Bovine Fibrinogen Research Articles

A murine experimental model of the pulmonary thrombotic effect induced by the venom of the snake Bothrops lanceolatus.

The venom of Bothrops lanceolatus, a viperid species endemic to the Lesser Antillean Island of Martinique, induces thrombosis in a number of patients. Previous clinical observations indicate that thrombotic events are more common in patients bitten by juvenile specimens. There is a need to develop an experimental model of this effect in order to study the mechanisms involved. The venoms of juvenile and adult specimens of B. lanceolatus were compared by (a) describing their proteome, (b) assessing their ability to induce thrombosis in a mouse model, and (c) evaluating their in vitro procoagulant activity and in vivo hemostasis alterations. Venom proteomes of juvenile and adult specimens were highly similar, albeit showing some differences. When injected by the intraperitoneal (i.p.) route, the venom of juvenile specimens induced the formation of abundant thrombi in the pulmonary vasculature, whereas this effect was less frequent in the case of adult venom. Thrombosis was not abrogated by the metalloproteinase inhibitor Batimastat. Both venoms showed a weak in vitro procoagulant effect on citrated mouse plasma and bovine fibrinogen. When administered intravenously (i.v.) venoms did not affect classical clotting tests (prothrombin time and activated partial thromboplastin time) but caused a partial drop in fibrinogen concentration. The venom of juvenile specimens induced partial alterations in some rotational thromboelastometry parameters after i.v. injection. When venoms were administered i.p., only minor alterations in classical clotting tests were observed with juvenile venom, and no changes occurred for either venom in rotational thromboelastometry parameters. Both juvenile and adult venoms induced a marked thrombocytopenia after i.p. injection. An experimental model of the thrombotic effect induced by B. lanceolatus venom was developed. This effect is more pronounced in the case of venom of juvenile specimens, despite the observation that juvenile and adult venom proteomes are similar. Adult and juvenile venoms do not induce a consumption coagulopathy characteristic of other Bothrops sp venoms. Both venoms induce a conspicuous thrombocytopenia. This experimental model reproduces the main clinical findings described in these envenomings and should be useful to understand the mechanisms of the thrombotic effect.

A green zwitterionic bi-continuous membranes prepared by dual-bath procedure for mitigating biofouling during bacterial removal from water

This work explores the possibility of forming green microfiltration antifouling membranes based on polyvinylidene fluoride (PVDF) and a zwitterionic copolymer derivative of sulfobetaine methacrylate (SBMA), utilizing gamma-valerolactone (GVL) as the solvent and a mixture of ethanol and water as non-solvents. Efforts were initially focused on adjusting the immersion time of the matrix polymer in alcohol alone to obtain a porous structure, and 15 s were sufficient to achieve a symmetric, open porous morphology. Next, the polymer/copolymer/solvent blend was characterized. While it did not show the characteristics of a fully miscible system, DLS and visual observations revealed the obtaining of a homogeneous (relatively narrow particle size) and transparent to translucent system. Thus, membranes could be cast, and were shown to be stable. The addition of the copolymer maintained the physical characteristics of microfiltration membranes, but an FT-IR analysis revealed its effect on the dominant crystalline polymorph (switching from β to α). The membrane wettability was enhanced in an important extent with a dynamic water contact angle (WCA) gradually falling to < 40° (while the virgin membrane's WCA remained about 138° all test long), and a hydration capacity reaching about 530 mg/cm3 against <10 mg/cm3 for the virgin membrane. It reflected on the antifouling abilities of the membranes as tested using bovine serum albumin, fibrinogen, Escherichia coli, and Stenotrophomonas maltophilia, with relative decreases in adsorption during static tests measured to be 95 %, 70 %, 91 % and 98 %, respectively. Finally, compared to the virgin membrane, the modified membrane featured improved flux recovery (32 % vs. 11 %), significantly higher rejection (98.9 % vs. 89.1 %) and lower irreversible fouling tendency (68 % vs. 89 %) during cyclic water/bacterial solution filtration in dead-end mode, making it potentially suitable for addressing bacterial pollution in wastewater.

Novel magnetic iron oxide-dextran sulphate nanocomposites as potential anticoagulants: Investigating interactions with blood components and assessing cytotoxicity

Examining the critical role of anticoagulants in medical practice, particularly their central function in preventing abnormal blood clotting, is of the utmost importance. However, the study of interactions between blood proteins and alternative anticoagulant nano-surfaces is still understood poorly. In this study, novel approach involving direct functionalisation of magnetic iron oxide nanoparticles (MNPs) as carriers with sulphated dextran (s-dext) is presented, with the aim of evaluating the potential of magnetically-responsive MNPs@s-dext as anticoagulants. The physicochemical characterisation of the synthesised MNPs@s-dext includes crystal structure analysis, morphology study, surface and electrokinetic properties, thermogravimetric analysis and magnetic properties` evaluation, which confirms the successful preparation of the nanocomposite with sulfonate groups. The anticoagulant potential of MNPs@s-dext was investigated using a standardised activated partial thromboplastin time (APTT) test and a modified APTT test with a quartz crystal microbalance with dissipation (QCM-D) which confirmed the anticoagulant effect. Time-resolved solid-liquid interactions between the MNPs@s-dext and model blood proteins bovine serum albumin and fibrinogen were also investigated, to gain insight into their hemocompatibility, and revealed protein-repellence of MNPs@s-dext against blood proteins. The study also addressed comprehensive cytotoxicity studies of prepared nanocomposites, and provided valuable insights into potential applicability of MNPs@s-dext as a promising magnetic anticoagulant in biomedical contexts.

Bioinspired Self-Adhesive Multifunctional Lubricated Coating for Biomedical Implant Applications.

In the realm of clinical applications, the concern surrounding biomedical device-related infections (BDI) is paramount. To mitigate the risk associated with BDI, enhancing surface characteristics such as lubrication and antibacterial efficacy is considered as a strategic approach. This study delineated the synthesis of a multifunctional copolymer, embodying self-adhesive, lubricating, and antibacterial properties, achieved through free radical polymerization and a carbodiimide coupling reaction. The copolymer was adeptly modified on the surface of stainless steel 316L (SS316L) substrates by employing a facile dip-coating technique. Comprehensive characterizations were performed by using an array of analytical techniques including Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy, optical interferometry, scanning electron microscopy, and atomic force microscopy. Nanoscale tribological assessments revealed a notable reduction in the value of the friction coefficient of the copolymer-coated SS316L substrates compared to bare SS316L samples. The coating demonstrated exceptional resistance to protein adsorption, as evidenced in protein contamination models employing bovine serum albumin and fibrinogen. The bactericidal efficacy of the copolymer-modified surfaces was significantly improved against pathogenic strains such as Staphylococcus aureus and Escherichia coli. Additionally, in vitro evaluations of blood compatibility and cellular compatibility underscored the remarkable anticoagulant performance and biocompatibility. Collectively, these findings indicated that the developed copolymer coating represented a promising candidate, with its facile modification approach, for augmenting lubrication and antifouling properties in the field of biomedical implant applications.

Isolation and characterization of a Mannheimiahaemolytica secreted serine protease that degrades sheep and bovine fibrinogen

Mannheimiahaemolytica is an opportunistic agent of the respiratory tract of bovines, a member of the Pasteurellaceae family, and the causal agent of fibrinous pleuropneumonia. This bacterium possesses different virulence factors, allowing it to colonize and infect its host. The present work describes the isolation and characterization of a serine protease secreted by M. haemolytica serotype 1. This protease was isolated from M. haemolytica cultured media by precipitation with 50 % methanol and ion exchange chromatography on DEAE-cellulose. It is a 70-kDa protease able to degrade sheep and bovine fibrinogen or porcine gelatin but not bovine IgG, hemoglobin, or casein. Mass spectrometric analysis indicates its identity with protease IV of M. haemolytica. The proteolytic activity was active between pH 5 and 9, with an optimal pH of 8. It was stable at 50 °C for 10 min but inactivated at 60 °C. The sera of bovines with chronic or acute pneumonia recognized this protease. Still, it showed no cross-reactivity with rabbit hyperimmune serum against the secreted metalloprotease from Actinobacilluspleuropneumoniae, another member of the Pasteurellaceae family. M. haemolytica secreted proteases could contribute to the pathogenesis of this bacterium through fibrinogen degradation, a characteristic of this fibrinous pleuropneumonia.

PEG-functionalized aliphatic polycarbonate brushes with self-polishing dynamic antifouling properties

Hydrophilic antifouling polymers provide excellent antifouling effects under usual short-term use conditions, but the long-term accumulation of contaminants causes them to lose their antifouling properties. To overcome this drawback, surface-initiated ring-opening graft polymerization (SI-ROP) was performed on the surface of the material by applying the cyclic carbide monomer 4′-(fluorosulfonyl)benzyl-5-methyl-2-oxo-1,3-dioxane-5-carboxylate (FMC), which contains a sulfonylfluoride group on the side chain, followed by a “sulfur(IV)-fluorine exchange” (SuFEx) post click modification reaction to link the hydrophilic polyethylene glycol (PEG) to the polyFMC (PFMC) brush, and a novel antifouling strategy for self-polishing dynamic antifouling surfaces was developed. The experimental results showed that the antifouling surface could effectively prevent the adsorption of proteins such as bovine serum albumin (BSA, ∼96.4%), fibrinogen (Fg, ∼87.8%) and lysozyme (Lyz ∼69.4%) as well as the adhesion of microorganisms such as the bacteria Staphylococcus aureus (S. aureus) (∼87.5%) and HeLa cells (∼67.2%). Moreover, the enzymatically self-polished surface still has excellent antifouling properties. Therefore, this modification method has potential applications in the field of biosensors and novel antifouling materials.

Isolation and Functional Characterization of Erythrofibrase: An Alfa-Fibrinogenase Enzyme from Trimeresurus erythrurus Venom of North-East India.

Green pit viper bites induce mild toxicity with painful local swelling, blistering, cellulitis, necrosis, ecchymosis and consumptive coagulopathy. Several bite cases of green pit vipers have been reported in several south-east Asian countries including the north-eastern region of India. The present study describes isolation and characterization of a haemostatically active protein from Trimeresurus erythrurus venom responsible for coagulopathy. Using a two-step chromatographic method, a snake venom serine protease erythrofibrase was purified to homogeneity. SDS-PAGE of erythrofibrase showed a single band of ~30 kDa in both reducing and non-reducing conditions. The primary structure of erythrofibrase was determined by ESI LC-MS/MS, and the partial sequence obtained showed 77% sequence similarity with other snake venom thrombin-like enzymes (SVTLEs). The partial sequence obtained had the typical 12 conserved cysteine residues, as well as the active site residues (His57, Asp102 and Ser195). Functionally, erythrofibrase showed direct fibrinogenolytic activity by degrading the Aα chain of bovine fibrinogen at a slow rate, which might be responsible for causing hypofibrinogenemia and incoagulable blood for several days in envenomated patients. Moreover, the inability of Indian polyvalent antivenom (manufactured by Premium Serum Pvt. Ltd., Maharashtra, India) to neutralize the thrombin-like and plasmin-like activity of erythrofibrase can be correlated with the clinical inefficacy of antivenom therapy. This is the first study reporting an α-fibrinogenase enzyme erythrofibrase from T. erythrurus venom, which is crucial for the pathophysiological manifestations observed in envenomated victims.

Comparative analysis of glimepiride interactions with bovine serum albumin and fibrinogen using fluorescence spectroscopy

Bovine Serum Albumin (BSA) and Fibrinogen (Fib) are the model proteins extensively used for investigating drug-protein interactions due to their distinct binding characteristics and structural similarities to human serum albumin. The present report examines the interactions between Glimepiride (GM) [a sulfonylurea used for Type 2 Diabetes mellitus management) and Fib And BSA, respectively. Fluorescence spectroscopy was employed to analyze quenching mechanisms and binding constants at 298 K and 290 K, respectively. The results indicate that GM binds to BSA and Fib via hydrophobic forces, with binding and quenching rate constants showing significant interactions. The binding constant (kb) due to the interaction between GM-Fib was found to be 1.297 × 104 Lmol-1 and 1.262× 104 Lmol-1 at 298 K and 290 K, respectively with Gibbs free energy (ΔG) equal to -5.609 Kcal/mol, and -3.627 Kcal/mol at 298 K and 290 K, respectively. The binding constant (kb) for the interaction between GM- BSA was determined to be 1.24 × 105 Lmol⁻¹ at 298 K and 9.896 × 103 Lmol⁻¹ at 290 K. The ΔG associated with these interactions is -6.947 Kcal/mol at 298 K and -5.302 Kcal/mol at 290 K. The thermodynamic parameters suggest spontaneous binding processes due to a negative value of ΔG. The results provide valuable insights into GM’s stability and interaction mechanisms with Fib and BSA, contributing to understanding drug-protein interactions essential for drug development.

Non-fibril amyloid aggregation at the air/water interface: self-adaptive pathway resulting in a 2D Janus nanofilm.

The amyloid states of proteins are implicated in several neurodegenerative diseases and bioadhesion processes. However, the classical amyloid fibrillization mechanism fails to adequately explain the formation of polymorphic aggregates and their adhesion to various surfaces. Herein, we report a non-fibril amyloid aggregation pathway, with disulfide-bond-reduced lysozyme (R-Lyz) as a model protein under quasi-physiological conditions. Very different from classical fibrillization, this pathway begins with the air-water interface (AWI) accelerated oligomerization of unfolded full-length protein, resulting in unique plate-like oligomers with self-adaptive ability, which can adjust their conformations to match various interfaces such as the asymmetric AWI and amyloid-protein film surface. The pathway enables a stepwise packing of the plate-like oligomers into a 2D Janus nanofilm, exhibiting a divergent distribution of hydrophilic/hydrophobic residues on opposite sides of the nanofilm. The resulting Janus nanofilm possesses a top-level Young's modulus (8.3 ± 0.6 GPa) among amyloid-based materials and exhibits adhesive strength two times higher (145 ± 81 kPa) than that of barnacle cement. Furthermore, we found that such an interface-directed pathway exists in several amyloidogenic proteins with a similar self-adaptive 2D-aggregation process, including bovine serum albumin, insulin, fibrinogen, hemoglobin, lactoferrin, and ovalbumin. Thus, our findings on the non-fibril self-adaptive mechanism for amyloid aggregation may shed light on polymorphic amyloid assembly and their adhesions through an alternative pathway.

Biochemistry of the Thrombin-Like Enzyme and Its Purification from Iranian Echis Carinatus Snake Venom: Its Interaction with Platelet Receptors.

Snake venoms are rich in valuable substances that have medical potential in the diagnosis and treatment of hemostatic diseases. The present paper was aimed at the purification and functional characterization basis of a thrombin-like enzyme and its role in the functioning of the coagulation cascade and platelet aggregation pathway. A thrombin-like serine protease was purified from the Iranian Echis carinatus venom (TLIECV), employing a one-step chromatographic procedure. This peptide was collected in high yield and purity by a single chromatographic step using RP-HPLC equipped with a C18 column. This peptide showed a 3000 Da molecular weight in gel-electrophoresis. Evidence in the SDS-PAGE gel has confirmed high recovery of fraction in optimal terms. Subsequently, this peptide was identified via its intact molecular mass and peptide mass fingerprint (PMF) using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS). Multiple sequence alignments were performed by ClustalW, the Bioedit software. Molegro Data Modeller (MDM) 3.0 software was used to predict the putative tertiary structure of the peptide. The enzyme possessed fibrinogenolytic, procoagulant, and aggregation inducer properties. Moreover, the SDS-PAGE (12%) was applied to examine fibrinogenolytic function. The purified enzyme degraded the Aα chain of fibrinogen while the Bβ and γ chains were not digested. According to that, the deficient human plasma in factor X and normal human plasma were also coagulated by TLIECV, it takes part in the common and intrinsic routes of the coagulation cascade. These findings proved that TLIECV is a serine protease identical to procoagulant thrombin-like snake venom proteases; however, it specifically releases the Aα chain of bovine fibrinogen. Because of its function to make up for the deficiency of factor X and its platelet aggregation inducer property, TLIECV could be considered a molecular impact to reveal the hemostasis mechanisms.

Preparation of aminated poly (L‐lactic acid) film coated with multilayers of mussel‐inspired heparin‐like carboxymethylcellulose sodium and chitosan for anticoagulant materials

AbstractIn this study, dopamine hydrochloride was grafted onto the sulfated carboxymethylcellulose sodium with different sulfated degree (DA‐g‐SCMCS) as heparin‐like polysaccharides. By studying the preliminary structure and coagulation time of DA‐g‐SCMCS, the DA‐g‐SCMC2 possessing good anticoagulant properties were selected to be assembled layer by layer on the surface of aminated poly (L‐lactic acid) (APLLA) film with chitosan (CS). The characterization properties including morphologies and wettability of DA‐g‐SCMC2/CS multilayers modified APLLA films were studied by scanning electron microscope and water contact angle instrument. Anticoagulation tests revealed that the (DA‐g‐SCMC2/CS)15 multilayers modified APLLA film experienced a decrease of 37.5% and 73.68% in the adsorption of bovine serum albumin (BSA) and fibrinogen (FBG), respectively. And the platelet adhesion ability was reduced by 92.42% compared with the APLLA film. The degree of platelet activation (PF4 and TAT) and hemolysis rates of modified APLLA films also decreased significantly. Besides, the modified APLLA films promoted the growth of L929 cells and exhibited remarkable anti‐inflammatory and antibacterial abilities. To summarize, the APLLA films modified with heparin‐like multifunctional DA‐g‐SCMC2/CS multilayers exhibit outstanding biocompatibility, cytocompatibility, anti‐inflammatory, and antibacterial characteristics, showcasing their potential as anticoagulation materials.

Tuning physical properties of polyurethane hard segments through extender chain length variation and doping of pentaerythritol and their effect on in vitro protein adsorption

AbstractThis study explores the synthesis and characterization of polyurethanes (PUs) derived from hexamethylene diisocyanate (HDI) and 1,n‐alkane diols with varying chain lengths (n = 4, 6, and 10). Additionally, pentaerythritol (PE) is introduced as a dopant in PU6 at different weight percentages (25%, 50%, 75%, and 1:1%w/w). The research encompasses a comprehensive analysis of PU properties, including morphology, crystallinity, surface area, porosity, thermal behavior, rheological properties, and electrical conductivity. Of particular interest is the evaluation of protein adsorption capabilities employing bovine serum albumin (BSA) and fibrinogen proteins in in vitro tests. The study emphasizes the crucial role played by the chain length between isocyanate and diol groups and the nature and strength of hydrogen bonds among chains in shaping the polymer's properties, especially crystallinity and biocompatibility. Among the synthesized PUs, PU6 emerges as the top performer in terms of crystallinity and biocompatibility. Furthermore, the addition of PE is found to act as a plasticizer, reducing the glass transition temperature (Tg) from 75°C to 31°C. Electrochemical Impedance Spectroscopy shows that doping influences charge transfer processes, rendering the material semi‐conducting, as evidenced by decreased conductance when adding silver nanoparticles to PU6. Notably, protein adsorption studies reveal that undoped PU6 displays superior protein resistance compared to its doped counterpart, with fibrinogen exhibiting a higher adsorption affinity than BSA. The study discusses a plausible mechanism underlying protein adsorption.

Thermoplastic polyurethane surface coated with polymer brushes for reduced protein and cell attachment.

The objective of this study was to coat negatively charged polymer brushes covalently onto the surface of thermoplastic polyurethane (TPU) using a simple conventional surface free-radical polymerization technique. The coated surfaces were assessed with contact angle, protein adsorption, cell adhesion and bacterial adhesion. Bovine serum albumin (BSA) and bovine fibrinogen (BFG) were used for protein adsorption evaluation. Mouse fibroblasts (NIH-3T3) and Pseudomonas aeruginosa (P. aeruginosa) were used to assess surface adhesion. Results show that the TPU surface modified with the attached polymer brushes exhibited significantly reduced contact angle, protein adsorption, and cell as well as bacterial adhesion, among which the negatively charged polymers showed the extremely low values in all the tests. Its contact angle is 5°, as compared to 70° for original TPU. Its BSA, BFG, 3T3 adhesion and P. aeruginosa adhesion were 93%, 84%, 92%, and 93% lower than original TPU. Furthermore, the TPU surface coated with negatively charged polymer brushes exhibited a hydrogel-like property. The results indicate that placing acrylic acids using a simple surface-initiated free-radical polymerization onto a TPU surface and then converting those to negative charges can be an effective and efficient route for fouling resistant applications.

Polyvinylchloride surface modified with polymer brushes for reduced protein, cell and bacteria attachment

AbstractThe objective of this study was to coat negatively charged polymer brushes by a covalent bond onto the surface of polyvinylchloride using a simple conventional surface free radical polymerisation technique. The coated surfaces were assessed with contact angle, protein adsorption, cell adhesion, and bacterial adhesion. Bovine serum albumin and bovine fibrinogen were used for protein adsorption evaluation. Mouse fibroblast (NIH‐3T3) cells and Pseudomonas aeruginosa were used to assess surface adhesion. Results show that the surface modified with all the attached polymers exhibited significantly reduced contact angle, protein adsorption, and cell as well as bacterial adhesion among which the negatively charged polymers showed extremely low values in all the tests. The negatively charged polymer shows its contact angle at 5° as compared to 70° for original polyvinylchloride. Its bovine serum albumin, bovine fibrinogen, 3T3 adhesion, and P. aeruginosa adhesion were 93%, 84%, 92%, and 92% lower than the original PVC, respectively. Furthermore, the polyvinylchloride surface coated with negatively charged polymer brushes exhibited a hydrogel‐like property. The results indicate that coating a polyvinylchloride surface with acrylic acids using a simple surface‐initiated free radical polymerisation and then converting those to negatively charged salts can be an effective and efficient route for fouling‐resistant applications.

Polyvinyl chloride surface coated with polymer brushes using a chain‐transfer reaction for enhanced fouling resistance

AbstractThis study reports coating negatively charged polymer brushes covalently onto the surface of polyvinylchloride (PVC) using a simple conventional surface free‐radical polymerization technique with help of a chain transfer agent. The coated surfaces were assessed with contact angle, protein adsorption, cell adhesion and bacterial adhesion. Bovine serum albumin (BSA) and bovine fibrinogen (BFG) were used for protein adsorption evaluation. Mouse fibroblast (NIH‐3 T3) cells and Pseudomonas aeruginosa (P. aeruginosa) were used to assess cell and bacterial adhesion. Results show that the surface modified with all the attached polymer brushes exhibited significantly reduced contact angle, protein adsorption, and cell as well as bacterial adhesion, among which the negatively charged polymers showed the extremely low values in all the tests. The negatively charged polymer shows its contact angle at 4.6° as compared to 70° for original PVC. Its BSA, BFG, 3 T3 adhesion and P. aeruginosa adhesion were 93%, 84%, 92%, and 95% lower than original PVC. Furthermore, the PVC surface coated with negatively charged polymer brushes exhibited a hydrogel‐like property. The results indicate placing acrylic acids using a simple surface‐initiated free‐radical polymerization with help of a chain transfer agent onto PVC surface and then converting the acids to negative charges can be an affective and efficient route for fouling resistant applications.

Antifouling Surfaces Based on Polyzwitterion Loop Brushes.

Antifouling surfaces have attracted increasing interest in recent years due to their potential application in various fields. In this work, we report a loop polyzwitterionic coating that exhibits excellent resistance to protein adsorption. Triblock and diblock copolymers of 2-[(2-hydroxyethyl)disulfanyl]ethyl methacrylate) (HSEMA) and 2-(dimethylamino)ethyl methacrylate) (DMAEMA) were synthesized by atom-transferred radical polymerization, followed by betainization of the DMAEMA block with 1,3-propane sultone and reduction of the disulfide bond in HSEMA to yield a triblock copolymer comprising a zwitterionic poly(sulfobetaine methacrylate) (PSBMA) midblock and poly(2-sulfanylethyl methacrylate) (PSEMA) terminal blocks as well as its diblock analogue that was of the same composition as the former and half the chain length. Both copolymers adsorbed to the gold substrate via the thiol groups in the terminal PSEMA block(s), creating loop and linear PSBMA brush coatings of comparable thickness, as revealed by X-ray photoelectron spectroscopy and ellipsometry. Adsorption of bovine serum albumin and fibrinogen as model proteins from solution to these surfaces was investigated by a quartz crystal microbalance with dissipation and confocal laser scanning microscopy (CLSM), and platelet and bacterial adhesions were assessed by scanning electron microscopy and CLSM. The results demonstrate that both linear and loop polyzwitterion brushes are excellent in resisting the adsorption of the foulants, and the loop brushes are superior to the linear analogues.

Enhancement of protein immobilization on polydimethylsiloxane using a synergistic combination of polydopamine and micropattern surface modification

Understanding protein interactions at biointerfaces is critical for the improved design of biomaterials and medical devices. Polydimethylsiloxane (PDMS) is used for numerous device applications, and surface modifications can enhance protein immobilization and the response to cells. A multifunctional approach combining topographical and biochemical modifications was applied to PDMS by fabricating 10–20 µm scale patterns onto PDMS surfaces and by coating with polydopamine (PDA). The modifications were confirmed by surface characterization and bovine serum albumin (BSA), fibrinogen (Fg), and fetuin-A (Fet-A) were radiolabeled with 125I. The amounts of protein attached to the surface before and after elution with sodium dodecyl sulfate (SDS) were quantified from single and complex multi-protein solutions to determine protein stability and competitive binding. The PDA coatings were the most stable and capable of immobilizing the highest levels of all proteins. Furthermore, combinations of PDA coatings with the smallest micropatterns provided an additional improvement, enhancing the amount immobilized and the stability. The adsorption of BSA and Fg from plasma demonstrated competitive binding and possible orientation changes, respectively. It was determined that Fet-A, a less studied protein, adsorbed from plasma at low levels, but the adsorption from fetal bovine serum (FBS) was significantly greater, providing important quantification data from radiolabeling that is relevant to many cell culture studies. Overall, combining topography and PDA modification has a synergistic effect on improving protein immobilization. These findings provide new insight on the quantities of proteins bound to PDMS and PDA coatings with implications for cell interactions in various biotechnology and medical applications.

Size Matters? A Comprehensive In Vitro Study of the Impact of Particle Size on the Toxicity of ZnO.

This study describes a comparative in vitro study of the toxicity behavior of zinc oxide (ZnO) nanoparticles and micro-sized particles. The study aimed to understand the impact of particle size on ZnO toxicity by characterizing the particles in different media, including cell culture media, human plasma, and protein solutions (bovine serum albumin and fibrinogen). The particles and their interactions with proteins were characterized in the study using a variety of methods, including atomic force microscopy (AFM), transmission electron microscopy (TEM), and dynamic light scattering (DLS). Hemolytic activity, coagulation time, and cell viability assays were used to assess ZnO toxicity. The results highlight the complex interactions between ZnO NPs and biological systems, including their aggregation behavior, hemolytic activity, protein corona formation, coagulation effects, and cytotoxicity. Additionally, the study indicates that ZnO nanoparticles are not more toxic than micro-sized particles, and the 50 nm particle results were, in general, the least toxic. Furthermore, the study found that, at low concentrations, no acute toxicity was observed. Overall, this study provides important insights into the toxicity behavior of ZnO particles and highlights that no direct relationship between nanometer size and toxicity can be directly attributed.

Toward Protein-Repellent Surface Coatings from Catechol-Containing Cationic Poly(2-ethyl-2-oxazoline)

Inspired by mussel proteins that enable surface binding in harsh marine environments, we envisioned a platform of protein-repellent macromolecules based on poly(2-ethyl-2-oxazoline) carrying catechol and cationic functional groups. To facilitate surface attachment, catechol units were installed by copolymerizing a functional comonomer, i.e., 2-(3,4-dimethoxyphenyl)-2-oxazoline, in a gradient fashion. Cationic units were introduced by partial acidic hydrolysis. The surface affinity of these polymers was probed using a quartz crystal microbalance with dissipation monitoring (QCM-D), and it was found that polymers with catechol units had a strong tendency to form surface-bound layers on different substrates, i.e., gold, iron, borosilicate, and polystyrene. While the neutral catechol-containing polymers showed strong, but uncontrolled binding, the ones with additional cationic units were able to form defined and durable polymer films. These coatings were able to prevent the attachment of different model proteins, i.e., bovine serum albumin (BSA), fibrinogen (FI), or lysozyme (LYZ). The herein-introduced platform offers straightforward access to nonfouling surface coatings using a biomimetic approach.

English

Actinobacillus seminis is a Gram-negative bacterium member of the Pasteurellaceae family. Even though it is part of the ovine microbiota it is also the causal agent of genital organs affections, particularly epididymitis and orchitis. Infections caused by this microorganism generate economic losses to the ovine industry due to impaired fertility and sterility in affected animals. Knowledge about virulence factors expressed by A. seminis is scarce. In the present work, we describe the expression of a metalloprotease secreted by A. seminis that can degrade bovine fibrinogen and immunoglobulin G and present homology with a carboxy-terminal protease from A. seminis. This metalloprotease presents an optimal activity at a pH between 6 and 7, is stable up to 60°C, inactive at higher temperatures, and completely inhibited by 30 mM EDTA. The expression of this proteolytic activity is controlled by temperature, calcium, and iron. Proteases degrading extracellular matrix components and molecules involved in the immune response could facilitate and improve host colonization and invasion by A. seminis