Novel fluorescent nano structures for bio-imaging of MCF-7 cells

Although nanomaterials have a wide area of application including targeted drug delivery, diagnostic imaging and controlled release, the design of nanomaterials having multimodal applications remains challenging. The recent trend in the field of nanomedicine revolves around development of in vivo dignostics and therapeutics that can be used as theranostics. In order to achieve this, we performed the optimization of the fabrication process at multiple levels.The major objective of the current work is to tune unique physicochemical properties in nanostructures through proper selection of fabrication methods, that can be used as therapeutic as well as diagnostic tools. Here we choose to develop a fluorescent imaging sensor based on metal oxide nanoparticles that can be used to study the nanoparticle uptake in cells and penetration of nanoparticles through tissue through laser scanning confocal microscopy [1]. Specifically, we choose to use semiconductor quantum dots or metal oxide nanoparticles as they can be designed to have optical, magnetic, and structural properties that are different from the same particles in their bulk form [2]. Here we choose to use ZnO nanoparticles as they are known to provide a targeted delivery which is beneficial for cancer patients as they can differentiate between cancer cell and normal cells, and hence avoid their adverse effects on normal cells.Secondly, we chose to use ZnO nanoparticles because of their excellent bio-compatibility [3]. Another feature of the proposed work is that the targeted approach requires minimal drug loading in contrary to large drug loading required in case of non-targeted approach [4]. The major novelty of the work lies in achieving the multimodalities in a single nanostructure, where we manipulated the optical properties of ZnO so that it can be used as therapeutic reagent as well as imaging sensor to study the cell uptake and penetration of particles. Here we propose a surfactant assisted method to synthsize the fluorescent ZnO nanoparticles. ZnO particles were formed using chemical synthesis method by mixing zinc nitrate hexahydrate and ammonium carbonate solutions along with a surfactant Tween 80 [5]. Since the objective of the work was to maximize the drug efficacy along with maintenance of the fluorescent level of the nanostructures, a series of experiment were performed for nanoparticle fabrication using various concentrations of Tween-80. The result clearly indicates that with increasing amount of Tween 80 present in the mixture, the morphology of the nanoparticles changed along with the fluorescence of the particles. The fluorescent nature of the particles increased with increase in Tween 80 which was evident from the confocal microscope imaging, the best being observed at Tween 80 concentration of 14% (v/v) (ZnOT14) . In the excitation wavelength of 488 nm and an emission range of 508-550 nm, the nanoparticles showed green fluorescence as shown in figure 1. The nanoparticles were further characterized by SEM, TEM, XRD and FTIR techniques. Images from the SEM and TEM show that with change in Tween 80 concentration, the size of the nanoparticles change as demonstrated in figure 2. XRD results confirm the formation of ZnO due to presence of prominent peaks at 31.9°, 34.6°, 36.61°, 47.88°, 68.34°, 69.1° which are in good agreement with JCPDS 89-0511. As per FTIR results, broad peak at 3500 cm -1 indicating the presence of hydroxyl groups intensified on addition of Tween 80, as the surfactant introduces additional hydroxyl groups on the surface of the nanoparticles. The ZnO nanoparticle with Tween 80 concentration of 14% (v/v) was found to be highly haemocompatible (0.031% haemolysis), indicating its non-toxicity. Secondly, the nanoparticles were used for imaging of MCF-7 cells which are a breast cancer cell line. Breast cancer is one of the most common cancer found in women. Breast cancer can lead to metastasis which is the secondary malignant growth from the primary cancer site. With progress in metastases of breast cancer, some organs like lungs, liver and bone get damaged [6]. Thus imaging of the cells by nanoparticles is important as it can lead to proper detection of the cancer tissues. Also the MCF-7 is an immortal cell line which do not proliferate indefinitely and can be grown in vitro. The cells were cultured in a media containing 44.5 ml Dulbecco's modified eagle's medium, 5 ml of 10% fetal bovine serum and 0.5 ml of 1% penstrap antibody in a 50 ml media. The cells were cultured for 4 days. Further the cells were treated with the synthesized ZnO nanoparticles and imaged using confocal microscope