pubs.acs.org/Langmuir © 2009 American Chemical Society
Gold Nanoparticles Prepared Using Cape Aloe Active Components )
Zeljka Krpetic,† Giorgio Scarı` ,‡ Enrico Caneva,§ Giovanna Speranza, and Francesca Porta*,† †
)
Dipartimento di Chimica Inorganica Metallorganica Analitica “Lamberto Malatesta”, University of Milan, Via Venezian 21, Milan, Italy, ‡Dipartimento di Biologia 7B, University of Milan, Via Celoria 26, Milan, Italy, § Centro Interdipartimentale Grandi Apparecchiature-CIGA and Dipartimento di Chimica Organica e Industriale, University of Milan, Via Golgi 19, Milan, Italy Received March 19, 2009. Revised Manuscript Received May 5, 2009 A novel use of two components of Cape aloe, aloin A and aloesin, acting as stabilizers in the preparation of gold and silver nanoparticles, is reported. Stable water-soluble particles of different size and shape are prepared by varying the reaction conditions, temperature, reaction time, and reducing agents. Characterization of the obtained particles is performed using UV-visible, attenuated total reflection Fourier transform infrared (ATR-FTIR), and 1H NMR spectroscopies and transmission electron microscopy (TEM). The efficient cellular uptake of 50 nm sized aloin A and aloesin stabilized gold particles into macrophages and HeLa cells was investigated, proposing these particles as nanovehicles.
Introduction Nanoscale gold materials have been exponentially developed in recent years.1-5 Nanoparticle synthesis using biological entities is already reported in the literature, including bacteria, yeast, fungi, and plants6-8 as clean, nontoxic, and environmentally acceptable routes. Many studies on plant use in nanobiotechnology that appear in the literature deal with controlled particle size formation. Different plants involved in both the intra- and extracellular preparation of silver and gold nanoparticles (GNPs) are reported, for example, oat (Avena sativa),9 lemongrass extract (Cymbopogon flexuosus),10-12 leguminous shrub (Sesbania drummondii),13 (Brassica juncea),14 neem leaf broth (Azadirachta indica),15 pine (Pinus desiflora), persimmon (Diopyros kaki), ginkgo (Ginko biloba), magnolia (Magnolia kobus), and platanus (Platanus orientalis).16 These reports mainly describe nanoparticle formation as a consequence of Au(III) reduction to Au(0) within *To whom correspondence should be addressed. Telephone: +390250314361. Fax: +390250314405. E-mail:
[email protected]. (1) Brust, M.; Bethell, D.; Kiely, C. J.; Schiffrin, D. J. Langmuir 1998, 14, 5425– 5429. (2) Martin, C. R.; Mitchell, D. T. Anal. Chem. 1998, 70, 322A–327A. (3) McConnell, W. P; Novak, J. P.; Brousseau, L. C.III; Fuiere, R. R.; Tenent, R. C.; Feldheim, D. L. J. Phys. Chem. B 2000, 104, 8925–8930. (4) Tanaka, K. Thin Solid Films 1999, 341, 120–125. (5) Troiani, H. E.; Camacho-Bragado, A.; Armendariz, V.; Gardea-Torresday, J. L.; Yacaman, M. J. Chem. Mater. 2003, 15, 1029–1031. (6) Mohanpuria, P.; Rana, N. K.; Yadav, S. K. J. Nanopart. Res. 2008, 10, 507– 517. (7) Sastry, M.; Ahmad, A.; Khan, M. I.; Kumar, R. Curr. Sci. 2003, 85, 162–170. (8) Gardea-Torresday, J.; Peralta-Videa, J. R.; Parsons, J. G.; Mokgalaka, N. S.; de la Rose; G. In Metal nanoclusters in catalysis and materials science: the issue of size control; Corain, B., Schimd, G., Toshima, N., Eds.; Elsevier: Amsterdam, 2008; chapter 28, pp 401-411. (9) Armendariz, V.; Herrera, I.; Peralta-Videa, J. R.; Jose-Yacaman, M.; Troiani, H.; Santiago, P.; Gardea-Torresdey, J. L. J. Nanopart. Res. 2004, 6, 377–382. (10) Shankar, S. S.; Rai, A.; Ahmad, A.; Sastry, M. Chem. Mater. 2005, 17, 566– 572. (11) Rai, A.; Singh, A.; Ahmad, A.; Sastry, M. Langmuir 2006, 22, 736–741. (12) Singh, A.; Chaudhari, M.; Sastry, M. Nanotechnology 2006, 17, 2399–2405. (13) Sharma, N. C.; Sahi, S. V.; Nath, S.; Parsons, J. G.; Gardea-Torresdey, J. L.; Pal, T. Environ. Sci. Technol. 2007, 41, 5137–5142. (14) Haverkamp, R. G.; Marshall, A. T.; van Agterveld, D. J. J. Nanopart. Res. 2007, 9, 697–700. (15) Shankar, S. S.; Rai, A.; Ahmad, A.; Sastry, M. J. Colloid Interface Sci. 2004, 275, 496–502. (16) Song, J. Y.; Kim, B. S. Bioprocess Biosyst. Eng. 2009, 32, 79–84.
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plant cells or tissues. In these particle preparation methods, leaf extracts are used as stabilizers and reducing agents, such as, Emblica Officinalis,17 Aloe vera,18 and Cinnamon camphora.19 It was presumed that the polyol components and water-soluble heterocyclic components were mainly responsible for the reduction of the silver or chloroaurate ions and the stabilization of the nanoparticles, respectively;6 only recently, gold nanoparticles were prepared using a selected molecule (apiin) extracted from henna leaf.20 Aloe vera leaf extract was used in the formation of gold nanotriangles, as a result of slow reduction on aqueous AuCl4- anions and the shape-directing effects of carbonyl compounds constituents of the plant extract. The colorless mucilaginous gel of Aloe leaves (mainly from Aloe vera) is widely used in various medical, cosmetic, and nutraceutical applications.21 Therapeutic uses of aloe gel are based on its anti-inflammatory, immunomodulating, and antibacterial activities which have been attributed to the polysaccharides, in particular acetylated mannan and glucomannan, and glycoproteins (lectins) present in the leaf pulp. In contrast, the dried exudate from the cut leaves (mainly from Aloe ferox Miller, known as Cape aloe) has been shown to be a complex mixture of secondary metabolites arising from the acetatemalonate pathway.21,22 Cape aloe is included in the main national pharmacopoeias as laxative activating peristalsis and is also used as a bittering agent in liqueur formulation. In addition, whole leaf extract Aloe species have been used for many years in folk medicine as generic chemopreventive and antitumor remedies.23 (17) Ankamwar, B.; Damle, C.; Absar, A.; Mural, S. J. Nanosci. Nanotechnol. 2005, 10, 1665–1671. (18) Chandran, S. P.; Chaudhary, M.; Pasricha, R.; Ahmad, A.; Sastry, M. Biotechnol. Prog. 2006, 22, 577–583. (19) Huang, J.; Li, Q.; Sun, D.; Lu, Y.; Su, Y.; Yang, X.; Wang, H.; Wang, Y.; Shao, W.; He, N.; Hong, J.; Chen, C. Nanotechnology 2007, 18, 105104–105115. (20) Kasthuri, J.; Veerapandian, S.; Rajendiran, N. Colloids Surf., B 2009, 68, 55–60. (21) Reynolds, T., Ed.; In Aloes-the genus Aloe; CRC Press, Boca Raton, London, New York, Washington, DC, 2004; Chapter 3. (22) Dogne, E.; Bisrat, D.; Viljoen, A.; Van Wyk, B.-E. Curr. Org. Chem. 2000, 4, 1055–1078. (23) Corsi, M.; Bertelli, A.; Gaja, G.; Fulgenzi, A.; Ferrero, M. Int. J. Tissue React. 1998, 20, 115–118.
Published on Web 06/08/2009
DOI: 10.1021/la9009674
7217
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Although, for the anthraquinone aloe-emodin, occurring in the plant in very small amounts, a selective activity against neuroectodermal tumors has been established by in vitro assays,24 the metabolites responsible for such antitumor activity as well as their mechanisms of action have not yet been identified. With the aim of preparing novel nanoparticles for biological applications, we utilized two active components of Cape aloe, aloin A and aloesin, as stabilizers for gold and silver nanoparticles. Aloins A and B, two diastereomeric C-glucosyl anthrones, are generally considered the purgative principles of Aloe exudates, responsible for its therapeutic activity.25 In addition, they have been shown responsible for its antihistamine26 and antipsoriatic action.27 5-Methylchromones (typically C-glucosylated), such as aloesin, exhibit antioxidant, free radical scavenging, and antiinflammatory activities both in vitro28 and in vivo.29 In order to prepare nanovehicles using natural molecules with specific therapeutic properties, we studied the preparation and characterization of aloin A and aloesin coated gold and silver nanoparticles. Various experimental conditions were planned to obtain differently sized and shaped nanoparticles. For biological applications, we planned a cell uptake study of designed, stable water-soluble aloin A/aloesin-gold hybrid systems involving sane (macrophages) and cancer cells (HeLa). Gold particles were characterized using UV-vis, ATR-FTIR, and 1H NMR spectroscopy, highlighting the interaction between gold and aloin A and aloesin. Although NMR studies of the particle ligand shell could be an issue, due to the very small content of organic material, we were able to perform the analysis using the HR-MAS 1H NMR technique.
Experimental Section 1. Spectroscopy and Microscopy. The samples were char-
acterized in solution by UV-vis, 1H NMR, and HR-MAS 1H NMR spectroscopy, and in the solid state by attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy and transmission electron microscopy (TEM) (see the Supporting Information for further details). Aloesin and aloin A were isolated from Cape aloe as reported in ref 32 and 25, respectively.
2. Preparation and Nanoparticle Characterization. 2.1. AuAloin A (6 nm) and Au-Aloesin (4 nm) by Borohydride Reduction. To 40 mL of Milli-Q (mQ) water, an aqueous solution of
NaAuCl4 (30 μmol; 0.116 M) was added. Under vigorous stirring, an aqueous solution of aloin A (3 μmol, 6 10-3 M) or aloesin (6 μmol, 8.7 10-3 M) was added. After 5 min, an aqueous solution of NaBH4 was added (60 μmol; 0.1M) and cherry red sol formed immediately. The obtained colloidal solution was left under stirring for a further 60 min to complete the reaction. The particles were purified by dialysis for 48 h (Sigma-Aldrich dry dialysis tubing).
2.2. Au-Aloin A (35 nm) and Au-Aloesin (45 nm) by Borohydride Reduction. The preparation is as described in section 2.1 using the following molar ratios between the components: aloin A or aloesin/NaBH4/NaAuCl4 =0.2:0.6:1 operating (24) Pecere, T.; Gazzola, M. V.; Mucignat, C.; Parolin, C.; Vecchia, F. D.; Cavaggioni, A.; Basso, G.; Diaspro, A.; Salvato, B.; Carli, M.; Palu, G. Cancer Res. 2000, 60, 2800–2804. (25) Manitto, P.; Monti, D.; Speranza, G. J. Chem. Soc., Perkin Trans. 1 1990, 1297–1300. (26) Yamamoto, I. J. Med. Soc. Toho Univ. 1970, 17, 361. (27) Muller, K. Gen. Pharmacol. 1996, 27, 1325–1335. (28) Yagi, A.; Kabash, A.; Okamura, N.; Haraguchi, H.; Moustafa, S. M.; Khalifa, T. I. Planta Med. 2002, 68, 957–960. (29) Speranza, G.; Morelli, C. F.; Tubaro, A.; Altinier, G.; Duri, L.; Manitto, P. Planta Med. 2005, 71, 79–81.
7218 DOI: 10.1021/la9009674
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Figure 1. Aloin A and aloesin molecule structures. at 55 °C for 22 h. The concentration of the reducing agent used was 0.01 M.
2.3. Au-Aloin A and Au-Aloesin by Citric Acid Reduction.
Operating at 55 °C, 10-15 nm sized particles have been obtained, while at 25 °C 40-50 nm sized particles have been obtained. To 40 mL of mQ water, an aqueous solution of NaAuCl4 (12.5 μmol; 1.54 10-2 M) was added at a desired temperature. Under stirring, an aqueous solution of aloin A or aloesin (5 μmol; 7.7 10-3 M) and an aqueous solution of citric acid were added (250 μmol; 1 M) and deep red sols were formed immediately. This was left under stirring for 48 h.
2.4. Au-Aloin A and Au-Aloesin (20-30 nm) by Ascorbic Acid Reduction. The preparation is as described in section 2.3 using the following molar ratio between the components: aloin A or aloesin/ascorbic acid/NaAuCl4 = 0.02:1:1 operating at 55 °C for 48 h. The concentration of the reducing agent used was 1 M.
2.5. Au-Aloin A and Au-Aloesin (14 nm) by Ligand Exchange. Fifteen nanometer citrate capped gold nanoparticles
were prepared by the Turkevich/Frens method.30,31 To 5 mL of citrate capped gold nanoparticles (CNPs =1.97 10-9 M), aloin A or aloesin aqueous solutions were added (1.5 mM). The amount of the stabilizing molecule was calculated to reach 10 000 molecules on the surface of each gold particle. The pH was adjusted to 10 by the addition of an aqueous solution of NaOH (0.5 M), and the mixture was stirred overnight. Subsequently, the particles were centrifuged (13 000 rpm, 15 min) and purified by three cycles of repetitive centrifugation and redispersion in fresh mQ water. 2.6. Ag-Aloin A and Ag-Aloesin (5 nm). The preparation is as described in section 2.1 using the following molar ratio between the components: aloin A or aloesin/AgNO3/NaBH4 = 0.2:1:10 operating under dinitrogen atmosphere at room temperature for 30 min. The concentration of the reducing agent used was 0.5 M. 3. Cell Culture and Nanoparticle Uptake. For the biological experiments, macrophage cells were extracted by an intraperitoneal wash of a CD1 mouse; the strain CD1 mice are bred in our Animal Care Facility. Peritoneal elicited macrophages were then incubated with gold nanoparticles (300 μL of 6 mM solution of gold nanoparticles added to 1 mL of extracted cells solution) for 30 min at 37 °C in a humidified atmosphere of 5% CO2 and subsequently centrifuged at 1300 rpm for 3 min. The cells were fixed onto microscopy cover glass slides and kept in a freezer/dark room before microscopy observations. HeLa cells were incubated with GNPs in a similar fashion.
Results and Discussion Recently, many papers in the literature appeared describing the biosynthesis of metallic nanoparticles using different plant extracts.9-21 In this context, we wondered whether specific metalplant active component adducts could exist during nanoparticle biosynthesis. Thus, one aim of this work was to discover novel intermediates, while the other was the biological application of hybrid materials as nanovehicles. (30) Turkevich, J.; Stevenson, P. C.; Hillier, J. J. Discuss. Faraday Soc. 1951, 11, 55–75. (31) Frens, G. Nature 1973, 241, 20–22.
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Figure 2. UV-vis spectra, TEM micrographs, and corresponding particle size distribution charts of Au-aloesin reduced by NaBH4 0.1 M, 25 °C (a, e, i); Au-aloesin reduced by NaBH4 0.01 M, 55 °C (b, f, j); Au-aloin A reduced by citric acid 1 M, 25 °C (c, g, k); and Au-aloin A prepared by ligand exchange reaction (d, h, l).
In our study, we chose to use two active components of Aloe isolated from a commercial sample of Cape aloe, namely, aloin A and aloesin (Figure 1).25,32 For the preparation of gold and silver nanoparticles in aqueous media, we used aloin A and aloesin molecules as stabilizers, employing sodium borohydride, citric acid, and ascorbic acid as reducing agents and NaAuCl4 and AgNO3 salts as metal precursors. The quantity of reducing agent and the reaction temperature were finely tuned to achieve differently sized and shaped metallic nanoparticles. The formation of the colloidal particles was followed by UVvis spectroscopy, and particle morphology was investigated by TEM. By varying both the concentration of sodium borohydride, from 0.1 to 0.01 M, and the reaction temperature, from 25 to 55 °C, we respectively obtained small and large sized spherical gold nanoparticles, using both aloin A (6 and 35 nm, respectively) and aloesin (4 and 45 nm, respectively) as stabilizing agents (see the Au-aloesin TEM images in Figure 2e (4 nm) and f (45 nm)). It is worth mentioning that it was possible to obtain large sized particles with sodium borohydride at 55 °C, because of the low concentration of NaBH4 (0.01 M). Particle growth of aloesin (45 nm) was monitored by UV-vis, and, in the case of sodium borohydride slow reduction (0.01 M), the increase in plasmon peak absorbance was progressive over 22 h (Figure 2b, 45 nm). Figure 2a shows in the UV-vis spectrum a plasmon peak absorbance due to the typical sodium borohydride fast reduction in the case of aloesin (4 nm). The use of citric acid (1 M) as reducing agent led, at 25 °C, to a mixture of differently shaped Au-aloin A (or Au-aloesin) coated
GNPs. After 48 h, the mixture consisted of spherical (50 nm) and large triangular nanoparticles or truncated triangles, as the result of slow citric acid reduction (Figure 2g, 50 nm). The UV-vis spectra present two plasmon peaks, centered at 528 and 975 nm (Figure 2c, 50 nm), in agreement with the TEM images reported in literature.11 For particle preparation, also ligand exchange between citrate coated GNPs and aloin A/aloesin molecules was utilized. In this way, 14 nm sized monodispersed and extremely stable spherical particles were obtained at room temperature after 24 h (see UVvis spectrum and TEM micrograph of Au-aloin A in Figure 2d and h). Aloin A and aloesin are able to stabilize spherical silver nanoparticles (5 nm) using sodium borohydride (0.5 M) at room temperature, under dinitrogen atmosphere. Within 30 min, a stable yellow colloidal solution was obtained, with a plasmon peak centered at 400 nm in the UV-vis spectrum, in accordance with literature data33 (Supporting Information). Prior to characterization, the aloin A and aloesin coated GNPs were purified of excess ligand by dialysis (48 h) in the case of small sized (
