GUSTAVO HENRIQUE COSTAVARCA

GUSTAVO HENRIQUE COSTAVARCA

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Mucoadhesive gellan gum-based and carboxymethyl cellulose -based hydrogels containing gemcitabine and papain for bladder cancer treatment
2023 - LIMA, CAROLINE S.A. de; RIAL-HERMIDA, MARIA I.; FREITAS, LUCAS F. de; PEREIRA-DA-MOTA, ANA F.; VIVERO-LOPEZ, MARIA; FERREIRA, ARYEL H.; KADŁUBOWSKI, SŁAWOMIR; VARCA, GUSTAVO H.C.; LUGAO, ADEMAR B.; ALVAREZ-LORENZO, CARMEN
Local treatment of bladder cancer faces several limitations such as short residence time or low permeation through urothelium tissue. The aim of this work was to develop patient-friendly mucoadhesive gel formulations combining gemcitabine and the enzyme papain for improved intravesical chemotherapy delivery. Hydrogels based on two different polysaccharides, gellan gum and sodium carboxymethylcellulose (CMC), were prepared with either native papain or papain nanoparticles (nanopapain) to explore for the first time their use as permeability enhancers through bladder tissue. Gel formulations were characterized regarding enzyme stability, rheological behavior, retention on bladder tissue and bioadhesion, drug release properties, permeation capacity, and biocompatibility. After 90 days of storage, the enzyme loaded in the CMC gels retained up to 83.5 ± 4.9 % of its activity in the absence of the drug, and up to 78.1 ± 5.3 with gemcitabine. The gels were mucoadhesive and the enzyme papain showed mucolytic action, which resulted in resistance against washing off from the urothelium and enhanced permeability of gemcitabine in the ex vivo tissue diffusion tests. Native papain shortened lag-time tissue penetration to 0.6 h and enhanced 2-fold drug permeability All formulations demonstrated pseudoplastic behavior and no irritability. Overall, the developed formulations have potential as an upgraded alternative to intravesical therapy for bladder cancer treatment.
Semi-solid pharmaceutical formulations for the delivery of papain nanoparticles
2020 - 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.
Enhancing the furosemide permeability by papain minitablets through a triple co-culture in vitro intestinal cell model
2020 - CORAZZA, FULVIO G.; ERNESTO, JULIA V.; NAMBU, FELIPE A.N.; CALIXTO, LEANDRO A.; VARCA, GUSTAVO H.C.; VIEIRA, DANIEL P.; LEITE-SILVA, VANIA R.; ANDREO-FILHO, NEWTON; LOPES, PATRICIA S.
The administration of medicines by the oral route is the most used approach for being very convenient. Although it is the most popular, this route also has absorption, and consequently, bioavailability limitations. In this sense, several pharmacotechnical strategies have been used to improve drug absorption, one of which is the use of permeation promoters. Papain is a very versatile plant enzyme that can be used as a permeation promoter of various active compounds. This study aimed to evaluate the safety of papain and the formulation of native papain minitablets to promote in vitro permeation of furosemide through an innovative biomimetic triple co-culture model of Caco-2, HT29-MTX, and Raji cells. Regarding permeation, furosemide and metaprolol concentrations are determined with HPLC; those are used to calculate Papp. Monolayer integrity was evaluated using TEER and Lucifer Yellow. In the presence of papain, TEER decreased two-fold and the Papp of furosemide increased six-fold. The results suggest that native papain minitablets can be used as therapeutic adjuvants to enhance the permeation of drugs significantly improving bioavailability.
Papain-cyclodextrin complexes as an intestinal permeation enhancer
2020 - 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.
Simultaneous intramolecular crosslinking and sterilization of papain nanoparticles by gamma radiation
2020 - 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.
The effects of radiation and experimental conditions over papain nanoparticle formation
2020 - 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.
The effect of radiation dose rate over the formation of protein-based nanoparticles for nanosized delivery of chemo and radiotherapeutics
2018 - VARCA, G.H.C.; FAZOLIN, G.N.; FERREIRA, A.H.; OLIVEIRA, J.P.R. de; MARQUES, F.; LUGAO, A.B.
Recent studies demonstrated the development of papain and bovine serum albumin nanoparticles using gamma radiation (10 kGy) in presence of 20-30% (v/v) ethanol. With the purpose of producing stable and well defined nanocarriers, this work aims to determine the influence of different dose rates over protein nanoparticle formation. For this purpose, papain and BSA nanoparticles were synthetized in phosphate buffer (50 mM, pH 7.2) and ethanol (20-30%, v/v) using a radiation dose of 10 kGy and dose rate of 0.8, 2, 5 and 10 kGy.h-1. After irradiation, samples were evaluated by dynamic light scattering, fluorescence and proteolytic activity to verify the size, secondary structure and monitoring of the enzymatic activity, respectively. For papain nanoparticles it was observed that the dose rate did not influence the particle size formation, however crosslinking evidenced by bityrosine showed that samples irradiated at 0.8 and 5 kGy.h-1 presented higher bityrosine levels. On the other hand, BSA nanoparticles presented different results if compared to papain NPs. Different dose rates caused different and non-linear size increase for each condition, following the order: 5 > 10 > 0.8 > 2 kGy.h-1. However, in terms of crosslinking formation, a linear increase was registered, as at 0.8 kGy.h-1 the smallest signal was achieved, whereas at 10 kGy.h- 1 the highest signal was recorded. In conclusion, BSA nanoparticles were more sensitive to different radiation dose rates than nanopapain. Optimized results in terms of size increase and higher bityrosine levels were observed for the samples irradiated at 5 kGy.h-1, in which nanoparticle formation will occur faster if compared to the synthesis carried out under distinct conditions. As final applications of the system concert their use for the delivery of chemo or radiotherapeutics, the loading of paclitaxel, a well-known chemotherapeutic agent, and radiolabeling with tecntetium- 99m, a radioisotope suitable for biomedical applications, have also been performed with high efficiency, thus demonstrating a proof of concept of such systems.
Desenvolvimento de nanocarreadores bioativos na forma sólida à base de papaína induzidas por radiação
2018 - FAZOLIN, G.N.; VARCA, G.H.C.; LUGAO, A.B.
A papaína, enzima proteolítica extraída do látex do fruto verde do mamão papaia adulto, é um produto de interesse médico por possuir características importantes como auxiliar na redução do crescimento bacteriano em escaras além de possuir alta bioatividade e ser um produto com potencial para carreamento de fármacos. Recentemente desenvolvida em nano escala, a nanopartícula de papaína foi sintetizada utilizando a técnica de síntese por radiação a fim de promover reticulação intermolecular e esterilização do meio, simultaneamente. Contudo, a papaína em meio aquoso sofre hidrolise e por este motivo sua aplicação como nanocarreador pode ser dificultada. Visando a utilização da nanopartícula de papaína como nanocarreador de fármacos, se faz necessário a obtenção da nanopartícula na forma sólida. Este trabalho tem o objetivo de estudar o comportamento das nanopartículas de papaína congeladas e liofilizadas além de sua estabilidade frente ao tempo. A síntese foi realizada utilizando tampão fosfato 50 Mm pH 7,4 e etanol como cosolvente, além de induzir a reticulação com radiação ionizante utilizando a dose de 10 kGy e taxa de dose de 5 kGy.h-1. Após a síntese e irradiação, as amostras foram congeladas a - 80ºC. O tamanho e a estrutura secundária foram avaliados através das técnicas de espalhamento dinâmico de luz e fluorescência, a atividade enzimática foi analisada utilizando substrato específico. A amostra de papaína nativa possui tamanho médio de 4 nm e a nanopapaína ± 7 nm, contudo, após a liofilização, observou-se que os tamanhos não são mantidos, apresentando ± 4 nm para ambas as amostras. Os rendimentos das amostras foram superiores a 90% para todas as condições e considerados satisfatórios. Utilizando açúcares como trehalose e beta-ciclodextrina (2% v/v), este rendimento subiu para 96-98%, porém, a bioatividade caiu cerca de 30% após liofilização e 40% nas amostras liofilizadas com açúcar, ou seja, o dobro do esperado. Sendo assim, concluiu-se que o uso do açúcar é satisfatório no aumento do rendimento porem é indispensável na concentração estudada.
Influência do cosolvente na formação de nanopartículas de papaína induzidas por radiação
2018 - FAZOLIN, G.N.; VARCA, G.H.C.; LUGAO, A.B.
Biomateriais à base de proteínas são de grande interesse biomédico e vem mostrando relevância nas linhas de pesquisas atuais por serem uma alternativa no carreamento de fármacos, além de apresentar efeitos colaterais reduzidos ou inexistentes. A papaína, enzima proteolítica utilizada neste trabalho, é uma alternativa no estudo de sistemas nano estruturados por se mostrar promissora no carreamento de fármacos. A técnica utilizada para síntese desta nanopartícula utiliza o etanol como cosolvente. Além de promover desolvatação na proteína, o etanol tem a função de proteger a enzima durante o processo de radiação – utilizado para reticulação e esterilização. Observou-se anteriormente que o etanol auxilia no aumento do tamanho da nanopartícula, contudo, não se sabe qual a influência deste cosolvente durante o processo. Sendo assim, o objetivo principal deste trabalho é estudar a influência do etanol nas nanopartículas de papaína. A síntese será realizada na presença (20%) e ausência do etanol, tampão fosfato 50 Mm pH 7,4 e radiação ionizante a 10 kGy durante 2 horas (5 kGy/hora). As amostras foram avaliadas através da técnica de espalhamento dinâmico de luz, fluorescência e bioatividade a fim de verificar o tamanho, a estrutura secundária e acompanhamento da atividade enzimática, respectivamente. Após a irradiação, as amostras foram filtradas, congeladas e liofilizadas por 48 horas. As amostras foram resuspendidas em duas condições: água deionizada e na presença de etanol. Observou-se quando resuspendidas em água, as amostras voltam ao tamanho nativo (± 4 nm) enquanto na presença de etanol este tamanho é mantido como no controle (± 7 nm). As análises de fluorescência demonstraram que a reticulação por radiação continuou existente e a bioatividade decaiu 30% em ambos os casos, quando comparada com a bioatividade inicial. Conclui-se que o etanol contribui para a formação do tamanho da nanopartícula durante a síntese porem quando evaporado perde-se a solvatação e mantem o aumento de tamanho somente promovido pela radiação.
In vitro permeability assay using an epithelial model of Caco-2/HT29-MTX/Raji-B cells
2018 - 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.