Biomaterial and cellular implants: foreign surfaces where immunity and coagulation meet.

Exposure of blood to a foreign surface in the form of a diagnostic or therapeutic biomaterial device or implanted cells or tissue elicits an immediate, evolutionarily conserved thromboinflammatory response from the host. Primarily designed to protect against invading organisms after an injury, this innate response features instantaneous activation of several blood-borne, highly interactive, well-orchestrated cascades and cellular events that limit bleeding, destroy and eliminate the foreign substance or cells, and promote healing and a return to homeostasis via delicately balanced regenerative processes. In the setting of blood-contacting synthetic or natural biomaterials and implantation of foreign cells or tissues, innate responses are robust, albeit highly context specific. Unfortunately, they tend to be less than adequately regulated by the host's natural anticoagulant or anti-inflammatory pathways, thereby jeopardizing the functional integrity of the device, as well as the health of the host. Strategies to achieve biocompatibility with a sustained return to homeostasis, particularly while the device remains in situ and functional, continue to elude scientists and clinicians. In this review, some of the complex mechanisms by which biomaterials and cellular transplants provide a "hub" for activation and amplification of coagulation and immunity, thromboinflammation, are discussed, with a view toward the development of innovative means of overcoming the innate challenges.

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.

An improved in vitro model for studying the structural and functional properties of the endothelial glycocalyx in arteries, capillaries and veins.

The endothelial glycocalyx is a dynamic structure integral to blood vessel hemodynamics and capable of tightly regulating a range of biological processes (ie, innate immunity, inflammation, and coagulation) through dynamic changes in its composition of the brush structure. Evaluating the specific roles of the endothelial glycocalyx under a range of pathophysiologic conditions has been a challenge in vitro as it is difficult to generate functional glycocalyces using commonly employed 2D cell culture models. We present a new multi-height microfluidic platform that promotes the growth of functional glycocalyces by eliciting unique shear stress forces over a continuous human umbilical vein endothelial cell monolayer at magnitudes that recapitulate the physical environment in arterial, capillary and venous regions of the vasculature. Following 72 hours of shear stress, unique glycocalyx structures formed within each region that were distinct from that observed in short (3 days) and long-term (21 days) static cell culture. The model demonstrated glycocalyx-specific properties that match the characteristics of the endothelium in arteries, capillaries and veins, with respect to surface protein expression, platelet adhesion, lymphocyte binding and nanoparticle uptake. With artery-to-capillary-to-vein transition on a continuous endothelial monolayer, this in vitro platform is an improved system over static cell culture for more effectively studying the role of the glycocalyx in endothelial biology and disease.

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.

The Mouse Heart Mitochondria N Terminome Provides Insights into ClpXP-Mediated Proteolysis.

The mammalian mitochondrial proteome consists of more than 1100 annotated proteins and their proteostasis is regulated by only a few ATP-dependent protease complexes. Technical advances in protein mass spectrometry allowed for detailed description of the mitoproteome from different species and tissues and their changes under specific conditions. However, protease-substrate relations within mitochondria are still poorly understood. Here, we combined Terminal Amine Isotope Labeling of Substrates (TAILS) N termini profiling of heart mitochondria proteomes isolated from wild type and Clpp-/- mice with a classical substrate-trapping screen using FLAG-tagged proteolytically active and inactive CLPP variants to identify new ClpXP substrates in mammalian mitochondria. Using TAILS, we identified N termini of more than 200 mitochondrial proteins. Expected N termini confirmed sequence determinants for mitochondrial targeting signal (MTS) cleavage and subsequent N-terminal processing after import, but the majority were protease-generated neo-N termini mapping to positions within the proteins. Quantitative comparison revealed widespread changes in protein processing patterns, including both strong increases or decreases in the abundance of specific neo-N termini, as well as an overall increase in the abundance of protease-generated neo-N termini in CLPP-deficient mitochondria that indicated altered mitochondrial proteostasis. Based on the combination of altered processing patterns, protein accumulation and stabilization in CLPP-deficient mice and interaction with CLPP, we identified OAT, HSPA9 and POLDIP2 and as novel bona fide ClpXP substrates. Finally, we propose that ClpXP participates in the cooperative degradation of UQCRC1. Together, our data provide the first landscape of the heart mitochondria N terminome and give further insights into regulatory and assisted proteolysis mediated by ClpXP.

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.

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.

Mucin-Inspired, High Molecular Weight Virus Binding Inhibitors Show Biphasic Binding Behavior to Influenza A Viruses.

Multivalent binding inhibitors are a promising new class of antivirals that prevent virus infections by inhibiting virus binding to cell membranes. The design of these inhibitors is challenging as many properties, for example, inhibitor size and functionalization with virus attachment factors, strongly influence the inhibition efficiency. Here, virus binding inhibitors are synthesized, the size and functionalization of which are inspired by mucins, which are naturally occurring glycosylated proteins with high molecular weight (MDa range) and interact efficiently with various viruses. Hyperbranched polyglycerols (hPGs) with molecular weights ranging between 10 and 2600 kDa are synthesized, thereby hitting the size of mucins and allowing for determining the impact of inhibitor size on the inhibition efficiency. The hPGs are functionalized with sialic acids and sulfates, as suggested from the structure of mucins, and their inhibition efficiency is determined by probing the inhibition of influenza A virus (IAV) binding to membranes using various methods. The largest, mucin-sized inhibitor shows potent inhibition at pm concentrations, while the inhibition efficiency decreases with decreasing the molecular weight. Interestingly, the concentration-dependent IAV inhibition shows a biphasic behavior, which is attributed to differences in the binding affinity of the inhibitors to the two IAV envelope proteins, neuraminidase, and hemagglutinin.

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.

Towards Robust Delivery of Antimicrobial Peptides to Combat Bacterial Resistance.

Antimicrobial peptides (AMPs), otherwise known as host defence peptides (HDPs), are naturally occurring biomolecules expressed by a large array of species across the phylogenetic kingdoms. They have great potential to combat microbial infections by directly killing or inhibiting bacterial activity and/or by modulating the immune response of the host. Due to their multimodal properties, broad spectrum activity, and minimal resistance generation, these peptides have emerged as a promising response to the rapidly concerning problem of multidrug resistance (MDR). However, their therapeutic efficacy is limited by a number of factors, including rapid degradation, systemic toxicity, and low bioavailability. As such, many strategies have been developed to mitigate these limitations, such as peptide modification and delivery vehicle conjugation/encapsulation. Oftentimes, however, particularly in the case of the latter, this can hinder the activity of the parent AMP. Here, we review current delivery strategies used for AMP formulation, focusing on methodologies utilized for targeted infection site release of AMPs. This specificity unites the improved biocompatibility of the delivery vehicle with the unhindered activity of the free AMP, providing a promising means to effectively translate AMP therapy into clinical practice.

Proteomic and N-Terminomic TAILS Analyses of Human Alveolar Bone Proteins: Improved Protein Extraction Methodology and LysargiNase Digestion Strategies Increase Proteome Coverage and Missing Protein Identification.

With 2129 proteins still classified by the Human Proteome Organisation Human Proteome Project (HPP) as "missing" without compelling evidence of protein existence (PE) in humans, we hypothesized that in-depth proteomic characterization of tissues that are technically challenging to access and extract would yield evidence for tissue-specific missing proteins. Paradoxically, although the skeleton is the most massive tissue system in humans, as one of the poorest characterized by proteomics, bone falls under the HPP umbrella term as a "rare tissue". Therefore, we aimed to optimize mineralized tissue protein extraction methodology and workflows for proteomic and data analyses of small quantities of healthy young adult human alveolar bone. Osteoid was solubilized by GuHCl extraction, with hydroxyapatite-bound proteins then released by ethylenediaminetetraacetic acid demineralization. A subsequent GuHCl solubilization extraction was followed by solid-phase digestion of the remaining insoluble cross-linked protein using trypsin and then 6 M urea dissolution incorporating LysC digestion. Bone extracts were digested in parallel using trypsin, LysargiNase, AspN, or GluC prior to liquid chromatography-mass spectrometry analysis. Terminal Amine Isotopic Labeling of Substrates was used to purify semitryptic peptides, identifying natural and proteolytic-cleaved neo N-termini of bone proteins. Our strategy enabled complete solubilization of the organic bone matrix leading to extensive categorization of bone proteins in different bone matrix extracts, and hence matrix compartments, for the first time. Moreover, this led to the high confidence identification of pannexin-3, a "missing protein", found only in the insoluble collagenous matrix and revealed for the first time by trypsin solid-phase digestion. We also found a singleton proteotypic peptide of another missing protein, meiosis inhibitor protein 1. We also identified 17 proteins classified in neXtprot as PE1 based on evidence other than from MS, termed non-MS PE1 proteins, including ≥9-mer proteotypic peptides of four proteins.

The proteome microenvironment determines the protective effect of preconditioning in cisplatin-induced acute kidney injury.

Acute kidney injury (AKI) leads to significant morbidity and mortality; unfortunately, strategies to prevent or treat AKI are lacking. In recent years, several preconditioning protocols have been shown to be effective in inducing organ protection in rodent models. Here, we characterized two of these interventions-caloric restriction and hypoxic preconditioning-in a mouse model of cisplatin-induced AKI and investigated the underlying mechanisms by acquisition of multi-layered omic data (transcriptome, proteome, N-degradome) and functional parameters in the same animals. Both preconditioning protocols markedly ameliorated cisplatin-induced loss of kidney function, and caloric restriction also induced lipid synthesis. Bioinformatic analysis revealed mRNA-independent proteome alterations affecting the extracellular space, mitochondria, and transporters. Interestingly, our analyses revealed a strong dissociation of protein and RNA expression after cisplatin treatment that showed a strong correlation with the degree of damage. N-degradomic analysis revealed that most posttranscriptional changes were determined by arginine-specific proteolytic processing. This included a characteristic cisplatin-activated complement signature that was prevented by preconditioning. Amyloid and acute-phase proteins within the cortical parenchyma showed a similar response. Extensive analysis of disease-associated molecular patterns suggested that transcription-independent deposition of amyloid P-component serum protein may be a key component in the microenvironmental contribution to kidney damage. This proof-of-principle study provides new insights into the pathogenesis of cisplatin-induced AKI and the molecular mechanisms underlying organ protection by correlating phenotypic and multi-layered omics data.

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.

Transcriptome analysis of signaling pathways of human peritoneal mesothelial cells in response to different osmotic agents in a peritoneal dialysis solution.

BACKGROUND: Glucose is a primary osmotic agent in peritoneal dialysis (PD) solutions, but its long-term use causes structural alteration of the peritoneal membrane (PM). Hyperbranched polyglycerol (HPG) is a promising alternative to glucose. This study was designed to compare the cellular responses of human peritoneal mesothelial cells (HPMCs) to these two different osmotic agents in a hypertonic solution using transcriptome analysis. METHODS: Cultured HPMCs were repeatedly exposed to HPG-based or Physioneal 40 (PYS, glucose 2.27%) hypertonic solutions. Transcriptome datasets were produced using Agilent SurePrint G3 Human GE 8 × 60 microarray. Cellular signaling pathways were examined by Ingenuity Pathway Analysis (IPA). Protein expression was examined by flow cytometry analysis and Western blotting. RESULTS: The HPG-containing solution was better tolerated compared with PYS, with less cell death and disruption of cell transcriptome. The levels of cell death in HPG- or PYS- exposed cells were positively correlated with the number of affected transcripts (HPG: 128 at day 3, 0 at day 7; PYS: 1799 at day 3, 212 at day 7). In addition to more affected "biosynthesis" and "cellular stress and death" pathways by PYS, both HPG and PYS commonly affected "sulfate biosynthesis", "unfolded protein response", "apoptosis signaling" and "NRF2-mediated oxidative stress response" pathways at day 3. PYS significantly up-regulated HLA-DMB and MMP12 in a time-dependent manner, and stimulated T cell adhesion to HPMCs. CONCLUSION: The lower cytotoxicity of hypertonic HPG solution is in agreement with its transient and minimal impact on the pathways for the "biosynthesis of cell constituents" and the "cellular stress and death". The significant up-regulation of HLA-DMB and MMP12 by PYS may be part of its initiation of immune response in the PM.

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.

Formalin-Fixed, Paraffin-Embedded Tissues (FFPE) as a Robust Source for the Profiling of Native and Protease-Generated Protein Amino Termini.

Dysregulated proteolysis represents a hallmark of numerous diseases. In recent years, increasing number of studies has begun looking at the protein termini in hope to unveil the physiological and pathological functions of proteases in clinical research. However, the availability of cryopreserved tissue specimens is often limited. Alternatively, formalin-fixed, paraffin-embedded (FFPE) tissues offer an invaluable resource for clinical research. Pathologically relevant tissues are often stored as FFPE, which represent the most abundant resource of archived human specimens. In this study, we established a robust workflow to investigate native and protease-generated protein N termini from FFPE specimens. We demonstrate comparable N-terminomes of cryopreserved and formalin-fixed tissue, thereby showing that formalin fixation/paraffin embedment does not proteolytically damage proteins. Accordingly, FFPE specimens are fully amenable to N-terminal analysis. Moreover, we demonstrate feasibility of FFPE-degradomics in a quantitative N-terminomic study of FFPE liver specimens from cathepsin L deficient or wild-type mice. Using a machine learning approach in combination with the previously determined cathepsin L specificity, we successfully identify a number of potential cathepsin L cleavage sites. Our study establishes FFPE specimens as a valuable alternative to cryopreserved tissues for degradomic studies.

N-Degradomic Analysis Reveals a Proteolytic Network Processing the Podocyte Cytoskeleton.

Regulated intracellular proteostasis, controlled in part by proteolysis, is essential in maintaining the integrity of podocytes and the glomerular filtration barrier of the kidney. We applied a novel proteomics technology that enables proteome-wide identification, mapping, and quantification of protein N-termini to comprehensively characterize cleaved podocyte proteins in the glomerulus in vivo We found evidence that defined proteolytic cleavage results in various proteoforms of important podocyte proteins, including those of podocin, nephrin, neph1, α-actinin-4, and vimentin. Quantitative mapping of N-termini demonstrated perturbation of protease action during podocyte injury in vitro, including diminished proteolysis of α-actinin-4. Differentially regulated protease substrates comprised cytoskeletal proteins as well as intermediate filaments. Determination of preferential protease motifs during podocyte damage indicated activation of caspase proteases and inhibition of arginine-specific proteases. Several proteolytic processes were clearly site-specific, were conserved across species, and could be confirmed by differential migration behavior of protein fragments in gel electrophoresis. Some of the proteolytic changes discovered in vitro also occurred in two in vivo models of podocyte damage (WT1 heterozygous knockout mice and puromycin aminonucleoside-treated rats). Thus, we provide direct and systems-level evidence that the slit diaphragm and podocyte cytoskeleton are regulated targets of proteolytic modification, which is altered upon podocyte damage.

Hemocompatibility of Degrading Polymeric Biomaterials: Degradable Polar Hydrophobic Ionic Polyurethane versus Poly(lactic-co-glycolic) Acid.

The use of degradable polymers in vascular tissue regeneration has sparked the need to characterize polymer biocompatibility during degradation. While tissue compatibility has been frequently addressed, studies on polymer hemocompatibility during degradation are limited. The current study evaluated the differences in hemocompatibility (platelet response, complement activation, and coagulation cascade initiation) between as-made and hydrolyzed poly(lactic-co-glycolic) acid (PLGA) and degradable polar hydrophobic ionic polyurethane (D-PHI). Platelet activation decreased (in whole blood) and platelet adhesion decreased (in blood without leukocytes) for degraded versus as-made PLGA. D-PHI showed minimal hemocompatibility changes over degradation. Leukocytes played a major role in mediating platelet activation for samples and controls, as well as influencing platelet-polymer adhesion on the degraded materials. This study demonstrates the importance of assessing the blood compatibility of biomaterials over the course of degradation since the associated changes in surface chemistry and physical state could significantly change biomaterial hemocompatibility.

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.

Nontransformed and Cancer Cells Can Utilize Different Endocytic Pathways To Internalize Dendritic Nanoparticle Variants: Implications on Nanocarrier Design.

Three hyperbranched polyglycerol nanoparticle (HPG NP) variants were synthesized and fluorescently labeled for the study of their cellular interactions. The polymeric nanoparticle that contains a hydrophobic core and a hydrophilic HPG shell, HPG-C10-HPG, is taken up faster by HT-29 cancer cells than nontransformed cells, while similar uptake rates are observed with both cell types for the nanoparticle HPG-C10-PEG that contains a hydrophobic core and a polyethylene glycol shell. The nanoparticle HPG-104, containing neither the hydrophobic core nor the polyethylene glycol shell, is taken up faster by nontransformed cells than HT-29 cells. Importantly, cancer and normal cells can utilize different endocytic mechanisms for the internalization of these HPG NPs. Both HPG-C10-HPG and HPG-C10-PEG are taken up by HT-29 cells through clathrin-mediated endocytosis and macropinocytosis. Nontransformed cells, however, take up HPG-C10-HPG and HPG-104 through macropinocytosis, while these cells utilize both clathrin-mediated endocytosis and macropinocytosis to internalize HPG-C10-PEG.