The major finding of this study is the formation of self-assembling HPF nanocomplexes by simply combining three FDA approved drugs, ferumoxytol, heparin and protamine directly into media containing cells, resulting in the labeling of stem cells and immune cells for cellular tracking and detection by MRI. Combining P with F results in the formation of large, polydisperse complexes that were not incorporated into cells. Neuwelt et al.13
reported that PF complexes were unable to label rat blood mononuclear cells. Although it is counterintuitive to add heparin to PF to facilitate endosomal incorporation in cells, the addition of heparin gives rise to the formation of HPF nanocomplexes that were endocytosed by cells. Heparin-based nanoparticles self-assemble with a variety of cationic molecules and have been used for drug delivery, tissue engineering or for prolonging circulating half-life of an agent26, 28, 31
When heparin and protamine are mixed together, they rapidly form complexes through electrostatic interactions25, 26, 29
. We observed that in RPMI, HP complexes form and then attracts F resulting in stable HPF nanocomplexes. Mixing HPF in an alternative order such as FHP also results in nanocomplexes that can be used for cell labeling; however, other combinations of the drugs (i.e., P:F:H) did not label cells. TEM of HP aggregates coated with colloidal iron demonstrated a dense ring of iron around a HP hypodense center29
, similar to HPF nanocomplexes. Further investigation is required to elucidate the ultrastructure and chemical composition of the HPF nanocomplexes in order to achieve effective iron loading of cells while preserving cell viability and function.
Heparin, protamine and ferumoxytol are routinely used for specific clinical indications. The concentrations of H, P and F used in this study results in intracellular concentrations of each drug that were substantially below recommended clinical doses. The HPF nanocomplexes have similar biochemical properties to SPION that have been shown to label cells and biodegrade through iron metabolic pathway16, 22, 23, 32
. We have also demonstrated that HPF-labeled NSC and BMSC remained PB positive for up to 28 days similar to observations in stem cells labeled with SPION16, 32
. Although further investigation is needed to determine any potential toxicity of delivering HPF-labeled cells in vivo
, ferumoxytol is likely to be metabolized similar to other SPIONs. As with previous findings of SPION-labeled cells, dilution of the endosomal HPF nanocomplexes will likely occur through cell division or via digestion33, 34
In this study, we were able to clearly visualize as few as 1000 cells at 8 days post intra-cerebral transplantation on T2* weighted images due to T2* shortening and the susceptibility blooming artifact associated with the HPF-labeled cells at 3 Tesla. The intracellular location of HPF nanocomplexes results in T2* shortening of water protons by causing magnetic field gradients in the region of interest. Hemorrhage at the site of injection may also cause T2* shortening and represents a limitation of interpreting MRI cell tracking studies of SPION labeled cells4, 14, 16
. Although the labeled cell death will occur overtime, we previously reported that approximately 10–20% of the iron label in transplanted cells maybe endocytosed by activated macrophagesbut minimally contributed to hypointense voxels on MRI35
The T2* weighted images employed in this study allowed for short acquisition times (approximately 10 minutes) without modification to the clinical MR scanner. Although the intracellular concentration of iron of HPF-labeled stem cells was less than cells labeled with SPION, the iron content is comparable to reports in which cells were labeled with other USPIO nanoparticles15, 18, 21, 36
. The intracellular iron content is dependent on the cell surface area and nuclear to cytoplasmic ratio with larger cells having the potential to uptake greater amounts of SPION4, 16
With the removal of the clinically available SPION contrast agents from the market, cell therapy trials that planned to incorporate magnetic cell labeling to monitor the cell migration were placed on hold or abandoned. Ferumoxytol is the only intravenous FDA-approved USPIO nanoparticle preparation that could be used to magnetically label and monitor the temporal spatial migration of infused or implanted cells by MRI. One of the major advantages of complexing ferumoxytol with heparin and protamine to label cells was that they were clinically used, therefore, extensive safety testing of the drugs should not be necessary and the time required for evaluating HPF for an investigative new drug application shortened. Although standard operating procedure protocols will need to be developed, the HPF labeling method presented in this study allows for the easy scale-up for cell labeling in current good manufacturing practice cell processing facility. Further optimization of the HPF protocol by modifying the amount of heparin (i.e., 1–3 IU ml−1), protamine (i.e., 30 – 60 μg ml−1) or increasing the amount of ferumoxytol (i.e., 50–100 μg ml−1) may result in higher intracellular iron concentrations, providing the basis for the rapid translation into clinical trials.