Olaparib Nanoparticles Potentiated Radio-sensitization Effects On Lung Cancer

objective:To investigate the in vivo radio-sensitization effects and its possible mechanism of Ola-NPs?MPEG-PCL-Olaparib?combined with radiotherapy on lung cancer.Methods:Ola-NPs was prepared by rotary evaporation method.The particle size and Zeta potential of Ola-NPs were measured using dynamic light scattering?NanoBrook90 Plus Zeta,Brookhaven,NY,USA?at 25°C.Morphologic characteristics of Ola-NPs were determined by transmission electron microscopy?TEM,Tecnai G2 F20,USA?.The filtered Ola-NPs solution was lyophilized and re-dissolved in deionized water.High performance liquid chromatography?HPLC?was used to detect the drug loading rate?DL?,entrapment efficiency?EE?and in vitro release of Ola-NPs.Then,the lung cancer xenograft model of nude mice was established in vivo.108 BALB/c nude mice?female,3 to 4 weeks old?were randomly divided into three groups?36 in each group?:RT group,Ola+RT group and Ola-NPs+RT group.Each group was divided into six subgroups?6 in each subgroup??0 Gy,2 Gy,6 Gy,10 Gy,14 Gy,18 Gy?according to the different radiation doses.When tumor measured 100–150 mm³,subgroups were made as follows:1)RT?a single dose ranges from 0 to 18 Gy?;2)Ola+RT?50 mg/kg/d,3 days,intravenous injection[IT],combined with RT?;3)Ola-NPs+RT?equivalent to 50 mg/kg/d Ola?.Each subgroup included six mice?n=6/subgroup?that received irradiation of 0,2,6,10,14,and 18 Gy,respectively.RT was given 30 minutes after the first time of using drug.The optimal dose of RT was calculated by monitoring tumor growth delay?TGD?.Tumor growth curves were prepared in accordance with the Gompertz model by using the following equation:y=V₀*exp?k*?1-exp?-a*X???,where y represented the TGD;V₀ represented the original tumor volume?100–150 mm³?;a and k are coefficients,and X indicated the RT dose.The sensitizing enhancement ratio?SER?of Ola and Ola-NPs were calculated by dose-response curve.The optimal RT dose was obtained from the SER.After calculating the optimal RT dose,the radio-sensitization effect and toxicity of Ola-NPs was further studied.In this study,an xenograft model of A549 lung cancer in nude mice was established.Tumor-bearing nude mice were randomly divided into the following six groups?n=12/group?:1)control?0.9%normal saline[NS]?;2)Ola;3)Ola-NPs;4)RT?a single dose of 10 Gy?;5)Ola+RT;6)Ola-NPs+RT.The volume change of tumors in each group was recorded,and the growth curve of tumors and the survival curve of nude mice in each group were drawn.Within 24-48 hours after the first administration,three nude mice in each group were randomly selected and killed.Tumor tissues were stripped to explore the expression of?-H2AX by immunohistochemical?IHC?method.On the 10th day after the first administration,three nude mice in each group were randomly selected and killed.Tumor tissues were stripped and divided into two parts.One part was used to detect cell cycle and apoptosis by flow cytometry,the other part was used to detect the expression of CC3,Ki-67 and CD-31 by IHC.The main organs such as heart,liver,spleen,lung and kidney of nude mice were dissected.The histological changes of tissue structure were observed by H&E staining to further understand the toxicity of drugs in vivo.Results:The prepared Ola-NPs showed a mean size of 31.96±1.54 nm and a higher entrapment efficiency?99.17±0.01%?.In vivo human non-small-cell lung cancer xenograft models,Ola-NPs group presented more obvious anti-tumor effect than control group,RT group and Ola group.When combined with radiotherapy,both Ola?1.66?and Ola-NPs?3.81?had radio-sensitization effects,but Ola-NPs shown significantly higher radio-sensitization effect than Ola?3.81vs 1.66?.The dose-response curve was calculated by by Gompertz tumor growth model,the optimal RT dose was calculated to be 10 Gy,which could be used for further research.Ola-NPs significantly inhibited the growth of tumors and prolonged the survival cycle of nude mice when compared with Ola and Control groups.When Ola-NPs was combined with radiotherapy?Ola-NPs+RT group?,it presented the best inhibition of the tumor growth and obtain the longest survival time of nude mice bearing tumors.At the same time,the toxicity of Ola-NPs in vivo was lower than that of other treatment groups,and the nude mice bearing tumors were better tolerance.Immunohistochemical?IHC?data shown that the positive rate of?-H2AX in Ola-NPs group was significantly higher than that in Ola group and Control group.When combined with radiotherapy?Ola-NPs+RT?,the highest positive rate of?-H2AX was found.CC3 reflects the apoptotic rate of cancer cells.The apoptotic rate of Ola-NPs+RT group was significantly higher than that of other treatment groups.The positive rate of Ki-67 in Ola-NPs group was significantly higher when compared with Ola group and Control group.The highest positive rate of Ki-67was possessed When combined with radiotherapy?Ola-NPs+RT?.CD-31positive cell in tumor tissue represents the number of microvessel density?MVD?.Ola-NPs group presented a lower number of MVD than Ola group and Control group.Ola-NPs+RT group had the lowest number of MVD when compared with other treatment groups.Flow cytometry was used to detect the apoptosis and cell cycle distribution of tumor cells.The results of apoptosis showed that Ola-NPs could increase the apoptotic rate of cancer cells compared with control group,Ola group and RT group.When combined with radiotherapy?Ola-NPs+RT?,Ola-NPs showed the strongest effect on promoting apoptosis of cancer cells.In cell cycle analysis,when combined chemotherapy with radiotherapy,cell cycle arrest was mainly reflected in G2/M phase.Compared with other treatment groups,the Ola-NPs+RT group possessed the significantly increased ratio of G2/M phase cells.Conclusion:Ola-NPs presented outstanding anti-tumor effect on human non-small-cell lung xenograft models.When combined with radiotherapy,it shown remarkable radio-sensitization effect and is expected to become an important drug system for effective in situ cancer therapy.