GUSTAVO HENRIQUE COSTAVARCA

GUSTAVO HENRIQUE COSTAVARCA

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Mucoadhesive drug delivery system with enhanced permeability capacity for intravesical therapy
2023 - LIMA, CAROLINE S.A. de; RIAL-HERMIDA, M.I.; FERREIRA, ARYEL H.; FREITAS, LUCAS F. de; VARCA, GUSTAVA H.C.; ALVAREZ-LORENZO, CARMEN; LUGAO, ADEMAR B.
Introduction and objective: Bladder cancer (BC) represents 3% of the new diagnosis of cancer. 70% of the patients present the non-muscle-invasive type of the disease [1]. This type of BC is usually treated by the transurethral resection of the visible tumor, followed by intravesical inmuno- or chemotherapy. The instillation of chemotherapy into the bladder is not totally efficient as it faces limitations such as short residence time and permeability restrictions of the bladder mucosa [2]. Thus, the main objective of this work was to develop drug delivery formulations for intravesical chemotherapy that enhance its residence time and permeation capacity. Methodology: Carboxymethylcellulose (CMC) and polyvinyl alcohol (PVA) polymers were used to prepare a physical hydrogel with mucoadhesive properties. Papain, a thiol proteolytic enzyme, was added to the formulations as a permeation enhancer. The mucoadhesive capacity of the formulation was evaluated by its interaction with mucin, bioadhesion strength and retention on bladder urothelium. Ex-vivo drug permeation was also elucidated using Franz cells to observe the papain effect. Results and discussion: The CMC + PVA formulations showed positive bioadhesion components, confirming their interaction with the urothelium. The significant decrease of ɳb in the formulation containing enzyme suggests that papain was cleaving the glycoproteins present in the mucin. On bioadhesion strength assays, the prepared formulations required a slightly higher detachment force than the control. In terms of work of adhesion, all formulations had values higher than those recorded for the control (p < 0.05), confirming that there were mucoadhesive interactions between the bladder tissue and the gels. Hydrogel retention assay revealed that the formulations had higher bioadhesion capacity than the control (FITC solution), especially after the third wash (15 mL). By drug permeation analysis, it was possible to observe that the lag time decreased for CMC + PVA hydrogels with and without papain in comparison to the control. At the timepoint of 2 h, the formulation containing papain presented the highest capacity of permeation. Conclusions: The biocompatible hydrogel containing papain as a permeability enhancer was an innovative approach for BC treatment to address the poor permeation of the tissue and short residence time. The formulation developed in this work could represent a very notable improvement in clinical practice, ensuring a longer retention time of the formulation in the bladder tissue, with a more sustained release of the drug and greater permeation capacity.
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.
Solubility study of Kraft lignin for the development of electrospun nanofibers
2022 - NOGUEIRA, K.M.; VARCA, J.O.; LIMA, C.S.; CRUZ, C.C. da; RIBEIRO, A.H.; FREITAS, L.F.; VARCA, G.H.; LUGAO, A.B.
Lignin is a high-volume byproduct of paper manufacturing which has been explored in many research fields, especially for the development of fiber and nanofiber for biomedical applications [1,2]. This work presents a solubility study performed through gravimetry for kraft lignin considering its application for the development of electrospun nanofibers [3]. In practical terms, lignin was solubilized in alkaline aqueous solution, dimethylformamide and dimethylsulfoxide, at concentrations of 10, 15 and 20% (w/v) and varying temperatures of 25, 50 and 75 ºC, under constant stirring. After solubilizing, the solution was filtered, and the insoluble fraction was dried in the oven at 100 ºC. At 25 ºC lignin was insoluble in all solvents tested, as predicted using Hansen solubility parameters. Although the increase in temperature promoted lignin solubilization in all solvents tested, at the highest temperature assayed, the solubilization was facilitated, presenting the smallest levels of the insoluble fraction. Lignin was soluble in all solvents tested, and optimum solubility conditions were achieved using 10% lignin solutions (w/v), without significant insoluble fraction, and therefore ideal concentration for the development of lignin based fibers.
A biological study of gelatin-PVA based scaffold functionalized with albumin for biomedical purposes
2022 - VARCA, J.O.; KLINGBEIL, F.; NOGUEIRA, K.M.; LIMA, C.S.; CRUZ, C.C. da; FREITAS, L.F.; VARCA, G.H.; MATHOR, M.B.; LUGAO, A.B.
Biomaterials have been designed for tissue reconstruction, bone regeneration and cell culture, and functionalized with presence of proteins, nanoparticles, peptides and other components to improve the biocompatibility for instance. This work shows a biological study of gelatin-PVA based scaffold with controlled pore size and functionalized with albumin for biomedical purposes. The in vitro study comprises cytotoxicity, cell adhesion and proliferation assessment. In practical terms, the gelatin-PVA scaffold crosslinked and sterilized by gamma radiation followed by freeze-drying was evaluated by cytotoxicity, adhesion and proliferation tests. The cytotoxicity results showed that the biomaterial produced was non-toxic, and adhesion and proliferation assays showed that the material was suitable for tissue engineering. The presence of albumin did not present a significant impact on the cell performance, at the assayed concentration.
Géis mucoadesivos para o tratamento do câncer de bexiga superficial
2022 - LIMA, C.S.; HERMIDA, M.R.; VARCA, G.C.; FREITAS, L.F.; LORENZO, C.A.; LUGAO, A.B.
A mucoadesividade tem sido estudada na área farmacêutica há mais de 50 anos e é uma propriedade explorada para aumentar o tempo de residência de um fármaco, bem como para melhorar a especificidade da entrega do ativo ao local desejado. Um sistema mucoadesivo que permite a absorção direta do fármaco e uma diminuição da taxa de excreção, consequentemente, tem-se uma maior biodisponibilidade do ativo associada à administração de doses menores, com menor frequência (Yan et al., 2017). O câncer de bexiga (CB) é uma das principais doenças que atacam o trato urinário e se dá pela proliferação anormal das células do tecido da parede interna da bexiga conhecida como urotélio (INCA, 2021). Sabe-se que o tratamento por quimioterapia intravesical do CB apresenta algumas limitações como a permeação do ativo no urotélio e o tempo de residência desse fármaco que é muito limitado devido ao efeito de diluição e lavagem da urina que acaba eliminando-o completamente. Dessa forma, o desenvolvimento de novos veículos mucoadesivos para o carreamento da quimioterapia pode ser uma opção de tratamento avançado (Kolawole et al., 2017). Neste trabalho, buscou-se preparar géis mucoadesivos a partir da goma gelana (0,1% m/v) e de uma blenda de carboximetilcelulose (2% m/v) e polivinil álcool (0,2% m/v), contendo um adjuvante para aumento de permeação (papaína), como potenciais alternativas para a quimioterapia intravesical. Foram realizados ensaios de reologia (frequency sweep para avaliação dos módulos de perda (G’’) e de armazenamento (G’)); força de adesão para estudo da capacidade de mucoadesão das formulações e, por fim, de citotoxidade em duas linhagens celulares, HUVEC (endotelial humana) e V79-4 (fibroblastos) . Os ensaios reológicos dos géis apresentam resultados característicos de comportamentos pseudoplásticos, isto é, com maior fluidez a altas frequências, e, portanto, com potencial para aplicação por seringa e cateter. Os ensaios de mucoadesão confirmaram que os polímeros escolhidos apresentam capacidade de interação com a mucina presente no urotélio, representando alternativas interessantes para o aumento do tempo de residência e da biodisponibilidade da terapia. Por fim, as formulações apresentaram pouca ou nenhuma toxicidade, mostrando potencial para aplicação biomédica.
The state of the art of theranostic nanomaterials for lung, breast, and prostate cancers
2021 - FREITAS, LUCAS F.; FERREIRA, ARYEL H.; THIPE, VELAPHI C.; VARCA, GUSTAVO H.C.; LIMA, CAROLINE S.A.; BATISTA, JORGE G.S.; RIELLO, FABIANE N.; NOGUEIRA, KAMILA; CRUZ, CASSIA P.C.; MENDES, GIOVANNA O.A.; RODRIGUES, ADRIANA S.; SOUSA, THAYNA S.; ALVES, VICTORIA M.; LUGAO, ADEMAR B.
The synthesis and engineering of nanomaterials offer more robust systems for the treatment of cancer, with technologies that combine therapy with imaging diagnostic tools in the so‐called nanotheranostics. Among the most studied systems, there are quantum dots, liposomes, polymeric nanoparticles, inorganic nanoparticles, magnetic nanoparticles, dendrimers, and gold nanoparticles. Most of the advantages of nanomaterials over the classic anticancer therapies come from their optimal size, which prevents the elimination by the kidneys and enhances their permeation in the tumor due to the abnormal blood vessels present in cancer tissues. Furthermore, the drug delivery and the contrast efficiency for imaging are enhanced, especially due to the increased surface area and the selective accumulation in the desired tissues. This property leads to the reduced drug dose necessary to exert the desired effect and for a longer action within the tumor. Finally, they are made so that there is no degradation into toxic byproducts and have a lower immune response triggering. In this article, we intend to review and discuss the state‐of‐the‐art regarding the use of nanomaterials as therapeutic and diagnostic tools for lung, breast, and prostate cancer, as they are among the most prevalent worldwide.
Hybrid gold-protein nanoparticles as radiosensitizers
2020 - FREITAS, L.F.; CRUZ, C.C. da; BATISTA, J.G.; VARCA, G.H.; LUGAO, A.B.; MATHOR, M.B.
Gold nanoparticles present unique optical properties which are dependent upon size and morphology, and consist on a differential interaction with radiation compared to the bulk material. Those nanoparticles can be modified in order to adjust their bioavailability and tissue-targeting, and one of the means to do so is by adsorbing one or more types of proteins onto their surface. Gamma radiation can be helpful in this regard, since it promotes intra- and intermolecular crosslinks in proteins and enables their adsorption onto the metallic nanoparticles’ surfaces. Here we present the results obtained for hybrid gold-protein nanoparticles as radiosensitizers. The nanoparticles were synthesized radiolytically by mixing 5 mmol L-1 NaAuCl4 with 1 mg mL-1 bovine serum albumin (BSA) or papain in the presence of 0.1 mol L-1 tert-butanol and 20% ethanol. The solutions were irradiated with 10 kGy in a multipurpose gamma irradiator (60Co source, 5 kGy per hour) for the radiolytic synthesis of the nanoparticles, and then the resulting red suspension was stored until use. 10^4 cells (MDA-MB-231 line) were seeded in 96-well plates and incubated with a 2:1 mixture of DMEM medium and nanoparticles suspension for 12 hours. Then, the wells were washed with sterile phosphate buffered saline, and fresh DMEM medium was added prior to irradiation in a gamma cell (60Co source, 0.6 kGy per hour) with 10, 30 and 50 Gy. 48 hours later, the cell viability was assessed by MTS assay. The results indicate that the radiation alone slightly stimulated the proliferation of the tumor cells, but this effect was more evident in the presence of gold-papain nanoparticles. The ablative effect due to radiosensitization was observed with 30 and 50 Gy for the cells incubated with gold-BSA nanoparticles, and 10 and 30 Gy for the cells incubated with gold-papain nanoparticles. This difference might be due to a more effective internalization or surface-attachment of nanoparticles when they are coated with papain, and one evidence for this assumption is the fact that the cell culture becomes red after the incubation with gold-papain nanoparticles. Therefore, protein-coated nanoparticles might be effective as radiosensitizers, depending on the coating and dose of radiation.
Synthesis and purification of albumin-based nanoparticles crosslinked by radiation
2020 - RIELLO, F.N.; VARCA, G.H.; LIMA, C.S.; FREITAS, L.F.; FERREIRA, A.H.; LUGAO, A.B.
Protein-based nanoparticles have been proved a promissing alternative for the loading and delivery of chemotherapeutic agents, radiopharmaceutics and other drugs of interests, constituting a less toxic therapeutic option due to its biocompatibility and low or null side effects. The use of radiation to crosslink or form covalent bonds enables the controll of the crosslinking process, without the need for crosslinking agents, as well as provides sterilizations simultaneously, withouth generating toxic compounds or products. The present work targets the synthesis an purification of albumin-based nanocarrier crosslinked by gamma radiation for biomedical applications. For such purpose, albumin nanoparticles were synthesized using BSA at 20% ethanol (v/v) in 50 mM phosphate buffer on an ice bath prior to and after irradiation. Samples were exposed to gamma radiation at a minimun absrobed dose of 10 kGy at 5kGy.h-1 and purified using a SuperdexTM 200 Increase 10/300GL for isolating the crosslinked protein (high molecular weight) from the native BSA. After the purification, the fractions were characterized by electrophoresis, Uv, fluorescence and dynamic light scaterring. The nanoparticles were obtained in the range of 25-40 nm and purified into fractions of high molecular weight and the native ones. The high molecular weight fractions presented increased bityrosine levels if compared to the fraction corresponded to the native BSA. The yields of nanoparticle formation remains to be determined, but our results provided a clear evidence of the formation of radiation-crosslinked BSA nanoparticles and the role of bityrosine in the nanoparticle assembly.
Development of Lignin/PEO nanofibers by electrospinning technique for tissue engineering application
2020 - NOGUEIRA, K.M.; VARCA, J.O.; LIMA, C.S.; CRUZ, C.C. da; RIBEIRO, A.H.; FREITAS, L.F.; VARCA, G.H.; LUGAO, A.B.
Lignin is a renewable carbon source and has been widely explored in different areas over the last years, especially in biomaterials such as dressings and other biomedical devices due its natural origin and low cost. Its chemical structure confers interesting properties such as antioxidant capacity, UV protection, bactericidal action and appropriate adsorption. Poly (ethylene oxide) (PEO) is used in electrospinning to facilitate the formation polymer fibers. The electrospinning technique has been largely explored in the bioengineering area towards designing nanomaterial with minimum defect and high surface area. The present work aimed the development of a lignin/PEO nanofiber by electrospinning technique. In practical terms, lignin/PEO solution was prepared following two different methods. In the first approach, polymer stock solutions were prepared in alkaline water by stirring at 70 °C. In the second, the polymer powders were mixed and dissolved together in dimethylformamide (DMF) under stirring at 80 °C. By both methods, PEO/lignin solutions were prepared at 10, 20 e 30% (w,v) solid content, at the ratios 99/1 and 95/5. For electrospinning parameters, the distance between ejector and plate collector was set to 15-20 cm, voltage to 20 kV and injection flow to 1 mL/h, chamber temperature to 40 °C and 30%. Nanofiber morphology was assessed by scanning electron microscopy and optical coherence tomography. Apparent porosity was measured by classical Archimedes method. Due to higher DMF dielectric constant compared to water, results showed that nanofibers made using DMF presented smaller beats formation and smaller fiber diameter. Nanofibers with higher solid content presented more uniform fibers with larger diameter. Nanofibers with higher lignin concentration presented larger number of beats and higher fiber diameter. However, lignin improved the system porosity in all cases. Further mechanical and biological experiments will be done, nevertheless, the nanofiber developed is a promising material to be applied in tissue engineering.
CMC and PVA hydrogel containing papain nanoparticles for drug delivery
2020 - LIMA, C.S.; VARCA, G.H.; OLIVEIRA, J.R.; NOGUEIRA, K.M.; SANTOS, F.A.; RIBEIRO, A.H.; LUGAO, A.B.; FREITAS, L.F.; ROGERO, S.O.
Four hydrogel formulations of Carboxymethylcellulose (CMC) and Poly (vinyl alcohol) (PVA) were prepared with native papain (AP and BP) and papain nanoparticles (AN and BN) for drug delivery. The formulations were evaluated for their preliminary stability, protein distribution in the matrix and cytotoxicity. Three methods for sterilization purposes were compared: irradiation by 60Co source, electron-beam and UV light. The preliminary stability test confirmed that the system was stable since there was no precipitation or alteration of the organoleptic properties of the samples in the evaluated period. The distribution of proteins in the hydrogel was very homogeneous in all the formulations. Quantification of the enzymatic activity of papain after contact with the gel showed that native papain maintained its activity high (86% and 93% for AP and BP gels, respectively), whereas there was a considerable drop in the activity of the papain nanoparticles to 60.54% and 69.44% for AP and BP gels, respectively. Such loss of activity is attributed to processing and/or process steps. The cell viability assay showed that the polymer matrix shows no cytotoxicity, corroborating with the literature, since the material is biocompatible. Thus, it is possible to affirm that the developed system presents potential for biomedical application, either as a vehicle of papain itself or for the transport of other drugs through complexation with papain nanoparticles. However, the need for further studies of stability, controlled release capacity and biocompatibility is required.