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In vivo behavior of near infrared-emitting quantum dots
Biomaterials 34 (2013) 4302e4308
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Biomaterials journal homepage: www.elsevier.com/locate/biomaterials
In vivo behavior of near infrared-emitting quantum dots Yimei Lu a, Yuanyuan Su a, *, Yanfeng Zhou a, Jie Wang a, Fei Peng a, Yiling Zhong a, Qing Huang b, Chunhai Fan b, Yao He a, * a
Institute of Functional Nano and Soft Materials (FUNSOM) and Jiangsu Key Laboratory for Carbon-based Functional Materials and Devices, Soochow University, Suzhou, Jiangsu 215123, China b Laboratory of Physical Biology, Shanghai Institute of Applied Physics, Chinese Academy of Sciences, Shanghai 201800, China
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Article history: Received 31 January 2013 Accepted 19 February 2013 Available online 13 March 2013
Near-infrared (NIR, 700e900 nm) fluorescent nanomaterials-based probes have shown major impacts on high-resolution and high-sensitivity bioimaging applications. Typically, NIR-emitting quantum dots (QDs) are highly promising as NIR bioprobes due to their unique optical properties. However, NIRemitting QDs-related in vivo behavior remains unknown at present, severely limiting their wideranging bioapplications. Herein, we investigate short- and long-term in vivo biodistribution, pharmacokinetics, and toxicity of the NIR-emitting QDs. Particularly, we reveal that the NIR-emitting QDs are initially accumulated in liver, spleen, and lung for short-time (0.5e4 h) post-injection, and then increasingly absorbed by kidney during long-time (4e94 days) blood circulation. Obviously timedependent biodistribution is observed: with time continues, most of NIR-emitting QDs are finally accumulated in liver and kidney; comparatively, less NIR-emitting QDs are observed in spleen, lung, and bone marrow. Furthermore, histological and biochemical analyses, and body weight measurements demonstrate that there is no overt toxicity of NIR-emitting QDs in mice even at long-time (94 days) exposure time. Our studies provide invaluable information for the design and development of NIRemitting QDs-based nanoprobes for biological and biomedical applications. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: Near infrared Quantum dots In vivo Biodistribution Toxicity
1. Introduction It is well known that near-infrared (NIR)-fluorescence imaging is widely recognized as an effective method for high-resolution and high-sensitivity bioimaging because of its minimized biological autofluorescence and increased penetration of emission light through tissues in the NIR window [1]. In recent years, nearinfrared (700e900 nm) fluorescent nanomaterials-based probes have shown major impacts on biomedical and biological applications [2e9]. Typically, NIR-emitting II-VI quantum dots (QDs) are considered as high-performance NIR bioprobes due to their unique optical merits (e.g., strong fluorescence, robust photostability, and size-tunable fluorescence, etc) [10e12]. For examples, Frangioni et al. demonstrated that injection of only 400 pmol of NIR-emitting CdTe (CdSe) core (shell) heterostructured QDs permitted sentinel lymph nodes 1 cm deep, and could be easily imaged in real time,
* Corresponding authors. Tel.: þ86 512 6588 0946; fax: þ86 512 6588 2846. E-mail addresses: [email protected] (Y. Su), [email protected], [email protected] (Y. He). 0142-9612/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biomaterials.2013.02.054
suggesting the potential of NIR-emitting QDs for biomedical imaging [4]. Gambhir and coworkers exploited intravital microscopy with subcellular resolution (w0.5 mm) to directly observe the tumor neovasculature in living subjects using arginine-glycineaspartic (RGD) peptides-conjugated NIR-emitting QDs. That study demonstrated the direct performance of RGD-NIR-QDs binding to tumor neovasculature via direct cellular-level visualization in living mice [8]. Of particular note, Chen et al. recently revealed that NIRemitting QDs allowed multiplex imaging of deeper tissues, which opens up perspectives for integrin-targeted NIR optical imaging and would have great potential in cancer diagnosis and management as well as imaging-guided surgery and therapy [9]. Very recently, we presented an example of aqueous synthesized NIRemitting CdTe QDs for in vivo tumor targeting. Our in vitro and in vivo imaging data demonstrated that the NIR-emitting aqQDs were especially suitable for highly spectrally and spatially resolved imaging in cells and animals [11]. These above reports strongly suggest NIR-emitting QDs as high-quality bioprobes for myriad bioimaging applications. Concerns on in vitro and in vivo biosafety of QDs have received increasingly intensive attention, which is of essential importance for wide-ranging bioapplications [13e21]. Sufficient in vitro studies
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Fig. 1. (A) Absorption and photoluminescence spectra of the prepared NIR-emitting QDs with the maximum emission wavelength of 720 nm. The hydrodynamic diameter of the NIR-emitting QDs is determined by DLS (B), and their corresponding TEM image is shown in (C). Inset shows a high-resolution TEM (HRTEM) image of a single QD with clear crystal lattices.
have suggested that the cytotoxicity of QDs is ascribed to release of toxic metals [14,15] and production of reactive oxygen species [16,17], which can be largely alleviated by surface modification (e.g. epitaxial growth of ZnS shell) [18]. However, preliminary studies revealed that, in comparison to obvious QDs-induced cytotoxicity, QDs produced much less side effects in vivo [19,20]. For examples, Chan et al. demonstrated in a pilot study that the organic synthesized QDs (orQDs) did not cause appreciable toxicity in vivo even over long-term periods (e.g. 80 days) [21]. Our recent investigation also showed that there was no overt toxicity of aqueous synthesized QDs (aqQDs) in mice [13]. Such significant conflict between the conclusions drawn from in vitro and in vivo studies is due to distinct diversity of in vitro and in vivo physiological conditions [22,23]. Typically, in comparison to relatively simple in vitro environment, in vivo systems are much complex. As a result, the interactions between the nanostructures and complicated biological components in vivo lead to unique biodistribution, clearance, immune response, and metabolism [21,24e26]. It is worthwhile to point out that, most of the above-mentioned QDs used for in vitro and in vivo studies emit the visible light (450e650 nm). There currently exists scanty information regarding in vitro and in vivo behaviors of the NIR-emitting QDs, which severely limits the QDsbased NIR bioimaging applications. In this work, by using our previously reported CdTe NIRemitting QDs as a model [11], we carry out a comprehensive investigation on in vivo behaviors of the NIR-emitting QDs, including their short- and long-term in vivo biodistribution, pharmacokinetics, and toxicity. Our results provide useful information concerning the targeted organs, distribution, and elimination characteristics of NIR-emitting QDs in vivo, which is valuable for the design and development of NIR-emitting QDs for wide-ranging biological and biomedical applications. 2. Experimental section 2.1. Preparation of NIR-emitting QDs Tellurium powder (99.99%) and CdCl2 (99.99%) are purchased from Aldrich. 3Mercaptopropionic acid (MPA) (98%) and NaBH4 (96%) are purchased from SigmaeAldrich. All chemicals are used without additional purification. All solutions are prepared using Milli-Q water (Millipore) as the solvent. The CdTe QDs are synthesized based on our previously reported protocol [11]. In brief, the CdTe precursor solution is obtained by adding freshly prepared NaHTe solution to N2-staurated CdCl2 solution at pH 8.4 in the presence of 3-mercaptopropionic acid (MPA). The molar ratio of Cd2þ/MPA/NaHTe is set as 1: 2.4: 0.5. By controlling the reaction time and temperature (170 C/5 min), we obtain NIR-emitting QDs with the maximum emission wavelengths of 720 nm (photoluminescence quantum yield (PLQY): w25%). After the microwave irradiation, the QDs samples are taken when
the temperature cools to lower than 50 C naturally. The microwave system NOVA used for synthesizing QDs is made by Preekem of Shanghai, China. The system operates at 2450 MHz frequency and works at 0e500 W power. Exclusive vitreous vessels with a volume of 15 or 20 ml are equipped for the system to provide security during reaction demanding high temperature and pressure. In order to exclude the influence of residual reagents such as MPA, Cd2þ and Te2- in solution, the samples are carefully purified before subsequent biological experiments. In details, 2-propanol is added dropwise under stirring until the sample solution becomes slightly turbid. The turbid dispersion is kept on stirring for 15 min, and then subjected to centrifugation. The precipitate is collected while the supernatant is added another portion of 2-propanol to obtain the second precipitated fraction. This procedure is repeated for three times. Afterward, these precipitate are washed by Milli-Q water for three times to adequately remove residual reagents from samples. Finally, the QDs samples are employed for the following studies. The NIR-emitting QDs are characterized by UV-vis absorption, photoluminescence (PL), transmission electronic microscopy (TEM), and dynamic light scatterer (DLS). All optical measurements are performed at room temperature under ambient air conditions. UV-vis absorption spectra are recorded with a Perkin Elmer Lambda 750 UV-vis-near-infrared spectrophotometer. PL measurement is performed using a HORIBA JOBTN YVON FLUOROMAX-4 spectrofluorimeter. The PLQY of samples is estimated using CY7 (PLQY: w28%) in an ethanol solution as a reference standard, which is freshly prepared to reduce the measurement error [13]. The TEM/HRTEM overview images are recorded using Philips CM 200 electron microscope operated at 200 kV. Light-scattering analysis is performed using a DynaPro Dynamic Light Scatterer (DLS). 2.2. Animal injection, weight measurements and sample collection Female BALB/c mice (7e8 weeks old), obtained from Shanghai SLAC Labs Animal CO. LTD, are given food and water ad libitum and housed in a 12 h/12 h light/dark cycle. To characterize the biodistribution and accumulation of the NIR-emitting QDs, 0.4 nmol QDs in a volume of 0.1 ml are intravenously injected into the tail vein (n ¼ 4 or 5 for each time point); these groups of mice constitute the test groups. BALB/c mice (n ¼ 4 or 5) with injection of physiologic saline are selected as the control group. At increasing time points after injection, mice are weighed and assessed for behavioral changes. And then animals are sacrificed by exsanguination. The heart, liver, spleen, lung, kidney, stomach, brain, bone, muscle, intestine, bladder, urine, and feces are collected. Histological samples are carefully removed with a sharp razor blade and placed directly into fixative. Note that: 1) Concentration of the NIRemitting QDs is calculated following a previously published method by Yu et al. [27e 30]. 2) A dose of 0.4 nmol per mouse (w24 g) is comparable to the reported dose for other in vivo QDs experiment [13]. 2.3. Pharmacokinetic analysis Dominant organ samples (e.g., heart, liver, spleen, lung, kidney, stomach, brain, bone, muscle, intestine, and bladder) are collected, weighed and digested with nitric acid in microwave digestion system (EXCEL, Preekem Scientific Instruments Co. Ltd, Shanghai, China). Urine is measured by volume, 20 ml urine equals to 20.43 mg by weighing and digested. The solution is diluted with Milli-Q water and the cadmium ions content is quantified with inductively coupled plasma-mass spectrometry (ICPMS) to determine the total cadmium in the original organs. The percent of injected dose per gram tissue (% ID/g) of NIR-emitting QDs in a specific tissue is calculated by the following equation [13,31]:
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%ID=g ¼
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ð½QD in tissue suspensionÞ ðvolume of tissue suspensionÞ ð½QD in injected solutionÞ ðvolume of injected QDÞ ðwet weight of tissueÞ
Fig. 2. In vivo biodistribution of the NIR-emitting QDs at serial time point post-intravenous injection: (A) 0.5 h, (B) 1 h, (C) 4 h, (D) 8 h, (E) 12 h, (F) 18 h, (G) 1 day, (H) 4 day, (I) 13 day, (J) 94 day, and (K) Control, respectively.
Y. Lu et al. / Biomaterials 34 (2013) 4302e4308 2.4. Serum biochemistry and histology Blood samples are harvested from the mice injected with NIR-emitting QDs for 0.5 h, 1 h, 4 h, 8 h, 12 h, 18 h, 1 day, 4 days, 13 days, and 94 days, and from the mice receiving physiologic saline injection. Blood is collected from the orbital sinus by quickly removing the eyeball from the socket with a pair of tissue forceps. Indicators for liver and kidney function (alkaline phosphatase (ALP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), total protein (TP), albumin/globulin (A/G), serum creatinine (Cr-S), and urea) are measured. Upon completion of the blood collection, mice are sacrificed. The heart, liver, spleen, lung, kidney, brain, intestine, and bladder are removed, and then embedded in paraffin, sectioned, and eventually stained with hematoxylin and eosin (HE staining), which are ready for examination using light microscopy.
3. Results and discussion 3.1. Characterization of NIR-emitting QDs NIR-emitting CdTe QDs prepared by using our previously reported microwave-assisted method are employed as a model in the following in vivo investigation [11]. Photoluminescent measurement reveals the maximum luminescent wavelength of NIRemitting QDs is 720 nm, and the UV spectrum indicates the NIRemitting QDs possess a resolved absorption peak (Fig. 1A). As measured by dynamic light scatterer (DLS) (Fig. 1B), the corresponding hydrodynamic diameter of NIR-emitting QDs in water is w8.7 nm, which is a bit larger than the size (w4.2 nm) determined by TEM (Fig. 1C). The slight deviation in diameter measured by DLS and TEM is attributed to different surface states of the samples under the tested conditions, which has been discussed in our previous work in a detailed way [13]. In brief, the NIR-emitting QDs samples are directly tested in the aqueous phase for DLS measurement; comparatively, water in the NIR-emitting QDs samples must be strictly removed in the TEM characterization. 3.2. In vivo biodistribution The cadmium concentration of the NIR-emitting QDs distributed in the tissues is measured by ICP-MS (Fig. 2 and Fig. 3). As shown in Fig. 2A, after 0.5-h intravenous injection, the NIR-emitting QDs are predominantly accumulated in the liver with 79.1% injected dose (ID)/g. Residual NIR-emitting QDs are mainly distributed in spleen (14.2% ID/g), lung (6.4% ID/g), and intestine (2.0% ID/g), respectively. Much lower levels (below 1% ID/g) are observed in the kidney, bone, heart, stomach, brain, muscle, and bladder. Nonetheless, cadmium is detected at levels exceeding those of the control animal in all
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tissues (Fig. 2K). The cadmium levels in feces and urine are comparable to those of control animals, and the distribution results of 1-h post-injection are similar to those of 0.5-h (Fig. 2B). After 4-h post-injection (p.i.), uptake of NIR-emitting QDs in the liver decreases to 45.8% ID/g, while uptakes in spleen and lung greatly increase to 146.9 and 23.6% ID/g, respectively (Fig. 2C). Residual NIR-emitting QDs are mainly distributed in bone (4.2% ID/g), kidney (2.6% ID/g), heart (1.1% ID/g), and stomach (1.2% ID/g), respectively. As time extends to 8 h, much more NIR-emitting QDs are located in the spleen (173.7% ID/g) and lung (64.7% ID/g), while uptake in the liver decreases to 24.8% ID/g (Fig. 2D). The cadmium level of NIRemitting QDs in the spleen reaches to the highest value after 12-h injection (232.2% ID/g) (Fig. 2E). Afterward, uptake in the spleen continuously decreases from 131.4 to 19.2% ID/g as time extends from 18 h to 94 days (Fig. 2F, Fig. 2J and Fig. 3A); while uptake in the liver (w30% ID/g) is relatively stable with time elongation (Fig. 2FeJ and Fig. 3B). It is worth pointing out that, uptake in the kidney continuously increases from 0.8 to 59.5% ID/g during the whole observation (0.5 h - 94 days, Fig. 2AeJ and Fig. 3C). The observed dominant accumulations in the liver, spleen, and lung are known to be ascribed to the clearance of nanoparticles from the blood by cells of the mononuclear phagocyte system (MPS) [26,32,33]. Notably, in comparison to relatively low visible QDs uptakes of the spleen (w2% in weight concentration) [13,33], much more NIR-emitting QDs (w25% in weight concentration) are distributed in the spleen in our experiment, suggesting that the spleen is an important organ for the NIR-emitting QDs metabolism. After long-time (e.g., 94 days) blood circulation, high accumulations of NIR-emitting QDs in the liver and kidney reveal that both liver metabolism and kidney metabolism make contribution to the NIR-emitting QDs elimination. It indicates that the liver and kidney fail to totally exhaust the accumulated NIR-emitting QDs, well consisting with the data shown in Fig. 3D and Fig. 3E, i.e., there are no obvious cadmium accumulation in urine and feces after shortand long-term intravenous injection. 3.3. Histology results In order to investigate the in vivo toxicity of the NIR-emitting QDs, a histological analysis of organs is performed to determine whether or not the NIR-emitting QDs themselves or their degradation products cause tissue damage, inflammation, or lesions. In our experiment, mice are sacrificed at 13- and 94-days postinjection. Afterward, eight representative organs, i.e., heart, liver,
Fig. 3. In vivo biodistribution of the NIR-emitting QDs in spleen (A), liver (B), kidney (C), urine (D), and feces (E) at serial time points post-injection.
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Fig. 4. Representative organic histology of the control (left column A-H) and the treated animals: middle column is for 13-day injection (I-P) and right column is for 94-day injection (QeX). Heart (He), liver (Li), spleen (Sp), lung (Lu), kidney (Ki), brain (Br), intestine (In), and bladder (Bl) are shown. Our analysis shows that organs do not exhibit obvious signs of toxicity.
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spleen, lung, kidney, brain, intestine, and bladder, are removed, embedded in paraffin, and then sectioned, and finally stained with hematoxylin and eosin (HE staining). The treated tissue slices are observed using light microscopy. As shown in Fig. 4, all the organs of the experiment mice are normal, preserving the same structures as those of the control group. Typically, no hydropic degeneration is observed according to the cardiac muscle tissues in the heart samples; hepatocytes in the liver samples are observed to be normal, and there are no signs of inflammatory response; no hyperplasia or pulmonary fibrosis is detected in the spleen or lung samples; the glomerulus structure in the kidney section is observed without difficulty; necrosis is not found in any of the histological samples analyzed. Overall, there are no apparent histopathological abnormalities or lesions related to treatment of mice with the NIRemitting QDs. 3.4. Serum biochemistry analysis While histology provides macroscopic and visual evidence, it is difficult to make a quantitative assessment of the QDs-induced in vivo toxicity. Thus, established serum biochemistry assays are performed in the following experiment. To confirm objective comparison, we define the mice younger than 2 months as young ones (Y) and those older than 3.5 months as old ones (O) in our case, since the age of mice has overt effects on the measurements. The five important hepatic indicators, i.e., alkaline phosphatase (ALP), aspartate transaminase (AST), alanine transaminase (ALT), total protein (TP), and albumin/globulin (A/G), are measured, showing no signs of liver injury (Fig. 5AeE). The two indicators of kidney function, i.e., serum creatinine (Cr-S) and urea, are also
Fig. 6. Changes in body weight obtained from mice injected with the NIR-emitting QDs (n ¼ 5, the experiment group) or physiologic saline (n ¼ 5, the control group), respectively.
normal (Fig. 5F,G). Importantly, the doses of these indicators are in the same level, similar to those of controls (Fig. 5H). In addition, little difference between the experiment and control groups is observed during long-term (94 days) measurements (Fig. 5I). We reason that the levels of Cd2þ ions presented in NIR-emitting QDstreated mice (18.5 mg Cd g1 in the kidney) are lower than the critical level (30 mg Cd g1 in the kidney) of small mammals [34e36]. Consequently, the in vivo toxicity is not evident for the NIR-emitting QDs, since they are insufficient to cause the toxicity.
Fig. 5. Serum biochemistry results of the mice treated with physiologic saline and NIR-emitting QDs. Results illustrate mean and standard deviation of ALP (A), AST (B), ALT (C), TP (D), albumin/globulin (A/G) (E), Cr-S (F), and urea (G), when the mice are exposed to NIR-emitting QDs for 0.5 h, 1 h, 4 h, 8 h, 12 h, 18 h, 1 day, 4 days, and 13 days. They are compared with young control mice injected with physiologic saline (denoted as Control-Y) (H). (I) results illustrate mean and standard deviation of ALP, AST, ALT, TP, albumin/globulin (A/G), Cr-S and urea when the mice are exposed to NIR-emitting QDs or physiologic saline for 94 days. They are compared with old control mice injected with physiologic saline (denoted as Control-O).
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3.5. Body weight measurements Body weight is also monitored in our study, because the fluctuation in body weight is recognized as a useful indicator for qualitatively assessing in vivo toxicity of agents. The NIR-emitting QDs in physiologic saline are administered to 5 BALB/c mice through tail vein injection, as an experiment group. Another five mice with injection of physiologic saline are compared as a control group. Fig. 6 presents the body weights of mice recorded during 94days post-injection. Significantly, over the whole period, the body weight of the mice in the experiment group normally increases from 23.28 1.99 g (0 day) to 25.98 1.38 g (94 days), which is nearly the same as that of the control group. It provides another accessorial demonstration that the NIR-emitting QDs produce no overt in vivo toxicity. 4. Conclusions In this paper we report on the in vivo behavior of NIR-emitting QDs, including short- and long-term in vivo biodistribution, pharmacokinetics, and toxicity. The NIR-emitting QDs initially accumulate in liver and spleen, at short-term (0.5e4 h) post-injection, and are increasingly distributed in the kidneys in the long-term (4e94 day). Moreover, there are no apparent histopathological abnormalities or lesions related to treatment of these animals with the NIR-emitting QDs. All measured biochemical markers are in the normal range and the body weight of the mice in experimental group increases in a similar manner to that of the control group. This data suggests minimal in vivo toxicity of the NIR-emitting QDs on the timescale considered, and provides invaluable information for the design and development of NIR-emitting QDs for biological applications, such as NIR fluorescence imaging-based tumor detection and therapy. Acknowledgments This work is supported by the National Basic Research Program of China (973 Program 2013CB934400, 2012CB932400, and 2013CB932600), NSFC (30900338, 51132006 and 51072126), and a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). References [1] Weissleder R. A clearer vision for in vivo imaging. Nat Biotechnol 2001;19: 316e7. [2] Yong KT. Mn-doped near-infrared quantum dots as multimodal targeted probes for pancreatic cancer imaging. Nanotechnology 2009;20:015102. [3] Michalet X, Pinaud FF, Bentolila LA, Tsay JM, Doose S, Li JJ, et al. Quantum dots for live cells, in vivo imaging, and diagnostics. Science 2005;307:538e44. [4] Kim S, Lim YT, Soltesz EG, Grand AMD, Lee J, Nakayama A, et al. Near-infrared fluorescent type II quantum dots for sentinel lymph node mapping. Nat Biotechnol 2004;22:93e7. [5] Zaheer A, Lenkinski RE, Mahmood A, Jones AG, Cantley LC, Frangioni JV. In vivo near-infrared fluorescence imaging of osteoblastic activity. Nat Biotechnol 2001;19:1148e54. [6] Morgan NY, English S, Chen W, Chernomordik V, Russo A, Smith PD, et al. Real time in vivo non-invasive optical imaging using near-infrared fluorescent quantum dots. Acad Radiol 2005;12:313e23. [7] Ohnishi S, Lomnes SJ, Laurence RG, Gogbashian A, Mariani G, Frangioni JV. Organic alternatives to quantum dots for intraoperative near-infrared fluorescent sentinel lymph node mapping. Mol Imaging 2005;4:172e81. [8] Smith BR, Cheng Z, De A, Koh AL, Sinclair R, Gambhir SS. Real-time intravital imaging of RGD-quantum dot binding to luminal endothelium in mouse tumor neovasculature. Nano Lett 2008;8:2599e606.
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In vivo behavior of near infrared-emitting quantum dots Yimei Lu a, Yuanyuan Su a, *, Yanfeng Zhou a, Jie Wang a, Fei Peng a, Yiling Zhong a, Qing Huang b, Chunhai Fan b, Yao He a, * a
Institute of Functional Nano and Soft Materials (FUNSOM) and Jiangsu Key Laboratory for Carbon-based Functional Materials and Devices, Soochow University, Suzhou, Jiangsu 215123, China b Laboratory of Physical Biology, Shanghai Institute of Applied Physics, Chinese Academy of Sciences, Shanghai 201800, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 31 January 2013 Accepted 19 February 2013 Available online 13 March 2013
Near-infrared (NIR, 700e900 nm) fluorescent nanomaterials-based probes have shown major impacts on high-resolution and high-sensitivity bioimaging applications. Typically, NIR-emitting quantum dots (QDs) are highly promising as NIR bioprobes due to their unique optical properties. However, NIRemitting QDs-related in vivo behavior remains unknown at present, severely limiting their wideranging bioapplications. Herein, we investigate short- and long-term in vivo biodistribution, pharmacokinetics, and toxicity of the NIR-emitting QDs. Particularly, we reveal that the NIR-emitting QDs are initially accumulated in liver, spleen, and lung for short-time (0.5e4 h) post-injection, and then increasingly absorbed by kidney during long-time (4e94 days) blood circulation. Obviously timedependent biodistribution is observed: with time continues, most of NIR-emitting QDs are finally accumulated in liver and kidney; comparatively, less NIR-emitting QDs are observed in spleen, lung, and bone marrow. Furthermore, histological and biochemical analyses, and body weight measurements demonstrate that there is no overt toxicity of NIR-emitting QDs in mice even at long-time (94 days) exposure time. Our studies provide invaluable information for the design and development of NIRemitting QDs-based nanoprobes for biological and biomedical applications. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: Near infrared Quantum dots In vivo Biodistribution Toxicity
1. Introduction It is well known that near-infrared (NIR)-fluorescence imaging is widely recognized as an effective method for high-resolution and high-sensitivity bioimaging because of its minimized biological autofluorescence and increased penetration of emission light through tissues in the NIR window [1]. In recent years, nearinfrared (700e900 nm) fluorescent nanomaterials-based probes have shown major impacts on biomedical and biological applications [2e9]. Typically, NIR-emitting II-VI quantum dots (QDs) are considered as high-performance NIR bioprobes due to their unique optical merits (e.g., strong fluorescence, robust photostability, and size-tunable fluorescence, etc) [10e12]. For examples, Frangioni et al. demonstrated that injection of only 400 pmol of NIR-emitting CdTe (CdSe) core (shell) heterostructured QDs permitted sentinel lymph nodes 1 cm deep, and could be easily imaged in real time,
* Corresponding authors. Tel.: þ86 512 6588 0946; fax: þ86 512 6588 2846. E-mail addresses: [email protected] (Y. Su), [email protected], [email protected] (Y. He). 0142-9612/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biomaterials.2013.02.054
suggesting the potential of NIR-emitting QDs for biomedical imaging [4]. Gambhir and coworkers exploited intravital microscopy with subcellular resolution (w0.5 mm) to directly observe the tumor neovasculature in living subjects using arginine-glycineaspartic (RGD) peptides-conjugated NIR-emitting QDs. That study demonstrated the direct performance of RGD-NIR-QDs binding to tumor neovasculature via direct cellular-level visualization in living mice [8]. Of particular note, Chen et al. recently revealed that NIRemitting QDs allowed multiplex imaging of deeper tissues, which opens up perspectives for integrin-targeted NIR optical imaging and would have great potential in cancer diagnosis and management as well as imaging-guided surgery and therapy [9]. Very recently, we presented an example of aqueous synthesized NIRemitting CdTe QDs for in vivo tumor targeting. Our in vitro and in vivo imaging data demonstrated that the NIR-emitting aqQDs were especially suitable for highly spectrally and spatially resolved imaging in cells and animals [11]. These above reports strongly suggest NIR-emitting QDs as high-quality bioprobes for myriad bioimaging applications. Concerns on in vitro and in vivo biosafety of QDs have received increasingly intensive attention, which is of essential importance for wide-ranging bioapplications [13e21]. Sufficient in vitro studies
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Fig. 1. (A) Absorption and photoluminescence spectra of the prepared NIR-emitting QDs with the maximum emission wavelength of 720 nm. The hydrodynamic diameter of the NIR-emitting QDs is determined by DLS (B), and their corresponding TEM image is shown in (C). Inset shows a high-resolution TEM (HRTEM) image of a single QD with clear crystal lattices.
have suggested that the cytotoxicity of QDs is ascribed to release of toxic metals [14,15] and production of reactive oxygen species [16,17], which can be largely alleviated by surface modification (e.g. epitaxial growth of ZnS shell) [18]. However, preliminary studies revealed that, in comparison to obvious QDs-induced cytotoxicity, QDs produced much less side effects in vivo [19,20]. For examples, Chan et al. demonstrated in a pilot study that the organic synthesized QDs (orQDs) did not cause appreciable toxicity in vivo even over long-term periods (e.g. 80 days) [21]. Our recent investigation also showed that there was no overt toxicity of aqueous synthesized QDs (aqQDs) in mice [13]. Such significant conflict between the conclusions drawn from in vitro and in vivo studies is due to distinct diversity of in vitro and in vivo physiological conditions [22,23]. Typically, in comparison to relatively simple in vitro environment, in vivo systems are much complex. As a result, the interactions between the nanostructures and complicated biological components in vivo lead to unique biodistribution, clearance, immune response, and metabolism [21,24e26]. It is worthwhile to point out that, most of the above-mentioned QDs used for in vitro and in vivo studies emit the visible light (450e650 nm). There currently exists scanty information regarding in vitro and in vivo behaviors of the NIR-emitting QDs, which severely limits the QDsbased NIR bioimaging applications. In this work, by using our previously reported CdTe NIRemitting QDs as a model [11], we carry out a comprehensive investigation on in vivo behaviors of the NIR-emitting QDs, including their short- and long-term in vivo biodistribution, pharmacokinetics, and toxicity. Our results provide useful information concerning the targeted organs, distribution, and elimination characteristics of NIR-emitting QDs in vivo, which is valuable for the design and development of NIR-emitting QDs for wide-ranging biological and biomedical applications. 2. Experimental section 2.1. Preparation of NIR-emitting QDs Tellurium powder (99.99%) and CdCl2 (99.99%) are purchased from Aldrich. 3Mercaptopropionic acid (MPA) (98%) and NaBH4 (96%) are purchased from SigmaeAldrich. All chemicals are used without additional purification. All solutions are prepared using Milli-Q water (Millipore) as the solvent. The CdTe QDs are synthesized based on our previously reported protocol [11]. In brief, the CdTe precursor solution is obtained by adding freshly prepared NaHTe solution to N2-staurated CdCl2 solution at pH 8.4 in the presence of 3-mercaptopropionic acid (MPA). The molar ratio of Cd2þ/MPA/NaHTe is set as 1: 2.4: 0.5. By controlling the reaction time and temperature (170 C/5 min), we obtain NIR-emitting QDs with the maximum emission wavelengths of 720 nm (photoluminescence quantum yield (PLQY): w25%). After the microwave irradiation, the QDs samples are taken when
the temperature cools to lower than 50 C naturally. The microwave system NOVA used for synthesizing QDs is made by Preekem of Shanghai, China. The system operates at 2450 MHz frequency and works at 0e500 W power. Exclusive vitreous vessels with a volume of 15 or 20 ml are equipped for the system to provide security during reaction demanding high temperature and pressure. In order to exclude the influence of residual reagents such as MPA, Cd2þ and Te2- in solution, the samples are carefully purified before subsequent biological experiments. In details, 2-propanol is added dropwise under stirring until the sample solution becomes slightly turbid. The turbid dispersion is kept on stirring for 15 min, and then subjected to centrifugation. The precipitate is collected while the supernatant is added another portion of 2-propanol to obtain the second precipitated fraction. This procedure is repeated for three times. Afterward, these precipitate are washed by Milli-Q water for three times to adequately remove residual reagents from samples. Finally, the QDs samples are employed for the following studies. The NIR-emitting QDs are characterized by UV-vis absorption, photoluminescence (PL), transmission electronic microscopy (TEM), and dynamic light scatterer (DLS). All optical measurements are performed at room temperature under ambient air conditions. UV-vis absorption spectra are recorded with a Perkin Elmer Lambda 750 UV-vis-near-infrared spectrophotometer. PL measurement is performed using a HORIBA JOBTN YVON FLUOROMAX-4 spectrofluorimeter. The PLQY of samples is estimated using CY7 (PLQY: w28%) in an ethanol solution as a reference standard, which is freshly prepared to reduce the measurement error [13]. The TEM/HRTEM overview images are recorded using Philips CM 200 electron microscope operated at 200 kV. Light-scattering analysis is performed using a DynaPro Dynamic Light Scatterer (DLS). 2.2. Animal injection, weight measurements and sample collection Female BALB/c mice (7e8 weeks old), obtained from Shanghai SLAC Labs Animal CO. LTD, are given food and water ad libitum and housed in a 12 h/12 h light/dark cycle. To characterize the biodistribution and accumulation of the NIR-emitting QDs, 0.4 nmol QDs in a volume of 0.1 ml are intravenously injected into the tail vein (n ¼ 4 or 5 for each time point); these groups of mice constitute the test groups. BALB/c mice (n ¼ 4 or 5) with injection of physiologic saline are selected as the control group. At increasing time points after injection, mice are weighed and assessed for behavioral changes. And then animals are sacrificed by exsanguination. The heart, liver, spleen, lung, kidney, stomach, brain, bone, muscle, intestine, bladder, urine, and feces are collected. Histological samples are carefully removed with a sharp razor blade and placed directly into fixative. Note that: 1) Concentration of the NIRemitting QDs is calculated following a previously published method by Yu et al. [27e 30]. 2) A dose of 0.4 nmol per mouse (w24 g) is comparable to the reported dose for other in vivo QDs experiment [13]. 2.3. Pharmacokinetic analysis Dominant organ samples (e.g., heart, liver, spleen, lung, kidney, stomach, brain, bone, muscle, intestine, and bladder) are collected, weighed and digested with nitric acid in microwave digestion system (EXCEL, Preekem Scientific Instruments Co. Ltd, Shanghai, China). Urine is measured by volume, 20 ml urine equals to 20.43 mg by weighing and digested. The solution is diluted with Milli-Q water and the cadmium ions content is quantified with inductively coupled plasma-mass spectrometry (ICPMS) to determine the total cadmium in the original organs. The percent of injected dose per gram tissue (% ID/g) of NIR-emitting QDs in a specific tissue is calculated by the following equation [13,31]:
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%ID=g ¼
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ð½QD in tissue suspensionÞ ðvolume of tissue suspensionÞ ð½QD in injected solutionÞ ðvolume of injected QDÞ ðwet weight of tissueÞ
Fig. 2. In vivo biodistribution of the NIR-emitting QDs at serial time point post-intravenous injection: (A) 0.5 h, (B) 1 h, (C) 4 h, (D) 8 h, (E) 12 h, (F) 18 h, (G) 1 day, (H) 4 day, (I) 13 day, (J) 94 day, and (K) Control, respectively.
Y. Lu et al. / Biomaterials 34 (2013) 4302e4308 2.4. Serum biochemistry and histology Blood samples are harvested from the mice injected with NIR-emitting QDs for 0.5 h, 1 h, 4 h, 8 h, 12 h, 18 h, 1 day, 4 days, 13 days, and 94 days, and from the mice receiving physiologic saline injection. Blood is collected from the orbital sinus by quickly removing the eyeball from the socket with a pair of tissue forceps. Indicators for liver and kidney function (alkaline phosphatase (ALP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), total protein (TP), albumin/globulin (A/G), serum creatinine (Cr-S), and urea) are measured. Upon completion of the blood collection, mice are sacrificed. The heart, liver, spleen, lung, kidney, brain, intestine, and bladder are removed, and then embedded in paraffin, sectioned, and eventually stained with hematoxylin and eosin (HE staining), which are ready for examination using light microscopy.
3. Results and discussion 3.1. Characterization of NIR-emitting QDs NIR-emitting CdTe QDs prepared by using our previously reported microwave-assisted method are employed as a model in the following in vivo investigation [11]. Photoluminescent measurement reveals the maximum luminescent wavelength of NIRemitting QDs is 720 nm, and the UV spectrum indicates the NIRemitting QDs possess a resolved absorption peak (Fig. 1A). As measured by dynamic light scatterer (DLS) (Fig. 1B), the corresponding hydrodynamic diameter of NIR-emitting QDs in water is w8.7 nm, which is a bit larger than the size (w4.2 nm) determined by TEM (Fig. 1C). The slight deviation in diameter measured by DLS and TEM is attributed to different surface states of the samples under the tested conditions, which has been discussed in our previous work in a detailed way [13]. In brief, the NIR-emitting QDs samples are directly tested in the aqueous phase for DLS measurement; comparatively, water in the NIR-emitting QDs samples must be strictly removed in the TEM characterization. 3.2. In vivo biodistribution The cadmium concentration of the NIR-emitting QDs distributed in the tissues is measured by ICP-MS (Fig. 2 and Fig. 3). As shown in Fig. 2A, after 0.5-h intravenous injection, the NIR-emitting QDs are predominantly accumulated in the liver with 79.1% injected dose (ID)/g. Residual NIR-emitting QDs are mainly distributed in spleen (14.2% ID/g), lung (6.4% ID/g), and intestine (2.0% ID/g), respectively. Much lower levels (below 1% ID/g) are observed in the kidney, bone, heart, stomach, brain, muscle, and bladder. Nonetheless, cadmium is detected at levels exceeding those of the control animal in all
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tissues (Fig. 2K). The cadmium levels in feces and urine are comparable to those of control animals, and the distribution results of 1-h post-injection are similar to those of 0.5-h (Fig. 2B). After 4-h post-injection (p.i.), uptake of NIR-emitting QDs in the liver decreases to 45.8% ID/g, while uptakes in spleen and lung greatly increase to 146.9 and 23.6% ID/g, respectively (Fig. 2C). Residual NIR-emitting QDs are mainly distributed in bone (4.2% ID/g), kidney (2.6% ID/g), heart (1.1% ID/g), and stomach (1.2% ID/g), respectively. As time extends to 8 h, much more NIR-emitting QDs are located in the spleen (173.7% ID/g) and lung (64.7% ID/g), while uptake in the liver decreases to 24.8% ID/g (Fig. 2D). The cadmium level of NIRemitting QDs in the spleen reaches to the highest value after 12-h injection (232.2% ID/g) (Fig. 2E). Afterward, uptake in the spleen continuously decreases from 131.4 to 19.2% ID/g as time extends from 18 h to 94 days (Fig. 2F, Fig. 2J and Fig. 3A); while uptake in the liver (w30% ID/g) is relatively stable with time elongation (Fig. 2FeJ and Fig. 3B). It is worth pointing out that, uptake in the kidney continuously increases from 0.8 to 59.5% ID/g during the whole observation (0.5 h - 94 days, Fig. 2AeJ and Fig. 3C). The observed dominant accumulations in the liver, spleen, and lung are known to be ascribed to the clearance of nanoparticles from the blood by cells of the mononuclear phagocyte system (MPS) [26,32,33]. Notably, in comparison to relatively low visible QDs uptakes of the spleen (w2% in weight concentration) [13,33], much more NIR-emitting QDs (w25% in weight concentration) are distributed in the spleen in our experiment, suggesting that the spleen is an important organ for the NIR-emitting QDs metabolism. After long-time (e.g., 94 days) blood circulation, high accumulations of NIR-emitting QDs in the liver and kidney reveal that both liver metabolism and kidney metabolism make contribution to the NIR-emitting QDs elimination. It indicates that the liver and kidney fail to totally exhaust the accumulated NIR-emitting QDs, well consisting with the data shown in Fig. 3D and Fig. 3E, i.e., there are no obvious cadmium accumulation in urine and feces after shortand long-term intravenous injection. 3.3. Histology results In order to investigate the in vivo toxicity of the NIR-emitting QDs, a histological analysis of organs is performed to determine whether or not the NIR-emitting QDs themselves or their degradation products cause tissue damage, inflammation, or lesions. In our experiment, mice are sacrificed at 13- and 94-days postinjection. Afterward, eight representative organs, i.e., heart, liver,
Fig. 3. In vivo biodistribution of the NIR-emitting QDs in spleen (A), liver (B), kidney (C), urine (D), and feces (E) at serial time points post-injection.
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Fig. 4. Representative organic histology of the control (left column A-H) and the treated animals: middle column is for 13-day injection (I-P) and right column is for 94-day injection (QeX). Heart (He), liver (Li), spleen (Sp), lung (Lu), kidney (Ki), brain (Br), intestine (In), and bladder (Bl) are shown. Our analysis shows that organs do not exhibit obvious signs of toxicity.
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spleen, lung, kidney, brain, intestine, and bladder, are removed, embedded in paraffin, and then sectioned, and finally stained with hematoxylin and eosin (HE staining). The treated tissue slices are observed using light microscopy. As shown in Fig. 4, all the organs of the experiment mice are normal, preserving the same structures as those of the control group. Typically, no hydropic degeneration is observed according to the cardiac muscle tissues in the heart samples; hepatocytes in the liver samples are observed to be normal, and there are no signs of inflammatory response; no hyperplasia or pulmonary fibrosis is detected in the spleen or lung samples; the glomerulus structure in the kidney section is observed without difficulty; necrosis is not found in any of the histological samples analyzed. Overall, there are no apparent histopathological abnormalities or lesions related to treatment of mice with the NIRemitting QDs. 3.4. Serum biochemistry analysis While histology provides macroscopic and visual evidence, it is difficult to make a quantitative assessment of the QDs-induced in vivo toxicity. Thus, established serum biochemistry assays are performed in the following experiment. To confirm objective comparison, we define the mice younger than 2 months as young ones (Y) and those older than 3.5 months as old ones (O) in our case, since the age of mice has overt effects on the measurements. The five important hepatic indicators, i.e., alkaline phosphatase (ALP), aspartate transaminase (AST), alanine transaminase (ALT), total protein (TP), and albumin/globulin (A/G), are measured, showing no signs of liver injury (Fig. 5AeE). The two indicators of kidney function, i.e., serum creatinine (Cr-S) and urea, are also
Fig. 6. Changes in body weight obtained from mice injected with the NIR-emitting QDs (n ¼ 5, the experiment group) or physiologic saline (n ¼ 5, the control group), respectively.
normal (Fig. 5F,G). Importantly, the doses of these indicators are in the same level, similar to those of controls (Fig. 5H). In addition, little difference between the experiment and control groups is observed during long-term (94 days) measurements (Fig. 5I). We reason that the levels of Cd2þ ions presented in NIR-emitting QDstreated mice (18.5 mg Cd g1 in the kidney) are lower than the critical level (30 mg Cd g1 in the kidney) of small mammals [34e36]. Consequently, the in vivo toxicity is not evident for the NIR-emitting QDs, since they are insufficient to cause the toxicity.
Fig. 5. Serum biochemistry results of the mice treated with physiologic saline and NIR-emitting QDs. Results illustrate mean and standard deviation of ALP (A), AST (B), ALT (C), TP (D), albumin/globulin (A/G) (E), Cr-S (F), and urea (G), when the mice are exposed to NIR-emitting QDs for 0.5 h, 1 h, 4 h, 8 h, 12 h, 18 h, 1 day, 4 days, and 13 days. They are compared with young control mice injected with physiologic saline (denoted as Control-Y) (H). (I) results illustrate mean and standard deviation of ALP, AST, ALT, TP, albumin/globulin (A/G), Cr-S and urea when the mice are exposed to NIR-emitting QDs or physiologic saline for 94 days. They are compared with old control mice injected with physiologic saline (denoted as Control-O).
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3.5. Body weight measurements Body weight is also monitored in our study, because the fluctuation in body weight is recognized as a useful indicator for qualitatively assessing in vivo toxicity of agents. The NIR-emitting QDs in physiologic saline are administered to 5 BALB/c mice through tail vein injection, as an experiment group. Another five mice with injection of physiologic saline are compared as a control group. Fig. 6 presents the body weights of mice recorded during 94days post-injection. Significantly, over the whole period, the body weight of the mice in the experiment group normally increases from 23.28 1.99 g (0 day) to 25.98 1.38 g (94 days), which is nearly the same as that of the control group. It provides another accessorial demonstration that the NIR-emitting QDs produce no overt in vivo toxicity. 4. Conclusions In this paper we report on the in vivo behavior of NIR-emitting QDs, including short- and long-term in vivo biodistribution, pharmacokinetics, and toxicity. The NIR-emitting QDs initially accumulate in liver and spleen, at short-term (0.5e4 h) post-injection, and are increasingly distributed in the kidneys in the long-term (4e94 day). Moreover, there are no apparent histopathological abnormalities or lesions related to treatment of these animals with the NIR-emitting QDs. All measured biochemical markers are in the normal range and the body weight of the mice in experimental group increases in a similar manner to that of the control group. This data suggests minimal in vivo toxicity of the NIR-emitting QDs on the timescale considered, and provides invaluable information for the design and development of NIR-emitting QDs for biological applications, such as NIR fluorescence imaging-based tumor detection and therapy. Acknowledgments This work is supported by the National Basic Research Program of China (973 Program 2013CB934400, 2012CB932400, and 2013CB932600), NSFC (30900338, 51132006 and 51072126), and a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). References [1] Weissleder R. A clearer vision for in vivo imaging. Nat Biotechnol 2001;19: 316e7. [2] Yong KT. Mn-doped near-infrared quantum dots as multimodal targeted probes for pancreatic cancer imaging. Nanotechnology 2009;20:015102. [3] Michalet X, Pinaud FF, Bentolila LA, Tsay JM, Doose S, Li JJ, et al. Quantum dots for live cells, in vivo imaging, and diagnostics. Science 2005;307:538e44. [4] Kim S, Lim YT, Soltesz EG, Grand AMD, Lee J, Nakayama A, et al. Near-infrared fluorescent type II quantum dots for sentinel lymph node mapping. Nat Biotechnol 2004;22:93e7. [5] Zaheer A, Lenkinski RE, Mahmood A, Jones AG, Cantley LC, Frangioni JV. In vivo near-infrared fluorescence imaging of osteoblastic activity. Nat Biotechnol 2001;19:1148e54. [6] Morgan NY, English S, Chen W, Chernomordik V, Russo A, Smith PD, et al. Real time in vivo non-invasive optical imaging using near-infrared fluorescent quantum dots. Acad Radiol 2005;12:313e23. [7] Ohnishi S, Lomnes SJ, Laurence RG, Gogbashian A, Mariani G, Frangioni JV. Organic alternatives to quantum dots for intraoperative near-infrared fluorescent sentinel lymph node mapping. Mol Imaging 2005;4:172e81. [8] Smith BR, Cheng Z, De A, Koh AL, Sinclair R, Gambhir SS. Real-time intravital imaging of RGD-quantum dot binding to luminal endothelium in mouse tumor neovasculature. Nano Lett 2008;8:2599e606.
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