Active Targeting Magnetoferritin Probe For Optical/Magnetic Multimodal Imaging

With the increasing incidence of cancer,people pay more and more attention to the early diagnosis of cancer.As an important means of early diagnosis of the tumor,including ultrasound,computer-assisted tomography(CT),magnetic resonance imaging(MRI)and optical imaging(OI),molecular imaging detection by labeling the key molecules during the continuous development of the tumor,a series of pathological changes and characteristics of the tumor can be directly observed,rather than merely showing changes in the end of the tumor.So this test means to provide more accurate and complete information on early detection of the tumor.MRI and optical imaging have both advantages and disadvantages in terms of temporal and spatial resolution,anatomical localization,detection sensitivity,etc.The spatial resolution of MRI is high,but the imaging sensitivity is poor;optical imaging has the higher sensitivity,but the tissue penetration is weak.However,although most of the molecular imaging techniques that utilize multifunctional nanoparticles have made great progress in high sensitivity and high specificity for the detection of tumors.These probes also have a few increasingly problems:(1)most quantum dots are composed of toxic heavy metal elements(such as Cd),these heavy metal elements often limit the application of quantum dots;(2)fluorescent dyes are easily gathered,the stability of fluorescent dyes is poor and easy to light bleaching;(3)multifunctional nanoparticles often need to be modified with target ligands(such as antibodies,peptides or aptamers,etc.)through the complex preparation process and expensive reagents to achieve the purpose of tumor targeting.These complex modifications tend to result in high consumption,low purity and biocompatibility.Therefore,it is an important problem to solve the problem of molecular imaging detection by constructing a self-target imaging probe with simple structure,good biocompatibility and high sensitivity.According to this,we constructed the active targeting optical / magnetic multimodal imaging probe(M-HFn/CDs)consist of the active targeting heavy chain ferritin(HFn),near-infrared fluorescent carbon dots(CDs)and magnetic iron oxide(Fe3O4)with magnetic resonance imaging capability to address these problems.Nitro-modified near infrared fluorescence(NIR fluorescence)CDs were synthesized by one-step hydrothermal synthesis,which had high fluorescence intensity,good water solubility,excellent optical stability and good biocompatibility.Fe3O4 nanoparticles were synthesized in the internal of the active targeting HFn,NIR fluorescence CDs was covalently coupled with the carboxyl group of HFn by amino group.Without any targeted reagent,the synthetic nanoprobe can specifically target to a variety of tumor cells(breast cancer,liver cancer,colon cancer,pancreatic cancer and other cancer cells)by the overexpressed transferrin Receptor(TfR1)on the surface of the tumor cells which achieved the tumor multimodal imaging(NIR fluorescence / MRI)and ultimately achieved the purpose of early diagnosis of tumor.The preparation process of M-HFn/CDs nanoprobe mainly includes:(1)synthesizing amino-modified CDs with NIR fluorescence properties by one-step hydrothermal synthesis method using citric acid as carbon source and ethylenediamine as nitrogen source.The NIR fluorescence properties was used for fluorescence imaging.(2)HFn was expressed by molecular biology method.The amino group on the surface of CDs was ligated with the carboxyl group on the HFn protein cage through amide bond.(3)In the cavity of HFn protein cage,magnetic Fe3O4 nanoparticles were obtained as a contrast agent for MRI.The particle size and zeta potential of each product was measured by particle size analyzer.The results show that the particle size of CDs is about 4 nm,and the distribution of particle is uniform.The PDI of the polydispersity coefficient is 0.217,zeta potential is about 8.50 mV.The positive performance indicates that the amino group is successfully modified on the surface of CDs.The average particle size of HFn is about 12 nm,PDI is 0.204,zeta potential is about-29 mV.M-HFn/CDs have an average particle size of about 51 nm,a PDI of 0.225,and an average zeta potential of about-28.6 mV.The fluorescence properties of CDs and M-HFn/CDs were investigated by fluorescence spectrophotometer.The results showed that the CDs and M-HFn/CDs had significant NIR fluorescence properties and good upconversion and down-conversion fluorescence characteristics.The strongest fluorescence intensity was at pH 6.0,which is compatible with the weakly acidic environment ofthe tumor,which indicated that the probe can be used for diagnosis of tumor.The magnetic Fe3O4 made M-HFn/CDs nanoprobe strong magnetic properties.MRI in vitro results indicated that M-HFn/CDs can be used for magnetic resonance imaging of T2 field,and the blackening effect is gradually increased with the increase concentration of M-HFn/CDs probe.In order to investigate the optical/magnetic multimodal imaging capability of M-HFn/CDs nanoprobe prepared by this study,we selected breast cancer cells(MCF-7,human)as a model for the in vitro tumor imaging ability of the probe.Including the cytotoxicity and uptake experiments of M-HFn/CDs probe.The cytotoxicity of CDs,HFn/CDs and M-HFn/CDs were investigated by standard SRB method.The results of SRB showed that in the set concentration range,CDs,HFn/CDs and M-HFn/CDs showed low cytotoxicity.Even at larger concentrations(800 ?g / mL),CDs still showed higher cell viability(90% or more).And after CDs conjugating to the surface of HFn,HFn/CDs and M-HFn/CDs groups had higher cell viability compared to the CDs group.The results of cytotoxicity showed that the M-HFn/CDs probe had a higher biocompatibility.The active targeting ability of M-HFn/CDs probes was investigated by MCF-7 cell uptake.Since the prepared NIR fluorescence CDs themselves had red fluorescence,the M-HFn/CDs probes could be investigated by observing the red fluorescence of CDs.HFn/CDs and M-HFn/CDs were more rapidly ingested by tumor cells compared to the individual CDs.The uptake of HFn/CDs and M-HFn/CDs during the same incubation time was significantly higher than that of CDs alone.The results of cell uptake indicated that the active targeting ability of HFn was more favorable for the rapid entry of M-HFn/CDs nanoprobe into tumor cells after coupling HFn.In vitro tumor imaging ability experiments showed that M-HFn/CDs could effectively target MCF-7 cells through the active targeting ability of HFn and showed significant red fluorescence for NIR fluorescence imaging of tumors.The in vitro MRI results of the M-HFn/CDs nanoprobe showed that the concentration-dependent blackening effect of M-HFn/CDs allowed the probe to be used as a contrast agent for tumor MRI diagnosis.