Biocellulose wound dressing comprising silver nanoparticles and method of preparation thereof

Abstract

A biocellulose wound dressing comprising silver nanoparticles comprises a biocellulose wound dressing, which comprises fibers and water, combined with silver nanoparticles and a preservative. In the preparation method, biocellulose is purified inside an autoclave by boiling the biocellulose in a sodium hydroxide solution, adjusting for a neutral pH, then soaking in an organic acid and storing in distilled water prior to use. Then, the biocellulose wound dressing comprising silver nanoparticles is prepared by soaking the obtained biocellulose wound dressing in a silver nanoparticle solution, autoclaving, then soaking in the preservative and sterilizing by gamma radiation.

Description

[0001] BIOCELLULOSE WOUND DRESSING COMPRISING SILVER NANOPARTICLES AND METHOD OF PREPARATION THEREOF

[0002] Field of the Invention

[0003] The present invention relates to the fields of polymer chemistry and biology, particularly to wound dressing comprising silver nanoparticles as antimicrobial agents, and methods for preparing such wound dressings.

[0004] Background of the Invention

[0005] Wound dressings are materials in direct contact with wounds that can expedite wound healing and help prevent pathogens like bacteria, viruses, fungi or parasites from entering the body through the wounds. Each wound dressing material plays an important role in wound healing process. At present, a variety of materials with different properties appropriate for different wound types can be used as wound dressings, such as:

[0006] ■ Gauze made from either natural or synthetic fibers. However, gauze cannot adsorb a large amount of exudate, such as blood or lymph, and does not possess a moisture-retention property useful for wounds. Also, gauze made from natural fibers leave their remnants on wounds that induce injuries when removed.

[0007] ■ Clear film made from polyurethane. Clear films possess a property that allows vapor and gas permeation, and can protect against contamination by pathogens. As the films are transparent, medical professionals are able to see wounds in treatment follow-up without the need to change wound dressings that would induce recurring injuries. However, the films cannot adsorb a large amount of exudate, such as blood or lymph, thus softening tissues around the wounds and delaying wound healing.

[0008] ■ Hydrofiber able to adsorb more exudate like blood or lymph, and act as a dressing for as long as the hydrofiber is not yet saturated. A disadvantage of this type of material is that it may obscure wound infections because it turns yellow and becomes stinky upon adsorbing lymph.

[0009] ■ Biocellulose or bacterial cellulose, which is pure cellulose nanofibers producible from bacterial cultured in appropriate culture media. Biocellulose has high purity; is resistant to chemicals; has large contact surface area; is highly water-absorbable, which indicates that biocellulose is “hydrophilic,” resulting in fibers in the biocellulose possessing a property to hold water up to 100 times higher than their own dry weight; and has highly organized structural arrangement. Moreover, biocellulose fibers are characterized by tightly packed weaving, hence the strength and high elasticity. Biocellulose is applied to the medical industry because bacterial cellulose fibers can expedite would healing process of the body and do not adhere to the wounds, thereby reducing pain upon applying or changing the wound dressings. Also, the many small pores of biocellulose help adsorb exudate, help stimulate would healing mechanisms of the body, do not interfere with re-epithelialization, reduce the chance of scar formation and keep the skin moist.

[0010] At present, several types of wound dressings have been developed to facilitate use with infected wounds through added disinfectants. For example, a Thai petty patent application number 2003001059 discloses preparation of biocellulose wound dressing comprising honey as an antimicrobial active ingredient where honey possesses a bacteriostatic property and expedites wound healing of the body. In addition, the plenty of sugar in honey helps inhibit bacterial growth, and honey dissolution by the lymph from wounds produces hydrogen peroxide, which degrades the bacterial cell wall, protein and nucleic acids. Also, honey helps reduce wound inflammation by dehydrating injured tissues, reducing swelling and stimulating lymphatic circulation.

[0011] A Thai patent application number 0601005003 describes a method of preparing fiber and cloth coated with silver nano, which is a broad-spectrum antimicrobial in the size of no more than 100 nanometers equivalent to DNA in the human body or approximately 10,000 times smaller than a human hair, hence the large surface area, which can make more contact with bacteria and is invisible to the naked eyes. The silver nano can act against bacteria, viruses and yeasts through silver nano atoms binding to the cell wall and cell membrane, inhibiting microbial enzymes and destroying the microbes eventually. The method of preparing fiber and cloth coated with silver nano according to this invention consists of a step of preparing the silver nano and a step of coating the fiber and cloth with a silver nano solution.

[0012] A petty patent application number 2103000748 describes an adhesive hydrogel wound dressing with silver nanoparticles enclosed in andrographolide molecules extractable from Andrographis medication that are an efficient bactericidal; can inhibit Escherichia coli, Burkholderia pseudomallei and Staphylococcus aureus,' help expedite wound healing; and reduce infection and inflammation.

[0013] A Thai patent application number 0901001945 describes materials with coagulation and bacteriostatic properties, such as cloth or strip, used as bandages, plasters, wound dressings, etc., for minor cuts, abrasion, puncture wounds or surgical wounds in order to protect against infection and help in coagulation. The said materials have a surface of at least one side coated with an aqueous combined solution of polysaccharide with a property to help expedite coagulation as can be selected from carboxymethylcellulose, carboxymethyl chitosan, sodium alginate and combination thereof, and a disinfectant. After drying, the coating substance is in the form of a thin film on the material that allows the material to maintain its softness and convolution, with the weight of the coating combined substance between 1-14 grams per square meter of the material. Materials coated with the said combined substance demonstrate microbial inhibition, in vitro blood coagulation and in vivo blood coagulation properties.

[0014] A US patent application number US 2015 / 0147410 Al discloses a wound dressing comprising biocellulose and silver nanoparticles, wherein the concentration of the said silver nanoparticles can be selected from 1,000 micrograms per no more than 100 square meters of the wound dressing, and the silver nanoparticles have localized surface plasma resonance maxima of 600-800 nanometers.

[0015] In addition, a research by the Faculty of Medicine, Siam University (source: https: / / e- research.siam.edu / wp-content / uploads / 2023 / 03 / Medicine-2022-Research-Report-Efficiency-of- hydrogel-based-wound-dressings-on-wound-healing-in-rats.pdf), studied efficiency of a wound dressing made from synthetic hydrogel containing curcumin, which is an active compound in turmeric, encapsulated by silver nanoparticles in would repair process in rats. The results showed that silver nanoparticle-encapsulated curcumin hydrogel could decrease the number of bacteria at the wounds and allowed good wound healing.

[0016] It can be seen that, after performing innovation data searches, prior inventions relating to a variety of wound dressings were found, indicating continuous commitment of innovators in developing wound dressings comprising a variety of materials and compositions for enhanced efficiency so that would dressings not only can prevent pathogens from entering the body through the wounds but also possess microbial inhibitory property, thus minimizing the chance of wound infection. Nevertheless, each of the said wound dressings usually possesses an inhibitory property against one particular type of microorganisms or has a weakness as regards its adopted material, which may not be able to adsorb exudate, delaying wound healing, or may induce wound infection because it absorbs lymph and then turns yellow and becomes stinky. This invention has, therefore, developed a wound dressing from biocellulose comprising silver nanoparticles with addition of a preservative composition as an unprecedented invention. It was proven that the product could inhibit growth of a variety of microorganisms, reduce the chance of wound infection. Also, the adopted biocellulose minimizes adherence to the wounds and helps adsorb exudate.

[0017] Summary of the Invention

[0018] The biocellulose wound dressing comprising silver nanoparticles comprises silver nanoparticles with diameter size of 50 to 100 nanometers, a preservative, and biocellulose that comprises fibers like those extractable from yeasts cultured in coconut water, and water. In the preparation method, the biocellulose is purified inside an autoclave by boiling it in a sodium hydroxide solution, adjusting the pH and then soaking the obtained biocellulose in an organic acid. Afterwards, the obtained biocellulose is stored in distilled water. Then, the biocellulose wound dressing comprising silver nanoparticles is prepared by soaking the purified biocellulose wound dressing in a silver nanoparticle solution, autoclaving and then soaking in the preservative. Then, sterilization is performed by gamma radiation.

[0019] The objective of this invention is to provide the biocellulose wound dressing comprising silver nanoparticles as well as the method of preparing the said dressing with an inhibitory property simultaneously against a variety of microorganisms, such as fungi, gram-positive bacteria, gramnegative bacteria and drug -resistant bacteria, which are clinically important species that include multidrug-resistant ones.

[0020] Detailed Description of the Invention

[0021] Unless specified otherwise, words of art and definitions appearing in this invention correspond with the generally used manner and are understandable by those with expertise in the said field.

[0022] The biocellulose wound dressing comprising silver nanoparticles according to this invention comprises:

[0023] ■ silver nanoparticles with a diameter size of 50 to 100 nanometers, 85 to 180 parts per million;

[0024] ■ preservative, 0.5 to 2% by volume, that can be selected from any one of chlorphenesin, glycerin and phenoxyethanol, or combination thereof; and

[0025] ■ biocellulose comprising 3 to 5% by weight fibers and 95 to 97% by weight water.

[0026] Preferably, the biocellulose wound dressing comprising silver nanoparticles according to this invention comprises:

[0027] ■ silver nanoparticles with a diameter size of 50 to 100 nanometers, 85-150 parts per million;

[0028] ■ preservative comprising chlorphenesin, glycerin and phenoxyethanol; and

[0029] ■ biocellulose comprising 3 to 5% by weight fibers and 95 to 97% by weight water, where the fibers are derived from bacterial synthesis using coconut water as a raw material.

[0030] Preferably, chlorphenesin quantity as a composition of the biocellulose wound dressing comprising silver nanoparticles according to this invention is in the range of 0.19 to 0.21% by weight.

[0031] Preferably, phenoxyethanol quantity for the biocellulose wound dressing comprising silver nanoparticles according to this invention is in the range of 0.69-0.21% by weight. Preferably, glycerin quantity for the biocellulose wound dressing comprising silver nanoparticles according to this invention is at 0.10% by weight.

[0032] The method of preparing the biocellulose wound dressing comprising silver nanoparticles according to this invention comprises steps of: a) purification of the biocellulose wound dressing by boiling the biocellulose in the sodium hydroxide (NaOH) solution inside an autoclave, adjustment for a neutral pH by boiling the obtained biocellulose in distilled water or deionized water inside an autoclave followed by soaking of the obtained biocellulose in an organic acid inside an autoclave once more; afterwards, the obtained biocellulose is stored in distilled water in preparation for the next step; b) soaking of the biocellulose wound dressing obtained from a) in the silver nanoparticle solution at an appropriate concentration followed by autoclaving for 1 hour; c) soaking of the biocellulose wound dressing comprising silver nanoparticles as obtained from b) in the preservative at an appropriate concentration at room temperature or 25-40 degrees Celsius; and d) sterilization of the biocellulose wound dressing comprising silver nanoparticles as obtained from c) by gamma radiation.

[0033] According to the preferred embodiment of this invention, the thickness of the biocellulose wound dressing comprising silver nanoparticles is in the range of 3 to 5 millimeters.

[0034] According to the preferred embodiment of this invention, the biocellulose is fibers extractable from yeasts cultured in coconut water.

[0035] According to the preferred embodiment of this invention, the preferred concentration of the sodium hydroxide solution for use in the purification step of the biocellulose wound dressing is 1 to 5% weight by volume.

[0036] According to the preferred embodiment of this invention, the autoclave temperature is in the range of 100 to 150 degrees Celsius.

[0037] According to the preferred embodiment of this invention, the concentration of the organic acid for use in the purification step of the biocellulose wound dressing is 1 to 5% by volume acetic acid.

[0038] According to the preferred embodiment of this invention, the preferred concentration of the silver nanoparticle solution is 150 to 350 parts per million.

[0039] According to the preferred embodiment of this invention, the preferred ratio of the biocellulose wound dressing volume to the silver nanoparticle solution volume is in the range of 1 :7 to 7:7. According to the preferred embodiment of this invention, the preferred concentration of the preservative is in the range of 0.5 to 1.5% by volume.

[0040] According to the preferred embodiment of this invention, the preferred ratio of the biocellulose wound dressing comprising silver nanoparticles volume to preservative solution volume is in the range of 1 :7 to 7:7.

[0041] According to an embodiment of this invention, the preservative can be selected from any one of chlorphenesin, glycerin and phenoxyethanol, or combination thereof. The preservative preferably comprises a combination of chlorphenesin, glycerin and phenoxyethanol.

[0042] According to the preferred embodiment of this invention, chlorphenesin is in the range of 0.19 to 0.21% by weight.

[0043] According to the preferred embodiment of this invention, phenoxyethanol is in the range of 0.69 to 0.71% by weight.

[0044] According to the preferred embodiment of this invention, glycerin is at 0.10% by weight.

[0045] According to the preferred embodiment of this invention, gamma radiation dose for the sterilization of the biocellulose wound dressing comprising silver nanoparticles is 15 to 50 kilo grays (kGy).

[0046] Then, antimicrobial activities of the biocellulose dressing comprising silver nanoparticles according to this invention were studied at 37 degrees Celsius by agar well diffusion, which is an initial method in antimicrobial tests that can readily be performed in a laboratory; can be applied to tests of anti -microorganism activities of substances derived from animals, plants and microorganisms; and of which results can be obtained rapidly.

[0047] The agar well diffusion method is achieved through diffusion of a substance to be tested for its actions in pores of agar with test microorganisms spreading across the agar surface. Should the substance to be tested have killing or inhibitory activities against the test microorganisms, clear zones will develop because of lack of growth (inhibition zone).

[0048] The examples provided hereafter will show clearer details of this invention and are not considered limiting the scope of this invention whatsoever.

[0049] Description of the invention as follows will be by exemplification to facilitate a clearer understanding and does not mean to limit invention results to those in the examples.

[0050] Example 1: purification of the biocellulose wound dressing

[0051] Purification of the biocellulose wound dressing is achieved by boiling the biocellulose in 1% weight by volume sodium hydroxide solution inside an autoclave at 115 degrees Celsius for 60 minutes, adjustment for a neutral pH by boiling the biocellulose in distilled water inside an autoclave at 115 degrees Celsius for 60 minutes, soaking of the biocellulose in 0.75% by volume acetic acid solution and then autoclaving once more at 115 degrees Celsius for 60 minutes. Afterwards, the obtained biocellulose is stored in distilled water in preparation for the next step. Example 2: preparation of the biocellulose wound dressing comprising silver nanoparticles

[0052] Preparation of the biocellulose wound dressing comprising silver nanoparticles by preparing the silver nanoparticle solution at a concentration of 300 parts per million; soaking the biocellulose sheets obtained from Example 1, using ratios of 100 25 -square-centimeter biocellulose sheets per 1,750 milliliters silver nanoparticle solution and 30 100-square-centimeter biocellulose sheets per 1,750 milliliters silver nanoparticle solution; and autoclaving at 121 degrees Celsius for 60 minutes

[0053] Example 3: soaking the biocellulose wound dressing comprising silver nanoparticles in the preservative comprising chlorphenesin, glycerin and phenoxyethanol

[0054] A 1% by volume preservative solution is prepared. The prepared biocellulose sheets from Example 2 are soaked, using ratios of 100 25 -square-centimeter biocellulose sheets per 1,750 milliliters preservative solution and 30 100-square-centimeter biocellulose sheets per 1,750 milliliters preservative solution, at room temperature for 12 hours.

[0055] Example 4: study of inhibitory activities of the biocellulose wound dressing comprising silver nanoparticles as prepared with the method according to this invention

[0056] This example demonstrates a test of inhibitory activities of the biocellulose wound dressing comprising silver nanoparticles as prepared according to this invention in comparison with a reference wound dressing in the market, using the agar well diffusion method. Test microorganisms selected for use in the study were:

[0057] • Candida albicans: a type of fungi that is found naturally, lives on the human body and causes a skin disease (representative of fungi);

[0058] • Escherichia coli: a type of gram-negative bacteria that can cause intestinal (gastrointestinal) infection (representative for test of activities against gram-negative bacteria);

[0059] • Pseudomonas aeruginosa: a type of gram-negative bacteria that can cause diseases in plants, animals and humans; is considered a clinically important species; and is multidrugresistant (representative of drug-resistant bacteria);

[0060] • Salmonella spp. : a type of gram -negative bacteria most commonly causing food poisoning; a small load of this bacteria in food can cause illness; and

[0061] • Staphylococcus aureus: a type of gram-positive bacteria that is part of normal flora of the skin and nasal cavity; it is a type of pathogens (representative for test of activities against gram-positive bacteria). The test adjusted quantities of the above microorganisms against the required McFarland standard concentration of McFarland No. 0.5. Sterile swabs were dipped into different culture tubes and then smeared on a standard culture medium for testing antimicrobials (Mueller Hinton agar: MHA). Then, samples of the biocellulose dressing comprising silver nanoparticles according to this invention were placed on the medium in comparison with the reference wound dressing in the market at 37 degrees Celsius for 24 hours, Then, results were recorded by measuring inhibition zone diameters on the medium that indicated inhibitory activities against the various types of microorganisms of the tested samples as shown in Table 1.

[0062] Table 1. Inhibition zone diameters on the surface of the medium.

[0063] Based on Table 1, it can be seen that, compared with the reference wound dressing in the market, the biocellulose wound dressing comprising silver nanoparticles according to this invention had inhibitory activities simultaneously against a variety of microorganisms - fungi, gram-positive bacteria, gram-negative bacteria and drug-resistant bacteria, which are considered a clinically important species and are resistant to multiple drugs - while the reference wound dressing possessed inhibitory activities against fewer types of microorganisms than the biocellulose dressing comprising silver nanoparticles according to this invention.

[0064] The material according to the intent of this invention is, therefore, created; that is, the biocellulose wound dressing comprising silver nanoparticles with an inhibitory property against a variety of microorganisms simultaneously.

[0065] Best Mode of the Invention

[0066] As described in Detailed Description of the Invention

Claims

Claims1. A biocellulose wound dressing comprising silver nanoparticles, comprising:• silver nanoparticles in an amount of 85 to 180 parts per million;• a preservative in an amount of 0.5 to 2% by weight; and• biocellulose comprising 3 to 5% by weight fibers and 95 to 97% by weight water; which is characterized in that the preservative can be selected from any one of chlorphenesin, glycerin and phenoxyethanol, or combination thereof; and wherein the preservative preferably comprises a combination of chlorphenesin, glycerin and phenoxyethanol.

2. The biocellulose wound dressing comprising silver nanoparticles according to claim 1, wherein the silver nanoparticles have a diameter size of 50 to 100 nanometers.

3. The biocellulose wound dressing comprising silver nanoparticles according to claim 1 or 2, wherein the preferred biocellulose is fibers extractable from yeasts cultured in coconut water.

4. The biocellulose wound dressing comprising silver nanoparticles according to any one of claims 1 to 3, wherein the preferred amount of chlorphenesin is in the range of 0.19 to 0.21% by weight.

5. The biocellulose wound dressing comprising silver nanoparticles according to any one of claims 1 to 4, wherein the preferred amount of phenoxyethanol is in the range of 0.69 to 0.71% by weight.

6. The biocellulose wound dressing comprising silver nanoparticles according to any one of claims 1 to 5, wherein the preferred amount of glycerin is at 0.10% by weight.

7. The biocellulose wound dressing comprising silver nanoparticles according to any one of claims 1 to 6, wherein the preferred thickness of the biocellulose wound dressing comprising silver nanoparticles is in the range of 3 to 5 millimeters.

8. A method of preparing biocellulose wound dressing comprising silver nanoparticles, comprising the following steps: a) purification of the biocellulose wound dressing by boiling the biocellulose in a sodium hydroxide (NaOH) solution inside an autoclave, adjustment for a neutral pH by boiling the obtained biocellulose in water inside an autoclave followed by soaking of the obtained biocellulose in an organic acid inside an autoclave once more; afterwards, the obtained biocellulose is stored in distilled water in preparation for the next step;b) soaking of the biocellulose wound dressing obtained from a) in a silver nanoparticle solution followed by autoclaving; c) soaking of the biocellulose wound dressing comprising silver nanoparticles as obtained from b) in a preservative at room temperature or 25-40 degrees Celsius; and d) sterilization of the biocellulose wound dressing comprising silver nanoparticles as obtained from c) by gamma radiation.

9. The method of preparing biocellulose wound dressing comprising silver nanoparticles according to claim 8, wherein the preferred biocellulose is fibers extractable from yeasts cultured in coconut water.

10. The method of preparing biocellulose wound dressing comprising silver nanoparticles according to claim 8 or 9, wherein the preferred sodium hydroxide concentration for use in the purification step of the biocellulose wound dressing is in the range of 1 to 5% weight by volume.

11. The method of preparing biocellulose wound dressing comprising silver nanoparticles according to any one of claims 8 to 10, wherein the preferred autoclave temperature is in the range of 100 to 150 degrees Celsius.

12. The method of preparing biocellulose wound dressing comprising silver nanoparticles according to any one of claims 8 to 11, wherein the preferred organic acid for use in the purification step of the biocellulose wound dressing is acetic acid.

13. The method of preparing biocellulose wound dressing comprising silver nanoparticles according to claim 12, wherein the preferred acetic acid concentration for use in the purification step of the biocellulose wound dressing is in the range of 1 to 5% by volume.

14. The method of preparing biocellulose wound dressing comprising silver nanoparticles according to any one of claims 8 to 13, wherein the preferred concentration of the silver nanoparticle solution is in the range of 150 to 350 parts per million.

15. The method of preparing biocellulose wound dressing comprising silver nanoparticles according to any one of claims 8 to 14, wherein the preferred ratio of biocellulose wound dressing volume to silver nanoparticle solution volume is in the range of 1 :7 to 7:7.

16. The method of preparing biocellulose wound dressing comprising silver nanoparticles according to any one of claims 8 to 15, wherein the preferred preservative concentration is in the range of 0.5 to 1.5% by volume.

17. The method of preparing biocellulose wound dressing comprising silver nanoparticles according to any one of claims 8 to 16, wherein the preferred ratio of biocellulose wounddressing comprising silver nanoparticles volume to preservative solution volume is in the range of 1 :7 to 7:7.

18. The method of preparing biocellulose wound dressing comprising silver nanoparticles according to any one of claims 8 to 17, wherein the preservative can be selected from chlorphenesin, glycerin and phenoxyethanol, or combination thereof; and wherein the preservative preferably comprises a combination of chlorphenesin, glycerin and phenoxyethanol.

19. The method of preparing biocellulose wound dressing comprising silver nanoparticles according to any one of claims 8 to 18, wherein the preferred amount of chlorphenesin is in the range of 0.19 to 0.21% by weight.

20. The method of preparing biocellulose wound dressing comprising silver nanoparticles according to any one of claims 8 to 19, wherein the preferred amount of phenoxyethanol is in the range of 0.69 to 0.71% by weight.

21. The method of preparing biocellulose wound dressing comprising silver nanoparticles according to any one of claims 8 to 20, wherein the preferred amount of glycerin is at 0.10% by weight.

22. The method of preparing biocellulose wound dressing comprising silver nanoparticles according to any one of claims 8 to 21, wherein the gamma radiation dose for sterilizing the biocellulose wound dressing comprising silver nanoparticles is 15 to 50 kilo grays (kGy).

23. The biocellulose wound dressing comprising silver nanoparticles as prepared by the method according to any one of claims 8 to 22, wherein the preferred thickness is in the range of 3 to 5 millimeters.

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