| Laryngeal carcinoma is one of the most common head and neckmalignant tumors, accounting for35.4%of the head and neck malignanttumors, whose incidence is at the third place among the head and necktumors. The laryngeal carcinoma is about1%in all the diagnosedcancers cancer diagnoses. Morever, the incidence of laryngeal carcinomahas been risingin recent years. At present, in case of combinationtreatment with surgery, radiation and chemotherapy, the five-yearsurvival rate is still unimproved, with treatment efficiency at80-90%inTis, T1and T2phase, whereas only40-50%in T3and T4phase oflaryngeal carcinoma. Although chemotherapy is increasingly acceptedby people for the preservation of laryngeal function, assurance of lifequality, traditional chemotherapy lacks of specificity and could lead toadverse side effects to normal tissues. In addition, treatment efficiency ispoorwhen dosages are insufficient in the targeted areas. Nowadays, thedevelopment of functional nanomaterials and its usage in drug deliverysystem are becoming a very important research field in cancer treatment. A variety of nano-drug delivery systems have been developed in targeteddrug delivery and controlled drug release, and have played an importantrole in improving the tumor lesion accumulation of antineoplastic drugsand reducing side effects.Among all the drug carrier materials, Mesoporous SilicaNanoparticles (MSN) have prominent high surface area and largepore volume, which endow them with exceptional capacity in drugstorage, and the massive silanolcontaining in the surface makes it easilyto be functionalized to allow better control over drug loading and release.The MSN based on mesoporous silica are equipped with “goalkeeper”,which are enormously appealing. It can regularly release drugs at aspecific time interval and required site by taking advantage oftransforming "goalkeeper" and observing stimuli response strategies. Toachieve effective and controlled release, choosing tumor-associatedbiological stimuli is a prerequisite. Most solid tumors have a weaklyacidic extracellular environment (pH=6.0to7.0) than normal tissuesand bloodstream (pH=7.2to7.4). The intracellular pH would be furtherreduced for the synergy caused by glycolysis induced hypoxia,especially in the endosome (pH=5.0to6.0) and lysosome (pH=4.0to5.0). Additionally, certain malignancies show a local characteristic ofdistinct hyperthermia. Based on above discussions, the pH value and thetemperature can be assumed to be the typical biological stimuli, applied in field of drug release. Methods and results: In our study, we preparedtargeted folic acid and pH/Thermo double response controlled drugcarrier of silica nanoparticles, loaded cisplatin, to testify that this kind ofdrug carrier could enhance apoptosis induced by cisplatin in laryngealcarcinoma Hep-2cells, in vivo and in vitro.Materials and Methods1. Preparation of targeted folic acid and pH/Thermo doubleresponse controlled drug carrier of silica nanoparticles and itsperformance testing.1.1Monodispersed MSNs were prepared by using a base-catalyzedsol–gel method, which was reported previously. The molar ratioofCTAB/TEOS/NaOH/H2O in the reaction mixture was1.0:8.16:2.55:4857.MSNs were prepared with the mean diameter of100nm, averagepore diameter of2.5nmand a specific surface area of786m2g-1.PowderX-ray diffraction (XRD) analyses in0°＜2θ＜10°showed that theas-synthesized MSNs exhibited a well-ordered porous structurewith ahexagonal arrangement.1.2The surface ofMSNs was functionalised by the reactionofmethacryloxypropyltrimethoxysilane (MPS) through carbon–carbon doublebonds, theresulting MSN-MPS was further grafted onto a polymer chain intheprocess of polymerization. Finally, N-isopropylacrylamide (NIPAM)andmethacrylic acid (MA) monomers were copolymerized by seeded precipitation polymerization using MSN-MPS as seeds,potassiumpersulfate (KPS) as the initiator, and N,N-methylenebisacrylamide(MBA) as the cross-linker, forming MSN@p(NIPAMco-MA) compositenanoparticles. The low-angle XRD patternsshowed that MSN@p(NIPAM-co-MA) still remained a mesoporous structure despite thelower intensity of XRD peaks, which may be ascribed to the cappingeffect. Fourier transform infrared (FTIR) spectra further provided clearevidence for the preparation procedure of the composite nanoparticles.Dynamic light scattering (DLS) indicated that MSN and MSN@p(NIPAM-co-MA) were both well distributed.1.3Conjugation of FA toMSN@p(NIPAM-co-MA) was completed bythe reaction betweenthe NH2groups of FA and COOH groups ofMSN@p(NIPAM-coMA). As shown in the UV/Vis spectra, indicatingthe successful conjugation of FA.The nanocarriers (MSN@p(NIPAM-co-MA)–FA) werestable and well distributed in10mM PBSfor one month withoutsignificant changes in their size and morphology.1.4We next investigated temperature/pH-responsive drug releasefromthe MSN@p(NIPAM-co-MA)–FA hybrid nanoparticles.Fluoresceinwasselected as a model drug to evaluate the loadingand controlled releasebehaviors, and the following cellularuptake due to its distinct fluorescenceemission. The results show that the prepared composite material show good dual response in both PH value and temperature sensitivity: when thebuffer solution is at high temperature and low pH value, it showed arapid, effective release responsewhile the buffer solution is at lowtemperature and high pH value, it showed slow release behavior atlowspeed.2. In vitro study2.1MTT Assay①T argeted folic acid andpH/Thermo double response controlleddrug carrier of silica nanoparticles (MSN@p(NIPAM-co-MA)-FA)would not inhibited the growth of laryngeal carcinoma Hep-2cells at theconcentration under100μg/ml.②T he group of cisplatin-loaded MSN@p(NIPAM-co-MA)-FAshowed a higher cytotoxicity compared with group of cisplatin alone andcisplatin-loaded MSN@p(NIPAM-co-MA), both with the sameconcentration of cisplatin.2.2Laser scanning confocal microscopy (LSCM)LSCM recorded that fluorescent agent loaded by MSN@p(NIPAM-co-MA)-FA can be absorbed by laryngeal carcinoma Hep-2cells, and the drugs can be successfully released in the cells.2.3Results by Flow Cytometry①U nder the same condition, FA-positive Hep-2cells have stronger ability to absorb cisplatin-loaded MSN@p(NIPAM-co-MA)-FA thanFA-negative A549cells.②Under the same concentration of loaded cisplatin, apoptosisinducing effect of cisplatin-loaded MSN@p(NIPAM-co-MA)-FA inHep-2cells is more obvious than cisplatin alone.3. In vivo studyThe inhibitory effect of cisplatin-loaded MSN@p(NIPAM-co-MA)-FAon nude mice inoculated of laryngeal carcinoma Hep-2cells is strongerthan the control group. Tunel assay demonstrated that cisplatin-loadedMSN@p(NIPAM-co-MA)-FA have stronger apoptosis inducing effect thancisplatin alone in tumor tissues. We used Western blot to detect theapoptosis factors, the results showed that, in the group of cisplatin-loadedMSN@p(NIPAM-co-MA)-A, the expression of p53and Bax is increasedthan that in the group of cisplatin alone and cisplatin-loaded MSN@p(NIPAM-co-MA), which proved an effective apoptosis inducing effectin Hep-2cells.Conclusion:In this study, we constructed functionalized silica nanoparticlescarried chemotherapeutics for the study of apoptosis inducing effect inlaryngeal carcinoma Hep-2cells for the first time, and we found thatMSN@p(NIPAM-co-MA)-FA as a carrier of chemotherapeutics cisplatin,could enhance the apoptosis inducing effect of chemotherapeutics and inhibitory effect on laryngeal carcinoma. |
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