Novel Water-soluble Photosensitizers As Antitumor Agents For Photodynamic Therapy

Tumor is a difficult problem for the medicinal world to conquer. Though a lot of progress have been achieved in traditional cancer therapeutic methods such as surgery, radiotherapy, chemotherapy and immunotherapy, however the practice demands more novel efficient approaches for caner treatment. Photodynamic therapy (PDT), a newly emerging non-invasive cancer treatment method is based on the combined interaction of photosensitizer, oxygen and light. While in this modality the crucial element is photosensitizer. Up to now the clinical photosensitizers still have some shortcomings, such as low cell uptake, slow metabolism, poor targeting and retarded phototoxicity, etc. Two new series of water-soluble photosensitive compounds synthesized in our laboratory are evaluated in this thesis:aminopolycarboxylic acid conjugated with tetraphenyl-porphyrins and saccharide conjugated with boron dipyrromethenes (BODIPY). The studies covers physical and chemical properties, in vitro and in vivo antitumor activity, antitumor mechanism. The goal of this work is to reveal structure-activity relationship of photosensitizers in these two chemical systems and to find high efficiency and low toxicity drug candidates for PDT.Part one:Aminopolycarboxglic acid complexes with tetraphenylporphyrinsCompounds A1-9:①High performance liquid chromatography(HPLC) was exployed to perform the quantitative analysis of compounds A1-9, the results indicated that the purity of compound A1-9were over95%.②The photobleaching rate, singlet oxygen yield and lipid-water partition coefficient were studied by UV spectrophotometry, the results showed compound A1-9was stable to light illumination, singlet oxygen yield was among0.41-0.61and lipid-water partition coefficient was among1.79-2.96.③The cell uptake experiment was conducted for compound A1-9in cell line Hep G2(human hepatoma), cell line HGC-27(human gastric cancer), cell line BGC-823(human gastric cancer) and cell line A549(human non-small cell lung cancer) by the spectrofluorimetry, the results showed that they were similar in the four tumor cell lines at condition of0.1-0.4μM/1×104cells in20μM dosage.④Cellular localization of compound A1-9in the four tumor cell lines were observed by fluorescence microscopy and confocal laser scanning microscope, the results showed they mainly located in the lysosomes.⑤MTT experiments in above four tumor cell lines showed dark toxicity of compound A1-9was low during1.25-20μM, however followed with laser irradiation, these compounds could significantly inhibit the proliferation of liver, gastric and lung cancer cells, the best phototoxicity compound was A7, whose maximum inhibition rate in the four tumor cells were above84%and IC50below0.89μM respectively.Compound A10is studied in more details as the hit compound:①The selective killing effect among tumor cells and normal cells were evaluated on cell line NCI-H460(human large cell lung cancer), cell line U-2OS (human osteosarcoma), cell line ECV-304(human umbilical vein endothelial), FB (human hypertrophic scar fibroblasts) and cell line HaCat (immortalized human epidermal), the results showed that A10-PDT could selectively kill tumor cells, whose maximum inhibition rates were above71%and IC50below6μM on two tumor cells, while maximum inhibition rates less than24%and IC50above20μM on three normal cells.②A10-PDT influenced on cell apoptosis and cell cycle were detected by flow cytometry, the results showed A10-PDT could make Hep G2cells blocked in G0/G1phase and induce cell line Hep G2and cell line BGC-823cells to apoptosis with dose-dependent relationship.③Generally pharmaceutical acts via interaction with protein, here BSA as model protein was chosen to investigate this interaction with hit compound A10by ITC and CD spectrum, the results showed hit compound A10could lead to the BSA secondary structural elements alpha-Helix content decrease and beta-Turn content increase via ensemble effect of hydrogen bonding, van der Waals force and electrostatic attraction.④Maximum dose as the index of acute toxicity on medicines was usually reported, here A10-PDT at2000mg/kg dose in Kunming mice showed A10-PDT had no risk of severe acute toxicity effects, while the working dose was20mg/kg. These results demonstrated A10-PDT is good enough for clinical application.⑤Mice bearing either H22liver cancer or Lewis lung cancer, nude mice bearing BGC-823respectively were used as animal model to evaluate A10-PDT in vivo, the results showed maximum tumor inhibition rate of A10-PDT on mice bearing Lewis lung cancer was81.52%, on H22liver cancer was95.15%at20mg/kg dosage. It could inhibit the tumor proliferation of nude mice bearing human gastric cancer BGC-823at5,10mg/kg respectively.⑥The tissue distribution of hit compound A10in tumor-bearing mice was explored by fluorescence imaging and frozen section method, the results showed after intravenous injection24h, the concentration of hit compound A10in the different organs decrease in the following order:tumor>liver>lung>kidney> spleen>heart, during12-48h the concentration of hit compound A10in tumor was at a high level. Part two:Saccharide group complexes with boron dipyrromethenesCompounds B1-6:①The photobleaching rate, singlet oxygen yield and lipid-water partition coefficient were studied by UV spectrophotometry, the results showed compound B1-6was stable to light illumination, singlet oxygen yield was among0.073-0.29and lipid-water partition coefficient was among0.37-2.69.②Cellular localization of compound B1-6in in cell line NCI-H460(human large cell lung cancer), cell line Hep G2(human hepatoma), cell line HGC-27(human gastric cancer), cell line LO2(human liver cell) were observed by fluorescence microscopy and confocal laser scanning microscope, the results showed these compounds located in both mitochondria and lysosomes.③MTT experiments in above four tumor cell lines showed compound B1-6followed with laser irradiation could significantly inhibit the proliferation of liver, gastric and lung cancer cells over80%at20μM dosage, while less than80%for liver cells. The above experiments indicated compound B3had special dark toxicity, whose maximum inhibition rate in the four tumor cells were above80%and IC50below0.18μM.Compound B3is studied in more details as the antitumor chemical agent:①The acute toxicity of hit compound B3was tested by karber method, the results showed LD50of hit compound B3was149.70mg/kg.②Mice bearing H22liver cancer and nude mice bearing Hep G2were used as animal model to evaluate hit compound B3in vivo, the results showed maximum tumor inhibition rate of hit compound B3on mice bearing H22liver cancer was69.21%at10mg/kg dosage, and it could inhibit the tumor proliferation of nude mice bearing human liver cancer Hep G2at5,10mg/kg dosage respectively.