A suspension of 1 × 106 CT-26 cells was subcutaneously injected into the flank of male Balb/c mice to induce colorectal tumors [57 (link)]. The UFRN's Committee on the Ethics of Animal Experiments accepted the procedure (CEUA, Permit Number: 170.020/2019).
Tumour growth was monitored daily with calliper and upon reaching 3-4 mm in diameter, animals were categorized into six groups (N=5, each group) and peritumorally treated three times within 15 days with: (1) Control (untreated) = 5 mg/kg saline solution; (2) Oxaliplatin (OXA) = 5 mg/kg; NP3 (RA)-CHO = 1.25 mg/Kg; (4) 1.25 mg/kg saline solution, previously treated (30 minutes before) with anti-PD-L1 inhibitor = 10F.9G2, 2.16 mg/kg; (5) NP3 (RA)-CHO = 1.25 mg/kg, previously treated (30 minutes before) with anti-PD-L1 inhibitor (10F.9G2, 2.16 mg/kg); (6) NP1 (OXA, RA)-CHO25 = 1.25 mg/kg, previously treated (30 minutes before) with anti-PD-L1 inhibitor (10F.9G2, 2.16 mg/kg). Prior administration of the anti-PD-L1 inhibitor was only given in the first treatment, with the others being administered only saline solution, NP3 (RA)-CHO or NP1 (OXA, RA)-CHO. In this line, some mice were treated with OXA, a standard chemotherapy to CRC, and with NP1 loaded with OXA+RA and coated with CHO [57 (link)]. They were used in this study as control groups to compare their effects to NP3, which contained RA but not OXA. During treatment the diameter of the tumours was measured every two days. Five days after the last treatment (day 19), the animals were euthanized and blood was collected from the cardiac cavity (for biochemical analysis), as well as the tumour for signalling pathway analysis of tumour progression by quantitative real-time (qRT)-PCR and immunohistochemistry. Lungs and liver were collected for histopathological analysis of metastasis using haematoxylin-eosin (H&E) staining and immunohistochemistry. Results were expressed as a growth curve from the average tumour volume (mm³) calculated by the following equation according to Oliveira et al. [57 (link)]: volume = (width x length²) x 0.52.
Tumour growth was monitored daily with calliper and upon reaching 3-4 mm in diameter, animals were categorized into six groups (N=5, each group) and peritumorally treated three times within 15 days with: (1) Control (untreated) = 5 mg/kg saline solution; (2) Oxaliplatin (OXA) = 5 mg/kg; NP3 (RA)-CHO = 1.25 mg/Kg; (4) 1.25 mg/kg saline solution, previously treated (30 minutes before) with anti-PD-L1 inhibitor = 10F.9G2, 2.16 mg/kg; (5) NP3 (RA)-CHO = 1.25 mg/kg, previously treated (30 minutes before) with anti-PD-L1 inhibitor (10F.9G2, 2.16 mg/kg); (6) NP1 (OXA, RA)-CHO25 = 1.25 mg/kg, previously treated (30 minutes before) with anti-PD-L1 inhibitor (10F.9G2, 2.16 mg/kg). Prior administration of the anti-PD-L1 inhibitor was only given in the first treatment, with the others being administered only saline solution, NP3 (RA)-CHO or NP1 (OXA, RA)-CHO. In this line, some mice were treated with OXA, a standard chemotherapy to CRC, and with NP1 loaded with OXA+RA and coated with CHO [57 (link)]. They were used in this study as control groups to compare their effects to NP3, which contained RA but not OXA. During treatment the diameter of the tumours was measured every two days. Five days after the last treatment (day 19), the animals were euthanized and blood was collected from the cardiac cavity (for biochemical analysis), as well as the tumour for signalling pathway analysis of tumour progression by quantitative real-time (qRT)-PCR and immunohistochemistry. Lungs and liver were collected for histopathological analysis of metastasis using haematoxylin-eosin (H&E) staining and immunohistochemistry. Results were expressed as a growth curve from the average tumour volume (mm³) calculated by the following equation according to Oliveira et al. [57 (link)]: volume = (width x length²) x 0.52.
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