Jeremy D. Kimmel

Acidic sweep gas with carbonic anhydrase coated hollow fiber membranes synergistically accelerates CO2 removal from blood.

UNLABELLED The use of extracorporeal carbon dioxide removal (ECCO2R) is well established as a therapy for patients suffering from acute respiratory failure. Development of next generation low blood flow (<500 mL/min) ECCO2R devices necessitates more efficient gas exchange devices. Since over 90% of blood CO2 is transported as bicarbonate (HCO3(-)), we previously reported development of a carbonic anhydrase (CA) immobilized bioactive hollow fiber membrane (HFM) which significantly accelerates CO2 removal from blood in model gas exchange devices by converting bicarbonate to CO2 directly at the HFM surface. This present study tested the hypothesis that dilute sulfur dioxide (SO2) in oxygen sweep gas could further increase CO2 removal by creating an acidic microenvironment within the diffusional boundary layer adjacent to the HFM surface, facilitating dehydration of bicarbonate to CO2. CA was covalently immobilized onto poly (methyl pentene) (PMP) HFMs through glutaraldehyde activated chitosan spacers, potted in model gas exchange devices (0.0151 m(2)) and tested for CO2 removal rate with oxygen (O2) sweep gas and a 2.2% SO2 in oxygen sweep gas mixture. Using pure O2 sweep gas, CA-PMP increased CO2 removal by 31% (258 mL/min/m(2)) compared to PMP (197 mL/min/m(2)) (P<0.05). Using 2.2% SO2 acidic sweep gas increased PMP CO2 removal by 17% (230 mL/min/m(2)) compared to pure oxygen sweep gas control (P<0.05); device outlet blood pH was 7.38 units. When employing both CA-PMP and 2.2% SO2 sweep gas, CO2 removal increased by 109% (411 mL/min/m(2)) (P<0.05); device outlet blood pH was 7.35 units. Dilute acidic sweep gas increases CO2 removal, and when used in combination with bioactive CA-HFMs has a synergistic effect to more than double CO2 removal while maintaining physiologic pH. Through these technologies the next generation of intravascular and paracorporeal respiratory assist devices can remove more CO2 with smaller blood contacting surface areas. STATEMENT OF SIGNIFICANCE A clinical need exists for more efficient respiratory assist devices which utilize low blood flow rates (<500 mL/min) to regulate blood CO2 in patients suffering from acute lung failure. Literature has demonstrated approaches to chemically increase hollow fiber membrane (HFM) CO2 removal efficiency by shifting equilibrium from bicarbonate to gaseous CO2, through either a bioactive carbonic anhydrase enzyme coating or bulk blood acidification with lactic acid. In this study we demonstrate a novel approach to local blood acidification using an acidified sweep gas in combination with a bioactive coating to more than double CO2 removal efficiency of HFM devices. To our knowledge, this is the first report assessing an acidic sweep gas to increase CO2 removal from blood using HFM devices.

Hollow fiber membrane modification with functional zwitterionic macromolecules for improved thromboresistance in artificial lungs.

Respiratory assist devices seek optimized performance in terms of gas transfer efficiency and thromboresistance to minimize device size and reduce complications associated with inadequate blood biocompatibility. The exchange of gas with blood occurs at the surface of the hollow fiber membranes (HFMs) used in these devices. In this study, three zwitterionic macromolecules were attached to HFM surfaces to putatively improve thromboresistance: (1) carboxyl-functionalized zwitterionic phosphorylcholine (PC) and (2) sulfobetaine (SB) macromolecules (mPC or mSB-COOH) prepared by a simple thiol-ene radical polymerization and (3) a low-molecular weight sulfobetaine (SB)-co-methacrylic acid (MA) block copolymer (SBMAb-COOH) prepared by reversible addition-fragmentation chain transfer (RAFT) polymerization. Each macromolecule type was covalently immobilized on an aminated commercial HFM (Celg-A) by a condensation reaction, and HFM surface composition changes were analyzed by X-ray photoelectron spectroscopy. Thrombotic deposition on the HFMs was investigated after contact with ovine blood in vitro. The removal of CO2 by the HFMs was also evaluated using a model respiratory assistance device. The HFMs conjugated with zwitterionic macromolecules (Celg-mPC, Celg-mSB, and Celg-SBMAb) showed expected increases in phosphorus or sulfur surface content. Celg-mPC and Celg-SBMAb experienced rates of platelet deposition significantly lower than those of unmodified (Celg-A, >95% reduction) and heparin-coated (>88% reduction) control HFMs. Smaller reductions were seen with Celg-mSB. The CO2 removal rate for Celg-SBMAb HFMs remained comparable to that of Celg-A. In contrast, the rate of removal of CO2 for heparin-coated HFMs was significantly reduced. The results demonstrate a promising approach to modifying HFMs using zwitterionic macromolecules for artificial lung devices with improved thromboresistance without degradation of gas transfer.

IL-6 Adsorption Dynamics in Hemoadsorption Beads Studied Using Confocal Laser Scanning Microscopy

Sepsis is characterized by a systemic inflammatory response caused by infection, and can result in organ failure and death. Removal of inflammatory mediators such as cytokines from the circulating blood is a promising treatment for severe sepsis. We are developing an extracorporeal hemoadsorption device to remove cytokines from the blood using biocompatible, polymer sorbent beads. In this study, we used confocal laser scanning microscopy (CLSM) to directly examine adsorption dynamics of a cytokine (IL-6) within hemoadsorption beads. Fluorescently labeled IL-6 was incubated with sorbent particles, and CLSM was used to quantify spatial adsorption profiles of IL-6 within the sorbent matrix. IL-6 adsorption was limited to the outer 15 microm of the sorbent particle over a relevant clinical time period, and intraparticle adsorption dynamics was modeled using classical adsorption/diffusion mechanisms. A single model parameter, alpha = q(max) K/D, was estimated by fitting CLSM intensity profiles to our mathematical model, where q(max) and K are Langmuir adsorption isotherm parameters, and D is the effective diffusion coefficient of IL-6 within the sorbent matrix. Given the large diameter of our sorbent beads (450 microm), less than 20% of available sorbent surface area participates in cytokine adsorption. Development of smaller beads may accelerate cytokine adsorption by maximizing available surface area per bead mass.

Effects of hemoadsorption with a novel adsorbent on sepsis: in vivo and in vitro study.

Background/Aims: Hemoadsorption may improve outcomes for sepsis by removing circulating cytokines. We tested a new sorbent used for hemoadsorption. Methods: CTR sorbent beads were filled into columns of three sizes: CTR0.5 (0.5 ml), CTR1 (1.0 ml) and CTR2 (2.0 ml) and tested using IL-6 capture in vitro. Next, rats were subjected to cecal ligation and puncture and randomly assigned to hemoadsorption with CTR0.5, CTR1, CTR2 or sham treatment. Plasma biomarkers were measured. Results: In vitro, IL-6 removal was accelerated with increasing bead mass. In vivo, TNF, IL-6, IL-10, high mobility group box1, and cystatin C were significantly lower 24 h after CTR2 treatment. Seven-day survival rate was 50, 64, 63, and 73% for the sham, CTR0.5, CTR1, CTR2, respectively. Conclusion: CTR appeared to have a favorable effect on kidney function despite no immediate effects on cytokine removal. However, CTR2 beads did result in a late decrease of cytokines.

Modeling competitive cytokine adsorption dynamics within hemoadsorption beads used to treat sepsis.

Extracorporeal blood purification is a promising therapeutic modality for sepsis, a potentially fatal, dysfunctional immunologic state caused by infection. Removal of inflammatory mediators such as cytokines from the blood may help attenuate hyper-inflammatory signaling during sepsis and improve patient outcomes. We are developing a hemoadsorption device to remove cytokines from the circulating blood using biocompatible, porous sorbent beads. In this work, we investigated whether competitive adsorption of serum solutes affects cytokine removal dynamics within the hemoadsorption beads. Confocal laser scanning microscopy (CLSM) was used to quantify intraparticle adsorption profiles of fluorescently labeled IL-6 in horse serum, and results were compared to predictions of a two component competitive adsorption model. Supraphysiologic IL-6 concentrations were necessary to obtain adequate CLSM signal, therefore unknown model parameters were fit to CLSM data at high IL-6 concentrations, and the fitted model was used to simulate cytokine adsorption behavior at physiologically relevant levels which were below the microscopy detection threshold. CLSM intraparticle IL-6 adsorption profiles agreed with predictions of the competitive adsorption model, indicating displacement of cytokine by high affinity serum solutes. However, competitive adsorption effects were predicted using the model to be negligible at physiologic cytokine concentrations associated with hemoadsorption therapy.

Characterizing accelerated capture of deoligomerized TNF within hemoadsorption beads used to treat sepsis

Sepsis is a systemic inflammatory response to infection, characterized by overexpression of cytokines in the circulating blood. Removal of cytokines and other inflammatory mediators from the blood may help attenuate systemic inflammation during sepsis and improve patient outcomes. In this work, we examined the dynamics of TNF capture within porous, polymeric sorbent beads used in a cytokine adsorption device. We sought to quantify how perturbation of TNF oligomeric structure accelerates TNF removal within the device. TNF was incubated with 10% DMSO for 24 h, which promoted complete monomerization of trimeric TNF, and accelerated TNF capture within the sorbent device compared with native TNF; removal halftime = 13.3 ± 1.5 min versus 112.8 ± 13.3 min, respectively. Intramolecular crosslinking stabilized the trimeric TNF structure and prevented DMSO monomerization. Results demonstrate that TNF is an unstable oligomeric molecule that can be dissociated into its smaller monomeric constituents to facilitate faster capture by hemoadsorption beads. Strategies to promote localized TNF deoligomerization at the sorbent surface may significantly accelerate TNF capture rates from the circulating blood using hemoadsorption as a treatment for sepsis. This concept could be extended to improve removal of other oligomeric molecules using size exclusion filtration materials for a variety of disease states.

Dynamics of Cytokine Capture Within Hemoadsorption Beads Used to Treat Sepsis

Sepsis is a serious medical condition characterized by systemic inflammation caused by infection, and affects more than 750,000 individuals per year in the US, with a mortality rate of approximately 30% [1]. The pathophysiology of sepsis is complex and not entirely understood, but is believed to be related to the dysfunction of multiple interdependent humoral mediator pathways, including redundant release of inflammatory cytokines [2]. Removal of both pro- and anti-inflammatory cytokines from the circulating blood is believed to be a promising therapy for severe sepsis [3]. We are developing an extracorporeal hemoadsorption device to remove cytokines from the blood using a novel, biocompatible, sorbent bead technology. A simple model was developed to characterize cytokine adsorption within hemoadsorption beads [4]. Despite rapid clearance of cytokines with hemoadsorption in an ex vivo murine sepsis model [5], our model analysis predicted that only the outer 20μm of each sorbent bead (avg diam = 450μm) adsorbed cytokine. In this work, we used in vitro column capture experiments and confocal laser scanning microscopy (CLSM) to examine cytokine adsorption dynamics within hemoadsorption beads.Copyright