Multifunctional Ferritin Nanoparticles As Theranostics For Imaging-guided Tumor Targeted Phototherapy

Objective:To develop functional ferritin nanoparticles as theranostics for imaging-guided tumor targeted phototherapy.Methods:Fn expression plasmid was constructed by inserting the gene sequence of targeted peptide CGKRK into the N-terminal of ferritin.After DNA sequencing verification,the plasmid was transformed into expression strain E.coli BL21(DE3).The product was purified with a Superdex 200(GE)column using AKTA explorer system.TFn and Fn were labeled with Cy5.5and then the tumor targeting efficiency of TFn was studied by in vitro and in vivo fluorescence imaging.The TFn was used as photosensitized carriers loading with the photosensitizer 556–Ph.TEM and DLS were used to observe and measure the particle size and morphology.The UV-vis-NIR absorption spectra were measured with a Genesys 10S UV-vis spectrophotometer.5×10~6 of MDA-MB-435 cells were subcutaneously injected into the right flank of 4-6 week old athymic nude mice.The mice were used for photoacoustic imaging to observe the accumulation effect of TFn@556-Ph nanoparticles on the tumor site.The potential for PTT/PDT of TFn@556-Ph was studied using the same animal model.After the therapy studies,the animals were sacrificed,and the toxicity effects of nanoparticles were tested by HE staining of major organs.Results:The TFn plasmid was successfully constructed,and E.Coli derived TFn was purified using AKTA explorer system with a size exclusion chromatography column.The high accumulation of TFn-Cy5.5 in tumor tissues was confirmed by in vivo fluorescence imaging.TFn could load photosensitizer 556-Ph with high encapsulation efficiency(84%).TEM images of TFn@556-Ph presented an explicit spherical nanocage with diameter around 14 nm,with the formation of 556-Ph inside Fn nanocages.TFn@556-Ph showed a laser-power-dependent temperature increase and reactive oxygen species generation under laser irradiation.As expected,a linear photoacoustic imaging signal amplification was observed with increased TFn@556-Ph concentration.In the group with TFn@556-Ph administration plus laser irradiation,all the tumors were completely eliminated,leaving scars at the original tumor locations.In this study,we did not notice obvious signs of toxic side effects in the TFn@556-Ph–treated cells,and neither death nor significant body weight drop was observed in the treated mice.No obvious abnormalities were found by hematoxylin and eosin(HE)staining of the major organs of the treated mice,both suggest safety of TFn@556-Ph nanoparticles.Conclusions:TFn engineered with homing peptide shows higher tumor uptake than Fn.556-Ph can be loaded into TFn nanocages with high efficiency.Following the guidance of photoacoustic imaging and fluorescence imaging,TFn@556-Ph was applied in phototherapy cancer treatment to achieve good cancer therapeutic efficiency with good biocompatibility from in vitro to in vivo.Such a nanobiotechnology holds great potential in clinical translation.