In vivo assessment in tumor-bearing animal models classically complements cell cultures data for comparing cancer treatment efficacies. For this reason, hTNBCSC and hOvCSC xenografts were implanted subcutaneously into immunodeficient mice as described in the Methods section with only 1000 cells given their highly tumorigenic nature. Three days later, the animals began receiving weekly IV bolus administration of either vehicle, docetaxel, or TH1902. Docetaxel was administered at a dose (15 mg/kg/week; for 3 cycles) in accordance with the estimated maximal tolerated dose (MTD) for mice, as well as at 1/4 of the MTD (3.75 mg/kg/week). TH1902 was administered at doses (35 and 8.75 mg/kg/week) which contained quantities of docetaxel equivalent to those in the two administrations of free docetaxel. From the size of the hTNBCSC and hOvCSC tumors, it is apparent that docetaxel had little impact on xenografts growth when administered neither at its MTD, for both hTNBCSC and hOvCSC, nor at 1/4 of this dosage as reflected by the growth curves. TH1902, when administered at a dosage equivalent to docetaxel at its MTD, provided greater tumor growth inhibition than did docetaxel for both xenograft models. Furthermore, higher dosage of administered TH1902 (up to 1.5 equivalent of docetaxel MTD) did not generate significant differences in terms of hOvCSC tumor inhibition without affecting mice body weights suggesting that TH1902, even at higher doses, is better tolerated compared to docetaxel. Then, in order to statistically compare the effects of docetaxel and TH1902 on tumor growth, the tumor sizes measured at the vehicle group endpoint were compared and statistically significant differences between the tumor sizes in vehicle-treated animals found in TH1902-treated animals for hTNBCSC and hOvCSC. Mouse body weight was used as an indicator of the morbidity associated with administration of docetaxel or TH1902. Administration of docetaxel at its MTD provoked a weight loss that approached 10% after the treatments, which is often observed in xenograft models with administration of docetaxel at this level. The body weights of animals treated with an equivalent quantity or a 1.5-fold equivalent of TH1902 were maintained at a roughly constant level throughout the experiment. The animals treated with vehicle recorded a slight weight gain (~5%) over this period while animals treated with the lower dosages of docetaxel or TH1902 were similar to the animals treated with vehicle. The body weight data indicates that TH1902 appears to be better tolerated than the equivalent quantity of free docetaxel, in addition to the fact that TH1902 is more efficacious than docetaxel when administered in vivo in the murine models of CSC tested.

In vivo assessment in tumor-bearing animal models classically complements cell cultures data for comparing cancer treatment efficacies. For this reason, hTNBCSC and hOvCSC xenografts were implanted subcutaneously into immunodeficient mice as described in the Methods section with only 1000 cells given their highly tumorigenic nature. Three days later, the animals began receiving weekly IV bolus administration of either vehicle, docetaxel, or TH1902. Docetaxel was administered at a dose (15 mg/kg/week; for 3 cycles) in accordance with the estimated maximal tolerated dose (MTD) for mice, as well as at 1/4 of the MTD (3.75 mg/kg/week). TH1902 was administered at doses (35 and 8.75 mg/kg/week) which contained quantities of docetaxel equivalent to those in the two administrations of free docetaxel. From the size of the hTNBCSC and hOvCSC tumors, it is apparent that docetaxel had little impact on xenografts growth when administered neither at its MTD, for both hTNBCSC and hOvCSC, nor at 1/4 of this dosage as reflected by the growth curves. TH1902, when administered at a dosage equivalent to docetaxel at its MTD, provided greater tumor growth inhibition than did docetaxel for both xenograft models. Furthermore, higher dosage of administered TH1902 (up to 1.5 equivalent of docetaxel MTD) did not generate significant differences in terms of hOvCSC tumor inhibition without affecting mice body weights suggesting that TH1902, even at higher doses, is better tolerated compared to docetaxel. Then, in order to statistically compare the effects of docetaxel and TH1902 on tumor growth, the tumor sizes measured at the vehicle group endpoint were compared and statistically significant differences between the tumor sizes in vehicle-treated animals found in TH1902-treated animals for hTNBCSC and hOvCSC. Mouse body weight was used as an indicator of the morbidity associated with administration of docetaxel or TH1902. Administration of docetaxel at its MTD provoked a weight loss that approached 10% after the treatments, which is often observed in xenograft models with administration of docetaxel at this level. The body weights of animals treated with an equivalent quantity or a 1.5-fold equivalent of TH1902 were maintained at a roughly constant level throughout the experiment. The animals treated with vehicle recorded a slight weight gain (~5%) over this period while animals treated with the lower dosages of docetaxel or TH1902 were similar to the animals treated with vehicle. The body weight data indicates that TH1902 appears to be better tolerated than the equivalent quantity of free docetaxel, in addition to the fact that TH1902 is more efficacious than docetaxel when administered in vivo in the murine models of CSC tested.

B16-F10 melanoma syngeneic tumors were generated, with tumor sizes monitored as described in the Methods section. Tumors in xenograft-bearing, vehicle-treated mice grew at an exponential rate. Partial inhibition of tumor growth was observed after IV administration of 15 mg/kg/wk docetaxel, whereas treatment with a docetaxel-equivalent quantity of TH1902 (35 mg/kg/wk) induced tumor regression after two treatments over the period measured. Due to rapid tumor growth, only two administrations of the test articles could be performed on a weekly schedule. B16-F10 melanoma tumors from the mice treated with either vehicle, docetaxel, or TH1902 were then excised, fixed in formalin, and immunohistochemically examined.

In the ES-2 study, mice treated with low and high doses of TH1902 significantly inhibited the growth of tumors by, respectively, 45% and 87%, whereas both docetaxel groups, at equivalent docetaxel content, produced little effect (Figure 6A and Table 1). Low doses of docetaxel and low and high doses of TH1902 were well-tolerated with slight weight gain, whereas three weekly cycles of treatments with high doses of docetaxel (the MTD for this agent in mice) produced a small weight loss when compared to initial mice weights

In the SKOV3 study, mice treated with low and high doses of TH1902 showed significant inhibitions of tumor growth where only high dose of docetaxel produced this effect (Figure 6B). When compared to vehicle endpoint (Day 28), low dose of TH1902 produced similar tumor growth inhibitions when compared to high dose of docetaxel (69% vs. 84%, respectively) while high dose of TH1902 induced stronger inhibition with slight regression of tumors (Table 1). Moreover, mice treated with high dose of TH1902 showed prolonged inhibitory effect (up to Day 46) where docetaxel-treated mice could not sustain this effect, which is marked by tumor regrowth (Figure 6B).

For mice bearing endometrial tumor xenografts, a similar dosage regimen to that for ovarian tumor xenografts was used as described previously for both docetaxel and TH1902. The growth of AN3-CA tumors was inhibited by both dosages of TH1902, whereas only a high dose of docetaxel could produce this effect. When compared to vehicle endpoint, low dose of TH1902 significantly inhibited the growth of AN3-CA tumors by 74%, whereas both high doses of docetaxel and TH1902 induced tumor regressions. Interestingly, a large portion of tumors (5 out of 6) within the high-dose TH1902 group were unmeasurable or remained in regression for a prolonged period (up to Day 54), whereas tumors in the equivalent docetaxel dose were unresponsive and regrew before the end of treatments. The two high doses were diminished by half for the final two treatments due to weight loss in the animals receiving high-dose docetaxel. Rapid weight loss was associated with administration of high dosage docetaxel, but this was reversed when the dosage was halved. In contrast, only mild weight loss was observed in animals administered with high-dosage TH1902.

In the SKOV3 study, mice treated with low and high doses of TH1902 showed significant inhibitions of tumor growth where only high dose of docetaxel produced this effect (Figure 6B). When compared to vehicle endpoint (Day 28), low dose of TH1902 produced similar tumor growth inhibitions when compared to high dose of docetaxel (69% vs. 84%, respectively) while high dose of TH1902 induced stronger inhibition with slight regression of tumors (Table 1). Moreover, mice treated with high dose of TH1902 showed prolonged inhibitory effect (up to Day 46) where docetaxel-treated mice could not sustain this effect, which is marked by tumor regrowth (Figure 6B).

In the ES-2 study, mice treated with low and high doses of TH1902 significantly inhibited the growth of tumors by, respectively, 45% and 87%, whereas both docetaxel groups, at equivalent docetaxel content, produced little effect (Figure 6A and Table 1). Low doses of docetaxel and low and high doses of TH1902 were well-tolerated with slight weight gain, whereas three weekly cycles of treatments with high doses of docetaxel (the MTD for this agent in mice) produced a small weight loss when compared to initial mice weights

For mice bearing endometrial tumor xenografts, a similar dosage regimen to that for ovarian tumor xenografts was used as described previously for both docetaxel and TH1902. The growth of AN3-CA tumors was inhibited by both dosages of TH1902, whereas only a high dose of docetaxel could produce this effect. When compared to vehicle endpoint, low dose of TH1902 significantly inhibited the growth of AN3-CA tumors by 74%, whereas both high doses of docetaxel and TH1902 induced tumor regressions. Interestingly, a large portion of tumors (5 out of 6) within the high-dose TH1902 group were unmeasurable or remained in regression for a prolonged period (up to Day 54), whereas tumors in the equivalent docetaxel dose were unresponsive and regrew before the end of treatments. The two high doses were diminished by half for the final two treatments due to weight loss in the animals receiving high-dose docetaxel. Rapid weight loss was associated with administration of high dosage docetaxel, but this was reversed when the dosage was halved. In contrast, only mild weight loss was observed in animals administered with high-dosage TH1902.

The use of intravenous administration of TH1902 was next investigated in the TNBC-derived MDA-MB-231 xenograft model. Mice subjected to docetaxel treatment were administered with three intravenous injections at 15 mg/kg/wk (MTD), whereas those treated with TH1902 received five injections of an equivalent dose of docetaxel at 35 mg/kg/wk. Similar to what was seen with the intraperitoneal administration protocol, a sustained decrease in tumor size was observed following intravenous administration of TH1902 until day 70, whereas a restart of tumor growth was observed at day 50 for the docetaxel-treated mice. When lower doses, equivalent to the quarter of the MTD, were used of docetaxel (3.75 mg/kg) and TH1902 (8.75 mg/kg), tumor growth as assessed by luminescence intensity was unaffected by intravenous administration of docetaxel, whereas it was significantly inhibited in the TH1902-treated group. This was further confirmed through the measurement of the tumor burden luminescence where TH1902 reduced it significantly in comparison to vehicle- or docetaxel-treated groups. Interestingly, body weight changes remained within endpoint limits in mice on intravenous administration of either docetaxel or TH1902 (data not shown). Similar conclusions were reached on testing another TNBC-derived HCC-70 xenograft model. In fact, administration of TH1902 at 8.75 mg/kg/wk led to a 93% inhibition of HCC-70 tumor growth as compared to 24% for docetaxel alone at an equivalent dose.

The in vivo efficacy of TH1902 and docetaxel against a TNBC xenograft model was next investigated in vivo. Thus, nude mice were implanted in the right flank with MDA-MB-231-luc cancer cells, and luminescence was measured to monitor tumor growth. Mice were treated with intraperitoneal injections of either docetaxel at the MTD of 15 mg/kg/wk, or with TH1902 at the maximal injectable dose of 50 mg/kg/wk, both for five cycles of treatment. Unlike the vehicle-treated control group, where the average tumor luminescence increased over time, a significant decline in luminescence intensity was observed, starting at day 5 in the TH1902-treated group. The level of luminescence intensity was also significantly lower in the free docetaxel-treated group, when compared to that in the vehicle-treated control group, but remained higher than that for the TH1902-treated group. Interestingly, tumor relapse was observed in the docetaxel-treated group beginning at day 46, whereas a sustained decrease of tumor volume was maintained in the TH1902-treated group until day 74, at which time point a complete disappearance of the tumor was achieved. Representative images of luminescence are shown for vehicle-, docetaxel-, or TH1902-treated mice at day 14 when the control group reached the tumor volume endpoint and at day 74 post-treatment at the end of the experiment. Quantification of the residual tumor burden at days 14 and 74 after docetaxel or TH1902 treatment was performed, and the analysis confirmed a much better in vivo TH1902 efficacy profile than was seen with unconjugated docetaxel. Given the lack of apoptotic or antiproliferative effects of TH19P01, no rationale supports its further assessment in vivo. As TH1902 is considered a new chemical entity, its best control condition therefore is the unconjugated docetaxel molecule itself. Body weight of mice on intraperitoneal administration of either docetaxel or TH1902 remained within endpoint limits (-20%, data not shown).

The use of intravenous administration of TH1902 was next investigated in the TNBC-derived MDA-MB-231 xenograft model. Mice subjected to docetaxel treatment were administered with three intravenous injections at 15 mg/kg/wk (MTD), whereas those treated with TH1902 received five injections of an equivalent dose of docetaxel at 35 mg/kg/wk. Similar to what was seen with the intraperitoneal administration protocol, a sustained decrease in tumor size was observed following intravenous administration of TH1902 until day 70, whereas a restart of tumor growth was observed at day 50 for the docetaxel-treated mice. When lower doses, equivalent to the quarter of the MTD, were used of docetaxel (3.75 mg/kg) and TH1902 (8.75 mg/kg), tumor growth as assessed by luminescence intensity was unaffected by intravenous administration of docetaxel, whereas it was significantly inhibited in the TH1902-treated group. This was further confirmed through the measurement of the tumor burden luminescence where TH1902 reduced it significantly in comparison to vehicle- or docetaxel-treated groups. Interestingly, body weight changes remained within endpoint limits in mice on intravenous administration of either docetaxel or TH1902 (data not shown). Similar conclusions were reached on testing another TNBC-derived HCC-70 xenograft model. In fact, administration of TH1902 at 8.75 mg/kg/wk led to a 93% inhibition of HCC-70 tumor growth as compared to 24% for docetaxel alone at an equivalent dose.

The use of intravenous administration of TH1902 was next investigated in the TNBC-derived MDA-MB-231 xenograft model. Mice subjected to docetaxel treatment were administered with three intravenous injections at 15 mg/kg/wk (MTD), whereas those treated with TH1902 received five injections of an equivalent dose of docetaxel at 35 mg/kg/wk. Similar to what was seen with the intraperitoneal administration protocol, a sustained decrease in tumor size was observed following intravenous administration of TH1902 until day 70, whereas a restart of tumor growth was observed at day 50 for the docetaxel-treated mice. When lower doses, equivalent to the quarter of the MTD, were used of docetaxel (3.75 mg/kg) and TH1902 (8.75 mg/kg), tumor growth as assessed by luminescence intensity was unaffected by intravenous administration of docetaxel, whereas it was significantly inhibited in the TH1902-treated group. This was further confirmed through the measurement of the tumor burden luminescence where TH1902 reduced it significantly in comparison to vehicle- or docetaxel-treated groups. Interestingly, body weight changes remained within endpoint limits in mice on intravenous administration of either docetaxel or TH1902 (data not shown). Similar conclusions were reached on testing another TNBC-derived HCC-70 xenograft model. In fact, administration of TH1902 at 8.75 mg/kg/wk led to a 93% inhibition of HCC-70 tumor growth as compared to 24% for docetaxel alone at an equivalent dose.

The use of intravenous administration of TH1902 was next investigated in the TNBC-derived MDA-MB-231 xenograft model. Mice subjected to docetaxel treatment were administered with three intravenous injections at 15 mg/kg/wk (MTD), whereas those treated with TH1902 received five injections of an equivalent dose of docetaxel at 35 mg/kg/wk. Similar to what was seen with the intraperitoneal administration protocol, a sustained decrease in tumor size was observed following intravenous administration of TH1902 until day 70, whereas a restart of tumor growth was observed at day 50 for the docetaxel-treated mice. When lower doses, equivalent to the quarter of the MTD, were used of docetaxel (3.75 mg/kg) and TH1902 (8.75 mg/kg), tumor growth as assessed by luminescence intensity was unaffected by intravenous administration of docetaxel, whereas it was significantly inhibited in the TH1902-treated group. This was further confirmed through the measurement of the tumor burden luminescence where TH1902 reduced it significantly in comparison to vehicle- or docetaxel-treated groups. Interestingly, body weight changes remained within endpoint limits in mice on intravenous administration of either docetaxel or TH1902 (data not shown). Similar conclusions were reached on testing another TNBC-derived HCC-70 xenograft model. In fact, administration of TH1902 at 8.75 mg/kg/wk led to a 93% inhibition of HCC-70 tumor growth as compared to 24% for docetaxel alone at an equivalent dose.

The in vivo efficacy of TH1902 and docetaxel against a TNBC xenograft model was next investigated in vivo. Thus, nude mice were implanted in the right flank with MDA-MB-231-luc cancer cells, and luminescence was measured to monitor tumor growth. Mice were treated with intraperitoneal injections of either docetaxel at the MTD of 15 mg/kg/wk, or with TH1902 at the maximal injectable dose of 50 mg/kg/wk, both for five cycles of treatment. Unlike the vehicle-treated control group, where the average tumor luminescence increased over time, a significant decline in luminescence intensity was observed, starting at day 5 in the TH1902-treated group. The level of luminescence intensity was also significantly lower in the free docetaxel-treated group, when compared to that in the vehicle-treated control group, but remained higher than that for the TH1902-treated group. Interestingly, tumor relapse was observed in the docetaxel-treated group beginning at day 46, whereas a sustained decrease of tumor volume was maintained in the TH1902-treated group until day 74, at which time point a complete disappearance of the tumor was achieved. Representative images of luminescence are shown for vehicle-, docetaxel-, or TH1902-treated mice at day 14 when the control group reached the tumor volume endpoint and at day 74 post-treatment at the end of the experiment. Quantification of the residual tumor burden at days 14 and 74 after docetaxel or TH1902 treatment was performed, and the analysis confirmed a much better in vivo TH1902 efficacy profile than was seen with unconjugated docetaxel. Given the lack of apoptotic or antiproliferative effects of TH19P01, no rationale supports its further assessment in vivo. As TH1902 is considered a new chemical entity, its best control condition therefore is the unconjugated docetaxel molecule itself. Body weight of mice on intraperitoneal administration of either docetaxel or TH1902 remained within endpoint limits (-20%, data not shown).

In vivo assessment in tumor-bearing animal models classically complements cell cultures data for comparing cancer treatment efficacies. For this reason, hTNBCSC and hOvCSC xenografts were implanted subcutaneously into immunodeficient mice as described in the Methods section with only 1000 cells given their highly tumorigenic nature. Three days later, the animals began receiving weekly IV bolus administration of either vehicle, docetaxel, or TH1902. Docetaxel was administered at a dose (15 mg/kg/week; for 3 cycles) in accordance with the estimated maximal tolerated dose (MTD) for mice, as well as at 1/4 of the MTD (3.75 mg/kg/week). TH1902 was administered at doses (35 and 8.75 mg/kg/week) which contained quantities of docetaxel equivalent to those in the two administrations of free docetaxel. From the size of the hTNBCSC and hOvCSC tumors, it is apparent that docetaxel had little impact on xenografts growth when administered neither at its MTD, for both hTNBCSC and hOvCSC, nor at 1/4 of this dosage as reflected by the growth curves. TH1902, when administered at a dosage equivalent to docetaxel at its MTD, provided greater tumor growth inhibition than did docetaxel for both xenograft models. Furthermore, higher dosage of administered TH1902 (up to 1.5 equivalent of docetaxel MTD) did not generate significant differences in terms of hOvCSC tumor inhibition without affecting mice body weights suggesting that TH1902, even at higher doses, is better tolerated compared to docetaxel. Then, in order to statistically compare the effects of docetaxel and TH1902 on tumor growth, the tumor sizes measured at the vehicle group endpoint were compared and statistically significant differences between the tumor sizes in vehicle-treated animals found in TH1902-treated animals for hTNBCSC and hOvCSC. Mouse body weight was used as an indicator of the morbidity associated with administration of docetaxel or TH1902. Administration of docetaxel at its MTD provoked a weight loss that approached 10% after the treatments, which is often observed in xenograft models with administration of docetaxel at this level. The body weights of animals treated with an equivalent quantity or a 1.5-fold equivalent of TH1902 were maintained at a roughly constant level throughout the experiment. The animals treated with vehicle recorded a slight weight gain (~5%) over this period while animals treated with the lower dosages of docetaxel or TH1902 were similar to the animals treated with vehicle. The body weight data indicates that TH1902 appears to be better tolerated than the equivalent quantity of free docetaxel, in addition to the fact that TH1902 is more efficacious than docetaxel when administered in vivo in the murine models of CSC tested.

In vivo assessment in tumor-bearing animal models classically complements cell cultures data for comparing cancer treatment efficacies. For this reason, hTNBCSC and hOvCSC xenografts were implanted subcutaneously into immunodeficient mice as described in the Methods section with only 1000 cells given their highly tumorigenic nature. Three days later, the animals began receiving weekly IV bolus administration of either vehicle, docetaxel, or TH1902. Docetaxel was administered at a dose (15 mg/kg/week; for 3 cycles) in accordance with the estimated maximal tolerated dose (MTD) for mice, as well as at 1/4 of the MTD (3.75 mg/kg/week). TH1902 was administered at doses (35 and 8.75 mg/kg/week) which contained quantities of docetaxel equivalent to those in the two administrations of free docetaxel. From the size of the hTNBCSC and hOvCSC tumors, it is apparent that docetaxel had little impact on xenografts growth when administered neither at its MTD, for both hTNBCSC and hOvCSC, nor at 1/4 of this dosage as reflected by the growth curves. TH1902, when administered at a dosage equivalent to docetaxel at its MTD, provided greater tumor growth inhibition than did docetaxel for both xenograft models. Furthermore, higher dosage of administered TH1902 (up to 1.5 equivalent of docetaxel MTD) did not generate significant differences in terms of hOvCSC tumor inhibition without affecting mice body weights suggesting that TH1902, even at higher doses, is better tolerated compared to docetaxel. Then, in order to statistically compare the effects of docetaxel and TH1902 on tumor growth, the tumor sizes measured at the vehicle group endpoint were compared and statistically significant differences between the tumor sizes in vehicle-treated animals found in TH1902-treated animals for hTNBCSC and hOvCSC. Mouse body weight was used as an indicator of the morbidity associated with administration of docetaxel or TH1902. Administration of docetaxel at its MTD provoked a weight loss that approached 10% after the treatments, which is often observed in xenograft models with administration of docetaxel at this level. The body weights of animals treated with an equivalent quantity or a 1.5-fold equivalent of TH1902 were maintained at a roughly constant level throughout the experiment. The animals treated with vehicle recorded a slight weight gain (~5%) over this period while animals treated with the lower dosages of docetaxel or TH1902 were similar to the animals treated with vehicle. The body weight data indicates that TH1902 appears to be better tolerated than the equivalent quantity of free docetaxel, in addition to the fact that TH1902 is more efficacious than docetaxel when administered in vivo in the murine models of CSC tested.

The in vivo efficacy of TH1902 and docetaxel against a TNBC xenograft model was next investigated in vivo. Thus, nude mice were implanted in the right flank with MDA-MB-231-luc cancer cells, and luminescence was measured to monitor tumor growth. Mice were treated with intraperitoneal injections of either docetaxel at the MTD of 15 mg/kg/wk, or with TH1902 at the maximal injectable dose of 50 mg/kg/wk, both for five cycles of treatment. Unlike the vehicle-treated control group, where the average tumor luminescence increased over time, a significant decline in luminescence intensity was observed, starting at day 5 in the TH1902-treated group. The level of luminescence intensity was also significantly lower in the free docetaxel-treated group, when compared to that in the vehicle-treated control group, but remained higher than that for the TH1902-treated group. Interestingly, tumor relapse was observed in the docetaxel-treated group beginning at day 46, whereas a sustained decrease of tumor volume was maintained in the TH1902-treated group until day 74, at which time point a complete disappearance of the tumor was achieved. Representative images of luminescence are shown for vehicle-, docetaxel-, or TH1902-treated mice at day 14 when the control group reached the tumor volume endpoint and at day 74 post-treatment at the end of the experiment. Quantification of the residual tumor burden at days 14 and 74 after docetaxel or TH1902 treatment was performed, and the analysis confirmed a much better in vivo TH1902 efficacy profile than was seen with unconjugated docetaxel. Given the lack of apoptotic or antiproliferative effects of TH19P01, no rationale supports its further assessment in vivo. As TH1902 is considered a new chemical entity, its best control condition therefore is the unconjugated docetaxel molecule itself. Body weight of mice on intraperitoneal administration of either docetaxel or TH1902 remained within endpoint limits (-20%, data not shown).

The use of intravenous administration of TH1902 was next investigated in the TNBC-derived MDA-MB-231 xenograft model. Mice subjected to docetaxel treatment were administered with three intravenous injections at 15 mg/kg/wk (MTD), whereas those treated with TH1902 received five injections of an equivalent dose of docetaxel at 35 mg/kg/wk. Similar to what was seen with the intraperitoneal administration protocol, a sustained decrease in tumor size was observed following intravenous administration of TH1902 until day 70, whereas a restart of tumor growth was observed at day 50 for the docetaxel-treated mice. When lower doses, equivalent to the quarter of the MTD, were used of docetaxel (3.75 mg/kg) and TH1902 (8.75 mg/kg), tumor growth as assessed by luminescence intensity was unaffected by intravenous administration of docetaxel, whereas it was significantly inhibited in the TH1902-treated group. This was further confirmed through the measurement of the tumor burden luminescence where TH1902 reduced it significantly in comparison to vehicle- or docetaxel-treated groups. Interestingly, body weight changes remained within endpoint limits in mice on intravenous administration of either docetaxel or TH1902 (data not shown). Similar conclusions were reached on testing another TNBC-derived HCC-70 xenograft model. In fact, administration of TH1902 at 8.75 mg/kg/wk led to a 93% inhibition of HCC-70 tumor growth as compared to 24% for docetaxel alone at an equivalent dose.

The use of intravenous administration of TH1902 was next investigated in the TNBC-derived MDA-MB-231 xenograft model. Mice subjected to docetaxel treatment were administered with three intravenous injections at 15 mg/kg/wk (MTD), whereas those treated with TH1902 received five injections of an equivalent dose of docetaxel at 35 mg/kg/wk. Similar to what was seen with the intraperitoneal administration protocol, a sustained decrease in tumor size was observed following intravenous administration of TH1902 until day 70, whereas a restart of tumor growth was observed at day 50 for the docetaxel-treated mice. When lower doses, equivalent to the quarter of the MTD, were used of docetaxel (3.75 mg/kg) and TH1902 (8.75 mg/kg), tumor growth as assessed by luminescence intensity was unaffected by intravenous administration of docetaxel, whereas it was significantly inhibited in the TH1902-treated group. This was further confirmed through the measurement of the tumor burden luminescence where TH1902 reduced it significantly in comparison to vehicle- or docetaxel-treated groups. Interestingly, body weight changes remained within endpoint limits in mice on intravenous administration of either docetaxel or TH1902 (data not shown). Similar conclusions were reached on testing another TNBC-derived HCC-70 xenograft model. In fact, administration of TH1902 at 8.75 mg/kg/wk led to a 93% inhibition of HCC-70 tumor growth as compared to 24% for docetaxel alone at an equivalent dose.

The use of intravenous administration of TH1902 was next investigated in the TNBC-derived MDA-MB-231 xenograft model. Mice subjected to docetaxel treatment were administered with three intravenous injections at 15 mg/kg/wk (MTD), whereas those treated with TH1902 received five injections of an equivalent dose of docetaxel at 35 mg/kg/wk. Similar to what was seen with the intraperitoneal administration protocol, a sustained decrease in tumor size was observed following intravenous administration of TH1902 until day 70, whereas a restart of tumor growth was observed at day 50 for the docetaxel-treated mice. When lower doses, equivalent to the quarter of the MTD, were used of docetaxel (3.75 mg/kg) and TH1902 (8.75 mg/kg), tumor growth as assessed by luminescence intensity was unaffected by intravenous administration of docetaxel, whereas it was significantly inhibited in the TH1902-treated group. This was further confirmed through the measurement of the tumor burden luminescence where TH1902 reduced it significantly in comparison to vehicle- or docetaxel-treated groups. Interestingly, body weight changes remained within endpoint limits in mice on intravenous administration of either docetaxel or TH1902 (data not shown). Similar conclusions were reached on testing another TNBC-derived HCC-70 xenograft model. In fact, administration of TH1902 at 8.75 mg/kg/wk led to a 93% inhibition of HCC-70 tumor growth as compared to 24% for docetaxel alone at an equivalent dose.

In vivo assessment in tumor-bearing animal models classically complements cell cultures data for comparing cancer treatment efficacies. For this reason, hTNBCSC and hOvCSC xenografts were implanted subcutaneously into immunodeficient mice as described in the Methods section with only 1000 cells given their highly tumorigenic nature. Three days later, the animals began receiving weekly IV bolus administration of either vehicle, docetaxel, or TH1902. Docetaxel was administered at a dose (15 mg/kg/week; for 3 cycles) in accordance with the estimated maximal tolerated dose (MTD) for mice, as well as at 1/4 of the MTD (3.75 mg/kg/week). TH1902 was administered at doses (35 and 8.75 mg/kg/week) which contained quantities of docetaxel equivalent to those in the two administrations of free docetaxel. From the size of the hTNBCSC and hOvCSC tumors, it is apparent that docetaxel had little impact on xenografts growth when administered neither at its MTD, for both hTNBCSC and hOvCSC, nor at 1/4 of this dosage as reflected by the growth curves. TH1902, when administered at a dosage equivalent to docetaxel at its MTD, provided greater tumor growth inhibition than did docetaxel for both xenograft models. Furthermore, higher dosage of administered TH1902 (up to 1.5 equivalent of docetaxel MTD) did not generate significant differences in terms of hOvCSC tumor inhibition without affecting mice body weights suggesting that TH1902, even at higher doses, is better tolerated compared to docetaxel. Then, in order to statistically compare the effects of docetaxel and TH1902 on tumor growth, the tumor sizes measured at the vehicle group endpoint were compared and statistically significant differences between the tumor sizes in vehicle-treated animals found in TH1902-treated animals for hTNBCSC and hOvCSC. Mouse body weight was used as an indicator of the morbidity associated with administration of docetaxel or TH1902. Administration of docetaxel at its MTD provoked a weight loss that approached 10% after the treatments, which is often observed in xenograft models with administration of docetaxel at this level. The body weights of animals treated with an equivalent quantity or a 1.5-fold equivalent of TH1902 were maintained at a roughly constant level throughout the experiment. The animals treated with vehicle recorded a slight weight gain (~5%) over this period while animals treated with the lower dosages of docetaxel or TH1902 were similar to the animals treated with vehicle. The body weight data indicates that TH1902 appears to be better tolerated than the equivalent quantity of free docetaxel, in addition to the fact that TH1902 is more efficacious than docetaxel when administered in vivo in the murine models of CSC tested.

The use of intravenous administration of TH1902 was next investigated in the TNBC-derived MDA-MB-231 xenograft model. Mice subjected to docetaxel treatment were administered with three intravenous injections at 15 mg/kg/wk (MTD), whereas those treated with TH1902 received five injections of an equivalent dose of docetaxel at 35 mg/kg/wk. Similar to what was seen with the intraperitoneal administration protocol, a sustained decrease in tumor size was observed following intravenous administration of TH1902 until day 70, whereas a restart of tumor growth was observed at day 50 for the docetaxel-treated mice. When lower doses, equivalent to the quarter of the MTD, were used of docetaxel (3.75 mg/kg) and TH1902 (8.75 mg/kg), tumor growth as assessed by luminescence intensity was unaffected by intravenous administration of docetaxel, whereas it was significantly inhibited in the TH1902-treated group. This was further confirmed through the measurement of the tumor burden luminescence where TH1902 reduced it significantly in comparison to vehicle- or docetaxel-treated groups. Interestingly, body weight changes remained within endpoint limits in mice on intravenous administration of either docetaxel or TH1902 (data not shown). Similar conclusions were reached on testing another TNBC-derived HCC-70 xenograft model. In fact, administration of TH1902 at 8.75 mg/kg/wk led to a 93% inhibition of HCC-70 tumor growth as compared to 24% for docetaxel alone at an equivalent dose.

The use of intravenous administration of TH1902 was next investigated in the TNBC-derived MDA-MB-231 xenograft model. Mice subjected to docetaxel treatment were administered with three intravenous injections at 15 mg/kg/wk (MTD), whereas those treated with TH1902 received five injections of an equivalent dose of docetaxel at 35 mg/kg/wk. Similar to what was seen with the intraperitoneal administration protocol, a sustained decrease in tumor size was observed following intravenous administration of TH1902 until day 70, whereas a restart of tumor growth was observed at day 50 for the docetaxel-treated mice. When lower doses, equivalent to the quarter of the MTD, were used of docetaxel (3.75 mg/kg) and TH1902 (8.75 mg/kg), tumor growth as assessed by luminescence intensity was unaffected by intravenous administration of docetaxel, whereas it was significantly inhibited in the TH1902-treated group. This was further confirmed through the measurement of the tumor burden luminescence where TH1902 reduced it significantly in comparison to vehicle- or docetaxel-treated groups. Interestingly, body weight changes remained within endpoint limits in mice on intravenous administration of either docetaxel or TH1902 (data not shown). Similar conclusions were reached on testing another TNBC-derived HCC-70 xenograft model. In fact, administration of TH1902 at 8.75 mg/kg/wk led to a 93% inhibition of HCC-70 tumor growth as compared to 24% for docetaxel alone at an equivalent dose.

The use of intravenous administration of TH1902 was next investigated in the TNBC-derived MDA-MB-231 xenograft model. Mice subjected to docetaxel treatment were administered with three intravenous injections at 15 mg/kg/wk (MTD), whereas those treated with TH1902 received five injections of an equivalent dose of docetaxel at 35 mg/kg/wk. Similar to what was seen with the intraperitoneal administration protocol, a sustained decrease in tumor size was observed following intravenous administration of TH1902 until day 70, whereas a restart of tumor growth was observed at day 50 for the docetaxel-treated mice. When lower doses, equivalent to the quarter of the MTD, were used of docetaxel (3.75 mg/kg) and TH1902 (8.75 mg/kg), tumor growth as assessed by luminescence intensity was unaffected by intravenous administration of docetaxel, whereas it was significantly inhibited in the TH1902-treated group. This was further confirmed through the measurement of the tumor burden luminescence where TH1902 reduced it significantly in comparison to vehicle- or docetaxel-treated groups. Interestingly, body weight changes remained within endpoint limits in mice on intravenous administration of either docetaxel or TH1902 (data not shown). Similar conclusions were reached on testing another TNBC-derived HCC-70 xenograft model. In fact, administration of TH1902 at 8.75 mg/kg/wk led to a 93% inhibition of HCC-70 tumor growth as compared to 24% for docetaxel alone at an equivalent dose.

To determine whether docetaxel or TH1902 inhibited TNBC cell proliferation, MTT assays were performed in MDA-MB-231 and HCC-70 cells exposed to various concentrations of TH19P01, docetaxel or TH1902. Inhibition of MDA-MB-231 cell proliferation was effectively triggered by both docetaxel and TH1902 compounds in a concentration-dependent manner whereas TH19P01 had no effect at the concentrations tested. The IC50 value of TH1902 was found to be comparable to that of docetaxel at low nM concentrations in both MDA-MB-231 and HCC-70 cells, which supports the rationale that conjugated docetaxel can indeed be released from TH1902 once internalized and exert its antiproliferative effect inside the targeted cancer cells. The effect of TH1902 on MBA-MB-231 cell-cycle arrest was also tested. The results show that more than 70% of treated cells were arrested in the G2/M phase of the cell cycle in comparison to vehicle-treated control cells where ~14% of the cells remained in the G2/M phase. While TH19P01 appears to be an inert peptide with no in vitro antiproliferative effects, these data confirm both the growth inhibitory effect of TH1902 and, more importantly, that the docetaxel anticancer potency was unaffected by its conjugation to the TH19P01 peptide.

Cells were thus exposed to various concentrations of TH1902 or docetaxel, and the effects on cell proliferation were measured using the MTT detection assay. It is apparent that both reagents exerted a cytotoxic effect with IC50 values of TH1902 comparable to that of docetaxel at low nM concentrations in the four cell lines tested. This supports the rationale that conjugated docetaxel can be released from TH1902 and exert its anti-proliferative effect inside the targeted cancer cells. Parallel experiment showed that cell proliferation was unaffected by the free peptide (TH19P01) at concentrations up to 1000 nM.

SORT1-positive SK-MEL-28 and B16-F10 melanoma cells were selected for testing the anti-proliferative effect of docetaxel and TH1902. When TH1902 biological effects were monitored, the half-maximal inhibitory concentration (IC50) of TH1902 was similar to that of docetaxel, averaging 0.38 vs. 0.39 nM, respectively, in human SK-MEL-28 cells, and 2.57 vs. 1.72 nM in murine B16-F10 cells.

Cells were thus exposed to various concentrations of TH1902 or docetaxel, and the effects on cell proliferation were measured using the MTT detection assay. It is apparent that both reagents exerted a cytotoxic effect with IC50 values of TH1902 comparable to that of docetaxel at low nM concentrations in the four cell lines tested. This supports the rationale that conjugated docetaxel can be released from TH1902 and exert its anti-proliferative effect inside the targeted cancer cells. Parallel experiment showed that cell proliferation was unaffected by the free peptide (TH19P01) at concentrations up to 1000 nM.

To determine whether docetaxel or TH1902 inhibited TNBC cell proliferation, MTT assays were performed in MDA-MB-231 and HCC-70 cells exposed to various concentrations of TH19P01, docetaxel or TH1902. Inhibition of MDA-MB-231 cell proliferation was effectively triggered by both docetaxel and TH1902 compounds in a concentration-dependent manner whereas TH19P01 had no effect at the concentrations tested. The IC50 value of TH1902 was found to be comparable to that of docetaxel at low nM concentrations in both MDA-MB-231 and HCC-70 cells, which supports the rationale that conjugated docetaxel can indeed be released from TH1902 once internalized and exert its antiproliferative effect inside the targeted cancer cells. The effect of TH1902 on MBA-MB-231 cell-cycle arrest was also tested. The results show that more than 70% of treated cells were arrested in the G2/M phase of the cell cycle in comparison to vehicle-treated control cells where ~14% of the cells remained in the G2/M phase. While TH19P01 appears to be an inert peptide with no in vitro antiproliferative effects, these data confirm both the growth inhibitory effect of TH1902 and, more importantly, that the docetaxel anticancer potency was unaffected by its conjugation to the TH19P01 peptide.

SORT1-positive SK-MEL-28 and B16-F10 melanoma cells were selected for testing the anti-proliferative effect of docetaxel and TH1902. When TH1902 biological effects were monitored, the half-maximal inhibitory concentration (IC50) of TH1902 was similar to that of docetaxel, averaging 0.38 vs. 0.39 nM, respectively, in human SK-MEL-28 cells, and 2.57 vs. 1.72 nM in murine B16-F10 cells.

Cells were thus exposed to various concentrations of TH1902 or docetaxel, and the effects on cell proliferation were measured using the MTT detection assay. It is apparent that both reagents exerted a cytotoxic effect with IC50 values of TH1902 comparable to that of docetaxel at low nM concentrations in the four cell lines tested. This supports the rationale that conjugated docetaxel can be released from TH1902 and exert its anti-proliferative effect inside the targeted cancer cells. Parallel experiment showed that cell proliferation was unaffected by the free peptide (TH19P01) at concentrations up to 1000 nM.

The impact of docetaxel and TH1902 on MDA-MB-231 cellular morphology was evaluated and visualized by light microscopy. A drastic change in cell shape was observed in both docetaxel- and TH1902-treated cells when compared to control cells. As already reported for docetaxel-treated human renal clear cell carcinoma, MDA-MB-231 cells treated with docetaxel appeared more contracted than control cells. Interestingly, TH1902-treated cell morphology appeared even smaller, less flattened, and presented greater inter-cell spacing in comparison to those treated with free docetaxel. This observation confirms the enhanced cytotoxic effect of TH1902 when compared to docetaxel. Docetaxel is further believed to exert its cytotoxic effects through both cell cycle regulation and apoptosis induction. MDA-MB-231 cells were therefore treated for 5 or 24 hours with various concentrations of either docetaxel or TH1902, followed by AnnexinV/PI staining. TH1902 increased MDA-MB-231 cell apoptosis in a dose-dependent manner when compared to docetaxel treatment. Less than 1% apoptotic cells were measured when cells were treated with 50 μM TH19P01 for 24 hours (data not shown). Overall, this suggests that, even within such a short time frame, receptor-mediated events account for the increased effects of TH1902. Finally, to confirm the role of SORT1 in TH1902 internalization and induced apoptosis, pharmacological inhibition of its apoptotic activity was measured after incubating MDA-MB-231 breast cancer cells with TH1902 in the presence or absence of the free unlabeled TH19P01 peptide or of one of the two different SORT1 ligands neurotensin or progranulin. The apoptosis assay showed that excess TH19P01 peptide, neurotensin or progranulin competitively reduced TH1902-induced cell death by 68%, 53% and 87%, respectively. This confirms the involvement of SORT1 in the internalization process of TH1902 leading to cell death in a TNBC cell model.

The impact of docetaxel and TH1902 on MDA-MB-231 cellular morphology was evaluated and visualized by light microscopy. A drastic change in cell shape was observed in both docetaxel- and TH1902-treated cells when compared to control cells. As already reported for docetaxel-treated human renal clear cell carcinoma, MDA-MB-231 cells treated with docetaxel appeared more contracted than control cells. Interestingly, TH1902-treated cell morphology appeared even smaller, less flattened, and presented greater inter-cell spacing in comparison to those treated with free docetaxel. This observation confirms the enhanced cytotoxic effect of TH1902 when compared to docetaxel. Docetaxel is further believed to exert its cytotoxic effects through both cell cycle regulation and apoptosis induction. MDA-MB-231 cells were therefore treated for 5 or 24 hours with various concentrations of either docetaxel or TH1902, followed by AnnexinV/PI staining. TH1902 increased MDA-MB-231 cell apoptosis in a dose-dependent manner when compared to docetaxel treatment. Less than 1% apoptotic cells were measured when cells were treated with 50 μM TH19P01 for 24 hours (data not shown). Overall, this suggests that, even within such a short time frame, receptor-mediated events account for the increased effects of TH1902. Finally, to confirm the role of SORT1 in TH1902 internalization and induced apoptosis, pharmacological inhibition of its apoptotic activity was measured after incubating MDA-MB-231 breast cancer cells with TH1902 in the presence or absence of the free unlabeled TH19P01 peptide or of one of the two different SORT1 ligands neurotensin or progranulin. The apoptosis assay showed that excess TH19P01 peptide, neurotensin or progranulin competitively reduced TH1902-induced cell death by 68%, 53% and 87%, respectively. This confirms the involvement of SORT1 in the internalization process of TH1902 leading to cell death in a TNBC cell model.

The impact of docetaxel and TH1902 on MDA-MB-231 cellular morphology was evaluated and visualized by light microscopy. A drastic change in cell shape was observed in both docetaxel- and TH1902-treated cells when compared to control cells. As already reported for docetaxel-treated human renal clear cell carcinoma, MDA-MB-231 cells treated with docetaxel appeared more contracted than control cells. Interestingly, TH1902-treated cell morphology appeared even smaller, less flattened, and presented greater inter-cell spacing in comparison to those treated with free docetaxel. This observation confirms the enhanced cytotoxic effect of TH1902 when compared to docetaxel. Docetaxel is further believed to exert its cytotoxic effects through both cell cycle regulation and apoptosis induction. MDA-MB-231 cells were therefore treated for 5 or 24 hours with various concentrations of either docetaxel or TH1902, followed by AnnexinV/PI staining. TH1902 increased MDA-MB-231 cell apoptosis in a dose-dependent manner when compared to docetaxel treatment. Less than 1% apoptotic cells were measured when cells were treated with 50 μM TH19P01 for 24 hours (data not shown). Overall, this suggests that, even within such a short time frame, receptor-mediated events account for the increased effects of TH1902. Finally, to confirm the role of SORT1 in TH1902 internalization and induced apoptosis, pharmacological inhibition of its apoptotic activity was measured after incubating MDA-MB-231 breast cancer cells with TH1902 in the presence or absence of the free unlabeled TH19P01 peptide or of one of the two different SORT1 ligands neurotensin or progranulin. The apoptosis assay showed that excess TH19P01 peptide, neurotensin or progranulin competitively reduced TH1902-induced cell death by 68%, 53% and 87%, respectively. This confirms the involvement of SORT1 in the internalization process of TH1902 leading to cell death in a TNBC cell model.

The impact of docetaxel and TH1902 on MDA-MB-231 cellular morphology was evaluated and visualized by light microscopy. A drastic change in cell shape was observed in both docetaxel- and TH1902-treated cells when compared to control cells. As already reported for docetaxel-treated human renal clear cell carcinoma, MDA-MB-231 cells treated with docetaxel appeared more contracted than control cells. Interestingly, TH1902-treated cell morphology appeared even smaller, less flattened, and presented greater inter-cell spacing in comparison to those treated with free docetaxel. This observation confirms the enhanced cytotoxic effect of TH1902 when compared to docetaxel. Docetaxel is further believed to exert its cytotoxic effects through both cell cycle regulation and apoptosis induction. MDA-MB-231 cells were therefore treated for 5 or 24 hours with various concentrations of either docetaxel or TH1902, followed by AnnexinV/PI staining. TH1902 increased MDA-MB-231 cell apoptosis in a dose-dependent manner when compared to docetaxel treatment. Less than 1% apoptotic cells were measured when cells were treated with 50 μM TH19P01 for 24 hours (data not shown). Overall, this suggests that, even within such a short time frame, receptor-mediated events account for the increased effects of TH1902. Finally, to confirm the role of SORT1 in TH1902 internalization and induced apoptosis, pharmacological inhibition of its apoptotic activity was measured after incubating MDA-MB-231 breast cancer cells with TH1902 in the presence or absence of the free unlabeled TH19P01 peptide or of one of the two different SORT1 ligands neurotensin or progranulin. The apoptosis assay showed that excess TH19P01 peptide, neurotensin or progranulin competitively reduced TH1902-induced cell death by 68%, 53% and 87%, respectively. This confirms the involvement of SORT1 in the internalization process of TH1902 leading to cell death in a TNBC cell model.

The results show that TH1902 induces more efficiently apoptosis in both cell lines when compared to docetaxel especially in SKOV3 cells. Overall, this suggests that receptor-mediated events appear to account for the increased effects of TH1902 within such a short time frame. To confirm the implication of SORT1 in TH1902 internalization and apoptosis induction, SORT1 was transiently silenced in ES-2 and SKOV3 cells.

The results show that TH1902 induces more efficiently apoptosis in both cell lines when compared to docetaxel especially in SKOV3 cells. Overall, this suggests that receptor-mediated events appear to account for the increased effects of TH1902 within such a short time frame. To confirm the implication of SORT1 in TH1902 internalization and apoptosis induction, SORT1 was transiently silenced in ES-2 and SKOV3 cells.

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)].

One of the limitations to the clinical use of AMPs as antimicrobial agents is their hemolytic activity which is associated with increased toxicity. Thus, as a first assessment of the MAAPC toxicity, we investigated whether the conjugates may cause lysis of human red blood cells (hRBCs). Notably, all MAAPCs exhibited negligible hemolytic activity against hRBCs (HC50 > 500 μM), which is the first indicator of the MAAPC safety toward eukaryotic cells. Bacterial and animal cell membranes significantly differ in composition. Indeed, microbial cell surfaces contain more anionic lipids (overall negatively charged) whereas mammalian cell membranes have more lipids with neutral zwitterionic head groups (overall neutrally charged). The MAAPCs were designed to selectively discriminate between bacterial and mammalian cells. The selectivity indexes (SIs), defined as HC50/MIC, for E. coli and S. aureus demonstrated great selectivity of all MAAPCs to bacteria over hRBCs, which implies that the MAAPCs are effective against bacteria without causing harm to human cells. MAAPC04 displayed the highest bacterial selectivity (SI > 640 and SI > 160 against E. coli and S. aureus, respectively) likely due to its reduced hydrophobic content.

One of the limitations to the clinical use of AMPs as antimicrobial agents is their hemolytic activity which is associated with increased toxicity. Thus, as a first assessment of the MAAPC toxicity, we investigated whether the conjugates may cause lysis of human red blood cells (hRBCs). Notably, all MAAPCs exhibited negligible hemolytic activity against hRBCs (HC50 > 500 μM), which is the first indicator of the MAAPC safety toward eukaryotic cells. Bacterial and animal cell membranes significantly differ in composition. Indeed, microbial cell surfaces contain more anionic lipids (overall negatively charged) whereas mammalian cell membranes have more lipids with neutral zwitterionic head groups (overall neutrally charged). The MAAPCs were designed to selectively discriminate between bacterial and mammalian cells. The selectivity indexes (SIs), defined as HC50/MIC, for E. coli and S. aureus demonstrated great selectivity of all MAAPCs to bacteria over hRBCs, which implies that the MAAPCs are effective against bacteria without causing harm to human cells. MAAPC04 displayed the highest bacterial selectivity (SI > 640 and SI > 160 against E. coli and S. aureus, respectively) likely due to its reduced hydrophobic content.

We first investigated the antimicrobial potency of these new MAAPCs by determining their minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) against Escherichia coli (E. coli MG1655) and Staphylococcus aureus (SA113) pathogens with a turbidity-based microdilution broth assay. Overall, all the MAAPCs display good antimicrobial activity and tend to be more selective to E. coli (MIC range 3.1-12.5 μM) over S. aureus (MIC range 12.5-25 μM), which follows the general observation that tobramycin is particularly active against Gram-negative organisms. Their MBC values are either equal to or slightly higher than their respective MIC values suggesting that the compounds not only are inhibiting bacterial growth but are also bactericidal. In contrast, the unconjugated peptides (P1-P4) exhibited no or little antimicrobial activity against E. coli and S. aureus. However, the MAAPCs did not show better activity than tobramycin, which is expected because our conjugates are not designed to target lab strains but instead to have high efficacy against clinically relevant pathogens such as persisters, resistant bacteria, and anaerobes that are impermeable to existing antibiotics. It should be noted that a mixture of the peptide itself with tobramycin does not enhance the antimicrobial activity compared to tobramycin alone, indicating there is no synergistic effect between the two entities when simply mixed. Whereas MAAPC05 showed similar activity against S. aureus in comparison to MAAPC01 (indicating that the terminus of conjugation did not greatly impact activity), it displayed a 2-fold increase of the MIC against E. coli. In contrast, MAAPC02, MAAPC03, and MAAPC04 displayed improved activity compared to MAAPC01. These results indicate the importance of the peptide transporter sequence as well as the conjugation site for tobramycin. Interestingly, MAAPC02 and MAAPC03 have nearly identical amino-acid composition as MAAPC01 (the only difference is a single glycine), but the amino acids are in a different sequence. In the MAAPC02 analog, the hydrophobic amino acids are clustered along one side of the -helical wheel of the peptide, which confers higher amphiphilic character and greater helical propensity. In the MAAPC03 analog, the peptide has the reversed sequence of MAAPC01. There are examples in the literature that the reversed analogs of antimicrobial peptides possess equal or enhanced antimicrobial activities. The amphiphilicity and peptide sequence orientation might play a role on the membrane-peptide interaction. However, further experiments are required to elucidate the reasons for this improved antibacterial activity.

We first investigated the antimicrobial potency of these new MAAPCs by determining their minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) against Escherichia coli (E. coli MG1655) and Staphylococcus aureus (SA113) pathogens with a turbidity-based microdilution broth assay. Overall, all the MAAPCs display good antimicrobial activity and tend to be more selective to E. coli (MIC range 3.1-12.5 μM) over S. aureus (MIC range 12.5-25 μM), which follows the general observation that tobramycin is particularly active against Gram-negative organisms. Their MBC values are either equal to or slightly higher than their respective MIC values suggesting that the compounds not only are inhibiting bacterial growth but are also bactericidal. In contrast, the unconjugated peptides (P1-P4) exhibited no or little antimicrobial activity against E. coli and S. aureus. However, the MAAPCs did not show better activity than tobramycin, which is expected because our conjugates are not designed to target lab strains but instead to have high efficacy against clinically relevant pathogens such as persisters, resistant bacteria, and anaerobes that are impermeable to existing antibiotics. It should be noted that a mixture of the peptide itself with tobramycin does not enhance the antimicrobial activity compared to tobramycin alone, indicating there is no synergistic effect between the two entities when simply mixed. Whereas MAAPC05 showed similar activity against S. aureus in comparison to MAAPC01 (indicating that the terminus of conjugation did not greatly impact activity), it displayed a 2-fold increase of the MIC against E. coli. In contrast, MAAPC02, MAAPC03, and MAAPC04 displayed improved activity compared to MAAPC01. These results indicate the importance of the peptide transporter sequence as well as the conjugation site for tobramycin. Interestingly, MAAPC02 and MAAPC03 have nearly identical amino-acid composition as MAAPC01 (the only difference is a single glycine), but the amino acids are in a different sequence. In the MAAPC02 analog, the hydrophobic amino acids are clustered along one side of the -helical wheel of the peptide, which confers higher amphiphilic character and greater helical propensity. In the MAAPC03 analog, the peptide has the reversed sequence of MAAPC01. There are examples in the literature that the reversed analogs of antimicrobial peptides possess equal or enhanced antimicrobial activities. The amphiphilicity and peptide sequence orientation might play a role on the membrane-peptide interaction. However, further experiments are required to elucidate the reasons for this improved antibacterial activity.

We first investigated the antimicrobial potency of these new MAAPCs by determining their minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) against Escherichia coli (E. coli MG1655) and Staphylococcus aureus (SA113) pathogens with a turbidity-based microdilution broth assay. Overall, all the MAAPCs display good antimicrobial activity and tend to be more selective to E. coli (MIC range 3.1-12.5 μM) over S. aureus (MIC range 12.5-25 μM), which follows the general observation that tobramycin is particularly active against Gram-negative organisms. Their MBC values are either equal to or slightly higher than their respective MIC values suggesting that the compounds not only are inhibiting bacterial growth but are also bactericidal. In contrast, the unconjugated peptides (P1-P4) exhibited no or little antimicrobial activity against E. coli and S. aureus. However, the MAAPCs did not show better activity than tobramycin, which is expected because our conjugates are not designed to target lab strains but instead to have high efficacy against clinically relevant pathogens such as persisters, resistant bacteria, and anaerobes that are impermeable to existing antibiotics. It should be noted that a mixture of the peptide itself with tobramycin does not enhance the antimicrobial activity compared to tobramycin alone, indicating there is no synergistic effect between the two entities when simply mixed. Whereas MAAPC05 showed similar activity against S. aureus in comparison to MAAPC01 (indicating that the terminus of conjugation did not greatly impact activity), it displayed a 2-fold increase of the MIC against E. coli. In contrast, MAAPC02, MAAPC03, and MAAPC04 displayed improved activity compared to MAAPC01. These results indicate the importance of the peptide transporter sequence as well as the conjugation site for tobramycin. Interestingly, MAAPC02 and MAAPC03 have nearly identical amino-acid composition as MAAPC01 (the only difference is a single glycine), but the amino acids are in a different sequence. In the MAAPC02 analog, the hydrophobic amino acids are clustered along one side of the -helical wheel of the peptide, which confers higher amphiphilic character and greater helical propensity. In the MAAPC03 analog, the peptide has the reversed sequence of MAAPC01. There are examples in the literature that the reversed analogs of antimicrobial peptides possess equal or enhanced antimicrobial activities. The amphiphilicity and peptide sequence orientation might play a role on the membrane-peptide interaction. However, further experiments are required to elucidate the reasons for this improved antibacterial activity.

We first investigated the antimicrobial potency of these new MAAPCs by determining their minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) against Escherichia coli (E. coli MG1655) and Staphylococcus aureus (SA113) pathogens with a turbidity-based microdilution broth assay. Overall, all the MAAPCs display good antimicrobial activity and tend to be more selective to E. coli (MIC range 3.1-12.5 μM) over S. aureus (MIC range 12.5-25 μM), which follows the general observation that tobramycin is particularly active against Gram-negative organisms. Their MBC values are either equal to or slightly higher than their respective MIC values suggesting that the compounds not only are inhibiting bacterial growth but are also bactericidal. In contrast, the unconjugated peptides (P1-P4) exhibited no or little antimicrobial activity against E. coli and S. aureus. However, the MAAPCs did not show better activity than tobramycin, which is expected because our conjugates are not designed to target lab strains but instead to have high efficacy against clinically relevant pathogens such as persisters, resistant bacteria, and anaerobes that are impermeable to existing antibiotics. It should be noted that a mixture of the peptide itself with tobramycin does not enhance the antimicrobial activity compared to tobramycin alone, indicating there is no synergistic effect between the two entities when simply mixed. Whereas MAAPC05 showed similar activity against S. aureus in comparison to MAAPC01 (indicating that the terminus of conjugation did not greatly impact activity), it displayed a 2-fold increase of the MIC against E. coli. In contrast, MAAPC02, MAAPC03, and MAAPC04 displayed improved activity compared to MAAPC01. These results indicate the importance of the peptide transporter sequence as well as the conjugation site for tobramycin. Interestingly, MAAPC02 and MAAPC03 have nearly identical amino-acid composition as MAAPC01 (the only difference is a single glycine), but the amino acids are in a different sequence. In the MAAPC02 analog, the hydrophobic amino acids are clustered along one side of the -helical wheel of the peptide, which confers higher amphiphilic character and greater helical propensity. In the MAAPC03 analog, the peptide has the reversed sequence of MAAPC01. There are examples in the literature that the reversed analogs of antimicrobial peptides possess equal or enhanced antimicrobial activities. The amphiphilicity and peptide sequence orientation might play a role on the membrane-peptide interaction. However, further experiments are required to elucidate the reasons for this improved antibacterial activity.

One of the limitations to the clinical use of AMPs as antimicrobial agents is their hemolytic activity which is associated with increased toxicity. Thus, as a first assessment of the MAAPC toxicity, we investigated whether the conjugates may cause lysis of human red blood cells (hRBCs). Notably, all MAAPCs exhibited negligible hemolytic activity against hRBCs (HC50 > 500 μM), which is the first indicator of the MAAPC safety toward eukaryotic cells. Bacterial and animal cell membranes significantly differ in composition. Indeed, microbial cell surfaces contain more anionic lipids (overall negatively charged) whereas mammalian cell membranes have more lipids with neutral zwitterionic head groups (overall neutrally charged). The MAAPCs were designed to selectively discriminate between bacterial and mammalian cells. The selectivity indexes (SIs), defined as HC50/MIC, for E. coli and S. aureus demonstrated great selectivity of all MAAPCs to bacteria over hRBCs, which implies that the MAAPCs are effective against bacteria without causing harm to human cells. MAAPC04 displayed the highest bacterial selectivity (SI > 640 and SI > 160 against E. coli and S. aureus, respectively) likely due to its reduced hydrophobic content.

One of the limitations to the clinical use of AMPs as antimicrobial agents is their hemolytic activity which is associated with increased toxicity. Thus, as a first assessment of the MAAPC toxicity, we investigated whether the conjugates may cause lysis of human red blood cells (hRBCs). Notably, all MAAPCs exhibited negligible hemolytic activity against hRBCs (HC50 > 500 μM), which is the first indicator of the MAAPC safety toward eukaryotic cells. Bacterial and animal cell membranes significantly differ in composition. Indeed, microbial cell surfaces contain more anionic lipids (overall negatively charged) whereas mammalian cell membranes have more lipids with neutral zwitterionic head groups (overall neutrally charged). The MAAPCs were designed to selectively discriminate between bacterial and mammalian cells. The selectivity indexes (SIs), defined as HC50/MIC, for E. coli and S. aureus demonstrated great selectivity of all MAAPCs to bacteria over hRBCs, which implies that the MAAPCs are effective against bacteria without causing harm to human cells. MAAPC04 displayed the highest bacterial selectivity (SI > 640 and SI > 160 against E. coli and S. aureus, respectively) likely due to its reduced hydrophobic content.

One of the limitations to the clinical use of AMPs as antimicrobial agents is their hemolytic activity which is associated with increased toxicity. Thus, as a first assessment of the MAAPC toxicity, we investigated whether the conjugates may cause lysis of human red blood cells (hRBCs). Notably, all MAAPCs exhibited negligible hemolytic activity against hRBCs (HC50 > 500 μM), which is the first indicator of the MAAPC safety toward eukaryotic cells. Bacterial and animal cell membranes significantly differ in composition. Indeed, microbial cell surfaces contain more anionic lipids (overall negatively charged) whereas mammalian cell membranes have more lipids with neutral zwitterionic head groups (overall neutrally charged). The MAAPCs were designed to selectively discriminate between bacterial and mammalian cells. The selectivity indexes (SIs), defined as HC50/MIC, for E. coli and S. aureus demonstrated great selectivity of all MAAPCs to bacteria over hRBCs, which implies that the MAAPCs are effective against bacteria without causing harm to human cells. MAAPC04 displayed the highest bacterial selectivity (SI > 640 and SI > 160 against E. coli and S. aureus, respectively) likely due to its reduced hydrophobic content.

We first investigated the antimicrobial potency of these new MAAPCs by determining their minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) against Escherichia coli (E. coli MG1655) and Staphylococcus aureus (SA113) pathogens with a turbidity-based microdilution broth assay. Overall, all the MAAPCs display good antimicrobial activity and tend to be more selective to E. coli (MIC range 3.1-12.5 μM) over S. aureus (MIC range 12.5-25 μM), which follows the general observation that tobramycin is particularly active against Gram-negative organisms. Their MBC values are either equal to or slightly higher than their respective MIC values suggesting that the compounds not only are inhibiting bacterial growth but are also bactericidal. In contrast, the unconjugated peptides (P1-P4) exhibited no or little antimicrobial activity against E. coli and S. aureus. However, the MAAPCs did not show better activity than tobramycin, which is expected because our conjugates are not designed to target lab strains but instead to have high efficacy against clinically relevant pathogens such as persisters, resistant bacteria, and anaerobes that are impermeable to existing antibiotics. It should be noted that a mixture of the peptide itself with tobramycin does not enhance the antimicrobial activity compared to tobramycin alone, indicating there is no synergistic effect between the two entities when simply mixed. Whereas MAAPC05 showed similar activity against S. aureus in comparison to MAAPC01 (indicating that the terminus of conjugation did not greatly impact activity), it displayed a 2-fold increase of the MIC against E. coli. In contrast, MAAPC02, MAAPC03, and MAAPC04 displayed improved activity compared to MAAPC01. These results indicate the importance of the peptide transporter sequence as well as the conjugation site for tobramycin. Interestingly, MAAPC02 and MAAPC03 have nearly identical amino-acid composition as MAAPC01 (the only difference is a single glycine), but the amino acids are in a different sequence. In the MAAPC02 analog, the hydrophobic amino acids are clustered along one side of the -helical wheel of the peptide, which confers higher amphiphilic character and greater helical propensity. In the MAAPC03 analog, the peptide has the reversed sequence of MAAPC01. There are examples in the literature that the reversed analogs of antimicrobial peptides possess equal or enhanced antimicrobial activities. The amphiphilicity and peptide sequence orientation might play a role on the membrane-peptide interaction. However, further experiments are required to elucidate the reasons for this improved antibacterial activity.

We first investigated the antimicrobial potency of these new MAAPCs by determining their minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) against Escherichia coli (E. coli MG1655) and Staphylococcus aureus (SA113) pathogens with a turbidity-based microdilution broth assay. Overall, all the MAAPCs display good antimicrobial activity and tend to be more selective to E. coli (MIC range 3.1-12.5 μM) over S. aureus (MIC range 12.5-25 μM), which follows the general observation that tobramycin is particularly active against Gram-negative organisms. Their MBC values are either equal to or slightly higher than their respective MIC values suggesting that the compounds not only are inhibiting bacterial growth but are also bactericidal. In contrast, the unconjugated peptides (P1-P4) exhibited no or little antimicrobial activity against E. coli and S. aureus. However, the MAAPCs did not show better activity than tobramycin, which is expected because our conjugates are not designed to target lab strains but instead to have high efficacy against clinically relevant pathogens such as persisters, resistant bacteria, and anaerobes that are impermeable to existing antibiotics. It should be noted that a mixture of the peptide itself with tobramycin does not enhance the antimicrobial activity compared to tobramycin alone, indicating there is no synergistic effect between the two entities when simply mixed. Whereas MAAPC05 showed similar activity against S. aureus in comparison to MAAPC01 (indicating that the terminus of conjugation did not greatly impact activity), it displayed a 2-fold increase of the MIC against E. coli. In contrast, MAAPC02, MAAPC03, and MAAPC04 displayed improved activity compared to MAAPC01. These results indicate the importance of the peptide transporter sequence as well as the conjugation site for tobramycin. Interestingly, MAAPC02 and MAAPC03 have nearly identical amino-acid composition as MAAPC01 (the only difference is a single glycine), but the amino acids are in a different sequence. In the MAAPC02 analog, the hydrophobic amino acids are clustered along one side of the -helical wheel of the peptide, which confers higher amphiphilic character and greater helical propensity. In the MAAPC03 analog, the peptide has the reversed sequence of MAAPC01. There are examples in the literature that the reversed analogs of antimicrobial peptides possess equal or enhanced antimicrobial activities. The amphiphilicity and peptide sequence orientation might play a role on the membrane-peptide interaction. However, further experiments are required to elucidate the reasons for this improved antibacterial activity.

We first investigated the antimicrobial potency of these new MAAPCs by determining their minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) against Escherichia coli (E. coli MG1655) and Staphylococcus aureus (SA113) pathogens with a turbidity-based microdilution broth assay. Overall, all the MAAPCs display good antimicrobial activity and tend to be more selective to E. coli (MIC range 3.1-12.5 μM) over S. aureus (MIC range 12.5-25 μM), which follows the general observation that tobramycin is particularly active against Gram-negative organisms. Their MBC values are either equal to or slightly higher than their respective MIC values suggesting that the compounds not only are inhibiting bacterial growth but are also bactericidal. In contrast, the unconjugated peptides (P1-P4) exhibited no or little antimicrobial activity against E. coli and S. aureus. However, the MAAPCs did not show better activity than tobramycin, which is expected because our conjugates are not designed to target lab strains but instead to have high efficacy against clinically relevant pathogens such as persisters, resistant bacteria, and anaerobes that are impermeable to existing antibiotics. It should be noted that a mixture of the peptide itself with tobramycin does not enhance the antimicrobial activity compared to tobramycin alone, indicating there is no synergistic effect between the two entities when simply mixed. Whereas MAAPC05 showed similar activity against S. aureus in comparison to MAAPC01 (indicating that the terminus of conjugation did not greatly impact activity), it displayed a 2-fold increase of the MIC against E. coli. In contrast, MAAPC02, MAAPC03, and MAAPC04 displayed improved activity compared to MAAPC01. These results indicate the importance of the peptide transporter sequence as well as the conjugation site for tobramycin. Interestingly, MAAPC02 and MAAPC03 have nearly identical amino-acid composition as MAAPC01 (the only difference is a single glycine), but the amino acids are in a different sequence. In the MAAPC02 analog, the hydrophobic amino acids are clustered along one side of the -helical wheel of the peptide, which confers higher amphiphilic character and greater helical propensity. In the MAAPC03 analog, the peptide has the reversed sequence of MAAPC01. There are examples in the literature that the reversed analogs of antimicrobial peptides possess equal or enhanced antimicrobial activities. The amphiphilicity and peptide sequence orientation might play a role on the membrane-peptide interaction. However, further experiments are required to elucidate the reasons for this improved antibacterial activity.

We first investigated the antimicrobial potency of these new MAAPCs by determining their minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) against Escherichia coli (E. coli MG1655) and Staphylococcus aureus (SA113) pathogens with a turbidity-based microdilution broth assay. Overall, all the MAAPCs display good antimicrobial activity and tend to be more selective to E. coli (MIC range 3.1-12.5 μM) over S. aureus (MIC range 12.5-25 μM), which follows the general observation that tobramycin is particularly active against Gram-negative organisms. Their MBC values are either equal to or slightly higher than their respective MIC values suggesting that the compounds not only are inhibiting bacterial growth but are also bactericidal. In contrast, the unconjugated peptides (P1-P4) exhibited no or little antimicrobial activity against E. coli and S. aureus. However, the MAAPCs did not show better activity than tobramycin, which is expected because our conjugates are not designed to target lab strains but instead to have high efficacy against clinically relevant pathogens such as persisters, resistant bacteria, and anaerobes that are impermeable to existing antibiotics. It should be noted that a mixture of the peptide itself with tobramycin does not enhance the antimicrobial activity compared to tobramycin alone, indicating there is no synergistic effect between the two entities when simply mixed. Whereas MAAPC05 showed similar activity against S. aureus in comparison to MAAPC01 (indicating that the terminus of conjugation did not greatly impact activity), it displayed a 2-fold increase of the MIC against E. coli. In contrast, MAAPC02, MAAPC03, and MAAPC04 displayed improved activity compared to MAAPC01. These results indicate the importance of the peptide transporter sequence as well as the conjugation site for tobramycin. Interestingly, MAAPC02 and MAAPC03 have nearly identical amino-acid composition as MAAPC01 (the only difference is a single glycine), but the amino acids are in a different sequence. In the MAAPC02 analog, the hydrophobic amino acids are clustered along one side of the -helical wheel of the peptide, which confers higher amphiphilic character and greater helical propensity. In the MAAPC03 analog, the peptide has the reversed sequence of MAAPC01. There are examples in the literature that the reversed analogs of antimicrobial peptides possess equal or enhanced antimicrobial activities. The amphiphilicity and peptide sequence orientation might play a role on the membrane-peptide interaction. However, further experiments are required to elucidate the reasons for this improved antibacterial activity.

One of the limitations to the clinical use of AMPs as antimicrobial agents is their hemolytic activity which is associated with increased toxicity. Thus, as a first assessment of the MAAPC toxicity, we investigated whether the conjugates may cause lysis of human red blood cells (hRBCs). Notably, all MAAPCs exhibited negligible hemolytic activity against hRBCs (HC50 > 500 μM), which is the first indicator of the MAAPC safety toward eukaryotic cells. Bacterial and animal cell membranes significantly differ in composition. Indeed, microbial cell surfaces contain more anionic lipids (overall negatively charged) whereas mammalian cell membranes have more lipids with neutral zwitterionic head groups (overall neutrally charged). The MAAPCs were designed to selectively discriminate between bacterial and mammalian cells. The selectivity indexes (SIs), defined as HC50/MIC, for E. coli and S. aureus demonstrated great selectivity of all MAAPCs to bacteria over hRBCs, which implies that the MAAPCs are effective against bacteria without causing harm to human cells. MAAPC04 displayed the highest bacterial selectivity (SI > 640 and SI > 160 against E. coli and S. aureus, respectively) likely due to its reduced hydrophobic content.

One of the limitations to the clinical use of AMPs as antimicrobial agents is their hemolytic activity which is associated with increased toxicity. Thus, as a first assessment of the MAAPC toxicity, we investigated whether the conjugates may cause lysis of human red blood cells (hRBCs). Notably, all MAAPCs exhibited negligible hemolytic activity against hRBCs (HC50 > 500 μM), which is the first indicator of the MAAPC safety toward eukaryotic cells. Bacterial and animal cell membranes significantly differ in composition. Indeed, microbial cell surfaces contain more anionic lipids (overall negatively charged) whereas mammalian cell membranes have more lipids with neutral zwitterionic head groups (overall neutrally charged). The MAAPCs were designed to selectively discriminate between bacterial and mammalian cells. The selectivity indexes (SIs), defined as HC50/MIC, for E. coli and S. aureus demonstrated great selectivity of all MAAPCs to bacteria over hRBCs, which implies that the MAAPCs are effective against bacteria without causing harm to human cells. MAAPC04 displayed the highest bacterial selectivity (SI > 640 and SI > 160 against E. coli and S. aureus, respectively) likely due to its reduced hydrophobic content.

One of the limitations to the clinical use of AMPs as antimicrobial agents is their hemolytic activity which is associated with increased toxicity. Thus, as a first assessment of the MAAPC toxicity, we investigated whether the conjugates may cause lysis of human red blood cells (hRBCs). Notably, all MAAPCs exhibited negligible hemolytic activity against hRBCs (HC50 > 500 μM), which is the first indicator of the MAAPC safety toward eukaryotic cells. Bacterial and animal cell membranes significantly differ in composition. Indeed, microbial cell surfaces contain more anionic lipids (overall negatively charged) whereas mammalian cell membranes have more lipids with neutral zwitterionic head groups (overall neutrally charged). The MAAPCs were designed to selectively discriminate between bacterial and mammalian cells. The selectivity indexes (SIs), defined as HC50/MIC, for E. coli and S. aureus demonstrated great selectivity of all MAAPCs to bacteria over hRBCs, which implies that the MAAPCs are effective against bacteria without causing harm to human cells. MAAPC04 displayed the highest bacterial selectivity (SI > 640 and SI > 160 against E. coli and S. aureus, respectively) likely due to its reduced hydrophobic content.

We first investigated the antimicrobial potency of these new MAAPCs by determining their minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) against Escherichia coli (E. coli MG1655) and Staphylococcus aureus (SA113) pathogens with a turbidity-based microdilution broth assay. Overall, all the MAAPCs display good antimicrobial activity and tend to be more selective to E. coli (MIC range 3.1-12.5 μM) over S. aureus (MIC range 12.5-25 μM), which follows the general observation that tobramycin is particularly active against Gram-negative organisms. Their MBC values are either equal to or slightly higher than their respective MIC values suggesting that the compounds not only are inhibiting bacterial growth but are also bactericidal. In contrast, the unconjugated peptides (P1-P4) exhibited no or little antimicrobial activity against E. coli and S. aureus. However, the MAAPCs did not show better activity than tobramycin, which is expected because our conjugates are not designed to target lab strains but instead to have high efficacy against clinically relevant pathogens such as persisters, resistant bacteria, and anaerobes that are impermeable to existing antibiotics. It should be noted that a mixture of the peptide itself with tobramycin does not enhance the antimicrobial activity compared to tobramycin alone, indicating there is no synergistic effect between the two entities when simply mixed. Whereas MAAPC05 showed similar activity against S. aureus in comparison to MAAPC01 (indicating that the terminus of conjugation did not greatly impact activity), it displayed a 2-fold increase of the MIC against E. coli. In contrast, MAAPC02, MAAPC03, and MAAPC04 displayed improved activity compared to MAAPC01. These results indicate the importance of the peptide transporter sequence as well as the conjugation site for tobramycin. Interestingly, MAAPC02 and MAAPC03 have nearly identical amino-acid composition as MAAPC01 (the only difference is a single glycine), but the amino acids are in a different sequence. In the MAAPC02 analog, the hydrophobic amino acids are clustered along one side of the -helical wheel of the peptide, which confers higher amphiphilic character and greater helical propensity. In the MAAPC03 analog, the peptide has the reversed sequence of MAAPC01. There are examples in the literature that the reversed analogs of antimicrobial peptides possess equal or enhanced antimicrobial activities. The amphiphilicity and peptide sequence orientation might play a role on the membrane-peptide interaction. However, further experiments are required to elucidate the reasons for this improved antibacterial activity.

We first investigated the antimicrobial potency of these new MAAPCs by determining their minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) against Escherichia coli (E. coli MG1655) and Staphylococcus aureus (SA113) pathogens with a turbidity-based microdilution broth assay. Overall, all the MAAPCs display good antimicrobial activity and tend to be more selective to E. coli (MIC range 3.1-12.5 μM) over S. aureus (MIC range 12.5-25 μM), which follows the general observation that tobramycin is particularly active against Gram-negative organisms. Their MBC values are either equal to or slightly higher than their respective MIC values suggesting that the compounds not only are inhibiting bacterial growth but are also bactericidal. In contrast, the unconjugated peptides (P1-P4) exhibited no or little antimicrobial activity against E. coli and S. aureus. However, the MAAPCs did not show better activity than tobramycin, which is expected because our conjugates are not designed to target lab strains but instead to have high efficacy against clinically relevant pathogens such as persisters, resistant bacteria, and anaerobes that are impermeable to existing antibiotics. It should be noted that a mixture of the peptide itself with tobramycin does not enhance the antimicrobial activity compared to tobramycin alone, indicating there is no synergistic effect between the two entities when simply mixed. Whereas MAAPC05 showed similar activity against S. aureus in comparison to MAAPC01 (indicating that the terminus of conjugation did not greatly impact activity), it displayed a 2-fold increase of the MIC against E. coli. In contrast, MAAPC02, MAAPC03, and MAAPC04 displayed improved activity compared to MAAPC01. These results indicate the importance of the peptide transporter sequence as well as the conjugation site for tobramycin. Interestingly, MAAPC02 and MAAPC03 have nearly identical amino-acid composition as MAAPC01 (the only difference is a single glycine), but the amino acids are in a different sequence. In the MAAPC02 analog, the hydrophobic amino acids are clustered along one side of the -helical wheel of the peptide, which confers higher amphiphilic character and greater helical propensity. In the MAAPC03 analog, the peptide has the reversed sequence of MAAPC01. There are examples in the literature that the reversed analogs of antimicrobial peptides possess equal or enhanced antimicrobial activities. The amphiphilicity and peptide sequence orientation might play a role on the membrane-peptide interaction. However, further experiments are required to elucidate the reasons for this improved antibacterial activity.

We first investigated the antimicrobial potency of these new MAAPCs by determining their minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) against Escherichia coli (E. coli MG1655) and Staphylococcus aureus (SA113) pathogens with a turbidity-based microdilution broth assay. Overall, all the MAAPCs display good antimicrobial activity and tend to be more selective to E. coli (MIC range 3.1-12.5 μM) over S. aureus (MIC range 12.5-25 μM), which follows the general observation that tobramycin is particularly active against Gram-negative organisms. Their MBC values are either equal to or slightly higher than their respective MIC values suggesting that the compounds not only are inhibiting bacterial growth but are also bactericidal. In contrast, the unconjugated peptides (P1-P4) exhibited no or little antimicrobial activity against E. coli and S. aureus. However, the MAAPCs did not show better activity than tobramycin, which is expected because our conjugates are not designed to target lab strains but instead to have high efficacy against clinically relevant pathogens such as persisters, resistant bacteria, and anaerobes that are impermeable to existing antibiotics. It should be noted that a mixture of the peptide itself with tobramycin does not enhance the antimicrobial activity compared to tobramycin alone, indicating there is no synergistic effect between the two entities when simply mixed. Whereas MAAPC05 showed similar activity against S. aureus in comparison to MAAPC01 (indicating that the terminus of conjugation did not greatly impact activity), it displayed a 2-fold increase of the MIC against E. coli. In contrast, MAAPC02, MAAPC03, and MAAPC04 displayed improved activity compared to MAAPC01. These results indicate the importance of the peptide transporter sequence as well as the conjugation site for tobramycin. Interestingly, MAAPC02 and MAAPC03 have nearly identical amino-acid composition as MAAPC01 (the only difference is a single glycine), but the amino acids are in a different sequence. In the MAAPC02 analog, the hydrophobic amino acids are clustered along one side of the -helical wheel of the peptide, which confers higher amphiphilic character and greater helical propensity. In the MAAPC03 analog, the peptide has the reversed sequence of MAAPC01. There are examples in the literature that the reversed analogs of antimicrobial peptides possess equal or enhanced antimicrobial activities. The amphiphilicity and peptide sequence orientation might play a role on the membrane-peptide interaction. However, further experiments are required to elucidate the reasons for this improved antibacterial activity.

We first investigated the antimicrobial potency of these new MAAPCs by determining their minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) against Escherichia coli (E. coli MG1655) and Staphylococcus aureus (SA113) pathogens with a turbidity-based microdilution broth assay. Overall, all the MAAPCs display good antimicrobial activity and tend to be more selective to E. coli (MIC range 3.1-12.5 μM) over S. aureus (MIC range 12.5-25 μM), which follows the general observation that tobramycin is particularly active against Gram-negative organisms. Their MBC values are either equal to or slightly higher than their respective MIC values suggesting that the compounds not only are inhibiting bacterial growth but are also bactericidal. In contrast, the unconjugated peptides (P1-P4) exhibited no or little antimicrobial activity against E. coli and S. aureus. However, the MAAPCs did not show better activity than tobramycin, which is expected because our conjugates are not designed to target lab strains but instead to have high efficacy against clinically relevant pathogens such as persisters, resistant bacteria, and anaerobes that are impermeable to existing antibiotics. It should be noted that a mixture of the peptide itself with tobramycin does not enhance the antimicrobial activity compared to tobramycin alone, indicating there is no synergistic effect between the two entities when simply mixed. Whereas MAAPC05 showed similar activity against S. aureus in comparison to MAAPC01 (indicating that the terminus of conjugation did not greatly impact activity), it displayed a 2-fold increase of the MIC against E. coli. In contrast, MAAPC02, MAAPC03, and MAAPC04 displayed improved activity compared to MAAPC01. These results indicate the importance of the peptide transporter sequence as well as the conjugation site for tobramycin. Interestingly, MAAPC02 and MAAPC03 have nearly identical amino-acid composition as MAAPC01 (the only difference is a single glycine), but the amino acids are in a different sequence. In the MAAPC02 analog, the hydrophobic amino acids are clustered along one side of the -helical wheel of the peptide, which confers higher amphiphilic character and greater helical propensity. In the MAAPC03 analog, the peptide has the reversed sequence of MAAPC01. There are examples in the literature that the reversed analogs of antimicrobial peptides possess equal or enhanced antimicrobial activities. The amphiphilicity and peptide sequence orientation might play a role on the membrane-peptide interaction. However, further experiments are required to elucidate the reasons for this improved antibacterial activity.

One of the limitations to the clinical use of AMPs as antimicrobial agents is their hemolytic activity which is associated with increased toxicity. Thus, as a first assessment of the MAAPC toxicity, we investigated whether the conjugates may cause lysis of human red blood cells (hRBCs). Notably, all MAAPCs exhibited negligible hemolytic activity against hRBCs (HC50 > 500 μM), which is the first indicator of the MAAPC safety toward eukaryotic cells. Bacterial and animal cell membranes significantly differ in composition. Indeed, microbial cell surfaces contain more anionic lipids (overall negatively charged) whereas mammalian cell membranes have more lipids with neutral zwitterionic head groups (overall neutrally charged). The MAAPCs were designed to selectively discriminate between bacterial and mammalian cells. The selectivity indexes (SIs), defined as HC50/MIC, for E. coli and S. aureus demonstrated great selectivity of all MAAPCs to bacteria over hRBCs, which implies that the MAAPCs are effective against bacteria without causing harm to human cells. MAAPC04 displayed the highest bacterial selectivity (SI > 640 and SI > 160 against E. coli and S. aureus, respectively) likely due to its reduced hydrophobic content.

One of the limitations to the clinical use of AMPs as antimicrobial agents is their hemolytic activity which is associated with increased toxicity. Thus, as a first assessment of the MAAPC toxicity, we investigated whether the conjugates may cause lysis of human red blood cells (hRBCs). Notably, all MAAPCs exhibited negligible hemolytic activity against hRBCs (HC50 > 500 μM), which is the first indicator of the MAAPC safety toward eukaryotic cells. Bacterial and animal cell membranes significantly differ in composition. Indeed, microbial cell surfaces contain more anionic lipids (overall negatively charged) whereas mammalian cell membranes have more lipids with neutral zwitterionic head groups (overall neutrally charged). The MAAPCs were designed to selectively discriminate between bacterial and mammalian cells. The selectivity indexes (SIs), defined as HC50/MIC, for E. coli and S. aureus demonstrated great selectivity of all MAAPCs to bacteria over hRBCs, which implies that the MAAPCs are effective against bacteria without causing harm to human cells. MAAPC04 displayed the highest bacterial selectivity (SI > 640 and SI > 160 against E. coli and S. aureus, respectively) likely due to its reduced hydrophobic content.

One of the limitations to the clinical use of AMPs as antimicrobial agents is their hemolytic activity which is associated with increased toxicity. Thus, as a first assessment of the MAAPC toxicity, we investigated whether the conjugates may cause lysis of human red blood cells (hRBCs). Notably, all MAAPCs exhibited negligible hemolytic activity against hRBCs (HC50 > 500 μM), which is the first indicator of the MAAPC safety toward eukaryotic cells. Bacterial and animal cell membranes significantly differ in composition. Indeed, microbial cell surfaces contain more anionic lipids (overall negatively charged) whereas mammalian cell membranes have more lipids with neutral zwitterionic head groups (overall neutrally charged). The MAAPCs were designed to selectively discriminate between bacterial and mammalian cells. The selectivity indexes (SIs), defined as HC50/MIC, for E. coli and S. aureus demonstrated great selectivity of all MAAPCs to bacteria over hRBCs, which implies that the MAAPCs are effective against bacteria without causing harm to human cells. MAAPC04 displayed the highest bacterial selectivity (SI > 640 and SI > 160 against E. coli and S. aureus, respectively) likely due to its reduced hydrophobic content.

We first investigated the antimicrobial potency of these new MAAPCs by determining their minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) against Escherichia coli (E. coli MG1655) and Staphylococcus aureus (SA113) pathogens with a turbidity-based microdilution broth assay. Overall, all the MAAPCs display good antimicrobial activity and tend to be more selective to E. coli (MIC range 3.1-12.5 μM) over S. aureus (MIC range 12.5-25 μM), which follows the general observation that tobramycin is particularly active against Gram-negative organisms. Their MBC values are either equal to or slightly higher than their respective MIC values suggesting that the compounds not only are inhibiting bacterial growth but are also bactericidal. In contrast, the unconjugated peptides (P1-P4) exhibited no or little antimicrobial activity against E. coli and S. aureus. However, the MAAPCs did not show better activity than tobramycin, which is expected because our conjugates are not designed to target lab strains but instead to have high efficacy against clinically relevant pathogens such as persisters, resistant bacteria, and anaerobes that are impermeable to existing antibiotics. It should be noted that a mixture of the peptide itself with tobramycin does not enhance the antimicrobial activity compared to tobramycin alone, indicating there is no synergistic effect between the two entities when simply mixed. Whereas MAAPC05 showed similar activity against S. aureus in comparison to MAAPC01 (indicating that the terminus of conjugation did not greatly impact activity), it displayed a 2-fold increase of the MIC against E. coli. In contrast, MAAPC02, MAAPC03, and MAAPC04 displayed improved activity compared to MAAPC01. These results indicate the importance of the peptide transporter sequence as well as the conjugation site for tobramycin. Interestingly, MAAPC02 and MAAPC03 have nearly identical amino-acid composition as MAAPC01 (the only difference is a single glycine), but the amino acids are in a different sequence. In the MAAPC02 analog, the hydrophobic amino acids are clustered along one side of the -helical wheel of the peptide, which confers higher amphiphilic character and greater helical propensity. In the MAAPC03 analog, the peptide has the reversed sequence of MAAPC01. There are examples in the literature that the reversed analogs of antimicrobial peptides possess equal or enhanced antimicrobial activities. The amphiphilicity and peptide sequence orientation might play a role on the membrane-peptide interaction. However, further experiments are required to elucidate the reasons for this improved antibacterial activity.

We first investigated the antimicrobial potency of these new MAAPCs by determining their minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) against Escherichia coli (E. coli MG1655) and Staphylococcus aureus (SA113) pathogens with a turbidity-based microdilution broth assay. Overall, all the MAAPCs display good antimicrobial activity and tend to be more selective to E. coli (MIC range 3.1-12.5 μM) over S. aureus (MIC range 12.5-25 μM), which follows the general observation that tobramycin is particularly active against Gram-negative organisms. Their MBC values are either equal to or slightly higher than their respective MIC values suggesting that the compounds not only are inhibiting bacterial growth but are also bactericidal. In contrast, the unconjugated peptides (P1-P4) exhibited no or little antimicrobial activity against E. coli and S. aureus. However, the MAAPCs did not show better activity than tobramycin, which is expected because our conjugates are not designed to target lab strains but instead to have high efficacy against clinically relevant pathogens such as persisters, resistant bacteria, and anaerobes that are impermeable to existing antibiotics. It should be noted that a mixture of the peptide itself with tobramycin does not enhance the antimicrobial activity compared to tobramycin alone, indicating there is no synergistic effect between the two entities when simply mixed. Whereas MAAPC05 showed similar activity against S. aureus in comparison to MAAPC01 (indicating that the terminus of conjugation did not greatly impact activity), it displayed a 2-fold increase of the MIC against E. coli. In contrast, MAAPC02, MAAPC03, and MAAPC04 displayed improved activity compared to MAAPC01. These results indicate the importance of the peptide transporter sequence as well as the conjugation site for tobramycin. Interestingly, MAAPC02 and MAAPC03 have nearly identical amino-acid composition as MAAPC01 (the only difference is a single glycine), but the amino acids are in a different sequence. In the MAAPC02 analog, the hydrophobic amino acids are clustered along one side of the -helical wheel of the peptide, which confers higher amphiphilic character and greater helical propensity. In the MAAPC03 analog, the peptide has the reversed sequence of MAAPC01. There are examples in the literature that the reversed analogs of antimicrobial peptides possess equal or enhanced antimicrobial activities. The amphiphilicity and peptide sequence orientation might play a role on the membrane-peptide interaction. However, further experiments are required to elucidate the reasons for this improved antibacterial activity.

We first investigated the antimicrobial potency of these new MAAPCs by determining their minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) against Escherichia coli (E. coli MG1655) and Staphylococcus aureus (SA113) pathogens with a turbidity-based microdilution broth assay. Overall, all the MAAPCs display good antimicrobial activity and tend to be more selective to E. coli (MIC range 3.1-12.5 μM) over S. aureus (MIC range 12.5-25 μM), which follows the general observation that tobramycin is particularly active against Gram-negative organisms. Their MBC values are either equal to or slightly higher than their respective MIC values suggesting that the compounds not only are inhibiting bacterial growth but are also bactericidal. In contrast, the unconjugated peptides (P1-P4) exhibited no or little antimicrobial activity against E. coli and S. aureus. However, the MAAPCs did not show better activity than tobramycin, which is expected because our conjugates are not designed to target lab strains but instead to have high efficacy against clinically relevant pathogens such as persisters, resistant bacteria, and anaerobes that are impermeable to existing antibiotics. It should be noted that a mixture of the peptide itself with tobramycin does not enhance the antimicrobial activity compared to tobramycin alone, indicating there is no synergistic effect between the two entities when simply mixed. Whereas MAAPC05 showed similar activity against S. aureus in comparison to MAAPC01 (indicating that the terminus of conjugation did not greatly impact activity), it displayed a 2-fold increase of the MIC against E. coli. In contrast, MAAPC02, MAAPC03, and MAAPC04 displayed improved activity compared to MAAPC01. These results indicate the importance of the peptide transporter sequence as well as the conjugation site for tobramycin. Interestingly, MAAPC02 and MAAPC03 have nearly identical amino-acid composition as MAAPC01 (the only difference is a single glycine), but the amino acids are in a different sequence. In the MAAPC02 analog, the hydrophobic amino acids are clustered along one side of the -helical wheel of the peptide, which confers higher amphiphilic character and greater helical propensity. In the MAAPC03 analog, the peptide has the reversed sequence of MAAPC01. There are examples in the literature that the reversed analogs of antimicrobial peptides possess equal or enhanced antimicrobial activities. The amphiphilicity and peptide sequence orientation might play a role on the membrane-peptide interaction. However, further experiments are required to elucidate the reasons for this improved antibacterial activity.

We first investigated the antimicrobial potency of these new MAAPCs by determining their minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) against Escherichia coli (E. coli MG1655) and Staphylococcus aureus (SA113) pathogens with a turbidity-based microdilution broth assay. Overall, all the MAAPCs display good antimicrobial activity and tend to be more selective to E. coli (MIC range 3.1-12.5 μM) over S. aureus (MIC range 12.5-25 μM), which follows the general observation that tobramycin is particularly active against Gram-negative organisms. Their MBC values are either equal to or slightly higher than their respective MIC values suggesting that the compounds not only are inhibiting bacterial growth but are also bactericidal. In contrast, the unconjugated peptides (P1-P4) exhibited no or little antimicrobial activity against E. coli and S. aureus. However, the MAAPCs did not show better activity than tobramycin, which is expected because our conjugates are not designed to target lab strains but instead to have high efficacy against clinically relevant pathogens such as persisters, resistant bacteria, and anaerobes that are impermeable to existing antibiotics. It should be noted that a mixture of the peptide itself with tobramycin does not enhance the antimicrobial activity compared to tobramycin alone, indicating there is no synergistic effect between the two entities when simply mixed. Whereas MAAPC05 showed similar activity against S. aureus in comparison to MAAPC01 (indicating that the terminus of conjugation did not greatly impact activity), it displayed a 2-fold increase of the MIC against E. coli. In contrast, MAAPC02, MAAPC03, and MAAPC04 displayed improved activity compared to MAAPC01. These results indicate the importance of the peptide transporter sequence as well as the conjugation site for tobramycin. Interestingly, MAAPC02 and MAAPC03 have nearly identical amino-acid composition as MAAPC01 (the only difference is a single glycine), but the amino acids are in a different sequence. In the MAAPC02 analog, the hydrophobic amino acids are clustered along one side of the -helical wheel of the peptide, which confers higher amphiphilic character and greater helical propensity. In the MAAPC03 analog, the peptide has the reversed sequence of MAAPC01. There are examples in the literature that the reversed analogs of antimicrobial peptides possess equal or enhanced antimicrobial activities. The amphiphilicity and peptide sequence orientation might play a role on the membrane-peptide interaction. However, further experiments are required to elucidate the reasons for this improved antibacterial activity.

One of the limitations to the clinical use of AMPs as antimicrobial agents is their hemolytic activity which is associated with increased toxicity. Thus, as a first assessment of the MAAPC toxicity, we investigated whether the conjugates may cause lysis of human red blood cells (hRBCs). Notably, all MAAPCs exhibited negligible hemolytic activity against hRBCs (HC50 > 500 μM), which is the first indicator of the MAAPC safety toward eukaryotic cells. Bacterial and animal cell membranes significantly differ in composition. Indeed, microbial cell surfaces contain more anionic lipids (overall negatively charged) whereas mammalian cell membranes have more lipids with neutral zwitterionic head groups (overall neutrally charged). The MAAPCs were designed to selectively discriminate between bacterial and mammalian cells. The selectivity indexes (SIs), defined as HC50/MIC, for E. coli and S. aureus demonstrated great selectivity of all MAAPCs to bacteria over hRBCs, which implies that the MAAPCs are effective against bacteria without causing harm to human cells. MAAPC04 displayed the highest bacterial selectivity (SI > 640 and SI > 160 against E. coli and S. aureus, respectively) likely due to its reduced hydrophobic content.

The tested compounds cytotoxic activity was determined using the MTT test, which is based on the ability to convert tetrazole salts to water insoluble formazan through mitochondrial dehydrogenases. Our results show a high cytotoxic effect on all pancreatic cancer cell lines. Exposing pancreatic cell lines MIA PaCa-2 and AsPC-1 to OGF-Gem decreased viability. Importantly, the OGF-Gem conjugate demonstrated a more pronounced cytotoxic effect against the metastatic pancreatic cancer cell line AsPC-1 compared to the commonly used chemotherapeutic agent. The results obtained for non-tumor-transformed cellsa human embryonic kidney line HEK-293 and human primary dermal fibroblast line HDFa presented a slight cytotoxicity effect from the OGF-Gem derivative within 3 days of incubation for all tested concentrations. Interestingly, an 80% reduction in HEK-293 cell viability was observed for the 100 nM Gem compared to the 100 nM OGF-Gem derivative. In HDFa cells, 100 nM Gem reduced viability to 35%, while the OGF-Gem conjugate slightly decreased the viability (to 75% viability) after 72 h of incubation. Based on the analysis of the results, concentrations of 3.125, 12.5, 50, and 100 nM, as well as an incubation time of 72 h, were selected for further experiments on the three pancreatic cancer cell lines.

The tested compounds cytotoxic activity was determined using the MTT test, which is based on the ability to convert tetrazole salts to water insoluble formazan through mitochondrial dehydrogenases. Our results show a high cytotoxic effect on all pancreatic cancer cell lines. Exposing pancreatic cell lines MIA PaCa-2 and AsPC-1 to OGF-Gem decreased viability. Importantly, the OGF-Gem conjugate demonstrated a more pronounced cytotoxic effect against the metastatic pancreatic cancer cell line AsPC-1 compared to the commonly used chemotherapeutic agent. The results obtained for non-tumor-transformed cellsa human embryonic kidney line HEK-293 and human primary dermal fibroblast line HDFa presented a slight cytotoxicity effect from the OGF-Gem derivative within 3 days of incubation for all tested concentrations. Interestingly, an 80% reduction in HEK-293 cell viability was observed for the 100 nM Gem compared to the 100 nM OGF-Gem derivative. In HDFa cells, 100 nM Gem reduced viability to 35%, while the OGF-Gem conjugate slightly decreased the viability (to 75% viability) after 72 h of incubation. Based on the analysis of the results, concentrations of 3.125, 12.5, 50, and 100 nM, as well as an incubation time of 72 h, were selected for further experiments on the three pancreatic cancer cell lines.

Furthermore, it was also observed that PDC shows time-dependent cytotoxicity against U87MG cells, as at 12 h drug treatment. PDC shows a slightly lower inhibitory effect on cell survival (EC50 = 25.82 ± 0.31 nM) than PTX alone (EC50 = 12.25 ± 0.13 nM), and CPP-SA alone shows higher EC50 = 54.37 ± 0.24 in U87MG cells. Besides this, it was previously observed that CPP-treated healthy VERO cells showed 88.05 ± 0.86% viable cells even using 10 uM concentration, indicating the specificity of CPP for glioblastoma cells. However, at 24 h incubation period, our PDC shows a significantly enhanced cell survival inhibitory effect with EC50 = 8.32 ± 0.09 nM, suggesting the delayed effect of PDC because of the time required for the intracellular pH to cause maximal cleavage of PTX from the PDC. Moreover, it also shows that CPP-SA has a lower cytotoxicity effect on glioblastoma cells compared with PTX alone and PDC, indicating its specificity as a carrier and selectivity for integrin receptors overexpressed on the cell surface. Additionally, we have also studied the potential of our novel-designed PDC to internalize into PTX-resistant glioblastoma (U87MG-PR) cells to show cytotoxic activity upon intracellular cleavage of PTX from CPP. The data presented in Figure 3b reveal approximately 15% viability increases in U87MG-PR cells compared with parent U87MG cells. It can be seen in the inset in Figure 3b that PTX alone (EC50 = 41.3 ± 1.5 nM) showed significantly 2-fold reduced cytotoxic activity in U87MG-PR cells in comparison with our novel-designed PDC having EC50 = 20.55 ± 1.02 nM. It was also observed in Figure 3b that at lower concentrations (0-10 nM) the viable cell count was ˜75%; however, it significantly decreased to ˜20% viability with an increase in concentration (40 nM) of the test samples, highlighting the dose-dependent cytotoxicity behavior of PDC in a 24 h incubation period.

Furthermore, it was also observed that PDC shows time-dependent cytotoxicity against U87MG cells, as at 12 h drug treatment. PDC shows a slightly lower inhibitory effect on cell survival (EC50 = 25.82 ± 0.31 nM) than PTX alone (EC50 = 12.25 ± 0.13 nM), and CPP-SA alone shows higher EC50 = 54.37 ± 0.24 in U87MG cells. Besides this, it was previously observed that CPP-treated healthy VERO cells showed 88.05 ± 0.86% viable cells even using 10 uM concentration, indicating the specificity of CPP for glioblastoma cells. However, at 24 h incubation period, our PDC shows a significantly enhanced cell survival inhibitory effect with EC50 = 8.32 ± 0.09 nM, suggesting the delayed effect of PDC because of the time required for the intracellular pH to cause maximal cleavage of PTX from the PDC. Moreover, it also shows that CPP-SA has a lower cytotoxicity effect on glioblastoma cells compared with PTX alone and PDC, indicating its specificity as a carrier and selectivity for integrin receptors overexpressed on the cell surface. Additionally, we have also studied the potential of our novel-designed PDC to internalize into PTX-resistant glioblastoma (U87MG-PR) cells to show cytotoxic activity upon intracellular cleavage of PTX from CPP. The data presented in Figure 3b reveal approximately 15% viability increases in U87MG-PR cells compared with parent U87MG cells. It can be seen in the inset in Figure 3b that PTX alone (EC50 = 41.3 ± 1.5 nM) showed significantly 2-fold reduced cytotoxic activity in U87MG-PR cells in comparison with our novel-designed PDC having EC50 = 20.55 ± 1.02 nM. It was also observed in Figure 3b that at lower concentrations (0-10 nM) the viable cell count was ˜75%; however, it significantly decreased to ˜20% viability with an increase in concentration (40 nM) of the test samples, highlighting the dose-dependent cytotoxicity behavior of PDC in a 24 h incubation period.