Enzyme prodrug therapy achieves site-specific, personalized physiological responses to the locally produced nitric oxide

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

Antibody-Drug Conjugates to Treat Bacterial Biofilms via Targeting and Extracellular Drug Release

The treatment of implant-associated bacterial infections and biofilms is an urgent medical need and a grand challenge because biofilms protect bacteria from the immune system and harbor antibiotic-tolerant persister cells. This need is addressed herein through an engineering of antibody-drug conjugates (ADCs) that contain an anti-neoplastic drug mitomycin C, which is also a potent antimicrobial against biofilms. The ADCs designed herein release the conjugated drug without cell entry, via a novel mechanism of drug release which likely involves an interaction of ADC with the thiols on the bacterial cell surface. ADCs targeted toward bacteria are superior by the afforded antimicrobial effects compared to the non-specific counterpart, in suspension and within biofilms, in vitro, and in an implant-associated murine osteomyelitis model in vivo. The results are important in developing ADC for a new area of application with a significant translational potential, and in addressing an urgent medical need of designing a treatment of bacterial biofilms

Substrate mediated enzyme prodrug therapy.

In this report, we detail Substrate Mediated Enzyme Prodrug Therapy (SMEPT) as a novel approach in drug delivery which relies on enzyme-functionalized cell culture substrates to achieve a localized conversion of benign prodrug(s) into active therapeutics with subsequent delivery to adhering cells or adjacent tissues. For proof-of-concept SMEPT, we use surface adhered micro-structured physical hydrogels based on poly(vinyl alcohol), β-glucuronidase enzyme and glucuronide prodrugs. We demonstrate enzymatic activity mediated by the assembled hydrogel samples and illustrate arms of control over rate of release of model fluorescent cargo. SMEPT was not impaired by adhering cells and afforded facile time - and dose - dependent uptake of the in situ generated fluorescent cargo by hepatic cells, HepG2. With the use of a glucuronide derivative of an anticancer drug, SN-38, SMEPT afforded a decrease in cell viability to a level similar to that achieved using parent drug. Finally, dose response was achieved using SMEPT and administration of judiciously chosen concentration of SN-38 glucuronide prodrug thus revealing external control over drug delivery using drug eluting surface. We believe that this highly adaptable concept will find use in diverse biomedical applications, specifically surface mediated drug delivery and tissue engineering

Utility of SMEPT in conversion of fluorogenic prodrug and cargo uptake is verified through quantification of fluorescence of hepatic cells cultured on the µS PVA hydrogels.

<p>Administration of FdG in the absence of enzyme led to a negligible change in the fluorescence of cultured cells (traces 1 : cells only, trace 2 : +FdG). SMEPT conditions (trace 3 : β-Glu immobilized within µS hydrogels, +FdG) afforded comparable level of fluorescence of the cultured cells as solution based administration (trace 4 : β-Glu and FdG are added to media above cultured cells), as quantified by flow cytometry analysis of harvested cells. In all cases, [FdG] = 2,5 µg/mL.</p

Dose response for HepG2 cells to the administered SN-38 glucuronide (for SMEPT and solution based EPT) or SN-38.

<p>Presented results (mean ± st.dev.) are average over at least 3 independent experiments, 3 replicates each.</p

Viability of HepG2 cells cultured on µS PVA thin films as quantified using Presto Blue viability assay and expressed relative to viability of these cells on tissue culture polystyrene at a matched initial cell seeding density.

<p>The cells were cultured on (A) pristine µS PVA hydrogels; (B) µS PVA thin films equipped with β-Glu; (C) enzyme-free µS PVA hydrogels in the presence of 1 µM SN-38 glucuronide; (D) enzyme-free µS PVA hydrogels, 1 µM SN-38; (E) µS PVA hydrogels in the presence of 1 µM SN-38 glucuronide and β-Glu added to the cell media (solution based enzyme prodrug therapy); (F) SMEPT conditions, i.e. β-Glu equipped µS PVA thin films in the presence of 1 µM SN-38 glucuronide. Presented results (mean ± st.dev.) are average over at least 3 independent experiments, 3 replicates each.</p

Time- and dose- dependent cellular internalization of the model fluorescent product, fluorescein, generated via SMEPT from its prodrug, FdG.

<p>Experimental conditions: 1 g/L enzyme in the polymer solution; initial concentration of FdG: 0.25 (red circles) and 0.025 (black squares) µg/mL. Presented results (mean ± st.dev.) are average over at least 3 independent experiments, 3 replicates each.</p

Conversion of the prodrug by the enzyme within a PVA hydrogel matrix is not impaired by the presence of serum, i.e. possible absorption of proteins, and adhesion of mammalian cells.

<p>Experimental conditions: 1 g/L β-Glu in polymer solution, 30 min reaction time. Presented results (mean ± st.dev.) are average over at least 3 independent experiments, 3 replicates each.</p

SMEPT offers several arms of control over the rate of generation and the overall amount of the product generated by the enzyme containing substrates: concentration of the enzyme in the gel (a), concentration of the added substrate (b) and time of enzymatic conversion (c).

<p>Experimental conditions: (a) FdG, 2,5 µg/mL, 30 min reaction time; (b): 1 g/L β-Glu in polymer solution, 30 min reaction time; c) FdG: 2,5 µg/mL; β-Glu: 1 (top), 0.1 (middle) and 0.01 (bottom) g/L, respectively. Presented results (mean ± st.dev.) are average over at least 3 independent experiments, 3 replicates each.</p