| Objective:To develop microsphere-hydrogel systems containing PTX microspheres,5-fluorouracil（5-FU）and cisplatin（DDP）.And to investigate the anti-tumor effect of microsphere-hydrogel systems on colorectal peritoneal carcinomatosis.Methods:In vitro,the triblock copolymer poly（ε-caprolactone）-poly（ethyleneglycol）-poly（ε-caprolactone）（PCL-PEG-PCL or PCEC）with theoretical molecular weight of 80000 was synthesized from PEG（Mn=4000）andε-caprolactone（ε-CL）by the ring-opening polymerization method using stannous octoate（Sn（Oct）2）as the catalyst.And PCEC was characterized by hydrogen nuclear magnetic resonance（1）H-NMRand Fourier transform infrared（FT-IR）analysis.PTX-microspheres were prepared by the oil-in-water emulsion solvent evaporation method,and the intraperitoneal injectable three-drug co-delivery microsphere-hydrogel system was constructed with PTX-microspheres,5-FU,DDP and hyaluronic acid（HA）.The particle size and surface morphology of the microspheres were analyzed by scanning electron microscopy（Hitachi,Japan）.Drug loading（DL）and encapsulation efficiency（EE）of PTX-microspheres were measured by high performance liquid chromatography（HPLC）.In vitro release of drug from PTX-microspheres and drug-loaded microsphere-hydrogel system was analyzed by means of dialysis.The cytotoxicity of blank PCEC microspheres and drug-loaded microsphere-hydrogel system was tested by methyl thiazolyltetrazolium（MTT）method.In vivo experiment,to evaluate the biocompatibility of blank microsphere-hydrogel system,it was injected subcutaneously into rats.The antitumor effect of the drug-loaded hydrogel was tested in bearing mouse model.The tumor-bearing mice were randomly divided into 4 groups（n=10each）:（1）normal saline（NS）,（2）blank microsphere-hydrogel,（3）free drugs（5-FU 20 mg/kg;PTX 5 mg/kg;DDP 1 mg/kg）,（4）drug-loaded microsphere-hydrogel（The drug dose was the same as that of free drugs group）.Tumor-bearing mice were intraperitoneally injected once a week with 200μL of the respective therapeutic agents,and their body weight was measured on alternate days.On the 14th day after treatment,tumor-bearing mice in the saline group began to die.Five mice per group were sacrificed,and the number of tumor nodules in the abdominal cavity and volume of ascites were measured.In addition,the intestinal,liver and lung tissues were collected for hematoxylin and eosin（H&E）staining to evaluate the local intestinal infiltration and hepatopulmonary metastasis of the tumor.At the same time,tumor cell proliferation was evaluated by Ki-67 immunostaining of tumor nodules in the abdominal cavity.The remaining five mice in each group were used to observe the survival duration and then draw the survival curve.Results:The synthesis of PCEC polymer was confirmed by 1 HNMR and FT-IR analysis,and its molecular weight（Mn）was calculated as 76430.The PTX-microspheres with 15%theoretical DL showed a better actual DL（12.84±0.15%）and higher EE（85.62±0.98%）.The in vitro release behavior showed that free drugs were released rapidly in a burst mode,while drugs were released slowly from microspheres and microsphere-hydrogel system.The cytotoxicity demonstrated that 84.58%±3.8%of the cells were viable in the presence of1000μg/m L blank PCEC microspheres,indicating low inherent cytotoxicity of these microspheres.And the inhibitory effect of drug loaded microsphere-hydrogel on CT26 cells was significantly higher than that of free drugs（P<0.05）in a dose-dependent manner.The results of cytotoxicity indicating that the therapeutic effect of the drugs was significantly enhanced when encapsulated in microsphere-hydrogel system.The local inflammatory response disappeared completely by 21 days after subcutaneous embedding of the drug-loaded hydrogel,indicating that the microsphere-hydrogel system prepared by PCEC microsphere and HA showed good biocompatibility in vivo.In vivo anti-tumor experiment,the mean number of abdominal tumor nodules in the drug-loaded microsphere-hydrogel group was a meagre 14±2.08compared to 88±5.86 in the normal saline group（P<0.05）,76±5.86 in the blank microsphere-hydrogel group（P<0.05）,and 29±4.04 in the free drugs injected group（P<0.05）.The average ascites volume in the drug-loaded microsphere-hydrogel group（0.04±0.02m L）was significantly lower than that in the saline group（1.29±0.17 m L,P<0.05）,the blank microsphere-hydrogel group（0.96±0.15 m L,P<0.05）and the free drugs group（0.11±0.03 m L P<0.05）.The median survival of mice in the drug-loaded microsphere-hydrogel group was 36 days,which was significantly longer than the 27 days,26 days,and 32 days of the saline group,the blank microsphere-hydrogel group,and the free drugs group.In addition,the results of Ki-67 immunohistochemistry showed that the tumor tissues of the drug-loaded microsphere-hydrogel group had significantly lower percentage of Ki-67 positive cells（29.84±6.78%）compared to that of the saline group（86.42±4.50%,P<0.05）,the blank microsphere-hydrogel group（84.05±4.27%,P<0.05）and the free drugs group（53.18±7.64%,P<0.05）.H&E staining indicated that tumor cell invasion into the intestinal muscle layer,liver/lung metastasis were observed in the saline group,the blank microsphere-hydrogel group and the free drugs group,while no obvious local intestinal infiltration and metastasis was observed in drug-loaded microsphere-hydrogel group.Conclusion:In this study,a microsphere-hydrogel multi-drug delivery system was developed by PCEC and HA,which showed sustained drug release,good biocompatibility,and significant anti-tumor effects.In vivo anti-tumor experiments demonstrated that the drug-loaded microsphere-hydrogel system can significantly reduce the formation of ascites,inhibit tumor growth and liver/lung metastasis,reduce systemic side effects,and prolong the survival time of colorectal peritoneal carcinomatosis mice through intraperitoneal administration.Therefore,the microsphere-hydrogel multi-drug drug delivery system has a good clinical application prospect and is expected to become a novel treatment tool for colorectal peritoneal carcinomatosis. |
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