Immobilized Carbonic Anhydrase on Hollow Fiber Membranes Accelerates CO(2) Removal from Blood.

Current artificial lungs and respiratory assist devices designed for carbon dioxide removal (CO(2)R) are limited in their efficiency due to the relatively small partial pressure difference across gas exchange membranes. To offset this underlying diffusional challenge, bioactive hollow fiber membranes (HFMs) increase the carbon dioxide diffusional gradient through the immobilized enzyme carbonic anhydrase (CA), which converts bicarbonate to CO(2) directly at the HFM surface. In this study, we tested the impact of CA-immobilization on HFM CO(2) removal efficiency and thromboresistance in blood. Fiber surface modification with radio frequency glow discharge (RFGD) introduced hydroxyl groups, which were activated by 1M CNBr while 1.5M TEA was added drop wise over the activation time course, then incubation with a CA solution covalently linked the enzyme to the surface. The bioactive HFMs were then potted in a model gas exchange device (0.0084 m(2)) and tested in a recirculation loop with a CO(2) inlet of 50mmHg under steady blood flow. Using an esterase activity assay, CNBr chemistry with TEA resulted in 0.99U of enzyme activity, a 3.3 fold increase in immobilized CA activity compared to our previous method. These bioactive HFMs demonstrated 108 ml/min/m(2) CO(2) removal rate, marking a 36% increase compared to unmodified HFMs (p < 0.001). Thromboresistance of CA-modified HFMs was assessed in terms of adherent platelets on surfaces by using lactate dehydrogenase (LDH) assay as well as scanning electron microscopy (SEM) analysis. Results indicated HFMs with CA modification had 95% less platelet deposition compared to unmodified HFM (p < 0.01). Overall these findings revealed increased CO(2) removal can be realized through bioactive HFMs, enabling a next generation of more efficient CO(2) removal intravascular and paracorporeal respiratory assist devices.

Hemocompatibility assessment of carbonic anhydrase modified hollow fiber membranes for artificial lungs.

Hollow fiber membrane (HFM)-based artificial lungs can require a large blood-contacting membrane surface area to provide adequate gas exchange. However, such a large surface area presents significant challenges to hemocompatibility. One method to improve carbon dioxide (CO(2)) transfer efficiency might be to immobilize carbonic anhydrase (CA) onto the surface of conventional HFMs. By catalyzing the dehydration of bicarbonate in blood, CA has been shown to facilitate diffusion of CO(2) toward the fiber membranes. This study evaluated the impact of surface modifying a commercially available microporous HFM-based artificial lung on fiber blood biocompatibility. A commercial poly(propylene) Celgard HFM surface was coated with a siloxane, grafted with amine groups, and then attached with CA which has been shown to facilitate diffusion of CO(2) toward the fiber membranes. Results following acute ovine blood contact indicated no significant reduction in platelet deposition or activation with the siloxane coating or the siloxane coating with grafted amines relative to base HFMs. However, HFMs with attached CA showed a significant reduction in both platelet deposition and activation compared with all other fiber types. These findings, along with the improved CO(2) transfer observed in CA modified fibers, suggest that its incorporation into HFM design may potentiate the design of a smaller, more biocompatible HFM-based artificial lung.

Surface modification of a titanium alloy with a phospholipid polymer prepared by a plasma-induced grafting technique to improve surface thromboresistance.

To improve the thromboresistance of a titanium alloy (TiAl(6)V(4)) surface which is currently utilized in several ventricular assist devices (VADs), a plasma-induced graft polymerization of 2-methacryloyloxyethyl phosphorylcholine (MPC) was carried out and poly(MPC) (PMPC) chains were covalently attached onto a TiAl(6)V(4) surface by a plasma induced technique. Cleaned TiAl(6)V(4) surfaces were pretreated with H(2)O-vapor-plasma and silanated with 3-methacryloylpropyltrimethoxysilane (MPS). Next, a plasma-induced graft polymerization with MPC was performed after the surfaces were pretreated with Ar plasma. Surface compositions were verified by X-ray photoelectron spectroscopy (XPS). In vitro blood biocompatibility was evaluated by contacting the modified surfaces with ovine blood under continuous mixing. Bulk phase platelet activation was quantified by flow cytometric analysis, and surfaces were observed with scanning electron microscopy after blood contact. XPS data demonstrated successful modification of the TiAl(6)V(4) surfaces with PMPC as evidenced by increased N and P on modified surfaces. Platelet deposition was markedly reduced on the PMPC grafted surfaces and platelet activation in blood that contacted the PMPC-grafted samples was significantly reduced relative to the unmodified TiAl(6)V(4) and polystyrene control surfaces. Durability studies under continuously mixed water suggested no change in surface modification over a 1-month period. This modification strategy shows promise for further investigation as a means to reduce the thromboembolic risk associated with the metallic blood-contacting surfaces of VADs and other cardiovascular devices under development.

Towards improved artificial lungs through biocatalysis.

Inefficient CO(2) removal due to limited diffusion represents a significant barrier in the development of artificial lungs and respiratory assist devices, which use hollow fiber membranes (HFMs) as the blood-gas interface and can require large blood-contacting membrane area. To offset the underlying diffusional challenge, "bioactive" HFMs that facilitate CO(2) diffusion were prepared via covalent immobilization of carbonic anhydrase (CA), an enzyme which catalyzes the conversion of bicarbonate in blood to CO(2), onto the surface of plasma-modified conventional HFMs. This study examines the impact of enzyme attachment on the diffusional properties and the rate of CO(2) removal of the bioactive membranes. Plasma deposition of surface reactive hydroxyls, to which CA could be attached, did not change gas permeance of the HFMs or generate membrane defects, as determined by scanning electron microscopy, when low plasma discharge power and short exposure times were employed. Cyanogen bromide activation of the surface hydroxyls and subsequent modification with CA resulted in near monolayer enzyme coverage (88%) on the membrane. The effect of increased plasma discharge power and exposure time on enzyme loading was negligible while gas permeance studies showed enzyme attachment did not impede CO(2) or O(2) diffusion. Furthermore, when employed in a model respiratory assist device, the bioactive membranes improved CO(2) removal rates by as much as 75% from physiological bicarbonate solutions with no enzyme leaching. These results demonstrate the potential of bioactive HFMs with immobilized CA to enhance CO(2) exchange in respiratory devices.

Synthetic blood group antigens for anti-A removal device and their interaction with monoclonal anti-A IgM.

Removal of blood group antibodies against the donor organ prior to ABO-incompatible transplantation can prevent episodes of hyperacute rejection. We are developing a specific antibody filter (SAF) device consisting of immobilized synthetic Atrisaccharide antigens conjugated to polyacrylamide (Atri-PAA) to selectively remove anti-A antibodies directly from whole blood. In this study, we evaluated eight anti-A IgM monoclonal antibodies (mAbs) using Enzyme-Linked Immunosorbent Assay (ELISA) to determine their specificity for binding to Atri-PAA. Five of the eight mAbs met our criteria for specificity by binding to Atri-PAA with at least five times greater affinity compared to the negative controls. These selected mAbs will be studied for their binding characteristics to Atri-PAA which will aid in the development of the SAF. The study of kinetics of antibody removal and quantification of antibody removal will be used in our mathematical model to maximize the antibody removal rate and binding capacity of the SAF.

Development of a membrane strip immunosensor utilizing ruthenium as an electro-chemiluminescent signal generator.

A photometric immunosensor that can be used for on-site diagnosis has been constructed. The sensor system was assembled by partially superimposing a nitrocellulose membrane strip (the lower) containing an immobilized antigen on the surface with a glass fiber membrane strip (the upper) including two electrodes on the opposite surfaces. To amplify the signal, we introduced a liposome, containing ruthenium molecules trapped in the core, chemically coupled to an antibody specific to the analyte (e.g. Legionella antigen). In the presence of the analyte, immune complexes were formed by antigen-antibody reactions upon addition of the immuno-liposome into a sample. This mixture was then absorbed by the capillary action from the bottom of the membrane strip. The liposome particles in the complexes were carried by a medium through the antigen pad without interaction, while free immuno-liposome was trapped by immune reactions on the pad surfaces. The aqueous medium influx into the glass pad dissolved a detergent pre-located within the compartment and the liposome rupture thereby released ruthenium molecules into the solution. The molecules were oxidized on the electrode surfaces and produced an electro-chemiluminescence (ECL) in proportion to the analyte concentration. The signal generation based on ECL resulted in an exponential dose-response pattern and the analyte detection limit of 2 ng/ml was approximately 10-fold more sensitive than that obtained from a conventional system.