| Inducing apoptosis of cancer cells is an effective means of cancer treatment,which is based on controlling or inducing the polar reaction of tumor cells to death.Among the many apoptotic protein factors,Bcl-2 family proteins are known to regulate the mitochondrial apoptotic pathway by controlling mitochondrial outer membrane permeabilization.The Bcl-2 family of proteins is divided into three subfamilies:anti-apoptotic Bcl-2 family proteins,pro-apoptotic multidomain Bcl-2 family proteins,and pro-apoptotic BH3-only proteins,which share homology only in the 15-to 25-residue BH3 structural domain with the other Bcl-2 family members.Thus,BH3 peptides from the Bcl-2 family and their analogs,on the other hand,exhibit unique apoptosis-activating functions.This antitumor peptide is intermediate between small molecule drugs（molecular weight<1000）and protein drugs,and it is a powerful alternative to traditional small molecule anticancer drugs.Since the beginning of this century,peptide drugs have become ideal candidates for alternative therapies due to their inherent high activity,small dosage,good solubility,excellent safety,uncomplicated synthesis and modification.However,it is undeniable that almost all antitumor peptides suffer from low metabolic stability,poor specificity for tumor cells,and poor permeability to tumor tissues and cell membranes,which seriously hinder the application of antitumor peptides in clinical therapy.Based on the above drawbacks of peptide drugs,the design and preparation of suitable carrier systems to deliver and ensure their entry into tumor cells to play their roles are of great research value in cancer therapy.In this paper,firstly,the Bax-BH3 expression plasmid of p LVX-m Cherry-N1 and the mutated control plasmid were constructed by recombinant technology for in vitro transfection experiments,and the cell survival of HEK-293T human embryonic kidney cells by the recombinant plasmid after transfection was explored.The results showed that the recombinant Bax-BH3 plasmid could induce apoptosis in a very short time after transfection.While the control plasmid did not cause cell damage after transfection after one key amino acid mutation based on the sequence of Bax-BH3,which indicated that the Bax-BH3 polypeptide has excellent pro-apoptotic activity and function,and can be used as an anti-tumor polypeptide drug candidate,and the polypeptide translated from the Bax-BH3 sequence was named BH3.Secondly,to address the drawbacks of low BH3 cell membrane permeability,poor stability against protease digestion,and low endosomal escape rate,MOF-based drug delivery carriers were designed and prepared,and zeolite imidazolium ester skeleton was synthesized-8.It is one of the most representative materials of MOFs,and its skeleton is a dodecahedral structure formed by linking the metal Zn2+with the N atoms in dimethylimidazole.In this paper,BH3@ZIF-8 nanomaterials were prepared and characterized by scanning electron microscopy and transmission electron microscopy;the materials were well dispersed,and the particle sizes of monodispersed nanoparticles were all around 70 nm,with good stability;the successful synthesis of ZIF-8 and the effective loading of BH3 were determined by X-ray diffraction,infrared,and surface potential measurements;and the protein assay by BCA was determined by The loading of BH3 in BH3@ZIF-8 NPs was 19.1%and the encapsulation rate was 22.9%.To evaluate the ex vivo and in vivo antitumor effects of BH3@ZIF-8 NPs,non-small cell lung cancer cells were selected and subjected to in vitro cytotoxicity assay,cellular uptake assay,mitochondrial membrane potential assay assay,and apoptosis assay by flow cytometry,as well as evaluated for cell migration and proliferation ability.Subsequently,a hormonal mouse model of cancer cells was established and administered to explore.The results showed that the BH3@ZIF-8 material exhibited excellent anti-tumor effects both in vivo and ex vivo,with the ability to promote tumor cell apoptosis,and at the same time inhibit the migration and proliferation ability of tumor cells.After the adoption of MOF as a drug delivery carrier,the present study continued with polymer composites as new nanodrug carriers to further construct a new delivery system for BH3 peptide transport.The new composites were characterized by transmission electron microscopy,etc.,and proved to be successfully synthesized and efficiently loaded with BH3 with 15.2%drug loading and 14.0%encapsulation rate.The results of in vitro and in vivo experiments in a loaded mouse model showed that the newly synthesized polymer composite-based nanodrug carriers can still successfully carry BH3 with high biosafety and have better efficacy in tumor therapy due to the addition of targeting factor modifications in the design.In order to investigate the anti-tumor mechanism of action of BH3,this thesis used reverse molecular docking method to predict its possible drug targets and mapped the protein interaction network and drug-target-disease network using BH3 polypeptide as a drug through Pharm Mapper,OMIM and Genecards databases.The core targets were screened out,and the binding modes of BH3 to the binding optimal targets were predicted to be mainly hydrogen bonding and hydrophobic interactions using reverse molecular docking technique.In summary,a recombinant plasmid with sequences originating from the Bax-BH3structural domain was constructed in this study,and its antitumor effect was verified after identification.Subsequently,the peptide translated from the target gene was synthesized,and two carrier systems,ZIF-8 and the polymeric nanomaterial RPPMMRA,were designed and applied respectively to explore its antitumor activity in vitro and in vivo after loading the BH3 peptide,and meanwhile,the potential antitumor mechanism of BH3 was investigated using reverse docking.The Bax-BH3peptide-based nanodrug delivery system designed and prepared in this thesis provides a new idea for the development of anticancer peptide drugs. |
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