Monolayer-Protected Gold Nanoparticles by the Self-Assembly of

Department of Chemistry, McGill University, 801 Sherbrooke Street West, Montreal, H3A2K6, Quebec, Canada ... Au/Block (4/1) nanoparticle dispersions i...
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Langmuir 2007, 23, 2126-2132

Monolayer-Protected Gold Nanoparticles by the Self-Assembly of Micellar Poly(ethylene oxide)-b-Poly(E-caprolactone) Block Copolymer Tony Azzam and Adi Eisenberg* Department of Chemistry, McGill UniVersity, 801 Sherbrooke Street West, Montreal, H3A2K6, Quebec, Canada ReceiVed September 20, 2006. In Final Form: NoVember 6, 2006 A study is presented of the preparation of gold nanoparticles incorporated into biodegradable micelles. Poly(ethylene oxide)-b-poly(-caprolactone) (PEO-b-PCL) copolymer was synthesized by ring-opening polymerization, and the hydroxyl end group of the PCL block was modified with thioctic acid using dicyclohexyl carbodiimide as the coupling reagent. The PEO-b-PCL-thioctate ester (TE) thus obtained was used in a later step to form monolayer protected gold nanoparticles via the thioctate spacer. Gold nanoparticles stabilized with the PEO-b-PCL block (named Au/Block (x/y), where x/y is the mole feed ratio between HAuCl4 and PEO-b-PCL-TE) were prepared and analyzed. Au/Block (1/1), Au/Block (2/1), and Au/Block (3/1) nanoparticles were found to form stable dispersions in the organic solvents commonly used to dissolve the unlabeled block copolymer. The average diameter of the nanoparticles was determined by transmission electron microscopy (TEM) and found to be 6 ( 2 nm. Au/Block (4/1) nanoparticle dispersions in organic solvents, on the other hand, were not stable and produced large gold clusters (50-100 nm). Cluster formation was attributed to the low grafting density of the block copolymer, which facilitates agglomeration. For Au/Block (12/1), along the same trend, only an insoluble product was isolated. Micelles in water were prepared by the slow addition of the dilute Au/Block solution in dimethylformamide into a large excess of water with vigorous stirring. Au/Block (1/1) and Au/Block (2/1) formed nanosized structures of 5-7 nm. TEM images of stained Au/Block (1/1) micelles, made in water, clearly showed the formation of core-shell structures. Au/Block (3/1) micelles, on the other hand, were not stable and large agglomerates a few microns in size were observed. The study focuses on the synthesis, characterization, and aggregation behavior of gold-loaded PEO-b-PCL block copolymer micelles, a potential system for drug delivery in conjunction with tissue and subcellular localization studies.

Introduction Synthetic copolymer materials have been used as the essential building blocks of drug carriers in the form of polymer-drug conjugates,1,2 block copolymer micelles,3-5 and micro(nano)particles.6 Among the various copolymers used in drug delivery, amphiphilic block copolymers are of great interest due to their unique structures and properties, which can be fine-tuned to enable the preparation of a drug carrier with the desired characteristics.7 In aqueous solution, the micellar aggregates consist of a hydrophobic core surrounded by a hydrophilic shell or corona. The core of the micelles acts as a microreservoir for the incorporation of lipophilic drugs, while the hydrophilic shell serves as the stabilizer. Lipophilic drugs and other agents can * Author to whom correspondence should be addressed. E-mail: [email protected]. Telephone: +1 514 3986934. Fax: +1 514 3983797. (1) Ulbrich, K.; Etrych, T.; Chytil, P.; Pechar, M.; Jelinkova, M.; Rihova, B. Polymeric anticancer drugs with pH-controlled activation. Int. J. Pharm. 2004, 277 (1-2), 63-72. (2) Duncan, R.; Vicent, M. J.; Greco, F.; Nicholson, R. I.; Polymer-drug conjugates: towards a novel approach for the treatment of endocrine-related cancer. Endocr.-Relat. Cancer 2005, 12 (Suppl. 1), S189-S199. (3) Allen, C.; Eisenberg, A.; Mrsic, J.; Maysinger, D. PCL-b-PEO micelles as a delivery vehicle for FK506: assessment of a functional recovery of crushed peripheral nerve. Drug DeliVery 2000, 7 (3), 139-145. (4) Kataoka, K.; Harada, A.; Nagasaki, Y. Block copolymer micelles for drug delivery: design, characterization and biological significance. AdV. Drug DeliVery ReV. 2001, 47 (1), 113-131. (5) Kabanov, A. V.; Batrakova, E. V.; Sriadibhatla, S.; Yang, Z.; Kelly, D. L.; Alakov, V. Y. Polymer genomics: shifting the gene and drug delivery paradigms. J. Controlled Release 2005, 101 (1-3), 259-271. (6) Reddy, L. H. Drug delivery to tumors: recent strategies. J. Pharm. Pharmacol. 2005, 57 (10), 1231-1242. (7) Lee, H.; Zeng, F.; Dunne, M.; Allen, C. Methoxy poly(ethylene glycol)block-poly(δ-valerolactone) copolymer micelles for formulation of hydrophobic drugs. Biomacromolecules 2005, 6 (6), 3119-3128. (8) Vasir, J. K.; Reddy, M. K.; Labhasetwar, V. D. Nanosystems in drug targeting: opportunities and challenges. Curr. Nanosci. 2005, 1 (1), 47-64.

be incorporated into the inner core of the polymeric micelles either by chemical conjugation or physical dispersion. In fact, many hydrophobic low molecular weight drugs have been successfully incorporated into the hydrophobic cores of various block copolymer micelles.8 In addition, the use of block copolymers containing ionic and nonionic blocks, in which the ionic blocks interact with oppositely charged agents to form the core of the complex micelles, has been studied extensively, in particular, in connection with the delivery of biologically active macromolecules such as proteins and DNA.9 In this group, poly(ethylene oxide)-b-poly(-caprolactone) (PEO-b-PCL) diblock copolymer micelles have been explored as a drug delivery system. PCL is a well-known biodegradable polymer that has been utilized in various biomedical applications because of its excellent biocompatibility and degradability. PEO-b-PCL based micelles have been found to be excellent drug carriers for lipophilic drugs, such as FK506, L-685,818, dihydrotestosterone and 17βestradiol.3,10,11 PEO serves as the hydrophilic block in the corona of the micelles. In fact, PEO is one of a few water-soluble polymers that have been widely used to improve the biocompatibilities of drug carriers. PEO is believed to prevent the uptake of the carriers by the reticuloendothelial system, which thus increases their lifetime in blood circulation.12 An understanding of carrier internalization into cellular and subcellular compartments is needed to develop drug delivery (9) Kabanov, A. V.; Sriadibhatla, S.; Alakhov, V. Y. Pluronic block copolymers for nonviral gene delivery. Polym. Gene DeliVery 2005, 313-328. (10) Allen, C.; Yu, Y.; Maysinger, D.; Eisenberg, A. Polycaprolactone-bpoly(ethylene oxide) block copolymer micelles as a novel drug delivery vehicle for neurotrophic agents FK506 and L-685,818. Bioconjugate Chem. 1998, 9 (5), 564-572. (11) Soo, P. L.; Lovric, J.; Davidson, P.; Maysinger, D.; Eisenberg, A. Polycaprolactone-block-poly(ethylene oxide) micelles: a nanodelivery system for 17β-estradiol. Mol. Pharm. 2005, 2 (6), 519-527.

10.1021/la0627563 CCC: $37.00 © 2007 American Chemical Society Published on Web 12/24/2006

Preparation of Monolayer-Protected Au Nanoparticles

systems.13,14 At present, little is known about the cellular internalization of polymeric micelles.15 Kabanov’s group reported that pluronic-based micelles can be internalized by an endocytotic pathway, and pluronic copolymers can increase drug absorption by inhibition of the P-glycoprotein drug efflux system in cells.16 In previous studies conducted by our group, PEO-b-PCL micelles were also shown to be internalized in PC12 cells via an endocytotic pathway.13,17 However, since the probes used in these studies were physically incorporated in the micelles, no direct evidence has been provided for the internalization of the polymer micelles themselves. Recently, fluorescently labeled PEO-b-PCL block copolymer was prepared by conjugating rhodamine to the end of the hydrophobic PCL block.18-20 Transmission electron microscopy (TEM) as well as dynamic light scattering (DLS) studies showed that the chemical labeling did not affect the morphology or the size of the micelles. Studies of the cellular uptake of the labeled micelles suggested that internalization proceed through a endocytotic pathway.18,19 An alternative for fluorophores for cellular labeling is the use of inert metallic quantum dots such as gold nanoparticles.21 The metallic nature of the gold nanoparticles offers enough contrast for imaging with electron microscopy. Since the resolution in electron microscopy is much better than in optical microscopy, biological structures can be visualized with a resolution of a few nanometers.22 Furthermore, the use of gold nanoparticles instead of fluorophores circumvents the drawbacks of photobleaching and blinking.23 Gold nanoparticles, in general, have been the subject of increasing attention for the past decade, particularly since Brust et al. introduced a simple method for the preparation of monodisperse gold nanoparticles stabilized with various thiolcontaining ligands.24 Gold nanoparticles are of special interest due to their potential applications in biomedical, electronic, and optical materials.25 In biomedical applications, gold nanoparticles (12) Caliceti, P.; Veronese, F. M. Pharmacokinetic and biodistribution properties of poly(ethylene glycol)-protein conjugates. AdV. Drug DeliVery ReV. 2003, 55 (10), 1261-1277. (13) Maysinger, D.; Berezovska, O.; Savic, R.; Lim Soo, P.; Eisenberg, A. Block copolymers modify the internalization of micelle-incorporated probes into neural cells. Biochim. Biophys. Acta: Mol. Cell Res. 2001, 1539 (3), 205-217. (14) Kabanov, A. V., Batrakova, E. V. New technologies for drug delivery across the blood brain barrier. Curr. Pharm. Des. 2004, 10 (12), 1355-1363. (15) Miller, D. W.; Batrakova, E. V.; Waltner, T. O.; Alakhov, V.; Kabanov, A. V. Interactions of pluronic block copolymers with brain microvessel endothelial cells: evidence of two potential pathways for drug absorption. Bioconjugate Chem. 1997, 8 (5), 649-657. (16) Batrakova, E. V.; Miller, D. W.; Li, S.; Alakhov, V. Y.; Kabanov, A. V.; Elmquist, W. F. Pluronic P85 enhances the delivery of digoxin to the brain: in vitro and in vivo studies. J. Pharmacol. Exp. Ther. 2001, 296 (2), 551-557. (17) Allen, C.; Yu, Y.; Eisenberg, A.; Maysinger, D. Cellular internalization of PCL20-b-PEO44 block copolymer micelles. Biochim. Biophys. Acta: Biomembr. 1999, 1421 (1), 32-38. (18) Luo, L.; Tam, J.; Maysinger, D.; Eisenberg, A. Cellular internalization of poly(ethylene oxide)-b-poly(-caprolactone) diblock copolymer micelles. Bioconjugate Chem. 2002, 13 (6), 1259-1265. (19) Savic, R.; Luo, L.; Eisenberg, A.; Maysinger, D. Micellar nanocontainers distribute to defined cytoplasmic organelles. Science (Washington, D.C.) 2003, 300 (5619), 615-618. (20) Savic, R.; Azzam, T.; Eisenberg, A.; Maysinger, D. Assessment of the integrity of poly(caprolactone)-b-poly(ethylene oxide) micelles under biological conditions: a fluorogenic-based approach. Langmuir 2006, 22 (8), 3570-3578. (21) Parak, W. J.; Pellegrino, T.; Plank, C. Labelling of cells with quantum dots. Nanotechnology 2005, 16 (2), R9-R25. (22) Koster, A. J.; Klumperman, J. Electron microscopy in cell biology: integrating structure and function. Nat. Cell Biol. 2003, 5, SS6-S10. (23) Hermann, R.; Walther, P.; Muller, M. Immunogold labeling in scanning electron microscopy. Histochem. Cell Biol. 1996, 106 (1), 31-39. (24) Brust, M.; Fink, J.; Bethell, D.; Schiffrin, D. J.; Kiely, C. Synthesis and reactions of functionalized gold nanoparticles. J. Chem. Soc., Chem. Commun. 1995, 16, 1655-1656. (25) Vossmeyer, T.; Guse, B.; Besnard, I.; Bauer, R. E.; Mullen, K.; Yasuda, A. Gold nanoparticle/polyphenylene dendrimer composite films. Preparation and vapor-sensing properties. AdV. Mater. (Weinheim, Ger.) 2002, 14 (3), 238-242.

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are often used to enhance the sensitivity of diagnostic assays,26 in radiotherapy,27 as well as in drug and gene delivery.28 Surface modification of gold nanoparticles is essential for cellular targeting. Such modification of antibodies and other targeting moieties is usually achieved by the adsorption or chemical conjugation of the ligand to the gold surface.29 Polymernanoparticles of core-shell structures were extensively studied by a number of groups. These studies involve the use of synthetic polymers for many potential applications such as chemical catalysis, lithography, optical properties, semiconductor quantum dots, and nanoscience, among others.30-33 In this paper, we report on the preparation of gold nanoparticles loaded into micelles made from biodegradable and biocompatible PEO-b-PCL block copolymer, which represent an alternative method for studies of subcellular localization. To our knowledge, this is the first attempt in using monolayer-protected gold nanoparticles for labeling micelles made from biodegradable and biocompatible components, which has potential application in drug delivery coupled with localization studies. Experimental Section Materials. Polyethylene oxide monomethyl ether (number average molecular weight, Mn ca. 2000), -caprolactone (99%), stannous (II) octoate (SnOct, ∼95%), thioctic acid (98%), 4-(dimethylamino) pyridine (DMAP, 99%), 1,3-dicyclohexyl carbodiimide (DCC, 99%), hydrogen tetrachloroaurate (III) trihydrate (HAuCl4‚3H2O, 99.9%), and phosphotungstic acid were obtained from Aldrich. A 0.25 M LiBH4 solution in tetrahydrofuran (THF) was freshly prepared by diluting commercial 2 M LiBH4 (Aldrich) with freshly distilled THF. PEO was dried by azeotropic distillation from toluene followed by storage under vacuum at 60 °C for 24 h. -Caprolactone was dried twice over CaH2 and distilled under reduced pressure just before use. Dichloromethane (CH2Cl2, 99% +, Aldrich) was purified by shaking with a few portions of sulfuric acid until the acid layer remained colorless. Then CH2Cl2 was washed with water (twice), washed with 5% NaHCO3, washed again with water, and left under anhydrous CaCl2 overnight. The CH2Cl2 was then mixed with CaH2 and distilled just before use. Triethylamine (TEA, 99.5%, Fluka) was treated with BaO for one week and distilled under vacuum just before use. THF (HPLC grade) was refluxed over sodium/ benzophenone ketyl and distilled under a nitrogen atmosphere. All other chemicals and solvents were of analytical grade and were used as received. Synthesis of PEO-b-PCL. PEO-b-PCL block copolymer was synthesized by ring-opening polymerization according to Bogdanov et al.34 Briefly, 6.0 mL of freshly distilled -caprolactone (54 mmol) (26) Goodman, C. M.; McCusker, C. D.; Yilmaz, T.; Rotello, V. M. Toxicity of gold nanoparticles functionalized with cationic and anionic side chains. Bioconjugate Chem. 2004, 15 (4), 897-900. (27) Hainfeld, J. F.; Slatkin, D.; Smilowitz, H. M. The use of gold nanoparticles to enhance radiotherapy in mice. Phys. Med. Biol. 2004, 49 (18), N309-N315. (28) Thomas, M.; Klibanov Alexander, M. Conjugation to gold nanoparticles enhances polyethylenimine’s transfer of plasmid DNA into mammalian cells. Proc. Natl. Acad. Sci. U.S.A. 2003, 100 (16), 9138-9143. (29) Lin, Z.; Su, X.; Mu, Y.; Jin, Q. Methods for labeling quantum dots to biomolecules. J. Nanosci. Nanotechnol. 2004, 4 (6), 641-645. (30) Yun, S.-H.; Sohn, B.-H.; Jung, J. C.; Zin, W.-C.; Ree, M.; Park, J. W. Micropatterning of a single layer of nanoparticles by lithographical methods with diblock copolymer micelles. Nanotechnology 2006, 17 (2), 450-454. (31) Genson, K. L.; Holzmueller, J.; Jiang, C.; Xu, J.; Gibson, J. D.; Zubarev, E. R.; Tsukruk, V. V. Langmuir-Blodgett monolayers of gold nanoparticles with amphiphilic shells from V-shaped binary polymer arms. Langmuir 2006, 22 (16), 7011-7015. (32) Shan, J.; Chen, J.; Nuopponen, M.; Viitala, T.; Jiang, H.; Peltonen, J.; Kauppinen, E.; Tenhu, H. Optical properties of thermally responsive amphiphilic gold nanoparticles protected with polymers. Langmuir 2006, 22 (2), 794-801. (33) Bennett, R. D.; Hart, A. J.; Cohen, R. E. Controlling the morphology of carbon nanotube films by varying the areal density of catalyst nanoclusters using block-copolymer micellar thin films. AdV. Mater. (Weinheim, Ger.) 2006, 18 (17), 2274-2279. (34) Bogdanov, B.; Vidts, A.; Van Deb Bulke, A.; Verbeeck, R.; Schacht, E. Synthesis and thermal properties of poly(ethylene glycol)-poly(-caprolactone) copolymers. Polymer 1998, 39 (8-9), 1631-1636.

2128 Langmuir, Vol. 23, No. 4, 2007 was added under a nitrogen atmosphere via a double-tipped needle into a 250 cm3 Schlenck flask equipped with a septum and containing a preweighed and dried 5 g (2.5 mmol) sample of the PEO macroinitiator. Then, 0.01 wt % of SnOct was added to the reaction mixture under N2, and the mixture was stirred at room temperature for ca. 10 min until complete dissolution of the initiator. The mixture was then cooled to liquid N2 temperature, evacuated for 1 h, sealed off, and heated to 135 °C with vigorous stirring. After 24 h, the resulting viscous colorless oil was cooled to room temperature, dissolved in ca. 50 mL of anhydrous THF, and precipitated by dropwise addition into a large excess (2 L) of cold hexanes. The white precipitate was collected by filtration and dried at 40 °C for 2 days under vacuum. The yield was 10.6 g (∼90%). 1H NMR (CDCl ): 1.3-1.75 (m, ∼138H); 2.25-2.4 (t, ∼46H); 3 3.38 (s, 3H); 3.6-3.8 (m, ∼180H); and 4.0-4.1 (t, ∼46 H) ppm. Mn (SEC): 7100 (polydispersity index (PDI) ) 1.33). Synthesis of PEO-b-PCL-TE. Thioctate ester (TE) conjugated to PEO-b-PCL copolymer was prepared according to the following procedure: 5 g of PEO-b-PCL copolymer (1.08 mmol), 1 g of thioctic acid (4.85 mmol), 55 mg of DMAP (0.45 mmol), and 0.125 mL of anhydrous TEA (0.9 mmol) were dissolved in 100 mL of anhydrous dichloromethane under a nitrogen atmosphere. The mixture was cooled in an ice bath, and 1 g (4.85 mmol) of DCC was added in one portion. The mixture was stirred at 0 °C for 1 h and left to stir at room temperature for 24 h. The precipitated byproduct (1,3dicyclohexyl urea) was removed by filtration, and the filtrate was dried in vacuum. The crude was redissolved in a small amount of dichloromethane and purified in a silica-gel column using dichloromethane/methanol (100/0 to 95/5) as gradient eluent. Fractions containing the pure PEO-b-PCL-TE were collected and combined, and the solvent was removed under reduced pressure. Finally, the polymer was dissolved in an excess of hot ethyl alcohol (∼0.5 L), cooled to room temperature, and stored at -20 °C for 2 days. The white precipitate obtained was filtered and dried in vacuum for 48 h. The yield was 3.2 g (∼60%). 1H NMR (see Results and Discussion). Mn (SEC): 7800 (PDI ) 1.31). Typical Preparation of Au/Block Nanoparticles. Glassware to be used for the preparation of Au nanoparticles was washed three times with aqua regia followed by copious amounts of Milli-Q water and finally dried in an oven at 130 °C for 48 h. The typical preparation of Au-polymer is as follows: 0.1 g of HAuCl4‚3H2O (0.254 mmol) was dissolved in 25 mL of anhydrous THF. To this solution was added 1.34 g of PEO-b-PCL-TE (∼1 equiv according to Mn (NMR)), and the mixture was stirred in the dark under nitrogen atmosphere and at room temperature for 12 h. Then, 3 mL (∼3 equiv to HAuCl4) of 0.25 M LiBH4 was added dropwise into the reaction mixture via an airtight and degassed syringe. Immediately after the addition of the reducing reagent, the color of the solution turned from light yellow to deep purple, indicating the formation of gold nanoparticles. The mixture was allowed to stir for another 3 h at room temperature and quenched by diluting the mixture with an excess of absolute ethyl alcohol (with a final volume of ca. 200 mL). Under the experimental conditions described here, the Au nanoparticles are insoluble in ethanol and precipitate, whereas the copolymer is soluble. The ethanolic layer was decanted, and the dissolution/precipitation/ decantation was repeated several times (up to 10) until no unassociated polymer was detected by thin-layer chromatography (TLC) (CHCl3/ CH3OH 9/1). Under this eluting condition, the copolymer has an Rf value of ca. 0.3 whereas the Rf value of the gold nanoparticles is nearly zero (strong spot near the origin), which is attributed to the high molecular weight of the stabilized nanoparticle. Further purification was conducted by resuspending the nanoparticles in THF and dialyzing against THF applying a 250 kDa molecular weight cut-off (MWCO) poly(vinylidene difluoride) (PVDF) membrane. PVDF membranes (from Spectrum Laboratories, Inc.) are solvent resistant and are specially designed for dialysis against organic solvents. The high cutoff of the membrane (250 kDa) ensures the removal of unassociated copolymer chains (Mn∼ 7 kDa). After the extensive dialysis in THF, the solvent was replaced by water to enable lyophilization of the product to obtain the final material in

Azzam and Eisenberg powder-form. The yield in this specific preparation was ∼550 mg (35%). Similar Au-polymer nanoparticles with varying polymer contents were prepared applying a similar protocol by changing the HAuCl4/polymer mole feed ratio. Preparation of Unlabeled and Gold-Labeled Micelles in Water. PEO-b-PCL and Au/Block micelles in water were prepared using a well-established method.35 Briefly, 0.5 wt % of stock solution of PEO-b-PCL or Au/Block was freshly prepared in dimethylformamide (DMF) and stirred overnight at room temperature to ensure complete dissolution. This solution was added slowly (25 µL/min) into a 4-fold excess of Milli-Q water with vigorous stirring. The mixture was allowed to stir for another hour at room temperature and dialyzed (12-14 k MWCO) against Milli-Q water. The water was changed every 1 h within the first 8 h and every 6 h within the following 24 h. The micelles were analyzed and characterized within a week after preparation. Characterization. The degrees of polymerization and polydispersity were measured by size-exclusion chromatography (SEC) in THF. A Waters 510 liquid chromatography pump, equipped with two (HR1 and HR4) Styragel columns connected in series, and a refractive index detector (Varian RI-4) were used at room temperature. Polystyrene standards (Scientific Polymer Products, Inc., NY) with a narrow molecular weight distribution were used for calibration. NMR spectra were recorded on a Varian XL-300 spectrometer. CDCl3 and d6-DMSO were used as solvents with TMS as the internal reference. TEM studies were conducted using a JEOL 2000FX instrument operating at an accelerating voltage of 80 kV. Dilute solutions of the polymer-gold in THF were deposited in copper grids coated with carbon (Electron Microscopy Science (EMS), Hatfield, PA). Excess solvent was swept away by touching the edge of the grids with a small piece of filter paper (Whatman-1). The grids were allowed to dry at ambient temperature for 24 h before measurements. Polymer-gold micelles in dilute aqueous solutions were deposited on copper grids that had been precoated with a thin film of Formvar (polyvinyl formal, EMS) and then coated with carbon. The grids were also allowed to dry at room temperature overnight. For staining, a drop of 2 wt % phosphotungstic acid (freshly prepared in Milli-Q water and filtered through a 0.2 µm filter membrane) was added to the dry samples on the grids. After 2 min, excess staining agent was swept away by filter paper, and the grids were further dried under ambient temperature. For unlabeled micelles (i.e., PEO-b-PCL without gold), an aqueous solution of uranyl diacetate (2 wt %) was used for staining. Better contrast was obtained with the free micelles using uranyl diacetate and with the gold-loaded micelles using phosphotungstic acid. TEM images were analyzed using SigmaScan Pro 4.0 software. The average diameter of the gold nanoparticles was obtained from measurements of at least 600 nanoparticles per sample. UV-vis spectra were recorded on a Varian Cary 50 spectrophotometer, between 300 and 800 nm wavelength. Dilute solutions of the gold nanoparticles (in organic or aqueous solution) were measured in quartz cuvettes, using pure solvent as a reference. DLS measurements were performed on a Brookhaven photon correlation spectrometer with a BI9000 AT digital correlator. The instrument is equipped with a compass 315M-150 laser (Coherent Technologies), which was used at a wavelength of 532 nm. Dustfree vials were used for the aqueous solutions, and measurements were made at 25 °C at an angle of 90 °C. The CONTIN algorithm was used to analyze the data. Thermogravimetric analyses (TGAs) of the Au nanoparticles were carried out on a TA Q500 instrument, with a Pt pan at a heating rate of 10 °C/min. The runs were performed under N2 gas, and the gas was switched to air at 550 °C for 10 min. At the end of the run, the (35) Vangeyte, P.; Gautier, S.; Jerome, R. About the methods of preparation of poly(ethylene oxide)-b-poly(-caprolactone) nanoparticles in water analysis by dynamic light scattering. Colloids Surf., A: Physicochem. Eng. Aspects 2004, 242 (1-3), 203-211.

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Scheme 1. Synthesis of the PEO-b-PCL Copolymer and the PEO-b-PCL-TE Conjugate

temperature was raised to 700 °C to ensure complete combustion of the organic material.36,37

Results and Discussion Synthesis of PEO-b-PCL and PEO-b-PCL-TE. PEO-bPCL block copolymer was synthesized as depicted in Scheme 1. The number average molecular weight (Mn) and PDI of the block copolymer determined by SEC were 7100 and 1.33, respectively. On the basis of the integrated values from NMR and the degree of polymerization of the narrowly polydisperse PEO block (45), the degree of polymerization of the PCL block was estimated to be 23.18 The calculated Mn from NMR was ca. 5000 and is in good agreement with the starting feed (24), which suggests that over 95% of -caprolactone monomer was reacted. The reason for the variation in the molecular weight between SEC and NMR is probably related to the differences in the hydrodynamic volume of the copolymer in THF compared to polystyrene standards. The terminal hydroxyl group of the PCL block was modified with thioctic acid using DCC as the coupling agent and TEA/DMAP as the catalysts (Scheme 1). thioctic acid, a naturally occurring disulfide, was used in this study as the anchoring spacer to the gold due to its safety and biocompatibility. It is known as an antioxidant agent and is frequently used in many biomedical applications.38-40 Purification of the PEO-bPCL-TE conjugate from unbound thioctic acid by simple precipitation in a nonsolvent (i.e., hexanes or petroleum ether) was not successful, as judged by TLC and NMR. The thioctic acid reagent can act as a stabilizer in the preparation of the gold nanoparticles;41,42 therefore, and in order to prevent the formation of gold nanoparticles protected with a mixture of the two ligands (i.e., PEO-b-PCL-TE and thioctic acid), purification of the PEOb-PCL-TE from unbound thioctic acid was crucial. A complete removal of the unbound thioctic acid from the reaction mixture was achieved by column chromatography (Experimental Section). (36) Corbierre, M. K.; Cameron, N. S.; Lennox, R. B.; Polymer-stabilized gold nanoparticles with high grafting densities. Langmuir 2004, 20 (7), 28672873. (37) Corbierre, M. K.; Cameron, N. S.; Sutton, M.; Laaziri, K.; Lennox, R. B. Gold nanoparticle/polymer nanocomposites: dispersion of nanoparticles as a function of capping agent molecular weight and grafting density. Langmuir 2005, 21 (13), 6063-6072. (38) Arivazhagan, P.; Ramanathan, K.; Panneerselvam, C. Effect of DL-Rlipoic acid on the status of lipid peroxidation and antioxidants in mitochondria of aged rats. J. Nutr. Biochem. 2001, 12 (1), 2-6. (39) Bast, A.; Haenen Guido, R. M. M. Lipoic acid: a multifunctional antioxidant. BioFactors (Oxford, Engl.) 2003, 17 (1-4), 207-213. (40) Smith, A. R.; Shenvi, S. V.; Widlansky, M.; Suh, J. H.; Hagen, T. M. Lipoic acid as a potential therapy for chronic diseases associated with oxidative stress. Curr. Med. Chem. 2004, 11 (9), 1135-1146. (41) Berchmans, S.; Thomas, P. J.; Rao, C. N. R. Novel effects of metal ion chelation on the properties of lipoic acid-capped Ag and Au nanoparticles. J. Phys. Chem. B 2002, 106 (18), 4647-4651. (42) Li, G.; Lauer, M.; Schulz, A.; Boettcher, C.; Li, F.; Fuhrhop, J.-H. Spherical and planar gold(0) nanoparticles with a rigid gold(I)-anion or a fluid gold(0)acetone surface. Langmuir 2003, 19 (16), 6483-6491.

Figure 1. 1H NMR of PEO-b-PCL-TE conjugate in CDCl3.

The efficiency of the conjugation of the thioctic acid to the block copolymer was judged by 1H NMR as shown in Figure 1. The chemical shift at 3.125-3.19 ppm (2H, b and c protons in PEOb-PCL-TE, Figure 1) is characteristic of the methylene group adjacent to the S-S bond of the thioctate moiety. The ratio of the integrated areas between the peak of this chemical shift (attached to the PCL block) and that of the terminal methoxy group of the PEO block (3H, a protons in PEO-b-PCL-TE, Figure 1) suggested that approximately 90% of the block copolymer chains may be functionalized. The Mn (SEC) of PEO-b-PCLTE was 7800 (PDI ) 1.31) and was not affected by the chemical modification. Synthesis and Characterization of Au/Block Nanoparticles. The size of the nanoparticles prepared by the reduction of the gold species normally depends on a number of parameters, such as the type of reducing agent and the loading of the metal precursor.43 The type of reducing agent determines the rate of nucleation and particle growth: slow reduction produces large particles, while fast reduction gives small particles. Preliminary attempts to prepare the gold-copolymer nanoparticles using a (43) Antonietti, M.; Grohn, F.; Hartmann, J.; Bronstein, L. Nonclassical shapes of noble-metal colloids by synthesis in microgel nanoreactors. Angew. Chem., Int. Ed. Engl. 1997, 36 (19), 2080-2083.

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Figure 2. TEM images of gold nanoparticles stabilized with PEO-b-PCL-TE at Au/Block mole ratios of 1/1 (a), 2/1 (b), 3/1 (c), 4/1 (d), and 12/1 (e). (f) Control experiment of Au/Block 1/1 prepared from unmodified block copolymer (i.e., without the TE moiety). All samples were prepared in THF (1 mg/mL) and allowed to stir overnight prior to the preparation of the samples.

strong reducing reagent, Li(Et)3BH (Superhydride), were not successful, probably due to successive and uncontrolled depolymerization via the ester linkages in the PCL block. The hydrolysis of the ester bonds in the PCL block leads to the formation of a thioctate group attached to a hydrophobic chain in the absence of PEO. The hydrophobic chain in water does not allow stable gold particles to form. On the other hand, the use of lithium borohydride (LiBH4) showed no PCL degradation even at elevated temperatures, as judged by SEC (data not shown). The synthesis of PEO-b-PCL-protected Au nanoparticles was accomplished in a single-phase method as described previously.44 A mixture of HAuCl4 and PEO-b-PCL-TE (1/1 to 12/1 mol ratio) was freshly prepared in dry THF and subsequently treated with excess of LiBH4 in THF. The nanoparticles were isolated by selective precipitation using ethyl alcohol as a nonsolvent for the modified Au nanoparticles. At room temperature, PEO-bPCL-TE is partially soluble in ethanol; therefore, the Au nanoparticles were dissolved several times in THF and subsequently precipitated in an excess of ethanol. Despite these efforts, free PEO-b-PCL-TE or PEO-b-PCL may still be present, even after repeated and successive purifications. Further purification was performed by extensive dialysis against THF (250 k MWCO PVDF membrane) followed by dialysis against water and lyophilization (Experimental Section). By this method, unassociated block copolymer was completely removed, as judged by TLC. TEM images of the Au/Block nanoparticles prepared in this study are shown in Figure 2. The stabilized gold nanoparticles are denoted as Au/Block (x/y), where the x/y ratio stands for the initial mole feed ratio between the aurate acid (HAuCl4) and the (44) Rowe, M. P.; Plass, K. E.; Kim, K.; Kurdak, C.; Zellers, E. T.; Matzger, A. J. Single-phase synthesis of functionalized gold nanoparticles. Chem. Mater. 2004, 16 (18), 3513-3517.

Figure 3. (a) UV-vis absorption spectra of Au/Block (1/1), Au/ Block (2/1), and Au/Block (3/1) nanoparticle dilute solutions in THF. (b) UV-vis spectra of the nanoparticles made in water (Experimental Section).

PEO-b-PCL-TE in a specific preparation. For example, Au/ Block (1/1) represents the nanoparticles prepared from a 1/1 mol ratio between the HAuCl4 and the copolymer. The nanoparticle

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Figure 4. TEM images of Au/Block (1/1) micelles (a) and Au/Block (2/1) micelles (b) made in water. (c) PEO-b-PCL micelles (without the gold) were negatively stained with uranyl diacetate (Experimental Section).

solutions were prepared from THF (1 mg/mL) and stirred overnight before testing to ensure maximum dissolution. At Au/ Block (1/1), nanoparticles with a narrow size distribution were obtained with an average diameter of 6.2 ( 1.4 nm (Figure 2a). Similar narrowly dispersed particles were obtained with Au/ Block (2/1) and Au/Block (3/1), with average diameters of 6.6 ( 1.4 and 6.4 ( 1.8 nm, respectively (Figure 2b,c). The polymer content of these nanoparticles, as determined by TGA, was 92% for Au/Block (1/1), 87% for Au/Block (2/1), and 82% for Au/Block (3/1) (Figure S8 in the Supporting Information). In fact, the differences in the percentages in polymer content of these nanoparticles were not significant, and therefore nearly similar sizes as well as plasmon peak maxima (λmax ) 520-526 nm) were obtained (Figure 3a). Au/Block (4/1) nanoparticles in THF, on the other hand, were not stable, and irreversible precipitation was observed. TEM observations of this sample showed the formation of relatively large gold clusters ca. 50100 nm in size, which agglomerate in time until complete precipitation (Figure 2d). The polymer content of this preparation was 77% with a surface plasmon peak of λmax ∼ 540 nm in THF (Figure S8 in the Supporting Information). This blue shift in the surface plasmon spectrum was explained by the formation of bulk gold during the agglomeration process.45 Au/Block (12/1), along the same trend, was insoluble, and only large gold aggregates a few microns in size were observed (Figure 2e). It is worth mentioning in this connection that the importance of disulfide anchoring was assessed by using unmodified block copolymer (e.g., without the disulfide moiety) as the stabilization agent. The isolated gold product was insoluble (Figure 2f) and contained only trace amounts of the block copolymer (∼5 wt %), as judged by TGA. Murray and co-workers reported on the grafting of Au nanoparticles with R-methoxy-ω-mercapto-poly(ethylene oxide) (PEO-SH, Mw ) 5000).46 In that study, the average diameter of the gold nanoparticles was 2.8 nm, and the calculated grafting density was ∼3 (chains/nm2). Similar grafting densities in the same range for PEO-SH (Mn ) 2100) and other polymers were recently reported by Lennox and co-workers.36 From the grafting density numbers for Au/Block (1/1), Au/Block (2/1), and Au/ Block (3/1), shown in Table S1 (Supporting Information), it appears that there is a significant percentage of chains that are not chemically anchored to the metal surface, and that are held

by hydrophobic-hydrophobic interactions with the bonded poly(caprolactone) chains. PCL is a semicrystalline polymer at ambient temperature;47 therefore, it is believed that the unbound polymer chains are strongly attached to the metal surface by cocrystallization with the PCL chains, which are chemically anchored to the metal surface. Au/Block (12/1), on the other hand, shows a reasonable grafting density (∼2.2 chains/nm2 assuming an identical ∼6 nm diameter, Table S1 in the Supporting Information), but its complete insolubility suggests that a number of the polymer chains in this system are “caught” in the clusters and are not contributing to its stabilization. Therefore, the unbonded polymer chains are probably also contributing to the stabilization of the gold nanoparticles by increasing the polymeric coverage around them. Preparation of Au/Block Micelles in Water. Au/Block micelles in water were prepared by the slow addition of a dilute Au/Block solution in good solvent into a large excess of water followed by dialysis, a method which is commonly used for amphiphilic block copolymers.48 A dilute solution of the Au/ Block nanoparticles was suspended in DMF and added dropwise into a large volume of water under vigorous stirring. Under these conditions, the PCL block forms the shell that surrounds the gold core, while the PEO block forms the corona. The micelles thus formed were purified from the organic solvent by dialysis against water. Figure 3b shows the UV-vis spectra of the Au/Block micelles in water. Au/Block (1/1) micelles showed a maximum absorption at a wavelength of 522 nm, which is similar to the plasmon peak maximum obtained using THF as the solvent (∼520 nm, Figure 3a). These micelles were freely soluble in water with no detectable aggregation. TEM images of Au/Block (1/1) prepared from water show nanoparticles with a narrow size distribution with an average diameter of 5.9 ( 1.7 nm (Figure 4a). This value is in good agreement with the average diameter obtained when THF was used as the solvent. The similarity of the sizes in water and THF also proves that, under the experimental conditions applied here, no aggregation was seen, and only micelles with a single gold core per micelle were observed. The stability of these micelles in water was monitored over 3 weeks, with no significant change in the surface plasmon peak (Figure S9 in the Supporting Information). Au/Block (2/1) micelles in water showed a slight blue shift in the plasmon peak (from 522 to 528 nm) and a 7 (

(45) Link, S.; El-Sayed, M. A. Size and temperature dependence of the plasmon absorption of colloidal gold nanoparticles. J. Phys. Chem. B 1999, 103 (21), 4212-4217. (46) Wuelfing, W. P.; Gross, S. M.; Miles, D. T.; Murray, R. W. Nanometer gold clusters protected by surface-bound monolayers of thiolated poly(ethylene glycol) polymer electrolyte. J. Am. Chem. Soc. 1998, 120 (48), 12696-12697.

(47) Li, S. M.; Chen, X. H.; Gross, R. A.; McCarthy, S. P. Hydrolytic degradation of PCL/PEO copolymers in alkaline media. J. Mater. Sci.: Mater. Med. 2000, 11 (4), 227-233. (48) Zhang, L.; Eisenberg, A. Multiple morphologies of “crew-cut” aggregates of polystyrene-b-poly(acrylic acid) block copolymers. Science (Washington, D.C.) 1995, 268 (5218), 1728-1731.

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6.0 ( 1.2 nm, similar to previous observations. The average diameter of the micelles (with the gold), however, was 20 ( 2 nm based on statistics accumulated on 300 micelles. The spacing distances between the gold nanoparticles cores shown in Figure 5a are proof that the nanoparticles are shielded by a ca. 7 nm shell. This polymeric coverage undoubtedly prevents the nanoparticles from aggregating. DLS analysis of Au/Block (1/1) micelles in water (Figure 5b) showed an average size of 35 nm and a narrow size distribution. The larger value from the DLS measurement relative to TEM is most likely due to the existence of a swollen PEO corona around the core, as well as the heavier weighting of the larger size units in DLS. Micelles made from Au/Block (2/1) nanoparticles in pure water showed an average diameter size of 40 nm and a narrow size distribution. Unlike TEM, DLS measurements are quantitative; therefore, the similarity in sizes for Au/Block (1/1) and Au/Block (2/1), as determined by DLS, suggests a nearly equal polymeric coverage around the metal core.

Conclusions A series of PEO-b-PCL micelles containing gold nanoparticles in the core were studied. PEO-b-PCL block copolymer modified with a disulfide moiety in the terminal position of the hydrophobic block was prepared and used as the ligand for the formation of stabilized gold nanoparticles. PCL is a well-known biodegradable polymer that has been utilized in various biomedical applications because of its excellent biocompatibility and degradability.49,50 When testing cellular the internalization of a particular delivery system (e.g., micelles made from PEO-b-PCL as in the present study), it is crucial that the labeled micelles should exhibit the same chemical and physical properties as the unlabeled or drugloaded ones. Figure 5. (a) TEM image of Au/Block (1/1) micelles prepared in water. The sample was stained with phosphotungstic acid before imaging (Experimental Section). (b) Size distribution histogram of Au/Block (1/1) micelles from DLS.

2 nm average diameter of the metal core (Figure 4b). The Au/B (3/1) micelles in water, on the other hand, were not stable, and a remarkable shift in the surface plasmon peak was observed (Figure 3b). The TEM image of this preparation showed large agglomerates a few microns in size (figure not shown). The weight percentage of the agglomerates was estimated to be as high as 90%, as judged by gravimetric analysis (data not shown). Au/Block (4/1) and Au/Block (12/1) nanoparticles in water were not investigated due to their limited solubility in DMF. Unlabeled micelles (i.e., those without gold cores) were also characterized by TEM (Figure 4c) after being stained with uranyl diacetate (Experimental Section). The average diameter of the unlabeled block copolymer micelles was calculated to be 27 ( 8 nm on the basis of statistics accumulated on 300 micelles. Figure 5a shows TEM images of Au/Block (1/1) micelles that were prepared in water after being stained with 2 wt % phosphotungstic acid (Experimental Section). The calculated average diameter of the gold nanoparticles was estimated to be (49) Geng, Y.; Discher, D. E. Hydrolytic degradation of poly(ethylene oxide)block-polycaprolactone worm micelles. J. Am. Chem. Soc. 2005, 127 (37), 1278012781. (50) Ahmed, F.; Discher, D. E. Self-porating polymersomes of PEG-PLA and PEG-PCL: hydrolysis-triggered controlled release vesicles. J. Controlled Release 2004, 96 (1), 37-53.

The stability of the gold nanoparticles in the present study was found to be related to the degree of polymeric coverage. High polymer content resulted in high solubility in common organic solvents for the block, whereas low polymer content resulted in low to limited solubility or even insolubility. The highly soluble Au/Block nanoparticles were used to form soluble micellar structures in water. TEM studies showed that the micelles prepared in water showed a core-shell structure with one gold core per micelle. Aggregates of these micelles were observed only for nanoparticles with a relatively low polymeric coverage. The current study presents a new approach for labeling biodegradable block copolymer micelles of potential biological applications, such as tissue and subcellular localization. The labeling technique is obviously applicable to a wide range of amphiphilic block copolymers of biomedical interest. Acknowledgment. We thank the Natural Science and Engineering Research Council of Canada (NSERC) for the support of this work. Supporting Information Available: Additional TEM images, histograms representing the core size distribution, and TGA of the nanoparticles. This material is available free of charge via the Internet at http://pubs.acs.org. LA0627563