The formation of an anti-restenotic/anti-thrombotic surface by immobilization of nitric oxide synthase on a metallic carrier.

Coronary stenosis due to atherosclerosis, the primary cause of coronary artery disease, is generally treated by balloon dilatation and stent implantation, which can result in damage to the endothelial lining of blood vessels. This leads to the restenosis of the lumen as a consequence of migration and proliferation of smooth muscle cells (SMCs). Nitric oxide (NO), which is produced and secreted by vascular endothelial cells (ECs), is a central anti-inflammatory and anti-atherogenic player in the vasculature. The goal of the present study was to develop an enzymatically active surface capable of converting the prodrug l-arginine, to the active drug, NO, thus providing a targeted drug delivery interface. NO synthase (NOS) was chemically immobilized on the surface of a stainless steel carrier with preservation of its activity. The ability of this functionalized NO-producing surface to prevent or delay processes involved in restenosis and thrombus formation was tested. This surface was found to significantly promote EC adhesion and proliferation while inhibiting that of SMCs. Furthermore, platelet adherence to this surface was markedly inhibited. Beyond the application considered here, this approach can be implemented for the local conversion of any systemically administered prodrug to the active drug, using catalysts attached to the surface of the implant.

A simple synthetic route to the formation of a block copolymer of poly(lactic-co-glycolic acid) and polylysine for the fabrication of functionalized, degradable structures for biomedical applications.

This article documents the formation of a block copolymer of poly(lactic-co-glycolic acid) and polylysine via a simple coupling technique using dicyclohexyl carbodiimide (DCC). The resulting polymer has been characterized via UV-Vis spectroscopy, GPC, (1)H NMR, and elemental analysis, is soluble in a wide variety of solvents, and is easily processable, making the technique a simple and practical one for the formation of functionalized, degradable block copolymers for the fabrication of functionalized structures for biomedical applications.

Sciatic nerve blockade with lipid-protein-sugar particles containing bupivacaine.

PURPOSE: To assess the efficacy of lipid-protein-sugar particles (LPSPs) in providing prolonged duration local anesthesia by percutaneous injection. METHODS: Bupivacaine-containing LPSPs were characterized and optimized in vitro. Male Sprague-Dawley rats were given sciatic nerve blocks with bupivacaine-containing LPSPs. Sensory and motor nerve blockade were measured in the hindpaw, as were contralateral functional deficits (a measure of systemic drug distribution). Poly(lactic-co-glycolic) acid (PLGA) microspheres were used as a reference. RESULTS: 10% (w/w) bupivacaine LPSPs (60% dipalmitoylphosphatidylcholine) were 4.4+/-0.39 microm in diameter, with a tap density of 0.11 +/-0.04 g/ml. These LPSPs and 50% (w/w) PLGA microspheres had comparable durations of sensory blockade (468+/-210 min vs. 706+/-344 min, p = 0.08), although the LPSPs produced a much lesser duration of motor blockade (508+/-258 min vs. 1062+/-456 min, p = 0.005). Systemic toxicity was minimal in both groups. CONCLUSIONS: LPSPs provide sensory blockade durations comparable to those from PLGA microspheres, with a smaller amount of drug loading. Motor blockade is shorter with LPSPs than with PLGA microspheres. LPSPs appear to be suitable for extended nerve blockade. Given their size and low density, they may be useful for topical anesthesia of the airway.

A biochemical logic gate using an enzyme and its inhibitor. 1. The inhibitor as switching element.

Molecular-scale logic systems will allow for further miniaturization of information processing assemblies and contribute to a better understanding of brain function. Of much interest are the pertinent biological systems, some of the basic components of which are biomolecular switching elements and enzyme-based logic gates. In this series of accounts, results of investigations are presented on the implementation of an enzyme/inhibitor logic gate operating under the rules of Boolean algebra. In this report (part 1 of the series), consideration is given to the experimental conditions-particularly the irradiation mode-that affect the performance of proflavine as inhibitor of alpha-chymotrypsin. Also, assessments are made on the reversibility of the process involved and the long-term stability of the system. Moreover, using a theoretical conformational analysis of proflavine and its reduction products, detailed features were established regarding their three-dimensional structure, partial charge distribution, and hydrophobicity. Accordingly, an understanding was reached as to the factors affecting the interaction between these compounds and the enzyme. In part 2 of this series, the actual implementation of an AND logic gate will be presented. This gate involves proflavine and a chemically derivatized alpha-chymotrypsin, and its operation relies on the conclusions reached in this report regarding the optimal mode for controlling the inhibitory activity of proflavine.

Dual enzyme multi-layer bioreactors: analytical modeling and experimental studies.

Enzymic reactors are developed for a variety of biomedical-biotechnological applications, including blood detoxification. For the latter, an appropriate approach is to use enzymes of the Mercapturic Acid Pathway. The first two enzymes of this pathway are Glutathione-S-Transferase (GST) and gamma-Glutamyl Transpeptidase (gamma GT). Earlier, the performance of an immobilized GST reactor was investigated experimentally and theoretically. Here, the analytical model was extended to describe a dual-enzyme continuous packed-bed reactor (DCP), in which the two enzymes (E1 and E2) are arranged in alternating layers. The performance of DCP reactors was first studied by numerical simulations, considering the effects of reactor configuration (i.e. number of enzyme layers), kinetic characteristics (K(m), Vmax, Kiq) and operational parameters (flow rate, substrates concentration). Results were obtained in terms of substrate and products concentration profiles along the reactor. The theoretical calculation were supplemented by experimental studies. In the latter GST (i.e. E1) and gamma GT (i.e. E2), were used when immobilized on porous beads, and the reactor was set up and operated in various configurations. It was found that the factors which mostly affect the performance of DCP systems are reactor configuration and extent of inhibition of E1 by its reaction product.

Microparticles of novel branched copolymers of lactic acid and amino acids: preparation and characterization.

The preparation and characterization of microparticles produced from a new class of functionalized, biodegradable, comblike graft copolymers is presented. The copolymers are polyester-polyamino acid hybrids, composed of a poly(L-lactic acid-co-L-lysine) (PLAL) backbone, and poly(L-lysine), poly(D,L-alanine) or poly(L-aspartic acid) side chains extending from the lysine residues of PLAL. The microparticles have been characterized with regard to their surface properties, morphology, and size. Thus, electron spectroscopy for chemical analysis data and results of Zeta potential measurements suggest that the polyamino acid side chains tend to concentrate at the surface of the particles. Also, analyses by environmental scanning electron microscopy and confocal scanning laser microscopy indicate that particles carrying poly(lysine) chains have an unusual porous structure, most probably due to the combined effects of the amphiphilic, polyelectrolyte, and chemical nature of the composing copolymer, as well as of the particular preparation technique employed. The capabilities of the microparticles to serve as carriers in controlled drug release and delivery devices were demonstrated by encapsulation and release of rhodamine B, a low molecular weight drug model.

Large porous particles for pulmonary drug delivery.

A new type of inhalation aerosol, characterized by particles of small mass density and large size, permitted the highly efficient delivery of inhaled therapeutics into the systemic circulation. Particles with mass densities less than 0.4 gram per cubic centimeter and mean diameters exceeding 5 micrometers were inspired deep into the lungs and escaped the lungs' natural clearance mechanisms until the inhaled particles delivered their therapeutic payload. Inhalation of large porous insulin particles resulted in elevated systemic levels of insulin and suppressed systemic glucose levels for 96 hours, whereas small nonporous insulin particles had this effect for only 4 hours. High systemic bioavailability of testosterone was also achieved by inhalation delivery of porous particles with a mean diameter (20 micrometers) approximately 10 times that of conventional inhaled therapeutic particles.

A molecular switch for biochemical logic gates: conformational studies.

This report presents the computer-assisted design of a molecular switching element, in which a molecular switch regulates the enzymatic activity of Ribonuclease A (RNase A). The molecular switch, an appropriately modified amino acid residue, is constructed with an electron donor group and an electron acceptor group, connected to one another with a conjugated double bond bridge. The switching mechanism is based on the azonium-hydrazo tautomerization, by which a charge separation induced in the excited state causes a rearrangement of the molecular electronic structure, resulting in the exchange of locations of single and double bonds. This rearrangement of bonds leads to different three-dimensional conformations of the switch. Using the electrostatically driven Monte Carlo (EDMC) method and the empirical conformational energy program for peptides (ECEPP/3) potential energy function, we carried out an exhaustive search of the conformational space of the switching element. The results of these calculations reveal two sets of conformations: in one set the access to the active site of the enzyme is preferentially blocked, while in the other set the active site is preferentially accessible. Integration of the designed element into biochemical logic gates operating under the rules of threshold value, and experimental implementation of this system, are considered.

Nanotechnology for biomaterials engineering: structural characterization of amphiphilic polymeric nanoparticles by 1H NMR spectroscopy.

Nanoparticles composed of diblock poly(D,L-lactide-co-glycolide)-poly(ethylene glycol) (PLGA-PEG) or a branched, multiblock PLA-(PEG)3 were prepared by the single emulsion technique. Results of previous studies of these nanoparticles suggested that their structure is of the core-corona type with a polyester core and an outer PEG coating. In the present study, 1H NMR spectroscopy was utilized to provide direct evidence of the structure of these nanoparticles suspended in an aqueous environment. The results confirm the existence of the core-corona structure under these conditions, and show that the PEG moieties extend out from the nanoparticle core into the aqueous environment, and exhibit chain mobility similar to that of PEG in solution.

Multimolecular process in a packed-bed immobilized enzyme reactor: numerical simulation and back-mixing effects.

In a previous report, we presented a new analytical model describing the performance of a packed-bed catalytic unit, where the reaction between two cosubstrates is catalyzed by an enzyme immobilized on a porous carrier. The model explicitly takes into account the changes in concentrations of both cosubstrates along the reactor, as well as the hydrodynamic regimen (i.e., back-mixing) prevailing in the packed bed. In the present report, and on the basis of the procedures developed, we present a detailed analysis of the performance of the reactor. With numerical simulations, the effects of internal diffusion limitations, the depth of the pores, the substrates' concentration in the feed, and kinetic parameters are evaluated. Particular attention is also given here to the back-mixing effects prevailing in the reactor. An experimental procedure for assessing their extent is described.

Cholesterol removal by haemoperfusion of whole blood in vivo.

Familial hypercholesterolaemia is caused by genetic defects in the cellular metabolism of cholesterol (C) and is characterized by high levels of low-density lipoproteins (LDL) and premature atherosclerosis. The C is carried in the plasma mainly as an LDL-C complex, and removal of the latter from plasma is highly desirable. This task can be achieved by selective haemoperfusion (HP), thereby eliminating the need for plasmapheresis. Agarose beads (2 per cent agarose, 0.85 to 1.4 mm in diameter) were prepared, and crosslinked with epichlorohydrin. Heparin and/or ethanolamine were subsequently attached. The beads thus obtained were found to be suitable for the removal of LDL-C from the whole blood of hypercholesterolaemic rabbits, using a simple HP technique. A single two-hour HP treatment with a 40 ml column packed with active agarose beads resulted in a 30 per cent decrease in the C plasma level in the experimental animals. Our previous, in vitro, studies with the plasma of hypercholesterolaemic patients showed a high selectivity of the beads for LDL. Yet when used with hypercholesterolaemic rabbits, a relatively high amount of HDL was also removed from the blood. This can be attributed to a significant difference between the structure of human hypercholesterolaemic lipoproteins and that of rabbits. Upon treatment of blood using the active agarose beads, no abnormalities in plasma and blood composition were detected, except for some prolongation of PT. It is to be hoped that this new system will replace the presently used and highly expensive plasmapheresis.

Enzyme-based hemoperfusion and blood treatment.

Enzyme-based artificial organs are being developed as metabolic assist devices. These are required when normal metabolism is impaired, or when the body is overloaded by undesired metabolites or toxins. The implementations of this approach for treating a genetic disease, and for metabolic support in liver failure are envisaged. The kinetic aspects and mass transfer characteristics of bioreactors for these systems are considered in detail.

A mechanistic model for the enzymic degradation of synthetic biopolymers.

The degradation pathway of a synthetic biopolymer, poly-N5-(2-hydroxyethyl)-L-glutamine, by papain under physiological conditions, was extensively investigated. The enzymic reaction was found to be rather complex: it progressively slows down, and comes to an end when the degradation fragments are tetrapeptides. In order to account for this phenomenon, a modified Michaelis-Menten kinetic model is proposed, in which Km is assumed to be dependent on the degree of polymerization of the macromolecular substrate, and therefore varies as the degradation reaction proceeds. The new model accurately describes all the experimental data, and allows one to predict the entire course of the reaction. The behaviour of the system considered is interpreted to represent a model of biological recognition at the molecular level.

Tailor-made agarose-based reactive beads for hemoperfusion and plasma perfusion.

Composite beads of approximately 1 mm diameter, made of crosslinked agarose and containing Fuller's Earth or zirconium oxide powders, were prepared and used in extracorporeal systems for blood detoxification. The former was used for the removal of Paraquat, while the latter was used to remove inorganic phosphate from hyperphosphatemic animals with or without acute renal failure. The high surface area of the powder, combined with the low resistance to diffusion in the crosslinked agarose matrix, are highly advantageous. The crosslinking provides high mechanical strength, heat stability, prolonged shelf life, good blood flow characteristics, and prevents the release of fine particles into the blood. Crosslinked agarose beads of 1 mm diameter, containing chemically-bound heparin were also prepared, and used as a model for direct contact removal of LDL-cholesterol from the blood of familial hypercholesterolemic patients by hemoperfusion. The high capacity of these beads (over 5 mg LDL/mL beads) indicates that this clinical modality can replace the highly expensive plasmapheresis procedure presently used.

In vivo evaluation of a composite sorbent for the treatment of paraquat intoxication by hemoperfusion.

In vivo evaluation of a new hemoperfusion (HP) device for Paraquat detoxification is reported. The key element of the extracorporeal system is a column packed with newly developed composite sorbent beads containing Fullers' Earth (FE) entrapped in crosslinked Agarose. The proposed HP system exhibits very good biocompatibility characteristics when conventional heparinization is supplemented by infusing 0.02 ml per min acid-citrate dextrose (ACD) solution per ml. perfused blood at the inlet to the HP column. No complications or abnormalities were detected in animals which were hemoperfused with the system described. It is suggested that the new device is effective and safe for clinical application.

Novel composite sorbent beads for paraquat removal by hemoperfusion.

The present report describes the development and performance of a column that is to be used for removal of paraquat from circulating blood in a hemoperfusion-type set-up. The key element of the system is a newly developed sorbent material containing fuller's earth entrapped in cross-linked agarose beads (Talosit). The technique produces a sorbent material exhibiting a very large active surface area while allowing for high mobility of paraquat molecules within the beads and favorable flow characteristics of packed column. Cross-linking of the agarose (by epichlorohydrin) also has a most beneficial effect on the mechanical strength of the beads as well as on their stability to sterilization in an autoclave. The composite beads exhibit good blood compatibility. A scanning electron microscope analysis of the beads showed no adherence of cellular blood components after contact with blood. Moreover, no significant changes in plasma composition had taken place when the beads were properly conditioned prior to contact with fresh human blood. A comparative study of paraquat removal from saline solution by the new beads and by cellulose-coated activated charcoal (Adsorba-300C) indicates a higher removal rate with the former. The results obtained so far with this new sorbent are very promising and extension of these studies to in vivo hemoperfusion is under way.

Removal of excess inorganic phosphate by haemoperfusion with composite beads.

New composite beads, made by encapsulating hydrous zirconium oxide powder in agarose, are evaluated in vitro and in vivo for the removal of inorganic phosphate from the blood. Phosphate adsorption is rather good and calcium removal can be controlled. Thus, a 250 to 300 ml column is capable of reducing phosphate plasma level by some 5 mg per cent, thus making it attractive for the treatment of acute renal failure. Biocompatibility tests showed that supplementing the standard heparinization procedure by adding some citrate, as ACD, to the extracorporeal circuit greatly improves platelet and leucocyte count. No adverse haemodynamic effects were noted in repeated haemoperfusions of five dogs.