Design Synthesis And Antitumor Activity Of E-selectin Targeted Podophyllotoxin Conjugate

E-selectin is highly expressed in vascular endothelial cells near tumors and is closely related to the occurrence,proliferation and metastasis of tumors.It is a target protein with significant potential for tumor therapy.In this project,a broad-spectrum small-molecule drug podophyllotoxin and E-selectin peptide ligands were used to construct and design an E-selectin-targeted small molecule anti-tumor drug coupling of"ligand-linking bridge-anti-tumor drug"（PEG-Pep-PODO）,and in-depth study of the targeting effect of E-selectin,the antitumor effect of podophyllotoxin,and the cytotoxicity of podophyllotoxin in the E-selectin target system were evaluated.The purpose of the research is to break through the limitations of existing anti-tumor drugs and develop new anti-tumor drugs with high efficiency,low toxicity and high targeting.Firstly,at the chemical level in vitro,after the E-selectin ligand（NH₂-CIELLQAR-COOH）was prepared by solid-phase synthesis technology,it was reacted with podophyllotoxin and polyethylene glycol through 6 steps to obtain a novel conjugated drug PEG-Pep-PODO.In vitro chemical characterization showed that the nanoparticles formed by PEG-Pep-PODO had a particle size of 108.70±0.26 nm,a potential of-6.41±0.47 m V,and the PDI value of 0.295.The release of podophyllotoxin in nanoparticles formed by PEG-Pep-PODO under different physiological conditions was detected by high performance liquid chromatography.In PBS aqueous solution with p H=7.4,the release rate of podophyllotoxin at 72 h was 67.90%.The release rate of podophyllotoxin in the PBS aqueous solution containing the final concentration of 4 m M GSH reached 100%in 48 h,which confirmed the high release rate of PEG-Pep-PODO in the simulated tumor microenvironment in vitro and relative stability under physiological conditions.Secondly,in this paper,the cytotoxicity of the target compound PEG-Pep-PODO on tumor cells and normal endothelial cell were analyzed by MTT method at the cell level,and the cell damage mechanism of PEG-Pep-PODO analyzed from the aspects of apoptosis and cell cycle.At the same time,the in vitro targeting effect of E-selectin ligand was explored through E-selectin-mediated cell adhesion model,and the potential ability of PEG-Pep-PODO to inhibit tumor cell metastasis and angiogenesis were further analyzed.After testing,PEG-Pep-PODO can significantly inhibit the proliferation of tumor cells in vitro.Except for the large cell lung cancer H460 cell line,it can exert a cytotoxic effect similar to that of podophyllotoxin monomer on other tumor cell lines.However,the cytotoxicity of PEG-Pep-PODO to normal endothelial cells HUVEC was significantly reduced,and the cell viability was 90.76%at 48 h,while the cell viability in the podophyllotoxin group was 21.02%at the same concentration.PEG-Pep-PODO can significantly inhibit cell adhesion and in vitro metastasis of MCF-7 tumor cells at the cellular level,with a metastasis inhibition rate of 78.53%.Finally,in this paper,a xenograft tumor model was established at the animal level to evaluate the in vivo pharmacodynamic effects of PEG-Pep-PODO,and combined with small animal imaging to explore the distribution and aggregation of the compound.The pharmacokinetics of PEG-Pep-PODO was analyzed by ICR mice to clarify the metabolism of PEG-Pep-PODO in mice.The results showed that the tumor inhibition rate of 10 mg/kg PEG-Pep-PODO reached 76.97%,and it could effectively maintain the body weight of mice while exerting tumor inhibitory effect with better safety than podophyllotoxin.The pharmacokinetics results showed that PEG-Pep-PODO was distributed faster,metabolized slower,and circulated more stable in mice compared with podophyllotoxin.In the fluorescence scale range of 1.40e⁸—7.57e⁸,the fluorescent substance indocyanine green ICG-conjugated PEG-Pep-ICG exhibited superior tumor targeting and aggregation effects,which further intuitively demonstrated the in vivo targeting effect of E-selectin polypeptide ligands.In conclusion,PEG-Pep-PODO can form a uniform nanoparticle structure in the aqueous environment.In vitro,it exhibited cytotoxicity similar to podophyllotoxin on multiple tumor cells at the cellular level,but the toxin was significantly reduced than podophyllin to normal endothelial cells with a certain"attenuating"effect.Meanwhile,PEG-Pep-PODO had significant E-selectin targeting at the cellular level and can effectively inhibit E-selectin-mediated cell adhesion.The superior anti-tumor and targeting effects of PEG-Pep-PODO had been further verified in vivo in tumor-bearing mice,with a tumor inhibition rate of 76.97%,and safety,pharmacokinetic properties and tumor vascularity targeting ability were obviously better than that of podophyllotoxin monomer.In this paper,a more comprehensive evaluation of PEG-Pep-PODO at biological and chemical perspectives had been carried out,which verified the conception of the multiple biological functions that at the beginning of the design of PEG-Pep-PODO,confirming the potential of PEG-Pep-PODO in the direction of targeting E-selectin antitumor drugs.