Size-dependent endocytosis of gold nanoparticles studied by three-dimensional mapping of plasmonic scattering images
© Wang et al; licensee BioMed Central Ltd. 2010
Received: 12 August 2010
Accepted: 20 December 2010
Published: 20 December 2010
Understanding the endocytosis process of gold nanoparticles (AuNPs) is important for the drug delivery and photodynamic therapy applications. The endocytosis in living cells is usually studied by fluorescent microscopy. The fluorescent labeling suffers from photobleaching. Besides, quantitative estimation of the cellular uptake is not easy. In this paper, the size-dependent endocytosis of AuNPs was investigated by using plasmonic scattering images without any labeling.
The scattering images of AuNPs and the vesicles were mapped by using an optical sectioning microscopy with dark-field illumination. AuNPs have large optical scatterings at 550-600 nm wavelengths due to localized surface plasmon resonances. Using an enhanced contrast between yellow and blue CCD images, AuNPs can be well distinguished from cellular organelles. The tracking of AuNPs coated with aptamers for surface mucin glycoprotein shows that AuNPs attached to extracellular matrix and moved towards center of the cell. Most 75-nm-AuNPs moved to the top of cells, while many 45-nm-AuNPs entered cells through endocytosis and accumulated in endocytic vesicles. The amounts of cellular uptake decreased with the increase of particle size.
We quantitatively studied the endocytosis of AuNPs with different sizes in various cancer cells. The plasmonic scattering images confirm the size-dependent endocytosis of AuNPs. The 45-nm-AuNP is better for drug delivery due to its higher uptake rate. On the other hand, large AuNPs are immobilized on the cell membrane. They can be used to reconstruct the cell morphology.
Gold nanoparticles (AuNPs) are important nanomaterials in biomedicine where they can be used to achieve drug delivery and photodynamic therapy [1–6]. For biomedical applications, a thorough understanding of the mechanisms of AuNP cellular entry and exit is required. In previous studies, the endocytosis of AuNPs was found to be not only dependent on the surface coating but also on particle size [7–12]. In these studies, AuNPs were observed by using electron microscopy or fluorescent optical microscopy. Several drawbacks are inherent in these methods, since cells are not alive when they are observed by electron microscopy, and fluorescent labelling suffers from problems with photobleaching. Long-term observation is not attainable by the fluorescent technique. Additionally, quantitative estimation of AuNP numbers in cells is not easy using fluorescent signals. In this paper, we present a label-free method for long-term tracking of the movement of AuNPs with different sizes. A three-dimensional (3D) image process was developed to identify the distribution of AuNPs. Using the 3D distribution, the uptake efficiencies for different sizes of AuNPs were compared.
Materials and methods
Dark-field optical sectioning microscopy
Cells and incubation
In the experiments, we studied the interactions of AuNPs with two kinds of cancer cells, non-small lung cancer cells (CL1-0) and HeLa cells. Both cells have lateral dimensions of about 20 μm and heights about 8 μm. These cells were cultured on cleaned glass slides with thin square chambers to hold the medium. They were maintained in RPMI medium (GIBCO) supplemented with 10% FBS (fetal bovine serum) (GIBCO) at 37°C in a humidified atmosphere. The cells were cultured for 24 hours to ensure that they adhered well onto the glass slides. The left image in Figure 2 shows a dark-field CCD image for a HeLa cell without any AuNPs. This image indicates that no colorful spots are in the cell. The ring patterns are the micrometre vesicles.
Fabrication of AuNPs
The parameters for making different sizes of gold nanoparticles
Gold nanoparticle size (nm)
5 ml, 38.8 mM
0.5 ml, 38.8 mM
0.4 ml, 38.8 mM
330 μl, 37%
The zeta potentials on the surface of gold nanoparticles before and after the surface modification of ssDNA. The ssDNAs carry negative charges that make the surface potentials more negative after the modification.
Size of gold nanoparticles (nm)
Zeta potential before ssDNA conjugated (mV)
Zeta potential after ssDNA conjugated (mV)
13 ± 2.6
-13.99 ± 1.75
-27.27 ± 1.03
45 ± 3.1
-17.83 ± 1.31
-28.69 ± 1.07
70 ± 4.9
-19.14 ± 1.48
-24.66 ± 1.88
110 ± 5.1
-10.25 ± 0.80
-19.48 ± 0.97
Surface modification of AuNPs
The surface modification is important for the endocytosis of AuNPs. For AuNPs without ligands, they cannot interact with cells. The unmodified AuNPs will be on the glass substrate . To make AuNPs interacting with cancer cells, we modified the AuNP surface with single-stranded DNA (ssDNA) sequences. The DNA sequence was SH-(CH2)10-GCAGTTGATCCTTTGGATACCCTGG, where the thiol group enabled covalent bonding between the ssDNA and gold surface. This ssDNA segment was an aptamer for cellular surface mucin glycoprotein (MUC1) which is over-expressed in the extracellular matrix of cancer cells [19–22]. It is noted that in the preparation of AuNPs, the sodium citrate acted as a reducing agent. The negatively-charged citrate ions were adsorbed on the gold nanoparticles, introducing negative surface charges. It is known that DNAs also carry negative charges. If DNA aptamers were immobilised on the AuNP surface, it made the surface charge more negative. We used a zeta-potential analyzer (Brookhaven 90Plus) to measure the surface potential. The electrostatic potential on the particle surface is called the zeta potential. In the measurement, we applied unit field strength (1 Volt per metre) to the AuNP solution. The electrophoretic mobility of AuNPs was measured based on dynamic light scattering. There are theories that link electrophoretic mobility with zeta potential. The calculated zeta potentials for different size of AuNPs are listed in Table 2. It can be seen that after the interaction with DNA aptamers, the AuNPs increased negative surface charges. It confirmed that the DNA aptamers were immobilised on the AuNP surface.
Preparation of AuNP aggregates in submicron holes
When endocytosis of AuNPs occurs, the AuNPs are wrapped by the vesicles. The vesicle size are most in submicron scale and the AuNPs in the vesicle are in aggregated form. To find the relation between the scattering optical intensity and number of AuNPs in the vesicle. We prepared 500-nm-diameter holes in a transparent film to mimic the vesicles. The transparent film was coated on a glass substrate. The glass surface was modified with 4-mercaptobenzoic acid, sodium borohydride, hydrogen peroxide (27.5 wt% solution in water) and 3-aminopropyltriethoxysilane (APTES) in order to immobilize the AuNPs . The sample was dipped in the AuNP solution. After six hours of interaction time, we washed the sample and measured the scattering images in water. The measured sample was then dried and observed by the SEM to identify the number of AuNPs in each hole.
Additional file 2:The movie for 70-nm-AuNPs and a HeLa cell. The images were observed by using the dark-field microscope. The CCD exposure time was 100 ms, the interval between images was 5 minutes and the overall recording time was 1.5 hours. (MOV 8 MB)
Additional file 3:The movie for 45-nm-AuNPs and a CL1-0 cell. The images were observed by using the dark-field microscope. The CCD exposure time was 100 ms, the interval between images was 5 minutes and the overall recording time was 2 hours. (MOV 8 MB)
3D distribution of AuNPs
Quantitative calculation of the endocytosis
The reason for size-dependent endocytosis of AuNPs can be explained by the thermodynamic model of the many-NP-cell system [24–26] for receptor-mediated endocytosis [27, 28]. There are two kinds of competitive energy important for endocytosis of nanoparticles (NPs). One is the binding energy between ligands and receptors. This energy refers to the amount of ligand-receptor interaction and the diffusion kinetics for the recruitment of receptors to the binding site. The other is the thermodynamic driving force for wrapping. The thermodynamic driving force refers to the amount of free energy required to drive the NPs into the cell. These two factors determined how fast and how many NPs are taken up by the cell. For NPs with a diameter smaller than 40 nm, the docking of a single small NP will not produce enough free energy to completely wrap the NPs on the surface of the membrane. This could prevent the uptake of the single NP by endocytosis. For the smaller NPs to go in, they must be clustered together and thus take a long diffusion time. Therefore, the uptake amount is much smaller than 50 nm NPs. For NPs with a diameter larger than 80 nm, endocytosis rarely occurs. The depletion of free receptors limits the ligand-receptor binding energy for forming a large membrane curvature. Almost all NPs are only partially wrapped in the membrane. Between both regions, the optimal NP diameter has been identified at which the cellular uptake of NPs is maximised [29–31]. The optimal diameter for AuNPs falls in the range of 40-60 nm for reasonable values of membrane bending rigidity and ligand-receptor binding energy.
In the optical scattering study of AuNPs and cells, we investigated particle sizes from 45 nm to 110 nm. AuNPs can be prepared as small as 5 nm. However, it is hard to identify small nanoparticles in the cells simply by using scattering images. As indicated in Eq. 1, the scattering cross-section is greatly reduced when particle diameter is reduced. For AuNPs with a diameter smaller than about 30 nm, the scattering signal will be smaller than the micron-sized vesicles and is hard to be identified. Therefore, the proposed 3D scattering method is suited only for medium-sized AuNPs. With this particle size, the scattering signals of vesicles and AuNPs are comparable. It should be noted that these medium-sized AuNPs are of great interest than other sizes of AuNPs for endocytosis studies. Previous experiments using TEM and fluorescence microscopy have all indicated that AuNPs with a size of 40-60 nm have the best cellular uptake efficiency [7–10]. For small and large AuNPs, most remain bound to the membrane. Hence, for drug delivery by AuNPs, this proposed method is very useful for long-term tracking of the process of endocytosis without any labelling.
We studied endocytosis of AuNPs with different sizes (45 nm, 70 nm and 110 nm) in various cells (the human cancer cell lines, CL1-0 and HeLa). Compared with previous methods using transmission electron microscopy and fluorescence microscopy, the proposed method provides a simple way to define whether AuNPs are in the cytoplasm or adhered to the membrane of living cells. Using the spectroscopic difference between AuNPs and cell organelles, a colour CCD with a simple post-process can easily identify the positions of AuNPs. From the 3D distributions of AuNPs, we have experimentally confirmed that endocytosis of AuNPs is size dependent. For the cells we studied, the optimal size for the uptake into cells was around 45 nm. These results suggest that a particle size of 45 nm has the highest efficiency for drug delivery by AuNPs. On the other hand, large AuNPs which remain bound to the cell membrane can be used to reconstruct the morphology of the cell.
This research is supported by the National Science Council, Taiwan (Grant No. 98-3112-B-001-022) and the Thematic Project of Academia Sinica, Taiwan.
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