External Trigger Free Charge Switchable Cationic Ligands in the Design of Safe and Effective Universal Heparin Antidote.

Thrombosis, the formation of blood clots within a blood vessel, can lead to severe complications including pulmonary embolism, cardiac arrest, and stroke. The most widely administered class of anticoagulants is heparin-based anticoagulants such as unfractionated heparin, low-molecular weight heparins (LMWHs), and fondaparinux. Protamine is the only FDA-approved heparin antidote. Protamine has limited efficacy neutralizing LMWHs and no reversal activity against fondaparinux. The use of protamine can lead to complications, including excessive bleeding, hypotension, and hypersensitivity, and has narrow therapeutic window. In this work, a new concept in the design of a universal heparin antidote: switchable protonation of cationic ligands, is presented. A library of macromolecular polyanion inhibitors (MPIs) is synthesized and screened to identify molecules that can neutralize all heparins with high selectivity and reduced toxicity. MPIs are developed by assembling cationic binding groups possessing switchable protonation states onto a polymer scaffold. By strategically selecting the identity and modulating the density of cationic binding groups on the polymer scaffold, a superior universal heparin reversal agent is developed with improved heparin-binding activity and increased hemocompatibility profiles leading to minimal effect on hemostasis. The activity of this heparin antidote is demonstrated using in vitro and in vivo studies.

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.

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.

Rheological and clot microstructure evaluation of heparin neutralization by UHRA and protamine.

The current study reports the use of small amplitude oscillatory rheometry to investigate the dynamics of blood clot formation upon heparin neutralization under three different oscillatory frequencies, two of which were mimicking physiological heart rates. We utilized two different heparin antidotes, namely protamine and newly developed universal heparin reversal agent (UHRA-7), at different concentrations to determine the quality of blood clot formed upon heparin neutralization by analyzing several key rheological parameters. Scanning electron microscopy (SEM) was used to determine the morphology and microstructure of the blood clot after heparin neutralization to support the rheological observations. The current study revealed that the structure of blood clots formed had significant differences when an oscillatory frequency that mimicked the physiological heart rate was used in comparison to a lower frequency commonly used in current clinical measurements. The limited working dose range for protamine and its intrinsic anticoagulation behaviour was observed. The neutralization profile of UHRA-7 showed a large window of activity. The global assessment of rheological parameters and microstructure of the clot together revealed additional details describing anticoagulant reversal and blood coagulation dynamics by relating the blood clot's fiber thickness and the oscillatory measurements, including storage modulus and blood clot's contractile force. Additionally, a mechanical characterization was conducted to provide a further assessment of blood coagulation using the rheological data.

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.

Design of Polyphosphate Inhibitors: A Molecular Dynamics Investigation on Polyethylene Glycol-Linked Cationic Binding Groups.

Inorganic polyphosphate (polyP) released by human platelets has recently been shown to activate blood clotting and identified as a potential target for the development of novel antithrombotics. Recent studies have shown that polymers with cationic binding groups (CBGs) inhibit polyP and attenuate thrombosis. However, a good molecular-level understanding of the binding mechanism is lacking for further drug development. While molecular dynamics (MD) simulation can provide molecule-level information, the time scale required to simulate these large biomacromolecules makes classical MD simulation impractical. To overcome this challenge, we employed metadynamics simulations with both all-atom and coarse-grained force fields. The force field parameters for polyethylene glycol (PEG) conjugated CBGs and polyP were developed to carry out coarse-grained MD simulations, which enabled simulations of these large biomacromolecules in a reasonable time scale. We found that the length of the PEG tail does not impact the interaction between the (PEG) n-CBG and polyP. As expected, increasing the number of the charged tertiary amine groups in the head group strengthens its binding to polyP. Our simulation shows that (PEG) n-CBG initially form aggregates, mostly with the PEG in the core and the hydrophilic CBG groups pointing toward water; then the aggregates approach the polyP and sandwich the polyP to form a complex. We found that the binding of (PEG) n-CBG remains intact against various lengths of polyP. Binding thermodynamics for two of the (PEG) n-CBG/polyP systems simulated were measured by isothermal titration calorimetry to confirm the key finding of the simulations that the length PEG tail does not influence ligand binding to polyP.

Approaches to prevent bleeding associated with anticoagulants: current status and recent developments.

Anticoagulants are widely used for the prophylaxis and treatment of cardiovascular disorders and to prevent blood clotting during surgeries. However, the major limitation associated with anticoagulant therapy is bleeding; all the current anticoagulants do have a bleeding risk. The propensity to bleed is much higher among the elderly population and patients with renal insufficiency. Therefore, there is an utmost and urgent clinical need for a highly efficient, nontoxic antidote with excellent anticoagulant reversal activity. This will significantly improve the safety of anticoagulation therapy. This review summarizes the current options and approaches to reverse anticoagulation activity of clinically used anticoagulants. We start with an introduction to thrombosis and then summarize the details of current clinically available anticoagulants and their mechanisms of action and limitations. This is followed by current practices in anticoagulant neutralization including the details of the only clinically approved unfractionated heparin antidote, protamine; recent advances in the development of antidotes against heparin-based drugs; and direct oral anticoagulants (DOACs).

Antimicrobial Peptide-Polymer Conjugates with High Activity: Influence of Polymer Molecular Weight and Peptide Sequence on Antimicrobial Activity, Proteolysis, and Biocompatibility.

We report the synthesis, characterization, activity, and biocompatibility of a novel series of antimicrobial peptide-polymer conjugates. Using parent peptide aurein 2.2, we designed a peptide array (∼100 peptides) with single and multiple W and R mutations and identified antimicrobial peptides (AMPs) with potent activity against Staphylococcus aureus (S. aureus). These novel AMPs were conjugated to hyperbranched polyglycerols (HPGs) of different molecular weights and number of peptides to improve their antimicrobial activity and toxicity. The cell and blood compatibility studies of these conjugates demonstrated better properties than those of the AMP alone. However, conjugates showed lower antimicrobial activity in comparison to that of peptides, as determined from minimal inhibition concentrations (MICs) against S. aureus, but considerably better than that of the available polymer-AMP conjugates in the literature. In addition to measuring MICs and characterizing the biocompatibility, circular dichroism spectroscopy was used to investigate the interaction of the novel conjugates with model bacterial biomembranes. Moreover, the novel conjugates were exposed to trypsin to evaluate their stability. It was found that the conjugates resist proteolysis in comparison with unprotected peptides. The peptide conjugates were active in serum and whole blood. Overall, the results show that combining a highly active AMP and low-molecular-weight HPG yields bioconjugates with excellent biocompatibility, MICs below 100 µg/mL, and proteolytic stability, which could potentially improve its utility for in vivo applications.

A Polymer Therapeutic Having Universal Heparin Reversal Activity: Molecular Design and Functional Mechanism.

Heparins are widely used to prevent blood clotting during surgeries and for the treatment of thrombosis. However, bleeding associated with heparin therapy is a concern. Protamine, the only approved antidote for unfractionated heparin (UFH) could cause adverse cardiovascular events. Here, we describe a unique molecular design used in the development of a synthetic dendritic polycation named as universal heparin reversal agent (UHRA), an antidote for all clinically used heparin anticoagulants. We elucidate the mechanistic basis for the selectivity of UHRA to heparins and its nontoxic nature. Isothermal titration calorimetry based binding studies of UHRAs having different methoxypolyethylene glycol (mPEG) brush structures with UFH as a function of solution conditions, including ionic strength, revealed that mPEG chains impose entropic penalty to the electrostatic binding. Binding studies confirm that, unlike protamine or N-UHRA (a truncated analogue of UHRA with no mPEG chains), the mPEG chains in UHRA avert nonspecific interactions with blood proteins and provide selectivity toward heparins through a combined steric repulsion and Donnan shielding effect (a balance of Fel and Fsteric). Clotting assays reveal that UHRA with mPEG chains did not adversely affect clotting, and neutralized UFH over a wide range of concentrations. Conversely, N-UHRA and protamine display intrinsic anticoagulant activity and showed a narrow concentration window for UFH neutralization. In addition, we found that mPEG chains regulate the size of antidote-UFH complexes, as revealed by atomic force microscopy and dynamic light scattering studies. UHRA molecules with mPEG chains formed smaller complexes with UFH, compared to N-UHRA and protamine. Finally, fluorescence and ELISA experiments show that UHRA disrupts antithrombin-UFH complexes to neutralize heparin's activity.

Alteration of blood clotting and lung damage by protamine are avoided using the heparin and polyphosphate inhibitor UHRA.

Anticoagulant therapy-associated bleeding and pathological thrombosis pose serious risks to hospitalized patients. Both complications could be mitigated by developing new therapeutics that safely neutralize anticoagulant activity and inhibit activators of the intrinsic blood clotting pathway, such as polyphosphate (polyP) and extracellular nucleic acids. The latter strategy could reduce the use of anticoagulants, potentially decreasing bleeding events. However, previously described cationic inhibitors of polyP and extracellular nucleic acids exhibit both nonspecific binding and adverse effects on blood clotting that limit their use. Indeed, the polycation used to counteract heparin-associated bleeding in surgical settings, protamine, exhibits adverse effects. To address these clinical shortcomings, we developed a synthetic polycation, Universal Heparin Reversal Agent (UHRA), which is nontoxic and can neutralize the anticoagulant activity of heparins and the prothrombotic activity of polyP. Sharply contrasting protamine, we show that UHRA does not interact with fibrinogen, affect fibrin polymerization during clot formation, or abrogate plasma clotting. Using scanning electron microscopy, confocal microscopy, and clot lysis assays, we confirm that UHRA does not incorporate into clots, and that clots are stable with normal fibrin morphology. Conversely, protamine binds to the fibrin clot, which could explain how protamine instigates clot lysis and increases bleeding after surgery. Finally, studies in mice reveal that UHRA reverses heparin anticoagulant activity without the lung injury seen with protamine. The data presented here illustrate that UHRA could be safely used as an antidote during adverse therapeutic modulation of hemostasis.

In vivo efficacy, toxicity and biodistribution of ultra-long circulating desferrioxamine based polymeric iron chelator.

Desferrioxamine (DFO) is currently in clinical use to remove iron from transfusion-dependent patients with ß-thalassemia major, sickle-cell anemia and the myelodysplastic syndromes. However, its short half-life, burdensome, subcutaneous mode of administration and propensity to cause neurotoxicity at high doses greatly hinder its use. Thus, developing an optimized version of DFO with extended half-life, and reduced toxicity is a major goal. Using high molecular weight (MW), non-toxic, hyperbranched polyglycerol with high functionality, we demonstrate that the efficacy of DFO can be tuned with considerable reduction in toxicity. Using zebrafish embryos and mice, we tested toxicity, iron removal efficacy with low dosing and the biodistribution of ultra-long circulating DFO (ULC-DFO) conjugates. There was no significant difference in the mortality and development of zebrafish embryos upon exposure to ULC-DFO. Similarly, body weights and serum lactate dehydrogenase levels in mice treated with ULC-DFO remained within the normal range throughout the tolerance study. Moreover, ULC-DFO is significantly more effective than low MW DFO in promoting iron removal both from organs and via urine in iron overloaded mice despite using a moderate, once-weekly dosing schedule. This is probably due to the extended circulation half-life of ULC-DFO. The MW of ULC-DFO influences the accumulation and biodistribution, with highest MW (637 KDa) associated with up to 12% accumulation in the liver. In contrast, ULC-DFO with MWs of 75 KDa and lower were associated with relatively low organ accumulation, indicating that biodistribution of ULC-DFO can be tuned. Since ULC-DFO has improved iron removal properties, longer plasma retention time and possesses excellent biocompatibility, it represents a polymer conjugate with high clinical utility in comparison to DFO for the treatment of transfusion dependent iron overload. More importantly, ULC-DFO is anticipated to reduce the requirement for prolonged subcutaneous infusion of DFO.

Toward Infection-Resistant Surfaces: Achieving High Antimicrobial Peptide Potency by Modulating the Functionality of Polymer Brush and Peptide.

Bacterial infection associated with indwelling medical devices and implants is a major clinical issue, and the prevention or treatment of such infections is challenging. Antimicrobial coatings offer a significant step toward addressing this important clinical problem. Antimicrobial coatings based on tethered antimicrobial peptides (AMPs) on hydrophilic polymer brushes have been shown to be one of the most promising strategies to avoid bacterial colonization and have demonstrated broad spectrum activity. Optimal combinations of the functionality of the polymer-brush-tethered AMPs are essential to maintaining long-term AMP activity on the surface. However, there is limited knowledge currently available on this topic. Here we report the development of potent antimicrobial coatings on implant surfaces by elucidating the roles of polymer brush chemistry and peptide structure on the overall antimicrobial activity of the coatings. We screened several combinations of polymer brush coatings and AMPs constructed on nanoparticles, titanium surfaces, and quartz slides on their antimicrobial activity and bacterial adhesion against Gram-positive and Gram-negative bacteria. Highly efficient killing of planktonic bacteria by the antimicrobial coatings on nanoparticle surfaces, as well as potent killing of adhered bacteria in the case of coatings on titanium surfaces, was observed. Remarkably, the antimicrobial activity of AMP-conjugated brush coatings demonstrated a clear dependence on the polymer brush chemistry and peptide structure, and optimization of these parameters is critical to achieving infection-resistant surfaces. By analyzing the interaction of polymer-brush-tethered AMPs with model lipid membranes using circular dichroism spectroscopy, we determined that the polymer brush chemistry has an influence on the extent of secondary structure change of tethered peptides before and after interaction with biomembranes. The peptide structure also has an influence on the density of conjugated peptides on polymer brush coatings and the resultant wettability of the coatings, and both of these factors contributed to the antimicrobial activity and bacterial adhesion of the coatings. Overall, this work highlights the importance of optimizing the functionality of the polymer brush to achieve infection-resistant surfaces and presents important insight into the design criteria for the selection of polymers and AMPs toward the development of potent antimicrobial coating on implants.

Affinity-based design of a synthetic universal reversal agent for heparin anticoagulants.

Heparin-based anticoagulant drugs have been widely used for the prevention of blood clotting during surgical procedures and for the treatment of thromboembolic events. However, bleeding risks associated with these anticoagulants demand continuous monitoring and neutralization with suitable antidotes. Protamine, the only clinically approved antidote to heparin, has shown adverse effects and ineffectiveness against low-molecular weight heparins and fondaparinux, a heparin-related medication. Alternative approaches based on cationic molecules and recombinant proteins have several drawbacks including limited efficacy, toxicity, immunogenicity, and high cost. Thus, there is an unmet clinical need for safer, rapid, predictable, and cost-effective anticoagulant-reversal agents for all clinically used heparins. We report a design strategy for a fully synthetic dendritic polymer-based universal heparin reversal agent (UHRA) that makes use of multivalent presentation of branched cationic heparin binding groups (HBGs). Optimization of the UHRA design was aided by isothermal titration calorimetry studies, biocompatibility evaluation, and heparin neutralization analysis. By controlling the scaffold's molecular weight, the nature of the protective shell, and the presentation of HBGs on the polymer scaffold, we arrived at lead UHRA molecules that completely neutralized the activity of all clinically used heparins. The optimized UHRA molecules demonstrated superior efficacy and safety profiles and mitigated heparin-induced bleeding in animal models. This new polymer therapeutic may benefit patients undergoing high-risk surgical procedures and has potential for the treatment of anticoagulant-related bleeding problems.

Nontoxic polyphosphate inhibitors reduce thrombosis while sparing hemostasis.

Polyphosphate (polyP) is secreted by activated platelets and has been shown to contribute to thrombosis, suggesting that it could be a novel antithrombotic target. Previously reported polyP inhibitors based on polycationic substances, such as polyethylenimine, polyamidoamine dendrimers, and polymyxin B, although they attenuate thrombosis, all have significant toxicity in vivo, likely due to the presence of multiple primary amines responsible for their polyP binding ability. In this study, we examined a novel class of nontoxic polycationic compounds initially designed as universal heparin reversal agents (UHRAs) to determine their ability to block polyP procoagulant activity and also to determine their utility as antithrombotic treatments. Several UHRA compounds strongly inhibited polyP procoagulant activity in vitro, and 4 were selected for further examination in mouse models of thrombosis and hemostasis. Compounds UHRA-9 and UHRA-10 significantly reduced arterial thrombosis in mice. In mouse tail bleeding tests, administration of UHRA-9 or UHRA-10 was associated with significantly less bleeding compared with therapeutically equivalent doses of heparin. Thus, these compounds offer a new platform for developing novel antithrombotic agents that target procoagulant anionic polymers such as polyP with reduced toxicity and bleeding side effects.