Chapter 2. Experimental details
2.4 In vitro experiments
2.4.4 In vitro photothermal therapy
2.4.4.1 Introduction to fluorescent Dyes
Staining is an auxiliary technique used in microscopy to enhance contrast in the microscopic image. Dyes are frequently used in biology to highlight structures in biological tissues for viewing, often with the aid of different microscopes.
YO-PRO-1 and PI (Propidium iodide) are used to observe the structures of cancer cells during the process this experiment.
YO-PRO-1 stain selectively passes through the plasma membranes of apoptotic cells and labels them with moderate green fluorescence. Necrotic cells are stained red-fluorescent with propidium iodide.
(i) YO-PRO-1: Molecular formula is C24H29I2N3O
Fig. 2.11 Absorption and fluorescence emission spectra of YO-PRO-1 bound to DNA.
(ii) Propidium Iodide: Molecular formula is C27H34I2N4
Fig. 2.12 Absorption and fluorescence emission spectra of propidium iodide bound to DNA.
Fig. 2.13 Fluorescent images of destroyed cell.
(a) Transparent pattern. (b) After the excitation, the emission signal (green) by YO-PRO-1 was detected. (c) After the excitation, the emission signal (red) by PI was detected. (d) Emerge signals of (a), (b), (c)
2.4.4.2
Confocal microscope and two-photon Iuminescence imaging (Zeiss LSM 510 META NLO DuoScan)I. Confocal Microscope: Improvement of fluorescence microscope image is the fundamental function of confocal microscope.
Compare with the conventional fluorescence microscope, confocal microscope uses laser instead of mercury lamp as its light source.
Conducted by the scanner mirrors, the laser beam excites
specimens and the detector detects the emission fluorescence signal of the specimen point-by-point and line-by-line. The spatial pinhole eliminates out-of-focus light in specimens that are thicker than the focal plane. It enables the reconstruction of three-dimensional structures from the obtained images.
Fig. 2.14 The difference between conventional and confocal microscope. The image indicates that the confocal image has better resolutions than the conventional image.
Fig. 2.15 Beam path of the excitation and emission light in a confocal laser-scanning microscope.
II. Two-photon Iuminescence image
Two-photon excitation is based on the idea that two photons of comparably lower energy than needed for one photon excitation can also excite a fluorophore in one quantum event. Each photon carries approximately half the energy necessary to excite the molecule. An excitation results in the subsequent emission of a fluorescence photon, typically at a higher energy than either of the two excitatory photons. The probability of the near-simultaneous absorption of two photons is extremely low. Therefore a high flux of
excitation photons is typically required, usually a femtosecond laser.
The most commonly used fluorophores have excitation spectra in the 400–500 nm range, whereas the laser used to excite the two-photon fluorescence lies in the ~700–1000 nm (infrared) range.
In this case, the surface plasmons absorb two near infrared photons simultaneously; it will absorb enough energy to transit into the excited state .The surface plasmons will then emit a single photon with wavelength that typically in the visible spectrum.
Because two photons are absorbed during the excitation of the surface plasmons, the probability for fluorescent emission from the surface plasmons increases quadratically with the excitation intensity.
Fig. 2.16 Differences between single photon and two-photon excitation.
Fig. 2.17 Two-Photon Iuminescence Images of gold nanorods. (a) Only the emission signal of AuNRs. (b) Signal by merge (a) and transparent light.
2.5 In Vivo Experiments
2.5.1 Animal model
[7, 8]Model: Ears of female Balb/c with ages 4 to 6 weeks. All animals are from BioLASCO Taiwan Co., Ltd
There are several advantages for setting the ears of mice as model 1. This model provides much more accurate for calculating the volume of the tumor, as comparing with the rear flank model of the mice.
2. Due to the thickness of ear is very thin, there is almost no limited of the laser depth.
3. It’s easier to observe the phenomenon of angiogenesis.
Fig. 2.18 Schematic of the ear structure
Fig. 2.19 Image of the histologic section of the mice ear
2.5.2 Anesthesia
A. Trichloroacetic acid (TCA) B. Ether
2.5.3 In vivo photothermal therapy
[9,10]2.5.3.1 Experimental processes I. Induce solid tumors
Prepare the suspended cancer cells ~106 in 30 µL medium, and inject it subcutaneously into the right and left ear of the female mice Balb/c, the mice were anesthetized with TCA.
II. Inject the PSS-AuNRs solutions into the solid tumors
When the tumors grew to approximately 30 mm3, PSS-AuNRs (10 µg in terms of gold nanorods amount), which were suspended in 10 µL DI water, were directly injected into the solid tumors of ears.
III. Photothermal therapy
After the injection of PSS-AuNRs for 22 hours, the mice’s ears were exposure under the 808 nm laser, power density 0.6 W/cm2 for 120 seconds.
IV. Observation of solid tumors size
Take photo by stereomicroscope and then import in software
“Image J”. Obtain the tumor size (volume) by calculating the pixels.
Fig. 2.20 Set-up of the in vivo photothermal therapy.
Fig. 2.21 Schematic of the in vivo photothermal therapy.
2.5.3.2 Microscope system
Observing the solid tumor by a stereomicroscope, Leica MZ16FA, The Leica MZ16 FA fluorescence stereomicroscope is the fluorescence stereomicroscope with greatest zoom capability (16:1), highest resolution (840 Lp/mm), highest magnification (115x with standard optics), a patented illumination/filter system for the most intense fluorescence on jet-black backgrounds, and a high-performance transmitted-light base for excellent relief contrast.
2.5.3.3 Estimation of tumor size The calculation of solid tumor size:
𝑇𝑢𝑚𝑜𝑟 𝑣𝑜𝑙𝑢𝑚𝑒 𝑚𝑚
!= 𝑎𝑟𝑒𝑎(𝑚𝑚
!)×𝑐(𝑚𝑚)× 𝜋 6
where c is the thickness of solid tumor.
The area was measured by importing the graph, which took by Leica MZ16FA, into software “Image J”. And the thickness was measured by vernier, take average for five times measurement.
2.5.3.4 Histological sections [11]
For fixation, place the whole specimen into Bouin for 24 to 72 hours.
Centrifuge the Bouin and re-disperse with 70 % alcohol for couple times, in order to remove the yellow-colored with 2,4,6-Trinitrophenol.
After the above procedure, dehydrate it by 80%, 85%, 90%, 95%, 100% alcohol in sequence. Paraffin-embedded after immersing the specimen in the toluene, then do the microtome cutting to the specimen with 5~6 µm thickness. Finally staining the specimen by the method called Gomori rapid one step trichome, and the last step is coverslipping with Rapid Mounting Media (Merk).
Chapter 3 Results and discussion
3.1 Characterizations of AuNRs
The AuNRs were prepared by the one pot synthesis method using hexadecyltrimethylammonium bromide (CTAB) as surfactants, CTAB–AuNRs. However, CTAB has been demonstrated to be a problem in application due to its cytotoxicity [Langmuir 22, 2 (2006)]. In order to make AuNRs suitable for for clinical purposes, additional surface modifications are necessary.
Here, polystyrenesulfonate sodium salt (PSS, 70 kDa) was, PSS–AuNRs are extremely stable at room temperature for several months. In order to figure out whether replacing the CTAB-coated (positive charge) with PSS-coated (negative charge) AuNRs, the zeta-potential measurements of CTAB-coated and PSS-coated AuNRs were also performed.
Fig. 3.4(a) and (b) show the results of zeta potential of CTAB- and PSS-coated AuNRs, respectively.
Fig. 3.1 (a) TEM image of PSS-AuNRs. (b) High resolution TEM image a single AuNR
Fig. 3.2 Absorption spectroscopy of CTAB-AuNRs and PSS-AuNRs.
Fig. 3.3(a) The zeta potential of CTAB-AuNRs is 45.2±13.8 mV.
Fig. 3.3(b) The zeta potential of PSS-AuNRs is -39.5±16.3 mV.
In addition, aliquots of AuNRs were also subjected to inductively coupled plasma optical mass spectroscopy (ICP-MS) to correlate the optical density (O.D.) with particle quantity. Comparison against a calibration line gave a gold content of ~20 µg/mL at O.D.
= 1. Fig. 3.5(a) and (b) show the calibration data and measured data respectively. And Fig. 3.5(c) shows the results of ICP-MS analysis of PSS-AuNRs.
Fig. 3.4(a) Calibration data of Au.
Fig. 3.4(b) Measured data of PSS-AuNRs.
Fig. 3.4(c) The results of ICP-MS measurement. The blue points are the standard curve of calibration, and the red points are the results of the analysis.
3.2 In vitro cancer cell photothermolysis mediated by AuNRs
Using a standard colorimetric cell viability assay (MTT assay), the cytotoxicity of the CTAB-AuNR and PSS-AuNRs were evaluated against EMT-6 breast tumor cells, as shown in Fig. 3.5.
The assay evidently verified that CTAB-AuNRs presented obviously cytotoxicity after incubation with the cells for 12, 24 and 48 h. In comparison, the viability of cells with PSS-AuNRs, was much higher than CTAB-AuNRs, confirming its biocompatibility with EMT-6 cells, and can thus be used in further studies pertaining to in vitro/in vivo imaging and photothermal therapeutics.
Fig. 3.5 Cell viability of EMT-6 cells exposed to CTAB- and PSS-coated AuNRs after (a) 12 h, (b) 24 h and (c) 48 h incubation, respectively. The viability of the cells was normalized with respect to a media-only control.
By using the inverted confocal microscope, LSM 510 META, the destruction of tumor cells triggered by PSS-AuNRs assisted with two photon laser excitation can be observed in situ real-time. The cell destruction process can be dynamic monitoring by using the fluorescent dyes, YO-PRO-1 (green coloring) and propidium iodide (red coloring). As the cell membrane becomes slightly permeable, it will enable YOPRO-1 but not propidium iodide to enter the cytoplasm. However, upon cell death, propidium iodide can
penetrate the comparatively leaky membranes. Neither of two dyes can penetrate viable cells. To realize the influence of different power density and different quantity of PSS-AuNRs on the destruction of cancer cells, two different energy fluences and quantity of PSS-AuNRs were applied to living cells in sequence.
The cell mortality was observed at energy fluence of 93 mJ/cm2. Results of the cells with AuNRs (N~30–40 clusters), under excitation at energy fluences of 24.2 and 5.9 mJ/cm2, are shown in the series of images of Fig. 3.6 and 3.7, respectively. Additionally, results of the cells with fewer AuNRs (N~2–15 clusters) under excitation at energy fluences of 24.2 and 5.9 mJ/cm2, are shown in the series of images in Fig. 3.8 and 3.9.
Fig. 3.6 Photothermolysis of the EMT-6 tumor cell triggered by PSS-AuNRs (N~30–40 clusters) under energy fluence 24.2 mJ/cm2. (a) Before excitation by laser, (b) After 0.6 s excitation by laser (g) (i), (ii), (iii), (iv) mean signal of AuNRs, YO-PRO-1, transparent light and propidium iodide respectively. From Fig. (e), The apoptosis of the tumors can be observed.
Fig. 3.7 Photothermolysis of the EMT-6 tumor cell triggered by PSS-AuNRs (N~30–40 clusters) under energy fluence 5.9 mJ/cm2. (a) Before excitation by laser, (b) (c) (d) (e) (f). After 0.6 s, 360 s, 720 s, 1080 s, 1860 s excitation by laser (g) (i), (ii), (iii), (iv) mean signal of AuNRs, YO-PRO-1, transparent light and propidium iodide respectively. From Fig. (g). The apoptosis of the tumors can be observed.
Fig. 3.8 Photothermolysis of the EMT-6 tumor cell triggered by PSS-AuNRs (N~2–15 clusters) under energy fluence 24.2 mJ/cm2. (a) Before excitation by laser, (b) (c) (d) (e) (f) After 0.6 s, 210 s, 630 s, 900 s, 1230 s excitation by laser (g) (i), (ii), (iii), (iv) mean signal of AuNRs, YO-PRO-1, transparent light and propidium iodide respectively. From Fig. (g), the apoptosis of the tumors can be observed.
Fig. 3.9 Photothermolysis of the EMT-6 tumor cell triggered by PSS-AuNRs (N~2–15 clusters) under energy fluence 5.9 mJ/cm2. (a) Before excitation by laser, (b) After 0.6 s excitation by laser (e) (i), (ii), (iii), (iv) mean signal of AuNRs, YO-PRO-1, transparent light and propidium iodide respectively. From Fig. (e), after observation for 1180 s, YO-PRO-1 and propidium iodide do not penetrate the comparatively leaky membranes. It figures that that the tumor cell is still alive instead of destruction by photothermolysis.
Fig. 3.10 The situation of cancer cells to the power after photothermal therapy.
3.3 In vivo tumor photothermal therapy
3.3.1 Power density test with the ears model
In order to ensure that the photothermal effect as the dominant cell-killing factor, power density test is the first priority to exclude other possible influences towards tumor cell viability. In experiments, each ear of the mouse was exposed with continue-wave laser (808 nm) for 2 minutes with different power density, 1.2 W/cm2, 1 W/cm2, 0.8 W/cm2, and 0.6 W/cm2. The results are shown in Fig. 3.11.
Fig. 3.11 Power test of non-treated mice ears. (a) 1.2 W/cm2; (b) 1 W/cm2; (c) 0.8 W/cm2; (d) 0.6 W/cm2. From these results, the limited power density of 0.8 W/cm2 was observed obviously.
3.3.2 Tumor growth curve
To analyze the therapeutic effect of the PSS-AuNRs on photothermal ablation of solid tumors, the tumor (EMT-6 cells, 1*106 cells in 30 µl PBS) was induced by intratumoral injection on the ear of female mice (Balb/c). When the tumor size grew to approximately 3 mm in diameter, PSS-Au NRs (~10 µg) were directly injected into mice’s ears. After 22~24 h, the mouse was anesthetized with ether, and then the tumor region was irradiated
with a laser for 2 minutes. In order to ensure the photothermolysis was the only factor that destroyed the solid tumors, setting relative control groups was necessary. There are three control groups: (i) tumors only, as shown in Fig. 3.12; (ii) tumors with exposure by 808 nm laser, 0.6 W/cm2, for 120 s, as shown in Fig. 3.13; (iii) tumors with PSS-AuNRs by intratumoral injection as shown in Fig. 3.14;
and there is one experimental group with PSS-AuNRs and exposed under power density 0.6 W/cm2 for 120 s, as shown in Fig. 3.15
Fig. 3.12 Control group (tumors only). (a)~(f) are before induced tumors, and after induced for 5 days, 8 days, 11 days, 15 days, 20 days, respectively.
Fig. 3.13 Control group (tumors exposed by laser). (a)~(f) are before induced tumors, and after induced for 5 days, 8 days, 11 days, 15 days, 20 days, respectively. Exposure with laser on the 12nd day, power density is 0.6 W/cm2, for 120 s. From this result, it can be observed that the laser was excluded as a factor, which destroys the solid tumors.
Fig. 3.14 Control group (tumors with PSS-AuNRs), Fig. (a)~(f) are after induced for 5 days, 8 days, 11 days, 15 days, 20 days, 24 days, respectively. 10 µg PSS-AuNRs were intratumorally injected into the solid tumor on the 11st day. From this result, it can be observed that the PSS-AuNRs was excluded as a cytotoxic factor.
Fig. 3.15 Experimental group, the tumors were exposed with laser on the 12nd day, power density is 0.6 W/cm2, for 120 seconds.
(a)~(g) are after induced for 5 days, 8 days, 11 days, 15 days, 20 days, 24 days, 28 days, respectively.
After calculation for the tumors size, the growth curve was presented in Fig. 3.16.
Fig. 3.16 Growth Curve.
The green-colored, blue-colored, black-colored and the red-colored symbol mean growth curve of tumors, tumors with laser, tumors with AuNRs and tumors with AuNRs under exposed by laser, respectively.
3.3.2 Histological study
Histological sections of tumor tissues from each group confirmed the successful destruction of tumor cells by the photothermal effect by PSS-AuNRs. Fig. 3.16 (a)~(f) show the results of the each group.
Fig. 3.17 Histological sections of tumor tissues (a) Normal cells (b) EMT-6 (c) EMT-6 under exposure with 0.6 w/cm2 laser for 2 min (d) EMT-6 with PSS-AuNRs injected (e) 1 day after photothermal treatment (f) 8 day after photothermal treatment
Fig. 3.17 shows that photothermal therapy can destroy the solid tumor structure and make severe destruction.
Chapter 4 Conclusion
All the experiments demonstrated the photothermal effect of PSS-AuNRs under NIR irradiation in destroying tumors cells in vitro and in vivo.
The results of photothermal therapy in in vitro experiments indicated that the destruction of tumors under photothermal therapy were related to the energy fluence of radiation as well as the clusters of PSS-AuNRs that tumors took up. Under the same energy fluence, the more clusters tumors took up, the severer the damage; within the same clusters, the higher energy the power fluence was, the greater the damage. According to the above results, the energy fluence for photothermal therapy, controlled within the medical safety level (100 mJ/cm2), was dependent on the amount of AuNRs taken up per cell.
When the solid tumors, where PSS-AuNRs were intratumorally injected, was irradiated by NIR laser, high thermal energy was generated from the optical excited PSS-AuNRs, which subsequently destroyed tumor cells in a noninvasive manner. The ability of photothermal therapy to completely eradicate the tumor cells is evident in the results shown in the growth curve and
histological sections of the tumor, yet the great amount of heat will also damage normal cells, like cartilage. In future practices of photothermal therapy, better experimental conditions should be the first priorities, including variables such as power density, exposure time, concentration of AuNRs, and model size.
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