| PART Ⅰ: Design and synthesis of VEGFR targeting peptide-drug conjugate and its effect on hepatoma cells and endothelial cells in vitroObjectives: To design and compound a new vascular endothelial growth factor receptor(VEGFR)targeting peptide-drug conjugate(QR-KLU)combining VEGFR targeting peptide(QR)and lytic peptide(KLU),and to explore the effect on hepatoma cells and endothelial cells under normoxic and hypoxia conditions.Methods: Peptides QR,KLU and QR-KLU were synthesized based on the solid phase peptide synthesis(SPPS).The biosafety of QR,KLU and QR-KLU peptides in blood was evaluated by hemolysis experiments,and the affinity of QR-KLU with VEGFR on the surface of endothelial cells and hepatoma cells was determined by fluorescence-activated cell sorting(FACS)analysis.Subsequently,CCK-8 and plate clone formation assay were used to assess the effects of QR-KLU on the proliferation of human liver cancer cell line Huh7,mouse hepatoma cell line Hepa1-6 and human umbilical vein endothelial cell line HUVEC,and the wound-healing assay was used to evaluate the effect of QR-KLU on the migration of the above cells.Flow cytometry was used to assess the effects of QR-KLU on the apoptosis and cell cycle of Huh7,Hepa1-6 and HUVEC cells,and the expression of HIF-1α and VEGF in Hepa1-6 cells were detected by western blot(WB).Results:(1)The evaluation of QR-KLU characterization,biosafety and VEGFR affinity:peptides QR,KLU and QR-KLU were successfully synthesized,and the molecular weights of the above three peptides were respectively 1634.9 g/mol,1621.2 g/mol and 3309.3 g/mol detected by electron spray ionization-mass spectrometry(ESI-MS).The hemolytic experiments proved that all three peptides were of no harm to red blood cell even at a high concentration,and the affinity experiments showed that QR-KLU had excellent affinity for HUVEC cells with high VEGFR expression.(2)Effects of QR-KLU on hepatoma cells and endothelial cells: in the CCK-8 assay,QR-KLU inhibited Huh7,Hepa1-6,and HUVEC proliferation in a dose-dependent manner under both normoxic and hypoxic conditions,and the inhibitory effect was better than that of KLU,which was further confirmed by plate clone formation assay.The results of wound-healing assay showed that both KLU and QR-KLU significantly inhibited the migration of Hepa1-6 and HUVEC cells under normoxic and hypoxia conditions,and the inhibitory ability of QR-KLU was better.Flow cytometry showed that both KLU and QR-KLU significantly promoted apoptosis in Huh7,Hepa1-6 and HUVEC cells under normoxic and hypoxic conditions,and the apoptosis ratio of QR-KLU was significantly higher than that of KLU.And QR-KLU could induce cell cycle arrest in the S phase on Huh7,Hepa1-6 and HUVEC cells,and the effect of QR-KLU is better than that of KLU.(3)Effects of QR-KLU on HIF-1α and VEGF expression in hepatoma cells: the results of WB showed that the expression of HIF-1α and VEGF in Hepa1-6 cells were significantly higher under hypoxic conditions,and QR-KLU could significantly reduce the high expression of VEGF caused by hypoxia.But under normoxic conditions,the inhibitory effect of QR-KLU on VEGF expression was not significant.Conclusion: The novel VEGFR targeting peptide-drug conjugate QR-KLU,with good biosafety and VEGFR targeting,could inhibit the proliferation and migration of hepatoma cells and endothelial cells under normoxic and hypoxic conditions,induce cell apoptosis and cycle arrest,and block the high expression of VEGF in hepatoma cells caused by hypoxia.PART Ⅱ: Evaluation of efficacy and mechanism of QR-KLU administered via TACE in the treatment of rabbit VX2 liver tumor modelObjectives: To evaluate the efficacy of QR-KLU administered via transarterial chemoembolization(TACE)in the treatment of VX2 rabbit hepatoma tumor model,and to investigate the mechanism of QR-KLU on anti-angiogenesis after TACE.Methods: A rabbit VX2 liver tumor model was constructed,and the rabbits were randomly divided into four groups: negative saline(NS)group,doxorubicin(DOX)group,KLU group and QR-KLU group.Magnetic resonance imaging(MRI)was used to assess the tumor growth rate.One week after TACE,four groups of tumor-bearing rabbits were sacrificed,and serum,tumor,heart,lung,kidney and spleen tissue samples were collected for efficacy and safety evaluation.Tumor necrosis rate was assessed by haematoxylin and eosin(H&E)staining,apoptosis rate was assessed by TUNEL fluorescent staining,and tumor cell proliferation was assessed by Ki67 immunohistochemistry(IHC)staining.The expression of HIF-1α was detected by IHC to evaluate the hypoxia of tumor tissues in each group,and the expression of VEGF and CD31 was detected to evaluate the effect of drugs on tumor angiogenesis.The liver and kidney functions of tumor-bearing rabbits in each group were detected by biochemical automatic analyzer.In addition,in order to evaluate the effect of each drug on the survival of tumor-bearing rabbits,survival analysis was also performed.Results:(1)Tumor prognosis and survival results: MRI showed that the tumor growth rates of DOX,KLU and QR-KLU group were significantly lower than that of NS group,and the tumor growth rate of QR-KLU group was significantly lower than that of DOX and KLU groups one week after TACE.H&E staining showed that the mean tumor necrosis rate was significantly higher in the DOX,KLU and QR-KLU groups than in the NS group,and the mean necrosis rate in the QR-KLU group was significantly higher than that in the DOX and KLU groups.The TUNEL staining and IHC staining of Ki67 showed that QR-KLU could significantly promote the apoptosis and inhibit the proliferation of hepatoma cells,which was better than DOX and KLU.The survival analysis showed that the median survival time of NS,DOX,KLU and QR-KLU groups were respectively 31.5 days,38 days,37.5 days and 47 days,and the cumulative survival rate of QR-KLU group was significantly higher than other groups.(2)Hypoxia,HIF-1α/VEGF pathway and angiogenesis: IHC staining showed that the expression of HIF-1α in DOX,KLU and QR-KLU groups were significantly higher than that in NS group.In addition,the expression of VEGF and the microvessel density(MVD)in the DOX and KLU groups were significantly higher than those in NS group,while the VEGF expression and MVD in QR-KLU group were significantly lower than those in the DOX and KLU groups.(3)Biosafety assessment: the liver and kidney functions were impaired by the embolization of TACE,but quickly returned to normal within one week.In addition,the administration of DOX,KLU,and QR-KLU did not cause morphological or organic damage to the main organs.Conclusion: Compared with DOX-TACE and KLU-TACE,QR-KLU-TACE could significantly inhibite the proliferation of VX2 liver tumors,promote their necrosis and apoptosis,and improve the survival of rabbits.TACE could promote tumor angiogenesis by upregulating the HIF-1α/VEGF pathway,and QR-KLU could significantly inhibit VEGF expression and angiogenesis induced by TACE.PART Ⅲ: Evaluation of efficacy and mechanism of QR-KLU combined with anti-PD-1 antibody in the treatment of mouse Hepa1-6 subcutaneous tumor modelObjectives: To evaluate the efficacy of QR-KLU combined with anti-PD-1 antibody in mouse Hepa1-6 subcutaneous tumor model and explore the synergistic mechanism.Methods: The Hepa1-6 subcutaneous tumor models were constructed in mice,they were randomly divided into the following six groups: phosphate buffered saline(PBS)group,KLU group,QR-KLU group,anti-PD-1 group,KLU+anti-PD-1 group and QR-KLU+anti-PD-1 group.The mice in each group were sarcrificed 11 days after treatment,and serum,tumor,heart,lung,liver,kidney and spleen tissue samples were collected for efficacy and safety evaluation.The tumor growth rate,necrosis rate and tumor cell proliferation of each group were compared.The hypoxia,VEGF expression and pericyte coverage rate of tumor vessels in each group were evaluated,the number of CD4~+ and CD8~+ T cells in tumor tissues was detected.Liver and kidney functions of each group were detected.In addition,survival analysis was performed in order to evaluate the effect of different drugs on the survival of tumor-bearing mice.Results:(1)Tumor prognosis and survival results: there was no significant difference in the baseline tumor volumes among groups,and the mean volume in QR-KLU+anti-PD-1 group was significantly lower than the other groups.H&E staining showed that the tumor necrosis rate in QR-KLU group was significantly higher than that in PBS and KLU groups,and the QR-KLU+anti-PD-1 group was significantly higher than QR-KLU and anti-PD-1 groups.IHC staining of Ki67 showed that the tumor cell proliferation rate in QR-KLU group was significantly lower than that in KLU group,while QR-KLU+anti-PD-1 group was significantly lower than QR-KLU and anti-PD-1 groups.The cumulative survival rate of QR-KLU+anti-PD-1 group was significantly higher than the other groups.(2)Hypoxia and tumor vessel normalization: compared with PBS group,the percentages of hypoxic region in KLU and QR-KLU groups were significantly higher,while QR-KLU group was significantly lower than KLU group.The VEGF expression in QR-KLU group was significantly lower than that in PBS and KLU groups,and QR-KLU+anti-PD-1 group was significantly lower than anti-PD-1 group.The staining of α-SMA and CD31 showed that pericyte coverage rate of tumor vessels was significantly higher in QR-KLU group than in PBS and KLU groups,and QR-KLU+anti-PD-1 group was significantly higher than anti-PD-1 group.(3)Immunomicroenvironment changes: The number of CD4~+ and CD8~+ T cells in QR-KLU group was significantly higher than those in PBS and KLU groups,and QR-KLU+anti-PD-1 group was significantly higher than QR-KLU and anti-PD-1 groups.ELISA showed that the TNF-α,IFN-γ and IL-2 expression in QR-KLU group were significantly higher than those in PBS group,QR-KLU+anti-PD-1 group was higher than QR-KLU and anti-PD-1 groups.In addition,the IL-10 expression of QR-KLU+anti-PD-1 group was significantly lower than the other groups.(4)Biosafety assessment: the mice body weight,liver and kidney functions were not significantly affected in each group,and the heart,lung,liver,kidney and spleen were not damaged morphologically or organicly.Conclusion: QR-KLU could significantly inhibit liver tumor proliferation,promote tumor necrosis and prolong the mice’s survival synergistically with anti-PD-1 antibody.QR-KLU could significantly inhibit tumor angiogenesis induced by hypoxia,regulate tumor vascular normalization,and promote intratumor CD8~+ T cell infiltration,activate the immunoactivating factors,while reduce the immunosuppressive factors synergistically with anti-PD-1 antibody. |
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