BIOACTIVE WOUND DRESSING

Abstract

A cost effective bacterial cellulose wound dressing includes a microbial-derived cellulose loaded with natural antibacterial and regenerative substances. This wound dressing is effective for the treatment of burns and chronic wounds. The dressing keeps the wound moist while absorbing exudates from the wound area. The dressings possess antibacterial qualities to protect the wound from infections and help the area to heal faster through the action of bioactive substances.

Claims

1. An antibacterial wound healing material, comprising: cellulose; a bioactive material; and an antibacterial agent; wherein the bioactive material is selected from Defensin-1 (a peptide), Royal Jelly and Royal Jelly extract; wherein the antibacterial agent is honey.

2. The wound healing material of claim 1, which is in the form of a dressing.

3. The wound healing material of claim 2, wherein the cellulose is bacterial cellulose.

4. The wound healing material of claim 3, wherein the cellulose is in the form of a membrane.

5. The wound healing material of claim 4, wherein the cellulose membrane is incorporated with Royal Jelly or Royal Jelly extract.

6. The wound healing material of claim 4, wherein the cellulose membrane is incorporated with Defensin-1.

7. The wound healing material of claim 1, wherein the Royal Jelly extract is preserved in honey wherein the concentration of RJ extract is 50 ug/ml at a ratio of 1:3 of RJ extract to Honey.

8. The wound healing material of claim 2, wherein the Royal Jelly extract is preserved in honey wherein the concentration of RJ extract is 50 ug/ml at a ratio of 1:3 of RJ extract to Honey.

9. The wound healing material of claim 3, wherein the Royal Jelly extract is preserved in honey wherein the concentration of RJ extract is 50 ug/ml at a ratio of 1:3 of RJ extract to Honey.

10. The wound healing material of claim 4, wherein the Royal Jelly extract is preserved in honey wherein the concentration of RJ extract is 50 ug/ml at a ratio of 1:3 of RJ extract to Honey.

11. The wound healing material of claim 5, wherein the Royal Jelly extract is preserved in honey wherein the concentration of RJ extract is 50 ug/ml at a ratio of 1:3 of RJ extract to Honey.

12. The wound healing material of claim 6, wherein the Royal Jelly extract is preserved in honey wherein the concentration of RJ extract is 50 ug/ml at a ratio of 1:3 of RJ extract to Honey.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0015] The accompanying drawings, which are included to provide a further understanding of the inventive concept, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the inventive concept, and, together with the description, serve to explain principles of the inventive concept.

[0016] FIGS. 1A and 1B illustrates the synthesized and purified BC wound dressings in accordance with an exemplary embodiment.

[0017] FIG. 2 Fourier transform infrared spectroscopy (FTIR) spectra of Bacterial cellulose according to an exemplary embodiment.

[0018] FIG. 3 Tensile strength measurement of Bacterial Cellulose according to an exemplary embodiment.

[0019] FIGS. 4A and 4B Field-emission scanning electron microscopy (FE-SEM) micrograph of dry Bacterial cellulose membrane according to an exemplary embodiment.

[0020] FIGS. 5A and 5B A typical antibacterial assay of Bacterial cellulose loaded with honey on S. aureus (5A) and E.coli bacterial strains (5B) according to an exemplary embodiment. BCM (Bacterial cellulose membranes loaded with honey), C (control pure BCM), +ve (honey), Std (standard tetracyclin antiniotic disc).

[0021] FIGS. 6A and 6B A typical cell proliferation assay of fibroblasts on Bacterial cellulose RJ Extract (6A) and pure RJ (6B) according to an exemplary embodiment.

[0022] FIGS. 7A and 7B A typical Bright field microscopic image of HaKaT (Skin cells) cultured for 3 days on Bacterial cellulose membrane according to an exemplary embodiment (7A) Fluorescent Microscopic Image labelled with DAPI nuclear stain (7B) according to an exemplary embodiment.

[0023] FIG. 8 A typical wound healing assay/scratch test of HaKaT cells on BC containing pure RJ 25 ug/ml, 50 ug/ml and 100 ug/ml. BC containing RJ Extract 25 ul, 50 ul and 100 ul according to an exemplary embodiment. Yellow markings show the area free of cells where no cell migration has occurred yet.

[0024] FIGS. 9A and 9B A typical quantitative analysis of the cell migration assay/scratch test of HaKaT cells on Bacterial cellulose RJ Extract (9A) and pure RJ (9B) according to an exemplary embodiment.

[0025] FIG. 10 A flow chart

DETAILED DESCRIPTION

Detailed Description of the Illustrated Embodiments

[0026] Exemplary embodiments relate to an antibacterial and fast healing wound dressing prepared through biosynthesis. Exemplary embodiments of the invention provide dressings made by bacteria through bacterial synthesis composed of pure cellulose polymer synthesized in the form of membranes. Method of preparation of the sheets employed the use of simple sweetened green tea as media with the provision of low pH through apple cider vinegar, as a result of 2-4-day fermentation, sheets were produced at the air water interface. These membranes were then purified to remove the impurities and bacteria by a simple treatment where sodium hydroxide has been used to remove living bacteria from the membranes to get pure cellulose nanofibrous membranes. These membranes are biomaterials to be used for biomedical applications such as wound dressings.

[0027] The BC membranes were impregnated with honey as a broad-spectrum antibacterial agent and RJ extract as a fast healing bioactive. These dressings may be used in various acute and chronic dermal applications. The method is a development of dressings through a process of biosynthesis and use of it as a carrier in which the organic and natural bee products as well a plant extracts were used as wound healing agents, thus enabling a simple, green, and cost effective route for the production of highly efficient wound healing biomedical material. Advantages include room temperatures biosynthesis at 21-30° C., with no need of special instruments or electricity to prepare dressings. Another advantage is that the honey and RJ extract were prepared by centrifugation to isolate the active protein (Defensin-1) which also tested to have twice as fast wound healing potential compared to the current BC membrane dressings loaded with normal saline. Moreover, the method of dressing preparation involves a negligible amount of waste as the inorganic and organic portion of the raw material are almost consumed totally or may be converted into valuable products.

[0028] FIG. 1 illustrates an exemplary cellulose membrane synthesized from sweetened green tea. Bacteria isolated from fermentation culture of symbiotic colony of bacteria and yeast were introduced in a culture broth containing sucrose and green tea extract in the presence of apple cider vinegar.

[0029] FIG. 10 is a flow chart illustrating aspects of the invention.

[0030] In block 100, the method provides preparation of broth. For example, the culture medium can be made from distilled water, to reduce the presence of unnecessary organic matter and other impurities. Green tea 2 gm/L containing antioxidants, polyphenols, flavonoids and vitamins to provide nitrogenous compounds and sugar 100 g/L is added to the culture medium. The bacteria isolated from a symbiotic colony of bacteria and yeast were inoculated at 10% v/v in the culture medium at room temperature 21-26° C. Organic apple cider vinegar was used as acidic content to reduce the pH of media to 3.4-4. The media was left to ferment into beakers at a volume 400 ml/1 L beaker for 2-3 days at static conditions covered with filter paper. The cellulose membranes were formed at the surface of media at air water interface. The resulting membrane ranging in thickness 1.5-5.6 mm is formed on the whole surface area of the beaker. In addition 3 L plastic container/9 cm petri plates can also be used to make variable sized membranes depending upon the time allowed for synthesis.

[0031] In block 100, the method provides purification of the synthesized BC membrane by soaking the BC in absolute alcohol for 1-2 min followed by boiling in 0.4% NaOH at 100° C. for 20 min twice.

[0032] Then the BC is washed three times with distilled water to remove all impurities. This BC is stored for further use by placing in 30% alcohol at 4° C.

[0033] In block 101, the method provides the analysis of BC membrane through FTIR, SEM and Mechanical Testing.

[0034] In block 102 the resulting purified cellulose membrane is sterilized for further use by soaking in 70% ethanol and subsequent washing with distilled water.

[0035] In blocks 103-105 the extracts of natural antibacterial and wound healing agents were prepared. Fresh high quality honey was sterilized by gamma irradiation and stored till further use in BC membrane at a concentration of 75% of the fluid content. The fresh pure organic RJ extract was prepared by diluting in sterile deionized water at a concentration of 100 mg/ml. The diluted RJ was then centrifuged at a speed of 4000 rpm for 15 min to isolate the protein content. This was named as RJ extract. Pure RJ was diluted to a concentration of 100 ug/ml. Pure concentrated curcumin extract was used at a concentration of 3-4 ug/ml. Arginine and Isoleucine amino acids were used at a concentration of 100 ug/ml.

[0036] In block 106 the method provides the loading of natural antibacterial and regenerative agents on the purified BC membranes. The RJ extract was incorporated in combination with honey by placing the BC membrane on the surface of the filter cup and removing 40-50% of water by using vacuum pump. The partially dehydrated membranes were then rehydrated by infusing pure RJ at a conc of 100 ug/ml. In another embodiment the BC was loaded with 100 ug/ml RJ aqueous extract. In another embodiment BC was loaded with honey and RJ aqueous extract at concentration of 750 ug/ml and 100 ug/ml respectively. The active ingredient may comprise 20-30% of honey with NS aqueous extract at a conc. of 100 ug/ml. In yet another embodiment the active ingredient may comprise curcumin or Nigella sativa extract at a conc. of 3-4 ug/ml. In other embodiments, it may be infused with amino acids L-Arginine and L-Isoleucine.

[0037] In block 107 the method provides sterilization of fast healing antibacterial BC dressings after packaging by gamma sterilization.

[0038] Honey is a natural bee product produced by either Apis dorsata, Apis cerana, or Apis florea, the three indigenous bee species of Pakistan or an imported Apis mallifera. In its concentrated form of invert sugar, glucose, fructose, amino acids, proline, aromatic substances, pigments waxes and pollen grains, unstable compounds such as enzymes, vitamins and sugars. The activity of enzymes, hydroxymethylfurfural (HMF), electrical conductivity makes it an effective wound healing substance. In addition, hyperosmotic nature, low pH and presence of hydrogen peroxide make it a potent antimicrobial component. Acacia honey inhibits growth of bacteria yeast and fungi better than other types of honey as the levels of total sugar, pH, HMF, invert sugar, proline, protein and essential nutrients indicate its high quality. Therefore, this honey is good for use as a topical treatment of antibiotic-resistant pathogenic strains. The quality of most natural raw honey of Pakistan fulfills all the requirements of international standards. Furthermore, the antimicrobial activity of honey against pathogenic bacteria, yeast and fungi confirmed that honey is a broad-spectrum antimicrobial agent and has been used for the healing of various types of wounds since ancient times. The honey used in this case was sourced from Karak area of Pakistan and was then gamma sterilized and kept in sterile tubes till further use.

[0039] Examples 1-7 below describe various experiments of synthesis and loading of bacterial cellulose dressings. Example 2 specifically uses fresh RJ loaded onto the purified BC membrane. Example 3 uses RJ aqueous extract as wound healing active. Example 4 uses a combination of RJ extract plus Honey as active wound healing and antibacterial factor. Example 5 uses NS aqueous extract as antibacterial factor loaded on the BC membrane. Example 6 Involves the use of NS and RJ extract as antibacterial and wound healing factor. Example 7 includes the use of amino acids L-Arginine and L-Isoleucine.

EXAMPLE 1

[0040] Example 1 uses the same processes where green tea extract, sugar and apple cider vinegar were used as media in place of Herman Schram media and inoculum of bacteria isolated from 10prior culture was given in a ratio of 10% v/v for synthesis of a Bacterial cellulose membrane instead of a symbiotic colony of bacteria and yeast.

EXAMPLE 2

[0041] Specifically uses fresh RJ loaded onto the purified BC membrane. The purified membranes were partially dehydrated by placing on the filter of a filter cup, then extra water was removed from the membrane using a vacuum pump. While still on the filter surface, the membrane was loaded with fresh RJ diluted in a concentration of 25-100 μg /ml.

EXAMPLE 3

[0042] Uses RJ aqueous extract as wound healing active. The purified membranes were partially dehydrated by placing on the filter of a filter cup, then using the vacuum pump, extra water was removed from the membrane. While still on the filter surface, the membrane was loaded with fresh RJ diluted in a concentration of 25 μl/ml or 50 μl/ml or 100 μl/ml.

EXAMPLE 4

[0043] A combination of RJ extract plus honey was used as active wound healing and antibacterial factor. The purified membranes were partially dehydrated by placing on the filter of a filter cup, then using the vacuum pump, extra water was removed from the membrane. While still on the filter surface, the membrane was loaded with fresh RJ diluted in a concentration of 25 μl/ml or 50 μl/ml or 100 μl/ml added with 75% w/v of pure gamma sterilized honey.

EXAMPLE 5

[0044] NS aqueous extract was used as antibacterial factor loaded on the BC membrane. The purified membranes were partially dehydrated by placing on the filter of a filter cup, then using the vacuum pump, extra water was removed from the membrane. While still on the filter surface, the membrane was loaded with NS aqueous extract (100 g Seeds of NS were weighed and cleaned grinded and added to 1000 ml distilled water in a conical flask for 24 h.) Then the debris was removed by filtration and extracts were used in diluted concentration of 25 μ/ml or 50 μl/ml or 100 μl/ml.

EXAMPLE 6

[0045] Involves the use of Curcumin as antibacterial and wound healing factor. Curcumin extract was used as antibacterial factor loaded on the BC membrane. The purified membranes were partially dehydrated by placing on the filter of a filter cup, then using the vacuum pump, extra water was removed from the membrane. While still on the filter surface, the membrane was loaded with Curcumin aqueous extract (concentration of 3-4 μl/ml). Added to this RJ extract was also loaded in concentrations of 100 μl/ml.

EXAMPLE 7

[0046] Includes the use of amino acids. The purified membranes were partially dehydrated by placing on the filter of a filter cup, then using the vacuum, pump extra water was removed from the membrane. While still on the filter surface, the membrane was loaded with L-Arginine, a non-essential amino acid that plays a key role in protein and amino acid synthesis. It is essential for efficient wound repair, and immune function. L-Arginine and L-Isoleucine is a necessary component of the process of tissue repair as inflammatory cells and fibroblasts use this within the wound for proliferation and as a source of energy, protein and nucleic acid synthesis and collagen deposition.

Fourier Transform Infrared Spectroscopic Analysis of Bacterial Cellulose

[0047] The FTIR spectra were recorded for Bacterial cellulose synthesized in sweetened green tea to identify the characteristics peaks as depicted in FIG. 2. Those assignments agree with the literature. Infrared bands attributed to OH-stretching are located at 3410 cm−1. The majority of the bands corresponding to CH-stretching are located at 2900 cm−1, and the expected absorption bands were in fact observed in the BC spectrum in FIG. 2. H—O—H bending of absorbed water is visible at 1642 cm−1. These data provide evidence of formation of pure bacterial cellulose.

Mechanical Strength Test

[0048] Characterization of the biomechanical properties of the fabricated membranes was investigated by a uniaxial tensile test using a electrodynamic fatigue testing system (Walter+bai ag, LFV-E 1.5 kN, Switzerland). See FIG. 3. To study the ultimate tensile strength, the dried biosynthesized BC membranes were cut keeping uniform width and length which were measured for each sample together with thickness, and then were clamped between two grips of a tensiometer. The length between the clamps was kept uniform for all samples and stress and strain were normalized by area and length of the sample respectively. A load cell of 45 N was used, A ramp test was applied at a rate of 0.1 mm/s and a displacement of 7 mm. Ultimate tensile strength was measured as the plateau at the first failure point. The data were obtained by plotting the max strength achieved before the suture ripped through the membrane as N. Youngs modulus was determined to be 154.2 MPa and ultimate tensile strength was calculated to be 19.69 MPa (FIG. 3). This is considered to be a very high strength for any biomaterial, to render the material easy to handle and apply on the wound area.

Scanning Electron Microscopic Analysis of Bacterial Cellulose

[0049] Scanning electron microscopy analysis of the membranes was done to see the morphology of the membranes in dried form. The membranes were nanofibrous and nanoporous as in FIG. 4A. Such nanoporous structure allows molecules and solutions to pass through but do not allow cells to pass. Therefore, these are the best candidates to cover the wounds from outside. The FIG. 4B shows the layered structure of BC. The lamellar sheet like microstructure is responsible for the high mechanical strength and crystallinity of the membranes.

Antibacterial Assay of Loaded BCM

[0050] Antibacterial effect of loaded BCM was tested through a zone of Inhibition Test, also called a Kirby-Bauer Test. This is a qualitative method to measure antibiotic resistance and to test the ability of substances to inhibit microbial growth. We tested the effect of honey loaded BCMs on S.aureus and E.coli ATCC strains as shown in FIG. 5. Both species of bacteria were inoculated in the sterile nutrient broth and incubated at 37° C. for 24 h. Petri dishes were prepared by pouring sterilized Mueller Hinton Agar medium and incubating at 37° C. for 24 h. Nutrient broth cultures of S.aureus and E coli bacteria were aseptically swabbed on sterile nutrient agar plates. Wells of 5 mm diameter were made aseptically in the inoculated plates. Standard (Tetracycline, 1 mg/ml) and Control (BCM) were also loaded into the respectively labeled wells. The plates were incubated at 37° C. for 24 h in upright position. The experiment was carried out in triplicate and the zone of inhibition was recorded.

In Vitro Evaluation

[0051] Cell cultures studies were done on the membranes using pre-osteoblast cell lines FIG. 6. In T75 culture flask NIH3T3 cells were expanded in the media that is prepared with a-MEM in which 100 μg/ml of Penicillin/Streptomycin were added and 10% FBS was supplemented, in a humidified incubator at a temperature of 37° C. with 5% CO2 and fresh media changes. After 2 to 3 days the cells were expanded and grown to 90% confluence. Afterwards these cells were detached using Trypsin-EDTA. Before seeding, the cells were counted and 50,000 cells were seeded on each sample in the 24 well plate to check the compatibility of the NIH3T3 cells with BC membranes and membranes loaded with different compounds. Before seeding, the samples were sterilized with 70% ethanol for 2 hours. The samples were washed 3 times with PBS at 15 minutes intervals and the cells were cultured on tissue culture plastic plates without membranes as control.

Alamar Blue Assay

[0052] After 3 days, absorbance measurements of Alamar Blue assay were taken to check the biocompatibility of cells with the BC membranes and the attachment of cells onto the membranes. Based on the metabolic activity of the cells, the Alamar blue has a redox indicator which changes from an oxidized (blue) form to a reduced (red) form as the substrate is taken up by the cells. Cell seeded samples were carefully washed with PBS and 0.5 ml of 1 mM Alamar Blue solution was added. After that, it was incubated for 3 to 4 hours at 37° C. Absorbance plate reader (PR4100 Absorbance Microplate Reader BIO RAD, UK) was used to measure the absorbance at 570 nm. Samples were fixated for DAPI staining and SEM analysis, after the results were taken from the plate reader. The results suggest that there was significantly higher cell proliferation in the cultures containing pure RJ at a concentration of 10 ug-100 ug/ml. This increased with increase in the concentration of RJ as shown in FIG. 6A. Similarly, the BCM containing RJ extract at a concentration of 20 ul/ml-60 ul/ml also show increasing trend in the proliferation of cells with increasing concentration of RJ extract in FIG. 6B. These results suggest that RJ and RJ extract both increase cell proliferation twice as much as without RJ.

Cell Attachment on BCM Through Fluorescence Microscopy

[0053] Fluorescence microscopy was done using 89404-464 VWR microscope having 240V with frequency of 50-60 Hz and a halogen lamp of 6V and 30W. The live cells were stained with DAPI and fluorescence images were taken after excitation with UV light with a 460 nm filter at 10×, magnification. The images clearly show cell attachment on membranes. The nuclei stained in blue are clearly seen on the surface of transparent wet BCM in FIGS. 7A and 7B, respectively. Therefore it can be concluded that BCM is a good biomaterial for cell attachment and proliferation, thus possesses regenerative potential.

Scratch Test (Wound Healing Assay)

[0054] The in vitro scratch assay is an easy, low-cost and well-developed method to measure cell migration in vitro. The skin cells HaKaT were selected as representative for the end user application on skin healing. The cells were seeded on 6 well culture plates to create a confluent monolayer. The dishes were incubated for approximately 6 h at 37° C., allowing cells to adhere and spread on the substrate completely. Then the cell monolayer was scraped in a straight line with a 1000 ul pipette tip. The debris was removed and washed once with DMEM and then DMEM supplemented with 25 50, and 100 ug of RJ and 25 ul, 50 ul and 100 ul of RJ aqueous extract was added. The edges of the scratch were marked with a black marker on the bottom of the dish to be used as reference point for image analysis, and images were then taken under an inverted microscope. The scratched dishes were incubated for 3 h, 6 h and 24 h and imaged again. The images acquired for each sample were further analyzed quantitatively by using computing software Image J. By comparing the images from time 0 to the last time point the distance of each scratch closure was obtained on the basis of the distances that are measured by software. The results, as depicted in FIGS. 8A and 8B, suggest a very fast cell migration in cells containing both pure RJ and RJ extract. This is clearly evident from the images taken after 3-6 and 24 hours post scratch (FIG. 8A). The area covered by the cells to cover the scratch suggests wound healing potential which was measured through image J software and was determined to be significantly higher than a control. There was clear evidence that a concentration of 25 ug/ml was best to activate the cell migration and thus healing compared to 50 and 100 ug/ml.

[0055] Similarly as in FIG. 8B, the purified RJ extract also determined a higher cell migration after just 3 h of scratch compared to the other two concentrations.