Thermosensitive Hydrogel In A Dual Drug Delivery System As A Novel Radio Sensitizer For The Treatment Of Cervical Cancer

objective: To investigate in vivo anti-tumor and radio sensitizing effects of a gel-based dual drug delivery system(PECE/DDP+MPEG-PCL/PTX,or PDMP)in a cervical cancer model and its possible mechanisms of action.Methods: MPEG-PCL/PTX micelles were successfully synthesized by a simple one-step solid dispersion method.Then a biodegradable and thermosensitive hydrogel PDMP was successfully developed to load MPEG-PCL/PTX and PECE/DDP and was characterized by HPLC,rheometer and laser particle size analysis.A xenograft cervical cancer model was used to investigate the radio sensitization effect of PDMP.Mice underwent PTX+DDP,PECE and PDMP treatment followed by single radiation doses ranging from 0Gy-20 Gy.Radio sensitization was analyzed by tumor regrowth delay(TGD).Sensitization enhancement ratio(SER)was calculated by doses needed to yield TGD in radiation alone and radiation plus drug treatment groups.The impact of irradiation and drugs on TGD was analyzed by Gompertz model,and the optimum radiation dose was used for further evaluation of anti-tumor and radio sensitizing effects.Mice underwent PTX+DDP,PECE,PDMP,RT,PTX+DDP+RT,PECE+RT and PDMP+RT.The growth of xenografted tumors was observed and the TGD of each group was calculated.3of the animals each group weresacrificed after 2 days of treatment,and tumor tissue was harvested for immunohistochemistry and western blot in order to evaluate ?-H2 AX and ATM.Then,3 of the animals each group were sacrificed after 10 days of treatment,and tumor tissue was harvested for immunohistochemistry and flow cytometry in order to evaluate tumor proliferation,tumor cell cycle redistribution and apoptosis.Their liver,lung,kidney,spleen and heart were harvested and H&E staining was performed in order to investigate in vivo toxicity of PDMP.The remaining animals in each group were used for further observation.Results: MPEG-PCL/PTX micelles have a good drug-loading(DL%,3.96±0.03%)and entrapment efficiency(EE%,99.18±0.65%).The PDMP hydrogel composite existed obvious thermal sensitivity which was in good agreement with the results of the blank PECE hydrogel.PDMP was effective at inhibiting tumor growth and prolonging the survival time of tumor-bearing nude mice compared with PTX+DDP<IT>,PTX+DD<IP>,PECE and control group.When those groups combined with radiation,PTX+DDP(IT)and PDMP can enhance radio sensitization compared as compared to irradiation alone.But,PDMP has a stronger radio sensitization effect than PTX+DDP(IT)(SER of 1.30 vs1.04).The in vivo study showed that the strongest radio sensitization was found in PDMP+RT group at 12 Gy.Therefore,12 Gy was chosen for further investigation.The PDMP+RT group showed the greatest efficacy in TGD and resulted in a higher survival rate than other RT groups andchemotherapeutic groups.The PDMP group exhibited a higher efficacy in the inhibition of tumor growth and the survival rate,when compared to the other chemotherapeutic groups.PDMP showed the highest expression of ?-H2 AX as compared to other chemotherapeutic groups.When chemotherapeutic groups were used in combination with irradiation,the expression of ?-H2 AX was increased in every group.The PDMP+RT group,however,exhibited significantly higher expression of ?-H2 AX than other irradiation groups.The quantity of MVD in the PDMP group was significantly lower than in other chemotherapeutic groups,and when combined with RT,PDMP+RT had the fewest number of MVD compared to other irradiation groups and chemotherapeutic groups.PDMP decreased the number of CD133 positive cells as compared to other chemotherapeutic groups and PDMP+RT had the fewest CD133 positive cells as compared to all groups.The higher ATM expression was detected in PDMP group compared with other chemotherapeutic groups,when chemotherapeutic groups were in combination with irradiation,the highest expression of ATM was detected in PDMP+RT group compared with all groups.Flow cytometry analysis revealed that PDMP increased the number of cells in G1 phase and led to higher apoptosis rate compared with other chemotherapeutic groups.PDMP+RT group demonstrated the highest proportion of cells in the G2 phase and led to the highest apoptosis rate compared with other groups.In addition,PDMP+RT group had the lowest ALDH1 expression.Conclusion:PDMP is a promising anti-tumor and radio sensitization reagent for the treatment of cervical carcinoma.