In this context, we developed a drug-bearing supramolecular hydrogel system to intratumourally (i.t.) deliver CDNs against malignant tumours to achieve cancer chemoimmunotherapy. Our strategy was to chemically conjugate the hydrophilic pep-tide moiety iRGD (a tumour-penetrating peptide that can bind to neuropilin-1 (NRP-1) and trigger tumour tissue penetration) to the hydrophobic anticancer drug CPT to form a self-assembling and self-formulating peptide-drug conjugate (diCPT-iRGD). In aqueous solution, the designed drug amphiphile spontaneously assembles into supramolecular nanotubes (NTs). The negatively charged STING agonist (c-di-AMP (CDA)) can condense on the surface of these positively charged NTs through electrostatic complexations. After injection into the tumour site, the CDA-NT solution can immediately form a hydrogel, functioning as a local reservoir for extended localized release of CDA and CPT to awaken both the innate and adaptive immune systems.

In this context, we developed a drug-bearing supramolecular hydrogel system to intratumourally (i.t.) deliver CDNs against malignant tumours to achieve cancer chemoimmunotherapy. Our strategy was to chemically conjugate the hydrophilic pep-tide moiety iRGD (a tumour-penetrating peptide that can bind to neuropilin-1 (NRP-1) and trigger tumour tissue penetration) to the hydrophobic anticancer drug CPT to form a self-assembling and self-formulating peptide-drug conjugate (diCPT-iRGD). In aqueous solution, the designed drug amphiphile spontaneously assembles into supramolecular nanotubes (NTs). The negatively charged STING agonist (c-di-AMP (CDA)) can condense on the surface of these positively charged NTs through electrostatic complexations. After injection into the tumour site, the CDA-NT solution can immediately form a hydrogel, functioning as a local reservoir for extended localized release of CDA and CPT to awaken both the innate and adaptive immune systems.

To enhance the affinity of the human epidermal growth receptor 2 (HER2) targeted peptide developed previously, bispecific fusion peptidesP1GCGT1andP1GCGCGT1were designed using anin silicoapproach. Molecular dynamic simulation showed that both peptides strongly interacted with HER2 domains II and IV. Compared with peptides targeting each single domain,P1GCGT1andP1GCGCGT1could bind to HER2 more significantly and targeted HER2-positive cells specifically. Additionally, both peptides were used to generate peptide-drug conjugates with camptothecin (CPT), among whichI-1andI-4were screened for enhanced cellular activity and selectivity. Biological evaluation demonstrated thatI-1andI-4induced cell apoptosis, promoted cell cycle arrestin S-phase, and inhibited Topo I activity. The binding affinity assay and confocal analysis revealed thatI-1andI-4were effective at targeting HER2. Moreover,I-1andI-4showed better stability than single targeting peptide and presented enhanced antitumor activity and safety than CPT in tumor-bearing mice.

To enhance the affinity of the human epidermal growth receptor 2 (HER2) targeted peptide developed previously, bispecific fusion peptidesP1GCGT1andP1GCGCGT1were designed using anin silicoapproach. Molecular dynamic simulation showed that both peptides strongly interacted with HER2 domains II and IV. Compared with peptides targeting each single domain,P1GCGT1andP1GCGCGT1could bind to HER2 more significantly and targeted HER2-positive cells specifically. Additionally, both peptides were used to generate peptide-drug conjugates with camptothecin (CPT), among whichI-1andI-4were screened for enhanced cellular activity and selectivity. Biological evaluation demonstrated thatI-1andI-4induced cell apoptosis, promoted cell cycle arrestin S-phase, and inhibited Topo I activity. The binding affinity assay and confocal analysis revealed thatI-1andI-4were effective at targeting HER2. Moreover,I-1andI-4showed better stability than single targeting peptide and presented enhanced antitumor activity and safety than CPT in tumor-bearing mice.

To enhance the affinity of the human epidermal growth receptor 2 (HER2) targeted peptide developed previously, bispecific fusion peptidesP1GCGT1andP1GCGCGT1were designed using anin silicoapproach. Molecular dynamic simulation showed that both peptides strongly interacted with HER2 domains II and IV. Compared with peptides targeting each single domain,P1GCGT1andP1GCGCGT1could bind to HER2 more significantly and targeted HER2-positive cells specifically. Additionally, both peptides were used to generate peptide-drug conjugates with camptothecin (CPT), among whichI-1andI-4were screened for enhanced cellular activity and selectivity. Biological evaluation demonstrated thatI-1andI-4induced cell apoptosis, promoted cell cycle arrestin S-phase, and inhibited Topo I activity. The binding affinity assay and confocal analysis revealed thatI-1andI-4were effective at targeting HER2. Moreover,I-1andI-4showed better stability than single targeting peptide and presented enhanced antitumor activity and safety than CPT in tumor-bearing mice.

To enhance the affinity of the human epidermal growth receptor 2 (HER2) targeted peptide developed previously, bispecific fusion peptidesP1GCGT1andP1GCGCGT1were designed using anin silicoapproach. Molecular dynamic simulation showed that both peptides strongly interacted with HER2 domains II and IV. Compared with peptides targeting each single domain,P1GCGT1andP1GCGCGT1could bind to HER2 more significantly and targeted HER2-positive cells specifically. Additionally, both peptides were used to generate peptide-drug conjugates with camptothecin (CPT), among whichI-1andI-4were screened for enhanced cellular activity and selectivity. Biological evaluation demonstrated thatI-1andI-4induced cell apoptosis, promoted cell cycle arrestin S-phase, and inhibited Topo I activity. The binding affinity assay and confocal analysis revealed thatI-1andI-4were effective at targeting HER2. Moreover,I-1andI-4showed better stability than single targeting peptide and presented enhanced antitumor activity and safety than CPT in tumor-bearing mice.

To enhance the affinity of the human epidermal growth receptor 2 (HER2) targeted peptide developed previously, bispecific fusion peptidesP1GCGT1andP1GCGCGT1were designed using anin silicoapproach. Molecular dynamic simulation showed that both peptides strongly interacted with HER2 domains II and IV. Compared with peptides targeting each single domain,P1GCGT1andP1GCGCGT1could bind to HER2 more significantly and targeted HER2-positive cells specifically. Additionally, both peptides were used to generate peptide-drug conjugates with camptothecin (CPT), among whichI-1andI-4were screened for enhanced cellular activity and selectivity. Biological evaluation demonstrated thatI-1andI-4induced cell apoptosis, promoted cell cycle arrestin S-phase, and inhibited Topo I activity. The binding affinity assay and confocal analysis revealed thatI-1andI-4were effective at targeting HER2. Moreover,I-1andI-4showed better stability than single targeting peptide and presented enhanced antitumor activity and safety than CPT in tumor-bearing mice.

In this study, a series of conjugates were designed and synthesized by fusing the SMAC peptide and P1 target peptide, and coupling with CPT, with the hope that they can play a synergistic role in promoting cell apoptosis. The in vitro anti-tumor activity study found that the better conjugate 4 had the best activity on HER2-positive cells, while it was less toxic to HER2-negative cells. From the experimental results, the coupling position of CPT affected the activity of the conjugates. Conjugates 1, 4 and 7, which connected the load in the same way, had different inhibitory activities on three HER2-positive cells, and among them, conjugate 4 showed the best activity. We speculated it is related to the releasing efficiency of free SMAC peptide and CPT. The subsequent biological activity evaluations of conjugate 4 confirmed that the introduction of the SMAC peptide sequence can enhance the activity of Caspase 3 and indeed play a role in promoting apoptosis. Conjugate 4 reduced the expression of XIAP and cIAP1 in SK-BR-3 cells in a concentration dependent manner. The in vivo activity study also fully proved that the antitumor activity as well as safety of conjugate 4 was superior to CPT. The pro-apoptotic effect of conjugate 4 is significantly superior to the C4 group without fused SMAC peptide and the C4+SMAC combination drug group. This indicates that that the introduced SMAC peptide exerts a synergistic anti-tumor effect. When CPT is engineered into PDCs molecules, its in vitro activity related to CPT can be retained but is somewhat diminished. This attenuation may be attributed to the fact that PDCs must undergo cellular internalization and release of CPT or its derivatives in the intracellular environment to exert anti-tumor activity.

In this study, a series of conjugates were designed and synthesized by fusing the SMAC peptide and P1 target peptide, and coupling with CPT, with the hope that they can play a synergistic role in promoting cell apoptosis. The in vitro anti-tumor activity study found that the better conjugate 4 had the best activity on HER2-positive cells, while it was less toxic to HER2-negative cells. From the experimental results, the coupling position of CPT affected the activity of the conjugates. Conjugates 1, 4 and 7, which connected the load in the same way, had different inhibitory activities on three HER2-positive cells, and among them, conjugate 4 showed the best activity. We speculated it is related to the releasing efficiency of free SMAC peptide and CPT. The subsequent biological activity evaluations of conjugate 4 confirmed that the introduction of the SMAC peptide sequence can enhance the activity of Caspase 3 and indeed play a role in promoting apoptosis. Conjugate 4 reduced the expression of XIAP and cIAP1 in SK-BR-3 cells in a concentration dependent manner. The in vivo activity study also fully proved that the antitumor activity as well as safety of conjugate 4 was superior to CPT. The pro-apoptotic effect of conjugate 4 is significantly superior to the C4 group without fused SMAC peptide and the C4+SMAC combination drug group. This indicates that that the introduced SMAC peptide exerts a synergistic anti-tumor effect. When CPT is engineered into PDCs molecules, its in vitro activity related to CPT can be retained but is somewhat diminished. This attenuation may be attributed to the fact that PDCs must undergo cellular internalization and release of CPT or its derivatives in the intracellular environment to exert anti-tumor activity.

In this study, a series of conjugates were designed and synthesized by fusing the SMAC peptide and P1 target peptide, and coupling with CPT, with the hope that they can play a synergistic role in promoting cell apoptosis. The in vitro anti-tumor activity study found that the better conjugate 4 had the best activity on HER2-positive cells, while it was less toxic to HER2-negative cells. From the experimental results, the coupling position of CPT affected the activity of the conjugates. Conjugates 1, 4 and 7, which connected the load in the same way, had different inhibitory activities on three HER2-positive cells, and among them, conjugate 4 showed the best activity. We speculated it is related to the releasing efficiency of free SMAC peptide and CPT. The subsequent biological activity evaluations of conjugate 4 confirmed that the introduction of the SMAC peptide sequence can enhance the activity of Caspase 3 and indeed play a role in promoting apoptosis. Conjugate 4 reduced the expression of XIAP and cIAP1 in SK-BR-3 cells in a concentration dependent manner. The in vivo activity study also fully proved that the antitumor activity as well as safety of conjugate 4 was superior to CPT. The pro-apoptotic effect of conjugate 4 is significantly superior to the C4 group without fused SMAC peptide and the C4+SMAC combination drug group. This indicates that that the introduced SMAC peptide exerts a synergistic anti-tumor effect. When CPT is engineered into PDCs molecules, its in vitro activity related to CPT can be retained but is somewhat diminished. This attenuation may be attributed to the fact that PDCs must undergo cellular internalization and release of CPT or its derivatives in the intracellular environment to exert anti-tumor activity.

In this study, a series of conjugates were designed and synthesized by fusing the SMAC peptide and P1 target peptide, and coupling with CPT, with the hope that they can play a synergistic role in promoting cell apoptosis. The in vitro anti-tumor activity study found that the better conjugate 4 had the best activity on HER2-positive cells, while it was less toxic to HER2-negative cells. From the experimental results, the coupling position of CPT affected the activity of the conjugates. Conjugates 1, 4 and 7, which connected the load in the same way, had different inhibitory activities on three HER2-positive cells, and among them, conjugate 4 showed the best activity. We speculated it is related to the releasing efficiency of free SMAC peptide and CPT. The subsequent biological activity evaluations of conjugate 4 confirmed that the introduction of the SMAC peptide sequence can enhance the activity of Caspase 3 and indeed play a role in promoting apoptosis. Conjugate 4 reduced the expression of XIAP and cIAP1 in SK-BR-3 cells in a concentration dependent manner. The in vivo activity study also fully proved that the antitumor activity as well as safety of conjugate 4 was superior to CPT. The pro-apoptotic effect of conjugate 4 is significantly superior to the C4 group without fused SMAC peptide and the C4+SMAC combination drug group. This indicates that that the introduced SMAC peptide exerts a synergistic anti-tumor effect. When CPT is engineered into PDCs molecules, its in vitro activity related to CPT can be retained but is somewhat diminished. This attenuation may be attributed to the fact that PDCs must undergo cellular internalization and release of CPT or its derivatives in the intracellular environment to exert anti-tumor activity.

In this study, a series of conjugates were designed and synthesized by fusing the SMAC peptide and P1 target peptide, and coupling with CPT, with the hope that they can play a synergistic role in promoting cell apoptosis. The in vitro anti-tumor activity study found that the better conjugate 4 had the best activity on HER2-positive cells, while it was less toxic to HER2-negative cells. From the experimental results, the coupling position of CPT affected the activity of the conjugates. Conjugates 1, 4 and 7, which connected the load in the same way, had different inhibitory activities on three HER2-positive cells, and among them, conjugate 4 showed the best activity. We speculated it is related to the releasing efficiency of free SMAC peptide and CPT. The subsequent biological activity evaluations of conjugate 4 confirmed that the introduction of the SMAC peptide sequence can enhance the activity of Caspase 3 and indeed play a role in promoting apoptosis. Conjugate 4 reduced the expression of XIAP and cIAP1 in SK-BR-3 cells in a concentration dependent manner. The in vivo activity study also fully proved that the antitumor activity as well as safety of conjugate 4 was superior to CPT. The pro-apoptotic effect of conjugate 4 is significantly superior to the C4 group without fused SMAC peptide and the C4+SMAC combination drug group. This indicates that that the introduced SMAC peptide exerts a synergistic anti-tumor effect. When CPT is engineered into PDCs molecules, its in vitro activity related to CPT can be retained but is somewhat diminished. This attenuation may be attributed to the fact that PDCs must undergo cellular internalization and release of CPT or its derivatives in the intracellular environment to exert anti-tumor activity.

In this study, a series of conjugates were designed and synthesized by fusing the SMAC peptide and P1 target peptide, and coupling with CPT, with the hope that they can play a synergistic role in promoting cell apoptosis. The in vitro anti-tumor activity study found that the better conjugate 4 had the best activity on HER2-positive cells, while it was less toxic to HER2-negative cells. From the experimental results, the coupling position of CPT affected the activity of the conjugates. Conjugates 1, 4 and 7, which connected the load in the same way, had different inhibitory activities on three HER2-positive cells, and among them, conjugate 4 showed the best activity. We speculated it is related to the releasing efficiency of free SMAC peptide and CPT. The subsequent biological activity evaluations of conjugate 4 confirmed that the introduction of the SMAC peptide sequence can enhance the activity of Caspase 3 and indeed play a role in promoting apoptosis. Conjugate 4 reduced the expression of XIAP and cIAP1 in SK-BR-3 cells in a concentration dependent manner. The in vivo activity study also fully proved that the antitumor activity as well as safety of conjugate 4 was superior to CPT. The pro-apoptotic effect of conjugate 4 is significantly superior to the C4 group without fused SMAC peptide and the C4+SMAC combination drug group. This indicates that that the introduced SMAC peptide exerts a synergistic anti-tumor effect. When CPT is engineered into PDCs molecules, its in vitro activity related to CPT can be retained but is somewhat diminished. This attenuation may be attributed to the fact that PDCs must undergo cellular internalization and release of CPT or its derivatives in the intracellular environment to exert anti-tumor activity.

In this study, a series of conjugates were designed and synthesized by fusing the SMAC peptide and P1 target peptide, and coupling with CPT, with the hope that they can play a synergistic role in promoting cell apoptosis. The in vitro anti-tumor activity study found that the better conjugate 4 had the best activity on HER2-positive cells, while it was less toxic to HER2-negative cells. From the experimental results, the coupling position of CPT affected the activity of the conjugates. Conjugates 1, 4 and 7, which connected the load in the same way, had different inhibitory activities on three HER2-positive cells, and among them, conjugate 4 showed the best activity. We speculated it is related to the releasing efficiency of free SMAC peptide and CPT. The subsequent biological activity evaluations of conjugate 4 confirmed that the introduction of the SMAC peptide sequence can enhance the activity of Caspase 3 and indeed play a role in promoting apoptosis. Conjugate 4 reduced the expression of XIAP and cIAP1 in SK-BR-3 cells in a concentration dependent manner. The in vivo activity study also fully proved that the antitumor activity as well as safety of conjugate 4 was superior to CPT. The pro-apoptotic effect of conjugate 4 is significantly superior to the C4 group without fused SMAC peptide and the C4+SMAC combination drug group. This indicates that that the introduced SMAC peptide exerts a synergistic anti-tumor effect. When CPT is engineered into PDCs molecules, its in vitro activity related to CPT can be retained but is somewhat diminished. This attenuation may be attributed to the fact that PDCs must undergo cellular internalization and release of CPT or its derivatives in the intracellular environment to exert anti-tumor activity.

In this study, a series of conjugates were designed and synthesized by fusing the SMAC peptide and P1 target peptide, and coupling with CPT, with the hope that they can play a synergistic role in promoting cell apoptosis. The in vitro anti-tumor activity study found that the better conjugate 4 had the best activity on HER2-positive cells, while it was less toxic to HER2-negative cells. From the experimental results, the coupling position of CPT affected the activity of the conjugates. Conjugates 1, 4 and 7, which connected the load in the same way, had different inhibitory activities on three HER2-positive cells, and among them, conjugate 4 showed the best activity. We speculated it is related to the releasing efficiency of free SMAC peptide and CPT. The subsequent biological activity evaluations of conjugate 4 confirmed that the introduction of the SMAC peptide sequence can enhance the activity of Caspase 3 and indeed play a role in promoting apoptosis. Conjugate 4 reduced the expression of XIAP and cIAP1 in SK-BR-3 cells in a concentration dependent manner. The in vivo activity study also fully proved that the antitumor activity as well as safety of conjugate 4 was superior to CPT. The pro-apoptotic effect of conjugate 4 is significantly superior to the C4 group without fused SMAC peptide and the C4+SMAC combination drug group. This indicates that that the introduced SMAC peptide exerts a synergistic anti-tumor effect. When CPT is engineered into PDCs molecules, its in vitro activity related to CPT can be retained but is somewhat diminished. This attenuation may be attributed to the fact that PDCs must undergo cellular internalization and release of CPT or its derivatives in the intracellular environment to exert anti-tumor activity.

In this study, a series of conjugates were designed and synthesized by fusing the SMAC peptide and P1 target peptide, and coupling with CPT, with the hope that they can play a synergistic role in promoting cell apoptosis. The in vitro anti-tumor activity study found that the better conjugate 4 had the best activity on HER2-positive cells, while it was less toxic to HER2-negative cells. From the experimental results, the coupling position of CPT affected the activity of the conjugates. Conjugates 1, 4 and 7, which connected the load in the same way, had different inhibitory activities on three HER2-positive cells, and among them, conjugate 4 showed the best activity. We speculated it is related to the releasing efficiency of free SMAC peptide and CPT. The subsequent biological activity evaluations of conjugate 4 confirmed that the introduction of the SMAC peptide sequence can enhance the activity of Caspase 3 and indeed play a role in promoting apoptosis. Conjugate 4 reduced the expression of XIAP and cIAP1 in SK-BR-3 cells in a concentration dependent manner. The in vivo activity study also fully proved that the antitumor activity as well as safety of conjugate 4 was superior to CPT. The pro-apoptotic effect of conjugate 4 is significantly superior to the C4 group without fused SMAC peptide and the C4+SMAC combination drug group. This indicates that that the introduced SMAC peptide exerts a synergistic anti-tumor effect. When CPT is engineered into PDCs molecules, its in vitro activity related to CPT can be retained but is somewhat diminished. This attenuation may be attributed to the fact that PDCs must undergo cellular internalization and release of CPT or its derivatives in the intracellular environment to exert anti-tumor activity.

In this study, a series of conjugates were designed and synthesized by fusing the SMAC peptide and P1 target peptide, and coupling with CPT, with the hope that they can play a synergistic role in promoting cell apoptosis. The in vitro anti-tumor activity study found that the better conjugate 4 had the best activity on HER2-positive cells, while it was less toxic to HER2-negative cells. From the experimental results, the coupling position of CPT affected the activity of the conjugates. Conjugates 1, 4 and 7, which connected the load in the same way, had different inhibitory activities on three HER2-positive cells, and among them, conjugate 4 showed the best activity. We speculated it is related to the releasing efficiency of free SMAC peptide and CPT. The subsequent biological activity evaluations of conjugate 4 confirmed that the introduction of the SMAC peptide sequence can enhance the activity of Caspase 3 and indeed play a role in promoting apoptosis. Conjugate 4 reduced the expression of XIAP and cIAP1 in SK-BR-3 cells in a concentration dependent manner. The in vivo activity study also fully proved that the antitumor activity as well as safety of conjugate 4 was superior to CPT. The pro-apoptotic effect of conjugate 4 is significantly superior to the C4 group without fused SMAC peptide and the C4+SMAC combination drug group. This indicates that that the introduced SMAC peptide exerts a synergistic anti-tumor effect. When CPT is engineered into PDCs molecules, its in vitro activity related to CPT can be retained but is somewhat diminished. This attenuation may be attributed to the fact that PDCs must undergo cellular internalization and release of CPT or its derivatives in the intracellular environment to exert anti-tumor activity.

In this study, a series of conjugates were designed and synthesized by fusing the SMAC peptide and P1 target peptide, and coupling with CPT, with the hope that they can play a synergistic role in promoting cell apoptosis. The in vitro anti-tumor activity study found that the better conjugate 4 had the best activity on HER2-positive cells, while it was less toxic to HER2-negative cells. From the experimental results, the coupling position of CPT affected the activity of the conjugates. Conjugates 1, 4 and 7, which connected the load in the same way, had different inhibitory activities on three HER2-positive cells, and among them, conjugate 4 showed the best activity. We speculated it is related to the releasing efficiency of free SMAC peptide and CPT. The subsequent biological activity evaluations of conjugate 4 confirmed that the introduction of the SMAC peptide sequence can enhance the activity of Caspase 3 and indeed play a role in promoting apoptosis. Conjugate 4 reduced the expression of XIAP and cIAP1 in SK-BR-3 cells in a concentration dependent manner. The in vivo activity study also fully proved that the antitumor activity as well as safety of conjugate 4 was superior to CPT. The pro-apoptotic effect of conjugate 4 is significantly superior to the C4 group without fused SMAC peptide and the C4+SMAC combination drug group. This indicates that that the introduced SMAC peptide exerts a synergistic anti-tumor effect. When CPT is engineered into PDCs molecules, its in vitro activity related to CPT can be retained but is somewhat diminished. This attenuation may be attributed to the fact that PDCs must undergo cellular internalization and release of CPT or its derivatives in the intracellular environment to exert anti-tumor activity.

In this study, a series of conjugates were designed and synthesized by fusing the SMAC peptide and P1 target peptide, and coupling with CPT, with the hope that they can play a synergistic role in promoting cell apoptosis. The in vitro anti-tumor activity study found that the better conjugate 4 had the best activity on HER2-positive cells, while it was less toxic to HER2-negative cells. From the experimental results, the coupling position of CPT affected the activity of the conjugates. Conjugates 1, 4 and 7, which connected the load in the same way, had different inhibitory activities on three HER2-positive cells, and among them, conjugate 4 showed the best activity. We speculated it is related to the releasing efficiency of free SMAC peptide and CPT. The subsequent biological activity evaluations of conjugate 4 confirmed that the introduction of the SMAC peptide sequence can enhance the activity of Caspase 3 and indeed play a role in promoting apoptosis. Conjugate 4 reduced the expression of XIAP and cIAP1 in SK-BR-3 cells in a concentration dependent manner. The in vivo activity study also fully proved that the antitumor activity as well as safety of conjugate 4 was superior to CPT. The pro-apoptotic effect of conjugate 4 is significantly superior to the C4 group without fused SMAC peptide and the C4+SMAC combination drug group. This indicates that that the introduced SMAC peptide exerts a synergistic anti-tumor effect. When CPT is engineered into PDCs molecules, its in vitro activity related to CPT can be retained but is somewhat diminished. This attenuation may be attributed to the fact that PDCs must undergo cellular internalization and release of CPT or its derivatives in the intracellular environment to exert anti-tumor activity.

In this study, a series of conjugates were designed and synthesized by fusing the SMAC peptide and P1 target peptide, and coupling with CPT, with the hope that they can play a synergistic role in promoting cell apoptosis. The in vitro anti-tumor activity study found that the better conjugate 4 had the best activity on HER2-positive cells, while it was less toxic to HER2-negative cells. From the experimental results, the coupling position of CPT affected the activity of the conjugates. Conjugates 1, 4 and 7, which connected the load in the same way, had different inhibitory activities on three HER2-positive cells, and among them, conjugate 4 showed the best activity. We speculated it is related to the releasing efficiency of free SMAC peptide and CPT. The subsequent biological activity evaluations of conjugate 4 confirmed that the introduction of the SMAC peptide sequence can enhance the activity of Caspase 3 and indeed play a role in promoting apoptosis. Conjugate 4 reduced the expression of XIAP and cIAP1 in SK-BR-3 cells in a concentration dependent manner. The in vivo activity study also fully proved that the antitumor activity as well as safety of conjugate 4 was superior to CPT. The pro-apoptotic effect of conjugate 4 is significantly superior to the C4 group without fused SMAC peptide and the C4+SMAC combination drug group. This indicates that that the introduced SMAC peptide exerts a synergistic anti-tumor effect. When CPT is engineered into PDCs molecules, its in vitro activity related to CPT can be retained but is somewhat diminished. This attenuation may be attributed to the fact that PDCs must undergo cellular internalization and release of CPT or its derivatives in the intracellular environment to exert anti-tumor activity.

In this study, a series of conjugates were designed and synthesized by fusing the SMAC peptide and P1 target peptide, and coupling with CPT, with the hope that they can play a synergistic role in promoting cell apoptosis. The in vitro anti-tumor activity study found that the better conjugate 4 had the best activity on HER2-positive cells, while it was less toxic to HER2-negative cells. From the experimental results, the coupling position of CPT affected the activity of the conjugates. Conjugates 1, 4 and 7, which connected the load in the same way, had different inhibitory activities on three HER2-positive cells, and among them, conjugate 4 showed the best activity. We speculated it is related to the releasing efficiency of free SMAC peptide and CPT. The subsequent biological activity evaluations of conjugate 4 confirmed that the introduction of the SMAC peptide sequence can enhance the activity of Caspase 3 and indeed play a role in promoting apoptosis. Conjugate 4 reduced the expression of XIAP and cIAP1 in SK-BR-3 cells in a concentration dependent manner. The in vivo activity study also fully proved that the antitumor activity as well as safety of conjugate 4 was superior to CPT. The pro-apoptotic effect of conjugate 4 is significantly superior to the C4 group without fused SMAC peptide and the C4+SMAC combination drug group. This indicates that that the introduced SMAC peptide exerts a synergistic anti-tumor effect. When CPT is engineered into PDCs molecules, its in vitro activity related to CPT can be retained but is somewhat diminished. This attenuation may be attributed to the fact that PDCs must undergo cellular internalization and release of CPT or its derivatives in the intracellular environment to exert anti-tumor activity.

In this study, a series of conjugates were designed and synthesized by fusing the SMAC peptide and P1 target peptide, and coupling with CPT, with the hope that they can play a synergistic role in promoting cell apoptosis. The in vitro anti-tumor activity study found that the better conjugate 4 had the best activity on HER2-positive cells, while it was less toxic to HER2-negative cells. From the experimental results, the coupling position of CPT affected the activity of the conjugates. Conjugates 1, 4 and 7, which connected the load in the same way, had different inhibitory activities on three HER2-positive cells, and among them, conjugate 4 showed the best activity. We speculated it is related to the releasing efficiency of free SMAC peptide and CPT. The subsequent biological activity evaluations of conjugate 4 confirmed that the introduction of the SMAC peptide sequence can enhance the activity of Caspase 3 and indeed play a role in promoting apoptosis. Conjugate 4 reduced the expression of XIAP and cIAP1 in SK-BR-3 cells in a concentration dependent manner. The in vivo activity study also fully proved that the antitumor activity as well as safety of conjugate 4 was superior to CPT. The pro-apoptotic effect of conjugate 4 is significantly superior to the C4 group without fused SMAC peptide and the C4+SMAC combination drug group. This indicates that that the introduced SMAC peptide exerts a synergistic anti-tumor effect. When CPT is engineered into PDCs molecules, its in vitro activity related to CPT can be retained but is somewhat diminished. This attenuation may be attributed to the fact that PDCs must undergo cellular internalization and release of CPT or its derivatives in the intracellular environment to exert anti-tumor activity.

To enhance the affinity of the human epidermal growth receptor 2 (HER2) targeted peptide developed previously, bispecific fusion peptidesP1GCGT1andP1GCGCGT1were designed using anin silicoapproach. Molecular dynamic simulation showed that both peptides strongly interacted with HER2 domains II and IV. Compared with peptides targeting each single domain,P1GCGT1andP1GCGCGT1could bind to HER2 more significantly and targeted HER2-positive cells specifically. Additionally, both peptides were used to generate peptide-drug conjugates with camptothecin (CPT), among whichI-1andI-4were screened for enhanced cellular activity and selectivity. Biological evaluation demonstrated thatI-1andI-4induced cell apoptosis, promoted cell cycle arrestin S-phase, and inhibited Topo I activity. The binding affinity assay and confocal analysis revealed thatI-1andI-4were effective at targeting HER2. Moreover,I-1andI-4showed better stability than single targeting peptide and presented enhanced antitumor activity and safety than CPT in tumor-bearing mice.

To enhance the affinity of the human epidermal growth receptor 2 (HER2) targeted peptide developed previously, bispecific fusion peptidesP1GCGT1andP1GCGCGT1were designed using anin silicoapproach. Molecular dynamic simulation showed that both peptides strongly interacted with HER2 domains II and IV. Compared with peptides targeting each single domain,P1GCGT1andP1GCGCGT1could bind to HER2 more significantly and targeted HER2-positive cells specifically. Additionally, both peptides were used to generate peptide-drug conjugates with camptothecin (CPT), among whichI-1andI-4were screened for enhanced cellular activity and selectivity. Biological evaluation demonstrated thatI-1andI-4induced cell apoptosis, promoted cell cycle arrestin S-phase, and inhibited Topo I activity. The binding affinity assay and confocal analysis revealed thatI-1andI-4were effective at targeting HER2. Moreover,I-1andI-4showed better stability than single targeting peptide and presented enhanced antitumor activity and safety than CPT in tumor-bearing mice.

To enhance the affinity of the human epidermal growth receptor 2 (HER2) targeted peptide developed previously, bispecific fusion peptidesP1GCGT1andP1GCGCGT1were designed using anin silicoapproach. Molecular dynamic simulation showed that both peptides strongly interacted with HER2 domains II and IV. Compared with peptides targeting each single domain,P1GCGT1andP1GCGCGT1could bind to HER2 more significantly and targeted HER2-positive cells specifically. Additionally, both peptides were used to generate peptide-drug conjugates with camptothecin (CPT), among whichI-1andI-4were screened for enhanced cellular activity and selectivity. Biological evaluation demonstrated thatI-1andI-4induced cell apoptosis, promoted cell cycle arrestin S-phase, and inhibited Topo I activity. The binding affinity assay and confocal analysis revealed thatI-1andI-4were effective at targeting HER2. Moreover,I-1andI-4showed better stability than single targeting peptide and presented enhanced antitumor activity and safety than CPT in tumor-bearing mice.

To enhance the affinity of the human epidermal growth receptor 2 (HER2) targeted peptide developed previously, bispecific fusion peptidesP1GCGT1andP1GCGCGT1were designed using anin silicoapproach. Molecular dynamic simulation showed that both peptides strongly interacted with HER2 domains II and IV. Compared with peptides targeting each single domain,P1GCGT1andP1GCGCGT1could bind to HER2 more significantly and targeted HER2-positive cells specifically. Additionally, both peptides were used to generate peptide-drug conjugates with camptothecin (CPT), among whichI-1andI-4were screened for enhanced cellular activity and selectivity. Biological evaluation demonstrated thatI-1andI-4induced cell apoptosis, promoted cell cycle arrestin S-phase, and inhibited Topo I activity. The binding affinity assay and confocal analysis revealed thatI-1andI-4were effective at targeting HER2. Moreover,I-1andI-4showed better stability than single targeting peptide and presented enhanced antitumor activity and safety than CPT in tumor-bearing mice.

To enhance the affinity of the human epidermal growth receptor 2 (HER2) targeted peptide developed previously, bispecific fusion peptidesP1GCGT1andP1GCGCGT1were designed using anin silicoapproach. Molecular dynamic simulation showed that both peptides strongly interacted with HER2 domains II and IV. Compared with peptides targeting each single domain,P1GCGT1andP1GCGCGT1could bind to HER2 more significantly and targeted HER2-positive cells specifically. Additionally, both peptides were used to generate peptide-drug conjugates with camptothecin (CPT), among whichI-1andI-4were screened for enhanced cellular activity and selectivity. Biological evaluation demonstrated thatI-1andI-4induced cell apoptosis, promoted cell cycle arrestin S-phase, and inhibited Topo I activity. The binding affinity assay and confocal analysis revealed thatI-1andI-4were effective at targeting HER2. Moreover,I-1andI-4showed better stability than single targeting peptide and presented enhanced antitumor activity and safety than CPT in tumor-bearing mice.

In this study, we developed novel melittin analogs with pH-responsive cell-penetrating and membrane-lytic activities by replacing arginines and lysines with histidines. Importantly, we found that the conjugation of cargoes to the N-terminus of melittin analogs decreased their cell-penetrating and membrane-lytic activity compared to the C-terminus, implying that the C-terminus of analogs is more suitable for cargo conjugation. After the attachment of CPT, CPT-AAM-1 and CPT-AAM-2 displayed obvious pH-responsive antitumor activity. CPT-AAM-1 and CPT-AAM-2 destroyed tumor cells through the release of CPT and membrane disruption. Compared with CPT-AAM-2, CPT-AAM-1 showed stronger antitumor activity under acidic conditions. Notably, CPT-AAM-1 significantly inhibited the tumor growth in vivo compared with AAM, AAM-1, and CPT. In addition, CPT-AAM-1 showed relatively low toxicity compared with melittin and CPT. Taken together, our results demonstrate that CPT-AAM-1, with efficient pH-responsive cell-penetrating and membrane-lytic activities, possesses significant therapeutic potential for tumor therapy. This study provides a novel strategy for the development of PDCs based on pH-responsive AMPs for oncology therapeutics.

In this study, we developed novel melittin analogs with pH-responsive cell-penetrating and membrane-lytic activities by replacing arginines and lysines with histidines. Importantly, we found that the conjugation of cargoes to the N-terminus of melittin analogs decreased their cell-penetrating and membrane-lytic activity compared to the C-terminus, implying that the C-terminus of analogs is more suitable for cargo conjugation. After the attachment of CPT, CPT-AAM-1 and CPT-AAM-2 displayed obvious pH-responsive antitumor activity. CPT-AAM-1 and CPT-AAM-2 destroyed tumor cells through the release of CPT and membrane disruption. Compared with CPT-AAM-2, CPT-AAM-1 showed stronger antitumor activity under acidic conditions. Notably, CPT-AAM-1 significantly inhibited the tumor growth in vivo compared with AAM, AAM-1, and CPT. In addition, CPT-AAM-1 showed relatively low toxicity compared with melittin and CPT. Taken together, our results demonstrate that CPT-AAM-1, with efficient pH-responsive cell-penetrating and membrane-lytic activities, possesses significant therapeutic potential for tumor therapy. This study provides a novel strategy for the development of PDCs based on pH-responsive AMPs for oncology therapeutics.

In this study, we developed novel melittin analogs with pH-responsive cell-penetrating and membrane-lytic activities by replacing arginines and lysines with histidines. Importantly, we found that the conjugation of cargoes to the N-terminus of melittin analogs decreased their cell-penetrating and membrane-lytic activity compared to the C-terminus, implying that the C-terminus of analogs is more suitable for cargo conjugation. After the attachment of CPT, CPT-AAM-1 and CPT-AAM-2 displayed obvious pH-responsive antitumor activity. CPT-AAM-1 and CPT-AAM-2 destroyed tumor cells through the release of CPT and membrane disruption. Compared with CPT-AAM-2, CPT-AAM-1 showed stronger antitumor activity under acidic conditions. Notably, CPT-AAM-1 significantly inhibited the tumor growth in vivo compared with AAM, AAM-1, and CPT. In addition, CPT-AAM-1 showed relatively low toxicity compared with melittin and CPT. Taken together, our results demonstrate that CPT-AAM-1, with efficient pH-responsive cell-penetrating and membrane-lytic activities, possesses significant therapeutic potential for tumor therapy. This study provides a novel strategy for the development of PDCs based on pH-responsive AMPs for oncology therapeutics.

In this study, we developed novel melittin analogs with pH-responsive cell-penetrating and membrane-lytic activities by replacing arginines and lysines with histidines. Importantly, we found that the conjugation of cargoes to the N-terminus of melittin analogs decreased their cell-penetrating and membrane-lytic activity compared to the C-terminus, implying that the C-terminus of analogs is more suitable for cargo conjugation. After the attachment of CPT, CPT-AAM-1 and CPT-AAM-2 displayed obvious pH-responsive antitumor activity. CPT-AAM-1 and CPT-AAM-2 destroyed tumor cells through the release of CPT and membrane disruption. Compared with CPT-AAM-2, CPT-AAM-1 showed stronger antitumor activity under acidic conditions. Notably, CPT-AAM-1 significantly inhibited the tumor growth in vivo compared with AAM, AAM-1, and CPT. In addition, CPT-AAM-1 showed relatively low toxicity compared with melittin and CPT. Taken together, our results demonstrate that CPT-AAM-1, with efficient pH-responsive cell-penetrating and membrane-lytic activities, possesses significant therapeutic potential for tumor therapy. This study provides a novel strategy for the development of PDCs based on pH-responsive AMPs for oncology therapeutics.

In this study, we developed novel melittin analogs with pH-responsive cell-penetrating and membrane-lytic activities by replacing arginines and lysines with histidines. Importantly, we found that the conjugation of cargoes to the N-terminus of melittin analogs decreased their cell-penetrating and membrane-lytic activity compared to the C-terminus, implying that the C-terminus of analogs is more suitable for cargo conjugation. After the attachment of CPT, CPT-AAM-1 and CPT-AAM-2 displayed obvious pH-responsive antitumor activity. CPT-AAM-1 and CPT-AAM-2 destroyed tumor cells through the release of CPT and membrane disruption. Compared with CPT-AAM-2, CPT-AAM-1 showed stronger antitumor activity under acidic conditions. Notably, CPT-AAM-1 significantly inhibited the tumor growth in vivo compared with AAM, AAM-1, and CPT. In addition, CPT-AAM-1 showed relatively low toxicity compared with melittin and CPT. Taken together, our results demonstrate that CPT-AAM-1, with efficient pH-responsive cell-penetrating and membrane-lytic activities, possesses significant therapeutic potential for tumor therapy. This study provides a novel strategy for the development of PDCs based on pH-responsive AMPs for oncology therapeutics.

In this study, we developed novel melittin analogs with pH-responsive cell-penetrating and membrane-lytic activities by replacing arginines and lysines with histidines. Importantly, we found that the conjugation of cargoes to the N-terminus of melittin analogs decreased their cell-penetrating and membrane-lytic activity compared to the C-terminus, implying that the C-terminus of analogs is more suitable for cargo conjugation. After the attachment of CPT, CPT-AAM-1 and CPT-AAM-2 displayed obvious pH-responsive antitumor activity. CPT-AAM-1 and CPT-AAM-2 destroyed tumor cells through the release of CPT and membrane disruption. Compared with CPT-AAM-2, CPT-AAM-1 showed stronger antitumor activity under acidic conditions. Notably, CPT-AAM-1 significantly inhibited the tumor growth in vivo compared with AAM, AAM-1, and CPT. In addition, CPT-AAM-1 showed relatively low toxicity compared with melittin and CPT. Taken together, our results demonstrate that CPT-AAM-1, with efficient pH-responsive cell-penetrating and membrane-lytic activities, possesses significant therapeutic potential for tumor therapy. This study provides a novel strategy for the development of PDCs based on pH-responsive AMPs for oncology therapeutics.

In this study, we developed novel melittin analogs with pH-responsive cell-penetrating and membrane-lytic activities by replacing arginines and lysines with histidines. Importantly, we found that the conjugation of cargoes to the N-terminus of melittin analogs decreased their cell-penetrating and membrane-lytic activity compared to the C-terminus, implying that the C-terminus of analogs is more suitable for cargo conjugation. After the attachment of CPT, CPT-AAM-1 and CPT-AAM-2 displayed obvious pH-responsive antitumor activity. CPT-AAM-1 and CPT-AAM-2 destroyed tumor cells through the release of CPT and membrane disruption. Compared with CPT-AAM-2, CPT-AAM-1 showed stronger antitumor activity under acidic conditions. Notably, CPT-AAM-1 significantly inhibited the tumor growth in vivo compared with AAM, AAM-1, and CPT. In addition, CPT-AAM-1 showed relatively low toxicity compared with melittin and CPT. Taken together, our results demonstrate that CPT-AAM-1, with efficient pH-responsive cell-penetrating and membrane-lytic activities, possesses significant therapeutic potential for tumor therapy. This study provides a novel strategy for the development of PDCs based on pH-responsive AMPs for oncology therapeutics.

In this study, we developed novel melittin analogs with pH-responsive cell-penetrating and membrane-lytic activities by replacing arginines and lysines with histidines. Importantly, we found that the conjugation of cargoes to the N-terminus of melittin analogs decreased their cell-penetrating and membrane-lytic activity compared to the C-terminus, implying that the C-terminus of analogs is more suitable for cargo conjugation. After the attachment of CPT, CPT-AAM-1 and CPT-AAM-2 displayed obvious pH-responsive antitumor activity. CPT-AAM-1 and CPT-AAM-2 destroyed tumor cells through the release of CPT and membrane disruption. Compared with CPT-AAM-2, CPT-AAM-1 showed stronger antitumor activity under acidic conditions. Notably, CPT-AAM-1 significantly inhibited the tumor growth in vivo compared with AAM, AAM-1, and CPT. In addition, CPT-AAM-1 showed relatively low toxicity compared with melittin and CPT. Taken together, our results demonstrate that CPT-AAM-1, with efficient pH-responsive cell-penetrating and membrane-lytic activities, possesses significant therapeutic potential for tumor therapy. This study provides a novel strategy for the development of PDCs based on pH-responsive AMPs for oncology therapeutics.

In this study, we developed novel melittin analogs with pH-responsive cell-penetrating and membrane-lytic activities by replacing arginines and lysines with histidines. Importantly, we found that the conjugation of cargoes to the N-terminus of melittin analogs decreased their cell-penetrating and membrane-lytic activity compared to the C-terminus, implying that the C-terminus of analogs is more suitable for cargo conjugation. After the attachment of CPT, CPT-AAM-1 and CPT-AAM-2 displayed obvious pH-responsive antitumor activity. CPT-AAM-1 and CPT-AAM-2 destroyed tumor cells through the release of CPT and membrane disruption. Compared with CPT-AAM-2, CPT-AAM-1 showed stronger antitumor activity under acidic conditions. Notably, CPT-AAM-1 significantly inhibited the tumor growth in vivo compared with AAM, AAM-1, and CPT. In addition, CPT-AAM-1 showed relatively low toxicity compared with melittin and CPT. Taken together, our results demonstrate that CPT-AAM-1, with efficient pH-responsive cell-penetrating and membrane-lytic activities, possesses significant therapeutic potential for tumor therapy. This study provides a novel strategy for the development of PDCs based on pH-responsive AMPs for oncology therapeutics.

In this study, we developed novel melittin analogs with pH-responsive cell-penetrating and membrane-lytic activities by replacing arginines and lysines with histidines. Importantly, we found that the conjugation of cargoes to the N-terminus of melittin analogs decreased their cell-penetrating and membrane-lytic activity compared to the C-terminus, implying that the C-terminus of analogs is more suitable for cargo conjugation. After the attachment of CPT, CPT-AAM-1 and CPT-AAM-2 displayed obvious pH-responsive antitumor activity. CPT-AAM-1 and CPT-AAM-2 destroyed tumor cells through the release of CPT and membrane disruption. Compared with CPT-AAM-2, CPT-AAM-1 showed stronger antitumor activity under acidic conditions. Notably, CPT-AAM-1 significantly inhibited the tumor growth in vivo compared with AAM, AAM-1, and CPT. In addition, CPT-AAM-1 showed relatively low toxicity compared with melittin and CPT. Taken together, our results demonstrate that CPT-AAM-1, with efficient pH-responsive cell-penetrating and membrane-lytic activities, possesses significant therapeutic potential for tumor therapy. This study provides a novel strategy for the development of PDCs based on pH-responsive AMPs for oncology therapeutics.