(F-215) FZD7-Targeted Nanoparticles to Enhance Chemosensitivity in Triple-Negative Breast Cancer

Introduction: Triple-negative breast cancer (TNBC) is an aggressive subtype of cancer that accounts for about 15% of all breast cancer cases and has a poorer short-term prognosis when compared to other subtypes.¹ Many current chemotherapeutics used to treat TNBC are nonspecific, and, therefore, cause off-target effects and fail to elicit a robust tumor response, which results in higher mortality and recurrence rates. Targeted therapy enabled by nanoparticle (NP)-based drug carriers may prove more effective against TNBC. One way to both target and therapeutically manipulate TNBC cells is by coating drug-loaded NPs with antibodies against Frizzled7 (FZD7) receptors, which are overexpressed in ~70% of TNBC cases.² When antibody nanoconjugates bind FZD7 receptors, it prevents their activation by extracellular Wnt ligands, leading to downstream inhibition of the Wnt signaling pathway which ordinarily promotes TNBC drug resistance, metastasis, and recurrence. Here, we demonstrate that FZD7 antibody-coated polymer NPs that are loaded with the chemotherapeutic doxorubicin (DOX) enable selective DOX delivery to TNBC cells versus non-cancerous breast epithelial cells and improve the drug’s potency compared to free delivery or delivery in non-targeted NPs.

Materials and Methods: DOX-loaded NPs (DOX-NPs) were synthesized by adding DOX-HCl (Sigma) to poly(lactide-co-glycolide)-polyethylene glycol-maleimide (PLGA-PEG; 20 kDa 50:50 PLGA, 5 kDa PEG; Nanosoft Polymers) dissolved in dichloromethane which was then added to 0.25% polyvinyl alcohol in a 1:3 ratio and probe sonicated. Anti-FZD7 antibodies (LSBio) were treated with tris(2-carboxyethyl) phosphine hydrochloride (TCEP; Sigma) to expose free thiol groups and facilitate binding with the maleimide groups on the NP surface. The NPs were incubated overnight with 30 µg TCEP-treated antibodies per mg PLGA and passivated with thiolated methoxy-PEG to synthesize FZD7-DOX-NPs. Control NPs were only passivated with the thiolated methoxy-PEG (PEG-DOX-NPs). A solution-based ELISA was used to quantify antibody loading on the NPs. The hydrodynamic diameter and zeta potential of the NPs were measured using Nanoparticle Tracking Analysis (NTA; Malvern NanoSight NS300) and dynamic light scattering (DLS; AntonPaar LiteSizer). Additionally, NP stability and drug release were analyzed in various conditions. All in vitro studies were performed in MDA-MB-231 human TNBC cells with a dosage of 5 µM DOX. Fluorescence microscopy (Zeiss Axioobserver Z1) was used to compare DOX accumulation after 4 and 24 hours in MDA-MB-231 cells after treatment with free DOX, PEG-DOX-NPs, or FZD7-DOX-NPs. Each treatment’s effect on MDA-MB-231 cellular metabolic activity and apoptosis was assessed by an MTT assay and an AnnexinV/SYTOX assay, respectively. Wnt inhibition in cells exposed to FZD7-targeted or control NPs for 24 hours was tested by measuring cell count.

Results, Conclusions, and Discussions: FZD7-DOX-NPs were successfully synthesized with a hydrodynamic diameter of 213 + 20.0 nm, an antibody loading of 1078 + 120 antibodies/NP, and DOX loading of 9.5 + 0.7 µg DOX/mg PLGA. Control, PEG-DOX-NPs had a diameter of 191 + 11.5 nm and encapsulated 13.6 + 4.6 µg DOX/mg PLGA. Each of these NP formulations had stable diameters and zeta potentials over 72 hours when placed in storage condition (water, 4°C) or physiological condition (10% FBS in PBS, 37°C, 100 rpm shaker). DOX release from the NPs was fastest when they were placed in acidic (pH 5.5) condition mimicking the tumor environment, with 81% of the encapsulated DOX being released from FZD7-DOX-NPs after 24 hours. When administered to MDA-MB-231 cells, FZD7-DOX-NPs inhibited metabolic activity to a greater extent than PEG-DOX-NPs, and the level of inhibition was significantly more than that achieved by free DOX (Figure 1). Quantification of nuclear DOX using fluorescence microscopy indicated a slower nuclear localization of DOX when encapsulated in NPs versus freely delivered (Figure 2). Preliminary experiments also indicate that MDA-MB-231 cells treated with FZD7-NPs containing no DOX induced a drastic drop in cell count for with only minor drops in cell counts for the control treatment groups, indicating that the antibody targeting is also affecting cell function. As a whole, these results demonstrate that FZD7-targeted nanocarriers have potential to increase the efficacy of conventional chemotherapies, such as DOX, which may ultimately lead to improved outcomes for TNBC patients.

Acknowledgements (Optional) : This work was supported by the National Institutes of Health (R01CA211925).

References (Optional) :

1. Yin, L., Duan, J.-J., Bian, X.-W. & Yu, S. Triple-negative breast cancer molecular subtyping and treatment progress. Breast Cancer Res 22, 61 (2020).
2. Yang, L. et al. FZD7 has a critical role in cell proliferation in triple negative breast cancer. Oncogene 30, 4437–4446 (2011).