Functional Layer-by-Layer Thin Films of Inducible Nitric Oxide (NO) Synthase Oxygenase and Polyethylenimine: Modulation of Enzyme Loading and NO-Release Activity

Abstract

Nitric oxide (NO) release counteracts platelet aggregation and prevents the thrombosis cascade in the inner walls of blood vessels. NO-release coatings also prevent thrombus formation on the surface of blood-contacting medical devices. Our previous work has shown that inducible nitric oxide synthase (iNOS) films release NO fluxes upon enzymatic conversion of the substrate l-arginine. In this work, we report on the modulation of enzyme loading in layer-by-layer (LbL) thin films of inducible nitric oxide synthase oxygenase (iNOSoxy) on polyethylenimine (PEI). The layer of iNOSoxy is electrostatically adsorbed onto the PEI layer. The pH of the iNOSoxy solution affects the amount of enzyme adsorbed. The overall negative surface charge of iNOSoxy in solution depends on the pH and hence determines the density of adsorbed protein on the positively charged PEI layer. We used buffered iNOSoxy solutions adjusted to pHs 8.6 and 7.0, while saline PEI solution was used at pH 7.0. Atomic force microscopy imaging of the outermost layer shows higher protein adsorption with iNOSoxy at pH 8.6 than with a solution of iNOSoxy at pH 7.0. Graphite electrodes with PEI/iNOSoxy films show higher catalytic currents for nitric oxide reduction mediated by iNOSoxy. The higher enzyme loading translates into higher NO flux when the enzyme-modified surface is exposed to a solution containing the substrate and a source of electrons. Spectrophotometric assays showed higher NO fluxes with iNOSoxy/PEI films built at pH 8.6 than with films built at pH 7.0. Fourier transform infrared analysis of iNOSoxy adsorbed on PEI at pH 8.6 and 7.0 shows structural differences of iNOSoxy in films, which explains the observed changes in enzymatic activity. Our findings show that pH provides a strategy to optimize the NOS loading and enzyme activity in NOS-based LbL thin films, which enables improved NO release with minimum layers of PEI/NOS.

Nitric Oxide Delivering Surfaces: An Overview of Functionalization Strategies and Efficiency Progress

“…Gunasekera and coworkers reached a higher NO flux of 0.3 nmol cm −2 min −1 by optimizing the quantity of iNOS immobilized. [88] It is interesting to notice that this value is close to the physiological level of NO production by human endothelial cells (0.4 nmol cm −2 min −1 ). [92] They studied an LbL organization allowing the immobilization of iNOS through electrostatic interactions with a cationic polyelectrolyte (poly(ethyleneimine)).…”

Micro‐ and Nanoscopic Imaging of Enzymatic Electrodes: A Review

“…This is consistent with an electrostatically driven immobilization of the enzyme, which is more negatively charged at higher pH. [70] AFM was also coupled to electrochemistry to reveal the impact of spatial organization at the nanometer scale to enzymatic activity of GOx immobilized on bacteriophage virus used as a platform on gold electrodes. [71] Surface coverage can also be deduced from STM images, as was done for Achromobacter xylosoxidans nitrite reductase (Ax NiR) [72] or Escherichia coli cytochrome c nitrite reductase (Ec cyt.c NiR).…”

“…After deposition of nitric oxide synthase oxygenase solution at pH 7 or 8.6 on positively charged polyethyleneimine (PEI) on HOPG, AFM imaging in ambient conditions showed more enzymatic clusters at higher pH. This is consistent with an electrostatically driven immobilization of the enzyme, which is more negatively charged at higher pH . AFM was also coupled to electrochemistry to reveal the impact of spatial organization at the nanometer scale to enzymatic activity of GOx immobilized on bacteriophage virus used as a platform on gold electrodes …”

Nitric Oxide-Producing Cardiovascular Stent Coatings for Prevention of Thrombosis and Restenosis

“…The coating materials possess the feasibility to directly attach or deposit onto the stents' surface and the ability to maintain a high local therapeutic concentration at specific site (Yang et al, 2017 ). Drugs and biomolecules such as paclitaxel (Palmerini et al, 2015 ), adhesive peptides (Wei et al, 2013 ), anti-CD 34 antibodies (Yoon et al, 2002 ), and nitric oxide (NO)-producing moieties (Gunasekera et al, 2018 ) are employed to improve the clinical behaviors of stents (see Table 1 for details). Although these molecules achieved good therapeutic effects, they still present some limitations.…”