Laser heating of gold nanoparticles: photothermal cancer cell therapy N.N. Nedyalkov1∗, P. A. Atanasov1, R. A. Toshkova2, E. G. Gardeva2, L. S. Yossifova2, M. T. Alexandrov2, D. Karashanova3 1
Institute of Electronics, Bulgarian Academy of Sciences, Tzarigradsko shousse 72, Sofia 1784, Bulgaria 2 Institute of Experimental Morphology, Pathology and Anthropology, Bulgarian Academy of Sciences, G. Bonchev Street, bl. 25, Sofia 1113, Bulgaria. 3 Institute of Optical materials and technologies, Bulgarian Academy of Sciences, G. Bonchev Street, Sofia 1113, Bulgaria.
ABSTRACT In this work an application of gold nanoparticles in in-vitro photothermal cancer cell therapy is demonstrated. Gold nanoparticles with different diameters – 40, 100 and 200 nm are mixed with HeLa cancer cells. After incubation, the nanoparticles are found to be deposited on the cell’s membrane or enter into the cells. Pulsed laser radiation at wavelength of 532 nm delivered by Nd:YAG system is used to irradiate the samples. The experiments are performed at fluences in the range from 50 mJ/cm2 up to the established safety standard for medical lasers of 100 mJ/cm2. The cell viability as a function of the particle dimensions and laser fluence is estimated. The nanoparticles heating and cooling dynamics is traced by a numerical model based on heat diffusion equation combined with Mie theory for calculation of the optical properties of nanoparticles. The particle response to the nanosecond laser heating is investigated experimentally as gold colloids are irradiated at different fluences. The threshold fluences for particle’s melting and boiling are defined. We show that at the presented fluence range the particles are decomposed into smaller fragments and even short irradiation time leads to decrease of cell viability.
Keywords: gold nanoparticles, photothermal cancer cell therapy 1. INTRODUCTION The increased development of fabrication methods for nanostructures nowadays opens growing efforts in designing of conceptually new applications1. Based on specific properties of matter in nanosized dimensions, nanostructures enters wide range of areas in modern life – from fundamental science of nano-word to commercial applications in cosmetics, sensor system development, medical treatment of diseases with high social impact. Nanosized structures are used in development of surface enhanced Raman spectroscopy2, scanning near field optical microscopy3, nanometer-scale optical twisters4 and applications in medicine5-7 for cancer cell therapy, cell imaging and targeted drug delivery. As a promising object of new applications nanoparticle of noble metals attract a growing interest. In these structures an efficient excitation of collective motion of the electron system (plasmons) can be realized when these interact with electromagnetic field8. The plasmons resonance wavelengths of noble metal nanoparticles are located in the near UV and visible spectral range, thus available laser sources can be used as excitation sources. The efficient plasmon excitation results in drastic enhancement of the coupling efficiency of the particle with the electromagnetic field. This is expressed by an enhancement of the absorption and scattering cross section at resonance wavelength. This enhancement may ensure absorption that is orders of magnitude higher than the conventional organic dyes used as absorbers in biophotonics applications9. In addition, gold nanoparticles are chemically stable, can be easily functionalized, and are not toxic. These properties make gold nanoparticles a promising alternative in photothermal therapy of cancer cells.9, 10 The plasmon resonance wavelength can be efficiently tuned in a wide spectral range by changing the particles size, shape, structure and local environment dielectric properties8. In this way working in tissue transparency window is possible when in-vitro applications are designed. The theoretical and experimental works9-12 devoted to applications of gold ∗
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[email protected] Biophotonics: Photonic Solutions for Better Health Care III, edited by Jürgen Popp, Wolfgang Drexler, Valery V. Tuchin, Dennis L. Matthews, Proc. of SPIE Vol. 8427, 84272P © 2012 SPIE · CCC code: 1605-7422/12/$18 · doi: 10.1117/12.921776 Proc. of SPIE Vol. 8427 84272P-1
nanoparticles in biophotonics show that particle heating up to temperatures high enough to cause cell damage can be achieved at laser fluences lower than the established safety standards. The cell killing mechanism could be related to irreversible biophysical and/or chemical effects, bubble formation and subsequent generation of strong stress waves that modify cell membrane or organelles 13, fragmentation of nanoparticles that produces fragments with high energy 14. The efficient and reliable application of metal nanoparticles in the field of biophotonics also needs information about the particle response to the laser radiation. Different theoretical and experimental studies are performed to investigate the particle melting, re-shaping and fragmentation when they are exposed to laser radiation15-18. Since these modifications will influence the electromagnetic field-particle interaction they will also influence the cell processing efficiency.14 In this work experimental results of gold nanoparticle-assisted photothermal therapy of HeLa human cancer cells are presented. The influence of the particle dimensions and incident laser parameters are studied. The evolution of laser-particle interaction is described in terms of particle temperature and particle phase changes at different fluences used. The obtained results demonstrate the ability of the method and show some optimal conditions for tumor cells treatment.
2.EXPERIMENTAL SETUP 2.1 MTT cytotoxicity assay HeLa cells were trypsinized by 0.25% Trypsin-EDTA and counted using hemocytometer. Cells were transferred to a 96well microtiter plate to ensure concentration 2x104 cells per well. After incubation overnight at 37oC in a humidified air with 5% CO2 to allow cells attachment, medium was changed and cells were treated with gold nanoparticles with various size (40, 100 and 200 nm in diameter) and incubated for another 24h in conditions mentioned above. Then the samples were irradiated with Nd:YAG laser system operated at λ = 532 nm and pulse duration of 15 ns. In these in-vitro experiments the choice of second harmonic of Nd:YAG laser is based on the maximal absorption of gold nanoparticles in this spectral range3. The repetition rate of the laser radiation was 10 Hz. Laser pulses with energy densities, F, from 30 to 100 mJ/cm2 were used in the presented experiment. The samples were irradiated for different time ranging between 5 and 40 s. After that the cells were incubated for 6 h and 24 h, at 37oC in a humidified air with 5% CO2. Control cells were also cultured at the same time. The effect of different treatments on cell viability was assessed by the MTT assay as referred by Mossmann19. After culture, cell number and viability were evaluated by measuring the mitochondrial-dependent conversion of the yellow tetrazolium salt MTT to purple formazan crystals by metabolic active cells. Each variant of treatment was assayed in sevenplicate. Briefly, the HeLa cells were washed twice with PBS (pH 7.4) and further incubated with 100 µl stock solution of MTT (Sigma Chemical Co.) at 37oC for 3 h; the supernatants were aspirated and 100 µl DMSO-Ethanol (1:1) were added to each well to dissolve the resulting formazan. MTT assay reading was performed using ELISA plate reader (TECAN). Significance testing was performed using one-way analysis of variance (ANOVA) followed by Bonferroni’s post hoc test. Values of *p
