Lumina series 3

The 4T1 tumour-bearing mice (n = 4) received BiRN nanoprobes intravenously for ratiometric fluorescence imaging to monitor tumour accumulation and internalisation of nanoparticles. The images were captured on an IVIS in vivo imaging system (Lumina Series III, PerkinElmer, USA) with time lapse using auto-exposure with a band-pass filter (λ_ex/λ_em: 620 ± 10 nm/670 ± 20 nm for Cy5, 780 ± 10 nm/845 ± 20 nm for Cy7.5). To demonstrate the broad application of BiRN and compare the difference between various tumours in accumulation and endocytosis, BiRN was administrated intravenously into other six tumour models mentioned above. Then, the mice were monitored by sequential acquisition at 670 and 845 nm at designated time-points. Mice were euthanized after the last imaging session, followed by resection of the tumour and organs. The dissected tumours and organs were fluorescently imaged immediately. The images of Cy5 and Cy7.5 channels were extracted using Living Image software (PerkinElmer) and the ratiometric images were generated by ImageJ software (NIH).

Orthotopic 4T1-Luc tumour-bearing mice (n = 32) with an average tumour volume of 50 mm³ were fluorescently imaged at 24 h post-administration of BiRN (20 mg kg⁻¹). The heterogeneity of EPR effect and internalisation amount of nanoparticles among tumours were quantified by the Cy7.5 and Cy5 signals over the entire tumour area, respectively. After imaging, three-quarters of the mice received the PDPA-PTX micelle with a single dose of 20 mg kg⁻¹ PTX by intravenous administration. For tumour progression monitoring, caliper measurements were performed every two days to monitor the tumour volume (V = Length × width²/2). Pulmonary metastasis was detected on 17th day by bioluminescence imaging with an IVIS in vivo imaging system (Lumina Series III, PerkinElmer, USA). At the end of the experiment, the GraphPad Prism 8 was used for correlation analysis between tumour progression and tumour accumulation as well as the internalisation of nanoparticles.