Nanodiamond-chymotrypsin and nanodiamond-papain conjugates, their synthesis and activity and visualization of their interaction with cells using optical and electron microscopy.

Two novel conjugates of detonation nanodiamonds (dNDs) with the proteolytic enzymes chymotrypsin and papain were synthesized. The synthesis was performed via functionalization of the dNDs' surface with acidic/alkali treatment followed by carbodiimide-mediated protein binding. Covalent binding of the enzymes was confirmed by Fourier transform infrared spectrographic analysis and high-performance liquid chromatography (HPLC) amino acid analysis. HPLC also proved the preservation of the enzymes' composition during synthesis. The same assay was used to determine the binding ratios. The ratios were 12% (mass to mass) for chymotrypsin and 7.4% for papain. The enzymatic activity of the conjugates was measured using chromogenic substrates and appeared to be approximately 40% of that of the native enzymes. The optimum pH values and stability under various conditions were determined. The sizes of resulting particles were measured using dynamic light scattering and direct electron microscopic observation. The enzyme conjugates were shown to be prone to aggregation, resulting in micrometer-sized particles. The ζ-potentials were measured and found to be positive for the conjugates. The conjugated enzymes were tested for biological activity using an in vitro model of cultured transformed human epithelial cells (HeLa cell line). It was shown that dND-conjugated enzymes effectively bind to the surface of the cells and that enzymes attack exposed proteins on the plasma membrane, including cell adhesion molecules. Incubation with conjugated enzymes results in morphological changes of the cells but does not affect cell viability, as judged by monitoring the cell division index and conducting ultrastructural studies. dNDs are internalized by the cells via endocytosis, being enclosed in forming coated vesicles by chance, and they accumulate in single membrane-bound vacuoles, presumably late endosomes/phagosomes, along with multimembranous onionlike structures. The authors propose a model of a stepwise conjugate binding to the cell membrane and gradual release of the enzymes.

Mass transfer mechanisms in high-performance membrane dialyzers.

Four dialyzers with super-high-flux membrane or high-performance membrane (HPM) with varying packing density of the hollow fiber (PDF) from 29.6 to 53.1% were investigated in aqueous in vitro experiments for the purpose of identifying the mass transfer mechanism for three test solutes. Clearances for relatively small test solutes (creatinine and vitamin B(12)) slowly increased with PDF and reached plateau since mass transfer mechanism of these solutes was diffusion limited. However, since unlike classic high-flux dialyzers a considerable amount of internal filtration ( > 20 ml/min) should occur in super-high-flux dialyzers even under relatively reduced blood and dialysis fluid flow rates, Q(B) = 200 ml/min and Q(D) = 500 ml/min, it should contribute to enhance the rate of mass transfer especially for those solutes that cannot be easily removed by diffusion, such as ß(2)-microglobulin or even larger toxic substances. For dialyzers with the HPM a module design becomes even more important for developing novel commercial products.

Encapsulation and release of alpha-chymotrypsin from poly(glycerol adipate-co-omega-pentadecalactone) microparticles.

Polymer-based microparticles are increasingly becoming of interest for a variety of applications including drug delivery. Recently poly(glycerol adipate) (PGA) and poly(glycol adipate-co-omega-pentadecalactone) have shown promise for delivery of dexamethasone phosphate and ibuprofen. In this paper the copolyester poly(glycol adipate-co-omega-pentadecalactone) was evaluated as a colloidal delivery system for encapsulated therapeutic proteins. Enzyme containing microparticles were prepared via the double water-in-oil-in-water (w/o/w) emulsion-solvent evaporation methodology. alpha-Chymotrypsin was used as a model proteolytic enzyme and its transfer was monitored during the emulsification process, in addition to in vitro release from formed particles. On average, 22.1 microg protein per 1 mg polymer was encapsulated, although gradual loss of activity of the protein, once released, was recorded. The work presented shows the potential of this polyester as a delivery system for enzymes via microparticles, with improvements to the system achievable via polymer and process optimization. The pendant hydroxyl groups on the polymer backbone provide future capacity for tailored alteration of the physical and chemical properties of the polymer, in addition to covalent attachment of various compounds.

Biodegradable nanoparticles of partially methylated fungal poly(beta-L-malic acid) as a novel protein delivery carrier.

The preparation of nanoparticles from 75% methylated poly(beta-L-malic acid) is described. Their degradation in aqueous environments was examined and the influence of pH and lipase on the rate of hydrolysis was evaluated. Six proteins were used to estimate the loading efficiency of the nanoparticles. The amount of protein retained in the nanoparticles was found to depend on the acid/basic character of the protein. Protein release from the loaded nanoparticles upon incubation in water under physiological conditions encompassed polymer hydrolysis and happened steadily within 3-10 d. The activity loss of entrapped alpha-chymotrypsin caused by loading and releasing depended on the method used for loading.

Effect of the molecular weight of poly(ethylene glycol) used as emulsifier on alpha-chymotrypsin stability upon encapsulation in PLGA microspheres.

Poly(ethylene glycol) (PEG) was used as emulsifier to prepare alpha-chymotrypsin-loaded poly(lactic-co-glycolic) acid (PLGA) microspheres by a solid-in-oil-in-water (s/o/w) technique. The effect of the molecular weight of PEG on protein stability was assessed by the determination of the amount of insoluble aggregates, the activity loss and the magnitude of structural perturbations. In addition, the effect of the molecular weight of PEG on the encapsulation efficiency, microsphere characteristics and release kinetics was investigated. X-ray photoelectron spectroscopy (XPS) was employed to characterize the surface chemistry of the microspheres. Microspheres were prepared using PEG with molecular weight of 6,000, 8,000, 10,000, 12,000 and 20,000. The results indicate that PEG 20,000 was the most effective emulsifier when producing alpha-chymotrypsin-loaded microspheres with respect to protein stability. The aggregate formation was decreased from 18% to 3%; the protein inactivation and the encapsulation-induced structural perturbations were largely prevented. XPS confirmed that PEG was largely located on the surface of microspheres. The molecular weight of PEG affected the microspheres' characteristics and release kinetics. Microspheres prepared with PEG 20,000 showed improved encapsulation efficiency (80%) and a continuous release (for 50 days) with the lowest amount of initial release. It is demonstrated that the selection of the optimum molecular weight of PEG when used as emulsifier in the preparation of microspheres is a critical factor in the development of sustained-release formulations for the delivery of proteins.

Drastic neuronal loss in vivo by beta-amyloid racemized at Ser(26) residue: conversion of non-toxic [D-Ser(26)]beta-amyloid 1-40 to toxic and proteinase-resistant fragments.

It is unclear how and when insoluble beta-amyloid in senile plaques exerts degenerative effects on distant hippocampal neurons in Alzheimer's disease. Racemization of Ser and Asp residues of insoluble beta-amyloid is a typical age-dependent process. In this study, we investigated the fibril formation activity and cytotoxic activity of beta-amyloid 1-40 racemized at the Asp or Ser residue. In contrast to beta-amyloid 1-40 and its derivative substituted with the D-Asp(1, 7 or 23) or D-Ser(8) residue, [D-Ser(26)]beta-amyloid 1-40 was non-toxic to PC12 cells, and did not exhibit significant fibril formation activity making it soluble. However, [D-Ser(26)]beta-amyloid 1-40, but not beta-amyloid 1-40, was converted in vitro to a potent neurotoxic and truncated peptide, [D-Ser(26)]beta-amyloid 25-35 or [D-Ser(26)]beta-amyloid 25-40, by chymotrypsin-like enzymes and aminopeptidase M. Soluble [D-Ser(26)]beta-amyloid 1-40 was injected into rat hippocampus with a non-toxic dose of ibotenic acid, an excitatory amino acid. Nissl staining and microtubule-associated protein-2 immunostaining revealed that [D-Ser(26)]beta-amyloid 1-40, as well as [D-Ser(26)]beta-amyloid 25-35, produced a drastic degeneration of the CA1 neurons with ibotenic acid although [D-Ser(26)]beta-amyloid 1-40 alone or ibotenic acid alone did not exert neuronal damage. This suggests the in vivo conversion of non-toxic [D-Ser(26)]beta-amyloid 1-40 to the toxic and truncated peptides which enhance the susceptibility of neurons to the excitatory amino acid.These results and the presence of [D-Ser(26)]beta-amyloid 25-35-like antigens in Alzheimer's disease brains suggest that soluble [D-Ser(26)]beta-amyloid 1-40, possibly formed during the aging process, is released from senile plaques, and converted by brain proteinases to truncated [D-Ser(26)]beta-amyloid 25-35(40)-like peptides, which degenerate hippocampal neurons by enhancing the susceptibility to excitatory amino acids in Alzheimer's disease brains. These findings may provide the basis for a new therapeutic approach to prevent the neurodegeneration in Alzheimer's disease.

Synthesis and antileukemic activity of chymotrypsin-activated derivatives of 3'-amino-2',3'-dideoxycytidine. (Synthetic nucleosides and nucleotides. XXXIII.

3'-Amino-2',3'-dideoxycytidine (8) was directly synthesized from 2'-deoxycytidine. 2',3'-Dideoxy-3'-(N-acyl-L-phenylalanylamino)cytidines (acyl = butoxycarbonyl (9a), acetyl (9b), benzoyl (9c), and n-hexanoyl (9d)) were synthesized as chymotrypsin-activated prodrugs of 8. This N-protection was required for activation by chymotrypsin to 8. In vitro, compound 8 showed high cytotoxic activity against P388 cells, but the prodrugs 9a-d were ineffective. In vivo, however, these prodrugs showed much higher activity than 8 in mice bearing P388 cells.

Adsorption and activation of zymogens at solid-liquid interfaces. I. Chymotrypsinogen on alkylamino modified silica derivatives.

Silica beads were modified with alkylamino groups of different lengths (C2, C4, C6, C8, and C10) and hydrophobicity. The relationship between surface structure and adsorption of chymotrypsinogen followed by its activation with trypsin at the solid-liquid interface was studied. From the adsorption isotherms, it follows that underivatized silica adsorbed chymotrypsinogen (CTG) well. The adsorption of CTG on alkylamino modified silicas appeared to correlate with the hydrophobicity of the latter. The longer the alkyl chains were, the higher was the amount of adsorbed CTG. The activation of adsorbed CTG with trypsin at the solid-liquid interface was a slower process when compared with the activation conducted in solution. Parallel experiments were performed with chymotrypsin (CT). The adsorption behavior was similar to that of CTG. The activity of adsorbed CT was inversely proportional to the hydrophobicity of the beads. These results correlated well with the desorption of CT after repeated washings. Repeated addition of substrate (Gly-Gly-Phe-NAp) to the CT covered beads resulted in the CT desorption. The higher the hydrophobicity of the beads was, the lower was the desorption of CT.

Fluorogenic bimane substrates with dabsyl group for endopeptidases; chymotrypsin, collagenase and thermolysin.

It was found that the fluorescence of 9,10-dioxa-syn-3,4,6,7-tetramethylbimane (bimane) can be quenched in the presence of dimethylaminoazobenzensulfonyl (Dabsyl) group. New combination of bimane (fluorophor) and dabsyl group (quencher) was applied to the syntheses of intramolecularly quenched fluorogenic substrates for hydrolytic enzymes. Bimane peptides containing dabsyl group were prepared, and were shown to be useful fluorogenic substrates for the assay of endopeptidases such as chymotrypsin, collagenase and thermolysin.

Fate of orally ingested enzymes in pancreatic insufficiency: comparison of two pancreatic enzyme preparations.

The effect on steatorrhoea of a pH-sensitive enteric-coated pancreatic preparation (Eurobiol 25,000) was compared with a conventional pancreatic enzyme preparation (Eurobiol) in six adult patients with exocrine pancreatic insufficiency. In addition, the fate of orally ingested pancreatic enzymes in the upper digestive tract was evaluated by measuring gastric and duodenal pH, amount of enzymes in the stomach, duodenal enzyme output, and fat absorption at the angle of Treitz for the 4 hours following a standard meal. When compared with placebo, Eurobiol and Eurobiol 25,000 reduced daily faecal fat excretion by 24% (not significant) and 43% (P less than 0.05), respectively. With the conventional preparation, enzyme output and fat absorption at the duodeno-jejunal flexure were significantly improved (P less than 0.05). Marked inter-individual differences in duodenal enzyme recovery (lipase 3% to 80%; chymotrypsin 26% to 100%) and, consequently, in the reduction of steatorrhoea (0% to 67%) were observed, with the gastric emptying rate emerging as a key determinant factor. With the enteric-coated preparation, enzyme output and fat absorption at the duodenojejunal flexure were not significantly improved. Discrepancy between the marked reduction of faecal fat excretion and the low duodenal enzyme recovery could indicate that enzyme delivery from microtablets occurs further down in the small bowel. Efficacy of enteric-coated preparations could be enhanced by adding unprotected enzymes, especially in patients with rapid gastric emptying.

[The cutaneous bioavailability of alpha-chymotrypsin from ointments]. / Cercetari privind biodisponibilitatea cutanata a alfa-chimotripsinei din unguente.

In view of improving the cutaneous bioavailability of alpha-chymotrypsin from ointments, the stability of this enzyme in two hydrophile ointment bases, macrogoli and carbopol, was tested. The cutaneous bioavailability of alpha-chymotrypsin in ointments, estimated by determining the anti-inflammatory action, underlines the important role played both by the ointment base and by the enzyme activating substances. By correlating the determinations of alpha-chymotrypsin enzymatic activity with the anti-inflammatory action a correlation between the manifested activity and the active substance stability was noticed.

Immobilization of enzymes on alumina by means of pyridoxal 5'-phosphate.

A number of proteases have been immobilized on alumina in a two-step procedure: the first step converted them into semisynthetic phosphoproteins which, in the second step, spontaneously bonded to alumina through their phosphate function. The immobilized enzymes thus obtained showed the physical properties typical of the inorganic carrier and a high activity on low molecular weight substrates.

Human hepatocytes exhibit receptors for alpha 2-macroglobulin and pregnancy zone protein-proteinase complexes.

Hepatocytes were isolated by application of the two-step collagenase technique to pieces of human liver. 125I-labelled alpha 2-macroglobulin-trypsin complex bound to hepatocytes at 4 degrees C with a half time of approximately 4.5 h. At near equilibrium half of the receptors were saturated at an alpha 2-macroglobulin-trypsin complex concentration of about 60 pmol 1(-1) and the Scatchard plot was linear. Dissociation of the labelled complex was slow (T1/2 = 24 h) at low receptor occupancies. At high receptor occupancies dissociation was biphasic with a rate constant (K-1) for the initial rapid phase of about 2.4 x 10(-2) min-1. Labelled alpha 2-macroglobulin-trypsin complex bound at 4 degrees C was rapidly internalized at 37 degrees C (T1/2 = 1.9 min), and in 3.5 h approximately 10% of the label was released into the medium in a trichloroacetic acid-soluble form. At 37 degrees C, 125I alpha 2-macroglobulin-trypsin was taken up by hepatocytes and trichloroacetic acid soluble radioactivity appeared in the medium following a sigmoidal curve. Similar results were obtained with 125I-pregnancy zone protein-chymotrypsin complex. At 4 degrees C, hepatocytes bound nearly equal amounts of labelled alpha 2-macroglobulin-trypsin and pregnancy zone protein-chymotrypsin complex, and a large excess (100 nmol 1(-1) of one of the macroglobulins could almost completely abolish binding of trace amounts (5-20 pmol 1(-1] of the other. The present findings strongly suggest that the hepatocyte is of major importance for removal of alpha 2-macroglobulin- and pregnancy zone protein-proteinase complex in humans, in agreement with previous results in rats and mice.