GUSTAVO HENRIQUE COSTAVARCA
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Artigo IPEN-doc 27583 Semi-solid pharmaceutical formulations for the delivery of papain nanoparticles2020 - LIMA, CAROLINE S.A. de; VARCA, JUSTINE P.R.O.; NOGUEIRA, KAMILA M.; FAZOLIN, GABRIELA N.; FREITAS, LUCAS F. de; SOUZA, ELISEU W. de; LUGAO, ADEMAR B.; VARCA, GUSTAVO H.C.Papain is a therapeutic enzyme with restricted applications due to associated allergenic reactions. Papain nanoparticles have shown to be safe for biomedical use, although a method for proper drug loading and release remains to be developed. Thus, the objective of this work was to develop and assess the stability of papain nanoparticles in a prototype semi-solid formulation suitable for dermatological or topical administrations. Papain nanoparticles of 7.0 ± 0.1 nm were synthesized and loaded into carboxymethylcellulose- and poly(vinyl alcohol)-based gels. The formulations were then assayed for preliminary stability, enzyme activity, cytotoxicity studies, and characterized according to their microstructures and protein distribution. The formulations were suitable for papain nanoparticle loading and provided a stable environment for the nanoparticles. The enzyme distribution along the gel matrix was homogeneous for all the formulations, and the proteolytic activity was preserved after the gel preparation. Both gels presented a slow release of the papain nanoparticles for four days. Cell viability assays revealed no potential cytotoxicity, and the presence of the nanoparticles did not alter the microstructure of the gel. The developed systems presented a potential for biomedical applications, either as drug delivery systems for papain nanoparticles and/or its complexes.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 24828 The impact of physical-chemical parameters over the formation of papain nanoparticles crosslinked by radiation2017 - FAZOLIN, GABRIELA N.; VARCA, GUSTAVO H.C.; LUGAO, ADEMAR B.With the advancement of research and use of nanotechnology in biomedical areas and application in different treatments the need for stable systems with low toxicity and null adverse effects becomes important. Within this context papain appears as an alternative biomolecule which is a proteolytic enzyme extract by the fruit of Carica papaya Linnaeus. Its already successfully applied in the pharmaceutical and biomedical area as a drug carrier and debriding agent for wounds. Recently, papain was developed in nanoscale using 50 Mm phosphate buffer (pH 7,2 ± 0,2), ethanol (20%, v/v) as cosolvent and 60Co as radiation source for gamma radiation (10 kGy) to promote protein crosslinking. This technique promotes papain nanoparticle sterilization and crosslinking in a single step showing that radiation is a promising technique for the development of nanoparticles for biomedical use. However, important parameters such as the influence of physical-chemical parameters over nanoparticle formation remains unexplored and unclear. Therefore, this work aimed to evaluate the influence of pH and temperature over papain nanoparticle formation including buffer molarity and different temperatures throughout the synthesis and irradiation process. Nanopapain was synthetized as above-mentioned however phosphate buffer was used in a range of 1 to 50 mM and ultrapure water in presence of ethanol and irradiated at 10 kGy. The influence of temperature was evaluated by performing the synthesis at -20, 0 and 20 °C. Additionally, it was verified the sterility of the material and its stability over time. The hydrodynamic radius and crosslinking were evaluated by dynamic light scattering and fluorescence measurements respectively. The pH range was influenced by buffer molarity, thus, lower molarities led to a final pH of 5 and the higher molarities around 7. Bityrosine formation was influenced by the pH range and increased when synthesis was performed near optimal pH (7 for papain), although particle size didn’t undergo any changes as a result of the pH changes. On the other hand, enzymatic activity was less affected by the process maintain about 80% of the native enzymatic activity only when synthesis was carried out in 50 mM phosphate buffer. With regard to temperature as the temperature increased the levels of crosslinking also increased in the order of 20>0ºC>-20ºC. In conclusion nanoparticle size was not influenced by the temperature shift. Concerning enzymatic activity samples irradiated under ice-cold conditions presented decreased bioactivity loss if compared to the samples irradiated at room temperature. After an evaluation of the results, the study revealed that samples synthetized with buffer molarity of 50 Mm (pH 7,2 ± 0,2) and 0ºC would promote higher crosslinking levels and less enzymatic activity decrease.