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Short Communication
One-step synthesized immunostimulatory oligonucleotides-functionalized quantum dots for simultaneous enhanced immunogenicity and cell imaging Yu Tao, Yan Zhang, Enguo Ju, Jinsong Ren ∗ , Xiaogang Qu State Key laboratory of Rare Earth Resources Utilization and Laboratory of Chemical Biology, Changchun Institute of Applied Chemistry, Graduate School of the Chinese Academy of Sciences, Chinese Academy of Sciences, Changchun, Jilin 130022, China
a r t i c l e
i n f o
Article history: Received 22 October 2014 Received in revised form 26 November 2014 Accepted 18 December 2014 Available online xxx Keywords: CpG motif Quantum dots Immunostimulation Oligodeoxynucleotides Cell imaging
a b s t r a c t Unmethylated cytosine–phosphate–guanine (CpG) dinucleotides, normally occur in natural bacterial and viral genomes, show strong immunostimulatory activities to invading pathogens and have found widespread applications in both basic research and clinical trials. For the first time, we design a simple one-step synthesis of CpG-functionalized quantum dots (QDs), combining fascinating features of enhanced immunogenicity and cell imaging. The induction of QDs can greatly increase CpG uptake ability by TLR9-positive cells and elevate CpG stability against nuclease degradation. What is more, the outstanding optical properties also suggest that the CpG-QDs can serve as promising optical probes for the evaluation of the cellular uptake efficiency of the CpG motifs. To our best knowledge, this is the first report to use a facile one-pot synthesis strategy that allows the CpG-functionalized QDs to be prepared, which are able to serve as both the potent platform for immunotherapy and the fluorescent probes for intracellular imaging. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Vaccines are considered to be one of the greatest inventions in human history, since they have led to dramatically improved healthcare and greatly reduced mortality in infectious diseases [1–3]. In recent years, due to safety concerns, vaccine research has shifted focus from the traditional live attenuated and inactivated pathogens toward the development of the recombinant protein antigen-based subunit vaccines [4,5], which however suffer from weak and short-lived humoral and cellular immune responses [6,7]. One aspect of vaccine optimization is to choose the immune adjuvant [8], which can efficiently enhance the immune response by stimulating antigen presenting cells, increasing the production of cytokines and chemokines [9,10]. As a novel immunostimulatory adjuvant, unmethylated cytosine–phosphate–guanine (CpG) oligodeoxynucleotides (ODNs) have been shown both in preclinical and clinical studies to promote Th1-responses [11,12]. Unmethylated CpG ODNs can be efficiently recognized by the mammalian immune system and internalized by the antigen-presenting cells through Toll-like receptor 9 (TLR9), inducing the release of a
∗ Corresponding author. Tel.: +86 431 85262625; fax: +86 431 85262625. E-mail address: [email protected] (J. Ren).
number of proinflammatory cytokines [13,14], such as interleukin IL-6 and tumor necrosis factors TNF-␣, which are able to stimulate a cascade of innate and adaptive immune responses. Therefore, CpG ODNs have long been considered as immunomodulators for therapeutic applications against cancers and infectious diseases [15]. Nevertheless, the application of CpG adjuvant as an immunostimulatory drug is largely prevented by the poor efficiency of cellular uptake and rapid degradation by nucleases [16,17]. A number of attempts have been made to enhance the stability or cellular uptake of CpG ODNs [17–25]. For instance, the nuclease-resistant phosphorothioate backbone has been applied to increase the stability of CpG ODNs [19,20]. Alternatively, various DNA assemblies and novel nanomaterials have emerged as effective carriers for CpG ODN delivery [21–25]. Although significant progress has been achieved in this field, the development of simple and straightforward methodology for the synthesis of CpG ODN carriers is still urgently needed. Over the past few decades, as a new class of fluorophores, semiconductor quantum dots (QDs) have attracted extensive research interest due to their unique photophysical properties, such as high fluorescence quantum yields, optically tunable light emissions, high stokes shifts, narrow and symmetric emission spectra, and superior stability against photobleaching [26–28]. Therefore, unlike bulk materials, QDs have shown a wide range of potential
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Please cite this article in press as: Y. Tao, et al., One-step synthesized immunostimulatory oligonucleotides-functionalized quantum dots for simultaneous enhanced immunogenicity and cell imaging, Colloids Surf. B: Biointerfaces (2015), http://dx.doi.org/10.1016/j.colsurfb.2014.12.037
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2.2. Cytokine assays
Fig. 1. Schematic showing the synthesis of the CpG-QDs and their immunostimulatory effect. (For interpretation of the references to color in text, the reader is referred to the web version of this article.)
applications, especially in light-emitting devices, sensory materials, immunoassays, and biological imaging [29,30]. However, the colloidal semiconductor nanocrystals are typically passivated with hydrophobic organic ligands during the synthesis [31]. So it is necessary either to replace these ligands or encapsulate the nanocrystals with hydrophilic moieties to make the lumiphores soluble in water, which requires sophisticated synthesis procedures. In addition, the biofunctionalization of QDs, which allows the fabrication of biosensors and targeting imaging agents, also needs time- and labor-intensive modification processes [32]. Therefore, developing a simple and general synthetic route is highly desirable for both fundamental studies and practical applications of this type of material. Herein, for the first time, we report a convenient, one-pot synthesis of CpG-functionalized QDs (CpG-QDs), which can elicit specific immunological responses and be used as probes for intracellular imaging. The method relies on the design of the chimeric oligonucleotides that consist of two different types of moieties: one portion is the phosphorothioates (ps), which preferentially serve as the ligand for QDs synthesis over phosphates (po) due to the higher affinity for metals, represented by the blue line, and the other is the CpG sequence that can effectively stimulate the immunocytes to secrete cytokines, represented by the pink line (Fig. 1). In this way, selective nanocrystal growth can be controlled on the ps backbones, while the CpG ODNs (po backbones) can maintain their native function to serve as the efficient immunostimulatory adjuvant. The introduction of QDs here offers several crucial benefits, such as the elevated intracellular delivery ability of CpG ODNs and the efficient protection of CpG from nuclease degradation, which lead to the remarkable immunostimulatory activity of CpG-QDs. What is more, the outstanding optical qualities also suggest that the CpGQDs can serve as excellent fluorescent probes for the estimation of the cellular uptake capability of the CpG ODNs. To our best knowledge, this is the first report to use a facile one-pot synthesis strategy that allows the CpG-functionalized QDs to be prepared, which are able to serve as both the robust platform for immunotherapy and the optical probes for cell imaging.
2. Materials and methods 2.1. Preparation of CpG-QDs CpG-QDs were prepared according to the previously reported method with minor modifications [33]. At first, NaBH4 (0.025 g) was allowed to react with tellurium powder (0.040 g) in 1 mL deionized water to produce sodium hydrogen telluride (NaHTe). This process was continued overnight at room temperature. Then, CdCl2 -GSH stock solution was made to dissolve 1.25 mM CdCl2 and 1.4 mM GSH in water and pH was adjusted to 9.0 by addition of NaOH. For a typical CdTe synthesis, 0.8 L of oxygen-free NaHTe solution was mixed with 400 L CdCl2 -GSH solution in a 1.5 mL eppendorf tube, and then DNA template solution containing nucleotides (300 nM) was added. The reaction was conducted at 100 ◦ C for 1 h and then gradually cooled to room temperature.
RAW264.7 cells were cultured in 6-well plates at a density of 5 × 105 cells/well. After 24 h incubation, cells were washed with 0.5 mL PBS before treatment with indicated conditions. Then, the medium was collected 8 h later for measurement of TNF-␣ secretions, or 24 h later for measurement of IL-6 secretions. And the supernatants were stored at −80 ◦ C until use. The levels of TNF␣ and IL-6 in the supernatants were determined with a sandwich enzyme-linked immunosorbent assay (ELISA) method according to protocols recommended by the manufacturer. 3. Results and discussion 3.1. Synthesis of CpG-QDs To verify the validity of this approach, we firstly prepared the CpG-QDs by a one-pot method [33–37]. The developed CpG-QDs exhibited strong green fluorescence signal, with an emission maximum at 560 nm (Fig. 2a). Interestingly, although low pH (pH < 5) diminished the luminescence of CpG-QDs, the emission of QDs was not perturbed at physiological pH and high ionic strength (Figs. S1 and S2), which undoubtedly confirmed the applicability of the CpGQDs for biological studies. As shown in Fig. 2b, the transmission electron microscopy (TEM) image clearly revealed that the obtained QDs were monodispersed and roughly spherical, with average size around 6.3 nm. Atomic force microscopy (AFM) was also recorded to confirm that the average size of CpG-QDs was about 6.5 nm (Fig. S3), consistent with TEM result. After that, the stability of CpG-QDs against nuclease digestion was evaluated. We cultured either CpG ODNs or CpG-QDs with 50% non-inactivated fetal bovine serum (FBS) [16,21]. Gel electrophoresis revealed that the band of CpGQDs largely remained over 8 h incubation (Fig. 2c), whereas CpG ODNs were almost entirely degraded in the same situation (Fig. S4), apparently confirming the excellent stability of the CpG-QDs. Interestingly, we noticed that the band of CpG-QDs was still able to be visualized with attenuated intensity after 24 h treatment. These results clearly demonstrated that the QDs had profound positive effects on the conformational stability of CpG ODNs. 3.2. Cell imaging of CpG-QDs Cytotoxicity was a crucial issue that needed to be addressed before the CpG-QDs were utilized for CpG delivery. To evaluate the cytotoxicity of the CpG-QDs, the relative viabilities of RAW264.7 cells were analyzed by the MTT assay treated with diverse concentrations of CpG-QDs (5–100 nM). As shown in Fig. 2d, there were more than 80% RAW264.7 cells viable for CpG-QDs even at the concentration of 50 nM, indicating that the CpG-QDs were stable and not prone to releasing components when introduced into biological media. These results suggested that the CpG-QDs were compatible for safe CpG delivery. Inspired by the low cellular toxicity and excellent fluorescence property, the CpG-QDs were then applied as the fluorescent probes to measure the cell uptake capacity of CpG ODNs. Fig. 3 shows the fluorescence images of RAW264.7 cells after treatment with the FAM-labeled CpG ODNs or CpG-QDs for 4 h. No fluorescence could be observed in cells treated with FAM-labeled CpG ODNs alone (Fig. 3a–d), which agreed with the facts that free ODNs could not freely penetrate through the cell membranes and were highly prone to be degraded by endogenous nucleases during delivery [38]. In contrast, as can be seen in Fig. 3e–h, significant green luminescent signal could be observed for RAW264.7 cells incubated with CpG-QDs, strongly indicating that the QDs could be used as attractive optical probes for cellular fluorescence imaging. The merging of brightfield and fluorescence images clearly clarified
Please cite this article in press as: Y. Tao, et al., One-step synthesized immunostimulatory oligonucleotides-functionalized quantum dots for simultaneous enhanced immunogenicity and cell imaging, Colloids Surf. B: Biointerfaces (2015), http://dx.doi.org/10.1016/j.colsurfb.2014.12.037
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Fig. 2. (a) Fluorescence excitation and emission spectra of the CpG-QDs. Inset: photograph of CpG-QDs solution exhibiting fluorescence under UV light. (b) TEM image of the CpG-QDs. Scale bar, 50 nm. Inset: size distribution histogram of the CpG-QDs. The total number of clusters counted for the histogram was 30. (c) Electrophoretic analysis of the stability of CpG-QDs. The CpG-QDs were incubated in 50% non-heat-inactivated fetal bovine serum (FBS) at 37 ◦ C for 2–24 h and then analyzed by gel electrophoresis. (d) Cytotoxicity of RAW264.7 cells evaluated by MTT assays after incubation with CpG-QDs with different concentrations for 24 h. The error bars represent variations among three independent measurements.
Fig. 3. Fluorescence images of RAW264.7 cells treated with FAM-labeled CpG ODNs (a–d) and CpG-QDs (e–h) for 4 h. The cell nucleus was indicated using Hoechst 33258. Scale bar = 10 m. (For interpretation of the references to color in text, the reader is referred to the web version of this article.)
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Fig. 4. Cytokines release from RAW264.7 cells stimulated by CpG-QDs. Comparison of (a) TNF-␣ and (b) IL-6 release stimulated by single stranded CpG ODNs (50 nM), CpG-ps ODNs (50 nM), QDs (50 nM) and CpG-QDs (50 nM). Error bars represent standard deviation of three independent measurements.
that the fluorescence was observed in the intracellular area, revealing that QDs could dramatically enhance the cellular uptake of CpG ODNs. These observations strongly suggested that CpG ODNs, as the bioconjugate ligands of QDs, were able to be effectively delivered into TLR9-positive immune cells. 3.3. Cytokine release from RAW264.7 cells stimulated by CpG-QDs Encouraged by these attractive merits of the CpG-QDs, we then investigated if the QDs could enhance the immunostimulatory effect of the CpG ODNs. We incubated the free CpG ODNs, CpGps ODNs, QDs and the CpG-QDs with RAW264.7 cells and used the enzyme-linked immunosorbent assay (ELISA) to determine the levels of secreted cytokines. As shown in Fig. 4a, as compared to CpG ODNs alone, the CpG-QDs induced significantly higher levels of TNF-␣, excelling that of CpG alone by more than 18 times. Additionally, QDs alone had minimal stimulating activity for the production of TNF-␣, thus implying that the observed immunostimulation effects were indeed induced by the CpG ODNs. Likewise, we also measured the secretion levels of IL-6 (Fig. 4b) in response to the stimulation of different treatments. The CpG-QDs induced substantially higher levels of IL-6 than the CpG control. It was appealing to observe that the CpG-QDs were capable to secrete cytokines with much higher efficiencies than CpG ODNs alone. This remarkable immunostimulatory activity of CpG-QDs might come from a combination of several important properties, the increased cell uptake of the CpG-QDs, the multivalency of CpG motifs on the surface of CpGQDs [21,33], and the enhanced stability and improved resistance to nuclease of surface-confined CpG ODNs. 4. Conclusion In summary, by appending CpG ODNs to the DNA template, we report a one-pot synthesis of CpG-functionalized QDs that show excellent immune activity while simultaneously perform as an imaging probe. The introduction of QDs brings about many unprecedented merits. Firstly, the QDs are simple to synthesize, and exhibit low cytotoxicity, which make them suitable for biomedical applications. Secondly, compared with CpG ODNs alone, the CpGQDs can remarkably enhance the cellular uptake efficiency of the CpG motifs. Thirdly, conjugation of CpG ODNs on QDs can efficiently protect them against degradation by nucleases, an influence that is highly significant for the immunogenicity of CpG ODNs. Finally, without the requirement of fluorophore labeling, the CpG-QDs with fascinating fluorescent qualities are also able to simultaneously act as promising fluorescent probes for bioimaging. Collectively, taken together, these findings suggest the CpG-QDs can be applied as effective immunostimulatory agents.
Acknowledgements The authors are grateful for the referee’s helpful comments on the manuscript. Financial support was provided by the National Basic Research Program of China (2011CB936004 and 2012CB720602) and the National Natural Science Foundation of China (grants 21210002, 91213302, 21431007, 91413111. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.colsurfb. 2014.12.037. References [1] J.B. Ulmer, U. Valley, R. Rappuoli, Nat. Biotechnol. 24 (2006) 1377–1383. [2] S.A. Plotkin, S.L. Plotkin, Nat. Rev. Microbiol. 9 (2011) 889–893. [3] D.W. Pack, A.S. Hoffman, S. Pun, P.S. Stayton, Nat. Rev. Drug Discov. 4 (2005) 581–593. [4] W. Byrd, A. de Lorimier, Z.-R. Zheng, F.J. Cassels, Adv. Drug Deliv. Rev. 57 (2005) 1362–1380. [5] B. Guy, Nat. Rev. Microbiol. 5 (2007) 505–517. [6] J.J. Moon, B. Huang, D.J. Irvine, Adv. Mater. 24 (2012) 3724–3746. [7] J.A. Hubbell, S.N. Thomas, M.A. Swartz, Nature 462 (2009) 449–460. [8] N. Mitter, C. Yu, K.T. Mody, A. Popat, D. Mahony, A.S. Cavallaro, Nanoscale 5 (2013) 5167–5179. [9] B.N. Lambrecht, M. Kool, M.A.M. Willart, H. Hammad, Curr. Opin. Immunol. 21 (2009) 23–29. [10] P.N. Gupta, S.P. Vyas, Colloids Surf. B 82 (2011) 118–125. [11] A.M. Krieg, Nucleic Acid Ther. 22 (2012) 77–89. [12] A.M. Krieg, Nat. Rev. Drug Discov. 5 (2006) 471–484. [13] D.M. Klinman, A.K. Yi, S.L. Beaucage, J. Conover, A.M. Krieg, Proc. Natl. Acad. Sci. U. S. A. 93 (1996) 2879–2883. [14] Y. Kumagai, O. Takeuchi, S. Akira, Adv. Drug Deliv. Rev. 60 (2008) 795–804. [15] D.M. Klinman, Nat. Rev. Immunol. 4 (2004) 249–259. [16] Y. Tao, Z. Li, E. Ju, J. Ren, X. Qu, Chem. Commun. 49 (2013) 6918–6920. [17] M. Wei, N. Chen, J. Li, M. Yin, L. Liang, Y. He, H. Song, C. Fan, Q. Huang, Angew. Chem. Int. Ed. 51 (2011) 1202–1206. [18] V. Sokolova, T. Knuschke, J. Buer, A.M. Westendorf, M. Epple, Acta Biomater. 7 (2011) 4029–4036. [19] H. Sands, L.J. Goreyferet, A.J. Cocuzza, F.W. Hobbs, D. Chidester, G.L. Trainor, Mol. Pharmacol. 45 (1994) 932–943. [20] S. Agrawal, Q. Zhao, Curr. Opin. Cell Biol. 2 (1998) 519–528. [21] J. Li, H. Pei, B. Zhu, L. Liang, M. Wei, Y. He, N. Chen, D. Li, Q. Huang, C.H. Fan, ACS Nano 5 (2011) 8783–8789. [22] K. Mohri, M. Nishikawa, N. Takahashi, T. Shiomi, N. Matsuoka, K. Ogawa, M. Endo, K. Hidaka, H. Sugiyama, Y. Takahashi, Y. Takakura, ACS Nano 6 (2012) 5931–5940. [23] V.J. Schüller, S. Heidegger, N. Sandholzer, P.C. Nickels, N.A. Suhartha, S. Endres, C. Bourquin, T. Liedl, ACS Nano 5 (2011) 9696–9702. [24] S. Rattanakiat, M. Nishikawa, H. Funabashi, D. Luo, Y. Takakura, Biomaterials 30 (2009) 5701–5706. [25] X. Ouyang, J. Li, H. Liu, B. Zhao, J. Yan, Y. Ma, S. Xiao, S. Song, Q. Huang, J. Chao, C. Fan, Small 9 (2013) 3082–3087. [26] M. Bruchez, M. Moronne, P. Gin, S. Weiss, A.P. Alivisatos, Science 281 (1998) 2013–2016.
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[27] S. Hinds, B.J. Taft, L. Levina, V. Sukhovatkin, C.J. Dooley, M.D. Roy, D.D. MacNeil, E.H. Sargent, S.O. Kelley, J. Am. Chem. Soc. 128 (2005) 64–65. [28] R. Freeman, T. Finder, I. Willner, Angew. Chem. Int. Ed. 48 (2009) 7818–7821. [29] W.C.W. Chan, S. Nie, Science 281 (1998) 2016–2018. [30] X. Gao, Y. Cui, R.M. Levenson, L.W.K. Chung, S. Nie, Nat. Biotechnol. 22 (2004) 969–976. [31] C.B. Murray, D.J. Norris, M.G. Bawendi, J. Am. Chem. Soc. 115 (1993) 8706–8715. [32] I.L. Medintz, H.T. Uyeda, E.R. Goldman, H. Mattoussi, Nat. Mater. 4 (2005) 435–446.
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Please cite this article in press as: Y. Tao, et al., One-step synthesized immunostimulatory oligonucleotides-functionalized quantum dots for simultaneous enhanced immunogenicity and cell imaging, Colloids Surf. B: Biointerfaces (2015), http://dx.doi.org/10.1016/j.colsurfb.2014.12.037
ARTICLE IN PRESS
COLSUB-6813; No. of Pages 5
Colloids and Surfaces B: Biointerfaces xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Short Communication
One-step synthesized immunostimulatory oligonucleotides-functionalized quantum dots for simultaneous enhanced immunogenicity and cell imaging Yu Tao, Yan Zhang, Enguo Ju, Jinsong Ren ∗ , Xiaogang Qu State Key laboratory of Rare Earth Resources Utilization and Laboratory of Chemical Biology, Changchun Institute of Applied Chemistry, Graduate School of the Chinese Academy of Sciences, Chinese Academy of Sciences, Changchun, Jilin 130022, China
a r t i c l e
i n f o
Article history: Received 22 October 2014 Received in revised form 26 November 2014 Accepted 18 December 2014 Available online xxx Keywords: CpG motif Quantum dots Immunostimulation Oligodeoxynucleotides Cell imaging
a b s t r a c t Unmethylated cytosine–phosphate–guanine (CpG) dinucleotides, normally occur in natural bacterial and viral genomes, show strong immunostimulatory activities to invading pathogens and have found widespread applications in both basic research and clinical trials. For the first time, we design a simple one-step synthesis of CpG-functionalized quantum dots (QDs), combining fascinating features of enhanced immunogenicity and cell imaging. The induction of QDs can greatly increase CpG uptake ability by TLR9-positive cells and elevate CpG stability against nuclease degradation. What is more, the outstanding optical properties also suggest that the CpG-QDs can serve as promising optical probes for the evaluation of the cellular uptake efficiency of the CpG motifs. To our best knowledge, this is the first report to use a facile one-pot synthesis strategy that allows the CpG-functionalized QDs to be prepared, which are able to serve as both the potent platform for immunotherapy and the fluorescent probes for intracellular imaging. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Vaccines are considered to be one of the greatest inventions in human history, since they have led to dramatically improved healthcare and greatly reduced mortality in infectious diseases [1–3]. In recent years, due to safety concerns, vaccine research has shifted focus from the traditional live attenuated and inactivated pathogens toward the development of the recombinant protein antigen-based subunit vaccines [4,5], which however suffer from weak and short-lived humoral and cellular immune responses [6,7]. One aspect of vaccine optimization is to choose the immune adjuvant [8], which can efficiently enhance the immune response by stimulating antigen presenting cells, increasing the production of cytokines and chemokines [9,10]. As a novel immunostimulatory adjuvant, unmethylated cytosine–phosphate–guanine (CpG) oligodeoxynucleotides (ODNs) have been shown both in preclinical and clinical studies to promote Th1-responses [11,12]. Unmethylated CpG ODNs can be efficiently recognized by the mammalian immune system and internalized by the antigen-presenting cells through Toll-like receptor 9 (TLR9), inducing the release of a
∗ Corresponding author. Tel.: +86 431 85262625; fax: +86 431 85262625. E-mail address: [email protected] (J. Ren).
number of proinflammatory cytokines [13,14], such as interleukin IL-6 and tumor necrosis factors TNF-␣, which are able to stimulate a cascade of innate and adaptive immune responses. Therefore, CpG ODNs have long been considered as immunomodulators for therapeutic applications against cancers and infectious diseases [15]. Nevertheless, the application of CpG adjuvant as an immunostimulatory drug is largely prevented by the poor efficiency of cellular uptake and rapid degradation by nucleases [16,17]. A number of attempts have been made to enhance the stability or cellular uptake of CpG ODNs [17–25]. For instance, the nuclease-resistant phosphorothioate backbone has been applied to increase the stability of CpG ODNs [19,20]. Alternatively, various DNA assemblies and novel nanomaterials have emerged as effective carriers for CpG ODN delivery [21–25]. Although significant progress has been achieved in this field, the development of simple and straightforward methodology for the synthesis of CpG ODN carriers is still urgently needed. Over the past few decades, as a new class of fluorophores, semiconductor quantum dots (QDs) have attracted extensive research interest due to their unique photophysical properties, such as high fluorescence quantum yields, optically tunable light emissions, high stokes shifts, narrow and symmetric emission spectra, and superior stability against photobleaching [26–28]. Therefore, unlike bulk materials, QDs have shown a wide range of potential
http://dx.doi.org/10.1016/j.colsurfb.2014.12.037 0927-7765/© 2014 Elsevier B.V. All rights reserved.
Please cite this article in press as: Y. Tao, et al., One-step synthesized immunostimulatory oligonucleotides-functionalized quantum dots for simultaneous enhanced immunogenicity and cell imaging, Colloids Surf. B: Biointerfaces (2015), http://dx.doi.org/10.1016/j.colsurfb.2014.12.037
G Model COLSUB-6813; No. of Pages 5
ARTICLE IN PRESS Y. Tao et al. / Colloids and Surfaces B: Biointerfaces xxx (2015) xxx–xxx
2
2.2. Cytokine assays
Fig. 1. Schematic showing the synthesis of the CpG-QDs and their immunostimulatory effect. (For interpretation of the references to color in text, the reader is referred to the web version of this article.)
applications, especially in light-emitting devices, sensory materials, immunoassays, and biological imaging [29,30]. However, the colloidal semiconductor nanocrystals are typically passivated with hydrophobic organic ligands during the synthesis [31]. So it is necessary either to replace these ligands or encapsulate the nanocrystals with hydrophilic moieties to make the lumiphores soluble in water, which requires sophisticated synthesis procedures. In addition, the biofunctionalization of QDs, which allows the fabrication of biosensors and targeting imaging agents, also needs time- and labor-intensive modification processes [32]. Therefore, developing a simple and general synthetic route is highly desirable for both fundamental studies and practical applications of this type of material. Herein, for the first time, we report a convenient, one-pot synthesis of CpG-functionalized QDs (CpG-QDs), which can elicit specific immunological responses and be used as probes for intracellular imaging. The method relies on the design of the chimeric oligonucleotides that consist of two different types of moieties: one portion is the phosphorothioates (ps), which preferentially serve as the ligand for QDs synthesis over phosphates (po) due to the higher affinity for metals, represented by the blue line, and the other is the CpG sequence that can effectively stimulate the immunocytes to secrete cytokines, represented by the pink line (Fig. 1). In this way, selective nanocrystal growth can be controlled on the ps backbones, while the CpG ODNs (po backbones) can maintain their native function to serve as the efficient immunostimulatory adjuvant. The introduction of QDs here offers several crucial benefits, such as the elevated intracellular delivery ability of CpG ODNs and the efficient protection of CpG from nuclease degradation, which lead to the remarkable immunostimulatory activity of CpG-QDs. What is more, the outstanding optical qualities also suggest that the CpGQDs can serve as excellent fluorescent probes for the estimation of the cellular uptake capability of the CpG ODNs. To our best knowledge, this is the first report to use a facile one-pot synthesis strategy that allows the CpG-functionalized QDs to be prepared, which are able to serve as both the robust platform for immunotherapy and the optical probes for cell imaging.
2. Materials and methods 2.1. Preparation of CpG-QDs CpG-QDs were prepared according to the previously reported method with minor modifications [33]. At first, NaBH4 (0.025 g) was allowed to react with tellurium powder (0.040 g) in 1 mL deionized water to produce sodium hydrogen telluride (NaHTe). This process was continued overnight at room temperature. Then, CdCl2 -GSH stock solution was made to dissolve 1.25 mM CdCl2 and 1.4 mM GSH in water and pH was adjusted to 9.0 by addition of NaOH. For a typical CdTe synthesis, 0.8 L of oxygen-free NaHTe solution was mixed with 400 L CdCl2 -GSH solution in a 1.5 mL eppendorf tube, and then DNA template solution containing nucleotides (300 nM) was added. The reaction was conducted at 100 ◦ C for 1 h and then gradually cooled to room temperature.
RAW264.7 cells were cultured in 6-well plates at a density of 5 × 105 cells/well. After 24 h incubation, cells were washed with 0.5 mL PBS before treatment with indicated conditions. Then, the medium was collected 8 h later for measurement of TNF-␣ secretions, or 24 h later for measurement of IL-6 secretions. And the supernatants were stored at −80 ◦ C until use. The levels of TNF␣ and IL-6 in the supernatants were determined with a sandwich enzyme-linked immunosorbent assay (ELISA) method according to protocols recommended by the manufacturer. 3. Results and discussion 3.1. Synthesis of CpG-QDs To verify the validity of this approach, we firstly prepared the CpG-QDs by a one-pot method [33–37]. The developed CpG-QDs exhibited strong green fluorescence signal, with an emission maximum at 560 nm (Fig. 2a). Interestingly, although low pH (pH < 5) diminished the luminescence of CpG-QDs, the emission of QDs was not perturbed at physiological pH and high ionic strength (Figs. S1 and S2), which undoubtedly confirmed the applicability of the CpGQDs for biological studies. As shown in Fig. 2b, the transmission electron microscopy (TEM) image clearly revealed that the obtained QDs were monodispersed and roughly spherical, with average size around 6.3 nm. Atomic force microscopy (AFM) was also recorded to confirm that the average size of CpG-QDs was about 6.5 nm (Fig. S3), consistent with TEM result. After that, the stability of CpG-QDs against nuclease digestion was evaluated. We cultured either CpG ODNs or CpG-QDs with 50% non-inactivated fetal bovine serum (FBS) [16,21]. Gel electrophoresis revealed that the band of CpGQDs largely remained over 8 h incubation (Fig. 2c), whereas CpG ODNs were almost entirely degraded in the same situation (Fig. S4), apparently confirming the excellent stability of the CpG-QDs. Interestingly, we noticed that the band of CpG-QDs was still able to be visualized with attenuated intensity after 24 h treatment. These results clearly demonstrated that the QDs had profound positive effects on the conformational stability of CpG ODNs. 3.2. Cell imaging of CpG-QDs Cytotoxicity was a crucial issue that needed to be addressed before the CpG-QDs were utilized for CpG delivery. To evaluate the cytotoxicity of the CpG-QDs, the relative viabilities of RAW264.7 cells were analyzed by the MTT assay treated with diverse concentrations of CpG-QDs (5–100 nM). As shown in Fig. 2d, there were more than 80% RAW264.7 cells viable for CpG-QDs even at the concentration of 50 nM, indicating that the CpG-QDs were stable and not prone to releasing components when introduced into biological media. These results suggested that the CpG-QDs were compatible for safe CpG delivery. Inspired by the low cellular toxicity and excellent fluorescence property, the CpG-QDs were then applied as the fluorescent probes to measure the cell uptake capacity of CpG ODNs. Fig. 3 shows the fluorescence images of RAW264.7 cells after treatment with the FAM-labeled CpG ODNs or CpG-QDs for 4 h. No fluorescence could be observed in cells treated with FAM-labeled CpG ODNs alone (Fig. 3a–d), which agreed with the facts that free ODNs could not freely penetrate through the cell membranes and were highly prone to be degraded by endogenous nucleases during delivery [38]. In contrast, as can be seen in Fig. 3e–h, significant green luminescent signal could be observed for RAW264.7 cells incubated with CpG-QDs, strongly indicating that the QDs could be used as attractive optical probes for cellular fluorescence imaging. The merging of brightfield and fluorescence images clearly clarified
Please cite this article in press as: Y. Tao, et al., One-step synthesized immunostimulatory oligonucleotides-functionalized quantum dots for simultaneous enhanced immunogenicity and cell imaging, Colloids Surf. B: Biointerfaces (2015), http://dx.doi.org/10.1016/j.colsurfb.2014.12.037
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Fig. 2. (a) Fluorescence excitation and emission spectra of the CpG-QDs. Inset: photograph of CpG-QDs solution exhibiting fluorescence under UV light. (b) TEM image of the CpG-QDs. Scale bar, 50 nm. Inset: size distribution histogram of the CpG-QDs. The total number of clusters counted for the histogram was 30. (c) Electrophoretic analysis of the stability of CpG-QDs. The CpG-QDs were incubated in 50% non-heat-inactivated fetal bovine serum (FBS) at 37 ◦ C for 2–24 h and then analyzed by gel electrophoresis. (d) Cytotoxicity of RAW264.7 cells evaluated by MTT assays after incubation with CpG-QDs with different concentrations for 24 h. The error bars represent variations among three independent measurements.
Fig. 3. Fluorescence images of RAW264.7 cells treated with FAM-labeled CpG ODNs (a–d) and CpG-QDs (e–h) for 4 h. The cell nucleus was indicated using Hoechst 33258. Scale bar = 10 m. (For interpretation of the references to color in text, the reader is referred to the web version of this article.)
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Fig. 4. Cytokines release from RAW264.7 cells stimulated by CpG-QDs. Comparison of (a) TNF-␣ and (b) IL-6 release stimulated by single stranded CpG ODNs (50 nM), CpG-ps ODNs (50 nM), QDs (50 nM) and CpG-QDs (50 nM). Error bars represent standard deviation of three independent measurements.
that the fluorescence was observed in the intracellular area, revealing that QDs could dramatically enhance the cellular uptake of CpG ODNs. These observations strongly suggested that CpG ODNs, as the bioconjugate ligands of QDs, were able to be effectively delivered into TLR9-positive immune cells. 3.3. Cytokine release from RAW264.7 cells stimulated by CpG-QDs Encouraged by these attractive merits of the CpG-QDs, we then investigated if the QDs could enhance the immunostimulatory effect of the CpG ODNs. We incubated the free CpG ODNs, CpGps ODNs, QDs and the CpG-QDs with RAW264.7 cells and used the enzyme-linked immunosorbent assay (ELISA) to determine the levels of secreted cytokines. As shown in Fig. 4a, as compared to CpG ODNs alone, the CpG-QDs induced significantly higher levels of TNF-␣, excelling that of CpG alone by more than 18 times. Additionally, QDs alone had minimal stimulating activity for the production of TNF-␣, thus implying that the observed immunostimulation effects were indeed induced by the CpG ODNs. Likewise, we also measured the secretion levels of IL-6 (Fig. 4b) in response to the stimulation of different treatments. The CpG-QDs induced substantially higher levels of IL-6 than the CpG control. It was appealing to observe that the CpG-QDs were capable to secrete cytokines with much higher efficiencies than CpG ODNs alone. This remarkable immunostimulatory activity of CpG-QDs might come from a combination of several important properties, the increased cell uptake of the CpG-QDs, the multivalency of CpG motifs on the surface of CpGQDs [21,33], and the enhanced stability and improved resistance to nuclease of surface-confined CpG ODNs. 4. Conclusion In summary, by appending CpG ODNs to the DNA template, we report a one-pot synthesis of CpG-functionalized QDs that show excellent immune activity while simultaneously perform as an imaging probe. The introduction of QDs brings about many unprecedented merits. Firstly, the QDs are simple to synthesize, and exhibit low cytotoxicity, which make them suitable for biomedical applications. Secondly, compared with CpG ODNs alone, the CpGQDs can remarkably enhance the cellular uptake efficiency of the CpG motifs. Thirdly, conjugation of CpG ODNs on QDs can efficiently protect them against degradation by nucleases, an influence that is highly significant for the immunogenicity of CpG ODNs. Finally, without the requirement of fluorophore labeling, the CpG-QDs with fascinating fluorescent qualities are also able to simultaneously act as promising fluorescent probes for bioimaging. Collectively, taken together, these findings suggest the CpG-QDs can be applied as effective immunostimulatory agents.
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