Preparation of a Ruthenium-Complex-Functionalized Two-Photon

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Biological and Medical Applications of Materials and Interfaces

Preparation of Ruthenium Complex-Functionalized Two-PhotonExcited Red Fluorescence Silicon Nanoparticles Composite for Target Fluorescence Imaging and Photodynamic Therapy in Vitro Ya-Kun Dou, Yue Shang, Xi-Wen He, Wen-You Li, Yu-Hao Li, and YuKui Zhang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b00288 • Publication Date (Web): 22 Mar 2019 Downloaded from http://pubs.acs.org on March 23, 2019

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ACS Applied Materials & Interfaces

Preparation of Ruthenium Complex-Functionalized Two-PhotonExcited Red Fluorescence Silicon Nanoparticles Composite for Target Fluorescence Imaging and Photodynamic Therapy in Vitro Ya-Kun Dou,† Yue Shang,‡ Xi-Wen He,† Wen-You Li,*,†,§ Yu-Hao Li,*,‡ and Yu-Kui Zhang†,║ †College

of Chemistry, Research Center for Analytical Sciences, State Key Laboratory of

Medicinal Chemical Biology, Tianjin Key Laboratory of Biosensing and Molecular Recognition, Nankai University, Tianjin 300071, China, [email protected]., Fax: +86-22-23502458 ‡Key

Laboratory of Tumor Microenvironment and Neurovascular Regulation, Nankai University

School of Medicine, Tianjin 300071, China, [email protected]., Fax: +86-22-23502554 §Collaborative

Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin

300071, China ║National

Chromatographic Research and Analysis Center, Dalian Institute of Chemical Physics,

Chinese Academy of Sciences, Dalian 116023, China

KEYWORDS: silicon nanoparticles composite, two-photon excitation, fluorescence resonance energy transfer, targeted fluorescence imaging, photodynamic therapy in vitro

ABSTRACT: Silicon nanoparticles (SiNPs), especially those emitting red fluorescence, have been widely applied in the field of bioimaging. However, harsh synthetic conditions and strong

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biological autofluorescence caused by short wavelength excitation restrict the further development of SiNPs in the field of biological applications. Here, we reported a method for synthesizing ruthenium complex-functionalized two-photon-excited red fluorescence silicon nanoparticles composite (SiNPs-Ru) based on fluorescence resonance energy transfer (FRET) under mild experimental conditions. In the prepared SiNPs-Ru composite, silicon nanoparticles synthesized by atmospheric pressure microwave-assisted synthesis served as fluorescence energy donor which had two-photon fluorescence properties, and tris(4,4’-dicarboxylic acid-2,2bipyridyl) ruthenium(II) dichloride (LRu) acted as fluorescence energy acceptor which could emit red fluorescence as well as had the ability to produce singlet-oxygen for photodynamic therapy. Therefore, the synthesized SiNPs-Ru could emit red fluorescence by two-photon excitation (TPE) based on fluorescence resonance energy transfer, which could effectively avoid the interference of biological autofluorescence. Fluorescence imaging tests in zebrafish and nude mice indicated that the as-prepared SiNPs-Ru could act as a new kind of fluorescence probe for fluorescence imaging in vivo. By coupling folic acid (FA) to SiNPs-Ru, the prepared composite (FA-SiNPs-Ru) could not only serve as targeted two-photon fluorescence imaging probe but also kill the cancer cells via photodynamic therapy (PDT) in vitro.

INTRODUCTION In recent years, as a new type of fluorescence probe, silicon nanoparticles (SiNPs) have obtained more and more attention in the field of bioimaging because of excellent optical properties, good biocompatibility, and lower toxicity than quantum dots (QDs) made up of II-VI or III-V elements such as CdTe QDs.1-6 It is well known that fluorescence probes which can emit


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red fluorescence and even fluorescence in the near infrared region are the goals that people pursue due to the strong tissue penetration ability and low tissue autofluorescence.7-9 Therefore, this kind of fluorescence probe can be better applied to bioluminescence imaging. But so far, the synthetic silicon nanoparticles often emit blue-green fluorescence,10-13 and silicon nanoparticles emitting red fluorescence are scarce. Recently, Zhong et al.14 prepared red luminescent SiNPs by using an established HF-assisted etching method, and a specialized microwave reactor at high reaction temperature (180-200°C) and high pressure was also used. Veinot’s group15 obtained silicon nanocrystals (Si-NCs) emitting red fluorescence in the anhydrous and anaerobic conditions via HF etching commercially available hydrogen silsesquioxane (HSQ) at high temperature (ca. 1100°C) to get unstable hydride terminated Si-NCs as precursor. By using HF, H2O2 and polyoxometalate (POM) as the catalyst in the electrochemical etching method, the unstable hydrogen sealed SiQDs as precursor was synthesized, then SiQDs emitting red fluorescence were obtained by Lee’s group.16 Nevertheless, these synthetic methods often need to be carried out under high temperature and high pressure conditions, or to use the dangerous substance HF in the etching process and producing unstable intermediates. Moreover, the synthesized nanoparticles were excited by relatively short wavelength light and were only used for fluorescence imaging of cells, but not in vivo fluorescence imaging. As we all know, nearinfrared (NIR) light excitation can deeply penetrate into living tissues and can effectively avoid the interference of autofluorescence from biological tissues as well.17,18 Therefore, it is a challenge to synthesize silicon nanoparticles that can emit red fluorescence with long wavelength light excitation under milder experimental conditions. Photodynamic therapy (PDT) has attracted wide attention as a kind of non-invasive medical technology.19-22 Compared with conventional chemotherapy and radiotherapy, PDT has


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significant selectivity in the treatment of various types of cancer and can reduce the incidence of side effects.23-26 Pyridine ruthenium and its derivatives have been used as optical imaging reagents or used in sensing monitoring applications because of their good optical properties.27-30 At the same time, it has also been found that its derivatives have the ability to produce singletoxygen to kill cancer cells for photodynamic therapy.31 In recent years, the use of Ru(II)polypyridyl complexes for PDT has aroused great interest.32-34 Herein, we reported a method for synthesizing ruthenium complex-functionalized two-photonexcited red fluorescence silicon nanoparticles composite (SiNPs-Ru) based on fluorescence resonance energy transfer (FRET) under mild experimental conditions. As far as we know, this is the first time that fluorescence SiNPs and ruthenium complexes have been combined for PDT. In the composite, silicon nanoparticles (SiNPs) act as the fluorescent energy donor and tris(4,4’dicarboxylic acid-2,2-bipyridyl) ruthenium(II) dichloride (LRu) acts as the fluorescence energy receptor. At the same time, folic acid (FA) was coupled to SiNPs-Ru to obtain a composite of FA-SiNPs-Ru with targeting ability. The composites can achieve two-photon excitation (TPE) and long wavelength emission (650 nm) under the action of FRET, which can effectively avoid the interference of biological autofluorescence. We have not only successfully applied the asprepared SiNPs-Ru and FA-SiNPs-Ru to in vivo fluorescence imaging, but also applied them to TPE fluorescence imaging and PDT in vitro.

EXPERIMENTAL SECTION Synthesis of the Silicon Nanoparticles (SiNPs). Add 8 mL glycerol to 25 mL round bottom flask, 0.3180 g citrate was added and stir 25 min in an argon atmosphere to remove oxygen, and then drip 3 mL 3-(2-Aminoethylamino) propyltrimethoxysilane into the flask and keep stirring


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for 10 min. The solution is then transferred to the microwave reactor and reacted at 180°C for 15 min to obtain the silicon nanoparticles (SiNPs). Preparation of SiNPs-Ru. 0.0098 g LRu was dissolved into 28 mL water, then 0.2588 g EDC and 0.1553 g NHS was added to 7 mL solution of LRu to activate LRu with stirring for 30 min. 2 mL SiNPs solution diluted 3 times with water was added to the activated LRu, reacting 12 h under the room temperature (25°C) and atmospheric pressure. Excess LRu and SiNPs could be removed by dialyzing through a dialysis membrane (1000 MW) to get the purified SiNPs-Ru. Then, the as-prepared SiNPs-Ru were kept in the dark and the solid product was obtained by freeze-drying for further applications. Preparation of FA-SiNPs-Ru. 12.5 mg folic acid (FA) was dissolved in 5 mL PBS buffer solution, and 13.4 mg EDC and 8.4 mg NHS were added to activate for 30 min. Then, 10 mL SiNPs-Ru solution was added, and the mixture was vigorously stirred for 12 h under the room temperature (25°C). Free FA and SiNPs-Ru was removed by dialysis (dialysis bag MW cutoff 1000) to obtain the purified FA-SiNPs-Ru. Other experimental parts, including chemicals and reagents, instrumentations, imaging experiments, cytotoxicity assay, etc., were presented detailedly in Supporting Information.

RESULTS AND DISCUSSION The Optimization of Reaction Conditions. The schematic illustration for the preparation of SiNPs-Ru and FA-SiNPs-Ru under mild experimental conditions and the biological applications in vitro and in vivo was shown in Scheme 1. First of all, we synthesized SiNPs referring to Ye’s method.11 SiNPs were synthesized by atmospheric pressure microwave-assisted syntheis method, in which 3-(2-aminoethylamino) propyltrimethoxysilane (DAMO) and sodium citrate were used


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as silicon source and reducing agent, respectively. The as-prepared SiNPs had two-photon fluorescence properties. Then, activated tris(4,4’-dicarboxylic acid-2,2-bipyridyl) ruthenium(II) dichloride (LRu) by EDC and NHS reacted with the as-prepared SiNPs to obtain silicon nanoparticles composite (SiNPs-Ru). The as-prepared SiNPs-Ru were capable of emitting red fluorescence by two-photon excitation under the action of FRET. Then through the action of EDC and NHS, SiNPs-Ru and folic acid (FA) were coupled together to obtain a composite of FA-SiNPs-Ru with targeting imaging ability. The as-prepared composites (SiNPs-Ru and FASiNPs-Ru) were applied to in vivo fluorescence imaging and two-photon fluorescence imaging and PDT in vitro. To obtain the optimal fluorescence properties of SiNPs-Ru, we optimized the synthetic reaction conditions. The conditions included two aspects: the dosage of donor SiNPs and the concentration of receptor LRu. The concentration of LRu was fixed to 0.3 mmol/L, and Figure S1a described the fluorescence spectra excited by 800 nm of SiNPs-Ru synthesized under different dosages of SiNPs. PL intensity of SiNPs gradually enhanced along with the increase of dosages of SiNPs from 0.5 mL to 6 mL (Figure S1b). And PL intensity of LRu was enhanced first from 0.5 mL to 2 mL and then weakened from 2 mL to 6 mL (Figure S1c). The dosages of SiNPs was fixed to 2 mL, the fluorescence spectra excited by 800 nm of SiNPs-Ru synthesized under different concentration of LRu was shown in Figure S2a. As shown in Figure S2b, we could see that PL intensity of SiNPs gradually decreased along with the increase of the concentration of LRu from 0.05 mmol/L to 0.5 mmol/L. PL intensity of LRu was increased firstly and reached a maximum when the concentration of LRu was 0.3 mmol/L and then decreased from 0.3 mmol/L to 0.5 mmol/L (Figure S2c). In summary, the as-prepared SiNPs-Ru was synthesized by


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atmospheric pressure microwave-assisted synthesis with the dosage of SiNPs of 2 mL and the concentration of LRu at 0.3 mmol/L under room temperature and atmospheric pressure for 12 h. Scheme 1. The Schematic Illustration of the Synthesis of SiNPs-Ru and FA-SiNPs-Ru and the Biological Applications in Vitro and in Vivo

The Structure Characterizations of SiNPs-Ru and FA-SiNPs-Ru. The high resolution transmission electron microscope (HETEM) images of SiNPs and SiNPs-Ru were shown in Figure 1a and Figure 1b, respectively. From these two images we could see that the as-prepared SiNPs and SiNPs-Ru had good monodispersity with sphericity. As shown the inset in Figure 1a, the lattice spacing of the as-prepared SiNPs was 0.32 nm, agreeing with the lattice of Si (111), which was consistent with the result reported in literatures.

10,35

From Figure S3, we have

respectively calculated the size distribution of more than 150 particles of SiNPs and SiNPs-Ru,


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and the average diameter size of SiNPs and SiNPs-Ru was 2.2 nm and 3.4 nm, respectively. The average hydrodynamic diameter of SiNPs measured by dynamic light scattering (DLS) was 2.8 nm (Figure 1c). And as shown in Figure 1d, the average hydrodynamic diameter of the asprepared SiNPs-Ru was 0.9 nm bigger than that of SiNPs and reached to 3.7 nm, indirectly demonstrating the successful synthesis of SiNPs-Ru. Compared with the results of hydrodynamic diameter of SiNPs and SiNPs-Ru, the sizes of SiNPs and SiNPs-Ru obtained from TEM are smaller than those from DLS. The results are consistent with those in the literatures.10,11,36 In order to prove the successful synthesis of SiNPs-Ru, FT-IR spectra of SiNPs and SiNPs-Ru were measured. As shown in Figure 1e, the broad absorbance peaks at 3270 and 3272 cm-1 were ascribed to the stretching vibration of the N-H bond. The absorbance peaks at 2932, 2929, 2880 and 2876 cm-1 were assigned to the antisymmetric stretching vibration of the C-H bond. The sharp absorbance peaks at 1060 cm-1 were attributed to the vibrational stretch of Si-O bond which was consistent with the reported literature.37 These absorbance peaks mentioned above existed both in the FT-IR spectra of SiNPs and SiNPs-Ru. Whereas, some absorption peaks only existed in the FT-IR of SiNPs-Ru. The absorbance peak at 1657 cm-1 ascribed to the stretching vibration of C=O in C=O-N, only existed in SiNPs-Ru, indicating the formation of O=C-N in SiNPs-Ru. In addition, the absorption peaks at 1727 cm-1 and 1437 cm-1 represented the stretching vibration of C=O in -COOH and C=N in pyridine, respectively, which proved the existence of pyridine and -COOH in SiNPs-Ru and indicated the existence of LRu in SiNPs-Ru. The results proved the generation of the O=C-N bond and the existence of LRu in SiNPs-Ru after the reaction between SiNPs and LRu. Therefore, the synthesis of SiNPs-Ru was successful. We also carried out zeta potential experiments to further prove the successful synthesis of SiNPs-Ru. As shown in Figure 1f, the zeta potential of SiNPs and LRu at neutral pH were 9.08±0.62 and -


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9.68 ±0.25 mV, respectively. The zeta potential of SiNPs-Ru generated after the reaction was 0.54 ± 0.04 mV. The changes in the zeta potential of each substance indicated the successful synthesis of SiNPs-Ru. 10,38,39

Figure 1. The structure characterizations. (a) HRTEM image of SiNPs (scale 20 nm), inset: HRTEM image of SiNPs (scale 5 nm). (b) HRTEM image of SiNPs-Ru. (c) DLS characterizations of SiNPs. (d) DLS characterizations of SiNPs-Ru. (e) FT-IR spectra of SiNPs, LRu and SiNPs-Ru. (f) The zeta potential of SiNPs, LRu and SiNPs-Ru at neutral pH.

We had also characterized FA-SiNPs-Ru by TEM, DLS, FT-IR and zeta potential. From Figure S4a, we could see that the average size of as-prepared FA-SiNPs-Ru calculated with more than 150 particles was 3.6 nm, and the average hydrodynamic diameter from DLS (Figure S4b) was 4.0 nm. In order to prove the formation of FA-SiNPs-Ru, we supplemented FT-IR spectra of FA


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and FA-SiNPs-Ru, and characterized zeta potential of FA-SiNPs-Ru. As shown in Figure S5a, FA-SiNPs-Ru had a similar FT-IR spectrum to that of FA, and the absorption peak at 1059 cm-1 in FT-IR spectrum of FA-SiNPs-Ru represented the Si-O bond.40 FT-IR spectra showed the presence of FA in the prepared FA-SiNPs-Ru.41 Therefore, the synthesis of FA-SiNPs-Ru was successful. At the same time, the zeta potential of SiNPs-Ru and FA-SiNPs-Ru were 0.54±0.04 mV and 10.05 ± 1.09 mV, respectively (Figure S5b). The changes in the zeta potential also indicated the successful synthesis of FA-SiNPs-Ru. 10,38,39

Figure 2. (a) Fluorescence excitation spectrum (black line) and PL spectrum of SiNPs (red line). (b) The excitation wavelength-independent emission spectra of SiNPs with the excitation wavelength varied from 300 to 430 nm. (c) Up-conversion fluorescence spectra of SiNPs with the excitation wavelength changed from 600 to 860 nm. (d) The linear relationship between twophoton luminescence intensity (TPL intensity) of SiNPs and the square of excitation laser power.


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The Optical Properties of SiNPs and SiNPs-Ru. We could see that the maximum emission wavelength of SiNPs was 490 nm under the excitation of 390 nm from Figure 2a. As shown in Figure S6a, with quinine sulfate as the reference material, the relative fluorescence quantum yield of SiNPs was up to 23.1%. The fluorescence lifetime of SiNPs was 14.39 ns (Figure S6b, Table S1), which was much longer than the biological autofluorescence, whose lifetimes were often shorter than 5 ns.10 As shown in Figure S7, the as-prepared SiNPs shown excellent chemical stability. In the wide pH range of 3-11, PL intensity of SiNPs had very small changes, demonstrating the excellent pH stability of SiNPs (Figure S7a). The as-prepared SiNPs also had fine NaCl salt stability when the concentration of NaCl increased from 0 to 100 mmol/L (Figure S7b). The synthesized SiNPs displayed excitation-independent property (Figure 2b). When the excitation wavelength was in the range of 300-430 nm, the emission wavelength of the synthesized SiNPs remained 490 nm, and did not change with the excitation wavelength. As shown shown in Figure 2c, SiNPs could be excited by light with long excitation wavelength (600-860 nm) and the emission wavelength remained unchanged at 490 nm, showing the property of up-conversion. From the experiments (Figure 2d), we proved that this up-conversion property originated from two-photon absorption process.11,42,43 The fluorescence intensity of SiNPs increased with the strengthen of the excitation laser power, and it showed a good linearity (R2=0.99937) with the square of the power (P2) (Figure 2d), indicating that the up-conversion fluorescence property of SiNPs was attributed to the two-photon absorption process.42-44 The excitation wavelength and emission wavelength of LRu were at 482 nm and 660 nm, respectively (Figure 3a). As shown in Figure 3b, the emission spectrum of SiNPs had a great overlap with the absorption spectrum of LRu at 470 nm. This result indicated that there was the possibility of FRET between SiNPs and LRu. As mentioned earlier, the synthesized SiNPs had


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two-photon fluorescence properties. Therefore, the above experimental results showed that the as-prepared SiNPs-Ru had the potential of two-photon excitation and emitting red fluorescence

Figure 3. (a) Fluorescence excitation spectrum (black line) and PL spectrum of LRu (red line). (b) UV−vis absorption spectrum of LRu (black line) and PL spectrum of SiNPs (red line). (c) PL spectra of SiNPs (black line), LRu (red line) and SiNPs-Ru (blue line) excited by light at 800 nm wavelength. (d) Photographs of SiNPs (left) and SiNPs-Ru (right) irradiated by 365 nm UV lamp.

through FRET. We have further confirmed the potential of long wavelength excitation and emitting red fluorescence of SiNPs-Ru through FRET. From Figure 3c we could see that when SiNPs and LRu were combined (SiNPs-Ru), the emission intensity of SiNPs decreased significantly and the emission intensity of LRu increased significantly when excited by light at


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800 nm wavelength. It could be concluded that FRET indeed occurred between SiNPs and LRu in the composite of SiNPs-Ru. Under 365 nm ultraviolet lamp irradiation, SiNPs emitted bright blue fluorescence and the as-prepared SiNPs-Ru emitted red fluorescence (Figure 3d).

Figure 4. Viability of HeLa cells cultured with different concentrations (0, 25, 50,100 and 200 μg/mL) of SiNPs-Ru for 6 h, 12 h and 24 h.

Cytotoxicity Assay of SiNPs-Ru and FA-SiNPs-Ru. MTT assay was used to assess the cytotoxicity of SiNPs-Ru in vitro. As shown in Figure 4, the cell viability showed a concentration-dependent manner at every culture time point (6, 12, and 24 h). The toxicity increased correspondingly with the prolongation of culture time. However, when cells were cultured with 200 μg/mL of SiNPs-Ru for 24 h, the cell viability remained above 80% and no statistical significance was found among groups at every culture time point. Similarly, the cytotoxicity of FA-SiNPs-Ru in vitro was very low. When cultured with different concentrations (0, 25, 50,100 and 200 μg/mL) of FA-SiNPs-Ru for 6, 12 and 24 h, the cell viability was still


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over 86% (Figure S8). The results demonstrated that SiNPs-Ru and FA-SiNPs-Ru showed very low cytotoxicity in vitro, and were suitable for cell fluorescence imaging.

Figure 5. (a) Bright-field and fluorescence pictures of zebrafish embryos exposed to SiNPs-Ru. Notes: Bright-field pictures (the above layer) and fluorescence pictures (the below layer) of embryos exposed to different concentrations (0, 25, 50,100 and 200 μg/mL) of SiNPs-Ru at 4 hpf. Scale bar: 300 μm. (b) Bright field and fluorescence pictures of zebrafish embryos exposed to SiNPs-Ru. Bright field images (the above layer) and red fluorescence images (the below layer) of embryos exposed to 100 μg/mL SiNPs-Ru at 8, 12, 24, 48, and 72 hpf. Scale bar: 300 μm.

Toxicity Test and Imaging of SiNPs-Ru in Zebrafish. In recent years, zebrafish has been widely used as an organism for in vivo imaging.45,46 We investigated the fluorescence imaging of


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SiNPs-Ru in zebrafish. Firstly, we conducted the survival rates, the hatching rates and phenotype observation experiments of zebrafish to explore the toxicity of SiNPs-Ru in zebrafish (Figure S9 and S10). In the control group, the survival rate of zebrafish embryos at 36 hpf was 86% (Figure S9a) and the hatching rate of zebrafish embryos at 72 hpf was 93% (Figure S9b). When cultured with different concentrations of SiNPs-Ru for the same time, the survival rate and the hatching rate showed the same trend. In the exposed group cultured with 200 μg/mL of SiNPs-Ru, the survival rate at 36 hpf and the hatching rate at 72 hpf were 70% and 78%, respectively. As shown in Figure S10, compared with the control group, no obvious malformation was observed in the treated groups (exposed to 25, 50, 100, 200 μg/mL of SiNPs-Ru) of embryos according to the standard at 6, 12, 24, 48, and 72 hpf. The results demonstrated that SiNPs-Ru had little effect on the development of zebrafish. Next, we carried out the fluorescence imaging experiments of SiNPs-Ru in zebrafish. Figure 5a showed the fluorescence imaging of zebrafish embryos which were exposed with different concentrations (25, 50, 100, 200 μg/mL) of SiNPs-Ru. The fluorescence intensity of SiNPs-Ru showed a concentration dependent nature in zebrafish embryos, and with the increase of the concentration, the fluorescence intensity increased gradually in zebrafish embryos. In the next experiments, the concentration of SiNPs-Ru was 100 μg/mL. As shown in Figure 5b, along with the development of zebrafish, SiNPs-Ru gradually entered into zebrafish body and mainly located in yolk sac. At the same time, the fluorescence intensity gradually weakened until nearly disappeared around 72 hpf (Figure 5b, the below layer). As we know, carbon dots were excreted by zebrafish through the digestive system.47,48 We suspected that SiNPs-Ru were excreted by zebrafish in the same way. Meanwhile, the zebrafish embryos exhibited the normal appearance at 72 hpf, which indicated that the as-prepared SiNPs-Ru had low toxicity in vivo.


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Figure 6. Time-dependent fluorescence imaging in vivo: nude mice were injected with LRu and SiNPs-Ru, and HeLa tumor-bearing mice were injected with FA-SiNPs-Ru via caudal vein.

In Vivo Imaging of SiNPs-Ru in Mice. We investigated the real-time fluorescence imaging of SiNPs-Ru in vivo, in which LRu was selected as a reference (Figure 6). When the mice in the control group were injected with LRu, LRu was not enriched in the liver of the nude mice, but was excreted quickly through bladder after 10 minutes of injection. However, when the nude mice were injected with SiNPs-Ru, the nanoparticles were first enriched in the liver of the nude mice. After 60 minutes of injection, the nanoparticles were gradually transferred to bladder and finally excreted through urine. At the same time, the fluorescence signal also disappeared. The results showed that SiNPs-Ru can circulate longer than LRu in vivo, probably because SiNPs-Ru were larger than LRu in size. Therefore, SiNPs-Ru were more suitable for fluorescence imaging in vivo than LRu. When HeLa tumor-bearing mice were injected with FA-SiNPs-Ru, the circulation of the composite of FA-SiNPs-Ru in the mice was the same as that of SiNPs-Ru; at the same time,


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FA-SiNPs-Ru had a relatively obvious enrichment in the tumor site, indicating that the asprepared FA-SiNPs-Ru was potential as a nanoprobe for targeting cancer cells in vivo fluorescence imaging.

Figure 7. (a) H&E images of heart, liver, lung, spleen, and kidney of the mice injected for 7 days in control group (injected with normal saline) and treatment groups (injected with SiNPs-Ru and FA-SiNPs-Ru, respectively). (b) The body weight trends of the mice injected with normal saline (n=3), SiNPs-Ru (n=3) and FA-SiNPs-Ru (n=3).

Toxicity Test of SiNPs-Ru and FA-SiNPs-Ru in Mice. In order to investigate the toxicity of SiNPs-Ru and FA-SiNPs-Ru in vivo, we carried out the test of H&E staining experiment of main organs and long-term body weight changes of the mice (Figure 7). As shown in Figure 7a, compared with the control group (injected with normal saline), no obvious tissue damage was


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detected in major organs (heart, liver, lung, spleen, and kidney) of the mice injected with SiNPsRu and FA-SiNPs-Ru, respectively. After 28 days of continuous culture, the same weight trends of the mice were found in the control group (injected with normal saline) and the treated groups (injected with SiNPs-Ru and FA-SiNPs-Ru, respectively) (Figure 7b). From the above results, we could see that the composites of SiNPs-Ru and FA-SiNPs-Ru exhibited low toxicity and high biocompatibility in vivo.

Figure 8. (a) Two-photon fluorescence images of HeLa cells incubated with 100 μg/mL of SiNPs-Ru and FA-SiNPs-Ru for 6 h. (b) Two-photon fluorescence images of HUWEC cells, L02 cells and HeLa cells incubated with 100 μg/mL of FA-SiNPs-Ru for 6 h.

Two-photon Target Imaging of FA-SiNPs-Ru in Vitro. In order to make SiNPs-Ru have the ability of targeted imaging, the as-prepared SiNPs-Ru were further bioconjugated with FA. As shown in Figure 8a, when HeLa cells were cultured for 6 h with the same concentration of SiNPs-Ru and FA-SiNPs-Ru, under the excitation of 800 nm two-photon laser, the cytoplasm


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and nucleus of the two groups of experimental cells were found to have fluorescence signals, but the fluorescence intensity of cultured with FA-SiNPs-Ru was significantly higher than that of SiNPs-Ru. The results indicated that the as-prepared SiNPs-Ru could enter cells by passive endocytosis and perform two-photon imaging as well. And FA-SiNPs-Ru showed the ability of active targeting imaging (Figure 8a). To further explore the active targeting ability of FA-SiNPsRu in cells, HUWEC cells (FA receptor low-expressed), L02 cells (FA receptor low-expressed) and HeLa cells (FA receptor over-expressed) were selected for in vitro imaging (Figure 8b). Under the excitation of 800 nm two-photon laser, obvious red fluorescence signals were found after HeLa cells cultured for 6 h with FA-SiNPs-Ru. FA-SiNPs- Ru were remarkably uptaken by HeLa cells and mainly localized in the cytoplasm and nucleus. In contrast, in HUWEC cells and L02 cells with low FA receptor expression, the fluorescence signals were obviously weaker than that of HeLa cell, indicating that FA-SiNPs-Ru had the ability to target fluorescence imaging with two-photon excitation. In addition, FA-SiNPs-Ru could not only enter the cytoplasm, but also enter the nucleus, which enabled FA-SiNPs-Ru to be better applied for PDT in vitro. In Vitro PDT in Living Cells. In the synthesized composites, the presence of LRu made the composites have the ability of photodynamic therapy. By comparing the cell viability or mortality of HeLa cells treated with different substances under illumination and non-illumination, we explored the PDT effect of the as-prepared composites. In Figure 9a, no obvious cytotoxicity was observed in cells cultured with different concentrations of LRu, SiNPs-Ru and FA-SiNPs-Ru in the absence of light, and the cell viability was over 80%. When irradiated by 655 nm laser for 10 minutes, the cell viability gradually decreased with the increase of the concentration of LRu, SiNPs-Ru and FA-SiNPs-Ru, and the cell viability of HeLa cells cultured with targeted FASiNPs-Ru was lower than those of cultured with LRu and SiNPs-Ru. When the concentration of


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FA-SiNPs-Ru was 150 μg/mL, the cell viability was only 53%. From the trypan blue staining experiments (Figure 9b), we could see more clearly that when cells were respectively treated with 150 μg/mL of LRu, SiNPs-Ru and FA-SiNPs-Ru, the cell death rates under illumination condition were significantly higher than those with no illumination condition. Meanwhile, the cell death rate of targeted FA-SiNPs-Ru was higher than that of LRu and SiNPs-Ru. The above results demonstrated that the as-prepared composites (SiNPs-Ru and FA-SiNPs-Ru) had the ability for photodynamic therapy, and FA-SiNPs-Ru had certain ability for targeting photodynamic therapy, but this ability needed to be improved.

Figure 9. PDT of as-prepared SiNPs-Ru and FA-SiNPs-Ru. (a) Viability of HeLa cells after incubated with LRu, SiNPs-Ru or FA-SiNPs-Ru under illumination or non-illumination for 10 min. (b) Microscopic photos of HeLa cells in each group (incubated with LRu, SiNPs-Ru or FASiNPs-Ru under illumination or non-illumination for 10 min) after trypan blue staining. Scale bars: 200 μm.


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CONCLUSION In summary, we reported a method for synthesizing ruthenium complex-functionalized twophoton-excited red fluorescence silicon nanoparticles composite (SiNPs-Ru) based on fluorescence resonance energy transfer under mild experimental conditions. In the composite, SiNPs with high fluorescence quantum yield and two-photon fluorescence properties acted as fluorescence energy donor and LRu with the red fluorescence emission and the ability of photodynamic therapy acted as fluorescence energy acceptor. We successfully applied SiNPs-Ru to in vivo fluorescence imaging in zebrafish and nude mice. When SiNPs-Ru and FA were coupled together, the resulting composite of FA-SiNPs-Ru had the two-photon excitation targeting imaging capability. At the same time, the obtained composites (SiNPs-Ru and FASiNPs-Ru) could be used not only for two-photon fluorescence imaging by FRET, but also for in vitro photodynamic therapy in living cells. Toxicity tests in vivo and in vitro showed that the obtained composites had low toxicity and excellent biocompatibility. The as-prepared SiNPs-Ru provided a novel synthesis method for the synthesis of red fluorescence silicon nanoparticles. Moreover, the application of silicon nanoparticles had been extended to the field of photodynamic therapy in vitro, and promoted the developments of further application of silicon nanoparticles in the biological field.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Experimental section, the optimization of synthetic reaction conditions, SiNPs-Ru the quantum yield and fluorescence lifetime of SiNPs, the chemical stability of SiNPs, toxicity test of SiNPs-


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Ru in vivo, imaging experiments in vitro and in vivo, and supplemental Table S1 and Figures S1−S10 (PDF).

AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected]. Fax: +86-22-23502458. *E-mail: [email protected]. Fax: +86-22-23502554. Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (nos. 21775077, 21475069 and 81671179) and the Tianjin Natural Science Foundation (nos. 16JCZDJC37200).

REFERENCES (1) Kirchner, C.; Liedl, T.; Kudera, S.; Pellegrino, T.; Javier, A. M.; Gaub, H. E.; Stölzle, S.; Fertig, N.; Parak, W. J. Cytotoxicity of Colloidal CdSe and CdSe/ZnS Nanoparticles. Nano Lett. 2005, 5, 331-338. (2) Zou, Jing.; Baldwin, R. K.; Pettigrew, K. A.; Kauzlarich, S. M. Solution Synthesis of Ultrastable Luminescent Siloxane-Coated Silicon Nanoparticles. Nano Lett. 2004, 4, 1181-11186. (3) Derfus, A. M.; Chan, W. C. W.; Bhatia, S. N. Probing the Cytotoxicity of Semiconductor Quantum Dots. Nano Lett. 2004, 4, 11-18.


22

Page 23 of 30 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


(4) Veinot, J. G. C. Synthesis, Surface Functionalization, and Properties of Freestanding Silicon Nanocrystals. Chem. Commun. 2006, 40, 4160-4168. (5) Rosso-Vasic, M.; Spruijt, E.; Popovic, Z.; Overgaag, K.; Lagen, B. V.; Grandidier, B.; Vanmaekelbergh, D.; Domnguez-Gutierrez, D.; Cola, L. D.; Zuilhof, H. Amine-Terminated Silicon Nanoparticles: Synthesis, Optical Properties and Their Use in Bioimaging. J. Mater. Chem. 2009, 19, 5926-5933. (6) Das, P.; Saha, A.; Maity, A. R.; Ray, S. C.; Jana, N. R. Silicon Nanoparticle Based Fluorescent Biological Label via Low Temperature Thermal Degradation of Chloroalkylsilane. Nanoscale 2013, 5, 5732-5737. (7) Altinoglu, E. I.; Adair, J. H. Near Infrared Imaging with Nanoparticles. Wires Nanomed Nanobi. 2010, 2, 461-477. (8) Pansare, V. J.; Hejazi, S.; Faenza, W. J.; Prud’homme, R. K. Review of Long-Wavelength Optical and NIR Imaging Materials: Contrast Agents, Fluorophores, and Multifunctional Nano Carriers. Chem. Mater. 2012, 24, 812-827. (9) Abdukayum, A.; Yang, C. X.; Zhao, Q.; Chen, J. T.; Dong, L. X.; Yan, X. P. Gadolinium Complexes Functionalized Persistent Luminescent Nanoparticles as a Multimodal Probe for Near-Infrared Luminescence and Magnetic Resonance Imaging in Vivo. Anal. Chem. 2014, 86, 4096-4101. (10) Wang, J.; Ye, D. X.; Liang, G. H.; Chang, J.; Kong, J. L.; Chen, J. Y. One-Step Synthesis of Water-Dispersible Silicon Nanoparticles and Their Use in Fluorescence Lifetime Imaging of Living Cells. J. Mater. Chem. B. 2014, 2, 4338-4345.


23


Page 24 of 30

(11) Ye, H. L.; Cai, S. J.; Li, S.; He, X. W.; Li, W. Y.; Li, Y. H.; Zhang, Y. K. One-Pot Microwave Synthesis of Water-Dispersible, High Fluorescence Silicon Nanoparticles and Their Imaging Applications in Vitro and in Vivo. Anal. Chem. 2016, 88, 11631-11638. (12) Wu, Y. Y.; Zhong, Y. L.; Chu, B. B.; Sun, B.; Song, B.; Wu, S. C.; Su, Y. Y.; He, Y. Plant-Derived Fluorescent Silicon Nanoparticles Featuring Excitation Wavelength-Dependent Fluorescence Spectra for Anti-Counterfeiting Applications. Chem. Commun. 2016, 52, 70477050. (13) Geng, X.; Li, Z. H.; Hu,Y. L.; Liu, H. F.; Sun, Y. Q.; Meng, H. M.; Wang,Y. W.; Qu, L. B.; Lin, Y. H. One-Pot Green Synthesis of Ultrabright N-Doped Fluorescent Silicon Nanoparticles for Cellular Imaging by Using Ethylenediaminetetraacetic Acid Disodium Salt as an Effective Reductant. ACS Appl. Mater. Interfaces. 2018, 10, 27979-27986. (14) Zhong, Y. L.; Peng, F.; Wei, X. P.; Zhou, Y. F.; Wang, J.; Jiang, X. X.; Su, Y. Y.; Su, S.; Lee, S. T.; He, Y. Microwave-Assisted Synthesis of Biofunctional and Fluorescent Silicon Nanoparticles Using Proteins as Hydrophilic Ligands. Angew. Chem., Int. Ed. 2012, 51, 84858489. (15) Dasog, M.; De los Reyes, G. B.; Titova, L. V.; Hegmann, F. A.; Veinot, J. G. C. Size vs Surface: Tuning the Photoluminescence of Freestanding Silicon Nanocrystals Across the Visible Spectrum via Surface Groups. ACS Nano 2014, 8, 9636-9648. (16) He, Y.; Kang, Z. H.; Li, Q. S.; Tsang, C. H. A.; Fan, C. H.; Lee, S. T. Ultrastable, Highly Fluorescent, and Water-Dispersed Silicon-Based Nanospheres as Cellular Probes. Angew. Chem., Int. Ed. 2009, 48, 128-132.


24

Page 25 of 30 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


(17) Wang, D.; Zhu, Lin.; Pu, Y.; Wang, J. X.; Chen, J. F.; Dai, L. M. Transferrin-coated Magnetic Upconversion Nanoparticles for Efficient Photodynamic Therapy with Near-Infrared Irradiation and Luminescence Bioimaging. Nanoscale 2017, 9, 11214-11221. (18) Zhou, J.; Liu, Z.; Li, F. Upconversion Nanophosphors for Small-Animal Imaging. Chem. Soc. Rev. 2012, 41, 1323-1349. (19) Ge, J. C.; Lan, M. H.; Zhou, B. J.; Liu, W. M.; Guo, L.; Wang, H.; Jia, Q. Y.; Niu, G. L.; Huang, X.; Zhou, H. Y.; Meng, X. M.; Wang, P. F.; Lee, C. S.; Zhang, W. J.; Han, X. D. A Graphene Quantum Dot Photodynamic Therapy Agent with High Singlet Oxygen Generation. Nat.Commun. 2014, 5, 4596-4603. (20) Park, J.; Jiang, Q.; Feng, D.; Mao, L.; Zhou, H. C. Size-Controlled Synthesis of Porphyrinic Metal–Organic Framework and Functionalization for Targeted Photodynamic Therapy. J. Am.Chem. Soc. 2016, 138, 3518-3525. (21) Lucky, S. S.; Soo, K. C.; Zhang, Y. Nanoparticles in Photodynamic Therapy. Chem. Rev. 2015, 115, 1990-2042. (22) Shen, Y. Z.; Tian, Q.; Sun, Y. D.; Xu, J. J.; Ye, D. J.; Chen, H. Y. ATP-Activatable Photosensitizer Enables Dual Fluorescence Imaging and Targeted Photodynamic Therapy of Tumor. Anal. Chem. 2017, 89, 13610-13617. (23) Detty, M. R.; Gibson, S. L.; Wagner, S. J. Current Clinical and Preclinical Photosensitizers for Use in Photodynamic Therapy. J. Med. Chem. 2004, 47, 3897-3915. (24) Celli, J. P.; Spring, B. Q.; Rizvi, I.; Evans, C. L.; Samkoe, K. S.; Verma, S.; Pogue, B. W.; Hasan, T. Imaging and Photodynamic Therapy: Mechanisms, Monitoring, and Optimization. Chem. Rev. 2010, 110, 2795-2838.


25


Page 26 of 30

(25) Tian, J. W.; Ding, L.; Xu, H. J.; Shen, Z.; Ju, H. X.; Jia, L.; Bao, L.; Yu, J. S. Cell-Specific and pH-Activatable Rubyrin-Loaded Nanoparticles for Highly Selective Near-Infrared Photodynamic Therapy against Cancer. J. Am. Chem. Soc. 2013, 135, 18850-18858. (26) Wang, R. G.; Zhao, M. Y.; Deng, D.; Ye, X.; Zhang, F.; Chen, H; Kong, J. L. An Intelligent and Biocompatible Photosensitizer Conjugated Silicon Quantum Dots–MnO2 Nanosystem for Fluorescence Imaging-Guided Efficient Photodynamic Therapy. J. Mater. Chem. B. 2018, 6, 4592-4601. (27) Kent, C. A.; Mehl, B. P.; Ma, L. P.; Papanikolas, J. M.; Meyer, T. J.; Lin, W. B. Energy Transfer Dynamics in Metal−Organic Frameworks. J. Am. Chem. Soc. 2010, 132, 12767-12769. (28) Liu, D. M.; Huxford, R. C.; Lin, W. B. Phosphorescent Nanoscale Coordination Polymers as Contrast Agents for Optical Imaging. Angew. Chem., Int. Ed. 2011, 50, 3696-3700. (29) Yin, X. B. Functional Nucleic Acids for Electrochemical and Electrochemiluminescent Sensing Applications. Trac-Trend. Anal.Chem. 2012, 33, 81-94. (30) Ru, J. X.; Tang, X. L.; Ju, Z. H.; Zhang, G. L.; Dou, W.; Mi, X. Q.; Wang, C. M.; Liu, W. S. Exploitation and Application of a Highly Sensitive Ru(II) Complex-Based Phosphorescent Chemodosimeter for Hg2+ in Aqueous Solutions and Living Cells. ACS Appl. Mater. Interface. 2015, 7, 4247-4256. (31) Wang, T.; Zabarska, N.; Wu, Y. Z.; Lamla, M.; Fischer, S.; Monczak, K.; Ng, D. Y. W.; Rau, S.; Weil, T. Receptor Selective Ruthenium-Somatostatin Photosensitizer for Cancer Targeted Photodynamic Applications. Chem. Commun. 2015, 51, 12552-12555. (32) Huang, H. Y.; Yu, B.; Zhang, P. Y.; Huang, J. J.; Chen, Y.; Gasser, G.; Ji, L. N.; Chao, H. Highly Charged Ruthenium(II) Polypyridyl Complexes as Lysosome-Localized Photosensitizers for Two-Photon Photodynamic Therapy. Angew. Chem., Int. Ed. 2015, 54, 14049-14052.


26

Page 27 of 30 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


(33) Mari, C.; Pierroz, V.; Ferrari, S.; Gasser, G. Combination of Ru(II) Complexes and Light: New Frontiers in Cancer Therapy. Chem. Sci.2015, 6, 2660-2686. (34) Padilla, R.; Rodriguez-Corrales, J. A.; Donohoe, L. E.; Winkel, B. S. J.; Brewer, K. J. A New Class of Ru(II) Polyazine Agents with Potential for Photodynamic Therapy. Chem. Commun. 2016, 52, 2705-2708. (35) Dou, Y. K.; Chen, Y.; He, X. W.; Li, W. Y.; Li, Y. H.; Zhang, Y. K. Synthesis of WaterDispersible Mn2+ Functionalized Silicon Nanoparticles under Room Temperature and Atmospheric Pressure for Fluorescence and Magnetic Resonance Dual-Modality Imaging. Anal. Chem. 2017, 89, 11286-11292. (36) Zhong, Y. L.; Peng, F.; Bao, F.; Wang, S. Y.; Ji, X. Y.; Yang, L.; Su, Y. Y.; Lee, S. T.; He, Y. Large-Scale Aqueous Synthesis of Fluorescent and Biocompatible Silicon Nanoparticles and Their Use as Highly Photostable Biological Probes. J. Am. Chem. Soc. 2013, 135, 8350-8356. (37) Atkins, T. M.; Cassidy, M. C.; Lee, M.; Ganguly, S.; Marcus, C. M.; Kauzlarich, S. M. Synthesis of Long T1 Silicon Nanoparticles for Hyperpolarized 29Si Magnetic Resonance Imaging. ACS Nano 2013, 7, 1609-1617. (38) Abdukayum, A.; Yang, C. X.; Zhao, Q.; Chen, J. T.; Dong, L. X.; Yan, X. P. Gadolinium Complexes Functionalized Persistent Luminescent Nanoparticles as a Multimodal Probe for Near-Infrared Luminescence and Magnetic Resonance Imaging in Vivo. Anal. Chem. 2014, 86, 4096-4101. (39) Wu, S. Q.; Chi, C. W.; Yang, C. X.; Yan, X. P. Penetrating Peptide-Bioconjugated Persistent Nanophosphors for Long-Term Tracking of Adipose-Derived Stem Cells with Superior Signal-to-Noise Ratio. Anal. Chem. 2016, 88, 4114-4121.


27


Page 28 of 30

(40) Zhong, Y. L.; Song, B.; Peng, F.; Wu, Y. Y.; Wu, S. C.; Su, Y. Y.; He, Y. In Situ Rapid Growth of Fluorescent Silicon Nanoparticles under Room Temperature and Atmospheric Pressure. Chem. Commun. 2016,52, 13444-13447. (41) Wang, Y.; Dai, C.; Yan, X. P. Fabrication of Folate Bioconjugated Near-infrared Fluorescent Silver Nanoclusters for Targeted in Vitro and in Vivo Bioimaging. Chem. Commun. 2014, 50, 14341-14344. (42) Cao, L.; Wang, X.; Meziani, M. J.; Lu, F. S.; Wang, H. F.; Luo, P. J.; Lin, Y.; Harruff, B. A.; Veca, L. M.; Murray, D.; Xie, S. Y.; Sun, Y. P. Carbon Dots for Multiphoton Bioimaging. J. Am. Chem. Soc. 2007, 129, 11318-11319. (43) Liu, Q.; Guo, B. D.; Rao, Z. Y.; Zhang, B. H.; Gong, J. R. Strong Two-Photon-Induced Fluorescence from Photostable, Biocompatible Nitrogen-Doped Graphene Quantum Dots for Cellular and Deep-Tissue Imaging. Nano Lett. 2013, 13, 2436-2441. (44) Zheng, X. Y.; Ananthanarayanan, A.; Luo, K. Q.; Chen, P. Glowing Graphene Quantum Dots and Carbon Dots: Properties, Syntheses, and Biological Applications. Small 2015, 11, 1620-1636. (45) Gerlai, R. Chapter 12-Associative Learning in Zebrafish (Danio rerio). Method Cell Biol. 2011, 101, 249-270. (46) Buckley, C. E.; Goldsmith, P.; Franklin, R. J. M. Zebrafish myelination: a transparent model for remyelination. Dis. Model. Mech. 2008, 1, 221-228. (47) Pham, D. H.; De Roo, B.; Nguyen, X. B.; Vervaele, M.; Kecskes, A.; Ny, A.; Copmans, D.; Vriens, H.; Locquet, J. P.; Hoet, P.; de Witte, P. A. Use of Zebrafish Larvae as a Multi-Endpoint Platform to Characterize the Toxicity Profile of Silica Nanoparticles. Sci. Rep. 2016, 6, 3714537157.


28

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(48) Shi, Q. Q.; Li, Y. H.; Xu, Y.; Wang, Y.; Yin, X. B.; He, X. W.; Zhang, Y. K. High-Yield and High-Solubility Nitrogen-Doped Carbon Dots: Formation, Fluorescence Mechanism and Imaging Application. RSC Adv. 2014, 4, 1563-1566.


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Table of content


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Preparation of a Ruthenium-Complex-Functionalized Two-Photon

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