Título da Dissertação: Nanofibras constituídas de policaprolactona e copolímero F-108 contendo curcumina como nova terapêutica para cicatrização de feridas

Orientadora: Profa. Dra. Graciette Matioli

Data da Defesa:27/02/2019



INTRODUCTION. Wound healing is a complex and dynamic process that can be negatively influenced by a number of factors, and in addition, current treatment options are usually limited and inefficient. The use of polymeric nanofibers in the form of scaffolds produced by the electrospinning technique has been the focus of several studies as a new therapy for the treatment of excitable wounds. The polymer known as polycaprolactone, poly (ε-caprolactone) (PCL), has attracted attention as a controlled drug delivery system, as absorbable surgical sutures and as three-dimensional membranes used in tissue engineering. However, its use is limited due to its low hydrophilicity, which compromises protein adsorption and cell adhesion. PEO-PPO-PEO triblock copolymer type micelles, known commercially as Pluronics®, have various applications for drug solubilization and have recently been employed as materials to improve the hydrophilic character of scaffolds developed by electro-spinning. Pluronic® F-108 has shown promising results for the formation of hydrophilized polymer blends, mainly because it presents high hydrophilicity. The nanofibers portray a technological advance among the forms of treatment of cutaneous wounds and their association to drugs of natural origin represents the most current in the area of development of therapeutics for the cicatrization. Curcumin is a natural yellow-orange polyphenol found in the Curcuma longa (turmeric) plant and exhibits great healing potential because of its numerous pharmacological properties. However, curcumin has low bioavailability, which limits its application. In order to improve the properties of curcumin and to enable its application in wound healing, its association with nanofibers has been explored.
AIMS. The objective of this work was to produce and characterize nanofibers composed of the polymer blend PCL/F-108 alone or associated with curcumin and to evaluate in vivo its action on the healing process of excisional skin wounds.
MATERIAL AND METHODS. The nanofibers were developed by the electrospinning technique. In order to obtain the PCL/F-108 nanofiber (control) PCL and F-108 were used and for drug association two nanofibers (m/V) were prepared in concentrations of 0.004% (PCL/F-108C4) and 0.7% (PCL/F-108C7) of curcumin. The characterization was performed by scanning electron microscopy (SEM), X-ray diffractometry (XRD) and fluorescence microscopy. To evaluate the efficacy of the nanofibers in the healing process, in vivo, analyzes of photoacoustic permeation and histological study were performed. Eighty male rats, Wistar, approximately 90 days old and weighing between 250-380 g, were used. Forty-eight hours before the experimental procedure, the animals were anesthetized intraperitoneally with a solution of 2% xylazine (1 mL/kg) and 10% ketamine (1 mL/kg) to perform the manual excision area, on the animal's back. For the manufacture of the excisional wounds, the animals were anesthetized again and two parallel demarcations were made to each other. Next, with scissors, the excision of the demarcated skin was performed. To determine the contraction rate the wounds were decalked on transparent films to obtain their initial and final area. The right side wounds were covered with PCL/F-108 nanofiber and left side wounds were coated with nanofibers combined with curcumin (PCL/F-108C4 or PCL/F-108C7). The wounds were evaluated daily for their macroscopic appearance. After 4, 7, 10 and 14 day periods, the animals were euthanized with an overdose of Thiopental (120 mg/kg, intraperitoneal) associated with 10 mg/mL (7 mg/kg) Lidocaine. In the animals used for the histological study, 0.5 mg/kg vincristine sulfate
was injected intravenously (penile vein) 2 h prior to death. Next, the skin samples containing the wounds were removed and fixed in 4% paraformaldehyde solution for 24 h and processed for inclusion in paraffin. Semi-serial sections 5 μm thick and stained with hematoxylin and eosin (HE) were used to study epidermal proliferation, morphometric analyses of the length and height of the neoformed epithelium, and to evaluate revascularization. They were also stained with Sirius red, using the Picrosírius technique, to quantify the area occupied by type I and III collagen fibers. The permeation of the nanofibres in the skin was determined by the technique of photoacoustic spectroscopy.
RESULTS AND DISCUSSION. The characterization techniques showed good evidence of the formation of nanofibers with the incorporation of curcumin. SEM analysis showed that PCL/F-108 (control) fiber had a mean diameter of 1630 nm, while in the condition containing 0.004% curcumin (PCL/F-108C4), the diameter decreased to 800 nm and in condition 0.7% (PCL/F-108C7) to 613 nm, suggesting that the higher concentration of curcumin caused a decrease in fiber thickness. Thicker fibers may be beneficial to tissue repair treatment as they provide better adhesion, organization, and cell proliferation results in the material. By XRD analysis, relevant diffraction peaks showed the ability of PCL to prevent the crystallization of curcumin and to allow the preparation of a homogeneous material. Fluorescence microscopy images showed fluorescence of the curcumin distributed throughout the fiber.
In the technique of photoacoustic spectroscopy, the Gaussian deconvolution allowed to observe the presence and performance of the nanofibers in all evaluated periods of healing. By the macroscopic observation it was possible to verify that all the nanofibers adhered to the wound, however, the nanofibers with curcumin adhered more easily, being absorbed within a few hours, besides presenting good moisture retention. The rate of wound contraction showed that all nanofibers aided wound contraction, even when there was no association with the drug. In the 14-day period, wounds treated with control nanofibers and combined with 0.04% and 0.7% curcumin gave a contraction degree of 92.2%, 95.6%, and 94.3%, respectively, indicating closure almost full of wounds.
The reepithelial analysis showed that at 4 days treatment with scaffolds with curcumin increased the migration of keratinocytes compared to wounds treated with the control nanofiber. At 7 and 10 days, wounds treated with PCL/F-108C7 nanofibre showed greater migration of keratinocytes along their margin, suggesting a stimulation in the migration of keratinocytes in the wounds treated with the nanofiber with the higher concentration of curcumin. The epidermal thickness measurement was taken at 10 and 14 days of repair in the wounds that were completely reepithelialized. The results showed that in the period of 14 days the wounds treated with curcumin showed an increase in the thickness in comparison to the wounds treated with the nanofiber control. This migratory incitation in wounds treated with nanofibres with curcumin can be attributed to the healing potential of curcumin, which provided a more accelerated epithelial repair.
Cellular proliferation in the neoformed epidermis was evaluated by the keratinocyte counting method blocked in metaphase and the results suggested that the application of polymer nanofibers, associated or not to curcumin, offered a favorable environment for cell growth. From the revascularization analysis, it was possible to observe that at 4 days there was an angiogenic stimulus in the wounds treated with the nanofiber containing the highest concentration of curcumin. In the other periods, the
number of vessels did not vary, showing similar action among all scaffolds evaluated in the revascularization process.
Fibroplasia showed that the nanofiber control aided the production of collagen, but it was the incorporation of curcumin that promoted a more organized deposition of collagen. The results suggested that PCL/F-108C4 nanofibre exerted a modulating action on collagen synthesis and that PCL/F-108C7 fiber stimulated the maturation of collagen fibers.
CONCLUSIONS. The characterization results evidenced the incorporation of curcumin in the nanofibres and, also, demonstrated that the electrospinning technique allowed the preparation of a homogeneous material. The analysis of photoacoustic permeability showed that the nanofibers permeated both in the epithelial and dermal phases, guaranteeing their action until the end of the treatment. The wound contraction was accelerated with the application of the nanofibres and the histological results showed that the application of the nanofibers benefited all stages of healing and guaranteed the delivery of the incorporated drug since with the application of nanofibers with curcumin the reepithelialization, proliferation, revascularization, and fibroplasia was stimulated. It is concluded that the use of PCL/F-108 nanofibers is an innovative and promising alternative for the treatment of excisional wounds and when associated with curcumin, may enhance its bioavailability and enable its delivery system in the healing treatment.
Keywords: nanofibres, scaffolds, polycaprolactone, Pluronic® F-108, curcumin, wound healing.

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