Site-specific Modification Of Apoferritin Based On The Host-guest Interaction

Ferritin,as an iron storage protein,is widely distributed in biological systems.The ordered structure of ferritin was consisted of 24 subunits to form supermolecule structure by non-colvent interaction.The demetallized form of ferritin,termed apoferritin,has received tremendous interests in biomedical applications such as drug delivery,cell imaging and disease diagnosis.Surface engineering by chemical functionalization provides a variety of merits,however,lacking efficient and convenient methods with site-accurate modification.The two-fold channel on the surface of ferritin can bind some organic molecule by non-colvently interaction termed as host-guest interaction.The channels on the shell of apoferritin remain unexplored in surface engineering.In this thesis,we designed a method to modify horse spleen apoferritin(abbreviated as HSAF,host)via host-guest interaction using a synthetic porphyrin derivative(termed TPA,guest)as a two-fold channel specific binding ligand.TPA has an oligo(ethyleneglycol)linker terminated with porphyrin an azide groups.The formation of supramolecular assembly between TPA and HSAF affords a versatile tool for conjugation with other desired functionalities via powerful click chemistry.In addition,the intercalation of TPA molecules in the two-fold channels(1:1 in stoichiometry)circumvents the burst release issue of apoferritin-based drug delivery system.The thesis mainly includes the following parts:Part 1:In this study,two Por-PEG-N₃ compounds containing different molecular weights of PEG was prepared.The compounds were complexed with HSAF by programmed injection and the final solution contained 10%acetonitrile in the solution,the one with shorter molecular weight(termed TPA)was selected for the next experiments in this thesis due to the satisfactory binding performance.The binding number was calculated to 12 according to the spectrophotometric analysis.The results from the iron uptake and iron release assays as well as arachidonic acid(ARA)-based competitive binding assay showed that TPA binds HSAF at the two-fold channel by host-guest interaction with the porphyrin ring situated deeply inside.The intercalative binding affinity of TPA toward the two-fold channel provides a new strategy for site-specific and quantitive engineering of HSAF surface.Part 2:Currently,surface modification of ferritin mainly includes covalent chemical reaction and genetic engineering.In this study,the terminal azide of TPA/HSAF complex(termed ^TPANP)was utilized to couple with DBCO-Cy5 by copper-free click reaction to form Cy5-labeled HSAF(termed as ^Cy5-TPANP).The MALDI-TOF characterization of ligand adduct showed the generation of Cy5-TPA.Efficient fluorescence resonance energy transfer(FRET)from porphyrin(energy donor)to Cy5(energy acceptor)occurred by exciting the porphyrin ring of Cy5-TPA.The cellular uptake and trafficking behavior of ^TPANP and ^Cy5-TPANP showed that they can be internalized efficiently and mainly distributed in lysosome.DBCO-Biotin was also utilized to react with ^TPANP to form ^Biotin-TPANP which showed enhanced cellular uptake efficiency.This kind of surface modification method can be achieved under mild condition,need no catalysis reagent and complicate purification procedures,and thus provide new possibilities for other functionalities of HSAF.Part 3:Ferritin was an ideal nanocarrier of chemotherapeutic drugs.The above results have demonstrated TPA is situated deeply at the two-fold channel of HSAF.In view of the interaction between TPA and subunits,TPA may play an important role in impacting on the stability of protein shell thus affecting the drug release kinetics.In this study,DOX was incorporated into apoferritin.The number of DOX encapsulated by HSAF was calculated as 40 per protein.The ^TPAHSAF/DOX nanocomposite was prepared using the programmed injection protocol.As compared to the HSAF/DOX system that was suffered from the burst release issue across broad p H ranges within the first 2 h,the ^TPAHSAF/DOX system exhibited much better stability.The DOX releasing percentage of ^TPAHSAF/DOX was about half of the percentage of HSAF/DOX at p H5.4 after 24 h.These results showed that TPA can regulate the drug release rates after binding with HSAF.Cell viability assay indicated that the ^TPAHSAF/DOX showed lower toxicity than HSAF/DOX and DOX with same concentration of DOX.The above study provides a new direction for the controlled drug release by using the interaction between TPA and the two-fold channel of apoferritin.