Dissecting the Factors Affecting the Fluorescence Stability of Quantum

Subscriber access provided by UNIV OF CALIFORNIA SAN DIEGO LIBRARIES

Article

Dissecting the factors affecting fluorescence stability of quantum dots in live cells Zhi-Gang Wang, Shu-Lin Liu, Yuan-Jun Hu, Zhi-Quan Tian, Bin Hu, Zhi-Ling Zhang, and Dai-Wen Pang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b01742 • Publication Date (Web): 21 Mar 2016 Downloaded from http://pubs.acs.org on March 23, 2016

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

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

Page 1 of 17

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

ACS Applied Materials & Interfaces

Dissecting the Factors Affecting Fluorescence Stability of Quantum Dots in Live Cells

Zhi-Gang Wang†, Shu-Lin Liu†, Yuan-Jun Hu, Zhi-Quan Tian, Bin Hu, Zhi-Ling Zhang, Dai-Wen Pang* Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), College of Chemistry and Molecular Sciences, State Key Laboratory of Virology, The Institute for Advanced Studies, and Wuhan Institute of Biotechnology, Wuhan University, Wuhan, 430072, P. R. China.

ABSTRACT: Labeling and imaging of live cells with quantum dots (QDs) has attracted great attention in biomedical field over the past two decades. Maintenance of the fluorescence of QDs in biological environment is crucial for performing long-term cell tracking to investigate the proliferation and functional evolution of cells. Cell-penetrating peptide trans-activator of transcription (TAT) is a well-studied peptide to efficiently enhance the transmembrane delivery. Here, we used TAT peptide-conjugated QDs (TAT-QDs) as a model system to examine the fluorescence stability of QDs in live cells. By confocal microscopy, we found that TAT-QDs were internalized into cells by endocytosis, and transported into the cytoplasm via mitochondria, Golgi apparatus and lysosomes. More importantly, the fluorescence of TAT-QDs in live cells was decreased mainly by cell proliferation, and low pH value in lysosomes could also lower the fluorescence intensity of intracellular QDs. Quantitative analysis of the amount of QDs in extracellular region and whole cells indicated that the exocytosis was not the primary cause of fluorescence decay of intracellular QDs. This work facilitates a better understanding of the fluorescence stability of QDs for cell imaging and long-term tracking in live cells. Also, it provides insights into the utility of TAT for transmembrane transportation, and the preparation and modification of QDs for cell imaging and tracking. KEYWORDS: quantum dots, trans-activator of transcription, fluorescence stability, influence factors, live cells

INTRODUCTION Quantum dots (QDs), known as fluorescent semiconductor nanocrystals, possess unique optical properties, such as extreme brightness and high photostability.1, 2 They can overcome the intrinsic limitations of organic dyes and fluorescent proteins, which have attracted considerable interest in many research fields, from biological imaging to medical diagnostics.3-9 With the development of water-solubilization and biofunctionalization of QDs, their properties have opened new

ACS Paragon Plus Environment


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

Page 2 of 17

possibilities for in-vivo application of QDs to image individual cells, to track cell development and fate, and further to assess cell distribution in live animals.10,

11

Thus, maintenance of the

fluorescence of QDs in biological environment is very crucial for performing long-term tracking to investigate the proliferation and functional evolution of cells. However, the fluorescence decay of QDs in live cells or organelles is a general problem, which hampers the application of QDs in vivo.12-15 For example, Summers et al. investigated the fluorescence stability of QDs loaded by endocytosis in live primary blood mononuclear cells, and found that the fluorescence decayed a lot after 1000 min.13 Sun et al. claimed that mercaptoacetic acid (MAA)-capped QDs is quenched in a low-pH environment in cellular vesicles.14 Pi et al. found a quick loss of QDs fluorescence in mouse embryonic stem cells, which may cause by degradation or excretion of QDs in cells rather than cell division.16 These studies mainly discussed the possible causes of fluorescence decay of QDs in live cells. To date, most studies used fluorometric methods for quantification of QDs to further evaluate fluorescence signal variation of QDs in live cells, which are susceptible to the background fluorescence or blinking of QDs. Thus, the researchers still need an unbiased method to comprehensively investigate the factors affecting the fluorescence signals of QDs, and to uncover which factors to what extent can influence the fluorescence stability of QDs in live cells. Cell-penetrating peptide trans-activator of transcription (TAT) is derived from human immunodeficiency virus-1, which is a well-studied peptide to efficiently enhance the transmembrane delivery.17-19 Due to its minimal cytotoxicity, TAT peptide has been reported to deliver QDs into the cytoplasm of living cells20, 21 and facilitate the blood-brain barrier penetration of QDs in vivo.22 Previous works have studied the delivery mechanisms of TAT peptide-conjugated QDs (TAT-QDs) in live cells.21, 23 Ruan et al. suggested the TAT-QDs were internalized into vesicles by active transport.21 Chen et al. used fluorescence imaging and flow cytometry methods to investigate the internalization pathway, and found that the TAT-QDs were internalized by lipid raft-dependent micropinocytosis.23 However, the distribution of TAT-QDs in cellular organelles is less reported, and the fluorescence stability of TAT-QDs in live cells is still rarely studied so far. In this work, we used TAT-QDs as a model system to examine the fluorescence stability of QDs in live cells. First, we used biotin-streptavidin interaction to conjugate CdSe/ZnS core-shell QDs with TAT directly. Fluorescence imaging was adopted to assess colocalization of QDs and dye-stained cellular organelles for analyzing the intracellular distribution of TAT-QDs. By flow cytometry, we studied the influence of cell proliferation and low pH on fluorescence stability. High-performance liquid chromatography coupled with inductively coupled plasma mass spectrometry (HPLC-ICP-MS) analysis allowed us to characterize the influence of the endocytosis on fluorescence decay of QDs. The results indicated that the fluorescence of TAT-QDs in live cells was decreased mainly by cell proliferation, and low pH value of lysosomes could also lower the fluorescence intensity of intracellular QDs. The exocytosis was not the primary cause of fluorescence decay of intracellular QDs. These results contributed to a better understanding on the preparation and modification of QDs applied in the cell imaging and further tracking researches.


Page 3 of 17

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


MATERIALS AND METHODS Characterization of TAT-QDs. The Sequence of biotinylated transactivator protein (biotinylated TAT, HD Biosciences Co. Ltd.) is RRRQRRKKRGYK-Biotin. TAT-QDs were prepared by incubating streptavidin-modified QDs (SA-QDs, Wuhan Jiayuan Quantum Dots Co. LTD) with biotinylated TAT at the molar ratio of 1:30 for 5 min at room temperature. The UV–vis spectra were recorded using a Shimadzu UV-2550 spectrophotometer. TEM images were acquired on a JEM 2010EF electron microscope. DLS measurements were performed on a Zetasizer Nano ZS90 (Malvern Instruments). Cell Culture and TAT-QDs Labeling. Hepatocellular carcinoma cells (HCCLM9) were cultured in the RPMI-1640 (Gibco) supplemented with 10% fetal bovine serum (Gibco), 100 units/mL penicillin G sodium and 100 µg/mL streptomycin sulfate in a humidified atmosphere of 5% CO2, 95% air at 37 oC. The cells were plated on a 35 mm glass-bottom Petri dish (NEST Corp) for 24 h before the experiments. The cells were washed with phosphate-buffered saline (PBS) buffer to remove excess mucus secretion and then incubated with 10 nM TAT-QDs under different culture conditions. Control experiments were carried out at the same conditions without biotinylated TAT in PBS buffer. Dye Staining and Drug Inhibitor. To stain the cytomembrane of live HCCLM9 cells, 5 µg/mL 3,3’-dioctadecyloxacarbocyanineperchlorate (DiO, Invitrogen) was added directly to the culture medium for 30 min at 37 °C. To stain the cell nucleus, 5µg/mL Hoechst 33342 was added into the culture medium for 30 min at 37 °C. For staining mitochondria, Golgi, and lysosomes, the cells labeled with TAT-QDs were stained with Mito-tracker green, Golgi-tracker red and Lyso-tracker green (Invitrogen) respectively. For the cell proliferation assay, HCCLM9 cells were trypsinized and resuspended into fresh culture medium and then labeled with carboxyfluorescein diacetate succinimidyl ester (CFDA SE, Beyotime Institute of Biotechnology) at a final concentration of 1.5 µmol/L according to the manufacturer’s directions. To inhibit the acidification of lysosomes, the cells incubated with TAT-QDs were cultured in the medium in the presence of 50 mM NH4Cl. MTT Assay. The HCCLM9 cells were cultured in 96 well plates for 24 h before incubate with TAT-QDs. After the 24 h incubation with TAT-QDs, the cells were treated with 5 mg/ml of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT, 20 µL/well, Sigma Chemical Co.) dissolved in PBS buffer for 4 h. Then the suspension in the 96 well plates was removed and the formazan crystals were dissolved in 200 µL DMSO. The 96 well plates were agitated on a plate shaker for 10 min and finally the absorbance spectra were measured at 490 nm, using the microplate spectrophotometer system (MULTISKAN MK3). Flow Cytometry. HCCLM9 cells were incubated with TAT-QDs at 37 °C. Subsequently, the cells were vigorously washed with PBS and trypsinized for 10 min at 37 °C. The cell suspensions were collected and centrifuged at 160 × g for 5 min and diluted with PBS. The flow cytometry was analyzed on an EPICS XL (Beckman Coulter) equipped with an argon laser. At least 10 000 cells



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

were measured for each sample, and further analysis was performed by using FlowJo software (TreeStar, Ashland). Flow cytometry of the cells without TAT-QDs or only with SA-QDs were performed as a negative control. ICP-MS Measurement. The stoke solution were prepared by dissolving powered CdCl2 into Milli-Q ultra-pure water (18.2MΩ/cm, Millipore, Molsheim, France) at a concentration of 0.1-50 ppb. The working curve was recorded by measuring the stoke solution using Agilent 7500 (Agilent Technologies, USA). The cells incubated with TAT-QDs and corresponding culture medium at 0-72 h were harvested to measure the whole cells and extracellular region of cadmium. Fluorescence Imaging. Fluorescence images were acquired by using a spinning-disk confocal microscope (Andor Revolution XD) equipped with an Olympus IX 81 microscope, a Nipkow disk-type confocal unit (CSU 22, Yokogawa) and an EMCCD (Andor iXon DV885K). Hoechst 33342, DiO/Mito-tracker green/Lyso Tracker green/QD525, and QD605/Golgi-tracker red were excited at 405 nm, 488 nm and 561 nm by DPSS lasers, respectively. The fluorescence signals were split into different channels using 447/60 nm, 525/50 nm, 617/73 nm band-pass emission filters. For simultaneous multiple-color imaging, fluorescence signals were detected separately with the EMCCD by the corresponding channels.

RESULTS AND DISCUSSION TAT-QDs for Live Cell Labeling Here, we used a general method to effectively and conveniently label live cells with QDs. Briefly, the biotinylated TAT can easily conjugate with streptavidin-modified QDs (SA-QDs) by biotin-streptavidin interaction. TEM images showed that the TAT-QDs were dispersed evenly after TAT conjugation, similar to the dispersion of SA-QDs (Figure S1A and S1B). We analyzed the size distributions of SA-QDs and TAT-QDs respectively, and found SA-QDs are the same size as TAT-QDs (Figure S1D and S1E). This indicated that the conjugations were non-aggregated and still maintained excellent fluorescence properties (Figure S1C). The changes of hydrodynamic diameters and zeta potentials clearly indicated that TAT was conjugated with QDs (Figure S1F). Then, TAT-QDs were incubated with the live HCCLM9 cells. As shown in Figure 1, we clearly observed the outlines of live cells by QDs signals, indicating that the TAT-mediated method can successfully realize the QDs labeling of live cells (Figure 1C). Additionally, we investigated the cells incubated with only SA-QDs by microscopy. The images showed very little fluorescence, suggesting that TAT played an important role on QDs labeling of live cells (Figure 1A and 1B). Further, we exploited flow cytometry to detect the labeling efficiency of TAT-QDs (Figure 1D). Almost all of the cells exhibited fluorescence signals, indicating that this labeling method possessed a very high labeling efficiency. Moreover, we investigated whether abundant TAT-QDs labeling exerted effect on cell viability. Thereby, MTT assay was used to test the cytotoxicity of


Page 4 of 17

Page 5 of 17

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


TAT-QDs (Figure S2). The HCCLM9 cells were exposed to TAT-QDs with the concentrations of 0-80 nM to test the influence of TAT-QDs on cell viability. As shown in Figure S2, almost 100% HCCLM cells maintained viability at the work concentration of 10 nM and 92% cells still retained viability even exposed at high concentration (80 nM) for 24 h. These results implied that the TAT-QDs labeling was low-toxicity and high-effective method for live-cell labeling.

Intracellular Distribution of TAT-QDs Understanding the distribution of QDs at different cellular organelles is not only essential for elucidating the transport pathway of TAT-QDs, but also for better cognizing the fluorescence intensity variation of TAT-QDs in live cells. Firstly, we used confocal microscopy to investigate the colocalization of TAT-QDs signals and dye-stained cellular organelles in live cells. QDs-labeled cells were cultured at 37 °C for 90 min, and then stained with a membrane dye (DiO) before imaging. We found that most of QDs signals were distributed in the cytoplasm, and colocalized with DiO-stained vesicles, illustrating that QDs may be trapped into vesicles and transported into cytoplasm by endocytosis (Figure 2A-2D). To determine whether the cells uptake of TAT-QDs is an energy-dependent endocytic process, we cultured the QDs-labeled cells at 4 °C for 30 min. As clearly shown in Figure S3, the cells uptake of TAT-QDs is largely blocked when the cells cultured at low temperature, suggesting that TAT-QDs transportation is an energy-dependent endocytic process.24 Next, we investigated the dynamic movements of TAT-QDs in live cells by real-time tracking. TAT-QDs were incubated with live cells stained by Hoechst 33342 (a nuclear dye) and DiO. We observed the colocalization of DiO signals and QDs signals in the cytoplasm, conforming that the TAT-QDs were indeed trapped into vesicles. Interestingly, we found that the vesicles containing QDs were fused together near the perinuclear region. Snapshots showed there are two separate vesicles containing QDs initially. Over times, the vesicles were merged together by the fusion process (Figure 2E). The results suggested that TAT-QDs were trapped in vesicles and then transported into the perinuclear region by endocytosis. According to the fusion events, we suspected that TAT-QDs may be delivered into some cell organelles in the perinuclear region. Based on previous reports, Golgi apparatus, mitochondria and lysosomes were the main intracellular organelles related to the transportation of internalized particles.25,

26

Thus, mitochondria, Golgi apparatus and lysosomes were investigated by

colocalization experiments. We incubated TAT-QDs (QD605) with cells, and then stained with mitochondria specific dye (Mito-tracker green). The fluorescence images showed that the signals of mitochondria were colocalized with QDs signals partly in the cells, providing evidence that mitochondria are one of cellular organelles involving TAT-QDs transportation in live cells (Figure 3A-3C). To examine whether QDs could be translocated with Golgi apparatus, we employed the Golgi tracker red dye to stain the cells preincubated with TAT-QDs (QD525). Our result exhibited that QDs were colocalized with Golgi apparatus, suggesting TAT-QDs were transported in the



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

cytoplasm via Golgi apparatus (Figure 3D-3F). Moreover, a striking phenomenon was observed that internalized TAT-QDs (QD605) were transported into lysosomes, which is clearly proved by colocalization between the signals of QDs and Lyso-tracker green dye (Figure 3G-3I). Taken together, the results suggested TAT-QDs were trapped into vesicles and transported in the cytoplasm via mitochondria, Golgi apparatus and lysosomes.

Fluorescence Stability of TAT-QDs in Live Cells In order to investigate the fluorescent stability of QDs in live cell, we first labeled the HCCLM9 cells with TAT-QDs, cultured the labeled cells for different time, and then checked the fluorescence signals of each cell by confocal microscopy and flow cytometry. The results showed that the florescence signals reduced to about 20% at 24 h culture, about 12% at 48 h, and 10% at 72 h, illustrating that the QDs signals decay dramatically during the first 24 h incubation (Figure 4). We speculated that there may be several influencing factors of QDs signals, including cell division, exocytosis, and pH value. In detail, compared with cellular components, QDs distributed in the cytosol is only a kind of inorganic materials without amplification ability. During cell proliferation, the one cell containing QDs can be divided into two separated cells. Thus, the QDs intensity of each cell will reduce a lot. Several researches reported that QDs was not stable in acidic environments.27, 28 The cells usually grow in pH 7 environment, but the pH value is only ~ 5 in cell lysosomes. Meanwhile, TAT-QDs were transported into cells by endocytosis, and the exocytosis may also occur for TAT-QDs, which can induce QDs to export into the extracellular environments, and impact the intensity of individual cells. Here, we characterized the fluorescent stability of QDs at the conditions of cell proliferation, low pH environments and exocytosis.

Influence of Cell Proliferation Carboxyfluorescein succinimidyl ester (CFDA SE) is a cell permeable dye for general fluorescent analysis of cell proliferation.29, 30After each cell division, the CFDA SE intensity halves in the daughter cells. To determine the influence of cell proliferation on fluorescent stability of QDs, we utilized CFDA SE to characterize the proliferation of cells. The intensity of CFDA SE reduced to about 50% after 24 h, 31% at 48 h, and 19% at 72 h (Figure 4D). By comparing the fluorescence changes of CFDA SE and TAT-QDs in live cells, we found that the fluorescence intensities of CFDA SE and TAT-QDs both decreased after long-time culture, suggesting the cell proliferation showed a great influence on fluorescence intensity changes. Meanwhile, the QDs intensity decreased more significantly, implying there are other factors causing the fluorescence intensity decrease of QDs.


Page 6 of 17

Page 7 of 17

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


Influence of Low pH Value To inspect the influence of pH value, we initially measured the fluorescence stability of QDs in the range of pH 5~8 with fluorescence spectrophotometer. As shown in fluorescence intensity vs. time plots, the fluorescence intensity of QDs was sensitive to environmental pH values, and the fluorescence intensity of QDs decreased significantly along with the decrease of pH values (Figure 5A). By the way, the emission spectra of QDs were recorded at different pH values (Figure 5B). The maximum emission wavelength of QDs kept consistent at different pH values, which indicated that the core of QDs showed no impairment even at low pH value. As mentioned above, QDs were transported to in the cytosol via lysosomes, in which the pH value is about 5. By two-color tracking, we monitored the QDs and lysosome signals simultaneously in live cells. We occasionally observed a phenomenon of fluorescence decay of QDs in lysosomes. As shown in Figure 5E, there existed strong QDs signals colocalized with lysosome signals initially. Later on, the QDs signals became weak after 8 min, and could not be observed at 12 minutes. These demonstrated that the fluorescence intensity of QDs decreased when QDs arrived in lysosomes, indicating that the low pH value of lysosomes or late endosomes may result in the fluorescence decay of QDs in cells. Next, we chose NH4Cl to inhibit the acidification of lysosomes in live cells.31 By flow cytometry measurement, we found that the fluorescence intensity of QDs in NH4Cl-treated cells was stronger than that in untreated cells at 24 h incubation (Figure 5C and 5D). The results suggested that the acidic environment resulted in the fluorescence intensity decay of QDs in live cells.

Influence of Exocytosis It is reported that TAT-QDs can be secreted from the tips of filopodia to the extracellular environments by typical exocytosis.21 To determine the influence of exocytosis on QDs intensity, we developed a quantitative method to measure the content of QDs in the whole cell and extracellular environment. Briefly, we labeled the cells, cultured with different time, then collected the medium and the labeled cells separately, and further analyzed the cadmium content by HPLC-ICP-MS respectively (Figure 6A). The amount of endogenous cadmium is negligible in cells, by compared that of CdSe/ZnS core-shell QDs. Thus, we used the amount of cadmium to represent the amount of QDs and investigated the change of QDs in extracellular region and whole cells at different culture time. HPLC-ICP-MS analysis verified that the content of QDs in extracellular region and whole cells showed little change within 72 h (Figure 6B). The results indicated that few QDs were expelled from cell body. Therefore, we considered that exocytosis is not the main reason causing the fluorescence intensity decay of QDs in live cells.



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

CONCLUSIONS Taken together, we have used TAT to conjugate QDs for labeling live cells. Our results indicated that this labeling method possesses high labeling efficiency. Confocal microscopy showed that TAT-QDs were internalized into cells by endocytosis, and transported in the cytoplasm via mitochondria, Golgi apparatus and lysosomes. More importantly, we studied the fluorescence variability of TAT-QDs in live cells. The QDs fluorescence reduced a lot after 24 h culture. Flow cytometry indicated that cell proliferation was the main factor of fluorescence decay of QDs in live cells, and low pH value of lysosomes can also lead to fluorescence intensity reduction of intracellular QDs. We further observed the dynamic process of fluorescence decay of QDs in lysosomes. HPLC-ICP-MS analysis suggested that the exocytosis was not the primary cause of fluorescence decay of intracellular QDs. These findings not only provided insights into the application of TAT for transmembrane transportation in biological field, but also contributed to a better understanding on fluorescence stability of QDs for cell imaging and further long-term tracking in vivo.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Characterization of the properties of TAT-QDs. Cell viability of TAT-QDs labeling under different concertation by MTT assay. Images of live cell labeled with TAT-QDs at 4°C.

AUTHOR INFORMATION Corresponding Authors

*E-mail: [email protected]. Author contribution †These authors contributed equally to this article. Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENTS This work was supported by the National Natural Science Foundation of China (No. 21535005), the National Basic Research Program of China (973 Program, No. 2011CB933600), the 111 Project (111-2-10), and Collaborative Innovation Center for Chemistry and Molecular Medicine.


Page 8 of 17

Page 9 of 17

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


REFERENCES 1.

Michalet, X.; Pinaud, F. F.; Bentolila, L. A.; Tsay, J. M.; Doose, S.; Li, J. J.; Sundaresan, G.; Wu, A. M.;

Gambhir, S. S.; Weiss, S., Quantum Dots for Live Cells, in Vivo Imaging, and Diagnostics. Science 2005, 307, 538-544. 2.

Resch-Genger, U.; Grabolle, M.; Cavaliere-Jaricot, S.; Nitschke, R.; Nann, T., Quantum Dots versus

Organic Dyes as Fluorescent Labels. Nat. Methods 2008, 5, 763-775. 3.

Wegner, K. D.; Hildebrandt, N., Quantum Dots: Bright and Versatile in Vitro and in Vivo

Fluorescence Imaging Biosensors. Chem. Soc. Rev. 2015, 44, 4792-4834. 4.

Fang, M.; Yuan, J. P.; Peng, C. W.; Pang, D. W.; Li, Y., Quantum Dots-Based in Situ Molecular

Imaging of Dynamic Changes of Collagen IV During Cancer Invasion. Biomaterials 2013, 34, 8708-8717. 5.

Wang, Z. G.; Liu, S. L.; Tian, Z. Q.; Zhang, Z. L.; Tang, H. W.; Pang, D. W., Myosin-Driven

Intercellular Transportation of Wheat Germ Agglutinin Mediated by Membrane Nanotubes between Human Lung Cancer Cells. ACS Nano 2012, 6, 10033-10041. 6.

Liu, S. L.; Zhang, Z. L.; Tian, Z. Q.; Zhao, H. S.; Liu, H.; Sun, E. Z.; Xiao, G. F.; Zhang, W.; Wang, H. Z.;

Pang, D. W., Effectively and Efficiently Dissecting the Infection of Influenza Virus by Quantum-Dot-Based Single-Particle Tracking. ACS Nano 2012, 6, 141-150. 7.

Kairdolf, B. A.; Smith, A. M.; Stokes, T. H.; Wang, M. D.; Young, A. N.; Nie, S., Semiconductor

Quantum Dots for Bioimaging and Biodiagnostic Applications. Annu. Rev. Anal. Chem. 2013, 6, 143-162. 8.

Wang, Z. G.; Liu, S. L.; Zhang, Z. L.; Tian, Z. Q.; Tang, H. W.; Pang, D. W., Exploring Sialic Acid

Receptors-Related Infection Behavior of Avian Influenza Virus in Human Bronchial Epithelial Cells by Single-Particle Tracking. Small 2014, 10, 2712-2720. 9.

Fang, M.; Peng, C. W.; Pang, D. W.; Li, Y., Quantum Dots for Cancer Research: Current Status,

Remaining Issues, and Future Perspectives. Cancer Biol. Med. 2012, 9, 151-163. 10. Jaiswal, J. K.; Mattoussi, H.; Mauro, J. M.; Simon, S. M., Long-Term Multiple Color Imaging of Live Cells Using Quantum Dot Bioconjugates. Nat. Biotechnol. 2003, 21, 47-51. 11. Gao, X.; Cui, Y.; Levenson, R. M.; Chung, L. W. K.; Nie, S., In Vivo Cancer Targeting and Imaging with Semiconductor Quantum Dots. Nat. Biotechnol. 2004, 22, 969-976. 12. Zhu, Z. J.; Yeh, Y. C.; Tang, R.; Yan, B.; Tamayo, J.; Vachet, R. W.; Rotello, V. M., Stability of Quantum Dots in Live Cells. Nat. Chem. 2011, 3, 963-968. 13. Summers, H. D.; Holton, M. D.; Rees, P.; Williams, P. M.; Thornton, C. A., Analysis of Quantum Dot Fluorescence Stability in Primary Blood Mononuclear Cells. Cytometry, Part A 2010, 77, 933-939. 14. Sun, Y. H.; Liu, Y. S.; Vernier, P. T.; Liang, C. H.; Chong, S. Y.; Marcu, L.; Gundersen, M. A., Photostability and pH Sensitivity of CdSe/ZnSe/ZnS Quantum Dots in Living Cells. Nanotechnology 2006, 17, 4469-4476. 15. Jiang, X.; Röcker, C.; Hafner, M.; Brandholt, S.; Dörlich, R. M.; Nienhaus, G. U., Endo-and Exocytosis of Zwitterionic Quantum Dot Nanoparticles by Live HeLa Cells. ACS Nano 2010, 4, 6787-6797. 16. Pi, Q. M.; Zhang, W. J.; Zhou, G. D.; Liu, W.; Cao, Y., Degradation or Excretion of Quantum Dots in Mouse Embryonic Stem Cells. BMC Biotechnol. 2010, 10, 36. 17. Fischer, R.; Fotin‐Mleczek, M.; Hufnagel, H.; Brock, R., Break on Through to the Other Side-Biophysics and Cell Biology Shed Light on Cell-Penetrating Peptides. ChemBioChem 2005, 6, 2126-2142.



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

18. Wadia, J. S.; Stan, R. V.; Dowdy, S. F., Transducible TAT-HA Fusogenic Peptide Enhances Escape of TAT-Fusion Proteins after Lipid Raft Macropinocytosis. Nat. Med. 2004, 10, 310-315. 19. Eguchi, A.; Akuta, T.; Okuyama, H.; Senda, T.; Yokoi, H.; Inokuchi, H.; Fujita, S.; Hayakawa, T.; Takeda, K.; Hasegawa, M., Protein Transduction Domain of HIV-1 Tat Protein Promotes Efficient Delivery of DNA into Mammalian Cells. J. Biol. Chem. 2001, 276, 26204-26210. 20. Santra, S.; Yang, H.; Holloway, P. H.; Stanley, J. T.; Mericle, R. A., Synthesis of Water-Dispersible Fluorescent, Radio-Opaque, and Paramagnetic CdS:Mn/ZnS Quantum Dots: A Multifunctional Probe for Bioimaging. J. Am. Chem. Soc. 2005, 127, 1656-1657. 21. Ruan, G.; Agrawal, A.; Marcus, A. I.; Nie, S., Imaging and Tracking of Tat Peptide-Conjugated Quantum Dots in Living Cells: New Insights into Nanoparticle Uptake, Intracellular Transport, and Vesicle Shedding. J. Am. Chem. Soc. 2007, 129, 14759-14766. 22. Santra, S.; Yang, H.; Stanley, J. T.; Holloway, P. H.; Moudgil, B. M.; Walter, G.; Mericle, R. A., Rapid and Effective Labeling of Brain Tissue Using TAT-Conjugated CdS:Mn/ZnS Quantum Dots. Chem. Commun. 2005, 25, 3144-3146. 23. Chen, B.; Liu, Q.; Zhang, Y.; Xu, L.; Fang, X., Transmembrane Delivery of the Cell-Penetrating Peptide Conjugated Semiconductor Quantum Dots. Langmuir 2008, 24, 11866-11871. 24. Kim, J.-S.; Yoon, T.-J.; Yu, K.-N.; Noh, M. S.; Woo, M.; Kim, B.-G.; Lee, K.-H.; Sohn, B.-H.; Park, S.-B.; Lee, J.-K., Cellular Uptake of Magnetic Nanoparticle Is Mediated through Energy-Dependent Endocytosis in A549 Cells. J. Vet. Sci. 2006, 7, 321-326. 25. Zhang, Y.; Mi, L.; Xiong, R.; Wang, P.-N.; Chen, J.-Y.; Yang, W.; Wang, C.; Peng, Q., Subcellular localization of Thiol-Capped CdTe Quantum Dots in Living Cells. Nanoscale Res. Lett. 2009, 4, 606-612. 26. Gao, X.; Wang, T.; Wu, B.; Chen, J.; Chen, J.; Yue, Y.; Dai, N.; Chen, H.; Jiang, X., Quantum Dots for Tracking Cellular Transport of Lectin-Functionalized Nanoparticles. Biochem. Biophys. Res. Commun. 2008, 377, 35-40. 27. Liu, Y.-S.; Sun, Y.; Vernier, P. T.; Liang, C.-H.; Chong, S. Y. C.; Gundersen, M. A., pH-Sensitive Photoluminescence of CdSe/ZnSe/ZnS Quantum Dots in Human Ovarian Cancer Cells. J. Phys. Chem. C 2007, 111, 2872-2878. 28. Durisic, N.; Godin, A. G.; Walters, D.; Grütter, P.; Wiseman, P. W.; Heyes, C. D., Probing the “Dark” Fraction of Core–Shell Quantum Dots by Ensemble and Single Particle pH-Dependent Spectroscopy. ACS Nano 2011, 5, 9062-9073. 29. Urbani, S.; Caporale, R.; Lombardini, L.; Bosi, A.; Saccardi, R., Use of CFDA-SE for Evaluating the in Vitro Proliferation Pattern of Human Mesenchymal Stem Cells. Cytotherapy 2006, 8, 243-253. 30. Lyons, A. B., Analysing Cell Division in Vivo and in Vitro Using Flow Cytometric Measurement of CFSE Dye Dilution. J. Immunol. Methods 2000, 243, 147-154. 31. Misinzo, G.; Delputte, P. L.; Nauwynck, H. J., Inhibition of Endosome-Lysosome System Acidification Enhances Porcine Circovirus 2 Infection of Porcine Epithelial Cells. J. Virol. 2008, 82, 1128-1135.


Page 10 of 17

Page 11 of 17

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


Figures:

Figure 1. Transactivator protein-conjugated quantum dots (TAT-QDs) binding to the cell surface. (A-C).The living HCCM9 cells (A) were incubated with only SA-QDs (B) and with TAT-QDs (C) at 37 °C for 30 min respectively. (D) Fluorescence positive percentage of the cells under different labeling conditions. Triple asterisks, P < 0.001. The scale bar represents 20 µm in panels A-C.



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

Figure 2. TAT-QDs were trapped into vesicles and transported to the perinuclear region of cells. The QDs-labeled cells were stained with DiO dye. Fluorescence images of DiO dye (A), TAT-QDs (B), and the merge image (C) are displayed. (D) Overlapped orthogonal slice view demonstrated the TAT-QDs were trapped into vesicles. (E) Snapshots showed TAT-QDs were merged together by vesicles fusion. Fluorescence images of Hoechst 33342 (blue), DiO dye (green), TAT-QDs (red) and the merge image are displayed. The white rectangular regions were magnified to better visualize the fusion process. The scale bars represent 10 µm in A-D, and 5µm in panel E. The time format is hours: minutes: seconds.


Page 12 of 17

Page 13 of 17

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


Figure 3. TAT-QDs were transported in the cytoplasm via mitochondria, Golgi apparatus and lysosomes. The QDs-labeled HCCLM9 cells were stained with Mito-tracker green dye, Golgi tracker Red and Lyso-tracker green. Fluorescence images of mitochondria (A), TAT-QDs (QD605) (B), and the merge image (C) are displayed. Fluorescence images of Golgi apparatus (D), TAT-QDs (QD525) (E), and the merge image (F) are displayed. Fluorescence images of lysosomes (G), TAT-QDs (QD605) (H), and the merge image (I) are displayed. The scale bar represents 10 µm in panels A-I.



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

Figure 4. Fluorescent intensity of TAT-QDs decreased as the culture time went by. The cells labeled with TAT-QDs were cultured for 0 h (A), 24 h (B) and 48 h (C), respectively. (D) Normalized fluorescence intensity of CFDA SE and TAT-QDs in living cells with different culture time. Single asterisk, P < 0.05, and double asterisks, P < 0.01. The scale bar represents 20 µm in panels A-C.


Page 14 of 17

Page 15 of 17

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


Figure 5. Fluorescence stability of QDs was influenced by low pH. Fluorescence intensity vs. time plots (A) and fluorescence spectra (B) of TAT-QDs under different pH values. (C) The cells incubated with TAT-QDs were analyzed by flow cytometry to evaluate the fluorescence of QDs under different culture condition. (D) The fluorescence positive percentage of cells incubated with TAT-QDs under different culture condition is displayed. Double asterisks, P < 0.01, and triple asterisks, P < 0.001. (E) Snapshots showed the fluorescence decay of TAT-QDs in lysosomes. Fluorescence images of lysosomes (green), TAT-QDs (red) and the merge image are displayed. The white circle regions in middle line of (E) were magnified to better visualize the fluorescence decay of QDs. The scale bar represents 10 µm in panel E. The time format is hours: minutes: seconds.



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

Figure 6. Content of cadmium in extracellular region and whole cells showed little change within 72 h. (A) The sketch showed the detection of cadmium in extracellular region and whole cells. (B) The normalized content of cadmium in extracellular region and whole cells at different culture time is displayed.


Page 16 of 17

Page 17 of 17

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


For Table of Contents Only


Dissecting the Factors Affecting the Fluorescence Stability of Quantum

Recommend Documents