| Objective:To explore a chitosan hydrogel?CS/DDP?-based drug delivery system for the in situ treatment of nasopharyngeal carcinoma in combination with RT,and investigated their synergistic antitumor efficacy and underlying mechanism of action.Methods:Firstly,an antitumor drug?the CS/DDP hydrogel?with sustained release properties was prepared with chitosan,?-glycerophosphate,and cisplatin in different ratios.Next,the temperature dependent sol-gel transition of CS/DDP hydrogel was characterized by the inverted tube test.The rheological behavior of the CS/DDP hydrogel was assessed at different temperatures using a rheometer.In vitro release studies,DDP concentration from the CS/DDP hydrogel and free DDP solution was determined by HPLC instrument with a reverse phase C18 column.Standard MTT assay was utilized to evaluate the in vitro cytotoxicity of CS hydrogel,DDP injections,and CS/DDP hydrogel on 5-8F cells with different concentration.The degradability and biocompatibility of CS hydrogel was studied by hematoxylin-eosin?H&E?.BALB/c mice were utilized to establish the murine NPC model,the 70 mice whose tumor volume were allotted into the seven treatment groups below?n=10 in per group?:?1?0.9%NaCl injection group?0.9%NaCl injection 120ml ip d1??2?DDP group?DDP 5mg/kg iv d1?,?3?CS/DDP group?CS/DDP 5mg/kg it d1?,?4?RT group?RT 3Gy/3F d2-4?,?5?CS+RT group?CS it 120ml d1+RT 3Gy/3F d2-4?,?6?DDP+RT group?DDP5mg/kg iv d1+RT 3Gy/3F d2-4?,and?7?CS/DDP+RT group?CS/DDP5mg/kg it d1+RT 3Gy/3F d2-4?.Then,the mice were injected intra-tumorally or intravenously with 120?l of the suitable preparation,and irradiated 16 hours later?i.e.the next day?as needed.On the first day after the end of treatment,three mice in each group were randomly selected to perform small animal PET/CT scan to assess the post-treatment tumor metabolism.Two animals of per group were randomly euthanized two days after irradiation,and the tumor tissues were processed for?-H2AX staining.After the mice were euthanized,their livers,lungs,kidneys,spleens,and hearts were isolated for H&E staining to evaluate the side effects.On day 5 after the start of treatment,three mice of each group were sacrificed,and their tissues were processed for cell cycle distribution,apoptosis,and other immuno-histochemical analyses to investigate the underlying mechanism of action of the synergistic antitumor efficacy.We measured tumor's length and width every other day from the day at which we started to treat to calculate tumor volume and plotted tumor growth curve for 5mice per group.And survival time was observed in each group for 5 mice after the end of treatment.Results:1.The CS/DDP hydrogel showed obvious thermo-sensitive property which was consistent with the results of the blank CS hydrogel.2.During the initial week post-injection,the CS hydrogel was largely intact and surrounded by excessive numbers inflammatory cells.But by 6weeks the hydrogel had almost completely degraded and was no longer visible.3.In vitro drug release experiment,the DDP release from CS/DDP hydrogel could be prolonged up to 6 days even longer,and no initial burst release was found at the beginning.The result indicated that CS/DDP hydrogel had an extented released property and thus provided a potential for sustained delivery drug.4.In cytotoxicity assay,when the concentration wasn't more than 0.5ng/ml,no significant difference was discovered between CS/DDP hydrogel and DDP injections.However,CS/DDP hydrogel displayed a stronger inhibitory effect than DDP injections at higher concentrations?P<0.05?.5.The time taken for tumor to reach four times its original volume was significantly longer in the CS/DDP+RT treated mice?31.4 days,p<0.01?compared to the mice receiving DDP+RT?23 days?,RT?18.4 days?,CS+RT?17.2days?,CS/DDP?13.4days?,DDP?12.6 days?and NS?10.8 days?.In addition,the median survival time of the mice showed similar trends.The median survival of the CS/DDP+RT group was 81 days,which was significantly longer?p<0.01?than the DDP+RT?72 days?,RT?51 days?,CS+RT?50 days?,CS/DDP?44 days?,DDP?43days?and NS groups?42 days?.There were no obvious differences in tumor growth and median survival time between the RT group and CS+RT group?51days vs 50 days,p>0.05?.6.When performing small animal PET/CT scan to assess the post-treatment tumor metabolism,the results displayed that the SUVmax values of the CS/DDP+RT,DDP+RT,RT,CS+RT,CS/DDP,DDP and NS groups were 1.23±0.09,1.47±0.05,1.57±0.09,1.6±0.16,1.7±0.08,1.83±0.18 and 1.9±0.22 respectively.The SUVmax value of the CS/DDP+RT group was lowest?n=3,p<0.05?,indicating that the NPC xenografts responded most favorably to the CS/DDP+RT regimen.7.In cell cycle and apoptosis analysis,The percentage of G2/M-phase cells in the CS/DDP+RT group was 72.61±1.17%,that was significantly higher?p<0.01?than that seen in the DDP+RT?65.13±1.47%?,RT?43.48±2.16%?,CS+RT?43.67±2.53%?,CS/DDP?35.51±1.77%?,DDP?33.94±1.71%?or NS groups?29.03±1.82%?.In addition,a significant increase in the percentage of apoptotic cells was seen in the CS/DDP+RT group?76.23±1.13%?,when compared to DDP+RT?71.48±1.73%,p<0.01?,RT?38.70±1.51%,p<0.01?,CS+RT?37.72±1.34%,p<0.01?,CS/DDP?33.25±1.39%,p<0.01?,DDP?31.56±1.31%,p<0.01?or NS groups?14.23±1.42%,p<0.01?.8.The tumor tissues in the CS/DDP+RT group showed significantly lower number of Ki-67 positive cells?22.4±4.77%,p<0.01?compared to the DDP+RT?39.8±3.42%?,RT?58.8±8.14%?,CS+RT?57.2±6.62%?,CS/DDP?75.3±4.12%?,DDP?78.16±6.25%?and NS groups?88.6±4.67%?.The MVD-CD31 value of the tumor tissues in CS/DDP+RT group?2.5±0.36%?was significantly lower?p<0.01?than that of DDP+RT?4.2±0.29%?,RT?4.9±0.25%?,CS+RT?4.8±0.80%?,CS/DDP?9.2±0.88%?,DDP?9.3±0.54%?and NS groups?10.4±0.36%?.Moreover,a significantly higher number of?-H2AX positive cells was observed in the CS/DDP+RT group?88.8±3.90%,P<0.01?compared to DDP+RT?69.1±4.26%?,RT?58.6±2.82%?,CS+RT?60.2±6.66%?,CS/DDP?43.4±5.03%?,DDP?45.6±3.79%?or NS groups?4.5±0.51%?.9.No severe side effects?appetite,behavioral change or body weight?were observed in any of the treatment groups.Conclusion:1.The CS/DDP hydrogel showed obvious thermo-sensitive property which was consistent with the results of the blank CS hydrogel.2.The CS hydrogel possessed preferable biocompatibility and biodegradability.The CS/DDP hydrogel had an extented released property,and thus provided a potential for sustained delivery drug.Furthermore,the treatment effect of DDP was strengthened when DDP was combined with CS hydrogel.All results above indicated that CS/DDP hydrogel provided a potential for sustained delivery drug.3.Compared to other groups,CS/DDP+RT obviously decreased the metabolism of tumor tissues,thus suppressing tumor growth and as well as prolonging the survival of tumor-bearing mice.The main mechanism was likely the increase in cancer cell apoptosis.Furthermore,the CS/DDP hydrogel in combination with RT also increased X-ray-induced DSBs and?-H2AX foci,induced G2/M phase arrest,inhibited cell proliferation by blocking Ki-67,and decreased CD31+micro-vessel density?MVD?.These results underscore the therapeutic potential of the combination of CS/DDP hydrogel and RT for localized NPC. |
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