Michael J. House

On T2* Magnetic Resonance and Cardiac Iron

Background— Measurement of myocardial iron is key to the clinical management of patients at risk of siderotic cardiomyopathy. The cardiovascular magnetic resonance relaxation parameter R2* (assessed clinically via its reciprocal, T2*) measured in the ventricular septum is used to assess cardiac iron, but iron calibration and distribution data in humans are limited. Methods and Results— Twelve human hearts were studied from transfusion-dependent patients after either death (heart failure, n=7; stroke, n=1) or transplantation for end-stage heart failure (n=4). After cardiovascular magnetic resonance R2* measurement, tissue iron concentration was measured in multiple samples of each heart with inductively coupled plasma atomic emission spectroscopy. Iron distribution throughout the heart showed no systematic variation between segments, but epicardial iron concentration was higher than in the endocardium. The mean±SD global myocardial iron causing severe heart failure in 10 patients was 5.98±2.42 mg/g dry weight (range, 3.19 to 9.50 mg/g), but in 1 outlier case of heart failure was 25.9 mg/g dry weight. Myocardial ln[R2*] was strongly linearly correlated with ln[Fe] (R2=0.910, P<0.001), leading to [Fe]=45.0×(T2*)−1.22 for the clinical calibration equation with [Fe] in milligrams per gram dry weight and T2* in milliseconds. Midventricular septal iron concentration and R2* were both highly representative of mean global myocardial iron. Conclusions— These data detail the iron distribution throughout the heart in iron overload and provide calibration in humans for cardiovascular magnetic resonance R2* against myocardial iron concentration. The iron values are of considerable interest in terms of the level of cardiac iron associated with iron-related death and indicate that the heart is more sensitive to iron loading than the liver. The results also validate the current clinical practice of monitoring cardiac iron in vivo by cardiovascular magnetic resonance of the midseptum.

Correlation of proton transverse relaxation rates (R2) with iron concentrations in postmortem brain tissue from alzheimer's disease patients

Iron accumulates in the Alzheimers disease (AD) brain and is directly associated with β‐amyloid pathology. The proton transverse relaxation rate (R2) has a strong linear relationship with iron concentrations in healthy brain tissue; however, an independent test of this relationship has not been extended to AD brain tissue. In this study in vitro single spin‐echo (SE) measurements were made on tissue samples from four human AD brains using a 4.7T MRI research scanner. R2 values were calculated for 14 cortical and subcortical gray matter (GM) and white matter (WM) regions. Atomic absorption spectroscopy was used to measure iron concentrations in the corresponding excised brain regions. Significant positive linear correlations were observed between R2 values and iron concentrations in GM regions assessed across individual tissue samples and data averaged by brain region. With the use of a predictive model for R2, a threshold iron concentration of 55 μg Fe/g wet tissue was determined above which R2 appears to be dominated by the affects of iron in AD brain tissue. High‐field MRI may therefore be a useful research tool for assessing brain iron changes associated with AD. Magn Reson Med 57:172–180, 2007.

Experimental validation of proton transverse relaxivity models for superparamagnetic nanoparticle MRI contrast agents

Analytical models of proton transverse relaxation rate enhancement by magnetic nanoparticles were tested by making measurements on model experimental systems in a field of 1.4 T. Proton relaxivities were measured for five aqueous suspensions of iron oxide (maghemite) nanoparticles with nominal mean particle sizes of 6, 8, 10, 11, and 13 nm. Proton relaxivity increased with mean particle size ranging from 13 s(-1) mM Fe(-1) for the 6 nm sample, up to 254 s(-1) mM Fe(-1) for the 13 nm sample. A strong correlation between the measured and predicted values of the relaxivity was observed, with the predicted values being consistently higher than the measured values. The results indicate that the models give a reasonable agreement with experimental results and hence can be used as the basis for the design of new magnetic resonance imaging contrast and labelling agents.

Multimodal analysis of PEI-mediated endocytosis of nanoparticles in neural cells.

Polymer nanoparticles are widely used as a highly generalizable tool to entrap a range of different drugs for controlled or site-specific release. However, despite numerous studies examining the kinetics of controlled release, the biological behavior of such nanoparticles remains poorly understood, particularly with respect to endocytosis and intracellular trafficking. We synthesized polyethylenimine-decorated polymer nanospheres (ca. 100-250 nm) of the type commonly used for drug release and used correlated electron microscopy, fluorescence spectroscopy and microscopy, and relaxometry to track endocytosis in neural cells. These capabilities provide insight into how polyethylenimine mediates the entry of nanoparticles into neural cells and show that polymer nanosphere uptake involves three distinct steps, namely, plasma membrane attachment, fluid-phase as well as clathrin- and caveolin-independent endocytosis, and progressive accumulation in membrane-bound intracellular vesicles. These findings provide detailed insight into how the intracellular delivery of nanoparticles is mediated by polyethylenimine, which is presently the most commonly used nonviral gene transfer agent. This fundamental knowledge may also assist in the preparation of next-generation nonviral vectors.

The impact of phlebotomy in nonalcoholic fatty liver disease: A prospective, randomized, controlled trial

Iron is implicated in the pathogenesis of liver injury and insulin resistance (IR) and thus phlebotomy has been proposed as a treatment for nonalcoholic fatty liver disease (NAFLD). We performed a prospective 6‐month randomized, controlled trial examining the impact of phlebotomy on the background of lifestyle advice in patients with NAFLD. Primary endpoints were hepatic steatosis (HS; quantified by magnetic resonance imaging) and liver injury (determined by alanine aminotransaminase [ALT] and cytokeratin‐18 [CK‐18]). Secondary endpoints included insulin resistance measured by the insulin sensitivity index (ISI) and homeostasis model of assessment (HOMA), and systemic lipid peroxidation determined by plasma F2‐isoprostane levels. A total of 74 subjects were randomized (33 phlebotomy and 41 control). The phlebotomy group underwent a median (range) of 7 (1‐19) venesection sessions and had a significantly greater reduction in ferritin levels over 6 months, compared to controls (−148 ± 114 vs. −38 ± 89 ng/mL; P < 0.001). At 6 months, there was no difference between phlebotomy and control groups in HS (17.7% vs. 15.5%; P = 0.4), serum ALT (36 vs. 46 IU/L; P = 0.4), or CK‐18 levels (175 vs. 196 U/L; P = 0.9). Similarly, there was no difference in end‐of‐study ISI (2.5 vs. 2.7; P = 0.9), HOMA (3.2 vs. 3.2; P = 0.6), or F2‐isoprostane levels (1,332 vs. 1,190 pmmol/L; P = 0.6) between phlebotomy and control groups. No differences in any endpoint were noted in patients with hyperferritinemia at baseline. Among patients undergoing phlebotomy, there was no correlation between number of phlebotomy sessions and change in HS, liver injury, or IR from baseline to end of study. Conclusion: Reduction in ferritin by phlebotomy does not improve liver enzymes, hepatic fat, or IR in subjects with NAFLD. (Hepatology 2015;61:1555–1564)

Toward Design of Magnetic Nanoparticle Clusters Stabilized by Biocompatible Diblock Copolymers for T2-Weighted MRI Contrast

We report the fabrication of magnetic particles comprised of clusters of iron oxide nanoparticles, 7.4 nm mean diameter, stabilized by a biocompatible, amphiphilic diblock copolymer, poly(ethylene oxide-b-D,L-lactide). Particles with quantitative incorporation of up to 40 wt % iron oxide and hydrodynamic sizes in the range of 80-170 nm were prepared. The particles consist of hydrophobically modified iron oxide nanoparticles within the core-forming polylactide block with the poly(ethylene oxide) forming a corona to afford aqueous dispersibility. The transverse relaxivities (r2) increased with average particle size and exceeded 200 s(-1) mM Fe(-1) at 1.4 T and 37 °C for iron oxide loadings above 30 wt %. These experimental relaxivities typically agreed to within 15% with the values predicted using analytical models of transverse relaxivity and cluster (particle core) size distributions derived from cryo-TEM measurements. Our results show that the theoretical models can be used for the rational design of biocompatible MRI contrast agents with tailored compositions and size distributions.

Continuously manufactured magnetic polymersomes-a versatile tool (not only) for targeted cancer therapy

Micromixer technology was used to prepare polymeric vesicles (Pluronic® L-121) dual loaded with the anti-cancer drug camptothecin and magnetic nanoparticles. Successful incorporation of the magnetic nanoparticles was confirmed by transmission electron microscopy. Dynamic light scattering measurements showed a relatively narrow size distribution of the hybrid polymersomes. Camptothecin polymersomes reduced the cell viability of prostate cancer cells (PC-3) measured after 72 h significantly, while drug-free polymersomes showed no cytotoxic effects. Covalent attachment of a cancer targeting peptide (bombesin) as well as a fluorescent label (Alexa Fluor® 647) to the hybrid polymersomes was performed and specific cell binding and internalization were shown by flow cytometry and confocal microscopy. Relaxometry measurements clearly demonstrated the capacity of magnetic polymersomes to generate significant T2-weighted MRI contrast and potentially allow for direct monitoring of the biodistribution of the polymersomes. Micromixer technology as an easy, fast and efficient way to manufacture hybrid polymersomes as theranostic drug delivery devices is a further step from basic research to personalized medicine.

The effect of polymer coatings on proton transverse relaxivities of aqueous suspensions of magnetic nanoparticles

Iron oxide magnetic nanoparticles are good candidates for magnetic resonance imaging (MRI) contrast agents due to their high magnetic susceptibilities. Here we investigate 19 polyether-coated magnetite nanoparticle systems comprising three series. All systems were synthesized from the same batch of magnetite nanoparticles. A different polyether was used for each series. Each series comprised systems with systematically varied polyether loadings per particle. A highly significant (p < 0.0001) linear correlation (r = 0.956) was found between the proton relaxivity and the intensity-weighted average diameter measured by dynamic light scattering in the 19 particle systems studied. The intensity-weighted average diameter measured by dynamic light scattering is sensitive to small number fractions of larger particles/aggregates. We conclude that the primary effect leading to differences in proton relaxivity between systems arises from the small degree of aggregation within the samples, which appears to be determined by the nature of the polymer and, for one system, the degree of polymer loading of the particles. For the polyether coatings used in this study, any changes in relaxivity from differences in water exclusion or diffusion rates caused by the polymer are minor in comparison with the changes in relaxivity resulting from variations in the degree of aggregation.

1.4T study of proton magnetic relaxation rates, iron concentrations, and plaque burden in Alzheimer's disease and control postmortem brain tissue.

We measured proton magnetic longitudinal (R1) and transverse (R2) relaxation rates at 1.4T, iron concentrations, water contents, and amyloid plaque densities in postmortem brain tissue samples from three Alzheimers disease (AD), two possible AD, and five control subjects. Iron concentrations and R1 were significantly higher in the temporal cortex region of our AD group compared to the controls. Frequency analyses showed that the observed trends of higher iron, R1, and R2 in AD gray matter regions were statistically significant. Simple regression models indicated that for AD and control gray matter the iron concentrations and water contents have significant linear correlations with R1 and R2. Multiple regression models based on iron concentrations and water contents were highly significant for all groups and tissue types and suggested that the effects of iron become more important in determining R1 and R2 in the AD samples. At 1.4T R1 and R2 are strongly affected by water content and to a lesser extent by variations in iron concentrations. The AD plaque density did not correlate with iron concentrations, water contents, R1, or R2, suggesting that increases in AD brain iron are not strongly related to the accumulation of amyloid plaques. Magn Reson Med, 2008.

Relationship between brain R2 and liver and serum iron concentrations in elderly men.

Studies of iron overload in humans and animals suggest that brain iron concentrations may be related in a regionally specific way to body iron status. However, few quantitative studies have investigated the associations between peripheral and regional brain iron in a normal elderly cohort. To examine these relationships, we used MRI to measure the proton transverse relaxation rate (R2) in 13 gray and white matter brain regions in 18 elderly men (average age, 75.5 years) with normal cognition. Brain R2 values were compared with liver iron concentrations measured using the FerriScan® MRI technique and serum iron indices. R2 values in high‐iron gray matter regions were significantly correlated (positively) with liver iron concentrations (globus pallidus, ventral pallidum) and serum transferrin saturation (caudate nucleus, globus pallidus, putamen) measured concurrently with brain R2, and with serum iron concentrations (caudate nucleus, globus pallidus) measured three years before the current study. Our results suggest that iron levels in specific gray matter brain regions are influenced by systemic iron status in elderly men. Magn Reson Med, 2010.