TY - JOUR
AU - Prasad, Paras, N.
AB - Abstract This contribution introduces the use of cadmium-free CuInS2quantum dots (QDs) for targeted and multiplexed optical imaging of tumors in mice. CuInS2/ZnS QDs were synthesized in a non-aqueous phase using the hot colloidal synthesis method. Previous challenges involving stable aqueous dispersion of highly luminescent CuInS2/ZnS QDs have been overcome by encapsulating them within functionalized phospholipid micelles, which also facilitated their conjugation with folic acid for targeted delivery. Luminescence signals of QDs of multiple colors were readily differentiated from background autofluorescence in whole animal optical imaging. In addition, two-photon excitation studies revealed that the prepared water-dispersible QDs are suitable for two-photonin vitro and in vivo imaging. This study demonstrates the important key steps in realizing of the potential of CuInS2 QDs as low-toxicity, photostable, cadmium-free and highly luminescent probes for cancer detection and sensing. Insight, innovation, integration Commonly used cadmium-based quantum dot (QD) formulations are suspected to result in biological toxicity at the cellular and tissue levels, as a result of their degradation in the biological environment. Although cadmium-free CuInS2/ZnS QDs have been proposed for biological applications, no studies are available in the literature to date reporting their biomedical use. Our research has developed a novel integrative strategy that produces bioconjugated CuInS2/ZnS QDs for targeted bioimaging applications, particularly in cancer diagnosis. CuInS2/ZnS QDs are expected to have a major impact on cancer research through development of biocompatible nanoformulations for safe and efficient targeted delivery. Employing folic acid-conjugated CuInS2/ZnS QDs, we find that tumor labelingin vivo can be achieved without showing any toxic effects, potentially revolutionizing early cancer diagnosis strategies. 1. Introduction The synthesis of non-toxic and bioconjugated quantum dots (QDs) remains an attractive and important biomedical research area for advancing the development of new cancer imaging, labeling, and sensing platforms.1–7 The motivation for engineering new QD systems is based on their unique tunable luminescence properties in comparison with conventional chromophores.8–11 The narrow emission peaks of QDs can be systematically tuned from visible to near-infrared by manipulating their size, composition, and shape.12 Also, QDs have a continuous absorption band behavior that allows a single laser light source to excite multicolored QDs simultaneously, which is a major advantage when compared to simultaneously exciting multiple organic dyes emitting at different wavelengths, which requires multiple light sources.7,13,14 More importantly, in sharp contrast to non-photostable organic dyes, QDs have extraordinary resistance towards photobleaching.15,16 All these attractive features of QDs have made them extremely promising candidates to be engineered as the new generation of optical probes for various immuno-assays, multiplex imaging of cancer cells, and in vivo cancer targeting and imaging studies, etc.17–20 However, their widespread biological use is severely limited by the presence of cadmium as the main ingredient in most commercial QD formulations. Many reports have shown that cadmium-based QDs were toxic at the tissue and cellular levels when their surfaces are not carefully functionalized.21,22 Therefore, the QD community is currently focused on developing cadmium-free QDs as safe and non-toxic probes for biological use, with potential for being translated into the clinic.23 CuInS2 is a direct band gap semiconductor material with a band gap of 1.45 eV. Thus, by tailoring their composition and size, it is possible to fabricate CuInS2 QDs that emit from the visible to near-infrared (NIR) region, with high quantum yield. More importantly, CuInS2 QDs are more suitable for biomedical imaging applications because the particles are free from toxic elements such as cadmium, lead, mercury, tellurium, and arsenic. Currently, several methods have been proposed in the literature for synthesizing high quality and monodispersed CuInS2 QDs in the non-aqueous phase.24–26 A very recent study has reported their transfer to the aqueous phase via a ligand exchange method and non-targeted biodistribution studies in vivo, where most of the QDs accumulated in the liver, spleen and lungs in a mouse.27 However, no studies are available in the literature regarding their bioconjugation for targeted delivery and bioimaging of cancer, both in vitro and in vivo. In this paper, we report a rapid and facile method for formulating CuInS2/ZnS QDs encapsulated within functionalized PEGylated phospholipid micelles, which not only renders their stable dispersion in biological fluids, but also facilitates their bioconjugation to folic acid (FA). FA is a well known tumor targeting molecule as its corresponding receptors (folate receptors) are known to be overexpressed in a variety of cancer cells.28 The engineered QD–FA bioconjugates are monodisperse in size, with a quantum yield as high as 35%, which is sufficient for bioimaging applications. We subsequently demonstrated a comparative study of tumor targeting and imaging using these QD–FA bioconjugates for labeling pancreatic tumor-bearing mice where the tumor cells are overexpressed with folate receptors. Toxicity studies reveal that the micelle-encapsulated QDs do not exhibit any toxicity at the cellular and tissue level. To the best of our knowledge, this is the first report using I-III-V semiconductor QDs as cadmium-free luminescence probes for targeted and multiplexed bioimaging. 2. Materials and methods 2.1. Materials Indium acetate, copper(iii) nitrate, oleylamine, oleic acid, sulfur, and dodecanethiol were purchased from Aldrich. DSPE-mPEG [1,2-diacyl-sn-glycero-3-phosphoethanolamine-N-methoxy(polyethylene glycol)] with average molecular weight of 5000 was purchased from Laysan Bio, Inc. All chemicals were used as received. All solvents (DMSO, hexane, toluene, and ethanol) were of reagent grade and were used without further purification. 2.2 Synthesis of CuInS2/ZnS QDs using hybrid synthesis approach The synthesis method was adapted from that presented by Xie et al.28 An oleylamine–sulfur solution was prepared by dissolving 0.1926 g of sulfur (6 mmol) in 6 mL of oleylamine. Separately, 1 mmol of indium acetate, 1 mmol copper nitrate, 2 mL of oleic acid, 3 mmol of stearic acid were dissolved in 15 mL of octadecene. The mixture solution was heated at 175 °C for 20–35 min under argon flow, then 10 mmol of dodecanethiol and 3 mL oleylamine–sulfur solution was injected under vigorous stirring into the hot reaction mixture. The reaction mixture was held at 175 °C and stirred for ∼10–15 min and then an aliquot was removed by syringe and injected into a large volume of toluene at room temperature to quench the reaction. The QDs were separated from the chloroform solution by the addition of ethanol and centrifugation. The NC precipitate could be redispersed in various organic solvents including hexane, toluene, and chloroform. The synthesis method for forming a ZnS shell on CuInS2quantum dots was adapted from Yong et al.21 A CuInS2quantum dot solution was prepared in advance by dispersing ∼0.4 g of QDs in ∼5 mL of chloroform. Separately, 2 mmol of zinc acetate, and 3 mmol of myristic acid were dissolved in 10 mL of oleic acid. The reaction mixture was heated to 150 °C for ∼30 min under an argon flow and then the nanocrystal solution was injected into the hot reaction mixture. After ∼15 min of heating, the reaction temperature was raised to 190 °C. Upon reaching the desired temperature, 1 mL of TOP-Sulfur was added dropwise into the reaction mixture. The reaction mixture was then held at ∼200 °C for 10 to 15 min and then an aliquot was removed via a syringe and was injected into a large volume of toluene at room temperature. The nanocrystals were separated from toluene solution by addition of ethanol and centrifugation. 2.3 Preparation of PEGylated phospholipid micelle-encapsulated CuInS2/ZnS QDs Briefly, ∼2 mL of QDs in chloroform dipersion (∼40 mg mL−1), ∼2 mL DSPE-mPEG in chloroform solution (∼20 mg mL−1), and ∼1 mL DSPE-PEG-carboxyl in chloroform solution (∼10 mg mL−1) were mixed together in a 25 mL round bottom flask. The mixture was gently stirred for 5 to 10 min. A Labconco rotory evaporator with a water bath of 25 °C was used to evaporate the organic solvent. The lipidic film, deposited on the reaction flask, was hydrated with 3 to 5 mL of HPLCwater. The resulting dispersion was filtered through a 0.45 μm membrane filter and kept at room temperature for further use. To remove the excess phospholipids from the micelle-encapsulated QD dispersion, the micelle-encapsulated QRs were further purified using centrifugation at 10 000 rpm for 15 min. The QD precipitate was then re-dispersed in 1 to 2 mL of HPLCwater. 2.4 Conjugation of micelle-encapsulated QDs with folic acid From the stock solution, 1 mL of the aqueous QD dispersion was mixed with 100 μL of 10 mM 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) solution and incubated for 5 min. Next, 100 μL of folic acid in DMSO solution (6 mg mL−1) was added into this mixture and stirred at room temperature for 1 h to allow the folic acid to covalently couple with the QDs. After one hour of stirring, the bioconjugated QDs were purified (removing excess byproducts) using centrifugation. The QD precipitate was re-dispersed in HPLCwater for in vivo studies. 2.5 Characterization of QDs The QD sizes were determined using a JEOL JEM-100cx transmission electron microscope with an accelerating voltage of 80 kV. The samples were prepared by drop-casting the QD dispersion onto an amorphous carbon coated 300 mesh copper grid, which was placed on filter paper to absorb the excess solvent. The hydrodynamic diameter of the aqueous dispersed QDs was measured using a 90Plus particle size analyzer (Brookhaven Instruments Corporation, USA). The UV-visible absorption and the fluorescence spectra of the QDs were acquired at room temperature by using a Shimadzu UV-3600 spectrophotometer and a Fluorolog-3 spectrofluorometer (Jobin Yvon, Longjumeau, France), respectively. Quantum yields (QYs) of the nanocrystal solutions were determined by comparing the integrated emission from the nanocrystals to that of the Coumarin 540A dye solution at normalized absorbance. Samples were diluted so that they were optically thin. Powder X-ray diffraction patterns were recorded using a Siemens D500 diffractometer, with Cu Kα radiation. A concentrated nanocrystal dispersion was drop cast onto a quartz plate for measurement. 2.6 Generation of xenografted tumors in mice Panc-1 (CRL-1469) cells line was obtained from American Type Tissue Collection (ATCC). The cells were maintained in a DMEM high glucose medium containing 10% FBS, 1 mM l-glutamine, 100 μg mL−1Kanamycin and 0.5 μg mL−1 amphotericin B in a 75 cm2 delta treated flask (Nunc A S−1, Rockslide, Denmark). An 85–90% confluent cell flask was rinsed 3 times with sterile DPBS (Sigma-Aldrich). 5 mL of a trypsin–EDTA (Sigma-Aldrich) solution was added to the flask and incubated at 37 °C and 5% CO2. Once the cells were released, 5 mL of medium was added to the trypsin, and the entire flask contents was removed and placed in a sterile 50 mL centrifuge tube. The cells were spun down at 2000 rpm for 5 min and the supernatant was removed. The cell pellet was re-suspended in 5 mL of fresh media for further in vivo tumor cell implantation use. 5 to 6 week old female athymic nude mice (Hsd:Athymic Nude-Foxn1nu) were obtained from Harlan Laboratories, Inc., and were allowed an acclimatization period of 1 week. The nude mice were housed in sterile M.I.C.E caging (Animal Care Systems, Centennial CO.) that contained sterile bedding, food and water. Animal care was set up in accordance with the guidelines of the Intuitional Animal Care and Use Committee (IACUC) at the University of Buffalo. Panc-1 (ATCC, CRL-1469) cells at a concentration of 2–3 × 106cells in a 100 μL suspension were mixed with equal volume of Matrigel (BD Biosciences) at 4 °C, and injected subcutaneously in one scapular region of the mice using a 1 mL Monoject tuberculin syringe with a 25g × 5/8” detachable needle (Tyco Healthcare Group, Mansfield, MA). Tumor growth was monitored every 48 h. When a tumor size of approximately 0.5–0.9 cm3 was obtained approximately 2 to 3 weeks post-transplantation of cells, the mice were processed for imaging studies. 2.7 Small animal imaging studies For the in vivo whole body tumor imaging study, athymic nude mice bearing subcutaneous Panc-1 tumors were intravenously injected with non-bioconjugated or folic acid-conjugated QDs (∼0.5 mg per 200 μL of QD bioconjugates per animal, 3 mice were used for each set of experiment). The mice were imaged at different time points starting from 1 to 24 h post-injection, using a Maestro in vivo imaging system (CRI, Inc., Woburn, MA; excitation filter: 445–490 nm, emission filter: 515 nm long-pass). The Maestro optical system consists of an optical head that includes a liquid crystal tunable filter (LCTF, with a bandwidth of 20 nm and a scanning wavelength range of 500–950 nm) with a custom-designed, spectrally optimized lens system that relays the image to a scientific-grade megapixel CCD. The CCD captured the images at each wavelength. The captured images (spectral cube, containing a spectrum at every pixel) can be loaded into the vendor software and analyzed. Spectra from the autofluorescence (from the skins, tissues, and food, coded green) and QD-associated luminescence signals (coded red) can be unmixed using the vendor software. In this study, a scanning wavelength range between 500 and 950 nm was used as recommended by the CRI instrument manual. 3. Results and discussion 3.1 Synthesis and characterization of micelle-encapsulated CuInS2/ZnS QDs Fig. 1 shows the TEM image of organic-dispersible CuInS2/ZnS QDs. The particle size is estimated to be 3 to 4 nm. The TEM images of these QDs following their aqueous dispersion via encapsulation within PEGylated phospholipids micelles are shown in Fig. 2. Fig. 2 shows a wide range of sizes of clusters for the CuInS2/ZnS QDs material. Careful analysis of Fig. 2a and b indicates the micelle size ranges from 40 nm to 90 nm. The larger clumps as shown in Fig. 2a are interpreted as micelle aggregates. Fig. 2b shows that about 8 to 25 QDs are encapsulated in a micelle. Fig. 2b shows the lattice fringes of many crystalline QDs within the micelle. The micelle-encapsulated QDs can be dispersed in biological buffers such as PBS and HEPES, without showing any quenching effects for more than a week. Fig. 3 presents the XRD pattern of CuInS2/ZnS QDs. All of the diffraction peaks from the samples can be indexed to the wurtzite CuInS2. EDS confirmed the presence of Cu, In, S, and Zn. Fig. 1 Open in new tabDownload slide TEM images of monodispersed organically-dispersible CuInS2/ZnS QDs at different magnification. Fig. 1 Open in new tabDownload slide TEM images of monodispersed organically-dispersible CuInS2/ZnS QDs at different magnification. Fig. 2 Open in new tabDownload slide TEM images of monodispersed phospholipid micelle-encapsulated CuInS2/ZnS QDs at different magnification. Fig. 2 Open in new tabDownload slide TEM images of monodispersed phospholipid micelle-encapsulated CuInS2/ZnS QDs at different magnification. Fig. 3 Open in new tabDownload slide XRD patterns of CuInS2/ZnS nanocrystals. Fig. 3 Open in new tabDownload slide XRD patterns of CuInS2/ZnS nanocrystals. In addition to TEM, EDS and XRD analysis, dynamic light scattering (DLS) was employed to determine the hydrodynamic diameter and stability of the micelle-encapsulated QDs, represented in Fig. 4. The average hydrodynamic diameter of the functionalized nanoparticles is estimated to be ∼86 nm, which corresponds well with the TEM result. The colloidal stability of the micelle-encapsulated nanoparticles in PBS was further evaluated using the DLS technique for one week. Less than 20% change in the particle size was observed in the hydrodynamic diameter during the period of measurement. Besides PBS, we have also tested these QDs dispersed in a serum containing cell-culture medium, which mimics human body fluids. No aggregation was observed for more than three days. More importantly, the quantum yield of QDs was maintained throughout the testing period. The quantum yield of CuInS2/ZnS QDs was estimated to be 35%. Fig. 4 Open in new tabDownload slide Hydrodynamic size distribution of micelle-encapsulated QDs in the aqueous suspension, measured by dynamic light scattering (DLS). Fig. 4 Open in new tabDownload slide Hydrodynamic size distribution of micelle-encapsulated QDs in the aqueous suspension, measured by dynamic light scattering (DLS). 3.2 Optical properties of CuInS2/ZnS QDs Fig. 5 shows the normalized one-photon (excitation at 532 nm) and two-photon (excitation at 1064 nm) excited photoluminescence spectra from the CuInS2/ZnS QDs dispersed in chloroform. The spectra were obtained using a grating spectrometer. Upon comparing these curves, the emission spectra under one- and two-photon excitation conditions are identical with an emission peak at ∼695 nm and a spectral bandwidth of ∼130 nm, indicating that they originate from the same upper emitting level(s) of this sample medium under two different excitation conditions. To verify that the observed photoluminescence is truly the result of one- and two-photon excitation at 532 and 1064 nm, respectively, the emission intensity was measured as a function of the input excitation intensity at these wavelengths. Fig. 6 and 7 show the measurements on a logarithmic scale. The one- and two-photon excitation shows a linear and quadratic dependence of the emission intensity on the excitation intensity. Therefore, on logarithmic scales, there should be a linear line with a slope of 1 for one-photon excitation and 2 for two-photon excitation, respectively. From Fig. 6, the slope of the best-fitting straight lines is 0.99, for data measured with 532 nm laser pulses, confirming the mechanism of one-photon excitation. Also, in Fig. 7, one can see that the slope of the best-fitting line is 2.01 for data measured with 1064 nm laser pulses, confirming the mechanism of two-photon excitation. Fig. 5 Open in new tabDownload slide One- and two-photon induced photoluminescence spectra of CuInS2/ZnS QDs dispersed in chloroform. Fig. 5 Open in new tabDownload slide One- and two-photon induced photoluminescence spectra of CuInS2/ZnS QDs dispersed in chloroform. Fig. 6 Open in new tabDownload slide Linear dependence of one-photon induced photoluminescence on the excitation intensity. Fig. 6 Open in new tabDownload slide Linear dependence of one-photon induced photoluminescence on the excitation intensity. Fig. 7 Open in new tabDownload slide Quadratic dependence of two-photon induced photoluminescence on the excitation intensity. Fig. 7 Open in new tabDownload slide Quadratic dependence of two-photon induced photoluminescence on the excitation intensity. In addition to spectral measurement of QDs in chloroform, we have also measured the normalized emission spectra of aqueous dispersed micelle-encapsulated CuInS2/ZnS QDs, shown in Fig. 8. Here, the obtained one- and two-photon excited emission spectra in an aqueous dispersion are similar to those obtained for QDs in chloroform. Therefore, the influence of solvent on the emission spectra of the CuInS2 QDs is insignificant. This result suggests that the nanoparticle formulation is suitable for both single and two-photon bioimaging studies. Fig. 8 Open in new tabDownload slide One- and two-photon induced photoluminescence spectra of micelle-encapsulated CuInS2/ZnS QDs dispersed in water. Fig. 8 Open in new tabDownload slide One- and two-photon induced photoluminescence spectra of micelle-encapsulated CuInS2/ZnS QDs dispersed in water. 3.3 In vitro cytotoxicity studies Owing to the absence of toxic heavy metals such as cadmium, lead and mercury as active ingredients, the micelle-encapsulated CuInS2/ZnS QDs are of extreme interest for biomedical applications, particularly for in vivo studies involving targeted cancer imaging. Prior to embarking on small animal imaging studies, we evaluated the potential cytotoxicity of these QDs in vitro in pancreatic cancer cells, using a cell viability (MTS) assay. Cytotoxicity studies of CdSe, CdTe, and CdSe/ZnS QDs with different sizes and surface coatings are well documented in the literature.29–31 However, similar studies have not been performed for CuInS2/ZnS QDs. Fig. 9 shows that cells treated with micelle-encapsulated QDs for 24 h maintained greater than 80% viability even at particle concentration as high as 195 μg mL−1, suggesting minimal cytotoxicity associated with these QDs. This result is in sharp contrast to previous reports involving similar studies using cadmium-based QDs, which have shown cytotoxicity within 24 h of treatment at much lower concentrations.32 Fig. 9 Open in new tabDownload slide Viability of Panc-1cells at 24 h of post-treatment in the presence of micelle-encapsulated CuInS2/ZnS QDs (filled circle) and CdTe QDs (opened circle). Fig. 9 Open in new tabDownload slide Viability of Panc-1cells at 24 h of post-treatment in the presence of micelle-encapsulated CuInS2/ZnS QDs (filled circle) and CdTe QDs (opened circle). To compare the cytotoxicity between cadmium-based and cadmium free QDs, cysteine-coated CdTe QDs were synthesized and used as a reference for MTS studies. As shown in Fig. 9, the particle concentrations corresponding to 50% cell viability (IC50) were roughly 100 μg mL−1 and 300 μg mL−1 for CdTe and CuInS2/ZnS QDs, respectively, in Panc-1cells. This means that even though CdTe QDs currently have a higher quantum yield (QY > 50%), the CuInS2/ZnS QDs (QY = 35%) can be loaded into cells at a higher concentration for bioimaging studies. Recently, we reported the preparation of micelle-encapsulated quantum rods for in vivo imaging.33 These anisotropic nanoparticles were observed to maintain their photoluminescence in animal body for several weeks without showing any ill effects. Previously, it was reported that micelle-encapsulated CdSe/ZnS QDs, with a single QD in a single micelle, can be fabricated within the size of 15 nm. In comparison here, our method has produced larger micelles with 8 to 25 QDs per micelle. Multiple QDs in a micelle may provide for more sensitive detection in biological systems than a single quantum dot micellar system would, thus increasing the sensitivity for in vitro and in vivo imaging as demonstrated by Erogbogbo et al.34 and Schroeder et al.35 We have previously demonstrated that these bioconjugates can be used as contrast agents for labelingcells and tissues, in vitro and in vivo.21,36 3.3 In vivo tumor targeting and imaging with functionalized CuInS2/ZnS QDs For this study, the carboxylated QDs were conjugated with folic acid for tumor targeting and imaging in small animals. We estimated that each QD has 70 to 100 carboxylgroups on the particle surface. The estimation method was adopted from our previous study. The total number of carboxylgroups on the particles surface can be varied by manipulating the ratio between DSPE-PEG-carboxyl and DSPE-PEG during the preparation of micelle-encapsulated QDs. The as-prepared bioconjugates was again examined by TEM. The size of QD bioconjugates was similar to that of non-conjugated QDs. The TEM analysis clearly indicated that no aggregation occurred during the conjugation process. To demonstrate the “proof of concept” of using the bioconjugated CuInS2/ZnS QDs for in vivo tumor targeting and imaging, the QD formulation was systemically administered in tumor-bearing mice by intravenous injection. After 30 min of injection, the mice were anesthetized for whole body luminescence imaging. The luminescence images were taken by employing the Maestro imaging system described in the experimental section. Fig. 10 shows the luminescence images of tumor-bearing mice intravenously injected with (a & b) bioconjugated and (c & d) non-bioconjugated QDs. As shown in Fig. 10, intense QD signals can be clearly detected from the tumor site of the mouse treated with bioconjugated QDs. Also, one can see that the QD signal (red) can be differentiated from the auto-fluorescence background (green). In contrast, little or no QD signal was obtained from the tumor area of the mouse treated with non-bioconjugated QDs, thus clearly demonstrating the tumor-specificity of the bioconjugated QDs. Fig. 11 shows the images of the resected tumor from the mouse injected with the folic acid-conjugated QDs formulation. The observation of red emission from the tumor of the mouse confirmed that folic acid-conjugation is necessary for in vivo tumor targeting in the present study. Fig. 10 Open in new tabDownload slide In vivo luminescence imaging of Panc-1 tumor-bearing mice (pointed to by white arrows) injected with ∼0.5 mg per 200 μL of folic acid-conjugated QDs (a & b) and non-bioconjugated QDs (c & d). All images were acquired under the same experimental conditions. The autofluorescence from tumor-bearing mice is coded green and the unmixed QD signal is coded red. Fig. 10 Open in new tabDownload slide In vivo luminescence imaging of Panc-1 tumor-bearing mice (pointed to by white arrows) injected with ∼0.5 mg per 200 μL of folic acid-conjugated QDs (a & b) and non-bioconjugated QDs (c & d). All images were acquired under the same experimental conditions. The autofluorescence from tumor-bearing mice is coded green and the unmixed QD signal is coded red. Fig. 11 Open in new tabDownload slide Ex vivo luminescence images of tumors, harvested 30 min post-injection from tumor-bearing mouse treated with bioconjugated QDs. Fig. 11 Open in new tabDownload slide Ex vivo luminescence images of tumors, harvested 30 min post-injection from tumor-bearing mouse treated with bioconjugated QDs. It is worth noting that the QD signal can be detected from the tumor as early as 15 min post-injection, although maximum uptake of QDs in the tumor matrix was achieved 30 min post-injection. Thus, we have chosen this particular time as our starting imaging point. We have noted that the QD signal was detected in the neck of the mice treated with both bioconjugated and non-bioconjugated QDs, which is probably a result of non-specific accumulation of the QDs. Also, physical evaluation was performed on the treated mice for more than 48 h post-injection. No changes were observed on the exploratory behavior, drinking, and eating of the mice, thus demonstrating the non-toxicity of our formulation. To study the biodistribution of the functionalized QDs, the treated tumor-bearing mice at one hour post-injection with the FA-conjugated QDs were sacrificed and their organs were removed and analyzed with the Maestro luminescence imaging system. In addition to their tumor-accumulation, the QDs were observed to be mainly accumulated in the liver and the spleen, indicating their rapid clearance from the blood via the reticuloendothelial system (RES) (data not shown). Some QD signal was also found in the lung and kidneys. Recently, Gao et al. prepared lectin-functionalized QDs for targeted brain tumor imaging.37 The authors found that the majority of the particles have accumulated in the lungs, liver, and kidney, and some signals were detected in the heart and spleen, 3 h post-injection. Our results are in good agreement with their observation. Our previous studies have demonstrated that particles accumulate in the liver or spleen, from where they are slowly removed via hepatobillary transport.38 Further investigations will be needed to determine the excretion time of the engineered micelle-encapsulated QDs formulation from the animal body. 3.4 In vivo multiplex imaging In addition to in vivo tumor imaging, functionalized CuInS2/ZnS QDs were used for whole body multiplex imaging. For this experiment, two different emission colors of CuInS2 QDs were injected in a mouse at different spots by subcutaneous injection. Fig. 12 shows the unmixed luminescence image of a live mouse after subcutaneous injections into the mouse back. High intensity luminescence from the QDs at different spots on the treated mouse can be easily observed and separated from one another. This proves that the micelle-encapsulated CuInS2 QDs can be used as a multicolored optical contrast agent for in vivo multiplex imaging. Fig. 12 Open in new tabDownload slide In vivo multiplex image of CuInS2/ZnS QDs subcutaneously injected at different locations in a live mouse. The back is injected with 640 (red) and 710 nm (blue) emitting CuInS2/ZnS quantum dots. The image is taken under 533 nm excitation in the Maestro imaging system. Fig. 12 Open in new tabDownload slide In vivo multiplex image of CuInS2/ZnS QDs subcutaneously injected at different locations in a live mouse. The back is injected with 640 (red) and 710 nm (blue) emitting CuInS2/ZnS quantum dots. The image is taken under 533 nm excitation in the Maestro imaging system. 3.5 Comparison between cadmium-based QDs and CuInS2/ZnS QDs To date, cadmium-based QDs are the most popular and widely used luminescence labels for in vitro and in vivo studies.39–43 However, the potential toxicity of these QDs remains a major roadblock for these particles to be successfully translated into clinical research.22,44,45 Several studies have suggested that these QDs could induce the toxicity when the particles are degraded and release cadmium ions to the biological environment.46,47 Therefore, many research groups are currently trying to overcome this challenge by two general approaches, which are either decorating the surface of cadmium-based QDs with biocompatible long lasting functional polymeric layer for preventing the breakdown of the particles, or designing cadmium-free QDs.8,48–52 In view of this, we have developed a formulation of functional CuInS2 as cadmium-free ultrasensitive probes to substitute cadmium-based QDs for some specific biological applications. We envision that the developed CuInS2 QDs here are more likely to complement the existing cadmium-based QDs, rather than replacing them because both types of QDs have their own limitations and advantages. In our approach, CuInS2 QDs can be fabricated without the need of using any toxic reagents and high temperature (>350 °C) environment. This is in sharp contrast to the synthesis of most common cadmium-based QDs, which involve toxic and explosive reagents, which requires for the QD precursors to be prepared and stored in vacuum condition for ensuring their high quality synthesis. More importantly, the quantum efficiency of high quality CuInS2/ZnS QDs is comparable with commonly used cadmium-based QDs, which make them appropriate probes for long term in vivo cancer screening and sensing. Also, by manipulating the composition of the particles, one can systematically tune the emission peak of the CuInS2 particles to the near-infrared region for enhanced signals in vivo, with significantly reduced background auto-fluorescence. Though there are many advantages of employing CuInS2 QDs for biological studies, there are some drawbacks as well. For example, the full-width half maximum CuInS2 QDs is around 140 nm, which is 2 to 3 times larger than that of the full-width half maximum of cadmium-based QDs. Also, the composition of copper, indium, sulfur in CuInS2 QD varies from particle to particle in one batch of synthesis process, which makes them difficult to isolate particles having the same composition. Further studies are required to solve these problems to further advance these functional CuInS2 QDs in biomedical applications. Summary In conclusion, we demonstrated the preparation of folic acid-conjugated CuInS2/ZnS QDs bioconjugates as powerful imaging probes for tumor targeting and imaging in live animals for the first time. The functional PEGylated phospholipid micelle-encapsulated QDs can be readily conjugated with cancer-specific biomolecules for targeted delivery to the cancerous area. By using a multichannel optical small animal imaging setup, we have demonstrated targeting the folate receptors which are overexpressed in tumor sites in vivo. More importantly, from the cytotoxicity cellular studies, the designed CuInS2/ZnS QDs were found to be more biocompatible than commonly used cadmium-based QDs. In addition, two-photon excitation studies for CuInS2 QDs suggest that these particles can be utilized for two-photon bioimaging applications. Thus, these biocompatible CuInS2/ZnS QDs are extremely promising candidates for advancing ultrasensitive cancer bioimaging and other medical applications. 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TI - Synthesis of ternary CuInS2/ZnS quantum dot bioconjugates and their applications for targeted cancer bioimaging
JF - Integrative Biology
DO - 10.1039/b916663g
DA - 2010-03-05
UR - https://www.deepdyve.com/lp/oxford-university-press/synthesis-of-ternary-cuins2-zns-quantum-dot-bioconjugates-and-their-iPHOByB2XC
SP - 121
EP - 129
VL - 2
IS - 2-3
DP - DeepDyve
ER -