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Gold-Labeled Block Copolymer Micelles Reveal Gold Aggregates at Multiple Subcellular Sites Patrick Lim Soo,† Stanislav N. Sidorov,‡ Jeannie Mui,§ Lyudmila M. Bronstein,| Hojatollah Vali,§,⊥ Adi Eisenberg,*,† and Dusica Maysinger*,# Department of Chemistry, McGill UniVersity, 801 Sherbrooke Street W., Montreal, Quebec, Canada H3A 2K6, NesmeyanoV Institute of Organoelement Compounds RAS, 28 VaViloV Str., Moscow, Russia 119991, Facility for Electron Microscopy Research, McGill UniVersity, 3640 UniVersity Street, Montreal, Quebec, Canada H3A 2B2, Department of Chemistry, Indiana UniVersity, 800 E. Kirkwood AVenue, Bloomington, Indiana 47405-7012, Department of Anatomy and Cell Biology, McGill UniVersity, 3640 UniVersity Street, Montreal, Quebec, Canada H3A 2B2, and Department of Pharmacology and Therapeutics, McGill UniVersity, 3655 Promenade Sir William Osler, Montreal, Quebec, Canada H3G 1Y6 ReceiVed NoVember 20, 2006. In Final Form: February 13, 2007 There is increasing interest in the usefulness of block copolymer micelles as drug delivery vehicles. However, their subcellular distribution has not been explored extensively, mostly because of the lack of adequately labeled block copolymers. In a previous study, we showed that fluorescently labeled block copolymer micelles entered living cells and co-localized with cytoplasmic organelles selectively labeled with fluorescent dyes. The details of the observed co-localizations were, however, limited by the resolution of the fluorescence approach, which is ca. 500 nm. Using transmission electron microscopy (TEM), we established time- and concentration-dependent subcellular distributions of gold-labeled micelles within human embryonic kidney (HEK 293) cells and human lung carcinoma (A549) cells. Gold particles were incorporated into poly(4-vinylpyridine)-block-poly(ethylene oxide) (P4VP21-b-PEO45) micelles. Data from dynamic light scattering (DLS) and TEM analyses revealed that the sizes of the gold particles ranged from 4 to 8 nm. The cells survived up to 24 h in the presence of low gold-labeled micelle concentrations (0.73 µg/mL), but cell death occurred at higher concentrations (i.e., kidney cells are more susceptible than lung cells). Over 24 h periods of equivalent exposure, lung cells internalized significantly more gold-incorporated micelles than kidney cells. Although micelles were added to the cell culture media as dispersed colloidal particles, the presence of serum in these media caused aggregation. These aggregates occurred mainly close to the cell plasma membrane at early times (5-10 min); however, at later times (24 h) aggregated particles were seen inside endosomes and lysozomes. Thus, goldincorporated (labeled) micelles can serve as a valuable extension of the fluorescence approach to visualizing the localization of micelles in subcellular compartments, improving the resolution by at least 20-fold.
Introduction Amphiphilic block copolymers are versatile materials being explored for applications in medical, cosmetic, environmental, and industrial fields.1 The recent developments of different synthetic techniques and the availability of numerous types of core and corona blocks with varying properties have provided a wide range of block copolymers. In typical micellar applications, these copolymers are composed of a hydrophobic core block and a hydrophilic corona block. Under appropriate conditions, these amphiphilic copolymers can produce different aggregates of a wide range of shapes and sizes (especially spheres, rods, * To whom correspondence should be addressed. (A.E.) E-mail:
[email protected]. Tel: 514-398-6934. Fax: 514-398-3797. (D.M.) E-mail:
[email protected]. Tel: 514-398-1264. Fax: 514-3986690. † Department of Chemistry, McGill University. ‡ Nesmeyanov Institute of Organoelement Compounds RAS. § Facility for Electron Microscopy Research, McGill University. | Indiana University. ⊥ Department of Anatomy and Cell Biology, McGill University. # Department of Pharmacology and Therapeutics, McGill University. (1) Hadjichristidis, N.; Pispas, S.; Floudas, G. A. Block Copolymers: Synthetic Strategies, Physical Properties, and Applications; Wiley & Sons, Inc.: Hoboken, NJ, 2003. (2) Zhang, L.; Eisenberg, A. Science 1995, 268, 1728-1731. (3) Antonietti, M.; Wenz, E.; Bronstein, L.; Seregina, M. AdV. Mater. 1995, 7, 1000-1005. (4) Shen, H.; Zhang, L.; Eisenberg, A. J. Am. Chem. Soc. 1999, 121, 27282740. (5) Zhang, Q.; Remsen, E. E.; Wooley, K. L. J. Am. Chem. Soc. 2000, 122, 3642-3651.
and vesicles).2-11 These aggregates can serve as versatile delivery vehicles for both hydrophobic and hydrophilic agents.12-23 (6) Alexandridis, P., Lindman, B., Eds. Amphiphilic Block Copolymers: SelfAssembly and Applications; Elsevier Science B.V.: Amsterdam, 2000. (7) Discher, D. E.; Eisenberg, A. Science 2002, 297, 967-973. (8) Jain, S.; Bates, F. S. Science 2003, 300, 460-464. (9) Wang, X.-S.; Arsenault, A.; Ozin, G. A.; Winnik, M. A.; Manners, I. J. Am. Chem. Soc. 2003, 125, 12686-12687. (10) Liu, G.; Yan, X.; Li, Z.; Zhou, J.; Duncan, S. J. Am. Chem. Soc. 2003, 125, 14039-14045. (11) Lim Soo, P.; Eisenberg, A. J. Polym. Sci., Part B: Polym. Phys. 2004, 42, 923-938. (12) Gref, R.; Minamitake, Y.; Peracchia, M. T.; Trubetskoy, V.; Torchilin, V.; Langer, R. Science 1994, 263, 1600-1603. (13) Kwon, G. S.; Naito, M.; Kataoka, K.; Yokoyama, M.; Sakurai, Y.; Okano, T. Colloids Surf., B 1994, 2, 429-434. (14) Allen, C.; Maysinger, D.; Eisenberg, A. Colloids Surf., B 1999, 16, 3-27. (15) Kataoka, K.; Matsumoto, T.; Yokoyama, M.; Okano, T.; Sakurai, Y.; Fukushima, S.; Okamoto, K.; Kwon, G. S. J. Controlled Release 2000, 64, 143153. (16) Torchilin, V. P. J. Controlled Release 2001, 73, 137-172. (17) Lavasanifar, A.; Samuel, J.; Kwon, G. S. AdV. Drug DeliVery ReV. 2002, 54, 169-190. (18) Rapoport, N.; Pitt, W. G.; Sun, H.; Nelson, J. L. J. Controlled Release 2003, 91, 85-95. (19) Jones, M.-C.; Ranger, M.; Leroux, J.-C. Bioconjugate Chem. 2003, 14, 774-781. (20) Riley, T.; Heald, C. R.; Stolnik, S.; Garnett, M. C.; Illum, L.; Davis, S. S.; King, S. M.; Heenan, R. K.; Purkiss, S. C.; Barlow, R. J.; Gellert, P. R.; Washington, C. Langmuir 2003, 19, 8428-8435. (21) Francis, M. F.; Piredda, M.; Winnik, F. M. J. Controlled Release 2003, 93, 59-68. (22) Tang, Y.; Liu, S. Y.; Armes, S. P.; Billingham, N. C. Biomacromolecules 2003, 4, 1636-1645. (23) Liu, J.; Xiao, Y.; Allen, C. J. Pharm. Sci. 2004, 93, 132-143.
10.1021/la063375s CCC: $37.00 © 2007 American Chemical Society Published on Web 03/29/2007
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Biocompatible block copolymers are particularly promising for use as drug delivery vehicles. Detection of the block copolymer micelles is crucial in determining their subcellular fate and the mechanism of entry into the cell. Typically, fluorescent molecules either incorporated into or attached to the micelles have been used.24-30 It has been proposed that polymeric micelles are internalized into cells by an endocytotic mechanism.24-26,29 This was supported by our earlier results that suggested that polycaprolactone-block-poly(ethylene oxide) micelles were internalized into rat adrenal pheochromocytoma (PC12) cells by endocytosis.31 More recent results obtained by confocal microscopy have shown that micelles labeled with rhodamine were localized in different organelles of PC12 and fibroblasts of Swiss mouse embryo cells.32 These fluorescence and confocal microscopy studies identified the co-localization of micelles with fluorescent labels for organelles with a resolution of a few hundred nanometers. This limitation of optical microscopy does not allow for the identification of individual micelles. Also, even though the use of fluorescent probes is simple and can be used for realtime imaging, photobleaching is another limitation.33 Heavy-metal-labeled materials, because of their high electron density and stability, could be incorporated into micelles to allow the visualization of individual micelles by transmission electron microscopy (TEM), which would pinpoint their location in vitro and in vivo with much better resolution than is achievable by optical microscopy. To date, however, there are no TEM reports showing the presence of individual micelles in subcellular compartments, although many groups have investigated the association between heavy metals and polymers for various applications.34-39 Block copolymers are particularly useful in this context for a number of reasons. For example, the block copolymers are effective as colloidal stabilizers, which allow for the control of particle size and the size distribution of metal particles; they can provide an environment that prevents corrosion and leakage of heavy metals, and they can also protect catalytic nanoparticles from deactivation.3,40,41 Block copolymer micelles containing a polystyrene core and a functional corona have been used to produce gold nanoparticles3,42 and cadmium sulfide
nanoparticles.43,44 Recently, other copolymer systems have also been used for the preparation of metal particles.45-47 For block copolymers, the selection of the core-forming block is important because a strong interaction between the polymer core and the metal precursor facilitates its incorporation into the micelle. Thus, gold-labeled micelles have been prepared using diblocks of either poly(2-vinylpyridine) (P2VP)48 or poly(4vinylpyridine) (P4VP)49 and poly(ethylene oxide) (PEO) in water. Gold nanoparticles formed from P2VP135-b-PEO350 micelles ranged in size from 1 to 4 nm, whereas gold nanoparticles formed from P4VP28-b-PEO45 ranged in size from 5 to 10 nm. The advantage of gold-labeled micelles with poly(ethylene oxide) coronas is that they allow for their preparation in water, an environmentally friendly option. Also, the ability of the PEO block to reduce the absorption of proteins and thus reduce the clearance of the micelles by the reticuloendothelial system in the body is important.50 We have chosen to use P4VP as the coreforming block because it coordinates a wide range of metal ions in the micelle core more strongly than does P2VP.3 The interaction with P4VP is less sterically hindered compared to that of P2VP, thus P4VP would result in a larger degree of gold incorporation and in more stable gold colloids.51 In this study, we prepared P4VP21-b-PEO45 micelles that contain single gold nanoparticles ranging in size from 4 to 8 nm within each individual micelle. Using these gold-labeled micelles, we applied a complementary approach to confocal microscopy, namely, transmission electron microscopy (TEM), to explore the internalization of the micelles into and their visualization in two different types of cell (human embyronic kidney (HEK 293) cells and human lung carcinoma (A549) cells) for different time periods (5 min to 24 h) and different concentrations (0.15 to 50 µg/mL). We now report TEM studies showing gold-labeled micelles predominantly in the subcellular compartments, specifically, endosomes and lysozomes; the micelles were conspicuously absent from the mitochondria. The findings demonstrate the usefulness of the gold-labeled block copolymers as a valuable tool in studies of the subcellular localization of micelles.
(24) Kabanov, A. V.; Slepnev, V. I.; Kuznetsova, L. E.; Batrakova, E. V.; Alakhov, V. Y.; Melik-Nubarov, N. S.; Sveshnikov, P. G.; Kabanov, V. A. Biochem. Int. 1992, 26, 1035-1042. (25) Batrakova, E. V.; Han, H.-Y.; Miller, D. W.; Kabanov, A. V. Pharm. Res. 1998, 15, 1525-1532. (26) Liaw, J.; Aoyagi, T.; Kataoka, K.; Sakurai, Y.; Okano, T. Pharm. Res. 1999, 16, 213-220. (27) Rapoport, N.; Marin, A. P.; Timoshin, A. A. Arch. Biochem. Biophys. 2000, 384, 100-108. (28) Liu, J.; Zhang, Q.; Remsen, E. E.; Wooley, K. L. Biomacromolecules 2001, 2, 362-368. (29) Zastre, J.; Jackson, J.; Bajwa, M.; Liggins, R.; Iqbal, F.; Burt, H. Eur. J. Pharm. Biopharm. 2002, 54, 299-309. (30) Zeng, F.; Lee, H.; Allen, C. Bioconjugate Chem. 2006, 17, 399-409. (31) Allen, C.; Yu, Y.; Eisenberg, A.; Maysinger, D. Biochim. Biophys. Acta 1999, 1421, 32-38. (32) Savic, R.; Luo, L.; Eisenberg, A.; Maysinger, D. Science 2003, 300, 615-618. (33) Stephens, D. J.; Allan, V. J. Science 2003, 300, 82-86. (34) Marinakos, S. M.; Novak, J. P.; Brousseau, L. C., III; House, A. B.; Edeki, E. M.; Feldhaus, J. C.; Feldheim, D. L. J. Am. Chem. Soc. 1999, 121, 8518-8522. (35) Otsuka, H.; Akiyama, Y.; Nagasaki, Y.; Kataoka, K. J. Am. Chem. Soc. 2001, 123, 8226-8230. (36) Ohno, K.; Koh, K.-m.; Tsujii, Y.; Fukuda, T. Macromolecules 2002, 35, 8989-8993. (37) Sun, Y.; Xia, Y. Science 2002, 298, 2176-2179. (38) Kamata, K.; Lu, Y.; Xia, Y. J. Am. Chem. Soc. 2003, 125, 2384-2385. (39) Corbierre, M. K.; Cameron, N. S.; Sutton, M.; Mochrie, S. G. J.; Lurio, L. B.; Ruehm, A.; Lennox, R. B. J. Am. Chem. Soc. 2001, 123, 10411-10412. (40) Mayer, A. B. R.; Mark, J. E. Colloid Polym. Sci. 1997, 275, 333-340. (41) Forster, S.; Antonietti, M. AdV. Mater. 1998, 10, 195-217. (42) Spatz, J. P.; Roescher, A.; Moeller, M. AdV. Mater. 1996, 8, 337-340.
Synthesis of P4VP21-b-PEO45 Copolymer. The P4VP21-b-PEO45 copolymer was synthesized using atom-transfer radical polymerization of the 4-vinylpyridine monomer52 and poly(ethylene oxide) with a 2-chloropropionyl-terminated group as a macroinitiator. A full description of the synthesis can be found in our previous publication.49 Preparation of Gold-Labeled Micelles. P4VP21-b-PEO45 copolymer was dissolved in water to form a solution with a concentration of 4 g/L. Gold(III) chloride (AuCl3 99.99+%, Aldrich) was added to the micelle solution to obtain a 4:1 molar ratio of 4-vinylpyridine to gold. When AuCl3 is dissolved in the aqueous solution, immediate
Experimental Section
(43) Moffitt, M.; Eisenberg, A. Chem. Mater. 1995, 7, 1178-1184. (44) Moffitt, M.; Vali, H.; Eisenberg, A. Chem. Mater. 1998, 10, 1021-1028. (45) Liu, S.; Weaver, J. V. M.; Save, M.; Armes, S. P. Langmuir 2002, 18, 8350-8357. (46) Jungmann, N.; Schmidt, M.; Maskos, M. Macromolecules 2003, 36, 39743979. (47) Bae, K. H.; Choi, S. H.; Park, S. Y.; Lee, Y.; Park, T. G. Langmuir 2006, 22, 6380-6384. (48) Bronstein, L. M.; Sidorov, S. N.; Valetsky, P. M.; Hartmann, J.; Coelfen, H.; Antonietti, M. Langmuir 1999, 15, 6256-6262. (49) Sidorov, S. N.; Bronstein, L. M.; Kabachii, Y. A.; Valetsky, P. M.; Lim Soo, P.; Maysinger, D.; Eisenberg, A. Langmuir 2004, 20, 3543-3550. (50) Lee, J. H.; Lee, H. B.; Andrade, J. D. Prog. Polym. Sci. 1995, 20, 10431079. (51) Mayer, A. B. R.; Mark, J. E. Eur. Polym. J. 1998, 34, 103-108. (52) Xia, J.; Zhang, X.; Matyjaszewski, K. Macromolecules 1999, 32, 35313533.
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Figure 1. Scheme of the preparation of gold-labeled micelles. hydrolysis leads to the formation of an acid, HAuCl3(OH).53 The 4VP units are protonated by the acid, thus electrostatic interaction between the AuCl3(OH)- ions and the protonated pyridine groups is the driving force for the incorporation of AuCl3(OH)- ions into the P4VP core, which is protected by the PEO corona. The resulting micellar solution was allowed to stir for 2 days. A 5-fold molar excess of hydrazine hydrate (N2H4‚H2O, 98+%) was then added to the solution, turning it from yellow to dark purple. The solution was allowed to stir for 1 day. Upon reduction of the gold ions inside the P4VP21-b-PEO45 micelles by hydrazine hydrate, a solution of micellelabeled gold particles was obtained, which we will refer to as goldlabeled micelles. For the internalization experiments, the micelles were filtered using a 0.2 µm Nalgene filter and then lyophilized to remove the water. To avoid contamination, the micelles were reconstituted in cell culture serum-free media corresponding to the cell type of interest (i.e., Roswell Park Memorial Institute 1640 medium for HEK 293 cells and Dulbecco’s modified Eagle’s medium for A549 cells). Characterization of Gold-Labeled Micelles. (i) UV-Vis. Measurements on the gold-labeled micelles were made on a Cary 50 Bio UV-visible spectrophotometer (Varian), equipped with two silicon diode detectors and a xenon flash lamp. (ii) Dynamic Light Scattering (DLS). The sizes and size distributions of the gold-labeled micelles were determined on a Brookhaven Instruments photon correlation spectrophotometer with a BI-9000AT digital correlator. The instrument was equipped with a compass 315M-150 laser (Coherent Technologies) that was used at a wavelength of 532 nm. Dust-free solution vials were used for the aqueous micelle solutions, and measurements were performed at an angle of 90° at room temperature. The CONTIN algorithm was used to analyze the DLS data. (iii) Transmission Electron Microscopy (TEM). The gold-labeled micelles were examined with a JEOL JEM-2000FX TEM operating at an accelerating voltage of 80 kV. Dilute solutions of the goldlabeled micelles were deposited onto 400-mesh copper grids (Electron Microscopy Sciences) that were precoated with a thin film of Formvar (poly(vinylformal)) and carbon. The samples were allowed to sit on the grids for a few seconds, and then a blotter was applied to remove the excess solution. The grids were then left overnight to air dry at room temperature. The samples were stained with uranyl acetate (for the PEO corona) for 2 min. Digital images were taken with a Gatan 792 Bioscan 1k × 1k wide angle multiscan CCD camera (JEM-2000 FX). Treatment of Cells with Gold-Labeled Micelles. (i) Cell Types. The A549 cells have characteristic lamellar bodies (i.e., loose and (53) Wilkinson, G. ComprehensiVe Coordination Chemistry : The Synthesis, Reactions, Properties, and Applications of Coordination Compounds; Pergamon Press: New York, 1987.
dense). The cells serve as a model for type II alveolar epithelial cells of the lung.54 In the case of HEK 293 cells, they are epitheloid in nature.55 Specific information on the cell culture of the HEK 293 and A549 cells and the cell culture treatment for the purposes of TEM can be found in Supporting Information. (ii) Cell Viability. Cells were plated in 24-well plates (1 × 104/ well). Twenty-four hours after the plating, cells were treated with gold-labeled micelles for 24 h. The media were removed, and the cells were harvested with trypsin, followed by staining with 0.4% trypan blue. The trypan blue-positive cells were counted using a hemocytometer and a phase contrast microscope. (iii) Mitochondrial ActiVity Assay. An MTT (3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetrazolium bromide) assay was used to assess the metabolic status of the HEK 293 and A549 cells in the presence of gold-labeled micelles.56,57 More detailed information about the MTT assay can be found in Supporting Information. (iV) Internalization of Gold-Labeled Micelles and Drug Treatments. Cells were plated in six-well plates (5 × 106 cells/well) and deprived of serum (for 4 h) before the addition of drugs or goldlabeled micelles. Maximal internalization of the micelles was determined in the absence of any drug. Cells in the other wells were treated with drug inhibitors as follows: sucrose (0.45 M for 30 min), methyl-β-cyclodextrin (10 mM for 1 h), and chlorpromazine (50 µM for 30 min). Absorbances were read at 540 nm, and the internalization was determined from the linear portion of the calibration curve. Time-dependent internalization was assessed between 5 min and 24 h. Inhibition experiments were performed at a fixed time (1 h) after treatment with micelles. All experiments were performed in triplicate (n ) 3). The results are expressed as mean values calculated from at least two independent experiments. Changes were considered to be different from controls when p < 0.05 (ANOVA). (V) TEM. For the study of the internalization of gold-labeled micelles into cells, ultrathin sections (70-100 nm) were prepared from a monolayer of cells using an Ultracut-E ultramicrotome (Reichert-Jung, Leica Microsystems, Austria). The ultrathin sections were transferred onto 200-mesh copper grids (Electron Microscopy Sciences). The grids were then doubly stained with uranyl acetate (negative stain for background) and Reynolds lead citrate stain (provides contrast for cellular membranes). (Vi) Energy-DispersiVe Spectrometry. Elemental microanalysis of the samples was performed using a Quartz XOne energy-dispersive spectrometer (Quartz Imaging Corporation, Canada). The electron (54) Lieber, M.; Smith, B.; Szakal, A.; Nelson-Rees, W.; Todaro, G. Int. J. Cancer 1976, 17, 62-70. (55) Simmons, N. L. Exp. Physiol. 1990, 75, 309-319. (56) Mosmann, T. J. Immunol. Methods 1983, 65, 55-63. (57) Denizot, F.; Lang, R. J. Immunol. Methods 1986, 89, 271-277.
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Figure 2. (a) Ultraviolet-visible spectra of the P4VP21-b-PEO45 copolymer micelles (-) and gold-labeled P4VP21-b-PEO45 micelles (--). (b) Transmission electron microscopy images of gold-labeled P4VP21-b-PEO45 micelles with original magnifications of (left) 170 000× and (right) 210 000×, both with uranyl acetate staining. One gold nanoparticle per micelle is seen. (c) Histogram of TEM images of gold nanoparticles in P4VP21-b-PEO45 micelles. (d) Histogram of TEM images of gold-labeled P4VP21-b-PEO45 micelles.
beam of the TEM was specifically positioned inside cell structures to confirm the location of gold-labeled micelles.
Results and Discussion Properties of Gold-Labeled Micelles. Poly(4-vinylpyridine)block-poly(ethylene oxide) (P4VP21-b-PEO45) copolymer was dissolved in water and yielded micelles by direct dissolution as shown in Figure 1. The UV-visible spectra of the yellowish solution of the copolymer micelles in water show a minimal absorbance contribution in the 300-800 nm region as shown in Figure 2a. The copolymer micelles absorb at around 200 nm. However, when gold particles are formed inside the micelles, there is a peak at 540 nm (Figure 2a) that corresponds to the plasmon band of the purple micellar solution, suggesting the formation of gold nanoparticles. The block copolymer serves as a stabilizer of the gold nanoparticles and controls their size. The size of the nonlabeled P4VP21-b-PEO45 micelles (21-24 nm) was previously reported by Sidorov et al.49 TEM was used to study the morphology directly and to identify the presence of gold particles. The gold particle is seen as a very dark circle in TEM, as shown in Figure 2b. The sample was additionally stained with uranyl acetate, which normally stains the PEO corona, so the micelle coronas are seen at higher magnification as lighter gray areas surrounding the darker gold nanoparticle. Also, a clear separation is seen between the individual gold particles, which could indicate the presence of polymer between the gold particles. The TEM images clearly show that each micelle contains a single gold nanoparticle.
We have estimated that there are approximately 3.3 × 103 gold atoms per micelle. If we assume that the particles are spherical, then we obtain a particle radius of ca. 2.5 nm, thus the diameter of a gold particle would be approximately 5 nm. (See Supporting Information for details.) This is consistent with the TEM images seen in Figure 2b. A histogram of the TEM data based on a sample size of 200 shows that the sizes of the gold particles also have a typical Gaussian distribution with a diameter of 6 ( 2 nm (average ( standard deviation) as seen in Figure 2c. The average diameter of the whole micelle was found by TEM to be 22 ( 4 nm as seen in Figure 2d. Dynamic light scattering (DLS) was also used to examine the size and size population distribution of the P4VP21-b-PEO45 gold-labeled micelles. The average diameter of the whole micelle was found to be ca. 18 nm, as calculated using Contin analysis by number. This corroborates the average diameter found by TEM to be 22 ( 4 nm. (See Supporting Information for the DLS graph.) Cell Viability and Internalization of Gold-Labeled Micelles. P4VP-b-PEO micelles were used as a tool to observe their localization in different cell types; however, the toxicity of the P4VP copolymer was of special interest. Because the P4VP units are readily adsorbed on the gold nanoparticle surface,58 we did not anticipate the rapid detachment of the block copolymer molecules in the cell. However, it is possible that some of the unimers enveloping the gold nanoparticles (or their aggregates) (58) Bronstein, L. M.; Chernyshov, D. M.; Volkov, I. O.; Ezernitskaya, M. G.; Valetsky, P. M.; Matveeva, V. G.; Sulman, E. M. J. Catal. 2000, 196, 302314.
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Figure 3. (a) Cell survival data after 24 h of exposure to gold-labeled micelles. (b) MTT assay: incubation of 0.73 µg/mL gold-labeled micelles in HEK 293 cells and lung A549 cells after 24 h. (c) Uptake of lung cells in the presence of gold-labeled micelles (2 µg/mL) for 24 h. (d) Inhibition of the internalization of gold-labeled micelles by drugs.
might have been present in the cytosolic compartment. As a result, some cytotoxicity might have been seen, most likely as a result of the lipophilic moiety of the block copolymer, P4VP. Nevertheless, to explore the toxicity aspect fully, additional information was needed concerning the concentration and time dependence of the internalization of the gold-labeled micelles into the cells. To determine if the gold-labeled micelles are biocompatible and if they are taken up by the cells, we tested them over a wide concentration range from 0.01 to 50 µg/mL. The optimal concentration range was between the 0.25 and 1 µg/mL as seen in Figure 3a. Micelles in the lower concentration range could not be detected either by spectrophotometry or TEM, and concentrations above 10 µg/mL were toxic. The viability of cells (refer to Figure 3b) was determined by cell counting using a trypan blue exclusion assay. Mitochondrial activity was determined by the reduction of MTT of both HEK 293 and lung A549 cells. A decrease in the viability and metabolic activity in the presence of gold-labeled micelles was time- and concentration-dependent. The cleavage of the yellow MTT salt by the mitochondrial enzyme succinate dehydrogenase results in a purple formazan product.57 As shown in Figure 3b, there was a greater than 86 ( 6% cell survival compared with untreated controls for the HEK 293 cells when exposed to the micelles for 24 h. Similarly, the MTT results for the lung 549 cells show that more than 91 ( 5% of the cells survived compared with untreated controls after exposure to 0.73 µg/mL gold-labeled micelles for 24 h. Both cell types survive at low micelle concentration (
