To test the in vivo efficacy and toxicity of BPP-PTX, an orthotopic mouse model was established. ACE-positive MDA-MB-468 cells were injected orthotopically into the mammary fat pad of female nude mice. After the inoculated tumors reached a mean volume of 100 mm³, they were randomly divided into four groups to ensure that the mean tumor volume was evaluated and treated for 28 days with PBS, plain PTX (2.4 μmol/kg, equivalent to 2.0 mg/kg), low-dose BPP-PTX (2.4 μmol/kg, equivalent to 4.9 mg/kg), and high-dose BPP-PTX (9.6 μmol/kg, equivalent to 19.6 mg/kg) every 4 days by i.p. injection. Tumor growth was assessed by measuring tumor volume every 4 days. On the fourth day of testing after the first injection, there was no significant difference in tumor volume compared with control and drug-treated groups. In the subsequent treatments, the tumors of the control mice grew significantly faster than those of mice treated with plain PTX and BPP-PTX. On day 28, mice treated with low-dose BPP-PTX showed an approximately 15% reduction in mean tumor volume compared with mice treated with plain PTX and a 54% reduction compared to controls (PBS). Meanwhile, the mean tumor volumes of mice treated with high-dose BPP-PTX were reduced by 62% compared with control animals and by approximately 30% compared to animals treated with plain PTX. Consistently, the mean tumor weight of mice with low-dose BPP-PTX treatment was 0.20 g (0.17, 0.24), which is lower than that of mice treated with plain PTX [0.26 g (0.21, 0.31)] and control mice [0.43 g (0.37, 0.49)]. The tumor weight of mice treated with high-dose BPP-PTX was significantly lower than that of the control and plain PTX-treated groups, with a mean tumor weight of only 0.16 g (0.14, 0.19). These results suggested that BPP-PTX has good tumor-suppression efficacy in vivo, even better than that of plain PTX.

To test the in vivo efficacy and toxicity of BPP-PTX, an orthotopic mouse model was established. ACE-positive MDA-MB-468 cells were injected orthotopically into the mammary fat pad of female nude mice. After the inoculated tumors reached a mean volume of 100 mm³, they were randomly divided into four groups to ensure that the mean tumor volume was evaluated and treated for 28 days with PBS, plain PTX (2.4 μmol/kg, equivalent to 2.0 mg/kg), low-dose BPP-PTX (2.4 μmol/kg, equivalent to 4.9 mg/kg), and high-dose BPP-PTX (9.6 μmol/kg, equivalent to 19.6 mg/kg) every 4 days by i.p. injection. Tumor growth was assessed by measuring tumor volume every 4 days. On the fourth day of testing after the first injection, there was no significant difference in tumor volume compared with control and drug-treated groups. In the subsequent treatments, the tumors of the control mice grew significantly faster than those of mice treated with plain PTX and BPP-PTX. On day 28, mice treated with low-dose BPP-PTX showed an approximately 15% reduction in mean tumor volume compared with mice treated with plain PTX and a 54% reduction compared to controls (PBS). Meanwhile, the mean tumor volumes of mice treated with high-dose BPP-PTX were reduced by 62% compared with control animals and by approximately 30% compared to animals treated with plain PTX. Consistently, the mean tumor weight of mice with low-dose BPP-PTX treatment was 0.20 g (0.17, 0.24), which is lower than that of mice treated with plain PTX [0.26 g (0.21, 0.31)] and control mice [0.43 g (0.37, 0.49)]. The tumor weight of mice treated with high-dose BPP-PTX was significantly lower than that of the control and plain PTX-treated groups, with a mean tumor weight of only 0.16 g (0.14, 0.19). These results suggested that BPP-PTX has good tumor-suppression efficacy in vivo, even better than that of plain PTX.

After obtaining positive in vitro results, BPP-PTX was then tested in vivo. To test the in vivo toxicity of the drug, the maximum tolerated dose (MTD) of BPP-PTX in Balb/c mice was determined. The single-dose MTD (acute toxicity) of plain PTX was 20 mg/kg (equivalent to 23.4 μmol/kg), similar to previous findings in the literature, while the single-dose MTD of BPP-PTX was 100 mg/kg (equivalent to 48.7 μmol/kg). The weight loss was more severe as the drug dose increased. However, the acute weight loss caused by high dose was temporary, with the body weight recovering to baseline levels within 15 days. Then, we evaluated the plasma pharmacokinetics profiles of plain PTX and BPP-PTX. Mice xenografted with MDA-MB-468 received intraperitoneal (i.p.) injections of 15 mg/kg PTX (equivalent to 17.6 μmol/kg) or 36.1 mg/kg BPP-PTX (equivalent to 17.6 μmol/kg). The plasma PTX concentration in the plain PTX-treated group reached peak levels at 1 h and then decreased rapidly, and it was quickly removed from the circulating system. In contrast, the plasma PTX concentration in the BPP-PTX-treated group remained high for an extended period after 1 h, suggesting that BPP-PTX could have a lower release rate in circulation.

After obtaining positive in vitro results, BPP-PTX was then tested in vivo. To test the in vivo toxicity of the drug, the maximum tolerated dose (MTD) of BPP-PTX in Balb/c mice was determined. The single-dose MTD (acute toxicity) of plain PTX was 20 mg/kg (equivalent to 23.4 μmol/kg), similar to previous findings in the literature, while the single-dose MTD of BPP-PTX was 100 mg/kg (equivalent to 48.7 μmol/kg). The weight loss was more severe as the drug dose increased. However, the acute weight loss caused by high dose was temporary, with the body weight recovering to baseline levels within 15 days. Then, we evaluated the plasma pharmacokinetics profiles of plain PTX and BPP-PTX. Mice xenografted with MDA-MB-468 received intraperitoneal (i.p.) injections of 15 mg/kg PTX (equivalent to 17.6 μmol/kg) or 36.1 mg/kg BPP-PTX (equivalent to 17.6 μmol/kg). The plasma PTX concentration in the plain PTX-treated group reached peak levels at 1 h and then decreased rapidly, and it was quickly removed from the circulating system. In contrast, the plasma PTX concentration in the BPP-PTX-treated group remained high for an extended period after 1 h, suggesting that BPP-PTX could have a lower release rate in circulation.

After obtaining positive in vitro results, BPP-PTX was then tested in vivo. To test the in vivo toxicity of the drug, the maximum tolerated dose (MTD) of BPP-PTX in Balb/c mice was determined. The single-dose MTD (acute toxicity) of plain PTX was 20 mg/kg (equivalent to 23.4 μmol/kg), similar to previous findings in the literature, while the single-dose MTD of BPP-PTX was 100 mg/kg (equivalent to 48.7 μmol/kg). The weight loss was more severe as the drug dose increased. However, the acute weight loss caused by high dose was temporary, with the body weight recovering to baseline levels within 15 days. Then, we evaluated the plasma pharmacokinetics profiles of plain PTX and BPP-PTX. Mice xenografted with MDA-MB-468 received intraperitoneal (i.p.) injections of 15 mg/kg PTX (equivalent to 17.6 μmol/kg) or 36.1 mg/kg BPP-PTX (equivalent to 17.6 μmol/kg). The plasma PTX concentration in the plain PTX-treated group reached peak levels at 1 h and then decreased rapidly, and it was quickly removed from the circulating system. In contrast, the plasma PTX concentration in the BPP-PTX-treated group remained high for an extended period after 1 h, suggesting that BPP-PTX could have a lower release rate in circulation.

After obtaining positive in vitro results, BPP-PTX was then tested in vivo. To test the in vivo toxicity of the drug, the maximum tolerated dose (MTD) of BPP-PTX in Balb/c mice was determined. The single-dose MTD (acute toxicity) of plain PTX was 20 mg/kg (equivalent to 23.4 μmol/kg), similar to previous findings in the literature, while the single-dose MTD of BPP-PTX was 100 mg/kg (equivalent to 48.7 μmol/kg). The weight loss was more severe as the drug dose increased. However, the acute weight loss caused by high dose was temporary, with the body weight recovering to baseline levels within 15 days. Then, we evaluated the plasma pharmacokinetics profiles of plain PTX and BPP-PTX. Mice xenografted with MDA-MB-468 received intraperitoneal (i.p.) injections of 15 mg/kg PTX (equivalent to 17.6 μmol/kg) or 36.1 mg/kg BPP-PTX (equivalent to 17.6 μmol/kg). The plasma PTX concentration in the plain PTX-treated group reached peak levels at 1 h and then decreased rapidly, and it was quickly removed from the circulating system. In contrast, the plasma PTX concentration in the BPP-PTX-treated group remained high for an extended period after 1 h, suggesting that BPP-PTX could have a lower release rate in circulation.

After obtaining positive in vitro results, BPP-PTX was then tested in vivo. To test the in vivo toxicity of the drug, the maximum tolerated dose (MTD) of BPP-PTX in Balb/c mice was determined. The single-dose MTD (acute toxicity) of plain PTX was 20 mg/kg (equivalent to 23.4 μmol/kg), similar to previous findings in the literature, while the single-dose MTD of BPP-PTX was 100 mg/kg (equivalent to 48.7 μmol/kg). The weight loss was more severe as the drug dose increased. However, the acute weight loss caused by high dose was temporary, with the body weight recovering to baseline levels within 15 days. Then, we evaluated the plasma pharmacokinetics profiles of plain PTX and BPP-PTX. Mice xenografted with MDA-MB-468 received intraperitoneal (i.p.) injections of 15 mg/kg PTX (equivalent to 17.6 μmol/kg) or 36.1 mg/kg BPP-PTX (equivalent to 17.6 μmol/kg). The plasma PTX concentration in the plain PTX-treated group reached peak levels at 1 h and then decreased rapidly, and it was quickly removed from the circulating system. In contrast, the plasma PTX concentration in the BPP-PTX-treated group remained high for an extended period after 1 h, suggesting that BPP-PTX could have a lower release rate in circulation.

After obtaining positive in vitro results, BPP-PTX was then tested in vivo. To test the in vivo toxicity of the drug, the maximum tolerated dose (MTD) of BPP-PTX in Balb/c mice was determined. The single-dose MTD (acute toxicity) of plain PTX was 20 mg/kg (equivalent to 23.4 μmol/kg), similar to previous findings in the literature, while the single-dose MTD of BPP-PTX was 100 mg/kg (equivalent to 48.7 μmol/kg). The weight loss was more severe as the drug dose increased. However, the acute weight loss caused by high dose was temporary, with the body weight recovering to baseline levels within 15 days. Then, we evaluated the plasma pharmacokinetics profiles of plain PTX and BPP-PTX. Mice xenografted with MDA-MB-468 received intraperitoneal (i.p.) injections of 15 mg/kg PTX (equivalent to 17.6 μmol/kg) or 36.1 mg/kg BPP-PTX (equivalent to 17.6 μmol/kg). The plasma PTX concentration in the plain PTX-treated group reached peak levels at 1 h and then decreased rapidly, and it was quickly removed from the circulating system. In contrast, the plasma PTX concentration in the BPP-PTX-treated group remained high for an extended period after 1 h, suggesting that BPP-PTX could have a lower release rate in circulation.

After obtaining positive in vitro results, BPP-PTX was then tested in vivo. To test the in vivo toxicity of the drug, the maximum tolerated dose (MTD) of BPP-PTX in Balb/c mice was determined. The single-dose MTD (acute toxicity) of plain PTX was 20 mg/kg (equivalent to 23.4 μmol/kg), similar to previous findings in the literature, while the single-dose MTD of BPP-PTX was 100 mg/kg (equivalent to 48.7 μmol/kg). The weight loss was more severe as the drug dose increased. However, the acute weight loss caused by high dose was temporary, with the body weight recovering to baseline levels within 15 days. Then, we evaluated the plasma pharmacokinetics profiles of plain PTX and BPP-PTX. Mice xenografted with MDA-MB-468 received intraperitoneal (i.p.) injections of 15 mg/kg PTX (equivalent to 17.6 μmol/kg) or 36.1 mg/kg BPP-PTX (equivalent to 17.6 μmol/kg). The plasma PTX concentration in the plain PTX-treated group reached peak levels at 1 h and then decreased rapidly, and it was quickly removed from the circulating system. In contrast, the plasma PTX concentration in the BPP-PTX-treated group remained high for an extended period after 1 h, suggesting that BPP-PTX could have a lower release rate in circulation.

To evaluate the in vitro antitumor activity, BPP-PTX, uncoupled peptide (BPP), and PTX were under cytotoxicity evaluation by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazole (MTT) assay in ACE-positive TNBC cell lines (MDA-MB-231 and MDA-MB-468) and ACE-negative cell lines (HEK293T). The free BPP peptide did not exhibit any cytotoxicity in all cell lines. In contrast, the IC50 value of BPP-PTX in ACE-negative HEK293T was 616.1 nM [95% CI, (242.7, 1564.0)], which was much higher than that of PTX in the same cell line {6.7 nM [95% CI, (5.3, 8.4)]}. Interestingly, the cytotoxicity of BPP-PTX was comparable with that of PTX in ACE-positive TNBC cell lines. In MDA-MB-231, the IC50 value of BPP-PTX was 9.5 nM [95% CI, (7.0, 12.8)], and that of PTX was 3.1 nM [95% CI, (2.8, 3.5)]. In MDA-MB-468, the IC50 value of BPP-PTX was 12.3 nM [95% CI, (6.8, 22.3)], and that of PTX was 3.0 nM [95% CI, (2.0, 4.7)].

To evaluate the in vitro antitumor activity, BPP-PTX, uncoupled peptide (BPP), and PTX were under cytotoxicity evaluation by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazole (MTT) assay in ACE-positive TNBC cell lines (MDA-MB-231 and MDA-MB-468) and ACE-negative cell lines (HEK293T). The free BPP peptide did not exhibit any cytotoxicity in all cell lines. In contrast, the IC50 value of BPP-PTX in ACE-negative HEK293T was 616.1 nM [95% CI, (242.7, 1564.0)], which was much higher than that of PTX in the same cell line {6.7 nM [95% CI, (5.3, 8.4)]}. Interestingly, the cytotoxicity of BPP-PTX was comparable with that of PTX in ACE-positive TNBC cell lines. In MDA-MB-231, the IC50 value of BPP-PTX was 9.5 nM [95% CI, (7.0, 12.8)], and that of PTX was 3.1 nM [95% CI, (2.8, 3.5)]. In MDA-MB-468, the IC50 value of BPP-PTX was 12.3 nM [95% CI, (6.8, 22.3)], and that of PTX was 3.0 nM [95% CI, (2.0, 4.7)].

To evaluate the in vitro antitumor activity, BPP-PTX, uncoupled peptide (BPP), and PTX were under cytotoxicity evaluation by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazole (MTT) assay in ACE-positive TNBC cell lines (MDA-MB-231 and MDA-MB-468) and ACE-negative cell lines (HEK293T). The free BPP peptide did not exhibit any cytotoxicity in all cell lines. In contrast, the IC50 value of BPP-PTX in ACE-negative HEK293T was 616.1 nM [95% CI, (242.7, 1564.0)], which was much higher than that of PTX in the same cell line {6.7 nM [95% CI, (5.3, 8.4)]}. Interestingly, the cytotoxicity of BPP-PTX was comparable with that of PTX in ACE-positive TNBC cell lines. In MDA-MB-231, the IC50 value of BPP-PTX was 9.5 nM [95% CI, (7.0, 12.8)], and that of PTX was 3.1 nM [95% CI, (2.8, 3.5)]. In MDA-MB-468, the IC50 value of BPP-PTX was 12.3 nM [95% CI, (6.8, 22.3)], and that of PTX was 3.0 nM [95% CI, (2.0, 4.7)].