Ales Prokop

Bioartificial Organs in the Twenty-first Century

Abstract: Bioartificial organs involve the design, modification, growth and maintenance of living tissues embedded in natural or synthetic scaffolds to enable them to perform complex biochemical functions, including adaptive control and the replacement of normal living tissues. Future directions in this area will lead to an abandonment of the trial‐and‐error implant optimization approach and a switch to the rational production of precisely formulated nanobiological devices. This will be accomplished with the help of three major thrusts: (1) use of molecularly manipulated nanostructured biomimetic materials; (2) application of microelectronic and nanoelectronic interfacing for sensing and control; and (3) application of drug delivery and medical nanosystems to induce, maintain, and replace a missing function that cannot be readily substituted with a living cell and to accelerate tissue regeneration. Biomimetics involves employment of microstructures and functional domains of organismal tissue function, correlation of processes and structures with physical and chemical processes, and use of this knowledge base to design and synthesize new materials for health applications. Nanostructured materials should involve biological materials (rather then synthetic ones) because their prefabricated structure is suitable for modular control of devices from existing materials. Nanostructured tools should encompass surface patterned molecular arrays, nanoscale synthetic scaffolding mimicking the cell‐extracellular matrix microenvironment, precise positioning of molecules with specific signals to provide microheterogeneity, composites of bioinorganic and organic molecules, molecular layering (coating), and molecular and supramolecular self‐assembly and self‐organization (template‐directed) assembly. The nanoelectronic interface includes electronic or optoelectronic biointerfaced devices based on individual cells, their aggregates and tissues, organelles, and molecules, such as enzyme‐based devices, transport and ion‐channel membrane proteins, and receptor‐ligand structures, including nanostructured semiconductor chips and microfluidic components. Delivery nanosystems encompass both water and lipid core vehicles (for hydrophilic and lipophilic components) of various geometries: liposomes, micelles, nanoparticles, lipid shells (as imaging and contrasting agents), solid nanosuspensions, lipid nanospheres, and coated film surfaces (molecular layering), all for use in delivering drugs, proteins, cell modifiers, and genes. Nanoelectronic interface and delivery nanosystems will be used for sensing, feedback, control, and analysis of function of bioartificial organs.

Hydrogel-based colloidal polymeric system for protein and drug delivery: Physical and chemical characterization, permeability control and applications

The use of polymeric nanoparticles as drug carriers is receiving an increasing amount of attention both in academia and industry. The development of suitable delivery systems for protein drugs with high molecular weights and short half-lives is of current interest. In addition, nanoparticles have a number of potential applications in drug and vaccine delivery as well as gene therapy applications. This article features a new production technology for nanoparticles comprised of multicomponent polymeric complexes that are candidates for delivery vehicles of biological molecules such as proteins and drugs. Materials science theory and practice provide the basis for the development of highly compacted structures that are insoluble in water and buffered media. Biocompatible and mostly natural polymers are fabricated into thermodynamically stable nanoparticles, in the absence of organic solvents, using two types of processing: batch and continuous. Careful choice of construction materials and the superposition of several interacting principles during their production allow for the customization of the physicochemical properties of the structures. Among the typical polymers used to assemble nanoparticles, different polysaccharides, natural amines and polyamines were investigated. The entrapped substances tested included proteins, antigens and small drug molecules. The size and charge of nanoparticles is considered to be of primary importance for application in biological systems. Detailed experiments in batch and continuous systems allowed time-dependent stoichiometric characterization of the production process and an understanding of fundamental assembly principles of such supramolecular structures. Continuous-flow production is shown to provide more consistent data in terms of product quality and consistency, with further possibilities of process development and commercialization. To control permeability, polydextran aldehyde, incorporated into the particle core, was used to enable physiologic cross-linking and long-term retention of substances that would otherwise rapidly leak out of the nanoparticles. Results of cross-linking experiments clearly demonstrated that the release rate could be substantially reduced, depending on the degree of cross-linking. For vaccine antigen delivery tests we measured an antibody production following subcutaneous and oral administration. The data indicated that only the cross-linked antigen was immunogenic when the oral route of administration was used. The data presented in this paper address primarily the utility of nanoparticulates for oral delivery of vaccine antigen. This novel technology is extensively discussed in contrast to other technologies, primarily water- and organic solvent-based. The usefulness is demonstrated using several examples, evaluating protein and small drug delivery.

Modification of the Cytosensor™ microphysiometer to simultaneously measure extracellular acidification and oxygen consumption rates

Abstract The Cytosensor™ microphysiometer has been modified by incorporating several platinum electrodes into the plunger head. The electrodes have been used to measure simultaneously the change in extracellular oxygen consumption rates and extracellular acidification rates during addition of various non-physiologic agents: 2-deoxy- d -glucose, sodium fluoride, paraoxon-ethyl, and antimycin A. Measurement of two parameters, oxygen and acidification, enables investigation of the aerobic and anaerobic metabolic consequences of these agents. Nafion membranes cast onto the platinum electrodes were useful in minimizing adsorption of cell-related interferents.

Purification of Polymers Used for Fabrication of an Immunoisolation Barriera

A multistep extraction procedure has been tested for purification of natural and semi-synthetic polymers used for fabrication of an immunoisolation barrier for implanting animal cells. This procedure, originally described by Klock et al. for alginates, has been adapted for other gelling polymers to remove pyrogens (endotoxins) and mitogens. Several other steps have also been tested, resulting in a new and simple procedure for polymer purification, giving satisfactory levels of contamination. Endotoxin levels have been quantified by means of chromogenic and gelclot LAL methods. A simple calculation of the endotoxin permissible levels shows that the quality of purified polymers exceeds FDA specifications for implantable polymers.

Towards Retrievable Vascularized Bioartificial Pancreas: Induction and Long-Lasting Stability of Polymeric Mesh Implant Vascularized with the Help of Acidic and Basic Fibroblast Growth Factors and Hydrogel Coating

We seek to improve existing methodologies for allogenic grafting of pancreatic islets. The lack of success of encapsulated transplanted islets inside the peritoneal cavity is presently attributed to poor vascularization of the implant. A thick, fibrotic capsule often surrounds the graft, limiting survival. We have tested the hypothesis that neovascularization of the graft material can be induced by the addition of proper angiogenic factors embedded within a polymeric coat. Biocompatible and nonresorbable meshes coated with hydrophilic polymers were implanted in rats and harvested after 1-, 6-, and 12-week intervals. The implant response was assessed by histological observations on the degree of vascularity, fibrosis, and inflammation. Macrostructural geometry of meshes was conducive to tissue ingrowth into the interstitial space between the mesh filaments. Hydrogel coating with incorporated acidic or basic FGF in an electrostatic complex with polyelectrolytes and/or with heparin provided a sustained slow release of the angiogenic growth factor. Anti-factor VIII and anti-collagen type IV antibodies and a GSL I-B4 lectin were used to measure the extent of vascularization. Vigorous and persistent vascularization radiated several hundred microns from the implant. The level of vascularization should provide a sufficient diffusion of nutrients and oxygen to implanted islets. Based on our observations, stable vascularization may require a sustained angiogenic signal to allow for the development of a permanent implant structure.

The effect of pH on the foam fractionation of β-glucosidase and cellulase

Abstract The surface tension–pH profile of β-glucosidase was established to determine its relationship to the corresponding profile of cellulase and to the foam fractionation of that cellulase. The goal of this work was to determine the optimal foaming points for both cellulase and cellobiase. This data may prove useful in the separation of certain components of cellulase, since the non-foaming hydrophilic β-glucosidase does not foam as well as the hydrophobic components of cellulase at low concentrations. A key finding from these experiments was that there are two local minima in the surface tension–pH trajectory for Trichoderma reesei cellulase, as contrasted to the usual single minimum. The lower of these minimum points corresponds to the cellulase isoelectric point. The double minimum surface tension–pH profile was also observed for cellobiase alone. The optimal foaming pH for cellobiase alone was determined to be around 10.5, while for cellulase it was between 6 and 9.

Foam fractionation of a dilute solution of bovine lactoferrin.

Lactoferrin (Lf), a protein found in human and bovine milk, tears, blood, and other secretory fluids, has been used to prevent infection from potential microbial pathogens by its ability to bind with iron (Fe3+). Currently, bovine lactoferrin can be purified from milk using ion exchange resin, which is a costly procedure making lactoferrin expensive. The purpose of this work was to investigate a low-cost foam fractionation process as the first step in separating lactoferrin from milk.

Effect of pH on the Startup of a Continuous Foam Fractionation Process Containing Ovalbumin

The effect of pH on the bubble size distribution, void fraction, and enrichment ratio of a continuous foam fractionation column containing ovalbumin was investigated. The bubble size and void fraction were measured using a photoelectric capillary probe for different solution pHs (3.5, 4.5, 6.5, and 9.7). The bubble diameters for pH 3.5 and 4.5 were the largest of the four pHs studied. At these two pHs, the foam was less stable and formed aggregates, leading to lower enrichment and mass recovery. For the nearly neutral pH 6.5 or the more basic pH 9.7, the bubble size was smaller and the foam was more stable, resulting in both high enrichment and high mass recovery. The void fraction was smallest for pH 6.5, but the effect of pH on void fraction was not significant. In the lower foam phase, the calculated specific area increased as the pH increased from 3.5 to 9.7, which may partially contribute to the higher enrichments at pH 6.5 and 9.7.

Variation of bubble size distribution in a protein foam fractionation column measured using a capillary probe with photoelectric sensors.

Bubble size is used to characterize not only bubble-specific interfacial area but also bubble coalescence in a foam column. The bubble size distributions were obtained in a continuous foam fractionation process for concentrating ovalbumin using a developed photoelectric probe. When the continuous process reached steady state, the bubble size distribution pattern remained stable. Bubble size distribution data above (+1 cm) or below (-1 cm) the bulk liquid-foam interface showed symmetry along the diameter of the column (14 cm ID). The bubble size distribution was affected by the column wall. The nearly constant protein concentration distribution across the column cross-section indicated that the bubble flow distribution approached a flat profile across the column. A log-normal bubble distribution pattern best fit the weighted range of bubbles in the column at column lengths above and below the liquid-foam interface. These observations may prove to be useful in understanding the mechanisms underlying the foam fractionation of proteins.

Effect of Bubble Size on Foam Fractionation of Ovalbumin

The bubble size distribution and void fraction (epsilong) (at two bulk liquid pool positions below the bulk liquid-foam interface and one lower foam phase position) in a continuous foam fractionation column containing ovalbumin were obtained using a photoelectric capillary probe. The bubble size and epsilong data were gathered for different operating conditions (including the changes in the superficial gas velocity and feed flow rate) at a feed solution of pH 6.5 and used to calculate the specific area, a, of the bubbles. Thus, local enrichment (ERl), values of ovalbumin could be estimated and compared with directly obtained experimental results. The ERl results were also correlated with the bubble size and epsilong to understand better the concentration mechanisms of foam fractionation. The high ERl in the lower foam phase was largely attributable to the abrupt increase in epsilong (from 0.25 to 0.75), or the a (from about 12 to 25 cm2/cm3) from the bulk liquid to the foam phase. These changes correspond with enhanced gravity drainage. With an increase in the superficial gas velocity, the bubble size increased and the a decreased in both the bulk liquid and lower foam phases, resulting in a decrease in the local experimentally determined enrichments at high superficial gas velocities. At intermediate feed flow rates, the bubble size reached the maximum. The epsilong and a, on the other hand, were the largest for the largest feed flow rate. The ERl in the lower foam phase was maximized at the lowest feed flow rate. It follows, therefore, that a alone is not sufficient to determine the magnitude of the ERl in the foam phase.