Toksičnost i kinetika otpuštanja insulina iz hormonaktivnih polisaharidnih vlakana

Abstract

In this paper an account of biocompatibility of a hormonactive fiber in the form of biologically active complex chitosan-insulin is presented. The behavior of artificial depots of insulin containing 180-330mg of insulin per gram of fiber has been studied. The artificial depot of insulin has been characterized from the standpoint of its behavior in physiological solution (in the culture of L929 cells and in organism of an experimental animal). In vitro conditions, the intensity of desorption depends on the nature of solution and on pH of medium. It has been shown that insulin is controllably delivered in physiological solutions in quantities of 1.3-1.6mg of insulin during 24 hours, at pH about 7. The quantity of insulin delivered has been assessed by UV Sprectrophotometry. Biocompatibility of hormone-active fibers in the form of artificial depot of insulin has been determined in pre-clinical conditions through determination of the degree of harmfulness (toxicity) to the organism of the experimental animal. Tests have been performed according to the matrix for testing of biocompatibility and according to recommendations and guides published in Guidelines ISO 10993. Noxiousness has been assessed by testing: citotoxicity, sensitivization and irritation. Qualitative and quantitative citotoxicity tests in direct contact on L929 cells culture have shown that a hormone-active fiber does not provoke the citotoxic effect. The results obtained have shown that quantities of controllably released insulin could satisfy basal needs of an organism and that the artificial depot does not show effects of toxicity. On the bases of the obtained results from tests and calculated index of sensitivitization it follows that the artificial depot of insulin does not cause sensitivization. Also, the results obtained by subcutaneous irritation testing showed that the fibrous artificial depot of insulin could be eveluated as a non-irritating bioactive material.

U ovom radu kao deo pretkliničkog ispitivanja biokompatibilnosti, ispitivana je toksičnost i efikasnost veštačkih depoa insulina. Veštački depoi insulina sadržali su 180-330 mg insulina po gramu vlakna hitozana. Cilj rada je bio: 1. odrediti kinetiku otpuštanja insulina iz hormonaktivnog vlakna u in vitro uslovima, 2. ispitati senzibilizaciju in vivo; 3. ispitati primarnu kutanu iritaciju in vivo i 4. ispitati citotoksični efekat in vitro. Metode: Količina otpuštenog insulina praćena je UV spektrofotometrijom. Pretklinička ispitivanja biokompatibilnosti hormonaktivnih vlakana izvršena su prema preporukama i vodičima publikovanih u Guidelines ISO 10993 kao i prema matriksu za ispitivanje biokompatibilnosti. Toksičnost je ispitana kroz testiranje: senzibilizacije i primarne kutane iritacije in vivo i testiranje citotoksičnosti (kvalitativno i kvantitativno) u kulturi L929 ćelija. Pokazano je da se insulin kontrolisano otpušta u fiziološkom rastvoru u količinama od 1.3-1.6 mg insulina u toku 24 sata, pri pH oko 7. Prema dobijenim rezultatima ispitivanja i izračunatog indeksa senzibilizcije sledi da veštački depo ne izaziva senzibilizaciju. Takođe, dobijeni rezultati ispitivanja primarne kutane iritacije su pokazali da se vlaknasti veštački depo insulina može oceniti kao neiritirajući bioaktivni materijal. Kvalitativni i kvantitativni testovi citotoksičnosti u direktnom kontaktu na kulturi ćelija L929 su pokazali da hormonaktivno vlakno ne izaziva citotoksični efekat. U cilju dalje karakterizacije biokompatibilnosti (toksičnosti i efikasnosti) hormonaktivnih polisahaidnih vlakana, dalja istraživanja su neophodna pre njihove kliničke primene.