MURILO ALISON VIGILATO

MURILO ALISON VIGILATO

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Ultrathin collagen and gelatin fibers
2023 - RODRIGUES, MURILO A.V.; MARTINS, VIRGINIA da C.A.; LUGAO, ADEMAR B.; MATTOSO, LUIZ H.C.; PLEPIS, ANA M. de G.
Introduction and objective: The main constituent of extracellular matrix (ECM) of various tissues is collagen, a biodegradable and biocompatible protein with excellent regenerative properties. A promising way to produce scaffolds, artificial ECM, involves the production of nanometric or submicrometric fibers, dimension of the natural fibers found in the ECM of many tissues. Solution blow spinning (SB-Spinning) allows the production of fibers with high feed rates and in situ deposition. Here we produce ultrathin collagen and gelatin (polymer obtained from collagen denaturation) fibers by SB-Spinning using solvents that preserve the integrity of the polymers and evaluate the morphology and properties of these fibers [1,2]. Methodology: Collagen (10 wt%) and gelatin (10 and 15 wt%) solutions were prepared in 90 wt% acetic acid under stirring overnight at room temperature. A glass syringe was used to spin 25 cm (for gelatin) or 20 cm (for collagen) away from the collector using 30 psi (gelatin) or 10 psi (collagen) of pressure at 3.6 to 10.8 mL/h for gelatin and at 3 to 6 mL/h for collagen. The fibers were characterized by scanning electron microscopy, differential scanning calorimetry and polyacrylamide gel electrophoresis. Results and discussion: Gelatin and collagen submicrometric fibers were produced from 90% acetic acid solutions, a benign solvent that allows the solubilization of high amounts of these polymers and present low toxicity with low cost, what reveals it as a promising benign solvent for this application. Gelatin fibers presented average diameters between 740 ± 299 nm and 909 ± 326 nm for 15% solutions and between 175 ± 64 nm and 196 ± 113 nm for 10% solutions, indicating the direct effect of polymer concentration on the diameter of fibers. Collagen fibers presented average diameters between 542 ± 185 nm and 543 ± 242 nm, being thicker than the gelatin fibers obtained with the same polymer concentration, an indicative of the preservation of the natural structure of these protein, the triple helix secondary structure. The preservation of collagen triple helix after the spinning process was confirmed by differential scanning calorimetry and gel electrophoresis results. Conclusions: Aqueous acetic acid was considered a good solvent for the solubilization of these collagenous proteins, being adequate to the production of biomaterials due to its low toxicity and ability to preserve the natural structure of collagen. It was also possible to understand the effect of some production parameters on the diameter of these fibers, an important step in the development of new biomimetic biomaterials produced by solution blow spinning.
Production of polycaprolactone/curcumin membranes and ITS effectiveness in cells growth, pH sensitivity and release of curcumin
2023 - SANTOS, LARISSA S. dos; RODRIGUES, MURILO A.V.; FREITAS, LUCAS F. de; FERREIRA, ARYEL H.; LUGAO, ADEMAR B.
Polyviniyl alcohol mats crosslinked with citric acid for regenerative medicine applications
2023 - SASDELLI, GABRIEL C.A.; RODRIGUES, MURILO A.V.; LUGAO, ADEMAR B.
Introduction: Currently, biomaterials have stood out in the medical field, seeking innovative solutions for wound healing. Papain, an enzyme with remarkable healing properties, shows promise in this context. However, its activity is compromised when in contact with water and at high temperatures. To overcome this challenge, this project has considered the use of polyvinyl alcohol (PVA) membranes, a low-toxicity, costeffective and hydrophilic polymer, offering an effective and safe approach to promote wound healing. However, the solubility of this polymer restrains its use, thus the aim of this project is to crosslink PVA fibers directly during solution blow spinning procedure. Methodology: Mats were produced using a Solution Blow Spinning (SBS) device with a rotary collector. The SBS device was 40 cm away from the collector and the solution was supplied with a pump at 3mL/h through a syringe using air at 6 psi of pressure. Mats were prepared using a 14 wt% PVA (65% high molecular weight/35% low molecular weight) solution in water and compared with solutions containing different concentrations of citric acid. The mechanical and rheological characterizations were done by tensile strength and viscosity tests, while infrared spectroscopy and scanning electron microscopy were employed to analyze fibers structure and morphology. Results and discussion: The mats produced in this study were consistently white, irrespective of citric acid presence. With increasing citric acid concentration, the mats showed improved resistance, making them more durable for various applications. The addition of citric acid also reduced mats water solubility, enhancing their long-term stability in different environments, notably biological fluids as exudate. Infrared spectroscopy analysis confirmed the crosslinking, while scanning electron microscopy revealed mats were composed by ultrafine fibers. These improved properties obtained by crosslinking make the mats highly suitable for practical applications of wound dressing, mainly by the use of safe crosslinking agent. The PVA solutions presented a higher viscosity with increasing citric acid content, presenting viscosity values of 0.9 ± 0.03 Pa s and 1.19 ± 0.07 Pa s for 0 wt% and 10 wt% of citric acid (in reference to the PVA mass) at 21,5 s-1. These findings hold significant promise for expanding crosslinked PVA mats usage in various types of wounds. Conclusions: Based on our results, it is possible to assert that with the addition of citric acid, which promoted polymer crosslinking, we obtained PVA dressings with ideal properties for wound use. Since the dressings are in perfect condition to withstand wound moisture, we can now consider the inclusion of papain in the mats, that can be stored dry and hydrated just in the moment of use, an attempt to preserve the hydrolysis of these enzyme.
Antitumoral efficacy of gold nanoparticles with polyphenols in breast cancer and metastatic cells in vitro
2023 - SPADREZANO, ISADORA; RODRIGUES, MURILO A.V.; VIEIRA, DANIEL P.; LUGAO, ADEMAR B.
Introduction and objective: Gold nanoparticles (AuNPs) present beneficial properties in cancer diagnosis and therapy, as they can be coated and biofunctionalized with bioactive molecules through surface modification using relatively non‐toxic reagents, enabling the reduction of metal ions, stabilization, and selective detection of cellular receptors [1]. The reduction of gold by phytochemicals to form nanoparticles represents promising methods in Green Nanotechnology. This study aims to compare the binding capacity and evaluate the antiproliferative potential of gold nanoparticles coated with tannic acid (TA‐AuNPs) against human breast tumor cell lines. Cytotoxicity was determined using the MTS method. Methodology: The synthesis of TA‐AuNPs was established through the chemical reduction, and the purification method was based on centrifugation, where the centrifuged sample (P1) was resuspended after removing and storing the supernatant (S1), thus generating three samples, including the non‐centrifuged (NC). The confirmation of TA‐AuNPs formation was achieved by UV‐Visible absorption spectroscopy, and the size determination of TA‐AuNPs was carried out using dynamic light scattering and Transmission Electron Microscopy (TEM) techniques. The Zeta potential was used to determine the stability of TA‐AuNPs. Human breast adenocarcinoma cell lines (MCF‐7 and MDA‐MB‐231) were used, and the cytotoxicity determination was performed using the MTS assay. Results and discussion: The synthesis of TA‐AuNPs showed a change in color, indicating the formation of spherical AuNPs. UV‐Visible analysis was performed for preliminary characterization, where the absorption band at a wavelength of 529 nm, correlated with the localized surface plasmon resonance band, indicates the presence of approximately 20 nm AuNPs in all samples. To evaluate the size of TA‐AuNPs, the samples were analyzed using the dynamic light scattering method. The hydrodynamic diameters measured indicate that centrifugation induces greater aggregation of the AuNPs. However, based on the polydispersity values ranging from 0.087 ‐ 0.186, the AuNPs showed uniform sizes. The Zeta potential was an effective indicator used as a criterion to classify and quantify stability, with the samples exhibiting magnitudes above 40 mV, indicating high stability. TEM images confirmed the presence of TA‐AuNPs in the samples and provided information about their dimensions, around approximately 20 nm with spherical morphology. The cytotoxicity assessment for validating TA‐AuNPs demonstrated that the coating plays a crucial role in the degree of internalization, and a higher quantity of specific receptors resulted in a greater internalization of TA‐ AuNPs. It was observed that there was a higher percentage of cell viability in MCF‐7 cells compared to MDA‐MB‐ 231 cells. Conclusions: The synthesis was efficient in obtaining TA‐AuNPs. The TA‐AuNPs showed a narrow size distribution, predominantly spherical morphology, and low PDI, indicating significant potential for biomedical applications in the healthcare field. The results satisfactorily and efficiently demonstrated that TA‐AuNPs are effective against breast cancer cells (MCF‐7) and exhibit higher efficiency in targeting metastatic breast cancer cells (MDA‐MB‐ 231), highlighting their significant potential for various medical applications in the field of nanomedicine.
Chitosan calcium phosphate
2022 - RODRIGUES, M.A.; MARTINS, V.C.; LUGAO, A.B.; PLEPIS, A.d.
Osseous reconstruction is a problem of interest for tissue engineering. One approach applied on this issue is the development of biomaterials used to induce osseointegration during osseous repair process, comprising materials from calcium phosphate bone cements to many different phosphorous-containing polymers. The incorporation of phosphorous-containing groups into natural or synthetic polymers as a way to induce the inorganic crystal nucleation and growth and to control its microstructure is an attempt to mimic the function of the organic phase of osseous tissues and promote bone tissue formation. In this study we analyze the effect of chitosan-calcium phosphate (CCP) salt on the mineralization of scaffolds of chitosan (CH). CH was extracted from squid pens and solubilized at 1% in H3PO4 9.4 mol L-1 and stirred for 1h (60ºC) to produce CCP. Then CCP was precipitated with CaCO3 until neutrality, washed with ethanol and dried at 50°C. The product was a yellowish solid soluble in water. A 1% CH solution was prepared by dissolution in 1% acetic acid. Chitosan/chitosan-calcium phosphate (CHCCP) gels were prepared by mixing 2.0 g of 1% CH solution to 0, 5 and 10 mg of CCP under stirring for 3 h, resulting respectively in CHCCP0, CHCCP5 and CHCCP10. Scaffolds were prepared by freeze-drying and the mineralization process was carried out by alternating soaking in 0.2 mol L-1 CaCl2 solution buffered with 0.05 mol L-1 Tris (pH 7.4) and 0.12 mol L-1 Na2HPO4 solution buffered with 0.05 mol L-1 Tris (pH 9.0) at 37°C for 30 minutes each, rinsing with water between the changes and repeating these cycle six times. CH and CHCCP5 before and after mineralization were analyzed by Fourier Transform Infrared spectroscopy. Scaffolds before and after mineralization were analyzed by thermogravimetric analysis and scanning electron microscope (SEM). The CH spectrum showed typical bands for this polymer, such as the O-H stretching band in 3400 cm-1 and the amide I (C=O stretching) at 1657 cm-1, bands also found in CHCCP5 spectra. A P-OH stretching band was observed at 571 and 561 cm-1 for CHCCP before and after mineralization, respectively, indicating the inclusion of phosphate groups in CH. Thermal behavior of scaffolds was characterized by three stages of mass loss; the first one (25-200°C) is associated with release of water and is higher with CCP, the second one (200–380°C) is due to degradation of CH structure and the third stage (380–650°C) is related to the carbonization of polymer. The residue values obtained at 750°C were used to determine the inorganic material contents produced in the mineralization, which were 28.4, 30.2 and 31.4% for CHCCP0, CHCCP5 and CHCCP10, respectively. These values indicate that the presence of CCP did not substantially increase the mineralization efficiency. SEM results showed fully mineralized surfaces with spherical aggregates, which presents a morphology of needle-like, a shape commonly observed in crystalline apatites.