Reactive Oxygen Species Auto-control Nanosystem Comprising Green Tea Catechin Derivatives And Melittin For Antitumor Therapy

Toxic peptides,such as melittin,scorpion venom peptide,snake venom peptide and so on,are the important components of traditional Chinese medicine.Multiple biological activities of toxic peptides,including anti-inflammatory,anti-bacterial,analgesic,anti-AIDS had been reported.Toxic peptides have been advocated to apply in the treatment of tumor because they can inhibit cell proliferation by multi-target effects or directly killing the cancer cell by lytic properties,resulting in the reverse of multidrug resistance.However,Toxic peptides are greatly limited to clinical applications because of the strong toxicity,nonspecific distribution and poor bioavailability in vivo.How to deliver toxic peptides to the tumor tissue safely and effectively without any side effects is the key of developing the drug delivery system of toxic peptides.In this study,melittin,an anticancer peptide,was selected as the model drug,and pEGCG was used as the carrier.PEGCG can self-assembly with melittin to form the pEGCG-melittin nanocomplex(NC)because of the affinity of pEGCG to protein.In order to deliver the NC to the tumor site stably,3-aminophenylboronic acid derivatized hyaluronic acid(pHA)is anchored to the surface of the NC to prepare pHA coated pEGCG-melittin nanocomplex(pHA-NC)by forming a ROS sensitive borate bond between boric acid and catechol hydroxyl.When the pHA-NC reached the tumor site through the EPR effect,HA shell was dissociated by highly expressed hyaluronidase(HAase)and reactive oxygen species(ROS)in tumor,resulting in the release of melittin.The melittin and pEGCG that had released from pHA-NC could further enlarge intracellular ROS signal and constructed an intracellular environment with high ROS levels,which ensure sustained and stable release of the drug.PEGCG was synthesized by Bayer reaction and the structural information were characterized by nuclear magnetic resonance spectroscopy,mass spectrometry,fourier transform infrared spectrometer and HPLC.Orthogonal experiment was used to select the optimal prescription of NC,and the particle size was 127.67 ± 5.17 nm,the PDI was 0.24±0.04,and the surface charge was 25.4 ± 1.8 mv in the optimal prescription.Transmission electron microscopy showed that NC was spherical with uniform size distribution.The interaction between melittin and pEGCG was confirmed by microscale thermophoresis and gel electrophoresis.The interaction of pEGCG and melittin was studied by monitoring the change of particle size after adding different competitors of interaction.The results showed that the hydrophobic interaction may be the main mode of interaction between,melittin and pEGCG,which was further confirmed by FRET experiment.In this study,hyaluronic acid(HA)was modified by 3-aminobenzene boronic acid,and the structure was confirmed by 1H NMR and 13C NMR.The ratio of the area of characteristic peaks in 1H NMR was used to calculate the content of 3-aminophenylboronic acid.The content of 3-aminophenylboronic acid was about 2.5%.The particle size of pHA-NC that prepared by optimal process was 141.06±1.16nm,the PDI was 0.13 ± 0.02,and the surface charge was-17.53±0.41mv.The transmission electron microscopy indicated that pHA-NC was spherical and had a shell-core structure.PHA-NC could remain stable in PBS,DMEM and 10%FBS.The formation of borate bond between pHA and pEGCG was confirmed by the fluorescence method.The release test showed that pHA-NC was able to respond to HAase and H2O2,resulting in the release of melittin.By monitoring the content of H2O2 in PBS,it was confirmed that pEGCG and NC could produce H2O2 in PBS at pH 7.4.However,after blocking by HA or pHA,the oxidation of pEGCG was prevented,which effectively reduced the formation of H2O2 and ensured the stable structure of pHA-NC.Cell research including the cell uptake,intracellular distribution,intracellular release,intracellular ROS levels,mitochondrial membrane potential,toxicity and apoptosis was carried out.The pHA-NC was taken up by clathrin-mediated and macropinocytosis pathway and CD44 receptor-mediated internalization.CLSM was used to observe the intracellular delivery of pHA-NC,the results demonstrated that pHA-NC can escape from lysosome rapidly.The FRET technique was used to monitor the intracellular release of pHA-NC,confirming the dissociation of pEGCG and melittin in the cells.Cytotoxicity and apoptosis experiments showed synergistic antitumor effects of melittin and pEGCG.The ability of melittin,pEGCG,NC and pHA-NC to amplify the ROS signal was confirmed by detecting the intracellular ROS levels.The ROS non-sensitive formulation HA-NC was prepared,and we confirmed that ROS-trigger release characteristics of pHA-NC enhanced the anti-tumor effect by comparing the ability of pHA-NC that induced apoptosis,produced ROS and reduced mitochondrial membrane potential with that of HA-NC.In this study,the B16F10 tumor-bearing mouse model was established.The tissue distribution of melittin,NC and pHA-NC in vivo was studied by living imaging technique.HA shell increased the tumor targeting of melittin and reduced the distribution in the liver.The results of pharmacokinetics study in vivo showed that the anticancer effects in vivo were pHA-NC>HA-NC>melittin>pHA-NCb>saline.Further,the safety evaluation showed that pHA-NC did not damage the heart,liver,spleen,lung,kidney and brain.What is more important is that pHA-NC almost completely shield the melittin toxicity,which breaking the limit that apply melittin to clinical therapy by intravenous injection because of hemolytic toxicity.