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CdS QDs and folate for tumor-targeted imaging
Biomaterials 35 (2014) 7881e7886
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Oligomeric nanoparticles functionalized with NIR-emitting CdTe/CdS QDs and folate for tumor-targeted imaging Yue Yuan, Jia Zhang, Linna An, Qinjingwen Cao, Yun Deng, Gaolin Liang* CAS Key Laboratory of Soft Matter Chemistry, Department of Chemistry & Collaborative Innovation Center of Suzhou Nano Science and Technology, University of Science and Technology of China, Hefei, Anhui 230026, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 31 March 2014 Accepted 23 May 2014 Available online 20 June 2014
We report herein the facile surface-functionalization of one type of biocompatible, oligomeric nanoparticles 1-NPs with NIR-emitting CdTe/CdS QDs and folate for tumor-targeted imaging in vivo. The eNH2 and eSH groups of cysteine residues on the 1-NPs were utilized to covalently conjugate CdTe/CdS QDs and Mal-FA to prepare the hybrid nanoparticles 1-NPs-QDs-FA. As-prepared 1-NPs-QDs-FA showed NIRfluorescence emission at 734 nm, selective uptake by FR-overexpressing tumor cells in vitro, and selective FR-overexpressing tumor-targeted imaging in vivo. This first example of oligomeric/inorganic hybrid nanoparticles provides people with new type of biomaterials for tumor-targeted imaging with high selectivity. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Oligomeric nanoparticles Quantum dots Hybrid nanoparticles Near-infrared Tumor-targeting Imaging
1. Introduction Near-infrared (NIR)-fluorescence probes for in vivo imaging have drawn great attention from the biomedical field in recent years due to the high penetration ability of their excitations (or emissions) through tissues in the NIR wavelength window (650e900 nm), while in the meantime maintaining a minimal biological autofluorescence background [1e3]. To date, many types of nanoparticle-based, highly-luminescent probes have been developed for NIR-fluorescence imaging [4e9]. Among them, quantum dots (QDs) with the highest quantum yields and unique size-tunable optical properties, are emerging as the most exciting and promising probes for in vivo NIR-fluorescence imaging [10e12]. However, to achieve a tissue/organ-targeted imaging, QDs need to be functionalized with targeting warheads before injection. For example, there are many methods reported to modify QDs with biomolecules such as folic acid (FA), DNA, affibody, peptide, or enzyme for tumor-targeted imaging [13e19]. But usually the biomolecular warheads could not be directly modified on the QDs' surface unless the QDs have been coated with stabilizers such as streptavidin [13], mercaptopropionic acid (MPA) [16], or bovine serum albumin (BSA) [17]. Moreover, to preserve
* Corresponding author. E-mail address: [email protected] (G. Liang). http://dx.doi.org/10.1016/j.biomaterials.2014.05.071 0142-9612/© 2014 Elsevier Ltd. All rights reserved.
their optical and colloidal stability in biological fluids, CdTe/ZnS core/shell QDs are always modified with polymers [20e23] or dendrimers [24,25]. Therefore, people always use carriers to load QDs for imaging applications. And inorganic nanoparticles with large sizes such as mesoporous silicon nanoparticles have been widely used for this purpose [26]. Inorganic nanoparticles as templates for QDs embedding have many advantages such as easier control of their sizes, prolonged retention of QDs in vivo, and multiple QDs modification [27,28]. Nevertheless, it is difficult for inorganic nanoparticles to be controlled functionalized with QDs together with targeting biomolecules. In contrast, organic nanoparticles (e.g., polymeric nanoparticles or oligomeric nanoparticles), which have abundant surface functional groups, good biological compatibility, and excellent operability, are ideal carriers to load QDs for tissue/organ-targeted imaging. Herein, we report one type of oligomeric nanoparticles (i.e., 1-NPs in Scheme 1) functionalized with NIR-emitting CdTe/CdS QDs and FA for tumor-targeted imaging. This type of oligomeric nanoparticles as carriers could not only prolong the retention times of the QDs as well as small molecule FA in vivo, but also provide reactive groups (i.e., eSH and eNH2) for multiple functionalization (e.g., functionalization with an anti-cancer drug or contrast agent together with the QDs and FA). The highly-luminescent NIR-emitting CdTe/ CdS core/shell QDs were chosen for this study because they are highly stable, water-soluble, and suitable for small animal imaging [16].
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Scheme 1. Schematic illustration of the preparative procedures of 1-NPs-QDs-FA.
To facilely synthesize a type of oligomeric nanoparticles with functional groups for CdTe/CdS QDs and FA conjugation, we chose a “click” condensation reaction between the 1,2-aminothiol group of a cysteine (Cys) motif and the cyano group on a 2cyanobenzothiazole (CBT) motif, recently developed by Rao and Liang [29e33]. In these literature, one simple monomer Cys(SEt)Lys [Cys(SEt)]-CBT (1) was reported which could undergo this condensation reaction upon reduction at pH 7.4 in water to yield amphiphilic oligomers and the oligomers self-assemble into oligomeric nanoparticles (i.e., 1-NPs) within 5 min. In detail, as shown in Scheme 1, upon reduction, the disulfide bonds on the Cys motifs of 1 are cleaved, exposing two reactive Cys residues. One of the cysteine residue condenses with the cyano group on another CBT motif to form oligomers of 1 (mostly dimers), which self-assemble into 1-NPs with abundant reactive Cys residues (-NH2 groups and eSH groups) on the surfaces of the nanoparticles. In this work, we used the amino groups (-NH2) on the surfaces of 1-NPs to conjugate with QDs using the classical 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride/N-hydroxysuccinimide (EDC/NHS) method to prepare the 1-NPs-QDs first. Then the thiol groups (-SH) on the 1-NPs-QDs were conjugated to the maleimide motifs of maleimide-folic acid (Mal-FA) via addition reaction to yield the oligomeric and inorganic hybrid nanoparticles 1-NPs-QDs-FA for folate receptor (FR)-overexpressing tumor-targeted imaging in vivo. 2. Materials and methods 2.1. Materials All the starting materials were obtained from Adamas or Sangon Biotech. Commercially available reagents were used without further purification, unless noted otherwise. All other chemicals were reagent grade or better. PBS buffer (0.01 M, pH ¼ 7.4) was prepared with pills purchased from Sangon Biotech Co., Ltd. (Shanghai, China). Ultrapure water (18.2 MU cm) was used throughout the experiment. Cells were obtained from Cell Bank of Chinese Academy of Sciences.
The spectra of electrospray ionization-mass spectrometry (ESI-MS) were recorded on an LCQ Advantage MAX ion trap mass spectrometer (Thermo Fisher). MALDITOF/TOF mass spectra were obtained on a time-of-flight Ultraflex II mass spectrometer (Bruker Daltonics). HPLC analyses were performed on an Agilent 1200 HPLC system equipped with a G1322A pump and in-line diode array UV detector using an Agilent Zorbax 300SB e C18 RP column with CH3CN (0.1% of trifluoroacetic acid (TFA)) and water (0.1% of TFA) as the eluant. 1H NMR spectra were obtained on a 300 MHz Bruker AV300. Fluorescence microscopic images were taken under a fluorescence microscope OLMPUS IX71. Confocal fluorescence images were recorded using a confocal laser microscope Olympus BX61W. Cells were routinely cultured in Dul-becco's modified Eagle's medium (DMEM, Hycolon) supplemented with 10% fetal bovine serum at 37 C, 5% CO2, and humid atmosphere. 4e6 week old (weighing 19~20 g) BALB/c nude mice were used for animal experiments. 2.2. Preparation of Cys(SEt)-Lys[Cys(SEt)]-CBT (1) 2-cyano-6-aminobenzothiazole (CBT) was synthesized following the literature method (White EH, Worther H, Seliger HH, McElroy WD. Amino analogs of firefly luciferin and biological activity thereof. J Am Chem Soc 1966; 88:2015e9). The preparation of compound 1 Cys(SEt)-Lys[Cys(SEt)]-CBT was synthesized following the literature method (Deng Y, Liu S, Mei K, Tang A, Cao C, Liang G. Multifunctional small molecule for controlled assembly of oligomeric nanoparticles and crosslinked polymers. Org Biomol Chem 2011; 9:6917e9). MS: calculated for C24H35N7O3S5 [(M þ H)þ]: 630.14831; obsvd. HR-MALDI-TOF/MS: m/z 630.14830 (Figure S1, Supporting Information). 2.3. Preparation of maleimide-folate (Mal-FA) The mixture of N-(2-Aminoethyl)maleimide trifluoroacetate salt (25.4 mg, 0.1 mmol), HBTU (41.7 mg, 0.1 mmol), HOBT (13.5 mg, 0.1 mmol) in DMF (2 mL) was stirred for 30 min in presence of DIPEA (11.1 mg, 0.1 mmol), then folic acid (FA, 53.0 mg, 0.12 mmol) dissolved in 1 mL of DMF was added into the mixture dropwise. After an overnight stirring, compound Mal-FA (39 mg, yield: 70%) was obtained after HPLC purification(Scheme S1, Supporting Information). 1H NMR of compound Mal-FA (d6-DMSO, 300 MHz, Figure S2, Supporting Information) d (ppm): 8.63 (d, J ¼ 1.62 Hz, 1H), 7.92 (d, J ¼ 6.25 Hz, 1H), 7.85 (d, J ¼ 7.92 Hz, 1H), 7.61 (d, J ¼ 8.72 Hz, 2H), 7.18 (s, 2H), 6.93 (d, J ¼ 3.29 Hz, 2H), 6.60 (m, 2H), 4.47 (s, 2H), 4.21 (s, 1H), 3.14 (m, 2H), 2.19 (m, 2H), 1.94 (m, 4H), 1.20 (s, 1H). MS: calculated for C25H26N9O7 [(M þ H)þ]: 564.20; obsvd. ESI/MS: m/z 564.02 (Figure S3, Supporting Information).
Y. Yuan et al. / Biomaterials 35 (2014) 7881e7886 2.4. Preparation of CdTe/CdS quantum dots CdTe/CdS quantum dots were synthesized following the literature method with minor modification (Chen LN, Wang J, Li WT, Han HY. Aqueous one-pot synthesis of bright and ultrasmall CdTe/CdS near-infrared-emitting quantum dots and their application for tumor targeting in vivo. Chem Commun 2012:48:4971e3). First, 74 mL MPA and 20 mg trisodium citrate were added to 25 mL CdCl2 solution (0.05 mmol) in succession. After that, the mixture was stirred for 5 min, followed by adding 0.1 M NaOH to tune the pH of the mixed solution to 11.2. Then, 4.4 mg Na2TeO3 and 20 mg NaBH4 were added into the mixture under rapid stirring. Finally, the reaction mixture was slowly heated to 90 C and stirred for 4 h. The obtained QDs was purified by the addition of ethanol and salt, then dispersed in water.
3. Results and discussion 3.1. Preparation and characterizations of 1-NPs-QDs-FA We began the study with reduction-controlled condensation of 1 and self-assembly of 1-NPs. At a concentration of 1 mM, 1 was dissolved in water containing 5% methanol. 5 min after adding 4 equiv. of tris(2-carboxyethyl)phosphine (TCEP) and adjusting the pH to 7.4 with sodium carbonate, we observed the solutions of 1 became turbid dispersions and their UV-vis spectra at 500e700 nm showed obvious increases, suggesting the aggregation of particles (Figure S4, Supporting Information). Dynamic light scattering (DLS) measurements indicated that as-formed 1-NPs have a mean dynamic diameter of 54.2 nm (Figure S5, Supporting Information). Transmission electron microscope (TEM) images of the 1-NPs indicated that the nanoparticles have diameters of 21e37 nm and tend to connect with each other to form fibrous structures (Fig. 1A). To 1 mL of 0.1 mg mL1 CdTe/CdS QDs solution, 40 mL of EDC (0.05 M
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solution in PBS) and an equal amount of NHS (0.05 M solution in PBS) were added and stirred for 15 min. After ultrafiltration under centrifugation (15,000 rpm, 10 min) with a YM-10 ultrafilter to remove excess EDC and NHS, the prepared QDs-NHS ester was redissolved with 1 mL of PBS buffer (5 mM). Then 1 mL of freshly prepared 1-NPs monodispersion was added into the QDs-NHS solution and stirred overnight. 1-NPs-QDs were obtained by centrifugation and washing with 1 mL phosphate buffered saline (PBS, 5 mM) for three times. TEM image of 1-NPs-QDs indicated that the nanoparticles have diameters of 24e42 nm (Fig. 1B). High resolution TEM (HR-TEM) image clearly shows the crystal lattice of CdTe/ CdS QDs, suggesting the success of QDs conjugation to the oligomeric nanoparticles 1-NPs (inset of Fig. 1B). 5 min after 4 equiv. of TCEP was added into 1 mL 1-NPs-QDs in water and the pH value was adjusted to 7.4 with sodium carbonate, 7 mL of Mal-FA (1 M in DMSO) was added and stirred for 2 h at room temperature to yield 1-NPs-QDs-FA after centrifugation and washing for three times. TEM image of 1-NPs-QDs-FA showed that nanoparticles have diameters of 25e44 nm (Fig. 1C), similar to those of 1-NPs-QDs in Fig. 1B. HR-TEM image of 1-NPs-QDs-FA consistently shows the crystal lattice of CdTe/CdS QDs, suggesting that Mal-FA modification does not induce the release of QDs from the oligomeric nanoparticles (inset of Fig. 1C). As-obtained 1-NPsQDs or 1-NPs-QDs-FA were redispersed in 200 mL water for all the experiments following. Firstly, after the successful preparations of 1-NPs-QDs and 1-NPs-QDs-FA, we measured their fluorescence spectra, together with 1-NPs. As shown in Fig. 1D, when excited at 600 nm, both 1-NPs-QDs and 1-NPs-QDs-FA showed emission maximum at 734 nm, with the intensity of the former slightly higher than the latter, suggesting the Mal-FA modification process
Fig. 1. (A) TEM image of 1-NPs. (B) TEM image of 1-NPs-QDs. Inset: HR-TEM graph of 1-NPs-QDs shows the crystal lattice of CdTe/CdS QDs. (C) TEM image of 1-NPs-QDs-FA. Inset: HR-TEM image of 1-NPs-QDs-FA shows the crystal lattice of CdTe/CdS QDs. (D) Fluorescence spectra of 1-NPs, 1-NPs-QDs, and 1-NPs-QDs-FA, respectively.
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induced a slight loss of the hybrid nanoparticles. This fluorescence emission maximum shows 8 nm-redshift to that of free CdTe/CdS QDs (Figure S6, Supporting Information), also suggesting the successful conjugation of the QDs to 1-NPs. The 1-NPs, as proposed, did not show any fluorescence emission within the range of 650e850 nm studied (Fig. 1D). 3.2. Cell imaging To ensure the feasibility of the NIR-emitting 1-NPs-QDs-FA for tumor-targeted imaging in vivo, we firstly applied them for FRoverexpressing tumor cell imaging in vitro (herein HepG2 cells [34]). Since the steric hindrance between conjugated QDs and FA might affect the targeting property of 1-NPs-QDs-FA, we prepared four stocks of 1-NPs-QDs-FA with different QDs/FA ratios and studied their fluorescence cell imaging. In detail, 1 mL of CdTe/CdS QDs at different concentrations (0.02 mg/mL, 0.1 mg/mL, 0.5 mg/ mL, and 1 mg/mL) was reacted with 40 mL of EDC/NHS (0.05 M solution in PBS) and centrifuged to prepare the QDs-NHS. As-prepared QDs-NHS then reacted with 1 mL of fresh 1-NPs to prepare 1NPs-QDs after centrifugation. Then the four stocks of 1-NPs-QDs were separately reacted with large excess of Mal-FA (7 mL, 1 M) and centrifuged to prepare four stocks of 1-NPs-QDs-FA with different QDs/FA ratios. As shown in Figure S7 (Supporting Information), the 1-NPs-QDs-FA prepared with 0.1 mg/mL CdTe/CdS QDs had the best fluorescence effect of cell imaging. Therefore, 0.1 mg/mL was chosen as the optimum concentration for CdTe/CdS QDs to prepare 1-NPs-QDs-FA for the following experiments. 5 mL of above 1-NPsQDs-FA solution was dissolved in 1 mL serum-free culture medium, then incubated with HepG2 cells for 1 h. As shown in Fig. 2, clearly we observed the NIR-fluorescence emission from the cells (left lane of Fig. 2). When 1-NPs-QDs-FA was incubated with FR-negative cell lines at the same condition (herein A549 cells [34]), fluorescence emission was hardly observed from the cells (left middle lane of Fig. 2). These results indicated that 1-NPs-QDs-FA are really FRtargeting. To exclude the possibility of that 1-NPs-QDs are also FR-targeting, we also incubated 1-NPs-QDs with HepG2 cells. As shown in the right middle lane of Fig. 2, very weak fluorescence was observed from the cells, suggesting the nanoparticles were uptaken via passive endocytosis. Interestingly, when the HepG2
cells were pretreated with 100 mM FA for 1 h before the incubation with 1-NPs-QDs-FA, the fluorescence was hardly to be observed (right lane of Fig. 2). This further indicated that the previous fluorescence emission from HepG2 cells in the left lane of Fig. 2 was actually resulted from the active cellular uptake of 1-NPs-QDs-FA via FA-FR interaction. To additionally prove that the fluorescence emission was come from the inside of the cells, we used a laser scanning confocal microscope to monitor the fluorescence of 1NPs-QDs-FA after incubation with HepG2 cells. The results indicated that 1-NPs-QDs-FA were actually inside the cells (Figure S8, Supporting Information). The HepG2 themselves, as supposed, did not show any autofluorescence at this condition (Figure S9, Supporting Information). Cytotoxicity of as-prepared 1-NPs-QDs-FA was assessed and the results shown that the dosage of 1-NPs-QDsFA for cell imaging (i.e., 5 mL of 1-NPs-QDs-FA in 1 mL culture medium) did not induce obvious cytotoxicity until 48 h (Figure S10, Supporting Information). 3.3. In vivo tumor imaging For the active tumor-targeting studies in vivo, each nude mouse was xenografted with A549 tumor in the left thigh and HepG2 tumor in the right thigh. Until the tumor sizes were within 5e10 mm in diameter, the nude mice were randomly divided into 3 groups (n ¼ 3 for each group). Probes 1-NPs-QDs-FA, 1-NPs-QDs, or QDs were respectively injected into these tumor-bearing nude mice in each group through tail veins and the mice were imaged for 4 h in a small animal imaging system. All animals received care in compliance with the guidelines outlined in the Guide for the Care and Use of Laboratory Animals. The procedures were approved by the University of Science and Technology of China Animal Care and Use Committee. As shown in Fig. 3A, for those nude mice injected with 1-NPs-QDs-FA after 4 h, strong NIR-fluorescence emissions with high contrast were observed from the HepG2 tumors in their right thighs (4 folds more than surrounding tissues). In contrast, the A549 tumors in the left thighs only showed very weak NIRemissions which were comparable to those from other organs or tissues. For those tumor-bearing nude mice injected with 1-NPsQDs or QDs, neither the HepG2 tumors nor the A549 tumors showed stronger NIR-fluorescence emissions than those from other
Fig. 2. Left, Fluorescence (upper, Texas Red channel) and overlay (lower, Differential interference contrast (DIC) with fluorescence) images of HepG2 cells after incubation with 5 mL 1-NPs-QDs-FA for 1 h. Left middle, Fluorescence (upper, Texas Red channel) and overlay (lower, DIC with fluorescence) images of A549 cells after incubation with 5 mL 1-NPs-QDsFA for 1 h. Right middle, Fluorescence (upper, Texas Red channel) and overlay (lower, DIC with fluorescence) images of HepG2 cells after incubation with 5 mL 1-NPs-QDs for 1 h. Right, Fluorescence (upper, Texas Red channel) and overlay (lower, DIC with fluorescence) images of HepG2 cells pre-treated with 100 mM FA for 1 h and then incubated with 5 mL 1NPs-QDs-FA for 1 h. Scare bar: 40 mm.
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Fig. 3. Noninvasive optical imaging of tumor-bearing nude mice at 4 h after intravenously injected with 100 mL 1-NPs-QDs-FA (A), 1-NPs-QDs (B), or QDs (C). Each mouse was xenografted with A549 tumor in left thigh (indicated with “A” by white arrow) and HepG2 tumor in right thigh (indicated with “H” by white arrow). Tumors are in the black circles.
organs, as shown in Fig. 3B&C. These results clearly indicated that our 1-NPs-QDs-FA could be effectively and selectively uptaken by FR-overexpressing tumor cells in vivo and applied for tumortargeted NIR-fluorescence imaging. 3.4. Ex vivo imaging To further validate that NIR-fluorescence was actually emitted from the tumors and show the uptakes of 1-NPs-QDs-FA by other organs, after the imaging of these mice, we sacrificed the mice and took out the organs from their bodies and conducted ex vivo imaging of these organs. As shown in Fig. 4, HepG2 tumors in mice injected with 1-NPs-QDs-FA showed highest NIR-fluorescence among the organs studied. The A549 tumors only exhibited weak NIR-fluorescence which was comparable to that of livers, kidneys, spleens, or hearts. The lungs showed the lowest fluorescence. For those mice injected with 1-NPs-QDs or QDs, the HepG2 tumors did not show obviously higher NIR-fluorescence than any other organs (Figure S11, Supporting Information), which was consistent with the imaging results in Fig. 3. The mean fluorescence intensity of organs in Fig. 3 and Figure S11 were shown in Figure S12 (Supporting Information).
4. Conclusions Using one AB2-type small molecule 1, we prepared oligomeric nanoparticles 1-NPs with a condensation reaction and selfassembly. With functional eNH2 and eSH groups on their surfaces, 1-NPs could be facilely conjugated with NIR-emitting CdTe/ CdS QDs and Mal-FA to synthesize oligomeric/inorganic hybrid nanoparticles 1-NPs-QDs-FA. As-prepared nanoparticles showed excellent targeting property to FR-expressing tumor cells in vitro and were successfully applied to selectively imaging FRoverexpressing tumors in vivo. Acknowledgments The authors are grateful to Dr. Ting Yue for his help with animal imaging. This work was supported by the National Natural Science Foundation of China (Grants 21175122, 91127036, and 21375121) and the Fundamental Research Funds for Central Universities (Grant WK2060190018). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.biomaterials.2014.05.071. References
Fig. 4. Ex vivo fluorescence image of different organs from tumor-bearing nude mouse intravenously injected with 100 mL 1-NPs-QDs-FA for 4 h in Fig. 3A.
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Oligomeric nanoparticles functionalized with NIR-emitting CdTe/CdS QDs and folate for tumor-targeted imaging Yue Yuan, Jia Zhang, Linna An, Qinjingwen Cao, Yun Deng, Gaolin Liang* CAS Key Laboratory of Soft Matter Chemistry, Department of Chemistry & Collaborative Innovation Center of Suzhou Nano Science and Technology, University of Science and Technology of China, Hefei, Anhui 230026, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 31 March 2014 Accepted 23 May 2014 Available online 20 June 2014
We report herein the facile surface-functionalization of one type of biocompatible, oligomeric nanoparticles 1-NPs with NIR-emitting CdTe/CdS QDs and folate for tumor-targeted imaging in vivo. The eNH2 and eSH groups of cysteine residues on the 1-NPs were utilized to covalently conjugate CdTe/CdS QDs and Mal-FA to prepare the hybrid nanoparticles 1-NPs-QDs-FA. As-prepared 1-NPs-QDs-FA showed NIRfluorescence emission at 734 nm, selective uptake by FR-overexpressing tumor cells in vitro, and selective FR-overexpressing tumor-targeted imaging in vivo. This first example of oligomeric/inorganic hybrid nanoparticles provides people with new type of biomaterials for tumor-targeted imaging with high selectivity. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Oligomeric nanoparticles Quantum dots Hybrid nanoparticles Near-infrared Tumor-targeting Imaging
1. Introduction Near-infrared (NIR)-fluorescence probes for in vivo imaging have drawn great attention from the biomedical field in recent years due to the high penetration ability of their excitations (or emissions) through tissues in the NIR wavelength window (650e900 nm), while in the meantime maintaining a minimal biological autofluorescence background [1e3]. To date, many types of nanoparticle-based, highly-luminescent probes have been developed for NIR-fluorescence imaging [4e9]. Among them, quantum dots (QDs) with the highest quantum yields and unique size-tunable optical properties, are emerging as the most exciting and promising probes for in vivo NIR-fluorescence imaging [10e12]. However, to achieve a tissue/organ-targeted imaging, QDs need to be functionalized with targeting warheads before injection. For example, there are many methods reported to modify QDs with biomolecules such as folic acid (FA), DNA, affibody, peptide, or enzyme for tumor-targeted imaging [13e19]. But usually the biomolecular warheads could not be directly modified on the QDs' surface unless the QDs have been coated with stabilizers such as streptavidin [13], mercaptopropionic acid (MPA) [16], or bovine serum albumin (BSA) [17]. Moreover, to preserve
* Corresponding author. E-mail address: [email protected] (G. Liang). http://dx.doi.org/10.1016/j.biomaterials.2014.05.071 0142-9612/© 2014 Elsevier Ltd. All rights reserved.
their optical and colloidal stability in biological fluids, CdTe/ZnS core/shell QDs are always modified with polymers [20e23] or dendrimers [24,25]. Therefore, people always use carriers to load QDs for imaging applications. And inorganic nanoparticles with large sizes such as mesoporous silicon nanoparticles have been widely used for this purpose [26]. Inorganic nanoparticles as templates for QDs embedding have many advantages such as easier control of their sizes, prolonged retention of QDs in vivo, and multiple QDs modification [27,28]. Nevertheless, it is difficult for inorganic nanoparticles to be controlled functionalized with QDs together with targeting biomolecules. In contrast, organic nanoparticles (e.g., polymeric nanoparticles or oligomeric nanoparticles), which have abundant surface functional groups, good biological compatibility, and excellent operability, are ideal carriers to load QDs for tissue/organ-targeted imaging. Herein, we report one type of oligomeric nanoparticles (i.e., 1-NPs in Scheme 1) functionalized with NIR-emitting CdTe/CdS QDs and FA for tumor-targeted imaging. This type of oligomeric nanoparticles as carriers could not only prolong the retention times of the QDs as well as small molecule FA in vivo, but also provide reactive groups (i.e., eSH and eNH2) for multiple functionalization (e.g., functionalization with an anti-cancer drug or contrast agent together with the QDs and FA). The highly-luminescent NIR-emitting CdTe/ CdS core/shell QDs were chosen for this study because they are highly stable, water-soluble, and suitable for small animal imaging [16].
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Scheme 1. Schematic illustration of the preparative procedures of 1-NPs-QDs-FA.
To facilely synthesize a type of oligomeric nanoparticles with functional groups for CdTe/CdS QDs and FA conjugation, we chose a “click” condensation reaction between the 1,2-aminothiol group of a cysteine (Cys) motif and the cyano group on a 2cyanobenzothiazole (CBT) motif, recently developed by Rao and Liang [29e33]. In these literature, one simple monomer Cys(SEt)Lys [Cys(SEt)]-CBT (1) was reported which could undergo this condensation reaction upon reduction at pH 7.4 in water to yield amphiphilic oligomers and the oligomers self-assemble into oligomeric nanoparticles (i.e., 1-NPs) within 5 min. In detail, as shown in Scheme 1, upon reduction, the disulfide bonds on the Cys motifs of 1 are cleaved, exposing two reactive Cys residues. One of the cysteine residue condenses with the cyano group on another CBT motif to form oligomers of 1 (mostly dimers), which self-assemble into 1-NPs with abundant reactive Cys residues (-NH2 groups and eSH groups) on the surfaces of the nanoparticles. In this work, we used the amino groups (-NH2) on the surfaces of 1-NPs to conjugate with QDs using the classical 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride/N-hydroxysuccinimide (EDC/NHS) method to prepare the 1-NPs-QDs first. Then the thiol groups (-SH) on the 1-NPs-QDs were conjugated to the maleimide motifs of maleimide-folic acid (Mal-FA) via addition reaction to yield the oligomeric and inorganic hybrid nanoparticles 1-NPs-QDs-FA for folate receptor (FR)-overexpressing tumor-targeted imaging in vivo. 2. Materials and methods 2.1. Materials All the starting materials were obtained from Adamas or Sangon Biotech. Commercially available reagents were used without further purification, unless noted otherwise. All other chemicals were reagent grade or better. PBS buffer (0.01 M, pH ¼ 7.4) was prepared with pills purchased from Sangon Biotech Co., Ltd. (Shanghai, China). Ultrapure water (18.2 MU cm) was used throughout the experiment. Cells were obtained from Cell Bank of Chinese Academy of Sciences.
The spectra of electrospray ionization-mass spectrometry (ESI-MS) were recorded on an LCQ Advantage MAX ion trap mass spectrometer (Thermo Fisher). MALDITOF/TOF mass spectra were obtained on a time-of-flight Ultraflex II mass spectrometer (Bruker Daltonics). HPLC analyses were performed on an Agilent 1200 HPLC system equipped with a G1322A pump and in-line diode array UV detector using an Agilent Zorbax 300SB e C18 RP column with CH3CN (0.1% of trifluoroacetic acid (TFA)) and water (0.1% of TFA) as the eluant. 1H NMR spectra were obtained on a 300 MHz Bruker AV300. Fluorescence microscopic images were taken under a fluorescence microscope OLMPUS IX71. Confocal fluorescence images were recorded using a confocal laser microscope Olympus BX61W. Cells were routinely cultured in Dul-becco's modified Eagle's medium (DMEM, Hycolon) supplemented with 10% fetal bovine serum at 37 C, 5% CO2, and humid atmosphere. 4e6 week old (weighing 19~20 g) BALB/c nude mice were used for animal experiments. 2.2. Preparation of Cys(SEt)-Lys[Cys(SEt)]-CBT (1) 2-cyano-6-aminobenzothiazole (CBT) was synthesized following the literature method (White EH, Worther H, Seliger HH, McElroy WD. Amino analogs of firefly luciferin and biological activity thereof. J Am Chem Soc 1966; 88:2015e9). The preparation of compound 1 Cys(SEt)-Lys[Cys(SEt)]-CBT was synthesized following the literature method (Deng Y, Liu S, Mei K, Tang A, Cao C, Liang G. Multifunctional small molecule for controlled assembly of oligomeric nanoparticles and crosslinked polymers. Org Biomol Chem 2011; 9:6917e9). MS: calculated for C24H35N7O3S5 [(M þ H)þ]: 630.14831; obsvd. HR-MALDI-TOF/MS: m/z 630.14830 (Figure S1, Supporting Information). 2.3. Preparation of maleimide-folate (Mal-FA) The mixture of N-(2-Aminoethyl)maleimide trifluoroacetate salt (25.4 mg, 0.1 mmol), HBTU (41.7 mg, 0.1 mmol), HOBT (13.5 mg, 0.1 mmol) in DMF (2 mL) was stirred for 30 min in presence of DIPEA (11.1 mg, 0.1 mmol), then folic acid (FA, 53.0 mg, 0.12 mmol) dissolved in 1 mL of DMF was added into the mixture dropwise. After an overnight stirring, compound Mal-FA (39 mg, yield: 70%) was obtained after HPLC purification(Scheme S1, Supporting Information). 1H NMR of compound Mal-FA (d6-DMSO, 300 MHz, Figure S2, Supporting Information) d (ppm): 8.63 (d, J ¼ 1.62 Hz, 1H), 7.92 (d, J ¼ 6.25 Hz, 1H), 7.85 (d, J ¼ 7.92 Hz, 1H), 7.61 (d, J ¼ 8.72 Hz, 2H), 7.18 (s, 2H), 6.93 (d, J ¼ 3.29 Hz, 2H), 6.60 (m, 2H), 4.47 (s, 2H), 4.21 (s, 1H), 3.14 (m, 2H), 2.19 (m, 2H), 1.94 (m, 4H), 1.20 (s, 1H). MS: calculated for C25H26N9O7 [(M þ H)þ]: 564.20; obsvd. ESI/MS: m/z 564.02 (Figure S3, Supporting Information).
Y. Yuan et al. / Biomaterials 35 (2014) 7881e7886 2.4. Preparation of CdTe/CdS quantum dots CdTe/CdS quantum dots were synthesized following the literature method with minor modification (Chen LN, Wang J, Li WT, Han HY. Aqueous one-pot synthesis of bright and ultrasmall CdTe/CdS near-infrared-emitting quantum dots and their application for tumor targeting in vivo. Chem Commun 2012:48:4971e3). First, 74 mL MPA and 20 mg trisodium citrate were added to 25 mL CdCl2 solution (0.05 mmol) in succession. After that, the mixture was stirred for 5 min, followed by adding 0.1 M NaOH to tune the pH of the mixed solution to 11.2. Then, 4.4 mg Na2TeO3 and 20 mg NaBH4 were added into the mixture under rapid stirring. Finally, the reaction mixture was slowly heated to 90 C and stirred for 4 h. The obtained QDs was purified by the addition of ethanol and salt, then dispersed in water.
3. Results and discussion 3.1. Preparation and characterizations of 1-NPs-QDs-FA We began the study with reduction-controlled condensation of 1 and self-assembly of 1-NPs. At a concentration of 1 mM, 1 was dissolved in water containing 5% methanol. 5 min after adding 4 equiv. of tris(2-carboxyethyl)phosphine (TCEP) and adjusting the pH to 7.4 with sodium carbonate, we observed the solutions of 1 became turbid dispersions and their UV-vis spectra at 500e700 nm showed obvious increases, suggesting the aggregation of particles (Figure S4, Supporting Information). Dynamic light scattering (DLS) measurements indicated that as-formed 1-NPs have a mean dynamic diameter of 54.2 nm (Figure S5, Supporting Information). Transmission electron microscope (TEM) images of the 1-NPs indicated that the nanoparticles have diameters of 21e37 nm and tend to connect with each other to form fibrous structures (Fig. 1A). To 1 mL of 0.1 mg mL1 CdTe/CdS QDs solution, 40 mL of EDC (0.05 M
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solution in PBS) and an equal amount of NHS (0.05 M solution in PBS) were added and stirred for 15 min. After ultrafiltration under centrifugation (15,000 rpm, 10 min) with a YM-10 ultrafilter to remove excess EDC and NHS, the prepared QDs-NHS ester was redissolved with 1 mL of PBS buffer (5 mM). Then 1 mL of freshly prepared 1-NPs monodispersion was added into the QDs-NHS solution and stirred overnight. 1-NPs-QDs were obtained by centrifugation and washing with 1 mL phosphate buffered saline (PBS, 5 mM) for three times. TEM image of 1-NPs-QDs indicated that the nanoparticles have diameters of 24e42 nm (Fig. 1B). High resolution TEM (HR-TEM) image clearly shows the crystal lattice of CdTe/ CdS QDs, suggesting the success of QDs conjugation to the oligomeric nanoparticles 1-NPs (inset of Fig. 1B). 5 min after 4 equiv. of TCEP was added into 1 mL 1-NPs-QDs in water and the pH value was adjusted to 7.4 with sodium carbonate, 7 mL of Mal-FA (1 M in DMSO) was added and stirred for 2 h at room temperature to yield 1-NPs-QDs-FA after centrifugation and washing for three times. TEM image of 1-NPs-QDs-FA showed that nanoparticles have diameters of 25e44 nm (Fig. 1C), similar to those of 1-NPs-QDs in Fig. 1B. HR-TEM image of 1-NPs-QDs-FA consistently shows the crystal lattice of CdTe/CdS QDs, suggesting that Mal-FA modification does not induce the release of QDs from the oligomeric nanoparticles (inset of Fig. 1C). As-obtained 1-NPsQDs or 1-NPs-QDs-FA were redispersed in 200 mL water for all the experiments following. Firstly, after the successful preparations of 1-NPs-QDs and 1-NPs-QDs-FA, we measured their fluorescence spectra, together with 1-NPs. As shown in Fig. 1D, when excited at 600 nm, both 1-NPs-QDs and 1-NPs-QDs-FA showed emission maximum at 734 nm, with the intensity of the former slightly higher than the latter, suggesting the Mal-FA modification process
Fig. 1. (A) TEM image of 1-NPs. (B) TEM image of 1-NPs-QDs. Inset: HR-TEM graph of 1-NPs-QDs shows the crystal lattice of CdTe/CdS QDs. (C) TEM image of 1-NPs-QDs-FA. Inset: HR-TEM image of 1-NPs-QDs-FA shows the crystal lattice of CdTe/CdS QDs. (D) Fluorescence spectra of 1-NPs, 1-NPs-QDs, and 1-NPs-QDs-FA, respectively.
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induced a slight loss of the hybrid nanoparticles. This fluorescence emission maximum shows 8 nm-redshift to that of free CdTe/CdS QDs (Figure S6, Supporting Information), also suggesting the successful conjugation of the QDs to 1-NPs. The 1-NPs, as proposed, did not show any fluorescence emission within the range of 650e850 nm studied (Fig. 1D). 3.2. Cell imaging To ensure the feasibility of the NIR-emitting 1-NPs-QDs-FA for tumor-targeted imaging in vivo, we firstly applied them for FRoverexpressing tumor cell imaging in vitro (herein HepG2 cells [34]). Since the steric hindrance between conjugated QDs and FA might affect the targeting property of 1-NPs-QDs-FA, we prepared four stocks of 1-NPs-QDs-FA with different QDs/FA ratios and studied their fluorescence cell imaging. In detail, 1 mL of CdTe/CdS QDs at different concentrations (0.02 mg/mL, 0.1 mg/mL, 0.5 mg/ mL, and 1 mg/mL) was reacted with 40 mL of EDC/NHS (0.05 M solution in PBS) and centrifuged to prepare the QDs-NHS. As-prepared QDs-NHS then reacted with 1 mL of fresh 1-NPs to prepare 1NPs-QDs after centrifugation. Then the four stocks of 1-NPs-QDs were separately reacted with large excess of Mal-FA (7 mL, 1 M) and centrifuged to prepare four stocks of 1-NPs-QDs-FA with different QDs/FA ratios. As shown in Figure S7 (Supporting Information), the 1-NPs-QDs-FA prepared with 0.1 mg/mL CdTe/CdS QDs had the best fluorescence effect of cell imaging. Therefore, 0.1 mg/mL was chosen as the optimum concentration for CdTe/CdS QDs to prepare 1-NPs-QDs-FA for the following experiments. 5 mL of above 1-NPsQDs-FA solution was dissolved in 1 mL serum-free culture medium, then incubated with HepG2 cells for 1 h. As shown in Fig. 2, clearly we observed the NIR-fluorescence emission from the cells (left lane of Fig. 2). When 1-NPs-QDs-FA was incubated with FR-negative cell lines at the same condition (herein A549 cells [34]), fluorescence emission was hardly observed from the cells (left middle lane of Fig. 2). These results indicated that 1-NPs-QDs-FA are really FRtargeting. To exclude the possibility of that 1-NPs-QDs are also FR-targeting, we also incubated 1-NPs-QDs with HepG2 cells. As shown in the right middle lane of Fig. 2, very weak fluorescence was observed from the cells, suggesting the nanoparticles were uptaken via passive endocytosis. Interestingly, when the HepG2
cells were pretreated with 100 mM FA for 1 h before the incubation with 1-NPs-QDs-FA, the fluorescence was hardly to be observed (right lane of Fig. 2). This further indicated that the previous fluorescence emission from HepG2 cells in the left lane of Fig. 2 was actually resulted from the active cellular uptake of 1-NPs-QDs-FA via FA-FR interaction. To additionally prove that the fluorescence emission was come from the inside of the cells, we used a laser scanning confocal microscope to monitor the fluorescence of 1NPs-QDs-FA after incubation with HepG2 cells. The results indicated that 1-NPs-QDs-FA were actually inside the cells (Figure S8, Supporting Information). The HepG2 themselves, as supposed, did not show any autofluorescence at this condition (Figure S9, Supporting Information). Cytotoxicity of as-prepared 1-NPs-QDs-FA was assessed and the results shown that the dosage of 1-NPs-QDsFA for cell imaging (i.e., 5 mL of 1-NPs-QDs-FA in 1 mL culture medium) did not induce obvious cytotoxicity until 48 h (Figure S10, Supporting Information). 3.3. In vivo tumor imaging For the active tumor-targeting studies in vivo, each nude mouse was xenografted with A549 tumor in the left thigh and HepG2 tumor in the right thigh. Until the tumor sizes were within 5e10 mm in diameter, the nude mice were randomly divided into 3 groups (n ¼ 3 for each group). Probes 1-NPs-QDs-FA, 1-NPs-QDs, or QDs were respectively injected into these tumor-bearing nude mice in each group through tail veins and the mice were imaged for 4 h in a small animal imaging system. All animals received care in compliance with the guidelines outlined in the Guide for the Care and Use of Laboratory Animals. The procedures were approved by the University of Science and Technology of China Animal Care and Use Committee. As shown in Fig. 3A, for those nude mice injected with 1-NPs-QDs-FA after 4 h, strong NIR-fluorescence emissions with high contrast were observed from the HepG2 tumors in their right thighs (4 folds more than surrounding tissues). In contrast, the A549 tumors in the left thighs only showed very weak NIRemissions which were comparable to those from other organs or tissues. For those tumor-bearing nude mice injected with 1-NPsQDs or QDs, neither the HepG2 tumors nor the A549 tumors showed stronger NIR-fluorescence emissions than those from other
Fig. 2. Left, Fluorescence (upper, Texas Red channel) and overlay (lower, Differential interference contrast (DIC) with fluorescence) images of HepG2 cells after incubation with 5 mL 1-NPs-QDs-FA for 1 h. Left middle, Fluorescence (upper, Texas Red channel) and overlay (lower, DIC with fluorescence) images of A549 cells after incubation with 5 mL 1-NPs-QDsFA for 1 h. Right middle, Fluorescence (upper, Texas Red channel) and overlay (lower, DIC with fluorescence) images of HepG2 cells after incubation with 5 mL 1-NPs-QDs for 1 h. Right, Fluorescence (upper, Texas Red channel) and overlay (lower, DIC with fluorescence) images of HepG2 cells pre-treated with 100 mM FA for 1 h and then incubated with 5 mL 1NPs-QDs-FA for 1 h. Scare bar: 40 mm.
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Fig. 3. Noninvasive optical imaging of tumor-bearing nude mice at 4 h after intravenously injected with 100 mL 1-NPs-QDs-FA (A), 1-NPs-QDs (B), or QDs (C). Each mouse was xenografted with A549 tumor in left thigh (indicated with “A” by white arrow) and HepG2 tumor in right thigh (indicated with “H” by white arrow). Tumors are in the black circles.
organs, as shown in Fig. 3B&C. These results clearly indicated that our 1-NPs-QDs-FA could be effectively and selectively uptaken by FR-overexpressing tumor cells in vivo and applied for tumortargeted NIR-fluorescence imaging. 3.4. Ex vivo imaging To further validate that NIR-fluorescence was actually emitted from the tumors and show the uptakes of 1-NPs-QDs-FA by other organs, after the imaging of these mice, we sacrificed the mice and took out the organs from their bodies and conducted ex vivo imaging of these organs. As shown in Fig. 4, HepG2 tumors in mice injected with 1-NPs-QDs-FA showed highest NIR-fluorescence among the organs studied. The A549 tumors only exhibited weak NIR-fluorescence which was comparable to that of livers, kidneys, spleens, or hearts. The lungs showed the lowest fluorescence. For those mice injected with 1-NPs-QDs or QDs, the HepG2 tumors did not show obviously higher NIR-fluorescence than any other organs (Figure S11, Supporting Information), which was consistent with the imaging results in Fig. 3. The mean fluorescence intensity of organs in Fig. 3 and Figure S11 were shown in Figure S12 (Supporting Information).
4. Conclusions Using one AB2-type small molecule 1, we prepared oligomeric nanoparticles 1-NPs with a condensation reaction and selfassembly. With functional eNH2 and eSH groups on their surfaces, 1-NPs could be facilely conjugated with NIR-emitting CdTe/ CdS QDs and Mal-FA to synthesize oligomeric/inorganic hybrid nanoparticles 1-NPs-QDs-FA. As-prepared nanoparticles showed excellent targeting property to FR-expressing tumor cells in vitro and were successfully applied to selectively imaging FRoverexpressing tumors in vivo. Acknowledgments The authors are grateful to Dr. Ting Yue for his help with animal imaging. This work was supported by the National Natural Science Foundation of China (Grants 21175122, 91127036, and 21375121) and the Fundamental Research Funds for Central Universities (Grant WK2060190018). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.biomaterials.2014.05.071. References
Fig. 4. Ex vivo fluorescence image of different organs from tumor-bearing nude mouse intravenously injected with 100 mL 1-NPs-QDs-FA for 4 h in Fig. 3A.
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