Polystyrene microsphere–ferritin conjugates: A robust phantom for correlation of relaxivity and size distribution

Iron overload is a serious health issue that results from transfusion-dependent anemias in thalassemia (1), hereditary disorders of iron metabolism such as hemochromatosis (2,3), cirrhosis (4), and viral hepatitis (5). Excess iron deposits in tissues as hemosiderin (aggregates of partially digested ferritin within lysosomes) that is associated with iron-catalyzed oxidative damage to proteins, lipids, and nucleic acids (6–10). Increased iron levels in the brain, heart, liver, lungs, endocrine organs, or other tissues eventually cause organ failure. This may lead to diabetes, cirrhosis, cardiac dysfunction, or even death (6–10). To prevent organ toxicity, iron must be efficiently regulated. Thus, treatment for hemochromatosis requires weekly phlebotomy until normal iron levels are obtained, followed by phlebotomy several times a year to maintain stasis (11), whereas transfusion-dependent iron overload requires iron chelation therapy to remove excess iron (12,13). Laboratory tests such as serum ferritin and serum transferrin saturation are often used to assess iron levels in the body. These serum tests are inexpensive and widely available, but isolated measurements cannot reliably diagnose iron overload (where serum ferritin levels have been shown to be only weakly correlated with iron load) and can be influenced by various factors such as inflammation and transfusion rate. Liver biopsy is currently the gold standard test because it is highly sensitive for liver iron levels, is quantitatively accurate, and provides a good estimate of iron burden in the body (11). However, this technique has several disadvantages. In addition to the pain associated with this invasive procedure, there is a significant risk of hemorrhage, infection, and damage to surrounding organs (14,15). Further, biopsies are prone to sampling error from uneven iron distribution. Finally, liver iron concentration may not correlate with iron levels in the rest of the body and is poorly correlated with cardiac iron deposition (16). A noninvasive method to quantify in vivo iron load would prove to be a significant clinical tool as it would avoid the complications associated with biopsies and sampling errors (17). One noninvasive technique to measure iron load is magnetic resonance imaging (MRI). MR scanners are widely available and able to determine pathological state of organs and iron content in areas besides the liver (18). Therefore, MR imaging is a promising alternative for noninvasive quantification of iron content. Numerous studies have been conducted to determine iron–relaxivity relationships in iron overload diseases. Transverse relaxation time (T2), effective transverse relaxation time (T2*) (17,19–21), and liver signal intensity ratios have been correlated to iron content in the liver (22–25). While MR imaging is a promising solution, there are inconsistent results from various centers, an inability to determine higher iron loads, and methodological inaccuracies, leading to inaccurate liver quantification (26–28). To overcome these barriers, an external reference MRI phantom would provide quantitative calibration between different exams, imaging sequences, patients, and scanners. However, due to the heterogeneous and complex nature of the liver and other biological tissues, phantoms possessing characteristics of an iron overloaded liver have been difficult to develop. Ideally, the MRI phantom should have background relaxation times similar to human tissue with homogeneous relaxation times throughout, be easy to handle, and possess long-term shelf life with regard to chemical and physical stabilities. Previously published phantoms include manganese- doped agarose (10) and ferritin-coated liposomes embedded in agarose (17). However, none of these studies take into account the size variability of the hemosiderin deposits and their effect on the interaction between the iron and water protons within the liver (28,29). Further, while the liposomes can be produced in various sizes, the ferritin is not covalently attached, leading to relatively unstable structures (they cannot withstand heat or high salt concentrations due to the electrostatic interaction between the ferritin and the liposome surface). Here, we describe the synthesis and relaxation properties of ferritin-coated latex microspheres that possess many similarities to hemosiderin including insolubility in aqueous media and 25- to 30-fold increase in relaxivity (r2) compared with ferritin in solution. Additionally, latex microspheres are available in a wide range of monodisperse sizes and can be functionalized for stable covalent protein attachment. This produces a chemically stable material whose synthesis is highly reproducible and is an excellent choice for studying the effects of ferritin size distribution on relaxation rate changes.