Recycled Synthesis of Whey-Protein-Capped Lead ... - ACS Publications

Letter pubs.acs.org/journal/ascecg

Recycled Synthesis of Whey-Protein-Capped Lead Sulfide Quantum Dots as the Second Near-Infrared Reporter for Bioimaging Application Jun Chen,† Yifei Kong,† Shaoqing Feng,‡ Chen Chen,§ Yan Wo,⊥ Wei Wang,∥ Yu Dong,† Ziying Wu,† Yunxia Li,*,† and Shiyi Chen*,† †

Department of Orthopedic Sports Medicine, Huashan Hospital, Fudan University, Shanghai 200040, China Department of Plastic and Reconstructive Surgery, Shanghai Ninth People’s Hospital, School of Medicine, Shanghai JiaoTong University, Shanghai 200011, China § Department of Orthopaedic Surgery, Shanghai Sixth People’s Hospital, Shanghai Jiaotong University School of Medicine, 600 Yishan Road, Shanghai 200233, China ⊥ Department of Human Anatomy, Histology and Embryology, School of Medicine, Shanghai JiaoTong University, Shanghai 200025, China ∥ Department of Gastric and Pancreatic Surgery, Sun Yat-sen University Cancer Center, State Key Laboratory of Oncology in South China, Guangzhou 510060, China ‡

S Supporting Information *

ABSTRACT: Owing to their high emission intensity and size-based tunability, lead sulfide (PbS) quantum dots (QDs) have been employed in a broad spectrum of biological applications especially for the second nearinfrared (NIR-II) biological imaging. However, the toxicity of Pb2+ ions remains an unavoidable risk factor during both preparation and biological applications. Here, we have developed an economical and environmentally friendly synthetic route with a recycle setup to prepare whey-protein-capped PbS QDs. Furthermore, the final Pb2+ ions were allowed to react with excess S2− ions and produce nontoxic precipitate in the end, which helped to avoid polluting the environment. After surface modification with protein, both our prepared QDs and the final reaction wastes demonstrated low toxicity in following both in vitro and in vivo experiments. Notably, the conversion efficiency of Pb2+ ions into PbS QDs in this study was almost 3 times than that of a one-time reaction without recycle. In addition, each batch of asprepared PbS QDs featured good stability and bright fluorescence at the NIR-II window in in vivo imaging experiments. Our work shows a novel green strategy for the sustainable synthesis of biocompatible QDs that contain heavy metal, especially for considering the follow-up biological applications. KEYWORDS: Quantum dots, Green chemistry, Recycled synthesis, Lead sulfide, In vivo imaging

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INTRODUCTION The development of quantum dots (QDs) for optical imaging is an area that has been widely studied in recent decades, because the advantages of QDs such as high quantum yield, good photostability and low toxicity make them an ideal luminescence probe for optical imaging.1−3 Recent progress reveals that the second near-infrared window (NIR-II, 1000−1400 nm) not only has extremely low autofluorescence and reduced photon scattering but also enables deep tissue penetration and low light scattering.4−7 Therefore, countless studies have been carried out in an endeavor to develop NIR-II QDs (e.g., PbS,8−10 Ag2Se11 and Ag2S6,12−14). Among these NIR-II QDs, PbS QDs have attracted considerable attention in recent years due to their extremely large bulk exciton Bohr radius (∼20 nm),15 which results in a © XXXX American Chemical Society

bandgap and absorption edge in the range from the visible to the NIR region.16−19 However, lead ions, a systemic toxicant, can adversely affect every organ system, especially the nervous system, and potentially disqualify PbS QDs for in vivo imaging applications.20,21 Hence, extensive work has been devoted to reducing the toxicity of PbS QDs. Owing to their unique metal ion affinity and excellent biocompatibility, biomolecules, such as DNA, proteins, enzymes and amino acids, have been proved in recent years to be useful tools for synthesizing biocompatible PbS QDs.9,22−25 These biomolecule-capped PbS QDs products exhibit low toxicity and Received: March 8, 2016 Revised: May 4, 2016

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DOI: 10.1021/acssuschemeng.6b00490 ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX

Letter

ACS Sustainable Chemistry & Engineering Scheme 1. Ilustration of LG-PbS QDs Recycled Synthesis

Figure 1. (A) Photoluminescence (PL) spectra of the as-prepared LG-PbS QDs after different reaction times. (B) UV−vis absorption spectra of asprepared LG-PbS QDs after different reaction times. Inset: Zoomed-in graph of the NIR absorption spectrum from 1000 to 1250 nm.

reacted with excess S2− and produced PbS precipitate, which prevented these reaction wastes from polluting the environment.

good water solubility, which is ascribed to the biomolecule layer coating around the surface of PbS QDs, preventing the leak of Pb2+ ions, and stabilizing them in the solution. More importantly, the surface passivation of these PbS QDs could effectively prevent oxidation during long-term air exposure, and increase photoluminescence efficiency as well as decrease photocharging,26 which benefits for following biological applications. In this study, we used the whey protein β-lactoglobulin (LG, molecular weight: ∼18 kDa), which acted as a template to stabilize PbS QDs by forming an inorganic−organic hybrid nanocomposite in the water phase. LG is recruited in this study because of its high water solubility and its structural features.27,28 Moreover, LG is also a natural carrier to entrap various nutrients,29,30 suggesting its good biosafety. More importantly, in this study, the potential hazard of excess Pb2+ ions in the reaction solution is seriously considered during the preparation of PbS QDs. Therefore, these excess Pb2+ ions in this work were separated and reused in order to improve the utilization of Pb2+ ions. Moreover, the final Pb2+ ions were

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RESULTS AND DISCUSSION As shown in Scheme 1, the synthesis was initiated by the mixing of two precursors, Pb(OAc)2 and LG, in a reaction vessel that was then placed in a microwave unit; (2) Na2S was quickly introduced into the above mixture under continuous stirring, and PbS QDs crystallized under the facilitation of microwave heating at 70 °C for 30 s; (3) the LG-capped PbS QDs (LG-PbS QDs) were prepared and exhibited NIR-II fluorescence when excited at 808 nm. (4) The freshly prepared LG-PbS QDs and the excess Pb2+ ions were separated by ultrafiltration tubes under maximum speed centrifugation (1, 3500g) for 15 min. After repeating those above steps for several times, the final Pb2+ ions were reacted with excess volumes of Na2S solution at 70 °C for 15 min, resulting in the formation of precipitate in the end. In this synthetic route, the number of recycle times is the critical factor for the conversion efficiency from Pb2+ ions to PbS B


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ACS Sustainable Chemistry & Engineering

Figure 2. (A) Quantum yields (QYs) of the as-prepared LG-PbS QDs after different reaction times. (B) XRD patterns of as-prepared LG-PbS QDs after different reaction times.

QYs in these samples decreased accompanied by the increasing reaction time. The crystalline structure and composition of PbS QDs after different reaction times were further examined by powder X-ray diffraction (XRD). As shown in Figure 2B, the powder XRD patterns for samples from all three batches clearly revealed that the crystalline structure of all batches of the as-prepared PbS QDs was consistent with that of bulk PbS (JCPDS: 05-0592). It was also observed that the intensity steadily increased as the times of reaction increased. Representative TEM images for the first, second and third batch of QDs, along with histograms of their sizes are provided in Figure 3. From Figure 3B,D,E, it can be observed that the sizes of QDs from each round of reaction varied from 4.7 to 5.8 nm. The inset provided in Figure 3A shows an observable single large quasi-spherical dot with lattice fringes. Interestingly, the sizes of the third batch of QDs displayed a bimodal distribution measured by dynamic light scattering (DLS), with one mode around 10 nm and a second mode around 100 nm (shown in Figure S2, Supporting Information). This phenomenon suggested that recycled reaction could influence not only the optical properties of the QDs but their size distribution as well. We then evaluated the products, including samples from all three batches of QDs and the final precipitate, for their cytotoxicity by exposing three cell lines to them: a human embryonic kidney cell line (293T), a human gastric epithelial cell line (GES-1) and a mouse-calvaria-derived cell line (MC3T3E1). Figure 4A shows the viability of 293T, GES-1 and MC3T3E1 cells after being incubated with the 1st, 2nd and 3rd batch of PbS QDs at a high concentration for 24 h. The cells without any treatment were taken as control. Clearly, no significant difference was observed between the QD-treated cells and untreated control groups. Importantly, more than 95% of the cells incubated with the final precipite remained viable, which clearly indicated that the final product after reaction had negligible negative effects on cell proliferation. The effects of these products on cell cycle distribution were studied via flow cytometric analysis. Figure 4B presents the percentages of 293T, GES-1 and MC3T3-E1 cells in Gap 1, S phase and Gap 2 after being treated with the QDs and the final products for 24 h compared to those percentages of the untreated control cells. Clearly, neither the QDs nor the final products had any obvious impact on cell cycle distribution, as can be seen in comparison to the control groups (Figure 4B). On the basis of these studies, we conclude that these LG-PbS QDs have good biocompatibility after their surface has been coated with

QDs. In Figure 1A, the PL intensity of LG-PbS QDs prepared after one, two and three times of reactions showed that the maximum emission peak of the QDs was red-shifted as the number of repeating times increased, which was 1163 nm for the first batch, 1287 nm for the second batch and 1311 nm for the third batch, respectively. The red-shift of QDs’ emission is mainly due to narrowing band gaps caused by the size growth of QDs at prolonged heating time. Interestingly, this red-shift of emission was accompanied by an increasing full width at half-maximum (fwhm) (Table S1), which showed that the sizes of QDs increased as the number of reactions increased. The large fwhm of QDs is mainly ascribe to the large size distribution. However, there was no detectable emission after the reaction was repeated for four times (shown in Figure S1, Supporting Information). Meanwhile, no PL emission was observed in the final PbS precipitate (shown in Figure S1, Supporting Information). In addition, the concentrations of the as-prepared LG-PbS QDs were calculated using the aforementioned method, which was about 150 nM for the first batch, 178 nM for the second batch and 228 nM for the third batch. Therefore, the reaction efficiency in this study was more than 3 times that in a one-time reaction, which indicated that the Pb2+ ions had been utilized to the maximum extent in this synthetic route. Figure 1B demonstrates the UV−vis-NIR absorbance spectra of the prepared LG-PbS QDs after different times of reaction, and reveals that all batches of prepared LG-PbS QDs had stronger absorbance in the visible region than in the NIR region, a phenomenon attributable to the quantum effects of QDs. Furthermore, the first excitonic absorption peak of these QDs was found around 1150 nm (shown in the inset of Figure 1B). Notably, the absorbance was gradually red-shifted with the increasing reaction times, which indicated that larger QDs were formed after more recycle times. Fluorescence quantum yields (QYs) of the different batches of QDs were determined by the aforementioned methods in order to make a direct comparison, with IR-26 as control (QYs = 0. 5%). As shown in Figure 2A, the first batch of QDs displayed the highest QYs (19.8 ± 0.5% at 1163 nm), with second batch and third batch exhibiting lower QYs at 17.5 ± 0.3% and 13.5 ± 0.2%, respectively. As is known, the QY of QDs is significantly influenced by the defects at their surfaces.31 When the size of QDs is too small, the reaction time is not long enough for efficient passivation of QD surface. On the other hand, when the QDs reaches the maximum QY, further reaction will probably cause detachment of ligands from the QD surface and result in larger amount of defects, thus decline the QY. In this study, the C


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Figure 3. Respective TEM images of (A) the 1st batch of LG-PbS QDs, (C) the 2nd batch of LG-PbS QDs, and (E) the 3rd batch of LG-PbS QDs. (B, D and F) Corresponding histograms with the average particle sizes are also shown. The right-up inset of panel A further illustrates the high-resolution TEM (HR-TEM) image of an individual QD. A typical LG-PbS QD is labeled by red dot-line in the TEM images.

LG, and the final products also have limited effects on normal cells and the environment.

A final set of experiments investigated whether LG-PbS QDs were suitable for in vivo imaging. In these experiments, LG-PbS D


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Figure 4. (A) MTT analysis and (B) cell cycle assay of 293T, MC3T3-E1 and GES-1 cells after incubating with various reaction times of products for 24 h.

Figure 5. (A) Comparison of NIR-II fluorescence images of the 3rd batch of LG-PbS QDs in nude mice at different times (0.5, 12 and 24 h). (B) Corresponding hematoxylin and eosin (H&E) stained tissue sections of the nude mice injected with LG-PbS QDs after 24 h.

QDs from each batch were injected respectively via the tail vein into nude mice that were then observed under the same NIR-II imaging conditions. As shown in Figure 5A, NIR-II fluorescence signals of the third batch of PbS QDs were detected 1 h postinjection, which was ascribed to the PL property of the LGPbS QDs and the ultralow autofluorescence background of mice in the NIR-II region. Notably, most fluorescence signals of our products disappeared 12 h postinjection, and no fluorescence signal was detected 24 h postinjection. Interestingly, fluorescence signals could be observed on mice injected with the first batch of PbS QDs 24 h postinjection but completely disappeared 48 h

postinjection (shown in Figure S3, Supporting Information). The rapid elimination of QDs from the mice was mainly ascribe to the ultrasmall size of our products. Moreover, considering the different QYs between the first, second and third batches of PbS QDs, these results clearly demonstrated that high QYs of QDs were critical to the quality of NIR-II in vivo imaging results, which confirmed that higher QYs led to higher spatial resolution of imaging. In addition, hematoxylin and eosin (H&E) staining was employed to investigate whether LG-PbS QDs would have any detrimental effects on some vital organs. As shown in Figure 5B, E


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the H&E assay indicated that there were no notable lesion, inflammation or other abnormalities in these main organs, including the spleen and the liver. These results illustrated that the toxicity of PbS QDs was negligible after they had been coated with biocompatible and hydrophilic protein.

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CONCLUSION In summary, we have developed an easy, economical, and environmentally friendly approach to synthesize high-NIR-IIemitting LG-PbS QDs in the aqueous phase by recycling the excess Pb2+ ions in the reaction solution. The synthesized LGPbS QDs showed no apparent cytotoxicity and were found to be effective imaging reporters, as illustrated in the in vivo imaging of mice. The final PbS precipitate produced via adding excess sulfide ions also proved to be noncytotoxic, which helped eliminate heavy metal ion pollutants to the environment. Intriguingly, we have found compelling evidence that excess Pb2+ ions in the reaction solution can be recycled into either useful QDs with the help of a protein or environmentally friendly products with S2− ions. Within a larger context, this work adds momentum to the researches aimed at converting excess heavy metal ions into useful and intriguing nanomaterials.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssuschemeng.6b00490. Experiment details, supporting table of emission peak, and size distribution and other in vivo imaging fluorescent figures of PbS QDs at different reaction times (PDF).

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AUTHOR INFORMATION

Corresponding Authors

*Prof. Yunxia Li. Tel: 0086 21 52888256. E-mail: liyunxia912@ aliyun.com *Prof. Shiyi Chen. Tel: 0086 21 52888256. E-mail: cshiyi@163. com. Author Contributions

The paper was written through contributions of all authors. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This project was supported by the National 863 Hi-tech Project (2015AA033703), National Natural Science Foundation of China (No. 81572108, 81301672, 81401771 and 81201773), The Royal Society-Internation Exchange scheme (2016 China NSFC cost-share), Natural Science Foundation of Shanghai Project (No. 12ZR1415800 and No. 15ZR1405000), China Postdoctoral Science Foundation (No. 2014M560296) and Innovation Program of Shanghai Municipal Education Commission (No. 15ZZ006).

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REFERENCES

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Recycled Synthesis of Whey-Protein-Capped Lead ... - ACS Publications

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