ZnS Quantum Dot

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Multifunctional Polymer ligand Interface CdZnSeS/ZnS Quantum Dot/Cy3-labeled Protein pairs as sensitive FRET sensors Hong Yu Yang, Yan Fu, Moon-Sun Jang, Yi Li, Jung Hee Lee, Heeyeop Chae, and Doo Sung Lee ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b12877 • Publication Date (Web): 08 Dec 2016 Downloaded from http://pubs.acs.org on December 15, 2016

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ACS Applied Materials & Interfaces

Multifunctional Polymer Ligand Interface CdZnSeS/ZnS Quantum Dot/Cy3-labeled Protein Pairs as Sensitive FRET Sensors Hong Yu Yang†,1, Yan Fu§,1, Moon-Sun Jang‡ , Yi Li†, Jung Hee Lee‡, Heeyeop Chae§,#,* and Doo Sung Lee†,*

†

Theranostic Macromolecules Research Center, School of Chemical Engineering,

Sungkyunkwan University, Suwon 440-746, Republic of Korea §

School of Chemical Engineering, Sungkyunkwan University, Suwon 440-746,

Republic of Korea ‡

Department of Radiology, Samsung Medical Center, Sungkyunkwan University

School of Medicine and Center for Molecular and Cellular Imaging, Samsung Biomedical Research Institute, Seoul 135-710, Republic of Korea #

Sungkyunkwan Advanced Institute of Nanotechnology (SAINT), Sungkyunkwan

University (SKKU), Suwon, 440-746, Republic of Korea

Keywords：： CdZnSeS/ZnS QDs, QDs-based FRET, clofazimine, sensor, in vivo imaging 1

ACS Paragon Plus Environment


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ABSTRACT High-quality CdZnSeS/ZnS alloyed core/thick-shell quantum dots (QDs) as energy donors were first exploited in Förster resonance energy transfer (FRET) applications. A highly efficient ligand-exchange method was used to prepare low toxicity, high quantum yield, stabile and biocompatible CdZnSeS/ZnS QDs densely capped with multifunctional polymer ligands containing dihydrolipoic acid (DHLA). The resulting QDs can be applied to construct QDs-based Förster resonance energy transfer (FRET) systems by their high affinity interaction with dye cyanine 3 (Cy3)-labeled human serum albumin (HSA). This QD-based FRET protein complex can serve as a sensitive sensor for probing the interaction of clofazimine with proteins using fluorescence spectroscopic techniques. The ability of FRET imaging both in vitro and in vivo not only reveals that the current FRET system can remain intact for 2 h but also confirms the potential of the FRET system to act as a nanocarrier for intracellular protein delivery or to serve as an imaging probe for cancer diagnosis.

2


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INTRODUCTION The development of a novel, sensitive and multifunctional sensor is one of the most important challenges in fluorescence-based techniques for the use in bio-diagnostics, protease detection assays, real-time cell monitoring and in vivo imaging.1-4 Specifically, quantum dot (QD) Förster resonance energy transfer-based (QD-based FRET) nanosensors have become an important tool to overcome a broad range of biomedical issues because these sensors not only exhibit spectroscopic ruling ability but also show the excellent photo and chemical stability of QDs.5-7 Recently, increasing attention has been paid to the use of QD-based FRET sensors to probe specific interactions of protein with ligands, which are important in the understanding of many biological events, such as the detection of analytes, immunoassays, viral infections and protein delivery.8-12 However, the development of high quantum yield (QY), highly stable, biocompatible, monodisperse and densely packed polymer ligand-capped QDs remains a great challenge and is necessary for protein binding and to develop a sensitive FRET sensor. To address these issues, alloyed core/thick-shell CdZnSeS/ZnS QDs as an energy donor was first introduced in FRET system because the ZnS thick shell of the cladding with nearly no defects that comprises ZnS shell-coated QDs has lower toxicity effect to cells, a narrow full-width at half-maximum (FWHM) and a greater photoluminescence (PL) QY (≥ 95%). Therefore, the use these QDs can greatly enhance the safety and sensitivity of a FRET system.13 Hydrophilic, biocompatible CdZnSeS/ZnS QDs have been developed using densely packed multifunctional polypeptide-based polymer ligand containing

3



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dihydrolipoic acid (DHLA), which serves as the precursor for sensing and imaging applications14 and provides a greater positively charged outer surface that results in a stronger affinity for the negatively charged proteins, siRNA or special receptors. Moreover, the addition of poly(ethylene glycol) as a hydrophilic shell effectively resists uptake by organs of the reticuloendothelial system (RES) and imposes stability and solubility.15 Organic molecular dyes and dye-labeled proteins as energy acceptors (A) have been widely used in a variety of QD-based FRET biophysical applications.16-19 As a rule, efficient FRET not only depends on the existence of spectral overlap between the donor and acceptor and the relative orientation of the donor-acceptor pairs but also strongly relies on the distance (r) between a QD donor and a proximal acceptor (typically less than 100 Å).20-21 Therefore, a major use of FRET is based on its distance-dependent properties, which can be applied for studying the association and dissociation of complex systems or the conformation change of a molecular because both result in changes to the distance (r) between the donor and acceptor,22-23 Therefore, this feature is used to further explore special interactions, such as multivalent interactions of immunodeficiency viruses and cell pathogen receptors24, the condensation and decondensation of plasmid DNA25, and the detection of protease activities 26-27 and even the detection special analytes.28 As the major carrier protein of blood plasma, human serum albumin (HSA) has been reported as a model protein for biophysical and physicochemical studies.29-30 HSA plays a key role in osmotic blood pressure and transports and distributes both 4


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endogenous and exogenous ligands, including metal cations, amino acids, and fatty acids.31 Drugs as other exogenous ligands, are transported in the circulatory system in the presence of a high concentration of HSA, and are known to have a high affinity for HSA. The interaction of drugs with plasma proteins has a significant influence on the drug absorption, distribution, and toxicity of drugs. Therefore, the development of a sensitive analytical tool for probing the interaction of drugs with protein will be crucial in addressing the future emerging challenges in the biomedical field. Clofazimine is a drug that is an effective treatment for multidrug-resistant tuberculosis and breast cancer; even in deadly cases,32-33 there has been an increasing interest in studying the interaction between clofazimine and HSA. However, few people have paid attention to the use of QD-based FRET sensors to investigate the conformational behavior of HSA changed by clofazimine. In this study, Cy3-labeled HSA was used as an acceptor to prepare QD-based FRET/Cy3-HSA complexes that acted as sensitive sensors for probing the interaction of clofazimine with HSA. The changes in the PL intensity of the QD donor were used to determine the protein conformational changes induced by clofazimine. This work has important significance in the application of this biosensor for drug transport, excretion and even in terms of therapeutic efficiency. We show that QD-based FRET/Cy3-HSA complexes serve as high sensitivity sensors for studying the interaction between HSA and clofazimine, and we also investigated the behavior of the FRET sensor in vitro and in vivo. Importantly, as expected, this is a complex system that may have more potential applications in 5



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intracellular protein delivery and in vivo imaging due to its excellent stability in biological medium and efficient cellular uptake. Moreover, we can design different types of FRET systems in a flexible and selective manner using similar strategies against various protein-ligand interactions, which will expand the application of this FRET system in the real -world. EXPERIMENTAL SECTION Materials. Cadmium acetate (Cd(acet)2, 99.9%), selenium (Se, 99.9%), zinc oxide [ZnO, 99.99%], Zn acetate dehydrate (Zn(CH3COO)2 · 2H2O), selenium (Se, 99.9%), sulfur (S, 99.9%), 1-octadecene (ODE, 90%), trioctylphosphine (TOP, 90%), oleic acid (OA, 90%), human serum albumin (HSA, fatty acid-free, Mn ≈ 66,500), L-glutamic

acid γ-benzyl ester, 1,1′-Carbonyldiimidazole (CDI, 99%), oleic acid (OA,

90%), lipoic acid (LA, 98%), 2-hydroxypyridine and anhydrous solvents including tetrahydrofuran (THF, ≥99.9%), chloroform (≥99.9%), N,N-dimethylformamide (DMF, ≥99.8%), and dimethyl sulfoxide (DMSO, ≥99%) were purchased from Sigma-Aldrich. Triphosgene and diethylenetriamine were obtained from TCI Co., Ltd. (Tokyo, Japan). Cyanine3 (Cy3) NHS ester, hexane, diethyl ether, ethanol and methoxy-ω-amino-poly (ethylene glycol) (mPEG-NH2, Mn = 5000) were obtained from Samchun Co., Ltd. and SunBio Co., Ltd (Korea). γ -benzyl L-glutamate N-carboxyanhydride (BLG-NCA) was synthesized according to the process described in our previous work.34

Synthesis

of

high-quality

CdZnSeS/ZnS

QDs.

Alloyed

core/thick-shell

6


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CdZnSeS/ZnS QDs were prepared according to the approach by Yang et al. with slight modifications.35 Briefly, 0.14 mmol of Cd(acet)2, 3.14 mmol of ZnO and 7 ml of OA were mixed in a three-neck flask, and the reaction was stirred at 150 °C for 30 min in a nitrogen (N2) environment. Then, 15 mL of 1-octadecene was injected as a non-coordinating solvent into the reaction at 150 °C and heated to 300 °C to form the cation precursor of Cd(OA) 2 and Zn(OA)2. A 2 mL of TOP containing Se (1.5 mmol) and S (2.5 mmol) was quickly added into the flask. Subsequently, the reaction was continued for 10 min to obtain alloyed core CdZnSeS QDs. A high-quality CdZnSeS/ZnS QDs was synthesized by growing the ZnS shell on the surface of the CdZnSeS QDs as a core based on the following process. A 2.8 ml of ODE in presences of S (1.8 mmol) was swiftly injected into the reactor and held for 12 min at 310 °C. Then, the mixture of OA and ODE with 2.86 mmol of Zn acetate dehydrate was added, and the temperature set at 270 °C; then, a 5 ml of TOP containing 10 mmol of S was added drop-wise for 10 min and held for 20 min to grow the ZnS shell. Finally, CdZnSeS/ZnS QDs were centrifuged using ethanol at 10,000 rpm, for 10 min. The final QDs were redispersed in chloroform at a concentration of 10 µM. The average particle sizes and morphology of CdZnSeS/ZnS QDs was determined by using a Zetasizer Nano series, ZS90 (Malvern Instrument) dynamic light scattering (DLS) and a high-resolution transmission electron microscope (HRTEM, JEM-ARM 200F) at 120 kV, respectively.

Synthesis

of

Polymer

ligand

backbone.

mPEG

functionalized 7



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poly(benzyl-L-glutamate) (abbreviated as mPEG-b-PBLG), which was the polymer ligand backbone, was prepared using the ring opening polymerization (ROP) of BLG-NCA in anhydrous chloroform at 27 °C for 72 hours under N2 using mPEG-NH2 as the macroinitiator at 40:1 molar ratio of the monomer to the initiator. After the reaction, the mixture was poured into a large amount of ether. Finally, the precipitated ligand was dried under vacuum for 24 h; the yield was 90%. Synthesis of mPEG functionalized poly( diethylenetriamine- dihydrolipoic acid-L-glutamate](mPEG-b-P(DA-DHLA)LG) polymer ligands. First, mPEG functionalized poly(diethylenetriamine)-L-glutamate (mPEG-b-PDALG) as a cationic polymer ligand was prepared using an aminolysis reaction. Briefly, mPEG-b-PBLG (1 g, 0.073 mmol) and 2-hydroxypyridine (0.833 g, 8.76 mmol) as a bifunctional catalyst were dissolved in 10 mL of DMF and stirred at 55 °C. Subsequently, 5 mL of diethylenetriamine were injected, and the reaction was vigorously stirred for 72 h under N2. The mixture solution was then precipitated in excess cold ether. The precipitate was dissolved in a 0.05 M HCl solution and dialyzed using the same amount of deionized water (DI water) (MWCO = 3500 Da) for 3 days. The final product was obtained after lyophilization with a yield of 73%. Second, lipoic acid (30 mg, 1.46 × 10-4 mol, 0.5 quivalent to total primary amine) and CDI (26 mg, 1.55 × 10-4 mol) were mixed in 10 mL of DMSO, and the reaction was carried out in purity N2 for 2 h in the dark. Afterward, 20 mL of DMSO containing mPEG-b-PDALG (200 mg) was injected into the above reaction and continuously stirred for 24 h. The resulting polymer was purified through dialysis for 3 days using a dialysis membrane 8


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(MWCO = 3500 Da) in DI water and was then lyophilized in the dark with a yield of 90%. Finally, the multifunctional polymer ligand mPEG-b-P(DA-DHLA)LG was obtained through reduction of the dithiolane group to dihydrolipoic acid (DHLA) in presence of NaBH4 reductant. Briefly, 200 mg of the mPEG-b-P(DA-LA)LG polymer ligand was dissolved in 30 mL of a 1 mg/mL NaBH4 aqueous solution and was stirred for 24 h under N2 in the dark. Then, the pH value of the precursor solution was adjusted to pH 7-8 with a 1 M HCl solution. The finally polymer ligand was purified by centrifugation (10000 rpm, 2 h) using an Amicon Ultra-15 centrifugal filter unit with a MWCO of 3.0 kDa with repeated DI water washing eight to ten times, the product was then lyophilized; the yield was 85%. Polymer

Ligand

Exchange

Process.

mPEG-b-P(DA-DHLA)LG-capped

CdZnSeS/ZnS QDs (abbreviated as QDs-PEG-PDDLG) was prepared via a slightly modified

ligand

exchange

(cap-exchange).36

Briefly,

100

mg

of

the

mPEG-b-P(DA-DHLA)LG polymer ligands were firstly dissolved in 7 mL DMSO and 3 mL CHCl3 dispersion of hydrophobic QDs was added. The mixture solution was then treated with a sonifier (40 min, setting: 20% amplitude, 6 s on and 2 s off). Subsequently, chloroform was removed by evaporation under vacuum, and the DMSO was completely substituted with deionized water by a dialysis membrane (MWCO: 3500 Da). The final solution was poured into centrifugal filter (Millipore, MWCO: 30,000 Da) and was centrifuged in DI water for 10 min at 10000 rpm to detach the displaced native ligands (TOP and OA) and residual polymer ligands. This process was repeated three times. The QD-PEG-PDDLG was obtained as an aqua powder 9



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after lyophilization and was redispersed in DI water for further studies. The average particle sizes and morphology of the QD-PEG-PDDLG were determined by DLS and FEI Tecnai F20 cryogenic transmission electron microscopy (Cryo-TEM) at 200 kV, respectively. Cy3 Labeling on human serum albumin (Cy3-HSA). To label with HSA, 500 mg of HSA and 20 mL of a PBS solution (pH 8.0) containing 5 mg of Cy3-NHS were stirred for 6 h at RT in the dark. The final solution was then dialyzed (cutoff: 3500 Da) against 10 mM PBS to remove the free Cy3-NHS. The Cy3-labeled HSA was obtained via lyophilization and redispersed in buffer to calculate the concentration of Cy3 labeling with HSA using a V-630 Bio UV−vis spectrophotometer at 548 nm. Complex

formation

between

QD-PEG-PDDLG

and

Cy3-HSA

(QD-PEG-PDDLG/Cy3-HSA). The appropriate amount of Cy3-HSA was added to a solution of QD-PEG-PDDLG (20 pmol) in PBS buffer (10 mM, pH 7.4) and was stirred for 2 h at RT in the dark to promote to formation the complex system. The molar ratios between Cy3 and QDs were discretely varied from 0 to 10, and the final product

was

stored

it

in

4

°C.

The

electrophoretic

mobility

of

the

QD-PEG-PDDLG/Cy3-HSA complex was be determined by a Zetasizer Nano series ZS90 (Malvern Instrument Inc., Worestershire, UK). Measurements of Fluorescence Lifetime. Time-resolved photoluminescence decay traces of the QDs were measured with a time-correlated single-photo counting spectrofluorometer (TCSPC, Horiba Jobin–Yvon FluoroLog 3). Briefly, the samples were excited using a pulsed laser with a 1 MHz repetition rate at 405 nm. The 10


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fluorescence decay was collected after passing through a 455 nm filter and emission was detected at 520 nm using the magic angle (54.7°) with the instrument response functions (IRFs ≈ 200 ps). Fluorescence resonance energy transfer between CdZnSeS/ZnS QD and Cy3 in the complex system. A steady-state fluorescence spectra was recorded using a FP-6200 spectrofluorometer, and the absorption spectra were collected from a V-630 Bio UV−vis spectrophotometer. The spectral overlap integral (J) between the CdZnSeS/ZnS QD donor and the Cy3 acceptor dye in units of cm3.M-1 can be defined as 37-38

I = J λ) = PL λ) ε λ)λ dλ (1) where I is the integral of the donor–acceptor spectral overlap over all wavelengths λ (cm), PLD-corr is the normalized fluorescence intensity of the QD donor and εA is the absorption extinction coefficient of the acceptor. The Förster distance (R0) is the separation distance of the donor (QD) to the acceptor at which E is 50%. R0 can be calculated using the following equation: R =

) !"#$ % &'

()/*

(2)

where nD is the refractive index of the sample medium, Avogadro’s number NA and QD is the fluorescence quantum yield (QY) of the donor [using Rhodamine 6G in ethanol as a reference standard]. We assumed that the fluorophores were randomly orientated such that the value of the orientation factor kp2 can be set to 2/3. I is the integral of the spectral overlap. The quantitative FRET efficiency E was measured by steady-state or the 11



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measurements of excited-state lifetime according to Eq. (3) E= 1−

. ' .

or E = 1 −

/ ' /

(3)

where FDA and FD are the fluorescence intensities of the donor in contact with acceptor or in isolation from acceptor; similarly, τD and τDA were designated excited state lifetime of the donor in the absence and presence of the acceptors The values of the donor to the acceptor separation distance (r) can be determined by the FRET efficiency using formula (4); the dye–acceptors are arrayed around a central QD donor so a centro-symmetric distribution was assumed. (where n is the ratio and number of dye acceptors per QD donor)

01 = R 2

3) /* 3

4

(4)

In vitro physiological stability of QD-PEG-PDDLG/Cy3-HSA. (1) The hydrodynamic particle size stability of QD-PEG-PDDLG/Cy3-HSA (mole ratio of Cy3/QD was 4:1, the final concentration of QD ~50 nM) incubation in different mediums, including, PBS buffer (pH 7.4), 0.5 M NaCl solution, DI water (pH 5.5), GSH (10 mM), RPMI 1640 (cell growth media) and RPMI (containing bovine serum and nutrient) at 37 °C were monitored by DLS at different time points. (2) The FRET stability of QD-PEG-PDDLG/Cy3-HSA incubation in FBS (Fetal Bovine Serum) at 37 °C with shaking was tested by a FP-6200 spectrofluorometer. PL intensity of QD donor and Cy3 acceptor were recorded using an excitation wavelength at 410 nm at predetermined time points. We defined ICy3-A/( IQD-D+ ICy3-A) as FRET ratio, where 12


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ICy3-A and IQD-D are the PL intensities of QD donor and Cy3 acceptor at 528 nm and 580 nm, respectively. The assay of QD-PEG-PDDLG/Cy3-HSA FRET sensor for sensing clofazimine. For the complex assay of the drug, different concentrations of clofazimine solution were first prepared with concentrations ranging from 20 µM to 1 mM by weight/volume (w/v) in a mixture solution of DI water and alcohol. Then, the different

solutions

were

treated

with

the

equal

amount

of

the

QD-PEG-PDDLG/Cy3-HSA sensor (mole ratio of Cy3/QD was 2:1 where the PL intensity of QD donor was recorded as I0), and then the solutions are shaken for 2 h at room temperature. The fluorescence spectra of each solution were collected using a 405 nm excitation and the changes in QD donor PL were recorded as Ii. Therefore, the changes in the FRET efficiency (E') may be expressed as follows: 567 =

89 8: 89

(5)

where I0 and Ii and are the PL intensities of the donor QDs relative to QD-PEG-PDDLG/Cy3-HSA complex not bound or bound at different concentration of clofazimine. In addition, the hydrodynamic size of QD-PEG-PDDLG/Cy3-HSA in response to clofazimine with varying concentration from 20 to 1000 µM was measured by DLS. Cell Culture. HeLa cells were obtained from the Korea Cell Line Bank (KCLB, Seoul, Korea). HeLa cells (1 × 104 per well) were plated onto 96-well plates and cultured in growth media (Gibco, Grand Island, NY) supplemented with 10 % of FBS and 1% penicillin–streptomycin for 24 h at 37 °C in atmosphere of 5% CO2 and 95% 13



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humidity. In Vitro Cytotoxicity Evaluation. The HeLa cells were cultured with a solution of the QD-PEG-PDDLG having different concentrations for 24 h and 48 h. The in vitro cytotoxicity of QD-PEG-PDDLG was evaluated using an MTT assay. The cell viability was then by measured using the UV absorbance at 520 nm and was detected with the Cell Counting Kit-8 (Dojindo) on the MTT-treated cell solution compared with the control cell solution. Cellular Imaging. The HeLa cells were treated with 500 µL of the QD-PEG-PDDLG/Cy3-HSA solution (4:1 molar ratio of Cy3/QD, ~ 50 nM) complexes for 1 h and 4 h. Fluorescence images were observed using a LSM780 confocal laser scanning microscope (Carl Zeiss, Germany) at X800 using a 405 nm laser excitation source. To obtain the FRET signal, the sensors were excited using a 405 nm laser and FRET signal was collected using a 580 nm emission filter. Donor QD signals were acquired at the same excitation source and using a 528 nm emission filter. The scale bar represents 10 µm. The lysosome escape capability of the QD-PEG-PDDLG as protein carrier was investigated using CLSM. Briefly, QD-PEG-PDDLG was incubated with the HeLa cells. The position of the lysosome was determined by staining with LysoTracker (red) for 30 min before harvesting the cells, and then cells were rinsed with cold PBS three times and nuclei was labeled using Hoechst 33342 for 10 mins at predetermined time interval (2 h and 4 h). Finally, fluorescence images of cells were collected under 420 nm excitation wavelengths for QD, 580 nm excitation wavelengths for LysoTracker and 360 nm wavelengths for 14


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Hoechst 33342 by CLSM assay. In Vivo imaging study. The MDA-MB-231 human breast cancer cell lines were used to establish a nude mouse (aged 6 to 8 week) tumor model, and all procedures were in accordance with the guidelines outlined of the Care and Use of Laboratory Animals of Korea at Samsung Biomedical Research Institute. The QD-PEG-PDDLG/Cy3-HSA complex (4:1 molar ratio of Cy3/QD complex, the concentration of QD ~0.1 µM) or QD-PEG-PDDLG (the concentration of QD ~0.1 µM)) were injected into the MDA-MB-231 mice via the tail vein. In vivo fluorescence imaging was performed at different time points post -injection (pre, 1 h and 2 h) using an IVIS 200 Imaging system (Xenogen, Caliper Life Science, MA, USA) with a self-fixed QD donor filter set (excitation = 430 nm) and a self-fixed acceptor Cy3 bandpass emission filter (emission = 580 nm). Identical illumination settings (lamp voltage) were used for all in vivo imaging experiments. At the end of the experiment, the mice were sacrificed, and the tumor and the major organs (heart, lung, kidney, spleen, and liver) were excised for ex vivo imaging. The fluorescence intensity was normalized as photons per second per centimeter squared per steradian (p/s/cm2/sr). Statistical analysis. Quantitative data can be analyzed as the mean ± SD from several separate experiments. The Student’s t-test was used to estimate statistical significance.

RESULTS AND DISCUSSION Synthesis and characterization of CdZnSeS/ZnS QDs The CdZnSeS nanocrystals as an alloyed core were first synthesized through a 15



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single-step method and were further used to produce CdZnSeS/ZnS QDs by consecutively over coating with additional ZnS on the surface of the CdZnSeS QDs. As a result, this CdZnSeS/ZnS QDs alloyed core/shell structure can dramatically improve the photoluminescence quantum yield (PL QY) to greater than 95% and exhibit a narrow full-width half-maximum at approximately 21 nm. Figure 1a shows the HR-TEM images of the CdZnSeS/ZnS QDs. A uniform spherical particle with lattice fringes was obtained, which reveals highly crystalline nature of these CdZnSeS/ZnS QDs. The average size of the CdZnSeS/ZnS QDs was estimated to be approximately 12.5 nm from the high magnification image, which agrees with the results from the DLS measurement (Figure 1b). Therefore, the above-mentioned properties of the CdZnSeS/ZnS QDs allow these QDs to serve as a superior donor for FRET applications. Design and synthesis of polymer ligand The multifunctional mPEG-b-P(DA-DHLA)LG) polymer ligands were designed and synthesized by applying a series of reactions (Scheme 1). Briefly, mPEG functionalized poly(benzyl-L-glutamate) (abbreviated as mPEG-b-PBLG) as a polymer ligand backbone was first synthesized through the ROP of BLG-NCA using mPEG-NH2 as a macroinitiator and the ligand architecture can be optimized by controlling the molar ratio of the initiator to the monomer. Figure S1 shows the characteristic proton peaks of mPEG (~ 3.65 ppm and ~ 3.41 ppm) and of the benzyl groups (~5.1 ppm and ~ 7.35 ppm) in the polymer, and the degree of polymerization was estimated from the integration ratio of the characteristic peak of the benzyl group 16


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(2H, δ ~ 5.10 ppm) and mPEG (4H, δ ~ 3.65 ppm) (shown in Table 1). Second, the side chains of the mPEG-b-PBLG were modified by replacing the benzyl groups with diethylenetriamine to prepare the cationic polymer ligand using an aminolysis reaction in the presence of bifunctional catalyst 2-hydroxypyridine, which has the ability to prevent chain interruption of the polymer ligand backbone.39 We measured the multiplet peaks range from ~3.1 ppm to 3.4 ppm, which belonged to the protons of the diethylenetriamine grafted on the side chain in the newly formed polymer (Figure S2). The conversion of benzyl groups can be estimated by comparing the integration values of the peak of the benzyl group at 5.10 and the result shown in Table 1. Third, lipoic acid (LA) moieties, as a precursor anchor, were coupled to the remaining amine group of diethylenetriamine using a CDI condensation strategy. The lipoic acid was successfully conjugated to the terminal amine groups of the diethylenetriamine in the polymer by observing the characteristic peaks of the lipoic acid at ~1.2-2.4 ppm and ~2.9 ppm in Figure S3. The degree of grafting was estimated by the integration ratio of the LA peak (2H, ~2.9 ppm) and the mPEG peak (4H, ~3.65 ppm), and the results are

shown

in

Table

1.

The

final,

multifunctional

polymer

ligand,

mPEG-b-P(DA-DHLA)LG (PEG-PDDLG ligand), was obtained through the reduction of the dithiolane group via the reductant NaBH4. Additional details on the polymer ligand synthesis and 1H NMR spectra of the intermediate of the polymer ligand are described in the Experimental Section and Figures S1-S3. Ligand exchange and characterization of the CdZnSeS/ZnS QD capped with mPEG-b-P(DA-DHLA)LG Compact,

stabile

and

biocompatible

mPEG-b-P(DA-DHLA)LG-capped 17



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CdZnSeS/ZnS QD (abbreviated as QD-PEG-PDDLG) has been developed by substituting CdZnSeS/ZnS QDs’ native hydrophobic ligands (OA and TOP) with DHLA-containing PEG-PDDLG polymer ligands using a direct ligand exchange approach (Scheme 2). The hydrophilic QD-PEG-PDDLG (in D2O) was characterized using 1H NMR spectroscopy. As shown in Figure 2a, the 1H NMR spectrum of QD-PEG-PDDLG clearly indicated the presence of a new polymer ligands through the observation of characteristic peaks at 1.3-2.4 ppm and 2.9 ppm, which were attributed to binding of the LA to the QDs. Figure 2a also confirms that the native’ ligand (TOP/OA) of the CdZnSeS/ZnS QDs was conspicuously absent due to detection of very weak 1H NMR signatures from TOP and OA at 0.89 and 1.23 ppm (the peaks are corresponding with the protons of TOP and OA in Figure S4). The results indicated that hydrophobic CdZnSeS/ZnS QDs were effectively transferred to the aqueous solution by removing the TOP/OA ligands and packing new PEG-PDDLG ligands to their surface. However, we should emphasize here that the use of this ligand-exchange strategy was used to surface functionalization the QDs rather than growing the ligands out of the polymer-encapsulation QDs within micelle-like structures. Because, the polymer ligand comprising the DHLA moieties has a strong chelating and binding abilities to the QDs surface to Zn (II) ions, and a similar process has been discussed in previous reports.40 A monodispersed QD-PEG-PDDLG can be further confirmed from the cryo-TEM images. Figure 2b shows that the PEG-PDDLG polymer ligand was tightly wrapped on the single QD’s surface with a homogeneous spherical structure, and its hydrodynamic size was 18


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estimated to be approximately 25 nm, which is in good agreement with the size extracted from the DLS measurements (Figure 2c). Compared with the size of the original CdZnSeS/ZnS QDs (~ 12.5 nm), the capping ligand thickness was estimated to be ~6.3 nm (Figure 2d), and the polydispersity index (PDI) and QY for the QD-PEG-PDDLG were measured and are summarized in Figure 2d. Moreover, the PL emission spectra of the QD-PEG-PDDLG dispersed in the buffer had profiles identical to those collected for the starting CdZnSeS/ZnS QDs in the presence of native ligand (TOP/OA) in solvent (Figure 2e) indicating that the CdZnSeS/ZnS QDs may be well protected following phase transfer by capping the PEG-PDDLG polymer ligand. The stability of QDs-PEG-PDDLG dispersed in PBS buffer (10 mM, pH = 7.4) was investigated

using

fluorescence

images.

The

results

suggested

that

the

QD-PEG-PDDLG dispersions had long-lasting stable for at least 6 months with no sign of aggregation or loss of fluorescence (Figure 2f). Evaluation of the QD-PEG-PDDLG cytotoxicity is essential for applying this material in a biological environment because of the existence of potential toxic effect of cadmium (Cd(II))-containing QDs. The viability of HeLa cells cultured with the QD-PEG-PDDLG was evaluated using the MTT assay. Figure 2g shows that the cell viability was greater than 85% at QDs concentration ranging from 0 to 200 µg/mL after 24 h of incubation, and the cells viability still excessed 80% after 48 h of treatment. To further investigate at what concentration that 80% of the HeLa cells were killed, we increased the concentration of QD-PEG-PDDLG. It could be seen in 19



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Figure 2g, even the concentration of QD-PEG-PDDLG is as high as 1000 µg/mL, about 20% of the cells still survived after incubation, indicating low cytotoxicity and good biocompatibility of QD-PEG-PDDLG. However, we should not be surprised at this result, because the thick-shell ZnS passivation of the CdZnSeS core QDs and the effective protection added from the PEG hydrophilic shells on the CdZnSeS/ZnS QDs can significantly reduce the toxicity inherently caused by the QDs’ core materials. Therefore, the above results are very important for use of QD-PEG-PDDLG to construct the FRET system and in in vivo imaging. The construction of the QD-PEG-PDDLG/Cy3-HSA complex The QD-PEG-PDDLG/Cy3-HSA complex was constructed using the electrostatic interaction between QD-PEG-PDDLG and Cy3-HSA (Scheme 2). As shown in Figure 3, the zeta potential measurements showed that naked Cy3-labeled HSA was negative and around -10 mV and the PEG-PDDLG polymer ligands were positive and around +44 mV. However, the zeta potential value of the QD-PEG-PDDLG was slightly decreased (+33 mV) because of the naked QDs with a negative charge. Therefore, the CdZnSeS/ZnS QDs capped with PEG-PDDLG polymers ligands can provide a quantitatively positive surface and can exhibit stronger affinity for the negatively charged

Cy3-labeled

HSA.

Moreover,

the

zeta

potential

value

of

the

QD-PEG-PDDLG/Cy3-HSA complex decreased from +33 mV to +3 mV when the molar ratio of Cy3 to QD was increased, which also indicated that this complex can effectively promote the cellular uptake by various cells due to the positively charged surface.41 Energy transfer between CdZnSeS/ZnS QDs and Cy3 in the complex system 20


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Efficient energy transfer can take place from the CdZnSeS/ZnS QD donor to the Cy3 acceptor (Scheme 2) because of the presence of remarkable spectral overlap between the emission spectra of CdZnSeS/ZnS QDs and the absorption spectrum of Cy3, as shown in Figure 4. The FRET between QD-PEG-PDDLG and Cy3-HSA was investigated by the fluorescence spectrum. Figure 5a shows that all emissions spectra that were collected for different Cy3/QD ratios. The fluorescence intensity of the QD donor is quenched remarkably by increasing the molar ratio of Cy3 to QD, and the fluorescence intensity of Cy3 acceptor at 570 nm shows a gradual increase. Importantly, the change in the emission intensities of the donor to the acceptor at the different molar ratios can be converted to FRET efficiency. Figure 5b shows that the FRET efficiency increased dramatically from 0 to greater than 90% with an increase in the number of Cy3 per QD. The greatest FRET efficiency of this complex was strongly dependent on the high QY of the CdZnSeS/ZnS QD and the complex’s stability. The occurrence of FRET was further confirmed using time-resolved PL measurements. Figure 5c shows the normalized PL lifetime decay curves of the QD-PEG-PDDLG before and after the addition of Cy3-HSA at different molar ratios, and the QD donor exhibits gradual decrease in the excited decay state of the QD. The average lifetime of the QD are estimated by fitting each decay curve, the results confirmed that average lifetimes of the QDs were quickly reduced from 9.96 to 3.66 ns with an increase in the molar ratio of Cy3 to QD, and the highest FRET efficiency based on lifetime measurement was calculated up to ~63% (Figure S5). Overall, these results suggest that the QD-PEG-PDDLG complex with Cy3-HSA indeed causes an 21



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Page 22 of 44

efficient energy transfer from the QD donor to the Cy3 acceptor. In addition, the FRET parameters (I, R0) in the final QD-PEG-PDDLG/Cy3-HSA complex system were calculated based on the assumption that the spatial orientation factor was κ2 was 2/3 and the experimental values for QD and nD, which were described previously,38 respectively. To obtain R0, I is first calculated from the integrated spectra from 450 to 700 nm as shown in Figure S6. Moreover, the average distance (r) between the QD and Cy3 is estimated to be 67.8 Å at different ratios ranging from 2 to 10, which is much smaller than 100 Å indicating highly efficient energy transfer between the QD donor and the Cy3 acceptor and the correlation parameters are shown in Table 2. In

vitro

physiological

stability

and

FRET

stability

of

QD-PEG-PDDLG/Cy3-HSA To investigate whether the interferences effects of QD-PEG-PDDLG/Cy3-HSA to counterions in biological medium is essential for biomedical applications, the stability of QD-PEG-PDDLG/Cy3-HSA was investigated by measuring its hydrodynamic size in various kinds of biological media including PBS buffer (pH 7.4), 0.5 M NaCl solution, DI water (pH 5.5), GSH (10 mM), cell growth media and RPMI containing bovine serum over 72 h. Figure 6a showed that QD-PEG-PDDLG/Cy3-HSA possess very high stability in different biological mediums in the presence of high ionic strength, high reducing agents and proteins. These results are very important for probing imaging and sensing of living cell and real application. In addition, the FRET stability of QD-PEG-PDDLG/Cy3-HSA cultured in FBS were further studied using an 22


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FRET ratio of ICy3-A/( IQD-D+ ICy3-A). Figure 6b shows that ICy3-A/( IQD-D+ ICy3-A) of QD-PEG-PDDLG/Cy3-HSA maintain a constant without the significantly difference over 72 h. Although the QD-PEG-PDDLG/Cy3-HSA presented the slightly positive surface charge and might from complex with the negative charge plasma proteins in blood flow, PEGylated ligands-capped QDs displayed highly compact, compatibility and excellent stability which could effective against non-specific adsorption of proteins.42-43 Sensor properties of the QD-PEG-PDDLG/Cy3-HSA complex with respect to the protein-clofazimine interaction The application of this sensor is based on the principle of Cy3-labeled HSA maintaing its original specificity in this complex, and the variation of the distance (r) between the donor and acceptor may be a significant influence on the conformational changes of HSA upon binding clofazimine. In this study, we determined the changes in QD donor PL with respect to I/I0 (the QD-PEG-PDDLG/Cy3-HSA complex was not exposed to the clofazimine solution) depending on the HSA conformational changes induced by clofazimine, and this variation can be converted into changes in the FRET efficiency (E’) of the complex. As shown in Figure 7, the data show that the I/I0 ratio values of the donor QD rapidly decrease when increasing the concentration of clofazimine from 20 µm to 500 µm and achieves a plateau when the clofazimine concentration ranges from 500 µm to 1 mM. As expected, the opposite trend occurred in the FRET efficiency. This trend change may be explained by the fact that Cy3-labeled HSA cannot provide enough binding sites to clofazimine with an increase in the concentration, and the conformational changes of HSA will gradually reach a 23



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Page 24 of 44

maximum. The reason of the changes of this FRET might be verified by DLS measurements.

As

shown

in

Figure

S7,

the

hydrodynamic

size

of

QD-PEG-PDDLG/Cy3-HSA slightly decreased with increase of clofazimine concentration (varying from 20 to 1000 µM). These results indicate that the volume of HSA was compacted upon binding clofazimine and also imply that a shortening of the distance (r) between the donor and the acceptor in the complex. Moreover, the average decreased ∆r value (change in the r value, namely, ∆r) can be estimated, which is shown in Table 2. As a result, the QD-PEG-PDDLG/Cy3-HSA complex can be used as a sensitive sensor for probing the interaction of drugs with plasma proteins. In vitro and in vivo behavior of the QD-PEG-PDDLG/Cy3-HSA FRET sensor To evaluate the FRET capability of the QD-PEG-PDDLG/Cy3-HSA sensor in cells, HeLa cells were incubated with the QD-PEG-PDDLG/Cy3-HSA sensor for 1 h and 4 h. Confocal laser scanning microscopy was used to acquire images of HeLa cells in the donor (QD, pseudocolored green) and the FRET (acceptor emission, pseudocolored red) channels by changing the filter sets, and these channels included the QD donor excitation and emission and the FRET (QD donor excitation and Cy3 acceptor emission). As shown in Figure 8a, the QD donor signals and the FRET signals were observed on the membrane of the cell after 1 h of incubation, and more FRET signals were found in the cells after 4 h of incubation because the sensor has a positive charge and can effectively increase cellular uptake and promote cell internalization. Moreover, large yellow regions (merge of green QDs donor fluorescence and red FRET acceptor fluorescence) were emerged in the cell after 4 h

24


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of incubation, showing that the QD-PEG-PDDLG/Cy3-HSA sensor can remain nearly intact. In addition, the fast lysosome escape ability is an important requirement for QD-PEG-PDDLG as potential protein carrier. This escape behavior can be investigated by CLSM. The CLSM images of HeLa cells treated with QD-PEG-PDDLG carriers were observed after 2 h and 4 h incubation. In the obtained images

of cells,

green,

red and

blue colors

represent fluorescence

of

QD-PEG-PDDLG, lysosomes, and nuclei, respectively, and yellow spots indicate the merge of green QD-PEG-PDDLG and red lysosomes. After 2 hour incubation, the co-localization of QD-PEG-PDDLG with lysosome as the yellow spots were shown in the cell interior and a small portion of the carriers escaped from the organelles (Figure. 8b), indicating the QD-PEG-PDDLG carriers were trapped in lysosomes. With increasing incubation time (4 h), cell images showed weak red fluorescence and dispersive green spots with almost no yellow spots (Figure 8b), demonstrating the escape ability of QD-PEG-PDDLG. The lysosome escape ability of QD-PEG-PDDLG might be due to the pH-dependent protonation of diethylenetriamine groups in the polymer ligand side chain44. Thus, this sensor can also potentially be used as a nanocarrier for intracellular protein delivery. The fate of the QD-PEG-PDDLG/Cy3-HSA FRET sensors in vivo were further assessed using an IVIS imaging system with a fixed excitation filter (donor QDs excitation: 430 nm) and an emission bandpass filter (FRET emission:580 nm). As shown in Figure 9a, with the assumption that the QD-PEG-PDDLG/Cy3-HSA sensor is intact in vivo, the FRET signal can be detected at 580 nm using a 430-nm excitation 25



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Page 26 of 44

filter. To confirm this hypothesis, the present FRET sensors were injected into nude mice bearing subcutaneous MDA-MB-231 human breast cancer tumors via tail vein injection. At 1 h post-injection, a weak FRET signal was observed in the tumor sites compared to pre-injection, and the FRET signal increased significantly 2 h post -injection (shown in Figure 9b). These results support the hypothesis that the FRET process can be monitored in vivo, and the QD-PEG-PDDLG/Cy3-HSA sensors remain intact for up to 2 h after injection. Tumor and other major visceral organs of the mice were extracted, and ex vivo FRET signals were verified under the same excitation and emission filters (Figure 9c). As observed in a comparison of the quantification assay in Figure 9d, the FRET signal in the tumor tissue was significantly greater than in other organs due to the small size of the QD-PEG-PDDLG/Cy3-HSA sensor, which increases the uptake by the tumor by enhancing the permeability and retention (EPR) effect.45 However, the slight FRET signal obtained in lung and kidney might be due to the rapid uptake and digestion by macrophages and the accompanying renal clearance. This demonstrates that the emission of the donor QD can be neglected at 580 nm using a 430 -nm excitation. QD-PEG-PDDLG was injected in the same mice model, and as expected, the fluorescence signal was not detected in vivo and ex vivo 2 h after injection (Figure S8ab). These results confirmed that the QD-PEG-PDDLG/Cy3-HSA sensor can not only be used as a stabile FRET sensor but can also be used as an imaging probe for cancer diagnosis.

26


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CONCLUSION High quality CdZnSeS/ZnS QDs were synthesized as energy donors. The CdZnSeS core QDs were over coated with ZnS shells, which led to a PL QY increase of 95% and a narrow FWHM (21 nm). Compact, stabile, biocompatible and hydrophilic CdZnSeS/ZnS QDs were developed by densely capping the QD with a multifunctional PEG-PDDLG polymer ligand via a direct ligand-exchange method. The resulting QD-PEG-PDDLG had lower cell toxicity demonstrated using an MTT assay and was used to construct a FRET system using the high affinity interaction between the QD-PEG-PDDLG and the Cy3-labeled HSA. The occurrence of FRET was assayed using the PL spectra and the time-resolved PL spectra. The QD-PEG-PDDLG/Cy3-HSA exhibited excellent stability under different biological media such as low pH, in high ionic strength media, in high reducing agents condition and in RPMI containing proteins. In this work, we explored the potential of the FRET sensor to be used to study the interaction of a protein and a drug based on the sensitivity to a change in the distance r between the donor and the acceptor. Additionally,

we

investigated

the

viability

and

stability

of

a

QD-PEG-PDDLG/Cy3-HSA FRET system as a sensitive sensor in vitro and in vivo. Moreover, we also believe that this novel and sensitive FRET sensor will have more potential applications in protein delivery and fluorescence imaging diagnostics as well as in other protein-ligand interactions. ASSOCIATED CONTENT Supporting Information

27



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Page 28 of 44

1

H NMR spectrum of mPEG-b-PBLG polymer ligand backbone, mPEG-b-PDALG polymer ligand and mPEG-b-P(DA-DHLA)LG polymer ligand. 1H NMR spectrum of hydrophobic CdZnSeS/ZnS QDs. FRET efficiencies of QDs-PEG-PDDLG/Cy3-HSA based on lifetime of donor. Spectral overlap function (J(λ)) of the QD-PEG-PDDLG/HSA-Cy3 FRET pair. Hydrodynamic size of the QD-PEG-PDDLG/HSA-Cy3 at different concentration of clofazimine. The in vivo fluorescence images of QD-PEG-PDDLG at before injection, 1h and 2 h after injected with 500 µL of QD-PEG-PDDLG and the fluorescence images of major tissues and tumor were excised from the mice after experiment. AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected] *E-mail: [email protected] Author Contributions 1

These authors are equally contributed to the article as co-first authors.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS This research was supported by the Basic Science Research Program through a National Research Foundation of Korea grant funded by the Korean Government (MEST) (20100027955) and the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (2012M3A9B6055205).

28


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REFERENCE (1) Wu, J.; Liu, W.; Ge, J.; Zhang, H.; Wang, P. New Sensing Mechanisms for Design of Fluorescent Chemosensors Emerging in Recent Years. Chem. Soc. Rev. 2011, 40, 3483-3495. (2) Lu, L.; Qian, Y. X.; Wang, L. H.; Ma, K. K.; Zhang, Y. D. Metal-Enhanced Fluorescence-Based Core-Shell Ag@SiO2 Nanoflares for Affinity Biosensing via Target-Induced Structure Switching of Aptamer. ACS Appl. Mater. Interfaces 2014, 6 (3), 1944-1950. (3) Jackson, R.; Oda, R. P.; Bhandari, R. K.; Mahon, S. B.; Brenner, M.; Rockwood, G. A.; Logue, B. A. Development of A Fluorescence-Based Sensor for Rapid Diagnosis of Cyanide Exposure. Anal. Chem. 2014, 86 (3), 1845-1852. (4) Yang, J. K.; Kwak, S. Y.; Jeon, S. J.; Lee, E.; Ju, J. M.; Kim, H. I.; Lee, Y. S.; Kim, J. H. Proteolytic Disassembly of Peptide-Mediated Graphene Oxide Assemblies for Turn-On Fluorescence Sensing of Proteases. Nanoscale 2016, 8, 12272-12281. (5) Stanisavljevic, M.; Krizkova, S.; Vaculovicova, M.; Kizek, R.; Adam, V. Quantum Dots-Fluorescence Resonance Energy Transfer-Based Nanosensors and Their Application. Biosens. Bioelectron. 2015, 74, 562-574. (6) Shi, J.; Tian, F.; Lyu, J.; Yang, M. Nanoparticle Based Fluorescence Resonance Energy Transfer (FRET) for Biosensing Applications. J. Mater. Chem. B 2015, 3, 6989-7005. (7) Onoshima, D.; Yukawa, H.; Baba, Y. Multifunctional Quantum Dots-Based Cancer Diagnostics and Stem Cell Therapeutics for Regenerative Medicine. Adv. Drug Delivery Rev. 2015, 95, 2-14. (8) Chu, X.; Dou, X.; Liang, R.; Li, M.; Kong, W.; Yang, X.; Luo, J.; Yang, M.; Zhao, M. A Self-Assembly Aptasensor Based on Thick-Shell Quantum Dots for Sensing of Ochratoxin A. Nanoscale 2016, 8, 4127-4133. (9) Yue, Z.; Lisdat, F.; Parak, W. J.; Hickey, S. G.; Tu, L. P.; Sabir, N.; Dorfs, D.; Bigall, N. C. Quantum-Dot-Based Photoelectrochemical Sensors for Chemical and Biological Detection. ACS Appl. Mater. Interfaces 2013, 5 (8), 2800-2814. (10) Long, F.; Gu, C.; Gu, A. Z.; Shi, H. Quantum Dot/Carrier-Protein/Haptens Conjugate as A Detection Nanobioprobe for FRET-Based Immunoassay of Small Analytes with All-Fiber Microfluidic Biosensing Platform. Anal. Chem. 2012, 84 (8), 3646-3653. (11) Medintz, I. L.; Pons, T.; Delehanty, J. B.; Susumu, K.; Brunel, F. M.; Dawson, P. E.; Mattoussi, H. Intracellular Delivery of Quantum Dot-Protein Cargos Mediated by Cell Penetrating Peptides. Bioconjugate Chem. 2008, 19 (9), 1785-1795. (12) Yeh, H. Y.; Yates, M. V.; Mulchandani, A.; Chen, W. Molecular Beacon-Quantum Dot-Au Nanoparticle Hybrid Nanoprobes for Visualizing Virus Replication in Living Cells. Chem. Commun. 2010, 46, 3914-3916. (13) Wegner, K. D.; Lanh, P. T.; Jennings, T.; Oh, E.; Jain, V.; Fairclough, S. M.; Smith, J. M.; Giovanelli, E.; Lequeux, N.; Pons, T.; Hildebrandt, N. Influence of Luminescence Quantum Yield, Surface Coating, and Functionalization of Quantum Dots on the Sensitivity of Time-Resolved FRET Bioassays. ACS Appl. Mater. Interfaces 2013, 5 (8), 2881-2892. (14) Palui, G.; Avellini, T.; Zhan, N.; Pan, F.; Gray, D.; Alabugin, I.; Mattoussi, H. Photoinduced Phase Transfer of Luminescent Quantum Dots to Polar and Aqueous Media. J. Am. Chem. Soc. 2012, 134 (39), 16370-16378. (15) Yaghini, E.; Pirker, K. F.; Kay, C. W. M.; Seifalian, A. M.; MacRobert, A. J. Quantification of Reactive Oxygen Species Generation by Photoexcitation of PEGylated Quantum Dots. Small 2014, 10 (24), 5106-5115. 29



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(16) Hadar, I.; Halivni, S.; Even-Dar, N. a.; Faust, A.; Banin, U. Dimensionality Effects on Fluorescence Resonance Energy Transfer Between Single Semiconductor Nanocrystals and Multiple Dye Acceptors. J. Phys. Chem. C 2015, 119 (7), 3849-3856. (17) Dennis, A. M.; Rhee, W. J.; Sotto, D.; Dublin, S. N.; Bao, G. Quantum Dot-Fluorescent Protein FRET Probes for Sensing Intracellular pH. ACS Nano 2012, 6 (4), 2917-2924. (18) Wu, P.; Hou, X. D.; Xu, J. J.; Chen, H. Y. Ratiometric Fluorescence, Electrochemiluminescence, and Photoelectrochemical Chemo/Biosensing Based on Semiconductor Quantum Dots. Nanoscale 2016, 8, 8427-8442. (19) Breger, J. C.; Sapsford, K. E.; Ganek, J.; Susumu, K.; Stewart, M. H.; Medintz, I. L. Detecting Kallikrein Proteolytic Activity with Peptide-Quantum Dot Nanosensors. ACS Appl. Mater. Interfaces 2014, 6 (14), 11529-11535. (20) Lee, J.; Brennan, M. B.; Wilton, R.; Rowland, C. E.; Rozhkova, E. A.; Forrester, S.; Hannah, D. C.; Carlson, J.; Shevchenko, E. V.; Schabacker, D. S.; Schaller, R. D. Fast, Ratiometric FRET from Quantum Dot Conjugated Stabilized Single Chain Variable Fragments for Quantitative Botulinum Neurotoxin Sensing. Nano Lett. 2015, 15 (10), 7161-7167. (21) Diaz, S. A.; Gillanders, F.; Jares-Erijman, E. A.; Jovin, T. M. Photoswitchable Semiconductor Nanocrystals with Self-Regulating Photochromic Forster Resonance Energy Transfer Acceptors. Nat. Commun. 2015, 6, 6036. (22) Kajihara, D.; Abe, R.; Iijima, I.; Komiyama, C.; Sisido, M.; Hohsaka, T. FRET Analysis of Protein Conformational Change Through Position-Specific Incorporation of Fluorescent Amino Acids. Nat. Methods 2006, 3, 923-929. (23) Boeneman, K.; Mei, B. C.; Dennis, A. M.; Bao, G.; Deschamps, J. R.; Mattoussi, H.; Medintz, I. L. Sensing Caspase 3 Activity with Quantum Dot-Fluorescent Protein Assemblies. J. Am. Chem. Soc. 2009, 131 (11), 3828-3829. (24) Guo, Y.; Sakonsinsiri, C.; Nehlmeier, I.; Fascione, M. A.; Zhang, H. Y.; Wang, W. L.; Pohlmann, S.; Turnbull, W. B.; Zhou, D. J. Compact, Polyvalent Mannose Quantum Dots as Sensitive, Ratiometric FRET Probes for Multivalent Protein-Ligand Interactions. Angew. Chem. Int. Ed. 2016, 55, 4738-4742. (25) Biju, V.; Anas, A.; Akita, H.; Shibu, E. S.; Itoh, T.; Harashima, H.; Ishikawa, M. FRET from Quantum Dots to Photodecompose Undesired Acceptors and Report the Condensation and Decondensation of Pasmid DNA. ACS Nano 2012, 6 (5), 3776-3788. (26) Dang, Y. Q.; Li, H. W.; Wu, Y. Construction of a Supramolecular Forster Resonance Energy Transfer System and Its Application Based on the Interaction between Cy3-Labeled Melittin and Phosphocholine Encapsulated Quantum Dots. ACS Appl. Mater. Interfaces 2012, 4 (3), 1267-1272. (27) He, X. W.; Ma, N. Biomimetic Synthesis of Fluorogenic Quantum Dots for Ultrasensitive Label-Free Detection of Protease Activities. Small 2013, 9, 2527-2531. (28) Loo, A. H.; Sofer, Z.; Bousa, D.; Ulbrich, P.; Bonanni, A.; Pumera, M. Carboxylic Carbon Quantum Dots as A Fluorescent Sensing Platform for DNA Detection. ACS Appl. Mater. Interfaces 2016, 8 (3), 1951-1957. (29) Abou-Zied, O. K.; Al-Lawatia, N.; Elstner, M.; Steinbrecher, T. B. Binding of Hydroxyquinoline Probes to Human Serum Albumin: Combining Molecular Modeling and Forster's Resonance Energy Transfer Spectroscopy to Understand Flexible Ligand Binding. J. Phys. Chem. B 2013, 117 (4), 1062-1074. (30) Yuan, J. L.; lv, Z.; Liu, Z. G.; Hu, Z.; Zou, G. L. Study on Interaction between Apigenin and Human Serum Albumin by Spectroscopy and Molecular Modeling. J. Photochem. Photobiol., A 2007, 191, 30


Page 31 of 44

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104-113. (31) Xiao, Q.; Huang, S.; Qi, Z. D.; Zhou, B.; He, Z. K.; Liu, Y. Conformation, Thermodynamics and Stoichiometry of HSA Adsorbed to Colloidal CdSe/ZnS Quantum Dots. Biochim. Biophys. Acta, Proteins Proteomics 2008, 1784 (7-8), 1020-1027. (32) Tyagi, S.; Ammerman, N. C.; Li, S. Y.; Adamson, J.; Converse, P. J.; Swanson, R. V.; Almeida, D. V.; Grosset, J. H. Clofazimine Shortens the Duration of The First-Line Treatment Regimen for Experimental Chemotherapy of Tuberculosis. Proc. Natl. Acad. Sci. U. S. A. 2015, 112 (3), 869-874. (33) Koval, A. V.; Vlasov, P.; Shichkova, P.; Khunderyakova, S.; Markov, Y.; Panchenko, J.; Volodina, A.; Kondrashov, F. A.; Katanaev, V. L. Anti-Leprosy Drug Clofazimine Inhibits Growth of Triple-Negative Breast Cancer Cells via Inhibition of Canonical Wnt Signaling. Biochem. Pharmacol. 2014, 87 (4), 571-578. (34) Yang, H. Y.; Jang, M. S.; Gao, G. H.; Lee, J. H.; Lee, D. S. pH-Responsive Biodegradable Polymeric Micelles with Anchors to Interface Magnetic Nanoparticles for MR Imaging in Detection of Cerebral Ischemic Area. Nanoscale 2016, 8, 12588-12598. (35) Lee, K. H.; Lee, J. H.; Kang, H. D.; Park, B.; Kwon, Y.; Ko, H.; Lee, C.; Lee, J.; Yang, H.Over 40 cd/A Efficient Green Quantum Dot Electroluminescent Device Comprising Uniquely Large-Sized Quantum Dots. ACS Nano 2014, 8 (5), 4893-4901. (36) Zhan, N.; Palui, G.; Mattoussi, H. Preparation of Compact Biocompatible Quantum Dots Using Multicoordinating Molecular-Scale Ligands Based on A Zwitterionic Hydrophilic Motif and Lipoic Acid Anchors. Nat. Protoc. 2015, 10, 859-874. (37) Medintz, I. L.; Mattoussi, H. Quantum Dot-Based Resonance Energy Transfer and Its Growing Application in Biology. Phys. Chem. Chem. Phys. 2009, 11, 17-45. (38) Clapp, A. R.; Medintz, I. L.; Mauro, J. M.; Fisher, B. R.; Bawendi, M. G.; Mattoussi, H. Fluorescence Resonance Energy Transfer Between Quantum Dot Donors and Dye-Labeled Protein Acceptors. J. Am. Chem. Soc. 2004, 126 (1), 301-310. (39) Yang, H. Y.; Jang, M. S.; Gao, G. H.; Lee, J. H.; Lee, D. S. Construction of Redox/pH Dual Stimuli-Responsive PEGylated Polymeric Micelles for Intracellular Doxorubicin Delivery in Liver Cancer. Polym. Chem. 2016, 7, 1813-1825. (40) Wang, W.; Kapur, A.; Ji, X.; Safi, M.; Palui, G.; Palomo, V.; Dawson, P. E.; Mattoussi, H. Photoligation of An Amphiphilic Polymer with Mixed Coordination Provides Compact and Reactive Quantum Dots. J. Am. Chem. Soc. 2015, 137 (16), 5438-5451. (41) Hu, L.; Mao, Z.; Gao, C. Colloidal Particles for Cellular Uptake and Delivery. J. Mate. Chem. 2009, 19, 3108-3115. (42) Zhang, H.; Feng, G.; Guo, Y.; Zhou, D. A Robust and Apecific Ratiometric Biosensing Using A Copper-Free Clicked Quantum Dot–DNA Aptamer Sensor. Nanoscale 2013, 5, 10307-10315. (43) Mattoussi, H.; Palui, G.; Na, H. B. Luminescent Quantum Dots as Platforms for Probing in Vitro and in Vivo Biological Processes. Adv. Drug Delivery Rev .2012, 64 (2), 138-166. (44) Kim, H. J.; Ishii, A.; Miyata, K.; Lee, Y.; Wu, S. R.; Oba, M.; Nishiyama, N.; Kataoka, K. Introduction of Stearoyl Moieties into A Biocompatible Cationic Polyaspartamide Derivative, PAsp(DET), with Endosomal Escaping Function for Enhanced siRNA-Mediated Gene Knockdown. J. Controlled Release 2010, 145 (2), 141-148. (45) Iyer, A. K.; Khaled, G.; Fang, J.; Maeda, H., Exploiting the Enhanced Permeability and Retention Effect for Tumor Targeting. Drug Discovery Today 2006, 11 (17), 812-818.

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Scheme 1. Synthesis process of multifunctional polymer ligand mPEG-b-P(DA-DHLA)LG and reducing the dithiolane group using reductive agent NaBH4

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Scheme 2. The preparation process of hydrophilic CdZnSeS/ZnS QDs by ligand exchange method and the constrction schemes of QDs-PEG-PDDLG/Cy3-labeled HSA FRET sensor. Intact FRET sensor emit light at the Cy3 emission wavelength (570-580 nm) upon QD excitation (405-430 nm).

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Figure 1. (a) HR-TEM images and (b) Size distribution of CdZnSeS/ZnS QDs.

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Figure 2. (a) 1H NMR spectrum of CdZnSeS/ZnS QDs capped with mPEG-b-P(DA-DHLA)LG dispersed in D2O. (b) Cryo-TEM images and (c) size distribution CdZnSeS/ZnS QDs-capped mPEG-b-P(DA-DHLA)LG. (d) Hydrodynamic radius (nm), polydispersity index (PDI), QY and Capping ligand thickness measured for CdZnSeS/ZnS QDs capped with mPEG-b-P(DA-DHLA)LG. (e) Normalized emission spectra of CdZnSeS/ZnS QDs emitting at 520 nm, before 35



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and after capped with mPEG-b-P(DA-DHLA)LG. (f) Stability tests of CdZnSeS/ZnS QDs-capped mPEG-b-P(DA-DHLA)LG dispersed in PBS solution during 6 months. (g) MTT viability tests of HeLa cells incubated for 24 h and 48 h with varying concentrations of QD-PEG-PDDLG (QDs emitting at 405 nm were used).

Figure 3. Zeta potential of HSA-Cy3, mPEG-b-P(DA-DHLA)LG polymer ligand and the QDs-PEG-PDDLG/Cy3-HSA complex system at different molar ratio of Cy3/QDs.

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Figure 4. Normalized fluorescence emission spectra of the QD-PEG-PDDLG and Cy3-labeled HSA, together with the absorption spectra of Cy3-labeled HSA.

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Figure 5. (a) Fluorescence emission spectra of QDs-PEG-PDDLG/Cy3-HSA with different molar ratio of Cy3/QDs (λex = 405 nm, where the emission of Cy3 is negligible). (b) FRET efficiencies of QDs-PEG-PDDLG/Cy3-HSA complex system deduce from the donor PL loss at different molar ratio of Cy3/QDs. (c) Fluorescence decay curves of the CdZnSeS/ZnS QDs emission at 520 nm (λex = 405 nm) with different molar ratio of Cy3/QDs.

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Figure 6. (a) In vitro hydrodynamic size stability of the the QD-PEG-PDDLG/Cy3-HSA in 0.5 M NaCl, 10 mM GSH, RPMI-1640, RPMI containing Bovine serum, PBS (pH 7.4) and pH 5.5 media; (b) The FRET stability of the QD-PEG-PDDLG/Cy3-HSA cultured in FBS at different time, ICy3-A/(IQD-D+ICy3-A) defined as the FRET ratio.

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Figure 7. Decreasing plot of QDs PL intensity (I/I0) and increasing plot of FRET efficiency (E’) in system as a function of clofazimine concentration. (λex = 420 nm, where the effects of clofazimine is negligible)

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Figure 8. (a) Confocal laser scanning microscopy FRET images of HeLa cells incubated with QD-PEG-PDDLG/HSA-Cy3 FRET sensors for 1 h and 4 h. The bright field, and the donor QDs fluorescence images, and red-FRET fluorescence images, and merge images (QD donor+Cy3 acceptor ) and merge images (FRET+Bright field) are shown from left to right. The scale bar is 10 µm. (b) CLSM images of HeLa cells incubated with QD-PEG-PDDLG for 2 h and 4h, respectively. The lysosomes and nuclei were stained with Lysotracter Red and Hoechst, respectively. The yellow spots indicate co-localization of QD-PEG-PDDLG (green) and lysosomes (red). The scale bar is 5 µm.

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Figure 9. (a) Schematic of in vivo FRET images by IVIS imaging system (setting: excitation filter: 430 nm; emission filter: 580 nm). (b) The FRET images were obtained at before injection, 1 h and 2 h after injected with 500 µL of QD-PEG-PDDLG/Cy3-HSA sensor. (c) Ex vivo FRET images of major tissues and tumor were excised from the mice after experiment. (d) A quantification of the ex vivo tissues and tumor were recorded as fluorescence intensity (p/s/cm2/sr). All data are represented as mean SD (n = 3); ‫ ٭٭‬is represented as significant (P

ZnS Quantum Dot

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