L. Henry Bryant

Magnetic intracellular labeling of mammalian cells by combining (FDA-approved) superparamagnetic iron oxide MR contrast agents and commonly used transfection agents.

Mammalian stem cells or other cells are being considered for use for infusion or transplantation into tissue for purposes of repair or for revascularization or therapeutic approaches (i.e., genetically altered cells) (1–5). Dextrancoated superparamagnetic iron oxide (SPIO) nanoparticles, a distinct class of MR contrast agents, cannot be used to efficiently label stem cells or other mammalian cells in vitro in their native unmodified form (6–8). Previously, we demonstrated that by conjugating antigenspecific internalizing monoclonal antibodies to the surface, dextran coating cells could be magnetically labeled during their normal expansion in culture (6). This magnetic labeling approach is limited because it requires the availability of an internalizing monoclonal antibody that recognizes a specific cell surface antigen. It is suitable only for labeling of cells that express the targeted receptor and is commonly species specific. Other approaches have involved the synthesis and modification of ultrasmall SPIO (USPIO or MION) particles with tat-proteins facilitating the incorporation into the cells, although this involves also a (synthetic) protein derivative (7,8). An alternative approach is the complex synthesis of a superparamagnetic iron oxide (SPIO) coated with dendrimers or “magnetodendrimers”, which will non-specifically label most mammalian cells (9). The dendrimer coating of the iron oxide nanoparticles provides the needed high affinity for cellular membranes for the construction of suitable cellular contrast agents, as dendrimers are commonly used as non-viral transfection agents (10,11). Magnetodendrimers can be used to efficiently and non-specifically label mammalian stem cells and cancer cells (12,13), however, they are not widely available or approved by the Food and Drug Administration (FDA). Over the past 10 years there has been significant research in developing new transfection agents (TA) including cationic peptides, dendrimers, poly-amines and lipids for nonviral transfection of DNA into the nucleus (10,14,15). These transfection agents are being developed to overcome the problem of the endosomal capture of the TA-DNA complex and inefficient release of the targeted material into the nucleus (14,15). TA’s are macromolecules with molecular weights from 1 to 10 kilo Dalton possessing an electrostatic charge. Based upon efficient labeling of mammalian cells by magnetodendrimers (12,13), we hypothesize that commercially available macromolecular transfection agents would coat via electrostatic interaction with dextran-coated iron oxide MR contrast agents and chaperon these nanoparticles into cells. We present here the magnetic labeling results of combining dendrimers and other commercially available TA’s with (FDA-approved) dextran-coated iron oxide MR contrast agents (Feridex and MION-46L). Acad Radiol 2002; 9(suppl 2):S484–S487

Self-assembling nanocomplexes by combining ferumoxytol, heparin and protamine for cell tracking by magnetic resonance imaging

We report on a new straightforward magnetic cell-labeling approach that combines three US Food and Drug Administration (FDA)-approved drugs—ferumoxytol, heparin and protamine—in serum-free medium to form self-assembling nanocomplexes that effectively label cells for in vivo magnetic resonance imaging (MRI). We observed that the ferumoxytol-heparin-protamine (HPF) nanocomplexes were stable in serum-free cell culture medium. HPF nanocomplexes show a threefold increase in T2 relaxivity compared to ferumoxytol. Electron microscopy showed internalized HPF in endosomes, which we confirmed by Prussian blue staining of labeled cells. There was no long-term effect or toxicity on cellular physiology or function of HPF-labeled hematopoietic stem cells, bone marrow stromal cells, neural stem cells or T cells when compared to controls. In vivo MRI detected 1,000 HPF-labeled cells implanted in rat brains. This HPF labeling method should facilitate the monitoring by MRI of infused or implanted cells in clinical trials.

Dendrimer-based MRI contrast agents: the effects of PEGylation on relaxivity and pharmacokinetics

UNLABELLED Polyethylene glycol (PEG) surface modification can make nanomaterials highly hydrophilic, reducing their sequestration in the reticuloendothelial system. In this study, polyamidoamine (PAMAM) dendrimers bearing gadolinium (Gd) chelates were PEGylated with different PEG-chain lengths, and the effects on paramagnetic and pharmacokinetic properties were evaluated. Specifically, Gd chelate-bearing PAMAM dendrimers (generations 4 and 5; G4 and G5) were conjugated with two different PEG chains (2 kDa and 5 kDa; 2k and 5k). Long PEG chains (5k) on the smaller (G4) dendrimer resulted in reduced relaxivity compared to non-PEGylated dendrimers, whereas short PEG chains (2k) on a larger (G5) dendrimer produced relaxivities comparable to non-PEGylated G4 dendrimers. The relaxivity of all PEGylated or lysine-conjugated dendrimers increased at higher temperature, whereas that of intact G4 Gd-PAMAM dendrimer decreased. All PEGylated dendrimers had minimal liver and kidney uptake and remained in circulation for at least 1 hour. Thus, surface-PEGylated Gd-PAMAM dendrimers showed decreased plasma clearance and prolonged retention in the blood pool. Shorter PEG, higher generation conjugates led to higher relaxivity. FROM THE CLINICAL EDITOR In this study, polyamidoamine dendrimers bearing gadolinium (Gd) chelates were PEGylated with different PEG-chain lengths, and the effects on paramagnetic and pharmacokinetic properties were evaluated.

Poly(amidoamine) Dendrimer Based MRI Contrast Agents Exhibiting Enhanced Relaxivities Derived via Metal Preligation Techniques

This report presents the preparation and characterization of three [Gd-C-DOTA](-1)-dendrimer assemblies by way of analysis, NMRD spectroscopy, and photon correlation spectroscopy (PCS). The metal-ligand chelates were preformed in alcohol media prior to conjugation to generation 4, 5, and 6 PAMAM dendrimers. The dendrimer-based agents were purified by Sephadex G-25 column chromatography. The combustion analysis, SE-HPLC, and UV-vis data indicated chelate to dendrimer ratios of 28:1, 61:1 and 115:1, respectively. Molar relaxivity measured at pH 7.4, 22 degrees C, and 3 T (29.6, 49.8, and 89.1 mM(-1) s(-1)) indicated the viability of conjugates as MRI contrast agents. 1/T(1) NMRD profiles were measured at 23 degrees C and indicated that at 22 MHz the 1/T(1) reached a plateau at 60, 85, and 140 mM(-1) s(-1) for the generation 4, 5, and 6 dendrimer conjugates, respectively. The PCS data showed the respective sizes of 5.2, 6.5, and 7.8 nm for G-4, 5, and 6 conjugates.

Gadolinium MRI contrast agents based on triazine dendrimers: relaxivity and in vivo pharmacokinetics.

Four gadolinium (Gd)-based macromolecular contrast agents, G3-(Gd-DOTA)(24), G5-(Gd-DOTA)(96), G3-(Gd-DTPA)(24), and G5-(Gd-DTPA)(96), were prepared that varied in the size of dendrimer (generation three and five), the type of chelate group (DTPA or DOTA), and the theoretical number of metalated chelates (24 and 96). Synthesis relied on a dichlorotriazine derivatized with a DOTA or DTPA ligand that was incorporated into the dendrimer and ultimately metalated with Gd ions. Paramagnetic characteristics and in vivo pharmacokinetics of all four contrast agents were investigated. The DOTA-containing agents, G3-(Gd-DOTA)(24) and G5-(Gd-DOTA)(96), demonstrated exceptionally high r1 relaxivity values at off-peak magnetic fields. Additionally, G5-(Gd-DOTA)(96) showed increased r1 relaxivity in serum compared to that in PBS, which was consistent with in vivo images. While G3-(Gd-DOTA)(24) and G3-(Gd-DTPA)(24) were rapidly excreted into the urine, G5-(Gd-DOTA)(96) and G5-(Gd-DTPA)(96) did not clear as quickly through the kidneys. Molecular simulation of the DOTA-containing dendrimers suggests that a majority of the metalated ligands are accessible to water. These triazine dendrimer-based MRI contrast agents exhibit several promising features such as high in vivo r1 relaxivity, desirable pharmacokinetics, and well-defined structure.

Functional MnO nanoclusters for efficient siRNA delivery

A non-viral gene delivery nanovehicle based on Alkyl-PEI2k capped MnO nanoclusters was synthesized via a simple, facile method and used for efficient siRNA delivery and magnetic resonance imaging.

Polyaspartic acid coated manganese oxide nanoparticles for efficient liver MRI

We report in this communication a simple, facile surface modification strategy to transfer hydrophobic manganese oxide nanoparticles (MONPs) into water by using polyaspartic acid (PASP). We systematically investigated the effect of the size of PASP-MONPs on MRI of normal liver and found that the particles with a core size of 10 nm exhibited greater enhancement than those with larger core sizes.

Rapid spectrophotometric technique for quantifying iron in cells labeled with superparamagnetic iron oxide nanoparticles: potential translation to the clinic

Labeling cells with superparamagnetic iron oxide (SPIO) nanoparticles provides the ability to track cells by magnetic resonance imaging. Quantifying intracellular iron concentration in SPIO labeled cells would allow for the comparison of agents and techniques used to magnetically label cells. Here we describe a rapid spectrophotometric technique (ST) to quantify iron content of SPIO-labeled cells, circumventing the previous requirement of an overnight acid digestion. Following lysis with 10% sodium dodecyl sulfate (SDS) of magnetically labeled cells, quantification of SPIO doped or labeled cells was performed using commonly available spectrophotometric instrument(s) by comparing absorptions at 370 and 750 nm with correction for turbidity of cellular products to determine the iron content of each sample. Standard curves demonstrated high linear correlation (R(2) = 0.998) between absorbance spectra of iron oxide nanoparticles and concentration in known SPIO-doped cells. Comparisons of the ST with inductively coupled plasma-mass spectroscopy (ICP-MS) or nuclear magnetic resonance relaxometric (R(2)) determinations of intracellular iron contents in SPIO containing samples resulted in significant linear correlation between the techniques (R(2) vs ST, R(2) > 0.992, p < 0.0001; ST vs ICP-MS, R(2) > 0.995, p < 0.0001) with the limit of detection of ST for iron = 0.66 µg ml(-1) for 10(6) cells ml(-1). We have developed a rapid straightforward protocol that does not require overnight acid digestion for quantifying iron oxide content in magnetically labeled cells using readily available analytic instrumentation that should greatly expedite advances in comparing SPIO agents and protocols for labeling cells.

Relaxometry, magnetometry, and EPR evidence for three magnetic phases in the MR contrast agent MION-46L

Magnetization curves, EPR spectra, and T1 and T2 nuclear magnetic relaxation times versus Larmor frequency have been measured for the MRI contrast agent MION-46L. The resulting data can be successfully explained by adding paramagnetic and antiferromagnetic terms to the known superparamagnetic component.

Pharmacokinetics of a High-Generation Dendrimer–Gd-DOTA

Dendrimers are three-dimensional branching polymers that have received much attention as transfection agents (1), drug delivery agents (2), and magnetic resonance (MR) contrast agents (3–5). To be used as MR contrast agents, dendrimers have been conjugated with paramagnetic metal ion complexes. The large number of paramagnetic metal ion complexes that can be covalently attached per dendrimer molecule, which depends on the dendrimer generation, has resulted in a new class of macromolecular MR contrast agents. Unlike other macromolecular-based MR contrast agents such as albumin, dextran, and poly-Llysine, dendrimers are discrete reagents with well-defined sizes and shapes. The paramagnetic metal ion complexes have included gadolinium chelated to the derivatized acyclic ligand of diethylenetriaminepentaacetic acid (DTPA) and the derivatized macrocylic ligands of 1,4,7,10-tetra