Controlled Release Of Melittin Based On Mesoporous Silica Nanoparticles

In recent years, the number of cancer patients is rising. The combination of surgery, radiotherapy and chemotherapy had played a certain role in improving the physical condition and the quality of malignant tumor patients. However, the toxicity and side effects of radiotherapy and chemotherapy still limited the therapeutic effect of malignant tumor. The anti-cancer effect of melittin has been validated by many scientists.Oral administration of melittin is not available because it is easy to be destroyed and degraded by gastric proteinases and acid. Meanwhile, melittin has hemolytic effect, and on-target melittin could break the cell membranes and cause the leakage of cellular content and result in the cell death. This study is to design a self-assembly targeted nano drug carrier based on mesoporous silica nanoparticles for the controlled release of melittin. This nano drug carrier was expected to reduce the lytic toxity and degradation of melittin, and represent a general delivery vehicle for natural biotoxins to exploit anticancer application.The first part of this subject completed the separation and purification of melittin, as well as qualitative and quantitative appraisal. We purified melittin by the three-step-column-chromatography through dextran gel Sephadex G-25, Sephadex G-50 and Sephadex G-75 and identified its hemolysis activity preliminarily. Then we identify the purified melittin according to the standard by means of HPLC with fluorescence detector. The results showed that the purity of melittin was more than 95%, and retained high hemolysis activity.The second part of this subject was to prepare large pore size mesoporous SiO2 nanoparticles (MSNs) and perform melittin-loaded MSNs adsorption experiments, and cell endocytosis experiment. Firstly, we prepared large pore size MSNs under the alkaline condition and use CTAB, TEOS and mesitylene as template, silicon source and expanding agent respectively. Then obtained MSNs were with average particle size of 300 ± 20 nm,average pore size of 7.79 nm,BET specific surface area of 341.13 m2/g, and pore volume of 0.66 cm3/g, its aperture is 5 nm bigger than MSNs without pore expanding. Secondly, we made use of amino, carboxyl, disulfide bond, a-cyclodextrin and other functional groups one by one to modify the surface of MSN. TEM, FTIR, nitrogen adsorption, Zeta potential and fluorescent amine methods were used to confirm each modification. The results suggested a successful modification of those functional groups on the surface of MSNs. Melittin’s adsorption experiment showed that MSNs could encapsulate melittin. The adsorption of melittin into MSN-NH2 and MSN-COOH was decreased, which suggested that melittin adsorption quantity will become less with further grafted function groups, and further functionalized modification has a certain effect on pore size and pore volume of MSNs. The larger pore size of MSN-S-S-NH2 had stronger adsorption ability. We evaluated the endocytose of particles with FITC labeled MSNs by using hepatocellular carcinoma HepG2. The number of endocytose particles increased as time goes on during the first 24 h.This indicated that the particles can be taked into hepatocellular carcinoma HepG2 cell.The third part of this subject, we designed a novel MSNs-controlled-release system, in which the "gate" was sealed by a-cyclodextrin self-assembly. To obtain PEG-functionalized MSNs (MSN-PEG), the amino of MSN-NH2 and the carboxyl of HO-PEG-COOH proceeded coupling reaction under the exists of catalyst of DCC and NHS. Then we obtained folic acid functionalized MSNs (FA-MSN) that can targeted folatereceptor on the surface of hepatocellular carcinoma HepG2 cell in the same way.TEM, FTIR, Zeta potential and DLS particle size were used to confirm whether PEG-and PEG-FA was grafted onto the surface of MSNs. Secondly, the reversible specific recognition between the a-cyclodextrin hydrophobic cavity and PEG polymer chains was applied to design a a-cyclodextrin based self-assembly controlled released system. We confirmed the self-assembly controlled release system is feasible by the enzyme lytic and adamantanamine competitive release experiment. Results indicated that a-cyclodextrin hydrophobic cavity can be used to form reversible self-assembly system with PEG chains under relatively mild conditions (It can conduct the process under physiological conditions with pH=7.4). The self-assembled system can represent a general carrier of bioactive polypeptides as melittin, to avoid the loss of polypeptide activity. We also evaluated the cell toxicity of MSNs in 48h. The cell viability is more than 94%,. and suggested that MSNs and surface functionalized MSNs have no obvious toxic effects on hepatocellular carcinoma HepG2 cell.