| Aims: To extract mesenchymal stem cells by tissue block adherence method,and extract exosomes from the supernatant of mesenchymal stem cells by ultracentrifugation to construct cyclic RGD(c RGD)polypeptide-modified triptolide(TPL)mesenchymal stem cells exosomal targeted drug delivery system(c RGD-EXO/TPL).The effect and the mechanism of this preparation on melanoma was evaluated through in vivo and in vitro experiments.Methods:1.The umbilical cord mesenchymal stem cells were extracted by the tissue block adherence method and identified by transmission electron microscopy and flow cytometry.Exosomes were isolated by ultracentrifugation and characterized by transmission electron microscopy and Western Blot.The binding ratio of c RGD peptides and exosomes was optimized by cell uptake fluorescence intensity.High-performance liquid chromatography(HPLC)was used to screen the optimal the ratio of TPL for drug loading and encapsulation efficiency.The particle size of the constructed drug delivery system was measured using particle size analyzer,and its release performance in vitro under different p H values was also studied by HPLC.2.Confocal microscopy was used to observe the tumor targeting of c RGD-EXO/TPL in vitro.The effect of amiloride,chlorpromazine,sodium azide,low temperature,and nystatin on the celluar c RGD-EXO/TPL uptake was also investigated.The effect of c RGD-EXO/TPL on the viability of A375 cells was evaluated by CCK-8.The effect of c RGD-EXO/TPL on the migration and invasion of A375 cells was observed using Transwell.The apoptosis kit and the cell cycle kit were used to detect the apoptosis-promoting effect of c RGD-EXO/TPL and the influence on the cell cycle.3.Nude mice model bearing human melanoma were constructed.c RGD-EXO was labeled using Di R fluorescent dye.The distribution of c RGD-EXO after intravenous injection was monitored through living imaging system.c RGD-EXO/TPL was administrated intravenously to treat tumor nude mice models to study its anti-tumor effect in vivo.Tumor tissues were stained with HE and Tunel to evaluate its canti-tumor effect.The safety of the c RGD-EXO/TPL was evaluated by HE staining of the heart,liver,spleen,lung and kidney of tumor-bearing nude mice.Results:1.Human umbilical cord mesenchymal stem cells were successfully extracted and consisted with the identification criteria for mesenchymal stem cells.Exosomes were successfully isolated from the supernatant of human umbilical cord mesenchymal stem cells.Transmission electron microscopy showed the typical ultrastructure of exosomes.Western blot showed the exosomal specific markers CD9,CD63 and TSG101.Flow cytometry results showed that the cellular uptake of exosomes reached the highest value when the binding ratio of exosomes to DSPE-PEG2000-c(RGDfk)was 1:1.HPLC experiments showed that when the ratio of exosomes to TPL was 1:1,the encapsulation rate of exosomes was12.07±1.89%.The particle size analyzer showed that the average particle size of EXO,c RGD-EXO and c RGD-EXO/TPL were 82.82 nm,110 nm and 160 nm,respectively.The cumulative release rates of c RGD-EXO/TPL under p H 7.4 and p H 5.5 condition within 24 h were 50.91% and 85.31%,respectively,indicating that the release of c RGD-EXO/TPL was more complete in an acidic environment.2.Confocal images demonstrated that c RGD could improve the internalization of exosomes by A375 cells.CCK8 results showed that c RGD-EXO/TPL can significantly inhibit the proliferation of A375 cells and reduce viability.Transwell results showed that c RGDEXO/TPL can effectively inhibit the migration and invasion of A375 cells.Cell cycle and apoptosis results showed that c RGD-EXO can arrest the S phase of A375 cells and promote cell apoptosis.3.In vivo imaging system was used to monitor the distribution of Di R labeled c RGDEXO/TPL in nude mice xenograft model,especially at the tumor site.After treatment with different preparations,the tumor growth of tumor-bearing nude mice was inhibited to varying degrees.Among them,the inhibitory effect of c RGD-EXO/TPL was the most significant.The HE and Tunel sections of tumor tissues also verified the results.From the HE stained sections of the heart,liver,spleen,lung,and kidney of tumor-bearing nude mice,it can be observed that c RGD-EXO/TPL can reduce the toxicity of TPL.The weight change of nude mice also verified this further.Conclusion: cRGD-modified engineered exosomal drug delivery system was successfully constructed.In vivo and in vitro experiments showed that the drug delivery system has good targeting and could enhance the therapeutic efficacy of TPL on tumors reduce TPL toxicity.This study provides new ideas and directions for the treatment of traditional Chinese medicine against malignant melanoma. |
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