Emodin diffusion in cancerous living cells studied by means of Raman spectroscopy enhanced by plasmon effects

MSc in Photonics PHOTONICSBCN Universitat Politècnica de Catalunya (UPC) Universitat Autònoma de Barcelona (UAB) Universitat de Barcelona (UB) Institut de Ciències Fotòniques (ICFO) http://www.photonicsbcn.eu Master in Photonics MASTER THESIS WORK EMODIN DIFFUSION IN CANCEROUS LIVING CELLS STUDIED BY MEANS OF RAMAN SPECTROSCOPY ENHANCED BY PLASMON EFFECTS Mónica Marro Sánchez Supervised by Prof. Dmitri Petrov, (ICFO, ICREA) Sponsored by Fundació CELLEX Barcelona Presented on date 9th June 2009 Registered at Emodin diffusion in cancerous living cells studied by means of SERS Emodin diffusion in cancerous living cells studied by means of Raman spectroscopy enhanced by plasmon effects. Mónica Marro Sánchez ICFO-Institut de Ciències Fotòniques, Mediterranean Technology Park, 08860 Castelldefels (Barcelona), Spain E-mail: [email protected] Abstract. Emodin (6-methyl-1,3,8-trihydroxyanthraquinone) is considered as a potential candidate for photodynamic therapy for the treatment of cancer. A success in using of the molecule depends among other factors on the knowledge how fast it penetrates through the cell membrane and then diffuses inside the cell. A spatial distribution of emodin inside cancerous cells is also an important factor. The aim of this study is to investigate the diffusion of emodin in cancerous cells by means of a non invasive and sensitive technique: Surface Enhanced Raman scattering which permits us to study the evolution of the small amount of emodin inside single living cells. The experimental results were compared with a theoretical model that simulated the Brownian diffusion of particles. From this comparison and comparing with previous results in fluorescence spectroscopy we have found that: for a long time, emodin is accumulated in the cytoplasm in a region close to the membrane. The result is discussed in terms of emodin being stored in cell organelles located in the cytoplasm. Keywords: Surface Enhanced Raman scattering, Brownian diffusion, emodin, cell, silver probe. 1. Introduction Emodin (figure 1) is a natural occurring anthraquinone present in plants (rhubarb and buckthorn) of common use in traditional medicine and has been shown to have a potent antitumor effect. Previous studies have confirmed that emodin is a natural anti-angiogenic compound [1, 2] and produces a phototoxic effect on tumor cells [1, 3, 4]. For this reason emodin is considered as a potential candidate for photodynamic therapy [5]. However there is little knowledge of how fast it penetrates through the cell membrane and then diffuses inside the cell and the spatial distribution of emodin inside cancerous cells, factors of strong importance for efficiently using emodin for cancer treatments. The aim of the present study is to investigate the diffusion of emodin in cancerous cells by means of a non invasive and sensitive technique: Surface Enhance Raman scattering. Previous studies have been carried out with fluorescence and liquid chromatography [6]. Our research is a complement and permits us to study the cell out of the region of absorption of the molecule. Furthermore, our experiment provides information about the dynamic behaviour of emodin in a specific region of the cell. In our study we present a new way for monitoring the drug diffusion into single living cells and we have extracted some new results that we report in the conclusions. First, the characteristics of Raman spectroscopy and SERS will be revised, followed by an explanation of the experimental setup and the methodology. Next, the experimental results will Emodin diffusion in cancerous living cells studied by means of SERS be discussed along with a program that simulates the brownian diffusion of particles with conditions similar to our experiment. Finally conclusion will be made comparing the results with the previous studies and with simulation and we will propose some new hypothesis of how emodin diffuses and distributes inside the cancerous cell. Figure 1. Chemical structure of emodin 1.1. Raman Spectroscopy Raman spectroscopy is a technique where the vibrational and rotational levels of a sample can be studied. It is based on the observation of inelastic scattering (Raman scattering). In Raman spectroscopy, the sample can be irradiated by a laser beam from UV to IR region (ν0), and the scattered light is analysed. The scattered light consists of two types: Rayleigh scattering, which is a elastic process and therefore the light has the same frequency as the incident beam (ν0), and Raman scattering which is inelastic, considerably weaker (1 over 107 incident photons are Raman scattered photons) and has frequencies different than the incident light. Raman scattering can be of two types: Stokes line with frequency ν0-νm and anti-Stokes line with ν0+νm (figure 2). In Raman Spectroscopy, the peaks are measured as a shift with respect to the incident beam frequency (ν0). It leads to a spectrum that is a fingerprint of the molecule used. Raman scattering can be explained by classical theory. The incident light is described as a electromagnetic wave (laser beam) where the electric field strength associated fluctuates with time (t) as: (1.1) where E0 is the vibrational amplitude and ν0 is the frequency of the incident light. The interaction of this incident light with a diatomic molecule results in an electric dipole moment P induced in the molecule: (1.2) which is proportional to the electric field strength with a proportionality constant α called polarizability. If the molecule has a vibrational frequency νm, the nuclear displacement q can be described as: (1.3) where q0 is the vibrational amplitude. In the case of small amplitude of vibration, α is a linear function of q: (1.4) where, α0 is the polarizability at the equilibrium position, and is the rate of change of α with respect to the change in q, evaluated at the equilibrium position. If we combine (1.2, 1.3 and 1.4), we obtain the dipole moment induced is: (1.5) From this equation it can be seen three terms: the first term represents an oscillating dipole that radiates light of frequency ν0 (Rayleigh scattering), the second term corresponds to the Raman scattering of frequency ν0 + νm (anti-Stokes) and the third term also represents Raman scattering with a frequency ν0νm (Stokes). From (1.5), if is equal to zero, the vibration is not Emodin diffusion in cancerous living cells studied by means of SERS Raman-active. Thus, the rate of change of polarizability (α) with the vibration must not to be zero for having a vibration Raman-active. Figure 2. Comparison of energy levels for the Stokes, Rayleigh and anti-Stokes Raman spectra. Figure 2 illustrates the different types of scattering in terms of the energy level in a molecule. In classical Raman spectroscopy, the energy of the excitation line is smaller than the first electronic excited state. The dotted line indicates a “virtual state” to distinguish it from the real excited state. According to Maxwell-Boltzmann distribution law, the probability to find a molecule in the ground state is larger than at excited vibrational states under normal conditions. Due to this, the Stokes (S) lines are stronger than the anti-Stokes (A) lines under normal conditions. As from both the same information can be obtained, normally only the Stokes side of the spectrum is measured because it gives stronger intensity. Resonance Raman (RR) occurs when the exciting light wavelength is near to the maximum absorption region of the studied sample. 1.2. Surface Enhanced Raman Spectroscopy Raman spectroscopy is a very useful technique in various research fields because provide us structural information of the sample used. However Raman signals are weak and therefore we are not able to investigate substances in low concentrations. One possible solution is surfaceenhanced Raman scattering. Within a few years, strongly enhanced Raman signals were found for many different molecules, which had been attached to “SERS-active substrates” [8]. These SERS-active substrates are different types of metallic structures with sizes on the order of tens of nanometers: colloidal silver or gold particles in the 10150 nm size range, silver or gold electrodes or evaporated films of these metals. By means of SERS-active substrates Raman signal can be enhanced by a factor of [9], depending on the applied method. In order to enhance the signal of a molecule, it has to be adsorbed on metal surface or placed a few nanometres of distance (20-50 nm) to the SERS-active substrate. Due to this we are able to acquire signal from a very specific point of our sample. The mechanism leading to the surface enhancement is not completely understood yet. In general there are two main contributions: the electromagnetic and the chemical charge transfer (CT) mechanism. 1.2.1 Electromagnetic enhancement. The electromagnetic contribution of the SERS enhancement is based on a plasmon excitation due to the incident laser light on the metal surface. This incident light excites the electrons in the metal which oscillate against the metal cores (called a surface plasmon). Having nanostructured surfaces, the oscillation of surface plasmon lead to an electromagnetic field, which reaches out of the metal surface, where the analyte is located. Therefore, in electromagnetic enhancement is not necessary to have attached the analyte. Due to the fact that the incident laser light has to excite the surface plasmon, it has to be adapted for the plasmon wavelength of each metal and the nanostructure of the metal surface. In order to accomplish this using the most common metals as SERS-active substrates (like silver and gold), SERS excitation lines cover mainly the visible spectral region up to the near infrared (NIR) between 450 and 1064 nm [10]. Excitation with UV light has also been reported [11]. 1.2.2 Chemical enhancement. Experiments revealed that there was a second enhancement mechanism [20]. A resonance Raman mechanism can explain the observations and there are two Emodin diffusion in cancerous living cells studied by means of SERS hypotesis (a) in the adsorbate the electronic states are shifted and broadened due to their interaction with the surface or (b) new electronic states are created because of the chemisorption and they serve as resonant intermediate states in Raman scattering. The results of experiments support the second hypothesis. Normally, the highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) of the adsorbate are symmetrically placed in energy with respect to the Fermi level of the metal. With this configuration charge-transfer between the metal and the molecule can occur at about half the energy of the intrinsic difference in energy levels of the adsorbate. Due to this charge transfer, in chemical enhancement the analyte has to be in contact with the metal surface. 2. Experiment and methods 2.1. Experimental setup Figure 3 represents the setup which consists of a 785 nm laser beam for Raman excitation with a power of about 5-7 mW at the sample. Firstly, laser light passes through a band pass filter and a beam expander and arrives to a notch filter. Notch filter has a cut-off frequency of 785nm and therefore transmission for this wavelength is zero. On the contrary the reflection is almost 100% for 785nm. From notch filter the light arrives to a 100x 1.3 numerical aperture oil immersion objective. The size of the focal spot was estimated to be 0.5 µm perpendicular to and 1 µm along the optical axis, i.e., less than the probe diameter. We use the backscattered geometry for collecting the Raman spectra. The backscattered light is collected by the objective and arrives to the dichroic mirror. There, a part of the light arrives to the CCD where we observe the real image from the microscope (figure 4). The rest of the light is reflected and passes through the Notch filter which finally removes the light with the same frequency than the excitation light. Our system has a confocal system formed by two lenses and one pinhole (150µm of diameter) that only allow passing light from the plane (confocal volume) [12] where the excitation laser is focused on the sample. Finally, the Raman scattered light is focused on the slit of the spectrometer (Acton Research, SpectraPro 2500i) and the Raman spectrum is recorded with a CCD camera with a spectral resolution of 3 cm-1. In our case we use a grating of 600 lines/mm and blazed on 750 nm which means that at a wavelength 750 nm the efficiency is maximum (70%) and therefore we have almost all the energy concentrated at the zero order (for this wavelength) and less energy in higher orders. Figure 3. Experimental setup The CCD of the spectrometer is cooled down to -100ºC and uses thermoelectric cooling. It uses the Peltier effect to create a heat flux between the junction of two different types of materials. A Peltier cooler, is a solid-state active heat pump which transfers heat from one side of the device to the other side against the temperature gradient, with consumption of electrical energy. During the experiment the sample is incubated in the sample holder with following conditions: a temperature of 37ºC and a controlled flux of 5% of CO2 and 40% of humidity. Emodin diffusion in cancerous living cells studied by means of SERS 2.2. Sample preparation Human glioma cells (U-87 MG line) were cultured as a monolayer and were grown in Dulbeccos modified Eagle medium (DMEM) containing L-glutamine (862 mg/L), sodium pyruvate (110 mg/L) and glucose (4500 mg/L), supplemented with 10 % fetal calf serum, penicillin (50 µg/mL) and streptomycin (50 µg/mL). Cells were maintained at 37 °C in a humidified 5 % CO2 atmosphere. One day prior to experiments, cells were plated in plastic petri dishes (35 mm with a #0 thick cover glass on the bottom) at an optimal density of 4 x 105 cells/mL and incubated with 2 µm size silver covered silica bead during the night. During this time cells absorb beads and they are stored in the cytoplasm near to the membrane. In the experiments silver covered silica beads were used as probes because silver colloids on the surface acted as SERS-active substrates. This Silver covered metal beads were prepared by modification of the silica surface with APTMS ((3-aminopropyl)trimethoxysilane). Silver colloid nanosize particles were prepared by well known method described by Lee and Meisel [13]. Modified beads were mixed with metal nanoparticles to obtain a homogeneous coverage of metal on the bead [14]. Figure 4. View of the cell with a bead inside. Left: the cell membrane of the cell is focused and we are not able to distinguish the bead. Right: Membrane is defocused and we can observe the bead. 2.3. Methodology In order to measure the diffusion of emodin inside the cell we used 2 µm silica bead covered with silver colloids [14] as SERS-active substrate for detecting and measuring the Raman spectra of emodin. Those beads were absorbed by the cell and stored close to the membrane in the cytoplasm. This technique permitted us to detect the evolution in time of the amount of emodin in a specific region of the living cell. In order to obtain better and contrasted results we used two methods: The cells were pre-incubated during 4 hours with a fresh culture medium containing 2 µM concentration of emodin. After incubation the cells were rewashed three times in a culture medium. During experiments, the cells were enclosed in the sample holder and the excitation beam is focused onto a bead situated inside of the cell. While the sample was incubated in the sample holder, the excitation beam was focused onto a bead situated inside of the cell. Later, a drop of emodin (2 µM) was added in the solution of the cells. At this time, we start to detect the presence of emodin in the region of interest. 2.4. Data analysis In the collected Raman spectra there are various types of noise. One of them is the background which is removed with a Mathematica software based on the established method [15]. In order to remove the spikes, select the desired peaks and plot the time dependence of the intensity of the emodin peaks we used Origin 7. 2.5 Simulation In a theoretical model we have modified a program from F.Salvat (Universitat de Barcelona, Facultat de Física, Estructura i constituents de la matèria) that simulates the Brownian motion Emodin diffusion in cancerous living cells studied by means of SERS of particles. We modified this software obtaining the diffusion of particles in a specific region (detection region) of a volume (cell and external media) and changing the conditions of the cell environment: temperature, diffusion coefficient and initial concentration of particles. 2.5.1. Brownian diffusion simulation In this software we simulate numerically the Brownian diffusion; it means the particles that suffer thermal fluctuations and friction. The most general equation to describe this type of movement is the Langevin equation, (2.1) where m is the mass of the particle, v his velocity and is the thermal noise. This last term is a random gaussian process. Here, we have considered the limit of high friction, in which the Langevin equation is reduced to: , (2.2) where is a random gaussian process with zero average and correlation (2.3) being the cartesian components of . The variance of each component is (2.4) The program simulates the equation 2.2 with a gaussian noise that has the property 2.3. In order to be able to visualize the results, we consider the movement in a plane. It can be prove that if we analyze the random trajectories of a high collectivity of particles: The mean value of the position remain constant The movement is diffusive, with a diffusion coefficient D whose components are: This is the Einstein’s relation, . The simulation algorithm is the following. We consider NP independent particles that depart the origin at the instant t=0 and their movement follow the equations 2.2 and 2.3. We calculate the evolution by measuring their displacement during small time steps Δt. In each step, the coordinates of the particle n are modified in accordance with: where the components Δx and Δy of the displacement are random gaussian numbers with zero average and variance equal to . 2. Results 3.1. Experimental results First of all, in order to identify and know the characteristic and better peaks for emodin in the samples, we obtained separately the Raman spectra of emodin M and the Raman spectra of cell, both with a metal covered bead in the confocal volume of the spectrometer (figure 5). From the graphs it can be observed that for 1170cm-1 and for 1334cm-1 Raman spectrum of emodin has peaks [16] as well as that the Raman spectrum of cell does not have peaks in this region. For this reason we have chosen these two peaks in order to analyze the behaviour of the quantity of emodin in the detection volume over time. Finally, by following the procedures reported in the methodology, we obtained the graphs of figure 6. The two methodologies that have been used permit us contrast the results. With the first method a considerable amount of data can be obtained because it is possible to make an average of various cells in one single experiment. However, experiments have to be carried out in a very short time due to the fact that emodin can diffuse out of the cell because we wash the sample and put new media without the presence of emodin. Emodin diffusion in cancerous living cells studied by means of SERS Figure 5. Raman spectrum of emodin ( M) (red line) and cell (black line) both measured in the presence of a metal covered bead. The second method describes the natural diffusion in a way more close to the reality and permits us to observe the behaviour of the Raman intensity in each minute. However it is not possible to do an average of various cells in the same experiment because the Raman excitation is carried out only in one single cell in each experiment. Therefore we obtain more temporal resolution but we have to perform more experiments and samples in order to have statistically significant results. Figure 6. Distribution of main Raman bands of emodin inside cell over incubation time following the first (a) and the second (b) method. Emodin diffusion in cancerous living cells studied by means of SERS 3.2. Simulation results In order to simulate as real situation with conditions similar to our problems, we defined a cell with 100 in size and the parameters of the configuration were: due to the fact that this is a common value for biomolecules like emodin diffusing in a liquid similar to water and with a temperature around 370C. Therefore: (with T=370C). 105 particles Membrane situated at 500 of the origin where the drop of particles starts to move and a region of detection of 1 of width. 2200 screenshots that corresponds to 308 minutes. The program simulates that at the initial time all the molecules are in the origin of coordinates. The region to be studied that represent the zone where the bead can detect the presence of emodin is situated at 500 of the origin and is 1 of width. We also simulated that the particles could not escape out of a volume of 3x3 cm. However, more sophisticated conditions could be imposed. From figure 7 it can be seen the results of this simulation. Figure 7. Theoretical simulation of diffusion of particles through the defined volume Other simulations with the same code were carried out in order to probe the different behaviour for different temperatures (different diffusion coefficients D). It can be seen from the graphs below (figure 8) that the higher the diffusion coefficient is, the more rapid the diffusion of particles is. Figure 8. Different behaviour for different diffusion coefficients. The studied region was situated at 10 µm of the origin with 5 µm of width. Left: number of particles that pass the studied region over time. Right: number of particles that we obtain in the region of study over time.