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Mannosylated semiconductor quantum dots for the labeling of macrophages
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Journal of Controlled Release 125 (2008) 131 – 136 www.elsevier.com/locate/jconrel
Note
Mannosylated semiconductor quantum dots for the labeling of macrophages Yuriko Higuchi, Machiko Oka, Shigeru Kawakami, Mitsuru Hashida ⁎ Department of Drug Delivery Research, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto, 606-8501, Japan Received 18 April 2007; accepted 3 October 2007 Available online 17 October 2007
Abstract Quantum dots show strong fluorescence emission and long stability compared with classical organic fluorescent dyes; therefore, quantum dots take the place of other dyes in Western blot, immunostaining and bioimaging. Since macrophage plays crucial roles in many pathophysiological processes, tracking macrophage migration, homing and fate is important for understanding the complex roles of macrophages in disease or developing disease diagnosis. Because of the high expression of mannose receptor on macrophage, mannosylation is an attractive strategy to label macrophage. In this study, using polyethylene-glycol (PEG) (M.W. 2,000; PEG2,000)-coated quantum dots, we prepared mannosylated PEG2,000 (Man-PEG2,000) quantum dots for labeling macrophage. The uptake characteristics of Man-PEG2,000 quantum dots were investigated by primary cultured peritoneal macrophages. The uptake of Man-PEG2,000 quantum dots was inhibited by an excess amount of mannose, suggesting mannose receptor-mediated uptake of Man-PEG2,000 quantum dots. The result of MTT assay suggested the extremely low cytotoxicity of Man-PEG2,000 quantum dots. In conclusion, the Man-PEG2,000 synthesized is safe and is taken up by macrophage mannose receptor recognition. © 2007 Elsevier B.V. All rights reserved. Keywords: Quantum dot; Imaging; Mannosylation; Macrophage; Receptor-mediated endocytosis
1. Introduction Quantum dots are semiconductor nanocrystals, which display very interesting optical properties such as single wavelength excitation, size-dependant narrow emission, high intensity of fluorescence, and low photobleaching. Because of strong fluorescence emission and the long stability of quantum dots compared with classical organic fluorescent dyes, quantum dots are taking the place of other dyes in Western blot, immunostaining and bioimaging [1,2]. To date, several bioconjugate types of quantum dots have been developed for living cell imaging, including folate [3] EGF [4] and biotin [5]. In vivo imaging also succeeded in preparing quantum dots labeled with peptide for lung [6] or tumor [7] and antibody for tumor targeting [8,9]. As for cell tracking, Voura et al. [10] prepared quantum dot-labeled tumor cells, which enable us to follow tumor cell extravasation in a living mouse. Therefore, quantum
⁎ Corresponding author. Tel.: +81 75 753 4525; fax: +81 75 753 4575. E-mail address: [email protected] (M. Hashida). 0168-3659/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.jconrel.2007.10.007
dots are attractive candidates for diagnosis by in vivo imaging without invasive operation. Macrophage plays crucial roles in many pathophysiological processes, including inflammation, cancer, organ rejection, autoimmune disease etc; therefore, tracking macrophage migration, homing and fate is important for understanding the complex roles of macrophages in disease or developing the disease diagnosis [11,12]. Recently, Wu et al. [13] demonstrated that labeling macrophage with μm-sized paramagnetic iron oxide in situ could detect acute cardiac rejection in rats using magnetic resonance imaging (MRI). However, the high magnetic field limits the application of MRI for patients who have metal in the body such as an artificial pacemaker and cochlear implant. Moreover, to create a high magnetic field large-scale expensive machine is necessary. Therefore, it is necessary to develop a simpler and more specific method for cell tracking of macrophages. Receptors for carbohydrates, such as asialoglycoprotein receptors on hepatocytes and mannose receptors on several macrophages and liver endothelial cells, recognize the corresponding sugars on the nonreducing terminal of sugar chains
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Y. Higuchi et al. / Journal of Controlled Release 125 (2008) 131–136
[14,15]. Therefore, sugar modification is an attractive tool for cellular targeted delivery of drugs and genes. We previously reported mannosylated protein [16,17], lipid particles [18,19] and macromolecules [20], and demonstrated that they could be delivered to macrophages by mannose receptor-mediated endocytosis in vivo. Therefore, mannosylation of quantum dots would be useful for macrophage detection in vivo. To date, mannosylated quantum dots were synthesized using the direct physical adhesion of sulfur atom to the colloidal core of quantum dots [21,22]. However, the cytotoxicity of the colloidal core in quantum dots complicates an appropriate coating for the application of quantum dots. Recently, RymanRasmussen et al. [23] revealed that quantum dots with NH2polyethylene-glycol (PEG) (M.W. 2,000; PEG2,000) significantly inhibited cytotoxicity and immune responses compared with quantum dots without NH2-PEG2,000 in cultured keratinocytes, suggesting that PEG coating is an effective approach for its safe use. For the application of mannosylated quantum dots for the detection of macrophages, it is also necessary to investigate the uptake characteristics of mannosylated quantum dots to cultured macrophages. In this study, mannosylated-PEG2,000 quantum dots (Man-PEG2,000 quantum dots) are synthesized for the labeling of macrophages (Fig. 1(A)). Then, the uptake characteristics of Man-PEG2,000 quantum dots were investigated in primary cultured peritoneal macrophages, which express mannose receptors. The results were compared with NH2PEG2,000 quantum dots as a control.
2. Materials and methods 2.1. Materials Qdot® 655 ITK™ amino (PEG2,000) quantum dots, OptiMEM® I and fetal bovine serum (FBS) were obtained from Invitrogen Co. (Carlsbad, CA, USA). An ULTRAFREE®-MC 10,000 NMWL filter unit was purchased from Millipore Corp. (Billerica, MA, USA). D(+)-Mannose was purchased from Wako Pure Chemical Industries, Ltd (Osaka, Japan). L-glutamine, penicillin, and streptomycin were purchased from Sigma Chemical Co., Inc. (St. Louis, MO, USA). DABCO (1,4-diazabicyclo-[2,2,2]octane) was purchased from Sigma Chemical Co., Inc. (St. Louis, MO, USA). DAPI (4',6Diamidine-2'-phenylindole dihydrochloride) was purchased from Roche Diagnostics (Indianapolis, IN, USA). RPMI 1640 medium was obtained from Nissui Pharmaceutical Co. (Tokyo, Japan). All other chemicals were of the highest purity available. 2.2. Animals Five-week-old female ICR mice (20–23 g) were purchased from the Shizuoka Agricultural Cooperative Association for Laboratory Animals (Shizuoka, Japan). All animal experiments were carried out in accordance with the Principles of Laboratory Animal Care as adopted and promulgated by the US National Institutes of Health and the Guidelines for Animal Experiments of Kyoto University. 2.3. Synthesis of Man-PEG2,000 quantum dots 2-imino-2-methoxyethyl-1-thiomannoside (IME-thiomannoside) was prepared as reported previously [24]. IMEthiomannoside (3.5 mg) was reacted with 0.5 ml 0.01 M sodium methoxide methanolic solution at room temperature for 2 h. The solvent was evaporated by centrifuging in vacuo overnight at room temperature. Fifty microliters of quantum dots was added to activate IME-thiomannoside. After vortexing for few minutes, the solution was left overnight at room temperature. The solution was filtrated with URTRAFREE filter (10,000 NMWL) by centrifuging at 6400 g for 10 min. PBS (50 μl) was added, and centrifuged again. Finally, 50 μl of PBS was added and Man-PEG2,000 quantum dots were collected. 2.4. Atomic force microscopic image
Fig. 1. Structure of NH2-PEG2,000 quantum dots and Man-PEG2,000 quantum dots (A), the atomic force microscopic image of Man-PEG2,000 quantum dots (B) and fluorescent intensity of Man-PEG2,000 quantum dots (closed circle) or NH2-PEG2,000 quantum dots (open circle) (C).
The structure of Man-PEG2,000 quantum dots was observed with a fluorescence-microscope-coupled atomic force microscope (NVB100, Olympus, Japan). Newly cleaved thin mica (thickness: ca. 30–50 μm) was closely stuck to a cover glass plate (Matsunami Glass, No.1, Japan). The sample droplet on the mica was removed by nitrogen-blowing, and the mica was washed with Millipore water, and dried again by nitrogenblowing. The mica surface was monitored by atomic force microscopy.
Y. Higuchi et al. / Journal of Controlled Release 125 (2008) 131–136
2.5. Culture of peritoneal macrophages Mouse-elicited peritoneal macrophages were cultured according to our previous report [25]. Briefly, peritoneal macrophages were obtained 4 days after intraperitoneal injection by 1 ml of 3% thioglycolate medium. Collected macrophages were plated on 24-well or 12-well cluster dishes at a density of 1.3 × 105 cells/cm2, respectively. Cells were cultured for 72 h with RPMI 1640 medium supplemented with 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin G, and 0.1 mg/ml streptomycin. Cells were maintained at 37 °C with 5% CO2 in a humidified incubator.
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Japan). The objective specifications were 40 × oil immersion and numerical aperture 1.0. To determine the stability, after 2 h incubation with 20 nM of Man-PEG2,000 quantum dots, cells were washed with PBS and cultured for indicated time with culture medium RPMI1640. 2.9. Statistical analysis Statistical analysis was performed using Student's paired t-test for two groups. P b 0.05 was considered to be indicative of statistical significance. 3. Results and discussion
2.6. Uptake experiment Primary peritoneal macrophages or HepG2 cells were plated on a 12-well cluster dish at a density of 1.3 × 105 cells/cm2 and cultivated in RPMI or DMEM culture medium. The culture medium was replaced with Opti-MEM I containing 20 nM NH2PEG2,000 quantum dots or Man-PEG2,000 quantum dots and cultured for the indicated time. After cells were washed twice with cold PBS, the incubation medium was replaced with 200 μl of PBS, and then cells were collected by a cell scraper. Cell intensity was counted by flow cytometry (Becton Dickinson Co., Inc., Franklin Lakes, NJ, USA) on the FL3-channel.
Previously we synthesized mannosylated protein using 2imino-2-methoxyethyl-1-thiomannoside [16,17]. Moreover,
2.7. Cytotoxicity of Man-PEG2,000 quantum dots The cytotoxicity of Man-PEG2,000 quantum dots was determined by MTT assay. Cells were plated on a 24-well cluster dish at a density of 1.3 × 105 cells/cm2 and cultivated in RPMI culture medium. Seventy-two hours later, the culture medium was replaced with Opti-MEM® I containing ManPEG2,000 quantum dots at the indicated concentration. After 24 h, cells were washed twice with cold PBS and added to 150 μl of 0.5 mg/ml MTT solution. Cells were incubated for 4 h at 37 °C in 5% CO2, a 10% SDS solution was added, and incubation continued overnight. Absorbance was measured at test and reference wavelengths of 570 and 660 nm, respectively, in a two-wavelength microplate photometer (Bio-Rad Model 550, Hercules, CA, USA). 2.8. Fluorescence microscopy study Isolated macrophages were cultured at a density of 1.3 × 105 cells/cm2 on a glass-bottom 12-well plate. After 72 h cultivation, the cells were incubated at 37 °C with 20 nM of Man-PEG2,000 quantum dots or NH2-PEG2,000 quantum. After 1 h incubation, the cells were washed five times with ice-cold PBS, then fixed with 4% paraformaldehyde and 0.01% glutaraldehyde in PBS (+) for 15 min on ice and washed 3 times with ice-cold PBS. For nuclear staining, fixed cells were incubated with 10 μg/ml DAPI at room temperature for 1 min. After washing twice with 1 ml icecold PBS, cover glasses were mounted on slide glasses with 50% glycerol–2.5% DABCO (1,4-diazabicyclo-[2,2,2]octane) (Sigma Chemical Co., Inc., St. Louis, MO, USA) in PBS. The samples were examined by Biozero microscopy (Keyence Corp., Osaka,
Fig. 2. Cellular association of Man-PEG2,000 quantum dots (closed circle) or NH2-PEG2,000 quantum dots (open circle) at 0.2, 2.0, 20 nM for 2 h (A) or at 20 nM for 0.5, 1.0 and 2.0 h (B) in primary cultured peritoneal macrophages at 37 °C. Cellular association of Man-PEG2,000 quantum dots (20 nM) with or without an excess amount of mannose (40 mM), or NH2-PEG2,000 quantum dots (20 nM) for 2 h in primary cultured peritoneal macrophages (■) or HepG2 cells (□) at 37 °C (C). Each value represents the mean ± S.D. or +S.D. (n = 3). There are statistically significant differences (⁎P b 0.05, ⁎⁎P b 0.01, ⁎⁎⁎P b 0.001 v.s. NH2-PEG2,000 quantum dots; ###P b 0.001 v.s. Man-PEG2,000 quantum dots with mannose.).
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we synthesized mannosylated lipid with 2-imino-2-methoxyethyl-1-thiomannoside, and prepared mannosylated liposome [18] and emulsion [19,25]. These mannosylated materials were taken up by macrophages through mannose receptor recognition [16–20]. In this study, we prepared Man-PEG2,000 quantum dots using 2-imino-2-methoxyethyl-1-thiomannoside, which could bind with amino residue; therefore, we selected PEG2,000 quantum dots. Synthesis of 2-imino-2methoxyethyl-1-thiomannoside and PEG2,000 quantum dots is one step in the water phase. Man-PEG2,000 quantum dots were not aggregated in water (Fig. 1(B)). The particle size of Man-PEG2,000 quantum dots was about 20–30 μm (Fig. 1 (B)). The fluorescence intensity of Man-PEG2,000 quantum dots was almost the same as that of quantum dots (Fig. 1(C)), suggesting that Man-PEG2,000 quantum dots maintain high intensity with bare quantum dots. In primary cultured peritoneal macrophages, the cellular uptake of Man-PEG2,000 quantum dots was enhanced depending the increase of concentration (Fig. 2 (A)) and incubation time (Fig. 2 (B)). FACS analysis (Fig. 2) and microscopic image (Fig. 3) revealed that the uptake of Man-PEG2,000 quantum dots by macrophages was 3 times higher than that of
Fig. 3. Intracellular distribution of NH2-PEG2,000 quantum dots (red) (A), or Man-PEG2,000 quantum dots (red) (B). After cells were washed with PBS and fixed with 4% paraformaldehyde, nuclei were stained by DAPI (blue).
Fig. 4. Cytotoxicity of Man-PEG2,000 quantum dots or NH2-PEG2,000 quantum dots in primary cultured peritoneal macrophages for 24 h at 37 °C measured by MTT assay. Each value represents the mean + S.D. (n = 3).
NH2-PEG2,000 quantum dots at 2 h. The cellular uptake of Man-PEG2,000 quantum dots by peritoneal macrophage, which express large number of mannose receptor, was significantly inhibited in the presence of an excess amount of mannose (Fig. 2 (C)). Moreover, the cellular uptake of PEG2,000 quantum dots was not affected by mannose and galactose (data not shown). On the other hand, the cellular uptake of ManPEG2,000 quantum dots by HepG2 cells, which express asialoglycoprotein receptor but not mannose receptor, was extremely lower than that by peritoneal macrophage and was not inhibited by excess amount of mannose (Fig. 2 (C)) These results suggested that Man-PEG2,000 quantum dots were efficiently taken up by mannose receptor-mediated endocytosis into macrophages.
Fig. 5. Intracellular stability of Man-PEG2,000 quantum dots (red). After incubation with Man-PEG2,000 quantum dots for 2 h, cells were washed with PBS and cultured in RPMI culture medium for 0 (A), 24 (B), 48 (C) and 96 h (D). Then cells were fixed with 4% paraformaldehyde, and nuclei were stained by DAPI (blue).
Y. Higuchi et al. / Journal of Controlled Release 125 (2008) 131–136
Ryman-Rasmussen et al. evaluated the cytotoxicity of quantum dots using primary cultured epidermal keratinocytes, and demonstrated that bare quantum dots show cytotoxicity survival of 70% by MTT assay at 24 h, but PEG-modified quantum dots (NH2-PEG2,000 quantum dots) do not show cytotoxicity (100% survival) at their maximum concentration of 20 nM [23]. Therefore, quantum dots are also modified by PEG2000 to reduce cytotoxicity into macrophages for labeling. In primary cultured peritoneal macrophages, NH2-PEG2,000 quantum dots and Man-PEG2,000 quantum dots do not show cytotoxicity at 24 h (20 nM) (Fig. 4). These observations of PEGylated quantum dots in macrophages agreed with the previous report on keratinocytes [23]. These observations support the view that PEGylation is an effective approach to reduce the cytotoxicity of quantum dots. For the use Man-PEG2,000 quantum dots in tracking macrophage in vivo, it is important that quantum dots taken up by cells were stably incorporated in the cells for the period of analysis. We evaluated how long Man-PEG2,000 quantum dots were maintained in the macrophage. After 2 h incubation, ManPEG2,000 quantum dots determined in the cell for at least 96 h slightly (Fig. 5). Although the intensity of Man-PEG2,000 quantum dots was kept for 48 h (Fig. 5), their intensity decreased depending incubation time; therefore optimization of dose and incubation time would be necessary for tracking cells in vivo. Recently, imaging of the lymphatic system was examined, especially of the sentinel lymph nodes for dissection decisionmaking in melanoma using SPECT. Since mannose-binding protein highly accumulated in lymph nodes, Vera et al. developed several types of radio-labeled mannosylated macromolecules, including (DTPA)-mannoyslated polylysine, mercaptoacetylglycylglycylglycine and (MAG3)-mannosyl-dextran [26] etc. Although SPECT diagnosis has high sensitivity, nonradioisotope diagnosis is currently suggested due to the difficulty of treatment and exposure to radiation. In this study, quantum dots were about 15 nm. Henze et al. reported that more than 5 nm particles are more selectively distributed to lymph nodes [27]. Therefore, Man-PEG2,000 quantum dots might be a candidate for a new lymph node-detecting agent in the future. In summary, we developed Man-PEG2,000 quantum dots and demonstrated that they were efficiently taken up by primary cultured peritoneal macrophages though mannose receptormediated endocytosis. Moreover, the cytotoxicity of quantum dots was extremely low by PEG2,000 modification. For the possibility of labeling macrophages in vivo, additional studies are in progress. Acknowledgement This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, and by Health and Labour Sciences Research Grants for Research on Advanced Medical Technology from the Ministry of Health, Labour and Welfare of Japan, and by the Radioisotope Research Center of Kyoto University. We acknowledge the technical support of
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Sumiko Araki of Department of Physics, Graduate School of Science, Kyoto University, Kyoto, Japan in performing the atomic force microscopic image. References [1] X. Michalet, F.F. Pinaud, L.A. Bentolila, J.M. Tsay, S. Doose, J.J. Li, G. Sundaresan, A.M. Wu, S.S. Gambhir, S. Weiss, Quantum dots for live cells, in vivo imaging, and diagnostics, Science 307 (5709) (2005) 538–544. [2] Y.P. Ho, H.H. Chen, K.W. Leong, T.H. Wang, Evaluating the intracellular stability and unpacking of DNA nanocomplexes by quantum dots-FRET, J. Control. Release 116 (1) (2006) 83–89. [3] D.J. Bharali, D.W. Lucey, H. Jayakumar, H.E. Pudavar, P.N. Prasad, Folate-receptor-mediated delivery of InP quantum dots for bioimaging using confocal and two-photon microscopy, J. Am. Chem. Soc. 127 (32) (2005) 11364–11371. [4] D.S. Lidke, P. Nagy, R. Heintzmann, D.J. Arndt-Jovin, J.N. Post, H.E. Grecco, E.A. Jares-Erijman, T.M. Jovin, Quantum dot ligands provide new insights into erbB/HER receptor-mediated signal transduction, Nat. Biotechnol. 22 (2) (2004) 198–203. [5] M.E. Akerman, W.C. Chan, P. Laakkonen, S.N. Bhatia, E. Ruoslahti, Nanocrystal targeting in vivo, Proc. Natl. Acad. Sci. U. S. A. 99 (20) (2002) 12617–12621. [6] M. Howarth, K. Takao, Y. Hayashi, A.Y. Ting, Targeting quantum dots to surface proteins in living cells with biotin ligase, Proc. Natl. Acad. Sci. U. S. A. 102 (21) (2005) 7583–7588. [7] W. Cai, D.W. Shin, K. Chen, O. Gheysens, Q. Cao, S.X. Wang, S.S. Gambhir, X. Chen, Peptide-labeled near-infrared quantum dots for imaging tumor vasculature in living subjects, Nano Lett. 6 (4) (2006) 669–676. [8] X. Gao, Y. Cui, R.M. Levenson, L.W. Chung, S. Nie, In vivo cancer targeting and imaging with semiconductor quantum dots, Nat. Biotechnol. 22 (8) (2004) 969–976. [9] H. Tada, H. Higuchi, T.M. Wanatabe, N. Ohuchi, In vivo real-time tracking of single quantum dots conjugated with monocle onal anti-HER2 antibody in tumors of mice, Cancer Res. 67 (3) (2007) 1138–1144. [10] E.B. Voura, J.K. Jaiswal, H. Mattoussi, S.M. Simon, Tracking metastatic tumor cell extravasation with quantum dot nanocrystals and fluorescence emission-scanning microscopy, Nat. Med. 10 (9) (2004) 993–998. [11] C. Sunderkotter, K. Steinbrink, M. Goebeler, R. Bhardwaj, C. Sorg, Macrophages and angiogenesis, J. Leukoc. Biol. 55 (3) (1994) 410–422. [12] Y. Higuchi, S. Kawakami, F. Yamashita, M. Hashida, The potential role of fucosylated cationic liposome/NFkappaB decoy complexes in the treatment of cytokine-related liver disease, Biomaterials 28 (3) (2007) 532–539. [13] Y.L. Wu, Q. Ye, L.M. Foley, T.K. Hitchens, K. Sato, J.B. Williams, C. Ho, In situ labeling of immune cells with iron oxide particles: an approach to detect organ rejection by cellular MRI, Proc. Natl. Acad. Sci. U. S. A. 103 (6) (2006) 1852–1857. [14] J. Kuiper, A. Brouwer, D.L. Knook, T.J.C. van Berkel, Kupffer and sinusoidal endothelial cells, in: I.M. Arias, J.L. Boyer, N. Fausto, W.B. Jakoby, D.A. Schachter, D.A. Shafritz (Eds.), The Liver: Biology and Pathobiology, third ed., Raven Press, New York, 1994, pp. 791–818. [15] K. Shigeta, S. Kawakami, Y. Higuchi, T. Okuda, H. Yagi, F. Yamashita, M. Hashida, Novel histidine-conjugated galactosylated cationic liposomes for efficient hepatocyte-selective gene transfer in human hepatoma HepG2 cells, J. Control. Release 118 (2) (2007) 262–270. [16] M. Nishikawa, Y. Ohtsubo, J. Ohno, T. Fujita, Y. Koyama, F. Yamashita, M. Hashida, H. Sezaki, Pharmacokinetics of receptor-mediated hepatic uptake of glycosylated albumin in mice, Int. J. Pharm. 85 (1–3) (1992) 75–85. [17] P. Opanasopit, K. Shirashi, M. Nishikawa, F. Yamashita, Y. Takakura, M. Hashida, In vivo recognition of mannosylated proteins by hepatic mannose receptors and mannan-binding protein, Am. J. Physiol. Gastrointest.: Liver Physiol. 280 (5) (2001) G879–G889. [18] S. Kawakami, J. Wong, A. Sato, Y. Hattori, F. Yamashita, M. Hashida, Biodistribution characteristics of mannosylated, fucosylated, and
136
[19]
[20]
[21] [22]
[23]
Y. Higuchi et al. / Journal of Controlled Release 125 (2008) 131–136 galactosylated liposomes in mice, Biochim. Biophys. Acta 1524 (2–3) (2000) 258–265. W. Yeeprae, S. Kawakami, Y. Higuchi, F. Yamashita, M. Hashida, Biodistribution characteristics of mannosylated and fucosylated O/W emulsions in mice, J. Drug Target. 13 (8–9) (2005) 479–487. M. Nishikawa, A. Kamijo, T. Fujita, Y. Takakura, H. Sezaki, M. Hashida, Synthesis and pharmacokinetics of a new liver-specific carrier, glycosylated carboxymethyl-dextran, and its application to drug targeting, Pharm. Res. 10 (9) (1993) 1253–1261. P. Babu, S. Sinha, A. Surolia, Sugar-quantum dot conjugates for a selective and sensitive detection of lectins, Bioconjug. Chem. 18 (1) (2007) 146–151. A. Robinson, J.M. Fang, P.T. Chou, K.W. Liao, R.M. Chu, S.J. Lee, Probing lectin and sperm with carbohydrate-modified quantum dots, Chem. biochem. 6 (10) (2005) 1899–1905. J.P. Ryman-Rasmussen, J.E. Riviere, N.A. Monteiro-Riviere, Surface coatings determine cytotoxicity and irritation potential of quantum dot
[24]
[25]
[26]
[27]
nanoparticles in epidermal keratinocytes, J. Invest. Dermatol. 127 (1) (2007) 143–153. Y.C. Lee, C.P. Stowell, M.J. Krantz, 2-Imino-2-methoxyethyl 1-thioglycosides: new reagents for attaching sugars to proteins, Biochemistry 15 (18) (1976) 3956–3963. W. Yeeprae, S. Kawakami, F. Yamashita, M. Hashida, Effect of mannose density on mannose receptor-mediated cellular uptake of mannosylated O/W emulsions by macrophages, J. Control. Release 114 (2) (2006) 193–201. H. Schöder, E.C. Glass, A.P. Pecking, J.K. Harness, A.M. Wallace, P. Hirnle, J.L. Alberini, D. Vilain, S.M. Larson, C.K. Hoh, D.R. Vera, Molecular targeting of the lymphovascular system for imaging and therapy, Cancer Metastasis Rev. 25 (2) (2006) 185–201. E. Henze, H.R. Schelbert, J.D. Collins, A. Najafi, J.R. Barrio, L.R. Bennett, Lymphoscintigraphy with Tc-99 m-labeled dextran, J. Nucl. Med. 23 (10) (1982) 923–929.
Journal of Controlled Release 125 (2008) 131 – 136 www.elsevier.com/locate/jconrel
Note
Mannosylated semiconductor quantum dots for the labeling of macrophages Yuriko Higuchi, Machiko Oka, Shigeru Kawakami, Mitsuru Hashida ⁎ Department of Drug Delivery Research, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto, 606-8501, Japan Received 18 April 2007; accepted 3 October 2007 Available online 17 October 2007
Abstract Quantum dots show strong fluorescence emission and long stability compared with classical organic fluorescent dyes; therefore, quantum dots take the place of other dyes in Western blot, immunostaining and bioimaging. Since macrophage plays crucial roles in many pathophysiological processes, tracking macrophage migration, homing and fate is important for understanding the complex roles of macrophages in disease or developing disease diagnosis. Because of the high expression of mannose receptor on macrophage, mannosylation is an attractive strategy to label macrophage. In this study, using polyethylene-glycol (PEG) (M.W. 2,000; PEG2,000)-coated quantum dots, we prepared mannosylated PEG2,000 (Man-PEG2,000) quantum dots for labeling macrophage. The uptake characteristics of Man-PEG2,000 quantum dots were investigated by primary cultured peritoneal macrophages. The uptake of Man-PEG2,000 quantum dots was inhibited by an excess amount of mannose, suggesting mannose receptor-mediated uptake of Man-PEG2,000 quantum dots. The result of MTT assay suggested the extremely low cytotoxicity of Man-PEG2,000 quantum dots. In conclusion, the Man-PEG2,000 synthesized is safe and is taken up by macrophage mannose receptor recognition. © 2007 Elsevier B.V. All rights reserved. Keywords: Quantum dot; Imaging; Mannosylation; Macrophage; Receptor-mediated endocytosis
1. Introduction Quantum dots are semiconductor nanocrystals, which display very interesting optical properties such as single wavelength excitation, size-dependant narrow emission, high intensity of fluorescence, and low photobleaching. Because of strong fluorescence emission and the long stability of quantum dots compared with classical organic fluorescent dyes, quantum dots are taking the place of other dyes in Western blot, immunostaining and bioimaging [1,2]. To date, several bioconjugate types of quantum dots have been developed for living cell imaging, including folate [3] EGF [4] and biotin [5]. In vivo imaging also succeeded in preparing quantum dots labeled with peptide for lung [6] or tumor [7] and antibody for tumor targeting [8,9]. As for cell tracking, Voura et al. [10] prepared quantum dot-labeled tumor cells, which enable us to follow tumor cell extravasation in a living mouse. Therefore, quantum
⁎ Corresponding author. Tel.: +81 75 753 4525; fax: +81 75 753 4575. E-mail address: [email protected] (M. Hashida). 0168-3659/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.jconrel.2007.10.007
dots are attractive candidates for diagnosis by in vivo imaging without invasive operation. Macrophage plays crucial roles in many pathophysiological processes, including inflammation, cancer, organ rejection, autoimmune disease etc; therefore, tracking macrophage migration, homing and fate is important for understanding the complex roles of macrophages in disease or developing the disease diagnosis [11,12]. Recently, Wu et al. [13] demonstrated that labeling macrophage with μm-sized paramagnetic iron oxide in situ could detect acute cardiac rejection in rats using magnetic resonance imaging (MRI). However, the high magnetic field limits the application of MRI for patients who have metal in the body such as an artificial pacemaker and cochlear implant. Moreover, to create a high magnetic field large-scale expensive machine is necessary. Therefore, it is necessary to develop a simpler and more specific method for cell tracking of macrophages. Receptors for carbohydrates, such as asialoglycoprotein receptors on hepatocytes and mannose receptors on several macrophages and liver endothelial cells, recognize the corresponding sugars on the nonreducing terminal of sugar chains
132
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[14,15]. Therefore, sugar modification is an attractive tool for cellular targeted delivery of drugs and genes. We previously reported mannosylated protein [16,17], lipid particles [18,19] and macromolecules [20], and demonstrated that they could be delivered to macrophages by mannose receptor-mediated endocytosis in vivo. Therefore, mannosylation of quantum dots would be useful for macrophage detection in vivo. To date, mannosylated quantum dots were synthesized using the direct physical adhesion of sulfur atom to the colloidal core of quantum dots [21,22]. However, the cytotoxicity of the colloidal core in quantum dots complicates an appropriate coating for the application of quantum dots. Recently, RymanRasmussen et al. [23] revealed that quantum dots with NH2polyethylene-glycol (PEG) (M.W. 2,000; PEG2,000) significantly inhibited cytotoxicity and immune responses compared with quantum dots without NH2-PEG2,000 in cultured keratinocytes, suggesting that PEG coating is an effective approach for its safe use. For the application of mannosylated quantum dots for the detection of macrophages, it is also necessary to investigate the uptake characteristics of mannosylated quantum dots to cultured macrophages. In this study, mannosylated-PEG2,000 quantum dots (Man-PEG2,000 quantum dots) are synthesized for the labeling of macrophages (Fig. 1(A)). Then, the uptake characteristics of Man-PEG2,000 quantum dots were investigated in primary cultured peritoneal macrophages, which express mannose receptors. The results were compared with NH2PEG2,000 quantum dots as a control.
2. Materials and methods 2.1. Materials Qdot® 655 ITK™ amino (PEG2,000) quantum dots, OptiMEM® I and fetal bovine serum (FBS) were obtained from Invitrogen Co. (Carlsbad, CA, USA). An ULTRAFREE®-MC 10,000 NMWL filter unit was purchased from Millipore Corp. (Billerica, MA, USA). D(+)-Mannose was purchased from Wako Pure Chemical Industries, Ltd (Osaka, Japan). L-glutamine, penicillin, and streptomycin were purchased from Sigma Chemical Co., Inc. (St. Louis, MO, USA). DABCO (1,4-diazabicyclo-[2,2,2]octane) was purchased from Sigma Chemical Co., Inc. (St. Louis, MO, USA). DAPI (4',6Diamidine-2'-phenylindole dihydrochloride) was purchased from Roche Diagnostics (Indianapolis, IN, USA). RPMI 1640 medium was obtained from Nissui Pharmaceutical Co. (Tokyo, Japan). All other chemicals were of the highest purity available. 2.2. Animals Five-week-old female ICR mice (20–23 g) were purchased from the Shizuoka Agricultural Cooperative Association for Laboratory Animals (Shizuoka, Japan). All animal experiments were carried out in accordance with the Principles of Laboratory Animal Care as adopted and promulgated by the US National Institutes of Health and the Guidelines for Animal Experiments of Kyoto University. 2.3. Synthesis of Man-PEG2,000 quantum dots 2-imino-2-methoxyethyl-1-thiomannoside (IME-thiomannoside) was prepared as reported previously [24]. IMEthiomannoside (3.5 mg) was reacted with 0.5 ml 0.01 M sodium methoxide methanolic solution at room temperature for 2 h. The solvent was evaporated by centrifuging in vacuo overnight at room temperature. Fifty microliters of quantum dots was added to activate IME-thiomannoside. After vortexing for few minutes, the solution was left overnight at room temperature. The solution was filtrated with URTRAFREE filter (10,000 NMWL) by centrifuging at 6400 g for 10 min. PBS (50 μl) was added, and centrifuged again. Finally, 50 μl of PBS was added and Man-PEG2,000 quantum dots were collected. 2.4. Atomic force microscopic image
Fig. 1. Structure of NH2-PEG2,000 quantum dots and Man-PEG2,000 quantum dots (A), the atomic force microscopic image of Man-PEG2,000 quantum dots (B) and fluorescent intensity of Man-PEG2,000 quantum dots (closed circle) or NH2-PEG2,000 quantum dots (open circle) (C).
The structure of Man-PEG2,000 quantum dots was observed with a fluorescence-microscope-coupled atomic force microscope (NVB100, Olympus, Japan). Newly cleaved thin mica (thickness: ca. 30–50 μm) was closely stuck to a cover glass plate (Matsunami Glass, No.1, Japan). The sample droplet on the mica was removed by nitrogen-blowing, and the mica was washed with Millipore water, and dried again by nitrogenblowing. The mica surface was monitored by atomic force microscopy.
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2.5. Culture of peritoneal macrophages Mouse-elicited peritoneal macrophages were cultured according to our previous report [25]. Briefly, peritoneal macrophages were obtained 4 days after intraperitoneal injection by 1 ml of 3% thioglycolate medium. Collected macrophages were plated on 24-well or 12-well cluster dishes at a density of 1.3 × 105 cells/cm2, respectively. Cells were cultured for 72 h with RPMI 1640 medium supplemented with 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin G, and 0.1 mg/ml streptomycin. Cells were maintained at 37 °C with 5% CO2 in a humidified incubator.
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Japan). The objective specifications were 40 × oil immersion and numerical aperture 1.0. To determine the stability, after 2 h incubation with 20 nM of Man-PEG2,000 quantum dots, cells were washed with PBS and cultured for indicated time with culture medium RPMI1640. 2.9. Statistical analysis Statistical analysis was performed using Student's paired t-test for two groups. P b 0.05 was considered to be indicative of statistical significance. 3. Results and discussion
2.6. Uptake experiment Primary peritoneal macrophages or HepG2 cells were plated on a 12-well cluster dish at a density of 1.3 × 105 cells/cm2 and cultivated in RPMI or DMEM culture medium. The culture medium was replaced with Opti-MEM I containing 20 nM NH2PEG2,000 quantum dots or Man-PEG2,000 quantum dots and cultured for the indicated time. After cells were washed twice with cold PBS, the incubation medium was replaced with 200 μl of PBS, and then cells were collected by a cell scraper. Cell intensity was counted by flow cytometry (Becton Dickinson Co., Inc., Franklin Lakes, NJ, USA) on the FL3-channel.
Previously we synthesized mannosylated protein using 2imino-2-methoxyethyl-1-thiomannoside [16,17]. Moreover,
2.7. Cytotoxicity of Man-PEG2,000 quantum dots The cytotoxicity of Man-PEG2,000 quantum dots was determined by MTT assay. Cells were plated on a 24-well cluster dish at a density of 1.3 × 105 cells/cm2 and cultivated in RPMI culture medium. Seventy-two hours later, the culture medium was replaced with Opti-MEM® I containing ManPEG2,000 quantum dots at the indicated concentration. After 24 h, cells were washed twice with cold PBS and added to 150 μl of 0.5 mg/ml MTT solution. Cells were incubated for 4 h at 37 °C in 5% CO2, a 10% SDS solution was added, and incubation continued overnight. Absorbance was measured at test and reference wavelengths of 570 and 660 nm, respectively, in a two-wavelength microplate photometer (Bio-Rad Model 550, Hercules, CA, USA). 2.8. Fluorescence microscopy study Isolated macrophages were cultured at a density of 1.3 × 105 cells/cm2 on a glass-bottom 12-well plate. After 72 h cultivation, the cells were incubated at 37 °C with 20 nM of Man-PEG2,000 quantum dots or NH2-PEG2,000 quantum. After 1 h incubation, the cells were washed five times with ice-cold PBS, then fixed with 4% paraformaldehyde and 0.01% glutaraldehyde in PBS (+) for 15 min on ice and washed 3 times with ice-cold PBS. For nuclear staining, fixed cells were incubated with 10 μg/ml DAPI at room temperature for 1 min. After washing twice with 1 ml icecold PBS, cover glasses were mounted on slide glasses with 50% glycerol–2.5% DABCO (1,4-diazabicyclo-[2,2,2]octane) (Sigma Chemical Co., Inc., St. Louis, MO, USA) in PBS. The samples were examined by Biozero microscopy (Keyence Corp., Osaka,
Fig. 2. Cellular association of Man-PEG2,000 quantum dots (closed circle) or NH2-PEG2,000 quantum dots (open circle) at 0.2, 2.0, 20 nM for 2 h (A) or at 20 nM for 0.5, 1.0 and 2.0 h (B) in primary cultured peritoneal macrophages at 37 °C. Cellular association of Man-PEG2,000 quantum dots (20 nM) with or without an excess amount of mannose (40 mM), or NH2-PEG2,000 quantum dots (20 nM) for 2 h in primary cultured peritoneal macrophages (■) or HepG2 cells (□) at 37 °C (C). Each value represents the mean ± S.D. or +S.D. (n = 3). There are statistically significant differences (⁎P b 0.05, ⁎⁎P b 0.01, ⁎⁎⁎P b 0.001 v.s. NH2-PEG2,000 quantum dots; ###P b 0.001 v.s. Man-PEG2,000 quantum dots with mannose.).
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we synthesized mannosylated lipid with 2-imino-2-methoxyethyl-1-thiomannoside, and prepared mannosylated liposome [18] and emulsion [19,25]. These mannosylated materials were taken up by macrophages through mannose receptor recognition [16–20]. In this study, we prepared Man-PEG2,000 quantum dots using 2-imino-2-methoxyethyl-1-thiomannoside, which could bind with amino residue; therefore, we selected PEG2,000 quantum dots. Synthesis of 2-imino-2methoxyethyl-1-thiomannoside and PEG2,000 quantum dots is one step in the water phase. Man-PEG2,000 quantum dots were not aggregated in water (Fig. 1(B)). The particle size of Man-PEG2,000 quantum dots was about 20–30 μm (Fig. 1 (B)). The fluorescence intensity of Man-PEG2,000 quantum dots was almost the same as that of quantum dots (Fig. 1(C)), suggesting that Man-PEG2,000 quantum dots maintain high intensity with bare quantum dots. In primary cultured peritoneal macrophages, the cellular uptake of Man-PEG2,000 quantum dots was enhanced depending the increase of concentration (Fig. 2 (A)) and incubation time (Fig. 2 (B)). FACS analysis (Fig. 2) and microscopic image (Fig. 3) revealed that the uptake of Man-PEG2,000 quantum dots by macrophages was 3 times higher than that of
Fig. 3. Intracellular distribution of NH2-PEG2,000 quantum dots (red) (A), or Man-PEG2,000 quantum dots (red) (B). After cells were washed with PBS and fixed with 4% paraformaldehyde, nuclei were stained by DAPI (blue).
Fig. 4. Cytotoxicity of Man-PEG2,000 quantum dots or NH2-PEG2,000 quantum dots in primary cultured peritoneal macrophages for 24 h at 37 °C measured by MTT assay. Each value represents the mean + S.D. (n = 3).
NH2-PEG2,000 quantum dots at 2 h. The cellular uptake of Man-PEG2,000 quantum dots by peritoneal macrophage, which express large number of mannose receptor, was significantly inhibited in the presence of an excess amount of mannose (Fig. 2 (C)). Moreover, the cellular uptake of PEG2,000 quantum dots was not affected by mannose and galactose (data not shown). On the other hand, the cellular uptake of ManPEG2,000 quantum dots by HepG2 cells, which express asialoglycoprotein receptor but not mannose receptor, was extremely lower than that by peritoneal macrophage and was not inhibited by excess amount of mannose (Fig. 2 (C)) These results suggested that Man-PEG2,000 quantum dots were efficiently taken up by mannose receptor-mediated endocytosis into macrophages.
Fig. 5. Intracellular stability of Man-PEG2,000 quantum dots (red). After incubation with Man-PEG2,000 quantum dots for 2 h, cells were washed with PBS and cultured in RPMI culture medium for 0 (A), 24 (B), 48 (C) and 96 h (D). Then cells were fixed with 4% paraformaldehyde, and nuclei were stained by DAPI (blue).
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Ryman-Rasmussen et al. evaluated the cytotoxicity of quantum dots using primary cultured epidermal keratinocytes, and demonstrated that bare quantum dots show cytotoxicity survival of 70% by MTT assay at 24 h, but PEG-modified quantum dots (NH2-PEG2,000 quantum dots) do not show cytotoxicity (100% survival) at their maximum concentration of 20 nM [23]. Therefore, quantum dots are also modified by PEG2000 to reduce cytotoxicity into macrophages for labeling. In primary cultured peritoneal macrophages, NH2-PEG2,000 quantum dots and Man-PEG2,000 quantum dots do not show cytotoxicity at 24 h (20 nM) (Fig. 4). These observations of PEGylated quantum dots in macrophages agreed with the previous report on keratinocytes [23]. These observations support the view that PEGylation is an effective approach to reduce the cytotoxicity of quantum dots. For the use Man-PEG2,000 quantum dots in tracking macrophage in vivo, it is important that quantum dots taken up by cells were stably incorporated in the cells for the period of analysis. We evaluated how long Man-PEG2,000 quantum dots were maintained in the macrophage. After 2 h incubation, ManPEG2,000 quantum dots determined in the cell for at least 96 h slightly (Fig. 5). Although the intensity of Man-PEG2,000 quantum dots was kept for 48 h (Fig. 5), their intensity decreased depending incubation time; therefore optimization of dose and incubation time would be necessary for tracking cells in vivo. Recently, imaging of the lymphatic system was examined, especially of the sentinel lymph nodes for dissection decisionmaking in melanoma using SPECT. Since mannose-binding protein highly accumulated in lymph nodes, Vera et al. developed several types of radio-labeled mannosylated macromolecules, including (DTPA)-mannoyslated polylysine, mercaptoacetylglycylglycylglycine and (MAG3)-mannosyl-dextran [26] etc. Although SPECT diagnosis has high sensitivity, nonradioisotope diagnosis is currently suggested due to the difficulty of treatment and exposure to radiation. In this study, quantum dots were about 15 nm. Henze et al. reported that more than 5 nm particles are more selectively distributed to lymph nodes [27]. Therefore, Man-PEG2,000 quantum dots might be a candidate for a new lymph node-detecting agent in the future. In summary, we developed Man-PEG2,000 quantum dots and demonstrated that they were efficiently taken up by primary cultured peritoneal macrophages though mannose receptormediated endocytosis. Moreover, the cytotoxicity of quantum dots was extremely low by PEG2,000 modification. For the possibility of labeling macrophages in vivo, additional studies are in progress. Acknowledgement This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, and by Health and Labour Sciences Research Grants for Research on Advanced Medical Technology from the Ministry of Health, Labour and Welfare of Japan, and by the Radioisotope Research Center of Kyoto University. We acknowledge the technical support of
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Sumiko Araki of Department of Physics, Graduate School of Science, Kyoto University, Kyoto, Japan in performing the atomic force microscopic image. References [1] X. Michalet, F.F. Pinaud, L.A. Bentolila, J.M. Tsay, S. Doose, J.J. Li, G. Sundaresan, A.M. Wu, S.S. Gambhir, S. Weiss, Quantum dots for live cells, in vivo imaging, and diagnostics, Science 307 (5709) (2005) 538–544. [2] Y.P. Ho, H.H. Chen, K.W. Leong, T.H. Wang, Evaluating the intracellular stability and unpacking of DNA nanocomplexes by quantum dots-FRET, J. Control. Release 116 (1) (2006) 83–89. [3] D.J. Bharali, D.W. Lucey, H. Jayakumar, H.E. Pudavar, P.N. Prasad, Folate-receptor-mediated delivery of InP quantum dots for bioimaging using confocal and two-photon microscopy, J. Am. Chem. Soc. 127 (32) (2005) 11364–11371. [4] D.S. Lidke, P. Nagy, R. Heintzmann, D.J. Arndt-Jovin, J.N. Post, H.E. Grecco, E.A. Jares-Erijman, T.M. Jovin, Quantum dot ligands provide new insights into erbB/HER receptor-mediated signal transduction, Nat. Biotechnol. 22 (2) (2004) 198–203. [5] M.E. Akerman, W.C. Chan, P. Laakkonen, S.N. Bhatia, E. Ruoslahti, Nanocrystal targeting in vivo, Proc. Natl. Acad. Sci. U. S. A. 99 (20) (2002) 12617–12621. [6] M. Howarth, K. Takao, Y. Hayashi, A.Y. Ting, Targeting quantum dots to surface proteins in living cells with biotin ligase, Proc. Natl. Acad. Sci. U. S. A. 102 (21) (2005) 7583–7588. [7] W. Cai, D.W. Shin, K. Chen, O. Gheysens, Q. Cao, S.X. Wang, S.S. Gambhir, X. Chen, Peptide-labeled near-infrared quantum dots for imaging tumor vasculature in living subjects, Nano Lett. 6 (4) (2006) 669–676. [8] X. Gao, Y. Cui, R.M. Levenson, L.W. Chung, S. Nie, In vivo cancer targeting and imaging with semiconductor quantum dots, Nat. Biotechnol. 22 (8) (2004) 969–976. [9] H. Tada, H. Higuchi, T.M. Wanatabe, N. Ohuchi, In vivo real-time tracking of single quantum dots conjugated with monocle onal anti-HER2 antibody in tumors of mice, Cancer Res. 67 (3) (2007) 1138–1144. [10] E.B. Voura, J.K. Jaiswal, H. Mattoussi, S.M. Simon, Tracking metastatic tumor cell extravasation with quantum dot nanocrystals and fluorescence emission-scanning microscopy, Nat. Med. 10 (9) (2004) 993–998. [11] C. Sunderkotter, K. Steinbrink, M. Goebeler, R. Bhardwaj, C. Sorg, Macrophages and angiogenesis, J. Leukoc. Biol. 55 (3) (1994) 410–422. [12] Y. Higuchi, S. Kawakami, F. Yamashita, M. Hashida, The potential role of fucosylated cationic liposome/NFkappaB decoy complexes in the treatment of cytokine-related liver disease, Biomaterials 28 (3) (2007) 532–539. [13] Y.L. Wu, Q. Ye, L.M. Foley, T.K. Hitchens, K. Sato, J.B. Williams, C. Ho, In situ labeling of immune cells with iron oxide particles: an approach to detect organ rejection by cellular MRI, Proc. Natl. Acad. Sci. U. S. A. 103 (6) (2006) 1852–1857. [14] J. Kuiper, A. Brouwer, D.L. Knook, T.J.C. van Berkel, Kupffer and sinusoidal endothelial cells, in: I.M. Arias, J.L. Boyer, N. Fausto, W.B. Jakoby, D.A. Schachter, D.A. Shafritz (Eds.), The Liver: Biology and Pathobiology, third ed., Raven Press, New York, 1994, pp. 791–818. [15] K. Shigeta, S. Kawakami, Y. Higuchi, T. Okuda, H. Yagi, F. Yamashita, M. Hashida, Novel histidine-conjugated galactosylated cationic liposomes for efficient hepatocyte-selective gene transfer in human hepatoma HepG2 cells, J. Control. Release 118 (2) (2007) 262–270. [16] M. Nishikawa, Y. Ohtsubo, J. Ohno, T. Fujita, Y. Koyama, F. Yamashita, M. Hashida, H. Sezaki, Pharmacokinetics of receptor-mediated hepatic uptake of glycosylated albumin in mice, Int. J. Pharm. 85 (1–3) (1992) 75–85. [17] P. Opanasopit, K. Shirashi, M. Nishikawa, F. Yamashita, Y. Takakura, M. Hashida, In vivo recognition of mannosylated proteins by hepatic mannose receptors and mannan-binding protein, Am. J. Physiol. Gastrointest.: Liver Physiol. 280 (5) (2001) G879–G889. [18] S. Kawakami, J. Wong, A. Sato, Y. Hattori, F. Yamashita, M. Hashida, Biodistribution characteristics of mannosylated, fucosylated, and
136
[19]
[20]
[21] [22]
[23]
Y. Higuchi et al. / Journal of Controlled Release 125 (2008) 131–136 galactosylated liposomes in mice, Biochim. Biophys. Acta 1524 (2–3) (2000) 258–265. W. Yeeprae, S. Kawakami, Y. Higuchi, F. Yamashita, M. Hashida, Biodistribution characteristics of mannosylated and fucosylated O/W emulsions in mice, J. Drug Target. 13 (8–9) (2005) 479–487. M. Nishikawa, A. Kamijo, T. Fujita, Y. Takakura, H. Sezaki, M. Hashida, Synthesis and pharmacokinetics of a new liver-specific carrier, glycosylated carboxymethyl-dextran, and its application to drug targeting, Pharm. Res. 10 (9) (1993) 1253–1261. P. Babu, S. Sinha, A. Surolia, Sugar-quantum dot conjugates for a selective and sensitive detection of lectins, Bioconjug. Chem. 18 (1) (2007) 146–151. A. Robinson, J.M. Fang, P.T. Chou, K.W. Liao, R.M. Chu, S.J. Lee, Probing lectin and sperm with carbohydrate-modified quantum dots, Chem. biochem. 6 (10) (2005) 1899–1905. J.P. Ryman-Rasmussen, J.E. Riviere, N.A. Monteiro-Riviere, Surface coatings determine cytotoxicity and irritation potential of quantum dot
[24]
[25]
[26]
[27]
nanoparticles in epidermal keratinocytes, J. Invest. Dermatol. 127 (1) (2007) 143–153. Y.C. Lee, C.P. Stowell, M.J. Krantz, 2-Imino-2-methoxyethyl 1-thioglycosides: new reagents for attaching sugars to proteins, Biochemistry 15 (18) (1976) 3956–3963. W. Yeeprae, S. Kawakami, F. Yamashita, M. Hashida, Effect of mannose density on mannose receptor-mediated cellular uptake of mannosylated O/W emulsions by macrophages, J. Control. Release 114 (2) (2006) 193–201. H. Schöder, E.C. Glass, A.P. Pecking, J.K. Harness, A.M. Wallace, P. Hirnle, J.L. Alberini, D. Vilain, S.M. Larson, C.K. Hoh, D.R. Vera, Molecular targeting of the lymphovascular system for imaging and therapy, Cancer Metastasis Rev. 25 (2) (2006) 185–201. E. Henze, H.R. Schelbert, J.D. Collins, A. Najafi, J.R. Barrio, L.R. Bennett, Lymphoscintigraphy with Tc-99 m-labeled dextran, J. Nucl. Med. 23 (10) (1982) 923–929.