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Serum-stable quantum dot--protein hybrid nanocapsules for optical bio-imaging
This content has been downloaded from IOPscience. Please scroll down to see the full text. 2014 Nanotechnology 25 175702 (http://iopscience.iop.org/0957-4484/25/17/175702) View the table of contents for this issue, or go to the journal homepage for more
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Nanotechnology Nanotechnology 25 (2014) 175702 (12pp)
doi:10.1088/0957-4484/25/17/175702
Serum-stable quantum dot–protein hybrid nanocapsules for optical bio-imaging Jeong Yu Lee1, Dong Heon Nam1, Mi Hwa Oh2, Youngsun Kim1, Hyung Seok Choi1, Duk Young Jeon1,3, Chan Beum Park1,3,4 and Yoon Sung Nam1,2,3,4 1
Department of Materials Science and Engineering, Korea Advanced Institute of Science and Technology, Daejeon 305-701, Republic of Korea 2 Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon 305-701, Republic of Korea 3 KAIST Institute for NanoCentury (KINC), Korea Advanced Institute of Science and Technology, Daejeon 305-701, Republic of Korea 4 KAIST Institute for BioCentury (KIB), Korea Advanced Institute of Science and Technology, Daejeon 305-701, Republic of Korea E-mail: [email protected] Received 23 December 2013, revised 27 February 2014 Accepted for publication 6 March 2014 Published 10 April 2014 Abstract
We introduce shell cross-linked protein/quantum dot (QD) hybrid nanocapsules as a serumstable systemic delivery nanocarrier for tumor-targeted in vivo bio-imaging applications. Highly luminescent, heavy-metal-free Cu0.3InS2/ZnS (CIS/ZnS) core-shell QDs are synthesized and mixed with amine-reactive six-armed poly(ethylene glycol) (PEG) in dichloromethane. Emulsification in an aqueous solution containing human serum albumin (HSA) results in shell cross-linked nanocapsules incorporating CIS/ZnS QDs, exhibiting high luminescence and excellent dispersion stability in a serum-containing medium. Folic acid is introduced as a tumortargeting ligand. The feasibility of tumor-targeted in vivo bio-imaging is demonstrated by measuring the fluorescence intensity of several major organs and tumor tissue after an intravenous tail vein injection of the nanocapsules into nude mice. The cytotoxicity of the QDloaded HSA-PEG nanocapsules is also examined in several types of cells. Our results show that the cellular uptake of the QDs is critical for cytotoxicity. Moreover, a significantly lower level of cell death is observed in the CIS/ZnS QDs compared to nanocapsules loaded with cadmiumbased QDs. This study suggests that the systemic tumor targeting of heavy-metal-free QDs using shell cross-linked HSA-PEG hybrid nanocapsules is a promising route for in vivo tumor diagnosis with reduced non-specific toxicity. S Online supplementary data available from stacks.iop.org/NANO/25/175702/mmedia Keywords: albumin, quantum dots, cytotoxicity, optical bio-imaging, targeted delivery (Some figures may appear in colour only in the online journal)
1. Introduction
nanocrystals have been used as a negative contrast agent for magnetic resonance imaging [5, 6]. Colloidal gold nanoparticles have been explored as an effective probe for computed tomography, surface-enhanced Raman scattering and photoacoustic tomography [7–9]. They have also been used to
Inorganic nanocrystals have been studied for a wide range of applications including biomedical imaging, diagnostics, biosensing and therapy [1–4]. For instance, iron oxide 0957-4484/14/175702+12$33.00
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© 2014 IOP Publishing Ltd Printed in the UK
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generate photothermal heating that can destroy tumor cells [10–12]. Semiconductor nanocrystals, known as quantum dots (QDs), have received increasing amounts of attention as a promising alternative to organic chromophores due to their high quantum efficiency, photochemical stability and spectral tunability from the visible to the near-infrared (NIR) regions [13, 14]. Accordingly, biomedical applications of QDs have been widely studied for photodynamic therapy, cell tracking and cancer detection [12, 15, 16]. However, the intrinsic toxicity derived from heavy metals (e.g., cadmium, tellurium and lead) has introduced some concern regarding their biological applicability [17]. Over the past few decades, significant efforts have been made to achieve desirable optical and electrical properties of various QDs, including binary II–VI and III–V compound (e.g., CdSe, CdTe, ZnSe, GaP, InAs and InP) and tertiary compound (e.g., CuInSe2, CuInS2, GaAsP, AgInS2 and AlGaAs) QDs [18–23]. In particular, heavy-metal-free QDs have gained an increasing amount of attention due to environmental and toxicological issues related to the applications of QDs in the areas of energy conversion and bio-imaging. One promising heavy-metal-free QD is CuInS2 (CIS), a I-IIIV semiconductor with an emission wavelength that can be modulated from the visible light to NIR regions with a high quantum yield [23–25]. It has been shown that CIS QDs, when transferred into an aqueous solution via ligand exchange or phospholipid micelle encapsulation, can be used for the in vivo imaging of major organs and regional lymph nodes [16, 23]. However, the in vivo application of CIS QDs for cancer diagnostics has not been well studied and has just started to be considered very recently [26]. In addition, no direct comparison of CIS QDs with Cd-based QDs in terms of cytotoxicity has been reported. In this work, we proposed a new tumor-targeting QDprotein hybrid nanostructure based on shell cross-linked nanocapsules (NCs) and CIS/ZnS QDs, and investigated the cytotoxicity and in vivo applicability of the nanoscale hybrid platform for tumor-targeted optical bio-imaging. The shells of the NCs were prepared by chemically cross-linking human serum albumin (HSA) with amine-reactive six-armed poly (ethylene glycol) (PEG), generating a stable interface for the in vivo application of the encapsulated QDs [27]. The dispersion stability, fluorescence characteristics and cytotoxicity of the QD-loaded NCs were examined. In particular, the cytotoxicity of CIS/ZnS QDs was directly compared with that of CdSe/ZnS QDs using four different cell lines. Finally, the tumor-targeted delivery of NCs loaded with CIS/ZnS QDs was investigated to demonstrate the in vivo feasibility of the heavy-metal-free QD-based bio-imaging system.
zinc stearate (10−12% Zn basis), zinc undecylenate, trioctylphosphine (TOP), PBS, fluorescamine, 4,6-diamidino-2phenylindole (DAPI) and propidium iodide (PI) were purchased from Sigma-Aldrich (St. Louis, MO, USA). 1-aminodecane was purchased from Tokyo Chemical Industry Co., Ltd (Tokyo, Japan). N-hydroxysuccinimide-functionalized six-arm-branched PEG (6-arm PEG-NHS, Mw = 15 kDa) was obtained from Sunbio Inc. (Walnut Creek, CA, USA). Heterobifunctionalized PEG with FA and a primary amine at each terminal end (FA-PEG-NH2, Mw = 3.4 kDa) was purchased from Nanocs Inc. (New York, NY, USA). Dulbecco’s modified Eagle’s medium (DMEM) and FBS were purchased from Invitrogen (Carlsbad, CA, USA) and smooth muscle cell basal medium (SmBM) from Lonza (Walkersville, MD, USA). A cell-counting kit-8 (CCK-8) and lactate dehydrogenase (LDH) assay kits were purchased from Dojindo laboratories (Kumamoto, Japan) and Sigma-Aldrich, respectively. All other chemicals and reagents were of reagent grade and used without further purification. 2.2. Preparation of quantum dots
A mixture of CuI (0.3 mmol), In(Ac)3 (1.0 mmol) and ODE (225.0 mmol) was maintained under a primary vacuum and backfilled with argon at room temperature. After the injection of DDT (42.0 mmol), the reaction mixture was heated to 210 °C and kept for 1 h. To form a ZnS shell, zinc stearate (2.0 mmol) and DDT (42.0 mmol) was added into the reaction mixture, and then kept at 225 °C for 3 h. The final product was purified with chloroform, methanol and acetone and redispersed in dichloromethane for further use. CdO (0.1 mmol) was mixed with OA (3.8 mmol) and ODE (32.0 mmol) and degassed under vacuum at room temperature for 30 min. The mixture was heated to 270 °C under argon atmosphere. When the mixture changes from a turbid red to a clear colorless solution, a stock solution of Se (0.38 mmol) dissolved in a mixture of TOP (1.8 mmol) and ODE (15 mmol) was swiftly injected into the flask. The temperature was adjusted to 230 °C and maintained for 3 min for further growth. To passivate the ZnS shell, DDT (4.2 mmol) and zinc undecylenate (0.49 mmol) dissolved in 1aminodecane (7.6 mmol) were injected at 250 °C. The injection of Zn and S stock solutions was repeated for 4 times every 30 min. The solution was then cooled to room temperature and purified by precipitation in an excess amount of ethanol. The final product was re-dispersed in dichloromethane for further use. 2.3. Preparation of quantum dot-loaded HSA-PEG NCs
The QD-loaded HSA-PEG (CIS/ZnS-HSA-PEG) NCs were synthesized using a modified emulsification/solvent evaporation method. In brief, 5 mg of QDs and 22.6 mg of six-armed PEG-NHS were first dissolved in 2 mL of dichloromethane. The mixture was added in a dropwise manner to a 10 mL HSA solution (1 mg mL−1, pH 9) saturated with dichloromethane, and then sonicated at ambient temperature for 5 min using a Branson Sonifier® 450 at a frequency of 20 kHz with a duty
2. Materials and methods 2.1. Materials
HSA (purity ⩾ 96%), copper (I) iodide (CuI), indium (III) acetate (In(Ac)3), cadmium oxide (CdO), selenium (Se), 1dodecanethiol (DDT), 1-octadecene (ODE), oleic acid (OA), 2
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cycle of 20 and an output control of 3.5. The prepared oil-inwater emulsion was transferred to a rotary evaporator, and the organic solvent was rapidly evaporated under reduced pressure at 30 °C. The produced HSA-PEG NCs were subjected to dialysis against deionized water (Mw cutoff of 100 kDa) to remove free residues and then stored at 4 °C until use.
supplemented with 10% fetal bovine serum (FBS) for 24 h at 37 °C. HCASMC cells (human coronary artery smooth muscle cells) were grown in SmBM supplemented with CC4149 for SmGM-2. The NCs were diluted using PBS to a wide range of the QD concentration (1 μg mL−1 ∼ 1 mg mL−1). The culture medium was replaced with FA-free, fresh medium containing different QD formulations, and further incubated for 24 h at 37 °C. The number of viable cells was determined by the CCK-8 cell viability assay. Cytotoxicity induced by the CIS/ZnS and CdSe/ZnS QDs was determined by LDH release into the culture medium. The plates were centrifuged at 250 × g and 4 °C for 4 min and an aliquot of 50 μL was taken to quantify the LDH. The activity of LDH in the medium was determined using a commercially available LDH toxicology assay kit (TOX7) from Sigma-Aldrich. The test was performed according to the manufacturer’s instructions.
2.4. Conjugation of folic acid to CIS/ZnS-HSA-PEG NCs
FA was conjugated to the QD-loaded HSA-PEG NCs using a hetero-bifunctional PEG (FA-PEG-NH2). 3 mg of FA-PEGNH2 was added to 5 mL deionized water containing the HSAPEG NCs (4 mg mL−1), followed by incubation at ambient temperature for one day. Unreacted materials were removed by dialysis (Mw cutoff of 10 kDa) against deionized water for one day. The amount of conjugated folic acid was determined using a Nanodrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilimington, DE, USA) [28].
2.8. In vivo tumor accumulation and biodistribution
2.5. Characterization of CIS/ZnS-HSA-PEG NCs
Female BALB/C nude mice (7–8 weeks in age, about 20 g) were housed in a pathogen-free environment at 4–5 mice/ cage. They were supplied with autoclaved and non-fluorescent mouse chow and water. All animal experiments were conducted in accordance with the guidelines provided by Institutional Animal Care and Use committee of KAIST. The mouse tumor model was developed by injecting 100 μL of HeLa cell suspension (3 × 107 cells) into the subcutaneous region of each mouse. Tumor growth was monitored daily until their volume reached 50 mm.3 Each mouse was intravenously injected with 200 μL of the CIS/ZnS-loaded HSAPEG NCs at a given dose of QD (1 mg kg−1 body weight). The CIS/ZnS-PEG QDs were used as a control. At predetermined time intervals, fluorescence images of each mouse were captured with the IVIS® Lumina imaging system (Caliper Life Sciences, Hopkinton, MA, USA) in a sequential acquisition mode. The fluorescence intensity was calculated from the obtained fluorescence signals using the software (Living Image® Software) provided by the manufacturer. A digital caliper was used to determine the perpendicular diameter of the tumor. The respective tumor volume was calculated from the following formula: tumor volume = 0.5 × major axis2 × minor axis. After 2 d, the mice were sacrificed and dissected to obtain the ex vivo fluorescence images of their organs and tumor.
The hydrodynamic diameter and zeta potential of the prepared HSA-PEG NCs was measured by using a Zeta-Plus analyzer (Brookhaven, NY, USA) in triplicate at a concentration of 1 mg mL−1 at 20 °C. The size and shape of the NCs were determined by using transmission electron microscopy (TEM). The 20 μL of NCs dispersed in aqueous solution (4 mg mL−1) was deposited on a Formvar/carbon-supported copper grid. The grid was dried in air for 12 h and observed using a JEM—2100 F HR TEM (JEOL, Tokyo, Japan) operated at 80 kV. The loading amount of the CIS/ZnS QD NCs was analyzed by using inductively coupled plasma atomic emission spectrometry (ICP-AES). Vis–UV light absorbance of the CIS/ZnS QDs and NCs was measured by using a Nanodrop spectrophotometer. Fluorescence intensity was determined using an F-7000 fluorescence spectrofluorometer (Hitachi High-technologies, Tokyo, Japan) with an excitation wavelength of 380 nm. The emission spectra were recorded from 400 to 800 nm at 37 °C. 2.6. Flow cytometry
HeLa cells were seeded in a 6-well plate at a density of 5 × 105 cells/well and incubated for 24 h. QD-incorporated NCs were treated in HeLa cells for 3 h in DMEM supplemented with 10% FBS. The cells were repeatedly washed with PBS three times and analyzed using flow cytometry (FACSCalibur, BD Biosciences, USA). The data were analyzed using the WinMDI 2 software program.
2.9. Statistical analysis
Statistical analysis was performed using a standard student ttest with a minimum confidence level of 0.05 for significant statistical difference. All experiments were performed in triplicate.
2.7. In vitro cytotoxicity evaluation
The inhibition of cell growth was examined to evaluate the cytotoxicity of the CIS/ZnS and CdSe/ZnS QDs. HeLa cells (human epithelial carcinoma cells), A549 cells (adenocarcinomic human alveolar basal epithelial cells) and PC3 cells (human prostate cancer cells) were plated over a 96-well plate at a density of 1 × 105 cells per well. The cells were grown in DMEM for HeLa cells or RPMI for A549 and PC3 cells
3. Results and discussion Heavy-metal-free CIS/ZnS QDs were synthesized with a hot injection method according to previously reported procedures [29]. A mixture of copper iodide and indium acetate was 3
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Figure 1. Schematic representation of the synthesis of the CIS/ZnS core-shell QDs by using a hot injection method (A) and TEM images of CIS/ZnS QDs dispersed in dichloromethane (B).
phenomenon has not yet been fully explained, the change in the donor–acceptor pair recombination between the quantized conduction band and the Cu vacancy acceptor seems to be an important factor that affects the change in the PLQY. In Cudeficient QDs, the Cu sites are preferentially occupied by indium atoms, which can act as donors, while Cu vacancies serve as acceptors [30, 31]. The crystal structure of the synthesized CIS/ZnS QDs was examined using x-ray diffraction analysis. The diffraction pattern was not significantly affected by the stoichiometric ratio of Cu to In (figure S3). The accurate phase of the CIS core could not be determined because the tetragonal chalcopyrite and cubic-zinc blend phases have a similar crystallographic profile [32]. With regard to the absence of heavy metals, the resulting CIS/ZnS QDs are very attractive for the in vivo imaging of molecules, cells, tissues and organs. For bio-imaging applications, the QDs need to be homogeneously dispersed in an aqueous milieu. Our previous study demonstrated that shell cross-linked NCs composed of HSA and six-armed PEG, called HSA-PEG hybrid NCs, can be an excellent nanocarrier for water-insoluble paclitaxel [27]. The hybrid NCs showed excellent dispersion stability in serum-containing media, allowing the efficient systemic, tumor-targeted delivery of a water-insoluble anticancer drug. In this work, the HSA-PEG
dissolved in octadecene and heated to 210 °C (figure 1(A)). As a capping ligand as well as a sulfur source, 1-dodecanethiol was injected into this mixture and the reaction solution was kept at the same temperature for 1 h. To form a shell structure, a mixture of zinc stearate and 1-dodecanethiol was injected into the reaction solution, heated to 225 °C and kept for 2 h under magnetic stirring. The resulting QDs were 3–4 nm in diameter, as observed by TEM. They could also be well dispersed in a non-polar organic solvent (e.g., chloroform, dichloromethane and heptane) (figure 1(B)). The emission wavelength of the CIS/ZnS QDs was in the range of 600–630 nm. The absorption spectrum of the prepared CIS/ ZnS QDs is shown in figure S1 (supporting information, available at stacks.iop.org/NANO/25/175702/mmedia), and the calculated photoluminescence quantum yield (PLQY) was about 20.4%. In addition, the presence of the ZnS shell induced a blue shift of the fluorescence emission as the maximum emission wavelength was changed from 690 nm to 610 nm (figure S2). The molar Cu/In and Cu/Zn ratios in the synthesized QDs were 0.31 and 0.14, respectively, as determined using ICP-AES. The CIS QDs, with a Cu/In molar ratio of 0.3, were synthesized because such a copper-deficient composition is known to have a very high PLQY [30]. Although the precise underlying mechanism of this 4
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Figure 2. Schematic illustration of the formation of the CIS/ZnS QDs-loaded shell cross-linked HSA-PEG NCs decorated with FA.
NCs were employed to encapsulate the CIS/ZnS QDs for tumor-targeted fluorescence imaging. Figure 2 illustrates the preparation procedures for the CIS/ZnS-loaded HSA-PEG NCs. Folic acid (FA) was introduced onto the surface of the NCs as a tumor-targeting moiety [33, 34]. The folic acid receptor (FR), a membrane protein having a high affinity to FA, is overexpressed on various human cancer cells (e.g., ovarian, lung, breast, endometrial, renal and myeloid hematopoietic cells), while its expression is highly restricted in normal tissues [35–37]. Therefore, FA has been widely used as a targeting ligand for various anti-cancer agents to avoid their non-specific translocation to normal tissues [38, 39]. The CIS/ZnS QD-loaded NCs were very stable in water, as clearly indicated by the apparent color change (figure S4A, supporting information, avilable at stacks.iop.org/NANO/25/ 175702/mmedia). The mean hydrodynamic diameter of the CIS/ZnS-HSA-PEG NCs was 91.3 ± 10.0 nm in Milli-Q water, as determined by dynamic light scattering (DLS) (figure 3(A)). In contrast, the diameter of the CIS/ZnS QDs dispersed using only six-armed PEG (denoted ‘CIS/ZnS-PEG NCs’) without HSA was 171.8 ± 19.0 nm. This result indicates that the presence of HSA is critical for the formation of stable NCs for the QDs. The surface of the CIS/ZnS-HSAPEG NCs can be chemically modified with various targeting ligands using the functional groups of HSA. For the tumortargeted delivery of the HSA-PEG NCs, FA was conjugated to the NCs using a hetero-bifunctional form of PEG (FAPEG-NH2, MW = 3.4 kDa). The calculated average FA-PEG grafting density on the HSA-PEG NCs was 5.7 × 103 PEG
chains per nanocapsule, as obtained by dividing the total number of FA-PEG-NH2 (4.5 × 1017) consumed for the conjugation to the NCs by the total number of the NCs (7.9 × 1013). The surface area of each nanocapsule, with a diameter of about 91.3 nm, was 2.6 × 104 nm2. Hence, one PEG chain was grafted per 5 nm2 surface of the NCs [27, 40]. The conjugation of FA-PEG increased the hydrodynamic diameter of the CIS/ZnS-HSA-PEG NCs from 91.3 ± 10.0 nm to 107.0 ± 12.2 nm. The hydrodynamic diameter of a linear PEG chain (3.4 × 103 g mol−1) is about 4.5 nm. The actual increase in the diameter of the NCs was about 15.7 nm, which was significantly larger than the value expected from the hydrodynamic diameter of PEG (4.5 nm × 2 = 9 nm). This may stem from the extension of the grafted PEG chains as they form a highly dense comb-like conformation on the surface of the NCs [41]. A zeta potential analysis showed that the CIS/ZnS-PEG exhibited a slightly negative surface charge (−15.6 ± 0.6 mV) (figure 3(B)). The CIS/ZnS-HSA-PEG NCs exhibited a more negative zeta potential value (−34.1 ± 1.7 mV) presumably on account of the intrinsic negative property of HSA, which has an isoelectric point of 5.05 [42]. Note that HSA-based nanoparticles generally show a zeta potential of about −40 mV at pH values between 7.2 and 10 [42]. The relatively low zeta potential value (−24.0 ± 2.1 mV) of the FA-conjugated CIS/ZnS-HSA-PEG (denoted as ‘CIS/ZnS-HSA-PEG-FA’) NCs may originate from the presence of the neutral PEG linkers of FA-PEGNH2. The long-term dispersion stability of CIS/ZnS-HSAPEG NCs in the presence of 10% FBS was examined by 5
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Figure 3. Characterization of the CIS/ZnS QDs. The hydrodynamic diameter (A) and zeta potential (B) of the CIS/ZnS QDs stabilized with six-armed PEG-NHS (a), CIS/ZnS -HSA-PEG NCs (b) and CIS/ZnS-HSA-PEG-FA NCs (c). The long-term dispersion stability of the CIS/ ZnS-PEG, CIS/ZnS-HSA-PEG and CIS/ZnS-HSA-PEG-FA NCs by measuring the time-dependent changes of hydrodynamic diameter (C) and absorbance at 500 nm (D) in a 10% serum-containing medium. The TEM images of the CIS/ZnS-HSA/PEG-FA NCs in deionized water (E).
in aqueous media containing serum proteins. The morphology of the CIS/ZnS-HSA-PEG-FA NCs was observed using TEM. While the CIS/ZnS QDs dispersed in DCM were evenly distributed on the grid, the CIS/ZnS-HSA-PEG NCs were well dispersed in the form of spherical nanoparticles of about 20–40 nm in diameter, containing numerous QDs with diameters of 3–4 nm (figure 3(E)). The size of the NCs is much smaller than the value determined by DLS, presumably because the dehydration caused the shrinkage of the NCs, as observed in a previous study [27].
monitoring the time-dependent changes of the hydrodynamic diameter and absorbance at 500 nm. The CIS/ZnS-HSA-PEG and CIS/ZnS-HSA-PEG-FA NCs showed no significant change in their size distribution for 10 d or in their absorbance for 20 d in phosphate-buffered saline (PBS) (figures 3(C) and (D)), whereas the size of the CIS/ZnS-PEG gradually increased under the same conditions. These results suggest that the HSA-PEG NCs maintained a stable structure due to the cross-linked shell composed of NHS-functionalized PEG and HSA, allowing the stable dispersion of the CIS/ZnS QDs 6
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Figure 4. Absorption spectra and photoluminescence with an excitation wavelength of 380 nm (A) of the CIS/ZnS QDs dispersed in
dichloromethane and the CIS/ZnS-PEG, CIS/ZnS-HSA-PEG and CIS/ZnS-HSA-PEG-FA NCs. Photographs of the CIS/ZnS QDs dispersed in dichloromethane (a), CIS/ZnS-PEG (b), CIS/ZnS-HSA-PEG NCs (c) and CIS/ZnS-HSA-PEG-FA NCs (d) under UV illumination at 365 nm immediately after preparation and 7 d of incubation at room temperature (B). (C) Luminescence signal images of the CIS/ZnS QDs dispersed in dichloromethane (a), CIS/ZnS-PEG (b), CIS/ZnS-HSA-PEG NCs (c) and CIS/ZnS-HSA-PEG-FA NCs (d) at the same concentration of the CIS/ZnS QDs (0.1 mg mL−1).
DCM. The reduced PLQY may have been caused by the increased medium polarity to which the QDs were exposed [9, 43, 44]. In general, fluorescence emission can be influenced by the dielectric constant and hydrogen bonding capability of the medium, which are likely to change when QDs are bound to proteins, nucleic acids or macromolecules [44, 45]. Figure 4(B) shows photographic images of CIS/ZnS QDs and the CIS/ZnS-loaded NCs visualized under UV light in the dark. After their preparation, all of the samples showed very good colloidal homogeneity in PBS. However, after a seven-day incubation period at room temperature, the CIS/ ZnS-PEG formed clearly observable agglomerates, while the
When the QDs were encapsulated within the HSA-PEG NCs, the emission peak showed a red shift to 650 nm. Moreover, the fluorescence emission intensity per CIS/ZnS QD decreased slightly (figure 4(A)). The relative peak intensity of the fluorescence emission from the CIS/ZnSHSA-PEG NCs was 78.0% compared to that of the CIS/ZnS QDs dispersed in DCM at the same concentration of CIS/ZnS QDs. The emission intensities were also measured at different concentrations of the CIS/ZnS QDs, which showed the same absorbance at the excitation wavelength (380 nm) (figure S5). The calculated PLQY in this condition showed a more dramatic decrease (44.7%) compared to the CIS/ZnS QDs in 7
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Figure 5. Flow cytometric analysis of the intensity of QD-mediated fluorescence in HeLa cells following the incubation with PBS (A), CIS/
ZnS-HSA-PEG NCs (B), CIS/ZnS-HSA-PEG-FA NCs (C) and CdSe-HSA-PEG-FA NCs for 12 h.
damage [47]. Cell viability is assayed quantitatively in a CCK-8 assay, in which the formation of formazan dye depends on the activity of the mitochondria [48]. HeLa cells (human epithelial carcinoma cells) and PC3 cells (human prostate cancer cells) were chosen as model cancer cells due to their relatively high expression of FRs. A549 cells (human adenocarcinomic alveolar basal epithelial cells) were also used as a negative control because of their FR deficiency. NCs loaded with the CdSe/ZnS QDs with and without FA were prepared and used as a control (denoted as ‘CdSe-HSAPEG NCs’ and ‘CdSe-HSA-PEG-FA NCs’, respectively). The absorption and fluorescence emission spectra of the CdSe/ZnS QDs are provided in figure S8. Several studies have shown acute toxicity originating from Cd-based QDs in tissue cultures [49, 50]. Cytotoxicity was determined after the exposure of cancer cells to the QD-loaded NCs for 12 h. In the LDH assay, when the cells were exposed to high QD doses (100–1,000 μg mL−1), HeLa cells showed cytotoxic responses only to the CdSe-HSA-PEG-FA NCs, while PC3 cells responded to all of the samples except for CIS/ZnSHSA-PEG NCs (figures 6(A)–(C)). The cytotoxic responses of HeLa and PC3 cells were significantly more sensitive to the FA-conjugated NCs, indicating that intracellular translocation is critical for the cytotoxic effects of the QDs. In particular, a dose-dependent increase of LDH leakage was observed in PC3 cells (figure 6(B)). The CdSe-HSA-PEG-FA NCs showed a greater LDH release by 1.6 times compared to the CIS/ZnS-HSA-PEG-FA NCs at a QD concentration of 1 mg mL−1. The CCK-8 assay also revealed the cytotoxic effects of QDs that were consistent with the LDH release results (figures 6(D), (E)). Loss of cell viability (74.6 ± 4.9%) was notable in the HeLa cells exposed to 1 mg mL−1 of the
CIS/ZnS-HSA-PEG and CIS/ZnS-HSA-PEG-FA NCs maintained their homogeneity. The CIS/ZnS-HSA-PEG-FA NCs showed blue fluorescence because the FA, which was conjugated to the surface of the CIS/ZnS-HSA-PEG NCs, has an emission band peaking at around 450 nm (figure S6) [35]. Different types of fluorescent optical images were taken using a charge-coupled-device (CCD) camera. The average radiance (photon/s/cm2/Sr) was obtained by drawing a region of interest (ROI) over appropriate glass wells. The CIS/ZnS QDs dispersed in DCM showed a heterogeneous distribution, and some of them were adhered to the surface of the glass wall. In contrast, the CIS/ZnS-HSA-PEG NCs showed a relatively homogeneous dispersion in PBS (figure 4(C)). The cellular uptake of the QD-loaded HSA-PEG NCs was measured by a fluorescence-activated cell sorting (FACS) analysis (figure 5). The conjugation of FA to the NCs increased the fluorescence intensity of HeLa cells, indicating more efficient cellular internalization of the NCs. There was no significant difference in the cellular uptake between the CdSe-HSA-PEG-FA and CIS/ZnS-HSA-PEG-FA NCs. Therefore, it was assumed that any differences in the cytotoxic effects of the tested samples must derive from the different QDs loaded with the NCs. To determine the impact of heavy metal species in the QDs on the level of cellular toxicity, two different cytotoxicity assays were conducted using three different cancer cell lines and smooth muscle cells. LDH release and CCK-8 assays were performed on cells treated with NCs which incorporated the QDs. LDH evaluates the integrity of the cell membrane using the cytoplasmic enzyme activity released from damaged cells [46]. The loss of intracellular LDH and its release into the culture medium is an indicator of irreversible cell death due to cell membrane 8
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Figure 6. Comparison of LDH release assay and cell viability in HeLa cells (A, D), PC3 cells (B, E) and A549 cells (C, F) after exposure to the CIS/ZnS-HSA-PEG, CIS/ZnS-HSA-PEG-FA, CdSe-HSA-PEG and CdSe-HSA-PEG-FA NCs for 12 h.
more sensitive to the FA conjugation because the prostatespecific membrane antigen could act as an FA transporter [51]. Prostate-specific membrane antigen provides prostate cancer cells with a growth advantage by increasing the cellular level of FA. A549 cells exhibited significantly higher cytotoxic responses for Cd-based QDs compared to CISbased QDs. However, A549 cells did not show any dependency on the FA conjugation owing to their FR deficiency. The LDH release from A549 cells was similar at different concentrations of the CIS/ZnS-HSA/PEG and CIS/ZnS-HSAPEG-FA NCs (figure 6(C)). The CIS/ZnS QDs showed no
CdSe-HSA-PEG-FA NCs, but no significant cytotoxic effect was observed for the CIS/ZnS-HSA-PEG NCs. The percentages of viable cells treated with the CIS/ZnS-loaded NCs decreased to 71.8%, while that with the CdSe-HSA-PEG NCs significantly decreased to 50.9% in PC3 cells. These results demonstrate that CIS/ZnS QDs are likely to be a safer alternative to CdSe/ZnS QDs. The differences in the cytotoxicities indicate intracellular effects rather than permanent cell membrane damage [47]. In general, the cytotoxicity of QDs is mainly caused by cadmium ions that are extracted from the nanoscale nanocrystals [17]. It was found that PC3 cells were 9
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Figure 7. (A) In vivo fluorescence images of a HeLa tumor-bearing mouse after intravenous injection of the CIS/ZnS QDs dispersed in 10% DMSO, CIS/ZnS-HSA-PEG NCs and CIS/ZnS-HSA-PEG-FA NCs in PBS. The area circled in red indicates the implanted tumor site. Whole body fluorescence images of each mouse were acquired from an IVIS® Xenogen imaging system. (B) Ex vivo images of major organs (heart, lung, kidney, spleen and liver) and a tumor harvested from the mice at 48 h post-injection. (C) Ex vivo distribution of the CIS/ZnS QDs expressed as the fluorescence intensity per gram of each excised organ.
formulations were detected as faint spots in the tumor region. To evaluate the spatial distribution of the CIS/ZnS QDs in the body more precisely, we obtained the ex vivo fluorescence images of the organs excised from the mice (figure 7(B)). The CIS/ZnS-HSA-PEG-FA NCs exhibited a very strong fluorescence signal in the tumor, while only marginal fluorescence was detected in the heart and kidney. A low fluorescence signal was observed in the lung, spleen and liver, indicating that the injected NCs were preferentially accumulated in the tumor with little non-specific translocation to normal tissues. Similarly, it was reported that PEGylated nanoparticles are scarcely recognized and cleared by the macrophages distributed in the liver [27]. The fluorescence intensity per gram of tumor increased strikingly due to the FA conjugation. The tumor targeting effect of FA suggests that the tumor tissues can be reliably monitored in vivo using FA-conjugated NCs incorporating heavy-metal-free CIS/ZnS QDs.
significant cytotoxic effect, even at high concentrations, whereas the CdSe/ZnS QDs induced a more significant LDH release, leading to cell death at high concentrations of the QDs. Finally, we investigated the feasibility of the CIS/ZnS QDs for in vivo tumor-targeted imaging. NCs containing 20 μg QDs were injected intravenously into the tail vein of female BALB/C nude mice. In vivo accumulation of the CIS/ ZnS-loaded NCs in tumor tissue was examined using an IVIS® Lumina imaging system (Caliper Life Sciences). Fluorescence imaging was performed using a laser excitation filter passband of 500–550 nm and a CCD camera with a onesecond exposure time, and the average fluorescence intensity, which is defined as the total photon count per unit area of a tumor, was determined. Figure 7(A) shows fluorescence images of the HeLa tumor-bearing mice 24 h after the injections of the CIS/ZnS QDs dispersed in 10% dimethyl sulfoxide, CIS/ZnS-HSA-PEG NCs and CIS/ZnS-HSA-PEG-FA NCs. The tumor, marked by a red circle, was easily visualized within a few hours of the injection, and the accumulation of QDs was observed 24 h after the injection. The injected CIS/ ZnS-HSA-PEG-FA NCs were efficiently directed to and stayed in the tumor tissue, whereas the CIS/ZnS QDs in other
4. Conclusion In this work, we introduced shell cross-linked HSA-PEG hybrid NCs incorporating heavy-metal-free CIS/ZnS QDs for 10
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tumor-targeted fluorescence imaging. The CIS/ZnS-HSAPEG NCs exhibited a high level of fluorescence intensity comparable to that in a non-polar organic solvent as well as excellent dispersion stability in a serum-containing medium. Surface modification of the NCs with FA effectively facilitated the tumor-targeted delivery of the QD-incorporated NCs. In contrast to Cd-based QDs, the CIS/ZnS QDs did not induce a significant level of cytotoxicity in cancer and smooth muscle cells. In addition to reduced cytotoxicity, the FAconjugated NCs loaded with the CIS/ZnS QDs were efficiently accumulated in tumor tissue with strong fluorescence intensity, demonstrating efficient tumor-targeted imaging. We expect that the shell cross-linked HSA-PEG hybrid NCs can be used as a promising nanocarrier platform for the stable systemic circulation of heavy-metal-free QDs and other functional molecules (e.g., anticancer drugs, iron oxide nanocrystals and other optical probes). Facile surface functionalization of the NC surface also provides a greater number of potential applications of shell cross-linked NCs for functional diagnosis and theranostics.
[9] Visaria R K, Griffin R J, Williams B W, Ebbini E S, Paciotti G F, Song C W and Bischof J C 2006 Enhancement of tumor thermal therapy using gold nanoparticle-assisted tumor necrosis factor-alpha delivery Mol. Cancer Ther. 5 1014–20 [10] Pissuwan D, Valenzuela S M and Cortie M B 2006 Therapeutic possibilities of plasmonically heated gold nanoparticles Trends Biotechnol. 24 62–7 [11] Moon H K, Lee S H and Choi H C 2009 In vivo near-infrared mediated tumor destruction by photothermal effect of carbon nanotubes ACS Nano. 3 3707–13 [12] Hessel C M, Pattani V P, Rasch M, Panthani M G, Koo B, Tunnell J W and Korgel B A 2011 Copper selenide nanocrystals for photothermal therapy Nano Lett. 11 2560–6 [13] Bruchez M Jr., Moronne M, Gin P, Weiss S and Alivisatos A P 1998 Semiconductor nanocrystals as fluorescent biological labels Science 281 2013–6 [14] Stroh M et al 2005 Quantum dots spectrally distinguish multiple species within the tumor milieu in vivo Nat. Med. 11 678–82 [15] Sen D, Deerinck T J, Ellisman M H, Parker I and Cahalan M D 2008 Quantum dots for tracking dendritic cells and priming an immune response in vitro and in vivo PLoS One 3 e3290 [16] Pons T, Pic E, Lequeux N, Cassette E, Bezdetnaya L, Guillemin F, Marchal F and Dubertret B 2010 Cadmiumfree CuInS2/ZnS quantum dots for sentinel lymph node imaging with reduced toxicity ACS Nano. 4 2531–8 [17] Derfus A M, Chan W C W and Bhatia S N 2004 Probing the cytotoxicity of semiconductor quantum dots Nano Lett. 4 11–18 [18] Kim S W, Zimmer J P, Ohnishi S, Tracy J B, Frangioni J V and Bawendi M G 2005 Engineering InAs(x)P(1 − x)/InP/ZnSe III-V alloyed core/shell quantum dots for the near-infrared J. Am. Chem. Soc. 127 10526–32 [19] Kim Y H, Jun Y W, Jun B H, Lee S M and Cheon J 2002 Sterically induced shape and crystalline phase control of GaP nanocrystals J. Am. Chem. Soc. 124 13656–7 [20] Malik M A, O’Brien P and Revaprasadu N 1999 A novel route for the preparation of CuSe and CuInSe2 nanoparticles Adv. Mater. 11 1441–4 [21] Sanguinetti S, Chiantoni G, Miotto A, Grilli E, Guzzi M, Henini M, Polimeni A, Patane A, Eaves L and Main P C 2000 Self-assembling of In(Ga)As/GaAs quantum dots on (N11) substrates: the (311)A case Micron 31 309–13 [22] Hamanaka Y, Ogawa T, Tsuzuki M and Kuzuya T 2011 Photoluminescence properties and its origin of AgInS2 quantum dots with chalcopyrite structure J. Phys. Chem. C 115 1786–92 [23] Li L, Daou T J, Texier I, Tran T K C, Nguyen Q L and Reiss P 2009 Highly luminescent CuInS(2)/ZnS core/shell nanocrystals: cadmium-free quantum dots for in vivo imaging Chem. Mater. 21 2422–9 [24] Castro S L, Bailey S G, Raffaelle R P, Banger K K and Hepp A F 2003 Nanocrystalline chalcopyrite materials (CuInS2 and CuInSe2) via low-temperature pyrolysis of molecular single-source precursors Chem. Mater. 15 3142–7 [25] Nanu M, Schoonman J and Goossens A 2005 Nanocomposite three-dimensional solar cells obtained by chemical spray deposition Nano Lett. 5 1716–9 [26] Guo W S, Chen N, Tu Y, Dong C H, Zhang B B, Hu C H and Chang J 2013 Synthesis of Zn-Cu-In-S/ZnS core/shell quantum dots with inhibited blue-shift photoluminescence and applications for tumor targeted bioimaging Theranostics 3 99–108 [27] Lee J Y, Bae K H, Kim J S, Nam Y S and Park T G 2011 Intracellular delivery of paclitaxel using oil-free, shell crosslinked HSA-multi-armed PEG nanocapsules Biomaterials 32 8635–44
Acknowledgements This research was financially supported by the Ministry of Health, Welfare and Family Affairs (A111552) and the Converging Research Center (2009-0082276) funded by the National Research Foundation, Republic of Korea.
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[41] Soong R and Macdonald P M 2008 Diffusion of PEG confined between lamellae of negatively magnetically aligned bicelles: pulsed field gradient 1H NMR measurements Langmuir 24 518–27 [42] Langer K, Balthasar S, Vogel V, Dinauer N, von Briesen H and Schubert D 2003 Optimization of the preparation process for human serum albumin (HSA) nanoparticles Int. J. Pharm. 257 169–80 [43] Bigger S W, Ghiggino K P, Meilak G A and Verity B 1992 Illustration of the principles of fluorometry. An apparatus and experiments specially designed for the teaching laboratory J. Chem. Edu. 69 675–7 [44] Maliwal B P, Hermetter A and Lakowicz J R 1986 A study of protein dynamics from anisotropy decays obtained by variable frequency phase-modulation fluorometry: internal motions of N-methylanthraniloyl melittin Biochim. Biophys. Acta. 873 173–81 [45] Lakowicz J R 1986 Fluorescence studies of structural fluctuations in macromolecules as observed by fluorescence spectroscopy in the time, lifetime, and frequency domains Methods Enzymol. 131 518–67 [46] Weyermann J, Lochmann D and Zimmer A 2005 A practical note on the use of cytotoxicity assays Int. J. Pharm. 288 369–76 [47] Fotakis G and Timbrell J A 2006 In vitro cytotoxicity assays: comparison of LDH, neutral red, MTT and protein assay in hepatoma cell lines following exposure to cadmium chloride Toxicol Lett. 160 171–7 [48] Chang Y, Yang S T, Liu J H, Dong E, Wang Y, Cao A, Liu Y and Wang H 2011 In vitro toxicity evaluation of graphene oxide on A549 cells Toxicology Lett. 200 201–10 [49] Su Y, He Y, Lu H, Sai L, Li Q, Li W, Wang L, Shen P, Huang Q and Fan C 2009 The cytotoxicity of cadmium based, aqueous phase—synthesized, quantum dots and its modulation by surface coating Biomaterials 30 19–25 [50] Hsieh M S, Shiao N H and Chan W H 2009 Cytotoxic effects of CdSe quantum dots on maturation of mouse oocytes, fertilization, and fetal development Int. J. Mol. Sci. 10 2122–35 [51] Yao V, Berkman C E, Choi J K, O’Keefe D S and Bacich D J 2010 Expression of prostate-specific membrane antigen (PSMA), increases cell folate uptake and proliferation and suggests a novel role for PSMA in the uptake of the nonpolyglutamated folate, folic acid Prostate 70 305–16
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Serum-stable quantum dot--protein hybrid nanocapsules for optical bio-imaging
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Nanotechnology Nanotechnology 25 (2014) 175702 (12pp)
doi:10.1088/0957-4484/25/17/175702
Serum-stable quantum dot–protein hybrid nanocapsules for optical bio-imaging Jeong Yu Lee1, Dong Heon Nam1, Mi Hwa Oh2, Youngsun Kim1, Hyung Seok Choi1, Duk Young Jeon1,3, Chan Beum Park1,3,4 and Yoon Sung Nam1,2,3,4 1
Department of Materials Science and Engineering, Korea Advanced Institute of Science and Technology, Daejeon 305-701, Republic of Korea 2 Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon 305-701, Republic of Korea 3 KAIST Institute for NanoCentury (KINC), Korea Advanced Institute of Science and Technology, Daejeon 305-701, Republic of Korea 4 KAIST Institute for BioCentury (KIB), Korea Advanced Institute of Science and Technology, Daejeon 305-701, Republic of Korea E-mail: [email protected] Received 23 December 2013, revised 27 February 2014 Accepted for publication 6 March 2014 Published 10 April 2014 Abstract
We introduce shell cross-linked protein/quantum dot (QD) hybrid nanocapsules as a serumstable systemic delivery nanocarrier for tumor-targeted in vivo bio-imaging applications. Highly luminescent, heavy-metal-free Cu0.3InS2/ZnS (CIS/ZnS) core-shell QDs are synthesized and mixed with amine-reactive six-armed poly(ethylene glycol) (PEG) in dichloromethane. Emulsification in an aqueous solution containing human serum albumin (HSA) results in shell cross-linked nanocapsules incorporating CIS/ZnS QDs, exhibiting high luminescence and excellent dispersion stability in a serum-containing medium. Folic acid is introduced as a tumortargeting ligand. The feasibility of tumor-targeted in vivo bio-imaging is demonstrated by measuring the fluorescence intensity of several major organs and tumor tissue after an intravenous tail vein injection of the nanocapsules into nude mice. The cytotoxicity of the QDloaded HSA-PEG nanocapsules is also examined in several types of cells. Our results show that the cellular uptake of the QDs is critical for cytotoxicity. Moreover, a significantly lower level of cell death is observed in the CIS/ZnS QDs compared to nanocapsules loaded with cadmiumbased QDs. This study suggests that the systemic tumor targeting of heavy-metal-free QDs using shell cross-linked HSA-PEG hybrid nanocapsules is a promising route for in vivo tumor diagnosis with reduced non-specific toxicity. S Online supplementary data available from stacks.iop.org/NANO/25/175702/mmedia Keywords: albumin, quantum dots, cytotoxicity, optical bio-imaging, targeted delivery (Some figures may appear in colour only in the online journal)
1. Introduction
nanocrystals have been used as a negative contrast agent for magnetic resonance imaging [5, 6]. Colloidal gold nanoparticles have been explored as an effective probe for computed tomography, surface-enhanced Raman scattering and photoacoustic tomography [7–9]. They have also been used to
Inorganic nanocrystals have been studied for a wide range of applications including biomedical imaging, diagnostics, biosensing and therapy [1–4]. For instance, iron oxide 0957-4484/14/175702+12$33.00
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generate photothermal heating that can destroy tumor cells [10–12]. Semiconductor nanocrystals, known as quantum dots (QDs), have received increasing amounts of attention as a promising alternative to organic chromophores due to their high quantum efficiency, photochemical stability and spectral tunability from the visible to the near-infrared (NIR) regions [13, 14]. Accordingly, biomedical applications of QDs have been widely studied for photodynamic therapy, cell tracking and cancer detection [12, 15, 16]. However, the intrinsic toxicity derived from heavy metals (e.g., cadmium, tellurium and lead) has introduced some concern regarding their biological applicability [17]. Over the past few decades, significant efforts have been made to achieve desirable optical and electrical properties of various QDs, including binary II–VI and III–V compound (e.g., CdSe, CdTe, ZnSe, GaP, InAs and InP) and tertiary compound (e.g., CuInSe2, CuInS2, GaAsP, AgInS2 and AlGaAs) QDs [18–23]. In particular, heavy-metal-free QDs have gained an increasing amount of attention due to environmental and toxicological issues related to the applications of QDs in the areas of energy conversion and bio-imaging. One promising heavy-metal-free QD is CuInS2 (CIS), a I-IIIV semiconductor with an emission wavelength that can be modulated from the visible light to NIR regions with a high quantum yield [23–25]. It has been shown that CIS QDs, when transferred into an aqueous solution via ligand exchange or phospholipid micelle encapsulation, can be used for the in vivo imaging of major organs and regional lymph nodes [16, 23]. However, the in vivo application of CIS QDs for cancer diagnostics has not been well studied and has just started to be considered very recently [26]. In addition, no direct comparison of CIS QDs with Cd-based QDs in terms of cytotoxicity has been reported. In this work, we proposed a new tumor-targeting QDprotein hybrid nanostructure based on shell cross-linked nanocapsules (NCs) and CIS/ZnS QDs, and investigated the cytotoxicity and in vivo applicability of the nanoscale hybrid platform for tumor-targeted optical bio-imaging. The shells of the NCs were prepared by chemically cross-linking human serum albumin (HSA) with amine-reactive six-armed poly (ethylene glycol) (PEG), generating a stable interface for the in vivo application of the encapsulated QDs [27]. The dispersion stability, fluorescence characteristics and cytotoxicity of the QD-loaded NCs were examined. In particular, the cytotoxicity of CIS/ZnS QDs was directly compared with that of CdSe/ZnS QDs using four different cell lines. Finally, the tumor-targeted delivery of NCs loaded with CIS/ZnS QDs was investigated to demonstrate the in vivo feasibility of the heavy-metal-free QD-based bio-imaging system.
zinc stearate (10−12% Zn basis), zinc undecylenate, trioctylphosphine (TOP), PBS, fluorescamine, 4,6-diamidino-2phenylindole (DAPI) and propidium iodide (PI) were purchased from Sigma-Aldrich (St. Louis, MO, USA). 1-aminodecane was purchased from Tokyo Chemical Industry Co., Ltd (Tokyo, Japan). N-hydroxysuccinimide-functionalized six-arm-branched PEG (6-arm PEG-NHS, Mw = 15 kDa) was obtained from Sunbio Inc. (Walnut Creek, CA, USA). Heterobifunctionalized PEG with FA and a primary amine at each terminal end (FA-PEG-NH2, Mw = 3.4 kDa) was purchased from Nanocs Inc. (New York, NY, USA). Dulbecco’s modified Eagle’s medium (DMEM) and FBS were purchased from Invitrogen (Carlsbad, CA, USA) and smooth muscle cell basal medium (SmBM) from Lonza (Walkersville, MD, USA). A cell-counting kit-8 (CCK-8) and lactate dehydrogenase (LDH) assay kits were purchased from Dojindo laboratories (Kumamoto, Japan) and Sigma-Aldrich, respectively. All other chemicals and reagents were of reagent grade and used without further purification. 2.2. Preparation of quantum dots
A mixture of CuI (0.3 mmol), In(Ac)3 (1.0 mmol) and ODE (225.0 mmol) was maintained under a primary vacuum and backfilled with argon at room temperature. After the injection of DDT (42.0 mmol), the reaction mixture was heated to 210 °C and kept for 1 h. To form a ZnS shell, zinc stearate (2.0 mmol) and DDT (42.0 mmol) was added into the reaction mixture, and then kept at 225 °C for 3 h. The final product was purified with chloroform, methanol and acetone and redispersed in dichloromethane for further use. CdO (0.1 mmol) was mixed with OA (3.8 mmol) and ODE (32.0 mmol) and degassed under vacuum at room temperature for 30 min. The mixture was heated to 270 °C under argon atmosphere. When the mixture changes from a turbid red to a clear colorless solution, a stock solution of Se (0.38 mmol) dissolved in a mixture of TOP (1.8 mmol) and ODE (15 mmol) was swiftly injected into the flask. The temperature was adjusted to 230 °C and maintained for 3 min for further growth. To passivate the ZnS shell, DDT (4.2 mmol) and zinc undecylenate (0.49 mmol) dissolved in 1aminodecane (7.6 mmol) were injected at 250 °C. The injection of Zn and S stock solutions was repeated for 4 times every 30 min. The solution was then cooled to room temperature and purified by precipitation in an excess amount of ethanol. The final product was re-dispersed in dichloromethane for further use. 2.3. Preparation of quantum dot-loaded HSA-PEG NCs
The QD-loaded HSA-PEG (CIS/ZnS-HSA-PEG) NCs were synthesized using a modified emulsification/solvent evaporation method. In brief, 5 mg of QDs and 22.6 mg of six-armed PEG-NHS were first dissolved in 2 mL of dichloromethane. The mixture was added in a dropwise manner to a 10 mL HSA solution (1 mg mL−1, pH 9) saturated with dichloromethane, and then sonicated at ambient temperature for 5 min using a Branson Sonifier® 450 at a frequency of 20 kHz with a duty
2. Materials and methods 2.1. Materials
HSA (purity ⩾ 96%), copper (I) iodide (CuI), indium (III) acetate (In(Ac)3), cadmium oxide (CdO), selenium (Se), 1dodecanethiol (DDT), 1-octadecene (ODE), oleic acid (OA), 2
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cycle of 20 and an output control of 3.5. The prepared oil-inwater emulsion was transferred to a rotary evaporator, and the organic solvent was rapidly evaporated under reduced pressure at 30 °C. The produced HSA-PEG NCs were subjected to dialysis against deionized water (Mw cutoff of 100 kDa) to remove free residues and then stored at 4 °C until use.
supplemented with 10% fetal bovine serum (FBS) for 24 h at 37 °C. HCASMC cells (human coronary artery smooth muscle cells) were grown in SmBM supplemented with CC4149 for SmGM-2. The NCs were diluted using PBS to a wide range of the QD concentration (1 μg mL−1 ∼ 1 mg mL−1). The culture medium was replaced with FA-free, fresh medium containing different QD formulations, and further incubated for 24 h at 37 °C. The number of viable cells was determined by the CCK-8 cell viability assay. Cytotoxicity induced by the CIS/ZnS and CdSe/ZnS QDs was determined by LDH release into the culture medium. The plates were centrifuged at 250 × g and 4 °C for 4 min and an aliquot of 50 μL was taken to quantify the LDH. The activity of LDH in the medium was determined using a commercially available LDH toxicology assay kit (TOX7) from Sigma-Aldrich. The test was performed according to the manufacturer’s instructions.
2.4. Conjugation of folic acid to CIS/ZnS-HSA-PEG NCs
FA was conjugated to the QD-loaded HSA-PEG NCs using a hetero-bifunctional PEG (FA-PEG-NH2). 3 mg of FA-PEGNH2 was added to 5 mL deionized water containing the HSAPEG NCs (4 mg mL−1), followed by incubation at ambient temperature for one day. Unreacted materials were removed by dialysis (Mw cutoff of 10 kDa) against deionized water for one day. The amount of conjugated folic acid was determined using a Nanodrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilimington, DE, USA) [28].
2.8. In vivo tumor accumulation and biodistribution
2.5. Characterization of CIS/ZnS-HSA-PEG NCs
Female BALB/C nude mice (7–8 weeks in age, about 20 g) were housed in a pathogen-free environment at 4–5 mice/ cage. They were supplied with autoclaved and non-fluorescent mouse chow and water. All animal experiments were conducted in accordance with the guidelines provided by Institutional Animal Care and Use committee of KAIST. The mouse tumor model was developed by injecting 100 μL of HeLa cell suspension (3 × 107 cells) into the subcutaneous region of each mouse. Tumor growth was monitored daily until their volume reached 50 mm.3 Each mouse was intravenously injected with 200 μL of the CIS/ZnS-loaded HSAPEG NCs at a given dose of QD (1 mg kg−1 body weight). The CIS/ZnS-PEG QDs were used as a control. At predetermined time intervals, fluorescence images of each mouse were captured with the IVIS® Lumina imaging system (Caliper Life Sciences, Hopkinton, MA, USA) in a sequential acquisition mode. The fluorescence intensity was calculated from the obtained fluorescence signals using the software (Living Image® Software) provided by the manufacturer. A digital caliper was used to determine the perpendicular diameter of the tumor. The respective tumor volume was calculated from the following formula: tumor volume = 0.5 × major axis2 × minor axis. After 2 d, the mice were sacrificed and dissected to obtain the ex vivo fluorescence images of their organs and tumor.
The hydrodynamic diameter and zeta potential of the prepared HSA-PEG NCs was measured by using a Zeta-Plus analyzer (Brookhaven, NY, USA) in triplicate at a concentration of 1 mg mL−1 at 20 °C. The size and shape of the NCs were determined by using transmission electron microscopy (TEM). The 20 μL of NCs dispersed in aqueous solution (4 mg mL−1) was deposited on a Formvar/carbon-supported copper grid. The grid was dried in air for 12 h and observed using a JEM—2100 F HR TEM (JEOL, Tokyo, Japan) operated at 80 kV. The loading amount of the CIS/ZnS QD NCs was analyzed by using inductively coupled plasma atomic emission spectrometry (ICP-AES). Vis–UV light absorbance of the CIS/ZnS QDs and NCs was measured by using a Nanodrop spectrophotometer. Fluorescence intensity was determined using an F-7000 fluorescence spectrofluorometer (Hitachi High-technologies, Tokyo, Japan) with an excitation wavelength of 380 nm. The emission spectra were recorded from 400 to 800 nm at 37 °C. 2.6. Flow cytometry
HeLa cells were seeded in a 6-well plate at a density of 5 × 105 cells/well and incubated for 24 h. QD-incorporated NCs were treated in HeLa cells for 3 h in DMEM supplemented with 10% FBS. The cells were repeatedly washed with PBS three times and analyzed using flow cytometry (FACSCalibur, BD Biosciences, USA). The data were analyzed using the WinMDI 2 software program.
2.9. Statistical analysis
Statistical analysis was performed using a standard student ttest with a minimum confidence level of 0.05 for significant statistical difference. All experiments were performed in triplicate.
2.7. In vitro cytotoxicity evaluation
The inhibition of cell growth was examined to evaluate the cytotoxicity of the CIS/ZnS and CdSe/ZnS QDs. HeLa cells (human epithelial carcinoma cells), A549 cells (adenocarcinomic human alveolar basal epithelial cells) and PC3 cells (human prostate cancer cells) were plated over a 96-well plate at a density of 1 × 105 cells per well. The cells were grown in DMEM for HeLa cells or RPMI for A549 and PC3 cells
3. Results and discussion Heavy-metal-free CIS/ZnS QDs were synthesized with a hot injection method according to previously reported procedures [29]. A mixture of copper iodide and indium acetate was 3
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Figure 1. Schematic representation of the synthesis of the CIS/ZnS core-shell QDs by using a hot injection method (A) and TEM images of CIS/ZnS QDs dispersed in dichloromethane (B).
phenomenon has not yet been fully explained, the change in the donor–acceptor pair recombination between the quantized conduction band and the Cu vacancy acceptor seems to be an important factor that affects the change in the PLQY. In Cudeficient QDs, the Cu sites are preferentially occupied by indium atoms, which can act as donors, while Cu vacancies serve as acceptors [30, 31]. The crystal structure of the synthesized CIS/ZnS QDs was examined using x-ray diffraction analysis. The diffraction pattern was not significantly affected by the stoichiometric ratio of Cu to In (figure S3). The accurate phase of the CIS core could not be determined because the tetragonal chalcopyrite and cubic-zinc blend phases have a similar crystallographic profile [32]. With regard to the absence of heavy metals, the resulting CIS/ZnS QDs are very attractive for the in vivo imaging of molecules, cells, tissues and organs. For bio-imaging applications, the QDs need to be homogeneously dispersed in an aqueous milieu. Our previous study demonstrated that shell cross-linked NCs composed of HSA and six-armed PEG, called HSA-PEG hybrid NCs, can be an excellent nanocarrier for water-insoluble paclitaxel [27]. The hybrid NCs showed excellent dispersion stability in serum-containing media, allowing the efficient systemic, tumor-targeted delivery of a water-insoluble anticancer drug. In this work, the HSA-PEG
dissolved in octadecene and heated to 210 °C (figure 1(A)). As a capping ligand as well as a sulfur source, 1-dodecanethiol was injected into this mixture and the reaction solution was kept at the same temperature for 1 h. To form a shell structure, a mixture of zinc stearate and 1-dodecanethiol was injected into the reaction solution, heated to 225 °C and kept for 2 h under magnetic stirring. The resulting QDs were 3–4 nm in diameter, as observed by TEM. They could also be well dispersed in a non-polar organic solvent (e.g., chloroform, dichloromethane and heptane) (figure 1(B)). The emission wavelength of the CIS/ZnS QDs was in the range of 600–630 nm. The absorption spectrum of the prepared CIS/ ZnS QDs is shown in figure S1 (supporting information, available at stacks.iop.org/NANO/25/175702/mmedia), and the calculated photoluminescence quantum yield (PLQY) was about 20.4%. In addition, the presence of the ZnS shell induced a blue shift of the fluorescence emission as the maximum emission wavelength was changed from 690 nm to 610 nm (figure S2). The molar Cu/In and Cu/Zn ratios in the synthesized QDs were 0.31 and 0.14, respectively, as determined using ICP-AES. The CIS QDs, with a Cu/In molar ratio of 0.3, were synthesized because such a copper-deficient composition is known to have a very high PLQY [30]. Although the precise underlying mechanism of this 4
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Figure 2. Schematic illustration of the formation of the CIS/ZnS QDs-loaded shell cross-linked HSA-PEG NCs decorated with FA.
NCs were employed to encapsulate the CIS/ZnS QDs for tumor-targeted fluorescence imaging. Figure 2 illustrates the preparation procedures for the CIS/ZnS-loaded HSA-PEG NCs. Folic acid (FA) was introduced onto the surface of the NCs as a tumor-targeting moiety [33, 34]. The folic acid receptor (FR), a membrane protein having a high affinity to FA, is overexpressed on various human cancer cells (e.g., ovarian, lung, breast, endometrial, renal and myeloid hematopoietic cells), while its expression is highly restricted in normal tissues [35–37]. Therefore, FA has been widely used as a targeting ligand for various anti-cancer agents to avoid their non-specific translocation to normal tissues [38, 39]. The CIS/ZnS QD-loaded NCs were very stable in water, as clearly indicated by the apparent color change (figure S4A, supporting information, avilable at stacks.iop.org/NANO/25/ 175702/mmedia). The mean hydrodynamic diameter of the CIS/ZnS-HSA-PEG NCs was 91.3 ± 10.0 nm in Milli-Q water, as determined by dynamic light scattering (DLS) (figure 3(A)). In contrast, the diameter of the CIS/ZnS QDs dispersed using only six-armed PEG (denoted ‘CIS/ZnS-PEG NCs’) without HSA was 171.8 ± 19.0 nm. This result indicates that the presence of HSA is critical for the formation of stable NCs for the QDs. The surface of the CIS/ZnS-HSAPEG NCs can be chemically modified with various targeting ligands using the functional groups of HSA. For the tumortargeted delivery of the HSA-PEG NCs, FA was conjugated to the NCs using a hetero-bifunctional form of PEG (FAPEG-NH2, MW = 3.4 kDa). The calculated average FA-PEG grafting density on the HSA-PEG NCs was 5.7 × 103 PEG
chains per nanocapsule, as obtained by dividing the total number of FA-PEG-NH2 (4.5 × 1017) consumed for the conjugation to the NCs by the total number of the NCs (7.9 × 1013). The surface area of each nanocapsule, with a diameter of about 91.3 nm, was 2.6 × 104 nm2. Hence, one PEG chain was grafted per 5 nm2 surface of the NCs [27, 40]. The conjugation of FA-PEG increased the hydrodynamic diameter of the CIS/ZnS-HSA-PEG NCs from 91.3 ± 10.0 nm to 107.0 ± 12.2 nm. The hydrodynamic diameter of a linear PEG chain (3.4 × 103 g mol−1) is about 4.5 nm. The actual increase in the diameter of the NCs was about 15.7 nm, which was significantly larger than the value expected from the hydrodynamic diameter of PEG (4.5 nm × 2 = 9 nm). This may stem from the extension of the grafted PEG chains as they form a highly dense comb-like conformation on the surface of the NCs [41]. A zeta potential analysis showed that the CIS/ZnS-PEG exhibited a slightly negative surface charge (−15.6 ± 0.6 mV) (figure 3(B)). The CIS/ZnS-HSA-PEG NCs exhibited a more negative zeta potential value (−34.1 ± 1.7 mV) presumably on account of the intrinsic negative property of HSA, which has an isoelectric point of 5.05 [42]. Note that HSA-based nanoparticles generally show a zeta potential of about −40 mV at pH values between 7.2 and 10 [42]. The relatively low zeta potential value (−24.0 ± 2.1 mV) of the FA-conjugated CIS/ZnS-HSA-PEG (denoted as ‘CIS/ZnS-HSA-PEG-FA’) NCs may originate from the presence of the neutral PEG linkers of FA-PEGNH2. The long-term dispersion stability of CIS/ZnS-HSAPEG NCs in the presence of 10% FBS was examined by 5
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Figure 3. Characterization of the CIS/ZnS QDs. The hydrodynamic diameter (A) and zeta potential (B) of the CIS/ZnS QDs stabilized with six-armed PEG-NHS (a), CIS/ZnS -HSA-PEG NCs (b) and CIS/ZnS-HSA-PEG-FA NCs (c). The long-term dispersion stability of the CIS/ ZnS-PEG, CIS/ZnS-HSA-PEG and CIS/ZnS-HSA-PEG-FA NCs by measuring the time-dependent changes of hydrodynamic diameter (C) and absorbance at 500 nm (D) in a 10% serum-containing medium. The TEM images of the CIS/ZnS-HSA/PEG-FA NCs in deionized water (E).
in aqueous media containing serum proteins. The morphology of the CIS/ZnS-HSA-PEG-FA NCs was observed using TEM. While the CIS/ZnS QDs dispersed in DCM were evenly distributed on the grid, the CIS/ZnS-HSA-PEG NCs were well dispersed in the form of spherical nanoparticles of about 20–40 nm in diameter, containing numerous QDs with diameters of 3–4 nm (figure 3(E)). The size of the NCs is much smaller than the value determined by DLS, presumably because the dehydration caused the shrinkage of the NCs, as observed in a previous study [27].
monitoring the time-dependent changes of the hydrodynamic diameter and absorbance at 500 nm. The CIS/ZnS-HSA-PEG and CIS/ZnS-HSA-PEG-FA NCs showed no significant change in their size distribution for 10 d or in their absorbance for 20 d in phosphate-buffered saline (PBS) (figures 3(C) and (D)), whereas the size of the CIS/ZnS-PEG gradually increased under the same conditions. These results suggest that the HSA-PEG NCs maintained a stable structure due to the cross-linked shell composed of NHS-functionalized PEG and HSA, allowing the stable dispersion of the CIS/ZnS QDs 6
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Figure 4. Absorption spectra and photoluminescence with an excitation wavelength of 380 nm (A) of the CIS/ZnS QDs dispersed in
dichloromethane and the CIS/ZnS-PEG, CIS/ZnS-HSA-PEG and CIS/ZnS-HSA-PEG-FA NCs. Photographs of the CIS/ZnS QDs dispersed in dichloromethane (a), CIS/ZnS-PEG (b), CIS/ZnS-HSA-PEG NCs (c) and CIS/ZnS-HSA-PEG-FA NCs (d) under UV illumination at 365 nm immediately after preparation and 7 d of incubation at room temperature (B). (C) Luminescence signal images of the CIS/ZnS QDs dispersed in dichloromethane (a), CIS/ZnS-PEG (b), CIS/ZnS-HSA-PEG NCs (c) and CIS/ZnS-HSA-PEG-FA NCs (d) at the same concentration of the CIS/ZnS QDs (0.1 mg mL−1).
DCM. The reduced PLQY may have been caused by the increased medium polarity to which the QDs were exposed [9, 43, 44]. In general, fluorescence emission can be influenced by the dielectric constant and hydrogen bonding capability of the medium, which are likely to change when QDs are bound to proteins, nucleic acids or macromolecules [44, 45]. Figure 4(B) shows photographic images of CIS/ZnS QDs and the CIS/ZnS-loaded NCs visualized under UV light in the dark. After their preparation, all of the samples showed very good colloidal homogeneity in PBS. However, after a seven-day incubation period at room temperature, the CIS/ ZnS-PEG formed clearly observable agglomerates, while the
When the QDs were encapsulated within the HSA-PEG NCs, the emission peak showed a red shift to 650 nm. Moreover, the fluorescence emission intensity per CIS/ZnS QD decreased slightly (figure 4(A)). The relative peak intensity of the fluorescence emission from the CIS/ZnSHSA-PEG NCs was 78.0% compared to that of the CIS/ZnS QDs dispersed in DCM at the same concentration of CIS/ZnS QDs. The emission intensities were also measured at different concentrations of the CIS/ZnS QDs, which showed the same absorbance at the excitation wavelength (380 nm) (figure S5). The calculated PLQY in this condition showed a more dramatic decrease (44.7%) compared to the CIS/ZnS QDs in 7
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Figure 5. Flow cytometric analysis of the intensity of QD-mediated fluorescence in HeLa cells following the incubation with PBS (A), CIS/
ZnS-HSA-PEG NCs (B), CIS/ZnS-HSA-PEG-FA NCs (C) and CdSe-HSA-PEG-FA NCs for 12 h.
damage [47]. Cell viability is assayed quantitatively in a CCK-8 assay, in which the formation of formazan dye depends on the activity of the mitochondria [48]. HeLa cells (human epithelial carcinoma cells) and PC3 cells (human prostate cancer cells) were chosen as model cancer cells due to their relatively high expression of FRs. A549 cells (human adenocarcinomic alveolar basal epithelial cells) were also used as a negative control because of their FR deficiency. NCs loaded with the CdSe/ZnS QDs with and without FA were prepared and used as a control (denoted as ‘CdSe-HSAPEG NCs’ and ‘CdSe-HSA-PEG-FA NCs’, respectively). The absorption and fluorescence emission spectra of the CdSe/ZnS QDs are provided in figure S8. Several studies have shown acute toxicity originating from Cd-based QDs in tissue cultures [49, 50]. Cytotoxicity was determined after the exposure of cancer cells to the QD-loaded NCs for 12 h. In the LDH assay, when the cells were exposed to high QD doses (100–1,000 μg mL−1), HeLa cells showed cytotoxic responses only to the CdSe-HSA-PEG-FA NCs, while PC3 cells responded to all of the samples except for CIS/ZnSHSA-PEG NCs (figures 6(A)–(C)). The cytotoxic responses of HeLa and PC3 cells were significantly more sensitive to the FA-conjugated NCs, indicating that intracellular translocation is critical for the cytotoxic effects of the QDs. In particular, a dose-dependent increase of LDH leakage was observed in PC3 cells (figure 6(B)). The CdSe-HSA-PEG-FA NCs showed a greater LDH release by 1.6 times compared to the CIS/ZnS-HSA-PEG-FA NCs at a QD concentration of 1 mg mL−1. The CCK-8 assay also revealed the cytotoxic effects of QDs that were consistent with the LDH release results (figures 6(D), (E)). Loss of cell viability (74.6 ± 4.9%) was notable in the HeLa cells exposed to 1 mg mL−1 of the
CIS/ZnS-HSA-PEG and CIS/ZnS-HSA-PEG-FA NCs maintained their homogeneity. The CIS/ZnS-HSA-PEG-FA NCs showed blue fluorescence because the FA, which was conjugated to the surface of the CIS/ZnS-HSA-PEG NCs, has an emission band peaking at around 450 nm (figure S6) [35]. Different types of fluorescent optical images were taken using a charge-coupled-device (CCD) camera. The average radiance (photon/s/cm2/Sr) was obtained by drawing a region of interest (ROI) over appropriate glass wells. The CIS/ZnS QDs dispersed in DCM showed a heterogeneous distribution, and some of them were adhered to the surface of the glass wall. In contrast, the CIS/ZnS-HSA-PEG NCs showed a relatively homogeneous dispersion in PBS (figure 4(C)). The cellular uptake of the QD-loaded HSA-PEG NCs was measured by a fluorescence-activated cell sorting (FACS) analysis (figure 5). The conjugation of FA to the NCs increased the fluorescence intensity of HeLa cells, indicating more efficient cellular internalization of the NCs. There was no significant difference in the cellular uptake between the CdSe-HSA-PEG-FA and CIS/ZnS-HSA-PEG-FA NCs. Therefore, it was assumed that any differences in the cytotoxic effects of the tested samples must derive from the different QDs loaded with the NCs. To determine the impact of heavy metal species in the QDs on the level of cellular toxicity, two different cytotoxicity assays were conducted using three different cancer cell lines and smooth muscle cells. LDH release and CCK-8 assays were performed on cells treated with NCs which incorporated the QDs. LDH evaluates the integrity of the cell membrane using the cytoplasmic enzyme activity released from damaged cells [46]. The loss of intracellular LDH and its release into the culture medium is an indicator of irreversible cell death due to cell membrane 8
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Figure 6. Comparison of LDH release assay and cell viability in HeLa cells (A, D), PC3 cells (B, E) and A549 cells (C, F) after exposure to the CIS/ZnS-HSA-PEG, CIS/ZnS-HSA-PEG-FA, CdSe-HSA-PEG and CdSe-HSA-PEG-FA NCs for 12 h.
more sensitive to the FA conjugation because the prostatespecific membrane antigen could act as an FA transporter [51]. Prostate-specific membrane antigen provides prostate cancer cells with a growth advantage by increasing the cellular level of FA. A549 cells exhibited significantly higher cytotoxic responses for Cd-based QDs compared to CISbased QDs. However, A549 cells did not show any dependency on the FA conjugation owing to their FR deficiency. The LDH release from A549 cells was similar at different concentrations of the CIS/ZnS-HSA/PEG and CIS/ZnS-HSAPEG-FA NCs (figure 6(C)). The CIS/ZnS QDs showed no
CdSe-HSA-PEG-FA NCs, but no significant cytotoxic effect was observed for the CIS/ZnS-HSA-PEG NCs. The percentages of viable cells treated with the CIS/ZnS-loaded NCs decreased to 71.8%, while that with the CdSe-HSA-PEG NCs significantly decreased to 50.9% in PC3 cells. These results demonstrate that CIS/ZnS QDs are likely to be a safer alternative to CdSe/ZnS QDs. The differences in the cytotoxicities indicate intracellular effects rather than permanent cell membrane damage [47]. In general, the cytotoxicity of QDs is mainly caused by cadmium ions that are extracted from the nanoscale nanocrystals [17]. It was found that PC3 cells were 9
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Figure 7. (A) In vivo fluorescence images of a HeLa tumor-bearing mouse after intravenous injection of the CIS/ZnS QDs dispersed in 10% DMSO, CIS/ZnS-HSA-PEG NCs and CIS/ZnS-HSA-PEG-FA NCs in PBS. The area circled in red indicates the implanted tumor site. Whole body fluorescence images of each mouse were acquired from an IVIS® Xenogen imaging system. (B) Ex vivo images of major organs (heart, lung, kidney, spleen and liver) and a tumor harvested from the mice at 48 h post-injection. (C) Ex vivo distribution of the CIS/ZnS QDs expressed as the fluorescence intensity per gram of each excised organ.
formulations were detected as faint spots in the tumor region. To evaluate the spatial distribution of the CIS/ZnS QDs in the body more precisely, we obtained the ex vivo fluorescence images of the organs excised from the mice (figure 7(B)). The CIS/ZnS-HSA-PEG-FA NCs exhibited a very strong fluorescence signal in the tumor, while only marginal fluorescence was detected in the heart and kidney. A low fluorescence signal was observed in the lung, spleen and liver, indicating that the injected NCs were preferentially accumulated in the tumor with little non-specific translocation to normal tissues. Similarly, it was reported that PEGylated nanoparticles are scarcely recognized and cleared by the macrophages distributed in the liver [27]. The fluorescence intensity per gram of tumor increased strikingly due to the FA conjugation. The tumor targeting effect of FA suggests that the tumor tissues can be reliably monitored in vivo using FA-conjugated NCs incorporating heavy-metal-free CIS/ZnS QDs.
significant cytotoxic effect, even at high concentrations, whereas the CdSe/ZnS QDs induced a more significant LDH release, leading to cell death at high concentrations of the QDs. Finally, we investigated the feasibility of the CIS/ZnS QDs for in vivo tumor-targeted imaging. NCs containing 20 μg QDs were injected intravenously into the tail vein of female BALB/C nude mice. In vivo accumulation of the CIS/ ZnS-loaded NCs in tumor tissue was examined using an IVIS® Lumina imaging system (Caliper Life Sciences). Fluorescence imaging was performed using a laser excitation filter passband of 500–550 nm and a CCD camera with a onesecond exposure time, and the average fluorescence intensity, which is defined as the total photon count per unit area of a tumor, was determined. Figure 7(A) shows fluorescence images of the HeLa tumor-bearing mice 24 h after the injections of the CIS/ZnS QDs dispersed in 10% dimethyl sulfoxide, CIS/ZnS-HSA-PEG NCs and CIS/ZnS-HSA-PEG-FA NCs. The tumor, marked by a red circle, was easily visualized within a few hours of the injection, and the accumulation of QDs was observed 24 h after the injection. The injected CIS/ ZnS-HSA-PEG-FA NCs were efficiently directed to and stayed in the tumor tissue, whereas the CIS/ZnS QDs in other
4. Conclusion In this work, we introduced shell cross-linked HSA-PEG hybrid NCs incorporating heavy-metal-free CIS/ZnS QDs for 10
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tumor-targeted fluorescence imaging. The CIS/ZnS-HSAPEG NCs exhibited a high level of fluorescence intensity comparable to that in a non-polar organic solvent as well as excellent dispersion stability in a serum-containing medium. Surface modification of the NCs with FA effectively facilitated the tumor-targeted delivery of the QD-incorporated NCs. In contrast to Cd-based QDs, the CIS/ZnS QDs did not induce a significant level of cytotoxicity in cancer and smooth muscle cells. In addition to reduced cytotoxicity, the FAconjugated NCs loaded with the CIS/ZnS QDs were efficiently accumulated in tumor tissue with strong fluorescence intensity, demonstrating efficient tumor-targeted imaging. We expect that the shell cross-linked HSA-PEG hybrid NCs can be used as a promising nanocarrier platform for the stable systemic circulation of heavy-metal-free QDs and other functional molecules (e.g., anticancer drugs, iron oxide nanocrystals and other optical probes). Facile surface functionalization of the NC surface also provides a greater number of potential applications of shell cross-linked NCs for functional diagnosis and theranostics.
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Acknowledgements This research was financially supported by the Ministry of Health, Welfare and Family Affairs (A111552) and the Converging Research Center (2009-0082276) funded by the National Research Foundation, Republic of Korea.
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