Nanoparticles Encapsulated ATM Antisense Oligonucleotides Enhace The Radiosensitivity Of Head And Neck Squamouse Carcinoma Of Mice In Vitro And In Vivo

Objective: (1) To explore the probability of PLGA nanoparticles as vectors to transfect antisense oligonucleotides in gene therapy for head and neck squamous carcinoma of mice.(2) To explore the mechanism of the radiosensitivity of head and neck squamouse carcinoma of mice by inhibiting the expression of ATM gene associated with radiation therapy in vitro and in vivo.Methods: (1) Biodegradable and biocompatible PLGA was used to prepare the nanoparticles encapsulated ATM oligonucleotides. Through identificating their physical and chemical characters, the stable technique was definited. (2) After nanoparticles encapsulated ATM oligonucleotides were transfected into SCCVII cells, the results of transfection were analysed with fluorescence microscope and flow cytometry. The expression of ATM mRNA and ATM protein were detected with real-time fluorescent quantu PCR and western blot respectively. (3) Associated with radiation therapy, the radiosensetivity of SCCVII cells was studied by clonogenic assay and MTT trials. The distribution of cell cycle and the ratio of cell apoptosis were tested by flow cytometry. (4) The model of head and neck squamouse carcinoma of C3H/He mice were developed through inoculating SCCVII cells into the floor of the mouth of the mice. (5) The grafts were treated with nanoparticles encapsulated ATM antisense or sense oligonucleotides by intratumor injection. Hematoxylin and eosin stain was carried to detect the character of histopathology , and immunohistochemical method was carried to detect the expression of ATM protein. (6) Associated with radiation therapy, the radiosensitivity was studied by observing the volume change of grafts and apoptosis of tumor cells by tunel trial.Results: (1) Nanoparticles encapsulated oligonucleotides were prepared resistantly. The mean diameter was 116±25.2nm with polydispersity less than 0.15. The encapsulating rate was 47.45% with capability to resist DNase I .(2) Compared with control group, the expression of ATM mRNA and ATM protein in SCCW cells were reduced statistically(p<0.05) after transfecting with ATM-ASODN-NP. (3)The results of clonogenic assay and MTT trials showed that the capability of SCCVII cells to clone and reproduce was inhibitated, while the treatment of ATM antisense oligonucleotides associated with radiation therapy. (4) In the treatment group, the results of flow cytometry showed that G2/M arrest was inhibitated and the apoptosis rate was increased. (5) Biological characteristics of head and neck squamous carcinoma were observed in SCCVII cells grafts of C3H/He mice. The results of immunohistochemical analysis showed that the expression of ATM protein in grafts was decreased statistically(p<0.05) after transfecting with ATM-ASODN-NP. (6) In the group of ATM antisense oligonucleotides associated with radiation therapy, the rate of apoptosis increased and the tumor growth rate decreased.Conclutions: (1) The technique to prepare the nanoparticles encapsulated oligonucleotides was stable. Nanoparticles used to gene therapy as vectors were feasible. (2) Through transfection of ATM-ASODN-NP, the expression of ATM mRNA and ATM protein were reduced and the radiosensitivity of SCCVII cells was increased associated with radiation therapy in vitro. (3) The SCCVII cells grafts of C3H/He mice had the biological characteristics of head and neck squamous carcinoma. The radiosensitivity of grafts was increased in vivo through the treatment of intratumor injection of ATM antisense oligonucleotides associated with radiation therapy.