It was demonstrated here, RCCD@NPs and RSSD@NPs exhibited significantly better tumor-growth inhibition compared with that of the free DM1, QCCD@NPs or QSSD@NPs (Figure (Figure4A).4A). RSSD@NPs showed the most suppression on B16 tumor up to 25 days, and resulted in a tumor volume 4 times smaller than the saline group at the end of the experiment. The final tumor volumes in various nano-DDS groups ranked from the greatest to the least: QCCD@NPs, QSSD@NPs>DM1>RCCD@NPs>RSSD@NPs. Almost the same trends were found in terms of tumor weight and tumor size (Figures (Figures4B,4B, C).

Remarkably, leucopenia and myelosuppression did not occur during the use of PDC. In addition, the values of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP), as the biomarkers of hepatic function, were all in the normal range in comparison to the saline group (Figure 5F-H). Meanwhile, normal renal function was also identified after treatment by monitoring CREA, UA, and BUN (Figure 5I-K).

It was demonstrated here, RCCD@NPs and RSSD@NPs exhibited significantly better tumor-growth inhibition compared with that of the free DM1, QCCD@NPs or QSSD@NPs (Figure (Figure4A).4A). RSSD@NPs showed the most suppression on B16 tumor up to 25 days, and resulted in a tumor volume 4 times smaller than the saline group at the end of the experiment. The final tumor volumes in various nano-DDS groups ranked from the greatest to the least: QCCD@NPs, QSSD@NPs>DM1>RCCD@NPs>RSSD@NPs. Almost the same trends were found in terms of tumor weight and tumor size (Figures (Figures4B,4B, C).

Remarkably, leucopenia and myelosuppression did not occur during the use of PDC. In addition, the values of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP), as the biomarkers of hepatic function, were all in the normal range in comparison to the saline group (Figure 5F-H). Meanwhile, normal renal function was also identified after treatment by monitoring CREA, UA, and BUN (Figure 5I-K).

Remarkably, leucopenia and myelosuppression did not occur during the use of PDC. In addition, the values of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP), as the biomarkers of hepatic function, were all in the normal range in comparison to the saline group (Figure 5F-H). Meanwhile, normal renal function was also identified after treatment by monitoring CREA, UA, and BUN (Figure 5I-K).

Remarkably, leucopenia and myelosuppression did not occur during the use of PDC. In addition, the values of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP), as the biomarkers of hepatic function, were all in the normal range in comparison to the saline group (Figure 5F-H). Meanwhile, normal renal function was also identified after treatment by monitoring CREA, UA, and BUN (Figure 5I-K).

Remarkably, leucopenia and myelosuppression did not occur during the use of PDC. In addition, the values of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP), as the biomarkers of hepatic function, were all in the normal range in comparison to the saline group (Figure 5F-H). Meanwhile, normal renal function was also identified after treatment by monitoring CREA, UA, and BUN (Figure 5I-K).

The results showed that the cytotoxicity of both conjugates 1 and 2 (IC50 = 1.3 and 2.2 uM, respectively), as well as the free Dox (IC50 = 1.5 uM) on MDA-MB-231 breast cancer cell line, were in the low micromolar range (Figure 4). For the breast cancer cell line MDA-MB-468, the free Dox (IC50 = 0.35 uM) was slightly more toxic compared to conjugates 1 (4.7 uM) and 2 (1.2 uM). For the non-cancerous cell line MCF 10A, the free Dox was highly toxic (IC50 = 0.24 uM) whereas conjugates 1 and 2 displayed much-reduced toxicity (IC50 = 38.6 and 15.1 uM, respectively).

Several PDCs have been prepared with peptide 18-4 and doxorubicin (Dox) using different linker chemistries such as ester, amide, succinimidyl thioether, or acyl hydrazone. In vitro results showed that these PDCs were highly specific toward breast cancer cells. PDCs displayed similar toxicity as free Dox toward the breast cancer cells and several-fold (7-40 times) less toxicity toward the non-cancerous cells such as MCF-10A and human umbilical vein endothelial cells (HUVECs). A peptide 18-4-Dox conjugate with amide/succinimidyl thioether linkage showed high selective toxicity toward triple negative breast cancer (TNBC) cell lines, i.e., MDA-MB-231 cells (IC50 1.3 ± 0.2 uM) and MDA-MB-468 cells (IC50 4.7 ± 0.3 uM) compared to the normal breast MCF10A cells (IC50 38.6 ± 1.1 uM). The linkage between the drug and the peptide was stable as the degradation half-life of peptide 18-4-Dox conjugate in the presence of human serum was found to be ˜18 h. Herein, we describe the first in vivo evidence for improved efficacy of this PDC targeting K1 receptor in an orthotopic TNBC mouse model. We also show a higher accumulation of PDC in TNBC tumors in mice, in accord with K1 overexpression in tumor over non-tumor tissues in MDA-MB-231 xenografted mice.

The results showed that the cytotoxicity of both conjugates 1 and 2 (IC50 = 1.3 and 2.2 uM, respectively), as well as the free Dox (IC50 = 1.5 uM) on MDA-MB-231 breast cancer cell line, were in the low micromolar range (Figure 4). For the breast cancer cell line MDA-MB-468, the free Dox (IC50 = 0.35 uM) was slightly more toxic compared to conjugates 1 (4.7 uM) and 2 (1.2 uM). For the non-cancerous cell line MCF 10A, the free Dox was highly toxic (IC50 = 0.24 uM) whereas conjugates 1 and 2 displayed much-reduced toxicity (IC50 = 38.6 and 15.1 uM, respectively).

Several PDCs have been prepared with peptide 18-4 and doxorubicin (Dox) using different linker chemistries such as ester, amide, succinimidyl thioether, or acyl hydrazone. In vitro results showed that these PDCs were highly specific toward breast cancer cells. PDCs displayed similar toxicity as free Dox toward the breast cancer cells and several-fold (7-40 times) less toxicity toward the non-cancerous cells such as MCF-10A and human umbilical vein endothelial cells (HUVECs). A peptide 18-4-Dox conjugate with amide/succinimidyl thioether linkage showed high selective toxicity toward triple negative breast cancer (TNBC) cell lines, i.e., MDA-MB-231 cells (IC50 1.3 ± 0.2 uM) and MDA-MB-468 cells (IC50 4.7 ± 0.3 uM) compared to the normal breast MCF10A cells (IC50 38.6 ± 1.1 uM). The linkage between the drug and the peptide was stable as the degradation half-life of peptide 18-4-Dox conjugate in the presence of human serum was found to be ˜18 h. Herein, we describe the first in vivo evidence for improved efficacy of this PDC targeting K1 receptor in an orthotopic TNBC mouse model. We also show a higher accumulation of PDC in TNBC tumors in mice, in accord with K1 overexpression in tumor over non-tumor tissues in MDA-MB-231 xenografted mice.

The results showed that the cytotoxicity of both conjugates 1 and 2 (IC50 = 1.3 and 2.2 uM, respectively), as well as the free Dox (IC50 = 1.5 uM) on MDA-MB-231 breast cancer cell line, were in the low micromolar range (Figure 4). For the breast cancer cell line MDA-MB-468, the free Dox (IC50 = 0.35 uM) was slightly more toxic compared to conjugates 1 (4.7 uM) and 2 (1.2 uM). For the non-cancerous cell line MCF 10A, the free Dox was highly toxic (IC50 = 0.24 uM) whereas conjugates 1 and 2 displayed much-reduced toxicity (IC50 = 38.6 and 15.1 uM, respectively).

Several PDCs have been prepared with peptide 18-4 and doxorubicin (Dox) using different linker chemistries such as ester, amide, succinimidyl thioether, or acyl hydrazone. In vitro results showed that these PDCs were highly specific toward breast cancer cells. PDCs displayed similar toxicity as free Dox toward the breast cancer cells and several-fold (7-40 times) less toxicity toward the non-cancerous cells such as MCF-10A and human umbilical vein endothelial cells (HUVECs). A peptide 18-4-Dox conjugate with amide/succinimidyl thioether linkage showed high selective toxicity toward triple negative breast cancer (TNBC) cell lines, i.e., MDA-MB-231 cells (IC50 1.3 ± 0.2 uM) and MDA-MB-468 cells (IC50 4.7 ± 0.3 uM) compared to the normal breast MCF10A cells (IC50 38.6 ± 1.1 uM). The linkage between the drug and the peptide was stable as the degradation half-life of peptide 18-4-Dox conjugate in the presence of human serum was found to be ˜18 h. Herein, we describe the first in vivo evidence for improved efficacy of this PDC targeting K1 receptor in an orthotopic TNBC mouse model. We also show a higher accumulation of PDC in TNBC tumors in mice, in accord with K1 overexpression in tumor over non-tumor tissues in MDA-MB-231 xenografted mice.

Analysis of dot-plots suggests that the rL-A9 peptide alone does not exert any significant cytotoxic effects on either HER2-positive, SKOV3 cells or HER2-negative, MDA-MB-231 cells at any of the investigated concentrations. In contrast, incubation of drug DOX with either of the cells resulted in enhanced cell death with a negligible viable population even at lower concentrations. However, it was observed that the peptide-drug conjugate, rL-A9-DOX had a concentration-dependent impact on cell death (SKOV3 cells). Notably, nearly half of the cell population died at 3.75 uM. At a concentration of 15 uM, there were only 5% viable SKOV3 cells. The higher percent of the viable cell population was observed at corresponding concentrations in the case of incubation of rL-A9-DOX with HER2-negative, MDA-MB-231 cells. Comparative data of viability in two different cell lines is presented in Figure 6 for the peptide-drug conjugate, rL-A9-DOX.

Analysis of dot-plots suggests that the rL-A9 peptide alone does not exert any significant cytotoxic effects on either HER2-positive, SKOV3 cells or HER2-negative, MDA-MB-231 cells at any of the investigated concentrations. In contrast, incubation of drug DOX with either of the cells resulted in enhanced cell death with a negligible viable population even at lower concentrations. However, it was observed that the peptide-drug conjugate, rL-A9-DOX had a concentration-dependent impact on cell death (SKOV3 cells). Notably, nearly half of the cell population died at 3.75 uM. At a concentration of 15 uM, there were only 5% viable SKOV3 cells. The higher percent of the viable cell population was observed at corresponding concentrations in the case of incubation of rL-A9-DOX with HER2-negative, MDA-MB-231 cells. Comparative data of viability in two different cell lines is presented in Figure 6 for the peptide-drug conjugate, rL-A9-DOX.

Analysis of dot-plots suggests that the rL-A9 peptide alone does not exert any significant cytotoxic effects on either HER2-positive, SKOV3 cells or HER2-negative, MDA-MB-231 cells at any of the investigated concentrations. In contrast, incubation of drug DOX with either of the cells resulted in enhanced cell death with a negligible viable population even at lower concentrations. However, it was observed that the peptide-drug conjugate, rL-A9-DOX had a concentration-dependent impact on cell death (SKOV3 cells). Notably, nearly half of the cell population died at 3.75 uM. At a concentration of 15 uM, there were only 5% viable SKOV3 cells. The higher percent of the viable cell population was observed at corresponding concentrations in the case of incubation of rL-A9-DOX with HER2-negative, MDA-MB-231 cells. Comparative data of viability in two different cell lines is presented in Figure 6 for the peptide-drug conjugate, rL-A9-DOX.

Analysis of dot-plots suggests that the rL-A9 peptide alone does not exert any significant cytotoxic effects on either HER2-positive, SKOV3 cells or HER2-negative, MDA-MB-231 cells at any of the investigated concentrations. In contrast, incubation of drug DOX with either of the cells resulted in enhanced cell death with a negligible viable population even at lower concentrations. However, it was observed that the peptide-drug conjugate, rL-A9-DOX had a concentration-dependent impact on cell death (SKOV3 cells). Notably, nearly half of the cell population died at 3.75 uM. At a concentration of 15 uM, there were only 5% viable SKOV3 cells. The higher percent of the viable cell population was observed at corresponding concentrations in the case of incubation of rL-A9-DOX with HER2-negative, MDA-MB-231 cells. Comparative data of viability in two different cell lines is presented in Figure 6 for the peptide-drug conjugate, rL-A9-DOX.

Analysis of dot-plots suggests that the rL-A9 peptide alone does not exert any significant cytotoxic effects on either HER2-positive, SKOV3 cells or HER2-negative, MDA-MB-231 cells at any of the investigated concentrations. In contrast, incubation of drug DOX with either of the cells resulted in enhanced cell death with a negligible viable population even at lower concentrations. However, it was observed that the peptide-drug conjugate, rL-A9-DOX had a concentration-dependent impact on cell death (SKOV3 cells). Notably, nearly half of the cell population died at 3.75 uM. At a concentration of 15 uM, there were only 5% viable SKOV3 cells. The higher percent of the viable cell population was observed at corresponding concentrations in the case of incubation of rL-A9-DOX with HER2-negative, MDA-MB-231 cells. Comparative data of viability in two different cell lines is presented in Figure 6 for the peptide-drug conjugate, rL-A9-DOX.

Analysis of dot-plots suggests that the rL-A9 peptide alone does not exert any significant cytotoxic effects on either HER2-positive, SKOV3 cells or HER2-negative, MDA-MB-231 cells at any of the investigated concentrations. In contrast, incubation of drug DOX with either of the cells resulted in enhanced cell death with a negligible viable population even at lower concentrations. However, it was observed that the peptide-drug conjugate, rL-A9-DOX had a concentration-dependent impact on cell death (SKOV3 cells). Notably, nearly half of the cell population died at 3.75 uM. At a concentration of 15 uM, there were only 5% viable SKOV3 cells. The higher percent of the viable cell population was observed at corresponding concentrations in the case of incubation of rL-A9-DOX with HER2-negative, MDA-MB-231 cells. Comparative data of viability in two different cell lines is presented in Figure 6 for the peptide-drug conjugate, rL-A9-DOX.

Analysis of dot-plots suggests that the rL-A9 peptide alone does not exert any significant cytotoxic effects on either HER2-positive, SKOV3 cells or HER2-negative, MDA-MB-231 cells at any of the investigated concentrations. In contrast, incubation of drug DOX with either of the cells resulted in enhanced cell death with a negligible viable population even at lower concentrations. However, it was observed that the peptide-drug conjugate, rL-A9-DOX had a concentration-dependent impact on cell death (SKOV3 cells). Notably, nearly half of the cell population died at 3.75 uM. At a concentration of 15 uM, there were only 5% viable SKOV3 cells. The higher percent of the viable cell population was observed at corresponding concentrations in the case of incubation of rL-A9-DOX with HER2-negative, MDA-MB-231 cells. Comparative data of viability in two different cell lines is presented in Figure 6 for the peptide-drug conjugate, rL-A9-DOX.

Analysis of dot-plots suggests that the rL-A9 peptide alone does not exert any significant cytotoxic effects on either HER2-positive, SKOV3 cells or HER2-negative, MDA-MB-231 cells at any of the investigated concentrations. In contrast, incubation of drug DOX with either of the cells resulted in enhanced cell death with a negligible viable population even at lower concentrations. However, it was observed that the peptide-drug conjugate, rL-A9-DOX had a concentration-dependent impact on cell death (SKOV3 cells). Notably, nearly half of the cell population died at 3.75 uM. At a concentration of 15 uM, there were only 5% viable SKOV3 cells. The higher percent of the viable cell population was observed at corresponding concentrations in the case of incubation of rL-A9-DOX with HER2-negative, MDA-MB-231 cells. Comparative data of viability in two different cell lines is presented in Figure 6 for the peptide-drug conjugate, rL-A9-DOX.

Analysis of dot-plots suggests that the rL-A9 peptide alone does not exert any significant cytotoxic effects on either HER2-positive, SKOV3 cells or HER2-negative, MDA-MB-231 cells at any of the investigated concentrations. In contrast, incubation of drug DOX with either of the cells resulted in enhanced cell death with a negligible viable population even at lower concentrations. However, it was observed that the peptide-drug conjugate, rL-A9-DOX had a concentration-dependent impact on cell death (SKOV3 cells). Notably, nearly half of the cell population died at 3.75 uM. At a concentration of 15 uM, there were only 5% viable SKOV3 cells. The higher percent of the viable cell population was observed at corresponding concentrations in the case of incubation of rL-A9-DOX with HER2-negative, MDA-MB-231 cells. Comparative data of viability in two different cell lines is presented in Figure 6 for the peptide-drug conjugate, rL-A9-DOX.

Analysis of dot-plots suggests that the rL-A9 peptide alone does not exert any significant cytotoxic effects on either HER2-positive, SKOV3 cells or HER2-negative, MDA-MB-231 cells at any of the investigated concentrations. In contrast, incubation of drug DOX with either of the cells resulted in enhanced cell death with a negligible viable population even at lower concentrations. However, it was observed that the peptide-drug conjugate, rL-A9-DOX had a concentration-dependent impact on cell death (SKOV3 cells). Notably, nearly half of the cell population died at 3.75 uM. At a concentration of 15 uM, there were only 5% viable SKOV3 cells. The higher percent of the viable cell population was observed at corresponding concentrations in the case of incubation of rL-A9-DOX with HER2-negative, MDA-MB-231 cells. Comparative data of viability in two different cell lines is presented in Figure 6 for the peptide-drug conjugate, rL-A9-DOX.

Analysis of dot-plots suggests that the rL-A9 peptide alone does not exert any significant cytotoxic effects on either HER2-positive, SKOV3 cells or HER2-negative, MDA-MB-231 cells at any of the investigated concentrations. In contrast, incubation of drug DOX with either of the cells resulted in enhanced cell death with a negligible viable population even at lower concentrations. However, it was observed that the peptide-drug conjugate, rL-A9-DOX had a concentration-dependent impact on cell death (SKOV3 cells). Notably, nearly half of the cell population died at 3.75 uM. At a concentration of 15 uM, there were only 5% viable SKOV3 cells. The higher percent of the viable cell population was observed at corresponding concentrations in the case of incubation of rL-A9-DOX with HER2-negative, MDA-MB-231 cells. Comparative data of viability in two different cell lines is presented in Figure 6 for the peptide-drug conjugate, rL-A9-DOX.

Analysis of dot-plots suggests that the rL-A9 peptide alone does not exert any significant cytotoxic effects on either HER2-positive, SKOV3 cells or HER2-negative, MDA-MB-231 cells at any of the investigated concentrations. In contrast, incubation of drug DOX with either of the cells resulted in enhanced cell death with a negligible viable population even at lower concentrations. However, it was observed that the peptide-drug conjugate, rL-A9-DOX had a concentration-dependent impact on cell death (SKOV3 cells). Notably, nearly half of the cell population died at 3.75 uM. At a concentration of 15 uM, there were only 5% viable SKOV3 cells. The higher percent of the viable cell population was observed at corresponding concentrations in the case of incubation of rL-A9-DOX with HER2-negative, MDA-MB-231 cells. Comparative data of viability in two different cell lines is presented in Figure 6 for the peptide-drug conjugate, rL-A9-DOX.