Microspheres Based Detoxification System Research Articles

Preparation and characterization of cellulose microspheres

The aim of this work was to synthesize and characterize cellulose microspheres with a particle size below 5 μm and narrow size distribution. After activation and functionalization with antibodies, these particles shall be applied as adsorbents in suspension-based extracorporeal blood purification systems, such as the Microspheres-Based Detoxification System. In the frame of this work such microspheres were developed and synthesized with reproducible properties. Besides using well-established methods for characterization of this kind of bead cellulose, additional procedures for the examination of its properties were developed and applied.

Functionalization and Application of Cellulose Microparticles as Adsorbents in Extracorporeal Blood Purification

AbstractTherapeutic apheresis is established as supportive therapy for various diseases, such as hypercholesterolemia, autoimmune diseases, liver failure, and sepsis. In combined membrane‐adsorption systems, the patient's plasma is continuously separated from whole blood by means of a hollow fiber filter, and pathogenic factors are removed from the plasma by selective or specific adsorbents. While adsorbent particles with a size range of 300–800 µm are used in conventional systems, we are currently developing a system based on adsorbent microparticles (1–5 µm), the Microspheres‐Based Detoxification System (MDS). The characteristics of the matrix used for immobilization of specific ligands influence the performance of the resulting adsorbents. Desirable matrix characteristics are an open porous structure with an inner surface accessible for target molecules, mechanical stability, narrow particle size distribution, and ease of derivatization. In addition, biocompatibility is a critical issue, since the particles are in direct contact with the patient's plasma. Cellulose represents an ideal support matrix, as it combines all the above‐mentioned features, and cellulosic polymers are widely applied in medicine and generally regarded as biocompatible. Cellulose microparticles can be activated using e.g. sodium periodate and functionalized with Polymyxin B or anti‐tumor necrosis factor (TNF) antibodies to generate specific adsorbents for endotoxins or TNF. In summary, cellulose microparticles represent an excellent matrix as basis for adsorbent development in blood purification.

Theoretical analysis of ferromagnetic microparticles in streaming liquid under the influence of external magnetic forces

The microsphere based detoxification system (MDS) is designed for high specific toxin removal in extracorporeal blood purification using functionalized microparticles. A thin wall hollow fiber membrane filter separates the microparticle–plasma suspension from the bloodstream. For patient safety, it is necessary to have a safety system to detect membrane ruptures that could lead to the release of microparticles into the bloodstream. A non-invasive optical detection system including a magnetic trap is developed to monitor the extracorporeal venous bloodstream for the presence of released microparticles. For detection, fluorescence-labeled ferromagnetic beads are suspended together with adsorbent particles in the MDS circuit. In case of a membrane rupture, the labeled particles would be released into the venous bloodstream and partly captured by the magnetic trap of the detector. A physical model based on fluidic, gravitational and magnetic forces was developed to simulate the motion and sedimentation of ferromagnetic particles in a magnetic trap. In detailed simulation runs, the concentrations of accumulated particles under different applied magnetic fields within the magnetic trap are shown. The simulation results are qualitatively compared with laboratory experiments and show excellent accordance. Additionally, the sensitivity of the particle detection system is proofed in a MDS laboratory experiment by simulation of a membrane rupture.

Alginate-Encapsulated Human Hepatoma C3A Cells for use in a Bioartificial Liver Device - The Hybrid-Mds

The aim of this study was to encapsulate C3A cells into alginate microcapsules with an average diameter of < or =100 microm, thus enabling them to be recirculated in a bioartificial liver device based on MDS (Microsphere-based Detoxification System) technology. The microcapsules have to be permeable for essential proteins such as albumin. C3A cells were encapsulated using alginate. The resulting alginate beads were coated with poly(diallyldimethylammoniumchloride) (pDADMAC) and poly(sodium-p-styrenesulfonate) (pSS). Their mechanical stability was tested by recirculation of the microcapsule suspension, while their permeability was determined by reverse-size exclusion chromatography and by the use of a confocal laser microscope. The metabolic activities of encapsulated C3A cells were compared to freely growing adherent C3A cells in static cultivation models. The metabolic functionality of encapsulated C3A cells in static conditions was compared to encapsulated C3A cells in a dynamic model. The mean diameter of the resulting microcapsules was 86 mum. Our experiments show that these microcapsules were permeable for albumin and the high flow rate of 600 ml/min in a dynamic model has no influence on the survival and the metabolic activities of the encapsulated cells during the tested time of 24 hours. Alginate microcapsules containing C3A cells can be used to produce albumin and growth factors in a bioartificial or hybrid liver support system. Thanks to their small diameter, the microcapsules in suspension can be recirculated in the MDS.

Thermodynamic Considerations in Solid Adsorption of Bound Solutes for Patient Support in Liver Failure

New detoxification modes of treatment for liver failure that use solid adsorbents to remove toxins bound to albumin in the patient bloodstream are entering clinical evaluations, frequently in head-to-head competition. While generally effective in reducing toxin concentration beyond that obtainable by conventional dialysis procedures, the solid adsorbent processes are largely the result of heuristic development. Understanding the principles and limitations inherent in competitive toxin binding, albumin versus solid adsorbent, will enhance the design process and, possibly, improve detoxification performance. An equilibrium thermodynamic analysis is presented for both the molecular adsorbent recirculating system (MARS) and fractionated plasma separation, adsorption, and dialysis system (Prometheus), two advanced systems with distinctly different operating modes but with similar equilibrium limitations. The Prometheus analysis also applies to two newer approaches: sorbent suspension reactor and microsphere-based detoxification system. Primary results from the thermodynamic analysis are that: (i) the solute-albumin binding constant is of minor importance to equilibrium once it exceeds about 10(5) L/mol; (ii) the Prometheus approach requires larger solid adsorbent columns than calculated by adsorbent solute capacity alone; and (iii) the albumin-containing recycle stream in the MARS approach is a major reservoir of removed toxin. A survey of published results indicates that MARS is operating under mass transfer control dictated by solute-albumin equilibrium in the recycle stream, and Prometheus is approaching equilibrium limits under current clinical protocols.

Magnetic Fluorescent Microparticles as Markers for Particle Transfer in Extracorporeal Blood Purification

The microspheres-based detoxification system (MDS) is a combined membrane-adsorption system for extracorporeal blood purification in which adsorbent microparticles are recirculated in an extracorporeal filtrate circuit. Because the plasma filter represents the only barrier between the adsorbents and the patient's blood, there is the potential risk of particle entrance into the patient in case of a membrane rupture. To guarantee first fault safety of the system required for clinical application, magnetic fluorescent microparticles are added as markers to the adsorbent circuit. Detection of these particles in the venous blood line results in immediate shutdown of the pumps. Magnetic beads were functionalized with cresyl violet and tested with an in vitro setup of the particle detector to assess the detection limit in different matrices (water versus blood) as well as the influence of flow rate and particle size on the signal. In addition, biocompatibility and influence of sterilization on the performance of the particles were assessed. Functionalization of the magnetic particles with cresyl violet yielded fluorescent particles that were stable at 4 degrees C for at least 12 months. No leakage of dye was detectable, and the particles were neither cytotoxic nor mutagenic. The particles could be steam sterilized without significant loss in fluorescence intensity. With an in vitro setup of the particle detector, 0.1 mg and 5 mg of particles were reproducibly detectable in water and blood, respectively.

Efficient Adsorption of Tumor Necrosis Factor with an in vitro Set-Up of the Microspheres-Based Detoxification System

Background: The Microsperes-Based Detoxification System (MDS) represents a flexible therapeutic option for various diseases, depending on the specificity of the adsorbents applied. A potential application of the MDS is the supportive therapy of sepsis. Methods: Microadsorbents for tumor necrosis factor (TNF) were prepared by immobilization of anti-TNF antibodies on cellulose (1–10 µm) and applied in an experimental set-up using a pool of human plasma (1 liter) spiked with TNF (800 pg/ml) and its soluble receptors (1,000 pg/ml each). Removal of TNF was compared using a plasma filter and the albumin-permeable filter Albuflow. Results: Addition of 4 (2) g of adsorbent to the filtrate circuit reduced TNF concentrations in the pool by 80% (64%). Removal rates did not differ significantly for the different filters. TNF adsorption was not influenced by the presence of excess TNF receptors. Conclusions: Concentrations of mediators can be efficiently modulated with the MDS using small quantities of specific adsorbents.

Comparison of Specific Adsorbents for Tumor Necrosis Factor-α and Therapeutic Anti-Tumor Necrosis Factor-α Antibodies: An in Vitro Sepsis Model

Tumor necrosis factor alpha (TNF-alpha), as a key mediator, represents a major point of attack in sepsis. Since it has been shown that systemic anti-TNF-alpha antibodies do not improve the situation of septic patients, the use of specific adsorption technology in the treatment of sepsis could have beneficial effects. Magnetic beads coated with polyclonal or with monoclonal anti-TNF-alpha antibodies were investigated in vitro in order to analyze their ability to prevent TNF-alpha induced adhesion of peripheral blood mononuclear cells (PBMCs) at human umbilical vein endothelial cells (HUVECs). Additionally, therapeutical monoclonal anti-TNF-alpha antibodies were proofed for inhibitory effects of TNF-alpha mediated activation of HUVECs. We have shown, in vitro, that beads coated with polyclonal or monoclonal anti-TNF-alpha antibodies were able to significantly reduce monocyte adhesion. It was possible to decrease monocyte adhesion from nearly 9% to 3% within 2 hours and from 18% to 2% within 6 hours of TNF-alpha treatment by the simultaneous use of beads coated with polyclonal anti-TNF-alpha antibodies. Beads coated with monoclonal anti-TNF-alpha antibodies could even prevent monocyte adhesion within the first 2 hours, and reduced monocyte adhesion to 2% during 6 hours of incubation with TNF-alpha. On the other hand, application of therapeutic anti-TNF-alpha antibodies showed no significant difference compared to the measured monocyte adhesion values of activated endothelial cells. Adsorption techniques using specific adsorbents, possibly used in MDS (Microspheres-Based Detoxification System), are efficient in specific reduction of TNF-alpha and pathophysiological consequences, since monocyte adhesion at activated HUVECs was shown to be reduced.

New Methods for Hemoglobin Detection in a Microparticle-Plasma Suspension

In the extracorporeal adsorption system, MDS (Microspheres based Detoxification System), micro-adsorbent particles measuring 1-25 micrometers circulate in a filtrate circuit for highly specific blood purification/adsorption. The MDS circuit containing the adsorbent microparticles is linked to the patient's blood line by a hollow fiber plasma filter. When the transmembrane pressure or the shear forces due to the red blood cells in the hollow fiber filter are too high, they can be damaged and hemoglobin will be released. In order to detect free hemoglobin (fHb) by optical means, we have designed a new flow-dynamic filter system, placed in the microadsorbent circuit for continuous separation of microparticles from the filtrate. In the flow dynamic filter, we use a high velocity liquid vortex to remove sedimentation and particle plugs on the filter membrane. In our investigations, 3 and 8 micron cellulose nitrate filter membranes for particle separation are used. The obtained particle free bypass filtrate flow rates are typically 0.5 and 0.8 ml/min respectively. The typical sensitivity for fHb detection by the applied noninvasive optical method is 0.15 g/dL. Medical safety regulations require a fail-safe mechanism for fHb detection which monitors the bypass filtrate flow in the flowdynamic filter and shuts down the system in case of membrane occlusion. The bypass filtrate flow is monitored by periodically occluding and releasing the bypass line by means of a clamp. The resulting back pressure profile gives information about the actual filtration rate. This safety principle was proven by statistical analysis and shows its clear functionality.

Fluidized Bed Adsorbent Systems for Extracorporeal Liver Support

Acute liver failure based on acute-on-chronic liver failure (AoCLF) or on acute severe damage of the liver caused by different etiologies includes complex mechanisms resulting in severe disturbances of principle liver functions. In order to compensate the liver's function of detoxification as efficiently as possible, fluidized bed absorbent systems have been designed. In these systems, small particles with specific adsorption properties for toxins related to acute liver failure are applied. A special technology based on adsorbents in suspension has been developed under the guidance of our group and is prepared for clinical application during the coming year. This technology is called microspheres-based detoxification system (MDS) and is based on microadsorbents with a diameter of 1-10 microm which are recirculated in suspension. The safety of the MDS is guaranteed by the use of fluorescently labeled magnetic microparticles, which in case of a membrane-leakage are detected in the blood circuit by an optical system equipped with a magnetic trap. In vitro tests with two kinds of microadsorbents (a combination of a hydrophobic neutral resin and an anion exchange resin) showed excellent efficiency of the system with respect to adsorption capacity as well as to the kinetics of elimination of albumin-bound substances (e.g. unconjugated bilirubin or cholic acid) and of non-protein-bound substances (e.g. phenol). Moreover, using a plasma filter or the Albuflow filter as membrane filters in the blood circuit, the MDS technology offers the possibility to remove inflammatory mediators such as tumor necrosis factor-alpha (TNF) by additional use of specific adsorbents.

Detection of Fluorescently Labeled Microparticles in Blood

Background: A microsphere-based detoxification system is an adsorption system, whereby microadsorbent particles having diameters of 1–20 µm circulate in an extracorporeal filtrate circle. A thin-wall hollow-fiber membrane filter separates the microparticle-plasma suspension from the bloodstream. For patient safety, it is necessary to have a means to detect membrane ruptures that could lead to a release of microparticles into the patient’s bloodstream. Methods: An optical detection system was developed to monitor the venous bloodstream for the presence of microparticles from the filtrate circuit. For detection purposes, cellulose microspheres, both ferromagnetic and fluorescence labeled, were included with the microsphere adsorbant particles. In the case of a membrane rupture, the labeled particles would also be released into the bloodstream. By illuminating a small volume of blood with an excitation wavelength (590 nm) of the fluorescence marker, the particles can be detected by their emission light at 620 nm. The detector sensitivity is increased by collecting the ferromagnetic and fluorescently labeled microparticles using a magnetic trap. The efficiency of magnetic trap arrangements was tested by adjusting the magnet placements. Results: In vitro experiments were performed by pumping whole blood and labeled microparticles through the fluorescence detector. The efficiency of a magnetic trap arrangement was determined. With an optimal trap setup, 5–10 µl of labeled microparticles can be clearly detected in streaming whole blood. Conclusion: An easy to handle microparticle detector was developed, ready for use in particle based blood detoxification systems. The microparticle detection system fulfills the medical and technical requirements to bring the MDS into clinical tests.

Patient safety technology for microadsorbent systems in extracorporeal blood purification.

Alternative technologies for extracorporeal blood purification systems based on microadsorbents in suspension are discussed. Principally, microadsorbents offer higher efficiency and flexibility when compared to conventional column-based adsorption systems. Systems already clinically employed (e.g., BioLogic DT) or close to clinical application (e.g., the microspheres-based detoxification system, MDS) are described. The MDS technology, in particular, is characterized by efficiency and a high degree of flexibility with respect to both the use of different adsorbents as well as the combination with hemodialysis/hemofiltration therapy. It was designed for continuous use in intensive-care units, but enables also the removal of low-density lipoprotein, fibrinogen, autoimmune antibodies, immune complexes, and other pathophysiologically relevant substances. Alternative anticoagulation regimes and safety systems on fluorescence sensor technology have recently been developed for the MDS and are presented in this paper.

In vitro cell culture systems as the basis for an extracorporeal blood purification strategy in multiorgan failure treatment.

Multiorgan failure (MOF) based on septic processes is very common but prognostically an extremely severe disease that has to be treated exclusively under intensive care conditions. Extracorporeal blood purification (ECBP) using specific and efficient systems such as the microspheres based detoxification system (MDS) (Artif Organs 1996;20:420) could improve significantly the situation of MOF in terms of the efficient removal of endotoxins as well as key mediators such as tumor necrosis factor alpha (TNF alpha). The purpose of the study was to test the effectiveness of endotoxin and cytokine removal to blunt cellular response. In terms of the in vitro principle methodology, isolated peripheral blood mononuclear cells (PBMC) were incubated with endotoxins and a selective endotoxin adsorbent, which was added at various times (immediately or 30, 60, 120, 240, or 360 min) after the onset of incubation. TNF alpha release of monocytes was measured following a standard procedure after 20 h. Human TNF alpha was incubated with cultured human endothelial cells with and without a specific TNF alpha adsorbent (polyclonal antibodies coated on polystyrene particles). The results showed that after the initial addition of endotoxins, the activation of monocytes can be stopped within 120 min by addition of endotoxin adsorbents. In addition, specific TNF alpha adsorbents are able to prevent intercellular adhesion molecule 1 (ICAM-1) expression of endothelial cells, therefore avoiding activation of endothelial cells. In conclusion, cell culture models are suitable to simulate cell interaction in MOF. Specific adsorbents are able to reduce or block pathophysiologically relevant cell interactions, and the time frame for effective ECBP seems to be very short, and therefore, efficiency must be high.

Microspheres Based Detoxification System: In Vitro Study and Mathematical Estimation of Filter Performance

Because of the closed plasma (secondary) circuit in the Microspheres based Detoxification System (MDS), a convective blood purification system, the same amount of filtrated plasma is backfiltrated into the blood circuit. Therefore, there is no direct way to determine the ultrafiltrate production rate, which is an important factor of efficiency. The only possible way to estimate the filtration properties of the filter is to consider pressure values. In this study the pressure distribution in the filter was investigated in vitro. To explain the results and to calculate inaccessible parameters, a mathematical model was established which also considered the asymmetric behaviour of the filter membrane. The result was a linear pressure gradient, agreement with the measurements was reasonably good (calculated primary pressure loss differs <13% from measured value when using mean measured filter resistance as model parameter). Linear pressure distribution offers the possibility of easily calculating the filtration length, a parameter which can be used to estimate the filter condition. The comparison between calculated filtration and backfiltration rates offers an instrument of control for these values.

Extracorporeal adsorbent-based strategies in sepsis

Gram-negative bacterial sepsis remains a challenging diagnostic and therapeutic dilemma to the practicing clinician. Bacterial-derived products (eg, gram-negative bacterial lipopolysaccharide or endotoxin) and host inflammatory mediators (eg, tumor necrosis factor-alpha and interleukin-1) are believed to play a pivotal role in the pathogenesis of sepsis and septic shock. Despite the many advances in the treatment of sepsis, mortality rates in septic patients remain high. Indeed, numerous clinical trials using biologically engineered immunotherapies targeting specific inflammatory mediators have proven unsuccessful. This lack of success has led to a renewed interest in blood purification techniques using extracorporeal therapies. During sepsis, circulating bacterial-derived products as well as inflammatory mediators can be reduced and/or eliminated by various extracorporeal adjunctive therapies such as plasma exchange, continuous renal replacement, and adsorbent-based therapies. Adsorbents have commonly been used orally for gastrointestinal removal of toxins or drugs. However, their potential use in sepsis has received little attention. The incorporation of adsorbents in hemoperfusion columns has allowed their use for the removal of toxic compounds from the circulatory system. Adsorbents developed for use in sepsis can bind toxins in a nonselective (eg, charcoal), selective (eg, polymyxin B-immobilized polystyrene-derivative fiber), or specific (eg, antibody-coated microsphere-based detoxification system) way. However, despite an explosive development in the experimental use of these promising therapies, randomized clinical trials are currently lacking. In summary, a multi-disciplinary complex therapeutic approach remains a prerequisite to the successful treatment of sepsis.

Extracorporeal removal of proinflammatory cytokines by specific absorption onto microspheres.

Elevated plasma concentrations of interleukin 1 beta (IL-1 beta) and tumor necrosis factor alpha (TNF alpha) derived from cellular stimulation have been found to correlate well with the systemic inflammatory response syndrome and clinical outcome of sepsis. Consequently, biologic inactivation and extracorporeal removal of these potent mediators gain increasing attractiveness as adjunctive therapeutic options. In realization of the latter strategy the authors developed specific adsorbents by covalently linking polyclonal antibodies against IL-1 beta and TNF alpha onto microspheres. The attachment process was characterized by high retention of antigen neutralizing activity. Batch testing of the adsorbents revealed specificity, biocompatibility, and high binding capacity (20.2 and 36.9 ng/mg of particles for IL-1 beta and TNF alpha, respectively). Employment of the particles in the Microspheres Based Detoxification System (MDS) resulted in efficient purification: human plasma spiked with recombinant IL-1 beta and TNF alpha (500 pg/ml) could be cleared at 42 ml/min (IL-1 beta) and 55 ml/min (TNF alpha) at a flow rate of 200 ml/min. These clearance rates are considerably higher than the values obtained with ultrafiltration. In conclusion, the microsphere technology allows efficient extracorporeal removal of cytokines from plasma. In addition, by combined application of IL-1 beta and TNF alpha binding particles and endotoxin adsorbents, such as cationically modified cellulose, it should be feasible to interfere with the complex pathobiochemical sequelae of sepsis.

Microspheres based detoxification system: a new method in convective blood purification.

A variety of protein-bound or hydrophobic substances, accumulating as a result of pathologic conditions such as exogenous or endogenous intoxications, are removed poorly by conventional detoxification methods because of low accessibility (hemodialysis), insufficient adsorption capabilities (hemosorption), low efficiency (peritoneal dialysis), or economic limitations (high-volume plasmapheresis). Combining advantages of existing methods with microspheric technology, a module-based system was designed. Major operating parameters of the latter can be modified to allow for adjustment to individual clinical situations. An extracorporeal blood circuit including a plasmafilter is combined with a secondary high-velocity plasma circuit driven by a centrifugal pump. Different microspheric adsorbers can be combined in one circuit or applied in sequence. Thus, a prolonged treatment can be tailored using specially designed selective adsorber materials. Comparing this system with existing methods (high-flux hemodialysis, molecular adsorbent recycling system), results from our in vitro studies and animal experiments demonstrate the superior efficiency of substance removal.

EXTRACORPOREAL REMOVAL OF PRO-INFLAMMATORY CYTOKINES BY SPECIFIC ADSORPTION ONTO MICROSPHERES

Elevated plasma concentrations of interleukin 1β (IL-1β) and tumor necrosis factor α (TNFα) derived from cellular stimulation have been found to correlate well with the systemic inflammatory response syndrome and clinical outcome of sepsis. Consequently, biologic inactivation and extracorporeal removal of these potent mediators gain increasing attractiveness as adjunctive therapeutic options. In realization of the latter strategy the authors developed specific adsorbents by covalently linking polyclonal antibodies against IL-1β and TNFα onto microspheres. The attachment process was characterized by high retention of antigen neutralizing activity. Batch testing of the adsorbents revealed specificity, biocompatibility, and high binding capacity (20.2 and 36.9 ng/mg of particles for IL-1β and TNFα, respectively). Employment of the particles in the Microspheres Based Detoxification System (MDS) resulted in efficient purification: human plasma spiked with recombinant IL-1β and TNFα (500 pg/ml) could be cleared at 42 ml/min (IL-1β) and 55 ml/min (TNFα) at a flow rate of 200 ml/min. These clearance rates are considerably higher than the values obtained with ultrafiltration. In conclusion, the microsphere technology allows efficient extracorporeal removal of cytokines from plasma. In addition, by combined application of IL-1β and TNFα binding particles and endotoxin adsorbents, such as cationically modified cellulose, it should be feasible to interfere with the complex pathobiochemical sequelae of sepsis.

The Microspheres based Detoxification System (MDS): A new extracorporeal blood purification technology based on recirculated microspherical adsorbent particles

Plasma sorption processes have so far been performed with filters and appropriate adsorption columns. In this paper, we introduce a newly developed plasma sorption system, which is based on the high adsorption capacity of microspheres in a recirculation system. The technology has been applied successfully in vitro to eliminate endotoxins, low density lipoproteins (LDL) and barbiturates from human plasma.