CZ20145A3 - Composition for preparation of modified gelatin nanofibres, nanofibers and their preparation - Google Patents

Abstract

The present invention provides a composition for preparing oxidized cellulose-containing gelatin nanofibers comprising gelatin and oxidized cellulose and / or an alkali metal, alkaline earth metal or ammonium oxidized cellulose salt, wherein the weight ratio of oxidized cellulose salt or oxidized cellulose to gelatin is in the range 0.1 to 2, and optionally further comprises a solvent selected from the group consisting of formic acid, acetic acid, and mixtures thereof. Further, the solution describes the method of preparation of nanofibres by spinning this composition and the nanofibres thus prepared.

Description

Composition for the preparation of modified gelatin nanofibers, nanofibers and method for their preparation

Technical area

The subject of the invention are antibacterial gelatin nanofibers modified with oxidized cellulose suitable for use in regenerative medicine of soft tissues as antibacterial hemostatic agents, wound dressings for moist healing, or in tissue engineering for skin regeneration, as well as compositions for their preparation and a method of preparation.

State of the art

Gelatin is a protein mixture obtained by partial hydrolysis of the natural protein collagen. It is biocompatible, biodegradable and has high absorption capacity and hemostatic effects. It is used in the food industry, pharmacy, and biomedicine, where it serves as a structural material for cells in tissue engineering. In addition to fluorine-based organic solvents (2,2,2-trifluoroethanol [Z. M. Huang, Y. Z. Zhang, S. Ramakrishna, C. T. Lim, Electrospinning and mechanical characterization of gelatin nanofibers. Polymer 45 (2004) 5361-5368], 1,1,1,3,3,3-hexafluoro-2-propanol [S.-H. Gu, Z.-M. Wang, J. Ren, C.-Y. Zhang, Electrospinning of gelatin I poly (L-lactide) blend and its characteristics for wound dressing. Material Science and Engineering C 29 (2009) 1822-1828]), electrostatic spinning from acetic or formic acid is used to prepare gelatin nanofibers [Petráš D. et al.: Safe nanofibers. Chemické Listy, 103 (2009) 1009 - 1016] and also ethanol/phosphate buffer mixtures [Z. Zha, W. Tend, V. Markle, Z. Dai, X. Wu, Fabrication of gelatin nanofibrous scaffolds using ethanol/phosphate buffer saline as a benign solvent. Biopolymers 97 (2012) 1026-1036].

Cellulose is a chemically polydisperse linear biopolymer (polysaccharide) composed of poly-(1,4)-D-glucose units with an asyndiotactic configuration. In order to obtain cellulose nanofibers, it is necessary to select a suitable solvent or solvent mixtures. The structure of cellulose contains very strong intramolecular hydrogen bonds, which makes it insoluble in common solvents [V. Kumar, T. Yang, HNO3/H3PO4NaNCh mediated oxidation of cellulose - preparation and characterization of bioabsorbable oxidized cellulose in high yields and with different levels of oxidation. Carbohydrate

Polymers 48 (2002) 403-412], Solvents suitable for dissolving pure cellulose are not volatile, therefore it is necessary to provide special coagulation procedures during spinning that remove the solvent from the electrostatically spun fibers. These solvents include the mixture of Nmethyl-morpholine N-oxide/water (nNMMO / H2O) and the mixture of lithium chloride / dimethylacetamide (LiCl / DMAc) [Μ. V. Frey: Electrospinning cellulose and cellulose derivatives. Polymer Reviews, 48 (2008) 378-391]. Other spinning options are listed in the following table no. 1.

Table 1: Possibilities of electrostatic spinning of pure cellulose.

Solvent References nNMM0/H ₂ 0 M. V. Frey: Polymer Reviews, 48 (2008) 378-391 Z. H. Huang et al.: Carbon, 40 (8) (2002) 1363-1367 C.W. Kim. et al.: Polymer, 47 (2006) 5097-5107 P. Kulpinski: J. Appl. Polymer Sci., 98 (4) (2005) 1855-1859 M.S. Khil et al.: Macromolecular Research, 13 (2005) 62-67 nNNMO M.S. Khil et al.: Macromolecular Research, 13 (2005) 62-67 P. Kulpinski: J. Appl. Polymer Sci., 103(3) (2007) 1473-1482 Ethylenediamine (ED)/Potassium Thiocyanate (KSCN) P. Kulpinski: J. Appl. Polymer Sci., 103(3) (2007) 1473-1482 A. Frenot et al.: J. Appl. Polymer Sci., 98 (4) (2005) 1855-1859 (LiCl)/DMAc M. V. Frey: Polymer Reviews, 48 (2008) 378-391 C.W. Kim. et al.: Polymer, 47 (2006) 5097-5107 C.W. Kim et al.: J. Polymer Sci. Part B-Polymer Physics, 43 (13) (2005)1673-1683 Viswanathan G. et al.: Biomacromolecules, 7 (2) (2006) 415-418 C.W. Kim. et al.: J. Polymer Sci. Part B: Polymer Physics, 40 (18)(2002)2119-2129 1-Butyl-3-methylimidazolium chloride or 1-ethyl-3-methylimidazolium benzoate Z. H. Huang et al.: Carbon, 40 (8) (2002) 1363-1367 C.W. Kim. et al.: J. Polymer Sci. Part B: Polymer Physics, 40 (18)(2002)2119-2129

The following table (Table 2) shows the possibilities of electrostatic spinning of cellulose derivatives. Cellulose derivatives are much more soluble than cellulose itself.

Table 2: Possibilities of electrostatic spinning of various cellulose derivatives.

Cellulose derivative Solvent References Cellulose acetate Acetone/water WK Son et al.: J. Polym. Sci. Polym. Phys. 42(2004) 5-11 W.K. Son et al.: Biomacromolecules.5 (2004) 197-201 WK Son et al.: Macromol. Rapid Commun. 25 (2004) 1632-1637 WK Son et al.: Carbohyd. Polym. 65 (2006) 430^134 Cellulose acetate Acetone/DMAc H. Qi et al.: Biomacromolecules. 7 (2006) 2327-2330 H. Liu et al.: J. Polym. Sci. Polym. Phys. 41 (2003) 953-964 Y. Wang et al.: J. Polym. Sci. Polym. Chem. 42 (2004) 4289-4299 P. Taepaiboon et al.: Eur. J. Pharm. Biopharm. 67 (2007) 387-397 S. Tungprapa et al.: Polymer. 48 (2007) 5030-5041 0. Suwantong et al.: Polymer. 48 (2007) 7546-7557 0. Suwnatong et al.: Polymer. 49 (2008) 4239^1247 Cellulose acetate Acetone/dimethylformamide (DMF)/trifluoroethanol Z. Ma et al.: J. Membr. Sci. 319 (2008) 23-28 Cellulose acetate Acetic acid/water S. 0. Han et al.: Mater. Lett. 62 (2008) 23-28 Cellulose acetate Acetone, chloroform, DMAc, DMF/dichloromethane (DCM), formic acid, methanol, pyridine, water S. Tungprapa et al.: Cellulose 14 (2007) 563-575 Cellulose triacetate Methylene chloride/ethanol S.O. Han et al.: Mater. Lett. 59 (2005) 2998-3001 Ethyl cellulose Tetrahydrofuran (THF)/DMAc X. Wu et al.: J. Appl. Polym. Sci. 97 (2005) 1292-1297

J. Y. Park et al.: J. Ind. Eng. Chem. 13 (2007) 1002-1008 Ethyl-cyanoethyl cellulose THF S. Zhao et al.: J. Appl. Polym. Sci. 91 (2004) 242-246 S. Zhao et al.: Cellulose 10 (2003) 405- 409 Hydroxypropylcellulose Water/ethanol A. Frenot et al.: J. Appl. Polymer Sci., 98 (4)(2005) 1855-1859 Hydroxypropyl methyl cellulose phthalate Acetone/ethanol M. Wang et al.: J. Appl. Polym. Sci. 103 (2007) 2177-2184 Methyl cellulose Water/ethanol H. Liu et al.: J. Polym. Sci. Polym. Phys. 41 (2003) 953-964 Acetate cellulose/hydroxyapatite Acetic acid/acetone A. Bishop et al.: Polym. Adv. Technol. 17(2006) 902-906 Cellulose acetate/PEG DMAc/acetone C. Chen et al.: Polymer. 48 (2007) 5202- 5207 Cellulose acetate/PEO DMF, DMF/dioxane L. Chen et al.: Polymer. 49 (2008) 1266—1275 L. Zhang et al.: Carbohyd. Polym. 71 (2008) 196-207 Carboxymethyl cellulose/PEO H. Liu et al.: J. Polym. Sci. Polym. Phys. 41 (2003) 953-964

Among the cellulose derivatives, we can include, among others, oxidized cellulose. It is completely biocompatible, bioresorbable and biodegradable, non-immunogenic and commercially readily available. Its bactericidal effect against the bacteria Staphylococcus epidermidis, Escherichia coli, Staphylococcus aureus, Proteus vulgaris, Beta streptococcus, Pseudomonas aeruginosa, Streptococcus faecalis, Bacteroides fragilis, Klebsiella aerogenes and Clostridium perfringens has been proven [M. Hora, E. Eret, T. Úrge, J. Klečka, Possibilities of tissue adhesives in kidney-sparing surgery for kidney tumors, Czech Urology, 2007, 147-153; www.bloodness.wz.cz/cz/ot.hemost.koag.htm]. It is used in healthcare, where it is mainly used as a local hemostatic agent, in the cosmetic and pharmaceutical industries as an additive to various preparations [V. Kumar, T. Yang, HNOs/FFPCU-NaNCh mediated oxidation of cellulose - preparation and characterization of bioabsorbable oxidized cellulose in high yields and with different levels of oxidation Carbohydrate Polymers 48 (2002) 403

412]. The hemostatic effect of oxidized cellulose is manifested by the formation of a clot, which is absorbed in approximately 1 to 2 weeks [M. Hora et al.: Česká urologie, 2007, 147-153].

Oxidized cellulose has not been spun yet. There is also no data that would indicate that the prepared nanofibrous material contains oxidized cellulose as an additive. Most often, underivitized cellulose or a derivative such as cellulose acetate is used for spinning, which is oxidized after spinning to form oxidized cellulose. For example, ultrafine fibers of oxidized cellulose were prepared by the method described by Son, Youk and Park [Biomacromolecules 5 (2004) 197-201]. The authors use the spinning of cellulose acetate, which is then deacetylated to cellulose. The cellulose is subsequently oxidized using a mixture of HNO3/H3PO4-NaNO2. Khil et al. [Macromolecular Research, 13 (2005) 62-67] electrostatically spun cellulose from a mixture of NMMO/H2O solvents. The obtained nanofibers were oxidized at 25 °C using a mixture of perfluorinated carbon (FC-84) and NO2. Then, the nanofibers were immersed in a 2-propanol/water mixture.

The essence of the invention

The subject of the present invention is a composition for the preparation of gelatin nanofibers containing oxidized cellulose, comprising gelatin and oxidized cellulose and/or a salt of oxidized cellulose with an alkali metal, alkaline earth metal or ammonium, wherein the weight ratio of the salt of oxidized cellulose or oxidized cellulose to gelatin is in the range of 0.1 to 2, preferably 1.

The composition for gelatin nanofibers preferably further comprises a solvent selected from the group consisting of formic acid, acetic acid and mixtures thereof.

The subject of the present invention is also a method for preparing nanofibers consisting in preparing a solution by mixing gelatin and oxidized cellulose or its salt with an alkali metal, alkaline earth metal or ammonium in a solvent selected from the group comprising formic acid, acetic acid and mixtures thereof. This solution is mixed on a magnetic stirrer. In a preferred embodiment, it can also be exposed to ultrasound. Spinning is carried out at laboratory temperature by known methods, preferably using a spinning apparatus, e.g. Nanospider technology.

The present invention also provides gelatin nanofibers comprising oxidized cellulose and/or its salt with an alkali metal, alkaline earth metal or ammonium, which can be prepared by spinning according to the present invention. Nanofibers are fibers having dimensions in the range of nanometers to hundreds of nanometers, i.e. preferably in the range of 1 nanometer to 1 micrometer. These nanofibers have significant bactericidal effects.

Description of the image

Fig. 1: ATR-FTIR spectrum of a spun sample according to the exemplary embodiment.

Fig. 2a: SEM image of sodium salt of oxidized cellulose in gelatin nanofibers according to the exemplary embodiment.

Fig. 2b: SEM image of the color elemental resolution of the obtained material according to the embodiment. Yellow is sodium (Na), green is nitrogen (N), blue is oxygen (O) and red is carbon (C).

Fig. 3: EDX spectrum of oxidized cellulose in gelatin nanofibers according to the exemplary embodiment.

Fig. 4: Bactericidal bioluminescence of a sample according to an exemplary embodiment.

Example of embodiment of the invention

20 ml of acetic acid (p.a., Penta), 8.3 wt. % gelatin (Sigma Aldrich from cowhide, type B) and 8.3 wt. % sodium oxidized cellulose (Syntesia, a. s., Czech Republic) were mixed. The mixture was then stirred on a magnetic stirrer for 19 hours. The mixture was left for 15 min in ultrasound. Spinning was carried out at ambient temperature (21.5 °C), at 60% relative humidity and a pressure of 97 kPa using a Nanospider Elmarco NSLAB 500 spinning apparatus.

The following table (Table 3) lists the spinning conditions.

Table 3: Experimental conditions for electrostatic spinning of the sample.

d [mm] U[kV] Ι[μΑ] Rotation [rpm] 150 47.9 3 5.5

d - electrode distance; U - applied voltage; I - passing current; rotation - speed of rotation of the electrode around its axis

The obtained nanofibers were studied using infrared spectroscopy (ATR-FTIR), see Fig. 1, the structure and morphology of the fibers were monitored using a scanning electron microscope (SEM), see Figures 2a and 2b, and elemental analysis (EDX), see Fig. 3. The bactericidal effects of the fibers were tested by direct inhibition of the growth of bioluminescent bacteria. ATR-FTIR analysis demonstrated the presence of an absorption band of sodium oxidized cellulose in the material. The spectrum cutout shows that the deposited nanofibers are a superposition of the spectra of gelatin itself and sodium oxidized cellulose. The absorption band in the region around 1400 cm' ¹ belongs to the vibration of the carboxyl group bound to carbon C6 of the pyranose ring of oxidized cellulose.

SEM analysis confirmed the obtaining of disordered gelatin nanofibers with a diameter of 262 ± 34 nanometers with homogeneously distributed oxidized cellulose, as demonstrated by EDX elemental analysis.

EDX analysis also confirmed the homogeneous, uniform distribution of Na ⁺ ions throughout the entire volume of the nanofibrous material, see Fig. 3.

The bactericidal activity of the obtained nanofibers was tested luminometrically. This method monitors the inhibition of bioluminescence during bacterial growth of plasmid-modified E. coli K12 bacteria.

In Fig. 4, the black solid line shows the control viability of the modified E. coli strain. The dotted curve corresponds to the viability of E. coli in the presence of gelatin alone, and the dashed line corresponds to the viability of E. coli in the presence of gelatin nanofibers with the addition of sodium salt of oxidized cellulose. It can be seen from Fig. 4 that the newly prepared material shows a permanent almost 90% inhibition of bacterial growth throughout the entire testing period.

Claims (4)

PATENT CLAIMS 1. A composition for the preparation of gelatin nanofibers containing oxidized cellulose, characterized in that it contains gelatin and oxidized cellulose and/or a salt of oxidized cellulose with an alkali metal, alkaline earth metal or ammonium, wherein the weight ratio of the salt of oxidized cellulose or oxidized cellulose to gelatin is in the range of 0.1 to 2, preferably 1. 2. The composition of claim 1, further comprising a solvent selected from the group consisting of formic acid, acetic acid, and mixtures thereof. 3. A method for preparing gelatin nanofibers containing oxidized cellulose, characterized in that a solution is prepared by mixing gelatin and oxidized cellulose or its salt with an alkali metal, alkaline earth metal or ammonium in a solvent selected from the group consisting of formic acid, acetic acid and mixtures thereof, and this solution is spun. 4. Gelatin nanofibers containing oxidized cellulose, characterized in that they contain gelatin and/or its salt with an alkali metal, alkaline earth metal or ammonium, and are preparable by the method according to claim 3.