To assess the anti-inflammatory efficacy of the conjugates, a carrageenan-induced acute inflammation model was developed in Balb/c male mice. Experimental mice were treated by injection of saline, free SIN, conjugate (L) or conjugate (C) in the tail vein at 1 h prior to the induction of paw edema. Photographs of the right hind paws indicated that swelling was milder in the mice treated with conjugate (C) and conjugate (L) than in the model group. Data for hind paw volumes suggested that conjugate (C) had the strongest therapeutic efficacy compared to that of the free drug SIN at 20.96 mg/kg and the model group. As previously reported, effective doses of SIN against experimental arthritis in vivo range from 15 mg/kg to 300 mg/kg body weight. Therefore, the dose of SIN was considered since the administration of 20.96 mg/kg is widely identified in the literature as a low dose for treating animals in arthritis mouse models without inducing side effects. The benefit of IV injections of conjugates at 90.96 mg/kg (eq. SIN) was clearly demonstrated by comparison with free SIN at the same molar dose. To verify the therapeutic efficacies of the conjugates, histological structures of paws were evaluated. Histological examination of the right hind paws revealed that the pedis skins contained massive lymphocyte, plasma cell and neutrophil infiltration in the paws of animals treated with saline. Animals treated with conjugates and free SIN showed less inflammatory cell infiltration, and conjugate (C) showed the mildest inflammation compared to the same dose of free SIN. To characterize the therapeutic efficacies of the conjugates more clearly, Image J software was used to statistically analyze the inflammatory cells in pathological sections (n = 7), and the results are shown in Fig. 6D. There were significant differences between the treatment groups and the model group (*p < 0.05, **p < 0.01, ***p < 0.001).

To assess the anti-inflammatory efficacy of the conjugates, a carrageenan-induced acute inflammation model was developed in Balb/c male mice. Experimental mice were treated by injection of saline, free SIN, conjugate (L) or conjugate (C) in the tail vein at 1 h prior to the induction of paw edema. Photographs of the right hind paws indicated that swelling was milder in the mice treated with conjugate (C) and conjugate (L) than in the model group. Data for hind paw volumes suggested that conjugate (C) had the strongest therapeutic efficacy compared to that of the free drug SIN at 20.96 mg/kg and the model group. As previously reported, effective doses of SIN against experimental arthritis in vivo range from 15 mg/kg to 300 mg/kg body weight. Therefore, the dose of SIN was considered since the administration of 20.96 mg/kg is widely identified in the literature as a low dose for treating animals in arthritis mouse models without inducing side effects. The benefit of IV injections of conjugates at 90.96 mg/kg (eq. SIN) was clearly demonstrated by comparison with free SIN at the same molar dose. To verify the therapeutic efficacies of the conjugates, histological structures of paws were evaluated. Histological examination of the right hind paws revealed that the pedis skins contained massive lymphocyte, plasma cell and neutrophil infiltration in the paws of animals treated with saline. Animals treated with conjugates and free SIN showed less inflammatory cell infiltration, and conjugate (C) showed the mildest inflammation compared to the same dose of free SIN. To characterize the therapeutic efficacies of the conjugates more clearly, Image J software was used to statistically analyze the inflammatory cells in pathological sections (n = 7), and the results are shown in Fig. 6D. There were significant differences between the treatment groups and the model group (*p < 0.05, **p < 0.01, ***p < 0.001).

To evaluated the in vitro anti-tumor activity of the conjugates, HER2-positive SK-BR-3, NCI-N87, HER2-negative MDA-MB-231 and normal MCF-10A cells were selected. As shown in Table 2, nearly all conjugates exhibited obvious anti-proliferation activity against SK-BR-3 cells, while most of the conjugates, except for Z6 and Z9, demonstrated different degrees of inhibitory activity on NCI-N87 cells. Taken together, Z8 and Z11 exhibited superior anti-tumor activity. The antiproliferative activity of Z8 against NCI-N87 cells (IC50 = 1.91 ± 0.71 uM) was comparable to that of CPT (IC50 = 2.10 ± 1.34 uM), while the antiproliferative activity of Z11 against SK-BR-3 cells (IC50 = 0.41 ± 0.13 uM) was comparable to that of CPT (IC50 = 0.26 ± 0.06 uM). Additionally, the IC50 values of these two conjugates against MDA-MB-231 and MCF-10A cells were significantly increased, showing their ability to selectively inhibit tumor cells. Based on the above results, Z8 and Z11, which exhibited better activity and apparent selectivity, were selected for subsequent studies.

To evaluated the in vitro anti-tumor activity of the conjugates, HER2-positive SK-BR-3, NCI-N87, HER2-negative MDA-MB-231 and normal MCF-10A cells were selected. As shown in Table 2, nearly all conjugates exhibited obvious anti-proliferation activity against SK-BR-3 cells, while most of the conjugates, except for Z6 and Z9, demonstrated different degrees of inhibitory activity on NCI-N87 cells. Taken together, Z8 and Z11 exhibited superior anti-tumor activity. The antiproliferative activity of Z8 against NCI-N87 cells (IC50 = 1.91 ± 0.71 uM) was comparable to that of CPT (IC50 = 2.10 ± 1.34 uM), while the antiproliferative activity of Z11 against SK-BR-3 cells (IC50 = 0.41 ± 0.13 uM) was comparable to that of CPT (IC50 = 0.26 ± 0.06 uM). Additionally, the IC50 values of these two conjugates against MDA-MB-231 and MCF-10A cells were significantly increased, showing their ability to selectively inhibit tumor cells. Based on the above results, Z8 and Z11, which exhibited better activity and apparent selectivity, were selected for subsequent studies.

To evaluated the in vitro anti-tumor activity of the conjugates, HER2-positive SK-BR-3, NCI-N87, HER2-negative MDA-MB-231 and normal MCF-10A cells were selected. As shown in Table 2, nearly all conjugates exhibited obvious anti-proliferation activity against SK-BR-3 cells, while most of the conjugates, except for Z6 and Z9, demonstrated different degrees of inhibitory activity on NCI-N87 cells. Taken together, Z8 and Z11 exhibited superior anti-tumor activity. The antiproliferative activity of Z8 against NCI-N87 cells (IC50 = 1.91 ± 0.71 uM) was comparable to that of CPT (IC50 = 2.10 ± 1.34 uM), while the antiproliferative activity of Z11 against SK-BR-3 cells (IC50 = 0.41 ± 0.13 uM) was comparable to that of CPT (IC50 = 0.26 ± 0.06 uM). Additionally, the IC50 values of these two conjugates against MDA-MB-231 and MCF-10A cells were significantly increased, showing their ability to selectively inhibit tumor cells. Based on the above results, Z8 and Z11, which exhibited better activity and apparent selectivity, were selected for subsequent studies.

To evaluated the in vitro anti-tumor activity of the conjugates, HER2-positive SK-BR-3, NCI-N87, HER2-negative MDA-MB-231 and normal MCF-10A cells were selected. As shown in Table 2, nearly all conjugates exhibited obvious anti-proliferation activity against SK-BR-3 cells, while most of the conjugates, except for Z6 and Z9, demonstrated different degrees of inhibitory activity on NCI-N87 cells. Taken together, Z8 and Z11 exhibited superior anti-tumor activity. The antiproliferative activity of Z8 against NCI-N87 cells (IC50 = 1.91 ± 0.71 uM) was comparable to that of CPT (IC50 = 2.10 ± 1.34 uM), while the antiproliferative activity of Z11 against SK-BR-3 cells (IC50 = 0.41 ± 0.13 uM) was comparable to that of CPT (IC50 = 0.26 ± 0.06 uM). Additionally, the IC50 values of these two conjugates against MDA-MB-231 and MCF-10A cells were significantly increased, showing their ability to selectively inhibit tumor cells. Based on the above results, Z8 and Z11, which exhibited better activity and apparent selectivity, were selected for subsequent studies.

The effect of the peptide conjugate QR-KLU on the proliferation of Huh7 and HUVEC cells was assessed by CCK8 assays. First, we investigated anti-proliferation ability of three peptides QR, KLU and QR-KLU on Huh7 and HUVEC cells with different concentrations. As shown in Fig. Fig.3A,3A, B, cells were treated with QR peptide, KLU peptide, QR-KLU peptide, DOX with different concentration respectively. The inhibition rate increased in a dose-dependent manner in KLU and QR-KLU groups in both cell lines. In HUVEC cells, peptide QR-KLU (IC50 10.7 ± 0.292 uM) showed more potent inhibition effect than KLU (IC50 33.8 ± 0.98 uM), and in Huh7 cells, QR-KLU (IC50 7.3 ± 0.74 uM) also showed more potent anti-tumor effect than KLU (IC50 36.27 ± 2.7 uM). Meanwhile, peptide QR showed negligible toxicity even under 80 uM.

The effect of the peptide conjugate QR-KLU on the proliferation of Huh7 and HUVEC cells was assessed by CCK8 assays. First, we investigated anti-proliferation ability of three peptides QR, KLU and QR-KLU on Huh7 and HUVEC cells with different concentrations. As shown in Fig. Fig.3A,3A, B, cells were treated with QR peptide, KLU peptide, QR-KLU peptide, DOX with different concentration respectively. The inhibition rate increased in a dose-dependent manner in KLU and QR-KLU groups in both cell lines. In HUVEC cells, peptide QR-KLU (IC50 10.7 ± 0.292 uM) showed more potent inhibition effect than KLU (IC50 33.8 ± 0.98 uM), and in Huh7 cells, QR-KLU (IC50 7.3 ± 0.74 uM) also showed more potent anti-tumor effect than KLU (IC50 36.27 ± 2.7 uM). Meanwhile, peptide QR showed negligible toxicity even under 80 uM.

The improved proapoptotic activity of QR-KLU was further confirmed by Annexin V-PI staining through FACS. Viable cells, early apoptotic cells, necrotic cells, late apoptotic cells were represented by Q4, Q3, Q2 and Q1 respectively. Peptide QR-KLU displayed a significant proapoptotic effect under 10 uM with apoptosis rate over 60% in Huh 7 cells and over 40% in HUVEC cells.

The improved proapoptotic activity of QR-KLU was further confirmed by Annexin V-PI staining through FACS. Viable cells, early apoptotic cells, necrotic cells, late apoptotic cells were represented by Q4, Q3, Q2 and Q1 respectively. Peptide QR-KLU displayed a significant proapoptotic effect under 10 uM with apoptosis rate over 60% in Huh 7 cells and over 40% in HUVEC cells.

NGR conjugate forms of both ibuprofen and naproxen showed improved activity when they were tested against SKOV-3 cell line which is positive for APN/CD13. Interestingly, both ibuprofen and naproxen show increased activity against this cell line when a six-carbon spacer is used for their conjugation to NGR. This is probably due to the less steric hindrance for NGR interaction with its binding protein on the cell surface. Ibuprofen-spacer-NGR and naproxen-spacer-NGR showed the same pattern of increased activity against HT-1080 tumor cells which this cell line show high expression of CD13. Surprisingly, NGR conjugates of both drugs without spacer did not show improved activity compared to the nonconjugated forms against this cell line. Therefore, it could be speculated that HT-1080 cells are more sensitive to the steric hindrance for interaction between NGR and its binding protein. None of the conjugates of ibuprofen and naproxen with or without spacer showed significantly improved activity against A2780 (as a tumor cell with normal RGD-binding protein) and OVCAR3. Therefore, it could be inferred again that the RGD motif is not qualified as a targeting tool for ibuprofen and naproxen.

NGR conjugate forms of both ibuprofen and naproxen showed improved activity when they were tested against SKOV-3 cell line which is positive for APN/CD13. Interestingly, both ibuprofen and naproxen show increased activity against this cell line when a six-carbon spacer is used for their conjugation to NGR. This is probably due to the less steric hindrance for NGR interaction with its binding protein on the cell surface. Ibuprofen-spacer-NGR and naproxen-spacer-NGR showed the same pattern of increased activity against HT-1080 tumor cells which this cell line show high expression of CD13. Surprisingly, NGR conjugates of both drugs without spacer did not show improved activity compared to the nonconjugated forms against this cell line. Therefore, it could be speculated that HT-1080 cells are more sensitive to the steric hindrance for interaction between NGR and its binding protein. None of the conjugates of ibuprofen and naproxen with or without spacer showed significantly improved activity against A2780 (as a tumor cell with normal RGD-binding protein) and OVCAR3. Therefore, it could be inferred again that the RGD motif is not qualified as a targeting tool for ibuprofen and naproxen.

NGR conjugate forms of both ibuprofen and naproxen showed improved activity when they were tested against SKOV-3 cell line which is positive for APN/CD13. Interestingly, both ibuprofen and naproxen show increased activity against this cell line when a six-carbon spacer is used for their conjugation to NGR. This is probably due to the less steric hindrance for NGR interaction with its binding protein on the cell surface. Ibuprofen-spacer-NGR and naproxen-spacer-NGR showed the same pattern of increased activity against HT-1080 tumor cells which this cell line show high expression of CD13. Surprisingly, NGR conjugates of both drugs without spacer did not show improved activity compared to the nonconjugated forms against this cell line. Therefore, it could be speculated that HT-1080 cells are more sensitive to the steric hindrance for interaction between NGR and its binding protein. None of the conjugates of ibuprofen and naproxen with or without spacer showed significantly improved activity against A2780 (as a tumor cell with normal RGD-binding protein) and OVCAR3. Therefore, it could be inferred again that the RGD motif is not qualified as a targeting tool for ibuprofen and naproxen.

NGR conjugate forms of both ibuprofen and naproxen showed improved activity when they were tested against SKOV-3 cell line which is positive for APN/CD13. Interestingly, both ibuprofen and naproxen show increased activity against this cell line when a six-carbon spacer is used for their conjugation to NGR. This is probably due to the less steric hindrance for NGR interaction with its binding protein on the cell surface. Ibuprofen-spacer-NGR and naproxen-spacer-NGR showed the same pattern of increased activity against HT-1080 tumor cells which this cell line show high expression of CD13. Surprisingly, NGR conjugates of both drugs without spacer did not show improved activity compared to the nonconjugated forms against this cell line. Therefore, it could be speculated that HT-1080 cells are more sensitive to the steric hindrance for interaction between NGR and its binding protein. None of the conjugates of ibuprofen and naproxen with or without spacer showed significantly improved activity against A2780 (as a tumor cell with normal RGD-binding protein) and OVCAR3. Therefore, it could be inferred again that the RGD motif is not qualified as a targeting tool for ibuprofen and naproxen.

NGR conjugate forms of both ibuprofen and naproxen showed improved activity when they were tested against SKOV-3 cell line which is positive for APN/CD13. Interestingly, both ibuprofen and naproxen show increased activity against this cell line when a six-carbon spacer is used for their conjugation to NGR. This is probably due to the less steric hindrance for NGR interaction with its binding protein on the cell surface. Ibuprofen-spacer-NGR and naproxen-spacer-NGR showed the same pattern of increased activity against HT-1080 tumor cells which this cell line show high expression of CD13. Surprisingly, NGR conjugates of both drugs without spacer did not show improved activity compared to the nonconjugated forms against this cell line. Therefore, it could be speculated that HT-1080 cells are more sensitive to the steric hindrance for interaction between NGR and its binding protein. None of the conjugates of ibuprofen and naproxen with or without spacer showed significantly improved activity against A2780 (as a tumor cell with normal RGD-binding protein) and OVCAR3. Therefore, it could be inferred again that the RGD motif is not qualified as a targeting tool for ibuprofen and naproxen.

NGR conjugate forms of both ibuprofen and naproxen showed improved activity when they were tested against SKOV-3 cell line which is positive for APN/CD13. Interestingly, both ibuprofen and naproxen show increased activity against this cell line when a six-carbon spacer is used for their conjugation to NGR. This is probably due to the less steric hindrance for NGR interaction with its binding protein on the cell surface. Ibuprofen-spacer-NGR and naproxen-spacer-NGR showed the same pattern of increased activity against HT-1080 tumor cells which this cell line show high expression of CD13. Surprisingly, NGR conjugates of both drugs without spacer did not show improved activity compared to the nonconjugated forms against this cell line. Therefore, it could be speculated that HT-1080 cells are more sensitive to the steric hindrance for interaction between NGR and its binding protein. None of the conjugates of ibuprofen and naproxen with or without spacer showed significantly improved activity against A2780 (as a tumor cell with normal RGD-binding protein) and OVCAR3. Therefore, it could be inferred again that the RGD motif is not qualified as a targeting tool for ibuprofen and naproxen.

NGR conjugate forms of both ibuprofen and naproxen showed improved activity when they were tested against SKOV-3 cell line which is positive for APN/CD13. Interestingly, both ibuprofen and naproxen show increased activity against this cell line when a six-carbon spacer is used for their conjugation to NGR. This is probably due to the less steric hindrance for NGR interaction with its binding protein on the cell surface. Ibuprofen-spacer-NGR and naproxen-spacer-NGR showed the same pattern of increased activity against HT-1080 tumor cells which this cell line show high expression of CD13. Surprisingly, NGR conjugates of both drugs without spacer did not show improved activity compared to the nonconjugated forms against this cell line. Therefore, it could be speculated that HT-1080 cells are more sensitive to the steric hindrance for interaction between NGR and its binding protein. None of the conjugates of ibuprofen and naproxen with or without spacer showed significantly improved activity against A2780 (as a tumor cell with normal RGD-binding protein) and OVCAR3. Therefore, it could be inferred again that the RGD motif is not qualified as a targeting tool for ibuprofen and naproxen.

NGR conjugate forms of both ibuprofen and naproxen showed improved activity when they were tested against SKOV-3 cell line which is positive for APN/CD13. Interestingly, both ibuprofen and naproxen show increased activity against this cell line when a six-carbon spacer is used for their conjugation to NGR. This is probably due to the less steric hindrance for NGR interaction with its binding protein on the cell surface. Ibuprofen-spacer-NGR and naproxen-spacer-NGR showed the same pattern of increased activity against HT-1080 tumor cells which this cell line show high expression of CD13. Surprisingly, NGR conjugates of both drugs without spacer did not show improved activity compared to the nonconjugated forms against this cell line. Therefore, it could be speculated that HT-1080 cells are more sensitive to the steric hindrance for interaction between NGR and its binding protein. None of the conjugates of ibuprofen and naproxen with or without spacer showed significantly improved activity against A2780 (as a tumor cell with normal RGD-binding protein) and OVCAR3. Therefore, it could be inferred again that the RGD motif is not qualified as a targeting tool for ibuprofen and naproxen.

NGR conjugate forms of both ibuprofen and naproxen showed improved activity when they were tested against SKOV-3 cell line which is positive for APN/CD13. Interestingly, both ibuprofen and naproxen show increased activity against this cell line when a six-carbon spacer is used for their conjugation to NGR. This is probably due to the less steric hindrance for NGR interaction with its binding protein on the cell surface. Ibuprofen-spacer-NGR and naproxen-spacer-NGR showed the same pattern of increased activity against HT-1080 tumor cells which this cell line show high expression of CD13. Surprisingly, NGR conjugates of both drugs without spacer did not show improved activity compared to the nonconjugated forms against this cell line. Therefore, it could be speculated that HT-1080 cells are more sensitive to the steric hindrance for interaction between NGR and its binding protein. None of the conjugates of ibuprofen and naproxen with or without spacer showed significantly improved activity against A2780 (as a tumor cell with normal RGD-binding protein) and OVCAR3. Therefore, it could be inferred again that the RGD motif is not qualified as a targeting tool for ibuprofen and naproxen.

NGR conjugate forms of both ibuprofen and naproxen showed improved activity when they were tested against SKOV-3 cell line which is positive for APN/CD13. Interestingly, both ibuprofen and naproxen show increased activity against this cell line when a six-carbon spacer is used for their conjugation to NGR. This is probably due to the less steric hindrance for NGR interaction with its binding protein on the cell surface. Ibuprofen-spacer-NGR and naproxen-spacer-NGR showed the same pattern of increased activity against HT-1080 tumor cells which this cell line show high expression of CD13. Surprisingly, NGR conjugates of both drugs without spacer did not show improved activity compared to the nonconjugated forms against this cell line. Therefore, it could be speculated that HT-1080 cells are more sensitive to the steric hindrance for interaction between NGR and its binding protein. None of the conjugates of ibuprofen and naproxen with or without spacer showed significantly improved activity against A2780 (as a tumor cell with normal RGD-binding protein) and OVCAR3. Therefore, it could be inferred again that the RGD motif is not qualified as a targeting tool for ibuprofen and naproxen.

NGR conjugate forms of both ibuprofen and naproxen showed improved activity when they were tested against SKOV-3 cell line which is positive for APN/CD13. Interestingly, both ibuprofen and naproxen show increased activity against this cell line when a six-carbon spacer is used for their conjugation to NGR. This is probably due to the less steric hindrance for NGR interaction with its binding protein on the cell surface. Ibuprofen-spacer-NGR and naproxen-spacer-NGR showed the same pattern of increased activity against HT-1080 tumor cells which this cell line show high expression of CD13. Surprisingly, NGR conjugates of both drugs without spacer did not show improved activity compared to the nonconjugated forms against this cell line. Therefore, it could be speculated that HT-1080 cells are more sensitive to the steric hindrance for interaction between NGR and its binding protein. None of the conjugates of ibuprofen and naproxen with or without spacer showed significantly improved activity against A2780 (as a tumor cell with normal RGD-binding protein) and OVCAR3. Therefore, it could be inferred again that the RGD motif is not qualified as a targeting tool for ibuprofen and naproxen.

NGR conjugate forms of both ibuprofen and naproxen showed improved activity when they were tested against SKOV-3 cell line which is positive for APN/CD13. Interestingly, both ibuprofen and naproxen show increased activity against this cell line when a six-carbon spacer is used for their conjugation to NGR. This is probably due to the less steric hindrance for NGR interaction with its binding protein on the cell surface. Ibuprofen-spacer-NGR and naproxen-spacer-NGR showed the same pattern of increased activity against HT-1080 tumor cells which this cell line show high expression of CD13. Surprisingly, NGR conjugates of both drugs without spacer did not show improved activity compared to the nonconjugated forms against this cell line. Therefore, it could be speculated that HT-1080 cells are more sensitive to the steric hindrance for interaction between NGR and its binding protein. None of the conjugates of ibuprofen and naproxen with or without spacer showed significantly improved activity against A2780 (as a tumor cell with normal RGD-binding protein) and OVCAR3. Therefore, it could be inferred again that the RGD motif is not qualified as a targeting tool for ibuprofen and naproxen.

NGR conjugate forms of both ibuprofen and naproxen showed improved activity when they were tested against SKOV-3 cell line which is positive for APN/CD13. Interestingly, both ibuprofen and naproxen show increased activity against this cell line when a six-carbon spacer is used for their conjugation to NGR. This is probably due to the less steric hindrance for NGR interaction with its binding protein on the cell surface. Ibuprofen-spacer-NGR and naproxen-spacer-NGR showed the same pattern of increased activity against HT-1080 tumor cells which this cell line show high expression of CD13. Surprisingly, NGR conjugates of both drugs without spacer did not show improved activity compared to the nonconjugated forms against this cell line. Therefore, it could be speculated that HT-1080 cells are more sensitive to the steric hindrance for interaction between NGR and its binding protein. None of the conjugates of ibuprofen and naproxen with or without spacer showed significantly improved activity against A2780 (as a tumor cell with normal RGD-binding protein) and OVCAR3. Therefore, it could be inferred again that the RGD motif is not qualified as a targeting tool for ibuprofen and naproxen.

NGR conjugate forms of both ibuprofen and naproxen showed improved activity when they were tested against SKOV-3 cell line which is positive for APN/CD13. Interestingly, both ibuprofen and naproxen show increased activity against this cell line when a six-carbon spacer is used for their conjugation to NGR. This is probably due to the less steric hindrance for NGR interaction with its binding protein on the cell surface. Ibuprofen-spacer-NGR and naproxen-spacer-NGR showed the same pattern of increased activity against HT-1080 tumor cells which this cell line show high expression of CD13. Surprisingly, NGR conjugates of both drugs without spacer did not show improved activity compared to the nonconjugated forms against this cell line. Therefore, it could be speculated that HT-1080 cells are more sensitive to the steric hindrance for interaction between NGR and its binding protein. None of the conjugates of ibuprofen and naproxen with or without spacer showed significantly improved activity against A2780 (as a tumor cell with normal RGD-binding protein) and OVCAR3. Therefore, it could be inferred again that the RGD motif is not qualified as a targeting tool for ibuprofen and naproxen.

NGR conjugate forms of both ibuprofen and naproxen showed improved activity when they were tested against SKOV-3 cell line which is positive for APN/CD13. Interestingly, both ibuprofen and naproxen show increased activity against this cell line when a six-carbon spacer is used for their conjugation to NGR. This is probably due to the less steric hindrance for NGR interaction with its binding protein on the cell surface. Ibuprofen-spacer-NGR and naproxen-spacer-NGR showed the same pattern of increased activity against HT-1080 tumor cells which this cell line show high expression of CD13. Surprisingly, NGR conjugates of both drugs without spacer did not show improved activity compared to the nonconjugated forms against this cell line. Therefore, it could be speculated that HT-1080 cells are more sensitive to the steric hindrance for interaction between NGR and its binding protein. None of the conjugates of ibuprofen and naproxen with or without spacer showed significantly improved activity against A2780 (as a tumor cell with normal RGD-binding protein) and OVCAR3. Therefore, it could be inferred again that the RGD motif is not qualified as a targeting tool for ibuprofen and naproxen.

NGR conjugate forms of both ibuprofen and naproxen showed improved activity when they were tested against SKOV-3 cell line which is positive for APN/CD13. Interestingly, both ibuprofen and naproxen show increased activity against this cell line when a six-carbon spacer is used for their conjugation to NGR. This is probably due to the less steric hindrance for NGR interaction with its binding protein on the cell surface. Ibuprofen-spacer-NGR and naproxen-spacer-NGR showed the same pattern of increased activity against HT-1080 tumor cells which this cell line show high expression of CD13. Surprisingly, NGR conjugates of both drugs without spacer did not show improved activity compared to the nonconjugated forms against this cell line. Therefore, it could be speculated that HT-1080 cells are more sensitive to the steric hindrance for interaction between NGR and its binding protein. None of the conjugates of ibuprofen and naproxen with or without spacer showed significantly improved activity against A2780 (as a tumor cell with normal RGD-binding protein) and OVCAR3. Therefore, it could be inferred again that the RGD motif is not qualified as a targeting tool for ibuprofen and naproxen.

NGR conjugate forms of both ibuprofen and naproxen showed improved activity when they were tested against SKOV-3 cell line which is positive for APN/CD13. Interestingly, both ibuprofen and naproxen show increased activity against this cell line when a six-carbon spacer is used for their conjugation to NGR. This is probably due to the less steric hindrance for NGR interaction with its binding protein on the cell surface. Ibuprofen-spacer-NGR and naproxen-spacer-NGR showed the same pattern of increased activity against HT-1080 tumor cells which this cell line show high expression of CD13. Surprisingly, NGR conjugates of both drugs without spacer did not show improved activity compared to the nonconjugated forms against this cell line. Therefore, it could be speculated that HT-1080 cells are more sensitive to the steric hindrance for interaction between NGR and its binding protein. None of the conjugates of ibuprofen and naproxen with or without spacer showed significantly improved activity against A2780 (as a tumor cell with normal RGD-binding protein) and OVCAR3. Therefore, it could be inferred again that the RGD motif is not qualified as a targeting tool for ibuprofen and naproxen.

NGR conjugate forms of both ibuprofen and naproxen showed improved activity when they were tested against SKOV-3 cell line which is positive for APN/CD13. Interestingly, both ibuprofen and naproxen show increased activity against this cell line when a six-carbon spacer is used for their conjugation to NGR. This is probably due to the less steric hindrance for NGR interaction with its binding protein on the cell surface. Ibuprofen-spacer-NGR and naproxen-spacer-NGR showed the same pattern of increased activity against HT-1080 tumor cells which this cell line show high expression of CD13. Surprisingly, NGR conjugates of both drugs without spacer did not show improved activity compared to the nonconjugated forms against this cell line. Therefore, it could be speculated that HT-1080 cells are more sensitive to the steric hindrance for interaction between NGR and its binding protein. None of the conjugates of ibuprofen and naproxen with or without spacer showed significantly improved activity against A2780 (as a tumor cell with normal RGD-binding protein) and OVCAR3. Therefore, it could be inferred again that the RGD motif is not qualified as a targeting tool for ibuprofen and naproxen.