The Preparation And Properties Of Phycocyanin-phthalocyanine Conjugates

Photodynamic Therapy(PDT)is rapidly developed in recent years as a new technology of selective treatment for proliferative lesion tissue.It has become one of the hotspots in the field of current anti-cancer research.The three elements of PDT are light,photosensitizer and oxygen,and the photosensitizer is the key factor.As the second generation of photosensitizern phthalocyanine attracted much more attention because it has good physical and chemical properties in near infrared region(650-750nm).However,because of the strong hydrophobicity of the macrocyclic conjugate system,phthalocyanine compounds generally showed low solubility in water,which hindered their practical applications.On the other hand,phycocyanin is a kind of porphyrin pigment protein with good solubility in water.Phycocyanins have been found to have photosensitive activities with the action spectrum at 550-650nm,but their photosensitive activities remains to be improved.In order to build complex photosensitizer with dual functional groups by using of the advantages of phthalocyanines and phycocyanins,this thesis mainly studied the preparation of phycocyanin-phthalocyanine conjugates and their spectrum properties and photodynamic activities.The main work and results are summarized as follows:1.The extraction and purification of phycocyaninSpirulina platensis was chose as the raw materials,because of its higher content of phycobiliprotein.First,the repeated freeze-thaw method was adopted to obtain the phycocyanin crude.The crude product was further purified by DEAE Sepharose Fast Flow exchange column and sephadex gel chromatography.Electrophoresis and spectrum results show that phycocyanin obtained has high purity,the spectral purity reaches more than 4.0,and the molecular weight is about 115 kDa.2.The synthesis and characterization of phthalocyaninesThree kinds of phthalocyanine were synthesized and characterized,they are 1-[4-(2-amino ethyl)phenoxy]phthalocyanine zinc(referred to as ZnPcNH2),1,8(11),15(18),22(25)-four(3-carboxyl phenoxy)zinc phthalocyanine(referred to as C4),1-[4-(2-carboxyl ethyl)phenoxy]phthalocyanine zinc(referred to as E1).The ZnPcNH2 phthalocyanine compound and its precursor were not reported in Literature.3.The synthesis of phycocyanin-phthalocyanine conjugatesWe have adopted non-covalent bonding and covalent bonding to build phycocyanin-phthalocyanine conjugates.For non-covalent bonding,the interaction between the phthalocyanine and phycocyanin was firstly studied by spectroscopy.Furthermore six kinds of phthalocyanines were selected to prepare non-covalent conjugates.As covalent bonding,two phthalocyanines modified by amino derivatives and two phthalocyanine modified by carboxyl derivatives were used to obtain four kinds of covalent conjugates by the methods of glutaraldehyde connection method and amide-forming reaction.At the same time,conjugates composition were analyzed through the spectrum.4.The properties research of phycocyanin-phthalocyanine conjugatesWe have studied the electronic absorption spectrum,fluorescence spectrum and existing states of conjugates in aqueous solution.And the photodynamic inhibitory activities of phycocyanin-phthalocyanine conjugates on human liver cancer cell HepG2 were evaluated.Results showed that the IC50 value of non-substituted zinc phthalocyanine-phycocyanin conjugate was 2.1?M,and the binding ratio of the conjugate was 8:1.The IC50 value of C4-phycocyanin conjugate was 0.93?M,and its binding ratio was 6:1.These two conjugates not only showed the significant photodynamic activities,but also visibly higher than phycocyanin and corresponding phthalocyanine,it is worth further researching.