Laser-Irradiation of Gold Nanoparticles Inside Living Cells: Photochemical Versus Photothermal Effects

Abstract

Gold nanorods are promising nanomaterials for biotechnology innovations that include photo assisted drug delivery, gene therapy, non-invasive cancer detection and therapy, and ultra-sensitive bio detection. Owing to their unique geometry, Au NRs exhibit surface plasmon modes in the near-infrared wavelength range, which is ideal for carrying out optical measurements in biological fluids and tissue. The present study consist of five parts which aim to produce gold nanorods which are suitable for bio medical applications. First part present the gold nanorod synthesis, where the seed-mediated growth method is the most popular method for gold nanorods synthesis, although it is a very complex, sensitive, and irreproducible chemical reaction. Moreover, the anisotropic growth mechanism of the gold nanorods is poorly understood. This limits the ability to control the gold nanorod synthesis procedure, and hence, the size and aspect ratio (length/width) and consequently, the wavelength of the longitudinal surface plasmon resonance (LSPR). The present study is based on varying the seed solution synthesis conditions instead of the growth solution synthesis conditions which are considered problematic. The aim is to investigate the factors that may help in optimizing the synthesis for better yield, reproducibility, optimal LSPR band position and minimize the synthesis time. The results show that often neglected parameters in the seed synthesis conditions affect the final product of the growth steps. These parameters include: (i) Concentration variations of seed solution constitutes (HAuCl4, CTAB, NaBH4 and/or Br-), (ii) Seed solution temperature variations, (iii) Mechanical variations during synthesis; include mixing speed and the method of adding NaBH4 to the seed solution, and (iv) Ageing of the seed solution. The variations in these parameters lead to subtle changes in the seed solutions, resulting in significantly different final products, as evidenced by changes in the position of the longitudinal plasmon band characterized by UV-VIS spectrophotometer and in nanorods TEM images. In this work a robust protocol for the entire procedure is presented in which the growth solution must be kept at a temperature of 270C, the mixing processes must be swirl, the addition rate of the NaBH4 to the HAuCl4/CTAB mixture must be slow and the aging time is 2 hours. This would produce a gold nanorod with LSPR at 800 nm which is considered the best candidate for biomedical applications. Second part discuss the gold nanorod functionalisation, since the CTAB gold nanorods are toxic, and for further in vitro and in vivo experiments the nanorods should be functionalized to be optically stable and biocompatible. In the present study, standard gold nanorods synthesized, which have LSPR position around 800 nm to be used for diagnostic/therapeutic applications. The gold nanorods were functionalized using both thiolated PEG and CALNN-TAT peptide sequence which known as cell penetrating peptide to ensure endocytosis of the gold nanorods inside Mesenchymal stem cells of mice (MSCD1). The characterization of functionalized gold nanorods were done using optical spectroscopy (UV-VIS), electron microscopy (TEM), zeta-potential and both microscopic FTIR and quantitative FTIR. Colloidal stability tests were done for (CTAB-GNRs, thiolated PEG-GNRs, PEG-TAT-GNRs and PEG-CALNN-TAT-GNRs). The colloidal stability tests show a good stability of PEG-CALNN-TAT gold nanorods. Third part show gold nanorod cellular uptake, where different gold nanorods were incubated in Mesenchymal stem cells of mice (MSCD1) to achieve cellular uptake. The cellular uptake was characterized using light microscope (with and without silver-enhanced staining), and transmission electron microscope (TEM). PEG-CALNN-TAT gold nanorods which have been modified by this study show good cellular uptake by endocytosis. To quantify the number of gold nanorods per cell for both different gold nanorods incubation concentrations and different incubation time, both UV-VIS and inductively coupled plasma mass spectrometry (ICP-MS) were used. Quantitative analysis gold nanorod cellular uptake were largely proportional to incubation concentrations and incubation time, quantitative results of different methods were in broad agreement. Fourth part present the photothermal effects resulted after laser irradiation of stem cells incubated with gold nanorods. The photothermal effects include two main applications first; photothermal cell killing, which was imaged using fluorescence microscope, hence photothermal cell death by photothermal was confirmed by temperature simulations, which are mainly based on the quantitative uptake results. Second; photoacoustic imaging using Multi-Spectral Optoacoustic Tomography (MSOT) as photothermal effect was done and it show success of PEG-CALNN-TAT GNRs to work as NIR contrast agent. In Fifth part studies of photochemical effect was presented as 1, 3-Diphenylisobenzofuran (DPBF) dye used to detect singlet oxygen produced by spherical gold nanoparticles and gold nanorods as a result of laser irradiation of gold nanorod/ nanoparticles solution mixed with DPBF. The results confirm that, gold nanorods do not photo generate singlet oxygen - most likely because of the PEG layer used as surface ligand.