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Bioconjugate Chem. 2005, 16, 494−496
Transferrin-Mediated Gold Nanoparticle Cellular Uptake Pei-Hui Yang,†,§ Xuesong Sun,† Jen-Fu Chiu,‡ Hongzhe Sun,*,† and Qing-Yu He*,† Department of Chemistry and Open Laboratory of Chemical Biology, and Institute of Molecular Biology, University of Hong Kong, Hong Kong . Received September 18, 2004; Revised Manuscript Received February 10, 2005
Targeted drug delivery is an important research area in specific therapy. Transferrin-conjugated nanoparticles are an attractive formulation as a vehicle for specific cellular uptake and targeted drug delivery. In this report, atomic force microscopy imaging was used to visualize the process of cellular uptake of transferrin-coupled gold nanoparticles on the surfaces of live cells for the first time. Highresolution images were captured, showing the endocytosis of transferrin-conjugated nanoparticles taking place during the process of internalization. This specific transferrin-mediated nanoparticle uptake was validated by confocal scanning imaging and transferrin competition experiments.
Targeted entry into cells is an important area of research in drug delivery and therapy (1-4). Site-specific delivery of drugs and therapeutics can significantly reduce drug toxicity and increase therapeutic effects. To realize efficient and specific cellular drug delivery, one strategy is to modify or conjugate drug with a ligand that can be efficiently taken up by target cells via receptormediated endocytosis (5). Transferrin (TF)1 is such a suitable “ligand” for conjugating drugs, since it can be specifically recognized and taken up by TF receptors actively expressed on the surface of various tumor cells (6). The transferrin-receptor interaction has therefore been exploited as a potential efficient pathway for the cellular uptake of drugs and genes (7-9). Nanoparticles have also been considered as effective delivery vehicles and extensively studied for delivering drugs, genes, diagnostics, and vaccines into cells of interest (10-12). Nanoparticles cross-linking with proteins have demonstrated better stability and efficiency for cellular uptake. Nanoparticle-TF conjugation is thus an attractive formulation for specific cellular uptake and targeted drug delivery. Atomic force microscopy (AFM) is a surface analytical technique that can generate nanoscale topography by scanning a fine silicon tip across the surface. Because it can perform high-resolution imaging under physiological conditions in a nondestructive manner, AFM has developed into a powerful tool for studying the structural details of biological systems (13, 14). While the use of * Correspondence to Dr. Qing-Yu He, Department of Chemistry, University of Hong Kong, Pokfulam, Hong Kong, China. Tel: (852)2299-0787, Fax: (852)2817-1006, E-mail:
[email protected]. † Department of Chemistry and Open Laboratory of Chemical Biology. ‡ Institute of Molecular Biology. § A visiting scholar from the Department of Chemistry, Jinan University, Guangzhou, China, and registered Ph.D. student in the Anhui Institute of Optics and Fine Mechanics, Chinese Academy of Sciences. 1 Abbreviations: TF, transferrin; Au-TF, transferrin-conjugated gold nanoparticles; holo-TF, natural full-length transferrin; AFM, atomic force microscopy; NPC, nasopharyngeal carcinoma cells; ICP-MS, inductive coupled plasma mass spectroscopy.
AFM to characterize the morphology of nanoparticles and biophysical properties of cells has previously been reported, the imaging of the nanoparticle uptake into cells is a rarely studied area. In this report we used AFM to visualize the process of cellular uptake of TF-conjugated gold nanoparticles on the surfaces of live cells for the first time. Gold nanoparticles were synthesized according to a modified literature method (15). These nanoparticles were characterized by transmission electron micrography and powder X-ray diffraction. They were then covalently coupled to TF molecules by mercaptoacetic acid (16). Measurements with dynamic laser light scattering showed that these nanoparticles have sizes of 20.2 ( 0.6 nm for Au alone and 96.7 ( 2.0 for TF-derivatized Au-TF particles, indicating that multiple coupling of Au to TF or aggregation for some of the Au-TF particles occurred. When the Au-TF nanoparticles were incubated with human nasopharyngeal carcinoma cells (NPC, SUNE1) (17) under physiologically relevant conditions, a phenomenon of nanoparticle internalization into cells was visualized by a multimode AFM with a Nanoscopy IIIa controller (Veeco/Digital Instruments, Santa Barbara, CA) (Figure 1). The AFM image captured after 5-h incubation shows that cellular uptake was partially proceeding, leading to a “bumpy” surface (Figure 1C). The process of the nanoparticle uptake by NPC cells can be further observed by AFM imaging in a “zoom in” scale. The highresolution image in Figure 1D demonstrated how the endocytosis is taking place. While a couple of clear holes indicated that the endocytosis may be just completed in these areas, cellular uptake halfway through the internalization process for some of the Au-TF nanoparticle cluster was vividly visualized. To our knowledge, this is the first time that the cellular process of nanoparticle uptake has been observed by AFM. To validate the nanoparticle internalization into NPC cells, a Zeiss 410 (Germany) confocal laser scanning microscope (excitation at wavelength 488 nm and emission at wavelength 515 nm) was utilized to capture the fluorescence images of the cells after incubation with fluorescence-labeled Au-TF nanoparticles. The emission of the labeled TF nanoparticles produces green fluorescence. Its position and intensity represent the location
10.1021/bc049775d CCC: $30.25 © 2005 American Chemical Society Published on Web 04/12/2005
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Figure 3. Nanoparticle concentrations at different ratio of holoTF verses Au-TF. [Au-TF] were fixed at 10 µM.
Figure 1. AFM images demonstrate Au-TF nanoparticle uptake by NPC cells. (A) A single NPC cell; (B) Au-TF nanoparticles on a NPC cell surface at the beginning of incubation; (C) NPC cell surface after incubation with Au-TF nanoparticles for 5 h; and (D) “zoom in” AFM image of cell surface after incubation with Au-TF for 5 h, showing nanoparticle uptake during the process of endocytosis.
Figure 2. Confocal cell images show that Au-TF nanoparticles were internalized into the cellular plasma of NPC cells via TFreceptor interaction pathway (5-h treatment). (A) Control cells without Au-TF nanoparticles; (B) cells treated with Au nanoparticles alone; (C) cells incubating with Au-TF nanoparticles; (D) cells treated with Au-albumin nanoparticles; (E and F) cells cotreated with different proportions of Au-TF verses holo-TF (1:2 and 1:5, respectively).
and relative concentration of the labeled TF nanoparticles. Figure 2 shows confocal images for cells treated under different conditions for 5 h. Control cells (Figure 2A) and cells treated with Au nanoparticles alone (Figure 2B) reasonably displayed little fluorescence. In contrast, very bright fluorescence was observed in cells incubated with Au-TF (Figure 2C), clearly indicating that alot of Au-TF nanoparticles were taken up into cells, with most of the internalized nanoparticles localizing in the cytoplasm compartment. At the same time, the NPC cells were still healthy after taking up the nanoparticles. Moreover, a control study showed that the fluorescencelabeled Au-TF particles did not have detectable changes
in sizes after incubation with the cell culture medium for 24 h, revealing the high stability of the covalently TFderivatized particles during the experiments. Since nanoparticle uptake into cells could go through different processes including phagocytosis, fluid-phase endocytosis, and receptor-mediated endocytosis, we performed control studies with fluorescence-labeled Aualbumin and competition experiments with natural fulllength transferrin (holo-TF) to verify whether the internalization of the TF-coupled nanoparticles was mediated via the specific TF-receptor interaction pathway. As shown in Figure 2D, cells incubated with Aualbumin displayed only little minor fluorescence, and compared to that in Figure 2C, fluorescence intensities significantly decreased in the cells cotreated with AuTF and excess holo-TF (Figure 2E,F). These results evidently implicate the specific nanoparticle cellular uptake through TF-receptor interaction. Other supporting evidence was derived from ICP-MS measurements of the nano-Au contents in cells treated with different proportions of holo-TF/Au-TF. Three different concentration ratios (0:1, 1:1, and 2:1) of holoTF verses Au-TF were introduced into the cell culture. Total cell lysates digested from the harvested NPC cells were used for determination of Au concentration in cells by ICP-MS (7500 series, Agilent). Figure 3 clearly shows that the Au concentration in the cells significantly decreased with regard to the increase of holo-TF concentration in the cell culture. These data, together with the above confocal results, strongly suggest that at least part of the TF-coupled nanoparticles were internalized into cells via TF-receptor mediated endocytosis. The TF-receptor cycling pathway has long been considered to be an effective “delivery system” into cells, and many cytotoxic agents have been conjugated to TF as potential anticancer therapeutics (6, 9). Our present data demonstrated that TF-receptor-mediated nanoparticle delivery into cells really occurred, and that the endocytosis process can be directly visualized through AFM imaging. In light of a recent study showing that TF binds the receptor ectodomain to form the TF-receptor complex (18), it is possible that TF conjugation with nanoparticles at certain positions is tolerable for the endocytosis, since the ectal binding mode of TF-receptor reduces space restrictions. That being the case, our present observations provide useful information for better design of nanodrugs and their targeted delivery.
496 Bioconjugate Chem., Vol. 16, No. 3, 2005 ACKNOWLEDGMENT
This work was partially supported by Hong Kong Research Grants Council Grants HKU 7227/02M (to Q.Y.H.), the Department of Chemistry, and the Areas of Excellence scheme of Hong Kong University Grants Committee. We thank Mr. Rong Chen for providing Au nanoparticles. Supporting Information Available: Experimental details. This material is available free of charge via the Internet at http://pubs.acs.org. LITERATURE CITED (1) Finley, R. S. (2003) Overview of targeted therapies for cancer. Am. J. Health Syst. Pharm. 60, S4-10. (2) Portlock, J. L., and Calos, M. P. (2003) Site-specific genomic strategies for gene therapy. Curr. Opin. Mol. Ther. 5, 376382. (3) Abou-Jawde, R., Choueiri, T., Alemany, C., and Mekhail, T. (2003) An overview of targeted treatments in cancer. Clin. Ther. 25, 2121-2137. (4) Weissig, V. (2003) Mitochondrial-targeted drug and DNA delivery. Crit. Rev. Ther. Drug Carrier Syst. 20, 1-62. (5) Russell-Jones, G. J. (2001) The potential use of receptormediated endocytosis for oral drug delivery. Adv. Drug Delivery Rev. 46, 59-73. (6) Qian, Z. M., Li, H., Sun, H., and Ho, K. (2002) Targeted drug delivery via the transferrin receptor-mediated endocytosis pathway. Pharmacol. Rev. 54, 561-587. (7) Li, H., and Qian, Z. M. (2002) Transferrin/transferrin receptor-mediated drug delivery. Med. Res. Rev. 22, 225-250.
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