Letter www.acsami.org
Living Cell Multilifetime Encoding Based on Lifetime-Tunable LatticeStrained Quantum Dots Li Zhang,† Chi Chen,† Wenjun Li, Guanhui Gao, Ping Gong, and Lintao Cai* Guangdong Key Laboratory of Nanomedicine, CAS Key Laboratory of Health Informatics, Shenzhen Bioactive Materials Engineering Lab for Medicine, Institute of Biomedicine and Biotechnology. Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, P. R. China S Supporting Information *
ABSTRACT: A series of functional quantum dots (QDs) with widely tunable nearinfrared fluorescence emission (620−750 nm) and lifetime (30−160 ns) were synthesized via lattice strain and showed excellent photo, colloid, pH, and lifetime stabilities. The welldefined targeting QDs were first developed for a living cell multilifetime encoding strategy to track and recognize specified tumor cell clusters dependent on lifetime distribution using fluorescence lifetime imaging microscopy.
KEYWORDS: quantum dots, fluorescence lifetime imaging, codes, living cells tracking, near-infrared-emitting
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encoding is very challenging for living cells imaging and tracking. Although fluorescence imaging using confocal laser scanning microscope has been widely developed for live-cells and longterm tracking,11 fluorescence lifetime-based encoding attracted much attention due to its unique properties that lifetime is independence of excitation light intensity and probe concentration.12−15 In our previous work, NIR-emitting QDs with long lifetime can be achieved through doping Cu2+ in host nanocrystals. Moreover, as-prepared QDs was used to fabricate two-dimensional codes based on multiemission and multilifetime, which demonstrated that fluorescence lifetime of QDs can be regarded as an excellent encoded parameter.15 However, Cu2+-doped QDs always have a second order of fluorescence lifetime, as the cocontribution from the band edge emission of host QDs and trap emission from dopants,15,16 which partly undermine and disturb the encoding effectiveness. Therefore, a robust and facile alternative for tunable lifetime should be highly desirable. Generally, fluorescence lifetime of QDs changes along with the increasing of particle size.17 Remarkably, compared with type I nanostructures, type II QDs have a lower band gap energy and longer excited-state lifetime.18,19 Interestingly, if QDs have a large mismatch between the core and shell crystal structures, and the thickness of the shell is much more than the
he unique identification and tracking of specific tumor cells in various populations and targeted living tumor cells for improved diagnosis and therapy is highly desirable and requires new research and development approaches.1 Despite the considerable requirement and expectation in various fields of life science, distinguishing one type of tumor cells and cell clusters in various complex cell environments is a great challenge. Quantum dots (QDs) are ideal precursors for fabricating encoding for biomedical diagnosis2 owing to their unique optical properties including high quantum yield, high molar extinction coefficients, broad absorption with narrow and symmetric photoluminescence spectra, large effective Stokes shifts, and exceptional resistance to photobleaching.3−6 By designing component and electron energy band structure, the QDs fluorescence emission spectrum can be tuned from ultraviolet (UV) to near-infrared (NIR) spectral ranges. Compared to the visible region, the NIR region is well-suited for bioimaging applications. For instance, ultrasmall-sized NIRemitting QDs offered great opportunities for in vitro and in vivo high-efficiency and high-sensitivity imaging applications.7,8 However, the full-width at half-maximum (fwhm) of NIRemitting QDs is consistently between 40 and 90 nm,9 which makes the NIR-emitting encoding exhibit limited capacity due to the overlay of emission spectra. Although high-quality core− shell CdSe/CdS QDs with narrow emission line widths (fwhm ∼20 nm) has been achieved through a slow growth rate of the shell, the photoluminescence still emit in the visible region, not NIR region.10 Thus, NIR-emitting QDs-based multiplex © XXXX American Chemical Society
Received: March 30, 2016 Accepted: May 18, 2016
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DOI: 10.1021/acsami.6b03795 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
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ACS Applied Materials & Interfaces
670) with high photoluminescence quantum yield (PL QY) (50−70%) were selected and fabricated multilifetime-based targeted living cells encoding. The principle of NIR-emitting living cells multilifetime encoding was illustrated in Scheme 1b, the relative intensity ratios at different lifetime were used for encoding purpose. For instance, an equal mixture of CPP30-LSQDs-670 and CPP30-LS-QDs-742 codes (101), an equal mixture of CPP30-LS-QDs-670 and CPP30-LS-QDs-700 codes (110), a mixture of CPP30-LS-QDs-700 and CPP30-LS-QDs742 with molar ratio of 1:2 codes (012). Furthermore, the targeted MCF-7 living cells encoding was demonstrated by incubating as-prepared CPP30-LS-QDs with the cocultured MCF-7 and C6 cells. To emphasize the lifetime-tunable advantage of LS-QDs, we investigated the optical properties of nonlattice-strained QDs and CPP30-functionalized LS-QDs by UV−vis spectrophotometer and fluorophotometer. According to Figure 1a, b, with increasing particle size, the fluorescence emission spectra of these two types of QDs were similarly red-shift from visible region to NIR region. However, the time-resolve decay curves of these two types of QDs were obviously different. It was clear that there was a significant increase in excited state lifetimes of LS-QDs. (Figure 1c, more details in Table S1 and S2) These
small core, the type I nanostructure can transform into type II nanostructure because of the strong quantum confinement effect and compressive strain in the core and the tensile strain in the shell.9 Thus, the strategy of lattice strain can be adopted to obtain lifetime-tunable QDs. As previously reported, CPP30 was a novel cell-penetrating peptide (CPP), obtained by mRNA display technology from MCF-7 cell line, which mediated by a dynamin-dependent and clatrin-independent endocytic pathway.20 The polyethylene glycol (PEG) may contribute to keeping the peptide from wrapping around the QDs surface as surrounding layer, and hexahistidine (His6) sequence is assumed to be in contact with the QDs surface and does not contribute to lateral extension.21 In this study, we prepared CPP30 functionalized QDs with widely tunable fluorescence emission (620−750 nm) and lifetime (30−160 ns) via lattice strain. Furthermore, as-prepared NIR-emitting QDs for living cells targeted (MCF-7) multilifetime encoding has been demonstrated by using fluorescence lifetime imaging microscopy (FLIM). Briefly, as shown in Scheme 1a, seed of latticeScheme 1. (a) Scheme for Synthesis of CPP30-LS-QDs for MCF-7 Cell Tracking (up) and Diagrams of Band Offsets (CB = conduction band, VB = valence band) for CdTe Ultrasmallcore, CdTe/CdS Core/Shell QDs, and LatticeStrained CdTe/CdS Core/Shell QDs (bottom); (b) Principle of NIR-Emitting Living Cells Multilifetime Encoding Based on Lifetime Distributiona
a
Large irregular shapes represent MCF-7 cells, in which small colored spheres represent QDs (red, CPP30-LS-QDs-742; green, CPP30-LSQDs-700; blue, CPP30-LS-QDs-670; the numbers of colored spheres (red, green, and blue) do not represent individual QDs, but are used to illustrate the fluorescence intensity levels.).
strained QDs (LS-QDs) was synthesized through slow growth of CdS shell on CdTe ultrasmall-core (ca. 2.1 nm Figure S1), then the CPP30 functionalized LS-QDs (CPP30-LS-QDs) was achieved by adding CPP30-PEG3-(His6-tag) in the process of capping CdS shell, which made it possesses the targeted MCF-7 cells. Then, the as-prepared CPP30 functionalized LS-QDs (CPP30-LS-QDs-742, CPP30-LS-QDs-700, CPP30-LS-QDs-
Figure 1. (a) Absorption spectra, (b) photoluminescence spectra, and (c) time-resolved fluorescence decay curves of CPP30-functionalized lattice-strained QDs (left) and nonlattice-strained QDs (right). B
DOI: 10.1021/acsami.6b03795 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
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ACS Applied Materials & Interfaces
cytotoxicities of LS-QDs, TAT-LS-QDs and CPP30-LS-QDs were tested with MCF-7 cells using Cell Counting Kit-8 (CCK8) assay. There were negligible effects on cell viabilities after 24 h incubation with three different types of QDs under various concentrations (up to 50 μg/mL). (Figure S8a) Besides, the average lifetime of three different types of QDs also showed negligible response in the absence and the presence of potential intracellular interfering biomolecules (BSA and glucose) and ions (HCO3−, K+, Mg2+, Mn2+). (Figure S8b) Thus, asprepared QDs can be an excellent precursor for living cells multilifetime encoding. To demonstrate the cellular uptake and specific tumor cells imaging, we performed confocal laser scanning microscope. MCF-7, HeLa, C6 and BEND3 cells were incubated with CPP30-LS-QDs-700, TAT-LS-QDs-620, and LS-QDs-700 solution for 2 h. As showed in Figure 2, compared to other
changes were caused by the spatial separation of holes into the core and electrons into the shell, as the result of the formation of Type II nanostructure, suggested by group of Nie9 and Liu.22 It was worth mentioning that LS-QDs also had a multiexponential decay, as the cocontribution from the band edge emission and surface state. However, the shorter lifetime only caused a slight decrease in average lifetime due to less contribution. To further clarify the morphology, composition, and structure of as-prepared QDs, we employed transmission electron microscopy (TEM), X-ray photoelectron spectroscopy (XPS), and powder X-ray diffraction (XRD). According to Figure S2, the mean diameter of CPP30-LS-QDs-670 was 5.49 ± 0.29 nm according to TEM image, and the morphology was nearly tetrahedral, being consistent with previous report.22 Moreover, the lattice fringes and ultrasmall core can be identified in the HRTEM image. XPS data further demonstrated the core/shell nanostructure. Photo energies of XPS can only analyze the certain depths elements from surface.23 In CdTe ultrasmall core and nonlattice-strained CdTe QDs-726, the characteristic peaks of Te 3d level can be clearly seen, but in CPP30-LS-QDs-742, the signal of Te 3d level visibly diminished. Meanwhile, the characteristic peaks of Cd 3d level in all samples were both distinct, indicating that Te ions were far from the surface and located in an ultrasmall core. It should be noted that the oxidation of CdTe QDs powder leaded to the formation of a tellurium oxide peak at higher energies than the main Te peak (Figure S3). XRD data (Figure S4) showed that as-prepared QDs were elucidated to be zincblende dominated structures. With increasing of shell thickness, the diffraction peak of CPP30-LS-QDs-742 was close to pure CdS phase. To confirm the as-prepared QDs can be successfully modified with the CPP30 through the interaction between His6-tag and Cd2+, we designed the sequences of biotin-PEG3(His6-tag) and TAT-PEG3-(His6-tag) (TAT peptide is a type of CPP with high solubility, diverse target range and nonselective internalization, which has been widely utilized.24) ) and used them to functionalize the as-prepared QDs. As shown in Figure S5, almost all of streptavidin microbeads were labeled with biotin functionalized LS-QDs and exhibited fluorescence, but no fluorescence was found on the microbeads mixed with QDs without biotin modification, which suggested the peptide sequences with His6-tag can be effectively functionalized QDs. Moreover, the hydrodynamic size increased from 6.52 ± 1.41 to 7.81 ± 1.73/10.12 ± 2.07 nm after LS-QDs-670 functionalized with TAT/CPP30-terminated peptide according to dynamic light scattering (DLS) measurements (Figure S6). To explore the potential application of as-prepared QDs for living cells multilifetime encoding, the photo, colloid, lifetime, and pH stabilities of LS-QDs, TAT-LS-QDs, and CPP30-LSQDs were evaluated, respectively. Little photobleaching was observed after continuously exciting the as-prepared QDs with a 400 nm laser for 120 min, and up to 98% of photoluminescence was preserved after one month incubation. Meanwhile, the fluorescence lifetime was nearly unchanged. More importantly, the fluorescence lifetime of QDs was also nearly invariant in buffers with different pH values (from 3.0 to 9.0), which means they are highly stable for living cells multilifetime encoding. (Figure S7) Additionally, two most problematic aspects as ideal precursors for living cells encoding are the cytotoxicity and the extremely crowded media. The
Figure 2. Cellular uptake and specific tumor cell imaging of CPP30LS-QDs-700 and TAT-LS-QDs-620 (scale bar, 25 μm).
cells, MCF-7 cells presented significantly strong red color in cytomembrane, indicating that CPP30-LS-QDs-700 could identify MCF-7 cells specifically. On the contrary, TAT-LSQDs-620 penetrated all the cells and presented blue color dramatically. But LS-QDs-700 without peptide modification can label no cells (Figure S9). This result can also further prove that the His6-tag could successfully attach the peptides to the QD surfaces. It should be noted that Figure 2 also showed uptake of C6 cells, it may be because the C6 cells have the same endocytic pathway, but much less than MCF-7 cells. To exhibit the as-prepared CPP30-LS-QDs using for living cells multilifetime encoding, we incubated first of all, MCF-7 cells with CPP30-LS-QDs-742, CPP30-LS-QDs-700, and CPP30-LS-QDs-670, and the lifetime of each CPP30-LS-QDs was 136 ± 1 ns, 102 ± 2 ns, and 63 ± 3 ns according to FLIM, respectively (Figure S10). Moreover, these lifetimes were defined as red (R), green (G), and blue (B), respectively. It should be noted that the lifetime obtained from FLIM was shorter than that from spectrofluorometry (The lifetime of CPP30-LS-QDs-742, CPP30-LS-QDs-700, and CPP30-LSC
DOI: 10.1021/acsami.6b03795 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
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ACS Applied Materials & Interfaces QDs-670 was 161 ± 1 ns, 139 ± 1 ns, and 61 ± 1 ns, respectively), which may be due to the different detection range. Then, for fabricating and recognizing the lifetime-based encoding, these three types of CPP30-LS-QDs with different molar ratio were incubated with MCF-7 cells. FLIM images and lifetime distribution of MCF-7 cells were presented in Figure 3,
experiment was carried out according to the protocol as described before. As showed in Figure 4, in despite of some
Figure 4. Specific living cells tracking of code-(120). Bright field (left), FLIM (middle), and lifetime distribution (right) (inset, unmixed FLIM image; pseudocolored green, CPP30-LS-QDs-700; pseudocolored blue, CPP30-LS-QDs-670; scale bar, 20 μm).
fluorescence signal from the C6 cells, the uptake is much more efficient for MCF-7 cells. It means that CPP30-LS-QDs have higher specificity to indentify and track MCF-7 cells in various population, and successfully fabricate the specific living cell encoding based on multilifetime and lifetime distribution. In conclusion, we synthesized CPP30-functionalized NIRemitting LS-QDs with widely tunable fluorescence emission (620−750 nm) and lifetime (30−160 ns) via lattice strain, which could uniquely identify and track MCF-7 cells in different cells mixture. Furthermore, the lifetime-tunable QDs were first-time used for fabricating of specific living cells encoding based on multilifetime and relative intensity. The asprepared QDs could also be used for cellular imaging and, in particular, for multiplexed detection using the different fluorescence lifetimes of the QDs to produce distinguishable optical codes. It will provide an alternative strategy for tracking and distinguishing one specified tumor cells in various complex cell environments.
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ASSOCIATED CONTENT
* Supporting Information S
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.6b03795. Details on the experiment process of synthesis and characterization of lifetime-tunable lattice-strained quantum dots and living cells multilifetime encoding; Figures S1−S10 and Tables S1 and S2 (PDF)
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected].
Figure 3. FLIM image (up) and lifetime distribution (bottom) of each NIR-QDs multilifetime encoded MCF-7 cells. ((a) Code-(101), (b) Code-(102), (c) Code-(104), (d) Code-(110), (e) Code-(120), (f) Code-(140), (g) Code-(012), (h) Code-(041), (i) Code-(061); inset, unmixed FLIM image; pseudocolored red, CPP30-LS-QDs-742; pseudocolored green, CPP30-LS-QDs-700; pseudocolored blue, CPP30-LS-QDs-670; scale bar, 30 μm).
Author Contributions †
L.Z. and C.C. contributed equally. C.C. conceived and designed the study, L.Z. performed the main experiments, C.C. and L.Z. analyzed the data, prepared all figures, and wrote the manuscript. L.C. guided and supervised the project. All authors discussed the results and commented on the manuscript.
defined pseudocolor of three different CPP30-LS-QDs can be clearly identified in MCF-7 cells. Here we showed nine representative codes, noteworthily based on our design we can achieve more RGB codes with RGB brightness (relative intensity ratios at different lifetime). Finally, to present the process of specific living cells multilifetime encoding, MCF-7 and C6 cells were cocultured in the same plate overnight, then incubated with well-defined mixed solution code-(120), FLIM
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The presented research was financially supported by the National Natural Science Foundation of China (Grant 51502333), the Research Foundation of Chinese Academy of D
DOI: 10.1021/acsami.6b03795 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
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Sciences (yz201439), the Guangdong Science and Technology Program (Grant 2012A061400013), the Shenzhen Science and Technology Program (Grant KQCX20140521115045447), SIAT Innovation Program for Excellent Young Researchers (201412), and Guangdong Innovation Research Team of Lowcost Healthcare.
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