Design And Synthesis Of Mitochondria-Targeting Near-Infrared Probes Based On The Inhibitor And Its Theranostic Application In Osteosarcoma

Objective As the most common malignant bone sarcoma,osteosarcoma(OS)is a severe threat to patients with poor prognoses.Currently,adjuvant chemotherapy combined with an adequate surgical margin is the primary treatment approach for OS.However,there has been no breakthrough in OS therapy since the introduction of adjuvant chemotherapy,and early diagnosis is crucial in improving the five-year survival of OS patients.However,current technologies for OS imaging have the disadvantages such as radiation,low temporal or spatial resolution,and cannot be applied to intraoperative real-time imaging.Hence,developing an osteosarcoma nearinfrared theranostic probe for simultaneous guiding surgery and chemotherapy can improve outcomes for osteosarcoma patients.Cyanine dyes,a class of organic fluorescent dyes,are always used for clinic and basic research.Some cationic,lipophilic cyanine dyes with a meso-Cl which can covalently bind to albumin,will preferentially accumulate in the mitochondria of the tumor,but these dyes show poor targeting when they were directly applied for tumor-targeted imaging.Moreover,compared to peptides or antibodies,small molecular inhibitors have some unique advantages as targeting ligands.Given these findings,to get excellent theranostic probes and explore the different outcomes of the modification on the dyes,we developed two candidate theranostic probes by conjugating BCL-2/MCL-1 dual inhibitor NAPd to two different heptamethine cyanine dyes.Our research will provide experimental evidence and reference for developing novel theranostic probes based on heptamethine cyanine dyes.Methods Two novel theranostic probes,NAP-808 and NAP-783,were synthesized by conjugating BCL-2/MCL-1 dual inhibitor to different heptamethine cyanine dyes.And the characterizations and photostability were detected.In vitro,CCK-8 and apoptosis assay were performed to evaluate the anti-tumor ability of probes and their parent structures.The cellular uptake and subcellular localization experiments were performed to evaluate the uptake mechanism of these probes.Then,the distributions of probes were assessed by whole-animal and ex vivo organ imaging at different time points after injection.And the imaging effects of probes and parent dyes were evaluated by in vivo imaging.Finally,we evaluated the anti-tumor ability of NAP-808,which showed the same effect as NAPd in vitro in osteosarcoma xenograft mice.Results Both NAP-808 and NAP-783 exhibited good photostability in the solvent,but NAP-783 showed a large blue-shift of emission wavelength while NAP-808 showed a small red-shift.Nap-808 demonstrated the best mitochondrial-target ability and similar tumor inhibition effect as the parent structure of inhibitor in osteosarcoma cell lines,but NAP-783,which meso-Cl was substituted by inhibitor,showed poor cell permeability,weak mitochondrial localization and anti-osteosarcoma ability in vitro.Moreover,the mitochondrial-target ability of NAP-808 could be compromised after being covalently bound to albumin.Consistent results were observed in vivo.Imaging results of osteosarcoma xenograft revealed that both NAP-808 and parent dyes could effectively accumulate in osteosarcoma tissue,while NAP-783 was rapidly cleared and could not actively target osteosarcoma.Finally,NAP-808 exhibited a stronger anti-OS ability than its parent structures with no obvious systemic toxicity.Conclusion NAP-808 exhibited good anti-osteosarcoma and targeting imaging ability in vitro and vivo,and it shall be a valuable candidate for targeted imaging and treatment of OS.Our results demonstrated that modifying the alkyl chain and more lipophilic dyes would help achieve tumor-targeted delivery and long-term retention,and enhance inhibitor anti-tumor activity.Moreover,inhibitor structure strengthens OS targeting and the probe’s retention ability.Targeting theranostic probe designed based on the inhibitor and heptamethine cyanine dye can restrict systemic toxic side effects compared with applying the inhibitor directly.In conclusion,NAP-808 can realize the integration of diagnosis and treatment of OS through near infra-red imaging and its anti-OS ability.Developing a novel drug delivery system based on the structure-activity relationship provides new ideas for facilitating the clinical practice of inhibitors with poor pharmacokinetics.