In situ mineralization of MnO2 on layer-by-layer polymer capsules to limit the accumulation of cytotoxic by-products resulting from enzymatic reactions

CADAVID VARGAS, J.F.; MARIN, EDURNE; LARRAGAÑA E, AITOR; SARRASUA, JOSÉ

Porto

Conferencia; 31st Annual Conference of the European Society for Biomaterials (ESB 2021); 2021

ntroductionThe use of enzymes has emerged as an attractive strategy in the fabrication of biomedical systems for therapeutic applications thanks to their specificity and efficacy. However, considering their susceptibility to undergo protease degradation and denaturation in body fluids, several encapsulation strategies have been considered to protect them from harsh environmental conditions and maintain their structural integrity [1].Glucose oxidase (GOx) catalyzes the reaction of the environmental glucose to yield gluconic acid and hydrogen peroxide (H2O2). Based on this reaction, a plethora of biomedical systems have been developed including insulin delivery capsules [2], glucose sensors [3], cancer cell targeting capsules [4] or antimicrobial capsules [1]. The cytotoxic H2O2 produced in the reaction has been exploited to combat cancerous cells and bacteria (Fig. 1a) [1,4]. However, in many other strategies, the accumulation of H2O2 can have a detrimental effect on the surrounding cells/tissues, as well as in the catalytic activity of the enzyme, thus limiting its biomedical applications (Fig. 1b) [2,3].To scavenge the produced H2O2, the use of MnO2 as antioxidant synthetic enzyme (nanozyme) has been placed in the spotlight of many investigations owing to its unique enzyme mimetic activity and stability.In this work, inspired by the compartmentalization strategies found at the cellular and subcellular level, we encapsulated GOx into layer-by-layer (LbL) polymer capsules. Their outer layer was further functionalized with an in situ created MnO2 shell to simultaneously regulate glucose levels and reduce the undesired production of H2O2 from the microenvironment (Fig. 1b).Experimental MethodsMicrocapsules were fabricated via the LbL approach following a well-established protocol [5]. GOx was preloaded in the inner cavity of the capsule through a co-precipititation method, to obtain GOx-loaded CaCO3 spherical microparticles. After the LbL process, using as polyelectrolytes poly (sodium 4-styrenesulfonate) (PSS) and poly (allylamine hydrochloride) (PAH), the MnO2 layer was created in situ via a mineralization process incubating different KMnO4 solutions (i.e., 2.5, 5, 10, 20, 50 and 100 mM) with the microparticle. After a morphological and physicochemical analysis of the fabricated capsules by means of scanning electron microscopy (SEM), ζ-potential, UV-vis and X-ray diffraction (XRD), the antioxidant capacity of the MnO2 layer and the glucose reduction capacity of the GOx was assessed. Finally, the protective effect of the capsules was studied in an in vitro model of human lung fibroblast cells (MRC-5).Results and DiscussionCapsules loaded with GOx and functionalized with the MnO2 shell were satisfactorily fabricated via the LbL approach and displayed a spherical morphology with sizes in the range of 3-5 microns (Fig. 2a). The alternate deposition of the layers was confirmed with ζ-potential measurements in which a charge reversal was observed as different polyelectrolytes were incorporated (Fig. 2b). The capsules efficiently scavenged H2O2 from solution at biologically relevant concentrations (10-50 µM H2O2) (Fig. 2c), particularly at the highest KMnO4 concentrations studied (i.e., 20, 50 and 100 mM). The activity of the encapsulated enzyme was preserved, as demonstrated by its ability to successfully reduce glucose from solution. The presence of naked (i.e., without the external MnO2 shell) capsules promoted H2O2-induced cell death at increasing glucose concentrations in the culture media. In contrast, the external MnO2 shell scavenged the overproduced H2O2, thus ensuring the survival of MRC-5 cells.ConclusionIn this work we fabricated polymer capsules via the LbL approach, which were able to reduce the glucose levels from the cellular microenvironment and simulatenously scavenge the undesired H2O2 created from the GOx reaction. These polymer capsules represent a promising strategy to protect enzymes from harsh environmental conditions maintaining their activity, while avoiding cell damage by reducing the effect of undesirable by-product accumulation