Hydrophilic polymers--biocompatibility testing in vitro.

Biocompatibility is one of the main prerequisites for safe use of medical devices. Estimation of cytotoxicity is a part of the initial evaluation laid down in ISO standards on biological evaluation of medical devices. Hydrophilic polymers (based on 2-hydroxyethyl methacrylate HEMA) doped by addition of selected additives with antioxidant and/or free radical scavenging potential (vitamin C and hindered amine stabilizer N-(2,2,6,6-tetramethylpiperidin-4-yl)methacrylamide) were tested in different in vitro systems (3T3 Balb/c cell culture and a 3D human skin model) for biocompatibility and suitability for use as wound dressings. The results of the 3T3 NRU cytotoxicity test using both the direct and indirect contact approaches and a 3D skin model modified irritation test (EpiDerm) confirmed high biocompatibility and good skin tolerance of both the basic polymers and those enriched with specific additives up to a balanced level. HEMA polymer showed a beneficial effect against cytotoxicity of an irritant (sodium dodecyl sulfate). The in vitro biocompatibility test results were confirmed by human local skin tolerance testing.

[The histopathologic picture of experimental poisoning with ethylmethacrylates]. / Histopatologický obraz experimentální intoxikace ethylmethakryláty.

Toxic effect of hydroxyethylmethacrylate, acetoxyethylmethacrylate and diethylenglycomethacrylate were studied in rats surviving as long as 1 to 20 days after intramuscular administration. Conspicuous lesion were found only in calf muscles at the site of application. Muscle fibre necroses with inflammatory reaction occurred repeatedly in animals surviving 1-2 days. Newly formed connective tissue replacing impaired muscle fibres was found in rats surviving 5 days. Intoxication related other lesion in rat organs were not identified.

Reactive oxygen species (ROS) generated by xanthine oxidase in the corneal epithelium and their potential participation in the damage of the corneal epithelium after prolonged use of contact lenses in rabbits.

Prolonged use of contact lenses (for 14 days) evoked an imbalance between the activity of xanthine oxidase (an enzyme belonging to reactive oxygen species-generating oxidases) and catalase (an enzyme belonging to reactive oxygen species-scavenging oxidases) in the corneal epithelium of rabbits. The activity of catalase decreased, while xanthine oxidase activity was very high. Of other enzymes studied in the corneal epithelium, the activities of xanthine oxidoreductase, glucoso-6-phosphate dehydrogenase and succinate dehydrogenase were decreased. In contrast, the activities of lactate dehydrogenase and lysosomal hydrolases (acid beta-galactosidase, dipeptidyl peptidase II) were increased and appeared in animals sacrificed immediately after contact lens removal. In rabbits sacrificed later (after 1 h), an additional increase of lactate dehydrogenase and lysosomal hydrolase activities developed in the superficial layers of the corneal epithelium. Catalase supplementation during use of contact lenses prevented both the significant decrease of catalase activity in the corneal epithelium and the development of additional epithelial damage. In contrast, topical treatment with 3-aminotriazole (an inhibitor of catalase) resulted in the nearly complete loss of catalase activity in the corneal epithelium and the appearance of more serious epithelial damage. We conclude that ROS generated by xanthine oxidase induce additional damage of the corneal epithelium related to the use of contact lenses.

[New approaches in clinical ophthalmology: UV absorbers and hindered amines covalently bound to contact lenses. Importance of enzyme histochemistry in studying their effectiveness in vivo]. / Nové prístupy vyuzitelné v klinické oftalmologii: UV absorbéry a stínené aminy kovalentne vázané v kontaktních cockách. Význam histochemie enzymu pro vysetrení jejich úcinnosti in vivo.

UV absorbers developed by us are covalently bound in hydrophilic contact lenses. This is great advantage because UV absorbers cannot be diluted from contact lenses. In laboratory investigations and "in vivo" experiments it was found that contact lenses containing UV absorbents prevent the eye against the damaging effect of UV irradiation (UVA, UVB, UVC rays). The second new approach (mentioned in this paper) is the use of Hindered Amine Light Stabilizer (4-methakryloylamino-2,2,6,6-tetramethylpiperidine) covalently bound in the hydrophilic contact or therapeutic lenses--for the healing of the damaged eye. In laboratory measurements the cleavage of hydrogen peroxide (of various concentrations) by hindered amine light stabilizer was confirmed. In "in vivo" experiments it was examined histochemically and morphologically that Hindered Amines Light Stabilizer bound in contact lenses favourable influenced the healing of the damaged anterior eye segment of rabbits. The amount as well as enzyme activation (lysosomal hydrolases) of inflammatory cells was significantly reduced in the anterior eye segment. It is suggested that Hindered Amine Light Stabilizer reacts with reactive oxygen species (oxyradicals and hydrogen peroxide) present in the damaged anterior eye segment (e.g. released from inflammatory cells). During this reaction intermediate products appear and the resulting product is a nitroxide radical. All reaction products as well as resulting product are covalently bound in hydrophilic material. For the development and also evaluation of the efficacy of both new approaches perspective for the use in clinical ophthalmology, biochemical investigations and histochemical methods are employed. Enzyme histochemistry is very important for the evaluation of the efficacy of new therapeutic approaches "in vivo" and therefore the particular attention is devoted to it in this communication.

Toxicity of monomers HEMA, DEGMA and AEMA, used in synthesis of hydrogels for medical applications.

Acute toxicity parameters (lethal doses) were evaluated and calculated after i.m. and i.p. administration of 2-hydroxyethyl methacrylate (HEMA), 2-(hydroxyethoxy)ethyl methacrylate (DEGMA) and 2-(acetoxy)ethyl methacrylate (AEMA) to rats. Our previous findings were revisited and corrected. Based on preceding standardization and physiological values study, some clinical chemical and hematological markers of in vivo subacute i.m. toxicity were followed. Hematological parameters revealed to be of low sensitivity to the toxic influence. Clinical chemical tests suggest the prevalent muscle cell injury.

[Distribution of 3H-labeled acetyl-HEMA monomer in rats]. / Distribuce 3H znaceného monomeru acetyl-HEMA u potkanu.

Original method of tritiation of acetyl-HEMA and its hydrogenated product was developed. Distribution and excretion of both compounds was examined in rat for up to 70 hrs. While the distribution in the tissues examined (liver, spleen, kidney, lung, muscle, heart, and skin) was nearly uniform, the excretion of acetyl-HEMA and its hydrogenated form differed: acetyl-HEMA in the 24. hour is excreted preferably into urine, the hydrogenated form in faeces. In addition to it the later form was absorbed from the application point (muscle) more rapidly. Distinct cumulation of activity (original compound or/and its metabolites) was observed in all tissues.

Disposition of lypophilized (methylmethacrylate-14C, 2-hydroxyethylmethacrylate, butylacrylate) nanoparticles in rats and their effect on zoxazolamine paralysis time.

The fate of lyophilized (methylmethacrylate-14C, 2-hydroxyethylmethacrylate, butylacrylate) nanoparticles was studied in male Wistar rats after p.o. administration. It was found that at least 4% of the dose of 14C was absorbed from the gastrointestinal tract after a single dose with these nanoparticles. Some radioactivity (less than 0.15% of dose) was found 7 d after administration in lung, spleen and liver. As expected excretion of the label was predominated via the feces. Ten d of p.o. treatment of rats with lyophilized nanoparticles (1 g/kg of body weight) was shown to prolong significantly zoxazolamine paralysis time. This result suggests that lyophilized nanoparticles decreased elimination of zoxazolamine.

Fate of 14C-terpolymer (methylmethacrylate-14C, 2-hydroxyethylmethacrylate, butylacrylate) nanoparticles after peroral administration to rats.

The fate of 14C-terpolymer (methylmethacrylate-14C, 2-hydroxyethylmethacrylate, butylacrylate) nanoparticles was studied in male Wistar rats after peroral administration. These nanoparticles may reach systemic circulation as evidenced by the plasma 14C level, excretion of the label in the urine, as well as organ label deposition. It was found that at least 2% of the dose of 14C was absorbed from the gastrointestinal tract. As expected, the radioactive nanoparticles were excreted predominantly via the feces. The amount of the label in the gastrointestinal tract, liver, and carcasses fell below the limit of detection on day seven after administration. However in the spleen and lung some slight radioactivity persisted after 7 d of experiment.

Determination of the interaction of lactate dehydrogenase with high-molecular-weight derivatives of AMP by affinity electrophoresis.

The interaction of lactate dehydrogenase with high-molecular-weight derivatives of AMP was studied by affinity electrophoresis in an alkaline buffer system and by means of kinetic measurements. AMP was coupled to synthetic hydroxypropylmethacrylamide copolymers through glycine, 6-aminohexanoic and 12-aminododecanoic spacer arms. The values of the dissociation constants (K) of the lactate dehydrogenase isoenzymes--immobilized AMP complexes determined by affinity electrophoresis decreased with increasing length of the spacer arm. Lactate dehydrogenase was competitively inhibited by high-molecular-weight derivatives of AMP; values of the inhibition constants (Ki) also depended on the spacer arm: the longer the spacer arm the stronger was the interaction between the enzyme and the inhibitor. Ki values for high-molecular weight derivatives of AMP were lower than those obtained for free AMP.

Reactive carriers of immobilized compounds.

Sphericanl macroporous reactive carriers capable of forming covalent bonds with amino acids and proteins were prepared by the suspension copolymerization of 2-hydroxyethyl methacrylate, ethylene dimethacrylate and p-nitrophenyl esters of methacrylic acid and methacryloyl derivatives of glycine, beta-alanine and epsilon-aminocaproic acid. The effect of the spacer length, pH and the type of the buffer used, concentration of reactive groups in the copolymer, concentration of the ligand and the participation of the hydrolytic and aminolytic reaction of p-nitrophenyl functional groups in the attachment of glycine, D,L-phenylalanine and serumalbumin was studied. Macroporous copolymers containing reactive functional groups can be used as active enzyme carriers, if their activity is not blocked by the presence of p-nitrophenol split off in the attachment reaction.