Anti-tumor Study Of Nanocarriers Based On Albumin And Its Fusion Proteins

The thesis work consists of two inter-related parts:1.A simple and efficient method of removing impurities from HSA and its fusion protein.Due to limited source and security risk of human plasma,researchers usually express recombinant HSA(rHSA)using genetic engineering by Pichia pastoris.As a transport protein,some impurities are wrapped into rHSA.We developed a simple and effective approach for the removal of rHSA impurities.In this technique,the recombinant HSA was adsorbed on a column,and was treated with 6 M urea and 30%alcohol to wash out the entrapped impurity.We utilized a series of technical methods to measure the functional properties of such treated rHSA.The results showed that our technique removed majority of the pigments embedded inside albumin,and our rHSA still retained bioactivity.In addition,the method was also used successfully for a series of albumin fusion proteins(e.g.ATF-HSA,PAItrap-HSA),demonstrating the wide applicability of this simple method.2.Antineoplastic study of nanocarnier based on albumin and its fusion proteinTraditional protein nanocarrier preparation technique contains thermal denaturation or chemical crosslinking using crosslinking agents,which causes protein nanocarnier to produce antigenicity and toxicity.To solve the problem,we developed a novel protein nanocarrier preparation method.Under the condition of adding nontoxic substance,human serum albumin(HSA)was formed into nanoparticles entrapped with hydrophobic photosensitizer(mono-substituted ?-carboxy phthalocyanine zinc,CPZ)by this method.In addition,we expressed a recombinant protein of albumin fused with ATF(amino terminal fragment of urokinase),which can specifically target to metastatic cancer cell with high-expression urokinase receptor(uPAR).The recombinant protein ATF-HSA was also successfully formed into nanoparticle entrapped with CPZ.Both protein nanoparticles(ATF-HSA:CPZ@NP and ATF-HSA:CPZ@NP)showed that not only high loading capability of CPZ,but good stability and bioactivity.Importantly,ATF-HSA:CPZ@NP disintegrated and released drug in the presence of receptor.Cell experiments demonstrated ATF-HSA:CPZ@NP caused high toxicity to cancer cells of high-expression urokinase receptor.Fluorescent molecular tomography(FMT)was applied to study the accumulation of two nanoparticles on tumor site,and showed that ATF-HSA:CPZ@NP HSA has higher level and better controlling release capacity than HSA:CPZ@NP in tumor bearing mice.In vivo experiments also demonstrated that ATF-HSA:CPZ@NP has a better therapeutic effect than HSA:CPZ@NP,likely due to uPAR-mediated active targeting effect.