Sami Khouri Salameh,1 Meghan Ruppel,2 Abdel Mamoon,3 and Lisa Miller2

Using Infrared Micro-Spectroscopy to Assess the Effectiveness of Indocyanine Green as a Photodynamic Therapy Agent for the Treatment of Skin Melanoma

1Ben Gurion University; 2Brookhaven National Laboratory; 3Egyptian Atomic Energy Commission

Abstract

The goal of this project was to assess the use of indocyanine green (ICG) as a photodynamic therapy (PDT) agent for skin melanoma. The research involved (1) determining the toxicity of the dye on human melanoma cells in culture, and (2) testing the impact of the dye as a PDT agent by activation with a Ti-Sapphire laser (830 nm). ICG is a promising PDT agent because it absorbs near infrared light, which has a long penetration depth in tissue. In addition, photoactivation of ICG results in the release of oxygen radicals that interact with the cell DNA causing damage to it, which leads to death of the cells. In order to monitor the cytotoxicity of the dye as well as the influence of photodynamic therapy, we use an infrared (IR) microscope that enables us to probe the nature and magnitude of the structural damage of the cell’s ingredients such as proteins, lipids, and nucleic acids. We also use a light microscope with trypan blue staining to monitor the fraction of dead cells in the culture. Results show that the dye can be toxic to the cells at high concentrations, but lower concentrations are not. We find that the optimal concentration is 100 µM which is the highest concentration useable where the ratio of dead cells is minimal. Results of PDT show that activation at 830 nm reveals chemical changes in the cells consistent with apoptosis, indicating that the dye has the potential to be an effective PDT agent.

Introduction

Ultraviolet light from the sun is the main cause of skin cancer melanoma. The occurrence of skin melanoma is becoming more common. One potential method for treating skin cancer is through light-sensitive topical agents. By applying these agents to the affected area, and activating them with light, the cancerous area can be preferentially treated, leaving the surrounding skin unaffected.

The photosensitive dye in our research is indocyanine green (ICG). The chemical structure is shown in Figure 1. The dye is activated by light in the near-infrared region, wavelengths that penetrate several millimeters into the skin. Activation of the dye produces oxygen radicals that can initiation apoptosis (i.e. cell death). Thus, the long penetration length and apoptosis-inducing action of this dye make it a promising photodynamic therapy agent for skin melanoma. The dye exists in two forms, monomeric and oligomeric, which give two different absorption maxima. The near infrared spectrum shows two main peaks at 778 nm and at 708 nm (Figure 3). The monomeric form absorbs at 778 nm (molar coefficient of 10800 M-1cm-1) and the oligomeric form absorbs at 708 nm.

The equilibrium between the two forms depends on the conditions of dye, such as pH, solvent, temperature, and concentration. The monomeric form of ICG decreases with the increasing ICG concentration and the absorbtion shifts toward the shorter wavelengths.

At the concentrations used in this experiment, the dye exists in primarily the monomeric form. Thus, we excite the dye with a Ti-Sapphire laser at 780 nm. Excitation causes the production of oxygen radicals that leads to cell death.

Theory of infrared absorption spectroscopy

IR radiation does not have enough energy to induce electronic transitions as seen with ultraviolet light. Absorption of IR arises from vibrational and rotational excitations in molecules.

For a molecule to absorb IR, the vibrations or rotations within a molecule must cause a net change in the dipole moment of the molecule. The alternating electrical field of the light interacts with fluctuations in the dipole moment of the molecule. If the frequency of the radiation matches the vibrational frequency of the molecule then radiation will be absorbed, causing a change in the amplitude of molecular vibration. The frequencies at which there are absorptions of IR radiation ("peaks" or "bands") can be correlated directly to bonds within the compound. Biological samples consist of materials such as proteins, lipids, and nucleic acids. Since they all have different chemical structures, they have unique IR spectra. A typical IR spectrum of a biological cell can be seen in Figure 4. The characteristic features of the spectrum are labeled. The absorbance features between 2800 – 3000 cm-1 arise primarily due to lipids in the sample. The peaks centered around 1650 and 1550 cm-1 are attributed to proteins, and the peaks around 1240 and 1100 cm-1 are assigned to nucleic acids.

Absorbance Lipid protein nucleic acid

4000 3500 3000 2500 2000 1500 1000

Frequency (cm-1)

Figure 4. A typical IR spectrum of a biological cell.

Materials and Methods

The sample: Our experiment used six cell cultures dishes that each contained about 0.5 ml of HTB-72 skin cancer cells/media. The cells were incubated in specific conditions at 370 C and 1 atm with 5% CO2, which are typical in vivo conditions for growing cells.

The dye: Indocyanine Green (ICG) was used in varying concentrations from 0 – 300 µM. The solution was prepared in PBS buffer under low light conditions so as not to activate the dye prematurely.

The cytotoxicity of the dye: ICG is toxic to cells at high concentrations. In order to avoid the killing of the cells as a result of excessive dose of the dye, we tested the cytotoxicity by adding different concentrations of the dye to the cells starting from 0 µM to 300 µM with intervals of 50 µM. We incubated the cultures with proper media in the dark for 24 hours and then we counted the fraction of dead cells using trypan blue staining.

Photodynamic effect measurements: After selecting the optimum concentration of dye, we irradiated the cells with laser light 2 at λex = 830 nm and at a fluence rate of 100 or 200 mW/cm with dose from 30 to 100 J/ cm2. After irradiating the cells they were incubated for 48 h. Trypan blue staining was performed and then the fraction of dead cells was determined.

Instrumentation

We used trypan blue staining, a hemocytometer, and light microscopy to count the fraction of dead cells for both monitoring the cytotoxicity of ICG and the effect of the PDT. We studied the effect of chemical composition on the cells after ICG treatment and PDT using a Perkin Elmer IR microscope. The method that we use for IR microspectroscopy is a Reflective mode which is the second most common mode after the Transmission one. In this mode the disadvantage is the loss up to half of the incident IR light because of the use of mirror for reflecting the reflected IR light (see Figure 5).

Figure 5. Reflection geometry of an infrared microscope.

For laser irradiation, we use a Ti-Sapphire green (780 nm) where this wavelength is close to IR part thing that allows deep penetrating into tissues.

Results and Discussion

The results of cell counting for the ICG cytotoxicity assay are found in Table 1.

Table 1: Dye concentration (µM) Living cells Dead cells Ratio (Dead/Total) Control 154 15 0.088 50 147 86 0.369 100 108 52 0.325 150 129 67 0.341 200 62 92 0.597 250 46 84 0.646 300 60 115 0.657

From the results, the cytotoxic effect of the cells can be seen. Without any dye, less than 10% of the cells died. With concentrations below 200 µM, approximately 1/3 of the cells died. Above 200 µM, over half the cells did not survive. Thus, we chose a concentration of 100µM for the PDT experiment, which is the highest concentration of dye that produced the lowest ratio of dead cells, which indicates that it is the optimum concetration with minimum cytotoxicity. Collection of IR spectra from the ICG-treated cells reveal the change of the chemical composition of the ICG treated cells that cannot be seen with the visible light microscope, where we can see just dead or alive cells.

IR micro-spectroscopy of the ICG-treated cells shows different peak absorbances that indicate different chemical structures in the cell itself. The spectra also show a change in the magnitude of the cell absorption due to the changing dye concentration. These results indicate that the dye changes the intensity and the health of the cell beyond certain concentration, which is predicted to be 100 µM because that concentration seems to be a optimum concentration with minimum toxicity as mentioned before.

Also, for cells containing a constant ICG concentration (100 µM) and laser-irradiated, we detect a changing/decrease of the absorbance and decrease of survival cells as a function of the laser dose that we would delived to the dyed cells (see Figure 6). 0 2 . 0 s t i n 5 U

1 . e 0 c n a b r o s 0 b 1 . A 0 5 0 . 0

3500 3000 2500 2000 1500 1000 Wavenumber cm-1

\\Spotlight\spectra\sami\Av2.0 Spectrum at -3063,15394,-791 Micrometers Aperture: 35,35 | Av. of 44 2005/8/5 \\Spotlight\spectra\sami\Av3.0 Spectrum at 457,11093,-781 Micrometers Aperture: 35,35 | Av. of 45 2005/8/5 \\Spotlight\spectra\sami\avg1.0 Spectrum at -8883,5881,58 Micrometers Aperture: 35,35 | Av. of 97 2005/8/4

Figure 6. Infrared spectra of irradiated cells with 100 µM ICG and 150 J laser power.

In Figure 6, we see different spectra for different doses of ICG or laser. The pink spectrum represents cells without ICG and laser, blue represents cells with 100 µM ICG and 150 J laser, and red represents cells with 100 µM and no laser. From these data we performed a cluster analysis (Figure 7) and find that unirradiated cells (dye, no laser) cluster closely to the control cells (no dye, no laser), whereas increasing laser dose separates the clusters as a function of increasing laser dose. Figure 7. Cluster analysis of PDT-treated cells.

Conclusions

We have seen that the ICG have an effect onto the cells and caused a damage and death of the cells for two reasons : (1) the cytotoxicity of the dye itself, and (2) by provoking the dye with light and emmiting oxigen radicals that destroy a different structures into the cells and leads to cell death.

References

Lisa M.Miller. Infrared Microspectroscopy and Imaging. Imaging at the NSLS Workshop, May 2002.

Urbanska K, Romanowska-Dixon B, Matuszak Z, Oszajca J, Nowak-Sliwinsk P, Stochel G. Indocyanine green as a prospective sensitizer for photodynamic therapy of melanomas. Acta Biochimica Polonica 49 (2), 387-391 (2002). www.shu.ac.uk/schools/sci/chem/tutorials/molspec

Acknowledgements

I would like to thank the US Department of Energy Initiative for the Middle East for the financial support during this training; the support and encouragement of Professor Brenda Laster and Dr. Herman Winick; Meghan Ruppel, Jeff Borack, Randy Smith, and Sydell Lamb for their guidance during the experimental aspects of this work. Dr. Lisa Miller, Dr. Vivian Stojanoff, and Dr. Zhong Zhong for receiving us in their groups. I'm grateful to the NSLS staff without which this training would not have been possible. The National Synchrotron Light Source is supported by the U.S. Department of Energy, Office of Science, and Office of Basic Energy Sciences, under Contract No. DE-AC02- 98CH10886.