| Objective:To investigate the anti-tumor effects of emodin in vivo and in vitro in in-situ hydrogel delivery system constructed with ion-complementary self-assembling peptide RADA16-I,providing theoretical and practical reference for solving the application problems of hydrophobic drugs,studying deeply on self-assembling peptide and developing new hydrophobic drug dosage forms.Methods:1.The self-assembling peptide RADA16-I-emodin colloidal suspension was prepared by magnetic stirring method,added into in vitro systems(PBS,0.9% NaCl or cells culture media)that mimic the in vivo conditions,and injected paratumorly or intratumorly to investigate formation of the hydrogels.2.Rabbit blood red blood cells were used to investigate the blood compatibility of the peptide RADA16-I.3.MTT assay was used to determine the proliferation inhibition effects of the peptide RADA16-I in situ hydrogel without emodin and RADA16-I-emodin in situ hydrogel delivery system on normal and cancer cells.4.The uptake of emodin in RADA16-I-emodin in situ hydrogel by tumor cells was qualitatively and quantitatively analyzed by immunofluorescence and flow cytometry.5.Flow cytometry was used to detect the effects of RADA16-I-emodin in situ hydrogel on tumor cell apoptosis and cycle.6.The effects of RADA16-I-emodin in situ hydrogel on tumor cell migration,invasion and cloning ability were investigated by Transwell and clone formation experiments.7.The xenograft models of Hepa1-6 liver cancer and LLC lung cancer in C57 mice were established and the antitumor effect of free emodin and RADA16-I-emodin in situ hydrogel on tumor-bearing mice were tested in vivo.The main investigation indicators included body weight,tumor volume,tumor inhibition rate;HE,TUNEL and Ki-67 staining were used to detect apoptosis and proliferation of tumor tissue cells;And HE staining was used to evaluate the toxicity of drugs to various organs and tissues.Results:1.The results of the hydrogel formation showed that the RADA16-I-emodin colloidal suspension could immediately form three-dimensional hydrogels in-situ in buffer solutions in vitro and para/intratumor tissues in vivo,and the hydrogels were resistant to dispersal to some extent.2.The hemolysis test results showed that the hemolytic rate of the self-assembling peptide RADA16-I was less than 5.0%,which met the biomedical requirements for the hemolytic rate of materials,indicating the good blood compatibility of the peptide material.3.The results of the cytotoxic tests showed that the viabilities of normal mouse cells(NCTC1469,MLE12,HC11)and mouse tumor cells(Hepa1-6,LLC,4T1,B16F10)treate with RADA16-I in situ hydrogel without emodin in a certain concentration range were all higher than 80% of that in control,indicating that no obvious cytotoxic effects werefound.4.Cell proliferation inhibition experiment showed that free emodin and RADA16-I-emodin in situ hydrogel had certain toxic effects on NCTC1469,MLE12 and HC11 normal cells,and the toxicity to cells in the hydrogel group was lower than that of free drugs,indicating that the in situ hydrogel delivery system could significantly reduce the toxic effects of drugs on normal cells.5.MTT assay of free emodin and RADA16-I-emodin in situ hydrogel on Hepa1-6,LLC,4T1,and B16F10 tumor cell proliferation inhibition results showed that the inhibitory effects on tumor cells increased in a concentration-and time-dependent manner,while the inhibitory effects of in situ hydrogel at low concentration were equivalent to that of free drug,and the inhibitory effects of in situ hydrogel at high concentration were significantly higher than that of free drug,indicating that the in situ hydrogel delivery system can maintain or enhance the antitumor effects of emodin.6.The results of laser confocal microscopy showed that the amounts of emodin delivered from the RADA16-I-emodin in situ hydrogels into tumor cells were significantly higher than that from free emodin in suspension without the peptide,and emodin(green fluorescence)was mainly distributed in cytoplasm and cytoplasm.7.Flow cytometry quantitative detection of cell uptake results showed that the drug intake of RDA16-I-emodin in situ hydrogels was significantly higher than that of free emodin(P<0.01),which was consistent with the results of laser confocal microscopy.8.Apoptosis test results showed that,compared with free emodin,RADA16-I-emodin in situ hydrogel could significantly induce cell apoptosis,and the difference between them was statistically significant(P<0.01).9.Cell cycle test results showed that,compared with free emodin,RADA16-I-emodin in situ hydrogel could significantly induce cell cycle arrest in G2/M phase.10.Migration,invasion and clone formation test results showed that,compared with free emodin,RADA16-I-emodin in situ hydrogel could significantly reduce the cell migration,invasion and cloning ability,and the differences between them were all statistically significant(P<0.01).11.The anti-tumor effect in tumor-bearing mice showed that the intratumor injection of RADA16-I-emodin suspension which formed hydrogels in situ and free emodin could both inhibit the growth of tumor cells to a certain extent,and the tumor inhibition rates of in situ hydrogels were higher than that of the free drug(P<0.05);and that the toxic effects of in situ hydrogels on the organs were all significantly lower than that of free emodin.Conclusion:The self-assembling peptide RADA16-I has good blood and cell compatibility;RADA16-I-emodin suspension-in situ hydrogel delivery system can reduce the toxic effects of emodin on normal cells,can also significantly enhance the interaction between drugs and tumor cells promoting the uptake of emodin by tumor cells,and can significantly improve the emodin-induced apoptosis of tumor cells.The anti-tumor studies have shown the potential of RADA16-I as a carrier of hydrophobic anti-tumor drugs and the suspension-in situ hydrogel delivery system constructed by RADA16-I can effectively improve the delivery of hydrophobic anti-tumor drugs and enhance or maintain their anti-tumor effects while reducing their toxic effects. |
PDF Full Text Request