Gold Nanoparticles associated to Bromelain-derived Cisteine-Proteases: Structural Characterization and Collagenolytic Activity.

Abstract

Bromelain (Bro) is a multiprotein complex extract (from the pineapple plant [Ananas comosus]) composed of at least eight cysteine-proteases. Bro is described as possessing anti-inflammatory, anti-coagulant and anti-tumor properties, including applications in the food, cosmetic and environmental industries. Concerning proteases applications, the increase of stability is considered a key factor, obtained, for example, by immobilization in nanoparticles (NPs). In addition, proteins can be used during the NP synthesis process, directely influencing the physico-chemical characteristics of the particles. In particular, gold nanoparticles (GNPs) have high biocompatibility and unique optical, electronic and catalytic properties. In order to synthesize and characterize new GNPs produced and immobilized with proteases, preliminary data from our group demonstrate that the complex Bro extract acts both as reducing agent and stabilizer during the process of GNP synthesis. In contrast, once in association with GNPs, Bro presented a significant increase in thermal stability and structural recovery when previously inactivated. When tested against collagen membranes, Bro-GNP activity resulted in uniques structural profiles in the membranes as observed under Electron Microscopy (SEM) and Infrared Absorption Spectroscopy (FTIR), suggesting the importance of individual Bromelain-derived proteases processing collagen. Since GNPs also have potential use for drug delivery, their association with proteases with collagenolytic activity points out the possibility to act direct in extracellular matrix, bringing interests to use Bro-GNPs in future anti-tumor treatments. Considering these significant effects, the present project aims to purify the possible eight proteolytic fractions of the Bro complex and to use them separately in the synthesis of new GNPs. The free forms of Bro (1-8) and the Bro(1-8)GNPs generated will be characterized structurally by Electron Transmission Microscopy and functionally using substrates as fluorogenic peptides and collagen membranes, which will be analyzed and compared through Confocal Laser Scanning Microscopy (CLSM), SEM and ATR-FTIR. Bro(1-8)GNPs produced will further be tested for cytotoxicity in non-tumor and tumor cells, essential assays for future directions of the project. Also, we will purify the four major cysteine proteases contained in commercial papain, as well perform the synthesis and initial characterization of derived nanoparticles - Pap(1-4)GNPs. Taken together, these data may represent advances in the characterization of proteases-GNPs complexes and will provide subsidies for biotechnological and pharmaceutical uses in these and other applications. (AU)