Loading Isorhamnetin Nanoparticles Was Investigated For The Anti-cancer Effect And Molecular Mechanism In Nasopharyngeal Carcinoma

Background and Objection:A novel pH-sensitive drug delivery system of mesoporous silica nanoparticles(MSNs)which were modified by polydopamine(PDA)for controlled release of Isorhamnetin(ISO)was prepared.The ISO release profiles of MSNs-ISO and MSNs-ISO@PDA were totally different,PDA was investigated for the effect on nanoparticle surface modification,MSNs-ISO@PDA was investigated for the anti-cancer effect and molecular mechanism in nasopharyngeal carcinoma.Materials and methods1.MaterialsTetraethylorthosilicate(TEOS),hexadecyl trimethyl ammonium bromide(CTAB),hydrochloride dopamine and hydrochloride desipramine were purchased from Sigma-Aldrich(St.Louis,MO,USA).Ammonium fluoride(NH4F)was purchased from Aladdin Industrial Co.,Ltd.(Shanghai,China).Acetonitrile and methanol were purchased from EMScience(HPLC grade,Mallinckrodt Baker,USA).Primary antibodies used in this work included LC3 antibody(rabbit source)was purchased from Cell Signaling Technology(Beverly,MA,USA).P62 antibody was purchased from Abmart Inc.(Shanghai,China).Beclin 1 was purchased from Cell Signaling Technology(Beverly,MA,USA)AKT,p-AKT,mTOR,p-mTOR,p70s6k,p-p70s6k antibodies were purchased from Santa Cruz(Santa Cruz,CA,USA).Human nasopharyngeal carcinoma cell line CNE-2 was purchased from American Type Culture Collection(ATCC).Water used throughout the studies was obtained from an ultrapure MilliQ water purification system(resistance>18 MU cm).All other chemicals of the highest available quality were commercially obtained and used as received.2.Methods2.1 MSNs were synthesized by dropping TEOS to a mixture of CTAB and NH4F,followed by reflux with ethanol and HCl to remove the surfactant CTAB.ISO and DOX were loaded and PDA coating were prepared for nanoparticle surface modification.MSNs-DOX@PDA and blank samples(MSNs@PDA)were also prepared by the same procedure.A series of instruments were used to measure the Characterization of the nanoparticles,and the drug loading content of MSNs-ISO@PDA was also analyzed.2.2 CNE-2 cells seeded into a 6-well dish,after treatment with FITC-MSNs-ISO and FITC-MSNs-ISO@PDA,respectively.We carried out confocal microscopy analysis to compare the cellular uptake efficiencies of FITC-MSNs-ISO and FITC-MSNs-ISO@PDA,the cell necrosis and apoptosis were determined using flow cytometer and MTT assay,the effects of MSNs@PDA,free ISO and MSNs-DOX@PDA on autophagy in CNE-2 cells were detected by Western blot,Real-time PCR and IF.2.3 Human nasopharyngeal carcinoma xenograft model was established bysubcutaneous inoculation of CNE-2 cell in nude mice.The xenografts were randomly divided into 4 groups:saline,MSNs@PDA,free ISO and MSNs-ISO@PDA.Theinhibition ability on the xenograf growth was evaluated.Furthermore,The systematic toxicity of MSNs-ISO@PDAin vivo was evaluated by body weight monitoring.Results:1.The sizes of MSNs,MSNs-ISO and MSNs-ISO@PDA were determined using dynamic light scattering(DLS).As shown in Table 1,the diameters of the MSNs,MSNs-ISO and MSNs-ISO@PDA were 146.54±4.55,152.45±3.74 and 168.53±4.66nm,respectively.The zeta potentials of MSNs,MSNs-ISO and MSNs-ISO@PDA were-20.11±4.25,-10.57±3.42 and-32.56±3.37 mV,respectively.The BET surface area was 246.46 m2/g,the pore volume was 0.57 cm3/g and,as evaluated by the BJH method,the most probable pore size was about 2.74 nm.Moreover,the pore size distribution of MSNs was rather narrow.With loading of ISO,the BET surface area and the most probable pore size decreased to 132.35 m2/g and 2.36 nm,respectively.The BET surface area of MSNs-ISO@PDA was 43.68 m2/g.However,the pore Schematic representation of MSNs-ISO@PDA synthesis.TEM image of MSNs and MSNs-ISO@PDA volumes were 0.15 and 0.11 cm3/g due to the coating of PDA onto the surface of ISO-loaded MSNs.The encapsulation efficiency of ISO in MSNs was about 96.63±3.42.2.The results suggested that cellular uptake of FITC-MSNs-ISO@PDA in CNE-2 cells was significantly higher than that of FITC-MSNs-ISO.Flow cytometry analysis also confirmed the enhanced cellular uptake of FITC-MSNs-ISO@PDA(Figure3B).This increase was probably due to PDA modified.Induction of pro-death autophagy through suppression of AKT-mTOR-p70S6K pathway by MSNs-ISO@PDA.3.MSNs-ISO@PDA demonstrated the best inhibition ability on the xenograf growth.tumor growth in the treatment groups was significantly slower than that in the saline-control group.Furthermore,The systematic toxicity of MSNs-ISO@PDA in vivo was evaluated by body weight monitoring.No statistically significant differences in body weight could be observed between the MSNs-ISO@PDA treated group and other groups(p>0.05,data not shown)Conclusion:In summary,a pH-sensitive drug delivery system involving mesoporous silica nanoparticles coated with PDA was successfully designed for the controlled release of ISO.Furthermore,MSNs-ISO@PDA exhibited a good anti-cancer efficacy through the induction of pro-death autophagy.The obtained results demonstrate that the ISO-loaded MSNs-ISO@PDA featured competitive advantages,including an excellent pH-sensitivity,suitable cellular uptake and therapeutic efficacy with low side effects.