Quaternary alloy quantum dots with widely tunable emission – a versatile system to fabricate dual-emission nanocomposites for bio-imaging

Abstract

Tuning the composition is an efficient strategy to control the photoluminescence (PL) emission of multiplex alloy quantum dots (QDs), just as the size is for binary QDs. Hence, in this paper, a quaternary alloy system was selected as a model. By controlling the composition, the quaternary QDs exhibit favorable, wide ranging composition-tuned PL emissions, while ZnS overcoating may further improve their PL quantum yields (QYs). Specifically, Zn–Ag–In–Se(ZAISe)/ZnS QDs have a tunable PL peak from 550 (green) to 820 (NIR) nm with up to 70% PL QY; the parameters for Zn–Cu–In–Se(ZCISe)/ZnS QDs are almost same as those for ZAISe/ZnS QDs. In addition, these quaternary QDs were proven to be versatile for bioimaging, serving as a promising alternative for Cd- and Pb-based QDs, by using biodegradable RGD-modified N-succinyl-N′-octyl-chitosan (RGD-SOC) micelles as the water transfer agent to fabricate dual-emission nanocomposites.

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Introduction

Optical imaging has been a crucial molecular imaging technique for preclinical and clinical assessments of biological and biochemical processes,^1,2 in which the optical signal in the visible region can be applied for in vitro microscopy of cells, while NIR light (700–900 nm) is quite useful for in vivo imaging of small animals because of its deeper penetration into tissue and reduced autofluorescence.^1–3 The applications of optical imaging methods are limited mainly by the development of new fluorescent probes. So far the fluorescent probes used include organic dyes, fluorescent proteins and inorganic quantum dots (QDs).^3–7 As a novel class of light-emitting material for optical imaging, inorganic QDs, especially highly luminescent binary II–VI or IV–VI ones, have been widely explored.^3–7 However, those QDs contain highly toxic elements (Cd, Hg, Pb, etc.). The release of these toxic components will induce unsolvable cytotoxicity. Thus, this has inspired further research into more biocompatible QDs.^6–11

In comparison with traditional organic dyes, inorganic QDs have many significant advantages such as the tunable emission, high PL quantum yield, broad excitation spectrum, and excellent photostability.^5–11 Among them, the tunability of PL emission peak is an important parameter for evaluating QDs. To tune the PL peak, a traditional straightforward way is to change the QD size.^12–15 For instance, decreasing the size of CdSe QDs could shift the PL wavelength continuously from 650 (red) to 420 nm (violet), covering the whole visible region.^12,13 The other efficient way is to change the composition of QDs,^16–30 e.g., quaternary Zn–Cu–In–S QDs.²⁹ Although the quaternary QDs had a similar particle size, their PL emission could be tuned from 520 (green) to 750 (NIR) nm with increasing the Cu/Zn ratio.²⁹ The widely tunable emission of the QDs helps to fabricate the QDs-based dual-emission systems (namely, multiplex optical encoding), which are significant for the high-throughput and fast detection of biological active molecules or processes.^31–36

The synthesis and spectral control of quaternary or ternary QDs is an obvious challenge due to the multiplex composition relative to binary ones with size-dependent emission.^16–30 In 2009, Peng et al. found that the dodecanethiol (DT) can act as the reactivity-controlling ligand to balance the reactivities of the cationic precursors and control their stoichiometric ratio in the nanocrystals.³⁷ Since then, ternary I-III-VI₂ QDs combining group I (Cu, Ag), group III (Ga, In), and group VI (S, Se) elements, have received increasing attention.^18–26 Besides, quaternary I-III-VI-based QDs such as Zn-I-III-VI nanostructures also are versatile systems which should possess wider composition-tuned PL emission than ternary ones (in these cases, the DT also are an efficient reactivity-controlling ligand for the related cationic precursors).^27–30 Here, noteworthy is the fact that the similar optoelectronic properties and the deficiency of highly toxic components make them suitable to alternate binary Cd- and Pb-based QDs for biomedical optical imaging.^21,26,30

In this work, we explored to synthesize Zn–Ag–In–Se/ZnS and Zn–Cu–In–Se/ZnS QDs, i.e., Zn-I-III-VI-based core/shell QDs based on the data of semiconductor bandgaps, in which the quaternary alloy systems may afford a wide composition-tunability, while the overcoating of the ZnS shell with a much larger bandgap may enhance their PL emission (AgInSe₂, E_g = 1.24 eV; CuInSe₂, E_g = 1.04 eV; ZnSe, E_g = 2.7 eV; ZnS, E_g = 3.6 eV).^12,27–30 The results confirm that as expected, the two quaternary Zn-I-III-VI-based core/shell QDs all exhibit wide tunable PL emission as well as high PL QYs. Meanwhile, these Cd-free Zn-I-III-VI-based QDs have been proven to have promising applications in multi-scale bioimaging as new fluorescent reagents, by using RGD-SOC micelles as a water transfer agent.

Experimental

Materials

Zinc acetate (Zn(Ac)₂, 99.99%), silver nitrate (AgNO₃, >98%), copper acetate (Cu(Ac)₂, 99.99%), indium acetate (In(Ac)₃, 99.99%), Se powder (100 mesh, 99.5%), sulfur powder (S, 99.5%), 1-octadecene (ODE, 90%), oleic acid (OA, 90%), 1-dodecanethiol (DT, 98%), oleylamine (OLA, 97%), chitosan (100 kDa), succinic anhydride (98%), octaldehyde (99%), monomeric cyclic RGD peptide, 1-ethyl-3(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI), N-hydroxysuccinimide (NHS). All above chemicals were used without further purification. The water used in all experiments had a resistivity higher than 18.2 MΩ cm.

Human malignant glioma cell line (U87MG) and human breast cancer cell line (MCF-7) (American Type Culture Collection, Manassas, VA, USA), athymic nude mice bearing U87MG tumors under armpit (male, 18–20 g; KeyGEN Biotech. Co. Ltd, Nanjing, China) and materials for cell culture were purchased from commercial sources.

Synthesis of oil-soluble quaternary ZnAgInSe/ZnS QDs

Quaternary ZAISe core QDs. For a typical synthetic reaction, Zn(Ac)₂ (0.1 mmol), AgNO₃ (0.04 mmol), and In(Ac)₃ (0.1 mmol) were mixed with 4 mL of ODE, 0.5 mL of DT and 50 μL of OA in a three-neck flask. Under mild stirring, the reaction mixture was slowly heated to 175 °C within 10 min under a N₂ atmosphere until a clear solution was formed. Meanwhile, the Se precursor solution was prepared by dissolving 0.27 mmol of Se powder in the mixture of OLA (0.5 mL) and DT (0.2 mL) with ultrasonication,³⁸ and then injected with a syringe. At this time, the solution turned immediately to dark. Subsequently, the reaction temperature was kept at 175 °C for 30 min to allow the growth of ZAISe core QDs.

ZAISe/ZnS core/shell QDs. Zn(AC)₂ (0.1 mmol) and sulphur powder (0.1 mmol) dissolved in the mixed solution of OLA and ODE (the ratio of OLA/ODE is 1/4, in all 0.5 mL) was injected into the above reaction solution containing ZAISe core QDs.^19,26,37,39 After that, the temperature was retained for about 30 min to promote the growth of ZAISe/ZnS core/shell QDs. At last, the flask was rapidly cooled to room temperature to harvest QDs products. The resulting core/shell QDs could be separated by ethanol precipitation, and re-dispersed in n-decane, toluene, hexane, chloroform, ether, etc. The QDs powder needed for XRD and XPS measurements were obtained by drying the precipitation at 50 °C. In this study, we explored intensively the effects of the Zn/Ag/In (Zn/Cu/In) feed ratios (core), the growth time and the amount of the Zn_shell on the core/shell QD synthesis.

Synthesis of oil-soluble quaternary ZnCuInSe/ZnS QDs

In general, the method for oil-soluble quaternary ZCISe/ZnS QDs is similar to that for ZAISe/ZnS counterparts described above except for Cu(Ac)₂ used to replace AgNO₃. Meanwhile, the Zn/Cu/In feed ratios (core) were optimized further to change the PL emission peaks of quaternary ZCISe core QDs and improve their PL intensities.

Characterization

A S2000 eight-channel optical fiber spectrophotometer (Ocean Optics corporation, USA), a X-Cite Series 120Q broadband light source (Lumen Dynamics Group Inc., Canada), a NL-FC-2.0-766 semiconductor laser (λ = 766 nm, Enlight, China), and a PerkinElmer UV/Vis spectrometer were employed to measure the optical spectra at room temperature. And PL QYs of the QDs were determined further following the previously reported method.^19,30

Transmission electron microscope operating at 200 kV (JEOL JEM-2100, Japan) or at 300 kV (JEOL JEM-3011, Japan) and laser particle size analyzer (LPSA) (Mastersizer 2000, Malvern, British) were combined to characterize the size and shape of samples. X-ray photoelectron spectroscopy (XPS, PHI5000 VersaProbe, ULVAC-PHI Inc., Japan) was used to detect the elemental compositions of QDs. Powder XRD experiment was performed on a Philips X'Pert PRO X-ray diffractometer to determine the crystal structure of QDs.

Synthesis of RGD peptide-modified SOC micelles

RGD peptide-modified SOC (N-succinyl-N′-octyl-chitosan) (RGD-SOC) micelles used for the water transfer of oil-soluble quaternary QDs were prepared following the two-step method reported previously: (i) synthesis of SOC micelles and (ii) subsequent surface modification with RGD peptide.^19,30 After further purification by dialysis (MWCO 10000) against deionized water, RGD-SOC micelles were obtained.

Water solubilization

The oil-soluble core/shell QDs (50 μL, ∼0.5 mg) (or the mixture of two different types of QDs with various emissions) prepared above could be transferred into water (1 mL) using RGD-SOC micelles (∼5 mg), according to our recently reported method.^19,30 Here, for both in vitro and in vivo imaging, oil-soluble ZAISe/ZnS QDs with red emission and oil-soluble ZCISe/ZnS QDs with NIR emission (the molar ratio of the ZAISe/ZnS QDs to the ZCISe/ZnS QDs is about 1 : 2) were simultaneously loaded into the RGD-SOC micelles, resulting in the dual-emission probes. After centrifugation, the obtained clear water solution of QDs-loaded RGD-SOC micelles was stored at room temperature (or at 4 °C) for subsequent research.

In vitro cell imaging

U87MG (or MCF-7) cells were seeded onto confocal Petri dishes containing 2 mL DMEM medium with 10% (v/v) calf serum, and then cultured at 37 °C in an atmosphere with 5% CO₂. After incubating for 24 h, 200 μL of the mixed QDs-loaded RGD-SOC micelles was added. After 2 h of co-incubation, the medium was removed and the cells attached on the bottom of the dishes were washed with phosphate buffered saline (PBS, pH = 7.4) for 3 times. Then, the fluorescence images of cells were acquired using the Olympus Fluoview 1000 laser confocal scanning microscope (LCSM, λ_ex = 405 nm).

In vivo animal imaging

Briefly, U87MG (or MCF-7) cells were injected subcutaneously into the armpit of each nude mouse. The mice would be ready for in vivo NIR imaging experiments when the tumors grew to 100–150 mm³ in volume. By this time, 200 μL of the mixed QDs-loaded RGD-SOC micelles ([QDs] = ∼10 μg per g body weight) was injected into each tumor-bearing nude mouse through tail vein. Then, the fluorescence images were obtained at various time points (0–10 h) post-injection with a homemade in vivo animal NIR imaging system.^19,30

The in vivo experimental protocols were approved by the Department of Science and Technology of Jiangsu Province and Jiangsu Association for Laboratory Animal. All animal experiments were carried out in accordance with the Laboratory Animal Management Rules of the Jiangsu Provincial People's Government (document no. 45, 2008).

Results and discussion

Synthesis of oil-soluble quaternary alloy QDs

In the framework of the colloidal chemistry approach, there are two main strategies for fabricating high-quality QDs with widely tunable PL emissions. Specifically, a straightforward way is to change their sizes, as achieved in the CdSe and CdTe QDs preparations (namely, the quantum size effect).^12–15 Recently, the other efficient approach developed is to tune the composition of QDs, e.g., Zn_xCd_1−xSe, Cu_xIn_(4−x)/3S₂, Cu_xIn_(4−x)/3Se₂, etc.^16–26 Compared with these ternary systems, quaternary ones should be more favorable for fabricating the widely tunable emissive QDs.^27–30 Among them, quaternary Zn-I-III-VI nanostructures are receiving increasing attention, based on the large differences on the bandgaps between the I-III-VI₂ and Zn-VI semiconductors. However, synthesizing these quaternary QDs is not facile owing to their more complicated compositions, compared to the binary and ternary semiconductors nanocrystals. As such, balancing the reactivity of the cationic precursors is critical for the control of the stoichiometric ratio among the Zn, the Group I and the Group III elements in a given sample. Luckily, the dodecanethiol (DT) has been proven to be an efficient reactivity-controlling ligand for the Group I elements (Cu and Ag) in these quaternary systems.^29,30,37

In this study, we developed a facile one-pot strategy for preparing oil-soluble quaternary ZAISe and ZCISe QDs (see the Methods section). Meanwhile, to further improve their PL QYs, ZnS, a stable nontoxic semiconductor with a larger band gap (E_g = 3.6 eV), was used to overcoat the quaternary QDs to form the corresponding core/shell nanostructures. The series of experimental data have indicated that the reaction temperature of 175 °C, the reaction time of 30 min and the DT amount of 0.7 mL (i.e., 0.7 = 0.5 + 0.2) are optimal for achieving highly luminescent quaternary ZAISe core QDs.³⁰ Next, major efforts were focused on the synthesis of the core/shell QDs and the composition-tunability of the quaternary cores. That is, the key experimental variables, including the growth time, the amount of the Zn_shell and the Zn/Ag/In feed ratios (core) were explored in detail by optical techniques, TEM, XRD, and XPS.

Fig. 1 shows absorption and PL spectra of initial quaternary ZAISe core QDs and the subsequent spectral evolutions during the growth of a ZnS shell. As shown, (i) for initial quaternary ZAISe core QDs, their absorption and PL spectra are observed to have no well-defined exciton absorption peak and be slightly broad, respectively. These observations, similar to previous reports,^27–30 might be the indication of the quaternary composition, which could be ascribed to the unique electronic properties and the inhomogeneity of the elemental distribution among various quaternary QDs in an ensemble.^37,40,41 Furthermore, the influence of the Zn/Ag/In feed ratios on the PL properties (PL peak position and PL intensity) of quaternary ZAISe core QDs was investigated and shown in ESI Fig. S1.†

Fig. 1 Evolution of both absorption (a) and PL (b) spectra of the resulting QDs during the growth of a ZnS shell, starting from the ZAISe core QDs synthesized at 175 °C with a Zn/Ag/In feed ratio of 1 : 0.4 : 1, in which the Zn_shell : Zn_core feed ratio was set to 1 : 1. (c) PL spectra of the formed QDs with the gradual deposition of different thicknesses of the ZnS shell around the core QDs used above. (d) PL spectra of ZAISe core QDs with various emission peaks (black) and the corresponding ZAISe/ZnS core/shell QDs (red): left, Zn_core/Ag/In/Zn_shell = 2 : 0.2 : 0.5 : 1; right, Zn_core/Ag/In/Zn_shell = 0 : 1 : 1 : 1. The two core QDs in panel (d) have weak PL emissions as compared to the core QDs in panel (a–c).

(ii) During the overcoating of the ZnS shell, the PL emission intensity of the QDs improved gradually (Fig. 1b and c), whereas their absorption spectra did not change significantly (Fig. 1a). This should be a strong indication of the epitaxial growth of ZnS shell, in which the surface defects of the core QDs, also known as centers for non-radiative decay, can be efficiently suppressed and thus the PL QY can be improved.^8–10,12,21 After 30 min of heating, the PL intensity of the QDs roughly doubled. Next, if further extending the growth time to 60 min, the PL intensity decreased slightly, probably because of the depletion of precursors for further growth. These features are consistent with those of typical ternary or quaternary core/shell QDs reported in previous literature.^8–10 Meanwhile, the amount of the Zn_shell precursor also was found to influence the PL intensity of the resulting QDs (Fig. 1c). In this case, the feed ratio of Zn_shell/Zn_core between 1 : 1 and 1.5 : 1 was favorable, and the highest PL QY achieved reached 70%. (iii) By combining the PL spectra in Fig. 1d and those in Fig. 1b and c, we noted that the increased magnitude of the PL intensity induced by the ZnS overcoating depends on the PL properties of initial ZAISe core QDs. In general, the increased magnitude is larger (∼4–5 times in Fig. 1d) when initial core QDs have weak PL emission. (iv) In addition, the addition of the Zn and S precursors resulted in a small spectral red-shift (several nanometers), which might indicate the growth of the ZnS shell, namely, the formation of the ZAISe/ZnS core/shell QDs. This phenomenon differs from previous reports,^19,21,41 in which during the shell coating of I-III-VI₂ QDs, serious cation exchange reaction often occurs and induces a blue shift of the PL spectra.

As shown in Fig. 1, the PL peak position of initial quaternary ZAISe core QDs will dominate that of the as-prepared core/shell QDs. Thus, to tune the PL wavelength of the ZAISe/ZnS QDs, the ZAISe cores with various PL emissions obtained by controlling the Zn/Ag/In feed ratio were used. Fig. 2 shows the resultant absorption and PL spectra (the Zn/Ag/In/Se molar ratios in samples S1–S6 are presented in Table S1†). Similarly, the PL wavelengths of the corresponding ZAISe/ZnS QDs can be tuned conveniently from ∼550 to 820 nm. Their PL QYs all are higher than 20% and the maximal value is close to 70%. These can be illustrated by the PL color of the resulting core/shell QDs, which covers most of the visible and NIR window with high brightness from green, yellow, orange, and red to the NIR (inset of Fig. 2b). These results have brought the quality of these QDs, especially their emissive properties – the wide tunability of PL peak and high PL QY, up to a level comparable to those of CdSe (or other binary II–VI semiconductor nanocrystals)^12–15 and ternary Cu–In–S-based QDs.^19,39 Hence, ZAISe/ZnS QDs without highly toxic (class A) elements as a component may compete with binary or ternary QDs both in light-emitting devices and biomedical fluorescent imaging and sensing in the future.

Fig. 2 (a) Absorption and (b) normalized PL spectra of the resulting ZAISe/ZnS core/shell QDs (S1–S6) prepared by tuning the Zn/Ag/In feed ratios for core QDs. Insets of panels (a) and (b) are the true-color digital photographs of the samples taken under room light and UV lamp, respectively (here, under UV lamp, the strong NIR fluorescence of the QDs cannot be seen by nude eyes, but could be detected by NIR-sensitive CCD camera, e.g., Fig. 6a).

To demonstrate further that quaternary alloy system has a wider composition-tuned PL emission relative to binary or ternary ones, next, we explored the synthesis of Zn–Cu–In–Se/ZnS QDs, since Cu and Ag all belong to group I elements and CuInSe₂ (E_g = 1.04 eV) has similar semiconductor bandgap with AgInSe₂ (E_g = 1.24 eV).^9,21,22 In general, the synthetic steps for quaternary ZCISe/ZnS QDs are similar to those for ZAISe/ZnS counterparts presented above except for Cu(Ac)₂ used to replace AgNO₃. The obtained experimental results were shown in Fig. 3. As shown, similarly, ZCISe/ZnS QDs also have wide composition-tuned PL emission in the range from 570 to 820 nm, and desired PL QYs. Furthermore, the additional data on the synthesis of ZCISe/ZnS QDs were given in Fig. S2–S4.†

Fig. 3 (a) Absorption and (b) normalized PL spectra of the resulting ZCISe/ZnS core/shell QDs prepared by tuning the Zn/Cu/In feed ratios for core QDs. Insets of panels (a) and (b) are the true-color digital photographs of the corresponding samples taken under room light and UV lamp, respectively.

At last, in this section, we employed TEM (Fig. 4a–c), powder XRD (Fig. 4d and S5†) and XPS (Fig. S6†) to characterize further the morphology, the crystal structure and the elemental compositions of ZAISe core and ZAISe/ZnS core/shell QDs. As presented in panels Fig. 4a and b, these nanocrystals are quasi-spherical, and in this case, a slight increase in the average size from ∼2.5 nm to ∼3.5 nm was observed upon ZnS shell growth, indicating that the shell should consist of two ZnS atomic layers.^19,21,37,41 Meanwhile, these TEM images also show that the ZAISe/ZnS core/shell QDs have a similar narrow size distribution to the core-alone QDs, which could demonstrate to some degree that the unique absorption and broad PL spectra observed in Fig. 1–3 are induced mainly by the intrinsic properties of quaternary nanocrystals rather than the wide size distribution. The HRTEM imaging (Fig. 4c) and XRD patterns (Fig. 4d) confirm that ZAISe core and ZAISe/ZnS core/shell QDs have a well cubic crystal structure.^22–26,30 Upon the overcoating of ZnS shell or the increase of the Zn content (or the decease of the Ag content) in core, the three major diffraction peaks systematically shifted toward higher angles (Fig. 4d and S5†). In addition, the core/shell structure of the ZAISe/ZnS QDs could be proven further by the data in Fig. S6† acquired by XPS.

Fig. 4 (a) TEM image of initial ZAISe core QDs, (b) TEM and (c) high-resolution TEM images of the as-prepared ZAISe/ZnS core/shell QDs (namely, the sample S3; the optical spectra of these samples are shown in Fig. 1a–c). (d) The XRD patterns of initial ZAISe core and as-prepared ZAISe/ZnS core/shell QDs (sample S3).

Based on the detailed characterizations in Fig. 2–4, we can conclude that oil-soluble ZAISe/ZnS and ZCISe/ZnS QDs with bright and widely tunable emissions have been prepared, and the overcoating of the ZnS shell with a much larger bandgap improves further their PL emission intensities. Here, it was found that the wide composition-tunability of the quaternary alloy systems, controlled by the Zn/Ag(or Cu)/In feed ratio, ensures the wide tunability of the PL peak position. This result helps to enrich our current understanding on the QDs synthesis that for quaternary Zn-I-III-VI semiconductor nanocrystals, their composition plays a crucial role in determining the PL peak position, just as the size for binary ones.^12–30

Oil-soluble quaternary alloy QDs for multi-scale bio-imaging via water transfer

As described above, the quaternary alloy QDs with bright and widely tunable emissions prepared are stabilized by DT, and thus are oil-soluble or hydrophobic. To explore further their applications in bio-imaging, in this work, RGD peptide-modified SOC (RGD-SOC) micelles were synthesized and used as the phase transfer agent (Fig. 5). The micelle should possess a hydrophobic core and a hydrophilic shell.^19,30,42,43 Here, the hydrophobic cores of the micelles were employed to upload the oil-soluble quaternary alloy QDs or their mixture due to the hydrophobic interaction. The PL properties of the uploaded QDs will determine the usage of the RGD-SOC-QDs (specifically, in Fig. 5, the RGD-SOC-QDs with visible and NIR emissions could be used for in vitro cell imaging and in vivo animal imaging, respectively). RGD-SOC micelles were selected based on the following reasons: (i) chitosan, as a natural biopolymer, has high density of amine groups on its backbone, as well as good biocompatibility and low cytotoxicity, which makes it suitable for the fabrication of the biocompatible micelle;^42,43 (ii) the RGD peptide has high affinity for α_vβ₃ integrin receptor over-expressed on the surfaces of specific malignant cells, and hence the modification of RGD peptide might endow the micelles with the ability to target tumor cells and issues;^44–46 (iii) it was found that the favorable water-solubility of RGD peptide affords the RGD-modified micelles with better solubility in water, and makes free RGD peptides hardly uploaded into the hydrophobic cores of micelles, compared with the folate used in our recent work.^19,30 Also noteworthy is the fact that RGD-SOC micelles are a promising targeted delivery system for hydrophobic anticancer drugs.⁴²

Fig. 5 Overall scheme for water transfer of oil-soluble QDs via the RGD-modified SOC micelles and subsequent application in multi-scale bioimaging. After chemical modification with succinic acid and octaldehyde, the obtained chitosan-based polymer molecules are amphiphilic which will spontaneously assemble into a spherical micelle.^19,30,42,43 Here, the RGD peptide was employed further to functionalize the surface of the SOC micelle.

Initially, we explored the water transfer of oil-soluble ZAISe/ZnS QDs with visible light emission via RGD-SOC micelles. The absorption and PL spectra obtained before and after water transfer showed that the RGD-SOC micelles used here should be a favorable phase transfer agent (Fig. S7†), since after water solubilization, most of the PL properties (e.g., PL peak and PL intensity) of initial oil-soluble QDs could be well retained. The corresponding digital photographs taken under room light and UV lamp also support this conclusion. Inspired by these results, next, we explored further the water transfer of the mixed oil-soluble QDs with RGD-SOC micelles. In previous studies, the mixture of different types of fluorescent QDs often was used to fabricate the optical encoding library,^31–35 where single-wavelength excitation with dual emission endows the particles with superior optical encoding capability for rapid and high-throughput multiplexed detection. In this study, two various types of fluorescent QDs having visible and NIR emissions respectively were used, in which the visible light-emitting QDs uploaded into the micelles were expected to qualify for in vitro cell imaging while the NIR-emitting QDs for in vivo animal imaging, as schemed in Fig. 5. Here, the molar ratio of the visible light-emitting QDs to the NIR-emitting QDs used is about 1 : 2. More NIR-emitting QDs were used to improve the PL intensity of the resulting mixed QDs–micelle nanocomposites in the NIR region.

Fig. 6a shows the obtained PL spectrum of the mixed oil-soluble QDs after water-solubilization with RGD-SOC micelles (the absorption spectrum is presented in Fig. S8†), and the corresponding optical images taken under different excitation light sources (the insets). As shown, the resulting mixed QDs-loaded RGD-SOC micelles possess the PL characteristics of both of these two types of QDs, defined as ‘dual emission probes’ here. And the dual emission probes exhibit strong fluorescence in both visible and NIR spectral range, indicating their potential for versatile applications in multi-scale bio-imaging, namely in vitro cell imaging and in vivo animal imaging.^3,19,22 Fig. 6b and c show further the shapes and sizes of initial RGD-SOC micelles and resulting mixed QDs-loaded micelles, characterized by TEM and dynamic light scattering (DLS). As compared with initial spherical micelles with hollow interiors, the micelles mixed with QDs are found to have enwrapped many QDs in their hydrophobic cores, although they possess similar size and size distribution. More interestingly, TEM image in the inset of Fig. 6c demonstrates further the simultaneous presence of both of two types of QDs in one hydrophobic core. In this case, the small ones (∼3.5 nm in diameter) should be assigned to the red-emitting ZAISe/ZnS QDs (Fig. 4b) and the large ones (∼6 nm in diameter) to NIR-emitting ZCISe/ZnS QDs (Fig. S4†). Hence, the data in Fig. 6 and S7† have confirmed that the oil-soluble QDs, and even the mixed oil-soluble QDs could be transferred into water successfully via the RGD-SOC micelles.

Fig. 6 (a) The PL spectrum of the mixed ZAISe/ZnS (the sample S3) and ZCISe/ZnS QDs (the sample S11) after water transfer using RGD-SOC micelles. The insets are the corresponding optical images taken under room light, UV lamp (λ_max = 365 nm) and NIR laser light (λ_max = 766 nm). Size distribution of (b) initial RGD-SOC micelles and (c) the resulting mixed QDs-loaded micelles by DLS. The corresponding TEM images are inserted in panels (b) and (c), respectively.

At last, we explored the potential application of the mixed QDs-loaded RGD-SOC micelles with dual emission in visible and NIR spectral range to multi-scale biomedical optical imaging, namely, in vitro (cell) and in vivo (animal) bioimaging. For targeted cell imaging, α_vβ₃ intergrin receptor-positive U87MG cells and α_vβ₃ intergrin receptor-negative MCF-7 cells were used and co-incubated with the dual emission probes. Also, mixed QDs-loaded SOC micelles without RGD modification were co-incubated with U87MG cells as control. The representative LCSM images obtained after 2 h of co-incubation are presented in Fig. 7. As shown, a significant number of mixed QDs-loaded RGD-SOC micelles were internalized by U87MG cells and mainly accumulated in the cytoplasms (Fig. 7a). Conversely, no significant fluorescence signals were detected in the sites of MCF-7 cells (Fig. 7b) and U87MG cells co-incubated with mixed QDs-loaded SOC micelles (Fig. 7c). These results indicate the necessity of the binding of RGD peptide and α_vβ₃ intergrin receptor in the endocytosis of these cells, and demonstrate that the as-prepared dual-emission nanocomposites should be a promising targeted system for in vitro cell imaging.

Fig. 7 The representative LCSM images of (a) U87MG cells and (b) MCF-7 cells co-incubated with mixed QDs-loaded RGD-SOC micelles, and (c) U87MG cells co-incubated with mixed QDs-loaded SOC micelles (λ_ex = 405 nm). These images were obtained after 2 h of co-incubation.

Subsequently, we investigated further the potential of the dual-emission nanocomposites for in vivo imaging. Using a self-built NIR fluorescence imaging system,^19,30 the in vivo distribution of the nanocomposites was monitored at different time points post injection (P.I.) straightly after they were injected into the U87MG (or MCF-7) tumor-bearing mice through tail vein. The obtained experimental results are presented in Fig. 8. As shown, the background fluorescence under the excitation of 766 nm laser light was quite weak. 2 h P.I., clear fluorescence signal at the liver site and a recognizable signal at the tumor site were detected. 4 h P.I., distinct accumulation of fluorescence signal could be detected at the tumor site, although the main signal was still at the liver site. After that, the signal intensity at all sites of the mouse body decreased. In contrast, no obvious fluorescence signal was detected in the tumor site of MCF-7 tumor-bearing mice during the whole detection process (Fig. S9†). These results from in vivo imaging experiments demonstrate the targeting capability of the mixed QDs-loaded RGD-SOC micelles, and thus the great potential of the quaternary alloy QDs for in vivo animal bio-imaging.

Fig. 8 The dynamic distribution of the mixed QDs-loaded RGD-SOC micelles in nude mice bearing α_vβ₃ intergrin receptor-positive U87MG tumors monitored using the NIR fluorescence imaging system (λ_ex = 766 nm).

Conclusions

In summary, quaternary alloy QDs, namely, ZAISe/ZnS and ZCISe/ZnS QDs have been synthesized, in which their PL emissions could be conveniently tuned from green to NIR by controlling the elemental composition, while the overcoating of ZnS further improved their PL QYs (highest PL QY up to 70%). Meanwhile, by using the RGD-SOC micelles as the phase transfer agent to fabricate water-soluble dual-emission nanocomposites, these quaternary alloy QDs exhibited versatile applications in multi-scale bioimaging, namely in vitro (cell) and in vivo (animal) bioimaging, as the promising substitutes for Cd- or Pb-based binary QDs.

Acknowledgements

This work was financially supported by the National Natural Science Foundation of China (81371627 and 81220108012), the Program for New Century Excellent Talents (NCET-12-0974) in University of the Ministry of Education of China, the Open Project Program of Jiangsu Key Laboratory of Drug Screening (JKLDS2015KF-03) and A Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

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Footnotes

† Electronic supplementary information (ESI) available: Additional characterizations of the quaternary alloy ZAISe/ZnS and ZCISe/ZnS QDs, SOC-RGD-QDs and in vivo imaging results. See DOI: 10.1039/c6ra07407c

‡ Jie Wang, Tao Deng and Dawei Deng contributed equally to this work.

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