Radiosensitization Of Drug-loaded Gelatinase-stimuli PEG-PEP-PCL Nanoparticles In Gastric Cancer

PurposeRadiotherapy is the main locoregional control modality for many types of unresectable tumors,including gastric cancer.Radiosensitizer will enhance the radiotherapy effect,and delivery of radiosensitizer by tumor-targeted nanoparticles will further improve the radiotherapy benefit.This dissertation focuses on two most prominent aspects of this area.Using docetaxel-loaded nanoparticles(DOC-NPs)based on gelatinase-stimuli strategy,we compared their radioenhancement efficacy with docetaxel(DOC)on GC in vitro and in vivo,and then explored the mechanism of the radiosensitization difference in vitro.We used gelatinase-stimuli nanoparticles to deliver miR-200c,which was reported as an inhibitor of cancer stem cells(CSCs)-like properties,evaluated its property as a potential radiosensitizer in GC cells,and then explore the relevance of the radiosensitization with inhibition of CSCs-like properties.MethodDocetaxel loading gelatinase-stimuli NPs were engineered.The gelatinase expression of GC cells and gastric epithelial cells were detected by Western blot.The cellular uptake difference of DOC-NPs between BGC823 cells and GES-1 cells were measured in cellular uptake assay.And then we compared the radiosensitization effect and cytotoxicity of both DOC-NPs and DOC between three GC cell lines and gastric epithelial cells GES-1 by the MTT assay and clonogenic assay.Subsequently,we investigated the mechanism of the radiosensitization difference between DOC-NPs and DOC from several radiosensitization associated factors,including G2/M arrest,reactive oxygen species,DNA double strand breaks and apoptosis,by flow cytometry and Western blot.When combined with radiotherapy,the tumor growth delay effect and body weight changes of DOC-NPs and DOC were compared in BGC823 xenograft.miR-200c loading gelatinase-stimuli NPs were engineered.We compared the cellular uptake difference of miR-200c NPs between gelatinases over-expressing BGC823 cells and gelatinases deficient GES-1 cells through cellular uptake assay,and then evaluated intracellular miR-200c expression after treated with miR-200c NPs in the two cell lines by real-time PCR assay.Subsequently,we measured the radioenhancement efficacy of miR-200c NPs in gelatinases over-expressing GC cells and gastric epithelial cells by the clonogenic assay and MTT assay.After the treatment of miR-200c NPs,the expressions of CSCs marker CD44 and epithelial marker E-cadherin were detected by real-time PCR and western blot in BGC823 cells.CD44 expressing BGC823 cells percentage is detected by flow cytometry.CSCs-like properties and CSCs associated radioresistance mechanisms were also investigated.ResultsCompared with three GC cell lines,both intracellular and excretory gelatinases(MMP-2/9)levels are much lower in GES-1 cells.In the cellular uptake assay,the cellular uptake amount of gelatinase-stimuli nanoparticles in BGC823 cells was significantly higher than that in GES-1 cells.In three gelatinases overexpressing GC cell lines,DOC-NPs exhibited significantly increased cytotoxicity compared with DOC(P<0.05).Meanwhile,in the normal cell line GES-1,both drugs showed similar inhibitory effect(P>0.05).Similarly,compared with DOC,radiosensitization of DOC-NPs was improved significantly(Sensitization enhancement ratio increased 1.09-fold to 1.24-fold,P<0.01)in all three gelatinase overexpressing GC cells,while increased slightly(1.02-fold,P=0.38)in gelatinase deficient normal gastric mucosa cells.The enhanced G2/M arrest,increased ROS,more effective DSBs and promoted apoptosis were observed when used DOC-NPs as a radiosensitizer.More importantly,under the same non-inhibitory concentration,the radiosensitization efficacy of DOC-NPs was more prominent than DOC in BGC823 xenograft;the results of body weight changes may reveal milder systemic toxicity of DOC-NPs.Through cellular uptake assay,we found that the fluorescence of Rhodamine-B loading gelatinase-stimuli NPs in gelatinases over-expressing BGC823 cells was significantly stronger than that in gelatinase deficient GES-1 cells.In BGC823 cells,significant miR-200c overexpression(40.3-fold over control)was observed in miR-200c NPs treatment.Meanwhile,only 3.7-fold increase of miR-200c expression was observed in miR-200c NPs treatment in GES-1 cells.Together,these results showed that gelatinase-stimuli NPs delivered the miR-200c into gelatinases over-expressing GC cells efficiently and selectively.Free miR-200c almost did not affect intracellular miR-200c expression in both BGC823 cells and GES-1 cells.Using gelatinase over-expressing BGC823 cells and gelatinase deficient GES-1 cells,it was demonstrated that the cellular uptake of NPs was dependent on expression level of gelatinases.miR-200c NPs augmented radiosensitivity significantly in three gastric cancer(GC)cells(Sensitization enhancement ratio ranging from 1.11 to 1.25),but slightly in GES-1(1.06).However,free miR-200c have no radiosensitization in all four cell lines.Accompany with the radioenhancement,miR-200c NPs reduced the expression of CSCs marker CD44 and the percentage of CD44+BGC823 cells,and upregulated E-cadherin mRNA and protein expression levels.Meanwhile,CSCs-like properties in BGC823 cells,including invasiveness and resistance to apoptosis,were suppressed when miR-200c NPs combined with radiotherapy.In addition,CSCs associated radioresistance mechanisms in BGC823 cells,involving reactive oxygen species level and DNA repair capacity,were attenuated by miR-200c NPs.ConclusionsIn gastric cancer,compared with DOC,DOC-NPs exhibited more efficient and more selective radiosensitization in vitro and in vivo,and the improved radiosensitization was associated with series of radiotherapy injury factors;nanoparticles formulation make inefficient free miR-200c become an efficient and selective radiosensitizer,which was associated with its CSCs-like properity inhibitory function.By understanding and manipulating the microenvironment difference between tumor and normal tissue,gelatinase-stimuli NPs could provide the tumor-targeted delivery for radiosensitizers.Radiosensitizers delivered by gelatinase-stimuli NPs will have improved radiosensitization on tumor,while have little effect on normal cells,having promise to further improve the radiotherapy benefit.Our study serves potentially superior platform for further study on gelatinase-mediated nanoscale delivery of other agents for radiosensitization;and makes some rational for its further clinical translational evaluation.