NANOFI BER MESH COMPRISING FUCOIDAN, METHOD AND USES THEREOF

TECHNICAL FIELD

The present disclosure relates to a functionalized nanofiber polymeric mesh for the repair and regeneration of tissues, in particular for use in the treatment of cancer. The functionalized nanofiber polymeric mesh may be used as a skin patch for melanoma treatment, namely as an adjuvant after tumor resection.

BACKGROUND

[0001] Current treatment modalities for cancer do not achieve the ideal therapeutic outcomes due to severe side effects experienced by cancer patients. In this sense, alternative approaches are required and, between the possibilities, there is an increasing interest in the use of natural compounds from marine resources as biologically active products. Brown algae are a source of polysaccharides that may present several biological responses. Among these marine origin materials, fucoidan has attracted enormous interest in the last recent years. Fucoidan is a polysaccharide consisting in a sequence of sulfated fucose residues, together with other sugars, namely uronic acids, with chemical structure depending on the specie and extraction parameters, among other factors. Different physicochemical properties such as molecular weight, carbohydrates composition, sulfation degree and pattern along fucoidan backbone have been related with its biological activities, such as antitumor, anti-angiogenic and anti-inflammatory. Particularly, fucoidan has been recognized as a potential antitumor agent for different types of cancer like breast, lung, colon and melanoma.

[0002] Melanoma represents about 1% of all skin tumors. However, it is the most aggressive and deadliest form of skin cancer, having a very poor prognosis when it becomes metastatic. Risk factors for melanoma include sunburns during childhood, genetic family history and intermittent exposure to strong sunlight. Current treatment modalities may be surgical resection, chemotherapy, immunotherapy and targeted therapy, depending on the tumor characteristics (size, site and genetic profile). To improve survival rates, the combination of different therapies is often recommended. The lack of specificity to tumor cells is one of the major limitations of current therapies that may result in adverse effects and reduced efficiency. Trying to overcome these limitations, the need for testing alternative strategies and compounds offers interesting possibilities.

[0003] Fucoidan has been reported to inhibit melanoma development and progression, namely in its soluble form. Crude commercial fucoidan extracts from Fucus vesiculosus and Sargassum sp. showed that, despite the demonstrated cell growth inhibition at low dosages, both extracts presented very similar cytotoxic profiles over melanoma cells. In another attempt, similar extracts were studied, with a comparable anti-proliferative trend, being crude commercial fucoidan more cytotoxic than the extracted fucoidan, for higher doses. It was also demonstrated that fucoidan reduced the proliferation of melanoma cells and melanin production in a dosedependent manner, producing alterations in cells morphology. The antitumor activity of fucoidan extracted from Undaria pinnatifida and Fucus evanescens was evaluated over different human cancer cell lines, namely colon, breast and melanoma. No cytotoxic effects were observed over a non-cancer mouse epidermal cell line, whereas more pronounced growth inhibition was observed for breast and melanoma cells. A recent study evaluated the toxicity of fucoidan from Fucus vesiculosus over B16 murine melanoma cells, showing growth inhibition by regulating specific proteins/enzymes expression levels. However, not all fucoidan extracts present this interesting and promising antitumor activity, relating its biological activity with its chemical structure. Indeed, some fucoidan extracts may present antitumor properties against some (not all) cell lines, as well as having different cytotoxic concentrations. In fact, despite the previous reports on the cytotoxic effect of different fucoidan extracts over melanoma cells, another commercially available fucoidan from Fucus vesiculosus was evaluated over five different uveal melanoma cells lines, not showing anti-tumorigenic effects.

[0004] Based on the discrepancy of published results, an alternative approach to face melanoma is needed, aiming to decrease the cytotoxic effects over non-cancer cells. [0005] These facts are disclosed in order to illustrate the technical problem addressed by the present disclosure.

GENERAL DESCRIPTION

[0006] The present disclosure relates to a tailored nanofiber mesh functionalized with fucoidan (NFM_Fu), administered as a skin patch for use in the treatment of melanoma in a more local, precise and effective way, as well as a complementary treatment after tumor excision.

[0007] In an embodiment, fucoidan was immobilized at the surface of electrospun nanofiber mesh (NFM_Fu), for the development of a therapeutic patch.

[0008] In an embodiment, the maximum immobilization concentration was 1.2 mg rnL- ¹ , determined by the Toluidine Blue Assay and confirmed by XPS. Furthermore, NFM_Fu is more hydrophilic than NFM, presenting a contact angle of 36°, lower than the 121° of the control condition. As so, the immobilization of fucoidan at the surface of NFM was successfully achieved.

[0009] In an embodiment, NFM_Fu was able to reduce human melanoma cells viability by 50% without affecting human dermal fibroblasts and keratinocytes, developing a more local strategy to treat melanoma that can overcome unwanted cytotoxic side effects.

[0010] In an embodiment, NFM_Fu membrane ormesh may be used as adjuvant treatment after tumor excision, to tackle eventual remaining melanoma cells. Furthermore, this therapy can be personalized according to the tumor characteristics by changing its size, shape and treatment duration.

[0011] The present disclosure relates to a nanofiber polymeric mesh for the repair and regeneration of tissues comprising a polymeric substrate and a fucoidan immobilized at the surface of the polymeric substrate.

[0012] In an embodiment, the nanofiber polymeric mesh comprises 0.6 mg mL ^-1 to 1.2 mg mL ^-1 of immobilized fucoidan. [0013] In an embodiment, the fucoidan is extracted from Fucus vesiculosus or Sargassum sp or Undaria pinnatifida, preferably from Fucus vesiculosus.

[0014] In an embodiment, the molecular weight of fucoidan ranges between 20 kDa to 150 kDa. In an embodiment, the sulfation degree of the fucoidan ranges from 9% to 40%.

[0015] In an embodiment, the polymeric substrate comprises polycaprolactone, polyglycolic acid, chitosan, alginate or mixtures thereof. In a preferred embodiment, the polymeric substrate comprises polycaprolactone.

[0016] In an embodiment, the mean porosity of the nanofiber polymeric mesh ranges from 83 to 91% and the mean pore size ranges from 1.2 to 12.37 pm.

[0017] In an embodiment, the fiber diameter of the disclosed nanofiber polymeric mesh ranges from 0.35 to 2 pm.

[0018] In an embodiment, the thickness of the membrane or mesh ranges from 40 to 70 pm.

[0019] In an embodiment, the surface area of the nanofiber polymeric mesh ranges from 15 to 90 cm ² /mg.

[0020] An aspect of the present disclosure relates to the use of the nanofiber polymeric mesh described in the present disclosure in the treatment of cancer. In an embodiment, the nanofiber polymeric mesh is for use in the treatment of melanoma, preferably as an adjuvant after tumor resection.

[0021] The present disclosure also relates to a patch, a membrane or a dressing comprising the described nanofiber polymeric mesh.

[0022] In an aspect, the present disclosure relates to a composition for use in the treatment of cancer comprising a polymeric substrate and immobilized fucoidan wherein the composition is administered in a skin patch.

[0023] The present disclosure also relates to a method to obtain a nanofiber polymeric mesh as described in the present disclosure comprising the following steps: preparing a polymeric electrospun membrane or mesh; activating the obtained membrane or mesh; functionalizing the membrane or mesh's surface by immersion in a solution of 1,6-hexanediamine; immersing the membrane or meshin a fucoidan solution for immobilizing the fucoidan at the mesh's surface; drying the membrane or mesh containing fucoidan, overnight; washing the membrane or mesh containing fucoidan.

[0024] In an embodiment, the electrospun membrane or mesh comprises polycaprolactone, polyglycolic acid, chitosan, alginate or mixtures thereof, preferably polycaprolactone.

[0025] In an embodiment, the fucoidan is extracted from Fucus vesiculosus or Sargassum sp. or Undaria pinnatifida, preferably from Fucus vesiculosus.

[0026] In an embodiment, the fucoidan solution comprises NaCI, phosphate buffered saline, ultra-pure water, cell culture medium, 2-(N-Morpholino)ethanesulfonic acid hydrate, or mixtures thereof. In an embodiment, the concentration of fucoidan in the fucoidan solution ranges from 2.5 - 10 mg/ml.

[0027] In an embodiment, the activation step comprises at least one of the following surface treatments: UV-ozone treatment, plasma treatment, flame, corona discharge, photons, electron beam, ion beam, X-ray, or gamma-ray treatment.

BRI EF DESCRIPTION OF THE DRAWINGS

[0028] The following figures provide preferred embodiments for illustrating the disclosure and should not be seen as limiting the scope of invention.

[0029] Figure 1. Diagrams of the quantification of immobilized fucoidan on the electrospun nanofiber meshes A). Change of color after fucoidan immobilization B) and afterToluidine Blue assay.

[0030] Figure 2. Diagrams of the water contact angle measurements for the electrospun nanofiber meshes without and with immobilized fucoidan at different concentrations.

[0031] Figure 3. XPS analysis: survey spectrum of CTR A); general spectra of Fu 10 B) and sulphur spectra of Fu 10 C).

[0032] Figure 4. Cell viability A) and representative SEM images B) of human melanoma cells cultured on NFMs with and without (CTR) fucoidan. [0033] Figure 5. Cell viability A) and representative SEM images B) of human keratinocytes cultured on NFMs with and without (CTR) fucoidan and control NFMs.

[0034] Figure 6. Cell viability A) and representative SEM images B) of human dermal fibroblasts (hDFs) cultured on NFMs with or without (CTR) fucoidan.

DETAILED DESCRI PTION

[0035] The present disclosure relates to a nanofiber polymeric mesh for the repair and regeneration of tissues comprising a polymeric substrate and fucoidan immobilized at the surface of the polymeric substrate.

[0036] One of the major disadvantages of some current cancer treatments is the fact that, besides affecting the tumor, they also have severe toxic effects over many healthy tissues. Therefore, when developing systems for melanoma treatment, there is the need to evaluate their cytotoxic effects not only over melanoma cells, but also over adjacent non-cancer cells.

[0037] In an embodiment, the fucoidan-based system described in the present disclosure was tested over primary human keratinocytes (most common cell type in the epidermis) and fibroblasts (most common cell type in the dermis), as well as, over a representative human melanoma cell line WM-115 (a cell line originated from the primary tumor with competence for metastasis) that has been used in different studies. In vitro results revealed a 50% decrease on melanoma cells' viability while maintain the viability of normal cells, which is an indication of the selectiveness and effectiveness of the developed functionalized nanofibrous membrane or mesh.

[0038] Example:

[0039] The following pertains to the preparation of electrospun polycaprolactone nanofiber meshes. A 15% (w/v) Polycaprolactone (PCL; Mw = 70 000-90 000) solution was prepared with an organic solvent mixture of chloroform and N,N- dimethylformamide at a 7:3 volume ratio. The PCL solution was electrospun by applying a voltage of 11 kV, a needle tip to ground collector distance of 18 cm, and a flow rate of 1 mL h" ¹ . After the complete processing of 1 mL of PCL solution, the nanofibrous mesh (NFM) was allowed to dry for 1 day. This processed NFMs were cut into samples of 1 x 1 cm ² for further assays.

[0040] For the activation of the NFM, an ultraviolet-ozone (UV-ozone) cleaner system was used (ProCleaner 220, Bioforce Nanoscience). Both sides of the electrospun NFMs were exposed for 90 seconds to UV-ozone irradiation. After this surface activation, amine groups (-NH2) were inserted at the surface of NFM by immersion in a 1 M 1,6- hexanediamine (HMD) solution for 1 h at 37 °C. Finally, the functionalized NFM were washed 3X with PBS.

[0041] The following pertains to the fucoidan immobilization on NFM. Fucoidan (Fu) from Fucus vesiculosus was dissolved in 0.1 M NaCI at different concentrations (Fu 2.5; Fu 5; Fu 7.5 and Fu 10 mg mL ¹ ) and 200 pL of each solution was added over functionalized NFM in a 24 well-plate. This reaction was performed during 8h and, after that, the solutions were removed and the biofunctionalized NFM were left to dry overnight. In the following day, NFM were washed twice. All steps were performed under sterile conditions.

[0042] Toluidine Blue (TBO) was used to quantify the amount of fucoidan immobilized into each NFM. Different concentrations of fucoidan were immobilized at the surface of NFM until its saturation concentration (10 mg mL ^-1 , according to the manufacture information). Each sample was immersed in 500 pL of TBO solution (0.1 M HCI, 20 mg NaCI, and 4 mg toluidine blue O chloride) for 4 h at room temperature. The TBO solution was removed and the biofunctionalized NFMs were washed until all unreacted TBO solution was removed. Afterwards, the biofunctionalized NFMs were immersed in 500 pL of a solution containing 0.1M NaCI and ethanol (1:4) for complete decoloration. The amount of TBO was assessed by measuring the absorbance of the supernatant at 530 nm using a microplate reader. The amount of fucoidan immobilized at the surface of each NFM was calculated from a standard curve established with fucoidan solutions at different concentrations (Fu 2.5; Fu 5; Fu 7.5 and Fu 10 mg mL ^-1 ). In this sense, after incubating fucoidan with TBO, the solutions were centrifuged and the unbound TBO was removed. The pellet was then resuspended with the above mentioned 0.1M NaCI and ethanol solution and read at 530 nm. [0043] In an embodiment, after the optimizing of the standard curve, the different concentrations were calculated and was observe that, by increasing the initial concentration of fucoidan, higher amounts are immobilized. The maximum immobilization capacity was around 1.2 mg mL ^-1 for FulO, twice the concentration of Fu2.5 with a minimum immobilization of around 0.6 mg mL ¹ (Figure 1 A). The lower concentrations Fu5 and Fu2.5 are the ones with higher immobilization percentages when compared to the initial concentration, 16% and 26%, respectively. FulO and Fu7.5 have similar immobilization percentages, of around 12%. Nevertheless, these are the two concentrations with more fucoidan immobilized on the NFM (Fu 7.5 - 0.9 mg mL ¹ ). The system seems not to have reached its maximum immobilization capacity (maximum fucoidan concentration tested was 10 mg mL ^-1 , which is the maximum solubility of fucoidan), but different initial concentrations lead to different percentages of immobilization.

[0044] In an embodiment, inn Figure 1 B) is possible to confirm the presence of fucoidan by observing the color change of the NFMs. Fucoidan in solution presents a light brown color and the membrane or mesh with fucoidan are more yellowish than the controls, which represent its original white color. After toluidine blue staining (Figure 1 C) it is clear that the NFMs that contain fucoidan becomes purple, whereas the control is not stained.

[0045] The following pertains to the measurement of the contact angle. Surface hydrophilicity of NFM and NFM Fu was measured as the static contact angle of a standard liquid (ultra-pure water, 3 pL), at room temperature, using a Contact Angle Equipment (OCA 15plus equipment, Germany and SCA-20 software). During every determination, a motor driven syringe was used to deposit a drop of liquid over the NFM surface. Measurements were recorded for each sample and the determinations were performed in triplicate.

[0046] In an embodiment, by increasing the concentration of immobilized fucoidan, NFMs presented a lower contact angle, being more hydrophilic (Figure 2). Electrospun NFMs without fucoidan presented a water contact angle of 121° ± 8. On the other hand, NFMs with immobilized fucoidan (FulO) were much more hydrophilic, presenting a water contact angle of 36° ± 8 (Table 1) Table 1 - Water contact angle measurements for the NFMs without and with immobilized fucoidan at different concentrations.

[0047] The following pertains to X-ray photoelectron spectroscopy (XPS) analysis. XPS was used to analyze the surface chemistry of the different NFMs with or without immobilized fucoidan. Analysis of the samples was performed using a Kratos Axis- Supra instrument controlled with ESCApe software. Due to the non-conductive nature of the samples it was necessary to use a co-axial electron neutralizer to minimize surface charging. The XPS measurements were carried out using monochromatic Al-Ka radiation (1486.6 eV). Photoelectrons were collected from a take-off angle of 90° relative to the sample surface. The measurement was done in a Constant Analyser Energy mode (CAE) with a 160 eV pass energy and 15 mA of emission current for survey spectra and 40 eV pass energy for high resolution spectra, also using an emission current of 15 mA. Charge referencing was done by setting the lower binding energy Cis photo peak at 285.0 eV Cis hydrocarbon peak. The chemical composition of the samples was examined by XPS surface measurements. The Cis, Ols, S2p and survey spectra were recorded using a Kratos Axis-Supra instrument. The residual vacuum in the X-Ray analysis chamber was maintained at around 4.5xl0 ⁸ torr. The samples were fixed to the sample holder with double sided carbon tape.

[0048] In an embodiment, from the quantitative analysis (Table 2) it was possible to observe that carbon and oxygen were present in all the NFMs with immobilized fucoidan, as well as in the control (without fucoidan), as expected since both elements are constituents of PCL and fucoidan chemical structures. With increasing fucoidan concentration for the functionalization of NFMs, there was a decrease in the atomic concentration of carbon and an increase of oxygen and particularly of sulphur, originated from the sulfate groups present on immobilized fucoidan. [0049] Table 2 - Elemental quantitative analysis of the surface of NFMs functionalized with different fucoidan concentrations.

[0050] A general spectrum of the control condition (Figure 3 A) indicates the presence of two main peaks representing carbon (291.40 eV) and oxygen (537.12 eV). For FulO (Figure 3 B), three main peaks are detected: carbon (297.70 eV), oxygen (544.47) and sulphur (176.0). After fucoidan immobilization, a peak starting at 177.95 eV and ending at 167.51 eV appears, which is attributed to the sulphur element (S2p) from the fucoidan chain, confirming fucoidan immobilization (Figure 3 C). The highest fucoidan concentrations presented the highest percentage of sulphur atoms, meaning that a higher concentration of fucoidan has been immobilized. Furthermore, the immobilization of fucoidan also increased the oxygen content due to the presence of this element in functional groups within polysaccharide structure, including the sulfates.

[0051] The following pertains to the biological assays and cell culture. Human melanoma cells (WM-115 cell line, ATCC) were cultured in Eagle's Minimum Essential Medium (EMEM 30-2003, ATCC), supplemented with 10% FBS (Alfagene), 1% pen/strep (100 U/100 pg mL" ¹ ; Life Technologies) and incubated at 37 °C in a humidified 5% CO2 atmosphere. Human dermal fibroblasts (hDFs, Gibco) were cultured in Medium 106 (Gibco) supplemented with 10% FBS (Alfagene), Pen/Strep (100 U/100 g mL" ¹ ; Life Technologies) and 2% Low Serum Growth Supplement (Life Technologies). Human keratinocytes (hKCs) were isolated and cultured in Keratinocyte-SFM (Life technologies) supplemented with 1% Pen/Strep (100 U/100 g mL" ¹ ; Life Technologies). The medium was changed every two days until cells reached confluence.

[0052] Cell seeding was performed onto fucoidan immobilized electrospun NFMs (NFM_Fu) by dropping a 50 pL cell suspension containing 50 000 cells per NFM_Fu. Cells were left to adhere for 4h before culture medium was added. NFMs subjected to surface activation, aminolysis, but without fucoidan immobilization were used as control (NFM_CTR). After 1, 3 and 7 days of culture, samples were collected for cell viability assay and SEM observation. Triplicates of each condition were used in three independent assays (n-3).

[0053] The metabolic activity was determined by the MTS assay (CellTiter 96 AQueous One Solution, Promega). At days 1, 3 and 7, the culture medium was removed and the samples were rinsed with sterile PBS. A mixture of culture medium and MTS reagent (5:1 volume ratio) was added to each condition as well as to the negative control comprising no cells. Samples were left to incubate for 3 h at 37 °C in a humidified 5% CO2 atmosphere. Thereafter, the absorbance of the MTS reaction medium from each sample was read in triplicate at 490 nm (Synergy HT, BioTEK).

[0054] The morphology and distribution of cells seeded on top of the NFM Fu or NFM_CTR was analyzed by Scanning Electron Microscopy (SEM). At each time point (1, 3 and 7 days), samples were collected and fixed with 2.5% glutaraldehyde. After fixation, samples were washed with PBS and dehydrated with increasing concentrations of ethanol, until 100% was reached. Samples were then left to dry overnight. In the following day, all samples were vacuum-coated with platinum mixture and observed at the SEM (JSM-6010 LV, JEOL, Japan). Photographs at xl50 and xlOOO magnifications were obtained.

[0055] In an embodiment, the viability of the melanoma cells was lower on the NFM_Fu condition than on the CTR from day 3 onward. At days 3 and 7, NFM_Fu induced a decrease in melanoma cells viability (Figure 4 A). Specifically, at day 3, around 70% of melanoma cells are viable, whereas at day 7 there is a viability of around 50% when compared with the CTR. These observations were corroborated by SEM micrographs (Figure 4 B). Cells were able to colonize the control NFM, covering its surface by showing increasing number of melanoma cells along the course of the 7 days. Oppositely, in the case of NFM_Fu condition this colonization of the mesh was not observed.

[0056] The immobilization of fucoidan on the NFM did not present cytotoxic effects over seeded keratinocytes (Figure 5 A). No significant differences were observed between the CTR and the NFM_Fu, for all time points. When analyzing the SEM micrographs, keratinocytes covered the NFMs surface, presenting no morphological differences between the CTR and the NFM_Fu conditions, along the 7 days of culture. NFM_Fu was also not cytotoxic for the fibroblasts (Figure 6). There were no significant differences between the CTR and the NFM_Fu regarding the viability for all time points. When analyzing the SEM micrographs, it is possible to observe that dermal fibroblasts covered the surface of the NFMs, forming a dense cell layer along time, and presenting no morphological differences between the two conditions.

[0057] The term "comprising" whenever used in this document is intended to indicate the presence of stated features, integers, steps, components, but not to preclude the presence or addition of one or more other features, integers, steps, components or groups thereof.

[0058] The disclosure should not be seen in any way restricted to the embodiments described and a person with ordinary skill in the art will foresee many possibilities to modifications thereof. The above described embodiments are combinable.

[0059] The following claims further set out particular embodiments of the disclosure.