Enzyme Prodrug Therapy Achieves Site-Specific, Personalized Physiological Responses to the Locally Produced Nitric Oxide

Abstract

Nitric oxide (NO) is a highly potent but short-lived endogenous radical with a wide spectrum of physiological activities. In this work, we developed an enzymatic approach to the site-specific synthesis of NO mediated by biocatalytic surface coatings. Multilayered polyelectrolyte films were optimized as host compartments for the immobilized β-galactosidase (β-Gal) enzyme through a screen of eight polycations and eight polyanions. The lead composition was used to achieve localized production of NO through the addition of β-Gal–NONOate, a prodrug that releases NO following enzymatic bioconversion. The resulting coatings afforded physiologically relevant flux of NO matching that of the healthy human endothelium. The antiproliferative effect due to the synthesized NO in cell culture was site-specific: within a multiwell dish with freely shared media and nutrients, a 10-fold inhibition of cell growth was achieved on top of the biocatalytic coatings compared to the immediately adjacent enzyme-free microwells. The physiological effect of NO produced via the enzyme prodrug therapy was validated ex vivo in isolated arteries through the measurement of vasodilation. Biocatalytic coatings were deposited on wires produced using alloys used in clinical practice and successfully mediated a NONOate concentration-dependent vasodilation in the small arteries of rats. The results of this study present an exciting opportunity to manufacture implantable biomaterials with physiological responses controlled to the desired level for personalized treatment.

Combatting implant-associated biofilms through localized drug synthesis

“…Alternatively, surface modification can be designed to be virtually permanent, using non-degradable polymers. In our prior studies, we engineered EPT into multilayered polyelectrolyte coatings to achieve localized synthesis of anti-proliferative drugs [ 22 ] and for delivery of nitric oxide [ 15 ], biomedical opportunities highly warranted in the context of cardiovascular stenting. We have also demonstrated that enzymes immobilized within such coatings exhibit negligible escape from the multilayered films and biocatalysis is confined to the surface coatings, being key to the desired site-specific production of therapeutics via EPT.…”

“…We have also demonstrated that enzymes immobilized within such coatings exhibit negligible escape from the multilayered films and biocatalysis is confined to the surface coatings, being key to the desired site-specific production of therapeutics via EPT. [ 15 , 22 , 40 ] Herein we propose that such nanometer-thin coatings can be deposited on the surface of an implant to prevent and/or treat bacterial biofilm ( Fig. 1 ).…”

“…EPT is a two-pronged approach that relies on i) an enzyme positioned at the site for therapeutic intervention, and ii) a corresponding prodrug that is inactive in the administered form but is activated by the enzyme. Benefits of EPT include the possibility to interactively change the drug dose through optimization of prodrug feed as a step towards personalized treatment; [ 15 ] performing combination therapy treatment and on-demand drug synthesis; [ 16 ] performing localized synthesis of drugs with limited stability in physiological media [ 15 ] for which conventional drug delivery measures are futile or challenging. Success of EPT depends on both, method of enzyme localization [ 14 ] and on the choice of the prodrug [ 17 ].…”

“…The latter approach is termed “substrate mediated EPT” (SMEPT, Fig. 1 ) and was engineered by us and others using hydrogel biomaterials, [ 16 ] [ 20 ] electrospun fibers, [ 21 ] and surface coatings [ 15 , 22 ] to achieve the synthesis of drugs with anti-proliferative and anti-inflammatory activity. [ 18 ] EPT has even been performed using bacteria to colonize tumor and therein express the enzyme for localized prodrug conversion [ 23 ].…”

Structural heterogeneity in polymeric nitric oxide donor nanoblended coatings for controlled release behaviors

“…Over the past tens of years, nitric oxide delivery carriers have developed into various biomedical application forms, such as nanoparticles, [1][2][3][4] thin lm coatings, 5,6 hydrogels, 7,8 metalorganic frameworks (MOFs). 9,10 Nitric oxide (NO) is an indispensable cell signaling gas molecule, which is generated by nitric oxide synthases (NOSs) by consuming L-arginine.…”