The Study Of Multimodal Imaging And Targeted Therapy With Ferritin Cage Nanocomposites

Only use chemotherapy often lead to little treatment in clinical cancer.Propose the method of combining diagnosis and treatment. Explore the integration of diagnosis and treatment for tumor therapy is an effective means to improve the success rate of cancer treatment.Because the advantages of the highly uniform,control assembly,easy to transform and to largely prepare,protein nano materials had attracted more and more scientists,attention in the field of nano biology.In this study,doxorubicin was wraped into the apoferritin（AFn） through self-assembly,which was prepared by removing the iron core of the ferritin.The composites had magnetic targeting and fluorescence labeling by functionalization and they were to control drug releasing by the p H variation.The research major content contain:（1） A multi-functional ferritin was structured for cancer theranostic applications;（2） Activity was evaluated in vitro based on MCF-7 cell lines as the model cells;（3） Based on B16-F10 melanoma cell lines of rat source,we constructed tumor-burdened mice model using the C57 female mices as the model to evaluated activity in vivo.To obtain the AFn/DOX composites, doxorubicin was wraped into the apoferritin（AFn） through self-assembly,which was prepared by removing the iron core of the ferritin.To make the composites can act as fluorescent probes and have cancer diagnosis function,the Cdots were decorated onto the surface of AFn/DOX by amido bond. Then, to realize the magnetic targeting and MR imaging, iron oxide nanoparticles（Fe₃O₄） were decorated onto the surface of AFn/DOX-Cdots composites,obtaining a multifunctional Fe₃O₄-AFn/DOX-Cdots nanocomposite with strong magnetic targeting ability,MR imaging ability and powerful fluorescence lables.Therefore,it makes drug have more uptake into tumor cells,this is directly relate to activity of tumor therapy.Fe₃O₄-AFn/DOX-Cdots nanocomposites were obtained by forming amido bond under the catalysis of EDC and NHS. Based on the results of single factor experiment,the formulation of composites were optimized by using magnetic-stir method,and ultimately determine the optimal prescription.Fe₃O₄-AFn/DOX-Cdots nanocomposite was characterized by ultraviolet and visible spectrophotometer,infrared spectrophotometer and fluorescence spectrophotometer and other methods.The average particle size of AFn was about 12±2.0nm by Nano-zs90 laser nano particle meter,PDI was about 0.2.The Cdots solution has a uniform size and the average particle size of Cdots was about 5±2.0nm by Nano-zs90 laser nano particle meter,PDI was about 0.2; With different p H,the fluorescence intensity of Cdots was shifty tested by fluorescence spectrophotometer.As the results show,there was the strongest fluorescence of Cdots at p H=4.0.As can be seen from TEM images,Fe₃O₄-AFn/DOX-Cdots had a ball-like structure,and it had uniform size distribution; We determined the drug loading efficiency through ultra-filtered centrifugation,DOX is measured by UV-vis and the drug loading efficiency of DOX was about 6.3%; At 37℃,DOX release in vitro was measured by bag filter method with p H=5.0 and p H=7.4.The results of DOX release in vitro showed that we can control the release rate of Fe₃O₄-AFn/DOX-Cdots by adjusting p H; The Fe₃O₄-AFn/DOX-Cdots displayed strong magnetic property; The nanocomposites can act as a T2 contrast agent for MR imaging,which revealed the concentration dependent darkening effect.Based on MCF-7 cell lines as the model cells in vitro, we study the cytotoxicity on tumor cells.The cytotoxicity of Fe₃O₄-AFn-Cdots was examined by MTT dyeing method to study the influence on MCF-7 cell.The results showed that the cytotoxicity of the Fe₃O₄-AFn-Cdots on MCF-7 cell could be ignored,so it can be considered to be nontoxic. Also, we investigated the the cytotoxicity of the Fe₃O₄-AFn/DOX-Cdots and the effect of concentration,acting time and magnetic field.The results showed that DOX with 50μg/ml for 72 h greatly enhanced the cytotoxicity and obviously higher than the other groups. Compared to Fe₃O₄-AFn/DOX-Cdots without magnet,Fe₃O₄-AFn/DOX-Cdots/magnet had more uptake into cells in cells uptake study,this proved Fe₃O₄ can be magnet targeting to promise drug delivery to cancers.To study Cdots as fluorescent probe,strong blue fluorescence were observed in MCF-7 cells incubated with Fe₃O₄-AFn-Cdots for 24 h.Based on B16-F10 melanoma cell lines of rat source, we constructed tumor-burdened mice model using the C57 female mices as the model to evaluated the anti-tumor properties of Fe₃O₄-AFn/DOX-Cdots in vivo,the result indicated Fe₃O₄-AFn/DOX-Cdots was easily to concentrate on tumor with magnetic targeting,and then DOX released from ferritin nanocage under acid environment around tumor,that make sure kill tumor cells.The research of pharmacokinetics in mices showed Fe₃O₄-AFn/DOX-Cdots significantly increased the blood circulation time of DOX in vivo.Moreover,An obvious darkening effect in the tumor was observed in T2-weighted MRI after injecting Fe₃O₄-AFn/DOX-Cdots with a magnet glued onto the tumor,the results suggest that the Fe₃O₄-AFn/DOX-Cdots can act as a sui Tab negative（T2） contrast agent in MRI applications.Systematic studies in vitro showed that Fe₃O₄-AFn/DOX-Cdots was effectively concentrate on MCF-7 cells and pass through cell membranes,which can carry largely DOX to get into cells.The acid environment around tumor made the ferritin nanocage unfold then DOX released from Fe₃O₄-AFn/DOX-Cdots to induce cells apoptosis and stop MCF-7 cells proliferating.Systematic studies in vivo showed that Fe₃O₄-AFn/DOX-Cdots had low toxicity and significantly increased the blood circulation time of DOX,Fe₃O₄-AFn/DOX-Cdots was easily to concentrate on tumor with magnetic field,then DOX was controlled release by the p H around.After injecting Fe₃O₄-AFn/DOX-Cdots,an obvious darkening effect in the tumor was observed in T2-weighted MRI,Fe₃O₄-AFn/DOX-Cdots can act as a sui Tab negative（T2） contrast agent in MRI applications.