Chemical Modification Of Bacteriochlorophyll And Characterization For Photodynamic Therapy

Photodynamic therapy(photodynamic therapy,PDT) is considered the most promising of new methods in the treatment of cancer since the 1980s.Photosensitizer is the main factor of photodynamic therapy.The improving of photodynamic therapy's proposal,development and application is around with the development of photosensitizers.At present,there are still some the shortcomings and problems of photosensitizers,which restricts the application of PDT.After treatment, there is still photosensitive side reactions as to photosensitizers,then need the dark time.And the absorption wavelength of photosensitizers is below 700 nm,thus,they limited the organizations penetration depth. More important,PDT of deep tumors or lesions larger tumors need the photosensitizer which is of the infrared/near-infrared wavelength absorption.In this paper,bacteriochlorophyll a in the photosynthetic bacteria is choosed as precursor,with the aim of research synthesis methods of metal-bacteriopheophytin a and bacteriochlorophyll a derivatives which are of the IR/near-infrared wavelength absorption as potential photosensitizers.(1) Use UV-Visible spectroscopy,HPLC and TLC as analysis means to get a effective extraction and isolation method of bacteriochlorophyll a: it is extracted with methanol from photosynthetic bacteria directly,then precipitated with dioxane ring and distilled water,finally isolated from the Silica and Sephadex LH-20 column chromatography.(2) Using UV-Visible spectroscopy,atomic absorption spectrometry, infrared spectroscopy combined with NMR as analysis and character- rization means,it has been optimized to zinc-bacteriopheophytin a synthesis method,and its product' structure has been characterized.The synthesis method has been successfully extended to metal-bacterio-pheophytin a kind compounds:add bacteriopheophytin a and corresponding metal acetate with the molal ratio 1/80,to dichloromethane/methanol reaction solvent(1/1,v/v),then heat to maintain refluxing for 3 h.At the same time,the reaction solution be filled with Nitrogen.After the reaction,the product isolated from the silica gel column chromatography.Finally the results indicate that the absorption wavelength of this kind compounds can be detected about 770 nm,effectively repair the existing photosensitizers' shortcomings.(3) Through monitoring and analysis the reaction,with UV-Visible spectra and NMR,the synthesis and separation method has been gotten to bacteriochlorophyll a cyclic anhydride compound called bacteriopurpurin-a propyl ester,and its structure has been characterized.The absorption wavelength of bacteriopurpurin-a propyl ester can arrive to 813 nm.However,for the current clinical trial purpurins-type photosensitizers,the most has a strong absorption peak among 650 nm and 700 nm.Obviously,the absorption wavelength has been red shift significantly.(4) Through monitoring and analysis the reaction,with UV-Visible spectra and NMR,the synthesis and separation method has been gotten to bacteriochlorophyll a N-cycloimides,and the product structure has been characterized.Its absorption wavelength can achieve 822 nm.(5) In the PDT treatment of Zn-BPhe a for HL-60 cells,by adding respectively quenching agent sodium azide,which has of relative specificity to singlet oxygen ¹O₂,and quenching agent D-mannitol,which has of relative specificity to hydroxyl radical(HR),in order to quenching of corresponding ROS generated from photodynamic reaction.At the same time,through methyl thiazolyl tetrazolium salt(MTT) assay with Annexin V and PI double staining flow cytometric detection results,the photodynamic reaction of Zn-Bphe a and its impact to HL-60 cells' death. The experimental results show that:The photodynamic reaction type of Zn-BPhe a for HL-60 cells may be typeâ…¡dominated;and in the ROS generated from photodynamic reaction,¹O₂ leading to cell necrosis, however,HR leading to cell apoptosis.(6) By measuring the changes of antioxidant enzyme activity, membrane fluidity and membrane closed degrees,before and after the photodynamic therapy(PDT) in HL-60 cell,which caused by zinc bacteriochlorophyll a,so as to understand the impact of PDT on HL-60 cell's membrane function and explore its mechanism of PDT.Using tetrazolium salt(MTT) and fluorescence polarization measure the cells' activity and cell membrane fluidity,kit is used to detect Na⁺,K⁺-ATPase activity and MDA.As a result,after PDT,the survival rate decreased dramatically,and the Na⁺,K⁺-ATPase activity decreased,MDA increased, fluorescence vibration degrees increased,viscosity increased.It is important signals of HL-60 cell injury for the changes above,and the cell membrane is also an important section for the photodynamic reaction of zinc bacteriochlorophyll a.