Nanoparticle Delivery of an Oligonucleotide Payload in a Glioblastoma Multiforme Animal Model

Summary

The blood-brain barrier is a significant hurdle in the delivery of therapies for glioblastoma, a disease for which there is no cure. Here, we report an in vivo image-guided iron oxide therapeutic nano platform that can bypass this physiological barrier by virtue of size and accumulate in the tumor.

Abstract

Glioblastoma multiforme (GBM) is the most common and aggressive form of primary brain malignancy for which there is no cure. The blood-brain barrier is a significant hurdle in the delivery of therapies to GBM. Reported here is an image-guided, iron oxide-based therapeutic delivery nano platform capable of bypassing this physiological barrier by virtue of size and accumulating in the tumor region, delivering its payload. This 25 nm nano platform consists of crosslinked dextran-coated iron oxide nanoparticles labeled with Cy5.5 fluorescent dye and containing antisense oligonucleotide as a payload. The magnetic iron oxide core enables tracking of the nanoparticles through in vivo magnetic resonance imaging, while Cy5.5 dye allows tracking by optical imaging. This report details the monitoring of the accumulation of this nanoparticle platform (termed MN-anti-miR10b) in orthotopically implanted glioblastoma tumors following intravenous injection. In addition, it provides insight into the in vivo delivery of RNA oligonucleotides, a problem that has hampered the translation of RNA therapeutics into the clinic.

Introduction

Glioblastoma multiforme (GBM) is the highest grade of astrocytoma for which there is virtually no cure. Approximately 15,000 people are diagnosed with glioblastoma annually, which has a dismal median survival of about 15 months and a 5-year survival rate of 5%¹. In the past decades, there has been marginal improvement in prognosis despite multiple efforts to advance therapeutic options. The current standard of care for GBM includes maximal surgical resection, when feasible, followed by radiotherapy and chemotherapy². Temozolomide (TMZ), the chemotherapy of choice, was the latest therapy for glioblastoma discovered to sho....

Protocol

All procedures involving animal subjects have been approved by the Michigan State University Institutional Animal Care and Use Committee (IACUC). Female outbred athymic nude mice were purchased from Jackson Labs (strain #007850) at 7 weeks of age and allowed to acclimate for 1 week prior to implantation surgery. Mice were approximately 21-25 g at the time of implant. U251 cells expressing firefly luciferase were generated and provided by Dr. Ana deCarvalho¹².

1. Cell culture and preparation for implantation

1. Culture luciferase-labeled, human glioblastoma cells, U251 in Dulbecco's Modif....

Results

MN-anti-miR10b was synthesized and characterized, as described previously¹¹. Transmission electron microscopy of MN-anti-miR10b shows the morphology and polydispersity of the nano platform (Figure 1B). This nano platform has an average size of 25.12 ± 0.34 nm with a zeta potential of 13.18 ± 1.47 mV (Figure 1C,D). In these studies, nude athymic mice were orthotopically implanted with U251 human glioblasto.......

Discussion

Several critical steps across the different methods of validating the accumulation of the nanoparticles across the BBB can be decisive for the success of the protocol. Beginning with the orthotopic implantation of GBM cells, it is important to ensure that the suture lines of the skull are visible after drying the bone; this aids in the accurate placement of the tumor cells. For drilling through the skull, it is best to apply light pressure to the drill site and begin drilling to make a shallow impression in the bone. Onc.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Athymic nude "J:NU" mice	Jackson Laboratory	RRID:IMSR_JAX:007850	Immunocompromised mouse model
0.25% Trypsin	Gibco	25200-056	Cell culture reagent for U251
1.7 mL microcentrifuge tube	DOT Scientific	RN1700-GMT	For tissue collection
10 µL, Neuros Syringe, Model 1701 RN, 33 gauge, Point Style 4	Hamilton	65460-06	Syringe for intracranial implantation of tumor cells
3M Vetbond	3M	1469SB	Tissue adhesive for surgical site closure
4% Paraformaldehyde	Thermo Scientific	J199943-K2	Tissue fixing solution
70% isopropoyl alcohol wipe	Cardinal	MW-APL	Topical antiseptic wipe for tumor implantation and tail vein injection
Aperio Versa	Leica		For scanning of stained tissue section slides
Betadine Surgical Scrub	Purdue	6761815101	Topical antiseptic for tumor implantation
BioSpec 70/30	Bruker		Magnetic resonance imaging scanner
Bone Wax	Medline	DYNJBW25	Bone wax for sealing implantation site
Burrs for Micro drill	F.S.T.	19007-05	Drill burr used to make hole in skull for tumor implantation
DAPI Fluoromount-G	SouthernBiotech	0100-20	Tissue mounting media containing DAPI stain
Dulbecco’s Modified Eagle Medium (DMEM)	Gibco	11995-065	Cell culture media for U251
Extra Fine Graefe Forceps	F.S.T.	11150-10	Sugical tool for tumor implantation
Fetal bovine serum	Corning	35-010-CV	Cell culture media supplement for U251
Fine Scissors - Sharp 10.5cm	F.S.T.	14060-10	Sugical tool for tumor implantation
Glydo (Lidocaine)	Sagent	673-76	Topical analgesic for surgical site
Ideal Micro Drill	CellPoint Scientific	67-1200A	Drill used to make hole in skull for tumor implantation
Insulin syringe 1CC 29G X 1/2"	Becton, Dickinson	324704	Syringe for D-Luciferin injection and tail vein injection of nanoparticles
Isoflurane	Covetrus	11695067772	Anethesia
Isoflurane vaporizer	SOMNI Scientific	VS6002	Anethesia apparatus
IVIS SpectrumCT In Vivo Imaging System	PerkinElmer/Revvity	128201	Bioluminescence and fluorescence imaging scanner
IVISbrite D-Luciferin Potassium Salt	PerkinElmer/Revvity	122799-100MG	Substrate for bioluminescence imaging
Ketaset (Ketamine)	Zoetis	10004027	Anesthetic for tumor implantation surgery
Ketofen (Ketoprofen)	Zoetis	10004031	Analgesic for tumor implantation surgery
Leica CM1950	Leica	CM1950	For cryosectioning of OCT-embedded samples
PBS	Gibco	14190-144	Cell culture reagent and cell suspension solution for implantation of U251
Penicillin-streptomycin	Gibco	15140-122	Antibiotic for cell culture media for U251
Puralube vet ointment	MWI Veterinary	27505	Opthalmic eye ointment for protection during tumor implantation
Ruler	F.S.T.	18000-30	Used to measure drill site for implanation
Tissue-Tek Cryomold  Intermediate 15 x 15 x 5 mm	Sakura	4566	Collection mold for collecting tissue samples
Tissue-Tek O.C.T. Compound	Sakura	4583	Freezing compound for collecting tissue samples
U-251 MG cell line human	Millapore Sigma	9063001	Human glioblastoma cell line
Xylazine Injectable Solution, 100 mg/ml	Covetrus	1XYL006	Paralytic for tumor implantation surgery