Polyethylene glycol functionalized gold nanoparticles: the influence of capping density on stability in various media
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- Manson, J., Kumar, D., Meenan, B.J. et al. Gold Bull (2011) 44: 99. doi:10.1007/s13404-011-0015-8
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Thiol-terminated polyethylene glycol (PEG) is commonly used to functionalize the surface of gold nanoparticles (AuNPs) in order to improve their in vivo stability and to avoid uptake by the reticular endothelial system. Although it has been reported that AuNPs functionalized with tethered PEG are stable in biological media, the influence of chain density remains unclear. This study investigates the influence of PEG capping density on the stability of washed and dried AuNPs in: water, phosphate-buffered saline solution (PBS), phosphate-buffered saline solution containing bovine serum albumin (PBS/BSA), and dichloromethane (DCM). PEG coating had a dramatic effect on stability enabling stable suspensions to be produced in all the media studied. A linear relationship was observed between capping density and stability in water and DCM with a somewhat lower stability observed in PBS and PBS/BSA. A maximum PEG loading level of ∼14 wt.% was achieved, equivalent to a PEG surface density of ∼1.13 chains/nm2.
KeywordsGold nanoparticle Drying Redispersion in media Thiolated poly(ethylene glycol)
Metallic nanoparticles are the subject of a concerted international research effort as novel platforms for the target-specific delivery of therapeutic agents. Gold nanoparticles (AuNPs) in particular are an excellent candidate for drug delivery vehicles due to their unique physical and chemical properties, enabling the transport and subsequent release of therapeutic payloads such as drugs or genetic materials to specific tissue sites. Their advantages include ease of synthesis in a range of monodisperse sizes from 1 to 150 nm, an essentially inert and non-toxic nature and an ability to be readily functionalized with targeting ligands and drugs. Payloads can be subsequently released at the required site by way of their photo-physical properties or by intercellular glutathione levels [1, 2, 3]. Active and passive targeting to a disease site such as a tumor can be achieved by attaching ligands such as tumor necrosis factor-α, or proteins onto the nanoparticles, or through size and charge effects [3, 4]. A wide range of molecules can be tethered onto the AuNP surface by means of a thiol (SH) group. AuNPs are also readily imaged which enables direct tracking of their fate within cells.
Gold nanoparticles can be synthesized in a range of shapes and sizes such as rods, triangles, cubes and wires [5, 6, 7, 8, 9]. They are generally synthesized chemically using sodium borohydride, sodium citrate or, more recently, hydroquinone reduction of a chloroauric acid (HAuCl4) solution [10, 11, 12, 13, 14, 15, 16]. The ratio of gold salt to reducing agent, type of reducing agent, and temperature play a critical role in determining the size and shape of the nanoparticles [17, 18]. In the case of sodium citrate reduction, the citrate acts as a loosely bound capping agent stabilizing the particles. Tetradecane, octadecane or dodecane thiols are often used as capping agents to stabilize AuNPs produced via sodium borohydride reduction [19, 20]. The biological response can be altered using capping agents such as polyethylene glycol (PEG) also referred to as polyethylene oxide (PEO) [20, 21], mercaptosuccinic acid, various proteins  or other biomolecules . Such functionalization has led to an extensive research effort investigating AuNPs as carriers for a range of biomolecules and drugs [22, 23, 24, 25, 26, 27].
While it is known that functionalizing AuNPs with PEG increases stability, both in vivo and in vitro, the influence of capping density and the effect that washing and drying has on resuspension in a range of media including organic solvents has not been previously reported. This study reveals the effect of PEG surface density on the aggregation behaviour of AuNPs in: water, an organic solvent dichloromethane (DCM) and the model biological media, phosphate-buffered saline (PBS) and PBS with bovine serum albumin (BSA).
Materials and methods
Prior to synthesis, all the glassware was washed with aqua regia, rinsed with distilled water and then dried overnight at 80–100°C. Magnetic flees were first sonicated in ethanol and then in de-ionized (DI) water for 15 min and dried before use. Chloroauric acid (HAuCl4.3H2O), PEG (5,000 Mw) and sodium citrate were obtained from Sigma Aldrich UK. The 0.01 wt.% chlorauric acid and 1 wt.% sodium citrate solutions were prepared using DI water, PBS, DCM (99.9%) and PBS with 0.05 wt% BSA, which were also obtained from Sigma Aldrich and used as received.
Synthesis and washing of gold nanoparticles
Chloroauric acid solution (200 ml of 0.01 wt.%) was heated to a rolling boil and refluxed in a 500-ml-round-bottom flask using a temperature-controlled hot plate with continuous stirring . A 4.5-ml aliquot of 1 wt.% sodium citrate solution was then added to the boiling chloroauric acid solution, and the heating was continued under reflux for 15 min to enable complete reaction. The solution was then allowed to cool to room temperature with continuous stirring yielding citrate-capped AuNPs. In order to produce PEG-capped AuNPs, various concentrations (3.6, 8.4, 16.8 and 25.2 μg per ml of as synthesized AuNP suspension) of 5,000 Mw PEG were added to the ‘as synthesized’ AuNP solutions at room temperature. After the required amount of PEG was added, the solution was stirred at room temperature for 2 h to allow for complete exchange of the citrate molecules with PEG. The AuNP solutions were then centrifuged using a Contifuge 17RS, Heraeus SEPATECH at 10,000 rpm for 90 min in 10 ml batches . Of the supernatant, 9.9 ml was then decanted, leaving the AuNP pellet at the bottom of the centrifuge tube. The volume was then made back up to 10 ml by adding 9.9 ml of DI water and agitated. This centrifugal washing process was repeated again to remove any unattached PEG or other reactants.
Dispersion of AuNPs in relevant media
After washing, both the citrate- and PEG-capped AuNPs were dried at 60°C for 3 days. The dried samples were sonicated for 15 min in a sonic bath and then redispersed in the required media at the original ‘as synthesized’ concentrations and further sonicated for 30 min. The media investigated were deionised H2O, PBS (137 mM NaCl, 2.7 mM KCl, 10 mM PBS at pH 7.4), PBS containing 0.05 wt.% BSA and DCM (99.9%). Dispersions of all five AuNP types investigated (one capped with citrate and four densities of PEG) were then prepared in each of the four media of interest.
UV–Visible spectra were recorded using a Perkin Elmer Lambda 11 in the range 400–600 nm at 0.1 nm increments, using 1 ml of sample solution in a 1.4-ml glass spectrophotometer cuvette (Sigma Aldrich). The UV–vis spectra of the ‘as synthesized’ sample of each type before washing, drying and redisperion were used as the reference spectra for the subsequent calculation of relative absorbance. After redispersion, UV–vis spectra were taken at five time points over a 24-h period, with three replicate spectra recorded.
Citrate and PEG (16.8 μg/ml)-capped AuNP samples were dried onto carbon-coated copper grids and imaged using a JEOL 2100 Transmission Electron Microscope at 200 kV. For SEM, gold nanoparticle solutions were dried onto titanium metal stubs and imaged at 20 kV using a FEI Helios NanoLab.
DLS was conducted using Nano ZetaSizer ZS Series. For size determination, 3 ml of each sample was measured in a disposable cuvette. For Zeta potential, samples were analysed using a standard zeta cuvette. The samples were analysed three times at 25°C.
Infrared spectra were obtained using a BIORAD FTS 3000MX Excalibur series spectrometer fitted with a DRIFTS accessory. Fifty millilitres of each sample type was centrifuged, and the pellets dried in a Teflon-coated dish at room temperature. The dried samples were then mixed with KBr to produce a fine powder which was pressed into discs. All spectra were recorded at a resolution of 4 cm−1 over a wave number range of 400–4,000 cm−1.
TGA was carried out using a TGA Q600 (TA Instruments) between 24°C and 800°C at 10°C/min under a nitrogen atmosphere flowing at 40 ml/min. Each ∼5-mg sample was prepared by drying the washed AuNP solutions. The amount of PEG attachment was calculated as the percentage weight loss occurring between 302°C and 450°C, which corresponds to the degradation of the PEG. The 1.8% weight loss recorded for the citrate-capped AuNPs over this temperature range was taken into account when calculating the levels of PEG attachment.
Results and discussion
Characterization of the AuNPs
UV–vis absorbance spectra for citrate-capped AuNPs displayed a characteristic peak at 519.4 nm compared to 519.5–520.6 nm for PEG-capped AuNPs. This peak relates to the surface plasmon, and an increase in intensity was observed as PEG functionalization density increased. This effect has been reported previously .
Effect of thiolated PEG on the characteristics of AuNPs
Amount of PEG (μg/ml)
PEG weight% from TGA
Diameter (nm) from TEM
DLS volume average diameter (nm)
Polydispersity index (PDI)
0 (citrate capped)
DLS measurement found that the surface charge (ζ potential) for the citrate-capped AuNP was −39.5 mV compared to −13.9 mV for the highest PEG loading, a result of steric shielding.
Colloid stability tests
Stable dispersions of washed and dried PEG coated gold nanoparticles were achieved in water, PBS, PBS containing BSA and DCM. PEG-coated AuNPs were also found to be stable at salt concentrations of 0.15–1 M, whereas citrate-capped AuNPs aggregated immediately under such conditions. In the case of redispersion in DCM and H2O, there is an approximately linear relationship between the amount of PEG attachment and stability. Lower levels of stability are observed in highly PEG-functionalized AuNPs after redispersion in PBS and PBS/BSA compared to DCM and H2O due to the effect of NaCl. It was observed that saturation of the AuNP surface with tethered PEG occurs at a PEG loading level of ∼14 wt.%, equivalent to ∼1.13 chains/nm2, and that the PEG chains adopt an elongated random coil configuration. This is well below the maximum theoretical packing density of ∼5 chains/nm2. Significant levels of stability enhancement were achieved at lower loading levels allowing for the co-functionalization of AuNPs with drugs or targeting groups for applications in assays and drug carrier systems.
The work was funded by the Department of Employment and Learning (DEL) as part of the Collaborative Centre for Functional Biomaterials (Project UU005). The authors wish to acknowledge Thomas Dooher who conducted the TEM analysis under the Science Foundation Ireland funded National Access Program (NAP Project 242). DLS measurements were conducted with the assistance of Yuri Rochev in NUI Galway.
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