery of Dual Anticancer Drugs. J. Am. Chem. Soc. 2017, 139, 2876–2879. (45) Hwang, D. W.; Hong, B. H.; Lee, D. S. Multifunctional Graphene Oxide for Bioimaging: 36


Page 36 of 39

Page 37 of 39 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


Emphasis on Biological Research. Eur. J. Nanomed. 2017, 9, 47–57. (46) Hua, X. W.; Bao, Y. W.; Wang, H. Y.; Chen, Z.; Wu, F. G. Bacteria-Derived Fluorescent Carbon Dots for Microbial Live/Dead Differentiation. Nanoscale 2017, 9, 2150–2161. (47) Tao, H. Q.; Yang, K.; Ma, Z.; Wan, J. M.; Zhang, Y. J.; Kang, Z. H.; Liu, Z. In Vivo NIR Fluorescence Imaging, Biodistribution, and Toxicology of Photoluminescent Carbon Dots Produced from Carbon Nanotubes and Graphite. Small 2012, 8, 281–290. (48) Sun, D.; Ban, R.; Zhang, P. H.; Wu, G. H.; Zhang, J. R.; Zhu, J. J. Hair Fiber as a Precursor for Synthesizing of Sulfur- and Nitrogen-Co-Doped Carbon Dots with Tunable Luminescence Properties. Carbon 2013, 64, 424–434. (49) Lu, S. Y.; Sui, L. Z.; Liu, J. J.; Zhu, S. J.; Chen, A. M..; Jin, M. X.; Yang, B. Near-Infrared Photoluminescent Polymer–Carbon Nanodots with Two-Photon Fluorescence. Adv. Mater. 2017, 29, 1603443. (50) Wang, Z. G.; Fu, B. S.; Zou, S. W.; Duan, B.; Chang, C . Y.; Yang, B.; Zhou, X.; Zhang, L. N. Facile Construction of Carbon Dots via Acid Catalytic Hydrothermal Method and Their Application for Target Imaging of Cancer Cells. Nano Res. 2016, 9, 214–223. (51) Qu, D.; Miao, X.; Wang, X. T.; Nie, C.; Li, Y. X.; Lu, L.; Sun, Z. C. Se & N Co-Doped Carbon Dots for Highperformance Fluorescence Imaging Agent of Angiography. J. Mater. Chem. B 2017, 5, 4988–4992. (52) Ding, H.; Cheng, L. W.; Ma, Y. Y.; Kong, J. L.; Xiong, H. M. Luminescent Carbon Quantum Dots and Their Application in Cell Imaging. New J. Chem. 2013, 37, 2515–2520. (53) Li, H. T.; He, X. D.; Kang, Z. H.; Huang, H.; Liu, Y.; Liu, J. L.; Lian, S. Y.; Tsang, C. H. A.; Yang, X. B.; Lee, S. T. Water-Soluble Fluorescent Carbon Quantum Dots and Photocatalyst Design. Angew. Chem., Int. Ed. 2010, 49, 4430–4434. (54) Zhao, A. D.; Chen, Z. W.; Zhao, C. Q.; Gao, N.; Ren, J. S.; Qu, X. G. Recent Advances in Bioapplications of C-Dots. Carbon 2015, 85, 309–327. (55) Liu, J.; Liu, Y.; Liu, N. Y.; Han, Y. Z.; Zhang, X.; Huang, H.; Lifshitz, Y.; Lee, S. T.; Zhong, J.; Kang, Z. H. Metal-Free Efficient Photocatalyst for Stable Visible Water Splitting via a Two-Electron Pathway. Science 2015, 347, 970–974. (56) Liu, Q.; Ma, C.; Liu, X. P.; Wei, Y. P.; Mao, C. J.; Zhu, J. J. A Novel Electrochemiluminescence Biosensor for the Detection of MicroRNAs Based on a DNA Functionalized Nitrogen Doped Carbon Quantum Dots as Signal Enhancers. Biosens. Bioelectron. 2017, 92, 273–279. (57) Li, H.; Kong, W. Q.; Liu, J.; Liu, N. Y.; Huang, H.; Liu, Y.; Kang, Z. H. Fluorescent N-Doped Carbon Dots for Both Cellular Imaging and Highly-Sensitive Catechol Detection. Carbon 2015, 91, 66–75. (58) Yang, J. J.; Zhang, X. D.; Ma, Y. H.; Gao, G.; Chen, X. K.; Jia, H. R.; Li, Y. H.; Chen, Z.; Wu, F. G. Carbon Dot-Based Platform for Simultaneous Bacteria Distinguishment and Antibacterial Applications. ACS Appl. Mater. Interfaces 2016, 8, 32170–32181. (59) Feng, T.; Ai, X. Z.; Ong, H. M.; Zhao, Y. L. Dual-Responsive Carbon Dots for Tumor Extracellular Microenvironment Triggered Targeting and Enhanced Anticancer Drug Delivery. ACS Appl. Mater. Interfaces 2016, 8, 18732−18740. (60) Feng, T.; Ai, X. Z.; An, G. H.; Yang, P. P.; Zhao, Y. L. Charge-Convertible Carbon Dots for Imaging-Guided Drug Delivery with Enhanced in Vivo Cancer Therapeutic Efficiency. ACS Nano 2016, 10, 4410–4420. (61) Hua, X. W.; Bao, Y. W.; Chen, Z.; Wu, F. G. Carbon Quantum Dots with Intrinsic Mitochondrial 37



Targeting Ability for Mitochondria-Based Theranostics. Nanoscale 2017, 9, 10948–10960. (62) Yang, J. J.; Gao, G.; Zhang, X. D.; Ma, Y. H.; Jia, H. R.; Jiang, Y. W.; Wang, Z. F.; Wu, F. G. Ultrasmall and Photostable Nanotheranostic Agents Based on Carbon Quantum Dots Passivated with Polyamine-Containing Organosilane Molecules. Nanoscale 2017, 9, 15441–15452. (63) Zheng, M.; Liu, S.; Li, J.; Qu, D.; Zhao, H. F.; Guan, X. G.; Hu, X. L.; Xie, Z. G.; Jing, X. B.; Sun, Z. C. Integrating Oxaliplatin with Highly Luminescent Carbon Dots: An Unprecedented Theranostic Agent for Personalized Medicine. Adv. Mater. 2014, 26, 3554–3560. (64) Miao, X.; Yan, X. L.; Qu, D.; Li, D. B.; Tao, F. F.; Sun, Z. C. Red Emissive Sulfur, Nitrogen Codoped Carbon Dots and Their Application in Ion Detection and Theraonostics. ACS Appl. Mater. Interfaces 2017, 9, 18549–18556. (65) Liu, J. J.; Lu, S. Y.; Tang, Q. L.; Zhang, K.; Yu, W. X.; Sun, H. C.; Yang, B. One-Step Hydrothermal Synthesis of Photoluminescent Carbon Nanodots with Selective Antibacterial Activity Against Porphyromonas Gingivalis. Nanoscale 2017, 9, 7135–7142. (66) Sun, S.; Zhang, L.; Jiang, K.; Wu, A. G.; Lin, H. W. Toward High-Efficient Red Emissive Carbon Dots: Facile Preparation, Unique Properties, and Applications as Multifunctional Theranostic Agents. Chem. Mater. 2016, 28, 8659−8668. (67) Ding, H.; Yu, S. B.; Wei, J. S.; Xiong, H. M. Full-Color Light-Emitting Carbon Dots with a Surface-State-Controlled Luminescence Mechanism. ACS Nano 2016, 10, 484–491. (68) Lu, F.; Yang, L.; Ding, Y. J.; Zhu, J. J. Highly Emissive Nd3+-Sensitized Multilayered Upconversion Nanoparticles for Effcient 795 nm Operated Photodynamic Therapy. Adv. Funct. Mater. 2016, 26, 4778–4785. (69) Zhu, W. W.; Dong, Z. L.; Fu, T. T.; Liu, J. J.; Chen, Q.; Li, Y. G.; Zhu, R.; Xu, L. G.; Liu, Z. Modulation of Hypoxia in Solid Tumor Microenvironment with MnO2 Nanoparticles to Enhance Photodynamic Therapy. Adv. Funct. Mater. 2016, 26, 5490–5498. (70) Gong, H.; Chao, Y.; Xiang, J.; Han, X.; Song, G. S.; Feng, L. Z.; Liu, J. J.; Yang, G. B.; Chen, Q.; Liu, Z. Hyaluronidase to Enhance Nanoparticle-Based Photodynamic Tumor Therapy. Nano Lett. 2016, 16, 2512–2521. (71) Li, M.; He, P.; Li, S. L.; Wang, X. Y.; Liu, L. B.; Lv, F. T.; Wang, S. Oligo(p-phenylenevinylene) Derivative-Incorporated and EnzymeResponsive Hybrid Hydrogel for Tumor Cell-Specific Imaging and Activatable Photodynamic Therapy. ACS Biomater. Sci. Eng. 2018, DOI: 10.1021/acsbiomaterials.7b00610.

38


Page 38 of 39

Page 39 of 39 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


TOC Graphic

39


Fluorescent Carbon Quantum Dots with Intrinsic ... - ACS Publications

Recommend Documents