GUSTAVO HENRIQUE COSTAVARCA
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Artigo IPEN-doc 26876 Papain-cyclodextrin complexes as an intestinal permeation enhancer2020 - CORAZZA, FULVIO G.; ERNESTO, JULIA V.; NAMBU, FELIPE A.N.; CARVALHO, LUMA R. de; LEITE-SILVA, VANIA R.; VARCA, GUSTAVO H.C.; CALIXTO, LEANDRO A.; VIEIRA, DANIEL P.; ANDREO-FILHO, NEWTON; LOPES, PATRICIA S.Oral drug delivery is the main route for drugs administration. However, some drugs have poor permeability across the intestinal barrier. Papain has been widely used in pharmaceutical applications due to its debridement properties and the ability to promote skin permeation of drugs. It is known that papain complexation with cyclodextrins improves its biological stability. In this paper, the ability of the native papain-cyclodextrin complexes is shown be an oral permeation enhancer to furosemide, using a Caco-2 monolayer system to evaluate drug permeability and apparent permeability coefficient. Analysis of the in vitro cytotoxicity over CHO–K1, Hep G2 and Caco-2 cell lines and genotoxicity over CHO–K1 and Hep G2 cell lines were also performed. Papaincyclodextrins complexes did not show any cytotoxicity above 31 μg/mL. No significant genotoxic damage was observed. Papain and cyclodextrin complexes induced almost 2.5-fold increase in furosemide permeation compared to controls, and maintenance of the paracellular integrity of the Caco-2 cells monolayer was confirmed. The papain complexes may be safely applied in pharmaceutical formulations, not only as a therapeutic agent but also as a strategic pharmaceutical adjuvant, promoting permeation of low oral permeability drugs.Artigo IPEN-doc 26883 Simultaneous intramolecular crosslinking and sterilization of papain nanoparticles by gamma radiation2020 - FAZOLIN, GABRIELA N.; VARCA, GUSTAVO H.C.; FREITAS, LUCAS F. de; ROKITA, BOZENA; KADLUBOWSKI, SLAWOMIR; LUGAO, ADEMAR B.Papain-based nanoparticles were recently developed using radiation technologies and proven effective to generate nanosized crosslinked papain particles with preserved enzymatic activity. The applications of such nanostructured systems are expected to be similar to native papain with considerable biopharmaceutical advantages and concern drug loading among other biotechnological applications. The nature of such crosslinks and the possibility to provide simultaneous sterilization have been hypothesized but remain not totally clarified. This manuscript advances the discussion on the radiation-induced synthesis of protein nanoparticles by approaching the nature of the crosslinking and the possible contribution of bityrosine linkages and disulfide bridges to the overall nanoparticle assembly as well as the feasibility of the simultaneous sterilization process under the preestablished conditions of processing. Papain nanoparticles were synthesized and characterized according to size, proteolytic activity, bityrosine, cysteine content and molecular weight by SDS-PAGE upon sonication at 40 kHz. Bacterial identification and the sterility tests were performed in accordance with ISO 11737 prior to and after inoculating 106 CFU of Corynebacterium xerosis. Our experiments evidenced the crosslinking of rather intra- than intermolecular nature and a contribution of cysteine bridges and bityrosine linkages to the stabilization and formation of the papain nanoparticle assembly. The technique was effective to promote simultaneous crosslinking and sterilization at the established conditions of processing and may be validated in accordance with the ISO 11137.Artigo IPEN-doc 25091 The effects of radiation and experimental conditions over papain nanoparticle formation2020 - FAZOLIN, GABRIELA N.; VARCA, GUSTAVO H.C.; KADLUBOWSKI, SLAWOMIR; SOWINSKI, SEBASTIAN; LUGAO, ADEMAR B.Papain is a natural enzyme extracted from the fruit of Carica papaya Linnaeus, successfully applied in the pharmaceutical area as a drug carrier and debridement agent for wounds. In recent studies papain nanoparticles were synthesized and crosslinked with the use of ionizing radiation in the search for biopharmaceutical advantages as well as the development of bioactive nanocarriers. This study addresses the effects of buffer molarity and irradiation conditions on papain nanoparticles formation. Nanoparticles were synthesized on ice bath using ethanol (20%, v/v) as a cosolvent and crosslinked by gamma radiation using a 60Co source. Experimental variables included the synthesis in deionized water and in 1, 10, 25 and 50mM phosphate buffer, under different temperatures of −20 °C, 0 °C and 20 °C before and throughout the irradiation period, and using radiation dose rates of 0.8, 2, 5 and 10 kGy h−1 to reach the dose of 10 kGy. Proteolytic activity was quantified using Nα- benzoyl-DL-arginine-p-nitroanilide hydrochloride. Nanoparticle size and crosslinking by means of bityrosine were evaluated by dynamic light scattering and fluorescence measurements, respectively. Buffer molarity and radiation dose rate were identified to influence bityrosine formation and proteolytic activity without impacting nanoparticle size. Variations in temperature impacted bityrosine formation exclusively. Optimized conditions for papain nanoparticle synthesis were achieved using 50mM phosphate buffer at the dose rate of 5 kGy h−1 and temperature of 0 °C throughout the process.Resumo IPEN-doc 25211 In vitro permeability assay using an epithelial model of Caco-2/HT29-MTX/Raji-B cells2018 - LOPES, P.S.; CORAZZA, F.G.; NAMBU, F.A.N.; ERNESTO, J.V.; VARCA, G.H.C.; LEITE-SILVA, V.R.; ANDREO-FILHO, N.The oral route is one of the main routes for administration of drugs, however, the gastrointestinal tract is a hostile environment due to pH variation, presence of several digestive enzymes and the intestinal barrier that undermines the permeation of drugs. The aim of this study was to evaluate the ability of papain complexed with β-cyclodextrin to enhancer the permeation of furosemide, as a model drug, in a triple co-culture of Caco-2, HT29-MTX and Raji cells. Papain and the papain-β-cyclodextrin complex were evaluated at 0.3, 0.7 and 1.0 μM and the biophysical integrity of the cell layer was evaluated by RET (Transepitelial Electrical Resistance) at 0, 4, 24, 48 and 72 hours. The epithelium was also stained using DAPI and Alexa Fluor™ 488 Phalloidin. HPLC was employed to quantification of furosemide. The RET results at initial time for all the samples and control were in a range of 267.63 to 318.28 Ω*cm2 and after 72 h this values were raised to a range of 365.14 to 492.64 Ω*cm2. There was a decrease of RET after samples’ application, nevertheless, the results showed that the epithelium presents a recovery, proportional to the time of cell replication, and that this recovery occurs in all samples tested with no significant statistical difference. The RET recovery implies that papain, complexed or not, was not able to kill the cells, corroborating the hypothesis that the action mechanism is the disruption of the tight junction. In addition, the triple co-culture presents a higher resistance to papain action, in comparison with the Caco-2 monolayer assays, emphasizing the importance of testing new drugs, potential candidates for oral formulations, in epitheliums that faithfully mimics what actually happens in in vivo systems. The fluorescent microscopy observation of the cells stained with DAPI and the junctions stained with Alexa Fluor™ 488 Phalloidin, showed that co-culture exhibits microvilli inherent to the intestinal tissue. The results obtained in the triple co-culture model bi-directional transport experiments confirmed the significant increase in furosemide transport indicating the importance of the paracellular route. In conclusion, the triple co-culture model was successfully standardized and papain complexed with β-cyclodextrin acts probably over the tight junctions enhancing the permeation of furosemide.