Langmuir 2008, 24, 3407-3411
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Effect of Freeze-Thawing on Lipid Bilayer-Protected Gold Nanoparticles Lixue Zhang, Peicai Li, Dan Li, Shaojun Guo, and Erkang Wang* State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, Jilin, 130022, China, and Graduate School of the Chinese Academy of Sciences, Beijing 100039, China ReceiVed NoVember 29, 2007 In this study, varieties of lipid bilayer-protected gold nanoparticles (AuNPs) were synthesized through a simple wet chemical method, and then the effect of freeze-thawing on the as-prepared AuNPs was investigated. The freezethawing process induced fusion or fission of lipid bilayers tethered on the AuNPs. The UV-vis spectra and transmission electron microscopy experiments revealed that the disruption of lipid bilayer structures on the nanoparticles led to the fusion or aggregation of AuNPs. The role of freeze-thawing in the evolution of lipid bilayer-protected AuNPs was studied. The addition of adequate sucrose, a well-known cryoprotectant, effectively prevented the fusion or aggregation of lipid bilayer-protected AuNPs undergoing the freeze-thawing process. The possible mechanism of sucrose preserving the integrity of the lipid bilayer-protected AuNPs was also discussed.
Introduction From both a fundamental and a practical point of view, the synthesis, characterization, and applications of nanomaterials have attracted enormous attention due to their novel electronic, optical, and thermal properties.1-3 For all applications of nanomaterials, their stability is always crucial. In some cases, the biological samples need to be freeze-dried for long-time preservation and storage;4,5 however, this process often has some side effects, such as the denaturation of proteins and the disruption of cell membranes.4-7 With the increasing applications of nanomaterials as bio-labels, biosensors, drug carriers, and gene vehicles, their stability during the freeze-drying or freezethawing process is worth studying.5 Recently, gold nanoparticles (AuNPs) frequently have been used in the biomedical fields because of their strong adsorption in the visible and near-infrared region and their distinguished photothermal properties.8-13 The stability of AuNPs is one of the most important phenomena to study. Many studies have revealed that laser irradiation,14 electron beam irradiation,15 * To whom correspondence should be addressed. Fax: 86-431-85689711; e-mail:
[email protected]. (1) Daniel, M. C.; Astruc, D. Chem. ReV. 2004, 104, 293. (2) Burda, C.; Chen, X. B.; Narayanan, R.; El-Sayed, M. A. Chem. ReV. 2005, 105, 1025. (3) Tasis, D.; Tagmatarchis, N.; Bianco, A.; Prato, M. Chem. ReV. 2006, 106, 1105. (4) Li, B.; Li, S.; Tan, Y. D.; Stolz, D. B.; Watkins, S. C.; Block, L. H.; Huang, L. J. Pharm. Sci. 2000, 89, 355. (5) Sameti, M.; Bohr, G.; Kumar, M.; Kneuer, C.; Bakowsky, U.; Nacken, M.; Schmidt, H.; Lehr, C. M. Int. J. Pharm. 2003, 266, 51. (6) Leslie, S. B.; Israeli, E.; Lighthart, B.; Crowe, J. H.; Crowe, L. M. Appl. EnViron. Microb. 1995, 61, 3592. (7) Abdelwahed, W.; Degobert, G.; Stainmesse, S.; Fessi, H. AdV. Drug DeliVery ReV. 2006, 58, 1688. (8) O’Neal, D. P.; Hirsch, L. R.; Halas, N. J.; Payne, J. D.; West, J. L. Cancer Lett. 2004, 209, 171. (9) Hirsch, L. R.; Stafford, R. J.; Bankson, J. A.; Sershen, S. R.; Rivera, B.; Price, R. E.; Hazle, J. D.; Halas, N. J.; West, J. L. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 13549. (10) Huang, X. H.; El-Sayed, I. H.; Qian, W.; El-Sayed, M. A. J. Am. Chem. Soc. 2006, 128, 2115. (11) Cognet, L.; Tardin, C.; Boyer, D.; Choquet, D.; Tamarat, P.; Lounis, B. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 11350. (12) Skirtach, A. G.; Dejugnat, C.; Braun, D.; Susha, A. S.; Rogach, A. L.; Parak, W. J.; Mohwald, H.; Sukhorukov, G. B. Nano Lett. 2005, 5, 1371. (13) Li, J. Y.; Wang, X. M.; Wang, C. X.; Chen, B. A.; Dai, Y. Y.; Zhang, R. Y.; Song, M.; Lv, G.; Fu, D. G. ChemMedChem 2007, 2, 374.
thermal treatment,16 and halide ions,17,18 etc. lead to the fragmentation or fusion or aggregation of AuNPs, and these approaches are useful in the preparation of designed nanostructures. However, only a few works concern the state of AuNPs in the ice phase. Several water-soluble AuNPs protected by some hydrophilic polymers19,20 or a pentapeptide ligand21 exhibiting good stability after a freeze-drying process have been reported; meanwhile, the citrate sodium capped AuNPs aggregated completely after the same process.19 Sodium acrylate-stabilized AuNPs have been used to prepare aligned gold patterns on a mica surface by a directional freezing method.22 More recently, Richardson and co-workers investigated the thermo-optical properties of AuNPs embedded in an ice matrix, in which they found that the AuNPs were not in the form of single particles but formed small complexes of various geometries.23 Nevertheless, the stability of AuNPs during the freezing process has yet to attract attention until now. On the other hand, lipid bilayer-modified nanomaterials, such as noble metal nanoparticles,24-28 semiconductor quantum (14) Fujiwara, H.; Yanagida, S.; Kamat, P. V. J. Phys. Chem. B 1999, 103, 2589. (15) Chen, Y.; Palmer, R. E.; Wilcoxon, J. P. Langmuir 2006, 22, 2851. (16) Fullam, S.; Cottell, D.; Rensmo, H.; Fitzmaurice, D. AdV. Mater. 2000, 12, 1430. (17) Cheng, W. L.; Dong, S. J.; Wang, E. K. Angew. Chem., Int. Ed. 2003, 42, 449. (18) Singh, S.; Pasricha, R.; Bhatta, U. M.; Satyam, P. V.; Sastry, M.; Prasad, B. L. V. J. Mater. Chem. 2007, 17, 1614. (19) Haba, Y.; Kojima, C.; Harada, A.; Ura, T.; Horinaka, H.; Kono, K. Langmuir 2007, 23, 5243. (20) Mangeney, C.; Ferrage, F.; Aujard, I.; Marchi-Artzner, V.; Jullien, L.; Ouari, O.; Rekai, E. D.; Laschewsky, A.; Vikholm, I.; Sadowski, J. W. J. Am. Chem. Soc. 2002, 124, 5811. (21) Levy, R.; Thanh, N. T. K.; Doty, R. C.; Hussain, I.; Nichols, R. J.; Schiffrin, D. J.; Brust, M.; Fernig, D. G. J. Am. Chem. Soc. 2004, 126, 10076. (22) Zhang, H. F.; Hussain, I.; Brust, M.; Butler, M. F.; Rannard, S. P.; Cooper, A. I. Nat. Mater. 2005, 4, 787. (23) Richardson, H. H.; Hickman, Z. N.; Govorov, A. O.; Thomas, A. C.; Zhang, W.; Kordesch, M. E. Nano Lett. 2006, 6, 783. (24) Patil, V.; Mayya, K. S.; Pradhan, S. D.; Sastry, M. J. Am. Chem. Soc. 1997, 119, 9281. (25) Fan, H. Y.; Leve, E.; Gabaldon, J.; Wright, A.; Haddad, R. E.; Brinker, C. J. AdV. Mater. 2005, 17, 2587. (26) He, P.; Urban, M. W. Biomacromolecules 2005, 6, 1224. (27) Takahashi, H.; Niidome, Y.; Niidome, T.; Kaneko, K.; Kawasaki, H.; Yamada, S. Langmuir 2006, 22, 2. (28) Zhang, L. X.; Sun, X. P.; Song, Y. H.; Jiang, X. E.; Dong, S. J.; Wang, E. K. Langmuir 2006, 22, 2838.
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dots,29-32 silica nanoparticles,33 and carbon nanotubes,34-36 have been extensively researched in recent years with respect to their water solubility, biocompatibility, and potential applications in many fields. It is believed that the lipid bilayers coated on the nanomaterials behave in the same way as biomembranes to a certain extent. This may lead us to use them as biomembrane models for the study of a wide variety of biological functions, such as membrane fusion, the interaction between proteins and cell membranes, and other processes in the fields of biophysics, chemistry, and medicine.33,34 Thus, aspects such as stability and fluidity of the lipid bilayer supported on the nanoscale substrate need to be examined. It is well-known that repeated freezethawing processes lead to the fusion or fission of liposomes, which change their sizes and permeability.37-41 Therefore, it is very interesting to make clear if a similar fusion or fission of the lipid bilayers, which are coated on the nanoscale substrate, induced by freeze-thawing also takes place and to what extent this will affect the AuNPs themselves. Here, we synthesized varieties of lipid bilayer-protected AuNPs, using didodecyldimethylammonium bromide (DDAB), cetyltrimethylammonium bromide (CTAB), dioctadecyldimethylammonium bromide (DODAB), and dimyristoyl phosphatidylglycerol (DMPG) via a simple wet chemical approach. The effect of freeze-thawing on the as-prepared AuNPs was investigated. It was found that freeze-thawing could induce fusion or fission of the lipid bilayers tethered on the nanoparticles, which may lead to the fusion or aggregation of AuNPs. However, the as-prepared DODAB lipid bilayer-protected AuNPs were still stable after several freeze-thawing cycles. Moreover, sucrose was examined for its cryoprotective activity during the freezethawing process of lipid bilayer-protected AuNPs. It was found that the addition of an appropriate amount of sucrose would effectively prevent the fusion or aggregation of AuNPs undergoing the freeze-thawing process.
Zhang et al. Scheme 1. Illustration of a Lipid Bilayer-Protected AuNP
mM, respectively. Then, the freshly prepared NaBH4 aqueous solution was added into the former stirred solution drop by drop. The asformed lipid bilayer-protected AuNP solutions were stored at room temperature for further use. To remove the uncoordinated lipid molecules from solution, the DODAB bilayer-protected AuNP solution was centrifuged 2 times at 10 000 rpm/min at room temperature for 10 min. The top solution was decanted off, and the bottom fraction of the AuNPs was saved. The resulting concentrated solution of AuNPs was then diluted to a suitable concentration. Freeze-Thawing Protocol. Typically, the sample was frozen at -20 °C in a refrigerator for at least 1 h and then thawed at room temperature with shaking or sonicating. For DODAB bilayerprotected AuNPs, the samples were thawed at room temperature first, and then the samples were treated at 50 °C with sonicating to make sure that complete rehydration of DODAB occurred. This operation was repeated several times. Another two different freezing protocols were adopted for DDAB bilayer-protected AuNPs at -80 °C for 1 h and in liquid nitrogen for 10 min. Measurements. UV-vis spectra of the samples were recorded using a Cary 500 Scan UV-vis/NIR spectrophotometer. The photographs were taken using a Canon A710 IS digital camera. Transmission electron microscopy (TEM) measurements were performed on a Hitachi H-8100 transmission electron microscope operated at an accelerating voltage of 200 kV.
Experimental Procedures
Results and Discussion
Chemicals. Chloroauric acid (HAuCl4) was purchased from the Shanghai Chemical Reagent Company. Sodium borohydride (NaBH4), DDAB, and CTAB were obtained from Aldrich. DODAB and DMPG were purchased from Sigma. Sucrose was obtained from the Beijing Chemical Industry. All reagents were used as received without further purification. The water used was purified through a Millipore system. Synthesis of Lipid Bilayer-Protected AuNPs. The various lipid bilayer-protected AuNPs were prepared according to the following procedure.28 First, the HAuCl4 aqueous solution and different lipid vesicle aqueous solutions were mixed together under vigorous stirring. The final concentrations of HAuCl4 and lipid were 0.3 mM and 1
We previously synthesized the DDAB bilayer-protected AuNPs through a facile one-step method.28 Here, we successfully extended this method to the preparation of some other lipid bilayer-protected AuNPs. Like DDAB, CTAB, DODAB, and DMPG act as effective protective reagents for the AuNPs. The schematic illustration of a lipid bilayer-protected AuNP is shown in Scheme 1. To study the effect of freeze-thawing on the lipid bilayerprotected AuNPs, we selected the DDAB bilayer-protected AuNPs as the model. As shown in Figure 1A, it was found that when the DDAB bilayer-protected AuNPs underwent several freeze-thawing cycles, the native surface plasmon band showed a gradual red shift and the absorbance in the red/infrared region increased with increasing the number of freeze-thawing cycles. The UV-vis data show that AuNPs redistributed in aqueous solution after freeze-thawing may aggregate or fuse together.1 The TEM experiments were performed to further characterize the effect of freeze-thawing on the morphology of DDAB bilayer-protected AuNPs. From the TEM image (Figure 1B), it can be seen that most of the as-prepared DDAB bilayer-protected AuNPs are in a spherical shape with a diameter of 5-8 nm and are well-separated from each other. However, after one freezethawing cycle, the morphology of the AuNPs varied greatly. Some large nanostructures, which were in the shape of triangular and truncated triangular plates, nanorods, and irregular nanoparticles, were clearly observed. Meanwhile, many smaller AuNPs still existed (Figure 1C). We suggest that the large nanostructures are derived from the fusion of the as-parepared DDAB bilayer-
(29) Fan, H. Y.; Leve, E. W.; Scullin, C.; Gabaldon, J.; Tallant, D.; Bunge, S.; Boyle, T.; Wilson, M. C.; Brinker, C. J. Nano Lett. 2005, 5, 645. (30) Geissbuehler, I.; Hovius, R.; Martinez, K. L.; Adrian, M.; Thampi, K. R.; Vogel, H. Angew. Chem., Int. Ed. 2005, 44, 1388. (31) Gopalakrishnan, G.; Danelon, C.; Izewska, P.; Prummer, M.; Bolinger, P. Y.; Geissbuhler, I.; Demurtas, D.; Dubochet, J.; Vogel, H. Angew. Chem., Int. Ed. 2006, 45, 5478. (32) Mulder, W. J. M.; Koole, R.; Brandwijk, R. J.; Storm, G.; Chin, P. T. K.; Strijkers, G. J.; Donega, C. D.; Nicolay, K.; Griffioen, A. W. Nano Lett. 2006, 6, 1. (33) Mornet, S.; Lambert, O.; Duguet, E.; Brisson, A. Nano Lett. 2005, 5, 281. (34) Zhou, X. J.; Moran-Mirabal, J. M.; Craighead, H. G.; McEuen, P. L. Nat. Nanotechnol. 2007, 2, 185. (35) Artyukhin, A. B.; Shestakov, A.; Harper, J.; Bakajin, O.; Stroeve, P.; Noy, A. J. Am. Chem. Soc. 2005, 127, 7538. (36) He, P.; Urban, M. W. Biomacromolecules 2005, 6, 2455. (37) Macdonald, R. C.; Jones, F. D.; Qiu, R. Z. Biochim. Biophys. Acta 1994, 1191, 362. (38) Oku, N.; MacDonald, R. C. Biochemistry 1983, 22, 855. (39) Sriwongsitanont, S.; Ueno, M. Chem. Pharm. Bull. 2004, 52, 641. (40) Sriwongsitanont, S.; Ueno, M. Colloid Polym. Sci. 2004, 282, 753. (41) Ueno, M.; Sriwongsitanont, S. Polymer 2005, 46, 1257.
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Figure 1. UV-vis spectra of DDAB bilayer-protected AuNPs (A): (a-e) after 0, 1, 2, 5, and 10 freeze-thawing cycles, respectively, and TEM image of DDAB bilayer-AuNPs after (B) 0, (C) 1, and (D) 10 freeze-thawing cycles, respectively.
protected AuNPs. After 10 freeze-thawing cycles, the TEM image is similar to one freeze-thawing cycle sample, except that the sizes and amount of the large nanostructures increased (Figure 1D). We also investigated the effect of different freezing protocols on DDAB bilayer-protected AuNPs. The UV-vis spectra show that different freezing protocols have a similar effect on the DDAB bilayer-protected AuNPs (Supporting Information). From both the UV-vis and the TEM results, it can be found that freeze-thawing indeed has a great effect on the DDAB bilayer-protected AuNPs. Sucrose, a popular cryoprotectant,6 was first tested for its use in the freezing of AuNPs. After thawing, the UV-vis spectra of DDAB bilayer-protected AuNPs with the addition of sucrose are shown in Figure 2A, which reveals that with an increasing concentration of sucrose, the degree of red shift of the surface plasmon band of the AuNPs decreased. When the concentration of sucrose is up to 50 mg/mL, there is no observable red shift after the freeze-thawing process. The TEM result (Figure 2B) confirms that the DDAB bilayer-protected AuNPs maintain their sizes, spherical shapes, and separation from each other without any aggregation after one freeze-thawing cycle with a sucrose concentration of 50 mg/mL. The stability of AuNPs increases markedly, and their coalescence and fusion during freezethawing can be prevented effectively by adding adequate sucrose. For CTAB and DMPG bilayer-protected AuNPs, analogous but rather dramatic changes of the UV-vis spectra and shape evolution appeared after freeze-thawing (Supporting Information). However, the DODAB bilayer-protected AuNPs exhibited an unusual stability during freeze-thawing. For DODAB bilayerprotected AuNPs, the peak of the plasmon resonance band is located at 540 nm (Figure 3A), and the TEM image shows that the AuNPs mainly range from 10 to 20 nm (Figure 3B). UV-vis spectra show no obvious change even after several freezethawing cycles without the addition of sucrose (Figure 3A). However, after removing the excess DODAB molecules in the
solution, the UV-vis spectra show that the AuNPs aggregated after a freeze-thawing cycle and that sucrose exhibited a protective activity on the DODAB bilayer-protected AuNPs even after the excess ligands were removed (Figure 4). Freeze-thawing is frequently used to prepare liposomes to homogenize their lipid composition,42,43 and it could be used to immobilize liposomes in the chromatographic gel beads.44 The lipid headgroup binds to water and forms a primary hydration shell, which is in rapid exchange with bulk water.45 The freezing process would remove the hydrogen bonded water from the lipid headgroup region of the lipid bilayer, which increases the headgroup packing, leading to an increase of van der Waals interactions between the acyl chains. The increase of van der Waals interactions between the acyl chains forces the lipid bilayer to undergo a phase transition: from the liquid crystalline to the gel phase. Upon thawing, the rehydration of the dry lipid forces it to undergo a reverse phase transition: from the gel phase back to the liquid crystalline phase, resulting in a leaky lipid bilayer.6 Otherwise, the solutes in the aqueous solution are concentrated close to the freezing front during the freezing process.22,38 For the DDAB bilayer-protected AuNP solution, its photograph after freezing shows a gray color in the ice phase instead of a wine-red color in the aqueous solution (Figure 5A,B). It is suggested that it is the freezing process that causes the aggregation of AuNPs, which should be attributed to the disruption of the lipid bilayer that is tethered on the AuNPs through weak electrostatic forces.28,46 Because the main phase transition temperature (Tm) (42) Mayer, L. D.; Hope, M. J.; Cullis, P. R.; Janoff, A. S. Biochim. Biophys. Acta 1985, 817, 193. (43) Traikia, M.; Warschawski, D. E.; Recouvreur, M.; Cartaud, J.; Devaux, P. F. Eur. Biophys. J. 2000, 29, 184. (44) Lundqvist, A.; Ocklind, G.; Haneskog, L.; Lundahl, P. J. Mol. Recognit. 1998, 11, 52. (45) Borle, F.; Seelig, J. Biochim. Biophys. Acta 1983, 735, 131. (46) Strauss, G.; Hauser, H. Proc. Natl. Acad. Sci. U.S.A. 1986, 83, 2422.
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Figure 2. UV-vis spectra of DDAB bilayer-protected AuNPs (A): (a) as-prepared and (b-e) after one freeze-thawing cycle with sucrose at a concentration of 0, 10, 25, and 50 mg/mL, respectively. (B) TEM image of DDAB bilayer-AuNPs after one freeze-thawing cycle with sucrose at a concentration of 50 mg/mL.
of DDAB is about 16 °C,47 the DDAB bilayers tethered on the AuNPs and DDAB vesicles are all in a liquid crystalline phase at room temperature; thus, the phase transition from the liquid crystalline to the gel phase during the freezing process would lead to the leaky lipid bilayer, easily leading to the disruption of the DDAB bilayer that decreases the repulsion between the AuNPs. Thus, the concentrated AuNPs, which have a high surface energy, lost the fine protection accompanying the disruption of lipid bilayers and tended to coalesce together. Upon thawing, the color of the sample slowly changed from gray to dark blue to purple (Figure 5C-E). This change indicates that the DDAB molecules formed a bilayer structure on the AuNPs again, which separated the adjacent or coalescent AuNPs into well-dispersed particles. Eventually, parts of the DDAB bilayer-protected AuNPs fused into large nanoparticles with different shapes, and the other AuNPs still maintained their sizes and shapes after a freezethawing process. The reason for the stability of DODAB bilayer-protected AuNPs during freeze-thawing may come from the intrinsic stability of DODAB lipid bilayers.48 As shown in Figure 6, when the DODAB bilayer-protected AuNP solution was frozen, it maintained a purple color. After thawing at room temperature (47) Feitosa, E.; Alves, F. R.; Niemiec, A.; Oliveira, M.; Castanheira, E. M. S.; Baptista, A. L. F. Langmuir 2006, 22, 3579. (48) Hubert, D. H. W.; Jung, M.; Frederik, P. M.; Bomans, P. H. H.; Meuldijk, J.; German, A. L. AdV. Mater. 2000, 12, 1286.
Zhang et al.
Figure 3. UV-vis spectra of DODAB bilayer-protected AuNPs (A): (a-c) after 0, 1, and 5 freeze-thawing cycles, respectively. (B) TEM images of DODAB bilayer-protected AuNPs.
Figure 4. UV-vis spectra of DODAB bilayer-protected AuNPs after being centrifuged: (a) before and (b) after one freeze-thawing cycle and (c) after one freeze-thawing cycle with sucrose at a concentration of 50 mg/mL.
with shaking, the DODAB bilayer-protected AuNP sample contained a purple precipitate, which was a clustering of DODAB and adjacent but not fused AuNPs, and at that time, the DODAB molecules were not completely rehydrated. The Tm of DODAB is about 43 °C;47 therefore, no phase transition occurred during the freezing process because the DODAB bilayer in the aqueous
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Figure 5. Photographs of DDAB bilayer-protected AuNPs: (A) as-synthesized, (B) the ice phase of AuNPs, (C-E) the evolution of AuNPs during the thawing process, and (F) the ice phase of AuNPs with sucrose at a concentration of 50 mg/mL.
occur around the lipid bilayer of AuNPs during freezing; therefore, the fusion or aggregation of AuNPs can be avoided as the sucrose prevents the lipid bilayer coatings from interacting with each other. However, the formation of hydrogen bonding between the sucrose and the cationic lipid is impossible because the headgroup of the cationic lipid lacks free electrons.49 For the as-prepared cationic lipid (i.e., DDAB, CTAB, and DODAB) bilayer-protected AuNPs, it is proposed that sucrose mainly plays the role of vitrifier during freeze-thawing. Figure 6. Photographs of DODAB bilayer-protected AuNPs: (A) as-synthesized, (B) the ice phase of AuNPs, and (C) after thawing at room temperature with shaking.
solution was in the gel phase at room temperature. It was suggested that the water molecules crystallize around the DODAB interface during the freezing process but that less of the DODAB bilayer would be leaky, leading to less disruption.49 Thus, the AuNPs themselves could keep their sizes and shapes during freezing. For the centrifuged DODAB bilayer-protected AuNPs, they aggregated after freeze-thawing, which may be attributed to the decrease of compactness of the lipid bilayer structure on the AuNPs after being redistributed in aqueous solution followed by removing the excess DODAB molecules in the solution. Disaccharides are frequently used to preserve the stability of liposomes and cell membranes during freeze-thawing and freeze-drying processes.6,50-53 A photograph of the DDAB bilayer-protected AuNPs with 50 mg/mL sucrose after freezing is shown in Figure 5F, and it still is a wine-red. This phenomenon demonstrates that the DDAB bilayer-protected AuNPs did not coalesce or aggregate together during the freezing process in the presence of sucrose. The mechanism by which sucrose preserves the original sizes and shapes of lipid bilayerprotected AuNPs is not completely understood. Previous studies have suggested that the addition of sucrose could form a rigid sugar-glass or vitrified network around the liposomes and thereby prevent lipid bilayer fusion or fission. Another possibility is that sucrose could interact with the lipid bilayer directly through hydrogen bonding, which forms a steric barrier to prevent bilayer apposition.4,46,49,50 It is believed that similar protective processes (49) Christensen, D.; Foged, C.; Rosenkrands, I.; Nielsen, H. M.; Andersen, P.; Agger, E. M. Biochim. Biophys. Acta 2007, 1768, 2120. (50) Anchordoguy, T. J.; Rudolph, A. S.; Carpenter, J. F.; Crowe, J. H. Cryobiology 1987, 24, 324. (51) Crowe, J. H.; Hoekstra, F. A.; Nguyen, K. H. N.; Crowe, L. M. Biochim. Biophys. Acta 1996, 1280, 187. (52) Sun, W. Q.; Leopold, A. C.; Crowe, L. M.; Crowe, J. H. Biophys. J. 1996, 70, 1769. (53) Wolfe, J.; Bryant, G. Cryobiology 1999, 39, 103.
Conclusion Several lipid bilayer-protected AuNPs were synthesized through a simple wet chemical method, and the effect of the freeze-thawing process on the lipid bilayer-protected AuNPs was investigated. On the basis of the aforementioned observations, the freeze-thawing process had great effects on the lipid bilayerprotected AuNPs. Unlike citrate sodium capped AuNPs and some hydrophilic polymer capped AuNPs,19,20 the lipid bilayerprotected AuNPs exhibited moderate stability during the freezethawing process. The lipid bilayer-protected AuNPs could not maintain their shapes (except the DODAB bilayer-protected AuNPs) and did not aggregate completely after freeze-thawing. This moderate stability should be attributed to the lipid bilayer structure of the protective reagents on the AuNPs, which is similar to a biomembrane structure. The lipid bilayer structures supported on the nanoscale substrate through weak electrostatic forces have a similar stability with the liposome during freeze-thawing, and the effect of freeze-thawing seems to be related to the intrinsic stability of the lipid bilayer. The data presented also confirm that sucrose in addition to its well-known ability to preserve the integrity of liposomes and solid lipid nanoparticles is able to protect the lipid bilayer-protected AuNPs during freeze-thawing. These findings are of great importance for studies of biomembranes supported on the nanoscale substrates, for drug and gene release, and for bio-labeling applications of lipid bilayer-protected nanomaterials. Acknowledgment. This work was supported by the National Natural Science Foundation of China by Grants 20335040 and 20575063 and the Chinese Academy of Sciences (KJCX2. YW.H09). Supporting Information Available: UV-vis spectra of DDAB bilayer-protected AuNPs that underwent different freezing protocols and experimental data of CTAB and DMPG bilayer-protected AuNPs. This material is available free of charge via the Internet at http:// pubs.acs.org. LA703737Q
