| BackgroundLaryngeal cancer is a common cancer of head and neck,its incidence increased recently. Currrently, surgery resection is the first choice of treatment laryngealcancer, but this option is often limited due to the anatomic location of the disease and potential consequence of a devastatedstructure of the throat; chemotherapy isoftenencountered with insensitive or drugresistance; radiotherapy has great toxic side effect. It is essential to find out new ways to improve the outcome of treatment of laryngeal cancer. Recently, researches have reported that calastrol a triterpenoid compound isolated from Chinese herbal medicine of Celas-traceae, has anti-cancer effects in many tumor types. However, its severe side effects and poor water solubility hamper celastrol for clinical therapeutic applications. Nanopartical vehicle drug delivery system has the potential to solve those problems. MSNs exhibited with exceptional properties for drug delivery because of its favorable biocompatibility, excellent physiochemical stability and its ease of surface multi-functionalization. Herein, we studied the character of our synthesized MSNs and tested the therapeutic effect of MSNs-loaded-celastrol for laryngeal cancer treatment in vitro, and in vivo. Research methods:MSNs were synthesized by chemical technology. Morphology and structure of the MSNs were investigated withtransmission electromicroscope(TEM) and scanning electron microscopy(SEM). Thehydrodynamic size of the samples was measured using dynamic light-scattering(DLS) techniques. The celastrol loading capacibility of MSNs was determined using an UV–Vis spectroscopy and the releasing kinetics of celastrol from MSNs was measuredby UV-4802 spectrophotometer. MSNs-loaded-celastrol as a drug delivery system for laryngeal cancer therapywas tested in vitro and in vivo. Results1. Synthesis and characterization ofmesoporous silica nanocarriersCharacterization ofthe synthesizedmesoporous silica nanoparticles herein showed a uniform morphology and highly ordered mesostructure.The transmission electron microscopy(TEM) image and scanning electron microscopy(SEM) image revealed that MSNs were in spherical shape, hadwell monodispersion in deionized water. Dynamic light- scattering(DLS) result shown an average diameter of ca.30 nm of the hydrodynamic distribution of MSNs 2. MSNs loading and releasing celastrol assayThe loading capacity property of MSNs nanocages as drug delivery systems was measured by spectroscopy technology.The drug loading efficiency LE(%) could reach 50% and the drug loading content was 25μg/mg meaning that 1.0mg of the MSNs could store approximately 25 μg of celastrol.The cumulative releasing of celastrol from the MSNs-celastrol exhibited a low pace releasing(less than 20% of the loaded amount) within 4h, which was attributed to the drugs weakly interacting on the outside of the silica nanorattles, and further sustained releasing from 8h to 96 h was attributed to the drugs being electrostatically absorbed(electrostatic interaction between the amino group in the matrix of silica nanoparticles and the carboxyl group of celastrol) in the mesopores of the silica nanoparticles. In the last phase, drug released from MSNs was at a very slow rate between 120 h to 168 h until celastrol was almost completely released. 3. MSNs-loaded celastrol anticancer effect in vitro and in vivoLaryngeal cancer HEp-2 cells were used for the study. HEp-2 cells were challenged with the same amount of dosage of free celestrol or MSNs loaded celestrol at indicated time points. The result shown that MSNs-celastrol inhibited HEp-2 growth more efficiently than that of free celastrol; MSNs-celastrol caused more HEp-2 cell arrested cell cycle in G2/M phase, resulted in higher rate of cell autoghagy and apoptosis. Encouraged by our in vitro results, we further evaluated the in vivo antitumor activity of MSNs-loaded celastrol system. Compared with vehicle control, MSNs-celastrol 1.0mg/kg and 1.75mg/kg celastrol group sresulted in 82.6% or 61% tumor growth inhibition, respectively; while, free celastrol 1.0mg/kg and 1.75mg/kg Results: VI groups resulted in 71.3% and 78.5%, respectively. However, the mice in the both free celastrol groups suffered noticeable total weight loss compared with MSNs-celastrol groups but the tumor sizes were smaller in MSNs-celastrol groups compared with that of free celastrol groups. These results indicated that MSNs-loaded cellastrol inhibited HEp-2 tumor cells growth more effective and less toxic than that of free celastrol groups. Our immunohistochemistry(IHC) results shown that higher rate of TUNEL positive cells in mouse tumor samples in MSNs-loaded groups than that of control vehicle and the free celastrol groups. Interestingly, Ki-67 results were reversely correlated with TUNEL results among the diferent groups. IHC staining result of ER-stress marker, BIP showed much positive stain of tumor cells treated with the MSNs-loaded celastrol relative to that of the control vehicle and celastrol only-treated tumor cells. 4. Molecular mechanism studyIt has been reported that celastrol inhibited cell proliferation of head and neck cancer via induction of ER-stress in the cells resulted in cellular apoptosis. HEp-2 cells were treated with celastrol(1.25μM) and 1.25μM. MSNs-loaded celastrol for 24 h.Western blot results indicated that both free celastrol and MSNs loaded celastrol induced the expression of proteins related to cellular autophagy. Intriguingly, MSNs-celastrol group induced a higher the expression of ER stress related protein and apoptotic protein than that of the cells treated with free celastrol.In order to perceiveshrinking sizes of celastrol-MSNs-treated tumors were tracking the same ways,we analysis ER stress related proteins and apoptosis related protein expression in the different groups animal tumor samples by western blot. As expected, the pattern of animal tumor samples was similar to the result tested in vitro. Conclusion: 1. An average diameter of 30 nm MSNswas successful synthesized, which have gooddispersibility and high stability property. 2. MSNs with high loading capability, especially in a sustained releasing of celastrolfrom them.3. MSNs-load celastrol inhibited HEp-2 cells proliferation more efficiently,comparing to that of free celastrol in vitro. MSNs-celastrol effectively inhibitedthe growth HEp-2 tumor in nude mouse with less side effects. The inhibitioneffect was the outcome of that MSNs-calstrol induced a higher ER stress in thecells resulted in cell apoptosis via autophagy. 4. MSNs possessed a useful biocompatibility and biodegradability. |
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