Design, Synthesis And Bio-application Of Water-soluble Perylenediimides

Perylenediimide derivatives (PDIs) are a class of polycyclic aromatic compounds with strong fluorescence, which display exceptional chemical, thermal, photochemical stability with high fluorescence quantum yields in organic solvents. To date, PDIs have been widely used in various fields, such as dye lasers, solar cells and photovoltaic devices. However, the poor solubility and very weak fluorescence of PDIs in an aqueous environment have hindered their utilization in the biological and medicinal fields. Therefore, the improvement of the water solubility of PDIs became a very important issue, which would benefit for their biological applications. In this thesis, a series of high-soluble and high fluorescent PDIs have been designed and synthesized, and their bio-applications have been studied. First, dendrimers, a class of perfectly branched polymers with well-defined three-dimensional shape and large number of cavities, have been widely used in the biological and medicinal fields. The previous reported PDI-cored dendrimers were water-soluble, biocompatible, neutral and with a nonionic character. To date, those PDI-cored dendrimers have not been explored as drug carriers or gene carriers. Therefore, we synthesized water-soluble PDI-cored cationic polyesters dendrimers with different generations. Their optical properties in water were investigated. The cell-penetrating abilities of these dendrimers and the gene transfection efficacies were assayed with live cells by fluorescent tracing the complexes of dendrimer and DNA. Second, the hydrophobic nature of outer polyesters leads to the relatively low water solubility of the previous PDI-cored dendrimers. And the primary amine-terminated PDI-cored polyester dendrimers easily undergo aminolysis of their ester groups during storage, thus they need to be treated with diluted HC1to yield their ammonium salts for better stability. Therefore, in this work, highly water-soluble poly(amido amine)(PAmAm) dendritic units were incorporated into the bay regions of PDIs and synthesized highly water-soluble PDI-cored poly(amido amine) dendrimer with peripheral amine groups (PDI-PAmAm). The optical properties of PDI-PAmAm in water were investigated. The cell-penetrating ability and the gene transfection efficacy of PDI-PAmAm were assayed both in vitro and in vivo. Third, PDIs contain a rigid and planar aromatic scaffold, however, to date, PDIs have not been explored as DNA intercalators with high enrichment in cell nuclei and to inhibit cancer cells and tumors. Therefore, we hypothesized that water-soluble PDIs could be designed as DNA intercalators. And their cellular distribution and anticancer activity were investigated. The main results of this thesis are as following:1. A series of water-soluble PDI-cored cationic polyester dendrimers with different generations (G1, G2, and G3) have been synthesized via "click" reaction. These dendrimers were fully characterized by NMR spectroscopy (’H NMR,13C NMR) and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) or gel permeation chromatography (GPC) analysis. The aggregation of the encapsulated PDI chromophores was noticeably suppressed by outer cationic dendrimers, thus leading to the enhancement of optical performances in water. Increased with the dendrimers generation, the fluorescence_quantum yields and excited state life-time of dendrimers were increased. TaliTM viabilty assay showed that pure dendrimers and dendrimers/DNA complexes exhibit lower toxicity against Drosophila S2cells. Cell-uptake experiments showed that all dendrimers were able to internalize into Drosophila S2cells, and the cellular uptake ability and the transfection efficacy of dendrimers increase with increasing dendrimers generation. These dendrimers have remakable gene transfection efficiency with low cytotoxicity, would be applied to live animal to explore the potential genetherapy in ongoing experiments.2. A highly water-soluble perylenediimide-cored poly(amido amine) dendrimers (PDI-PAmAm) with peripheral amine groups via a Michael addition reaction has been synthesized. It was fully characterized by NMR spectroscopy (1H NMR,13C NMR) and GPC analysis. The aggregation of the encapsulated PDI chromophore was noticeably suppressed by outer PAmAm shells, thus leading to the enhancement of optical performance of PDI in water. PDI-PAmAm showed excellent water solubility and high FQY in aqueous solution. Gel retardation test showed that PDI-PAmAm can prevent DNA band from moving in the gel at an N/P>1. The mean diameters of the complexes were found to be in the range of100-200nm. The Zeta potentials of PDI-PAmAm/DNA complex increased with the increase of N/P ratios, indicating the efficient binding of PDI-PAmAm and DNA and facilitates cellular uptake. TaliTM viabilty assay showed that PDI-PAmAm and PDI-PAmAm/DNA complexes exhibit lower toxicity against Drosophila S2cells. Cell-uptake experiments showed that PDI-PAmAm can be rapidly internalized into Drosophila S2live cells at low concentration (2μM), while higher concentration was required when as-synthetic cationic polyester dendrimers were applied inside live cells. The in vitro gene transfection studies in Drosophila S2cells showed that PDI-PAmAm showed effective transfection activity at all N/P ratios. The gene delivery efficacy of PDI-PAmAm was better than that of the previous reported fluorescent nanoparticle (FNP) and cationic polyester dendrimers (G1and G2). The in vivo conditions test showed that PDI-PAmAm, as an efficient gene carrier delivered dsRNA, rapidly entered into living insect cells resulting in inhibition of insect pests’development. PDI-PAmAm shows great performance of gene transfection and gene interference with fluorescent traceable in both in vitro and in vivo experiments.3. Five water-soluble ionic perylene derivatives were designed and synthesized as DNA intercalators. They were fully characterized by’H NMR and13C NMR spectroscopy. In this study, we chose the perylene-based DNA intercalator (PBDI) as a research object, which bearing two tertiary ammonium salts in the imides. PBDI showed excellent water solubility and high FQY in aqueous solution. Absorption, emission and CD spectral analyses as well as viscosity measurements demonstrated that PBDI can efficiently intercalate into the base pairs of double-stranded DNA and serves as an effective DNA intercalating agent. The fluorescence quenching technique suggested that PBDI was a more effective intercalator than commercial DNA intercalator amonafide, and the apparent binding constant (Kb) of PBDI and amonafide with DNA were1.89×106and1.02×105M-1, respectively. The cellular distribution of PBDI showed that PBDI was exclusively distributed in the cell nuclei in all tested tissues, the PBDI staining pattern was essentially coincident with the commercial nuclear dye4’,6-diamidino-2-phenylindole (DAPI),this implies that PBDI can accumulate in the cell nuclei. MTT assay showed that PBDI was superior to amonafide in suppressing cancer cells and tumors because of the excellent water solubility and specific nuclear accumulation of PBDI. The in vivo experiments supported that the novel DNA intercalator PBDI was a potentially efficient drug with high capacity for the suppression of cancer cells and tumors. PBDI is the first example of PDIs designed as DNA intercalators that efficiently localize to cell nuclei and inhibits cancer cells and tumors.