TECHNICAL FIELD

[0001] The present disclosure generally relates to biopolymer nanostructures, particularly to a method for stabilizing biopolymer nanostructures, and more particularly to a method for stabilizing protonated biopolymer nanostructures in an aqueous medium by water vapor treatment.

BACKGROUND ART

[0002] Biopolymer nanostructures may be synthesized using an acidic solution of a biopolymer. Acidic solvents may dissolve biopolymer by protonating the biopolymer and solubilize the biopolymer. However, there is a major obstacle in utilizing protonated biopolymer nanostructures in aqueous media. The protonated biopolymer nanostructures are not stable in aqueous environments and lose their structures upon exposure to water due to remaining acidic solvents and formation of biopolymer salts in their structures. Therefore, stabilization of the protonated biopolymer nanostructures is necessary for utilizing these structures in aqueous environments.

[0003] Conventional approaches for stabilization of the protonated biopolymer nanostructures are cross-linking and neutralization by alkali solutions as described in US 2014/0046236 Al and V. Sencadas et al. (Journal of polymer testing, 2012, volume 31, issue 8, pages 1062-1069). Chemical cross-linkers such as glutaraldehyde (GA) stabilize the biopolymer nanostructures by forming imine bonds between biopolymer molecules and GA molecules. However, cross-linking the biopolymer nanostructures may lead to structural deformation and makes the biopolymer nanostructures brittle. In addition, chemical crosslinking of the biopolymer nanostructures may increase a toxicity risk of the cross-linked biopolymer nanostructures in biological systems.

[0004] On the other hand, neutralization of the protonated biopolymer nanostructures by alkali solutions, such as NaOH, K2C03, Na2C03, and NH4 may lead to morphological changes and structural shrinkage which negatively affect the physicochemical properties of the biopolymer nanostructures. Moreover, in the neutralization process of the protonated biopolymer nanostructures by alkali solutions, the biopolymer nanostructures have to be submerged in a neutralizing solution. Consequently, any loaded component into the biopolymer nanostructures such as drugs and biological compounds may undesirably be released during the neutralization process.

[0005] Therefore, there is a need for an efficient, simple, and biocompatible method for fabricating stabilized biopolymer nanostructures which are stable in aqueous media. Moreover, there is a need for an efficient and cost-effective method for stabilizing protonated biopolymer nanostructures without any structural deformations and cellular toxicity.

SUMMARY OF THE DISCLOSURE

[0006] This summary is intended to provide an overview of the subject matter of this patent, and is not intended to identify essential elements or key elements of the subject matter, nor is it intended to be used to determine the scope of the claimed implementations. The proper scope of this patent may be ascertained from the claims set forth below in view of the detailed description below and the drawings.

[0007] In one general aspect, the present disclosure describes a method for fabricating stabilized biopolymer nanostructures in aqueous media. The method may include preparing a protonated biopolymer by mixing a biopolymer with an acidic solution, forming a protonated biopolymer nanostructure using the protonated biopolymer, and forming a stabilized biopolymer nanostructure by exposing the protonated biopolymer nanostructure to water vapor through passing water vapor at a temperature between 40 °C and 100 °C across the protonated biopolymer nanostructure for a time period between 30 minutes and 120 minutes.

[0008] The above general aspect may include one or more of the following features. In some exemplary embodiments, forming the protonated biopolymer may include forming the protonated biopolymer by mixing at least one of chitosan, collagen, gelatin, polyethylene oxide, polyvinyl alcohol, hyaluronic acid, and combinations thereof with the acidic solution. In some exemplary embodiments, forming the protonated biopolymer may include forming the protonated biopolymer by mixing polyethylene oxide (PEO) and chitosan with a weight ratio between about 1:4 w/w and about 5:5 w/w.

[0009] According to some exemplary embodiments, the acidic solution may include at least one of acetic acid, trifluoroacetic acid, lactic acid, citric acid, formic acid, malic acid, and combinations thereof. In some exemplary embodiments, the acidic solution may have a concentration between about 1% (w/w) and about 100% (w/w). In some exemplary embodiments, the water vapor may include at least one of vapor of distilled water and vapor of an aqueous buffer with a pH level of at least about 7.

[0010] According to some exemplary embodiments, the protonated biopolymer nanostructure may include at least one of nanofibers, nanoparticles, porous structures, and combinations thereof. In some exemplary embodiments, the method may further include drying the stabilized biopolymer nanostructure at room temperature. In an exemplary embodiment, the stabilized biopolymer nanostructure may be stable in an aqueous medium for at least 28 days.

[0011] According to some exemplary embodiments, fabricating the protonated biopolymer nanostructure using the protonated biopolymer may include fabricating the protonated biopolymer nanostructure using at least one of electro spinning, force spinning, freeze-drying, phase separation, and combinations thereof. In some exemplary embodiments, the stabilized biopolymer nanostructure may further include a bioactive agent. In an exemplary embodiment, the bioactive agent may include an antimicrobial agent, a therapeutic agent, a healing agent, a growth factor, a drug, and combinations thereof.

[0012] In another general aspect, the present disclosure describes a method for stabilizing protonated biopolymer nanostructures in an aqueous medium. The method may include neutralizing a protonated biopolymer nanostructure by passing water vapor at a temperature between 40 °C and 100 °C through the protonated biopolymer nanostructure for a time period between 30 minutes and 120 minutes.

[0013] In another general aspect, the present disclosure describes a method for fabricating stabilized chitosan nanostructures. The method may include preparing a plurality of protonated chitosan molecules by mixing chitosan with an acidic solution, forming a protonated chitosan nanostructure using the plurality of protonated chitosan molecules, and stabilizing the protonated chitosan nanostructure by exposing the protonated chitosan nanostructure to water vapor at a temperature between 40 °C and 100 °C for a time period between 30 minutes and 120 minutes.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The drawing figures depict one or more implementations in accord with the present teachings, by way of example only, not by way of limitation. In the figures, like reference numerals refer to the same or similar elements. [0015] FIG. 1 illustrates a method for stabilizing a biopolymer nanostructure in an aqueous medium, consistent with one or more exemplary embodiments of the present disclosure.

[0016] FIG. 2A illustrates a scanning electron microscopy (SEM) micrograph of untreated chitosan nanofibers, consistent with one or more exemplary embodiments of the present disclosure.

[0017] FIG. 2B illustrates a scanning electron microscopy (SEM) micrograph of chitosan nanofibers cross-linked by glutaraldehyde (GA), consistent with one or more exemplary embodiments of the present disclosure.

[0018] FIG. 2C illustrates an SEM micrograph of chitosan nanofibers treated with water vapor for a time period of about 30 minutes, consistent with one or more exemplary embodiments of the present disclosure.

[0019] FIG. 2D illustrates an SEM micrograph of chitosan nanofibers treated with water vapor for a time period of about 60 minutes, consistent with one or more exemplary embodiments of the present disclosure.

[0020] FIG. 2E illustrates an SEM micrograph of chitosan nanofibers treated with water vapor for a time period of about 120 minutes, consistent with one or more exemplary embodiments of the present disclosure.

[0021] FIG. 3A illustrates an SEM micrograph of untreated chitosan nanofibers after immersing in phosphate-buffered saline (PBS) for a time period of about 2 hours, consistent with one or more exemplary embodiments of the present disclosure.

[0022] FIG. 3B illustrates an SEM micrograph of chitosan nanofibers cross-linked by glutaraldehyde (GA) after immersing in phosphate-buffered saline (PBS) for a time period of about 2 hours, consistent with one or more exemplary embodiments of the present disclosure.

[0023] FIG. 3C illustrates SEM micrographs of water vapor-treated chitosan nanofibers after immersing in phosphate-buffered saline (PBS) for a time period of about 2 hours, consistent with one or more exemplary embodiments of the present disclosure.

[0024] FIG. 3D illustrates an SEM micrograph of water vapor-treated chitosan nanofibers after immersing in phosphate -buffered saline (PBS) for a time period of about 28 days, consistent with one or more exemplary embodiments of the present disclosure.

[0025] FIG. 4 illustrates Fourier-transform infrared (FTIR) spectra of chitosan powder, untreated chitosan nanofibers, and chitosan nanofibers treated with water vapor, consistent with one or more exemplary embodiments of the present disclosure. [0026] FIG. 5 illustrates strain-stress curves of untreated chitosan nanofibers, water vapor- treated chitosan nanofibers, and glutaraldehyde cross-linked chitosan nanofibers, consistent with one or more exemplary embodiments of the present disclosure.

[0027] FIG. 6 illustrates viability of cells cultured on untreated chitosan nanofibers, water vapor-treated chitosan nanofibers, and glutaraldehyde cross-linked chitosan nanofibers, consistent with one or more exemplary embodiments of the present disclosure.

[0028] FIG. 7 A illustrates morphology of a cell cultured on a glutaraldehyde cross-linked chitosan nanofibrous scaffold, consistent with one or more exemplary embodiments of the present disclosure.

[0029] FIG. 7B illustrates morphology of a cell cultured on a water vapor-treated chitosan nanofibrous scaffold, consistent with one or more exemplary embodiments of the present disclosure.

DESCRIPTION OF EMBODIMENTS

[0030] In the following detailed description, numerous specific details are set forth by way of examples in order to provide a thorough understanding of the relevant teachings. However, it should be apparent that the present teachings may be practiced without such details. In other instances, well known methods, procedures, components, and/or circuitry have been described at a relatively high-level, without detail, in order to avoid unnecessarily obscuring aspects of the present teachings.

[0031] Structural instability of protonated biopolymer nanostructures which are synthesized using acidic solutions of the biopolymers is a major obstacle for utilizing these protonated biopolymer nanostructures in aqueous media and limits their applications. Cross-linking and neutralization by alkali solution are known approaches for stabilizing the protonated biopolymer nanostructures. However, these conventional methods have several disadvantages, such as structural deformations, toxicity, complex process, and low efficiency.

[0032] In order to overcome above-mentioned shortcomings of the conventional methods, disclosed herein is a simple and feasible method for fabricating stabilized biopolymer nanostructures in aqueous media and an exemplary efficient and safe method for stabilizing protonated biopolymer nanostructures using water-vapor treatment. Utilization of water- vapor treatment may neutralize the protonated biopolymer nanostructure by deprotonating protonated biopolymer nanostructures which improve the structural consistency of the biopolymer nanostructures in contact with aqueous media.

[0033] FIG. 1 is a method 100 for fabricating stabilized biopolymer nanostructures in aqueous media. Method 100 may include preparing a protonated biopolymer by mixing a biopolymer with an acidic solution (step 102), forming a protonated biopolymer nanostructure using the protonated biopolymer (step 104), and forming a stabilized biopolymer nanostructure by exposing the protonated biopolymer nanostructure to water vapor through passing water vapor across the protonated biopolymer nanostructure (step 106).

[0034] Step 102 may include forming the protonated biopolymer by mixing the biopolymer with the acidic solution. In some exemplary implementations, forming the protonated biopolymer may include forming the protonated biopolymer by mixing at least one of chitosan, collagen, gelatin, polyethylene oxide, polyvinyl alcohol, hyaluronic acid, and combinations thereof with the acidic solution.

[0035] In some exemplary embodiments, forming the protonated biopolymer may include forming the protonated biopolymer by mixing polyethylene oxide (PEO) and chitosan with a weight ratio between about 1:4 w/w and about 5:5 w/w. In one or more exemplary embodiments, the acidic solution may include at least one of acetic acid, trifluoroacetic acid, lactic acid, citric acid, formic acid, malic acid, and combinations thereof. In some exemplary embodiments, the acidic solution may have a concentration between about 1% (w/w) and about 100% (w/w).

[0036] Step 104 may include forming the protonated biopolymer nanostructure using the protonated biopolymer. In some exemplary embodiments, the protonated biopolymer nanostructure may include at least one of nanofibers, nanoparticles, porous structures, and combinations thereof. In some exemplary embodiments, fabricating the protonated biopolymer nanostructure using the protonated biopolymer may include fabricating the protonated biopolymer nanostructure using at least one of electro spinning, force spinning, freeze-drying, phase separation, and combinations thereof.

[0037] Step 106 may include forming the stabilized biopolymer nanostructure by exposing the protonated biopolymer nanostructure to the water vapor through passing water vapor across the protonated biopolymer nanostructure. In some exemplary embodiments, exposing the protonated biopolymer nanostructure to water vapor may include passing water vapor at a temperature between 40 °C and 100 °C through the protonated biopolymer nanostructure for a time period between 30 minutes and 120 minutes. In some exemplary embodiments, the water vapor may include at least one of vapor of distilled water and vapor of an aqueous buffer with a pH level of at least about 7.

[0038] In some exemplary implementations, the method may further include drying the stabilized biopolymer nanostructure at room temperature. In an exemplary embodiment, the stabilized biopolymer nanostructure may be stable in an aqueous medium for at least about 28 days. In some exemplary embodiments, the stabilized biopolymer nanostructure may further include a bioactive agent, such as an antimicrobial agent, a therapeutic agent, a healing agent, a growth factor, a drug, and combinations thereof.

EXAMPLES

[0039] EXAMPLE 1: FABRICATING AN EXEMPLARY STABILIZED CHITOSAN NANOSTRUCTURE IN AQUEOUS MEDIA

[0040] In this example, an exemplary stabilized chitosan nanostructure was fabricated through the steps of preparing an acidic solution of chitosan/PEO, forming a protonated chitosan/PEO nanostructure using the acidic solution of chitosan/PEO, and stabilizing the protonated chitosan/PEO nanostructure by exposing the protonated chitosan nanostructure to water vapor.

[0041] At first, the acidic solution of chitosan/PEO was prepared as follows. A chitosan solution with a concentration of about 2.5 % w/w and a polyethylene oxide (PEO) solution with a concentration of about 2.5 % w/w was prepared by dissolving chitosan and PEO powder in diluted acetic acid with a concentration of about 80 %w/w and stirring for a time period of about 24 hour at room temperature to obtain homogeneous solutions. Then, the solutions of chitosan and PEO was mixed with a ratio of 4:1 (chitosan: PEO) to obtain the PEO/chitosan solution and stirred gently overnight at room temperature.

[0042] After that, the protonated chitosan (CS)/PEO nanostructure was formed by electro spinning the acidic solution of chitosan/PEO. The electro spinning processes were carried out using an electrospinning apparatus by filling the acidic solution of CS/PEO in a 10 ml syringe that was connected to an 18-gauge stainless steel needle. A syringe pump fed the solution to the needle tip at an injection rate of about 1.0 ml/hour. [0043] During electro spinning, the applied voltage was about 20 kV and the needle to collector distance was set at about 14 cm. The chitosan/PEO nanofibers were collected on an aluminum foil covered rotating cylindrical drum at a speed of about 500 round per minute (rpm). The chitosan/PEO nanofibers were removed from collector surface after 1 hour of spinning and preserved for further treatment and investigations.

[0044] In the last step, stabilized CS/PEO nanofibers were fabricated by stabilizing the protonated chitosan/PEO nanofibers through water vapor treatment. The as-spun CS/PEO nanofibers were exposed to water vapor in a cylindrical glass chamber. The glass chamber was filled with 10 ml deionized (DI) water at a pH level of about 7, and the as-spun CS/PEO nanofibers which had been cut into 6x4 cm ² slices were placed in the chamber above the preheated DI water.

[0045] After sealing of chamber with aluminum foil, it was put into a precision compact mechanical convection oven to achieve intended temperature. The temperature was set at 40 °C, 50 °C, 60 °C, and 70 °C for time durations of 30 minutes, 60 minutes, and 120 minutes. Water-vapor treatment of protonated CS/PEO nanofibers may lead to neutralization and deprotonation of the protonated CS/PEO nanofibers. After water-vapor treatment, the CS/PEO nanofibers preserve their structure after exposure to aqueous media.

[0046] Furthermore, in order to evaluate the efficiency of water vapor treatment in stabilizing the protonated CS/PEO nanofibers, the protonated CS/PEO nanofibers were stabilized by cross-linking. The protonated CS/PEO nanofibers were cross-linked in a bounded desiccator chamber containing 20 mL of glutaraldehyde (GA) liquid. Then, the protonated CS/PEO nanofibers were put at the top of the GA for at least 24 hours and the CS/PEO nanofibers were cross-linked as a result of contacting with vaporized GA at room temperature.

[0047] FIG. 2A illustrates a scanning electron microscopy (SEM) micrograph of untreated chitosan/PEO nanofibers, consistent with one or more exemplary embodiments of the present disclosure. Referring to FIG. 2A, the SEM micrograph of untreated chitosan/PEO nanofibers showed randomly oriented nanofibrous structure without any beads and with a fiber mean diameter of about 210+42 nm. Also, the untreated CS/PEO nanofibers had a white color.

[0048] FIG. 2B illustrates an SEM micrograph of chitosan/PEO nanofibers cross-linked by glutaraldehyde (GA), consistent with one or more exemplary embodiments of the present disclosure. Referring to FIG. 2B, the total fibrous structure of CS/PEO nanofibers was preserved after cross-linking by GA. However, the average diameter of CS/PEO nanofibers was increased to 269+39.8 nm after cross-linking. Also, the white color of untreated CS/PEO nanofibers had been changed to yellow after cross-linking by GA.

[0049] FIG. 2C illustrates an SEM micrograph of chitosan nanofibers treated with water vapor at a temperature of about 60 °C for a time period of about 30 minutes, consistent with one or more exemplary embodiments of the present disclosure. FIG. 2D illustrates an SEM micrograph of chitosan nanofibers treated with water vapor at a temperature of about 60 °C for a time period of about 60 minutes, consistent with one or more exemplary embodiments of the present disclosure. FIG. 2E illustrates an SEM micrograph of chitosan nanofibers treated with water vapor at a temperature of about 60 °C for a time period of about 120 minutes, consistent with one or more exemplary embodiments of the present disclosure.

[0050] Referring to FIGs. 2C-2E, preservation of nanofibrous structure was also observed for water vapor-treated CS/PEO nanofibers at all temperature and incubation times. No considerable increase in average diameter of CS/PEO nanofibers was observed. Also, there was not any change in color for all water vapor-treated CS/PEO nanofibers in comparison with the untreated CS/PEO nanofibers.

[0051] EXAMPLE 2: STRUCTURAL STABILITY OF THE EXEMPLARY

STABILIZED CHITOSAN NANOSTRUCTURE

[0052] In this example, structural stability and morphology alteration of the exemplary stabilized chitosan nanostructure in an aqueous medium was investigated by SEM images of untreated chitosan nanofibers, GA-cross-linked chitosan nanofibers, and water vapor-treated chitosan nanofibers before and after immersing in PBS for 2 hours at room temperature.

[0053] FIG. 3A shows an SEM micrograph of untreated chitosan nanofibers after immersing in phosphate-buffered saline (PBS) for a time period of about 2 hours, consistent with one or more exemplary embodiments of the present disclosure. Referring to FIG. 3A, the SEM image revealed that untreated chitosan nanofibers completely lost their fibrous structure in contact with the aqueous medium. Destruction of the fibrous structure of untreated chitosan nanofibers after exposure to the aqueous medium may be due to the dissolving of formed chitosan acetate salts and remnant acetic acid in the chitosan nanofibers.

[0054] FIG. 3B shows an SEM micrograph of chitosan nanofibers cross-linked by glutaraldehyde (GA) after immersing in phosphate-buffered saline (PBS) for a time period of about 2 hours, consistent with one or more exemplary embodiments of the present disclosure. Referring to FIG. 3B, GA-cross-linked chitosan nanofibers showed complete structural stability after immersing in PBS for 2 hours.

[0055] FIG. 3C shows SEM micrographs of different water vapor-treated chitosan nanofibers after immersing in PBS for a time period of about 2 hours, consistent with one or more exemplary embodiments of the present disclosure. Referring to FIG. 3C, the nanofibrous structure of the water vapor-treated chitosan nanofibers were preserved after immersing in PBS for 2 hours.

[0056] FIG. 3D shows an SEM micrograph of water vapor-treated chitosan nanofibers after immersing in phosphate-buffered saline (PBS) for a time period of about 28 days, consistent with one or more exemplary embodiments of the present disclosure. Referring to FIG. 3D, the nanofibrous structure of the water vapor-treated chitosan nanofibers were preserved after immersing in PBS for 28 hours. The structure of water vapor-treated chitosan nanofibers remained to a large extent even after prolonged submersion in PBS.

[0057] EXAMPLE 3: INFRARED SPECTROSCOPY OF THE EXEMPLARY STABILIZED CHITOSAN NANOSTRUCTURE

[0058] In this example, the exemplary stabilized chitosan (CS) nanostructure was characterized using Fourier-transform infrared (FTIR) spectroscopy at wavelengths from 400 cm ^-1 to 4000 cm ^-1 for 30 times by an FTIR spectroscope with a 2 cm ^-1 spectral resolution. FIG. 4 shows FTIR spectra of chitosan powder 400, untreated chitosan nanofibers 404, and treated chitosan nanofibers 402 with water vapor at a temperature of about 60 °C for 1 hour, consistent with one or more exemplary embodiments of the present disclosure.

[0059] Referring to FIG. 4, all spectra exhibited peaks at wavelengths between about 1000 cm ¹ and about 1150 cm ¹ and at a wavelength of about 897 cm ¹ which are assigned to the saccharine structure of chitosan. Spectra of treated chitosan nanofibers 402 and untreated chitosan nanofibers 404 chitosan mats showed a peak at a wavelength of about 1623 cm ¹ which can be assigned to carbonyl stretching of the amide (I) in the -CONH- group. Spectrum of chitosan powder 400 showed a peak around a wavelength of about 1607 cm ¹ which can be attributed to N-H deformation of amine groups of chitosan. This peak may have overlapped with the peak of amid (I).

[0060] Referring again to FIG. 4, spectrum of untreated chitosan nanofibers 404 showed the amide II peak around a wavelength of about 1570 cm ¹ which indicates that synthesizing chitosan nanofibers using acetic acid could result in chitosan nanofibers in which chitosan was changed to chitosan acetate. However, the amide II peak around a wavelength of about 1570 cm ¹ were absent in spectrum of treated chitosan nanofibers 402 which indicates that the acetate salt of chitosan was disappeared after water vapor treatment of chitosan nanofibers. Therefore, water vapor treatment converted chitosan acetate molecules to chitosan molecules with free amine groups.

[0061] EXAMPLE 4: MECHANICAL CHARACTERISTICS OF THE EXEMPLARY STABILIZED CHITOSAN NANOSTRUCTURE

[0062] In this example, mechanical characteristics of the exemplary stabilized chitosan nanostructure, such as stress at break point, the percentage of elongation at break (eb), and elastic Young's modulus (E) were investigated by uniaxial mechanical testing at a crosshead speed of about 10 mm/minute. The test was performed with an applied load cell of about 50 N at room temperature. Prior to the test, each sample was cut in the rectangular form of 3x1 cm following a cardboard pattern of the same dimensions and the test was repeated 5 times for each sample.

[0063] FIG. 5 shows strain-stress curves of untreated chitosan nanofibers 500, glutaraldehyde (GA)-cross-linked chitosan nanofibers (GAC) 502, and water vapor-treated chitosan nanofibers (CS60) 504 at a temperature of about 60 °C for 1 hour, consistent with one or more exemplary embodiments of the present disclosure.

[0064] TABLE. 1 shows the mechanical properties such as stress at break point, the percentage of elongation at break (eb), and elastic Young's modulus (E), ultimate tensile strength (UTS) of different chitosan nanofibers which were obtained according to the stress- strain curves.

[0065] TABLE. 1 : Mechanical properties of different chitosan nanofibers

Sample Elastic modulus UTS Elongation at

(MPa) (MPa) break (%)

Untreated chitosan nanofibers 65+11 6.15+1.59 16.93+2.6

Water vapor-treated chitosan 120+23 10.61+1.89 9.2+1.41

nanofibers

GA-cross-linked chitosan 40+4 1.27+0.35 3.64+0.88 nanofibers [0066] Referring to FIG. 5 and TABLE. 1, the GA-cross-linked chitosan nanofibers 502 revealed the lowest elongation at break in comparison with untreated chitosan nanofibers 500 and water vapor-treated chitosan nanofibers 504 which is related to fragileness of chitosan nanofibers after crosslinking with GA. In addition, the ultimate tensile strength (UTS) of GA- cross-linked CS nanofibers 502 was 1.3 MPa which was considerably less than untreated CS nanofibers 500 which was about 6.15 MPa.

[0067] Referring again to FIG. 5 and TABLE. 1, the elastic module of GA-cross-linked chitosan nanofibers 502 was about 40 MPa, which was extremely lower than untreated chitosan nanofibers 500 and water vapor-treated chitosan nanofibers 504. Moreover, in contrast to GA-cross-linked chitosan nanofibers 502, the ultimate tensile strength (UTS) and elastic module of water vapor-treated chitosan nanofibers 504 were higher than untreated CS nanofibers 500 due to water vapor treatment.

[0068] EXAMPLE 5: BIOCOMPATIBILITY OF THE EXEMPLARY STABILIZED CHITOSAN NANOSTRUCTURE

[0069] One of the major applications of biopolymer nanofibers is a scaffold of cells for tissue engineering. In this example, biocompatibility of the exemplary stabilized chitosan nanostructure was studied using a 2,5-diphenyltetrazolium bromide (MTT) proliferation assay. Normal human dermal fibroblast (NHDF) cell line was used in this study. NHDF is a non-malignant adherent cell line which can be highly reproducible and easily interacted with various types of biomaterials.

[0070] At first, NHDF cells were cultured in DMEM/ F-12 Ham medium modified with fetal bovine serum (FBS) at a volume ratio of about 10% and penicillin/streptomycin (50 U/ml penicillin and 50 pg/ml streptomycin) at a volume ratio of about 1%. Cultured cells were incubated in standard conditions at a temperature of about 37 °C temperature and a humidity of about 5% and they were sub-cultured every 2 or 3 days.

[0071] After that, chitosan nanofibrous scaffolds were produced from different chitosan nanofibers and were placed under ultraviolet (UV) irradiation for at least lhour to be sterilized prior to use in in-vitro studies. Moreover, the chitosan nanofibrous scaffolds were washed in culture media of DMEM/ F-12 Ham medium before any in-vitro test. After that, the cells were cultured on the chitosan nanofibrous scaffolds and MTT proliferation assay was conducted to determine the viability of NHDF cells cultured on the chitosan nanofibrous scaffolds. [0072] FIG. 6 shows viability of cells cultured on untreated chitosan nanofibrous scaffold (control), water vapor-treated chitosan nanofibrous scaffold at a temperature of about 60 °C for 1 hour (CS60), and glutaraldehyde (GA) cross-linked chitosan nanofibrous scaffold (GAC) at different incubation times, consistent with one or more exemplary embodiments of the present disclosure. Referring to FIG. 6, no significant discrepancy was observed in the proliferation of cultured cells on water vapor-treated chitosan nanofibrous scaffold (CS60) and GA-cross -linked nanofibrous scaffold (GAC) in comparison with untreated chitosan nanofibrous scaffold (control) in the first day of incubation.

[0073] Although the proliferation of cells was considerably enhanced in the second and fourth day of incubation in all scaffolds, the GA-cross-linked nanofibrous scaffold (GAC) showed significantly lower cellular viability in comparison with the untreated chitosan nanofibrous scaffold (control) and water vapor-treated chitosan nanofibrous scaffold (CS60). Crosslinking by GA may cause some cytotoxic effects because of the unreacted aldehyde groups that were remained within the structure of cross-linked chitosan nanofibrous scaffolds.

[0074] In addition, during the crosslinking of CS nanofibers, free amine (NH2) groups would be occupied which contributes to less cell adhesion and growth. The intrinsic cytotoxicity of aldehyde residues that were incorporated into the GA-cross-linked chitosan nanofibrous scaffold as well as lacking free amine group on the surface of the chitosan nanofibers fibers hinders proliferation of the cells on GAC scaffolds in a long-term period, for example, 4 days.

[0075] EXAMPLE 6: CELL ADHESION ASSAY OF THE EXEMPLARY STABILIZED CHITOSAN NANOSTRUCTURE

[0076] In this example, cell adhesion assay of the exemplary stabilized chitosan nanofibrous scaffold was conducted by studying morphology, adhesion, and spreading of cultured cells on different chitosan nanofibrous scaffold. At first, water vapor-treated chitosan nanofibrous scaffold and GA-cross-linked chitosan nanofibrous scaffold were cut into pieces with a size of about 0.8 cmx0.8 cm and sterilized under UV light for lhour. The scaffold pieces were washed by culture medium and stuck on the bottom of wells of a 48-well plate. Then, about 2xl0 ⁴ cells per well in a total volume of 300 pl were cultured on the surface of scaffolds. Cultured cells were incubated at a temperature of about 37°C and a humidity of about 5%.

[0077] After 6 hours of incubation, the supernatant of each well was taken out and the scaffolds were mildly washed with phosphate-buffered saline (PBS). Afterward, adherent cells on chitosan nanofibrous scaffold were fixed by glutaraldehyde (GA) grade I with a concentration of about 2.5% (w/w) in PBS at room temperature for 1 hour. The fixation was followed by removing GA solution and rinsing scaffolds by PBS for three times. In the final step, the chitosan nanofibrous scaffold was dehydrated by a series of ethanol solutions and dried at room temperature.

[0078] FIG. 7 A shows the morphology of a cell cultured on glutaraldehyde cross-linked chitosan nanofibrous scaffold, consistent with one or more exemplary embodiments of the present disclosure. FIG. 7B shows the morphology of a cell cultured on water vapor-treated chitosan nanofibrous scaffold, consistent with one or more exemplary embodiments of the present disclosure.

[0079] Referring to FIGs. 7A and 7B, cultured cell 700 on GA-cross-linked chitosan nanofibrous scaffold had spherical morphology with some small filopodia and without any considerable extension of cell mass which expresses at an early stage of cell adhesion. The occupation of free amine groups by GA is an inappropriate factor for adherence of the cells to GA-cross-linked chitosan nanofibrous scaffold. Contrariwise, an excellent cell-scaffold interaction was observed in SEM micrograph of water vapor-treated chitosan nanofibrous scaffold as shown in FIG. 7B. Cultured cell 702 was completely spread on the surface of chitosan nanofibrous scaffold and the dendritic entanglements were integrated with the adjacent chitosan nanofibers.

INDUSTRIAL APPLICABILITY

[0080] Applicants have found that the exemplary method for fabricating water-resistant biopolymer nanostructures and the exemplary method for stabilizing protonated biopolymer nanostructures in an aqueous medium of the present disclosure are particularly suited for industrial applications. By way of example, industrial applications may include biomedical industry for tissue engineering, producing transdermal patches, and drug delivery. Moreover, the exemplary methods of the present disclosure may be used for filtration of heavy metals.

[0081] While the foregoing has described what are considered to be the best mode and/or other examples, it is understood that various modifications may be made therein and that the subject matter disclosed herein may be implemented in various forms and examples, and that the teachings may be applied in numerous applications, only some of which have been described herein. It is intended by the following claims to claim any and all applications, modifications and variations that fall within the true scope of the present teachings.

[0082] Unless otherwise stated, all measurements, values, ratings, positions, magnitudes, sizes, and other specifications that are set forth in this specification, including in the claims that follow, are approximate, not exact. They are intended to have a reasonable range that is consistent with the functions to which they relate and with what is customary in the art to which they pertain.

[0083] The scope of protection is limited solely by the claims that now follow. That scope is intended and should be interpreted to be as broad as is consistent with the ordinary meaning of the language that is used in the claims when interpreted in light of this specification and the prosecution history that follows and to encompass all structural and functional equivalents. Notwithstanding, none of the claims are intended to embrace subject matter that fails to satisfy the requirement of Sections 101, 102, or 103 of the Patent Act, nor should they be interpreted in such a way. Any unintended embracement of such subject matter is hereby disclaimed.

[0084] Except as stated immediately above, nothing that has been stated or illustrated is intended or should be interpreted to cause a dedication of any component, step, feature, object, benefit, advantage, or equivalent to the public, regardless of whether it is or is not recited in the claims.

[0085] It will be understood that the terms and expressions used herein have the ordinary meaning as is accorded to such terms and expressions with respect to their corresponding respective areas of inquiry and study except where specific meanings have otherwise been set forth herein. Relational terms such as first and second and the like may be used solely to distinguish one entity or action from another without necessarily requiring or implying any actual such relationship or order between such entities or actions. The terms“comprises,” “comprising,” or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. An element proceeded by“a” or“an” does not, without further constraints, preclude the existence of additional identical elements in the process, method, article, or apparatus that comprises the element. [0086] The Abstract of the Disclosure is provided to allow the reader to quickly ascertain the nature of the technical disclosure. It is submitted with the understanding that it will not be used to interpret or limit the scope or meaning of the claims. In addition, in the foregoing Detailed Description, it can be seen that various features are grouped together in various implementations. This is for purposes of streamlining the disclosure, and is not to be interpreted as reflecting an intention that the claimed implementations require more features than are expressly recited in each claim. Rather, as the following claims reflect, inventive subject matter lies in less than all features of a single disclosed implementation. Thus, the following claims are hereby incorporated into the Detailed Description, with each claim standing on its own as a separately claimed subject matter.

[0087] While various implementations have been described, the description is intended to be exemplary, rather than limiting and it will be apparent to those of ordinary skill in the art that many more implementations and implementations are possible that are within the scope of the implementations. Although many possible combinations of features are shown in the accompanying figures and discussed in this detailed description, many other combinations of the disclosed features are possible. Any feature of any implementation may be used in combination with or substituted for any other feature or element in any other implementation unless specifically restricted. Therefore, it will be understood that any of the features shown and/or discussed in the present disclosure may be implemented together in any suitable combination. Accordingly, the implementations are not to be restricted except in light of the attached claims and their equivalents. Also, various modifications and changes may be made within the scope of the attached claims.