Abstract
ABSTRACT|The discovery and development of extrinsic fluorophores, also known as optical probes, was an essential step in understanding the structure and function of biological systems. In fluorescence sensing, organic dyes are used very commonly as optical probes because of their structural diversity, immense variation of chemical, photochemical and spectroscopic properties, versatility, easy availability, and low cost. However, these dyes also have lots of disadvantages like small Stokes shift, short fluorescence lifetime, tendency to degrade or photo-bleach etc. Lanthanide metal complexes are novel class of optical probes that have several advantages over conventional organic dyes. Lanthanide complexes have a large Stokes shift, extremely long luminescence lifetimes, two lanthanides can be employed concurrently for monitoring biological processes simultaneously, ideal for time gated studies, higher quantum yield and are insensitive to photo-bleaching. The main purpose of the present study was to characterize novel lanthanide metal ion chelates for their physicochemical, aerodynamic and in-vitro cellular properties. The novel lanthanide complexes were synthesized using triflate salts of Europium, Dysprosium, Samarium and Terbium lanthanide metals. These lanthanide metal salts were chelated by 1, 4-substituted triazole ligands. The ligands were synthesized using a cycloaddition reaction known as Husigen cycloaddition reaction or Click Chemistry. These complexes were characterized for their solubility, integrity, stability, fluorescence, aerosol properties and cellular toxicity. The lanthanide complexes were found to be fluorescent with their characteristic color (Eu as orange, Dy as blue, Sm as red and Tb as green) under black light. Solubility of 1 mg of complex was checked in 1 mL of variety of common organic solvents. The complexes were assayed for its limited solubility in water/organic solutions. The complexes were insoluble in water. The water insoluble complexes can be solubilized by using mixture of water and organic solvents. Integrity and purity of complexes were analyzed using ESI-MS, UPLC, DSC and TGA. A stock solution of all the complexes containing microgram of samples in a water/acetonitrile mixture (15:85 v/v) were characterized by using electro spray ionization mass spectroscopy (ESI-MS) using a QTrap3200 Mass Spectrometer (Software: Analyst). All the complexes were scanned in the range of m/z = 200 to 1700 and fragmentation pattern was observed. Observed parent mass missing one triflate ions was found out to be same as theoretical mass confirming the successful synthesis of the complexes. 50 µg/mL concentrations of ligand and complex were characterized using a Waters AcquityH UPLC (Software: Empower). The absorbance was measured at 260 nm. The concentration gradient varies from 80% organic at 0 minutes to 20% organic at 1.5 minutes and again comes to 80% organic at 1.9 minutes. Different retention time of complex and ligand in UPLC confirms the integrity of the complexes. Each complex (3-5 mg) was analyzed for their melting points and weight loss due to solvents/moisture content using Shimadzu DSC-60 and Shimadzu TGA-50 respectively. DSC and TGA data also shows that the complexes and ligand are in their pure forms. DSC data proved the integrity of the complexes and confirmed that the ligand and complex are separate entity. The stability and fluorescence properties like Stokes shift, resistance to photo-bleach and quantum yield were analyzed using SynergyH1 Hybrid Multi Mode Microplate Reader (Software: Gen5). The complexes have Stokes shift >150 nm and mean RFU values in thousands that confirmed higher quantum yield. The complexes were found to be stable and resistant to photo-bleaching for 12 hours. Also, the complexes were found to be stable and resistant to photo-bleaching when exposed to pH range of 1-11 for 72 hours. Europium and terbium complexes were nebulized at the rate of 15 L/min for 14 minutes in the NGI to obtain MMAD and % FPF. NGI data confirmed that the complexes can be aerosolized and retain their fluorescence even after nebulization. Cellular cytotoxicity was determined on A-549 lung cancer cell line using MTT cytotoxicity assay. The complexes showed dose dependent cytotoxic effect. Percent viability values were higher for 24 hour incubation than 72 hour incubation. The ease of preparation of complexes, variable fluorescent colors, large Stokes shift, pH stability, resistance to photobleaching, stability to aerosolization, stability to cell culture assay conditions and lack of toxicity make these complexes attractive as a fluorescent probes. However, experiments to improve their water solubility, incorporation into nanoparticles and their uptake in the cells needs to be done.