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Direct Facile Approach to the Fabrication of Chitosan-Gold Hybrid Nanospheres Rui Guo,† Leyang Zhang,† Zhenshu Zhu,† and Xiqun Jiang*,†,‡ Laboratory of Mesoscopic Chemistry and Department of Polymer Science & Engineering, College of Chemistry & Chemical Engineering, and Jiangsu ProVincial Laboratory for Nanotechnology, Nanjing UniVersity, Nanjing, 210093, PR China ReceiVed October 4, 2007. In Final Form: January 5, 2008 Chitosan-gold hybrid nanospheres were prepared through a direct facile approach that utilized cross-linked composite nanospheres consisting of low-molecular-weight chitosan (LWCS) and ethylenediaminetetraacetic acid (EDTA) as a precursor reaction system. EDTA was employed not only to construct the counterion interaction-based composite nanospheres with the cationic chitosan but also as the reductant for subsequent in situ gold salt reduction within the LWCS-EDTA composite nanospheres. This approach elegantly ensured that each and every nanosphere was loaded with gold nanoparticles and no nonembedded free gold nanoparticles would exist in the dispersing medium. Moreover, becauseof the noncovalent interaction between LWCS and EDTA, the EDTA reductant can be easily removed from the cross-linked nanospheres, and “pure” chitosan-gold hybrid nanospheres can be obtained. The obtained chitosangold hybrid nanospheres were found to have a tunable size and good dispersing stability within a wide pH range. The embedded gold nanoparticles were in the range from several to several tens of nanometers, which may be useful for sensing and imaging. Morphology studies indicated that most of the loaded gold nanoparticles were located in the interior of the hybrid nanospheres. Taking into account the good biocompatibilities of LWCS, abundant functional (amino) groups in chitosan, and the mild preparation conditions, we find that the chitosan-gold hybrid nanospheres prepared here may have tremendous potential in advanced biomedical applications.
Introduction In recent years, polymeric nanospheres, especially biocompatible ones, have found wide-spread application in drug delivery, gene therapy, and so forth.1,2 Meanwhile, many inorganic nanomaterials are also showing their strength in fields ranging from catalysis to sensing and imaging.3,4 Consequently, because of the combined and synergic properties arising from both the polymeric nanospheres and inorganic nanomaterials, polymerinorganic hybrid nanospheres have attracted more and more attention in recent years.5-19 Among the inorganic materials * To whom correspondence should be addressed. E-mail: jiangx@ nju.edu.cn. Fax: 86 25 83317761. † Laboratory of Mesoscopic Chemistry and Department of Polymer Science & Engineering. ‡ Jiangsu Provincial Laboratory for Nanotechnology. (1) Edlund, U.; Albertsson, A. C. AdV. Polym. Sci. 2002, 157, 67. (2) Panyam, J.; Labhasetwar, V. AdV. Drug DeliVery ReV. 2003, 55, 329. (3) Daniel, M.; Astruc, D. Chem. ReV. 2004, 104, 293. (4) Michalet, X.; Pinaud, F. F.; Bentolila, L. A.; Tsay, J. M.; Doose, S.; Li, J. J.; Sundaresan, G.; Wu, A. M.; Gambhir, S. S.; Weiss, S. Science 2005, 307, 538. (5) Crooks, R.; Zhao, M.; Sun, L.; Chechik, V.; Yeung, L. Acc. Chem. Res. 2001, 34, 181. (6) Godovsky, D. Y. AdV. Polym. Sci. 2000, 153, 162. (7) Zhang, J. G.; Coombs, N.; Kumacheva, E.; Lin, Y. K.; Sargent, E. H. AdV. Mater. 2002, 14, 1756. (8) Ding, Y.; Hu, Y.; Jiang, X.; Zhang, L.; Yang, C. Angew. Chem., Int. Ed. 2004, 43, 6369. (9) Ding, Y.; Hu, Y.; Zhang, L.; Chen, Y.; Jiang, X. Biomacromolecules 2006, 7, 1766. (10) Perkin, K.; Turner, J.; Wooley, K.; Mann, S. Nano Lett. 2005, 5, 1457. (11) Cen, L.; Neoh, K.; Kang, E. AdV. Mater. 2005, 17, 1656. (12) Kang, Y.; Taton, T. Angew. Chem., Int. Ed. 2005, 44, 409. (13) Liu, S.; Zhang, Z.; Han, M. AdV. Mater. 2005, 17, 1862. (14) Kim, D.; Kang, S.; Kong, B.; Kim, W.; Paik, H.; Choi, H.; Choi, I. Macromol. Chem. Phys. 2005, 206, 1941. (15) Singh, N.; Lyon, L. Chem. Mater. 2007, 19, 719. (16) Corbierre, M.; Cameron, N.; Sutton, M.; Mochrie, S.; Lurio, L.; Ruhm, A.; Lennox, R. J. Am. Chem. Soc. 2001, 123, 10411. (17) Sharma, G.; Ballauff, M. Macromol. Rapid Commun. 2004, 25, 547. (18) Suzuki, D.; Kawaguchi, H. Langmuir 2005, 21, 12016. (19) Suzuki, D.; Kawaguchi, H. Langmuir 2006, 22, 3818.
commonly employed in the construction of such hybrid nanospheres, gold nanoparticles are of particular interest because of their unique optical and electronic characteristics as well as their excellent biocompatibility.3 Gold nanoparticles with different shapes and sizes have been widely used in fundamental research, catalysis, biosensing, and, very recently, the diagnosis and treatment of cancer.3,20,21 By hybridization of gold nanoparticles into biocompatible polymeric nanospheres, the resultant hybrid system is expected to be of great use simultaneously in advanced biomedical applications such as bioreactors, drug delivery, and diagnosis. However, despite the fact that both gold nanoparticles3 and polymeric nanospheres1,2 could be readily obtained through many established methods, it is indeed not an easy task to prepare polymer-gold hybrid nanospheres effectively. So far, there are mainly two kinds of approaches available for the synthesis of such hybrid nanospheres. The first one comprises two steps: the preparation of gold nanoparticles and the subsequent encapsulation of the gold nanoparticles with polymers.12-15 However, this strategy requires that either the gold nanoparticles be surface modified7 or the polymers have special groups with good affinity to gold.15,16 Moreover, there would inevitably be a fraction of empty polymer nanospheres without any gold loading.12,13 The second approach is to synthesize polymer nanospheres first and then add Au salt to the system, after which the Au salt adsorbed on the polymer nanospheres is reduced in situ with an additional mild reducing agent.17-19 Nevertheless, in contrast to the first strategy, this approach requires that the polymer nanospheres have sufficient affinity with the gold salt and usually leaves nonembedded gold nanoparticles in the dispersing medium as the result of the reduction of Au salt outside of polymer nanospheres. Obviously, both of the above-mentioned approaches (20) El-Sayed, I.; Huang, X.; El-Sayed, M. Nano Lett. 2005, 5, 829. (21) Huang, X.; El-Sayed, I.; Qian, W.; El-Sayed, M. J. Am. Chem. Soc. 2006, 128, 2115.
10.1021/la703080j CCC: $40.75 © 2008 American Chemical Society Published on Web 02/22/2008
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Figure 1. Schematic illustration of the preparation of chitosangold hybrid nanospheres.
need laborious materials synthesis and an extra separation process to obtain “pure” polymer-gold hybrid nanospheres. Herein, we report a direct facile approach to the fabrication of pure biocompatible polymer-gold hybrid nanospheres through a precursor polymer/reductant composite nanoparticulate reaction system. Low-molecular-weight water-soluble chitosan (LWCS) was chosen as the polymer moiety, considering its good biocompatibility,22,23 and ethylenediaminetetraacetic acid (EDTA) was employed as the reductant moiety, utilizing its reducing ability.24 To prepare chitosan-gold hybrid nanospheres, we first synthesized cross-linked LWCS-EDTA composite nanospheres on the basis of electrostatic interaction using a novel nonsolvent (ethanol)-aided counterion complexation method. Then the obtained composite nanospheres were subjected to reaction with HAuCl4. Because EDTA, the reductant in this system, was one of the components that construct the LWCS-EDTA composite nanospheres, the chemical reduction of gold salt could occur only within the particle interior, which elegantly ensured that each and every nanosphere was loaded with gold nanoparticles and no free gold nanoparticles were generated out of the nanospheres in the dispersing medium. Moreover, thanks to the noncovalent interaction between LWCS and EDTA and also the small-molecule nature of EDTA, the EDTA reductant can be easily removed from the cross-linked nanospheres by dialysis against water after completing its role so that pure chitosangold hybrid nanospheres can be directly obtained with no need for complicated materials synthesis or extra separation (Figure 1). Considering that both LWCS and EDTA have good biocompatibility and the preparation is simple and mild without harsh organic solvents, the obtained hybrid system is appealing for biomedical purposes.
Guo et al. aided counterion complexation method was used. Briefly, 50 mg of LWCS were first dissolved in 10 mL of water. A certain amount of EDTA was then added to the LWCS aqueous solution and stirred until dissolution. Then, ethanol (nonsolvent) was added dropwise to the system under vigorous stirring, and the clear solution turned opalescent when the concentration of ethanol exceeded a certain critical value, implying the formation of colloidal particles. After that, 30 µL of GA solution (25%) was used to cross link the obtained nanospheres at room temperature for 4 h. The cross-linked nanospheres were purified using centrifugation, followed by redispersing the sediment into ethanol solution (40% v/v). Preparation of Chitosan-Gold Hybrid Nanoparticles. The suspension (2 mL) containing LWCS-EDTA composite nanospheres with a concentration of 0.25% w/v was mixed with 12.5, 25, 50, and 100 µL of HAuCl4 solution (1% w/w) and placed into a water bath at 70 °C under stirring. The mixture was allowed to react for 10 min, after which it was slowly cooled and dialyzed against pure water for 24 h to remove the EDTA molecules. Dynamic Light Scattering (DLS) Analysis. The mean diameter and size distribution of the prepared nanospheres were determined by DLS using a Brookhaven BI-9000AT instrument (Brookhaven Instruments Corporation). The turbidity of the solution was also monitored on the basis of the light scattering intensity reported in the DLS analysis. Each sample was adjusted to a concentration of 0.05% (w/v) in filtered water and sonicated before measuring. All results were the average of triplicate measurements. UV and FTIR. The UV absorption spectra were recorded on a Shimadzu UV3100 spectrophotometer (Shimadzu, Japan). Samples were freeze dried prior to FTIR and XRD analyses. FTIR spectra were collected using a Bruke IFS66V vacuum-type spectrometer (Bruke, Germany). Morphological Studies. Morphological studies of the hybrid nanospheres were carried out using transmission electron microscopy (TEM) (JEM-100S, JEOL, Japan), scanning electron microscopy (SEM) (Philips), and atomic force microscopy (AFM) (SPI 3800, Seiko Instruments Inc., Japan). For TEM observations, 1 drop of properly diluted sample was placed on a copper grid covered with a nitrocellulose membrane and air dried before examination. For SEM and AFM observations, a drop of the suspension was placed on a clean silicon wafer and air dried. The silicon wafer with nanospheres on it was subjected to AFM analysis directly using a 20 µm scanner in tapping mode, whereas in the case of SEM, the sample wafer was coated with a thin layer of gold prior to observation.
Results and Discussion
Materials. Water-soluble chitosan (LWCS) with an average molecular weight (Mn) of 5000 was purchased from Yuhuan Biomedical Company (Zhejiang, China) and used without further purification. EDTA (Sigma), glutaraldehyde (GA) (Sigma), and HAuCl4 (Aldrich) were used as received. All other ingredients were analytical grade unless otherwise stated. Preparation of LWCS-EDTA Composite Nanospheres. To prepare LWCS-EDTA composite nanospheres, a novel nonsolvent-
Being a cationic biopolymer bearing amino groups, chitosan chains can undergo counterion condensation in aqueous solution with many multivalent anionic substances, including poly(acrylic acid) and EDTA,8,9,25,26 to form colloidal aggregates. In our case, however, LWCS could not form an insoluble counterion complex in water with anionic EDTA probably because of its extremely small molecular weight (Mn ) 5000). Therefore, to prepare the LWCS-EDTA composite nanospheres, we used a novel nonsolvent-aided counterion complexation method in which ethanol was introduced to facilitate counterion condensation between LWCS and EDTA. With the addition of ethanol, a nonsolvent for both chitosan and EDTA, the chitosan chains and EDTA molecules may get closer because of the desolvation of chitosan, resulting in increased counterion interactions. Besides, the introduction of a nonsolvent may also help the counterion complex to assemble into colloidal particles. Figure 2a depicts the change in the scattering intensity of the solution containing both LWCS (0.5% w/v) and EDTA (0.17% w/v) as a function of ethanol concentration in the system. It can be found that with the addition of ethanol the scattering intensity of the solution experienced a
(22) Richardson, S.; Kolbe, H.; Duncan, R. Int. J. Pharm. 1999, 178, 231. (23) Chae, S.; Jang, M.; Nah, J. J. Controlled Release 2005, 102, 383. (24) Sarac, A. S. Prog. Polym. Sci. 1999, 24, 1149.
(25) Hu, Y.; Chen, Y.; Chen, Q.; Zhang, L.; Jiang, X.; Yang, C. Polymer 2005, 46, 12703. (26) Rana, R.; Murthy, V.; Yu, J.; Wong, M. AdV. Mater. 2005, 17, 1145.
Experimental Section
Fabrication of Chitosan-Gold Hybrid Nanospheres
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Figure 2. (a) Change in scattering intensity of the solution containing both chitosan (0.5% w/v) and EDTA (0.17% w/v) as a function of ethanol concentration in comparison with that of the solutions of EDTA and of chitosan. (b) log-normal size distributions of three batches of the LWCS-EDTA composite nanospheres obtained using different weight ratios of LWCS to EDTA (9, LWCS/EDTA ) 10:1; 2, LWCS/EDTA ) 5:1; [, LWCS/EDTA ) 3:1). (c, d) Typical TEM micrographs of the LWCS-EDTA composite nanospheres with different sizes: 80 and 230 nm, respectively.
sudden increase as the result of nanosphere formation when the percentage of added ethanol reached a certain critical value (about 37%). In contrast, neither the individual chitosan solution nor the individual EDTA solution (pH 10, adjusted with NaOH) had such an increase in scattering intensity, and no nanospheres were detected even if the ethanol fraction exceeded 50%, suggesting that the increase in scattering intensity did result from the counterion complexation between LWCS and EDTA and not just the simple precipitation of chitosan or EDTA caused by the addition of a nonsolvent. The counterion complex structure was also confirmed by FTIR analysis, as will be discussed later. Because the counterion condensation between LWCS and EDTA is a process determined by the electrostatic interaction between two oppositely charged substances, the ratio of the positively charged LWCS to the negatively charged EDTA plays a key role in the formation of LWCS-EDTA composite nanospheres. We found that when the ratio of LWCS to EDTA in the initial aqueous solution was increased the concentration of ethanol required to induce the counterion condensation between LWCS and EDTA also increased accordingly (Supporting Information) whereas the diameter of the resultant LWCS-EDTA composite nanospheres decreased as depicted in Figure 2b, rendering this system highly flexible in terms of size control. Figure 2c,d shows TEM micrographs of the cross-linked composite nanospheres of two typical size distributions: 80 and 230 nm, respectively, as determined by DLS. It can be seen that the nanospheres had a spherical shape and smooth outline with nearly uniform size that was slightly smaller than the DLS result because of the dehydration of the nanospheres. Though EDTA is famous for its outstanding chelating ability, which has already been utilized in the construction the electrostatic interaction-based LWCS-EDTA composite nanospheres, it is
its reducing ability that we are exploiting in the in situ reduction of gold salt. Previous work has demonstrated that EDTA could be oxidized through decarboxylation by highly oxidative substances;24 however, there is no documentation about the reduction of gold salt using EDTA as the reductant. Therefore, a preliminary experiment was conducted to confirm the reducing ability of EDTA in our system. We found that the color of the aqueous solution containing HAuCl4 and EDTA turned from yellow to red and finally to deep blue after being heated to 70 °C in a water bath within 5 min, as a consequence of the formation of submicrometer gold particles. Figure 3a shows the TEM result of the obtained gold nanoparticles. It can be seen that the gold nanoparticles had a size of several nanometers with irregular shape and severe aggregation. The UV spectrum of the suspension containing these gold nanospheres shown in Figure 3b exhibits a broad absorption peak from 550 to 650 nm, which is a result of the interactions between neighboring nonspherical gold nanoparticles,18 correlating well with the observed deep-blue color. Thus, EDTA can on one hand act as a building block of the composite nanospheres and on the other hand can act as the reductant in the nanoparticulate reaction system. Though the mechanism behind the fast reduction of gold salt in the presence of EDTA, which may be somewhat beyond the scope of the present study, is still not clear, the results obtained here do provide the prerequisite for the subsequent preparation of chitosan-gold hybrid nanospheres through the approach proposed here. For the preparation of chitosan-gold hybrid nanospheres, LWCS-EDTA composite nanospheres dispersed in 40% ethanol solution were mixed with a predetermined amount of HAuCl4, and the mixture was heated to 70 °C by a water bath to convert Au salt in situ into gold nanoparticles using the EDTA molecules as the reductant. The color of the solution turned from yellow
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Figure 4. FTIR spectra of chitosan, EDTA, and hybrid nanospheres before and after dialysis.
Figure 3. (a) TEM micrograph of the gold nanoparticles prepared by the reduction of AuCl4- with EDTA. The bar corresponds to 100 nm. (b) UV spectrum of the solution containing gold nanoparticles in part a.
to purple-red rapidly, implying that Au salt had been reduced and nanosized gold nanoparticles were formed, which was verified by UV, XRD (details in Supporting Information), and TEM analyses. It should also be mentioned that either GA or ethanol cannot reduce Au salt under the same conditions. Besides, although chitosan has been reported to be able to reduce gold salt,27,28 parallel experiments in pure chitosan nanospheres indicated that the reducing ability of chitosan is too limited compared with EDTA and that the fast reduction of the Au salt should be mainly attributed to EDTA in our case (Supporting Information). However, both the good affinity of chitosan with gold salt and the potential stabilization effect offered by chitosan moieties for gold nanoparticles are also favorable factors in the fabrication of chitosan-gold hybrid nanospheres through our approach. After the reaction, remaining EDTA inside the nanospheres could be easily removed by dialysis against water as indicated by the FTIR results, which is an additional advantageous feature of the strategy reported here. By this means, pure chitosan-gold hybrid nanospheres were obtained directly. Figure 4 shows the FTIR spectra of chitosan and EDTA along with the hybrid nanospheres before and after dialysis, which provide further information regarding the structure of the resultant nanospheres. It could be found that the absorption peak of the (27) Huang, H.; Yang, X. Carbohydrate Res. 2004, 339, 2627. (28) Wang, B.; Chen, K.; Jiang, S.; Reincke, F.; Tong, W.; Wang, D.; Gao, C. Biomacromolecules 2006, 7, 1203.
carbonyl group in EDTA (1690 cm-1) shifts to a lower wavenumber of 1630 cm-1 in the spectrum of the hybrid nanospheres without dialysis because of the ionization of the carboxyls in EDTA molecules. Besides, the amino band at 1560 cm-1, which appears in the spectrum of pure chitosan, disappears in the case of nanospheres, suggesting that the amino groups are in their protonated state whose absorption peak (around 1620 cm-1) may be overlapped by that of the carbonyl groups in the nanospheres (1630 cm-1). These changes in the IR spectrum indicate that the carboxyl groups on EDTA molecules exist in their ionized state and form ionic bonds with the protonated amino groups of chitosan, which corroborates the proposed counterion complex structure of the LWCS-EDTA composite nanospheres. In the spectrum of chitosan-gold hybrid nanospheres after dialysis, the amino band at 1560 cm-1 shifted to a higher wavenumber in the form of a shoulder peak at 1590 cm-1, which implies an interaction between the amine groups of chitosan and the Au nanoparticles that are also present in the hybrid nanospheres.29 Also noteworthy is that in the spectra of hybrid nanospheres the characteristic absorption peak of EDTA decreased significantly after dialysis, implying that the EDTA molecules could still diffuse into the aqueous medium freely and be removed from the cross-linked chitosan matrix. The removal of EDTA by dialysis against water was also proved by titration of the extracted EDTA (Supporting Information). Therefore, the cross-linking of the chitosan matrix improves the structural stability of the nanospheres. The easy removal of EDTA by dialysis is actually a favorable feature and has been fully utilized to purify the hybrid nanospheres after the in situ reduction of Au salt as mentioned in the above text. Figure 5a shows the UV-vis spectra of several batches of the chitosan-gold hybrid nanospheres prepared with different amounts of Au salt feedings. All samples displayed a characteristic absorption peak above 520 nm, which was assigned to the surface plasma excitation of gold nanoparticles.3 The sample obtained with the lowest HAuCl4 feeding (2.5% w/w, chitosan based) exhibited an absorption peak at 525 nm (λmax) whereas samples with higher HAuCl4 feeding showed a small red shift of λmax that is probably due to the increased average gold particle size. Figure 5b shows a typical TEM micrograph of the chitosan-gold hybrid (29) Santos, D.; Goulet, P.; Pieczonka, N.; Oliveira, O.; Aroca, R. Langmuir 2004, 20, 10273.
Fabrication of Chitosan-Gold Hybrid Nanospheres
Figure 5. (a) UV-vis spectra of solutions containing chitosangold hybrid nanospheres prepared with different HAuCl4 feedings (chitosan-weight-based). (b) TEM micrograph of chitosan-gold hybrid nanospheres prepared using the LWCS-EDTA composite nanospheres in Figure 2d as the precursor reaction system with a HAuCl4 feeding of 10% w/w (chitosan-based). (c-f) TEM micrographs of a single typical chitosan-gold hybrid nanosphere prepared with different HAuCl4 feedings (bar ) 100 nm; c, 2.5%; d, 5%; e, 10%; and f, 20%, chitosan-weight-based).
nanospheres after dialysis. The markedly higher electron density of gold enables the direct visualization of the existence of gold nanoparticles within the chitosan nanospheres. It is apparent that nanospheres’ shape and size were not substantially affected after gold loading owing to the good structural stability of the crosslinked chitosan matrix. Consequently, size control of the chitosan-gold hybrid nanospheres can be realized by using LWCS-EDTA composite nanospheres with the desired size distribution as the precursor nanoparticulate reaction system (data not shown). More importantly, as also shown in Figure 5b, each and every chitosan nanosphere was loaded with gold nanoparticles, and no gold nanoparticles could be found out of the hybrid nanospheres, highlighting the advantage of the approach proposed here. Figure 5c-f shows the TEM micrographs of a single typical chitosan-gold hybrid nanosphere prepared with different HAuCl4 feedings at higher magnification. We can clearly see the differences in the average size and quantity of the loaded gold nanoparticles, which correlate well with the UV-vis results. Thermogravimetric analysis (TGA) indicated that the actual goldto-chitosan ratio (5.84% w/w) in the final hybrid nanospheres was in good agreement with the feeding value (5.79% w/w), suggesting that all the added gold salt was effectively reduced into gold nanoparticles within LWCS-EDTA composite nanospheres (Supporting Information). The embedded gold nanoparticles had a heterogeneous size distribution ranging from several to several tens of nanometers and were in a separate state because the chitosan matrix could efficiently prevent the alreadyformed gold nanoparticles from mutual contact during the reduction of Au salt. According to our previous studies, the interior of a counterion complex nanosphere is mainly made of the hydrophobic electrostatic complex whereas uncomplexed polymers construct
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Figure 6. (a) SEM micrograph (bar ) 1 µm) and (b) AFM image (bar ) 200 nm) of the chitosan-gold hybrid nanospheres in Figure 5b.
the nanospheres’ outer shell.8,25,30 That means that all EDTA molecules (the reducing agent) should be located inside of the chitosan-EDTA composite nanospheres. Therefore, only those AuCl4- anions that have diffused to the composite nanospheres will be in contact with the reductive EDTA, which guarantees that the reduction of gold salt would start from inside the nanospheres’ outer layer and that all polymeric nanospheres can be loaded with gold nanoparticles providing the amount of gold salt is sufficient. Additionally, in our system, the nanospheres’ surface property seems not to be affected by gold loading because the zeta potentials of both the as-prepared empty chitosan nanospheres and chitosan-gold hybrid nanospheres were about 30 mV (as a result of the presence of uncomplexed protonated chitosan molecules), which can also support the above conclusion that the nanospheres’ outermost layer is composed of uncomplexed protonated chitosan and gold nanoparticles are mainly generated inside the outer surface of the nanospheres. A closer look at the TEM images of the chitosan-gold hybrid nanospheres reveals that some gold nanoparticles seem to extrude out of the nanospheres’ surface, which may be explained by the fact that the space inside the nanosphere is relatively limited and certain gold nanoparticles have to find new room for their size expansion with the further reduction of Au salt. SEM and AFM observations were carried out to investigate further the surface morphology of the chitosan-gold hybrid nanospheres. As shown in the SEM micrograph (Figure 6a), these hybrid nanospheres had a smooth surface of a relatively uniform size. The AFM image in Figure 6b provides more detailed information about the nanospheres’ surface. Once again, a smooth surface was observed for all of the nanospheres, suggesting that the majority of the (30) Guo, R.; Zhang, L.; Cao, Y.; Ding, Y.; Jiang, X. Biomacromolecules 2007, 8, 843.
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Figure 7. Hydrodynamic diameter of the chitosan-gold hybrid nanospheres as a function of medium pH.
gold nanoparticles were located in the inner part of the chitosangold hybrid nanospheres. For conventional chitosan-based colloidal systems, deprotonation of the amino groups at neutral pH or above would lead to particle agglomeration25 and would thus hamper their use in biomedical applications. However, because the chitosan with a low molecular weight (5000 Da) used here is water-soluble in a wide pH range from 2 to 12, the dispersing stability problem was successfully addressed. Even those chitosan chains in the outermost layer that are deprotonated still provide certain steric stabilization for the obtained chitosan-gold hybrid nanospheres at elevated medium pH. Figure 7 shows the mean hydrodynamic diameters of the chitosan-gold hybrid nanospheres (initial diameter 230 nm) at different pH values. It can be seen that no significant variation in the hydrodynamic diameter of the hybrid nanospheres is observed in media with pH values ranging from 2 to 12, providing solid evidence of the superior dispersing stability of such chitosan-gold hybrid system. Such good dispersing stability within a wide pH range will be useful in further unlocking the potential of these hybrid nanospheres in biomedical applications.
Conclusions Chitosan-gold hybrid nanospheres were prepared using crosslinked LWCS-EDTA composite nanospheres as a precursor
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reaction system. EDTA was used not only to construct the counterion interaction-based composite nanospheres with chitosan but also as the reductant for subsequent in situ gold salt reduction within the LWCS-EDTA composite nanospheres. Additionally, EDTA can be easily removed by dialysis after exerting its reducing ability. This approach elegantly ensures that each nanosphere is loaded with gold nanoparticles and no nonembedded gold nanoparticles would exist. More importantly, this approach successfully circumvents both laborious materials synthesis and tedious separation processes that are commonly associated with conventional strategies for the fabrication of hybrid nanomaterials. Detailed characterizations revealed that the obtained hybrid nanospheres had a tunable size and good dispersing stability within a wide pH range. The embedded gold nanoparticles were in the range from several to several tens of nanometers, which may be useful for sensing and imaging,3 whereas the submicrometer chitosan matrix can also be loaded with other active agents to make the resultant hybrid nanospheres multifunctional. Taking into account of the good biocompatibilities of LWCS, abundant functional (amino) groups in chitosan, and the mild preparation conditions, the chitosan-gold hybrid nanospheres prepared here may have tremendous potential in advanced biomedical applications. In addition, by taking advantage of EDTA’s strong chelating ability with various multivalent metal ions, this approach is also envisaged to be applicable to the facile preparation of many other hybrid nanomaterials. Acknowledgment. This work is supported by the Natural Science Foundation of China (nos. 50625311 and 50573031), the 973 Program of MOST (no. 2003CB615600), and the Cultivation Fund of the Key Scientific and Technical Innovation Project, Ministry of Education of China (no. 707028). Supporting Information Available: Additional experimental details and results as described in the text. This material is available free of charge via the Internet at http://pubs.acs.org. LA703080J
