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

Abstract

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.

Figure 1

A. Diagram of a model respiratory device employed for measuring CO₂ removal rates of unmodified and modified HFMs. Bovine blood or PBS was perfused over the outside of the fibers while oxygen sweep gas was passed through the fiber lumens in the opposite direction. B. Experimental setup for the in vitro CO₂ gas exchange assessment. Both the blood reservoir and de-oxygenator employed the use of a heat exchanger to maintain blood temperature at 37C°.

Figure 2

Esterase activity units of CA-modified HFMs. Traditional CNBr (Kaar et al.) activation reflects the activity level resulting from our previous work. CNBr/TEA activation demonstrates the enhanced activity of the optimized method. *p < 0.005

Figure 3

Increasing immobilized CA activity results in a proportional increase in CO₂ removal from PBS. (N=2)

Figure 4

CO₂ removal by unmodified (control) and modified (CA immobilized) HFMs in bovine blood in a model respiratory assist device (N=4). The modified fibers demonstrated a 36% increase in CO₂ removal capacity. *p <0.005

Figure 5

Platelet deposition onto surfaces after contact with ovine blood for 2 h as determined by a lactate dehydrogenase (LDH) assay (N=3).

Figure 6

SEM images of testing hollow fiber membrane surfaces after contact with heparinized ovine blood for 2 h at 37°C. (A) Unmodified PMP; (B) CA-modified PMP. Images were recorded using an accelerating voltage of 5 kV at a magnification of 1,000× (scale bar = 10 μm).