Water-Containing Hydrogel Composition Comprising Elemental Silver Particles

Abstract

The invention relates to a water-containing hydrogel composition comprising elemental silver particles and to a multi-layered wound dressing comprising the hydrogel composition. The multi-layered wound dressing is used especially in the inflammation and/or granulation phases in the treatment of, for example, burns and/or chronic wounds. In addition, the invention relates to a process for preparing the hydrogel composition.

Claims

1. A water-containing hydrogel composition comprising elemental silver particles with an average particle diameter of 5-20 nm, determined by transmission electron microscopy (TEM), and a particle size distribution of D9025 nm, determined by laser diffractometry, wherein the silver content, based on the total weight of the hydrogel composition, is between 15 and 500 ppm.

2. The water-containing hydrogel composition as claimed in claim 1, wherein the average particle diameter is 9-18 nm.

3. The water-containing hydrogel composition as claimed in claim 1, wherein the silver content, based on the total weight of the hydrogel composition, is between 25 and 250 ppm.

4. The water-containing hydrogel composition as claimed in claim 1, wherein the hydrogel composition comprises a hydrophilic polyurethane foam.

5. The water-containing hydrogel composition as claimed in claim 1, wherein the hydrogel composition comprises a polyurethane-polyurea copolymer.

6. The water-containing hydrogel composition as claimed in claim 1, wherein the hydrogel composition further comprises a non-ionic surfactant.

7. The water-containing hydrogel composition as claimed in claim 6, wherein the surfactant is selected from Tween 20, polyethylene glycol, sorbitol and polyvinyl pyrrolidone, or mixtures thereof.

8. The water-containing hydrogel composition as claimed in claim 1, further comprising 5 to 30% by weight, based on the total weight of the hydrogel composition, of a polyhydric alcohol selected from propylene glycol and/or glycerol.

9. The water-containing hydrogel composition as claimed in claim 1, consisting of 6 to 20% by weight of a prepolymer with isophorone diisocyanate ends, 4 to 15% by weight of a diamine based on polyethylene oxide, 15 to 20% by weight propylene glycol and/or glycerol, 40 to 70% by weight water and 25 to 250 ppm elemental silver with an average particle diameter of 9-18 nm, all weight statements based on the total weight of the hydrogel composition.

10. A multi-layered wound dressing, comprising at least one water-impermeable and water vapour-impermeable support layer, an absorbent layer and a layer comprising the water-containing hydrogel composition as claimed in claim 1.

11. The multi-layered wound dressing as claimed in claim 10, wherein the absorbent layer comprises a hydrophilic polyurethane foam and the hydrogel.

12. The multi-layered wound dressing as claimed in claim 11, wherein the surface of the hydrophilic polyurethane foam is at least partially impregnated with the hydrogel.

13. The multi-layered wound dressing as claimed in claim 10, wherein the wound dressing is used in the inflammation phase and/or the granulation phase of wound healing.

14. A method for treating a burn and/or a chronic wound comprising administering the multi-layered wound dressing as claimed in claim 10 to said burn and/or chronic wound.

15. A process for preparing a hydrogel composition, comprising reacting a mixture comprising: (a) a polyamine, (b) optionally a non-ionic surfactant, (c) further optionally a polyhydric alcohol selected from propylene glycol and/or glycerol, (d) elemental silver particles with an average particle diameter of 5-20 nm, determined by transmission electron microscopy (TEM), and (e) water, with an aliphatic diisocyanate polymer in order to form a water-containing hydrogel composition in which the silver content, based on the total weight of the hydrogel composition, is between 15 and 500 ppm.

16. The process for preparing a hydrogel composition as claimed in claim 15, wherein the average particle diameter is 9-18 nm.

17. The process for preparing a hydrogel composition as claimed in claim 15, wherein the silver content, based on the total weight of the hydrogel composition, is between 25 and 250 ppm.

18. The process for preparing a hydrogel composition as claimed in claim 15, wherein the hydrogel composition comprises a hydrophilic polyurethane foam.

19. The process for preparing a hydrogel composition as claimed in claim 15, wherein the hydrogel composition comprises a polyurethane-polyurea copolymer.

20. The process for preparing a hydrogel composition as claimed in claim 15, wherein the hydrogel composition further comprises a non-ionic surfactant.

Description

DESCRIPTION OF THE FIGURES

[0070] FIG. 1: Release of silver nanoparticles from the hydrogel into the aqueous medium

[0071] FIG. 2: Release of silver ions from the hydrogel into the aqueous medium

[0072] FIG. 3A/B: Antimicrobial activity of the hydrogel at different silver contents

[0073] FIG. 4: Cytotoxicity and cell compatibility of the hydrogel at different silver contents

[0074] FIG. 5A/B: Absorption capacity of a hydrogel of the invention at different silver contents

[0075] FIG. 6A/B: Dehydration of a hydrogel of the invention at different silver contents

EXAMPLES

[0076] Preparation of a Hydrogel Composition

[0077] In a first step, a mixture of 52.5% by weight polyamine (Jeffamin ED-2003, Huntsman; Everberg, Belgium) and 47.5% by weight water is mixed. Of that mixture, 132.5 g are mixed with 200 g glycerol, 567.5 g water and the corresponding amount of an aqueous silver nanoparticle suspension (Agpure W10, RAS materials GmbH, Regensburg, Germany) with a nominal silver content of 10% by weight, a particle size distribution of D99<20 nm and an average particle size of 15 nm. 100 g of an IPDI-based prepolymer (Aquapol PI-13000-3; Carpenter; Richmond, USA) are added to that mixture. The components are thoroughly blended, the still liquid gel is portioned into petri dishes and polymerises completely there.

[0078] Jeffamin Mix:

TABLE-US-00001 Jeffamin ED-2003 Huntsman; Everberg, Belgium 52.5% by weight Aqua purificata water treatment plant 47.5% by weight

[0079] Aquapol:

TABLE-US-00002 Aquapol PI-13000-3 Carpenter; Richmond, USA 100.0% by weight

[0080] Components for 1 kg hydrogel:

TABLE-US-00003 Chemical Amount weighed in [g] Proportion of gel [%] glycerol 200 20 water 567.5 56.75 Jeffamin mix 132.5 13.25 Aquapol 100 10 silver 0.0125 to 0.25 0.00125 to 0.025 (12.5 ppm to 250 ppm)

[0081] Measuring Methods

[0082] Nanoparticle Release

[0083] Production of the Calibration Curve

[0084] In order to investigate the migration of the silver nanoparticles from the gel into the aqueous medium, the eluate of the ion release is analysed by means of a photometer. The nanoparticles have an absorption peak in the visible range at approx. 410 nm. In order to be able to determine the concentration of the silver nanoparticles in the eluate, standards in concentrations of 50; 25; 10; 1; 0.1 mg Ag Pure W 10/kg demineralised water are measured, with which a calibration curve can be produced.

[0085] Determining the Silver Nanoparticles Released

[0086] In order to determine the ion release, test pieces with a diameter of 50 mm are punched out, removed from the petri dish and weighed. The gels are transferred to 100 mL Erlenmeyer flasks, 30 mL water (HPLG grade) is added, and sealed with a ground-glass stopper. After that the samples are incubated at room temperature on a shaker for 24 hours at 130 rpm. After incubation for 24 hours, the extinction of the eluate at 410 nm is measured in the photometer. Polystyrene cuvettes are used.

[0087] Ion Release

[0088] In order to determine the ion release, test pieces with a diameter of 50 mm are punched out, removed from the petri dish and weighed. The gels are transferred to 100 mL Erlenmeyer flasks, 30 mL water (HPLG grade) is added, and sealed with a ground-glass stopper. After that the samples are incubated at room temperature on a shaker for 24 hours at 130 rpm. Following that, approx. 5-6 mL of the eluates are pipetted into brown vials and mixed with 20 L concentrated nitric acid for stabilisation purposes. The samples are examined by IPC-MS in accordance with EN ISO 17294-2 (E29).

[0089] Release Kinetics

[0090] In order to observe the release of the silver nanoparticles or silver ions over a longer period, the corresponding measurements are determined at assay times of 2; 4; 6; 8; 24; 48 and 72 hours. Test pieces with a diameter of 50 mm are punched out of the AgNP gels, drawn from the petri dish and weighed. After that, the gels are treated as described in the corresponding sections.

[0091] Efficacy Studies

[0092] Working Cultures and Media

[0093] For the zone of inhibition and soft agar method, 24-hour cultures of the bacterial strain Staphylococcus aureus DSM 346 are used. For this purpose, a cryosphere is transferred to 9 mL caso broth and incubated in the incubator at 37 C. for 24 hours. The suspension is visually inspected for high turbidity and hence good growth.

[0094] Pour Plate Method

[0095] The viable bacteria are evaluated by counting the colony forming units (CFU). A defined quantity of bacterial suspension is mixed in warm caso agar and counted after incubation at the appropriate temperature. It is presumed that each bacterial cell forms a colony on the plate.

[0096] In the pour plate method, appropriate dilution levels of the samples in steps of 10 (1:10) are prepared and 1 mL thereof in each case is poured into an empty petri dish. After that approx. 20 mL are poured onto caso agar in the petri dish heated to 45 C. and the plate is swung in a figure-of-8-shaped movement so that the bacterial cells are distributed evenly in the agar. The plates are stored at room temperature until they have solidified. Finally, they are incubated in the incubator for approx. 24 hours at 37 C. and the bacteria colonies formed are counted. For evaluation purposes, the CFU/mL are calculated.

[0097] CFU: colonies counted

[0098] DF soft agar: dilution factor soft agar

[0099] DF D/e neutraliser: dilution factor soft agar

[0100] DF plate pour: dilution factor of the plates counted

CFU/mL=counted CFU*DF soft agar*DF D/eneutraliser*DF plate pour

[0101] Zone of Inhibition Assay

[0102] In order to obtain a first impression of the antimicrobial efficacy of the AgNP gels, the samples are placed on freshly inoculated agar plates and assessed as to whether the samples inhibit the growth of the bacteria. For the zone of inhibition method, circular test pieces with a diameter of 30 mm are punched out. 5 mL of a bacteria suspension, 1 mL suspended in 150 mL liquid caso agar, are pipetted onto finished caso plates, and then it is necessary to wait until the plates are cured. The plates must be used within an hour so that the bacterial growth does not begin before the test samples are placed on them. The test pieces are placed with the active side facing the agar plate and pressed down so that the sample has good contact with the plate, and are incubated in the incubator overnight at 37 C. After that, the zone of inhibition which forms is measured.

H=(Dd)/2

[0103] H=inhibition zone [mm]

[0104] D=total diameter [mm]

[0105] d=diameter of the sample [mm]

[0106] Soft Agar Test

[0107] In order to obtain more precise results than with the zone of inhibition assay, a defined concentration of bacteria in soft agar is placed on the samples to be tested, and the CFU/mL are assessed after a test period of four hours.

[0108] 100 mL soft agar are heated to 45 C. and inoculated with 1 mL of the bacteria suspension. Sample pieces measuring 2.5 cm2.5 cm are punched out with a punch and transferred to empty petri dishes with sterile tweezers. 1 mL of the inoculated soft agar is placed on the surface of the sample pieces with a pipette, care being taken to ensure that the soft agar does not flow down. The latter solidifies at room temperature after approx. 10 minutes. In addition, a positive control is prepared, for this purpose, 1 mL of inoculated soft agar is pipetted into a 50 mL Falcon tube, and 3 mL strength Ringer's solution is added so that the soft agar does not dry out. The inoculated sample pieces are incubated in the incubator overnight at 37 C. After the test period of four hours, 20 mL Dey-Engley neutralising broth is added in order to bind any free silver ions still present. For the 0-hour value, 1 mL of the inoculated soft agar is pipetted directly into the 20 mL D/E neutralising broth.

[0109] The batches are treated for one minute in an ultrasonic bath. After that, suitable dilution series are prepared with strength Ringer's solution and plated with the pour plate method.

Geometrical mean of the sample=(log x.sub.1+log x.sub.2+log x.sub.3)/(3)

Geometrical mean of the control=(log xk.sub.1+log xk.sub.2+log xk.sub.3)/(3)

Log level reduction=log level controllog level sample

[0110] Measuring the Humidity Loss (Dehydration)

[0111] The loss of weight over a specific period of time at a defined temperature is described as the humidity loss. The humidity loss is calculated according to the following equation and is stated in the unit g/g:

humidity loss=final weight/initial weight

[0112] Measuring the Absorption Capacity

[0113] In order to measure the absorption capacity, gel samples with a diameter of 5 cm are punched out. After that, they are placed in a glass beaker with V=300 ml deionised water. Then they are weighed again at certain intervals. The absorption capacity is calculated according to the following equation and is stated in the unit g/g:

absorption capacity=(final weightinitial weight)/initial weight

[0114] Test for Cell Compatibility

[0115] The tests for cell compatibility were performed in accordance with DIN EN ISO 10993-5 and the methodological instructions of the Department for Functional Materials in Medicine and Dentistry: BioLab 973302, 042901, 964702 and 964805 and comprise measurements of cell growth, metabolic activity and protein content.

[0116] The hydrogels were supplied sterile in petri dishes. For the assay, 0.1 g/ml culture medium was weighed into each of the samples.

[0117] The cell activity, the cell count and the protein concentration were examined three times per sample in four parallel batches each. The elution time was 48 h. the incubation of the cells with the eluates also 48 h. [0118] Cell line: L 929 CC1 Murine fibroblasts (American Type Culture Collection. Rockeville Md., USA). [0119] Culture medium: DMEM (Dulbecco's mod. Eagle's medium) according to VA BioLab 042901 for the preliminary culture and elution. [0120] Negative control: polystyrene (Nunc GmbH & Co KG, Wiesbaden). [0121] Positive control: Vekoplan KT PVC plates (Konig GmbH, Wendelstein).

[0122] Per sample, three eluates from each hydrogel were tested, which were prepared on different test days. For this purpose, the hydrogels were cut through in the middle in the petri dishes with a sterile scalpel and transferred to a sterile 50 ml reaction vessel. Per 0.1 g sample, 1 ml elution medium was added to the hydrogels, and these were then eluted in the incubator for 48 h at 37 C. and 5% CO.sub.2. In order to remove from the eluates any suspended matter present, the samples were centrifuged for 5 min at 4,000 rpm after incubation and filtered through a filter (pore size 0.2 m).

[0123] The cells were seeded in a concentration of 50,000 cells/ml, preculturing was at 37 C. and 5% CO.sub.2 for 24 h. After that, the DMEM medium added during seeding was withdrawn, and the cells were each covered with 1 ml eluate in a concentration of 100%. As a negative control, DMEM medium was incubated in a 50 ml Falcon tube for 48 h like the samples; the eluate from the plastic discs in a concentration of 100% was used as a positive control. After 48 hours of incubation, the cell activity, the cell count and the total protein content were determined.

[0124] Cell Growth (Cell Counting)

[0125] Cell counting was performed after the enzymatic detachment of the cells by means of Accutase with the aid of the cell counter.

[0126] Vitality Test Via Metabolic Activity (WST)

[0127] The vitality was tested with tetrazolium salt, WST 1, Roche Diagnostics GmbH Mannheim, in accordance with the manufacturer's instructions. WST 1 is reacted by succinate dehydrogenase (an enzyme of the citric acid cycle) in the mitochondria of the metabolically active cells to yield coloured formazan and measured photometrically. The absorption values(OD), determined at 450 nm and 620 nm, correlate with the breathing activity of the cultured cells.

[0128] Protein Content (Lowry)

[0129] The protein content was tested with the DC Protein Assay, BIO-RAD GmbH Munich, in accordance with the manufacturer's instructions. The Lowry method of determining protein is based on the reduction of Cu(II) to Cu(I) by the aromatic tyrosine-tryptophan residues of proteins. In a further step, the copper-protein complex reduces a phosphomolybdic acid/phosphotungstate reagent to molybdenum or tungsten blue respectively. The extinction of this intense blue colouring is measured photometrically at 750 nm. The protein concentration can be determined by conducting a standard series at the same time.

[0130] Acceptance and Evaluation

[0131] The classification of the assessment ranges for acceptance and evaluation was performed in line with DIN EN ISO 7405 and the definition of the inhibition dose (ID 50: dose at which 50% of the cells are inhibited in their growth) (literature: Allgemeine Pharmakologie und Toxikologie, Henschler, ed.: Forth Wolfgang; Spektrum akad. Verl. Heidelberg; 7th ed. 1996). A cell growth of 0-29% is characterised as strong growth inhibition, a cell growth of 30-59% as moderate inhibition and a cell growth of 60-79% as weak inhibition compared to the control. Cell growth rates of between 80 and 100% indicate uninhibited cell growth.

[0132] A cell activity of 0-29% is characterised as highly reduced metabolic activity, a cell activity of 30-59% as moderately reduced metabolic activity and a cell activity of 60-79% as slightly reduced metabolic activity compared to the control. Cell activity rates of between 80 and 100% indicate no reduction in metabolic activity.

[0133] A protein concentration of 0-34% is characterised as a highly reduced protein content, a protein concentration of 35-69% as a moderately reduced protein content compared to the control. Protein concentrations of between 70 and 100% indicate no reduction in protein content.

[0134] The PS value in the presentation of the results corresponds to the polystyrene negative control.

Example 1: Release of Silver Particles and Silver Ions from the Gel into the Aqueous Medium

[0135] The migration of silver nanoparticles and silver ions from a hydrogel prepared in the manner described above into the aqueous medium was examined by determining the release kinetics. It was established that no detectable quantities of silver particles were released into the aqueous medium (FIG. 1). The quantity of silver ions released was 50 to 300 g/L Ag+ per 1 g hydrogel, depending on the silver content, as shown in FIG. 2. In simulated wound exudate, a release of silver ions in a range of up to 250 mg/kg hydrogel (250 ppm) was identified.

Example 2: Antimicrobial Activity of the Hydrogel at Different Silver Contents

[0136] In zone of inhibition assays, a significant inhibition of the growth of Staphylococcus aureus was already observed as of a silver content of 25 mg/kg hydrogel (FIG. 3A. In the range of 25 to 250 mg/kg hydrogel, the inhibition reached 8 log levels (FIG. 3B).

Example 3: Cytotoxicity and Cell Compatibility of the Hydrogel at Different Silver Contents

[0137] The cytotoxicity and cell compatibility of the hydrogel at different silver contents was established by determining the cell activity, the cell count and the protein concentration, and by means of a vitality test of the metabolic activity (WST). In all the tests, good cell compatibility with low cytotoxicity was established (FIG. 4).

Example 4: Absorption Capacity of a Hydrogel of the Invention at Different Silver Contents

[0138] By measuring the absorption capacity of a hydrogel of the invention at different silver contents over a period of up to 24 h, it was not possible to find any change in the absorption capacity depending on the silver content outside any statistical fluctuations (FIGS. 5A and 5B).

Example 5: Dehydration of a Hydrogel of the Invention at Different Silver Contents

[0139] By measuring the dehydration of a hydrogel of the invention at different silver contents over a period of 24 h, it was not possible to find any change in the dehydration depending on the silver content outside any statistical fluctuations (FIGS. 6A and 6B).

[0140] It was thus possible by means of the examples shown to demonstrate that by providing a hydrogel composition of the invention, a sufficiently large release of silver ions is possible which ensures effective antimicrobial activity during the treatment of wounds. At the same time, however, the release of silver particles and hence their absorption into the human body is prevented effectively, so that any side-effects caused by this are effectively avoided or can be prevented.

[0141] It was also possible to show that the silver particles contained in the hydrogel do not have any influence on its absorption capacity or dehydration, and hence do not impair the function of a wound dressing containing the hydrogel.

[0142] By providing the hydrogel composition of the invention, it was thus possible to provide a system for wound therapy with which the treatment of wounds can be performed as effectively as possible and which exhibits high antimicrobial activity. In this way, wound healing can be effectively promoted and accelerated. The wound therapy systems exhibit great wearing comfort and have effective antimicrobial efficacy even when worn for a long time.