Biofunctionalized Prussian Blue Nanoparticles for Multimodal Molecular Imaging Applications

Summary

This protocol describes the synthesis of biofunctionalized Prussian blue nanoparticles and their use as multimodal, molecular imaging agents. The nanoparticles have a core-shell design where gadolinium or manganese ions within the nanoparticle core generate MRI contrast. The biofunctional shell contains fluorophores for fluorescence imaging and targeting ligands for molecular targeting.

Abstract

Multimodal, molecular imaging allows the visualization of biological processes at cellular, subcellular, and molecular-level resolutions using multiple, complementary imaging techniques. These imaging agents facilitate the real-time assessment of pathways and mechanisms in vivo, which enhance both diagnostic and therapeutic efficacy. This article presents the protocol for the synthesis of biofunctionalized Prussian blue nanoparticles (PB NPs) - a novel class of agents for use in multimodal, molecular imaging applications. The imaging modalities incorporated in the nanoparticles, fluorescence imaging and magnetic resonance imaging (MRI), have complementary features. The PB NPs possess a core-shell design where gadolinium and manganese ions incorporated within the interstitial spaces of the PB lattice generate MRI contrast, both in T₁ and T₂-weighted sequences. The PB NPs are coated with fluorescent avidin using electrostatic self-assembly, which enables fluorescence imaging. The avidin-coated nanoparticles are modified with biotinylated ligands that confer molecular targeting capabilities to the nanoparticles. The stability and toxicity of the nanoparticles are measured, as well as their MRI relaxivities. The multimodal, molecular imaging capabilities of these biofunctionalized PB NPs are then demonstrated by using them for fluorescence imaging and molecular MRI in vitro.

Introduction

Molecular imaging is the non-invasive and targeted visualization of biological processes at the cellular, subcellular, and molecular levels¹. Molecular imaging permits a specimen to remain in its native microenvironment while its endogenous pathways and mechanisms are assessed in real-time. Typically, molecular imaging involves the administration of an exogenous imaging agent in the form of a small molecule, macromolecule, or nanoparticle to visualize, target, and trace relevant physiological processes being studied². The various imaging modalities that have been explored in molecular imaging include MRI, CT, PET, SPECT, ultrasound, photoacoustic....

Protocol

1. Synthesis of PB NPs, GdPB, and MnPB

Synthesis of the nanoparticles (PB NPs, GdPB, or MnPB) is achieved using a one-pot synthesis scheme by performing the steps detailed below:

1. Prepare solution 'A' containing 5 ml of 5 mM potassium hexacyanoferrate (II) in deionized (DI) water. Depending on the type of nanoparticle being synthesized — PB NPs, GdPB, or MnPB, prepare solution 'B' as follows:
   1. For PB NPs: prepare 10 ml of a solution containing 2.5 mM iron (III) chloride in DI water.
   2. For GdPB NPs: prepare 10 ml of a solution containing 2.5 mM each of gadolinium (III) nitrate and iron (III) ch....

Results

Using the one-pot synthesis scheme, nanoparticles of PB NPs (mean diameter 78.8 nm, polydispersity index (PDI) = 0.230; calculated by the dynamic light scattering instrument), GdPB (mean diameter 164.2 nm, PDI = 0.102), or MnPB (mean diameter 122.4 nm, PDI = 0.124) that are monodisperse (as measured by DLS) can be consistently synthesized (Figure 2A). The measured zeta potentials of the synthesized nanoparticles are less than -30 mV (Figure 2B), indicating moderate stability of the parti.......

Discussion

This article has presented the methods for the synthesis of a novel class of multimodal, molecular imaging agents based on biofunctionalized Prussian blue nanoparticles. The molecular imaging modalities incorporated into the nanoparticles are fluorescence imaging and molecular MRI, due to their complementary features. The biofunctionalized Prussian blue nanoparticles have a core-shell design. The key steps in the synthesis of these nanoparticles are the: 1) one-pot synthesis which yields the cores that are comprised of P.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Potassium hexacyanoferrate (II) trihydrate (K4Fe(CN)6·3H2O)	Sigma-Aldrich	P9387
Manganese (II) chloride tetrahydrate (MnCl2·4H2O)	Sigma-Aldrich	221279
Gadolinium (III) nitrate hexahydrate (Gd(NO3)3·6H2O)	Sigma-Aldrich	211591
Iron (III) chloride hexahydrate (FeCl3·6H2O)	Sigma-Aldrich	236489
Sodium chloride (NaCl)	Sigma-Aldrich	S9888
Anti-NG2 Chondroitin Sulfate Proteoglycan, Biotin Conjugate Antibody	Millipore	AB5320
Biotinylated Anti-Human Eotaxin-3	Peprotech	500-P156GBT
Neuro-2a Cell Line	ATCC	CCL-131
BSG D10 Cell Line	Lab stock	---
OE21 Cell Line	Sigma-Aldrich	96062201
SUDIPG1 Neurospheres	Lab stock	---
Eol-1 Cell Line	Sigma-Aldrich	94042252
Poly(L-lysine) hydrobromide	Sigma-Aldrich	P1399
Formaldehyde	Sigma-Aldrich	F8775
Bovine serum albumin	Sigma-Aldrich	A2153
Aminoactinomycin D	Sigma-Aldrich	A9400
Triton X-100	Sigma-Aldrich	X100
CellTrace Calcein Red-Orange, AM	Life Technologies	C34851
Avidin-Alexa Fluor 488	Life Technologies	A21370
Centrifuge	Eppendorf	5424
Peristaltic Pump	Instech	P270
Zetasizer Nano ZS	Malvern	ZEN3600
Sonicator	QSonica	Q125
Hot Plate/Magnetic Stirrer	VWR	97042-642
Ultra Clean Aluminum Foil	VWR	89107-732
Vortex Mixer	VWR	58816-121
1.7 ml conical microcentrifuge tubes	VWR	87003-295
15 ml conical centrifuge tubes	VWR	21008-918
Tube holders	VWR	82024-342
Disposable plastic cuvettes	VWR	7000-590 (/586)
Zetasizer capillary cell	VWR	DTS1070
Centrifugal Filters, 0.2 micrometer spin column	VWR	82031-356
96-well cell culture tray	VWR	29442-056
Trypsin EDTA 0.25% solution 1x	JR Scientific	82702
Cell Culture Grade PBS (1x)	Life Technologies	10010023
XTT Cell Proliferation Assay Kit	Trevigen	4891-025-K
T75 Flask	89092-700	VWR
Dulbecco's Modified Eagle's Medium	Biowhitaker	12-604Q
Fetal Bovine Serum	Life Technologies	10437-010
Pen-Strep 1x	Life Technologies	15070063
Fluoview FV1200 Confocal Laser Scanning Microscope	Olympus	FV1200
Chambered Microscope Slides	Thermo Scientific	154534
Micro Cover Glasses, Square, No. 1.5	VWR	48366-227
Microscope Slides	VWR	16004-368
RPMI	Sigma-Aldrich	R8758
Agarose	Sigma-Aldrich	A9539
FACSCalibur Flow Cytometer	BD Biosciences
3 T Clinical MRI Magnet	GE Healthcare
100 ml round-bottom flask