Introduction: The development of nanotherapeutics for cancer therapy is a challenging task due to the complexity, heterogeneity, and diversity of cancers.¹ Nano-scale drug delivery platforms are widely used for passive targeted drug delivery since they facilitate the enhanced permeability and retention (EPR) effect.² However, active targeting can be more efficient compared to the conventional passive targeted drug delivery systems since it specifically and selectively targets malignant cells. The use of nanoparticles modified by target-specific bioligands facilitates both passive and active targeting increasing the therapeutic index and minimizing systemic toxicities. In this study, we used MRI-detectable ultra-small superparamagnetic iron oxide (USPIO) nanoparticles and conjugated them with anti-PSMA 5D3 mAb and DM1 anti-tubulin agent to develop a targeted 5D3-USPIO-DM1 drug delivery system for PSMA-positive prostate cancer (PC) therapy. Labeling USPIO with fluorophores enables multimodality in vivo detection of the drug delivery system, 5D3-USPIO-DM1-CF750, by optical and MR imaging. 5D3 mAb has improved PSMA binding affinity and internalization characteristics compared to other existing anti-PSMA mAbs.^3-5 USPIO, as a drug delivery platform, combines high loading capacity and biocompatibility with strong MRI contrast.^6-7

Methods: In the synthesis, first, 5D3 mAb was functionalized with pegylated trans-cyclooctene (TCO) groups. The USPIO (Ocean NanoTech, Inc. Surface functionality: Amine groups, Diameter 10 nm) was functionalized with tetrazine (Tz) click chemistry reactive group and MCC linker. Then the product Tz-USPIO-MCC nanoparticles were conjugated with anti-PSMA 5D3-TCO by TCO-Tz inverse electron-demand Diels-Alder reaction (IEDDA) click reaction. The 5D3-USPIO-MCC was further conjugated with mertansine (DM1), an anti-tubulin agent, as the chemotherapeutic drug to obtain 5D3-USPIO-DM1. The product, 5D3-USPIO-DM1 was labeled with near-infrared (NIR) fluorophore, CF-750, for in vivo and ex vivo optical imaging. The final product, USPIO-5D3-DM1-CF750 was characterized by transmission electron microscopy (TEM), dynamic light scattering (DLS), and SDS-PAGE. The 5D3-USPIO-DM1 was evaluated in vitro using PSMA(+) PC3-PIP and PSMA(-) PC3-Flu cells for cellular uptake and cytotoxicity. The tumor uptake, therapeutic efficacy, and toxicological effects were determined in PSMA(+/-) bilateral human PC subcutaneous xenograft mouse models.

Results: The conjugation was confirmed by the SDS-PAGE (Figure 1A) and the diameter of the drug delivery system of approximately 13 nm was not significantly increased compared to unconjugated USPIO (Figure 1B). As shown in Figure 1C, the Prussian blue staining study demonstrated the specific and efficient cellular uptake of 5D3-USPIO-DM1 in PSMA(+) PC cells. We also observed significantly higher tumor uptake by PSMA(+) tumors compared to PSMA(-) tumors by in vivo optical Xenogen imaging. The therapeutic study results exhibited significant control of PSMA(+) tumor growth by 5D3-USPIO-DM1-CF750 compared to the PSMA(-) tumors in human PC xenograft mouse models without significant toxicities in the liver and kidneys. In vivo T1/T2 MR images showed considerably higher contrast of 5D3-USPIO-DM1-CF750 in PSMA(+) tumors.

Conclusions: In this study, we have developed an efficient drug delivery system utilizing both active and passive targeting strategies using USPIO and anti-PSMA 5D3 mAb. This delivery system with DM1 therapeutic cargo shows a significant reduction of PSMA(+) tumor growth in human PC xenograft mouse models without significant toxicities in the liver and kidneys. The system is integrated with imaging agents and therefore it can be further developed as a theranostic for prostate cancer therapy.

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Figure 1. Development of anti-PSMA 5D3-USPIO-DM1. (A) SDS-PAGE study. (B) TEM images of (a) USPIO and (b) 5D3-USPIO-DM1-CF750. (C) Prussian Blue staining of 5D3-USPIO-DM1 treated (a) PSMA (+) and (b) PSMA (-) cells. (D) In vivo tumor uptake of 5D3-USPIO-DM1-CF750. Post-injection Xenogen optical images (a) after 1h and (b) after 24 h. (E) Therapeutic efficacy of 5D3-USPIO-DM1-CF750 in PSMA(+) tumors against unconjugated USPIO and untreated mice as controls. (F) Therapeutic efficacy of 5D3-USPIO-DM1-CF750 in PSMA(-) tumors against unconjugated USPIO and untreated mice as controls. (G) MRI of (Panel: a) T1-weighted and (Panel: b) T2-weighted images of PSMA(+) tumors (yellow arrows) and PSMA(-) tumors (blue arrows) pre-scan and 24 h after treatment with 5D3-USPIO-DM1-CF750. 

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