Development of novel tumor-targeted theranostic nanoparticles activated by membrane-type matrix metalloproteinases for combined cancer magnetic resonance imaging and therapy

Abstract

A major drawback with current cancer therapy is the prevalence of unrequired dose-limiting toxicity to non-cancerous tissues and organs, which is further compounded by a limited ability to rapidly and easily monitor drug delivery, pharmacodynamics and therapeutic response. In this report, the design and characterization of novel multifunctional "theranostic" nanoparticles (TNPs) is described for enzyme-specific drug activation at tumor sites and simultaneous in vivo magnetic resonance imaging (MRI) of drug delivery. TNPs are synthesized by conjugation of FDA-approved iron oxide nanoparticles ferumoxytol to an MMP-activatable peptide conjugate of azademethylcolchicine (ICT), creating CLIO-ICTs (TNPs). Significant cell death is observed in TNP-treated MMP-14 positive MMTV-PyMT breast cancer cells in vitro, but not MMP-14 negative fibroblasts or cells treated with ferumoxytol alone. Intravenous administration of TNPs to MMTV-PyMT tumor-bearing mice and subsequent MRI demonstrates significant tumor selective accumulation of the TNP, an observation confirmed by histopathology. Treatment with CLIO-ICTs induces a significant antitumor effect and tumor necrosis, a response not observed with ferumoxytol. Furthermore, no toxicity or cell death is observed in normal tissues following treatment with CLIO-ICTs, ICT, or ferumoxytol. These findings demonstrate proof of concept for a new nanotemplate that integrates tumor specificity, drug delivery and in vivo imaging into a single TNP entity through attachment of enzyme-activated prodrugs onto magnetic nanoparticles. This novel approach holds the potential to significantly improve targeted cancer therapies, and ultimately enable personalized therapy regimens.

Keywords: MMP-14; MR imaging; cancer therapy; iron oxide; nanoparticles; theranostic.

Figure 1

(A) Schematic representation of theranostic nanoparticle (TNP) activation by MMP-14: the IO NP core is shown in orange; the prodrug ICT is shown in red, and after MMP-14 activation, its product is shown in magenta; the peptide linker is shown in blue, and the FITC is shown in green. (B) Synthesis of the theranostic nanoparticles. FITC (shown in green) is linked to the TNP via the amino group of cysteine.

Figure 2

(A) Absorption spectra of the TNPs and their components (a.u. = arbitrary units) and the emission spectrum of CLIO-ICT (excitation wavelength is 350nm). ICT absoption is scaled up 5× to avoid the overlap. All spectra recorded at the same ICT emission; Only CLIO (cross-linked iron oxide with amino groups) absorption was measured at the same Fe concentration (0.02 mM) as for CLIO-ICT (ICT concentration is 0.094 mM); (B) A representative transmission electron microscopy image of CLIO-ICT. Inset shows crystalline iron oxide core of a single nanoparticle; (C) CLIO-ICT activation by MMP-14 in PBS buffer for 30 min and analyzed by HPLC. Mass spectrum of the indicated peak confirmed the presence of product of TNP cleavage by MMP-14. See experimental section for details.

Figure 3

(A) Caspase assay: PyMT, 4T1, human dermal fibroblasts, endothelial cells, and macrophages were incubated with PBS, Ferumoxtyol only, CLIO-ICT, and ICT only. After incubation the assay was run for 4 hr – readings taken every 5 min. Cells incubated with CLIO-ICT along with those incubated with ICT showed more fluorescence (more cell death) than those incubated with Ferumoxytol only and PBS only. Cells incubated with ICT only showed similar levels of fluorescence to that of CLIO-ICT, but showed a plateau after 60 min; (B) qPCR of MMP-14 expression of MMTV-PyMT, 4T1 and human dermal fibroblasts; (C) PyMT tumor sizes were measured daily for 7 days after intravenous injection of PBS, Ferumoxytol, CLIO-ICT and ICT. The tumor size increases in that of the PBS and Ferumoxtyol administered subjects and decreases in the CLIO-ICT and ICT cases. See experimental section for more details.

Figure 4

(A) Axial T2-weighted MR images (TR 2500 ms, TE 80 ms) of MMTV-PyMT mammary tumors before and after a single intravenous injection: 0.6 M (Fe) solution of ferumoxytol (0.5 mmol Fe/kg), 0.4 M (Fe) solution of CLIO-ICT (0.75 mmol Fe/kg and 1.0 μmol/kg of ICT), 0.29 mM solution of ICT (1.0 μmol/kg), or PBS(1.0 μl/gm). Contrast agent accumulation is noted as a negative (dark) signal enhancement of the tumors; (B) MR signal enhancement data in tumors corresponding to Figure 4 quantified as ΔR₂ = (R₂pre − R₂post). Data are displayed as mean data of n=6 tumors in each group for 1h and 24h time points.

Figure 5

(A) MMP-14 negative fibroblasts: H&E stained histologic sections of fibroblasts treated with CLIO-ICT and ICT showing no necrosis (both images taken at 40X magnification); (B) TNP-induced cell death in MMTV-PyMT tumors. H&E panels: CLIO-ICT treated tumor demonstrating diffuse necrosis (200X magnification); ICT treated tumor with predominately viable tumor cells and a subset of cells undergoing necrosis (200X magnification); Ferumoxytol treated tumor with diffuse viability and no necrosis (100X magnification, inset: 400X magnification); (C) Iron panels: Scattered CLIO-ICT treated tumor and rare admixed histiocytes contain blue pigment indicating cytoplasmic iron deposition (200X magnification); ICT treated tumor shows no cytoplasmic iron deposition, scattered iron laden histiocytes serve as an internal positive control (200X magnification); Ferumoxytol treated tumor show cytoplasmic iron deposition, scattered iron laden histiocytiocytes serve as an internal positive control (200X magnification). (D) fluorescence microscopy showing FITC signal for CLIO-ICT and ICT but no signal for Ferumoxytol. (E) Caspase-3 panels: CLIO-ICT and ICT treated tumors show Cy3 labeling throughout the samples; Ferumoxytol treated tumor shows few areas with weak Cy3 fluorescence (4X magnification).

Figure 6

CLIO-ICTs do not cause toxic effects in normal organs. Above histopathologies show no significant necrosis of normal organs on H&E staining of the Heart, Kidney, Spleen, Brain, Bone Marrow and Liver.