| Objective:Based on the self-assembly properties of FFKY(FY4)and the aggregation-induced emission(AIE)technology,a GSH/p H dual-sensitive nanocarrier,polyethylene glycoldisulfide-FY4-hydra-tetrastyrene(PEG-Pep-TPE),was constructed in this study to detect the release of doxorubicin dynamically,and achieve the synergistic anticancer efficacy with DOX.Meanwhile,combined with macrophage immunotherapy,FY4 was modified with CD47 targeting peptide(4N1K),and a hypoxia-responsive nanocarrier,4N1K-FY4-azobenzene-PEG(4N1K-FY4-Azo-PEG,PAP),was constructed for anti-tumor growth and metastasis research.Methods:Combining the low p H,high GSH,and low oxygen characteristics of tumor tissues,one end of the self-assembling peptide FY4 was connected to a hydrophilic polymer PEG,and the other end was separately connected to an aggregation-induced luminescence(AIE)molecule(TPE-CHO)or a target for CD47(4N1K)to prepared PEG-Pep-TPE or PAP finally.The structure of conjugates was confirmed by nuclear magnetic resonance(NMR),infrared spectroscopy(IR),mass spectrometry(MS),and its particle size was characterized by DLS.GSH / p H responsiveness of PEG-Pep-TPE NPs was investigated by transmission electron microscope(TEM);the optical properties such as AIE / FRET of PEG-Pep-TPE NPs were investigated by a fluorescence spectrophotometer and ultraviolet-visible spectrophotometer(UV/NIS);Confocal receptor photobleaching,spectroscopy,and sensitized emission methods were used to detect and quantify the change of FRET signals in A549 cells to monitor the cellular uptake of nanoprobes and intracellular drug molecule release intuitively.Finally,MTT and CLSM assay was used to evaluate synergistic cytotoxicity as well as its mechanism between the peptide nanofibers and doxorubicin.PAP NPs were constructed by combining macrophage immunity.Microscopic observation and flow cytometry were used to study the abilities of PAP NPs in blocking CD47 and promoting macrophage phagocytosis on B16F10 cell model in vitro,so as to investigate its inhibitory effect on tumor cell invasion and metastasis.C57 mice bearing B16F10 melanoma models was established to investigate the effect of PAP NPs in inhibiting tumor growth and metastasis and evaluate related factors in tumors.Results:GSH/p H-responsive PEG-Pep-TPE NPs and hypoxic-responsive PAP NPs were successfully prepared.The morphology was uniform and spherical,and the particle size was about 150 nm.Optical properties measured in vitro and CLSM results confirmed that the PEG-Pep-TPE NPs has AIE/FRET effect and can monitor drug release dynamically.Cytotoxicity experiment showed that peptide self-assembling fibers and DOX could achieve the synergistic anticancer efficacy.CLSM results,flow cytometry analysis and in vivo tumor suppression studies implied that the PAP NPs can effectively block CD47 expressed on the surface of tumor cells,so as to promote the phagocytosis of macrophages;Combined with low-dose DOX,PAP NPs could significantly inhibit tumor growth and achieved the same anti-tumor effect produced by multiple chemotherapy,thereby reducing its toxic side effects;Scratch experiments in vitro and tumor metastasis inhibition in vivo showed that PAP NPs could effectively inhibit tumor angiogenesis,up-regulate the expression of E-cadherin,and selfassemble to form fibers at the tumor site,which effectively inhibit tumor invasion and metastasis.Conclusion:PEG-Pep-TPE NPs were equipped with GSH / p H responsiveness and AIE / FRET effects,so as to monitor drug release dynamically,and achieve synergistic cytotoxicity with DOX in the final;Hypoxia-responsive PAP NPs could self-assemble to form fibers at the tumor site and effectively block the CD47,thereby promoting the phagocytosis of tumor cells by macrophages,and ultimately inhibit tumor growth and metastasis. |
