MO752: Doping Polysulfone Dialysis Membranes with Human Neutrophil Elastase Inhibitors—A Proof-of-Concept Study
Authors: Rocha Susana , Rebelo Rute , Amorim Célia , Moreira Rui , Santos-Silva Alice , Conceição Branco Da Silva Montenegro Maria 
Published: May 2022

Abstract

BACKGROUND AND AIMS
The recurrent contact of blood with the haemodialysis (HD) membrane during the treatment procedure, triggers neutrophil activation, leading to an increase of blood levels of neutrophil elastase that contributes to the chronic inflammatory response experienced by end-stage renal disease patients with a consequent increase of morbidity and mortality. Based on our group's experience in developing flat-sheet polysulfone (PSF) membranes by using the spin-coating and phase-inversion methods [1], and in the synthesis of human neutrophil elastase inhibitors (HNEIs), namely 4-oxo-β-lactam-based compounds [2], the successful doping of PSF membranes with HNEIs has been achieved [3]. These PSF-HNEI biomaterials have proven to be bioactive and biocompatible, and they included in house synthetized HNEIs alongside Sivelestat (SIV, a second generation HNEI) PSF membranes [3]. In this study, we took a step forward aiming to test the efficacy of such modified membranes when exposed to biological conditions (human plasma).

METHOD
SIV (Abcam) and an in house synthesized 4-oxo-β-lactam-based compound (D4L-2) [2], from our library, were used. The IC50 of both HNEIs in HEPES buffer was evaluated by performing an HNE activity assay [2]. PSF membranes were prepared as described elsewhere [1] and doped with each HNEI by adsorption [3]. In 3 independent assays, triplicates of PSF membrane circles (Ø = 0.6 cm) were incubated with HNEIs vehicle (2.5% DMSO) or with 10–2000 nM SIV or D4L-2 during 3 h at 25°C, for HNEIs adsorption. Afterwards, all PSF-HNEI membranes were incubated in diluted plasma (50% PBS, pH 7.4) or HEPES buffer for 3 h at 37°C. The bioactivity of PSF-HNEI membranes was assessed before and after incubation using the HNE activity assay [2]. The value obtained for PSF membranes incubated with HNEIs vehicle was used to normalize the results (0% HNE inhibition).

RESULTS
The IC50 of SIV and D4L-2 in solution were 32.7 and 18.1 nM, respectively. Before incubation with either plasma or HEPES buffer, the HNE inhibition capacity of both PSF-HNEI membranes increased in a concentration-dependent manner, reflecting the HNEIs solutions used during the adsorption process. The highest HNE inhibition ability was presented by PSF-D4L-2 membranes (91.7% ± 0.9% versus 35.3% ± 5.0%). After incubation, PSF-HNEIs membranes immersed in HEPES buffer showed a similar HNE inhibition (84.5% ± 2.2% and 34.0% ± 6.2% for D4L-2 and SIV, respectively). However, the modified membranes incubated in plasma showed a significant decrease in HNE inhibition (D4L-2: 23.7% ± 1.6% and SIV: 7.0% ± 2.1%).

CONCLUSION
D4L-2 showed the best performance (lower IC50 value and higher HNE inhibitory capacity of doped membranes), which is in accordance with following the assumption that the 4-oxo-β-lactam-based inhibitors are more stable molecules and present higher specificity for HNE. However, both PSF-HNEI biomaterials seem to have fulfiled the objective for which they were developed, since, after being incubated in plasma, they presented a significant decrease in the capacity to inhibit HNE, showing that the HNEIs immobilized in the membranes had captured/bound to the HNE present in the plasma sample.