RESUMO
Biocatalytic surface coatings are prepared herein for localized synthesis of drugs and their on-demand, site-specific delivery to adhering cells. This novel approach is based on the incorporation of an enzyme into multilayered polymer coatings to accomplish enzyme-prodrug therapy (EPT). The build-up of enzyme-containing multilayered coatings is characterized and correlations are drawn between the multilayer film assembly conditions and the enzymatic activity of the resulting coatings. Therapeutic effect elicited by the substrate mediated EPT (SMEPT) strategy is investigated using a prodrug for an anticancer agent, SN-38. The performance of biocatalytic coatings under flow conditions is investigated and it is demonstrated that EPT allows synthesizing the drugs on-demand, at the time desired and in a controllable amount to suit particular applications. Finally, using cells cultured in sequentially connected flow chambers, it is demonstrated that SMEPT affords a site-specific drug delivery, that is, exerts a higher therapeutic effect in cells adhering directly to the biocatalytic coatings than in the cells cultured "downstream". Taken together, these data illustrate biomedical opportunities made possible by engineering tools of EPT into multilayered polymer coatings and present a novel, highly versatile tool for surface mediated drug delivery.
Assuntos
Biocatálise , Técnicas de Cultura de Células/métodos , Materiais Revestidos Biocompatíveis/farmacologia , Preparações Farmacêuticas/síntese química , Polímeros/farmacologia , Camptotecina/análogos & derivados , Camptotecina/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Glucuronidase/metabolismo , Células Hep G2 , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Irinotecano , Perfusão , Pró-Fármacos/síntese química , Técnicas de Microbalança de Cristal de QuartzoRESUMO
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 ß-galactosidase (ß-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 ß-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.
Assuntos
Óxido Nítrico/análise , Animais , Endotélio Vascular , Inibidores Enzimáticos , Enzimas Imobilizadas , Humanos , Pró-Fármacos , Ratos , Vasodilatação , beta-GalactosidaseRESUMO
In this work, we characterize physical hydrogels based on poly(vinyl alcohol), PVA, as intelligent biointerfaces for surface-mediated drug delivery. Specifically, we assemble microstructured (µS) surface adhered hydrogels via noncryogenic gelation of PVA, namely polymer coagulation using sodium sulfate (Na(2)SO(4)). We present systematic investigation of concentrations of Na(2)SO(4) as a tool of control over assembly of µS PVA hydrogels and quantify polymer losses and retention within the hydrogels. For polymer quantification, we use custom-made PVA with single terminal thiol group in a form of mixed disulfide with Ellman's reagent which provides for a facile UV-vis assay of polymer content in coagulation baths, subsequent washes in physiological buffer, and within the hydrogel phase. Polymer coagulation using varied concentrations of sodium sulfate afforded biointerfaces with controlled elasticity for potential uses in investigating mechano-sensitive effects of mammalian cell culture. For surface mediated drug delivery, we propose a novel concept termed Substrate Mediated Enzyme Prodrug Therapy (SMEPT) and characterize µS PVA hydrogels as reservoirs for enzymatic cargo. Assembled functional interfaces are used as matrices for cell culture and delivery of anticancer drug achieved through administration of a benign prodrug, its conversion into an active therapeutic within the hydrogel phase, and subsequent internalization by adhered hepatic cells. Taken together, the presented data contribute significantly to the development of novel matrices for surface-mediated drug delivery and other biomedical applications.