Anti-Folate Receptor 1 Antibody-Conjugated Copper Sulfide Nanoparticles for In Vivo Multispectral Optoacoustic Tomography

329 Objectives: Copper sulfide nanoparticles (CuS NPs) have emerged as one of the most promising theranostic nanoplatforms for treating cancer, due to their relatively small size, facile chemistry, intrinsic biodegradability and excellent photothermal properties. Herein, we designed an anti-folate receptor 1 antibody-conjugated CuS NP (CuS-PEG-αFOLR1) through click chemistry. Our goal was to investigate its in vitro and in vivo targeting towards FOLR1-postive tumor (SKOV3 ovarian cancer) and its application in multispectral optoacoustic tomography (MSOT) as a novel nonradioactive cancer diagnosis paradigm. Methods: Polyethylene glycol (PEG)-coated CuS NPs were synthesized via one-step reaction using CuCl2 and Na2S as the precursors and mPEG-SH and NH2-PEG-SH as the surfactants at 95 oC. As-prepared CuS-PEG-NH2 was conjugated with a click chemistry compound trans-cyclooctene (TCO), while αFOLR1 and its isotype control IgG were conjugated with the corresponding click chemistry compound tetrazine (Tz). The resulting CuS-PEG-TCO was subsequently reacted with αFOLR1-Tz and IgG-Tz to form CuS-PEG-αFOLR1 or CuS-PEG-IgG for in vitro and in vivo experiments. The CuS nanoconjugates were characterized by transmission electron microscopy (TEM), dynamic light scattering (DLS) and UV-VIS-NIR spectrometry. Fluorescence-activated cell sorting (FACS) was performed to validate the targeting specificity of αFOLR1 and CuS-PEG-αFOLR1 in vitro. MSOT was carried out to noninvasively monitor the biodistribution of CuS-PEG-αFOLR1 and CuS-PEG-IgG and evaluate their tumor targeting efficacy in SKOV3 ovarian tumor-bearing mice. The imaging results were validated by histological analysis. Results: CuS-PEG-αFOLR1 and CuS-PEG-IgG were successfully synthesized with narrow size range (14~16 nm in diameter). After incubating with SKOV3 cells at 4 oC for one hour, αFOLR1 exhibited ~30 times higher cell uptake than its isotype control IgG, while CuS-PEG-αFOLR1 displayed ~3 times higher uptake than that of CuS-PEG-IgG. Successful blocking with an excess amount of αFOLR1 further confirmed the targeting specificity. In vivo MSOT studies revealed a significant enhancement of tumor uptake of CuS-PEG-αFOLR1 at various wavelengths (922 ± 116, 1052 ± 216,1136 ± 361 and 1050 ± 213 ΔMSOT a.u. at 800, 850, 900 and 950 nm), which was significantly higher than that of CuS-PEG-IgG (156 ± 241, 126 ± 263,143 ± 96 and 243 ± 307 ΔMSOT a.u. at 800, 850, 900 and 950 nm). In vitro and ex vivo histological examinations further confirmed the specificity of CuS-PEG-αFOLR1 for folate receptor 1 overexpressed cancer. Conclusion: In this study, we reported a novel nonradioactive αFOLR1-conjugated CuS NP, capable of targeting FOLR1 overexpressing SKOV3 tumor both in vitro and in vivo. MSOT imaging revealed significantly enhanced tumor uptake of CuS-PEG-αFOLR1 compared with CuS-PEG-IgG. The data presented here provides evidence for establishing CuS-PEG-αFOLR1 as a potential nanoplatform for molecular imaging and cancer theranostics. Acknowledgements:Supported in part by John S. Dunn Foundation.