Preparation Of Pro-apoptotic Peptide-doxorubicin Conjugate And Its Anti-cervical Research

Doxorubicin（Dox）is an antibiotic with a broad-spectrum antitumor effect which displayed remarkable efficacy in a variety of tumors such as malignant lymphoma,lung cancer,and breast cancer.However,doxorubicin has toxic side effects in normal tissues,especially in heart tissues.In the past two decades,peptide-drug conjugates（PDCs）has attracted great attention in the field of anticancer drug development due to the reduced toxic and side effects and improved the anticancer efficacy.Until now,a variety of functional peptides such as cell-penetrating peptides,tumor-targeting peptides,and tumor microenvironment-responsive peptides have been used to develop a variety of PDCs for cancer treatment.PDCs could significantly improve the therapeutic effect by increasing the targeted delivery efficiency of drugs.Pro-apoptotic peptide（KLAK）is an antitumor peptide that can induce programmed cell death by disrupting mitochondrial membranes.In this study,we developed a conjugate（KLAK-Dox）based on Dox and KLAK peptides for the efficeient treatment of cervical cancer.We explored the acid-sensitive properties of pro-apoptotic peptide-doxorubicin（KLAK-Dox）conjugates.And we investigated the endocytosis of KLAK-Dox conjugates by tumor cells and its antitumor effects in vitro and in vivo.In this study,KLAK-Dox conjugates was prepared by a Michael addition reaction using the thiol group of the terminal cysteine of KLAK peptide（KLAKLAK）₂ and maleimide-functionalized doxorubicin（DOXO-EMCH）.Mass spectrometry,infrared spectroscopy and high performance liquid chromatography were used to characterize KLAK-Dox conjugates.The acid-responsive release characteristics of KLAK-Dox conjugates was detected via dialysis.Confocal laser scanning microscopy（CLSM）and flow cytometry were utilized to examine the endocytosis of KLAK-Dox on tumor cells.The cytotoxicity of KLAK-Dox conjugates was determined by MTT method and live/dead cell staining method.We used the characteristics of doxorubicin autofluorescence to detect the distribution of drugs in vivo through the CRI Maestro ^TMM imager;we utilized cervical cancer-bearing mouse models to test its antitumor effect and toxic side effects in vivo.In this study,KLAK-Dox conjugate based on a pro-apoptotic peptide-doxorubicin was successfully prepared,and more than 80%of Dox was released in phosphate buffer saline at pH5.5,while only 10%was released in phosphate buffer saline at pH 7.4.Fluorescence imaging and flow cytometry showed that KLAK-Dox can be rapidly endocytosed by HeLa cells.MTT and live/dead cell staining results showed that KLAK-Dox conjugates have stronger killing effects than Dox,KLAK,and KLAK+Dox in tumor cells.The distribution of conjugate in vivo showed that KLAK-Dox accumulated more in tumor tissues than small molecule doxorubicin group after6 hours of tail vein administration.In vivo anti-tumor results showed that the effects of free Dox,KLAK,Dox+KLAK and KLAK-Dox conjugates on inhibiting tumor growth were 32.50%,22.25%,38.08%and 61.22%,respectively.The above results showed that the KLAK-Dox group has a stronger tumor suppressive effect and lower systemic toxicity than the small molecule doxorubicin showed by the weight change curve of mice.Our study suggested that PDCs prepared with pro-apoptotic peptide is a highly effective and low toxicity anticancer drug.