Current artificial lungs and respiratory assist devices designed for carbon dioxide removal (CO2R) 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 CO2 directly at the HFM surface. In this study, we tested the impact of CA-immobilization on HFM CO2 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 m2) and tested in a recirculation loop with a CO2 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/m2 CO2 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 CO2 removal can be realized through bioactive HFMs, enabling a next generation of more efficient CO2 removal intravascular and paracorporeal respiratory assist devices.
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 as the blood-gas interface and can require large blood-contacting membrane area. To offset the underlying diffusional challenge, "bioactive" hollow fiber membranes that facilitate CO 2 diffusion were prepared via covalent immobilization of carbonic anhydrase, an enzyme which catalyzes the conversion of bicarbonate in blood to CO 2 , onto the surface of plasma-modified conventional hollow fiber membranes. 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 carbonic anhydrase could be attached, did not change gas permeance of the hollow fiber membranes 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 carbonic anhydrase 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 hollow fiber membranes with immobilized carbonic anhydrase to enhance CO 2 exchange in respiratory devices.
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.
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