We report on a novel and straightforward magnetic cell labeling approach that combines three FDA-approved drugs ferumoxytol (F) heparin (H) and protamine (P) in serum free media to form self-assembling nanocomplexes that effectively label cells for MRI. the monitoring by MRI of infused or implanted cells in clinical trials. and through electrostatic interactions25 and have been used to facilitate intracellular drug delivery26 27 Combining heparin protamine and ferumoxytol results in the formation of a self-assembling nanocomplex (HPF) that was characterized and used to label stem cells or immune cells for MRI. Labeling cells with HPF was nontoxic to cells and therefore should facilitate the rapid translation of this technique to clinical trials. RESULTS Chemical characterization of HPF nanocomplexes The chemical characteristics of heparin28 protamine25-29 ferumoxytol9-11 13 Tmem10 24 and various combinations of the brokers are as follows; the HPF nanocomplexes at the ratio of H (2 IU ml?1): P (60 μg ml?1): F (50 μg ml?1) used to magnetically label cells had a zeta potential (ζ) of 14.1 ± 3.43 mV and size of 204 nm in water and a ζ of ?10.9 ± 0.0 and size of 153.6 nm in RPMI at 37 °C (Fig. 1a and Supplementary Table 1). Transmission Electron Microscopy (TEM) micrographs of the HPF nanocomplexes reveal F as electron dense iron nanoparticles coating the clear HP aggregates in an ovoid shape of approximately 150-200 nm in diameter (Fig. 1b c). Physique 1 Characteristics of self-assembling heparin (H) protamine (P) and ferumoxytol (F) nanocomplexes Cell labeling and iron content Approximately 100% of the HPF-labeled cells were Prussian blue (PB) or PB-DAB positive on histology (Fig. 2a-j). The internalization of HPF in endosomes was confirmed by TEM (Fig. 3) with HPF appearing as electron dense iron oxide nanoparticles that are approximately 6-8 nm in size. HPF was not observed around the cell membrane following cell washes. To determine the longevity of intracellular iron NSC BMSC and T-cells were labeled with HPF and were either allowed to proliferate and divided or were produced to confluence and exhibited contact inhibition. Labeled cells were stained at multiple time points to determine the presence of intracellular iron (Supplementary Figs. 1 and 2). PB positive T-cells NSC or BMSC could be detected for 7 or 14 days when repeatedly cultured whereas PB-DAB positive were detected for 21 days (NSC) and 28 days (BMSC) when produced to confluence. Physique 2 Representative light microscopy images of DAB-enhanced Prussian blue (PB)-stained HPF-labeled human stem or immune cells Physique 3 Internalization and encapsulation of HPF nanocomplexes in HPF-labeled cells The average iron content per cell was as follows: BMSC = 2.12 ± 0.11 picograms (pg); NSC= 2.8 ± 1.19 pg; HSC = 1.33 ± 0.2 pg; T-cells= 0.73 ± 0.25 pg; Lysionotin and Monocytes= 2.56 ± 1.1 pg. The iron content of unlabeled cells contained 0.0-0.5 pg cell?1 which was significantly different from labeled cells (p<0.05). We were unable to label cells with ferumoxytol alone or when combined with protamine over a wide range of ratios. HPF cells: toxicity phenotype differentiation and function There were no substantial differences in the rate of apoptosis increases in reactive oxygen species (ROS) viability or proliferation 1-4 days following labeling with HPF for all those cell types as compared to Lysionotin controls (Fig. 4). A slight decrease was observed in numbers of NSC (6%) and T-cells (10%) immediately after Lysionotin cell collection (Fig. 4a) and in proliferation of NSC and HSC at days 3-4 compared to control cells (Fig. 4b). The proliferative capacity recovered overtime when the cells were assessed at Day 7 for NSC and Day 30 for Lysionotin HSC. Phenotypic analyses of HPF-labeled and unlabeled BMSC surface markers were positive for surface markers CD90 CD73 CD105 (Supplementary Fig. 3). In this study HPF-labeled BMSCs were cryo-preserved and subsequently thawed for analysis. These results revealed that there were no effects on cellular viability (Supplementary Fig. 3a) or surface markers (Supplementary Fig. 3b-d) after freeze-thaw cycle indicating that it is not necessary to immediately label cells prior to use. The differentiation potential of Lysionotin HPF-labeled BMSCs towards adipogenic and osteogenic lineages exhibited no differences when compared to controls.