While after the administration of SynB3PVGLIGPTX, there were few differences in the tumor signals between day 7 and day 14 (Figure 6A). At all later days, the differences in log (BLI) between the SynB3PVGLIGPTX and control group were significantly increased (***p < 0.001, Figure 6B), in which the mouse treated with SynB3PVGLIGPTX yielded a notably weaker bioluminescence radiance and a concurrently smaller bioluminescent area than those in the control group. On day 28, mice treated with SynB3PVGLIGPTX achieved the highest tumor inhibition rate (91.40±0.57%), which was 2.27fold and 1.30fold higher than that of the PTX and TMZ groups, respectively (***p < 0.001, Figure 6C). From the results of the bioluminescence assays, we concluded that SynB3PVGLIGPTX could sufficiently suppress the growth of intracerebral tumors in vivo after 28 days compared with the inhibition rate yielded by TMZ or PTX (***p < 0.001, Figure 6C). In addition, the weight and overall survival period of mice administered with SynB3PVGLIGPTX (15 mg/5 mL kg-1, i.v.) were significantly higher than those in the control group (*p < 0.05, **p < 0.01, ***p < 0.001, Figure 6D,,E).E). Again, the separation in survival curves was notable among the four groups, with survival medians equal to 23, 29, and 25 for the control group, TMZ group and PTX group, respectively. In contrast, there was no death in the SynB3PVGLIGPTX group across the assays. Especially concerning was that, the administration concentration of SynB3PVGLIGPTX was 15 mg/5 mL kg-1, which is equivalent to 5.25 μmol/5 mL kg-1. Theoretically, after SynB3PVGLIGPTX at this concentration is completely hydrolyzed by MMP2, the concentration of free PTX released is 4.49 mg/5 mL kg-1, which is only onethird of the concentration administered in the PTX group. The experimental results show that, compared with the group given a high dose of PTX (15 mg/5 mL kg-1), the tumor volume of mice given the nanoconjugate (SynB3PVGLIGPTX) was significantly reduced, and its overall survival was also significantly prolonged, which indicates that SynB3PVGLIGPTX has a significant antiglioma activity in vivo. Accordingly, SynB3PVGLIGPTX, as a PDC that combined PTX to a dualfunctional peptide consisting of SynB3 and a MMP2sensitive peptide, could observably improve survival and decrease weight loss over the PTX monotherapy in vivo, and significantly overmatched the mice treated with TMZ.

While after the administration of SynB3PVGLIGPTX, there were few differences in the tumor signals between day 7 and day 14 (Figure 6A). At all later days, the differences in log (BLI) between the SynB3PVGLIGPTX and control group were significantly increased (***p < 0.001, Figure 6B), in which the mouse treated with SynB3PVGLIGPTX yielded a notably weaker bioluminescence radiance and a concurrently smaller bioluminescent area than those in the control group. On day 28, mice treated with SynB3PVGLIGPTX achieved the highest tumor inhibition rate (91.40±0.57%), which was 2.27fold and 1.30fold higher than that of the PTX and TMZ groups, respectively (***p < 0.001, Figure 6C). From the results of the bioluminescence assays, we concluded that SynB3PVGLIGPTX could sufficiently suppress the growth of intracerebral tumors in vivo after 28 days compared with the inhibition rate yielded by TMZ or PTX (***p < 0.001, Figure 6C). In addition, the weight and overall survival period of mice administered with SynB3PVGLIGPTX (15 mg/5 mL kg-1, i.v.) were significantly higher than those in the control group (*p < 0.05, **p < 0.01, ***p < 0.001, Figure 6D,,E).E). Again, the separation in survival curves was notable among the four groups, with survival medians equal to 23, 29, and 25 for the control group, TMZ group and PTX group, respectively. In contrast, there was no death in the SynB3PVGLIGPTX group across the assays. Especially concerning was that, the administration concentration of SynB3PVGLIGPTX was 15 mg/5 mL kg-1, which is equivalent to 5.25 μmol/5 mL kg-1. Theoretically, after SynB3PVGLIGPTX at this concentration is completely hydrolyzed by MMP2, the concentration of free PTX released is 4.49 mg/5 mL kg-1, which is only onethird of the concentration administered in the PTX group. The experimental results show that, compared with the group given a high dose of PTX (15 mg/5 mL kg-1), the tumor volume of mice given the nanoconjugate (SynB3PVGLIGPTX) was significantly reduced, and its overall survival was also significantly prolonged, which indicates that SynB3PVGLIGPTX has a significant antiglioma activity in vivo. Accordingly, SynB3PVGLIGPTX, as a PDC that combined PTX to a dualfunctional peptide consisting of SynB3 and a MMP2sensitive peptide, could observably improve survival and decrease weight loss over the PTX monotherapy in vivo, and significantly overmatched the mice treated with TMZ.

While after the administration of SynB3PVGLIGPTX, there were few differences in the tumor signals between day 7 and day 14 (Figure 6A). At all later days, the differences in log (BLI) between the SynB3PVGLIGPTX and control group were significantly increased (***p < 0.001, Figure 6B), in which the mouse treated with SynB3PVGLIGPTX yielded a notably weaker bioluminescence radiance and a concurrently smaller bioluminescent area than those in the control group. On day 28, mice treated with SynB3PVGLIGPTX achieved the highest tumor inhibition rate (91.40±0.57%), which was 2.27fold and 1.30fold higher than that of the PTX and TMZ groups, respectively (***p < 0.001, Figure 6C). From the results of the bioluminescence assays, we concluded that SynB3PVGLIGPTX could sufficiently suppress the growth of intracerebral tumors in vivo after 28 days compared with the inhibition rate yielded by TMZ or PTX (***p < 0.001, Figure 6C). In addition, the weight and overall survival period of mice administered with SynB3PVGLIGPTX (15 mg/5 mL kg-1, i.v.) were significantly higher than those in the control group (*p < 0.05, **p < 0.01, ***p < 0.001, Figure 6D,,E).E). Again, the separation in survival curves was notable among the four groups, with survival medians equal to 23, 29, and 25 for the control group, TMZ group and PTX group, respectively. In contrast, there was no death in the SynB3PVGLIGPTX group across the assays. Especially concerning was that, the administration concentration of SynB3PVGLIGPTX was 15 mg/5 mL kg-1, which is equivalent to 5.25 μmol/5 mL kg-1. Theoretically, after SynB3PVGLIGPTX at this concentration is completely hydrolyzed by MMP2, the concentration of free PTX released is 4.49 mg/5 mL kg-1, which is only onethird of the concentration administered in the PTX group. The experimental results show that, compared with the group given a high dose of PTX (15 mg/5 mL kg-1), the tumor volume of mice given the nanoconjugate (SynB3PVGLIGPTX) was significantly reduced, and its overall survival was also significantly prolonged, which indicates that SynB3PVGLIGPTX has a significant antiglioma activity in vivo. Accordingly, SynB3PVGLIGPTX, as a PDC that combined PTX to a dualfunctional peptide consisting of SynB3 and a MMP2sensitive peptide, could observably improve survival and decrease weight loss over the PTX monotherapy in vivo, and significantly overmatched the mice treated with TMZ.

However, in the MMP2 siRNAtransfected U87MG and U251, SynB3PVGLIGPTX could result in an extremely lower inhibitory effect on cell proliferation than that treated at the same concentration ranges (25 x 10^-6, 50 x 10^-6, 75 x 10^-6, and 100 x 10^-6 M) in the U87MG and U251 cell lines (Figure 5G,,H).H). The absence of MMP2 manifested as an obviously reducing trend of IC50 of SynB3PVGLIGPTX in both cell lines (Table 3). These results indicated that SynB3PVGLIGPTX could only inhibit cell proliferation based on the presence of MMP2, which suggested that SynB3PVGLIGPTX performs a specific inhibitory action on the proliferation of GBM cells. In addition, in order to further verify the mechanism underlying the specific inhibitory activity of SynB3PVGLIGPTX on GBM cell proliferation, a supplementary experiment was performed using SynB3PTXtreated cells as inactive control groups. The results showed that the inhibition rates of SynB3PTX without an MMP2sensitive linker (PVGLIG) in U87MG and U251 were extremely low, at a nearzero level. Based on these observations, we confirmed that SynB3PVGLIGPTX has a specific antigrowth activity, that is, only in the presence of MMP2, the sensitive linker (PVGLIG) contained in SynB3PVGLIGPTX can be specifically hydrolyzed to release PTX, thereby inhibiting the proliferation of GBM cells.

However, in the MMP2 siRNAtransfected U87MG and U251, SynB3PVGLIGPTX could result in an extremely lower inhibitory effect on cell proliferation than that treated at the same concentration ranges (25 x 10^-6, 50 x 10^-6, 75 x 10^-6, and 100 x 10^-6 M) in the U87MG and U251 cell lines (Figure 5G,,H).H). The absence of MMP2 manifested as an obviously reducing trend of IC50 of SynB3PVGLIGPTX in both cell lines (Table 3). These results indicated that SynB3PVGLIGPTX could only inhibit cell proliferation based on the presence of MMP2, which suggested that SynB3PVGLIGPTX performs a specific inhibitory action on the proliferation of GBM cells. In addition, in order to further verify the mechanism underlying the specific inhibitory activity of SynB3PVGLIGPTX on GBM cell proliferation, a supplementary experiment was performed using SynB3PTXtreated cells as inactive control groups. The results showed that the inhibition rates of SynB3PTX without an MMP2sensitive linker (PVGLIG) in U87MG and U251 were extremely low, at a nearzero level. Based on these observations, we confirmed that SynB3PVGLIGPTX has a specific antigrowth activity, that is, only in the presence of MMP2, the sensitive linker (PVGLIG) contained in SynB3PVGLIGPTX can be specifically hydrolyzed to release PTX, thereby inhibiting the proliferation of GBM cells.

However, in the MMP2 siRNAtransfected U87MG and U251, SynB3PVGLIGPTX could result in an extremely lower inhibitory effect on cell proliferation than that treated at the same concentration ranges (25 x 10^-6, 50 x 10^-6, 75 x 10^-6, and 100 x 10^-6 M) in the U87MG and U251 cell lines (Figure 5G,,H).H). The absence of MMP2 manifested as an obviously reducing trend of IC50 of SynB3PVGLIGPTX in both cell lines (Table 3). These results indicated that SynB3PVGLIGPTX could only inhibit cell proliferation based on the presence of MMP2, which suggested that SynB3PVGLIGPTX performs a specific inhibitory action on the proliferation of GBM cells. In addition, in order to further verify the mechanism underlying the specific inhibitory activity of SynB3PVGLIGPTX on GBM cell proliferation, a supplementary experiment was performed using SynB3PTXtreated cells as inactive control groups. The results showed that the inhibition rates of SynB3PTX without an MMP2sensitive linker (PVGLIG) in U87MG and U251 were extremely low, at a nearzero level. Based on these observations, we confirmed that SynB3PVGLIGPTX has a specific antigrowth activity, that is, only in the presence of MMP2, the sensitive linker (PVGLIG) contained in SynB3PVGLIGPTX can be specifically hydrolyzed to release PTX, thereby inhibiting the proliferation of GBM cells.

However, in the MMP2 siRNAtransfected U87MG and U251, SynB3PVGLIGPTX could result in an extremely lower inhibitory effect on cell proliferation than that treated at the same concentration ranges (25 x 10^-6, 50 x 10^-6, 75 x 10^-6, and 100 x 10^-6 M) in the U87MG and U251 cell lines (Figure 5G,,H).H). The absence of MMP2 manifested as an obviously reducing trend of IC50 of SynB3PVGLIGPTX in both cell lines (Table 3). These results indicated that SynB3PVGLIGPTX could only inhibit cell proliferation based on the presence of MMP2, which suggested that SynB3PVGLIGPTX performs a specific inhibitory action on the proliferation of GBM cells. In addition, in order to further verify the mechanism underlying the specific inhibitory activity of SynB3PVGLIGPTX on GBM cell proliferation, a supplementary experiment was performed using SynB3PTXtreated cells as inactive control groups. The results showed that the inhibition rates of SynB3PTX without an MMP2sensitive linker (PVGLIG) in U87MG and U251 were extremely low, at a nearzero level. Based on these observations, we confirmed that SynB3PVGLIGPTX has a specific antigrowth activity, that is, only in the presence of MMP2, the sensitive linker (PVGLIG) contained in SynB3PVGLIGPTX can be specifically hydrolyzed to release PTX, thereby inhibiting the proliferation of GBM cells.

However, in the MMP2 siRNAtransfected U87MG and U251, SynB3PVGLIGPTX could result in an extremely lower inhibitory effect on cell proliferation than that treated at the same concentration ranges (25 x 10^-6, 50 x 10^-6, 75 x 10^-6, and 100 x 10^-6 M) in the U87MG and U251 cell lines (Figure 5G,,H).H). The absence of MMP2 manifested as an obviously reducing trend of IC50 of SynB3PVGLIGPTX in both cell lines (Table 3). These results indicated that SynB3PVGLIGPTX could only inhibit cell proliferation based on the presence of MMP2, which suggested that SynB3PVGLIGPTX performs a specific inhibitory action on the proliferation of GBM cells. In addition, in order to further verify the mechanism underlying the specific inhibitory activity of SynB3PVGLIGPTX on GBM cell proliferation, a supplementary experiment was performed using SynB3PTXtreated cells as inactive control groups. The results showed that the inhibition rates of SynB3PTX without an MMP2sensitive linker (PVGLIG) in U87MG and U251 were extremely low, at a nearzero level. Based on these observations, we confirmed that SynB3PVGLIGPTX has a specific antigrowth activity, that is, only in the presence of MMP2, the sensitive linker (PVGLIG) contained in SynB3PVGLIGPTX can be specifically hydrolyzed to release PTX, thereby inhibiting the proliferation of GBM cells.

However, in the MMP2 siRNAtransfected U87MG and U251, SynB3PVGLIGPTX could result in an extremely lower inhibitory effect on cell proliferation than that treated at the same concentration ranges (25 x 10^-6, 50 x 10^-6, 75 x 10^-6, and 100 x 10^-6 M) in the U87MG and U251 cell lines (Figure 5G,,H).H). The absence of MMP2 manifested as an obviously reducing trend of IC50 of SynB3PVGLIGPTX in both cell lines (Table 3). These results indicated that SynB3PVGLIGPTX could only inhibit cell proliferation based on the presence of MMP2, which suggested that SynB3PVGLIGPTX performs a specific inhibitory action on the proliferation of GBM cells. In addition, in order to further verify the mechanism underlying the specific inhibitory activity of SynB3PVGLIGPTX on GBM cell proliferation, a supplementary experiment was performed using SynB3PTXtreated cells as inactive control groups. The results showed that the inhibition rates of SynB3PTX without an MMP2sensitive linker (PVGLIG) in U87MG and U251 were extremely low, at a nearzero level. Based on these observations, we confirmed that SynB3PVGLIGPTX has a specific antigrowth activity, that is, only in the presence of MMP2, the sensitive linker (PVGLIG) contained in SynB3PVGLIGPTX can be specifically hydrolyzed to release PTX, thereby inhibiting the proliferation of GBM cells.

However, in the MMP2 siRNAtransfected U87MG and U251, SynB3PVGLIGPTX could result in an extremely lower inhibitory effect on cell proliferation than that treated at the same concentration ranges (25 x 10^-6, 50 x 10^-6, 75 x 10^-6, and 100 x 10^-6 M) in the U87MG and U251 cell lines (Figure 5G,,H).H). The absence of MMP2 manifested as an obviously reducing trend of IC50 of SynB3PVGLIGPTX in both cell lines (Table 3). These results indicated that SynB3PVGLIGPTX could only inhibit cell proliferation based on the presence of MMP2, which suggested that SynB3PVGLIGPTX performs a specific inhibitory action on the proliferation of GBM cells. In addition, in order to further verify the mechanism underlying the specific inhibitory activity of SynB3PVGLIGPTX on GBM cell proliferation, a supplementary experiment was performed using SynB3PTXtreated cells as inactive control groups. The results showed that the inhibition rates of SynB3PTX without an MMP2sensitive linker (PVGLIG) in U87MG and U251 were extremely low, at a nearzero level. Based on these observations, we confirmed that SynB3PVGLIGPTX has a specific antigrowth activity, that is, only in the presence of MMP2, the sensitive linker (PVGLIG) contained in SynB3PVGLIGPTX can be specifically hydrolyzed to release PTX, thereby inhibiting the proliferation of GBM cells.

However, in the MMP2 siRNAtransfected U87MG and U251, SynB3PVGLIGPTX could result in an extremely lower inhibitory effect on cell proliferation than that treated at the same concentration ranges (25 x 10^-6, 50 x 10^-6, 75 x 10^-6, and 100 x 10^-6 M) in the U87MG and U251 cell lines (Figure 5G,,H).H). The absence of MMP2 manifested as an obviously reducing trend of IC50 of SynB3PVGLIGPTX in both cell lines (Table 3). These results indicated that SynB3PVGLIGPTX could only inhibit cell proliferation based on the presence of MMP2, which suggested that SynB3PVGLIGPTX performs a specific inhibitory action on the proliferation of GBM cells. In addition, in order to further verify the mechanism underlying the specific inhibitory activity of SynB3PVGLIGPTX on GBM cell proliferation, a supplementary experiment was performed using SynB3PTXtreated cells as inactive control groups. The results showed that the inhibition rates of SynB3PTX without an MMP2sensitive linker (PVGLIG) in U87MG and U251 were extremely low, at a nearzero level. Based on these observations, we confirmed that SynB3PVGLIGPTX has a specific antigrowth activity, that is, only in the presence of MMP2, the sensitive linker (PVGLIG) contained in SynB3PVGLIGPTX can be specifically hydrolyzed to release PTX, thereby inhibiting the proliferation of GBM cells.

However, in the MMP2 siRNAtransfected U87MG and U251, SynB3PVGLIGPTX could result in an extremely lower inhibitory effect on cell proliferation than that treated at the same concentration ranges (25 x 10^-6, 50 x 10^-6, 75 x 10^-6, and 100 x 10^-6 M) in the U87MG and U251 cell lines (Figure 5G,,H).H). The absence of MMP2 manifested as an obviously reducing trend of IC50 of SynB3PVGLIGPTX in both cell lines (Table 3). These results indicated that SynB3PVGLIGPTX could only inhibit cell proliferation based on the presence of MMP2, which suggested that SynB3PVGLIGPTX performs a specific inhibitory action on the proliferation of GBM cells. In addition, in order to further verify the mechanism underlying the specific inhibitory activity of SynB3PVGLIGPTX on GBM cell proliferation, a supplementary experiment was performed using SynB3PTXtreated cells as inactive control groups. The results showed that the inhibition rates of SynB3PTX without an MMP2sensitive linker (PVGLIG) in U87MG and U251 were extremely low, at a nearzero level. Based on these observations, we confirmed that SynB3PVGLIGPTX has a specific antigrowth activity, that is, only in the presence of MMP2, the sensitive linker (PVGLIG) contained in SynB3PVGLIGPTX can be specifically hydrolyzed to release PTX, thereby inhibiting the proliferation of GBM cells.

However, in the MMP2 siRNAtransfected U87MG and U251, SynB3PVGLIGPTX could result in an extremely lower inhibitory effect on cell proliferation than that treated at the same concentration ranges (25 x 10^-6, 50 x 10^-6, 75 x 10^-6, and 100 x 10^-6 M) in the U87MG and U251 cell lines (Figure 5G,,H).H). The absence of MMP2 manifested as an obviously reducing trend of IC50 of SynB3PVGLIGPTX in both cell lines (Table 3). These results indicated that SynB3PVGLIGPTX could only inhibit cell proliferation based on the presence of MMP2, which suggested that SynB3PVGLIGPTX performs a specific inhibitory action on the proliferation of GBM cells. In addition, in order to further verify the mechanism underlying the specific inhibitory activity of SynB3PVGLIGPTX on GBM cell proliferation, a supplementary experiment was performed using SynB3PTXtreated cells as inactive control groups. The results showed that the inhibition rates of SynB3PTX without an MMP2sensitive linker (PVGLIG) in U87MG and U251 were extremely low, at a nearzero level. Based on these observations, we confirmed that SynB3PVGLIGPTX has a specific antigrowth activity, that is, only in the presence of MMP2, the sensitive linker (PVGLIG) contained in SynB3PVGLIGPTX can be specifically hydrolyzed to release PTX, thereby inhibiting the proliferation of GBM cells.

However, in the MMP2 siRNAtransfected U87MG and U251, SynB3PVGLIGPTX could result in an extremely lower inhibitory effect on cell proliferation than that treated at the same concentration ranges (25 x 10^-6, 50 x 10^-6, 75 x 10^-6, and 100 x 10^-6 M) in the U87MG and U251 cell lines (Figure 5G,,H).H). The absence of MMP2 manifested as an obviously reducing trend of IC50 of SynB3PVGLIGPTX in both cell lines (Table 3). These results indicated that SynB3PVGLIGPTX could only inhibit cell proliferation based on the presence of MMP2, which suggested that SynB3PVGLIGPTX performs a specific inhibitory action on the proliferation of GBM cells. In addition, in order to further verify the mechanism underlying the specific inhibitory activity of SynB3PVGLIGPTX on GBM cell proliferation, a supplementary experiment was performed using SynB3PTXtreated cells as inactive control groups. The results showed that the inhibition rates of SynB3PTX without an MMP2sensitive linker (PVGLIG) in U87MG and U251 were extremely low, at a nearzero level. Based on these observations, we confirmed that SynB3PVGLIGPTX has a specific antigrowth activity, that is, only in the presence of MMP2, the sensitive linker (PVGLIG) contained in SynB3PVGLIGPTX can be specifically hydrolyzed to release PTX, thereby inhibiting the proliferation of GBM cells.

However, in the MMP2 siRNAtransfected U87MG and U251, SynB3PVGLIGPTX could result in an extremely lower inhibitory effect on cell proliferation than that treated at the same concentration ranges (25 x 10^-6, 50 x 10^-6, 75 x 10^-6, and 100 x 10^-6 M) in the U87MG and U251 cell lines (Figure 5G,,H).H). The absence of MMP2 manifested as an obviously reducing trend of IC50 of SynB3PVGLIGPTX in both cell lines (Table 3). These results indicated that SynB3PVGLIGPTX could only inhibit cell proliferation based on the presence of MMP2, which suggested that SynB3PVGLIGPTX performs a specific inhibitory action on the proliferation of GBM cells. In addition, in order to further verify the mechanism underlying the specific inhibitory activity of SynB3PVGLIGPTX on GBM cell proliferation, a supplementary experiment was performed using SynB3PTXtreated cells as inactive control groups. The results showed that the inhibition rates of SynB3PTX without an MMP2sensitive linker (PVGLIG) in U87MG and U251 were extremely low, at a nearzero level. Based on these observations, we confirmed that SynB3PVGLIGPTX has a specific antigrowth activity, that is, only in the presence of MMP2, the sensitive linker (PVGLIG) contained in SynB3PVGLIGPTX can be specifically hydrolyzed to release PTX, thereby inhibiting the proliferation of GBM cells.