Apoferritin-Based Delivery of Metal Complex Drugs

Author:Li Xiao

Supervisor:feng fu de


Degree Year:2019





Functional metal complexes and metal-based drugs are increasingly used in the field of biomedicine,but there are also practical problems in its use such as poor biocompatibility and non-targeting delivery.Ferritin,a metal-binding functional protein that is ubiquitous in organisms to maintain iron balance in physiological conditions,is commonly used in targeted drug delivery systems.In this thesis,we explored the effective entrapment of different types of functional metal complexes or metal-based drugs in ferritin nanocage,and explored potential applications of these functionalized protein nanocomposites in cell uptake,imaging and cancer therapy.The thesis mainly includes the following four parts:Part Ⅰ,three Ru complexes with different hydrophilicity were synthesized,encapsulated into apoferritin nanocages(termed Ru-NP)and delivered into cells for imaging.In this part,a series of Ru complexes were incorporated into apoferritin in the presence of organic solvent to prevent formation of aggregates.The hydrophobic environment of apoferritin inner cavity was revealed by the "light switch" effect of caged Ru complexes possessing dppz ligand(Ru-dppz).Ru-NPs exhibited uniform shape and good stability in water.At elevated temperature(37℃),Ru complexes released with a half lifetime(t1/2)of about 6 hours.Cell viability assay indicated that the non-specific toxicity of the Ru complexes was notably reduced after caging by apoferritin.Modification of folate acid in the ferritin shell increased tumor cell-targeting capability and enhanced cellular uptake efficiency of Ru-NPs.The delivery method paves a way for resolving the problems of delivering hydrophobic metal complexes that suffer from poor solubility and biocompatibility,and expands application of ferritin in the biomedical field.Part Ⅱ,Roussin’s black salt(RBS),a nitric oxide(NO)donor that is sensitive to water and light,was caged into apoferritin to form a light-controlled NO delivery system.Apoferritin is known as an iron storage protein,which provides an opportunity to study the binding affinity of iron complexes with apoferritin,particularly the interior side of apoferritin.Apoferritm was found to be capable of accumulating a large number of RBS moieties in its spacious cavity,significantly stabilize the Fe-NO bond by shielding water contact and conserve the light sensitivity for controlled NO release under low dose of light irradiation in aqueous media or in living cells.Phototoxicity of cells were controlled by white light flux.To increase the sensitivity of delivery system to the longer wavelength region of light,a metal porphyrin complex ZnPPIX was co-loaded in the protein cage and employed as an energy donor to transfer absorbed energy to RBS and induce dissociation of Fe-NO bond.Thereby NO releasing was achieved by excitation of the multicomponent nanocomposites at 580 nm,which widen the activation window of RBS in visible light region.This is a simple and efficient delivery system and promising for light-controlled release of gas signaling molecules.Part Ⅲ,a method for stabilization of a nucleic acid methylation agent,MTIC which is recognized as the key intermediate of temozolomide degradation,by copper ion is established in both solution and apoferritin.As a first line drug for treatment of glioblastoma(GBM),temozolomide has been widely used as a prodrug of MTIC by rapid hydrolysis into MTIC under basic pH.However,in most cases under physiological pH,conversion of temozolomide into MTIC is slow and inefficient,which limits application of temozolomide in other medications.In the present study,copper ion,other than any other metal ions,was confirmed to coordinate with MTIC and form a new metal drug Cu-MTIC that was stable in aqueous solution at physiological pH.Interestingly,the metal drug protected by apoferritin was stable against both alkaline and acidic pH,and could be selectively activated by the presence of glutathione which is the most abundant endogenous thiol species in cells.In addition,a MTIC delivery system was developed by in situ generation of the metal drug in apoferritin via coordination interaction,with high loading efficiency(up to 92.9%based on original TMZ)and capacity(up to 185 MTIC per apoferritin).The delivery system could induce apoptosis of both temozolomide-sensitive and temozolomide-resistant cells with reduced IC50 values,and importantly,exhibits tumor accumulation capability and anti-cancer effects with low hepatotoxicity in in vivo tests.The system has potential application in solving temozolomide resistance and improving GBM treatment efficacy.Part IV,by virtue of the copper binding property,apoferritin is developed as a carrier of Cu(Ⅱ)-diethyldithiocarbamate(CuET)that is derived from disulflram.Disulfiram,well known as an anti-alcohol drug,is under a quantity of clinical trials for its anticancer effects particularly in combination use with Cu(Ⅱ).CuET exhibits excellent redox stability in the presence of glutathione,cysteine and hydrogen peroxide,however,poorly soluble in water and lacking targeting capability that arise a concern with non-specific toxicity.The composites formed by incorporation of CuET into apoferritin were fully characterized in structures.Apoptosis of malignant cells was induced at very low concentration of composites.In addition,the antitumor effect was enhanced by co-encapsulation of doxorubicin and diethyldithiocarbamate in apoferritin,facilitated by the presence of copper coordination with loading efficiency>97%.The apoferritin-based delivery systems provide a solid foundation for targeted delivery and cotrolled releasing of metal drugs in combination therapy.