Gold Nanoparticles Enhance the Anti-Leukemia Action of a 6

In the last step of the preparation, 50 mL of a water solution containing a 2:1 molar ratio ... Cell cultures of K-562 maintained at a density of 2 ×...
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Langmuir 2008, 24, 568-574

Gold Nanoparticles Enhance the Anti-Leukemia Action of a 6-Mercaptopurine Chemotherapeutic Agent Paul Podsiadlo,†,| Vladimir A. Sinani,†,| Joong Hwan Bahng,‡ Nadine Wong Shi Kam,† Jungwoo Lee,‡ and Nicholas A. Kotov*,†,‡,§ Departments of Chemical Engineering, Biomedical Engineering, and Materials Science and Engineering, UniVersity of Michigan, Ann Arbor, Michigan 48109 ReceiVed September 7, 2007. In Final Form: October 17, 2007

6-Mercaptopurine and its riboside derivatives are some of the most widely utilized anti-leukemic and anti-inflammatory drugs. Their short biological half-life and severe side effects limit their use. A new delivery method for these drugs based on 4-5 nm gold nanoparticles can potentially resolve these issues. We have found substantial enhancement of the antiproliferative effect against K-562 leukemia cells of Au nanoparticles bearing 6-mercaptopurine-9-β-Dribofuranoside compared to the same drug in typically administered free form. The improvement was attributed to enhanced intracellular transport followed by the subsequent release in lysosomes. Enhanced activity and nanoparticle carriers will make possible the reduction of the overall concentration of the drug, renal clearance, and, thus, side effects. The nanoparticles with mercaptopurine also showed excellent stability over 1 year without loss of inhibitory activity.

Introduction 6-Mercaptopurine (6-MP) is one of the most widely utilized drugs for the treatment of human leukemias and many other diseases: inflammatory bowel disease, systemic lupus erythematosus, and rheumatoid arthritis.1,2 The development of 6-MP and its derivatives has emanated from a Nobel-prize-winning work by Ellion and Hitchings3 in the 1950s,4-6 and its development was a breakthrough in the treatment of the leukemias. Other purine derivatives have also been prepared based on this work, and they are now used in the prevention of organ transplant rejection (azathioprine), gout and hyperuricemia (allopurinol), and herpes virus infections (acyclovir).1,7-12 The antiproliferative mechanism of action of 6-MP and other purine antimetabolites has been determined to be through the inhibition of de noVo purine synthesis and incorporation into DNA and RNA (see the Supporting Information, Figure S.2). This requires efficient delivery and intracellular uptake of the drug by cancerous cells. Initial therapy with 6-MP constituted oral administration of the drug; however, poor absorption led to wider use of intravenous administration. Intravenous treatment showed much improved efficacy of 6-MP; however, the drug * To whom correspondence should be addressed. Telephone: (734) 7638768. Fax: (734) 764-7453. E-mail: [email protected]. † Department of Chemical Engineering. ‡ Department of Biomedical Engineering. § Department of Materials Science and Engineering. | These authors have contributed equally to this work. (1) Elion, G. B. Science 1989, 244, 41. (2) Cheok, M. H.; Evans, W. E. Nat. ReV. Cancer 2006, 6, 117. (3) Elion, G. B. Prix Nobel 1989, 267. (4) Hitchings, G.; Elion, G. B.; Falco, E. A.; Russell, P. B.; VanderWerff, H. Ann. N. Y. Acad. Sci. 1950, 52, 1318. (5) Elion, G. B.; Burgi, E.; Hitchings, G. J. Am. Chem. Soc. 1952, 74, 411. (6) Skipper, H. E.; Thomson, J. R.; Elion, G. B.; Hitchings, G. H. Cancer Res. 1954, 14, 294. (7) Hitchings, G. H.; Elion, G. B. Acc. Chem. Res. 1969, 2, 202. (8) Pacher, P.; Nivorozhkin, A.; Szabo, C. Pharmacol. ReV. 2006, 58, 87. (9) Van Scoik, K. G.; Johnson, C. A.; Porter, W. R. Drug Metab. ReV. 1985, 16, 157. (10) Coen, D. M. In Alpha HerpesViruses; Sandri-Goldin, R. M., Ed.; Caister Academic Press: Norwich, U.K., 2006; p 361. (11) Sampedro, A.; Perez, M.; Rodriguez, J.; Camacho, E. New Approaches in the Use of Antibiotics; Research Signpost: Trivandrum, India, 2003; p 169. (12) Elion, G. B. J. Med. Virol. 1993, 2.

has a short plasma half-life and it is rapidly eliminated through the renal system, thereby greatly reducing its curative effect. Resolution of this problem can provide a more potent drug and potentially reduce side effects. The renal system acts as a filter that passes through molecules below a certain size. So, to prevent elimination of a drug from the bloodstream by the kidneys, the active molecules should have molecular weight above ∼5,000 Da. In terms of the average hydrodynamic diameter of organic molecules, it corresponds to ∼3 nm.13 This limitation can be obviated by using nanoparticle delivery platforms, for instance, multiple-emulsions, chemical modification, liposome entrapment, and polymeric microsphere entrapment.13,14 However, as the diameter of the delivery particles increases, they become the target of different cells, which belong to the reticular endothelial system (RES) responsible for the removal of foreign particles from the blood. Unlike the kidneys, the RES is targeting fairly large particles aboVe the size of typical proteins. RES activity typically starts with opsonization, or coating of the particles with special proteins, opsonins.15 After that, the particles can be recognized by macrophages and transported into the liver. Particle size plays an important role in RES activity.13,16,17 The universal trend is that the smaller particles have a substantially longer lifetime in the blood than the larger particles.16,18-22 For instance, 300 nm polymer particles have a typical blood clearance time of a few minutes, while small particles (13) Owens, D. E.; Peppas, N. A. Int. J. Pharm. 2006, 307, 93. (14) LaVan, D. A.; McGuire, T.; Langer, R. Nat. Biotechnol. 2003, 21, 1184. (15) Vonarbourg, A.; Passirani, C.; Saulnier, P.; Benoit, J. P. Biomaterials 2006, 27, 4356. (16) Fang, C.; Shi, B.; Pei, Y. Y.; Hong, M. H.; Wu, J.; Chen, H. Z. Eur. J. Pharm. Sci. 2006, 27, 27. (17) Sun, X.; Rossin, R.; Turner, J. L.; Becker, M. L.; Joralemon, M. J.; Welch, M. J.; Wooley, K. L. Biomacromolecules 2005, 6, 2541. (18) Kim, J. S.; Yoon, T. J.; Yu, K. N.; Kim, B. G.; Park, S. J.; Kim, H. W.; Lee, K. H.; Park, S. B.; Lee, J. K.; Cho, M. H. Toxicol. Sci. 2006, 89, 338. (19) Chittimalla, C.; Zammut-Italiano, L.; Zuber, G.; Behr, J. P. J. Am. Chem. Soc. 2005, 127, 11436. (20) Gref, R.; Couvreur, P. In Encyclopedia of Nanoscience and Nanotechnology; Nalwa, H. S., Ed.; American Scientific: Valencia, CA, 2004; Vol. 10, p 83. (21) Balogh, L. P.; Nigavekar, S. S.; Cook, A. C.; Minc, L.; Khan, M. K. PharmaChem 2003, 2, 94. (22) Banerjee, T.; Mitra, S.; Kumar Singh, A.; Kumar Sharma, R.; Maitra, A. Int. J. Pharm. 2002, 243, 93.

10.1021/la702782k CCC: $40.75 © 2008 American Chemical Society Published on Web 12/05/2007

Anti-Leukemia Action of 6-MPR-AuNPs

(both polymeric and inorganic) of 10-20 nm may have a lifetime in the blood from a few hours to several days.16,18-22 As well, the change of particle size from 240 to 80 nm showed an extension of the half-life of the particles in mice and a drug in it (TNFR) by 24-fold from 28.2 min to 11.33 h.16 So, between the renal and RES systems, there must be an optimum particle diameter that will prevent renal filtration and will avoid immediate capture by the RES. This size will correspond to the maximum residence time in the blood, which, in turn, can be further tuned by the surface chemistry of the particles, for example, by PEG modification. The technology of size control is very well developed for Au nanoparticles (AuNPs). Thiols such as 6-MP and similar ones, such as 6-mercaptopurine-9-β-D-ribofuranoside (6-MPR, a prodrug of 6-MP), provide very convenient molecules that can be loaded on the surfaces of AuNPs through sulfur-gold bonds known for their strength. Some of the pioneering work on the preparation of thiol-stabilized AuNPs was shown by Giersig and Mulvaney in the early 1990s.23 Since then, AuNPs have been receiving substantial attention for drug delivery, with some of the examples including photodynamic cancer therapy24-27 and delivery of proteins, peptides, and oligonucleotides, among others.28-33 Cancer cell targeting and imaging with AuNPs has also been explored in recent years.34-43 The important characteristics of AuNPs for this application include biocompatibility, solubility under physiological conditions, ease of surface functionalization, and distinct optical properties.31-33 The preparation of 6-MP loaded AuNPs has been recently presented,44 and their inhibitory effect against bacteria has been further evaluated.45 In our own work, we have further shown a robust (23) Giersig, M.; Mulvaney, P. Langmuir 1993, 9, 3408. (24) Wieder, M. E.; Hone, D. C.; Cook, M. J.; Handsley, M. M.; Gavrilovic, J.; Russell, D. A. Photochem. Photobiol. 2006, 5, 727. (25) El-Sayed, I. H.; Huang, X.; El-Sayed, M. A. Cancer Lett. 2006, 239, 129. (26) Huang, X.; Jain, P. K.; El-Sayed, I. H.; El-Saved, M. A. Photochem. Photobiol. 2006, 82, 412. (27) Huang, X.; El-Sayed, I. H.; Qian, W.; El-Sayed, M. A. J. Am. Chem. Soc. 2006, 128, 2115. (28) Oishi, M.; Nakaogami, J.; Ishii, T.; Nagasaki, Y. Chem. Lett. 2006, 35, 1046. (29) Mukherjee, P.; Bhattacharya, R.; Mukhopadhyay, D. J. Biomed. Nanotechnol. 2005, 1, 224. (30) Bhattacharya, R.; Mukherjee, P.; Xiong, Z.; Atala, A.; Soker, S.; Mukhopadhyay, D. Nano Lett. 2004, 4, 2479. (31) Paciotti, G. F.; Kingston, D. G. I.; Tamarkin, L. Drug DeV. Res. 2006, 67, 47. (32) Han, G.; Ghosh, P.; De, M.; Rotello, V. M. NanoBiotechnology 2007, 3, 40. (33) Han, G.; Ghosh, P.; Rotello, V. M. Nanomedicine 2007, 2, 113. (34) Sokolov, K.; Follen, M.; Aaron, J.; Pavlova, I.; Malpica, A.; Lotan, R.; Richards-Kortum, R. Cancer Res. 2003, 63, 1999. (35) Aaron, J.; Nitin, N.; Travis, K.; Kumar, S.; Collier, T.; Park, S. Y.; JoseYacaman, M.; Coghlan, L.; Follen, M.; Richards-Kortum, R.; Sokolov, K. J. Biomed. Opt. 2007, 12, 034007-1-034007/11. (36) Bhattacharya, R.; Patra, C. R.; Earl, A.; Wang, S.; Katarya, A.; Lu, L.; Kizhakkedathu, J. N.; Yaszemski, M. J.; Greipp, P. R.; Mukhopadhyay, D.; Mukherjee, P. Nanomedicine 2007, 3, 224. (37) Shi, X.; Wang, S.; Meshinchi, S.; Van Antwerp, M. E.; Bi, X.; Lee, I.; Baker, J. R., Jr. Small 2007, 3, 1245. (38) Lee, S.; Kim, S.; Choo, J.; Shin, S. Y.; Lee, Y. H.; Choi, H. Y.; Ha, S.; Kang, K.; Oh, C. H. Anal. Chem. 2007, 79, 916. (39) Eghtedari, M.; Oraevsky, A.; Copland, J. A.; Kotov, N. A.; Conjusteau, A.; Motamedi, M. Nano Lett. 2007, 7, 1914. (40) Mallidi, S.; Larson, T.; Aaron, J.; Sokolov, K.; Emelianov, S. Opt. Express 2007, 15, 6583. (41) El-Sayed, I. H.; Huang, X.; El-Sayed, M. A. Nano Lett. 2005, 5, 829. (42) El-Sayed, I.; Huang, X.; Macheret, F.; Humstoe, J. O.; Kramer, R.; ElSayed, M. Technol. Cancer Res. Treat. 2007, 6, 403. (43) Rahman, M.; bd-El-Barr, M.; Mack, V.; Tkaczyk, T.; Sokolov, K.; Richards-Kortum, R.; Descour, M. Gynecol. Oncol. 2005, 99, S112. (44) Viudez, A. J.; Madueno, R.; Pineda, T.; Blazquez, M. J. Phys. Chem. B 2006, 110, 17840. (45) Selvaraj, V.; Alagar, M.; Hamerton, I. Electrochim. Acta 2006, 52, 1152.

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method for the preparation of 6-MPR loaded AuNPs with good luminescence properties.46 While the 6-MP loaded AuNPs have been found to have some level of inhibition against bacterial cultures, mammalian cells are very different. The question is whether 6-MPR or other small drugs that may be loaded onto AuNPs to escape fast blood clearance will retain their activity. Looking at the metabolism of mercaptopurines and the strength of Au-S bonds (see the Supporting Information, Figure S.2), one will arrive to the conclusion that the immobilization of mercaptopurines on gold must prevent its activity as an inhibitor of the de noVo synthesis of purines because it will lose its ability to be incorporated in DNA and RNA. Quite surprisingly, this is not correct. In this publication, we demonstrate that 6-MPR carried by AuNPs does not lose its in Vitro anticancer activity. Despite all expectations, we see an increase of anticancer activity when 6-MPR is immobilized on gold. The intracellular metabolism of gold-bound 6-MPR may not be completely understood at the moment. Nevertheless, these findings open the road for substantial improvement of the curative effects of 6-MPR by optimizing the particle size and extending its blood clearance time. Experimental Section Materials. 6-Mercaptopurine-9-β-D-ribofuranoside (6-MPR) was purchased from Fluka. Sodium citrate decahydrate, tetrachloroauric(III) acid trihydrate (Au(III)), L-cysteine (Cys), and sodium borohydride were obtained from Sigma-Aldrich and utilized without further purification. Fluorescein isothiocyanate isomer I (FITC) was obtained from Sigma-Aldrich. The FM 4-64 membranes and endosome marker were obtained from Molecular Probes Inc. Dialysis membranes with a MW cutoff of 1000 Da were obtained from Spectrum Laboratories Inc. Ultrapure water (Barnstead) with 18.2 MΩ/cm was used for all experiments. AuNP Synthesis. Aqueous solutions of AuNPs and AuNPs modified by 6-MPR (6-MPR-AuNPs) were prepared by sodium borohydride reduction of tetrachloroauric(III) acid solutions.46 Briefly, 0.5 mL of 1 wt % tetrachloroauric(III) acid solution was diluted in 50 mL of ultrapure water and stirred for 1 min. Subsequently, 0.5 mL of 3.4 mM sodium citrate solution was added, and the resulting solution was then stirred for 1 min. Afterward, gold was reduced by addition of 0.5 mL of 0.075 wt % sodium borohydride in 3.4 mM sodium citrate solution. In the end, the resulting AuNP suspension was diluted with an additional 50 mL of ultrapure water prior to use to keep the final concentration approximately the same as that for the 6-MPR-AuNPs. In the preparation of 6-MPR-AuNPs, as the last step, 50 mL of a water solution containing 5 mg (17.5 µmol) of 6-MPR was added to the stirred reaction vessel containing AuNPs 30 s after sodium borohydride. Variations of this procedure with earlier additions of 6-MPR may also be tested, if necessary. The resulting red solutions of AuNPs and 6-MPR-AuNPs were purified by two-step centrifugation. First, at the speed of 40 000 rpm (gr max ) 190 000), 20 mL of the AuNP solution was settled at the bottom of the tube with 1 h of centrifugation. Following removal of the supernatant, while taking special care to prevent disturbing of the precipitate, the nanoparticles were redispersed in 20 mL of water and centrifuged one more time under the same conditions. After removal of the second supernatant, the precipitate was redispersed in 3 mL of water and stored at 4 °C. FITC-Labeled 6-MPR/Cys-AuNP Synthesis. In the first stage, an aqueous solution of AuNPs with a mixed stabilizer of 6-MPR and Cys was prepared following the same method as that in the 6-MPRAuNP synthesis. In the last step of the preparation, 50 mL of a water solution containing a 2:1 molar ratio mixture of 6-MPR (11.7 µmol) and Cys (5.8 µmol) was added to the reaction vessel instead of pure 6-MPR. The resulting solution was dialyzed against ultrapure water for 3 days and then further purified and concentrated by high-speed (46) Sinani, V. A.; Podsiadlo, P.; Lee, J.; Kotov, N. A.; Kempa, K. Int. J. Nanotechnol. 2007, 4, 239.

570 Langmuir, Vol. 24, No. 2, 2008 centrifugation to a final volume of 20 mL. To the resulting dispersion, a 1 mL solution containing 0.58 µmol of FITC was added, and, after adjusting the pH to ∼8, the mixture was allowed to stir at room temperature overnight in darkness. At this stage, the FITC fluorescent probe was conjugated to the Cys stabilizers via condensation of the isothiocyanate group of FITC and the primary amine of the Cys groups.47 On the following day, the solution was placed in a dialysis bag and allowed to purify against ultrapure water until no more FITC was eluting (∼1 week). The resulting NP solution was stored until further use in a refrigerator, protected from ambient light. Cell Culture. K-562 human chronic myeloid leukemia cells (American Type Culture Collection) were grown as a suspension culture at 37 °C in a humidified, 5% CO2 atmosphere incubator. The culture was maintained in 90% RPMI 1640 with L-glutamine medium (Cambrex), 10% fetal bovine serum (HyClone Lab, Inc.), supplemented with 1% penicillin/streptomycin antibiotics (10 000 units/ mL/10 000 ug/mL; Sigma). The doubling time for a culture was ∼30-40 h. Growth Inhibitory Effect Against K-562 Cells. K-562 cells were treated with aliquots of tested substances, and the growth of these cells was compared against controls. The series of cell suspensions were incubated in 24-well plates, with 2 mL of suspension in each well. Each experiment was conducted in triplicate. Cells were counted under a light microscope using a hemacytometer. Flow Cytometry. Cell cultures of K-562 maintained at a density of 2 × 105 cells/mL were treated with tested solutions (2, 5, 10, 20, and 40 µL of tested solutions were added to 2 mL of cell suspension) and allowed to incubate for 72 h. Cells were collected by centrifugation (1000 rpm in a microfuge for 3 min), washed in cold phosphate-buffered saline (PBS), and resuspended in 1 mL of the same buffer in 5 mL polystyrene tubes (Falcon, Becton-Dickinson). Subsequently, 1 µL of YO-PRO-1 stock solution and 1 µL of propidium iodide were added to each 1 mL of resuspended cell suspension (Vybrant Apoptosis Assay kit 4; Molecular Probes, Inc.) and the cells were incubated for 20-30 min on ice in the dark. After staining, apoptotic cells showed green fluorescence, dead cells showed green and red fluorescence, and live cells showed little or no fluorescence. These populations were measured by using a flow cytometer (FACSCalibur, Becton-Dickinson) that uses the 488 nm line of an argon ion laser for excitation. Fluorescence was detected with 530/30 bp and 590/32 bp filters. A total of 10 000 cells were measured for each histogram. Apoptosis of K-562 cells was induced by treatment with 10 µM camptothecin (Sigma) for 4 h. Necrosis was induced by hyperthermia; cells were heated at 56 °C for 1 h and incubated for 1 h at 37 °C. Prepared apoptotic and necrotic cells were used as standards to optimize the instrument settings. Imaging of Cells Incubated with FITC-Labeled AuNPs. For the purpose of imaging, 100 µL of the FTIC-labeled AuNP solution was added to 1 mL of the K-562 cell suspension at an initial concentration of 2.5 × 105 cells/mL. The suspension was then placed in a 37 °C incubator. At the specified time intervals, 100 µL of the suspension was withdrawn, washed twice by centrifugation and resuspension with fresh media, and imaged with a confocal microscope. Similarly, for the cells incubated at low temperature, after mixing with the FITC-labeled AuNPs, the suspension was placed in a 4 °C refrigerator and 100 µL samples were withdrawn from the stack at specified intervals. For the purpose of better visualization, the FM 4-64 marker48,49 was used to stain the cell membranes. All confocal images were taken immediately after the incubation and washing steps. A total of 20 µL of the cell suspension was dropped onto a glass cover slide and imaged by using a Zeiss LSM 510 confocal microscope. Transmission Electron Microscopy. After 48 h of incubation of 6-MPR-AuNPs with the suspension of K-562 cells, the medium was removed and aliquoted into ultracentrifuge tubes. The resuspended pellet was mixed with an equal volume of 5% glutaraldehyde (47) Hermanson, G. T., Ed. Bioconjugate Techniques; Academic Press: San Diego, CA, 1995. (48) Silverstein, S. C.; Steinman, R. M.; Cohn, Z. A. Annu. ReV. Biochem. 1977, 46, 669. (49) Vida, T. A.; Emr, S. D. J. Cell Biol. 1995, 128, 779.

Podsiadlo et al. and fixed overnight in the refrigerator. Glutaraldehyde was removed by centrifugation, after which 0.1 M sodium cacodylate buffer pH 7.4 was added for 20 min. The procedure was repeated two more times. Next, 1% sodium cacodylate-buffered osmium tetroxide was added and the cells were fixed for 2 h. Osmium tetroxide was removed by centrifugation and replaced with 0.1 M sodium cacodylate buffer for 20 min. This was again repeated two times, after which the cells were left in the refrigerator in the last wash overnight. The cells were then dehydrated with a series of ethyl alcohol dilutions (50%, 70%, 90%, 95%, and 100%) three times for 20 min each step. The cells were then placed in a 1:1 mixture of 100% ethyl alcohol and LR White. The tubes were filled completely and placed into the refrigerator overnight. On the next day, the 1:1 mixture was removed and replaced with 100% LR White. The tubes were again completely filled and placed into the refrigerator overnight. On the following day, the mixture was replaced with 100% LR White and kept for 24 h. The cells were embedded, and the old LR White was removed and replaced with fresh LR White. Polymerization took place in -20 atm and at 70 °C for 24 h. The sample was sectioned with a Diatome diamond knife on a Sorvall MT 5000 ultramicrotome. The 60-90 nm thick sections were stained with 2% aqueous uranyl acetate and Reynold’s lead citrate and observed at 80 kV in a JEOL JEM-100 CX II transmission electron microscope. Atomic Force Microscopy. Atomic force microscopy (AFM) imaging was performed using a Nanoscope III (Digital Instruments/ Veeco, Santa Barbara, CA) instrument. AFM images were obtained in the tapping mode with standard Si/N tips.

Results and Discussion Aqueous solutions of citrate stabilized AuNPs were prepared by sodium borohydride reduction of tetrachloroauric(III) acid solution in the presence of sodium citrate (see the Experimental section) as described in our previous publication.46 6-MPR-AuNPs were obtained by modification of the AuNPs through addition of 6-MPR into the reaction vessel immediately (within 30 s) after AuNP nucleation. Thus, prepared 6-MPR-AuNPs were characterized with the use of transmission electron microscopy (TEM, Figure 1) and atomic force microscopy (AFM, see the Experimental section). The result of this synthesis protocol was the formation of positively charged NPs with an average diameter between 4 and 5 nm, which is about 50% larger than the cutoff size of the renal system.46 For comparison, as we have presented previously, the same procedure when carried out without 6-MPR addition resulted in the formation of NPs with a mean diameter of about 30 nm. By using cyanide digestion of AuNPs, it was established that, in the described synthetic procedure, ∼50% of 6-MPR molecules taken for the synthesis are associated with AuNPs. Correlating the obtained value with standard 6-MPR solutions, it was determined that the concentration of Au-bound 6-MPR is ∼25 µg/mL in the as prepared dispersion (see the Supporting Information) We have also established by gel filtration chromatography that there is no appreciable detachment of 6-MPR from the surfaces of the AuNPs. This information enabled us to prepare solutions of 6-MPR and 6-MPR-AuNPs with equal cumulative concentrations of the drug to compare its effectiveness in the free form and in the immobilized form on AuNPs. To test the growth-inhibitory effect of 6-MPR-AuNPs, we have evaluated it in Vitro against K-562 human chronic myeloid leukemia cells using pure 6-MPR and citrate stabilized AuNPs as benchmarks. Citrate stabilized AuNPs were shown to have little effect on this particular cell line as described by Connor et al., and hence, we have used them for proper comparison.50 In addition, we have prepared a 6-MPR-Au(III) complex to (50) Connor, E. E.; Mwamuka, J.; Gole, A.; Murphy, C. J.; Wyatt, M. D. Small 2005, 1, 325.

Anti-Leukemia Action of 6-MPR-AuNPs

Figure 1. Gold nanoparticles stabilized with 6-mercaptopurine9-β-D-ribofuranoside. (A) Chemical formula of 6-MPR. (B) Schematic of 6-MPR stabilization of AuNPs. Simple geometrical calculations based on the available surface atoms and/or geometry of the 6-MPR moiety indicates that there might be 100-350 molecules of 6-MPR on the surfaces of the AuNPs. (C) Transmission electron micrograph of 6-MPR-AuNPs.

compare its effectiveness in case any dissociation and oxidation of gold would occur under biological conditions. K-562 cells at an initial density of 2.5 × 105 cells/mL were incubated with citrate stabilized AuNPs, neat gold tetrachloroaureate, neat 6-MPR, Au(III)-6-MPR, and 6-MPR-AuNPs for 72 h (Figure 2). After the incubation period, cells were counted using a hemocytometer. Neither plain AuNPs nor Au(III) solutions, which were used as a part of control experiments, had visible antiproliferative or cytotoxic effects on the cells (Figure 2A). On the other hand, the plain solution of 6-MPR, the 6-MPRAu(III) complex, and the suspension of 6-MPR-AuNPs exerted a significant antiproliferative effect and suppressed the growth of leukemia cells in the given concentrations. Clear visualization of the inhibitory effect can be seen from the optical microscopy images (Figure 2B-E). Unexpectedly, 6-MPR-AuNPs displayed

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Figure 2. Comparative inhibition of K-562 human chronic myeloid leukemia cells by different derivatives of AuNPs and 6-MPR. (A) Antiproliferative effects of substances indicated below the data bar against leukemia cells after 72 h of incubation. Concentration of 6-MPR in solutions is 1.8 × 10-6 M; concentration of Au(III) is 2.5 × 10-7 M. (B-E) Representative optical microscopy images of cell culture dishes treated with different derivatives of AuNPs and 6-MPR: (B) negative control (no 6-MPR), (C) cells treated with freely dissolved 6-MPR; (D) cells with citrate stabilized AuNPs, and (E) cells treated with 6-MPR-AuNPs. All experiments were performed in triplicate. Images were taken after 72 h of incubation. Total concentration of 6-MPR in solutions in (B-E) was 8.8 × 10-7 M. Magnification is 50×.

increased inhibition efficiency over the free drug without the carrier (Figure 2). In all likelihood, processing of the bound mercaptopurine by guanosine monophosphate synthetase, which is an important step in thiopurine metabolism, must be frustrated because of the steric hindrance from the Au surfaces. To verify the equal or higher activity of 6-MPR immobilized on AuNPs, we have compared the effectiveness of 6-MPR and 6-MPR-AuNPs at varying cumulative concentrations of the drug. The inhibitory effect of 6-MPR-AuNPs was reliably observed to be stronger than that of the plain solution of 6-MPR at all concentrations in the range between 1.8 × 10-7 and 1.8 × 10-6 M (Figure 3). Further evaluation of the cell cultures remaining after 72 h of incubation by flow cytometry with apoptosis and necrosis specific stains showed that a relatively low percentage of cells were either necrotic or apoptotic for both 6-MPR and 6-MPR-AuNPs (Table 1). Although the percentage of apoptotic cells for 6-MPR-AuNPs seemed to be consistently higher than that for plain 6-MPR, it is most likely within the experimental error. These data correlate well with the hypothesis that 6-MPR-AuNPs have the mechanism

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Figure 3. Antiproliferative effects of 6-MPR and 6-MPR-AuNPs on K-562 human chronic myeloid leukemia cells. (A) Cell count after 72 h of incubation at specific concentrations of 6-MPR and 6-MPR-AuNPs. Initial cell concentration was 5 × 105 cells/mL. (B) Comparative effects of 6-MPR and 6-MPR-AuNPs stored for 1 year at ambient conditions. Cell count after 72 h of incubation at specified concentrations of 6-MPR, 6-MPR-AuNPs, and AuNPs. Initial cell concentration was 2.25 × 105 cells/mL.

of anti-leukemia action similar to that of 6-MPR. It also becomes quite clear that 6-MPR-AuNPs act via the proliferationsuppression mechanism as was very well established for 6-MPR rather than by a dramatic increase in cell death rate via either necrosis or apoptosis. Notably, the inhibitory activity of the particles has also shown remarkable stability over time. NPs which were stored for over 1 year in a light-protected solution at 20 °C have shown a equally high potency as that of the freshly prepared material (Figure 3B). Under equal conditions, 6-MPR oxidizes quite quickly and loses its activity. Besides obvious practical convenience associated with long shelf life, this observation underscores again the fact that the biological activity of AuNPs is not associated with unbound or loosely adsorbed drug molecules which can be oxidized in solution over time. Current understanding of the anti-leukemia action of 6-MPR involves (a) feedback inhibition of the first enzyme in de noVo ribonucleotide synthesis, (b) inhibition of purine ribonucleotide interconversion, and (c) indirect incorporation into DNA after intracellular conversion to 6-thioguanine. All of these require detachment of 6-MPR from the AuNPs to participate in these processes.1 Detailed understanding of the metabolism of mer-

Figure 4. Cross-sectional TEM images of K-562 cells incubated for 72 h with 6-MPR-AuNPs: (A) image of the entire cell and (B) magnification of the selected vesicle. Image is rotated by 90°. 6-MPRAuNPs are visible as black spots both inside the vesicle and throughout the cell (indicated with arrows).

captopurines on AuNPs will require substantial research effort going beyond one publication. Nevertheless, we can suggest a role of the AuNPs that is consistent with the observations and the known mechanism of leukemia inhibition by 6-MPR. It includes the intracellular transport of the drug on the carrier and its eventual detachment inside the cell. Both free mercaptopurines and 6-MPR-AuNPs need to penetrate the cell membrane. Measurements of the electrokinetic

Anti-Leukemia Action of 6-MPR-AuNPs

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Figure 5. FITC-labeled 6-MPR/Cys-AuNPs: (A) schematic structure of a AuNP with 6-MPR and Cys-FITC conjugate bound on its surface; (B and C) optical images of FITC-labeled 6-MPR/Cys-AuNPs under ambient light and UV illumination; (D-G) laser scanning confocal microscopy images of K-562 cells following incubation at 37 °C with FITC-labeled 6-MPR/Cys-AuNPs for (D) 0 h, (E) 2 h, (F) 4 h, and (G) 16 h (FITC ) green; FM 4-64 membrane dye48,49 ) red); and (H and I) confocal microscopy images of K-562 cells following incubation at 4 °C for (H) 1 h and (I) 2 h. Initial cell concentration was 2.5 × 105 cells/mL. Intensity of the FITC excitation laser source in (I) has been substantially increased to determine any presence of the dye inside of the cells. The small level of the green color in (I) is mostly due to autofluorescence. Table 1. Percentage of Apoptotic and Necrotic K-562 Cells after Treatment with Tested Solutions for 72 h 6-MPR-AuNP

6-MPR

Au(III)-6MPR

[6-MPR] (×106 M)

necrotic cells (%)

apoptotic cells (%)

necrotic cells (%)

apoptotic cells (%)

necrotic cells (%)

apoptotic cells (%)

0.44 0.88 1.8 3.5

5 ( 0.5 8.8 ( 0.9 8.9 ( 0.9 11.6 ( 1.2

1.4 ( 0.15 1.8 ( 0.2 2.1 ( 0.2 2.4 ( 0.2

4.3 ( 0.4 6.1 ( 0.6 8.6 ( 0.9 12 ( 1.2

1.1 ( 0.1 0.9 ( 0.1 1.6 ( 0.2 1.9 ( 0.2

4.7 ( 0.5 6.5 ( 0.7 8.3 ( 0.8 11.9 ( 1.2

2 ( 0.2 1.7 ( 0.2 2.2 ( 0.2 1.9 ( 0.2

potential, ξ, of the 6-MPR-stabilized AuNPs revealed that they are positively charged with ξ ) +19 mV. The source of the positive charge is most likely a small number of adsorbed Au ions on the surfaces of the AuNPs. Compared to the slightly negatively charged or neutral mercaptopurine, the positive charge of the 6-MPR-AuNPs strongly facilitates permeation through the negatively charged cell membranes. The same principle was put in the foundation of many polyelectrolyte drug carriers. The small size of the NPs (4-5 nm) also supports the efficient intracellular transport. Evidence for the successful accumulation of large quantities of 6-MPR-AuNPs inside the cell can be clearly seen in the transmission electron microscopy images (Figure 4). While the arrows in Figure 4B point to a few aggregates, a careful look shows that this fragment of the cell is filled with hundreds of NPs which are visible as small black dots owing to their very small size. The particles can often be found localized inside the endocytotic vesicles, as well as throughout the cytoplasm. Similarly, analysis of cross sections revealed a high density of well dispersed NPs, suggesting efficient transport. Further investigation of the transport mechanism revealed that endocytosis indeed does play a significant role in delivering the NPs to the cell interior. While we have shown in our previous publication that the 6-MPR-AuNPs have good luminescence properties, we found that the strength of the signal was insufficient

for imaging and also the cell culture media quenched the luminescence of the NPs. For the purpose of visualizing the process, we have synthesized AuNPs with mixed surface stabilizers of 6-MPR and L-cysteine (Cys) in a molar ratio of 2:1 (see the Experimental section) followed by covalent functionalization of Cys with the fluorescent dye fluorescein isothiocyanate isomer I (FITC) in a 10:1 molar ratio of Cys/FITC (Figure 5A-C). Incubation of the cells with FITC-labeled 6-MPR/CysAuNPs at 37 °C showed a gradual increase of fluorescence inside the cells (Figure 5D-G). Conversely, incubation of the cells at 4 °C showed little or no fluorescence (Figure 5H and I) which is consistent with the blockage of the endocytosis process.48,49 Enhanced intracellular transport may be beneficial, but it still cannot explain the availability of the drug for the de noVo synthesis taking place in the cytoplasm. We believe that the intrinsic similarity of the NPs to many nanoscale biological objects such as proteins,51 viruses, and parts of other cells allows the endocytosed NPs to be transferred to the lysosomes for digestion. The interior of the lysosomes is much more acidic, that is, pH 4.8, than the surrounding cytosol or blood, that is, pH 7. At low pH conditions, the thiol group of mercaptopurine is protonated (51) Tang, Z.; Zhang, Z.; Wang, Y.; Glotzer, S. C.; Kotov, N. A. Science 2006, 314 (5797) 274.

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and the Au-S bond is severed, releasing the free drug, which diffuses through the cell similarly to the free mercaptopurine. Proteins and other products of lysis present in the lysosomes quickly coat the surface of Au and prevent the readsorption of 6-MPR when the NPs are released in the cytoplasm. Also, S-containing amino acids and other biomolecules with thiol groups, such as glutathione and proteins, can simply replace 6-MPR from the Au surface with a similar consequence of 6-MPR release. The latter process was confirmed experimentally by observing a sharp luminescence decrease of the natural fluorescence of 6-MPR AuNPs (no FITC)46 upon addition of 3 µM L-cysteine. For comparison, the blood plasma concentrations of glutathione and L-cysteine are 5 and 10 µM, respectively. The intracellular concentrations of each are on the order of 5 mM for glutathione and 0.5 mM for L-cysteine.

Conclusions The nanometer-scale AuNPs appear to provide a flexible platform for the delivery of drugs with thiol functionalities. Despite the strong Au-S bond, NP carriers may not reduce their activity but they can even enhance it due to increased transport through the cell membrane and eventual drug release in lysosomes. Importantly, control over particle size affords fine optimization of the blood residence time, preventing fast elimination of the small drugs by the kidneys while avoiding scavenging of the

Podsiadlo et al.

carrier by the RES. This study is one of the first steps in understanding the metabolism of AuNPs in the cells. Yet, it demonstrates that important reservations about potential drug activity immobilized on AuNPs may not be prohibitive. In general, the AuNP approach is likely to be particularly useful for small molecule drugs. In addition to the points made above, the small drugs can be loaded in high density on the NP surface and protected from the activity of blood enzymes. If combined with targeting molecules, they will provide a formidable weapon against a wide range of diseases. The delivery mechanism of drug loaded NPs to the cancerous cells including some protection against premature release should be further developed. Acknowledgment. N.A.K. thanks AFOSR, NSF, DARPA, and NRL for the support of this research. P.P. thanks the Fannie and John Hertz Foundation for support of his work through a graduate fellowship. N.W.S.K. thanks the Michigan Society of Fellows for support of her research. The authors thank C. R. Iacovella for help with the graphics. Supporting Information Available: Procedure for and results of the determination of 6-MPR loading on AuNPs, biochemical pathway of 6-MP and 6-MPR metabolism, and AFM characterization of the 6-MPR AuNP sizes. This material is available free of charge via the Internet at http://pubs.acs.org. LA702782K