Biocompatibility assessment of silk nanoparticles: hemocompatibility and internalization by human blood cells.

Many nanoparticles are designed for use as potential nanomedicines for parenteral administration. However, emerging evidence suggests that hemocompatibility is important, but is highly particle- and test-bed dependent. Thus, knowledge of bulk material properties does not predict the hemocompatibility of uncharacterized nanoparticles, including silk nanoparticles. This study compares the hemocompatibility of silk versus silica nanoparticles, using whole human blood under quasi-static and flow conditions. Substantial hemocompatibility differences are noted for some nanoparticles in quasi-static versus dynamic studies; i.e., the inflammatory response to silk nanoparticles is significantly lower under flow versus quasi-static conditions. Silk nanoparticles also have very low coagulant properties - an observation that scales from the macro- to the nano-level. These nanoparticle hemocompatibility studies are complemented by preliminary live cell measurements to evaluate the endocytosis and trafficking of nanoparticles in human blood cells. Overall, this study demonstrates that nanoparticle hemocompatibility is affected by several factors, including the test bed design.

Adsorbed polymer conjugates to adaptively inhibit blood coagulation activation by medical membranes.

Adaptive drug release can combat coagulation and inflammation activation at the blood-material interface with minimized side effects. For that purpose, poly(styrene-alt-maleic-anhydride) copolymers were conjugated to heparin via coagulation-responsive linker peptides and shown to tightly adsorb onto poly(ethersulfone) (PES)-surfaces from aqueous solutions as monolayers. Coagulation-responsive release of unfractionated as well as low molecular weight heparins from the respective coatings was demonstrated to be functionally beneficial in human plasma and whole blood incubation with faster release kinetics resulting in stronger anticoagulant effects. Coated poly(ethersulfone)/poly(vinylpyrrolidone) (PES/PVP) flat membranes proved the technology to offer an easy, effective and robust anticoagulant interfacial functionalization of hemodialysis membranes. In perspective, the modularity of the adaptive release system will be used for inhibiting multiple activation processes.

Advanced in vitro hemocompatibility assessment of biomaterials using a new flow incubation system.

Physiologically relevant in vitro hemocompatibility assessment of biomaterials remains challenging. We present a new setup that enables standardized whole blood incubation of biomedical materials under flow. A blood volume of 2 mL is recirculated over test surfaces in a custom-made parallel plate incubation system to determine the activation of hemostasis and inflammation. Controlled physiological shear rates between 125 s-1 and 1250 s-1 and minimized contact to air are combined with a natural-like pumping process. A unique feature of this setup allows tracing adhesion of blood cells to test surfaces microscopically in situ. Validation testing was performed in comparison to previously applied whole blood incubation methodologies. Experiments with the newly developed setup showed that even small obstacles to blood flow activate blood (independent of materials-induced blood activation levels); that adhesion of blood cells to biomaterials equilibrates within 5 to 10 min; that high shear rates (1250 compared to 375 s-1) induce platelet activation; and that hemolysis, platelet factor 4 (PF4) release and platelet loss - but not thrombin formation - depend on shear rate (within the range investigated, 125 to 1250 s-1).

A New In Vitro Blood Flow Model for the Realistic Evaluation of Antimicrobial Surfaces.

Device-associated bloodstream infections can cause serious medical problems and cost-intensive postinfection management, defining a need for more effective antimicrobial coatings. Newly developed coatings often show reduced bacterial colonization and high hemocompatibility in established in vitro tests, but fail in animal studies or clinical trials. The poor predictive power of these models is attributed to inadequate representation of in vivo conditions. Herein, a new single-pass blood flow model, with simultaneous incubation of the test surface with bacteria and freshly-drawn human blood, is presented. The flow model is validated by comparative analysis of a recently developed set of antiadhesive and contact-killing polymer coatings, and the corresponding uncoated polycarbonate surfaces. The results confirm the model's ability to differentiate the antimicrobial activities of the studied surfaces. Blood activation data correlate with bacterial surface coverage: low bacterial adhesion is associated with low inflammation and hemostasis. Shear stress correlates inversely with bacterial colonization, especially on antiadhesive surfaces. The introduced model is concluded to enable the evaluation of novel antimicrobial materials under in vivo-like conditions, capturing interactions between bacteria and biomaterials surfaces in the presence of key components of the ex vivo host response.

Bioresponsive starPEG-heparin hydrogel coatings on vascular stents for enhanced hemocompatibility.

Hydrogel coatings can improve the biocompatibility of medical devices. However, stable surface bonding and homogeneity of hydrogel coatings are often challenging. This study exploits the benefits of biohybrid hydrogels of crosslinked four-armed poly(ethylene glycol) and heparin to enhance the hemocompatibility of cobalt­chromium (CoCr) vascular stents. A bonding layer of dual silane and poly(ethylene-alt-maleic anhydride) (PEMA) treatment was applied to the stent to provide covalent immobilization and hydrophilicity for the homogeneous spreading of the hydrogel. A spray coating technology was used to distribute the aqueous solution of the reactive hydrogel precursors onto the sub-millimeter struts of the stents, where the solution polymerized to a homogeneous hydrogel film. The coating was mechanically stable on the stent after ethanol dehydration, and the stents could be stored in a dry state. The homogeneity and stability of the coating during stent expansion were verified. Quasistatic and dynamic whole blood incubation experiments showed substantial suppression of the pro-coagulant and inflammatory activity of the bare metal by the coating. Translation of the technology to industrial coating devices and future surface modification of stents with anti-inflammatory hydrogels are discussed.

Poly(2-alkyl-2-oxazoline)-Heparin Hydrogels-Expanding the Physicochemical Parameter Space of Biohybrid Materials.

Poly(ethylene glycol) (PEG)-glycosaminoglycan (GAG) hydrogel networks are established as very versatile biomaterials. Herein, the synthetic gel component of the biohybrid materials is systematically varied by combining different poly(2-alkyl-2-oxazolines) (POx) with heparin applying a Michael-type addition crosslinking scheme: POx of gradated hydrophilicity and temperature-responsiveness provides polymer networks of distinctly different stiffness and swelling. Adjusting the mechanical properties and the GAG concentration of the gels to similar values allows for modulating the release of GAG-binding growth factors (VEGF165 and PDGF-BB) by the choice of the POx and its temperature-dependent conformation. Adsorption of fibronectin, growth of fibroblasts, and bacterial adhesion scale with the hydrophobicity of the gel-incorporated POx. In vitro hemocompatibility tests with freshly drawn human whole blood show advantages of POx-based gels compared to the PEG-based reference materials. Biohybrid POx hydrogels can therefore enable biomedical technologies requiring GAG-based materials with customized and switchable physicochemical characteristics.

Epigallocatechin gallate mediated sandwich-like coating for mimicking endothelium with sustained therapeutic nitric oxide generation and heparin release.

In-stent restenosis after stenting is generally characterized by an inflammatory response, excessive proliferation of smooth muscle cells, and delayed healing of the endothelium layer. In this study, inspired by catechol/gallol surface chemistry, a sandwich-like layer-by-layer (LBL) coating was developed using chitosan and heparin as polyelectrolytes, along with the embedding of an epigallocatechin gallate/copper (EGCG/Cu) complex. The embedding of EGCG stabilized the coating by various intermolecular interactions in the LBL coating (e.g., π-π stacking, weak intermolecular crosslinking, and enriched hydrogen bonding) and supported the sustained release of the cargo heparin over 90 days. This design enabled a biomimetic endothelium function in terms of the sustained release of heparin and continuous in situ generation of nitric oxide, driven by the catalytic decomposition of endogenous S-nitrostothiols by copper ions. The result showed enhanced durability of anticoagulation and suppressed inflammatory response. Moreover, the "sandwich-like" coating supported the growth of endothelial cells and up-regulated the protein expression of vascular endothelial growth factor, while effectively suppressing the proliferation and migration of smooth muscle cells (SMCs) via the up-regulation of cyclic guanosine monophosphate. Ex vivo and in vivo experiments demonstrated the effectiveness of the sandwich-like coating in preventing thrombosis formation, suppressing the growth of SMCs, reducing the infiltration and activation of inflammatory cells, and ultimately achieving rapid in situ endothelialization. Hence, the EGCG-assisted sandwich-like coating might be used as a robust and versatile surface modification strategy for implantable cardiovascular devices.

The innate immune response of self-assembling silk fibroin hydrogels.

Silk has a long track record of use in humans, and recent advances in silk fibroin processing have opened up new material formats. However, these new formats and their applications have subsequently created a need to ascertain their biocompatibility. Therefore, the present aim was to quantify the haemocompatibility and inflammatory response of silk fibroin hydrogels. This work demonstrated that self-assembled silk fibroin hydrogels, as one of the most clinically relevant new formats, induced very low blood coagulation and platelet activation but elevated the inflammatory response of human whole blood in vitro. In vivo bioluminescence imaging of neutrophils and macrophages showed an acute, but mild, local inflammatory response which was lower than or similar to that induced by polyethylene glycol, a benchmark material. The time-dependent local immune response in vivo was corroborated by histology, immunofluorescence and murine whole blood analyses. Overall, this study confirms that silk fibroin hydrogels induce a similar immune response to that of PEG hydrogels, while also demonstrating the power of non-invasive bioluminescence imaging for monitoring tissue responses.

Sperm Micromotors for Cargo Delivery through Flowing Blood.

Micromotors are recognized as promising candidates for untethered micromanipulation and targeted cargo delivery in complex biological environments. However, their feasibility in the circulatory system has been limited due to the low thrust force exhibited by many of the reported synthetic micromotors, which is not sufficient to overcome the high flow and complex composition of blood. Here we present a hybrid sperm micromotor that can actively swim against flowing blood (continuous and pulsatile) and perform the function of heparin cargo delivery. In this biohybrid system, the sperm flagellum provides a high propulsion force while the synthetic microstructure serves for magnetic guidance and cargo transport. Moreover, single sperm micromotors can assemble into a train-like carrier after magnetization, allowing the transport of multiple sperm or medical cargoes to the area of interest, serving as potential anticoagulant agents to treat blood clots or other diseases in the circulatory system.

Protein Component of Oyster Glycogen Nanoparticles: An Anchor Point for Functionalization.

Biosourced nanoparticles have a range of desirable properties for therapeutic applications, including biodegradability and low immunogenicity. Glycogen, a natural polysaccharide nanoparticle, has garnered much interest as a component of advanced therapeutic materials. However, functionalizing glycogen for use as a therapeutic material typically involves synthetic approaches that can negatively affect the intrinsic physiological properties of glycogen. Herein, the protein component of glycogen is examined as an anchor point for the photopolymerization of functional poly(N-isopropylacrylamide) (PNIPAM) polymers. Oyster glycogen (OG) nanoparticles partially degrade to smaller spherical particles in the presence of protease enzymes, reflecting a population of surface-bound proteins on the polysaccharide. The grafting of PNIPAM to the native protein component of OG produces OG-PNIPAM nanoparticles of ∼45 nm in diameter and 6.2 MDa in molecular weight. PNIPAM endows the nanoparticles with temperature-responsive aggregation properties that are controllable and reversible and that can be removed by the biodegradation of the protein. The OG-PNIPAM nanoparticles retain the native biodegradability of glycogen. Whole blood incubation assays revealed that the OG-PNIPAM nanoparticles have a low cell association and inflammatory response similar to that of OG. The reported strategy provides functionalized glycogen nanomaterials that retain their inherent biodegradability and low immune cell association.

Protein adsorption dynamics to polymer surfaces revisited-A multisystems approach.

Performance and safety of materials in contact with living matter are determined by sequential and competitive protein adsorption. However, cause and consequences of these processes remain hard to be generalized and predicted. In a new attempt to address that challenge, the authors compared and analyzed the protein adsorption and displacement on various thoroughly characterized polymer substrates using a combination of surface-sensitive techniques. A multiple linear regression approach was applied to model the dependence of protein adsorption, desorption, and exchange dynamics on protein and surface characteristics. While the analysis confirmed that protein properties primarily govern the observed adsorption and retention phenomena and hydrophobicity as well as surface charge are the most relevant polymer surface properties, the authors have identified several protein-surface combinations that deviate from these patterns and deserve further investigation.

Biomimetic engineering endothelium-like coating on cardiovascular stent through heparin and nitric oxide-generating compound synergistic modification strategy.

Co-immobilization of two or more molecules with different and complementary functions to prevent thrombosis, suppress smooth muscle cell (SMC) proliferation, and support endothelial cell (EC) growth is generally considered to be promising for the re-endothelialization on cardiovascular stents. However, integration of molecules with distinct therapeutic effects does not necessarily result in synergistic physiological functions due to the lack of interactions among them, limiting their practical efficacy. Herein, we apply heparin and nitric oxide (NO), two key molecules of the physiological functions of endothelium, to develop an endothelium-mimetic coating. Such coating is achieved by sequential conjugation of heparin and the NO-generating compound selenocystamine (SeCA) on an amine-bearing film of plasma polymerized allylamine. The resulting surface combines the anti-coagulant (anti-FXa) function provided by the heparin and the anti-platelet activity of the catalytically produced NO. It also endows the stents with the ability to simultaneously up-regulate α-smooth muscle actin (α-SMA) expression and to increase cyclic guanylate monophosphate (cGMP) synthesis of SMC, thereby significantly promoting their contractile phenotype and suppressing their proliferation. Importantly, this endothelium-biomimetic coating creates a favorable microenvironment for EC over SMC. These features impressively improve the antithrombogenicity, re-endothelialization and anti-restenosis of vascular stents in vivo.

The blood compatibility challenge. Part 3: Material associated activation of blood cascades and cells.

Following protein adsorption/activation which is the first step after the contact of material surfaces and whole blood (part 2), fibrinogen is converted to fibrin and platelets become activated and assembled in the form of a thrombus. This thrombus formation is the key feature that needs to be minimized in the creation of materials with low thrombogenicity. Further aspects of blood compatibility that are important on their own are complement and leukocyte activation which are also important drivers of thrombus formation. Hence this review summarizes the state of knowledge on all of these cascades and cells and their interactions. For each cascade or cell type, the chapter distinguishes statements which are in widespread agreement from statements where there is less of a consensus. STATEMENT OF SIGNIFICANCE: This paper is part 3 of a series of 4 reviews discussing the problem of biomaterial associated thrombogenicity. The objective was to highlight features of broad agreement and provide commentary on those aspects of the problem that were subject to dispute. We hope that future investigators will update these reviews as new scholarship resolves the uncertainties of today.

The blood compatibility challenge. Part 4: Surface modification for hemocompatible materials: Passive and active approaches to guide blood-material interactions.

Biomedical devices in the blood flow disturb the fine-tuned balance of pro- and anti-coagulant factors in blood and vessel wall. Numerous technologies have been suggested to reduce coagulant and inflammatory responses of the body towards the device material, ranging from camouflage effects to permanent activity and further to a responsive interaction with the host systems. However, not all types of modification are suitable for all types of medical products. This review has a focus on application-oriented considerations of hemocompatible surface fittings. Thus, passive versus bioactive modifications are discussed along with the control of protein adsorption, stability of the immobilization, and the type of bioactive substance, biological or synthetic. Further considerations are related to the target system, whether enzymes or cells should be addressed in arterial or venous system, or whether the blood vessel wall is addressed. Recent developments like feedback controlled or self-renewing systems for drug release or addressing cellular regulation pathways of blood platelets and endothelial cells are paradigms for a generation of blood contacting devices, which are hemocompatible by cooperation with the host system. STATEMENT OF SIGNIFICANCE: This paper is part 4 of a series of 4 reviews discussing the problem of biomaterial associated thrombogenicity. The objective was to highlight features of broad agreement and provide commentary on those aspects of the problem that were subject to dispute. We hope that future investigators will update these reviews as new scholarship resolves the uncertainties of today.

Polydopamine Modified TiO2 Nanotube Arrays for Long-Term Controlled Elution of Bivalirudin and Improved Hemocompatibility.

Sustained and controllable release characteristics are pivotal factors for novel drug delivery technologies. TiO2 nanotube arrays prepared by self-ordering electrochemical anodization are attractive for the development of biomedical devices for local drug delivery applications. In this work, several layers of polydopamine (PDA) were deposited to functionalize TiO2 nanotube arrays. The anticoagulant drug bivalirudin (BVLD) was used as a model drug. PDA extended the release period of BVLD and maintained a sustained release kinetic. Depending on the number of PDA layers, the release characteristics of BVLD improved, as there was a reduced burst release (from 45% to 11%) and extended overall release period from 40 days to more than 300 days in the case of 5 layers. Besides, the BVLD loaded 5-layer PDA coating maintained the high bioactivity of BVLD and effectively reduced the thrombosis formation by inhibition of the adhesion and denaturation of fibrinogen, platelets, and other blood components. Both in vitro and ex vivo blood evaluation results demonstrated that this coating significantly improved the hemocompatibility. These results confirmed the capability of PDA fitted TiO2 nanotube systems to be applied for local drug delivery over an extended period with well retained bioactivity and predictable release kinetics.

Assembly of Metal-Phenolic/Catecholamine Networks for Synergistically Anti-Inflammatory, Antimicrobial, and Anticoagulant Coatings.

The development of a facile and versatile strategy to endow surfaces with synergistically anti-inflammatory, antimicrobial, and anticoagulant functions is of particular significance for blood-contacting biomaterials and medical devices. In this work, we report a simple and environmentally friendly "one-pot" method inspired by byssal cuticle chemistry, namely, [Fe(dopa)3] coordination chemistry for assembly of copper ions (Cu2+) and plant polyphenol (tannic acid)/catecholamine (dopamine or norepinephrine) to form metal-phenolic/catecholamine network-based coatings. This one-pot method enabled us to easily develop a multifunctional surface based on the combination of the characteristic functions of metal ions and plant polyphenol or catecholamine. The residual phenolic hydroxyl groups on the coatings imparted the modified surface with excellent antioxidant and anti-inflammatory functions. The robust chelation of copper ions to the metal-phenolic/catecholamine networks provided not only durable antibacterial property but also glutathione peroxidase like catalytic capability to continuously and controllably produce antithrombotic nitric oxide by catalyzing endogenous S-nitrothiol. The biological functions of such coatings could be well regulated by adjusting the ratios of the feed concentration of Cu2+ ions to plant polyphenol or catecholamine. We envision that our simple, multifunctional, and bioinspired coating strategy can hold great application promise for bioengineering blood-contacting devices.

In vitro blood reactivity to hydroxylated and non-hydroxylated polymer surfaces.

Complement activation on hydroxyl-group-bearing surfaces is regarded as the main reason for granulocyte activation in applications of blood-contacting medical devices such as extracorporeal blood purification. However, the factors inducing the cell adhesion so far remained ambiguous. For a dedicated research, whole blood was incubated with a set of structurally similar polymer coatings on glass with either hydroxy or ether functionalities. By co-incubation of an activating with a non-activating surface, the reaction of granulocytes activated by complement fragments on non-activating surfaces could be evaluated. As expected, hydroxyl-terminated polymer layers induced much higher levels of complement activation than those with ether functionalities. Leukocyte activation, as measured by the expression of CD11b, correlated closely with the presence of free complement fragment C5a. However, adhesion of leukocytes was rather associated with the adsorption of activated fragments of C3 than with the activation level of the cells. Moreover, it was found that adsorbed quantities of fibrin and fibrinogen had little influence on leukocyte adhesion. It is concluded that the activation of leukocytes is triggered by soluble complement factors such as C5a while their adhesion on hydroxy-bearing surfaces is mainly triggered by the presence of surface-bound complement fragment C3b.

Sulfated glycopolymer thin films - preparation, characterization, and biological activity.

The impact of heparinoid characteristics on model surfaces obtained from immobilization of sole sulfate groups as well as sulfated glycosides, sulfated cellulose, and definite heparin has been investigated. The obtained layers were physico-chemically characterized regarding film thickness, chemical composition, wettability, and surface morphology. Antithrombin adsorption, studied by fluorescence labeling, revealed a strong dependence on the presence of glycosidic structures and on the molecular weight of the grafted saccharide. On contact with whole blood, the coatings resulted in a diminished plasmatic and cellular coagulation in vitro, which did not reflect well the antithrombin binding. Therefore, more complex activating pathways are discussed.

Neutrophil extracellular trap formation upon exposure of hydrophobic materials to human whole blood causes thrombogenic reactions.

Neutrophil extracellular trap (NET) formation, a reaction of the innate immune system to fight pathogens, was shown to be involved in thrombus formation. In the present study blood-contacting biomaterials with graded surface characteristics were investigated as a potential cause of NET formation on medical devices. Surface properties are known to govern protein adsorption, cell adhesion and ultimately the activation of several other host defense pathways - potentially also the formation of NETs. Model materials of defined hydrophilic or hydrophobic properties (glass, and thin films of poly(ethylene-alt-maleic anhydride), self-assembled monolayers of methyl terminated alkanethiols, and Teflon AF™) were incubated either with isolated human granulocytes after pre-adsorption with plasma proteins or with human whole blood. NET formation - detected as extracellular DNA, citrullinated histones, elastase and reactive oxygen species (ROS) - was observed on hydrophobic surfaces. Furthermore, NET formation on the hydrophobic surface Teflon AF™ resulted in elevated thrombin generation in hirudin-anticoagulated whole blood, but not in heparinized whole blood. Disintegration of surface-bound NETs by DNase treatment resulted in significantly lower pro-coagulant effects. Thus, NET formation can contribute to the thrombogenicity of clinically applied hydrophobic materials, suggesting NETosis as well as NET surface anchorage as new targets of anticoagulation strategies.

Adaptive release of heparin from anticoagulant hydrogels triggered by different blood coagulation factors.

Feedback-controlled anticoagulant hydrogels were formed by crosslinking the anticoagulant heparin with star-shaped poly(ethylene glycol) using peptide linkers, which are selectively cleaved by different activated blood coagulation factors acting as proteolytic enzymes. Various cleavable peptide units, differing either in their thrombin turnover rates or in their responsiveness to factors activated earlier in the course of blood coagulation, were used for the formation of the biohybrid materials. Release triggered by the early coagulation factors Xa (FXa) or FXIIa/kallikrein was shown to enhance the efficiency of the released anticoagulant. Furthermore, FXa-cleavable gels enabled a faster release of heparin, which was attributed to the lower affinity of the factor for heparin. Combining early and fast responses, FXa-cleavable gels were shown to provide anticoagulant protection of biomaterial surfaces at low levels of released heparin in human whole-blood incubation experiments. The results demonstrate the potential for employing biomolecular circuits in the design of functional biomaterials to tailor the adaptive delivery of bioactive molecules.