We decided to select some of the described peptides bearing the mentioned substitutions and having affirmed affinity for GRP-R in the low nanomolar range, together with the original BBN, and use them as targeting moiety to deliver a cytotoxic payload, daunorubicin (Dau), to prostate and breast cancers. Compared to the BBN sequence, our targeting peptides are elongated by a D-Phe in position 6 and comprise substitutions in positions 11, 13 and 14. Starting from these variations, we have also generated a new sequence: [D-Phe6, β-Ala11, Sta13, Nle14]BBN. The bombesin analogues have been published in the works of different research groups throughout many years, however, a direct comparison between them in terms of drug delivery has never been done. Once the targeting peptides bind to receptors that are overexpressed on the surface of cancer cells, such as GRP-R, the PDC can be carried inside the cells via receptor-mediated endocytosis. Briefly, the receptor-ligand complex internalises via a special, coated vesicle that later fuses with an early endosome and matures into a late endosome. After the late endosome fuses with a lysosome, the PDC is also digested, the drug is released into the cytoplasm and binds to its target compartment inside the cell. To ensure this release, we have decided to insert cathepsin B cleavable tetrapeptide linkers, namely either GFLG or LRRY, between the homing peptide and Dau. Indeed, cathepsin B is highly expressed by lysosomes and can cleave between Gly-Phe and Leu-Arg, liberating the active metabolites Dau=Aoa-Gly-OH or Dau=Aoa-Leu-OH. Dau is part of the family of anthracyclines and elicits its cytostatic activity by intercalating between DNA base pairs. This prevents the topoisomerase II from resealing the DNA double helix, resulting in reduced cell proliferation. Its attachment to a peptide can be easily performed through an oxime linkage in a very simple and straightforward reaction that occurs between an aminooxy moiety and the C13 ketone on Dau. The high yields of the reaction are advantageous, especially when it comes to the production of an increased amount of conjugate for in vivo studies. Furthermore, thanks to its intrinsic fluorescence, it is possible to evaluate the internalisation of the Dau-conjugates by fluorescence-activated cell sorting (FACS) and confocal laser scanning microscopy (CLSM), without the need to produce alternative peptide conjugates. To the best of our knowledge, so far, the delivery of a payload by bombesin-related peptides has only been studied using fluorescent molecules such as TAMRA, radioligands or nanocarriers. Drugs were attached to the peptides less often, and mainly using the original or truncated bombesin sequence instead of more promising analogues, or ones that are non-selective for GRP-R. Oppositely, investigating features such as the cellular uptake directly, through a conjugate attached to a chemotherapeutic, gives the most realistic picture of the success of a homing peptide acting as a drug carrier. Thus, the generated structures provide valuable tools for the selection of targeting peptides, aiming to the further development of new tumour-selective conjugates containing different linker-payload systems.

We decided to select some of the described peptides bearing the mentioned substitutions and having affirmed affinity for GRP-R in the low nanomolar range, together with the original BBN, and use them as targeting moiety to deliver a cytotoxic payload, daunorubicin (Dau), to prostate and breast cancers. Compared to the BBN sequence, our targeting peptides are elongated by a D-Phe in position 6 and comprise substitutions in positions 11, 13 and 14. Starting from these variations, we have also generated a new sequence: [D-Phe6, β-Ala11, Sta13, Nle14]BBN. The bombesin analogues have been published in the works of different research groups throughout many years, however, a direct comparison between them in terms of drug delivery has never been done. Once the targeting peptides bind to receptors that are overexpressed on the surface of cancer cells, such as GRP-R, the PDC can be carried inside the cells via receptor-mediated endocytosis. Briefly, the receptor-ligand complex internalises via a special, coated vesicle that later fuses with an early endosome and matures into a late endosome. After the late endosome fuses with a lysosome, the PDC is also digested, the drug is released into the cytoplasm and binds to its target compartment inside the cell. To ensure this release, we have decided to insert cathepsin B cleavable tetrapeptide linkers, namely either GFLG or LRRY, between the homing peptide and Dau. Indeed, cathepsin B is highly expressed by lysosomes and can cleave between Gly-Phe and Leu-Arg, liberating the active metabolites Dau=Aoa-Gly-OH or Dau=Aoa-Leu-OH. Dau is part of the family of anthracyclines and elicits its cytostatic activity by intercalating between DNA base pairs. This prevents the topoisomerase II from resealing the DNA double helix, resulting in reduced cell proliferation. Its attachment to a peptide can be easily performed through an oxime linkage in a very simple and straightforward reaction that occurs between an aminooxy moiety and the C13 ketone on Dau. The high yields of the reaction are advantageous, especially when it comes to the production of an increased amount of conjugate for in vivo studies. Furthermore, thanks to its intrinsic fluorescence, it is possible to evaluate the internalisation of the Dau-conjugates by fluorescence-activated cell sorting (FACS) and confocal laser scanning microscopy (CLSM), without the need to produce alternative peptide conjugates. To the best of our knowledge, so far, the delivery of a payload by bombesin-related peptides has only been studied using fluorescent molecules such as TAMRA, radioligands or nanocarriers. Drugs were attached to the peptides less often, and mainly using the original or truncated bombesin sequence instead of more promising analogues, or ones that are non-selective for GRP-R. Oppositely, investigating features such as the cellular uptake directly, through a conjugate attached to a chemotherapeutic, gives the most realistic picture of the success of a homing peptide acting as a drug carrier. Thus, the generated structures provide valuable tools for the selection of targeting peptides, aiming to the further development of new tumour-selective conjugates containing different linker-payload systems.

We decided to select some of the described peptides bearing the mentioned substitutions and having affirmed affinity for GRP-R in the low nanomolar range, together with the original BBN, and use them as targeting moiety to deliver a cytotoxic payload, daunorubicin (Dau), to prostate and breast cancers. Compared to the BBN sequence, our targeting peptides are elongated by a D-Phe in position 6 and comprise substitutions in positions 11, 13 and 14. Starting from these variations, we have also generated a new sequence: [D-Phe6, β-Ala11, Sta13, Nle14]BBN. The bombesin analogues have been published in the works of different research groups throughout many years, however, a direct comparison between them in terms of drug delivery has never been done. Once the targeting peptides bind to receptors that are overexpressed on the surface of cancer cells, such as GRP-R, the PDC can be carried inside the cells via receptor-mediated endocytosis. Briefly, the receptor-ligand complex internalises via a special, coated vesicle that later fuses with an early endosome and matures into a late endosome. After the late endosome fuses with a lysosome, the PDC is also digested, the drug is released into the cytoplasm and binds to its target compartment inside the cell. To ensure this release, we have decided to insert cathepsin B cleavable tetrapeptide linkers, namely either GFLG or LRRY, between the homing peptide and Dau. Indeed, cathepsin B is highly expressed by lysosomes and can cleave between Gly-Phe and Leu-Arg, liberating the active metabolites Dau=Aoa-Gly-OH or Dau=Aoa-Leu-OH. Dau is part of the family of anthracyclines and elicits its cytostatic activity by intercalating between DNA base pairs. This prevents the topoisomerase II from resealing the DNA double helix, resulting in reduced cell proliferation. Its attachment to a peptide can be easily performed through an oxime linkage in a very simple and straightforward reaction that occurs between an aminooxy moiety and the C13 ketone on Dau. The high yields of the reaction are advantageous, especially when it comes to the production of an increased amount of conjugate for in vivo studies. Furthermore, thanks to its intrinsic fluorescence, it is possible to evaluate the internalisation of the Dau-conjugates by fluorescence-activated cell sorting (FACS) and confocal laser scanning microscopy (CLSM), without the need to produce alternative peptide conjugates. To the best of our knowledge, so far, the delivery of a payload by bombesin-related peptides has only been studied using fluorescent molecules such as TAMRA, radioligands or nanocarriers. Drugs were attached to the peptides less often, and mainly using the original or truncated bombesin sequence instead of more promising analogues, or ones that are non-selective for GRP-R. Oppositely, investigating features such as the cellular uptake directly, through a conjugate attached to a chemotherapeutic, gives the most realistic picture of the success of a homing peptide acting as a drug carrier. Thus, the generated structures provide valuable tools for the selection of targeting peptides, aiming to the further development of new tumour-selective conjugates containing different linker-payload systems.

We decided to select some of the described peptides bearing the mentioned substitutions and having affirmed affinity for GRP-R in the low nanomolar range, together with the original BBN, and use them as targeting moiety to deliver a cytotoxic payload, daunorubicin (Dau), to prostate and breast cancers. Compared to the BBN sequence, our targeting peptides are elongated by a D-Phe in position 6 and comprise substitutions in positions 11, 13 and 14. Starting from these variations, we have also generated a new sequence: [D-Phe6, β-Ala11, Sta13, Nle14]BBN. The bombesin analogues have been published in the works of different research groups throughout many years, however, a direct comparison between them in terms of drug delivery has never been done. Once the targeting peptides bind to receptors that are overexpressed on the surface of cancer cells, such as GRP-R, the PDC can be carried inside the cells via receptor-mediated endocytosis. Briefly, the receptor-ligand complex internalises via a special, coated vesicle that later fuses with an early endosome and matures into a late endosome. After the late endosome fuses with a lysosome, the PDC is also digested, the drug is released into the cytoplasm and binds to its target compartment inside the cell. To ensure this release, we have decided to insert cathepsin B cleavable tetrapeptide linkers, namely either GFLG or LRRY, between the homing peptide and Dau. Indeed, cathepsin B is highly expressed by lysosomes and can cleave between Gly-Phe and Leu-Arg, liberating the active metabolites Dau=Aoa-Gly-OH or Dau=Aoa-Leu-OH. Dau is part of the family of anthracyclines and elicits its cytostatic activity by intercalating between DNA base pairs. This prevents the topoisomerase II from resealing the DNA double helix, resulting in reduced cell proliferation. Its attachment to a peptide can be easily performed through an oxime linkage in a very simple and straightforward reaction that occurs between an aminooxy moiety and the C13 ketone on Dau. The high yields of the reaction are advantageous, especially when it comes to the production of an increased amount of conjugate for in vivo studies. Furthermore, thanks to its intrinsic fluorescence, it is possible to evaluate the internalisation of the Dau-conjugates by fluorescence-activated cell sorting (FACS) and confocal laser scanning microscopy (CLSM), without the need to produce alternative peptide conjugates. To the best of our knowledge, so far, the delivery of a payload by bombesin-related peptides has only been studied using fluorescent molecules such as TAMRA, radioligands or nanocarriers. Drugs were attached to the peptides less often, and mainly using the original or truncated bombesin sequence instead of more promising analogues, or ones that are non-selective for GRP-R. Oppositely, investigating features such as the cellular uptake directly, through a conjugate attached to a chemotherapeutic, gives the most realistic picture of the success of a homing peptide acting as a drug carrier. Thus, the generated structures provide valuable tools for the selection of targeting peptides, aiming to the further development of new tumour-selective conjugates containing different linker-payload systems.

We decided to select some of the described peptides bearing the mentioned substitutions and having affirmed affinity for GRP-R in the low nanomolar range, together with the original BBN, and use them as targeting moiety to deliver a cytotoxic payload, daunorubicin (Dau), to prostate and breast cancers. Compared to the BBN sequence, our targeting peptides are elongated by a D-Phe in position 6 and comprise substitutions in positions 11, 13 and 14. Starting from these variations, we have also generated a new sequence: [D-Phe6, β-Ala11, Sta13, Nle14]BBN. The bombesin analogues have been published in the works of different research groups throughout many years, however, a direct comparison between them in terms of drug delivery has never been done. Once the targeting peptides bind to receptors that are overexpressed on the surface of cancer cells, such as GRP-R, the PDC can be carried inside the cells via receptor-mediated endocytosis. Briefly, the receptor-ligand complex internalises via a special, coated vesicle that later fuses with an early endosome and matures into a late endosome. After the late endosome fuses with a lysosome, the PDC is also digested, the drug is released into the cytoplasm and binds to its target compartment inside the cell. To ensure this release, we have decided to insert cathepsin B cleavable tetrapeptide linkers, namely either GFLG or LRRY, between the homing peptide and Dau. Indeed, cathepsin B is highly expressed by lysosomes and can cleave between Gly-Phe and Leu-Arg, liberating the active metabolites Dau=Aoa-Gly-OH or Dau=Aoa-Leu-OH. Dau is part of the family of anthracyclines and elicits its cytostatic activity by intercalating between DNA base pairs. This prevents the topoisomerase II from resealing the DNA double helix, resulting in reduced cell proliferation. Its attachment to a peptide can be easily performed through an oxime linkage in a very simple and straightforward reaction that occurs between an aminooxy moiety and the C13 ketone on Dau. The high yields of the reaction are advantageous, especially when it comes to the production of an increased amount of conjugate for in vivo studies. Furthermore, thanks to its intrinsic fluorescence, it is possible to evaluate the internalisation of the Dau-conjugates by fluorescence-activated cell sorting (FACS) and confocal laser scanning microscopy (CLSM), without the need to produce alternative peptide conjugates. To the best of our knowledge, so far, the delivery of a payload by bombesin-related peptides has only been studied using fluorescent molecules such as TAMRA, radioligands or nanocarriers. Drugs were attached to the peptides less often, and mainly using the original or truncated bombesin sequence instead of more promising analogues, or ones that are non-selective for GRP-R. Oppositely, investigating features such as the cellular uptake directly, through a conjugate attached to a chemotherapeutic, gives the most realistic picture of the success of a homing peptide acting as a drug carrier. Thus, the generated structures provide valuable tools for the selection of targeting peptides, aiming to the further development of new tumour-selective conjugates containing different linker-payload systems.

We decided to select some of the described peptides bearing the mentioned substitutions and having affirmed affinity for GRP-R in the low nanomolar range, together with the original BBN, and use them as targeting moiety to deliver a cytotoxic payload, daunorubicin (Dau), to prostate and breast cancers. Compared to the BBN sequence, our targeting peptides are elongated by a D-Phe in position 6 and comprise substitutions in positions 11, 13 and 14. Starting from these variations, we have also generated a new sequence: [D-Phe6, β-Ala11, Sta13, Nle14]BBN. The bombesin analogues have been published in the works of different research groups throughout many years, however, a direct comparison between them in terms of drug delivery has never been done. Once the targeting peptides bind to receptors that are overexpressed on the surface of cancer cells, such as GRP-R, the PDC can be carried inside the cells via receptor-mediated endocytosis. Briefly, the receptor-ligand complex internalises via a special, coated vesicle that later fuses with an early endosome and matures into a late endosome. After the late endosome fuses with a lysosome, the PDC is also digested, the drug is released into the cytoplasm and binds to its target compartment inside the cell. To ensure this release, we have decided to insert cathepsin B cleavable tetrapeptide linkers, namely either GFLG or LRRY, between the homing peptide and Dau. Indeed, cathepsin B is highly expressed by lysosomes and can cleave between Gly-Phe and Leu-Arg, liberating the active metabolites Dau=Aoa-Gly-OH or Dau=Aoa-Leu-OH. Dau is part of the family of anthracyclines and elicits its cytostatic activity by intercalating between DNA base pairs. This prevents the topoisomerase II from resealing the DNA double helix, resulting in reduced cell proliferation. Its attachment to a peptide can be easily performed through an oxime linkage in a very simple and straightforward reaction that occurs between an aminooxy moiety and the C13 ketone on Dau. The high yields of the reaction are advantageous, especially when it comes to the production of an increased amount of conjugate for in vivo studies. Furthermore, thanks to its intrinsic fluorescence, it is possible to evaluate the internalisation of the Dau-conjugates by fluorescence-activated cell sorting (FACS) and confocal laser scanning microscopy (CLSM), without the need to produce alternative peptide conjugates. To the best of our knowledge, so far, the delivery of a payload by bombesin-related peptides has only been studied using fluorescent molecules such as TAMRA, radioligands or nanocarriers. Drugs were attached to the peptides less often, and mainly using the original or truncated bombesin sequence instead of more promising analogues, or ones that are non-selective for GRP-R. Oppositely, investigating features such as the cellular uptake directly, through a conjugate attached to a chemotherapeutic, gives the most realistic picture of the success of a homing peptide acting as a drug carrier. Thus, the generated structures provide valuable tools for the selection of targeting peptides, aiming to the further development of new tumour-selective conjugates containing different linker-payload systems.

We decided to select some of the described peptides bearing the mentioned substitutions and having affirmed affinity for GRP-R in the low nanomolar range, together with the original BBN, and use them as targeting moiety to deliver a cytotoxic payload, daunorubicin (Dau), to prostate and breast cancers. Compared to the BBN sequence, our targeting peptides are elongated by a D-Phe in position 6 and comprise substitutions in positions 11, 13 and 14. Starting from these variations, we have also generated a new sequence: [D-Phe6, β-Ala11, Sta13, Nle14]BBN. The bombesin analogues have been published in the works of different research groups throughout many years, however, a direct comparison between them in terms of drug delivery has never been done. Once the targeting peptides bind to receptors that are overexpressed on the surface of cancer cells, such as GRP-R, the PDC can be carried inside the cells via receptor-mediated endocytosis. Briefly, the receptor-ligand complex internalises via a special, coated vesicle that later fuses with an early endosome and matures into a late endosome. After the late endosome fuses with a lysosome, the PDC is also digested, the drug is released into the cytoplasm and binds to its target compartment inside the cell. To ensure this release, we have decided to insert cathepsin B cleavable tetrapeptide linkers, namely either GFLG or LRRY, between the homing peptide and Dau. Indeed, cathepsin B is highly expressed by lysosomes and can cleave between Gly-Phe and Leu-Arg, liberating the active metabolites Dau=Aoa-Gly-OH or Dau=Aoa-Leu-OH. Dau is part of the family of anthracyclines and elicits its cytostatic activity by intercalating between DNA base pairs. This prevents the topoisomerase II from resealing the DNA double helix, resulting in reduced cell proliferation. Its attachment to a peptide can be easily performed through an oxime linkage in a very simple and straightforward reaction that occurs between an aminooxy moiety and the C13 ketone on Dau. The high yields of the reaction are advantageous, especially when it comes to the production of an increased amount of conjugate for in vivo studies. Furthermore, thanks to its intrinsic fluorescence, it is possible to evaluate the internalisation of the Dau-conjugates by fluorescence-activated cell sorting (FACS) and confocal laser scanning microscopy (CLSM), without the need to produce alternative peptide conjugates. To the best of our knowledge, so far, the delivery of a payload by bombesin-related peptides has only been studied using fluorescent molecules such as TAMRA, radioligands or nanocarriers. Drugs were attached to the peptides less often, and mainly using the original or truncated bombesin sequence instead of more promising analogues, or ones that are non-selective for GRP-R. Oppositely, investigating features such as the cellular uptake directly, through a conjugate attached to a chemotherapeutic, gives the most realistic picture of the success of a homing peptide acting as a drug carrier. Thus, the generated structures provide valuable tools for the selection of targeting peptides, aiming to the further development of new tumour-selective conjugates containing different linker-payload systems.

We decided to select some of the described peptides bearing the mentioned substitutions and having affirmed affinity for GRP-R in the low nanomolar range, together with the original BBN, and use them as targeting moiety to deliver a cytotoxic payload, daunorubicin (Dau), to prostate and breast cancers. Compared to the BBN sequence, our targeting peptides are elongated by a D-Phe in position 6 and comprise substitutions in positions 11, 13 and 14. Starting from these variations, we have also generated a new sequence: [D-Phe6, β-Ala11, Sta13, Nle14]BBN. The bombesin analogues have been published in the works of different research groups throughout many years, however, a direct comparison between them in terms of drug delivery has never been done. Once the targeting peptides bind to receptors that are overexpressed on the surface of cancer cells, such as GRP-R, the PDC can be carried inside the cells via receptor-mediated endocytosis. Briefly, the receptor-ligand complex internalises via a special, coated vesicle that later fuses with an early endosome and matures into a late endosome. After the late endosome fuses with a lysosome, the PDC is also digested, the drug is released into the cytoplasm and binds to its target compartment inside the cell. To ensure this release, we have decided to insert cathepsin B cleavable tetrapeptide linkers, namely either GFLG or LRRY, between the homing peptide and Dau. Indeed, cathepsin B is highly expressed by lysosomes and can cleave between Gly-Phe and Leu-Arg, liberating the active metabolites Dau=Aoa-Gly-OH or Dau=Aoa-Leu-OH. Dau is part of the family of anthracyclines and elicits its cytostatic activity by intercalating between DNA base pairs. This prevents the topoisomerase II from resealing the DNA double helix, resulting in reduced cell proliferation. Its attachment to a peptide can be easily performed through an oxime linkage in a very simple and straightforward reaction that occurs between an aminooxy moiety and the C13 ketone on Dau. The high yields of the reaction are advantageous, especially when it comes to the production of an increased amount of conjugate for in vivo studies. Furthermore, thanks to its intrinsic fluorescence, it is possible to evaluate the internalisation of the Dau-conjugates by fluorescence-activated cell sorting (FACS) and confocal laser scanning microscopy (CLSM), without the need to produce alternative peptide conjugates. To the best of our knowledge, so far, the delivery of a payload by bombesin-related peptides has only been studied using fluorescent molecules such as TAMRA, radioligands or nanocarriers. Drugs were attached to the peptides less often, and mainly using the original or truncated bombesin sequence instead of more promising analogues, or ones that are non-selective for GRP-R. Oppositely, investigating features such as the cellular uptake directly, through a conjugate attached to a chemotherapeutic, gives the most realistic picture of the success of a homing peptide acting as a drug carrier. Thus, the generated structures provide valuable tools for the selection of targeting peptides, aiming to the further development of new tumour-selective conjugates containing different linker-payload systems.

We decided to select some of the described peptides bearing the mentioned substitutions and having affirmed affinity for GRP-R in the low nanomolar range, together with the original BBN, and use them as targeting moiety to deliver a cytotoxic payload, daunorubicin (Dau), to prostate and breast cancers. Compared to the BBN sequence, our targeting peptides are elongated by a D-Phe in position 6 and comprise substitutions in positions 11, 13 and 14. Starting from these variations, we have also generated a new sequence: [D-Phe6, β-Ala11, Sta13, Nle14]BBN. The bombesin analogues have been published in the works of different research groups throughout many years, however, a direct comparison between them in terms of drug delivery has never been done. Once the targeting peptides bind to receptors that are overexpressed on the surface of cancer cells, such as GRP-R, the PDC can be carried inside the cells via receptor-mediated endocytosis. Briefly, the receptor-ligand complex internalises via a special, coated vesicle that later fuses with an early endosome and matures into a late endosome. After the late endosome fuses with a lysosome, the PDC is also digested, the drug is released into the cytoplasm and binds to its target compartment inside the cell. To ensure this release, we have decided to insert cathepsin B cleavable tetrapeptide linkers, namely either GFLG or LRRY, between the homing peptide and Dau. Indeed, cathepsin B is highly expressed by lysosomes and can cleave between Gly-Phe and Leu-Arg, liberating the active metabolites Dau=Aoa-Gly-OH or Dau=Aoa-Leu-OH. Dau is part of the family of anthracyclines and elicits its cytostatic activity by intercalating between DNA base pairs. This prevents the topoisomerase II from resealing the DNA double helix, resulting in reduced cell proliferation. Its attachment to a peptide can be easily performed through an oxime linkage in a very simple and straightforward reaction that occurs between an aminooxy moiety and the C13 ketone on Dau. The high yields of the reaction are advantageous, especially when it comes to the production of an increased amount of conjugate for in vivo studies. Furthermore, thanks to its intrinsic fluorescence, it is possible to evaluate the internalisation of the Dau-conjugates by fluorescence-activated cell sorting (FACS) and confocal laser scanning microscopy (CLSM), without the need to produce alternative peptide conjugates. To the best of our knowledge, so far, the delivery of a payload by bombesin-related peptides has only been studied using fluorescent molecules such as TAMRA, radioligands or nanocarriers. Drugs were attached to the peptides less often, and mainly using the original or truncated bombesin sequence instead of more promising analogues, or ones that are non-selective for GRP-R. Oppositely, investigating features such as the cellular uptake directly, through a conjugate attached to a chemotherapeutic, gives the most realistic picture of the success of a homing peptide acting as a drug carrier. Thus, the generated structures provide valuable tools for the selection of targeting peptides, aiming to the further development of new tumour-selective conjugates containing different linker-payload systems.

We decided to select some of the described peptides bearing the mentioned substitutions and having affirmed affinity for GRP-R in the low nanomolar range, together with the original BBN, and use them as targeting moiety to deliver a cytotoxic payload, daunorubicin (Dau), to prostate and breast cancers. Compared to the BBN sequence, our targeting peptides are elongated by a D-Phe in position 6 and comprise substitutions in positions 11, 13 and 14. Starting from these variations, we have also generated a new sequence: [D-Phe6, β-Ala11, Sta13, Nle14]BBN. The bombesin analogues have been published in the works of different research groups throughout many years, however, a direct comparison between them in terms of drug delivery has never been done. Once the targeting peptides bind to receptors that are overexpressed on the surface of cancer cells, such as GRP-R, the PDC can be carried inside the cells via receptor-mediated endocytosis. Briefly, the receptor-ligand complex internalises via a special, coated vesicle that later fuses with an early endosome and matures into a late endosome. After the late endosome fuses with a lysosome, the PDC is also digested, the drug is released into the cytoplasm and binds to its target compartment inside the cell. To ensure this release, we have decided to insert cathepsin B cleavable tetrapeptide linkers, namely either GFLG or LRRY, between the homing peptide and Dau. Indeed, cathepsin B is highly expressed by lysosomes and can cleave between Gly-Phe and Leu-Arg, liberating the active metabolites Dau=Aoa-Gly-OH or Dau=Aoa-Leu-OH. Dau is part of the family of anthracyclines and elicits its cytostatic activity by intercalating between DNA base pairs. This prevents the topoisomerase II from resealing the DNA double helix, resulting in reduced cell proliferation. Its attachment to a peptide can be easily performed through an oxime linkage in a very simple and straightforward reaction that occurs between an aminooxy moiety and the C13 ketone on Dau. The high yields of the reaction are advantageous, especially when it comes to the production of an increased amount of conjugate for in vivo studies. Furthermore, thanks to its intrinsic fluorescence, it is possible to evaluate the internalisation of the Dau-conjugates by fluorescence-activated cell sorting (FACS) and confocal laser scanning microscopy (CLSM), without the need to produce alternative peptide conjugates. To the best of our knowledge, so far, the delivery of a payload by bombesin-related peptides has only been studied using fluorescent molecules such as TAMRA, radioligands or nanocarriers. Drugs were attached to the peptides less often, and mainly using the original or truncated bombesin sequence instead of more promising analogues, or ones that are non-selective for GRP-R. Oppositely, investigating features such as the cellular uptake directly, through a conjugate attached to a chemotherapeutic, gives the most realistic picture of the success of a homing peptide acting as a drug carrier. Thus, the generated structures provide valuable tools for the selection of targeting peptides, aiming to the further development of new tumour-selective conjugates containing different linker-payload systems.

We decided to select some of the described peptides bearing the mentioned substitutions and having affirmed affinity for GRP-R in the low nanomolar range, together with the original BBN, and use them as targeting moiety to deliver a cytotoxic payload, daunorubicin (Dau), to prostate and breast cancers. Compared to the BBN sequence, our targeting peptides are elongated by a D-Phe in position 6 and comprise substitutions in positions 11, 13 and 14. Starting from these variations, we have also generated a new sequence: [D-Phe6, β-Ala11, Sta13, Nle14]BBN. The bombesin analogues have been published in the works of different research groups throughout many years, however, a direct comparison between them in terms of drug delivery has never been done. Once the targeting peptides bind to receptors that are overexpressed on the surface of cancer cells, such as GRP-R, the PDC can be carried inside the cells via receptor-mediated endocytosis. Briefly, the receptor-ligand complex internalises via a special, coated vesicle that later fuses with an early endosome and matures into a late endosome. After the late endosome fuses with a lysosome, the PDC is also digested, the drug is released into the cytoplasm and binds to its target compartment inside the cell. To ensure this release, we have decided to insert cathepsin B cleavable tetrapeptide linkers, namely either GFLG or LRRY, between the homing peptide and Dau. Indeed, cathepsin B is highly expressed by lysosomes and can cleave between Gly-Phe and Leu-Arg, liberating the active metabolites Dau=Aoa-Gly-OH or Dau=Aoa-Leu-OH. Dau is part of the family of anthracyclines and elicits its cytostatic activity by intercalating between DNA base pairs. This prevents the topoisomerase II from resealing the DNA double helix, resulting in reduced cell proliferation. Its attachment to a peptide can be easily performed through an oxime linkage in a very simple and straightforward reaction that occurs between an aminooxy moiety and the C13 ketone on Dau. The high yields of the reaction are advantageous, especially when it comes to the production of an increased amount of conjugate for in vivo studies. Furthermore, thanks to its intrinsic fluorescence, it is possible to evaluate the internalisation of the Dau-conjugates by fluorescence-activated cell sorting (FACS) and confocal laser scanning microscopy (CLSM), without the need to produce alternative peptide conjugates. To the best of our knowledge, so far, the delivery of a payload by bombesin-related peptides has only been studied using fluorescent molecules such as TAMRA, radioligands or nanocarriers. Drugs were attached to the peptides less often, and mainly using the original or truncated bombesin sequence instead of more promising analogues, or ones that are non-selective for GRP-R. Oppositely, investigating features such as the cellular uptake directly, through a conjugate attached to a chemotherapeutic, gives the most realistic picture of the success of a homing peptide acting as a drug carrier. Thus, the generated structures provide valuable tools for the selection of targeting peptides, aiming to the further development of new tumour-selective conjugates containing different linker-payload systems.

We decided to select some of the described peptides bearing the mentioned substitutions and having affirmed affinity for GRP-R in the low nanomolar range, together with the original BBN, and use them as targeting moiety to deliver a cytotoxic payload, daunorubicin (Dau), to prostate and breast cancers. Compared to the BBN sequence, our targeting peptides are elongated by a D-Phe in position 6 and comprise substitutions in positions 11, 13 and 14. Starting from these variations, we have also generated a new sequence: [D-Phe6, β-Ala11, Sta13, Nle14]BBN. The bombesin analogues have been published in the works of different research groups throughout many years, however, a direct comparison between them in terms of drug delivery has never been done. Once the targeting peptides bind to receptors that are overexpressed on the surface of cancer cells, such as GRP-R, the PDC can be carried inside the cells via receptor-mediated endocytosis. Briefly, the receptor-ligand complex internalises via a special, coated vesicle that later fuses with an early endosome and matures into a late endosome. After the late endosome fuses with a lysosome, the PDC is also digested, the drug is released into the cytoplasm and binds to its target compartment inside the cell. To ensure this release, we have decided to insert cathepsin B cleavable tetrapeptide linkers, namely either GFLG or LRRY, between the homing peptide and Dau. Indeed, cathepsin B is highly expressed by lysosomes and can cleave between Gly-Phe and Leu-Arg, liberating the active metabolites Dau=Aoa-Gly-OH or Dau=Aoa-Leu-OH. Dau is part of the family of anthracyclines and elicits its cytostatic activity by intercalating between DNA base pairs. This prevents the topoisomerase II from resealing the DNA double helix, resulting in reduced cell proliferation. Its attachment to a peptide can be easily performed through an oxime linkage in a very simple and straightforward reaction that occurs between an aminooxy moiety and the C13 ketone on Dau. The high yields of the reaction are advantageous, especially when it comes to the production of an increased amount of conjugate for in vivo studies. Furthermore, thanks to its intrinsic fluorescence, it is possible to evaluate the internalisation of the Dau-conjugates by fluorescence-activated cell sorting (FACS) and confocal laser scanning microscopy (CLSM), without the need to produce alternative peptide conjugates. To the best of our knowledge, so far, the delivery of a payload by bombesin-related peptides has only been studied using fluorescent molecules such as TAMRA, radioligands or nanocarriers. Drugs were attached to the peptides less often, and mainly using the original or truncated bombesin sequence instead of more promising analogues, or ones that are non-selective for GRP-R. Oppositely, investigating features such as the cellular uptake directly, through a conjugate attached to a chemotherapeutic, gives the most realistic picture of the success of a homing peptide acting as a drug carrier. Thus, the generated structures provide valuable tools for the selection of targeting peptides, aiming to the further development of new tumour-selective conjugates containing different linker-payload systems.

We decided to select some of the described peptides bearing the mentioned substitutions and having affirmed affinity for GRP-R in the low nanomolar range, together with the original BBN, and use them as targeting moiety to deliver a cytotoxic payload, daunorubicin (Dau), to prostate and breast cancers. Compared to the BBN sequence, our targeting peptides are elongated by a D-Phe in position 6 and comprise substitutions in positions 11, 13 and 14. Starting from these variations, we have also generated a new sequence: [D-Phe6, β-Ala11, Sta13, Nle14]BBN. The bombesin analogues have been published in the works of different research groups throughout many years, however, a direct comparison between them in terms of drug delivery has never been done. Once the targeting peptides bind to receptors that are overexpressed on the surface of cancer cells, such as GRP-R, the PDC can be carried inside the cells via receptor-mediated endocytosis. Briefly, the receptor-ligand complex internalises via a special, coated vesicle that later fuses with an early endosome and matures into a late endosome. After the late endosome fuses with a lysosome, the PDC is also digested, the drug is released into the cytoplasm and binds to its target compartment inside the cell. To ensure this release, we have decided to insert cathepsin B cleavable tetrapeptide linkers, namely either GFLG or LRRY, between the homing peptide and Dau. Indeed, cathepsin B is highly expressed by lysosomes and can cleave between Gly-Phe and Leu-Arg, liberating the active metabolites Dau=Aoa-Gly-OH or Dau=Aoa-Leu-OH. Dau is part of the family of anthracyclines and elicits its cytostatic activity by intercalating between DNA base pairs. This prevents the topoisomerase II from resealing the DNA double helix, resulting in reduced cell proliferation. Its attachment to a peptide can be easily performed through an oxime linkage in a very simple and straightforward reaction that occurs between an aminooxy moiety and the C13 ketone on Dau. The high yields of the reaction are advantageous, especially when it comes to the production of an increased amount of conjugate for in vivo studies. Furthermore, thanks to its intrinsic fluorescence, it is possible to evaluate the internalisation of the Dau-conjugates by fluorescence-activated cell sorting (FACS) and confocal laser scanning microscopy (CLSM), without the need to produce alternative peptide conjugates. To the best of our knowledge, so far, the delivery of a payload by bombesin-related peptides has only been studied using fluorescent molecules such as TAMRA, radioligands or nanocarriers. Drugs were attached to the peptides less often, and mainly using the original or truncated bombesin sequence instead of more promising analogues, or ones that are non-selective for GRP-R. Oppositely, investigating features such as the cellular uptake directly, through a conjugate attached to a chemotherapeutic, gives the most realistic picture of the success of a homing peptide acting as a drug carrier. Thus, the generated structures provide valuable tools for the selection of targeting peptides, aiming to the further development of new tumour-selective conjugates containing different linker-payload systems.

We decided to select some of the described peptides bearing the mentioned substitutions and having affirmed affinity for GRP-R in the low nanomolar range, together with the original BBN, and use them as targeting moiety to deliver a cytotoxic payload, daunorubicin (Dau), to prostate and breast cancers. Compared to the BBN sequence, our targeting peptides are elongated by a D-Phe in position 6 and comprise substitutions in positions 11, 13 and 14. Starting from these variations, we have also generated a new sequence: [D-Phe6, β-Ala11, Sta13, Nle14]BBN. The bombesin analogues have been published in the works of different research groups throughout many years, however, a direct comparison between them in terms of drug delivery has never been done. Once the targeting peptides bind to receptors that are overexpressed on the surface of cancer cells, such as GRP-R, the PDC can be carried inside the cells via receptor-mediated endocytosis. Briefly, the receptor-ligand complex internalises via a special, coated vesicle that later fuses with an early endosome and matures into a late endosome. After the late endosome fuses with a lysosome, the PDC is also digested, the drug is released into the cytoplasm and binds to its target compartment inside the cell. To ensure this release, we have decided to insert cathepsin B cleavable tetrapeptide linkers, namely either GFLG or LRRY, between the homing peptide and Dau. Indeed, cathepsin B is highly expressed by lysosomes and can cleave between Gly-Phe and Leu-Arg, liberating the active metabolites Dau=Aoa-Gly-OH or Dau=Aoa-Leu-OH. Dau is part of the family of anthracyclines and elicits its cytostatic activity by intercalating between DNA base pairs. This prevents the topoisomerase II from resealing the DNA double helix, resulting in reduced cell proliferation. Its attachment to a peptide can be easily performed through an oxime linkage in a very simple and straightforward reaction that occurs between an aminooxy moiety and the C13 ketone on Dau. The high yields of the reaction are advantageous, especially when it comes to the production of an increased amount of conjugate for in vivo studies. Furthermore, thanks to its intrinsic fluorescence, it is possible to evaluate the internalisation of the Dau-conjugates by fluorescence-activated cell sorting (FACS) and confocal laser scanning microscopy (CLSM), without the need to produce alternative peptide conjugates. To the best of our knowledge, so far, the delivery of a payload by bombesin-related peptides has only been studied using fluorescent molecules such as TAMRA, radioligands or nanocarriers. Drugs were attached to the peptides less often, and mainly using the original or truncated bombesin sequence instead of more promising analogues, or ones that are non-selective for GRP-R. Oppositely, investigating features such as the cellular uptake directly, through a conjugate attached to a chemotherapeutic, gives the most realistic picture of the success of a homing peptide acting as a drug carrier. Thus, the generated structures provide valuable tools for the selection of targeting peptides, aiming to the further development of new tumour-selective conjugates containing different linker-payload systems.

We decided to select some of the described peptides bearing the mentioned substitutions and having affirmed affinity for GRP-R in the low nanomolar range, together with the original BBN, and use them as targeting moiety to deliver a cytotoxic payload, daunorubicin (Dau), to prostate and breast cancers. Compared to the BBN sequence, our targeting peptides are elongated by a D-Phe in position 6 and comprise substitutions in positions 11, 13 and 14. Starting from these variations, we have also generated a new sequence: [D-Phe6, β-Ala11, Sta13, Nle14]BBN. The bombesin analogues have been published in the works of different research groups throughout many years, however, a direct comparison between them in terms of drug delivery has never been done. Once the targeting peptides bind to receptors that are overexpressed on the surface of cancer cells, such as GRP-R, the PDC can be carried inside the cells via receptor-mediated endocytosis. Briefly, the receptor-ligand complex internalises via a special, coated vesicle that later fuses with an early endosome and matures into a late endosome. After the late endosome fuses with a lysosome, the PDC is also digested, the drug is released into the cytoplasm and binds to its target compartment inside the cell. To ensure this release, we have decided to insert cathepsin B cleavable tetrapeptide linkers, namely either GFLG or LRRY, between the homing peptide and Dau. Indeed, cathepsin B is highly expressed by lysosomes and can cleave between Gly-Phe and Leu-Arg, liberating the active metabolites Dau=Aoa-Gly-OH or Dau=Aoa-Leu-OH. Dau is part of the family of anthracyclines and elicits its cytostatic activity by intercalating between DNA base pairs. This prevents the topoisomerase II from resealing the DNA double helix, resulting in reduced cell proliferation. Its attachment to a peptide can be easily performed through an oxime linkage in a very simple and straightforward reaction that occurs between an aminooxy moiety and the C13 ketone on Dau. The high yields of the reaction are advantageous, especially when it comes to the production of an increased amount of conjugate for in vivo studies. Furthermore, thanks to its intrinsic fluorescence, it is possible to evaluate the internalisation of the Dau-conjugates by fluorescence-activated cell sorting (FACS) and confocal laser scanning microscopy (CLSM), without the need to produce alternative peptide conjugates. To the best of our knowledge, so far, the delivery of a payload by bombesin-related peptides has only been studied using fluorescent molecules such as TAMRA, radioligands or nanocarriers. Drugs were attached to the peptides less often, and mainly using the original or truncated bombesin sequence instead of more promising analogues, or ones that are non-selective for GRP-R. Oppositely, investigating features such as the cellular uptake directly, through a conjugate attached to a chemotherapeutic, gives the most realistic picture of the success of a homing peptide acting as a drug carrier. Thus, the generated structures provide valuable tools for the selection of targeting peptides, aiming to the further development of new tumour-selective conjugates containing different linker-payload systems.

We decided to select some of the described peptides bearing the mentioned substitutions and having affirmed affinity for GRP-R in the low nanomolar range, together with the original BBN, and use them as targeting moiety to deliver a cytotoxic payload, daunorubicin (Dau), to prostate and breast cancers. Compared to the BBN sequence, our targeting peptides are elongated by a D-Phe in position 6 and comprise substitutions in positions 11, 13 and 14. Starting from these variations, we have also generated a new sequence: [D-Phe6, β-Ala11, Sta13, Nle14]BBN. The bombesin analogues have been published in the works of different research groups throughout many years, however, a direct comparison between them in terms of drug delivery has never been done. Once the targeting peptides bind to receptors that are overexpressed on the surface of cancer cells, such as GRP-R, the PDC can be carried inside the cells via receptor-mediated endocytosis. Briefly, the receptor-ligand complex internalises via a special, coated vesicle that later fuses with an early endosome and matures into a late endosome. After the late endosome fuses with a lysosome, the PDC is also digested, the drug is released into the cytoplasm and binds to its target compartment inside the cell. To ensure this release, we have decided to insert cathepsin B cleavable tetrapeptide linkers, namely either GFLG or LRRY, between the homing peptide and Dau. Indeed, cathepsin B is highly expressed by lysosomes and can cleave between Gly-Phe and Leu-Arg, liberating the active metabolites Dau=Aoa-Gly-OH or Dau=Aoa-Leu-OH. Dau is part of the family of anthracyclines and elicits its cytostatic activity by intercalating between DNA base pairs. This prevents the topoisomerase II from resealing the DNA double helix, resulting in reduced cell proliferation. Its attachment to a peptide can be easily performed through an oxime linkage in a very simple and straightforward reaction that occurs between an aminooxy moiety and the C13 ketone on Dau. The high yields of the reaction are advantageous, especially when it comes to the production of an increased amount of conjugate for in vivo studies. Furthermore, thanks to its intrinsic fluorescence, it is possible to evaluate the internalisation of the Dau-conjugates by fluorescence-activated cell sorting (FACS) and confocal laser scanning microscopy (CLSM), without the need to produce alternative peptide conjugates. To the best of our knowledge, so far, the delivery of a payload by bombesin-related peptides has only been studied using fluorescent molecules such as TAMRA, radioligands or nanocarriers. Drugs were attached to the peptides less often, and mainly using the original or truncated bombesin sequence instead of more promising analogues, or ones that are non-selective for GRP-R. Oppositely, investigating features such as the cellular uptake directly, through a conjugate attached to a chemotherapeutic, gives the most realistic picture of the success of a homing peptide acting as a drug carrier. Thus, the generated structures provide valuable tools for the selection of targeting peptides, aiming to the further development of new tumour-selective conjugates containing different linker-payload systems.

We decided to select some of the described peptides bearing the mentioned substitutions and having affirmed affinity for GRP-R in the low nanomolar range, together with the original BBN, and use them as targeting moiety to deliver a cytotoxic payload, daunorubicin (Dau), to prostate and breast cancers. Compared to the BBN sequence, our targeting peptides are elongated by a D-Phe in position 6 and comprise substitutions in positions 11, 13 and 14. Starting from these variations, we have also generated a new sequence: [D-Phe6, β-Ala11, Sta13, Nle14]BBN. The bombesin analogues have been published in the works of different research groups throughout many years, however, a direct comparison between them in terms of drug delivery has never been done. Once the targeting peptides bind to receptors that are overexpressed on the surface of cancer cells, such as GRP-R, the PDC can be carried inside the cells via receptor-mediated endocytosis. Briefly, the receptor-ligand complex internalises via a special, coated vesicle that later fuses with an early endosome and matures into a late endosome. After the late endosome fuses with a lysosome, the PDC is also digested, the drug is released into the cytoplasm and binds to its target compartment inside the cell. To ensure this release, we have decided to insert cathepsin B cleavable tetrapeptide linkers, namely either GFLG or LRRY, between the homing peptide and Dau. Indeed, cathepsin B is highly expressed by lysosomes and can cleave between Gly-Phe and Leu-Arg, liberating the active metabolites Dau=Aoa-Gly-OH or Dau=Aoa-Leu-OH. Dau is part of the family of anthracyclines and elicits its cytostatic activity by intercalating between DNA base pairs. This prevents the topoisomerase II from resealing the DNA double helix, resulting in reduced cell proliferation. Its attachment to a peptide can be easily performed through an oxime linkage in a very simple and straightforward reaction that occurs between an aminooxy moiety and the C13 ketone on Dau. The high yields of the reaction are advantageous, especially when it comes to the production of an increased amount of conjugate for in vivo studies. Furthermore, thanks to its intrinsic fluorescence, it is possible to evaluate the internalisation of the Dau-conjugates by fluorescence-activated cell sorting (FACS) and confocal laser scanning microscopy (CLSM), without the need to produce alternative peptide conjugates. To the best of our knowledge, so far, the delivery of a payload by bombesin-related peptides has only been studied using fluorescent molecules such as TAMRA, radioligands or nanocarriers. Drugs were attached to the peptides less often, and mainly using the original or truncated bombesin sequence instead of more promising analogues, or ones that are non-selective for GRP-R. Oppositely, investigating features such as the cellular uptake directly, through a conjugate attached to a chemotherapeutic, gives the most realistic picture of the success of a homing peptide acting as a drug carrier. Thus, the generated structures provide valuable tools for the selection of targeting peptides, aiming to the further development of new tumour-selective conjugates containing different linker-payload systems.

We decided to select some of the described peptides bearing the mentioned substitutions and having affirmed affinity for GRP-R in the low nanomolar range, together with the original BBN, and use them as targeting moiety to deliver a cytotoxic payload, daunorubicin (Dau), to prostate and breast cancers. Compared to the BBN sequence, our targeting peptides are elongated by a D-Phe in position 6 and comprise substitutions in positions 11, 13 and 14. Starting from these variations, we have also generated a new sequence: [D-Phe6, β-Ala11, Sta13, Nle14]BBN. The bombesin analogues have been published in the works of different research groups throughout many years, however, a direct comparison between them in terms of drug delivery has never been done. Once the targeting peptides bind to receptors that are overexpressed on the surface of cancer cells, such as GRP-R, the PDC can be carried inside the cells via receptor-mediated endocytosis. Briefly, the receptor-ligand complex internalises via a special, coated vesicle that later fuses with an early endosome and matures into a late endosome. After the late endosome fuses with a lysosome, the PDC is also digested, the drug is released into the cytoplasm and binds to its target compartment inside the cell. To ensure this release, we have decided to insert cathepsin B cleavable tetrapeptide linkers, namely either GFLG or LRRY, between the homing peptide and Dau. Indeed, cathepsin B is highly expressed by lysosomes and can cleave between Gly-Phe and Leu-Arg, liberating the active metabolites Dau=Aoa-Gly-OH or Dau=Aoa-Leu-OH. Dau is part of the family of anthracyclines and elicits its cytostatic activity by intercalating between DNA base pairs. This prevents the topoisomerase II from resealing the DNA double helix, resulting in reduced cell proliferation. Its attachment to a peptide can be easily performed through an oxime linkage in a very simple and straightforward reaction that occurs between an aminooxy moiety and the C13 ketone on Dau. The high yields of the reaction are advantageous, especially when it comes to the production of an increased amount of conjugate for in vivo studies. Furthermore, thanks to its intrinsic fluorescence, it is possible to evaluate the internalisation of the Dau-conjugates by fluorescence-activated cell sorting (FACS) and confocal laser scanning microscopy (CLSM), without the need to produce alternative peptide conjugates. To the best of our knowledge, so far, the delivery of a payload by bombesin-related peptides has only been studied using fluorescent molecules such as TAMRA, radioligands or nanocarriers. Drugs were attached to the peptides less often, and mainly using the original or truncated bombesin sequence instead of more promising analogues, or ones that are non-selective for GRP-R. Oppositely, investigating features such as the cellular uptake directly, through a conjugate attached to a chemotherapeutic, gives the most realistic picture of the success of a homing peptide acting as a drug carrier. Thus, the generated structures provide valuable tools for the selection of targeting peptides, aiming to the further development of new tumour-selective conjugates containing different linker-payload systems.

We decided to select some of the described peptides bearing the mentioned substitutions and having affirmed affinity for GRP-R in the low nanomolar range, together with the original BBN, and use them as targeting moiety to deliver a cytotoxic payload, daunorubicin (Dau), to prostate and breast cancers. Compared to the BBN sequence, our targeting peptides are elongated by a D-Phe in position 6 and comprise substitutions in positions 11, 13 and 14. Starting from these variations, we have also generated a new sequence: [D-Phe6, β-Ala11, Sta13, Nle14]BBN. The bombesin analogues have been published in the works of different research groups throughout many years, however, a direct comparison between them in terms of drug delivery has never been done. Once the targeting peptides bind to receptors that are overexpressed on the surface of cancer cells, such as GRP-R, the PDC can be carried inside the cells via receptor-mediated endocytosis. Briefly, the receptor-ligand complex internalises via a special, coated vesicle that later fuses with an early endosome and matures into a late endosome. After the late endosome fuses with a lysosome, the PDC is also digested, the drug is released into the cytoplasm and binds to its target compartment inside the cell. To ensure this release, we have decided to insert cathepsin B cleavable tetrapeptide linkers, namely either GFLG or LRRY, between the homing peptide and Dau. Indeed, cathepsin B is highly expressed by lysosomes and can cleave between Gly-Phe and Leu-Arg, liberating the active metabolites Dau=Aoa-Gly-OH or Dau=Aoa-Leu-OH. Dau is part of the family of anthracyclines and elicits its cytostatic activity by intercalating between DNA base pairs. This prevents the topoisomerase II from resealing the DNA double helix, resulting in reduced cell proliferation. Its attachment to a peptide can be easily performed through an oxime linkage in a very simple and straightforward reaction that occurs between an aminooxy moiety and the C13 ketone on Dau. The high yields of the reaction are advantageous, especially when it comes to the production of an increased amount of conjugate for in vivo studies. Furthermore, thanks to its intrinsic fluorescence, it is possible to evaluate the internalisation of the Dau-conjugates by fluorescence-activated cell sorting (FACS) and confocal laser scanning microscopy (CLSM), without the need to produce alternative peptide conjugates. To the best of our knowledge, so far, the delivery of a payload by bombesin-related peptides has only been studied using fluorescent molecules such as TAMRA, radioligands or nanocarriers. Drugs were attached to the peptides less often, and mainly using the original or truncated bombesin sequence instead of more promising analogues, or ones that are non-selective for GRP-R. Oppositely, investigating features such as the cellular uptake directly, through a conjugate attached to a chemotherapeutic, gives the most realistic picture of the success of a homing peptide acting as a drug carrier. Thus, the generated structures provide valuable tools for the selection of targeting peptides, aiming to the further development of new tumour-selective conjugates containing different linker-payload systems.

We decided to select some of the described peptides bearing the mentioned substitutions and having affirmed affinity for GRP-R in the low nanomolar range, together with the original BBN, and use them as targeting moiety to deliver a cytotoxic payload, daunorubicin (Dau), to prostate and breast cancers. Compared to the BBN sequence, our targeting peptides are elongated by a D-Phe in position 6 and comprise substitutions in positions 11, 13 and 14. Starting from these variations, we have also generated a new sequence: [D-Phe6, β-Ala11, Sta13, Nle14]BBN. The bombesin analogues have been published in the works of different research groups throughout many years, however, a direct comparison between them in terms of drug delivery has never been done. Once the targeting peptides bind to receptors that are overexpressed on the surface of cancer cells, such as GRP-R, the PDC can be carried inside the cells via receptor-mediated endocytosis. Briefly, the receptor-ligand complex internalises via a special, coated vesicle that later fuses with an early endosome and matures into a late endosome. After the late endosome fuses with a lysosome, the PDC is also digested, the drug is released into the cytoplasm and binds to its target compartment inside the cell. To ensure this release, we have decided to insert cathepsin B cleavable tetrapeptide linkers, namely either GFLG or LRRY, between the homing peptide and Dau. Indeed, cathepsin B is highly expressed by lysosomes and can cleave between Gly-Phe and Leu-Arg, liberating the active metabolites Dau=Aoa-Gly-OH or Dau=Aoa-Leu-OH. Dau is part of the family of anthracyclines and elicits its cytostatic activity by intercalating between DNA base pairs. This prevents the topoisomerase II from resealing the DNA double helix, resulting in reduced cell proliferation. Its attachment to a peptide can be easily performed through an oxime linkage in a very simple and straightforward reaction that occurs between an aminooxy moiety and the C13 ketone on Dau. The high yields of the reaction are advantageous, especially when it comes to the production of an increased amount of conjugate for in vivo studies. Furthermore, thanks to its intrinsic fluorescence, it is possible to evaluate the internalisation of the Dau-conjugates by fluorescence-activated cell sorting (FACS) and confocal laser scanning microscopy (CLSM), without the need to produce alternative peptide conjugates. To the best of our knowledge, so far, the delivery of a payload by bombesin-related peptides has only been studied using fluorescent molecules such as TAMRA, radioligands or nanocarriers. Drugs were attached to the peptides less often, and mainly using the original or truncated bombesin sequence instead of more promising analogues, or ones that are non-selective for GRP-R. Oppositely, investigating features such as the cellular uptake directly, through a conjugate attached to a chemotherapeutic, gives the most realistic picture of the success of a homing peptide acting as a drug carrier. Thus, the generated structures provide valuable tools for the selection of targeting peptides, aiming to the further development of new tumour-selective conjugates containing different linker-payload systems.

We decided to select some of the described peptides bearing the mentioned substitutions and having affirmed affinity for GRP-R in the low nanomolar range, together with the original BBN, and use them as targeting moiety to deliver a cytotoxic payload, daunorubicin (Dau), to prostate and breast cancers. Compared to the BBN sequence, our targeting peptides are elongated by a D-Phe in position 6 and comprise substitutions in positions 11, 13 and 14. Starting from these variations, we have also generated a new sequence: [D-Phe6, β-Ala11, Sta13, Nle14]BBN. The bombesin analogues have been published in the works of different research groups throughout many years, however, a direct comparison between them in terms of drug delivery has never been done. Once the targeting peptides bind to receptors that are overexpressed on the surface of cancer cells, such as GRP-R, the PDC can be carried inside the cells via receptor-mediated endocytosis. Briefly, the receptor-ligand complex internalises via a special, coated vesicle that later fuses with an early endosome and matures into a late endosome. After the late endosome fuses with a lysosome, the PDC is also digested, the drug is released into the cytoplasm and binds to its target compartment inside the cell. To ensure this release, we have decided to insert cathepsin B cleavable tetrapeptide linkers, namely either GFLG or LRRY, between the homing peptide and Dau. Indeed, cathepsin B is highly expressed by lysosomes and can cleave between Gly-Phe and Leu-Arg, liberating the active metabolites Dau=Aoa-Gly-OH or Dau=Aoa-Leu-OH. Dau is part of the family of anthracyclines and elicits its cytostatic activity by intercalating between DNA base pairs. This prevents the topoisomerase II from resealing the DNA double helix, resulting in reduced cell proliferation. Its attachment to a peptide can be easily performed through an oxime linkage in a very simple and straightforward reaction that occurs between an aminooxy moiety and the C13 ketone on Dau. The high yields of the reaction are advantageous, especially when it comes to the production of an increased amount of conjugate for in vivo studies. Furthermore, thanks to its intrinsic fluorescence, it is possible to evaluate the internalisation of the Dau-conjugates by fluorescence-activated cell sorting (FACS) and confocal laser scanning microscopy (CLSM), without the need to produce alternative peptide conjugates. To the best of our knowledge, so far, the delivery of a payload by bombesin-related peptides has only been studied using fluorescent molecules such as TAMRA, radioligands or nanocarriers. Drugs were attached to the peptides less often, and mainly using the original or truncated bombesin sequence instead of more promising analogues, or ones that are non-selective for GRP-R. Oppositely, investigating features such as the cellular uptake directly, through a conjugate attached to a chemotherapeutic, gives the most realistic picture of the success of a homing peptide acting as a drug carrier. Thus, the generated structures provide valuable tools for the selection of targeting peptides, aiming to the further development of new tumour-selective conjugates containing different linker-payload systems.

We decided to select some of the described peptides bearing the mentioned substitutions and having affirmed affinity for GRP-R in the low nanomolar range, together with the original BBN, and use them as targeting moiety to deliver a cytotoxic payload, daunorubicin (Dau), to prostate and breast cancers. Compared to the BBN sequence, our targeting peptides are elongated by a D-Phe in position 6 and comprise substitutions in positions 11, 13 and 14. Starting from these variations, we have also generated a new sequence: [D-Phe6, β-Ala11, Sta13, Nle14]BBN. The bombesin analogues have been published in the works of different research groups throughout many years, however, a direct comparison between them in terms of drug delivery has never been done. Once the targeting peptides bind to receptors that are overexpressed on the surface of cancer cells, such as GRP-R, the PDC can be carried inside the cells via receptor-mediated endocytosis. Briefly, the receptor-ligand complex internalises via a special, coated vesicle that later fuses with an early endosome and matures into a late endosome. After the late endosome fuses with a lysosome, the PDC is also digested, the drug is released into the cytoplasm and binds to its target compartment inside the cell. To ensure this release, we have decided to insert cathepsin B cleavable tetrapeptide linkers, namely either GFLG or LRRY, between the homing peptide and Dau. Indeed, cathepsin B is highly expressed by lysosomes and can cleave between Gly-Phe and Leu-Arg, liberating the active metabolites Dau=Aoa-Gly-OH or Dau=Aoa-Leu-OH. Dau is part of the family of anthracyclines and elicits its cytostatic activity by intercalating between DNA base pairs. This prevents the topoisomerase II from resealing the DNA double helix, resulting in reduced cell proliferation. Its attachment to a peptide can be easily performed through an oxime linkage in a very simple and straightforward reaction that occurs between an aminooxy moiety and the C13 ketone on Dau. The high yields of the reaction are advantageous, especially when it comes to the production of an increased amount of conjugate for in vivo studies. Furthermore, thanks to its intrinsic fluorescence, it is possible to evaluate the internalisation of the Dau-conjugates by fluorescence-activated cell sorting (FACS) and confocal laser scanning microscopy (CLSM), without the need to produce alternative peptide conjugates. To the best of our knowledge, so far, the delivery of a payload by bombesin-related peptides has only been studied using fluorescent molecules such as TAMRA, radioligands or nanocarriers. Drugs were attached to the peptides less often, and mainly using the original or truncated bombesin sequence instead of more promising analogues, or ones that are non-selective for GRP-R. Oppositely, investigating features such as the cellular uptake directly, through a conjugate attached to a chemotherapeutic, gives the most realistic picture of the success of a homing peptide acting as a drug carrier. Thus, the generated structures provide valuable tools for the selection of targeting peptides, aiming to the further development of new tumour-selective conjugates containing different linker-payload systems.

We decided to select some of the described peptides bearing the mentioned substitutions and having affirmed affinity for GRP-R in the low nanomolar range, together with the original BBN, and use them as targeting moiety to deliver a cytotoxic payload, daunorubicin (Dau), to prostate and breast cancers. Compared to the BBN sequence, our targeting peptides are elongated by a D-Phe in position 6 and comprise substitutions in positions 11, 13 and 14. Starting from these variations, we have also generated a new sequence: [D-Phe6, β-Ala11, Sta13, Nle14]BBN. The bombesin analogues have been published in the works of different research groups throughout many years, however, a direct comparison between them in terms of drug delivery has never been done. Once the targeting peptides bind to receptors that are overexpressed on the surface of cancer cells, such as GRP-R, the PDC can be carried inside the cells via receptor-mediated endocytosis. Briefly, the receptor-ligand complex internalises via a special, coated vesicle that later fuses with an early endosome and matures into a late endosome. After the late endosome fuses with a lysosome, the PDC is also digested, the drug is released into the cytoplasm and binds to its target compartment inside the cell. To ensure this release, we have decided to insert cathepsin B cleavable tetrapeptide linkers, namely either GFLG or LRRY, between the homing peptide and Dau. Indeed, cathepsin B is highly expressed by lysosomes and can cleave between Gly-Phe and Leu-Arg, liberating the active metabolites Dau=Aoa-Gly-OH or Dau=Aoa-Leu-OH. Dau is part of the family of anthracyclines and elicits its cytostatic activity by intercalating between DNA base pairs. This prevents the topoisomerase II from resealing the DNA double helix, resulting in reduced cell proliferation. Its attachment to a peptide can be easily performed through an oxime linkage in a very simple and straightforward reaction that occurs between an aminooxy moiety and the C13 ketone on Dau. The high yields of the reaction are advantageous, especially when it comes to the production of an increased amount of conjugate for in vivo studies. Furthermore, thanks to its intrinsic fluorescence, it is possible to evaluate the internalisation of the Dau-conjugates by fluorescence-activated cell sorting (FACS) and confocal laser scanning microscopy (CLSM), without the need to produce alternative peptide conjugates. To the best of our knowledge, so far, the delivery of a payload by bombesin-related peptides has only been studied using fluorescent molecules such as TAMRA, radioligands or nanocarriers. Drugs were attached to the peptides less often, and mainly using the original or truncated bombesin sequence instead of more promising analogues, or ones that are non-selective for GRP-R. Oppositely, investigating features such as the cellular uptake directly, through a conjugate attached to a chemotherapeutic, gives the most realistic picture of the success of a homing peptide acting as a drug carrier. Thus, the generated structures provide valuable tools for the selection of targeting peptides, aiming to the further development of new tumour-selective conjugates containing different linker-payload systems.

We decided to select some of the described peptides bearing the mentioned substitutions and having affirmed affinity for GRP-R in the low nanomolar range, together with the original BBN, and use them as targeting moiety to deliver a cytotoxic payload, daunorubicin (Dau), to prostate and breast cancers. Compared to the BBN sequence, our targeting peptides are elongated by a D-Phe in position 6 and comprise substitutions in positions 11, 13 and 14. Starting from these variations, we have also generated a new sequence: [D-Phe6, β-Ala11, Sta13, Nle14]BBN. The bombesin analogues have been published in the works of different research groups throughout many years, however, a direct comparison between them in terms of drug delivery has never been done. Once the targeting peptides bind to receptors that are overexpressed on the surface of cancer cells, such as GRP-R, the PDC can be carried inside the cells via receptor-mediated endocytosis. Briefly, the receptor-ligand complex internalises via a special, coated vesicle that later fuses with an early endosome and matures into a late endosome. After the late endosome fuses with a lysosome, the PDC is also digested, the drug is released into the cytoplasm and binds to its target compartment inside the cell. To ensure this release, we have decided to insert cathepsin B cleavable tetrapeptide linkers, namely either GFLG or LRRY, between the homing peptide and Dau. Indeed, cathepsin B is highly expressed by lysosomes and can cleave between Gly-Phe and Leu-Arg, liberating the active metabolites Dau=Aoa-Gly-OH or Dau=Aoa-Leu-OH. Dau is part of the family of anthracyclines and elicits its cytostatic activity by intercalating between DNA base pairs. This prevents the topoisomerase II from resealing the DNA double helix, resulting in reduced cell proliferation. Its attachment to a peptide can be easily performed through an oxime linkage in a very simple and straightforward reaction that occurs between an aminooxy moiety and the C13 ketone on Dau. The high yields of the reaction are advantageous, especially when it comes to the production of an increased amount of conjugate for in vivo studies. Furthermore, thanks to its intrinsic fluorescence, it is possible to evaluate the internalisation of the Dau-conjugates by fluorescence-activated cell sorting (FACS) and confocal laser scanning microscopy (CLSM), without the need to produce alternative peptide conjugates. To the best of our knowledge, so far, the delivery of a payload by bombesin-related peptides has only been studied using fluorescent molecules such as TAMRA, radioligands or nanocarriers. Drugs were attached to the peptides less often, and mainly using the original or truncated bombesin sequence instead of more promising analogues, or ones that are non-selective for GRP-R. Oppositely, investigating features such as the cellular uptake directly, through a conjugate attached to a chemotherapeutic, gives the most realistic picture of the success of a homing peptide acting as a drug carrier. Thus, the generated structures provide valuable tools for the selection of targeting peptides, aiming to the further development of new tumour-selective conjugates containing different linker-payload systems.

We decided to select some of the described peptides bearing the mentioned substitutions and having affirmed affinity for GRP-R in the low nanomolar range, together with the original BBN, and use them as targeting moiety to deliver a cytotoxic payload, daunorubicin (Dau), to prostate and breast cancers. Compared to the BBN sequence, our targeting peptides are elongated by a D-Phe in position 6 and comprise substitutions in positions 11, 13 and 14. Starting from these variations, we have also generated a new sequence: [D-Phe6, β-Ala11, Sta13, Nle14]BBN. The bombesin analogues have been published in the works of different research groups throughout many years, however, a direct comparison between them in terms of drug delivery has never been done. Once the targeting peptides bind to receptors that are overexpressed on the surface of cancer cells, such as GRP-R, the PDC can be carried inside the cells via receptor-mediated endocytosis. Briefly, the receptor-ligand complex internalises via a special, coated vesicle that later fuses with an early endosome and matures into a late endosome. After the late endosome fuses with a lysosome, the PDC is also digested, the drug is released into the cytoplasm and binds to its target compartment inside the cell. To ensure this release, we have decided to insert cathepsin B cleavable tetrapeptide linkers, namely either GFLG or LRRY, between the homing peptide and Dau. Indeed, cathepsin B is highly expressed by lysosomes and can cleave between Gly-Phe and Leu-Arg, liberating the active metabolites Dau=Aoa-Gly-OH or Dau=Aoa-Leu-OH. Dau is part of the family of anthracyclines and elicits its cytostatic activity by intercalating between DNA base pairs. This prevents the topoisomerase II from resealing the DNA double helix, resulting in reduced cell proliferation. Its attachment to a peptide can be easily performed through an oxime linkage in a very simple and straightforward reaction that occurs between an aminooxy moiety and the C13 ketone on Dau. The high yields of the reaction are advantageous, especially when it comes to the production of an increased amount of conjugate for in vivo studies. Furthermore, thanks to its intrinsic fluorescence, it is possible to evaluate the internalisation of the Dau-conjugates by fluorescence-activated cell sorting (FACS) and confocal laser scanning microscopy (CLSM), without the need to produce alternative peptide conjugates. To the best of our knowledge, so far, the delivery of a payload by bombesin-related peptides has only been studied using fluorescent molecules such as TAMRA, radioligands or nanocarriers. Drugs were attached to the peptides less often, and mainly using the original or truncated bombesin sequence instead of more promising analogues, or ones that are non-selective for GRP-R. Oppositely, investigating features such as the cellular uptake directly, through a conjugate attached to a chemotherapeutic, gives the most realistic picture of the success of a homing peptide acting as a drug carrier. Thus, the generated structures provide valuable tools for the selection of targeting peptides, aiming to the further development of new tumour-selective conjugates containing different linker-payload systems.

We decided to select some of the described peptides bearing the mentioned substitutions and having affirmed affinity for GRP-R in the low nanomolar range, together with the original BBN, and use them as targeting moiety to deliver a cytotoxic payload, daunorubicin (Dau), to prostate and breast cancers. Compared to the BBN sequence, our targeting peptides are elongated by a D-Phe in position 6 and comprise substitutions in positions 11, 13 and 14. Starting from these variations, we have also generated a new sequence: [D-Phe6, β-Ala11, Sta13, Nle14]BBN. The bombesin analogues have been published in the works of different research groups throughout many years, however, a direct comparison between them in terms of drug delivery has never been done. Once the targeting peptides bind to receptors that are overexpressed on the surface of cancer cells, such as GRP-R, the PDC can be carried inside the cells via receptor-mediated endocytosis. Briefly, the receptor-ligand complex internalises via a special, coated vesicle that later fuses with an early endosome and matures into a late endosome. After the late endosome fuses with a lysosome, the PDC is also digested, the drug is released into the cytoplasm and binds to its target compartment inside the cell. To ensure this release, we have decided to insert cathepsin B cleavable tetrapeptide linkers, namely either GFLG or LRRY, between the homing peptide and Dau. Indeed, cathepsin B is highly expressed by lysosomes and can cleave between Gly-Phe and Leu-Arg, liberating the active metabolites Dau=Aoa-Gly-OH or Dau=Aoa-Leu-OH. Dau is part of the family of anthracyclines and elicits its cytostatic activity by intercalating between DNA base pairs. This prevents the topoisomerase II from resealing the DNA double helix, resulting in reduced cell proliferation. Its attachment to a peptide can be easily performed through an oxime linkage in a very simple and straightforward reaction that occurs between an aminooxy moiety and the C13 ketone on Dau. The high yields of the reaction are advantageous, especially when it comes to the production of an increased amount of conjugate for in vivo studies. Furthermore, thanks to its intrinsic fluorescence, it is possible to evaluate the internalisation of the Dau-conjugates by fluorescence-activated cell sorting (FACS) and confocal laser scanning microscopy (CLSM), without the need to produce alternative peptide conjugates. To the best of our knowledge, so far, the delivery of a payload by bombesin-related peptides has only been studied using fluorescent molecules such as TAMRA, radioligands or nanocarriers. Drugs were attached to the peptides less often, and mainly using the original or truncated bombesin sequence instead of more promising analogues, or ones that are non-selective for GRP-R. Oppositely, investigating features such as the cellular uptake directly, through a conjugate attached to a chemotherapeutic, gives the most realistic picture of the success of a homing peptide acting as a drug carrier. Thus, the generated structures provide valuable tools for the selection of targeting peptides, aiming to the further development of new tumour-selective conjugates containing different linker-payload systems.

We decided to select some of the described peptides bearing the mentioned substitutions and having affirmed affinity for GRP-R in the low nanomolar range, together with the original BBN, and use them as targeting moiety to deliver a cytotoxic payload, daunorubicin (Dau), to prostate and breast cancers. Compared to the BBN sequence, our targeting peptides are elongated by a D-Phe in position 6 and comprise substitutions in positions 11, 13 and 14. Starting from these variations, we have also generated a new sequence: [D-Phe6, β-Ala11, Sta13, Nle14]BBN. The bombesin analogues have been published in the works of different research groups throughout many years, however, a direct comparison between them in terms of drug delivery has never been done. Once the targeting peptides bind to receptors that are overexpressed on the surface of cancer cells, such as GRP-R, the PDC can be carried inside the cells via receptor-mediated endocytosis. Briefly, the receptor-ligand complex internalises via a special, coated vesicle that later fuses with an early endosome and matures into a late endosome. After the late endosome fuses with a lysosome, the PDC is also digested, the drug is released into the cytoplasm and binds to its target compartment inside the cell. To ensure this release, we have decided to insert cathepsin B cleavable tetrapeptide linkers, namely either GFLG or LRRY, between the homing peptide and Dau. Indeed, cathepsin B is highly expressed by lysosomes and can cleave between Gly-Phe and Leu-Arg, liberating the active metabolites Dau=Aoa-Gly-OH or Dau=Aoa-Leu-OH. Dau is part of the family of anthracyclines and elicits its cytostatic activity by intercalating between DNA base pairs. This prevents the topoisomerase II from resealing the DNA double helix, resulting in reduced cell proliferation. Its attachment to a peptide can be easily performed through an oxime linkage in a very simple and straightforward reaction that occurs between an aminooxy moiety and the C13 ketone on Dau. The high yields of the reaction are advantageous, especially when it comes to the production of an increased amount of conjugate for in vivo studies. Furthermore, thanks to its intrinsic fluorescence, it is possible to evaluate the internalisation of the Dau-conjugates by fluorescence-activated cell sorting (FACS) and confocal laser scanning microscopy (CLSM), without the need to produce alternative peptide conjugates. To the best of our knowledge, so far, the delivery of a payload by bombesin-related peptides has only been studied using fluorescent molecules such as TAMRA, radioligands or nanocarriers. Drugs were attached to the peptides less often, and mainly using the original or truncated bombesin sequence instead of more promising analogues, or ones that are non-selective for GRP-R. Oppositely, investigating features such as the cellular uptake directly, through a conjugate attached to a chemotherapeutic, gives the most realistic picture of the success of a homing peptide acting as a drug carrier. Thus, the generated structures provide valuable tools for the selection of targeting peptides, aiming to the further development of new tumour-selective conjugates containing different linker-payload systems.

We decided to select some of the described peptides bearing the mentioned substitutions and having affirmed affinity for GRP-R in the low nanomolar range, together with the original BBN, and use them as targeting moiety to deliver a cytotoxic payload, daunorubicin (Dau), to prostate and breast cancers. Compared to the BBN sequence, our targeting peptides are elongated by a D-Phe in position 6 and comprise substitutions in positions 11, 13 and 14. Starting from these variations, we have also generated a new sequence: [D-Phe6, β-Ala11, Sta13, Nle14]BBN. The bombesin analogues have been published in the works of different research groups throughout many years, however, a direct comparison between them in terms of drug delivery has never been done. Once the targeting peptides bind to receptors that are overexpressed on the surface of cancer cells, such as GRP-R, the PDC can be carried inside the cells via receptor-mediated endocytosis. Briefly, the receptor-ligand complex internalises via a special, coated vesicle that later fuses with an early endosome and matures into a late endosome. After the late endosome fuses with a lysosome, the PDC is also digested, the drug is released into the cytoplasm and binds to its target compartment inside the cell. To ensure this release, we have decided to insert cathepsin B cleavable tetrapeptide linkers, namely either GFLG or LRRY, between the homing peptide and Dau. Indeed, cathepsin B is highly expressed by lysosomes and can cleave between Gly-Phe and Leu-Arg, liberating the active metabolites Dau=Aoa-Gly-OH or Dau=Aoa-Leu-OH. Dau is part of the family of anthracyclines and elicits its cytostatic activity by intercalating between DNA base pairs. This prevents the topoisomerase II from resealing the DNA double helix, resulting in reduced cell proliferation. Its attachment to a peptide can be easily performed through an oxime linkage in a very simple and straightforward reaction that occurs between an aminooxy moiety and the C13 ketone on Dau. The high yields of the reaction are advantageous, especially when it comes to the production of an increased amount of conjugate for in vivo studies. Furthermore, thanks to its intrinsic fluorescence, it is possible to evaluate the internalisation of the Dau-conjugates by fluorescence-activated cell sorting (FACS) and confocal laser scanning microscopy (CLSM), without the need to produce alternative peptide conjugates. To the best of our knowledge, so far, the delivery of a payload by bombesin-related peptides has only been studied using fluorescent molecules such as TAMRA, radioligands or nanocarriers. Drugs were attached to the peptides less often, and mainly using the original or truncated bombesin sequence instead of more promising analogues, or ones that are non-selective for GRP-R. Oppositely, investigating features such as the cellular uptake directly, through a conjugate attached to a chemotherapeutic, gives the most realistic picture of the success of a homing peptide acting as a drug carrier. Thus, the generated structures provide valuable tools for the selection of targeting peptides, aiming to the further development of new tumour-selective conjugates containing different linker-payload systems.

We decided to select some of the described peptides bearing the mentioned substitutions and having affirmed affinity for GRP-R in the low nanomolar range, together with the original BBN, and use them as targeting moiety to deliver a cytotoxic payload, daunorubicin (Dau), to prostate and breast cancers. Compared to the BBN sequence, our targeting peptides are elongated by a D-Phe in position 6 and comprise substitutions in positions 11, 13 and 14. Starting from these variations, we have also generated a new sequence: [D-Phe6, β-Ala11, Sta13, Nle14]BBN. The bombesin analogues have been published in the works of different research groups throughout many years, however, a direct comparison between them in terms of drug delivery has never been done. Once the targeting peptides bind to receptors that are overexpressed on the surface of cancer cells, such as GRP-R, the PDC can be carried inside the cells via receptor-mediated endocytosis. Briefly, the receptor-ligand complex internalises via a special, coated vesicle that later fuses with an early endosome and matures into a late endosome. After the late endosome fuses with a lysosome, the PDC is also digested, the drug is released into the cytoplasm and binds to its target compartment inside the cell. To ensure this release, we have decided to insert cathepsin B cleavable tetrapeptide linkers, namely either GFLG or LRRY, between the homing peptide and Dau. Indeed, cathepsin B is highly expressed by lysosomes and can cleave between Gly-Phe and Leu-Arg, liberating the active metabolites Dau=Aoa-Gly-OH or Dau=Aoa-Leu-OH. Dau is part of the family of anthracyclines and elicits its cytostatic activity by intercalating between DNA base pairs. This prevents the topoisomerase II from resealing the DNA double helix, resulting in reduced cell proliferation. Its attachment to a peptide can be easily performed through an oxime linkage in a very simple and straightforward reaction that occurs between an aminooxy moiety and the C13 ketone on Dau. The high yields of the reaction are advantageous, especially when it comes to the production of an increased amount of conjugate for in vivo studies. Furthermore, thanks to its intrinsic fluorescence, it is possible to evaluate the internalisation of the Dau-conjugates by fluorescence-activated cell sorting (FACS) and confocal laser scanning microscopy (CLSM), without the need to produce alternative peptide conjugates. To the best of our knowledge, so far, the delivery of a payload by bombesin-related peptides has only been studied using fluorescent molecules such as TAMRA, radioligands or nanocarriers. Drugs were attached to the peptides less often, and mainly using the original or truncated bombesin sequence instead of more promising analogues, or ones that are non-selective for GRP-R. Oppositely, investigating features such as the cellular uptake directly, through a conjugate attached to a chemotherapeutic, gives the most realistic picture of the success of a homing peptide acting as a drug carrier. Thus, the generated structures provide valuable tools for the selection of targeting peptides, aiming to the further development of new tumour-selective conjugates containing different linker-payload systems.

We decided to select some of the described peptides bearing the mentioned substitutions and having affirmed affinity for GRP-R in the low nanomolar range, together with the original BBN, and use them as targeting moiety to deliver a cytotoxic payload, daunorubicin (Dau), to prostate and breast cancers. Compared to the BBN sequence, our targeting peptides are elongated by a D-Phe in position 6 and comprise substitutions in positions 11, 13 and 14. Starting from these variations, we have also generated a new sequence: [D-Phe6, β-Ala11, Sta13, Nle14]BBN. The bombesin analogues have been published in the works of different research groups throughout many years, however, a direct comparison between them in terms of drug delivery has never been done. Once the targeting peptides bind to receptors that are overexpressed on the surface of cancer cells, such as GRP-R, the PDC can be carried inside the cells via receptor-mediated endocytosis. Briefly, the receptor-ligand complex internalises via a special, coated vesicle that later fuses with an early endosome and matures into a late endosome. After the late endosome fuses with a lysosome, the PDC is also digested, the drug is released into the cytoplasm and binds to its target compartment inside the cell. To ensure this release, we have decided to insert cathepsin B cleavable tetrapeptide linkers, namely either GFLG or LRRY, between the homing peptide and Dau. Indeed, cathepsin B is highly expressed by lysosomes and can cleave between Gly-Phe and Leu-Arg, liberating the active metabolites Dau=Aoa-Gly-OH or Dau=Aoa-Leu-OH. Dau is part of the family of anthracyclines and elicits its cytostatic activity by intercalating between DNA base pairs. This prevents the topoisomerase II from resealing the DNA double helix, resulting in reduced cell proliferation. Its attachment to a peptide can be easily performed through an oxime linkage in a very simple and straightforward reaction that occurs between an aminooxy moiety and the C13 ketone on Dau. The high yields of the reaction are advantageous, especially when it comes to the production of an increased amount of conjugate for in vivo studies. Furthermore, thanks to its intrinsic fluorescence, it is possible to evaluate the internalisation of the Dau-conjugates by fluorescence-activated cell sorting (FACS) and confocal laser scanning microscopy (CLSM), without the need to produce alternative peptide conjugates. To the best of our knowledge, so far, the delivery of a payload by bombesin-related peptides has only been studied using fluorescent molecules such as TAMRA, radioligands or nanocarriers. Drugs were attached to the peptides less often, and mainly using the original or truncated bombesin sequence instead of more promising analogues, or ones that are non-selective for GRP-R. Oppositely, investigating features such as the cellular uptake directly, through a conjugate attached to a chemotherapeutic, gives the most realistic picture of the success of a homing peptide acting as a drug carrier. Thus, the generated structures provide valuable tools for the selection of targeting peptides, aiming to the further development of new tumour-selective conjugates containing different linker-payload systems.

As observed, the cathepsin B labile spacer (GFLG), which was incorporated between the targeting peptide and the daunomycin to ensure the efficient release of the active metabolite, highly increases the hydrophobicity of the conjugates. As demonstrated, the solubility problem of these conjugates can be solved by hydrophilic polymer coupling, not only by using the well-known PEG but also by using the amino-monofunctional HbPG. To the best of our knowledge, 1:1 covalent peptide-polymer conjugates with well-defined monofunctional HbPG have been reported here for the first time. The results of the in vitro cell viability and cellular uptake measurements on HT-29 human colon adenocarcinoma cells prove that the HbPG and the PEG highly influenced the biological activity of the drug-peptide-polymer conjugates. In both peptide conjugate series, one GE11 and one D4 targeting peptide-based conjugate were found with outstanding cellular uptake and cytotoxicity values, namely Dau[double bond, length as m-dash]Aoa-GFLG-GE11-PEG and Dau[double bond, length as m-dash]Aoa-GFLG-D4-G5-HbPG. According to our results, the PEG is suitable for longer targeting peptides (e.g. GE11), but the G5 spacer is not suitable irrespective of the length of the peptide because it may decrease the biological effect by increasing the flexibility of the polymer and shading of the targeting moiety. In contrast, the use of the hydrophilic hyperbranched polyglycerol (HbPG) is advantageous for short targeting peptides (e.g. D4) but only with a G5 spacer, which provides accessibility of the peptide for receptor binding and cellular uptake resulting in outstanding cytotoxicity.

In the previous study, we focused on the discovery of novel cell targeting peptides, leading to the identification of the preferred HER2-targeting peptide P1 (NPNWGRSWYNQRFK) derived from pertuzumab, with the Kd of 5.70 10-6 M. Here, we designed and synthesized a series of PDCs choosing Camptothecin (CPT) as the payload and P1 as the peptide carrier. We aimed to discover novel PDC molecules with potent inhibitory activity and selectivity targeting HER2 for effective cancer treatment. Conjugate Z8, which balanced the activity and the biosafety, showed good antiproliferative activity (IC50 = 1.91 ± 0.71 M), comparable to that of CPT (IC50 = 2.10 ± 1.34 M), against NCI-N87 cells. Moreover, subsequent verification tests indicated that Z8 operated through a mechanism similar to CPT, but the results from in vivo anti-tumor activity assay suggested that Z8 exhibited greater safety compared to CPT to some extent. These findings highlight Z8 as a promising candidate for further investigation in the treatment of HER2-positive tumors.

In the previous study, we focused on the discovery of novel cell targeting peptides, leading to the identification of the preferred HER2-targeting peptide P1 (NPNWGRSWYNQRFK) derived from pertuzumab, with the Kd of 5.70 10-6 M. Here, we designed and synthesized a series of PDCs choosing Camptothecin (CPT) as the payload and P1 as the peptide carrier. We aimed to discover novel PDC molecules with potent inhibitory activity and selectivity targeting HER2 for effective cancer treatment. Conjugate Z8, which balanced the activity and the biosafety, showed good antiproliferative activity (IC50 = 1.91 ± 0.71 M), comparable to that of CPT (IC50 = 2.10 ± 1.34 M), against NCI-N87 cells. Moreover, subsequent verification tests indicated that Z8 operated through a mechanism similar to CPT, but the results from in vivo anti-tumor activity assay suggested that Z8 exhibited greater safety compared to CPT to some extent. These findings highlight Z8 as a promising candidate for further investigation in the treatment of HER2-positive tumors.

In the previous study, we focused on the discovery of novel cell targeting peptides, leading to the identification of the preferred HER2-targeting peptide P1 (NPNWGRSWYNQRFK) derived from pertuzumab, with the Kd of 5.70 10-6 M. Here, we designed and synthesized a series of PDCs choosing Camptothecin (CPT) as the payload and P1 as the peptide carrier. We aimed to discover novel PDC molecules with potent inhibitory activity and selectivity targeting HER2 for effective cancer treatment. Conjugate Z8, which balanced the activity and the biosafety, showed good antiproliferative activity (IC50 = 1.91 ± 0.71 M), comparable to that of CPT (IC50 = 2.10 ± 1.34 M), against NCI-N87 cells. Moreover, subsequent verification tests indicated that Z8 operated through a mechanism similar to CPT, but the results from in vivo anti-tumor activity assay suggested that Z8 exhibited greater safety compared to CPT to some extent. These findings highlight Z8 as a promising candidate for further investigation in the treatment of HER2-positive tumors.

In the previous study, we focused on the discovery of novel cell targeting peptides, leading to the identification of the preferred HER2-targeting peptide P1 (NPNWGRSWYNQRFK) derived from pertuzumab, with the Kd of 5.70 10-6 M. Here, we designed and synthesized a series of PDCs choosing Camptothecin (CPT) as the payload and P1 as the peptide carrier. We aimed to discover novel PDC molecules with potent inhibitory activity and selectivity targeting HER2 for effective cancer treatment. Conjugate Z8, which balanced the activity and the biosafety, showed good antiproliferative activity (IC50 = 1.91 ± 0.71 M), comparable to that of CPT (IC50 = 2.10 ± 1.34 M), against NCI-N87 cells. Moreover, subsequent verification tests indicated that Z8 operated through a mechanism similar to CPT, but the results from in vivo anti-tumor activity assay suggested that Z8 exhibited greater safety compared to CPT to some extent. These findings highlight Z8 as a promising candidate for further investigation in the treatment of HER2-positive tumors.

As observed, the cathepsin B labile spacer (GFLG), which was incorporated between the targeting peptide and the daunomycin to ensure the efficient release of the active metabolite, highly increases the hydrophobicity of the conjugates. As demonstrated, the solubility problem of these conjugates can be solved by hydrophilic polymer coupling, not only by using the well-known PEG but also by using the amino-monofunctional HbPG. To the best of our knowledge, 1:1 covalent peptide-polymer conjugates with well-defined monofunctional HbPG have been reported here for the first time. The results of the in vitro cell viability and cellular uptake measurements on HT-29 human colon adenocarcinoma cells prove that the HbPG and the PEG highly influenced the biological activity of the drug-peptide-polymer conjugates. In both peptide conjugate series, one GE11 and one D4 targeting peptide-based conjugate were found with outstanding cellular uptake and cytotoxicity values, namely Dau[double bond, length as m-dash]Aoa-GFLG-GE11-PEG and Dau[double bond, length as m-dash]Aoa-GFLG-D4-G5-HbPG. According to our results, the PEG is suitable for longer targeting peptides (e.g. GE11), but the G5 spacer is not suitable irrespective of the length of the peptide because it may decrease the biological effect by increasing the flexibility of the polymer and shading of the targeting moiety. In contrast, the use of the hydrophilic hyperbranched polyglycerol (HbPG) is advantageous for short targeting peptides (e.g. D4) but only with a G5 spacer, which provides accessibility of the peptide for receptor binding and cellular uptake resulting in outstanding cytotoxicity.

In conclusion, we developed a robust and regioselective rhodium-catalyzed methodology for C(7)-H Trp maleimidation. This reaction served as an efficient tool for peptide/drug modification, ligation, and particularly peptide cyclization, confirming its promising potential in pharmaceutical chemistry and drug synthesis. Notably, this catalytical system is not limited by the Trp position in the peptides. We also demonstrated that tryptophan-substituted maleimide could be used as an effective click functional group to rapidly react with sulfhydryl groups. Moreover, the introduced N-pivaloyl directing group and protecting groups of the peptides could be removed in a single step, providing a more convenient approach compared to the previous methods, which require multi-step removal of the corresponding directing groups and peptide protection groups. Additionally, cyclic peptide 10a exhibited excellent binding affinity to integrin vβ3, indicating its good drug-like properties. With rational design, RGD- GFLG -DOX, which is a stapled PDC, displayed higher selectivity, stronger binding affinity and better cell penetrability than the more commonly used DOX. The proposed strategy for rapid preparation of stapled peptides is expected to further improve PDC formulation.

In conclusion, we developed a robust and regioselective rhodium-catalyzed methodology for C(7)-H Trp maleimidation. This reaction served as an efficient tool for peptide/drug modification, ligation, and particularly peptide cyclization, confirming its promising potential in pharmaceutical chemistry and drug synthesis. Notably, this catalytical system is not limited by the Trp position in the peptides. We also demonstrated that tryptophan-substituted maleimide could be used as an effective click functional group to rapidly react with sulfhydryl groups. Moreover, the introduced N-pivaloyl directing group and protecting groups of the peptides could be removed in a single step, providing a more convenient approach compared to the previous methods, which require multi-step removal of the corresponding directing groups and peptide protection groups. Additionally, cyclic peptide 10a exhibited excellent binding affinity to integrin vβ3, indicating its good drug-like properties. With rational design, RGD- GFLG -DOX, which is a stapled PDC, displayed higher selectivity, stronger binding affinity and better cell penetrability than the more commonly used DOX. The proposed strategy for rapid preparation of stapled peptides is expected to further improve PDC formulation.

In conclusion, we developed a robust and regioselective rhodium-catalyzed methodology for C(7)-H Trp maleimidation. This reaction served as an efficient tool for peptide/drug modification, ligation, and particularly peptide cyclization, confirming its promising potential in pharmaceutical chemistry and drug synthesis. Notably, this catalytical system is not limited by the Trp position in the peptides. We also demonstrated that tryptophan-substituted maleimide could be used as an effective click functional group to rapidly react with sulfhydryl groups. Moreover, the introduced N-pivaloyl directing group and protecting groups of the peptides could be removed in a single step, providing a more convenient approach compared to the previous methods, which require multi-step removal of the corresponding directing groups and peptide protection groups. Additionally, cyclic peptide 10a exhibited excellent binding affinity to integrin vβ3, indicating its good drug-like properties. With rational design, RGD- GFLG -DOX, which is a stapled PDC, displayed higher selectivity, stronger binding affinity and better cell penetrability than the more commonly used DOX. The proposed strategy for rapid preparation of stapled peptides is expected to further improve PDC formulation.

In conclusion, we developed a robust and regioselective rhodium-catalyzed methodology for C(7)-H Trp maleimidation. This reaction served as an efficient tool for peptide/drug modification, ligation, and particularly peptide cyclization, confirming its promising potential in pharmaceutical chemistry and drug synthesis. Notably, this catalytical system is not limited by the Trp position in the peptides. We also demonstrated that tryptophan-substituted maleimide could be used as an effective click functional group to rapidly react with sulfhydryl groups. Moreover, the introduced N-pivaloyl directing group and protecting groups of the peptides could be removed in a single step, providing a more convenient approach compared to the previous methods, which require multi-step removal of the corresponding directing groups and peptide protection groups. Additionally, cyclic peptide 10a exhibited excellent binding affinity to integrin vβ3, indicating its good drug-like properties. With rational design, RGD- GFLG -DOX, which is a stapled PDC, displayed higher selectivity, stronger binding affinity and better cell penetrability than the more commonly used DOX. The proposed strategy for rapid preparation of stapled peptides is expected to further improve PDC formulation.

In conclusion, we developed a robust and regioselective rhodium-catalyzed methodology for C(7)-H Trp maleimidation. This reaction served as an efficient tool for peptide/drug modification, ligation, and particularly peptide cyclization, confirming its promising potential in pharmaceutical chemistry and drug synthesis. Notably, this catalytical system is not limited by the Trp position in the peptides. We also demonstrated that tryptophan-substituted maleimide could be used as an effective click functional group to rapidly react with sulfhydryl groups. Moreover, the introduced N-pivaloyl directing group and protecting groups of the peptides could be removed in a single step, providing a more convenient approach compared to the previous methods, which require multi-step removal of the corresponding directing groups and peptide protection groups. Additionally, cyclic peptide 10a exhibited excellent binding affinity to integrin vβ3, indicating its good drug-like properties. With rational design, RGD- GFLG -DOX, which is a stapled PDC, displayed higher selectivity, stronger binding affinity and better cell penetrability than the more commonly used DOX. The proposed strategy for rapid preparation of stapled peptides is expected to further improve PDC formulation.

In conclusion, we developed a robust and regioselective rhodium-catalyzed methodology for C(7)-H Trp maleimidation. This reaction served as an efficient tool for peptide/drug modification, ligation, and particularly peptide cyclization, confirming its promising potential in pharmaceutical chemistry and drug synthesis. Notably, this catalytical system is not limited by the Trp position in the peptides. We also demonstrated that tryptophan-substituted maleimide could be used as an effective click functional group to rapidly react with sulfhydryl groups. Moreover, the introduced N-pivaloyl directing group and protecting groups of the peptides could be removed in a single step, providing a more convenient approach compared to the previous methods, which require multi-step removal of the corresponding directing groups and peptide protection groups. Additionally, cyclic peptide 10a exhibited excellent binding affinity to integrin vβ3, indicating its good drug-like properties. With rational design, RGD- GFLG -DOX, which is a stapled PDC, displayed higher selectivity, stronger binding affinity and better cell penetrability than the more commonly used DOX. The proposed strategy for rapid preparation of stapled peptides is expected to further improve PDC formulation.

As observed, the cathepsin B labile spacer (GFLG), which was incorporated between the targeting peptide and the daunomycin to ensure the efficient release of the active metabolite, highly increases the hydrophobicity of the conjugates. As demonstrated, the solubility problem of these conjugates can be solved by hydrophilic polymer coupling, not only by using the well-known PEG but also by using the amino-monofunctional HbPG. To the best of our knowledge, 1:1 covalent peptide-polymer conjugates with well-defined monofunctional HbPG have been reported here for the first time. The results of the in vitro cell viability and cellular uptake measurements on HT-29 human colon adenocarcinoma cells prove that the HbPG and the PEG highly influenced the biological activity of the drug-peptide-polymer conjugates. In both peptide conjugate series, one GE11 and one D4 targeting peptide-based conjugate were found with outstanding cellular uptake and cytotoxicity values, namely Dau[double bond, length as m-dash]Aoa-GFLG-GE11-PEG and Dau[double bond, length as m-dash]Aoa-GFLG-D4-G5-HbPG. According to our results, the PEG is suitable for longer targeting peptides (e.g. GE11), but the G5 spacer is not suitable irrespective of the length of the peptide because it may decrease the biological effect by increasing the flexibility of the polymer and shading of the targeting moiety. In contrast, the use of the hydrophilic hyperbranched polyglycerol (HbPG) is advantageous for short targeting peptides (e.g. D4) but only with a G5 spacer, which provides accessibility of the peptide for receptor binding and cellular uptake resulting in outstanding cytotoxicity.

As observed, the cathepsin B labile spacer (GFLG), which was incorporated between the targeting peptide and the daunomycin to ensure the efficient release of the active metabolite, highly increases the hydrophobicity of the conjugates. As demonstrated, the solubility problem of these conjugates can be solved by hydrophilic polymer coupling, not only by using the well-known PEG but also by using the amino-monofunctional HbPG. To the best of our knowledge, 1:1 covalent peptide-polymer conjugates with well-defined monofunctional HbPG have been reported here for the first time. The results of the in vitro cell viability and cellular uptake measurements on HT-29 human colon adenocarcinoma cells prove that the HbPG and the PEG highly influenced the biological activity of the drug-peptide-polymer conjugates. In both peptide conjugate series, one GE11 and one D4 targeting peptide-based conjugate were found with outstanding cellular uptake and cytotoxicity values, namely Dau[double bond, length as m-dash]Aoa-GFLG-GE11-PEG and Dau[double bond, length as m-dash]Aoa-GFLG-D4-G5-HbPG. According to our results, the PEG is suitable for longer targeting peptides (e.g. GE11), but the G5 spacer is not suitable irrespective of the length of the peptide because it may decrease the biological effect by increasing the flexibility of the polymer and shading of the targeting moiety. In contrast, the use of the hydrophilic hyperbranched polyglycerol (HbPG) is advantageous for short targeting peptides (e.g. D4) but only with a G5 spacer, which provides accessibility of the peptide for receptor binding and cellular uptake resulting in outstanding cytotoxicity.

As observed, the cathepsin B labile spacer (GFLG), which was incorporated between the targeting peptide and the daunomycin to ensure the efficient release of the active metabolite, highly increases the hydrophobicity of the conjugates. As demonstrated, the solubility problem of these conjugates can be solved by hydrophilic polymer coupling, not only by using the well-known PEG but also by using the amino-monofunctional HbPG. To the best of our knowledge, 1:1 covalent peptide-polymer conjugates with well-defined monofunctional HbPG have been reported here for the first time. The results of the in vitro cell viability and cellular uptake measurements on HT-29 human colon adenocarcinoma cells prove that the HbPG and the PEG highly influenced the biological activity of the drug-peptide-polymer conjugates. In both peptide conjugate series, one GE11 and one D4 targeting peptide-based conjugate were found with outstanding cellular uptake and cytotoxicity values, namely Dau[double bond, length as m-dash]Aoa-GFLG-GE11-PEG and Dau[double bond, length as m-dash]Aoa-GFLG-D4-G5-HbPG. According to our results, the PEG is suitable for longer targeting peptides (e.g. GE11), but the G5 spacer is not suitable irrespective of the length of the peptide because it may decrease the biological effect by increasing the flexibility of the polymer and shading of the targeting moiety. In contrast, the use of the hydrophilic hyperbranched polyglycerol (HbPG) is advantageous for short targeting peptides (e.g. D4) but only with a G5 spacer, which provides accessibility of the peptide for receptor binding and cellular uptake resulting in outstanding cytotoxicity.

As observed, the cathepsin B labile spacer (GFLG), which was incorporated between the targeting peptide and the daunomycin to ensure the efficient release of the active metabolite, highly increases the hydrophobicity of the conjugates. As demonstrated, the solubility problem of these conjugates can be solved by hydrophilic polymer coupling, not only by using the well-known PEG but also by using the amino-monofunctional HbPG. To the best of our knowledge, 1:1 covalent peptide-polymer conjugates with well-defined monofunctional HbPG have been reported here for the first time. The results of the in vitro cell viability and cellular uptake measurements on HT-29 human colon adenocarcinoma cells prove that the HbPG and the PEG highly influenced the biological activity of the drug-peptide-polymer conjugates. In both peptide conjugate series, one GE11 and one D4 targeting peptide-based conjugate were found with outstanding cellular uptake and cytotoxicity values, namely Dau[double bond, length as m-dash]Aoa-GFLG-GE11-PEG and Dau[double bond, length as m-dash]Aoa-GFLG-D4-G5-HbPG. According to our results, the PEG is suitable for longer targeting peptides (e.g. GE11), but the G5 spacer is not suitable irrespective of the length of the peptide because it may decrease the biological effect by increasing the flexibility of the polymer and shading of the targeting moiety. In contrast, the use of the hydrophilic hyperbranched polyglycerol (HbPG) is advantageous for short targeting peptides (e.g. D4) but only with a G5 spacer, which provides accessibility of the peptide for receptor binding and cellular uptake resulting in outstanding cytotoxicity.

As observed, the cathepsin B labile spacer (GFLG), which was incorporated between the targeting peptide and the daunomycin to ensure the efficient release of the active metabolite, highly increases the hydrophobicity of the conjugates. As demonstrated, the solubility problem of these conjugates can be solved by hydrophilic polymer coupling, not only by using the well-known PEG but also by using the amino-monofunctional HbPG. To the best of our knowledge, 1:1 covalent peptide-polymer conjugates with well-defined monofunctional HbPG have been reported here for the first time. The results of the in vitro cell viability and cellular uptake measurements on HT-29 human colon adenocarcinoma cells prove that the HbPG and the PEG highly influenced the biological activity of the drug-peptide-polymer conjugates. In both peptide conjugate series, one GE11 and one D4 targeting peptide-based conjugate were found with outstanding cellular uptake and cytotoxicity values, namely Dau[double bond, length as m-dash]Aoa-GFLG-GE11-PEG and Dau[double bond, length as m-dash]Aoa-GFLG-D4-G5-HbPG. According to our results, the PEG is suitable for longer targeting peptides (e.g. GE11), but the G5 spacer is not suitable irrespective of the length of the peptide because it may decrease the biological effect by increasing the flexibility of the polymer and shading of the targeting moiety. In contrast, the use of the hydrophilic hyperbranched polyglycerol (HbPG) is advantageous for short targeting peptides (e.g. D4) but only with a G5 spacer, which provides accessibility of the peptide for receptor binding and cellular uptake resulting in outstanding cytotoxicity.

The blood-brain barrier (BBB) is a semipermeable system, and, therefore, most of the active substances are poorly transported through this barrier, resulting in decreased therapeutic effects. Angiopep-2 (TFFYGGSRGKRNNFKTEEY) is a peptide ligand of low-density lipoprotein receptor-related protein-1 (LRP1), which can cross the BBB via receptor-mediated transcytosis and simultaneously target glioblastomas. Angiopep-2 contains three amino groups that have previously been used to produce drug-peptide conjugates, although the role and importance of each position have not yet been investigated. Thus, we studied the number and position of drug molecules in Angiopep-2 based conjugates. Conjugates containing one, two, and three daunomycin molecules conjugated via oxime linkage in all possible variations were prepared. The in vitro cytostatic effect and cellular uptake of the conjugates were investigated on U87 human glioblastoma cells. Degradation studies in the presence of rat liver lysosomal homogenates were also performed in order for us to better understand the structure-activity relationship and to determine the smallest metabolites. Conjugates with the best cytostatic effects had a drug molecule at the N-terminus. We demonstrated that the increasing number of drug molecules does not necessarily increase the efficacy of the conjugates, and proved that modification of the different conjugation sites results in differing biological effectiveness.

As observed, the cathepsin B labile spacer (GFLG), which was incorporated between the targeting peptide and the daunomycin to ensure the efficient release of the active metabolite, highly increases the hydrophobicity of the conjugates. As demonstrated, the solubility problem of these conjugates can be solved by hydrophilic polymer coupling, not only by using the well-known PEG but also by using the amino-monofunctional HbPG. To the best of our knowledge, 1:1 covalent peptide-polymer conjugates with well-defined monofunctional HbPG have been reported here for the first time. The results of the in vitro cell viability and cellular uptake measurements on HT-29 human colon adenocarcinoma cells prove that the HbPG and the PEG highly influenced the biological activity of the drug-peptide-polymer conjugates. In both peptide conjugate series, one GE11 and one D4 targeting peptide-based conjugate were found with outstanding cellular uptake and cytotoxicity values, namely Dau[double bond, length as m-dash]Aoa-GFLG-GE11-PEG and Dau[double bond, length as m-dash]Aoa-GFLG-D4-G5-HbPG. According to our results, the PEG is suitable for longer targeting peptides (e.g. GE11), but the G5 spacer is not suitable irrespective of the length of the peptide because it may decrease the biological effect by increasing the flexibility of the polymer and shading of the targeting moiety. In contrast, the use of the hydrophilic hyperbranched polyglycerol (HbPG) is advantageous for short targeting peptides (e.g. D4) but only with a G5 spacer, which provides accessibility of the peptide for receptor binding and cellular uptake resulting in outstanding cytotoxicity.