Dual-Targeted Delivery of Nanoparticles Encapsulating Paclitaxel and Everolimus: a Novel Strategy to Overcome Breast Cancer Receptor Heterogeneity

Abstract

Purpose: The intratumoral heterogeneity observed in breast cancer (BC), in particular with regard to cell surface receptor expression, can hinder the success of many targeted cancer therapies. The development of novel therapeutic agents that target more than one receptor can overcome this inherent property of tumors and can facilitate their selective internalization in cancer cells. The goal of this study is to develop a drug combination-loaded nanoparticle (NP) formulation that is actively-targeted to HER2 and EGFR receptors on BC cells.

Methods: A polymeric NP formulation was prepared which co-encapsulated a synergistic combination of the chemotherapeutic agent, paclitaxel (PTX), and the mTOR inhibitor, everolimus (EVER), and is targeted to HER2 and EGFR receptors on BC cells using antibody Fab fragments as the targeting moieties. The physicochemical characteristics of the dual-targeted formulation (Dual-NP) were evaluated, along with its cytotoxic profile (in both, monolayer and 3D BC models), as well as the degree of cellular uptake in HER2_high/EGFR_mod and HER2_neg/EGFR_low BC cells.

Results: Dual-NPs were found to have significantly higher cytotoxicity relative to HER2 mono-targeted (T-NPs) and untargeted NPs (UT-NPs) in HER2_high/EGFR_mod monolayer BC cells after 72 h exposure, while no significant difference was observed in HER2_neg/EGFR_low cells. However, in the HER2_high/EGFR_mod spheroids, the cytotoxicity of Dual-NPs was comparable to that of T-NPs. This was thought to be attributed to the previously reported downregulation of EGFR in 3D in comparison to 2D BC models. Dual-NPs had significantly higher cellular uptake relative to UT-NPs and T-NPs in HER2_high/EGFR_mod BC cells after 24 h exposure, whereas in the HER2_neg/EGFR_low cells, the increase in cellular uptake of the Dual-NPs was not as high as the level achieved in the HER2_high/EGFR_mod cells. Blocking HER2 and EGFR significantly reduced the uptake of T-NPs and Dual-NPs in the HER2_high/EGFR_mod BC cells, demonstrating specific binding to both EGFR and HER2.

Conclusions: The dual-targeting strategy developed in this study in conjunction with a potentially promising delivery vector for a synergistic combination therapy can overcome receptor heterogeneity, yielding significant improvements in the cytotoxicity and cellular uptake in BC cells.

Keywords: combination therapy; dual-targeting; everolimus; nanoparticles; paclitaxel.