Chapter 5 Cytotoxicity of Nanowires
5.4 Conclusion
In summary, we have investigated the cytotoxicity of the micrometer long gold nanowires with four different aspect ratios and four different surface modifications. It was found that the serum coated nanowires exhibited the least cytotoxicity with a LD50 value around 150 µg/ml, which was less than those measured for the smaller gold nanoparticles. All other surface functionalized nanowires possessed some degree of toxicity, which depended on the surface charge. Among them, the mercapto acid modified nanowires were the most toxic nanowires. For the same type of surface modification, HeLa cell, which can grow in suspension, were found to be more resistant to the addition of the nanowires solution. As for the nanowires with different aspect ratio, the cytotoxicity experiments indicated that nanowires with different aspect ratios exhibited the same degree of toxicity. However, the uptake efficiency for the shorter nanowires was measured to be higher than the longer nanowires. Therefore, we concluded that the internalized nanowires with higher aspect ratio were more toxic than the shorter one, which explained that the LD50 value for the nanowires was lower than that of the low aspect ratio nanorods. This conclusion also agreed with the cytotoxicity experiment for the spherical nanoparticle where the nanowires were found to be more toxic than the spherical nanoparticles.
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Figure 1. The cytotoxicity of gold nanowires with various surface modifications for (A) fibroblast cells and (B) HeLa cell.
Figure 2. The cytotoxicity of different sizes of gold nanowires modified with mecapto acid for (A) fibroblast cells (B) HeLa cell.
(A)
Figure 3. The cytotoxicity of 250nm gold nonoparticles at different concentrations for (A) fibroblast cells (B) HeLa cell.
Figure 4. The uptake of aminothiol modified gold nanowires with four different lengths in (A) fibroblast cell and (B) HeLa cell. 0.5µm (solid circles), 1.8 µm (open circles), 4.8µm (solid triangles), 8.6 µm (open triangles).
Chapter 6 Nanowire as Living Cell Probes
6.1. Introduction
In the advent of nanotechnology, various nanomaterials with distinct properties have synthesized routinely. To take the advantages of the novel properties of these nanomaterials in the field of biological studies, it is very important to investigate the interaction between the nanomaterials and the biological objects such as cells. Recently, there have been increasing research activities in developing novel functionalized nanomaterials for biological applications, especially for the cellular study. These nanomaterials have been designed to enter the cells efficiently and to escape endosomal/lysosomal complexes while the local environment within the living cells can be revealed by these nanomaterials. For this purpose, the nanomaterials must remain stable in the intracellular environment and not disturb the cell’s normal biochemical functioning.
Several types of nanomaterials such as magnetic, polymeric, metallic, semiconductor nanoparticles or nanowires have been introduced for the cellular study. For example, Goodman et al. have focused the capability of gold nanoparticles to incorporate secondary tags such as peptides to target specific cell types [1]. In their study, the gold nanoparticles have been functionalized with cationic and anionic side chains. The carboxylated modification on the gold nanoparticles was found to be nontoxic to the cells. In contrast, the cationic side chain bound to the gold nanoparticles exhibited moderately toxicity. It was found
that the toxicity of gold nanoparticles was related to their interactions with the cell membranes. In another study, Derfus et al. have explored the intracellular delivery of quantum dots (QD) for live cell labeling and organelle tracking [2].The QDs modified with polyethylene glycol (PEG) were mixed with different transfection reagents and then were delivered to the interior of the HeLa cells. The flow cytometry was used to quantify the amount of QD delivered to the cells. It was found that QDs often tended to accumulate in vesicles and distributed non-homogeneously in the cytoplasm [3]. Therefore, to investigate the subcellular localization, the QDs were modified with 23 mer nuclear localization sequence peptide and a 28 mer mitochondrial localization sequence peptide allowing tracking QDs to the nucleus and mitochondrial respectively.
As regarding to metallic nanoparticles, recent studies have shown that it was possible to use metallic nanoparticle for targeted nuclear delivery. For example, Tkachenko and coworkers investigated that the multifunctional gold nanoparticle –peptide complexes for nuclear targeting [4]. The 20nm diameter gold particle was modified with bovine serum albumin (BSA) bound with various cellular targeting peptides. These nanoparticles must have carried both receptor-mediated endocytosis (REM) and nuclear localization signal (NLS) peptides to enter HepG2 cells and perform nuclear localization. Non-spherical nanorods also have been demonstrated capable of conducting gene delivery. It was shown that the conjugation of DNA plasmid and targeting ligands can be achieved simultaneously in a
spatially defined manner. For example, Salem and coworkers have demonstrated that it was possible to transfer gene using bifunctional Au/Ni nanorods [5]. In their approach, the carboxylate end group was first attached to the Ni segment. Subsequently, the plasmids were bound to the protonated amines on the surface of nickel segment by electrostatic interactions.
The transferrin was bound to the gold segment of the nanorods through thiolate linkage. These dual-functionalized Au/Ni nanorods were used in a vitro transfection experiment using human embryonic Kindney (HEK 293) mammalian cell line.
To optimize non-viral gene delivery systems, Akita and co-workers have developed a novel technique for quantitative three-dimensional analysis of the intracellular trafficking of the plasmid DNA [6]. They evaluated the fraction of the plasmid DNA in cytosol, endosome/lysosome, and nucleus in the cell based on the z-series images sequentially obtained by confocal laser scanning microscopy. Lacerda et al. have investigated the interactions between mammalian cells and ammonium-functionalized single-walled carbon nanotubes [7]. Using this approach, they could track the luminescence signal of SWNT-NH3+
by using confocal laser scanning microscopy. Recently, Lu et al. have developed fluorescein isothiocyanate (FITC)-incorporated silica core-shell iron oxide nanoparticles as bifuntional contrast agents [8]. These dye molecules could be easily integrated into a silica cell, and silica is known to be biocompatible and resistant to biodegradation in the biological environment.
Moreover, these nanoparticle complexes exhibited both fluorescent and magnetic properties to
serve as good molecular imaging probe to monitor cell trafficking.
In this thesis, we have introduced functionalized gold nanowires as new probes that took the advantage of the nanowires-thiol group conjugation. Using various types of functionalization, it was possible to probe the local environment inside the cells by confocal microscopy.
6.2 Experimental
6.2.1 Preparation of cells for confocal analysis
HeLa cells (105/ml) were cultured in minimal essential media in 35 mm glass bottom dishes coating with poly-D-lysin. After 24 hours of incubation, the media was removed and the cells were washed by PBS solution. For visualization purpose, the plasmid DNA coated gold nanowires were mixed with 50nM YOTO-1 dye (Molecular Probe, Invitrogen) for 20 minutes in the media without serum. The mixture solution was added to cells culture and incubated for 24 hours. Subsequently, the cells were stained with Image iT LIVE plasma Membrane and Nuclear Labeling Kit (Molecular Probe, Invitrogen ) for 20 minutes.
6.2.2 Transfection experiment
Fibroblast cell were seeded onto 35 mm glass bottom culture dishes at a density of 2×
105. After 24 hours, the cells were washed and supplied with 0.5 ml fresh growth medium.
The plasmid used in this study was pAcGFP1-Actin (BD), which expresses green fluorescence protein (GFP). The surface modified gold nanowires (4×105) was mixed with 5.6 µg of Plasmid DNA for 20 minutes. The mixture solution was added into cell culture and
incubated for 24hour. The behavior of fibroblast cells with plasmid coated nanowires was monitored under a laser scanning confocal microscope (Olympus, Fluroview 300).
6.2.3 Intracellular behavior monitored by the Lysosensor yellow/Blue dye
Generally, fibroblast cell were seeded at a density 105 in 1ml DMEM medium with 10%
serum. Amino-functionalized gold nanowires were prepared as described previously. An appropriate amount of 2 µm long amino-modified gold nanowires (105) was mixed with 75
nM Lysosensor yellow/Blue dye DND-160 (Molecular Probe, Invitrogen) for 10 minutes.
After the sample was centrifuged at 5000 rpm for 2 minutes, the supernatant was removed.
The nanowire/dye complexes were then added to a 0.5 ml growth medium with fibroblast cell and incubated for 12 hours.
6.3 Results and discussion
It has been shown that the nanoparticles with diameter less than 100 nm can be internalized easily by cells through the endocytosis pathway [9]; therefore, smaller gold nanoparticles have been used as the carriers of the genetic materials through electrostatic or covalent
interaction for gene therapy, which has been demonstrated to be very effective and not very toxic (LD50 was about 750 µg/ml) [10-12] However, it is not known that the micrometer long metallic nanowires can be internalized by cells without damaging the cells, which is important
if the nanowires are to be utilized for probing the local environment inside a living cell. To observe the internalization process of nanowires, 4.5 µm long gold nanowires (200 nm in
diameter) coated with serum were added into a glass bottom culture dish containing HeLa cells, which were placed in a CO2 incubator on an inverted microscope. The phase contrast images of the cells and the nanowires were recorded by a CCD camera every 10 minutes.
Shown in figure 1 is the image of a gold nanowire sitting outside the cell. When the cell migrated around the glass surface, the nanowire was enclosed inside the cell as shown in figure 1(a) to (d). As the result of this enclosure, the nanowire was internalized by the cells which took about four hours to complete.
To investigate the capability of delivering DNA molecules into the cells by the nanowires, 5 µm long nanowires were first functionalized with aminothiols, which covered the nanowire
surfaces with positive charges [13]. Then the negatively charged plasmid DNA molecules were attached to the nanowires through electrostatic interaction. For visualization purposes, the plasmid DNA on the nanowire surfaces were further labeled with YOYO-1, which emitted a strong green fluorescence when bound to a double strain DNA, and the cells were stained with Image iT LIVE Plasma Membrane and Nuclear Labeling Kit (Invitrogen). Shown in
figure 2 (A) the nanowire was clearly visualized. The nanowires were discovered intracellularly at the perinuclear region. This was thought to directly indicate that nanowire
traffic intracellularly, the nanowire seems the ability to travel intracellularly around the cell cytoplasm form outside intact cell. Figure 2 (B) z-stack imaging data obtained from the 5µm
functionalizd gold nanowires coated with plasmid and labeled with YOYO-1 dye. A series of the z- stack imaging indicated that the intracellular and perinuclear localization of the nanowire signal was at the same plane of focus as the nuclear stain. We have demonstrated that nanowire themselves are capable of internalizing and trafficking within cell for several hour. Moreover, at a dose of up to high concentrated ( > 106/ml) of nanowires cell easily death was observed by confocal microscopy.
From these two measurements, two conclusions can be drawn: first, the micrometer long nanowires can be readily internalized by the cells despite of their relative large size. Secondly,
from the sectioning images, it is evident that the surface of individual nanowires could be visualized with sub-µm resolution, which may allow the use of functionalized multi-segment
nanowires to probe the microenvironment inside the cells.
To demonstrate that the surface functionalized nanowires can carry the plasmids into the cytoplasm, it is important to investigate the functionality of plasmids on the gold nanowires after entering the cell. To study the functionality of the plasmids, a green fluorescence protein (GFP) expressing plasmid (pAcGFP1-Actin, BD) was coated on the gold nanowires modified
by aminothiols and incubated with the fibroblast cells for 24 hours. Shown in figure 3 is a combined fluorescence and DIC image of gold nanowires with GFP expressing plasmids inside a fibroblast cell. It can be clearly seen that there were still several gold nanowires remained inside the fibroblast cell after 24 hours of incubation. The fact that the whole cell exhibited a strong green fluorescence signal indicated that the plasmids on the nanowires were still functional and capable of expressing GFP. This experiment confirmed that the micrometer long aminothiol modified gold nanowires can not only protect plasmid DNA molecules from degradation but also release plasmid NDA molecules inside the cells.
To investigate the behavior of the surface modified nanowires inside the cells, DIC images
of the living cells were monitored in an incubator on an Olympus Fluoview 300 confocal microscope. Figure 4 (A) shows the image of the 5 µm long alkanethiol modified gold
nanowires internalized by the HeLa cells after 24 hours of incubation. The density of the nanowires was about 5 x 104 nanowire/ml and the cell density was about 105 cell/ml. From the
DIC image, it can be seen that the nanowires were internalized unevenly among cells. When a 2 µm long nanowire solution at a concentration of 5x105 nanowire/ml was used, several gold
nanowires could be found inside most of the HeLa cells as seen in figure 4 (B). In some occasion, nanowires aggregation in a single cell was observed similar to the one shown in figure 4 (C). The exact origin of such behavior is not known at this time. At this concentration, more than 50% of the HeLa cells were not viable, which could be seen by the decrease in the
cell density. This observation also agreed with the results of the uptake measurement in chapter 5 figure 4 (B), which the shorter nanowires exhibited higher uptake efficiency. When a serum coated gold nanowire solution at 2 x106 nanowire/ml were added to the culture dish containing 3T3 cells, most cells were found to be attached on the surfaces. At this concentration, more than 70% the cells were still viable and on average each cell internalized about 10 nanowires as shown in figure 5 (A) and (B). This result also agreed with the uptake measurement for the 3T3 cells as shown in figure 4 (A) in chapter 5.
For gold nanoparticles with size smaller than 100 nm, it is believed that the nanoparticles entered the cells via the receptor mediated endocytosis pathway [14]. However, it is not clear which mechanism dominates the cellular uptake behavior of the micrometer long nanowires.
In this experiment, we have monitored the internationalization process of nanowires through a confocal microscope. In most cases, we have found the nanowires entering the cell with one end pointing to the cell similar to the one shown in figure 1. It is reasonable that the smaller dimension of the nanowires has higher probability to be taken by the cells. Once one end of the nanowire was internalized by the cell, the rest of the nanowire would be surrounded by the cell membrane and finally the whole nanowire was taken by the cell. As long as the nanowires stayed inside the cell, they could explore the space inside the cytoplasm. In most of the time, we have found the nanowires maneuvered along the edge of the cell. More studies are needed
to understand the detail mechanism of the endocytosis pathway for the micrometer long nanowires [15].
To demonstrate that it is possible to monitor the local environment of the nanowires, the LysoSensor Yellow/Blue DND-160 (Invitrogen), was incubated with the 2 µm long
amino-modified gold nanowires for 10 minutes. The nanowire solution was then incubated with 3T3 cells for 24 hours. It can be clearly seen that both LysoSensor and nanowires can be visualized simultaneously on a confocal microscope. Shown in figure 6 are the confocal images of the LysoSensor coated nanowires inside the 3T3 cells. Since LysoSensor Yellow/Blue exhibited pH dependent dual emission spectra where the emission in 500-600 nm region (artificial red color in figure 6 (B) was observed in the acidic environment and the fluorescence in 410-500 nm region (artificial green color in figure 6) was observed in the less acidic environment, LysoSensor can be used to probe acidic organelles such as endosomes or lysosomes. The colocalization of the nanowires and the green fluorescence signals indicated that all the nanowires were located in less acidic compartments, presumably endosomes, as a result of endocytosis process of the nanowires. By monitoring both the DIC image of the nanowires and confocal image of the probe molecules as a function of time, the evolution of the local environment around the nanowires can be explored. In general, this approach can be extended to other types of probe molecules or biological assays.
6.4 Conclusion
In summary, we have demonstrated that the 5 µm long nanowires with surface
modifications are capable of delivering plasmid DNA molecules, which has been visualized and recorded on an optical microscope. In addition, LysoSensor was attached to the nanowires revealing the local environment of the nanowires. By monitoring the color change of the Lysosensor on the nanowires, it was found the nanowires stayed in the less acidic environment indicting that nanowires never escaped from the endosome/lysosome complexes.
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Figure 1.The phase contrast image of the serum coated with gold nanowires entering HeLa cell. The bar is 20 µm.
(A)
(B)
Figure 2. Multiple stained confocal image of HeLa cell. (A).stacked laser scanning confocal image of nanowires in HeLa cell. (B) z-stack imaging obtained from the 5µm functionalizd
gold nanowires coated with plasmid and labeled with YOYO-1 dye.
Figure 3. The combined DIC and fluorescence image of plasmid coated nanowires and fibroblast cell expressing green fluorescence proteins. Nanowires are indicated by the red circles. The bar is 20 µm.
Figure 4.DIC images of the alkanethiol modified gold nanowires in the HeLa cells at different nanowire concentrations. (A) 5×104 nanowire/ml. Bar:20µm (B) 5×105 nanowire/ml. Bar:10 µm (C) nanowires aggregation in a single HeLa cell. Bar:10 µm
Figure 5. The DIC images of the serum coated gold nanowires in the fibroblast cells at 106 nanowire/ml nanowire concentrations. (A) the nanowires distribution in a single cell. (B) the nanowires distribution among cells. The bar is 20 µm.
(B)
Figure 6. (A) The DIC image (B) combined confocal image of the gold nanowires inside Hela cells. The bar is 20 µm.
Chapter 7 Nanowire for Gene Delivery
7.1 Introduction
Gene therapy is becoming an important strategy in the treatment of various diseases such as AIDS, cancer. The challenge of in vivo gene therapy is to develop safe and efficient gene delivery system. For the gene therapy to be used in the clinical applications, it requires the development of efficient DNA delivery vehicle that can be synthesized both easily and in large quantities. It has been demonstrated that the viral vectors can provide the most efficient
Gene therapy is becoming an important strategy in the treatment of various diseases such as AIDS, cancer. The challenge of in vivo gene therapy is to develop safe and efficient gene delivery system. For the gene therapy to be used in the clinical applications, it requires the development of efficient DNA delivery vehicle that can be synthesized both easily and in large quantities. It has been demonstrated that the viral vectors can provide the most efficient