Abstract 1962: Engineering of EGFR-targeted nanoparticles for triple negative breast cancer therapy

Abstract

Abstract The human epidermal growth factor receptor (EGFR) is expressed at high levels in at least 20% of breast cancers overall, but in 60-70% of patients with triple-negative breast cancer (TNBC), which makes EGFR a potential treatment target. Herein we developed a novel therapy for TNBC that utilizes anti-EGFR monoclonal antibody (mAb)-conjugated nanodiamonds (NDs) to specifically deliver paclitaxel (PTX) to breast cancer cells over-expressing EGFR. NDs, 2-8 nm diameter carbon nanoparticles, have recently emerged as promising drug carriers. In this study, we have synthesized a versatile ND construct that incorporates anti-EGFR mAb, fluorescent imaging agent and paclitaxel for multimodal imaging, targeting and treatment of TNBC. Sulfo-LC-SPDP was attached on to an aminated ND surface (ND-NH2), yielding sulfhydryl-reactive NDs (ND-SPDP). Thiolated paclitaxel-oligonucleotide (PTX-DNA) conjugates and thiolated mAbs were then simultaneously attached to ND-SPDPs, forming PTX-DNA/mAb@NDs. For the PTX-DNA conjugate, a fluorescein-labeled dT-nucleotide was inserted within the thiolated 20-mer poly-dT strand for cellular imaging and PTX loading quantification. The resultant nanoparticles could form highly stable suspensions in aqueous solutions. Dithiothreitol treatment detaches PTX-DNA and thiolated mAbs from the ND surface. About 2.2 ± 0.4 nmol of PTX and 53.5 ± 2.1 µg of mAb were loaded on per mg of PTX-DNA/mAb@NDs, while 3.1 ± 0.7 nmol of PTX for per mg of PTX-DNA@NDs. The targeting capacity of PTX-DNA/mAb@NDs was tested through flow cytometry analysis and confocal microscopy within 2 breast cancer cells of contrasting EGFR expression. Internalization of PTX-DNA@NDs and PTX-DNA/mAb@NDs was nearly equivalent within basal EGFR expressing MCF7 cells. However, when EGFR-overexpressing MDA-MB-231 cells were treated with PTX-DNA/mAb@NDs, there was a nearly 150% enhancement in the fluorescence signal as compared to that of PTX-DNA@NDs. Internalization of anti-EGFR-conjugated NDs within MDA-MB-231 cells was competitively inhibited by the addition of EGF. MTT assays were performed comparing free PTX with untargeted and targeted versions of PTX-DNA@NDs at equivalent PTX dosages in both cells. Enhanced cytotoxicity was observed in escalating-dose studies after 48 h treatment of PTX-DNA@NDs and PTX-DNA/mAb@NDs across all concentrations. Within MDA-MB-231 cells, anti-EGFR linked conjugates had an IC50 value of 43.8 nM, a nearly two-fold increase in efficacy compared to untargeted conjugates (83.3 nM). The data suggests that targeting through the mAb moiety increases specificity and internalization, which subsequently enhances therapeutic activity. Intravenous injection of NDs (up to 1.2 mg) didn't induce any toxicity and systemic immune response in mice, attesting to NDs as a biocompatible nanocarrier platform. In vivo efficacy of PTX-DNA/mAb@NDs will be further investigated in tumor-bearing mice. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1962. doi:1538-7445.AM2012-1962