INHIBITORS OF INORGANIC POLYPHOSPHATE AND NUCLEIC ACIDS ATTENUATE IN VITRO THROMBIN GENERATION IN PLASMA FROM TRAUMA PATIENTS.

ABSTRACT: Background: Inorganic polyphosphate (polyP) is a procoagulant polyanion. We assessed the impact of polyP inhibition on thrombin generation after trauma using the novel polyP antagonists, macromolecular polyanion inhibitor 8 (MPI 8), and universal heparin reversal agent 8 (UHRA-8). Methods: Plasma thrombin generation (calibrated automated thrombogram, CAT), in 56 trauma patients and 39 controls +/- MPI 8 and UHRA-8 (50 µg/mL), was expressed as lag time (LT, minutes), peak height (PH, nM), and time to peak (ttPeak, minutes), with change in LT (ΔLT) and change in ttPeak (ΔttPeak) quantified. Results expressed in median and quartiles [Q1, Q3], Wilcoxon matched-pairs testing, P < 0.05 significant. Results: Trauma patients had greater baseline PH than controls (182.9 [121.0, 255.2]; 120.5 [62.1, 174.8], P < 0.001). MPI 8 treatment prolonged LT and ttPeak in trauma (7.20 [5.88, 8.75]; 6.46 [5.45, 8.93], P = 0.020; 11.28 [8.96, 13.14]; 11.00 [8.95, 12.94], P = 0.029) and controls (7.67 [6.67, 10.50]; 6.33 [5.33, 8.00], P < 0.001; 13.33 [11.67, 15.33]; 11.67 [10.33, 13.33], P < 0.001). UHRA-8 treatment prolonged LT and ttPeak and decreased PH in trauma (9.09 [7.45, 11.33]; 6.46 [5.45, 8.93]; 14.02 [11.78, 17.08]; 11.00 [8.95, 12.94]; 117.4 [74.5, 178.6]; 182.9 [121.0, 255.2]) and controls (9.83 [8.00, 12.33]; 6.33 [5.33, 8.00]; 16.67 [14.33, 20.00]; 11.67 [10.33, 13.33]; 55.3 [30.2, 95.9]; 120.5 [62.1, 174.8]), all P < 0.001. Inhibitor effects were greater for controls (greater ΔLT and ΔttPeak for both inhibitors, P < 0.001). Conclusion: PolyP inhibition attenuates thrombin generation, though to a lesser degree in trauma than in controls, suggesting that polyP contributes to accelerated thrombin generation after trauma.

Development of an iron overload HepG2 cell model using ferrous ammonium citrate.

Cell-based iron overload models provide tremendous utility for the investigations into the pathogenesis of different diseases as well as assessing efficacy of various therapeutic strategies. In the literature, establishing such models vary widely with regards to cell lines, iron source, iron treatment conditions and duration. Due to this diversity, researchers reported significant differences in the measured outcomes, either in cellular function or response to a stimulus. Herein, we report the process required to establish an iron overload HepG2 cell model to achieve a consistent and reproducible results such that the literature can strive towards a consensus. Iron loading in cells was achieved with 50 µM of iron every 24 h for 2 days, followed by an additional 24 h of maintenance in fresh media. We demonstrated that iron overloaded cells had significantly increased ROS generation, labile and total iron whilst having various cellular functions resemble cells without iron overload. The present report addresses key pitfalls with regards to the lack of consensus currently present in the literature.

Inhibitors of Polyphosphate and Neutrophil Extracellular Traps.

The contact pathway of blood clotting has received intense interest in recent years as studies have linked it to thrombosis, inflammation, and innate immunity. Because the contact pathway plays little to no role in normal hemostasis, it has emerged as a potential target for safer thromboprotection, relative to currently approved antithrombotic drugs which all target the final common pathway of blood clotting. Research since the mid-2000s has identified polyphosphate, DNA, and RNA as important triggers of the contact pathway with roles in thrombosis, although these molecules also modulate blood clotting and inflammation via mechanisms other than the contact pathway of the clotting cascade. The most significant source of extracellular DNA in many disease settings is in the form of neutrophil extracellular traps (NETs), which have been shown to contribute to incidence and severity of thrombosis. This review summarizes known roles of extracellular polyphosphate and nucleic acids in thrombosis, with an emphasis on novel agents under current development that target the prothrombotic activities of polyphosphate and NETs.

Smart thrombosis inhibitors without bleeding side effects via charge tunable ligand design.

Current treatments to prevent thrombosis, namely anticoagulants and platelets antagonists, remain complicated by the persistent risk of bleeding. Improved therapeutic strategies that diminish this risk would have a huge clinical impact. Antithrombotic agents that neutralize and inhibit polyphosphate (polyP) can be a powerful approach towards such a goal. Here, we report a design concept towards polyP inhibition, termed macromolecular polyanion inhibitors (MPI), with high binding affinity and specificity. Lead antithrombotic candidates are identified through a library screening of molecules which possess low charge density at physiological pH but which increase their charge upon binding to polyP, providing a smart way to enhance their activity and selectivity. The lead MPI candidates demonstrates antithrombotic activity in mouse models of thrombosis, does not give rise to bleeding, and is well tolerated in mice even at very high doses. The developed inhibitor is anticipated to open avenues in thrombosis prevention without bleeding risk, a challenge not addressed by current therapies.

What's fishy about protamine? Clinical use, adverse reactions, and potential alternatives.

Protamine, a highly basic protein isolated from salmon sperm, is the only clinically available agent to reverse the anticoagulation of unfractionated heparin. Following intravenous administration, protamine binds to heparin in a nonspecific electrostatic interaction to reverse its anticoagulant effects. In clinical use, protamine is routinely administered to reverse high-dose heparin anticoagulation in cardiovascular procedures, including cardiac surgery with cardiopulmonary bypass. Despite the lack of supportive evidence regarding protamine's effectiveness to reverse low-molecular-weight heparin, it is recommended in guidelines with low-quality evidence. Different dosing strategies have been reported for reversing heparin in cardiac surgical patients based on empiric dosing, pharmacokinetics, or point-of-care measurements of heparin levels. Protamine administration is associated with a spectrum of adverse reactions that range from vasodilation to life-threatening cardiopulmonary dysfunction and shock. The life-threatening responses appear to be hypersensitivity reactions due to immunoglobulin E and/or immunoglobulin G antibodies. However, protamine and heparin-protamine complexes can activate complement inflammatory pathways and inhibit other coagulation factors. Although alternative agents for reversing heparin are not currently available for clinical use, additional research continues evaluating novel therapeutic approaches.

Sulfated Hyperbranched and Linear Polyglycerols Modulate HMGB1 and Morphological Plasticity in Neural Cells.

The objective of this study was to establish if polyglycerols with sulfate or sialic acid functional groups interact with high mobility group box 1 (HMGB1), and if so, which polyglycerol could prevent loss of morphological plasticity in excitatory neurons in the hippocampus. Considering that HMGB1 binds to heparan sulfate and that heparan sulfate has structural similarities with dendritic polyglycerol sulfates (dPGS), we performed the experiments to show if polyglycerols can mimic heparin functions by addressing the following questions: (1) do dendritic and linear polyglycerols interact with the alarmin molecule HMGB1? (2) Does dPGS interaction with HMGB1 influence the redox status of HMGB1? (3) Can dPGS prevent the loss of dendritic spines in organotypic cultures challenged with lipopolysaccharide (LPS)? LPS plays a critical role in infections with Gram-negative bacteria and is commonly used to test candidate therapeutic agents for inflammation and endotoxemia. Pathologically high LPS concentrations and other stressful stimuli cause HMGB1 release and post-translational modifications. We hypothesized that (i) electrostatic interactions of hyperbranched and linear polysulfated polyglycerols with HMGB1 will likely involve sites similar to those of heparan sulfate. (ii) dPGS can normalize HMGB1 compartmentalization in microglia exposed to LPS and prevent dendritic spine loss in the excitatory hippocampal neurons. We performed immunocytochemistry and biochemical analyses combined with confocal microscopy to determine cellular and extracellular locations of HMGB1 and morphological plasticity. Our results suggest that dPGS interacts with HMGB1 similarly to heparan sulfate. Hyperbranched dPGS and linear sulfated polymers prevent dendritic spine loss in hippocampal excitatory neurons. MS/MS analyses reveal that dPGS-HMGB1 interactions result in fully oxidized HMGB1 at critical cysteine residues (Cys23, Cys45, and Cys106). Triply oxidized HMGB1 leads to the loss of its pro-inflammatory action and could participate in dPGS-mediated spine loss prevention. LPG-Sia exposure to HMGB1 results in the oxidation of Cys23 and Cys106 but does not normalize spine density.

HyClear: A Novel Tissue Clearing Solution for One-Step Clearing of Microtissues.

3-D cell cultures are being increasingly used as in vitro models are capable of better mimicry of in vivo tissues, particularly in drug screenings where mass transfer limitations can affect the cancer biology and response to drugs. Three-dimensional microscopy techniques, such as confocal and multiphoton microscopy, have been used to elucidate data from 3-D cell cultures and whole organs, but their reach inside the 3-D tissues is restrained by the light scattering of the tissues, limiting their effective reach to 100-200 µm, which is simply not enough. Tissue clearing protocols, developed mostly for larger specimens usually involve multiple steps of viscous liquid submersion, and are not easily adaptable for much smaller spheroids and organoids. In this work, we have developed a novel tissue clearing solution tailored for small spheroids and organoids. Our tissue clearing protocol, called HyClear, uses a mixture of DMSO, HPG and urea to allow for one-step tissue clearing of spheroids and organoids, and is compatible with high-throughput screening studies due to its speed and simplicity. We have shown that our tissue clearing agent is superior to many of the commonly used tissue clearing agents and allows for elucidating better quality data from drug screening experiments.

Active transport nanochelators for the reduction of liver iron burden in iron overload.

Liver dysfunction and failure account for a major portion of premature deaths in patients suffering from various iron associated pathogeneses, particularly primary and secondary iron overload disorders, despite intensive treatment. The liver is a central player in iron homeostasis and a major iron storage organ, and currently, there are no active approaches for the excretion of excess liver iron. Herein, we report a new method for the rapid reduction of iron burden in iron overload diseases by developing a new class of liver targeted nanochelators with favorable pharmacokinetics and biodistribution. The new nanochelators bypass the reticuloendothelial system and specifically target hepatocytes without non-specific accumulation in other organs. The targeted nanochelators bound and neutralized excess iron in the liver and from the vasculature and, eventually leading to rapid hepatobiliary excretion of labile iron. Further, these rapidly excreted nanochelators did not induce toxicity in the liver, were highly cytocompatible in both iron overload and non-loaded conditions, and were promising in mitigating iron triggered free radical oxidative damage. These studies provide key insights into the development of organ targeted nanochelating systems and the rapid reduction of iron burden in vivo. This methodology allows for further development of nanotherapeutics for specific iron overload diseases.

Hydrophilic Polymer-Guided Polycatecholamine Assembly and Surface Deposition.

Mussel-inspired surface chemistry based on polycatecholamines and polyphenols has been widely applied as a facile and universal method for modifying surfaces. Specifically, the catecholamine-assisted codeposition as a one-step strategy is a versatile strategy used to impart surface functionalities. Despite successful incorporation of numerous functional agents, very little understanding has emerged over the years regarding the mechanism behind their coassembly and codeposition. Here, we employed six different ultrahigh molecular weight hydrophilic polymers of diverse chemistry and architecture and three catecholamines and a polyphenol for investigating the coassembly and codeposition process. The chemistry of the polymers is found to influence the strength of the interaction between the polycatecholamine and the hydrophilic polymers, thus playing an important role in the aqueous self-assembly in solution to nanoaggregates, its formation kinetics, steric stabilization, and surface deposition. Additionally, the codeposition method was used as a platform for developing antifouling and antibiofilm coatings and evaluating their efficiency. Both the chemistry of hydrophilic polymers and the type of the catecholamine influence the antibiofilm properties of the coating. Our studies demonstrated that significant opportunities exist to further define the surface coating process and polycatecholamine self-assembly process by altering the polycatecholamine-hydrophilic polymer interactions.

Influence of Steric Shield on Biocompatibility and Antithrombotic Activity of Dendritic Polyphosphate Inhibitor.

The polyanion, inorganic polyphosphate (polyP), is a procoagulant molecule which has become a promising therapeutic target in the development of antithrombotics. Neutralizing polyP's prothrombotic activity using polycationic inhibitors is one of the viable strategies to design new polyP inhibitors. However, in this approach, a fine balance between the electrostatic interaction of polyP and the inhibitor is needed. Any unprotected polycations are known to interact with negatively charged blood components, potentially resulting in platelet activation, cellular toxicity, and bleeding. Thus, designing potent polycationic polyP inhibitors with good biocompatibility is a major challenge. Building on our previous research on universal heparin reversal agent (UHRA), we report polyP inhibitors with a modified steric shield design. The molecular weight, number of cationic binding groups, and the length of the polyethylene glycol (PEG) chains were varied to arrive at the desired inhibitor. We studied two different PEG lengths (mPEG-750 versus mPEG-350) on the polyglycerol scaffold and investigated their influence on biocompatibility and polyP neutralization activity. The polyP inhibitor with mPEG-750 brush layer, mPEG750 UHRA-10, showed superior biocompatibility compared to its mPEG-350 analogs by a number of measured parameters without losing its neutralization activity. An increase in cationic binding groups (25 groups in mPEG750 UHRA-8 and 32 in mPEG750 UHRA-10 [HC]) did not alter the neutralization activity, which suggested that the mPEG-750 shield layer provides significant protection of cationic binding groups and thus helps to minimize unwanted nonspecific interactions. Furthermore, these modified polyP inhibitors are highly biocompatible compared to conventional polycations that have been previously used as polyP inhibitors (e.g., PAMAM dendrimers and polyethylenimine). Through this study, we demonstrated the importance of the design of steric shield toward highly biocompatible polyP inhibitors. This approach can be exploited in the design of highly biocompatible macromolecular inhibitors.

Blood Compatibility of Hydrophilic Polyphosphoesters.

Polyphosphoesters (PPEs) are a class of versatile degradable polymers. Despite the high potential of this class of polymers in biomedical applications, little is known about their blood interaction and compatibility. We evaluated the hemocompatibility of water-soluble PPEs (with different hydrophilicities and molar masses) and PPE-coated model nanocarriers. Overall, we identified high hemocompatibility of PPEs, comparable to poly(ethylene glycol) (PEG), currently used for many applications in nanomedicine. Hydrophilic PPEs caused no significant changes in blood coagulation, negligible platelet activation, the absence of red blood cells lysis, or aggregation. However, when a more hydrophobic copolymer was studied, some changes in the whole blood clot strength at the highest concentration were detected, but only concentrations above that are typically used for biomedical applications. Also, the PPE-coated model nanocarriers showed high hemocompatibility. These results contribute to defining hydrophilic PPEs as a promising platform for degradable and biocompatible materials in the biomedical field.

Prevention of vascular-allograft rejection by protecting the endothelial glycocalyx with immunosuppressive polymers.

Systemic immunosuppression for the mitigation of immune rejection after organ transplantation causes adverse side effects and constrains the long-term benefits of the transplanted graft. Here we show that protecting the endothelial glycocalyx in vascular allografts via the enzymatic ligation of immunosuppressive glycopolymers under cold-storage conditions attenuates the acute and chronic rejection of the grafts after transplantation in the absence of systemic immunosuppression. In syngeneic and allogeneic mice that received kidney transplants, the steric and immunosuppressive properties of the ligated polymers largely protected the transplanted grafts from ischaemic reperfusion injury, and from immune-cell adhesion and thereby immunocytotoxicity. Polymer-mediated shielding of the endothelial glycocalyx following organ procurement should be compatible with clinical procedures for transplant preservation and perfusion, and may reduce the damage and rejection of transplanted organs after surgery.

Role of Iron in the Molecular Pathogenesis of Diseases and Therapeutic Opportunities.

Iron is an essential mineral that serves as a prosthetic group for a variety of proteins involved in vital cellular processes. The iron economy within humans is highly conserved in that there is no proper iron excretion pathway. Therefore, iron homeostasis is highly evolved to coordinate iron acquisition, storage, transport, and recycling efficiently. A disturbance in this state can result in excess iron burden in which an ensuing iron-mediated generation of reactive oxygen species imparts widespread oxidative damage to proteins, lipids, and DNA. On the contrary, problems in iron deficiency either due to genetic or nutritional causes can lead to a number of iron deficiency disorders. Iron chelation strategies have been in the works since the early 1900s, and they still remain the most viable therapeutic approach to mitigate the toxic side effects of excess iron. Intense investigations on improving the efficacy of chelation strategies while being well tolerated and accepted by patients have been a particular focus for many researchers over the past 30 years. Moreover, recent advances in our understanding on the role of iron in the pathogenesis of different diseases (both in iron overload and iron deficiency conditions) motivate the need to develop new therapeutics. We summarized recent investigations into the role of iron in health and disease conditions, iron chelation, and iron delivery strategies. Information regarding small molecule as well as macromolecular approaches and how they are employed within different disease pathogenesis such as primary and secondary iron overload diseases, cancer, diabetes, neurodegenerative diseases, infections, and in iron deficiency is provided.

MANTI: Automated Annotation of Protein N-Termini for Rapid Interpretation of N-Terminome Data Sets.

Site-specific proteolytic processing is an important, irreversible post-translational protein modification with implications in many diseases. Enrichment of protein N-terminal peptides followed by mass spectrometry-based identification and quantification enables proteome-wide characterization of proteolytic processes and protease substrates but is challenged by the lack of specific annotation tools. A common problem is, for example, ambiguous matches of identified peptides to multiple protein entries in the databases used for identification. We developed MaxQuant Advanced N-termini Interpreter (MANTI), a standalone Perl software with an optional graphical user interface that validates and annotates N-terminal peptides identified by database searches with the popular MaxQuant software package by integrating information from multiple data sources. MANTI utilizes diverse annotation information in a multistep decision process to assign a conservative preferred protein entry for each N-terminal peptide, enabling automated classification according to the likely origin and determines significant changes in N-terminal peptide abundance. Auxiliary R scripts included in the software package summarize and visualize key aspects of the data. To showcase the utility of MANTI, we generated two large-scale TAILS N-terminome data sets from two different animal models of chemically and genetically induced kidney disease, puromycin adenonucleoside-treated rats (PAN), and heterozygous Wilms Tumor protein 1 mice (WT1). MANTI enabled rapid validation and autonomous annotation of >10 000 identified terminal peptides, revealing novel proteolytic proteoforms in 905 and 644 proteins, respectively. Quantitative analysis indicated that proteolytic activities with similar sequence specificity are involved in the pathogenesis of kidney injury and proteinuria in both models, whereas coagulation processes and complement activation were specifically induced after chemical injury.

A facile colorimetric method for the quantification of labile iron pool and total iron in cells and tissue specimens.

Quantification of iron is an important step to assess the iron burden in patients suffering from iron overload diseases, as well as tremendous value in understanding the underlying role of iron in the pathophysiology of these diseases. Current iron determination of total or labile iron, requires extensive sample handling and specialized instruments, whilst being time consuming and laborious. Moreover, there is minimal to no overlap between total iron and labile iron quantification methodologies-i.e. requiring entirely separate protocols, techniques and instruments. Herein, we report a unified-ferene (u-ferene) assay that enables a 2-in-1 quantification of both labile and total iron from the same preparation of a biological specimen. We demonstrate that labile iron concentrations determined from the u-ferene assay is in agreement with confocal laser scanning microscopy techniques employed within the literature. Further, this assay offers the same sensitivity as the current gold standard, inductively coupled plasma mass spectrometry (ICP-MS), for total iron measurements. The new u-ferene assay will have tremendous value for the wider scientific community as it offers an economic and readily accessible method for convenient 2-in-1 measurement of total and labile iron from biological samples, whilst maintaining the precision and sensitivity, as compared to ICP-MS.

Understanding the Interaction of Polyelectrolyte Architectures with Proteins and Biosystems.

The counterions neutralizing the charges on polyelectrolytes such as DNA or heparin may dissociate in water and greatly influence the interaction of such polyelectrolytes with biomolecules, particularly proteins. In this Review we give an overview of studies on the interaction of proteins with polyelectrolytes and how this knowledge can be used for medical applications. Counterion release was identified as the main driving force for the binding of proteins to polyelectrolytes: Patches of positive charge become multivalent counterions of the polyelectrolyte and lead to the release of counterions from the polyelectrolyte and a concomitant increase in entropy. This is shown from investigations on the interaction of proteins with natural and synthetic polyelectrolytes. Special emphasis is paid to sulfated dendritic polyglycerols (dPGS). The Review demonstrates that we are moving to a better understanding of charge-charge interactions in systems of biological relevance. Research along these lines will aid and promote the design of synthetic polyelectrolytes for medical applications.

Evaluation of hyperbranched polyglycerol for cold perfusion and storage of donor kidneys in a pig model of kidney autotransplantation.

Hyperbranched polyglycerol (HPG) is a biocompatible polyether polymer that is a potential colloid component in a preservation solution for suppressing interstitial edema during cold storage of a donor organ. This study evaluated the outcomes of kidney transplants after cold perfusion and storage with a HPG-based preservation solution (HPGS) in a pig model of kidney autotransplantation. The left kidneys of farm pigs (weighing 35-45 kg) were perfused with and stored in either cold HPGS or standard UW solution (UWS), followed by transplantation to the right side after right nephrectomy. The survival and function of transplants were determined by the urine output, and serum creatinine (SCr) and blood urea nitrogen (BUN) of recipients. Transplant injury was examined by histological analysis. Here, we showed that there was no significant difference between HPGS and UWS in the prevention of tissue edema, but HPGS was more effective than UWS for initial blood washout of kidney perfusion and for the prevention of cold ischemia injury during cold storage. After autotransplantation, the kidneys preserved with HPGS (HPG group) had better functional recovery than those with UWS (UW group), indicated by significantly more urine output and lower levels of SCr and BUN. The survived grafts in HPG group had less tissue damage than those in UW group. In conclusion, as compared to the UWS the HPGS has less negative impact on kidney cold ischemia during cold storage, resulting in improving immediate functional recovery after transplantation, suggesting that HPG is a promising colloid for donor kidney preservation.

Polyglycerol-Based Macromolecular Iron Chelator Adjuvants for Antibiotics To Treat Drug-Resistant Bacteria.

Iron is an essential micronutrient for life. Its redox activity is a key component in a plethora of vital enzymatic reactions that take place in processes such as drug metabolism, DNA synthesis, steroid synthesis, gene regulation, and cellular respiration (oxygen transport and the electron transport chain). Bacteria are highly dependent on iron for their survival and growth and have specific mechanisms to acquire iron. Limiting the availability of iron to bacteria, thereby preventing their growth, provides new opportunities to treat infection in the era of the persistent rise of antibiotic-resistant bacteria. In this work, we have developed macromolecular iron chelators, conjugates of a high-affinity iron chelator (HBEDS) with polyglycerol, in an attempt to sequester iron uptake by bacteria to limit their growth in order to enhance antibiotic activity. The new macromolecular chelators are successful in slowing the growth of Staphylococcus aureus and worked as an efficient bacteriostatic against S. aureus. Further, these cytocompatible macrochelators acted as effective adjuvants to prevent bacterial growth when used in conjunction with antibiotics. The adjuvant activity of the macrochelators depends on their molecular weight and the chelator density on these molecules. These selective macro iron(III) chelators are highly efficient in growth inhibition and killing of methicillin-resistant S. aureus in conjunction with a low concentration of rifampicin.

Targeting Biological Polyanions in Blood: Strategies toward the Design of Therapeutics.

In this Review, we highlight well-described and emerging polyanions, and the way these molecules can be targeted in the design of potential therapeutics (synthetic and biologics) with applications in thrombosis and hemostasis. It is important to strike a balance between bleeding and clotting. In thrombosis, unwanted blood clots are formed in the lumen of a blood vessel, obstructing the flow of blood through the circulatory system. Over many years of research, several polyanionic biopolymers that can either impede (anticoagulant) or promote (procoagulant) blood clotting have been identified. Mediators impeding blood clotting, including polyanionic polysaccharides such as heparins and heparin mimics, are widely used as antithrombotics, although they impart adverse complications such as bleeding. Emerging synthetic polycations and well-described cationic proteins that are specifically designed to neutralize the biological activity of heparins to prevent bleeding complications are discussed. On the other hand, there is growing evidence that several polyanions bear a procoagulant nature in blood; polyphosphate (polyP), neutrophil extracellular traps (NETs), extracellular RNA, and cell-free DNA are shown to promote blood clotting. Recent research highlights the use of polycations and enzymes that either inhibit or cleave these procoagulant polyanions and demonstrates the proof-of-concept design of new antithrombotics without bleeding side effects. Additional studies have shown that some of these procoagulant polyanions can be used as a hemostat to prevent bleeding in an emergency. There are significant opportunities for chemists in the design of new inhibitors and agents with improved selectivity toward these biological polyanions, furthering the development of novel therapeutics.

Design of Safe Nanotherapeutics for the Excretion of Excess Systemic Toxic Iron.

Chronic transfusion of red blood cells (RBCs) to patients with ß-thalassemia, sickle cell disease, and other acquired anemic disorders generates significant amounts of bioactive iron deposits in the body. The inactivation and excretion of redox active iron(III) from the blood pool and organs are critical to prevent organ damage, and are the focus of iron chelation therapy (ICT) using low molecular weight Fe(III) specific chelators. However, the current ICT is suboptimal because of the short circulation time of chelators, toxicity, severe side effects, difficult regime of administration, and patient noncompliance. To address this issue, we have designed long circulating and biodegradable nanoconjugates with enhanced circulation time and well-defined biodegradability to improve iron excretion and avoid nonspecific organ accumulation. A series of iron chelating nanoconjugates were generated with deferoxamine (DFO) as the iron(III) specific chelator using polymer scaffolds containing structurally different acidic pH sensitive ketal groups. The type of degradation linkages used in the polymer scaffold significantly influenced the vascular residence time, biodistribution, and mode of excretion of chelators in mice. Remarkably, the conjugate, BGD-60 (140 kDa; R h, 10.6 nm; cyclic ketal), exhibited the long circulation half-life (t 1/2ß, 64 h), a 768-fold increase compared to DFO, and showed minimal polymer accumulation in major organs. The nanoconjugates were found to be nontoxic and excreted iron significantly better than DFO in iron overloaded mice. BGD-60 showed greater iron mobilization from plasma (p = 0.0390), spleen (p < 0.0001), and pancreas (p < 0.0001) whereas BDD-200 (340 kDa; R h, 13.7 nm; linear ketal) mobilized iron significantly better from the spleen, liver, and pancreas (p < 0.0001, p < 0.0001, and p < 0.0001, respectively) compared to DFO at equivalent doses. The nanoconjugate's favorable long blood circulation time, biodegradability, and iron excretion profiles highlight their potential for future clinical translation.