ZnS Quantum Dots for In vitro and In vivo Bioimaging - American

Subscriber access provided by Columbia Univ Libraries

Article

High-Quality CuInS2/ZnS Quantum Dots for in vitro and in vivo Bioimaging Dawei Deng, Yuqi Chen, Jie Cao, Junmei Tian, Zhiyu Qian, Samuel Achilefu, and Yueqing Gu Chem. Mater., Just Accepted Manuscript • DOI: 10.1021/cm3015594 • Publication Date (Web): 23 Jul 2012 Downloaded from http://pubs.acs.org on July 29, 2012

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.

Chemistry of Materials 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 25

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

Chemistry of Materials

High-Quality CuInS2/ZnS Quantum Dots for in vitro and in vivo Bioimaging Dawei Deng,†,‡ Yuqi Chen,‡ Jie Cao,‡ Junmei Tian,‡ Zhiyu Qian,§ Samuel Achilefu,*,|| and Yueqing Gu*,†,‡ †

‡

State Key Laboratory of Natural Medicines, and School of Life Science and Technology, China Pharmaceutical

University, Nanjing 210009, China §

Department of Biomedical Engineering, School of Automation, Nanjing University of Aeronautics and

Astronautics, Nanjing 210016, China ||

Department of Radiology, School of Medicine, Washington University, St. Louis, Missouri, USA

* Corresponding author. Fax: +86 25 83271046 E-mail: [email protected] (D. W. Deng), [email protected] (Y. Q. Gu), [email protected] (S. Achilefu)

1

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 25

ABSTRACT: The exploration of biocompatible quantum dots (QDs) for biomedical imaging

is currently one of the fastest growing fields of nanotechnology. This strategy overcomes the intrinsic toxicity of well-developed II-VI and other semiconductor QDs (Cd, Hg, Pb, Se, Te, As, etc.) that remains a major obstacle to their clinical use. In this report, we synthesized high-quality CuInS2/ZnS (CIS/ZnS) QDs without using conventional toxic heavy metals. These QDs exhibited improved photoluminescence (PL) properties, with tunable emission peaks ranging from 550 to 800 nm and a maximum PL quantum yield (QY) up to 80%. Next, we explored the effective loading of the prepared oil-soluble CIS/ZnS QDs using biodegradable folate-modified N-succinyl-N´-octyl chitosan (FA-SOC) micelles. Targeting efficacy of the resulting QDs-loaded micelles to tumors using in vitro and in vivo optical imaging techniques was also investigated. The results show that the micelle platform allowed successful formulation of these oil-soluble QDs in water, while retaining the morphology, crystal structure, and PL of the initial CIS/ZnS QDs. This study demonstrates the versatility of using the biocompatible CIS/ZnS QDs across different spatial scales (in vitro cell imaging and in vivo small animal imaging) for multicolor biological imaging applications.

KEYWORDS: CuInS2/ZnS, Quantum Dots, Micelles, In vitro Imaging, In vivo Imaging

2


Page 3 of 25

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


Introduction Medical imaging techniques, such as computed tomography (CT), magnetic resonance imaging (MRI), positron emission tomography (PET), ultrasound imaging, and optical imaging, play key roles in disease diagnosis.1,2 Each imaging technique (or modality) can bring unique information to molecular medicine. Unlike other imaging modalities, optical imaging uses low energy radiation in the visible or near-infrared (NIR) regions of light to assess biological processes.3–5 As a result, this optical method is widely used for biological imaging across multi-spatial scale that includes cells, tissues and small animals. Nearly all the optical imaging methods reported in this thematic issue use a fluorescence signal for molecular imaging because of the high detection sensitivity.1–5 Specifically, visible light can be used for in vitro cell imaging, while red and NIR light (650–900 nm) is suitable for in vivo tissue or whole-body imaging of small animals because of the deeper penetration of NIR light in tissue. Hence, optical imaging is widely used in preclinical studies as a workhorse of molecular imaging. Over the past years, the development of new fluorescent semiconductor QDs for optical imaging has attracted much attention.6–8 Compared with the use of organic dyes as imaging probes, QDs are a novel class of materials with unique optical and electronic properties, such as size-tunable PL, high PL quantum yield (QY), sharp and symmetrical fluorescence peak, broad excitation spectrum, large Stokes shift, multi-color fluorescence with a single-wavelength excitation source, and high resistance to photobleaching. Unfortunately, most of the highly luminescent QDs currently used for biomedical imaging are composed of toxic elements (Cd, Hg, Pb, Se, Te, As, etc.).9–12 Several groups have shown that these toxic components strongly influence the cytotoxic effect of QDs, due to their eventual release into the cellular environment. This represents a major obstacle to the clinical use of QDs and has motivated the development of new biocompatible QDs based on the use of III–V or I–III–VI2 materials with relatively low toxicity.13–16 CuInS2 is a I–III–VI2 semiconductor with a direct band gap of 1.5 eV, corresponding to an 830 nm emission wavelength, and does not contain highly toxic heavy metals.16 Therefore, this material might be a promising candidate for optical imaging, which offers the opportunity to develop semiconductor QDs without the toxicity limitations encountered by II–VI QDs, especially at low concentrations. In addition, this class of QDs provides multi-color PL emission ranging 3



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 4 of 25

from the visible to the NIR wavelengths. Recently, several colloidal chemistry approaches have been employed to synthesize CuInS2 or other I–III–VI2 QDs.16–21 However, most of the previous syntheses were still relatively complicated, and reported applications focused on their high potential in solar energy conversion or light emitting diodes.22 To our knowledge, only a few demonstrations has been recently reported for in vivo imaging using these QDs.23,24 Nanoscale targeted micelles are undoubtedly of special interest for the targeted delivery of hydrophobic diagnostic and therapeutic agents (or drugs) to diseased tissues, which may increase the therapeutic effect and correspondingly minimize potential adverse effect.25–27 Chitosan, an abundant natural biopolymer, is a favorable choice for the fabrication of targeted nanoscale drug delivery micelles due to the high biocompatibility, low toxicity, and bio-degradability.28−31 The high content of amine groups in the backbone of chitosan endows chitosan-based micelles with various multifunctional surface modifications. In the previous studies, optical imaging techniques have been widely applied to visualize the tumor targeting ability of the drug carriers by using organic fluorescent probes, such as fluorescein, rhodamine, Texas Red, and cyanine dyes.32−35 Most of the high-quality QDs are synthesized in non-polar organic solvents, and therefore have a hydrophobic surface, which limits their dissolution in aqueous medium for biological applications.5–8,36 However, the micelles self-assembled from amphiphilic molecules have a hydrophobic core and a hydrophilic shell.25–33 Thus, the hydrophobic cores of the micelles provide a stable environment to load hydrophobic (i.e., oil-soluble) QDs due to the favorable hydrophobic interactions. Capitalizing on this phenomenon, we synthesized oil-soluble CuInS2 QDs via a non-injection approach by systematically optimizing the effects of the reaction time, the amount of dodecanethiol (DDT), the temperature, the Cu:In precursor ratio, and the ZnS shell coating. The resulting CIS/ZnS core/shell QDs exhibit brighter PL emission with a wider tunable range (from yellow to NIR) than previously reported QDs.16−21 Using FA-SOC micelles, we demonstrate in this study that these QDs can be effectively solubilized in aqueous medium. By in vitro and in vivo optical imaging techniques, we also demonstrate for the first time the use of these QDs as a versatile luminescent probe for evaluating the tumor targeting of micelles in cells and small animals.

4


Page 5 of 25

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


Experimental Section Chemicals. Copper acetate (Cu(Ac)2, 99.99%), indium acetate (In(Ac)3, 99.99%), zinc acetate (Zn(Ac)2, 99.99%), 1-dodecanethiol (DDT, 98%), 1-octadecene (ODE, 90%), oleic acid (OA, 90%), oleylamine (OAm, 97%), n-decane (98%), rhodamine B (RhB), Cypate, chitosan with deacetylation degree of 90% (100 KDa, 95%), succinic anhydride (98%), octaldehyde (99%), folic acid

(or

folate), N,N'-dicyclohexylcarbodiimide (DCC), N-hydroxysuccinimide

(NHS),

fluorescein isothiocyanate (FITC), (folate-free) RPMI 1640 medium, calf serum, penicillin, streptomycin, trypsin, and EDTA were purchased from commercial sources. The water used in all experiments had a resistivity higher than 18.2 MΩ·cm. All chemicals were used without further purification. Human hepatoma cell line (Bel-7402), human lung carcinoma cell line (A549), and human embryonic lung fibroblast cell line (HELF) were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). Athymic nude mice (nu/nu, half male and half female; aged 4–6 weeks and weighed at 18–22 g) were purchased from Charles River Laboratories (Shanghai, China). Synthesis of CIS Core QDs. For a typical synthetic reaction, In(Ac)3 (0.2 mmol) and Cu(Ac)2 (0.2 mmol) were mixed with 2.0 mL (8.35 mmol) of DDT, 3 mL of ODE and 0.3 mL of OA in a 50 mL three-neck flask. The reaction mixture was heated to 100 ºC for 10 min under N2 flow with mild stirring until a clear solution was formed. The reaction temperature was then raised to 230 ºC (215–280 ºC) with a rate of 10–15 ºC/min and retained at this temperature to allow the growth of CIS QDs. As the temperature increased, the color of the reaction solution progressively changed from slight yellow to yellow, red, and finally black, indicating nucleation and subsequent growth of CIS QDs. Aliquots of the sample were taken at different time intervals and injected into cold n-decane to terminate growth of QDs for UV-Vis absorbance and PL measurements without any size sorting. After completion of quantum dot growth, the reaction mixture was allowed to cool to 50 ºC, and an equal volume of n-decane was added thereafter. We explored the effect of the growth time, the amount of DDT, the reaction temperature, and the Cu/In molar ratio on the synthesis of CIS core QDs. Synthesis of CIS/ZnS Core/shell QDs. The synthesized CIS core QD growth solution was used directly without intermediate purification step. The reaction temperature was kept within a range 5



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 6 of 25

of 215–230 °C for the subsequent growth of a ZnS shell. A Zn precursor solution was prepared by dissolving 2 mmol of Zn(Ac)2 in ODE/oleylamine (the ratio of ODE/OAm is 4/1, in all 5 mL) at 160 °C under N2 flow, and stored at 50 °C for use. The ZnS coating was accomplished by injecting the Zn precursor solution into the QD growth solution with a syringe. Subsequently, the temperature was maintained between 215–230 °C for about 30 min to allow the growth of ZnS shell. To monitor the reaction, aliquots were taken at different time intervals, and optical spectra were recorded for each aliquot. In addition, the effect of the precursor Zn:Cu feed ratio was also investigated by injecting 2.4 mL of Zn precursor solution in 6 batches with a time interval of 15 min. The resulting QDs were isolated by precipitating with ethanol, centrifuging and decanting the supernatant. The residue was re-dispersed in n-decane, hexane or chloroform. XRD, TEM and XPS measurements were performed to characterize the crystallinity, size, morphology and elemental composition of the QDs. Synthesis

of

Folate-modified

N-succinyl-N′-octyl-chitosan

Micelles.

Folate-modified

N-succinyl-N′-octyl-chitosan (FA-SOC) micelles were synthesized using a two-step method reported previously (see Figure 1b).37,38 The Preparation of SOC Micelles: Briefly, chitosan (1 g) was reacted with succinic anhydride (3 g) in acetone (17 mL) for 48 h with constant stirring at room temperature to produce N-succinyl-chitosan, followed by precipitation with 1 M NaOH solution, centrifugation, and washing procedures using alcohol. After decantation, the precipitate of N-succinyl-chitosan was dried under vacuum at 60°C overnight. Next, N-succinyl-chitosan (1 g) was further reacted with octaldehyde (1.02 g) in acetic acid solution. After constant stirring for 4 h, NaBH4 (0.16 g) solution was added dropwise to the mixture and reacted for another 12 h, followed by neutralization with 1 M NaOH and purification by dialysis (molecular weight cut off (MWCO) 10000). After lyophilization, the SOC powder was obtained. The Folate Modification of SOC Micelles: The γ-COOH group of folic acid (22 mg) was activated by DCC/NHS catalyst systems (molar ratio of folate:DCC:NHS = 1:1.2:2) in anhydrous dimethyl sulfoxide (DMSO, 2.5 mL) under mild stirring in the dark for 12 h at room temperature. Then, the activated folic acid was added dropwise into the SOC solution, and reacted with the free amino group on the surface of micelles to form folate-modified SOC micelles. The product was purified by dialysis (MWCO 10000) against distilled water for subsequent study. As shown in Figure 1b, 6


Page 7 of 25

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


the degree of modification of the amino groups in the chitosan molecule by octaldehyde, folate and succinic anhydride is denoted by the ratio of L:J:m:(n-m-L-J), which is close to 4:1:3:2. In addition, to demonstrate the potential of CIS/ZnS QDs as fluorescent probe in cell imaging of micelles, FA-SOC micelles were further conjugated with FITC. QD Solubilization in Water with FA-SOC Micelles. The CIS/ZnS QDs was transferred into water by micelle encapsulation: Typically, 50 µL of the core/shell QDs (~0.8 mg) was precipitated with ethanol to remove excess unreacted precursors and re-dispersed in 50 µL of chloroform. The QDs were then mixed with 1 mL of FA-SOC micelles (10 mg) water solution by sonication in cold-water bath. At room temperature, the chloroform was gradually evaporated, yielding a slightly turbid solution. After centrifugation, the limpid QDs-loaded FA-SOC micelles in water solution was obtained and kept at room temperature for further use. Characterization of QDs and QDs-loaded Micelles. An S2000 eight-channel optical fiber spectrophotometer (Ocean Optics corporation, America), a broadband light source (X-Cite Series 120Q, Lumen Dynamics Group Inc., Canada) and an NL-FC-2.0-763 semiconductor laser (λ=766 nm, Enlight, China) were utilized for the detection of red and NIR fluorescence spectrum. A Shimadzu RF-5301 PC spectrofluorimeter was used to record the PL emission in the visible region. A 754-PC UV–Vis spectrophotometer (JH 754PC, Shanghai, China) was employed for the measurement of UV–Vis spectrum. PL QY of QDs in n-decane was calculated by comparing their integrated emission to that of rhodamine B (QY=90%, absolute ethanol, λex=515 nm, 10 °C) or cypate (QY, 12%).39,40 Optical densities of all solutions at the excitation wavelength were less than 0.3 in order to measure truly the PL spectra of QDs (for the quantum yield determination, optical density used was below 0.1 to avoid reabsorption effects). All optical measurements were performed at room temperature. TEM images were taken on a JEOL JEM-2100 transmission electron microscope with an acceleration voltage of 200 kV. Carbon-coated nickel grids were dipped in the decane solution to deposit QDs onto the film. Elemental compositions of QDs were determined by energy-dispersive spectroscopy (EDS) and X-ray photoelectron spectroscopy (XPS, PHI5000 VersaProbe, ULVAC-PHI Inc., Japan). Powder XRD measurement was carried out using a Philips X’Pert PRO X-ray diffractometer. XRD samples were prepared by evaporating the concentrated QD ethanol suspension on a small glass plate. The size and morphology of micelles and QDs-loaded micelles 7



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 8 of 25

were characterized by Mastersizer 2000 Laser Particle Size Analyzer (LPSA, Malvern, British), combined with TEM. . In vitro Cell Microscopy Imaging. Bel-7402 and A549 cells were cultured in folate-free RPMI 1640 medium with 10% (v/v) calf serum at 37 °C (5% CO2) and grown in a 24-well plate. After seeding for 24 h, the medium was aspirated, and then 0.5 mL of fresh folate-free RPMI 1640 medium containing QDs-loaded FA-SOC micelles ([QDs]=~10 µg/mL) was added to the wells. After incubation for 9 h, the medium was aspirated and the cells were washed with 1× PBS three times for 5 min each. To further monitor the position of the micelles in living cells, we measured the green fluorescence of FA-SOC micelles labeled with FITC (FITC fluorescence was used in combination with the PL of the quantum dots). The fluorescence images of the cells were obtained with an Olympus Fluoview 300 confocal laser scanning system with 488 nm argon laser excitation. In vivo NIR Fluorescence Imaging. In vivo NIR Fluorescence imaging of the QDs-loaded FA-SOC micelles in tumor-bearing nude mice was performed with an in-house-built small animal NIR imaging system, which has been described in detail in our previous reports.41,42 All animal studies conform to the policies in the Animal Management Rules of the Ministry of Health of the People’s Republic of China (document no. 55, 2001) and the guidelines for the Care and Use of Laboratory Animals of China Pharmaceutical University. Briefly, the Bel-7402 or A549 cells were injected subcutaneously in the left armpit of each nude mouse. After the tumors reached about 0.5 cm in diameter, the mice were used for in vivo imaging studies. In a typical imaging experiment, mice (n=3 for each group) were injected intravenously with QDs-loaded FA-SOC micelles (the dose of QDs, ~8 µg/g body weight), and imaged at various time points (0–12 h) post-injection, typically, 4 h post-injection. All fluorescence images were acquired with 300 ms exposure.

Results and Discussion In the framework of the colloidal chemistry approach, there are two main strategies for fabricating high quality CIS QDs. They are the hot-injection method that requires the fast precursor (i.e., sulfur powder) injection,17–19 and the non-injection approach based on the use of the anion precursor (i.e., dodecanethiol) as both ligand and sulfur source.20,21 Comparing these two methods, an obvious advantage over the hot-injection method is that the non-injection approach 8


Page 9 of 25

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


does not require the addition of sulfur precursor. This minimizes the number of reagents and simplifies the steps, used in the synthesis. Hence, we explored the various synthetic parameters affecting the quality of the CuInS2-based QDs using the non-injection approach. As shown in the general scheme (Figure 1a), we loaded the prepared CIS/ZnS core/shell QDs into the hydrophobic core of the FA-SOC micelles because they exhibit bright fluorescence in a wide emission range from yellow, orange, and red to NIR. Finally, by in vitro confocal microscopy and in vivo NIR fluorescence imaging, we confirmed that these highly luminescent QDs can be used as a versatile optical probe to evaluate the targeting of micelles to tumor cells and tissue. Figure 1. (a) General scheme for the formation of (FITC-tagged) QDs-loaded FA-SOC micelles. (b) Synthesis of folate-modified N-succinyl-N´-octyl chitosan. Synthesis and Optical Properties of CIS and CIS/ZnS QDs. An obvious challenge for the synthesis of I–III–VI semiconductors nanocrystals is their ternary composition, compared to the common binary II–VI and III–V semiconductors nanocrystals. As such, balancing the reactivity of two cationic precursors is critical for the control of the stoichiometric ratio between the Group I and the Group III elements in a given sample. In 2009, Peng et al. reported that the control of the Cu:In stoichiometric ratio in the nanocrystals can be achieved by replacing the commonly used fatty acid with the reactivity-controlling ligands for Cu, dodecanethiol.17 Since then, several efficient methods have been developed for the synthesis of high quality ternary CuInS2 QDs.18–24 In this study, we developed a facile method based on previous reports for the preparation of CIS core QDs.17,20 The mixed system of Cu(Ac)2, In(Ac)3 and dodecanethiol (DDT) in 1-octadecene was selected as the starting reagents, where DDT served as both the sulfur source and the stabilizing ligand for the formed QDs, thereby eliminating the use of additional sulfur source (such as commonly used elemental sulfur 17–19). As the temperature increased, the color of the reaction solution correspondingly first changed from turbid to clear, and then from slight yellow to yellow, red, and finally black, indicating the nucleation and subsequent growth of CuInS2 QDs. In addition to the reaction time, other crucial experimental variables, such as the amount of DDT, the reaction temperature, and the Cu/In feed molar ratio, were also orderly varied to systematically investigate the influence on the PL properties of the resulting CIS QDs. The series of results obtained are shown in Figures S1 and S2. The absorption spectra of the 9



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 10 of 25

resulting CIS QDs were similar to those of typical I–III–VI semiconductor QDs, exhibiting a broad shoulder with a trail in the long-wavelength direction. In contrast to previous reports,18,23 the typical PL spectra from this study are narrow and characterized by well-defined single peaks, suggesting structural homogeneity of the QDs. Our findings can be summarized as follows: (1) There is an optimal growth time for achieving the strongest PL emission (Figure S1). In fact, prolonging the reaction time may not be a favorable way for tuning the PL emission of CIS QDs. (2) The amount of DDT was found to play a more important role in determining the PL emission intensity rather than the peak position (Figure S2a). The optimal amount of DDT is 2 mL under our reaction conditions. (3) The PL peak position of QDs was mainly controlled by the reaction temperature (Figure S2b). By increasing the temperature from 215 to 280 ºC, the PL peak red-shifted gradually from 675 to 785 nm. (4) The Cu:In feed ratio also influences strongly the QD PL properties (Figure S2c). To our knowledge, this influence has not been investigated intensively in the previous studies.16–21 By varying the ratio of Cu:In from 1:1 to 0.15:1.3, the QD PL peak position could be further tuned from 700 to 655 nm. Further decrease in the feed ratio does favor stronger PL intensity. For instance, the plain CIS QDs with the Cu:In feed ratio of 0.5:1.17 routinely exhibit PL QY of 15%, while the record value is up to 18%. As described above, high quality CIS QDs have been synthesized by refining the reaction conditions, such as the reaction time, the amount of DDT, the temperature, and the Cu:In feed ratio. Figure 2 shows the normalized PL spectra of the resulting CIS QDs, indicating that the tunable emission of the plain CIS QDs can cover both the visible and near-infrared window (from 620 to 870 nm) with a typical narrow bandwidth (90–120 nm). Interestingly, bright fluorescence of the prepared CIS QD samples was observed under a UV lamp (inset of Figure 2), confirming that the resulting plain CIS QDs had favorable QY. This is evidenced by the determined maximum PL QY of up to 15–18%, which is superior to previously reported 3–5%. Although the PL wavelength can also be tuned by a programmed heating process, the QDs obtained in the initial stage may have no practical use due to the low PL (Figure S3). Figure 2. PL spectra of the prepared oil-soluble CIS QDs. Inset is the digital photographs of the samples taken under a UV lamp. The fluorescence of the QDs became darker when the emission wavelength gradually red-shifted out of the visible region.

10


Page 11 of 25

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


Surface passivation of QDs by means of the epitaxial overgrowth of another inorganic shell material with a higher band gap has been proven to be a versatile approach to improve the PL QY and stability of quantum dots. Although recent studies using diverse methods to achieve good optical properties have been reported, the synthetic procedures are still relatively complicated.20 To further improve the PL QY of the CIS QDs in our case, ZnS, rather than CdS, was chosen as the shell material based on the following reasons: (i) it has a much larger band gap (Eg = 3.6 eV) relative to CIS (Eg = 1.5 eV), exhibiting a type I band alignment with CIS that results in a more effective surface passivation; (ii) the crystal structure of CIS can be described as a derivative of the ZnS zinc blende structure, in which the zinc positions are occupied by Cu and In, respectively, and the lattice mismatch between CIS and ZnS is relatively low (2–3%); (iii) most importantly, ZnS is a near-ideal shell material due to its chemical stability and nontoxic character.18,23 We adapted the method reported by Zhong et al.18 to develop a one-pot approach for fabricating the CIS/ZnS core/shell structure in situ (i.e., in the initial CIS QD reaction mixture). Because of the large excess of DDT (sulfur precursor) used in the preparation of CIS core QDs, no additional sulfur source was added in the coating procedure of ZnS shell. To avoid the formation of separate ZnS nanoparticles, Zn stock solution was injected after the temperature of the reaction mixture was lowered to 230 ºC or below. In this segment of the study, we focused on the influence of the growth time and the precursor Zn:Cu feed ratio on the PL properties of the formed CIS/ZnS core/shell QDs. Figures 3a and 3b show the temporal evolutions of both absorption and PL spectra during the growth of a ZnS shell, where the Zn:Cu feed ratio was set to 4:1. Although the overcoating with ZnS did not affect absorption spectra significantly, it resulted in a dramatic improvement of the PL emission intensity. In the initial 20 min of growth time, the PL QY increased from ~13% to 60% – up to ~5 times. Further extending the growth time to 30 min did not significantly change the PL QY. We also observed that the precursor Zn:Cu feed ratio plays a key role in determining the PL properties of the QDs (Figure 3c). As the Zn stock solution was injected continuously in batches, PL QY of the resulting core/shell QDs increased gradually. When the ratio of Zn:Cu is 5:1, the highest PL QY are achieved and reached 80%. This QY is among the highest reported for ternary CIS QDs to date,16–24 and may pave the way for the use of CIS QDs in many applications such as light-emitting devices or fluorescent biological labeling and sensing. In addition, the overgrowth 11



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 12 of 25

with ZnS leads to blue-shifted PL spectrum in all cases, which is likely indicative of etching of the core material under the shell growth conditions.21 The etching or the cation exchange can reduce the core size, resulting in an increase of the band-gap energy. Figure 3. Evolution of both (a) absorption and (b) PL spectra of the resulting QDs during the growth of a ZnS shell, starting from the CIS core QDs synthesized at 215 ºC with a Cu:In feed ratio of 0.5:1.17. Inset of panel b is the digital photographs of the corresponding samples taken under UV light. (c) Evolution of PL spectra of the formed QDs with the deposition of the ZnS shell, starting from the core QDs synthesized at 240 ºC with a Cu:In feed ratio of 0.8:1.07. Absorption and PL spectra of the resulting CIS/ZnS core/shell QDs starting from different CIS core QDs are shown in Figure 4. As noted above, the PL wavelength of the CIS core can be conveniently tuned from 620 to 870 nm by refining the reaction conditions (Figure 2). Similarly, the PL wavelengths of the resulting CIS/ZnS core/shell structure can also be tuned correspondingly from 550 to 800 nm by varying the starting QDs core. This is illustrated by the PL color of the resulting CIS/ZnS QDs, which covers most of the visible and near-infrared window with high brightness from yellow, orange, and red to the NIR (Figure 4b). The PL QYs of the CIS/ZnS core/shell QDs that emit in the range from 550 to 700 nm are more than 50%, while the maximum value is as high as 80% before decreasing gradually to ~20% as the emission wavelength further red-shifts toward 800 nm. The above results suggest that these core/shell QDs exhibited a single, well-defined emission band, with a slightly higher PL QY up to about 80% and a wider emission peak range from 550 to 800 nm than those synthesized in recent studies.20–24 Besides, the formation of core/shell structure also improves the storage stability of the CIS QDs. Figure 4. Absorption (a) and PL (b) spectra of the resulting CIS/ZnS core/shell QDs. Inset of panel b is the digital photographs of the corresponding samples taken under UV lamp. We employed TEM and powder XRD to characterize the morphology and crystal structure of CIS core and CIS/ZnS core/shell QDs (Figure S4 and S5). As expected, a visible increase in size (from ~2.5 nm to ~4 nm) was observed upon shell growth. The CIS/ZnS core/shell QDs contain roughly 3–4 monolayers of ZnS shell, resulting in a slight broadening of the QD size distribution compared to the core-alone QDs. The experimental results from HRTEM imaging, SAED analysis and XRD measurement all indicate that high-quality CIS core and CIS/ZnS core/shell QDs have a

12


Page 13 of 25

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


cubic structure. We also investigated the elemental compositions of the CIS core QDs and the corresponding CIS/ZnS core/shell QDs and confirmed the results by XPS and EDX analyses (Figures S6 and S7). It was found that the chemical compositions of these QDs were consistent with the expected stoichiometries, which could be controlled by tuning the precursor ratio.

Loading and Water Transfer of CIS/ZnS QDs with Folate-modified SOC Micelles. Folate-modified N-succinyl-N´-octyl chitosan (FA-SOC) micelles synthesized in our recent report are a promising drug carrier system.37 Therefore, FA-SOC micelles were used to assess whether the highly luminescent CIS/ZnS QDs we prepared can act as a new fluorescent probe for evaluating tumor targeting of drug carriers. Here, FA-SOC micelles were considered as a drug carrier model. Detailed scheme for the formation of the micelles and water transfer of oil-soluble CIS/ZnS QDs with micelles is shown in Figure 1. After chemically modifying chitosan with succinic acid and octaldehyde, the amphiphilic polymer molecules formed spontaneously assembled into a spherical micelle having a hydrophobic core and a hydrophilic shell.37,38 Due to the coordinate interaction between thiol groups and cations on the particle surface, the CIS/ZnS QDs capped by one monolayer of DDT molecules are hydrophobic. Thus, the hydrophobic interaction may entrap spontaneously the QDs into the hydrophobic cores of micelles, where the loading content of FA-SOC micelles for hydrophobic QDs could be up to ~20% (the loading content=mass of the QDs loaded in micelles/mass of the QDs-loaded micelles × 100%). Size distributions and morphologies of the initial FA-SOC micelles and the resultant QDs-loaded micelles were first examined using dynamic light scattering (DLS) and TEM. As shown in Figures 5a and 5b, the loading of oil-soluble CIS/ZnS QDs into FA-SOC micelles has no major effect on the average hydrodynamic size of micelles (~200 nm). The TEM characterization clearly indicates the formation of the QDs/micelle nanocomposites (NCs), where spherical micelles loaded with QDs (~150 nm in diameter) were observed (Fig. 5c). The hydrophobic CIS/ZnS QDs (~4 nm in diameter) were located in the hydrophobic cores of micelles, and their size, morphology and crystal structure were retained. The decrease in the TEM diameter with respect to the hydrodynamic diameter is due to drying of the micelles in the process of TEM sample preparation.

13



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 14 of 25

Figure 5. Size distributions of (a) the initial FA-SOC micelles and (b) the resulting QDs-loaded micelles measured by DSL. (c) Typical TEM image of the QDs-loaded micelles. Insets of panel c are high-resolution TEM image (top) and corresponding SAED pattern (middle) of the CIS/ZnS QDs encapsulated in micelles, and higher magnification TEM image of a QDs/micelle nanocomposite (bottom), respectively. The PL spectra of the initial oil-soluble CIS/ZnS QDs and the resulting water-soluble QDs-loaded FA-SOC micelles are shown in Figure 6a. After transferring the product into water via micelle encapsulation, the PL intensity decreased by about 40%, accompanied by a visible red-shift of the emission peak from 560 to 590 nm. For clarity, the corresponding photographs of the two studied solutions taken under room light and UV light excitation are shown in the inset of Figure 6a. A similar decrease in PL intensity was also observed when using other oil-soluble CIS/ZnS QDs with different emission wavelength as the starting material (Figure 6b). Nonetheless, since the absolute value of initial QY for CIS/ZnS QDs is high (see Figure 4, where the maximum value of QY is as high as 80%), the retained QY value for the resulting QDs-loaded micelles after transfer into water is still sufficiently high for their use in biological applications. Moreover, the bright PL of the QDs-loaded micelles in water can be retained for 3–5 days without observable quenching, which further favors their potential use as unique materials for biomedical optical imaging. Figure 6. PL spectra of (a) representative visible light-emitting CIS/ZnS QDs and (b) two representative NIR-emitting QDs in chloroform and after transfer into water via FA-SOC micelles. Insets of panel a are the corresponding photographs of the initial QDs and the resulting QDs-loaded micelles taken under room light and UV light excitation. In vitro and in vivo Fluorescence Imaging Using CIS/ZnS QDs. In the previous study, we used organic dyes as fluorescent probes to confirm that folate modification can enhance the targeting ability of SOC micelles to folate receptor-positive Bel-7402 tumor.37 In that case, two different dyes must be used: green-emitting fluorescein (or other visible light-emitting dye) was loaded for in vitro cell imaging, and NIR light-emitting cypate for in vivo tumor targeting evaluation. Here, we note that the CIS/ZnS QDs exhibited strong PL emission in the wide range of 550–800 nm, and could be successfully transferred into water using FA-SOC micelles, as described above. Hence, we explored the utilization of these heavy-metal-free QDs for in vitro as well as in vivo targeting of FA-SOC micelles. Before optical imaging, we briefly assessed the cytotoxicity of the 14


Page 15 of 25

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


QDs-loaded FA-SOC micelles by using standard MTT assay (Figure S8). The results showed that the cytotoxicity of the QDs-loaded micelles is low due to many factors, including the use of the elements with low toxicity. To determine the potential of using these highly luminescent CIS/ZnS QDs as optical probes for in vitro fluorescence imaging of the micelles distribution in living cells, FA receptor-positive Bel-7402 cells were co-incubated with 625 nm emitting QDs-loaded FA-SOC micelles. The FA-SOC micelles were also labeled with green fluorescent dye (see Figure S9) in order to visualize the position of micelles in cells. As shown in Figure 7, numerous large and punctuate fluorescent structures were found in all observed cells, and the green fluorescence signals co-localized with the red ones (the green and red fluorescence signals are attributed to fluorescein and the encapsulated QDs, respectively). In particular, the overlay of the fluorescence and bright field images in Figure 7d indicates clearly their high labeling specificity on FA receptor expressing Bel-7402 cells. On the contrary, a dramatic reduction in the fluorescence signals were observed when FA receptor-negative A549 cells were used as the control. These data further confirm that FA-SOC micelles can efficiently target FA receptor-positive cells. It is important to note that the green fluorescent organic dye molecules (fluorescein) can dissociate from the backbone of micelles. Subsequently, free dye molecules might diffuse into and stain the cells (Figure 7b) due to good water-solubility and small molecular weight. This problem is occasionally encountered when using organic dyes as fluorescent probes for cell imaging. Figure 7. Optical microscopy of live FA receptor-positive Bel-7402 cells incubated with FITC-labeled and QDs-loaded micelles: (a) differential interference contrast (DIC), (b) fluorescein, (c) 625 nm emitting QDs-loaded micelles, (d) the merged image of the fluorescence images and DIC image. λex = 488 nm; Scale bar: 30 µm. In addition to in vitro fluorescence microscopy, we also explored the performance of the highly fluorescent CIS/Zn QDs for in vivo tracking of the micelles in mouse, where NIR fluorescent QDs (for instance, em: 800 nm) were used instead of visible-light emitting QDs. The NIR QDs-loaded micelles were injected into tumor-bearing nude mice via the tail vein. The in vivo fluorescence images were captured with the NIR imaging system at different time points (Figure 8). Before tail-vein injection, the background fluorescence at 766 nm excitation was very weak (Figure 8a). At 4 h post injection, an obvious accumulation of fluorescence at the tumor site 15



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 16 of 25

and the liver was observed for the FA receptor-positive Bel-7402 tumor-bearing nude mice (Figure 8b). However, administering the same dose of NIR QDs-loaded micelles to the nude mice bearing FA receptor-negative A549 tumors did not result in obvious fluorescence signal at the tumor site at the same 4 h post-injection (Figure 8c). These results from in vivo imaging further confirm the high targeting ability of FA-SOC micelles to FA receptor-positive Bel-7402 tumor. Figure 8. NIR fluorescence images of tumor-bearing mice after intravenous injection with 800 nm emitting QDs-loaded FA-SOC micelles for 4 h: (a) before tail-vein injection, (b) FA receptor-positive Bel-7402 tumor-bearing nude mouse, (c) FA receptor-negative A549 tumor-bearing nude mouse. The tumor site is indicated by white circle. To demonstrate, for the first time, the capability for in vivo multiplex imaging, two different NIR-emitting QDs-loaded micelles were administered into the mouse through subcutaneous injection (PL spectra of the two QDs-loaded micelles are shown in Figure 6b). Before injection, the background fluorescence obtained at 660 nm excitation and captured with a 700 nm long pass filter was very weak (Figure 9a). However, the two legs (Figure 9b, pseudo-color) injected with the nanocomposites (NCs) became much brighter than other parts of the body. Further spectral analysis (Figure S10) confirmed that the fluorescence signal of the bright region in the leg originated from the injected QDs/micelle NCs. When using an 800 nm long pass filter (and 766 nm excitation), the recorded fluorescence signal of the right leg was practically insignificant, while the fluorescence signal of the leg remained very bright (Figures 9c and 9d). This preliminary result indicates that CIS/ZnS QDs could serve as an excellent bioprobes for multiplexed imaging of molecular targets in vivo by selecting the appropriate excitation wavelength or filter. Figure 9. Multiplex NIR fluorescence imaging of mouse administered with two different NIR-emitting QDs-loaded micelles by subcutaneous injection (the right leg, 720 nm-emitting NCs; the left leg, 800 nm-emitting NCs): (a) before injection, (b) λex = 660 nm, a 700 nm long pass filter, (c) λex = 660 nm, an 800 nm long pass filter, (d) λex = 766 nm, an 800 nm long pass filter.

Caution: Although we did not observe the obvious toxicity of the CIS/ZnS QDs in cells and mice at the low injection dose used, detailed acute and chronic toxicity study is needed to comprehensively determine the toxic effects of these new materials. Accordingly, the use of these QDs for in vivo imaging experiments should be limited to mice or other small animals at low doses. Conclusion By systematically refining the reaction conditions, highly PL CIS/ZnS QDs without any toxic 16


Page 17 of 25

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


heavy metals were synthesized via a facile non-injection approach. The QDs not only exhibit brighter PL emission, but also have a wide tunable PL, ranging from yellow to NIR. With FA-SOC micelles, these oil-soluble QDs can be effectively formulated in water, where the morphology, crystal structure, and major PL emission of the initial CIS/ZnS QDs were retained. Using in vitro and in vivo optical imaging techniques, the less cytotoxic CIS/ZnS QDs prepared were shown, for the first time, to be a versatile fluorescent probe for (multicolor) biomedical imaging in different spatial scales (i.e., in vitro cell imaging and in vivo small animal imaging). The ensemble of the results provides an accessible and straightforward platform to evaluate the tumor-targeting properties of the CIS/ZnS QDs micelles.

Acknowledgements This work was financially supported by the National Natural Science Foundation of China (No. 30800257, No. 30970776, No. 31050110123 and No. 81071194), the Natural Science Foundation of Jiangsu Province (No. BK2011634), and the Fundamental Research Funds for the Central Universities (Program No. JKP2011017). Supporting Information Supporting results mentioned in the text (PDF) include absorption and PL spectra of CIS QDs obtained under different conditions; high-resolution TEM images, XRD patterns, SAED pattern, XPS spectra, and EDX spectra of QDs; PL spectrum of FITC-labeled FA-SOC micelles loaded with red CIS/ZnS QDs; PL spectra of the bright site in the leg. This material is available free of charge via the Internet at http://pubs.acs.org.

References (1) Achilefu, S. Chem. Rev. 2010, 110, 2575–2578. (2) Willmann, J. K.; van Bruggen, N.; Dinkelborg, L. M.; Gambhir, S. S. Nat. Rev. Drug Discov. 2008, 7, 591–607. (3) Becker, A.; Hessenius, C.; Licha, K.; Ebert, B.; Sukowski, U.; Semmler, W.; Wiedenmann, B.; Grötzinger, C. Nat Biotechnol. 2001, 19, 327–331. (4) Edwards, W. B.; Xu, B.; Akers, W.; Cheney, P. P.; Liang, K.; Rogers, B. E.; Anderson, C. J.; Achilefu, S. Bioconjugate Chem. 2008, 19, 192–200. (5) Law, W.C.; Yong, K.T.; Roy, I.; Ding, H.; Hu, R.; Zhao, W.W.; Prasad, P. N. Small 2009, 5, 1302–1310. (6) 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. Science 2005, 307, 538–544. 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

Page 18 of 25

(7) Kim, S.; Lim, Y. T.; Soltesz, E. G.; De Grand, A. M.; Lee, J.; Nakayama, A.; Parker, J. A.; Mihaljevic, T.; Laurence, R. G.; Dor, D. M.; Cohn, L. H.; Bawendi, M. G.; Frangioni, J. V. Nat Biotechnol. 2004, 22, 93–97. (8) Gao, X. H.; Cui, Y. Y.; Levenson, R. M. ; Chung, L. W. K. ; Nie, S. M. Nat Biotechnol. 2004, 22, 969–976. (9) Lewinski, N.; Colvin, V.; Drezek, R. Small 2008, 4, 26–49. (10) Chen, L. D.; Liu, J.; Yu, X. F.; He, M.; Pei, X. F.; Tang, Z. Y.; Wang, Q. Q.; Pang, D. W.; Li, Y. Biomaterials 2008, 29, 4170–4176. (11) Smith, A. M.; Duan, H. W.; Mohs, A. M.; Nie, S. M. Adv. Drug Delivery Rev. 2008, 60, 1226–1240. (12) Deng, D. W.; Xia, J. F.; Cao, J.; Qu, L. Z.; Tian, J. M.; Qian, Z. Y.; Gu Y. Q.; Gu, Z. Z. J. Colloid Interface Sci. 2012, 367, 234–240. (13) Xie, R.G.; Peng, X.G. J. Am. Chem. Soc. 2009, 131, 10645–10651. (14) Allen, P. M.; Liu, W.; Chauhan, V. P.; Lee, J.; Ting, A. Y.; Fukumura, D.; Jain, R. K.; Bawendi, M. G. J. Am. Chem. Soc. 2010, 132, 470–471. (15) Park, J.; Dvoracek, C.; Lee, K. H.; Galloway, J. F.; Bhang, H. C.; Pomper, M. G.; Searson, P. C. Small 2011, 7, 3148–3152. (16) Zhong, H. Z.; Zhou, Y.; Ye, M. F. ; He, Y. J.; Ye, J. P.; He, C.; Yang, C. H.; Li, Y. F. Chem. Mater. 2008, 20, 6434–6443. (17) Xie, R. G.; Rutherford, M.; Peng, X. G. J. Am. Chem. Soc. 2009, 131, 5691–5697. (18) Zhang, W. J.; Zhong, X. H. Inorg. Chem. 2011, 50, 4065–4072. (19) Zhang, J.; Xie, R. G.; Yang, W. S. Chem. Mater. 2011, 23, 3357–3361. (20) Li, L.; Pandey, A.; Werder, D. J.; Khanal, B. P.; Pietryga, J. M.; Klimov, V. I. J. Am. Chem. Soc. 2011, 133, 1176–1179. (21) Park, J.; Kim, S. W. J. Mater. Chem. 2011, 21, 3745–3750. (22) Song, W. S.; Yang, H. Chem. Mater. 2012, 24, 1961–1967. (23) Li, L.; Daou, T. J.; Texier, I.; Chi, T. T. K.; Liem, N. Q.; Reiss, P. Chem. Mater. 2009, 21, 2422–2429. (24) Pons, T.; Pic, E.; Lequeux, N.; Cassette, E.; Bezdetnaya, L.; Guillemin, F.; Marchal, F.; Dubertret, B. ACS Nano, 2010, 4, 2531–2538. 18


Page 19 of 25

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


(25) Croy, S. R.; Kwon, G. S. Curr. Pharm. Design. 2006, 12, 4669–4684. (26) Zhang, L.; Gu, F. X.; Chan, J. M.; Wang, A. Z.; Langer, R. S.; Farokhzad, O. C. Clin. Pharmacol. Ther. 2008, 83, 761–769. (27) Nasongkla, N.; Bey, E.; Ren, J.; Ai, H.; Khemtong, C.; Guthi, J. S.; Chin, S. F.; Sherry, A. D. Boothman, D. A.; Gao, J. Nano Lett. 2006, 6, 2427–2430. (28) Zhang, C.; Qu, G. W.; Sun, Y. J.; Wu, X. L.; Yao, Z.; Guo, Q. L.; Ding, Q. L.; Yuan, S. T.; Shen, Z. L.; Ping, Q. N.; Zhou, H. P. Biomaterials 2008, 29, 1233–1241. (29) Bhattarai, N.; Gunn, J.; Zhang, M. Adv. Drug Deliv. Rev. 2010, 62, 83–99. (30) Mao, S.; Sun, W.; Kissel, T. Adv. Drug Deliv. Rev. 2010, 62, 12–27. (31) Hua, D. B.; Jiang, J. L.; Kuang, L. J.; Jiang, J.; Zheng, W.; Liang, H. J. Macromolecules 2011, 44, 1298–1302. (32) Min, K. H.; Kim, J. H.; Bae, S. M.; Shin, H.; Kim, M. S.; Park, S.; Lee, H.; Park, R.W.; Kim, I. S.; Kim, K.; Kwon, I. C.; Jeong, S. Y.; Lee, D. S. J. Control. Release 2010, 144, 259–266. (33) Shan, L. L.; Xue, J. P.; Guo, J.; Qian, Z. Y.; Achilefu, S.; Gu, Y.Q. Bioconjugate Chem. 2011, 22, 567–581. (34) Shan, L. L.; Cui, S. S.; Du, C. L.; Wan, S. N.; Qian, Z. Y. Achilefu, S.; Gu, Y. Q. Biomaterials 2012, 33, 146–162. (35) Chen, J.; Chen, H. Y.; Cui, S. S.; Xue, B. ; Tian, J. M.; Achilefu, S.; Gu, Y. Q. J. Mater. Chem. 2012, 22, 5770–5783. (36) Anderson, R. E.; Chan, W. C. W. ACS Nano 2008, 2, 1341–1352. (37) Zhu, H. Y.; Liu, F.; Guo, J.; Xue, J. P.; Qian, Z. Y.; Gu, Y. Q. Carbohyd. Polym. 2011, 86, 1118–1129. (38) Cui, S. S.; Chen, H. Y.; Zhu, H. Y.; Tian, J. M.; Chi, X. M.; Qian, Z. Y.; Achilefu, S.; Gu Y. Q. J. Mater. Chem. 2012, 22, 4861–4873. (39) Kubin, R. F.; Fletcher, A. N. J. Lumin. 1982, 27, 455–462. (40) Ye, Y. P.; Bloch, S.; Xu, B.; Achilefu, S. Bioconjugate Chem. 2008, 19, 225–234. (41) Zhang, J.; Chen, H. Y.; Xu, L.; Gu, Y. Q. J. Control. Release 2008, 131, 34–40. (42) Zhang, J.; Deng, D. W.; Qian, Z. Y.; Liu, F.; Chen, X. Y.; An, L. X.; Gu, Y. Q. Pharmacol. Res. 2010, 27, 46–55. 19



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 20 of 25

Captions for Figures Figure 1. (a) General scheme for the formation of (FITC-tagged) QDs-loaded FA-SOC micelles. (b) Synthesis of folate-modified N-succinyl-N´-octyl chitosan. Figure 2. PL spectra of the prepared oil-soluble CIS QDs. Inset is the digital photographs of the samples taken under a UV lamp. The fluorescence of the QDs became darker when the emission wavelength gradually red-shifted out of the visible region. Figure 3. Evolution of both (a) absorption and (b) PL spectra of the resulting QDs during the growth of a ZnS shell, starting from the CIS core QDs synthesized at 215 ºC with a Cu:In feed ratio of 0.5:1.17. Inset of panel b is the digital photographs of the corresponding samples taken under UV light. (c) Evolution of PL spectra of the formed QDs with the deposition of the ZnS shell, starting from the core QDs synthesized at 240 ºC with a Cu:In feed ratio of 0.8:1.07. Figure 4. Absorption (a) and PL (b) spectra of the resulting CIS/ZnS core/shell QDs. Inset of panel b is the digital photographs of the corresponding samples taken under UV lamp. Figure 5. Size distributions of (a) the initial FA-SOC micelles and (b) the resulting QDs-loaded micelles measured by DSL. (c) Typical TEM image of the QDs-loaded micelles. Insets of panel c are high-resolution TEM image (top) and corresponding SAED pattern (middle) of the CIS/ZnS QDs encapsulated in micelles, and higher magnification TEM image of a QDs/micelle nanocomposite (bottom), respectively. Figure 6. PL spectra of (a) representative visible light-emitting CIS/ZnS QDs and (b) two representative NIR-emitting QDs in chloroform and after transfer into water via FA-SOC micelles. Insets of panel a are the corresponding photographs of the initial QDs and the resulting QDs-loaded micelles taken under room light and UV light excitation. Figure 7. Optical microscopy of live FA receptor-positive Bel-7402 cells incubated with FITC-labeled and QDs-loaded micelles: (a) differential interference contrast (DIC), (b) fluorescein, (c) 625 nm emitting QDs-loaded micelles, (d) the merged image of the fluorescence images and DIC image. λex = 488 nm; Scale bar: 30 µm. Figure 8. NIR fluorescence images of tumor-bearing mice after intravenous injection with 800 nm emitting QDs-loaded FA-SOC micelles for 4 h: (a) before tail-vein injection, (b) FA receptor-positive Bel-7402 tumor-bearing nude mouse, (c) FA receptor-negative A549 tumor-bearing nude mouse. The tumor site is indicated by white circle. Figure 9. Multiplex NIR fluorescence imaging of mouse administered with two different NIR-emitting QDs-loaded micelles by subcutaneous injection (the right leg, 720 nm-emitting NCs; the left leg, 800 nm-emitting NCs): (a) before injection, (b) λex = 660 nm, a 700 nm long pass filter, (c) λex = 660 nm, an 800 nm long pass filter, (d) λex = 766 nm, an 800 nm long pass filter.

20


Page 21 of 25

Figure 1.

Figure 2.

PL Intensity (a.u.)

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


500

600

700

800

900

1000

1100

Wavelength (nm)

21



Figure 3. 0.6

1000

initial core QDs 5 min, core/shell QDs 10 20 30

0.4

PL Intensity (a.u.)

Absorbance

a

0.2

0.0 400

500

600

700

575 nm, QY=60%

b 500

0 400

800

initial core QDs 5 min, core/shell QDs 10 20 30

630 nm, QY=13%

500

600

700

800

900

1000

Wavelength (nm)

Wavelength (nm)

PL Intensity (a.u.)

2500

c

630 nm, QY = 80%

2000

1500

1000

702 nm, QY = 15%

500

500

600

700

800

Zn : Cu feed ratio 0 1 2 3 4 5 6 900

1000

1100

Wavelength (nm)

a

400

How to prepare high-quality CIS QDs? 1. Selecting the optimal growth time 2. Using the optimal amount of DDT 3. Tuning the teaction temperature 4. Varing the Cu/In feed ratio 5. ZnS shell coating

500

600

700

Wavelength (nm)

800

900

PL Intensity (a.u.)

Figure 4.

Absorbance

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 22 of 25

400

b

500

600

700

800

900

1000 1100

Wavelength (nm)

22


Page 23 of 25

Figure 5.

a

50

0

0

b

100

Particle Counts

Particle counts

100

100

200

300

400

50

0

500

100

200

300

400

500

Hydrodynamic Diameter (nm)

Hydrodynamic Diameter (nm)

a

initial oil-soluble QDs in CHCl3 QDs-loaded micelles in H2O

500

600

700

800

Wavelength (nm)

900

1000

PL Intensity (a.u.)

Figure 6.

PL Intensity (a.u.)

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


500

b

in CHCl3

in H2O

600

700

800

900

1000

1100

Wavelength (nm)

23



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 24 of 25

Figure 7.

Figure 8.

Figure 9.

24


Page 25 of 25

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


By in vitro and in vivo optical imaging techniques, less cytotoxic, oil-soluble, highly luminescent CuInS2/ZnS QDs with tunable emission from 550 to 800 nm have been firstly proven to be a versatile fluorescent probe for (multicolor) bioimaging of micelles across different spatial scales, that is, in vitro cell imaging and in vivo small animal imaging. 204x176mm (96 x 96 DPI)


ZnS Quantum Dots for In vitro and In vivo Bioimaging - American

Recommend Documents