Monitoring of Nanodrug Accumulation in Murine Breast Cancer Metastases

Summary

Here, we describe the protocol for in vivo delivery of magnetic iron oxide nanoparticles carrying RNA oligomers to metastatic breast cancer in animal models, providing a clinically viable approach for the therapeutic silencing of oncogenic nucleic acids.

Abstract

Metastatic breast cancer is a devastating disease with very limited therapeutic options, calling for new therapeutic strategies. Oncogenic miRNAs have been shown to be associated with the metastatic potential of breast cancer and are implicated in tumor cell migration, invasion, and viability. However, it can be difficult to deliver an inhibitory RNA molecule to the tissue of interest. To overcome this challenge and deliver active antisense oligonucleotides to tumors, we utilized magnetic iron oxide nanoparticles as a delivery platform. These nanoparticles target tissues with increased vascular permeability, such as sites of inflammation or cancer. Delivery of these nanoparticles can be monitored in vivo by magnetic resonance imaging (MRI) due to their magnetic properties. Translation of this therapeutic approach into the clinic will be more accessible because of its compatibility with this relevant imaging modality. They can also be labeled with other imaging reporters such as a Cy5.5 near-infrared optical dye for correlative optical imaging and fluorescence microscopy. Here, we demonstrate that nanoparticles labeled with Cy5.5 and conjugated to therapeutic oligomers targeting oncogenic miRNA-10b (termed MN-anti-miR10b, or "nanodrug") administered intravenously accumulate in metastatic sites, opening a possibility for therapeutic intervention of metastatic breast cancer.

Introduction

Despite many advances in the treatment of breast cancer, clinical options for metastatic disease remain limited. Patients commonly receive therapies targeted against drivers identified in the primary tumor, such as estrogen or HER2, but these drivers are not always conserved in metastases, rendering therapy ineffective¹. Other systemic therapies, such as chemotherapy, are non-specific and known for their side effects. To develop effective options for the treatment of metastatic breast cancer, it is important to consider the biological drivers that allow cancer cells to spread and colonize distant sites. One of these drivers is miR-10b, an oncog....

Protocol

The Michigan State University Institutional Animal Care and Use Committee (IACUC) has approved all procedures involving animal subjects. Values for calculations are summarized in Table 1.

1. Key steps of MN-anti-miR10b synthesis

NOTE: Details of the MN-anti-miR10b synthesis have been described previously^9,10,11.

1. Prepare the magn.......

Discussion

Nanoparticles have great potential for cancer treatment. Here, we showed that a Cy5.5-conjugated MNP carrier can reach cancer tissues to deliver therapeutic oligonucleotides in a murine model of metastatic breast cancer. The ability to administer the nanodrug systemically while still achieving considerable accumulation in cancer tissues offers tremendous advantages over many existing ASO delivery methods, which commonly require local and often invasive administration. As target specificity is imperative to patient safety.......

Materials

Name	Company	Catalog Number	Comments
Agilent 5800 ICP-OES	Agilent	5800 ICP-OES	For ICP-OES
Ammonium hydroxide	Thermo Fisher Scientific Inc	458680025	For nanodrug synthesis
Athymic nude "J:NU" mice	Jackson Laboratory	RRID:IMSR_JAX:007850	Immunocompromised mouse model
Betadine Surgical Scrub	Purdue	6761815101	For tumor resection
Cotton Tipped Applicators	Puritan	S-18991	For tumor resection
Crile Hemostats - Straight	F.S.T.	13004-14	For tumor resection
Cy5.5-NHS ester	Abcam	ab146455	For nanodrug synthesis
Dulbecco’s Modified Eagle Medium (DMEM)	Gibco	11995-065	For cell culture of MDA-MB-231
Eclipse 50i Clinical Microscope	Nikon	50i-B	For imaging of cryosections
Epichlorohydrin	Thermo Fisher Scientific Inc	117780250	For nanodrug synthesis
Extra Fine Graefe Forceps	F.S.T.	11150-10	For tumor resection and metastasis dissection
Fe standard	Inorganic Ventures	CGFE1-500ML	For ICP-OES
Fetal bovine serum	Corning	35-010-CV	For cell culture of MDA-MB-231
Fine Scissors - Sharp 10.5cm	F.S.T.	14060-10	For tumor resection and metastasis dissection
Flask (T-75)	Corning	430641U	For cell culture of MDA-MB-231
HNO3 nitric acid (70%, trace metal grade)	Fisher Chemical	A509P212	For ICP-OES
Insulin syringe 1 CC 29 G x 1/2"	Becton, Dickinson	324704	For tumor implant
Isoflurane	Covetrus	11695067772	For mouse anesthetization
Isoflurane vaporizer	SOMNI Scientific	VS6002	For mouse anesthetization
Isopropyl alcohol (70%) wipe	Cardinal	MW-APL	For tumor resection
IVIS SpectrumCT In Vivo Imaging System	PerkinElmer/Revvity	128201	For bioluminescence imaging
IVISbrite D-Luciferin Potassium Salt	PerkinElmer/Revvity	122799-100MG	For bioluminescence imaging
Ketofen (ketoprofen)	Zoetis	10004031	For tumor resection
Leica CM1950	Leica	CM1950	For cryosectioning of OCT-embedded samples
MARS 6 microwave digestion system	CEM	MARS 6	For ICP-OES
Matrigel, growth factor-reduced	Corning	354230	For tumor implant of MDA-MB-231
MDA-MB-231-luc-D3H2LN	PerkinElmer/Revvity	119369	For mouse model of spontaneous metastasis
Metal-free polypropylene 15 mL conical tubes	Labcon	31343450019	For ICP-OES
Microcentrifuge tube (1.7 mL)	DOT Scientific	RN1700-GMT	For metastasis sample collection
N-succinimidyl 3-[2-pyridyldithio]-propionate (SPDP)	Thermo Fisher Scientific Inc	21857	For nanodrug synthesis
PBS	Gibco	14190-144	For cell culture and tumor implant of MDA-MB-231
Penicillin-streptomycin	Gibco	15140-122	For cell culture of MDA-MB-231
Puralube vet ointment	MWI Veterinary	27505	For tumor resection
Sodium hydroxide	Thermo Fisher Scientific Inc	3728-70	For nanodrug synthesis
Tissue-Tek Cryomold Intermediate 15 x 15 x 5 mm	Sakura	4566	For metastasis sample collection
Tissue-Tek O.C.T. Compound	Sakura	4583	For metastasis sample collection
Tris(2-carboxyethyl)phosphine (TCEP)	Thermo Fisher Scientific Inc	T2556	For nanodrug synthesis
Trypsin, 0.25%	Gibco	25200-056	For cell culture of MDA-MB-231
Vicryl PLUS (Antibacterial) violet 27" RB-1 taper	Ethicon	VCP303H	For tumor resection