Protocol

NIR-II FPs based on recombinant albumin under mild conditions for rapidly assessing BTB disruption

Considering both non-covalent affinity and covalent binding ability, we ultimately chose rHSA-C476G, the optimal recombinant HSA, to construct the FPs for assessing BTB integrity (Fig. 1A). With the mutation of Cys476 to Gly476, the modified protein cavity acted as an improved microreactor that covalently bound to CO-1080 under mild conditions. Here, BTB-damaged mice injected with CO-1080@rHSA were used as the treated group, while both BTB-damaged and normal mice injected with CO-1080@HSA were classified as the control groups. All the FPs were incubated at room temperature for 2 h. NIR-II whole-body imaging and testicular imaging of the treated and control groups revealed that CO-1080@rHSA outperformed CO-1080@HSA in assessing BTB integrity, both in terms of brightness and TSR (Fig 1, B to E, and Fig. 2). Notably, the brightness of BTB-damaged testes showed a statistically significant difference from that of normal testes at early as 1 h after CO-1080@rHSA injection. Similar to the effect observed with CO-1080@HSA incubated at 60 °C for 2 h in testicular imaging, there was no overlap in the brightness of BTB-damaged and normal testes at 6 h of CO-1080 injection, when the TSR reached 4.3. CO-1080@rHSA retained the advantage of NIR-II FPs based on exogenous albumin for rapidly assessing BTB integrity and had similar brightness and TSR to CO-1080@HSA (incubated at 60 °C for 2 h) for in vivo imaging. Notably, it is unique advantage lies in its ability to be prepared by simple mixing of CO-1080 and recombinant HSA without the need for heating.

Fig. 1 Optimized recombinant HSAconstructed by site-directed mutagenesis strategy for efficiently assessing BTB integrity. (A) Schematic of the construction of rHSA-C476G based on genetic recombination technology. Comparison of (B) NIR-II whole-body imaging and (C) testicular imaging in BTB-damaged mice injected with CO-1080@rHSA as the treated group, BTB-damaged mice injected with CO-1080@HSA and normal mice injected with CO-1080@HSA as control groups at specific time points post-injection (>1200 nm collection, 200 ms, n=3 for each group). (D) Brightness measurement of testis and skin of the treated and control groups at various time points post-injection (>1200 nm, 200 ms, n=6 for each group). (E) Statistical analysis of quantitative differences on the testis and skin of treated and control groups at various time points post-injection and comparison of significant differences in the effectiveness of CO-1080@rHSA and CO-1080 on identifying BTB disruption at 6 h after injection (mean ± SD, n=6 for each group). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Some schematic diagrams were designed using BioRender software. The protein structure was generated by the Protein Data Bank (PDB). Here, CO-1080@rHSA and CO-1080@HSA for all NIR-II bioimaging was incubated at room temperature for 2 h and the injection dose was 200 μL (200 μM).

Fig. 2. Statistical analysis of quantitative differences on the testis and skin at various time points post-injection and comparison of significant differences in the effectiveness of CO-1080@rHSA and CO-1080 on identifying BTB disruption (mean ± SD, n=6 for each group). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

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Experimental Design Assistant (EDA) Report