Biotechnologically-produced cellulose-containing article for dermatological use

Abstract

A cellulose-containing article for treating an area of skin, wherein the article comprises BNC in an amount of at least 1% by weight and at most 15% by weight, comprises fluid in an amount of at least 85% by weight and at most 99% by weight, has an average thickness of at least 0.5 mm and at most 8 mm, wherein the BNC is of microbial origin.

Claims

1. A method of manufacturing an article containing biotechnologically produced nanostructured cellulose (BNC), comprising: providing a BNC non-woven in a continuous semi-static process, including producing BNC from a bacterial culture in a cultivation medium, and providing the article, the article comprising BNC of microbial origin in an amount of at least 1% by weight and at most 15% by weight; and fluid in an amount of at least 85% by weight and at most 99% by weight; and wherein the article comprises carbonyl groups in an amount of less than 8.0 μmol/g.

2. The method according to claim 1, wherein the fluid comprises water.

3. The method according to claim 1, comprising the step of: adding cultivation medium and/or constituents of the cultivation medium during bacterial culture.

4. The method according to claim 1, comprising the step of: harvesting or removing BNC non-woven from the bacterial culture during BNC synthesis.

5. The method according to claim 4, wherein harvesting or removing BNC non-woven is done stepwise or continuously.

6. The method according to claim 1, wherein providing the BNC non-woven comprises, providing a reaction vessel comprising cultivation medium; inoculating the cultivation medium with a BNC-producing bacterial strain; and bacterial synthesis of BNC in the reaction vessel.

7. The method according to claim 1, wherein a cell count is from 10.sup.4 to 10.sup.7 cells/ml of cultivation medium during the culture.

8. The method according to claim 1, wherein the cultivation medium comprises a carbon source, a nitrogen source and/or a vitamin source.

9. The method according to claim 1, wherein the cultivation medium comprises a buffer.

10. The method according to claim 8, wherein the cultivation medium comprises the carbon source in an amount of at least 10 g/l.

11. The method according to claim 8, wherein the cultivation medium comprises the nitrogen source in an amount of at least 2 g/l.

12. The method according to claim 8, wherein the cultivation medium comprises the vitamin source in an amount of at least 2 g/l.

13. The method according to claim 1, wherein a cultivation temperature is at least 20° C. and/or at most 36° C.

14. The method according to claim 1, wherein a cultivation time is at least 1 day.

15. The method according to claim 1, wherein a culture volume is at least 10,000 ml.

16. The method according to claim 1, wherein a synthesis area is at least 10 m.sup.2.

17. The method according to claim 1, wherein a weight average molecular weight of the BNC is at most 1,000,000 g/mol.

18. The method according to claim 1, wherein providing the article includes adapting the article's shape to an area of skin, including the face, a part of the face, the mouth, forehead, or the eyes.

19. The method according to claim 1, wherein providing the article includes cutting the non-woven to the desired shape.

20. The method according to claim 19, wherein cutting is performed with a fluid jet.

21. The method according to claim 20, wherein the fluid jet has a diameter of from 100 to 300 μm.

22. The method according to claim 1, comprising sterilizing the article.

23. The method according to claim 22, wherein sterilizing is e-beam sterilization.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The present invention will be described in further detail with reference to the drawings from which further features, embodiments and advantages may be taken, and in which:

(2) FIGS. 1A to 1G illustrate cutting patterns of cellulose-containing articles according to the present invention. Particularly, FIGS. 1A and 1B show the cutting pattern of an inventive cellulose-containing articles adapted to be applied on the face (face mask); FIG. 1C shows the cutting pattern of an inventive cellulose-containing article adapted to be applied on or around the mouth (mouth mask); FIG. 1D shows the cutting pattern of an inventive cellulose-containing article adapted to be applied on the forehead (forehead mask); FIG. 1E shows the cutting pattern of an inventive cellulose-containing article in the form of a 10 cm×10 cm overlay; FIG. 1F shows the cutting pattern of an inventive cellulose-containing article adapted to be applied on the eyes (eye mask); and FIG. 1G shows the cutting pattern of an inventive cellulose-containing article adapted to be applied on single eyes (eye pads).

(3) The face mask as shown in FIGS. 1A and 1B may particularly be provided with an average thickness of about 2 mm or with an average thickness of about 0.8 to 1 mm. The face mask may have a width of 185 mm and a height of 175 mm. Alternatively, the face mask may be provided with a width of 240 mm and a height of 175 mm.

(4) The mouth mask as shown in FIG. 1C may particularly be provided with an average thickness of about 1 mm. Alternatively the mouth mask may be provided with an average thickness of about 2 mm. The mouth mask may have a width of 125 mm and a height of 85 mm.

(5) The forehead mask as shown in FIG. 1D may particularly be provided with an average thickness of about 1 mm. Alternatively the forehead mask may be provided with an average thickness of about 2 mm. The forehead mask may have a width of 190 mm and a height of 60 mm.

(6) The overlay as shown in FIG. 1E may particularly be provided with an average thickness of about 2 mm. Alternatively the overlay may be provided with an average thickness of about 0.8 to 1 mm. The overlay may have a width of 100 mm and a height of 100 mm.

(7) The eye mask as shown in FIG. 1F may particularly be provided with an average thickness of about 2 mm. Alternatively the eye mask may be provided with an average thickness of about 1 mm. The eye mask may have a width of 191 mm and a height of 60 mm.

(8) The eye pads as shown in FIG. 1G may particularly be provided with an average thickness of about 1.0 to 2.0 mm. The eye pads may have each a width of 75 mm and a height of 35 mm.

(9) FIG. 2 shows the CP/MAS 13C-spectra of cellulose of the strain ATCC 11142 (upper panel) and of the strain DSM 14666 (middle panel) manufactured using a continuous semi-static cellulose manufacturing process in comparison to the comparative example (lower panel).

DETAILED DESCRIPTION OF THE INVENTION

Example 1

(10) The cellulose-containing article according to the present invention and manufactured using a semi-static continuous process was compared with regard to its material characteristics to a cellulose-containing article manufactured using a static and discontinuous culture (comparative example).

(11) Molecular Structure

(12) The molecular structure of the article according to the present invention was determined using gel permeation chromatography (GPC).

(13) For this purpose cellulose of the strain ATCC 11142 was used. The comparative example was produced using a static cellulose manufacturing process. The article according to the present invention was produced using a continuous semi-static cellulose manufacturing process.

(14) The GPC-measurement was performed using 0.9% (m/v) LiCl/DMAc-solution according to Röder et al (RÖDER T, MORGENSTERN B, SCHELOSKY N, GATTER O: Solutions of cellulose in N,N-dimethylacetamide/LiCl by light scattering methods. Polymer (2001), 42/16, 6765-73.) with dissolved dry-frozen BC samples and was performed using four serial GPC-columns (PL Gel ALS, 20 μm, 7.5×300 mm) and using three detectors (Fluorescence, MALLS and Refraction index). An 0.9% (m/v) LiCl/DMAc-solution was used as the eluent. Filtration was performed using an 0.02 μm-filter. The flow rate was 1 ml/min, the injection volume was 100 μl and the running time was 45 min. For labeling of the carbonyl groups a fluorescence marker was used and measured fluorometrically. For evaluation the program CS53_76-79 according to Röhrling et al. (RÖHRLING J, POI I HAST A, ROSENAU T, LANGE T, EBNER G, SIXTA H, KOS-MA P A: Novel method for the determination of carbonyl groups in cellulosics by fluorescence labelling. 1. Method development. Biomacromolecules (2002), 3, 959-68) was used.

(15) Table 1 shows the results. Particularly, the molecular characteristics are shown in table 1 of an article according to batches of articles according to the present invention (sample 1 and sample 2), and one comparative example.

(16) TABLE-US-00001 TABLE 1 Car- Carbon- bonyl- ylend- Mn DP.sub.n M.sub.w PDI groups groups [g/mol] [M.sub.n/M.sub.0] [g/mol] [M.sub.w/M.sub.n] [μmol/g] [μmol/g] compar- 308300 1902 1069000 3.5 8.50 3.24 ative example sample 1 305200 1882  775800 2.5 5.61 3.28 sample 2 355900 2195  627100 1.8 3.46 2.80

(17) Thereby, the amount of carbonyl-end-groups was determined based on DPn.

(18) The present inventors have surprisingly found that the length of the cellulose chains of samples 1 and 2, manufactured using a continuous semi-static process are more uniform than the cellulose chains of the comparative example, manufactured with a static culturing. This is particularly reflected by the relatively low PDI-value of sample 1 and sample 2. The comparative example, however, comprises a mixture of long- and short chains of cellulose. This also explains the differences shown between the theoretical and the experimentally determined value of the amounts of carbonyl groups

(19) Near Structure

(20) The near structure of the article according to the present invention was determined using NMR.

(21) For this purpose cellulose of the strain ATCC 11142 and of the strain DSM 14666 was used. The comparative example was produced as a wet fleece using a static cellulose manufacturing process. The article according to the present invention was produced as a wet fleece using a continuous semi-static cellulose manufacturing process.

(22) For determination of cellulose lα and lβ as well as the crystallinity of never-dried BC-samples solid-state-13C-NMR-spectroscopy was performed. The 13C-CP-MAS having a TPPM decoupling (4 mm High-Kopf) was performed using a 400 MHz-Avance II-Spectrometer of Bruker, at a static magnetic field of 9,4 T. The rotational frequency in the measurement of the sample was 5 kHz and the relaxation time (the time between the scans) was 2 seconds.

(23) FIG. 2 shows the CP/MAS 13C-spectra of cellulose of the strain ATCC 11142 (upper panel) and of the strain DSM 14666 (middle panel) manufactured using a continuous semi-static cellulose manufacturing process in comparison to the comparative example (lower panel). It is immediately apparent therefrom that also in the CP/MAS 13C-spectra the samples from the continuous semi-static manufacturing method differ in the samples from the static manufacturing method. Table 2 below shows the content of cellulose lα and lβ, respectively, and the crystallinity Ic based on the CP/MAS 13C NMR.

(24) The results of the NMR experiment confirm the results achieved by the above-described GPC-analysis.

(25) TABLE-US-00002 TABLE 2 strain Iα [%] Iβ [%] Iα/Iβ Ic [%] DSM comp. example 43 20 2, 2 86 14666 inventive sample 40 17 2, 4 86 ATCC comp. example 35 15 2, 2 81 11142 inventive sample 34 14 2, 4 82

(26) Supra Molecular Structure

(27) The supra molecular structure was determined using REM in 2,000-fold magnification after labeling with leading carbon and subsequent gold sputtering. Electron-microscope Leica S440i, with tungsten cathode to maximum of 30 kV, scintillation-SE-detector, 4-quadrantfield semi-conductor RE-detector. The respective BNC fleeces of cellulose of the strain ATCC 11142 and of the strain DSM 14666 were freeze-dried and subsequently subjected to REM. It was found that independent of the utilized strain the supra molecular structure of the inventive samples (continuous semi-static cellulose manufacturing process) was indistinguishable from the supra molecular structure of the comparative samples (static cellulose manufacturing process).

(28) Surface Structure

(29) The surface structure of cellulose-containing articles is of importance, particularly if applied as wound dressing or cosmetic product. The surface structure was analyzed using laser scanning microscopy-(LSM). For this purpose BNC-fleeces of strains ATCC 11142 and DSM 14666, respectively, were produced in a static process and in a semi-static continuous process, respectively.

(30) Hot pressing of the samples was performed at 120° C. for 10 min (d≤50 μm) or 20 min (d≥50 μm) using Yellow Press 4050/Schulze Thermal Transfer Press.

(31) LSM-pictures of the upper surfaces and the lower surfaces of wet fleeces of the bacterial strain ATCC 11142 produced in a semi-static continuous process and in a static process revealed that the surface structure of fleeces produced in a semi-static continuous process was indistinguishable of the surface structure of fleeces produced in a static process.

(32) Water Absorption Capacity, Water Retention Capacity and Tensile Strength

(33) BNC by nature forms a hydro gel, which results in its characteristic liquid affinity of water or other organic solutions. Thereby, the water absorption capacity and the water retention capacity of the BNC-articles are important features.

(34) After purification of the BNC-samples and consecutive washing steps using a.dest until the washing water was neutral (determined with unitestpaper), the average weight of the never-dried samples was determined.

(35) The WRC was determined using standardized conditions as described in Jayme & Rothamel (JAYME G, ROTHAMEL L: Composition of the extractives obtained from black poplarwood and of those found in the resulting sulfite and sulfate pulps. Cellulose-Chemie (1944), 22, 88-96). The samples to be determined were cut into pieces of 0.5 cm2. The never-dried BNC-samples were centrifuged for 15 min, at 4000 U/min (rpm) and the wet weight was determined. 4000 rpm correspond to about 1788 g. After air drying at 100° C. in a drying chamber to constant weight, the WRC was determined using the quotation
WRC=(mass wet−mass dry)/mass wet×100%

(36) The re-quelling of the dried samples was performed at 30° C. for 2 hrs. in a.dest.

(37) Table 3 shows the water absorption capacity (WAC) and the water retention capacity (WRC) of wet BNC fleeces of the respective bacterial strain manufactured in a semi-static continuous process and in a static process, respectively.

(38) TABLE-US-00003 TABLE 3 Strain manufacturing method WAC [%] WRC [%] DSM 14666 static 16 900 ± 1.520 853 ± 94 DSM 14666 semi-static, continuous 14.000 ± 1.260 900 ± 99 ATCC 11142 static 13.195 ± 1.310 815 ± 85 ATCC 11142 semi-static, continuous 11.003 ± 1.083 781 ± 83

(39) Furthermore the tensile strength was determined. The tensile strength is a preferable measure for the uniformity of the BNC. The BNC fleeces were hot pressed. The BNC fleeces manufactured in a semi-static continuous process revealed a higher tensile strength compared with the BNC fleeces manufactured in a static process. BNC fleeces manufactured in a static process showed a tensile strength of 252 MPa, whereas BNC fleeces manufactured in a semi-static continuous process showed a bending tensile strength of 312 MPa.

(40) The features of the present invention disclosed in the specification, the claims, examples and/or the figures may both separately and in any combination thereof be material for realizing the invention in various forms thereof.