Encapsulation of gold nanoparticles into self-assembling protein nanoparticles
© Yang and Burkhard; licensee BioMed Central Ltd. 2012
Received: 17 July 2012
Accepted: 23 October 2012
Published: 31 October 2012
Gold nanoparticles are useful tools for biological applications due to their attractive physical and chemical properties. Their applications can be further expanded when they are functionalized with biological molecules. The biological molecules not only provide the interfaces for interactions between nanoparticles and biological environment, but also contribute their biological functions to the nanoparticles. Therefore, we used self-assembling protein nanoparticles (SAPNs) to encapsulate gold nanoparticles. The protein nanoparticles are formed upon self-assembly of a protein chain that is composed of a pentameric coiled-coil domain at the N-terminus and trimeric coiled-coil domain at the C-terminus. The self-assembling protein nanoparticles form a central cavity of about 10 nm in size, which is ideal for the encapsulation of gold nanoparticles with similar sizes.
We have used SAPNs to encapsulate several commercially available gold nanoparticles. The hydrodynamic size and the surface coating of gold nanoparticles are two important factors influencing successful encapsulation by the SAPNs. Gold nanoparticles with a hydrodynamic size of less than 15 nm can successfully be encapsulated. Gold nanoparticles with citrate coating appear to have stronger interactions with the proteins, which can interfere with the formation of regular protein nanoparticles. Upon encapsulation gold nanoparticles with polymer coating interfere less strongly with the ability of the SAPNs to assemble into nanoparticles. Although the central cavity of the SAPNs carries an overall charge, the electrostatic interaction appears to be less critical for the efficient encapsulation of gold nanoparticles into the protein nanoparticles.
The SAPNs can be used to encapsulate gold nanoparticles. The SAPNs can be further functionalized by engineering functional peptides or proteins to either their N- or C-termini. Therefore encapsulation of gold nanoparticles into SAPNs can provide a useful platform to generate a multifunctional biodevices.
Due to their unique size-dependent properties, inorganic nanoparticles and their applications in the life sciences have been a topic of dramatically increasing interest over the last several years [1, 2]. Gold nanoparticles (GNPs) are the most commonly used inorganic nanoparticles for biological applications [2, 3], because of their attractive physical and chemical properties . GNPs have been mainly used for labeling and visualizing applications as they can strongly absorb and scatter visible light. This is because of their surface plasmon resonance . GNPs are often used as contrast agents for transmission electron microscopy and X-ray imaging because of their ability to scatter electrons and X-rays efficiently . GNPs generate heat when they absorb light, which enables their potential in photo-thermal therapeutic applications [7, 8]. GNPs are also promising as drug and gene delivery vehicles . For example, they have been used as nano-bullets for gene guns . In addition, GNPs are inert and relatively biocompatible . They can easily be synthesized and conjugated with biological molecules in a straightforward manner .
The uses of GNPs in biological applications have demonstrated the importance of the conjugation of GNPs with biological molecules [12, 13]. The biological molecules not only provide the interfaces for interactions between nanoparticles and biological environment, but also contribute their biological functions, such as tumor cell targeting , cell penetration , antibody-antigen recognition , and many others. Furthermore, biological molecules, such as DNA, can serve as platform for assembly and organization of GNPs [1, 17]. Due to their well-defined surface chemistry , GNPs can be modified and functionalized with a wide variety of biological molecules, such as peptides , proteins , oligonucleotides [1, 17], carbohydrates , and even whole viral capsids [21–26]. Although several publications reported that GNPs with a defined number of attached molecules per particle could be obtained using sorting techniques [27, 28], no protocols for controlling the exact number of attached molecules per gold particles have yet been established [29, 30]. The viral capsids provide an ordered and controlled platform for conjugation with GNPs [31, 32]. However, the relatively stringent structure of the viral capsids limits their further functionalization via fusion with functional peptides .
Encapsulation of citrate-coated GNPs by P6c SAPNs
GNPs of the three different sizes of 5, 10 and 15 nm with citrate surface coating were used to test the encapsulation capability of the P6c SAPNs. Negatively stained TEM images (Additional file 1: Figure S1a-c) revealed that the thickness of the organic layer around the GNPs was approximately 1 nm manifested by the uranyl acetate staining as a bright ring around the GNPs. The 5 nm GNPs had an average hydrodynamic size of 7.2 nm, while the 10 nm GNPs had an average hydrodynamic size of 14.8 nm (Additional file 1: Figure S1d). The difference between the acclaimed GNP sizes and measured hydrodynamic sizes is attributed to the thickness of the citrate layer and the adsorbed water layer.
In order to avoid aggregation of the 5 nm GNPs, a refolding buffer containing only 10 mM NaCl was used. The 5 nm GNPs were shown to be stable in the refolding buffer containing 10 mM NaCl. However, the protein shells around the encapsulated GNPs became irregular (Figure 2c), which implies that the integrity of the P6c SAPNs was disturbed by the stronger interaction between the protein and the GNPs in buffers with lower ionic strength.
Figure 2d shows the light scattering results of the encapsulation samples in buffers with three different salt concentrations. DLS results show that the average hydrodynamic size of the sample prepared in 150 mM NaCl buffer was larger than those prepared in 75 and 10 mM NaCl buffers. As the samples were mixtures of free GNPs, empty P6c SAPNs, and the GNPs encapsulated by P6c SAPNs, light scattering will yield the average hydrodynamic sizes of all the three kinds of particles. The larger average hydrodynamic size in 150 mM NaCl buffer might be due to the aggregation of the GNPs and/or the larger P6c SAPNs .
Figure 3c shows the effect of using lower protein concentration during encapsulation. Although the shapes of the protein shells was more spherical and regular, the thickness of the protein shells around the encapsulated GNPs in Figure 3c was approximately 4.5 nm, which was much thinner than those shown in Figures 3a and b. The difference in the thickness of the protein shells could be due either to insufficient amount of protein for the formation of complete protein shells or to the collapse of the protein on the surface of GNPs.
DLS results (Figure 3d) show that the average hydrodynamic sizes of the encapsulated samples slightly decreased with decreasing ratio of protein to GNPs. When an excess of P6c was used (Figure 3d, black and red line), the average hydrodynamic sizes are actually the average of the empty SAPNs and encapsulated GNPs. TEM images suggested (Figure 3c) that when insufficient amount of protein was used, the protein shells around the encapsulated GNPs became thinner. Therefore the average hydrodynamic size of the encapsulation sample with 0.025 mg/ml P6c and ~4.7×10-3 nmol/ml GNPs became smaller. There was a peak around 11 nm (Figure 3d, blue line) present in the encapsulation sample with an excess of GNPs, which is close to the hydrodynamic size of free GNPs. The smaller peak suggests that free GNPs were present in the solution due to an excess of GNPs.
Encapsulation of PEG-coated GNPs by P6c SAPNs
PEG-coated GNPs of two gold core sizes of 5 and 10 nm were purchased from Nanocs and used for encapsulation. The TEM images of the PEG-coated GNPs with 1% uranyl acetate staining are shown in Additional file 2: Figure S2. The light aureole around the black dots (gold cores) proves the presence of PEG layers coating on the surface of the gold core. Although the shapes of the polymer layers were not regular, the whole particle sizes were considerably larger than the core sizes of the GNPs. Additional file 2: Figure S2c shows the dynamic light scattering profiles of the two PEG-coated GNPs. The hydrodynamic size was 18.6 nm and 21.6 nm for the PEG-coated GNPs with the gold core sizes of 5 nm and 10 nm, respectively. The large hydrodynamic sizes are mainly attributed to the PEG layers and a little to the adsorbed water layer.
Encapsulation of polymer-coated GNPs with carboxyl or amine surface functional groups by P6c SAPNs
The P6c SAPNs have a positively charged central cavity due to its arginine residues (R61 in P6c) as shown in a computer model (Figure 1c). Electrostatic interactions between the central cavity and the surface charges from GNPs might exist. Therefore GNPs with different surface charges were tested for encapsulation. The polymer-coated GNPs with carboxyl surface functional groups were purchased from Ocean NanoTech, Inc. The GNPs were coated with amphiphilic polymer bearing carboxyl functional groups. The size of the inorganic core was about 5 nm. The thickness of the organic layers was about 4 nm, as shown in the negatively stained TEM images (Additional file 3: Figure S3a). Dynamic light scattering (Additional file 3: Figure S3c) showed that the hydrodynamic size of the GNPs was 15 nm.
The polymer-coated GNPs with amine surface functional groups were also purchased from Ocean NanoTech, Inc. The GNPs were coated with amphiphilic polymer and PEG coating. Their surface functional group is amine. The size of the inorganic core was about 6 nm. The thickness of the organic layers was about 6 nm (Additional file 3: Figure S3b). Dynamic light scattering (Additional file 3: Figure S3c) showed that the hydrodynamic size of the GNPs was 19.6 nm.
Encapsulation of GNPs by P11c SAPNs
The P6c protein has an arginine residue (R61 in P6c, Figure 1a) next to its two-glycine linker, which results in a positively charged central cavity within the P6c SAPN after refolding. When a GNP is encapsulated into the P6c SAPN, the residues around the two-glycine linker are probably in contact with the surface of the GNP (Figure 1e). The environments in the central cavity might affect the encapsulation of GNPs. In order to test the effect of the possible electrostatic interactions between the cavity and the encapsulated GNP, the arginine residue was mutated to a glutamic acid residue (E61 in P11c, Figure 1b). Therefore, P11c SAPNs presumably have an overall negatively charged central cavity.
Our previous work  suggested that the majority of the P6c SAPNs are T = 3-like icosahedral particles. Therefore, there might be a size limit for GNPs to be encapsulated. The encapsulation results for the citrate-coated GNPs of three different sizes show that GNPs with hydrodynamic size smaller than about 15 nm (citrate-coated GNPs with 5 nm and 10 nm core sizes) can be encapsulated, although there are aggregation problems with the GNPs in the buffers containing high salt concentrations. The aggregation problem is likely due to increased hydrophobic interactions driven by higher salt concentration. Similarly, the polymer-coated GNPs with carboxyl surface functional groups can be successfully encapsulated into SAPNs, as this type of GNPs also has a hydrodynamic size of 15 nm. On the contrary, the failures in encapsulation of the PEG-coated GNPs (Figure 5) and the polymer-coated GNPs with amine functional groups (Figure 6b) can be attributed to their large hydrodynamic sizes; the three types of GNPs have average hydrodynamic sizes ranging from 18.6 to 21.6 nm.
The success of encapsulation of GNPs into SAPNs allows further functionalization by fusing functional peptides to the nanoparticle-forming protein chains. The SAPNs can present the functional peptides on its surface in an ordered and repetitive ways, when the functional peptides were fused to the termini of the protein chains.
Protein Expression and Purification
The modified pPEP-T vector  was kindly provided by the M. E. Müller Institute, Basel, Switzerland. The genes encoding P6c and P11c protein were placed between NcoI and EcoRI restriction sites. The plasmids were then transformed into the Escherichia Coli strain BL21(DE3)pLysS expression cells (Novagen, Madison, WI, USA). The bacteria were incubated at 37°C in Luria Broth (LB) medium in the presence of 200 mg/ml ampicillin and 30 mg/ml chloramphenicol. Expression was induced by adding 1 mM isopropyl β-D-thiogalactopyranoside. After 3 hours of expression, the bacteria were collected by centrifugation at 4000 g for 15 min. The bacterial pellet was resuspended and lysed in a lysis buffer (9 M urea, 100 mM NaH2PO4, 10 mM Tris, 10 mM β-mercaptoethanol, and pH 8.0) by sonication. The cell debris was removed by centrifugation at 305000 g for 45 min. The supernatant was then incubated with Ni-NTA Agarose beads (Qiagen, Valencia, CA, USA) overnight and then loaded into a column. The protein contaminants were removed by washing the column sequentially with pH buffers 6.3, 5.9 and 5.0, which contain 9 M urea, 100 mM NaH2PO4, 20 mM sodium citrate, 10 mM imidazole and 10 mM β-mercaptoethanol. The P6c proteins were then eluted by the elution buffer containing 9 M urea, 100 mM NaH2PO4, 10 mM Tris, 500 mM imidazole, 10 mM β-mercaptoethanol and pH 8.0. The purity of the P6c proteins was verified by sodium dodecyl sulfate polyacrylamide gel electrophoresis.
Protein refolding procedure
The P6c protein was first denatured in a urea-containing buffer (9 M urea, 20 mM HEPES, 150 mM NaCl, 5% Glycerol, pH 7.5), and then concentrated to 1 mg/ml. The protein was refolded by adding it drop wise to the refolding buffer (20 mM HEPES, 150 mM NaCl, 5% Glycerol, pH 7.5), until the protein concentration reached a concentration of 0.05 mg/ml. The samples were then dialyzed overnight in the refolding buffer to remove the remaining urea.
The citrated-coated gold nanoparticles of the three gold core sizes of 5, 10 and 15 nm, respectively, were purchased from Nanocs Inc., New York, USA. The stock concentrations of the 5, 10 and 15 nm citrate-coated gold nanoparticles were approximately 0.083, 0.0095 and 0.0023 nmole/ml, respectively.
The PEG-coated gold nanoparticles with two gold core sizes of 5 and 10 nm were purchased from Nanocs Inc., New York, USA. The stock concentrations of the 5 and 10 nm PEG-coated gold nanoparticles were approximately 0.083 and 0.0095 nmole/ml, respectively.
The polymer-coated gold nanoparticles with carboxyl or amine surface functional groups were purchased from Ocean NanoTech Inc., AR, USA. Both polymer-coated gold nanoparticle had 5 nm gold cores. The gold nanoparticles with carboxylic acid groups were coated with dodecanethiol and a monolayer of amphiphilic polymer. The zeta potential of these gold nanoparticles is −30 mV to −50 mV (provided by the supplier). The concentration of the gold nanoparticles with carboxylic acid groups was about 5 mg/ml, which gives a concentration of approximately 6.7 nmole/ml. The gold nanoparticles with amine groups were coated with amphiphilic polymer and PEG. The zeta potential of these gold nanoparticles is −10 mV to +10 mV (provided by the supplier). The concentration of the gold nanoparticles with amine groups was about 1 mg/ml, which gives a concentration of approximately 1.3 nmole/ml.
Encapsulation of citrate-coated gold nanoparticles by P6c SAPNs
The P6c proteins were first denatured in the denaturing buffer (9 M urea, 20 mM HEPES pH 7.5, 150 mM NaCl, 5% glycerol). Then the denatured proteins were concentrated to about 1 mg/ml using the Amicon centrifuge filter (5000 MWCO, Millipore, MA, USA).
The 5 nm citrate-coated nanoparticles were diluted in the refolding buffer to a concentration of approximately 0.0047 nmole/ml. Three different refolding buffers were used for dilution of gold nanoparticles. The refolding buffers were composed of 20 mM HEPES pH 7.5, 5% glycerol, and 10, 75, and 150 mM NaCl, respectively. Then, the denatured P6c protein solution (~1 mg/ml) was added drop wise to the GNP-refolding buffer until the protein concentration reached a concentration of 0.05 mg/ml (3.96 nmole/ml) in the final protein-GNP solution. The protein-GNP solution was then dialyzed overnight against the buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 5% glycerol) to remove the remaining urea.
The encapsulation procedures for the 10 nm citrate-coated gold nanoparticles were similar to that for the 5 nm citrate-coated gold nanoparticles. The 10 nm citrate-coated gold nanoparticles were first diluted in the refolding buffer containing 10 mM HEPES pH 7.5, 75 mM NaCl, and 5% glycerol. Then, the denatured P6c protein solution (~1 mg/ml) was added drop wise to the GNP-refolding buffer until the protein concentration reached a concentration of 0.05 mg/ml (3.96 nmole/ml) in the final protein-GNP solution. The protein-GNP solution was then dialyzed overnight against the buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 5% glycerol) to remove the remaining urea. Three different molar ratios of gold nanoparticles to proteins were used for the encapsulation: (a) The P6c protein concentration was 0.05 mg/ml (approximately 4 nmol/ml). The 10 nm GNPs concentration was 4.7×10-4 nmol/ml. (b) The P6c protein concentration was 0.05 mg/ml. The 10 nm GNPs concentration was 4.7×10-3 nmol/ml. (c) The P6c protein concentration was 0.025 mg/ml. The 10 nm GNPs concentration was 4.7×10-3 nmol/ml.
The encapsulation procedures for the 15 nm citrate-coated gold nanoparticles were also similar to that for the 5 nm citrate-coated gold nanoparticles. The 15 nm citrate-coated gold nanoparticles were first dissolved in the refolding buffer containing 10 mM HEPES pH 7.5, 75 mM NaCl, and 5% glycerol. Then, the denatured P6c protein solution (~1 mg/ml) was added drop wise to the GNP-refolding buffer, until the protein concentration reached a concentration of 0.05 mg/ml (3.96 nmole/ml) in the final protein-GNP solution. The protein-GNP solution was then dialyzed overnight against the buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 5% glycerol) to remove the remaining urea. Two different molar ratios of gold nanoparticles to proteins were used for the encapsulation: (a) The P6c protein concentration was 0.05 mg/ml (approximately 4 nmol/ml). The 10 nm GNPs concentration was approximately 2.3×10-3 nmol/ml. (b) The P6c protein concentration was 0.025 mg/ml. The 10 nm GNPs concentration was approximately 2.3×10-3 nmol/ml.
Encapsulation of PEG-coated gold nanoparticles by P6c SAPNs
The PEG-coated gold nanoparticles were first diluted in the refolding buffer containing 10 mM HEPES pH 7.5, 75 mM NaCl, and 5% glycerol to a concentration of approximately 0.0047 nmole/ml. Then, the denatured P6c protein solution (~1 mg/ml) was added drop wise to the GNP-refolding buffer until the protein concentration reached 0.05 mg/ml (3.96 nmole/ml) in the final protein-GNP solution. The protein-GNP solution was then dialyzed overnight against the buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 5% glycerol) to remove the remaining urea.
Encapsulation of polymer-coated gold nanoparticles with carboxyl or amine surface functional groups by P6c SAPNs
The polymer-coated gold nanoparticles were diluted to approximately 0.01 nmole/ml in the refolding buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 5% glycerol). Then, the denatured P6c protein solution (~1 mg/ml) was added drop wise to the GNP-refolding buffer, until the protein concentration reached a concentration of 0.05 mg/ml (3.96 nmole/ml) in the final protein-GNP solution. The protein-GNP solution was then dialyzed overnight against the buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 5% glycerol) to remove the remaining urea.
Encapsulation of gold nanoparticles by P11c SAPNs
The encapsulation procedures for gold nanoparticles by the P11c SAPNs were similar to the procedures for the P6c SAPNs. P11c SAPNs were used for the encapsulation of all three kinds of gold nanoparticles mentioned above. The concentration of the P11c proteins was kept as 0.05 mg/ml for all the encapsulation samples. In the final encapsulation samples, approximately 0.0047 nmole/ml of the citrate-coated gold nanoparticles were used. The concentrations of the polymer-coated gold nanoparticles were also approximately 0.01 nmole/ml in their encapsulation samples.
Dynamic light scattering
The hydrodynamic diameter was determined with a Malvern Zetasizer Nano S equipped with a 633 nm laser. Hellma Quartz cuvettes with a 3 mm light path and centre 9.65 mm were used (Cat. No. 105.251.005-QS). The measurements were performed at 20°C using 80 μl samples. All the samples were filtered once using 0.1 μm Millex-VV filter (Millipore, MA, USA) before measurement. The volume-average hydrodynamic sizes were reported by the Malvern DTS software, version 6.01.
Transmission Electron Microscopy
A drop of 5 μl sample was placed on a 400 mesh copper grid coated with Formvar/carbon film (Electron Microscopy Sciences, PA, USA) for 1 min. The grid was washed sequentially by three drops of 5 μl distilled water. Then the sample was negatively stained with a drop of 5 μl 1% uranyl acetate (SPI Supplies, PA, USA) for 1 min. Excess stain solution was removed by Whatman filter paper, before the grid was slowly dried at room temperature. Electron micrographs were taken with an FEI Tecnai T12 transmission electron microscope at an accelerating voltage of 80 kV.
The TEM images were first inspected with Photoshop CS4 (Adobe, San Jose, CA). The particles were selected and filled manually using the selection tools in Photoshop CS4, omitting the very small particles or background (area less than 50 nm2) and large aggregates (area larger than 5000 nm2). Image analysis was then performed with the public domain software ImageJ . Then, the Feret diameter obtained by ImageJ was used to describe the size of the particles.
Support by the UConn Research Foundation for this work is gratefully acknowledged.
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