Dual-functional Carbon Dots Labeled Heavy Chain Ferritin For Self-targeting And Chemo-photodynamic Therapy

Cancer has become the greatest threat to human health,and people have paid more attention to the early diagnosis of cancer.In the past few years,the imaging-mediated treatment of cancers has rapidly developed,which has paved a new way for the treatment of cancer.Among them,the combination of imaging and treatment has attracted a great deal of attention.And,nano-theranostics agents can be accurately transported to the tumor site by the use of targeting nano-carriers,thus achieving accurate diagnosis and treatment.Therefore,we have constructed H-ferritin-targeting theranostics system combining imaging-guided chemotherapy with photodynamic therapy for the diagnosis and treatment of tumors.In this study,high quantum yield of carbon dots(CDs)with good stability and biocompatibility was synthesized by the reflux of citric acid and formamide,and the CDs could emit bright red fluorescence with the excitation of green light.Moreover,the CDs produce reactive oxygen species(ROS)under 532 nm laser irradiated,which would kill tumor cells by damaging nuclear DNA,thus playing the role of photodynamic therapy.The targeted drug delivery system was constructed using heavy chain ferritin(HFn)as the carrier,and coupling with dual-functional CDs by chemical bond in the external.Doxorubicin(DOX)was encapsulated into HFn nanocages by utilizing the feature that the ferritin nanocage is dissociated into subunits in solution at pH 2.0,and it tends to reassemble by slowly raising the pH to 7.5.HFn could target a variety of tumor cells without targeted reagents modification due to the specifical bind of HFn to the overexpressed transferrin receptor(TfR1)on the surface of tumor cells.The morphology of CDs was observed by transmission electron microscope(TEM).The morphology of the CDs was spherical with a diameter of about 7 nm,which was consistent with the result of dynamic light scattering.Fluorescence spectra of CDs showed that the emission wavelength of the CDs increased with the increasing of excitation wavelength and the maximum emission wavelength could reach the red region with good fluorescence properties.DPBF was used as a probe to detect the generation of extracellular ROS,and the results demonstrated that CDs possessed the characteristic of producing ROS with 532 nm laser irradiation.Furthermore,the production of ROS would not been influenced when coupling with the HFn.The characterization of the pharmaceutics showed that the particle size of HFn(DOX)/CDs was about 26 nm with distributing uniformly.The encapsulation efficiency of DOX can reach 80% by optimizing the loading ratio of carrier to drug.The cumulative rate of release of HFn(DOX)/CDs was determined by dialysis at pH 7.4 and 5.0.The results showed that the cumulative release percentage of the pharmaceutics was 78.0% at pH 5.0,which was higher than that at pH 7.4.The results indicated that HFn(DOX)/CDs can release the drug in a weakly acidic environment,accumulating in the tumor site,so as to restrain the growth of tumor.In this study,the cellular uptake and tumor targeting experiments,inhibition assay were investigated by selecting human breast cancer cells(MCF-7)as model cells in vitro.The results of the effects of CDs and HFn/CDs on tumor and normal cells viability revealed that the carrier had almost no effect on the cell survival rate.The detect results of reactive oxygen species assay kit revealed that the production of ROS by CDs was increased with the extension of laser irradiation time and intracellular ROS detection in the groups with the existence of HFn(DOX)/CDs showed the production of ROS would not been influenced when coupling with the HFn.The results of single cell gel electrophoresis confirmed the ROS produced by CDs could damage nuclear DNA of tumor cells,thus creating stronger effect on tumor cells with the simultaneous action of DOX.The results of cell-targeting experiments indicated the superior targeting of HFn.Thus,HFn could carry most drugs to tumor cells with favourable targeting capability.Cell uptake experiments showed that CDs could be applied to red-emissive imaging,and the nanoparticles HFn(DOX)/CDs could translocate into the cell nucleus after DNA damage caused by partial release of DOX in cytoplasm,enhancing the nucleus damage of the drug.The results of cell inhibition assays certificated that the group HFn(DOX)/CDs with laser irradiation possessed much better therapeutic efficacy compared with the group HFn(DOX)/CDs without laser irradiation,indicating that the combination of chemotherapy and photodynamic therapy would have a better therapeutic efficacy.In this study,female Kunming mice and S180 ascites tumor cell lines were selected to establish an animal model in vivo.In vivo imaging experiments illustrated that the red-emissive CDs could be used as fluorescent probes for imaging in vivo.The results of in vivo imaging in the HFn(DOX)/CDs group showed that the nanoparticles mainly accumulated at the tumor site,which further proved the targeting ability of HFn.The results of pharmacodynamics experiments showed that the growth of tumors was inhibited to some extent in the group CDs with laser irradiation,indicating that the ROS generated by CDs can be used for the treatment of tumors.The inhibition effect of HFn(DOX)/CDs group on tumors was much better than that in DOX group.The reason might be that HFn could target tumor tissue,delivering large amount of drug to tumor sites,and DOX were mainly released at tumor site.And DOX can effect on nuclear DNA and induce cell apoptosis,thus inhibiting tumor growth.When HFn(DOX)/CDs were irradiated by 532 nm laser,the ROS generated by CDs can simultaneously damage nuclear DNA with DOX,enhancing the damage of nucleus and the inhibition effect compared with group HFn(DOX)/CD without laser irradiation.