BIOCHEMICAL AND HISTOLOGICAL EFFECTS OF DIFFERENT LIGHT DOSES ON HYPERTHERMIC THERAPY WITH GOLD NANORODS Lucas F. de Freitas 1 , Clóvis Grecco 2 , Lilian T. Moriyama 2 , Cristina Kurachi 2 , Virgínia. C. A. Martins 3 , Ana M. G. Plepis 1,3 1 Programa de Pós Graduação Interunidades Bioengenharia – FMRP/EESC/IQSC – Universidade de São Paulo. 2 Instituto de Física de São Carlos, Universidade de São Paulo. 3 Instituto de Química de São Carlos, Universidade de São Paulo. e-mail: [email protected]ABSTRACT. To investigate the effect of different light doses applied on tumors containing gold nanorods, as well as to characterize the damage in different cell structures, nanorods were synthesized by a seeding method, centrifuged and washed to eliminate soluble CTAB. Those nanoparticles were used for hyperthermic treatment of Ehrlich tumor in mice. Control group mice did not receive nanoparticles and were not irradiated with laser, while N group mice received nanorods without being irradiated with laser. L group mice did not receive nanoparticles, but were irradiated with laser, while H group mice received both nanorods and laser irradiation. The light doses used were 216 or 600 J cm -2 . After irradiation, the tumors were collected and subjected to biochemical tests to investigate oxidative stress on cell membranes and to evaluate the total antioxidant capacity of cells, or were subjected to histological analysis. Results show that the laser itself is responsible for membrane oxidative damage, regardless the presence of gold nanorods, but the nanoparticles are important to oxidative stress generation intracellularly. The intensity of histological damage is directly dependent on the light dose applied and on the presence of nanorods. Key words: Nanorods, Gold, Cancer, Laser Dose, Hyperthermia. 1. INTRODUCTION. Hyperthermic therapy is a novel treatment intervention considered less invasive and has less side effects than the conventional anticancer therapies, such as chemotherapy and radiotherapy. Hyperthermia can be achieved directly by the interaction of an energy source, which can be ultrasound, light or a magnetic field, with the target tissue. In vitro experiments confirmed that just 10 expositions of 60 µs to a near infrared laser with power of 7 mW are enough to cause severe oxidative stress (HUFF et al, 2007), leading to great
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BIOCHEMICAL AND HISTOLOGICAL EFFECTS OF DIFFERENT LIGHT DOSES ON HYPERTHERMIC THERAPY WITH GOLD
NANORODS
Lucas F. de Freitas1, Clóvis Grecco2, Lilian T. Moriyama2, Cristina Kurachi2, Virgínia. C. A. Martins3, Ana M. G. Plepis1,3
1Programa de Pós Graduação Interunidades Bioengenharia – FMRP/EESC/IQSC – Universidade de São Paulo.2Instituto de Física de São Carlos, Universidade de São Paulo.3Instituto de Química de São Carlos, Universidade de São Paulo.
ABSTRACT. To investigate the effect of different light doses applied on tumors containing gold nanorods, as well as to characterize the damage in different cell structures, nanorods were synthesized by a seeding method, centrifuged and washed to eliminate soluble CTAB. Those nanoparticles were used for hyperthermic treatment of Ehrlich tumor in mice. Control group mice did not receive nanoparticles and were not irradiated with laser, while N group mice received nanorods without being irradiated with laser. L group mice did not receive nanoparticles, but were irradiated with laser, while H group mice received both nanorods and laser irradiation. The light doses used were 216 or 600 J cm -2. After irradiation, the tumors were collected and subjected to biochemical tests to investigate oxidative stress on cell membranes and to evaluate the total antioxidant capacity of cells, or were subjected to histological analysis. Results show that the laser itself is responsible for membrane oxidative damage, regardless the presence of gold nanorods, but the nanoparticles are important to oxidative stress generation intracellularly. The intensity of histological damage is directly dependent on the light dose applied and on the presence of nanorods.
Hyperthermic therapy is a novel treatment intervention considered less invasive and has less side effects than the conventional anticancer therapies, such as chemotherapy and radiotherapy. Hyperthermia can be achieved directly by the interaction of an energy source, which can be ultrasound, light or a magnetic field, with the target tissue. In vitro experiments confirmed that just 10 expositions of 60 µs to a near infrared laser with power of 7 mW are enough to cause severe oxidative stress (HUFF et al, 2007), leading to great membrane damage, without affecting neighbor healthy cells (LAPOTKO et al., 2006). Hyperthermia occur more easily and more effectively, however, by means of the use of some devices, mostly metallic nanoparticles (NADEJDA et al., 2009).
In vitro and in vivo data show that the cellular damage caused by hyperthermia is proportional to the temperature achieved with the treatment (usually around 42 – 47ºC) and the time spent with the irradiation. Sometimes the damage occurs directly on the target cells, but it can occur also on the blood vessels, diminishing the blood flow and leading to acidosis and hypoxia (HILDEBRANDT et al., 2002).
Cell death, either by apoptosis or necrosis, can occur in consequence of hyperthermic interventions due to alteration on membrane permeability and conformation, usually by membrane blebbing (LAPOTKO et al., 2006) as well as by impaired fusion of DNA
fragments of cells during S phase of cell cycle (BULLIVANT, 2008) or denaturation of cytoskeleton proteins (HILDEBRANDT et al., 2002).
Gold nanoparticles are the most used devices for hyperthermic therapy regarding its properties which include low intrinsic toxicity, conformational flexibility and surface plasmon resonance with light on the visible and near infrared region (HIRSCH et al., 2006). The particles can assume a wide range of conformations, such as silica cores covered with gold (gold nanoshells), gold nanospheres, nanorods or nanowires. Silica nanospheres covered with a thin layer of gold have shown to be effective on the eradication of murine carcinoma (O’NEAL et al., 2004), while gold nanospheres successfully eliminated about 87% of malignant cells in patients with leukemia, sparing healthy neighboring cells (LAPOTKO et al., 2006).
From all the conformations available for gold nanoparticles to assume, one of the most reliable is the nanorod, which is known to increase Raman scattering and fluorescence signals (WEI et al., 2004), but its easy synthesis, usually by a simple seed mediated method (MURPHY et al., 2002), and its photothermal properties are the most interesting characteristics for researchers. Nanorods have a bigger area of interaction with light per volume unit than the other conformations, demanding less energy to generate the necessary heat through light-induced surface plasmon resonance (HUFF et al., 2007, VON MALTZAHN et al., 2007).
In this paper we propose to investigate two different doses of radiation on hyperthermic treatment using gold nanorods and their biochemical and histological consequences on a murine carcinoma.
2. METHODS.
EXPERIMENTAL ANIMALS:
We used 60 male Swiss mice, divided in four experimental groups: L group was composed of 10 mice which were irradiated with 216 J cm-2 of laser and other 10 which were irradiated with 600 J cm-2, none of them received nanorods; N group consisted of 10 mice which received nanorods, but were not irradiated with laser; H group was formed by 20 mice injected with nanorods 10 of which were irradiated with 216 J cm-2 and the last 10 were irradiated with 600 J cm-2 of laser; finally, CONTROL group was composed of 10 mice which didn’t receive nanorods and were not irradiated with laser.
EHRLICH CARCINOMA
A suspension of 2x106 Ehrlich carcinoma cells was inoculated subcutaneously on the back of mice (KURODA et al., 1976). The tumor grew for 10 days before the hyperthermic treatment could be performed in group H.
GOLD NANORODS SYNTHESIS
Gold nanorods were synthesized by a seed mediated method (MURPHY et al., 2002). Basically, gold seeds were synthesized by adding 5 mL of a 0.2 mol L-1 CTAB solution with 5 mL of a 5x10-4 mol L-1 auric chloride solution. The solution was gently stirred, and then 600 μL of a 0.01 mol L-1 sodium borohydride was added, followed by an additional two minutes stirring. The resultant solution was left to stand at 25ºC for six hours in absence of light, and the final seeds suspension was then stocked in dark flasks. Nanorods were
synthesized by mixing 200 μL of a 0.004 mol L-1 silver nitrate solution with 5 mL of a 0.2 mol L-1 CTAB and 5 mL of 0.001 mol L-1 auric chloride. After stirring, 70 μL of 0.0788 mol L-1 ascorbic acid was added in order to reduce gold. Finally, 12 μL of seeds suspension were added, and the nanorods growth occurred during 15 minutes at 27º C in the dark. The growth of nanorods can be confirmed when the solution goes from colorless to bordeaux within this time.
HYPERTHERMIC TREATMENT
After 10 days of tumor growth, the H group mice were treated with 100 μL of nanorods suspension at a 0.5 optical density, injected through the tail vein. L group and control animals received 100 μL of sterile saline solution. N group mice received the nanorods on the same day as H group. Nanorods accumulated in the tumor during 72 hours, and after this incubation period mice were anesthetized (10 mg xilazine and 100 mg ketamine per kg of body weight) to proceed with the laser irradiation. The tumors of L and H group mice was irradiated with near infrared (808 nm, 720 mW cm-2 or 2 W cm-2) laser for 5 minutes. These laser intensities were chosen regarding some protocols available on the literature (O’NEAL et al., 2004, VON MALTZAHN et al., 2009). The temperatures achieved with hyperthermic therapy were assessed, and after the treatment the tumor mass was collected after mice euthanasia by cervical dislocation and used for biochemical tests and histological analysis.
HYSTOLOGICAL ANALYSIS
Tumors of three mice of groups N and CONTROL, plus six tumors of mice from groups L and H (three irradiated with 720 mW cm-2 and three irradiated with 2 W cm-2 from each group) were fixed with formaldehyde 10% for 24 hours and then included in paraffin. Sections of 7 µm were made from the included tumors, dyed with Haematoxylin-Eosin and analyzed in optical microscope.
In order to evaluate the oxidative membrane damage caused by hyperthermia, a chemiluminescence assay was performed with homogenates obtained from those tumors that were not used for histological analysis. Basically, the tumors were homogenized in physiologic phosphate 10% buffer, added to tert-butyl hydroperoxide, which emits light as long as it reacts with previously oxidized lipids, and analyzed in a luminometer for 60 minutes (FLECHA et al., 1990). From the resultant graph it is possible to assess and compare the oxidative damage from the different experimental groups.
TOTAL RADICAL-TRAPPING ANTIOXIDANT PARAMETER (TRAP) ASSAY
With the TRAP assay it is possible to assess the total cellular response to an oxidative damage. The same homogenate was used for this test, which basically consists in adding the compound 2,2’-azo-bis-(2-amidinopropane) (ABAP), which produces peroxyl radicals at a constant rate, and observe for how long are the sample’s antioxidants able to inhibit ABAP’s radical production. The total antioxidant activity of the samples can be elicited comparing the
data of the samples with the inhibition time of an antioxidant with known concentration, such as Trolox® (an hydrophilic analogue of vitamin E) (WAYNER et al., 1985).
STATISTICAL ANALYSIS
Two-Way ANOVA was performed, with Bonferroni as a post test, to analyze chemiluminescence data. TRAP results were analyzed statistically with One-Way ANOVA. All results were considered statistically significant when p value was less than 0.05.
3. RESULTS.
HYPERTHERMIA
When a dose of 216 J cm-2 was applied to the tumors, the temperature increase mean was 15ºC, most of the tumors being heated from 31ºC to 46ºC. The temperature of the surrounding tissue increased 5ºC. In the absence of nanoparticles (group L), the tumors were heated from 32ºC to 42ºC, while the surrounding health area temperature increased from 31ºC to 33ºC (figure 1).
Figure 1. Temperature of tumors during hyperthermic treatment.
With the dose of 600 J cm-2 the heating became much more intense. The laser itself applied in the absence of nanoparticles on the tumor of group L animals was enough to increase the temperature from 32ºC to 52ºC, against no temperature modification observed in the mouth. When the tumors of H group were irradiated, they were heated from 32ºC to 79ºC, against an 11ºC elevation on the surrounding health tissues (figure 2). It was observed a mild reduction on tumor volume of group H animals, as well as a loss of color at the local of irradiation, probably due to ischemia following the hyperthermic stimulus.
Figure 2. Temperature of tumors during hyperthermic treatment.
HISTOLOGICAL ANALYSIS
A typical histological section of Ehrlich tumor obtained from a Control mouse is shown in figure 3. Intense pleomorphism, nuclear alterations and moderated necrosis can be observed. When N group tumors were analyzed, there was no difference observed in the histological sections (figure 4), but the analysis of L group tumors which received 216 J cm -2
light dose revealed a mild augment on necrosis (figure 5). This augment was much more intense on the sections of H group tumors irradiated with 600 J cm-2 light dose. The severe tumor parenchyma destruction with intense caseous necrosis can be seen in figure 6.
Figure 3. Histological section of Ehrlich tumor from Control mouse. CN: Caseous necrosis
Figure 4. Histological section of Ehrlich tumor from N group mouse.
CN
Figure 5. Histological section of Ehrlich tumor from L group mouse.
Figure 6. Histological section of Ehrlich tumor from H group mouse irradiated with 216 J cm-2
light dose.
The cellular damage observed in the sections of L group animals irradiated with 600 J cm-2 light dose were similar to those observed for H group treated with 216 J cm -2 (figure 7), while the tumor destruction was almost complete in H group mice when irradiated with the higher light dose, as can be observed in figure 8. There are very few cell nucleus, most of them with necrotic characteristics, and many areas of blood congestion (figure 9).
Figure 7. Histological section of Ehrlich tumor from L group mouse irradiated with 600 J cm-2 light dose.
Figure 8. Histological section of Ehrlich tumor from H group mouse irradiated with 600 J cm-2
light dose.
Figure 9. Histological section of Ehrlich tumor from H group mouse irradiated with 600 J cm-2 light dose showing an area of blood congestion.
The only curves statistically different from control mice ones were those from L and H groups irradiated with 600 J cm-2 light dose, but they were not different one from the other, leading to the conclusion that the presence of nanoparticles doesn’t play a key role on the membrane damage. The figures 10 and 11 show the obtained results.
Figura 10. Membrane lipoperoxidation demonstrated by a chemiluminescence assay (light dose of 216 J cm-2): (—■—) Controls, (—●—) Group N, (—▼—) Group L (—▲—) Group H.
Figura 11. Membrane lipoperoxidation demonstrated by a chemiluminescence assay (light dose of 600 J cm-2): (—■—) Controls, (—●—) Group N, (—▼—) Group L (—▲—) Group H.
TOTAL RADICAL-TRAPPING ANTIOXIDANT PARAMETER (TRAP) ASSAY
The total antioxidant capacity of tumors from H group mice treated with 600 J cm -2
was the only one statistically different from controls (figures 12 and 13). The data leads to the conclusion that the presence of nanoparticles plays an important role on the development of a severe oxidative stress on the target cells, mainly with higher doses of light.
Figure 12. Total Antioxidant Capacity of mice treated with 216 J cm-2 light dose.
Figure 13. Total Antioxidant Capacity of mice treated with 600 J cm-2 light dose.
4. DISCUSSION.
Novel and more effective forms of cancer diagnosis and therapeutic interventions with less side effects are the aim of researchers, and promising results have been obtained with treatments based on magnetic fields and light interaction with organic tissues (HILDEBRANDT et al., 2002; NADEJDA et al., 2009). Photothermal ablation using gold nanorods is one of those promising novel therapies, and is based on the heat generated through light excitation of gold nanorods present on the target tissue (WEI et al, 2004; VON MALTZAHN et al., 2007).
The main cell death pathway activated with hyperthermic treatment observed was necrosis, as can be seen with the histological sections. The amount of necrotic cells depends directly on the light dose applied on the tumor. It is known that hyperthermia alters membrane conformation and proprieties, mainly through membrane blebbing (TIRLAPUR et al., 2001; HILDEBRANDT et al., 2002). Vitamin E (alpha tocopherol) is one of the molecules present on cell membranes, and can be down regulated after hyperthermia, fact that corroborates to necrosis initiation (MUTAKU et al., 2002).
The results of this work show that the laser itself is capable of generating oxidative damage on biological membranes, and the intensity of lipoperoxidation depends on the laser power used. Indeed, previous data on the literature confirm that irradiation with near infrared laser calibrated on 7 mW is enough to generate reactive oxygen species and compromise membrane integrity (TIRLAPUR et al., 2001). The presence of nanorods doesn’t change the chemiluminescence profile observed for treated tumors, reinforcing the conclusion that laser irradiation – regardless the presence of nanorods – is the main mechanism of damage generation on membranes.
On the other hand, the presence of nanorods is mandatory to generate oxidative stress in a whole cell level. The mobilization of intracellular antioxidants in order to face the oxidative stress generated with hyperthermia is more intense after irradiation of nanorods with a dose of light of 600 J cm-2, as expected after a huge elevation in temperature (HALLIWELL; GUTTERIDGE, 2006).
5. CONCLUSION.
Biochemical and histological data point necrosis as the main cell death pathway activated with hyperthermic therapy, with the membrane damages being caused by laser irradiation itself, while intracellular damages being caused by the heat generated by nanorods activation with near infrared laser.
6. AKNOWLEDGEMENTS
This research project was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo – FAPESP.
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