Study On Novel Anti-tumor Drug Delivery System Based On Functionalized Carbon Dots

Carbon dots(CDs)with a small size of less than 10 nm have drawn intensive research interest since they were first reported in 2004 due to their excellent biocompatibility and fascinating optical properties,and now CDs have been widely applied in chemistry,biology,optics,electronics and other related fields.In this study,the strong blue fluorescent pegylated CDs with high quantum yield have been prepared by one step hydrothermal treatment of citric acid,ethylenediamine and PEG.In the previous studies,CDs were mainly localized in the cytoplasm and cell membrane,and few CDs could enter the nucleus for imaging.To address this issue,the nuclear localization signal(NLS)peptide was employed to modified CDs(NLSCDs)for nucleus targeted imaging.Then the anti-tumor drug doxorubicin(DOX)was covalently conjugated to NLS-CDs(DOX-CDs)through a pH-sensitive hydrazone bond using hydrazinobenzoic acid as a linker.This work highlights the potential of NLS-CDs in constructing an intelligent nano medicine with integration of targeting,real-time imaging,and therapeutic functions.The in vitro release behavior,chemical structure,and surface morphology of the complexes were examined systematically.The cytotoxicity,cellular uptake,and effect of DOX-CDs on cell apoptosis were investigated using MTT method,confocal fluorescence microscopy,and flow cytometry.Furthermore,the in vivo therapeutic efficacy of the nanocomplexes was evaluated in tumor-bearing mice.The in vivo non-invasive imaging was performed to monitor the in vivo distribution of DOX-CDs.The pegylated CDs with a quantum yield of 75%were synthesized by one step hydrothermal treatment and characterized by Fourier transform infrared(FT-IR)spectroscopy,nuclear magnetic resonance(NMR)spectra,X-ray photoelectron(XPS)spectroscopy and other techniques.The photoluminescence property of CDs was evaluated by UV-vis and fluorescence spectra.The results showed that the as prepared CDs exhibited well-dispersed spherical morphology.The pale yellow aqueous solution of CDs displayed strong blue light under irradiation with a 365 nm UV lamp and the maximum excitation and emission wavelength of the solution were 350 and 450 nm,respectively.The NLS modified CDs were fabricated for nucleus imaging.The MTT method was utilized to assess the in vitro cytotoxicity of NLS-CDs using Hep G2,MCF7,A549,and L929 cells.The cellular localization imaging of NLS-CDs into MCF7 and A549 cells was observed by confocal scanning laser microscope.The cellular uptake,cell apoptosis were evaluated toward A549 cells.Moreover,the in vivo toxicity of NLS-CDs was investigated in mice.MTT results showed that the NLS-CDs exhibited very low cytotoxicity at the tested dose and the cell imaging revealed that NLS-CDs could be internalized into the nucleus of MCF7 and A549 cells successfully.The uptake of NLS-CDs displayed a time-dependent manner and the high dose of NLS-CDs could induce early apoptosis.The in vivo toxicity evaluation showed that the blood biochemistry and blood routine were normal,and no obvious histopathological change was observed in the major organs,which demonstrated the low toxicity of NLS-CDs in vivo.Based on the unique properties of NLS-CDs,the anti-tumor drug DOX was coupled with NLS-CDs to construct multifunctional nanocomplexes DOX-CDs.The in vitro release profile of DOX-CDs was investigated in various dissolution media and the cytotoxicity,cell imaging,cellular uptake,and cell apoptosis were also studied.The as prepared nanocomplexes displayed well-dispersed spherical morphology with a mean statistical diameter of 6.1 nm and the release was in a pH-sensitive manner.The cytotoxicity of DOX-CDs was significantly lower than that of DOX at the concentration of below 1 μg·mL-1.DOX-CDs exhibited an enhanced cellular uptake compared with DOX,and the nanocomplexes could localize to the nucleus.The results of cell apoptosis revealed that DOX-CDs could effectively induce apoptosis and decrease the number of necrotic cells,which suggested that the nanocomplexes may possess great potential in cancer treatment.In vivo antitumor activity of DOX-CDs was assessed in A549 xenograft mice and H22 tumor-bearing mice were employed to investigate the pharmacodynamics further.Histological analysis was carried out to evaluate the in vivo toxicity of DOX-CDs.The effect of DOX-CDs on cell proliferation and apoptosis was investigated by immunohistochemical assay.The distribution of DOX-CDs in mice was monitored using real time in vivo fluorescence imaging.The results presented that DOX-CDs could suppress tumor growth effectively and the tumor growth inhibition rate was 60.9%.Furthermore,the nanocomplexes could reduce the heart toxicity of DOX according to the H&E staining.More importantly,DOX-CDs could inhibit tumor cell proliferation and induce tumor cell apoptosis in vivo.The in vivo non-invasive image showed that the nanocomplexes mainly distributed in the bladder area after intravenous injection of DOX-CDs,with the time increasing,DOX-CDs accumulated at the tumor sites gradually due to the EPR effect.The ex vivo optical images of tumor and major organs revealed that the strong fluorescence was observed in tumor and kidney,which indicated the excellent tumor-targeting capability of DOX-CDs.