pubs.acs.org/Langmuir © 2009 American Chemical Society
Gold Nanoparticles Stabilized by Thermosensitive Diblock Copolymers of Poly(ethylene glycol) and Polyphosphoester You-Yong Yuan,†,‡ Xi-Qiu Liu,§ Yu-Cai Wang,‡ and Jun Wang*,†,§ †
Hefei National Laboratory for Physical Sciences at Microscale, ‡Department of Polymer Science and Engineering, and §School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230027, People’s Republic of China Received March 31, 2009. Revised Manuscript Received May 7, 2009 Aqueous dispersions of thermosensitive gold nanoparticles protected by diblock copolymers of poly(ethylene glycol) and polyphosphoester were prepared and studied. Diblock copolymers MPEG-b-P(EEP-co-PEP) with different compositions that are composed of monomethoxy poly(ethylene glycol), random copolymer of ethyl ethylene phosphate (EEP), and isopropyl ethylene phosphate (PEP) were synthesized by ring-opening polymerization in bulk. Thioctic acid was then conjugated to the terminal hydroxyl group of the polyphosphoester block by esterification. Gold nanoparticles were then prepared by a one-step method and showed core-shell structure with an average gold core diameter of about 10 nm surrounded by a MPEG-b-P(EEP-co-PEP) shell with a thickness of about 30 nm. These polymer stabilized gold nanoparticles are reversibly thermosensitive in aqueous medium, exhibiting tunable collapse temperatures which are dependent on the composition of the diblock copolymers. Methyl tetrazolium (MTT) assay against HEK 293 cells demonstrated that these gold nanoparticles are with good biocompatibility. These gold nanoparticles protected by thermosensitive diblock copolymers with tunable collapse temperature are expected to be useful for biomedical applications.
Introduction Increased attention has been paid to gold nanoparticles in biomedical applications for enhancing diagnostic sensitivity in radiotherapy1 and as carriers for drug and gene delivery2-8 owing to their low toxicity, stability, and versatility of surface functionalities. Gold nanoparticles protected by synthetic polymers are envisioned to be superior to polymeric micelles,9 whereas the latter have shown potential as drug delivery vehicles.10,11 Stimuli-responsive polymers, exhibiting sharp property changes to a small or modest variation of environmental conditions (e.g., temperature, light, salt concentration, or pH), can be utilized for the development of “smart” drug delivery systems.12-14 Many stimuli-responsive polymers, particularly pH sensitive (e.g., poly(4-vinylpyridine)) and thermosensitive *Corresponding author. Fax: +86 551 360 0402. E-mail: jwang699@ ustc.edu.cn. (1) Hyukjin, L.; Kyuri, L.; In Kyoung, K.; Tae Gwan, P. Biomaterials 2008, 29, 4709–4718. (2) Ghosh, P.; Han, G.; De, M.; Kim, C. K.; Rotello, V. M. Adv. Drug Delivery Rev. 2008, 60, 1307–1315. (3) Kang, Y. J.; Taton, T. A. Angew. Chem., Int. Ed. 2005, 44, 409–412. (4) Gibson, J. D.; Khanal, B. P.; Zubarev, E. R. J. Am. Chem. Soc. 2007, 129, 11653–11661. (5) Murakami, T.; Tsuchida, K. Mini-Rev. Med. Chem. 2008, 8, 175–183. (6) Joshi, H. M.; Bhumkar, D. R.; Joshi, K.; Pokharkar, V.; Sastry, M. Langmuir 2006, 22, 300–305. (7) Qin, J.; Jo, Y. S.; Ihm, J. E.; Kim, D. K.; Muhammed, M. Langmuir 2005, 21, 9346–9351. (8) Karg, M.; Pastoriza-Santos, I.; Liz-Marzan, L. M.; Hellweg, T. ChemPhysChem 2006, 7, 2298–2301. (9) Javakhishvili, I.; Hvilsted, S. Biomacromolecules 2009, 10, 74–81. (10) Park, J. H.; Lee, S.; Kim, J. H.; Park, K.; Kim, K.; Kwon, I. C. Prog. Polym. Sci. 2008, 33, 113–137. (11) Qiu, L. Y.; Zheng, C.; Jin, Y.; Zhu, K. J. E. Expert Opin. Ther. Pat. 2007, 17, 819–830. (12) Bajpai, A. K.; Shukla, S. K.; Bhanu, S.; Kankane, S. Prog. Polym. Sci. 2008, 33, 1088–1118. (13) Ganta, S.; Devalapally, H.; Shahiwala, A.; Amiji, M. J. Controlled Release 2008, 126, 187–204. (14) Schmaljohann, D. Adv. Drug Delivery Rev. 2006, 58, 1655–1670.
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polymers (e.g., poly(N-isopropylacrylamide) or Pluronic F127), have been coated on gold nanoparticles to obtain responsive nanoparticles.15-19 Gold nanoparticles can also be dually responsive by coating with thermo- and pH-responsive polymers, such as poly(methacrylic acid)-b-poly(N-isopropylacrylamide) and hyperbranched polyelectrolytes.20,21 Gold nanoparticles stabilized by polymers are generally prepared by “graft-from” and “graft-to” methods in addition to postmodification of preformed gold nanoparticles and physisorption as described in the literature.22 The “graft-from” strategy can be achieved by chain initiation from active sites attached to the gold nanoparticle surfaces.17,23 For example, it is useful to prepare polymer stabilized gold clusters through controlled radical polymerization that enables control of the molecular weight and molecular weight distribution of grafted polymers. In the “graft-to” strategy, thiol group end-capped or disulfide-contained polymers are used as the stabilizer during the generation of gold nanoparticles instead of using alkanethiol ligands in a traditional synthesis process.9,16,24,25
(15) Li, D. X.; He, Q.; Yang, Y.; Mohwald, H.; Li, J. B. Macromolecules 2008, 41, 7254–7256. (16) Zhu, M. Q.; Wang, L. Q.; Exarhos, G. J.; Li, A. D. Q. J. Am. Chem. Soc. 2004, 126, 2656–2657. (17) Li, D. X.; Cui, Y.; Wang, K. W.; He, Q.; Yan, X. H.; Li, J. B. Adv. Funct. Mater. 2007, 17, 3134–3140. (18) Bae, K. H.; Choi, S. H.; Park, S. Y.; Lee, Y.; Park, T. G. Langmuir 2006, 22, 6380–6384. (19) Shan, J.; Nuopponen, M.; Jiang, H.; Viitala, T.; Kauppinen, E.; Kontturi, K.; Tenhu, H. Macromolecules 2005, 38, 2918–2926. (20) Shen, Y.; Kuang, M.; Shen, Z.; Nieberle, J.; Duan, H. W.; Frey, H. Angew. Chem., Int. Ed. 2008, 47, 2227–2230. (21) Nuopponen, M.; Tenhu, H. Langmuir 2007, 23, 5352–5357. (22) Shan, J.; Tenhu, H. Chem. Commun. 2007, 4580–4598. (23) Wang, H.; Chen, Y.; Li, X. Y.; Liu, Y. Mol. Pharmaceutics 2007, 4, 189– 198. (24) Shan, J.; Chen, H.; Nuopponen, M.; Viitala, T.; Jiang, H.; Peltonen, J.; Kauppinen, E.; Tenhu, H. Langmuir 2006, 22, 794–801. (25) Azzam, T.; Eisenberg, A. Langmuir 2007, 23, 2126–2132.
Published on Web 06/01/2009
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Scheme 1. Synthesis Route of Thioctate Ester of MPEG-b-P(EEP-co-PEP) and Schematic Illustration of Synthesis of Gold Nanoparticles with a Gold Core, a PPE Inner Shell, and a MPEG Outer Corona and Its Thermoinduced Size Change
Previous studies have shown that polyphosphoesters (PPEs) with repeated ethyl ethylene phosphate and isopropyl ethylene phosphate are thermosensitive in aqueous solution, and more importantly, the lower critical solution temperatures are dependent on the composition and molecular weights.26 Block copolymers of such PPEs with poly(ethylene glycol) or poly(ε-caprolactone) also exhibit tunable thermosensitivity.27 On the other hand, PPEs have received considerable attention in biomedical applications due to their biodegradability, biocompatibility, and pendant functional ability in addition to their variable backbone structure.28,29 In this study, we aimed to synthesize thermosensitive gold nanoparticles stabilized by diblock copolymers of poly(ethylene glycol) and PPEs (MPEG-b-PPE) for potential biomedical applications. Considering well-defined and thermosensitive PPEs can only be synthesized by ring-opening polymerization (ROP),30,31 whereas ROP is sensitive to moisture and impurities, which needs high precaution and is difficult to perform on the surface of gold nanoparticles,32 the “graft-to” method was then taken in this study. We synthesized the thioctate esters of MPEG-b-P(EEP-co-PEP), which were thermosensitive diblock copolymers of poly(ethylene glycol) and PPE with a disulfide end moiety. The polymers were further used as the stabilizer to synthesize nanoparticles with a gold core, a PPE inner shell, and a MPEG outer corona (Scheme 1). The thermosensitivity and (26) Iwasaki, Y.; Wachiralarpphaithoon, C.; Akiyoshi, K. Macromolecules 2007, 40, 8136–8138. (27) Wang, Y. C.; Tang, L. Y.; Li, Y.; Wang, J. Biomacromolecules 2009, 10, 66– 73. (28) Zhao, Z.; Wang, J.; Mao, H. Q.; Leong, K. W. Adv. Drug Delivery Rev. 2003, 55, 483–499. (29) Wang, J.; Mao, H. Q.; Leong, K. W. J. Am. Chem. Soc. 2001, 123, 9480– 9481. (30) Chen, D. P.; Wang, J. Macromolecules 2006, 39, 473–475. (31) Iwasaki, Y.; Wachiralarpphaithoon, C.; Akiyoshi, K. Macromolecules 2007, 40, 8136–8138. (32) Kotal, A.; Mandal, T. K.; Walt, D. R. J. Polym. Sci., Part A: Polym. Chem. 2005, 43, 3631–3642.
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cytotoxicity of the obtained polymer stabilized gold nanoparticles were also studied.
Experimental Section Materials. Ethyl ethylene phosphate (EEP, 2-ethoxy-2-oxo1,3,2-dioxaphospholane) and isopropyl ethylene phosphate (PEP, 2-isopropoxy-2-oxo-1,3,2-dioxaphospholane) were synthesized by a method described previously and distilled under reduced pressure just before use.30 Monomethoxy poly(ethylene glycol) (MPEG, Mn = 2000 g mol-1, Acros Organics) was dried by azeotropic distillation from anhydrous toluene. Stannous octoate (Sn(Oct)2, Sinopharm Chemical Reagent, China) was purified according to a method described in the literature.33 Methanol and N,N-dimethylformamide (DMF) were dried over calcium hydride for 24 h at room temperature, followed by distillation just before use. Dichloromethane was refluxed over P2O5 for 24 h and distilled before use. Thioctic acid, 4-dimethylaminopyridine (DMAP), 1,3-dicyclohexyl carbodiimide (DCC), hydrogen tetrachloroaurate(III) trihydrate (HAuCl4 3 3H2O), and sodium borohydride (NaBH4) were obtained from Beijing Chemical Reagent, China and used as received. All other reagents and solvents were of analytical grade and used as received. Synthesis of Thioctate Ester of MPEG-b-P(EEP-coPEP) (Scheme 1). Thioctate esters of MPEG-b-P(EEP-coPEP) were synthesized in two steps. First, block copolymers of MPEG and PPE were prepared in bulk at 90 °C according to our previously reported procedure.27 Three diblock copolymers with different compositions were synthesized, and the compositions are shown in Table 1. In the second step, thioctic acid was conjugated to MPEG-b-P(EEP-co-PEP) according to the following typical procedure: 2.0 g of MPEG-b-P(EEP235-co-PEP20) and 30 mg (3 equiv) of thioctic acid were dissolved in 25 mL of dichloromethane, and then DCC (20 mg, 2 equiv) and DMAP (11.9 mg, 2 equiv) were added under a nitrogen atmosphere. The mixture was stirred at room temperature for 2 days. The (33) Kricheldorf, H. R.; Kreiser-Saunders, I.; Stricker, A. Macromolecules 2000, 33, 702–709.
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Table 1. Composition and Molecular Weight Distribution of Block Copolymers of MPEG and Polyphosphoester code MPEG-b-PEEP224 MPEG-bP(EEP235-co-PEP20) MPEG-bP(EEP220-co-PEP37)
feed molar ratio DP of EEP/PEP/MPEG PEEP/PPEPa
Mnb
PDIc
300/0/1 300/30/1
224/0 235/20
36 050 1.35 41 040 1.40
300/60/1
220/37
41 590 1.39
a
1
Degree of polymerization (DP) of EEP and PEP was determined by H NMR. b Determined by 1H NMR. c Determined by GPC.
precipitate (1,3-dicyclohexyl urea, DCU) was removed by filtration, and the filtrate was concentrated under vacuum and precipitated into diethyl ether at room temperature twice. The polymer was dialyzed extensively (molecular weight cutoff of 2000) against distilled water, which was followed by lyophilization. The other two thioctate esters were synthesized by a similar method.
Preparation of Polymer Stabilized Gold Nanoparticles. Glassware to be used for the preparation of gold nanoparticles was washed three times with aqua regia followed by copious amounts of Milli-Q water and dried. In a typical example, thioctate ester of MPEG-b-P(EEP235-co-PEP20) (1.0 g) and HAuCl4 3 3H2O (5 mg) were dissolved in 25 mL of methanol and stirred overnight in the dark at room temperature, and then 1 mL of 0.10 mol L-1 NaBH4 in DMF was added dropwise under vigorous stirring. The mixture was stirred for another 6 h at room temperature. The obtained gold nanoparticles were purified first by centrifugation (5000g) and then dialysis (molecular weight cutoff 100 000) against distilled water at 4 °C. The aqueous dispersion was lyophilized. Gold nanoparticles decorated with the other two polymers were prepared according to a similar procedure. Characterization. Number and weight average molecular weights (Mn and Mw) and molecular weight distributions (polydispersity index, PDI = Mw/Mn) were determined by gel permeation chromatography (GPC) measurements on a Waters 1515 GPC system, which was equipped with a Waters 2414 refractive index detector and three Waters Styragel high resolution columns (HR4, HR2, and HR1: effective molecular weight range = 5000500 000, 500-20 000, and 100-5000, respectively). HPLC grade chloroform was purchased from J.T. Baker and used as the eluent at 40 °C, delivered at a flow rate of 1.0 mL min-1. Monodispersed polystyrene standards obtained from Waters Co. with a molecular weight range of 1310-(5.51 104) were used to generate the calibration curve. A Bruker AV300 NMR spectrometer was used for the 1 H NMR analyses to determine the structure and composition of the block copolymers. The size and size distribution of nanoparticles in aqueous solution were measured by dynamic light scattering (DLS) carried out on a Malvern Zetasizer Nano ZS90 instrument with a He-Ne laser (633 nm) and 90° collecting optics. All samples were prepared in aqueous solution and filtered through Millipore 0.45 μm filters prior to measurements. The aqueous solution was kept in the thermostat of the apparatus at various temperatures for 20 min to reach equilibrium prior to measurements. The data were analyzed by Malvern Dispersion Technology software 4.20. The optical absorbance of gold nanoparticles in saline (1 wt %) at various temperatures was measured at 600 nm with a UV-2802 PC (UNICO, China) spectrophotometer. The sample cell was thermostatted in a refrigerated circulator bath for 20 min at different temperatures prior to measurements. Transmission electron microscopy (TEM) measurements were performed on a JEOL 2010 transmission electron microscope with an accelerating voltage of 200 kV. All of the samples were stained with phosphotungstic acid before imaging. For staining, a drop of 1 wt % phosphotungstic acid (freshly prepared in Milli-Q 10300 DOI: 10.1021/la901120x
water) was added to the solution. We prepared the samples by pipetting a drop of the solution onto a 230 mesh copper grid coated with carbon and allowing the sample to dry in air or in an oven at 44 °C before measurements. Methyl Tetrazolium (MTT) Assay. The relative cytotoxicity of nanoparticles was assessed with a MTT viability assay against HEK 293 cells. The cells were seeded in 96-well plates at 20 000 cells per well in 100 μL of complete Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum, supplemented with 50 units mL-1 penicillin and 50 units mL-1 streptomycin, and incubated at 37 °C in a 5% CO2 atmosphere for 24 h, followed by removing culture medium and adding the solution of nanoparticles (100 μL in complete DMEM medium) at different concentrations (0-1 mg mL-1). After 2 days of incubation, 25 μL of MTT stock solution (5 mg mL-1 in PBS) was added to each well to achieve a final concentration of 1 mg mL-1, with the exception of the wells designated as blank, to which 25 μL of phosphate buffered saline (PBS) was added. After incubation for an additional 2 h, 100 μL of extraction buffer (20% SDS in 50% DMF, pH 4.7, prepared at 37 °C) was added to the wells, and they were incubated overnight at 37 °C. The solution was mixed, and the absorbance of the solution was measured at 570 nm using a Bio-Rad 680 microplate reader. The cell viability was normalized to that of HEK 293 cells cultured in medium without nanoparticles.
Results and Discussion Syntheses of Thioctate Esters of MPEG-b-P(EEP-coPEP) (Scheme 1). We have previously reported that block polymers of poly(ethylene glycol) with poly(ethyl ethylene phosphate) or its random copolymer with poly(isopropyl ethylene phosphate) are thermosensitive in aqueous solution, whereas the thermotransition temperature can be well controlled by adjusting the composition of polyphosphoester or the length of PEG. It has also been demonstrated that such block copolymers are biocompatible with cells and muscular tissue and do not induce significant hemolysis and plasma protein precipitation.27 In this study, aiming to synthesize thermosensitive polymer stabilized gold nanoparticles for potential biomedical applications, we first synthesized three block copolymers using MPEG with a number average molecular weight of 2000 as the initiator and Sn(Oct)2 as the catalyst according to a procedure described previously. The compositions of the block copolymers are listed in Table 1, which were obtained by similar analyses as described previously.27 Thioctic acid, which is a naturally existing acid with good biocompatibility, was then conjugated to the terminal hydroxyl group of the polyphosphoester block by typical esterification (Scheme 1). DCC was used as the coupling agent, and DMAP was used as the catalyst. After removing DCU, which precipitated in the reaction, unreacted thioctic acid was removed by precipitating the resulting polymer in diethyl ether twice. It was further dialyzed against water to remove residual DMAP. Figure 1 shows a comparison of 1H NMR spectra of MPEG-b-P(EEP235-coPEP20) and its thioctate ester. In comparison with that of MPEG-b-P(EEP235-co-PEP20), characteristic resonances of thioctate ester protons are clearly observed, shown at 3.20, 2.35, 1.68, and 1.58 ppm, which are assigned to the protons of thioctate ester as labeled in Figure 1. By comparing the integral ratio of the proton signals at 2.35 ppm with that of the methylene protons from the MPEG block at 3.65 ppm, the end-capping efficiency of MPEG-b-P(EEP235-co-PEP20) is estimated to be 79.4%. Endcapping efficiencies of MPEG-b-PEEP224 and MPEG-b-P(EEP220-co-PEP37) are 80.1% and 75.2%, respectively. Synthesis and Characterization of Polymer Stabilized Gold Nanoparticles. To produce the block copolymer stabilized Langmuir 2009, 25(17), 10298–10304
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Figure 1. 1H NMR spectra of (A) MPEG-b-P(EEP235-co-PEP20) and (B) its thioctate ester (in CDCl3).
gold nanoparticles, a mixture of thioctate ester of MPEG-b-P(EEP-co-PEP) and HAuCl4 3 3H2O was freshly prepared in methanol and stirred overnight in the dark at room temperature. After being treated with excess NaBH4 in DMF, the solution was allowed to stir at room temperature for another 6 h to ensure the complete reduction of HAuCl4 3 3H2O and thioctate ester of MPEG-b-P (EEP-co-PEP). Upon addition of NaBH4, the solution turned into purple immediately, indicating the formation of small gold nanoparticles.21 Purification of the nanoparticles was performed first by centrifugation at 5000g to remove a small portion of large aggregates, and then the supernatant was concentrated by rotary evaporation and dialyzed against water at 4°C to remove unmodified MPEG-b-P(EEP-co-PEP), unreacted thioctate ester of MPEG-b-P(EEP-co-PEP), and NaBH4. The gold nanoparticles stabilized by block copolymers were obtained by lyophilization. Surface plasmon resonances are commonly determined to characterize Cu, Ag, and Au nanoparticles. The electric field of the incoming radiation induces the formation of a dipole in the nanoparticle, and there is a restoring force that tries to compensate it, so that a unique resonance frequency matches this electron oscillation within the nanoparticle.34 In a typical UV-vis absorption spectrum of a resulting transparent and brownish aqueous solution of gold nanoparticles stabilized by MPEG-b-P(EEP235co-PEP20) (Figure 2A), an absorbance at 514 nm was observed, which is attributed to the surface plasmon resonance (SPR) characteristic of the metallic gold clusters. It indicates the formation of nanosized gold nanoparticles in aqueous solution.15 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.25,35-37 The type of reducing agent determines the rate (34) Liz-Marzan, L. M. Langmuir 2006, 22(1), 32–41. (35) Antonietti, M.; Wenz, E.; Bronstein, L. Adv. Mater. 1995, 7, 1000. (36) Sidorov, S. N.; Bronstein, L. M.; Kabachii, Y. A.; Valetsky, P. M.; Soo, P. L.; Maysinger, D.; Eisenberg, A. Langmuir 2004, 20, 3543–3550. (37) Grzelczak, M.; Perez-Juste, J.; Mulvaney, P.; Liz-Marzan, L. M. Chem. Soc. Rev. 2008, 37, 1783–1791.
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Figure 2. (A) UV-vis absorption spectrum and (B) TEM image of gold nanoparticles stabilized by MPEG-b-P(EEP235-co-PEP20) at 25 °C (scale bar is 60 nm). DOI: 10.1021/la901120x
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of nucleation and particle growth: slow reduction produces large particles, while fast reduction gives small particles. We used NaBH4 as the reducing agent in this study, which leads to a fast rate of nucleation and small gold cores. For example, Javakhishvili and Hvilsted prepared gold nanoparticles protected by a diblock copolymer of poly(ε-caprolactone) and poly(acrylic acid) and observed the diameter of gold cores was about 9.0 ( 3.1 nm.9 Similarly, a TEM image obtained at 25 °C, shown in Figure 2B, also demonstrated the presence of a small gold core. Inspection of the TEM image revealed a spherical core-shell structure of the polymer stabilized gold nanoparticles, and the average diameter of the gold cores was less than 10 nm, which were surrounded by a polymer shell with a thickness of about 30 nm. Thermosensitivity of Polymer Stabilized Gold Nanoparticles. Polymers with thermosensitive properties have been reviewed by Hudson and Gil, and Tsvetanov et al.38,39 As the most widely investigated thermosensitive polymer, poly(N-isopropylacrylamide) shows a rapid response to temperature change and has been one of the earliest thermosensitive polymers to be coated on gold nanoparticles.16 However, development of thermosensitive gold nanoparticles with tunable lower critical solution temperature (LCST) by coating the nanoparticles with thermoresponsive polymers will be more attractive.20,40,41 Moreover, novel thermosensitive polymers with tunable collapse temperature as well as biodegradability and biocompatibility will be beneficial for potential biomedical applications. It has been demonstrated that block copolymers of MPEG and some polyphosphoesters are thermosensitive in aqueous solution and the LCST can be tuned by adjusting the chemical compositions. Those block copolymers have also exhibited good biocompatibility both in vitro and in vivo.27 Figure 3A shows the temperature dependence of transmittances of aqueous solutions (1 wt % in saline) of the thioctate ester of block copolymers MPEG-b-PEEP224, MPEG-bP(EEP235-co-PEP20), and MPEG-b-P(EEP220-co-PEP37), which have cloud points (Cp’s) at 49.6, 37.5, and 30.9 °C, respectively. Incorporation of the hydrophobic component (PEP unit) led to a decreased Cp, which is in agreement with our previous observations. An increase of temperature would cause the disruption of hydrogen bonding and dehydration, making the PEEP more hydrophobic, and it is assumed that dehydration of the polymer preferably occurred with the addition of the more hydrophobic PEP unit.27 Herein, we observed that gold nanoparticles stabilized by the block copolymer of MPEG and polyphosphoesters possessed similar thermoresponsive properties. As shown in Figure 3B, the transmittance of aqueous dispersions of gold nanoparticles at a concentration of 1 wt % in saline exhibited a rapid response to temperature changes. The solution was transparent at temperatures lower than the collapse temperature. However, as the temperature increased, the solution tuned to be turbid and the transmittance was dramatically reduced in response to the increased temperatures, owing to the predominant hydrophobicity over the hydrophilicity driven by the phase transition of PPE blocks. More importantly, the Cp, which is defined as the temperature exhibiting a 50% decrease in optical transmittance of an aqueous solution at 600 nm, is dependent on the polymer composition. From Figure 3B, it is observed that gold (38) Gil, E. S.; Hudson, S. A. Prog. Polym. Sci. 2004, 29, 1173–1222. (39) Dimitrov, I.; Trzebicka, B.; Muller, A. H. E.; Dworak, A.; Tsvetanov, C. B. Prog. Polym. Sci. 2007, 32, 1275–1343. (40) Lutz, J. F.; Akdemir, O.; Hoth, A. J. Am. Chem. Soc. 2006, 128, 13046– 13047. (41) Zou, Y. Q.; Brooks, D. E.; Kizhakkedathu, J. N. Macromolecules 2008, 41, 5393–5405.
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Figure 3. Temperature-dependence of transmittances of aqueous solution (1 wt % in saline) of the thioctate ester of the block copolymer (A) and gold nanoparticles stabilized by the block copolymer (B): (2) MPEG-b-PEEP224; (b) MPEG-b-P(EEP235co-PEP20); and (9) MPEG-b-P(EEP220-co-PEP37).
nanoparticles stabilized by MPEG-b-PEEP224 showed a cloud point at 44.5 °C, whereas coating MPEG-b-P(EEP235-co-PEP20) and MPEG-b-P(EEP220-co-PEP37) to gold nanoparticles led to significantly decreased cloud points. With 7.8% of PEP in MPEG-b-P(EEP235-co-PEP20), the Cp decreased to 33.3 °C, but more PEP components in MPEG-b-P(EEP220-co-PEP37) (14.4% of PEP) led to a much lower Cp at 28.0 °C. At this tested concentration (1 wt %), the association of the thermosensitive polymer of gold nanoparticles is likely to be dominant, which is consistent with the observation by the other group.20 Figure 4 shows the transmittance response of gold nanoparticles stabilized by MPEG-b-P(EEP235-co-PEP20) under several heating-cooling cycles between 25 and 50 °C. The result indicates that the suspension is transparent at 25 °C and opaque at 50 °C. However, the opaque suspension does not precipitate, and this temperature-dependent clear-opaque transition of the thermosensitive gold nanoparticles is completely reversible. The change in size of the block copolymer stabilized gold nanoparticles at different temperatures is verified by DLS measurements obtained at below and above the collapse temperature. As an example shown in Figure 5, the results suggest that the hydrodynamic radius of gold nanoparticles stabilized by MPEGb-P(EEP235-co-PEP20) is about 86 nm on average at 25 °C. This value is slightly larger than that observed by TEM owning to the significant contribution of solvent, which also indicates the swollen corona around the core in an aqueous solution. The Langmuir 2009, 25(17), 10298–10304
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Figure 4. Reversible transmittance curve of gold nanoparticles stabilized by MPEG-b-P(EEP235-co-PEP20) during heating-cooling cycles between 25 and 50 °C.
Figure 5. Size change of gold nanoparticles stabilized by MPEG-1
b-P(EEP235-co-PEP20) (0.1 mg mL ) in response to the temperature of the aqueous solution.
diameter at 44 °C was found to be 40 nm in average, reflecting a collapse of the shell around gold nanoparticles when the temperature is higher than its LCST. As schematically illustrated in Scheme 1, at lower temperature, the block copolymer chains should be fully extended, leading to larger size. When the temperature increased above the LSCT, the inner PPE blocks became hydrophobic and collapsed onto the gold surface, causing the decrease of the hydrodynamic radius. It is worth noting that no measurable particle aggregation occurred at this temperature and 0.1 mg mL-1, which should be a function of the outer MPEG corona. Also, the TEM image of gold nanoparticles at 44 °C in Figure 6 shows a smaller diameter than that at room temperature. Such thermoresponsibility of block copolymer stabilized gold nanoparticles only altered the thickness of the polymer coat layer at lower concentration, indicating that the intrachain “coil-toglobule” transition is dominant, which is similar to the observation by Li et al.17 Cytocompatibility of Polymer Stabilized Gold Nanoparticles. Biocompatibility is highly desired in biomedical applications. In fact, PEG is one of a few water-soluble polymers that Langmuir 2009, 25(17), 10298–10304
Figure 6. TEM image of gold nanoparticles stabilized by MPEGb-P(EEP235-co-PEP20) at 44 °C (scale bar is 60 nm).
Figure 7. Cytocompatibility of gold nanoparticles stabilized by (1) MPEG-b-P(EEP235-co-PEP20) to HEK293 cells as compared with (9) sodium dodecyl sulfate.
have been widely used to improve the biocompatibilities of drug carriers. It can also prevent recognition by the reticuloendothelial system, thus preventing preliminary elimination of the nanoparticles from the bloodstream, which results in prolonged periods of nanoparticle circulation time.42 Previous studies have demonstrated the biocompatibility of block copolymers of MPEG with polyphosphoesters.27 The cytotoxicity of gold nanoparticles stabilized by MPEG-b-P(EEP235-co-PEP20) to HEK293 cells was evaluated by MTT assay in this study. Figure 7 shows the viability of cells treated with the nanoparticles at different concentrations for two days, which was compared with those treated with sodium dodecyl sulfate. It indicates that more than 85% of cells cultured with gold nanoparticles stabilized by MPEG-b-P(EEP235-coPEP20) remained viable when the concentration was up to 1.0 mg mL-1, whereas there were few live cells left in the treatment with sodium dodecyl sulfate at the same concentration. Lower concentrations of gold nanoparticles stabilized by (42) Otsuka, H.; Nagasaki, Y.; Kataoka, K. Adv. Drug Delivery Rev. 2003, 55, 403–419.
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MPEG-b-P(EEP235-co-PEP20) did not exhibit any cytotoxicity to HEK293 cells, which suggests its good cytocompatibility.
Conclusions Gold nanoparticles protected by thermosensitive diblock copolymers with a tunable collapse temperature were prepared and studied. Three diblock copolymers composed of MPEG and PPE with different PEP compositions were prepared, and thioctic acid was then conjugated to the terminal hydroxyl group of the PPE block by esterification. The nanoparticles showed gold cores with a diameter of about 10 and polymer shells of 30 nm thick, exhibiting a structure with thermosensitive block polyphosphoester bounded to the particle surface and hydrophilic MPEG as the
10304 DOI: 10.1021/la901120x
outer corona. The collapse temperature was tunable, ranging from 28.0 to 44.5 °C, depending on the relative ratio of the PEP component. DLS measurements also confirmed the thermosensitive property of the gold nanoparticles. These gold nanoparticles protected by thermosensitive diblock copolymer are biocompatible with HEK293 cells and may have potential for application in cancer diagnosis and therapeutics. Acknowledgment. This work was supported by grants from the National Natural Science Foundation of China (50733003), the Ministry of Sciences and Technology of the People’s Republic of China (2006CB933300, 2009CB930300), and the “Bairen” Program of the Chinese Academy of Sciences.
Langmuir 2009, 25(17), 10298–10304
