Controlled Release Of Melittin For Hepatocell-Ular Carcinoma HepG2 Based On Mesoporous Silica Nanoparticles

Melittin is the main component of bioactivities and pharmacological activities in bee venom,which could effectively kill a variety of cancer cells.However,melittin is a polypeptide composed with 26 amino acid residues.So melittin could be decomposed by pepsin and peptidase and lose related activities rooting of the structural damage of melittin.Also because of its nonspecific hemolysis effect and unable to target diseased tissues transporting in vivo,melittin has serious side effects on organism's normal tissues and organs under the treatment of doses.In recent years,target controlled release drug exposures to more attention.Mesoporous silica nanoparticles have well biocompatibility,tuned mesoporous size distribution and homogeneous structure,large specific surface area(>900 m2/g)and pore volume(>0.9 cm3/g)and a variety of functional groups modifications of internal and external surface given the abundant silanol groups(Si-OH).We can load multiple drugs within the channel,which not only can protect drugs,bioactivity and slowly release drugs,but also can target lesion location and thus control the release of drugs under the condition of low toxic side effects,to improve the persistence and effectiveness of drugs.In this paper,we designed a target controlled release nano-drug delivery system of self-assembled seal.Base on MSNs rigid channel structure,not only melittin activities could be protected which may avoid melittin to be degraded,but also could reduce its nonspecific hemolysis side effects when it is applied to management in vivo later,exerting a novel idea for natural biological toxin application in anti-cancer-targeted drug delivery.In this paper,based on surfactant CTAB as structure-directing agent,TEOS as organic silicon source,mixing mesitylene as pore expanding agents,we synthesized mesoporous silica MSNs,which average diameter is about 3 nm,particle size is about 200 nm,333.56 m2/g and 0.22 cm3/g for BET specific surface and pore volume,respectively.Given that melittin shows a positive charge in PBS(pH=7.4)solution,APTES was modified on the surface of MSNs to exclude protein adsorbed on the external surface of MSN-NH2 by electrostatic interaction,thus the results on loading capacity difference of melittin could be attributed to interior pore voids role of MSN-NH2,Then MSN-NH2 reacted with slilyl reagents to modify-NH2,-PO3 and-COOH groups on the surface of internal channel of MSN-NH2,respectively.These are inner-P03-MSN-NH2,inner-NH2-MSN-NH2 and inner-COOH-MSN-NH2.We researched pore of MSN-NH2 modified with different functionalized groups to effect on melittin loading capacity in the channel.We determined super-natant melittin before and after adsorbed at different time periods until equilibtation by HPLC-IFD.Results indicated that inner-COOH-MSN-NH2 has the best adsorption quantity and capacity for melittin.And then we studied the inner-COOH-MSN-NH2 with different pore size to effect on adsorption quantity of melittin.In addition,we synthesized inner-COOH-MSN-NH2 with the larger pore size,which average diameter is about 7 nm,particle size is about 200 nm,341.13 m2/g and 0.70 cm3/g for BET specific surface and pore volume,respectively.Both of them adsorbed melittin in different concentrations until equilibration.Result indicated that the increased pore size and the concentration of melittin,the increased loading quantity.In addition,as the steps MSN synthesized above,we synthesized mesoporous silica MSN,which average diameter is about 4.54 nm,particle size is about 200 nm,353.64 m2/g and 0.49 cm3/g for BET specific surface and pore volume,respectively.The surface of MSN was modified by a variety of functional groups to obtain MSN-S-S-PEG-FA nanoparticles which exerts the properties of well biocompatibility,GSH redox triggered and specific target the surface folate receptor of hepato-cellular carcinoma HepG2 cells.Characterized by relative techniques,MSNs adsorbed melittin in pH=7.4 0.01 M PBS buffer solution,and self-assembly by forming inclusion of ?-CD hydrophobic cavity and polymer PEG chain through hydrophobic interaction to seal the opening pore of MSNs nanoparticles.The results which GSH trigger controlled-release of melittin in pH?7.4 0.01M PBS buffer solution shown that disulfide linkage redox and ?-CD self-assembly seal operated.Hemo-lysis assays proved that melittin released from MSNs nanoparticles still possesses higher biological activity compared with the free melittin before adsorbed,and at the same time,proved that the fashion of ?-CD self-assembly to seal has no damage to melittin activity compared with click chemistry to seal the orifices by crosslinking agent EDC+NHS or NHS.We further used fluores-cence microscope to prove that MSNs labeled by fluorescein sodium could be targeted and endocytosed soon by HepG2 cells.Based on this phenomenon,we hypothesized that MSNs labeled by fluorescein sodium could be taked into intracellular by HepG2 cells' surface folate receptors mediation,and then high level GSH in cytoplasm redox to crack disulfide bond to open the "gate-like effect",?-CD entities and release the "cargo" fluorescein sodium.Given the nanoparticles could be entered into intracellular,we further determined the survival rate of hepatocellular carcinoma HepG2 incubated with melittin/MSNs nanoparticles by MTT assays.Results showed that melittin/MSNs nanoparticles could effectively inhibit cancer cells'proliferation.Based on the results,we hypothesized that melittin/MSNs nanoparticles could be entered into intracellular and break disulfide bond to open the "gate-like effect" ?-CD entities and release the "cargo" melittin,thus could significant inhibit cancer cell proliferation.