In vivo assessment of dual-function submicron textured nitric oxide releasing catheters in a 7-day rabbit model.

Catheter-induced thrombosis is a major contributor to infectious and mechanical complications of biomaterials that lead to device failure. Herein, a dualfunction submicron textured nitric oxide (NO)-releasing catheter was developed. The hemocompatibility and antithrombotic activity of vascular catheters were evaluated in both 20 h in vitro blood loop and 7 d in vivo rabbit model. Surface characterization assessments via atomic force microscopy show the durability of the submicron pattern after incorporation of NO donor S-nitroso-N-acetylpenicillamine (SNAP). The SNAP-doped catheters exhibited prolonged and controlled NO release mimicking the levels released by endothelium. Fabricated catheters showed cytocompatibility when evaluated against BJ human fibroblast cell lines. After 20h in vitro evaluation of catheters in a blood loop, textured-NO catheters exhibited a 13-times reduction in surface thrombus formation compared to the control catheters, which had 83% of the total area covered by clots. After the 7 d in vivo rabbit model, analysis on the catheter surface was examined via scanning electron microscopy, where significant reduction of platelet adhesion, fibrin mesh, and thrombi can be observed on the NO-releasing textured surfaces. Moreover, compared to relative controls, a 63% reduction in the degree of thrombus formation within the jugular vein was observed. Decreased levels of fibrotic tissue decomposition on the jugular vein and reduced platelet adhesion and thrombus formation on the texture of the NO-releasing catheter surface are indications of mitigated foreign body response. This study demonstrated a biocompatible and robust dual-functioning textured NO PU catheter in limiting fouling-induced complications for longer-term blood-contacting device applications. STATEMENT OF SIGNIFICANCE: Catheter-induced thrombosis is a major contributor to infectious and mechanical complications of biomaterials that lead to device failure. This study demonstrated a robust, biocompatible, dual-functioning textured nitric oxide (NO) polyurethane catheter in limiting fouling-induced complications for longer-term blood-contacting device applications. The fabricated catheters exhibited prolonged and controlled NO release that mimics endothelium levels. After the 7 d in vivo model, a significant reduction in platelet adhesion, fibrin mesh, and thrombi was observed on the NO-releasing textured catheters, along with decreased levels of fibrotic tissue decomposition on the jugular vein. Results illustrate that NO-textured catheter surface mitigates foreign body response.

In Vitro and In Vivo Assessment of the Infection Resistance and Biocompatibility of Small-Molecule-Modified Polyurethane Biomaterials.

Bacterial intracellular nucleotide second messenger signaling is involved in biofilm formation and regulates biofilm development. Interference with the bacterial nucleotide second messenger signaling provides a novel approach to control biofilm formation and limit microbial infection in medical devices. In this study, we tethered small-molecule derivatives of 4-arylazo-3,5-diamino-1H-pyrazole on polyurethane biomaterial surfaces and measured the biofilm resistance and initial biocompatibility of modified biomaterials in in vitro and in vivo settings. Results showed that small-molecule-modified surfaces significantly reduced the Staphylococcal epidermidis biofilm formation compared to unmodified surfaces and decreased the nucleotide levels of c-di-AMP in biofilm cells, suggesting that the tethered small molecules interfere with intracellular nucleotide signaling and inhibit biofilm formation. The hemocompatibility assay showed that the modified polyurethane films did not induce platelet activation or red blood cell hemolysis but significantly reduced plasma coagulation and platelet adhesion. The cytocompatibility assay with fibroblast cells showed that small-molecule-modified surfaces were noncytotoxic and cells appeared to be proliferating and growing on modified surfaces. In a 7-day subcutaneous infection rat model, the polymer samples were implanted in Wistar rats and inoculated with bacteria or PBS. Results show that modified polyurethane significantly reduced bacteria by ∼2.5 log units over unmodified films, and the modified polymers did not lead to additional irritation/toxicity to the animal tissues. Taken together, the results demonstrated that small molecules tethered on polymer surfaces remain active, and the modified polymers are biocompatible and resistant to microbial infection in vitro and in vivo.

FXII contact activation products have an inhibitory effect on αFXIIa.

It is accepted that the contact activation complex of the intrinsic pathway of blood coagulation cascade produces active enzymes that lead to plasma coagulation following biomaterial contact. In this study, FXII was activated through contact with hydrophilic glass beads and hydrophobic octadecyltrichlorosilane-modified glass beads from neat buffer solutions. These FXII contact activation products generated from material interaction were found to suppress the procoagulant activity of exogenous αFXIIa, and this inhibition was dependent on surface wettability and the concentration of exogenous αFXIIa. Higher relative inhibition rates were generally observed at low concentrations of αFXIIa (1-2 µg/mL) while both hydrophobic and hydrophilic materials showed similar inhibition levels (~39%) at high concentrations of αFXIIa (20 µg/mL). The presence of prekallikrein in the activation system increased the amount of FXIIa produced during FXII contact activation, and also suppressed the apparent levels of inhibitors on hydrophilic surfaces, while having no effect on apparent levels of inhibitors on hydrophobic surface. The combination of FXII contact activation products and activator surfaces was found to dramatically increase inhibition of αFXIIa activity compared to the activation products alone, regardless of activator surface wettability and the presence of prekallikrein. This finding of inhibitors in the suite of proteins generated by contact activation provides additional knowledge into the complex series of interactions that occur when plasma comes into contact with material surfaces.

In vitro evaluation of blood plasma coagulation responses to four medical-grade polyurethane polymers.

Segmented polyurethane (PU) block copolymers are widely used in implantable cardiovascular medical devices due to their good biocompatibility and excellent mechanical properties. More specifically, PU Biospan MS/0.4 was used in ventricular assist devices over the past decades. However, this product is being discontinued and it has become necessary to find an alternative PU biomaterial for application in cardiovascular devices. One important criterion for assessing cardiac biomaterials is blood compatibility. In this study, we characterized the surface properties of four medical-grade PU biomaterials: Biospan MS/0.4, BioSpan S, BioSpan 2F, and CarboSil 20 80A, including surface chemistry, topography, microphase separation structure and wettability, and then measured the blood plasma coagulation responses using bovine and human blood plasma. Results showed that BioSpan 2F contains high amounts of fluorine and has the lowest surface free energy while the other materials have surfaces with silicone present. An in vitro coagulation assay shows that these materials demonstrated improved blood coagulation responses compared to the polystyrene control and there were no significant differences in coagulation time among all PU biomaterials. The chromogenic assay showed all PU materials led to low FXII contact activation, and there were no significant differences in FXII contact activation, consistent with plasma coagulation responses.

Surfaces modified with small molecules that interfere with nucleotide signaling reduce Staphylococcus epidermidis biofilm and increase the efficacy of ciprofloxacin.

Staphylococcus epidermidis are common bacteria associated with biofilm related infections on implanted medical devices. Antibiotics are often used in combating such infections, but they may lose their efficacy in the presence of biofilms. Bacterial intracellular nucleotide second messenger signaling plays an important role in biofilm formation, and interference with the nucleotide signaling pathways provides a possible way to control biofilm formation and to increase biofilm susceptibility to antibiotic therapy. This study synthesized small molecule derivates of 4-arylazo-3,5-diamino-1 H-pyrazole (named as SP02 and SP03) and found these molecules inhibited S. epidermidis biofilm formation and induced biofilm dispersal. Analysis of bacterial nucleotide signaling molecules showed that both SP02 and SP03 significantly reduced cyclic dimeric adenosine monophosphate (c-di-AMP) levels in S. epidermidis at doses as low as 25 µM while having significant effects on multiple nucleotides signaling including cyclic dimeric guanosine monophosphate (c-di-GMP), c-di-AMP, and cyclic adenosine monophosphate (cAMP) at high doses (100 µM or greater). We then tethered these small molecules to polyurethane (PU) biomaterial surfaces and investigated biofilm formation on the modified surfaces. Results showed that the modified surfaces significantly inhibited biofilm formation during 24 h and 7-day incubations. The antibiotic ciprofloxacin was used to treat these biofilms and the efficacy of the antibiotic (2 µg/mL) was found to increase from 94.8% on unmodified PU surfaces to > 99.9% on both SP02 and SP03 modified surfaces (>3 log units). Results demonstrated the feasibility of tethering small molecules that interfere with nucleotide signaling onto polymeric biomaterial surfaces and in a way that interrupts biofilm formation and increases antibiotic efficacy for S. epidermidis infections.

Nucleotide Messenger Signaling of Staphylococci in Responding to Nitric Oxide - Releasing Biomaterials.

Nitric oxide (NO) releasing biomaterials are a promising approach against medical device associated microbial infection. In contrast to the bacteria-killing effects of NO at high concentrations, NO at low concentrations serves as an important signaling molecule to inhibit biofilm formation or disperse mature biofilms by regulating the intracellular nucleotide second messenger signaling network such as cyclic dimeric guanosine monophosphate (c-di-GMP) for many Gram-negative bacterial strains. However, Gram-positive staphylococcal bacteria are the most commonly diagnosed microbial infections on indwelling devices, but much less is known about the nucleotide messengers and their response to NO as well as the mechanism by which NO inhibits biofilm formation. This study investigated the cyclic nucleotide second messengers c-di-GMP, cyclic dimeric adenosine monophosphate (c-di-AMP), and cyclic adenosine monophosphate (cAMP) in both Staphylococcus aureus (S. aureus) Newman D2C and Staphylococcus epidermidis (S. epidermidis) RP62A after incubating with S-nitroso-N-acetylpenicillamine (SNAP, NO donor) impregnated polyurethane (PU) films. Results demonstrated that NO release from the polymer films significantly reduced the c-di-GMP levels in S. aureus planktonic and sessile cells, and these bacteria showed inhibited biofilm formation. However, the effect of NO release on c-di-GMP in S. epidermidis was weak, but rather, S. epidermidis showed significant reduction in c-di-AMP levels in response to NO release and also showed reduced biofilm formation. Results strongly suggest that NO regulates the nucleotide second messenger signaling network in different ways for these two bacteria, but for both bacteria, these changes in signaling affect the formations of biofilms. These findings provide cues to understand the mechanism of Staphylococcus biofilm inhibition by NO and suggest novel targets for antibiofilm interventions.

Crosslinkable fluorophenoxy-substituted poly[bis(octafluoropentoxy) phosphazene] biomaterials with improved antimicrobial effect and hemocompatibility.

Biomaterial-associated microbial infection is one of the most frequent and severe complications associated with the use of biomaterials in medical devices. In previous studies, we developed new fluorinated polyphosphazenes, poly[bis(octafluoropentoxy) phosphazene] (OFP) and crosslinkable OFP (X-OFP), and demonstrated the inhibition of bacterial adhesion and biofilm formation, thereby controlling microbial infection. In this study, two additional fluorinated polyphosphazenes (PPs, defined as LS02 and LS03) with fluorophenoxy-substituted side groups, 4-fluorophenoxy and 4-(trifluoromethyl)phenoxy, respectively, based on X-OFP general structure, were synthesized and applied as coatings on stainless steel. The linkage of fluorophenoxy groups to the P-N backbone of PPs was found to increase the surface stiffness and significantly reduced Staphylococcus bacterial adhesion and inhibited biofilm formation. It also significantly reduced microbial infection compared to OFP, our prior X-OFPs or poly[bis(trifluoroethoxy) phosphazene] (TFE). The biofilm experiments show that the newly synthesized PPs LS02 and LS03 are biofilm free up to 28 days. Plasma coagulation and platelet adhesion/activation experiments also demonstrated that new PPs containing fluorophenoxy side groups are hemocompatible. The development of new crosslinkable fluorinated PPs containing fluorophenoxy-substituted side groups provides a new generation of polyphosphazene materials for medical devices with improved resistance to microbial infections and thrombosis formation.

Submicron topography design for controlling staphylococcal bacterial adhesion and biofilm formation.

Surface topography modification with nano- or micro-textured structures has been an efficient approach to inhibit microbial adhesion and biofilm formation and thereby to prevent biomaterial-associated infection without modification of surface chemistry/bulk properties of materials and without causing antibiotic resistance. This manuscript focuses on submicron-textured patterns with ordered arrays of pillars on polyurethane (PU) biomaterial surfaces in an effort to understand the effects of surface pillar features and surface properties on adhesion and colonization responses of two staphylococcal strains. Five submicron patterns with a variety of pillar dimensions were designed and fabricated on PU film surfaces and bacterial adhesion and biofilm formation of Staphylococcal strains (Staphylococcus epidermidis RP62A and Staphylococcus aureus Newman D2C) were characterized. Results show that all submicron textured surface significantly reduced bacterial adhesion and inhibited biofilm formation, and bacterial adhesion linearly decreased with the reduction in top surface area fraction. Surface wettability did not show a linear correlation with bacterial adhesion, suggesting that surface contact area dominates bacterial adhesion. From this, it appears that the design of textured patterns should minimize surface area fraction to reduce the bacterial interaction with surfaces but in a way that ensures the mechanical strength of pillars in order to avoid collapse. These findings may provide a rationale for design of polymer surfaces for antifouling medical devices.

Surface Texturing and Combinatorial Approaches to Improve Biocompatibility of Implanted Biomaterials.

Biomaterial associated microbial infection and blood thrombosis are two of the barriers that inhibit the successful use of implantable medical devices in modern healthcare. Modification of surface topography is a promising approach to combat microbial infection and thrombosis without altering bulk material properties necessary for device function and without contributing to bacterial antibiotic resistance. Similarly, the use of other antimicrobial techniques such as grafting poly(ethylene glycol) (PEG) and nitric oxide (NO) release also improve the biocompatibility of biomaterials. In this review, we discuss the development of surface texturing techniques utilizing ordered submicron-size pillars for controlling bacterial adhesion and biofilm formation, and we present combinatorial approaches utilizing surface texturing in combination with poly(ethylene glycol) (PEG) grafting and NO release to improve the biocompatibility of biomaterials. The manuscript also discusses efforts towards understanding the molecular mechanisms of bacterial adhesion responses to the surface texturing and NO releasing biomaterials, focusing on experimental aspects of the approach.

Inhibition of bacterial adhesion and biofilm formation by a textured fluorinated alkoxyphosphazene surface.

The utilization of biomaterials in implanted blood-contacting medical devices often induces a persistent problem of microbial infection, which results from bacterial adhesion and biofilm formation on the surface of biomaterials. In this research, we developed new fluorinated alkoxyphosphazene materials, specifically poly[bis(octafluoropentoxy) phosphazene] (OFP) and crosslinkable OFP (X-OFP), with improved mechanical properties, and further modified the surface topography with ordered pillars to improve the antibacterial properties. Three X-OFP materials, X-OFP3.3, X-OFP8.1, X-OFP13.6, with different crosslinking densities were synthesized, and textured films with patterns of 500/500/600 nm (diameter/spacing/height) were fabricated via a two stage soft lithography molding process. Experiments with 3 bacterial strains: Staphylococcal epidermidis, Staphylococcal aureus, and Pseudomonas aeruginosa showed that bacterial adhesion coefficients were significantly lower on OFP and X-OFP smooth surfaces than on the polyurethane biomaterial, and surface texturing further reduced bacterial adhesion due to the reduction in accessible surface contact area. Furthermore the anti-bacterial adhesion effect shows a positive relationship with the crosslinking degree. Biofilm formation on the substrates was examined using a CDC biofilm reactor for 7 days and no biofilm formation was observed on textured X-OFP biomaterials. The results suggested that the combination of fluorocarbon chemistry and submicron topography modification in textured X-OFP materials may provide a practical approach to improve the biocompatibility of current biomaterials with significant reduction in risk of pathogenic infection.

New cross-linkable poly[bis(octafluoropentoxy) phosphazene] biomaterials: Synthesis, surface characterization, bacterial adhesion, and plasma coagulation responses.

Biomaterial-associated microbial infection and thrombosis represent major issues to the success of long-term use of implantable blood-contacting medical devices. The development of new poly[bis(octafluoropentoxy) phosphazene (OFP) biomaterials provides new routes for combatting microbial infection and thrombosis. However, the limited mechanical properties of OFP to date render them unsuitable for application in medical devices and inhibit any attempts at subsequent surface topography modification. In this study, we synthesized cross-linkable OFPs (X-OFPs) with the different degrees of cross-linking in an effort to improve the mechanical properties. The results showed that the surface chemistry and surface topography of X-OFPs do not change significantly, but the surface mechanical stiffness increased after cross-linking. Atomic force microscopic phase images showed that the polymer phase separation structures changed due to cross-linking. Experiments with three bacterial strains: Staphylococcal epidermidis, Staphylococcal aureus, and Pseudomonas aeruginosa showed that bacterial adhesion was significantly decreased on the OFP and X-OFPs for both the pre-cross-linked and cross-linked as compared to polyurethane biomaterials. Furthermore, bacterial adhesions were lower on X-OFP surfaces than on pre-cross-linked materials, suggesting that mechanical stiffness is an important parameter influencing bacterial adhesion. Blood plasma coagulation responses revealed longer coagulation times for OFP and X-OFP materials than on polyurethanes, indicating that the new cross-linked OFPs are resistant to plasma coagulation compared to currently used polyurethane biomaterials.

Blood coagulation response and bacterial adhesion to biomimetic polyurethane biomaterials prepared with surface texturing and nitric oxide release.

A dual functional polyurethane (PU) film that mimics aspects of blood vessel inner surfaces by combining surface texturing and nitric oxide (NO) release was fabricated through a soft lithography two-stage replication process. The fabrication of submicron textures on the polymer surface was followed by solvent impregnation with the NO donor, S-nitroso-N-acetylpenicillamine (SNAP). An in vitro plasma coagulation assay showed that the biomimetic surface significantly increased the plasma coagulation time and also exhibited reduced platelet adhesion and activation, thereby reducing the risk of blood coagulation and thrombosis. A contact activation assay for coagulation factor XII (FXII) demonstrated that both NO release and surface texturing also reduced FXII contact activation, which contributes to the inhibition of plasma coagulation. The biomimetic surface was also evaluated for bacterial adhesion in plasma and results demonstrate that this combined strategy enables a synergistic effect to reduce bacterial adhesion of Staphylococcus epidermidis, Staphylococcus aureus, and Pseudomonas aeruginosa microorganisms. The results strongly suggest that the biomimetic modification with surface texturing and NO release provides an effective approach to improve the biocompatibility of polymeric materials in combating thrombosis and microbial infection. STATEMENT OF SIGNIFICANCE: (1) Developed a dual functional polyurethane (PU) film that mimics blood vessel inner surface by combining surface texturing and nitric oxide (NO) release for combatting biomaterial associated thrombosis and microbial infection. (2) Studied the blood coagulation response and bacterial adhesion to such biomimetic PU surfaces, and demonstrated that the combination of surface texturing and NO release synergistically reduced the platelet adhesion and bacterial adhesion in plasma, providing an effective approach to improve the biocompatibility of biomaterials used in blood-contacting medical devices. (3) The NO releasing surface significantly inhibits the plasma coagulation via the reduction of contact activation of FXII, indicating the multifunctional roles of NO in improving the biocompatibility of biomaterials in blood-contacting medical devices.

Increased circulating microparticles in streptozotocin-induced diabetes propagate inflammation contributing to microvascular dysfunction.

KEY POINTS: Circulating microparticles (MPs) are elevated in many cardiovascular diseases and have been considered as biomarkers of disease prognosis; however, current knowledge of MP functions has been mainly derived from in vitro studies and their precise impact on vascular inflammation and disease progression remains obscure. Using a diabetic rat model, we identified a >130-fold increase in MPs in plasma of diabetic rats compared to normal rats, the majority of which circulated as aggregates, expressing multiple cell markers and largely externalized phosphatidylserine; vascular images illustrate MP biogenesis and their manifestations in microvessels of diabetic rats. Using combined single microvessel perfusion and systemic cross-transfusion approaches, we delineated how diabetic MPs propagate inflammation in the vasculature and transform normal microvessels into an inflammatory phenotype observed in the microvessels of diabetic rats. Our observations derived from animal studies resembling conditions in diabetic patients, providing a mechanistic insight into MP-mediated pathogenesis of diabetes-associated multi-organ microvascular dysfunction. ABSTRACT: In various cardiovascular diseases, microparticles (MPs), the membrane-derived vesicles released during cell activation, are markedly increased in the circulation. These MPs have been recognized to play diverse roles in the regulation of cellular functions. However, current knowledge of MP function has been largely derived from in vitro studies. The precise impact of disease-induced MPs on vascular inflammation and disease progression remains obscure. In this study we investigated the biogenesis, profile and functional roles of circulating MPs using a streptozotocin-induced diabetic rat model with well-characterized microvascular functions. Our study revealed a >130-fold increase in MPs in the plasma of diabetic rats compared to normal rats. The majority of these MPs originate from platelets, leukocytes and endothelial cells (ECs), and circulate as aggregates. Diabetic MPs show greater externalized phosphatidylserine (PS) than normal MPs. When diabetic plasma or isolated diabetic MPs were perfused into normal microvessels or systemically transfused into normal rats, MPs immediately adhered to endothelium and subsequently mediated leukocyte adhesion. These microvessels then exhibited augmented permeability responses to inflammatory mediators, replicating the microvascular manifestations observed in diabetic rats. These effects were abrogated when MPs were removed from diabetic plasma or when diabetic MPs were pre-coated with a lipid-binding protein, annexin V, suggesting externalized PS to be key in mediating MP interactions with endothelium and leukocytes. Our study demonstrated that the elevated MPs in diabetic plasma are actively involved in the propagation of vascular inflammation through their adhesive surfaces, providing mechanistic insight into the pathogenesis of multi-organ vascular dysfunction that commonly occurs in diabetic patients.

Liquid Biopsy of Vitreous Reveals an Abundant Vesicle Population Consistent With the Size and Morphology of Exosomes.

PURPOSE: To investigate the molecular components of the vitreous in order to better understand retinal physiology and disease. METHODS: Vitreous was acquired from patients undergoing vitrectomy for macular hole and/or epiretinal membrane, postmortem donors, and C57BL/6J mice. Unbiased proteomic analysis was performed via electrospray ionization tandem mass spectrometry (MS/MS). Gene ontology analysis was performed and results were confirmed with transmission electron microscopy, atomic force microscopy, and nanoparticle tracking analysis (NTA). RESULTS: Proteomic analysis of vitreous obtained prior to vitrectomy identified a total of 1121 unique proteins. Gene ontology analysis revealed that 62.6% of the vitreous proteins were associated with the gene ontology term "extracellular exosome." Ultrastructural analyses, Western blot, and NTA confirmed the presence of an abundant population of vesicles consistent with the size and morphology of exosomes in human vitreous. The concentrations of vitreous vesicles in vitrectomy patients, postmortem donors, and mice were 1.3, 35, and 9 billion/mL, respectively. CONCLUSIONS: Overall, these data strongly suggest that information-rich exosomes are a major constituent of the vitreous. The abundance of these vesicles and the presence of retinal proteins imply a dynamic interaction between the vitreous and retina. Future studies will be required to identify the cellular origin of vitreal exosomes as well as to assess the potential role of these vesicles in retinal disease and treatment. TRANSLATIONAL RELEVANCE: The identification of vitreous exosomes lays the groundwork for a transformed understanding of pathophysiology and treatment mechanisms in retinal disease, and further validates the use of vitreous as a proximal biofluid of the retina.

A new textured polyphosphazene biomaterial with improved blood coagulation and microbial infection responses.

A new poly[bis(octafluoropentoxy) phosphazene] (OFP) was synthesized for the purpose of blood contacting medical devices. OFP was further either developed into crosslinkable polyphosphazene (X-OFP) or blended with polyurethane (PU) as the mixture (OFP/PU) for improvement of mechanical property of polyphosphazene polymers. All the materials were fabricated as smooth films or further textured with submicron pillars for the assay of antimicrobial and antithrombotic properties. Results showed that crosslinkable OFP (X-OFP) and blends of OFP/PU successfully improved the mechanical strength of OFP and fewer defects of pillars were found on the textured polyphosphazene surfaces. The antithrombotic experiments showed that polyphosphazene OFP materials reduced human Factor XII activation and platelet adhesion, thereby being resistant to plasma coagulation and thrombosis. The bacterial adhesion and biofilm experiments demonstrated that OFP materials inhibited staphylococcal bacterial adhesion and biofilm formation. The surface texturing further reduced the platelet adhesion and bacterial adhesion, and inhibited biofilm formation up to 23 days. The data suggested that textured OFP materials may provide a practical approach to improve the biocompatibility of current biomaterials in the application of blood contacting medical devices with significant reduction in risk of pathogenic infection and thrombosis. STATEMENT OF SIGNIFICANCE: The thromboembolic events and microbial infection have been the significant barriers for the long term use of biomaterials in blood-contacting medical devices. The development of new materials with multiple functions including anti-thrombosis and antibacterial surfaces is a high research priority. This study synthesized new biostable and biocompatible polyphosphazene polymers, poly[bis(octafluoropentoxy)phosphazene] (OFP) and crosslinkable OFP, and successfully improved the mechanical strength of polyphosphazenes. Polymers were fabricated into textured films with submicron pillars on the surfaces. The antimicrobial and antithrombotic assays demonstrated that new materials combined with surface physical modification have significant reduction in risk of pathogenic infection and thrombosis, and improve the biocompatibility of current biomaterials in the application of blood-contacting medical devices. It would be interest to biomaterials and bioengineering related communities.

Antimicrobial nitric oxide releasing surfaces based on S-nitroso-N-acetylpenicillamine impregnated polymers combined with submicron-textured surface topography.

A novel dual functioning antimicrobial CarboSil 20 80A polymer material that combines physical topographical surface modification and nitric oxide (NO) release is prepared and evaluated for its efficacy in reducing bacterial adhesion in vitro. The new biomaterial is created via a soft lithography two-stage replication process to induce submicron textures on its surface, followed by solvent impregnation with the NO donor, S-nitroso-N-acetylpenicillamine (SNAP), to obtain long-term (up to 38 d) NO release. The NO releasing textured polymer surface is evaluated against four bacteria commonly known to cause infections in hospital settings and the results demonstrate that the combined strategy enables a synergistic effect on reducing the bacterial adhesion of Staphylococcus epidermidis and Pseudomonas aeruginosa bacteria.

Interleukin-13 conjugated quantum dots for identification of glioma initiating cells and their extracellular vesicles.

Cadmium selenide (CdSe) based quantum dots modified with polyethylene glycol and chemically linked to interleukin-13 (IL13) were prepared with the aim of identifying the high affinity receptor (IL13Rα2) which is expressed in glioma stem cells and exosomes secreted by these cancer stem cells. IL13 conjugated quantum dots (IL13QD) were thoroughly characterized for their physicochemical properties including particle size and surface morphology. Furthermore, the specific binding of the IL13QD to glioma cells and to glioma stem cells (GSC) was verified using a competitive binding study. The exosomes were isolated from the GSC conditioned medium and the expression of IL13Rα2 in the GSC and exosomes was verified. The binding property of IL13QD to the tumor associated exosomes was initially confirmed by transmission electron microscopy. The force of attraction between the quantum dots and U251 glioma cells and the exosomes was investigated by atomic force microscopy, which indicated a higher force of binding interaction between the IL13QD and IL13Rα2 expressing glioma cells and exosomes secreted by glioma stem cells. Flow cytometry of the IL13QD and exosomes from the culture media and cerebrospinal fluid (CSF) of patients with glioma tumors indicated a distinctly populated complex pattern different from that of non-targeted quantum dots and bovine serum albumin (BSA) conjugated quantum dots confirming specific binding potential of the IL13QD to the tumor associated exosomes. The results of this study demonstrate that IL13QD can serve as an ex vivo marker for glioma stem cells and exosomes that can inform diagnosis and prognosis of patients harboring malignant disease. STATEMENT OF SIGNIFICANCE: Functionalized quantum dots are flexible semiconductor nanomaterials which have an immense application in biomedical research. In particular, when they are functionalized with biomolecules like proteins or antibodies, they have the specialized ability to detect the expression of receptors and antigens in cells and tissues. In this study we designed a cytokine (interleukin-13) functionalized quantum dot to detect a cancer associated receptor expressed in cancer stem cells and the extracellular vesicles (exosomes) secreted by the cancer cells themselves. The binding pattern of these cytokine modified quantum dots to the cancer stem cells and exosomes alters the physical properties of the complex in the fixed and suspended form. This altered binding pattern can be monitored by a variety of techniques, including transmission electron microscopy, atomic force microscopy and flow cytometry, and subsequent characterization of this quantum dot binding profile provides useful data that can be utilized as a fingerprint to detect cancer disease progression. This type of functionalized quantum dot fingerprint is especially useful for invasive cancers including brain and other metastatic cancers and may allow for earlier detection of disease progression or recurrence, thus saving the lives of patients suffering from this devastating disease.

Surface dependent contact activation of factor XII and blood plasma coagulation induced by mixed thiol surfaces.

Studies of the activation of FXII in both platelet poor plasma and in neat buffer solutions were undertaken for a series of mixed thiol self-assembled monolayers spanning a broad range of water wettability. A wide spectrum of carboxyl/methyl-, hydroxyl/methyl-, and amine/methyl-thiol modified surfaces were prepared, characterized, and then utilized as the procoagulant materials in a series of FXII activation studies. X-ray photoelectron spectroscopy was utilized to verify the sample surface's thiol composition and contact angles measured to determine the sample surface's wettability. These samples were then used in in vitro coagulation assays using a 50% mixture of recalcified plasma in phosphate buffered saline. Alternatively, the samples were placed into purified FXII solutions for 30 min to assess FXII activation in neat buffer solution. Plasma coagulation studies supported a strong role for anionic surfaces in contact activation, in line with the traditional models of coagulation, while the activation results in neat buffer solution demonstrated that FXIIa production is related to surface wettability with minimum levels of enzyme activation observed at midrange wettabilities, and no statistically distinguishable differences in FXII activation seen between highly wettable and highly nonwettable surfaces. Results demonstrated that the composition of the solution and the surface properties of the material all contribute to the observation of contact activation, and the activation of FXII is not specific to anionic surfaces as has been long believed.

Inhibition of bacterial adhesion and biofilm formation by dual functional textured and nitric oxide releasing surfaces.

In separate prior studies, physical topographic surface modification or nitric oxide (NO) release has been demonstrated to each be an effective approach to inhibit and control bacterial adhesion and biofilm formation on polymeric surfaces. Such approaches can prevent biomaterial-associated infection without causing the antibiotic resistance of the strain. In this work, both techniques were successfully integrated and applied to a polyurethane (PU) biomaterial surface that bears ordered pillar topographies (400/400nm and 500/500nm patterns) at the top surface and a S-nitroso-N-acetylpenicillamine (SNAP, NO donor) doped sub-layer in the middle, via a soft lithography two-stage replication process. Upon placing the SNAP textured PU films into PBS at 37°C, the decomposition of SNAP within polymer film initiates NO release with a lifetime of up to 10days at flux levels >0.5×10-10molmin-1cm-2 for a textured polyurethane layer containing 15wt% SNAP. The textured surface reduces the accessible surface area and the opportunity of bacteria-surface interaction, while the NO release from the same surface further inhibits bacterial growth and biofilm formation. Such dual functionality surfaces are shown to provide a synergistic effect on inhibition of Staphylococcus epidermidis bacterial adhesion that is significantly greater than the inhibition of bacterial adhesion achieved by either single treatment approach alone. Longer term experiments to observe biofilm formation demonstrate that the SNAP doped-textured PU surface can inhibit the biofilm formation for >28d and provide a practical approach to improve the biocompatibility of current biomimetic biomaterials and thereby reduce the risk of pathogenic infection. STATEMENT OF SIGNIFICANCE: Microbial infection remains a significant barrier to development and implementation of advanced blood-contacting medical devices. Clearly, determining how to design and control material properties that can reduce microbial infection is a central question to biomaterial researchers. In separate prior studies, physical topographic surface modification or nitric oxide (NO) release has been demonstrated to each be an effective approach to inhibit and control bacterial adhesion and biofilm formation on polymeric surfaces. Such approaches can prevent biomaterial-associated infection without causing antibiotic resistance of the bacterial strain. However, efficiency of antimicrobial properties of each approach is still limited and far from sufficient for widespread clinical use. This work successfully integrates both techniques and applies them to a polyurethane (PU) biomaterial surface that bears dual functions, surface topographic modification and NO release. The former reduces the surface contact area and changes surface wettability, resulting in reduction of bacterial adhesion, and NO release further inhibits bacteria growth. Such dual functionalized surfaces provide a synergistic effect on inhibition of Staphylococcus epidermidis bacterial adhesion that is significantly greater than the inhibition of bacterial adhesion achieved by either single treatment approach alone. Furthermore, longer-term experiments demonstrate that the dual functionalized surfaces can inhibit biofilm formation for >28days. The success of this work provides a practical approach to improve the biocompatibility of current biomaterials and thereby reduce the risk of pathogenic infection.

Reduction of Endothelial Nitric Oxide Increases the Adhesiveness of Constitutive Endothelial Membrane ICAM-1 through Src-Mediated Phosphorylation.

Nitric oxide (NO) is a known anti-adhesive molecule that prevents platelet aggregation and leukocyte adhesion to endothelial cells (ECs). The mechanism has been attributed to its role in the regulation of adhesion molecules on leukocytes and the adhesive properties of platelets. Our previous study conducted in rat venules found that reduction of EC basal NO synthesis caused EC ICAM-1-mediated firm adhesion of leukocytes within 10-30 min. This quick response occurred in the absence of alterations of adhesion molecules on leukocytes and also opposes the classical pattern of ICAM-1-mediated leukocyte adhesion that requires protein synthesis and occurs hours after stimulation. The objective of this study is to investigate the underlying mechanisms of reduced basal NO-induced EC-mediated rapid leukocyte adhesion observed in intact microvessels. The relative levels of ICAM-1 at different cell regions and their activation status were determined with cellular fractionation and western blot using cultured human umbilical vein ECs. ICAM-1 adhesiveness was determined by immunoprecipitation in non-denatured proteins to assess the changes in ICAM-1 binding to its inhibitory antibody, mAb1A29, and antibody against total ICAM-1 with and without NO reduction. The adhesion strength of EC ICAM-1 was assessed by atomic force microscopy (AFM) on live cells. Results showed that reduction of EC basal NO caused by the application of caveolin-1 scaffolding domain (AP-CAV) or NOS inhibitor, L-NMMA, for 30 min significantly increased phosphorylated ICAM-1 and its binding to mAb1A29 in the absence of altered ICAM-1 expression and its distribution at subcellular regions. The Src inhibitor, PP1, inhibited NO reduction-induced increases in ICAM-1 phosphorylation and adhesive binding. AFM detected significant increases in the binding force between AP-CAV-treated ECs and mAb1A29-coated probes. These results demonstrated that reduced EC basal NO lead to a rapid increase in ICAM-1 adhesive binding via Src-mediated phosphorylation without de novo protein synthesis and translocation. This study suggests that a NO-dependent conformational change of constitutive EC membrane ICAM-1 might be the mechanism of rapid ICAM-1 dependent leukocyte adhesion observed in vivo. This new mechanistic insight provides a better understanding of EC/leukocyte interaction-mediated vascular inflammation under many disease conditions that encounter reduced basal NO in the circulation system.