Polymeric nanoparticles enhance the ability of deferoxamine to

Aug 7, 2018 - Polymeric nanoparticles enhance the ability of deferoxamine to deplete hepatic and systemic iron. Shanshan Guo , Gang Liu , David M Fraz...
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Polymeric nanoparticles enhance the ability of deferoxamine to deplete hepatic and systemic iron Shanshan Guo, Gang Liu, David M Frazer, Tianqing Liu, Linhao You, Jiaqi Xu, Yongwei Wang, Gregory J Anderson, and Guangjun Nie Nano Lett., Just Accepted Manuscript • DOI: 10.1021/acs.nanolett.8b02428 • Publication Date (Web): 07 Aug 2018 Downloaded from http://pubs.acs.org on August 7, 2018

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Polymeric nanoparticles enhance the ability of deferoxamine to deplete hepatic and systemic iron

Shanshan Guo1,2,3, Gang Liu1, David M Frazer2, Tianqing Liu2, Linhao You1, Jiaqi Xu1, 3, Yongwei Wang1,3, Gregory J. Anderson2* and Guangjun Nie1,3*

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CAS Key Laboratory for Biological Effects of Nanomaterials and Nanosafety, CAS

Center for Excellence in Nanoscience, National Center for Nanoscience and Technology, Beijing, 100190, P. R. China 2

Iron Metabolism Laboratory, QIMR Berghofer Medical Research Institute, Brisbane

Queensland, 4006, Australia 3

University of Chinese Academy of Sciences, Beijing, 100049, P. R. China

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ABSTRACT

Chelators are commonly used to remove excess iron in iron-loading disorders. Deferoxamine (DFO) is an effective and safe iron chelator, but an onerous parenteral administration regimen limits its routine use. To develop more effective methods for delivering iron chelators, we examined whether amphiphilic copolymer nanoparticles (NPs), could deliver DFO more efficiently. Physical characterization showed a uniform and stable preparation of DFO nanoparticles (DFO-NPs) with an average diameter of 105.3 nm. In macrophage (RAW264.7) and hepatoma (HepG2) cell lines, DFO-NPs proved more effective at depleting iron than free DFO. In wild-type mice previously loaded with iron dextran, as well as Hbbth3/+ and Hfe-/- mice, which are predisposed to iron loading, DFO-NPs (40 mg/kg DFO; alternate days; 4 weeks) reduced hepatic iron levels by 71, 46 and 37% respectively, whereas the equivalent values for free DFO were 53, 7 and 15%. Staining for tissue iron and urinary iron excretion confirmed these findings. Pharmacokinetic analysis showed that NP-encapsulated DFO had a much longer elimination half-life than free DFO (48.63 ± 28.80 vs 1.46 ± 0.59 h), and that DFO-NPs could be readily taken up by tissues, and, in particular, by hepatic Kupffer cells. In vitro, DFO-NPs were less toxic to several cell lines than free DFO, and in vivo they did not elicit any specific inflammatory responses or histological changes. Our results suggest that using a nanoformulation of DFO is a valuable strategy for improving its efficiency as an iron chelator and that this could broaden its clinical use for the treatment of human iron overload disorders.

KEY WORDS Nanoformulation, deferoxamine, iron overload, iron chelation, macrophages, half-life

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Iron overload disorders represent an important class of human diseases.1, 2 Some, such as hereditary hemochromatosis, result from increased dietary iron absorption (primary iron loading),3, 4 while others can result from defects in red blood cell production and the need for frequent blood transfusions (secondary iron loading).5, 6 The common feature of both types of iron loading is the inappropriate accumulation of iron in vital body organs including the liver, heart, spleen and endocrine glands.1 Although iron is an essential nutrient, it is also toxic when present in excess, so patients with iron loading diseases can suffer severe organ damage and its clinical consequences.2, 7 Iron removal is a key therapeutic strategy for iron loading disorders.1, 7, 8 For the commonest form of iron loading, HFE-related hemochromatosis, iron depletion can be achieved by bleeding the patients repeatedly.9 For β-thalassemia, however, bleeding is not feasible and such patients must be treated with iron chelating agents.10-12 After binding iron, the iron-chelator complex is secreted in the urine and/or feces. Current chelator treatments are effective, but remain suboptimal.8, 12, 13 The chelator that has been most extensively used clinically is DFO, however, its short half-life means that it must be administered by infusion over long periods of time (8-12 h per day, 5 days per week).14, 15 This onerous administration regimen has meant that orally active iron chelators, of which two are in routine clinical use, are now more frequently employed.15,

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Nevertheless, DFO appears to be the most

effective and safest of the approved chelators. The first oral iron chelator approved for clinical use, Deferiprone, is also an effective chelator, but its ability to deplete liver iron removal is not optimal, 17 and it has been associated with some side effects, such as agranulocytosis and cytopenia

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and hepatotoxicity.20, 21 The other oral iron

chelator, Deferasirox can lead to gastrointestinal disturbances, skin rash and increased serum creatinine.22 With the aim of developing more effective methods for delivering iron chelators, and particularly DFO, we have examined whether amphiphilic copolymer nanoparticles (NPs), which can prolong the blood circulation time of their cargo and can passively target to the liver, spleen and other organs, can be used to deliver DFO efficiently. 3

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DFO-loaded NPs proved more effective at removing iron from iron dextran loaded wild-type mice, as well as Hfe-/- and Hbbth3/+ mice, murine models of hemochromatosis and β-thalassemia respectively. Nanoparticle-encapsulated DFO had a longer half-life than free DFO, and was associated with increased urinary iron excretion. Thus DFO-containing nanoparticles show considerable potential for the treatment of iron overload without causing significant side effects. NPs encapsulating DFO were generated using the double emulsion method from mPEG-PLGA monomers (Figure 1a). The average size of the NPs was 105.3 nm, with more than 90% of the NPs between 50 nm and 200 nm (Figure 1b). TEM showed the spherical, core-shell structure of the NPs (Figure 1b). These NPs proved to be highly stable, and no changes in size were observed after incubation at pH 4.4, 5.3 or 7.4 at 37oC for 3 days (Figure 1c). We also examined the encapsulation efficiency of DFO-NPs based on the specific absorption peak of FO at 430 nm (Figure 1d). The encapsulation efficiency was > 50% when the co-polymer to DFO weight ratio was higher than 20:1 (Figure 1e). In vitro drug release profiles showed that DFO is released more rapidly from the NPs at pH 4.4 than at pH 7.4 (Figure 1f). After 150 h, more than 90% of the DFO originally encapsulated in the NPs was released. We also carried out a series of studies to show that (a) DFO did not bind significant amounts of iron during the NP encapsulation process (Figure S1a,b), (b) we could accurately quantitate DFO levels in DFO-NPs (Figure S1c) and (c) there was insufficient available iron in any of the solutions we used (including tissue culture medium containing 10% FBS) to occupy more than 0.5% of the DFO (Figure S2). This is important to ensure that dose equivalence between free DFO and NP-encapsulated DFO is retained in both in vitro and in vivo studies. The relative iron removal efficacy of DFO-NPs and free DFO was first tested in vitro in RAW264.7 and HepG2 cells. Both the cellular iron content (Figures S3a,c) and ferritin levels (Figures S3b,d) of cells treated with DFO-NPs were markedly lower 4

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than those treated with free DFO. TFR1 levels were increased with iron chelation to similar levels using both free DFO and DFO-NPs (Figures S3b,d). We carried out similar studies after pre-loading the cells with iron and found similar results (Figure S4). To evaluate the efficacy of DFO-NPs in iron overloaded mice, we first established an iron overload model by injecting wild-type (C57BL/6J) mice with iron dextran (0.3 mg/kg; i.p.) on alternate days for one week. The characteristics of the model are described in Figure S5. To assess the efficacy of DFO-NPs in iron removal, iron dextran loaded mice were treated via tail vein injection with saline, DFO (40 mg/kg), B-NPs or DFO-NPs (40 mg/kg DFO) every second day for 4 weeks. As shown in Figure 2a, DFO-NPs were approximately twice as effective at removing iron as free DFO. This was demonstrated by both a significant reduction in iron content (Figure 2a,b) and reduced stainable iron (Figure 2c) in a range of tissues. We extended this work to include two genetic mouse models of iron loading: Hbbth3/+mice, a model of the β-thalassemia intermedia; and Hfe-/- mice, a model of HFE-related haemochromatosis. Free DFO treatment led to a slight reduction in iron levels in the liver and spleen of Hbbth3/+ (Figures 2d,e) and Hfe-/- mice (Figures 2f,g), but this was not statistically significant in Hbbth3/+animals. It should be noted that, in contrast to iron dextran-loaded mice, each of these two mouse strains will continue to absorb higher than average levels of dietary iron throughout the treatment period, and this may have led to a relative blunting of the response. Despite this, DFO-NPs had a much stronger effect in reducing liver and spleen iron levels in each model than free DFO, as demonstrated by both non-heme iron determination (Figure 2d-g) and stainable iron (Figure S6a). After DFO binds iron, the resulting ferrioxamine is excreted via the urine.23-25 To investigate urinary excretion, free DFO and DFO-NPs at equivalent doses were 5

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administered to 6 week-old iron dextran loaded mice. Urinary iron excretion was greatly stimulated following chelator administration (Figure 2h). The initial rate of excretion was much greater with free DFO than with DFO-NPs, but the total amount of iron excreted was significantly greater with the nanoparticle formulation. These data are consistent with DFO-NPs having a longer half-life than free DFO. DFO has a short circulation half-life (t1/2) of approximately 20 min in humans and 5 min in mice,26,

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and must be infused 8-12 h daily to maintain a therapeutic

concentration in the blood.14, 15 To determine the half-life of NP-derived DFO, we examined the plasma concentration of DFO by HPLC following intravenous injection of either free DFO or DFO-NPs in male mice. Plasma DFO decreased quickly in the first hour after the administration of free drug to become essentially undetectable (Figure 3a). These data were fitted to a standard two compartment open model to calculate the pharmacokinetic parameters. The distribution half-life (t1/2α) of DFO was 0.12 ± 0.06 h and the elimination half-life (t1/2β) was 1.46 ± 0.59 h. In contrast, the t1/2α of DFO in DFO-NP treated mice was 1.13 ± 0.83 h and the t1/2β was 48.63 ± 28.80 h. These data indicate that the NP formulation prolongs the retention of DFO. Additional pharmacokinetic parameters are shown in Table S1. The area under the curve (AUC0-∞) for the DFO group is 0.50 ± 0.49 mg h L-1 while the AUC for the DFO-NPs group is 1.03 ± 0.13 mg h L-1. The difference in AUC values likely reflects the rapid excretion of free DFO within the first 5 min,

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hence the AUC is an

underestimate of the true value. We also demonstrated that little DFO was released from NPs when they were incubated with normal serum (approximately 20% after 2 h; Figure 3b) suggesting that significant amounts of DFO were unlikely to be released from NPs before they entered the organs. When DFO-NPs were incubated with serum from iron dextran loaded mice, only 0.4% FO could be detected when the NPs were subsequently analyzed, suggesting that DFO contained within NPs was not accessible to external iron. To further understand the fate of DFO administered as either the free drug or encapsulated in NPs, we analyzed the tissue distribution of DFO at different time 6

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intervals after administration. DFO in NPs, but not free DFO, showed rapid and strong accumulation in the liver (reaching a peak at 6 h) followed by a slow decline (Figure 3c), consistent with strong tissue penetration of the NPs. NP-derived DFO also accumulated in the kidney, but much more slowly (Figure 3d). We also investigated the relative distribution of DFO between five major organs. Five minutes after the mice were treated with free DFO, 80% of the remaining drug in the organs was in the kidney, but only 4% was in the liver (Figure 3e). In contrast, for the DFO-NP group, 70% of the remaining DFO was in the liver and 12% of the DFO was in the kidney at 2 h. The proportion of DFO in the liver remained high after both 6 h (66%) and 12 h (30%) (Figure 3e). An important part of understanding the mechanism of DFO-NPs is determining their distribution in vivo. Enhanced tissue accumulation of the NPs is likely a major reason for the prolonged half-life of DFO in the NP group. We thus encapsulated the fluorescent dye Rho B into mPEG-PLGA nanoparticles and administered them either i.p. or i.v. into mice. NPs without RhoB and free RhoB were administered to separate animals as controls. Fluorescent signals were observed by biophotonic imaging 0.5, 3 and 24 h after injection. After 24 h, we collected the heart, liver, spleen, lungs and kidneys for ex vivo imaging. Higher levels of fluorescence were noted in the animals when the RhoB-NPs were administered i.v. (Figure 4a). At the end of the experiment, both nanoparticle groups (i.p. and i.v.) showed stronger tissue signals than the control group (Figure 4a), particularly in the liver and kidney, indicating relative retention of the NP encapsulated dye within various tissues. These studies confirm that the NPs are able to prolong the retention of their cargo in the liver and kidney. To study the cellular distribution of polymeric NPs within the liver, we isolated hepatocytes and Kupffer cells from C57BL/6J mice 1 h after treatment with saline, B-NPs or NPs encapsulating the dye propidium iodide (PI-NPs). Flow cytometry analysis showed that there was much greater accumulation of PI (and hence NPs) in Kupffer cells than in hepatocytes (Figure 4b), consistent with the known phagocytic propensity of macrophages.28-30 7

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To examine the cellular and subcellular distribution of mPEG-PLGA-NPs in more detail, we administered a single dose of either saline or a nanoformulation containing electron-dense gold nanoparticles (mPEG-PLGA-Au-NPs) i.v. to C57BL/6J mice. At 0.5 or 1 h after injection, liver tissue was collected, processed and examined by TEM (Figure 4c). Strong accumulation of Au nanoparticles, and hence mPEG-PLGA-NPs was observed predominantly in Kupffer cells over the first half hour, with a few mPEG-PLGA-Au-NPs in hepatocytes. However, over time, an increasing number of Au NPs were observed in hepatocytes, consistent with intra-hepatic transfer of the NPs from macrophages to parenchymal cells. To investigate the fate of mPEG-PLGA NPs following uptake by macrophages, we encapsulated the green dye fluorescein isothiocyanate (FITC) and the red fluorescent dye RhoB into the hydrophilic and hydrophobic layers of the NPs, respectively (RhoB-FITC-NPs). Then we monitored RAW264.7 cells treated with either free RhoB or FITC, or RhoB-FITC-NPs using confocal microscopy (Figure 4d). In cells treated with free RhoB and FITC at 0.5 h, separate fluorescent red and green signals were observed, while in cells treated with RhoB-FITC-NPs, extensive overlap of the signals was observed after 0.5 h, but by 3 h the degree of overlap had diminished considerably. These data are consistent with the NPs degrading over time. In vitro studies with RAW264.7 cells (Figure 5a) and HepG2 cell lines (Figure 5b) showed that both free DFO and DFO-NPs led to a significant reduction in cell viability as the chelator concentration was increased, but that greater toxicity was observed with free DFO. This suggests that the free drug might be more rapidly depleting iron than NP-encapsulated DFO, although it is also possible that DFO has some inherent toxicity that is independent of its iron chelating ability. Although the NPs and DFO individually have low inherent toxicity in vivo, the safety and toxicity of the DFO-NP combination must be investigated. To determine whether DFO-NPs were able to induce an inflammatory (innate immune) response, we examined the plasma levels of several cytokines (IL-6, TNF-α and IFN-γ) (Figures 8

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5c-e). None of the analyses have suggested that DFO-NPs are able to induce an innate immune response. Hematoxylin and eosin (H&E) staining of various tissues after treatment of control, iron dextran-loaded, Hbbth3/+ or Hfe-/- mice with free DFO, B-NPs or DFO-NPs revealed no overt tissue damage in any group (Figure 5f; Figure S6b). In this study, we have examined whether encapsulating the iron chelator DFO within PEG-PLGA nanoparticles could improve its efficacy as an iron removal agent. Both in vitro and in vivo, DFO-NPs proved more effective than free DFO at removing cellular and tissue iron. For the in vivo studies, we used iron dextran loaded wild-type mice and two genetic mouse models of iron overload, Hbbth3/+, a model for β-thalassemia intermedia, and Hfe-/-, a model for HFE-associated hereditary hemochromatosis. In iron dextran loaded mice and Hbbth3/+ mice, strong Kupffer cell iron loading is observed in the liver,31, 32 whilst in Hfe-/- mice, hepatic iron loading is predominantly in hepatocytes.33 Irrespective of its intrahepatic distribution, DFO-NPs were much more effective at removing iron than free DFO. The nanoparticle encapsulation extended the half-life of the iron chelator, as well as facilitating its tissue accumulation (particularly within macrophages), and either of these (or a combination of them) could explain its increased efficacy. DFO is a highly effective iron chelator with a favorable safety profile, but its short circulation half-life (20 - 30 min) means that onerous and prolonged subcutaneous infusion must be used to make it therapeutically useful.4 Thus, there has been considerable interest in developing new formulations of DFO with an extended half-life14,

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. Two strategies that have been used to achieve this are chemical

modification of DFO to make it more hydrophobic,34 or aggregating DFO with, or binding it to, non-toxic dendritic polymers.9-11, 35 In the former, a range of molecules (e.g. adamantyl groups) have been attached to DFO in a position that does not interfere with its ability to chelate iron.7 However, they can increase the hydrophobicity of the chelator and, at least in vitro, improve its tissue penetration and iron removing ability several fold. The second approach is not to modify the chelator 9

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per se, but to improve its formulation such that its half-life is increased. Conjugating DFO to high molecular weight biocompatible polymers8-11 can lead to an impressive increase in the half-life of up to several hundred-fold. Interestingly, the major benefit of such DFO-conjugates seems to be a reduction in toxicity (relative to free DFO),10,11 with increases in iron removal capacity being disproportionately small relative to the increase in half-life. Since DFO is increasingly being used in combination therapy with an oral iron chelator, there is considerable clinical benefit in developing new DFO formulations to enable the chelator to be delivered as efficiently as possible In the present study, we incorporated DFO into polymeric nanoparticles. In addition to prolonging the half-life of DFO, we were able to take advantage of the natural propensity of polymer nanoparticles to passively target to the liver to increase the efficacy of iron chelation therapy.28, 29, 36, 37 We found that urinary iron excretion was somewhat slower with NP-encapsulated DFO than with free DFO, but the NP formulation ultimately removed twice as much iron as an equivalent dose of free DFO. Although the NP-encapsulated chelator acts somewhat more slowly than the parent compound, in the context of iron loading diseases, where the treatments are characteristically long-term, this is not a practical limitation. Intravenously injected, non-stealth, colloidal carriers, such as liposomes and polymeric nanospheres, can be rapidly recognized and taken up by Kupffer cells in the liver.28-30 Consistent with this, we tracked the nanoparticles by encapsulating the fluorescent dye RhoB, and found that most mPEG-PLGA NPs accumulated in the liver, with some in the lung and kidney. When liver cells were isolated and either examined by flow cytometry (after the administration of PI encapsulated mPEG-PLGA NPs) or by TEM (after injection of mPEG-PLGA-Au-NPs) preferential accumulation of the NPs in Kupffer cells was observed. In addition, the intracellular degradation of PLGA NPs was demonstrated using a macrophage cell line. Our finding that DFO release is enhanced at low pH, as has been observed by others for drug release from PLGA NPs,38, 39 is consistent with the liberation of DFO in the low pH environment of lysosomes.40 These results are also consistent with mPEG-PLGA 10

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NPs delivering DFO to Kupffer cells, where it can be released to chelate iron, not only from within the Kupffer cells, but also potentially from surrounding hepatocytes and other non-parenchymal cells. While some studies have suggested that PEGylation may reduce the recognition of NPs by cells of the reticuloendothelial system,41 our studies have clearly shown that NP uptake by Kupffer cells is more than sufficient and other studies have shown that PEGylated NPs can still be internalized by macrophages.42-45 Although we have produced strong evidence that DFO-NPs have the capacity to remove iron from tissues, their precise mode of action is difficult to define. Our demonstration that DFO-NPs were equally efficient at removing iron from livers irrespective of whether the iron was in Kupffer cells or hepatocytes, suggests that multiple mechanisms could be involved. Some DFO is likely released from the NPs in the circulation where it may chelate non-transferrin-bound serum iron (NTBI). If the NPs acted as a reservoir from which DFO is released over time, then this could potentially make a significant contribution to iron removal. However, since the intact NPs will be taken up by macrophages relatively rapidly, we do not expect that DFO-NPs act as a long-term circulating reservoir of the chelator. Another possibility is that the DFO we detected in the plasma is largely the result of breakdown of NPs within Kupffer cells and other macrophages, followed by its release into the extracellular environment. In this case DFO could chelate both intracellular iron, as that is the iron it would see first after being released from the NPs, and plasma NTBI. Overall, we believe that such a combination of intracellular and extracellular chelation mechanisms is most likely. An important aspect of a therapy is its potential to cause adverse off-target effects. We first demonstrated in vitro that DFO-NPs proved less toxic than free DFO even though they removed more iron. This is likely due to the relative rate at which the different formulations facilitate iron release, with the more rapid release associated with free DFO being more detrimental to the cells. In preliminary safety studies in vivo, we found no evidence that DFO-NP treatment was associated with any altered 11

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tissue architecture or inflammatory response, giving confidence that nanoparticle encapsulation is both an effective and safe way of delivering DFO. Although there are potential safety risks associated with any type of NP,45 there is an extensive literature to show that PLGA NPs represent an extremely safe means of drug delivery.46 At present, any DFO formulation must be administered intravenously and, in view of this, DFO is unlikely to be the first choice iron chelator for the majority of patients with iron loading disease. One of the two oral chelators is used in preference due to their ease of administration. However, DFO-NPs could prove beneficial for the relatively small number of patients who cannot tolerate oral therapy, as the increased half-life when compared to free DFO would reduce the overall treatment time. The reduced toxicity of DFO-NPs may also allow higher doses to be delivered in these patients, further reducing the time required for infusion. In addition, multiple studies have demonstrated that a combination of DFO with one of the orally available iron chelators is a very effective means of treating patients with high iron loads and is more effective than treating with any single oral chelator47, 48. As such combination chelation strategies are increasingly being used, the development of more efficient delivery methods for DFO, such as DFO-NP formulations, can further enhance patient treatment. In summary, we have demonstrated that the encapsulation of DFO within polymeric nanoparticles is an effective and safe way to deliver the iron chelator. DFO-NPs likely act via several pathways, but the passive targeting of the NPs to macrophages, and particularly to Kupffer cells in the liver, appears to be most significant. While some release of DFO from intact NPs in the plasma could occur, the most likely mode of action involves the direct chelation of iron from the Kupffer cells themselves, as well as from surrounding cells, following degradation of the DFO-NPs and release of the chelator. After DFO binds iron within the cells, it can be exported into the circulation and subsequently excreted, largely in the urine. The delivery of DFO in a nanoformulation is not without its limitations. For example, the encapsulation of DFO adds time, and hence cost, to the preparation of the chelator formulation, and there is a 12

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practical limit on how much chelator can be incorporated. However, the effects of these limitations can be minimized by scaling up and optimizing the packaging process. The ability of DFO-NPs to accumulate in the liver and facilitate hepatic iron removal is a particularly favorable characteristic which has the potential to make this formulation more effective clinically than existing chelator treatments. Overall, the high efficacy and low toxicity of NP-mediated chelator delivery makes it a strong candidate for the therapy of iron loading diseases in the future.

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Figure 1. Synthesis and characterization of DFO-NPs. (a) Schematic illustration of the synthesis of biodegradable amphiphilic copolymer NPs loaded with DFO using an improved double emulsion method. (b) The size of the DFO-NPs was determined by dynamic light scattering (DLS), TEM was used to show the core-shell structure of the NPs. Scale bar: 200 nm. (c) Stability of the NPs was studied at 37oC at varying pH values. At daily intervals up to 3 d, the diameter of the NPs was assessed using DLS (n = 3). (d) The UV-vis profile of ferrioxamine, which shows an absorption maximum at 430 nm. DFO could be quantified by adding iron and measuring the intensity of the absorbance at this characteristic peak. (e) Effects of the co-polymer/DFO weight ratio on the encapsulation efficiency of DFO (n = 3). (f) Release of DFO from 14

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mPEG-PLGA nanoparticles at pH 4.4 (black) or pH 7.4 (red) in PBS at room temperature (n = 3).

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Figure 2. Efficacy of DFO-NPs in vivo. Three mouse models of iron loading were used: iron dextran-treated wild-type mice, Hbbth3/+ mice and Hfe-/- mice. All models were treated with saline, free DFO, B-NPs or DFO-NPs. Liver and spleen iron concentrations were measured in iron dextran loaded male mice (a, b; n = 4), Hbbth3/+ female mice (d, e; n = 7) and Hfe-/- female mice (f, g; n = 7) following various treatments. DFO-NPs consistently removed more iron from the tissues than free DFO. (c) Perls’ Prussian Blue staining for iron in the liver and spleen of iron dextran-loaded mice after treatment with DFO or DFO-NPs. Yellow arrows indicate iron deposited in 16

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Kupffer cells. The untreated group represents tissue from mice without iron dextran loading. Scale bar for liver: 100 µm; scale bar for spleen: 200 µm. (h) Cumulative urinary excretion of iron at various time points after iron dextran-loaded mice were given a single dose of DFO or DFO-NPs (n = 6). *p < 0.05; **p < 0.01; ***p < 0.001. LIC, liver iron concentration; SIC, splenic iron concentration.

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Figure 3. Circulation half-life, pharmacokinetics and tissue distribution of DFO and DFO-NPs in mice. (a) Plasma concentrations of DFO at various times after the administration of DFO (blue) or DFO-NPs (red) to mice (n = 4). DFO has a very short half-life, as expected, whereas DFO-NPs allow DFO concentrations in the circulation to remain relatively high for a much longer time. (b) To determine whether DFO could be released from DFO-NPs in plasma, the drug-containing NPs were incubated with plasma in vivo for 2 h and the level of DFO was determined in both the plasma and the NPs. Only about 20% of the DFO was released from the NPs over this time. ***p < 0.001; n = 3. (c) DFO concentrations in the liver at various time intervals following the administration of DFO or DFO-NPs (n = 4). Extensive hepatic accumulation of DFO-NPs was observed. (d) DFO concentrations in the kidney at different intervals following the administration of DFO or DFO-NPs (n = 4). Extensive tissue accumulation of DFO-NPs was observed, but in this case, the rise was progressive over the first 10 h before reaching a plateau. (e) Relative DFO distribution between five major organs 5 min after administering free DFO, or 2 , 6 and 12 h after administering DFO-NPs (n = 4).

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Figure 4. Specific accumulation of PLGA-NPs in Kupffer cells in mice and drug release in a macrophage cell line. (a) Saline (i.v.), free Rhodamine B (i.v.), B-NPs (i.v.) or Rhodamine-labeled mPEG-PLGA-NPs (i.v. or i.p.) were administered to 19

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wild-type mice. In vivo fluorescence imaging of the mice was carried out for up to 24 h, after which the animals were euthanized and major organs were removed for ex vivo imaging. Most treatments were intravenous, but some animals were treated with Rho-NPs intraperitoneally. NP-encapsulated Rho B was retained much longer in the body than free Rho B, with ex vivo imaging showing particularly high accumulation in the liver and kidney. (b) To investigate where the NPs were localized in the liver, hepatic cell suspensions were prepared and analyzed by flow cytometry 1 h after injection of mice with B-NPs or PI-NPs. Separation of hepatocytes and Kupffer cells showed that most PI signal was associated with the Kupffer cell fraction (high PI and high F4/80 staining), consistent with macrophage internalization of the NPs. Hepatocytes and Kupffer cells were stained with Alexa 633-labeled anti-albumin and anti F4/80-FITC. (c) High resolution TEM images of liver fractions 0.5 and 1 h following administration of mPEG-PLGA NPs, containing 15 nm gold nanoparticles. Red arrows indicate Au NPs in the cytoplasm of Kupffer cells (KC) and hepatocytes (HEP). (d) In vitro fluorescence images of RAW264.7 cells after incubation with RhoB-FITC-NPs to measure degradation of the NPs. After 0.5 h, the yellow-green fluorescence in the cells suggests that the two fluorophores are closely juxtaposed, i.e., the NPs are intact. However, by 3 h (bottom row), the red and green signals have clearly separated, consistent with breakdown of the NPs. Scale bars: Black bar represents 50 µm for the first four column and white bar represent 10 µm for the last column.

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Figure 5. In vitro and in vivo safety of DFO-NPs. The viability of RAW264.7 (a) and HepG2 (b) cells was assessed by CCK-8 analysis following treatment with either free DFO or DFO-NPs (n = 5). (c-e) Pro-inflammatory cytokine levels in the plasma of mice treated with DFO, B-NPs or DFO-NPs. With the exception of the saline only group, mice were first loaded with iron dextran before treatment with chelator formulations. Results for (c) IL-6, (d) TNF-α and (e) IFN-γ (n = 6) are shown. *p < 0.05; ***p < 0.001. (f) Following their last treatment, the mice used in (c-e) were euthanized and sections from various organs were stained with H&E to assess tissue morphology. Scale bars: 100 µm.

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ASSOCIATED CONTENT Supporting Information The following file is available free of charge. Experimental Section, Table S1, and Figures S1-S7 (PDF)

AUTHOR INFORMATION Corresponding Authors *Guangjun Nie, E-mail: [email protected] *Gregory J. Anderson, E-mail: [email protected]

Author Contributions S.G., G.J.A. and G.N. conceived the project, designed the experiments and wrote the manuscript. S.G. performed the majority of the experiments and data analysis. G.L. contributed to the conception of the project. D.M.F. helped with designation of experiments and analysis of data. T.L., L.Y., J.X. and Y.W. provided assistance with some of the experimental work. All authors discussed the results and commented on the manuscript. Notes The authors declare no competing financial interests.

ACKNOWLEDGMENT This work was supported by the National Basic Research Plan of China (2018YFA0208900), the National Natural Science Foundation of China (31730032, 31471035), a Key Research Project of Frontier Science of the Chinese Academy of Sciences (QYZDJ-SSW-SLH022), an Australian National Health and Medical 22

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Research Council Project Grant (APP1107095), a Queensland-Chinese Academy of Sciences (Q-CAS) Collaborative Science Fund Grant (QCAS03916-17RD6) and the Beijing Municipal Science & Technology Commission (Z161100000116035). GJA was supported by a Senior Research Fellowship from the National Health and Medical Research Council of Australia (APP1024230).

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Graphic for Table of Contents Incorporation of the iron chelator deferoxamine (DFO) into polymeric nanoparticles increases its efficiency as an iron removal agent by enhancing its tissue penetration and prolonging its half-life. Hepatic Kupffer cells are a major target of DFO-NPs, making them attractive agents for removing iron from the liver, a major site of iron accumulation in iron loading disorders.

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