Nanodiamond-mitoxantrone complexes enhance drug retention in chemoresistant breast cancer cells

Abstract

Chemoresistance is a prevalent issue that accounts for the vast majority of treatment failure outcomes in metastatic cancer. Among the mechanisms of resistance that markedly decrease treatment efficacy, the efflux of drug compounds by ATP-binding cassette (ABC) transporter proteins can impair adequate drug retention by cancer cells required for therapeutic cytotoxic activity. Of note, ABC transporters are capable of effluxing several classes of drugs that are clinical standards, including the anthracyclines such as doxorubicin, as well as anthracenediones such as mitoxantrone. To address this challenge, a spectrum of nanomaterials has been evaluated for improved drug retention and enhanced efficacy. Nanodiamonds (NDs) are emerging as a promising nanomaterial platform because they integrate several important properties into a single agent. These include a uniquely faceted truncated octahedral architecture that enables potent drug binding and dispersibility in water, scalably processed ND particles with uniform diameters of approximately 5 nm, and a demonstrated ability to improve drug tolerance while delaying tumor growth in multiple preclinical models, among others. This work describes a ND-mitoxantrone complex that can be rapidly synthesized and mediates marked improvements in drug efficacy. Comprehensive complex characterization reveals a complex with favorable drug delivery properties that is capable of improving drug retention and efficacy in an MDA-MB-231-luc-D3H2LN (MDA-MB-231) triple negative breast cancer cell line that was lentivirally transduced for resistance against mitoxantrone. Findings from this study support the further evaluation of ND-MTX in preclinical dose escalation and safety studies toward potentially clinical validation.

Figure 1

Physical characterization of ND–MTX. (A) Schematic diagram of MTX loading onto ND to form ND–MTX conjugate. (B) Standard curve of MTX loading efficiency on ND. (C) Particle size distribution (i) and zeta potential (ii) of ND and ND–MTX. (D) FITR spectra of ND, MTX, and ND–MTX. The FTIR spectra of ND–MTX showed distinctively C=C–H out of plane bending vibration at 820–840 cm^–1, C=C stretching vibration at 1560 and 1603 cm^–1, and C=O stretching vibration at 1628 cm^–1 compared with that of ND. Those peaks are matched well with MTX.

Figure 2

Release profile of MTX from ND–MTX conjugate. (A) In vitro release profile of MTX from ND–MTX complexes under various pH conditions. MTX elution was evaluated over a period of 72 h under pH 2, 4, 7, 10, 12, and pH 7 + 50% FBS conditions. MTX release behaviors displayed as percentage of total loaded MTX. (B) Release profile of MTX in 1:1 and 1:10 (DMEM (+50% FBS)/PBS). Higher FBS concentrations enhanced the release of MTX from ND–MTX. MTX release behaviors displayed as percentage of total loaded MTX.

Figure 3

ABC transporter protein expression in breast cancer cells. (A) Gene expression analysis of ABCG2 (left panel) and protein analysis of ABCG2 compared to control β-actin (right panel) in breast cancer cells (MDA-MB-231 and MDA-MB-231-ABCG2). (B) Gene expression analysis of two other major drug transporter proteins (ABCB1 and ABCC1) of the ABC transporter family in breast cancer cells (MDA-MB-231 and MDA-MB-231-ABCG2).*, p < 0.05; ***, p < 0.001.

Figure 4

In vitro efficacy of MTX and ND–MTX in breast cancer cells. (A) Dose–response curves of MDA-MB-231 and MDA-MB-231-ABCG2 after exposure to a range of MTX or ND–MTX concentrations. (B) Fold change in IC₅₀ values of MTX and ND–MTX for MDA-MB-231-ABCG2 cells relative to MDA-MB-231 control cells.

Figure 5

Conjugation of MTX to ND prolongs MTX retention in breast cancer cells. (A) Representative images of MTX retention at (i) 6 h and (ii) 24 h after 1 h of treatment with IC₅₀ concentrations of MTX and ND–MTX. ND–MTX treatment at 24 h showed significant MTX retention (indicated by red arrows). (B) Quantitative scores of MTX retention efficiency at (i) 6 h and (ii) 24 h after 1 h of treatment with IC₅₀ concentrations of MTX and ND–MTX.**, p < 0.01; *, p < 0.05. Scale bar represents 50 μm.