SUCROSE ACETATE ISOBUTYRATE-BASED COMPOSITIONS, METHODS AND USES THEREOF

Abstract

The present disclosure relates to a composition for the release of the bioactive substance comprising: sucrose acetate isobutyrate dissolved in an ionic liquid and an additive selected from the list consisting of: chitin, silk fibroin, cellulose, alginate, chitosan, gellan gum, dextrin, collagen, guar gum, carregeenan, heparin, kefiran, or mixtures thereof. By taking advantage of the properties of an ionic liquid (IL), in particular 1-butyl-imidazolium acetate (BMIMAc), it was possible to achieve a good dissolution of SAIB, which combined with chitin and/or silk, natural polymers, allows the development of the structures with different shape and sizes.

Claims

1. A composition for the release of a bioactive substance, the composition comprising: sucrose acetate isobutyrate dissolved in an ionic liquid; and an additive selected from group consisting of chitin, silk fibroin, cellulose, alginate, chitosan, gellan gum, dextrin, collagen, guar gum, carregeenan, heparin, kefiran, and mixtures thereof.

2. The composition of claim 1, wherein the composition comprises: 10-60% (w/V) of sucrose acetate isobutyrate; and 40-90% (w/V) of the additive.

3. The composition of claim 1, wherein the composition comprises 30-50% (w/V) of sucrose acetate isobutyrate.

4. (canceled)

5. The composition of claim 1, wherein the composition comprises 55-65% (w/V) of the additive.

6. The composition of claim 1, wherein the additive is dissolved in the ionic liquid.

7. (canceled)

8. The composition of claim 1, wherein the ionic liquid is selected from the group consisting of: 1-butyl-3-methylimidazolium chloride, 1-butyl-3-methyl imidazolium acetate, 1-allyl-3-methylimidazolium bromide, 1-ethyl-3-methylimidazolium dimethyl phosphate, 1-carboxymethyl-3-methylimidazolium hydrochloride, 1-ethyl-3-methylimidazolium chloride, 1-ethyl-3-methylimidazolium acetate, and mixtures thereof.

9. The composition of claim 1, further comprising a ceramic additive, wherein the ceramic additive is selected from hydroxyapatite, bioglass, silicon substituted hydroxyapatite (HAp), tri-calcium phosphate (TCP), or combinations thereof.

10. (canceled)

11. The composition of claim 9, wherein the release of the bioactive substance is a sustained release and the sustained release is 1-2 months, and wherein the bioactive substance is selected from the group consisting of: a molecule, antibiotic, growth factor, nanoparticles and micro-particles, and mixtures thereof.

12. (canceled)

13. The composition of claim 11, wherein the antibiotic is bacitracin, erythromycin, polymyxin, vancomycin, gentamycin, kanamycin, neomycin, amoxicillin streptomycin, rapamycin, streptomycin, polymyxin, colistin, tyrocidine, gramicidin, cyclosporin, or mixtures thereof.

14. The composition of claim 11, wherein the growth factor is an epidermal growth factor, platelet-derived growth factor, growth hormone releasing factor, platelet derived growth factor, nerve growth factor, or mixtures thereof.

15. The composition of claim 11, wherein the nanoparticles are dendrimers nanoparticles, in particular polyamidoamine (PAMAM).

16. The composition of claim 11, wherein the microparticles are systems of synthetic polymers selected from the group consisting of: poly(lactic-co-glycolic acid) (PLGA), poly(lactic acid) (PLA), natural polymers, and mixtures thereof.

17. (canceled)

18. A composite comprising the composition of claim 1, wherein the composite is a gel, a membrane or a scaffold, and wherein the scaffold has a pore sized of 0.5-50 m.

19. (canceled)

20. (canceled)

21. The composition of claim 1, wherein the composition is suitable for treating or preventing bone disease or defect, cartilage disease or defect, cornea disease or defect, skin disease or defect, vascular tissue disease or defect, peripheral nerve disease or defect, spinal cord disease or defect, brain diseases or defect or wound healing.

22. (canceled)

23. (canceled)

24. (canceled)

25. A method of producing a composition or a composite, the method comprising the following steps: dissolving sucrose acetate isobutyrate in an ionic liquid; dissolving an additive selected from the group consisting of a polysaccharide, a protein, and mixtures thereof in the ionic liquid; mixing the dissolved sucrose acetate isobutyrate and the dissolved additive in a solution; homogenizing the solution; transferring the solution to a mold; solidifying the solution; removing the ionic liquid; and washing of the composition or the composite.

26. The method of claim 25, comprising a further step of freeze-drying the composition or the composite for obtaining a scaffold, wherein the composite is a gel.

27. The method of claim 25, wherein the step of mixing is carried out at 60-120 C.

28. The method of claim 25, wherein the step of mixing is carried out for 5-60 min.

29. The method of claim 25, wherein the step of solidifying is carried out at 80 C.-25 C.

30. The method of claim 25, wherein the step of solidifying is carried out with a solvent that is selected from the group consisting of: water, isopropanol, ethanol, tetraglycol (glycofurol), benzyl alcohol, dimethyl sulfoxide, ethyl lactate, ethyl acetate, triacetin, N-methylpyrrolidone, propylene carbonate, glycerol formal, isopropylideneglycerol, and mixtures thereof.

31. The method of claim 30, wherein the solvent is water or water:isopropanol (1:1).

32. The method of claim 25, wherein the ionic liquid is selected from the group consisting of: 1-butyl-3-methylimidazolium chloride, 1-butyl-3-methyl imidazolium acetate, 1-allyl-3-methylimidazolium bromide, 1-ethyl-3-methylimidazolium dimethyl phosphate, 1-carboxymethyl-3-methylimidazolium hydrochloride, 1-ethyl-3-methylimidazolium chloride, 1-ethyl-3-methylimidazolium acetate, and mixtures thereof.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0056] The following figures provide preferred embodiments for illustrating the description and should not be seen as limiting the scope of invention.

[0057] FIG. 1. Sucrose acetate isobutyrate C.sub.40H.sub.62O.sub.19.

[0058] FIG. 2. Schematic representation of the production steps, together with some example images.

[0059] FIGS. 3A-3B. Conductivity measurements on aliquots of the washing step solvents, obtained after immersion of the structures with 150 rpm of agitation, on the first day (black bars), second day (white bars) and 5.sup.th day (gray bars), wherein for FIG. 3A, SAIB/Chitin A stands for SAIB processed with [bmim][Ac] and mixed with Chitin processed with [bmim][Ac] in water (solvent A), SAIB/Chitin B stands for SAIB processed with [bmim][Ac] and mixed with Chitin processed with [bmim][Ac] in water:isopropanol (1:1) (solvent B), Chitin A stands for chitin processed with [bmim][Ac] in water (solvent A); Chitin B stands for chitin processed with [bmim][Ac] in water:isopropanol (1:1)(solvent B). In the same way, for FIG. 3B, SF B and C stands for silk fibroin (SF) scaffolds processed with solvent B (isopropanol:water 1:1) and C (methanol:water 1:1) and SAIB:SF 1:1, SAIB:SF 1:2 and SAIB:SF 2:1 the different proportions of SAIB to SF, in the same solvents B and C. Data represent the meanstandard deviation (significant differences p<0.05, two-way ANOVA).

[0060] FIG. 4. FTIR spectra of SAIB/Chitin and Chitin samples processed with [bmim][Ac] in water (A), and in water:isopropanol (1:1) (B).

[0061] FIG. 5. XRD patterns of SAIB/Chitin and Chitin samples processed with BMIMAc, in water (A) and in water:isopropanol (1:1)(B).

[0062] FIGS. 6A-6B. FIG. 6A: SEM images of surface and cross-section (C/S) of the scaffolds:SAIB:Chitin and Chitin samples processed with BMIMAc, in water (A) and in water:isopropanol (B). FIG. 6B: SEM images of surface and cross section (C/S) of the scaffolds with different proportions of SAIL:Silk fibroin (1:1, 1:2 and 2:1) processed with BMIMAc, in water:isopropanol (B) and water:methanol (C).

[0063] FIGS. 7A-7B. Swelling behavior of the SAIB/chitin (FIG. 7A) and SAIB/SF (FIG. 7B) scaffolds. Data represent the meanstandard deviation (*** denotes the statistical significant differences p<0.003, two-way ANOVA).

[0064] FIGS. 8A-8D. Pore size distribution of chitin scaffolds and SAIB/Chitin based scaffolds: chitin A (FIG. 8A), chitin B (FIG. 8B), SAIB/chitin-A (FIG. 8C) and SAIB/chitin B (FIG. 8D). Data obtained from the micro-CT analysis.

[0065] FIGS. 9A-9C. SAIB scaffolds cytotoxicity. FIG. 9A: Cells' growth of hASCs cultured during 72 hours directly with SAIB/Chitin A and Chitin A scaffolds. Results are present as percentage in relation to CTRL. Data is presented as meanSEM. FIG. 9B: Quantification of hASCs' proliferation upon culture directly with SAIB/Chitin A and Chitin A scaffolds along 72 h of culture. Data is presented as meanSEM. * denotes statistically significant differences (p<0.05). FIG. 9C: Cells' damage assessed by F-actin staining (cytoskeleton, red) and counterstained with DAPI staining (nuclei, blue), during 72 hours of culturing (scale bar: 100 m).

[0066] FIG. 10. Chitin and SAIB/chitin based membranes photos, before and after genipin crosslinking.

[0067] FIG. 11. Conductivity measurements measured during IL removal, on the first day (black bars), second day (gray bars) and 5.sup.th day (white bars).

[0068] FIG. 12. SEM images of the crosslinked chitin and SAIB/chitin based membranes.

[0069]

TABLE-US-00001 TABLE 1 Adhesivity values obtained for Chitin and SAIB/Chitin gels (in water, solvent SAMPLE ADHESIVITY (N .Math. s) SAIB/CHITIN A 0.135 0.003 CHITIN A 0.035 0.002

TABLE-US-00002 TABLE 2 Morphological features of the chitin and chitin/SAIB- based scaffolds from microCT essays. INTER- POROS- CONNEC- MEAN WALL MEAN PORE ITY TIVITY THICKNESS SIZE SAMPLE (%) (%) (M) (M) CHITIN A 88.58 98.52 20.52 101.4 CHITIN B 81.74 95.61 23.96 77.59 CHITIN/ 46.57 14.53 56.34 57.80 SAIB A CHITIN/ 57.43 54.40 50.06 60.21 SAIB B

DETAILED DESCRIPTION

[0070] The present disclosure relates to a composition for the release of the bioactive substance comprising: sucrose acetate isobutyrate dissolved in an ionic liquid and an additive selected from the list consisting of: chitin, silk fibroin, cellulose, alginate, chitosan, gellan gum, dextrin, collagen, guar gum, carregeenan, heparin, kefiran, or mixtures thereof. By taking advantage of the properties of an ionic liquid (IL), in particular 1-butyl-imidazolium acetate (BMIMAc), it was possible to achieve a good dissolution of SAIB, which combined with chitin and/or silk, natural polymers, allows the development of the structures with different shape and sizes.

[0071] In an embodiment, the materials used were the following: chitin from crab shells (practical grade; Sigma Aldrich) with a degree of N-acetylation of 57.9%, determined by elemental analysis, were ground through a Wiley Mill (model 4, Thomas) and stored in plastic bottles. Ground chitin (106 m) was used throughout the experiments to obtain reproducible results. SAIB and the ionic liquid (IL), 1-Butyl-3-methylimidazolium chloride [bmim][Cl] and 1-butyl-3-methyl imidazolium acetate [bmim][Ac], used without further purification, were obtained from Sigma Aldrich. Silk fibroin from cocoons of B. mori was kindly supplied by the APPACDM (Castelo Branco, Portugal) Genipin was purchased from Wako Chemicals. All other chemicals were reagent grade and were used as received.

[0072] In an embodiment, the preparation of the SAIB/chitin based structures was carried out as follows: SAIB (0.4 g.Math.mL.sup.1) and chitin (0.01 g.Math.mL.sup.1) were dissolved individually in [bmim][Ac], at 75 C. and 95 C., respectively, and then homogenized in the ratio 50/50 by adding the SAIB/IL solution to the chitin/IL solution. After homogenization (about 15 min), the solution was transferred to silicone molds (d=8 mm, h=2 mm), followed by gelation of the systems through their immersion in water (solvent A) or water:isopropanol (1:1, solvent B) to obtain the chitin/SAIB gels. Moreover, membranes were also produced using bigger molds (d=3 cm). The [bmim][Ac] removal procedure was first carried out by immersing the materials in the solvents A and B for 48 hours followed by Soxhlet extraction with the same solvents. In the optimization stage, the Soxlet was replaced by a coarser but equally effective procedure. After 48 h in the molds, and immersed in the solvents, the structures were placed in caped flasks at 150 rpm with daily solvent change (5 days). During this washing step, aliquots were collected to measure the conductivity and thus follow the removal of the ionic liquids throughout time. This method is schematically represented on FIG. 1.

[0073] A similar procedure was used and optimized to prepare SAIB/Silk fibroin (SAIB/SF) structures. Briefly, SAIB (0.4 g.Math.mL.sup.1) and SF (0.1 g.Math.mL.sup.1) were dissolved individually in [bmim][Ac], at 75 C. and 60 C., respectively, and then homogenized in the ratios 1:1, 1:2 and 2:1 SAIB:SF by adding the SAIB/IL solution to the SF/IL solution. After homogenization (about 15 min), the solution was transferred to silicone molds (d=8 mm, h=2 mm), followed by gelation of the systems at 20 C. for 2 hours. Further, the systems [bmim][Ac] removal procedure and beta sheet formation were carried out by immersing the systems in water/isopropanol 1:1 (solvent B), water/methanol 1:4 and water.

[0074] Moreover, taking advantage of the acquired knowledge on the developed SAIB/chitin structures and also of the genipin properties, genipin crosslinked SAIB/chitin membranes were also produced. The developed and optimized procedure started by using SAIB (0.4 g.Math.mL.sup.1) and chitin (0.1 g.Math.mL.sup.1) dissolved individually in [bmim][Ac], at 75 C. and 95 C., respectively, and then homogenized in the ratio 50/50 by adding the SAIB/IL solution to the chitin/IL solution. After homogenization (about 15 min), the solution was transferred to small petri dishes, followed by gelation of the systems at 20 C during 2 hours. To create crosslinked SAIB/chitin membranes, the systems were immersed in genipin solutions (10 mM, 20 mM), prepared using ethanol/water, for 24 hours at room temperature (RT) and 4 C. After that, the materials were immersed in water (solvent A) or water:isopropanol (1:1, solvent B) to obtain the crosslinked chitin/SAIB gels. During IL removal, aliquots were collected to measure the conductivity and thus follow the removal of the ionic liquids throughout time. In an embodiment, to obtain SAIB/Chitin and SAIB/SF based scaffolds, SAIB/Chitin and SAIB/SF gels were freeze-dried.

[0075] In an embodiment, to obtain porous SAIB/Chitin based membranes the crosslinked chitin/SAIB gels were freeze-dried.

[0076] In an embodiment, the physicochemical characterization of the SAIB/Chitin, SAIB/SF and porous SAIB/Chitin membranescaffolds was carried out.

[0077] In an embodiment, the conductivities of the collected aliquots, of the washing solvents throughout time, were measured using a conductivimeter (INOLAB, Multi-level 3) with a Sonda WTW TetraCon 325.

[0078] In an embodiment, Fourier transform infrared spectroscopy (FTIR) was carried out. The specimens to be analyzed were powdered and mixed with potassium bromide, milled and molded into a transparent pellet using a press (Pike, USA). Transmission spectra were acquired on an IR Prestige-21 spectrometer (Shimadzu, Japan), using 32 scans, a resolution of 4 cm.sup.1 and a wavenumber ranging between 4000 cm.sup.1 and 500 cm.sup.1.

[0079] In an embodiment, X-ray diffraction (XRD) patterns were determined. The qualitative analyses of crystalline phases presented on the hydrogels were assessed by XRD using a conventional Bragg-Brentano diffractometer (Bruker D8 Advance DaVinci, Germany) equipped with CuK radiation, produced at 40 kV and 40 mA. Data sets were collected in the 2 range of 5-60 with a step size of 0.02 and is for each step.

[0080] In an embodiment, scanning electron microscopy (SEM) was carried out. The freeze-dried samples were attached to aluminum stubs using a carbon tape and coated with platinum in a sputter coater (Model EM ACE600, Leica, Germany). The morphology images were obtained on a SEM (JSM-6010LV, JEOL, Japan), featuring integrated energy dispersive spectroscopy (EDS) (INCAx-Act, PentaFET Precision, Oxford Instruments).

[0081] In an embodiment, the microstructure of the prepared SAIB/chitin-based scaffolds was evaluated using a high-resolution X-ray microtomography system Skyscan 1072 scanner (Skyscan, Kontich, Belgium). Samples were scanned using a pixel size of 8.79 mm x/y/z and an X-ray source fixed at 40 keV of energy and 248 mA of current. After the acquisition, reconstructed grey-scale images were converted into binary images by using a dynamic threshold of 40-255. Then, the binary images were used for morphometric analysis (CT Analyzer v1.12.0.0, SkyScan, Kontich, Belgium) by quantification of porosity, mean pore size, mean wall thickness and interconnectivity. In an embodiment, swelling tests were made. The dry samples weight was measured (W.sub.D) and, after immerse in PBS and remove the excess of water (by softly tapping the surface with filter paper), the samples wet weight (W.sub.S) was measured. The samples were rehydrated until equilibrium. The degree of swelling was determined by calculating (W.sub.SW.sub.D)/W.sub.D. Each experiment time point was repeated 3 times.

[0082] In an embodiment, pull away experiments were performed using a Kinexus pro+ rheometer (Malvern Instruments, UK), using the acquisition software rSpace. The measuring system used in these experiments was equipped with stainless steel (316 grade) plate-plate system. These experiments involved loading a sample and then pulling away the upper plate at a defined gap speed (1 mm.Math.s.sup.1), with a contact time of 2 s and a contact force of 1 N. The resultant normal force was then recorded as function of gap and was used to determine the adhesion properties (area under the force-gap curve).

[0083] In an embodiment, cell growth was assessed using the CellTiter 96 AQueous One Solution Cell Proliferation Assay (MTS, Promega, USA). At each time point, 24 hours, 48 hours and 72 hours cells were incubated with 20% V/V of MTS in culture medium without phenol red (Sigma Aldrich, USA) for 3 hours at 37 C. The supernatant was then transferred to a new 96-well plate and absorbance measurements were carried out using a microplate reader (Biotek Synergy HT) at 490 nm.

[0084] In an embodiment, cell damage was studied through F-actin staining. For that, cells were washed with phosphate buffer saline (PBS, Sigma Aldrich, USA), fixed with 10% Neutral Buffered Formalin (ThermoFisher Scientific, USA) for 15 minutes and permeabilized for 5 minutes with 0.1% V/V Triton X-100 (Sigma Aldrich, USA) in PBS. Afterwards, samples were incubated for 30 minutes in 1% (w/V) of BSA (Sigma Aldrich, USA) in PBS to block unspecific binding. F-actin filaments were stained with Phalloidin-Tetramethylrhodamine B isothiocyanate (1:40, Sigma Aldrich, USA) and nuclei were counterstained with 1:5000 of the stock of 4,6-Diamidino-2-phenyindole, dilactate solution (DAPI, 1 mg/mL, Biotium, USA). Samples were analysed under a fluorescence inverted microscope (Zeiss Axio observer).

[0085] In an embodiment, cytotoxicity screening of the SAIB/Chitin based scaffolds were performed. Human adipose derived stem cells (hASCs) were obtained from human adipose tissue after liposuction procedure, which was performed at Hospital da Prelada (Porto, Portugal), after patient's informed consent and under a collaboration protocol approved by the ethical committees of both institutions. In order to isolate the hASCs, the adipose tissue was submitted to the action of 0.05% collagenase type II (Sigma Aldrich, USA), under agitation for 1 hour at 37 C. Then, it was filtered with a strainer and centrifuged at 800 G for 10 minutes. After discard the supernatant, pellets were re-suspended in PBS and centrifuged at 350 G for 5 min. Finally, the cell pellet was re-suspended in Minimum Essential Media (-MEM, Gibco, UK), supplemented with 10% fetal bovine serum (FBS, Invitrogen, USA), and 1% antibiotic/antimycotic (Invitrogen, USA). Cultures were maintained at 37 C. under a humidified atmosphere of 5% V/VCO.sub.2 in air. hASCs were selected by plastic adherence and passage at 80% confluence. In the different studies hASCs in passage 4 were used.

[0086] In an embodiment, the evaluation of cytotoxicity of SAIB/Chitin based scaffolds was performed as described in the ISO 10993-12, using hASCs. First, hASCs were seeded in each well of a 96-well plate at a density of 3000 cells per cm.sup.2. After 24 hours of culturing, the SAIB/Chitin based scaffolds produced using solvent A and solvent B (SAIB/Chitin A and SAIB/Chitin B, respectively) were added to the top of cells. As control, Chitin scaffolds produced using solvent A and B were tested (Chitin A and Chitin B, respectively). Additionally, a negative control (Ctrl) was prepared composed of hASCs without addition of scaffolds and a positive control (Ctrl+) composed of Triton X-100 at a concentration of 1% in culture medium. Cultures were maintained at 37 C. under a humidified atmosphere of 5% V/V CO.sub.2 in air. Finally, at 24, 48 and 72 hours of culture, cell growth and cell damage were analysed as described previously.

[0087] In an embodiment, cell morphology: whole-mounted samples were evaluated. At different time points scaffolds were recovered and F-actin was stained to evaluate cell morphology. For that, samples were washed with PBS, fixed with 10% Neutral Buffered Formalin for 15 minutes and permeabilized for 5 minutes with 0.1% V/VTriton X-100 in PBS. F-actin filaments were stained with Phalloidin-Tetramethylrhodamine B isothiocyanate (1:80) and nuclei were counterstained with DAPI (1:5000). Controls analyzed under a fluorescence inverted microscope (Zeiss Axio observer) and samples were analyzed using confocal microscopy (Leica TCS SP8).

[0088] In an embodiment, statistical analyses was performed using GraphPad Prism 6.0 software. The non-parametric Mann-Whitney test was used to compare two groups, whereas comparison between more than two groups was performed using the Kruskal-Wallis test followed by Dunn's comparison test. Additionally, a two-way ANOVA followed by Tukey's multiple comparisons test was used every time that studies involved two independent variables. The critical level of statistical significance chosen was p<0.05. Data of 3 experiments are presented as meanSEM. In an embodiment, the physicochemical characterization of scaffolds and membranes produced and herein disclosed was carried out as follow.

[0089] In an embodiment, the SAIB/Chitin based scaffolds were produced as herein described and schematically shown on FIG. 2. The preliminary experiments showed that SAIB not only is able to dissolve in [bmim][Ac] but also to blend with other compounds dissolved in IL, in particular chitin/IL or silk fibroin/IL.

[0090] In an embodiment, it was also observed that SAIB does not dissolve in [bmim][Cl] neither is able to form gels alone in [bmim][Ac]. Concerning the used solvents A (water) and B (water:isopropanol 1:1), those were chosen, after testing ethanol, methanol and others, concerning what was visible to the naked eye (e.g. do not dissolve the gel, able to clean the ionic liquid, . . . ). Therefore, the subsequent experiments were performed by mixing SAIB/IL in Chitin/IL (95 C., 15 min), after the dissolution step where SAIB and Chitin were separately dissolved in the IL (18 hours approximately). It was noticed that, mixing SAIB/IL in Chitin/IL and mixing Chitin/IL in SAIB/IL was slightly different, apparently having the first a better mechanical behavior in the end of the process. Temperatures for the first 2 hours of molding were also tested, room temperature and 4 C. showed weaker gels that disintegrate during the washing step (FIG. 2) while at 20 C. the gels showed a better behavior maintaining the shape unbroken.

[0091] In an embodiment, in the washing step, using the Soxhlet installation and procedure (1 L solvent per day), the gels were cleaned within 3 days, while when just placed in 50 mL-flasks (50 mL solvent), it takes 8 days. Though, in 100 mL-flasks with agitation (150 rpm) the samples were clean of ionic liquid within 5 days, which make a good compromise between time and efficiency, especially in the optimization stage, since it is conceivable to clean several lots of samples (with 5 mL of solvent per gel) at once, using 100 mL per day (FIG. 3A).

[0092] In an embodiment, the SAIB/SF gels were produced and washed following the same previous procedure (FIG. 2) and the conductivity measurements were also performed (FIG. 3B), where the solvent B is a mixture of water:isopropanol (1:1) and C is a mixture of water:methanol (1:1) are the used washing solvents.

[0093] From FIG. 4, it is possible to observe the FTIR spectra of SAIB/Chitin mixture and Chitin samples, processed with [bmim][Ac] in water (A) and in water:isopropanol (1:1)(B).

[0094] In an embodiment, the FTIR spectra of [bmim][Ac] and the native chitin used in this disclosure was already reported. Comparing those, with the ones obtained for the produced scaffolds it is possible to observe that the absorption bands characteristic for [bmim][Ac] (1400 cm.sup.1, 1180 cm.sup.1 and 1020 cm.sup.1) are not present on the studied samples, appointing to a good removal of the ionic liquid. Concerning the native chitin spectra, it is possible to observe the most characteristic bands and easier to detect on the amide region (specifically, at 1655 cm.sup.1 and 1564 cm.sup.1). These peaks, together with one more vanished at around 1628 cm.sup.1, are assigned to the stretching of the CO groups bonded to NH groups of the adjacent chain, the stretching of the CO groups diverged by forming an additional hydrogen bond to the primary OH groups of the same chain, and NH deformations, respectively [4]. Concerning the pure SAIB spectra, a strong IR absorption denotes a relevant peak for SAIB identification, at 1744 cm.sup.1. The carbonyl (CO) absorption between 1690-1760 cm.sup.1 indicates either an aldehyde, ketone, carboxylic acid, ester, amide, anhydride or acyl halide. Concerning the SAIB structure, this strong band is relative to its ester carbonyl stretching absorption.

[0095] In an embodiment, comparing the scaffolds analyzed, Chitin/IL scaffolds prepared in (A) water or in (B) the mixture of water and isopropanol (1:1) (Chitin A and Chitin B, FIG. 4) are very similar. Though, the blends of SAIB/IL and Chitin/IL in both solvents are quite different regarding the chitin presence. In fact, in water seams that the SAIB bands are more intense, while in the mixture water:isopropanol the presence of chitin is more remarked. That could be explained by the SAIB good solubility in organic solvents. Thus, besides SAIB/Chitin A and SAIB/Chitin B being prepared with the same proportions of SAIB/IL and Chitin/IL, some of the SAIB in the solvent B was lost, while in the solvent A it remained and more intense peaks are perceived.

[0096] In an embodiment, the XRD patterns of the scaffolds are depicted in FIG. 5.

[0097] In an embodiment, FIG. 5, it can be observed the main characteristic peaks of the chitin crystalline phase [5] at 2=9.1, 19.3 and 26.4, associated to (020), (110) and (013) crystallographic planes, respectively. The peaks at 9.1 and 19.3 can be specifically endorsed to glucosamine sequences and N-acetyl-D-glucosamine monomers of chitin [4]. It can also be observed that the presence of SAIB in the scaffolds can affect the crystallinity. This effect is particularly observed in SAIB/Chitin A scaffold, where no SAIB has been lost to the solvent and therefore SAIB is more concentrated. This is in accordance with the literature, where SAIB is known as a monocrystalline ester, being thus anticipated a reduction on the crystallinity of the mixture.

[0098] In an embodiment, the morphology of the scaffolds (mixtures of chitin/IL or SF/IL and SAIB/IL and just chitin/IL), in solvents A, B and C (SAIB/chitin A, SAIB/chitin B, Chitin A and Chitin B or even SAIB/SF B and SAIB/SF C), was observed by SEM, for the surface (S) and also for the cross-sections (C/S) (FIGS. 6A and 6B).

[0099] In an embodiment, the obtained micrographs (FIGS. 6A-6B) suggest that the scaffolds with SAIB (A and B, FIG. 6A) are denser than the ones without it. Nonetheless, the sample SAIB/chitin B (B1 and B2, FIG. 6A), possibly due to the already referred slight loss of SAIB to the isopropanol, has some opening cracks as well as some fibrous regions (B1 2000, FIGS. 6A-6B). Moreover, the cross-section of the sample B1 shows a morphology that resembles the typical morphology of aerogels. The observed pore size of the samples also revealed the effect of both compounds. In fact, when SAIB is present bigger pore sizes are observed 8026/5419 m (length/width) for SAIB/Chitin A, 3623/2820 m but also another group of sizes 1.61.1/1.20.8 m (length/width) for SAIB/Chitin B. Nevertheless, for chitin in water (Chitin A) the bigger pores were observed 203135/10289 m (length/width), mainly due to the solvent used, water. When the solvent is the mixture water:isopropanol (1:1) smaller pores are obtained for chitin (Chitin B), as well as a better distribution, 0.55+0.29/0.400.23 m (length/width).

[0100] In an embodiment, both silk and SAIB/SF based scaffolds were successfully produced using the methodology applied to SAIB/chitin based materials. However, the solvents used in the gelation and IL removal were changed to promote the beta sheet formation into the materials. The SEM images of the cross sections of the scaffolds (FIG. 6B) indicated that both silk and SAIB/SF have an open structure. Differences in the morphology of the scaffolds were observed probably due to the partial dissolution of the SAIB during the processing.

[0101] In an embodiment, the swelling procedure for SAIB/chitin and SAIB/SF scaffolds was also performed at the timepoints of 15 min, 7 h and 24 h. FIGS. 7A and 7B shows the swelling ratio obtained for each sample. In an embodiment, the swelling studies on the scaffolds produced were performed, in PBS, to understand the water uptake ability. It is possible to observe a significant difference between the SAIB/Chitin and the Chitin. It is possible to observe that the presence of SAIB prevented the entrance of water on the structure (FIG. 7A).

[0102] In an embodiment, the water uptake of the SAIB/SF scaffolds (FIG. 7B) showed different swelling degree. In fact, these results could be related to 3-sheet crystalline content within the matrices, which may have a significant role in their stability when incubated in PBS. Once again, the SAIB presence showed to prevent the entrance of water resulting in the stabilization of the water uptake throughout time.

[0103] In an embodiment, pull away tests to Chitin and SAIB/Chitin gels (in water, solvent A) allowed us to state that the presence of SAIB contributes to an increase of adhesivity (4 times), Table 1. This feature could be useful to help bacterial cells for adhesion to biological surfaces and biofilm formation. The advances in biofilm formation knowledge, coupled with emerging engineered biomaterials, provide many potential platforms and strategies to prevent or significantly reduce biofilm infections.

[0104] In an embodiment, micro-CT analysis was used to obtain quantitative information of the 3D architecture of the chitin and chitin/SAIB based scaffolds (Table x2). Table 2 demonstrates that chitin scaffolds presented both high porosity and interconnectivity. Moreover, the values of porosity and interconnectivity of SAIB/chitin-based scaffolds were lower than chitin scaffolds. The differences on morphological features of SAIB/chitin-based scaffolds can be associated to partial dissolution of SAIB during their processing. The micro-CT morphometric analysis revealed a widespread pore size as shown in FIGS. 8A-8D. Overall, the morphological features of the developed scaffolds may help homogeneous cell distribution and transfer of nutrients effectively, as well as to facilitate cell growth within the 3D porous structures.

[0105] In an embodiment, to assess cytotoxicity of SAIB scaffolds by direct contact, hASCs were used to provide a more physiologically relevant environment. In FIGS. 9A-9C, it is depicted cell growth evaluated by metabolic activity determination using MTS assay, cell proliferation assessed by dsDNA quantification, and cell damage observed by F-actin staining, along the time of culture. In relation to cells growth (FIG. 9A), no statistical differences were observed along the time of culture between SAIB/Chitin A and Chitin A. But it was noticeable that cells' metabolic activity was lower after 24 h of culture when comparing with CTRL, reaching values similar after 48 h, but decreasing again to initial values after 72 h. In other hand, considering dsDNA quantification (FIG. 9B), it was observed an opposite trend. In fact, it is evident that cells' proliferation rates were higher after 24 h in contact with SAIB/Chitin A and Chitin A comparing with CTRL, but they decreased after 48 h. After 72 h of culture cells' proliferation increased and were similar to all conditions. These observations indicated that during 48 h cells were adapting to the scaffolds, which make them more metabolic active and less proliferative. Nevertheless, after 72 h of culture, cells were completely adapted, as demonstrated by the decrease on their metabolic activity and increase on proliferative rates. Additionally, F-actin staining (FIG. 9C) showed that the culturing of cells directly with each scaffold did not promote any visible cell damage, corroborating previous observations. Worth mentioning, as Ctrl+ was equal to zero it was withdrawn from the results to simplify its analysis, only CTRL was kept.

[0106] In an embodiment, chemical crosslinking on SAIB/chitin membranes was investigated as an approach to increase the stability of the blends which will imply in an improvement of their physical properties. Genipin, a natural crosslinker, was chosen to perform the crosslinking reactions on SAIB/chitin membranes. Genipin can react with amino, carboxyl and hydroxyl groups. In our work, we hypothesize that genipin will react with amino groups present into chitin. Both genipin crosslinked chitin and SAIB/chitin-based membranes were successfully prepared as shown in FIG. 10. The results showed that crosslinked membranes presented colour changes during the reaction, namely from slight yellowish color (chitin membrane) and brown colour (SAIB/chitin) to green, then to dark green.

[0107] In an embodiment, the membranes were almost clean after 3 days (FIG. 11), and a complete removal was achieved after 6 days. Considering that polymers like chitin contain amino groups that react with genipin in a moderate reaction, all reactions were performed for 24 hours at RT and 37 C. It seems that the extent of the crosslinking was affected by key factors such as genipin concentration (10 mM, 20 mM) and reaction temperature (RT, 37 C.), where crosslinked membranes prepared at 37 C. in both genipin concentrations could have a higher degree of crosslinking.

[0108] In an embodiment, the SEM images of the cross-sections of the membranes (FIG. 12) showed that they have a porous formation that could be interesting for tissue engineering applications. Therefore, it is reasonable to assume that genipin may serve as a crosslinker to produce porous crosslinked chitin and SAIB/chitin membranes. The present disclosure relates to an innovative technology by green processing SAIB. The produced 3D scaffolds were physicochemical and biologically characterized. By taking advantage of the properties of the used ionic liquid ([BMIM][Ac]) it was possible to achieve a good dissolution of SAIB, chitin and silk fibroin. It was possible to efficiently remove the ionic liquid. The presence of Chitin, Silk fibroin and SAIB is easily detected in the FTIR spectra obtained. Moreover, the aerogel morphology was possible to obtain in certain conditions. Furthermore, SAIB contributed to decrease the crystallinity of the scaffolds. It was found that in the biological outcomes of the SAIB/chitin scaffolds demonstrated herein reveals a positive effect of the structures over cell adhesion and proliferation.

[0109] These results clearly show the potential of the used approach. The shown characteristics prove the applicability of the products in skin and cartilage repair.

[0110] 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.

[0111] It will be appreciated by those of ordinary skill in the art that unless otherwise indicated herein, the particular sequence of steps described is illustrative only and can be varied without departing from the disclosure. Thus, unless otherwise stated the steps described are so unordered meaning that, when possible, the steps can be performed in any convenient or desirable order.

[0112] 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.

[0113] The above described embodiments are combinable.

[0114] The following claims further set out particular embodiments of the disclosure.

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