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.