Rationale: Although immunotherapy through programmed death 1 / programmed death ligand 1 (PD-1/PD-L1) checkpoint blockade has shown impressive clinical outcomes, not all patients respond to it. Recent studies have demonstrated that the expression level of PD-L1 in tumors is one of the factors that correlate with the PD-1/PD-L1 checkpoint blockade therapy. Herein, a novel ⁶⁸Ga-labeled nanobody tracer, ⁶⁸Ga-NOTA-Nb109, was designed and developed for specific and noninvasive imaging of PD-L1 expression in the melanoma-bearing mice model. Methods: The nanobody Nb109 was labeled with the radionuclide ⁶⁸Ga through a NOTA chelator. In vitro binding assay was performed to assess the affinity and binding epitope of Nb109 to PD-L1. The clinical application value of ⁶⁸Ga-NOTA-Nb109 was evaluated by the stability assay, biodistribution and pharmacokinetics studies as well as positron emission tomography (PET) imaging, autoradiography and immunohistochemical staining studies on the tumor-bearing models with different PD-L1 expression. Results: The tracer ⁶⁸Ga-NOTA-Nb109 was obtained with the radiochemical yield > 95% and the radiochemical purity > 98% in 10 min. It showed a high specific affinity for PD-L1 with the equilibrium dissociation constant (KD) of 2.9 × 10-9 M. A competitive binding assay indicated a different binding epitope for Nb109 compared with PD-1 and PD-L1 antibody. All the biodistribution, PET imaging, autoradiography and immunohistochemical staining studies revealed that the tracer ⁶⁸Ga-NOTA-Nb109 specifically accumulated in A375-hPD-L1 tumor with a maximum uptake of 5.0 ± 0.35 %ID/g at 1 h. Conclusion: The tracer ⁶⁸Ga-NOTA-Nb109 holds great potential for noninvasive PET imaging of the PD-L1 status in tumors and timely evaluating the therapeutic effect of immune checkpoint targeting treatment.

Five Ru(II)–N-heterocyclic carbenes (NHC) (1–5) were synthesized by reacting the appropriately substituted imidazolium chlorides with Ag₂O, forming the NHC-silver chloride in situ followed by transmetalation with dimeric p-cymene ruthenium(II) dichloride. All the complexes were characterized by NMR and ESI-MS, and complex 1 was also characterized by single-crystal X-ray diffraction. The IC₅₀ values of these five complexes were determined by the MTT-based assay on four human cancer cell lines, SKOV-3 (ovarian), PC-3 (prostate), MDA-MB-231 (breast) and EC109 (esophagus). The cytotoxicities of these complexes changed from a moderate effect to a fine one, corresponding to the increasing lipophilicity order of the complex of 2 < 1 < 3 < 4 < 5 (0.91, 0.88, 1.36, 1.85 and 2.62 for 1–5 respectively). Complex 5 showed the most cytotoxicity with the IC₅₀ values 10.3 ± 0.3 μM for SKOV-3, 2.9 ± 0.1 μM for PC-3, 8.2 ± 0.6 μM for MDA-MB-231, 6.4 ± 0.2 μM for EC109 cell lines. Due to the superior cytotoxicity of complex 5 against the PC-3 cell lines, further biological evaluations were carried out to elucidate its action mechanism. The morphologic changes and cell cycle analysis showed that complex 5 can inhibit PC-3 cell lines by inducing cell cycle arrest at the G2/M phase. The DNA binding experiments further demonstrate that complex 5 has a better binding ability for DNA (K_b = 2.2 × 10⁶ M⁻¹) than complexes 1–4 (3.8 × 10⁵, 7.0 × 10⁵, 5.7 × 10⁵, and 1.9 × 10⁵ respectively).

Nanocarriers are widely used for delivering drugs to tumors and their development is progressing steadily. In this study, a pH sensitive mesoporous silica nanocarrier, RuNHC@MSNs-CTS-Biotin (CTS = chitosan), is developed for the targeted delivery and controlled release of a ruthenium(II) N-heterocyclic carbene (RuNHC) complex. The RuNHC@MSNs-CTS-Biotin nanoparticles were composed of RuNHC loaded mesoporous silica nanoparticles (MSNs) coated with chitosan-biotin (CTS-Biotin) conjugates. CTS traps the RuNHC complex inside the mesopores and biotin is used as a targeting ligand to improve specific cell uptake. The particle size of RuNHC@MSNs-CTS-Biotin was around 90 nm with a zeta potential of 12.0 mV and the RuNHC loading capacity was 26.31%. The release of RuNHC from RuNHC@MSNs-CTS-Biotin was in a pH-dependent manner, and it exhibited a 59.71% terminal release ratio at pH 5.0, but almost no release under neutral conditions (pH 7.4). Its in vitro cellular uptake and anticancer activity revealed that RuNHC@MSNs-CTS-Biotin could be selectively internalized in cancer cells by biotin receptor-mediated endocytosis and this resulted in a significant improvement in anticancer activities as compared with the RuNHC complex. This multifunctional nanocarrier system provides a promising platform for the development of precisely controllable cancer therapy.