Targeting CD133~+ Laryngeal Carcinoma Cells With Chemotherapeutic Drugs And SiRNA Against ABCG2 Mediated By Thermos/pH-sensitive Mesoporous Silica Nanoparticles

Laryngeal cancer is one of the most common malignancies in head and neck region,and laryngeal cancer patients account for 1/3 of all the patients with head and neck malignant tumors.The estimation of newly diagnosed laryngeal cancers was 156877 worldwide in 2012,which caused 83376 deaths.In clinic,the treatment for laryngeal carcinoma regime remains the same of surgical removal followed with chemotherapy,with severe problems in patients' social adaptability due to pathological and psychological disorders caused by postoperative malfunction or completely loss of phonation.Furthermore,local recurrence and metastasis are frequently observed among patients.Chemotherapy is a better choice in reserving laryngeal function and structure.However,the development of multidrug resistance(MDR)in tumor cells has significantly reduced the efficiency of the current chemotherapeutic regime.Scientists has already proved that CD133~+ Hep-2 laryngeal cancer cells are responsible for multidrug resistance due to elevated expression of ABCG2.By conjugating polymers with thermos/p H-coupling sensitivity onto mesoporous silica as “valves” and folic acid molecules as tumor targeting moiety,we have previously designed a tumor-targeted drug delivery system with dual-responsive property.Furthermore,positively charged MSN nanocarriers could electrostatically interact with negatively charged siRNA to form siRNA-nanoparticle complexes.This novel drug nanocarrier,MSNs with thermo/p H-coupling sensitivity and site-specificity,has great potential in anti-tumor treatment.Methods and results:Thermo/pH sensitive folic acid loaded MSNs was used to form drug-nanoparticle complexes and siRNA-nanoparticle complexes.Chemotherapeutic drugs include cisplatin,5-Fu and paclitaxel.The anti-tumor effect of drug-loaded MSNs and siRNA-loaded MSNs was tested under certain pHvalue and temperature by extracellular,intracellular and in vivo studies.1.Preparation of siRNA-MSNs and its nucleic acid uptake efficiency.1.1 Preparation of DNA-MSNs.Due to the low stability of RNA against enzymatic degradation,DNA oligo duplex was used to mimic siRNA duplex to determine the capacity and to establish the conditions for siRNA loading to MSNs.We confirmed that the condition for DNA oligo duplex to get loaded onto MSNs is to incubate them together for 6 hours under room temperature.1.2 The uptake and release of DNA from MSNs and the DNA safety test.To test the oligo loading capacity of MSN and whether MSN binding could protect oligo from nuclease degradation,oligo-loaded MSN and free oligo in the supernatant were both incubated with DNase overnight.To examine how much oligo was still bound to MSN after overnight DNase treatment,MSN was collected by centrifugation.And oligo was released from MSN by adding Na Cl and then shaking for 30 min.Samples were examined by agarose gel electrophoresis.1.3 Three candidate siRNA.Three candidate siRNA were synthesized by a company.Loading and releasing processes of siRNA were the same with methods above.And the siRNA segment with the highest uptake-releasing rate was selected to be used in further studies.2.In vitro studies 2.1 CD133~+ laryngeal cancer cells sorting.For flow cytometry sorting and analysis,cell suspensions were incubated with phycoerythrin(PE)-conjugated CD133/2 antibody,and CD133~+ and CD133-cells were sorted out on a BD FACSAria flow cytometer.2.2 Cellular uptake of siRNA-MSN-FA.CD133~+ cells were incubated with FA-loaded MSN-siRNA at 37 for 3 hours.Then cells were visualized using a confocal laser scanning microscope.Green fluorescence was presented in the cytoplasm,indicating successful intracellular uptake of the nanoparticles.2.3 Analysis of expression of ABCG2 protein by western blot.Western blot analysis was performed to detect the expression of ABCG2 in CD133~+ cancer cells treated by normal saline,MSN,or siRNA-loaded MSN.The results showed that the level of ABCG2 protein in siRNA-loaded MSN group was significantly reduced than that in MSN group.2.4 In vitro cytotoxicity of differently loaded MSNs against Hep-2 cells.A quantitative MTT assay was used for testing the in vitro cytotoxicity of MSN particles loaded with different combinations of chemotherapeutic drugs and siRNA again CD133~+ cancer cells.The cells were treated with 1)cisplatin-loaded MSN,2)combination of cisplatin–loaded MSN and siRNA-loaded MSN,3)5-Fu–loaded MSN,4)combination of 5-Fu–loaded MSN and siRNA-loaded MSN,5)paclitaxel–loaded MSN,6)combination of paclitaxel–loaded MSN and siRNA-loaded MSN.The mean concentration of nanoparticles that was cytotoxic to 50% of the cells(IC50)was calculated.Data showed that the viability of CD133~+ cells decreased more significantly in cells treated with the combination of drug-MSN and siRNA-MSN compared with those incubated with drug-MSN only.3.In vivo animal studies 3.1 In vivo studies were performed on 5-week-old adult female BALB/c-nu/nu mice.Each mouse was injected subcutaneously with CD133~+ Hep-2 cells.When tumors reached an average volume of 200 mm3,the mice were randomly divided into eight groups and received 1)normal saline,2)pure MSN,3)cisplatin-loaded MSN,4)5-Fu–loaded MSN,5)paclitaxel–loaded MSN,6)combination of cisplatin–loaded MSN and siRNA-loaded MSN,7)combination of 5-Fu–loaded MSN and siRNA-loaded MSN,8)combination of paclitaxel–loaded MSN and siRNA-loaded MSN,respectively.Two weeks after the treatment,shortest and longest of tumors were attained to draw a tumor growth inhibition graph,from which we could conclude that the mice treated only with normal saline and pure MSN exhibited a rapid increase in tumor volume.In contrast,mice treated with drug-loaded MSN combined with siRNA-MSN showed significant reductions in tumor sizes,although the anti-cancer drugs-loaded MSNs group and in combination with siRNA against ABCG2 group had similar tumor growth curves.3.2 In situ TUNEL assay.Apoptotic cells in tumor sections were detected by terminal deoxynucleotidyl transferase d UTP nick end labeling(TUNEL).The numbers of total cancer cells and TUNEL-positive cells in specimens were manually counted in five randomly selected fields under light microscopic analysis.The apoptosis index(AI)was expressed as the ratio of TUNEL-positive cells to total cells.TUNEL staining showed that the AI was significantly higher in the combination groups compared with the drug-MSN only groups.Discussion:This study use the p H/thermo-coupling sensitive mesoporous silica nanoparticles loaded with folic acid as a gene and drug carrier.The studies above manifested the possibility for MSNs to be used as a gene and chemotherapeutic drug carrier,and the combination treatment of drugs-MSN and siRNA-MSN knocked down ABCG2,enhanced the efficacy of chemotherapeutics,and reduced MDR.