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A biologically friendly single step method for gold nanoparticle formation
Colloids and Surfaces B: Biointerfaces 85 (2011) 330–337
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Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
A biologically friendly single step method for gold nanoparticle formation Damyanti Sharma ∗ Ian Wark Research Institute, University of South Australia, Mawson Lakes Campus, Mawson Lakes, Adelaide, SA 5095, Australia
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Article history: Received 6 December 2010 Accepted 5 March 2011 Available online 16 March 2011 Keywords: Gold nanoparticles Vesicles Lecithin Lamellar phases Sonication
a b s t r a c t There has been a keen interest for developing a biologically friendly approach for the preparation of gold nanoparticles for their application reasons. A biocompatible, quick and single step method is established for the preparation of gold nanoparticles in lecithin (Egg phosphatidylcholine)/water systems where lecithin itself acts as a reductant for hydrogen tetrachloro aurate (HAuCl4 ) to form the gold nanoparticles. Small gold nanoparticles (5–7 nm in diameter) were prepared in lamellar phases formed by lecithin within 6–7 h of HAuCl4 addition. Sonication of aqueous mixture of lecithin/HAuCl4 reduces the time of reduction process to seconds when a sonicator with probe (100 W) is used. Most of the particles are found attached to lecithin structures and are comparatively large in size. Some 10 nm particles are found attached to small lecithin vesicles (∼100 nm) formed during sonication. The nanoparticles formed were stabilized by an anionic surfactant sodium dodecylsulfate (SDS) which proved to be a good stabilizer, the nanoparticles being stable up to six months. To the best of our knowledge, this is the first report where a biological surfactant lecithin itself has acted as a reductant and no other chemical reductants were required for the gold nanoparticle formation. Particles were characterized by Uv–vis spectroscopy, transmission electron microscopy (TEM) and dynamic light scattering (DLS). Lamellar phases were characterized by a polarizing microscope. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Ever since Faraday’s [1] work on colloidal gold sols, gold nanoparticles have been of particular interest because of their unique applications in catalysis [2], biomedical materials [3–5], optical devices [6], and electronics [7]. The most important being the presence of distinctive absorption bands in visible region, due to the surface plasmon oscillation of free electrons [8,9]. A number of synthesis protocols have been developed for the formation of gold nanoparticles over a size range, both in aqueous medium [10] and in non-polar organic solvents [11]. Most of these protocols involve sodium borohydride or sodium citrate as reductant [10,11]. The use of microemulsions [12], copolymer micelles [13], surfactants [14–17], membranes, dendrimers and other amphiphiles has also been used for the synthesis [7,18] of stabilized AuNPs in the presence or absence of thiol ligands [7,12–18]. In some cases, these approaches has certain obvious disadvantages, such as the use of organic solvents, the generation of small molecule byproducts from reducing agents and the multiple steps required during the whole reduction process which involves harsh chemicals.
∗ Corresponding author. Present address: ARC Centre of Excellence for Electromaterials Science, Intelligent Polymer Research Institute, AIIM Facility, Innovation Campus, University of Wollongong, Wollongong, NSW 2522, Australia. Tel.: +61 2 42214872. E-mail address: [email protected] 0927-7765/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2011.03.005
Currently there is a growing need to develop a single step, quick and biologically friendly nanoparticle synthesis protocols. As a result researchers have been looking for different types of biological systems for inspiration. It has been recently demonstrated that gold nanoparticles can be synthesized by employing certain microorganisms like bacteria and fungi [19–23]. Not only gold, but silver nanoparticles [23–25] have also been produced using some microorganisms. Particles have been produced intracellularly as well as extracellularly. In any case, the exact mechanism of formation of nanoparticles is still not understood. It has been suggested that certain proteins are released into the solution during reduction process and these proteins act as reductant for the formation of gold and silver nanoparticles. In another report the authors have reported the synthesis of stable gold hydrosol by the reduction of chloroaurate ions by aspartic acid [26]. Not only microorganisms like bacteria and yeast but also the some other biomasses like from plants also play an important role in the synthesis of gold and silver nanoparticles. Jose-Yacaman and co-workers have prepared Gold [27] Silver nanoparticles [28] in alfa alfa plants. Silver nanoparticles have also been prepared in geranium leaf extracts [29]. When it comes to biologically friendly or biocompatible systems, phopholipids are of great importance because of their rich phase behavior and structural relationship to biological membranes. Lecithin is generally the major fraction (more than 50%) of the total phospholipid occurring in biological membranes. Since
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lecithin molecule posses a phosphate and a trimethylammonium group separated by two methylene groups, its structure allows two ionic forms: one in which the separation of charges is maximal and the other in which a reduced separation of charges results from an internal salt linkage between the phosphate and the trimethylammonium groups in the same molecule [30]. These ionic charges can be of considerable interest for different reactions to be carried out. Since lecithin is a fully biocompatible substance, it is widely used in every day life as surfactant, and mixtures of lecithin, water, and oils are adopted in human and animal food, medicine, cosmetics and pharmaceuticals [31] and can be a promising material suitable for engineering biocompatible nanomaterials. There are not many reports about involving different phospholipids directly in the synthesis protocol for gold nanoparticles. In most of the cases they have been used as stabilizers or for coating presynthesized nanoparticles. Takahashi et al. [32] has recently reported that replacing cetyltrimethylammonium bromide (CTAB) with phosphatidylcholine can diminish the cytotoxicity of CTAB on gold nanorod surfaces and is a good candidate for suppressing aggregation of nanorods after extraction of CTAB. More recently, biocompatible double-hydrophilic block copolymers containing poly [2-(methacryloyloxy) ethyl phosphoryl choline block have been used for synthesis of gold and magnetite nanoparticles [33,34]. He and Urban [35] has reported Phopholipid (1,2-dipalmitoyl-sn-glycero-3-phosphothio-ethanol) stabilized gold using NaBH4 as a reductant. Koetz et al. [36] has used a phophatidylcholine based biocompatible reverse microemulsion system for the preparation of barium sulfate nanoparticles. Wu et al [37] has coated single-walled carbon nanotubes with different phospholipids which can be useful in biosensing or further fictionalization with antioxidants and monoclonal antibodies may be utilized in nanomedicine. Regev et al. [38,39] has reported the preparation of gold nanoparticles in phosphatidylcholine (S100® )-monoolein multilamellar vesicles where monoolein acts as a reductant. Same group of researchers have prepared gold fractal structures in sheared lamellar phases of the similar system [40]. The researches have argued that in this case monoolein acts as a reductant and lecithin acts as a catalyst. All the existing reports about gold nanoparticle formation in phospholipid containing systems use either a mild or conventional chemical reductant. We have used lecithin, as a reductant for gold and silver nanoparticle formation as no other foreign material was added during the reduction process. Small (5–7 nm) gold nanoparticles are formed in lamellar structures formed by lecithin and are stable up to 28 h. Sonicating the lecithin/HAuCl4 mixtures reduces the time of reduction process to seconds. Two methods of sonication have been used. A bath type sonicator and the other sonicator with a probe. The sonicator with probe is more effective as it reduces the time of preparation and produces comparatively small nanoparticles. Nanoparticles are found attached to the structures formed by lecithin. Most of the nanoparticles are somehow attached to lecithin and some aggregating with each other. To overcome this, an anionic surfactant sodium dodecylsulfate (SDS) and tri-sodium citrate were tried for stabilization. SDS stabilized particles are well dispersed and are stable over six months. Surprisingly the SDS stabilized particles are smaller in size (from 7 to 20 nm) and all the particles being spherical in shape as compared to citrate stabilized (data not shown) or without stabilizer samples. In another attempt, Silver nanoparticles could also be prepared in lecithin/water system without using any external reductant (data not shown). Preparation of silver nanoparticles in lecithin/water system is the first report to the best of our knowledge.
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2. Materials and methods 2.1. Materials HAuCl4 was purchased and used as received from Aldrich. AgNO3 (AR grade, 99.9%) was received from BDH, Australia. Egg Phosphatidylcholine (EPC) (99.0%) and Dimeristoyalphosphatidylcholine (DMPC) 99%, palmitoyl-2-oleoyl-sn-glycero-3phosphocholine (POPC) 99%, and 1,2-dioleoyl-sn-glycero-3phosphocholine (DOPC) 98% were purchased from Avanti Polar Lipids and were used without further purification. SDS (>99.0%) was obtained from Sigma–Aldrich and was used as received. Ethanol and other chemicals were AR grade from BDH chemicals. Mili Q water was used through out the experiments. 2.2. Gold/silver nanoparticles preparation in lamellar phases Lamellar phases of lecithin were prepared by placing pasty lecithin on a glass slide and slowly adding aqueous HAuCl4 /orAgNO3 with the help of a syringe. Glass slides were covered with cover slips and left standing undisturbed for the reduction process. Lamellar phases and gold/or silver nanoparticles were formed spontaneously on glass slide. Different concentrations of HAuCl4 (1–10 mM) were tried for optimization and it was found that 10 mM produces the reddish pink color on glass slide on a larger area than the lower concentrations used. Lower concentrations rather formed the purple color with a tinge of pink at some places on glass slide. 50 mM of AgNO3 was added to prepare the silver nanoparticle. 2.3. Gold nanoparticle preparation on sonication A multi-wave ultrasonic generator (Labsonic◦ L, B Braun Diessel Biotech.) and a submersible transducer with a solid titanium probe tip of 19 mm diameter were used for ultrasonic irradiation at 20 kHz with an output of 100 W. The reactions were carried out in a 1.5 ml measuring tubes. Pasty lecithin was weighed directly into eppendorf tubes and required volume of aqueous HAuCl4 was added. The samples were shaken by hand for few minutes to disperse lecithin and then sonicated until the reddish or pinkish color appeared. A bath type sonicator Soniclean120 T (Transtek Systems) with at 50/50 Hz with an input of 50 W was also used for some experiments. 2.4. Polarizing microscopy Nikon 80i microscope with polarizer and phase contrast was used to characterize the lamellar phases formed by lecithin. Images were captured at cross polars by using a Nikon CCD camera. 2.5. UV–vis spectroscopy UV–vis spectra were taken using Cary 1 Spectrophotometer in the UV–vis region to find the location and intensity of the surface Plasmon resonance peaks for the nanoparticles at room temperature. The spectra were taken by directly transferring the final dispersion from sonicated samples into cuvettes. UV–vis spectra of gold nanoparticle containing lamellar phases prepared on glass slide samples were recorded on glass slides without disturbing the original sample. No dilution was made before the measurements. 2.6. Transmission electron microscopy (TEM) TEM observations were performed on Philips CM 100 transmission electron microscope. Samples for imaging were prepared by simply placing a small drop of the lecithin/nanoparticle dispersion
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25 ◦ C at an angle of 90◦ .The light source is a water cooled Lexel Argon ion laser. 3. Results and discussion 3.1. Formation of gold nanoparticles in lamellar phases of lecithin
Fig. 1. (a) UV–vis spectra of Gold nanoparticles prepared in lecithin/water system forming lamellar phases on a glass slide. (b) Polarizing microscope image showing the lamellar structures and characteristic color due to gold nanoparticles associated with the lamellar phases in lecithin/water.
on a carbon coated 200 mesh copper grid. The excess of solution was removed with a filter paper. The grids were left standing in air for 30 min for drying and film formation before the measurements. Gold nanoparticle/Lamellar samples were diluted with water or ethanol to prepare dilute dispersion just before the TEM grid preparation. 2.7. Dynamic Light Scattering (DLS) Dynamic light scattering measurements were performed on a Brookhaven Instruments photon correlation spectrometer with BI 200SM goniometer and BI-2030 autocorrelator. Dust free vials were used for the aqueous solutions and measurements were done at
Fig. 1b shows the lamellar phases formed by lecithin/water system under a polarizing microscope. The characteristic reddish color shows the formation of nanoparticles in lamellar phases. The color under the cover slips starts appearing after 5 h of HAuCl4 addition to pasty lecithin on a glass slide. Intensity of color keeps increasing with up to 7 h and no change is observed after that. So we have assumed that it takes 7 h for complete reduction of HAuCl4 by lecithin. Different concentrations of HAuCl4 (1–10 mM) were tried to see the effect on reduction process and to optimize the HAuCl4 concentration for the formation of gold nanoparticles. Lower concentrations of HAuCl4 produce purple color on glass slides with little reddish color at some places on the glass slides. 10 mM HAuCl4 was found to produce the reddish color on glass slide with purple color at some places. This sample was left under the microscope for 28 h and it was found that there is no change in lamellar structures as well as in the intensity of color (Fig. 1b). This indicates that gold nanoparticles associated with lamellar structures are stable at least for 28 h. Fig. 1a shows the Uv–vis spectra of the 10 mM HAuCl4 solutions with lecithin after 7 h of HAuCl4 addition. The characteristic surface Plasmon band appears at 540. Homogeneous spherical gold nanoparticles are known to produce the surface Plasmon band at 520 nm. In the present system, the peak maximum for absorbance was found at 540 nm instead of 520 nm, which might be due to collective absorption of reddish and purple color (indicating presence of large gold nanoparticles) on the glass slide. These results indicated that Au3+ has been reduced to Au0 by lecithin as no other foreign material was added to the system during the reduction process. To gain an insight into exact shape and size of the particles formed, TEM measurements were performed. Fig. 2a shows the TEM image of gold nanoparticles. The image was taken by slightly touching the grid to sample on glass slide to know the original shape, size and location of the nanoparticles formed as such. All the particles formed are spherical in shape and 5–7 nm in size. Particles are fairly monodisperse in size and shape and all the particles were attached to lecithin structures. No aggregation was observed. To understand if particles grow in size when detached of lecithin, samples were dissolved in ethanol and TEM images were taken by putting a drop of the dispersion on TEM grids (Fig. 2b). TEM image shows that the particles grew in size range of 20–50 nm in ethanol and were found aggregating. Some different shapes other than spherical particle were also observed. Mylenic structures aligning nanoparticles were also found (Fig. 2c). It seems that the particles came out of the lamellar structures and were aligned by the water flowing through the channels in mylinic structures as most of the particles were found outside mylinic structures and some scattered on them. To get an insight into the mechanism of formation of gold nanoparticles and the role of phosphocholine head group, a synthetic lipid dimeristoyal phosphatidylcholine (DMPC) was also tried with the HAuCl4 on a glass slide which produced reddish purple color after 12 days. DMPC is similar in head group to lecithin but no double bond in the alkyl chain. The extent of reduction (appearance of color on glass slide) was lesser and took much longer time (12 days) for reduction process in comparison to lecithin. This gives us an indication that the head group phosphocholine initiates the reduction process and rules out the role of impurities.
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Fig. 2. (a) TEM Images of gold nanoparticles formed in lecithin lamellar phases (images taken by touching the grid to sample on glass slide), scale bar—200 nm. (b) TEM Images of gold nanoparticles formed in lecithin lamellar phases (images taken by dissolving the sample in ethanol), scale bar—1 m. (c) Presence of mylinic structures and alignment of gold nanoparticles (image taken by dissolving the sample in ethanol), scale bar—1 m.
Lipids with one double bond in alkyl chain with the same phosphocholine head group, 1-palmitoyl-2-oleoyl-sn-glycero-3phosphocholine (POPC) and two double bonds 1,2-dioleoylsn-glycero-3-phosphocholine (DOPC) could also form the gold nanoparticles and the reduction process took the almost similar time as in case of lecithin. This gives us an indication that the polar head group phosphocholine initiates the process of reduction which can be enhanced by the presence of ethylene groups in alkyl chain. Further saturation (presence of two double bonds) does not affect the reduction process further. This further clarifies the role of phosphocholine group in the reduction process. A further support for our observations can be found from the reported literature [33] where S100® a mixture of phosphatidyle cholines from fat free soybean consisting mainly of linoleic phophatidylcholine has been accounted as a catalyst for the reduction process of KAuCl4 by monoolein to form gold nanoparticles. It has been suggested that a blue-green color appears with adding S100® indicating the formation of gold nanoparticles. The IR studies show that the nanoparticles interact with head group area, with the acyl group, and with the ethylene groups of the lipid chains. It has been suggested that quaternary ammonium groups play an important part in the reduction process. Reduction by impurities alone present in the phophatidylcholine has been ruled out by the authors. As in our system no other foreign material has been added to Lecithin/water system during the reduction process, we conclude that the lecithin itself acts as a reductant.
A further study in our case needs to be done to find out the exact mechanism of formation of nanoparticles. 3.2. Gold nanoparticle formation during sonication After the formation of nanoparticles in lamellar phases, we attempted to prepare them in lecithin vesicles by sonicating the dispersion of lecithin and HAuCl4 . Two types of sonicators were used; bath type sonicator and a sonicator with a probe. Ultrasonication has been used to prepare vesicles in the literature. 3.2.1. Using a bath type sonicator Fig. 3a shows the UV–vis spectra of 3 mM HAuCl4 with different lecithin concentrations after sonication with a bath type sonicator. Peaks between 540 and 554 indicate the presence of gold nanoparticles prepared in the system. It takes 45–60 min of sonication time for the appearance of pink color which is quite low as compared to the time of reduction in lamellar phases without sonication. This can be due to the cavitation and formation of radicals by ultrasonic irradiation which could have further accelerated the reduction process. Sonochemistry is used to enhance or alter the chemical reactions. There have been reports about the formation of gold nanoparticles in an ultrasound field, with chemicals such as alcohols and surfactants used to enhance the radical formation [41–45]. Dynamic light scattering show broad distribution of particles ranging from 40 to 600 nm size (Fig. 3b). TEM measurements were also done to gain a further insight into the distribution pro-
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Fig. 3. (a) UV–vis spectra of gold nanoparticles in lecithin/water system after sonication with bath type sonicator. (b) Particle size distribution by DLS in the system. (c) TEM micrograph of gold nanoparticles prepared in the system, scale bar–1 m.
file of nanoparticles. TEM image confirms the polydispersity of the particles sitting on lecithin structures (Fig. 3c). Particles are polydisperse and aggregating with each other. Most of the particles are spherical in shape but some non spherical shapes and triangular shapes are also observed. DLS results are in good agreement with the TEM results as TEM also shows the bigger particle size up to 50–200 nm gold nanoparticles sitting on up to 600 nm wide lecithin structures. 3.2.2. Using a sonicator with a probe Different concentrations of lecithin and HAuCl4 were tried systematically with sonicator with probe. Samples become pink after 45 seconds to few minutes time of sonication depending on the concentration of lecithin. Time for reduction process is comparatively smaller in comparison to reduction time by using Soniclean. This result is also in good agreement with the literature recognizing that smaller is the intensity of the ultrasound; the slower is the rate of reduction for gold (III) in presence of 2-propanol [43]. Fig. 4a shows the UV–vis spectra of 5 mM HAuCl4 with different lecithin
concentrations (1–5 mM). A Plasmon resonance band is observed for all lecithin concentrations between 548 and 561 nm which indicates the presence of large nanoparticles or aggregation of particles. The spectra also indicate that at all the lecithin concentrations a good amount of unreacted HAuCl4 is left (band at 350 nm) in the dispersion which does not diminish with time (data not shown). We do not see any of the peaks around 350 nm in all lecithin concentrations HAuCl4 is reduced to 3 mM (Fig. 4b). This shows that the reduction process is complete for this concentration of HAuCl4 with all the lecithin concentrations tried (2, 3 and 5 mM). 3 mM lecithin + 3 mM HAuCl4 was chosen for further experiments to form equimolar dispersions. DLS results (Fig. 4c) show a bimodal distribution of particles in 60–400 nm size range. TEM images were taken to gain further insight into the size distribution of particles. From TEM images (Fig. 4d) it is clear that some 10 nm gold nanoparticles are attached to lecithin vesicles of around 100–150 nm. But most of the particles were sitting outside aggregating with each other. The particles outside the vesicles are 50 nm in size. And the aggregates are
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Fig. 4. UV–vis spectra of (a) 5.0 mM (b) 3.0 HAuCl4 with different [lecithin] (c) DLS particle size profile for 3 mM EPC + 3 mM HAuCl4 (d) TEM image showing the 10 nm particles attached to lecithin vesiclesand, scale bar—200 nm (e) UV–vis spectra of 1 mM aqueous HAuCl4 after 8 min of sonication with a sonicator with probe.
around 200 nm in size. A comparatively small size of nanoparticles by this method of sonication may be due to the increasing rate of reduction of HAuCl4 which has also been demonstrated by Okitsu et al. [43]. Sonication of 1 mM aqueous HAuCl4 up to 8 min with the sonicator with probe at 2 min time intervals does not show
any change in initial pale color of HAuCl4. There is a blue shift observed in the UV–vis absorption peak from 351 nm to 343 nm and a decrease in absorption intensity from 2.25 to 1.6 (Fig. 4e) but no peak is observed between 400 and 800 nm indicating that no gold nanoparticles have been formed in the solution on sonication.
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acted as a reductant. Sonication of lecithin/aqHAuCl4 dispersions reduces the time of reduction. Nanoparticles are found to be attached to the lamellar structures and vesicles formed by lecithin. Gold nanoparticle containing vesicles can be used as biological markers for vesicle/liposome uptake in cells. SDS was found to be a good stabilizer. SDS stabilized particles are well dispersed and are stable over six months. This work is a step towards using biologically friendly approach for the nanoparticle formation. Acknowledgements This work was financially supported by Australian Research Council under the scheme Nano and Biomaterials Centre, Ian Wark Research Institute, University of South Australia. I also thank Prof Roger Horn for his guidance and helping me initiate this work. We are thankful to Adelaide Microscopy for providing Transmission Electron Microscopy facility for imaging. References
Fig. 5. (a) UV–vis spectra of lecithin/HAuCl4 in presence and absence of SDS after six months. (b) TEM image of gold nanoparticles formed in presence of SDS, 2 h after the sample preparation, scale bar—100 nm.
When SDS was added to the lecithin/HAuCl4 dispersion before sonication, the final dispersions were stable up to six months and did not show a change in color or the precipitation. Dispersions without SDS show black precipitate after a week time. The UV–vis spectra of without SDS and with SDS spectra is shown in Fig. 5a. The sample without SDS does not show any peak in around 500–600 nm range indicating that no nanoparticles are present in the dispersion while sample with SDS still have the absorption peak at 544 nm. TEM observations show that the nanoparticles formed in SDS/lecithin/HAuCl4 system are much smaller in size (5–10 nm) (Fig. 5b) and were well dispersed. No aggregates were present in the system. SDS is known to interact with lecithin bilayers by partitioning between the monomers in the bilayer and imparting a net negative charge to the system [46]. The negatively charged sulfate head groups may be responsible for the stabilization of nanoparticles by electrostatic charge repulsion. Wang et al. [47] has reported that SDS acts as a reductant when forming gold nanoparticles in lecithin/surfactant system. This might be the reason for observing very small nanoparticles when SDS is present with lecithin. SDS might be enhancing the reduction power and leads to small nanoparticle formation. 4. Conclusion In conclusion, for the first time gold nanoparticles have been formed in lamellar phases formed by lecithin where lecithin itself
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Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
A biologically friendly single step method for gold nanoparticle formation Damyanti Sharma ∗ Ian Wark Research Institute, University of South Australia, Mawson Lakes Campus, Mawson Lakes, Adelaide, SA 5095, Australia
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Article history: Received 6 December 2010 Accepted 5 March 2011 Available online 16 March 2011 Keywords: Gold nanoparticles Vesicles Lecithin Lamellar phases Sonication
a b s t r a c t There has been a keen interest for developing a biologically friendly approach for the preparation of gold nanoparticles for their application reasons. A biocompatible, quick and single step method is established for the preparation of gold nanoparticles in lecithin (Egg phosphatidylcholine)/water systems where lecithin itself acts as a reductant for hydrogen tetrachloro aurate (HAuCl4 ) to form the gold nanoparticles. Small gold nanoparticles (5–7 nm in diameter) were prepared in lamellar phases formed by lecithin within 6–7 h of HAuCl4 addition. Sonication of aqueous mixture of lecithin/HAuCl4 reduces the time of reduction process to seconds when a sonicator with probe (100 W) is used. Most of the particles are found attached to lecithin structures and are comparatively large in size. Some 10 nm particles are found attached to small lecithin vesicles (∼100 nm) formed during sonication. The nanoparticles formed were stabilized by an anionic surfactant sodium dodecylsulfate (SDS) which proved to be a good stabilizer, the nanoparticles being stable up to six months. To the best of our knowledge, this is the first report where a biological surfactant lecithin itself has acted as a reductant and no other chemical reductants were required for the gold nanoparticle formation. Particles were characterized by Uv–vis spectroscopy, transmission electron microscopy (TEM) and dynamic light scattering (DLS). Lamellar phases were characterized by a polarizing microscope. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Ever since Faraday’s [1] work on colloidal gold sols, gold nanoparticles have been of particular interest because of their unique applications in catalysis [2], biomedical materials [3–5], optical devices [6], and electronics [7]. The most important being the presence of distinctive absorption bands in visible region, due to the surface plasmon oscillation of free electrons [8,9]. A number of synthesis protocols have been developed for the formation of gold nanoparticles over a size range, both in aqueous medium [10] and in non-polar organic solvents [11]. Most of these protocols involve sodium borohydride or sodium citrate as reductant [10,11]. The use of microemulsions [12], copolymer micelles [13], surfactants [14–17], membranes, dendrimers and other amphiphiles has also been used for the synthesis [7,18] of stabilized AuNPs in the presence or absence of thiol ligands [7,12–18]. In some cases, these approaches has certain obvious disadvantages, such as the use of organic solvents, the generation of small molecule byproducts from reducing agents and the multiple steps required during the whole reduction process which involves harsh chemicals.
∗ Corresponding author. Present address: ARC Centre of Excellence for Electromaterials Science, Intelligent Polymer Research Institute, AIIM Facility, Innovation Campus, University of Wollongong, Wollongong, NSW 2522, Australia. Tel.: +61 2 42214872. E-mail address: [email protected] 0927-7765/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2011.03.005
Currently there is a growing need to develop a single step, quick and biologically friendly nanoparticle synthesis protocols. As a result researchers have been looking for different types of biological systems for inspiration. It has been recently demonstrated that gold nanoparticles can be synthesized by employing certain microorganisms like bacteria and fungi [19–23]. Not only gold, but silver nanoparticles [23–25] have also been produced using some microorganisms. Particles have been produced intracellularly as well as extracellularly. In any case, the exact mechanism of formation of nanoparticles is still not understood. It has been suggested that certain proteins are released into the solution during reduction process and these proteins act as reductant for the formation of gold and silver nanoparticles. In another report the authors have reported the synthesis of stable gold hydrosol by the reduction of chloroaurate ions by aspartic acid [26]. Not only microorganisms like bacteria and yeast but also the some other biomasses like from plants also play an important role in the synthesis of gold and silver nanoparticles. Jose-Yacaman and co-workers have prepared Gold [27] Silver nanoparticles [28] in alfa alfa plants. Silver nanoparticles have also been prepared in geranium leaf extracts [29]. When it comes to biologically friendly or biocompatible systems, phopholipids are of great importance because of their rich phase behavior and structural relationship to biological membranes. Lecithin is generally the major fraction (more than 50%) of the total phospholipid occurring in biological membranes. Since
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lecithin molecule posses a phosphate and a trimethylammonium group separated by two methylene groups, its structure allows two ionic forms: one in which the separation of charges is maximal and the other in which a reduced separation of charges results from an internal salt linkage between the phosphate and the trimethylammonium groups in the same molecule [30]. These ionic charges can be of considerable interest for different reactions to be carried out. Since lecithin is a fully biocompatible substance, it is widely used in every day life as surfactant, and mixtures of lecithin, water, and oils are adopted in human and animal food, medicine, cosmetics and pharmaceuticals [31] and can be a promising material suitable for engineering biocompatible nanomaterials. There are not many reports about involving different phospholipids directly in the synthesis protocol for gold nanoparticles. In most of the cases they have been used as stabilizers or for coating presynthesized nanoparticles. Takahashi et al. [32] has recently reported that replacing cetyltrimethylammonium bromide (CTAB) with phosphatidylcholine can diminish the cytotoxicity of CTAB on gold nanorod surfaces and is a good candidate for suppressing aggregation of nanorods after extraction of CTAB. More recently, biocompatible double-hydrophilic block copolymers containing poly [2-(methacryloyloxy) ethyl phosphoryl choline block have been used for synthesis of gold and magnetite nanoparticles [33,34]. He and Urban [35] has reported Phopholipid (1,2-dipalmitoyl-sn-glycero-3-phosphothio-ethanol) stabilized gold using NaBH4 as a reductant. Koetz et al. [36] has used a phophatidylcholine based biocompatible reverse microemulsion system for the preparation of barium sulfate nanoparticles. Wu et al [37] has coated single-walled carbon nanotubes with different phospholipids which can be useful in biosensing or further fictionalization with antioxidants and monoclonal antibodies may be utilized in nanomedicine. Regev et al. [38,39] has reported the preparation of gold nanoparticles in phosphatidylcholine (S100® )-monoolein multilamellar vesicles where monoolein acts as a reductant. Same group of researchers have prepared gold fractal structures in sheared lamellar phases of the similar system [40]. The researches have argued that in this case monoolein acts as a reductant and lecithin acts as a catalyst. All the existing reports about gold nanoparticle formation in phospholipid containing systems use either a mild or conventional chemical reductant. We have used lecithin, as a reductant for gold and silver nanoparticle formation as no other foreign material was added during the reduction process. Small (5–7 nm) gold nanoparticles are formed in lamellar structures formed by lecithin and are stable up to 28 h. Sonicating the lecithin/HAuCl4 mixtures reduces the time of reduction process to seconds. Two methods of sonication have been used. A bath type sonicator and the other sonicator with a probe. The sonicator with probe is more effective as it reduces the time of preparation and produces comparatively small nanoparticles. Nanoparticles are found attached to the structures formed by lecithin. Most of the nanoparticles are somehow attached to lecithin and some aggregating with each other. To overcome this, an anionic surfactant sodium dodecylsulfate (SDS) and tri-sodium citrate were tried for stabilization. SDS stabilized particles are well dispersed and are stable over six months. Surprisingly the SDS stabilized particles are smaller in size (from 7 to 20 nm) and all the particles being spherical in shape as compared to citrate stabilized (data not shown) or without stabilizer samples. In another attempt, Silver nanoparticles could also be prepared in lecithin/water system without using any external reductant (data not shown). Preparation of silver nanoparticles in lecithin/water system is the first report to the best of our knowledge.
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2. Materials and methods 2.1. Materials HAuCl4 was purchased and used as received from Aldrich. AgNO3 (AR grade, 99.9%) was received from BDH, Australia. Egg Phosphatidylcholine (EPC) (99.0%) and Dimeristoyalphosphatidylcholine (DMPC) 99%, palmitoyl-2-oleoyl-sn-glycero-3phosphocholine (POPC) 99%, and 1,2-dioleoyl-sn-glycero-3phosphocholine (DOPC) 98% were purchased from Avanti Polar Lipids and were used without further purification. SDS (>99.0%) was obtained from Sigma–Aldrich and was used as received. Ethanol and other chemicals were AR grade from BDH chemicals. Mili Q water was used through out the experiments. 2.2. Gold/silver nanoparticles preparation in lamellar phases Lamellar phases of lecithin were prepared by placing pasty lecithin on a glass slide and slowly adding aqueous HAuCl4 /orAgNO3 with the help of a syringe. Glass slides were covered with cover slips and left standing undisturbed for the reduction process. Lamellar phases and gold/or silver nanoparticles were formed spontaneously on glass slide. Different concentrations of HAuCl4 (1–10 mM) were tried for optimization and it was found that 10 mM produces the reddish pink color on glass slide on a larger area than the lower concentrations used. Lower concentrations rather formed the purple color with a tinge of pink at some places on glass slide. 50 mM of AgNO3 was added to prepare the silver nanoparticle. 2.3. Gold nanoparticle preparation on sonication A multi-wave ultrasonic generator (Labsonic◦ L, B Braun Diessel Biotech.) and a submersible transducer with a solid titanium probe tip of 19 mm diameter were used for ultrasonic irradiation at 20 kHz with an output of 100 W. The reactions were carried out in a 1.5 ml measuring tubes. Pasty lecithin was weighed directly into eppendorf tubes and required volume of aqueous HAuCl4 was added. The samples were shaken by hand for few minutes to disperse lecithin and then sonicated until the reddish or pinkish color appeared. A bath type sonicator Soniclean120 T (Transtek Systems) with at 50/50 Hz with an input of 50 W was also used for some experiments. 2.4. Polarizing microscopy Nikon 80i microscope with polarizer and phase contrast was used to characterize the lamellar phases formed by lecithin. Images were captured at cross polars by using a Nikon CCD camera. 2.5. UV–vis spectroscopy UV–vis spectra were taken using Cary 1 Spectrophotometer in the UV–vis region to find the location and intensity of the surface Plasmon resonance peaks for the nanoparticles at room temperature. The spectra were taken by directly transferring the final dispersion from sonicated samples into cuvettes. UV–vis spectra of gold nanoparticle containing lamellar phases prepared on glass slide samples were recorded on glass slides without disturbing the original sample. No dilution was made before the measurements. 2.6. Transmission electron microscopy (TEM) TEM observations were performed on Philips CM 100 transmission electron microscope. Samples for imaging were prepared by simply placing a small drop of the lecithin/nanoparticle dispersion
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25 ◦ C at an angle of 90◦ .The light source is a water cooled Lexel Argon ion laser. 3. Results and discussion 3.1. Formation of gold nanoparticles in lamellar phases of lecithin
Fig. 1. (a) UV–vis spectra of Gold nanoparticles prepared in lecithin/water system forming lamellar phases on a glass slide. (b) Polarizing microscope image showing the lamellar structures and characteristic color due to gold nanoparticles associated with the lamellar phases in lecithin/water.
on a carbon coated 200 mesh copper grid. The excess of solution was removed with a filter paper. The grids were left standing in air for 30 min for drying and film formation before the measurements. Gold nanoparticle/Lamellar samples were diluted with water or ethanol to prepare dilute dispersion just before the TEM grid preparation. 2.7. Dynamic Light Scattering (DLS) Dynamic light scattering measurements were performed on a Brookhaven Instruments photon correlation spectrometer with BI 200SM goniometer and BI-2030 autocorrelator. Dust free vials were used for the aqueous solutions and measurements were done at
Fig. 1b shows the lamellar phases formed by lecithin/water system under a polarizing microscope. The characteristic reddish color shows the formation of nanoparticles in lamellar phases. The color under the cover slips starts appearing after 5 h of HAuCl4 addition to pasty lecithin on a glass slide. Intensity of color keeps increasing with up to 7 h and no change is observed after that. So we have assumed that it takes 7 h for complete reduction of HAuCl4 by lecithin. Different concentrations of HAuCl4 (1–10 mM) were tried to see the effect on reduction process and to optimize the HAuCl4 concentration for the formation of gold nanoparticles. Lower concentrations of HAuCl4 produce purple color on glass slides with little reddish color at some places on the glass slides. 10 mM HAuCl4 was found to produce the reddish color on glass slide with purple color at some places. This sample was left under the microscope for 28 h and it was found that there is no change in lamellar structures as well as in the intensity of color (Fig. 1b). This indicates that gold nanoparticles associated with lamellar structures are stable at least for 28 h. Fig. 1a shows the Uv–vis spectra of the 10 mM HAuCl4 solutions with lecithin after 7 h of HAuCl4 addition. The characteristic surface Plasmon band appears at 540. Homogeneous spherical gold nanoparticles are known to produce the surface Plasmon band at 520 nm. In the present system, the peak maximum for absorbance was found at 540 nm instead of 520 nm, which might be due to collective absorption of reddish and purple color (indicating presence of large gold nanoparticles) on the glass slide. These results indicated that Au3+ has been reduced to Au0 by lecithin as no other foreign material was added to the system during the reduction process. To gain an insight into exact shape and size of the particles formed, TEM measurements were performed. Fig. 2a shows the TEM image of gold nanoparticles. The image was taken by slightly touching the grid to sample on glass slide to know the original shape, size and location of the nanoparticles formed as such. All the particles formed are spherical in shape and 5–7 nm in size. Particles are fairly monodisperse in size and shape and all the particles were attached to lecithin structures. No aggregation was observed. To understand if particles grow in size when detached of lecithin, samples were dissolved in ethanol and TEM images were taken by putting a drop of the dispersion on TEM grids (Fig. 2b). TEM image shows that the particles grew in size range of 20–50 nm in ethanol and were found aggregating. Some different shapes other than spherical particle were also observed. Mylenic structures aligning nanoparticles were also found (Fig. 2c). It seems that the particles came out of the lamellar structures and were aligned by the water flowing through the channels in mylinic structures as most of the particles were found outside mylinic structures and some scattered on them. To get an insight into the mechanism of formation of gold nanoparticles and the role of phosphocholine head group, a synthetic lipid dimeristoyal phosphatidylcholine (DMPC) was also tried with the HAuCl4 on a glass slide which produced reddish purple color after 12 days. DMPC is similar in head group to lecithin but no double bond in the alkyl chain. The extent of reduction (appearance of color on glass slide) was lesser and took much longer time (12 days) for reduction process in comparison to lecithin. This gives us an indication that the head group phosphocholine initiates the reduction process and rules out the role of impurities.
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Fig. 2. (a) TEM Images of gold nanoparticles formed in lecithin lamellar phases (images taken by touching the grid to sample on glass slide), scale bar—200 nm. (b) TEM Images of gold nanoparticles formed in lecithin lamellar phases (images taken by dissolving the sample in ethanol), scale bar—1 m. (c) Presence of mylinic structures and alignment of gold nanoparticles (image taken by dissolving the sample in ethanol), scale bar—1 m.
Lipids with one double bond in alkyl chain with the same phosphocholine head group, 1-palmitoyl-2-oleoyl-sn-glycero-3phosphocholine (POPC) and two double bonds 1,2-dioleoylsn-glycero-3-phosphocholine (DOPC) could also form the gold nanoparticles and the reduction process took the almost similar time as in case of lecithin. This gives us an indication that the polar head group phosphocholine initiates the process of reduction which can be enhanced by the presence of ethylene groups in alkyl chain. Further saturation (presence of two double bonds) does not affect the reduction process further. This further clarifies the role of phosphocholine group in the reduction process. A further support for our observations can be found from the reported literature [33] where S100® a mixture of phosphatidyle cholines from fat free soybean consisting mainly of linoleic phophatidylcholine has been accounted as a catalyst for the reduction process of KAuCl4 by monoolein to form gold nanoparticles. It has been suggested that a blue-green color appears with adding S100® indicating the formation of gold nanoparticles. The IR studies show that the nanoparticles interact with head group area, with the acyl group, and with the ethylene groups of the lipid chains. It has been suggested that quaternary ammonium groups play an important part in the reduction process. Reduction by impurities alone present in the phophatidylcholine has been ruled out by the authors. As in our system no other foreign material has been added to Lecithin/water system during the reduction process, we conclude that the lecithin itself acts as a reductant.
A further study in our case needs to be done to find out the exact mechanism of formation of nanoparticles. 3.2. Gold nanoparticle formation during sonication After the formation of nanoparticles in lamellar phases, we attempted to prepare them in lecithin vesicles by sonicating the dispersion of lecithin and HAuCl4 . Two types of sonicators were used; bath type sonicator and a sonicator with a probe. Ultrasonication has been used to prepare vesicles in the literature. 3.2.1. Using a bath type sonicator Fig. 3a shows the UV–vis spectra of 3 mM HAuCl4 with different lecithin concentrations after sonication with a bath type sonicator. Peaks between 540 and 554 indicate the presence of gold nanoparticles prepared in the system. It takes 45–60 min of sonication time for the appearance of pink color which is quite low as compared to the time of reduction in lamellar phases without sonication. This can be due to the cavitation and formation of radicals by ultrasonic irradiation which could have further accelerated the reduction process. Sonochemistry is used to enhance or alter the chemical reactions. There have been reports about the formation of gold nanoparticles in an ultrasound field, with chemicals such as alcohols and surfactants used to enhance the radical formation [41–45]. Dynamic light scattering show broad distribution of particles ranging from 40 to 600 nm size (Fig. 3b). TEM measurements were also done to gain a further insight into the distribution pro-
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Fig. 3. (a) UV–vis spectra of gold nanoparticles in lecithin/water system after sonication with bath type sonicator. (b) Particle size distribution by DLS in the system. (c) TEM micrograph of gold nanoparticles prepared in the system, scale bar–1 m.
file of nanoparticles. TEM image confirms the polydispersity of the particles sitting on lecithin structures (Fig. 3c). Particles are polydisperse and aggregating with each other. Most of the particles are spherical in shape but some non spherical shapes and triangular shapes are also observed. DLS results are in good agreement with the TEM results as TEM also shows the bigger particle size up to 50–200 nm gold nanoparticles sitting on up to 600 nm wide lecithin structures. 3.2.2. Using a sonicator with a probe Different concentrations of lecithin and HAuCl4 were tried systematically with sonicator with probe. Samples become pink after 45 seconds to few minutes time of sonication depending on the concentration of lecithin. Time for reduction process is comparatively smaller in comparison to reduction time by using Soniclean. This result is also in good agreement with the literature recognizing that smaller is the intensity of the ultrasound; the slower is the rate of reduction for gold (III) in presence of 2-propanol [43]. Fig. 4a shows the UV–vis spectra of 5 mM HAuCl4 with different lecithin
concentrations (1–5 mM). A Plasmon resonance band is observed for all lecithin concentrations between 548 and 561 nm which indicates the presence of large nanoparticles or aggregation of particles. The spectra also indicate that at all the lecithin concentrations a good amount of unreacted HAuCl4 is left (band at 350 nm) in the dispersion which does not diminish with time (data not shown). We do not see any of the peaks around 350 nm in all lecithin concentrations HAuCl4 is reduced to 3 mM (Fig. 4b). This shows that the reduction process is complete for this concentration of HAuCl4 with all the lecithin concentrations tried (2, 3 and 5 mM). 3 mM lecithin + 3 mM HAuCl4 was chosen for further experiments to form equimolar dispersions. DLS results (Fig. 4c) show a bimodal distribution of particles in 60–400 nm size range. TEM images were taken to gain further insight into the size distribution of particles. From TEM images (Fig. 4d) it is clear that some 10 nm gold nanoparticles are attached to lecithin vesicles of around 100–150 nm. But most of the particles were sitting outside aggregating with each other. The particles outside the vesicles are 50 nm in size. And the aggregates are
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Fig. 4. UV–vis spectra of (a) 5.0 mM (b) 3.0 HAuCl4 with different [lecithin] (c) DLS particle size profile for 3 mM EPC + 3 mM HAuCl4 (d) TEM image showing the 10 nm particles attached to lecithin vesiclesand, scale bar—200 nm (e) UV–vis spectra of 1 mM aqueous HAuCl4 after 8 min of sonication with a sonicator with probe.
around 200 nm in size. A comparatively small size of nanoparticles by this method of sonication may be due to the increasing rate of reduction of HAuCl4 which has also been demonstrated by Okitsu et al. [43]. Sonication of 1 mM aqueous HAuCl4 up to 8 min with the sonicator with probe at 2 min time intervals does not show
any change in initial pale color of HAuCl4. There is a blue shift observed in the UV–vis absorption peak from 351 nm to 343 nm and a decrease in absorption intensity from 2.25 to 1.6 (Fig. 4e) but no peak is observed between 400 and 800 nm indicating that no gold nanoparticles have been formed in the solution on sonication.
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acted as a reductant. Sonication of lecithin/aqHAuCl4 dispersions reduces the time of reduction. Nanoparticles are found to be attached to the lamellar structures and vesicles formed by lecithin. Gold nanoparticle containing vesicles can be used as biological markers for vesicle/liposome uptake in cells. SDS was found to be a good stabilizer. SDS stabilized particles are well dispersed and are stable over six months. This work is a step towards using biologically friendly approach for the nanoparticle formation. Acknowledgements This work was financially supported by Australian Research Council under the scheme Nano and Biomaterials Centre, Ian Wark Research Institute, University of South Australia. I also thank Prof Roger Horn for his guidance and helping me initiate this work. We are thankful to Adelaide Microscopy for providing Transmission Electron Microscopy facility for imaging. References
Fig. 5. (a) UV–vis spectra of lecithin/HAuCl4 in presence and absence of SDS after six months. (b) TEM image of gold nanoparticles formed in presence of SDS, 2 h after the sample preparation, scale bar—100 nm.
When SDS was added to the lecithin/HAuCl4 dispersion before sonication, the final dispersions were stable up to six months and did not show a change in color or the precipitation. Dispersions without SDS show black precipitate after a week time. The UV–vis spectra of without SDS and with SDS spectra is shown in Fig. 5a. The sample without SDS does not show any peak in around 500–600 nm range indicating that no nanoparticles are present in the dispersion while sample with SDS still have the absorption peak at 544 nm. TEM observations show that the nanoparticles formed in SDS/lecithin/HAuCl4 system are much smaller in size (5–10 nm) (Fig. 5b) and were well dispersed. No aggregates were present in the system. SDS is known to interact with lecithin bilayers by partitioning between the monomers in the bilayer and imparting a net negative charge to the system [46]. The negatively charged sulfate head groups may be responsible for the stabilization of nanoparticles by electrostatic charge repulsion. Wang et al. [47] has reported that SDS acts as a reductant when forming gold nanoparticles in lecithin/surfactant system. This might be the reason for observing very small nanoparticles when SDS is present with lecithin. SDS might be enhancing the reduction power and leads to small nanoparticle formation. 4. Conclusion In conclusion, for the first time gold nanoparticles have been formed in lamellar phases formed by lecithin where lecithin itself
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