Preparation And Spectroscopic Properties Of Carboxy-containing Zinc Phthalocyanines And Their Covalent Albumin Conjugates

Photodynamic Therapy (PDT) is a new modality for cancer therapy treatment.Anticancer photosensitizer is the key element of photodynamic therapy. The firstgeneraton photosensitizers for clinical use are mainly Hematoporphyrin derivativeswhich represent Photofrin, whereras their gigantic disadvantage are that effectiveingredients are complex, dark cytoxicity is high, etc. Thus it is important to seek newphotosensitizer. The research of phthalocyanines as the second generationphotosensitizer receives extensive interests for their high absorption in photodynamictherapy spectrum, strong photosensitivity, low dark toxicity, and easy to chemicallymodify, etc. It is a hot point in this field this days in researching structure-activityrelationship, action mechanism and effective method to increase photoactivity andtarget tissue selectivity of phthalocyanine type photosensitizer.The dissertation firstly synthesizes a series of carboxy-containing substitutedphthalocyanine zincs, then conjugated these phthalocyanines to albumin (BovineSerum Albumin and Human Serum Albumin) through amide formation way, preparedseventeen high water-soluble and unreported phthalocyanine-albumin covalentconjugates, purified by methods such as gel permeation chromatography, andcharacterized by infra-red spectrum, fluorescence spectrum, electronic absorptionspectrum and Comassic bright blue method, etc. It finds that there are nearly nodifferent effect of molar ratios of phthalocyanine-albumin covalent conjugates onsubstiturion type, substitution position and number of carboxy groups inphthalocyanine cycle. The covalent conjugates are obtained whose molar ratio isabout6~7：1, when10equivalent of phthalocyanine reacts with1equivalent ofalbumin.The dissertation compares carboxy-containing substituted phthalocyanine zincswith their phthalocyanine-albumin covalent conjugates in spectrum property andexistent state in Phosphate buffer solution. It indicates that the conjugates have moreobvious phthalocyanine monomer spectrum characteristic than their corresponding free phthalocyanine, when the substituted phthalocyanine covalently bind to albuminmacromolecule. There are effect of carboxy substitution position in phthalocyanine onspectrum change with conjugation of phthalocyanine and albumin. α positionsubstitution has more phthalocyanine monomer percentage than β positionsubstitution. The covalent albumin conjuagtes of α-substituted phthalocyanine1,2,3and tetra α-substituted phthalocyanine4,5exibit mainly phthalocyanine monomerspectrum characteristic, with Q-band maximum absorption in681~706nmapproximately. The covalent albumin conjuagte of eight carboxy substitutedphthalocyanine zinc8also exibits phthalocyanine monomer spectrum characteristicmainly, with Q-band maximum absorption in about677nm. The covalent albuminconjuagte of sixteen carboxy substituted phthalocyanine zinc9exibits phthalocyaninemonomer spectrum characteristic, with Q-band maximum absorption in about681nm,whose molar extinction coefficient are about2.0105mol-1·L·cm-1.The dissertation investigates the effect of solution pH on spectrum property andexistent state of carboxy-containing substituted phthalocyanine zincs and theirphthalocyanine-albumin covalent conjugates. It finds that the spectrum property andexistent state of free carboxy-containing substituted phthalocyanine change with pHof PBS solution. In general, when solution is acid (pH2.0), the free phthalocyaninedisplays highly aggregated characteristic absorption (the absorption band becomeswide to a great extent). The obvious disaggregation occurs when pH accending(Q-band absorbance becomes higher and sharpper). The situation embodies obviouslyin α substituted carboxy-phthalocyanine3,4,5and sixteen carboxy substitutedphthalocyanine9. But the phthalocyanine Q-band characteristic pectrum property andexistent state in all phthalocyanine-albumin covalent conjugates do not change withsystem pH, when carboxy-containing substituted phthalocyanine covalently bind toalbumin framework. It would inspire the design of the photosensitizer that has highpH capacity and is protein target-mediated.The dissertation also explores the noncovalent interaction with monosubstitutedphthalocyanine2and albumin, compares the effect of conjugation way (covalentconjugation and noncovalent conjugation) on spectrum property and existent state ofsubstituted phthalocyanine. It shows that there is strong noncovalent interactionbetween phthalocyanine2and albumin, and the binding constant is about1.0105mol-1·L. The binding sites competition experiment indicates that the noncovalentinterzction site locates in subdomain ⅠB of human serum albumin. The phthalocyanine conjugated with albumin, no matter covalent conjugate or noncovalentconjugate, both displays more obvious monomer chacteristic absorption thancorresponding free phthalocyanine, which is a property beneficial to photodynamictherapy. Covalent conjugate leads to Q-band maximum absorption of phthalocyanine2red-shift about5nm, while noncovalent conjugate does not cause red-shift.Comparatively, the red-shift of action spectrum for photodynamic therapy is abeneficial change, because the longer wavelength the better for the light permeation tohuman tissue.