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The Study on Biochemical Synthesis and Regulation of Three Functional Molecule for Medicine

Author: WangMengLiang
Tutor: LiuDianSheng
School: Shanxi University
Course: Inorganic Chemistry
Keywords: metal bacterial chlorophyll photodynamictherapy(PDT) photosensitizer tumour
CLC: TQ460.1
Type: PhD thesis
Year: 2006
Downloads: 80
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Abstract

Nowadays, photodynamic therapy （PDT） has become a hot point which has been broadly employed to diagnoses and therapy tumorous and nontumorous diseases. Photo sensitizer plays an important role in PDT. The optical characteristics of photo sensitizer have the directly influences on curative effect of PDT. The characteristic absorption of the second-generation photo sensitizer locates in 670 nm or 690nm. However, there are many disadvantages of the second-generation photo sensitizer to therapy cancers such as the poor penetrable ability in tissues, the wide absorption spectrum in visible range and the obvious side-effects. In our study, we have synthesized some new photo sensitizers which have characteristic absorption in 750 nm. Because of their great penetrable ability, the new photo sensitizer can be directly used to disguise and therapy tumorous and nontumorous diseases in tissues. Moreover, the side-effects have decreased to some extent. The mechanism of PDT and the bioactivities of the new photo sensitizers have been studied in the research as well.METHODS AND RESULTS:（1） We have studied the effects of light, O₂, substrates and other factors on the biosynthesis of bacteriochlorophyll by Rhodobacter sphaeroides. The bacteria are cultured with strong light and at the low concentration of O₂. Glycin, α-ketoglutaric acid and trans-butene diacid at the optimal concentration are contained in the medium. And the concentration of Fe²⁺ is lower than 1mg/L. After cultured in growth medium for 50h, then the bacteria have been inoculated in liquid medium. The maximum bacteria chlorophyll （12μmol/L） has been determined at 100h.（2） Bacterial chlorophyll is be extracted by supersonic in ethanol, absorbed by French chalk, separated with DEAE-sepharose CL-6B（2.7 × 6.5 ）and sepharose CL-6B（2.7×18）. Bchla50mg/100g （wet weight） is obtained. Bacterial chlorophyll is identified by otherreferences.（3） The synthesis of transition metal （Cu,Zn,Co,Ni） and transition metal was investigated using chlorophyll as ligand. There were five complexes that were worked out through reaction of metal and deprivating-Mg bacterial chlorophyll in the organic solvent. The ultra violet-visual spectrum and the fluorescence spectrum of these compounds which were analyzed detailedly comparing with deprivating-Mg bacterial chlorophyll and plant deprivating-Mg bacterial chlorophyll. The results showed that the main absorption peaks of deprivating-Mg bacterial chlorophyll in infrared region and ultraviolet band wander from 410nm and 670nm in absorption peaks of plant deprivating-Mg bacterial chlorophyll to 410nm and 670nm respective. The absorption peaks of transition metal bacterial chlorophyll also have some wandering comparing with plant deprivating-Mg bacterial chlorophyll. The wandering of the absorption peaks of cu- bacterial chlorophyll is marked in those compounds, which have several nanometers. The fluorescence absorption peaks of transition metal bacterial chlorophyll deviate a little to short-wave region. The results show that the molecule flatness is some decrease. Thus the transition metal is embedded in the circle molecule.（4） The K562 and HL-60 cell that were incubated in CO2 container were inhibited by the four metal complex of bacterial chlorophyll in intro, which were detected by ELISA. It indicates that they have the strong inhibition on the two tumorous cell. When the pharmic concentration was 10 μg/ml, the inhibited rate arrived at 82.87% and 84.93%,respectively;when 200 μg /ml,the most inhibited rate was 100%.It also indicates that the inhibited rate has been markedly increased after the light ,but because of the condition limit ,we only employ the common daylight lamp as light source. If the light source turn to the special excitating one that was aborbed by bacterial chlorophyll, the effect has become better.（5） The modes and activities of the interaction of DNA and metal complexes of Bacteriochlorophyll （Cu, Zn, Co, Ni）, which have been prepared by extraction and synthesis reaction, have been discussed according to the ultraviolet-visual spectrum and nucleic acid electrophoresis. It indicates that the system of DNA and metal bacterial chlorophyll have strong the interaction .The system of metal Bacteriochlorophyll and DNA made the absorption peak of porphyrin Einstein shift ,which arrived at 788.8nm.Theelectrophoresis indicates that the interaction of bacteria chlorophyll and DNA has at least two combining way （EB competed and non-EB competed ）.Bacteriochlorophyll have strong cutting effection to DNA in illumination and there have a large of little DNA fragments. Metal complexs of bacteriochlorophyll and DNA have the best strong cutting effection in the near infrared ray up 700nm. If the metal complexes of bacteriochlorophyll were used in tumour diagnoses and therapy （photo dynamical therapeutics）, then we can use the near infrared ray up 750nm as remedial light house,which will greatly increase penetrable ability of light ray in tissue, can directly be used in diagnoses and therapy ,and decrease photosensitive reaction after the therapy at the same time.（6） Effect of different reactive oxygen species on K562 cells death mediated by MBC-PDT: The K562 cell were treated by MBC-PDT, The rate of apoptotic or necrotic cells was respectively detected on flow cytometry through double staining of Annexin-V and popodium iodide after PDT 6 or 24 hours. Results showed that MBC-PDT could induced K562 cell death through necrosis and apoptosis. hi an early phase after PDT, Sodium azide（the singlet oxygen quencher） could protected K562 cell from necrosis induced by MBC-PDT, whereas . D-Mannitol （the hydroxyl radical quencher） protected K562 cell from apoptosis also induced by MBC-PDT.（7） The method inoculate K562 tumor cell into rat was adopted, fluorescence microscope and spectrophoto metry were used to measure the distributing disciplinarian of bacterial chlorophyll in the tumor tissue, liver, kidney and spleen. The study showed that: the tumor which fluoresce in group IT was higher than in group IP after Bchl was injected in it, reach to 10-49 times, moreover, fluorescence out of cell was relatively strong, the cell fluoresce in liver, spleen, kidney were lower than group IP, the result was same as Amano. These were showed that the photosensitizer in the tumor cell was increased when Bchl was injected straightly, at the same time, distributing of it in the normal tissue was decreased. The distribution of Bhl in smear of tumor, normal tissue and check tissue were not appeared. When injected in after 2 days. The percentage of fluoresce in tumor cell was 0.83% that was the highest one, fluoresce out of cell was weak. In group IT, strong fluorescence appeared in both inside and outside of tumor, it was evidence higher than group IP, on the first day reached to 9.83%, on the second and third day reached to 20.02%and 17.75% which were the highest. On the third and forth and fifth day, the percentage of fluoresce in Spleen and liver in group IT was higher than group IP, the other were evidently lower than group IP. Exposed in sunlight for 40 minutes, the check group had not changed, the eyes of rats brightly, In group IT, only one rat had eye conjunctive dropsy, the other five rats had not abnormality changes, in group IP, six rats appeared evident eyes conjunctive dropsy. On the second day, one rat in group IP had bad state, the other five rats were dead. The rat only had eyes conjunctive dropsy in group IT had bad state, the other five rat were the same as check group,and had good state.（8） Use Lecithin polyethylene pyrrolidone as material, multiphase lipid of bacterial chlorophyll was prepared by the method of ultrasonic decentralization, moreover, the hot stability and light stability were studied, the study indicated that: the multiphase lipid was composed of single room lipid which particle size was less than lum, little excessive room lipid and little type of o/w emulsion, it were a mixed particulate disperse system, all were inside the colloid disperse system. According to electrical conductivity and temperature curve, the phase of transform temperature was 50 °C, have higher level hot stability. The hot degradation appeared a first-order reaction when multiphase lipid metal bacterial chlorophyll was below 85 °C, and the reaction was inhabited by multiphase lipid. The envelope rate of multiphase lipid was 52%.

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