pubs.acs.org/Langmuir © 2010 American Chemical Society
Creation of Highly Stable Selenium Nanoparticles Capped with Hyperbranched Polysaccharide in Water Yifeng Zhang, Jianguo Wang, and Lina Zhang* Department of Chemistry, Wuhan University, Wuhan 430072, China Received August 26, 2010. Revised Manuscript Received October 2, 2010 Water-dispersible selenium nanoparticles (SeNPs) were created by using natural hyperbranched polysaccharide (HBP) as the stabilizer and capping agent under extremely safe conditions. The structure, morphology, size, and stability of the nanocomposites were investigated by transmission electron microscopy (TEM), atomic force microscopy (AFM), and static and dynamic light scattering (DLS) measurements. The results revealed that the spherical selenium nanoparticles (mean particle size of about 24 nm) were ligated with HBP to form nanocomposites (Se-HBP) in aqueous solution and were stable for over one month. In our findings, supported by the results of FTIR, TEM, AFM, and DLS, SeNPs were capped with the HBP macromolecules, as a result of strong physical adsorption of OH groups on Se surfaces, leading to a highly stable structure of Se nanoparticles in water. This work provided reaction sites for the complexation between HBP and Se to fabricate well-dispersed Se nanoparticles in aqueous system with potential bioapplications.
Introduction Nanoscale materials with large surface areas and small size effects exhibit improved bioactivities, and they have become potential candidates for biomedical applications that could open new avenues of development.1-6 In particular, selenium nanoparticles (SeNPs) have excellent bioavailability, high biological activity, and low toxicity.7,8 It is noted that trace mineral selenium is also an essential nutrient of fundamental importance to human health. It has been confirmed that selenium can improve the activities of the selenoenzyme such as selenium-dependent glutathione peroxidases (Se-GSH-Px) which act as a function of redox centers and prevent free radicals from damaging cells and tissues in vivo.9 Selenium has additional important health effects particularly in relation to the immune response and cancer prevention, which are almost certainly not exclusively linked to these enzymatic functions.9,10 Therefore, Se nanoparticles have potential application in biological and nanotechnological fields. It has been reported that inorganic nanoparticles can be encapsulated or stabilized by synthetic polymer such as dendrimers,11-14 *Corresponding author: Tel þ86-27-87219274; Fax þ86-27-68762005; e-mail
[email protected]. (1) Latham, A. H.; Williams, M. E. Acc. Chem. Res. 2008, 41, 411–420. (2) Gao, J.; Gu, H.; Xu, B. Acc. Chem. Res. 2009, 42, 1097–1107. (3) Rosi, N. L.; Mirkin, C. A. Chem. Rev. 2005, 105, 1547–1562. (4) Moghimi, S. M.; Hunter, A. C.; Murray, J. C. Pharmacol. Rev. 2001, 53, 283– 318. (5) Panyam, J.; Labhasetwar, V. Adv. Drug Delivery Rev. 2003, 55, 329–347. (6) Gupta, A. K.; Gupta, M. Biomaterials 2005, 26, 3995–4021. (7) Zhang, J.; Gao, X.; Zhang, L. BioFactors 2001, 15, 27–38. (8) Wang, H.; Zhang, J.; Yu, H. Free Radical Biol. Med. 2007, 42, 1524–1533. (9) Rayman, M. P. Lancet 2000, 356, 233–241. (10) WHO working group. Environ. Health Criteria 1987, 58, 306. (11) Esumi, K.; Suzuki, A.; Aihara, N.; Usui, K.; Torigoe, K. Langmuir 1998, 14, 3157–3159. (12) Hanus, L. H.; Sooklal, K.; Murphy, C. J.; Ploehn, H. J. Langmuir 2000, 16, 2621–2626. (13) Scott, R. W.; Wilson, O. M.; Oh, S. K.; Kenik, E. A.; Crooks, R. M. J. Am. Chem. Soc. 2004, 126, 15583–15591. (14) Scott, R. W.; Wilson, O. M.; Crooks, R. M. J. Phys. Chem. B 2005, 109, 692–704. (15) Aymonier, C.; Schlotterbeck, U.; Antonietti, L.; Zacharias, P.; Thomann, R.; Tiller, J. C.; Mecking, S. Chem. Commun. 2002, 24, 3018–3019.
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hyperbranched polymers,15-19 and linear polymers. It is noted that the past decade has witnessed growing interests in the creation of nanometer-scale inorganic and hybrid materials using biological systems as catalysts, microreactors, and supporting templates.20-22 The advantages of biomacromolecule-enabled synthesis of inorganic materials as compared with traditional chemical synthesis include room temperature, aqueous environment, and neutral pH.23-25 The syntheses of biomolecule-stabilized SeNPs are of particular interests due to the potential biological activities. Shen and co-workers have prepared SeNPs with average sizes of 36 nm through a simple, one-step method without the aid of any surfactant to synthesize dextran/Se nanocomposites for nanomedicine application.26 Zheng et al. have reported large-scale synthesis of SeNPs with average sizes ranging from 59 to 220 nm by introducing L-cysteine and U. pinnatifida polysaccharide into the water-phase redox system via an environment-friendly route for the biomedical application.27,28 Further, gold nanoparticles capped with short peptides have been synthesized.29 Biomacromolecules have been used as soft templates (16) Garamus, V. M.; Maksimova, T.; Richtering, W.; Aymonier, C.; Thomann, R.; Antonietti, L.; Mecking, S. Macromolecules 2004, 37, 7893–7900. (17) Ho, C. H.; Tobis, J.; Sprich, C.; Thomann, R.; Tiller, J. C. Adv. Mater. 2004, 16, 957–961. (18) Rybak, B. M.; Ornatska, M.; Bergman, K. N.; Genson, K. L.; Tsukruk, V. V. Langmuir 2006, 22, 1027–1037. (19) Zhang, Y.; Peng, H.; Huang, W.; Zhou, Y.; Zhang, X.; Yan, D. J. Phys. Chem. C 2008, 112, 2330–2336. (20) Raveendran, P.; Fu, J.; Wallen, S. L. J. Am. Chem. Soc. 2003, 125(46), 13940–13941. (21) Gao, X.; Zhang, J.; Zhang, L. Adv. Mater. 2002, 14, 290–293. (22) Cai, J.; Kimura, S.; Wada, M.; Kuga, S. Biomacromolecules 2009, 10, 87–94. (23) Li, C.; Kaplan, D. L. Curr. Opin. Solid. State. Mater. Sci. 2003, 7, 265–271. (24) Bill, J. Adv. Sci. Technol. 2006, 45, 643–651. (25) Kharlampieva, E.; Slocik, J. M.; Singamaneni, S.; Poulsen, N.; Kroger, N.; Naik, R. R.; Tsukruk, V. V. Adv. Funct. Mater. 2009, 19, 2303–2311. (26) Shen, Y.; Wang, X.; Xie, A.; Huang, L.; Zhu, J.; Chen, L. Mater. Chem. Phys. 2008, 109, 534–540. (27) Chen, T.; Wong, Y.; Zheng, W.; Bai, Y.; Huang, L. Colloids Surf., B 2008, 67, 26–31. (28) Li, Q.; Chen, T.; Yang, F.; Liu, J.; Zheng, W. Mater. Lett. 2010, 64, 614– 617. (29) Serizawa, T.; Hirai, Y.; Aizawa, M. Langmuir 2009, 25, 12229–12234.
Published on Web 10/21/2010
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for controlling inorganic crystal nucleation and growth.30-34 In view of these literatures, inorganic nanoparticles-biomacromolecule composite formed via a “green” route will be very important for the biomedical applications. Recently, inorganic nanoparticles synthesized by hyperbranched amphiphilic copolymer and dendrimers have been much studied. However, the formation of Se-hyperbranched polysaccharide composite and the quantitative research on the stability of Se-hyperbranched polysaccharide composite have been scarcely reported. Natural polysaccharides are not only involved in a wide variety of life activities of the cell but also participate in the mineralization process of plants and animals. It is worth noting that hyperbranched polysaccharides are especially prone to dissolution in water, and they show tendencies of readily binding to the cell surface receptors due to the large number of terminal hydroxyl groups and high specific surface area. In our laboratory, two kinds of water-soluble hyperbranched polysaccharide (HBP) had been extracted from the sclerotia of Pleurotus tuber-regium and Rhizoma Panacis Japonici,35,36 and they exhibit significant in vivo and in vitro antitumor activities. Encouraged by these findings, we tried to create nano-selenium in the presence of the hyperbranched polysaccharide in aqueous systems to prepare welldispersed selenium nanoparticles under extremely safe conditions. In the present work, SeNPs were synthesized via a facile redox system in the presence of the water-soluble HBP as stabilizer and capping agent. It is not hard to imagine that the hyperbranched polysaccharide not only can play the capping role for the creation of nanoparticles but also supply a shell to prevent aggregation of the grown nanoparticles. The structure and stability of Se-HBP nanocomposites were characterized by transmission electron microscope (TEM), atomic force microscopy (AFM), laser light scattering measurements, and viscometry, and the influence of HBP on the formation of SeNPs was also investigated. The objective of this work is to provide a novel “green” pathway to prepare hyperbranched polysaccharide-capped Se nanoparticles as well as to clarify the formation mechanism of Se-HBP nanocomposites.
Experimental Section Materials. Selenious acid and ascorbic acid were purchased from Tianjin Chemical Reagent Institute and Sinopharm Chemical Reagent Co., Ltd., respectively, and were used as received without further purification. The water used in all experiments was ultrapure by a Milli-Q water purification system from Millipore. Water-soluble hyperbranched β-D-glucan with β-(1f6), β-(1f4), β-(1f3) and β-(1, 4, 6)-linked residues having degree of branching (DB) of 57.6% was extracted from sclerotia of Pleurotus tuber-regium (gifts from Department of Biology in the Chinese University of Hong Kong) according to our previous method.35 The defatted sclerotia powder of Pleurotus tuber-regium was immersed stepwise in 0.9% aqueous NaCl at 20 and 80 C overnight to remove the extract and then was stirred overnight at 120 C to extract the hyperbranched β-D-glucan. After centrifugation, the supernatant was obtained, coded as TM3. The TM3 sample formed two phases when it was cooled to room (30) Bauerlein, E. Angew. Chem., Int. Ed. 2003, 42, 614–641. (31) Arias, J. L.; Fernandez, M. S. Chem. Rev. 2008, 108, 4475–4482. (32) Cusack, M.; Freer, A. Chem. Rev. 2008, 108, 4433–4454. (33) Dickerson, M. B.; Sandhage, K. H.; Naik, R. R. Chem. Rev. 2008, 108, 4935–4978. (34) Palmer, L. C.; Newcomb, C. J.; Kaltz, S. R.; Spoerke, E. D.; Stupp, S. I. Chem. Rev. 2008, 108, 4754–4783. (35) Tao, Y.; Zhang, L. Biopolymers 2006, 83, 414–423. (36) Huang, Z.; Huang, Y.; Li, X.; Zhang, L. Carbohydr. Polym. 2009, 78, 596– 601.
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temperature, coded as TM3a and TM3b. The TM3b supernatant was purified by decolorization and free protein removal. The resulting polysaccharide solution was precipitated by absolute ethanol and recovered by centrifugation to remove the supernatant. The precipitate was dissolved in distilled water, then dialyzed, and finally lyophilized to obtain the purified TM3b sample, coded as HBP in this study. The structural formula diagram for HBP is shown in Figure S1 in the Supporting Information. Preparation of Selenium Nanoparticles. Into a 100 mL Erlenmeyer flask, aqueous hyperbranched polysaccharide solution (0.2%, 45 mL) was added, and it was then mixed with 600 μL of 0.1 M selenious acid and 11.4 mL of water under magnetic stirring overnight. 3 mL of aqueous ascorbic acid solution (0.1 M) was added dropwise into the resulting mixture, and then it was stirred further at room temperature. The reacted product was dialyzed by using regenerated cellulose tubes (Mw cutoff 8000) against ultrapure water for 2 days. The solution was subjected to centrifugation at 4000 rpm for 20 min and finally was freezedried with a lyophilizer (Christ Alpha 1-2, Osterode am Harz, Germany) to obtain the nanoparticle-polysaccharide composites (red powder), coded as Se-HBP. Characterization and Measurements. Transmission electron microscopy (TEM) of the dilute solution of nanocomposites was carried out with a JEOL JEM-2010 (HT) electron microscope at an accelerating voltage of 200 kV. The high-resolution transmission electron microscopy (HRTEM) image was taken on a JEOL JEM 2010 FEF (UHR) microscope at 200 kV. Fouriertransform infrared spectra (FT-IR) of the samples were recorded on a Nicolet 170SX FT-IR (Spectrum One, Perkin-Elmer Co.) spectrometer in the range 4000-400 cm-1 using the KBr-disk method. A modified commercial instrument consisted of an ALV/DLS/ SLS-5000E light scattering goniometer (ALV/CGS-8F, ALV, Germany) with vertically polarized incident light of wavelength 632.8 nm from a He-Ne laser equipped with an ALV/LSE-5003 light scattering electronics, and a multiple tau digital correlator was used to measure the molecular weight and the size of HBP and its nanocomposites. Their dilute solutions were prepared by mixing the required amounts of HBP, Se-HBP nanocomposites, and ultrapure water in a flask by stirring gently at room temperature overnight. The solutions with different concentration were prepared by sequentially diluting the stock solution of 1.015 10-3 and 1.008 10-3 g/mL. All of the dilute solutions were finally filtered directly into the cylindrical light-scattering cuvettes using 0.22 μm pore size syringe filters (PTFE, Whatman, Inc., England). The refractive index increment (dn/dc) at 632.8 nm and 25 C for HBP and Se-HBP in water was determined by using an Optilab refractometer (Dawn-DSP, Wyatt Technology Co., Santa Barbara, CA) to be 0.135 and 0.142 mL/g, respectively. For the static laser light scattering (SLS), the scattering light intensity of the Se-HBP solution in water was measured by a laser light scattering instrument at an angular ranging from 30 to 150 with an interval of 10 at 25 C. The weight-average molecular weight (Mw) and radius of gyration (Rg) of the Se-HBP samples in water were calculated by Kc 1 1 = 1 þ Rg 2 q2 þ 2A2 c Rθ Mw 3
ð1Þ
where K = 4π2n2(dn/dc)2/(NAλ04) and the magnitude of the scattering vector q = 4πn sin(θ/2)/λ0, with NA, n, and λ0 as Avogadro’s number, refractive index of the solvent (1.332 at 25 C for the water), and wavelength of the light in vacuum, respectively. A2 is the second virial coefficient. By measuring the Rθ at different c and q, the values of Mw, A2, and Rg can be obtained from the Zimm plot. Dynamic light scattering (DLS) was used to characterize the hydrodynamic radii (Rh) of Se/HBP in water at 25 C. The precisely measured intensity-intensity time correlation function G(2)(q,τ) in the self-beating mode can be related to the normalized Langmuir 2010, 26(22), 17617–17623
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Scheme 1. Reaction of Selenious Acid with Ascorbic Acid in Water at Room Temperature
field-field autocorrelation function g(1)(q,τ) via the Siegert relation as37,38 Gð2Þ ðq, τÞ ¼ A½1 þ βjgð1Þ ðq, τÞj2
ð2Þ
where A is the measured baseline and β is a constant related to the coherence of the detected optics. For a polydisperse system, g(1)(q,τ) is related to the distribution of the characteristic line width G(Γ) by39,40 Z ¥ jgð1Þ ðq, τÞj ¼ GðΓÞe - Γτ dΓ ð3Þ 0 (1)
Thus, g (q,τ) can be converted to a line-width distribution G(Γ) by the CONTIN Laplace inversion algorithm in the correlator according to eq 2. For a pure diffusive relaxation, Γ is related to the translational diffusion coefficient (D), and G(Γ) can be converted to a translation diffusion coefficient distribution G(D) by Γ ¼ Dq2
ð4Þ
and a hydrodynamic radius distribution, f(Rh), by the StokesEinstein equation Rh ¼
kB T 6πη0 D
ð5Þ
where kB, T, and η0 denote the Boltzmann constant, absolute temperature, and the solvent viscosity, respectively. The intrinsic viscosity ([η]) of Se-HBP aqueous solutions was measured at 25 C by using an Ubbelohde capillary viscometer. All of the solutions had the same original concentration of 3 10-3 g/mL. The kinetic energy correction was always found to be negligible. The Huggins and Kraemer equations were used to estimate the [η] value as ηsp =c ¼ ½η þ k½η2 c
ð6Þ
ln ηr =c ¼ ½η - β½η2 c
ð7Þ
where ηsp/c is the reduced viscosity, ln ηr/c is the inherent viscosity, c is the polymer concentration, and k and β are constants for a given polymer in the desired conditions. For atomic force microscopic (AFM) measurements, the solutions were filtered through a 0.22 μm filter (NYL, 13 mm Syringe filter, Whatman Inc. England) and were diluted with ultrapure water to a polymer concentration of 10 μg/mL. The specimen was examined using a Picoscan AFM (Molecular Imaging, Tempe, AZ) in a MAC Mode with commercial MAClever II tips (Molecular Imaging), with a spring constant of 0.95 N/m. A piezoelectric scanner with a range up to 6 μm was used for the image. A 10 μL drop of the HBP or Se-HBP aqueous solution with concentration of 10 μg/mL was deposited onto freshly cleaved mica and allowed to dry in air for 1.5 h at room temperature in a small covered Petri dish prior to imaging. The measurement was (37) Trappe, V.; Bauer, J.; Weissm€uller, M.; Burchard, W. Macromolecules 1997, 30, 2365–2372. (38) Hanselmann, R.; Burchard, W.; Ehrat, M.; Widmer, H. M. Macromolecules 1996, 29, 3277–3282. (39) Berne, B.; Pecora, R. Dynamic Light Scattering; Plenum Press: New York, 1976. (40) Chu, B. Laser Light Scattering, 2nd ed.; Academic Press: New York, 1991.
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Figure 1. (a) Typical TEM image of selenium nanoparticles in the HBP aqueous solution. (b) Histogram showing the size distribution of the selenium nanoparticles (the mean particle size d = 24.1 nm and standard deviation σ = 2.2 nm); the total number of particles counted for the histogram is 186. (c) HRTEM of an individual particle and the lattice fringes marked representatively is about 0.54 nm. (d) Typical EDS spectrum from HRTEM. performed in air at ambient pressure and humidity, and the image was stored as 256 256 point arrays.
Results and Discussion Formation of Se Nanoparticles. In this process, selenious acid was first well-intermixed into aqueous HBP solution. Subsequently, the ascorbic acid solution was added dropwise into the resultant system and reacted with selenious acid to form Se nanoparticles (SeNPs). The reaction of selenious acid with ascorbic acid in water was formulated in Scheme 1.41 The reaction could be clearly observed by monitoring color changes. The solution color changed gradually from colorless to pale yellow and then to yellow, and it finally turned into orange-red. The appearance of characteristic red color indicated an occurrence of the reaction to form either amorphous or monoclinic selenium particles occurred since trigonal selenium is black.42 After reacting for 30 min, the sample solution was removed for the TEM observation. Figure 1 shows the TEM image of the thus-synthesized selenium nanoparticles (a) and the size distribution of the nanoparticles (b) in the HBP aqueous solution. The results revealed that the selenium nanoparticles existed as well-dispersed spherical particles in the solution. By counting more than 180 particles in several TEM images, the statistical results showed that the mean particle size d = 24.1 nm and standard deviation σ = 2.2 nm. The HRTEM image and the corresponding energydispersive spectrum (EDS) are shown in Figure 1c,d. The clear lattice fringes indicated a crystal structure of the particles, confirming that these nanoparticles were purely made up of selenium. Therefore, the nanoparticles were typically monoclinic selenium, indicating the successful synthesis of Se particle in the HBP aqueous solution. (41) Mees, D. R.; Pysto, W.; Tarcha, P. J. J. Colloid Interface Sci. 1995, 170, 254–260. (42) Johnson, J. A.; Saboungi, M.; Thiyagarajan, P.; Csencsits, R.; Meisel, D. J. Phys. Chem. B 1999, 103, 59–63.
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Figure 2. Photographs of the results for 2-day storage after the redox reaction in the absence (a) and presence (b) of HBP.
Figure 2 shows the photographs of SeNPs aqueous solution after 2 days of storage in the absence and presence of HBP. The SeNPs solution generated in the presence of HBP exhibited orange-red color due to the nanosize effects and was quite stable, transparent, and lucent without any discernible precipitation for at least 30 days. However, brick-red particles were precipitated at the bottom of the cuvette when SeNPs were prepared in the absence of HBP, as shown in Figure 2a. This could be due to the high surface energy of SeNPs which led to their aggregation, resulting in significant precipitation. Therefore, the presence of the polysaccharides played a crucial role in both the formation and the improvement of the stability of the Se nanoparticles. The results suggested that the natural hyperbranched polysaccharides (HBP) with the large number of terminal hydroxyl groups and high specific surface area possessed strong attraction to some elements such as Se. In our findings, the selenium atoms in the selenous acid could associate with the polysaccharide hydroxyl groups through intermolecular hydrogen bonds (O-H 3 3 3 Se).43,44 During the preparation process of SeNPs, the precursor selenious acid in the HBP molecular microenvironment was reduced to elemental selenium, and the selenium atoms aggregated into selenium particles and immediately grew as the reaction went on. Finally, the Se surface was strongly adsorbed and passivated by the polysaccharide molecules, resulting in the formation of a uniform and spherical morphology. Size and Morphology of Se-HBP Nanocomposites. For the TEM measurement, a droplet of orange-red solution containing the Se-HBP nanocomposites was dropped onto a carboncoated copper grid, then stained by 0.1% (w/v) phosphotungstic acid aqueous solution, and dried in the air for 10 min. Simultaneous visualization of SeNPs and their stabilized polysaccharides under the TEM can be achieved with negative staining, where phosphotungstic acid was introduced onto the specimen to enhance the contrast. Figure S2 (Supporting Information) shows the TEM image of stained Se-HBP. The Se-HBP nanocomposites were observed from the dark zone, with mean size of 220 nm. This particle size was much larger than the sum of the native HBP35 and SeNP. This suggested that SeNPs were associated with the (43) Green, D. C.; Eichhorn, B. W. J. Solid State Chem. 1995, 120, 12–16. (44) Krebs, B.; M€uller, H. Z. Anorg. Allg. Chem. 1983, 496, 47–57.
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polysaccharides to form a large inclusion complexes. A strong interaction between HBP and the ripened SeNPs prevented Se self-aggregation, leading to the stabilization of uniform spherical Se particles. To further confirm that the two kinds of particles were packed closely to form spherical composites, AFM was used to observe their morphology and size in water. Figure 3a,b shows the MAC mode AFM images of the HBP and the Se-HBP nanocomposites aqueous solutions drop-cast on mica. The corresponding height of arrow-marked particle in Figure 3a,b is shown in Figure 3c,d, respectively. In order to reduce the influence of tip broadening effects, the full width at half-maximum (fwhm) analysis was used to determine the diameters of particles. The result indicated that the mean size of Se-HBP nanocomposites was about 200 nm, obviously larger than that of HBP. It was noted that the nanocomposites size by AFM was close to those obtained by TEM measurement. The results provided strong evidence for the formation of the globular structure associated with HBP and Se nanoparticles. To determine the reason why the SeNPs and HBP could be bound, the interaction between the two kinds of particles was studied by infrared spectroscopy. The FT-IR spectra of HBP and the Se-HBP nanocomposites are shown in Figure 4a,b. The characteristic absorption peak of hydroxyl group (OH) of SeHBP nanocomposites at 3389 cm-1 was lower than that of HBP (3416 cm-1), but the peak shape hardly changed. The peak in SeHBP nanocomposites shifted to lower wavenumber, compared with the native HBP, indicating a decrease of free OH groups in HBP. Namely, some OH groups of HBP were ligated with Se. It is quite plausible that a simple adsorption of OH groups of HBP on the Se surfaces occurred here, leading to the association between SeNPs and HBP. To provide the evidence of the complexation between SeNPs and HBP, the molecular weight and size of HBP and Se-HBP in water were determined by light scattering. Figure S3 (Supporting Information) shows the typical Zimm plots of HBP and Se-HBP in aqueous solution at 25 C. From the Zimm plot of HBP, the extrapolations of [Kc/Rθ]cf0,qf0 led to Mw of 1.44 107 g/mol and Rg of 78.8 nm. The Mw value for Se-HBP nanocomposites remarkably increased from 1.44 107 g/mol for the native HBP to 23.5 107 g/mol. However, the value of Rg increased only slightly to 97.2 nm. Moreover, the increasing extent of the particle size was in good agreement with that obtained by AFM measurement. This indicated that the aggregates formed from HBP existed in Se-HBP aqueous solution. The apparent mean aggregation number (Nagg) can be estimated by Nagg ¼
Mw, Se-HBP Mw, HBP
ð8Þ
where Mw,Se-HBP and Mw,HBP are the weight-average molecular weight of the Se-HBP nanocomposites and HBP in water, respectively. The molecular weight contribution of SeNP can be ignored because of its small size; thus, the resulting value of Nagg was calculated to be around 16. It is well-known that Rg reflects the space occupied by the polymer chains. Compared with HBP, the Rg value of Se-HBP in water showed no significant increase, suggesting that the two kinds of particles (Se and HBP) closely packed to each other and collapsed to form a more compact globule. Dynamic light scattering (DLS) is a powerful technique for the analysis of particle size and the size distribution of the nanoscale (45) Duan, H.; Kuang, M.; Wang, J.; Chen, D.; Jiang, M. J. Phys. Chem. B 2004, 108, 550–555.
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Figure 3. 3D AFM images of HBP (a) and Se-HBP (b); the bird’s-eye view is shown in the lower left corner. The corresponding height of arrow-marked particle is shown in (c) and (d).
Figure 4. FT-IR spectra of HBP (a) and Se-HBP (b).
particles in solution, and such studies have been performed by many research groups.45,46 Figure 5 shows the f(Rh) profiles of the two samples in aqueous solution with concentration of 5.0 10-4 g/mL at 25 C and θ = 90. As shown in Figure 5a, the f(Rh) exhibited only one peak, and the corresponding Rh value of HBP was about 78 nm. However, there were two peaks in the Se-HBP aqueous solution; they were located at around 14 nm (labeled as peak 1) and 114 nm (labeled as peak 2), as shown in Figure 5b. The results suggested that there were two components in the solution: one was the noncapped selenium nanoparticles, which were very small, and another component was the HBPcapped selenium nanoparticles, namely Se-HBP nanocomposites. The predominant species in the solution existed as Se-HBP nanocomposites. The corresponding Rh values of the components are summarized in Table 1. It was noted that the SeNPs size
(46) Zhou, K.; Li, J.; Lu, Y.; Zhang, G.; Xie, Z.; Wu, C. Macromolecules 2009, 42, 7146–7154.
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Figure 5. Hydrodynamic radius distribution [f(Rh)] of HBP (a) and Se-HBP (b) in aqueous solution at a fixed concentration of 5.0 10-4 g/mL at 25 C and shifted by A.
obtained by TEM was about 24 nm, which was comparable to that (2Rh = 28 nm) from DLS. It is known that Rh is regarded as a quantity to characterize the dimension of macromolecules in solution taking into account the hydrodynamic interactions. The structure-sensitive dimensionless parameter F (= Rg/Rh) can be used as a qualitative determination of the chain architecture and conformation for polymer in solutions. For a uniform and nondraining sphere, F is about 0.8, and for a loosely connected hyperbranched chain or aggregate, F is about 1.0.47 In our laboratory, the F value of a hyperbranched polysaccharide (TM3a) extracted from sceleria of Pleurotus tuber-rigum in 0.25 M LiCl/DMSO was determined to be in the range from 0.79 to 1.69 for 1.9 105 to 2.06 107 of weightaverage molecular weight, and the hyperbranched polysaccharides existed as spheres in the solution.48 The F values of the HBP and (47) Niu, A.; Liaw, D.; Sang, H.; Wu, C. Macromolecules 2000, 33, 3492–3494. (48) Tao, Y.; Zhang, L.; Yan, F.; Wu, X. Biomacromolecules 2007, 8, 2321–2328.
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Zhang et al. Table 1. Molecular Parameters of the HBP and Se-HBP Nanocomposites in Water at 25 C
samples
Mw 10-7 (g mol-1)
A2 (mol dm3 g-2)
Rg (nm)
Rh (nm)
F (Rg/Rh)
HBP nanocomposites
1.44 23.5
-5.81 10-9 1.16 10-9
78.8 97.2
78.1 114
1.0 0.8
Figure 7. Time dependences of the hydrodynamic radius (Rh) and the intrinsic viscosity ([η]) for the Se-HBP aqueous solution at a fixed concentration of 0.9 10-3 g/mL and 25 C.
Figure 6. Typical intensity-intensity time correlation functions
[G (2)(τ)] of the Se-HBP aqueous solution (0.9 10-3 g/mL) with increasing storage time at θ = 90 and T = 25 C (a) and the corresponding hydrodynamic radius distributions [f(Rh)] calculated from G(2)(τ) by using a Laplace inversion program (CONTIN) in the correlator and shifted by A (b).
Se-HBP samples were calculated and are listed in Table 1. The F value for the Se-HBP nanocomposites was 0.8, indicating that the Se-HBP aggregates existed as compact spheres in the water system. This conclusion was consistent with the above-mentioned results from the Mw and the Rg values as well as the TEM and AFM results. This further confirmed the existence of strong association between SeNPs and HBP molecules, leading to the compact aggregates. Stability of the Se-HBP Nanocomposites. The stability of the Se-HBP nanocomposites was investigated by DLS measurements and viscometry to examine the changes of the properties of the nanoparticles with time. Figure 6a shows the intensity-intensity time correlation functions (G(2)(τ)) of the Se-HBP in the aqueous solution with increasing storage time. It is well-known that if the relaxation is purely diffusive, the time correlation functions should be superimposed on each other. As clearly shown in Figure 6a, the intensity-intensity time correlation functions exhibited singleexponential decay and had only one fast relaxation mode with the same relaxation time. Moreover, at different storage time, these correlation functions were indeed overlapping with each other, indicating that the observed fast mode of scattering objects in the solution was unchanged, thus showing no significant dependence on the standing time. Therefore, the Se-HBP aqueous solution was a stable homogeneous system. Figure 6b shows the hydrodynamic 17622 DOI: 10.1021/la1033959
radius distributions of Se-HBP at different storage time. The Rh values for the Se-HBP solutions with time progress hardly changed, further suggesting that the Se-HBP nanocomposite solution was stable. Figure 7 shows the dependence of the hydrodynamic radius (Rh) and the intrinsic viscosity ([η]) of Se-HBP in water at 25 C on the storage time (t). Clearly, the Rh and [η] values of Se-HBP in water displayed no significant change with an increase of the storage time. During the whole storage period of the Se-HBP solution, the Rh value remained constant, indicating that stable nanocomposite aggregated with HBP and Se nanoparticles. The results were supported by the observation that the Se-HBP solution was clear, and no macroscopic precipitation was detected even after 2 months. Usually, the [η] value is a measure of the hydrodynamic volume of macromolecules.49 It is generally accepted that macromolecule conformation can be evaluated basically by the value of [η]. The [η] value also reflects the expanded extent of the polymer chain.50,51 As shown in Figure 7, the [η] value was constant with respect to the storage time and was at a low value of ∼5 mL/g. This value is very low compared with that of the hyperbranched polysaccharides. This indicated that the Se-HBP nanocomposites existed as compact spheres in the aqueous solution, supporting the conclusion mentioned above. Schematic Adsorption of HBP on SeNPs Surfaces. In view of the above findings, we proposed a schematic model to describe the Se-HBP nanocomposites, as shown in Figure 8. When precursor H2SeO3 was dispersed in the HBP aqueous solution, it was reduced by ascorbic acid to create elemental selenium. Subsequently, further reduction of H2SeO3 increased the number of selenium atoms, and they aggregated into the SeNPs nanoparticles. This proposed scheme was supported by the results shown in Figures 1 and 2. The HBP macromolecules in the solution could be ligated easily with SeNPs through physical (49) Mansfield, M. L.; Douglas, J. F.; Irfan, S.; Kang, E. H. Macromolecules 2007, 40, 2575–2589. (50) Michell, J. R. In Polysaccharide in Foods; Blanshard, J. M. V., Mitchell, J. R., Eds.; Butterworths: Boston, MA, 1979; pp 51-71. (51) Lapasin, R.; Pricl, S. Rheology of Polysaccharide Systems. Rheology of Industrial Polysaccharides, Theory and Applications; Blackie: Glasgow, 1995; pp 250-494.
Langmuir 2010, 26(22), 17617–17623
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Article
Figure 8. Schematic model of the Se-HBP nanocomposites during the formation process.
adsorption. As demonstrated in Figures 2-5, the interaction between HBP and SeNPs was strong, leading to good waterdispersible nanoparticles. It was not hard to imagine that the SeNPs surface could be easily capped with HBP. HBP as capping agent supplied not only reaction sites for the creation of Se nanoparticles but also a shell to prevent aggregation of the grown nanoparticles. SeNPs were capped with the HBP macromolecules, as a result of strong physical adsorption of OH groups of HBP on Se surfaces, to form more compact and stable globular nanocomposites, supported by the results in Figures 5-7 as well as in Table 1.
Conclusions Natural hyperbranched polysaccharides (HBP) have a large number of terminal hydroxyl groups and high specific surface area, leading to strong adsorption on some elements. Selenium nanoparticles (SeNPs) with the mean particle size of 24 nm were created successfully in water system via a novel synthetic pathway of HBP-capped reaction sites under extremely safe conditions. The results from TEM, AFM, and light scattering revealed that SeNPs were capped by the HBP molecules to prevent aggregation of the grown nanoparticles, leading to good dispersion of the Se nanoparticles in water. HBP played an important role as capping
Langmuir 2010, 26(22), 17617–17623
reagents in the formation and stabilization of the Se-HBP nanocomposites, as a result of the strong physical adsorption of HBP on SeNPs surfaces. The hyperbranched polysaccharides supplied not only reaction sites for the creation of Se nanoparticles but also a shell to protect the nanoparticle structure, leading to water-dispersible nanoparticles. This is a simple method for the preparation of the organic-inorganic hybrid composites with high safety, biocompatibility, and biodegradability, which are very important in the field of life science and technology. Acknowledgment. This work was supported by National Basic Research Program of China (973 Program, 2010CB732203), National Supporting Project for Science and Technology (2006BAF02A09), and the National Natural Science Foundation of China (20874079). We acknowledged the Center of Nanoscience and Nanotechnology and the Center for Electron Microscope of Wuhan University for their technical support. Supporting Information Available: Pictures of structural formula diagram for HBP, TEM image, and Zimm plot. This material is available free of charge via the Internet at http:// pubs.acs.org.
DOI: 10.1021/la1033959
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