Construction Of Melittin@ZIF-8 Nanoparticle Drug Delivery System And Its Anti-Tumor Activity Study

The natural biotoxin melittin?MLT?is the main component of bee venom.It is an amphiphilic polypeptide consisting of 26 amino acids with a wide range of pharmacological effects,such as anti-inflammatory,antibacterial,antiviral,analgesic,anti-radiation and so on,especially in anti-tumor,MLT has unique advantages.It can directly attack tumor cells,increase the gap of cell membrane by lysing tumor cell membrane,destroy the integrity of phospholipid membrane,and subsequently induce cell death.In addition,after intracellular delivery,MLT also can act on the cytomembranes of the internal organelles in a similar manner,inducing biochemical changes or transcriptional regulation.MLT exerts various impacts on functions of cancer cells,including inhibiting cancer cell proliferations,inducing apoptosis,suppressing tumor metastasis and angiogenesis,and blocking cell cycle,providing broad prospects for the future cancer treatment.However,the hemolysis and non-specific cytotoxicity of MLT,as well as the characters of variability and rapid degradation,have seriously hampered its clinical application.Therefore,how to achieve attenuation and synergy is of great significance for its clinical application,which can effectively deliver MLT to tumor tissues and reduce its toxicity to normal tissue cells.The nanoparticle drug delivery system can effectively exert the protective effect of materials on drugs,assist drugs to cross the mucosal barrier,enhance the accumulation of drugs at the target site through the enhanced permeability and retention?EPR?effect,change the distribution of drugs in body,and reduce the systemic toxicity,which has become an important direction of modern drug research.The method of nanocarrier delivery is also considered to be the best strategy to improve the therapeutic effect of MLT.Metal-organic frameworks?MOFs?is a new type of organic-inorganic hybrid porous materials.Compared with traditional nanomaterials,MOFs possess large specific surface area,high porosity,tunable pore size,simple preparation,and easy functionalization,so they have been widely used in catalysis.chemical sensing,gas adsorption and other fields,especially in drug loading and delivery.Among them,Zeolitic imidazolate frameworks-8?ZIF-8?,formed by zinc ions and 2-methylimidazole,is the most representative one.In addition to the general advantages of MOFs,ZIF-8itself has better properties,such as better drug loading capacity,higher thermal stability and chemical stability,and good biocompatibility.More importantly,ZIF-8 material also possess pH-sensitive behavior,which maintains structural stability under neutral physiological conditions,and rapidly decomposes in the weakly acidic cell environment of tumor tissue.It can realize the controlled and precise drug release in the tumor site,and meet people's requirements on drug loading,stability and drug release for the new nanoparticle drug delivery system.Based on the above characteristics,we chose ZIF-8nanomaterial as the drug delivery vector for MLT to achieve the attenuated and synergistic treatment.Our research includes three fields of the work as follows:1.Preparation and characterization of MLT@ZIF-8 NPsIn this work,the green synthesis method of the"one-pot stirring method"of melittin@ZIF-8 nanoparticles?MLT@ZIF-8 NPs?was established under the conditions of ensuring the water system.The formed MLT@ZIF-8 NPs have been proved to possess uniform rhombic dodecahedral shapes and monodisperse particles size with the mean diameter of approximately 100 nm through SEM,TEM and DLS.The assay methods of PXRD,FTIR and Zeta potential measurement were used to confirm the successful synthesis of ZIF-8 crystals and the existence of MLT in MLT@ZIF-8 NPs.The protein loading and encapsulation efficiency of the MLT@ZIF-8 NPs were calculated to be 6.9 wt%and 25.5%through a standard BCA protein quantitative method and TGA analysis.The drug release experiments of MLT@ZIF-8 NPs under different pH conditions showed that MLT@ZIF-8 NPs remained stable under neutral physiological conditions?pH 7.4?,and the release rate of MLT was very slow,while the drug release rate is significantly increased in the slightly acidic environment?pH 6.8and 5.5?,suggesting that ZIF-8 material has excellent pH sensitivity,this material would disintegrate in a slightly acidic environment,and effectively release the loaded drug.The stability test of MLT@ZIF-8 NPs in room temperature aqueous solution and 37^oC simulated physiological conditions?PBS buffer containing 10%FBS?confirmed that MLT@ZIF-8 NPs could remain their hydrodynamic diameter and PDI basically unchanged for 7 days in these two solutions,indicating that MLT@ZIF-8 NPs possess favorable dimensional stability.Meanwhile,the in vitro hemolysis assay results showed that free MLT itself had strong hemolytic properties,which could cause about 90%hemolysis at the concentration of 8?g/mL,whereas MLT@ZIF-8 NPs still exhibited minimal hemolysis?8%?even MLT concentration reached 128?g/mL,implying that the method of encapsulating MLT with ZIF-8 nanomaterial can effectively reduce the hemolytic activity of MLT and improve the drug safety.2.Evaluation of the anti-tumor activity in vitro of MLT@ZIF-8 NPs and its mechanism studyTo evaluate the anti-tumor effects in vitro of MLT@ZIF-8 NPs,MTT assay was used to detect the effect of MLT@ZIF-8 NPs on tumor cell proliferating activity.The results showed that MLT@ZIF-8 NPs loaded with the same amount of MLT exhibited higher inhibition efficiency for the proliferation of both tumor cells?A549 cells and HeLa cells?than free MLT.The IC₅₀ values of MLT@ZIF-8 NPs and free MLT for A549 cells were 6.7 and 8.2?g/mL,respectively,and for HeLa cells were 4.2 and 4.8?g/mL,indicating that loading MLT into the framework of ZIF-8 could effectively enhance its killing effect on tumor cells.This study investigated the cell uptake and apoptosis induction of MLT@ZIF-8 NPs by confocal laser scanning microscopy and flow cytometry analysis.The results showed that MLT@ZIF-8 NPs could be efficiently taken into A549 tumor cells,and the endocytosis behavior was in time-dependent and dose-dependent manner.Meanwhile,MLT@ZIF-8 NPs also could induce apoptosis of A549 cells,and the apoptosis ratio increased significantly with the increasing concentration,whereas the cell changes were mainly necrosis after treatment with free MLT,suggesting that the effects of free MLT and MLT@ZIF-8 NPs on cancer cells are different.To further explore the anti-tumor mechanism of MLT@ZIF-8 NPs,gene chip technology was used to study the gene expression changes of A549 cells before and after MLT@ZIF-8 NPs treatment,and we identified 3383 differentially expressed genes,including 1945 up-regulated genes and 1438 down-regulated genes.We next performed Gene Ontology?GO?and KEGG?Kyoto Encyclopedia of Genes and Genomes?pathway analysis for 3383 differentially expressed genes caused by ZIF-8@MLT NPs.Among them,GO enrichment analysis comprehensively summarized the functional information of differentially expressed genes from three aspects:biological processes,cellular components,and molecular function.The results of KEGG pathway analysis showed that Pathways in cancer possessed the highest percentage of differentially expressed genes,and p53 and PI3K genes were included in these genes,suggesting that p53 and AKT pathways might be involved in the regulation of MLT@ZIF-8 NPs on A549 cells.Subsequently,Western Blot was used to detect the protein expression levels of p53 and AKT pathway in A549 cells after MLT@ZIF-8 NPs treatment.The results showed that after MLT@ZIF-8 NPs treatment,p53 protein expression was up-regulated,Bax,Cleaved caspase-3,Cleaved caspase-9 and Cytc expression were up-regulated,Bcl-2 protein expression was down-regulated,Bax/Bcl-2 ratio was up-regulated,PI3K and p-AKT protein expression was down-regulated,and p-AKT/AKT ratio was down-regulated,which was consistent with the results of gene chip analysis,validating that PI3K/Akt-regulated p53 signal pathway is related to the inhibitory effect of MLT@ZIF-8 NPs on the proliferation of A549 tumor cells and the molecular mechanism of inducing apoptosis.3.Study of the anti-tumor activity in vivo of MLT@ZIF-8 NPsIn this study,we established two kinds of mice tumor models?BALB/c nude mice model with immunodeficiency and Kunming mice model with normal immune system?to evaluate the inhibitory effect of MLT@ZIF-8 NPs on tumor growth in vivo after intravenous administration,and the safety of nanodrugs in vivo was evaluated through blood physiological and biochemical indicators detection.The results showed that MLT@ZIF-8 NPs could significantly inhibited the growth of tumors in vivo in both two mice tumor models,and MLT@ZIF-8 NPs exhibited better anti-tumor effect than free MLT.The tumor inhibition rates of MLT@ZIF-8 NPs and free MLT in BALB/c nude mice model were 84%and 59%,respectively,and in Kunming mice model were 71%and 49%,suggesting that loading MLT into the framework of ZIF-8 could also effectively enhance its anti-tumor efficiency in vivo.Hematoxylin and eosin?H&E?staining of tumor regions in BALB/c nude mice model and Kunming mice model showed that there were different degrees of tumor necrosis-like changes both in MLT treated group and MLT@ZIF-8 NPs treated group.And in these two groups,MLT@ZIF-8 NPs treated group had the most dead cells in the tumor tissues,and the tumor necrosis was the most serious,indicating that MLT@ZIF-8 NPs possessed more potent killing effect on tumor tissues in vivo.The results of physiological and biochemical detection showed that the hematological parameters and serum biochemistry indicators of mice at different time points after MLT@ZIF-8 NPs administration were all in the normal range,demonstrating that MLT@ZIF-8 NPs had no obvious side effects at the current dose,and didn't affect the normal function of major organs,so they could be safely and effectively used for anti-tumor therapy in vivo.In conclusion,we successfully constructed the pH-responsive melittin@ZIF-8nanoparticle drug delivery system based on a new type of nanoporous material ZIF-8.The formed MLT@ZIF-8 NPs not only solve the problems of hemolysis and non-specific effects of MLT,but also efficiently enhance its anti-tumor effects in vitro and in vivo,and PI3K/Akt-regulated p53 signal pathway is related to the anti-tumor effect of MLT@ZIF-8 NPs.This work provides new insights and development directions for the application of natural biological toxin MLT as an anti-tumor drug,and also laid the foundation for the development of other biotoxin resources and their attenuation and synergy study.