Photochemical conjugation of mammalian cells to polymeric supports and membranes: a kinetic study.

Abstract

A commercial polyester acrylate prepolymer, to which a 2:3 wt/wt ratio of tripropyleneglycol diacrylate was added to increase photopolymerization rate, was employed as photochemical conjugating agent, through photografting. 1,2-Diphenyl,2,2-dimethoxyethanone was added as standard photoinitiator (7.0 wt/wt%), together with varying amounts (0.003-4.0 wt/wt%) of some proprietary photocatalytic systems, based on the following photocatalysts: mu-peroxo-bis[N,N'-ethylene-bis(salicylideneiminato)cobalt(I II) (I), vanadium(V) triethoxide (II), and a synergic mixture of vanadium(V) tri-t-butoxide and tri-i-propoxide (III). A homogeneous suspension containing (10 +/- 2) x 10(5) human thyroid follicular cells per milliliter of photochemically reacting medium was photografted, at a surface density of 6.5 +/- 0.7 mg.cm-2 of diacrylate prepolymer mixture, onto polystyrene plates or onto commercial microfiltration and ultrafiltration membranes consisting of nonwoven cellulose tissues with known porosities varying between 5 and 30 microns and in photografted polyester acrylate-based membranes with a cutoff of 50 +/- 5 KD. Bioconjugation yields, as a function of photografting time, were measured gravimetrically and by multiple internal reflection IR spectroscopy. Three series of experiments were performed: 1) measurements of graft yields of the prepolymer, and of the parallel disappearance of double bonds, in the absence of mammalian cells; 2) the same as 1), in the presence of thyroid follicular cells; 3) the same as 2), but with the photoinitiating system formed by the standard photoinitiator alone, with no photocatalyst. Results show that if a suitable photocatalyst is not added, no practical conjugation is possible. An appropriate choice of the photocatalytic system and of its concentration allows reduction of irradiation times (e.g., by a factor of about 2 x 10(4) calculated as the mean lifetime ratio, between the uncatalyzed system and that with 0.1 wt% of [III]), thus minimizing cell inactivation and/or improving responsiveness to the bioassay. From this point of view, photoactivity of (III) is outstanding. The very small, but clearly perceptible, influence of polymeric support on bioconjugation is also commented upon.